PYRROLIDINE COMPOUNDS WITH Lp(a) LOWERING ACTIVITY

Abstract

The present disclosure provides compounds of the formula: and their pharmaceutically acceptable salts, and compounds of the formula: and their pharmaceutically acceptable salts, as well as pharmaceutical compositions comprising these compounds and their use in the treatment of cardiovascular disease and elevated Lp(a) plasma levels.

Description

Pyrrolidine Compounds with Lp(a) Lowering Activity FIELD OF THE INVENTION

[0001] This invention relates to pyrrolidine compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions, and therapeutic uses of the compounds, in particular their use in lowering lipoprotein(a) (Lp(a)) plasma levels. BACKGROUND OF THE INVENTION

[0002] There have been significant advances in treating cardiovascular disease (CVD). Despite treatment advances, patients continue to experience cardiovascular disease events such as angina, myocardial infarction, and stroke, which if untreated, lead to death. Lipid disorder or dyslipidemia remains a major risk factor for CVD. Lipid disorders can be divided into four general risk factors: elevated low-density lipoprotein cholesterol (LDL-c), low high-density lipoprotein cholesterol (HDL-c), elevated triglycerides (TG), and elevated Lp(a). There are a variety of treatment regimens targeting elevated LDL-c, low HDL-c, and elevated triglycerides. There are few approved treatment options for patients with elevated Lp(a) concentrations. In some cases, apheresis may be used to filter the blood to remove LDL and Lp(a); however, the effects are temporary and typically need to be repeated every two weeks. There is currently no pharmaceutical treatment approved specifically to lower Lp(a) levels.

[0003] Lp(a) is a genetically determined, independent cardiovascular risk factor. Elevated serum Lp(a) levels greater than 50 mg / dL or 125 nmol / L found in ~20% of the population confer at least a 1.6-fold risk of a first cardiovascular event (Kronenberg, F. Clin. Res. Cardiol. Suppl. 14, 5-12 (2019)) and a >1.42-fold increase risk of a second event (Madsen, C. M. et al. Arterioscler. Thromb. Vasc. Biol.40, 255-266 (2020)). Lp(a) may exhibit both prothrombotic and antithrombotic properties, and atherogenic and atherothrombotic properties. Lp(a) may inhibit fibrinolysis and accumulate in the vascular wall inducing thrombogenesis and atherosclerotic lesions. Plasma levels of Lp(a) vary substantially among individuals. Unlike the other risk factors, Lp(a) plasma levels do not vary significantly with diet and exercise.

[0004] Lp(a) resembles LDL-c in that it includes an LDL lipid core with the attendant apolipoprotein B (apoB), but unlike LDL-c, Lp(a) also contains a unique apolipoprotein(a) (apo(a)) bound to the apoB via disulfide bond. Apo(a) is synthesized in the liver. The assemblyof Lp(a) from apo(a) and LDL particles can occur in hepatocytes, on the cell wall, or in plasma. Inhibition of the assembly of the LDL particle with apo(a) reduces Lp(a) levels.

[0005] WO2020 / 247429 discloses compounds which inhibit the formation of Lp(a) by blocking the interaction between Apo(a) and ApoB.

[0006] Additional treatment options are desired for patients suffering from cardiovascular diseases and, in particular, patients suffering from lipid disorders or dyslipidemia. There is a need for additional treatment options for patients whose cardiovascular risks are not adequately managed using current standard of care therapies, such as, diet, exercise and / or the use of one or more drugs such as statins, fibrates, and niacin. More particularly, there is a need for further, alternative pharmaceutically acceptable compounds which inhibit the formation of Lp(a) and thus, reduce plasma Lp(a) levels. SUMMARY OF THE INVENTION

[0007] Accordingly, in a first aspect, there is provided a compound of Formula I:wherein L is C2-C6alkylene, C2-C6alkenylene or -(CH2)nNHCONH(CH2)n-; n at each occurrence is independently 2 to 4; Y at each occurrence is independently CH2, O or S; Z at each occurrence is independently H, C1-C4 alkyl, OH or cyclopropyl; or a pharmaceutically acceptable salt thereof.

[0008] In a second aspect, there is provided a compound of Formula II:IIwherein L is C2-C3alkylene or C2-C3alkenylene; R1is -NHC(O)R2, -NHC(O)NHR2, COOH, -C(O)NH-cyclopropyl,, -O-phenyl, -NH-phenyl or phenyl, wherein the phenyl is optionally substituted with one or two substituents selected from halo and OCH3; R2is C1-C3 alkyl; Y is CH2, O or S; Z is H, C1-C4alkyl, OH or cyclopropyl; or a pharmaceutically acceptable salt thereof.

[0009] In a third aspect, there is provided a pharmaceutical composition comprising a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0010] In a fourth aspect, there is provided a method of treating cardiovascular disease in a patient comprising administering to the patient an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0011] In a fifth aspect, there is provided a method of treating elevated Lp(a) plasma levels in a patient comprising administering to the patient an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0012] In a sixth aspect, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0013] In a seventh aspect, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in the treatment of cardiovascular disease.

[0014] In an eighth aspect, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in the treatment of elevated Lp(a) plasma levels. DETAILED DESCRIPTION OF THE INVENTION

[0015] In an embodiment of a compound of Formula I, Z at each occurrence is H.

[0016] In an embodiment of a compound of Formula I, Y at each occurrence is CH2.

[0017] In an embodiment, there is provided a compound of Formula Ia:or a pharmaceutically acceptable salt thereof. Formula I encompasses Formula Ia and reference to Formula I below, for example in the methods of treatment and therapeutic uses, is also to be read as a reference to Formula Ia.

[0018] In an embodiment of a compound of Formula I, L is C2alkylene. In an alternative embodiment of a compound of Formula I, L is C2alkenylene. In a further alternative embodiment of a compound of Formula I, L is -(CH2)3NHCONH(CH2)3-.

[0019] In an embodiment, the compound of Formula I is selected from:or a pharmaceutically acceptable salt thereof.

[0020] In an embodiment of a compound of Formula II, Z is H.

[0021] In an embodiment of a compound of Formula II, Y is CH2.

[0022] In an embodiment, there is provided a compound of Formula IIa:IIa or a pharmaceutically acceptable salt thereof. Formula II encompasses Formula IIa and reference to Formula II below, for example in the methods of treatment and therapeutic uses, is also to be read as a reference to Formula IIa.

[0023] In an embodiment of a compound of Formula II, R1is -NHC(O)CH3,

[0024] In an embodiment, the compound of Formula II is selected from:or a pharmaceutically acceptable salt thereof.

[0025] In an embodiment, there is provided a method of treating a patient in need of treatment for cardiovascular disease, comprising administering an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof. In an embodiment, there is provided a method of treating a patient in need of treatment for elevated Lp(a) plasma levels, comprising administering an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0026] In an embodiment, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in therapy.

[0027] In an embodiment, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in the treatment of cardiovascular disease. In an embodiment, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in treating elevated Lp(a) plasma levels.

[0028] In an embodiment, there is provided the use of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cardiovascular disease. In an embodiment, there is provided the use of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of elevated Lp(a) plasma levels.

[0029] The term “C1-Cnalkyl” refers to a straight or branched chain saturated hydrocarbon containing 1 to n carbon atoms. Examples of a C1-C4 alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, and tert-butyl. Examples of a C1-C3alkyl group include, but are not limited to, methyl, ethyl and propyl.

[0030] The term “C2-C6 alkylene” refers to a bivalent straight or branched chain saturated hydrocarbon containing 2 to 6 carbon atoms. The term “C2-C3 alkylene” refers to a bivalent straight or branched chain saturated hydrocarbon containing 2 to 3 carbon atoms.

[0031] The term “C2-C6 alkenylene” refers to a bivalent straight or branched chain hydrocarbon containing 2 to 6 carbon atoms and at least one double bond. The term “C2-C3 alkenylene” refers to a bivalent straight or branched chain hydrocarbon containing 2 to 3 carbon atoms and at least one double bond.

[0032] As used herein, the term “elevated Lp(a) plasma levels” means a plasma level of Lp(a) that is equal to or above about 50 mg / dL. A compound provided herein may be used in treatment to reduce Lp(a) plasma levels.

[0033] The term “pharmaceutically acceptable salt” as used herein refers a salt of a compound that is acceptable for clinical and / or veterinary use. Examples of pharmaceutically acceptable salts and common methodology for preparing them can be found in “Handbook of Pharmaceutical Salts: Properties, Selection and Use” P. Stahl, et al., 2nd Revised Edition, Wiley- VCH, 2011 and S.M. Berge, et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19. In particular, the compounds of Formula I or II may be a zwitterion, a mono-, di, or tri-acid addition salt.

[0034] The pharmaceutical compositions for the present invention may be prepared using pharmaceutically acceptable additives. The term “pharmaceutically acceptable” refers to one or more carriers, diluents, and / or excipients that are compatible with the other components of the composition and not pharmaceutically deleterious to the patient. Examples of pharmaceutical compositions and processes for their preparation are well known to the skilled artisan, and can befound, for example, in “Remington: The Science and Practice of Pharmacy”, Loyd, V., et al. Eds., 22nd Ed., Mack Publishing Co., 2012.

[0035] As used herein, the term “effective amount” refers to a dosage amount that is effective in treating a disorder. The effective amount for a particular patient can be determined by a skilled health professional.

[0036] As used herein, the terms “treating”, “to treat”, or “treatment”, includes slowing, reducing, preventing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. As used herein, “treating cardiovascular disease” means slowing, reducing, preventing, or reversing the progression of heart or blood vessel disease.

[0037] As used herein, the term "patient" refers to a mammal. Preferably, the patient is a human.

[0038] Pharmaceutical compositions can be formulated as a tablet or capsule for oral administration, a solution for oral administration, or an injectable solution. In an embodiment the composition is suitable for oral administration.

[0039] The present invention contemplates all individual enantiomers, diastereomers, mixtures thereof, and racemates, of compounds of Formula I and II, and pharmaceutically acceptable salts thereof.

[0040] Certain abbreviations may refer to the following: “ACN” refers to acetonitrile; “Apo” refers to Apolipoprotein; “BOC” refers to tert-butoxycarbonyl; “DCM” refers to dichloromethane; “DMA” refers dimethylacetamide; “DMEM” refers to Dulbecco’s Modified Eagle’s Medium; “DMF” refers to dimethylformamide; “DMSO” refers to dimethyl sulfoxide; “EACA” refers to epsilon-aminocaproic acid or 6-aminocaproic acid; “ELISA” refers to enzyme- linked immunosorbent assay; “EtOAc” refers to ethyl acetate; “FBS” refers to Fetal Bovine Serum; “HEC” refers to hydroxy ethyl cellulose; “HEK” refers to human embryonic kidney; “HepG2” refers to a human hepatoma cell line; “HEPES” refers to 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid; “HLB” refers to hydrophilic-lipophilic balance; “hr / s” refers to hour / hours; “HRP” refers to Horseradish Peroxidase; “IC50” refers to the concentration of an agent that produces 50% of the maximal inhibitory response possible for that agent; “MeCN” refers to acetonitrile; “min” refers to minute / s; “MTBE” refers to methyl tert-butyl ether; “RT” refers to room temperature; “TEA” refers to triethylamine; “TFA” refers to trifluoroacetic acid; “THF” refers to tetrahydrofuran; and “TMB” refers to 3,3’,5,5’-teramethylbenzidine.

[0041] Individual isomers, enantiomers, and diastereomers may be separated or resolved by one of ordinary skill in the art at any convenient point in the synthesis of compounds listed below, by methods known to the artisan, such as selective crystallization techniques or chiral chromatography.

[0042] A compound of Formula I or II is readily converted to and may be isolated as a pharmaceutically acceptable salt. Salt formation can occur upon the addition of a pharmaceutically acceptable acid to form the acid addition salt or by the addition of a pharmaceutically acceptable base to form a base addition salt. Salts can also form simultaneously upon deprotection of a nitrogen or oxygen, i.e., removing the protecting group. Examples, reactions and conditions for salt formation are known to the skilled artisan.

[0043] The compounds of Formula I or II or any depicted formulae, or salts thereof, may be prepared by a variety of procedures, some of which are illustrated in the Preparations and Examples below. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different routes, to prepare compounds or salts of the present invention. The products of each step in the Preparations below can be recovered by conventional methods, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization.

[0044] In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations, and examples are provided to further illustrate the invention. Compounds of afore-depicted formulae, or salts thereof may be prepared by using starting materials or intermediates with the corresponding desired stereochemical configuration.

[0045] In Scheme 1, Step A depicts conversion of compound (1) to compound (2). Compound (1) is reacted with (E)-1,4-dibromobut-2-ene in the presence of lithium bis(trimethylsilyl)amide as a base and a solvent such as THF to give compound (2).

[0046] Step B depicts formation of compound (3) by reacting compound (2) with potassium acetate and a solvent such as MeCN.

[0047] Step C shows a reaction in which oxazolidinone in compound (3) is hydrolyzed by using LiOH / H2O2 in the presence of a solvent such as THF to give compound (4).

[0048] Step D depicts formation of a tert-butyl ester, compound (5), by reacting compound (4) with 2-tert-butyl-1,3-diisopropylisourea in the presence of a solvent such as 2- methyltetrahydrofuran.

[0049] Step E shows converting compound (5) to compound (6) in the presence of an iridium catalyst and a solvent such as acetone.

[0050] Step F depicts oxidation of the aldehyde in compound (6) to give compound (7) by use of selenium (IV) oxide in the presence of H2O2. In step 2F, compound (7) is converted to compound (7a) (compound of Formula II) by acid deprotection of compound (7) using excess HCl in diethyl ether to give the HCl salt compound.

[0051] Step G shows a process of reacting compound (7) with a cyclopropylamine in the presence of ethyl chloroformate and a solvent such as DCM to give compound (8). In Step 2G, compound (8) is converted to compound (8a) (compound of Formula II) by acid deprotection of compound (8) using excess HCl in diethyl ether to give the HCl salt compound.

[0052] In Scheme 2, Step A, aldehyde compound (6) is reduced to alcohol compound (9) using a reducing agent such as sodium borohydride in an alcohol solvent. In Step B, alcohol compound (9) is converted to mesylate (10) using methanesulfonyl chloride in the presence of a base such as TEA in a solvent such as DCM. In Step C, compound (10) is reacted with an aniline (optionally substituted with one or two substituents (depicted by “R”), selected from halo and OCH3) using a base that may include potassium carbonate and sodium iodide, and a solvent such as DCM to give compound (11). BOC-deprotection using HCl of (11) in Step D gives compound (11a) (compound of Formula II).

[0053] Mesylate (10) can alternatively be reacted in Step E with potassium cyanide in a solvent such as DMF to give cyano compound (13). Reaction with HCl in Step F converts the cyano group to a carboxylic acid and removes the BOC groups to give compound (13a) (compound of Formula II).

[0054] Mesylate (10) can also be reacted in Step G with pyrazole in presence of a base such as potassium carbonate, and a solvent such as MeCN to give compound (12), then BOC deprotected in Step H to give compound (12a) (compound of Formula II). Scheme 3

[0055] In Scheme 3, Step A depicts a process of converting compound (14) to compound (15) by reacting compound (14) with compound (1) in the presence of lithium bis(trimethylsilyl)amide, and a solvent such as THF to give compound (15).

[0056] Step B shows a reaction in which oxazolidinone in compound (15) is cleaved by using LiOH / H2O2 in the presence of a solvent such as THF to give compound (16).

[0057] Step C shows acid deprotection by use of excess HCl and diethyl ether to give the HCl salt, compound (17) (compound of Formula II).

[0058] Step D shows an alternative route in which the double bond in compound (16) is reduced by hydrogenation with a palladium catalyst and hydrogen gas in an alcohol solvent such as ethanol to give compound (31).

[0059] Step E shows the acid deprotection of compound (31) by use of excess HCl and diethyl ether to give the HCl salt, compound (32) (compound of Formula II). Scheme 4

[0060] In Scheme 4, Step A depicts a reductive amination reaction, in which, compound (6) is converted to compound (18) by reacting with dibenzylamine in the presence of sodium triacetoxyborohydride, and a solvent such as DCM to give compound (18).

[0061] Step B shows converting compound (18) to compound (19) in a hydrogenation reaction, in the presence of a palladium on carbon catalyst, and a solvent such as ethanol.

[0062] Step C shows converting the amine in compound (19) to amide in compound (21) through a reaction with an acid chloride [R2-C(O)Cl], a base such as TEA, and a solvent such as DCM.

[0063] Step D shows formation of compound (20) by reacting two equivalents of compound (19) with one equivalent of 1,1'-carbonyldiimidazole and a solvent such as THF.

[0064] Step E shows a reaction of an isocyanate (R2-N=C=O) with amine in compound (19) in the presence of a base such as TEA, and a solvent such as DCM to give compound (22).

[0065] Steps 2C, 2D, and 2E depict BOC removal of compounds (21), (20), and (22), respectively by use of excess HCl to give HCl salt compounds (24) (compound of Formula II), (23) (compound of Formula I), and (25) (compound of Formula II) respectively. R2is as defined in Formula II. Scheme 5

[0066] In Scheme 5, Step A depicts alkylation of compound (1) to give compound (26). Compound (1) is reacted with allyl bromide in the presence of lithium bis(trimethylsilyl)amide as a base and a solvent such as THF to give compound (26).

[0067] Step B shows a hydrolysis reaction in which oxazolidinone in compound (26) is cleaved by using LiOH / H2O2in the presence of a solvent such as THF to give compound (27).

[0068] Step C shows an esterification reaction in which compound (27) is reacted with N,N- dimethylformamide di-tert-butyl acetal in the presence of a solvent such as toluene to give compound (28).

[0069] Step D shows a metathesis reaction in formation of compound (29) by contacting compound (28) with Grubbs catalyst 2nd generation and a solvent such as DCM.

[0070] Step E depicts acid deprotection of compound (29) by use of excess HCl and diethyl ether to give the HCl salt, compound (30) (compound of Formula II). Preparation 1 tert-Butyl (3R)-3-[2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-2-oxo-ethyl]pyrrolidine-1- carboxylate

[0071] TEA (56.5 g, 77.9 mL, 559 mmol) was added to a solution of (R)-N-boc-3- pyrrolidineacetic acid (CAS Registry No.204688-60-8; 53.8 g, 235 mmol) in THF (540 mL) maintained at 10 °C. After 5 min, pivaloyl chloride (33.7 g, 34.2 mL, 279 mmol) was added. After 15 min, lithium chloride (11.8 g, 279 mmol) in THF (540 mL) and (4S)-4- benzyloxazolidin-2-one (40.0 g, 223 mmol) were added. The mixture was allowed to warm to RT and stirred 24 hrs. After 24 hrs., in aqueous HC1 (500 mL) was added and the organic phase separated from the aqueous phase. The organic phase was washed with in aqueous NaOH (500 mL) and saturated aqueous NaCl (500 mL), dried over MgSO4, filtered, and concentrated under vacuum. The residue was suspended in a mixture of MeOH and H2O (1:2, 575 mL) and stirred at RT for 16-18 hrs. The solid was filtered off, washed with hexanes (2 x 150 mL), and dried to give the title compound (65.7 g, 76%) as a white solid. ES / MS (m / z): 333 [M+H]-tert-butyl.Preparation 2 tert-Butyl (3R)-3-[(E,1S)-5-acetoxy-1-[(4S)-4-benzyl-2-oxo-oxazolidine-3-carbonyl]pent-3- enyl]pyrrolidine-1-carboxylate

[0072] tert-Butyl (3R)-3-[(E,1S)-1-[(4S)-4-benzyl-2-oxo-oxazolidine-3-carbonyl]-5-bromo- pent-3-enyl]pyrrolidine-1-carboxylate (5.59 g, 10.7 mmol) was dissolved in MeCN (75 mL) and potassium acetate (1.6 g, 16 mmol) was added. The mixture was stirred at 70 °C for 16-18 hrs. The solvent was evaporated, the residue dissolved in diethyl ether and washed with H2O. The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to give the title compound as a pale-yellow foamy material (5.36 g, 99+%). ES / MS (m / z): 401 (M+H-Boc). Preparation 3 tert-Butyl (3R)-3-[(E,1S)-1-tert-butoxycarbonyl-5-hydroxy-pent-3-enyl]pyrrolidine-1-

[0073] (E,2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-6-hydroxy-hex-4-enoic acid (0.28 g, 0.935 mmol) was purged under N2and then, 2-methyltetrahydrofuran (5.5 mL) and 2- tert-butyl-1,3-diisopropylisourea (830 µL) were added and the reaction mixture kept stirring at RT for 16-18 hrs. Additional 2-tert-butyl-1,3-diisopropylisourea was added (150 mL) and the reaction continued stirring at RT for about 4 hrs. The reaction was then diluted with EtOAc andfiltered through diatomaceous earth. The solid was further washed with diethyl ether (x3) and the filtrate evaporated. The residue was put again under three more cycles of solubilization in diethyl ether, filtration, then dried under vacuum. The material was then purified with silica gel chromatography eluting with DCM in EtOAc (3:1) to give the title compound (0.26 g, 70%) as a dense yellow oil. ES / MS m / z: 298 (M-H, negative ionization mode). Preparation 4 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-oxo-pentyl]pyrrolidine-1-carboxylate

[0074] tert-Butyl (3R)-3-[(E,1S)-1-tert-butoxycarbonyl-5-hydroxy-pent-3-enyl]pyrrolidine- 1-carboxylate (0.195 g, 0.56 mmol) was dissolved in acetone and H2O. Dichloro(pentamethylcyclopentadienyl)iridium(III) dimer catalyst (0.006 g, 0.008mmol) was added and the reaction mixture kept stirring at RT for 16-18 hrs. Acetone was evaporated and the reside was partitioned between diethyl ether and H2O. After separation, the organics were dried over Na2SO4, evaporated, and dried under vacuum for about 1 hr., to give of pale-yellow dense oil (0.186 g). The crude product was purified by silica gel chromatography eluting with DCM in EtOAc (9:1), evaporated, and dried under vacuum to give the title compound (0.14 g, 75%) as a very dense pale-yellow oil. ES / MS m / z: 244 (M+H- 2x tert-butyl). Preparation 5 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-hydroxy-pentyl]pyrrolidine-1-carboxylate

[0075] tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-oxo-pentyl]pyrrolidine-1- carboxylate (0.58 g, 1.631 mmol) was dissolved in EtOH (7 mL). Sodium borohydride (0.049 g, 1.29 mmol) was added, and the reaction mixture stirred at RT for 0.5 hrs. The reaction was diluted with EtOAc and washed with H2O and aqueous NH4Cl. The organics were dried over Na2SO4, evaporated, and dried under vacuum to give of pale-yellow dense oil (0.55 g). The crude product was purified using silica gel chromatography, eluting with DCM in EtOAc (2.7:1), evaporated, and dried under vacuum to give the title compound (0.48 g, 80%) as a dense pale- yellow oil. ES / MS m / z: 246 (M+H- 2x tert-butyl). Preparation 6 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-methylsulfonyloxy-pentyl]pyrrolidine-1- carboxylate

[0076] tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-hydroxy-pentyl]pyrrolidine-1- carboxylate (0.24 g, 0.671 mmol) and TEA (160 µL) were dissolved in dry DCM, under N2. The mixture was cooled to 0 °C and methanesulfonyl chloride (70 µL) was added drop wise. The reaction mixture was kept stirring at 0 °C for 1 hr. The reaction was diluted with DCM and washed with H2O. After separation, the organics were dried over Na2SO4, evaporated, and dried under vacuum to give the title compound as a pale-yellow very dense oil (0.29 g, 90%). ES / MS m / z: 324 (M+H- 2x tert-butyl). Preparation 7 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(3-fluoro-5-methoxy-anilino)pentyl]pyrrolidine- 1-carboxylate

[0077] 3-Fluoro-5-methoxy-aniline and tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5- methylsulfonyloxy-pentyl]pyrrolidine-1-carboxylate (0.162 g, 0.371 mmol) along with potassium carbonate (0.075 g, 0.542 mmol) and sodium iodide (0.008 g, 0.053 mmol) were purged under N2. DMF was added and the reaction mixture heated at 85 º C for 16-18 hrs. The reaction was poured into H2O and extracted with diethyl ether. The organics were washed with H2O (x2), dried over Na2SO4, evaporated, and dried under vacuum. The crude product was purified by with silica gel chromatography, eluting with DCM in diethyl ether (95:5) to give a very dense pale- yellow oil (0.07 g). The product was repurified by reverse phase chromatography (12 g column; A: water, B: MeCN; 70% 2CVs, 70-80% 4CVs, 80% 4CVs) to give the title compound as a dense pale-yellow oil (0.038 g, 22.91%). ES / MS m / z: 481 (M+H). Preparation 8 (5S)-6-tert-Butoxy-5-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-6-oxo-hexanoic acid

[0078] To a solution of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-oxo- pentyl]pyrrolidine-1-carboxylate (0.15 g, 0.422 mmol) in THF (2 mL) was added selenium (IV) dioxide (0.003 g, 0.270 mmol) followed by H2O2 (34% aqueous, 100 µL) and the reaction mixture was heated at 70 °C for about 2.5 hrs. The reaction was diluted with EtOAc and washed with H2O. After separation, the organics were washed with H2O, dried over Na2SO4, evaporated, and dried under vacuum to give transparent dense oil (160 mg). The crude product was purified by silica gel chromatography eluting with DCM in diethyl ether (9:1), evaporated, and driedunder vacuum to give the title compound as a dense transparent oil (0.125 g, 80%). ES / MS m / z: 260 (M+H- 2x tert-butyl). Preparation 9 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(cyclopropylamino)-5-oxo-pentyl]pyrrolidine-1-

[0079] A solution of (5S)-6-tert-butoxy-5-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-6- oxo-hexanoic acid (0.215 g, 0.579 mmol) and TEA (170 µL) in dry DCM (3.5 mL) was purged under N2. Ethyl chloroformate (80 µL) was added drop wise and the mixture stirred for 0.75 hrs. Next, cyclopropylamine (60 µL) was added drop wise and the reaction mixture was kept stirring at RT for about 2.5 hrs. The reaction was diluted with DCM and washed with H2O plus 10% Na2CO3. After separation of phases, the organic phase was washed with H2O, dried over Na2SO4, evaporated, and dried under vacuum to give a pale-yellow dense oil (250 mg). The crude product was purified by silica gel chromatography eluting with DCM in diethyl ether (4:1), evaporated, and dried under vacuum to give the title compound (0.13 g, 55%) as a very dense pale-yellow oil. ES / MS m / z: 411 (M+H). Preparation 10 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-cyano-pentyl]pyrrolidine-1-carboxylate

[0080] tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-methylsulfonyloxy- pentyl]pyrrolidine-1-carboxylate (0.146 g) and potassium cyanide (0.028 g, 0.335 mmol) were purged under N2. Dry DMF (1.2 mL) was added, and the reaction mixture heated at 90 ºC for 16- 18 hrs. The reaction was diluted with diethyl ether and washed with H2O. After separation, the organics were washed with H2O (x2), dried over Na2SO4, evaporated, and dried under vacuum to give a pale-yellow dense oil (80 mg). The crude was purified by silica gel chromatography eluting with DCM in EtOAc (95:5); and dried under vacuum to give the title compound (0.058 g, 47%) as a very dense transparent oil. ES / MS m / z: 255 (M+H- 2x tert-butyl).tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-pyrazol-1-yl-pentyl]pyrrolidine-1-carboxylate

[0081] A mixture of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-methylsulfonyloxy- pentyl]pyrrolidine-1-carboxylate (0.155 g, 0.356 mmol) and pyrazole (0.04 g, 0.588 mmol) along with potassium carbonate (0.095 g, 0.687 mmol) were purged under N2in refrigerant. MeCN (2.5 mL) was added, and the reaction mixture was heated at 75 ºC for 16-18 hrs., under N2 atmosphere for about 16 hrs. The reaction continued heating for 16-18 hrs., under N2 atmosphere at 80 °C. The reaction was then diluted with EtOAc and washed with H2O. After separation, the organics were dried over Na2SO4, evaporated, and dried under vacuum to give a very pale-yellow dense oil (150 mg). The crude was purified by silica gel chromatography eluting with DCM in EtOAc (9:1); solvent evaporated, and residue dried under vacuum to give the title compound (0.071 g, 50%) as a very dense transparent oil. ES / MS m / z: 408 (M+H). Preparation 12 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(ethylcarbamoylamino)pentyl]pyrrolidine-1- carboxylate

[0082] A mixture of tert-butyl (3R)-3-[(1S)-5-amino-1-tert-butoxycarbonyl- pentyl]pyrrolidine-1-carboxylate (0.145 g, 0.407 mmol) and TEA (120 µL) were dissolved in dry DCM, under N2 atmosphere. Isocyanoethane (60 µL) was then added dropwise, and the reaction mixture stirred at RT for about 1.5 hrs. The reaction was diluted with more DCM and washed with H2O. The organics were dried over Na2SO4, evaporated, and dried under vacuum to give a dense pale-yellow oil (190 mg). The crude product was purified by silica gel chromatography eluting with DCM in MeOH (65:35). The solvent was evaporated, and the residue dried under vacuum to give the title compound (0.124 g, 80%) as a very dense transparent oil. ES / MS m / z: 428 (M+H). Preparation 13 1-[(E)-4-Bromobut-2-enoxy]-3-fluoro-5-methoxy-benzene

[0083] A mixture of (E)-1,4-dibromobut-2-ene (CAS Registry No.821-06-7; 0.00192 g, 1.92 mmol) and 3-fluoro-5-methoxy-phenol (CAS Registry No.850793-25-8; 0.833 g) were dissolved in MeCN (50 mL) under N2 atmosphere in a refrigerant. Potassium carbonate (0.00135 g, 0.0097 mmol) was added in a single portion and the final reaction mixture was stirred at 60º C for about 4.5 hrs. The solvent was evaporated, and the residue was partitioned between diethyl ether and H2O. After separation of phases, the organics were washed with water, dried over Na2SO4, filtered, and evaporated to give a pale-yellow dense oil (2 g). The crude product was purified by silica gel chromatography eluting with DCM in hexane (1:9) to give the title compound (0.702 g, 45%) as a very pale-yellow oil. 1H NMR (400.13 MHz, CDCl3) d 6.29-6.25 (m, 3H), 6.14-6.09(m, 1H), 6.02-5.96 (m, 1H), 4.53 (dd, J= 1.1, 5.1 Hz, 2H), 4.01 (dd, J= 0.8, 7.3 Hz, 2H), 3.79 (s, 3H). Preparation 14 2-[(E)-4-Bromobut-2-enyl]isoindolin-1-one

[0084] (E)-1,4-Dibromobut-2-ene (CAS Registry No.821-06-7; 1.12 g) was dissolved in dry THF (20 mL) under N2 atmosphere. Then, sodium hydride (60% mass mineral oil, 0.175 g) was added at RT and the mixture was stirred for 0.25 hrs. Solid isoindolin-1-one (CAS Registry No.480-91-1; 0.551 g, 4.13 mmol) was added in a single portion and the reaction mixture kept stirring at RT for about 4.5 hrs. The reaction was diluted with diethyl ether, washed with H2O (x2), dried over Na2SO4, evaporated, and stirred under vacuum to give a yellow solid (1 g) crude residue. The crude product was purified by silica gel chromatography eluting with EtOAc (100 mL). EtOAc was evaporated and the residue dried under vacuum to give the title compound (0.22 g, 19%) as a very dense pale-yellow oil. ES / MS m / z: 266, 268 (M+H). Preparation 15 3-[(E)-4-Bromobut-2-enyl]-1,3-benzoxazol-2-one

[0085] In a refrigerator, (E)-1,4-dibromobut-2-ene (CAS Registry No.821-06-7; 1.5 g, 7.02 mmol) and 3H-1,3-benzoxazol-2-one (CAS Registry No.59-49-4; 0.69 g) were dissolved in MeCN (40 mL) under N2. Then, potassium carbonate (0.99 g, 7.16 mmol) was added in a single portion and the reaction mixture stirred at 50 ºC for about 4.25 hrs. The solvent was evaporated, and the residue was partitioned between diethyl ether and water. After separation of phases, theorganics were washed with H2O, dried over Na2SO4, filtered, and evaporated to give crude as brown solid (1.5 g). The crude product was purified by silica gel chromatography eluting with DCM in hexane (3:1) to give the title compound (0.72 g, 50%) as a very pale-yellow solid. ES / MS m / z: 268, 270 (M+H). Preparation 16 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(dibenzylamino)pentyl]pyrrolidine-1-

[0086] A mixture of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-oxo-pentyl]pyrrolidine- 1-carboxylate (0.15 g, 0.52 mmol) and dibenzylamine (0.12 mL, 0.61 mmol) was dissolved in DCM (3 mL) and stirred at 30 min at RT. Sodium triacetoxyborohydride (0.175 g, 0.80 mmol) was added in a single portion and stirred at RT for 16-18 hrs. The mixture was diluted with DCM, an aqueous solution of NaHCO3added and the phases separated. The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography eluting with 5% diethyl ether in DCM to give the title compound (0.17 g, 54% yield) as a dense pale-yellow oil. ES / MS (m / z): 537 (M+H). Preparation 17 tert-Butyl (3R)-3-[(1S)-5-amino-1-tert-butoxycarbonyl-pentyl]pyrrolidine-1-carboxylate

[0087] tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(dibenzylamino)pentyl]pyrrolidine- 1-carboxylate (0.170 g, 0.30 mmol) was dissolved in EtOH (3 mL) and purged with N2. Palladium on carbon (5% w / w, 140 mg, 0.06 mmol) was added and purged with hydrogen. The mixture was pressurized with 70 psi of hydrogen and stirred at RT for 4.5 hrs., filtered through a pad of diatomaceous earth and washed with DCM. The filtrate was evaporated to give the title compound (0.12 g, 99+% yield) as a dense dark-brown oil. ES / MS (m / z): 357 (M+H). Preparation 18 tert-Butyl (3R)-3-[(1S)-5-[[(5S)-6-tert-butoxy-5-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-6- oxo-hexyl]carbamoylamino]-1-tert-butoxycarbonyl-pentyl]pyrrolidine-1-carboxylate

[0088] tert-Butyl (3R)-3-[(1S)-5-amino-1-tert-butoxycarbonyl-pentyl]pyrrolidine-1- carboxylate (0.12 g, 0.29 mmol) was dissolved in dry THF (3 mL) under N2.1,1'- Carbonyldiimidazole (0.025 g, 0.15 mmol) was added and stirred at 70 °C for 5 hrs. The reaction mixture was poured into water and extracted with diethyl ether (x2). The combined organics were washed with H2O, and dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography eluting sequentially with DCM and diethyl ether (3:1) and EtOAc to give the title compound (0.050 g, 49%).1H NMR (400.21 MHz, CDCl3): 3.67-3.37 (m, 4H), 3.32-3.07 (m, 6H), 2.99-2.81 (m, 2H), 2.35-2.21 (m, 2H), 2.18-2.08 (m, 2H), 1.99-1.86 (m, 2H), 1.69-1.23 (m, 50H). Preparation 19 ditert-Butyl (E,2S,7S)-2,7-bis[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]oct-4-enedioate

[0089] tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonylbut-3-enyl]pyrrolidine-1-carboxylate (0.204 g, 0.627 mmol) was purged under N2atmosphere in a refrigerant. Then, dry DCM was added followed by Grubbs Catalyst® M2a (C848, CAS registry # 246047-72-3, 0.055 g, 0.065 mmol). Next, the reaction mixture was heated at reflux for 16-18 hrs. The solvent was diluted with DCM and washed with H2O. The organics were dried over Na2SO4and evaporated to dryness to give a dark brown dense oil (0.3 g). The residue was purified by silica gel chromatography eluting with DCM in EtOAc (95:5). The solvent was evaporated, and the residue dried under vacuum to give the title compound (0.11 g, 55%) as a brown foamy substance. ES / MS m / z: 623 (M+H). Preparation 20 ditert-Butyl (2S,7S)-2,7-bis[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]octanedioate

[0090] A mixture of ditert-butyl (E,2S,7S)-2,7-bis[(3R)-1-tert-butoxycarbonylpyrrolidin-3- yl]oct-4-enedioate (400 mg, 0.64 mmol) and palladium catalyst (5 mass% on carbon, 700 mg, 0.33 mmol) was purged under nitrogen atmosphere. EtOH (7 mL) was added and the mixturewas purged with hydrogen with vigorous stirring. The mixture was stirred for 2 h at RT under a balloon of hydrogen gas, then filtered through a pad of diatomaceous earth. The pad was washed with DCM and the filtrate was evaporated. The residue was dissolved in DCM and dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography using 10% EtOAc in DCM to give the title compound (365 mg, 91%) as a white foamy solid. ES-MS m / z 525 (M+H-Boc). Preparation 21 tert-Butyl (3R)-3-[(E,1S)-1-[(4S)-4-benzyl-2-oxo-oxazolidine-3-carbonyl]-5-(3-fluoro-5- methoxy-phenoxy)pent-3-enyl]pyrrolidine-1-carboxylate

[0091] tert-Butyl (3R)-3-[2-[(4S)-4-benzyl-2-oxo-oxazolidin-3-yl]-2-oxo-ethyl]pyrrolidine- 1-carboxylate (0.815 g; 2.1 mmol) was dissolved in dry THF (9 mL) under N2and cooled down to approximately 0 ºC. Lithium bis(trimethylsilyl)amide (2.3 mL, 1M) was added dropwise and the mixture kept stirring for about 1 hr. Solid 1-[(E)-4-bromobut-2-enoxy]-3-fluoro-5-methoxy- benzene (0.607 g, 2.21 mmol) was added in a single portion and the reaction mixture continued stirring at 0 °C for about 1 hr., and then, put at RT for about 16-18 hrs. The reaction was poured into saturated NaHCO3 and extracted with diethyl ether. The organics were further washed with H2O, dried over Na2SO4, and solvent evaporated to give dense yellow oil (1.25 g). The crude material was purified by silica gel chromatography eluting with DCM in EtOAc (96:4). The solvent was evaporated, and the residue dried under vacuum to give the title compound (0.53 g, 40%) as a white foamy substance. ES / MS m / z: 483 (M+H-Boc).

[0092] Preparations 22-26 in Table 1 were prepared in a manner analogous to the method of Preparation 21 using the corresponding appropriate reagents with the reaction time adjusted to reach completion. Table 1Preparation 27 (E,2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-6-hydroxy-hex-4-enoic acid

[0093] A solution of tert-butyl (3R)-3-[(E,1S)-5-acetoxy-1-[(4S)-4-benzyl-2-oxo- oxazolidine-3-carbonyl]pent-3-enyl]pyrrolidine-1-carboxylate (5.36 g, 10.7 mmol) in THF (80 mL) was cooled down to 0 ºC. H2O2 (34 wt%, 9.5 mL, 110 mmol) was added followed by a solution of lithium hydroxide (750 mg, 30.7 mmol) in H2O (14 mL). The reaction mixture was stirred for 16-18 hrs., while reaching RT. The mixture was then poured into H2O and EtOAc and 1M aqueous HCl added and shaken vigorously. The phases were separated, and the organic phase washed with H2O (x2). The organic phase was dried over Na2SO4, filtered, and concentratedunder vacuum. The residue was purified by silica gel chromatography eluting with 3% MeOH in DCM with 0.25% acetic acid to give a dense oil containing a small amount of acetic acid. The residue was then dissolved in DCM and washed with H2O (x2). The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum to give the title compound (2.1 g) as a dense oil. ES / MS (m / z): 298 (M-H).

[0094] Preparations 28-33 in Table 2 were prepared in a manner analogous to the method of Preparation 27 using the corresponding appropriate reagents with the reaction time adjusted to reach completion. Table 2Preparation 32 (2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-5-phenyl-pentanoic acid

[0095] A solution of (E,2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-5-phenyl-pent-4- enoic acid (155 mg, 0.449 mmol) in ethanol (7 mL) was purged with nitrogen, then palladium (5mass% on carbon, 205 mg, 0.096 mmol) was added. The reaction was purged with hydrogen then stirred under a balloon of hydrogen gas for 3.5 h. The mixture was filtered through a pad of diatomaceous earth, rinsed with DCM, and the filtrate was concentrated under reduced pressure. The residue was taken up in DCM, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound (149 mg, 96%) as a white foam. ES-MS m / z 292 (M+H-tert- butyl). Preparation 34 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonylbut-3-enyl]pyrrolidine-1-carboxylate

[0096] To a solution of (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]pent-4-enoic acid (D, 10.5 mmol, 10.5 mmol, 2.83 g) in toluene (105 mL) at 80 ºC was added N,N- dimethylformamide di-tert-butyl acetal (42.0 mmol, 90 mass%, 11.2 mL, 9.49 g) and the mixture was heated at 80ºC for 1 h. More N,N-dimethylformamide di-tert-butyl acetal (42.0 mmol, 90 mass%, 11.2 mL, 9.49 g) was added and the mixture was heated at 80ºC for 3 hr. The reaction mixture was concentrated and the residue was purified by silica gel chromatography (gradient of 5-20% acetone in hexanes) to give the title compound (1.8 g, 53%) as colorless oil. ES-MS m / z 348 (M+Na+). Preparation 35 tert-Butyl (3R)-3-[(1S)-5-acetamido-1-tert-butoxycarbonyl-pentyl]pyrrolidine-1-carboxylate

[0097] To a mixture of tert-butyl (3R)-3-[(1S)-5-amino-1-tert-butoxycarbonyl- pentyl]pyrrolidine-1-carboxylate (110 mg, 0.308 mmol) and TEA (100 µL, 72.5 mg, 0.72 mmol)in DCM (2 mL) was added acetyl chloride (25 µL, 27.5 mg, 0.35 mmol) dropwise and the mixture was stirred at room temperature for 2 h. The mixture was diluted with DCM and washed with water. The organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography using a gradient of 33-100% diethyl ether in DCM to give the title compound (75 mg, 68%) as a pale yellow oil. ES-MS m / z 299 (M-BOC+H). Preparation 36 tert-Butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(3-fluoro-5-methoxy- phenoxy)pentyl]pyrrolidine-1-carboxylate

[0098] A mixture of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-hydroxy- pentyl]pyrrolidine-1-carboxylate (265 mg, 0.74 mmol), 3-fluoro-5-methoxy-phenol (129 mg, 0.91 mmol), and triphenylphosphine (241 mg, 0.92 mmol) was purged under a nitrogen atmosphere, then dry THF (4 mL) was added and the mixture was cooled in an ice / water bath. Diisopropyl azodicarboxylate (190 µL, 196 mg, 0.97 mmol) was added dropwise and the reaction was stirred with cooling for 0.5 h. The reaction was stirred overnight at room temperature, then poured into 10% aqueous Na2CO3 and extracted with diethyl ether. The organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography DCM as eluent, then re-purifed twice by reversed phase HPLC to give the title compound (96 mg, 27%) as a brown oil. ES-MS m / z 382 (M-BOC+H). Preparation 37 (2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-6-(2-oxo-1,3-benzoxazol-3-yl)hexanoic acid

[0099] A mixture of (E,2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-6-(2-oxo-1,3- benzoxazol-3-yl)hex-4-enoic acid (52 mg, 0.12 mmol) in ethanol (2 mL) was purged with nitrogen. Then, palladium (5 mass% on carbon, 75 mg, 0.035 mmol) was added and the reaction was purged with hydrogen and kept under a balloon of hydrogen for 2 h. The mixture was filtered through a pad of diatomaceous earth and the pad was washed with DCM. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography using 3% MeOH in DCM to give the title compound (42 mg, 82%) as a white foam. ES-MS m / z 319 (M-BOC+H). Example 1 (2S)-6-(3-Fluoro-5-methoxy-anilino)-2-[(3R)-pyrrolidin-3-yl]hexanoic acid; dihydrochloride

[0100] Concentrated HCl (35 wt% aqueous, 0.3 mL) was added to a solution of tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-(3-fluoro-5-methoxy-anilino)pentyl]pyrrolidine-1- carboxylate (0.038 g, 0.079 mmol) in diethyl ether (3 mL). The reaction mixture was stirred at RT for 16-18 hrs. The ethereal phase was eliminated with pipette and the aqueous phase washed with diethyl ether (x2). H2O was then evaporated with a N2 stream and dried under vacuum to give the title compound (0.029 g, 95.17 %) as a pale-brown solid. ES / MS m / z: 325 (M+H)

[0101] Examples 2-17 in Table 3 were prepared in a manner essentially as described in Example 1 using the appropriate BOC and tert-butyl ester protected starting material,simultaneously deprotecting the amine and acid functional groups, or using the appropriate BOC protected starting material which bears an unprotected carboxylic acid. Table 3a. Starting material used: tert-butyl (3R)-3-[(1S)-1-tert-butoxycarbonyl-5-cyano- pentyl]pyrrolidine-1-carboxylate In vitro Lp(a) Assembly Assay

[0102] The ability of compounds to inhibit the formation of Lp(a) particles in vitro was assessed by a cell-free assembly assay. Conditioned media (DMEM supplemented with 10% FBS, 20 mM HEPES, and 1x penicillin / streptomycin) was collected from confluent wild-type HepG2 cells (a source of endogenously expressed ApoB) and from a HEK293 stable cell line expressing human Apo(a) protein containing 17 Kringle repeats (selected on 1 mg / ml geneticin) after 24 h of culture at 37 °C and 5% CO2. An in vitro assembly assay was conducted by combining equal parts of HepG2 and HEK293 conditioned media with the test compounds added in dilution series (final concentration 0.01~100 nM). The reaction was incubated at 37 °C for 2 hrs and then stopped with the addition of 6-aminocaproic acid (EACA) to a final concentration of 150 mM. Lp(a) was detected using a sandwich ELISA with an anti-Lp(a) capture antibody and an HRP -conjugated anti-ApoB detection antibody. The ELISA was developed using TMB, stopped using 1 N sulfuric acid, and the signal was read at 450 nm on a Molecular Devices plate reader. The % inhibition of Lp(a) formed for each test condition was determined with an assembly reaction having no inhibitor present (with matched DMSO concentration at 1%) set to0% inhibition, and an assembly reaction with a minimal amount of the HepG2 conditioned media present (50-fold dilution) set to 100% inhibition. Data were fitted to a 4-parameter curve to determine the IC50 values summarized in Table 4. Addition of the Example test compound to conditioned media containing ApoB and Apo(a) lead to concentration-dependent inhibition of Lp(a) formation in vitro, as summarized in Table 4. The results indicate that these compounds inhibit the assembly of Lp(a) from Apo(a) and the LDL particle.

Claims

CLAIMS We claim:

1. A compound of the formula:wherein L is C2-C6 alkylene, C2-C6 alkenylene or -(CH2)nNHCONH(CH2)n-; n at each occurrence is independently 2 to 4; Y at each occurrence is independently CH2, O or S; Z at each occurrence is independently H, C1-C4 alkyl, OH or cyclopropyl; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein Z at each occurrence is H, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or claim 2, wherein Y at each occurrence is CH2, or a pharmaceutically acceptable salt thereof.

4. The compound according to any one of claims 1 to 3, wherein L is C2alkylene, or a pharmaceutically acceptable salt thereof.

5. The compound according to any one of claims 1 to 3, wherein L is C2alkenylene, or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1 to 3, wherein L is -(CH2)3NHCONH(CH2)3-, or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 1, wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.

8. A compound of the formula:wherein L1is C2-C3alkylene or C2-C3alkenylene; R1is -NHC(O)R2, -NHC(O)NHR2, COOH, -C(O)NH-cyclopropyl,, ,, -O-phenyl, -NH-phenyl or phenyl, wherein the phenyl is optionally substituted with one or two substituents selected from halo and OCH3; R2is C1-C3alkyl; Y is CH2, O or S;Z is H, C1-C4 alkyl, OH or cyclopropyl; or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 8, wherein Z is H, or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 8 or claim 9, wherein Y is CH2, or a pharmaceutically acceptable salt thereof.

11. The compound according to any one of claims 8 to 10, wherein R1is -NHC(O)CH3, -NHC(O)NHCH2CH3, COOH, -C(O)NH-cyclopropyl,phenyl, or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 11 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

13. A method of treating cardiovascular disease in a patient comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

14. A method of treating elevated Lp(a) plasma levels in a patient comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

15. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, for use in therapy.

16. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, for use in the treatment of cardiovascular disease.

17. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, for use in the treatment of elevated Lp(a) plasma levels.

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