EA040816B1 - GLP-1 RECEPTOR AGONISTS AND THEIR USE - Google Patents

Abstract

6-carboxylic acids of benzimidazoles and 4-aza-, 5-aza- and 7-azabenzimidazoles (formula I) are proposed as GLP-1R agonists, methods for producing said compounds and methods involving the administration of said compounds to mammals in need thereof.

Description

Field of invention

6-carboxylic acids of benzimidazoles are proposed as GLP-1R agonists, methods for obtaining said compounds and methods involving the administration of said compounds to a mammal in need thereof.

Prior art

Diabetes is a major public health concern due to its increasing prevalence and associated health risks. The disease is characterized by high blood glucose levels caused by problems with insulin production, insulin action, or both. There are two main forms of diabetes: type 1 diabetes and type 2 diabetes. Type 1 diabetes (T1D) occurs when the body’s immune system destroys the beta cells of the pancreas, the only cells in the body that produce the hormone insulin, which regulates blood glucose levels. People with type 1 diabetes must take insulin by injection or pump to survive. Type 2 diabetes (commonly referred to as T2DM) usually begins with either insulin resistance or insufficient insulin production to maintain acceptable glucose levels.

Currently, various pharmacological approaches are available for the treatment of hyperglycemia and subsequently T2DM (Hampp, C. et al. Use of Antidiabetic Drugs in U.S., 2003-2012, Diabetes Care 2014, 37, 1367-1374). These approaches can be grouped into six main classes, each of which acts through different primary mechanisms: (A) insulin secretagogues, including sulfonylureas (eg, glipizide, glimepiride, glyburide), meglitinides (eg, nateglidine, repaglidine), dipeptidyl peptidase IV (DPP-IV) inhibitors (eg, sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin, saxogliptin), and glucagon-like protein-1 receptor (GLP-1R) agonists (eg, liraglutide, albiglutide, exenatide, lixisenatide, dulaglutide, semaglutide), which enhance insulin secretion by acting on pancreatic beta cells. Sulfonylureas and meglitinides have limited efficacy and tolerability, cause weight gain, and frequently induce hypoglycemia. DPP-IV inhibitors have limited efficacy. Commercially available GLP-1R agonists are peptides administered by subcutaneous injection. Liraglutide has been additionally approved for the treatment of obesity. (B) Biguanides (eg, metformin) are thought to act primarily by decreasing hepatic glucose production. Biguanides frequently cause gastrointestinal upset and lactic acidosis, which also limits their use. (C) Alpha-glucosidase inhibitors (eg, acarbose) decrease intestinal absorption of glucose. These agents frequently cause gastrointestinal upset. (D) Thiazolidinediones (eg, pioglitazone, rosiglitazone) act on a specific receptor (peroxisome proliferator-activated receptor-gamma) in the liver, muscle, and adipose tissue. They regulate lipid metabolism, subsequently enhancing the response of these tissues to the actions of insulin. Frequent use of these drugs may lead to weight gain and may cause oedema and anemia. (E) Insulin is used in more severe cases, either alone or in combination with the above agents, and their frequent use may also lead to weight gain and carries a risk of hypoglycemia. (F) Sodium-glucose cotransporter 2 (SGLT2) inhibitors (eg, dapagliflozin, empagliflozin, canagliflozin, ertugliflozin) inhibit glucose reabsorption in the kidneys and thereby lower blood glucose levels. This emerging class of drugs may be associated with ketoacidosis and urinary tract infections.

However, with the exception of GLP-1R agonists and SGLT2 inhibitors, these drugs have limited efficacy and do not address the underlying problems, deterioration of β-cell function and worsening associated obesity.

Obesity is a chronic disease that is highly prevalent in modern society and is associated with numerous medical problems, including hypertension, hypercholesterolemia, and coronary heart disease. It is also closely associated with T2DM and insulin resistance, which is usually accompanied by hyperinsulinemia or hyperglycemia, or both. In addition, T2DM is associated with a two- to four-fold increase in the risk of coronary artery disease. Currently, the only treatment that eliminates obesity with high efficacy is bariatric surgery, but this treatment is expensive and risky. Pharmacological interventions are usually less effective and are associated with side effects. Therefore, there is a clear need for more effective pharmacological interventions with fewer side effects and convenient administration.

Although T2DM is most commonly associated with hyperglycemia and insulin resistance, other diseases associated with T2DM include hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, obesity, dyslipidemia, hypertension, hyperinsulinemia, and nonalcoholic fatty liver disease (NAFLD).

NAFLD is the hepatic manifestation of the metabolic syndrome and represents a spectrum of liver conditions that includes steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and, increasingly, hepatocellular carcinoma. NAFLD and NASH are considered the major fatty liver diseases because they occur in the largest proportion of individuals with elevated hepatic lipid levels. The severity of NAFLD/NASH depends on the presence of lipid, inflammatory cell infiltrate, hepatocyte ballooning, and the degree of fibrosis. Although not all individuals with steatosis develop NASH, a significant proportion do.

GLP-1 is a 30 amino acid long incretin hormone secreted by L cells in the intestine in response to food ingestion. GLP-1 has been shown to stimulate insulin secretion in a physiological and glucose-dependent manner, decrease glucagon secretion, inhibit gastric emptying, reduce appetite, and stimulate beta cell proliferation. In non-clinical experiments, GLP-1 stimulates ongoing beta cell competence by stimulating the transcription of genes important for glucose-dependent insulin secretion and by stimulating beta cell neogenesis (Meier, et al. Biodrugs. 2003; 17(2):93-102).

In healthy individuals, GLP-1 plays an important role in regulating postprandial blood glucose levels by stimulating glucose-dependent insulin secretion from the pancreas, resulting in increased peripheral glucose absorption. GLP-1 also suppresses glucagon secretion, resulting in decreased hepatic glucose production. In addition, GLP-1 delays gastric emptying and slows small intestinal motility, delaying food absorption. In individuals with T2DM, the normal postprandial rise in GLP-1 is absent or decreased (Vilsboll T, et al. Diabetes. 2001. 50; 609-613).

Hoist (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728) report that GLP-1 receptor agonists such as GLP-1, liraglutide, and exendin-4 have 3 major pharmacological activities to improve glycemic control in patients with T2DM by lowering fasting and postprandial glucose (FPG and PPG): (1) increasing glucose-dependent insulin secretion (improving first and second phase), (2) glucagon suppression activity in hyperglycemic states, (3) slowing the rate of gastric emptying resulting in slower absorption of glucose from meals.

There remains a need for easily implementable prevention and/or treatment of cardiometabolic and associated diseases.

Brief description of graphic materials

Fig. 1 is an observed X-ray powder diffraction pattern for the anhydrous (anhydrate) crystalline form (Form 1) of the tris salt of the compound of Example 7.

Fig. 2 is an observed X-ray powder diffraction pattern for the anhydrous (anhydrate) crystalline form (Form A) of the tris salt of the compound of Example 10.

Detailed description of the invention

The present invention relates to a compound of formula I

I or a pharmaceutically acceptable salt thereof, wherein R is F; p is 0 or 1; ring A is phenyl or pyridinyl;

m is 0, 1, 2, or 3;

each R ¹ is independently selected from halogen, -CN, -C _1-3 alkylα or -OC _1-3 alkyl, wherein the alkyl of C _1-3 alkyl and OC _1-3 αalkyl is substituted with 0-3 F atoms;

R ² is H or -C _1-3 alkyl, wherein alkyl is substituted with 0-1 OH;

X-L represents N-CH2 or cyclopropyl;

Y represents CH or N;

R ⁴ is -C1-3alkyl, -C0-3alkylene-C3-6cycloalkyl, -C0-3alkylene-R ⁵ or -C1-3alkylene-R ⁶ , wherein said alkyl may be substituted, when valence permits, with 0-3 substituents independently selected from 0-3 F atoms and 0-1 substituents selected from -C0-1alkylene-OR ^O , -SO2-N(R ⁿ ) ₂ , -C(O)N(R ⁿ )2, -N(C=O)(R ⁿ ) and -N(R ⁿ )2;

R ⁵ is a 4-6 membered heterocycloalkyl selected from oxetanyl, tetrahydrofuranyl, morpholinyl, 1,3-oxazolidinyl and pyrrolidinyl, wherein said heterocycloalkyl may be substituted, when valence permits, with 0-2 substituents independently selected from: 0-1 oxo (=O);

R ⁶ is a 5-6 membered heteroaryl selected from oxazolyl, imidazolyl, pyridinyl, pyrazolyl and triazolyl, wherein said heteroaryl may be substituted, when valence permits, with 0-2 substituents independently selected from:

0-2 -C _1-3 alkyls, wherein the alkyl may be substituted, when valence permits, with 0-3 substituents independently selected from:

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0-1 -OR ^O ;

each R ^O is independently H or -C _1-3 alkyl;

each RN is independently H or -C _1-3 alkyl;

each of Z ¹ , Z ² and Z ³ is -CR ^Z ; and each R ^Z is independently H, F, Cl or -CH ₃ ; or

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-{2[methyl(methylsulfonyl)amino]ethyl}-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof.

Another embodiment relates to a compound of formula II

or a pharmaceutically acceptable salt thereof, where

R represents F;

p is 0 or 1;

ring A is phenyl or pyridinyl;

m is 0, 1 or 2;

each R ¹ is independently selected from halogen, -CN, -C _1-3 alkyl or -OC _1-3 alkyl, wherein the alkyl of C _1-3 alkyl and OC _1-3 alkyl is substituted with 0-3 F atoms;

R ² is H or CH ₃ ;

X-L represents N-CH2 or cyclopropyl;

Y represents CH or N;

Z ³ represents -CR ^Z ; and

R ^Z represents H or -CH ₃ .

Another embodiment relates to a compound of formula III

or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl or pyridinyl;

m is 0, 1 or 2;

each R ¹ is independently selected from F, Cl, or -CN;

R ² is H or CH ₃ ; and

Y represents CH or N.

Another embodiment relates to a compound of formula IV

or a pharmaceutically acceptable salt thereof, wherein m is 0, 1 or 2; each R ¹ is independently selected from F, Cl and -CN; R ² is H or CH ₃ ; and Y is CH or N.

Another embodiment relates to a compound of formula V

or a pharmaceutically acceptable salt thereof, where m is 0 or 1;

R ¹ is F, Cl or -CN;

R ² is H or CH ₃ ; and

Y represents CH or N.

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Another embodiment relates to compounds of formula IV or formula V, wherein the phenyl or pyridinyl ring A has one R ¹ in the para position relative to the carbon atom of said phenyl or pyridinyl attached to the dioxolane, to form:

or pharmaceutically acceptable salts thereof, wherein each R ¹ is independently selected from F, Cl and -CN;

R ² is H or CH ₃ ; and

Y represents CH or N.

Another embodiment relates to compounds of other embodiments, such as compounds of formula I or II or pharmaceutically acceptable salts thereof, wherein XL is N-CH2; and Y is CH or N. Of the embodiments described herein, in such a case, X is N and L is _CH2 .

Another embodiment relates to compounds of other embodiments, such as compounds of formula I or II or pharmaceutically acceptable salts thereof, wherein X-L is cyclopropyl; and Y is N.

In embodiments where X-L is cyclopropyl, the compounds of formula I or II provide:

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein R ⁴ is -CH2CH2OCH3, C1-3alkylene-R ⁵ or C1-3alkylene-R ⁶ , or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of formula II, III, IV or V, wherein R ⁴ is as defined for the compound of formula I, or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein R ⁴ is -C1-3alkyl, wherein said alkyl may be substituted, when valence allows, with 0-1 substituents selected from -C0-1alkylene-OR ^O and -N(R ⁿ )2, or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein R ⁴ is -(CH ₂ ) ₂ OCH ₃ or -(CH ₂ ) ₂ N(CH ₃ ) ₂ , or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein R ⁴ is -CH2-R ⁵ , wherein R ⁵ is 4-5-membered heterocycloalkyl, or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heterocycloalkyl is

where heterocycloalkyl is unsubstituted.

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Another embodiment relates to compounds of formula I, II, III, IV or V, wherein -CH2-R ⁵ and the nitrogen atom to which R ⁴ is attached provide:

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein R ⁴ is -CH2-R ⁶ , wherein R ⁶ is a 5-membered heteroaryl, wherein said heteroaryl may be substituted, when valence permits, with 0-2 substituents independently selected from: 0-1 -CH ₃ , -CH ₂ CH ₃ or -CH ₂ CH ₂ OCH ₃ ; or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heteroaryl is

wherein said heteroaryl may be substituted, when valence permits, with 0-2 substituents, for example substituting a hydrogen atom, independently selected from:

0-2 -C _1-3 alkyls, wherein the alkyl may be substituted, when valence permits, with 0-3 substituents independently selected from:

0-1 -ORO;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heteroaryl is

wherein said heteroaryl may be substituted, when valence permits, with 0-1 -C _1-2 alkyl substituents, wherein alkyl may be substituted, when valence permits, with 0-3 substituents independently selected from: 0-1 -ORO; and each RO is independently H or -C _1-3 alkyl; or pharmaceutically acceptable salts thereof. It is understood that any substituent may replace H on the carbon or nitrogen being substituted. Non-limiting examples of substituted heteroaryls are:

It is clear that H is replaced by a substituent, for example R ^6s (a substituent provided on any heteroaryl R ⁶ ), to form:

wherein R ^6s is -C _1-2 alkyl, wherein the alkyl may be substituted, when valence permits, with 0-3 substituents independently selected from: 0-1 -ORO; and each RO is independently H or -C _1-3 alkyl.

Another embodiment relates to compounds of formula I, II, III, IV or V, wherein heteroaryl is k N-Ci.sal kyl d/. ^OR or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of other embodiments herein, such as compounds of formula I, II, III, IV or V, wherein R ^Z is H; or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of other embodiments herein, such as compounds of formula I, II, III, IV or V, wherein each R ¹ is independently F, Cl or -CN; or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of other embodiments herein, such as compounds of formula I, II, III, IV or V, wherein R ⁴ is -CH ₂ -R ⁵ ; or pharmaceutically acceptable salts thereof.

Another embodiment refers to compounds of other embodiments in this document, such as

- 5 040816 compounds of formulas I, II, III, IV or V, where R ⁴ is -CH2-R ⁶ , or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of other embodiments herein, such as compounds of formula I, II, III, IV or V, wherein the compound is a free acid.

Another embodiment refers to any embodiment of the compounds of formula I, II, III, IV or V, wherein ring A and R2 provide

or their pharmaceutically acceptable salts, where

R represents F;

p is 0 or 1;

m is 0, 1, or 2; and each R ¹ is independently selected from halogen, -CN, -C ₁ . ₃ alkyl, and -OC ₁ . ₃ alkyl, wherein the alkyl of the C ₁ . ₃ alkyl and OC ₁ . ₃ alkyl is substituted with 0-3 F atoms.

Another embodiment relates to compounds of the invention wherein R ² is H; or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of the invention, wherein the compound is

2-( { 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1-yl } methyl)-1 |[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or

2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7fluoro-1-[(2,8)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is

2-( { 4-((2 8)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-1 - [(2 8)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylic acid or

2-( { 4-((2 8)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-7-fluoro-1 -[(2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention wherein R ² is CH ₃ ; or pharmaceutically acceptable salts thereof.

Another embodiment relates to compounds of the invention, wherein the compound is

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-1 - [(2 8)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2,8)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid;

2-({ 4-(2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-1 - [(2 8)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-3-(1,3-oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-((1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-(1,3-oxazol-4-ylmethyl)-1-benzomidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-(pyridin-3-ylmethyl)-1H-benzimidazole-6-carboxylic acid;

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2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-(1,3-oxazol-5-ylmethyl)-1//-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-[(1-ethyl-1H-1,2,3-triazol-5-yl)methyl]-1H-benzamide azole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1//-benzimidazole-6-carboxylic acid;

2-({ 4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1-[(25)-oxetan-2-ylmethyl]-1/7-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylic acid or 2-({4-((28)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is 2-({4-[(28)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or

2-({4-[(28)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-7-fluoro-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is 2-({4-((28)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid;

2-( { 4-((2 8)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl] piperidin1-yl } methyl)-1-[(2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid or

2-( { 4-((2 8)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl } methyl)-1-((1-ethyl-1 H-imidazol-5-yl)methyl]-1 H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is 2-({4-[(2R)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid;

2-({ 4-[(2R)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl } methyl)-1-[(2,8)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl } methyl)-1-((1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is 2-({4-[2-(4-cyano-2-fluorophenyl)-2*-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2,8)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, wherein the chirality 2* is from C56;

2-({ 4-(2-(5-chloropyridin-2-yl)-2*-methyl-1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1-[(2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid, where the chirality 2* is derived from P9;

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2-({4-[2-(4-chloro-2-fluorophenyl)-2*-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid, where the chirality 2* is derived from 17;

2-({4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2*-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, where the chirality of 2* is from 96; or

2-({4-[2-(4-cyano-2-fluorophenyl)-2*-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl} methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylic acid, where the chirality 2* is from C82;

or their pharmaceutically acceptable salts.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof, wherein the salt is the tris-salt.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid as a free acid.

Another embodiment includes a compound that is

or a pharmaceutically acceptable salt thereof.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt, wherein the salt is the tris-salt {the tris-salt of this compound is also known as 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate 1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium}.

In some embodiments, the present invention provides a crystalline form of the anhydrous tris-salt of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid. In some further embodiments, the crystalline form of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid, anhydrous (anhydrate) tris salt is designated Form I, which is characterized by its unique solid state properties with respect to, e.g., X-ray powder diffraction (XRPD), as described herein (substantially as depicted in FIG. 1). In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising at least two characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1 ±0.2°; 14.7 ±0.2°; and 16.9 ±0.2° in 2θ units. In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising at least three characteristic peaks selected from the peaks at 3.7 ±0.2°; 7.3 ±0.2°; 8.5 ±0.2°; 10.1 ±0.2°; 14.7 ±0.2°; and 16.9 ±0.2° in 2θ units. In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising at least four characteristic peaks selected from the peaks at 3.7 ±0.2°; 7.3 ±0.2°; 8.5 ±0.2°; 10.1 ±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units. In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising at least five characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising characteristic peaks at 3.7±0.2° and 7.3±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising peaks at 3.7±0.2°; 7.3±0.2°, and 14.7±0.2° in 2θ units. In some further embodiments, Form I exhibits an X-ray powder diffraction pattern which further comprises at least one peak selected from peaks at 8.5±0.2°; 10.1±0.2°, and 16.9±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising peaks at 3.7±0.2°; 7.3±0.2°; 14.7±0.2°, and 16.9±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2°; and 16.9±0.2° in 2θ units.

- 8 040816

In some embodiments, Form I exhibits an X-ray powder diffraction pattern substantially as shown in Fig. 1. A list of diffraction peaks, expressed in units of degrees 2θ, and relative intensities with a relative intensity >3.0% is presented below in Table X1.

As is well known in the field of powder diffraction, the relative intensities of peaks (reflections) can vary depending on the sample preparation method used, the sample setup procedure, and the specific instrument. Furthermore, variations in instrument readings and other factors can affect the 2-theta values. Consequently, the XRD values of peaks can vary over a range of plus or minus approximately 0.2°.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid as a free acid.

Another embodiment includes a compound that is

or a pharmaceutically acceptable salt thereof.

Another embodiment includes a compound which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]1H-benzimidazole-6-carboxylic acid, 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or 2({4-[(2R)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid as free acid.

Another embodiment includes a compound which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]1H-benzimidazole-6-carboxylic acid, 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or 2({4-[(2R)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; or a pharmaceutically acceptable salt thereof, wherein the salt is a tris-salt.

Another embodiment includes a compound which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]1H-benzimidazole-6-carboxylic acid, DIAST-X2:

or a pharmaceutically acceptable salt thereof. In some further embodiments, the present invention provides a compound which is 2-({4-[2-(5-chloropyridin2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid, DIAST-X2, or a tris-salt [i.e., 1,3-dihydroxy-2(hydroxymethyl)propan-2-amine salt] thereof. The chiral center on the left side of the compound structure is marked as abs to indicate that the chiral center has only one stereoconfiguration (i.e., not a racemate at that chiral center).

In some embodiments, the present invention provides a crystalline form of 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid tris-salt anhydrous, DIAST-X2. In some further embodiments, a crystalline form of 2-({4-[2-(5-chloropyridin-2-yl)2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid tris salt, anhydrous (anhydrate), DIAST-X2, is designated Form A, which is characterized by its unique solid state properties with respect to, for example, X-ray powder diffraction (XRPD), described herein (substantially as depicted in FIG. 2). In some embodiments, Form A exhibits an X-ray powder diffraction pattern comprising at least two characteristic peaks selected from the peaks at 7.7 ± 0.2°; 15.2 ± 0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units. In some embodiments, Form A exhibits an X-ray powder diffraction pattern comprising at least three characteristic peaks selected from the peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units. In some embodiments, Form A exhibits an X-ray powder diffraction pattern comprising characteristic peaks selected from the peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising characteristic peaks at 7.7±0.2° and 17.6±0.2° in 2θ units.

In some embodiments, Form A exhibits an X-ray powder diffraction pattern comprising peaks at 7.7±0.2°; 15.2±0.2°, and 17.6±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising peaks at 7.7±0.2°; 15.2±0.2°, and 15.7±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern comprising peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2°, and 17.6±0.2° in 2θ units.

In some embodiments, Form A exhibits an X-ray powder diffraction pattern substantially as shown in Fig. 2. A list of diffraction peaks, expressed in units of degrees 20, and relative intensities with a relative intensity >3.0% is presented above in Table X2.

As is well known in the field of powder diffraction, the relative intensities of peaks (reflections) can vary depending on the sample preparation method used, the sample setup procedure, and the specific instrument. Furthermore, variations in instrument readings and other factors can affect the 2-theta values. Consequently, the XRD values of peaks can vary over a range of plus or minus approximately 0.2°.

In another embodiment of the invention there is provided a pharmaceutical composition comprising a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, in admixture with at least one pharmaceutically acceptable excipient. This will include a pharmaceutical composition comprising a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, in admixture with at least one pharmaceutically acceptable excipient and one or more other therapeutic agents contemplated herein.

Also disclosed herein: a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use as a medicament; a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use in the prevention and/or treatment of the cardiometabolic and associated diseases contemplated herein, including T2DM, prediabetes, NASH and cardiovascular disease; a method of treating a disease for which a GLP-1R agonist is indicated, in a subject in need of such prevention and/or treatment, comprising administering to the subject a therapeutically effective amount of a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein; the use of a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for the manufacture of a medicament for the treatment of a disease or condition in which a GLP-1R agonist is indicated; a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use in the treatment of a disease or condition in which a GLP-1R agonist is indicated; or a pharmaceutical composition for the treatment of a disease or condition in which a GLP-1R agonist is indicated, comprising a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein.

Each exemplified compound or pharmaceutically acceptable salt thereof may be claimed individually or grouped together in any combination with any number of each and every embodiment described herein.

Also disclosed is a pharmaceutical composition comprising a compound of formula I, II, III, IV or V or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use in the treatment and/or prevention of cardiometabolic and associated diseases contemplated herein, including T2DM, prediabetes, NASH and cardiovascular disease.

Also disclosed is the use of compounds of formula I, II, III, IV or V or pharmaceutically acceptable salts thereof, as defined in any of the embodiments described herein, in the treatment and/or prevention of cardiometabolic and associated diseases including diabetes (T1D and/or T2DM, including prediabetes), idiopathic T1D (type 1b), latent autoimmune diabetes of adults (LADA), early onset T2DM (EOD), atypical diabetes of the young (YOAD), maturity-onset diabetes of the young (MODY), diabetes associated with malnutrition, gestational diabetes, hyperglycemia, insulin resistance, hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, kidney disease (e.g. acute kidney injury, renal tubular dysfunction, proinflammatory changes in the proximal renal tubule), diabetic retinopathy, adipocyte dysfunction, visceral fat deposition, sleep apnea, obesity (including hypothalamic obesity and monogenic

- 10 040816 obesity) and related comorbidities (eg, osteoarthritis and urinary incontinence), eating disorders (including binge eating disorder, bulimia nervosa, and syndromic obesity such as Prader-Willi and Bardet-Biedl syndromes), weight gain due to use of other agents (eg, due to use of steroids and antipsychotics), excessive sugar craving, dyslipidemia (including hyperlipidemia, hyperglyceridemia, elevated total cholesterol, high LDL (low-density lipoprotein) cholesterol and low HDL (high-density lipoprotein) cholesterol, hyperinsulinemia, NAFLD (including related diseases such as steatosis, NASH, fibrosis, cirrhosis, and hepatocellular carcinoma), cardiovascular disease, atherosclerosis (including coronary artery disease), peripheral vascular disease, hypertension, endothelial dysfunction, vascular incompatibility disorder, congestive heart failure, myocardial infarction (e.g., necrosis and apoptosis), stroke, hemorrhagic stroke, ischemic stroke, traumatic brain injury, pulmonary hypertension, postangioplasty restenosis, intermittent claudication, postprandial lipemia, metabolic acidosis, ketosis, arthritis, osteoporosis, Parkinson's disease, left ventricular hypertrophy, peripheral arterial disease, macular degeneration, cataract, glomerulosclerosis, chronic renal failure, metabolic syndrome, syndrome X, premenstrual syndrome, angina pectoris, thrombosis, atherosclerosis, transient ischemic attacks, vascular restenosis, impaired glucose metabolism, impaired fasting glucose states, hyperuricemia, gout, erectile dysfunction, skin and connective tissue disorders, psoriasis, foot ulcers, ulcerative colitis, hyperapolipoprotein B-lipoproteinemia, Alzheimer's disease, schizophrenia, cognitive decline, inflammatory bowel disease, short bowel syndrome, Crohn's disease, colitis, irritable bowel syndrome, in the prevention or treatment of polycystic ovary syndrome and the treatment of addiction (e.g. alcohol and/or drug addiction).

The term alkyl, as used herein, means a monovalent straight or branched chain hydrocarbon group of the formula -C _n H( _2n+ i). Non-limiting examples include methyl, ethyl, propyl, butyl, 2-methyl-propyl, 1,1-dimethylethyl, pentyl, and hexyl.

The term alkylene, as used herein, means a straight or branched chain divalent hydrocarbon group of the formula -C _n H _2n -. Non-limiting examples include ethylene and propylene.

The term cycloalkyl, as used herein, means a cyclic monovalent hydrocarbon group of the formula -C _n H( _2n - ₁ ) containing at least three carbon atoms. Non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term halogen as used in this document refers to fluoride, chloride, bromide, or iodide.

The term heterocycloalkyl as used herein refers to a cycloalkyl group in which one or more ring methylene groups (-CH2-) are substituted with a group selected from -O-, -S-, or a nitrogen atom, wherein the nitrogen atom may provide the point of attachment or may be substituted as provided in each embodiment. In cases where the nitrogen atom provides the point of attachment, the heterocycloalkyl structure depiction will have a hydrogen on said nitrogen atom. Typically, heterocycloalkyl may be substituted, when valence permits, with 0-2 substituents independently selected from oxo, -CN, halogen, alkyl, and -Oalkyl, and the alkyl may be further substituted. It should be noted that in the case of zero substitution, heterocycloalkyl is unsubstituted.

The term heteroaryl as used herein refers to a monocyclic aromatic hydrocarbon containing from 5 to 6 carbon atoms in which at least one ring carbon atom is replaced by a heteroatom selected from an oxygen atom, a nitrogen atom and a sulfur atom. Such a heteroaryl group may be attached via a ring carbon atom or, if valence allows, via a ring nitrogen atom. Typically, heteroaryl may be substituted, when valence allows, with 0-2 substituents independently selected from halogen, OH, alkyl, Oalkyl group and amino (e.g., NH2, NH-alkyl, N(alkyl) ₂ ), and alkyl may be further substituted. It should be noted that in the case of zero substitution, heteroaryl is unsubstituted.

Room temperature: room temperature (from 15 to 25°C).

Methanol: MeOH.

Ethanol: EtOH.

Isopropanol: iPrOH.

Ethyl acetate: EtOAc.

Tetrahydrofuran: THF.

Toluene: PhCH ₃ .

Cesium carbonate: Cs ₂ CO ₃ .

Lithium bis(trimethylsilyl)amide: LiHMDS.

sodium tert-butoxide: NaOtBu.

- 11 040816 Potassium tert-Butoxide: KOtBu.

Lithium diisopropylamide: LDA.

Triethylamine: Et ₃ N.

N,N-Diisopropylethylamine: DIPEA.

Potassium carbonate: K2CO3.

Dimethylformamide: DMF.

Dimethylacetamide: DMAc.

Dimethyl sulfoxide: DMSO.

N-Methyl-2-pyrrolidinone: NMP.

Sodium hydride: NaH.

Trifluoroacetic acid: TFA.

Trifluoroacetic anhydride: TFAA.

Acetic anhydride: Ac2O.

Dichloromethane: DCM.

1,2-Dichloroethane: DCE.

Hydrochloric acid: HCl.

1,8-Diazabicyclo[5.4.0]undec-7-ene: DBU.

Borane-dimethyl sulfide complex: BH ₃ -DMS.

Borane-tetrahydrofuran complex: BH ₃ -THF.

Lithium aluminum hydride: LAH.

Acetic acid: AcOH.

Acetonitrile: MeCN.

para-Toluenesulfonic acid: pTSA.

Dibenzylidinacetone: DBA.

2,2'-Bis(diphenylphosphino)-1,1'-binaphthalene: BINAP.

1,1'-Ferrocenediyl-bis(diphenylphosphine): dppf.

1,3- Bis(diphenylphosphino)propane: DPPP.

3-Chloroperbenzoic acid: m-CPBA.

tert-Butyl methyl ether: MTBE.

Methanesulfonyl: Ms.

N-Methylpyrrolidinone: NMP.

Thin layer chromatography: TLC.

Supercritical fluid chromatography: SFC.

4-(Dimethylamino)pyridine: DMAP.

tert-Butyloxycarbonyl: Vos.

1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate: HATU.

Petroleum ether: PE.

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate: HBTU.

2-Amino-2-(hydroxymethyl)propane-1,3-diol: tris.

Tris(dibenzylideneacetone)dipalladium: Pd ₂ (dba) ₃ .

1H nuclear magnetic resonance (NMR) spectra were consistent with the proposed structures in all cases. Characteristic chemical shifts (δ) are given in parts per million (ppm) relative to the residual proton signal in the deuterated solvent ( _CHCl3 at 7.27 ppm; CD2HOD at 3.31 ppm; MeCN at 1.94 ppm; DMSO at 2.50 ppm) and are reported using standard abbreviations for major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broadened. The symbol ^Λ indicates that the area of the 1H NMR peak was inferred because the peak was partially obscured by the water peak. The symbol λλ indicates that the area of the 1H NMR peak was inferred because the peak was partially obscured by the solvent peak.

The wavy line “' ^ch '” used in this document denotes the point of attachment of a substituent to another group.

The compounds and intermediates described below have been named using the naming convention provided by ACD/ChemSketch 2012, File Version C10H41, Build 69045 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The naming convention provided by ACD/ChemSketch 2012 is well known to those skilled in the art, and the naming convention provided by ACD/ChemSketch 2012 is generally considered to be consistent with the IUPAC (International Union of Pure and Applied Chemistry) recommendations for Nomenclature of Organic Chemistry and the CAS (Chemical Abstract Service) identifier rules. It should be noted that chemical names may have parentheses only or may have parentheses and square brackets. Stereochemical descriptors may also appear in different places within the name itself, depending on the naming convention. One skilled in the art will recognize these placements and recognize that they refer to the same chemical structure.

Pharmaceutically acceptable salts of the compounds of formula I include acid addition salts and base addition salts.

Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, glyceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, 1,5-naphthalenedisulfonic acid salts acids and xinafoate.

Suitable base addition salts are formed from bases that form non-toxic salts. Examples include aluminum salts, salts with arginine, benzathine, calcium salts, salts with choline, diethylamine, bis(2-hydroxyethyl)amine (diolamine), glycine, lysine, magnesium salts, salts with meglumine, 2-aminoethanol (olamine), potassium, sodium salts, salts with 2-amino-2(hydroxymethyl)propane-1,3-diol (tromethamine), and zinc salts.

Hemisalts of acids and bases can also be formed, such as hemisulfates and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable salts of the compounds of formula I can be prepared by one or more of three methods:

(1) by reacting a compound of formula I with the desired acid or the desired base;

(2) by removing an acid- or base-labile protecting group from a suitable precursor of a compound of formula I or by ring opening a suitable cyclic precursor, for example a lactone or lactam, using the desired acid or the desired base; or (3) by converting one salt of a compound of formula I into another by reaction with a suitable acid or a suitable base or by using a suitable ion exchange column.

All three reactions are usually carried out in solution. The resulting salt may precipitate and may be collected by filtration or may be isolated by evaporation of the solvent. The degree of ionization of the resulting salt may vary from fully ionized to nearly unionized.

The compounds of formula I and their pharmaceutically acceptable salts may exist in unsolvated form and in solvated forms. The term solvate is used herein to describe a molecular complex comprising a compound of formula I or a pharmaceutically acceptable salt thereof and one or more molecules of a pharmaceutically acceptable solvent, such as ethanol.

The term hydrate is used when the specified solvent is water.

The currently accepted classification system for organic hydrates is one that defines free hydrates, channel-type hydrates, or metal-ion coordinated hydrates (see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995)). Free hydrates are hydrates in which the water molecules are isolated from direct contact with each other by the organic molecules lying between them. In channel-type hydrates, the water molecules lie in lattice channels where they are adjacent to other water molecules. In metal-ion coordinated hydrates, the water molecules are bound to a metal ion.

When the solvent or water is strongly bound, then the complex will have a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, as in the case of channel solvates and hygroscopic compounds, then the water/solvent content may depend on humidity and drying conditions. In such cases, lack of stoichiometry will be the norm.

Also included within the scope of the present invention are multi-component complexes (other than salts and solvates) in which the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are generally defined as crystalline complexes of neutral molecular constituents that are bound together by non-covalent interactions, but may also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallization, solvent recrystallization, or physical comminution of the components together (see Chem. Commun., 17, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004)). For a general review of multi-component complexes, see J Pharm. Sci, 64(8), 1269-1288, Haleblian (August 1975).

The compounds of the invention may exist in a continuum of solid states ranging from completely amorphous to completely crystalline. The term amorphous refers to the state in which

- 13 040816 in which the substance has no long-range order at the molecular level and, depending on the temperature, can exhibit the physical properties of a solid or a liquid. Such substances usually do not give characteristic X-ray powder diffraction patterns and, while simultaneously exhibiting the properties of a solid, are more formally characterized as a liquid. When heated, a transition from the properties of a solid to those of a liquid occurs, which is characterized by a change of state, usually of the second order (vitrification). The term crystalline refers to a solid phase in which the substance has a regular, ordered internal structure at the molecular level and gives a characteristic X-ray powder diffraction pattern with defined peaks. Such substances, when heated sufficiently, will also exhibit the properties of a liquid, but the transition from the solid state to the liquid is characterized by a phase transition, usually of the first order (melting point).

Compounds of formula I may also exist in a mesomorphic state (mesophase or liquid crystal) when exposed to appropriate conditions. The mesomorphic state is an intermediate state between the true crystalline state and the true liquid state (either a melt or a solution). Mesomorphism that occurs as a result of a change in temperature is classified as thermotropic, and mesomorphism that occurs as a result of the addition of a second component, such as water or another solvent, is classified as lyotropic. Compounds that can form lyotropic mesophases are classified as amphiphilic and consist of molecules that contain an ionic (such as -COO ^- Na ⁺ , -COO-K+, or -SO3 ^- Na ⁺ ) or non-ionic (such as -N ^- N ⁺ (CH3) ₃ ) polar end group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and A. Stuart, ^4th Edition (Edward Arnold, 1970).

The compounds of formula I may exhibit polymorphism and/or one or more types of isomerism (e.g. optical, geometric or tautomeric isomerism). The compounds of formula I may also be labeled with isotopes. Such variations are intended for the compounds of formula I defined as such based on their structural features and are therefore within the scope of the invention.

Compounds of formula I containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. If a compound of formula I contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. If structural isomers are capable of interconversion due to a low energy barrier, tautomeric isomerism (tautomerism) may occur. This may take the form of proton tautomerism in compounds of formula I containing, for example, an imino, keto or oxime group, or so-called valence tautomerism in compounds that contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.

Pharmaceutically acceptable salts of the compounds of formula I may also contain a counterion that is optically active (e.g. d-lactate or l-lysine) or racemic (e.g. dl-tartrate or dl-arginine).

Cis/trans isomers can be separated by standard methods known to those skilled in the art, such as chromatography and fractional crystallization.

Standard methods for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). Alternatively, a racemic precursor containing a chiral ester can be resolved by enzymatic resolution (see, for example, Int J Mol Sci 29682-29716 by A. C. L. M. Carvaho et al. (2015)). In case the compound of formula I contains an acidic or basic moiety, a salt can be formed with an optically pure base or an optically pure acid, for example with l-phenylethylamine or tartaric acid. The resulting diastereomeric mixture can be separated by fractional crystallization, and one or both diastereomeric salts can be converted to the corresponding pure enantiomer(s) using methods known to those skilled in the art. Alternatively, the racemate (or racemic precursor) can be covalently reacted with a suitable optically active compound, for example an alcohol, an amine or benzyl chloride. The resulting diastereomeric mixture can be separated by chromatography and/or fractional crystallization by methods known in the art to afford the individual diastereomers as single enantiomers with 2 or more chiral centers. The chiral compounds of formula I (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume isopropanol, typically 2 to 20%, and from 0 to 5% by volume alkylamine, typically 0.1% diethylamine. Concentration of the eluate yields an enriched mixture. Chiral chromatography using sub- and supercritical fluids can be used. Chiral chromatography techniques useful in some embodiments of the present invention are known in the art (see, e.g., Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography

- 14 040816 with Packed Columns), pp. 223-249, and references cited therein). In some of the relevant examples cited in this description, the columns were obtained from Chiral Technologies, Inc, West

Chester, Pennsylvania, USA, a subsidiary of Daicel® Chemical Industries, Ltd., Tokyo, Japan.

When any racemate crystallizes, two different types of crystals may form. The first type is the racemic compound (true racemate) mentioned above, where one homogeneous crystal form is formed, containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate, where two crystal forms are formed in equimolar amounts, each containing a single enantiomer. Although both crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to a true racemate. Racemic mixtures can be separated by standard techniques known to those skilled in the art (see, for example, Stereochemistry of Organic Compounds by E. L. Eliel and S. H. Wilen (Wiley, 1994)).

It should be emphasized that although the compounds of formula I are depicted in this description in a single tautomeric form, all possible tautomeric forms are within the scope of the invention.

The present invention encompasses all pharmaceutically acceptable isotopically labeled compounds of formula I in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number that predominates in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen such as ² H and ³ H, carbon such as ⁿ C, ¹³ C and ¹⁴ C, chlorine such as ³⁶ Cl, fluorine such as ¹⁸ F, iodine such as ¹²³ I and ¹²⁵ I, nitrogen such as ¹³ N and ¹⁵ N, oxygen such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus such as ³² P, and sulfur such as ³⁵ S.

Certain isotopically labeled compounds of formula I, such as those with an incorporated radioactive isotope, are useful in tissue distribution studies of drugs and/or substrates. The radioactive isotopes tritium, i.e., ³ H, and carbon-14, i.e., ¹⁴ C, are particularly useful for this purpose because of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ² H, may provide some therapeutic benefits due to greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements.

Substitution with positron-emitting isotopes such as ^nC , ^18F , ^15O , and ^13N may be useful in positron emission tomography (PET) studies to examine receptor occupancy by substrate.

Isotopically labeled compounds of formula I can generally be prepared by standard methods known to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using an isotopically labeled reagent in place of the unlabeled reagent previously used.

Pharmaceutically acceptable solvates according to the invention include those solvates in which the crystallization solvent may be substituted with an isotope, for example D2O, d ₆ -acetone, d ₆ -DMSO.

One mode of carrying out the invention is to administer a compound of formula I in the form of a prodrug. Thus, certain derivatives of the compound of formula I, which themselves may have little or no pharmacological activity, may be converted, when administered into or to the body, into a compound of formula I having the desired activity, for example by hydrolytic cleavage, in particular by hydrolytic cleavage stimulated by an esterase or peptidase enzyme. Such derivatives are called prodrugs. Further information on the use of prodrugs can be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (Ed. E. B. Roche, American Pharmaceutical Association). Reference may also be made to Nature Reviews/Drug Discovery, 2008, 7, 355, and Current Opinion in Drug Discovery and Development, 2007, 10, 550.

Prodrugs according to the invention can be obtained, for example, by replacing the corresponding functional groups present in the compounds of formula I with certain moieties known to those skilled in the art as promoieties, which are described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985) and Y. M. Choi-Sledeski and C. G. Wermuth, Designing Prodrugs and Bioprecursors in Practice of Medicinal Chemistry, (Fourth Edition), Chapter 28, 657-696 (Elsevier, 2015).

Thus, a prodrug according to the invention is (a) an ester or amide derivative of a carboxyl group in a compound of formula I; (b) an ester, carbonate, carbamate, phosphate or ether derivative of a hydroxyl group in a compound of formula I; (c) an amide, imine, carbamate or amine derivative of an amino group in a compound of formula I; (d) an oxime or imine derivative of a carbonyl group in a compound of formula I; or (e) a methyl, primary alcohol or aldehyde group which can be metabolically oxidized to a carboxylic acid group in a compound of formula I.

Some specific examples of prodrugs according to the invention include:

(1) if the compound of formula I contains a carboxyl functional group (-COOH), then an ester thereof, such as a compound in which the hydrogen of the carboxylic acid functional group of the compound of formula I is replaced by C1- _C8 alkyl (e.g. ethyl) or by a (C1- _C8 alkyl)C(=O) _OCH2 group (e.g. 'BuC(=O) _OCH2- );

(2) if the compound of formula I contains an alcohol functional group (-OH), then its ester, such as a compound in which the hydrogen of the alcohol functional group of the compound of formula I is replaced by a -CO(C1- _C8 alkyl) group (e.g. methylcarbonyl), or the alcohol is esterified with an amino acid;

(3) if the compound of formula I contains an alcohol functional group (-OH), then its ether, such as a compound in which the hydrogen of the alcohol functional group of the compound of formula I is replaced by a group (C ₁ -C ₈ alkyl)C(=O)OCH ₂ - or -CH ₂ OP(=O)(OH) ₂ ;

(4) if the compound of formula I contains an alcohol functional group (-OH), then its phosphate, such as a compound in which the hydrogen of the alcohol functional group of the compound of formula I is replaced by a group -P(=O)(OH) ₂ , or -P(=O)(ONa) ₂ , or -P(=O)(O ^- ) ₂ Ca ²⁺ ;

(5) if the compound of formula I contains a functional primary or secondary amino group (-NH2 or -NHR, where R ^ H), then its amide, for example a compound in which, depending on the circumstances, one or both hydrogen atoms of the functional amino group of the compound of formula I is/are substituted by (C1- _C10 )alkanoyl, -COCH2NH2, or the amino group is derivatized with an amino acid;

(6) if the compound of formula I contains a functional primary or secondary amino group (-NH2 or -NHR, where R ^ H), then its amine, for example a compound in which, depending on the circumstances, one or both hydrogen atoms of the functional amino group of the compound of formula I is/are substituted by the group -CH2OP(=O)(OH)2;

(7) wherein the carboxyl group in the compound of formula I is substituted by a methyl group, a -CH2OH group or an aldehyde group.

Certain compounds of formula I may themselves act as prodrugs of other compounds of formula I. It is also possible for two compounds of formula I to be linked together to form a prodrug. In some circumstances, a prodrug of a compound of formula I may be created by internally linking two functional groups in a compound of formula I, such as by forming a lactone.

References to compounds of formula I include the compounds themselves and prodrugs thereof. The invention encompasses such compounds of formula I as well as pharmaceutically acceptable salts of such compounds and pharmaceutically acceptable solvates of said compounds and salts.

Administration and dosage

Typically, a compound of the invention is administered in an amount effective to treat a condition as described herein. The compounds of the invention may be administered as the compound itself or, alternatively, as a pharmaceutically acceptable salt. For purposes of administration and dosage, the compound itself or a pharmaceutically acceptable salt thereof will be referred to simply as compounds of the invention.

The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition suitable for such route and in a dose effective for the intended treatment. The compounds of the invention may be administered orally, rectally, vaginally, parenterally or topically.

The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be used, in which the compound enters the bloodstream directly from the oral cavity.

In another embodiment, the compounds of the invention can be administered directly into the bloodstream, into a muscle, or into an internal organ. Suitable routes of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion devices.

In another embodiment, the compounds of the invention can also be administered locally to the skin or mucous membrane, i.e. dermally or transdermally. In another embodiment, the compounds of the invention can also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention can be administered rectally or vaginally. In another embodiment, the compounds of the invention can also be administered directly to the eye or ear.

The dosage regimen for the compounds of the invention and/or compositions containing said compounds is determined by taking into account a number of factors including the type, age, body weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound used. Therefore, the dosage regimen can vary within very wide limits. In one embodiment, the total daily dose of a compound of the invention is typically from about 0.001 to about 100 mg/kg (i.e. mg of compound of the invention per kg of body weight) for the treatment of the conditions discussed herein. In another embodiment, the total daily dose of a compound of the invention is from about 0.01 to about 30 mg/kg, and in another embodiment from about 0.03 to about 10 mg/kg, and in yet another embodiment from about 0.1 to about 3. It is not uncommon for the administration of a compound of the invention to be repeated several times a day (usually not more than 4 times). Multiple daily doses can usually be used to increase the total daily dose if required.

For oral administration, the compositions can be provided in the form of tablets containing 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 30.0, 50.0, 75.0, 100, 125, 150, 175, 200, 250 and 500 mg of the active ingredient for symptomatic dosage adjustment for the patient. The drug typically contains from about 0.01 to about 500 mg of the active ingredient or, in another embodiment, from about 1 to about 100 mg of the active ingredient. Intravenously administered doses can range from about 0.01 to about 10 mg/kg/min at a constant infusion rate.

Suitable subjects of the invention include mammalian subjects. In one embodiment, suitable subjects are humans. Human subjects may be of either sex and at any stage of development.

Pharmaceutical compositions

In another embodiment, the invention encompasses pharmaceutical compositions. Such pharmaceutical compositions comprise a compound of the invention present with a pharmaceutically acceptable carrier. Other physiologically active substances may also be present. As used herein, the term pharmaceutically acceptable carrier encompasses any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof, and may have isotonic agents in the composition, for example, sugars, sodium chloride, or polyalcohols such as mannitol or sorbitol. Pharmaceutically acceptable substances such as wetting agents or minor amounts of excipients such as wetting agents or emulsifiers, preservatives or buffers that enhance the shelf life or potency of the antibody or antibody portion.

The compositions of the present invention can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g. injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form depends on the intended route of administration and therapeutic use.

Typical compositions are in the form of injection or infusion solutions, such as compositions similar to those used for passive immunization of humans with antibodies, typically. One route of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the antibody is administered by intravenous infusion or injection. In another embodiment, the antibody is administered by intramuscular or subcutaneous injection.

For oral administration, the solid dosage form may be presented, for example, in discrete units such as hard or soft capsules, pills, cachets, troches or tablets, each containing a predetermined amount of at least one compound of the invention. In another embodiment, oral administration may be in the form of a powder or granules. In another embodiment, the oral dosage form is sublingual, such as, for example, a troche. In such solid dosage forms, the compounds of formula I are typically combined with one or more excipients. Such capsules or tablets may be controlled-release preparations. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents or may be formulated with an enteric coating.

In another embodiment, oral administration can be carried out in a liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions can also contain auxiliary substances such as wetting, emulsifying, suspending, correcting (e.g., sweetening) agents and/or flavoring agents.

In another embodiment, the invention encompasses a parenteral dosage form. Parenteral administration includes, for example, subcutaneous injections, intravenous injections, intraperitoneal, intramuscular injections, intrasternal injections and infusion. Injectable preparations (i.e., sterile injectable aqueous or oily suspensions) can be prepared by known

- 17 040816 in this area by methods using suitable dispersing, wetting and/or suspending agents.

In another embodiment, the invention encompasses a topical dosage form. Topical administration includes, for example, transdermal administration, such as by means of transdermal patches or iontophoresis devices, intraocular administration, or intranasal administration, or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments and creams. The topical composition may contain a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of the invention are administered by means of a transdermal device, then the administration will be carried out using a patch of either a reservoir type or a porous membrane or solid matrix type. Topical compositions for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be included (see, e.g., B. C. Finnin and T. M. Morgan, J. Pharm. Sci., vol. 88, p. 955-958, 1999).

Forms of compositions for topical administration to the eye include, for example, eye drops, wherein the compound of the invention is dissolved or suspended in a suitable carrier. A typical composition suitable for administration to the eyes and ears may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted sterile saline. Other forms of composition suitable for administration to the eyes and ears include ointments, biodegradable (i.e. absorbable gel sponges, collagen) and non-biodegradable (i.e. silicone) implants, wafers, lenses and particulate or vesicular systems such as niosomes or liposomes. A polymer such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulose polymer such as hydroxypropyl methylcellulose, hydroxyethylcellulose or methylcellulose, or a heteropolysaccharide polymer such as gellan gum may be included in the composition together with a preservative such as benzalkonium chloride. Such compositions may also be delivered by iontophoresis.

For intranasal or inhalation administration, the compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump-operated nebulizer container squeezed or pumped by the patient, or as an aerosol spray from a pressurized container or nebulizer using a suitable propellant. Compositions suitable for intranasal administration are typically administered in the form of a dry powder (either alone or as a mixture, such as a dry mixture with lactose, or as a mixture of component particles, such as a mixture with phospholipids such as phosphatidylcholine) from a dry powder inhaler or an aerosol spray from a pressurized container, pump, nebulizer, atomizer (preferably an atomizer using electrohydrodynamics to create a fine mist) or nebulizer, with or without the use of a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. The powder for intranasal use may contain a bioadhesive agent, such as chitosan or cyclodextrin.

In another embodiment, the invention encompasses a rectal dosage form. Such a rectal dosage form may be, for example, a suppository. Cocoa butter is a traditional suppository base, but various other alternatives may be used if appropriate.

Other carrier materials and routes of administration known in the pharmaceutical art may also be used. The pharmaceutical compositions of the invention may be prepared by any of the generally known methods of pharmacy, such as effective formulation and administration methods. The above considerations with respect to effective formulations and administration methods are known in the art and are described in standard textbooks. The preparation of drugs is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

Co-administration

The compounds of the invention may be used alone or in combination with other therapeutic agents. The invention provides any uses, methods or compositions as defined herein, wherein a compound of any embodiment of formula I described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or said salt, is used in combination with one or more other therapeutic agents discussed herein. This combination will include a pharmaceutical composition for the treatment of a disease or condition for which a GLP-1R agonist is indicated, comprising a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, and one or more

- 18 040816 than any other therapeutic agent discussed in this document.

Administration of two or more compounds in combination means that all compounds are administered close enough in time that each can produce a biological effect within the same time period. The presence of one agent may alter the biological effects of the other compound(s). Two or more compounds may be administered simultaneously, in parallel, or sequentially. In addition, simultaneous administration may be accomplished by mixing the compounds prior to administration or by administering the compounds at the same time but as separate dosage forms at the same site or at different sites of administration.

The phrases concurrent administration, co-administration, simultaneous administration and administration simultaneously mean that the compounds are administered in combination.

Also provided are methods of treatment comprising administering the compounds of the present invention in combination with one or more other pharmaceutical agents, wherein the one or more other pharmaceutical agents may be selected from the agents discussed herein.

The compounds of the present invention can be administered with an antidiabetic agent including, but not limited to, a biguanide (e.g., metformin), a sulfonylurea (e.g., tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, gliclopyramide, glimepiride, or glipizide), a thiazolidinedione (e.g., pioglitazone, rosiglitazone, or lobeglitazone), a glitazar (e.g., saroglitazar, aleglitazar, muraglitazar, or tesaglitazar), a meglitinide (e.g., nateglinide, repaglinide), a dipeptidyl peptidase 4 (DPP-4) inhibitor (e.g., sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, dutogliptin, or omarigliptin), a glitazone (e.g., pioglitazone, rosiglitazone, balaglitazone, rivoglitazone, or lobeglitazone), a sodium glucose cotransporter 2 (SGLT2) inhibitor (e.g., empagliflozin, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, or ertugliflozin), an SGLTL1 inhibitor, a GPR40 agonist (FFAR1/FFA1 agonist, e.g., fasiglyfam), glucose-dependent insulinotropic peptide (GIP) and its analogues, an alpha-glucosidase inhibitor (e.g., voglibose, acarbose, or miglitol), or insulin or an insulin analogue, including pharmaceutically acceptable salts of the specifically named agents and pharmaceutically acceptable solvates of the agents mentioned and salts.

The compounds of the present invention can be administered with an anti-obesity agent including, but not limited to, peptide YY or an analogue thereof, a neuropeptide Y receptor type 2 (NPYR2) agonist, an NPYR1 or NPYR5 antagonist, a cannabinoid receptor type 1 (CB1R) antagonist, a lipase inhibitor (e.g., orlistat), human proislet peptide (HIP), a melanocortin 4 receptor agonist (e.g., setmelanotide), a melanin-concentrating hormone 1 receptor antagonist, a farnesoid X receptor (FXR) agonist (e.g., obeticholic acid), zonisamide, phentermine (alone or in combination with topiramate), a norepinephrine/dopamine reuptake inhibitor (e.g., bupropion), an opioid receptor antagonist (e.g., naltrexone), a combination norepinephrine/dopamine reuptake inhibitor and opioid receptor antagonist (eg, the combination of bupropion and naltrexone), GDF-15 analog, sibutramine, cholecystokinin agonist, amylin and its analogs (eg, pramlintide), leptin and its analogs (eg, metroleptin), serotonergic agent (eg, lorcaserin), methionine aminopeptidase 2 (MetAP2) inhibitor (eg, beloranib or ZGN-1061), phendimetrazine, diethylpropion, benzphetamine, SGLT2 inhibitor (eg, empagliflozin, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate, or ertugliflozin), SGLTL1 inhibitor, dual SGLT2/SGLT1, a fibroblast growth factor receptor (FGFR) modulator, an AMP-activated protein kinase (AMPK) activator, biotin, a MAS receptor modulator, or a glucagon receptor agonist (alone or in combination with another GLP-1R agonist, such as liraglutide, exenatide, dulaglutide, albiglutide, lixisenatide, or semaglutide), including pharmaceutically acceptable salts of the specifically named agents and pharmaceutically acceptable solvates of the agents and salts.

The compounds of the present invention can be administered with an agent for treating NASH, including, but not limited to, PF-05221304, an FXR agonist (e.g., obeticholic acid), a PPAR α/δ agonist (e.g., elafibranor), a synthetic fatty acid-bile acid conjugate (e.g., aramchol), a caspase inhibitor (e.g., emricasan), an anti-lysyl oxidase homolog 2 (LOXL2) monoclonal antibody (e.g., simtuzumab), a galectin 3 inhibitor (e.g., GR-MD-02), a MAPK5 inhibitor (e.g., GS-4997), a dual chemokine receptor 2 (CCR2) and CCR5 antagonist (e.g., cenicriviroc), a fibroblast growth factor 21 (FGF21) agonist (e.g., BMS-986036), a leukotriene D4 receptor (LTD4) inhibitor (e.g., tipelukast), niacin analog (e.g., ARI 3037MO), ASBT inhibitor (e.g., volixibat), acetyl CoA carboxylase (ACC) inhibitor (e.g., NDI 010976), ketohexokinase (KHK) inhibitor, diacylglyceryl acyltransferase 2 (DGAT2) inhibitor, CB1 receptor antagonist, anti-CB1R antibody, or apoptosis signal-regulated kinase 1 (ASK1) inhibitor, including pharmaceutically acceptable salts of the specifically named agents and pharmaceutically acceptable solvates of said agents and salts.

Some specific compounds that can be used in combination with the compound

- 19 040 816 compounds of the present invention for treating the diseases or disorders described herein (e.g., NASH) include: 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid, which is an example of a selective ACC (acetyl-coenzyme A carboxylase) inhibitor and was prepared as the free acid in Example 9 of U.S. Patent No. 8,859,577, which is U.S. National Phase I International Application PCT/GB2011/054P9, the disclosures of which are incorporated herein by reference in their entirety for all purposes; crystalline forms of 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid, including the anhydrous mono-tris form (Form 1) and the trihydrate mono-tris salt (Form 2), are described in International Application PCT/IB2018/058966, the disclosure of which is incorporated herein by reference in its entirety for all purposes; (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide, or a pharmaceutically acceptable salt and crystalline solid forms (Form 1 and Form 2) thereof, which is an example of a DGAT2 (diacylglycerol O-acyltransferase 2) inhibitor described in Example 1 of U.S. Patent No. 1,0071,992, the disclosure of which is incorporated herein by reference in its entirety for all purposes; [(1R,5S,6R)3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6yl]acetic acid or a pharmaceutically acceptable salt thereof (including its crystalline free acid form), which is an example of a ketohexokinase inhibitor and is described in Example 4 of U.S. Patent No. 9,809,579, the disclosure of which is incorporated herein by reference in its entirety for all purposes; and the FXR agonist tropifexor or a pharmaceutically acceptable salt thereof, which is described in Example 1-1B of U.S. Patent No. 9,150,568, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

These agents and compounds of the invention can be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, etc. The specific dosage regimen, i.e., dose, time of administration, and frequency, will depend on the particular individual and the individual's medical history.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and may include buffers such as phosphate buffer, citrate buffer, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or Ig (immunoglobulins); hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethyleneglycol (PEG).

Liposomes containing these agents and/or the compounds of the invention are prepared by methods known in the art, such as those described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be prepared by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters of defined pore sizes to obtain liposomes of the desired diameter.

These agents and/or compounds of the invention may also be incorporated into microcapsules obtained, for example, by coacervation or interfacial polymerization techniques, such as hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

Sustained release preparations may be used. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing a compound of formula I, II, III, IV or V in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol, polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-E-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic acid and glycolic acid such as the copolymers used in LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic acid and glycolic acid and leuprolide acetate), sucrose acetate isobutyrate, and poly-O-(-)-3-hydroxybutyric acid.

Compositions intended for intravenous administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. The compounds of the invention are typically placed in a container having a sterile access port, such as an intravenous solution bag or a vessel having a stopper pierced by a hypodermic needle.

Suitable emulsions may be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient is either dissolved in a pre-mixed emulsion composition or, alternatively, it may be dissolved in an oil (e.g., soybean oil, sunflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and the emulsion formed by mixing with a phospholipid (e.g., egg phospholipids, soy phospholipids or soy lecithin) and water. It will be appreciated that other ingredients, e.g., glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, e.g., 5 to 20%. The fat emulsion may contain fat droplets of 0.1 to 1.0 µm, especially 0.1 to 0.5 µm, and may have a pH in the range of 5.5 to 8.0.

Compositions in the form of an emulsion may be emulsions that are prepared by mixing a compound of the invention with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients, as set out above. In some embodiments, the compositions are administered orally or nasal respiratory route for local or systemic effect. Compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized using gases. Nebulized solutions can be inhaled directly from a nebulizer, or the nebulizer can be attached to a face mask, tent or intermittent positive pressure breathing machine. Compositions in the form of a solution, suspension or powder can be administered preferably orally or nasally from devices that deliver the composition accordingly.

Sets

In another aspect of the invention, kits are disclosed comprising a compound of formula I, II, III, IV or V or a pharmaceutical composition comprising a compound of formula I, II, III, IV or V according to the invention. The kit may include, in addition to the compound of formula I, II or III according to the invention or a pharmaceutical composition comprising it, diagnostic or therapeutic agents. The kit may also include instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit includes a compound of formula I, II, III, IV or V or a pharmaceutical composition comprising it and a diagnostic agent. In other embodiments, the kit includes a compound of formula I, II, III, IV or V or a pharmaceutical composition comprising it.

In yet another embodiment, the invention encompasses kits that are suitable for use in performing the treatment methods described herein. In one embodiment, the kit comprises a first dosage form comprising one or more compounds of the invention in amounts sufficient to perform the methods of the invention. In another embodiment, the kit comprises a first dosage form comprising one or more compounds of the invention in amounts sufficient to perform the methods of the invention, and a dosing container.

Receiving

Compounds of formula I, II, III, IV, or V can be prepared by the general and specific methods described below using the ordinary general knowledge of one skilled in the art of organic chemistry. Such ordinary general knowledge can be found in standard reference works such as Comprehensive Organic Chemistry, Ed. Barton and Ollis, Elsevier; Comprehensive Organic Transformations: A Guide to Functional Group Preparations, Larock, John Wiley and Sons; and Compendium of Organic Synthetic Methods, Vol. IXII (published by Wiley-Interscience). The starting materials used are commercially available or can be prepared by routine methods known in the art.

It should be noted that in the preparation of compounds of formula I, II, III, IV or V, some of the preparation methods described herein require protection of a terminal functional group (e.g., primary amine, secondary amine, carboxyl in the precursors of compounds of formula I). The need for such protection will depend on the nature of the terminal functional group and the conditions of the preparation methods. The need for such protection will be readily determined by one skilled in the art. The use of such protection/deprotection methods is also within the skill of the art. A general description of protecting groups and their use is given in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

For example, some compounds contain a primary amine functionality or a carboxyl functionality that may interfere with reactions at other sites on the molecule if left unprotected. Accordingly, such functional groups may be protected by an appropriate protecting group that can be removed in a subsequent step. Suitable protecting groups for amine and carboxylic acid protection include those commonly used in peptide synthesis (such as N-tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9-fluorenylmethyleneoxycarbonyl (Fmoc) for amines and lower alkyl or benzyl esters for carboxylic acids), which are generally not chemically reactive under the reaction conditions described and can generally be removed without chemically altering another functional group in the compounds of formula I.

The schemes described below are intended to provide a general description of the methodology used in the preparation of the compounds of the present invention. Some compounds of the present invention may contain a single chiral center and multiple chiral centers with the stereochemical designation (R) or (S). One skilled in the art will appreciate that all synthetic transformations can be carried out in a similar manner regardless of whether the materials are enantioenriched or racemic. Moreover, resolution to the desired optically active species can take place at any desired point in the reaction sequence using well-known methods, such as those described herein and in the chemical literature. For example, intermediates (e.g., S4, S7, S8, S24, S40, and S41) and final products (e.g., S25 and S42) can be isolated using chiral chromatographic methods. Alternatively, chiral salts can be used to isolate enantiomerically enriched intermediates and final compounds.

In the schemes below, the variables X, Y, ^Z1 , ^Z2 , ^Z3 , R, ^R1 , ^R2 , ^R3 , ^R4 , m, p, and q are as described herein for compounds of formula I, II, III, IV, or V, unless otherwise noted. In the schemes below, ^X1 , ^X2 , ^X3 , and ^X4 may each independently represent a leaving group such as any alkyl or aryl sulfonate (e.g., mesylate, tosylate, or triflate), or a halogen, or any other group that can be substituted by an amine or used in a metal-mediated coupling reaction. ^X4 may also represent a protected carboxyl group (i.e., an ester). When the protecting group is identified as Pg ¹ , then it may be an alkylamine protecting group such as benzyl, benzhydryl or the like; a carbamate protecting group such as Boc, Cbz or the like; or an amide protecting group such as trifluoroacetamide. When the protecting group is identified as Pg ² , then it may be an acid protecting group such as methyl, ethyl, benzyl, t-butyl or the like. When the protecting group is identified as Pg ³ , then it may be an alcohol protecting group such as trimethylsilylethoxyethyl; or an acyl group such as acetyl, benzoyl or the like; or a trialkylsilyl group such as trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyl or the like. R ^2a is H or -C1.2alkyl, wherein alkyl may have 0-1 OH. R ^4a is _C1.2alkyl , _{C0_2alkylene} - _{C3_6cycloalkyl} ^{, C0.2alkyleneZ5} _or C1.2αalkylene- ^R6 , wherein said alkyl, alkylene or cycloalkyl may independently be substituted, when valence permits, with 0-3 _F atoms and _0-1 substituent independently selected from _C0.1alkylene - ^ORO and -N(R _n ⁾ ₂ .

A substituted piperidine S8, where R ² = H and Y = CH, can be prepared as shown in Scheme 1. The aryl or heteroaryl bromide S1 can be treated with an alkyl lithium, such as butyl lithium or tert-butyl lithium, to give an aryl or heteroaryl lithium compound, which can be reacted with an aldehyde S2 to give a diol S3. Other aryl or heteroaryl organometallic reagents, such as, but not limited to, Grignard reagents, can also be used in the preparation of S3. The reaction is typically carried out at a temperature of about -70 °C. The diol S3 can then be oxidized with NaIO4 to give the acetal S4 (R ² = H). Compound S4 can then be reacted with a substituted boronic acid or borate ester (S5) in the presence of a palladium catalyst and a ligand complex via a Suzuki reaction (Maluenda and Navarro, Molecules, 2015, 20, 7528-7557) to give a compound of general formula S6. The reduction of the olefin to give a compound of general structure S7 can be carried out under a hydrogen atmosphere (15-100 psi _H2 ) in an alcoholic solvent such as MeOH or EtOH, or alternatively in an aprotic organic solvent such as EtOAc or THF, in the presence of a suitable catalyst such as palladium on carbon, Pd(OH)2 on carbon (Pearlman's catalyst), PtO2 (Adams' catalyst), or tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst). Hydrogen transfer hydrogenation reagents such as ammonium formate or dihydrobenzene or the like can be used using a suitable catalyst. Alternatively, the reduction may be accomplished by alternative methods known to those skilled in the art using reagents such as triethylsilane or other silanes, under acid or metal catalysis, or metal reducing agents such as magnesium or

- 22 040816 the like. Alternatively, the olefin can be functionalized by methods known to those skilled in the art to introduce R ³ groups. For example, the olefin can be hydroborated to produce an alcohol, which can be alkylated or further converted to a nitrile, F or alkyl group. Removal of Pg ¹ can be accomplished by many methods described in the literature to produce S8 amines.

Scheme 1

Scheme 2 shows an alternative preparation of compounds of general structure S4. The reaction of appropriately substituted diols of general structure S9 with aldehydes or ketones of general structure S10a in the presence of an acid such as p-toluenesulfonic acid or pyridinium p-toluenesulfonate in an aprotic organic solvent such as toluene or benzene can provide compounds of general structure S4. The reaction mixture is typically heated to reflux using a Dean-Stark trap to azeotropically remove water. Diols S9 can also be reacted with cyclic (dashed line present) or acyclic (dashed line absent) acetals or ketals of general structure S10b under acid catalysis. The same applies to cyclic or acyclic thioacetals or thioketals of general structure S10c upon exposure to a mercuric salt, mild oxidizing agents or alkylating reagents to afford compounds S4. Alternatively, diols of general formula S9 can be reacted with an appropriately substituted alkyne S11 in an aprotic solvent such as toluene in the presence of triruthenium dodecacarbonyl at a temperature of about 100 °C to afford compounds of general structure S4, where R ² = CH2R ^2a . In cases where R ² contains an alcohol functionality such as CH2OH, an alcohol protecting group (Pg ³ ), such as acetate, can be introduced into compounds of general structure S10. The protecting group can then be removed in a subsequent step. Intermediate S4 can then be further modified using the methods described for Scheme 1 to afford amines of general structure S8.

Scheme 2

As shown in Scheme 3, the conversion of S4 to compounds of general structure S7, where Y = N, can be accomplished by a method such as the CN Buchwald-Hartwig coupling between compounds of general structure S4 and an appropriately substituted and protected piperazine S12 in the presence of a palladium or copper catalyst and a ligand complex. These reactions are typically carried out at 0-110 °C in aprotic organic solvents such as, but not limited to, 1,4-dioxane and _PhCH3 , with the addition of a base such as _Cs2CO3 , _LiHMDS , or NaOtBu. Removal of ^Pg1 to give amines S8, where Y = N, can be accomplished by many methods described in the literature.

Scheme 3

Amino compounds S8 prepared by the methods depicted in Schemes 1-3 can be alkylated with a protected 2- _bromoacetate in the presence of a suitable base such as _K2CO3 , _Et3N , NaH, or LiHMDS in a polar aprotic solvent such as, but not limited to, DMF, DMAc, DMSO, or NMP to afford compounds of general structure S13 (X=N, L= _CH2 ). Standard ester hydrolysis can be accomplished to afford acids S14. When ^Pg2 is tert-butyl, standard acid deprotection methods such as TFA/DCM, HCl/1,4-dioxane, HCl/EtOAc, or other suitable conditions can be used to afford acids S14. If ^Pg2 is methyl or ethyl, standard base deprotection methods such as aqueous NaOH in methanol or ethanol or other suitable conditions can be used to prepare the S14 acids.

Scheme 4

Compounds of general structure S15 (Scheme 5) can be reacted with amines R ⁴ NH2 in the presence of bases such as sodium, potassium or cesium carbonate, bicarbonate, hydroxide, acetate or an organic amine base such as Et3N, DIPEA, DBU and the like, in a polar aprotic solvent such as, but not limited to, THF, DMF, DMAc, DMSO or NMP, or in a protic solvent such as water, MeOH, EtOH or iPrOH or a mixture thereof, to afford compounds of general structure S16. It should be noted that if an example provides R ⁴ with a resolved enantiomeric center, the other enantiomer or a racemic mixture thereof can be obtained by selecting the appropriate starting material. Preferred X ³ substituents include F, Cl and Br, preferred X ⁴ groups include Cl, Br and -CO2-Pg ² . Reduction of the nitro group can be accomplished by hydrogenation under 1-6 atm _H2 with a metal catalyst such as palladium on carbon or Raney nickel in a protic solvent such as MeOH or EtOH or in an aprotic solvent such as DMF, THF or EtOAc. Alternatively, the nitro group can be reduced with iron, zinc, _SnCl2 or another suitable metal in an acidic medium such as 1N HC, AcOH or in aqueous _NH4Cl in THF or methanol to give compounds of general structure S17 (Scheme 5a). Compounds such as S18 can be acylated with acyl halides in a standard manner or with carboxylates in standard amide coupling protocols to give compounds S19. Reduction to S20 compounds can be accomplished under standard conditions with reducing agents such as LAH, or _BH3- THF or BH3-DMS (Scheme 5b).

Scheme 5

E) r4r4 χ3 z3 _X 4 HN Z^ χ4 HN^Z^X ⁴

1% - 1% - 1% o ₂ n^^ o ₂ n^^h

S15 S16S17 ^b) r ⁴ ^or h ₂ n^^x ⁴ HN^^X ⁴ hn^^x ⁴ j T —- TT —-TT

H ₂ NNR ^z H2N NR ^z H2N NR ^z

S18 S19 S20

The diamino compounds S17 and S20 prepared by the methods shown in Schemes 5a and 5b, collectively designated as diamine S21 (Scheme 6), can be acylated with acids of general structure S14 under standard amide coupling conditions to afford amides S22, which will exist as a mixture of 100% S22a to 100% S22b. This mixture of amines S22 can be cyclized to afford compounds of general structure S23 by a variety of methods. The amines S22 can be heated with a dehydrating agent such as _T3P® or an alkyl alcohol such as n-butanol under microwave irradiation (10-60 min at 120-180°C) to afford compounds S23. Alternatively, the mixture of compounds S22 can be heated under acidic conditions such as AcOH at 60-100 °C or under basic conditions such as aqueous NaOH or KOH in 1,4-dioxane at 60-100 °C to afford S23. Compounds of general structure S23 (X ⁴ = C, Br, or I) can be converted to esters of structure S24 by palladium catalyzed carbonylation under 15-100 psi carbon monoxide at 20-100 °C using an appropriate alcohol such as MeOH or EtOH or another alkyl alcohol. Hydrolysis of ester S24 can be accomplished as depicted in Scheme 4 to afford acids S25. For S22 compounds where X ⁴ = CO2-Pg ² , the conversion to ester S24 occurs under conditions similar to those described previously, except that the alkaline cyclization method is used, where compound S25 can be isolated directly from the reaction mixture. For S24 compounds where X ⁴ is CO ₂ tBu, removal of the acid protecting group of S25 can be accomplished under the acidic conditions shown in Scheme 4.

- 24 040816

Alternatively, for S24 compounds where ^Pg2 is C1- _C8 alkyl such as methyl, ethyl, hexyl or octyl, removal of the protecting ester group can be accomplished using a variety of enzymes including esterases, proteases, peptidases, lipases and glycosidases, which are well known to those skilled in the art. Hydrolysis can also be accomplished by treating the ester with an aqueous solution of 1,5,7-triazabicyclo[4.4.0]dec-5-ene at rt.

Scheme 6

R ⁴

Additionally, diamine S21 can be converted to 2-chloromethylbenzimidazole S26 (Scheme 7) by several routes. Treatment with 2-chloroacetyl chloride or chloroacetic anhydride in an aprotic solvent such as 1,4-dioxane followed by heating at 40-100 °C for 2-18 h can provide the desired benzimidazole S26, where Z ¹ , Z ² , and Z ³ are CH. In cases where Z ¹ , Z ² and Z ³ are not all CR ^z , after treatment with 2-chloroacetyl chloride in an aprotic solvent such as 1,4-dioxane for a period of 30 min to 4 h, the solvent is exchanged with an acidic medium such as AcOH or TFA, followed by heating at 40-100 °C for 2-18 h to provide the desired compound S26. The diamine S21 can also be treated with chloroacetic anhydride at 0-80 °C in an aprotic solvent such as, but not limited to, 1,4-dioxane, THF or MeCN, followed by heating at 60-100 °C for 2-18 h to provide the desired compound S26. Additionally, the diamine S21 can be treated with 2-chloro-1,1,1-trimethoxyethane in an aprotic solvent such as, but not limited to, 1,4-dioxane, THF, or MeCN, or in a protic solvent such as MeOH or EtOH, in the presence of an acid catalyst such as pTSA, at 20-100 °C. Alternatively, the diamines S21 can be heated at 100-180 °C with 2-hydroxyacetic acid in an aprotic solvent such as, but not limited to, mesitylene, to produce a hydroxymethyl intermediate. Conversion of the hydroxymethyl group to the chloromethyl compound S26 can be accomplished by standard methods involving treatment with SOCl2 in an aprotic solvent. Compounds of general structure S26 can be reacted with compounds S8 in the presence of bases such as sodium, potassium or cesium carbonate, sodium, potassium or cesium bicarbonate, NaH or an organic amine base such as Et3N, DIPEA, DBU, etc., in a polar aprotic solvent such as, but not limited to, THF, MeCN, DMF, DMAc, DMSO or NMP to give compounds S23 (X ⁴ = C, Br, I) or compounds S24 (X ⁴ = CO2-Pg ² ), which are then used to give compounds S25 by the methods shown in Scheme 6.

Scheme 7

R ⁴ N^Z ³ X ⁴ S8 IS21 - 3 ; ₂ --------- S24 - S25

Cl N^ _Z 1 ^Z _X 4 ₌ co ₂ -Рд ²

S26

S8 S26 --------- S23

X ⁴ = CI, Bg, I

Alternatively (Scheme 8), compounds of general structure S26 can be reacted with appropriately substituted and protected piperazines S12 in the presence of bases such as sodium, potassium or cesium carbonate, sodium, potassium or cesium bicarbonate, NaH or an organic amine base such as _Et3N , DIPEA, DBU, etc., in a polar aprotic solvent such as, but not limited to, THF, MeCN, DMF, DMAc, DMSO or NMP to afford compounds S27 (Scheme 8). Removal of ^Pg1 can be accomplished by many methods described in the literature to afford amines S28. Conversion to compounds of general structure S23 (X ⁴ = C, Br, or I) or S24 (X ⁴ = CO ₂ -Pg ² ) can be accomplished by a method such as the CN Buchwald-Hartwig coupling between compounds of general structure S4, and as previously shown in Scheme 3. Compounds of general structure S23 or S24 can then be used to prepare compounds of structure S25 by the methods shown in Scheme 6.

Scheme 8

^{R4R4 ​}

S12 -Z ³ __uz S4 ^S26 ζ 1 ^N \Y ^_ _χ 4 N^/VX ⁴ * S23 or S24 ^g (R ³ )q Z ¹ =Z ² (R ³ )q Z ¹ =Z ²

S27 _S28

Compounds of general structure S25 can also be prepared as shown in Scheme 9. The diol S9 can be protected to form S29. The trimethylsilylethoxymethyl group is the preferred protecting group. Protection of the diol as the corresponding acetal, such as formaldehyde acetal, is also preferred. Compound S29 can then be reacted with a substituted boronic acid or borate ester (S5) and an olefin, followed by reduction by the methods shown in Scheme 1, to give a compound of general formula S31, where Y = CH. Alternatively, compound S29 can be coupled with piperazines of general structure S12 using the methods outlined in Scheme 3 to afford S31, where Y = N. Compounds of general structure S31 can be deprotected and then coupled with S26 to afford compounds of general structure S33 using the methods outlined in Scheme 7. Alternatively, compounds of general structure S33 can be prepared from S32 by converting S32 to the corresponding N-acetic acid derivative and subsequent condensation with diamines S21, as outlined in Scheme 4 and Scheme 6. Deprotection of S33 using methods known to those skilled in the art can provide diols of general structure S34, which can then be reacted with alkynes of general structure S11 using the methods outlined in Scheme 2 to afford S23 or S24. Alternatively, compound S34 can be converted to compound S23 or S24 using aldehydes, ketones or derivatives thereof as shown in Scheme 2. Compounds of general structure S23 or S24 can then be used to prepare compounds of structure S25 by the methods shown in Scheme 6.

Scheme 9

OH Rd ³ O Rd ³ O γΥ ⁹

HO^A^Br ^Pg3O ^ ^YBrS5

TJ ---kJ ---kJ ^{( )q} (R)p ^ (R)p ^ (R)p'^ ^x

S9 S29 S30

S11

---------► S23 or S24

Compounds of general structure S24 and general structure S33, where Y = N and XL = cyclopropyl, can be prepared as shown in Scheme 10. The protected piperidinone S35 can be homologated to the unsaturated ester S36 using methods well known to those skilled in the art. For example, Horner-Wadsworth-Emmons olefination of S42 with a phosphonate such as ethyl (diethoxyphosphoryl) acetate that has been deprotonated with a strong base such as lithium, sodium, or potassium tert-butoxide can provide S36. The reaction is typically carried out in an aprotic solvent such as THF or DME at a temperature of from about 0 to -50 °C. Conversion of S36 to the cyclopropane derivative S37 can be accomplished by treatment with sulfoxonium ylide prepared from trimethylsulfoxonium iodide and a base such as potassium tert-butoxide or sodium hydride. Deprotection of S37 and subsequent coupling of the resulting carboxylic acid S38 with S21, where X ⁴ = CO ₂ Pg ² , using the methods presented in

- 26 040816 Scheme 6, can provide compounds of general formula S39. Deprotection of S39 and coupling with S4 using the methods shown in Scheme 3 can provide compounds of general structure S24, where Y = N and X-L is cyclopropyl. Compounds of general structure S24 can then be used to prepare compounds of structure S25 using the methods shown in Scheme 6. Alternatively, S40 can be reacted with S29 using the methods shown in Scheme 3 to provide S33, where Y = N and X-L = cyclopropyl. Compounds of general structure S33 can then be used to prepare compounds of structure S25 using the methods shown in Scheme 6 and Scheme 9.

Scheme 10

Alternatively, compounds of general structure S25, where Y = N and XL is cyclopropyl, can be prepared as shown in Scheme 11. Removal of Pg ¹ from S37 provides the piperidine derivative S43. Coupling of S43 with S4 in a manner similar to that shown in Scheme 3 provides S13, where Y = N and XL is cyclopropyl. Deprotection can then provide compounds of general structure S14, which can then be used to prepare S25, as shown in Scheme 6.

Scheme 11

Examples

The following illustrates the synthesis of non-limiting compounds of the present invention. Additional compounds within the scope of this invention can be prepared using the methods illustrated in these examples, either alone or in combination with methods known in the art.

Experiments were typically performed under an inert atmosphere (nitrogen or argon), particularly when oxygen- or moisture-sensitive reagents or intermediates were used. Commercial solvents and reagents were typically used without further purification. Anhydrous solvents were used when practical, typically AcroSeal® products from Acros Organics, Aldrich® Sure/Seal™ from Sigma-Aldrich, or DriSolv® products from EMD Chemicals. Otherwise, commercial solvents were passed through columns packed with 4A molecular sieves until the following QC standards were achieved for water: a) <100 ppm for dichloromethane, toluene, N,N-dimethylformamide, and tetrahydrofuran; b) <180 ppm for dichloromethane, toluene, N,N-dimethylformamide, and tetrahydrofuran; for methanol, ethanol, 1,4-dioxane and diisopropylamine. For very sensitive reactions, solvents were further treated with sodium metal, calcium hydride or molecular sieves and distilled immediately before use. Typically, products were dried under vacuum before further reactions or biological testing. Mass spectrometric data were obtained on either liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure chemical ionization (APCI) or gas chromatography-mass spectrometry (GC-MS) instruments. The symbol ♦ indicates that a chlorine isotope peak pattern was observed in the mass spectrum.

Chiral resolution was used to separate the enantiomers or diastereomers of certain intermediates to prepare the compounds of the invention. When chiral resolution was accomplished, the separated enantiomers were designated as ENT-1 or ENT-2 (or DIAST-1 or DIAST-2) in the order of their elution. In some embodiments, the enantiomers designated as ENT-1 or ENT-2 may be used as starting materials to prepare other enantiomers or diastereomers. In such situations, the resulting enantiomers are designated as ENT-X1 and ENT-X2, respectively, according to their starting materials; similarly, the resulting diastereomers are designated as DIAST-X1 and DIAST-X2, respectively (or DIAST- according to their starting materials. DIAST-Y and DIAST-Z nomenclature is used analogously in syntheses using multiple intermediates.

- 27 040816 connections.

For compounds with two chiral centers, the stereoisomers at each stereocenter were separated at different times. The designation ENT-1 or ENT-2 (or DIAST-1 or DIAST-2) of an intermediate or exemplified compound refers to the elution order of the separation performed at that step. It is understood that when stereoisomers at a chiral center are separated in a compound with two or more centers, the separated enantiomers are diastereomers of each other. By way of example, but not limitation, the compounds of Examples 15 and 16 have two chiral centers. The chiral center of the cyclopropyl moiety was separated when intermediate C36 was separated into ENT-1, yielding intermediate P17, and ENT-2, yielding intermediate P18. P18 was then used to prepare C70, which has a single stereoisomer enriched at the chiral cyclopropyl carbon and a mixture of stereoisomers at the dioxolane carbon. C70 was then resolved into DIAST-Y1 at the dioxolane carbon to give intermediate C71 and DIAST-Y2 at the dioxolane carbon to give intermediate C72, where these intermediates are enriched in a single stereoisomer. C71 was then used to prepare the compound of Example 15, which is identified by the name 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct-1-yl}-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X1, trifluoroacetate salt [from P18 via C71]. In these preparations, after the mixture has been separated, the chiral center is identified by the abs label near that center, implying that the separated enantiomers may not be enantiomerically pure. Typically, the enriched enantiomer at each chiral center constitutes >90% of the isolated material. Preferably, the enriched enantiomer at each center constitutes >98% of the mixture.

In some examples, the optical rotation of the enantiomer was measured using a polarimeter. According to the rotation data (or specific rotation data), the enantiomer with right-handed rotation was designated as the (+) enantiomer, and the enantiomer with left-handed rotation was designated as the (-) enantiomer. Racemic compounds are indicated by either the absence of depicted or described stereochemistry or the presence of (+/-) near the structure; in this latter case, the indicated stereochemistry represents the relative (rather than absolute) configuration of the substituents of the compound.

Reactions through detectable intermediates were typically followed by LCMS and continued until complete conversion before addition of subsequent reagents. For syntheses referenced in other Examples or Methods, reaction conditions (reaction time and temperature) may vary. Reactions were typically followed by thin layer chromatography or mass spectrometry and work-up when appropriate. Purifications may vary from experiment to experiment: typically, solvents and solvent ratios used for eluents/gradients were chosen to obtain appropriate Rf values or retention times. All starting materials in the Preparations and Examples are either commercially available or can be prepared by methods known in the art or as described herein.

Preparation of P1. tert-Butyl 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine-1carboxylate (P1)

Step 1. Synthesis of 2-bromo-6-[(4-chloro-2-fluorophenyl)(hydroxy)methyl]phenol (C1).

This experiment was performed in two batches of equal scale, n-butyl lithium (2.5 M solution in hexanes; 32.8 mL, 82.0 mmol) was slowly added to a -70 °C solution of 1-bromo-4-chloro-2-fluorobenzene (17.2 g, 82.1 mmol) in diethyl ether (100 mL), maintaining the reaction temperature below -60 °C. After stirring the reaction mixture at -70 °C for 20 min, a solution of 3-bromo-2-hydroxybenzaldehyde (5.5 g, 27 mmol) in diethyl ether (100 mL) was slowly added, maintaining the reaction temperature below -60 °C. After stirring for an additional 1 h at -70 °C, the reaction mixture was quenched by the addition of aqueous ammonium chloride (50 mL) at -70 °C and the resulting mixture was diluted with water (100 mL). The two batches were combined at this time and extracted with ethyl acetate (400 mL). The organic layer was washed with saturated aqueous sodium chloride (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 7% ethyl acetate in petroleum ether) gave Cl as a white solid. Overall yield: 15.7 g, 47.4 mmol, 88%.

1H NMR (400 MHz, chloroform-d) δ 7.44 (dd, J=8.0, 1.5 Hz, 1H), 7.37 (dd, J=8.1, 8.1 Hz, 1H), 7.15 (br dd, J= 8.5, 2.1 Hz, 1H), 7.12-7.05 (m, 2H), 6.80 (dd, J=7.8, 7.8 Hz, 1H), 6.78 (s, 1H), 6.31 (d, J=4.8 Hz, 1H), 3.02 (br d, J=4.9 Hz, 1H ).

Step 2. Synthesis of 4-bromo-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxole (C2).

To a solution of Cl (15.7 g, 47.4 mmol) in methanol (450 mL) was added a solution of sodium periodate (25.4 g, 119 mmol) in water (105 mL), and the reaction mixture was stirred at 30 °C for 16 h, after which it was concentrated in vacuo. After diluting the residue with dichloromethane (500 mL), it was washed with water (500 mL). The dichloromethane solution was then dried over sodium sulfate, filtered and concentrated in vacuo. Purification by chromatography on silica gel (eluent: petroleum ether) gave C2 as a white solid. Yield: 10.0 g, 30.3 mmol, 64%. The following 1H NMR data were obtained from an experiment carried out in the same manner but on a smaller scale.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.67-7.61 (m, 2H), 7.50 (s, 1H), 7.43 (br dd, J=8, 2 Hz, 1H), 7.09 (dd, J= 8.3, 1.1 Hz, 1H), 7.01 (dd, J=7.9, 1.1 Hz, 1H), 6.86 (dd, J=8.1, 8.1 Hz, 1H).

Step 3. Synthesis of tert-butyl-4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]-3,6dihydropyridin-1(2H)-carboxylate (C3).

A reaction flask containing a suspension of C2 (8.00 g, 24.3 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (9.01 g, 29.1 mmol), sodium carbonate (5.15 g, 48.6 mmol), and [1,D-bis(diphenylphosphino)ferrocene]dichloropalladium(II) [Pd(dppf) _Cl2 ; 888 mg, 1.21 mmol] in 1,4-dioxane (80 mL) and water (32 mL) was evacuated and backfilled with nitrogen. This vacuum cycle was repeated twice and the reaction mixture was then stirred at 90 °C for 16 h. After removal of the solvent in vacuo, the residue was partitioned between ethyl acetate (200 mL) and water (200 mL). The organic layer was washed with saturated aqueous sodium chloride solution (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. Silica gel chromatography (gradient: 0 to 4.3% ethyl acetate in petroleum ether) gave the product, which was combined with the material obtained from a similar reaction using C2 (2.00 g, 6.07 mmol) to give C3 as a light yellow gum. Overall yield: 10.3 g, 23.8 mmol, 78%.

1H NMR (400 MHz, chloroform-d) δ 7.53 (dd, J=8.3, 7.8 Hz, 1H), 7.23-7.16 (m, 3H), 6.88-6.83 (m, 2H), 6.81-6.76 (m, 1H), 6.34-6.28 (br m, 1H), 4.10-4.05 (m, 2H), 3.61 (br dd, J=6.5 Hz, 2H), 2.59-2.50 (brm, 2H), 1.48 (s, 9H).

Step 4. Synthesis of tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine-1carboxylate (P1).

A solution of C3 (10.3 g, 23.8 mmol) and tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst; 1.54 g, 1.66 mmol) in methanol (100 mL) was stirred at 50 °C under hydrogen atmosphere (45 psi) for 18 h. The reaction mixture was then filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure and chromatographed on silica gel (gradient: 0 to 9% ethyl acetate in petroleum ether). The resulting material was combined with the material obtained from a similar reaction carried out using C3 (1.67 g, 3.87 mmol) to give P1 as a colorless gum. Overall yield: 10.3 g, 23.7 mmol, 86%. LC-MS m/z 456.1 [M+Na+].

1H NMR (400 MHz, chloroform-d) δ 7.52 (dd, J=8.5, 7.6 Hz, 1H), 7.23-7.17 (m, 2H), 7.16 (s, 1H), 6.83 (dd, J=7.8 , 7.8 Hz, 1H), 6.78-6.69 (m, 2H), 4.35-4.10 (br m, 2H), 2.89-2.71 (m, 3H), 1.89-1.77 (m, 2H), 1.771.63 (m, 2H), 1.47 (s, 9H).

Preparation of P2. tert-Butyl 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1carboxylate (P2) on

Pd(dppf) _CI2

_Na2CO3 ( _{Ph3P ) 3RhCI}

Step 1. Synthesis of 4-bromo-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxole (C4).

To a solution of 3-bromobenzene-1,2-diol (330 g, 1.75 mol) in toluene (1.5 L) were added 1-(4-chloro-2-fluorophenyl)ethanone (316 g, 1.83 mol) and p-toluenesulfonic acid (6.02 g, 35.0 mmol). The reaction apparatus was equipped with a Dean-Stark trap and the reaction mixture was heated at 140 °C for 60 h, after which the solution was concentrated in vacuo and purified by chromatography on silica gel (eluent:

- 29 040816 petroleum ether); C4 was obtained as a mixture of yellow oil and solid. Yield: 158 g, 460 mmol, 26%.

1H NMR (400 MHz, chloroform-d): δ 7.54 (dd, J=8.4, 8.4 Hz, 1H), 7.17-7.10 (m, 2H), 6.95 (dd, J=7.9, 1.4

Hz, 1H), 6.75 (dd, component of the ABX system, J=7.8, 1.4 Hz, 1H), 6.70 (dd, component of the ABX system, J=7.9,

7.9 Hz, 1H), 2.11 (d, J=1.1 Hz, 3H).

Step 2. Synthesis of tert-butyl-4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-3,6dihydropyridine-(2H)-carboxylate (C5) .

tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)carboxylate (62 g, 200 mmol) and sodium carbonate (100 g, 940 mmol) were added to a solution of C4 (58.0 g, 169 mmol) in 1,4-dioxane (600 mL). After addition of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (6.0 g, 8.2 mmol), the reaction mixture was heated to 90 °C and stirred for 16 h. Then water (500 mL) was added and the resulting mixture was extracted with ethyl acetate (2 x 500 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (2 x 500 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (gradient: 0 to 9% ethyl acetate in petroleum ether) gave C5 as a yellow oil. Yield: 56.0 g, 126 mmol, 75%.

1H NMR (400 MHz, chloroform-d) δ 7.50 (dd, J=8.2, 8.2 Hz, 1H), 7.17-7.09 (m, 2H), 6.83-6.77 (m, 2H), 6.74 (dd, component of the ABX system, J=5.4, 3.6 Hz, 1H), 6.39-6.33 (br m, 1H), 4.14-4.08 (m, 2H), 3.70-3.56 (m, 2H), 2.66-2.45 (m, 2H), 2.07 (d, J=1.1 Hz, 3H), 1.50 (s, 9H).

Step 3. Synthesis of tert-butyl-4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine 1-carboxylate (P2).

To a solution of C5 (56.0 g, 126 mmol) in methanol (200 mL) was added tris(triphenylphosphine)rhodium(1) chloride (Wilkinson's catalyst; 8.10 g, 8.75 mmol), and the reaction mixture was heated to 50 °C for 18 h under hydrogen atmosphere (45 psi). It was then cooled to 25 °C and filtered through diatomaceous earth. The filtrate was concentrated in vacuo and purified twice by silica gel chromatography (first column: gradient: 0 to 9% ethyl acetate in petroleum ether; second column: gradient: 0 to 2% ethyl acetate in petroleum ether) to give P2 as a yellow solid. Yield: 37.0 g, 82.6 mmol, 66%. LC-MS m/z 392.1 [(M - 2-methylprop-1-ene)+H]+.

1H NMR (400 MHz, chloroform-d) δ 7.51 (dd, J=8.3, 8.0 Hz, 1H), 7.17-7.09 (m, 2H), 6.77 (dd, component of the ABC system, J=7.8, 7, 8 Hz, 1H), 6.70 (dd, ABC system component, J=1.1, 1.3 Hz, 1H), 6.66 (dd, ABC system component, J=7.8, 1.3 Hz, 1H), 4.37-4.13 (br m, 2H), 2.92-2.73 (m, 3H), 2.05 (d, J= 1.1 Hz, 3H), 1.901.63 (m, 4H), 1.49 (s, 9H).

Preparation of P3. 4-[(2S)-2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine paratoluenesulfonate salt (P3)

Step 1. Isolation of tert-butyl 4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate (C6) and tert-butyl 4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate (C7).

Separation of P2 (75.2 g, 168 mmol) into its constituent enantiomers was performed by SFC [column: Chiral Technologies Chiralpak AD-H, 5 μm; mobile phase: 4:1 carbon dioxide/(2-propanol containing 0.2% 1-aminopropan-2-ol)]. The first eluting compound was designated C6, and the second eluting enantiomer was designated C7. These absolute configurations were established on the basis of single crystal X-ray diffraction data on C8, a derivative of C6 (see below).

C6 - Yield: 38.0 g, 84.8 mmol, 50%. Retention time 3.64 min [column: Chiral Technologies Chiralpak AD-H, 4.6 x 250 mm, 5 μm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.2% 1-aminopropan-2-ol; gradient: 5% B over 1.00 min, then 5 to 60% B over 8.00 min; flow rate: 3.0 mL/min; backpressure: 120 bar].

C7 - Yield: 36.8 g, 82.2 mmol, 49%. Retention time 4.19 min (analytical SFC conditions,

- 30 040816 identical to the conditions used for C6).

Step 2. Synthesis of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine paratoluenesulfonate salt (P3).

A solution of C7 (1.62 g, 3.62 mmol) in ethyl acetate (36 mL) was treated with paratoluenesulfonic acid monohydrate (791 mg, 4.16 mmol) and heated at 45 °C. After 23 h, the reaction mixture was allowed to cool to room temperature and the solid was collected by filtration. It was washed with a mixture of ethyl acetate and heptane (1:1, 2 x 15 mL) to give P3 as a white solid. Yield: 1.37 g, 2.63 mmol, 73%. LC-MS m/z 348.1 ♦ [M+H]+.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.53 (v br s, 1H), 8.29 (v br s, 1H), 7.65-7.55 (m, 2H), 7.47 (d, J=8.1 Hz, 2H), 7.35 (dd, J=8.4, 2.0 Hz, 1H), 7.11 (d, J=7.8 Hz, 2H), 6.88-6.81 (m, 2H), 6.75-6.68 (m, 1H), 3.42-3.33 (m, 2H), 3.11-2.93 (m, 3H), 2.29 (s, 3H), 2.03 (s, 3H), 1.98-1.82 (m, 4H).

Conversion of C6 to 4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine methanesulfonate salt (C8) for determination of absolute stereochemistry

C6C8 p-Toluenesulfonic acid (377 mg, 2.19 mmol) was added to a solution of C6 (490 mg, 1.09 mmol) in ethyl acetate (5.5 mL), and the reaction mixture was stirred at room temperature overnight. After dilution with additional ethyl acetate, the reaction mixture was washed successively with aqueous sodium bicarbonate, water, and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. Yield: 375 mg, 1.08 mmol, 99%.

¹ H NMR (400 MHz, methanol-d ₄ ) δ 7.59 (dd, J=8.3, 8.3 Hz, 1H), 7.27 (dd, J=10.9, 2.0 Hz, 1H), 7.20 (br dd, J=8.4, 2.1 Hz, 1H), 6.81-6.75 (m, 1H), 6.74-6.67 (m, 2H), 3.18-3.09 (m, 2H), 2.88-2.77 (m, 1H), 2.77-2.67 (m, 2H), 2.02 (d, J=0.7 Hz, 3H), 1.85-1.73 (m, 4H).

A 0.1 M solution of this free base (4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine) in ethyl acetate was prepared and subjected to salt screening. Only the formation of the methanesulfonate salt is described here. A mixture of methanesulfonic acid (25 μL, 39 μmol) and substrate solution (0.1 M; 0.25 mL, 25 μmol) was stirred overnight. Sufficient methanol was then added to dissolve the solid present and ethyl acetate (3 mL) was added. The resulting solution was allowed to evaporate slowly without stirring to give crystals of C8; one of these crystals was used for the single crystal X-ray structure determination described below.

X-ray structural analysis of a C8 single crystal.

X-ray structural analysis of a single crystal.

Data collection was performed on a Bruker D8 Quest diffractometer at room temperature. Data collection consisted of omega and phi scans.

The structure was solved by internal phasing using the SHELX software package in the orthorhombic space group class P212121. The structure was then refined by least squares in the full matrix approximation. All non-hydrogen atoms were identified and refined using anisotropic substitution parameters.

The formation of the methanesulfonate salt was confirmed by proton transfer N1 H1X 04.

Hydrogen atoms located on nitrogen and oxygen were identified from the difference Fourier map and refined with limited distances. The remaining hydrogen atoms were placed in calculated positions and left on their carrier atoms. The final refinement included isotropic substitution parameters for all hydrogen atoms.

The analysis of the absolute structure using probabilistic methods (Hooft, 2008) was performed using PLATON (Spek). The results show that the absolute structure was determined correctly; the probability that the structure is accurate is calculated to be 100% by this method. The Hooft parameter is given as 0.02 with an esd (estimated standard deviation) of 0.0012, and the Parson parameter is given as 0.07 with an esd of 0.009. The absolute configuration at C7 was confirmed as (R).

The asymmetric unit consists of one molecule of protonated C8 free base and one molecule of deprotonated methanesulfonic acid. The final R-index was 4.6%. Finite difference Fourier analysis revealed no missing or mismatched electron density.

The relevant crystal, data collection and refinement information are summarized in Table A. Atomic coordinates, bond lengths, bond angles and substitution parameters are listed in Tables B–D.

- 31 040816

Software and information sources.

SHELXTL, Version 5.1, Bruker AXS, 1997.

PLATON, A. L. Spek, J. Appl. Christ. 2003, 36, 7-13.

MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R.

Taylor, M. Towler, and J. van de Streek, J. Appl. Christ. 2006, 39, 453-457.

OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Christ. 2009, 42, 339-341.

R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Christ. 2008, 41, 96-103.

H. D. Flack, Acta Cryst. 1983, A39, 867-881.

Table A

Crystal data and structure refinement for C8

Empirical formula Molecular mass by formula Temperature Wavelength C20H23CIFNO5S 443.90 296(2)K 1.54178 A Crystal system Space group Unit cell parameters orthorhombic P212121 a = 6.5348(5) A a = 90° b = 9.3688(7) A β = 99° c = 35.214(3) A y = 90° Volume 2155.9(3) A ³ Z 4 Density (calculated) Absorption coefficient A(000) 1.368 mg/ ^m3 2.823 mm' ¹ 928 Crystal dimensions Theta range for data collection 0.480 x 0.100 x 0.040 mm ³ from 2.509 to 70.483° Index ranges Collected reflections Independent reflections -l<=h<=7, -11<=К=8, -42<=/<=42 16311 4035 [R _int = 0.0638] Completion to theta = 67.679° 99.0% Absorption correction Refinement method Empirical full-matrix least squares method for F ² Data/constraints/parameters Goodness-of-fit criterion F ² Final R indices [Ι>2σ(Ι)] R indices (all data) Absolute structural 4035/2/271 0.832 RI = 0.0463, wR2 = 0.1227 RI = 0.0507, wR2 = 0.1294 -0.003(18) parameter Extinction coefficient Maximum diff, peak and gap 0.0051(6) 0.256 and -0.305 e.A' ³

- 32 040816

Table B

Atomic coordinates (x 104) and equivalent isotropic substitution parameters (A ² x10 ³ ) for C8. U(eq) is defined as one third of the trace of the orthogonalized tensor Ui ^j x y z U(eq)

S(l) 3842(2) 9910(1) 5317(1) 57(1) Cl(l) -1625(2) -718(1) 6588(1) 80(1) 0(1) 6138(4) 3727(3) 6876(1) 53(1) F(l) 639(5) 3071(4) 7503(1) 89(1) 0(2) 3445(4) 5043(3) 7117(1) 57(1) 0(4) 2909(6) 11013(4) 5082(1) 78(1) 0(3) 3708(7) 10299(4) 5708(1) 83(1) N(l) 10461(5) 2909(4) 5493(1) 56(1) C(9) 5652(6) 4826(4) 6629(1) 44(1) C(l) 3361(7) 1662(4) 6697(1) 53(1) C(6) 2957(6) 2523(4) 7012(1) 49(1) C(10) 4075(6) 5613(4) 6776(1) 47(1) C(14) 6628(6) 5138(4) 6294(1) 47(1) 0(5) 5833(7) 9578(4) 5179(1) 96(1) C(15) 8265(6) 4182(4) 6130(1) 49(1) C(5) 1105(7) 2270(5) 7190(1) 59(1) C(16) 7309(6) 3048(5) 5874(1) 54(1) C(2) 1971(7) 670(4) 6567(1) 55(1) C(4) -286(7) 1288(5) 7080(1) 64(1) C(7) 4448(6) 3667(4) 7142(1) 52(1) C(13) 5876(8) 6374(5) 6113(1) 60(1) C(11) 3359(7) 6819(4) 6602(1) 57(1) C(8) 5296(8) 3485(6) 7537(1) 64(1) C(19) 9905(7) 4976(6) 5902(1) 67(1) C(17) 8902(7) 2063(5) 5702(1) 59(1) C(12) 4316(8) 7178(5) 6263(1) 65(1) C(3) 150(7) 497(4) 6756(1) 56(1) C(18) 11476(7) 3977(6) 5738(1) 73(1) C(20) 2328(14) 8399(7) 5260(2) 117(3)

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Table C

Bond lengths [A] and angles [°] for C8

S(l)-O(5) 1,423(4) C(4)-C(3) 1,389(6) S(l)-O(3) 1,428(3) C(4)-H(4) 0,9300 S(l)-O(4) 1,458(3) C(7)-C(8) 1,506(6) S(l)-C(20) 1,738(6) C(13)-C(12) 1,373(7) Cl(l)-C(3) 1,729(5) C(13)-H(13) 0,9300 O(l)-C(9) 1,385(4) C(ll)-C(12) 1,388(7) O(l)-C(7) 1,449(4) C(ll)-H(ll) 0,9300 F(l)-C(5) 1,367(4) C(8)-H(8A) 0,9600 O(2)-C(10) 1,376(4) C(8)-H(8B) 0,9600 O(2)-C(7) 1,449(4) C(8)-H(8C) 0,9600 N(l)-C(18) 1,478(6) C(19)-C(18) 1,505(7) N(l)-C(17) 1,486(5) C(19)-H(19A) 0,9700 N(1)-H(1X) 0.99(2) C(19)-H(19B) 0,9700 N(1)-H(1Y) 0.97(2) C(17)-H(17A) 0,9700 C(9)-C(10) 1,369(5) C(17)-H(17B) 0,9700 C(9)-C(14) 1,375(5) C(12)-H(12) 0,9300

C(l)-C(2) 1,378(6) C(18)-H(18A) 0,9700 C(l)-C(6) 1,395(5) C(18)-H(18B) 0,9700 C(l)-H(l) 0,9300 C(20)-H(20A) 0,9600 C(6)-C(5) 1,384(6) C(20)-H(20B) 0,9600 C(6)-C(7) 1,519(6) C(20)-H(20C) 0,9600 C(10)-C(ll) 1,369(5) C(14)-C(13) 1,409(6) O(5)-S(l)-O(3) 116.2(3) C(14)-C(15) 1,509(5) O(5)-S(l)-O(4) 110,1(2) C(15)-C(16) 1,527(5) O(3)-S(l)-O(4) 109.9(2) C(15)-C(19) 1,531(6) O(5)-S(l)-C(20) 107.6(4) C(15)-H(15) 0,9800 O(3)-S(l)-C(20) 106.6(3) C(5)-C(4) 1,351(7) O(4)-S(l)-C(20) 105.9(4) C(16)-C(17) 1,518(6) C(9)-O(l)-C(7) 105.0(3) C(16)-H(16A) 0,9700 C(10)-0(2)-0(7) 105.2(3) C(16)-H(16B) 0,9700 C(18)-N(l)-C(17) 112.3(3) C(2)-C(3) 1,372(6) C(18)-N(1)-H(1X) 107(3) C(2)-H(2) 0,9300 C(17)-N(1)-H(1X) 113(3) C(18)-N(1)-H(1Y) 113(3) C(l)-C(2)-H(2) 120.3 C(17)-N(1)-H(1Y) 103(3) 0(5)-0(4)-0(3) 117.5(4) H(1X)-N(1)-H(1Y) 108(4) C(5)-C(4)-H(4) 121.2 C(10)-C(9)-C(14) 124.1(3) C(3)-C(4)-H(4) 121.2 C(10)-C(9)-O(l) 109.6(3) 0(1)-0(7)-0(2) 105.7(3) C(14)-C(9)-O(l) 126.3(3) 0(1)-0(7)-0(8) 108.7(3) C(2)-C(l)-C(6) 121.9(4) 0(2)-0(7)-0(8) 108.8(3) C(2)-C(l)-H(l) 119,0 0(1)-0(7)-0(6) 108.7(3) C(6)-C(l)-H(l) 119,0 0(2)-0(7)-0(6) 108.6(3) C(5)-C(6)-C(l) 115.3(4) 0(8)-0(7)-0(6) 115.8(3) C(5)-C(6)-C(7) 123.0(3) 0(12)-0(13)-0(14) 122.4(4) C(l)-C(6)-C(7) 121.7(4) C(12)-C(13)-H(13) 118.8 C(9)-C(10)-C(ll) 122,1(4) C(14)-C(13)-H(13) 118.8 C(9)-C(l 0)-0(2) 110.3(3) C(10)-C(ll)-C(12) 115.6(4) C(11)-C(10)-0(2) 127.5(4) 0(10)-0(1 l)-H(ll) 122.2

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C(9)-C(14)-C(13) 113.6(4) C(12)-C(11)-H(11) 122.2 C(9)-C(14)-C(15) 122,1(3) C(7)-C(8)-H(8A) 109.5 C(13)-C(14)-C(15) 124.2(3) C(7)-C(8)-H(8B) 109.5 C(14)-C(15)-C(16) 110.4(3) H(8A)-C(8)-H(8B) 109.5 C(14)-C(15)-C(19) 114.1(3) C(7)-C(8)-H(8C) 109.5 C(16)-C(15)-C(19) 108.4(3) H(8A)-C(8)-H(8C) 109.5 C(14)-C(15)-H(15) 107.9 H(8B)-C(8)-H(8C) 109.5 C(16)-C(15)-H(15) 107.9 C(18)-C(19)-C(15) 112.2(4) C(19)-C(15)-H(15) 107.9 C(18)-C(19)-H(19A) 109.2 C(4)-C(5)-F(l) 117.2(4) C(15)-C(19)-H(19A) 109.2 C(4)-C(5)-C(6) 125.0(4) C(18)-C(19)-H(19B) 109.2 F(l)-C(5)-C(6) 117.9(4) C(15)-C(19)-H(19B) 109.2 C(17)-C(16)-C(15) 112.2(3) N(19A)-C(19)-N(19B) 107.9 C(17)-C(16)-H(16A) 109.2 N(l)-C(17)-C(16) 110,1(3) C(15)-C(16)-H(16A) 109.2 N(1)-C(17)-H(17A) 109.6 C(17)-C(16)-H(16B) 109.2 C(16)-C(17)-H(17A) 109.6 C(15)-C(16)-H(16B) 109.2 N(1)-C(17)-H(17B) 109.6 N(16A)-C(16)- 107.9 C(16)-C(17)-H(17B) 109.6 N(16V) C(3)-C(2)-C(1) 119.4(4) H(17A)-C(17)-H(17B) 108.2 C(3)-C(2)-H(2) 120.3 C(13)-C(12)-C(11) 122,1(4) C(1)-C(2)-H(2) 120.3 C(13)-C(12)-H(12) 118.9 C(11)-C(12)-H(12) 118.9 C(2)-C(3)-C(4) 120.8(4) H(18A)-C(18)-H(18B) 108.2 C(2)-C(3)-C1(1) 119.6(3) S(l)-C(20)-H(20A) 109.5 C(4)-C(3)-C1(1) 119.6(3) S(l)-C(20)-H(20B) 109.5 N(l)-C(18)-C(19) 109.9(3) N(20A)-C(20)-N(20V) 109.5 N(1)-C(18)-H(18A) 109.7 S(l)-C(20)-H(20C) 109.5 C(19)-C(18)-H(18A) 109.7 N(20A)-C(20)-N(20C) 109.5 N(1)-C(18)-H(18B) 109.7 H(20B)-C(20)-H(20C) 109.5 C(19)-C(18)-H(18B) 109.7

Symmetry transformations used to form equivalent atoms.

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Table D

Anisotropic substitution parameters (A ² x10 ³ ) for C8. The exponent of the anisotropic substitution factor takes the form:

-2π ² [ή ² a* ² U ^u + ... + 2 hka* b* U ¹² ]

and ¹¹ and ²² and ³³ and ²³ and ¹³ and ¹² S(l) 73(1) 48(1) 48(1) -2(1) 7(1) -1(1) C1(1) 81(1) 78(1) 81(1) -8(1) 1(1) -8(1) 0(1) 54(1) 50(1) 56(1) 14(1) Yu(1) 17(1) F(l) 83(2) 103(2) 79(2) -40(2) 38(2) -6(2) 0(2) 66(2) 49(1) 54(1) 2(1) And(1) 18(1) 0(4) 87(2) 84(2) 64(2) 19(2) 17(2) 21(2) 0(3) 122(3) 80(2) 47(1) -3(1) 7(2) -13(2) N(l) 47(2) 73(2) 48(2) 7(2) 3(1) And(2) C(9) 51(2) 38(2) 44(2) 2(1) -7(1) 2(2) C(1) 63(2) 46(2) 50(2) 5(2) 21(2) 13(2) C(6) 55(2) 47(2) 45(2) 5(1) And(2) 19(2) C(10) 55(2) 39(2) 46(2) -5(1) -4(2) 6(2) C(14) 54(2) 46(2) 42(2) 0(1) -9(2) -5(2) 0(5) 88(2) 88(3) 113(3) -24(2) 13(2) 21(2) C(15) 47(2) 61(2) 40(2) 3(2) -3(1) -2(2) C(5) 60(2) 62(2) 54(2) -6(2) 19(2) 13(2) C(16) 43(2) 53(2) 65(2) -4(2) 8(2) -6(2) C(2) 72(3) 49(2) 45(2) 2(2) 16(2) 14(2) C(4) 57(2) 68(3) 65(2) -3(2) 23(2) 6(2) C(7) 54(2) 50(2) 51(2) 7(2) 12(2) 16(2) C(13) 81(3) 54(2) 46(2) 9(2) -4(2) 4(2) C(11) 70(3) 46(2) 54(2) -8(2) -14(2) 17(2) C(8) 69(3) 71(3) 51(2) 4(2) 4(2) 15(2) C(19) 54(2) 78(3) 70(3) -13(2) 2(2) -25(2) C(17) 54(2) 57(2) 67(2) -3(2) 8(2) 3(2) C(12) 96(3) 43(2) 56(2) 5(2) -14(2) 13(2) C(3) 64(2) 52(2) 52(2) 4(2) 2(2) 14(2) C(18) 43(2) 103(4) 73(3) 7(3) 3(2) -18(2) C(20) 153(7) 87(4) 110(5) -14(4) -6(5) -57(5)

Preparation of P3. Di-para-toluoyl-L-tartrate salt of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine di-para-toluoyl-L-tartrate salt (P3, di-para-toluoyl-L-tartrate salt)

A solution of C13, free base (519 mg, 1.49 mmol) and di-p-toluoyl-L-tartaric acid (278 mg, 0.719 mmol) in acetonitrile (7.5 mL) was stirred at 50 °C for 1.5 h. The mixture was allowed to cool to room temperature at 0.2 °C per minute. After 15 h at room temperature, the mixture was heated to 65 °C and acetonitrile (15 mL) was charged. The mixture was allowed to cool to room temperature at 0.2 °C per minute. After 15 h at room temperature, the mixture was heated to 54 °C. After 3 h, the solid was collected by filtration and dried in a vacuum oven at 35 °C under nitrogen to give P3, di-p-toluoyl-L-tartrate salt, as a white solid (217 mg, 0.296 mmol, 20%, 82% ee).

A solution of P3, di-p-toluoyl-L-tartrate salt, (217 mg, 0.296 mmol, 82% ee) in acetonitrile (8.0 mL) at 50 °C was allowed to cool to room temperature at 0.2 °C per minute. After 15 h, the solid was collected by filtration and dried in a vacuum oven at 35 °C under nitrogen to give P3, di-p-toluoyl-L-tartrate salt, as a white solid (190 mg, 0.259 mmol, 88% ee, 88%

- 36 040816 ee). LC-MS m/z 348.1 ♦ [M+H]+.

1H NMR (400 MHz, DMSC-d ₆ ) δ 8.9-8.5 (br s, 2H), 7.79 (d, J=8.1 Hz, 4H), 7.64-7.54 (br, 2H), 7.34 (dd, J=8.4, 2.1 Hz, 1H), 7.26 (d, J=8.0 Hz, 4H), 6.87-6.78 (m, 2H), 6.69 (dd, J=6.7, 2.5 Hz, 1H), 5.58 (s, 2H), 3.37-3.28 (m, 2H, putative; partially hidden by water peak), 3.05-2.89 (m, 3H), 2.33 (s, 6H), 2.02 (s, 3H), 1.92-1.80 (m, 4H). Retention time: Peak 1 (4.97 min, minor) and Peak 2 (5.31 min, major) {Column: Chiralpak IC-U 3.0x50 mm, 1.6 μm; Mobile phase A: Carbon dioxide; Mobile phase B: 0.1% isopropylamine in methanol; Gradient: 10% B for 5.00 min, then 45% B for 0.6 min; Flow rate: 1.7 mL/min; Back pressure: 130 bar}.

Preparation of P4. tert-Butyl 4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate (P4)

A suspension of P2 (2.00 g, 4.46 mmol), zinc cyanide (734 mg, 6.25 mol), zinc (70.1 mg, 1.07 mmol), 1,1'-bis(diphenylphosphino)ferrocene (dppf; 198 mg, 0.357 mmol) and tris(dibenzylideneacetone)dipalladium(0) (164 mg, 0.179 mmol) in N,N-dimethylacetamide (20 ml) was stirred at 120 °C for 16 h, after which it was filtered. The filtrate was mixed with water (50 ml) and extracted with ethyl acetate (3 x 50 ml). The combined organic layers were then washed successively with water (30 ml) and a saturated aqueous solution of sodium chloride (20 ml) and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 30% ethyl acetate in petroleum ether) gave a solid which was treated with acetonitrile (15 mL) and water (15 mL) and lyophilized. This afforded P4 as a light yellow solid. Yield: 1.17 g, 2.67 mmol, 60%. LC-MS m/z 461.3 [M+Na ⁺ ].

¹ H NMR (400 MHz, chloroform-d) 7.71 (dd, J=1.1, 7.6 Hz, 1H), 7.45 (dd, J=8.0, 1.6 Hz, 1H), 7.42 (dd, J=10.0, 1.5 Hz, 1H), 6.79 (dd, component of the ABC system, J=1.1, 7.6 Hz, 1H), 6.72 (dd, component of the ABC system, J=7.8, 1.3 Hz, 1H), 6.68 (dd, component of the ABC system, J=7.8, 1.3 Hz, 1H), 4.37-4.14 (br m, 2H), 2.91-2.73 (m, 3H), 2.07 (d, J=1.1 Hz, 3H), 1.89-1.62 (m, 4H), 1.49 (s, 9H).

Preparation of P5 and P6. 4-Bromo-2-phenyl-1,3-benzodioxole, ENT-1, (P5) and 4-bromo-2-phenyl-1,3-benzodioxole, ENT-2, (P6)

R5 R6

Step 1. Synthesis of 2-bromo-6-[hydroxy(phenyl)methyl]phenol (C9).

Phenyllithium (1.9 M solution in 1-butoxybutane; 78.5 mL, 149 mmol) was slowly added to a -70 °C solution of 3-bromo-2-hydroxybenzaldehyde (10.0 g, 49.7 mmol) in tetrahydrofuran (70 mL) at a rate that maintained the reaction temperature below -60 °C. The resulting suspension was stirred at -70 °C for 1 h and then allowed to warm to room temperature overnight before being poured into 0 °C aqueous ammonium chloride (30 mL). This mixture was extracted with ethyl acetate (3 x 30 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (30 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 5% ethyl acetate in petroleum ether) gave C9 as a yellow solid. Yield: 6.11 g, 21.9 mmol, 44%.

1H NMR (400 MHz, chloroform-d) δ 7.45-7.28 (m, 6H), 7.22-7.18 (m, 1H), 7.06 (br d, J=7.7 Hz, 1H), 6.77 (dd, J= 7.9, 7.8 Hz, 1H), 6.06 (br s, 1H), 2.89 (br s, 1H).

Step 2. Synthesis of 4-bromo-2-phenyl-1,3-benzodioxole (C10).

To a solution of C9 (6.11 g, 21.9 mmol) in methanol (370 ml) was added a solution of sodium periodate (11.7 g, 54.7 mmol) in water (175 ml). The reaction mixture was stirred at 30 °C for 40 h, after which most of the methanol was removed by concentration in vacuo. The resulting mixture was extracted with dichloromethane (5 x 100 ml) and the combined organic layers were washed successively with aqueous sodium sulfite solution (100 ml) and saturated aqueous sodium chloride solution (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. Chromatography on silica gel (eluent: petroleum ether) gave C10 as a colourless oil. Yield: 4.50 g,

- 37 040816

16.2 mmol, 74%. LC-MS m/z 278.5 (bromine isotope peaks were observed) [M+H]+.

'H NMR (400 MHz, chloroform-J) δ 7.62-7.57 (m, 2H), 7.49-7.43 (m, 3H), 7.04 (s, 1H), 7.00 (dd, J=8.0, 1.4 Hz, 1H), 6.79 (dd, ABX system component, J=7.8, 1.4 Hz, 1H), 6.75 (dd, ABX system component, J=7.9, 7.8 Hz, 1H).

Step 3. Isolation of 4-bromo-2-phenyl-1,3-benzodioxole, ENT-1, (P5) and 4-bromo-2-phenyl-1,3-benzodioxole, ENT-2, (P6).

The enantiomers comprising C10 (5.00 g, 18.0 mmol) were separated using SFC [column: Chiral Technologies ChiralCel OD, 10 μm; mobile phase: 3:1 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer was designated ENT-1 (P5) and the second eluting enantiomer was designated ENT-2 (P6); both were obtained as yellow oils.

P5 Yield: 2.20 g, 7.94 mmol, 44%. LC-MS m/z 277.0 (bromine isotope peaks observed) [M+H]+.

‘H NMR (400 MHz, chloroform-d) δ 7.63-7.55 (m, 2H), 7.51-7.42 (m, 3H), 7.04 (s, 1H), 7.00 (dd, J=8.0, 1.3 Hz, 1H), 6.80 (dd, component of the ABX system, J=7.8, 1.4 Hz, 1H), 6.75 (dd, component of the ABX system, J=7.9, 7.8 Hz, 1H). Retention time 3.28 min (column: Chiral Technologies ChiralCel OD-H, 4.6x150 mm, 5 μm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5 to 40% B over 5.5 min; flow rate: 2.5 ml/min).

P6 Yield: 2.00 g, 7.22 mmol, 40%. LC-MS m/z 276.9 (bromine isotope peaks observed) [M+H]+.

‘H NMR (400 MHz, chloroform-d) δ 7.63-7.55 (m, 2H), 7.50-7.42 (m, 3H), 7.04 (s, 1H), 7.00 (dd, J=8.0, 1.4 Hz, 1H), 6.80 (dd, component of the ABX system, J=7.8, 1.4 Hz, 1H), 6.75 (dd, component of the ABX system, J=7.9, 7.9 Hz, 1H). Retention time 3.73 min (analytical conditions identical to those used for P5).

Preparation of P7. tert-Butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1carboxylate (P7)

C11

Step 1. Synthesis of 2-(4-bromo-2-methyl-1,3-benzodioxol-2-yl)-5-chloropyridine (C11).

A mixture of 5-chloro-2-ethynylpyridine (1.80 g, 13.1 mmol), 3-bromobenzene-1,2-diol (2.47 g, 13.1 mmol), and triruthenium dodecacarbonyl (167 mg, 0.261 mmol) in toluene (25 mL) was degassed for 1 min and then heated at 100 °C for 16 h. The reaction mixture was diluted with ethyl acetate (30 mL) and filtered through a pad of diatomaceous earth. The filtrate was concentrated in vacuo and purified by silica gel chromatography (gradient: 0 to 1% ethyl acetate in petroleum ether) to give C11 as a yellow oil. Yield: 1.73 g, 5.30 mmol, 40%. LC-MS m/z 325.6 (a pattern with peaks of bromine and chlorine isotopes was observed) [M+H]+.

‘H NMR (400 MHz, chloroform-J) δ 8.63 (dd, J=2.4, 0.7 Hz, 1H), 7.71 (dd, ABX system component, J=8.4, 2.4 Hz, 1H), 7.60 (dd, ABX system component, J=8.4, 0.7 Hz, 1H), 6.97 (dd, J=8.0, 1.4 Hz, 1H), 6.76 (dd, ABX system component, J=7.8, 1.4 Hz, 1H), 6.72 (dd, ABX system component, J=8.0, 7.8 Hz, 1H), 2.10 (s, 3H).

Step 2. Synthesis of tert-butyl-4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]-3,6dihydropyridine-(2H)-carboxylate (C12) .

[1,G-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (388 mg, 0.530 mmol) was added to a suspension of C11 (1.73 g, 5.30 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.64 g, 5.30 mmol), and cesium carbonate (5.18 g, 15.9 mmol) in 1,4-dioxane (35 mL) and water (6 mL). The reaction mixture was stirred at 90 °C for 4 h and then diluted with ethyl acetate (30 mL) and water (5 mL). The organic layer was concentrated in vacuo and the residue was chromatographed on silica gel (gradient: 0 to 5% ethyl acetate in petroleum ether) to afford C12 as a yellow gum. Yield: 1.85 g, 4.31 mmol, 81%. LC-MS m/z 451.04 [M+Na+].

'H NMR (400 MHz, chloroform-d) δ 8.62 (dd, J=2.5, 0.8 Hz, 1H), 7.69 (dd, component of the ABX system, J=8.4, 2.4 Hz, 1H), 7.57 ( dd, component of the ABX system, J=8.4, 0.8 Hz, 1H), 6.84-6.79 (m, 2H), 6.78-6.73 (m, 1H), 6.39-6.33 (br m, 1H), 4.13-4.07 (m, 2H), 3.68-3.58 (m, 2H), 2.60-2.51 ( br m, 2H), 2.07 (s, 3H), 1.49 (s, 9H).

- 38 040816

Step 3. Synthesis of tert-butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate (P7).

A solution of C12 (2.61 g, 6.08 mmol) and tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst; 563 mg, 0.608 mmol) in methanol (100 ml) was degassed under vacuum and then purged with hydrogen;

This vacuum-purge cycle was repeated a total of three times. The reaction mixture was then stirred at 60°C under hydrogen (50 psi) for 16 h, after which it was filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel chromatography (gradient: 0 to 10% ethyl acetate in petroleum ether). This material was combined with the material obtained from a similar hydrogenation of C12 (110 mg, 0.256 mmol) to give P7 as a light yellow gum. Overall yield: 2.05 g, 4.76 mmol, 75%. LCMS m/z 431.3^ [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.62 (d, J=2.3 Hz, 1H), 7.69 (dd, component of the ABX system, J=8.4, 2.4 Hz, 1H), 7.57 (d, half of the AB quartet, J=8.4 Hz, 1H), 6.79 (dd, component of the ABC system, J=7.8, 7.7 Hz, 1H), 6.72 (dd, component of the ABC system, J=7.8, 1.3 Hz, 1H), 6.68 (br d, component of the ABC system, J=7.9 Hz, 1H), 4.32-4.12 (br m, 2H), 2.91-2.73 (m, 3H), 2.05 (s, 3H), 1.90-1.62 (m, 4H), 1.48 (s, 9H).

Preparation of P8 and P9. tert-Butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate, ENT-1, (P8) and tert-Butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate, ENT-2, (P9)

Separation of P7 (500 mg, 1.16 mmol) into its component enantiomers was accomplished using SFC {column: Phenomenex Lux Amylose-1, 5 μm; mobile phase: 9:1 carbon dioxide/[2-propanol containing 0.2% (7 M ammonia in methanol)]}. The first eluting enantiomer was designated ENT-1 (P8) and the second eluting enantiomer was designated ENT-2 (P9).

P8 Yield: 228 mg, 0.529 mmol, 46%. Retention time: 4.00 min {Column: Phenomenex Lux Amy lose-1, 4.6 x 250 mm, 5 μm; Mobile phase A: carbon dioxide; Mobile phase B: [2-propanol containing 0.2% (7 M ammonia in methanol)]; Gradient: 5% B over 1.00 min, then 5 to 60% B over 8.00 min; Flow rate: 3.0 mL/min; Back pressure: 120 bar}.

P9 Yield: 229 mg, 0.531 mmol, 46%. Retention time 4.50 min (analytical conditions identical to those used for P8).

Preparation of P10. {4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}acetic acid (P10)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine paratoluenesulfonate salt (C13).

A solution of P2 (5.0 g, 11 mmol) and p-toluenesulfonic acid (4.81 g, 27.9 mmol) in ethyl acetate (100 mL) was stirred at 60 °C for 2 h and then concentrated in vacuo to give C13 as a yellow gum. This material was carried directly to the next step. LC-MS m/z 347.9 ♦ [M+H] ⁺ .

Step 2. Synthesis of ethyl {4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}acetate (C14).

Potassium carbonate (7.71 g, 55.8 mmol) and ethyl bromoacetate (1.86 g, 11.2 mmol) were added to a solution of C13 (from the previous step; <11 mmol) in acetonitrile (150 mL), and the reaction mixture was stirred at 55 °C for 16 h. It was then filtered, and the filtrate was concentrated in vacuo and purified by silica gel chromatography (gradient: 0 to 30% ethyl acetate in petroleum ether) to give C14 in

- 39 040816 as a yellow resin. According to ¹ H NMR analysis, this substance was not quite pure. Yield: 3.57 g, 8.23 mmol, 75% in 2 steps.

¹ H NMR (400 MHz, chloroform-d), C14 peaks only: δ 7.52 (dd, J=8.4, 8.0 Hz, 1H), 7.17-7.07 (m, 2H), 6.77 (dd, component of the ABC system, J=7.8, 7.8 Hz, 1H), 6.72-6.67 (m, 2H), 4.21 (q, J=7.1 Hz, 2H), 3.27 (s, 2H), 3.07 (m, 2H), 2.70 (tt, J=12.1, 3.8 Hz, 1H), 2.35 (ddd, J=11.5, 11.5, 2.7 Hz, 2H), 2.04 (d, J=1.1 Hz, 3H), 2.021.76 (m, 4H), 1.29 (t, J=7.1 Hz, 3H).

Step 3. Synthesis of {4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}acetic acid (P10).

A solution of C14 (3.57 g, 8.23 mmol) and aqueous sodium hydroxide (3 M; 13.7 mL, 41.1 mmol) in a mixture of methanol (80 mL) and tetrahydrofuran (40 mL) was stirred at 25 °C for 16 h. After removing the solvents in vacuo, the aqueous residue was acidified to pH 7 by adding 1 M hydrochloric acid and then extracted with a mixture of dichloromethane and methanol (10:1, 2 x 100 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give P10 as a yellow solid. Yield: 2.95 g, 7.27 mmol, 88%. LC-MS m/z 406.2^ [M+H]+.

¹ H NMR (400 MHz, methanol^) δ 7.61 (dd, J=8.3, 8.3 Hz, 1H), 7.29 (dd, J=10.9, 2.0 Hz, 1H), 7.22 (ddd, J=8.4, 2.0, 0.8 Hz, 1H), 6.82 (dd, component of the ABC system, J=8.3, 7.1 Hz, 1H), 6.78-6.72 (m, 2H), 3.65-3.54 (br m, 2H), 3.51 (s, 2H), 3.04-2.88 (m, 3H), 2.23-2.07 (m, 2H), 2.07-1.93 (m, 2H), 2.04 (d, J=1.1 Hz, 3H).

Preparation of P11. Methyl 2-(chloromethyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (P11)

P11, HCl salt

Step 1. Synthesis of methyl 3-[(2-methoxyethyl)amino]-4-nitrobenzoate (C15).

To a colorless solution of methyl 3-fluoro-4-nitrobenzoate (50 g, 250 mmol) in tetrahydrofuran (400 mL) was added triethylamine (40.7 g, 402 mmol, 55.8 mL), then 2-methoxyethanamine (30.2 g, 402 mmol) in tetrahydrofuran (100 mL) was added dropwise at room temperature. The resulting yellow solution was stirred at 55 °C for 18 h. The solution was cooled to room temperature and concentrated under reduced pressure to remove tetrahydrofuran. The resulting yellow solid was dissolved in ethyl acetate (800 mL) and washed with saturated aqueous ammonium chloride (250 mL). The aqueous phase was separated and extracted with ethyl acetate (200 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (3 x 250 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give C15 (60.2 g, 94%) as a yellow solid.

¹ H NMR (600 MHz, chloroform-d) δ 8.23 (d, 1H), 8.17 (br s, 1H), 7.58 (d, 1H), 7.25 (dd, 1H), 3.95 (s, 3H), 3.69-3.73 (m, 2H), 3.56 (m, 2H), 3.45 (s, 3H); LC-MS m/z 255.4 [M+H] ⁺ .

Step 2. Synthesis of methyl 4-amino-3-[(2-methoxyethyl)amino]benzoate (C16).

Pd/C (10 g, 94 mmol) was added to a solution of C15 (30 g, 118 mmol) in methanol (500 mL). The reaction mixture was stirred at room temperature under 15 psi hydrogen for 18 h. The black suspension was filtered through diatomaceous earth and the filter cake was washed with methanol (500 mL).

The combined filtrates were concentrated in vacuo to give C16 (26.5 g, quantitative yield) as a brown oil that solidified on standing.

¹ H NMR (400 MHz, chloroform-d) δ 7.48 (dd, 1H), 7.36 (d, 1H), 6.69 (d, 1H), 3.87 (s, 3H), 3.77 (br s, 2H), 3.68 (t, 2H), 3.41 (s, 3H), 3.32 (t, 2H); LC-MS m/z 224.7 [M+H] ⁺ .

Step 3. Synthesis of methyl 2-(chloromethyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (P11).

To a solution of C16 (5.00 g, 22.3 mmol) in tetrahydrofuran (100 mL) was added 2-chloro-1,1,1-trimethoxyethane (3.31 mL, 24.6 mmol), followed by the addition of p-toluenesulfonic acid monohydrate (84.8 mg, 0.446 mmol). The reaction mixture was heated at 45 °C for 5 h, after which it was concentrated in vacuo; the residual oil was dissolved in ethyl acetate (10 mL) and heated to solution. The solution was stirred slowly while cooling to room temperature overnight. The precipitate was collected by filtration and washed with heptane to give P11 as a gray solid. Yield: 5.73 g, 20.3 mmol, 91%.

^1H NMR (600 MHz, chloroform-d) δ 8.12 (br s, 1H), 8.01 (br d, J=8.6 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H),

- 40 040816

4.96 (s, 2H), 4.52 (t, J=5.1 Hz, 2H), 3.96 (s, 3H), 3.74 (t, J=5.1 Hz, 2H), 3.28 (s, 3H).

Step 4. Synthesis of methyl 2-(chloromethyl)-1-(2-methoxyethyl)-III-benzimidazole-6-carboxylate hydrochloride salt (P11, HCl salt).

A solution of C16 (5.0 g, 24 mmol) in 1,4-dioxane (100 mL) was heated to 100 °C, a solution of chloroacetic anhydride (4.1 g, 24.5 mmol) in 1,4-dioxane (60 mL) was added via an addition funnel over a period of 10 h, and the reaction mixture was stirred for an additional 12 h at 100 °C. The next day, the reaction mixture was cooled to room temperature, and 1,4-dioxane was removed under reduced pressure. The crude reaction mixture was dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate. The ethyl acetate layer was separated, dried over sodium sulfate, and filtered. A solution of 4 M hydrogen chloride in 1,4-dioxane (1.1 equiv.) was added to the ethyl acetate solution with constant stirring. The hydrochloride salt of P11 precipitated as a pale yellow solid. The suspension was stirred for 1 h, and the hydrochloride salt of P11 was then collected by filtration to give a yellow solid (6.1 g, 86%).

1H NMR (600 MHz, CD3OD) δ 8.64 (s, 1H), 8.30 (d, 1H), 7.92 (d, 1H), 5.32 (s, 2H), 4.84 (m, 2H), 3.99 (s, 3H), 3.83 (t, 2H), 3.31 (s, 3H). LC-MS m/z 283.2 [M+H]+.

Obtaining carboxylate (P12)

Methyl-1 -(2-methoxyethyl)-2-(piperazin-1 -ylmethyl)-1 H-benzimidazole-6P12.

Step 1. Synthesis of methyl 2-{[4-(tert-butoxycarbonyl)piperazin-1-yl]methyl}-1-(2-methoxyethyl)1H-benzimidazole-6-carboxylate (C17).

Compound P11 (1.59 g, 5.62 mmol) was added to a mixture of tert-butyl piperazine-1-carboxylate (1.00 g, 5.37 mmol) and potassium carbonate (2.97 g, 21.5 mmol) in acetonitrile (15 mL) at 15 °C and the reaction mixture was stirred at 55 °C for 12 h. It was then combined with the analogous product from a reaction carried out using P11 and tert-butyl piperazine-1-carboxylate (200 mg, 1.07 mmol) and the mixture was filtered. After concentration of the filtrate in vacuo, the residue was purified by silica gel chromatography (gradient: 0 to 60% ethyl acetate in petroleum ether) to give C17 as a pale yellow solid. Overall yield: 2.30 g, 5.32 mmol, 83%. LC-MS m/z 433.0 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.12 (d, J=1.5 Hz, 1H), 7.96 (dd, J=8.4, 1.5 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H), 4.58 (t, J=5.4 Hz, 2H), 3.95 (s, 3H), 3.89 (s, 2H), 3.73 (t, J=5.4 Hz, 2H), 3.46-3.37 (br m, 4H), 3.28 (s, 3H), 2.54-2.44 (br m, 4H), 1.45 (s, 9H).

Step 2. Synthesis of methyl 1-(2-methoxyethyl)-2-(piperazin-1-ylmethyl)-1H-benzimidazole-6carboxylate (P12).

To a solution of C17 (2.30 g, 5.32 mmol) in dichloromethane (80 mL) was added a solution of hydrogen chloride in ethyl acetate (20 mL). The reaction mixture was stirred at 20 °C for 2 h and then concentrated in vacuo. The residue was diluted with water (20 mL), adjusted to pH 9-10 with saturated aqueous sodium bicarbonate and extracted with ethyl acetate-methanol (10:1, 15 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to give P12 as a pale yellow solid. Yield: 1.68 g, 5.05 mmol, 95%. LC-MS m/z 332.8 [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.13 (br s, 1H), 7.96 (br d, J=8.5 Hz, 1H), 7.72 (d, J=8.5 Hz, 1H), 4.59 (t, J=5.5 Hz, 2H), 3.95 (s, 3H), 3.86 (s, 2H), 3.75 (t, J=5.5 Hz, 2H), 3.29 (s, 3H), 2.87 (t, J=4.8 Hz, 4H), 2.50 (br m, 4H).

Preparation of P13. 6-Bromo-2-(chloromethyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine (P13)

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Step 1. Synthesis of 5-bromo-N-(2-methoxyethyl)-2-nitropyridin-3-amine (C18).

A solution of 5-bromo-3-fluoro-2-nitropyridine (400 mg, 1.81 mmol) and 2-methoxyethanamine (408 mg, 5.43 mmol) in tetrahydrofuran (10 mL) was stirred at 25 °C for 2 h, after which it was diluted with ethyl acetate (100 mL) and washed with water (50 mL). The organic layer was washed with saturated aqueous sodium chloride solution (50 mL), dried over magnesium sulfate, filtered and concentrated to give C18 as a yellow solid. Yield: 430 mg, 1.56 mmol, 86%.

Step 2. Synthesis of 5-bromo-N ³ -(2-methoxyethyl)pyridine-2,3-diamine (C19).

A solution of C18 (430 mg, 1.56 mmol), ammonium chloride (833 mg, 15.6 mmol) and iron powder (870 mg, 15.6 mmol) in a mixture of methanol (10 mL) and water (2 mL) was stirred at 80 °C for 30 min. The resulting suspension was poured into water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to give C19 as a brown solid. Yield: 350 mg, 1.42 mmol, 91%.

¹ H NMR (400 MHz, chloroform-d) δ 7.63 (d, J=2.1 Hz, 1H), 6.88 (d, J=2.0 Hz, 1H), 4.33-4.19 (br s, 2H), 3.65 (dd, J=5.6, 4.6 Hz, 2H), 3.40 (s, 3H), 3.22 (br t, J=5 Hz, 2H).

Step 3. Synthesis of 6-bromo-2-(chloromethyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine (P13).

A solution of C19 (400 mg, 1.63 mmol) in 1,4-dioxane (8 mL) was treated with chloroacetyl chloride (0.284 mL, 3.57 mmol) and stirred at room temperature until LCMS analysis showed complete conversion of C19 to the intermediate amide. After removal of 1,4-dioxane in vacuo, the residue was dissolved in trifluoroacetic acid (8 mL) and heated at 80 °C for 18 h, after which the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting oil was dissolved in ethyl acetate (50 mL) and neutralized by the addition of saturated aqueous sodium bicarbonate. The aqueous layer was extracted with ethyl acetate (20 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (gradient: 0 to 80% ethyl acetate in heptane) gave P13 as a solid. Yield: 176 mg, 0.578 mmol, 35%. LC-MS m/z 306.1 (bromine and chlorine isotope peaks observed) [M+H]+.

1H NMR (600 MHz, chloroform-d) δ 8.58 (br s, 1H), 7.89 (br s, 1H), 4.92 (s, 2H), 4.44 (t, J=5.0 Hz, 2H), 3.71 (t, J=5.0 Hz, 2H), 3.28 (s, 3H).

Preparation of P14. Methyl 2-{[4-(2,3-dihydroxyphenyl)piperidin-1-yl]methyl}-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylate (P14)

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Step 1. Synthesis of [(3-bromobenzene-1,2-diyl)bis(oxymethanediyloxyethane-2,1diyl)]bis(trimethylsilane) (C20).

This reaction was carried out in two batches of identical scale. N,N-Diisopropylethylamine (37.8 mL, 217 mmol) was added dropwise to a solution of 3-bromobenzene-1,2-diol (10.0 g, 52.9 mmol) in tetrahydrofuran (300 mL). After stirring the mixture for 10 min at 20 °C, [2(chloromethoxy)ethyl](trimethyl)silane (19.2 mL, 108 mmol) was added dropwise over 5 min, and stirring was continued for 16 h at room temperature (18 °C). N,N-Diisopropylethylamine (27.6 mL, 158 mmol) was added again, then [2(chloromethoxy)ethyl](trimethyl)silane (14.0 mL, 79.1 mmol) was added dropwise at room temperature (18°C). After an additional 2.5 h at room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo. At this point, the crude products from the two batches were combined and purified by silica gel chromatography (gradient: 0 to 7% ethyl acetate in petroleum ether) to give C20 as a colorless oil. This material was not entirely pure according to 1H NMR analysis. Overall yield: 22.9 g, 50.9 mmol, 48%.

¹ H NMR (400 MHz, chloroform-d), C20 peaks only: δ 7.19 (dd, J=8.1, 1.5 Hz, 1H), 7.12 (dd, J=8.3, 1.4 Hz, 1H), 6.90 (dd, J=8.2, 8.2 Hz, 1H), 5.26-5.19 (m, 4H), 4.00-3.92 (m, 2H), 3.80-3.73 (m, 2H), 1.00-0.91 (m, 4H), 0.03 (s, 9H), 0.00 (s, 9H).

Step 2. Synthesis of tert-butyl-4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)-3,6dihydropyridine-1(2H)-carboxylate (C21).

A reaction vessel containing a suspension of C2O (6.11 g, 13.6 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (5.04 g, 16.3 mmol), aqueous sodium carbonate solution (1 M; 40.8 mL, 40.8 mmol) and [1,1'bis(diphenylphosphino)ferrocene]dichloropalladium(II) (497 mg, 0.679 mmol) in 1,4-dioxane (100 mL) was evacuated and backfilled with nitrogen. This vacuum cycle was repeated twice and the reaction mixture was then stirred at 85 °C for 16 h, after which the reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Purification by silica gel chromatography (gradient: 0 to 8% methanol in dichloromethane) gave C21 as a yellow oil. Yield: 5.47 g, 9.91 mmol, 73%.

1H NMR (600 MHz, chloroform-d) δ 7.10 (br d, J=8.2 Hz, 1H), 6.98 (dd, J=7.9, 7.9 Hz, 1H), 6.81 (br d, J=1 ,1 Hz, 1H), 5.79 (br s, 1H), 5.23 (s, 2H), 5.07 (s, 2H), 4.03 (br s, 2H), 3.83-3.74 (m, 4H), 3.59 (br s, 2H), 2.52 (br s, 2H), 1.49 (s, 9H), 1.01-0.89 (m, 4H), 0.01 (s, 9H), 0.01 (s, 9H).

Step 3. Synthesis of tert-butyl 4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)piperidine-1-carboxylate (C22).

A solution of C21 (12.5 g, 22.6 mmol) in methanol (300 mL) was treated with 10% palladium on carbon (2.94 g, 2.76 mmol) and hydrogenated for 16 h at 40 psi and 25 °C. LC-MS analysis at this time point indicated conversion to the product: LC-MS m/z 576.0 [M+Na+]. After filtering the reaction mixture and washing the filter cake with methanol (2 x 100 mL), the combined filtrates were concentrated in vacuo to afford C22 as a colorless oil. Yield: 11.2 g, 20.1 mmol, 89%.

1H NMR (400 MHz, chloroform-d) δ 7.05-6.97 (m, 2H), 6.83 (dd, J=6.9, 2.5 Hz, 1H), 5.22 (s, 2H), 5.13 (s, 2H), 4.38-4.10 (br m, 2H), 3.90-3.82 (m, 2H), 3.81-3.73 (m, 2H), 3.22 (tt, J=12.2, 3.5 Hz, 1H), 2.79 (br dd, J=12.8, 12.8 Hz, 2H), 1.78 (br d, J=13 Hz, 2H), 1.65-1.52 (m, 2H), 1.48 (s, 9H), 1.04-0.91 (m, 4H), 0.03 (s, 9H),

- 43 040816

0.00 (s, 9H).

Step 4. Synthesis of 4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)piperidine (C23).

To a room temperature (15 °C) solution of C22 (7.23 g, 13.0 mmol) in dichloromethane (90 mL) was added 2,6-dimethylpyridine (2.39 g, 22.3 mmol), followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (3.80 g, 17.1 mmol). The reaction mixture was stirred at 15 °C for 16 h, after which additional 2,6-dimethylpyridine (909 mg, 8.48 mmol) and trimethylsilyl trifluoromethanesulfonate (1.45 g, 6.52 mmol) were added. After stirring at room temperature (15 °C) for an additional 5 h, LC-MS analysis of the reaction mixture indicated the presence of the product: LC-MS m/z 454.1 [M+H]+. The reaction mixture was concentrated in vacuo and the residue was washed successively with aqueous ammonium chloride (3 x 100 ml) and saturated aqueous sodium chloride (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to give C23 as a brown oil (6.6 g). This material was carried directly to the next step.

Step 5. Synthesis of methyl-2-{[4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)piperidin-1yl]methyl}-1-(2-methoxyethyl)-1 H-benzimidazole -6-carboxylate (C24).

To a solution of C23 (from the previous step; 6.6 g, <13 mmol) in acetonitrile (150 mL) was added P11 (3.08 g, 10.9 mmol), then potassium carbonate (10.1 g, 73.1 mmol) was added and the reaction mixture was stirred at room temperature (15 °C) for 16 h. LC-MS analysis at this time indicated the presence of product: LC-MS m/z 700.2 [M+H] ⁺ . The reaction mixture was filtered and the filtrate was concentrated in vacuo. Purification by silica gel chromatography (gradient: 34 to 56% ethyl acetate in petroleum ether) gave C24 as a yellow oil. Yield: 5.4 g, 7.7 mmol, 59% over 2 steps.

¹ H NMR (400 MHz, chloroform-d) δ 8.16-8.12 (m, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H), 7.04-6.96 (m, 2H), 6.86 (dd, J=6.7, 2.6 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.63 (t, J=5.5 Hz, 2H), 3.95 (s, 3H), 3.93-3.83 (m, 4H), 3.80-3.72 (m, 4H), 3.31 (s, 3H), 3.17-3.06 (m, 1H), 2.99 (br d, J=11.2 Hz, 2H), 2.35-2.22 (m, 2H), 1.81 (br d, half of AB quartet, J=12.6 Hz, 2H), 1.75-1.61 (m, 2H), 1.04-0.91 (m, 4H), 0.05 (s, 9H), -0.01 (s, 9H).

Step 6. Synthesis of methyl 2-{[4-(2,3-dihydroxyphenyl)piperidin-1-yl]methyl}-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylate (P14).

A solution of hydrogen chloride in 1,4-dioxane (4 M; 96 mL, 384 mmol) was added to a room temperature (18 °C) solution of C24 (6.40 g, 9.14 mmol) in 1,4-dioxane (120 mL). After complete addition, the reaction mixture was stirred at room temperature (18 °C) for 16 h, combined with the product from a similar reaction carried out using C24 (1.00 g, 1.43 mmol), and concentrated in vacuo. The residue was treated with a mixture of dichloromethane and methanol (20:1, 150 mL) and stirred at room temperature (18 °C) for 1 h, after which the solid (4.85 g) was collected by filtration. This material was treated with water (100 mL) and the mixture was adjusted to pH 7–8 by adding aqueous sodium bicarbonate solution, stirred at room temperature (18 °C) for 30 min and filtered. The filter cake was washed with water (2 x 20 mL), then mixed with methanol (100 mL) and concentrated in vacuo. The resulting material was treated with petroleum ether (100 mL) and stirred at room temperature (18 °C) for 30 min. After filtration, the filter cake was mixed with toluene (30 mL) and concentrated in vacuo to give P14 as a grey solid. Overall yield: 2.92 g, 6.64 mmol, 63%. LC-MS m/z 440.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.21 (d, J=1.6 Hz, 1H), 7.81 (dd, J=8.5, 1.6 Hz, 1H), 7.66 (d, J=8, 5 Hz, 1H), 6.64-6.51 (m, 3H), 4.63 (t, J=5.3 Hz, 2H), 3.88 (s, 3H), 3.84 (s, 2H), 3.75 (t, J=5.3 Hz, 2H), 3.22 (s, 3H), 2.97-2.78 (m , 3Н), 2.18 (br dd, J=11, 11 Hz, 2Н), 1.75-1.64 (m, 2H), 1.64-1.49 (m, 2H).

Receiving P15. Methyl 2-(chloromethyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (P15)

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szo

This entire reaction sequence was carried out on a large scale. Typically, the reactors were evacuated to -0.08 to -0.05 MPa before reactions were carried out and reagents were added, and then backfilled with nitrogen to normal pressure. This process was typically repeated 3 times, and the oxygen content was then assessed to ensure that it was <1.0%. For the extraction and washing processes of the organic layers, the mixtures were typically stirred for 15-60 min and then allowed to settle for 15-60 min before the layers were separated.

Step 1. Synthesis of (2S)-2-[(benzyloxy)methyl]oxetane (C25).

This reaction was carried out in three batches of approximately equal size. A 2000 L glass lined reactor was charged with 2-methylpropan-2-ol (774.7 kg). Potassium tert-butoxide (157.3 kg, 1402 mol) was added via the solids loading funnel and the mixture was stirred for 30 min. Trimethylsulfoxonium iodide (308.2 kg, 1400 mol) was then added in the same manner and the reaction mixture was heated at 55-65 °C for 2-3 h, after which (28)-2-[(benzyloxy)methyl]oxirane (92.1 kg, 561 mol) was added at a rate of 5 to 20 kg per hour. After the reaction mixture was maintained at 55-65 °C for 25 h, it was cooled to 25-35 °C and filtered through diatomaceous earth (18.4 kg). The filter cake was washed with tert-butyl methyl ether (3 x 340 kg) and the combined filtrates were transferred to a 5000 L reactor, treated with purified water (921 kg) and stirred for 15-30 min at 1530 °C. The organic layer was then washed twice with a solution of sodium chloride (230.4 kg) in purified water (920.5 kg) and concentrated under reduced pressure (< -0.08 MPa) at <45 °C. Nheptane (187 kg) was added and the resulting mixture was concentrated under reduced pressure (< -0.08 MPa) at <45 °C. The organic phase was purified by chromatography on silica gel (280 kg) with sodium chloride (18.5 kg) at the top of the column. The crude material was loaded onto the column using n-heptane (513 kg) and then eluted with a mixture of n-heptane (688.7 kg) and ethyl acetate (64.4 kg). The three batches were combined to give C25 as a light yellow oil with 85% purity (189.7 kg, 906 mmol, 54%).

1H NMR (400 MHz, chloroform-d), only C25 peaks: δ 7.40-7.32 (m, 4H), 7.32-7.27 (m, 1H), 4.98 (dddd, J=8.1, 6.7, 4.9, 3.7 Hz , 1H), 4.72-4.55 (m, 4H), 3.67 (dd, ABX system component, J=11.0, 4.9 Hz, 1H), 3.62 (dd, ABX system component, J=11.0, 3.7 Hz, 1H), 2.72-2.53 (m, 2H).

Step 2. Synthesis of (2S)-oxetan-2-ylmethanol (C26).

10% palladium on carbon (30.7 kg) was added via a charging funnel to a 10-30°C solution of 85% pure C25 (from the previous step; 185.3 kg, 884.8 mol) in tetrahydrofuran (1270 kg) in a 3000 L stainless steel autoclave reactor. The charging funnel was rinsed with purified water and tetrahydrofuran (143 kg), and the rinses were added to the reaction mixture. After purging the reactor contents with nitrogen, it was similarly purged with hydrogen, increasing the pressure to 0.3-0.5 MPa and then dropping to 0.05 MPa. This hydrogen purging was repeated 5 times, after which the hydrogen pressure was increased to 0.3-0.4 MPa. The reaction mixture was then heated to 35-45°C. After 13 h, during which the hydrogen pressure was maintained at 0.3-0.5 MPa, the mixture was vented to 0.05 MPa and flushed with nitrogen five times, increasing the pressure to 0.15-0.2 MPa and then dropping to 0.05 MPa. After cooling the mixture to 10-25°C, it was filtered and the reactor was washed with tetrahydrofuran (2 x 321 kg). The filter cake was soaked twice with the washing liquid and then filtered. Concentration under reduced pressure (< -0.06 MPa) was carried out at < 40°C to give C26 (62.2 kg, 706 mol, 80%) in tetrahydrofuran (251 kg).

Step 3. Synthesis of (2S)-oxetan-2-ylmethyl-4-methylbenzenesulfonate (C27).

4-(Dimethylamino)pyridine (17.5 kg, 143 mol) was added to a solution of C26 (from the previous step; 62.2 kg, 706 mol) in tetrahydrofuran (251 kg) and triethylamine (92.7 kg, 916 mol) in dichloromethane (1240 kg) at 10-25 °C. After 30 min, p-toluenesulfonyl chloride (174.8 kg, 916.9 mol) was added portionwise at 20-40 min intervals and the reaction mixture was stirred at 15-25 °C for 16 h and 20 min. Purified water (190 kg) was added and after stirring, the organic layer was washed with an aqueous solution of sodium bicarbonate (prepared from 53.8 kg of sodium bicarbonate and 622 kg of purified water) and then washed with an aqueous solution of ammonium chloride (prepared from 230 ammonium chloride and 624 kg of purified water). After a final wash with purified water (311 kg), the organic layer was filtered through a steel Nutsche filter pre-loaded with silica gel (60.2 kg). The filter cake was soaked in dichloromethane (311 kg) for 20 min and then

- 45 040816 was filtered. The combined filtrates were concentrated under reduced pressure (< -0.05 MPa) and < 40 °C until 330-400 L remained. Tetrahydrofuran (311 kg) was then added at 15-30 °C and the mixture was concentrated in the same manner to a final volume of 330-400 L. The addition of tetrahydrofuran and concentration were repeated again to a volume of 330-400 L to give a light yellow solution of C27 (167.6 kg, 692 mmol, 98%) in tetrahydrofuran (251.8 kg).

1H NMR (400 MHz, chloroform-d), only C27 peaks: δ 7.81 (d, J=8.4 Hz, 2H), 7.34 (d, J=8.1 Hz, 2H), 4.91 (ddt, J=8.0, 6.7, 3.9 Hz, 1H), 4.62-4.55 (m, 1H), 4.53-4.45 (m, 1H), 4.14 (d, J=3.9 Hz, 2H), 2.75-2.63 (m, 1H), 2.60-2.49 (m, 1H), 2.44 (s, 3H).

Step 4. Synthesis of (2S)-2-(azidomethyl)oxetane (C28).

K,K-Dimethylformamide (473 kg), sodium azide (34.7 kg, 534 mol), and potassium iodide (5.2 kg, 31 mol) were combined in a 3000 L glass-lined reactor at 10-25°C. After adding C27 (83.5 kg, 344.6 mol) in tetrahydrofuran (125.4 kg), the reaction mixture was heated to 55-65°C for 17 h and 40 min, after which it was cooled to 25-35°C and nitrogen was bubbled from the bottom valve for 15 min. Tert-butyl methyl ether (623 kg) and purified water (840 kg) were then added, and the resulting aqueous layer was extracted twice with tert-butyl methyl ether (312 kg and 294 kg). The combined organic layers were washed with purified water (2x419 kg) maintaining the temperature at 10-25°C to give C28 (31.2 kg, 276 mol, 80%) in a solution of the above organic layer (1236.8 kg).

Step 5. Synthesis of 1-[(2S)-oxetan-2-yl]methanamine (C29).

10% palladium on carbon (3.7 kg) was added via a feed funnel to a 10-30°C solution of C28 [from the previous step; 1264 kg (31.1 kg C28, 275 mol)] in tetrahydrofuran (328 kg) in a 3000 L steel autoclave reactor. The feed funnel was rinsed with tetrahydrofuran (32 kg) and the rinse liquid was added to the reaction mixture. After purging the reactor contents with nitrogen, it was similarly purged with hydrogen, increasing the pressure from 0.05 to 0.15 MPa and then venting to 0.03-0.04 MPa. This hydrogen purge was repeated 5 times, after which the hydrogen pressure was increased to 0.05-0.07 MPa. The reaction temperature was increased to 25-33 °C and the hydrogen pressure was maintained at 0.05-0.15 MPa for 22 h, replacing the hydrogen every 3-5 h. The mixture was then purged with nitrogen five times, increasing the pressure to 0.15-0.2 MPa and then venting to 0.05 MPa. After filtration, tetrahydrofuran (92 kg and 93 kg) was used to rinse the reactor and then soak the filter cake. The combined filtrates were concentrated under reduced pressure (< -0.07 MPa) and < 45 °C to give C29 (18.0 kg, 207 mol, 75%) in tetrahydrofuran (57.8 kg).

¹ H NMR (400 MHz, DMSO-d6), only C29 peaks: δ 4.62 (ddt, J=7.6, 6.6, 5.1 Hz, 1H), 4.49 (ddd, J=8.6, 7.3, 5.6 Hz, 1H), 4.37 (dt, J=9.1, 5.9 Hz, 1H), 2.69 (d, J=5.1 Hz, 2H), 2.55-2.49 (m, 1H), 2.39 (m, 1H).

Step 6. Synthesis of methyl 4-nitro-3-{[(2S)-oxetan-2-ylmethyl]amino}benzoate (C30).

Potassium carbonate (58.1 kg, 420 mol) was added to a solution of methyl 3-fluoro-4-nitrobenzoate (54.8 kg, 275 mol) in tetrahydrofuran (148 kg) in a 100 L glass-lined reactor and the mixture was stirred for 10 min. A solution of C29 (29.3 kg, 336 mol) in tetrahydrofuran (212.9 kg) was added and the reaction mixture was stirred at 20-30 °C for 12 h, after which ethyl acetate (151 kg) was added and the mixture was filtered through silica gel (29 kg). The filter cake was washed with ethyl acetate (150 kg and 151 kg) and the combined filtrates were concentrated under reduced pressure (< -0.08 MPa) and < 45 °C to a volume of 222-281 L. After cooling the mixture to 10-30°C, n-heptane (189 kg) was added, stirring was carried out for 20 min, and the mixture was concentrated under reduced pressure (< -0.08 MPa) and <45°C to a volume of 222 L. n-heptane (181 kg) was again added to the mixture at a standard rate of 100-300 kg per hour, and stirring was continued for 20 min. Samples of the mixture were analyzed until the residual tetrahydrofuran was <5% and the residual ethyl acetate was 10%-13%. The mixture was heated to 40-45°C and stirred for 1 h, after which it was cooled to 15-25°C at a rate of 5-10°C per hour and then stirred at 15-25°C for 1 h. After filtration using a steel centrifuge, a filter cake was obtained, which was washed with a mixture of ethyl acetate (5.0 kg) and n-heptane (34 kg) and then stirred with tetrahydrofuran (724 kg) at 10-30°C for 15 min. After filtration, a yellow solid consisting mainly of C30 (57.3 kg, 210 mol, 76%) was obtained.

1H NMR (400 MHz, DMSO-d6) 8.34 (t, J=5.8 Hz, 1H), 8.14 (d, J=8.9 Hz, 1H), 7.63 (d, J=1.7 Hz, 1H), 7.13 (dd, J=8.9, 1.8 Hz, 1H), 4.99 (dddd, J=7.7, 6.7, 5.3, 4.1 Hz, 1H), 4.55 (ddd, J=8.6, 7.3, 5.8 Hz, 1H), 4.43 (dt, J=9.1, 6.0 Hz, 1H), 3.87 (s, 3H), 3.67-3.61 (m, 2H), 2.67 (dddd, J=11.1, 8.6, 7.7, 6.2 Hz, 1H), 2.57-2.47 (m, 1H).

Step 7. Synthesis of methyl 2-(chloromethyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6carboxylate (P15).

A solution of C30 (from the previous step; 51.8 kg, 190 mol) in tetrahydrofuran (678 kg) in a 3000 L autoclave reactor was treated with 10% palladium on carbon (5.2 kg) at 10-30 °C. The loading tube was flushed with tetrahydrofuran (46 kg), and the flushing liquid was added to the reaction mixture. After purging the reactor contents with nitrogen, it was similarly purged with hydrogen, increasing the pressure to 0.1-0.2 MPa and then dropping to 0.02-0.05 MPa. This hydrogen purging was repeated 5 times, after which the hydrogen pressure was increased to 0.1-0.25 MPa. The reaction mixture was stirred at 20-30 °C and

- 46 040816 Every 2-3 h the mixture was purged with nitrogen three times and then purged with hydrogen five times; after each final hydrogen change the hydrogen pressure was increased to 0.1-0.25 MPa. After 11.25 h of reaction time the reaction mixture was vented to normal pressure and purged with nitrogen five times, increasing the pressure to 0.15-0.2 MPa and then dropping to 0.05 MPa. The mixture was then filtered and the filter cake was washed twice with tetrahydrofuran (64 kg and 63 kg). The combined washings and filtrate were concentrated under reduced pressure (< -0.08 MPa) and <40 °C to a volume of 128-160 L. Tetrahydrofuran (169 kg) was added and the mixture was concentrated again to a volume of 128-160 L; This process was repeated a total of 4 times to obtain a solution of the intermediate methyl 4-amino-3-{[(2S)oxetan-2-ylmethyl]amino}benzoate.

Tetrahydrofuran (150 kg) was added to this solution, then 2-chloro-1,1,1-trimethoxyethane (35.1 kg, 227 mol) and p-toluenesulfonic acid monohydrate (1.8 kg, 9.5 mol) were added. After stirring the reaction mixture for 25 min, it was heated at 40-45°C for 5 h, after which it was concentrated under reduced pressure to a volume of 135-181 L. 2-Propanol (142 kg) was added, and the mixture was again concentrated to a volume of 135-181 L, after which 2-propanol (36.5 kg) and purified water (90 kg) were added, and stirring was continued until a solution was obtained. This solution was filtered using an in-line liquid filter and then treated with purified water (447 kg) at a standard rate of 150-400 kg per hour at 20-40°C. After cooling the mixture to 20-30°C, it was stirred for 2 h and the solid was collected by filtration using a centrifuge. The filter cake was washed with a solution of 2-propanol (20.5 kg) and purified water (154 kg); after drying, P15 was obtained as a white solid (32.1 kg, 109 mol, 57%).

¹ H NMR (400 MHz, chloroform-d) δ 8.14-8.11 (m, 1H), 8.01 (dd, J=8.5, 1.1 Hz, 1H), 7.79 (br d, J=8.6 Hz, 1H), 5.26-5.18 (m, 1H), 5.04 (s, 2H), 4.66-4.58 (m, 2H), 4.53 (dd, component of the ABX system, J=15.7, 2.7 Hz, 1H), 4.34 (dt, J=9.1, 6.0 Hz, 1H), 3.96 (s, 3H), 2.82-2.71 (m, 1H), 2.48-2.37 (m, 1H).

Receiving P16. Methyl 2-(chloromethyl)-1-methyl-1H-benzimidazole-6-carboxylate (P16)

Methyl 4-amino-3-(methylamino)benzoate (206 mg, 1.14 mmol) was dissolved in 1,4-dioxane (11.5 mL) and treated with chloroacetyl chloride (109 μL, 1.37 mmol). The mixture was stirred at 100 °C for 3 h and cooled to room temperature. Triethylamine (0.8 mL, 7 mmol) and heptane (10 mL) were added and filtered. The filtrate was concentrated under reduced pressure and the crude material was purified by silica gel chromatography (eluent: 40% ethyl acetate in heptane) to give 120 mg of P16 (44%).

1H NMR (400 MHz, chloroform-d) δ 8.14 (s, 1H), 8.01 (d, 1H), 7.78 (d, 1H), 4.87 (s, 2H), 3.97 (s, 3H), 3.94 (s, 3H); LC-MS m/z 239.1 [M+H]+.

Preparation of P17 and P18. Methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate, ENT-1 (P17) and methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate, ENT-2 (P18)

Step 1. Synthesis of tert-butyl 4-(2-ethoxy-2-oxoethylidene)piperidine-1-carboxylate (C31).

- 47 040816

A solution of potassium tert-butoxide (65.9 g, 587 mmol) in tetrahydrofuran (500 mL) was added to a 0 °C solution of ethyl (diethoxyphosphoryl)acetate (132 g, 589 mmol) in tetrahydrofuran (500 mL), and the resulting suspension was stirred at 0 °C for 1 h, after which it was cooled to 50 °C. A solution of tert-butyl 4-oxopiperidine-1-carboxylate (90.0 g, 452 mmol) in tetrahydrofuran (1.5 L) was added dropwise at -50 °C, and the reaction mixture was then allowed to warm slowly to 20 °C and then stirred for 16 h at 20 °C. After adding water (1 L), the mixture was concentrated in vacuo to remove tetrahydrofuran. The aqueous residue was extracted with ethyl acetate (2 x 800 mL) and the combined organic layers were washed with saturated aqueous sodium chloride solution (500 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting material was washed several times with petroleum ether (200 mL) to give C31 as a white solid. Yield: 95.0 g, 353 mmol, 78%.

¹ H NMR (400 MHz, chloroform-d) δ 5.71 (s, 1H), 4.16 (q, J=7.2 Hz, 2H), 3.55-3.43 (m, 4H), 2.94 (br t, J=5.5 Hz, 2H), 2.28 (br t, J=5.5 Hz, 2H), 1.47 (s, 9H), 1.28 (t, J=7.0 Hz, 3H).

Step 2. Synthesis of 6-tert-butyl-1-ethyl-6-azaspiro[2.5]octane-1,6-dicarboxylate (C32).

To a solution of trimethylsulfoxonium iodide (140 g, 636 mmol) in dimethyl sulfoxide (800 mL) was added potassium tert-butoxide (71.2 g, 634 mmol) in one portion at 20 °C. After stirring the reaction mixture at 20 °C for 1.5 h, a solution of C31 (95.0 g, 353 mmol) in dimethyl sulfoxide (800 mL) was added dropwise and stirring was continued at 20 °C for 16 h. Then, a saturated aqueous solution of sodium chloride (2.0 L) was added. The resulting mixture was neutralized by adding ammonium chloride and extracted with ethyl acetate (3.0 L). The combined organic layers were washed successively with water (2 x 1.0 L) and saturated aqueous sodium chloride solution (2.0 L), dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (eluent: 10:1 petroleum ether/ethyl acetate) gave C32 as a yellow oil. 1H NMR analysis showed that a foreign aliphatic compound was present. Yield: 80 g, 280 mmol, 79%.

1H NMR (400 MHz, chloroform-d), only C32 peaks: δ 4.19-4.09 (m, 2H), 3.55-3.39 (m, 3H), 3.27 (ddd, J=13.0, 7.0, 4.5 Hz, 1H), 1.76-1.64 (m, 2H), 1.56 (dd, J=8.0, 5.5 Hz, 1H, putative; partially hidden by the water peak), 1.47 (s, 9H), 1.47-1.37 (m, 2H), 1.27 (t, J=7.0 Hz, 3H), 1.17 (dd, J=5.0, 5.0 Hz, 1H), 0.93 (dd, J=8.0, 4.5 Hz, 1H).

Step 3. Synthesis of 6-(tert-butoxycarbonyl)-6-azaspiro[2.5]octane-1-carboxylic acid (C33).

To a mixture of C32 (80 g, 280 mmol) in tetrahydrofuran (500 mL) and water (500 mL) was added lithium hydroxide monohydrate (37.4 g, 891 mmol) in one portion. The reaction mixture was stirred at 25 °C for 16 h, after which it was diluted with water (600 mL) and washed with ethyl acetate (3 x 300 mL). The organic layers were discarded and the aqueous layer was acidified to pH 3-4 with 6 M hydrochloric acid. The resulting mixture was extracted with ethyl acetate (3 x 600 mL) and the combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Trituration of the residue with petroleum ether (300 mL) gave C33 as a white solid. Yield: 42.0 g, 164 mmol, 59%. LC-MS m/z 278.2 [M+Na+].

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.15-12.03 (br s, 1H), 3.43-3.25 (m, 3H, putative; partially hidden by water peak), 3.23-3.12 (m, 1H), 1.64-1.50 (m, 2H), 1.52 (dd, J=7.5, 5.5 Hz, 1H), 1.39 (s, 9H), 1.39-1.28 (m, 2H), 0.96-0.88 (m, 2H).

Step 4. Synthesis of tert-butyl-1-({4-(methoxycarbonyl)-2-[(2-methoxyethyl)amino]phenyl}carbamoyl)-6-azaspiro[2.5]octane-6-carboxylate (C34).

A solution of C33 (570 mg, 2.23 mmol), C16 (500 mg, 2.23 mmol) and O-(1-azabenzotriazol-1-yl)-N,N,N',N'tetramethyluronium hexafluorophosphate (HATU; 1.27 g, 3.34 mmol) in N,N-dimethylformamide (10 ml) was stirred at 30 °C for 30 min, after which triethylamine (902 mg, 8.91 mmol) was added and stirring was continued at 30 °C for 16 h. The reaction mixture was then poured into water (60 ml) and extracted with ethyl acetate (3 x 50 ml). The combined organic layers were washed with saturated aqueous sodium chloride solution (3 x 50 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (eluent: 1:1 petroleum ether/ethyl acetate) gave C34 as a brown oil, which was carried directly to the next step.

Step 5. Synthesis of methyl 2-[6-(tert-butoxycarbonyl)-6-azaspiro[2.5]oct-1-yl]-1-(2-methoxyethyl)1 H-benzimidazole-6-carboxylate (C35).

A solution of C34 (from the previous step, <2.23 mmol) in acetic acid (15 mL) was stirred at 50 °C for 16 h and then concentrated in vacuo to give C35 as a brown oil. This material was used directly in the next step. LC-MS m/z 444.1 [M+H] ⁺ .

Step 6. Synthesis of methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (C36).

Trifluoroacetic acid (5 ml) was added to a solution of C35 (from the previous step; <2.23 mmol) in dichloromethane (10 ml), and the reaction mixture was stirred at 25 °C for 2 h. After removal of the solvents in vacuo, the residue was made basic by adding a saturated aqueous solution of carbonate

- 48 040816 potassium (40 ml) and extracted with a mixture of dichloromethane and methanol (10:1, 3 x 40 ml). The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and chromatographed on silica gel (eluent: 10:1:0.1 dichloromethane/methanol/concentrated ammonium hydroxide) to give C36 as a yellow solid. Yield: 640 mg, 1.86 mmol, 83% over three steps.

LC-MS m/z 344.1 [M+H]+.

Step 7. Isolation of methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate ENT-1 (P17) and methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate ENT-2 (P18).

Separation of C36 (630 mg, 1.83 mmol) into its component enantiomers was accomplished using SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 55:45 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting peak was designated ENT-1 (P17) and the second eluting enantiomer was designated ENT-2 (P18); both were isolated as pale yellow solids.

P17 Yield: 300 mg, 0.874 mmol, 48%. LC-MS m/z 344.1 [M+H]+. Retention time: 5.10 min (column: Chiral Technologies Chiralpak AD-3, 4.6x150 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B over 5.5 min, then hold at 40% B for 3.0 min; flow rate: 2.5 mL/min).

P18 Yield: 240 mg, 0.699 mmol, 38%. LC-MS m/z 344.1 [M+H]+. Retention time: 7.35 min (analytical conditions identical to those used for P17).

Preparation of P19. Methyl 4-amino-3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}benzoate (P19) o

C37 R19

Step 1. Synthesis of methyl 3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}-4-nitrobenzoate (C37).

Triethylamine (3.65 mL, 26.2 mmol) was added to a solution of methyl 3-fluoro-4-nitrobenzoate (1.00 g, 5.02 mmol) and 1-(1-ethyl-3-imidazol-5-yl)methanamine dihydrochloride salt (1.00 g, 5.05 mmol) in a mixture of tetrahydrofuran (12 mL) and methanol (8 mL). The reaction mixture was stirred at 60 °C for 40 h, after which it was concentrated in vacuo and purified by silica gel chromatography (gradient: 0 to 2% methanol in dichloromethane) to give C37 as an orange solid. Yield: 1.27 g, 4.17 mmol, 83%.

¹ H NMR (400 MHz, chloroform-d) δ 8.24 (d, J=8.8 Hz, 1H), 7.98-7.91 (m, 1H), 7.68 (d, J=1.7 Hz, 1H), 7.57 (br s, 1H), 7.33 (dd, J=8.8, 1.7 Hz, 1H), 7.11 (br s, 1H), 4.53 (d, J=4.9 Hz, 2H), 3.99 (q, J=7.3 Hz, 2H), 3.95 (s, 3H), 1.47 (t, J=7.3 Hz, 3H).

Step 2. Synthesis of methyl 4-amino-3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}benzoate (P19).

A mixture of wet palladium on carbon (144 mg) and C37 (412 mg, 1.35 mmol) in methanol (13 mL) was stirred under a hydrogen balloon for 16 h at 25 °C. The reaction mixture was then filtered through a pad of diatomaceous earth and the filtrate was concentrated in vacuo to give P19 as a gray solid. Yield: 340 mg, 1.24 mmol, 92%.

1H NMR (400 MHz, methanol-d ₄ ) δ 7.66 (br s, 1H), 7.38-7.29 (m, 2H), 6.97 (br s, 1H), 6.67 (d, J=7.9 Hz, 1H), 4.35 (s, 2H), 4.11 (q, J=7.3 Hz, 2H), 3.81 (s, 3H), 1.44 (t, J=7.3 Hz, 3H).

Preparation of P20. Methyl 4-amino-3-(methylamino)benzoate (P20)

D1 P20

Step 1. Synthesis of methyl 3-(methylamino)-4-nitrobenzoate (D1).

To a solution of methyl 3-fluoro-4-nitrobenzoate (5.10 g, 25.6 mmol) in tetrahydrofuran (60 mL) was added methylamine (38.4 mL, 76.8 mmol, 2 M in tetrahydrofuran) dropwise over 10 min. Immediately after the addition, the pale yellow solution turned deep orange, and it was stirred for 2 h at room temperature. The reaction mixture was then diluted with diethyl ether (100 mL), and the organic layer was washed successively with water (50 mL) and saturated aqueous sodium chloride solution (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 5.26 g of methyl 3-(methylamino)-4-nitrobenzoate (98%) as a deep orange solid. LC-MS m/z 211.1 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.22 (d, J=8.9 Hz, 1H), 8.00 (br s, 1H), 7.56 (d, J=1.7 Hz, 1H), 7.25 (dd, J=8.9, 1.7 Hz, 1H, predicted; partially hidden by solvent peak), 3.95 (s, 3H), 3.09 (d, J=5.1 Hz, 3H).

Stage 2. Synthesis of methyl 4-amino-3-(methylamino)benzoate (P20).

- 49 040816

A solution of D1 (5.26 g, 25.0 mmol) in ethanol (150 mL) was added to a 500 mL Parr® flask pre-loaded with 10% palladium on carbon (50% water; 1 g). The mixture was shaken under 50 psi hydrogen for 1 h at room temperature, after which it was filtered and the filter cake was washed with ethanol (100 mL). The filtrate was concentrated under reduced pressure to give 4.38 g of P20 (97%) as an off-white solid. LCMS m/z 181.1 [M+H]+.

^1H NMR (400 MHz, chloroform-d) δ 7.46 (dd, J=8.0, 1.9 Hz, 1H), 7.34 (d, J=1.8 Hz, 1H), 6.68 (d, J=8, 0 Hz, 1H), 3.87 (s, 3H), 3.72 (br s, 2H), 3.21 (br s, 1H), 2.91 (s, 3H).

Preparation of P21 and P22. 5-Bromo-N ³ -methylpyridine-2,3-diamine (P21) and 5-Bromo-N ³ ,6-dimethylpyridine2,3-diamine (P22) n n n ₃ u ii η p ₃ ь Tj d η ₂ ν^ν^ h ₂ n^n^ch ₃

Ρ21 Ρ22

Intermediate P21 was synthesized according to the procedure described in the literature (Choi, J. Y. et al. J. Med. Chem. 2012, 55, 852-870). Intermediate P22 was synthesized using the same method.

Preparation of P23. Methyl 2-(chloromethyl)-[(1-methyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6carboxylate (P23)

D4 P23

Step 1. Synthesis of methyl 3-{[(1-methyl-1H-imidazol-5-yl)methyl]amino}-4-nitrobenzoate (D2).

To a colourless solution of methyl 3-fluoro-4-nitrobenzoate (1.0 g, 5.0 mmol) in N,N-dimethylformamide (10 mL) were slowly added 1-(1-methyl-1H-imidazol-5-yl)methanamine (670 mg, 6.0 mmol) and triethylamine (762 mg, 7.53 mmol). The reaction mixture was stirred at 60 °C for 16 h, after which it was poured into water (30 mL) and extracted with dichloromethane (3 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by chromatography on silica gel (eluent: 20% methanol in dichloromethane). The resulting yellow solid was triturated with 30:1 petroleum ether/ethyl acetate to give D2 (1.2 g, 82%) as a yellow solid. LC-MS m/z 290.9 [M+H] ⁺ .

1H NMR (400 MHz, chloroform-d) δ 8.25 (d, J=8.9 Hz, 1H), 7.98-7.92 (m, 1H), 7.70 (d, J=1.7 Hz, 1H), 7.49 (s, 1H), 7.34 (dd, J=8.9, 1.7 Hz, 1H), 7.12 (s, 1H), 4.54 (d, J=5.0 Hz, 2H), 3.96 (s, 3H), 3.67 (s, 3H).

Step 2. Synthesis of methyl 4-amino-3-{[(1-methyl-1H-imidazol-5-yl)methyl]amino}benzoate (D3).

To a suspension of D2 (5.46 g, 18.8 mmol) in methanol (160 mL) was added wet 10% palladium on carbon (1 g). The mixture was stirred under 1 atm hydrogen for 36 h at 20 °C. The reaction mixture was filtered and the filter cake was washed with methanol (200 mL). The filtrate was concentrated under reduced pressure to give D3 (4.8 g, 98%) as a brown solid. LCMS m/z 260.9 [M+H] ⁺ .

1H NMR (400 MHz, DMSC-d6) δ 7.56 (s, 1H), 7.18 (br d, J=8.1 Hz, 1H), 7.12 (brs, 1H), 6.87 (s, 1H), 6.55 (d , J=8.2 Hz, 1H), 5.50 (s, 2H), 4.84 (t, J=5.2 Hz, 1H), 4.23 (d, J=5.0 Hz, 2H), 3.73 (s, 3H), 3.63 (s, 3H).

Step 3. Synthesis of methyl 2-(hydroxymethyl)-1-[(1-methyl-1H-imidαzol-5-yl)methyl]-1Hbenzimidazole-6-carboxylate (D4).

A mixture of D3 (780 mg, 3.00 mmol) and 2-hydroxyacetic acid (342 mg, 4.49 mmol) in 1,3,5-trimethylbenzene (8 mL) was stirred at 140 °C for 14 h and at 25 °C for 48 h. The clear yellow solution was decanted to give a brown residue, which was dissolved in methanol (50 mL) and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (eluent: 20% methanol in dichloromethane) to give D4 (318 mg, 35%) as a yellow foam. LCMS m/z 300.9 [M+H] ⁺ .

1H NMR (400 MHz, DMSC-d6) δ 8.13-8.11 (m, 1H), 7.83 (dd, J=8.4, 1.6 Hz, 1H), 7.71 (d, J=8.5 Hz, 1H), 7.59 (s, 1H), 6.58 (s, 1H), 5.69 (s, 2H), 4.75 (s, 2H), 3.84 (s, 3H), 3.53 (s, 3H).

Step 4. Synthesis of methyl-2-(chloromethyl)-1-[(1-methyl-1H-imidazol-5-yl)methyl]-1H-benzimidazol-6

- 50 040816 carboxylate (P23).

To a suspension of D4 (500 mg, 1.66 mmol) in dichloromethane (10 mL) and N,N-dimethylformamide (3 mL) was added thionyl chloride (990 mg, 0.60 mL, 8.32 mmol) dropwise at room temperature. The reaction mixture was stirred at room temperature for 1 h, then concentrated under reduced pressure. The resulting brown residue was triturated with dichloromethane (10 mL). The solid was collected by filtration and washed with dichloromethane (5 mL) to give P23 (431 mg, 73%) as an off-white solid. LCMS m/z 318.9^ [M+H]+.

¹ H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.31 (s, 1H), 7.93 (br d, J=8.5 Hz, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.11 (s, 1H), 5.92 (s, 2H), 5.13 (s, 2H), 3.87 (s, 3H), 3.87 (s, 3H).

Preparation of P24. 5-Chloro-2-(chloromethyl)-3-methyl-3H-imidazo[4,5-b]pyridine (P24)

Step 1. Synthesis of 6-chloro-N-methyl-3-nitropyridin-2-amine (D5).

To a suspension of 2,6-dichloro-3-nitropyridine (200 g, 1.04 mol) and _Na2CO3 (132 g, 1.24 mol) in ethanol (1 _L ) was added a solution of methylamine in tetrahydrofuran (2.0 M; 622 mL, 1.24 mol) dropwise at 0 °C with a syringe. After complete addition, the reaction mixture was stirred at 18 °C for 6 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to give a yellow solid. This crude material was purified by silica gel chromatography (gradient: 0 to 5% ethyl acetate in petroleum ether) to give D5 (158 g, 81% yield) as a yellow solid.

1H NMR (400 MHz, DMSO-d6) δ 8.72 (br s, 1H), 8.41 (d, J=8.6 Hz, 1H), 6.76 (d, J=8.6 Hz, 1H), 3.00 (d, J=4.8 Hz, 3H).

Step 2. Synthesis of 6-χλορ-Ν-methylpyridine-2,3-diamine (D6).

To a mixture of D5 (15.8 g, 84.2 mmol) in acetic acid (100 mL) was added iron powder (15.4 g, 276 mmol). The reaction mixture was stirred at 80 °C for 3 h, then cooled to room temperature and filtered. The filter cake was washed with ethyl acetate (2 x 100). The combined organic layers were concentrated under reduced pressure and the crude material was purified by silica gel chromatography (eluent: 1:1 ethyl acetate/petroleum ether) to give D6 (8.40 g, 63% yield) as a brown solid.

1H NMR (400 MHz, chloroform-d) δ 6.79 (d, J=1.1 Hz, 1H), 6.49 (d, J=7.7 Hz, 1H), 3.00 (s, 3H).

Step 3. Synthesis of 5-chloro-2-(chloromethyl)-3-methyl-3H-imidazo[4,5-b]pyridine (P24).

To a solution of D6 (50.0 g, 317 mmol) in 1,4-dioxane (1.2 L) was added chloroacetyl chloride (55.5 mL, 698 mmol) and the reaction mixture was stirred at 15 °C for 50 min. It was then concentrated under reduced pressure to give a brown solid, which was taken up in trifluoroacetic acid (1.2 L) and stirred at 80 °C for 60 h. The mixture was concentrated under reduced pressure to give a brown oil, which was diluted with ethyl acetate (1 L) and neutralized by the addition of saturated aqueous sodium bicarbonate. When the evolution of carbon dioxide had subsided, the layers were separated and the aqueous layer was extracted with ethyl acetate (200 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (gradient: 10 to 25% ethyl acetate in petroleum ether) to give P24 (61.0 g, 79% yield) as a yellow solid. LC-MS m/z 215.7 (dichloro isotope pattern observed) [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.13 (d, J=8.3 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 5.11 (s, 2H), 3.84 (s, 3H).

Examples 1 and 2.

2-({4-[2-(4-Chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1-(2-methoxyethyl)1H-benzimidazole-6-carboxylic acid ENT-X1 trifluoroacetate salt (1) [from C39]; and 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid ENT-X2 trifluoroacetate salt (2) [from C40]

- 51 040816

Step 1. Synthesis of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1yl}methyl)-1-(2-methoxyethyl)- 1 H-benzimidazole-6-carboxylate (C3 8).

This experiment was performed in two batches of identical scale. A reaction vessel containing a mixture of C2 (500 mg, 1.52 mmol), P12 (530 mg, 1.59 mmol), [2',6'-bis(propan-2-yloxy)biphenyl-2-yl](dicyclohexyl)phosphane (Ruphos; 142 mg, 0.304 mmol), tris(dibenzylideneacetone)dipalladium(0) (139 mg, 0.152 mmol), and cesium carbonate (1.48 g, 4.54 mmol) in toluene (15 mL) was evacuated and backfilled with nitrogen. This evacuation cycle was repeated twice, after which the reaction mixture was stirred at

100°C for 16 h, combined with the second crop and filtered. The filtrate was concentrated and the residue was subjected to silica gel chromatography (gradient: 0 to 60% ethyl acetate in petroleum ether) followed by preparative thin layer chromatography (eluent: 1:1 petroleum ether/ethyl acetate) to afford C38 as a pale yellow solid. Overall yield: 600 mg, 1.03 mmol, 34%. LC-MS m/z 581.0 ♦ [M+H]+.

Step 2. Isolation of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate ENT-1 (C39) and methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate ENT-2 (C40).

Separation of C38 (780 mg, 1.34 mmol) into its component enantiomers was accomplished using SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer, designated ENT1 (C39), was obtained as a white solid. Yield: 282 mg, 0.485 mmol, 36%. LC-MS m/z 581.0^ [M+H]+. Retention time 1.90 min (column: Chiral Technologies Chiralpak AD-3, 4.6 x 50 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5% B for 0.20 min, then 5 to 40% B over 1.4 min, then hold at 40% B for 1.05 min; flow rate: 4.0 ml per minute).

The second eluting enantiomer, designated ENT-2 (C40), was subjected to a second purification using SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. This afforded C40 as a pale brown solid. Yield: 280 mg, 0.482 mmol, 36%. LC-MS m/z 581.0^ [M+H]+. Retention time 2.18 min (analytical conditions identical to those used for C39).

Step 3. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid ENT-X1 trifluoroacetate salt (1) [from C39].

An aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added to a solution of C39 (70 mg, 0.12 mmol) in a mixture of methanol (3 ml) and tetrahydrofuran (3 ml). After stirring the reaction mixture at 25 °C for 16 h, an aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added again, and

- 52 040816 stirring was continued for another 20 h. The reaction mixture was then adjusted to pH 7 with 1 M hydrochloric acid and then concentrated in vacuo to remove methanol and tetrahydrofuran. The residue was adjusted to pH 5-6 with trifluoroacetic acid and then purified by reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 30 to 60% B) to afford 1 as a white solid. Yield: 40.5 mg, 59.5 μmol, 50%. LC-MS m/z 567.0^ [M+H]+.

1H NMR (400 MHz, methanol-d ₄ ) δ 8.37 (br s, 1H), 8.07 (dd, J=8.5, 1.5 Hz, 1H), 7.79 (d, J=8.6 Hz, 1H), 7.59 (dd, J=8.0, 8.0 Hz, 1H), 7.34 (dd, J=10.2, 2.0 Hz, 1H), 7.30 (br dd, J=8.3, 2.0 Hz, 1H), 7.22 (s, 1H), 6.87 (dd, J=8.1, 8.1 Hz, 1H), 6.63 (brd, J=8 Hz, 1H), 6.60 (br d, J=8 Hz, 1H), 4.70 (s, 2H), 4.65 (t, J=4.8 Hz, 2H), 3.75 (t, J=4.8 Hz, 2H), 3.59-3.42 (m, 8H), 3.29 (s, 3H).

Step 4. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid ENT-X2 trifluoroacetate salt (2) [from C40].

An aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added to a solution of C40 (69 mg, 0.12 mmol) in a mixture of methanol (3 ml) and tetrahydrofuran (3 ml). After stirring the reaction mixture at 25 °C for 16 h, an aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added again and stirring was continued for another 20 h. The reaction mixture was adjusted to pH 7 by adding 1 M hydrochloric acid and then concentrated in vacuo to remove methanol and tetrahydrofuran. The residue was adjusted to pH 5-6 with trifluoroacetic acid and then purified by reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 30 to 60% B) to afford 2 as a white solid. Yield: 22.9 mg, 33.6 μmol, 28%. LC-MS m/z 567.0^ [M+H] ⁺ .

'and NMR (400 MHz, methanol) δ 8.40-8.35 (m, 1H), 8.07 (dd, J=8.6, 1.5 Hz, 1H), 7.79 (d, J=8.6 Hz, 1H), 7.59 (dd, J=8.0, 8.0 Hz, 1H), 7.35 (dd, J=10.2, 2.0 Hz, 1H), 7.31 (br dd, J=8, 2 Hz, 1H), 7.22 (s, 1H), 6.87 (dd, J=8.3, 8.0 Hz, 1H), 6.63 (br d, J=8 Hz, 1H), 6.60 (br d, J=8 Hz, 1H), 4.68 (s, 2H), 4.65 (t, J=4.9 Hz, 2H), 3.76 (t, J=4.8 Hz, 2H), 3.57-3.40 (m, 8H), 3.29 (s, 3H).

Example 3.

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1H-imidazo[4,5-b]pyridine-6-carboxylic acid trifluoroacetate salt (3)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine (C13, free base).

To a solution of P2 (300 mg, 0.670 mmol) in ethyl acetate (3.5 ml) was added toluenesulfonic acid vapor monohydrate (318 mg, 1.67 mmol). The reaction mixture was stirred at 60 °C for 1 h, after which it was made basic by adding saturated aqueous potassium carbonate solution (20 ml) and extracted with a mixture of dichloromethane and methanol (10:1, 3 x 50 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo to give C13, the free base, as a brown solid. Yield: 230 mg, 0.661 mmol, 99%.

Step 2. Synthesis of 6-bromo-2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine (C41).

A suspension of C13, free base (130 mg, 0.374 mmol), P13 (130 mg, 0.427 mmol) and potassium carbonate (172 mg, 1.24 mmol) in acetonitrile (2 mL) was stirred at 50 °C for 16 h. The reaction mixture was then purified by preparative thin layer chromatography (eluent: ethyl acetate) to afford C41 as a brown oil. Yield: 114 mg, 0.185 mmol, 49%. LC-MS m/z 617.1 (bromine and chlorine isotope peaks observed) [M+H]+.

Step 3. Synthesis of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine-6-carboxylate (C42).

A solution of C41 (114 mg, 0.185 mmol), 1,3-bis(diphenylphosphino)propane (15.3 mg, 37.1 μmol), palladium(II) acetate (8.3 mg, 37 μmol) and triethylamine (187 mg, 1.85 mmol) in a mixture of methanol (5 mL) and N,N-dimethylformamide (1 mL) was stirred at 80 °C under carbon monoxide (50 psi) for 16 h. After diluting the reaction mixture with ethyl acetate (50 mL), it was washed with saturated aqueous sodium chloride solution (2 x 50 mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by preparative thin layer chromatography (eluent: ethyl acetate) gave C42 as a colorless oil. Yield: 60.0 mg, 0.101 mmol, 55%. LC-MS m/z 617.2 (a pattern with chlorine isotope peaks [M+Na ⁺ ] was observed).

Step 4. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine-6-carboxylic acid trifluoroacetate salt (3).

To a solution of C42 (60.0 mg, 0.101 mmol) in methanol (2.0 mL) was added aqueous sodium hydroxide solution (3 M; 1.0 mL, 3.0 mmol) and the reaction mixture was stirred at 20 °C for 2 h. It was then adjusted to pH 7 with 1 M hydrochloric acid and extracted with a mixture of dichloromethane and methanol (10:1, 3 x 30 mL). The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and purified using reversed-phase HPLC (column: Boston Green ODS, 5 μm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 10 to 95% B) to give 3 as a white solid. Yield: 29.6 mg, 42.6 µmol, 42%. LC-MS m/z 581.0^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-d4) δ 9.13 (d, J=1.9 Hz, 1H), 8.74 (d, J=1.9 Hz, 1H), 7.63 (dd, J=8.3, 8.3 Hz, 1H), 7.30 (dd, J=10.9, 2.0 Hz, 1H), 7.24 (ddd, J=8.4, 2.0, 0.7 Hz, 1H), 6.89-6.84 (m, 1H), 6.82-6.77 (m, 2H), 4.98-4.89 (m, 2H, presumed; most of it is hidden by the water peak), 4.64 (t, J=4.8 Hz, 2H), 4.04-3.92 (br m, 2H), 3.75 (dd, J=5.4, 4.2 Hz, 2H), 3.51-3.39 (m, 2H), 3.31 (s, 3H), 3.19-3.06 (m, 1H), 2.41-2.24 (m, 2H), 2.24-2.12 (m, 2H), 2.06 (d,J=1.0 Hz, 3H).

Examples 4 and 5.

2-({4-[(2R)-2-(4-Chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (4) and 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (5)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine trifluoroacetate salt (C43).

Trifluoroacetic acid (1.3 ml) was added to a solution of P1 (300 mg, 0.691 mmol) in dichloromethane (5 ml). The reaction mixture was stirred at 29 °C for 2 h, after which it was concentrated in vacuo to give C43 as a brown oil, which was used directly in the next step.

Step 2. Synthesis of methyl-2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C44) and methyl-2-({4-[(2S)-2-(4chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1 -yl} methyl)-1-[(28)-oxetane -2-ylmethyl]-1Hbenzimidazol-6-carboxylate (C45).

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To a solution of C43 (from the previous step, <0.691 mmol) in acetonitrile (10 ml) was added P15 (204 mg, 0.692 mmol), followed by the addition of potassium carbonate (956 mg, 6.92 mmol). The reaction mixture was stirred at 29 °C for 16 h, after which it was filtered; the filtrate was concentrated in vacuo to give a residue, which was purified by preparative thin layer chromatography (eluent: 2:1 petroleum ether/ethyl acetate) to give a mixture of diastereomeric products as a yellow gum (178 mg). Separation of the two products was accomplished by SFC [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 55:45 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)]. The first eluting diastereomer, obtained as a yellow oil, was designated C44. Yield: 44.3 mg, 74.8 μmol, 11% over 2 steps. LC-MS m/z 592.1 ♦ [M+H]+. Retention time 4.26 min (column: Chiral Technologies ChiralCel OD-3, 4.6 x 100 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5 to 40% B over 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 mL/min).

The second eluting diastereomer was subjected to a second purification by SFC [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 3:2 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)] to afford the second eluting diastereomer as a colourless oil designated C45. Yield: 38 mg, 64 μmol, 9% over 2 steps. LCMS m/z 592.1^ [M+H] ⁺ . Retention time 4.41 min (analytical conditions identical to those used for C44).

The absolute stereochemistries reported for dioxolane were assigned by correlation of the activity of 5 with that of 5, the free acid synthesized from intermediate C48; the absolute stereochemistry of this intermediate was determined by single crystal X-ray diffraction (see below) of C49, the hemisulfate salt of C48.

Step 3. Synthesis of ammonium 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (4).

An aqueous solution of lithium hydroxide (2 M; 0.80 mL, 1.6 mmol) was added to a solution of C44 (44.3 mg, 74.8 μmol) in a mixture of methanol (1 mL) and tetrahydrofuran (1 mL), and the reaction mixture was stirred at 26 °C for 3 h. It was then adjusted to pH 7 with trifluoroacetic acid, and the resulting mixture was concentrated in vacuo and purified by reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 30 to 50% B) to give 4 as a white solid. Yield: 26.6 mg, 44.7 μmol, 60%. LCMS m/z 578.0^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-^) δ 8.31 (d, J=1.4 Hz, 1H), 7.96 (dd, J=8.5, 1.6 Hz, 1H), 7.66 (d, J=8.5 Hz, 1H), 7.57 (dd, J=8.0, 8.0 Hz, 1H), 7.34 (dd, J=10.1, 2.0 Hz, 1H), 7.29 (br dd, J=8.3, 2.0 Hz, 1H), 7.20 (s, 1H), 6.86-6.79 (m, 1H), 6.77 (br dd, component of the ABC system, J=7.9, 1.3 Hz, 1H), 6.73 (dd, component of the ABC system, J=7.5, 1.4 Hz, 1H), 5.29-5.18 (m, 1H), 4.9-4.78 (m, 1H, putative; partially hidden by the water peak), 4.68 (dd, J=15.3, 2.7 Hz, 1H), 4.54 (td, J=8.0, 5.9 Hz, 1H), 4.44 (dt, J=9.2, 5.9 Hz, 1H), 4.02 (AB quartet, J _AB = 13.9 Hz, Av _ab = 49.0 Hz, 2H), 3.18-3.08 (m, 1H), 3.05-2.96 (m, 1H), 2.81-2.68 (m, 2H), 2.56-2.45 (m, 1H), 2.45-2.30 (m, 2H), 2.03-1.88 (m, 2H), 1.88-1.79 (m, 2H).

Step 4. Synthesis of ammonium 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (5).

An aqueous solution of lithium hydroxide (2 M; 0.80 mL, 1.6 mmol) was added to a solution of C45 (38 mg, 64 μmol) in a mixture of methanol (1 mL) and tetrahydrofuran (1 mL), and the reaction mixture was stirred at 24 °C for 2.5 h. It was then adjusted to pH 7 with 1 M hydrochloric acid, and the resulting mixture was concentrated in vacuo and purified by reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 29 to 49% B) to give 5 as a white solid. Yield: 27.9 mg, 46.9 μmol, 73%. LC-MS m/z 577.9^ [M+H] ⁺ .

1H NMR (400 MHz, methanol-d ₄ ) δ 8.32 (d, J=1.4 Hz, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.66 (d, J=8.5 Hz, 1H), 7.56 (dd, J=8.0, 8.0 Hz, 1H), 7.34 (dd, J=10.2, 2.0 Hz, 1H), 7.29 (br dd, J=8.3, 2.0 Hz, 1H), 7.20 (s, 1H), 6.85-6.80 (m, 1H), 6.77 (dd, component of the ABC system, J=8.0, 1.3 Hz, 1H), 6.73 (dd, component of the ABC system, J=7.5, 1.4 Hz, 1H), 5.30-5.20 (m, 1H), 4.9-4.79 (m, 1H), putative; partially hidden by the water peak), 4.68 (dd, J=15.4, 2.7 Hz, 1H), 4.62-4.54 (m, 1H), 4.44 (dt, J=9.2, 5.9 Hz, 1H), 4.02 (AB quartet, J _AB = 13.9 Hz, Av _ab = 44.6 Hz, 2H), 3.18-3.09 (m, 1H), 3.06-2.97 (m, 1H), 2.80-2.67 (m, 2H), 2.55-2.30 (m, 3H), 2.02-1.78 (m, 4H).

Alternative Synthesis of Example 5, Free Acid 2-({4-[(2S)-2-(4-Chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid (5, Free Acid)

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5" free acid

Stage 1. Isolation of tert-butyl 4-[(2R)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4yl]piperidine-1-carboxylate (C46) and tert-butyl 4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4yl]piperidine-1-carboxylate (C47).

Separation of P1 (10 g, 23 mmol) into its component enantiomers was accomplished using reversed-phase HPLC [column: Phenomenex Lux Amylose-1, 5 μm; mobile phase: 9:1 carbon dioxide/(2-propanol containing 0.2% 1-aminopropan-2-ol)]. The first eluting enantiomer was designated C46 and the second eluting enantiomer was designated C47; both were obtained as a colorless oil. The absolute stereochemistry reported for C46 and C47 was assigned from the X-ray diffraction data on a single crystal of C49, which was synthesized from C47 (see below).

C46 Yield: 4.47 g, 10.3 mmol, 45%. Retention time: 3.98 min [column: Phenomenex Lux Amylose-1, 4.6 x 250 mm, 5 μm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.2% 1-aminopropan-2-ol; gradient: 5% B over 1.00 min, then 5 to 60% B over 8.00 min; flow rate: 3.0 ml/min; backpressure: 120 bar].

C47 Yield: 4.49 g, 10.3 mmol, 45%. Retention time: 4.32 min (analytical SFC conditions identical to those used for C46).

Step 2. Synthesis of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine (C48).

p-Toluenesulfonic acid monohydrate (566 mg, 2.98 mmol) was added to a solution of C47 (1.12 g, 2.58 mmol) in ethyl acetate (26 mL). After heating the reaction mixture at 45 °C for 16 h, it was concentrated in vacuo, dissolved in ethyl acetate, and washed with saturated aqueous sodium bicarbonate. The aqueous layers were extracted with ethyl acetate, and the combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give C48 as a foamy white solid (947 mg), LCMS m/z 334.0^ [M+H]+. A portion of this material, which still contained some p-toluenesulfonic acid, was used in the synthesis of C50 below.

A second crop of foamy white solid (440 mg) was dissolved in ethyl acetate (25 mL) and washed with saturated aqueous sodium bicarbonate (2 x 15 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo to give C48 (350 mg) as a colorless oil that no longer contained p-toluenesulfonic acid. Corrected yield: 350 mg, 1.05 mmol, 88%.

‘H NMR (400 MHz, chloroform-d) δ 7.53 (dd, J=8.4, 7.8 Hz, 1H), 7.22-7.13 (m, 3H), 6.87-6.80 (m, 1H), 6.79-6.71 (m, 2H), 3.23-3.14 (m, 2H), 2.86-2.69 (m, 3H), 1.90-1.68 (m, 4H).

Step 3. Synthesis of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine hemisulfate salt (C49).

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A 0.1 M solution of C48 (colourless oil from the step above) in ethyl acetate was prepared and salt screened. Only the formation of the sulfate salt is described here. A mixture of sulfuric acid (25 μmol) and substrate solution (0.1 M, 250 μl, 25 μmol) was heated to 45 °C for 1 h, allowed to cool to room temperature and stirred for 15 h. The resulting suspension was treated with methanol (approximately 150 μl) to form a solution. This solution was allowed to evaporate slowly overnight until approximately 50 μl of solvent remained. One of the resulting crystals was analyzed by single crystal X-ray diffraction, giving the absolute stereochemistry as shown.

X-ray structural analysis of a C49 single crystal.

X-ray structural analysis of a single crystal.

Data collection was performed on a Bruker D8 Venture diffractometer at room temperature. Data collection consisted of omega and phi scans.

The structure was solved stepwise using the SHELX software package in the triclinic space group class P1. The structure was then refined by least squares in the full matrix approximation. All non-hydrogen atoms were identified and refined using anisotropic substitution parameters.

Hydrogen atoms located on nitrogen and oxygen were identified from the difference Fourier map and refined with limited distances. The remaining hydrogen atoms were placed in calculated positions and left on their carrier atoms. The final refinement included isotropic substitution parameters for all hydrogen atoms.

The asymmetric unit consists of two molecules of protonated C48, one molecule of doubly deprotonated sulfuric acid, and one molecule of fully occupied water. The structure is thus a hemisulfate salt and a hemihydrate. The chlorofluorophenyl ring is disordered and modeled with 60/40 occupancy, with the ring rotated at two positions.

Absolute structure analysis using probabilistic methods (Hooft, 2008) was performed using PLATON (Spek). The results show that the absolute structure was determined correctly; this method calculated that the probability that the structure is accurate is 100%. The Hooft parameter is given as 0.061 with an esd (estimated standard deviation) of 0.004, and the Parson parameter is given as 0.063 with an esd of 0.0050.07.

The final R-index was 3.1%. The final difference Fourier transform revealed no missing or inappropriate electron density.

The relevant crystal, data collection and refinement information are summarized in Table E. Atomic coordinates, bond lengths, bond angles and substitution parameters are listed in Tables F–H.

Software and information sources.

SHELXTL, Version 5.1, Bruker AXS, 1997.

PLATON, A. L. Spek, J. Appl. Christ. 2003, 36, 7-13.

MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R.

Taylor, M. Towler, and J. van de Streek, J. Appl. Christ. 2006, 39, 453-457.

OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H.

Puschmann, J. Appl. Christ. 2009, 42, 339-341.

R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Christ. 2008, 41, 96-103.

H. D. Flack, Acta Cryst. 1983, A39, 867-881.

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Table E

Crystallographic data and structure refinement for C49

Empirical formula C36H38CI2F2N2O9S Molecular weight according to the formula 783,64 Temperature 296(2)K Wavelength 1.54178 A Crystal system Triclinic Space group P1 Dimensions of the unit cell a = 5.9095(2) A a = 86.5910(10)° b = 6.1712(2) Κβ = 89.3680(10)° c = 25.6096(8) A y = 75.7680(10)° Volume 903.68(5)A ³ Z 1 Density (calculated) 1,440 mg/ ^m3 Absorption coefficient 2,743 mm' ¹ F(000) 408 Crystal dimensions 0.380 x 0.120 x 0.080 mm ³ Theta range for data collection from 3.458 to 72.096° Index ranges ~7<=h<=7, ~7<=k<=7, -31 <=/<=31 Collected reflections 24619 Independent reflections 6399 \R _mt = 0.0323]] Total to theta = 67.679° 96.6% Absorption correction Empirical Refinement method Full-matrix method of least squares by F ² Data/constraints/parameters 6399/9/495 Goodness of fit criterion for F ² 1,014 Finite R indices \Ι>2σ(Ι)] RI = 0.0305, wR2 = 0.0805 R indices (all data) RI = 0.0310, wR2 = 0.0810 Absolute structure parameter 0.058(4) Extinction coefficient η/a (no data) Maximum diff, peak and gap 0.167 and -0.184 e. A' ³

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Table F

Atomic coordinates (x 104) and equivalent parameters of isotropic substitution (A ² x10 ³ ) for C49.

U(eq.) is defined as one third of the trace of the orthogonalized U ^j tensor.

1 1 ^x 1 ^at 1 ^z 1 U(eq) S(l) 8968(1) 2512(1) 4774(1) 33(1) C1(1) 2534(3) 7001(5) 9863(1) 161(1) F(l) 9192(9) 7761(7) 8721(2) 95(1) C(1) 7533(7) 6719(7) 8821(1) 72(1) C(2) 6041(9) 7355(8) 9230(2) 92(1) C(3) 4428(8) 6206(10) 9350(2) 93(2) C(4) 4276(8) 4392(9) 9082(2) 86(1) C(5) 5801(7) 3784(7) 8678(1) 69(1) C(6) 7444(6) 4930(5) 8533(1) 56(1) C1(G) 2534(3) 7001(5) 9863(1) 161(1) F(l') 6045(13) 1811(12) 8450(3) 95(1) C(G) 5801(7) 3784(7) 8678(1) 69(1) C(2') 4276(8) 4392(9) 9082(2) 86(1) C(Z') 4428(8) 6206(10) 9350(2) 93(2) C(4') 6041(9) 7355(8) 9230(2) 92(1) C(5') 7533(7) 6719(7) 8821(1) 72(1) C(b') 7444(6) 4930(5) 8533(1) 56(1) C1(2) -2047(5) 12265(3) 154(1) 157(1) F(2) -2662(7) 5436(7) 1220(2) 92(1) C(19) -1591(6) 7059(7) 1154(1) 68(1) C(20) -2327(8) 8653(9) 752(2) 88(1) C(21) -1157(9) 10260(8) 665(2) 88(1) C(22) 728(9) 10361(7) 964(2) 80(1) C(23) 1431(6) 8731(6) 1364(1) 65(1) C(24) 274(5) 7058(5) 1472(1) 54(1) C1(2') -2047(5) 12265(3) 154(1) 157(1) F(2') 3433(15) 8441(16) 1630(4) 92(1)

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C(19') 1431(6) 8731(6) 1364(1) 65(1) C(20') 728(9) 10361(7) 964(2) 80(1) C(2G) -1157(9) 10260(8) 665(2) 88(1) C(22') -2327(8) 8653(9) 752(2) 88(1) C(23') -1591(6) 7059(7) 1154(1) 68(1) C(24') 274(5) 7058(5) 1472(1) 54(1) N(l) 4370(3) 2950(4) 5713(1) 41(1) N(2) 4133(4) 8236(3) 4386(1) 42(1) 0(1) 10923(4) 2331(5) 8233(1) 77(1) 0(2) 7874(4) 3730(4) 7651(1) 64(1) 0(3) 1766(4) 6201(4) 2352(1) 64(1) 0(4) 2966(5) 3591(4) 1729(1) 75(1) 0(5) 9024(3) 2305(3) 4214(1) 50(1) 0(6) 7650(4) 989(3) 5024(1) 63(1) 0(7) 11358(3) 1934(4) 4982(1) 64(1) 0(8) 7789(3) 4827(3) 4909(1) 46(1) O(1W) 10276(4) 6879(4) 5537(1) 54(1) C(7) 9086(6) 4293(6) 8090(1) 63(1) C(8) 9234(4) 1745(5) 7490(1) 44(1) C(9) 11056(5) 930(6) 7834(1) 54(1) C(10) 12654(5) -1059(6) 7768(1) 62(1) C(ll) 12316(5) -2213(6) 7338(1) 58(1) C(12) 10459(4) -1405(5) 6994(1) 47(1) C(13) 8826(4) 623(4) 7066(1) 38(1) C(14) 6762(4) 1637(4) 6711(1) 37(1) 0(15) 7243(4) 3516(4) 6343(1) 42(1) 0(16) 5126(4) 4639(4) 6009(1) 44(1) 0(17) 3883(5) 1105(5) 6056(1) 50(1) 0(18) 5997(4) -38(4) 6386(1) 41(1) 0(25) 996(6) 5296(6) 1900(1) 60(1) C(26) 3848(5) 4738(4) 2505(1) 45(1) C(27) 4542(6) 3183(5) 2133(1) 52(1) C(28) 6579(6) 1567(5) 2178(1) 56(1) C(29) 7932(6) 1577(5) 2620(1) 56(1) C(30) 7236(5) 3123(5) 2992(1) 51(1) 0(31) 5126(5) 4786(4) 2944(1) 42(1) C(32) 4261(4) 6474(4) 3352(1) 39(1) C(33) 6145(5) 7543(5) 3544(1) 51(1) C(34) 5139(5) 9272(4) 3932(1) 50(1) C(35) 2313(5) 7116(5) 4227(1) 49(1) C(36) 3263(4) 5420(4) 3826(1) 42(1)

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Table G

Bond lengths [A] and angles [°] for C49

S(l)-O(5) 1,4463(18) S(l)-O(7) 1,4668(19) S(l)-O(6) 1,475(2) S(l)-O(8) 1,4863(18) Cl(l)-C(3) 1,731(4) F(l)-C(l) 1,314(6) C(l)-C(6) 1,375(5) C(l)-C(2) 1,374(6) C(2)-C(3) 1,343(8) C(2)-H(2) 0,9300 C(3)-C(4) 1,369(8) C(4)-C(5) 1,373(6) C(4)-H(4) 0,9300 C(5)-C(6) 1,370(5) C(5)-H(5) 0,9300 C(6)-C(7) 1,493(5) C1(G)-C(3') 1,731(4) F(l')-C(l') 1,357(8) C(l')-C(6') 1,370(5) C(G)-C(2') 1,373(6) C(2')-C(3') 1,369(8)

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C(2')-H(2') 0,9300 C(3')-C(4') 1,343(8) C(4')-C(5') 1,374(6) C(4')-H(4') 0,9300 C(5')-C(6') 1,375(5) C(5')-H(5') 0,9300 C(6')-C(7) 1,493(5) C1(2)-C(21) 1,739(4) F(2)-C(19) 1,312(5) C(19)-C(24) 1,378(5) C(19)-C(20) 1,378(6) C(20)-C(21) 1,348(7) C(20)-H(20) 0,9300 C(21)-C(22) 1,375(7) C(22)-C(23) 1,384(6) C(22)-H(22) 0,9300 C(23)-C(24) 1,385(5) C(23)-H(23) 0,9300 C(24)-C(25) 1,485(5) C1(2')-C(2G) 1,739(4) F(2')-C(19') 1,340(9) C(19')-C(20') 1,384(6) C(19')-C(24') 1,385(5) C(20')-C(2G) 1,375(7) C(20')-H(20') 0,9300 C(2G)-C(22') 1,348(7) C(22')-C(23') 1,378(6) C(22')-H(22') 0,9300 C(23')-C(24') 1,378(5) C(23')-H(23') 0,9300 C(24')-C(25) 1,485(5) N(l)-C(17) 1,480(4) N(l)-C(16) 1,480(3)

- 62 040816

N(1)-H(1X) 0.95(2) N(1)-H(1Y) 0.97(2) N(2)-C(34) 1,483(4) N(2)-C(35) 1,487(4) N(2)-H(2X) 0.96(2) N(2)-H(2Y) 0.99(2) O(l)-C(9) 1,368(4) O(l)-C(7) 1,445(4) O(2)-C(8) 1,373(3) O(2)-C(7) 1,443(3) O(3)-C(26) 1,380(3) O(3)-C(25) 1,440(3) O(4)-C(27) 1,369(4) O(4)-C(25) 1,447(4) O(1W)-H(1WX) 0.93(2) O(1W)-H(1WY) 0.94(2) C(7)-H(7) 0,9800 C(8)-C(9) 1,374(4) C(8)-C(13) 1,376(4) C(9)-C(10) 1,370(5) C(10)-C(ll) 1,387(5) C(10)-H(10) 0,9300 C(ll)-C(12) 1,390(4) C(ll)-H(ll) 0,9300 C(12)-C(13) 1,400(4) C(12)-H(12) 0,9300 C(13)-C(14) 1,514(3) C(14)-C(18) 1,518(3) C(14)-C(15) 1,528(3) C(14)-H(14) 0,9800 C(15)-C(16) 1,518(3) C(15)-H(15A) 0,9700 C(15)-H(15B) 0,9700

- 63 040816

C(16)-H(16A) 0,9700 C(16)-H(16B) 0,9700 C(17)-C(18) 1,513(4) C(17)-H(17A) 0,9700 C(17)-H(17B) 0,9700 C(18)-H(18A) 0,9700 C(18)-H(18B) 0,9700 C(25)-H(25) 0,9800 C(26)-C(31) 1,367(4) C(26)-C(27) 1,379(3) C(27)-C(28) 1,363(4) C(28)-C(29) 1,394(5) C(28)-H(28) 0,9300 C(29)-C(30) 1,376(4) C(29)-H(29) 0,9300 C(30)-C(31) 1,408(4) C(ZO)-N(ZO) 0,9300 C(31)-C(32) 1,514(3) C(32)-C(33) 1,527(4) C(32)-C(36) 1,524(3) C(32)-H(32) 0,9800 C(33)-C(34) 1,510(4) C(33)-H(33A) 0,9700 C(33)-H(33B) 0,9700 C(34)-H(34A) 0,9700 C(34)-H(34B) 0,9700 C(35)-C(36) 1,515(3) C(35)-N(35A) 0,9700 C(35)-H(35B) 0,9700 C(36)-H(36A) 0,9700 C(36)-H(36B) 0,9700 O(5)-S(l)-O(7) 109.68(13) O(5)-S(l)-O(6) 109.65(13)

- 64 040816

O(7)-S(1)-0(6) 109.45(15) O(5)-S(l)-O(8) 111,22(11) O(7)-S(1)-0(8) 109,11(11) O(6)-S(1)-0(8) 107.69(11) F(l)-C(l)-C(6) 118.6(4) F(l)-C(l)-C(2) 119,1(4) C(6)-C(1)-0(2) 122,1(4) C(3)-C(2)-C(l) 118.9(4) C(3)-C(2)-H(2) 120,5 C(l)-C(2)-H(2) 120,5 C(2)-C(3)-C(4) 121.6(4) C(2)-C(3)-C1(1) 119.3(4) C(4)-C(3)-C1(1) 119,1(5) C(3)-C(4)-C(5) 118.2(5) C(3)-C(4)-H(4) 120.9 C(5)-C(4)-H(4) 120.9 C(6)-C(5)-C(4) 122.4(4) C(6)-C(5)-H(5) 118.8 C(4)-C(5)-H(5) 118.8 C(5)-C(6)-C(l) 116.7(3) C(5)-C(6)-C(7) 122.7(3) C(l)-C(6)-C(7) 120.6(3) F(l')-C(r)-C(6') 114.7(4) F(l’)-C(l')-C(2’) 122,1(5) C(6')-C(G)-C(2') 122.4(4) C(3')-C(2')-C(G) 118.2(5) C(3')-C(2')-H(2') 120.9 C(G)-C(2')-H(2') 120.9 C(4')-C(3')-C(2') 121.6(4) C(4')-C(3')-C1(G) 119.3(4) C(2')-C(3')-C1(G) 119,1(5) C(3')-C(4')-C(5') 118.9(4) C(3')-C(4')-H(4') 120,5

- 65 040816

C(5')-C(4')-H(4') 120,5 C(6')-C(5')-C(4') 122,1(4) C(6')-C(5')-H(5') 118.9 C(4')-C(5')-H(5') 118.9 C(G)-C(6')-C(5') 116.7(3) C(G)-C(6')-C(7) 122.7(3) C(5')-C(6')-C(7) 120.6(3) F(2)-C(19)-C(24) 119.3(4) F(2)-C(19)-C(20) 118,1(4) C(24)-C(19)-C(20) 122.5(4) C(21)-C(20)-C(19) 118.4(4) C(21)-C(20)-H(20) 120.8 C(19)-C(20)-H(20) 120.8 C(20)-C(21)-C(22) 122.4(4) C(20)-C(21)-C1(2) 118.9(4) C(22)-C(21)-C1(2) 118.7(4) C(21)-C(22)-C(23) 117.8(4) C(21)-C(22)-H(22) 121,1 C(23)-C(22)-H(22) 121,1 C(22)-C(23)-C(24) 122.0(4) C(22)-C(23)-H(23) 119,0 C(24)-C(23)-H(23) 119,0 C(19)-C(24)-C(23) 116.8(3) C(19)-C(24)-C(25) 120.3(3) C(23)-C(24)-C(25) 122.9(3) F(2')-C(19')-C(20') 123.5(5) F(2')-C(19')-C(24') 113.9(5) C(20')-C(19')-C(24') 122.0(4) C(2G)-C(20')-C(19') 117.8(4) C(21')-C(20')-H(20') 121,1 C(19')-C(20')-H(20') 121,1 C(22')-C(2G)-C(20') 122.4(4) C(22')-C(2G)-C1(2') 118.9(4)

- 66 040816

C(20')-C(2G)-C1(2') 118.7(4) C(2G)-C(22')-C(23') 118.4(4) C(2G)-C(22')-H(22') 120.8 C(23')-C(22')-H(22') 120.8 C(24')-C(23')-C(22') 122.5(4) C(24')-C(23')-H(23') 118.7 C(22')-C(23')-H(23') 118.7 C(23')-C(24')-C(19') 116.8(3) C(23')-C(24')-C(25) 120.3(3) C(19')-C(24')-C(25) 122.9(3) C(17)-N(l)-C(16) 112.6(2) C(17)-N(1)-H(1X) 110.7(19) C(16)-N(1)-H(1X) 108(2) C(17)-N(1)-H(1Y) 108(2) C(16)-N(1)-H(1Y) 112.4(19) H(1X)-N(1)-H(1Y) 105(3) C(34)-N(2)-C(35) 112.2(2) C(34)-N(2)-H(2X) 109.7(19) C(35)-N(2)-H(2X) 109.7(19) C(34)-N(2)-H(2Y) 107.7(19) C(35)-N(2)-H(2Y) 110.8(19) H(2X)-N(2)-H(2Y) 107(3) C(9)-O(l)-C(7) 106.0(2) C(8)-O(2)-C(7) 105.9(2) C(26)-O(3)-C(25) 105.9(2) C(27)-O(4)-C(25) 105.7(2) H( 1 WX)-O( 1 W)-H( 1W Y) 105(4) O(2)-C(7)-O(l) 106.5(3) O(2)-C(7)-C(6) 110.4(3) O(l)-C(7)-C(6) 111.2(3) O(2)-C(7)-C(6') 110.4(3) O(l)-C(7)-C(6') 111.2(3) O(2)-C(7)-H(7) 109.6

- 67 040816

O(l)-C(7)-H(7) 109.6 C(6)-C(7)-H(7) 109.6 C(9)-C(8)-O(2) 110.0(2) C(9)-C(8)-C(13) 123.4(2) O(2)-C(8)-C(13) 126.6(2) O(1)-C(9)-C(U) 128,1(3) O(1)-C(9)-C(8) 110,1(3) C(10)-C(9)-C(8) 121.7(3) C(9)-C(10)-C(11) 116.3(3) C(9)-C(10)-H(10) 121.8 C(11)-C(10)-H(10) 121.8 C(10)-C(11)-C(12) 122.0(3) C(10)-C(11)-H(11) 119,0 C(12)-C(11)-H(11) 119,0 C(11)-C(12)-C(13) 121.3(3) C(11)-C(12)-H(12) 119.4 C(13)-C(12)-H(12) 119.4 C(8)-C(13)-C(12) 115.3(2) C(8)-C(13)-C(14) 119.8(2) C(12)-C(13)-C(14) 124.9(2) C(13)-C(14)-C(18) 114.2(2) C(13)-C(14)-C(15) 111.38(19) C(18)-C(14)-C(15) 108.70(19) C(13)-C(14)-H(14) 107.4 C(18)-C(14)-H(14) 107.4 C(15)-C(14)-H(14) 107.4 C(16)-C(15)-C(14) 111.7(2) C(16)-C(15)-H(15A) 109.3 C(14)-C(15)-H(15A) 109.3 C(16)-C(15)-H(15B) 109.3 C(14)-C(15)-H(15B) 109.3 N(15A)-C(15)-N(15B) 107.9 N(l)-C(16)-C(15) 109.9(2)

- 68 040816

N(1)-C(16)-H(16A) 109.7 C(15)-C(16)-H(16A) 109.7 N(1)-C(16)-H(16B) 109.7 C(15)-C(16)-H(16B) 109.7 H(16A)-C(16)-H(16B) 108.2 N(l)-C(17)-C(18) 110.94(19) N(1)-C(17)-H(17A) 109.5 C(18)-C(17)-H(17A) 109.5 N(1)-C(17)-H(17B) 109.5 C(18)-C(17)-H(17B) 109.5 H(17A)-C(17)-H(17B) 108,0 C(17)-C(18)-C(14) 110.6(2) C(17)-C(18)-H(18A) 109.5 C(14)-C(18)-H(18A) 109.5 C(17)-C(18)-H(18B) 109.5 C(14)-C(18)-H(18B) 109.5 H(18A)-C(18)-H(18B) 108.1 O(3)-C(25)-O(4) 106.6(2) O(3)-C(25)-C(24') 111.0(3) O(4)-C(25)-C(24') 109.4(3) O(3)-C(25)-C(24) 111.0(3) O(4)-C(25)-C(24) 109.4(3) O(3)-C(25)-H(25) 109.9 O(4)-C(25)-H(25) 109.9 C(24)-C(25)-H(25) 109.9 C(31)-C(26)-C(27) 123.2(3) C(31)-C(26)-O(3) 127.3(2) C(27)-C(26)-O(3) 109.5(2) C(28)-C(27)-O(4) 127.7(2) C(28)-C(27)-C(26) 121.9(3) O(4)-C(27)-C(26) 110.3(2) C(27)-C(28)-C(29) 116.3(2) C(27)-C(28)-H(28) 121.9

- 69 040816

C(29)-C(28)-H(28) 121.9 C(30)-C(29)-C(28) 121.8(3) C(30)-C(29)-H(29) 119.1 C(28)-C(29)-H(29) 119.1 C(29)-C(30)-C(31) 121.7(3) C(29)-C(30)-H(30) 119.2 C(31)-C(30)-H(30) 119.2 C(26)-C(31)-C(30) 115,1(2) C(26)-C(31)-C(32) 121.5(2) C(30)-C(31)-C(32) 123.4(2) C(31)-C(32)-C(33) 113.3(2) C(31)-C(32)-C(36) 111.48(19) C(33)-C(32)-C(36) 108.02(19) C(31)-C(32)-H(32) 107.9 C(33)-C(32)-H(32) 107.9 C(36)-C(32)-H(32) 107.9 C(34)-C(33)-C(32) 110.5(2) C(34)-C(33)-H(33A) 109.6 C(32)-C(33)-H(33A) 109.6 C(34)-C(33)-H(33B) 109.6 C(32)-C(33)-H(33B) 109.6 H(33A)-C(33)-H(33B) 108.1 N(2)-C(34)-C(33) 110.6(2) N(2)-C(34)-H(34A) 109.5 C(33)-C(34)-H(34A) 109.5 N(2)-C(34)-H(34B) 109.5 C(33)-C(34)-H(34B) 109.5 H(34A)-C(34)-H(34B) 108.1 N(2)-C(35)-C(36) 110.71(19) N(2)-C(35)-H(35A) 109.5 C(36)-C(35)-H(35A) 109.5 N(2)-C(35)-H(35B) 109.5 C(36)-C(35)-H(35V) H(35A)-C(35)-H(35V) 109.5 108.1 C(35)-C(36)-C(32) 111.9(2) C(35)-C(36)-H(36A) 109.2 C(32)-C(36)-H(36A) 109.2 C(35)-C(36)-H(36B) 109.2 C(32)-C(36)-H(36B) 109.2 N(36A)-C(36)-N(36B) 107.9

Symmetry transformations used to create equivalent atoms.

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Table H

Anisotropic substitution parameters (A ² x10 ³ ) for C49.

The exponent of the anisotropic substitution factor takes the form: ^{-2π a} * U + ... + 2hka*b*U ].

and ¹¹ U ²² and ³³ and ²³ and ¹³ and ¹² S(l) 32(1) 32(1) 32(1) -3(1) -2(1) -1(1) Cl(l) 107(1) 258(2) 90(1) -63(1) 19(1) 19(1) F(l) 1I(2) 91(2) 98(2) -30(2) 6(2) -46(2) C(l) 81(2) 71(2) 60(2) -20(2) -16(2) -6(2) C(2) 100(3) 92(3) 74(3) -42(2) -16(2) 7(2) C(3) 70(2) 134(4) 53(2) -27(2) -7(2) 19(3) C(4) 71(2) 116(3) 67(2) 0(2) -1(2) -16(2) C(5) 75(2) 70(2) 59(2) -I(2) -7(2) -Yu(2) C(6) 65(2) 54(2) 42(1) -8(1) -18(1) -1(1) C1(G) 107(1) 258(2) 90(1) -63(1) 19(1) 19(1) F(l') 1I(2) 91(2) 98(2) -30(2) 6(2) -46(2) C(l') 75(2) 70(2) 59(2) -I(2) -7(2) -Yu(2) C(2') 71(2) 116(3) 67(2) 0(2) -1(2) -16(2) C(Z') 70(2) 134(4) 53(2) -27(2) -7(2) 19(3) C(4') 100(3) 92(3) 74(3) -42(2) -16(2) 7(2) C(5') 81(2) 71(2) 60(2) -20(2) -16(2) -6(2) C(b') 65(2) 54(2) 42(1) -8(1) -18(1) -1(1) C1(2) 243(2) 1U(1) 80(1) 12(1) -39(1) 26(1) F(2) 88(2) 106(2) 93(2) -12(2) -22(2) -44(2)

- 71 040816

C(19) 62(2) 77(2) 62(2) -26(2) -12(2) -5(2) C(20) 85(3) 98(3) 66(2) -20(2) -31(2) Yu(2) C(21) 117(3) 74(3) 51(2) -I(2) -Yu(2) 18(2) C(22) 104(3) 70(2) 60(2) -9(2) 8(2) -8(2) C(23) 58(2) 73(2) 60(2) -13(2) -3(1) -6(2) C(24) 50(2) 60(2) 47(2) -23(1) -4(1) -2(1) C1(2') 243(2) 1U(1) 80(1) 12(1) -39(1) 26(1) F(2') 88(2) 106(2) 93(2) -12(2) -22(2) -44(2) C(19') 58(2) 73(2) 60(2) -13(2) -3(1) -6(2) C(20') 104(3) 70(2) 60(2) -9(2) 8(2) -8(2) C(2G) 117(3) 74(3) 51(2) -I(2) -Yu(2) 18(2) C(22') 85(3) 98(3) 66(2) -20(2) -31(2) Yu(2) C(23') 62(2) 77(2) 62(2) -26(2) -12(2) -5(2) C(24') 50(2) 60(2) 47(2) -23(1) -4(1) -2(1) N(l) 30(1) 59(1) 32(1) -3(1) -4(1) -7(1) N(2) 49(1) 38(1) 37(1) -and(1) -5(1) 0(1) 0(1) 58(1) 107(2) 55(1) -23(1) -26(1) 6(1) 0(2) 64(1) 66(1) 50(1) -21(1) -23(1) 12(1) 0(3) 66(1) 62(1) 52(1) -27(1) -19(1) And(1) 0(4) 92(2) 64(1) 56(1) -32(1) -20(1) Yu(1) 0(5) 62(1) 51(1) 34(1) -5(1) -2(1) -9(1) 0(6) 76(1) 43(1) 70(1) -4(1) 32(1) -14(1) 0(7) 45(1) 68(1) 69(1) -29(1) -22(1) 13(1) 0(8) 45(1) 35(1) 53(1) -9(1) -4(1) 2(1) O(1W) 56(1) 50(1) 51(1) -3(1) -12(1) 1(1) C(7) 68(2) 73(2) 45(2) -12(1) -14(1) -12(2) C(8) 38(1) 51(1) 36(1) -4(1) -3(1) 0(1) C(9) 42(1) 76(2) 39(1) -1(1) -9(1) -4(1) C(10) 38(1) 87(2) 48(2) Yu(1) -8(1) 6(1) C(11) 45(1) 60(2) 55(2) 9(1) 2(1) 13(1) C(12) 41(1) 46(1) 47(1) 0(1) 3(1) 0(1) C(13) 34(1) 43(1) 34(1) 2(1) -1(1) -4(1) C(14) 30(1) 44(1) 31(1) -4(1) -1(1) 0(1) C(15) 41(1) 38(1) 45(1) 0(1) -12(1) -7(1) C(16) 44(1) 43(1) 39(1) -3(1) -6(1) 4(1) C(17) 39(1) 73(2) 42(1) -1(1) -3(1) -23(1) C(18) 41(1) 46(1) 39(1) -4(1) 2(1) -14(1) C(25) 65(2) 62(2) 51(2) -22(1) -9(1) -8(1) C(26) 55(1) 37(1) 37(1) -8(1) 1(1) -2(1) C(27) 72(2) 41(1) 39(1) -9(1) -2(1) -6(1) C(28) 79(2) 39(1) 43(1) -ю(1) And(1) 1(1) C(29) 62(2) 45(2) 48(2) -2(1) 7(1) 8(1) C(30) 58(2) 45(2) 42(1) -1(1) -1(1) 1(1) C(31) 54(1) 34(1) 34(1) -4(1) 2(1) -4(1) C(32) 50(1) 30(1) 33(1) -4(1) -6(1) 0(1) C(33) 63(2) 45(1) 54(2) -9(1) 17(1) -28(1) C(34) 59(2) 38(1) 58(2) -9(1) -1(1) -22(1) C(35) 46(1) 46(1) 56(2) -17(1) 16(1) -and(1) C(36) 39(1) 36(1) 53(1) -15(1) 12(1) -13(1)

Step 4. Synthesis of methyl-2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylate (C50).

Solution C48 (500 mg of foamy white solid from the step described above, adjusted

- 72 040816 (p-toluenesulfonic acid: 1.25 mmol) in acetonitrile (6 mL) were treated with N,N-diisopropylethylamine (0.68 mL, 3.9 mmol) and stirred for 5 min at 45 °C. After addition of P15 (319 mg, 1.08 mmol), stirring at 45 °C was continued for 7.25 h, after which the reaction mixture was diluted with water (6 mL) and acetonitrile (2 mL) at 45 °C. The resulting heterogeneous mixture was allowed to cool to room temperature and stirred for 72 h. Water (5 mL) was added again and after an additional 30 min of stirring, the solid was collected by filtration and washed with a mixture of acetonitrile and water (15:85, 3 x 5 mL) to give C50 as a white solid with a slight pink tint. Yield: 605 mg, 1.02 mmol, 82%. LC-MS m/z 592.0^ [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.17 (d, J=1.6 Hz, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.73 (d, J=8.4 Hz, 1H), 7.51 (dd, J=8.0, 8.0 Hz, 1H), 7.19 (br s, 1H), 7.18-7.14 (m, 2H), 6.85-6.79 (m, 1H), 6.76-6.71 (m, 2H), 5.26-5.18 (m, 1H), 4.73 (dd, component of the ABX system, J=15.3, 5.9 Hz, 1H), 4.67 (dd, component of the ABX system, J=15.3, 3.5 Hz, 1H), 4.63-4.55 (m, 1H), 4.38 (ddd, J=9.1, 6.0, 5.9 Hz, 1H), 3.94 (s, 5H), 3.03-2.89 (m, 2H), 2.77-2.65 (m, 2H), 2.51-2.39 (m, 1H), 2.34-2.20 (m, 2H), 1.91-1.76 (m, 4H).

Step 5. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid (5, free acid).

A suspension of C50 (595 mg, 1.00 mmol) in methanol (10 ml) was heated to 45 °C and treated with aqueous sodium hydroxide (1 M; 2.01 ml, 2.01 mmol). After 21 h at 45 °C, the reaction mixture was allowed to cool to room temperature and then treated with aqueous citric acid (1 M, 1 ml), which adjusted the pH to 5-6. Water (10 ml) was added and the mixture was stirred for 1 h, after which the solid was collected by filtration. It was washed with a mixture of methanol and water (1:10, 3 x 5 ml) to give a solid (433 mg). A portion of this material (300 mg) was stirred with a mixture of heptane and ethyl acetate (1:3, 5 mL) at 40 °C for 1 h. After cooling to room temperature with stirring, the solid was collected by filtration and washed with a mixture of heptane and ethyl acetate (3:1, 3 x 3 mL) to give 5, the free acid, as a white solid. Yield: 260 mg, 0.450 mmol, corresponding to 65% for the entire reaction. LC-MS m/z 578.0^ [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.75 (v br s, 1H), 8.26 (br s, 1H), 7.79 (dd, J=8.4, 1.6 Hz, 1H), 7.66-7.56 (m, 3H), 7.40 (dd, J=8.3, 2.0 Hz, 1H), 7.35 (s, 1H), 6.87-6.75 (m, 3H), 5.13-5.03 (m, 1H), 4.76 (dd, component of the ABX system, J=15.3, 7.2 Hz, 1H), 4.62 (dd, component of the ABX system, J=15.2, 2.8 Hz, 1H), 4.46-4.38 (m, 1H), 4.34 (ddd, J=9.0, 5.9, 5.8 Hz, 1H), 3.84 (AB quartet, J _AB = 13.5 Hz, Av _ab = 67.7 Hz, 2H), 3.00 (br d, J=11.2 Hz, 1H), 2.84 br (d, J=11.3 Hz, 1H), 2.71-2.56 (m, 2H), 2.45-2.34 (m, 1H), 2.28-2.08 (m, 2H), 1.84-1.65 (m, 4H).

This compound was determined to have the same absolute configuration as Example 5 above by comparing its biological activity with that of both 4 and 5: in Assay 2, this sample of Compound 5, the free acid, exhibited an _EC50 of 25 nM (geometric mean of 3 replicates). The activity in Assay 2 for the ammonium salt of Example 4 and the compound of Example 5 was >20,000 nM (2 replicates) and 20 nM (geometric mean of 3 replicates), respectively.

Synthesis of Example Compound 5,1,3-Dihydroxy-2-(hydroxymethyl)propan-2-aminium salt 2-({4[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2ylmethyl]-1H-benzimidazole-6-carboxylate 1,3-hydroxy-2-(hydroxymethyl)propan-2-aminium (5,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt) he he ⁴⁼⁷ he

5, free acid

5,1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium salt

A mixture of 5, the free acid, (0.50 g, 0.86 mmol) in tetrahydrofuran (4 ml) was treated with an aqueous solution of 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris, 1.0 M; 0.5 ml, 1.0 mmol). After 20 h, the mixture was concentrated in vacuo with ethanol (2 x 6 ml). The mixture was treated with ethanol (4 ml). After stirring for 48 h, the solid was collected by filtration, washed with ethanol (2 x 10 ml) and dried in vacuo to give 5,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt as a white solid. Yield: 410 mg, 0.586 mmol, 68%.

1H NMR (600 MHz, DMSO-d ₆ ), characteristic peaks: δ 8.19 (s, 1H), 7.78 (br d, J=8.4 Hz, 1H), 7.62-7.58 (m, 2H), 7.55 (d, J=8.3 Hz, 1H), 7.40 (dd, J=8.4, 2.0 Hz, 1H), 7.35 (s, 1H), 6.85-6.80 (m, 2H), 6.79 (dd, J=6.9, 2.4 Hz, 1H), 5.11-5.05 (m, 1H), 4.73 (dd, J=15.2, 7.2 Hz, 1H), 4.60 (dd, J=15.3, 2.9 Hz, 1H), 4.454.39 (m, 1H), 4.34 (ddd, J=9.0, 6.0, 5.8 Hz, 1H), 3.91 (d, J=13.5 Hz, 1H), 3.74 (d, J=13.5 Hz, 1H), 2.99 (br d, J=11.1 Hz, 1H), 2.85 (br d, J=11.3 Hz, 1H), 2.68-2.59 (m, 2H), 2.44-2.37 (m, 1H), 2.25-2.18 (m, 1H), 2.17-2.10 (m, 1H), 1.80-1.69 (m, 4H). T.pl. = 168-178°C.

- 73 040816

Examples 6 and 7.

2-({4-[(2R)-2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S)-oxetan-2-ylmethyl]-1H-ammonium benzimidazole-6-carboxylate (6) and 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-ammonium benzimidazole-6-carboxylate (7)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine paratoluenesulfonate salt (C13).

A solution of P2 (150 mg, 0.335 mmol) and p-toluenesulfonic acid monohydrate (159 mg, 0.836 mmol) in ethyl acetate (2.0 mL) was stirred at 60 °C for 3.5 h. The reaction mixture was concentrated in vacuo to give C13 as a brown oil, which was used directly in the next step. LC-MS m/z 348.1 ♦ [M+H]+.

Step 2. Synthesis of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C51).

To a suspension of C13 (from the previous step; <0.335 mmol) and potassium carbonate (232 mg, 1.68 mmol) in acetonitrile (5.0 mL) was added P15 (99.1 mg, 0.336 mmol). The reaction mixture was stirred at 60 °C for 10 h, then filtered and the filtrate concentrated in vacuo. After combining the residue (390 mg) with the material obtained from a similar reaction carried out using C13 (<0.11 mmol), it was diluted with water (20 mL) and extracted with a mixture of dichloromethane and methanol (10:1, 3 x 30 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo and subjected to preparative thin layer chromatography (eluent: 1:1 dichloromethane/methanol) to afford C51, a mixture of diastereomers, as a colorless oil. Overall yield: 80.6 mg, 0.133 mmol, 30% over 2 steps. LC-MS m/z 606.2^ [M+H] ⁺ .

Step 3. Isolation of methyl 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C52) and methyl 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C53).

Separation of C51 (180 mg, 0.297 mmol) into its component diastereomers was accomplished by repeated SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting diastereomer was designated C52. Yield: 61.2 mg, 0.101 mmol, 34%. LC-MS m/z 627.9^ [M+Na ⁺ ]. Retention time: 5.03 min (column: Chiral Technologies Chiralpak AD-3, 4.6 x 150 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B over 5.5 min, then hold at 40% B for 3.0 min; flow rate: 2.5 ml/min).

The second eluting diastereomer was designated C53. This material was found to be contaminated with the corresponding ethyl ester. It was carried forward to the hydrolysis step (to create 7) as this mixture. Yield: 40.0 mg, 66.0 µmol, 22%. LC-MS m/z 606.0^ [M+H] ⁺ . Retention time: 5.19 min (analytical conditions identical to those used in

- 74 040816 for C52).

The reported absolute stereochemistry with respect to dioxolane was assigned by correlation of the activity of 7 with that of 7, the free acid synthesized from intermediate P3 (see Alternative Synthesis of Example Compound 7, Free Acid, below). The absolute stereochemistry of P3 was established by single crystal X-ray diffraction analysis of compound C8 (see above).

Step 4. Synthesis of ammonium 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (6).

An aqueous solution of lithium hydroxide (2 M; 0.990 mL, 1.98 mmol) was added to a solution of C52 (60 mg, 99 μmol) in a mixture of methanol (1.0 mL) and tetrahydrofuran (1.0 mL), and the reaction mixture was stirred at 20 °C for 16 h. Trifluoroacetic acid was added until the reaction mixture reached pH 7, after which the mixture was concentrated in vacuo and the residue was purified using reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 29 to 49% B) to give 6 as a white solid. Yield: 14.4 mg, 23.6 μmol, 24%. LC-MS m/z 592.0^ [M+H]+.

1H NMR (400 MHz, methanol-d ₄ ), characteristic peaks: δ 8.35 (d, J=1.3 Hz, 1H), 7.97 (dd, J=8.5, 1.5 Hz, 1H), 7.67 (d, J=8.5 Hz, 1H), 7.58 (dd, J=8.3, 8.3 Hz, 1H), 7.28 (dd, J=10.9, 2.0 Hz, 1H), 7.21 (br dd, J=8.4, 1.9 Hz, 1H), 6.81-6.75 (m, 1H), 6.74-6.68 (m, 2H), 5.33-5.25 (m, 1H), 4.72 (dd, J=15.4, 2.7 Hz, 1H), 4.49 (dt, J=9.1, 6.0 Hz, 1H), 4.03 (AB quartet, Jab = 13.9 Hz, Δυλβ = 47.8 Hz, 2H), 3.14 (br d, J=11 Hz, 1H), 3.02 (br d, J=11.5 Hz, 1H), 2.88-2.78 (m, 1H), 2.77-2.68 (m, 1H), 2.60-2.50 (m, 1H), 2.47-2.32 (m, 2H), 2.03 (d, J=1.1 Hz, 3H), 2.01-1.87 (m, 2H), 1.87-1.78 (brm, 2H).

Step 5. Synthesis of ammonium 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (7).

Aqueous lithium hydroxide solution (2 M; 0.642 mL, 1.28 mmol) was added to a solution of C53 (38.9 mg, 64.2 μmol) in a mixture of methanol (1.0 mL) and tetrahydrofuran (1.0 mL). After stirring the reaction mixture at 20 °C for 16 h, it was adjusted to pH 7 with trifluoroacetic acid, concentrated in vacuo and purified using reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 0 to 80% B) to give 7 as a white solid. Yield: 25.1 mg, 41.2 μmol, 64%. LC-MS m/z 591.9^ [M+H] ⁺ .

1H NMR (400 MHz, methanol-d ₄ ), characteristic peaks: δ 8.34 (d, J=1.5 Hz, 1H), 7.98 (dd, J=8.5, 1.6 Hz, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.58 (dd, J=8.3, 8.3 Hz, 1H), 7.28 (dd, J=10.9, 2.0 Hz, 1H), 7.20 (br dd, J=8.4, 1.9 Hz, 1H), 6.81-6.74 (m, 1H), 6.74-6.67 (m , 2H), 5.33-5.23 (m, 1H), 4.73 (dd, J=15.4, 2.7 Hz, 1H), 4.68-4.61 (m, 1H), 4.48 (dt, J=9.1, 5.9 Hz, 1H), 4.05 (AB quartet, Jab = 13.9 Hz, Avab = 44.1 Hz, 2H), 3.15 (br d, J=11.7 Hz, 1H), 3.03 (br d, J=11.6 Hz, 1H), 2.87-2.69 (m, 2H), 2.60-2.49 (m, 1H), 2.48-2.33 (m , 2H), 2.03 (br s, 3H), 2.01-1.77 (m, 4H).

An alternative synthesis of Example 7, the free acid 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid (7, free acid)

7" free acid

Step 1. Synthesis of methyl-2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)- 1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C53).

N,N-Diisopropylethylamine (15.1 mL, 86.9 mmol) was added to a mixture of P3 (8.22 g, 15.8 mmol) in acetonitrile (185 mL). After stirring for 5 min, P15 (4.57 g, 15.5 mmol) was added and the reaction mixture was heated at 45 °C. After 4 h, the reaction mixture was concentrated in vacuo to half its original volume and the resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water (50 mL), dried over magnesium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (gradient: 50 to 100% ethyl acetate in heptane) gave C53 as a white solid. Yield: 8.4 g, 13.9 mmol, 88%. LC-MS m/z 606.1 ♦ [M+H] ⁺ .

- 75 040816

1H NMR (600 MHz, DMSO-d ₆ ) δ 8.30 (s, 1H), 7.82 (br d, J=8.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.58- 7.53 (m, 2H), 7.33 (dd, J=8.4, 2.1 Hz, 1H), 6.80-6.76 (m, 2H), 6.76-6.72 (m, 1H), 5.14-5.07 (m, 1H), 4.81 (dd, J=15.2, 7.2 Hz, 1H), 4.67 (dd, J=15.3, 2.8 Hz, 1H), 4.51-4.44 (m, 1H), 4.37 (ddd, J=8.9, 5.9, 5.9 Hz, 1H), 3.97 (d, J=13.6 Hz, 1H), 3.87 (s, 3H), 3.78 (d, J=13.5 Hz, 1H), 3.02 (br d, J=11.1 Hz, 1H), 2.86 (br d, J=11.3 Hz, 1H), 2.74-2.60 (m, 2H), 2.48-2.41 (m, 1H), 2.29-2.22 (m, 1H), 2.21-2.14 (m, 1H), 2.02 (s, 3H), 1.83-1.73 ( m, 2H), 1.73-1.64 (m, 2H).

Step 2. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid (7, free acid).

A mixture of C53 (8.40 g, 14.0 mmol) in methanol (135 ml) was heated to 45 °C and treated with aqueous sodium hydroxide (1 M; 27.7 ml, 27.7 mmol). After 20 h, the reaction mixture was concentrated in vacuo to half its original volume. The resulting mixture was diluted with water (100 ml) and aqueous citric acid (1 M, 15 ml) was used to adjust the pH to 5–6. The resulting solid was filtered, washed with water (2 x 15 ml) and transferred to a separatory funnel as a solution in ethyl acetate (50 ml); residual water was removed in this way. The organic layer was dried over magnesium sulfate, filtered, combined with four crops previously prepared by a similar procedure (the amounts of C53 used in these reactions were 987 mg, 1.63 mmol; 1.15 g, 1.90 mmol; 8.57 g, 14.1 mmol; and 12.6 g, 20.8 mmol) and concentrated in vacuo. The resulting sticky solid was treated with 10% ethyl acetate in heptane (500 mL). After 4 h, the solid was collected by filtration and washed with 10% ethyl acetate in heptane (2 x 25 mL) to give 7, the free acid, as a white solid. Yield 29.4 g, 0.527 mmol, 74% for the combined reactions. LC-MS 592.2 ♦ [M+H]+.

1H NMR (600 MHz, DMSO-d ₆ ): δ 12.74 (br s, 1H), 8.28 (s, 1H), 7.80 (br d, J=8.4 Hz, 1H), 7.64 (d, J=8 ,4 Hz, 1H), 7.59-7.52 (m, 2H), 7.33 (dd, J=8.4, 2.1 Hz, 1H), 6.81-6.76 (m, 2H), 6.76-6.72 (m, 1H), 5.14-5.07 (m, 1H), 4.79 (dd, J=15.3, 7.3 Hz, 1H), 4.65 (dd, J=15.2, 2.8 Hz, 1H), 4.51-4.45 (m, 1H), 4.38 (ddd, J=9.0, 5.9, 5.9 Hz, 1H), 3.96 (br d, J=13.6 Hz, 1H), 3.78 (br d, J=13.5 Hz , 1H), 3.02 (br d, J=11.1 Hz, 1H), 2.86 (br d, J=11.1 Hz, 1H), 2.74-2.60 (m, 2H), 2.48-2.41 (m, 1H), 2.29-2.21 (m, 1H), 2.21-2.14 (m, 1H), 2.02 (s, 3H), 1.83-1.74 (m, 2H), 1.74-1.64 (m, 2H).

This compound was determined to have the same absolute configuration as the above compound of Example 7 by comparing its biological activity with that of both 6 and 7: in Assay 2, this sample of compound 7, the free acid, exhibited an EC ₅₀ of 4.3 nM (geometric mean of 3 replicates). The activities in Assay 2 for the ammonium salt of compound of Example 6 and compound of Example 7 were 2400 nM (geometric mean of 5 replicates) and 2.9 nM (geometric mean of 8 replicates), respectively.

Synthesis of 7S-1. Synthesis of Example Compound 7,1,3-Dihydroxy-2-(hydroxymethyl)propan-2-aminium Salt of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate 1,3-ihydroxy-2-(hydroxymethyl)propan-2-aminium (7,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt)

7,1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium salt

A mixture of 7, the free acid, (2.00 g, 3.38 mmol) in tetrahydrofuran (16 mL) was treated with an aqueous solution of 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris, 1.0 M; 3.55 mL, 3.55 mmol). After 18 h, the reaction mixture was concentrated in vacuo and treated with ethanol (30 mL). After stirring this mixture for 23 h, the solid was collected by filtration and washed with ethyl acetate (2 x 10 mL) to give 7, 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt as a white solid. Yield: 1.41 g, 1.98 mmol, 59%. LCMS m/z 592.3^ [M+H] ⁺ .

1H NMR (600 MHz, DMSO-d ₆ ), characteristic peaks: δ 8.20 (s, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.59-7.52 (m, 3H), 7.33 (br d, J=8.5 Hz, 1H), 6.81-6.72 (m, 3H), 5.14-5.07 (m, 1H), 4.76 (dd, J=15.2, 7.2 Hz, 1H), 4.63 (br d, J=15.4 Hz, 1H), 4.50-4.44 (m, 1H), 4.37 (ddd, J=8.9, 5.9, 5.9 Hz, 1H), 3.94 (d, J=13.4 Hz, 1H), 3.76 (d, J=13.4 Hz, 1H), 3.01 (br d, J=11.1 Hz, 1H), 2.86 (br d, J=11.2 Hz, 1H), 2.73-2.60 (m, 2H), 2.5-2.41 (m, 1H), 2.27-2.20 (m, 1H), 2.20-2.13 (m, 1H), 2.02 (s, 3H), 1.83-1.64 (m, 4H). T.pl. = 175-180°C.

Synthesis of 7S-2. An alternative synthesis of the example compound 7,1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium salt. A 3.3 M solution of 2-amino-2-(hydroxymethyl)-1,3-propanediol (1.0 equiv, 1.93 L) in water was added to a solution of 7, the free acid, (3.74 kg) in isopropanol (20 L) at 65°C. Additional isopropanol (19 L) was added, followed by methanol (19 L), maintaining the temperature at 65°C. The mixture was slowly cooled to 45°C over 2 h, then held at 45°C for

- 76 040816 for at least 12 h. The mixture was then cooled to 5°C over 3 h, then held at 5°C for at least 3 h. The mixture was then filtered and the solid collected and washed with ethyl acetate (2 x 10 mL) to give 7,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt as a white solid (yield: 3.64 kg, 80.9%). LC-MS and 1H NMR data were obtained that were essentially the same as in the synthesis of 7S-1 presented above.

Obtaining X-ray powder diffraction (XRPD) data for Form I of the compound of Example 7, 1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt.

The white solid of the tris salt of Example 7 (from Preparation 7S-1 and Preparation 7S-2) was analyzed by X-ray powder diffraction and found to be a crystalline solid (designated Form I of this anhydrous crystalline form). Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavor diffractometer equipped with a Cu source. The slit was set to 15 mm continuous illumination. The diffracted radiation was detected with a PSD-Lynx Eye detector with the PSD (position sensitive detector) detector window set to 2.99 degrees. The X-ray tube voltage and current were set to 40 kV and 40 mA, respectively. Data were collected at Cu wavelength (CuKa = 1.5418 λ) in a theta-theta goniometer from 3.0 to 40.0 degrees 2-theta in 0.01 degree steps and 1.0 s time steps. The anti-scatter screen was set at 1.5 mm. Samples were rotated for data collection. Samples were prepared by placing them in a low background silicone holder and rotating for data collection.

Data were collected using DIFFRAC Plus software and analyzed using EVA diffract plus software. The XRD data file was not processed prior to peak searching. Using the peak searching algorithm in EVA software, peaks selected with a threshold of 1 were used to create preliminary peak assignments. To ensure reliability, adjustments were made manually; the output of the automated assignments was visually inspected and peak positions were adjusted to the maximum peak value. Typically, peaks with a relative intensity >3% were selected. Typically, peaks that were not resolved or did not match the noise were not selected. The typical uncertainty associated with peak position in the XRD pattern specified in USP is up to +/- 0.2° 2-theta (USP-941). One diffraction pattern was consistently observed and is shown in Fig. 1. The list of diffraction peaks expressed in 2θ degrees and relative intensities with relative intensity >3.0% XRD from the sample obtained as a result of synthesis of 7S-2 is presented above in Table X1.

Table X1

Angle (2-theta) 3.7 7.3 8.1 8.5 Yu,1 13.6 14.7 15.2 15,5 15.9 16.9 17.4 17.7 17.9 18.9

Relative intensity (%) 74.3 83.3 12.5 6.5 6,6 3.5 49.8 7.9 28.7 18.3 60.8 26.3 And,4 13.5 75.4

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19.7 18.7 20.2 100,0 20.9 24.8 21,5 14.8 22.2 31.7 22.9 10.1 23.5 34.6 23.7 8.2 24.4 6.5 24.9 8.7 25.2 6.4 25.9 14.7 26.4 48.6 26.7 12.5 27,5 15.8 27.9 6.1 28.3 10,5 29,5 15,5 29.8 12.6 30.2 12.1 30.9 3,4 31.7 16.4 33.3 17.2 34,0 14.9 35.8 4.8 37,5 3.2 38.6 5.3

Examples 8 and 9.

2-({4-[2-(4-Cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X1 (8) [from C56]; and 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (9) [from C57]

NC

CH ₃

C56 C57

Step 1. Synthesis of 3-fluoro-4-[2-methyl-4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]benzonitrile para-toluenesulfonate salt (C54).

To a solution of P4 (161 mg, 0.367 mmol) in ethyl acetate (8 mL) was added p-toluenesulfonic acid (158 mg, 0.919 mmol), and the reaction mixture was stirred at 65°C for 16 h. Removal of the solvent in vacuo gave C54 as a dark yellow gum. This material was carried directly to the next step.

Step 2. Synthesis of methyl 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C55).

To a solution of C54 (from the previous step; <0.367 mmol) in acetonitrile (3.7 mL) was added potassium carbonate (219 mg, 1.58 mmol), followed by addition of P15 (115 mg, 0.390 mmol). The reaction mixture was stirred at 50 °C for 20 h, then diluted with ethyl acetate (10 mL) and filtered. The filter cake was washed with ethyl acetate (3 x 10 mL), and the combined filtrates were concentrated in vacuo. Silica gel chromatography (gradient: 0 to 100% ethyl acetate in petroleum ether) afforded C55 as a dark yellow oil. Yield: 191.0 mg, 0.320 mmol, 87% over 2 steps. LCMS m/z 619.1 [M+Na+].

Step 3. Isolation of methyl 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl} methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate ENT-1 (C56) and methyl 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl} methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate ENT-2 (C57).

Separation of C55 (191 mg, 0.320 mmol) into its component stereoisomers with dioxolane was accomplished by SFC [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 3:2 carbon dioxide/2-propanol]. The first eluting isomer, obtained as a white gum, was designated ENT-1 (C56). Yield: 114 mg; this material contained residual ethanol. LC-MS m/z 597.1 [M+H] ⁺ . Retention time 4.40 min (column: Chiral Technologies ChiralCel OD-3, 4.6x100 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.05% diethylamine; gradient: 5 to 40% B over 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 ml/min).

The second eluting isomer was further purified by SFC [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 55:45 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)] to give a colourless gum which was designated ENT-2 (C57). Yield: 50 mg, 83.8 μmol, 26%. LC-MS m/z 597.1 [M+H] ⁺ . Retention time 4.74 min (analytical conditions identical to those used for C56).

Step 4. Synthesis of 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X1 (8) [from C56].

A solution of C56 (114 mg, 0.191 mmol) in acetonitrile (10 mL) was treated with an aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.97 M, 394 μL, 0.382 mmol), and the reaction mixture was stirred at room temperature for 23 h. An aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.97 M, 394 μL, 0.382 mmol) was added again and stirring was continued for 6 h, after which the pH was carefully adjusted to 7-8 with 1 M hydrochloric acid. After removal of volatiles in vacuo, purification was performed using reversed-phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 30 to 50% B) to afford 8 as a white solid. Yield: 22.2 mg, 38.1 μmol, 20%. LC-MS m/z 583.3 [M+H] ⁺ .

^1H NMR (400 MHz, methanol^): δ 8.19 (d, J=1.4 Hz, 1H), 7.94 (dd, J=8.4, 1.5 Hz, 1H), 7.77 (dd, J=7.7, 7.7 Hz, 1H), 7.64 (dd, J=10.6, 1.6 Hz, 1H), 7.58 (d, J=8.4 Hz, 1H), 7.57 (dd, J=8.0, 1.5 Hz, 1H), 6.81-6.75 (m, 1H), 6.75-6.68 (m, 2H) , 5.34-5.25 (m, 1H), 4.73 (dd, J=15.3, 3.0 Hz, 1H), 4.67-4.59 (m, 1H), 4.49 (dt, J=9.2, 6.0 Hz, 1H), 3.96 (AB quartet, J _AB = 13.7 Hz, Av _ab = 41.2 Hz, 2H), 3.06 (br d, J= 11 Hz, 1H), 2.95 (br d, J=11 Hz, 1H), 2.87-2.76 (m, 1H), 2.71 (tt, J=12.0, 3.9 Hz, 1H), 2.61-2.50 (m, 1H), 2.36-2.21 (m, 2H), 2.06 (s, 3H), 1.95-1.72 (m, 4H).

Step 5. Synthesis of 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (9) [from C57].

A solution of C57 (50 mg, 84 μmol) in acetonitrile (10 ml) was treated with an aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.97 M; 173 μl, 0.168 mmol). The reaction mixture was stirred at room temperature (ca. 25 °C) for 16 h, after which additional aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.97 M; 173 μl, 0.168 mmol) was added and stirring was continued at 25 °C for 29 h. The reaction mixture was then carefully adjusted to pH 7-8 with 1 M hydrochloric acid. The resulting mixture was concentrated in vacuo and subjected to reversed-phase HPLC (column: Xtimate™ C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 27 to 67% B) to afford 9 as a white solid. Yield: 18.0 mg, 30.9 μmol, 37%. LC-MS m/z 583.3 [M+H] ⁺ .

1H NMR (400 MHz, methanol-d ₄ ) δ 8.36-8.33 (m, 1H), 7.97 (dd, J=8.5, 1.5 Hz, 1H), 7.78 (dd, J=7.7, 7.7 Hz, 1H), 7.70-7.63 (m, 2H), 7.57 (dd, J=8.0, 1.5 Hz, 1H), 6.83-6.76 (m, 1H), 6.76-6.71 (m, 2H), 5.34-5.25 (m, 1H), 4.95-4.85 (m, 1H, putative; partially hidden by the water peak), 4.73 (dd, component of the ABX system, J=15.3, 2.7 Hz, 1H), 4.68-4.60 (m, 1H), 4.50 (dt, J=9.2, 6.0 Hz, 1H), 4.02 (AV quartet, J _AB = 13.8 Hz, Av _ab =

- 79 040816

48.2 Hz, 2H), 3.13 (br d, J=11 Hz, 1H), 3.01 (br d, J=11.5 Hz, 1H), 2.89-2.78 (m, 1H), 2.78-2.68 (m , 1H), 2.602.50 (m, 1H), 2.45-2.30 (m, 2H), 2.07 (br s, 3H), 2.00-1.86 (m, 2H), 1.83 (m, 2H).

Example 10.

2-({4-[2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetane- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (10) [from P9] but J ³ ci

H ₃

P9

C58

Step 1. Synthesis of 5-chloro-2-[2-methyl-4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]pyridine, ENT-X2, p-toluenesulfonate salt (C58) [from P9].

A solution of P9 (228 mg, 0.529 mmol) in ethyl acetate (2.7 mL) was treated with paratoluenesulfonic acid monohydrate (116 mg, 0.610 mmol) and the reaction mixture was heated at 50 °C for 16 h. It was then stirred at room temperature overnight, after which the precipitate was collected by filtration and washed with a mixture of ethyl acetate and heptane (1:1, 2 x 20 mL) to give C58 as a white solid. Yield: 227 mg, 0.451 mmol, 85%. LC-MS m/z 331.0^ [M+H]+.

1H NMR (400 MHz, DMSO-d6): δ 8.73 (d, J=2.4 Hz, 1H), 8.61-8.46 (br m, 1H), 8.35-8.18 (br m, 1H), 8.02 (dd, J=8.5, 2.5 Hz, 1H), 7.64 (d, J=8.5 Hz, 1H), 7.47 (d, J=7.8, 2H), 7.11 (d, J=7.8 Hz, 2H), 6.89-6.81 (m, 2H), 6.72 (pentet, J=4.0 Hz, 1H), 3.45-3.27 (m, 2H, putative; partially hidden by water peak), 3.10-2.91 (m, 3H), 2.28 (s, 3H), 2.02 (s, 3H), 1.97-1.80 (m, 4H).

Step 2. Synthesis of methyl 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate, DIAST-Y2 (C59) [from P9].

N,N-Diisopropylethylamine (0.234 mL, 1.34 mmol) was added to a solution of C58 (225 mg, 0.447 mmol) in acetonitrile (2.2 mL). After stirring this mixture for 5 min at 45 °C, P15 (120 mg, 0.407 mmol) was added and stirring was continued at 45 °C for 16 h, after which P15 (11 mg, 37 μmol) was added again. After stirring for a further 3 h, the reaction mixture was treated with water (2.5 mL) and allowed to cool to room temperature. More water (5 mL) was added and the resulting suspension was stirred for 2 h, after which the solid was collected by filtration and washed with a mixture of acetonitrile and water (15:85, 3 x 5 mL) to give C59 as an off-white solid (252 mg). This material contained some N,N-diisopropylethylamine by 1H NMR analysis and was carried directly to the next step. LCMS m/z 589.1 ♦ [M+H] ⁺ .

¹ H NMR (400 MHz, chloroform-d) 8.61 (d, J=2.3 Hz, 1H), 8.18 (d, J=1.5 Hz, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.67 (dd, ABX system component, J=8.4, 2.4 Hz, 1H), 7.59-7.51 (m, 1H), 6.82-6.75 (m, 1H), 6.74-6.66 (m, 2H), 5.28-5.19 (m, 1H), 4.75 (dd, ABX system component, J=15.3, 6.0 Hz, 1H), 4.68 (dd, ABX system component, J=15.3, 3.4 Hz, 1H), 4.67-4.58 (m, 1H), 4.41 (ddd, J=9.1, 5.9, 5.9 Hz, 1H), 3.95 (s, 2H), 3.95 (s, 3H), 3.07-2.89 (m, 2H), 2.81-2.69 (m, 2H), 2.53-2.41 (m, 1H), 2.37-2.22 (m, 2H), 2.05 (s, 3H), 1.93-1.74 (m, 4H).

Step 3. Synthesis of 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (10) [from P9].

A suspension of C59 (from the previous step; 250 mg, <0.407 mmol) in methanol (2 ml) was heated to 40 °C, after which aqueous sodium hydroxide solution (1 M; 0.81 ml, 0.81 mmol) was added. After 17 h, the reaction mixture was allowed to cool to room temperature and the pH was adjusted to 5-6 with 1 M aqueous citric acid solution. The resulting mixture was diluted with water (2 ml), stirred for 2 h and extracted with ethyl acetate (3 x 5 ml). The combined organic layers were washed with saturated aqueous sodium chloride solution (5 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give a foamy solid. This material was taken up in a mixture of ethyl acetate and heptane (1:1, 4 ml), heated to 50 °C and then allowed to cool with stirring overnight.

- 80 040816

Filtration gave 10 as a white solid. Yield: 179 mg, 0.311 mmol, 76% over 2 steps. LC-MS m/z 575.1 ♦ [M+H]+.

^1H NMR (400 MHz, DMSO-d6) δ 12.73 (br s, 1H), 8.71 (d, J=2.5 Hz, 1H), 8.27 (d, J=1.5 Hz, 1H), 8.00 ( dd, J=8.5, 2.5 Hz, 1H), 7.80 (dd, J=8.4, 1.6 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.60 (d, J=8.5 Hz, 1H), 6.83-6.72 (m, 3H), 5.14 -5.06 (m, 1H), 4.77 (dd, component of the ABX system, J=15.2, 7.2 Hz, 1H), 4.63 (dd, component of the ABX system, J=15.2, 2.8 Hz, 1H), 4.50-4.42 (m, 1H), 4.37 (ddd, J=9.0, 5.9, 5.9 Hz, 1H), 3.85 (AV quartet, JAB = 13.6 Hz , ΔνAB = 71.5 Hz, 2H), 3.01 (br d, J=11.2 Hz, 1H), 2.85 (br d, J=11.2 Hz, 1H), 2.74-2.57 (m, 2H), 2.472.38 (m, 1H), 2.29-2.10 (m, 2H ), 2.01 (s, 3H), 1.81-1.64 (m, 4H).

Synthesis of 10S-1. Synthesis of Example Compound 10,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt Synthesis of 1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium 2-({4-[2-(5-chloropyridin-2-s)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate, DIAST-X2 (10,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt) [from P9].

10,1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium salt

A mixture of 10 (1.54 g, 2.68 mmol) in tetrahydrofuran (10 mL) was treated with an aqueous solution of 2-amino2-(hydroxymethyl)propane-1,3-diol (Tris, 1.0 M; 2.81 mL, 2.81 mmol). After 24 h, the reaction mixture was concentrated in vacuo with ethanol (2 x 50 mL). The residue was triturated with ethanol (15 mL). After stirring for 20 h, the solid was collected by filtration and washed with cold ethanol (5 mL) to give 10, 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt as a white solid. Yield: 1.41 g, 2.03 mmol, 76%. LCMS m/z 575.3^ [M+H] ⁺ .

1H NMR (600 MHz, DMSO-d ₆ ) δ 8.71 (d, J=2.5 Hz, 1H), 8.21 (br s, 1H), 8.00 (dd, J=8.5, 2.5 Hz, 1H), 7.79 (br d, J=8.4 Hz, 1H), 7.60 (d, J=8.5 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 6.82-6.73 (m, 3H), 5.13-5.07 (m, 1H), 4.74 (dd, J=15.3, 7.2 Hz, 1H), 4.61 (dd, J=15.3, 2.9 Hz, 1H), 4.49-4.43 (m, 1H), 4.37 (ddd, J=9.0, 5.9, 5.9 Hz, 1H), 3.93 (d, J=13.6 Hz, 1H), 3.75 (d, J=13.5 Hz, 1H), 3.01 (br d , J=11.3 Hz, 1H), 2.86 (br d, J=11.4 Hz, 1H), 2.732.59 (m, 2H), 2.48-2.37 (m, 1H), 2.27-2.20 (m, 1H), 2.19-2.12 (m, 1H), 2.01 (s, 3H), 1.82-1.66 (m, 4H ). T.pl. = 184-190°C.

Synthesis of 10S-2. An alternative synthesis of the example compound 10,1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium salt.

A mixture of 10 (8.80 g, 15.3 mmol) in 2-methyltetrahydrofuran (90 mL) was concentrated in vacuo on a rotary evaporator in a water bath at 37 °C until the total volume was reduced to about 54 mL. Isopropanol (90 mL) was added to the mixture and then the resulting mixture was concentrated again to a volume of about 54 mL. Isopropanol (135 mL) was added to the mixture, then aqueous tris-amine (3 M; 5.0 mL, 0.98 equiv) was added. The resulting mixture/solution was stirred at ambient temperature and a solid precipitated within about 15 min.

The mixture was then stirred at ambient temperature for an additional 5 h. The resulting mixture/suspension was cooled to 0 °C and the cooled suspension was stirred for approximately 2 h more. The suspension was filtered and washed with cold isopropanol (3 x 15 mL). The collected solid was allowed to air dry on a collecting funnel for approximately 90 min and then transferred to a vacuum oven to dry overnight. After approximately 16 h at 50 °C/23 mmHg (with a gentle nitrogen stream), 8.66 g of 10,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt was obtained as a white solid; 99.8 area % by UPLC (yield: 12.5 mmol, 81%). LC-MS and 1H NMR data were obtained which were essentially the same as those reported above in the synthesis of 10S-1.

Obtaining X-ray Powder Diffraction (XRD) Data for Form A of the Compound of Example 10, 1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt (also known as Form A of the anhydrous tris salt of the compound of Example 10)

The white solid of the tris salt of Example 10 (from Preparation 10S-1 and Preparation 10S-2) was analyzed by X-ray powder diffraction and found to be a crystalline solid (designated Form A). Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavor diffractometer equipped with a Cu source. The slit was set to 15 mm continuous illumination. Diffracted radiation was detected with a PSD-Lynx Eye detector with the PSD (position sensitive detector) detector window set to 2.99 degrees. The voltage and current across the X-ray tube were set to

- 81 040816 at 40 kV and 40 mA, respectively. Data were collected at Cu wavelength (CuKa = 1.5418 λ) in a theta-theta goniometer from 3.0 to 40.0 degrees 2-theta in 0.01 degree steps and 1.0 second time steps. The anti-scatter screen was set to 1.5 mm. Samples were rotated for data collection. Samples were prepared by placing them in a low background silicone holder and rotating for data collection. Data were collected using DIFFRAC Plus software and analyzed with EVA diffract plus software. The X-ray diffraction data file was unprocessed prior to peak searching. Using the peak searching algorithm in EVA software, peaks selected with a threshold of 1 were used to create preliminary peak assignments. Adjustments were made manually to ensure reliability; The output of the automated assignments was visually inspected and peak positions were adjusted to the maximum peak magnitude. Peaks with relative intensities >3% were typically selected. Typically, peaks that were not resolved or did not match the noise were not selected. The typical uncertainty associated with peak position in a PXRD pattern specified by USP is up to +/- 0.2° 2-theta (USP-941). One diffraction pattern was consistently observed and is shown in Fig. 1. A list of diffraction peaks, expressed in degrees 2θ, and relative intensities with relative intensities >3.0% PXRD from the sample obtained from the synthesis of 7S-2 are presented in Table X2.

Example 11.

1-(2-Methoxyethyl)-2-({4-[2-methyl-2-(pyridin-3-yl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)1H-benzimidazole-6-carboxylic acid formate salt (11)

- 82 040816

This entire synthesis sequence was carried out in a library format.

Step 1. Synthesis of methyl-1-(2-methoxyethyl)-2-({4-[2-methyl-2-(pyridin-3-yl)-1,3-benzodioxol-4yl]piperidin-1 -yl}methyl )-1 H-benzimidazole-b-carboxylate (C60).

A mixture of P14 (44 mg, 100 μmol) and 3-ethynylpyridine (21 mg, 200 μmol) in toluene (800 μl) was treated with sodium bicarbonate (100 μmol) followed by triruthenium dodecacarbonyl (6 mg, 9 μmol). The reaction vessel was then capped and shaken at 120°C for 16 h. Removal of the solvent using a Speedvac® concentrator afforded compound C60, which was carried directly to the next step.

Step 2. Synthesis of 1-(2-methoxyethyl)-2-({4-[2-methyl-2-(pyridin-3-yl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1H-benzimidazole-6-carboxylic acid formate salt (11).

Aqueous sodium hydroxide solution (1.0 M; 200 µL, 200 µmol) was added to a solution of C60 (from the previous step, <100 µmol) in a mixture of methanol (400 µL) and tetrahydrofuran (400 µL). The reaction vessel was capped and shaken at 80 °C for 16 h, after which the reaction mixture was evaporated using a Speedvac® concentrator and purified using reversed-phase HPLC (column: Agela Durashell C18, 5 µm; mobile phase A: 0.225% formic acid in water; mobile phase B: acetonitrile; gradient: 12 to 52% B) to give 11. Yield: 2.2 mg, 4.2 µmol, 4% over 2 steps. LCMS m/z 529 [M+H]+. Retention time: 2.47 min (column: Waters XBridge C18, 2.1x50 mm, 5 μm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 1 to 5% B over 0.6 min; 5 to 100% B over 3.4 min; flow rate: 0.8 ml/min).

Example 12.

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[2(dimethylamino)ethyl]-1H-benzimidazole-6-carboxylic acid (12)

C64 12

This entire synthesis sequence was carried out in a library format.

Step 1. Synthesis of methyl 3-{[2-(dimethylamino)ethyl]amino}-4-nitrobenzoate (C61).

Methyl 3-fluoro-4-nitrobenzoate (0.2 M in N,N-dimethylformamide; 1 ml, 200 μmol) was treated with N,N-dimethylethane-1,2-diamine (18 mg, 200 μmol) and N,N-diisopropylethylamine (78 mg, 600 μmol). The reaction vessel was then capped and shaken at 50 °C for 16 h, after which the reaction mixture was evaporated using a Speedvac® concentrator to give C61. This material was carried directly to the next step.

Step 2. Synthesis of methyl 4-amino-3-{[2-(dimethylamino)ethyl]amino}benzoate (C62).

Zinc dust was activated with dilute hydrochloric acid. Methanol (2 mL) was added to C61 (from the previous step, <200 μmol), followed by the addition of an aqueous solution of calcium chloride (1.0 M; 200 μL,

- 83 040816

200 μmol) and activated zinc dust (130 mg, 2.0 mmol). The reaction vessel was capped and shaken at 70 °C for 16 h, after which the reaction mixture was filtered. The filtrate was concentrated using a Speedvac® and the residue was taken up in water (2 mL) and then extracted with ethyl acetate (2 x 3 mL). The combined organic layers were evaporated using a Speedvac® to afford compound C62 (estimated 150 μmol), which was used directly in the next step.

Step 3. Synthesis of methyl 4-[({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl} acetyl)amino]-3-{[2-(dimethylamino)ethyl]amino} benzoate (C63).

Compound P10 (41 mg, 100 μmol) was added to C62 (from the previous step, approximately 150 μmol) and this mixture was treated with a solution of N,N-dimethylacetamide 2-hydroxypyridine 1-oxide and 1[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.1 M each; 1 mL, 100 μmol each). N,N-Diisopropylethylamine (39 mg, 300 μmol) was then added and the reaction vessel was capped and shaken at 50 °C for 16 h. The reaction mixture was then concentrated using a Speedvac® concentrator and purified by preparative thin layer chromatography to afford compound C63, which was directly carried on to the next step.

Step 4. Synthesis of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[2-(dimethylamino)ethyl]-1H-benzimidazole-6-carboxylate (C64).

A mixture of acetic acid (500 µl) and C63 (from the previous step, <100 µmol) was shaken in a closed vessel at 150°C for 2 h, after which the reaction mixture was evaporated using a Speedvac® concentrator. The resulting C64 compound was directly carried on to the next step.

Step 5. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[2-(dimethylamino)ethyl]-1H-benzimidazole-6-carboxylic acid (12).

A solution of C64 (from the previous step, <100 μmol) in ethanol (500 μL) was treated with aqueous lithium hydroxide (2.0 M; 500 μL, 1 mmol) and the reaction mixture was shaken at 50 °C for 2 h in a sealed vessel. After adjusting the pH of the mixture to 7 with 1.0 M hydrochloric acid, the resulting mixture was concentrated using a Speedvac® concentrator and then purified by reversed-phase HPLC [column: Agela Durashell C18, 5 μm; mobile phase A: ammonium hydroxide in water (pH 10); mobile phase B: acetonitrile; gradient: 25 to 65% B] to give 12. Yield: 7.0 mg, 12 μmol, 12% over 3 steps. LC-MS m/z 593 [M+H]+. Retention time: 2.45 min (column: Waters XBridge C18, 2.1x50 mm, 5 μm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 10 to 100% B over 4.0 min; flow rate: 0.8 mL/min).

Example 13.

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-3-(1,3oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid (13)

Step 1. Synthesis of methyl 6-[(1,3-oxazol-2-ylmethyl)amino]-5-nitropyridine-2-carboxylate (C65).

Triethylamine (532 mg, 5.26 mmol) was added to a suspension of 1-(1,3-oxazol-2-yl)methanamine hydrochloride salt (236 mg, 1.75 mmol) and methyl 6-chloro-5-nitropyridine-2-carboxylate (386 mg, 1.78 mmol) in tetrahydrofuran (5 ml). After stirring the reaction mixture at 25°C for 14 h, it was poured into water (30 ml) and extracted with dichloromethane (2 x 50 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. After chromatography on silica gel

- 84 040816 (gradient: 0 to 5% methanol in dichloromethane) gave C65 as a yellow solid. Yield: 310 mg, 1.11 mmol, 63%. LC-MS m/z 278.7 [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.69-8.61 (br m, 1H), 8.58 (d, J=8.4 Hz, 1H), 7.65 (d, J=0.8 Hz, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.11 (d, J=1.0 Hz, 1H), 5.07 (d, J=5.3 Hz, 2H), 3.97 (s, 3H).

The rest of the synthesis sequence was carried out in library format.

Step 2. Synthesis of methyl 5-amino-6-[(1,3-oxazol-2-ylmethyl)amino]pyridine-2-carboxylate (C66).

Aqueous ammonium chloride solution (5.0 M; 400 μL, 2.0 mmol) followed by activated zinc (131 mg, 2.0 mmol) was added to a solution of C65 (56 mg, 200 μmol) in methanol (2.0 mL). The reaction vessel was then capped and shaken at 30 °C for 16 h, after which the reaction mixture was filtered. The filtrate was concentrated using a Speedvac® concentrator, then mixed with water (1.0 mL) and extracted with dichloromethane (3 x 1.0 mL). The combined organic layers were evaporated using a Speedvac® concentrator to afford compound C66, which was directly carried on to the next step.

Step 3. Synthesis of methyl 5-[({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}acetyl)amino]-6-[(1,3-oxazol-2-ylmethyl)amino]pyridine-2-carboxylate (C67).

A mixture of P10 (81 mg, 200 μmol) and C66 (from the previous step, <200 μmol) was mixed with N,N-dimethylacetamide and then treated with N,N-diisopropylethylamine (100 μL, 600 μmol). A solution containing 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.24 M) and 2-hydroxypyridine 1-oxide (0.1 M) in N,N-dimethylacetamide (1.0 mL, containing 240 μmol 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride and 100 μmol 2-hydroxypyridine 1-oxide) was added and the reaction vessel was capped and shaken at 50 °C for 16 h. The volatiles were then removed using a Speedvac® concentrator and the residue was subjected to preparative thin layer chromatography to afford compound C67, which was carried directly to the next step.

Step 4. Synthesis of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidinyl}methyl)-3-(1,3 -oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylate (C68).

A mixture of acetic acid (1.0 mL) and C67 (from the previous step, <200 μmol) was shaken at 150 °C for 2 h, after which the reaction mixture was evaporated using a Speedvac® concentrator. The resulting compound C68 was used directly in the next step.

Step 5. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-3-(1,3-oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid (13).

An aqueous solution of lithium hydroxide (2.0 M; 1.0 mL, 2.0 mmol) was added to the mixture of C68 (from the previous step, <200 μmol) in tetrahydrofuran (1.0 mL). After addition of methanol (500 μL), the reaction vessel was capped and shaken at 50 °C for 16 h. After removal of volatiles using a Speedvac® concentrator, dimethyl sulfoxide (1.0 mL) was added and the pH was adjusted to 7-8 with concentrated hydrochloric acid. The resulting mixture was purified using reversed-phase HPLC [column: Agela Durashell C18, 5 μm; mobile phase A: ammonium hydroxide in water (pH 10); mobile phase B: acetonitrile; gradient: 24 to 64% B] to give 13. Yield: 3.9 mg, 6.5 μmol, 3% over 4 steps. LC-MS m/z 604 [M+H] ⁺ . Retention time: 3.14 min (column: Waters XBridge C18, 2.1 x 50 mm, 5 μm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 1 to 5% B over 0.6 min; 5 to 100% B over 3.4 min; flow rate: 0.8 mL per minute).

Example 14.

2-({4-[(2S)-2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1methyl-1H-benzimidazole-6-carboxylic acid (14)

SLEEP

O-SNz

CH ₃ CH;

_CH3N

Step 1. Synthesis of methyl-2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidin-1 -yl}methyl)- 1 -methyl-1 H-benzimidazole-6-carboxylate (C69).

N,N-Diisopropylethylamine (683 µl, 3.92 mmol) was added to a mixture of P3 (680 mg, 1.31 mmol) in acetonitrile (5.2 ml). This mixture was stirred for 5 min at 45°C, after which P16 (319 mg, 1.34 mmol) was added. Stirring was continued at 45°C for 2.75 h, and then water (6 ml) was added.

- 85 040816 after which the reaction mixture was allowed to cool to room temperature and stirred for 30 min. The solid was collected by filtration and washed with a mixture of acetonitrile and water (1:4, 3x5 mL) to give C69 as a white solid. Yield: 635 mg, 1.15 mmol, 88%. LCMS m/z 550.1 ♦ [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.15-8.12 (m, 1H), 7.97 (dd, J=8.5, 1.6 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.50 (dd, J=8.2, 8.2 Hz, 1H), 7.16-7.07 (m, 2H), 6.79-6.73 (m, 1H), 6.72-6.65 (m, 2H), 3.98 (s, 3H), 3.96 (s, 3H), 3.88 (s, 2H), 3.04- 2.93 (m, 2H), 2.76-2.66 (m, 1H), 2.37-2.25 (m, 2H), 2.04 (br s, 3H), 1.89-1.78 (m, 4H).

Step 2. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-methyl-1H-benzimidazole-6-carboxylic acid (14).

A mixture of C69 (600 mg, 1.09 mmol) in methanol (11 mL) was heated to 45 °C and then treated with aqueous sodium hydroxide (1 M; 2.2 mL, 2.2 mmol). After 24 h, the reaction mixture was adjusted to pH 56 by adding aqueous citric acid (1 M; 1.1 mL) and then diluted with water (10 mL). The resulting mixture was allowed to cool to room temperature and stirred for 1 h, after which the precipitated solid was collected by filtration and washed with a mixture of methanol and water (1:4; 3 x 5 mL). This gave 14 as a white solid. Yield: 535 mg, 0.998 mmol, 92%. LC-MS m/z 536.1 ♦ [M+H] ⁺ .

1H NMR (400 MHz, DMSC-d6) δ 8.16 (d, J=1.5 Hz, 1H), 7.81 (dd, J=8.4, 1.6 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.59-7.52 (m, 2H), 7.33 (dd, J=8.3, 2.1 Hz, 1H), 6.81-6.70 (m, 3H), 3.94 (s, 3H), 3.84 (s, 2H), 3.01-2.91 (m, 2H), 2.70-2.59 (m, 1h), 2.28-2.16 (m, 2H), 2.02 (s, 3H), 1.73 (m, 4H).

Examples 15 and 16 -(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X1, trifluoroacetate salt (15) [from P18 via C71] and 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}-1 -(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X2, trifluoroacetate salt (16) [from P18 via C72] ^NC %

C4 ΒΙΝΑΡ θ ⁷ θ ( ^{mixture of} diastereomers)

С71 (from Р18) ) С72 (from Р18)

Step 1. Synthesis of methyl-2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}- 1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (C70) [from P18].

A mixture of P18 (240 mg, 0.699 mmol), C4 (275 mg, 0.800 mmol), cesium carbonate (455 mg, 1.40 mmol), tris(dibenzylideneacetone)dipalladium(0) (40.0 mg, 43.7 μmol) and 1,1'-binaphthalene-2,2'diylbis(diphenylphosphane) (BINAP; 52.2 mg, 83.8 μmol) in toluene (5 mL) was degassed with nitrogen for 5 min and then stirred at 90 °C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. Preparative thin layer chromatography (eluent: 1:1 petroleum ether/ethyl acetate) gave C70, a mixture of diastereomers, as a yellow oil. Yield: 165 mg, 0.272 mmol, 39%. LC-MS m/z 628.14 [M+Na ⁺ ].

Step 2. Isolation of methyl 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct-1-yl}-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate, DIAST-Y1 (C71) [from P18], and methyl 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct-1-yl}-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate, DIAST-Y2 (C72) [from P18].

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Separation of stereoisomers by dioxolane in C70 (165 mg, 0.272 mmol) was carried out using SFC

[Column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting isomer was designated DIAST-Y1 (C71) and the second eluting isomer was designated DIAST-Y2 (C72); both were isolated as a white solid.

C71 Yield: 55.0 mg, 90.7 μmol, 33%. LC-MS m/z 605.9^ [M+H]+. Retention time 4.47 min (column: Chiral Technologies Chiralpak AD-3, 4.6x100 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B over 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 mL/min).

C72 Yield: 58.0 mg, 95.7 μmol, 35%. LC-MS m/z 628.0^ [M+Na ⁺ ]. Retention time 4.88 min (analytical conditions identical to those used for C71).

Step 3. Synthesis of 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct1-yl}-1- (2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X1, trifluoroacetate salt (15) [from P18 through C71].

To a solution of C71 (55.0 mg, 90.7 μmol) in a mixture of methanol (2.0 mL) and tetrahydrofuran (1.0 mL) was added aqueous sodium hydroxide solution (3 M; 1.0 mL, 3.0 mmol). After stirring the reaction mixture at 20 °C for 2 h, the pH was adjusted to 7 with 1 M hydrochloric acid and the resulting mixture was extracted with a mixture of dichloromethane and methanol (10:1, 3 x 30 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. After reversed-phase HPLC (column: Boston Green ODS, 5 μm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 10 to 95% B), 15 was obtained as a white solid. Yield: 35.8 mg, 50.7 µmol, 56%.

LC-MS m/z 592.3 ♦ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol^) δ 8.46 (s, 1H), 8.21 (d, J=8.6 Hz, 1H), 7.78 (d, J=8.6 Hz, 1H), 7.54 (dd, J=8.3, 8.3 Hz, 1H), 7.16-7.08 (m, 2H), 6.76 (dd, J=8.2, 8.1 Hz, 1H), 6.55-6.47 (m, 2H), 4.9-4.70 (m, 2H, putative; partially hidden by water peak), 3.82 (t, J=4.9 Hz, 2H), 3.66-3.56 (m, 1H), 3.50-3.41 (m, 1H), 3.19-3.09 (m, 1H), 3.15 (s, 3H), 3.08-2.99 (m, 1H), 2.63-2.57 (m, 1H), 2.27-2.17 (m, 1H), 2.01 (s, 3H), 1.761.66 (m, 2H), 1.62-1.50 (m, 2H), 1.35-1.26 (m, 1H).

Step 4. Synthesis of 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct1-yl}-1- (2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X2, trifluoroacetate salt (16) [from P18 through C72].

To a solution of C72 (58.0 mg, 95.7 μmol) in a mixture of methanol (2.0 mL) and tetrahydrofuran (1.0 mL) was added aqueous sodium hydroxide solution (3 M; 1.0 mL, 3.0 mmol). After stirring the reaction mixture at 20 °C for 2 h, the pH was adjusted to 7 with 1 M hydrochloric acid and the resulting mixture was extracted with a mixture of dichloromethane and methanol (10:1, 3 x 30 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. After reversed-phase HPLC (column: Boston Green ODS, 5 μm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 35 to 95% B) 16 was obtained as a white solid. Yield: 33.4 mg, 47.3 μmol, 49%. LC-MS m/z 592.2^ [M+H] ⁺ .

1H NMR (400 MHz, methanol-d4) δ 8.53-8.50 (m, 1H), 8.25 (dd, J=8.6, 1.4 Hz, 1H), 7.80 (br d, J=8.6 Hz, 1H), 7.57 (dd, J=8.4, 8.2 Hz, 1H), 7.25 (dd, J=10.8, 2.0 Hz, 1H), 7.19 (br dd, J=8.4, 2.1 Hz, 1H), 6.77 (dd, J=8.2, 8.1 Hz, 1H), 6.55-6.50 (m, 2H), 4.9-4.72 (m, 2H, putative; partially obscured by water peak), 3.933.80 (m, 2H), 3.68-3.58 (m, 1H), 3.41-3.3 (m, 1H, presumed; partially hidden by the solvent peak), 3.25 (s, 3H), 3.22-3.12 (m, 1H), 3.07-2.97 (m, 1H), 2.67 (dd, J=8.3, 5.8 Hz, 1H), 2.28-2.17 (m, 1H), 2.01 (d, J=1.0 Hz, 3H), 1.86-1.71 (m, 2H), 1.69-1.56 (m, 2H), 1.36-1.26 (m, 1H).

Examples 17 and 18.

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl} methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-ammonium carboxylate, ENT-1 (17), and 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl} methyl)-1-[(1-ethyl-1H-imidazol-5yl)methyl]-1H-benzimidazole-6-ammonium carboxylate, ENT-2 (18)

- 87 040816

CH ₃ CH ₃

Step 1. Synthesis of methyl 4-[({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl} acetyl)amino]-3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino} benzoate (C73).

O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (566 mg, 1.49 mmol) was added to a mixture of P19 (340 mg, 1.24 mmol) in N,N-dimethylformamide (10 ml) and the mixture was stirred at 25 °C for 10 min. A solution of P10 (503 mg, 1.24 mmol) and N,N-diisopropylethylamine (615 μl, 3.53 mmol) in N,N-dimethylformamide (7.7 ml) was then added and the reaction mixture was stirred at 25 °C for 16 h, after which it was poured into water (10 ml) and extracted with ethyl acetate (3 x 50 ml). The combined organic layers were washed successively with aqueous ammonium chloride (3 x 20 mL) and saturated aqueous sodium chloride (2 x 20 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel chromatography (gradient: 0 to 5% methanol in ethyl acetate) afforded C73 as a pale brown gum. Yield: 316 mg, 0.477 mmol, 38%. LC-MS m/z 662.2^ [M+H]+.

Step 2. Synthesis of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylate (C74).

A solution of C73 (316 mg, 0.477 mmol) in acetic acid (14 mL) was stirred at 55 °C for 16 h. The solvent was removed under high vacuum and the residue was purified by preparative thin layer chromatography (eluent: 10:1 dichloromethane/methanol) to give C74 as a colorless oil. Yield: 200 mg, 0.310 mmol, 65%. LC-MS m/z 644.3^ [M+H] ⁺ .

Step 3. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-ammonium benzimidazole-6-carboxylate, ENT-1 (17), and 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5-yl)methyl]-1H-ammonium benzimidazole-6-carboxylate, ENT-2 (18).

A mixture of C74 (150 mg, 0.233 mmol) and aqueous sodium hydroxide solution (2 M; 233 μl, 0.466 mmol) in a mixture of methanol (3 ml) and tetrahydrofuran (3 ml) was stirred at 45 °C for 16 h. After adjusting the pH of the reaction mixture to 7 with 1 M hydrochloric acid, it was concentrated in vacuo to give a mixture of 17 and 18. These enantiomers were separated by SFC [column: Chiral Technologies ChiralCel OD, 10 μm; mobile phase: 1:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer was designated ENT-1 (17) and the second eluting enantiomer was designated ENT-2 (18); both were isolated as a white solid.

Yield: 45.0 mg, 69.5 μmol, 30%. LC-MS m/z 630.3^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-d ₄ ) δ 8.15 (br s, 1H), 8.00 (br d, J=8.4 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J=8.2 Hz, 1H), 7.56 (dd, J=8.3, 8.3 Hz, 1H), 7.28 (dd, J=10.9, 2.0 Hz, 1H), 7.21 (dd, J=8.3, 2.1 Hz, 1H), 6.77 (dd, component of the ABC system, J=8.0, 7.7 Hz, 1H), 6.69 (dd, component of the ABC system, J=7.8, 1.2 Hz, 1H), 6.67-6.60 (m, 2H), 5.82 (s, 2H), 4.12 (q, J=7.2 Hz, 2H), 3.89 (AB quartet, Jab = 14.3 Hz, Avab = 6.9 Hz, 2H), 3.00-2.90 (m, 2H), 2.74-2.64 (m, 1H), 2.32-2.21 (m, 2H), 2.02 (s, 3H), 1.82-1.61 (m, 4H), 1.29 (t, J=7.3 Hz, 3H).

Retention time 5.66 min (column: Chiral Technologies Chiralpak AD-3, 4.6 x 150 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5 to 40% B over 5.5 min, then hold at 40% B for 3.0 min; flow rate: 2.5 ml/min).

Yield: 32.8 mg, 50.7 µmol, 22%. LC-MS m/z 630.3^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-d4) δ 8.15 (s, 1H), 8.00 (d, J=8.5 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J=8.5 Hz, 1H), 7.56 (dd, J=8.3, 8.3 Hz, 1H), 7.28 (dd, J=10.9, 2.0 Hz, 1H), 7.21 (dd, J=8.3, 2.0 Hz, 1H), 6.77 (dd, compo-

- 88 040816 (dd, ABC system component, J=7.8, 7.8 Hz, 1H), 6.69 (dd, ABC system component, J=7.9, 1.2 Hz, 1H), 6.67-6.60 (m,

2H), 5.82 (s, 2H), 4.12 (q, J=7.3 Hz, 2H), 3.89 (AB quartet, Jab = 14.1 Hz, Avab = 7.4 Hz, 2H), 3.01-2.90 ( m,

2H), 2.74-2.63 (m, 1H), 2.31-2.21 (m, 2H), 2.02 (s, 3H), 1.82-1.60 (m, 4H), 1.29 (t, J=7.3 Hz, 3H). Retention time 5.34 min (analytical SFC conditions identical to those used for 17).

The compounds listed in Table 1 were prepared using procedures similar to those described in the examples identified in Table 2, using the appropriate intermediate(s) identified in Table 2. The compounds were purified using the methods discussed herein. The final compounds could be isolated as neutral compounds or as acid or base salts.

Table 1

Structure and IUPAC name for compounds of examples 19-102

Ex. No. Structure IUPAC name 19 ^ch o s ^>0 p ^ν · CF ₃ COOH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperazin-1-yl}methyl)-1-(2methoxyethyl)-1/7-benzimidazole-6carboxylic acid trifluoroacetate salt 20 Ъ- X · CF3COOH ENT-X2 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1-benzimidazole-6-carboxylic acid, ENT-X2, trifluoroacetate salt [from C77; note 1 in Table 2] 21 0- ^F S CF3COOH ENT-X1 2-( { 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1 -(2-methoxyethyl)- 1-benzimidazole-6-carboxylic acid, ENT-X1, trifluoroacetate salt, [from C76; note 1 in Table 2] 22 ^x o O S ау-о 9 ^Ν Ό% _η · CF3COOH ENT-X1 1-(2-methoxyethyl)-2-{[4-(2-phenyl-1,3-benzodioxol-4-yl)piperazin-1- yl]methyl}-1/7-azole-6-carboxylic acid benzimide, ENT-X1, trifluoroacetate salt [from P5] 23 o s ^abs )~O '“'OH • CF3COOH ENT-X2 1-(2-methoxyethyl)-2-{[4-(2-phenyl-1,3-benzodioxol-4-yl)piperazin-1- yl]methyl}-III-azole-6-carboxylic acid benzimide, ENT-X2, trifluoroacetate salt [from P6] 24 ^ch o ^ci rr s XvGVw Xd \=/ън • CF3C00H 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl] piperidin-1-yl } methyl)-1-(2methoxyethyl)-1H-benzamide azole-6-carboxylic acid trifluoroacetate salt 25 ^c 'XKl l X ^abs/O • CF3COOH DIAST-Z2 2-{6-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}-1-(2methoxyethyl)-1H-azole-6-carboxylic acid benzimide, DIAST-Z2, trifluoroacetate salt [from P17 through C79; note 2 in Table 2]

- 89 040816

26 CI^^F > XX s abs/O f^ZAabs ' • CF3COOH DIAST-Z1 2-{6-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5 ]oct-1-yl }-1-(2methoxyethyl)-1/7-azole-6-carboxylic acid benzimide, DIAST-Z1, trifluoroacetate salt [from P17 through C78; note 2 in Table 2] 27 NC \ t> ^F s ΛΟ ί^Ν·%<4 ^O U<A> LDu/ OH • CF3COOH 2-({4-[2-(4-cyano-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid trifluoroacetate salt 28 9^3 ⁴ o & S AOTKf 1 J ^^OH ·HCOOH 1-(2-methoxyethyl)-2-[(4-{2-methyl-2-[3 (trifluoromethyl)phenyl]-1,3- benzodioxol-4-yl} piperidin-1 yl)methyl]-III-benzimidazole-6-carboxylic acid formate salt 29 ^x o TT ^= ⁷ OH · HCOOH 2-( { 4-[2-(4-ethylphenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1 -(2-methoxyethyl)- 1Hbenzimidazole-6-carboxylic acid formate salt 30 f ⁴ O ύι s •HCOOH 2-({4-[2-(3-fluoro-4-methoxyphenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1H-benzamide azole-6-carboxylic acid formate salt 31 XrX °y\A> rL/y^ ⁰ 1(^1 '^(DH •HCOOH 2-( { 4-[2-(3-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl } methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid formate salt

- 90 040816

32 °yVL ν-MT ⁵ LJ he nsoon 1-(2-methoxyethyl)-2-({ 4-[2-(4methoxyphenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)- 1/7-benzamide azole-6-carboxylic acid formate salt 33 ^x o ^CI IX S TU \=/οη •nsoon 2-( { 4-[2-(4-chlorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1 -(2-methoxyethyl)- 1Hbenzimidazole-6-carboxylic acid formate salt 34 ΟγΟ y jb 9 o ζ 2-({4-[2-(4-cyanophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid 35 ^h o ϊ Τ \=/ he nsoon 2-({4-[2-(2-fluoro-4-methoxyphenyl)-2- methyl-1,3-benzodioxol-4- yl] piperidin-1-yl } methyl)-1 -(2- methoxyethyl)- 1/7-benzamide azole-6-carboxylic acid formate salt 36 tu 9=/^ · nsoon 1-(2-methoxyethyl)-2-({ 4-[2-methyl-2-(6methylpyridin-2-yl)-1,3- benzodioxol-4-yl]piperidin-1- yl } methyl)- 1/7-benzamide azole-6-carboxylic acid formate salt 37 । ^ch ? gg° T2G ° ⁿ 1-(2-methoxyethyl)-2-({ 4-[2-(2methoxyphenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)-1H-benzimidazole-6-carboxylic acid

- 91 040816

38 ^ch o ^f Ol S chips ·nsoon 2-({4-[2-(4-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid formate salt 39 0 + %hop>n° UX \ ₌ /\ _on •nsoon 1-(2-methoxyethyl)-2-({ 4-[2-(3 methoxyphenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1H-benzamide azole-6-carboxylic acid formate salt 40 'XxL' UJ HE •UNNC 1-(2-methoxyethyl)-2-[(4-{2-methyl-2-[4- (trifluoromethyl)phenyl]-1,3- benzodioxol-4-yl} piperidin-1 yl)methyl]-1H-benzimidazole-6-carboxylic acid formate salt 41 1 ΑχθΊ%4> |Q| \=/he 2-({4-[2-(3,4-difluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid 42 A. '° ·nsoon 1-(2-methoxyethyl)-2-({ 4-[2-methyl-2-(6methylpyridin-3-yl)-1,3- benzodioxol-4-yl]piperidin-1 yl } methyl)- 1//-benzamide azole-6-carboxylic acid formate salt 43 0 Clx^^F < Yli ' P ϊ T OH 1 - { 2- [acetyl(methyl)amino]ethyl } -2({4-[2-(4-chloro-2-fluorophenyl)-2-methyl- 1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)- 1H-benzimidazole-6-carboxylic acid

- 92 040816

44 0 Ac ΟΆ o ^o hAA>nAv< VJ he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2(morpholin-4-yl)ethyl]-\Nbenzimidazole-6-carboxylic acid 45 SK F A GT ^N / X^J ^^^ΟΗ 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1 (pyridin-2-ylmethyl)-III-benzimidazole-6-carboxylic acid 46 ΟγΟ °?A э V U 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1-[2-(2oxo-1,3-oxazolide-3-yl)ethyl]-1Hbenzimidazole-6-carboxylic acid 47 ^X N^ °Ao X Ti / Aq ACH o °chDA> n/W XJj A=/on 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2(dimethylsulfamoyl)ethyl]-1Hbenzimidazole-6-carboxylic acid 48 ΟγΟ A X l o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2-(2oxopyrrolidin-1-yl)ethyl]-\Nbenzimidazole-6-carboxylic acid 49 <An ^C 'A Ao o °чААА Νγ [TJ \=/ he 1-[2-(acetylamino)ethyl]-2-({ 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl } methyl)-III-benzimidazole-6-carboxylic acid

- 93 040816

50 P N ^c 'XIi / Wushu [GD \=/on 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1-yl } methyl)-1 - [2-( 1Nimidazol-1-yl)ethyl]- \N- benzimidazole-6-carboxylic acid 51 <Τ^ ^Ν >Λ /° оШДЭ n-Ч Уч yj \=/ he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-2-yl)methyl]-1Hbenzimidazole-6-carboxylic acid 52 Y Y ^C 'YY ^F ^7-0 θ °y\A> N-Uuch Hjr OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2(methylamino)-2-oxoethyl]-\Nbenzimidazole-6-carboxylic acid 53 a CI^^F <4-о ,Ο °\A/M му У4 \=/ он 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1-yl } methyl)-1-[2-(\Npyrazol-1-yl)ethyl]-1H-benzimidazole-6-carboxylic acid 54 0^0 ^v t J~' o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1-yl } methyl)-1-[3-(\H1,2,4-triazol-1-yl)propyl]-\Hbenzimidazole-6-carboxylic acid 55 ΟγΟ .у у ч о 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2-(1 methyl-1N-imidazol-4-yl)ethyl]-1Nbenzimidazole-6-carboxylic acid

- 94 040816

56 ^CI X4 lljr he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1(tetrahydrofuran-3-yl methyl)-1Hbenzimidazole-6-carboxylic acid 57 οίγγΡ d~°l Р [ΪΤ \=/ OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(1 methyl- 1H-1,2,4-triazol-5-yl)methyl] 1H-benzimidazole-6-carboxylic acid 58 ^c w OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-4-ylmethyl)-1H-benzimidazole-6-carboxylic acid 59 y ₀ θ'τΐι s X^J \= ^/Z oh 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[3(d-methylamino)-3-oxopropyl]-\Nbenzimidazole-6-carboxylic acid 60 A '5 3~^ ^r 0^0 I 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1 - [2-( 1 methyl-1/7-1,2,3-triazol-4-yl )ethyl]1Н-benzimidazole-6-carboxylic acid 61 ^s| g+| \Ύ \=/ he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1(tetrahydrofuran-3-yl)-1Hbenzimidazole-6-carboxylic acid

- 95 040816

62 ΟγΟ S O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2-(2methyl- 1R-imidazol-1-yl)ethyl]-1Rbenzimidazole-6-carboxylic acid 63 o rfS 0^0 I 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(1 methyl-1H-1,2,3-triazol-4-yl)methyl]1H-benzimidazole-6-carboxylic acid 64 J ar o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(27?)tetrahydrofuran-2-ylmethyl]-1Ybenzimidazole-6-carboxylic acid 65 о A. €>° Ф 0^0 HZ 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1 (pyridin-3-ylmethyl)-III-benzimidazole-6-carboxylic acid 66 OyO A a o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2(dimethylamino)-2-oxoethyl]-\Nbenzimidazole-6-carboxylic acid 67 °γδ J о 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1-[2(pyrrolidin-1-yl)ethyl]-\Nbenzimidazole-6-carboxylic acid

- 96 040816

68 0 Μ Vz- ⁿ 'NH ei^^F \=< Y Τι 7 °y\A> OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-{[3 (methoxymethyl)-3-pyrazol-5yl]methyl}-1N-benzimidazole-6-carboxylic acid 69 OyO & o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-5-ylmethyl)-1H-benzimidazole-6-carboxylic acid 70 Cl F XL Ά ΛνΟΊΟ-uch ⁰ CT 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1-yl}methyl)-1-{[4-(2methoxyethyl)-4H-1,2,4-triazol-3 yl]methyl}-1H-benzimidazole-6-carboxylic acid 71 0 N T^r V=/ OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-{2[methyl(methylsulfonyl)amino]ethyl}1H-benzimidazole-6-carboxylic acid 72 rr, Y ^0H tjj ^^^OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(1hydroxycyclobutyl)methyl]-1Ybenzimidazole-6-carboxylic acid

- 97 040816

73 Cly^F XVWv TT\=/oh • CF3COOH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1//pyrazol-4-ylmethyl)-1H- benzimidazole-6-carboxylic acid trifluoroacetate salt 74 ⁿ Cnh T ,° °xAAJ N-# УЧ Ϊ Y \=/o _H CF3COOH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[2-(\Nimidazol-2-yl)ethyl]-\N- benzimidazole-6-carboxylic acid trifluoroacetate salt 75 OyO Λ o o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2hydroxyethyl)-1//-benzimidazole-6-carboxylic acid 76 ΟγΟ A o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(1-ethyl177-1 ,2,3-triazol-5-yl)methyl]-\Nbenzimidazole-6-carboxylic acid 77 ΟγΟ ..U and 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(1 methyl- 1//-imidazol-4-yl)methyl]-\Nbenzimidazole-6-carboxylic acid 78 J Сг г о 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 - [(4methyl-4R-1,2, 4-triazol-3-yl)methyl]1R-benzimidazole-6-carboxylic

- 98 040816 acid

2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1-[(25)tetrahydrofuran-2-ylmethyl]-1R-benzimidazole-6-carboxylic acid 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1-(1,3-oxazol-2-ylmethyl)-1R-benzimidazole-6-carboxylic acid 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-3 -(1,3-oxazol-5-ylmethyl)-3//-imidazo[4,5/>]pyridine-5-carboxylic acid 1 -(2-methoxyethyl)-2-{[4-(2-methyl-2-phenyl-1,3-benzodioxol-4yl)piperidin-1-yl]methyl} -\Nbenzimidazole-6-carboxylic acid formate salt

2-( { 4- [2-(2-chloro-4-methoxyphenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1-yl} methyl)-1-(2-methoxyethyl)-3-benzimidazole-6-carboxylic acid formate salt 1-(2-methoxyethyl)-2-( {4- [2-methyl-2-(4-methylphenyl)-1,3-benzodioxol-4-yl] piperidin-1-yl } methyl)-\Nbenzimidazole-6-carboxylic acid formate salt

- 99 040816

85 OTs ΓΟΆ ρ |ί Τ ^= ⁷ he HCOOH 1-(2-methoxyethyl)-2-({ 4-[2-methyl-2-(3 methylphenyl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1Hbenzimidazole-6-carboxylic acid formate salt 86 ^χ ο ry XT ^= ⁷ OH · HCOOH 2-({ 4-[2-(2-chlorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1 yl}methyl)-1-(2-methoxyethyl)-1-benzimidazole-6-carboxylic acid formate salt 87 θΝ ^ch o ώι s QWQXyy UD ^= ⁷ on •HCOOH 2-({4-[2-(3-cyanophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid formate salt 88 ^x o N-# UD' [ϊ Ύ ^= ⁷ OH •HCOOH 1-(2-methoxyethyl)-2-({ 4-[2-methyl-2-(2methylphenyl)-1,3-benzodioxol-4yl]piperidin-1-yl }methyl)-1Hbenzimidazole-6-carboxylic acid formate salt 89 ^hop । S OU GP*N R °uUU^ Uch O ^= ⁷ on • CF ₃ COOH ENT-X2 1-(2-methoxyethyl)-2-({ 4-[2-methyl-2(pyridin-2-yl)-1,3-benzodioxol-4yl]piperidin-1-yl }methyl)-1Hbenzimidazole-6-carboxylic acid, ENT-X2, trifluoroacetate salt [from C81; note 7 in Table 2] 90 ^ch o Qi S · CF3COOH ENT-X1 1-(2-methoxyethyl)-2-({ 4-[2-methyl-2(pyridin-2-yl)-1,3-benzodioxol-4yl]piperidin-1-yl }methyl)-1Hbenzimidazole-6-carboxylic acid, ENT-X1, trifluoroacetate salt [from C80; note 7 in Table 2]

- 100 040816

91 ^x o αγγ \ AHA. ρ I Τ 0' νη ₄ ⁺ ΕΝΤ-Χ1 2-({4-[2-(5-chloropyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1/7-benzimidazole-6-ammonium carboxylate, ENT-X1 [from P8] 92 ⁴ ο ^CI OG S УтОУуХ Цу \=/ 0- nh ₄ ⁺ ENT-X2 2-({4-[2-(5-chloropyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1H-benzimidazole-6-ammonium carboxylate, ENT-X2 [from P9] 93 ^Nc n, X T'N'%rj' ^N K _n θγΑγΟ Zug O'NH ₄ ⁺ ENT-X1 2-({4-[2-(5-cyanopyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1//-benzimidazole-6-ammonium carboxylate, ENT-X1 [from P8] 94 ^nc Oq '° 9 T ^x N ^x %f ^N v °ууУ> N-Ууу <У ONH ₄ ⁺ ENT-X2 2-({4-[2-(5-cyanopyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1H-benzimidazole-6-ammonium carboxylate, ENT-X2 [from P9] 95 ^CI Y^N oХ Lif I 7 %аА> IK/ TU '“'OH nsoon DIAST-X1 2-({4-[2-(5-chloropyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(26)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X1 [from P8] 96 °Όί ”7 DI AST-1 2-({4-[2-(4-chloro-2-fluorophenyl)-7fluoro-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(26)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-1 [note 10 in Table 2]

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97 oL T Τι 7 chn. ^F DI AST-2 2-({4-[2-(4-chloro-2-fluorophenyl)-7fluoro-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(25)oxetan-2-ylmethyl]-1N-benzimidazole-6-carboxylic acid, DIAST-2 [note 10 in Table 2] 98 ^NC tXi [Г^Г \=/o- NH ₄ ⁺ ENT-X2 2-({4-[2-(4-cyano-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-ammonium carboxylate, ENT-X2 [from C83; note 12 in Table 2] 99 W Ц YwTv O' nh ₄ ⁺ ENT-X1 2-({4-[2-(4-cyano-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-ammonium carboxylate, ENT-X1 [from C82; note 12 in Table 2] 100 ^CI \^Y ^F oY TI] ch ₃ 7 • 1/2 citric acid 2-({4-[(25)-2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-7-fluoro-1[(25)-oxetan-2-ylmethyl]-1Ybenzimidazole-6-carboxylic acid hemicitrate salt [from RE] 101 oX L ί ^H 7 °γ\Α> ν·Λ7 OH •1/2 citric acid 2-({4-[(25)-2-(4-chloro-2-fluorophenyl)1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(25)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid hemicitrate salt [from C48] 102 ^H z% ¥T \=/oh · CF3C00H 2-({4-[2-(hydroxymethyl)-2-phenyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-\Nbenzimidazole-6-carboxylic acid trifluoroacetate salt

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Table 2

Method of preparation and physicochemical data for compounds of examples 19-102

Ex. No. Way 'H NMR (400 MHz, methanol-^) δ; mass spectrum, registered ion m/z [M+H] ⁺ or HPLC retention time; mass spectrum m/z [M+H] ⁺ (unless otherwise stated) 19 Examples 15 and 16; C4, P12 8.39 (br s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.3, 8.3 Hz, 1H ), 7.28 (dd, J = 10.9, 2.1 Hz, 1H), 7.22 (dd, J = 8.4, 2.0 Hz, 1H), 6.83 (dd, J = 8.1, 8.1 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 6.55 (d, J = 8, 4 Hz, 1H), 4.73 (s, 2H), 4.66 (t, J = 4.9 Hz, 2H), 3.77 (t, J = 4.8 Hz, 2N), 3.59-3.43 (t, 8N), 3.30 (s, ZN ^LL ), 2.05 (s, ZN); 581.0 20 Examples 4 and 5 ¹ ; C43, P11 8.34-8.31 (m, 1H), 8.03 (dd, 7=8.5,1.5 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.0, 8.0 Hz, 1H), 7.36 (dd, J = 10.2, 1.9 Hz, 1H), 7.31 (dd, J = 8.4, 1.8 Hz, 1H), 7.25 (s, 1H), 6.90 (dd, ABC system component, J = 8.9, 6.6 Hz, 1H), 6.86-6.80 (w, 2H ), 4.79 (s, 2H), 4.60 (br t, J = 4.8 Hz, 2H), 3.95-3.85 (m, 2H), 3.74 (dd, J = 5.3, 4.2 Hz, 2H), 3.44-3.33 (m, 2H), 3.28 (s, ZN), 3.15-3.05 (m, 1H), 2.37-2.12 (m, 4H); 566.04 21 Examples 4 and 5 ¹ ; C43, P11 8.32 (dd, J= 1.6, 0.7 Hz, 1H), 8.03 (dd, J= 8.5, 1.5 Hz, 1H), 7.78 (dd, J= 8.5, 0.7 Hz, 1H), 7.60 ( dd, J= 8.1, 7.9 Hz, 1H), 7.36 (dd, J= 10.2, 2.0 Hz, 1H), 7.31 (brdd,7=8.3, 1.8 Hz, 1H), 7.25 (s, 1H), 6.90 (dd, ABC system component, J= 8.8, 6.7 Hz, 1H), 6.87-6.80 (m, 2H), 4.79 (s, 2H), 4.60 (t, J= 4.8 Hz, 2H), 3.90 (br d, J= 12.3 Hz, 2H), 3.74 (dd, J= 5.3, 4.2 Hz, 2H), 3.38 (br dd, J= 12.6, 12.5 Hz, 2H), 3.28 ( s, ZN), 3.10 (tt, J= 11.9, 4.0 Hz, 1H), 2.37-2.11 (m, 4H); 566.04 22 Examples 1 and 2; P12, P5 8.37 (d, J= 1.5 Hz, 1H), 8.07 (dd, J= 8.5, 1.5 Hz, 1H), 7.79 (d, J= 8.6 Hz, 1H), 7.60-7.54 (m, 2H), 7.50-7.42 (m, 3H), 6.98 (s, 1H), 6.86 (dd, 7=8.1, 8.1 Hz, 1H), 6.61 (dd,7=7.9, 0.9 Hz, 1H), 6.59 (dd, 7= 8.4, 0.9 Hz, 1H), 4.73 ( s, 2H), 4.64 (t, 7= 4.8 Hz, 2H), 3.75 (dd, 7= 5.4, 4.3 Hz, 2H), 3.61-3.44 (m, 8H), 3.28 (s, 3H); 515.1 23 Examples 1 and 2; P12, P6 8.37 (br s, 1H), 8.07 (dd, 7= 8.6, 1.5 Hz, 1H), 7.79 (d, 7= 8.5 Hz, 1H), 7.61-7.54 (m, 2H), 7.51-7.42 (m, 3H), 6.98 (s, 1H), 6.86 (dd, 7= 8.2, 8.1 Hz, 1H), 6.61 (br d, 7= 8 Hz, 1H), 6.59 (br d, 7= 8.5 Hz, 1H), 4.69 (s, 2H), 4.64 (t , 7= 4.9 Hz, 2H), 3.75 (t, 7= 4.9 Hz, 2H), 3.59-3.43 (m, 8H), 3.29 (s, 3H); 515.1 24 Examples 4 and 5; C13, P11 8.33 (dd, 7= 1.5, 0.6 Hz, 1H), 8.03 (dd, 7= 8.5, 1.5 Hz, 1H), 7.79 (dd, 7= 8.5, 0.5 Hz, 1H), 7.62 ( dd, 7= 8.4, 8.3 Hz, 1H), 7.29 (dd, 7= 10.9, 2.0 Hz, 1H), 7.22 (ddd, 7= 8.4, 2.0, 0.7 Hz, 1H), 6.88-6.82 (m, 1H), 6.82-6.76 (m, 2H), 4.83 ( s, 2H), 4.63 (t, 7= 4.8 Hz, 2H), 3.98-3.88 (m, 2H), 3.75 (dd, 7= 5.3, 4.2 Hz, 2H), 3.47-3.36 (m, 2H), 3.31 (s, ЗН ^LL ), 3.10 (tt, 7= 12.0, 4.1 Hz, 1H ), 2.36-2.10 (m, 4H), 2.05 (d, 7= 1.0 Hz, ЗН); 580.14 25 Examples 15 and 16 ² ; C4, P17 8.51 (dd, 7= 1.5, 0.7 Hz, 1H), 8.25 (dd, 7= 8.6, 1.4 Hz, 1H), 7.79 (dd, 7= 8.6, 0.7 Hz, 1H), 7.57 ( dd, 7= 8.3, 8.3 Hz, 1H), 7.25 (dd, 7= 10.8, 2.0 Hz, 1H), 7.19 (ddd, 7= 8.4, 2.0, 0.7 Hz, 1H), 6.80-6.73 (m, 1H), 6.55-6.50 (m, 2H), 4.9- 4.73 (m, 2N ^L ), 3.92-3.81 (m, 2H), 3.66-3.58 (m, 1H), 3.41-3.3 (m, 1H ^LL ), 3.25 (s, 3H), 3.20-3.12 (m, 1H), 3.05-2.97 (m, 1H), 2.70-2.63 (m, 1H), 2.27-2.17 (m, 1H), 2.01 (d,7= 1.0 Hz, ZN), 1.84-1.71 (m, 2H), 1.67-1.58 (m, 2H), 1.31 (br d,7= 13 Hz, 1H); 592.34

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26 Examples 15 and 16 ² ; C4, P17 8.53-8.50 (m, 1H), 8.26 (dd, J= 8.6, 1.4 Hz, 1H), 7.81 (d, J= 8.6 Hz, 1H), 7.55 (dd, 7=8.3, 8.2 Hz, 1H), 7.16-7.08 (m, 2H), 6.77 (dd, J= 8.3, 7.9 Hz, 1H), 6.52 (br d, J= 8.3 Hz, 1H), 6.51 (br d, J= 7.7 Hz, 1H), 4.9-4.74 (m, 2H ^L ), 3.83 (t, J= 4.8 Hz, 2H), 3.68-3.60 (m, 1H), 3.54-3.46 (m, 1H), 3.18-3.09 (m, 1H), 3.14 (s, 3H), 3.09-3.01 (m, 1H), 2.69-2.62 (m, 1H), 2.31-2.21 (m, 1H), 2.01 (br s , 3H), 1.78-1.69 (m, 2H), 1.63-1.52 (m, 2H), 1.33-1.25 (m, 1H); 592.34 27 Examples 4 and 5 ³ ; P11 8.32 (br s, 1H), 8.02 (dd, J= 8.5, 1.5 Hz, 1H), 7.82-7.76 (m, 2H), 7.73 (br d, J= 10.0 Hz, 1H), 7.67 ( br d, J= 8.0 Hz, 1H), 7.35 (s, 1H), 6.96-6.89 (m, 1H), 6.88-6.83 (m, 2H), 4.76 (s, 2H), 4.61 (t, J= 4.8 Hz, 2H), 3.87 (br d, J= 12.3 Hz , 2H), 3.74 (t, J= 4.8 Hz, 2H), 3.39-3.3 (m, 2N ^LL ), 3.29 (s, 3H), 3.15-3.05 (m, 1H), 2.35-2.10 (m, 4H); 557.1 28 Example 11; P14 3.08 min ⁴ ; 596 29 Example 11; P14 3.12 min ⁴ ; 556 30 Example 11; P14 2.90 min ⁴ ; 576 31 Example 11; P14 2.92 min ⁴ ; 546 32 Example 11; P14 2.88 min ⁴ ; 558 33 Example 11; P14 3.04 min ⁴ ; 562 34 Example 11; P14 2.99 min ⁵ ; 553 35 Example 11; P14 2.92 min ⁴ ; 576 36 Example 11; P14 2.81 min ⁵ ; 543 37 Example 11; P14 2.90 min ⁴ ; 558 38 Example 11; P14 2.91 min ⁴ ; 546 39 Example 11; P14 2.89 min ⁴ ; 558 40 Example 11; P14 3.11 min ⁴ ; 596 41 Example 11; P14 2.97 min ⁴ ; 564 42 Example 11; P14 2.40 min ⁵ ; 543 43 Example 12; P10 2.70 min ⁴ ; 621 44 Example 12; P10 2.49 min ⁴ ; 635

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45 Example 12; PIO 2.79 min ⁴ ; 613 46 Example 12; PIO 2.71 min ⁴ ; 635 47 Example 12; PIO 2.85 min ⁴ ; 657 48 Example 12; PIO 2.71 min ⁴ ; 633 49 Example 12; PIO 2.66 min ⁴ ; 607 50 Example 12; PIO 2.43 min ⁴ ; 616 51 Example 12; PIO 2.74 min ⁴ ; 630 52 Example 12; PIO 2.73 min ⁴ ; 593 53 Example 12; PIO 2.79 min ⁴ ; 616 54 Example 12; PIO 2.67 min ⁴ ; 631 55 Example 12; PIO 2.44 min ⁴ ; 630 56 Example 12; PIO 2.77 min ⁴ ; 606 57 Example 12; PIO 2.72 min ⁴ ; 617 58 Example 12; PIO 2.78 min ⁴ ; 603 59 Example 12; PIO 2.82 min ⁴ ; 621 60 Example 12; PIO 2.74 min ⁴ ; 631 61 Example 12; PIO 2.76 min ⁴ ; 592 62 Example 12; PIO 2.45 min ⁴ ; 630 63 Example 12; PIO 2.78 min ⁴ ; 617 64 Example 12; PIO 2.84 min ⁴ ; 606 65 Example 12; PIO 2.56 min ⁴ ; 613 66 Example 12; PIO 2.75 min ⁴ ; 607 67 Example 12; PIO 2.48 min ⁴ ; 619 68 Example 12; PIO 2.75 min ⁴ ; 646 69 Example 12; PIO 2.73 min ⁴ ; 603 70 Example 12; PIO 2.86 min ⁵ ; 661 71 Example 12; PIO 2.77 min ⁴ ; 657 72 Example 12; PIO 2.79 min ⁴ ; 606 73 Example 12; PIO 2.70 min ⁴ ; 602 74 Example 12; PIO 2.45 min ⁴ ; 616 75 Example 13; PIO 2.92 min ⁴ ; 566

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76 Example 13; PIO 2.99 min ⁴ ; 631 77 Example 13; PIO 2.94 min ⁴ ; 616 78 Example 13; PIO 3.08 min ⁵ ; 617 79 Example 13; PIO 3.09 min ⁴ ; 606 80 Example 13; PIO 3.02 min ⁴ ; 603 81 Example 13; PIO 3.10 min ⁵ ; 604 82 Example 11; P14 2.87 min ⁴ ; 528 83 Example 11; P14 3.00 min ⁴ ; 592 84 Example 11; P14 2.99 min ⁴ ; 542 85 Example 11; P14 2.98 min ⁴ ; 542 86 Example 11; P14 2.97 min ⁴ ; 562 87 Example 11; P14 2.97 min ⁵ ; 553 88 Example 11; P14 2.90 min ⁴ ; 542 89 Examples 4 and 5 ^{6 7} ; P11 8.63 (ddd, J= 4.9, 1.8, 0.9 Hz, 1H), 8.34 (dd, J= 1.6, 0.7 Hz, 1H), 8.03 (dd, J= 8.5, 1.5 Hz, 1H), 7.90 (ddd, J= 7.8, 7.8, 1.7 Hz, 1H), 7.80 (dd, 7=8.5, 0.7 Hz, 1H), 7.74 (ddd, 7= 7.9, 1.1, 1.0 Hz, 1H), 7.45 (ddd, 7= 7.6, 4.9, 1.2 Hz, 1H), 6.88-6.83 (w, 1H), 6.83-6.76 (m, 2H), 4.83 (s, 2H), 4.63 (t, 7= 4.8 Hz, 2H), 3.99-3.88 (m, 2H), 3.75 (dd, 7= 5.3, 4.2 Hz, 2H), 3.45-3.34 (w, 2H), 3.31 (s, ZN), 3.15-3.03 (m, 1Н), 2.41-2.20 (m, 2Н), 2.192.08 (m, 2Н), 2.05 (s, ЗН); 529.3 90 Examples 4 and 5 ^{6 7} ; P11 8.63 (ddd, 7= 4.9, 1.8, 0.9 Hz, 1H), 8.34 (dd, 7= 1.6, 0.7 Hz, 1H), 8.04 (dd, 7= 8.5, 1.5 Hz, 1H), 7.90 (ddd, 7= 7.8, 7.8, 1.7 Hz, 1H), 7.80 (dd, 7=8.5, 0.7 Hz, 1H), 7.73 (ddd, 7= 8.0, 1.1, 1.0 Hz, 1H), 7.45 (ddd, 7= 7.6, 4.9, 1.2 Hz, 1H), 6.88-6.83 (w, 1H), 6.83-6.75 (m, 2H), 4.83 (s, 2H), 4.63 (t, 7= 4.9 Hz, 2H), 3.98-3.88 (m, 2H), 3.75 (t, 7= 4.8 Hz, 2H), 3.44-3.34 (m, 2H), 3.32 (s, ZN ^LL ), 3.15-3.03 (m, 1Н), 2.40-2.19 (m, 2Н), 2.18-2.08 (m, 2Н), 2.05 (s, ЗН); 529.3 91 Examples 6 and 7; P8, P11 8.59 (d, 7= 2.4 Hz, 1H), 8.26 (d, 7= 1.4 Hz, 1H), 7.96 (dd, 7 = 8.5, 1.5 Hz, 1H), 7.87 (dd, 7= 8.5, 2.5 Hz, 1H), 7.68-7.61 (w, 2H), 6.83-6.75 (m, 1H), 6.75-6.67 (t, 2H), 4.67 (t, 7= 5.2 Hz,

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2H), 4.00 (s, 2H), 3.82 (t, J= 5.1 Hz, 2H), 3.29 (s, ZN), 3.133.05 (m, 2H), 2.81-2.70 (m, 1H), 2.45 -2.34 (m, 2N), 2.01 (s, ZN), 1.98-1.77 (m, 4H); 563.34 92 Examples 6 and 7; P9, P11 8.59 (d, J= 2.3 Hz, 1H), 8.26 (d, J= 1.4 Hz, 1H), 7.96 (dd, J= 8.5, 1.5 Hz, 1H), 7.87 (dd, J= 8.5, 2.5 Hz, 1H), 7.68-7.62 (w, 2H), 6.82-6.76 (m, 1H), 6.74-6.68 (m, 2H), 4.67 (t, J= 5.2 Hz, 2H), 4.00 (s, 2H), 3.82 (t, J= 5.1 Hz, 2N), 3.29 (s, ZN), 3.133.04 (m, 2N), 2.76 (tt, J= 11.8, 4 Hz, 1H), 2.45-2.34 (m, 2H), 2.01 (s, ZN), 1.97-1.78 (m, 4H); 563.34 93 Examples 8 and 9 ⁸ ; P8, P11 8.97 (dd, 7=2.1, 0.9 Hz, 1H), 8.27-8.25 (m, 1H), 8.21 (dd, J= 8.2, 2.1 Hz, 1H), 7.96 (dd, J= 8.5, 1.5 Hz, 1H), 7.81 (dd, J= 8.3, 0.9 Hz, 1H), 7.64 (d, J= 8, 6 Hz, 1H), 6.83-6.77 (m, 1H), 6.76-6.68 (m, 2H), 4.68 (t, J= 5.2 Hz, 2H), 3.95 (s, 2H), 3.83 (t, J= 5.2 Hz, 2H), 3.30 (s, ZN), 3.08-2.99 (m, 2H), 2.79-2.69 (t, 1H), 2.39-2.28 (t, 2H), 2.04 (s, ZN), 1.96-1.76 (t, 4H); 554.4 94 Examples 8 and 9 ⁸ ; P9, P11 8.97 (dd, J= 2.2, 0.9 Hz, 1H), 8.26 (br s, 1H), 8.21 (dd, J= 8.2, 2.1 Hz, 1H), 7.96 (dd, J= 8.4, 1, 4 Hz, 1H), 7.81 (dd, J= 8.2, 0.9 Hz, 1H), 7.64 (br d, J= 8.5 Hz, 1H), 6.83-6.77 (m, 1H), 6.766.69 (m, 2H), 4.68 (t, J= 5.3 Hz, 2H), 3.95 (s, 2H), 3.83 (t, J= 5.2 Hz, 2H), 3.30 (s, 3H), 3.08-2.99 (m, 2H), 2.79-2.69 (m, 1H), 2.39-2.28 (m, 2H), 2.04 (s, 3H), 1.96-1.76 (m, 4H); 554.4 95 Example 10; P8, P15 8.61 (dd, 7=2.5, 0.7 Hz, 1H), 8.41 (s, 1H), 8.33 (dd,7 = 1.6, 0.7 Hz, 1H), 7.97 (dd, 7= 8.5, 1.5 Hz, 1H), 7.88 (dd, 7= 8.5, 2.5 Hz, 1H), 7.66 (dd, 7 = 8.5, 0.6 Hz, 1H), 7.65 (dd, 7 = 8.5, 0.7 Hz, 1H), 6.82-6.77 (m, 1H), 6.76-6.69 (m, 2H), 5.32-5.24 (m, 1H), 4.9-4.83 (m, 1Н ^L ), 4.71 (dd, 7= 15.4, 2.6 Hz, 1H), 4.65-4.58 (m, 1H), 4.48 (ddd, 7= 9.2, 6.0, 5.9 Hz, 1H), 4.03 (AB quartet, 7ab = 13.9 Hz , Avab = 49.7 Hz, 2H), 3.18-3.11 (m, 1H), 3.06-2.98 (m, 1H), 2.87-2.69 (m, 2H), 2.60-2.49 (m, 1H), 2.46-2.31 (m, 2H), 2.02 (s, 3H ), 1.98-1.79 (m, 4H); 574.94 96 Examples 6 and 7 ^9,10 ; p ₁₅ 7.01 min ¹¹ ; 610.54

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97 Examples 6 and 7 ⁹ ,1°; p ₁₅ 7.89 min ¹¹ ; 610.54 98 C54 ¹² 8.25-8.23 (m, 1H), 8.00 (dd, J= 8.5, 1.5 Hz, 1H), 7.89 (d, J= 0.9 Hz, 1H), 7.76 (dd, J= 7.9, 7.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.64 (dd, J = 10.6, 1.5 Hz, 1H), 7.57 (dd, J = 8.0, 1.5 Hz, 1H), 7.14 (d, J = 0.9 Hz, 1H), 6.78 (dd, component ABC system, J = 7.9, 7.8 Hz, 1H), 6.70 (dd, ABC system component, J = 7.8, 1.2 Hz, 1H), 6.66 (br d, component of the ABC system, J = 7.9 Hz, 1H), 5.94 (AB quartet, Dav = 17.2 Hz, Avab = 6.5 Hz, 2H), 3.96 (s, 2H), 3.02-2.92 (m, 2H), 2.74-2.63 (m, 1H), 2.31-2.21 (m, 2H), 2.05 (br s, 3H), 1.80-1.58 (m, 4H); 594.3 99 C54 ¹² 8.23 (d, J= 1.4 Hz, 1H), 8.00 (dd, J= 8.5, 1.5 Hz, 1H), 7.89 (d, J = 0.9 Hz, 1H), 7.76 (dd, J= 7.9, 7.6 Hz, 1H), 7.69 (d, J= 8.5 Hz, 1H), 7.64 (dd, J = 10.6, 1.5 Hz, 1H), 7.57 (dd, J = 8.1, 1.5 Hz, 1H), 7.14 (d, J = 0.9 Hz, 1H), 6.78 (dd, ABC system component, J = 7.8, 7.8 Hz, 1H), 6.70 (dd, ABC system component, J = 7.8, 1.2 Hz, 1H), 6.66 (br d, component of the ABC system, J = 7.9 Hz, 1H), 5.94 (AB quartet, Dav = 17.1 Hz, Avab = 6.6 Hz, 2H), 3.96 (s, 2H), 3.01- 2.92 (m, 2H), 2.74-2.63 (m, 1H), 2.30-2.20 (m, 2H), 2.05 (br s, 3H), 1.80-1.58 (m, 4H); 594.3 100 Example 7, free acid ¹³ ; P3, C29 characteristic peaks: 7.80 (dd, D= 8.5, 6.6 Hz, 1H), 7.59 (dd, D= 8.3, 8.3 Hz, 1H), 7.51 (d, D= 8.6 Hz, 1H), 7.28 (dd, D = 10.9, 2.0 Hz, 1H), 7.21 (brddV=8.4, 2.0 Hz, 1H), 6.83-6.77 (t, 1H), 6.76-6.71 (t, 2H), 5.32-5.23 (t, 1H), 4.99 (dd, D= 15.5, 7, 1 Hz, 1H), 4.79 (dd, D= 15.6, 2.8 Hz, 1H), 4.72-4.63 (t, 1H), 4.47 (ddd, D= 9.1, 6.0, 6.0 Hz, 1H), 4.31 (AV quartet, Jab = 14.4 Hz, Avab = 33.3 Hz, 2H), 3.40 (br d , D = 11.9 Hz, 1H), 2.92-2.65 (w, 4H), 2.82 (AB quartet, Jab = 15.5 Hz, Avab = 37.5 Hz, 2H), 2.612.49 (m, 1H), 2.13-1.87 (t, 4H), 2.04 (s, ZN); 610.04 101 Example 5, free acid ¹³ ;C48, characteristic peaks: 7.79 (dd, J = 8.5, 6.6 Hz, 1H), 7.57 (dd, J = 8.0, 8.0 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.35 (dd, J = 10.2, 1.9 Hz, 1H), 7.30 (br d, J = 8.4 Hz, 1H), 7.22 (s, 1H), 6.88- C29 6.82 (m, 1H), 6.82-6.74 (m, 2H), 5.30-5.21 (m, 1H), 4.95 (dd, J = 15.4, 7.1 Hz, 1H), 4.77(brd, J = 15.1 Hz, 1H), 4.67-4.59 (m, 1H), 4.44 (ddd, J = 9.1, 5.9, 5.9 Hz, 1H), 4.28 (AB quartet, Dav = 14.4 Hz, Avab = 31.7 Hz, 2H), 3.37 (br d, J = 12.3 Hz, 1H ^ΛΛ ), 2.92-2.61 (m, 4H), 2.82 (AB quartet, Dav = 15.6 Hz, Avab = 37.1 Hz, 2H), 2.58-2.47 (m, 1H), 2.12-1.89 (m, 4H); 596.14 102 Examples 4 and 5 ¹⁴ ; P11 8.34 (dd, D = 1.6, 0.7 Hz, 1H), 8.04 (4fD = 8.5, 1.5 Hz, 1H), 7.80 (dd, J = 8.5, 0.7 Hz, 1H), 7.66-7.60 ( m, 2N), 7.46-7.36 (t, ZN), 6.84-6.76 (t, 2H), 6.74 (dd, D= 7.2, 2.0 Hz, 1H), 4.84 (s, 2H), 4.63 (t, D= 4.7 Hz, 2H), 4.01-3.91 ( t, 4H), 3.76 (dd, D= 5.3, 4.2 Hz, 2H), 3.47-3.37 (t, 2H), 3.32 (s, ZN), 3.19-3.08 (t, 1N), 2.412.26 (t, 2N), 2.26-2.13 (t, 2N); 544.2

^l area calculated, peak partially hidden by water peak ^ll area calculated, peak partially hidden by solvent peak ♦a picture with chlorine isotope peaks was observed

1. Racemic methyl ester [methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate] was separated into its constituent enantiomers by SFC [column: Chiral Technologies ChiralCel OD-H, 5 μm; mobile phase: 7:3 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer, ENT-1 (C76), was used in the synthesis of Example 21, and the second eluting enantiomer, ENT-2 (C77), was converted to Example 20. C76 retention time: 5.72 min (column: Chiral Technologies Chiralpak OD-3, 4.6 x 150 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.05% diethylamine; gradient: 5 to 40% B over 5.5 min, then hold at 40% B for 3.0 min; flow rate: 2.5 ml/min). C77 retention time: 6.01 min (analytical SFC conditions identical to those used for C76).

- 108 040816

2. The methyl ester (methyl 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct-1-yl}-1-(2-methoxyethyl)-1/3-benzimidazole-6-carboxylate), obtained by coupling of C4 and P17, was separated into its constituent dioxolane components by SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting isomer, DIAST-1 (C78), was converted to the compound of Example 26; This material was shown by 1H NMR to be an enantiomer of Example 15. The second eluting isomer, DIAST-2 (C79), was used in the synthesis of Example 25; this material was shown by 1H NMR to be an enantiomer of Example 16. C78 retention time: 3.60 min (column: Chiral Technologies Chiralpak AD-3, 4.6 x 100 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B over 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 ml per minute). C79 retention time: 3.82 min (analytical SFC conditions identical to those used for C78).

3. 4-(4-Bromo-1,3-benzodioxol-2-yl)-3-fluorobenzonitrile was prepared by treating 3-fluoro-4-formylbenzonitrile and 3-bromobenzene-1,2-diol with p-toluenesulfonic acid in toluene with removal of water using a Dean-Stark apparatus. This material was then reacted with [1(tert-butoxycarbonyl)piperidin-4-yl](iodo)zinc in the presence of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and copper(T) iodide, followed by ester cleavage using p-toluenesulfonic acid to give the desired 3-fluoro-4[4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]benzonitrile.

4. Analytical HPLC conditions: Column: Waters XBridge C18, 2.1 x 50 mm, 5 μm; Mobile phase A: 0.0375% trifluoroacetic acid in water; Mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; Gradient: 10 to 100% B in 4.0 min; Flow rate: 0.8 ml/min.

5. Analytical HPLC conditions: Column: Waters XBridge C18, 2.1 x 50 mm, 5 μm; Mobile phase A: 0.0375% trifluoroacetic acid in water; Mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; Gradient: 1 to 5% B in 0.6 min; 5 to 100% B in 3.4 min; Flow rate: 0.8 ml/min.

6. tert-Butyl 4-[2-methyl-2-(pyridin-2-yl)-1,3-benzodioxol-4-yl]-3,6-dihydropyridine-1(2H)carboxylate was synthesized from 3-bromobenzene-1,2-diol and 2-ethynylpyridine using the procedure described for the synthesis of C12 in Preparation P7. Hydrogenation over palladium on carbon followed by treatment with hydrogen chloride in ethyl acetate afforded the desired 2-[2-methyl-4(piperidin-4-yl)-1,3-benzodioxol-2-yl]pyridine hydrochloride salt.

7. Racemic methyl ester [methyl 1-(2-methoxyethyl)-2-({4-[2-methyl-2-(pyridin-2-yl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1H-benzimidazole-6-carboxylate] was separated into its constituent enantiomers by SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer ENT-1 (C80) was used in the synthesis of Example 90, and the second eluting enantiomer ENT-2 (C81) was converted to Example 89. C80 retention time: 4.11 min (column: Chiral Technologies Chiralpak AD-3, 4.6 x 100 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B over 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 ml/min). C81 retention time: 4.62 min (analytical SFC conditions identical to those used for C80).

8. The transformation of P8 and P9 into the corresponding cyano-substituted derivatives was carried out using the method described for the synthesis of P4 from P2 in Preparation of P4.

9. Treatment of 1-(4-chloro-2-fluorophenyl)ethanone with trimethyl orthoformate and para-toluenesulfonic acid afforded 4-chloro-1-(1,1-dimethoxyethyl)-2-fluorobenzene, which was reacted with 3-bromo-6-fluorobenzene-1,2-diol in the presence of para-toluenesulfonic acid to give 4-bromo-2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxole. This material was converted to the desired tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4yl]piperidine-1-carboxylate using the method described in Preparation of P1 for the synthesis of P1 from C2.

10. Separation of the dioxolane stereoisomers in 96 and 97 was accomplished using SFC [column: Chiral Technologies Chiralpak IG, 5 μm; mobile phase: 3:1 carbon dioxide/(2-propanol containing 0.2% ammonium hydroxide)]. The first eluting isomer was designated DIAST-1 (96) and the second eluting isomer was designated DIAST-2 (97).

11. Conditions for analytical SFC: Column: Chiral Technologies Chiralpak IG, 4.6x100 mm, 5 µm; Mobile phase: 7:3 Carbon dioxide/(2-propanol containing 0.2% ammonium hydroxide); Flow rate: 1.5 ml/min; Back pressure: 150 bar.

12. tert-Butyl 2-(chloromethyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylate was synthesized from tert-butyl 3-fluoro-4-nitrobenzoate and 1-(1,3-oxazol-2-yl)methanamine using the method described for the synthesis of P11. Subsequent reaction with C54 was carried out using triethylamine to give tert-butyl 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1

- 109 040816 (yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylate, which was separated into its constituent enantiomers using SFC [column: Chiral Technologies ChiralCel OD-H, 5 μm; mobile phase: 55:45 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer ENT-1 (C82) was used in the synthesis of 99, and the second eluting enantiomer ENT-2 (C83) was converted to 98. C82 retention time: 1.47 min (column: Chiral Technologies Chiralpak OD-3, 4.6 x 50 mm, 3 μm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5% B over 0.2 min, then 5 to 40% B over 1.4 min, then hold at 40% B for 1.05 min; flow rate: 4 ml min-1). C83 retention time: 1.85 min (analytical SFC conditions identical to those used for C82).

13. The reaction of 1-bromo-2,3-difluoro-4-nitrobenzene with copper(T) cyanide in 1-methylpyrrolidin-2-one at elevated temperature gave 2,3-difluoro-4-nitrobenzonitrile, which was treated with thionyl chloride and methanol to give methyl 2,3-difluoro-4-nitrobenzoate. This material was converted using C29 to the desired methyl 2-(chloromethyl)-7-fluoro-1[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate by the method described in Preparation of P11 for the synthesis of P11 from methyl 3-fluoro-4-nitrobenzoate.

14. The required [2-phenyl-4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]methanol was synthesized from 2-oxo-2-phenylethyl acetate in a manner similar to that described for the synthesis of C13.

CHO GLP-1R Clone H6 - analysis 1.

GLP-1R-mediated agonist activity was determined by performing a cellular functional assay using a cAMP (cyclic adenosine monophosphate) assay kit (cAMP HI Range Assay Kit; CisBio catalog #62AM6PEJ) by HTRF (homogeneous time-resolved fluorescence), which measures cellular cAMP levels. This assay is a competitive immunoassay between native cAMP produced by cells and exogenous d2-labeled cAMP. Binding of the indicator is visualized with a cryptate-labeled monoclonal anti-cAMP antibody. The specific signal (i.e., energy transfer) is inversely proportional to the concentration of cAMP in either the standard or experimental sample.

The human GLP-1R coding sequence (NCBI reference sequence NP_002053.3, including the naturally occurring Gly168Ser variant) was subcloned into pcDNA3 (Invitrogen) and a cell line stably expressing the receptor was isolated (designated Clone H6). Saturation binding assays (filtration assay technique) using ¹²⁵ I-GLP-1 ₇ _ ₃₆ (Perkin Elmer) showed that plasma membranes derived from this cell line express GLP-1R at high density (K _d : 0.4 nM, B _max : 1900 fmol/mg protein).

Cells were removed from cryopreservation, resuspended in 40 ml Dulbecco's phosphate-buffered saline (DPBS - Lonza Cat # 17-512Q) and centrifuged at 800xg for 5 min at 22°C. The cell pellet was then resuspended in 10 ml of growth medium [DMEM/F12 1:1 mixture with HEPES, L-Gln, 500 ml (DMEM/F12 Lonza catalog #12-719F), 10% heat-inactivated fetal bovine serum (Gibco catalog #16140-071), 5 ml 100X Pen-Strep (Gibco catalog #15140-122), 5 ml 100X L-glutamine (Gibco catalog #25030-081), and 500 μg/ml Geneticin (G418) (Invitrogen #10131035)]. A 1 ml sample of the cell suspension in the growth medium was counted on a Becton Dickinson ViCell to determine cell viability and cell count. per ml. The remaining cell suspension was then diluted with growth medium to deliver 2000 live cells per well using a Matrix Combi Multidrop reagent dispenser, and the cells were dispensed into a white 384-well tissue culture-treated assay plate (Corning 3570). The assay plate was then incubated for 48 h at 37°C in a humidified environment containing 5% carbon dioxide.

Varying concentrations of each compound to be tested (in DMSO) were diluted in assay buffer HBSS (Hank's Balanced Salt Solution) with calcium/magnesium (Lonza/BioWhittaker catalog #10-527F)/0.1% BSA (Sigma Aldrich catalog #A7409-1L)/20 mM HEPES (Lonza/BioWhittaker catalog #17-737E) containing 100 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma catalog #I5879). The final DMSO concentration was 1%.

After 48 h, the growth medium was removed from the wells of the plate and the cells were treated with 20 μl of the compound serially diluted in assay buffer for 30 min at 37°C in a humidified environment containing 5% carbon dioxide. After incubation for 30 min, 10 μl of d2-labeled cAMP and 10 μl of anti-cAMP antibody (both diluted 1:20 in cell lysis buffer as described in the manufacturer's assay protocol) were added to each well of the assay plate. The plates were then incubated at room temperature and after 60 min, the HTRF signal changes were read on an Envision 2104 multichannel plate reader using an excitation wavelength of 330 nm and emission wavelengths of 615 and 665 nm. Raw data were converted to nM cAMP by interpolation from a cAMP standard curve (as described by the manufacturer in the assay protocol) and the percent effect was determined relative to the saturating concentration of the full agonist GLP-17.36 (1 μM) contained in each plate. EC ₅₀ determinations were made from agonist dose-response curves analyzed by a curve-fitting program using a 4-parameter logarithmic dose-response equation.

CHO GLP-1R Clone C6 - analysis 2.

GLP-1R-mediated agonist activity was determined by performing a cellular functional assay using a cAMP HI Range Assay Kit (CisBio catalog #62AM6PEJ) by HTRF (homogeneous time-resolved fluorescence), which measures cellular cAMP levels. This assay is a competitive immunoassay between native cAMP produced by cells and exogenous d2-labeled cAMP. Binding of the indicator is visualized with a cryptate-labeled monoclonal anti-cAMP antibody. The specific signal (i.e., energy transfer) is inversely proportional to the concentration of cAMP in either the standard or experimental sample.

The human GLP-1R coding sequence (NCBI reference sequence NP_002053.3, including the naturally occurring Leu260Phe variant) was subcloned into pcDNA5-FRTTO and a clonal CHO cell line stably expressing the receptor at low density was isolated using the Flp-In™ T-Rex™ System as described by the manufacturer (ThermoFisher). Saturation binding assays (filtration assay technique) using ¹²⁵ I-GLP-1 (Perkin Elmer) showed that plasma membranes derived from this cell line (designated Clone C6) express GLP-1R at low density (Kd: 0.3 nM, B _max : 240 fmol/mg protein) compared to the Clone H6 cell line.

Cells were removed from cryopreservation, resuspended in 40 ml Dulbecco's phosphate-buffered saline (DPBS - Lonza catalog number 17-512Q) and centrifuged at 800 x g for 5 min at 22°C. DPBS was aspirated and the cell pellet was resuspended in 10 ml complete growth medium (DMEM:F12 1:1 mixture with HEPES, L-Gln, 500 ml (DMEM/F12 Lonza catalog #12-719F), 10% heat-inactivated fetal bovine serum (Gibco catalog #16140-071), 5 ml 100X Pen-Strep (Gibco catalog #15140-122), 5 ml 100X L-glutamine (Gibco catalog #25030-081), 700 μg/ml hygromycin (Invitrogen catalog #10687010), and 15 μg/ml blasticidin (Gibco catalog #R21001). 1 ml of the cell suspension sample in growth medium was counted on a Becton Dickinson ViCell to determine cell viability and count the number of cells per ml. The remaining cell suspension was then diluted with growth medium to obtain 1600 viable cells per well using a Matrix Combi Multidrop reagent dispenser, and the cells were dispensed into a white 384-well tissue culture-treated assay plate (Corning 3570). The assay plate was then incubated for 48 h at 37°C in a humidified environment (95% O2, 5% CO2).

Varying concentrations of each compound to be tested (in DMSO) were diluted in assay buffer (HBSS with calcium/magnesium (Lonza/BioWhittaker catalog #10527F)/0.1% BSA (Sigma Aldrich catalog #A7409-1L)/20 mM HEPES (Lonza/BioWhittaker catalog #17-737E) containing 100 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma catalog #15879). The final DMSO concentration was 1%.

After 48 h, the growth medium was removed from the wells of the plate and the cells were treated with 20 μl of the compound serially diluted in assay buffer for 30 min at 37°C in a humidified environment (95% _O2 , 5% _CO2 ). After incubation for 30 min, 10 μl of d2-labeled cAMP and 10 μl of anti-cAMP antibody (both diluted 1:20 in cell lysis buffer as described in the manufacturer's assay protocol) were added to each well of the assay plate. The plates were then incubated at room temperature and after 60 min, HTRF signal changes were read on an Envision 2104 multichannel plate reader using an excitation wavelength of 330 nm and emission wavelengths of 615 and 665 nm. Raw data were converted to nM cAMP by interpolation from a cAMP standard curve (as described by the manufacturer in the assay protocol) and the percent effect was determined relative to the saturating concentration of the full GLP-1 agonist (1 μM) contained in each plate. EC ₅₀ determinations were made from agonist dose-response curves analyzed by a curve-fitting program using a 4-parameter logarithmic dose-response equation.

Table 3 presents the assay data to two (2) significant digits as the geometric mean (EC50) and arithmetic mean (Emax) for the specified number of replicates (Number). An empty cell indicates that no data are available for the given example or Emax was not calculated.

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Table 3

Biological activity of compounds of examples 1-102

Ex. No. Analysis 1 EU ₅₀ (nM) Analysis 1 Yeshah (%) Analysis 1 Quantity Analysis 2 EC ₅₀ (nM) Analysis 2 Emax (%) Analysis 2 Quantity 1 880 99 3 >20000 1 2* 6,6 81 5 260 100 4 3 1,3 94 3 45 120 3 4 1600 87 3 >20000 1 5** 1,3 89 6 23 97 7 6 140 89 7 2400 89 5 u** 0.26 98 3 3.1 93 12 g*** 0,30 92 6 3.6 91 6 2*** 73 88 9 1600 90 4 0.96 99 5 17 96 8 AND 290 78 3 12 29 83 3 690 92 3 13 4,5 95 3 38 110 3 14 7 95 6 79 85 5 15 >18000 100 3 >20000 1 16 7,7 90 3 120 64 3 17 0,079 97 3 1,1 96 4

18 210 97 3 1000 87 3 19 1,2 87 3 25 100 3 20 17 85 3 270 100 3 21 >20000 1 >20000 1 22 >20000 1 23 680 76 3 24 1,4 82 3 49 110 3 25 >20000 1 >20000 1 26 >20000 1 >20000 1 27 61 98 3 1000 100 3 28 480 87 3 29 5.3 87 4 150 93 3 30 45 86 4 1100 77 4 31 190 88 3 1900 65 3 32 18 86 3 450 87 3 33 2.6 85 3 100 86 3 34 7,8 98 3 110 88 3 35 6,6 86 3 170 89 3 36 760 85 3 37 81 100 3 1000 83 3 38 10 87 3 240 73 3 39 200 83 3 40 14 88 3 130 73 3 41 91 78 3 2000 74 2 42 120 93 3 1700 83 3 43 3.5 88 4 65 86 3 44 160 78 4 45 9.9 81 3 220 79 3 46 5.2 95 4 57 96 3 47 42 75 3 1400 76 4 48 14 81 3 280 73 3

- 112 040816

49 230 93 3 50 12 87 4 140 92 4 51 19 80 3 280 81 3 52 32 85 3 570 80 3 53 3.1 87 3 52 84 4 54 18 82 3 160 64 3 55 74 81 3 1100 50 3 56 1,2 87 4 And 81 3 57 15 86 3 500 98 3 58 4 98 3 23 88 4 59 74 85 3 680 53 3 60 15 82 3 240 60 3 61 10 79 3 240 85 3 62 2,2 94 3 82 95 3 63 5.2 91 3 66 96 3 64 9.2 94 3 91 80 3 65 1,2 99 3 AND 99 6 66 51 82 3 850 74 3 67 710 83 3 68 73 89 3 1200 94 3 69 10 100 3 8.3 98 3 70 2.8 100 4 97 100 4 71 6.8 80 4 74 80 3 72 14 76 3 310 80 3 73 1,7 98 3 10 100 3 74 460 90 3 75 65 82 3 1000 71 3 76 0.77 93 3 7.6 100 3 77 53 89 3 1700 92 3 78 4,5 89 4 78 100 3 79 1,4 85 3 21 85 3

- 113 040816

80 1,1 87 3 6.9 96 4 81 29 BY 3 54 BY 3 82 47 83 3 1000 83 3 83 3,4 85 4 44 88 4 84 9.1 93 3 100 86 3 85 230 80 3 86 24 91 z 410 100 3 87 570 89 3 88 17 86 3 360 91 3 89 130 85 3 2900 87 3 90 >20000 1 91 14000 100 3 >20000 1 92 4.2 90 5 72 83 3 93 >6500 84 5 >20000 1 94 12 89 5 360 87 3 220 77 3 >13000 5 96 1,1 85 3 AND 93 4 97 14 86 3 140 93 4 98 50 97 3 440 95 3 99 2.8 99 4 5.4 91 2 100 7.6 99 1 101 19 74 1 102 600 86 4

* Tested as ammonium salt and trifluoroacetate salt ** Tested as ammonium salt and 1,3-dihydroxy-2-(hydroxymethyl)propan-2aminium (Tris) salt and as free acid ** Tested as ammonium salt and free acid ** ^ Tested as formate salt and free acid

All patents, patent applications, and references cited in this specification are hereby incorporated by reference in their entirety.

Claims (14)

CLAIM 1. Compound of formula I or a pharmaceutically acceptable salt thereof, wherein R is F; p is 0 or 1; ring A is phenyl or pyridinyl; m is 0, 1, 2, or 3; each R ¹ is independently selected from halogen, -CN, -O _1-3 alkyl, or -OC _1-3 alkyl, where alkyl O _1-3 alkyl and OC _1-3 alkyl are substituted with 0-3 F atoms; R ² is H or -C _1-3 alkyl, where alkyl is substituted with 0-1 OH; X-L is N-CH2 or cyclopropyl; Y is CH or N; R ⁴ is -C1-3 alkyl, -C0-3 alkylene-C3-6 cycloalkyl, -C0-3 alkylene-R ⁵ or -C1-3 alkylene-R ⁶ , wherein said alkyl may be substituted, when valence permits, with 0-3 substituents, independently selected from 0-3 F atoms and 0-1 substituent selected from -C0-1 alkylene-OR ^O , -SO2-N(R ⁿ ) ₂ , -C(O)N(R ⁿ )2, -N(C =O)(R ⁿ ) and -N(R ⁿ )2; R ⁵ is a 4-6 membered heterocycloalkyl selected from oxetanyl, tetrahydrofuranyl, morpholinyl, 1,3-oxazolidinyl and pyrrolidinyl, wherein said heterocycloalkyl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-1 oxo (=O); - 114 040816 R ⁶ is a 5-6 membered heteroaryl selected from oxazolyl, imidazolyl, pyridinyl, pyrazolyl and triazolyl, wherein said heteroaryl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-2 -C _1-3 alkyl, where the alkyl may be substituted, when valency permits, with 0-3 substituents independently selected from: 0-1 -ORO; each RO is independently H or —C _1-3 alkyl; each R ⁿ is independently H or —C _1-3 alkyl; each of Z ¹ , Z ² and Z ³ is -CR ^Z ; and each R ^Z is independently H, F, Cl, or —CH ₃ ; or 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-{2[methyl(methylsulfonyl )amino]ethyl}-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, which is a compound of formula II II or a pharmaceutically acceptable salt thereof, wherein R is F; p is 0 or 1; ring A is phenyl or pyridinyl; m is 0, 1 or 2; each R ¹ is independently selected from halogen, -CN, -C _1-3 alkyl, or -OC _1-3 alkyl, where C _1-3 alkyl and OC _1-3 alkyl are substituted with 0-3 F atoms; R ² is H or CH ₃ ; X-L is N-CH2 or cyclopropyl; Y is CH or N; Z ³ is -CR ^Z ; And R ^Z is H or -CH ₃ . 3. The compound according to claim 1 or 2, which is a compound of formula III III or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl or pyridinyl; m is 0, 1 or 2; each R ¹ is independently selected from F, Cl or -CN; R ² is H or CH ₃ ; And Y is CH or N. 4. The compound according to any one of claims 1-3, where R ⁴ is -CH2-R ⁵ , where R ⁵ is a 4-5 membered heterocycloalkyl selected from oxetanyl, tetrahydrofuranyl, 1,3-oxazolidinyl and pyrrolidinyl, where said heterocycloalkyl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-1 oxo (=O); or a pharmaceutically acceptable salt thereof. 5. The compound according to any one of claims 1-3, where R ⁴ is -CH2-R ⁶ , where R ⁶ is a 5-membered heteroaryl selected from oxazolyl, imidazolyl, pyrazolyl and triazolyl, where said heteroaryl may be substituted, when valency permits, 0-2 substituents independently selected from: 0-1 -CH3, -CH2CH3 or -CH2CH2OCH3; or a pharmaceutically acceptable salt thereof. 6. A compound according to any one of claims 1 to 5, wherein R ² is CH ₃ , or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 1, which is 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2ylmethyl] - 1 H-benzimidazole-6-carboxylic acid; - 115 040816 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]nineridin-1-yl}methyl)-1-[(2S)-oxetane2 -ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-3-(1,3oxazol-2 -ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl1 H -imidazol-5-yl)methyl]-1 H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-4 -ylmethyl)-1 H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(pyridin3-ylmethyl)- III-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-5 -ylmethyl)-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl1H- 1,2,3-triazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-2 -ylmethyl)-1 H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-2 -ylmethyl)-1 H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro1- [(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 1ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2R)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1 [(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2R)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, or 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 1ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof. 8. The compound according to claim 1, which is or a pharmaceutically acceptable salt thereof. 9. The compound according to claim 1, which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, or - 116 040816 2-({4-[(2K)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[ (2S)oxetan-2-ylmethyl]-1 H-benzimidazol-6-carboxylic acid; or a pharmaceutically acceptable salt thereof, wherein the salt is a tris salt. 10. The compound according to claim 1 which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1-yl}methyl) -1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2: or a pharmaceutically acceptable salt thereof. I. The compound according to claim 1, which is a crystalline form (Form I) of anhydrous 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt 2-({4-[(28)-2-(4 -chloro-2-fluorophenyl)-2-methyl1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6carboxylic an acid having an x-ray powder diffraction pattern (CuKa radiation) containing at least two characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5+0.2°; 10.1+0.2°; 14.7+0.2° and 16.9+0.2° in units of 20. 12. The compound according to claim 1, which is a crystalline form (Form A) of anhydrous 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt of 2-({4-[2-(5-chloropyridine-2 -yl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST- X2 having an x-ray powder diffraction (CuKa radiation) pattern containing at least two characteristic peaks selected from peaks at 7.7+0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in units of 20. 13. A pharmaceutical composition containing (1) a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, and (2) a pharmaceutically acceptable excipient. 14. Use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, for the treatment of a disease or disorder selected from the group consisting of T2DM (type 2 diabetes mellitus), hyperglycemia, insulin resistance, hepatic insulin resistance, intolerance to glucose, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, obesity, dyslipidemia, hyperinsulinemia, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), fibrosis, cirrhosis and hepatocellular carcinoma. 15. The use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, for the treatment of a disease or condition for which a GLP-1R (glucagon-like peptide-1 receptor) agonist is indicated.