HK1209126B - Ring constrained analogs as arginase inhibitors - Google Patents

Description

Ring-constrained analogs as arginase inhibitors

Background

The present invention relates generally to inhibitors of arginase and their use for treating pathological conditions. Two isoforms of arginase have been identified to date. Arginase i (arg i) expressed in the cytosol and arginase ii (arg ii) expressed in the mitochondria. Arginase plays an important role in the regulation of the level of free arginine in cells, together with the NOS enzyme.

Arginase has been shown to play a role in a variety of pathological conditions. These pathological conditions include, for example, erectile dysfunction, pulmonary hypertension, atherosclerosis, renal disease, asthma, T cell dysfunction, ischemia reperfusion injury, neurodegenerative disease, wound healing and fibrotic disease. Although the mechanism of action of arginases in these disease states remains the subject of ongoing research, several studies have shown that arginases are generally upregulated during pathological disease states.

For example, it is hypothesized that upregulation of arginase activity results in decreased levels of arginine, which in turn decreases the levels of NO, a physiologically important signaling molecule required for cell division, stimulates enhanced blood flow, and is used to control muscle and nerve signal transduction.

Besides the effect of regulating NO levels, arginase also affects the production of important polyamines (such as putrescine, spermidine and spermine). Ornithine is produced when arginine is consumed by arginase. Ornithine is subsequently converted to putrescine, spermidine and spermine via ornithine decarboxylase, spermidine synthase and spermine synthase, respectively. Thus, arginase controls physiological signaling events by controlling intracellular levels of polyamine signal transduction proteins. See king J-Y (Wang, J-Y); and mini-carbazo R.A (Casero, Jr, R.A.); hamana Press (Humana Press), tosowa (Totowa), NJ (NJ), 2006.

Thus, these results indicate the role of inhibitors of arginase as candidate therapeutic agents for the treatment of various disease states. The present invention provides compounds that are inhibitors of arginase activity and methods of using the compounds of the present invention in therapy.

Disclosure of Invention

The present invention provides certain boron-containing compounds that are inhibitors of arginase activity. The invention also provides methods of treatment using the compounds of the invention. Thus, in one embodiment, the compounds of the present invention and their pharmaceutically acceptable formulations are provided as therapeutic agents, which are capable of inhibiting arginase activity. The compounds and pharmaceutical formulations according to the present invention are useful for treating a variety of diseases and conditions, including, but not limited to, pulmonary hypertension, Erectile Dysfunction (ED), hypertension, atherosclerosis, nephropathy, asthma, T cell dysfunction, ischemia reperfusion injury, neurodegenerative diseases, wound healing, and fibrotic diseases.

In one embodiment, the present invention provides a compound selected from the following table:

the invention also encompasses pharmaceutically acceptable salts, stereoisomers, tautomers or prodrugs of said compounds.

In another embodiment, the present invention provides a pharmaceutical composition comprising a compound selected from the compounds of the above tables or a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug of a compound of the present invention, and a pharmaceutically acceptable carrier.

In one embodiment, the present invention also provides a method for inhibiting arginase I, arginase II, or a combination thereof in a cell comprising contacting the cell with at least one compound selected from the above tables. According to another embodiment, the present invention provides a method for treating or preventing a disease or condition associated with expression or activity of arginase I, arginase II, or a combination thereof in a subject, comprising administering to the subject a therapeutically effective amount of at least one compound selected from the above tables.

The compounds of the present invention and pharmaceutical formulations thereof may also be useful in the treatment of a variety of disorders including, but not limited to, cardiovascular disorders, sexual disorders, wound healing disorders, gastrointestinal disorders, autoimmune disorders, immune disorders, infections, pulmonary disorders, fibrotic disorders, and hemolytic disorders.

Detailed Description

The compounds of the invention are inhibitors of arginase I and arginase II activity. Accordingly, the compounds of the present invention are candidate therapeutic agents for the treatment of diseases and disorders associated with an imbalance in cellular arginase.

The compounds of the invention may exist in various isomeric forms, including configurational, geometric and conformational isomers, including, for example, cis or trans conformations. The compounds of the invention may also exist in one or more tautomeric forms, including individual tautomers and mixtures of tautomers. The term "isomer" is intended to encompass all isomeric forms of the compounds of the present invention, including tautomeric forms of the compounds.

Some of the compounds of the present invention contain one or more chiral centers. Because of the presence of asymmetric centers, certain compounds of the present invention may exist as enantiomers and diastereomers or mixtures thereof, including racemic mixtures. Optical isomers of the compounds of the present invention may be obtained by known techniques, such as asymmetric synthesis, chiral chromatography, simulated moving bed techniques or by chemical separation of stereoisomers via the use of optically active resolving agents.

Unless otherwise indicated, "stereoisomer" means one stereoisomer of a compound that is substantially free of other stereoisomers of the compound. Thus, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. Typical stereoisomerically pure compounds contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.

If there is a discrepancy between the structure depicted and the name given to that structure, then the structure depicted controls. Furthermore, if the stereochemistry of a structure or a portion of a structure is not indicated, for example, in bold or dashed lines, the structure or portion of the structure is to be understood as encompassing all stereoisomers of it. However, in some cases where more than one chiral center is present, structures and names may be represented in a single enantiomer to help describe the relative stereochemistry. One skilled in the art of organic synthesis will know whether a compound is prepared in a single enantiomeric form by the method used to prepare it.

"pharmaceutically acceptable salts" are pharmaceutically acceptable organic or inorganic acid or base salts of the compounds of the present invention. Representative pharmaceutically acceptable salts include, for example, alkali metal salts, alkaline earth metal salts, ammonium salts, water-soluble and water-insoluble salts such as acetate, astraganesulfonate (4, 4-diaminostilbene-2, 2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsylate, carbonate, chloride, citrate, clavulanate (clavulanate), dihydrochloride, edetate, edisylate, etonate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycollylalyazinoaslinate (glyphosate), hexafluorophosphate, hexylresorcinate (hexylresorcinonate), hydrabamine (hydrabamine), hydronium, hydrochloride, hydroxynaphthoate, iodide, and mixtures thereof, Isosulfurized hydroxy acid salts, lactate salts, lactobionate salts, laurate salts, malate salts, maleate salts, mandelate salts, methanesulfonate salts, methyl bromide salts, methyl nitrate salts, methylsulfate salts, mucate salts, naphthalenesulfonate salts, nitrate salts, N-methylglucamine ammonium salts, 3-hydroxy-2-naphthoate salts, oleate salts, oxalate salts, palmitate salts, pamoate salts (1, 1-methylene-bis-2-hydroxy-3-naphthoate salts, embonate salts (embonate salts)), pantothenate salts, phosphate/diphosphate salts, picrate salts, polygalacturonate salts, propionate salts, p-toluenesulfonate salts, salicylate salts, stearate salts, subacetate salts, succinate salts, sulfate salts, sulfosalicylate salts, suramate salts (sumate salts), tannate salts, tartrate salts, theachlorate salts (teoclate salts), Tosylate, triethyl iodide and valerate. Pharmaceutically acceptable salts may have more than one charged atom in their structure. In this case, the pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

The terms "treating", "treating" and "treatment" refer to ameliorating or eradicating a disease or a symptom associated with a disease. In certain embodiments, the terms refer to minimizing the spread or progression of the disease by administering one or more prophylactic or therapeutic agents to a patient suffering from the disease.

The terms "preventing (preventing, suppressing, and suppressing)" refer to preventing the onset, recurrence, or spread of a disease in a patient as a result of administration of a prophylactic or therapeutic agent.

The term "effective amount" refers to an amount of a compound of the present invention or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of disease or to delay or minimize symptoms associated with disease. Furthermore, for the compounds of the present invention, a therapeutically effective amount means the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or prevention of the disease, alone or in combination with other therapies. When used in conjunction with a compound of the present invention, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease, or enhances the therapeutic efficacy of or synergizes with another therapeutic agent.

The term "modulate, modulation, etc." refers to the ability of a compound of the invention to increase or decrease the function or activity of, for example, arginase I or arginase II. "modulation" in various forms is intended to encompass inhibition, antagonism, partial antagonism, activation, agonism and/or partial agonism of the activity associated with arginase. Arginase inhibitors are compounds that, for example, bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate signal transduction. The ability of a compound to modulate arginase activity can be demonstrated in an enzymatic assay or a cell-based assay.

"patient" includes animals such as humans, cattle, horses, sheep, lambs, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits or guinea pigs. The animal can be a mammal, such as a non-primate and a primate (e.g., monkey and human). In one embodiment, the patient is a human, such as a human infant, child, adolescent or adult.

The term "prodrug" refers to a precursor of a drug, which is a compound that must undergo a chemical transformation by a metabolic process after administration to a patient before becoming an active pharmacological agent. Exemplary prodrugs of the compounds of the invention are esters, pinenes, dioxolanes and amides.

Compounds of the invention

The present invention provides small molecule therapeutics that are potent inhibitors of arginase I and II activity. Exemplary compounds of the invention are shown in table 1 below. Although some exemplary compounds are depicted using stereochemistry, it is to be understood that the present invention includes all possible stereoisomers, such as diastereomers, of the compounds.

TABLE 1

Pharmaceutical compositions and dosages

The present invention is directed, in part, to pharmaceutical formulations of the compounds of the invention, and the use of the formulations of the invention for treating disease states associated with an imbalance in arginase activity or inappropriate function of arginase. Accordingly, in one embodiment, the present invention provides a pharmaceutical composition comprising a compound selected from table 2, or a salt, solvate, stereoisomer, tautomer, or prodrug thereof, and a pharmaceutically acceptable carrier.

TABLE 2

In one aspect, the invention provides combination therapy in which a patient or individual in need of therapy is administered a formulation of a compound of the invention in combination with one or more other compounds having similar or different biological activities.

According to one aspect of the combination therapy routine, a therapeutically effective dose of a compound of the present invention and a therapeutically effective dose of a combination drug may be administered separately to a patient or individual in need thereof. One skilled in the art will recognize that these two doses may be administered within hours or days of each other, or that these two doses may be administered together.

Exemplary disease conditions to which combination therapy according to the present invention may be administered include any of the conditions described in more detail below. These conditions include, but are not limited to, heart disease, hypertension, sexual dysfunction, gastric disorders, autoimmune disorders, parasitic infections, pulmonary disorders, smooth muscle relaxation disorders, asthma, and hemolytic disorders.

Suitable compounds that may be used in combination with the compounds of the present invention include (but are not limited to):

erectile dysfunction: sildenafil (sildenafil), vardenafil (vardenafil), tadalafil (tadalafil) and alprostadil (alprostadil).

Pulmonary hypertension/hypertension: epoprostenol (epoprostenol), iloprost (iloprost), bosentan (bosentan), amlodipine (amlodipine), diltiazem (dilizem), nifedipine (nifedipine), ambrisentan (ambrisentan) and warfarin (warfarin).

Asthma: fluticasone (fluticasone), budesonide (budesonide), mometasone (mometasone), flunisolide (flunisolide), beclomethasone (beclomethasone), montelukast (montelukast), zafirlukast (zafirlukast), zileuton (zileuton), salmeterol (salmeterol), formoterol (formoterol), theophylline (theophylline), salbutamol (albuterol), levalbuterol (levalbuterol), pirbuterol (pirbuterol), ipratropium (ipratropium), prednisone (prednisone), methylprednisolone (methylprednisolone), omazumab (omab), corticosteroid (corticosterosteroid), and glycopyrric acid (cromolyn).

Atherosclerosis: atorvastatin (atorvastatin), lovastatin (lovastatin), simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), rosuvastatin (rosuvastatin), gemfibrozil (gemfibrozil), fenofibrate (fenofibrate), nicotinic acid (nicotinic acid), clopidogrel (clopidogrel).

The present invention also provides a pharmaceutically suitable composition comprising a therapeutically effective amount of one or more compounds of the present invention, or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or prodrug thereof, in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains one or more other therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, coloring, buffering or flavoring agents (flavoring agents), according to accepted pharmaceutical compounding practices.

The compositions of the present invention may be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

Compositions suitable for oral administration according to the present invention include, but are not limited to, tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

Within the scope of the present invention are pharmaceutical compositions suitable for a single unit dose comprising a compound of the present invention, a pharmaceutically acceptable stereoisomer, prodrug, salt, solvate, hydrate or tautomer thereof, and a pharmaceutically acceptable carrier.

Compositions of the invention suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For example, liquid formulations of the compounds of the present invention contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations of arginase inhibitor.

For tablet compositions, tablets are made using a mixture of the active ingredient with non-toxic pharmaceutically acceptable excipients. Examples of such excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained therapeutic effect over a desired period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin; or in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

For aqueous suspensions, the compounds of the invention are mixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include, but are not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

Oral suspensions may also contain dispersing or wetting agents such as a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids (for example polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (for example heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (for example polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (for example polyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate; one or more colorants; one or more flavoring agents; and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil; or mineral oil, such as liquid paraffin, or mixtures of these substances. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth; naturally occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. These formulations may also contain a demulcent, a preservative and flavoring and coloring agents. Pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanol. Acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (e.g., oleic acid) may be used in the preparation of injectables.

The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. The materials are cocoa butter and polyethylene glycols.

Compositions for parenteral administration are administered in a sterile medium. Parenteral formulations can be suspensions or solutions containing dissolved drug, depending on the vehicle used and the concentration of the drug in the formulation. Adjuvants such as local anesthetics, preservatives, and buffering agents can also be added to the parenteral composition.

Synthesis of Compounds

Generally, intermediates and target compounds containing chiral centers are specified in a stereospecific manner. This designation is used primarily to distinguish relative stereochemistry and is not indicative of optical purity. It will be apparent to those skilled in the art of organic synthesis which compounds are optically pure depending on the method used to prepare the compounds.

In addition, the compounds described below may also be isolated as hydrates or salts (e.g., hydrochloride salts), but are not necessarily so indicated. The compounds described in this invention are typically named using common names, IUPAC names, or names generated using the naming algorithm in chemdraw 10.0.

Example 1: preparation of (1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- (((2, 3-dihydro-1H-inden-2-yl) amino) methyl) cyclopentanecarboxylic acid

Step 1: the method A comprises the following steps: 2-Oxocyclopentanecarboxylic acid allyl ester (transesterification)

To a stirred solution of methyl 2-oxocyclopentanecarboxylate (4.26g, 30mmol) and allyl alcohol (10.2mL, 150mmol) in dry toluene (25mL) was added zinc powder (0.40g, 6 mmol). After the mixture was refluxed for 48h, it was cooled to room temperature and the suspension was filtered. Washing with tolueneThe cake was filtered and the combined filtrates were concentrated to give allyl 2-oxocyclopentanecarboxylate as a colorless oil (5.01g, 99%).¹H NMR(CDCl₃，300MHz)5.89(ddt，J₁＝15.9Hz，J₂＝10.5Hz，J₃＝4.8Hz，1H)，5.33(dtd，J₁＝15.9Hz，J₂＝2.7Hz，J₃＝1.4Hz，1H)，5.23(dtd，J₁＝10.5Hz，J₂＝2.7Hz，J₃1.4Hz, 1H), 4.83-4.75(m, 1H), 3.18(t, J ═ 9.0Hz, 1H), 2.41-2.23(m, 4H), 2.22-2.07(m, 1H), 1.94-1.80(m, 1H); MS (+ CI): for C₉H₁₂O₃m/z: the expected value is 168.1; experimental value 169.1(M + H)⁺。

Step 1: the method B comprises the following steps: 2-Oxocyclopentanecarboxylic acid allyl ester (Dieckman)

To a stirred solution of diallyl adipate (4.53g, 20mmol) in anhydrous tetrahydrofuran (100mL) at 0 deg.C was added lithium bis (trimethylsilyl) amide (40mL, 1.0N in THF, 40 mmol). After the addition was complete, the solution was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was cooled again to 0 ℃ and acidified by the addition of acetic acid (2.53mL, 44mmol) in a dropwise manner. The addition of acetic acid produced a cloudy mixture, which was allowed to warm to room temperature and filtered. The obtained filtrate was concentrated, dissolved in a minimum amount of dichloromethane and purified by flash column chromatography (silica gel, dichloromethane) to obtain allyl 2-oxocyclopentanecarboxylate as a colorless oil (2.62g, 78%). The NMR spectrum of the purified product was the same as that observed for allyl 2-oxocyclopentanecarboxylate prepared using method a.

Step 2: synthesis of 2-allylcyclopentanone

A stirred solution of palladium (II) acetate (51mg, 0.23mmol) and triphenylphosphine (0.24g, 0.9mmol) in dry THF (20mL) was added under a nitrogen atmosphereHeat to 65 ℃. To the hot solution was added a solution of allyl 2-oxocyclopentanecarboxylate (2.52g, 15mmol) in anhydrous THF. After stirring at 65 ℃ for 45 minutes, the reaction mixture was cooled and concentrated. The resulting residual yellow oil was dissolved in a minimum amount of dichloromethane and purified by flash column chromatography (silica gel, dichloromethane) to afford 2-allylcyclopentanone (1.32g, 71%) as a colorless oil.¹HNMR(CDCl₃，300MHz)5.72(ddt，J₁＝17.1Hz，J₂＝10.2Hz，J₃＝7.2Hz，1H)，5.09-4.98(m，2H)，2.55-2.46(m，1H)，2.35-2.22(m，1H)，2.22-1.91(m，5H)，1.87-1.70(m，1H)，1.63-1.48(m，1H)。

And step 3: synthesis of 2-phenylseleno-5- (propen-3-yl) cyclopentanone, mixture of isomers

A solution of 2- (propen-3-yl) cyclopentanone (12.4g, 100mmol) in anhydrous tetrahydrofuran (100mL) was cooled to-70 ℃ under an inert atmosphere of nitrogen to this cold solution 1N lithium bis (trimethylsilyl) amide in tetrahydrofuran (200mL, 200mmol) was added at a rate effective to maintain the temperature of the reaction mixture below-55 ℃ once addition was complete, the mixture was stirred at-60 ℃ to-70 ℃ for an additional hour, then a second solution of phenylselenochloride (19.5g, 102mmol) in anhydrous tetrahydrofuran (50mL) was added dropwise and the reaction mixture was stirred at-60 ℃ to-70 ℃ for an additional 30 minutes, then the reaction was warmed to 0 ℃ and quenched by adding a mixture of ethyl acetate (500mL) and 5% aqueous citric acid solution (200mL) while stirring the reaction mixture rapidly, after separation of the organic and aqueous layers, the aqueous layers were extracted with ethyl acetate (2 × 100mL) and the combined organic layers were washed with brine (200mL), washed with MgSO 5% MgSO 4, and the aqueous layers were extracted with magnesium chloride (MgSO 4mL)₄) Dried and concentrated in vacuo. The residue was dissolved in heptane and chromatographed using a column of silica gel (ca. 600ml) and 2: 1 heptane/dichloromethane solution as the initial eluent. Then, the elution solution is changedA1: 1 heptane/dichloromethane mixture was obtained to give the title compound as a pale yellow oil (19.7g, 71%). NMR (CDCl)₃): 7.40-7.50(m, 2H), 7.05-7.25(m, 3H), 5.50-5.70(m, 1H), 4.80-4.95(m, 2H), 3.45-3.75(m, 1H), 2.30-2.50(m, 1H), 1.80-2.25(m, 5H), 1.50-1.75(m, 1H). MS (M + 1): 279.1/280.9 (2 major isotopes for Se).

And 4, step 4: synthesis of 5- (propen-3-yl) cyclopent-2-enone

An ice-cold (3 ℃) solution of 2-phenylseleno-5- (propen-3-yl) cyclopentanone (mixture of isomers, (12.0g, 43mmol)) in dichloromethane (200mL) was stirred in a 1L round-bottomed flask prepared for boiling over constraints. To this solution was added saturated aqueous ammonium chloride (45mL), followed by dropwise addition of 30% aqueous hydrogen peroxide (22 mL). The reaction mixture was then slowly warmed to room temperature and intermittently cooled as needed to prevent excessive foaming and boil-over. After stirring at room temperature for another hour, the solution was washed with water (100mL), followed by stirring with 10% aqueous sodium thiosulfate pentahydrate (75mL) for 10 minutes, and then the aqueous layer and the organic layer were separated. The organic solution was washed with saturated aqueous sodium bicarbonate and brine (75mL each) using Na₂SO₄Dried and concentrated to a volume of about 30 mL. Purification of the crude reaction was achieved by loading the crude mixture onto a silica gel column (about 400cc) using dichloromethane as the eluting solvent. The appropriate fractions were concentrated to give 5- (propen-3-yl) cyclopent-2-enone as a very pale yellow oil (3.95g, 75%). NMR (CDCl)₃)：7.61(m，1H)，6.12(m，1H)，5.60-5.75(m，1H)，4.90-5.05(m，2H)，2.70-2.80(m，1H)，2.45-2.55(m，1H)，2.30-2.40(m，2H)，2.05-2.15(m，1H)。

And 5: synthesis of 3-nitromethyl-5- (propen-3-yl) cyclopentanone, mixture of isomers

A stirred solution of 5- (propen-3-yl) cyclopent-2-enone (0.428g, 3.5mmol) in nitromethane (2mL) under nitrogen with550A-OH resin (0.80g which had previously been rinsed with methanol and partially air dried) was treated and heated to 60 ℃ for 2 hours. The mixture was cooled to room temperature, diluted with dichloromethane (20mL) and filtered. The filtrate was concentrated in vacuo, redissolved in a minimum amount of dichloromethane, and loaded onto a silica gel column (about 100 mL). Elution with dichloromethane gave the title compound as a colorless oil (0.368g, 57%). NMR (CDCl)₃)：5.65-5.80(m，1H)，5.00-5.15(m，2H)，4.40-4.50(m，2H)，2.85-3.15(m，1H)，2.30-2.70(m，4H)，1.90-2.20(m，3H)。MS(M+1)：183.9。

Step 6: synthesis of 1-acetamido-3-nitromethyl-5- (propen-3-yl) cyclopentanecarboxylic acid, tert-butyramide, (isomers A and B)

To a stirring solution of 3-nitromethyl-5- (propen-3-yl) cyclopentanone (mixture of isomers (0.366g, 2.0mmol)) in 2, 2, 2-trifluoroethanol (1.5mL) under an inert nitrogen atmosphere were added ammonium acetate (0.617g, 8mmol) and tert-butylisonitrile (0.68mL, 6.0mmol), and the reaction mixture was stirred at room temperature for 2 days. The mixture was then diluted with dichloromethane (20mL) and loaded directly onto a silica gel column (approximately 250 mL). The two cyclopentane-tert-butyl carboxamide isomers (isomers 1 and 2) in cis conformation with the acetylamino and allyl substituents eluted first, followed by isomer a (122mg, 19%) and then isomer B as a white solid (195mg, 30%).

For isomer a: NMR (CDCl)₃)：6.12(br s，2H)，5.65-5.80(m，1H)，5.00-5.15(m，2H)，4.53(d，J＝7Hz，1H)，4.35-4.50(m，1H)，2.80-3.00(m，1H)，2.45-2.60(m，1H)，2.25-2.35(m，2H)，1.90-2.20(m，2H)，2.00(s，3H)，1.20-1.60(m，2H)，1.34(s，9H)。MS(M+1)：326.0。

For isomer B: NMR (CDCl)₃)：6.05-6.15(m，2H)，5.65-5.80(m，1H)，5.00-5.15(m，2H)，4.43(d，J＝6.5Hz，2H)，2.90-3.10(m，2H)，2.40-2.50(m，1H)，2.20-2.30(m，1H)，2.00(s，3H)，1.70-2.00(m，4H)，1.35(s，9H)。MS(M+1)：326.0。

And 7: synthesis of (1S, 2S, 4S) -1-acetylamino-N- (tert-butyl) -4- (nitromethyl) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide

To a stirred solution of 1-acetamido-3-nitromethyl-5- (propen-3-yl) cyclopentanecarboxylic acid, tert-butyramide, isomer B (0.179g, 0.55mmol) in dry dichloromethane (5mL) under nitrogen was added chloro-1, 5-cyclooctadieneiridium dimer (12mg, 0.018mmol) and(14mg, 0.035 mmol). After stirring for 30 minutes, the reaction mixture was cooled to-25 ℃. Pinacolborane (0.123mL, 0.85mmo1) was then added dropwise via syringe and the reaction mixture was gradually warmed to 0 ℃ (ice bath temperature) and then allowed to gradually warm to room temperature overnight (18 hours). The reaction was quenched by the addition of water (3mL), stirred at room temperature for 20 minutes, and then extracted twice with ethyl acetate (25mL and 10mL, respectively). Combination of Chinese herbsThe organic layer was washed sequentially with water and brine (20mL each) and MgSO₄And (5) drying. After concentration in vacuo, the crude product was recrystallized from acetonitrile (batch 2) to yield 0.173g (69%) of the title compound as a white solid. NMR (CDCl)₃)：6.11(br s，1H)，5.94(br s，1H)，4.41(d，J＝7Hz，2H)，3.00-3.15(m，1H)，2.93(dd，J＝14Hz，9.5Hz，1H)，2.25-2.35(m，1H)，2.00(s，3H)，1.65-1.85(m，3H)，1.15-1.50(m，4H)，1.34(s，9H)，1.24(s，12H)，0.65-0.85(m，2H)。MS(M+1)：453.7。

And 8: synthesis of (1S, 2S, 4S) -1-acetylamino-4- (aminomethyl) -N- (tert-butyl) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide

To a stirring solution of (1S, 2S, 4S) -1-acetylamino-N- (tert-butyl) -4- (nitromethyl) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide (0.907g, 2.0mmol) in a mixture of tetrahydrofuran (20mL), ethyl acetate (15mL) and ethanol (5mL) was added Raney nickel (Raney Kel) (1.2g) under nitrogen. The reaction mixture was then purged with hydrogen and stirred at room temperature under a hydrogen atmosphere for 6 hours. At the end of this period, the mixture is purged with nitrogen and then carefully passed throughAfter rinsing the filter cake with ethyl acetate, the combined filtrates were concentrated in vacuo to give the title compound as a white solid (0.841g, 99%). NMR (CDCl)₃)：6.98(br s，1H)，6.93(br s，1H)，2.55-2.70(m，3H)，2.44(m，1H)，2.24(m，1H)，1.91(m，3H)，1.50-1.65(m，3H)，1.20-1.45(m，4H)，1.26(s，9H)，1.66(s，12H)，0.60-0.75(m，2H)。MS(M+1)：424.4。

And step 9: synthesis of (1S, 2S, 4S) -1-amino-4- ((benzylamino) methyl) -2- (3-boronopropyl) cyclopentanecarboxylic acid

To a stirring solution of benzaldehyde (43mg, 0.40mmol) in methanol (3.5mL) were added (1S, 2S, 4S) -1-acetylamino-4- (aminomethyl) -N- (tert-butyl) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide (148mg, 0.35mmol) and glacial acetic acid (one drop). The reaction mixture was stirred at 50 ℃ for 1 hour, then cooled using an ice bath, followed by addition of sodium borohydride (17mg, 0.45 mmol). After stirring for a further hour (1h) at 3 ℃, the reaction was allowed to warm to room temperature and stirred for a further 20 minutes. After the reaction was quenched with water (1mL), the crude product was treated with a 2: 1 mixture of concentrated hydrochloric acid: glacial acetic acid: water (8mL) in a pressure bottle, stirred at 60 ℃ for 2 hours, and then capped and stirred at 130 ℃ for a further 18 hours. Subsequently, the reaction mixture was cooled to room temperature, and the pressure-resistant bottle was uncapped. The crude mixture was diluted with water (20mL), extracted with dichloromethane (20mL) and concentrated in vacuo. The obtained residue was treated with water (20mL) and concentrated three times to remove excess HCl. Next, the crude reaction mixture was dissolved in water (40mL) and used550A-OH resin (3g, which had been washed with methanol prior to use.) after stirring for 40 minutes, the reaction mixture was filtered and the resin was washed twice with water, methanol and dichloromethane in sequence after washing, the resin was stirred and filtered with 1N HCl (15mL × 4.) the combined filtrates were concentrated and the residue was treated with water (20mL) and the aqueous mixture was then concentrated three times to remove excess HCl the crude product was purified by HPLC followed by the formation of the hydrochloride salt to give the title compound as a hygroscopic white foam (71.4mg, 50%). NMR (D)₂O)7.40(br s，5H)，4.17(br s，2H)，3.02(d，J＝5.5Hz，2H)，2.70(m，1H)，2.51(m，1H)，2.15(m，1H)，1.80(m，2H)，1.55(m，1H)，1.30-1.45(m，2H)，1.20(m，1H)，1.05(m，1H)，0.60-0.75(m，2H)。MS(M+1)：335.5；MS(M-H₂O+1)：317.4；MS(M-2H₂O+1)：299.3。

Example 2: preparation of (1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- (((((4 '-chloro- [1, 1' -biphenyl ] -4-yl) methyl) amino) methyl) cyclopentanecarboxylic acid

(1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- ((((4 '-chloro- [1, 1' -biphenylyl) propyl) amide]-4-yl) methyl) amino) methyl) cyclopentanecarboxylic acid was prepared in a similar manner to that set forth in example 1, except that 4 '-chloro- [1, 1' -biphenyl was used in step 9]-4-Formaldehyde was used as aldehyde. NMR (D)₂O)7.65(d，J＝6Hz，2H)，7.56(d，J＝6Hz，2H)，7.47(d，J＝6Hz，2H)，7.41(d，J＝6Hz，2H)，4.20(m，2H)，3.03(m，2H)，2.70(m，1H)，2.51(m，1H)，2.10(m，1H)，1.75(m，2H)，1.52(m，1H)，1.25-1.45(m，2H)，1.16(m，1H)，1.04(m，1H)，0.55-0.70(m，2H)。MS(M+1)：445.3；MS(M-H₂O+1)：427.6；MS(M-2H₂O+1)：409.4。

Example 3: preparation of (1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- (((2, 3-dihydro-1H-inden-2-yl) amino) methyl) cyclopentanecarboxylic acid

(1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- (((2, 3-dihydro-1H-inden-2-yl) amino) methyl) cyclopentanecarboxylic acid was prepared in a similar manner to that set forth in example 1, except that 1H-inden-2 (3H) -one was used as the ketone in step 9. NMR (D)₂O)7.15-7.25(m，4H)，3.46(m，1H)，3.35(dd，J＝12.5Hz，5.5Hz，2H)，3.00-3.15(m，4H)，2.72(m，1H)，2.55(m，1H)，2.20(m，1H)，1.85(m，2H)，1.60(m，1H)，1.35-1.50(m，2H)，1.25(s，1H)，1.05-1.15(m，1H)，0.60-0.75(m，2H)。MS(M+1)：361.3；MS(M-H₂O+1)：343.3；MS(M-2H₂O+1)：325.4。

Example 4: preparation of (1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- (((1, 2, 3, 4-tetrahydronaphthalen-2-yl) amino) methyl) cyclopentanecarboxylic acid

(1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- (((1, 2, 3, 4-tetrahydronaphthalen-2-yl) amino) methyl) cyclopentanecarboxylic acid was prepared in a similar manner to that set forth in example 1, except that 3, 4-dihydronaphthalen-2 (1H) -one was used as the ketone in step 9. NMR (D)₂O)7.05-7.15(m，4H)，3.50(m，1H)，3.21(m，1H)，3.15(d，J＝5.5Hz，2H)，2.80-2.95(m，3H)，2.73(m，1H)，2.55(m，1H)，2.20(m，2H)，1.85(m，2H)，1.75(m，1H)，1.58(m，1H)，1.30-1.50(m，2H)，1.25(s，1H)，1.10(m，1H)，0.60-0.75(m，2H)。MS(M+1)：375.6；MS(M-H₂O+1)：357.5；MS(M-2H₂O+1)：339.4。

Example 5: preparation of (1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- ((cyclobutylamino) methyl) cyclopentanecarboxylic acid

(1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- ((cyclobutylamino) methyl) cyclopentanecarboxylic acid was prepared in a similar manner to that set forth in example 1, except that cyclobutanone was used as the ketone in step 9. And one and two instances of the cyclobutane moiety were isolated from the same reaction. NMR (D)₂O)3.67(m，1H)，2.89(d，J＝5.5Hz，2H)，2.66(m，1H)，2.50(m，1H)，2.15-2.25(m，3H)，2.00-2.10(m，2H)，1.70-1.85(m，4H)，1.53(m，1H)，1.30-1.50(m，2H)，1.23(m，1H)，1.09(m，1H)，0.60-0.75(m，2H).MS(M+1)：299.6；MS(M-H₂O+1)：281.4；MS(M-2H₂O+1)：263.4。

Example 6: preparation of (1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- ((dicyclobutylamino) methyl) cyclopentanecarboxylic acid

(1S, 2S, 4S) -1-amino-2- (3-boronopropyl) -4- ((dicyclobutylamino) methyl) cyclopentanecarboxylic acid was prepared in a similar manner to that set forth in example 1, except that cyclobutanone was used as the ketone in step 9. Both the mono-and di-cyclobutane products were isolated from the same reaction. NMR3.65-3.75(m, 2H), 2.90-3.05(m, 2H), 2.78(m, 1H), 2.54(m, 1H), 2.05-2.30(m, 8H), 1.60-1.90(m, 6H), 1.53(m, 1H), 1.44(m, 2H), 1.25(m, 1H), 0.85-1.15(m, 2H), 0.60-0.75 (2H). MS (M + 1): 353.5; MS (M-H)₂O+1)：335.6；MS(M-2H₂O+1)：317.5。

Example 7: preparation of (1S, 2S) -2- (3-boronopropyl) -1- (methylamino) cyclopentanecarboxylic acid

Step 1: synthesis of (1S, 2R) -2-allyl-N- (tert-butyl) -1- (N-methylacetamido) cyclopentanecarboxamide

To a round bottom flask containing 2- (propen-3-yl) cyclopentanone (0.745g, 6.0mmol) was added a premixed slurry of 8N methylamine/ethanol (3.0mL, 24mmol) and glacial acetic acid (1.37mL, 24mmol) in trifluoroethanol (3 mL). The reaction mixture was stirred for 30 min and then treated with tert-butylisonitrile (2.04mL, 18 mmol). After stirring for 2 days, the reaction mixture was diluted with dichloromethane (10mL) and silica was usedThe gel column (175mL) was chromatographed. Gradient elution with 20%, 50% and 80% mixtures of ethyl acetate and heptane yielded the title compound as a white crystalline solid (559mg, 33%). NMR (CDCl)₃)：5.82(br s，1H)，5.70(m，1H)，4.90-5.00(m，2H)，2.96(s，3H)，2.63(m，1H)，2.37(m，1H)，2.26(m，1H)，2.04(s，3H)，1.60-1.85(m，4H)，1.40-1.60(m，2H)，1.24(s，9H)。MS(M+1)：281.4；MS(M+Na)：303.4。

Step 2: synthesis of (1S, 2S) -N- (tert-butyl) -1- (N-methylacetamido) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide

To a solution of (1S, 2R) -2-allyl-N- (tert-butyl) -1- (N-methylacetamido) cyclopentanecarboxamide (0.561g, 2.00mmol) in anhydrous dichloromethane (20mL) maintained under an inert nitrogen atmosphere were added chloro-1, 5-cyclooctadieneiridium dimer (48mg, 0.071mmol) and 1, 2-bis (diphenylphosphino) ethane (57mg, 0.143 mmol). The reaction was stirred for 30 minutes and then cooled to-25 ℃. Pinacolborane (0.44mL, 3.0mmol) was added dropwise to the cold mixture and the mixture was allowed to slowly reach room temperature after the addition of pinacolborane. After stirring at room temperature for 18 hours, water (12mL) was added to the reaction mixture and stirring was continued for another 30 minutes. The mixture was then extracted with ethyl acetate (75mL followed by 25 mL). The combined organic layers were washed sequentially with water and brine (50mL each) and MgSO₄Dried and then concentrated under vacuum. The obtained residue was dissolved in warm heptane and loaded onto a silica gel column (175mL) initially eluted with a solvent mixture comprising 70% ethyl acetate/heptane followed by ethyl acetate to give the title compound as a white solid (0.611g, 75%). NMR (CDCl)₃)：5.64(br s，1H)，2.94(s，3H)，2.68(m，1H)，2.22(m，1H)，2.02(s，3H)，1.85(m，1H)，1.75(m，1H)，1.35-1.60(m，5H)，1.05-1.30(m，23H)，0.65-0.80(m，2H)。MS(m+1)：409.5；MS(m+1)：431.5。

And step 3: synthesis of (1S, 2S) -2- (3-boronopropyl) -1- (methylamino) cyclopentanecarboxylic acid

A solution of (1S, 2S) -N- (tert-butyl) -1- (N-methylacetamido) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan.2-yl) propyl) cyclopentanecarboxamide (0.600g, 1.47mmol) was hydrolyzed in a similar manner to that described in step 9 of example 1 to give the title compound as a light amber glass (256mg, 66%). NMR (D)₂O)2.61(s，3H)，2.30(m，1H)，1.95-2.15(m，2H)，1.80-1.95(m，2H)，1.70(m，1H)，1.35-1.50(m，3H)，1.24(m，1H)，1.00-1.15(m，1H)，0.60-0.75(m，2H)。MS(M+1)：230.4；MS(M-H₂O+1)：212.2；MS(M-2H₂O+1)：194.2。

Example 8: preparation of (1S, 2S) -1-amino-2- (3-boronopropyl) cyclopentanecarboxylic acid

Step 1: synthesis of (1S, 2R) -2-allyl-N- (tert-butyl) -1- (N- ((S) -1-phenylethyl) acetylamino) cyclopentanecarboxamide

To a stirring solution of 2- (propen-3-yl) cyclopentanone (0.745g, 6.0mmol) in 2, 2, 2-trifluoroethanol (5mL) under a nitrogen atmosphere was added (S) - α -methylbenzylamine (3.1mL, 24mmol), glacial acetic acid (1.38mL, 24mmol), and tert-butylisonitrile (2.04mL, 18 mmol). after stirring at room temperature for five days and then at 60 ℃ for an additional 2 days, the mixture was concentrated in vacuo, taken up in water (50mL) and extracted with ethyl acetate (75mL, then 50 mL). the combined organic layers were washed with brine (75mL), usingMgSO₄Dried and concentrated in vacuo. The residual oil was dissolved in a minimum amount of dichloromethane and the crude material was loaded onto a silica gel column (175 mL). The crude mixture was purified by eluting the column with 20% ethyl acetate/heptane and then using a solvent mixture containing 30% ethyl acetate/heptane to give the title compound as a pale yellow viscous oil as a single enantiomer (0.341g, 15%). NMR (CDCl)₃)：7.46(m，2H)，7.31(m，2H)，7.19(m，1H)，6.27(m，1H)，5.67(m，1H)，4.90(m，2H)，4.80(br s，1H)，2.88(m，1H)，2.43(m，2H)，1.87(m，2H)，1.50-1.80(m，10H)，1.30(s，9H)。MS(M+1)：371.1；MS(M+Na)：393.4。

Step 2: synthesis of (1S, 2S) -N- (tert-butyl) -1- (N- ((S) -1-phenylethyl) acetylamino) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide

A solution of (1S, 2R) -2-allyl-N- (tert-butyl) -1- (N- ((S) -1-phenylethyl) acetylamino) cyclopentanecarboxamide (0.322g, 0.87mmol) in dry dichloromethane (8mL) maintained under an inert nitrogen atmosphere was treated with chloro-1, 5-cyclooctadieneiridium dimer (20.5mg, 0.030mmol) and 1, 2-bis (diphenylphosphino) ethane (24.3mg, 0.060 mmol). After stirring for 30 minutes, the reaction mixture was cooled to-30 ℃. Next, pinacolborane (0.19mL, 1.3mmol) was added dropwise to the cold reaction mixture. After addition of pinacolborane, the mixture was allowed to warm slowly to room temperature and stirred for an additional 18 hours. Next, water (4mL) was added to the reaction and the mixture was stirred for an additional 30 minutes. The crude product was extracted with ethyl acetate (30mL followed by 20 mL). The combined organic layers were washed sequentially with water and brine (20mL each) and MgSO₄Dried and then concentrated in vacuo. The residue obtained was dissolved in a minimum amount of dichloromethane and loaded onto a silica gel column (50mL) washed with 40% ethyl acetate/heptaneTo give the title compound as a colorless viscous oil (285mg, 66%). NMR (CDCl)₃)：7.47(m，2H)，7.29(m，2H)，7.15-7.22(m，1H)，6.13(br s，1H)，4.74(br s，1H)，2.92(m，1H)，2.30(m，1H)，1.98(m，1H)，1.40-1.80(m，11H)，1.05-1.30(m，23H)，0.60-0.75(m，2H)。MS(m+1)：499.6；MS(m+1)：521.7。

And step 3: synthesis of (1S, 2S) -1-amino-2- (3-boronopropyl) cyclopentanecarboxylic acid

A cold (-50 ℃ C.) (1S, 2S) -N- (tert-butyl) -1- (N- ((S) -1-phenylethyl) acetylamino) -2- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) cyclopentanecarboxamide (0.400g, 0.802mmol) in anhydrous tetrahydrofuran (5mL) was combined (in small portions) gradually over a period of several minutes under an inert nitrogen atmosphere with liquid ammonia (20mL) and lithium wire (0.14g, 20 mmol). after stirring for 1.5 hours at-40 ℃ to-50 ℃, the dark blue reaction was quenched with solid ammonium chloride, slowly warmed to room temperature, and the residual ammonia was driven off using nitrogen gas, then water (3mL) was added to the reaction flask and the mixture was extracted with dichloromethane (3 × 30 mL.) the combined organic layers were extracted with Na₂SO₄Dried and concentrated in vacuo, followed by HPLC purification to give the title compound as a white foam (100mg, 50%). NMR (D)₂O)2.32(m，1H)，2.00(m，2H)，1.77-1.90(m，2H)，1.70(m，1H)，1.35-1.50(m，3H)，1.24(m，1H)，1.12(m，1H)，0.60-0.75(m，2H)。MS(M+1)：216.3；MS(M-H₂O+1)：198.2；MS(M-2H₂O+1)：180.3。

Example 9: preparation of (3R, 4S) -3-amino-4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

Step 1: synthesis of tert-butyl (3R, 4S) -3- (tert-butylcarbamoyl) -3- (N- ((S) -1-phenylethyl) acetylamino) -4- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine-1-carboxylate

To a stirring solution of tert-butyl (S) -3-allyl-4-oxopyrrolidine-1-carboxylate (0.451g, 2.0mmol) in 2, 2, 2-trifluoroethanol (1.5mL) under nitrogen was added (S) - α -methylbenzylamine (1.03mL, 8mmol), glacial acetic acid (0.46mL, 8mmol) and tert-butylisonitrile (0.68mL, 6 mmol). The reaction mixture was stirred at room temperature for three days and then at 60 ℃ for 6 hours.the mixture was then diluted with dichloromethane (15mL) and added directly to a silica gel column (175 mL). gradient elution of the column using 20%, 30% and 40% ethyl acetate/heptane gave a mixture of the unresolvable intermediate diastereoisomers (0.477g, 51%, 2: 1 mixture). The mixture of diastereoisomers (0.472g, 1.00mmol) in anhydrous dichloromethane (5mL) was added under inert atmosphere to this mixture, followed by addition of dichloromethane-1.025 mmol) and, after the mixture was slowly stirred to a stirring solution of iridium-1-carboxylate, the mixture was added to a reaction mixture of iridium-1-borane, followed by addition of aqueous solution, stirring, followed by addition of sodium chloride-18 mmol, stirring, followed by addition of sodium chloride-2-1-ethyl acetate, stirring, addition of the reaction mixture to a slow addition of sodium chloride-17-bis-bromoborane, followed by addition of sodium chloride-2-1-ethyl acetate, stirring, and addition of the reaction mixture, followed by addition of the reaction mixture, stirring, and addition of sodium chloride, followed by addition of sodium chloride, and addition₄Dried and then concentrated in vacuo. The obtained residue was dissolved in heptane containing a small amount of ethyl acetate and loaded onto a silica gel column (175 cc). Purification was achieved by eluting the column with a mixture of ethyl acetate and heptane using the following concentrations: 25% ethyl acetate/heptane, then 30% ethyl acetate/heptane, and finally 35% ethyl acetate/heptane to give the title compound as a colorless foam (289mg, 48%). NMR (CDCl)₃)：7.70(d，J-7.5Hz，2H)，7.35(t，J＝7.5Hz，2H)，7.25(m，1H)，6.00(m，1H)，4.88(m，1H)，4.40-4.70(m，1H)，3.80(m，1H)，3.10-3.30(m，2H)，2.70(m，1H)，1.00-1.80(m，40H)，0.60-0.80(m，2H)。MS(M+1)：600.2。

Step 2: synthesis of (3R, 4S) -3-amino-4- (3-boronopropyl) pyrrolidine-3-carboxylic acid

(S, S) -4- (N-acetyl-N- (1S-phenylethyl) amino) -4- (tert-butylamino) carbonyl-3- (3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) propyl) pyrrolidine (0.165g, 0.275mmol) was purified by HPLC to give the title compound (61mg, 77%) as a white solid. No additional procedure for removal of phenethyl groups from the amines was required. NMR (D)₂O)3.86(d，J＝12.5Hz，1H)，3.70(dd，J₁＝11.5，J₂＝8.5Hz，1H)，3.42(d，J＝12.5Hz，1H)，3.15-3.30(m，1H)，2.45-2.60(m，1H)，1.50-1.65(m，1H)，1.10-1.40(m，3H)，0.60-0.75(m，2H)。MS(M+1)：216.9；MS(M-H₂O+1)：199.0；MS(M-2H₂O+1)：180.9。

Route of administration and dosing regimen

Although there is ample evidence that arginase inhibition is relevant to the treatment of various diseases and conditions, only a limited number of compounds capable of inhibiting arginase activity are known. Accordingly, the present invention provides compounds and pharmaceutical compositions thereof that are useful in treating individuals suffering from such diseases or conditions as described more generally above.

The compounds or compositions of the present invention can be formulated as described above and are suitable for administration to a subject in a therapeutically effective amount in any number of ways. The therapeutically effective amount of a compound of the invention may depend on the amount and type of excipients used, the amount and specific type of active ingredient in the dosage form, and the route by which the compound is administered to the patient. However, a typical dosage form of the invention comprises a compound, or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof, and a pharmaceutically acceptable carrier.

Typical dosage levels of the compounds of the invention will generally range from about 0.001 to about 100mg/kg of patient body weight per day, which may be administered in single or multiple doses. Exemplary dosages are from about 0.01 to about 25 mg/kg/day or from about 0.05 to about 10 mg/kg/day. In other embodiments, the dosage level is from about 0.01 to about 25 mg/kg/day, from about 0.05 to about 10 mg/kg/day, or from about 0.1 to about 5 mg/kg/day.

The dose is typically in the range of about 0.1 mg/day to about 2000 mg/day, administered as a single dose once a day or as divided doses throughout the day, optionally taken with food. In one embodiment, the daily dose is administered in divided doses twice daily. Daily dosages may range from about 5 mg/day to about 500 mg/day, for example, between about 10 mg/day and about 300 mg/day. In administering the patient, treatment may be initiated at a lower dose (perhaps about 1mg to about 25mg) and increased as necessary to about 200 mg/day to about 2000 mg/day, administered as a single dose or divided into multiple doses depending on the overall response of the patient.

Depending on the disease to be treated and the condition of the subject, the compounds of the invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation, nasal, vaginal, rectal, sublingual or topical (e.g., transdermal, topical) routes of administration. The compounds may be formulated, individually or together, in suitable dosage unit formulations for each route of administration containing the usual non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles as described above. The invention also encompasses administration of a compound of the invention in a depot formulation wherein the active ingredient is released over a predetermined period of time.

Method and use

The compounds of the invention are useful for inhibiting the expression or activity of arginase I, arginase II, or a combination of these enzymes. The enzymes of the arginase family play an important role in the regulation of the physiological levels of L-arginine, a precursor of the signaling molecule Nitric Oxide (NO), and in the regulation of the levels of L-ornithine, a precursor of certain polyamines of important physiological signal transduction proteins.

More specifically, the present invention provides methods and uses for inhibiting arginase I, arginase II, or a combination thereof in a cell comprising contacting the cell with at least one compound of the present invention or a composition thereof as described herein. In some embodiments, the present invention provides methods of treating or preventing a disease or condition associated with expression or activity of arginase I, arginase II, or a combination thereof in a subject.

For example, the disease or condition is selected from the group consisting of: cardiovascular disorders, gastrointestinal disorders, sexual disorders, pulmonary disorders, immune disorders, infections, autoimmune disorders, pulmonary disorders, and hemolytic disorders.

According to one embodiment, the compounds of the present invention are candidate therapeutic agents useful for the treatment of cardiovascular disorders, such as diseases or disorders selected from the group consisting of: hypertension (including systemic hypertension, Pulmonary Arterial Hypertension (PAH), plateau pulmonary hypertension), Ischemia Reperfusion (IR) injury, myocardial infarction, atherosclerosis.

Exemplary conditions that can be treated using the compounds of the present invention are diseases or conditions selected from the group consisting of: peyronie's Disease and Erectile Dysfunction (ED).

In one embodiment, the arginase inhibitor of the present invention is suitable for treating a lung disorder selected from the group consisting of: chemically induced pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, Chronic Obstructive Pulmonary Disease (COPD), and asthma.

The compounds of the invention may also be used to treat gastrointestinal disorders, such as diseases or conditions selected from the group consisting of: gastrointestinal motility disorders, gastric cancer, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and gastric ulcers.

Transplantation of organs (such as liver, kidney and heart) increases the risk of ischemia reperfusion Injury (IR). The compounds of the invention are useful for protecting transplanted organs from IR during transplantation.

According to one embodiment of the invention, the compounds of the invention are useful for the treatment of autoimmune disorders. Exemplary diseases or conditions include, but are not limited to, encephalomyelitis, multiple sclerosis, antiphospholipid syndrome 1, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyradiculoneuropathy, dermatitis herpetiformis, dermatomyositis, myasthenia gravis, pemphigus, rheumatoid arthritis, stiff person syndrome, type 1 diabetes, ankylosing spondylitis, Paroxysmal Nocturnal Hemoglobinuria (PNH), paroxysmal cryohemoglobinuria, severe idiopathic autoimmune hemolytic anemia, and Goodpasture's syndrome.

The arginase inhibitors of the present invention may also be used to treat immune disorders, such as diseases or conditions selected from the group consisting of: myeloid-derived suppressor cell (MDSC) -mediated T cell dysfunction, Human Immunodeficiency Virus (HIV), autoimmune encephalomyelitis, and ABO-non-transfusion-responsive.

In one embodiment, the compounds of the invention are useful as candidate therapeutic agents for treating a subject suffering from a hemolytic disorder. Exemplary diseases or conditions include, but are not limited to, sickle-cell disease, thalassemia, hereditary spherocytosis, polycythemia oris, microangiopathic hemolytic anemia, pyruvate kinase deficiency, infection-induced anemia, cardiopulmonary bypass, and mechanical heart valve-induced anemia and chemically-induced anemia.

Other exemplary disease conditions in which the compounds described herein are candidate therapeutic agents are inflammation, psoriasis, leishmaniasis (leishmaniasis), neurodegenerative disease, wound healing, Hepatitis B Virus (HBV), helicobacter pylori (h. pylori) infection, fibrotic disease, arthritis, candidiasis, periodontal disease, keloids, adenoid tonsillar disease, african narcolepsy, and Chagas' disease.

Advantageously, the compounds of the invention are particularly useful for treating a disease or condition selected from the group consisting of: pulmonary Arterial Hypertension (PAH), Erectile Dysfunction (ED), hypertension, myocardial infarction, atherosclerosis, nephropathy, asthma, inflammation, psoriasis, immune responses, T cell dysfunction (such as myeloid-derived suppressor cell (MDSC) -mediated T cell dysfunction), leishmaniasis, ischemia reperfusion injury, sickle cell disease, neurodegenerative disease, wound healing, Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), helicobacter pylori infection, and fibrotic diseases (such as cystic fibrosis). In addition, the compounds described herein are useful for protecting organs, such as during organ transplantation.

In one embodiment, the subject receiving treatment is a mammal. For example, the methods and uses described herein are suitable for human medical use. Alternatively, the methods and uses are also suitable for a veterinary setting, where individuals include (but are not limited to) dogs, cats, horses, cattle, sheep, lambs, and reptiles.

The following are more detailed descriptions of diseases and conditions.

Erectile dysfunction

The observation of differences in arginase activity in the penis of young mice versus older mice led to the following conclusions: arginase may play a role in Erectile Dysfunction (ED). In this context, Changen (Champion) et al (am. J. Physiol. Heart and circulation physiology) 292: 340-351 (2006) and the biochemical and biophysical research communication (biochem. and Biophys. research microorganisms), 283: 923-27 (2001)) observed increased mRNA expression levels and arginase proteins and decreased activity of constitutively active NOS in aged mice.

Nitric oxide is associated with non-adrenergic, non-cholinergic neurotransmission that results in smooth muscle relaxation in the corpus cavernosum to achieve penile erection (New England Journal of medicine, 326, (1992)), and thus erectile dysfunction can be generally treated by increasing Nitric Oxide (NO) levels in the penile tissue. The increase in Nitric Oxide (NO) levels in the tissue can be achieved by inhibiting arginase activity in penile tissue of an aged individual. In other words, it has been postulated that arginase depletes the cell pool of free L-arginine available to NOS, resulting in lower Nitric Oxide (NO) levels and erectile dysfunction. See Christianson et al, (chemical research review (Acc. chem. Res.), 38: 191-S201, (2005)) and (Nature Structural Biol., 6 (11): 1043-S1047, (1999)). Therefore, inhibitors of arginase may play a role in the treatment of erectile dysfunction.

Pulmonary hypertension

Alterations in arginine metabolism have been suggested to be associated with the pathogenesis of pulmonary hypertension (Xu), et al, union of the american society for biology (fasebj.), 18: 1746-48, 2004). The proposal is based in part on the following findings: arginase II expression and arginase activity are significantly increased in pulmonary artery endothelial cells obtained from lung explants of patients with class I pulmonary hypertension.

In addition, secondary pulmonary hypertension is becoming one of the leading causes of mortality and morbidity in patients with hemolytic anemia (such as thalassemia and sickle cell disease). The underlying cause of secondary pulmonary hypertension is impaired nitric oxide bioavailability caused by post-hemolytic arginase release that reduces the pool of free arginine required for Nitric Oxide (NO) synthesis. Thus, inhibition of arginase activity may provide a potential therapeutic approach for the treatment of pulmonary hypertension.

Hypertension (hypertension)

Xuw et al, journal of the american society for biology, journal 2004, 14, 1746-8, suggested a fundamental role for arginase II in blood pressure regulation. In this context, high levels of vascular arginase are associated with a concomitant decrease in vascular Nitric Oxide (NO) in hypertensive animals. For example, a rat that is genetically predisposed to hypertension (i.e., spontaneously)Hypertensive rats), but administration of the antihypertensive agent hydralazine (hydralazine) reduced blood pressure while the expression level of vascular arginase was reduced, thereby indicating a strong correlation between arginase activity and blood pressure (fibrate (Berthelot) et al, Life Sciences, 80: 1128-34, (2008)). Similar administration of the known arginase inhibitor N in spontaneously hypertensive rats^ω-hydroxy-n-L-arginine (n-NOHA) lowers blood pressure and improves the vascular response of resistant vessels to blood flow and blood pressure, thereby highlighting inhibitors of arginase as candidate therapeutics for the treatment of hypertension (demux (Demougeot) et al, journal of hypertension (j. hypertension), 26: 1110-18, (2008)).

Arginase also plays a role in reflex cutaneous hypertension by reducing the cellular level of Nitric Oxide (NO). Nitric oxide causes vasodilation and the level of Nitric Oxide (NO) is normally increased or decreased to keep blood pressure at physiologically acceptable levels. Kenny (Kenny) et al (journal of Physiology, 581(2007)863-872) postulate that reflex vasodilation in hypertensive individuals may attenuate arginase inhibition, thereby suggesting an role of arginase inhibitors for the treatment of hypertension.

Asthma (asthma)

Arginase activity is also associated with airway hyperresponsiveness in asthma. For example, arginase I is upregulated in human asthmatic patients and mice with acute and chronic asthma, while the levels of arginase II and NOS isoforms remain unchanged (Scott et al, am. j. physiol. lungcell. physiol.) 296: 911-920 (2009)). In addition, methacholine (methacholine) -induced central airway reactivity in the murine chronic model was attenuated following administration of the arginase inhibitor S- (2-boronoethyl) -L-cysteine. The similarity between the expression profiles of ARG I in humans and mice with chronic asthma suggests that compounds capable of inhibiting arginase activity are candidate therapeutic agents for the treatment of asthma.

Additional evidence suggests additional correlations between increased arginase activity in lung tissue and disease progression in asthmatic patients, such as up-regulation of genes involved in the metabolism of cationic amino acids in asthmatic mice, including arginase I and II (rosenberg et al, journal of clinical research (j. clin. invest.), 111: 1863-74(2003) and milrs (sources) et al, reviews on drug experts (Expert opin. investig Drugs), 14 (10: 12211231 (2005)).

In addition, the levels of all amino acids were lower in the plasma of asthmatic patients compared to normal individuals, but the levels of arginine were significantly lower in the plasma (Morris et al, am. J. Respir. Crit Care Med.), 170: 148- "154, (2004)). Thus, arginase activity is significantly increased in the plasma of asthmatic patients, where an increased level of arginase activity contributes to a lower bioavailability of plasma arginine, resulting in a deficiency of Nitric Oxide (NO), which is responsible for the excessive airway responses in asthmatic patients.

Inflammation(s)

Arginase activity is also associated with autoimmune inflammation (Chen) et al, Immunology (Immunology), 110: 141-148, (2003)). The authors identified up-regulation of expression levels of the ARG I gene in murine spinal cord cells from animals undergoing Experimental Autoimmune Encephalomyelitis (EAE). However, administration of the arginase inhibitor amino-6-boronohexanoic Acid (ABH) resulted in a more mild form of EAE occurring in the animals compared to control animals. These results indicate a therapeutic effect of arginase inhibitors for the treatment of autoimmune encephalomyelitis.

Furthermore, Holuovitz et al, (American J. Physiol gastroenterological Liver physiology (Physiol), 292: G1323-36, (2007)) suggest a role for arginase in vascular pathophysiological conditions. For example, these authors state that there is a loss of Nitric Oxide (NO) production in the long-term inflamed intestinal vessels in patients with Irritable Bowel Disease (IBD), crohn's disease, and ulcerative colitis. Loss of Nitric Oxide (NO) production is associated with upregulation of arginase expression and activity that reduces arginine levels, thereby preventing Nitric Oxide Synthase (NOs) from synthesizing Nitric Oxide (NO). Thus, inhibitors of arginase activity may be candidate therapeutic agents for the treatment of vascular pathophysiological conditions.

Ischemia reperfusion

Arginase inhibition is also proposed to play a cardioprotective role during ischemia reperfusion. More specifically, inhibition of arginase prevents myocardial infarction through a mechanism that may depend on NOS activity and the resulting bioavailability of Nitric Oxide (NO) (Burnaw et al, (Cardiovascular Research, 85: 147-154 (2010)).

Myocardial infarction and atherosclerosis

Polymorphisms in arginase I are associated with increased risk of myocardial infarction and development of intimal-media thickening in the carotid arteries, which is considered a reliable indicator of atherosclerosis and other coronary artery diseases (brusseau et al, (journal of medical Genetics (j.med Genetics), 44: 526-, increased levels of ARGII in atherosclerotic mice indicate the role of inhibitors of arginase as candidate therapeutics for treating atherosclerosis.

In addition, studies by Ming et al (recent Reports of Hypertension, 54: 54-59, (2006)) indicate that upregulation of arginase, but not endothelial Nitric Oxide (NO) dysfunction, plays an important role in cardiovascular disorders, including atherosclerosis. The association of arginase with cardiovascular disease is further supported by the following observations: upregulation of ARGI and ARGII activity in cardiomyocytes, which in turn negatively affects NOS activity and myocardial contractility. (see Margelis et al, J.physiol.USA-Heart & circulatory physiology, 290: 1756-62, (2006)).

Immune response

The arginine/Nitric Oxide (NO) pathway may also play a role in immune responses, such as following organ transplantation. For example, it is hypothesized that reperfusion of orthotopic liver grafts results in a significant increase in the level of ornithine due to upregulation of arginase activity in the graft (scax (Tsikas) et al, (Nitric oxide, 20: 61-67, (2009)). increased levels of hydrolytic and proteolytic enzymes in the graft may lead to less favorable outcomes for the transplanted organ.

Psoriasis disease

Arginase has been shown to play a role in the pathogenesis of psoriasis. For example, ARG I is highly expressed in hyperproliferative psoriasis and is in fact responsible for the down-regulation of Nitric Oxide (NO), which is an inhibitor of cell proliferation by competing with the common substrate L-arginine, as postulated by d. The renewed work of Abelian (Abeyakrthi) et al (British J.Dermatology), (2010)) and Burkhett et al (WO/2007/005620) supports the finding of lower Nitric Oxide (NO) levels in keratinocytes of psoriatic patients. Abelian et al found that keratinocytes in psoriatic patients were poorly differentiated and hyperproliferated. It is postulated that the poor differentiation is caused by a low level of Nitric Oxide (NO) not due to poor expression of NOs but due to overexpression of arginase competing with NOs for the substrate L-arginine. Thus, inhibition of arginase may provide therapeutic relief from psoriasis.

Wound healing

Under normal physiological conditions, Nitric Oxide (NO) plays an important role in promoting wound healing. For example, Herster (Hulst) et al, (nitric oxide, 21: 175-. Immediately after injury, it is desirable to increase the tissue level of Nitric Oxide (NO) in order to promote angiogenesis and cell proliferation, which are vital for healing. Therefore, inhibitors of arginase are useful as therapeutic agents for the treatment of wounds, as the compounds increase tissue levels of Nitric Oxide (NO). Soxh (South) et al (Experimental Dermatology, 29: 664-668(2004)) provided more support for the use of arginase inhibitors as candidate therapeutics for treating wounds, and they found that arginase I increased 5-fold in chronic wounds, such as skin erosion and blistering.

Cystic fibrosis

Cystic Fibrosis (CF) is a multisystemic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Common symptoms of CF are persistent lung infection, dyspnea, pancreatic insufficiency, and elevated sweat chloride levels. CF can be fatal if left untreated, with lung diseases caused by mucus plugging and decreased mucociliary clearance being the leading cause of morbidity and mortality.

It has been asserted that patients with Cystic Fibrosis (CF) have increased plasma and sialarginase activity with concomitant reduction in plasma 1-arginine levels (H. Grasemann et al, J. Respiro & Critical Care Med., 172(12) (2005) 1523-. However, an increase in arginase activity results in lower physiological levels of Nitric Oxide (NO), which can cause a decrease in airway obstructive lung function in patients with Cystic Fibrosis (CF).

Electric field-induced smooth muscle relaxation stimulation in the airways of the mouse CF model was impaired and administration of 1-arginine and NO reversed this effect, as suggested by m. mohanna (m. mhanna), et al, american journal of respiratory and molecular biology (am.j. respir. cell mol. biol.), 24 (2001) 621-. Grisman et al found a positive correlation between lung function and the concentration of NO and NO metabolites released in saliva of CF patients (Grisman H; Mikler E (Michler, E.); Vallotte M (M.); Ratjen, F.), pediatric Pulmonol 1997, 24, 173-7).

Taken together, these results indicate that increased arginase activity in CF causes NO deficiency and lung obstruction in CF by limiting NOS availability of 1-arginine. Therefore, inhibitors of arginase activity are candidate therapeutic agents for the treatment of Cystic Fibrosis (CF).

Organ protection

Another therapeutic approach for the compounds of the present invention is to protect an organ during transport of the organ from a donor to the site where it is transplanted into a recipient. Ischemia reperfusion Injury (IR) due to exposure of the transplanted organ to warm ischemia (time from donor until flushing with preservation medium) and cold ischemia (cryopreservation) for a period of time is often observed in patients undergoing transplant surgery. Ischemia reperfusion Injury (IR) and the accompanying primary graft dysfunction and/or acute or chronic rejection are due to altered cellular activity of the L-arginine/NO pathway.

It has been proposed that arginase 1 and arginase 2 be released from apoptotic endothelial cells and kidney cells within the first 24 hours of organ removal from the body. To counteract the released arginase, L-arginine is added to the preservation medium. Results from dog kidney transplants indicate that the addition of L-arginine reduces the incidence and severity of ischemia, resulting in lower levels of MDA at 1 hour post-transplantation and lower levels of BUN and serum creatinine during the first 72 hours. See alcassap S (Erkasap, S.); astes E (Ates, E.), journal of renal disease and dialysis kidney transplantation (Nephrol DialTransplant) 2000, 15, 1224-7.

For dog lung transplantation, similar results were observed over a 24 hour period when lungs were stored in University of Wisconsin (University of Wisconsin) solution supplemented with L-arginine. Severe (Yen) et al observed that the addition of L-arginine to storage media increased Lung endothelial protection and decreased the incidence of ischemia compared to controls stored in media not containing L-arginine (Zhu Y (Chu, Y); Wu Y.C (Wu, Y.C.; week Y.C. (Chou, Y.C.); percept H.Y (Chueh, H.Y), Liu HP (Liu HP), Zhu JJ (Chu JJ), Lin PJ (Lin PJ), journal of cardiopulmonary transplantation (J Heart Lung Transplant.)2004, 23, 592-8).

Koch et al allegedly, when heart was preserved in HTK solution with L-arginine and N- α -acetyl-histidine, myocardial contractility and relaxation in rat myocardium after transplantation were improved (Koch a), ladovich t (radovits t), loganashan s (logatahan s), seck fu (sack fu), kake m (karck m), shabo GB (Szab usa GB.), report on transplantation (Transplant Proc.)2009, 41, 2592-4).

Thus, the addition of an arginase inhibitor may be a candidate therapeutic regimen to prevent and/or reduce the incidence and risk of ischemia reperfusion injury by synergistically increasing the organ protective effects of the preservation medium. The arginase inhibitors of the present invention are useful as therapeutic agents for preserving organs under conditions where the number of available organs suitable for transplantation is small and the organs are lost and damaged due to ischemic attack, in order to increase the organ availability by reducing the amount of ischemia-reperfusion injury during organ transplantation.

Leishmaniasis

Leishmaniasis is caused by protozoa and manifests as cutaneous leishmaniasis (i.e., skin infections that cause low-pigmented nodules) and visceral leishmaniasis (more severe, affecting internal organs). Arginase is postulated to play a role in disease progression as parasites rely on arginase to synthesize cellular polyamines essential for pathogenesis. Thus, inhibition of arginase reduces cellular parasite burden and promotes increased Nitric Oxide (NO) levels, thereby enhancing parasite clearance. See Liu FY (Liew FY) et al, European journal of immunology (Eur J Immunol), 21(1991)2489, Immunostat V (Inieta V) et al, Parasite immunology (Parasite Immunol.), 24(2002) 113-. Thus, the compounds of the present invention are useful as therapeutic agents for the treatment of leishmaniasis.

Myeloid Derived Suppressor Cell (MDSC)

MDSCs are potent immunomodulators that limit immune responses via several pathways, such as depletion of L-arginine via release of arginase 1 into the microenvironment (Rodriguez 2009 Cancer study (Cancer Res)), MHC-restricted inhibition (nagareri s (nagaraj s), guprata k (gupta k), pisarev (pisarev v), canalski L (kinarsky L), sielman s (sherman s), kang L (kang L), heberer dl (herber dl), seneke j (schneck j), gareliveryvie dl (gabrilovich dl), nature medicine (Nat Med) 2007, 13, 828-35), induced T regulatory cells (selafenib p (serafini p), gerberney s (mgebroff s), nebruonelli k (borreli L), borreli I (borreli jl), borreli jl (borrorrei) and IL production (rogrue jc) 49, gerei jc, gerue L, gerei L) via release of arginase 1 into the microenvironment, joxas gc (gonzalez gc), zhang l (zhang l), labelain g (ibrahim g), kelly jj (kelly jj), gustafsen mp (gustafson mp), lin y (lin y), dietz ab (dietz ab), fossa pa (forsyth pa), wara vw (yong vw), parner IF (Parney IF.), neurooncology (NeuroOncol 2010, 12, 351-65) (sinha p), clemetvk (clements vk), bungartsk (buntsk), albeda sm (albelda sm), Ostrand-roberg S (ostran-roberg S, journal of immunology, 179, 9783-9783).

Tumorigenesis is postulated to be accompanied by an increase in the number of MDSCs (both peripheral and infiltrating within the tumor). See aldard b (almand b), clark ji (clark ji), nikitina e (nikitina e), faneberg j (van Beynen j), engley nr (english nr), neet sc (knight sc), cabberen dp (carbon dp), garrilovich DI (Gabrilovich DI.), immunology journal 2001, 166, 678-89, and drape rayleigh D (Gabrilovich D.) for natural review: immunology (Nat Rev Immunol.)2004, 4, 941-52.Treatment of tumor bearing mice with established chemotherapeutic agents, such as gemcitabine (gemcitabine) and 5-Fluorouracil (5-Fluorouracil), abrogates MDSC immunosuppression and results in delayed tumor growth. See, respectively, lehk (le hk), grem l (graham l), chare (cha e), morales jk (morales jk), mangrove mh (manjili mh), bel HD (Bear HD), international immunopharmacology (Int Immunopharmacol) 2009, 9, 900-9 and wenston j (vincent j), milano g (mignot g), salman f (chalmin f), ladori s (ladoire s), brussel m (uchard m), sheffrio a (chevriaux a), martin f (martin f), alpeh l (apetoh l), rapebuire C (C m), schroe l (r a), martin f (r f), alpeh l (apeto l), rapel C (C)Guillingelli F. (Ghiringhelli F.), cancer study 2010, 70, 3052-61. Furthermore, inhibition of arginase 1 enhances anti-tumor immunity by decreasing MDSC function. Thus, inhibitors of arginase (e.g., compounds of the invention) reduce or delay tumor growth and may be used in combination with established anti-cancer agents to treat cancer.

Helicobacter pylori (Helicobacter pylori, H. pylori)

Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa. Bacterial colonization can lead to acute or chronic gastritis and is highly associated with peptic ulcer disease and gastric cancer. The observation that addition of L-arginine to co-cultures of h.pylori and macrophages increases Nitric Oxide (NO) -mediated killing of h.pylori (charvedi r), (chaturvedi r), acem m (asim m), liuyi nd (lewis nd), algold hm (algoodhm), carvel tl (cover tl), aury (kim py), wilson kt (wilson kt), infection and immunity (infection Immun.)2007, 75, 4305-15) supports the hypothesis that bacterial arginase competes with macrophage arginase for free arginine required for Nitric Oxide (NO) synthesis. See, Gofibrate AP (Gobert AP), Migji DJ (McGee DJ), Alertal M (Akhtar M), Mentz GL (Mendz GL), Newton JC (Newton JC), Zheng Y (Cheng Y), Mobuli HL (Mobley HL), Wilson KT., Proc Natacad Sci USA 2001, 98, 13844-9. L-arginine is required for T cell activation and rapid bacterial clearance from infected cells. By depleting the free L-arginine pool in vivo, h.pylori reduced arginine-induced CD3 ζ expression on T cells and prevented T cell activation and proliferation. See, sabalata j (zabalata j), mikiji dj (mcgee dj), zie ah (zea ah), ehrondes CP (Hern index CP), rodriegs pc (rodriguez pc), sela ra (sierra ra), corea p (corea p), geoya ac (ochoa ac), journal of immunology 2004, 173, 586-93.

However, inhibition of bacterial arginase using the known inhibitor NOHA re-establishes CD3 expression on T cells (sapalata J2004) and enhances macrophage NO production, thereby promoting macrophage-mediated clearance of bacteria from infected cells. See Charles Wedi R, acem M, Liuyi ND, Alder HM, carveol TL, gold PY, Wilson KT, infection and immunity 2007, 75, 4305-15.

Furthermore, Liuyi et al have suggested a role for arginase II in H.pylori infection. For example, these authors indicated that argII-/-primary macrophages incubated with H.pylori extracts showed enhanced NO production and correspondingly increased (about 15%) NO-mediated bacterial cell killing (Liuyi ND, Acimem, Barry DP (Barry DP), CingeK (Singh K), Texulaet T (de Sablet T), Bochester JL (Boucher JL), Gofibrate AP, Charpy Weddir R, Wilson KT., J Immunol 2010, 184, 2572-82). Thus, inhibitors of arginase activity may be candidate therapeutic agents for the treatment of vascular pathophysiological conditions. Therefore, inhibitors of arginase activity may be candidate therapeutic agents for the treatment of helicobacter pylori infection and for the treatment of gastric ulcers, peptic ulcers and cancer.

Sickle Cell Disease (SCD)

Sickle Cell Disease (SCD) or sickle cell anemia or sickle cell disease (drenocytosis) is a genetic blood disorder characterized by red blood cells that assume an abnormal, non-deforming sickle shape. Sickling reduces the flexibility of the cells and increases the risk of complications. An increase in the concentration of Reactive Oxygen Species (ROS) in the circulation leads to blood cell adhesion and NO depletion, which results in poor vasodilation or failure of the blood vessels to vasodilate. The inability to vasodilate and increased blood cell adhesion in SCD leads to vaso-occlusive crisis and pain.

Low levels of plasma L-arginine are commonly detected in patients with SCD (morris cr), garcinia gj (kato gj), poliomyelia vich m (poljakovic m), wang x (wang x), blakewed wc (blackwelder wc), sakakfv (sachdev v), samson sl (hazen sl), viscinki ep (vichinsky ep), morris SM (morris SM jr), glade temperature mt. (Gladwin MT.), journal of the american medical society (JAMA) 2005, 294, 81-90). According to these authors, lysis of red blood cells (RBC's) in patients with SCD causes arginase release and subsequent reduction of physiological L-arginine levels. This series of biological events reduces the physiological concentration of Nitric Oxide (NO), a signaling molecule that plays a role in vasodilation. Other biological events also limit the bioavailability of NO. These include, for example, the uncoupling of Nitric Oxide Synthase (NOS) and the subsequent reduction of physiological NO levels, and superoxide (O)^-2) The reactive oxygen species react with NO to chelate the latter to ONOO^-。

Based on these observations, the inventors propose inhibitors of arginase, particularly arginase I inhibitors, as candidate therapeutic agents for patients with sickle cell disease. As described above, SCD causes eNOS uncoupling due to low physiological levels of L-arginine. However, inhibition of arginase present in the blood circulation can solve this problem by increasing the physiological level of L-arginine, a substrate of endothelial nitric oxide synthase (eNOS). Importantly, the inventors propose that this series of events enhances endothelial function and alleviates the vasoconstriction associated with SCD.

Human Immunodeficiency Virus (HIV)

HIV is caused by a virus that infects CD4+ helper T cells and causes severe lymphopenia, making infected individuals susceptible to opportunistic infections. Although antiretroviral therapy (ART) is widely used against HIV infection, the widespread use of antiretroviral drugs has led to the development of HIV resistant strains.

There is a correlation between the activity of arginase in patients with HIV and the severity of the HIV disease. That is, increased arginase activity has been linked to increased viral titers in HIV patients. These patients also showed reduced serum arginine levels and reduced levels of CD4+/CD8+ cells.

Taken together, these observations suggest the role of arginase inhibitors (e.g., compounds of formula I or II) as candidate therapeutic agents for the treatment of HIV infection.

Chronic Hepatitis B Virus (HBV)

Chronic hepatitis b infection is a viral disease transmitted by contact with infected body fluids. Chronic HBV infection is characterized by liver inflammation and jaundice, and if left untreated, can cause cirrhosis, which can progress to hepatocellular carcinoma. However, currently used antiviral drugs have a low efficacy against chronic HBV infection. Serum and liver homogenates from patients with chronic HBV infection show reduced levels of arginine and increased arginase activity. In addition, for infected patients, increased arginase activity is associated with an impaired Cytotoxic T Lymphocyte (CTL) response (with reduced IL-2 production and CD3z expression).

However, supplementation of serum arginine to physiologically acceptable levels restores CD3z and IL-2 expression, suggesting a role for arginase inhibitors as potential therapeutic agents for the treatment of chronic HBV infection.

Inhibition of arginase

The compounds of the invention inhibit human arginase i (arg i) and arginase ii (arg ii) as demonstrated by ex vivo assays as described by the published protocols (bargo (Baggio) et al, journal of pharmacology and experimental therapeutics (j. pharmacol. exp. ther.), 1999, 290, 1409-. The assay determines the inhibition required to reduce arginase activity by 50%Concentration of formulation (IC)₅₀)。

Assay protocol

The inhibition of arginase I (ARG I) and arginase II (ARG II) by the compounds of the present invention was followed by spectrophotometric measurements at 530 nm. The compounds to be tested were dissolved in DMSO at an initial concentration that was 50 times the final concentration in its cuvette. Mu.l of the stock solution was diluted in 90. mu.l of assay buffer comprising 0.1M sodium phosphate buffer (pH 7.4) containing 130mM NaCl to which Ovalbumin (OVA) was added at a concentration of 1 mg/ml. Solutions of arginase I and II were prepared in 100mM sodium phosphate buffer (pH 7.4) containing 1mg/ml OVA to give a final arginase stock solution at a final concentration of 100 ng/ml.

To each well of a 96-well microtiter plate, 40. mu.l of enzyme, 10. mu.l of the compound of the invention and 10. mu.l of enzyme substrate (L-arginine + manganese sulfate) were added. For wells used as positive controls, only the enzyme and its substrate were added, while wells used as negative controls contained only manganese sulfate.

After incubating the microtiter plate at 37 ℃ for 60 minutes, 150. mu.l of urea reagent obtained by combining reagents A and B in equal proportions (1: 1) was added to each well of the microtiter plate to terminate the reaction. The urea reagent was prepared just prior to use by combining reagent a (10 mM o-phthalaldehyde and 0.4% polyoxyethylene (23) lauryl ether (w/v) in 1.8M sulfuric acid) with reagent B (1.3 mM primaquine diphosphate, 0.4% polyoxyethylene (23) lauryl ether (w/v), 130mM boric acid in 3.6mM sulfuric acid). After quenching the reaction mixture, the microtiter plate was allowed to stand at room temperature for an additional 10 minutes for color development. Inhibition of arginase was calculated by measuring the Optical Density (OD) of the reaction mixture at 530nm and normalizing the OD values to the percent inhibition observed in the control. The normalized OD was then used by comparing the normalized OD values to log [ concentration ]]Plotting and Generation of dose-response curves Using regression analysis to calculate IC₅₀The value is obtained.

Table 3 below shows the efficacy of the compounds of the invention in amounts of 1 to 5The table is ranked, i.e., the most potent compound is designated 1 and the least potent compound is designated 5. Thus, potency value 1 refers to IC₅₀A compound of the invention with a value in the range of 0.1nM to 25 nM; potency value 2 refers to IC₅₀A compound of the invention with a value in the range of 26nM to 100 nM; compounds with potency value 3 show IC in the range of 101nM to 500nM₅₀A value; will IC₅₀Compounds of the invention with values in the range of 501nM to 1500nM were assigned potency values of 4 and IC was assigned₅₀Compounds with values greater than 1501nM were assigned an efficacy value of 5.

TABLE 3

^aOrder of potency (highest-lowest): 1-0.1 nM → 25 nM; 2-26 nM → 100 nM; 101nM → 500 nM; 4 ═ 501nM → 1500 nM; and 5 1501nM → higher.

The foregoing examples are intended to illustrate certain embodiments of the invention, which is defined solely by the claims. In addition, all publications cited herein are incorporated by reference as if fully set forth herein.

Claims (20)

1. A compound selected from the following table:

or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition, comprising:

(i) a therapeutically effective amount of at least one compound according to claim 1, or a pharmaceutically acceptable salt thereof; and

(ii) a pharmaceutically acceptable carrier.

3. Use of at least one compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for inhibiting arginase I, arginase II, or a combination thereof in a cell.

4. Use of at least one compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating or preventing a disease or condition associated with expression or activity of arginase I, arginase II, or a combination thereof in a subject.

5. The use of claim 4, wherein the disease or condition is selected from the group consisting of: cardiovascular disorders, sexual disorders, wound healing disorders, gastrointestinal disorders, immunological disorders, infections, pulmonary disorders, and hemolytic disorders.

6. The use of claim 5, wherein the disease or condition is a cardiovascular disorder selected from the group consisting of: systemic hypertension, pulmonary arterial hypertension PAH, high altitude pulmonary hypertension, ischemia reperfusion IR injury, myocardial infarction, and atherosclerosis.

7. The use of claim 6, wherein the disease or condition is Pulmonary Arterial Hypertension (PAH).

8. The use of claim 6, wherein the disease or condition is myocardial infarction or atherosclerosis.

9. The use of claim 5, wherein the disease or condition is a pulmonary disorder selected from the group consisting of: chemically induced pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease, COPD, and asthma.

10. The use of claim 5, wherein the disease or condition is an immune disorder selected from the group consisting of: myeloid-derived suppressor cells MDSC-mediated T cell dysfunction, human immunodeficiency virus HIV, autoimmune encephalomyelitis, and ABO do not respond to transfusions.

11. The use of claim 10, wherein the disease or condition is myeloid-derived suppressor cell MDSC-mediated T cell dysfunction.

12. The use of claim 5, wherein the disease or condition is a hemolytic disorder selected from the group consisting of: sickle cell disease, thalassemia, hereditary spherocytosis, polycythemia orosa, microangiopathic hemolytic anemia, pyruvate kinase deficiency, infection-induced anemia, cardiopulmonary bypass, and mechanical heart valve-induced anemia and chemical-induced anemia.

13. The use of claim 5, wherein the disease or condition is a gastrointestinal disorder selected from the group consisting of: gastrointestinal motility disorders, gastric cancer, inflammatory bowel disease, crohn's disease, ulcerative colitis, and gastric ulcers.

14. The use of claim 5, wherein the disease or condition is a disorder selected from the group consisting of: peyronie's disease and erectile dysfunction.

15. The use of claim 5, wherein the disease or condition is ischemia-reperfusion IR injury selected from the group consisting of: liver IR, kidney IR and myocardium IR.

16. The use of claim 4, wherein the disease or condition is selected from the group consisting of: inflammation of renal disease, psoriasis, leishmaniasis, neurodegenerative disease, wound healing, human immunodeficiency virus HIV, hepatitis B virus HBV, infection by helicobacter pylori, fibrotic disease, arthritis, candidiasis, periodontal disease, keloids, adenoid tonsillar disease, african narcolepsy and chagas disease.

17. The use of claim 16, wherein the disease or condition is a wound healing disorder selected from the group consisting of: infected and uninfected wounds healed.

18. The use of claim 4, wherein the subject is a mammal selected from the group consisting of: humans, dogs, cats, horses, cattle, sheep and lambs.

19. The use of claim 4, wherein the disease or condition is an autoimmune disorder.

20. The use of claim 19, wherein the autoimmune disorder is a disorder selected from the group consisting of: encephalomyelitis, multiple sclerosis, antiphospholipid syndrome, autoimmune hemolytic anemia, chronic inflammatory demyelinating polyradiculoneuropathy, dermatitis herpetiformis, dermatomyositis, myasthenia gravis, pemphigus, rheumatoid arthritis, stiff person syndrome, type 1 diabetes, ankylosing spondylitis, paroxysmal nocturnal hemoglobinuria PNH, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, and Goodpasture's syndrome.