EA045074B1 - PYRROLIDINE COMPOUNDS - Google Patents

Description

The present invention relates to pyrrolidine compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions and therapeutic uses of the same.

Significant advances have been made in the treatment of cardiovascular diseases (CVD). Despite advances in treatment, patients continue to experience cardiovascular disease events such as angina, myocardial infarction, and stroke, which lead to death if left untreated. Impaired lipid metabolism 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-density lipoprotein cholesterol (HDL-C), elevated triglycerides (TG), and elevated lipoprotein(a) (LP(a) )). There are various treatment regimens that target 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 can be used to filter the blood to remove LDL and LDL(a); however, the effects are temporary and, as a rule, the procedure must be repeated every two weeks. There is no approved drug treatment to lower Lp(a) levels. The physiological function of LA(a) is complex; however, elevated plasma Lp(a) levels have been reported to be an independent risk factor for CVD. There is a need for drug treatment of patients with elevated levels of lipid(a)1.

Additional treatment options are desirable for patients suffering from cardiovascular diseases, and in particular for patients suffering from lipid disorders or dyslipidemia. There is a need for additional treatment options for patients whose cardiovascular risks are not sufficiently modifiable with current standard treatments, such as diet, exercise, and/or the use of one or more medications, such as statins, fibrates, and niacin. The present invention provides another treatment option for patients suffering from CVD. There is a need for pharmaceutically acceptable compounds and treatment options to reduce plasma levels of Lp(a). A compound of formula I' is proposed:

H/ ^r3

Where

L is selected from the group consisting of -CH2NHCH2-, -CH2NH-, -NH-, -S-, -S(O)-, -S(O)2-, -O-, -OCH2-,

Ceutically acceptable salt.

A compound of formula I is proposed:

its pharmaceutically acceptable salt. Proposed compound of formula 1:

- 1 045074

or a pharmaceutically acceptable salt thereof.

In one embodiment, a compound of formula 2 is provided:

or a pharmaceutically acceptable salt thereof.

In one embodiment, a compound of Formula I', I, 1, or Formula 2 is provided, wherein said compound is a pharmaceutically acceptable salt. In one embodiment, a compound of Formula I', Formula 1, or Formula 2 is provided, wherein said compound is a hydrochloride salt. In one embodiment, a compound of Formula 1 or Formula 2 is provided, wherein said compound is a tetrahydrochloride salt.

In one embodiment, a compound of Formula I', Formula 1, or Formula 2 is provided, wherein said compound is a hydrochloride salt selected from the group consisting of monohydrochloride, dihydrochloride, trihydrochloride, and tetrahydrochloride. In one embodiment, a compound of Formula 1 or Formula 2 is provided as a zwitterion.

A compound of formula I' is proposed, where L is selected from the group consisting of -CH2NHCH2-, -CH2NH-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -O-, - OCH2-, -OCH2CH2O- and -NHSO2NH-; or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where each of R ¹ and R ³ represents H; and L is selected from the group consisting of -CH2NHCH2-, -CH2NH-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -O-, -OCH ₂ -, -OCH2CH2O - and -NHSO2NH-; or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where each of R ¹ and R ³ represents H; R ² and R ⁴ are each CH ₃ ; and L is selected from the group consisting of -CH2NHCH2-, -CH2NH-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -O-, -OCH2-, -OCH2CH2O- and -NHSO2NH-; or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where each of R ¹ and R ³ represents H; R ² and R ⁴ are each H; and L is selected from the group consisting of -CH ₂ NHCH ₂ -, -CH ₂ NH-, -NH-, -S-, -S(O)-, -S(O)2-, -O-, -OCH2 -, -OCH2CH2O- and -NHSO2NH-; or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where L is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where each of R ¹ , R ³ and R ⁵ represents

N; and L is selected from the group consisting of

or its pharmaceutical 2 045074 ceutically acceptable salt.

A compound of formula I' is proposed, where each of R ¹ , R ³ and R ⁵ represents H; each of R ² ,

R ⁴ and R ⁶ represent H; or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where each of R ¹ , R ³ and R ⁵ represents H; and each of

R ² , R ⁴ and R ⁶ are CH ₃ ; or a pharmaceutically acceptable salt thereof.

A compound of formula I' is proposed, where L is

-CH2-N-CH2-

R ¹ , R ³ and R ⁵ are each H; each of R ² ,

R ⁴ and R ⁶ represents CH ₃ ; or a pharmaceutically acceptable salt thereof.

In one embodiment, a pharmaceutical composition is provided comprising a compound of Formula I', Formula I, Formula 1 or Formula 2, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent or excipient.

In one embodiment, a method of treating a patient in need of treatment for a cardiovascular disease is provided, comprising administering an effective amount of a compound selected from the group consisting of Formula I', Formulas 1 and 2, or a pharmaceutically acceptable salt thereof. In one embodiment, a method of treating a patient in need of treatment for a cardiovascular disease is provided, comprising administering an effective amount of a compound of formula I'' or a pharmaceutically acceptable salt thereof. In one embodiment, a method of treating a patient in need of treatment of elevated plasma levels of Lp(a) is provided, comprising administering an effective amount of a compound selected from the group consisting of Formula I', Formula 1 and Formula 2, or a pharmaceutically acceptable salt thereof. In one embodiment, a method of treating a patient in need of treatment of elevated plasma levels of Lp(a) is provided, comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

In one embodiment, a compound selected from the group consisting of Formula I', Formulas 1 and 2, or a pharmaceutically acceptable salt thereof, for use in therapy is provided. In one embodiment, a compound of Formula I or a pharmaceutically acceptable salt thereof is provided for use in therapy.

In one embodiment, a compound selected from the group consisting of Formula I', Formulas 1 and 2, or a pharmaceutically acceptable salt thereof, is provided for use in the treatment of a cardiovascular disease. In one embodiment, a compound of formula I'' or a pharmaceutically acceptable salt thereof is provided for use in the treatment of a cardiovascular disease.

In one embodiment, a compound selected from the group consisting of Formula I', Formulas 1 and 2, or a pharmaceutically acceptable salt thereof, is provided for use in the treatment of elevated plasma levels of Lp(a).

One embodiment provides the use of a compound selected from the group consisting of Formula I', Formula 1 and Formula 2, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of a cardiovascular disease.

LP(a) can exhibit both prothrombotic and antithrombotic properties, as well as atherothrombotic properties. LP(a) can inhibit fibrinolysis and accumulate in the vascular wall, causing thrombus formation and atherosclerotic lesions. Plasma levels of Lp(a) vary significantly between individuals. In contrast to other risk factors, plasma Lp(a) levels do not vary significantly with diet and exercise. Plasma levels of Lp(a) may be associated with genetic predisposition.

Lp(a) is similar to LDL-C in that it includes the lipid core of LDL with accompanying apolipoprotein B (apoB), but unlike LDL-C, Lp(a) also contains a unique apolipoprotein(a) (apo(a) ), linked to apoB via a disulfide bond. Apo(a) is synthesized in the liver. Assembly of lipo(a) from apo(a) particles and LDL can occur in hepatocytes, on the cell wall, or in plasma. Inhibition of LDL particle assembly by apo(a) can reduce Lp(a) levels.

As used herein, the term elevated plasma Lp(a) levels means a plasma level that is equal to or greater than about 50 mg/dL. The compound provided herein can be used for treatment to reduce plasma levels of Lp(a).

As used herein, the term pharmaceutically acceptable salt refers to a salt of a compound that is acceptable for clinical and/or veterinary use. Examples of pharmaceutically acceptable salts and the general methodology for their preparation can be found in Handbook of Pharmaceutical Salts: Properties, Selection and Use by P. Stahl, et al., 2nd edition, Wiley-VCH, 2011, and S.M. Berge, et al., Pharmaceutical Salts, Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19. The compounds of formulas I', I, 1 or formula 2 may be a zwitterionic salt, a salt of addition of one, two or three acids. The compounds of formula I', I, 1 or formula 2 may be one, two or three base addition salts.

The pharmaceutical compositions of the present invention can 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 are not harmful to the patient from a pharmaceutical point of view. Examples of pharmaceutical compositions and methods for their preparation are well known to those skilled in the art and are presented, for example, in Remington: The Science and Practice of Pharmacy, Loyd, V., et al. ed., 22nd ed., Mack Publishing Co., 2012.

As used herein, the term effective amount refers to the amount of dose that is effective in treating a disorder. The effective amount for a particular patient can be determined by a qualified healthcare professional.

As used herein, the terms treatment process, treat, or cure include slowing, preventing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. As used herein, the term treating a cardiovascular disease means slowing, reducing, preventing, or reversing the progression of a heart or blood vessel disease. A method of treating myocardial infarction is proposed, comprising administering a compound of formula I', formula 1 or formula 2 to a patient in need thereof.

As used herein, the term patient refers to a mammal. Preferably the patient is a human.

The pharmaceutical compositions may be formulated in the form of a tablet or capsule for oral administration, an oral solution or an injection. In one embodiment, the composition is suitable for oral administration.

Some abbreviations are defined as follows: Apo refers to apolipoprotein; BOC refers to tert-butoxycarbonyl; BSA refers to bovine serum albumin; DAD refers to a diode array detector; DCM refers to dichloromethane or methylene chloride; di. refers to diasteriomeric excess; DMEA refers to dimethylethylamine; DMEM refers to Dulbecco's modified Eagle's medium; DMF refers to K,K-dimethylformamide; DMSO refers to dimethyl sulfoxide; e.i. refers to enantiomeric excess; EACA refers to epsilon-aminocaproic acid or 6-aminocaproic acid; ELISA refers to an enzyme-linked immunosorbent assay; eq. refers to equivalents; Et ₂ O refers to diethyl ether; EtOAc refers to ethyl acetate; EtOH refers to ethanol or ethyl alcohol; Etc. refers to example; FBS refers to fetal bovine serum; HEC refers to hydroxyethylcellulose; HEK refers to the human embryonic kidney; HepG2 is a human hepatoma cell line; HEPES refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC refers to high-performance liquid chromatography; HRP refers to horseradish peroxidase; IC50 refers to the concentration of an agent that produces 50% of the maximum inhibitory response possible for that agent; min refers to a minute or minutes; MeOH refers to methanol or methyl alcohol; MTBE refers to methyl tert-butyl ether; RP-HPLC/MS refers to reverse-phase high-performance liquid chromatography with mass spectrometry; RT refers to room temperature; SLC refers to supercritical liquid chromatography; SPA refers to scintillation proximity analysis; t( _R ) refers to retention time; THF refers to tetrahydrofuran; TMB refers to 3,3',5,5'-teramethylbenzidine, and Tris refers to tris(hydroxymethyl)aminomethane.

The individual isomers, enantiomers and diastereomers can be isolated or separated by those skilled in the art at any convenient point in the synthesis of the compounds listed below by methods known to those skilled in the art, such as selective crystallization techniques or chiral chromatography.

A compound of formula I', formula I, formula 1 or formula 2 is readily converted into a pharmaceutically acceptable salt and can be isolated as such a salt. Salt formation can occur by adding a pharmaceutically acceptable acid to form an acid addition salt or by adding a pharmaceutically acceptable base to form a base addition salt. Salts can also form simultaneously when nitrogen or oxygen is deprotected, that is, when the protecting group is removed. Examples, reactions and salt formation conditions are known to one skilled in the art.

Compounds selected from the group consisting of Formula I', Formula I'', Formula 1 and Formula 2, or salts thereof, can be prepared by various methods, some of which are illustrated in the Preparation Examples and Examples sections below. The specific synthetic steps for each of the described methods can be combined in various ways or in combination with steps from other methods to produce the compounds or salts of the present invention. Products of each

- 4 045074 steps in the Preparation Examples section can be isolated by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration and crystallization.

In the following diagrams, all substituents, unless otherwise noted, are as previously defined. The reagents and starting materials are generally available to those skilled in the art. Without limiting the scope of the invention, the following diagrams, production methods and examples are presented to further illustrate the invention. The compounds of formula I', I, formula 1 and formula 2, or salts thereof, can be prepared using starting materials or intermediates with the corresponding desired stereochemical configuration.

Scheme 1 shows the preparation of intermediates that can provide access to the compounds of the present invention. Protected pyrrolidin-3-ylacetic acid derivative A is first converted to acyloxazolidinone B. This is achieved by first converting A to the acid chloride and reacting with (4S)-4-benzyloxazolidin-2-one in the presence of lithium chloride at 10°C. Alkylation of acyloxazolidinone B with a benzyl bromide derivative and a base such as lithium bis(trimethylsilyl)amide at 0°C affords intermediate C in a diastereoselective manner. It is known to those skilled in the art that the stereochemistry of oxazolidinone substitution affects the diastereoselectivity of the alkylation, and that the use of an oxazolidinone derivative B with the opposite or racemic stereochemical configuration in this synthesis can result in either the opposite diastereoselectivity or no diastereoselectivity. The conversion of acyloxazolidinone C to acid intermediate D is accomplished with aqueous LiOH and H ₂ O ₂ in THF at 5 to 15°C. Acidic intermediate D is optionally isolated as an ammonium salt. Acidic intermediate D is protected, for example, as tert-butyl ether by reaction with tert-butyl-1,3-diisopropyl isourea at elevated temperature to give intermediate E.

Scheme 1 also shows the conversion of acid A to methyl ester F, which is achieved by reacting A with iodomethane in the presence of a carbonate base. Intermediate F is then alkylated with a benzyl bromide derivative using a base such as lithium bis(trimethylsilyl)amide at -78°C to give intermediate G. Intermediate G is again alkylated with iodomethane using a base such as lithium bis(trimethylsilyl)amide. at -78°C, and the ester is then hydrolyzed with sodium hydroxide at elevated temperature to produce acid intermediate H. Acid intermediate H is protected, for example, as tert-butyl ester by reaction with tert-butyl-1,3diisopropylisourea at elevated temperature to obtain intermediate I.

In particular, intermediates D, E, H and I, where R ^a represents bromine or -NO2, are particularly suitable for further transformations in the preparation of compounds of formula I'. Intermediate D, where R ^a is -H, can be prepared either by alkylation of B with benzyl bromide followed by hydrolysis of an acyloxazolidinone, or by stirring intermediate D, where R ^a is bromine with carbon-supported palladium under a hydrogen atmosphere. When R ^a is -H, deprotection of the pyrrolidine nitrogen in intermediate D provides compounds of formula I.

- 5 045074

Scheme 1.

Scheme 2 shows the conversion of the key intermediate (J, prepared as described in Scheme 1) into the penultimate compounds of the present invention. Bromide is converted to aldehyde K using synthesis gas (1:1 CO/H ₂ ), palladium(II) acetate, butyldi-1adamantylphosphine and N,N,N',N'-tetramethylethylenediamine at elevated temperature. The aldehyde K is then converted to a mixture of N and O by reductive amination of K (2 or 3 equivalents, respectively) with ammonium and a reducing agent such as sodium triacetoxyborohydride or sodium cyanoborohydride, and then N and O are separated by chromatography. Alternatively, dimeric compound O is prepared by converting aldehyde K to aldoxime L, reducing L to amine M by flow hydrogenation using a nickel sponge catalyst at elevated temperature, followed by reductive amination of amine M with aldehyde K. Intermediate P is prepared by reductive amination of the dimeric intermediate O compounds with 3-fluoro-5-methoxybenzaldehyde.

Scheme 2.

R ^b and R ^c independently = -Н or -СНз

Scheme 3 shows an additional use of bromide intermediate J to prepare the penultimate compounds of the present invention. Bromide J is converted to boronic acid Q using tetrahydroxydiboron, chloro(2-dicyclohexylphosphino-2',4',6'-tripropyl-1,G-biphenyl)(2'-amino-1,G-biphenyl-2-yl) palladium(P), X-PHOS and potassium acetate at elevated temperature. Boronic acid Q is then converted to phenol T using H ₂ O ₂ at 5°C. Phenol T is coupled to bromide J using copper(I) iodide, N,N-dimethylglycine hydrochloride and

- 6 045074 cesium carbonate at elevated temperature to give diphenyl ether U. Phenol T also reacts with 1,2-dibromoethane and carbonate base at elevated temperature to give V. Aldehyde intermediate K (derived from bromide J, as described in diagram 2) reduced with sodium borohydride at 0°C to alcohol R, which is then subjected to the Mitsunobu reaction to give intermediate S.

Scheme 3.

R ^b and R ^c independently = -Н or -СНз

Scheme 4 shows an additional use of bromide intermediate J to prepare the penultimate compounds of the present invention. Two equivalents of bromide J are combined with potassium thioacetate using bis(dibenzylideneacetone)palladium, tribasic potassium phosphate), 1,1'-bis(diphenylphosphino)ferrocene to produce diphenyl sulfide W. Sulfide W is then converted to sulfone X or sulfoxide Y using 1 or 2 equivalents of metachloroperoxybenzoic acid, respectively.

- 7 045074

Scheme 4.

Scheme 5 shows the use of nitro intermediate Z (prepared as in Scheme 1) to prepare the penultimate compounds of the present invention. The intermediate nitro compound Z is reduced to aniline AA in the presence of a catalyst under a hydrogen atmosphere. Aniline AA undergoes reductive amination with aldehyde K (prepared as in Scheme 2) using a reducing agent such as sodium triacetoxyborohydride to give CC. Buchwald reaction between aniline AA and bromide BB (prepared as in Scheme 1) using methanesulfonate [(2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)- 2(2'-amino-1,G-biphenyl)]palladium(P) (BrettPhos Pd G3) and potassium carbonate at elevated temperature allows one to obtain the diphenylamine derivative DD. Two equivalents of aniline AA also react with the adduct of 1,4-diazabicyclo[2.2.2]octane-bis(sulfur dioxide) (DABSO) and iodine at elevated temperature to produce sulfonamide EE.

- 8 045074

Scheme 5.

General deprotection of intermediates N, O, P, S, U, V, W, X, Y, CC, DD and EE from Schemes 2-5 allows one to obtain compounds of formula I'. If the pyrrolidine protecting group (Pg ¹ in Schemes 1-5) is -BOC and the ester (Pg ² in Schemes 1-5) is tert-butyl ether, the overall deprotection is carried out in one step using a solution of HCl in organic a solvent such as diethyl ether, dioxane or isopropanol. After deprotection, the pyrrolidine nitrogen in the compounds of formula I' can be methylated by reductive amination with paraformaldehyde and sodium triacetoxyborohydride.

Receipt example 1.

tert-Butyl-(3R)-3-[2-[(4S)-4-benzyl-2-oxooxazolidin-3-yl]-2-oxoethyl]pyrrolidine-1-carboxylate.

Add triethylamine (56.5 g, 77.9 mL, 559 mmol, 2.5 eq.) to a solution of 2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]acetic acid (53.8 g, 235 mmol, 1.05 eq.) in THF (540 ml) maintained at 10°C. After 5 min, pivaloyl chloride (33.7 g, 34.2 mL, 279 mmol, 1.25 eq.) was added. After 15 min, add lithium chloride (11.8 g, 279 mmol, 1.25 eq.) in THF (540 ml) and (4S)-4benzyloxazolidin-2-one (40.0 g, 223 mmol, 1 eq.) . Allow the mixture to warm to RT and stir for 24 hours. After 24 hours, add 1 N. aqueous HCl (500 ml) and separate the organic phase from the aqueous phase. The organic phase is washed with 1 N. aqueous NaOH (500 ml) and saturated aqueous NaCl (500 ml), dry over MgSO ₄ , filter and concentrate the solution in vacuo. The precipitate is suspended in MeOH/water (1:2, 575 ml) and stirred at RT overnight. Filter the solid, wash with hexanes (2x150 ml) and dry the solid to give the title compound (65.7 g, 76%). ER/MS (m/z): 333 (M+H-tert-butyl), ¹ H NMR (400.13 MHz, CDCl ₃ ) δ 7.38-7.28 (m, 3H), 7.25- 7.20 (m, 2H), 4.73-4.70 (m, 1H), 4.27-4.19 (m, 2H), 3.75-3.66 (m, 1H), 3, 55-3.48 (m, 1H), 3.38-3.30 (m, 2H), 3.112.96 (m, 3H), 2.84-2.76 (m, 1H), 2.74- 2.65 (m, 1H), 2.14-2.11 (m, 1H), 1.64-1.58 (m, 1H), 1.49 (s, 9H).

- 9 045074

Receipt example 2.

tert-Butyl-(3S)-3-[2-[(4R)-4-benzyl-2-oxooxazolidin-3-yl]-2-oxoethyl]pyrrolidine-1-carboxylate.

The title compound was prepared substantially as described in Preparation Example 1 using 2-[(3S)-1-tert-butoxycarbonylpyrrolidin-3-yl]acetic acid and (4R)-4benzyloxazolidin-2-one. Purify the product by silica gel chromatography using a gradient of 10 to 50% EtOAc in hexanes. ES/MS (m/z): 333 (M+H-tert-butyl).

Receipt example 3.

tert-Butyl-(3R)-3-(2-methoxy-2-oxoethyl)pyrrolidine-1-carboxylate.

Add iodomethane (2 mol/L in MTBE, 240 ml, 480 mmol, 1.1 eq.) to a solution of 2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]acetic acid (100 g, 436 mmol) in DMF (800 ml) at CT. Add potassium carbonate (90.4 g, 654 mmol, 1.5 eq) and stir the mixture for 4 hours. Add water (1.5 L) and extract with MTBE (3 L). The organic phase is washed with ice/water (3 x 500 ml), dry the organic phase over MgSO4, filter and concentrate the solution under reduced pressure to give the title compound (103 g, 97%). ^1H NMR (300 MHz, CDC13) 5 3.68 (s, 3H), 3.65-3.53 (m, 1H), 3.52-3.36 (m, 1H), 3.35-3 .23 (m, 1H), 3.03-2.84 (m, 1H), 2.64-2.49 (m, 1H), 2.45-2.30 (m, 2H), 2.13 -1.97 (m, 1H), 1.65-1.48 (m, 1H), 1.45 (s, 9H).

Receipt example 4.

tert-Butyl-(3R)-3-[1-[(3-bromophenyl)methyl]-2-methoxy-2-oxoethyl]pyrrolidine-1-carboxylate.

To a solution of tert-butyl-(3R)-3-(2-methoxy-2-oxoethyl)pyrrolidine-1-carboxylate (7 g, 28 mmol) in THF (93 ml) at -78°C add bis(trimethylsilyl)amide lithium (1 M solution in THF, 33.5 ml, 33.5 mmol, 1.2 eq.). The mixture was stirred at -78°C for 1 hour. A solution of 3-bromobenzyl bromide (8.37 g, 33.5 mmol, 1.2 eq.) in THF (5 ml) was added. Allow the mixture to warm to RT and stir overnight. Quench the mixture with saturated aqueous NH ₄ Cl and extract with EtOAc. The organic layers are washed with saturated aqueous NaCl, dried over MgSO ₄ , filtered and evaporated to dryness. The residue was purified by silica gel chromatography using a gradient of 10 to 40% EtOAc in hexanes to give a mixture of diastereomers of the title compound (8.4 g, 73%) as a yellow oil. ES/MS (m/z): 356, 358 (M+H-tert-butyl).

Receipt example 5.

3-(3-Bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-2-methylpropanoic acid.

- 10 045074

To a solution of tert-butyl-(3R)-3-[1-[(3-bromophenyl)methyl]-2-methoxy-2-oxoethyl]pyrrolidine-1 carboxylate (8.4 g, 20 mmol) in THF (100 ml) under nitrogen atmosphere and at -78°C add lithium bis(trimethylsilyl)amide (1 M solution in THF, 41 ml, 41 mmol, 2 eq.). Stir the reaction mixture at -78°C for 2 hours. Add iodomethane (25 ml, 410 mmol, 20 eq.) and allow the mixture to warm to RT.

Stir the mixture overnight. Add saturated aqueous NH4Cl and extract with EtOAc. The organic layer was washed with saturated aqueous NaCl and dried over MgSO ₄ , filtered and the solvent was removed in vacuo. The residue is dissolved in MeOH (80 ml) and THF (80 ml), then sodium hydroxide (5 M solution in water, 81 ml, 410 mmol, 20 eq.) is added and the resulting mixture is heated at 60°C for 3 days. Allow the mixture to cool to RT, add HCl (1 N aqueous solution) to bring the pH of the mixture to 2-3. Extract the aqueous layer with EtOAc. The organic layer is dried over MgSO ₄ , filtered and concentrated in vacuo. The precipitate is subjected to chiral SLC using the following parameters: Chiralpak® AD column (25x3 cm, 5 µm); mobile phase solvent A=CO2, solvent

B=MeOH+0.2% v/v DMEA; gradient - isocratic 80:20 A:B; flow rate - 120 ml/min). Isomer 1 (1.7 g, 27%) and isomer 2 (3.4 g, 41%) of the title compound were obtained as a white solid. ES/MS (m/z): 356 / 358 (M+H-tert-butyl).

Receipt example 6.

tert-Butyl-(3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxooxazolidin-3-yl]-1-[(3-bromophenyl)methyl]-2oxoethyl]pyrrolidin- 1-carboxylate.

Add a solution of lithium bis(trimethylsilyl)amide (1M in THF, 818 mL, 818 mmol, 1.2 eq.) over 29 min at 0°C to a solution of tert-butyl-(3R)-3-[2-[(4S )-4-benzyl-2-oxooxazolidin-3-yl]-2oxoethyl]pyrrolidine-1-carboxylate (265 g, 682 mmol) in THF (1325 mL) in a 3-L 3-neck round-bottom flask in an ice bath under nitrogen. with mechanical stirring. The mixture is stirred for 47 minutes at 0.6°C. A solution of 1-bromo-3-(bromomethyl)benzene (190 g, 760 mmol, 1.12 eq) in THF (450 ml) was then added over 28 minutes, raising the reaction temperature to 5.1°C. Allow the mixture to warm to RT with stirring overnight. Cool the reaction mixture using an ice/water bath, then add saturated aqueous NH4Cl (1 L) in 4 portions at a rate to maintain the reaction temperature below 21°C. Water (1 L) was added to the mixture and extracted with MTBE (3.5 L). The organic layer was washed with a mixture of water (1 L) and saturated aqueous NaCl (500 ml), then saturated aqueous NaCl (500 ml). Organic substances are dried over Na ₂ SO ₄ , filtered, then concentrated in vacuo. Hexanes (1 L) were added to the residue and concentrated in vacuo, then dried under high vacuum overnight to give the title compound as an orange oil (416 g, >100%), purity estimated at 90 wt.% based on theoretical output. ES/MS (m/z): 501/503 (M+H-tert-butyl).

Receipt example 7.

tert-Butyl-(3S)-3-[(1R)-2-[(4R)-4-benzyl-2-oxooxazolidin-3-yl]-1-[(3-bromophenyl)methyl]-2oxoethyl]pyrrolidin- 1-carboxylate.

- 11 045074

The title compound was prepared substantially as described in Preparation Example 6 using tert-butyl(3S)-3-[2-[(4R)-4-benzyl-2-oxooxazolidin-3-yl]-2- oxoethyl]pyrrolidine-1carboxylate. ES/MS (m/z): 501, 503 (M+H-tert-butyl).

Receipt example 8.

tert-Butyl-(3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxooxazolidin-3-yl]-1-[(3-nitrophenyl)methyl]-2oxoethyl]pyrrolidin- 1-carboxylate.

Add lithium bis(thimethylsilyl)amide (1.0 M in THF, 46 mL, 46 mmol, 1.2 eq.) to the solution of tert-butyl-(3R)-3-[2-[(4S)-4-benzyl-2 -oxooxazolidin-3-yl]-2-oxoethyl]pyrrolidine-1-carboxylate (15 g, 39 mmol, 1 eq.) in THF (75 ml) at -20°C. Stir the mixture at -20°C for 20 minutes. Add a solution of 1-(bromomethyl)-3-nitrobenzene (9.17 g, 42.5 mmol, 1.1 eq.) in THF (45 ml). Stir the solution for 2 hours and allow it to warm to RT. The mixture is diluted with MTBE and quenched with a saturated aqueous solution of NH ₄ Cl. The phases are separated and the aqueous phase is extracted with MTBE. The organic extracts are combined and the organics are washed successively with water and saturated aqueous NaCl. Dry over Na ₂ SO ₄ , filter and concentrate the filtrate in vacuo. The precipitate is triturated in a mixture of MeOH/H ₂ O (2:1, 150 ml). Stir the suspension overnight. Filter to collect the solid and wash with hexanes. Dry the solid in vacuo at 40°C to give the title compound (19 g, 88%). ES/MS (m/z): 468 (M+H-tert-butyl).

Receipt example 9.

tert-Butyl-(3R)-3-[(1S)-1-benzyl-2-[(4S)-4-benzyl-2-okCOOksazolidin-3-yl]-2-oxoethyl]pyrrolidine-1carboxylate.

The title compound was prepared with a purity of 76% essentially as described in Preparation Example 6 using benzyl bromide. ES/MS (m/z): 423 (M+H-tert-butyl).

Example of receiving 10.

(2S)-3 -(3-Bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid.

Add hydrogen peroxide solution (0.88 M in water, 105 mL, 926 mmol, 1.5 eq.) in one portion to the mechanically stirred mixture of tert-butyl-(3R)-3-[(1S)-2-[(4S )-4-benzyl-2-oxooxazolidin-3yl]-1-[(3-bromophenyl)methyl]-2-oxoethyl]pyrrolidine-1-carboxylate (90 wt% purity, 381 g, 615 mmol) in THF (4 l) and cool to 8.6°C in a 12 L three-neck round bottom flask in an ice/water bath. Add a solution of lithium hydroxide monohydrate (38.7 g, 923 mmol, 1.5 eq.) in water

- 12 045074 (930 ml) for 25 minutes, increasing the reaction temperature to 12.8°C. The reaction mixture is stirred for 2.5 hours, then cooled to 5.7°C. Add a solution of sodium bisulfite (129.4 g, 1244 mmol, 2.02 eq.) in water (2 L) over 40 minutes, raising the reaction temperature to 14.7°C. Add aqueous NaOH (5N) to raise the pH of the reaction mixture to >12, then add water (1 L) and MTBE (4 L). Separate the layers and extract the aqueous layer with MTBE (2 L). Organic substances are combined, extracted with water (1 L), then the specified aqueous extraction is added to the main aqueous solution. Mix the aqueous solution with MTBE (3 L) and cool the mixture to 5°C. Add aqueous hydrochloric acid (5N) to adjust the pH of the mixture to 3. Separate the layers and wash the organic layer with a mixture of saturated aqueous NaCl (1L) and water (500ml). The organic layers are dried over Na ₂ SO ₄ , filtered and concentrated in vacuo at 40°C. The residue was dried under high vacuum to give the title compound (221.5 g, 90%) as a white solid. ES/MS (m/z): 342/344 (M+H-tert-butyl).

Receipt example 11.

Ammonium;(2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoate.

Mix (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid (237.6 g, 596.5 mmol) and MTBE (3801 ml) with formation of a cloudy mixture with solids. Filter the mixture through 2 glass fiber filter papers and add ammonium (7 M) in MeOH (128 mL, 896 mmol, 1.5 eq) to the filtrate under nitrogen with mechanical stirring. A white solid precipitates and the mixture becomes very thick. MTBE (800 ml) was added to the mixture to obtain a flowable suspension. The mixture was stirred at RT for 1.5 hours, then cooled from 5 to 0°C in an ice/acetone bath and stirred for 1.5 hours. The solid was vacuum filtered through a propylene mat and a 3 L porous glass funnel and wash the solid with MTBE (1 L), then continue applying vacuum at RT overnight under nitrogen. Combine the solid with two more batches of material prepared in a similar manner, starting with (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3yl]propanoic acid (47.3 g, 113 mmol and 53.0 g, 120 mmol). Suspend the solid in acetonitrile (4300 ml) and stir the mixture at 23°C with mechanical stirring under nitrogen atmosphere overnight. The solid was vacuum filtered and washed with acetonitrile (500 mL) and MTBE (1 L), then vacuum was applied for 4 hours at RT under nitrogen to obtain the title compound as a white powder (249.5 g, 77%). ES/MS (m/z): 342/344 (M+H-tert-butyl).

Receipt example 12.

Ammonium; (2R)-3-(3-bromophenyl)-2-[(3S)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoate.

The title compound was prepared substantially as described in Preparation Example 10 using Ίret-butyl-(3S)-3-[(1R)-2-[(4R)-4-benzyl-2-oxooxazolidine-3- yl]-1-[(3-bromophenyl)methyl]2-oxoethyl]pyrrolidine-1-carboxylate followed by the formation of ammonium salt as described in Preparation Example 11. ES/MS (m/z): 342, 344 (M+ N-tert-butyl).

Receipt example 13.

(2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-3-(3-nitrophenyl)propanoic acid.

The title compound is prepared substantially as described in Preparation Example 10.

- 13 045074 using tert-butyl-(3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxooxazolidin-3-yl]-1-[(3-nicrophenyl)methyl] -2-oxoethyl]pyrrolidine-1-carboxylate, skipping the step of adding aqueous sodium bisulfite during the reaction. ES/MS (m/z): 309 (M+H-tert-butyl).

Receipt example 14.

(2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-3-phenylpropanoic acid.

The title compound was prepared substantially as described in Preparation Example 10 using tert-butyl-(3R)-3-[(1S)-1-benzyl-2-[(4S)-4-benzyl-2- oxooxazolidin-3-yl]-2oxoethyl]pyrrolidine-1-carboxylate. The product is purified by trituration with a 1:1 MeOH:water mixture. ES/MS (m/z): 264 (M+H-tert-butyl).

Receipt example 15.

(2R)-2-[(3S)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-3-phenylpropanoic acid.

Dissolve ammonium (2R)-3-(3-bromophenyl)-2-[(3S)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoate (830 mg, 2.08 mmol) in EtOH (20 ml) and add palladium on a carbon support (10 wt.%, 222 mg, 0.208 mmol, 0.1 eq.). The mixture was stirred under a hydrogen atmosphere at RT overnight. The reaction mixture is filtered through a layer of diatomite. Concentrate the filtrate and purify the residue by silica gel chromatography using a gradient of 10 to 40% EtOAc in hexanes with 1% acetic acid to give the title compound (560 mg, 84%) as a white solid. ES/MS (m/z): 264 (M+H-tert-butyl).

Receipt example 16.

tert-Butyl-(3R)-3-[(1S)-1-[(3-bromophenyl)methyl]-2-Ίret-butoxy-2-oxoethyl]pyrrolidine-1-carboxylate.

Ammonium is added to the reactor; (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin3-yl]propanoate (500 g, 1210 mmol), 2-methyltetrahydrofuran (4000 ml) and then a solution of KHSO4 (1M in water, 3000 ml). The mixture is stirred for 30 minutes, during which time pH 2-3 is measured. The phases of the reaction mixture are separated and the aqueous layer is extracted with 2-methyltetrahydrofuran (1000 ml).

Combine the organic phases and wash them with saturated aqueous NaCl. The organic phase is dried over MgSO ₄ and filtered. Transfer the solution to the reactor and add 2-tert-butyl-1,3diisopropylisourea (618.2 g, 3024 mmol, 2.5 eq.). Stir the mixture at 65°C for three hours and add more 2-tert-butyl-1,3-diisopropylisourea (247.3 g, 1210 mmol, 1 eq.). The mixture is stirred at 65°C overnight. The mixture is cooled to RT. Filter off the solid and wash the organic layer with saturated aqueous NaHCO ₃ (1000 ml). Dry the organic layer over MgSO ₄ , filter and concentrate in vacuo. MTBE (2000 ml) was added to the precipitate and the solid was filtered off. Concentrate the filtrate to obtain the title compound (483 g, 88%) as a white solid. ER/MS (m/z): 342/344 (M+H- 2x tert-butyl).

Receipt example 17.

tert-Butyl-(3R)-3-[1-[(3-bromophenyl)methyl]-2-tert-butoxy-1-methyl-2-oxoethyl]pyrrolidine-1 carboxylate.

- 14 045074

To a solution of 3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-2-methylpropanoic acid, isomer 2 (3.4 g, 8.2 mmol) in 2-methyltetrahydrofuran (33 ml) add 2-tert-butyl-1,3diisopropylisourea (5.1 g, 5.7 ml, 25 mmol, 3 eq.) and heat the mixture at 55°C for 3.5 hours. Add additional 2 -tert-butyl-1,3-diisopropylisourea (5.1 g, 5.7 ml, 25 mmol, 3 eq.) and continue heating the reaction mixture at 55°C for 1.5 hours. Add additional 2-tert -butyl-1,3-diisopropylisourea (2.5 g, 2.9 ml, 12 mmol, 1.5 eq.) and continue to heat the reaction mixture at 55°C overnight. Filter off the white solid and wash with MTBE, then evaporate the filtrate to dryness. The residue was purified by silica gel chromatography using a gradient of 0 to 100% EtOAc in hexanes to give the title compound (3.6 g, 93%) as a yellow oil. ER/MS (m/z): 356, 358 [M+H-(2xtert-butyl)].

Receipt example 18.

tert-Butyl-(3R)-3-[( 1 S)-2-tert-butoxy-1-[(3-nitrophenyl)methyl]-2-oxoethyl]pyrrolidine-1 carboxylate.

The title compound was prepared substantially as described in Preparation Example 17 using (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-(3-nitrophenyl)propanoic acid. and carry out the reaction at 80°C in toluene as the reaction solvent. The crude product is purified by silica gel chromatography using a gradient of 5 to 20% EtOAc in hexanes. ER/MS (m/z): 309 [M+H-(2xtert-butyl)].

Receipt example 19.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-formylphenyl)methyl]-2-oxoethyl]pyrrolidine-1carboxylate.

Add a solution of tert-butyl-(3R)-3-[(1S)-1-[(3-bromophenyl)methyl]-2-tert-butoxy-2-oxoethyl]pyrrolidine-1-carboxylate (300 g, 660 mmol) to the Parr reactor ) in toluene (3000 ml) followed by palladium(II) acetate (7.41 g, 33.0 mmol, 0.05 eq), butyldi-1-adamantylphosphine (24.92 g, 66.02 mmol, 0 .1 eq.) and N,N,N',N'-tetramethylethylenediamine (115 g, 149 ml, 990 mmol, 1.5 eq.). Create a synthetic gas pressure in the mixture of 70 psig. inch (CO/H2 1:1) and stir at 100°C overnight. The mixture is cooled to RT and the solvent is evaporated to dryness. The precipitate was dissolved in EtOAc and filtered through silica gel to give the title compound (273 g, 97%) as an orange oil. ER/MS (m/z): 426 (M+Na).

Example of getting 20.

tert-Butyl-(3R)-3-[2-tert-butoxy-1-[(3-formylphenyl)methyl]-1-methyl-2-oxoethyl]pyrrolidine-1carboxylate.

- 15 045074

The title compound was prepared substantially as described in Preparation Example 19 using tert-bugyl-(3R)-3-[1-[(3-bromophenyl)methyl]-2-tert-butoxy-1-methyl- 2-oxoethyl]pyrrolidine 1-carboxylate. The crude product is purified by silica gel chromatography using a gradient of 10 to 30% acetone in hexanes. ES/MS (m/z): 262 (M+H-tert-butyl-BOC).

Receipt example 21.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[hydroxyiminomethyl]phenyl]methyl]-2oxoethyl]pyrrolidine-1-carboxylate.

Mix tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-formylphenyl)methyl]-2-oxoethyl]pyrrolidine 1-carboxylate (50 g, 105 mmol, 85% purity ), EtOH (400 ml), pyridine (17.0 ml, 211 mmol, 2 eq.) and hydroxylamine hydrochloride (10.98 g, 158.0 mmol, 1.5 eq.). The mixture is stirred at RT for 2 hours. The solvent is evaporated, KHSO ₄ (1 M solution in water, 300 ml) and MTBE (500 ml) are added. Separate the organic layer, wash the organic layer with saturated aqueous NaHCO ₃ , filter and dry over MgSO ₄ . Filter through Celite® and concentrate to dryness to give the title compound as a yellow oil (49 g, 98% yield, 88% w/w purity). ER/MS (m/z): 319 (M+H-Boc).

Receipt example 22.

tert-Butyl-(3R)-3-[(1S)-1-[[3-(aminomethyl)phenyl]methyl]-2-tert-butoxy-2-oxoethyl]pyrrolidine-1carboxylate.

Prepare a FlowCAT high pressure flow chemical reactor (HEL Ltd) with a stainless steel packed bed reactor column (inner diameter 1.2 cm x length 10 cm) containing a sponge nickel catalyst (2 g) and glass beads (6 g, 212 to 300 µm ). The reactor is equipped with gas and liquid flow regulators and a heating jacket around the column. The column is equilibrated by washing with MeOH and hydrogen gas with the following parameters: liquid flow rate - 4 ml/min; flow rate H ₂ - 60 ml/min; pressure - 50 Bar; the temperature of the reactor column jacket is 120°C (the internal reaction temperature is maintained at 50°C).

Dissolve tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[hydroxyiminomethyl]phenyl]methyl]-2oxoethyl]pyrrolidine-1-carboxylate (49 g, 103 mmol, purity 88% w/w) in ammonium (7 M solution in MeOH, 606 ml) and washed through the reactor column along with hydrogen gas using column equilibration parameters, collecting the eluate from the reactor column. Wash the column again with ammonium (7 M solution in MeOH) for 20 minutes, collecting the eluate from the reactor column. Combine the eluate fractions from the reactor column and concentrate the mixture in vacuo to give the title compound as an oil (46.9 g, 88% yield, 78% w/w purity). ER/MS (m/z): 405 (M+H).

Receipt example 23.

tert-Butyl-(3R)-3-[(1S)-1-[(3-aminophenyl)methyl]-2-tert-butoxy-2-oxoethyl]pyrrolidine-1-16 045074 carboxylate.

Add carbon-supported palladium (10% w/w, 380 mg, 0.36 mmol, 0.05 eq.) to the stirred solution of tert-butyl-(3R)-3-[(1S)-2-tert- butoxy-1-[(3-nitrophenyl)methyl]-2-oxoethyl]pyrrolidine 1-carboxylate (3 g, 7.1 mmol) in EtOAc (71 ml) under nitrogen. The mixture is purged with hydrogen and stirred under a hydrogen atmosphere at RT overnight. The reaction mixture is filtered through a layer of diatomite. Concentrate the filtrate in vacuo to give the title compound (2.62 g, 94%) as a white solid. ER/MS (m/z): 291 (M+H-BOC).

Example of receiving 24.

tert-Butyl-(3R)-3-[(1S)-2-Ίret-butoxy-1-[[3-(hydroxymethyl)phenyl]methyl]-2-oxoethyl]pyrrolidine-1carboxylate.

Add sodium borohydride (0.208 g, 5.50 mmol, 1.2 eq.) to the solution of tert-butyl-(3R)-3-[(1S)-2 tert-butoxy-1-(3-formylphenyl)methyl]-2 -oxoethyl]pyrrolidine-1-carboxylate (1.85 g, 4.58 mmol) in MeOH (25 ml) at 0°C. After 10 minutes, the solvent is evaporated. Add saturated aqueous NaHCO3 and extract the aqueous layer with EtOAc. Wash the organic layer with water and a saturated aqueous NaCl solution.

The organic layers were dried over MgSO ₄ and concentrated in vacuo to give the title compound (2.94 g, 97.5%) as a colorless oil. ER/MS (m/z): 428 (M+Na).

Example of receiving 25.

[3-[(2S)-3-tert-Butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]boronic acid.

Mix tert-butyl-(3R)-3-[(1S)-1-[(3-bromophenyl)methyl]-2-m-tert-butoxy-2-oxoethyl]pyrrolidine-1carboxylate (16.4 g, 36.1 mmol ), tetrahydroxydiboron (5.00 g, 54.1 mmol, 1.5 eq.), chloro-(2dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2' -amino-1,1'-biphenylyl)]palladium (II) (XPhos Pd G2, 0.145 g, 0.180 mmol, 0.005 eq.), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos, 0.176 g , 0.361 mmol, 0.01 eq.), potassium acetate (10.6 g, 108 mmol, 3 eq.), EtOH (246 ml) and ethylene glycol (6.10 ml, 108 mmol, 3 eq.). Purge with nitrogen for 5 minutes. The mixture is stirred at 90°C overnight. The reaction mixture was cooled to RT, 2-methyltetrahydrofuran (158 ml) was added and filtered. Concentrate and use the precipitate from Preparation Example 26 without further purification. ES/MS (m/z): 308 (M+H-2x tert-butyl).

Receipt example 26.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-hydroxyphenyl)methyl]-2-oxoethyl]pyrrolidine-1carboxylate.

- 17 045074

To a solution of [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3oxopropyl]phenyl]boronic acid (3.8 g, 8.4 mmol ) in THF (42 ml) add a solution of hydrogen peroxide (30% in water, 9.1 ml, 84 mmol, 10 eq.). The reaction mixture was stirred at RT overnight. Slowly quench the reaction by adding sodium bisulfite solution (20% w/v in water, 120 ml). Extract the mixture with EtOAc twice and wash the combined organic layers with saturated aqueous NaCl. The organic layers are dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of 10 to 40% acetone in hexanes to give the title compound (3.05 g, 90%) as a beige solid. ES/MS (m/z): 280 (M+H-2x tert-butyl).

Receipt example 27.

Cret-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3-tert-butoxy-2-[(3R) -1-Ίret-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidine-1 carboxylate.

Add tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-formylphenyl)methyl]-2-oxoethyl]pyrrolidine 1-carboxylate (48 g, 100 mmol, 85% purity w/w, 1.1 eq), isopropanol (328 ml) and tert-butyl-(3R)3-[(18)-1-[[3-(aminomethyl)phenyl]methyl]-2-tert -butoxy-2-oxoethyl]pyrrolidine-1-carboxylate (46.9 g, 93 mmol, 78% purity, 1 eq.) into a round bottom flask. The mixture was stirred at RT for 1 hour. The mixture was cooled to 0°C and sodium triacetoxyborohydride (59 g, 280 mmol, 3 eq.) was added and the mixture was stirred at RT for 2 days. Remove the solvent under reduced pressure to obtain a precipitate. Add water (200 ml), saturated aqueous NaHCO ₃ (300 ml) and EtOAc (500 ml). The organic layer was separated and purified by silica gel chromatography using a gradient of 50 to 100% EtOAc in hexanes to give the title compound as a colorless oil (36 g, 42% yield, 85% w/w purity). ER/MS (m/z): 792 (M+H).

Receipt example 28.

tert-Butyl-(3R)-3-[(1S)-1-[[3-[[bis[[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert- butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tert-butoxy-2oxoethyl]pyrrolidine-1-carboxylate and tert-butyl-(3R)-3-[( 1S)-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3-tertbutoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3- oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidine-1-carboxylate.

- 18 045074

Method 1 for preparing the title compounds as a mixture followed by separation by chromatography.

Add tert-butyl(3R)-3-[(1S)-2-Ίret-butoxy-1-[(3-formylphenyl)methyl]-2oxoethyl]pyrrolidine-1-carboxylate (222 g, 550 mmol) to a round bottom flask. , 2-propanol (888 ml) and ammonium solution (2M in 2propanol, 302.6 ml, 605.2 mmol, 1.1 eq.). Cool the mixture to 0-5°C using an ice bath. Add sodium triacetoxyborohydride (116.6 g, 550.2 mmol, 1 eq.) in four portions, allowing 40 minutes between additions of each portion. Stir the mixture at RT overnight. Evaporate the solvent to dryness. Add water (200 ml), aqueous K2HPO4 (300 ml) to the precipitate and extract the aqueous layer with MTBE (2x500 ml). The organic layer is dried over MgSO ₄ , filtered and concentrated to dryness. The residue is purified by silica gel chromatography using a gradient of 20 to 80% EtOAc in hexanes to give tert-butyl-(3R)-3-[(1S)-1-[[3-[[bis[[3-[ (2S)-3-tert-butoxy-2-[(3R)-1-tertbutoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tertbutoxy-2-oxoethyl ]pyrrolidine-1-carboxylate (57.8 g, 27%) as a white solid. 1H-NMR (400 MHz, CDC13) δ 7.30-7.24 (m, 6H), 7.12 (s, 3H), 7.04 (s, 3H), 3.75-3.43 (m , 12H), 3.30-3.21 (m, 3H), 3.102.96 (m, 3H), 2.89-2.76 (m, 6H), 2.49 (d, J=4.7 Hz, 3H), 2.37 (dd, J=7.2, 14.4 Hz, 3H), 1.98-1.90 (m, 3H), 1.741.61 (m, 3H), 1.48 (s, 27H), 1.22 (s, 27H).

The silica gel chromatography described above also provides tert-butyl-(3R)-3[(1S)-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3- tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3oxopropyl] phenyl] methyl] amino] methyl] phenyl] methyl] -2-oxoethyl] pyrrolidin-1-carboxylate (82.50 g, 34%) in the form of a colorless oil. ES/MS (m/z): 792 (M+H), HPLC purity 90 wt%.

Method 2 for obtaining tert-butyl-(3R)-3-[(1S)-1-[[3-[[bis[[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tertbutoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tertbutoxy-2-oxoethyl]pyrrolidine-1-carboxylate.

Add tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3-Ίret-butoxy-2-[( 3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate (purity 90 wt.%, 81.5 g, 92.6 mmol), tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3-formylphenyl)methyl]-2-oxoethyl]pyrrolidine-1-carboxylate (purity 85 wt.%, 50.6 g, 106 mmol, 1.15 eq.), 2-propanol (652 ml) and acetic acid (5.31 ml, 92.6 mmol, 1 eq.) and stir the mixture for 30 min. Sodium triacetoxyborohydride (2 eq., 185 mmol, 39.3 g) was added to the mixture and stirred at RT for 2 hours, then the reaction mixture was concentrated in vacuo. Water (200 ml) and MTBE (300 ml) are added to the precipitate, and then concentrated aqueous ammonium hydroxide is added to adjust the pH to 9-10. The organic phase is separated and dried over MgSO ₄ , filtered and concentrated to dryness. The residue is purified by silica gel chromatography using a gradient of 20 to 40% EtOAc in hexanes to give tert-butyl-(3R)-3-[(1S)-1-[[3-[[bis[[3[( 2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tert-butoxy- 2-oxoethyl]pyrrolidine-1-carboxylate (89 g, 82%) as a white solid.

Receipt example 29.

tert-Butyl-(3R)-3-[(1S)-2-Ίret-butoxy-1-[[3-[[[3-[(2S)-3-tert-butoxy-2-[(3R)- 1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl] methyl-[(3-fluoro-5-methoxyphenyl)methyl] amino]meyl]phenyl]methyl]-2-oxoethyl]pyrrolidine-1-carboxylate.

- 19 045074

Mix tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3-Ίtert-butoxy-2-[(3R )-1-Ίret butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate (36 g, 39 mmol, 85% purity), 3-fluoro-5-methoxybenzaldehyde (6.684 g, 42.49 mmol, 1.1 eq.), isopropanol (288 ml) and sodium triacetoxyborohydride (16.38 g, 77.26 mmol, 2 eq.), and stir the mixture at RT for 2 hours. The solvent is evaporated. Add saturated aqueous NaHCO ₃ (500 ml) and extract the aqueous layer with EtOAc (500 ml). The organic layer is dried over MgSO ₄ then filtered and concentrated to dryness. Purify by silica gel chromatography using a gradient of 10 to 40% EtOAc in hexanes to give the title compound as a colorless oil (32 g, 76% yield, 85% w/w purity). ER/MS (m/z): 931 (M+H).

Example of getting 30.

tert-butyl-(3R)-3-[1-[[3-[[bis[[3-[3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3yl]-2-methyl -3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tert-butoxy-1-methyl-2oxoethyl]pyrrolidine-1-carboxylate.

Prepare the title compound substantially as described in Preparation Example 28 using tert-butyl-(3R)-3-[2-tert-bugoxy-1-[(3-formylphenyl)methyl]-1-methyl -2-oxoethyl]pyrrolidine-1-carboxylate. The crude material is purified by silica gel chromatography using a gradient of 20 to 40% EtOAc in hexanes. ¹ H NMR (400.21 MHz, d ₆ -DMSO): δ 7.27-7.19 (m, 6H), 7.13 (s, 3H), 7.08-7.00 (m, 3H) , 3.57-3.24 (m, 9H, in solvent), 3.22-2.98 (m, 9H), 2.63-2.38 (m, 9H, in solvent), 1.86- 1.68 (m, 6H), 1.40 (s, 27H), 1.31 (s, 27H), 0.93 (s, 9H).

Receipt example 31.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1- tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenoxy]phenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate.

- 20 045074

Mix tert-butyl-(3R)-3-[(1S)-1-[(3-bromophenyl)methyl]-2-tert-butoxy-2-oxoethyl]pyrrolidine-1 carboxylate (150 mg, 0.330 mmol), tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-hydroxyphenyl)methyl]2-oxoethyl]pyrrolidine-1-carboxylate (0.155 g, 0.396 mmol, 1.2 eq.), copper iodide (6 mg, 0.03 mmol, 0.1 eq.), N,N-dimethylglycine hydrochloride (0.0138 g, 0.0990 mmol, 0.3 eq.) and cesium carbonate (0.215 g, 0.660 mmol, 2 eq.) in DMF (2.3 ml). The mixture is stirred under nitrogen atmosphere at 110°C overnight. The reaction mixture is cooled, then filtered through a pad of diatomaceous earth, washing with DCM and MeOH. The mixture is concentrated in vacuo. The resulting precipitate is purified by RP-HPLC/MS using the following parameters: column - XBridge™ C18 (19x100 mm, 5 µm); mobile phase - solvent A = 20 mM ammonium bicarbonate in water (pH 9), solvent B = acetonitrile; gradient - from 0:100 to 100:0 V: A; flow rate -25 ml/min. The title compound is obtained (43 mg, 17%). ER/MS (m/z): 665 (M+H-BOC).

Receipt example 32.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[2-[3-[(2S)-3-tert-butoxy-2-[(3R) -1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenoxy]ethoxy]phenyl]methyl]-2-oxoethyl]pyrrolidin-1 carboxylate.

Dissolve tert-butyl-(3R)-3-[(1S)-2-Ίret-butoxy-1-[(3-hydroxyphenyl)methyl]-2-oxoethyl]pyrrolidine-1-carboxylate (275 mg, 0.70 mmol ) in DMF (1.2 ml). Cesium carbonate (285 mg, 0.86 mmol) and 1,2-dibromoethane (0.030 mL, 0.34 mmol) were added and stirred at RT for 3 days. Over the next 9 days, add three additional portions of cesium carbonate or potassium carbonate and 1,2 dibromoethane and gradually increase the temperature to 70 and 110°C. The mixture is cooled at RT and saturated aqueous NH4Cl is added. Extract the aqueous layer with EtOAc. The organic phase is washed with saturated aqueous NaCl solution and water. The organic phase is dried over MgSO ₄ , filtered and the solution is concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of 0 to 30% EtOAc in hexanes to give the title compound (33.4 mg, 11%) as a colorless oil. ER/MS (m/z): 709 (M+H-BOC).

Receipt example 33.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenoxy]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate.

Add triphenylphosphine (0.5733 g, 2.164 mmol, 1.5 eq.) to the tert-butyl-(3R)-3-[(1S)-2 tert-butoxy-1-[[3-(hydroxymethyl)phenyl]methyl solution ]-2-oxoethyl]pyrrolidine-1-carboxylate (585 mg, 1.443 mmol) and tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-hydroxyphenyl)methyl] -2-oxoethyl]pyrrolidine-1-carboxylate (0.8472 g, 2.164 mmol, 1.5 eq.) in THF (14 ml). Purge with nitrogen and then add diethyl azodicarboxylate (0.34 ml, 2.2 mmol, 1.5 eq.) dropwise. The mixture is stirred overnight at RT. Filter the reaction mixture through a pad of diatomaceous earth, then wash with DCM and MeOH. The mixture is concentrated in vacuo. The resulting precipitate is purified using SLC with the following parameters: column - Chiracel® OD (5 μm, 2x25 cm); mobile phase - solvent A = CO2, solvent B = MeOH + DMEA (1.0% v/v); gradient - isocratic 80:20 A:B; flow rate - 80 ml/min; pressure - 120 bar; column temperature - 40°C. The title compound is obtained (315 mg, 28%). ER/MS (m/z): 679 (M+H-BOC).

Receipt example 34.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1- tert-butoxycarbonyl-21 045074 pyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfanylphenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate.

Add dry toluene (0.9 ml) and acetone (1.8 ml) to the mixture of tert-butyl-(3R)-3-[(1S)-1-[(3bromophenyl)methyl]-2-tert-butoxy-2 -oxoethyl]pyrrolidine-1-carboxylate (796 mg, 1.75 mmol), tribasic potassium phosphate (229 mg, 1.06 mmol), palladium bis(dibenzylideneacetone) (50 mg, 0.09 mmol), 1.1' bis(diphenylphosphino)ferrocene (70 mg, 0.12 mmol) and potassium thioacetate (103 mg, 0.90 mmol). The resulting mixture is sonicated for five minutes under a nitrogen atmosphere, then stirred at 110°C for 6 hours. Saturated aqueous NH ₄ Cl and EtOAc are added. Wash the organic layer with water. The organic phase is dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of 0 to 40% EtOAc in hexanes to give the title compound as a yellow oil (370 mg, 52%). ER/MS (m/z): 781 (M+H).

Example of receiving 35.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1- tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfinylphenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate.

Dissolve tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfanylphenyl]methyl]-2-oxoethyl]pyrrolidine-1-carboxylate (111 mg, 0.14 mmol) in DCM (2 ml). Add 3-chloroperoxybenzoic acid (34 mg, 0.14 mmol, 1 eq.). The mixture was stirred at RT for 3 hours. Add saturated aqueous NaHCO ₃ followed by DCM and separate the layers. The organic phase is washed with NaOH solution (3% w/v in water) and water. The organic phase is dried over MgSO ₄ , then filtered and the solution is concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient of 0 to 60% EtOAc in hexanes to give the title compound (97.2 mg, 87%) as a colorless oil. ER/MS (m/z): 697 (M+H-BOC).

Receipt example 36.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1- tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfonylphenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate.

The compound was prepared in a manner substantially similar to that of Preparation Example 35, but using 2.5 equivalents of 3-chloroperoxybenzoic acid. The crude product was purified by silica gel chromatography using a gradient of 20 to 60% EtOAc in hexanes to give the title compound (116.6 mg, 82%) as a white solid. ER/MS (m/z): 713 (M+H-BOC).

Receipt example 37.

tert-Butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]anilino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidin-1-carboxylate.

- 22 045074

Add sodium triacetoxyborohydride (543 mg, 2.56 mmol, 2 eq.) to the solution of tert-butyl-(3R)-3[(1S)-1-[(3-aminophenyl)methyl]-2-tert-butoxy-2 -oxoethyl]pyrrolidine-1-carboxylate (500 mg, 1.28 mmol), tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[(3-formulphenyl)methyl]- 2-oxoethyl]pyrrolidine-1carboxylate (620 mg, 1.54 mmol, 1.2 eq.) and acetic acid (220 μl, 3.84 mmol, 3 eq.). Stir at RT for 4 days. Add saturated aqueous NaHCO ₃ and extract the aqueous layer with DCM. Dry the combined organic layers over MgSO ₄ , filter and concentrate the solution in vacuo. The residue is purified by silica gel chromatography using a gradient of 10 to 50% EtOAc in hexanes to give the title compound (850 mg, 72%). ER/MS (m/z): 778 (M+H).

Receipt example 38.

(2S)-3-[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]anilino]phenyl]- 2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid.

Mix (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid (8 g, 20 mmol) in 1,4-dioxane (160 ml), tert-butyl-(3R)-3-[(1S)-1-[(3-aminophenyl)methyl]-2-tert-butoxy-2-oxoethyl]pyrrolidine-1-carboxylate (8.628 g, 22.09 mmol, 1. 1 equiv.) and potassium carbonate (4 equiv., 11.22 g, 80.34 mmol). The mixture is heated at 60°C for 15 minutes under a nitrogen atmosphere. Add [(2dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)] palladium (II) methanesulfonate ) (BrettPhos Pd G3, 0.371 g, 0.402 mmol, 0.02 eq.) and heat the reaction mixture at 100°C with stirring overnight. The reaction mixture is cooled to room temperature, diluted with EtOAc, and the pH of the mixture is adjusted to less than 3 by adding aqueous HCl (1 N). The aqueous layer is extracted three times with Me-THF. Wash the combined organic layers with saturated aqueous NaCl. Dry the organic phase over MgSO ₄ and concentrate the solution in vacuo. Purify the residue by chromatography on silica gel, eluting with a gradient of 0-100% EtOAc in hexanes to give the title compound (9.5 g, 67%). ER/MS (m/z): 608 (M+H-BOC).

Receipt example 39.

Di-tert-butyl-3,3'-((2S,2'S)-((sulfonylbis(azandiyl))bis(3,1-phenylene))bis(3-(tert-butoxy)-3oxopropane-1,2 - diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate).

Cool to 0°C a solution of 1,4-diazabicyclo[2.2.2]octane-bis(sulfur dioxide) adduct (DABSO, 0.6279 g, 2.561 mmol, 2 eq.) in acetonitrile (12.8 ml). Add iodine (0.4875 g, 1.921 mmol, 1.5 eq). The mixture is stirred at 0°C for 15 min, and then tert-butyl-(3R)-3-[(1S)-1-[(3

- 23 045074 aminophenyl)methyl]-2-tert-butoxy-2-oxoethyl]pyrrolidine-1-carboxylate (500 mg, 1.280 mmol). Heat the reaction mixture to 80°C overnight. The reaction mixture is cooled, filtered through a pad of diatomaceous earth and washed with DCM and MeOH. The filtrate is concentrated in vacuo. Purify the residue using RP-HPLC/MS with the following parameters: column -XBridge™ C18 (19x 100 mm, 5 µm); mobile phase - solvent A = 20 mM aqueous ammonium bicarbonate (pH 9), solvent B = acetonitrile; gradient - isocratic 80:20 A:B; flow rate - 25 ml/min; CT. The title compound (195 mg, 18%) is obtained as a pale yellow solid. ER/MS (m/z): 743 (M+NVOC).

Example 1.

(2S)-3-[3-[[Bis[[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]methyl]amino]methyl]phenyl] -2[(3R)-nirrolidin-3-yl]propanoic acid; tetrahydrochloride.

N

Add hydrochloric acid solution (4M in 1,4-dioxane, 67 mL, 270 mmol, 20 eq.) to tert-butyl(3R)-3-[(1S)-1-[[3-[[bis[[3 -[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3oxopropyl]phenyl]methyl] amino]methyl]phenyl] methyl]-2-tert-butoxy -2-oxoethyl]pyrrolidine-1 carboxylate (15.7 g, 13.3 mmol) and stir the mixture at 40°C overnight. The mixture is cooled to RT and then concentrated to dryness. Dissolve the precipitate in water (40 ml) and lyophilize. The resulting solid was redissolved in water (40 ml) and lyophilized to give the title compound (8.6 g, 75%) as a white foam. ER/MS (m/z): 711 (M+H); 1H-NMR (500 MHz, D2O) δ 7.357.10 (m, 12H), 4.25-4.17 (m, 6H), 3.55 (dd, J=8.1, 11.5 Hz, 3H ), 3.38-3.33 (m, 3H), 3.22-3.15 (m, 3H), 3.032.87 (m, 12H), 2.56-2.45 (m, 3H), 2.12-2.08 (m, 3H), 1.73-1.63 (m, 3H).

Example 2.

(2S)-3-[3-[[bis[[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]methyl]amino]methyl]phenyl] -2[(3R)-nirrolidin-3-yl]propanoic acid.

H

ABOUT

Add tert-butyl-(3R)-3-[(1S)-1-[[3-[[bis[[3-[(2S)-3-tert-butoxy-2[(3R)-1) to a round bottom flask -tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]2-tert-butoxy-2-oxoethyl]pyrrolidine-1-carboxylate (499.3 g, 423.3 mmol), 1,4-dioxane (1997 ml) and hydrochloric acid solution (12 M in water, 529.1 ml, 15 eq.). The mixture is stirred at 40°C for 1 hour and then the mixture is concentrated in vacuo to remove 1,4-dioxane, resulting in an aqueous suspension. The mixture is filtered through a propylene filter to remove insoluble particles. Adjust the pH of the filtrate to 9-10 using NaOH solution (2M in water). Stir the mixture at RT overnight. Slowly filter off the resulting solid using a paper filter (slow filtration, use low vacuum). The solid was washed with water and dried under vacuum at 45°C to obtain the title compound (281 g, 88%) as a white crystalline solid. ER/MS (m/z): 711 (M+H); 1H-NMR (500 MHz, D2O) δ 7.33 (t, J=7.6 Hz, 3H), 7.27 (d, J=7.8 Hz, 3H), 7.13 (d, J= 7.8 Hz, 3H), 7.09 (s, 3H), 4.20 (s, 6H), 3.54 (dd, J=7.9, 11.6 Hz, 3H), 3.393.34 ( m, 3H), 3.23-3.17 (m, 3H), 3.02-2.98 (m, 3H), 2.84 (dd, J=4.6, 13.7 Hz, 3H) , 2.76 (dd, J=10.6, 13.3 Hz, 3H), 2.60 (td, J=9.9, 4.8 Hz, 3H), 2.48 (td, J=17 ,3, 9.6 Hz, 3H), 2.12-2.07 (m, 3H), 1.73-1.65 (m, 3H).

Example 3.

(2S)-3-[3-[[[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]methylamino]methyl]phenyl]-2-24 045074

[(3R)-pyrrolidin-3-yl]propanoic acid; trihydrochloride.

Prepare the title compound essentially as described in Example 1 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[[[3-[ (2S)-3]-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl] amino]methyl]phenyl] methyl]-2oxoethyl]pyrrolidin-1- carboxylate and 2 M HCl in Et ₂ O at RT. ER/MS (m/z): 480 (M+H).

Example 4 (2S)-3-[3-[[[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]methyl-[(3-fluoro- 5methoxyphenyl)methyl]amino]methyl]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid.

Prepare the title compound essentially as described in Example 2 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[[3-[ (2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl-[(3-fluoro-5-methoxyphenyl)methyl] amino] methyl]phenyl]methyl]-2-oxoethyl]pyrrolidine-1-carboxylate. ER/MS (m/z): 618 (M+H).

Example 5.

(2S)-3-[3-[[[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]methyl-[(3-fluoro-5- methoxyphenyl)methyl]amino]methyl]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; trihydrochloride.

Mix tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3-tert-butoxy-2-[(3R )-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]methyl-[(3-fluoro-5-methoxyphenyl)methyl]amino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidine-1-carboxylate ( 541 mg, 0.582 mmol), HCl (2 M solution in diethyl ether, 5.8 ml) and water (0.5 ml). The mixture was stirred at RT for 5 hours. The solvent was removed under reduced pressure to give the title compound (434 mg, 102%). ER/MS (m/z): 618 (M+H).

Example 6.

3-[3-[[bis[[3-[2-carboxy-2-[(3R)-pyrrolidin-3-yl]propyl]phenyl]methyl]amino]methyl]phenyl]-2methyl-2-[(3R )-pyrrolidin-3-yl]propanoic acid; tetrahydrochloride.

- 25 045074

Mix tert-butyl-(3R)-3-[1-[[3-[[bis[[3-[3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]- 2-methyl-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tert-butoxy-1methyl-2-oxoethyl]pyrrolidine-1-carboxylate (275 mg, 0.225 mmol) in DCM (1, 4 ml) and add HCl (2 M solution in Et ₂ O, 3.4 ml, 6.7 mmol, 30 eq.). Stir the mixture at RT overnight. Drain the reaction solvent from the white solid and dissolve the solid in water. The mixture was evaporated to dryness under a stream of nitrogen, then dried in vacuo at 40°C to obtain the title compound (190 mg, 86%) as a white solid. ER/MS (m/z): 753 (M+H).

Example 7.

(2S)-3-[3-[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenoxy]phenyl]-2-[(3R)-pyrrolidin- 3yl]propanoic acid; dihydrochloride.

The title compound is prepared substantially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S )-3-tert-butoxy]]-2-[(3R)-1-tertbutoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenoxy]phenyl]methyl]-2-oxoethyl]pyrrolidin-1 carboxylate. ER/MS (m/z): 453 (M+H).

Example 8.

(2S)-3-[3-[2-[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenoxy]ethoxy]phenyl]-2-[( 3R)pyrrolidin-3-yl]propanoic acid; dihydrochloride.

The title compound is prepared essentially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[2-[3- [(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenoxy]ethoxy]phenyl]methyl]-2-oxoethyl]pyrrolidine-1carboxylate. The product is isolated by trituration with MTBE. ER/MS (m/z): 497 (M+H).

Example 9.

(2S)-3-[3-[[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenoxy]methyl]phenyl]-2-[(3R) pyrrolidin-3-yl]propanoic acid; dihydrochloride.

Prepare the title compound essentially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[3-[( 2S)-3-tert-butoxy-2-[(3R)-1-tertbutoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenoxy]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidine1-carboxylate. ER/MS (m/z): 467 (M+H).

Example 10.

(2S)-3-[3-[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]sulfanylphenyl]-2-[(3R)pyrrolidin-3 -yl]propanoic acid; dihydrochloride.

- 26 045074

The title compound is prepared substantially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S )-3-tert-butoxy]]-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfanylphenyl]methyl]-2-oxoethyl]pyrrolidin-1 carboxylate. Isolate the product by trituration with Et ₂ O. ER/MS (m/z): 469 (M+H).

Example 11.

(2S)-3-[3-[3-[(2S)-2-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]sulfinylphenyl]-2-[(3R)pyrrolidine -3-yl]propanoic acid; dihydrochloride.

The title compound is prepared substantially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S )-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfinylphenyl]methyl]-2-oxoethyl]pyrrolidin-1 carboxylate. The product is isolated by trituration with Et ₂ O followed by RP-HPLC/MS using the following parameters: column - Agilent ZORBAX Bonus RP; mobile phase - solvent A = 0.05% trifluoroacetic acid in water (pH 2.5), solvent B = acetonitrile + 0.05% trifluoroacetic acid; gradient - from 5 to 30% solvent B in solvent A; Flow rate: 25 ml/min. Place the product in a mixture of HCl (10% w/v) and water, then evaporate the solvents under a stream of nitrogen at 40°C. ER/MS (m/z): 485 (M+H).

Example 12.

(2S)-3-[3-[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]phenyl]sulfonylphenyl]-2-[(3R)pyrrolidin-3 -yl]propanoic acid; dichlorohydride.

The title compound is prepared substantially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-[(2S )-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]phenyl]sulfonylphenyl]methyl]-2-oxoethyl]pyrrolidin-1 carboxylate. Isolate the product by trituration with Et ₂ O. ER/MS (m/z): 501 (M+H).

Example 13.

(2S)-3-[3-[[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]anilino]methyl]phenyl]-2-[(3R) pyrrolidin-3-yl]propanoic acid; trihydrochloride.

Prepare the title compound essentially as described in Example 6 using tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[[3-[(2S )-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-oxopropyl]anilino]methyl]phenyl]methyl]-2-oxoethyl]pyrrolidine 1-carboxylate. After decanting the product, it is purified using RP-HPLC/MS using the following parameters: column - Waters™ XBridge™ C18 (19x100 mm, 5 µm); mobile phase solvent A = 20 mM NH ₄ HCO ₃ in water, solvent B = acetonitrile; flow rate 25 ml/min; gradient - from 5:95 to 25:75 V:A. Dissolve the purified material in aqueous HCl (1 N), stir at RT for 1 hour, evaporate the solvent under a stream of nitrogen and dry the solid in vacuo at

- 27 045074

40°C. ER/MS (m/z): 466 (M+H).

Example 14.

(2S)-3-[3-[3-[(2S)-2-Carboxy-2-[(3R)-pyrrolidin-3-yl]ethyl]anilino]phenyl]-2-[(3R)-pyrrolidin- 3yl]propanoic acid; dihydrochloride.

Mix (2S)-3-[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3oxopropyl]anilino]phenyl]-2 -[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid (9 g, 12.7 mmol), isopropanol (27 ml) and HCl (5.5 M solution in isopropanol) and stir at RT for 2.5 hours. The reaction mixture is heated to 60°C for 2.5 hours, then cooled to RT and stirred for 3 days. The reaction mixture is again heated to 60°C for 2 hours, cooled to RT and the reaction mixture is concentrated in vacuo to dryness. The solid precipitate is triturated with MTBE under ultrasonication, filtered and washed with MTBE, then the solid is dried in a vacuum. The solid is mixed with concentrated aqueous HCl and heated to 80°C overnight, then cooled to RT and concentrated in vacuo to dryness. The residue is dissolved in a minimum amount of water and the pH is adjusted to 7.5 by adding aqueous NaOH. The mixture is stirred at room temperature for 3 hours, then the precipitated solid is filtered off. Dissolve the solid in aqueous HCl (1N) and stir for 15 minutes at room temperature, then remove the water in vacuo. The residue was dried in vacuo at 45°C overnight to give the title compound (5.4 g, 81%). ER/MS (m/z): 452 (M+H).

Example 15.

(2S)-3-[3-[3-[(2S)-2-Carboxy-2-[(3R)-1-methylpyrrolidin-3-yl]ethyl]anilino]phenyl]-2-[(3R)- 1methylpyrrolidin-3-yl]propanoic acid; dihydrochloride.

Add paraformaldehyde (89 mg, 0.95 mmol) to the (2S)-3-[3-[3-[(2S)-2-carboxy-2-[(3R)pyrrolidin-3-yl]ethyl]anilino] suspension phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; dihydrochloride (100 mg, 0.19 mmol) in MeOH (1.9 ml).

Stir at RT for 15 minutes. Add sodium triacetoxyborohydride (202 mg, 0.95 mmol) and stir at RT for 16 hours. The mixture is concentrated in vacuo. The precipitate is purified by reverse phase chromatography (column: Claricep C-series silica _C18 ) using a gradient of 5 to 25% acetonitrile in aqueous NH ₄ CO ₃ (pH 9). Dissolve the purified material in aqueous hydrochloric acid (1 N, 1 ml) and stir at RT for 6 hours. The solution is concentrated in vacuo to give the title compound (30 mg, 27%). ER/MS (m/z): 480 (M+H).

Example 16.

(2S,2'S)-3,3'-[Sulfonylbis(azandiyl-3,1-phenylene)]bis{2-[(3R)-pyrrolidin-3-yl]propanoic acid};dihydrochloride.

Prepare the title compound essentially as described in Example 6 using di-tert-butyl-3,3'-((2S,2'S)-((sulfonylbis(azandiyl))bis(3,1-phenylene) )bis(3-(tert-butoxy)-3oxopropane-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate). ER/MS (m/z): 531 (M+H).

Example 17.

(2S)-3-Phenyl-2-[(3R)-pyrrolidin-3-yl]propanoic acid; hydrochloride

Add HCl (5.5 M in isopropanol, 511 mL, 2.81 mol, 13 eq.) to the solution of (2S)-2-[(3R)-1-tert-28 045074 butoxycarbonylpyrrolidin-3-yl]-3- phenylpropanoic acid (69 g, 216 mmol) in isopropanol (207 ml) and stir at RT overnight. The mixture is diluted with a 2:1 mixture of MTBE:hexanes (900 ml) and stirred for 10 minutes. The suspension is filtered and the solid is washed 1:1

MTBE:hexanes (100 ml). Dry the solid in vacuo at 50°C to give the title compound (51.3 g, 93%). ER/MS (m/z): 220 (M+H).

Example 18.

(2R)-3-Phenyl-2-[(3S)-pyrrolidin-3-yl]propanoic acid; hydrochloride

The title compound was prepared substantially as described in Example 6 using (2R)-2-[(3S)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-phenylpropanoic acid. The isolated product is stirred with HCl (2M in ether) and water for 3 hours, concentrated in vacuo, then triturated with MTBE and dried in vacuo at 40°C. ER/MS (m/z): 220 (M+H).

Synthesis of radiolabeled and cold (i.e., non-radiolabeled) standard for in vitro Apo(a) binding assay.

Example of getting 40.

-(3-Benzyloxyphenyl)imidazolidin-2-one.

Add argon-sparged DMF (20 ml) to a mixture of ethylene urea (1.4 g, 16 mmol), 1-benzyloxy-3-iodobenzene (4.9 g, 16 mmol, 1.0 eq.), copper iodide (0.61 g, 3.1 mmol, 0.20 eq.), potassium dihydrogen phosphate (4.2 g, 31 mmol, 2.0 eq.). N,N'dimethylethylenediamine (0.33 ml, 0.28 g, 3.1 mmol, 0.20 eq.) was added to the resulting suspension. Heat the blue suspension in a microwave reactor at 120°C for 3 hours. Cool the reaction mixture and filter through a pad of silica gel, rinsing the layer with EtOAc. The filtrate is concentrated. The residue was purified by silica gel chromatography using a 0 to 100% gradient of 5:20:75 MeOH:acetone:EtOAc in hexanes to give the title compound (1.10 g, 26%) as a yellow solid. ER/MS (m/z): 269 (M+H).

Receipt example 41.

(2S)-3-[3-[3-(3-Benzyloxyphenyl)-2-oxoimidazolidin-1-yl]phenyl]-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3 -yl]propanoic acid.

HE

To a mixture of 1-(3-benzyloxyphenyl)imidazolidin-2-one (3.33 g, 12.4 mmol, 1.50 eq.) and ammonium; (2S)3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoate (3.30 g) under nitrogen atmosphere add acetonitrile (41 ml) and DMF (to improve solubility ). Pass a constant flow of argon through the suspension for 15 minutes. Add potassium carbonate (3.75 g, 26.8 mmol, 3.24 eq), copper iodide (0.316 g, 1.66 mmol, 0.2 eq) and N,N'-dimethylethylenediamine (0.360 ml, 3 .31 mmol, 0.4 eq.). Heat the sealed vessel to 100°C over the weekend. Add water and extract the aqueous layer twice with EtOAc. Acidify the aqueous phase with 0.5 N. HCl in water and extract the aqueous layer with EtOAc, DCM and then EtOAc. Dry the combined organic phases over MgSO ₄ , filter and evaporate to dryness. The residue is purified by silica gel chromatography using a gradient of 10 to 60% (1% acetic acid/acetone) in hexanes to give the title compound (4 g, 82%). ER/MS (m/z): 486 (M+H-BOC).

Receipt example 42.

tert-Butyl-(3R)-3-[(1S)-1-[[3-[3-(3-benzyloxyphenyl)-2-oxoimidazolidin-1-yl]phenyl]methyl]-2tert-butoxy-2-oxoethyl ]pyrrolidine-1-carboxylate.

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To a solution of (2S)-3-[3-[3-(3-benzyloxyphenyl)-2-oxoimidazolidin-1-yl]phenyl]-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid (4 .0 g, 6.8 mmol) in toluene (68 ml) at 70°C, add N,N-dimethylformamide di-tert-butylacetal (15 ml, 55 mmol, 8.0 eq.) and heat the reaction mixture at 70 °C overnight. Purify the residue by chromatography on silica gel, eluting with a gradient of 0-50% EtOAc in hexanes to give the title compound (1.56 g, 33%). ER/MS (m/z): 542 (M+H-BOC).

Receipt example 43.

tert-Butyl-(3R)-3-[( 18)-2-tert-butoxy-1-[[3-[3-(3-hydroxyphenyl)-2-oxoimidazolidin-1 yl]phenyl] methyl] -2 - oxoethyl]pyrrolidine-1-carboxylate.

Dissolve tert-butyl-(3R)-3-[(1S)-1-[[3-[3-(3-benzyloxyphenyl)-2-oxoimidazolidin-1yl]phenyl]methyl]-2-tert-butoxy-2- oxoethyl]pyrrolidine-1-carboxylate (1.56 g, 2.43 mmol) in THF (80 ml) and add palladium hydroxide (0.7 g, 0.99 mmol, 0.41 eq). The reaction mixture is degassed under vacuum and then exposed to hydrogen gas three times. Stir the reaction mixture at RT under a hydrogen atmosphere (1 atm) for 4 hours. Concentrate the reaction mixture and purify the residue by chromatography on silica gel using a gradient of 0 to 50% EtOAc in hexanes to give the title compound (1 .19 g, 87%). ER/MS (m/z): 452 (M+H-BOC).

Receiving example 44.

tert-Butyl-(3R)-3-[( 1 S)-2-Ίret-butoxy-2-oxo-1 -[[3-[2-oxo-3-[3 -(tritrithiomethoxy)phenyl] imidazolidine-1 -yl]phenyl]methyl]ethyl]pyrrolidine-1-carboxylate.

To a solution of tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[3-(3-hydroxyphenyl)-2-oxoimidazolidin-1 yl]phenyl]methyl]- 2-oxoethyl]pyrrolidine-1-carboxylate (4 mg, 0.007 mmol) in DMF (0.5 ml) was added to cesium carbonate (10 mg, 0.03 mmol). Stir the solution at RT for 10 minutes. [ ³ H]methylnosylate (50 mCi) was added and stirred at RT for 1.5 h. The reaction mixture was purified by RP-HPLC/MS using the following parameters: column - Phenomenex® Gemini® C18 (250x10 mm); mobile phase - solvent A = water + trifluoroacetic acid (0.1%), solvent B = acetonitrile + trifluoroacetic acid (0.1%); gradient - from 50 to 100% B in 50 minutes; flow rate - 3 ml/min. The purified product was dissolved in EtOH and proceeded to Preparation Example 45 without further description.

Example of receiving 45.

(2S)-3-[3-[2-Oxo-3-[3-(trithiomethoxy)phenyl]imidazolidin-1-yl]phenyl]-2-[(3R)-pyrrolidin-3yl]propanoic acid; hydrochloride

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Remove the solvent from tert-butyl-(3R)-3-[(1S)-2-tert-butoxy-2-oxo-1-[[3-[2-oxo-3-[3(tritrithiomethoxy)phenyl]imidazolidine- 1-yl]phenyl]methyl]ethyl]pyrrolidine-1-carboxylate in vacuo and dissolve the precipitate in hydrogen chloride (4 M solution in 1,4-dioxane). Stir the mixture at RT overnight. The reaction mixture is purified by RP-HPLC/MS using the following parameters: column - Phenomenex® Gemini® C18 (250x10 mm); mobile phase - solvent A = water + trifluoroacetic acid (0.1%), solvent B = acetonitrile + trifluoroacetic acid (0.1%); gradient from 10 to 70% B in A in 60 minutes; flow rate - 3 ml/min. The purified product is dissolved in ethanol. Mass spectrometry shows a spectrum that is consistent with inactive material [ER/MS (m/z): 410 (M+H)] and a specific activity of 63 Ci/mmol.

Receipt example 46.

1-(3-Methoxyphenyl)imidazolidin-2-one.

The title compound was prepared substantially as described for Preparation Example 40 using 1-iodo-3-methoxybenzene and tribasic potassium phosphate as the base. Use normal heat to heat the reaction to 120°C for 16 hours. The reaction is diluted with EtOAc, filtered through a pad of Celite®, and the organic layers are washed with water, aqueous NH ₄ OH, and saturated aqueous NaCl. The crude material is purified by silica gel chromatography using a gradient of 20 to 100% EtOAc in hexanes. ER/MS (m/z): 193 (M+H).

Receipt example 47.

(2S)-2-[(3R)-1-tert-Butoxycarbonylpyrrolidin-3-yl]-3-[3-[3 -(3-methoxyphenyl)-2-oxoimidazolidin1-yl]phenyl]propanoic acid.

Mix (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]propanoic acid (190 mg, 0.4770 mmol), 1-(3-methoxyphenyl)imidazolidine -2-one (0.2751 g, 1.431 mmol, 3 eq.), [(2-di-tert-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)-2-( 2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate (tBuXPhos-Pd-G3, 0.0379 g, 0.0477 mmol, 0.1 eq.) and sodium tert-butoxide (0.1418 g, 1.431 mmol, 3 eq.) in 1,4-dioxane (4.8 ml) and stir the mixture under nitrogen at 100°C overnight. The mixture is diluted with EtOAc and acidified with aqueous HCl (1 N). The mixture is filtered through a pad of Celite®, the layers are separated, and the organic layers are dried over MgSO ₄ . Filter and concentrate the organic layers, then purify the precipitate by reverse phase flash chromatography (C18 silica column) using a gradient of 40 to 70% acetonitrile in aqueous NH ₄ CO ₃ (pH 9) to give title compound (70 mg, 28%) as a white solid. Obtain additional amount of the title compound (90 mg, 36%) by HPLC/MS using the following parameters: column - Waters™ XBridge™ C18 (19x100 mm, 5 μm); mobile phase - solvent A = 20 mM NH4HCO3 in water, solvent B = acetonitrile; flow rate 25 ml/min; gradient - from 30:70 to 50:50 V:A. ER/MS (m/z): 510 (M+H).

Receipt example 48.

(2S)-3-[3-[2-Oxo-3-[3-(methoxy)phenyl]imidazolidin-1-yl]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; hydrochloride

The title compound was prepared substantially as described in Preparation Example 45 using (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]-3-[3-[3-(3 -methoxyphenyl)-2

- 31 045074 oxoimidazolidin-1-yl]phenyl]propanoic acid. ER/MS (m/z): 410 (M+H).

Biological analyses.

In vitro Apo(a) binding assay.

The in vitro binding affinity of the compounds to the target human Apo(a) protein was tested in a competitive binding assay. Human Apo(a) protein containing 17 Kringle repeats is affinity purified from conditioned media of transiently transfected HEK-293F cells. All reagents are prepared in assay buffer containing 50 mM Tris-HCl, pH 7.4, 0.1% BSA. The binding assay is performed by adding 50 µl of each of (1) test compound in a dilution series (final concentration 0.32~10000 nM), (2) Apo(a) protein (6 ng/well) to each clear-bottom plate well. , (3) re-suspended polyvinyltoluene wheat germ agglutinin SPA beads, polyvinyltoluene (20 mg/ml) and (4) tritium-labeled radioligand (2S)-3-[3-[2-okco-3-[3(trithiomethoxy)phenyl ]imidazolidin-1-yl]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; hydrochloride (final concentration 0.52 nM). The plates are incubated for 60 minutes at RT and counted on a TRILUX LSC. Nonspecific binding, defined as binding in the presence of 10 μM cold (i.e., non-radiolabeled) ligand: (2S)-3-[3-[2-okco-3-[3(methoxy)phenyl]imidazolidin-1-yl ]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; hydrochloride is subtracted to determine specific binding. Data are analyzed by fitting to a standard single site binding model and the IC ₅₀ is determined for an example of the test compound. These results are summarized in table. 1 and indicate that Examples of test compounds bind to human Apo(a) protein. Inhibition of LDL particle assembly with apo(a) through binding to the Apo(a) protein maintains a decrease in LDL(a) levels.

Table 1

Example IC50*(nM) 1 <0.314 (n=1) 3 2.13 ± 0.159 (n=2) 6 <0.314 (n=1) 14 <0.314 (n=1) 17 152 ±21.3 (n=2) 18 126 ± 5.97 (n=2)

*Geometric mean ± SEM (n)

In vitro LP assembly assay (a).

The ability of compounds to inhibit the formation of drug(a) particles in vitro was assessed using a cell-free assembly assay. Conditioned media (DMEM supplemented with 10% FBS, 20 mM HEPES, and 1x penicillin/streptomycin) were collected from confluent wild-type HepG2 cells (the source of endogenously expressed ApoB) and from a stable HEK293 cell line expressing the human Apo(a) protein containing 17 repeats Kringle (selected at 1 mg/ml geneticin), after 24 h of culture at 37°C and 5% CO2. The in vitro assembly assay is performed by combining equal parts of HepG2 and HEK293 conditioned medium with test compounds added in a dilution series (final concentration 0.01~100 nM). The reaction mixture was incubated at 37°C for 2 h and then stopped by adding 6-aminocaproic acid (EACA) to a final concentration of 150 mM. LP(a) is detected using a sandwich ELISA with an anti-LP(a) capture antibody and an anti-ApoB detection antibody conjugated to HRP. ELISA is carried out using TMB, stopped with 1 N. sulfuric acid, and the signal was read at 450 nm on a Molecular Devices plate reader. The % LP(a) inhibition produced for each test condition was determined using an assembly reaction without the presence of inhibitor (with an appropriate DMSO concentration of 1%) set at 0% inhibition, and an assembly reaction with a minimum amount of HepG2 in conditioned medium (50 -fold dilution) set to 100% inhibition. The data are fitted to a 4-parameter curve to determine the IC ₅₀ values summarized in Table. 2. Addition of an example of the test compound to conditioned medium containing ApoB and Apo(a) results in a concentration-dependent inhibition of LP(a) formation in vitro, as shown in Table. 2. The results show that these compounds inhibit the assembly of Lp(a) from Apo(a) and LDL particles.

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table 2

Example IC50*(nM) 1 0.0963 ± 0.0233 (n=9) 3 0.934 ± 0.306 (n=2) 5 0.17 ±0.0124 (n=4) 6 0.108 ±0.0149 (n=4) 7 0.372 ± 0.0464 (n=8) 8 0.288 ± 0.19 (n=4) 9 0.359 ±0.0292 (n=2) 10 0.36 ± 0.119 (n=4) AND 0.396 ±0.241 (n=4) 12 0.235 ±0.0509 (n=2) 13 2.39 ±0.61 (n=2) 14 0.348 ± 0.0984 15 54.6 ±9.15 (n=3) 16 0.563 ± 0.119 (n=2) 17 1690 ±402 18 635 ±165(n=9)

*Geometric mean ± SEM (n)

Inhibition of LP(a) in mice in vivo.

The ability of the compounds to reduce the equilibrium concentration of LP(a) in vivo is assessed using a transgenic mouse model capable of producing humanized LP(a) particles. The in vivo effects of drug-degrading compounds (a) were tested on 7-17-month-old female double transgenic mice expressing human apoB-100 and human apo(a) containing 17 Kringle repeats: B6.SJL-Tg (APOB)1102Sgy Tg( Alb-LPA)32Arte. Mice are maintained on a standard light cycle (12 hours light/12 hours dark), room temperature 72±8°F and relative humidity 30~70%, with free access to water and a normal diet (Harlan Tecklad diet, 2014). .). Mice are randomized into treatment groups (n=5/group) 3-5 days before the study based on body weight and initial plasma Lp(a) concentration using BRAT (Block Randomized Allocation Tool) for the study. Mice are administered orally (or subcutaneously, if indicated) with vehicle (oral vehicle dose: 10 mL/kg, 1% HEC, 0.25% Tween80, 0.01% antifoam; vehicle subcutaneous dose: 5 mL/kg saline), or test compound in different doses twice a day (6:30 and 15:30) for 5 days. Blood is collected into heparin-coated capillary tubes through a tail incision. Twenty microliters of each blood sample was transferred to a DBS card (Whatman catalog number: WB12 9243) for drug exposure analysis. The remaining blood samples are centrifuged to separate the plasma. Plasma Lp(a) concentrations are measured using a sandwich ELISA as described for the in vitro Lp(a) assembly assay. The % inhibition for each dosage group is determined using the mean Lp(a) of the vehicle control group set to 0% inhibition. In table Figure 3 shows the results of dose-response studies in which tail blood samples were collected on day 3, 8 hours after the morning oral dose. Calculations of ED50 (effective dose to inhibit 50% of LP(a)), as determined by threshold minimum effective dose analysis. In table Figure 4 shows the results of single-dose studies in which tail blood samples were collected on day 3, 4 hours (or 8 hours if indicated) after the morning oral dose (or subcutaneous dose if indicated). These results, shown in table. 3 and 4 demonstrate that the compounds are effective in reducing plasma Lp(a) levels in vivo, supporting the notion that the compounds can be used to reduce plasma Lp(a) concentrations.

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Table 3

Example EDso (mg/kg) ¹ LP inhibition (a) (% at 30 mg/kg) ² 1 3.3 + 0.53 91 + 0.73 14 14.6 + 2.48 72 ± 2.23 17 20.6+1.79 60 + 3.46 18 7.6 ± 2.43 68+ 1.82

1. ED50 ± standard error

2. (average ± SEM)

Table 4

Example Dose (mg/kg) LA inhibition (a) (%) ¹ 5 3 43.8 ± 4.24 7 zo ² 44.6 ± 5.0 9 thirty 56.5 ± 2.77 10 thirty 43.6 + 4.6 12 thirty 50 + 3.82 ³

1. Average ± SEM

2. Subcutaneous dosing

3. Samples taken 8 hours after oral administration

Claims (24)

1. Compound formula where L is selected from the group consisting of -CH ₂ NHCH ₂ -, -CH2NH-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -O-, -ch ₂ - n-ch ₂ — 5 R—N -OCH2-, -OCH2CH2O-, -NHSO2NH-, each R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ independently selected from the group consisting of H and CH ₃ ; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, characterized in that R ¹ represents H and R ³ represents H; or a pharmaceutically acceptable salt thereof. 3. The compound according to claim 1, characterized in that R ⁵ represents H; or a pharmaceutically acceptable salt thereof. 4. The compound according to claim 1, characterized in that R ² represents H and R ⁴ represents H; or a pharmaceutically acceptable salt thereof. 5. The compound according to claim 1, characterized in that R ⁶ represents H; or a pharmaceutically acceptable salt thereof. 6. A compound according to claim 1, characterized in that R ² represents CH3 and R ⁴ represents CH ₃ ; or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 1, characterized in that R ² represents CH ₃ , R ⁴ represents CH3 and R ⁶ represents CH3; or a pharmaceutically acceptable salt thereof. - 34 045074 8. A compound according to claim 1, wherein L is selected from the group consisting of -CH2NHCH2-, -CH2NH-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -O-, -OCH2-, -OCH2CH2O- and -NHSO2NH-; or a pharmaceutically acceptable salt thereof. 9. The compound according to claim 1, characterized in that L is -ch ₂ -n-ch ₂ o R—NJ, T ^j^ ^r6 no^o or a pharmaceutically acceptable salt thereof. 10. The connection according to claim 1, characterized in that said connection is or a pharmaceutically acceptable salt thereof. 11. The connection according to claim 10, characterized in that said connection is or a pharmaceutically acceptable salt thereof. 12. A compound according to claim 11, characterized in that said compound is a hydrochloride salt. 13. A compound according to claim 12, characterized in that said compound is a tetrahydrochloride salt. 14. A pharmaceutical composition containing a compound according to claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient. 15. Use of the composition according to claim 14 for the treatment of cardiovascular disease. 16. Use of a composition according to claim 14 for reducing elevated levels of lipoprotein (a) (LP(a)) in plasma. 17. A method of treating a cardiovascular disease in a patient in need thereof, comprising administering an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof. 18. A method of reducing elevated plasma levels of LP(a), comprising administering an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof. 19. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the treatment of cardiovascular disease. 20. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for reducing elevated plasma levels of Lp(a). 21. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of a cardiovascular disease. 22. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof as a medicament, said medicament being suitable for reducing elevated plasma levels of Lp(a). - 35 045074 CO ₂ H 23. A compound of formula or a pharmaceutically acceptable salt thereof. 24. A pharmaceutical composition containing a compound according to claim 23 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.