EA043952B1 - PCSK9 ANTAGONIST COMPOUNDS - Google Patents

Description

Cross reference to related application

This application claims benefit to US Application No. 62/687913, filed June 21, 2018, which is incorporated herein by reference in its entirety.

State of the art to which the invention relates

Identification of compounds and/or agents that are effective in the treatment of cardiovascular diseases is highly desirable. In clinical trials, reductions in LDL cholesterol levels were directly associated with the incidence of coronary events; Law et al., 2003 BMJ 326:1423-1427. Moderate lifelong reductions in plasma LDL-cholesterol levels have been found to correlate with significant reductions 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 incidence of non-lipid-related cardiovascular risk factors; higher. Thus, controlled monitoring of LDL cholesterol levels would be greatly beneficial.

Proprotein convertase subtilisin-kexin type 9 (hereafter referred to as PCSK9), also known as neuronal apoptosis-regulated convertase 1 (NARC-1), is a protease K-like subtilase identified as the 9th member of the secretory subtilase family; see Seidah et al., 2003 PNAS 100:928-933. PCSK9 belongs to the family of mammalian proprotein convertases, 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. Investigation of transcriptional regulation of PCSK9 demonstrated that it is regulated by sterol regulatory element binding proteins (SREBPs), as seen for other genes involved in cholesterol metabolism; Maxwell et al., 2003 J. Lipid Res. 44:2109-2119, which is typical of other genes involved in lipoprotein metabolism; Dubuc et al., 2004 Arterioscler. Thromb. Vasc. Biol 24:1454–1459. Statins have been shown to increase PCSK9 expression similar to the cholesterol-lowering effects of drugs; higher. Moreover, the PCSK9 promoters have been shown to possess two conserved regions involved in cholesterol regulation: the sterol regulatory element and the Sp1 region; higher.

Meanwhile, in the endoplasmic reticulum, PCSK9, as its only catalytic activity, performs autocleavage between residues Gln-152 and Ser-153; see Naureckiene et al., 2003 Arch. Biochem. Biophys. 420:55-67; Seidah et al., 2003 Proc. Natl. Acad. Sci. U.S.A. 100:928-933. The prodomain remains tightly associated with the catalytic domain during subsequent transport through the trans-Golgi network. Maturation by autocleavage has been demonstrated to be key for PCSK9 secretion and subsequent extracellular function (see Benjannet et al., 2012 J. Biol. Chem. 287:33745–33755). Thus, several lines of evidence demonstrate that PCSK9, in particular, reduces the amount of LDLR protein in the liver and thus reduces the liver's ability to remove LDL cholesterol from the bloodstream.

Adenovirus-mediated overexpression of PCSK9 in mouse liver results in the accumulation of circulating LDL-C due to a pronounced loss of hepatic LDLR protein with no effect on LDLR mRNA levels; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875; Maxwell & Breslow, 2004 PNAS 101:7100-7105; Park et al., 2004 J. Biol. Chem. 279:50630-50638; and Lalanne et al., 2005 J. Lipid Res. 46:13121319. The effect of PCSK9 overexpression on increasing LDL-C levels in the circulation of mice is entirely dependent on LDLR expression, again indicating that the regulation of LDL-C by PCSK9 is mediated by downregulation of LDLR protein. Consistent with these data, mice lacking PCSK9, or in which PCSK9 mRNA is reduced by antisense oligonucleotide inhibitors, have higher levels of LDLR protein in the liver and a higher 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, reduction of PCSK9 levels in cultured human hepatocytes by siRNA also results in higher LDLR protein levels and increased LDL-C uptake capacity; 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 the action of PCSK9 leads to increased LDL-C levels by decreasing LDLR protein levels.

A number of mutations in the PCSK9 gene are also significantly associated with autosomal dominant hypercholesterolemia (ADH), an inherited metabolic disorder characterized by a marked increase in plasma levels of low-density lipoprotein (LDL) particles, which can lead to premature cardiovascular failure; see Abifadel et al., 2003 Nature Genetics 34:154156; Timms et al., 2004 Hum. Genet. 114:349-353; Leren, 2004 Clin. Genet. 65:419-422. A later published study of the S127R mutation by Abifadel et al., above, showed that patients carrying the mutation exhibited higher plasma total cholesterol and apoB100 levels associated with (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 conversion of these lipoproteins; Ouguerram et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1448–1453.

Thus, there can be no doubt that PCSK9 plays a role in the regulation of LDL. Expression or

- 1043952 activation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and corresponding inhibition or absence of PCSK9 expression is associated with reduced plasma levels of LDL cholesterol. Reduced LDL cholesterol levels associated with PCSK9 sequence variation were found to provide protection against coronary heart disease;

Cohen, 2006 N. Engl. J. Med. 354:1264-1272.

Thus, the identification of compounds and/or agents effective for the treatment of cardiovascular diseases is highly desirable, including antagonism of the role of PCSK9 in the regulation of LDL, however, generally, since PCSK9 circulates in the blood and has a moderate binding affinity for LDL receptors on the cellular surfaces, previous attempts to exploit this mechanism to treat diseases associated with high serum LDL levels have focused on the use of large biomolecules, such as antibodies. Thus, there is a lack of publications showing activity against its target using small peptides or small molecules to inhibit PCSK9, see for example Zhang et al., 2014 J. Biol. Chemistry, 289(2): 942-955. Moreover, there are few compounds that can be formulated into a dosage form for the oral route of administration of such compounds, which is highly desirable for providing therapy for conditions in which regulation of PCSK9 activity may play a role.

The present invention addresses these interests by providing PCSK9 antagonists that can conceivably be used to inhibit the activity of PCSK9 and the corresponding role that PCSK9 plays in various conditions for which administration of a PCSK9 antagonist provides therapy.

The essence of the invention

In one aspect, the invention relates to a compound of formula I

Formula I, where

X is H, F, Cl or Br;

R ¹ selected from:

(a) -H; or (b) -(CH ₂ )zR ^14A , where z is 1-6 and R ^14A is:

(i) -H;

(ii) -NH2;

(iii) -N%;

(iv) -N ⁺ (H3C)3;

(v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14B where R ^14B is: -NH2; -N ⁺ H ₃ ; -N(CH ₃ ) ₂ or -N ⁺ (CH3)3;

(vi) -NH-C(O)-[(CH2)y12-O-]2-(CH2)y13R ¹⁴ B, where y12 and y13 are not both equal to 2 and are independently equal to 2 to 4; And

R ^14B represents: -NH2; -N ⁺ H3; -N(CH3)2; or -N ⁺ (CH3) ₃ ;

(vii) -NH-C(O)-(CH ₂ ) _y R ^14C where y = 1-6 and R ^14C is -O-(CH2)za-N ⁺ (CH3) ₃ where za is 3 or 4; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y=1-6 and R ^14C is:

(ai) -O-(CH2)2-N ⁺ (CH ₃ ) ₃ ;

(aii) -N ⁺ (CH ₃ ) ₃ ; or

- 2 043952

(aiii) part of the formula:

R ² selected from:

(a) -H; and (b) -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is selected from: (i) -H;

(ii) -NH2;

(iii) -N+Нз;

(iv) -К ⁺ (НзС)з;

(v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14B where R ^14B is: -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ or K ⁺ (CH3)3;

(Vi) -NH-C(O)-[(CH2) _y 12-O-]2-(CH2)y13R ^14B , where y12 and y13 are not both equal to 2 and are independently equal to 2 to 4; And

R ^14b represents: -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ or -N ⁺ (CH ₃ ) ₃ ;

(vii) -NH-C(O)-(CH ₂ )yR ^14C , where y = 1-6 and R ^14C is -O-(CH2)zb-N ⁺ (CH3) ₃ , where zb is 3 or 4 ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C , where, y=1-6 and R ^14C represents:

(ai) -O-(CH2)2-N ⁺ (CH3)3;

(aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3 or -(CH ₂ ) _1-4 -OCH ₃ ;

o but no ^z .

(aiii) part of the formula: ' or ' ^I ICuVD ⁷ ( / _y НСс (aiv) part of the formula: ' where y ^14Cb and y ^14Сс are equal from 1 to 4;

or

R ¹ and R ² can be linked together to form part of the formula: where

^G1 , ^RG1a and ^RG1b are defined as follows:

(a) G ¹ represents the linker part of the formula:

where n ^q1 is from 1 to 6, m ^q1 is 0, 1 or 2 and together the values of n ^q1 and m ^q1 are chosen so that the length of the linker part that they define does not exceed a total of 8 carbon and/or oxygen atoms making up the chain , including a carbon atom in the chain that forms the carbonyl part;

R ^G1a is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms; And

R ^G1b selected from:

(i) parts of the formula:

And

(ii) parts of the formula:

- 3 043952 (b) G ¹ represents the linker part of the formula:

where n ^q2 is 0, 1 or 2, m ^q2 is from 1 to 6, and together the values of n ^q2 and m ^q2 are chosen so that the length of the linker part that they define does not exceed a total of 8 carbon and/or oxygen atoms that make up a chain including a carbon atom in the chain that forms a carbonyl moiety; R ^G1a selected from:

(i) parts of the formula:

and (ii) parts of the formula:

R ^G1b is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms;

R ⁸ represents -CH ₃ or part of the formula:

where R ^8a represents -H or linear, branched or cyclic alkyl of up to four carbon atoms;

A selected from:

(a) parts of the formula:

(b) -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y is from 1 to 6;

(c) parts of the formula:

' where A ^b1 represents:

(i) part of the formula:

CH ₂ ' where x is from 1 to 6; or (ii) part of the formula:

’ where y is from 1 to 5;

(d) parts of the formula: -CH ₂ -(CH ₂ )mO-(CH ₂ ) _n -, where m=1-5, and n=0 or 1-4;

B represents:

(a) communication;

(b) -(CH ₂ )1.4; or

(c) part of the formula:

D represents:

- 4 043952

O, (a) part of the formula: ' where E represents -CH ₂ - or (CH ₂ ) ₂ -4-O-, and A and B are as defined above;

straight branched or cyclic alkyl of up to four carbon atoms, and A and B are as defined above, or any pharmaceutically acceptable salt thereof.

In a further embodiment, the invention provides a compound of formula I wherein X is F, or any pharmaceutically acceptable salt thereof. In some embodiments, it is preferred that D is part of the formula:

'where E is -CH2- or -( _CH2 ) ₂ -O-, and A and B are as defined herein.

In some embodiments, it is preferred that D is part of the form ft ---

About ly: 'where E represents -CH2- or -(CH ₂ ) ₂ -O-, and A and B are as defined herein.

In some embodiments, it is preferred that D is part of the

description. In some embodiments, it is preferred that D is part

- 5 043952 formula: ’ where A and B are as defined herein. In some embodiments, it is preferred that D is

ABOUT

O ^L

part of the formula: 'where A and B are as defined herein. In some embodiments, it is preferred that D represents

is part of the formula: 'where A and B are as defined herein. In some embodiments, it is preferred that D is

constituted part of the formula: 'where A and B are as defined herein.

In some embodiments, where R ¹ and R ² are joined together and, together with the peptide ring to which they are attached, form a cyclic structure, it is preferred that R ¹ and R ² form part of the structure:

In one embodiment, the present invention relates to pharmaceutical compositions containing a compound of the invention, for example, a compound of formula I, and at least one pharmaceutical excipient, preferably a composition intended for oral administration.

In one aspect, the present invention provides a method of antagonizing PCSK9 to provide therapy for disease states associated with PCSK9 activity, for example, atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or related cardiovascular disease and cardiometabolic conditions, by administering to an individual to the person 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 of the Invention

The description that follows uses a generally accepted representation of the structure and includes the stereochemical designation of certain asymmetric carbon centers.

Thus, the structural representation of the compounds of the invention includes the conventional stereochemical designation for some of the asymmetric carbon centers present in the illustrative compounds. Thus, in such cases, solid black wedge bonds indicate bonds protruding from the image plane, shaded wedge bonds indicate bonds descending into the image plane, and a wavy line attached to the carbon atom having a double bond indicates that possible both are included. cis and trans orientations. As is generally accepted, simple solid lines indicate all spatial configurations for the depicted connection. Thus, when there is no specific stereochemical designation, the image provides all the stereochemical and spatial orientations of the structural features.

As shown in the examples of the invention and as mentioned above, specific asymmetric carbon centers are structurally depicted using conventional solid wedge and dotted wedge bond representations. For the most part, absolute configuration has not been determined for illustrative compounds, but has been assigned by analogy to specific illustrative compounds of known stereochemical configuration (determined by X-ray crystallography) prepared using the same or similar reaction conditions and starting reagents and isolated with using the same chromatographic conditions. Thus, specific reference to the configurations structurally presented herein is intended to identify specific compounds obtained that have an excess of one particular stereoisomer and is not necessarily provided herein to make statements about the absolute determination of the stereochemical structure of the specified compound, unless otherwise indicated in the data provided. .

It will be understood that, when isomeric mixtures are prepared, the preparation of the individual stereoisomers in significant percentages of enantiomeric excess can be accomplished, if desired, by separating the mixture using specialized techniques, for example, by chromatography or crystallization, or by using stereochemically homogeneous starting materials for the synthesis described. or through stereoselective synthesis. Optionally, derivatization can be carried out before the separation of stereoisomers. The separation of the mixture of stereoisomers can be carried out at an intermediate stage during the synthesis of the compound of formula I, or it can be carried out on the final racemic product.

When specified herein, absolute stereochemistry is determined by x-ray crystallography of crystalline products or crystalline intermediates that are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration. Unless a specific isomer, salt, solvate (including hydrates) or solvated salt of such racemate, enantiomer or diastereomer is indicated, the present invention covers all such isomers, as well as salts, solvates (including hydrates) and solvated salts of such racemates, enantiomers, diastereomers and their mixtures.

The present invention also covers isotopically labeled compounds of the present invention that are structurally identical to the compounds described herein except for the fact that a statistically significant percentage of one or more atoms in this form of the compound is replaced by atoms having an atomic mass or mass number different from atomic mass or mass number of the most abundant isotope commonly found in nature, thereby changing the natural isotope content of the compound of the invention. The present invention is intended to include all suitable isotopic variants of the compounds of formula I.

Examples of isotopes that may preferably be included in the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine, fluorine and chlorine, for example, but not limited to: ^2H , ^3H , ^nC , ^13C , 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 I. It will be understood that other isotopes may also be included by known methods.

In particular, certain isotopically labeled compounds of the invention (eg, compounds designated ³ H, 11 C and ¹⁴ C) are found to be particularly useful in tissue distribution assays of compounds and/or substrates using various known methods. In addition, the compounds of the invention involving isotopic substitution include various isotopic forms of hydrogen (H), including protium (1H) and deuterium ( ^2H or D). Protium is the predominant isotope of hydrogen in nature. Deuterium enrichment may provide certain therapeutic benefits, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterizing biological samples. Isotopically enriched compounds of formula I can be prepared without undue experimentation by conventional methods well known to those skilled in the art or by methods similar to those described in the schemes and examples herein, using suitable isotopically enriched reagents and/or intermediates.

When a wavy line is at the end of a conventional bond (as opposed to joining two atoms in a structure), it indicates the point of attachment to the structure, for example:

^C M indicates that the secondary butyl moiety is linked through a methylene group through a bond that has a wavy line at the end. When a letter designation is used to indicate a moiety of a substituent, a dash is used to indicate the point of binding to the specified substrate, for example, -CH ₂ -C(O)-CH ₂ C1 indicates that the acetyl chloride moiety is linked through the methylene moiety of that moiety.

When any variable (for example, n, Ra, Rb, etc.) appears more than once in any component or in Formula I, its definition in each case is independent of its definition in each other case, unless otherwise specified at the place of definition. One skilled in the art will understand that the choice of combinations of various substituents defined in the structural representation, i.e. R ¹ , R ^A , is carried out in accordance with well-known principles of connection and stability of chemical structures, and combinations of substituents and/or variables are permissible, only

- 7 043952 if such combinations lead to stable connections.

A stable compound is a compound that can be prepared and isolated and whose structure and properties are retained and can remain substantially unchanged for a period of time sufficient to permit use of the compound for the purposes described herein (e.g., therapeutic administration to an individual) . The compounds of the present invention are limited to the stable compounds covered by Formula I.

When any variable or part is expressed in range form, such as (-CH ₂ -)1_4, both ends of the specified range are included (i.e. 1 and 4 in this example), as well as all integers in between (i.e. 2 and 3 in the example).

The term halogen includes fluorine, chlorine, bromine and iodine unless specifically indicated at the place of reference.

As used herein, the term individuals (alternatively patients) refers to an animal, preferably a mammal, and in particular a human or non-human animal, including farm animals 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 individual is preferably a human. As used herein, the term administration and variations thereof (eg, administering a compound) with respect to a compound of Formula I means providing the compound or a pharmaceutically acceptable salt thereof to an individual in need of treatment.

As mentioned above, in one aspect, the present invention relates to the provision of compounds of Formula I, or pharmaceutically acceptable salts thereof, that have PCSK9 function antagonistic properties.

In one embodiment, the compounds of Formula I have the structure of Formula IA:

Formula IA, where

R ¹ selected from:

(a) -H; or (b) -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is:

(i) -H;

(ii) -NH2;

(iii) -N ⁺ H3;

(iv) -N ⁺ (HaC)3;

(v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14B where R ^14B is: -NH2; -N ⁺ H ₃ ; -N(CH ₃ ) ₂ or N ⁺ (CH3)3;

(vi) -NH-C(O)-[(CH ₂ ) _y12 -O-]1_ ₄ -(CH ₂ ) _y13 R ^14B , preferably -NH-C(O)-[(CH ₂ ) _y12 -O- ] ₂ -(CH ₂ ) _y13 R ^14B , where y12 and y13 are both 2 and independently range from 2 to 4; And

R ^14b represents: -NH2; -N ⁺ H3; -N(CH3)2 or -N ⁺ (CH3) ₃ ;

(vii) -NH-C(O)-(CH ₂ ) _y R ^14C where y = 1-6 and R ^14C is -O-(CH2)za-N ⁺ (CH3) ₃ where za is 3 or 4; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y=1-6 and R ^14C is:

(ai) -O-(CH ₂ ) ₂ -N ⁺ (CH ₃ ) ₃ ;

(aii) -N ⁺ (CH ₃ ) ₃ ; or

BUT (aiii) part of the formula:

- 8 043952

R ² selected from:

(a) -H; and (b) -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is selected from:

(i) -H;

(ii) -NH2;

(iii) -N ⁺ H3;

(iv) -N ⁺ (H3C)3;

(v) -NH-C(O)-[(CH ₂ ) ₂ -O-] _1-4 -(CH ₂ ) ₂ R ^14B , preferably -NH-C(O)-[(CH2)2-O- ]2-(CH2)2R ^14B , where R ^14B represents: -NH2; -N ⁺ H3; -N(CH3)2 or -N ⁺ (CH3) ₃ ;

(vi) -NHC(O)-[(CH ₂ )y1,-O-] ₂ -(CH ₂ )y ₁ 3R ^14B , where 12 and y13 are both equal to 2 and independently range from 2 to 4; And

R ^14b represents -NH2; -N ⁺ H3; -N(CH3)2 or -N ⁺ (CH3) ₃ ;

(vii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y=1-6 and R ^14C or 4; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y = 1-6 and R ^14C is -O-(CH ₂ ) _zb -N ⁺ (CH ₃ ) ₃ , where zb equals 3 represents:

(ai) -O-(CH2)2-N ⁺ (CH3)3;

(aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3 or -(CH ₂ ) _1-4 -OCH ₃ ;

(aiv) part of the formula: or (aiii) part of the formula:

' or

where Y ^14Cb and Y ^14Cc are equal to 1-4;

R ¹ and R ² can be linked together to form part of the formula: where

^G1 , ^RG1a and ^RG1b are defined as follows:

(a) G ¹ represents the linker part of the formula:

where n ^q1 is equal to from 1 to 6, m ^q1 is equal to 0, 1 or 2 and together the values of n ^q1 and

m ^q1 are chosen so that the length of the linker part that they define does not exceed a total of 8 carbon and/or oxygen atoms making up the chain, including the carbon atom in the chain that forms the carbonyl part;

R ^G1a is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms; And

R ^G1b selected from:

(i) parts of the formula:

(ii) parts of the formula:

or (b) G ¹ represents the linker part of the formula:

where n ^q2 is equal to 0, 1 or 2, m ^q2 is equal to from 1 to 6, and together the values of n ^q2 and m ^q2 are chosen so that the length

- the 9,043,952 linker moiety they define does not exceed a total of 8 carbon and/or oxygen atoms constituting a chain including the carbon atom in the chain that forms the carbonyl moiety;

R ^G1a selected from:

(i) parts of the formula:

and (ii) parts of the formula:

R ^G1b is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms; R ⁸ represents -CH ₃ or

part of the formula:

where R ^8a represents -H or linear, branched or cyclic alkyl of up to four carbon atoms; A selected from:

(a) parts of the formula:

(b) -CH ₂ ^- (CH ₂ ) _y -CH ₂ -, where y is from 1 to 6;

A ³¹ (c) parts of the formula:

' where A ^b1 represents:

(i) part of the formula:

’ where x is from 1 to 6; or

(ii) part of the formula:

’ where y is from 1 to 5;

(d) parts of the formula: -CH ₂ -(CH ₂ ) _m -O-(CH ₂ ) _n -, where m=1-5, and n=0 or 1-4;

B represents:

(a) communication;

(b) -(CH ₂ ) _1-4 ; or

(c) part of the formula:

D represents:

IN

O(a) part of the formula:

or -(CH ₂ ) _2-4 -O-, and A and B are as defined above;

' where E represents -CH ₂ - 10 043952

linear branched or cyclic alkyl of up to four carbon atoms, and A and B are as defined above, or any pharmaceutically acceptable salt thereof.

In one embodiment of compounds of formula IA, R ¹ is -(CH ₂ ) _z -R ^14A , where z is 1-6, and R ^14A is:

(i) -H;

(ii) -NH2;

(iii) -N ⁺ H ₃ ; or (iv) -К ⁺ (НзС)з;

R ² is -(CH ₂ ) _z -R ^14A where z is 1-6 and R ^14A is selected from:

(i) -H;

(ii) -NH2;

(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 represents -NH2; -N ⁺ H3; -N(CH3)2; or -N ⁺ (CH3)3;

(iv) -NH-C(O)-[(CH2) _y 12-O-]2-(CH2) _y 13R ^14B , where y12 and y13 are both 2 and independently range from 2 to 4; And

R ^14B represents: -NH2; -N ⁺ H3; -N(CH3)2 or -N ⁺ (CH3) ₃ ;

(v) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is -O-(CH2)zb-N ⁺ (CH3) ₃ where zb is 3 or 4; and (vi) -NH-C(O)-(CH ₂ ) _y R ^14C , where y=1-6 and R ^14C is:

(ai) -O-(CH2)2-N ⁺ (CH3)3;

(aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3 or -(CH ₂ ) _1-4 -OCH ₃ ;

(aiv) part of the formula:

(aiii) part of the formula: ’ or

ABOUT

where Y ^14Cb and Y ^14Cc are equal from 1 to 4;

or

Ρ ^ΰρ

ABOUT

R ⁸ represents -CH ₃ or part of the formula: ' where R ^8a represents -H or linear, branched or cyclic alkyl of up to four carbon atoms; A selected from:

- 11 043952

(a) parts of the formula:

(b) -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y is from 1 to 6;

(c) part of the formula:

Λ ³¹ (i) part of the formula:

' where A ^b1 represents:

, where x is from 1 to 6; or (ii) part of the formula:

, where y is from 1 to 5; and (d) part of the formula: -CH ₂ -(CH ₂ ) _m -O-(CH ₂ )n-, where m=1-5, and n=0 or 1-4;

B represents:

(a) -(CH ₂ )1-4; or

(b) part of the formula:

D represents:

a linear, branched or cyclic alkyl group of up to four carbon atoms, and A and B are as defined above, or a pharmaceutically acceptable salt of any of them.

In one embodiment, a compound of formula IA, D is part of the formula:

where E represents -CH2- or -(CH ₂ ) ₂ -O-, and A and B are as defined above in Formula IA.

In one embodiment of a compound of Formula IA, A is:

(a) -(CH2)6;

- 12 043952 (b) part of the formula:

where x is from 1 to 3; or (c) part of the formula:

In another embodiment of a compound of formula IA, R ² is:

(a) -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is:

(a) -H;

(b) -СНз;

(c) -NH2;

(d) -N+Нз;

(e) -NWb;

(f) -NH-C(O)-[(CH ₂ ) _2-4 -O-] _2-4 -(CH ₂ ) _2-4 R ^14B where R ^14B is: -NH2; -N ⁺ H ₃ ; -N(CH ₃ )2 or ^- N ^(C H3)3 ^; (g) -NH-C(O)-[(CH ₂ ) _y R ^14C , where y=1-6 and R ^14C is:

(ai) -O-(CH2)2-4-N ⁺ (CH3)3;

(aii) -N ⁺ (CH ₃ ) ₃ ; or (aiii) part of the formula:

’ or (b) part of the formula

In the following embodiment of a compound of formula IA, R ¹ is selected from:

(a) -H;

(b) -(CH ₂ ) _z -R ^14A where z is 1-6 and R ^14A is:

(i) -H;

(ii) -N ⁺ H ₃ ; or (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2-N ⁺ (CH3)3.

In another embodiment of the compound of formula IA, A is -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y is 3-5. In a further embodiment, A is -(CH ₂ ) ₆ .

In another embodiment of a compound of formula IA, B is part of the formula:

In another embodiment of the compound of Formula IA, R ¹ is -(CH2)zR ^14A , where z is 1-6, and R ^14A is -H. In another embodiment of the compound of Formula IA, R ¹ is -(CH2) _z -R ^14A , where z is 1 and R ^14A is -H.

In another embodiment of a compound of formula IA, R ² is -(CH2)zR ^14A , where z is 1-6, and R ^14A is -NH-C(O)-(CH2) _y R ^14C , where y=1 -6 and R ^14C is N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3.

In another embodiment of a compound of Formula IA, R ⁸ is part of the formula:

- 13 043952

R“ ^P

Q' where R ^8a represents -H or linear alkyl of up to four carbon atoms.

^R03

Q

In the following embodiment, R ⁸ is part of the formula: ' where

R ^8b represents -H, -CH ₃ or -C(CH ₃ ) ₃ .

In some embodiments, it is preferred that the compounds of Formula I have the structure of Formula II or Formula IIA, or a pharmaceutically acceptable salt formula thereof:

Formula PA, where A, R ¹ and R ² are as defined above in Formula IA, and B ¹ is -(CH ₂ ) _0-2 , and D ¹ is selected from:

- 14 043952

In some embodiments of Formula II or Formula IIA, it is preferred that D ¹

was part of the formula:

In some embodiments of Formula II or Formula IIA, it is preferable that D ¹

was part of the formula:

In some embodiments of Formula II or Formula IIA, it is preferred that D ¹

was part of the formula:

In some embodiments of Formula II or Formula IIA, it is preferable that D ¹

was part of the formula:

In some embodiments, the compound of Formula I is preferably a compound of Formula IID:

(a) part of the formula:

, or where D ² represents:

(b) part of the formula:

In some embodiments, the compound of Formula I is preferably a compound of Formula IIE:

- 15 043952

Formula IIE. In some embodiments, the compound of formula I is preferably a compound of formula III:

where A, R ¹ and R ² are as defined above in Formula IA, and D ² represents the part

formulas:

In some embodiments of Formula III, it is preferred that D ² be

part of the formula:

In some embodiments of Formula III, it is preferred that D ² be

part of the formula:

In some embodiments of Formula III, it is preferred that D ² be

- 16 043952

part of the formula:

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

Formula IV, wherein A, R ¹ and R ² are as defined above in Formula IA. In some embodiments, the compound of formula I is a compound of formula V:

Where

A, B, R ¹ and R ² are as defined above in Formula IA; And

- 17 043952

In some embodiments of Formula I, Formula IA, Formula II, Formula IIA, Formula III, Formula IV, or Formula V, it is preferred that A is part of the formula: -(CH ₂ ) _ya , where ya is from 4 to 6. B In some embodiments of Formula I, Formula IA, Formula II, or Formula IIA, it is preferred that A is part of the formula: -CH ₂ -(CH ₂ ) _ma -O-(CH ₂ ) _na -, where ma is 2 or 3 and na is 0 or 1. In some embodiments of Formula III, Formula IV, or Formula V, it is preferred that A is part of the formula: -CH ₂ -(CH ₂ ) _ma -O-(CH ₂ ) _na -, where ma is equal to 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, it is preferable that A

was part of the formula:

'where yb is from 1 to 3. In some embodiments of Formula I, Formula IA, Formula II, Formula IIA, Formula III, Formula IV

or formula V, preferably A is part of the formula:

Also within the framework of the present invention, compounds of formula I are provided as 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, Ex16, 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-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, Ex56, Ex-57, Ex-58, Ex-59, Ex-60 and Ex-61, or any pharmaceutically acceptable salt thereof. These compounds, which are described in table. 1 are also referred to herein as compounds of the invention.

- 18 043952

Table 1

- 19 043952

- 20 043952

- 21 043952

- 22 043952

-23 043952

where A- represents a pharmaceutically acceptable anion.

The term salt(s) and its use in the expression pharmaceutically acceptable salts as used herein includes any of the following: acid salts formed with inorganic and/or organic acids, basic salts formed with inorganic and/or organic acids. bases, zwyterionic and quaternary ammonium complexes. Salts of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art, for example, by reacting a compound of the invention with an amount of acid or base, such as an equivalent amount, in a medium, such as a medium in which the salt precipitates, or in aqueous medium followed by lyophilization.

The compounds of the invention contain tricoordinated nitrogen atoms, for example primary, secondary or tertiary amino moieties, where, as is known, one pair of electrons present on the nitrogen atom can be protonated with an appropriate acid or alkylated with an appropriate reagent, for example an alkyl bromide, under appropriate reaction conditions, with the formation of a tetracoordinated charged nitrogen stabilized by an anion formed in the process, for example, a halogen ion or a conjugate base. Thus, the compounds of the invention can be obtained in free base form or isolated in the form of a quaternary complex or a salt complex. In some cases, where there is a corresponding acidic proton close to the basic nitrogen, the formation of a zwitterionic complex is possible. As this term is used herein, salts of the compounds of the invention are all of acid salts formed with inorganic and/or organic acids, basic salts formed with inorganic and/or organic bases, salts that include a zwitterionic element, for example, where the compound contains both a basic part, for example, but not limited to, a nitrogen atom, for example, an amine, pyridine or imidazole, and an acidic part, for example, but not limited to, a carboxylic acid, and quaternary ammonium complexes are included within the scope of the compounds of the invention described in the present description.

Thus, the structural representation of the compounds of the invention in either free base form, salt form, zwitterionic form, or quaternary ammonium form also includes all other forms of such compounds discussed above. Thus, one aspect of the invention relates to providing the compounds of the invention in the form of a pharmaceutically acceptable salt, zwitterionic complex or quaternary ammonium complex. Those skilled in the art will recognize cases in which the compounds of the invention may form such complexes, including cases where the tetracoordinate nitrogen may be quaternized or protonated and the charged nitrogen form is stabilized by an anion. The term pharmaceutically acceptable salt refers to a salt (including a quaternary ammonium complex and an internal salt such as a zwitterionic complex) that has potency similar to or greater than that of the free base form of the compound, and that is not biologically or otherwise undesirable (e.g., is neither toxic or otherwise harmful to its recipient).

The formation of pharmaceutically useful salts from basic (or acidic) pharmaceutical compounds is described, 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, D.C. on their website); and R. 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. These documents are incorporated herein by reference.

The present invention relates to both the free base forms of the compounds of the invention and all available salts, including salts that are generally accepted as safe for use in the preparation of pharmaceutical formulations and salts that can be prepared by those skilled in the art and then classified as generally recognized as safe for use in the preparation of pharmaceutical formulations, referred to herein as pharmaceutically acceptable salts. As will be appreciated, compounds in free base form can be obtained by controlling the isolation conditions of the compound during synthesis or by neutralization or ion exchange from salt forms of the compounds of the invention.

Examples of pharmaceutically acceptable acid salts include, but are not limited to, acetates, including trifluoroacetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentane propionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates , glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2hydroxyethanesulfonates, lactates, maleates, methanesulfonates, methylsulfates, 2-naphthalene sulfonates, nicotinates, nitrates, oxalates, pamoates, pectinates, persulfates, 3-phenyl propionates, phosphates, picrates , pivalates, propionates, salicylates, succinates, sulfates, sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates (also known as tosylates), undecanoates, and the like.

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, dicyclohexylamines, hydro-rabamines (formed with N,N-bis(dehydroabiethyl)ethylenediamine), N-methyl-O-glucamines, N-methylD-glucamides, tert-butylamines , piperazine, phenylcyclohexylamine, choline, tromethamine and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups can be converted to ammonium ion or quaternized by means 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 sulfates), long chain halides (e.g. , decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (for example, benzyl and phenethyl bromides), and others.

The term pharmaceutically acceptable anion refers to an anion suitable to form a pharmaceutically acceptable salt.

The following examples of pharmaceutically acceptable salts that can be used within the scope of the present invention include, but are not limited to, fluoride, chloride, bromide and iodide.

In general, the salts of a compound are intended to be pharmaceutically acceptable salts within the scope of the invention.

The terms purified, purified form, or isolated and purified form for a compound refer to the physical state of said compound after isolation from a synthesis process or natural source, or a combination thereof. Thus, the terms purified, in purified form, or in isolated and purified form for a compound refer to the physical state of said compound after being prepared by a purification process or by methods described herein or well known to one skilled in the art, and in sufficient purity to be characterized by standard analytical methods described herein or well known to one skilled in the art. The compounds of the invention include any form of the compound, including in situ in the reaction mixture, as well as isolated and purified form obtained by standard methods. Also included are polymeric forms of the compounds of the invention and their solvates and prodrugs.

Certain compounds of the invention may exist in various tautomeric forms, for example, but not limited to, keto/enol tautomeric forms, imine amine tautomeric forms, and, for example, heteroaromatic forms, such as the following parts:

Likewise, unless otherwise indicated, providing a structural representation of any tautomeric form of a compound that exhibits tautomerism is intended to include all such tautomeric forms of the compound. Thus, while the compounds of the invention, their salts and solvates, and their prodrugs may exist in different tautomeric forms or in equilibrium among such forms, all such forms of the compound are embraced and included within the scope of the invention.

In another aspect, the present invention relates to pharmaceutical compositions containing one or more compounds of the invention. As used herein, the term pharmaceutical composition includes at least one pharmaceutically active compound and at least one excipient and is intended to cover both the combination of specific ingredients in specific amounts and any product that results, directly or indirectly, combining specific ingredients in specific quantities.

As will be understood by one skilled in the art, excipients are any component that adapts the composition to a particular route of administration or facilitates processing of the composition into dosage form without itself exhibiting an active pharmaceutical effect. Typically, the compositions contain more than one excipient depending on the route of administration and the characteristics of the active substance administered. Examples of excipients that impart properties to the composition that facilitate storage or processing include, but are not limited to, lubricants or compression aids in powdered medicinal products intended for tabletting and emulsion stabilizers in compositions in which the active substance is present in the form of an emulsion . Examples of excipients that tailor the composition to the desired route of administration include, for example, but are not limited to, for oral administration: absorption enhancers to promote absorption from the gastrointestinal tract, for transdermal or transmucosal administration: penetration enhancers, e.g. used in adhesive skin patches or compositions for buccal administration.

Regardless of the function that the excipients perform in the composition, the excipients are collectively referred to herein as a carrier. Typically, the formulations may contain up to about 95 percent active ingredient and the balance carrier, although formulations can be prepared in other ratios. In general, suitable pharmaceutical compositions contain

- 26 043952 a current concentration of the active substance, such that an effective amount of the PCSK9 antagonist can be provided in an individual dosage form of an acceptable volume, based on the route of administration, so that it can provide a therapeutic serum level of the active substance for an acceptable period of time in the individual to whom the composition is administered, and that the composition retains biological activity when stored at a temperature within an acceptable range for an acceptable period of time.

The pharmaceutical composition, as used within the scope of the invention, refers to both a bulk composition, i.e. formulated material that has not yet been formed into individual dosage units for administration, and to the composition contained in the individual dosage units.

While the compositions of the invention may be used in bulk form, it will be understood that for most applications the compositions may be included in a dosage form providing individual units suitable for administration to a patient, each dosage form containing an amount of the selected composition that contains an effective amount of said one or more compounds of formula I. Examples of suitable dosage forms include, but are not limited to, (i) dosage forms adapted for oral administration, for example, a liquid, gel, powder, solid or semi-solid pharmaceutical composition that is encapsulated or compressed into a tablet and may further contain one or more coatings that modify its release properties, for example, coatings that provide delayed release or formulations that have sustained release properties; (ii) dosage forms adapted for administration through oral tissues, for example, a rapidly dissolving tablet, lozenge, solution, gel, sachet or needle matrix suitable for providing intramucosal administration; (iii) dosage forms adapted for administration through the nasal mucosa or upper respiratory cavity, for example, a solution, suspension or emulsion formulation for spraying into the nose or respiratory tract; (iv) dosage forms adapted for transdermal administration, for example, patch, cream or gel; (v) dosage forms adapted for intradermal administration, for example, microneedle array; (vi) dosage forms adapted for intravenous (IV) infusion, for example, over an extended period, using an IV infusion pump; (vii) dosage forms adapted for intramuscular (IM) administration, for example, an injection solution or suspension, and which can be adapted to form a depot having sustained release properties; (viii) dosage forms adapted for intravenous (IV) drip administration, for example, a solution or suspension, for example, as an IV solution or concentrate for administration in a saline IV bag; (ix) dosage forms adapted for subcutaneous administration, including over an extended period of time, by implantation of a rod or other device from which the compound diffuses into the surrounding tissue and thereby provides a continuous serum therapeutic level; or (x) dosage forms adapted for delivery through the rectal or vaginal mucosa, such as a suppository.

Pharmaceutical compositions may be solid, semi-solid or liquid. Formulations in solid, semi-solid and liquid form can be adapted to various modes of administration, examples of which include, but are not limited to, powders, dispersible granules, mini-tablets, dragees, which can be used, for example, for tableting, encapsulation or direct administration. In addition, preparations in liquid form include, but are not limited to, solutions, suspensions and emulsions, which, for example, but not exclusively, can be used to prepare formulations for oral, inhalation or intravenous (IV) administration, for example but not limited to, administration by IV drip or infusion pump, intramuscular (IM) injection, such as a bolus that is released over an extended period, direct IV injection, or adapted for subcutaneous routes of administration.

Other routes of administration that may be contemplated include intranasal administration, or administration to certain other mucous membranes. Formulations prepared for administration to various mucous membranes may also include additional components that adapt them for such administration, for example, viscosity modifiers.

Although in some embodiments, compositions suitable for administration in solid dosage form, such as a tablet or a fast-melting oral dissolving formulation, are preferred for administration of a compound of the invention or a salt thereof, a composition of the invention may be formulated for administration via other routes mentioned higher. Examples include aerosol preparations, for example, suitable for administration by inhalation or nasal mucosa, and may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable propellant, for example, an inert compressed gas, for example nitrogen. Also included are preparations in solid form that are intended to be converted, immediately before use, into a suspension or solution, for example, for oral or parenteral administration. Examples of such solid forms include, but are not limited to, lyophilized formulations and liquid formulations adsorbed onto a solid adsorbent medium.

- 27 043952

For example, the compounds of the invention may also be delivered by the transdermal or transmucosal route, for example, from a liquid form, suppository, cream, foam, gel or rapidly dissolving solid form. It will be understood that transdermal compositions may also take the form of creams, lotions, aerosols and/or emulsions and may be provided in unit dosage form that includes a transdermal patch known in the art, for example, a patch that includes either a matrix containing a pharmaceutically active compound, or reservoir, which contains a solid or liquid form of a pharmaceutically active compound.

Examples of pharmaceutically acceptable carriers and methods for producing the various compositions mentioned above can be found in A. Gennaro (ed.), Remington: The Science and Practice of Pharmacy, ^20th Edition, (2000), Lippincott Williams & Wilkins, Baltimore, MD . Additional examples of publications related to compilation can be found in the publications below. Pharmaceutical compositions can be formulated by any of 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 & 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.

In another aspect, the present invention relates to methods of using PCSK9-specific antagonist compounds described herein to antagonize PCSK9 function, which are further described below. Use of the term antagonism throughout this application refers to providing the affected tissue(s) with a substance that opposes, inhibits, neutralizes or reduces one or more functions of PCSK9 in the affected tissue. Inhibition or antagonism of one or more PCSK9-associated functional properties can be readily determined using methodologies known in the art (see, for example, Barak & Webb, 1981 J. Cell Biol. 90:595-604; Stephan & Yurachek, 1993 J Lipid Res 34:325330; and McNamara et al., 2006 Clinica Chimica Acta 369:158-167), as well as the methodologies described herein. Inhibition or antagonism may result in a reduction in PCSK9 activity relative to the activity observed in the absence of an antagonist or, for example, the activity observed when a control antagonist with an unrelated specificity is present. Preferably, the PCSK9-specific antagonist of the present invention is an antagonist of PCSK9 function such that there is a reduction of at least 10% of a measurable parameter, including, but not limited to, the activity described herein, and more preferably, a decrease in the measurable parameter by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%. Such inhibition/antagonism of PCSK9 function is particularly effective in cases where the functioning of PCSK9 at least partially contributes to a particular phenotype, disease, disorder or condition that negatively affects the individual.

In one aspect, the present invention provides a method for antagonizing the activity of PCSK9, which includes contacting a cell, population of cells, or tissue sample that is capable of being influenced by PCSK9 (i.e., that express and/or contain LDL receptors) with a specific PCSK9- a specific antagonist described herein, under conditions that allow said antagonist to bind to PCSK9 when present and inhibit PCSK9 inhibition of cellular uptake of LDL. In some embodiments, the present invention includes methods wherein the cell is a human cell. Additional embodiments of the present invention include methods wherein the cell is a mouse cell.

In one aspect, the present invention provides a method of providing antagonism of PCSK9 activity in an individual, which comprises administering to the individual a therapeutically effective amount of a PCSK9-specific antagonist of the present invention. In some embodiments, methods of providing antagonism of PCSK9 function are for treating, as defined herein, a PCSK9-associated disease, disorder or condition or, alternatively, for providing therapy for a disease, disorder or condition that would benefit from the effects of PCSK9 antagonists.

The present invention thus covers the use of PCSK9-specific antagonists described herein in various treatments where antagonism of PCSK9 function is desired. As used herein, the term treatment method refers to a course of action resulting in a change in at least one symptom of a disease, which may be prophylactic or therapeutic in nature. In some embodiments, the present invention provides a method of treating a condition associated and/or attributable to PCSK9 activity, or a condition where PCSK9 function 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 pharmaceutical thereof. acceptable salt. In some embodiments, the condition may be atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or a related cardiovascular disease and cardiometabolic condition, or may be a disease or condition in which PCSK9 activity is contraindicated.

Methods of treatment in accordance with the present invention include administering to an individual a therapeutically (or prophylactically) effective amount of a PCSK9-specific antagonist of the present invention. The use of the terms therapeutically effective or prophylactically effective in amount refers to the amount required at the intended dosage to achieve the desired therapeutic and/or prophylactic effect over the desired period of time. The desired effect may be, for example, alleviation, mitigation, reduction, or elimination of at least one symptom associated with the condition being treated. These amounts may vary, as one skilled in the art will appreciate, depending on various factors, including, but not limited to, the disease state, age, sex and weight of the individual, and the ability of the PC8K9-specific antagonist to induce the desired effect in the individual. The response may be documented through in vitro assays, in vivo studies in non-human animals, and/or further confirmed in clinical trials.

In some embodiments, it is preferable to administer the PCSK9 antagonist compound of the invention in the form of a pharmaceutical composition as described herein.

Dosing of antagonist therapeutic agents is within the ability of one skilled in the art, see, for example, Lederman et al., 1991 Int. J Cancer 47:659–664; Bagshawe et al., 1991 Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922, and varies based on a number of factors, such as, but not limited to, the factors mentioned above, including the patient's condition, the area being treated, the route of administration and the desired treatment , for example, prevention or emergency treatment, etc. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe an effective therapeutic amount of the antagonist.

An individual may need or desire treatment for an existing disease or medical condition. As used herein, an individual's need for treatment for an existing condition encompasses both the determination of need by a medical professional and the individual's desire to receive such treatment. When a compound or a salt thereof is provided in combination with other active substances, administration and variations thereof in each case means providing the compound or a salt thereof and other substances together or simultaneously or in separate administrations over a period of time. When the agents in combination are administered simultaneously, they may be administered together in a single composition or they may be administered separately. It will be appreciated that the combination of active agents may be a single composition containing all the active agents, or a plurality of compositions each containing one or more active agents. In the case of two active substances, the combination may be either a single composition containing both active substances, or two separate compositions, each containing one of the substances; in the case of three active substances, the combination may be either one composition containing all three substances, or three separate compositions, each containing one of the substances, or two compositions, one containing two substances and the other containing a third substance, etc. .d.

The compositions and combinations of the present invention are suitably administered in effective amounts. The term effective amount means an amount of active compound sufficient to provide antagonism of PCSK9 and thereby induce a desired response (i.e., induction of a therapeutic response in the treatment or management of conditions associated with or influenced by PCSK9 function, including, but not limited to them, atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome and related cardiovascular disease and cardiometabolic conditions in animal or human).

The actual dosage used may vary depending on the needs of the patient and the severity of the condition being treated. Determination of the appropriate dosage regimen for a particular condition is within the ability of one skilled in the art, e.g., as described in standard literature, e.g., as described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), Physician's Desk Reference, ^56th Edition, 2002 (published by Medical Economics company, Inc. Montvale, NJ 07645-1742), or Physician's Desk Reference, ^57th Edition, 2003 (published by Thompson PDR, Montvale, NJ 076451742); the contents of which are incorporated herein by reference. For convenience, the total daily dosage may be divided and may be administered in portions throughout the day as needed, or may be delivered continuously.

The PCSK9-specific antagonist can be administered to an individual by any route of administration known in the art, including, but not limited to, oral administration, injection (specific embodiments of which include intravenous, subcutaneous, intraperitoneal, or intramuscular injection), or administration by inhalation, intranasal or topical administration, either alone or in combination with other agents intended to assist in the treatment of the individual. The PCSK9-specific antagonist can also be administered through injection devices, pen syringes, needleless devices, and subcutaneous patch delivery systems. The route of administration should be determined taking into account a number of factors known to one skilled in the art, including, but not limited to, the desired physicochemical characteristics of the drug.

One or more additional pharmacologically active substances can be administered in combination with a compound of formula I. Additional active substance (or substances) means a pharmaceutically active substance (or substances) that is active in the body, including prodrugs that are converted to a pharmaceutically active form after administration, that are different from the compound of formula I, and also includes a free acid, a free base and pharmaceutically acceptable salts of these additional active substances. In general, any suitable additional active agent or agents, including, but not limited to, antihypertensive agents, antiatherosclerotic agents, such as lipid-modifying compounds, antidiabetic agents, and/or anti-obesity agents, can be used in any combination with a compound of formula I in a single dosage formulation (fixed dose combination of drugs), or may be administered to an individual in one or more separate dosage formulations, allowing simultaneous or sequential administration of active substances (co-administration of individual active substances).

Examples of additional active agents that may be used include, but are not limited to, angiotensin-converting enzyme inhibitors (e.g., alacepril, benazepril, captopril, ceronapril, cilazapril, delacepril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril or trandolapril), angiotensin II receptor antagonists (eg, losartan, i.e. COZAAR®, valsartan, candesartan, olmesartan, telmesartan and any of these drugs used in combination with a hydrochlorothiazide such as HYZAAR®) ; neutral endopeptidase inhibitors (eg, thiorphan and phosphoramidone), aldosterone antagonists, aldosterone synthase inhibitors, renin inhibitors (eg, urea derivatives and di- and tri-peptides (see US Patent No. 5116835), amino acids and derivatives (US Patents 5095119 and 5104869) , amino acid chains linked by non-peptide bonds (US Patent 5,114,937), di- and tri-peptide derivatives, peptidylaminodiols and peptidyl betaaminoacylaminodiol carbamates, and small molecule renin inhibitors (including diolsulfonamides and sulfinyls), N-morpholino derivatives, N-heterocyclic alcohols and pyrolimidazolones; also pepstatin derivatives and fluorine and chlorine derivatives of statin-containing peptides, enalcrein, RO 425892, A 65317, CP 80794, ES 1005, ES 8891, SQ 34017, aliskiren (2(S),4(S),5(S) ,7(S)-N-(2-carbamoyl-2methylpropyl)-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 (eg, sildenafil, tadalfil and vardenafil), vasodilators, calcium channel blockers (eg, amlodipine, nifedipine, verapamil, diltiazem, gallopamil, niludipine, nimodipine, nicardipine), potassium channel activators (eg, nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam), diuretics (eg, hydrochlorothiazide), sympatholytics, beta-adrenergic blockers (eg, propranolol, atenolol, bisoprolol, carvedilol, metoprolol or metoprolol tartrate), alpha -adrenergic blockers (for example, doxazosin, prazosin or alpha-methyldopa), central alpha-adrenergic agonists, peripheral vasodilators (for example, hydralazine); lipid-lowering agents, for example, HMGCoA reductase inhibitors such as simvastatin and lovastatin, which are marketed as ZOCOR® and MEVACOR® in lactone prodrug form and function as inhibitors after administration, and pharmaceutically acceptable salts of dihydroxy HMG-CoA reductase inhibitors open-ring acids such as atorvastatin (specifically the calcium salt sold as LIPITOR®), rosuvastatin (specifically the calcium salt marketed as CRESTOR®), pravastatin (specifically the sodium salt marketed as PRAVACHOL®), fluvastatin (specifically the sodium salt sold as LESCOL®), crivastatin and pitavastatin; a cholesterol absorption inhibitor such as ezetimibe (ZETIA®) and ezetimibe in combination with any other lipid-lowering agents, such as the HMG-CoA reductase inhibitors described above, and in particular with simvastatin (VYTORIN®) or atorvastatin calcium; niacin in immediate release or controlled release forms and/or with an HMG-CoA reductase inhibitor; niacin receptor agonists such as acipimox and acitran, as well as partial niacin receptor agonists; metabolism-modifying agents including insulin and insulin mimetics (eg, insulin degludec, insulin glargine, insulin lispro), dipeptidyl peptidase IV (DPP-4) inhibitors (eg, sitagliptin, alogliptin, omarigliptin, linagliptin, vildagliptin); insulin sensitizers including (i) PPARy agonists such as glitazones (eg, pioglitazone, AMG 131, MBX2044, mitoglitazone, lobeglitazone, IDR-105, rosiglitazone and balaglitazone), and other PPAR ligands including (1) dual agonists

- 30 043952 PPARa/γ compounds (for example, ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar and naveglitazar); (2) PPARa agonists, such as fenofibric acid derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), (3) selective PPARy modulators (SPPARyM), (e.g., such as those described in WO 02/060388, WO 02/08188, WO 2004/019869, WO 2004/020409, WO 2004/020408, and WO 2004/066963); and (4) partial PPARy agonists; (ii) biguanides, such as metformin and pharmaceutically acceptable salts thereof, in particular metformin hydrochloride, and extended release formulations thereof, such as Glumetza™, Fortamet™ and GlucophageXR™; and (iii) protein tyrosine phosphatase-1B (PTP-1B) inhibitors (eg, ISIS-113715 and TTP814); insulin or insulin analogues (eg, insulin detemir, insulin glulisine, insulin degludec, insulin glargine, insulin lispro and inhalation formulations of each); leptin and leptin derivatives and agonists; amylin and amylin analogues (eg pramlitinide); sulfonylureas, non-sulfonylurea insulin secretagogues (eg, tolbutamide, glyburide, glipizide, glimepiride, mitiglinide, meglitinides, nateglinide and repaglinide); α-glucosidase inhibitors (eg acarbose, voglibose and miglitol); glucagon receptor antagonists (eg, MK-3577, MK-0893, LY2409021 and KT6-971); incretin mimetics such as GLP-1, GLP-1 analogs, derivatives and mimetics; and GLP-1 receptor agonists (eg, dulaglutide, semaglutide, albiglutide, exenatide, liraglutide, lixisenatide, taspoglutide, CJC-1131 and BIM-51077, including its intranasal, transdermal and once-weekly formulations); bile acid sequestrants (for example, colestilaan, colestimide, colesevalam hydrochloride, colestipol, cholestyramine and dialkylaminoalkyl derivatives of cross-linked dextran), acyl CoA:cholesterol acyltransferase inhibitors (for example, avasimib); anti-obesity compounds; agents intended for use in inflammatory conditions, such as aspirin, non-steroidal anti-inflammatory drugs or NSAIDs, glucocorticoids and selective cyclooxygenase-2 or COX-2 inhibitors; glucokinase activators (GKA) (eg, AZD6370); 11 β-hydroxysteroid dehydrogenase type 1 inhibitors (eg, such as those described in US Pat. No. 6,730,690 and LY-2523199); CETP inhibitors (eg, anacetrapib, torcetrapib and evacetrapib); fructose-1,6-bisphosphatase inhibitors (eg, such as those described in US Pat. Nos. 6,054,587; 6,110,903; 6,284,748; 6,399,782 and 6,489,476); acetyl-CoA carboxylase 1 or 2 inhibitors (ACC1 or ACC2); activators of AMP-activated protein kinase (AMPK); other G protein coupled receptor agonists: (i) GPR-109, (ii) GPR-119 (eg, MBX2982 and PSN821), and (iii) GPR-40 (eg, TAK875); SSTR3 antagonists (eg, such as those described in WO 2009/001836); neuromedin U receptor agonists (eg, such as those described in WO 2009/042053, including, but not limited to, neuromedin S (NMS)); SCD modulators; GPR-105 antagonists (eg, such as those described in WO 2009/000087); SGLT inhibitors (eg, ASP1941, SGLT-3, empagliflozin, dapagliflozin, canagliflozin, BI-10773, ertugliflozin, remogloflozin, TS-071, tofogliflozin, ipragliflozin and LX-4211); inhibitors of acyl-coenzyme A: diacylglycerol acyltransferase 1 and 2 (DGAT-1 and DGAT-2); fatty acid synthase inhibitors; acyl-coenzyme A:monoacylglycerol acyltransferase 1 and 2 inhibitors (MGAT-1 and MGAT-2); TGR5 receptor agonists (also known as GPBAR1, BG37, GPCR19, GPR131 and M-BAR); ileal bile acid transporter inhibitors; PACAR, PACAR mimetics, and PACAR receptor 3 agonists; PPAR agonists; protein tyrosine phosphatase-1B (PTP-1B) inhibitors; antibodies against IL-1b (eg, HOMA052 and canakinumab); and bromocriptine mesylate and immediate release formulations thereof; or with other drugs useful for the treatment of the above conditions or disorders, including free acid, free base and pharmaceutically acceptable salt forms of the above active compounds, whenever chemically possible.

The compounds of the present invention can be readily prepared by the following reaction schemes and examples, or modifications thereof, using readily available starting materials, reagents, and conventional synthetic techniques. Known variants can also be used in these reactions. A C18 column was used to purify the compounds using reverse phase chromatography (either HPLC or MPLC as indicated below). Other methods for preparing the compounds of the invention will be readily apparent to one skilled in the art from the following reaction schemes and examples. The abbreviations below may be used in illustrative diagrams and/or examples of the present description.

ACN is acetonitrile.

AcOH is acetic acid.

AcO ^- NH ₄ is ammonium acetate.

Boc ₂ O is di-tert-butyl dicarbonate.

Bn represents benzyl.

BnBr is benzyl bromide.

BzCl is benzoyl chloride.

CBr ₄ is perbromomethane.

Cbz-Cl is benzyl chloroformate.

- 31 043952

DBU is 1,8-diazabicyclo[5.4.0]udnet-7-ene.

DCC is dicyclohexylcarbodiimide.

DCE is 1,2-dichloroethane.

DCM is dichloromethane.

DEA is N,N-diethylamine.

DIAD is (E)-diisopropyldiazene-1,2-dicarboxylate.

DIEA or DIPEA is N,N-diisopropylethylamine.

DMAP is 4-dimethylaminopyridine.

DMF is N,N-dimethylformamide.

DMSO is dimethyl sulfoxide.

EA or EtOAc is ethyl acetate.

EtOH represents ethanol.

Et ₂ O is diethyl ether.

Fmoc is a fluorenylmethyloxycarbonyl protecting group.

Fmoc-Cl is (9H-fluoro-9-yl)methylcarbonochloridate.

Fmoc-D-Dap(Boc)-OH is N-alpha-(9-fluorenylmethyloxycarbonyl)-N-beta-tert-butyloxycarbonyl-D-2,3-diaminopropionic acid.

Fmoc-Osu is an N-hydroxysuccinimide ester of Fmoc.

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

HPLC is high performance liquid chromatography.

IPA is isopropyl alcohol.

LiOH is lithium hydroxide.

LC/MS is liquid chromatography-mass spectrometry.

Me ₃ N is trimethylamine.

MeOH is methanol.

MPLC is medium pressure liquid chromatography.

MsCl is methanesulfonyl chloride.

NaBH(OAc) ₃ is sodium triacetoxyborohydride.

NMR stands for nuclear magnetic resonance.

NsCl is 4-nitrobenzene-1-sulfonyl chloride.

PE represents petroleum ether.

Pd ₂ (dba) ₃ (HCCl ₃ ) is a tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct.

PPh ₃ is triphenylphosphine.

PdCl ₂ (dppf) or Pd(ii)(dppf)Cl ₂ is dichloro[1,1'-bis(diphenylphosphino)ferrocene] palladium(II).

Pd(dppf)Cl ₂ CH ₂ Cl ₂ is a dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(P)dichloromethane adduct.

Pd(PPh ₃ ) ₄ is tetrakis(triphenylphosphine)palladium.

PPTS is pyridinium p-toluenesulfonate.

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

Rt, or kt, or ct is room temperature.

tBuOAc is tert-butyl acetate.

TEA is triethylamine.

TFA is trifluoroacetic acid.

TFE is tetrafluoroethylene.

THF is tetrahydrofuran.

Tf2O is trifluoromethanesulfonic anhydride.

Teoc-OSu is 2,5-dioxopyrrolidin-1-yl (2-(trimethylsilyl)ethyl) carbonate.

TBAF is tetrabutylammonium fluoride.

TMS is tetramethylsilane.

Zhan catalyst 1B is dichloro(1,3-bis(2,4,6-trimethylphenyl)-2imidazolidinylidene)((5-((dimethylamino)sulfonyl)-2-(1-methylethoxy-O)phenyl)methylene-C) ruthenium(II) (also described as 1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene[2(isopropoxy)-5-(N,N-dimethylaminosulfonyl)phenyl[ methyleneruthenium(II)).

- 32 043952

Example 1. Preparation of Ex-01 and Ex-51.

ABOUT

Ex-01 (free base illustrated).

ABOUT

Ex-51 (free base illustrated).

- 33 043952

Salt forms of compounds Ex-01 and Ex-51 were prepared from intermediates 76 and 86 (preparation described below) according to the following scheme:

- 34 043952

formic acid

Stage A: preparation of intermediate 89.

To a solution of 76 (1.56 g, 1.917 mmol, preparation described below) and 86 (1.506 g, 1.936 mmol, preparation described 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, then partitioned between EtOAc (300 ml) and brine (100 ml). The organic phase was washed with brine (2x100 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column using MeOH/DCM as elution solvents to give 89. LC/MS: (M+1) +: 1573.8.

Step B: preparation of intermediate 90.

A solution of 89 (0.68 g, 0.432 mmol) in DCM (500 ml) and acetic acid (40 ml) was bubbled with N ₂ for 20 min, and then catalyst Zhan-1b (0.222 g, 0.302 mmol) was added. 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 by a silica gel column using MeOH/DCM as elution solvents to give 90 (cis/trans mixture). LC/MS: (M+1) ⁺ : 1545.7.

Stage C: preparation of intermediate 91.

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 hour, 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 (a mixture of cis and trans). LC/MS: (M+1) ⁺ : 1324.0.

Stage D: preparation of intermediate 92.

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

Step E: preparation of intermediate 93.

To a solution of 92 (700 mg, 0.388 mmol) in THF (20 ml), MeOH (6 ml) and water (6 ml) at 0°C, a 1 N aqueous solution of LiOH (3.11 ml, 3.11 mmol) was added dropwise. , and the resulting solution was stirred at 0°C for 23 hours. The solution was neutralized by adding 1 N HCl to pH 7-8, 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 elution solvents to give 93 in TFA salt form. To a solution of 93 in the form of TFA salt (427 mg, 0.257 mmol) in water (70 ml) and acetonitrile (70 ml) at 0°C was added dropwise 0.1 N HCl (13.5 ml, 1.350 mmol), the resulting solution stirred at 0°C for 5 min and then lyophilized to give 93 as the HCl salt. LC/MS: (M+1) ⁺ : 1567.1.

Step F: preparation of intermediate 94.

To a solution of 93 as HCl salt (200 mg, 0.125 mmol) in DMF (30 mL) 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) and then DIEA (0.065 ml, 0.374 mmol) was added. The resulting solution was stirred at room temperature. for 1 h, the volatiles were rotary evaporated and the resulting DMF solution was purified by reverse phase MPLC using acetonitrile (0.05% TFA)/water (0.05% TFA) as elution solvents to give 94.LC /MS: (M+1) ⁺ : 1549.2.

Step G: preparation of intermediate 95.

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 hydrogenated at room temperature. through an H ₂ bottle for 1 hour. The mixture was filtered through celite and the filtrate was concentrated to give intermediate 95. LC/MS: (M+1) ⁺ : 1550.9.

Stage H: receipt of Ex-01.

Intermediate 95 from the previous step (26 mg, 0.017 mmol) 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 4 N HCl in dioxane (0.042 ml, 0.168 mmol), and concentrated again to give Ex-01 as the crude product. Crude Ex-01 was purified by reverse phase HPLC using acetonitrile (0.1% formic acid)/water (0.1% formic acid) as elution solvents to obtain the formic acid salt form of Ex-01. LC/MS: (M+1) ⁺ : 1394.4.

Stage I: obtaining Ex-51.

To a solution of intermediate 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 min and then concentrated. The residue was dissolved in DCM (2 ml), treated with HCl (4 N in dioxane) (0.048 ml, 0.194 mmol) and concentrated to give Ex-51 as the 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 give the formic acid salt form Ex-51. LC/MS: (M+1) ⁺ : 1392.0.

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

- 36 043952

Preparation of intermediate 68, used to prepare intermediate 76.

Stage A: preparation of intermediate 65.

To a suspension of (2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid (5.32 g, 40.6 mmol) in dioxane (100 ml) at 0° C. was added sodium hydroxide (122 ml, 122 mmol), and then benzyl chloroformate (6.50 mL, 44.6 mmol) was added dropwise. The resulting suspension was stirred at 0°C for 5 hours. After removal of volatiles, the aqueous phase was acidified to pH 3, then distributed between 30% IPA/DCM (200 ml) and brine (50 ml), and then the aqueous phase was further extracted with 30% IPA/DCM (2x100 ml). The combined organic phases were dried over Na ₂ SO ₄ and concentrated to give (2S,3S)-1((benzyloxy)carbonyl)-3-hydroxypyrrolidine-2-carboxylic acid (65). LC/MS: (M+1)+: 266.1.

Stage B: preparation of intermediate 66.

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

Stage C: preparation of intermediate 67.

A solution of 66 (4.81 g, 17.22 mmol) in DCM (200 mL) was bubbled with N2 for 30 min and then rhodium(n) acetate dimer (0.761 g, 1.722 mmol) was added. The mixture was cooled in an ice bath and tert-butyldiazoacetate (3.58 mL, 25.8 mmol) was added dropwise at 0°C. The resulting mixture was stirred at 0°C for 1.5 hours. The reaction was quenched by adding water (100 ml), the mixture was extracted with DCM (3x100 ml), the combined organic phase was dried over Na2SO4, concentrated and the residue was purified by reverse phase MPLC using acetonitrile (0. 05% TFA)/water (0.05% TFA) as elution solvents. The fraction containing the product was concentrated and the aqueous phase was extracted with DCM (2x100 ml). The combined organic phase was dried over Na ₂ SO ₄ and concentrated to give 67. LC/MS: (M+1) ⁺ : 394.2.

Stage D: preparation of intermediate 68.

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 hydrogenated using an H ₂ 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.

- 37 043952

Preparation of intermediate 76.

Stage E

Stage A: preparation of intermediate 69.

To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3 -(5-fluoro-1 N-indol-3-yl)propionic acid (3 g, 6 .75 mmol) in THF (20 ml), MeOH (10 ml) and water (20.00 ml) at 0°C, NaOH (20.25 ml, 20.25 mmol) was added and the resulting solution was stirred at ambient temperature in for 4 hours, and then the volatile substances were evaporated. Dioxane (50 ml) and water (20 ml) were added to the aqueous mixture, the resulting solution was cooled to 0° C. and Boc ₂ O (1.881 ml, 8.10 mmol) was added to the above solution. The resulting solution was stirred at 0°C for 3 hours, volatiles were removed and the aqueous phase was extracted with Et ₂ O (3x40 ml), acidified to pH 3, then extracted with DCM

- 38 043952 (3x100 ml) followed by 30% IPA/DCM (2x80 ml). The combined organic phase was dried over Na ₂ SO ₄ and concentrated to give 69. LC/MS: (M+1)+: 322.9.

Step B: preparation of intermediate 70.

To a solution of 69 (2.079 g, 6.45 mmol) in DMF (40 ml) at 0°C was added 60% NaH in hexane (0.568 g, 14.19 mmol) and the resulting solution was stirred at 0°C for 50 min. and then allyl bromide (1.172 mL, 13.54 mmol) was added dropwise. The resulting solution was stirred at 0°C for 1.5 hours and then quenched by adding 1 N HCl (approximately 3.68 ml). The solution was then partitioned between EtOAc (200 ml) and water (100 ml), the organic phase was washed with brine (2x100 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column using MeOH/DCM as elution solvents with obtaining 70. LC/MS: (M+1) ⁺ : 363.0.

Stage C: preparation of intermediate 71.

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 (3x100 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column using EtOAc/hexane as elution solvents to give 71. LC/MS: (M+1) ⁺ : 604.2.

Stage D: preparation of intermediate 72.

To a solution of 71 (2.83 g, 4.69 mmol) in CH ₂ Cl ₂ (20 ml) and tBuOAc (30 ml) at 0°C was added methanesulfonic acid (1.218 ml, 18.75 mmol), and the resulting solution was stirred at 0°C for 16.5 hours and then at ambient temperature for 2.5 hours. Solution (72) was used directly in the next step. LC/MS: (M+1) ⁺ : 504.2.

Step E: preparation of intermediate 73.

To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)propionic acid (2, 66 g, 5.16 mmol) in DMF (10 ml), HATU (1.961 g, 5.16 mmol) and DIEA (5.32 ml, 30.5 mmol) were added and the resulting solution was stirred at room temperature. for 30 min and then added to solution 72 prepared as described above in an ice bath. The resulting solution was stirred at ambient temperature for 1 hour. 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 elution solvents. The collected fractions were concentrated on a rotary evaporator to give 73. LC/MS: (M+1) ⁺ : 1002.1.

Step F: Preparation of Intermediate 74.

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 h and then concentrated. The residue was dissolved in DCM (10 ml), treated with 4 N HCl in dioxane (3.23 ml, 12.91 mmol), then concentrated and the residue was dissolved in acetonitrile (100 ml)/water (50 ml) and lyophilized to give 74. LC/MS: (M+1) ⁺ : 846.1.

Step G: preparation of intermediate 75.

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) and then DIEA (1.692 ml, 9.69 mmol) was added dropwise. 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 solvents. Fractions containing product were concentrated and the aqueous layer was partitioned between DCM (200 ml) and sat. NaHCO ₃ (200 ml). The aqueous phase was extracted with DCM (2x100 ml) and the combined organic phase was dried over Na2SO4 and concentrated to give 75. LC/MS: (M+1) ⁺ : 828.1.

Step H: preparation of intermediate 76.

To a solution of 75 (1.93 g, 2.331 mmol) in THF (60 ml), MeOH (30 ml) and water (20 ml) at 0°C was added dropwise 1 N aqueous LiOH (9.9 ml, 9.90 mmol ) and the resulting solution was stirred at 0°C for 16 hours and then quenched by adding 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 (3x100 ml). The combined organic phase was dried over Na ₂ SO ₄ , concentrated and the residue was purified by a silica gel column using MeOH/DCM as elution solvents to give 76. LC/MS: (M+1) ⁺ : 814.2.

An alternative method for preparing intermediate 75b and intermediate 76B from it:

- 39 043952

Substantially the general procedure described above for the preparation of intermediate 75 was followed, except that step B was omitted and a final step G was included, replacing the Fmoc protecting group with a Boc protecting group, thereby yielding intermediate 75a. The methods for 75a and 76b are described below.

Step C: Preparation of intermediate 71a.

To a solution of 69 (5.00 g, 15.5 mmol) in DMF (40.0 ml) at -50°C was added 68, HATU (5.90 g, 15.5 mmol) and DIEA (4.01 g, 31.0 mmol), and the reaction mixture was stirred at -50°C for 3 hours. 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 an acetonitrile/water+0 gradient .01% ammonium bicarbonate) to obtain 71a. LCMS (ESI): calculated for C ₂₈ H ₃₈ FN ₃ O ₈ [M+H]+: 564.3, found 564.2.

Step D: Preparation of Intermediate 72a.

To a solution of 2 N HCl in dioxane (100 ml) and THF (100 ml) at room temperature. 71a (8.40 g, 14.9 mmol) was added and the reaction mixture was stirred for 5 hours. The final solution was concentrated under reduced pressure to give 72a. LCMS (ESI): calculated for C ₂₃ H ₃₁ ClFN ₃ O ₆ [M-HC1+H] ⁺ : 464.2, found 464.3.

Step E: Preparation of intermediate 73a.

To a solution of 72a (800 mg, 1.60 mmol) in DMF (10.0 ml) at -50°C was added (S)-2-((((9H-fluoren-9yl)methoxy)carbonyl)amino)-3 -(3-(((tert-butoxycarbonyl)amino)methyl)phenyl)propionic acid (827 mg, 1.60 mmol), HATU (608 mg, 1.60 mmol) and DIEA (620 mg, 4.80 mmol) and the mixture was stirred at -50°C for 3 hours. The resulting solution was diluted with water (50 ml) and the aqueous layer was extracted with EtOAc (3x100 ml). The combined organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with a gradient of 1%-60% EtOAc in PE) to give 73a. LCMS (ESI): calculated for C53H60FN5O11 [M+H]+: 962.4, found 962.6.

Step F: Preparation of Intermediate 74a.

To a solution of 73 a (3.00 g, 3.12 mmol) in DCM (15.0 ml) at room temperature. TFA (15.0 ml) was added and the reaction mixture was stirred for 1 hour at room temperature.

The resulting solution was concentrated under reduced pressure and then co-evaporated with toluene and DCM to give 74a. LCMS (ESI): calculated for C46H45F4N5O11 [M - TFA+H]+: 806.3,

- 40 043952 found 806.7.

Step G: Preparation of intermediate 75a.

To a solution of 74a (4.00 g, 4.35 mmol) in DMF (150 ml) at room temperature. HATU (1.65 g, 4.35 mmol) was added 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 .T. for 3 hours. The resulting solution was quenched with water (5 ml), concentrated under reduced pressure and the residue was purified by C18 reverse phase column chromatography (eluting with acetonitrile/water+0.05% TFA gradient) to give the Fmoc protected intermediate. LCMS (ESI): calculated for C _M II. ₁₂ FN5O ₈ [M+H]+: 788.3, found 788.9.

To a solution of the Fmoc-protected intermediate described immediately above (200 mg, 0.250 mmol) in DCM (5.00 ml) at RT. piperidine (1.25 ml) was added and the reaction solution was stirred for 1 hour. 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 in DCM) to give the amine intermediate. LCMS (ESI): calculated for C ₂₉ H ₃₂ FN ₅ O ₆ [M+H]+: 566.2, found 566.3.

To a solution of the amine intermediate described immediately above (2.86 g, 5.06 mmol) in THF (30.0 mL) and water (30.0 mL) at RT. Boc ₂ O (2.21 g, 10.1 mmol) and sodium bicarbonate (1.70 g, 20.2 mmol) were added and the reaction mixture was stirred for 3 hours. The final solution was diluted with water (50 ml) and extracted with EtOAc ( 3x100 ml). The combined organic layer was washed with brine (3x100 ml), dried over anhydrous Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with a gradient of 0%-5% MeOH in DCM) to give 75a. LCMS (ESI): calculated for C ₃₄ H ₄₀ FN ₅ O ₈ [M+H]+: 666.3, found 666.5; 1H-NMR (300 MHz, CD3OD) δ 7.35-7.08 (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).

Step H: Preparation of intermediate 75b.

To a solution of 75a (0.665 g, 0.99 mmol) in DMF (0.5 ml) was added Cs ₂ CO ₃ (1.11 g, 3.40 mmol) and 3bromoprop-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 a 50% sat. solution. brine/10% citric acid and then extracted with ethyl acetate (2x20 ml). The organic layer was washed with brine (3x20 ml), dried over anhydrous MgSO ₄ and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a gradient of 1%-5% MeOH in DCM. Fractions containing intermediate 75b were combined and concentrated to give the title compound. LCMS (ESI): calculated for C37H44FN5O8 [M+H]+: 706.3, found 706.3.

Step I: Preparation of Intermediate 76B.

Hydrolysis of 75b with LiOH followed conditions similar to those described for intermediate 93 to yield 76B.

Preparation of intermediate 77B.

- 41 043952

Step A: Preparation of Intermediate 77A.

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-hydrochloride (2-aminoethyl)benzylcarbamate (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), and then the reaction mixture was allowed to warm to room temperature. and stirred for 2 hours. The final mixture was diluted with water, extracted with EtOAc, washed with brine, dried over MgSO ₄ and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (eluting with a gradient of 060% EtOAc in hexane) to give 77A. MS (ESI): m/z (M+H)+ 584.5.

Step B: Preparation of Intermediate 77B.

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

Preparation of intermediate 86.

⁸⁶

Stage A: preparation of intermediate 77.

To a solution of (S)-(9H-fluoren-9-yl)methyl 2-((4-(aminomethyl)phenethyl)carbamoyl)-2-42 043952 methylpyrrolidin-1-carboxylate hydrochloride (77B) (5.87 g, 11 .29 mmol) in DCM (140 ml) at 0°C, DIEA (5.91 ml, 33.9 mmol) and CBZ-Cl (1.726 ml, 11.85 mmol) were added dropwise, and the resulting solution was stirred at 0°C within 4 hours

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 phase was dried over Na ₂ SO ₄ and the residue was purified on a silica gel column using EtOAc/hexane as eluents. solvents to give 77. LC/MS: (M+1)+: 618.3.

Stage B: preparation of intermediate 78.

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 aqueous NaOH (45.0 mL, 45.0 mmol), and the resulting solution was stirred at room temperature. for 2 hours. The volatiles were evaporated and dioxane (200 ml) and Boc ₂ O (2.508 ml, 10.80 mmol) in dioxane (20 ml) were added to the aqueous solution. The resulting mixture was stirred at a temperature from 0°C to room temperature. during the night. Volatiles were evaporated on a rotary evaporator and the aqueous phase was extracted with DCM (3x150 ml). The combined organic phase was dried over Na ₂ SO ₄ , concentrated and the residue was purified by a silica gel column using EtOAc/hexane as elution solvents to give 78. LC/MS: (M+1) ⁺ : 496.2.

Stage C: preparation of intermediate 79.

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 hydrogenated through an H ₂ cylinder at room temperature. for 1.5 hours. The mixture was filtered through celite and the filtrate was concentrated to give 79. LC/MS: (M+1) ⁺ : 362.2.

Step D: Preparation of Intermediate 80.

To a solution of 79 (2.58 g, 7.14 mmol) in DMF (15 ml) was added pent-4-en-1-yl 4methylbenzenesulfonate (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 hours. 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 elution solvents to give the TFA salt product, which was further partitioned between DCM (100 ml) and 1 N aqueous NaOH solution (50 ml). The aqueous phase was further extracted with DCM (2x50 ml), the combined organic phase was dried over Na ₂ SO ₄ and concentrated to give 80. LC/MS: (M+1)+: 430.3.

Step E: preparation of intermediate 81.

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 hour. The solution was partitioned between EtOAc (200 ml) and brine (100 ml), the organic phase was washed with brine (2x100 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column using EtOAc/hexane in as elution solvents to obtain 81. LC/MS: (M+1) ⁺ : 544.2.

Step F: preparation of intermediate 82.

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 hours and the mixture was concentrated to give 82. LC/MS: (M+1) ⁺ : 444.2.

Step G: preparation of intermediate 83.

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. within 50 min. The solution was partitioned between EtOAc (200 ml) and brine (100 ml), the organic phase was washed with brine (2x100 ml), the combined organic phase was dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column using EtOAc/hexane as eluents solvents to obtain 83. LC/MS: (M+1) ⁺ : 843.4.

Step H: preparation of intermediate 84.

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 hour and then concentrated. The residue was resuspended in acetonitrile (20 ml) and re-concentrated, the cycle was repeated once, and the residue was further dried under high vacuum to give 84. LC/MS: (M+1) ⁺ : 621.3.

Stage I: preparation of intermediate 85.

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 hour. The solution was partitioned between EtOAc (200 ml) and brine (100 ml), the organic phase was washed with brine (2x100 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column using EtOAc/hexane in as elution solvents to obtain 85. LC/MS: (M+1) ⁺ : 1000.2.

Step J: Preparation of Intermediate 86.

To a solution of 85 obtained in the previous step (1.94 g, 1.940 mmol) in acetonitrile (20 ml)

- 43 043952 piperidine (0.960 ml, 9.70 mmol) was added 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), 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.

Preparation of intermediate 88.

Step A - synthesis of intermediate 87.

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

Step B - synthesis of intermediate 88.

Resin 87 (4.5 g, 2.475 mmol) was manually deprotected using 5% piperidine in DMF (30 ml) for 30 min, filtered, re-treated with 5% piperidine in DMF (30 ml) for an additional 30 min , filtered and then rinsed with DMF and DCM and dried. 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) were then manually added to the resin over 2 h, then filtered, rinsed with DMF and DCM, and then dried. The resin was then treated with 10% AcOH and TFE in DCM (60 ml) for 90 min, filtered and the filtrate concentrated to give 88. LC/MS: [2M+H] ⁺ =995.01.

Example 1A. Alternative synthesis of Ex-01 and production of Ex-25 from it.

Compound Ex-01 above can be prepared by an alternative method, and from Ex-01 compound Ex-25 can be prepared according to the following scheme:

- 44 043952

- 45 043952

Int-cdS

- 46 043952

Stage A - synthesis of the intermediate compound Int-cd1.

To a solution of Int-3c (synthesis from intermediate 107 described below) (7.09 g, 10.33 mmol) in DMF (45 ml) at 0°C was added Int-2d (3.63 g, 9.84 mmol , preparation described below) and HATU (3.74 g, 9.84 mmol)), followed by DIPEA in 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 at 0°C with brine and extracted with EtOAc. The combined organic fractions were washed with brine, dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with a gradient of 1% to 80% ethyl acetate in petroleum ether) to give Int-cd1. LC/MS: [M+l]+=1000.5.

Stage B - synthesis of the intermediate compound Int-cd2.

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 hours. The mixture was concentrated, the residue was redissolved in DCM/acetonitrile (1:1, 20 ml), concentrated again, and the residue was dried in vacuo to give Int-cd2 as the crude product. LC/MS: (M+1)+=778.5.

Step C - synthesis of the intermediate Int-cd3.

To a solution of 76 (preparation shown in Example 1 above) (2.45 g, 3.01 mmol) and Int-cd2 (2.69 g, 3.46 mmol) in DMF (70 ml) at 0°C was added HATU (1.37 g, 3.61 mmol) and then DIEA (1.05 ml, 6.02 mmol). The resulting solution was stirred at room temperature. for 50 minutes, then distributed between

- 47 043952

EtOAc (500 ml) and brine (200 ml). The organic phase was washed with brine (2x200 ml), dried over

Na ₂ SO ₄ was concentrated and the residue was purified by silica gel column chromatography (eluting with a gradient of 1%-5% MeOH in DCM) to give Int-cd3. LC/MS: (M+1)+=1574.7.

Step D - synthesis of the intermediate Int-cd4.

A solution of Int-cd3 (1.91 g, 1.21 mmol) in DCM (1500 ml) and acetic acid (30 ml) at room temperature was bubbled with N2 for 30 min, and then catalyst Zhan-1B (0.445 g, 0.607) was added mmol). The resulting mixture was further bubbled with N2 at room temperature for 30 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 by silica gel column chromatography (eluting with a gradient of 1-5% MeOH in DCM) to give Int-cd4 (as a mixture of cis and trans olefins). LC/MS: (M+1)+=1546.8.

Step E - synthesis of the intermediate Int-cd5.

Piperidine (1.76 mL, 17.8 mmol) was added to a solution of Int-cd4 (a mixture of cis and trans olefins) (5.49 g, 3.55 mmol) in DCM (20 mL) and acetonitrile (50 mL). . The resulting solution was stirred at room temperature. for 2 hours, then concentrated and the residue was suspended in acetonitrile (20 ml) and concentrated again. The residue was then dried in vacuo to give Int-cd5 (as a mixture of cis and trans olefins) as a crude mixture. LC/MS: (M+1) ⁺ =1323.8.

Step F - synthesis of the intermediate Int-cd6.

To a solution of Int-cd5 (mixture of cis and trans olefins) (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 HATU (1.76 g, 4.62 mmol) and DIEA (1.55 ml, 8.88 mmol) were added. The resulting solution was warmed to room temperature and stirred for 1 hour and then partitioned between EtOAc (300 ml) and brine (200 ml). The aqueous phase was extracted with EtOAc (200 ml), the EtOAc phase was combined and washed with brine (3x200 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified by silica gel column chromatography (eluting with a gradient of 1%-5% MeOH in DCM) to give Int -cd6 (as a mixture of cis and trans olefins). LC/MS: (M+1) ⁺ =1802.8.

Step G - synthesis of the intermediate Int-cd7.

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

Stage H - synthesis of the intermediate compound Int-cd8.

To a solution of Int-cd7, the HCl salt obtained in the previous step, (1.01 g, 0.630 mmol) in DMF (50 ml) and DCM (1300 ml) were 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 hours, 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 (3x100 ml), dried over Na ₂ SO ₄ , concentrated, and the residue was purified by silica gel column chromatography (eluting with a gradient of 1%-10% MeOH in DCM) to give Int -cd8 (as a mixture of cis and trans olefins). LC/MS: (M+1) ⁺ =1548.8.

Stage I - synthesis of the intermediate Int-cd9.

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

Stage J - synthesis of compound Ex-01 in the form of HCl salt.

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 give the product as a solid. This solid was redissolved in acetonitrile (200 ml) and water (100 ml), and 1 N aqueous HCl 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 minutes, then frozen and lyophilized to obtain Ex-01 in the form of the HCl salt. LC/MS: (M+1) ⁺ =1394.7.

Step K - synthesis of the compound of Example Ex-25 in the form of a TFA salt.

- 48 043952

HATU (254 mg, 0.669 mmol) and DIEA (425 µl,

2.433 mmol). The resulting solution was stirred at room temperature. for 1 hour and then quenched by adding 1.2 ml of water. The mixture was filtered and the filtrate was purified by C18 column chromatography (eluting with an acetonitrile (0.05% TFA)/water (0.05% TFA) gradient) to give Ex-25 in the form of the TFA salt. LC/MS: M ⁺ =1550.6.

Step L: Preparation of the compound of Example Ex-25 in the form of the Cl salt.

The two columns were packed with 73.6 g of AG MP-1 chloride form ion exchange resin (catalog # 141-1841 BIO-RAD) for a total of 36.8 g of resin in each column. Each column was washed with water (2x80 ml) and then with 20% acetonitrile in water (2x100 ml). A solution of Ex-25, the TFA salt obtained in the previous step (737 mg, 0.443 mmol) in 20% acetonitrile in water (100 ml) was uniformly loaded into two resin columns, then elution was carried out in each column with 20% acetonitrile in water (130 ml). The eluents were combined, frozen and lyophilized to obtain Ex-25 in chloride form. LC/MS: M ⁺ =1550.6.

The following is a description of a number of intermediates that are useful in the synthesis of Ex-01 and Ex-25 described above.

Preparation of intermediate compound Int-1d.

Intermediate Int-1d was prepared from starting materials according to the following scheme:

Stage A - synthesis of Int-1da.

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) at 0°C was added DIPEA (7.03 ml, 40.2 mmol) and the mixture was stirred at 0°C for 2 hours and then kept in the refrigerator overnight. The mixture was quenched at room temperature with water and extracted with EtOAc. The combined organic fractions were washed with half brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with a hexane/EtOAc gradient) to give Int1da. LC/MS: [M+H] ⁺ =512.3.

Stage B - synthesis of Int-1d.

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) at room temperature, solid sodium hydroxide (0.691 g, 17.27 mmol) was added. The mixture was stirred at room temperature overnight. The mixture was concentrated, acidified with 0.5 N to pH ~2 (~40 ml), extracted three times with EtOAc, washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified by C18 column chromatography (eluting with acetonitrile/water+0.1% TFA gradient) to give Int-1d. LC/MS: [M+H] ⁺ =498.25.

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

Preparation of the intermediate compound Int-2d.

The intermediate Int-2d, useful as a linker for the preparation of the compounds of the invention, was prepared according to the following scheme:

- 49 043952

Stage C

Stage A - synthesis of Int-2da.

4-bromobenzaldehyde solution (15.00 g, 81 mmol), potassium tert-butyl N-[2-(trifluoroboranuidyl)ethyl]carbamate (20.97 g, 84 mmol), cesium carbonate (52.8 g, 162 mmol) and 1,1'bis(diphenylphosphino)ferrocene-palladium(II)-dichloromethane dichloride complex (Pd(II)(dppf)Cl ₂ , 1.99 g, 2.43 mmol) in degassed toluene (250 ml) and water ( 85 ml) was heated to 76°C and stirred overnight. The mixture was quenched at room temperature with semi-saturated aqueous ammonium chloride and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with a DCM/EtOAc gradient) to give Int-2da. LC/MS: (M-56+1)+=193.0.

Stage B - synthesis of Int-2db.

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

Stage C - synthesis of Int-2dc.

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, then quenched with a saturated aqueous solution of NaHCO ₃ (10 ml). The mixture was partitioned between EtOAc (500 ml) and saturated aqueous NaHCO ₃ (200 ml), the organic phase was washed with brine (3x200 ml), dried over Na ₂ SO ₄ , concentrated and the residue was purified on a silica gel column (eluting with a hexane/EtOAc gradient) with receipt of Int-2dc. LC/MS: (M+1)+=433.4.

Stage D - synthesis of Int-2d.

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

Preparation of Int-3c from 107 used for the synthesis of Ex-01 and Ex-25 described above.

The intermediate compound Int-3c was prepared according to the following scheme:

- 50 043952

lnt-3c

Stage A - synthesis of Int-Zsa.

To solution 107 (the preparation of which is described in the description of the synthesis of 109 and which was further used for intermediate 116, which was 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) at room temperature, 2N lithium hydroxide monohydrate (43.3 ml, 87 mmol) was added and the mixture was heated to 45°C and stirred overnight to obtain Int-3ca as a crude solution. LC/MS: (M+1)+=464.3. The reaction mixture was cooled to 0°C and treated with 1 M HCl (40 ml). The mixture was directly used for the next step.

Stage B - synthesis of Int-3c.

NaHCO ₃ (1.725 g, 20.54 mmol) and Fmoc-OSu (3.81 g, 11.30 mmol) were added to the crude Int-3ca obtained in the previous step at 0°C. The reaction mixture was stirred at 0°C for 2 hours, treated with 1 M HCl (20.5 ml) and extracted with ethyl acetate (2x200 ml). The combined organic layers were washed with brine (2x100 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%-5% MeOH in DCM to give Int-3c. LC/MS: (M+1)+=686.4.

Preparation of intermediate Int-4b.

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

Stage A - synthesis of Int-4ba from tert-butyl-3-(2-hydroxyethoxy)propanoate.

To a solution of tert-butyl 3-(2-hydroxyethoxy)propanoate (500.0 mg, 2.63 mmol) in DCM (2 ml) was added CBr ₄ (1395 mg, 4.21 mmol) and PPh ₃ (965 mg, 3 .68 mmol) at 0°C. The mixture was stirred at room temperature for 2 hours. 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 in petroleum ether. Fractions containing the desired product were combined and concentrated to give tert-butyl 6-bromohexanoate. The thus obtained solution of tert-butyl 6bromohexanoate (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 obtain Int-4ba. LC/MS: M ⁺ =230.3.

Stage B - synthesis of Int-4b.

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

The preparation of intermediate Int-2d is described above for the preparation of Ex-01 and Ex-25. This part of the molecule can be described as a linker that cyclizes the lower peptide ring having substituents R ¹ , R ² and R ⁸ . Other similar linkers can be used instead of Int-2d. Below is a description of other linkers that can be used to prepare the compounds of the invention examples described herein.

Preparation of intermediate compound Int-2e.

Intermediate Int-2e, useful as a linker for the preparation of compounds of the invention, was prepared according to the following scheme:

- 51 043952

IntZed

Stage A - synthesis of the intermediate compound Int-2ea.

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 for 14 hours at 40°C. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with a 1%-40% gradient of EtOAc in PE) to give Int-2ea. LC/MS: (M+Na)+:=484.4.

Stage B - synthesis of intermediate compound Int-2eb.

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

Step C - synthesis of intermediate compound Int-2ec.

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

Step D - Synthesis of the Int-2ed intermediate

To a solution of Int-2ec (4.50 g, 9.67 mmol) in DMF (150 ml) was added K ₂ CO ₃ (2.01 g, 14.5 mmol) and 3-bromoprop-1-ene (1.41 g, 11.6 mmol). The reaction mixture was stirred for 5 hours at room temperature, then diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with a gradient of 1%-50% EtOAc in PE) to give Int-2ed. LC/MS: (M+H)+:=506.2.

Step E - synthesis of intermediate compound Int-2e.

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 2mercaptoethanol (2.08 g, 26.7 mmol). The reaction mixture was stirred for 14 h at room temperature and then purified by C18 column chromatography (column: 330 g; mobile phase A: water/0.05% TFA, mobile phase B: ACN; flow rate: 85 ml/min; gradient: from 10% B to 20% B over 15 min, from 20% B to 45% B over 15 min, detector: UV 210 nm; Rt=20 min) to obtain Int2e. LC/MS: (M+H) ⁺ :=321.2. ¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.21-7.13 (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=7.1 Hz, 2H), 1.41 (s, 9H).

Preparation of intermediate compound Int-2f-1.

The intermediate Int-2f-1, useful as a linker for the preparation of the compounds of the invention, was prepared according to the following scheme:

- 52 043952

lnt-2fb-2

I nt-2f b- racemic cttoe

Stage A - synthesis of the intermediate compound Int-2fa.

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

Step B - synthesis of the intermediate compound Int-2fb (racemate) and separation into enantiomers Int2/b-1 and Int-2fb-2.

To a solution of Int-2fa (20.0 g, 65.9 mmol) in dry DCM (200 ml) was added slowly but-3-en1-ylmagnesium bromide (15.7 g, 99 mmol) at -48°C under nitrogen protection . The mixture was stirred at -48°C for 2 hours, then quenched with saturated aqueous NH ₄ Cl (400 ml) and extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and the resulting residue (containing the racemic mixture of Int2fb) was purified by silica gel column chromatography (eluting with a gradient of 1%-35% EtOAc in PE) to give Int-2fb-1 and Int-2fb-2. LC/MS: (M+H)+:=344.0, 346.0.

Step C - synthesis of intermediate compound Int-2fc-1.

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

Step D - synthesis of intermediate Int-2fd-1.

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. This 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 silica gel column chromatography (eluting with a gradient of 1%-10% EtOAc in PE) to give Int-2fd-1. LC/MS: (M+Na+CH3CN)+:=447.3, 449.3.

- 53 043952

Step E - synthesis of the intermediate compound Int-2fe-1.

To a solution of Int-2fd-1 (15.1 g, 37.3 mmol), (2-((tert-butoxycarbonyl)amino)ethyl)potassium trifluoroborate (18.7 g, 74.6 mmol), Cs ₂ CO ₃ (36.5 g, 112 mmol) in toluene (285 ml) and water (95 ml) was added PdCl ₂ (dppf) (1.37 g, 1.87 mmol) under nitrogen protection. The mixture was stirred at 80°C for 40 hours. The resulting solution was quenched with water and extracted with EtOAc. The combined organic layers were dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography over silica gel (eluting with a gradient of 1%-40% EtOAc in PE) to give Int2fe-1. LC/MS: (M+Na)+:=471.4.

Step F - synthesis of intermediate Int-2f-1.

To a solution of Int-2fe-1 (10.6 g, 22.4 mmol) in THF (100 mL) was added 1 N TBAF in THF (44.9 mL, 44.9 mmol). This mixture was stirred at room temperature for 16 hours and then quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with a gradient of 1%-70% EtOAc in PE) followed by C18 column chromatography (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 over 10 min, 20% B to 45% B over 10 min, 45% B to 70% B over 20 min, detector: UV 210 nm; Rt=25 min) to obtain Int2f-1. LC/MS: (M+H)+:=305.1. 1H-NMR (300 MHz, CD3OD) δ 7.27-7.16 (m, 4H), 5.85-5.75 (m, 1H), 5.00-4.85 (m, 2H), 3 .79 (t, J=7.0 Hz, 1H), 3.32-3.21 (m, 2H), 2.75 (t, J=7.4 Hz, 2H), 2.98-1, 72 (m, 4H), 1.42 (s, 9H).

Example 2. Preparation of Ex-50 and Ex-52.

Compound Ex-50 was prepared according to the scheme below from compound Ex-01, the preparation of which is described herein in Example 1, by reacting it under appropriate conditions with intermediate Int 32, prepared according to the following scheme:

- 54 043952

Step A: Preparation of intermediate Int-32A.

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)-3oxopropoxy)ethoxy)-N,N,N-trimethylethanaminium bromide (Int 32A). LC/MS: (M) ⁺ : 276.5.

Stage B: preparation of intermediate Int-32.

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

Preparation of Example Compound Ex-50.

To a solution of Ex-01 (crude) (17.4 mg, 0.012 mmol) and 2-(2-(2-carboxyethoxy)ethoxy)N,N,N-trimethylethanaminium bromide (Int-32) (4.49 mg, 0.015 mmol) in DMF (2 mL), HATU (5.69 mg, 0.015 mmol) and DIEA (6.54 μL, 0.037 mmol) were added 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 obtain Ex-50. LC/MS: M ⁺ =1596.3.

- 55 043952

Preparation of Example Compound Ex-52.

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

Example 3. Preparation of Ex-53, Ex-54 and Ex-55.

Compounds Ex-53, Ex-54 and Ex-55 were prepared similarly to the preparation of the compounds described above from intermediate 115 (preparation described below), according to the following schemes and description of the synthesis:

- 56 043952

HATU/DIEA/DMF

Step A - synthesis of intermediate 116.

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

Stage B - synthesis of compound Ex-54.

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 hour. 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 4 N HCl in dioxane (100 μl), and then concentrated to give Ex-54 as the HCl salt. LC/MS: M = 1383.44.

Stage C - synthesis of compound Ex-55.

The compound of Example Ex-55 was prepared in formate form from Ex-54 in a manner identical to that described in Example 2 for the synthesis of compound Ex-50. LC/MS: M = 1583.69.

Stage D - synthesis of compound Ex-53.

Example Ex-53 was prepared in HC1 salt form from intermediate 116 in a manner identical to that described in Example 1 for the synthesis of Ex-51. LC/MS: M = 1381.33.

The preparation of intermediates necessary to provide intermediate 115, from which compounds Ex-53, Ex-54 and Ex-55 are ultimately prepared, is described in the synthetic schemes and methods below for preparing intermediate 103.

Step A - synthesis of intermediate 100.

Solution of 4-bromo-2-hydroxybenzaldehyde (3.00 g, 14.92 mmol), potassium tert-butyl N-[2trifluoroboraniudyl)ethyl]carbamate (3.82 g, 15.22 mmol), cesium carbonate (17.02 g, 52.2 mmol) and 1,G-bis(diphenylphosphino)ferrocenepalladium(P)-dichloromethane dichloride complex (0.611 g, 0.746 mmol) in degassed toluene (45 ml) and water (15 ml) were heated to 75°C and stirred overnight. The mixture was quenched at room temperature with semi-saturated aqueous sodium bicarbonate and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with 99:1 to 60:40 hexane/EtOAc) to give 100. LC/MS: (M-55) = 210.25.

Step B - synthesis of intermediate 101.

To a solution of 100 (1.70 g, 6.41 mmol) and allyl bromide (0.832 ml, 9.61 mmol) in DMF (10 ml) at room temperature

-57043952 at sodium temperature, potassium carbonate (1.328 g, 9.61 mmol) was added and the mixture was heated at 50°C and stirred for 1 hour. The mixture was quenched at room temperature with semisaturated aqueous sodium bicarbonate and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with 99:1 to 70:30 hexane/EtOAc) to give 101. LC/MS: (M-55) ⁺ =250.29.

Step C - synthesis of intermediate 102.

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

Step D - synthesis of intermediate 103.

To a solution of 102 (220 mg, 0.718 mmol) and monomethyl succinic acid (114 mg, 0.862 mmol) in DMF (4 mL) at room temperature were added HATU (300 mg, 0.790 mmol) and DIPEA (0.314 mL, 1.795 mmol) and the mixture was stirred for 30 minutes. The mixture was quenched at room temperature with saturated aqueous sodium bicarbonate and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with 99:1 to 30:70 hexane/EtOAc) to give an intermediate which was treated with 20% TFA in DCM for 1 hour. The reaction mixture was concentrated and then treated with 4 N HCl (1 .5 ml) and concentrated to give 103. LC/MS: (M+H) ⁺ =321.29.

Preparation of intermediate 109.

Step A - synthesis of intermediate 104.

To a stirred solution of methyl (S)-2-methylpyrrolidine-2-carboxylate hydrochloride (7.00 g, 39 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxyphenyl)propionic 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 mixture was quenched with 10% aqueous LiCl and extracted with EtOAc. The organic extract was washed with a 10% aqueous solution of LiCl and dried over MgSO ₄ . The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (eluting with 80:20 to 40:60 hexane/EtOAc) to give 104.

Step B - synthesis of intermediate 105.

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

- 58 043952 further cleaning.

Step C - synthesis of intermediate 106.

To a solution of 105 (13.2 g, 37.0 mmol) and N-((benzuloxy)carbonyl)-O-(tert-butyl)-L-threonine (18.15 g, 37.0 mmol) in DMF at 0 °C DIPEA (16.15 mL, 92 mmol) was added followed by HATU (18.28 g, 48.1 mmol). The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was quenched with 10% aqueous LiCl and extracted with EtOAc. The organic extract was washed with a 10% aqueous solution of LiCl and dried over MgSO ₄ . The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (eluting with 80:20 to 40:60 hexane/EtOAc) to give 106.

Step D - synthesis of intermediate 107.

A 10% Pd/C slurry was added to a solution of 106 (16.5 g, 27.0 mmol) in MeOH and the mixture was hydrogenated at 20 psi. inch for 4 hours. 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.

Step E - synthesis of intermediate 108.

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 hours and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with 100:0 to 40:60 hexane/EtOAc) to give 108.

Step F - synthesis of intermediate 109.

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

Preparation of intermediates 110 to 115.

Step A - synthesis of intermediate 110.

Stage A

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 allowed to warm to room temperature and stirred for 30 min. The mixture was quenched at 0°C with saturated aqueous sodium bicarbonate and extracted with EtOAc. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with 99:1 hexane/EtOAc-EtOH 3:1 to EtOAc-EtOH 3:1) to give 110. LC/MS: (M+H) ⁺ =866.21.

- 59 043952

Steps B and C - synthesis of intermediates 111 and 112.

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

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) at 0°C was added DIPEA (0.128 ml, 0.734 mmol) and HATU (123 mg, 0.323 mmol), and the mixture was stirred for 30 minutes. The mixture was quenched at 0°C with brine and extracted with EtOAc. The combined organic fractions were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluting with 99:1 to 30:70 hexane/EtOAc-EtOH 3-1 followed by 99:1 to 70:30 DCM/MeOH) to give 112. LC/MS: (M +H) ⁺ =1506.11.

Step D - synthesis of intermediate 113.

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

Step E - synthesis of intermediate 114.

- 60 043952

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 co-evaporated with acetonitrile three times to obtain crude material. To a slurry of that crude material (119 mg, 0.095 mmol) and intermediate 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 C18 column chromatography (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. Main isomer LC/MS: (M) ⁺ =1735.28; LC/MS (minor isomer): (M) ⁺ =1735.25.

Step F - synthesis of intermediate 115.

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

As described above for the preparation of Ex-50 from Ex-01, and Ex-55 from Ex-54 by reacting their amide ^R2 in the respective starting compounds with an acidic substituent precursor, the following intermediates can be used in similar reactions to obtain the suitable compounds according to invention.

Synthesis of R1/ ^R2 substituent precursors.

Preparation of 5-carboxy-N-(3-methoxypropyl)-N,N-dimethylpentane-1-aminium chloride (intermediate Z-1a).

Step A: Preparation of intermediate Z-1.

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 hours. The resulting mixture was concentrated under reduced pressure to obtain Z-1. LC/MS: (M-Br) ⁺ =288.4. ¹ H-NMR (300 MHz, CDCb): δ 3.76-3.47 (m, 6H), 3.38 (d, J=28.9 Hz, 9H), 2.25 (t, J=7 .2 Hz, 2H), 2.12-1.95 (m, 2H), 1.87-1.55 (m, 4H), 1.45 (s, 11H).

Step B: synthesis of intermediate Z-la.

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

Preparation of intermediate Z-2b.

Step A: Preparation of intermediate Z-2.

- 61 043952

Dimethylamine (2M in THF) (7.96 mL, 15.93 mmol) was added to a stirred solution of tert-butyl 6-bromohexanoate (1.0 g, 3.98 mmol) in THF (10 mL). The reaction mixture was stirred at room temperature for 16 hours. 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 in DCM. Fractions containing the desired product were combined and concentrated to give Z-2. LC/MS: (M+H)+=216.2. ¹ H-NMR (300 MHz, CDCl ₃ ): δ 2.35-2.17 (m, J=8.5, 6.5 Hz, 10H), 1.67-1.47 (m, 4H), 1.45 (s, 9H), 1.42 -1.23 (m, 2H).

Step B: Preparation of intermediate Z-2a.

To a stirred solution of Z-2 (250 mg, 1.161 mmol) in ACN (1 mL) was added 1-bromo-2methoxyethane (645 mg, 4.64 mmol). The reaction mixture was stirred at 50°C for 16 hours. The resulting mixture was concentrated under reduced pressure to obtain Z-2a. LC/MS: (M-Br) ⁺ =274.3. 1HNMR (300 MHz, CDOj): δ 4.02-3.80 (m, 4H), 3.70-3.54 (m, 2H), 3.42 (d, J=13.0 Hz, 9H) , 2.24 (t, J=7.2 Hz, 2H), 1.85-1.75 (m, 2H), 1.72-1.55 (m, 2H), 1.44 (s, 11H ).

Step C: Preparation of intermediate Z-2b.

To a stirred solution of Z-2a (450 mg, 1.270 mmol) in DCM (0.5 mL) was added 4 M HCl in dioxane (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure. The residue was redissolved in DCM (5 ml) and concentrated under reduced pressure to give Z-2b. LC/MS: ^-0)+=218.3.

Preparation of intermediate Z-3b.

Step A: Preparation of intermediate Z-3.

To a solution of tert-butyl 3-(2-hydroxyethoxy)propanoate (500.0 mg, 2.63 mmol) in DCM (2 ml) was added CBr ₄ (1395 mg, 4.21 mmol) and PPh ₃ (965 mg, 3 .68 mmol) at 0°C. The mixture was stirred at room temperature for 2 hours. 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 in PE. Fractions containing the desired product were combined and concentrated to give Z-3. ¹ H-NMR (400 MHz, CDOj): δ 3.78 (dt, J=11.1, 6.3 Hz, 4H), 3.47 (t, J=6.3 Hz, 2H), 2, 53 (t, J=6.4 Hz, 2H), 1.48 (s, 9H).

Step B: synthesis of intermediate Z-3a.

Trimethylamine (955 mg, 5.33 mmol) (33% Wt, in EtOH) was added to a stirred solution of Z-3 tert-butyl 3-(2-bromoethoxy)propanoate (450 mg, 1.778 mmol) in ACN (2 mL). The reaction mixture was stirred at 50°C for 16 hours. The resulting mixture was concentrated under reduced pressure to obtain Z-3a. LC/MS: (M-Br) ⁺ =232.3. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 5.32 (s, 1H), 4.04-3.94 (m, 4H), 3.73 (t, J=5.7 Hz, 2H) , 3.50 (s, 10H), 2.50 (t, J=5.7 Hz, 2H), 1.44 (s, 9H).

Step C: synthesis of intermediate Z-3b.

To a solution of Z-3a (550 mg, 1.761 mmol) in DCM (0.6 mL) was added 4 M HCl in dioxane (2.5 mL) at room temperature. The mixture was stirred at room temperature for 4 hours. 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 obtain Z-3b. LC/MS: (M-C1) ⁺ =176.2.

Preparation of intermediate Z-4b.

- 62 043952

Step A: Preparation of intermediate Z-4.

To a solution of DIAD (1.755 mL, 9.03 mmol) in THF (30 mL) was added Ph ₃ P (2.368 g, 9.03 mmol). The mixture was stirred at room temperature for 10 min, then methyl 2-(3hydroxyphenyl)acetate (1.0 g, 6.02 mmol) and 3-(dimethylamino)propan-1-ol (0.931 g, 9.03 mmol). The mixture was stirred at 50°C for 1 hour. 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 in DCM. Fractions containing the desired product were combined and concentrated to give Z-4. LC/MS: (M+H) ⁺ =252.2. 1H-NMR (300 MHz, CDC13): δ 7.23-7.18 (m, 1H), 6.82 (td, J=8.7, 4.1 Hz, 3H), 4.01 (t, J=6.4 Hz, 2H), 3.69 (s, 3H), 3.59 (s, 2H), 2.46 (t, J=7.3 Hz, 2H), 2.27 (s, 6H), 1.97 (dt, J=7.9, 6.5 Hz, 2H).

Step B: Preparation of intermediate Z-4a.

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 hour. The resulting solution was concentrated under reduced pressure to give -Z-4a. LC/MS: (MI) ⁺ =266.2.

Step C: Preparation of intermediate Z-4b.

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). This mixture was stirred at room temperature for 2 hours. The pH of the solution was adjusted to 4 with HCl (1 M) and the solution was concentrated under reduced pressure. The crude product was purified by C18 reverse phase chromatography (mobile phase A: water, mobile phase B: ACN; flow rate: 60 ml/min; gradient: 1% B to 25% B over 25 min; 25% B to 95% B for 15 min; 95% B to 95% B for 10 min) to obtain Z-4b. LC/MS: (M-C1) ⁺ =252.2. ¹ H-NMR (300 MHz, CD3OD): δ 7.21 (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).

- 63 043952

Example 4. Preparation of Ex-23.

Step A: Preparation of Intermediate S-1b.

(2S,3R,4S,5S,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate S-1a (5 g, 12.81 mmol) was added to the solvent 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 saturated aqueous sodium hydrogen carbonate solution, cooled on ice, and the mixture was extracted with DCM (3x60 ml). The organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated, and then the crude product was purified by silica gel flash chromatography (eluting with 0~30% EtOAc/PE) to give S-1b.

Step B: Preparation of Intermediate S-1c.

To a solution of S-1b (4.5 g, 10.94 mmol) in dry DMF (45 ml) was added sodium azide (0.854 g, 13.13 mmol) and the reaction mixture was stirred at 18°C for 30 min. The reaction mixture was diluted with water (30 ml) and extracted with EtOAc (3x80 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 give S-1c. To a solution of S-1b (4.5 g, 10.94 mmol) in dry DMF (45 ml) was added sodium azide (0.854 g, 13.13 mmol) and the reaction mixture was stirred at 18°C for 30 min. The reaction mixture was diluted with water (30 ml) and extracted with EtOAc (3x80 ml). The organic layer was dried over Na2SO4 and evaporated to dryness. The crude product was purified by flash chromatography on silica gel (eluting with 0~30% EtOAc/PE) to give S-1c.

Step C: Preparation of Intermediate S-1d.

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 with air and filled with H2 at 50 psi. inch (345 kPa). The reaction mixture was stirred at 18°C for 5 hours. The reaction mixture was diluted with EtOAc, filtered through celite and concentrated to give S-1d, which was used for the next step.

Step D: Preparation of Intermediate S-1e.

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 Et ₃ N (0.939 ml, 6. 74 mmol). The reaction mixture was stirred for 3 hours until the starting material was completely consumed, and then evaporated. The resulting crude product was purified by flash chromatography on silica gel (eluting with 0~10% DCM/MeOH) to give S-1e. MS

- 64 043952 (ESI): m/z (M+H)+ 448.1. Sh-NMR (400 MHz, CDCl ₃ ) δ: 6.48 (d, J=9.04 Hz, 1H), 5.43 (s, 1H), 5.24 (t, J=8.93

Hz, 1H), 5.07-5.17 (m, 2H), 4.08-4.18 (m, 2H), 4.04 (kv, J=6.69 Hz, 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).

Stage E: obtaining Ex-23.

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 hour. The reaction mixture was quenched dropwise with 1 N LiOH (2.58 ml, 2.58 mmol), the resulting solution was stirred at room temperature. for 2 hours and then filtered and the filtrate was purified on a C18 reverse phase HPLC column using a gradient of 29-34% acetonitrile (0.05% TFA) in water (0.05% TFA) to give Ex-23. LC/MS: [M+1] ⁺ =1657.1.

Example 5. Preparation of Ex-14.

Ex-14

Compound Ex-14 was prepared in a manner similar to those described in Example 1, but using different linkers and alternative synthesis steps. The synthesis of these linkers, alternative steps, and basic assembly are described below.

Preparation of intermediate compound Int-2g.

The intermediate Int-2g, useful as a linker for the preparation of compounds of the invention, was prepared according to the following scheme:

- 65 043952

Stage A - synthesis of Int-2gb.

Sodium triacetoxyhydroborate (1.06 g, 5 mmol) and the mixture was stirred for 2 hours. The reaction mixture was slowly quenched with saturated aqueous NaHCO ₃ and then extracted with DCM and washed with brine. The combined organic layers were dried over MgSO ₄ and concentrated to give Int-2gb. LC/MS: (M+1)+=320.3.

Stage B - synthesis of Int-2gc.

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

Stage C - synthesis of Int-2g.

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 hours. The mixture was concentrated under reduced pressure to obtain Int-2g. LC/MS: (M+1)+=334.3.

Preparation of intermediate compounds 70B and 76C.

Step A - synthesis of intermediate 70B.

To a solution of 69B (1.5 g, 4.46 mmol) in DMF (17.8 ml) at 0°C was added 95% NaH (0.141 g, 5.56 mmol), and the resulting solution was stirred at 0°C for 20 min, and then 3-bromoprop1-yne (80% in toluene) (0.596 ml, 5.35 mmol) was added dropwise. An aqueous solution of lithium hydroxide (2 M) (3345 μL, 6.69 mmol) was added dropwise to the resulting solution. The reaction mixture was stirred at room temperature for 2 hours, filtered and purified by reverse phase HPLC (eluting with acetonitrile/water+0.1% TFA) to give 70B. LC/MS: (M+1) ⁺ : 361.0, (M+Na) ⁺ : 383.0.

Stage B - synthesis of intermediate 76C.

Conversion of 70B to intermediate 76C was carried out using procedures similar to those described for the preparation of intermediate 76, C-H steps. LC/MS: [M+1] ⁺ =812.16.

- 66 043952

Assembling example Ex-14 into a connection:

- 67 043952

Step A - synthesis of intermediate 117.

Intermediate 117 was prepared from intermediates Int-2g and 76C according to procedures similar to those described in Example 1 and 1A. More specifically, Int-2g was functionalized using the same reagents and procedures used to prepare intermediate 77B, steps A-B, then developed the following procedures to prepare intermediate 86, steps G-J, and then finally coupled to intermediate 76C following the procedures for the preparation of Example 1A, Steps B-C, to give 117. LC/MS: (M+1)+: 1573.36.

Step B - synthesis of intermediate 118.

Tetrakis(acetonitrile)copper(I) hexafluorophosphate (51.6 mg, 0.138 mmol), tris[(1-benzyl-1H-1,2,3triazol-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 bubbled with nitrogen and then heated to 50°C. Intermediate 117 (435.3 mg, 0.277 mmol) was added to the reaction mixture as a solid. After 1 hour, the reaction mixture was treated with aqueous buffer at pH 4 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 give intermediate 118. LC/MS: (M+1) ⁺ : 1573.2.

Stage C - synthesis of Ex-14.

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

Using the synthetic schemes described above, and as will be appreciated, in some cases with appropriate substitution of certain intermediates, including the use of alternative spacers known to those skilled in the art, the preparation of which may be described above, the following compounds of the invention were prepared , given in table. 2 below. In addition, alternative salt forms of the compounds of the present invention may also be described herein.

table 2

- 69 043952

- 70 043952

- 71 043952

- 72 043952

- 73 043952

- 74 043952

- 75 043952

- 76 043952

- 77 043952

- 78 043952

- 79 043952

- 80 043952

- 81 043952

- 82 043952

- 83 043952

- 84 043952

- 85 043952

Ex-58, salt C1 . o ^nL ^CI V-7 _n ^f _h -£>n AQ ., Up to ⁰ ϊ - D ° YES A 1594.93 Ex-59, salt C1 o =( A ΖΞ O T / ' ° /=\ ¹ A VV - - o X 1593.7 Ex-60, salt C1 , o A aJJ H V oVV NH η γγΊ ^'V ⁰ oAn η A ⁿ ^aA_ DO. , ϊ Up to ⁰ DD) ” nmuA^A A o Chaa 1 LAJ 1607.4 Ex-61, salt C1 | ^Cl 0 C cnA 1 i»V ^N A 1 Ί А·,·^ ⁰ J CA.NH _c 5 _n [ Г h V A ^F A AA 1 Η Ί A ^'· NH %/ > 1 .,- ⁰ А о 4 ¹¹ n ^{0 N} DD A o H-A IIN^ ⁰ tlj 1592.4

Definition of activity.

Individual compounds of the invention were subjected to one or more assays

- 86 043952 their activity in relation to antagonism of PCSK9 activity.

The following is a description of the assay methods used to determine the PCSK9 antagonism activity of the compounds of the invention and any described comparison compounds. Biotinylated PCSK9 was obtained from a commercial source.

TR-FRET for LDLR.

The TR-FRET assay for PCSK9 measures the interaction between PCSK9 and LDLR. A solution containing 40 nM biotinylated PCSK9 + 10 nM Lance ULight Streptavidin is prepared in 50 mM HEPES, pH 7.4, with 0.15 M NaCl, 5 mM CaCl ₂ , 0.01% BSA, and 0.01% surfactant substance P20. A separate solution containing 40 nM rhLDLR-6xHis + 10 nM Eu-W1024 anti-6xHis is prepared in the same buffer system. Echo is used to transfer 0.750 ml of compound to the assay plate followed by the addition of 15 µl PCSK9+Ulight and 15 µl LDLR+Eu. The final assay volume is 30,750 µL containing 20 nM PCSK9, 5 nM Ulight, 20 nM LDLR and 5 nM Eu. The reaction mixture is incubated at room temperature for at least two hours and then fluorescence measurements are taken using the Envision Multilabel Reader. CD values are determined by fitting the data to a sigmoid dose-response curve using nonlinear regression. The number of counts (B counts) of europium-labeled LDLR is determined to determine whether the compounds adversely affect LDLR. A decrease in the number of B pulses likely indicates false-positive inhibition.

TR-FRET with Alexa FRET standard.

The standard Alexa FRET assay for PCSK9 measures the interaction between PCSK9 and AlexaFluor647 (AF)-labeled cyclic peptide, reagent A (K _D =83 nM).

A solution containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (StrepEu) is prepared in 50 mM HEPES pH 7.4, with 0.15 M NaCl, 5 mM CaCl ₂ , 0.01% BSA and 0.01% surfactant P20. A separate solution containing 40 nM AlexaFluor-labeled cyclic peptide was prepared in the same buffer system. Echo is used to transfer 0.750 ml of compound to the assay plate followed by the addition of 15 μl of PCSK9+Stept-Eu and 15 μl of AF peptide. The final assay volume is 30,750 μl containing 0.5 nM PCSK9, 1.25 nM Strep-Eu and 20 nM cyclic AF peptide. The reaction mixture is incubated at room temperature for at least two hours and then fluorescence measurements are taken using the Envision Multilabel Reader. IC50 values are determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. Ki is then calculated from the IC ₅₀ and K _D of the cyclic peptide AF. The number of counts (B counts) of europium-labeled PCSK9 is determined to determine whether the compounds adversely affect PCSK9. A decrease in the number of B pulses likely indicates false-positive inhibition. The data from this procedure is described as A=numeric value (nanomolar)

Reagent A was prepared according to the following method.

- 87 043952

Step A - synthesis of the intermediate compound Int-A.

The peptide was synthesized at 0.250 mmol scale on a Liberty Blue CEM microwave synthesis apparatus using Fmoc/tBu chemistry on PS Rink MBHA amide resin, 0.32 mmol g' ¹ . Assembly was performed using single additions using 4 equiv. 0.2 M Fmoc protected amino acid in DMF, 4 eq. 0.5 M HATU in DMF, 4 eq. 2 M DIPEA (double attachment for Tyr). Fmoc deprotection cycles were performed using 20% v/v. piperidine in DMF.

The sequence of Fmoc-protected amino acids and structural elements used is as follows.

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

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

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

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

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

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

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

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

9. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-M-(tert-butoxycarbonyl)b-lysine.

10. 3-(Tritylthio)propionic acid.

At the end of assembly, the resin was washed with DMF, MeOH, DCM, Et ₂ O. The peptide was cleaved from the solid support using 50 ml of TFA solution (v/v) (91% TFA, 5% H ₂ O, 4% TIPS) for approximately 1.5 hours at room temperature. The resin was filtered, washed with TFA and brine, concentrated to dryness and lyophilized. Lyophilization provided the intermediate Int. A (399 mg), which was used as crude material in the next step. LCMS, analytically calculated for C ₆₁ H ₇₅ F ₂ N ₁₅ O ₁₃ S ₂ : 1328.48, found: 1328.2 (M+1)+.

Step B - synthesis of intermediate Int-B: as described for reagent B.

Purification was carried out by RP-HPLC (Waters Deltapak C4, dual cassette, 40x100 mm, 15 μm, 300 A; 15% to 35% ACN/water + 0.1% TFA modifier for 20 min). The collected fractions were lyophilized to yield 35 mg of intermediate Int-B. LCMS analytically calculated for C69H81F2N15O13S2: 1430.62; found: 1430.9 (M+1) ⁺ .

Step C - synthesis of reagent A compound: as described for reagent B.

LCMS analytically calculated for C105H122F2N17O26S6 ³ ·: 2268.58; 1135.8 (M+2) ²⁺ .

Alexa FRET Plus TR-FRET.

The Alexa FRET Plus assay for PCSK9 detects the interaction between PCSK9 and AlexaFluor647 (AF)-labeled cyclic peptide, reagent B (K _D =35 nM). A solution containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) is prepared in 50 mM HEPES pH 7.4, with 0.15 M NaCl, 5 mM CaCl2, 0.01% BSA and 0.01 % surfactant P20. A separate solution containing 1920 nM AlexaFluor-labeled cyclic peptide was prepared in the same buffer system. Echo is used to transfer 0.075 μl of compound plus 0.675 μl of DMSO into each well of the assay plate, followed by the addition of 15 μl of PCSK9+Stept-Eu and 15 μl of AF peptide. The final assay volume is 30,750 μl containing 0.5 nM PCSK9, 1.25 nM Strep-Eu and 960 nM cyclic AF peptide. The reaction mixture is incubated at room temperature for at least two hours and then fluorescence measurements are taken using the Envision Multilabel Reader. IC ₅₀ values are determined by fitting the data to a sigmoidal dose-response curve

- 88 043952 using nonlinear regression. IC50 values are determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. Ki is then calculated from the IC50 and KD of cyclic peptide AF. The number of counts (B counts) of europium-labeled PCSK9 is determined to determine whether the compounds adversely affect PCSK9. A decrease in the number of B pulses likely indicates false-positive inhibition. The data from this procedure is described as P=numeric value (nanomolar)

Reagent B was prepared according to the following procedure.

Reagent B

Step A - synthesis of the intermediate compound Int-A.

The peptide was synthesized at 0.250 mmol scale on a Liberty Blue CEM microwave synthesis apparatus using Fmoc/tBu chemistry on PS Rink MBHA amide resin, 0.32 mmol g' ¹ . Assembly was performed using single addition using 4 eq. 0.2 M Fmoc protected amino acid in DMF, 4 eq. 1 M oxime in DMF, 4 eq. 0.5 Ν,N-diisopropylcarbodiimide (DIC) (double addition for Y01). Fmoc deprotection cycles were performed using 20% v/v. piperidine in DMF.

The sequence of the Fmoc-protected amino acids and structural elements used is as follows.

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

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

3. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propionic acid.

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

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

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

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

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

9. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N ⁶ -(tert-butoxycarbonyl)-L-lysine.

10. 3-(Tritylthio)propionic acid.

At the end of assembly, the resin was washed with DMF, MeOH, DCM, Et ₂ O. The peptide was cleaved from the solid support using 50 ml of TFA solution (v/v) (91% TFA, 5% H ₂ O, 4% TIPS) for approx. 89 043952 but 1.5 hours at room temperature. The resin was filtered, washed with TFA and brine, concentrated to dryness and lyophilized. Lyophilization provided the intermediate Int.

A (300 mg), which was used as crude material in the next step. LCMS, analytically calculated for C ₆₃ H ₇₉ F ₂ N ₁₅ O ₁₃ S ₂ : 1356.53, found: 1356.9 (M+1)+.

Stage B - synthesis of the intermediate compound Int-B.

The crude compound Int-A (0.22 mmol) was redissolved in 24 ml DMF. 6 mL of 1 M aqueous sodium bicarbonate was added to raise the pH to 7. Then 0.26 mmol of 1,3 bis(bromomethyl)benzene (0.1 M in DMF) was added dropwise. The reaction mixture was left stirring at room temperature for 20 min, quenched with TFA (pH to 3-4), and then concentrated in vacuo to give crude Int-B, which was purified by RP-HPLC (Waters XBridge, C18, 50x150 mm, 5 µm, 130A; from 25% to 40% ACN/water + modifier 0.1% TFA for 20 min). The collected fractions were lyophilized to yield 35 mg of intermediate Int-B. LCMS analytically calculated for C ₇₁ H ₈₅ F ₂ N ₁₅ O ₁₃ S ₂ : 1458.67; found: 1458.8 (M+1) ⁺

Stage C - synthesis of compound reagent B.

Intermediate Int-B (15 mg) was dissolved in 0.2 ml dry DMSO. Then 15 mg of ALEXAFLUOR 647NHS Ester (A37566, Life technology) dissolved in 1.5 ml of dry DMSO was added. 20 µl of dry DIPEA was added. The reaction mixture was left stirring at room temperature for 12 hours under a nitrogen atmosphere in the dark. It was quenched with TFA (pH to 3-4) and purified by HPLC (Dr Maish, Reprosil Gold C18, 250x20 mm, 120A, 10 µm; 20% to 35% 0.1% TFA in ACN/0.1% TFA in H ₂ O, for 20 minutes, then from 35% to 40% for 5 minutes at a flow rate of 20 ml/min). The collected fractions were lyophilized to yield 16.1 mg of compound reagent B. LCMS analytically calculated for C ₁₀₇ H ₁₂₆ F ₂ N ₁₇ O ₂₆ S6 ^3- : 2296.64; found: 1150.6 (M+2) ²⁺ .

Activity data obtained by one or both of the techniques described above are described for individual exemplary compounds of the invention in the following format.

Example No.: A (TR Fret standard)=numeric value; P (Alexa Fret plus TR Fret standard)=numerical value/It should be noted that all values stated are nanomolar.

Alexa FRET Ultra TR-FRET.

The Alexa FRET Ultra assay for PCSK9 measures the interaction between PCSK9 and AlexaFluor647 (AF)-labeled cyclic peptide, reagent B (K _D =0.99 nM). A solution containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) is prepared in 50 mM HEPES pH 7.4, with 0.15 M NaCl, 5 mM CaCl ₂ , 0.01% BSA and 0. 01% surfactant P20. A separate solution containing 1920 nM AlexaFluor-labeled cyclic peptide was prepared in the same buffer system. Echo is used to transfer 0.015 μl of compound plus 0.735 μl of DMSO into each well of the assay plate, followed by the addition of 15 μl of PCSK9+Stept-Eu and 15 μl of AF peptide. The final assay volume is 30,750 μl containing 0.5 nM PCSK9, 1.25 nM Strep-Eu and 960 nM cyclic AF peptide. The reaction mixture is incubated at room temperature for at least two hours and then fluorescence measurements are taken using the Envision Multilabel Reader. IC ₅₀ values are determined by fitting the data to a sigmoidal dose-response curve using nonlinear regression. Ki is then calculated from the IC ₅₀ and KD of cyclic peptide AF. The number of counts (B counts) of europium-labeled PCSK9 is determined to determine whether the compounds adversely affect PCSK9. A decrease in the number of B pulses likely indicates false-positive inhibition. Data from this procedure are described as Ki Ultra=numeric value (data presented are nanomolar).

The following compounds were evaluated as shown in table. 2, using the protocol described above, with the results presented:

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 Ki Plus=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

-

Claims (35)

Ultra-0.01519/ Ex-23 Ki Plus=0.00938, Ki Ultra=0.00239/ Ex-24 Ki Plus=0.00812, Ki Ultra-0.00767/ Ex-25 Ki Plus=0.01127, Ki Ultra-0.00463/ Ex-26 Ki Plus < 0.00558, Ki Ultra-0.002754/ Ex-27 Ki Plus < 0.00558, Ki Ultra-0.00301/ Ex-28 Ki Plus < 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-38 Ki Plus-0.00904, Ki Ultra=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 Ki Plus=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, Ki Ultra-O.00359/ Ex-53 Ki Standard <1.26, Ki Plus-0.09629, Ki Ultra-0.21500/ Ex54 Ki Standard <1.26, Ki Plus=0.36720, Ki Ultra-0.48390/ Ex-55 Ki Standard <1.26, Ki Plus=0.07240, Ki Ultra-O.23 800/ Ex-56 Ki Standard <1.257, Ki Plus=O.02237, Ki Ultra=O.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=O.00788, Ki Ultra-O.004959/ Ex-60 Ki Plus-0.006515, Ki Ultra-0.005312/Ex-61 Ki Plus-0.00747, Ki Ultra-O.006543. CLAIM 1. Compound of formula I Formula I, where X is H, F, Cl or Br; R ¹ is selected from (a) -H; or (b) -(CH ₂ ) _z -R ^14A where z is 1-6 and R ^14A is (i) -H; (ii) -NH2; (iii) -N+Нз; (iv) -N ⁺ (H3C)3; (v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14b where R ^14B is -NH2; -N ⁺ H ₃ ; -N(CH ₃ )2 -N ⁺ (CH3)3; (vi) -NH-C(O)-[(CH ₂ ) _y 12-O-] ₂ -(CH ₂ ) _yl 3R ^14B , where y12 and y13 are not both equal to 2 and are independently equal to 2 to 4; And R ^14b represents -NH2; -N ⁺ H3; -N(CH3)2 or -N ⁺ (CH3) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y = 1-6 and R ^14C is -O-(CH2)3.4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is (ai) -O-(CH2)2-N ⁺ (CH3)3; (aii) -N ⁺ (CH3)3; or or - 91 043952 (aiii) the formula moiety R ² is selected from (a) -H; and (b) -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is selected from (i) -H; ^{(ii) -} NH2 ^; (iii) -N ⁺ Ha; (iv) -К ⁺ (НзС)з; (v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14B where R ^14B is -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ or -K ⁺ (CH3)3; (vi) -NH-C(O)-[(CH2)y12-O-]2-(CH2)y13R ^14B , where y12 and y13 are not both equal to 2 and are independently equal to 2 to 4; And R ^14B represents -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ or -N ⁺ (CH ₃ ) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y = 1-6 and R ^14C is -O-(CH2)3.4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y=1-6 and R ^14C represents (ai) -O-(CH2)2-K ⁺ (CH3)3; (aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3 or -(CH ₂ ) _1-4 -OCH ₃ ; (aiii) part of the formula ’ or (aiv) part of the formula where y ^14Cb and y ^14Сс are equal from 1 to 4; or R ¹ and R ² can be linked together to form part of the formula Where ^G1 , ^RG1a and ^RG1b are defined as follows: (a) G ¹ represents the linker part of the formula: where n ^q1 is from 1 to 6, m ^q1 is 0, 1 or 2 and together the values of n ^q1 and m ^q1 are chosen so that the length of the linker part that they define does not exceed a total of 8 carbon and/or oxygen atoms making up the chain , including a carbon atom in the chain that forms the carbonyl part; R ^G1a is selected from (i) -H; and (ii) alkyl of up to 4 carbon atoms; And R ^G1b selected from (i) part of the formula And - 92 043952 (ii) parts of the formula (b) G ¹ represents the linker part of the formula is equal to 0, 1 or 2, m ^q2 is equal to 1 to 6, and together the values of n ^q2 wherein n ^q2 and m ^q2 are chosen so that the length of the linker portion they define does not exceed a total of 8 carbon and/or oxygen atoms constituting a chain including the carbon atom in the chain that forms the carbonyl portion; R ^G1a selected from (i) part of the formula and (ii) parts of the formula: R ^G1b is selected from (i) -H; and (ii) alkyl of up to 4 carbon atoms; R ⁸ represents -CH3 or part of the formula where R ^8a represents -H or a linear, branched or cyclic alkyl of up to four carbon atoms; A selected from (a) parts of formula (b) -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y is from 1 to 6; (c) parts of the formula where A ^b1 represents (i) part of the formula where x is from 1 to 6; or (ii) part of the formula where y is from 1 to 5; - 93 043952 (d) parts of the formula: -CH ₂ -(CH ₂ ) _m -O-(CH ₂ ) _n -, where m=1-5 and n=0 or 1-4; B represents (a) bond; (b) -(CH2)1-4; or (c) part of formula D is (a) part of formula or -(CH ₂ ) _2-4 -O-, and A and B are as defined above; where E represents -CH ₂ - (b) part of the formula ' where A and B are as defined above; (c) part of the formula ' where n ^a is equal to 1, 2 or 3, m ^a is equal to 2, 3 or 4, and n ^a +m ^a is >3, and where A and B are as defined above; (d) a portion of the formula 'wherein R ^34b is -H or a linear branched or cyclic alkyl of up to four carbon atoms, and A and B are as defined above, or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, wherein X represents F, or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 2, wherein D is part of the formula where E represents -CH2- or -(CH2)2-O-, or a pharmaceutically acceptable salt thereof. 4. The compound according to claim 2 where D represents part of the formula where E represents -CH ₂ - or -(CH ₂ ) ₂ -O-, or a pharmaceutically acceptable salt thereof. 5. A compound according to claim 2, wherein D is part of the formula - 94 043952 or a pharmaceutically acceptable salt thereof. 6. A compound according to claim 2, wherein D is part of the formula or a pharmaceutically acceptable salt thereof. 7. A compound according to claim 2, wherein D is part of the formula or a pharmaceutically acceptable salt thereof. 8. A compound according to claim 2, wherein D is part of the formula or a pharmaceutically acceptable salt thereof. 9. A compound according to claim 2, wherein D is part of the formula or a pharmaceutically acceptable salt thereof. 10. A compound according to claim 2, wherein D is part of the formula or a pharmaceutically acceptable salt thereof. 11. A compound according to claim 2, where R ¹ and R ² are joined together by part of the formula so that together with the cyclopeptide to which R ¹ and R ² are linked, they form a cyclic structure, or a pharmaceutically acceptable salt thereof. 12. A compound according to claim 2, having the structure of formula IIB, or a pharmaceutically acceptable salt thereof - 95 043952 its pharmaceutically acceptable where D ² is (a) part of the formula 13. A compound according to claim 2 having the structure of formula IIC, or a salt - 96 043952 salt salt salt (b) part of the formula 14. A compound according to claim 2, having a structure of formula IID, or a pharmaceutically acceptable form thereof or where D ² represents its pharmaceutically acceptable 16. A compound according to claim 2, having a structure of formula IIF, or a pharmaceutically acceptable form thereof - 97 043952 ABOUT Formula IIF. 17. A compound according to any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof, where A represents (a) -(CH ₂ )6; (b) part of the formula sn _? ’ where x is from 1 to 3; or (c) part of the formula 18. A compound according to any one of claims 1-10 and 12-17 or a pharmaceutically acceptable salt thereof, wherein R ² is (a) -(CH ₂ ) _z -R ^14A , where z is 1-6, and R ^14A is by itself (a) -H; (b) -NH2; (c) -N ⁺ H3; (d) -N ⁺ (H3C)3; (e) -NH-C(O)-[(CH ₂ ) _2-4 -O-]2-(CH ₂ ) ₂ - ₄ R ^14B where R ^14B is -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ or -N ⁺ (CH3)3; (f) -NH-C(O)-[(CH ₂ )yR ^14C , where y=1-6 and R ^14C is (ai) -O-(CH2)2-4-N ⁺ (CH3)3; (aii) -N ⁺ (CH ₃ ) ₃ ; or (aiii) part of the formula 19. A compound according to any one of claims 1-10 and 12-18 or a pharmaceutically acceptable salt thereof, where R ¹ is selected from (a) -H; (b) -(CH ₂ ) _z -R ^14A where z is 1-6 and R ^14A is (i) -H; (ii) -N ⁺ H ₃ ; or (iii)-NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ -N ⁺ (CH ₃ ) ₃ . 20. A compound according to any one of claims 1-11 and 17-19 or a pharmaceutically acceptable salt thereof, where R ⁸ - 98 043952 represents part of the formula where R ^8b represents -H, -CH3 or -C(CH ₃ ) ₃ . 21. The compound according to claim 1, which is selected from the group consisting of - 99 043952 - 100 043952 - 101 043952 - 102 043952 - 103 043952 - 104 043952 - 105 043952 - 106 043952 - 107 043952 - 108 043952 - 109 043952 - 110 043952 - 111 043952 - 112 043952 - 113 043952 - 114 043952 - 115 043952 - 116 043952 - 117 043952 or any other pharmaceutically acceptable salt thereof. 22. A pharmaceutical composition containing at least one compound according to any one of claims 1 to 21, or a pharmaceutically acceptable salt or free base form thereof, and at least one pharmaceutically acceptable excipient. 23. A method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of the composition according to claim 22. 24. A method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 21. 25. A compound according to claim 15, having the structure of formula IIE, or a pharmaceutically acceptable salt thereof The formula is NOT where R ¹ is selected from - 118 043952 (a) -N; or (b) -(CH ₂ ) _z -R ^14A where z is 1-6 and R ^14A is (i) -H; (ii)-NH2; (iii) -N ⁺ H ₃ ; (iv) -N ⁺ (H ₃ C) ₃ ; (v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14b where R ^14B is -NH2; -N ⁺ H3; -N(CH3)2; or ^-N (CH3)3 ^; (Vi) -NH-C(O)-[(CH2) _y 12-O-]2-(CH2) _y 13R ^14B where y12 and y13 are both not 2 and independently represent 2-4; And R ^14b represents: -NH2; -N ⁺ H3; -N(CH3)2; or -N ⁺ (CH3) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C where, y=1-6 and R ^14C represents -O-(CH2)3_4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is (ai) -O-(CH2)2-N ⁺ (CH3)3; (aii) -N ⁺ (CH3)3; R ² is selected from (a) -H; and (b) -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is selected from (i) -H; (ii) -NH2; (iii) -N ⁺ H ₃ ; (iv) -N ⁺ (H3C)3; (v) -NH-C(O)-[(CH ₂ ) ₂ -O-] ₂ -(CH ₂ ) ₂ R ^14b where R ^14B is -NH2; -N ⁺ H ₃ ; -N(CH ₃ )2; or -N ⁺ (CH3)3; (vi) -NH-C(O)-[(CH2)y12-O-]2-(CH2)y13R ^14B where y12 and y13 are both not 2 and independently represent 2 to 4; And R ^14b represents -NH2; -N ⁺ H3; -N(CH3)2; or -N ⁺ (CH3) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y = 1-6 and R ^14C represents -O-(CH2)3_4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is (ai) -O-(CH2)2-N ⁺ (CH3)3; (aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca represents -CH3 or -(CH ₂ ) ₁ _ ₄ -OCH ₃ ; A is selected from (a) -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y represents 1-6; (b) part of the formula ' where A ^b1 represents (i) part of the formula CH ₂ (ii) part of the formula , where x represents 1-6; or , where y represents 1-5; and (c) a portion of the formula -CH ₂ -(CH ₂ )mO-(CH ₂ ) _n -, where m=1-5 and n=0 or 1-4. 26. The compound according to claim 25, or a pharmaceutically acceptable salt thereof, where R ¹ is -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is (i) -H; (ii) -NH2; (iii) -N ⁺ H ₃ ; (iv) -N ⁺ (H3C)3; (v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R ^14B , where R ^14B is -NH2; -N+Ш -N(CH ₃ )2; -N ⁺ (CH3)3; (vi) -NH-C(O)-[(CH2)y12-O-]2-(CH2)y13R ^14B where y12 and y13 are both not 2 and independently are 2-4; And R ^14b represents -NH2; -N ⁺ H3; -N(CH3)2; or -N ⁺ (CH3) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y = 1-6 and R ^14C represents -O-(CH2)3_4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is (ai) -O-(CH2)2-N ⁺ (CH3)3; (aii) -N ⁺ (CH3)3; R ² is -(CH ₂ ) _z -R ^14A where z is 1-6 and R ^14A is selected from or - 119 043952 (i) -H; (ii) -NH2; (iii) -N ⁺ H ₃ ; (iv) -NWf; (v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R ^14B where R ^14B is -NH2; -N%; -N(CH3b; or -N'(CI th; (vi) -NH-C(O)-[(CH2) _y i2-O-]2—(CH2) _y i3R ^14B where y12 and y13 are both not 2 and independently represent 2 to 4; And R ^14B represents -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ ; or -N ⁺ (CH ₃ ) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is -O-(CH2)3-4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C , where y = 1-6 and R ^14C is (ai) -O-(CH ₂ ) ₂ -N ⁺ (CH ₃ ) ₃ ; (aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3 or -(CH ₂ ) _1-4 -OCH ₃ ; A represents -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y represents 1-6. 27. The compound according to claim 26, or a pharmaceutically acceptable salt thereof, where R ¹ is -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is (i) -H; R ² is -(CH ₂ ) _z -R ^14A , where z is 1-6 and R ^14A is selected from (i) -H; (ii) -NH2; (iii) -\H _; ; (iv) -N ⁺ (H3C)3; (v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R ^14B , where R ^14B is -NH2; -N%; -N(CH3h; or -N ⁺ (CH3)3; (vi) -NH-C(O)-[(CH2)y12-O-]2-(CH2)y13R ^14B where y12 and y13 are both not 2 and independently represent 2 to 4; And R ^14B represents -NH2; -N ⁺ H3; -N(CH ₃ ) ₂ ; or -N ⁺ (CH ₃ ) ₃ ; (vii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is -O-(CH2)3-4-N ⁺ (CH3) ₃ ; and (viii) -NH-C(O)-(CH ₂ ) _y R ^14C where y=1-6 and R ^14C is (ai) -O-(CH2)2-N ⁺ (CH3)3; (aii) -N ⁺ (CH3)2R ^14ca , where R ^14ca is -CH3 or -(CH ₂ ) _1-4 -OCH ₃ ; A represents -CH ₂ -(CH ₂ ) _y -CH ₂ -, where y represents 1-6. 28. A compound selected from the group consisting of - 120 043952 - 121 043952 - 122 043952 - 123 043952 - 124 043952 - 125 043952 - 126 043952 - 127 043952 - 128 043952 where A- represents a pharmaceutically acceptable anion. 29. A compound having a structure where ^A is a pharmaceutically acceptable anion. 30. A pharmaceutical composition containing a compound according to claim 29 and at least one pharmaceutically acceptable excipient. 31. A method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of the composition according to claim 30. 32. The connection according to claim 29, where the connection is - 129 043952 33. A pharmaceutical composition containing a compound according to claim 32 and at least one pharmaceutically acceptable excipient. 34. A method for treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of the composition according to claim 33. 35. The connection according to claim 13, where D ² represents 36. The compound according to claim 14, where D ² represents (b) part of the formula (a) part of the formula Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2 - 130-