PCSK9 antagonist compounds

Abstract

Disclosed are compounds of Formula I or salts thereof, wherein A, B, D, X, R1, R2, and R8 are as defined herein, and wherein the compounds have PCSK9 antagonistic properties. Also described are pharmaceutical formulations comprising the compounds of Formula I or salts thereof, and methods for treating cardiovascular diseases and conditions associated with PCSK9 activity, such as atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, or related cardiovascular diseases and cardiometabolic conditions. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. application No. 62 / 687,913, filed on June 21, 2018, the entire contents of which are incorporated herein by reference. Background Art

[0003] The identification of compounds and / or agents that are effective in treating patients with cardiovascular disease is highly desirable. In clinical trials, reductions in LDL cholesterol levels have been directly correlated with the rate of coronary events; Law et al., 2003 BMJ 326:1423-1427. Modest lifelong reductions in plasma LDL cholesterol levels have been found to be associated with a substantial reduction in the incidence of coronary events; Cohen et al., 2006 N. Engl. J. Med. 354:1264-1272. This is true even in populations with a high prevalence of non-lipid-related cardiovascular risk factors; ibid. Therefore, significant benefits would be derived from the management of LDL cholesterol levels.

[0004] Proprotein convertase subtilisin-kexin type 9 (hereinafter referred to as "PCSK9"), also known as neural apoptosis-regulated convertase 1 ("NARC-1"), is a proteinase K-class subtilase identified as the ninth member of the secretory subtilase family; see Seidah et al., 2003 PNAS 100:928-933. PCSK9 belongs to the mammalian proprotein convertase family of serine proteases and contains an N-terminal signal sequence, a prodomain, a catalytic domain, and a C-terminal domain; see Seidah et al., 2012 Nat. Rev. Drug Discov. 11:367–383. Studies of PCSK9 transcriptional regulation suggest that it is regulated by sterol regulatory element-binding proteins ("SREBPs"), as seen with other genes involved in cholesterol metabolism; Maxwell et al., 2003 J. Lipid Res. 44:2109-2119, and with other genes involved in lipoprotein metabolism; Dubuc et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1454-1459. Statins have been shown to upregulate PCSK9 expression in a manner attributed to the cholesterol-lowering effects of the drugs; ibid. Furthermore, the PCSK9 promoter has been shown to possess two conserved sites involved in cholesterol regulation: the sterol regulatory element and the Sp1 site; ibid.

[0005] When in the endoplasmic reticulum, PCSK9 performs self-cleavage between residues Gln-152 and Ser-153 with its only catalytic activity; see Naureckiene et al., 2003 Arch. Biochem. Biophys. 420: 55–67; Seidah et al., 2003 Proc. Natl. Acad. Sci. USA 100: 928–933. During continuous migration through the trans-Golgi network, the prodomain remains closely associated with the catalytic domain. Maturation via self-cleavage has been shown to be crucial for PCSK9 secretion and subsequent extracellular function (see Benjannet et al., 2012 J. Biol. Chem. 287: 33745–33755). Thus, several lines of evidence suggest that PCSK9, in particular, reduces the amount of hepatic LDLR protein and, therefore, impairs the liver's ability to remove LDL cholesterol from the circulation.

[0006] Adenovirus-mediated overexpression of PCSK9 in mouse livers resulted in accumulation of circulating LDL-C due to a dramatic loss of hepatic LDLR protein, with no effect on LDLR mRNA levels (Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875; Maxwell and Breslow, 2004 PNAS 101:7100-7105; Park et al., 2004 J. Biol. Chem. 279:50630-50638; and Lalanne et al., 2005 J. Lipid Res. 46:1312-1319). The effect of PCSK9 overexpression on elevated circulating LDL-C levels in mice was entirely dependent on LDLR expression, again indicating that LDL-C regulation by PCSK9 is mediated through downregulation of LDLR protein. Consistent with these findings, mice lacking PCSK9 or in which PCSK9 mRNA has been reduced by antisense oligonucleotide inhibitors have higher levels of hepatic LDLR protein and a greater ability to clear circulating LDL-C; Rashid et al., 2005 PNAS 102:5374-5379; and Graham et al., 2007 J. Lipid Res. 48(4):763-767. In addition, reducing PCSK9 content in cultured human hepatocytes by siRNA also resulted in higher LDLR protein levels and increased ability to absorb LDL-C; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875; and Lalanne et al., 2005 J. Lipid Res. 46:1312-1319. Taken together, these data indicate that PCSK9 acts by reducing LDLR protein levels, leading to increased LDL-C.

[0007] A number of mutations in the gene PCSK9 have also been conclusively associated with autosomal dominant hypercholesterolemia ("ADH"), an inherited metabolic disorder characterized by significantly elevated low-density lipoprotein ("LDL") particles in the plasma, which can lead to premature cardiovascular failure; see Abifadel et al., 2003 Nature Genetics 34:154-156; Timms et al., 2004 Hum. Genet. 114:349-353; Leren, 2004 Clin. Genet. 65:419-422. A later published study on the S127R mutation by Abifadel et al. (supra) reported that patients carrying this mutation exhibited higher total cholesterol and apoB100 in the plasma, which was attributed to (1) overproduction of apoB100-containing lipoproteins such as low-density lipoprotein ("LDL"), very low-density lipoprotein ("VLDL"), and intermediate-density lipoprotein ("IDL"), and (2) an associated decrease in the clearance or turnover of these lipoproteins; Ouguerram et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1448-1453.

[0008] Therefore, it is undeniable that PCSK9 plays a role in regulating LDL. Expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and corresponding inhibition or loss of PCSK9 expression is associated with decreased plasma levels of LDL cholesterol. It has been found that reduced LDL cholesterol levels associated with sequence variations in PCSK9 confer protection against coronary heart disease; Cohen, 2006 N. Engl. J. Med. 354: 1264-1272.

[0009] Therefore, the identification of compounds and / or agents that are effective in treating cardiovascular disease is highly desirable, including antagonism of PCSK9 in LDL regulation. However, generally, because PCSK9 circulates in the blood and has a modest binding affinity for cell surface LDL receptors, attempts to exploit this mechanism in treating diseases associated with high serum LDL levels have focused on the use of large biomolecules, such as antibodies. Consequently, there are few publications that reflect the use of small peptides or small molecules to inhibit PCSK9 activity for this purpose, see, for example, Zhang et al., 2014 J. Biol. Chemistry, 289(2):942-955. Furthermore, there are very few compounds that are suitable for formulation into dosage forms that utilize the oral route of administration of such compounds, a route that would be highly desirable for providing treatments for conditions in which modulation of PCSK9 activity can play a role.

[0010] The present invention advances these interests by providing antagonists of PCSK9 that are believed to have utility for inhibiting the activity of PCSK9 and the corresponding roles played by PCSK9 in various pathologies for which administration of PCSK9 antagonists provides treatment. Summary of the Invention

[0011] In one aspect, the present invention provides a compound of formula I:

[0012]

[0013] in:

[0014] X is H, F, Cl or Br;

[0015] R 1 Selected from:

[0016] (a)-H; or

[0017] (b)-(CH2) z -R 14A , where z is 1-6, and R 14A for:

[0018] (i)-H;

[0019] (ii)–NH2;

[0020] (iii)-N + H3;

[0021] (iv)-N + (H3C)3;

[0022] (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0023] (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B , wherein: y12 and y13 are not both 2 and are independently 2 to 4; R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0024] (vii)-NH-C(O)-(CH2) y R 14C, where y = 1 to 6 and R 14C -O-(CH2) za -N + (CH3)3, wherein za is 3 or 4; and

[0025] (viii)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for:

[0026] (ai)–O-(CH2)2-N + (CH3)3;

[0027] (aii)-N + (CH3)3; or

[0028] (aiii) a portion of the following formula:

[0029]

[0030] R 2 Selected from:

[0031] (a)-H; and

[0032] (b)-(CH2) z -R 14A , where z is 1-6, and R 14A Selected from:

[0033] (i)-H;

[0034] (ii)–NH2;

[0035] (iii)-N + H3;

[0036] (iv)-N + (H3C)3;

[0037] (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0038] (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B , wherein: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N+ H3; -N(CH3)2; or -N + (CH3)3;

[0039] (vii)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C -O-(CH2) zb -N + (CH3)3, wherein zb is 3 or 4; and

[0040] (viii)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for:

[0041] (ai)–O-(CH2)2-N + (CH3)3;

[0042] (aii)-N + (CH3)2R 14ca , where R 14ca is –CH3 or –(CH2) 1-4 -OCH3;

[0043] (aiii) a portion of the following formula:

[0044] or

[0045] (aiv) the part of the following formula:

[0046]

[0047] where R 14Cb and R 14Cc is 1 to 4; or

[0048] R 1 With R 2 can be combined together to form parts of the formula:

[0049] in:

[0050] G 1 、R G1a and R G1b The definition is as follows:

[0051] (a)G 1 is the linker part of the following formula:

[0052]

[0053] where n q1 1 to 6, m q1 is 0, 1 or 2, and n is selected at the same time q1 and m q1 such that the length of the linker moiety it defines does not exceed a total of 8 carbon atoms and / or oxygen atoms, including the chain of carbon atoms in the chain forming the carbonyl moiety;

[0054] R G1a is selected from: (i) -H; and (ii) an alkyl group of up to 4 carbon atoms; and

[0055] R G1b Selected from:

[0056] (i) the part of the formula:

[0057] and

[0058] (ii) a portion of the formula:

[0059] or

[0060] (b)G 1 is the connector portion of the following formula:

[0061]

[0062] where n q2 is 0, 1, or 2, m q2 1 to 6, and select n q2 and m q2 such that the length of the linker moiety it defines does not exceed a total of 8 carbon atoms and / or oxygen atoms, including the chain of carbon atoms in the chain forming the carbonyl moiety;

[0063] R G1a Selected from:

[0064] (i) the part of the formula:

[0065] and

[0066] (ii) a portion of the formula:

[0067] and

[0068] R G1b is selected from: (i) -H; and (ii) an alkyl group of up to 4 carbon atoms;

[0069] R 8 is -CH3 or a portion of the formula:

[0070]

[0071] where R 8a is -H, or a linear, branched or cyclic alkyl group of up to four carbon atoms;

[0072] A is selected from:

[0073] (a) The part of the following formula:

[0074]

[0075] (b)-CH2-(CH2) y -CH2-, wherein y is 1 to 6;

[0076] (c) the part of the formula:

[0077] Among them A b1 for:

[0078] (i) the part of the formula:

[0079] where x is 1 to 6; or

[0080] (ii) a portion of the formula:

[0081] Where y is 1 to 5;

[0082] (d) a moiety of the formula: -CH2-(CH2) m -O-(CH2) n -, wherein m=1 to 5, and n=0 or 1 to 4;

[0083] B is:

[0084] (a) key;

[0085] (b)–(CH2) 1-4 ;or

[0086] (c) the part of the formula:

[0087]

[0088] D is:

[0089] (a) The part of the following formula:

[0090]

[0091] Where E is -CH2- or -(CH2) 2-4 -O-, and A and B are as defined above;

[0092] (b) The part of the following formula:

[0093]

[0094] wherein A and B are as defined above;

[0095] (c) the part of the formula:

[0096]

[0097] where n a is 1, 2 or 3, m a is 2, 3, or 4, and n a +m a is ≥3, and wherein A and B are as defined above;

[0098] (d) the part of the formula:

[0099]

[0100] Among them, R 34b is -H or a linear, branched or cyclic alkyl group of up to four carbon atoms, and A and B are as defined above,

[0101] or any pharmaceutically acceptable salt thereof.

[0102] In another embodiment, the present invention provides a compound of formula I, wherein X is F, or any pharmaceutically acceptable salt thereof. In some embodiments, D is preferably a moiety of the following formula:

[0103]

[0104] wherein E is -CH2- or -(CH2)2-O-, and A and B are as defined herein.

[0105] In some embodiments, D is preferably a moiety of the formula:

[0106]

[0107] wherein E is -CH2- or -(CH2)2-O-, and A and B are as defined herein.

[0108] In some embodiments, D is preferably a moiety of the formula:

[0109]

[0110] wherein A and B are as defined herein.

[0111] In some embodiments, D is preferably a moiety of the formula:

[0112]

[0113] wherein A and B are as defined herein.

[0114] In some embodiments, D is preferably a moiety of the formula:

[0115]

[0116] wherein A and B are as defined herein.

[0117] In some embodiments, D is preferably a moiety of the formula:

[0118]

[0119] wherein A and B are as defined herein.

[0120] In some embodiments, D is preferably a moiety of the formula:

[0121]

[0122] wherein A and B are as defined herein.

[0123] In some embodiments, D is preferably a moiety of the formula:

[0124]

[0125] wherein A and B are as defined herein.

[0126] In which R 1 and R 2 Together with the peptide ring to which it is connected, thereby forming a ring structure, in some embodiments, R 1 and R 2 Preferably forming part of the following structure:

[0127]

[0128] In one embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention (eg, a compound of Formula I) and at least one pharmaceutical excipient, preferably a composition for oral administration.

[0129] In one aspect, the present invention provides a method for antagonizing PCSK9 in providing therapy for a disease state associated with PCSK9 activity, such as atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, or related cardiovascular diseases and cardiometabolic conditions, by administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I or a salt thereof, preferably in the form of a pharmaceutical composition. DETAILED DESCRIPTION

[0130] In the following description, known structural representations are employed and include conventional stereochemical notations for certain asymmetric carbon centers.

[0131] Thus, the structural representations of the compounds of the present invention include conventional stereochemical notation for some of the asymmetric carbon centers shown in the example compounds. Thus, in such cases, a solid black "wedge" bond represents a bond protruding from the plane of the reproducing medium, a "hashed wedge" bond represents a bond descending in the plane of the reproducing medium, and a "wavy" line to which a double-bonded carbon is attached indicates that both cis and trans orientations are possible. As is conventional, a plain solid line represents all spatial configurations of the depicted bond. Thus, when no specific stereochemical notation is provided, this representation encompasses all stereochemical and spatial orientations of a structural feature.

[0132] As shown in the examples of the present invention and as mentioned above, specific asymmetric carbon centers are structurally represented using a combination of known "solid wedges" and "hashed wedges." In most cases, the absolute configuration of the example compounds has not yet been determined, but has been assigned by simulation to specific example compounds with known stereochemical configurations (determined by X-ray crystallography) prepared using the same or similar reaction conditions and starting reagents and separated under the same chromatographic conditions. Therefore, unless otherwise indicated in the presented data, the specific assignment of configurations presented in the structures herein is intended to identify that a specific compound prepared has an excess of one specific stereoisomer and is not necessarily presented herein as a statement of the absolute determination of the stereochemical structure of the compound.

[0133] It will be appreciated that when an isomeric mixture is obtained, the preparation of the individual stereoisomers can be carried out as needed in the form of a significant percentage of enantiomeric excess by separating the mixture using conventional methods, such as chromatography or crystallization, or by using stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally, derivatization can be performed before separating the stereoisomers. Separation of the stereoisomer mixture can be carried out at an intermediate step during the synthesis of the compound of formula I or it can be carried out on the final racemic product.

[0134] Where indicated herein, absolute stereochemistry is determined by X-ray crystallization of crystalline products or crystalline intermediates, which are optionally derivatized with reagents containing stereocenters of known configuration. Unless a specific isomer, salt, solvate (including hydrate) or solvated salt of such racemates, enantiomers or diastereomers is indicated, the invention includes all such isomers and salts, solvates (including hydrates) and solvated salts of such racemates, enantiomers, diastereomers and mixtures thereof.

[0135] The present invention also includes isotopically labeled compounds of the invention that are structurally identical to those described herein, but in which a statistically significant percentage of one or more atoms in that form of the compound are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number of the most abundant isotope normally found in nature, thereby altering the naturally occurring abundance of that isotope present in the compounds of the invention. The present invention is intended to include all suitable isotopic variations of the compounds of Formula I.

[0136] Examples of isotopes that may be preferably incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine, fluorine and chlorine, such as but not limited to: 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F and 36 Cl, 123 I and 125 1. It will be appreciated that other isotopes may also be incorporated by known means.

[0137] In particular, certain isotopically labeled compounds of the present invention (e.g., 3 H. 11 C and 14 C-labeled ones) are believed to be particularly useful in compound and / or substrate tissue distribution assays using a variety of known techniques. In addition, the compounds of the present invention encompass isotopic substitutions, which include different isotopic forms of hydrogen (H), including protium ( 1 H) and deuterium ( 2 H or D). Protium is the predominant hydrogen isotope found in nature. Deuterium enrichment can provide certain therapeutic advantages, such as increased in vivo half-life or reduced dosage requirements, or can provide compounds useful as standards for characterizing biological samples. Isotopically enriched compounds within Formula I can be prepared without undue experimentation by known techniques familiar to those skilled in the art, or by methods analogous to those described in the schemes and examples herein, using appropriate isotopically enriched reagents and / or intermediates.

[0138] Where a wavy line terminates a conventional bond (as opposed to connecting two atoms within a structure), it indicates the point of attachment to the structure, for example:

[0139]

[0140] Indicates that the sec-butyl moiety is attached via a methylene group via a bond terminated by a wavy line. When letter symbols are used to depict substituent moieties, a dash is used to indicate the point of attachment to the indicated substrate, for example: -CH2-C(O)-CH2Cl indicates that the chloroacetyl moiety is attached via the methylene portion of the moiety.

[0141] Unless otherwise specified at a definition point, when any variable (e.g., n, R a 、R b When , ... 1 、R A The choice of combinations of substituents and / or variables will be chosen consistent with well-known principles of chemical structure connectivity and stability, and combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0142] A "stable" compound is one that can be prepared and isolated, and whose structure and properties remain or can be caused to remain substantially unchanged for a period of time sufficient to allow the compound to be used for the purposes described herein (e.g., therapeutic administration to a subject). The compounds of the present invention are limited to stable compounds encompassed by Formula I.

[0143] When any variable or moiety is expressed in the form of a range, such as (-CH2-) 1-4 , which includes the two extreme values ​​of the specified range (i.e., 1 and 4 in the example) and all integer values ​​in between (i.e., 2 and 3 in the example).

[0144] Unless otherwise specified at the point of use, the term "halogen" includes fluorine, chlorine, bromine and iodine. As used herein, the term "subject" (or "patient") refers to an animal, preferably a mammal, and particularly a human or non-human animal including livestock and domestic animals, including but not limited to cattle, horses, sheep, pigs, goats, rabbits, cats, dogs and other mammals in need of treatment. In some embodiments, the subject is preferably a human. As used herein, the term "administering" and variants thereof (e.g., "administering" a compound) with reference to a compound of Formula I means providing the compound or a pharmaceutically acceptable salt thereof to a subject in need of treatment.

[0145] As mentioned above, in one aspect, the present invention includes providing a compound of Formula I or a pharmaceutically acceptable salt thereof having the property of antagonizing PCSK9 function.

[0146] In one embodiment, the compound of Formula I has the structure of Formula IA:

[0147]

[0148] in:

[0149] R 1 Selected from:

[0150] (a)-H; or

[0151] (b)-(CH2) z -R 14A , where z is 1-6, and R 14A for:

[0152] (i)-H;

[0153] (ii)–NH2;

[0154] (iii)-N + H3;

[0155] (iv)-N + (H3C)3;

[0156] (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0157] (vi)-NH-C(O)-[(CH2) y12 -O-] 1-4 -(CH2) y13 R 14B , preferably -NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B , wherein: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0158] (vii)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C -O-(CH2) za -N + (CH3)3, wherein za is 3 or 4; and

[0159] (viii)-NH-C(O)-(CH2) y R 14C, where y = 1 to 6 and R 14C for:

[0160] (ai)–O-(CH2)2-N + (CH3)3;

[0161] (aii)-N + (CH3)3; or

[0162] (aiii) a portion of the following formula:

[0163]

[0164] R 2 Selected from:

[0165] (a)-H; and

[0166] (b)-(CH2) z -R 14A , where z is 1-6, and R 14A Selected from:

[0167] (i)-H;

[0168] (ii)–NH2;

[0169] (iii)-N + H3;

[0170] (iv)-N + (H3C)3;

[0171] (v)-NH-C(O)-[(CH2)2-O-] 1-4 -(CH2)2R 14B , preferably -NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0172] (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B , wherein: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0173] (vii)-NH-C(O)-(CH2)y R 14C , where y = 1 to 6 and R 14C -O-(CH2) zb -N + (CH3)3, wherein zb is 3 or 4; and

[0174] (viii)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for:

[0175] (ai)–O-(CH2)2-N + (CH3)3;

[0176] (aii)-N + (CH3)2R 14ca , where R 14ca is –CH3 or –(CH2) 1-4 -OCH3;

[0177] (aiii) the part of the formula:

[0178] or

[0179] (aiv) the part of the formula:

[0180]

[0181] where Y 14Cb and Y 14Cc is 1 to 4; or

[0182] R 1 With R 2 can be combined together to form parts of the formula:

[0183]

[0184] in:

[0185] G 1 、R G1a and R G1b The definition is as follows:

[0186] (a)G 1 is the connector portion of the following formula:

[0187]

[0188] where n q1 1 to 6, m q1 is 0, 1, or 2, and n is selected at the same time q1 and m q1such that the length of the linker moiety it defines does not exceed a total of 8 carbon atoms and / or oxygen atoms, the linker moiety comprising the chain including the carbon atoms in the chain forming the carbonyl moiety;

[0189] R G1a is selected from: (i) -H; and (ii) an alkyl group of up to 4 carbon atoms; and

[0190] R G1b Selected from:

[0191] (i) the part of the formula:

[0192] and

[0193] (ii) a portion of the formula:

[0194] or

[0195] (b)G 1 is the connector portion of the following formula:

[0196]

[0197] where n q2 is 0, 1, or 2, m q2 1 to 6, and select n q2 and m q2 such that the length of the linker moiety it defines does not exceed a total of 8 carbon atoms and / or oxygen atoms, the linker moiety comprising the chain including the carbon atoms in the chain forming the carbonyl moiety;

[0198] R G1a Selected from:

[0199] (i) the part of the formula:

[0200] and

[0201] (ii) a portion of the formula: and

[0202] R G1b is selected from: (i) -H; and (ii) an alkyl group of up to 4 carbon atoms;

[0203] R 8 is –CH3 or a portion of the formula:

[0204]

[0205] where R 8a is -H, or a linear, branched or cyclic alkyl group of up to four carbon atoms;

[0206] A is selected from:

[0207] (a) The part of the following formula:

[0208]

[0209] (b)-CH 2- (CH2) y -CH2-, wherein y is 1 to 6;

[0210] (c) the part of the formula: Among them A b1 for:

[0211] (i) the part of the formula:

[0212]

[0213] where x is 1 to 6; or

[0214] (ii) a portion of the formula:

[0215]

[0216] Where y is 1 to 5;

[0217] (d) a moiety of the formula: -CH2-(CH2) m -O-(CH2) n -, wherein m=1 to 5, and n=0 or 1 to 4;

[0218] B is:

[0219] (a) key;

[0220] (b)–(CH2) 1-4 ;or

[0221] (c) the part of the formula:

[0222]

[0223] D is:

[0224] (a) The part of the following formula:

[0225]

[0226] Where E is -CH2- or -(CH2) 2-4 -O-, and A and B are as defined above;

[0227] (b) The part of the following formula:

[0228]

[0229] wherein A and B are as defined above;

[0230] (c) the part of the formula:

[0231]

[0232] where n a is 1, 2 or 3, m a is 2, 3, or 4, and n a +m a is at least 3, and wherein A and B are as defined above;

[0233] (d) the part of the formula:

[0234]

[0235] Among them, R 34b is -H or a linear, branched or cyclic alkyl group of up to four carbon atoms, and A and B are as defined above,

[0236] or any pharmaceutically acceptable salt thereof.

[0237] In one embodiment of the compound of Formula IA, R 1 -(CH2) z -R 14A , where z is 1-6, and R 14A for:

[0238] (i)-H;

[0239] (ii)–NH2;

[0240] (iii)-N + H3; or

[0241] (iv)-N + (H3C)3;

[0242] R 2 -(CH2) z -R 14A , where z is 1-6, and R 14A Selected from:

[0243] (i)-H;

[0244] (ii)–NH2;

[0245] (iii) -NH-C(O)-[(CH2)2-O-] 1-4 -(CH2)2R 14B , preferably -NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N+ H3; -N(CH3)2; or -N + (CH3)3;

[0246] (iv)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B , wherein: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0247] (v)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C -O-(CH2) zb -N + (CH3)3, wherein zb is 3 or 4; and

[0248] (vi)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for:

[0249] (ai)–O-(CH2)2-N + (CH3)3;

[0250] (aii)-N + (CH3)2R 14ca , where R 14ca is –CH3 or –(CH2) 1-4 -OCH3;

[0251] (aiii) the part of the formula:

[0252] or

[0253] (aiv) the part of the formula:

[0254]

[0255] where Y 14Cb and Y 14Cc is 1 to 4; or

[0256] R 8 is -CH3 or a portion of the formula:

[0257]

[0258] where R8a is -H, or a straight-chain, branched or cyclic alkyl group of up to four carbon atoms;

[0259] A is selected from:

[0260] (a) The part of the following formula:

[0261]

[0262] (b)-CH2-(CH2) y -CH2-, wherein y is 1 to 6;

[0263] (c) the part of the formula: Among them A b1 for:

[0264] (i) the part of the formula:

[0265]

[0266] where x is 1 to 6; or

[0267] (ii) a portion of the formula:

[0268]

[0269] where y is 1 to 5; and

[0270] (d) a moiety of the formula: -CH2-(CH2) m -O-(CH2) n -, wherein m=1 to 5, and n=0 or 1 to 4;

[0271] B is:

[0272] (a)–(CH2) 1-4 ;or

[0273] (b) The part of the following formula:

[0274]

[0275] D is:

[0276] (a) The part of the following formula:

[0277]

[0278] Where E is -CH2- or -(CH2) 2-4 -O-, and A and B are as defined above;

[0279] (b) The part of the following formula:

[0280]

[0281] wherein A and B are as defined above; or

[0282] (c) the part of the formula:

[0283]

[0284] Among them, R 34b is -H or a linear, branched or cyclic alkyl group of up to four carbon atoms, and A and B are as defined above,

[0285] or any pharmaceutically acceptable salt thereof.

[0286] In one embodiment of the compound of formula IA, D is a moiety of the formula:

[0287]

[0288] wherein E is -CH2- or -(CH2)2-O-, and A and B are as defined above in Formula IA.

[0289] In one embodiment of the compound of formula IA, A is:

[0290] (a)–(CH2)6;

[0291] (b) The part of the following formula:

[0292]

[0293] where x is 1 to 3; or

[0294] (c) the part of the formula:

[0295]

[0296] In another embodiment of the compound of Formula IA, R 2 for:

[0297] (a)-(CH2) z -R 14A , where z is 1-6, and R 14A for:

[0298] (a)-H;

[0299] (b)–CH3;

[0300] (c)–NH2;

[0301] (d)-N + H3;

[0302] (e)-N + (H3C)3;

[0303] (f)-NH-C(O)-[(CH2) 2-4 -O-] 2-4 -(CH2) 2-4 R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0304] (g)-NH-C(O)-[(CH2) y R 14C , where y = 1 to 6 and R 14C for:

[0305] (ai)–O-(CH2) 2-4 -N + (CH3)3;

[0306] (aii)-N + (CH3)3; or

[0307] (aiii) a portion of the following formula:

[0308] or

[0309] (b) The part of the following formula

[0310]

[0311] In another embodiment of the compound of Formula IA, R 1 Selected from:

[0312] (a)-H;

[0313] (b)-(CH2) z -R 14A , where z is 1-6, and R 14A for:

[0314] (i)-H;

[0315] (ii)-N + H3; or

[0316] (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2-N + (CH3)3.

[0317] In another embodiment of the compound of formula IA, A is -CH2-(CH2) y -CH2-, wherein y is 3 to 5. In another embodiment, A is -(CH2)6.

[0318] In another embodiment of the compounds of formula IA, B is a moiety of the formula:

[0319]

[0320] In another embodiment of the compound of Formula IA, R 1 -(CH2) z -R 14A , where z is 1-6, and R 14A In another embodiment of the compound of formula IA, R 1 -(CH2) z -R 14A , where z is 1 and R 14A is -H.

[0321] In another embodiment of the compound of Formula IA, R 2 -(CH2) z -R 14A , where z is 1-6, and R 14A -NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C -N + (CH3)2R 14ca , where R 14ca is -CH3.

[0322] In another embodiment of the compound of Formula IA, R 8 is the part of the following formula:

[0323]

[0324] where R 8a is -H or a straight chain alkyl group of up to four carbon atoms. 8 is the part of the following formula:

[0325]

[0326] where R 8b It is -H, -CH3 or -C(CH3)3.

[0327] In some embodiments, the compound of Formula I preferably has the structure of Formula II or Formula IIA, or a pharmaceutically acceptable salt thereof:

[0328]

[0329]

[0330] Among them, A, R 1 and R2 As defined in Formula IA above, and B 1 -(CH2) 0-2 , and D 1 Selected from:

[0331] a) The part of the following formula:

[0332] and

[0333] b) the part of the following formula:

[0334]

[0335] In some embodiments of Formula II or Formula IIA, D 1 Preferably it is a portion of the formula: In some embodiments of Formula II or Formula IIA, D 1 Preferably it is a portion of the formula: In some embodiments of Formula II or Formula IIA, D 1 Preferably it is a portion of the formula: In some embodiments of Formula II or Formula IIA, D 1 Preferably it is a portion of the formula:

[0336] In some embodiments, the compound of formula I is preferably a compound of formula III:

[0337]

[0338] Among them, A, R 1 and R 2 As defined in Formula IA above, and D 2 is the part of the following formula:

[0339]

[0340] In some embodiments of Formula III, D 2 Preferably it is a portion of the formula: In some embodiments of Formula III, D 2 Preferably it is a portion of the formula: In some embodiments of Formula III, D 2 Preferably it is a portion of the formula:

[0341] In some embodiments, the compound of formula I is preferably a compound of formula IV:

[0342]

[0343] Among them, A, R 1 and R2 As defined above in Formula IA.

[0344] In some embodiments, the compound of formula I is a compound of formula V:

[0345]

[0346] in

[0347] A, B, R 1 and R 2 As defined above in Formula IA; and

[0348] D 2 for:

[0349] (a) The part of the following formula:

[0350]

[0351] (b) The part of the following formula:

[0352]

[0353] (c) the part of the formula:

[0354] or

[0355] (d) the part of the formula:

[0356]

[0357] In some embodiments of Formula I, Formula IA, Formula II, Formula IIA, Formula III, Formula IV, or Formula V, A is preferably a moiety of the formula: —(CH 2 ) ya , wherein ya is 4 to 6. In some embodiments of Formula I, Formula IA, Formula II or Formula IIA, A is preferably a moiety of the formula: -CH2-(CH2) ma -O-(CH2) na -, wherein ma is 2 or 3 and na is 0 or 1. In some embodiments of Formula III, Formula IV or Formula V, A is preferably a moiety of the formula: -CH2-(CH2) ma -O-(CH2) na -, wherein ma is 2 or 4 and na is 0, 1 or 2. In some embodiments of Formula I, Formula IA, Formula II, Formula IIA, Formula III, Formula IV or Formula V, A is preferably a moiety of the formula:

[0358]

[0359] wherein yb is 1 to 3. In some embodiments of Formula I, Formula IA, Formula II, Formula IIA, Formula III, Formula IV, or Formula V, A is preferably a moiety of the formula:

[0360]

[0361] Also provided herein as compounds of formula I are compounds Ex-1, Ex-2, Ex-3, Ex-4, Ex-5, Ex-6, Ex-7, Ex-8, Ex-9, Ex-10, Ex-11, Ex-12, Ex-13, Ex-14, Ex-15, Ex-16, Ex-17, Ex-18, Ex-19, Ex-20, Ex-21, Ex-22, Ex-23, Ex-24, Ex-25, Ex-26, Ex-27, Ex-28, Ex-29, Ex-30, Ex-31, Ex-32, Ex-33, Ex-34, Ex-35, Ex-36, Ex-37, Ex-38, Ex-39, Ex-40, Ex-41, Ex-42, Ex-43, Ex-44, Ex-45, Ex-46, Ex-47, Ex-48, Ex-49, Ex-50, Ex-51, Ex-52, Ex-53, Ex-54, Ex-55, Ex-56, Ex-57, Ex-58, Ex-59, Ex-60, Ex-61, Ex-62, Ex-63, Ex-64, Ex-65, Ex-66, Ex-67, Ex-68, Ex-69, Ex-70, Ex-71, Ex-72, Ex-73, Ex-74, Ex-75, Ex-76, Ex-77, Ex-78, Ex-79, Ex-80, Ex-81, Ex-82, Ex-83, Ex-84, Ex-85, Ex-86, Ex-87 -27, Ex-28, Ex-29, Ex-31, Ex-35, Ex-36, Ex-38, Ex-39, Ex-40, Ex-41, Ex-44, Ex-47, Ex-48, Ex-49, Ex-50, Ex-51, Ex-52, Ex-53, Ex-54, Ex-55, Ex-56, Ex-57, Ex-58, Ex-59, Ex-60 and Ex-61, or any pharmaceutically acceptable salt thereof. The compounds disclosed in Table 1 are also referred to herein as "compounds of the present invention."

[0362] Table 1

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377] Among them A - It is a pharmaceutically acceptable anion.

[0378] As used herein, the term "salt" and its use in the phrase "pharmaceutically acceptable salt" include any of the following: acid salts formed with inorganic and / or organic acids, base salts formed with inorganic and / or organic bases, zwitterions, and quaternary ammonium complexes. The salts of the compounds of the present invention can be formed by methods known to those of ordinary skill in the art, for example, by reacting the compounds of the present invention with a certain amount (e.g., a certain equivalent amount) of an acid or base in a medium such as a salt precipitate or an aqueous medium, followed by lyophilization.

[0379] The compounds of the present invention contain a tricoordinate nitrogen atom, such as a primary, secondary, or tertiary amino moiety, wherein, as is known, the lone electron pair present on the nitrogen atom can be protonated with an appropriate acid or alkylated with an appropriate reagent (e.g., an alkyl bromide) under appropriate reaction conditions to provide a tetracoordinate charged nitrogen stabilized by an anion (e.g., a halide ion or a conjugate base) generated in the process. Thus, the compounds of the present invention can be prepared in the form of a free base or isolated in the form of a quaternary complex or salt complex. In some cases, it is possible to form a zwitterionic complex in which a suitable acidic proton is close to the basic nitrogen. When the term is used herein, salts of the compounds of the present invention are included within the scope of the compounds of the present invention described herein, whether they are acid salts formed with inorganic and / or organic acids, base salts formed with inorganic and / or organic bases, salts formed with zwitterionic properties (e.g., where the compound contains both a basic moiety, such as, but not limited to, a nitrogen atom, such as, an amine, pyridine, or imidazole; and an acidic moiety, such as, but not limited to, a carboxylic acid), and quaternary ammonium complexes.

[0380] Thus, whether in free base form, salt form, zwitterion formation or quaternary ammonium form, the structural representation of the compounds of the present invention also includes all other forms of such compounds discussed above. Thus, one aspect of the present invention is to provide the compounds of the present invention in the form of pharmaceutically acceptable salts, zwitterionic complexes or quaternary ammonium complexes. Those skilled in the art will recognize those instances in which the compounds of the present invention can form such complexes, including instances in which the four-coordinate nitrogen can be quaternized or protonated and the charged nitrogen form is stabilized by an associated anion. The term "pharmaceutically acceptable salt" refers to salts (including quaternary ammonium complexes and inner salts such as zwitterionic complexes) that possess effects similar to or greater than the free base form of the compound and that are not biologically or otherwise undesirable (e.g., neither toxic nor otherwise harmful to the recipient thereof).

[0381] The formation of pharmaceutically useful salts from basic (or acidic) drug compounds is discussed, for example, by S. Berge et al., Journal of Pharmaceutical Sciences (1977) 66(1): 1-19; P. Gould, International J. of Pharmaceutics (1986) 33: 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; in The Orange Book (Food & Drug Administration, Washington, DC, on its website); and P. Heinrich Stahl, Camille G. Wermuth (eds.), Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (2002) Int'l. Union of Pure and Applied Chemistry, pp. 330-331. The disclosures of these are incorporated herein by reference.

[0382] The present invention encompasses both the free base form and all available salts of the compounds of the present invention, including salts generally recognized as safe for use in the preparation of pharmaceutical formulations, as well as those currently within the purview of those skilled in the art that can be formed and subsequently classified as "generally recognized as safe" for use in the preparation of pharmaceutical formulations, referred to herein as "pharmaceutically acceptable salts." It will be appreciated that the free base compounds can be prepared by controlling the conditions for isolation of the compounds during synthesis or by neutralization and ion exchange from the salt forms of the compounds of the present invention.

[0383] Examples of pharmaceutically acceptable acid salts include, but are not limited to, acetate (including trifluoroacetate), adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gluconoheptate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, methylsulfate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate (such as those mentioned herein), tartrate, thiocyanate, toluenesulfonate (also known as tosylate), undecanoate, and the like.

[0384] Examples of pharmaceutically acceptable base salts include, but are not limited to, ammonium salts, alkali metal salts (such as sodium, lithium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts, aluminum salts, zinc salts), salts with organic bases (e.g., organic amines) (such as benzathines, diethylamine, dicyclohexylamine, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butylamine, piperazine, phenylcyclohexyl-amine, choline, tromethamine), salts with amino acids (such as arginine, lysine), and the like. Basic nitrogen-containing groups can be converted to ammonium ions or quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfate), long chain halides (e.g., decyl, dodecyl, tetradecyl, and octadecyl chlorides, bromides, and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and other agents.

[0385] The term "pharmaceutically acceptable anion" refers to anions that are suitable for forming pharmaceutically acceptable salts.

[0386] Other examples of pharmaceutically acceptable salts that may be used with the present invention include, but are not limited to, fluoride, chloride, bromide, and iodide.

[0387] Generally, salts of the compounds are intended to be pharmaceutically acceptable salts within the scope of the present invention.

[0388] The terms "purified," "in purified form," or "in isolated and purified form" of a compound refer to the physical state of the compound after isolation from a synthetic method or natural source, or a combination thereof. Thus, the terms "purified," "in purified form," or "in isolated and purified form" of a compound refer to the physical state of the compound after obtaining from a purification method or a method described herein or known to those skilled in the art and characterized by standard analytical techniques described herein or known to those skilled in the art to a sufficient degree of purity. The compounds of the present invention include any form of the compound, including isolated and purified forms obtained in an in situ reaction mixture as well as by conventional techniques. Also included are polymorphic forms of the compounds of the present invention, solvates, and prodrugs thereof.

[0389] Certain compounds of the present invention may exist in different tautomeric forms, such as, but not limited to, keto / enol tautomeric forms, imine-enamine tautomeric forms, and heteroaromatic forms, such as, for example:

[0390]

[0391] In the same manner, unless otherwise indicated, any structural representation presenting a tautomeric form of a compound exhibiting tautomerism is intended to include all such tautomeric forms of the compound. Thus, when a compound of the present invention, its salt, and its solvate and prodrug can exist in different tautomeric forms or be in equilibrium among such forms, all such forms of the compound are encompassed and included within the scope of the present invention.

[0392] In another aspect, the present invention provides pharmaceutical compositions comprising one or more compounds of the present invention. As used herein, the term "pharmaceutical composition" comprises at least one pharmaceutically active compound and at least one excipient, and is intended to encompass both the combination of the specified ingredients in the specified amounts and any product resulting, directly or indirectly, from the combination of the specified ingredients in the specified amounts.

[0393] It will be understood by those of ordinary skill in the art that an excipient is any ingredient that adapts a composition to a specific route of administration or helps process the composition into a dosage form without itself exerting an active drug effect. Typically, depending on the route of administration and the properties of the active agent being administered, the composition comprises more than one excipient. Examples of excipients that impart properties to the composition that make it easier to handle or process include, but are not limited to, lubricants or compression aids in powdered medicaments intended for tableting and emulsion stabilizers in the composition (wherein the active agent is present in the form of an emulsion). Examples of excipients that adapt the composition to the desired route of administration are, for example, but not limited to, absorption enhancers that promote absorption from the gastrointestinal tract for oral administration; penetration enhancers, such as those used for adhesive skin "patches" or compositions for buccal administration, for transdermal or transmucosal administration.

[0394] Although functional excipients are implemented in the form of compositions, excipients are collectively referred to herein as "carriers." Typically, a formulation may contain up to about 95% active ingredient and the balance carrier, although formulations may be prepared in varying ratios. In general, an acceptable pharmaceutical composition contains an active agent at a suitable concentration to provide an effective amount of the PCSK9 antagonist in a single dosage form of an acceptable volume based on the route of administration, such that the pharmaceutical composition can provide therapeutic serum levels of the active agent in a subject to whom the composition is administered for an acceptable period of time, and the composition will maintain biological activity during storage within an acceptable temperature range for an acceptable period of time.

[0395] As used herein, a pharmaceutical composition refers to both the bulk composition, ie, the formulated material that has not yet been formed into individual dosage units for administration, and the composition contained within the individual dosage units.

[0396] While the compositions of the present invention may be employed in bulk form, it will be appreciated that for most applications the compositions will be incorporated into dosage forms providing individual units suitable for administration to a patient, each dosage form containing an amount of the selected composition containing an effective amount of the one or more compounds of Formula I. Examples of suitable dosage forms include, but are not limited to, dosage forms suitable for: (i) oral administration, such as liquid, gel, powder, solid or semisolid pharmaceutical compositions that are loaded into capsules or compressed into tablets and may further comprise one or more coatings that modify their release characteristics (e.g., coatings that impart delayed release) or formulations having delayed release characteristics; (ii) dosage forms suitable for administration through the tissues of the oral cavity, such as fast-dissolving tablets, lozenges, solutions, gels, sachets or needle arrays suitable for providing intramucosal administration; (iii) dosage forms suitable for administration via the nasal mucosa or upper respiratory tract cavities, such as solutions, suspensions or emulsion formulations for dispersion in the nose or respiratory tract; (iv) dosage forms suitable for transdermal administration, such as patches, creams or gels; (v) dosage forms suitable for intradermal administration, such as microneedle arrays. (vi) a dosage form suitable for intravenous (IV) infusion, e.g., over an extended period of time using an IV infusion pump; (vii) a dosage form suitable for intramuscular administration (IM), e.g., an injectable solution or suspension, which may be suitable for forming a reservoir with delayed release properties; (viii) a dosage form suitable for intravenous (IV) drip administration, e.g., a solution or suspension, e.g., as an IV solution or concentrate to be injected into a saline IV bag; (ix) a dosage form suitable for subcutaneous administration, including administration over an extended period of time by implantation of a rod or other device that diffuses the compound into the surrounding tissue and thereby provides sustained serum therapeutic levels; or (x) a dosage form suitable for delivery via the rectal or vaginal mucosa, e.g., a suppository.

[0397] The pharmaceutical composition can be solid, semi-solid or liquid. Solid, semi-solid and liquid form preparations are applicable to a variety of modes of administration, and examples of the preparations include but are not limited to powders, dispersible particles, mini-tablets, beads that can be used for tableting, encapsulation or direct application. In addition, liquid form preparations include but are not limited to solutions, suspensions and emulsions, which preparations can be used, for example but not exclusively, in the form of preparations intended for ingestion, inhalation or intravenous administration (IV) (such as but not limited to via IV drip or infusion pump, intramuscular injection (IM) administration (such as through extended duration release of the drug group (bolus)), direct IV injection) or suitable for subcutaneous administration route.

[0398] Other routes of administration that may be considered include intranasal administration or administration to some other mucosal membrane. Formulations prepared for various mucosal administrations may also include additional components to make them suitable for such administration, such as viscosity modifiers.

[0399] Although in some embodiments, compositions suitable for solid oral dosage forms (such as tablets or fast-melt oral dissolving preparations) are preferred routes of administration for the compounds of the present invention or their salts, the compositions of the present invention can be formulated to be administered via other approaches mentioned above. Examples include, for example, aerosol formulations suitable for administration via inhalation or via nasal mucosa, which may include solutions and solids in powder form, which may be combined with pharmaceutically acceptable propellants such as inert compressed gases (such as nitrogen). Furthermore, it is intended to be immediately converted into a solid form preparation such as a suspension or solution for oral or parenteral administration before use. Examples of such solid forms include, but are not limited to, freeze-dried formulations and liquid preparations adsorbed into a solid absorbent medium.

[0400] For example, the compounds of the present invention can also be delivered transdermally or transmucosally, for example, in liquid, suppository, cream, foam, gel or fast dissolving solid form. It should be understood that transdermal compositions can also take the form of creams, emulsions, sprays and / or lotions and can be provided in unit dosage form as any transdermal patch known in the art, such as a patch incorporating a matrix comprising the pharmaceutically active compound or a reservoir comprising a pharmaceutically active compound in solid or liquid form.

[0401] Examples of pharmaceutically acceptable carriers and methods of making the various compositions mentioned above can be found in A. Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., (2000), Lippincott Williams & Wilkins, Baltimore, MD. Other examples of solutions to formulation problems can be found in the following publications: Pharmaceutical compositions can be formulated by a number of strategies known in the art, see, for example, McGoff and Scher, 2000 Solution Formulation of Proteins / Peptides:—McNally, EJ, ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions.—Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Taylor and Francis; pp. 145-177; Akers et al., 2002, Pharm. Biotechnol. 14:47-127.

[0402] In another aspect, the present invention provides methods of antagonizing PCSK9 function using the PCSK9-specific antagonist compounds described herein; the methods are further described below. The term "antagonize" as used throughout this application refers to a substance that provides an effect that antagonizes PCSK9 in the affected tissue, inhibits, counteracts, neutralizes, or reduces one or more functions of PCSK9 in the affected tissue. Inhibition or antagonism of one or more of the functional properties associated with PCSK9 can be readily determined according to methods known in the art (see, for example, Barak and Webb, 1981 J. Cell Biol. 90: 595-604; Stephan & Yurachek, 1993 J. Lipid Res. 34: 325-330; and McNamara et al., 2006 Clinica Chimica Acta 369: 158-167) and those described herein. Inhibition or antagonism will achieve a reduction in PCSK9 activity relative to that seen in the absence of the antagonist or, for example, relative to the activity observed in the presence of a control antagonist with irrelevant specificity. Preferably, the PCSK9-specific antagonists of the present invention antagonize PCSK9 function to the point where a measured parameter (including but not limited to an activity disclosed herein) is reduced by at least 10%, and more preferably, a measured parameter is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%. Such inhibition / antagonism of PCSK9 function is particularly effective in those instances where PCSK9 function contributes, at least in part, to a particular phenotype, disease, disorder, or condition that negatively impacts an individual.

[0403] In one aspect, the present invention provides a method for antagonizing the activity of PCSK9, the method comprising contacting a cell, cell population, or tissue sample that is susceptible to PCSK9 (i.e., that expresses and / or contains an LDL receptor) with a PCSK9-specific antagonist disclosed herein under conditions that allow the antagonist to bind to PCSK9 (when present) and inhibit PCSK9's inhibition of cellular LDL uptake. Some embodiments of the present invention include such methods wherein the cell is a human cell. Additional embodiments of the present invention include such methods wherein the cell is a mouse cell.

[0404] In one aspect, the present invention provides a method for antagonizing the activity of PCSK9 in a subject, the method comprising administering to the subject a therapeutically effective amount of a PCSK9-specific antagonist of the present invention. In some embodiments, the method for antagonizing PCSK9 function is used as defined herein for the treatment of a PCSK9-associated disease, disorder, or condition, or alternatively for providing therapy in a disease, disorder, or condition that may benefit from the action of a PCSK9 antagonist.

[0405] Thus, the present invention encompasses the use of the PCSK9-specific antagonists described herein in various therapeutic methods requiring antagonism of PCSK9 function. As used herein, the term "therapeutic method" relates to a process of action that results in a change in at least one symptom of a disease state that may be prophylactic or therapeutic in nature. In some embodiments, the present invention relates to a method of treating a condition associated with and / or attributed to PCSK9 activity, or a condition in which the function of PCSK9 is contraindicated for a particular individual, the method comprising administering to the individual a therapeutically effective amount of a PCSK9 antagonist compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the condition may be atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, or related cardiovascular diseases and cardiometabolic conditions, or may be a disease state or condition in which PCSK9 activity is contraindicated.

[0406] The treatment methods according to the present invention comprise administering to an individual a therapeutically (or prophylactically) effective amount of a PCSK9 specific antagonist of the present invention. The term "therapeutically effective" or "prophylactically effective" with reference to the use of a certain amount refers to the amount necessary at the intended dose to achieve the desired therapeutic and / or prophylactic effect for the desired period of time. The desired effect may, for example, be the alleviation, reduction, reduction or cessation of at least one symptom associated with the condition being treated. As will be appreciated by those skilled in the art, these amounts will vary depending on various factors, including but not limited to the disease state, age, sex and weight of the individual and the ability of the PCSK9 specific antagonist to induce the desired effect in the individual. The response can be documented by in vitro assays, in vivo non-human animal studies and / or further supported by clinical trials.

[0407] In some embodiments, it is preferred to administer the PCSK9 antagonist compounds of the present invention in the form of pharmaceutical compositions as described herein.

[0408] The administration of antagonist therapy is well within the capabilities of those skilled in the art, see, for example, Lederman et al., 1991 Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922, and will vary based on many factors, such as, but not limited to, those mentioned above, including the condition of the patient, the area to be treated, the route of administration, and the treatment desired (e.g., prophylactic or acute treatment), etc. A physician or veterinarian of ordinary skill can readily determine and prescribe a therapeutically effective amount of the antagonist.

[0409] A subject may need or desire treatment for an existing disease or medical condition. As used herein, a subject who is "in need" of treatment for an existing condition encompasses both the need for determination by a medical professional and the need for such treatment. When a compound or its salt is provided in combination with one or more other active agents, "administering" and variations thereof are each understood to include providing the compound or its salt and other agents contemporaneously or simultaneously or over a course of separate administration over a period of time. When the combined agents are administered simultaneously, they may be administered together in a single composition or they may be administered separately. It should be understood that a "combination" of active agents may be a single composition containing all active agents or multiple compositions each containing one or more of the active agents. In the case of two active agents, the combination may be a single composition comprising the two agents or two separate compositions each containing one of the agents; in the case of three active agents, the composition may be a single composition comprising all three agents, three separate compositions each containing one of the agents, or two compositions, one of which contains two of the agents and the other contains the third agent; and so on.

[0410] The compositions and combinations of the present invention are suitable for administration in an effective amount. The term "effective amount" means an amount of active compound sufficient to antagonize PCSK9 and thereby induce the response sought (i.e., a therapeutic response induced in the treatment or management of a condition associated with or affected by PCSK9 function, including but not limited to atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, and related cardiovascular diseases and cardiometabolic conditions in animals or humans).

[0411] The actual dosage used may vary depending on the patient's needs and the severity of the condition being treated. The determination of an appropriate dosage regimen for a particular situation is within the skill of the art, for example, as described in standard literature, for example, in the "Physicians' Desk Reference" (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), the Physician's Desk Reference, 56th edition, 2002 (published by Medical Economics company, Inc. Montvale, NJ 07645-1742), or the Physician's Desk Reference, 57th edition, 2003 (published by Thompson PDR, Montvale, NJ 07645-1742); the disclosures of which are incorporated herein by reference. For convenience, the total daily dose may be divided into several portions and administered in batches or continuously as needed that day.

[0412] PCSK9 specific antagonists can be administered to an individual by any route of administration known in the art, alone or in combination with other agents designed to assist in the treatment of an individual, including but not limited to oral administration, administration by injection (specific examples of which include intravenous, subcutaneous, intraperitoneal or intramuscular injection) or by inhalation, intranasal or topical administration. PCSK9 specific antagonists can also be administered by injection devices, syringe pens, needle-free devices; and subcutaneous patch delivery systems. The route of administration should be determined based on a variety of considerations known to those skilled in the art, including but not limited to the desired physicochemical properties of the treatment.

[0413] One or more other pharmacologically active agents can be administered in combination with the compound of formula I. Additional active agent (or agent) is intended to mean a pharmaceutically active agent (or agent) having activity in vivo, including a prodrug (which is different from the compound of formula I) that is converted into a pharmaceutically active form after administration, and also including free acids, free bases, and pharmaceutically acceptable salts of the additional active agent. Typically, any suitable one or more additional active agents (including but not limited to antihypertensive agents, antiatherosclerotic agents (such as lipid-modified compounds), antidiabetic agents, and / or antiobesity agents) can be used with any combination (fixed-dose drug combination) of the compound of formula I in a single dosage form, or can be administered to a subject in one or more separate dosage forms, which allow simultaneous or sequential administration of active agents (co-administration of separate active agents).

[0414] Examples of additional active agents that may be employed include, but are not limited to, angiotensin converting enzyme inhibitors (e.g., alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, or trandolapril), angiotensin II receptor antagonists (e.g., losartan, i.e., valsartan, candesartan, olmesartan, telmesartan, and any of these drugs used in combination with hydrochlorothiazide, such as neutral endopeptidase inhibitors (e.g., thiorphan and phospho-dipeptides), aldosterone antagonists, aldosterone synthase inhibitors, renin inhibitors (e.g., urea derivatives of dipeptides and tripeptides (see U.S. Pat. No. 5,116,835), amino acids and derivatives (U.S. Pat. Nos. 5,095,119 and 5,104,869), amino acid chains linked by non-peptide bonds (U.S. Pat. No. 5,114,937), dipeptide and tripeptide derivatives, peptidylaminodiols and peptidyl β-aminoacylaminodiol carbamates and small molecule renin inhibitors (including diol sulfonamides and sulfenyls), N-morpholino derivatives, N-heterocyclic alcohols, and pyrroloimidazolones;In addition, pepstatin peptide derivatives and fluorine and chlorine derivatives of statone peptides, enalkrein, RO 42-5892, A 65317, CP 80794, ES 1005, ES 8891, SQ 34017, aliskiren (2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamide hemifumarate) SPP600, SPP630 and SPP635), endothelin receptor antagonists, phosphodiesterase-5 inhibitors (such as sildenafil) , tadalafil, and vardenafil), vasodilators, calcium channel blockers (e.g., amlodipine, nifedipine, verapamil, diltiazem, gallopamil, niludipine, nimodipins, nicardipine, cardipine), potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam), diuretics (e.g., hydrochlorothiazide), sympatholytics, beta-adrenergic blocking drugs (e.g., propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartrate), alpha-adrenergic blocking drugs (e.g., doxazocin, prazosin, or alpha-methyldopa), central alpha-adrenergic agonists, peripheral vasodilators (e.g., hydralazine); lipid-lowering agents, such as HMG-CoA reductase inhibitors, as and Simvastatin and lovastatin, which are sold as lactone prodrugs and act as inhibitors after administration, and pharmaceutically acceptable salts of dihydroxy-secoke acid HMG-CoA reductase inhibitors, such as atorvastatin (particularly as calcium salts sold), rosuvastatin (especially as calcium salts sold), pravastatin (especially as sodium salt), fluvastatin (especially as sodium salt), crivastatin, and pitavastatin; cholesterol absorption inhibitors, such as ezetimibe and ezetimibe in combination with any other lipid-lowering agent such as the above-mentioned HMG-CoA reductase inhibitors, and in particular with simvastatin or in combination with atorvastatin calcium; niacin in immediate-release or controlled-release form and / or with an HMG-CoA reductase inhibitor; niacin receptor agonists, such as acipimox and acifuran, and niacin receptor partial agonists; metabolism-altering agents, including insulin and insulin mimetics (e.g., degludec insulin, glargine insulin, lispro insulin), dipeptidyl peptidase-IV (DPP-4) inhibitors (e.g., sitagliptin, alogliptin, omarigliptin, linagliptin, vildagliptin); insulin sensitizers, including (i) PPARγ agonists, such as glitazones (e.g., pioglitazone, AMG 131, MBX2044, mitoglitazone, lobeglitazone, IDR-105, rosiglitazone, and balaglitazone) and other PPAR ligands, including (1) PPARα / γ dual agonists (e.g., ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar, and naveglitazar); (2) PPARα agonists, such as fenofibric acid; acid) derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), (3) selective PPARγ modulators (SPPARγM's), (e.g., such as those disclosed in WO 02 / 060388, WO 02 / 08188, WO 2004 / 019869, WO 2004 / 020409, WO 2004 / 020408 and WO 2004 / 066963); and (4) PPARγ partial agonists; (ii) biguanides, such as metformin and pharmaceutically acceptable salts thereof, in particular metformin hydrochloride, and sustained-release formulations thereof, such as Glumetza TM 、Fortamet TM and GlucophageXR TMand (iii) protein tyrosine phosphatase-1B (PTP-1B) inhibitors (e.g., ISIS-113715 and TTP814); insulin or insulin analogs (e.g., insulin detemir, insulin glulisine, insulin degludec, insulin glargine, insulin lispro, and inhaled formulations of each); leptin and leptin derivatives and agonists; amylin and amylin analogs (e.g., pramlintide); sulfonylurea and non-sulfonylurea insulin secretagogues (e.g., tolbutamide, glyburide, glipizide, glimepiride); glimepiride, mitiglinide, meglitinides, nateglinide, and repaglinide); α-glucosidase inhibitors (e.g., acarbose, voglibose, and miglitol); glucagon receptor antagonists (e.g., MK-3577, MK-0893, LY-2409021, and KT6-971); incretin mimetics, such as GLP-1, GLP-1 analogs, derivatives, and mimetics; and GLP-1 receptor agonists. agonists (e.g., dulaglutide, semaglutide, albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide, CJC-1131, and BIM-51077, including their intranasal, transdermal, and once-weekly formulations); bile acid sequestrants (e.g., colestilan, colestiamide, colesevalam hydrochloride, colestipol, stipol), cholestyramine, and dialkylaminoalkyl derivatives of cross-linked polydextrose), acyl CoA: cholesterol acyltransferase inhibitors, (e.g., avasimibe); weight loss compounds; agents intended for use in inflammatory conditions, such as aspirin, nonsteroidal anti-inflammatory drugs or NSAIDs, glucocorticoids, and selective cyclooxygenase-2 or COX-2 inhibitors; glucokinase activators (GKA) (e.g., AZD6370); inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (e.g., such as those disclosed in U.S. Pat. No. 6,730,690 and LY-2523199);CETP inhibitors (e.g., anacetrapib, torcetrapib, and evacetrapib); inhibitors of fructose 1,6-bisphosphatase (e.g., such as those disclosed in U.S. Pat. Nos. 6,054,587; 6,110,903; 6,284,748; 6,399,782; and 6,489,476); inhibitors of acetyl CoA carboxylase 1 or 2 (ACC1 or ACC2); AMP-activated protein kinase (AMPK) activators; G protein Other agonists of leukocyte-coupled receptors: (i) GPR-109, (ii) GPR-119 (e.g., MBX2982 and PSN821), and (iii) GPR-40 (e.g., TAK875); SSTR3 antagonists (e.g., such as those disclosed in WO2009 / 001836); neuromedin U receptor agonists (e.g., such as those disclosed in WO2009 / 042053, including but not limited to neuromedin S (NMS)); SCD modulators; GPR-105 antagonists (e.g., such as those disclosed in WO2009 / 042053, including but not limited to neuromedin S (NMS)); 2009 / 000087); SGLT inhibitors (e.g., ASP1941, SGLT-3, empagliflozin, dapagliflozin, canagliflozin, BI-10773, ertugliflozin, remogloflozin, TS-071, tofogliflozin, ipragliflozin, and LX-4211); inhibitors of acyl-CoA:diacylglycerol acyltransferases 1 and 2 (DGAT-1 and DGAT-2); inhibitors of fatty acid synthase; acyl-CoA:monoacylglycerol acyltransferase 1 and 2 (MGAT-1 and MGAT-2); agonists of the TGR5 receptor (also known as GPBAR1, BG37, GPCR19, GPR131 and M-BAR); ileal bile acid transporter inhibitors; PACAP, PACAP mimetics and PACAP receptor 3 agonists; PPAR agonists; protein tyrosine phosphatase-1B (PTP-1B) inhibitors; IL-1b antibodies (e.g., XOMA052 and canakinumab); and bromocriptine mesylate and its rapid-release formulations; or in combination with other drugs that are beneficial for treating the above-mentioned conditions or disorders, including free acid, free base and chemically possible pharmaceutically acceptable salt forms of the above active agents.

[0415] Compound of the present invention can be easily prepared according to the following reaction scheme and examples or its modification, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to utilize known variants. For using reverse phase chromatography (HPLC or MPLC, as described below) to purify the compound, a C18 column is used. In view of the following reaction scheme and examples, it will be apparent to those of ordinary skill in the art that other methods for preparing the compound of the present invention will be. The following abbreviations can be used in the exemplary schemes and / or examples herein.

[0416] ACN is acetonitrile

[0417] AcOH is acetic acid

[0418] AcO - NH4 is ammonium acetate

[0419] Boc2O is di-tert-butyl dicarbonate

[0420] Bn is benzyl

[0421] BnBr is benzyl bromide

[0422] BzCl is benzoyl chloride

[0423] CBr4 is methyl bromide

[0424] Cbz-Cl is benzyl chloroformate

[0425] DBU is 1,8-diazabicyclo[5.4.0]undec-7-ene

[0426] DCC is dicyclohexylcarbodiimide

[0427] DCE is 1,2-dichloroethane

[0428] DCM is dichloromethane

[0429] DEA is N,N-diethylamine

[0430] DIAD is diisopropyl (E)-diazene-1,2-dicarboxylate

[0431] DIEA or DIPEA is N,N-diisopropylethylamine

[0432] DMAP stands for 4-dimethylaminopyridine

[0433] DMF is N,N-dimethylformamide

[0434] DMSO is dimethyl sulfoxide

[0435] EA or EtOAc is ethyl acetate

[0436] EtOH is ethanol

[0437] Et2O is diethyl ether

[0438] Fmoc is a fluorenylmethoxycarbonyl protecting group

[0439] Fmoc-Cl is (9H-fluoren-9-yl)methyl chloroformate

[0440] Fmoc-D-Dap(Boc)-OH is N-α-(9-fluorenylmethoxycarbonyl)-N-β-tert-butoxycarbonyl-D-2,3-diaminopropionic acid

[0441] Fmoc-Osu is Fmoc N-hydroxysuccinimide ester

[0442] HATU is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0443] HPLC stands for high performance liquid chromatography

[0444] IPA is isopropyl alcohol

[0445] LiOH is lithium hydroxide

[0446] LC / MS stands for liquid chromatography-mass spectrometry

[0447] Me3N is trimethylamine

[0448] MeOH is methanol

[0449] MPLC stands for medium pressure liquid chromatography

[0450] MsCl is methanesulfonyl chloride

[0451] NaBH(OAc)3 is sodium triacetoxyborohydride

[0452] NMR stands for nuclear magnetic resonance

[0453] NsCl is 4-nitrobenzene-1-sulfonyl chloride

[0454] PE is petroleum ether

[0455] Pd2(dba)3(HCCl3) is tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct

[0456] PPh3 is triphenylphosphine

[0457] PdCl2(dppf) or Pd(ii)(dppf)Cl2 is dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II)

[0458] Pd(dppf)Cl2CH2Cl2 is dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct

[0459] Pd(PPh3)4 is tetrakis(triphenylphosphine)palladium

[0460] PPT s Pyridinium p-toluenesulfonate

[0461] [Rh(OAc)2]2 is a rhodium(II) acetate dimer

[0462] RT or rt or rt is room temperature

[0463] tBuOAc is tert-butyl acetate

[0464] TEA is triethylamine

[0465] TFA is trifluoroacetic acid

[0466] TFE is tetrafluoroethylene

[0467] THF is tetrahydrofuran

[0468] Tf2O is trifluoromethanesulfonic anhydride

[0469] Teoc-OSu is 2,5-dioxopyrrolidin-1-yl (2-(trimethylsilyl)ethyl) carbonate

[0470] TBAF is tetrabutylammonium fluoride

[0471] TMS is tetramethylsilane

[0472] Zhan's catalyst 1B is dichloro(1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)((5-((dimethylamino)sulfonyl)-2-(1-methylethoxy-O)phenyl)methylene-C)ruthenium(II)

[0473] [Also described as 1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene[2-(isopropoxy)-5-(N,N-dimethylaminosulfonyl)phenyl[methyleneruthenium(II) dichloride]

[0474] Example 1 Preparation of Ex-01 and Ex-51

[0475]

[0476] Ex-01, (Exemplary free base)

[0477]

[0478] Ex-51, (Exemplary free base)

[0479] The salt forms of compounds Ex-01 and Ex-51 were prepared from intermediates 76 and 86 (prepared below) according to the following scheme:

[0480]

[0481]

[0482]

[0483] Step A: Preparation of Intermediate 89

[0484] To a solution of 76 (1.56 g, 1.917 mmol, prepared below) and 86 (1.506 g, 1.936 mmol, prepared below) in DMF (40 ml) was added HATU (0.802 g, 2.108 mmol) and DIEA (0.670 ml, 3.83 mmol). The resulting solution was stirred at room temperature for 50 min and then partitioned between EtOAc (300 mL) and brine (100 mL). The organic phase was washed with brine (2×100 mL), dried over Na 2 SO 4 , concentrated, and the residue was purified on a silica gel column using MeOH / DCM as the eluting solvent to give 89. LC / MS: (M+1) + :1573.8.

[0485] Step B: Preparation of Intermediate 90

[0486] A solution of 89 (0.68 g, 0.432 mmol) in DCM (500 ml) and acetic acid (40 mL) was bubbled with N2 for 20 min, followed by the addition of Jan's catalyst-1b (0.222 g, 0.302 mmol). The resulting mixture was further bubbled with N2 for 20 min and then heated at 55 ° C for 5 h. After cooling to room temperature, the mixture was filtered through celite, the filtrate was concentrated, and the residue was purified on a silica gel column using MeOH / DCM as eluting solvent to give 90 (cis / trans mixture). LC / MS: (M+1) + :1545.7.

[0487] Step C: Preparation of Intermediate 91

[0488] To a solution of 90 (cis / trans mixture) (65 mg, 0.042 mmol) in acetonitrile (2 ml) was added piperidine (0.042 ml, 0.420 mmol). The resulting solution was stirred at room temperature for 1 h, then concentrated and the residue was dissolved in acetonitrile (4 mL) and concentrated again. The residue was further dried under high vacuum for 30 min to give 91 (cis and trans mixture). LC / MS: (M+1) + :1324.0.

[0489] Step D: Preparation of Intermediate 92

[0490] To a solution of 91 (cis / trans mixture) (548 mg, 0.414 mmol) and 88 (227 mg, 0.455 mmol, prepared below) in DMF (10 ml) was added HATU (181 mg, 0.476 mmol) and DIEA (0.166 ml, 0.952 mmol) at 0° C. The resulting solution was stirred at 0° C. for 1 h and purified by reverse phase MPLC on a C18 column using acetonitrile (0.05% TFA) / water (0.05% TFA) as the eluting solvent to give 92 (cis / trans mixture). LC / MS: (M+1) + :1803.5.

[0491] Step E: Preparation of Intermediate 93

[0492] To a solution of 92 (700 mg, 0.388 mmol) in THF (20 ml), MeOH (6 ml) and water (6 ml) was added 1 N aqueous LiOH (3.11 ml, 3.11 mmol) dropwise at 0° C., and the resulting solution was stirred at 0° C. for 23 h. The solution was neutralized to pH 7-8 by addition of 1 N HCl, the volatiles were evaporated, the aqueous phase was acidified to pH 5, and the mixture was purified by reverse phase MPLC on a C18 column using acetonitrile (0.05% TFA) / water (0.05% TFA) as the eluting solvent to afford 93 as a TFA salt. To a solution of 93 TFA salt (427 mg, 0.257 mmol) in water (70 mL) and acetonitrile (70 ml) was added 0.1 N HCl (13.5 ml, 1.350 mmol) dropwise at 0° C., the resulting solution was stirred at 0° C. for 5 min, and then lyophilized to afford 93 as an HCl salt. LC / MS:(M+1) + :1567.1.

[0493] Step F: Preparation of Intermediate 94

[0494] To a solution of 93 (200 mg, 0.125 mmol) in DMF (30 mL) as an HCl salt was added HATU (56.9 mg, 0.150 mmol). The resulting solution was stirred at room temperature for 30 min, then diluted with DCM (400 mL), followed by the addition of DIEA (0.065 mL, 0.374 mmol). The resulting solution was stirred at room temperature for 1 h, the volatiles were evaporated on a rotary evaporator, and the resulting DMF solution was purified by reverse phase MPLC using acetonitrile (0.05% TFA) / water (0.05% TFA) as the eluting solvent to give 94. LC / MS: (M + 1) + :1549.2.

[0495] Step G: Preparation of Intermediate 95

[0496] To a solution of 94 (14 mg, 9.04 μmol) in MeOH (20 ml) was added 10% Pd / C (1.924 mg, 1.808 μmol), and the resulting mixture was subjected to hydrogenation via H2 balloon at room temperature for 1 h. The mixture was filtered through celite and the filtrate was concentrated to give intermediate compound 95. LC / MS: (M+1) + :1550.9.

[0497] Step H: Preparation of Ex-01

[0498] The intermediate compound 95 (26 mg, 0.017 mmol) prepared in the previous step was dissolved in DCM (2 ml). TFA (6 mL, 78 mmol) was added to this solution and the resulting solution was stirred at room temperature for 30 min, then concentrated and the residue was dissolved in DCM (3 mL) and treated with 4N HCl / dioxane (0.042 mL, 0.168 mmol) and concentrated again to give Ex-01 in the form of a crude product. The crude material Ex-01 was purified by reverse phase HPLC using acetonitrile (0.1% formic acid) / water (0.1% formic acid) as the eluting solvent to provide Ex-01 in the form of a formate salt. LC / MS: (M+1) + :1394.4.

[0499] Step I: Preparation of Ex-51

[0500] To a solution of intermediate compound 94 (30 mg, 0.019 mmol) in DCM (2 ml) was added TFA (4 ml, 51.9 mmol), and the reaction mixture was stirred at ambient temperature for 30 minutes and then concentrated. The residue was dissolved in DCM (2 mL), treated with HCl (4N in dioxane) (0.048 ml, 0.194 mmol), and concentrated to produce Ex-51 in the form of an HCl salt. The compound was purified by reverse phase HPLC using acetonitrile (0.1% formic acid) / water (0.1% formic acid) as the mobile phase to provide Ex-51 in the form of a formate salt. LC / MS: (M+1) + :1392.0.

[0501] The following schemes and procedures were used to prepare intermediates 76, 86 and 88 used in the procedures described above.

[0502] Preparation of Intermediate 68 for the preparation of Intermediate 76

[0503]

[0504] Step A: Preparation of intermediate compound 65

[0505] To a suspension of (2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid (5.32 g, 40.6 mmol) in dioxane (100 ml) was added sodium hydroxide (122 ml, 122 mmol) followed by the dropwise addition of benzyl chloroformate (6.50 ml, 44.6 mmol) at 0°C. The resulting suspension was stirred at 0°C for 5 h. After removal of volatiles, the aqueous phase was acidified to pH 3 and then partitioned between 30% IPA / DCM (200 mL) and brine (50 mL). The aqueous phase was further extracted with 30% IPA / DCM (2×100 mL). The combined organic phases were dried over Na2SO4 and concentrated to give (2S,3S)-1-((benzyloxy)carbonyl)-3-hydroxypyrrolidine-2-carboxylic acid (65). LC / MS: (M+1) + :266.1.

[0506] Step B: Preparation of intermediate compound 66

[0507] To a solution of 65 (7.48 g, 28.2 mmol) in MeOH (80 ml) was added TMS-diazomethane (70.5 ml, 141 mmol) dropwise, and the resulting solution was stirred at room temperature for 10 min, then quenched by the addition of acetic acid (about 400 uL). The solution was concentrated, and the residue was purified on a silica gel column using EtOAc / hexane as the eluting solvent to give 66. LC / MS: (M+1) + :280.1.

[0508] Step C: Preparation of intermediate compound 67

[0509] A solution of 66 (4.81 g, 17.22 mmol) in DCM (200 mL) was bubbled with N2 for 30 min, followed by the addition of rhodium (ii) acetate dimer (0.761 g, 1.722 mmol). The mixture was cooled in an ice-water bath, and tert-butyl diazoacetate (3.58 mL, 25.8 mmol) was added dropwise at 0 ° C. The resulting mixture was stirred at 0 ° C for 1.5 h. The reaction was quenched by adding water (100 mL), the mixture was extracted with DCM (3 × 100 mL), the combined organic phases were dried over Na2SO4, concentrated, and the residue was purified by reversed-phase MPLC using acetonitrile (0.05% TFA) / water (0.05% TFA) as elution solvent. The fraction containing the product was concentrated and the aqueous phase was extracted with DCM (2 × 100 mL). The combined organic phases were dried over Na2SO4 and concentrated to give 67. LC / MS: (M+1) + :394.2.

[0510] Step D: Preparation of intermediate compound 68

[0511] To a solution of 67 (3.72 g, 9.46 mmol) in MeOH (80 ml) was added 10% Pd / C (0.805 g, 0.756 mmol) and the resulting mixture was subjected to hydrogenation via a H2 balloon at ambient temperature for 2 hours and then filtered through Celite. The filtrate was concentrated to give 68. LC / MS: (M+1) + :259.9.

[0512] Preparation of intermediate compound 76

[0513]

[0514] Step A: Preparation of Intermediate 69

[0515] To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1H-indol-3-yl)propanoic acid (3 g, 6.75 mmol) in THF (20 ml), MeOH (10 mL) and water (20.00 ml) was added NaOH (20.25 ml, 20.25 mmol) at 0°C and the resulting solution was stirred at ambient temperature for 4 hours before evaporating the volatiles. Dioxane (50 ml) and water (20 mL) were added to the aqueous mixture, the resulting solution was cooled to 0°C and BocO (1.881 ml, 8.10 mmol) was added to the above solution. The resulting solution was stirred at 0°C for 3 hours, the volatiles were removed and the aqueous phase was extracted with EtO (3 x 40 mL), acidified to pH 3, then extracted with DCM (3 x 100 mL) and then with 30% IPA / DCM (2 x 80 mL). The combined organic phases were dried over Na2SO4 and concentrated to give 69. LC / MS: (M+1) + :322.9.

[0516] Step B: Preparation of Intermediate 70

[0517] To a solution of 69 (2.079 g, 6.45 mmol) in DMF (40 ml) was added 60% NaH / hexane (0.568 g, 14.19 mmol) at 0°C and the resulting solution was stirred at 0°C for 50 min before allyl bromide (1.172 mL, 13.54 mmol) was added dropwise. The resulting solution was stirred at 0°C for 1.5 h and then quenched by the addition of 1N HCl (approximately 3.68 mL). The solution was then partitioned between EtOAc (200 mL) and water (100 mL), and the organic phase was washed with brine (2×100 mL), dried over Na 2 SO 4 , concentrated, and the residue was purified on a silica gel column using MeOH / DCM as the eluting solvent to give 70. LC / MS: (M+1) + :363.0.

[0518] Step C: Preparation of Intermediate 71

[0519] To a solution of 70 (2.239 g, 6.18 mmol) and 68 (1.842 g, 7.11 mmol) in DMF (30 ml) was added HATU (2.82 g, 7.41 mmol) and DIEA (2.59 ml, 14.83 mmol), and the resulting solution was stirred at ambient temperature for 1 hour. The mixture was partitioned between EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine (3×100 mL), dried over Na 2 SO 4 , concentrated, and the residue was purified on a silica gel column using EtOAc / hexane as the eluting solvent to give 71. LC / MS: (M+1)+ :604.2.

[0520] Step D: Preparation of Intermediate 72

[0521] To a solution of 71 (2.83 g, 4.69 mmol) in CH2Cl2 (20 ml) and tBuOAc (30 ml) was added methanesulfonic acid (1.218 ml, 18.75 mmol) at 0°C and the resulting solution was stirred at 0°C for 16.5 h and then at ambient temperature for 2.5 h. The solution (72) was used directly in the next step. LC / MS: (M+1) + :504.2.

[0522] Step E: Preparation of Intermediate 73

[0523] To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)propanoic acid (2.66 g, 5.16 mmol) in DMF (10 ml) was added HATU (1.961 g, 5.16 mmol) and DIEA (5.32 ml, 30.5 mmol), and the resulting solution was stirred at room temperature for 30 min and then added to the ice-cold bath of the solution of 72 prepared above. The resulting solution was stirred at ambient temperature for 1 hour. The volatiles were evaporated on a rotary evaporator, and the residue was purified by reverse phase MPLC using acetonitrile (0.05% TFA) / water (0.05% TFA) as the eluting solvent. The collected fractions were concentrated on a rotary evaporator to give 73. LC / MS: (M+1) + :1002.1.

[0524] Step F: Preparation of Intermediate 74

[0525] To a solution of 73 (3.235 g, 3.23 mmol) in DCM (4 ml) was added TFA (7.46 ml, 97 mmol) and the resulting solution was stirred at ambient temperature for 1 hour and then concentrated. The residue was dissolved in DCM (10 mL), treated with 4N HCl / dioxane (3.23 ml, 12.91 mmol), then concentrated, and the residue was dissolved in acetonitrile (100 mL) / water (50 mL) and lyophilized to provide 74. LC / MS: (M+1) + :846.1.

[0526] Step G: Preparation of Intermediate 75

[0527] To a solution of 74 (2.85 g, 3.23 mmol) in DMF (45 ml) was added HATU (1.474 g, 3.88 mmol), and the resulting solution was stirred at ambient temperature for 30 min, then diluted with DCM (600 ml), followed by dropwise addition of DIEA (1.692 ml, 9.69 mmol). The resulting solution was stirred at ambient temperature for 1 hour. The solution was concentrated, and the residue was purified by reverse phase MPLC on a C18 column using acetonitrile (0.05% TFA) / water (0.05% TFA) as elution solvent. The fractions containing the product were concentrated, and the aqueous layer was partitioned between DCM (200 mL) and saturated NaHCO 3 (200 mL). The aqueous phase was extracted with DCM (2×100 mL), and the combined organic phases were dried over Na 2 SO 4 and concentrated to give 75. LC / MS: (M+1) + :828.1.

[0528] Step H: Preparation of intermediate compound 76

[0529] To a solution of 75 (1.93 g, 2.331 mmol) in THF (60 ml), MeOH (30 ml) and water (20 ml) was added 1N aqueous LiOH (9.9 ml, 9.90 mmol) dropwise at 0°C and the resulting solution was stirred at 0°C for 16 h before being quenched by the addition of HCl (1 N, 9.9 mL). The volatiles were evaporated on a rotary evaporator and acetone (60 ml), sodium carbonate (0.371 g, 3.50 mmol) and Fmoc-Osu (0.802 g, 2.378 mmol) were added to the above solution at 0°C. The resulting solution was stirred at 0°C for 6 h, the volatiles were evaporated on a rotary evaporator, the aqueous phase was acidified to pH 4 and then extracted with 30% IPA / DCM (3×100 mL). The combined organic phases were dried over NaSO, concentrated and the residue was purified on a silica gel column using MeOH / DCM as the eluting solvent to give 76. LC / MS:(M+1) + :814.2.

[0530] Intermediate compounds 75b and 76B were prepared from the following alternative

[0531] The general procedure described above for the preparation of intermediate compound 75 was generally followed, except that elimination step B and final step G also involved the replacement of the Fmoc protecting group with a Boc protecting group, thereby providing intermediate compound 75a. The procedures for 75a and 76B are described below.

[0532] Step C: Preparation of intermediate compound 71a

[0533] To a solution of 69 (5.00 g, 15.5 mmol) in DMF (40.0 mL) was added 68, HATU (5.90 g, 15.5 mmol) and DIEA (4.01 g, 31.0 mmol) at -50°C, and the reaction mixture was stirred at -50°C for 3 h. The final solution was quenched with water (5 mL), concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography on C18 (eluting with a gradient of acetonitrile / water + 0.01% ammonium bicarbonate) to provide 71a. 28 H 38 LCMS (ESI) calcd for FN3O8[M+H]+: 564.3, found 564.2.

[0534] Step D: Preparation of intermediate compound 72a

[0535] To a solution of 2N HCl / dioxane (100 mL) and THF (100 mL) was added 71a (8.40 g, 14.9 mmol) at room temperature and the reaction mixture was stirred for 5 h. The final solution was concentrated under reduced pressure to provide 72a. 23 H 31 LCMS (ESI) calculated value of ClFN3O6[M-HCl+H]+: 464.2, found value 464.3.

[0536] Step E: Preparation of intermediate compound 73a

[0537] To a solution of 72a (800 mg, 1.60 mmol) in DMF (10.0 mL) was added (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)propanoic acid (827 mg, 1.60 mmol), HATU (608 mg, 1.60 mmol) and DIEA (620 mg, 4.80 mmol) at -50°C, and the mixture was stirred at -50°C for 3 h. The resulting solution was diluted with water (50 mL) and the aqueous layer was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluted with a gradient of 1% to 60% EtOAc / PE) to give 73a. 53 H 60 FN5O 11 LCMS (ESI) calcd. for [M+H]<+> 962.4, found 962.6.

[0538] Step F: Preparation of intermediate compound 74a

[0539] To a solution of 73a (3.00 g, 3.12 mmol) in DCM (15.0 mL) was added TFA (15.0 mL) at room temperature and the reaction mixture was stirred at room temperature for 1 h. The resulting solution was concentrated under reduced pressure and co-evaporated with toluene and DCM to give 74a. 46 H 45 F4N5O 11 LCMS (ESI) calcd for [M-TFA+H]+: 806.3, found 806.7.

[0540] Step G: Preparation of intermediate compound 75a

[0541] To a solution of 74a (4.00 g, 4.35 mmol) in DMF (150 mL) was added HATU (1.65 g, 4.35 mmol) at room temperature, and the reaction solution was stirred for 0.5 h. The solution was diluted with DCM (450 mL) and DIEA (1.69 g, 13.1 mmol), and then stirred at room temperature for 3 h. The resulting solution was quenched with water (5 mL), concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography on C18 (eluting with a gradient of acetonitrile / water + 0.05% TFA) to provide the Fmoc-protected intermediate. 44 H 42 LCMS (ESI) calcd for FN5O8[M+H]+: 788.3, ​​found 788.9.

[0542] To a solution of the above Fmoc-protected intermediate (200 mg, 0.250 mmol) in DCM (5.00 mL) was added piperidine (1.25 mL) at room temperature and the reaction solution was stirred for 1 h. The final solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluting with a gradient of 0%-5% MeOH / DCM) to provide the amine intermediate. 29 H 32 LCMS (ESI) calculated value for FN5O6 [M+H]+: 566.2, found value 566.3.

[0543] To a solution of the above amine intermediate (2.86 g, 5.06 mmol) in THF (30.0 mL) and water (30.0 mL) was added Boc2O (2.21 g, 10.1 mmol) and sodium bicarbonate (1.70 g, 20.2 mmol) at room temperature, and the reaction was stirred for 3 h. The final solution was diluted with water (50 mL) and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluted with a gradient of 0%-5% MeOH / DCM) to provide 75a. C34 H 40 LCMS (ESI) calculated value of FN5O8[M+H]+: 666.3, found value 666.5; 1 H NMR (300MHz, CD3OD) δ7.35-7.08(m,6H),6.95-6.79(m,2H),5.01-4.91(m,1H),4.72-4.56(m,2H),4. 45-4.38(m,1H),4.45-4.38(m,5H),3.69(s,3H),3.32-2.92(m,5H),2.14-1.82(m,2H),1.47(s,9H).

[0544] Step H: Preparation of intermediate compound 75b

[0545] To a solution of 75a (0.665 g, 0.99 mmol) in DMF (0.5 mL) was added Cs2CO3 (1.11 g, 3.40 mmol) and 3-bromoprop-1-ene (0.43 g, 3.55 mmol) at 0°C. The reaction mixture was stirred at room temperature for 16 hours and poured into 5 mL of 50% saturated brine / 10% citric acid solution, then extracted with ethyl acetate (2×20 mL). The organic layer was washed with brine (3×20 mL), dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with a 1%-5% gradient of MeOH / DCM. The fractions containing intermediate compound 75b were combined and concentrated to give the title compound. C 37 H 44 FN5O8[M+H] + LCMS (ESI) calcd. 706.3, found 706.3.

[0546] Step I: Preparation of intermediate compound 76B

[0547] Hydrolysis of 75b with LiOH following conditions similar to those described in the preparation of intermediate 93 provided 76B.

[0548] Preparation of intermediate 77B

[0549]

[0550] Step A: Preparation of Intermediate 77A

[0551] To a solution of (S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-methylpyrrolidine-2-carboxylic acid (6.16 g, 17.54 mmol) and tert-butyl 4-(2-aminoethyl)benzylcarbamate hydrochloride (5.03 g, 17.54 mmol) in DMF (140 ml) at 0°C was added HATU (8.00 g, 21.05 mmol) and DIPEA (9.16 ml, 52.6 mmol), then the reaction was allowed to warm to room temperature and stirred for 2 h. The final mixture was diluted with water, extracted with EtOAc, washed with brine, dried over MgSO4, and filtered. The filtrate was concentrated, and the residue was purified by column chromatography on silica gel (eluting with a 0-60% EtOAc / hexane gradient) to afford 77A. MS (ESI): m / z (M+H)+ 584.5.

[0552] Step B: Preparation of Intermediate 77B

[0553] To a solution of 77A (8.92 g, 15.28 mmol) in DCM (40 ml) was added HCl 4N / dioxane (15.28 ml, 61.1 mmol) and the resulting solution was stirred at room temperature overnight. The mixture was concentrated to give 77B. MS (ESI): m / z (M+H) + 484.3.

[0554] Preparation of intermediate 86

[0555]

[0556] Step A: Preparation of Intermediate 77

[0557] To a solution of (S)-(9H-fluoren-9-yl)methyl 2-((4-(aminomethyl)phenethyl)carbamoyl)-2-methylpyrrolidine-1-carboxylate hydrochloride (77B) (5.87 g, 11.29 mmol) in DCM (140 ml) was added DIEA (5.91 ml, 33.9 mmol) and CBZ-Cl (1.726 ml, 11.85 mmol) dropwise at 0°C, and the resulting solution was stirred at 0°C for 4 h. The reaction solution was partitioned between water (200 mL) and DCM (200 mL), the aqueous phase was extracted with DCM (100 mL), the combined organic phases were dried over Na2SO4, and the residue was purified on a silica gel column using EtOAc / hexane as the eluting solvent to give 77. LC / MS: (M+1) + :618.3.

[0558] Step B: Preparation of Intermediate 78

[0559] To a solution of 77 (5.56 g, 9.00 mmol) in THF (100 ml), water (50 ml) and MeOH (30 ml) was added 1 N NaOH aqueous solution (45.0 ml, 45.0 mmol), and the resulting solution was stirred at room temperature for 2 h. The volatiles were evaporated, and dioxane (200 ml) and dioxane (20 mL) containing Boc2O (2.508 ml, 10.80 mmol) were added to the aqueous solution. The resulting mixture was stirred from 0 ° C to room temperature overnight. The volatiles were evaporated on a rotary evaporator, and the aqueous phase was extracted with DCM (3×150 mL). The combined organic phases were dried over Na2SO4, concentrated, and the residue was purified on a silica gel column using EtOAc / hexane as eluting solvent to give 78. LC / MS: (M+1) + :496.2.

[0560] Step C: Preparation of Intermediate 79

[0561] To a solution of 78 (4.04 g, 8.15 mmol) in MeOH (100 ml) was added 10% Pd / C (0.867 g, 0.815 mmol), and the resulting mixture was subjected to hydrogenation via a H2 balloon at room temperature for 1.5 h. The mixture was filtered through celite, and the filtrate was concentrated to give 79. LC / MS: (M+1) + :362.2.

[0562] Step D: Preparation of Intermediate 80

[0563] To a solution of 79 (2.58 g, 7.14 mmol) in DMF (15 ml) was added 4-methylbenzenesulfonic acid pent-4-en-1-yl ester (0.858 g, 3.57 mmol) and K2CO3 (1.973 g, 14.27 mmol), and the resulting mixture was heated at 80 ° C for 6 h. After cooling to room temperature, the mixture was filtered, and the filtrate was purified by reverse phase MPLC using acetonitrile (0.05% TFA) / water (0.05% TFA) as the eluting solvent to give the product as a TFA salt, which was further partitioned between DCM (100 mL) and 1N NaOH aqueous solution (50 mL). The aqueous phase was further extracted with DCM (2×50 mL), and the combined organic phases were dried over Na2SO4 and concentrated to give 80. LC / MS: (M+1) + :430.3.

[0564] Step E: Preparation of Intermediate 81

[0565] To a solution of 80 (0.95 g, 2.211 mmol) in DMF (15 ml) was added 4-methoxy-4-oxobutanoic acid (0.321 g, 2.433 mmol), HATU (1.009 g, 2.65 mmol) and DIEA (0.927 ml, 5.31 mmol), and the resulting solution was stirred at room temperature for 1 h. The solution was partitioned between EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine (2×100 mL), dried over Na 2 SO 4 , concentrated, and the residue was purified on a silica gel column using EtOAc / hexane as the eluting solvent to give 81. LC / MS: (M+1) + :544.2.

[0566] Step F: Preparation of Intermediate 82

[0567] To a solution of 81 (1.165 g, 2.143 mmol) in DCM (12 ml) was added HCl (4 N in dioxane) (5.36 ml, 21.43 mmol). The resulting solution was stirred at room temperature for 3 h, and the mixture was concentrated to give 82. LC / MS: (M+1) + :444.2.

[0568] Step G: Preparation of Intermediate 83

[0569] To a solution of 82 (1.003 g, 2.089 mmol) in DMF (20 ml) was added Fmoc-L-Tyr(Me)-OH (0.959 g, 2.298 mmol), HATU (0.914 g, 2.403 mmol) and DIEA (1.095 ml, 6.27 mmol), and the resulting solution was stirred at room temperature for 50 min. The solution was partitioned between EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine (2×100 mL), the combined organic phases were dried over Na 2 SO 4 , concentrated, and the residue was purified on a silica gel column using EtOAc / hexane as the eluting solvent to give 83. LC / MS: (M+1) + :843.4.

[0570] Step H: Preparation of Intermediate 84

[0571] To a solution of 83 (1.63 g, 1.934 mmol) in acetonitrile (10 ml) was added piperidine (0.574 ml, 5.80 mmol) and the resulting solution was stirred at room temperature for 1 h and then concentrated. The residue was resuspended in acetonitrile (20 mL) and concentrated again, the cycle was repeated once, and the residue was further dried under high vacuum to give 84. LC / MS: (M+1) + :621.3.

[0572] Step I: Preparation of Intermediate 85

[0573] To a solution of 84 (1.2 g, 1.933 mmol) in DMF (15 ml) was added Fmoc-L-Thr(tBu)-OH (0.922 g, 2.320 mmol), HATU (0.919 g, 2.416 mmol) and DIEA (0.844 ml, 4.83 mmol), and the resulting solution was stirred at room temperature for 1 h. The solution was partitioned between EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine (2×100 mL), dried over Na 2 SO 4 , concentrated, and the residue was purified on a silica gel column using EtOAc / hexane as the eluting solvent to give 85. LC / MS: (M+1) + :1000.2.

[0574] Step J: Preparation of Intermediate 86

[0575] To a solution of 85 (1.94 g, 1.940 mmol) prepared in the previous step in acetonitrile (20 ml) was added piperidine (0.960 ml, 9.70 mmol) and the resulting solution was stirred at room temperature for 30 min and then concentrated. The residue was redissolved in DCM / acetonitrile (1:1, 20 mL) and then concentrated again. The cycle was repeated once and the residue was dried under high vacuum to give 86. LC / MS: (M+1) + :778.3.

[0576] Preparation of intermediate 88

[0577]

[0578] Step A - Synthesis of Intermediate 87

[0579] To 2-chloro-2-chlorotrityl resin 1-1.5 mmol / g (7.0 g, 1-1.5 mmol / g) was added dry DCM (45 ml). The resin was shaken for 20 min, followed by the addition of half of DIPEA 0.17 N in DCM (3.67 ml, 21.00 mmol), Fmoc-D-Dap(Boc)-OH (3.28 g, 7.70 mmol), and then the remainder of DIPEA 0.17 N in DCM (3.67 ml, 21.00 mmol). The resin was shaken overnight at room temperature, rinsed with DCM, and dried. The resin was then quenched with 5% DIPEA and 10% MeOH in DCM (80 mL), shaken for 2 h, then filtered, rinsed with DCM (3x), DMF (3x), and DCM (3x), and then dried under vacuum to provide resin 87, which was used as is in the next step.

[0580] Step B - Synthesis of Intermediate 88

[0581] Resin 87 (4.5 g, 2.475 mmol) was manually deprotected with 5% piperidine / DMF (30 ml) for 30 min, filtered, treated with 5% piperidine / DMF (30 ml) for another 30 min, filtered, then rinsed with DMF and DCM and dried. The resin was then manually coupled with Fmoc-Ala-OH (1.541 g, 4.95 mmol), HATU (1.694 g, 4.46 mmol) and DIPEA (1.729 ml, 9.90 mmol) in DMF (30 ml) for 2 h, then filtered, rinsed with DMF and DCM, and dried. The resin was then treated with 10% AcOH and TFE in DCM (60 ml) for 90 min, filtered, and the filtrate concentrated to provide 88. LC / MS: [2M+H] + =995.01.

[0582] Example 1A - Alternative Synthesis of Ex-01 and Preparation of Ex-25 Therefrom:

[0583] Compound Ex-01 presented above can be prepared alternatively, and compound Ex-25 can be prepared from Ex-01 according to the following scheme:

[0584]

[0585]

[0586]

[0587]

[0588] Step A - Synthesis of Intermediate Int-cd1

[0589] To a solution of Int-3c (synthesized from intermediate 107 described below) (7.09 g, 10.33 mmol) in DMF (45 ml) was added Int-2d (3.63 g, 9.84 mmol, prepared as described below) and HATU (3.74 g, 9.84 mmol) at 0°C, followed by DIPEA / DMF (6.87 ml, 39.4 mmol), and the mixture was allowed to warm to room temperature and stirred for 1 h. The mixture was quenched with brine and extracted with EtOAc at 0°C. The combined organic fractions were washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with a gradient of 1% to 80% ethyl acetate / petroleum ether) to afford Int-cd1. LC / MS: [M+1]+ = 1000.5.

[0590] Step B - Synthesis of Intermediate Int-cd2

[0591] To a solution of Int-cd1 (3.48 g, 3.48 mmol) in acetonitrile (50 ml) was added piperidine (1.72 ml, 17.40 mmol) and the resulting solution was stirred at room temperature for 3 h. The mixture was concentrated, the residue was redissolved in DCM / acetonitrile (1:1, 20 mL), concentrated again, and the residue was dried under vacuum to give Int-cd2 as a crude product. LC / MS: (M+1) + =778.5.

[0592] Step C - Synthesis of Intermediate Int-cd3

[0593] To a solution of 76 (prepared as described above in Example 1) (2.45 g, 3.01 mmol) and Int-cd2 (2.69 g, 3.46 mmol) in DMF (70 ml) was added HATU (1.37 g, 3.61 mmol) followed by DIEA (1.05 ml, 6.02 mmol) at 0°C. The resulting solution was stirred at room temperature for 50 min and then partitioned between EtOAc (500 mL) and brine (200 mL). The organic phase was washed with brine (2 x 200 mL), dried over Na2SO4, concentrated, and the residue was purified by column chromatography on silica gel (eluting with a gradient of 1% to 5% MeOH / DCM) to afford Int-cd3. LC / MS: (M+1) + =1574.7.

[0594] Step D - Synthesis of Intermediate Int-cd4

[0595] A room temperature solution of Int-cd3 (1.91 g, 1.21 mmol) in DCM (1500 ml) and acetic acid (30 mL) was bubbled with N2 for 30 min, followed by the addition of Jan's catalyst-1B (0.445 g, 0.607 mmol). The resulting mixture was further bubbled with N2 for 30 min at room temperature and then heated at 55 ° C for 5 h. After cooling to room temperature, the mixture was filtered through celite, the filtrate was concentrated, and the residue was purified by column chromatography on silica gel (eluting with a gradient of 1%-5% MeOH / DCM) to give Int-cd4 (in the form of a mixture of cis and trans olefins). LC / MS: (M+1) + =1546.8.

[0596] Step E - Synthesis of Intermediate Int-cd5

[0597] To a solution of Int-cd4 (a mixture of cis and trans alkenes) (5.49 g, 3.55 mmol) in DCM (20 ml) and acetonitrile (50 ml) was added piperidine (1.76 ml, 17.8 mmol). The resulting solution was stirred at room temperature for 2 h, then concentrated, and the residue was suspended in acetonitrile (20 ml) and concentrated again. The residue was then dried under vacuum to obtain Int-cd5 (a mixture of cis and trans alkenes) in the form of a crude mixture. LC / MS: (M+1) + =1323.8.

[0598] Step F-Synthesis of Intermediate Int-cd6

[0599] To a solution of Int-cd5 (a mixture of cis and trans alkenes) (4.70 g, 3.55 mmol) and Int-1d (2.21 g, 4.44 mmol, preparation described below) in DMF (70 ml) at 0°C was added HATU (1.76 g, 4.62 mmol) and DIEA (1.55 ml, 8.88 mmol). The resulting solution was warmed to room temperature and stirred for 1 h, then partitioned between EtOAc (300 mL) and brine (200 mL). The aqueous phase was extracted with EtOAc (200 mL), the EtOAc phases were combined and washed with brine (3 x 200 mL), dried over Na2SO4, concentrated, and the residue was purified by column chromatography on silica gel (eluting with a gradient of 1% to 5% MeOH / DCM) to give Int-cd6 (as a mixture of cis and trans alkenes). LC / MS: (M+1) + =1802.8

[0600] Step G - Synthesis of Intermediate Int-cd7

[0601] To a solution of Int-cd6 (as a mixture of cis- and trans-olefins) (5.41 g, 3.00 mmol) in THF (100 ml), MeOH (30 ml) and water (30 ml) was added 1N aqueous LiOH (24.0 ml, 24.0 mmol) dropwise at 0° C., and the resulting solution was stirred for 3 h at 0° C. The mixture was neutralized by adding 1N HCl at 0° C., the volatiles were evaporated, and the aqueous layer was neutralized to pH 5 by 1N HCl. The mixture was then frozen and lyophilized, and the residue was purified by column chromatography on C18 eluting with a gradient of acetonitrile (0.05% TFA) / water (0.05% TFA) to give Int-cd7 (as a mixture of cis- and trans-olefins) as a TFA salt. To the thus obtained Int-cd7 TFA salt (as a mixture of cis and trans olefins) in acetonitrile (750 mL) and water (450 mL) was added dropwise 0.1 N HCl aqueous solution (150 ml, 15.00 mmol) at 0°C, and the resulting solution was stirred at 0°C for 5 min, frozen and lyophilized to obtain Int-cd7 (as a mixture of cis and trans olefins) as an HCl salt. LC / MS: (M+1) + =1566.6.

[0602] Step H - Synthesis of Intermediate Int-cd8

[0603] To a solution of the Int-cd7 HCl salt (1.01 g, 0.630 mmol) obtained from the previous step in DMF (50 ml) and DCM (1300 ml) was added DIEA (0.330 ml, 1.890 mmol) and HATU (0.287 g, 0.756 mmol). The resulting solution was stirred at room temperature for 2 h, the volatiles were evaporated, and the residue was partitioned between EtOAc (400 mL) and brine (200 mL). The aqueous phase was extracted with EtOAc (300 mL), the combined organic layers were washed with brine (3×100 mL), dried over Na 2 SO 4 , concentrated, and the residue was purified by column chromatography on silica gel (eluting with a gradient of 1%-10% MeOH / DCM) to give Int-cd8 as a mixture of cis and trans olefins. LC / MS: (M+1) + =1548.8.

[0604] Step I - Synthesis of Intermediate Int-cd9

[0605] To a solution of Int-cd8 (1.22 g, 0.788 mmol) obtained in the previous step in MeOH (100 ml) was added 10% Pd / C (0.645 g, 0.607 mmol) and the resulting mixture was hydrogenated via a H2 balloon at ambient temperature for 7 hours. After 7 hours, the reaction was filtered through celite, the filtrate was concentrated, and the residue was purified by column chromatography on silica gel (eluting with a gradient of 1% to 10% MeOH / DCM) to give Int-cd9. LC / MS: (M+1) + =1550.9. Step J - Synthesis of compound Ex-01 in the form of HCl salt

[0606] To a solution of Int-cd9 (1.14 g, 0.735 mmol) in DCM (6 ml) was added TFA (12 ml, 156 mmol), and the resulting solution was stirred at ambient temperature for 30 min. The mixture was then concentrated, and the residue was dissolved in DCM (20 ml) and toluene (20 ml). The resulting mixture was concentrated, and the residue was redissolved in DCM (20 ml) and treated with HCl (4 N in dioxane) (0.919 ml, 3.68 mmol). The resulting mixture was concentrated to obtain a product in solid form. This solid product was redissolved in acetonitrile (200 ml) and water (100 ml), and 1 N HCl aqueous solution (3.68 ml, 3.68 mmol) was added dropwise to the above solution at 0 ° C. The resulting solution was stirred at 0 ° C for 2 min, then frozen and lyophilized to obtain Ex-01 in the form of HCl salt. LC / MS: (M+1) + =1394.7.

[0607] Step K - Synthesis of Example Ex-25 as a TFA salt

[0608] To a solution of Ex-01 HCl salt (870 mg, 0.608 mmol) and Int-4b (170 mg, 0.669 mmol, prepared as described below) in DMF (1.2 ml) and water (0.6 ml) was added HATU (254 mg, 0.669 mmol) and DIEA (425 μl, 2.433 mmol). The resulting solution was stirred at room temperature for 1 h and then quenched by adding 1.2 mL of water. The mixture was filtered and the filtrate was purified by column chromatography on C18 (eluting with a gradient of acetonitrile (0.05% TFA) / water (0.05% TFA)) to afford Ex-25 as a TFA salt. LC / MS: M + =1550.6.

[0609] Step L - Preparation of Example Ex-25 as a Cl Salt

[0610] 73.6 g of AG MP-1 ion exchange resin chloride form (lot 141-1841 BIO-RAD) was packed into two columns, with a total of 36.8 g of resin in each column. Each column was washed with water (2×80 ml) followed by 20% acetonitrile / water (2×100 ml). A solution of Ex-25 TFA salt (737 mg, 0.443 mmol) prepared in the previous step in 20% acetonitrile / water (100 mL) was evenly loaded onto both resin columns, and each column was then eluted with 20% acetonitrile / water (130 ml). The eluates were combined, frozen, and lyophilized to obtain Ex-25 in chloride form. LC / MS: M + =1550.6.

[0611] Various intermediates usefully employed in the syntheses of Ex-01 and Ex-25 described in the immediately preceding text are described below.

[0612] Preparation of intermediate Int-1d

[0613] The intermediate compound Int-1d was prepared from the starting materials according to the following scheme:

[0614]

[0615] Step A - Synthesis of Int-1da

[0616] To a solution of D-Dap(Boc)-OMe HCl salt (4.10 g, 16.10 mmol), Fmoc-Ala-OH (5.01 g, 16.10 mmol) and HATU (6.43 g, 16.90 mmol) in DMF (40 ml) was added DIPEA (7.03 ml, 40.2 mmol) at 0°C and the mixture was stirred at 0°C for 2 h and then stored in the refrigerator overnight. The mixture was quenched with water at room temperature and extracted with EtOAc. The combined organic fractions were washed with half brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with a gradient of hexane / EtOAc) to give Int-1da. LC / MS: [M+H]+ = 512.3.

[0617] Step B—Synthesis of Int-1d

[0618] To a solution of Int-1da (8.03 g, 15.70 mmol) and 0.8 N calcium chloride (19.62 ml, 15.70 mmol) in water (40 ml) and 2-propanol (120 ml) was added solid sodium hydroxide (0.691 g, 17.27 mmol) at room temperature. The mixture was stirred at room temperature overnight. The mixture was concentrated, acidified to pH ~2 with 0.5 N (~40 mL), immediately extracted with EtOAc, washed with brine, dried over Na2SO4, and concentrated. The residue was purified by column chromatography on C18 (eluting with a gradient of acetonitrile / water + 0.1% TFA) to afford Int-1d. LC / MS: [M+H]+ = 498.25.

[0619] The preparation of intermediate Int-1d is described above for use in the preparation of Ex-01 and Ex-25. This portion of the molecule can be described as a "linker," which cyclizes the lower peptide ring into the higher peptide ring. Other similar "linkers" can be used to replace Int-1d by varying the spacer used in the synthesis, including but not limited to the use of Dap and D-Ala.

[0620] Preparation of intermediate Int-2d

[0621] Intermediate Int-2d, which can be used as a "linker" in preparing the compounds of the present invention, is prepared according to the following scheme:

[0622]

[0623] Step A - Synthesis of Int-2da

[0624] A solution of 4-bromobenzaldehyde (15.00 g, 81 mmol), potassium tert-butyl N-[2-(trifluoroboryl)ethyl]carbamate (20.97 g, 84 mmol), cesium carbonate (52.8 g, 162 mmol) and 1,1'-bis(diphenylphosphino)ferrocene-dichloropalladium(II) dichloromethane complex (Pd(II)(dppf)Cl2, 1.99 g, 2.43 mmol) in degassed toluene (250 ml) and water (85 ml) was warmed to 76 ° C and stirred overnight. The mixture was quenched with half-saturated aqueous ammonium chloride solution at room temperature and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with a gradient of DCM / EtOAc) to give

[0625] Int-2da. LC / MS: (M-56+1) + =193.0.

[0626] Step B - Synthesis of Int-2db

[0627] To a solution of Int-2da (12.9 g, 51.7 mmol) and pent-4-ene-1-amine (6.61 g, 78 mmol) in DCM (120 ml) and AcOH (3 ml) was added sodium triacetoxyborohydride (32.9 g, 155 mmol) portionwise at room temperature in a water bath, and the mixture was stirred for 30 min. The reaction was slowly quenched with 3 ml of water at 0 ° C, poured into 1N NaOH (500 ml), stirred for 15 min, then extracted with DCM, dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (eluting with a gradient of DCM / MeOH) to give Int-2db. LC / MS: (M+1) + =319.2.

[0628] Step C - Synthesis of Int-2dc

[0629] To a solution of Int-2db (8.48 g, 20.77 mmol) and 4-methoxy-4-oxobutanoic acid (3.02 g, 22.85 mmol) in DMF (40 ml) was added HATU (9.48 g, 24.92 mmol) and DIPEA (8.71 ml, 49.8 mmol). The resulting solution was stirred at room temperature for 1 hour and then quenched with saturated aqueous NaHCO3 (10 mL). The mixture was partitioned between EtOAc (500 mL) and saturated aqueous NaHCO3 (200 mL). The organic phase was washed with brine (3×200 mL), dried over Na2SO4, concentrated, and the residue was purified on a silica gel column (eluted with a gradient of hexane / EtOAc) to give Int-2dc. LC / MS: (M+1) + =433.4.

[0630] Step D - Synthesis of Int-2d

[0631] To a solution of Int-2dc (2.9 g, 6.70 mmol) in DCM (15 mL) was added 4M HCl / dioxane (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure to give methyl 4-((4-(2-aminoethyl)benzyl)(pent-4-en-1-yl)amino)-4-oxobutanoate hydrochloride (Int-2d). LC / MS [M-HCl+H] + =333.3.

[0632] Int-3c was prepared from 107 used in the synthesis of Ex-01 and Ex-25 described above.

[0633] Intermediate Int-3c was prepared according to the following scheme:

[0634]

[0635] Step A - Synthesis of Int-3ca

[0636] To a solution of 107 (whose preparation was presented in the synthesis of 109 and later used in 116, the intermediate compound used in the synthesis of Ex-53, Ex-54, and Ex-55 in Example 3 below) (10.34 g, 21.65 mmol) in THF (100 ml) was added 2N lithium hydroxide monohydrate (43.3 ml, 87 mmol) at room temperature, and the mixture was warmed to 45° C. and stirred overnight to give Int-3ca as a crude solution. LC / MS: (M+1) + =464.3. The reaction mixture was cooled to 0°C and treated with 1M HCl (40 mL). The mixture was used directly in the next step.

[0637] Step B - Synthesis of Int-3c

[0638] To the crude Int-3ca prepared in the previous step was added NaHCO 3 (1.725 g, 20.54 mmol) and Fmoc-OSu (3.81 g, 11.30 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 2 h, treated with 1 M HCl (20.5 mL), and extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with brine (2×100 mL) and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with a gradient of 2% to 5% MeOH / DCM to give Int-3c. LC / MS: (M+1) + =686.4.

[0639] Preparation of intermediate Int-4b

[0640] Intermediate Int-4b was prepared according to the following scheme:

[0641]

[0642] Step A - Synthesis of Int-4ba from tert-butyl 3-(2-hydroxyethoxy)propionate

[0643] To a solution of tert-butyl 3-(2-hydroxyethoxy)propanoate (500.0 mg, 2.63 mmol) in DCM (2 mL) was added CBr4 (1395 mg, 4.21 mmol) and PPh3 (965 mg, 3.68 mmol) at 0 ° C. The mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluting with a gradient of 1%-15% ethyl acetate / petroleum ether. The fractions containing the desired product were combined and concentrated to give tert-butyl 6-bromohexanoate. A solution of tert-butyl 6-bromohexanoate (5 g, 19.91 mmol) in acetonitrile (10 ml) was treated with trimethylamine (13.56 ml, 59.7 mmol) and the resulting solution was heated at 50 ° C overnight. The solution was concentrated to give Int-4ba. LC / MS: M + =230.3.

[0644] Step B - Synthesis of Int-4b

[0645] To a solution of Int-4ba (6.8 g, 21.92 mmol) in DCM (6 ml) was added 4N HCl / dioxane (27.4 ml, 110 mmol) and the resulting solution was stirred at room temperature for 3 h. The mixture was then concentrated to give Int-4b. LC / MS: M + =174.3.

[0646] The preparation of intermediate Int-2d is described above for use in the preparation of Ex-01 and Ex-25. This portion of the molecule can be described as a "linker" which will carry R 1 、R 2 and R 8 The lower peptide ring of the substituent is cyclized. Other similar "linkers" can be used in place of Int-2d. The following is a description of other "linkers" that can be used to prepare the embodiments of the invention described herein.

[0647] Preparation of intermediate Int-2e

[0648] The intermediate Int-2e, which can be used as a "linker" in preparing the compounds of the present invention, is prepared according to the following scheme:

[0649]

[0650]

[0651] Step A - Synthesis of Intermediate Int-2ea

[0652] To a solution of tert-butyl (2-(3-oxoisoindolin-5-yl)ethyl)carbamate (1.60 g, 5.79 mmol) in DCE (20 mL) was added NsCl (1.93 g, 8.69 mmol), triethylamine (1.76 g, 17.4 mmol) and DMAP (0.141 g, 1.16 mmol). The reaction mixture was stirred at 40 ° C for 14 h. The reaction was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with a gradient of 1%-40% EtOAc / PE) to give Int-2ea. LC / MS: (M+Na) + :=484.4.

[0653] Step B - Synthesis of Intermediate Int-2eb

[0654] To a solution of Int-2ea (11.3 g, 24.5 mmol) in THF (100 mL) and water (100 mL) was added LiOH (1.76 g, 73.5 mmol). The reaction mixture was stirred at 25 ° C for 5 h, and then the resulting solution was adjusted to pH 4-5 by HCl (1 M). The solution was extracted with EtOAc and the combined organic layers were washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluted with a gradient of 1%-6% MeOH / DCM) to give Int-2eb. LC / MS: (M+Na) + :=502.2.

[0655] Step C - Synthesis of Intermediate Int-2ec

[0656] To a solution of Int-2eb (1.70 g, 3.55 mmol) in THF (8 mL) was added borane (0.147 g, 10.6 mmol) at 0 ° C. The reaction mixture was stirred at 25 ° C for 14 h, and the resulting solution was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluting with a gradient of 1%-50% EtOAc / PE) to give Int-2ec. LC / MS: (M+NH4] + =483.2.

[0657] Step D - Synthesis of Intermediate Int-2ed

[0658] To a solution of Int-2ec (4.50 g, 9.67 mmol) in DMF (150 mL) was added K2CO3 (2.01 g, 14.5 mmol) and 3-bromoprop-1-ene (1.41 g, 11.6 mmol). The reaction mixture was stirred at room temperature for 5 h, then diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluted with a gradient of 1%-50% EtOAc / PE) to give Int-2ed. LC / MS: (M+H) + :=506.2.

[0659] Step E - Synthesis of Intermediate Int-2e

[0660] To a solution of Int-2ed (4.50 g, 8.90 mmol) in DMF (35 mL) was added DBU (1.35 g, 8.90 mmol) and 2-mercaptoethanol (2.08 g, 26.7 mmol). The reaction mixture was stirred at room temperature for 14 h and then purified by column chromatography on C18 (column: 330 g; mobile phase A: water / 0.05% TFA, mobile phase B: ACN; flow rate: 85 mL / min; gradient: 10% B to 20% B in 15 min, 20% B to 45% B in 15 min; detector: UV 210 nm; Rt = 20 min) to afford Int-2e. LC / MS: (M+H) + :=321.2.1H NMR (300MHz, CDCl3) δ7.21-7.13(m,2H),7.10-7.01(m,1H),5.96-5.79(m,1H),5.30-5.09 (m,2H),4.58(s,2H),3.84(s,2H),3.43-3.19(m,4H),2.76(t,J=7.1Hz,2H),1.41(s,9H).

[0661] Preparation of intermediate Int-2f-1

[0662] The intermediate Int-2f-1, which can be used as a "linker" in the preparation of the compounds of the present invention, is prepared according to the following scheme:

[0663]

[0664]

[0665] Step A - Synthesis of Intermediate Int-2fa

[0666] To a solution of 4-bromobenzaldehyde (20.0 g, 108 mmol), (S)-2-methylpropane-2-sulfinamide (12.5 g, 103 mmol), and MgSO4 (130 g, 1081 mmol) in DCM (225 mL) was added pyridine 4-methylbenzenesulfonate (1.35 g, 5.40 mmol) under nitrogen. The mixture was stirred at 25°C for 72 h, then the resulting solution was filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (eluted with a gradient of 1% to 15% EtOAc / PE) to afford Int-2fa. LC / MS: (M+H) + :=287.9,289.9.

[0667] Step B - Synthesis of intermediate Int-2fb (racemate) and separation into enantiomers Int-2fb-1 and Int-2fb-2

[0668] To a solution of Int-2fa (20.0 g, 65.9 mmol) in anhydrous DCM (200 mL) was slowly added but-3-en-1-ylmagnesium bromide (15.7 g, 99 mmol) under nitrogen at -48 ° C. The mixture was stirred for 2 h at -48 ° C, then quenched with a saturated aqueous solution of NH4Cl (400 mL) and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue (containing the Int-2fb racemic mixture) was purified by column chromatography on silica gel (eluted with a gradient of 1%-35% EtOAc / PE) to obtain Int-2fb-1 and Int-2fb-2. LC / MS: (M+H) + :=344.0,346.0.

[0669] Step C - Synthesis of Intermediate Int-2fc-1

[0670] To a solution of HCl (100 mL, 4 N in 1,4-dioxane) at room temperature was added Int-2fb-1 (17.0 g, 46.9 mmol). The reaction solution was stirred for 1 h and then concentrated under reduced pressure to afford Int-2fc-1. LC / MS: (M+H-HCl) + :=240.0,242.0.

[0671] Step D - Synthesis of Intermediate Int-2fd-1

[0672] To a solution of Int-2fc-1 (8.20 g, 28.2 mmol) and Teoc-OSu (8.03 g, 31.0 mmol) in 1,4-dioxane (200 mL) was added TEA (8.55 g, 84 mmol) at 25 ° C. The mixture was stirred for 2 hours, then quenched with water and extracted with petroleum ether (PE). The combined organic layers were concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluted with a gradient of 1%-10% EtOAc / PE) to give Int-2fd-1. LC / MS: (M+Na+CH3CN) + :=447.3,449.3.

[0673] Step E - Synthesis of Intermediate Int-2fe-1

[0674] To a solution of Int-2fd-1 (15.1 g, 37.3 mmol), potassium (2-((tert-butoxycarbonyl)amino)ethyl)trifluoroborate (18.7 g, 74.6 mmol), Cs2CO3 (36.5 g, 112 mmol) in toluene (285 mL) and water (95 mL) was added PdCl2(dppf) (1.37 g, 1.87 mmol) under nitrogen. The mixture was stirred at 80°C for 40 h. The resulting solution was quenched with water and extracted with EtOAc. The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluted with a gradient of 1%-40% EtOAc / PE) to give Int-2fe-1. LC / MS: (M+Na) + :=471.4.

[0675] Step F-Synthesis of Intermediate Int-2f-1

[0676] To a solution of Int-2fe1 (10.6 g, 22.4 mmol) in THF (100 mL) was added 1N TBAF / THF (44.9 mL, 44.9 mmol). This mixture was stirred at room temperature for 16 h, then quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluting with a gradient of 1%-70% EtOAc / PE) followed by column chromatography on C18 (column: 330 g; mobile phase A: water (10 mm NH4HCO3), mobile phase B: ACN; flow rate: 80 mL / min; gradient: 10% B to 10% B in 10 min, 20% B to 45% B in 10 min, 45% B to 70% B in 20 min; detector: UV 210 nm; Rt = 25 min) to provide Int-2f-1. LC / MS: (M+H) + :=305.1.1H NMR(300MHz,CD3OD)δ7.27-7.16(m,4H),5.85-5.75(m,1H),5.00-4.85(m,2H),3.79(t, J=7.0Hz,1H),3.32-3.21(m,2H),2.75(t,J=7.4Hz,2H),2.98-1.72(m,4H),1.42(s,9H).

[0677] Example 2 Preparation of Ex-50 and Ex-52

[0678]

[0679] Compound Ex-50 was prepared according to the following scheme from compound Ex-01 (whose preparation is described herein in Example 1) by reacting it under appropriate conditions with intermediate Int32 prepared according to the following scheme:

[0680]

[0681] Step A: Preparation of intermediate Int-32A

[0682] To a solution of tert-butyl 3-(2-(2-bromoethoxy)ethoxy)propanoate (5 g, 16.82 mmol) in acetonitrile (10 ml) was added trimethylamine (33% in ethanol, 11.46 ml, 50.5 mmol) and the resulting solution was heated at 50° C. overnight. The solution was concentrated to give 2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)-N,N,N-trimethylethanolammonium bromide (Int 32A). LC / MS: (M) + :276.5.

[0683] Step B: Preparation of intermediate Int-32

[0684] To a solution of 2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)-N,N,N-trimethylethanolammonium bromide (Int-32A) (5.99 g, 16.81 mmol) in DCM (20 ml) was added HCl (4N in dioxane) (21.01 ml, 84 mmol) and the resulting solution was stirred at room temperature overnight. The solution was concentrated to give 2-(2-(2-carboxyethoxy)ethoxy)-N,N,N-trimethylethanolammonium bromide (Int-32). LC / MS: (M) + :220.1.

[0685] Preparation Example Compound Ex-50

[0686]

[0687] To a solution of Ex-01 (crude) (17.4 mg, 0.012 mmol) and 2-(2-(2-carboxyethoxy)ethoxy)-N,N,N-trimethylethanolammonium bromide (Int-32) (4.49 mg, 0.015 mmol) in DMF (2 ml) were added HATU (5.69 mg, 0.015 mmol) and DIEA (6.54 μl, 0.037 mmol), and the resulting solution was stirred at room temperature for 50 min and then purified by reverse phase HPLC using acetonitrile (0.1% formic acid) / water (0.1% formic acid) as the mobile phase to give Ex-50. LC / MS: M + =1596.3.

[0688] Preparation Example Compound Ex-52

[0689] Compound Ex-52 was prepared in a similar manner to compound Ex-50, except that Ex-51 was used instead of Ex-01. Ex-52 was purified using reverse phase HPLC according to the method described herein. LC / MS: M+ = 1593.8.

[0690] Example 3 Preparation of Ex-53, Ex-54 and Ex-55

[0691]

[0692]

[0693] Compounds Ex-53, Ex-54 and Ex-55 were prepared from intermediate 115 (prepared as described below) in a similar manner to the compounds described above according to the following scheme and synthetic description:

[0694] Step A - Synthesis of Intermediate 116

[0695] To a solution of 115 (66.3 mg, 0.044 mmol) in DMF (1.5 ml), DCM (10 ml) and water (0.5 ml) was added DIPEA (0.030 ml, 0.173 mmol) followed by HATU (18.50 mg, 0.049 mmol) at 0° C. and the mixture was stirred for 30 min. The mixture was concentrated in vacuo and directly purified by column chromatography on C18 (30 g, eluted with acetonitrile + 0.05% TFA / water + 0.05% TFA 90:10 to 40:60) to provide 116 as a mixture of E and Z isomers and 116 as pure fractions of E or Z isomers. LC / MS (major isomer) LC / MS: M + =1481.19; LC / MS (minor isomer): LC / MS: M + =1480.

[0696] Step B-Synthesis of Compound Ex-54

[0697] A solution of 116 (25.1 mg, 0.017 mmol) and Pd-C 10% (3.61 mg, 3.39 μmol) in MeOH (10 ml) was hydrogenated at 1 atm for 1 h. The reaction mixture was filtered through celite and concentrated. The residue was treated with DCM / TFA 1:1 for 30 min, then concentrated, treated with 4N HCl / dioxane (100 uL), and then concentrated to provide Ex-54 as an HCl salt. LC / MS: M + =1383.44.

[0698] Step C-Synthesis of Compound Ex-55

[0699] Example compound Ex-55 was prepared from Ex-54 as a formate salt in a manner consistent with that described for the synthesis of compound Ex-50 in Example 2. LC / MS: M + =1583.69. Step D-Synthesis of Compound Ex-53

[0700] Example compound Ex-53 was prepared from intermediate compound 116 as HCl salt in a manner consistent with that described for the synthesis of compound Ex-51 in Example 1. LC / MS: M + =1381.33.

[0701] The preparation of the intermediates required to provide intermediate compound 115 (from which compounds Ex-53, Ex-54 and Ex-55 were ultimately prepared) is described in the following schemes and syntheses, which begin with the preparation of intermediate compound 103.

[0702]

[0703] Step A - Synthesis of Intermediate 100

[0704] A solution of 4-bromo-2-hydroxybenzaldehyde (3.00 g, 14.92 mmol), potassium tert-butyl N-[2-trifluoroboryl)ethyl]carbamate (3.82 g, 15.22 mmol), cesium carbonate (17.02 g, 52.2 mmol) and 1,1'-bis(diphenylphosphino)ferrocene-dichloropalladium(ii) dichloromethane complex (0.611 g, 0.746 mmol) in degassed toluene (45 ml) and water (15 ml) was warmed to 75 ° C and stirred overnight. The mixture was quenched by half-saturated sodium bicarbonate aqueous solution at room temperature and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with hexane / EtOAc 99: 1 to 60: 40) to obtain 100. LC / MS: (M-55) + =210.25.

[0705] Step B - Synthesis of Intermediate 101

[0706] To a solution of 100 (1.70 g, 6.41 mmol) and allyl bromide (0.832 ml, 9.61 mmol) in DMF (10 ml) was added potassium carbonate (1.328 g, 9.61 mmol) at room temperature, and the mixture was warmed to 50 ° C and stirred for 1 h. The mixture was quenched by half-saturated aqueous sodium bicarbonate solution and extracted with EtOAc at room temperature. The combined organic fractions were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with hexane / EtOAc 99: 1 to 70: 30) to obtain 101. LC / MS: (M-55) + =250.29.

[0707] Step C - Synthesis of Intermediate 102

[0708] To a solution of 101 (1.76 g, 5.76 mmol), 4A molecular sieves (2 g) and ammonium acetate (4.44 g, 57.6 mmol) prepared in the previous step in MeOH (100 ml) was added sodium cyanoborohydride (0.380 g, 6.05 mmol) at room temperature, and the mixture was shaken overnight. The mixture was concentrated, quenched with water at room temperature and extracted with DCM. The combined organic fractions were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with DCM / MeOH 99:1 to 30:70) to provide 102. LC / MS: (2M+H) + =613.56.

[0709] Step D - Synthesis of Intermediate 103

[0710] To a solution of 102 (220 mg, 0.718 mmol) and monomethyl succinate (114 mg, 0.862 mmol) in DMF (4 ml) was added HATU (300 mg, 0.790 mmol) and DIPEA (0.314 ml, 1.795 mmol) at room temperature, and the mixture was stirred for 30 min. The mixture was quenched by saturated aqueous sodium bicarbonate solution at room temperature and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with hexane / EtOAc 99: 1 to 30: 70) to obtain an intermediate, which was treated with 20% TFA / DCM for 1 h. The reactant was concentrated, then treated with 4N HCl (1.5 mL) and concentrated to provide 103. LC / MS: (M+H) + =321.29.

[0711] Preparation of intermediate 109

[0712]

[0713] Step A - Synthesis of Intermediate 104

[0714] To a stirred solution of (S)-2-methylpyrrolidine-2-carboxylic acid methyl ester hydrochloride (7.00 g, 39 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxyphenyl)propanoic acid (12.08 g, 40.9 mmol) in DMF (100 ml) at 0° C. was added DIPEA (17.01 ml, 97.0 mmol) followed by HATU (19.26 g, 50.7 mmol). The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with 10% aqueous LiCl solution and extracted with EtOAc. The organic extract was washed with 10% aqueous LiCl solution and dried over MgSO 4. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel (eluting with hexane / EtOAc 80:20 to 40:60) to provide 104.

[0715] Step B - Synthesis of Intermediate 105

[0716] To a solution of 104 (16.4 g, 39.0 mmol) in EtOAc (100 ml) was added 4N HCl / dioxane (48.8 ml, 195 mmol). The resulting mixture was stirred at room temperature for 18 h and concentrated under reduced pressure to give 105 which was used in the next step without further purification.

[0717] Step C - Synthesis of Intermediate 106

[0718] To a solution of 105 (13.2 g, 37.0 mmol) and N-((benzyloxy)carbonyl)-O-(tert-butyl)-L-threonine (18.15 g, 37.0 mmol) in DMF was added DIPEA (16.15 ml, 92 mmol) followed by HATU (18.28 g, 48.1 mmol) at 0°C. The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with 10% aqueous LiCl solution and extracted with EtOAc. The organic extract was washed with 10% aqueous LiCl solution and dried over MgSO4. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel (eluting with hexane / EtOAc 80:20 to 40:60) to provide 106.

[0719] Step D - Synthesis of Intermediate 107

[0720] To a solution of 106 (16.5 g, 27.0 mmol) in MeOH was added a slurry of 10% Pd / C and the mixture was hydrogenated at 20 psi for 4 h. The reaction mixture was filtered through celite and concentrated under reduced pressure. The crude product was then redissolved in DCM, and the solution was filtered through a 2 μm filter and concentrated to give 107.

[0721] Step E - Synthesis of Intermediate 108

[0722] To a solution of 107 (3.2 g, 6.70 mmol) in DCM was added DIPEA (1.52 ml, 8.71 mmol) followed by di-tert-butyl dicarbonate (1.90 g, 8.71 mmol). The resulting mixture was stirred at room temperature for 2 h and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with hexane / EtOAc 100:0 to 40:60) to give 108.

[0723] Step F - Synthesis of Intermediate 109

[0724] A solution of 108 (1.43 g, 2.475 mmol) and 1N LiOH aqueous solution (9.90 ml, 9.90 mmol) in THF (15 ml) and MeOH (15 ml) was warmed to 45 ° C and stirred for 4 h, then stirred at 32 ° C for 48 h. The reaction was concentrated, quenched with 0.5 M aqueous hydrochloric acid at 0 ° C until pH ~ 2-3, and extracted with EtOAc. The combined organic fractions were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with hexane / EtOAc-EtOH 99: 1 to EtOAc-EtOH 3: 1) to give 109. LC / MS: (M + H) + =564.49.

[0725] Preparation of intermediate compounds 110 to 115

[0726] Step A - Synthesis of Intermediate 110

[0727]

[0728] A solution of 109 (217 mg, 0.385 mmol), HATU (133 mg, 0.350 mmol) and DIPEA (0.245 ml, 1.400 mmol) in DMF (2.5 ml) was treated with 103 (217 mg, 0.385 mmol) at 0 ° C, and the mixture was warmed to room temperature and stirred for 30 min. The mixture was quenched with saturated aqueous sodium bicarbonate solution at 0 ° C and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with hexane / EtOAc-EtOH 3: 1 99: 1 to EtOAc-EtOH 3: 1) to obtain 110. LC / MS: (M+H) + =866.21.

[0729] Step B and Step C - Synthesis of Intermediate Compounds 111 and 112

[0730]

[0731] To a solution of 110 (249 mg, 0.288 mmol) in DCM (1 ml) was added HCl 4N / dioxane (0.359 ml, 1.438 mmol) at room temperature, and the mixture was stirred for 6 h, then concentrated to provide 111. LC / MS: (M+H) + =710.19.

[0732] To a solution of 111 (219 mg, 0.293 mmol) and 76 (232 mg, 0.285 mmol) in DMF (3 ml) and water (0.15 ml) was added DIPEA (0.128 ml, 0.734 mmol) and HATU (123 mg, 0.323 mmol) at 0 ° C, and the mixture was stirred for 30 min. The mixture was quenched with brine and extracted with EtOAc at 0 ° C. The combined organic fractions were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with hexane / EtOAc-EtOH3-1 99: 1 to 30: 70, then with DCM / MeOH 99: 1 to 70: 30) to give 112. LC / MS: (M+H) + =1506.11.

[0733] Step D - Synthesis of Intermediate 113

[0734]

[0735] To a solution of 112 (173 mg, 0.115 mmol) in DCM (180 ml) and AcOH (15 ml) degassed with nitrogen for 30 min was added Jan's catalyst (59.0 mg, 0.080 mmol), and the mixture was warmed to 50 ° C and stirred for 3 h. The mixture was filtered through celite, washed with DCM, and then concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with DCM / MeOH 99: 1 to 80: 20) to give 113 as a mixture of E and Z isomers. LC / MS (major isomer): (M) + =1477.80; LC / MS (minor isomer): (M) + =1478.28.

[0736] Step E - Synthesis of Intermediate 114

[0737]

[0738] To a solution of 113 (141 mg, 0.095 mmol) in acetonitrile (2 ml) was added piperidine (0.066 ml, 0.668 mmol), and the mixture was stirred for 45 min. The mixture was concentrated in vacuo and immediately co-evaporated with acetonitrile to give the crude material. To a slurry of this crude material (119 mg, 0.095 mmol) and intermediate compound 88 (52.0 mg, 0.105 mmol) in DMF (2 ml) and water (0.1 ml) at 0° C. was added HATU (39.7 mg, 0.105 mmol) and DIPEA (0.037 ml, 0.209 mmol), and the mixture was stirred for 30 min. The mixture was purified by column chromatography on C18 (eluting with acetonitrile + 0.05% TFA / water + 0.05% TFA 90:10 to 30:70) to give 114 as a mixture of E and Z isomers. LC / MS major isomer: (M) + =1735.28; LC / MS (minor isomer): (M) + =1735.25.

[0739] Step F - Synthesis of Intermediate Compound 115

[0740]

[0741] To a solution of 114 (134 mg, 0.077 mmol) in THF (1.5 ml) and MeOH (1.5 ml) was added 1 N aqueous LiOH (0.386 ml, 0.386 mmol) dropwise at 0°C, and the mixture was stirred for 2 h. The reaction was treated dropwise with 0.5 N HCl at 0°C until pH ~7, concentrated from the organic solvent, and the slurry was then dissolved with ~1 mL of DMF and directly purified by column chromatography on C18 (eluted with acetonitrile + 0.05% TFA / water + 0.05% TFA 90:10 to 50:50) to give 115 as a mixture of E and Z isomers. LC / MS major isomer: (M) + =1498.71; LC / MS (minor isomer): (M) + =1499.48.

[0742] As described above in Ex-01 and Ex-54 by making the respective starting compounds R 2 As described for the reaction of amides with acidic substituent precursors to prepare Ex-50 and Ex-55, the following intermediate compounds can be similarly reacted to provide useful compounds of the present invention.

[0743] Synthetic R 1 / R 2 Substituent precursors:

[0744] Preparation of 5-carboxy-N-(3-methoxypropyl)-N,N-dimethylpentane-1-ammonium chloride (Intermediate Z-1a)

[0745] Step A: Preparation of Intermediate Z-1

[0746]

[0747] To a stirred solution of tert-butyl 6-(dimethylamino)hexanoate (300 mg, 1.393 mmol) in acetonitrile (1 mL) was added 1-bromo-3-methoxypropane (853 mg, 5.57 mmol). The reaction mixture was stirred at 50 ° C for 16 h. The resulting mixture was concentrated under reduced pressure to give Z-1. LC / MS: (M-Br) + =288.4. 1 H NMR (300MHz, CDCl3): δ3.76-3.47(m,6H),3.38(d,J=28.9Hz,9H),2.25(t,J=7.2Hz,2H),2.12-1.95(m,2H),1.87-1.55(m,4H),1.45(s,11H).

[0748] Step B: Synthesis of intermediate compound Z-1a

[0749]

[0750] To a stirred solution of Z-1 (460 mg, 1.249 mmol) in DCM (0.5 mL) was added 4 M HCl / dioxane (2 mL) at room temperature. The reaction mixture was stirred for 4 h at room temperature and concentrated under reduced pressure. The residue was redissolved in DCM (5 mL) and concentrated under reduced pressure to give the intermediate compound Z-1a. LC / MS: (M-Cl) + =232.3.

[0751] Preparation of intermediate Z-2b

[0752] Step A: Preparation of intermediate Z-2

[0753]

[0754] To a stirred solution of tert-butyl 6-bromohexanoate (1.0 g, 3.98 mmol) in THF (10 mL) was added dimethylamine (2 M in THF) (7.96 mL, 15.93 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with a gradient of 1%-15% MeOH / DCM. The fractions containing the desired product were combined and concentrated to give Z-2. LC / MS: (M+H) + =216.2.1 H NMR (300MHz, CDCl3): δ2.35-2.17 (m, J=8.5, 6.5Hz, 10H), 1.67-1.47 (m, 4H), 1.45 (s, 9H), 1.42-1.23 (m, 2H).

[0755] Step B: Preparation of intermediate Z-2a

[0756]

[0757] To a stirred solution of Z-2 (250 mg, 1.161 mmol) in ACN (1 mL) was added 1-bromo-2-methoxyethane (645 mg, 4.64 mmol). The reaction mixture was stirred at 50 °C for 16 h. The resulting mixture was concentrated under reduced pressure to afford Z-2a. LC / MS: (M-Br) + =274.3. 1 H NMR (300MHz, CDCl3): δ4.02-3.80 (m, 4H), 3.70-3.54 (m, 2H), 3.42 (d, J = 13.0Hz, 9H), 2.24 (t, J = 7.2Hz, 2H), 1.85-1.75 (m, 2H), 1.72-1.55 (m, 2H), 1.44 (s, 11H).

[0758] Step C: Preparation of intermediate Z-2b

[0759]

[0760] To a stirred solution of Z-2a (450 mg, 1.270 mmol) in DCM (0.5 mL) was added 4 M HCl / dioxane (2 mL) at room temperature. The reaction mixture was stirred for 4 h at room temperature and concentrated under reduced pressure. The residue was redissolved in DCM (5 mL) and concentrated under reduced pressure to give Z-2b. LC / MS: (M-Cl) + =218.3.

[0761] Preparation of intermediate Z-3b

[0762] Step A: Preparation of intermediate Z-3

[0763]

[0764] To a solution of tert-butyl 3-(2-hydroxyethoxy)propionate (500.0 mg, 2.63 mmol) in DCM (2 mL) was added CBr (1395 mg, 4.21 mmol) and PPh (965 mg, 3.68 mmol) at 0°C. The mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with a gradient of 1%-15% EA / PE. The fractions containing the desired product were combined and concentrated to give Z-3. 1 H NMR (400MHz, CDCl3): δ3.78 (dt, J=11.1, 6.3Hz, 4H), 3.47 (t, J=6.3Hz, 2H), 2.53 (t, J=6.4Hz, 2H), 1.48 (s, 9H).

[0765] Step B: Synthesis of intermediate Z-3a

[0766]

[0767] To a stirred solution of tert-butyl 3-(2-bromoethoxy)propanoate Z-3 (450 mg, 1.778 mmol) in ACN (2 mL) was added trimethylamine (955 mg, 5.33 mmol) (33% Wt in EtOH). The reaction mixture was stirred at 50 ° C for 16 h. The resulting mixture was concentrated under reduced pressure to give Z-3a. LC / MS: (M-Br) + =232.3. 1 H NMR (400MHz, CDCl3): δ5.32 (s, 1H), 4.04-3.94 (m, 4H), 3.73 (t, J = 5.7Hz, 2H), 3.50 (s, 10H), 2.50 (t, J = 5.7Hz, 2H), 1.44 (s, 9H).

[0768] Step C: Synthesis of intermediate Z-3b

[0769]

[0770] To a solution of Z-3a (550 mg, 1.761 mmol) in DCM (0.6 mL) was added 4 M HCl / dioxane (2.5 mL) at room temperature. The mixture was stirred at room temperature for 4 h. The resulting mixture was concentrated under reduced pressure, and the residue was redissolved in DCM (3 mL) and toluene (3 mL). The mixture was then concentrated under reduced pressure to give Z-3b. LC / MS: (M-Cl) + =176.2.

[0771] Preparation of intermediate Z-4b

[0772] Step A: Preparation of intermediate Z-4

[0773]

[0774] To a solution of DIAD (1.755 mL, 9.03 mmol) in THF (30 mL) was added Ph3P (2.368 g, 9.03 mmol). The mixture was stirred at room temperature for 10 min, and then 2-(3-hydroxyphenyl)acetic acid methyl ester (1.0 g, 6.02 mmol) and 3-(dimethylamino)propan-1-ol (0.931 g, 9.03 mmol) were added to the solution. The mixture was stirred at 50 ° C for 1 h. The resulting solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with a gradient of 1%-10% MeOH / DCM. The fractions containing the desired product were combined and concentrated to give Z-4. LC / MS: (M+H) + =252.2. 1 H NMR (300MHz, CDCl3): δ7.23-7.18(m,1H),6.82(td,J=8.7,4.1Hz,3H),4.01(t,J=6.4Hz,2H) ,3.69(s,3H),3.59(s,2H),2.46(t,J=7.3Hz,2H),2.27(s,6H),1.97(dt,J=7.9,6.5Hz,2H).

[0775] Step B: Preparation of intermediate Z-4a

[0776]

[0777] To a solution of Z-4 (600 mg, 2.268 mmol) in ACN (12 mL) was added MeI (1.288 g, 9.07 mmol). The mixture was stirred at room temperature for 1 h. The resulting solution was concentrated under reduced pressure to give Z-4a. LC / MS: (MI) + =266.2.

[0778] Step C: Preparation of intermediate Z-4b

[0779]

[0780] To a solution of Z-4a (800 mg, 1.729 mmol) in THF (12 mL) was added 2 M LiOH (1.729 mL, 3.46 mmol). The mixture was stirred at room temperature for 2 h. The pH of the solution was adjusted to 4 by HCl (1 M), and the solution was concentrated under reduced pressure. The crude product was purified by reverse phase chromatography on C18 (mobile phase A: water, mobile phase B: ACN; flow rate: 60 mL / min; gradient: 1% B to 25% B in 25 min; 25% B to 95% B in 15 min; 95% B to 95% B in 10 min) to give Z-4b. LC / MS: (M-Cl) + =252.2. 1 H NMR (300MHz, CD3OD): δ7.21 (t, J = 7.9 Hz, 1H), 6.95-6.75 (m, 3H), 4.12 (t, J = 5.7 Hz, 2H), 3.63-3.50 (m, 4H), 3.18 (s, 9H), 2.35-2.20 (m, 2H).

[0781] Example 4 Preparation of Ex-23

[0782]

[0783] Step A: Preparation of intermediate S-1b

[0784] (2S, 3R, 4S, 5S, 6R) -6- (acetoxymethyl) tetrahydro -2H- pyran -2,3,4,5- tetraacetic acid tetrayl ester S-1a (5 g, 12.81 mmol) was added to a solvent of 48% HBr (7.25 mL, 64.0 mmol) in AcOH and DCM (40 mL) at 0 ° C, and the mixture was stirred at 0 ° C for 1 hour. The reaction mixture was poured into a saturated aqueous sodium bicarbonate solution cooled on ice, and the mixture was extracted with DCM (3 × 60 mL). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated, and the crude product was purified by flash chromatography on silica gel (eluted with 0-30% EtOAc / PE) to S-1b.

[0785] Step B: Preparation of intermediate S-1c

[0786] To a solution of S-1b (4.5 g, 10.94 mmol) in anhydrous DMF (45 mL) was added sodium azide (0.854 g, 13.13 mmol), and the reaction was stirred at 18° C. for 30 min. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3×80 mL). The organic layer was dried over Na 2 SO 4 and evaporated to dryness. The crude product was purified by flash chromatography on silica gel (eluting with 0-30% EtOAc / PE) to afford S-1c.

[0787] Step C: Preparation of intermediate S-1d

[0788] To a solution of S-1c (3.15 g, 8.44 mmol) in EtOH (60 mL) was added 10% Pd-C (0.898 g, 0.844 mmol). The reaction vessel was purged from air and filled with H at 50 psi. The reaction was stirred at 18 ° C for 5 h. The reaction mixture was diluted with EtOAc, filtered through celite, and concentrated to give S-1d for the next step.

[0789] Step D: Preparation of intermediate S-1e

[0790] To a solution of S-1d (2.34 g, 6.74 mmol) in anhydrous THF (20 mL) was added dihydrofuran-2,5-dione (0.742 g, 7.41 mmol) and Et3N (0.939 mL, 6.74 mmol). The reaction was stirred for 3 h until the starting material was completely consumed and then evaporated. The crude product was purified by flash chromatography on silica gel (eluted with 0-10% DCM / MeOH) to afford S-1e. MS (ESI): m / z (M+H) + 448.1. 1HNMR (400MHz, CDCl3) δ: 6.48 (d, J = 9.04Hz, 1H), 5.43 (s, 1H), 5.24 (t, J = 8.93Hz, 1H), 5.07-5.17 (m, 2H), 4.08-4.1 8(m,2H),4.04(q,J=6.69Hz,1H),2.72-2.84(m,1H),2.58-2.69(m,2H), 2.43-2.53(m,2H),2.15(s,3H),2.06(s,3H),2.04(s,4H),2.00(s,3H).

[0791] Step E: Preparation of Ex-23

[0792] To a solution of Ex-01 (300 mg, 0.215 mmol) and S-1e (115 mg, 0.258 mmol) in DMF (8 ml) and water (0.4 ml) was added DIEA (0.150 ml, 0.860 mmol) and HATU (98 mg, 0.258 mmol), and the resulting solution was stirred at room temperature for 1 h. The reaction was quenched dropwise with 1N LiOH (2.58 ml, 2.58 mmol), and the resulting solution was stirred at room temperature for 2 h, then filtered, and the filtrate was purified on a reverse phase HPLC C18 column using a 29% to 34% gradient of acetonitrile (0.05% TFA) / water (0.05% TFA) to afford Ex-23. LC / MS: [M+1] = 1657.1.

[0793] Example 5 Preparation of Ex-14

[0794]

[0795] Compound Ex-14 was prepared in a manner similar to the procedure described in Example 1, but using different "linkers" and alternative synthetic steps. The synthesis of these "linkers", alternative steps and primary assembly are described below.

[0796] Preparation of intermediate Int-2g

[0797] Intermediate Int-2g, which can be used as a "linker" in preparing the compounds of the present invention, is prepared according to the following scheme:

[0798]

[0799]

[0800] Step A - Synthesis of Int-2gb

[0801] To a solution of Int-2da (0.5 g, 2 mmol) and 2-azidoethanolamine, HCl (0.246 g, 2 mmol) in THF (16 ml) was added sodium triacetoxyborohydride (1.06 g, 5 mmol) in a water bath at room temperature, and the mixture was stirred for 2 h. The reaction was slowly quenched with saturated aqueous NaHCO3, then extracted with DCM and washed with brine. The combined organic layers were dried over MgSO4 and concentrated to give Int-2gb. LC / MS: (M+1) + =320.3.

[0802] Step B - Synthesis of Int-2gc

[0803] To a solution of Int-2gb (0.64 g, 2 mmol) and monomethyl succinate (0.3 g, 2.3 mmol) in DMF (4 ml) and DCM (8 ml) was added HATU (0.914 g, 2.4 mmol) and DIPEA (0.7 ml, 4.01 mmol) at -15 ° C. The resulting solution was stirred for 2 hours at -15 ° C., then quenched with water and concentrated. The residue was purified by reverse phase chromatography on C18 (eluted with acetonitrile / water + 0.1% TFA) to give Int-2gc. LC / MS: (M+1) + =434.3.

[0804] Step C - Synthesis of Int-2g

[0805] To a solution of Int-2gc (0.52 g, 1.2 mmol) in DCM (9 mL) was added TFA (3 mL, 38.9 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure to give Int-2g. LC / MS: (M+1) + =334.3.

[0806] Preparation of intermediate compounds 70B and 76C

[0807]

[0808] Step A - Synthesis of Intermediate 70B

[0809] To a solution of 69B (1.5 g, 4.46 mmol) in DMF (17.8 ml) was added 95% NaH (0.141 g, 5.56 mmol) at 0°C and the resulting solution was stirred at 0°C for 20 min, followed by the dropwise addition of 3-bromoprop-1-yne (80% in toluene) (0.596 ml, 5.35 mmol). To the resulting solution was added aqueous lithium hydroxide (2 M) (3345 μl, 6.69 mmol). The reaction was stirred at room temperature for 2 h, filtered, and purified by reverse phase HPLC (eluting with acetonitrile / water + 0.1% TFA) to afford 70B. LC / MS: (M+1) + :361.0,(M+Na) + :383.0.

[0810] Step B - Synthesis of Intermediate 76C

[0811] Conversion of 70B to intermediate 76C was carried out according to procedures similar to those described in preparation steps C to H of intermediate 76. LC / MS: [M+1]+ = 812.16.

[0812] Assembled as Example Ex-14:

[0813]

[0814]

[0815] Step A - Synthesis of Intermediate 117

[0816] Intermediate 117 was prepared from intermediates Int-2g and 76C according to procedures similar to those described in Examples 1 and 1A. More specifically, Int-2g was functionalized following the reagents and procedures of Preparation Steps A to B of Intermediate 77B, further detailed as the following procedures of Preparation Steps G to J of Intermediate 86, and then finally coupled with Intermediate 76C following the procedures of Preparation Steps B to C of Example 1A to provide 117. LC / MS: (M+1)+: 1573.36.

[0817] Step B - Synthesis of Intermediate 118

[0818] Tetrakis(acetonitrile)copper(I)hexafluorophosphate (51.6 mg, 0.138 mmol), tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine (73.4 mg, 0.138 mmol) and sodium ascorbate (137 mg, 0.692 mmol) in BuOH (185.00 mL) / water (93 mL) was sparged with nitrogen and then heated at 50° C. Intermediate 117 (435.3 mg, 0.277 mmol) was added to the reaction as a solid. After 1 h, the reaction was treated with pH 4 aqueous buffer and extracted with EtOAc. The combined organic layers were evaporated and the residue was purified by reverse phase chromatography (eluting with a gradient of acetonitrile / water + 0.1% formic acid) to provide intermediate 118. LC / MS: (M+1)+: 1573.2.

[0819] Step C - Synthesis of Ex-14

[0820] Example Ex-14 was synthesized from intermediate 118 according to procedures similar to those described in Example 1, including the use of alternative spacers for assembly. LC / MS: [M+1]+ = 1621.01.

[0821] Using the synthetic schemes described above, and as will be appreciated, in some cases where appropriate substitutions were made to certain intermediates, including the use of alternative spacers apparent to those skilled in the art, the following compounds of the invention, listed below in Table 2, whose preparation may be described above, were prepared. Additionally, alternative salt forms of the compounds of the invention may also be described herein:

[0822] Table 2

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842] Activity assay

[0843] Selected compounds of the present invention were subjected to one or more of the following procedures to determine their activity in antagonizing PCSK9 activity.

[0844] The following is a description of the assay used to determine the compounds of the invention and any reported comparator compounds for PCSK9 antagonism. Biotinylated PCSK9 was commercially available.

[0845] LDLR TR-FRET

[0846] The PCSK9 TR-FRET assay measures the interaction between PCSK9 and LDLR. A solution containing 40 nM biotinylated PCSK9 and 10 nM Lance ULight Streptavidin was prepared in 50 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.01% BSA, and 0.01% surfactant P20. A separate solution containing 40 nM rhLDLR-6xHis and 10 nM Eu-W1024 anti-6xHis was prepared in the same buffer system. An Echo was used to transfer 0.750 μl of compound to the assay plate, followed by the addition of 15 μl of PCSK9+Ulight and 15 μl of LDLR+Eu. The final assay volume was 30.750 μl, containing 20 nM PCSK9, 5 nM Ulight, 20 nM LDLR, and 5 nM Eu. The reaction was incubated at room temperature for at least two hours, and then fluorescence was measured using an Envision Multilabel reader. IC50 values ​​were determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. Europium-labeled LDLR counts (B counts) were tracked to observe whether the compound adversely affected LDLR. A decrease in B counts could indicate a false positive for inhibition.

[0847] AlexaFRET Standard TR-FRET

[0848] PCSK9 Alexa FRET standard assay for PCSK9 and AlexaFluor647 (AF) labeled cyclic peptide (reagent A (K D =83nM)) were measured. A solution containing 1nM biotinylated PCSK9 + 2.5nM Lance Streptavidin Europium (Strep-Eu) was prepared in 50mM HEPES pH 7.4, 0.15M NaCl, 5mM CaCl2, 0.01% BSA, and 0.01% surfactant P20. A separate solution containing 40nM AlexaFluor-labeled cyclic peptide was prepared in the same buffer system. An Echo was used to transfer 0.750ul of compound to the assay plate, followed by the addition of 15ul of PCSK9 + Stept-Eu and 15ul of AF peptide. The final assay volume was 30.750ul, containing 0.5nM PCSK9, 1.25nM Strep-Eu, and 20nM AF cyclic peptide. The reaction was incubated at room temperature for at least two hours, and then fluorescence was measured using an Envision Multilabel reader. IC50 values ​​were determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. DTo calculate Ki. Europium-labeled PCSK9 counts (B counts) are tracked to see if the compound is adversely affecting PCSK9. A decrease in B counts may indicate a false positive for inhibition. Data from this program are reported as "A = 'Number' (nanomoles)"

[0849] Prepare Reagent A as follows:

[0850]

[0851] Step A-Synthesis of Intermediate Compound Int-A

[0852] PS Rink-Amide MBHA resin (0.32 mmol g) was synthesized using Fmoc / tBu chemistry on a CEM Liberty Blue Microwave synthesizer. -1 The peptide was synthesized on a 0.250 mmol scale using a single coupling using 4 equivalents of the Fmoc-protected amino acid 0.2 M / DMF, 4 equivalents of 0.5 M HATU / DMF, and 4 equivalents of 2 M DIPEA (double coupling for Tyr). Fmoc deprotection cycles were performed using 20% ​​(v / v) piperidine / DMF.

[0853] The sequences of the Fmoc-protected amino acids and building blocks used were:

[0854] 1. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-trityl-L-cysteine

[0855] 2. (S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpyrrolidine-2-carboxylic acid

[0856] 3. (((9H-fluoren-9-yl)methoxy)carbonyl)-L-tyrosine

[0857] 4. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-trityl-L-histidine

[0858] 5. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid

[0859] 6. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1H-indol-3-yl)propanoic acid

[0860] 7. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1H-indol-3-yl)propanoic acid

[0861] 8. (((9H-fluoren-9-yl)methoxy)carbonyl)glycine

[0862] 9.N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 -(tert-Butoxycarbonyl)-L-lysine

[0863] 10. 3-(Triphenylmethylthio)propionic acid

[0864] At the end of the assembly, the resin was washed with DMF, MeOH, DCM, and Et2O. The peptide was cleaved from the solid support using 50 ml of a TFA solution (v / v) (91% TFA, 5% H2O, 4% TIPS) at room temperature for approximately 1.5 hours. The resin was filtered, washed with TFA, and the solution was concentrated to dryness and lyophilized. Lyophilization gave the intermediate compound Int.A (399 mg), which was used crude in the next step. LCMS analysis calculated for C61H75F2N15O13S2: 1328.48, found: 1328.2 (M+1) +

[0865] Step B - Synthesis of intermediate compound Int-B: as described for reagent B

[0866] Purification was performed by RP-HPLC (Waters Deltapak C4, dual cartridge, 40×100 mm, 15 μm, 300 Å; 15% to 35% ACN / water with 0.1% TFA modifier over 20 min). The collected fractions were lyophilized to give 35 mg of intermediate compound Int-B. LCMS analysis calculated for C69H81F2N15O13S2: 1430.62; found: 1430.9 (M+1) +

[0867] Step C - Synthesis of Compound Reagent A: As described for Reagent B

[0868] C105H122F2N17O26S6 3- LCMS analysis calculated value: 2268.58; 1135.8 (M+2) 2+

[0869] Alexa FRET Plus TR-FRET

[0870] The PCSK9 Alexa FRET Plus assay is a method for the binding of PCSK9 to AlexaFluor 647 (AF)-labeled cyclic peptides (Reagent B (K D=35nM)) were measured. A solution containing 1nM biotinylated PCSK9 + 2.5nM Lance Streptavidin Europium (Strep-Eu) was prepared in 50mM HEPES pH 7.4, 0.15M NaCl, 5mM CaCl2, 0.01% BSA, and 0.01% surfactant P20. A separate solution containing 1920nM AlexaFluor-labeled cyclic peptide was prepared in the same buffer system. An Echo was used to transfer 0.075ul of compound plus 0.675ul of DMSO to each well of the assay plate, followed by the addition of 15ul of PCSK9 + Stept-Eu and 15ul of AF peptide. The final assay volume was 30.750ul, containing 0.5nM PCSK9, 1.25nM Strep-Eu, and 960nM AF cyclic peptide. The reaction was incubated at room temperature for at least two hours, and then fluorescence was measured using an Envision Multilabel reader. IC50 values ​​were determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. D To calculate Ki. Europium-labeled PCSK9 counts (B counts) are tracked to see if the compound adversely affects PCSK9. A decrease in B counts may indicate a false positive for inhibition. Data from this procedure are reported as "P = 'Number' (nanomolar)"

[0871] Reagent B was prepared by the following procedure.

[0872]

[0873]

[0874] Step A-Synthesis of Intermediate Compound Int-A

[0875] PS Rink-Amide MBHA resin (0.32 mmol g) was synthesized using Fmoc / tBu chemistry on a CEM Liberty Blue Microwave synthesizer. -1 The peptide was synthesized on a 0.250 mmol scale using a single coupling using 4 equivalents of an Fmoc-protected amino acid at 0.2 M in DMF, 4 equivalents of 1 M Oxyme in DMF, and 4 equivalents of 0.5 M N,N-diisopropylcarbodiimide (DIC) (double coupling of Y01). Fmoc deprotection cycles were performed using 20% ​​(v / v) piperidine in DMF.

[0876] The sequences of the Fmoc-protected amino acids and building blocks used were:

[0877] 1. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-trityl-L-cysteine

[0878] 2. (S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpyrrolidine-2-carboxylic acid

[0879] 3. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propanoic acid

[0880] 4. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-trityl-L-histidine

[0881] 5. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid

[0882] 6. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1H-indol-3-yl)propanoic acid

[0883] 7. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1H-indol-3-yl)propanoic acid

[0884] 8. (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanine

[0885] 9.N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 -(tert-Butoxycarbonyl)-L-lysine

[0886] 10. 3-(Triphenylmethylthio)propionic acid

[0887] At the end of the assembly, the resin was washed with DMF, MeOH, DCM, and Et2O. The peptide was cleaved from the solid support using 50 ml of a TFA solution (v / v) (91% TFA, 5% H2O, 4% TIPS) at room temperature for about 1.5 hours. The resin was filtered, washed with TFA, and the solution was concentrated to dryness and lyophilized. Lyophilization gave the intermediate compound Int.A (300 mg), which was used as crude material in the next step. LCMS analysis calculated for C63H79F2N15O13S2: 1356.53, found: 1356.9 (M+1) +

[0888] Step B-Synthesis of Intermediate Compound Int-B

[0889] The crude material Int-A (0.22 mmol) was redissolved in 24 ml of DMF. 6 ml of 1 M aqueous sodium bicarbonate solution was added to raise the pH to 7. 0.26 mmol of 1,3-bis(bromomethyl)benzene (0.1 M in DMF) was then added dropwise. The reaction was allowed to stand at room temperature with stirring for 20 min, quenched with TFA (pH 3-4), and then concentrated in vacuo to provide crude Int-B, which was purified by RP-HPLC (Waters XBridge, C18, 50×150 mm, 5 μm, 130A; 25% to 40% ACN / water + 0.1% TFA modifier over 20 min). The collected fractions were lyophilized to give 35 mg of the intermediate compound Int-B. LCMS analysis calculated for C71H85F2N15O13S2: 1458.67; found: 1458.8 (M+1) +

[0890] Step C-Synthesis of Compound Reagent B

[0891] The intermediate compound Int-B (15 mg) was dissolved in 0.2 ml of anhydrous DMSO. Then 15 mg of ALEXAFLUOR 647 NHS ester (A37566, Life Technology) dissolved in 1.5 ml of anhydrous DMSO was added. 20 uL of anhydrous DIPEA was added. The reaction was allowed to stand under stirring at room temperature in the dark for 12 h under a nitrogen atmosphere. It was quenched with TFA (pH 3-4) and the product was analyzed by RP-HPLC (Dr Maish, Reprosil Gold C18, 250×20 mm, 10 μm; 20% to 35% 0.1% TFA / ACN / 0.1% TFA / H2O over 20 min, then 35% to 40% over 5 min at a flow rate of 20 mL / min). The collected fractions were lyophilized to give 16.1 mg of compound reagent B. C107H126F2N17O26S6 3- LCMS analysis value: 2296.64; found value: 1150.6 (M+2) 2+

[0892] Activity data obtained by one or both of the above procedures are reported for selected example compounds of the present invention in the following format:

[0893] Example No.: A (standard TR FRET) = 'value'; P (Alexa Fret plus standard TR FRET) = 'value' / , Note that all reported values ​​are in nanomolar.

[0894] Alexa FRET Ultra TR-FRET

[0895] PCSK9 Alexa FRET Ultra assay for PCSK9 and AlexaFluor647 (AF) labeled cyclic peptide (Reagent B (K D =0.99nM)) were measured. A solution containing 1nM biotinylated PCSK9 + 2.5nM Lance Streptavidin Europium (Strep-Eu) was prepared in 50mM HEPES pH 7.4, 0.15M NaCl, 5mM CaCl2, 0.01% BSA, and 0.01% surfactant P20. A separate solution containing 1920nM AlexaFluor labeled cyclic peptide was prepared in the same buffer system. An Echo was used to transfer 0.015ul of compound plus 0.735ul of DMSO to each well of the assay plate, followed by the addition of 15ul of PCSK9 + Stept-Eu and 15ul of AF peptide. The final assay volume was 30.750ul, which contained 0.5nM PCSK9, 1.25nM Strep-Eu, and 960nM AF cyclic peptide. The reaction was incubated at room temperature for at least two hours, and then fluorescence was measured using an Envision Multilabel reader. IC50 values ​​were determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. The IC50 values ​​were then calculated based on the K values ​​of the AF cyclic peptides and the IC50 values ​​of the AF cyclic peptides. D To calculate Ki. Europium-labeled PCSK9 counts (B counts) are tracked to see if the compound adversely affects PCSK9. A decrease in B counts may indicate a false positive for inhibition. Data from this program are reported as "Ki Ultra = 'Numerical' (data reported are nanomolar)"

[0896] The following compounds, as shown in Table 2, were evaluated using the protocol described above, with the following results:

[0897] Ex-01 Ki Plus=<0.00558,Ki Ultra=0.0046 / Ex-02 Ki Plus=0.00558,Ki Ultra=0.005933 / Ex-03 Ki Plus=0.02535,Ki Ultra=0.06803 / Ex-04 Ki Plus≤0.00558,Ki Ultra=0.004711 / Ex-05 Ki Plus=0.009621,Ki Ultra=0.03296 / Ex-06 Ki Plus=0.00568,Ki Ultra=0.003424 / Ex-07 Ki Plus=0.05914,Ki Ultra=0.06753 / Ex-08 Ki Plus=0.01574,Ki Ultra=0.06832 / Ex-09 Ki Plus=0.09189,Ki Ultra=0.247 / Ex-10 Ki Plus=0.005743,Ki Ultra=0.02489 / Ex-11 Ki Plus=0.04334,Ki Ultra=0.2067 / Ex-12 Ki Plus=0.01448,Ki Ultra=0.02247 / Ex-13 Ki Plus=0.1454,Ki Ultra=0.4772 / Ex-14 Ki Plus=0.01605,Ki Ultra=0.02099 / Ex-15 Ki Plus=0.1027,Ki Ultra=0.2601 / Ex-16 Ki Plus=0.01423,Ki Ultra=0.05141 / Ex-17 Ki Plus≤0.00558,Ki Ultra=0.0028 / Ex-18 Ki Plus=0.03356,Ki Ultra=0.1183 / Ex-19 KiPlus=0.01662,Ki Ultra=0.01204 / Ex-20 Ki Plus=0.01303,Ki Ultra=0.01711 / Ex-21 Ki Plus=0.005692,Ki Ultra=0.001264 / Ex-22 Ki Plus=0.00926,Ki Ultra=0.01519 / Ex-23 Ki Plus=0.00938,Ki Ultra=0.00239 / Ex-24 Ki Plus=0.00812,KiUltra=0.00767 / Ex-25 Ki Plus=0.01127,Ki Ultra=0.00463 / Ex-26Ki Plus≤0.00558,Ki Ultra=0.002754 / Ex-27 Ki Plus≤0.00558,Ki Ultra=0.00301 / Ex-28 KiPlus≤0.00558,Ki Ultra=0.00078 / Ex-29 Ki Plus=0.00981,Ki Ultra=0.00614 / Ex-31 Ki Plus<0.00558,Ki Ultra=0.00074 / Ex-35 Ki Plus=0.04652,Ki Ultra=0.08434 / Ex-36 Ki Plus=0.00762,Ki Ultra=0.00507 / Ex-38Ki Plus=0.00904,KiUltra=0.01416 / Ex-39 Ki Plus=0.00716,Ki Ultra=0.00414 / Ex-40 Ki Plus=0.30800,Ki Ultra=0.86010 / Ex-41 Ki Plus=0.00697, Ki Ultra=0.00628 / Ex-44 KiPlus=0.01445, Ki Ultra=0.02194 / Ex-47 Ki Plus=0.01474, Ki Ultra=0.01193 / Ex-48 Ki Plus=0.01169, Ki Ultra=0.01545 / Ex-49 Ki Plus=0.00716, Ki Ultra=0.00414 / Ex-50 Ki Standard<1.26, Ki Plus=0.01052, Ki Ultra=0.00443 / Ex-51 Ki Standard<1.26, Ki Plus<0.00558, Ki Ultra=0.00597 / Ex-52 Ki Standard<1.26, Ki Plus<0.00558, KiUltra=0.00359 / Ex-53 Ki standard<1.26, Ki Plus=0.09629, Ki Ultra=0.21500 / Ex-54 Ki standard<1.26, Ki Plus=0.36720, Ki Ultra=0.48390 / Ex-55 Ki standard<1.26, Ki Plus=0.07240, Ki Ultra=0.23800 / Ex-56 Ki standard<1.257, Ki Plus=0.02237, Ki Ultra=0.00481 / Ex-57 Ki standard<1.257, Ki Plus<0.00558, Ki Ultra=0.00162 / Ex-58 Ki standard<1.257, Ki Plus = 0.00773, Ki Ultra = 0.00196 / Ex-59 Ki Plus=0.00788,Ki Ultra=0.004959 / Ex-60 Ki Plus=0.006515,Ki Ultra=0.005312 / Ex-61Ki Plus=0.00747,KiUltra=0.006543。.

Claims

1. Compounds of formula I: in: X is H, F, Cl or Br; R 1 Selected from: (a)-H; or (b)-(CH2) z -R 14A , where z is 1-6, and R 14A for: (i)-H; (ii)-N + (H3C)3; and (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; R 2 Selected from: (a)-H; and (b)-(CH2) z -R 14A , where z is 1-6, and R 14A Selected from: (i)-H; (ii)–NH2; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; and (iv)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for: (ai)–O-(CH2)2-N + (CH3)3; (aii)-N + (CH3)2R 14ca , where R 14ca for –CH3 or –(CH2) 1-4 -OCH3; (aiii) a portion of the following formula: or R 1 With R 2 can be combined together to form parts of the formula: in G 1 、R G1a and R G1b The definition is as follows: (a)G 1 is the connector portion of the following formula: where n q1 1 to 6, m q1 is 0, and n is selected at the same time q1 and m q1 such that the length of the linker moiety they define does not exceed a total of 6 carbon atoms and / or oxygen atoms, including the chain of carbon atoms included in the chain forming the carbonyl moiety; R G1a is -H; and R G1b for: The following part: or (b)G 1 is the connector portion of the following formula: where n q2 is 0, m q2 1 to 6, and select n q2 and m q2 such that the length of the linker moiety they define does not exceed a total of 6 carbon atoms and / or oxygen atoms, including the chain of carbon atoms included in the chain forming the carbonyl moiety; R G1a for: The following part: and R G1b is -H; R 8 is -CH3 or a portion of the formula: where R 8a is -H; A is selected from: (a) The part of the following formula: (b)-CH 2- (CH2) y -CH2-, wherein y is 1 to 6; (c) the part of the formula: Among them A b1 for: (i) the part of the formula: where x is 1 to 6; or (ii) a portion of the formula: where y is 1 to 5; and (d) a moiety of the formula: -CH2-(CH2) m -O-(CH2) n -, wherein m=1 to 5, and n=0 or 1 to 4; B is: (a)–(CH2) 0-2 -;or (b) The part of the following formula: D is: (a) The part of the following formula: Where E is -CH2- or -(CH2) 2-4 -O-, and A and B are as defined above; (b) The part of the following formula: wherein A and B are as defined above; or (c) the part of the formula: Among them, R 34b is -H, and A and B are as defined above, or any pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is F.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein D is a moiety of the formula: in, E is -CH2- or -(CH2)2-O-.

4. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein D is a moiety of the formula: in, E is -CH2- or -(CH2)2-O-.

5. The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein D is a moiety of the formula:

6. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein D is a moiety of the formula:

7. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein D is a moiety of the formula:

8. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein D is a moiety of the formula:

9. The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are combined to form the following parts: So that together with R 1 and R 2 The attached cyclic peptides together form a ring structure.

10. The compound of claim 2 or a pharmaceutically acceptable salt thereof, which has the structure of Formula IIB: B 1 =–((CH2) 0-2 )-;and D 1 for: The following part:

11. The compound of claim 2 or a pharmaceutically acceptable salt thereof, which has a structure of formula IIC: in D 2 for: The following part: or The following part:

12. The compound of claim 2 or a pharmaceutically acceptable salt thereof, which has a structure of formula IID: Among them D 2 for: (a) The part of the following formula: or (b) The part of the following formula:

13. The compound of claim 2 or a pharmaceutically acceptable salt thereof, which has a structure of Formula IIE:

14. The compound of claim 2 or a pharmaceutically acceptable salt thereof, which has a structure of formula IIF:

15. The compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein A is: (a)–(CH2)5; (b) The part of the following formula: where x is 1 to 3; or (c) the part of the formula:

16. The compound of any one of claims 1 to 8 or 10 to 15, or a pharmaceutically acceptable salt thereof, wherein R 2 for: -(CH2) z -R 14A ,in: z is 1-6, and R 14A for: (a)-H; (b)–NH2; (c)-NH-C(O)-[(CH2) 2-4 -O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; (d)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for: (ai)–O-(CH2) 2-4 -N + (CH3)3; (aii)-N + (CH3)3; or (aiii) a portion of the following formula:

17. The compound of any one of claims 1 to 8 or 10 to 16, or a pharmaceutically acceptable salt thereof, wherein R 1 Selected from: (a)-H; (b)-(CH2) z -R 14A ,in: z is 1-6, and R 14A for: (i)-H; or (ii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2-N + (CH3)3。 18. The compound of any one of claims 1 to 9 or 15 to 17, or a pharmaceutically acceptable salt thereof, wherein R 8 is the part of the following formula: where R 8b is –H.

19. A compound selected from the group consisting of: or any other pharmaceutically acceptable salt form thereof.

20. A composition comprising at least one compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

21. Use of a therapeutically effective amount of the composition of claim 20 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

22. Use of a therapeutically effective amount of a compound according to any one of claims 1 to 19 for the preparation of a medicament for the treatment of hypercholesterolemia in a patient in need thereof.

23. The compound of claim 13, which has a structure of Formula IIE, or a pharmaceutically acceptable salt thereof: in: R 1 Selected from: (a)-H; or (b)-(CH2) z -R 14A , where z is 1-6, and R 14A for: (i)-H; (ii)-N + (H3C)3; or (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; R 2 Selected from: (a)-H; and (b)-(CH2) z -R 14A , where z is 1-6, and R 14A Selected from: (i)-H; (ii)–NH2; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; and (iv)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for: (ai)–O-(CH2)2-N + (CH3)3; (aii)-N + (CH3)2R 14ca , where R 14ca for –CH3 or –(CH2) 1-4 -OCH3; A is selected from: (a)-CH 2- (CH2) y -CH2-, wherein y is 1 to 6; (b) The part of the following formula: Among them A b1 for: (i) the part of the formula: where x is 1 to 6; or (ii) a portion of the formula: Where y is 1 to 5; (c) a moiety of the formula: -CH2-(CH2) m -O-(CH2) n -, wherein m=1 to 5, and n=0 or 1 to 4.

24. The compound of claim 23, or a pharmaceutically acceptable salt thereof, wherein R 1 -(CH2) z -R 14A ,in: z is 1-6, and R 14A for: (i)-H; (ii)-N + (H3C)3; or (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; R 2 -(CH2) z -R 14A , where z is 1-6, and R 14A Selected from: (i)-H; (ii)–NH2; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; and (iv)-NH-C(O)-(CH2) y R 14C , where y = 1 to 6 and R 14C for: (ai)–O-(CH2)2-N + (CH3)3; (aii)-N + (CH3)2R 14ca , where R 14ca for –CH3 or –(CH2) 1-4 -OCH3; A is -CH 2- (CH2) y -CH2-, wherein y is 1 to 6.

25. The compound of claim 24, or a pharmaceutically acceptable salt thereof, wherein R 1 -(CH2) z -R 14A ,in: z is 1-6, and R 14A for: (i)-H; R 2 -(CH2) z -R 14A , where z is 1-6, and R 14A Selected from: (i) -H; or (ii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; and A is -CH 2- (CH2) y -CH2-, wherein y is 1 to 6.

26. A compound selected from the group consisting of: Among them A - It is a pharmaceutically acceptable anion.

27. Compounds of the following structure: Among them A - It is a pharmaceutically acceptable anion.

28. A composition comprising the compound of claim 27, and at least one pharmaceutically acceptable excipient.

29. Use of a therapeutically effective amount of the composition of claim 28 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

30. The compound of claim 27, wherein the compound is:

31. A composition comprising the compound of claim 30, and at least one pharmaceutically acceptable excipient.

32. Use of a therapeutically effective amount of the composition of claim 31 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

33. Compounds of the following structure: Among them A - It is a pharmaceutically acceptable anion.

34. A composition comprising the compound of claim 33, and at least one pharmaceutically acceptable excipient.

35. Use of a therapeutically effective amount of the composition of claim 34 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

36. The compound of claim 33, wherein the compound is:

37. A composition comprising the compound of claim 36, and at least one pharmaceutically acceptable excipient.

38. Use of a therapeutically effective amount of the composition of claim 37 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

39. Compounds of the following structure: Among them A - It is a pharmaceutically acceptable anion.

40. A composition comprising a compound according to claim 39, and at least one pharmaceutically acceptable excipient.

41. Use of a therapeutically effective amount of the composition of claim 40 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

42. The compound of claim 39, wherein the compound is:

43. A composition comprising the compound of claim 42, and at least one pharmaceutically acceptable excipient.

44. Use of a therapeutically effective amount of the composition of claim 43 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

45. Compounds having the following structure:

46. ​​A composition comprising the compound of claim 45, and at least one pharmaceutically acceptable excipient.

47. Use of a therapeutically effective amount of the composition of claim 46 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

48. Compounds having the following structure: Among them A - It is a pharmaceutically acceptable anion.

49. A composition comprising a compound according to claim 48, and at least one pharmaceutically acceptable excipient.

50. Use of a therapeutically effective amount of the composition of claim 49 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

51. The compound of claim 48, wherein the compound is:

52. A composition comprising a compound according to claim 51, and at least one pharmaceutically acceptable excipient.

53. Use of a therapeutically effective amount of the composition of claim 52 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

54. Compounds having the following structure: Among them A - It is a pharmaceutically acceptable anion.

55. A composition comprising the compound of claim 54, and at least one pharmaceutically acceptable excipient.

56. Use of a therapeutically effective amount of the composition of claim 55 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

57. The compound of claim 54, wherein the compound is:

58. A composition comprising the compound of claim 57, and at least one pharmaceutically acceptable excipient.

59. Use of a therapeutically effective amount of the composition of claim 58 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

60. Compounds having the following structure: Among them A - It is a pharmaceutically acceptable anion.

61. A composition comprising a compound according to claim 60, and at least one pharmaceutically acceptable excipient.

62. Use of a therapeutically effective amount of the composition of claim 61 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

63. The compound of claim 60, wherein the compound is:

64. A composition comprising the compound of claim 63, and at least one pharmaceutically acceptable excipient.

65. Use of a therapeutically effective amount of the composition of claim 64 in the preparation of a medicament for treating hypercholesterolemia in a patient in need thereof.

Images