Pyrrolidine compounds

Abstract

To provide compounds for use in the treatment of cardiovascular diseases.SOLUTION: A compound of formula (I'), or a pharmaceutically acceptable salt thereof, is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to pyrrolidine compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions, and these to the therapeutic use of the compounds.

[0002] Great progress has been made in the treatment of cardiovascular disease (CVD). Despite advances in treatment, patients continue to experience cardiovascular disease events such as angina pectoris, myocardial infarction, and stroke, and if left untreated it leads to death. Lipid disorders or dyslipidemia remain a major risk factor for CVD. Lipid disorders are divided into four general risk factors: elevated low-density lipoprotein cholesterol (L DL-c), decreased high-density lipoprotein cholesterol (HDL-c), trigly ceride (TG) elevation, and lipoprotein(a) (Lp(a)) elevation. Various therapeutic regimens targeting high LDL-c, low HDL-c, and high triglycerides are available. There are few approved treatment options for patients with elevated Lp(a) concentrations. In some cases, apheresis is used to filter blood to remove LDL and Lp(a) , but the effect is temporary and must typically be repeated every two weeks. There are no approved pharmaceutical therapies for reducing Lp(a) levels. Although the physiological function of Lp(a) is complex, elevated plasma Lp(a) levels are an independent risk factor for CVD, as reported. Drug treatment is needed for patients with elevated Lp(a).

[0003] Additional treatment options are desired for patients suffering from cardiovascular disease, particularly patients suffering from lipid disorders or dyslipidemia. Dietary therapy, exercise, and / or statins, fibra ​Additional therapeutic options are needed for patients whose cardiovascular risk is not adequately managed using current standard-of-care treatments, such as the use of one or more drugs including statins, niacin, etc. The present invention provides an alternative therapeutic option for patients suffering from CVD. There is a need for pharmaceutically acceptable compounds and therapeutic options for lowering plasma Lp(a) levels.

[0004] A compound of Formula I', wherein

化

化

[0005] A compound of Formula I''

化

[0006] A compound of Formula 1,

化

[0007] ​​ One embodiment provides a compound of Formula 2,

Chemical Formula

[0008] In one embodiment, the compound is a compound of Formula I', I'', 1 or Formula 2, and the compound is a pharmaceutically acceptable salt. In one embodiment, the compound is a compound of Formula I', Formula 1, or Formula 2, and the compound is a hydrochloride salt. In one embodiment, the compound is a compound of Formula 1 or Formula 2, and the compound is a tetrahydrochloride salt.

[0009] In one embodiment, the compound is a compound of Formula I', Formula 1, or Formula 2, and the compound is a hydrochloride salt selected from the group consisting of monohydrochloride, dihydrochloride, trihydrochloride, and tetrahydrochloride. In one embodiment, the embodiment provides the compound of Formula 1 or Formula 2 as a zwitterion.

[0010] Provided is a compound of Formula I', wherein L is selected from the group consisting of -CH2NHCH2-, -CH2NH-, -N H-, -S-, -S(O)-, -S(O)2-, -O-, -OCH2-, -OCH2CH 2O-, and -NHSO2NH-, or a pharmaceutically acceptable salt thereof.

[0011] Provided is a compound of Formula I', wherein R 1 and R 3 are each H, and L is -CH 2 NHCH 2 2-, -CH2NH-, -NH-, -S-, -S(O)-, -S(O)2-, - O-, -OCH2-, -OCH2CH2O-, and -NHSO2NH-, or a pharmaceutically acceptable salt thereof is provided.

[0012] A compound of formula I', wherein R 1 and R 3 H and R 2 and R 4 Each of these is CH3, and L is -CH2NHCH2- 、 -CH2NH-, -NH -、- S-、-S(O)-、-S(O)2 -, -O-, -OCH2-, -OCH2CH2O-, A compound selected from the group consisting of -NHSO2NH-, or a pharmaceutically acceptable compound thereof. Salt is provided.

[0013] A compound of formula I', wherein R 1 and R 3 H and R 2 and R 4 Each of these is H, and L is -CH 2 NHCH 2 -, -CH 2 NH-, -NH-, -S- -S(O)-, -S(O) 2 -, -O-, -OCH2-, -OCH2CH2O-, -N A compound selected from the group consisting of HSO2NH-, or a pharmaceutically acceptable salt thereof It will be provided.

[0014] A compound of formula I', where L is [ka] A compound, or a pharmaceutically acceptable salt thereof, is provided, selected from the group consisting of the following.

[0015] A compound of formula I', wherein R 1 , R 3 , and R 5 These are H and L are [ka] A compound, or a pharmaceutically acceptable salt thereof, is provided, selected from the group consisting of the following.

[0016] A compound of formula I', wherein R 1 , R 3 , and R 5 H and R 2 , R 4 , and R 6 A compound, or a pharmaceutically acceptable salt thereof, is provided, wherein each of the elements is H. It will be done.

[0017] A compound of formula I', wherein R 1 , R 3 , and R 5 H and R 2 , R 4 , and R 6 Compounds, or pharmaceutically acceptable salts thereof, in which each of the groups is CH3 It will be provided.

[0018] A compound of formula I', wherein L is [ka] And R 1 , R 3 , and R 5 H and R 2 , R 4 , and R 6 But each A compound or a pharmaceutically acceptable salt thereof, which is CH3, is provided.

[0019] In one embodiment, a compound of formula I', formula I'', formula 1, or formula 2, or its pharmaceutically acceptable properties A salt that is acceptable in the form of a pharmaceutically acceptable carrier, diluent, or excipient. It is a pharmaceutical composition containing the following:

[0020] In one embodiment, this is a method for treating patients who require treatment for cardiovascular disease. This is an effective amount of a compound selected from the group consisting of formulas I', 1, and 2, or the drug thereof. This includes administering a scientifically acceptable salt. In one embodiment, the treatment of cardiovascular disease is required. This is a method of treating patients, and this method involves an effective amount of the compound of formula I'', or the drug. This includes administering a scientifically acceptable salt. In one embodiment, above Lp(a) plasma levels This is a method for treating patients who require treatment for asthma, and this method involves an effective amount of formula I', formula 1 , or administer a compound selected from the group consisting of formula 2, or a pharmaceutically acceptable salt thereof. This includes, in one embodiment, a patient who requires treatment for elevated Lp(a) plasma levels. A method for treating the condition, wherein the method comprises an effective amount of the compound of formula I'', or the pharmaceutically acceptable compound thereof. This includes administering the salt.

[0021] In one embodiment, a selection is made from the group consisting of formulas I', 1, and 2 for use in treatment. A compound, or a pharmaceutically acceptable salt thereof. In one embodiment, used for therapeutic purposes. A compound of formula I'', or a pharmaceutically acceptable salt thereof, for use.

[0022] In one embodiment, formulas I', 1, and 2 are used for the treatment of cardiovascular disease. A compound selected from the group, or a pharmaceutically acceptable salt thereof. In one embodiment This refers to compounds of formula I'', or their pharmaceutically acceptable compounds, for use in the treatment of cardiovascular diseases. It is a tolerable salt.

[0023] In one embodiment, formulas I' and 1 are used for treating elevated Lp(a) plasma levels. Compounds selected from the group consisting of , and 2, or pharmaceutically acceptable salts thereof. ru.

[0024] In one embodiment, in the manufacture of a drug for cardiovascular disease, the formulas I', 1, and 2 This involves the use of a compound selected from the group, or a pharmaceutically acceptable salt thereof.

[0025] Lp(a) possesses both pro-thrombus formation and anti-thrombotic properties, as well as atherothrombotic properties. It may exhibit certain properties. Lp(a) inhibits fibrinolysis and accumulates in the blood vessel wall, leading to thrombus formation and thrombus formation. It can induce loamy arteriosclerotic lesions. Plasma levels of Lp(a) vary substantially between individuals. Unlike other risk factors, Lp(a) plasma levels are significantly altered by diet and exercise. It does not change. Lp(a) plasma levels may be related to genetic predisposition.

[0026] Lp(a) contains an LDL lipid core that includes the associated apolipoprotein B (apoB). It is similar to LDL-c in that it contains disulfide, but unlike LDL-c, Lp(a) is disulfide. A unique apolipoprotein (a) (apo(a)) bound to apoB via an id bond. It contains. apo(a) is synthesized in the liver. L from apo(a) and LDL particles p(a) aggregates can develop within hepatocytes, on the cell wall, or in plasma. Inhibition of particle aggregation with apo(a) can reduce Lp(a) levels.

[0027] As used herein, the term “elevated Lp(a) plasma levels” refers to approximately 50 m This refers to plasma levels of g / dL or greater. The compounds provided herein are Lp(a) plasma. It can be used in treatments to lower levels.

[0028] As used herein, the term “pharmaceutically acceptable salt” refers to clinical and / or This refers to salts of compounds that are acceptable for veterinary use. It also refers to pharmaceutically acceptable salts and their preparation. An example of a common methodology for doing so is "Handbook of Pharmaceuti cal Salts:Properties,Selection and Use”P .Stahl,et al.,2nd Revised Edition,Wiley- VCH, 2011 and SMBerge, et al., “Pharmaceuti Cal Salts”, Journal of Pharmaceutical Sci. This can be found in ences, 1977, 66(1), 1-19. Formulas I', I'', 1 also The compound of formula 2 may be a zwitterion, monoacid, diacid, or triacid addition salt. Formulas I', I The compounds of formula '', 1, or 2 may be monobase, dibase, or tribase addition salts.

[0029] The pharmaceutical compositions of the present invention can be prepared using pharmaceutically acceptable additives. The term "pharmaceutically acceptable" means that the composition is compatible with other components and is not pharmaceutically harmful to the patient. Refers to one or more carriers, diluents, and / or excipients that are not part of a pharmaceutical composition and so Examples of processes for these preparations are well known to those skilled in the art, for example, “Remingto n:The Science and Practice of Pharmacy”, Loyd, V.,et al.Eds.,22nd Ed.,Mack Publish This can be found in ing Co., 2012.

[0030] As used herein, the term “effective dose” means an effective dose for treating a disorder. This refers to the dosage. The effective dose for a particular patient can be determined by a skilled healthcare professional. ru.

[0031] As used herein, “to treat,” “to heal,” or “to treat” The term refers to slowing or reducing the progression or severity of existing symptoms, impairments, conditions, or diseases. This includes, preventing, or reversing. As used herein, "cardiac blood "Treating vascular diseases" means slowing or reducing the progression of heart or vascular disease, or preventing it. It means to prevent or reverse the compound of formula I', formula 1, or formula 2. A method for treating myocardial infarction is provided, which includes administering a drug to patients who require it.

[0032] As used herein, the term “patient” refers to a mammal. Preferably, A person is a human being.

[0033] Pharmaceutical compositions include tablets or capsules for oral administration, solutions for oral administration, and Alternatively, it can be formulated as an injectable solution. In one embodiment, the composition is suitable for oral administration. be.

[0034] Certain abbreviations are defined as follows: "Apo" refers to apolipoprotein, and "B "OC" refers to tert-butoxycarbonyl, and "BSA" refers to bovine serum albumin. "DAD" refers to a diode array detector, and "DCM" refers to dichloromethane... "T" refers to methylene chloride, "de" refers to diastereomer excess, and "DMEA" is "Dimethylethylamine" refers to dimethylethylamine, while "DMEM" refers to Dulbecco's Improved Eagle Medium. "DMF" refers to N,N-dimethylformamide, and "DMSO" refers to dimethyl sulfide. "Hoxide" refers to the enantiomer excess, "ee" refers to the epsilon-amino acid It refers to caproic acid or 6-aminocaproic acid, and "ELISA" stands for Enzyme-Linked Immunoadsorption. "Et2O" refers to diethyl ether. "Equiv" refers to an equivalent, and "Et2O" refers to diethyl ether. ".''" refers to ethyl acetate, and "EtOH" refers to ethanol or ethyl acetate. "Ex" refers to alcohol, "FBS" refers to fetal bovine serum, "HEC" refers to hydroxyethylcellulose, and "HEK" refers to human fetal kidney. "HepG2" refers to a human hepatocellular carcinoma cell line, and "HEPES" refers to 4-(2-hydroxyethyl acetate). This refers to (Cyl)-1-piperazineethanesulfonic acid, and "HPLC" stands for High-Performance Liquid Chromatography. Raffy refers to horseradish peroxidase, and "IC" refers to horseradish peroxidase. 50 "teeth, This refers to the concentration of the drug that produces 50% of the maximum inhibitory response possible with that drug, and "min" means minute. It refers to (singular or plural), and "MeOH" refers to methanol or methyl alcohol. "MTBE" refers to methyl tert-butyl ether, and "RP-HPLC / MS" "RT" refers to reverse-phase high-performance liquid chromatography with mass spectrometry, and "RT" refers to room temperature. "SFC" stands for supercritical fluid chromatography, and "SPA" stands for scintillation This refers to a proximity assay, and "t (R) " refers to the retention time, and "THF" stands for tetrahydro "T" refers to furan, "TMB" refers to 3,3',5,5'-teramethylbenzidine, and "T "Ris" refers to tris(hydroxymethyl)aminomethane.

[0035] Individual isomers, enantiomers, and diastereomers are listed below by those skilled in the art. At any convenient point in the synthesis of the compound to be synthesized, selective crystallization techniques or chiral crystallization can be used. They may be separated or divided by methods such as rhomography.

[0036] Compounds of formula I', formula I'', formula 1, or formula 2 are readily convertible and pharmaceutically acceptable. It can be isolated as a salt. Salt formation is achieved by adding a pharmaceutically acceptable acid. Forming a salt, or forming a base addition salt by adding a pharmaceutically acceptable base. This can be done by salting. Salts can also be used to deprotect nitrogen or oxygen, i.e., remove the protecting group. They can be formed simultaneously at the same time. Examples of salt formation, reactions, and conditions are known to those skilled in the art. ru.

[0037] Compounds selected from the group consisting of formulas I', I'', 1, and 2, or those The salt can be prepared by various procedures, some of which are described below in preparation and execution. For example, the following routes are used to prepare each of the compounds or salts of the present invention. Specific synthesis steps can be combined in different ways, or combined with steps in different paths. It may be done. The products of each step in the following preparations may be extracted, evaporated, precipitated, and chromatographed. It can be recovered by conventional methods, including roughing, filtration, grinding, and crystallization.

[0038] In the following scheme, all substituents are already defined unless otherwise specified. That is correct. The reagents and starting materials are readily available to those skilled in the art. Without limiting the scope, the following schemes, preparations, and examples further illustrate the present invention. Provided for demonstration purposes: Compounds of formulas I', I'', formula 1, and formula 2, or salts thereof. This is achieved by using a starting material or intermediate having the corresponding desired stereochemical configuration. It can be prepared by doing so.

[0039] Scheme 1 shows the preparation of an intermediate that may provide access to the compound of the present invention. Protected The pyrrolidine-3-ylacetic acid derivative A is first converted to acyloxazolidinone B. This first converts A to an acid chloride, and then, in the presence of lithium chloride at 10°C, (4S)- This is achieved by reacting with 4-benzyloxazolidine-2-one. Acid derivatives and bases such as lithium bis(trimethylsilyl)amide at 0°C Alkylation of luoxazolidinone B yields intermediate C in a diastereoselective manner. Those skilled in the art will know that the stereochemistry of oxazolidinone substitution affects the diastereoselectivity of alkylation. This affects the oxazolidi of the opposite stereochemistry or racemic stereochemistry in this synthesis. The use of non-derivative B gives either opposite or diastereoselectivity, respectively. It is recognized that this is possible. The conversion of acyloxazolidinone C to acid intermediate D occurs in THF. This is achieved using an aqueous solution of LiOH and H2O2 at 5-15°C. Acid intermediate D is optional. It is isolated as an ammonium salt. Acid intermediate D is, for example, tert-butyl at high temperatures. By reacting with -1,3-diisopropylisourea to obtain intermediate E, tert-br It is protected as a chill ester.

[0040] Scheme 1 also shows the conversion from acid A to methyl ester F, which is a carbonate base This is achieved by reacting A with iodomethane in the presence of - At 78°C, benzyl bromide is induced using a base such as lithium bis(trimethylsilyl)amide. Alkylation with a conductor yields intermediate G. Intermediate G is subjected to lithium bis(trimmed) at -78°C. Re-alkylated with iodomethane using a base such as ethylsilylamide, then E Tel is hydrolyzed with sodium hydroxide at high temperature to obtain the acidic intermediate H. The acidic intermediate H is, for example, Then, by reaction with tert-butyl-1,3-diisopropylisourea at high temperatures, It is protected as an rt-butyl ester to obtain intermediate I.

[0041] In particular, R a Intermediates D, E, H, and I, where is bromine or -NO2, are the compounds of formula I'. It is particularly useful for further transformations in the preparation of materials. a Intermediate D, which is -H, is bromide Alkylation of B in dinyl, followed by hydrolysis of acyloxazolidinone, Taha R a The intermediate D, which is a bromide, is stirred with palladium on carbon under a hydrogen atmosphere. It can be prepared by either of the following methods. a If -H, the pyrroligi on intermediate D Deprotecting the nitrogen yields the compound of formula I. [ka]

[0042] Scheme 2 is an important intermediate (J, prepared as described in Scheme 1) This invention demonstrates the conversion to the second-to-last compound. The bromide is synthesized in synthesis gas (1:1 ratio). CO / H2), palladium(II) acetate, butyldi-1-adamantylphosphine, and By using N,N,N',N'-tetramethylethylenediamine at high temperatures, aldehyde K It is converted. Then, aldehyde K is converted into ammonia and sodium triacetoxyhydride. Reductive amination of K with reducing agents such as thorium or sodium borocyanohydride ( They are converted into a mixture of N and O by (2 or 3 equivalents each), and then N and O It is separated by chromatography. Alternatively, the dimer compound O is separated from the aldehyde K. Converted to aldoxime L, and then flow hydrogenated at high temperature using a sponge nickel catalyst. L is reduced to amine M, and then the amine M is reductively aminated by aldehyde K. It is produced. Intermediate P is a dimer intermediate with 3-fluoro-5-methoxybenzaldehyde. It is prepared by reductive amination of body O. [ka]

[0043] Scheme 3 is a method for preparing the second-to-last compound of the present invention using bromide intermediate J. The following uses are indicated. Bromide J is tetrahydroxydiborone, chloro(2-disic Rohexylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl (2'-amino-1,1'-biphenyl-2-yl)palladium(II), X-PHO It is converted to boronic acid Q using S and potassium acetate. Boronic acid Q is then converted to 5 At °C, it is converted to phenol T using H2O2. Phenol T is converted to copper iodide at high temperatures. (I) Using N,N-dimethylglycine hydrochloride and cesium carbonate, bromide J is bonded. Combined to obtain biphenyl ether U. Phenol T is also converted at high temperature to 1,2-dibromoate It reacts with tan and carbonate to obtain V. Aldehyde intermediate K (odor as described in Scheme 2). The compound J (prepared from the alcohol) is reduced to alcohol R with sodium borohydride at 0°C, and then the Mitsunobu reaction Upon receiving the response, an intermediate S is obtained. [ka]

[0044] Scheme 4 is a method for preparing the second-to-last compound of the present invention using bromide intermediate J. The following usage is indicated. Two equivalents of bromide J are mixed with bis(dibenzylideneacetone)paradiol. Uses um, tripotassium phosphate, and 1,1'-bis(diphenylphosphino)ferrocene. Then, it is coupled with potassium thioacetate to obtain diphenyl sulfide W. Sulfide W is then... Then, add 1 equivalent or 2 equivalents of meta to either sulfone X or sulfoxide Y. It is converted using chloroperoxybenzoic acid. [ka]

[0045] Scheme 5 is a nitro intermediate Z(S) for preparing the second to last compound of the present invention. The use of the nitro intermediate Z (prepared as Chiem 1) is shown. The catalyst is under a hydrogen atmosphere. It is reduced to aniline AA in the presence of [unclear]. Aniline AA is reduced to sodium triacetoxyboron hydride. Reduction with aldehyde K (prepared as in Scheme 2) using a reducing agent such as lium. It undergoes amination to obtain CC. [(2-dicyclohexylphosphino-3,6-dimeth Xy-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-A Mino-1,1'-biphenyl) Palladium(II) methanesulfonate (BrettPh Aniline AA and bromide BB (s) using os Pd G3) and potassium carbonate at high temperatures Diphenylamine derivatives are obtained by the Buchwald reaction between (prepared as Chiem 1) and Obtain DD. Two equivalents of aniline AA are also obtained at high temperatures as 1,4-diazabicyclo[2.2.2] Octanbis(sulfur dioxide) adduct (DABSO) reacts with iodine to form sulfamide. Obtain EE. [ka]

[0046] Intermediates N, O, P, S, U, V, W, X, Y, CC, DD, and E for schemes 2-5 The compound of formula I' is obtained by the overall deprotection of E. (Scheme 1-5) Pg 1 ) is -BOC, and ester (Pg of schemes 1-5) 2 ) is tert-butyl If it is an ester, the overall deprotection is diethyl ether, dioxane, or isop This is achieved in a single step using a solution of HCl in an organic solvent such as ropanol. Deprotection Then, the pyrrolidine nitrogen in the compound of formula I' is paraformaldehyde and triacetone. It can be methylated by reductive amination with sodium borohydride.

[0047] Preparation 1 tert-butyl(3R)-3-[2-[(4S)-4-benzyl-2-oxo-oxa [Zolidine-3-yl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] Triethylamine (56.5g, 77.9mL, 559mmol, 2.5 equivalents) 2-[(3R)-1-tert-butoxycal in THF (540 mL) maintained at 0°C Dissolve bonylpyrrolidine-3-yl acetate (53.8g, 235mmol, 1.05 equivalents) Add to the solution. After 5 minutes, pivaloyl chloride (33.7g, 34.2mL, 279 mmol) Add 1.25 equivalents. After 15 minutes, the lithium chloride (11. (8g, 279 mmol, 1.25 equivalents) and (4S)-4-benzyloxazolidine- Add 2-one (40.0 g, 223 mmol, 1 equivalent). Warm the mixture to room temperature. Stir for 24 hours. After 24 hours, add 500 mL of 1N HCl aqueous solution to separate the organic phase. Separate the organic phase from the aqueous phase. Dissolve the organic phase in 1N NaOH aqueous solution (500 mL) and saturated NaCl aqueous solution. Wash with solution (500 mL), dry on MgSO4, filter, and concentrate the solution under vacuum. Suspend the residue in a mixture of MeOH / water (1:2, 575 mL) and stir overnight at room temperature. The solids were filtered off, washed with hexane (2 × 150 mL), and the solids were dried and labeled. A composite (65.7g, 76%) was obtained. ES / MS (m / z): 333 (M + H - tert -butyl), 1 H NMR (400.13MHz, CDCl3)δ7.38-7.28( 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), 3.11-2.96(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).

[0048] Preparation 2 tert-butyl(3S)-3-[2-[(4R)-4-benzyl-2-oxo-oxa [Zolidine-3-yl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] 2-[(3S)-1-tert-butoxycarbonylpyrrolidine-3-yl]acetic acid Using bi(4R)-4-benzyloxazolidine-2-one, essentially prepare as described in Preparation 1. Prepare the title compound as described. Prepare a gradient of 10-50% butyl in hexane. The product is purified by silica gel chromatography. ES / MS (m / z ):333(M+H-tert-butyl)

[0049] Preparation 3 tert-butyl(3R)-3-(2-methoxy-2-oxo-ethyl)pyrrolidine-1 -carboxylate [ka] Iodomethane (2 mol / L in MTBE, 240 mL, 480 mmol, 1.1 equivalents) 2-[(3R)-1-tert-butoxycarbonylpyrro in DMF (800 mL) Add to a solution of lysine-3-yl acetate (100 g, 436 mmol) at room temperature. Add lium (90.4g, 654 mmol, 1.5 equivalents) and steep the resulting mixture for 4 hours. Stir. Add water (1.5L) and extract with MTBE (3L). Remove the organic phase with ice / water (3L). Wash with 500 mL, dry the organic phase with MgSO4, filter, and concentrate under reduced pressure. The title compound (103g, 97%) was obtained. 1 1H NMR (300 MHz, CDCl3) δ 3.68(S, 3H), 3.65-3.53(m, 1H), 3.52-3.36(m, 1 H), 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, 1 H), 1.65-1.48(m, 1H), 1.45(S, 9H).

[0050] Preparation 4 tert-butyl(3R)-3-[1-[(3-bromophenyl)methyl]-2-methyl [C-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-(2-methoxy- in THF (93 mL) at -78°C Solution of 2-oxo-ethyl)pyrrolidine-1-carboxylate (7g, 28 mmol) Lithium bis(trimethylsilyl)amide (1M solution in THF, 33.5 mL, 3 Add 3.5 mmol (1.2 equivalents). Stir the mixture at -78°C for 1 hour and remove 3-3 bromide. Bromobenzyl (8.37 g, 33.5 mmol, 1.2 equivalents) in THF (5 mL) solution Add [ingredient]. Warm the mixture to room temperature and stir overnight. Mix the mixture with saturated NH4Cl aqueous solution. Enchymate and extract with ammonium compounds. Wash the organic matter with saturated NaCl aqueous solution and place on MgSO4. Dry, filter, and evaporate to dryness. A gradient of 10-40% siRNA in hexane is used. The residue is purified by silica gel chromatography to obtain the diastere compound of the title compound. A mixture of Omar (8.4g, 73%) is obtained as a yellow oily substance. ES / MS (m / z): 356, 358 (M+H-tert-butyl).

[0051] Preparation 5 3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyro Lysine-3-yl]-2-methylpropanoic acid [ka] tert-butyl(3R)-3-[ in THF (100 mL) under nitrogen and at -78°C 1-[(3-bromophenyl)methyl]-2-methoxy-2-oxo-ethyl)pyrrolidine In a solution of ¹⁴-1-carboxylate (8.4 g, 20 mmol), lithium bis(trimmed) Add thylsilylamide (1M solution in THF, 41 mL, 41 mmol, 2 equivalents). Stir the reaction mixture at -78°C for 2 hours. Iodomethane (25 mL, 410 mmol, 2 Add (0 equivalents) and warm the mixture to room temperature. Stir the mixture overnight. Saturated NH4Cl water Add the solution and extract with siRNA. Wash the organic layer with saturated NaCl aqueous solution and MgSO 4. Dry on the surface, filter, and remove the solvent under vacuum. Remove the residue with MeOH (80 mL) and Dissolve in THF (80 mL), then sodium hydroxide (5 M aqueous solution, 81 mL, 41 Add 0 mmol (20 equivalents) and heat the resulting mixture at 60°C for 3 days. Cool to room temperature, add HCl (1N aqueous solution), and adjust the pH of the mixture to 2-3. The layers are extracted with thio. The organic layer is dried over MgSO4, filtered, and concentrated under vacuum. The residue was subjected to chiral SFC under the following parameters: column-Chiralp ak(registered trademark)AD (25 × 3 cm, 5 μm), mobile phase - solvent A = CO2, solvent B = M DMEA with eOH + 0.2% v / v, gradient - isocratic 80:20 A:B, flow rate -120 mL / min). Isomer 1 (1.7 g, 27%) and isomer 2 (3. 4g, 41%) is obtained as a white solid. ES / MS (m / z): 356 / 358 (M+ H-tert-butyl).

[0052] Preparation 6 tert-butyl(3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxy [S-oxazolidine-3-yl]-1-[(3-bromophenyl)methyl]-2-oxo -ethyl]pyrrolidine-1-carboxylate [ka] Lithium bis(trimethylsilyl)amide (1M in THF, 818mL, 818mmo) A solution of 1.2 equivalents (1 liter) is placed in a 3 L three-necked round-bottom flask in an ice bath under nitrogen. (1325 mL) contains tert-butyl(3R)-3-[2-[(4S)-4-benzyl -2-oxo-oxazolidine-3-yl]-2-oxo-ethyl]pyrrolidine-1-ca Add 265g, 682 mmol of ruboxylate to a 0°C solution while mechanically stirring. The mixture was added over 9 minutes. The mixture was stirred at 0.6°C for 47 minutes. Then, THF (450m) L) 1-bromo-3-(bromomethyl)benzene (190g, 760mmol, 1. Add 12 equivalents of the solution over 28 minutes to raise the reaction temperature to 5.1°C. Stir overnight. While warming the mixture to room temperature, cool the reaction mixture using an ice / water bath, then warm the reaction temperature. Add a saturated aqueous solution of NH4Cl (1L) in four separate portions at a rate that maintains the temperature below 21°C. Added. Water (1 L) was added to the mixture and extracted with MTBE (3.5 L). The organic layer was then watered. Wash with a mixture of (1 L) and saturated NaCl aqueous solution (500 mL), then wash with saturated NaCl aqueous solution. Wash with solution (500 mL). Dry the organic matter over Na2SO4, filter, and then Concentrate in a vacuum. Add hexane (1L) to the residue and concentrate in a vacuum, then proceed to the next step under high vacuum. After drying overnight, the title compound was obtained as an orange oily substance (416 g, >100%). The purity is estimated to be 90% by weight based on the theoretical yield. ES / MS(m / z):501 / 503 (M+H-tert-butyl).

[0053] Preparation 7 tert-butyl(3S)-3-[(1R)-2-[(4R)-4-benzyl-2-oxy [S-oxazolidine-3-yl]-1-[(3-bromophenyl)methyl]-2-oxo -ethyl]pyrrolidine-1-carboxylate [ka] The title compound is tert-butyl(3S)-3-[2-[(4R)-4-benzyl-2 -Oxo-oxazolidine-3-yl]-2-oxo-ethyl]pyrrolidine-1-carb Prepare substantially as described in Preparation 6 using xylate. ES / MS(m / z): 501, 503 (M+H-tert-butyl).

[0054] Preparation 8 tert-butyl(3R)-3-[(1S)-2-[(4S)-4-benzyl-2-oxy [S-oxazolidine-3-yl]-1-[(3-nitrophenyl)methyl]-2-oxo -ethyl]pyrrolidine-1-carboxylate [ka] Lithium bis(methylsilyl)amide (1.0 M in THF, 46 mL, 46 mmol) , 1.2 equivalents) in THF (75 mL) tert-butyl(3R)-3-[2-[( 4S)-4-benzyl-2-oxo-oxazolidine-3-yl]-2-oxo-ethyl In a solution of pyrrolidine-1-carboxylate (15g, 39 mmol, 1 equivalent), -2 It was added at 0°C. The mixture was stirred at -20°C for 20 minutes. 1-( in THF (45 mL) Bromomethyl)-3-nitrobenzene (9.17g, 42.5mmol, 1.1 equivalents) Add the solution. Stir the solution for 2 hours and warm it to room temperature. Dilute the mixture with MTBE and fill to saturate. Quench with an aqueous solution of NH4Cl. Separate the phases and extract the aqueous phase with MTBE. Organic extraction. The materials are combined, and the organic matter is sequentially washed with water and saturated NaCl aqueous solution. Dry on Na2SO4. Dry, filter, and concentrate the filtrate in a vacuum. The residue is a mixture of MeOH / H2O (2:1, Mix with 150 mL. Stir the slurry overnight. Filter to collect the solids and hexane. Wash with [method]. Dry the solid material under vacuum at 40°C to obtain the title compound (19g, 88%). ES / MS (m / z): 468 (M+H-tert-butyl).

[0055] Preparation 9 tert-butyl(3R)-3-[(1S)-1-benzyl-2-[(4S)-4-benzyl [Zyl-2-oxo-oxazolidine-3-yl]-2-oxo-ethyl]pyrrolidine-1 -carboxylate [ka] Using benzyl bromide, prepare essentially as described in Preparation 6, with a purity of 76%. Prepare the compound in question. ES / MS (m / z): 423 (M+H-tert-butyl).

[0056] Preparation 10 (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxycal Bonylpyrrolidine-3-yl]propanoic acid [ka] Prepare a hydrogen peroxide solution (0.88 M in water, 105 mL, 926 mmol, 1.5 equivalents), tert-butyl(3R)-3-[(1S)-2-[(4S)-4- [Benzyl-2-oxo-oxazolidine-3-yl]-1-[(3-bromophenyl)methyl [Tyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate (purity 90% by weight, 3 Add to a mechanically stirred mixture of 81 g (615 mmol) in one step, using an ice / water bath. Cool to 8.6°C in a 12L three-necked round-bottom flask. Lithium hydroxide in water (930mL) A solution of 38.7g, 923 mmol, 1.5 equivalents of ruthone monohydrate is added over 25 minutes. Raise the reaction temperature to 12.8°C. Stir the reactants for 2.5 hours, then cool to 5.7°C. Sodium bisulfite (129.4g, 1244mmol, 2.02%) in water (2L) Add the equivalent volume of the solution over 40 minutes to raise the reaction temperature to 14.7°C. Add an aqueous solution of N) to raise the pH of the reaction mixture to >12, then add water (1 L) and MT. Add BE (4 L). Separate the layers and extract the aqueous layer with MTBE (2 L). Organic matter Combine the ingredients, extract with water (1L), and then add this aqueous extract to the bulk aqueous solution. Stir with MTBE (3L) and cool the mixture to 5°C. Add aqueous hydrochloric acid (5N). Bring the pH of the mixture to 3. Separate the layers and add saturated NaCl aqueous solution (1L) and water (50%). Wash the organic layer with a mixture of (0 mL). Dry the organic material over Na2SO4 and filter it. The mixture is concentrated in a vacuum at 40°C. The residue is dried under high vacuum to obtain the title compound (221.5g). 90% is obtained as a white solid. ES / MS(m / z):342 / 344(M+H -tert-butyl).

[0057] Preparation 11 Ammonium; (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert -Butoxycarbonylpyrrolidine-3-yl]propanoate [ka] (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxyca Rubonylpyrrolidine-3-yl]propanoic acid (237.6g, 596.5mmol) and M Mix with TBE (3801 mL) to obtain a turbid mixture containing solids. Dissolve the mixture in two sheets. Filtered through glass fiber filter paper, the ammonia (7M) in MeOH (128 mL, 896 mm) was obtained. Add ol (1.5 equivalents) to the filtrate while mechanically stirring under nitrogen. A white solid precipitate will form. The mixture becomes very concentrated. Add MTBE (800 mL) to the mixture to achieve free flow. Obtain a slurry. Stir the mixture at room temperature for 1.5 hours, then cool it in an ice / acetone bath at -5 to 0°C. Cool to °C and stir for 1.5 hours. Pass through a propylene mat and a 3L glass frit funnel. The solids are filtered by vacuum filtration, the solids are rinsed with MTBE (1L), and then nitrogen filtration is performed. Apply vacuum overnight at room temperature under a blanket. (2S)-3-(3 -bromophenyl)-2-[(3R)-1-tert-butoxycarbonylpyrrolidine- Two more batches of material prepared in a similar manner, starting with 3-yl]propanoic acid (47 Combine with 0.3g, 113mmol, and 53.0g, 120mmol. Solids Suspend in acetonitrile (4300 mL) and mechanically stir overnight under nitrogen at 23°C. Stir the mixture. Filter out the solids by vacuum filtration, and add acetonitrile (500 mL). Wash with MTBE (1L), then vacuum at room temperature for 4 hours under a nitrogen blanket. Continued use yields the title compound as a white powder (249.5 g, 77%). ES / MS( m / z): 342 / 344 (M+H-tert-butyl).

[0058] Preparation 12 Ammonium; (2R)-3-(3-bromophenyl)-2-[(3S)-1-tert -Butoxycarbonylpyrrolidine-3-yl]propanoate [ka] tert-butyl(3S)-3-[(1R)-2-[(4R)-4-benzyl-2-O [xo-oxazolidine-3-yl]-1-[(3-bromophenyl)methyl]-2-oxy Essentially, preparation 10 is prepared using so-ethyl]pyrrolidine-1-carboxylate. Prepare the title compound as described, and then prepare the ammonia as described in Preparation 11. Forms a methyl salt. ES / MS (m / z): 342, 344 (M+H-tert-butyl) .

[0059] Preparation 13 (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl] -3-(3-nitrophenyl)propanoic acid [ka] tert-butyl(3R)-3-[(1S)-2-[(4S)-4-benzyl-2-o [xo-oxazolidine-3-yl]-1-[(3-nitrophenyl)methyl]-2-oxy Using so-ethyl]pyrrolidine-1-carboxylate, bisulfite was added as a post-reaction treatment. By omitting the step of adding the aqueous thorium solution, the preparation essentially proceeds as described in Preparation 10. Prepare the compound in question. ES / MS (m / z): 309 (M+H-tert-butyl).

[0060] Preparation 14 (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl] -3-phenyl-propanoic acid [ka] tert-butyl(3R)-3-[(1S)-1-benzyl-2-[(4S)-4-benzyl [2-oxo-oxazolidine-3-yl]-2-oxo-ethyl]pyrrolidine- Using 1-carboxylate, the title compound is essentially prepared as described in Preparation 10. Prepare the solution. Purify the product by kneading with a 1:1 MeOH:water mixture. ES / MS(m / z):264(M+H-tert-butyl).

[0061] Preparation 15 (2R)-2-[(3S)-1-tert-butoxycarbonylpyrrolidine-3-yl] -3-phenyl-propanoic acid [ka] Ammonium; (2R)-3-(3-bromophenyl)-2-[(3S)-1-ter t-Butoxycarbonylpyrrolidine-3-yl]propanoate (830 mg, 2.08 mmol) is dissolved in EtOH (20 mL), and palladium (10% w / w, 22) is placed on carbon. Add 2 mg (0.208 mmol, 0.1 equivalent). Heat the mixture under a hydrogen balloon. Stir overnight at room temperature. Filter the reaction mixture through a diatomaceous earth pad. Concentrate the filtrate and add 1% acetic acid. Silica gel chromatography using a 10-40% gradient in hexane with added [ingredient] Further purification of the residue yields the title compound (560 mg, 84%) as a white solid. ES MS(m / z):264(M+H-tert-butyl).

[0062] Preparation 16 tert-butyl(3R)-3-[(1S)-1-[(3-bromophenyl)methyl]- 2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] Ammonium in the reactor; (2S)-3-(3-bromophenyl)-2-[(3R)-1 -tert-butoxycarbonylpyrrolidine-3-yl]propanoate (500g, 1 210 mmol), 2-methyltetrahydrofuran (4000 mL), then KHSO4 Add the solution (1M water, 3000mL). Stir the mixture for 30 minutes, during which time the pH will be It is measured as 2-3. Separate the phases of the reaction mixture and extract 2-methyltetrahydrofuran from the aqueous layer. Extract using 1000 mL of solution. Combine the organic phases and wash them with saturated NaCl aqueous solution. The organic phase is dried over MgSO4 and filtered. The solution is transferred to the reactor and 2-tert -Butyl-1,3-diisopropylisourea (618.2g, 3024mmol, 2.5 Add an equivalent amount. Stir the mixture at 65°C for 3 hours, and then add 2-tert-butyl-1, Add 3-diisopropyl isourea (247.3 g, 1210 mmol, 1 equivalent). Stir the mixture at 65°C overnight. Cool the mixture to room temperature. Filter out the solids and leave the other layer. Wash with saturated NaHCO3 aqueous solution (1000 mL). Dry the organic layer on MgSO4. Filter and concentrate in a vacuum. Add MTBE (2000 mL) to the residue and filter out the solids. The filtrate is concentrated to obtain the title compound (483 g, 88%) as a white solid. S / MS (m / z): 342 / 344 (M + H - 2 × tert-butyl)

[0063] Preparation 17 tert-butyl(3R)-3-[1-[(3-bromophenyl)methyl]-2-ter t-Butoxy-1-methyl-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] 3-(3-bromophenyl)-2-[ in 2-methyltetrahydrofuran (33 mL) (3R)-1-tert-butoxycarbonylpyrrolidine-3-yl]-2-methyl- In a solution of ropanoic acid and isomer 2 (3.4 g, 8.2 mmol), 2-tert-butyl-1 ,3-Diisopropylisourea (5.1g, 5.7mL, 25 mmol, 3 equivalents) was added. Then, heat the mixture at 55°C for 3.5 hours. Add 2-tert-butyl-1,3-di Add sopropyl isourea (5.1 g, 5.7 mL, 25 mmol, 3 equivalents) and heat at 55°C. Continue heating the reaction mixture for 1.5 hours. Then add 2-tert-butyl-1,3-diisopropyl Add pyruisourea (2.5g, 2.9mL, 12 mmol, 1.5 equivalents) and heat at 55°C. Continue heating the reaction mixture overnight. Filter off the white solid, wash with MTBE, and then steam the filtrate. Drying is performed using silica gel in hexane with a 0-100% sorbate gradient. The residue was purified by matrixing, and the title compound (3.6g, 93%) was obtained as a yellow oily substance. Obtained as follows: ES / MS(m / z):356, 358[M+H-(2×tert-butyl] )].

[0064] Preparation 18 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3- Trophenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl Using ]-3-(3-nitrophenyl)propanoic acid, in toluene as the reaction solvent, The reaction is carried out at 80°C to prepare the title compound essentially as described in Preparation 17. Silica gel chromatography using a 5-20% sorbate gradient in hexane. The crude product is purified by ES / MS (m / z): 309[M+H-(2×ter (t-butyl)

[0065] Preparation 19 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3-H Lumilphenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] In a parr reactor, tert-butyl (3R) in toluene (3000 mL) -3-[(1S)-1-[(3-bromophenyl)methyl]-2-tert-butoxy- 2-Oxo-ethyl]pyrrolidine-1-carboxylate (300g, 660mmol) The solution, followed by palladium(II) acetate (7.41g, 33.0 mmol, 0.05 equivalents) ), butyldi-1-adamantylphosphine (24.92g, 66.02mmol, 0. 1 equivalent) and N,N,N',N'-tetramethylethylenediamine (115g, 149 Add (mL, 990 mmol, 1.5 equivalents) to the mixture. Stir the mixture at 70 psi synthesis gas (CO2). Pressurize with H21:1) and stir overnight at 100°C. Cool the mixture to room temperature and evaporate the solvent. Dry the residue. Dissolve the residue in acetyl and filter through silica gel to obtain the title compound (273). g, 97%) is obtained as an orange oily substance. ES / MS (m / z): 426 (M+N a).

[0066] Preparation 20 tert-butyl(3R)-3-[2-tert-butoxy-1-[(3-formylfe [Nyl)methyl]-1-methyl-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[1-[(3-bromophenyl)methyl]-2-te rt-butoxy-1-methyl-2-oxo-ethyl]pyrrolidine-1-carboxylate Using this, the title compound is prepared essentially as described in Preparation 19. Hexa Crude cells are produced by silica gel chromatography using a gradient of 10-30% acetone in the solution. The product is purified. ES / MS (m / z): 262 (M+H-tert-butyl-BOC) .

[0067] Preparation 21 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ [Hydroxyiminomethyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1- Carboxylate [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3- Formylphenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate (50 g, 105 mmol, purity 85%), EtOH (400 mL), pyridine (17. 0 mL, 211 mmol, 2 equivalents) and hydroxylamine hydrochloride (10.98 g, 1 58.0 mmol, 1.5 equivalents) are mixed. The mixture is stirred at room temperature for 2 hours. The solvent is evapor ated, then KHSO4 (1 M aqueous solution, 300 mL) and MTBE (500 mL) are added . The organic layer is separated, washed with a saturated aqueous NaHCO3 solution, filtered, and dried over MgSO4 . After filtration through Celite (registered trademark), the filtrate is concentrated to dryness to give the title compound as a yellow oily substance (49 g, yield 98%, purity 88% w / w). ES / MS (m / z): 319 (M+H-Boc)

[0068] Preparation 22 tert-butyl (3R)-3-[(1S)-1-[[3-(aminomethyl)phenyl] methyl]-2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carboxy late

Chemical Formula

[0069] ​​ tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [Hydroxyiminomethyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1 -Carboxylate (49g, 103 mmol, purity 88% w / w) to ammonia (Me Dissolve in OH (7M solution, 606 mL) and use column equilibrium parameters to measure hydrogen gas The eluate is then flushed through the reactor column and collected from the reactor column. The reactor was then flushed again with ammonia (7M solution in MeOH) for 20 minutes, and the reactor was cleaned. The eluate from the eluate is collected. The reactor column eluate fractions are combined, and the mixture is concentrated in a vacuum. The title compound was obtained as an oily substance (46.9 g, 88% yield, 78% w / w purity). ES / MS (m / z): 405 (M+H).

[0070] Preparation 23 tert-butyl(3R)-3-[(1S)-1-[(3-aminophenyl)methyl]- 2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] Palladium on carbon (10% w / w, 380 mg, 0.36 mmol, 0.05 equivalent) This is tert-butyl(3R)-3-[(1S)-2-te in SiO(71mL). rt-butoxy-1-[(3-nitrophenyl)methyl]-2-oxo-ethyl]pylori A stirred solution of din-1-carboxylate (3 g, 7.1 mmol) under a nitrogen atmosphere Add it. Purge the mixture with hydrogen and stir overnight at room temperature under a hydrogen balloon. The reaction mixture is filtered through a moss pad. The filtrate is concentrated under vacuum to obtain the title compound (2.62 g, 94%) is obtained as a white solid. ES / MS (m / z): 291 (M+H-BO C).

[0071] Preparation 24 tert-butyl (3R)-3-[(1S)-2-tert-butoxy-1-[[3-( hydroxymethyl)phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carbo xylate

Chemical Formula

[0072] Preparation 25 [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy carbonylpyrrolidin-3-yl]-3-oxo-propyl]phenyl]boronic acid

Chemical Formula

[0073] Preparation 26 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3-butyl [Droxyphenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] [3-[(2S)-3-tert-butoxy-2-[(3R) in THF (42 mL) -1-tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl] A solution of phenylboronic acid (3.8 g, 8.4 mmol) is mixed with a solution of hydrogen peroxide (3 ml in water). Add 0%, 9.1 mL, 84 mmol, 10 equivalents. Stir the reaction mixture at room temperature overnight. Add a solution of sodium bisulfite (20% w / v in water, 120 mL) to react with the product. Quench slowly. Extract the mixture twice with ethyl acetate, and combine the organic matter with saturated sodium. Wash with an aqueous solution of Cl. Dry the organic matter over Na2SO4, filter it, and concentrate it in a vacuum. Silica gel chromatography using a gradient of 10-40% acetone in hexane. Further purification of the residue yielded the title compound (3.05 g, 90%) as a beige solid. ES / MS (m / z): 280 (M + H - 2x tert-butyl)

[0074] Preparation 27 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ [[[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-but [Xycarbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]methyl] Minomethylphenylmethyl-2-oxoethylpyrrolidine-1-carboxy rate [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3- Formylphenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate (48g, 100mmol, 85% w / w purity, 1.1 equivalents), isopropanol (32 8 mL) and tert-butyl(3R)-3-[(1S)-1-[[3-(aminomethic acid) [Phenyl]methyl]-2-tert-butoxy-2-oxo-ethyl]pyrrolidine- 1-carboxylate (46.9g, 93 mmol, 78% purity, 1 equivalent) in a round-bottom fraser Place in a container. Stir the mixture at room temperature for 1 hour. Cool the mixture to 0°C and add triacetoxyhydrate. Add sodium borohydride (59g, 280 mmol, 3 equivalents) and let the mixture rise to room temperature for 2 minutes. Stir for 2 days. Remove the solvent under reduced pressure. Add water (200 mL) and saturated NaHCO3 aqueous solution. Add 300 mL of ethyl acetate and 500 mL of ethyl acetate. Separate the organic layer and soak it in hexane. Silica gel chromatography using a 50-100% phosphate gradient is used to obtain precision Prepared to obtain the title compound as a colorless oil (36g, 42% yield, 85% w / w purity). ES / MS (m / z): 792 (M+H).

[0075] Preparation 28 tert-butyl(3R)-3-[(1S)-1-[[3-[[bis[[3-[(2S) -3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyllo [Zin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl [L]methyl]-2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carb xylate and tert-butyl(3R)-3-[(1S)-2-tert-butoxy -1-[[3-[[[[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl]fer [Nyl]methyl]amino]methyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine -1-carboxylate [ka] A method for preparing the title compound as a mixture and then separating it by chromatography. 1 In a round-bottom flask, tert-butyl(3R)-3-[(1S)-2-tert-butyx C-1-[(3-formylphenyl)methyl]-2-oxo-ethyl]pyrrolidine-1- Carboxylate (222g, 550mmol), 2-propanol (888mL) ammonia solution (2M in 2-propanol, 302.6 mL, 605.2 mmol, 1 Add 0.1 equivalent. Cool the mixture to 0-5°C in an ice bath. Boron triacetoxyhydride Add sodium phosphate (116.6g, 550.2 mmol, 1 equivalent) in four separate additions. Add the mixture at 40-minute intervals. Stir the mixture at room temperature overnight. Evaporate the solvent to dryness. Add water (200 mL) and K2HPO4 aqueous solution (300 mL) to the residue and perform MTB Extract the aqueous layer with E (2 × 500 mL). Dry the organic layer with MgSO4, filter, and concentrate. Dry to dryness. Use a silica gel chromatograph with a 20-80% SiO2 gradient in hexane. The residue was purified by tography and obtained tert-butyl(3R)-3-[(1S)-1- [[3-[[biS[[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl]fer [Nyl]methyl]amino]methyl]phenyl]methyl]-2-tert-butoxy-2-o Xo-ethyl]pyrrolidine-1-carboxylate (57.8g, 27%) is a white solid. To obtain as such. 1 H-NMR (400MHz, CDCl3)δ7.30-7.24(m, 6 H), 7.12(S, 3H), 7.04(S, 3H), 3.75-3.43(m, 12H) ), 3.30-3.21(m, 3H), 3.10-2.96(m, 3H), 2.89-2 .76(m, 6H), 2.49(d, J=4.7Hz, 3H), 2.37(dd, J=7 .2, 14.4Hz, 3H), 1.98-1.90(m, 3H), 1.74-1.61( m, 3H), 1.48(S, 27H), 1.22(S, 27H).

[0076] From the above silica gel chromatography, tert-butyl(3R)-3-[(1S )-2-tert-butoxy-1-[[3-[[[[3-[(2S)-3-tert-bu Toxy-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl]- 3-Oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2- Oxo-ethyl]pyrrolidine-1-carboxylate (82.50g, 34%) is also It is obtained as a colorless oil. ES / MS (m / z): 792 (M+H), HPLC: 90 wt Indicates purity in percentage.

[0077] tert-butyl(3R)-3-[(1S)-1-[[3-[[bis[[3-[(2S) -3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyllo [Zin-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl [L]methyl]-2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carb Method 2 for preparing xylates In a round-bottom flask, add tert-butyl(3R)-3-[(1S)-2-tert-butoxy -1-[[3-[[[[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl]fer [Nyl]methyl]amino]methyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine -1-carboxylate (90% by weight purity, 81.5g, 92.6 mmol), ter t-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3-formyl Phenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate (purity 8 5% by weight, 50.6g, 106 mmol, 1.15 equivalents), 2-propanol (652mg) Place the mixture in (L) and acetic acid (5.31 mL, 92.6 mmol, 1 equivalent) and steep for 30 minutes. Stir. Sodium triacetoxyborohydride (2 equivalents, 185 mmol, 39.3 Add g) to the mixture, stir at room temperature for 2 hours, then concentrate the reaction mixture under vacuum. Water (200 mL) and MTBE (300 mL) were added to the residue, followed by concentrated ammonium hydroxide. Add an aqueous solution of um to adjust the pH to 9-10. Separate the organic phase and dry it on MgSO4. Filter, concentrate, and dry. Use a gradient of 20-40% siRNA in hexane. The residue was purified by silica gel chromatography and tert-butyl(3R)-3 -[(1S)-1-[[3-[[biS[[3-[(2S)-3-tert-butoxy- 2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl]-3-oxy So-propyl]phenyl]methyl]amino]methyl]phenyl]methyl]-2-tert -Butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate (89g, 82%) ) is obtained as a white solid.

[0078] Preparation 29 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ [[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy [Cicarbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]methyl-[( 3-Fluoro-5-methoxyphenyl)methyl]aminomethyl]phenyl]methyl] -2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [[[[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-bu Toxycarbonylpyrrolidine-3-yl-3-oxopropylphenylmethyl [amino]methyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboc Silate (36g, 39mmol, 85% purity), 3-fluoro-5-methoxybenzalkonium Ludehyde (6.684g, 42.49mmol, 1.1 equivalents), Isopropanol (28 8 mL) and sodium triacetoxyborhydride (16.38 g, 77.26 ml) Mix ol (2 equivalents) and stir the mixture at room temperature for 2 hours. Evaporate the solvent. Saturated Na Add 500 mL of HCO3 aqueous solution and extract the aqueous layer with ethyl acetate (500 mL). The organic layer is dried over MgSO4, then filtered and concentrated to dryness. 10- Purified by silica gel chromatography using a 40% acetone gradient, and then presented. The compound in question was obtained as a colorless oil (32 g, 76% yield, 85% w / w purity). ES / MS (m / z): 931 (M+H).

[0079] Preparation 30 tert-butyl(3R)-3-[1-[[3-[[bis[[3-[3-tert-but Xy-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl]-2 -methyl-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl]methyl]methyl [Lu]-2-tert-butoxy-1-methyl-2-oxo-ethyl]pyrrolidine-1-ca Lu Boxylate [ka] tert-butyl(3R)-3-[2-tert-butoxy-1-[(3-formilf [[Phenyl)methyl]-1-methyl-2-oxo-ethyl]pyrrolidine-1-carboxylate Using the hexagonal compound, the title compound is prepared essentially as described in Preparation 28. Silica gel chromatography using a gradient of 20-40% phosphate in the sun The crude substance is then purified. 1 H NMR (400.21MHz, d6-DMSO): δ7.2 7-7.19(m, 6H), 7.13(S, 3H), 7.08-7.00(m, 3H), 3.57-3.24 (m, 9H, under solvent), 3.22-2.98 (m, 9H), 2.63 -2.38(m, 9H, under solvent), 1.86-1.68(m, 6H), 1.40(S, 2 7H), 1.31(S, 27H), 0.93(S, 9H).

[0080] Preparation 31 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ 3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxyca [Rubonylpyrrolidine-3-yl]-3-oxopropyl]phenoxy]phenyl]methyl [L]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[(1S)-1-[(3-bromophenyl)methyl] -2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate ( 150 mg, 0.330 mmol), tert-butyl(3R)-3-[(1S)-2- tert-butoxy-1-[(3-hydroxyphenyl)methyl]-2-oxo-ethyl Pyrrolidine-1-carboxylate (0.155g, 0.396mmol, 1.2 equivalents) ), cuprous iodide (6 mg, 0.03 mmol, 0.1 equivalent), N,N-dimethylglycyrrhetinol Calcium hydrochloride (0.0138g, 0.0990mmol, 0.3 equivalents) and cesium carbonate ( Mix 0.215 g (0.660 mmol, 2 equivalents) in DMF (2.3 mL). The mixture is stirred overnight at 110°C under nitrogen. The reaction mixture is cooled and then passed through a diatomaceous earth pad. Filter and wash with DCM and MeOH. Concentrate the filtrate under vacuum. The following parameters Using the following method, the resulting residue is purified by RP-HPLC / MS: Column X Bridge(TM) C18 (19 x 100 mm, 5 μm), mobile phase - solvent A = 20 in water mM ammonium bicarbonate (pH 9), solvent B = acetonitrile, gradient -0:100~1 B:A in 00:0, flow rate -25 mL / min. The title compound (43 mg, 17%) was obtained. ES MS (m / z): 665 (M + H - BOC).

[0081] Preparation 32 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ 2-[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-but [Xycarbonylpyrrolidine-3-yl]-3-oxopropyl]phenoxy]ethoxy ]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3- [Hydroxyphenyl)methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate Dissolve 275 mg (0.70 mmol) of cesium carbonate in 1.2 mL of DMF. Mu (285 mg, 0.86 mmol) and 1,2-dibromoethane (0.030 mL, Add 0.34 mmol) and stir at room temperature for 3 days. Over the next 9 days, add cesium carbonate or Potassium carbonate and 1,2-dibromoethane were added three more times, and the temperature was raised to 70°C and The temperature is gradually increased to 110°C. This mixture is cooled to room temperature, and a saturated aqueous solution of NH4Cl is added. Add the following: Extract the aqueous layer with ELISA. Wash the organic phase with saturated NaCl aqueous solution and water. The organic phase is dried over MgSO4, filtered, and the solution is concentrated in vacuum. 0-3 hexane The residue was purified by silica gel chromatography using a 0% ammonium gradient. Then, the title compound (33.4 mg, 11%) is obtained as a colorless oil. ES / MS (m / z):709(M+H-BOC).

[0082] Preparation 33 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy Carbonylpyrrolidine-3-yl]-3-oxopropyl]phenoxy]methyl]phenoxy [nyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] Triphenylphosphine (0.5733g, 2.164 mmol, 1.5 equivalents) is administered to T tert-butyl(3R)-3-[(1S)-2-tert-butyrate in HF (14 mL) Xy-1-[[3-(hydroxymethyl)phenyl]methyl]-2-oxo-ethyl]p Loridine-1-carboxylate (585 mg, 1.443 mmol) and tert- Butyl(3R)-3-[(1S)-2-tert-butoxy-1-[(3-hydroxyf 2-oxo-ethyl]pyrrolidine-1-calcium methyl ethanol-THF (14 mL) Add to a solution of benzene (0.8472 g, 2.164 mmol, 1.5 equivalents). After purging with nitrogen, add diethyl azodicarboxylate (0.34 mL, 2.2 mmol, 1 Add 0.5 equivalents dropwise. Stir the mixture overnight. Filter the reaction through a diatomaceous earth pad. Wash with DCM and MeOH. Concentrate the filtrate under vacuum. Use the following parameters: The residue obtained by SFC is purified: Column - Chiracel(registered trademark) OD( (5 μm, 2 x 25 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. Title compound (315 mg, 28%). ES / MS (m / z): 679 (M+H-BOC).

[0083] Preparation 34 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ 3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxyca [Rubonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]sulfanylphenyl [nyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] Dry toluene (0.9 mL) and acetone (1.8 mL) are mixed with tert-butyl (3 R)-3-[(1S)-1-[(3-bromophenyl)methyl]-2-tert-butoxy [C-2-oxo-ethyl]pyrrolidine-1-carboxylate (796 mg, 1.75 mg) (mol), tripotassium phosphate (229 mg, 1.06 mmol), bis(dibenzylide) Palladium (50 mg, 0.09 mmol), 1,1'-bis(diphenyl) Phosphinoferrocene (70 mg, 0.12 mmol) and potassium thioacetate (10 Add to a mixture of 3 mg and 0.90 mmol. Allow the resulting mixture to stand under a nitrogen atmosphere for 5 minutes. The mixture is subjected to ultrasonic treatment, followed by stirring at 110°C for 6 hours. Saturated NH4Cl aqueous solution and EtO Add Ac. Wash the organic layer with water. Dry the organic layer over MgSO4 and filter it. Concentrated in a vacuum. Silica gel using a 0-40% phosphate gradient in hexane. The residue was purified by chromatography to obtain the title compound as a yellow oily substance (370 mg, 52%). ES / MS(m / z):781(M+H).

[0084] Preparation 35 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ 3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxyca [Rovonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]sulfinylphenyl [nyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [3-[(2S)-3-tert-butoxy-2-[(3R)-1-in DCM (2 mL) [tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl]fer [nyl]sulfanylphenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carb Dissolve xylate (111 mg, 0.14 mmol) in DCM (2 mL). 3-C Add loloperoxybenzoic acid (34 mg, 0.14 mmol, 1 equivalent). Mixture Stir at room temperature for 3 hours. Add saturated NaHCO3 aqueous solution, followed by DCM, and separate the layers. Wash the organic phase with NaOH solution (3% w / v in water) and water. Place the organic phase on MgSO4. Dry the solution, then filter it, and concentrate the solution in a vacuum. 0-60% EtOA in hexane. The residue was purified by silica gel chromatography using a gradient of c to obtain the title compound. (97.2 mg, 87%) is obtained as a colorless oil. ES / MS (m / z): 697( M+H-BOC).

[0085] Preparation 36 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ 3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxyca [Rovonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]sulfonylphenyl [Methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] The compound is prepared by a method essentially similar to that of Preparation 35, but with 2.5 equivalents of 3- Use chloroperoxybenzoic acid. Use a gradient of 20-60% acetoxide in hexane. The crude product was purified by silica gel chromatography to obtain the title compound (116). 6 mg, 82% is obtained as a white solid. ES / MS (m / z): 713 (M + H- BOC).

[0086] Preparation 37 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy Carbonylpyrrolidine-3-yl]-3-oxopropyl]anilino]methyl]phenyl [Methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] Sodium triacetoxyborhydride (543 mg, 2.56 mmol, 2 equivalents) tert-butyl(3R)-3-[(1S)-1-[(3-aminophenyl)methyl] -2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate ( 500 mg, 1.28 mmol), tert-butyl(3R)-3-[(1S)-2-t ert-butoxy-1-[(3-formylphenyl)methyl]-2-oxo-ethyl]p Loridine-1-carboxylate (620 mg, 1.54 mmol, 1.2 equivalents) and Add to a solution of acetic acid (220 μL, 3.84 mmol, 3 equivalents). Stir at room temperature for 4 days. Add saturated NaHCO3 aqueous solution and extract the aqueous layer with DCM. The combined organic layer is Mg Dry over SO4, filter, and concentrate the solution under vacuum. Add 10-50% Et in hexane. The residue was purified by silica gel chromatography using an OAc gradient, and the title compound was obtained. Obtained substance (850 mg, 72%). ES / MS (m / z): 778 (M+H).

[0087] Preparation 38 (2S)-3-[3-[3-[(2S)-3-tert-butoxy-2-[(3R)-1 -tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl]ani [Lino]phenyl]-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3 -yl]propanoic acid [ka] (2S)-3-(3-bromophenyl)-2- in 1,4-dioxane (160 mL) [(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl]propanoic acid (8 g, 20 mmol), tert-butyl(3R)-3-[(1S)-1-[(3-amino Phenyl)methyl]-2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1- Carboxylate (8.628 g, 22.09 mmol, 1.1 equivalents) and potassium carbonate Mix in 4 equivalents (11.22 g, 80.34 mmol). Stir the mixture under nitrogen for 60°C. Heat at °C for 15 minutes. [(2-di-cyclohexylphosphino-3,6-dimethoxy-2 ',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1 ,1'-Biphenyl) Palladium(II) Methanesulfonate BrettPhos Pd Add G3 (0.371g, 0.402 mmol, 0.02 equivalents) and stir overnight. The reaction mixture is heated to 100°C. The reaction mixture is cooled to room temperature and diluted with HCl. Adjust the pH of the mixture to less than 3 by adding solution (1N). Dilute the water with Me-THF. Extract the layers three times. Wash the combined organic matter with saturated NaCl aqueous solution. Mix the organic matter with MgSO 4. Dry on the surface and concentrate the solution in a vacuum. Gradient of 0-100% siRNA in hexane. The residue was purified by silica gel chromatography using the following method: title compound (9.5g) 67% is obtained. ES / MS(m / z):608(M+H-BOC)

[0088] Preparation 39 Di-tert-butyl 3,3'-((2S,2'S)-((sulfonylbis(azanezi) Bis(3,1-phenylene)bis(3-(tert-butoxy)-3-oxop Ropan-1,2-diyl))(3R,3'R)-bis(pyrrolidine-1-carboxylate) to) [ka] 1,4-diazabicyclo[2.2.2]octane in acetonitrile (12.8 mL) Bis(sulfur dioxide) adduct (DABSO, 0.6279g, 2.561 mmol, 2 equivalents) Cool the solution of iodine (0.4875g, 1.921 mmol, 1.5 equivalents) to 0°C. Add ). Stir the mixture at 0°C for 15 minutes, then add tert-butyl(3R)-3- [(1S)-1-[(3-aminophenyl)methyl]-2-tert-butoxy-2-o Xo-ethyl]pyrrolidine-1-carboxylate (500 mg, 1,280 mmol) Add [ingredient]. Heat the reaction mixture overnight at 80°C. Cool the reaction mixture and pass it through a diatomaceous earth pad. Filter and wash the pads with DCM and MeOH. Concentrate the filtrate under vacuum. The following pads Purify the residue using RP-HPLC / MS with a column: column-XBridge( (Trademark) C18 (19 x 100 mm, 5 μm), mobile phase - solvent A = 20 mM ammonium bicarbonate Aqueous solution of nium (pH 9), solvent B = acetonitrile, gradient - isocratic 80:20 A:B, flow rate -25 mL / min, room temperature. The title compound (195 mg, 18%) was found to be a pale yellow solid. It is obtained as a physical substance. ES / MS (m / z): 743 (M + H - BOC).

[0089] Example 1 (2S)-3-[3-[[bis[[3-[(2S)-2-carboxy-2-[(3R)- Pyrrolidine-3-ylethyl]phenyl]methyl]aminomethyl]phenyl]-2- [(3R)-pyrrolidine-3-yl]propanoic acid, tetrahydrochloride [ka] Prepare a hydrochloric acid solution (4M in 1,4-dioxane, 67 mL, 270 mmol, 20 equivalents), tert-butyl(3R)-3-[(1S)-1-[[3-[[biS[[3-[(2S )-3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonylpyro Lysine-3-yl]-3-oxopropyl]phenyl]methyl]amino]methyl]phenyl [Nyl]methyl]-2-tert-butoxy-2-oxo-ethyl]pyrrolidine-1-cal Add to 15.7 g of benzene (13.3 mmol) and stir the mixture overnight at 40°C. Cool the mixture to room temperature, then concentrate it to dryness. Dissolve the residue in water (40 mL) and freeze. Dry the mixture. Dissolve the resulting solid in water (40 mL) again and freeze-dry it to form the title compound. The substance (8.6g, 75%) is obtained as a white foam. ES / MS (m / z): 711(M +H); 1 H-NMR (500 MHz, D2O) δ7.35-7.10 (m, 12H) , 4.25-4.17(m, 6H), 3.55(dd, J=8.1, 11.5 Hz, 3 H), 3.38-3.33(m, 3H), 3.22-3.15(m, 3H), 3.03- 2.87(m, 12H), 2.56-2.45(m, 3H), 2.12-2.08(m, 3H), 1.73-1.63 (m, 3H).

[0090] Example 2 (2S)-3-[3-[[bis[[3-[(2S)-2-carboxy-2-[(3R)- Pyrrolidine-3-ylethyl]phenyl]methyl]aminomethyl]phenyl]-2- [(3R)-pyrrolidine-3-yl]propanoic acid [ka] In a round-bottom flask, tert-butyl(3R)-3-[(1S)-1-[[3-[[bi S[[3-[(2S)-3-tert-butOxy-2-[(3R)-1-tert- Butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]methyl [amino]methyl]phenyl]methyl]-2-tert-butoxy-2-oxo-ethyl Pyrrolidine-1-carboxylate (499.3g, 423.3mmol), 1,4- Dioxane (1997 mL) and hydrochloric acid solution (12 M water, 529.1 mL, 15 equivalents) Add ). Stir the mixture at 40°C for 1 hour, then concentrate the mixture in a vacuum to obtain 1,4- Remove the dioxane to obtain an aqueous slurry. Pass the mixture through a propylene filter. Filter to remove insoluble particles. Use a NaOH solution (2M in water) to adjust the pH of the filtrate. Adjust to 9-10. Stir this mixture at room temperature overnight. Using filter paper, remove the obtained solid The material is slowly filtered (using slow filtration and low vacuum). The solid is washed with water and vacuumed at 45°C. By drying, the title compound (281 g, 88%) is obtained as a white crystalline solid. ES / MS (m / z): 711 (M+H); 1 H-NMR (500 MHz, D2O) δ7.3 3(t, J=7.6Hz, 3H), 7.27(d, J=7.8Hz, 3H), 7.13( d, J=7.8Hz, 3H), 7.09(s, 3H), 4.20(s, 6H), 3.54 (dd, J=7.9, 11.6Hz, 3H), 3.39-3.34(m, 3H), 3.2 3-3.17(m, 3H), 3.02-2.98(m, 3H), 2.84(dd, J=4 .6, 13.7Hz, 3H), 2.76(dd, J=10.6, 13.3Hz, 3H), 2.60(td, J=9.9, 4.8Hz, 3H), 2.48(td, J=17.3, 9 .6Hz, 3H), 2.12-2.07(m, 3H), 1.73-1.65(m, 3H) .

[0091] Example 3 (2S)-3-[3-[[[3-[(2S)-2-carboxy-2-[(3R)-pyloric acid [3-yl]ethyl]phenyl]methylamino]methyl]phenyl]-2-[(3R )-Pyrrolidine-3-yl]propanoic acid, trihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [[[[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-bu Toxycarbonylpyrrolidine-3-yl-3-oxopropylphenylmethyl [amino]methyl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboc Using 2M HCl in silate and Et2O at room temperature, the title compound was essentially carried out. Prepare as described in Example 1. ES / MS (m / z): 480 (M+H).

[0092] Example 4 (2S)-3-[3-[[[3-[(2S)-2-carboxy-2-[(3R)-pyloric acid [3-Ilethyl]phenyl]methyl-[(3-fluoro-5-methoxy-phenyl] [(3R)-pyrrolidine-3-yl]p Ropanic acid [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [[[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-but [Xycarbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]methyl-[ (3-fluoro-5-methoxyphenyl)methyl]aminomethyl]phenyl]methyl Using ]-2-oxo-ethyl]pyrrolidine-1-carboxylate, the title compound Prepare as described in Example 2. ES / MS (m / z): 618 (M+H).

[0093] Example 5 (2S)-3-[3-[[[3-[(2S)-2-carboxy-2-[(3R)-pyloric acid [3-Ilethyl]phenyl]methyl-[(3-fluoro-5-methoxy-phenyl] [(3R)-pyrrolidine-3-yl]p Ropanic acid, trihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [[[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-but [Xycarbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]methyl-[ (3-fluoro-5-methoxyphenyl)methyl]aminomethyl]phenyl]methyl ]-2-oxo-ethyl]pyrrolidine-1-carboxylate (541 mg, 0.582 mmol), HCl (2M solution in diethyl ether, 5.8 mL), and water (0.5 mL) Mix the following: Stir the mixture at room temperature for 5 hours. Remove the solvent under reduced pressure and extract the title compound ( 434 mg, 102% obtained. ES / MS (m / z): 618 (M+H).

[0094] Example 6 3-[3-[[bis[[3-[2-carboxy-2-[(3R)-pyrrolidine-3-yl ]propyl]phenyl]methyl]amino]methyl]phenyl]-2-methyl-2-[(3 R)-Pyrrolidine-3-yl]propanoic acid, tetrahydrochloride [ka] tert-butyl(3R)-3-[1-[[3-[[bis[ [3-[3-tert-butoxy-2-[(3R)-1-tert-butoxycarbonyl Pyrrolidine-3-yl]-2-methyl-3-oxopropyl]phenyl]methyl]amine [no]methyl]phenyl]methyl]2-tert-butoxy-1-methyl-2-oxo- Ethyl]pyrrolidine-1-carboxylate (275 mg, 0.225 mmol) and Add HCl (2M solution in Et2O, 3.4 mL, 6.7 mmol, 30 equivalents). Stir the mixture at room temperature overnight. Decant the reaction solvent from the white solid and dissolve the solid in water. To resolve, the mixture is evaporated to dryness under a nitrogen stream, then dried in a vacuum at 40°C, and the title is formed. The mixture (190 mg, 86%) is obtained as a white solid. ES / MS (m / z): 753 M+H).

[0095] Example 7 (2S)-3-[3-[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [-3-yl]ethyl]phenoxy]phenyl]-2-[(3R)-pyrrolidine-3-yl] ] Propanoic acid, dihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy Carbonylpyrrolidine-3-yl]-3-oxopropyl]phenoxy]phenyl]methyl Using [Tyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate, the title compound Prepare the material essentially as described in Example 6. ES / MS(m / z):453(M +H).

[0096] Example 8 (2S)-3-[3-[2-[3-[(2S)-2-carboxy-2-[(3R)-pyro Lysine-3-yl]ethyl]phenoxy]ethoxy]phenyl]-2-[(3R)-pyro Lysine-3-yl]propanoic acid, dihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [2-[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-bu Toxycarbonylpyrrolidine-3-yl]-3-oxopropyl]phenoxy]ethoxy Using phenylmethyl-2-oxoethyl pyrrolidine-1-carboxylate Using this method, the title compound is prepared essentially as described in Example 6. Mix with MTBE. The product is isolated by this method. ES / MS (m / z): 497 (M+H).

[0097] Example 9 (2S)-3-[3-[[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [3-yl]ethyl]phenoxy]methyl]phenyl]-2-[(3R)-pyrrolidine] -3-yl]propanoic acid, dihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy [Cicarbonylpyrrolidine-3-yl]-3-oxopropyl]phenoxy]methyl] Using [enylmethyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate Prepare the title compound essentially as described in Example 6. ES / MS (m / z): 467 (M+H).

[0098] Example 10 (2S)-3-[3-[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [-3-yl]ethyl]phenyl]sulfanylphenyl]-2-[(3R)-pyrrolidine] -3-yl]propanoic acid, dihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy Carbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]sulfanyl Using [enylmethyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate The title compound is prepared essentially as described in Example 6. It is kneaded with Et2O. The product is isolated by the following method: ES / MS (m / z): 469 (M+H).

[0099] Example 11 (2S)-3-[3-[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [-3-yl]ethyl]phenyl]sulfinylphenyl]-2-[(3R)-pyrrolidine] -3-yl]propanoic acid, dihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy Carbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]sulfinyl Using [enylmethyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate The title compound is prepared essentially as described in Example 6. The product is kneaded with Et2O. Next, isolate the product by RP-HPLC / MS using the following parameters: Column: Agilent ZORBAX Bonus RP, Mobile phase: Solvent A = 0 in water. 0.5% trifluoroacetic acid (pH 2.5), solvent B = acetonitrile + 0.05% tri Fluoroacetic acid, gradient - solvent B at 5-30% in solvent A, flow rate: 25 mL / min. HCl(1 The product is taken into a mixture of a 0% w / v aqueous solution and water, and the solvent is evaporated at 40°C under a nitrogen stream. To cause. ES / MS (m / z): 485 (M+H).

[0100] Example 12 (2S)-3-[3-[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine -3-yl]ethyl]phenyl]sulfonylphenyl]-2-[(3R)-pyrrolidine- 3-yl]propanoic acid, dihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy Carbonylpyrrolidine-3-yl]-3-oxopropyl]phenyl]sulfonylphenyl Using [nyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate, The title compound is prepared essentially as described in Example 6. It is kneaded with Et2O. The product is then isolated. ES / MS (m / z): 501 (M+H).

[0101] Example 13 (2S)-3-[3-[[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [3-yl]ethyl]anilino]methyl]phenyl]-2-[(3R)-pyrrolidine- 3-yl]propanoic acid, trihydrochloride [ka] tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3- [[3-[(2S)-3-tert-butoxy-2-[(3R)-1-tert-butoxy [Cicarbonylpyrrolidine-3-yl]-3-oxopropyl]anilino]methyl]phen Using [nyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboxylate, The title compound is prepared essentially as described in Example 6. After decanting the product, Purify by RP-HPLC / MS using the following parameters: Column - Waters (Trademark) XBridge (Trademark) C18 (19 × 100 mm, 5 μm), Mobile phase - Solvent A = 20 mM NH4HCO3 in water, solvent B = acetonitrile, flow rate 25 mL / min, gradient - B:A for 5:95~25:75. Dissolve the purified substance in an aqueous HCl solution (1N) at room temperature. Stir for 1 hour, evaporate the solvent under a nitrogen stream, and dry the solids in a vacuum at 40°C. S / MS (m / z): 466 (M+H).

[0102] Example 14 (2S)-3-[3-[[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [(3R)-pyrrolidine-3-yl]ethyl]anilino]phenyl]-2-[(3R)-pyrrolidine-3-yl] ] Propanoic acid, dihydrochloride [ka] (2S)-3-[3-[3-[(2S)-3-tert-butoxy-2-[(3R)- 1-tert-butoxycarbonylpyrrolidine-3-yl]-3-oxopropyl] [Nilino]phenyl]-2-[(3R)-1-tert-butoxycarbonylpyrrolidine- 3-yl]propanoic acid (9g, 12.7mmol), isopropanol (27mL), Mix with HCl (a 5.5 M solution in isopropanol) and stir at room temperature for 2.5 hours. The reaction mixture is heated to 60°C for 2.5 hours, then cooled to room temperature and stirred for 3 days. Heat again to 60°C for 2 hours, cool to room temperature, and dry the reaction mixture in a vacuum. Ultrasonic treatment Using a method, the solid residue is kneaded with MTBE, filtered, rinsed with MTBE, and then the solids Dry in a vacuum. Mix the solid with concentrated hydrochloric acid solution, heat at 80°C overnight, then at room temperature. Cool to a low temperature and concentrate to dryness in a vacuum. Dissolve the residue in the minimum amount of water and add an aqueous NaOH solution. This adjusts the pH to 7.5. Stir the mixture at room temperature for 3 hours, then allow it to precipitate. Filter out the solids. Dissolve the solids in an aqueous HCl solution (1N) and stir at room temperature for 15 minutes. Then, remove the water in a vacuum. Dry the residue overnight in a vacuum at 45°C to obtain the title compound (5 Obtain 0.4g, 81%. ES / MS (m / z): 452 (M+H).

[0103] Example 15 (2S)-3-[3-[[3-[(2S)-2-carboxy-2-[(3R)-1-meth Lupyrolidine-3-yl]ethyl]anilino]phenyl]-2-[(3R)-1-methyl] Pyrrolidine-3-yl]propanoic acid, dihydrochloride [ka] Paraformaldehyde (89 mg, 0.95 mmol) in MeOH (1.9 mL) (2S)-3-[3-[3-[(2S)-2-carboxy-2-[(3R)-pyrrolidine [(3R)-pyrrolidine-3-yl]ethyl]anilino]phenyl]-2-[(3R)-pyrrolidine-3-yl] ] Propanic acid, dihydrochloride (100 mg, 0.19 mmol) MeOH (1.9 mL) Add to the turbidity. Stir at room temperature for 15 minutes. Sodium triacetoxyborohydride (2 Add 0.2 mg (0.95 mmol) and stir at room temperature for 16 hours. Concentrate the solution under vacuum. Reverse-phase chromatography (column: Claricep C series silica bonded to silica). Ta C 18Using ), a gradient of 5-25% acetonitrile in aqueous NH4CO3 (pH 9) was observed. The residue was purified using [method / tool ​​name]. The purified substance was dissolved in an aqueous hydrochloric acid solution (1N, 1 mL) at room temperature. Stir for 6 hours. Concentrate the solution under vacuum to obtain the title compound (30 mg, 27% yield). ES / MS (m / z): 480 (M+H).

[0104] Example 16 (2S,2'S)-3,3'-[sulfonylbis(azandiyl-3,1-phenylene) ]bis{2-[(3R)-pyrrolidine-3-yl]propanoic acid}, dihydrochloride [ka] Di-tert-butyl 3,3'-((2S,2'S)-((sulfonylbis(azandyl) Il))bis(3,1-phenylene))bis(3-(tert-butoxy)-3-oxo Propane-1,2-diyl))(3R,3'R)-bis(pyrroridine-1-carboxyle Prepare the title compound as described in Example 6 using ES / MS(m). / z):531(M+H).

[0105] Example 17 (2S)-3-phenyl-2-[(3R)-pyrrolidine-3-yl]propanoic acid, hydrochloride [ka] HCl (5.5 M in isopropanol, 511 mL, 2.81 mol, 13 equivalents) (2S)-2-[(3R)-1-tert-butoxy in sopropanol (207 mL) Carbonylpyrrolidine-3-yl]-3-phenyl-propanoic acid (69g, 216mmo) Add to solution l) and stir overnight at room temperature. Mix the mixture in a 2:1 ratio of MTBE:hexane (90°C). Dilute with the mixture (0 mL) and stir for 10 minutes. Filter the suspension and remove the solids in a 1:1 ratio. Wash with BE:hexane (100 mL). Dry the solids under vacuum at 50°C, then label. The compound (51.3g, 93%) was obtained. ES / MS (m / z): 220 (M+H).

[0106] Example 18 (2R)-3-phenyl-2-[(3S)-pyrrolidine-3-yl]propanoic acid, hydrochloride [ka] (2R)-2-[(3S)-1-tert-butoxycarbonylpyrrolidine-3-yl Using ]-3-phenyl-propanoic acid, the title compound is essentially as described in Example 6. Prepare the product. Stir the isolated product with HCl (2M in ether) and water for 3 hours and extract the true Concentrate in air, then knead with MTBE, and dry in vacuum at 40°C. ES / MS(m / z):220(M+H).

[0107] Radiolabeling and low temperature (i.e., non-radioactive) for in vitro Apo(a) binding assays Labeling) Standard synthesis Preparation 40 1-(3-benzyloxyphenyl)imidazolidined-2-one [ka] 20 mL of argon-spurged DMF, ethylene urea (1.4 g, 16 mmol) l), 1-benzyloxy-3-iodobenzene (4.9g, 16 mmol, 1.0 equivalent) ), cuprous iodide (0.61g, 3.1 mmol, 0.20 equivalents), potassium dihydrogen phosphate Add to the mixture of (4.2g, 31 mmol, 2.0 equivalents). Add N to the resulting suspension. ,N'-dimethylethylenediamine (0.33 mL, 0.28 g, 3.1 mmol, 0. Add 20 equivalents. Heat the blue suspension in a microwave reactor at 120°C for 3 hours. The reaction mixture is cooled, filtered through a silica gel pad, and the pad is flushed with Â. Concentrate the filtrate. Mix 5:20:75 MeOH:acetone:siRNA in hexane. The residue was purified by silica gel chromatography using a gradient of 0-100%. The title compound (1.10 g, 26%) was obtained as a yellow solid. ES / MS (m / z): 2 69(M+H)

[0108] Preparation 41 (2S)-3-[3-[3-(3-benzyloxyphenyl)-2-oxo-imidazoly [Zin-1-yl]phenyl]-2-[(3R)-1-tert-butoxycarbonylpyro Lysine-3-yl]propanoic acid [ka] 1-(3-benzyloxyphenyl)imidazolidined-2-one (3.33g, 12. 4 mmol, 1.50 equivalents) and ammonium; (2S)-3-(3-bromophenyl)- 2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl]propanoe To the mixture with (3.30 g), under a nitrogen atmosphere, add acetonitrile (41 mL) and Add DMF (to aid solubility). Apply a steady flow of argon to this suspension for 15 minutes. Whisk until frothy. Potassium carbonate (3.75g, 26.8mmol, 3.24 equivalents), iodide monocopper (0.316g, 1.66 mmol, 0.2 equivalents) and N,N'-dimethylethylene Add diamine (0.360 mL, 3.31 mmol, 0.4 equivalents). Close the container tightly. Heat at 100°C over the weekend. Add water and extract the aqueous layer twice with ethyl acetate. The phase is acidified with 0.5N HCl in water, and the aqueous layer is divided into siRNA, DCM, and then siRNA. Extraction is performed. The combined organic phase is dried on MgSO4, filtered, and evaporated to dryness. Silica gel chromatography using 10-60% (1% acetic acid / acetone) in a SAN solution. The residue was purified to obtain the title compound (4g, 82%). ES / MS(m / z):4 86 (M+H-BOC).

[0109] Preparation 42 tert-butyl(3R)-3-[(1S)-1-[[3-[3-(3-benzyloxy Phenyl)-2-oxoimidazolidin-1-yl]phenyl]methyl]-2-ter t-Butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate [ka] (2S)-3-[3-[3-(3-benzyloxyphenyl) in toluene (68 mL) )-2-oxoimidazolidin-1-yl]phenyl]-2-[(3R)-1-ter t-Butoxycarbonylpyrrolidine-3-yl]propanoic acid (4.0g, 6.8mmol) In the solution of ), add N,N-dimethylformamide di-tert-butylacetal ( Add 15 mL, 55 mmol, 8.0 equivalents, and heat the reaction mixture at 70°C overnight. Silica gel chromatography using a 0-50% phosphate gradient in the sun leaves The residue was purified to obtain the title compound (1.56 g, 33%). ES / MS (m / z): 54 2(M+H-BOC).

[0110] Preparation 43 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-1-[[3-[ 3-(3-hydroxyphenyl)-2-oxo-imidazolidin-1-yl]phenyl] Methyl 2-oxoethyl pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[(1S)-1-[[3-[3-(3-benzyl oxy [Ciphenyl]-2-oxo-imidazolidin-1-yl]phenyl]methyl]-2-te rt-butoxy-2-oxo-ethyl]pyrrolidine-1-carboxylate (1.56g) Dissolve 2.43 mmol of palladium hydroxide (0.7 g) in THF (80 mL), Add 0.99 mmol (0.41 equivalents). Degas the reaction mixture under vacuum, then add hydrogen gas. Expose to the sun three times. Stir the reaction mixture at room temperature under hydrogen (1 atmosphere) for 4 hours. Concentrate the reaction mixture. Silica gel chromatography using a 0-50% butyl gradient in hexane Further purification of the residue yielded the title compound (1.19 g, 87%). ES / MS (m / z) :452(M+H-BOC).

[0111] Preparation 44 tert-butyl(3R)-3-[(1S)-2-tert-butoxy-2-oxo-1 -[[3-[2-oxo-3-[3-(tritrithiomethoxy)phenyl]imidazolid [1-yl]phenyl]methyl]ethyl]pyrrolidine-1-carboxylate [ka] tert-butyl(3R)-3-[(1S)-2-tert in DMF (0.5 mL) -Butoxy-1-[[3-[3-(3-hydroxyphenyl)-2-oxo-imidazoly [Zin-1-yl]phenyl]methyl]-2-oxo-ethyl]pyrrolidine-1-carboc A solution of silate (4 mg, 0.007 mmol) is mixed with cesium carbonate (10 mg, 0.03 mmol). Add mmol). Stir the solution at room temperature for 10 minutes. 3 [H]methylnosilate (5 Add 0 mCi) and stir at room temperature for 1.5 hours. Use the following parameters to RP- Purify the reaction mixture by HPLC / MS: Column - Phenomenex (registered trademark) ) Gemini(registered trademark) C18 (250×10mm) is used in the mobile phase-solvent A = water + triglycerides. Fluoroacetic acid (0.1%), Solvent B = Acetonitrile + Trifluoroacetic acid (0.1%) Gradient A to B (50-100%) for 50 minutes or more, flow rate - 3 mL / min. Purified product to Et It is dissolved in OH and the process proceeds to preparation 45 without further characterization.

[0112] Preparation 45 (2S)-3-[3-[2-oxo-3-[3-(tritrithiomethoxy)phenyl] Midazolidine-1-yl]phenyl]-2-[(3R)-pyrrolidine-3-yl]propane Acids, hydrochloride [ka] In a vacuum, 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 Remove the solvent from the mixture and dissolve the residue in hydrogen chloride (a 4M solution in 1,4-dioxane). Stir this mixture overnight at room temperature. Use the following parameters to perform RP-HPLC / M Purify the reaction mixture using S: Column - Phenomenex(registered trademark) Gemin i(registered trademark) C18 (250 x 10 mm) is used in the mobile phase-solvent A = water + trifluoroacetic acid. (0.1%), Solvent B = Acetonitrile + Trifluoroacetic acid (0.1%), Gradient - A 1 Dissolve the purified product in ethanol for 60 minutes or more at a flow rate of -3 mL / min in 0-70% B solution. Mass spectrometry is performed on inert substances [ES / MS(m / z):410(M+H)] and 63 It provides a spectrum consistent with the specific activity of Ci / mmol.

[0113] Preparation 46 1-(3-methoxyphenyl)imidazolidined-2-one [ka] Using 1-iodo-3-methoxybenzene and tripotassium phosphate as a base, Essentially, the title compound is prepared as described in Preparation 40. Using conventional heating Then, heat the reaction mixture at 120°C for 16 hours. Dilute with acetate and Celite (registered trademark). Filter the organic matter through the pad (standard), and dissolve it in water, NH4OH aqueous solution, and saturated NaCl aqueous solution. The reaction products are prepared by washing with a liquid. 20-100% EtOA in hexane. The crude substance is purified by silica gel chromatography using a gradient of c. ES / MS (m / z): 193 (M+H).

[0114] Preparation 47 (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl] -3-[3-[3-(3-methoxyphenyl)-2-oxoimidazolidined-1-yl] ]phenyl]propanoic acid [ka] (2S)-3-(3-bromophenyl)-2-[(3R)-1-tert-butoxyca Rubonylpyrrolidine-3-yl]propanoic acid (190 mg, 0.4770 mmol), 1 -(3-methoxyphenyl)imidazolidined-2-one (0.2751g, 1.431m) (mol, 3 equivalents), [(2-di-tert-butylphosphin-2',4',6'-tri Isopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl) ] Palladium(II) methanesulfonate (tBuXPhos-Pd-G3, 0.0379 (g, 0.0477 mmol, 0.1 equivalents) and tert-butoxide sodium (0. (1418g, 1.431 mmol, 3 equivalents) in 1,4-dioxane (4.8mL) Mix the mixture and stir it overnight at 100°C under a nitrogen atmosphere. Dilute the mixture with toluene. Acidify with an aqueous HCl solution (1N). Mix through a Celite® pad. The material is filtered, the layers are separated, and the organic matter is dried on MgSO4. The organic matter is filtered and concentrated. Next, using a gradient of 40-70% acetonitrile in an NH4CO3 aqueous solution (pH 9) The residue is purified using reverse-phase flash chromatography (silica-bonded C18 column). The title compound (70 mg, 28%) is then obtained as a white solid. The following parameters are used. Then, an additional amount (90 mg, 36%) of the title compound is obtained by RP-HPLC / MS: Ram-Waters(TM) XBridge(TM) C18(19×100mm, 5μm ), mobile phase - solvent A = 20 mM NH4HCO3 in water, solvent B = acetonitrile, flow rate 2 5 mL / min, gradient -30:70 to 50:50, B:A. ES / MS (m / z): 510( M+H).

[0115] Preparation 48 (2S)-3-[3-[2-oxo-3-[3-(methoxy)phenyl]imidazolidine -1-yl]phenyl]-2-[(3R)-pyrrolidine-3-yl]propanoic acid, hydrochloride [ka] (2S)-2-[(3R)-1-tert-butoxycarbonylpyrrolidine-3-yl ]-3-[3-[3-(3-methoxyphenyl)-2-oxo-imidazolidin-1-i Using [phenyl]propanoic acid, essentially prepare as described in the title of preparation 45. Prepare the compound. ES / MS (m / z): 410 (M+H).

[0116] Biological assays In vitro Apo(a) binding assay The in vitro binding affinity of a compound to the target human Apo(a) protein is compared. Test by binding assay. Human Apo(a) taint containing 17 clingle repeats. The protein was converted from the conditioned medium of transiently transfected HEK-293F cells to an affinity protein. Purify the solution. All reagents should be prepared with 50 mM Tris-HCl pH 7.4 and 0.1% B Prepare in assay buffer containing SA. The binding assay is performed on each of the clear-bottom plates. (1) Dilution series of test compounds (final concentration 0.32~10000nM), (2) Ap o(a) Protein (6 ng / well), (3) Resuspended wheat germ agglutinin polyvinyl Luen SPA beads (20 mg / mL), and (4) radioactive ligand, tritium labeled. (2S)-3-[3-[2-oxo-3-[3-(tritrithiomethoxy)phenyl] Midazolidine-1-yl]phenyl]-2-[(3R)-pyrrolidine-3-yl]propane This is done by adding 50 μl each of nic acid and hydrochloride (final concentration 0.52 nM). Incubate the plate at room temperature for 60 minutes and count it using a TRILUX LSC. Nonspecific binding, binding in the presence of 10 μM of low-temperature (i.e., non-radioactively labeled) ligand. It is defined as (2S)-3-[3-[2-oxo-3-[3-(methoxy)phenyl [Imidazolidine-1-yl]phenyl]-2-[(3R)-pyrrolidine-3-yl]p Subtracting ropanoic acid and hydrochloride salts determines specific binding. Using a standard single-site binding model... By adapting the data, the IC of exemplary test compounds is analyzed. 50 To decide. The results are summarized in Table 1, showing examples of test compounds binding to the human Apo(a) protein. This indicates that L is produced by apo(a) via binding to the Apo(a) protein. Inhibition of DL particle aggregation supports a decrease in Lp(a) levels. [Table 1]

[0117] In vitro Lp(a) aggregation assay The compound's ability to inhibit Lp(a) particle formation in vitro is relevant to cell-free aggregation assays. Therefore, it is evaluated. Acclimatization medium (10% FBS, 20 mM HEPES, and 1x penicillin) DMEM supplemented with sylin / streptomycin was incubated at 37°C and 5% CO2 for 24 hours. After culturing for a certain period, confluent wild-type HepG2 cells (with endogenously expressed ApoB) From the source, and expressing human Apo(a) containing 17 kringle repeats Recovered from HEK293 stable cell lines (selected with 1 mg / ml of Geneticin) Dilute equal volumes of HepG2 and HEK293 acclimatized medium in a series of dilutions (final concentration 0.01-10). By combining it with a test compound added at 0 nM, an in vitro assembly assay can be performed. Apply the solution. Incubate the reaction at 37°C for 2 hours, then add 6-aminocaproic acid (EAC). Add A) to a final concentration of 150 mM and stop. Lp(a) is an anti-Lp(a) scavenging agent. It was detected using a sandwich ELISA with the body and an HRP-conjugated anti-ApoB detection antibody. The ELISA is colorimetrically developed using TMB and stopped using 1N sulfuric acid, and the signal is signaled. This is read at 450nm using a Molecular Devices plate reader. The percentage of Lp(a) inhibition formed under each test condition is when no inhibitor is present (DMSO concentration 1 Set the aggregation reaction (corresponding to %) to 0% inhibition, and the minimum amount of HepG2 conditioned medium present ( Set the 50-fold dilution to 100% inhibition. The ICs summarized in Table 2 are as follows. 50 To determine the value Therefore, the data is fitted to a four-parameter curve. As summarized in Table 2, ApoB The addition of exemplary test compounds to conditioned media containing Apo(a) is in vitro. This results in concentration-dependent inhibition of Lp(a) formation. The result is that these compounds inhibit Apo(a) This also indicates that it inhibits the aggregation of Lp(a) from LDL particles. [Table 2]

[0118] In vivo Lp(a) inhibition in mice The ability of compounds to lower steady-state Lp(a) levels in vivo is that of humanized Lp(a Evaluate using a transgenic mouse model capable of producing particles. Lp(a) The in vivo effect of disruptor compounds is observed in human apoB-100 and 17 Klings. Female double transgender individuals aged 7-17 months expressing human apo(a) containing rulipito Test with a phenic mouse: B6.SJL-Tg(APOB)1102Sgy Tg(A lb-LPA)32Arte. The mouse uses a standard light cycle (12 hours light / 12 hours). During the dark period, they are raised at a room temperature of 72±8°F and a relative humidity of 30-70%, with water and regular solid feed. Make it available for free intake (Harlan Tecklad diet 2014). Uss used BRAT (Block Random Assignment Tool) for the study, and weight and Based on baseline plasma Lp(a) concentration, the treatment group (n=5 / group) was determined 3-5 days prior to the study. Mice will be randomized. They will be given a vehicle (oral administration vehicle: 10 mL / kg, 1% H EC, 0.25% Tween80, 0.01% antifoaming agent, subcutaneous administration vehicle: 5 mL / (kg of physiological saline) or the test compound in various doses, twice a day (6:30 am and 3:00 am). :30pm) Administer orally (or subcutaneously, if indicated) for 5 days. Blood is taken from the tail. The blood is collected through the bloodstream into a heparin-coated capillary tube. A 20 μL sample is placed on a DBS card (Whatman catalog number) for drug exposure analysis. Attach to WB12 9243). The remaining blood sample is placed far away to separate the plasma. Cardiac isolation was performed. Plasma Lp(a) concentrations were described in the in vitro Lp(a) aggregate assay. The measurement is performed using sandwich ELISA. The inhibition percentage for each dose group is equal to the average of the vehicle control group. The average Lp(a) level is set to 0% inhibition for determination. Table 3 shows the results of oral administration on the morning of day 3. This shows the results of a dose-response study in which tail hemorrhage samples were taken 8 hours later. Ed 50(50 The calculated effective amount (%) to inhibit % of Lp(a) is determined by the analysis of the minimum effective amount. Table 4 shows the oral (or subcutaneous, if indicated) dosage for the morning of day 3. Single-dose study in which tail hemorrhage samples were collected after hours (or 8 hours if specified). The results shown in Tables 3 and 4 indicate that the compound was effective in plasma in vivo. The compound was shown to be effective in lowering Lp(a) levels, and the compound is effective in lowering Lp(a) plasma concentration. I support the proposition that it can be used to reduce [something]. [Table 3] [Table 4]

Claims

[Claim 1] The following formula: (In the formula, R ｂ is H or CH ３ ; R ｅ is a protecting group, R ｆ is a protecting group, R ｄ These are H, Br, CHO, and NO ２ A compound of (selected from the group consisting of) or a pharmaceutically acceptable salt thereof.

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