HK1149261B - Pyrrolidine derivatives - Google Patents

Description

Pyrrolidine compounds

This application claims priority from U.S. provisional patent application No.61/033,877, filed on 5.3.2008, the contents of which are incorporated herein by reference in their entirety.

Background

Dipeptidyl peptidase-IV (DPP-IV) is a member of the prolyl peptidase family that cleaves specific dipeptides in the penultimate position from the amino terminus of proteins. Dipeptidyl peptidase-IV causes rapid degradation of glucagon-like peptide-1 (GLP-1), wherein glucagon-like peptide-1 is an intestinal hormone produced by intestinal endocrine L-cells (intestinal endocrine L-cells) upon ingestion of food.

GLP-1 then inhibits glucagon secretion and stimulates glucose-dependent insulin release from the pancreas (Zander M, et al. Lancet 2002, 359: 824-. Inhibition of DPP-IV has been reported to result in increased insulin secretion, decreased blood glucose levels, and improved islet beta-cell function (Pederson R.A., et al. diabetes 1998, 47: 1253-. DPP-IV inhibitors are thus candidates for potential agents for the treatment of type ii diabetes.

Recent studies have shown that DPP-IV inhibitors are potential inhibitors of dipeptidyl peptidase VIII (DPP-VIII), another member of the prolyl peptidase family, whereas inhibition of DPP-VIII leads to side effects such as toxicity and thrombocytopenia (Diabetes, 2005, 54: 2988-. Thus, DPP-IV inhibitors are preferably those having little or no inhibitory activity against DPP-VIII as candidates for agents for treating type II diabetes.

Disclosure of Invention

The present invention is based on the surprising discovery that pyrrolidine compounds (pyrrolidine compounds) have the efficacy of inhibiting DPP-IV.

In one aspect, the present invention relates to pyrrolidine compounds of the formula (I):

wherein R is¹、R²、R³、R⁴、R⁵And R⁶Each independently hydrogen, halogen (halo), nitro, cyano, amino, hydroxy, alkyl, haloalkyl, alkoxy, aryloxy, aralkyl, cyclyl, heterocyclyl, aryl or heteroaryl; r⁷Is alkyl or heteroaryl, and R⁸Is hydrogen or alkyl; or R⁷And R⁸Together with the nitrogen atom to which they are bound form a 3-to 10-membered monocyclic or bicyclic ring, optionally via halogen, CN, NO₂-OR ', alkyl, aryl, heteroaryl, haloalkyl, hydroxyalkyl, alkoxyalkyl, -C (O) R', -SR ', -S (O) R', -S (O)₂R ', -NR ' R ", -C (O) OR ', -C (O) NR ' R", -OC (O), R ', -NR ' C (O) R ", -NR ' C (O) OR", OR-R ' C (O) NR "R '; r ', R "and R'" are each independently hydrogen, alkyl or aryl; m and n are each independently 0, 1, 2 or 3; x is NR^aWherein R is^aIs hydrogen, alkyl or aryl.

The compounds of formula (I) may also have one or more of the following characteristics: x is NH; m is 1; n is 1; each R¹And R²Is H; each R³And R⁴Is alkyl (such as methyl); each R⁵And R⁶Is H; and R⁷And R⁸Is alkyl, or R⁷And R⁸Together with the nitrogen atom to which they are bound form a 3-to 10-membered monocyclic or bicyclic ring, optionally via halogen, CN, NO₂-OR ', alkyl, aryl, heteroaryl, haloalkyl, hydroxyalkyl, alkoxyalkyl, -C (O) R', -SR ', -S (O) R', -S (O)₂R’、-NR’R”、-C(O)OR’A saturated OR unsaturated ring substituted by, -C (O) NR 'R ", -OC (O) R', -NR 'C (O) R", -NR' C (O) OR ", OR-R 'C (O) NR" R'; r ', R "and R'" are each independently hydrogen, alkyl or aryl. Examples of such rings include, but are not limited to, saturated or unsaturated pyrrolidinyl (pyrrolidinyl), thiazolidinyl (thiazolidinyl), piperidinyl (piperidyl), morpholinyl (morpholinoyl), thiomorpholinyl (thiomorpholinyl), piperazinyl (piperidyl), 1, 2, 3, 6-tetrahydropyridinyl (1, 2, 3, 6-tetrahydropyridinyl), isoindolyl (isoindolinyl), and 7-azabicyclo [2.2.1 ] 7]Heptane-7-yl (7-azabicyclo [ 2.2.1)]heptan-7-yl)。

Another aspect of the present invention relates to a pyrrolidine compound of the following formula (II):

wherein R is¹Is H or CN; r²、R³、R⁴、R⁵And R⁶Each independently hydrogen, halogen, nitro, cyano, amino, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, aralkyl, cyclyl, heterocyclyl, aryl, or heteroaryl; r⁷Is hydrogen, alkyl, hydroxyalkyl or alkoxyalkyl; m is 0, 1, 2, 3, 4 or 5; n is 0, 1 or 2; w is CR^aR^a’、NR^aO or S, wherein R^aAnd R^a' are each independently hydrogen, halogen, alkyl or aryl; and X is O, S or CR^b(NR^b’^b") wherein R is^b、R^b' and R^bEach "is independently hydrogen, alkyl or aryl.

The compound of formula (II) may also include one or more of the following features: w is CR^aR^a’；R¹Is CN; x is CH (NH)₂) (ii) a n is 1; r³And R⁴Each independently is hydrogen or alkyl; each R⁵And R⁶Is H; and R⁷Is alkyl (e.g. methyl)Hydroxyalkyl (e.g., hydroxymethyl), or alkoxyalkyl (e.g., methoxymethyl).

The following are exemplary compounds of the invention:

the term "alkyl" refers to straight or branched hydrocarbon groups, which include 1-10 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. The term "alkoxy" refers to-O-alkyl. The term "alkoxyalkyl" refers to an alkyl group substituted with one or more alkoxy groups. The term "haloalkyl" refers to an alkyl group substituted with one or more halogens. The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups.

The term "aryl" refers to C₆-monocyclic aromatic ring structure, C₁₀-a bicyclic aromatic ring structure, C₁₄Tricyclic aromatic ring structures, wherein each ring may have 1 to 4 substituents. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. The term "aryloxy" refers to-O-aryl. The term "aralkyl" refers to an alkyl group substituted with an aryl group.

The term "cyclyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group having from 3 to 12 carbon atoms. Examples of cyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term "heteroaryl" refers to aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic structures having one or more heteroatoms (e.g., O, N or S). Examples of heteroaryl groups include pyridyl (pyridyl), furyl (furyl), imidazolyl (imidazoyl), benzimidazolyl (benzimidazolyl), pyrimidinyl (pyrimidinyl), thienyl (thienyl), quinolyl (quinolyl), indolyl (indolyl), and thiazolyl (thiazolyl). The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group.

The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic structure having one or more heteroatoms (e.g., O, N or S). Examples of heterocyclyl groups include, but are not limited to, piperazinyl (piperazinyl), pyrrolidinyl (pyrrolidinyl), dioxanyl (dioxanyl), morpholinyl (morpholinonyl), and tetrahydrofuranyl (tetrahydrofuranyl).

The alkyl, cyclic, heterocyclic, aryl, heteroaryl, aralkyl, heteroaralkyl, alkoxy, and aryloxy groups described herein may include substituted and unsubstituted groups. Examples of substituents include, but are not limited to, halogen, hydroxyl, amino, cyano, nitro, thiol (mercapto), alkoxycarbonyl (alkyloxycarbonyl), amido (amidoo), carboxyl (carboxy), alkylsulfonyl (alkanesulfonyl), alkylcarbonyl (alkylcarbonyl), ureido (carbomid), carbamoyl (carbomyl), carboxyl (carboxyl), thioureido (thioureido), thiocyanato (thiocyanato), sulfonamido (sulfonimide), alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cyclic, heterocyclic, wherein alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cyclic, and heterocyclic may be further substituted.

Monocyclic rings described herein are substituted or unsubstituted, but may not be fused to another aromatic or non-aromatic ring.

The pyrrolidine compounds described above include pharmaceutically acceptable salts and prodrugs thereof, as appropriate. The salts may be formed between a positively charged ionic group (e.g., ammonium group) and a negatively charged counterion (e.g., trifluoroacetate) in the pyrrolidine compound. Similarly, negatively charged ionic groups (e.g., carboxylate salts) in pyrrolidine compounds can also form salts with positively charged counterions (e.g., sodium, potassium, calcium, or magnesium). The pyrrolidine compounds may include a non-aromatic double bond and one or more asymmetric centers. Thus, they may be racemic mixtures (racemic mixures), single enantiomers, individual diastereomers, mixtures of diastereomers, and cis (cis-) or trans (trans-) isomeric forms. All of these isomers are contemplated.

The pyrrolidine compounds are useful for inhibiting DPP-IV. Accordingly, another aspect of the present invention relates to a method for inhibiting DPP-IV using one or more pyrrolidine compounds. The compounds of the present invention are also useful for treating type II diabetes, since inhibition of DPP-IV results in a decrease in blood glucose levels and an increase in insulin secretion. Accordingly, the invention also includes a method of treating type II diabetes by administering to a subject in need thereof an effective amount of one or more pyrrolidine compounds.

The scope of the invention also includes a pharmaceutical composition comprising one or more of the pyrrolidine compounds described above, the use of such compositions in the treatment of type ii diabetes, and the preparation of medicaments for use in the foregoing treatments.

The details of various embodiments of the invention are disclosed below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Detailed Description

The pyrrolidine compounds of the invention can be synthesized by methods well known in the art. Exemplary methods for synthesizing these compounds are shown in schemes 1-3 below.

Scheme 1

Scheme 1 shows the synthetic route for the compounds of formula (I). The starting material (A) is an N-terminally protected beta-amino acid which is reacted with an amine (W) in the presence of a coupling agent (e.g., N- (3-dimethylaminopropyl) -N-ethylcarbodiimide, EDC) and subsequently deprotected to give an amide (B) having a free amino group. Next, the amide (B) is coupled with pyrrolidine (C) to form the target compound (D). The N-terminally protected beta-amino acids (A) and the pyrrolidines (C) can be prepared by known methods. Please see, e.g., j.med.chem.2006, 49, 373; med chem.1988, 31, 92; med chem.2002, 45, 2362; and bioorg.med.chem.2004, 12, 6053.

(scheme 2)

Scheme 2 shows the synthetic route for the compound of formula (II). In this scheme, the starting material is an amino-substituted dicarboxylic acid (K) in which the amino group and one of the two carboxyl groups are protected. The compound (K) is coupled with an amine to produce a compound (L), which is then hydrolyzed to obtain an acid (M). The acid (M) is coupled with L-prolinamide to obtain the compound (N). After the compound (N) is subjected to dehydration reaction, the amino protecting group is removed to obtain the target product (O). Certain compounds used in the above syntheses may be prepared by methods well known in the art. See, e.g., bioorg.med.chem.2004, 12, 6053.

The above-described flow diagrams are merely illustrative. The skilled artisan can modify or not modify the schemes of the schemes to synthesize all pyrrolidine compounds of the invention. Synthetic chemical Transformations and protecting group methodologies (protection and deprotection) for the synthesis of pyrrolidine compounds are known in the art and are described, for example, in r.larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3^ndEd, John Wiley and Sons (1999); fieser and M.Fieser, Fieser and Fieser' sRegenes for Organic Synthesiss, John Wiley and Sons (1994); paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and its successors.

The pyrrolidine compound thus obtained can be further purified by column chromatography, high pressure liquid chromatography or crystallization.

The present invention encompasses a method of inhibiting DPP-IV by contacting it with an effective amount of one or more pyrrolidine compounds described above. The invention also encompasses a method of treating type II diabetes by administering to a subject in need thereof an effective amount of one or more pyrrolidine compounds described above. The term "treating" refers to the application or administration of a pyrrolidine compound to a subject having, exhibiting symptoms of, or susceptible to type ii diabetes to achieve a cure, alleviate, alter, remove, ameliorate, enhance, or affect the symptoms of type ii diabetes, or susceptible to type ii diabetes. The term "effective amount" refers to an amount of the pyrrolidine compound that produces the desired therapeutic effect in the recipient to which it is administered. As known to those skilled in the art, effective dosages will vary depending upon the route of administration, the use of excipients, and the potential for combining other therapeutic agents (e.g., the use of other active agents).

To practice the treatment methods of the present invention, pharmaceutical compositions having one or more of the above pyrrolidine compounds may be administered parenterally, orally, nasally, rectally, topically, sublingually, or the like. "parenteral administration" as used herein refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, aortic, intrasynovial, thoracic, intraspinal, intralesional or intracranial injection, as well as other suitable infusion techniques.

Sterile injectable compositions may be solutions or suspensions in nontoxic intravenous diluents or solvents, such as 1, 3-butanediol. Among the acceptable carrier and solvent types that may be employed are mannitol (mannitol) and water. In addition, fixed oils are conventionally used as solvents or suspending media (e.g., synthetic mono-or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions (polyoxyethylated). These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants (e.g., Tweens or Spans) or other similar emulsifying agents or biopotency enhancers (commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms) may also be used for formulation purposes.

The composition for oral administration is any orally acceptable dosage form comprising: capsules, tablets, emulsifier and aqueous suspension, dispersant and solvent. In the case of tablets, the carrier used is typically lactose or corn starch. Lubricating agents, such as magnesium stearate, are also typical additives. For oral administration in capsule form, effective diluents include lactose and dried corn starch. When administered orally as an aqueous suspension or emulsion, the active ingredient may be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, suitable sweeteners, flavors or colors may be added.

Nasal aerosol or inhalant compositions may be prepared according to techniques known in the art of pharmaceutical formulation. For example, the composition may be prepared as a solution in saline, employing benzyl alcohol (benzyl alcohol) or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Compositions with active pyrrolidine compounds may also be administered rectally in the form of suppositories.

The carrier in the pharmaceutical composition must be "acceptable" in the sense that it must be compatible with the active ingredient in the composition (and more preferably, have the function of stabilizing the active ingredient) and not injurious to the recipient to be treated. One or more solubilizing agents may serve as pharmaceutical excipients for delivery of the active pyrrolidine compounds. Other examples of carriers include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate and D & C Yellow # 10.

The pyrrolidine compounds of the invention can be used alone or in combination with another diabetes drug for the treatment of type ii diabetes. Examples of diabetes drugs include, but are not limited to, insulin secretagogues (sulfonylureas or benzoic acid derivatives), insulin sensitizers (thiazolidinediones), biguanides, or α -glucosidase inhibitors.

Pyrrolidine compounds of the invention can be initially screened for one or more therapeutic activities (e.g., inhibition of DPP-IV) via in vitro assays. The compounds with high activity in the primary screening can be further screened for their efficacy by in vivo analysis. For example, a test compound can be administered to an animal (e.g., a rat model) suffering from type II diabetes to achieve its therapeutic effect. Appropriate dosage ranges and routes of delivery may also be assessed based on the results of the assay.

The following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, practice the present invention in its entirety. All publications cited herein are incorporated herein by reference in their entirety.

EXAMPLE 1 Synthesis of (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino) -acetyl ] -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-pyrrolidinin-1-yl-propyllamino) -acetyl ] -4-fluoro-pyrrolidinin-2-carbonitrile, Compound 1]

(1) Preparation of 3-amino-3-methyl-1-pyrrolidin-1-yl-butan-1-one, trifluoroacetic acid [ 3-amino-3-methyl-1-pyrrolidinin-1-yl-butan-1-one, trifluoric acid ]

Adding N-ethyl-N' - (3-dimethyl)Aminopropyl) carbodiimide hydrochloride (EDC, 0.77g, 4mmol) was added to 3- (tert-butoxycarbonylamino) -3-methylbutyric acid (3- (tert-butoxycarbylamino) -3-methylbutanamino-3-methylbutanamic acid, 0.87g, 4mmol), pyrrolidine (0.28g, 4mmol) and 1-hydroxybenzotriazole hydrate (HOBt hydrate, 0.54g, 4mmol) in CH₂Cl₂(10 mL). The reaction mixture was stirred at room temperature for 12 hours and with CH₂Cl₂Diluted (40mL), and washed with a saturated aqueous solution of sodium hydrogencarbonate (20mL), a 0.5N aqueous solution of citric acid (20mL) and brine (20 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product as a thick oil. The crude product was purified by flash chromatography (silica gel, 40% EtOAc in hexane) to give N-Boc (protecting group) -protected amine (1.03g) as a colorless oil.

A solution of the above amine in trifluoroacetic acid (TFA, 2mL) was stirred at room temperature for 10 minutes and concentrated under vacuum to give 3-amino-3-methyl-1-pyrrolidin-1-yl-butan-1-one trifluoroacetic acid (3-amino-3-methyl-1-pyrrolidinin-1-yl-butan-1-one trifluoric acid, 1.08g, 95% overall yield) as a colorless oil, which was used in the next step without further purification.

(2) Preparation of (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino) -acetyl ] -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-pyrrolidinin-1-yl-pyrrolidino) -acetyl ] -4-fluoro-pyrrolidinine-2-carbonitrile, Compound 1]

Potassium carbonate (0.55g, 4mmol) was added to a stirred solution of 3-amino-3-methyl-1-pyrrolidin-1-yl-butan-1-one trifluoroacetic acid (0.28g, 1mmol) dissolved in anhydrous tetrahydrofuran (5 mL). After stirring at room temperature for 1 hour, the mixture was filtered through Celite (Celite pad) and washed with ethyl acetate (5 mL). (2S, 4S) -1- (2-Bromoacetyl) -4-fluoro-2-cyano-pyrrolidine ((2S, 4S) -1- (2-bromoacetyl) -4-fluoropyrrolidine-2-carbonitrile, 0.12g, 0.5mmol) was added to the filtrate, and the reaction mixture was stirred under nitrogen at room temperature for 12 hours. Removing most of the solvent under reduced pressure, and using CH as residue₂Cl₂(20mL) and H₂The phases were separated O (5 mL). Reuse of CH in aqueous layer₂Cl₂Extraction was performed (10 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product as a thick oil. By chromatography (silica gel, 4 to 10% CH)₃OH/CH₂Cl₂Gradient), the crude product was purified to obtain compound 1 as a white solid (0.12g, 74% overall yield).

¹H NMR(CDCl₃300MHz, δ): (2/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 5.53(d, J ═ 9.0Hz, 1/3H), 5.49(t, J ═ 3.3Hz, 1/3H), 5.40(t, J ═ 3.3Hz, 1/6H), 5.32(t, J ═ 3.3Hz, 1/3H), 5.22(t, J ═ 3.3Hz, 1/6H), 4.95(d, J ═ 9.0Hz, 2/3H), 4.04-3.52(m, 2H), 3.48-3.39(m, 6H), 2.70(t, J ═ 15.9Hz, 1/3H), 2.62(t, J ═ 15.9Hz, 2/3H), 2.44-2.26(m, 3H, pplet 2.1, 1.79), 1.79 g (m, 3H, 1.79H);

MS(ES⁺)m/z calcd.for C₁₆H₂₅FN₄O₂：324.39；found：325.2(M+H)，347.2(M+Na).

EXAMPLE 2 Synthesis of (2S, 4S) -1- [2- (1, 1-dimethyl-3-morpholin-4-yl-3-oxo-propylamino) -acetyl ] -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- [2- (1, 1-dimethyl-3-morpholino-4-yl-3-oxo-propyllamino) -acetyl ] -4-fluoro-pyrolidine-2-carbonitrile, Compound 2]

The preparation of compound 2 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixtures of trans- (trans-)/cis- (cis-) amide isomers) 5.50(t, J ═ 3.3Hz, 1/3H), 5.42(t, J ═ 3.3Hz, 1/6H), 5.38(d, J ═ 9.0Hz, 1/3H), 5.32(t, J ═ 3.3Hz, 1/3H), 5.23(t, J ═ 3.3Hz, 1/6H), 4.94(d, J ═ 9.0Hz, 2/3H), 4.12-3.35(m,12H，overlapped singlet at 3.41)，2.70(t，J＝15.6Hz，1/3H)，2.65(t，J＝15.6Hz，2/3H)，2.50-2.18(m，3H)，1.20(s，6H)；

MS(ES⁺)m/z calcd.for C₁₆H₂₅FN₄O₃：340.39；found：341.2(M+H)，363.2(M+Na).

EXAMPLE 3 Synthesis of 3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethylamino ] -3-methyl-N-pyridin-3-yl-butanamide [3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethyl-lamino ] -3-methyl-N-pyrindin-3-yl-butanamide, Compound 3]

The preparation of compound 3 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (3/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 11.12(brs, 3/4H), 11.01(brs, 1/4H), 8.66-8.63(m, 1H), 8.28(d, J ═ 4.5Hz, 1H), 8.22-8.14(m, 1H), 7.26-7.21(m, 1H), 5.53(t, J ═ 3.3Hz, 3/8H), 5.46(t, J ═ 3.3Hz, 1/8H), 5.36(t, J ═ 3.3Hz, 3/8H), 5.29(t, J ═ 3.3Hz, 1/8H), 4.98(d, J ═ 9.3Hz, 3/4H), 4.80(d, J ke ═ 9.3Hz, 1/4H), 3.97 (t, 3.3H), 19.7H, 367H), 4.98 (t, J ═ 9.3Hz, 3H, 3/4H), 4.80(d, J ke ═ 9.3, 1/4H, 3, 3.97, 3.2H, 19H, 362, 16, 16.79H, 16H, 3671H, j ═ 15.3Hz, 3/4H), 2.50-2.39(m, 3H, overlapped2singlet at 2.45, 2.44), 1.26(s, 3H), 1.24(s, 3H);

MS(ES⁺)m/z calcd.for C₁₇H₂₂FN₅O₂：347.39；found：348.2(M+H)，370.2(M+Na).

EXAMPLE 4 Synthesis of (2S, 4S) -4-fluoro-1- {2- [3- ((R) -3-hydroxy-pyrrolidin-1-yl) -1, 1-dimethyl-3-oxo-propylamino ] -acetyl } -2-cyano-pyrrolidine [ (2S, 4S) -4-fluoro-1- {2- [3- ((R) -3-hydroxy-pyrrolidin-1-yl) -1, 1-dimethyl-3-oxo-propyllamino ] -acetyl } -pyrrolidine-2-carbonitrile, Compound 4]

The preparation of compound 4 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 5.50(t, J ═ 3.3Hz, 1/3H), 5.40(t, J ═ 3.3Hz, 1/6H), 5.38(d, J ═ 9.0Hz, 1/3H), 5.32(t, J ═ 3.3Hz, 1/3H), 5.23(t, J ═ 3.3Hz, 1/6H), 4.94(d, J ═ 9.0Hz, 2/3H), 4.47-4.21(m, 1H), 3.99-3.22(m, 8H), 2.84(brs, OH), 2.69(t, J ═ 15.3Hz, 1/3H), 2.61(t, J ═ 15.3, 2/3H), 2.50-2.15(m, 3H), 2.04 (m, 3H), 2.1H, 1.3H, 89(m, 1H), 1.3H, 1H);

MS(ES⁺)m/z calcd.for C₁₆H₂₅FN₄O₃：340.39；found：341.2(M+H)，363.2(M+Na).

EXAMPLE 5 Synthesis of (2S, 4S) -4-fluoro-1- (2- (2-methyl-4- (4- (methylsulfonyl) piperazin-1-yl) -4-oxobutan-2-ylamino) acetyl) -2-cyano-pyrrolidine [ (2S, 4S) -4-fluoro-1- (2- (2-methyl-4- (4- (methylsulfonyl) piperazin-1-yl) -4-oxobutan-2-ylamino) acetyl) pyrolidine-2-carbonitrile, Compound 5]

The preparation of compound 5 is analogous to that described in example 1.

EXAMPLE 6 Synthesis of (2S, 4S) -4-fluoro-1- (2- (4- ((R) -3-fluoropyrrolidin-1-yl) -2-methyl-4-oxobutan-2-ylamino) acetyl) -2-cyano-pyrrolidine [ (2S, 4S) -4-fluoro-1- (2- (4- ((R) -3-fluoropyrrolidin-1-yl) -2-methyl-4-oxobutan-2-ylamino) acetyl) pyrolidine-2-carboninic Trile, Compound 6]

The preparation of compound 6 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (3/1 mixtures of trans- (trans-)/cis- (cis-) amide isomers) 5.50(t, J ═ 3.0Hz, 3/8H), 5.41(t, J ═ 3.0Hz, 1/8H), 5.3(t, J ═ 3.0Hz, 3/8H), 5.25(t, J ═ 3.0Hz, 1/8H), 5.23(d, J ═ 9.3Hz, 1/4H), 4.92(d, J ═ 9.3Hz, 3/4H), 4.00-3.40(m, 8H, overlappe)d singlet at 3.46)，3.31-3.20(m，4H)，2.80(s，9/4H)，2.78(s，3/4H)，2.69(t，J＝15.6Hz，1/4H)，2.64(t，J＝15.6Hz，3/4H)，2.54-2.23(m，3H，overlapped singlet at 2.51)，1.24(s，6H)；

MS(ES⁺)m/z calcd.for C₁₇H₂₈FN₅O₄S：417.50；found：418.2(M+H)，440.1(M+Na).

EXAMPLE 7 Synthesis of 3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethylamino ] -3-methyl-N- (5-methyl-thiazol-2-yl) -butanamide [3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethyl-lamino ] -3-methyl-N- (5-methyl-thiazol-2-yl) -butyramide, Compound 7]

The preparation of compound 7 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (3/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 6.48(s, 1H), 5.53(t, J ═ 3.3Hz, 3/8H), 5.45(t, J ═ 3.3Hz, 1/8H), 5.36(t, J ═ 3.3Hz, 3/8H), 5.32(t, J ═ 3.3Hz, 1/8H), 4.89(d, J ═ 9.0Hz, 3/4H), 4.87(d, J ═ 9.0Hz, 1/4H), 4.12-3.63(m, 2H), 3.52-3.39(m, 2H), 2.75(t, J ═ 15.6Hz, 1/4H), 2.70(t, J ═ 15.6, 3/4H), 2.60-2.26 (pph, 6, 2, 31, 22H), 2.31H, 31, 22, 31H);

MS(ES⁺)m/z calcd.for C₁₆H₂₂FN₅O₂S：367.44；found：368.1(M+H)，390.1(M+Na).

EXAMPLE 8 Synthesis of 3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethylamino ] -N- (6-methoxy-pyridin-3-yl) -3-methyl-butanamide [3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethyl-lamino ] -N- (6-methoxy-pyridin-3-yl) -3-methyl-butanamide, Compound 8]

The preparation of compound 8 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (3/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 10.76(brs, 3/4H), 10.59(brs, 1/4H), 8.29-8.26(m, 1H), 7.96(dd, J ═ 9.0, 3.0Hz, 1H), 6.70(d, J ═ 9.0Hz, 1H), 5.54(t, J ═ 3.6Hz, 3/8H), 5.46(t, J ═ 3.6Hz, 1/8H), 5.36(t, J ═ 3.6Hz, 3/8H), 5.29(t, J ═ 3.6Hz, 1/8H), 4.98(d, J ═ 9.0Hz, 3/4H), 4.80(d, J ═ 9.0Hz, 1/4H), 4.13-3.13, ov7H, 4.98(d, J ═ 9.0Hz, 3.79H), 3.79 (16H, 3g ═ 3.7H, 3.79 (16H), j ═ 15.3Hz, 3/4H), 2.49-2.23(m, 3H, overlapped2singlet at 2.44, 2.42), 1.26(s, 3H), 1.24(s, 3H);

MS(ES⁺)m/z calcd.for C₁₈H₂₄FN₅O₃：377.41；found：378.2(M+H)，400.1(M+Na).

EXAMPLE 9 Synthesis of (2S, 4S) -1- {2- [3- (3, 6-dihydro-2H-pyridin-1-yl) -1, 1-dimethyl-3-oxo-propylamino ] -acetyl } -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- {2- [3- (3, 6-dihydro-2H-pyridin-1-yl) -1, 1-dimethyl-3-oxo-propyllamino ] -acetyl } -4-fluoro-2-pyrrolidino } -4-fluoro-pyrrolidine-2-carbonitrile, Compound 9]

The preparation of compound 9 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixtures of trans (trans-)/cis (cis-) amide isomers) 5.89-5.77(m, 1H), 5.69-5.60(m, 1H), 5.49(t, J ═ 3.3Hz, 1/3H), 5.46(d, J ═ 9.3Hz, 1/3H), 5.40(t, J ═ 3.3Hz, 1/6H), 5.31(t, J ═ 3.3Hz, 1/3H), 5.22(t, J ═ 3.3, 1/6H), 4.92(d, J ═ 9.3Hz, 2/3H), 4.02-3.39(m, 8H), 2.67(t, J ═ 15.3Hz, 1/3H), 2.61(t, J ═ 15.3Hz, 2/3H), 2.48-2.12(m, 3H), 1.19H, 1H), 1.18H (s ═ 3H);

MS(ES⁺)m/z calcd.for C₁₇H₂₅FN₄O₂：336.40；found：337.2(M+H)，359.2(M+Na).

EXAMPLE 10 Synthesis of (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-thiazol-3-yl-propylamino) -acetyl ] -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-thiazolidin-3-yl-propyllamino) -acetyl ] -4-fluoro-pyrolidine-2-carbonitrile, Compound 10]

The preparation of compound 10 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 5.52(t, J ═ 3.3Hz, 1/3H), 5.43(t, J ═ 3.3Hz, 1/6H), 5.36(t, J ═ 3.3Hz, 1/3H), 5.33(d, J ═ 9.0Hz, 1/3H), 5.25(t, J ═ 3.3Hz, 1/6H), 4.96(d, J ═ 9.0Hz, 2/3H), 4.57(s, 1H), 4.51(s, 1H), 4.02-3.04(m, 6H, overlappled singat 3.43), 3.10(t, J ═ 6.3Hz, 1H), 3.00(t, J ═ 6.3, 1H), 2.75(t, t ═ 2.3, t ═ 2.75H), 15.75 (t, 15.75H, 865H), 3.47 (t, 8H, 3651H, 3647H, 36H), 1.23(s, 6H);

MS(ES⁺)m/z calcd.for C₁₅H₂₃FN₄O₂S：342.43；found：343.1(M+H)，365.1(M+Na).

EXAMPLE 11 Synthesis of (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-piperidin-1-yl-propylamino) -acetyl ] -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- [2- (1, 1-dimethyl-3-oxo-3-piperidine-1-yl-propyalino) -acetyl ] -4-fluoro-pyrolidine-2-carbonitrile, Compound 11]

The preparation of compound 11 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixtures of trans- (trans-)/cis- (cis-) amide isomers) 5.48(t, J ═ 3.3Hz, 1/3H), 5.45(d, J ═ 9.3Hz, 1/3H), 5.39(t, J ═ 3.3Hz, 1/6H), 5.30(t, J ═ 3.3Hz, 1/3H), 5.22(t, J ═ 3).3Hz，1/6H)，4.93(d，J＝9.3Hz，2/3H)，4.00-3.35(m，8H)，2.67(t，J＝15.6Hz，1/3H)，2.60(t，J＝15.6Hz，2/3H)，2.50-2.20(m，3H，overlapped singlet at 2.41)，1.64-1.44(m，6H)，1.18(s，6H)；

MS(ES⁺)rm/z calcd.for C₁₇H₂₇FN₄O₂：338.42；found：339.2(M+H)，361.2(M+Na).

EXAMPLE 12 Synthesis of ethyl 4- {3- [2- ((2S, S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethylamino ] -3-methyl-butyryl } -piperazine-1-carboxylate [4- {3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethyl-lamino ] -3-methyl-butyryl } -piperazine-1-carboxylic acid ethyl ester, Compound 12]

The preparation of compound 12 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixture of trans- (trans-)/cis- (cis-) amide isomers) 5.51(t, J ═ 3.3Hz, 1/3H), 5.42(t, J ═ 3.3Hz, 1/6H), 5.36(d, J ═ 9.0Hz, 1/3H), 5.34(t, J ═ 3.3Hz, 1/3H), 5.25(t, J ═ 3.3Hz, 1/6H), 4.96(d, J ═ 9.0Hz, 2/3H), 4.16(q, J ═ 6.9Hz, 2H), 4.19-3.38(m, 12H), 2.72(t, J ═ 15.3Hz, 1/3H), 2.65(t, J ═ 15.3Hz, 2/3H), 2.53-2.22(m, 3H, 2 ovt, 2.43H), 2.46 (pperlich), 1.6H, 27H), 1.9H, 27H, 1H, 27H, and 1H.

MS(ES⁺)m/z calcd.for C₁₉H₃₀FN₅O₄：411.47；found：412.2(M+H)，434.2(M+Na).

EXAMPLE 13 Synthesis of 3- [2- ((2S, S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethylamino ] -N-cyclopentyl-3-methyl-butanamide [3- [2- ((2S, 4S) -2-cyano-4-fluoro-pyrrolidin-1-yl) -2-oxo-ethylamido ] -N-cyclopentyl-3-methyl-butyramide, Compound 13]

The preparation of compound 13 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (4/1 mixtures of trans- (trans-)/cis- (cis-) amide isomers) 7.94(d, J ═ 7.2Hz, 4/5H), 7.59(d, J ═ 7.2Hz, 1/5H), 5.51(t, J ═ 3.3Hz, 2/5H), 5.4(t, J ═ 3.3Hz, 1/10H), 5.34(t, J ═ 3.3Hz, 2/5H), 5.25(t, J ═ 3.3Hz, 1/10H), 5.02(d, J ═ 9.0Hz, 1/5H), 4.92(d, J ═ 9.0Hz, 2H), 4.20-4.07(m, 1H), 4.03-3.27 (m, 4H), 2.73(t, J ═ 15.6, 23H, 732H), 15.68 (t, t ═ 1H), 44.50H, 4/5.68H, 44 (m, 1H), 44.48H, 44H, 3.50H, 4H) 1.43-1.31(m, 2H), 1.15(s, 6H).

MS(ES⁺)m/z calcd.for C₁₇H₂₇FN₄O₂：338.42；found：339.4(M+H)，361.4(M+Na).

EXAMPLE 14 Synthesis of (2S, 4S) -4-fluoro-1- {2- [3- ((S) -2-hydroxymethyl-pyrrolidin-1-yl) -1, 1-dimethyl-3-oxo-propylamino ] -acetyl } -2-cyano-pyrrolidine [ (2S, 4S) -4-fluoro-1- {2- [3- ((S) -2-hydroxymethyl-pyrrolidin-1-yl) -1, 1-dimethyl-3-oxo-propyllamino ] -acetyl } -pyrrolidine-2-carbonitile, Compound 14]

The preparation of compound 14 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixtures of trans- (trans-)/cis- (cis-) amide isomers) 5.50(t, J ═ 3.3Hz, 1/3H), 5.41(t, J ═ 3.3Hz, 1/6H), 5.37(d, J ═ 9.3Hz, 1/3H), 5.32(t, J ═ 3.3Hz, 1/3H), 5.22(t, J ═ 3.3Hz, 1/6H), 4.95(d, J ═ 9.0Hz, 2/3H), 4.26-4.19(m, 1H), 4.03-3.37(m, 8H, overlapsignet 3.43), 2.70(t, J ═ 15.6Hz, 1/3H), 2.63(t, J ═ 15.6Hz, 2/3H), 2.55 (OH, 2.55H), 2.70(t, J ═ 15.6Hz, 2/3H), 2.55H, 2.42H, 1.79 (1, 3.5.3H), 6H) (ii) a

MS(ES⁺)m/z calcd.for C₁₇H₂₇FN₄O₃：354.42；found：355.2(M+H)，377.2(M+Na).

EXAMPLE 15 Synthesis of (2S, 4S) -1- {2- [1, 1-dimethyl-3- ((R) -2-methyl-pyrrolidin-1-yl) -3-oxo-propylamino ] -acetyl } -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- {2- [1, 1-dimethyl-3- ((R) -2-methyl-pyrrolidin-1-yl) -3-oxo-propylamino ] -acetyl } -4-fluoro-2-cyano-pyrrolidine } -4-fluoro-pyrrolidine-2-carbonitrile, Compound 15]

The preparation of compound 15 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (2/1 mixtures of trans (trans-)/cis (cis-) amide isomers) 5.52-5.47(m, 2/3H), 5.39(t, J ═ 3.3Hz, 1/6H), 5.31(t, J ═ 3.3Hz, 1/3H), 5.22(t, J ═ 3.3Hz, 1/6H), 4.92(d, J ═ 9.3Hz, 2/3H), 4.19-3.31(m, 6H, overlappedsinglet at 3.39), 2.78-2.19(m, 5H), 2.02-1.83(m, 3H), 1.68-1.49(m, 1H), 1.19-1.12(m, 9H);

MS(ES⁺)m/z calcd.for C₁₇H₂₇FN₄O₂：338.42；found：339.4(M+H)，361.4 (M+Na).

EXAMPLE 16 Synthesis of (2S, 4S) -1- {2- [3- (1, 3-dihydro-isoindol-2-yl) -1, 1-dimethyl-3-oxo-propylamino ] -acetyl } -4-fluoro-2-cyano-pyrrolidine [ (2S, 4S) -1- {2- [3- (1, 3-dihydro-isoindol-2-yl) -1, 1-dimethyl-3-oxo-propyllamino ] -acetyl } -4-fluoro-2-cyano-pyrrolidine-, Compound 16]

The preparation of compound 16 is analogous to that described in example 1.

¹H NMR(CDCl₃300MHz, δ): (3/1 mixtures of trans- (trans-)/cis- (cis-) amide isomers) 7.30-7.16(m, 4H), 5.52(d, J ═ 9.3Hz, 1/4H), 5.50(t, J ═ 3.3Hz, 3/8H), 5.40(t, J ═ 3.3Hz, 1/8H), 5.31(t, J ═ 3.3Hz, 3/8H), 5.23(t, J ═ 3.3Hz, 1/8H), 4.94(d, J ═ 9Hz, 3/4H), 4.83(s, 2H), 4.78(s, 2H), 4.15-3.35(m, 4H, overlaid singet 3.45), 2.70(t,J＝15.3Hz，1/4H)，2.64(t，J＝15.3Hz，3/4H)，2.59-2.18(m，3H)，1.22(s，6H)；

MS(ES⁺)m/z calcd.for C₂₀H₂₅FN₄O₂：372.44；found：373.2(M+H)，395.2(M+Na).

EXAMPLES 17-20 Synthesis of Compounds 17-20

The preparation of compounds 17-20 is analogous to that described in example 1.

EXAMPLE 21 Synthesis of (2S) -1- [2-amino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoyl ] -2-cyano-pyrrolidine trifluoroacetic acid [ (2S) -1- [2-amino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoyl ] -pyrrolidine-2-carbonitrifluoroacetic acid, Compound 21]

(1) Preparation of 2-tert-Butoxycarbonylamino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoic acid methyl ester [ 2-tert-butoxyarylamido-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanecarboxylic acid methyl ester ]

EDC (0.77g, 4mmol) was added to 4- (tert-butoxycarbonylamino) -5-methoxy-5-oxopentanoic acid (4- (tert-butoxyarylamino) -5-methoxy-5-oxopentanoic acid, 1.05g, 4mmol), (S) -2- (methoxymethyl) -pyrrolidine (0.46g, 4mmol) and 1-hydroxybenzotriazole hydrate (HOBt hydrate, 0.54g, 4mmol) in CH₂Cl₂(10 mL). The reaction mixture was stirred at room temperature for 12 hours and with CH₂Cl₂Diluted (40mL), washed sequentially with saturated aqueous sodium bicarbonate (20mL), 0.5N aqueous citric acid (20mL) and brine (20mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure to give the crude product as a thick oil. The crude product was purified by flash chromatography (silica gel, 50% EtOAc in hexane) to afford the title compound (1.36g, 95%) as a colorless oil.

(2) Preparation of 2-tert-Butoxycarbonylamino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoic acid [ 2-tert-butoxyarylamino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoic acid ]

2N aqueous sodium hydroxide (20mL) was added to 20mL methanol (CH)₃OH) methyl 2-tert-butoxycarbonylamino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-valerate (2-tert-butoxyarylamido-5- ((S) -2-methylymethyl-pyrrolidin-1-yl) -5-oxo-pentanic acid methyl ester, 0.72g, 2mmol) in a stirred solution. After stirring at room temperature for 12 hours, a 6N aqueous hydrochloric acid solution was added at 0 ℃, and the mixture was acidified to pH 4. Under reduced pressure, most of the methanol is removed and the residue is freed from CH₂Cl₂(20mL) and H₂O (20mL) was used for phase separation. Reuse of CH in aqueous layer₂Cl₂Extraction was performed (20 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound (0.54g, 79%) as a foamy solid, which was used in the next step without further purification.

(3) Preparation of tert-butyl-1- ((S) -2-carbamoylpyrrolidin-1-yl) -5- ((S) -2- (methoxymethyl) pyrrolidin-1-yl) -1, 5-dioxopentan-2-ylcarbamate [ tert-butyl-1- ((S) -2-carbarylpyrrolidin-1-yl) -5- ((S) -2- (methoxymethyl) pyrrolidin-1-yl) -1, 5-dioxoentan-2-ylcarbamate ]

EDC (0.19g, 1mmol) was added to 2-tert-butoxycarbonylamino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoic acid (2-tert-butoxy carbonamino-5- ((S) -2-methoxy-pyrrolidin-1-yl) -5-oxo-pentanic acid, 0.34g, 1mmol), L-prolinamide (0.11g, 1mmol) and HOBt hydrate (0.14g, 1mmol) in CH₂Cl₂(5 mL). The reaction mixture was stirred at room temperature for 12 hours and with CH₂Cl₂Diluted (20mL), and washed sequentially with saturated aqueous sodium bicarbonate (10mL), 0.5N aqueous citric acid (10mL), and brine (10mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude product as a thick oil. By flash chromatography (silica gel, 2 to 8% CH)₃OH/CH₂Cl₂Gradient), the crude product was purified to obtain the title compound as a foamy solid (0.36g, 81%).

(4) Preparation of (2S) -1- [2-amino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl-5-oxo-pentanoyl ] -2-cyano-pyrrolidine trifluoroacetate [ (2S) -1- [2-amino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoyl ] -pyrrolidine-2-carbonitrifluoroacetic acid, Compound 21]

Phosphoryl chloride (phosphoryl chloride, 0.32g, 2.1mmol) was added to a mixture of tert-butyl-1- ((S) -2-carbamoylpyrrolidin-1-yl) -5- ((S) -2- (methoxymethyl) pyrrolidin-1-yl) -1, 5-dioxopent-2-ylcarbamate (tert-butyl-1- ((S) -2-carbamoylpyrrolidin-1-yl) -5- ((S) -2- (methoxymethyl) pyrrolidin-1-yl) -1, 5-dioxospan-2-ylcarbamate, 0.36g, 0.8mmol) and imidazole (0.68g, 1mmol) in pyridine (4mL) at-20 ℃. The slurry mixture was stirred at-20 ℃ for 1 hour, warmed to room temperature, and concentrated in vacuo. The residue is CH₂Cl₂The phases were separated (10mL) and 0.5N aqueous citric acid (10 mL). Reuse of CH in aqueous layer₂Cl₂Extraction was performed (10 mL). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give a light yellow oil. The crude oil was purified by chromatography (silica gel, 50% ethyl acetate/dichloromethane) to give a colorless oil.

A solution of the above oil and trifluoroacetic acid (TFA, 1mL) was stirred at room temperature for 10 minutes and concentrated under vacuum to afford compound 21 as a foamy solid (0.27g, 77%).

MS(ES⁺)m/z calcd.for C₁₆H₂₆N₄O₃：322.40；found：323.6(M+H)，345.6(M+Na).

EXAMPLE 22 Synthesis of (2S) -1- [2-amino-5- ((S) -2-hydroxymethyl-pyrrolidin-1-yl) -5-oxo-pentanoyl ] -2-cyano-pyrrolidine trifluoroacetic acid [ (2S) -1- [2-amino-5- ((S) -2-hydroxymethy-lin-1-yl) -5-oxo-pentanoyl ] -pyrrolidine-2-carbonitrifluoroacetic acid, Compound 22]

The preparation of compound 22 is analogous to that described in example 21.

MS(ES⁺)m/z calcd.for C₁₅H₂₄N₄O₃：308.38；found：309.1(M+H)，331.1(M+Na).

EXAMPLE 23 Synthesis of (2S) -1- [2-amino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -3, 3-dimethyl-5-oxo-pentanoyl ] -2-cyano-pyrrolidine trifluoroacetic acid [ (2S) -1- [2-amino-5- ((S) -2-methoxymethyl-pyrrolidin-1-yl) -3, 3-dimethyl-5-oxo-pentanoyl ] -pyrrolidine-2-carbonitrile trifluoric acid acetic acid, Compound 23]

The preparation of compound 23 is analogous to that described in example 21.

MS(ES⁺)m/z calcd.for C₁₈H₃₀N₄O₃：350.46；found：351.6(M+H)，373.7(M+Na).

EXAMPLE 24 Synthesis of (2S) -1- [2-amino-5- ((R) -2-methyl-pyrrolidin-1-yl) -5-oxo-pentanoyl ] -2-cyano-pyrrolidine trifluoroacetic acid [ (2S) -1- [2-amino-5- ((R) -2-methyl-pyrrolidin-1-yl) -5-oxo-pentanoyl ] -pyrolidin-2-carbonitrifluoroacetic acid, Compound 24]

The preparation of compound 24 is analogous to that described in example 21.

MS(ES⁺)m/z calcd.for C₁₈H₃₀N₄O₃：292.38；found：293.6(M+H)，315.6(M+Na).

EXAMPLE 25 Synthesis of (2S) -1- (2-amino-5- ((S) -2- (hydroxymethyl) pyrrolidin-1-yl) -3, 3-dimethyl-5-oxopentanoyl) -2-cyano-pyrrolidine trifluoroacetic acid [ (2S) -1- (2-amino-5- ((S) -2- (hydroxymethyl) pyrrolidin-1-yl) -3, 3-dimethyl-5-oxolanyl) pyrrolidine-2-carbonitrile trifluoroacetic acid, Compound 25]

The preparation of compound 25 is analogous to that described in example 21.

Example 26

Using a method similar to Biochemistry, 2006, 45: 7006-7012 DPP-IV is purified from human serum and insect cells.

Using a similar j.biol.chem.2006, 28: 138653-138662 DPP-VIII was purified from baculovirus-infected sfa cells.

The purity of DPP-IV or DPP-VIII is checked by SDS-PAGE, followed by staining with Coomassie Brilliant blue (comassie blue) or silver staining. The concentrations of DPP-IV and DPP-VIII were determined by the Bradford method using BSA as a standard (Bradford, M.M (1976) anal. biochem.72, 248-254.).

Compounds 1-24 were tested for their inhibitory efficacy against DPP-IV as follows: for each compound, eight dilutions in sequence (final concentration: 0.0046-10. mu. mol/l) were prepared and used. Mu.l of DPP-IV (in Tris, 40mM, pH 8.3) and 10. mu.l of test compound (in Tris containing 1% DMSO) were incubated in 37 ℃ dishes for 10 minutes. Mu.l of Gly-Pro-7-amino-4-methylcoumarin was added to the above solution (final concentration of 150. mu.M) and incubated at 37 ℃ for 1 hour. Continuous observation of 96-well fluorescent discs (Victor)²V) release of 7-amino-4-methylcoumarin and record data of the end point of the inhibition reaction. From the result, the IC is calculated₅₀The value is obtained.

Compounds 1-24 were likewise tested for their inhibitory efficacy against DPP-VIII. PBS (137mM NaCl, 2.7mM KCl, 1.4mM KH)₂PO₄，4.3mM Na₂HPO₄pH7.4) and 1. mu.l of test compound in DMSO at 37 ℃ for 10 minutes. Mu.l Gly-Pro-p-nitroaniline (final concentration 2.5mM) was added. The resulting solution was incubated at 37 ℃ for 30-45 minutes. The reaction was observed and measured at OD 405 nm. From the result, the IC is calculated₅₀The value is obtained.

All test compounds for inhibition of DPP-IV (derived from human blood)Clear or insect cells) have low IC₅₀Values, while high IC for inhibition of DPP-VIII (derived from baculovirus-infected sfa cells)₅₀The value is obtained. Some of the test compounds had a relatively high ratio of IC to inhibit DPP-VIII₅₀IC for inhibiting DPP-IV in value ratio₅₀Values such as 100, or even higher ratios. Therefore, the compounds 1 to 24 have extremely high selectivity for inhibiting DPP-IV rather than DPP-VIII.

Other embodiments

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

From the foregoing, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the following claims. For example, compounds structurally similar to the pyrrolidine compounds of the invention can be prepared, screened for DPP-IV inhibitory activity, and used to treat type II diabetes, and the inventors therefore, additional embodiments are also included within the scope of the claims.

Claims (15)

1. A compound of the formula:

wherein the content of the first and second substances,

R^1、R²、R³、R⁴、R⁵and R⁶Each independently is hydrogen or methyl;

R⁷is alkyl or heteroaryl, and R⁸Is hydrogen or alkyl; or R⁷And R⁸Taken together with the nitrogen atom to which they are bound as a 3-to 10-membered monocyclic OR bicyclic ring, optionally substituted by halogen, -OR', haloalkyl, hydroxyalkyl, alkoxyalkyl, OR-S (O)₂R' substituted saturated or unsaturated ring; r' is hydrogen or alkyl;

m and n are 1; and is

X is NH;

wherein the alkyl group is a straight or branched hydrocarbon group comprising 1 to 10 carbon atoms;

the haloalkyl is an alkyl substituted with one or more halogens;

the hydroxyalkyl group is an alkyl group substituted with one or more hydroxyl groups;

the alkoxyalkyl group is an alkyl group substituted with one or more alkoxy groups;

said alkoxy is-O-alkyl; and

the heteroaryl is pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, or thiazolyl.

2. The compound of claim 1, wherein each R is³And R⁴Is methyl.

3. The compound of claim 1, wherein each R is¹、R²、R⁵And R⁶Is H.

4. The compound of claim 3, wherein each R is⁷And R⁸Is methyl.

5. A compound of claim 3, wherein R⁷And R⁸Together with the nitrogen atom to which they are bound, form a 3-to 10-membered monocyclic OR bicyclic ring, optionally substituted by halogen, -OR', haloalkyl, hydroxyalkyl, alkoxyalkyl, OR-S (O)₂R ' is a saturated or unsaturated ring substituted with R ' and R ' is hydrogen or alkyl;

wherein the alkyl group is a straight or branched hydrocarbon group comprising 1 to 10 carbon atoms;

the haloalkyl is an alkyl substituted with one or more halogens;

the hydroxyalkyl group is an alkyl group substituted with one or more hydroxyl groups;

the alkoxyalkyl group is an alkyl group substituted with one or more alkoxy groups;

the alkoxy group is an-O-alkyl group.

6. The compound of claim 1, wherein each R is¹And R²Is H.

7. The compound of claim 1, wherein each R is⁵And R⁶Is H.

8. The compound of claim 1, wherein each R is⁷And R⁸Is methyl.

9. The compound of claim 1, wherein R⁷And R⁸Together with the nitrogen atom to which they are bound, form a 3-to 10-membered monocyclic OR bicyclic ring, optionally substituted by halogen, -OR', haloalkyl, hydroxyalkyl, alkoxyalkyl, OR-S (O)₂R ' is a saturated or unsaturated ring substituted with R ' and R ' is hydrogen or alkyl;

wherein the alkyl group is a straight or branched hydrocarbon group comprising 1 to 10 carbon atoms;

the haloalkyl is an alkyl substituted with one or more halogens;

the hydroxyalkyl group is an alkyl group substituted with one or more hydroxyl groups;

the alkoxyalkyl group is an alkyl group substituted with one or more alkoxy groups;

the alkoxy group is an-O-alkyl group.

10. A compound, wherein the compound is one of the following compounds 1 to 20:

11. a compound of the formula:

wherein the content of the first and second substances,

R¹is CN;

R³、R⁴、R⁵and R⁶Each independently is hydrogen or methyl;

R⁷is methyl, hydroxymethyl or methoxymethyl;

n is 1;

w is CH₂(ii) a And

x is CH (NH)₂)。

12. The compound of claim 11, wherein each R is⁵And R⁶Is H.

13. The compound of claim 11, wherein the compound is one of the following compounds 21 to 25:

14. use of a compound of claim 1 for the manufacture of a medicament for the treatment of type ii diabetes.

15. Use of a compound of claim 11 for the preparation of a pharmaceutical composition for the treatment of type ii diabetes.