(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

......................

International Bureau (10) International Publication Number

(43) International Publication Date WO 2019/246349 Al
26 December 2019 (26.12.2019) WI P Ο I PCT

(51) International Patent Classification: WALJI, Abbas, M.; c/o Merck Sharp & Dohme Corp,
C07K 7/06 (2006.01) C12N9/64 (2006.01) 770 SumneytownPike, WestPoint, PA 19486 (US). NAIR,
A61K 38/00 (2006.01) Anikumar, G.; c/o Merck Sharp & Dohme Corp, 2000
Galloping Hill Road, Kenilworth, NJ 07033 (US). DING,
(21) International Application Number:
Fa-Xiang; c/o Merck Sharp & Dohme Corp., 2000 Gal­
PCT/US2019/038155
loping Hill Road, Kenilworth, NJ 07033 (US). BIANCHI,
(22) International Filing Date: Elisabetta; c/o Irbm Science Park S.p.A., Via Pontina
20 June 2019 (20.06.2019) Km 30, 600-0040 Pomezia (IT). BRANCA, Danila; c/o
Irbm Science Park S.p.A., Via Pontina Km 30, 600-0400
(25) Filing Language: English
Pomezia (IT). WU, Chengwei; c/o Merck Sharp & Dohme
(26) Publication Language: English Corp., 770 SumneytownPike, WestPoint, PA 19486 (US).
XIONG, Yusheng; c/o Merck Sharp & Dohme Corp., 2000
(30) Priority Data:
Galloping Hill Road, Kenilworth, NJ 07033 (US). HA,
62/687,913 21 June 2018 (21.06.2018) US
Sookhee, Nicole; c/o Merck Sharp & Dohme Corp., 2000
(71) Applicant: MERCK SHARP & DOHME CORP. Galloping Hill Road, Kenilworth, NJ 07033 (US). LIU,
[US/US]; 126 East Lincoln Avenue, Rahway, NJ Jian; c/o Merck Sharp & Dohme Corp., 2000 Galloping
07065-0907 (US). Hill Road, Kenilworth, NJ 07033 (US). BOGA, Sobhana,
Babu; c/o Merck Sharp & Dohme Corp., 2000 Galloping
(72) Inventors: WOOD, Harold, B.; c/o Merck Sharp & Hill Road, Kenilworth, NJ 07033 (US).
Dohme Corp, 2000 Galloping Hill Road, Kenilworth, NJ
07033 (US). JOSIEN, Hubert, B.; c/o Merck Sharp & (74) Agent: TRINQUE, Brian, C. et al.; Lathrop Gage LLP, 28
Dohme Corp, 2000 Galloping Hill Road, Kenilworth, NJ State Street, 7th Floor, Boston, MA 02109 (US).
07033 (US). TUCKER, Thomas, Joseph; c/o Merck Sharp (81) Designated States (unless otherwise indicated, for every
& Dohme Corp, 770 Sumneytown Pike, West Point, PA kind of national protection available)'. AE, AG, AL, AM,
19486 (US). KEREKES, Angela, Dawn; c/o Merck Sharp AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
& Dohme Corp., 2000 Galloping Hill Road, Kenilworth, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
NJ 07033 (US). TONG, Ling; c/o Merck Sharp & Dohme DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
Corp, 770 Sumneytown Pike, West Point, PA 19486 (US). HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,

(54) Title: PCSK9 ANTAGONIST COMPOUNDS

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIN
2019/246349 A l

(57) Abstract: Disclosed are compounds of Formula I, or a salt thereof: where A, B, D, X, Rl, R2 and R8 are as defined herein, which
compounds have properties for antagonizing PCSK9. Also described are pharmaceutical formulations comprising the compounds of
Formula I or their salts, and methods of treating cardiovascular disease and conditions related to PCSK9 activity, e.g. atherosclerosis,
hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, or related cardiovascular disease and
cardiometabolic conditions.
wo

[Continued on next page]

 WO 2019/246349 Al IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
KM, ML, MR, NE, SN, TD, TG).

Published:
— with international search report (Art. 21(3))
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments (Rule 48.2(h))
 WO 2019/246349 PCT/US2019/038155

PCSK9 ANTAGONIST COMPOUNDS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Application No. 62/687,913, filed
June 21,2018, which is incorporated herein by reference in its entirety.
5

BACKGROUND OF THE INVENTION

The identification of compounds and/or agents that are effective in the treatment of
cardiovascular affliction is highly desirable. In clinical trials, reductions in LDL cholesterol
levels have been directly related to the rate of coronary events; Law et al., 2003 BMJ
10 326:1423-1427. The moderate lifelong reduction in plasma LDL cholesterol levels was
found to correlate with a substantial reduction in the incidence of coronary events; Cohen et
al., 2006 N. Engl. J. Med. 354:1264-1272. This was the case even in populations with a
high prevalence of non-lipid-related cardiovascular risk factors; supra. Accordingly, there is
great benefit to be reaped from the managed control of LDL cholesterol levels.
15 Proprotein convertase subtilisin-kexin type 9 (hereinafter called “PCSK9”), also
known as neural apoptosis-regulated convertase 1 (“NARC-1”), is a proteinase 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 mammalian proprotein convertase family
of serine proteases and contains an N-terminal signal sequence, a prodomain, a catalytic
20 domain, and a C-terminal domain; see Seidah et al., 2012 Nat. Rev. Drug Discov. 11:367­
383. A study of PCSK9 transcriptional regulation demonstrated that it is regulated by sterol
regulatory element-binding proteins (“SREBP”), as seen with other genes involved in
cholesterol metabolism; Maxwell et al., 2003 J. Lipid Res. 44:2109-2119, as is typical of
other genes implicated in lipoprotein metabolism; Dubucefa/., 2004 Arterioscler. Thromb.

25 Vase. Biol. 24:1454-1459. Statins have been shown to upregulate PCSK9 expression in a
1
 WO 2019/246349 PCT/US2019/038155

manner attributed to the cholesterol-lowering effects of the drugs; supra. Moreover, it has
been shown that PCSK9 promoters possess two conserved sites involved in cholesterol
regulation, a sterol regulatory element and an Sp1 site; supra.

While in the endoplasmic reticulum, PCSK9 performs as its only catalytic activity an
5 autocleavage between residues Gln-152 and Ser-153; see Naureckiene et al., 2003 Arch.
Biochem. Biophys. 420:55-67; Seidah etal., 2003 Proc. Natl. Acad. Sci. U. S. A. 100:928-
933. The prodomain remains tightly associated with the catalytic domain during subsequent
trafficking through the frans-Golgi network. The maturation via autocleavage has been
demonstrated to be critical for PCSK9 secretion and subsequent extracellular function (see
10 Benjannet et al., 2012 J. Biol. Chem. 287:33745-33755). Accordingly, several lines of
evidence demonstrate that PCSK9, in particular, lowers the amount of hepatic LDLR protein
and thus compromises the liver's ability to remove LDL cholesterol from the circulation.
Adenovirus-mediated overexpression of PCSK9 in the liver of mice results in the
accumulation of circulating LDL-C due to a dramatic loss of hepatic LDLR protein, with no
15 effect on LDLR mRNA levels; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875;
Maxwell & Breslow, 2004 PNAS 101:7100-7105; Park etal., 2004 J. Biol. Chem.

279:50630-50638; and Lalanne etal., 2005 J. Lipid Res. 46:1312-1319. The effect of
PCSK9 overexpression on raising circulating LDL-C levels in mice is completely dependent
on the expression of LDLR, again, indicating that the regulation of LDL-C by PCSK9 is
20 mediated through downregulation of LDLR protein. In agreement with these findings, mice
lacking PCSK9 or in which PCSK9 mRNA has been lowered by antisense oligonucleotide
inhibitors have higher levels of hepatic LDLR protein and a greater ability to clear circulating
LDL-C; Rashid etal., 2005 PNAS 102:5374-5379; and Graham etal., 2007 J. Lipid Res.
48(4):763-767. In addition, lowering PCSK9 levels in cultured human hepatocytes by
25 siRNA also results in higher LDLR protein levels and an increased ability to take up LDL-C;

2
 WO 2019/246349 PCT/US2019/038155

Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875; and Lalanne etal., 2005 J. Lipid
Res. 46:1312-1319. Together, these data indicate that PCSK9 action leads to increased
LDL-C by lowering LDLR protein levels.
A number of mutations in the gene PCSK9 have also been conclusively associated
5 with autosomal dominant hypercholesterolemia (“ADH”), an inherited metabolism disorder
characterized by marked elevations of low density lipoprotein (“LDL”) particles in the

plasma which can lead to premature cardiovascular failure; see Abifadel et al., 2003 Nature
Genetics 34:154-156; Timms et al., 2004 Hum. Genet. 114:349-353; Leren, 2004 Clin.
Genet. 65:419-422. A later-published study on the S127R mutation of Abifadel etal.,
10 supra, reported that patients carrying such a mutation exhibited higher total cholesterol and
apoB100 in the plasma attributed to (1) an 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 reduction in clearance or

conversion of said lipoproteins; Ouguerram etal., 2004 Arterioscler. Thromb. Vase. Biol.
15 24:1448-1453.
Accordingly, there can be no doubt that PCSK9 plays a role in the regulation of LDL.
The expression or upregulation of PCSK9 is associated with increased plasma levels of
LDL cholesterol, and the corresponding inhibition or lack of expression of PCSK9 is
associated with reduced LDL cholesterol plasma levels. Decreased levels of LDL
20 cholesterol associated with sequence variations in PCSK9 have been found to confer
protection against coronary heart disease; Cohen, 2006 N. Engl. J. Med. 354:1264-1272.
Thus, identification of compounds and/or agents effective in the treatment of
cardiovascular affliction is highly desirable, including antagonism of PCSK9’s role in LDL

regulation, however, in general, because PCSK9 circulates in blood and has modest
25 binding affinity to cell surface LDL receptors here-to-fore attempts to utilize this mechanism

3
 WO 2019/246349 PCT/US2019/038155

in treatment of diseases related to high serum LDL levels have been focused on the use of
large biomolecules, for example, antibodies. Accordingly, there is scant publication
reflecting activity toward this target using small peptides or small molecules to inhibit
PCSK9, see for example, Zhang etal., 2014 J. Biol. Chemistry, 289(2): 942-955.
5 Moreover, there is a paucity of compounds which are amenable to formulation into a
dosage form for utilizing an oral administration route of dosing such compounds, a route
which would be highly desirable for the provision of therapy for conditions in which
regulation of the activities of PCSK9 could play a role.
The present invention advances these interests by providing antagonists of PCSK9
10 which are believed to be of use for inhibiting the activities of PCSK9 and the corresponding
role PCSK9 plays in various conditions for which the administration of a PCSK9 antagonist
provides therapy.

SUMMARY OF THE INVENTION

In one aspect the invention provides a compound of Formula I:

15
Formula I,

wherein:
X is H, F, Cl or Br;
20 R1 is selected from:
4
 WO 2019/246349 PCT/US2019/038155

(a) -H; or
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
(i) -H;
(ii) -NH2;
5 (iii) -N+H3;
(iv) -N+(H3C)3;
(v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vi) -NH-C(O)-[(CH2)yi2-O-]2-(CH2)yi3R14B wherein:
10 yi2 and yi3 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C
is -O-(CH2)za-N+(CH3)3, wherein za is 3 or 4; and
(viii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
15 (ai) -O-(CH2)2-N+(CH3)3;
(aii) -N+(CH3)3; or
(aiii) a moiety of the formula:

R2 is selected from:
20 (a) -H; and
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is selected from:
(i) -H;
(ii) -NH2;

5
 WO 2019/246349 PCT/US2019/038155

(iii) -N+H3;
(iv) -N+(H3C)3;
(v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
5 (vi) -NH-C(O)-[(CH2)yi2-O-]2-(CH2)yi3R14B wherein:
yi2 and yi3 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C
is -O-(CH2)zb-N+(CH3)3, wherein zb is 3 or 4; and
io
10 (viii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
(ai) -O-(CH2)2-N+(CH3)3;
(aii) -N+(CH3)2R14ca, wherein R14ca is -CH3 or-(CH2)i-4-OCH3;
(aiii) a moiety of the formula:
OH

OH

HO ; or
(aiv) a moiety of the formula:
o
,N+(CH3)3

14Cc

where R14Cb and R14Ccare 1 to 4; or

R1 and R2 may be bonded together to form a moiety of the formula:

RG1a RG1b , wherein:

6
 WO 2019/246349 PCT/US2019/038155

G1 RG1a anc| RG1b are cjefjnec| as follows:

(a) G1 is a linker moiety of the formula:

wherein nq1 is 1 to 6, mq1 is 0, 1 or 2 and together the value of nq1 and mq1 are
5 selected such that the length of the linker moiety they define does not exceed
a total of 8 carbon and/or oxygen atoms comprising the chain including the
carbon atom in the chain that forms the carbonyl moiety;
RG1a is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms; and
RG1b is selected from:
10 (i) a moiety of the formula:

(ii) a moiety of the formula:

(b) G1 is a linker moiety of the formula:

wherein nq2 is 0, 1 or 2, mq2 is 1 to 6, and together the value of nq2 and mq2
are selected such that the length of the linker moiety they define does not
exceed a total of 8 carbon and/or oxygen atoms comprising the chain
including the carbon atom in the chain that forms the carbonyl moiety;

7
 WO 2019/246349 PCT/US2019/038155

RG1a is selected from:

(i) a moiety of the formula:

(ii) a moiety of the formula:

RG1b is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms;
R8 is -CH3 or a moiety of the formula:
Rea

ch3

wherein R8a is -H, or a linear, branched or cyclic alkyl of up to four carbon atoms;
10 A is selected from:
(a) a moiety of the formula:

(b) -CH2-(CH2)y-CH2- wherein y is 1 to 6;

(c) a moiety of the formula:

15 wherein Ab1 is:

(i) a moiety of the formula:

8
 WO 2019/246349 PCT/US2019/038155

ch2

wherein x is 1 to 6; or
a moiety of the formula:

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

B is:
a bond;
(b) —(CH2)i-4; or
(c) a moiety of the formula:

10

D is:
a moiety of the Formula:

B N

15 wherein E is -CH2- or -(CH2)2-4-O-, and A and B are as defined above;

(b) a moiety of the formula:

B
N
H

wherein A and B are as defined above;

9
 WO 2019/246349 PCT/US2019/038155

(c) a moiety of the formula:

wherein na is 1,2, or 3, ma is 2, 3, or 4, and na + ma is £ 3, and wherein A and B

are as defined above;
5 (d) a moiety of the formula:

wherein, R34b is -H or a liner, branched or cyclic alkyl of up to four carbon atoms,

and A and B are as defined above,
or a pharmaceutically acceptable salt of any thereof.
10 In a further embodiment, the invention provides a compound of Formula I, wherein X
is F, or a pharmaceutically acceptable salt of any thereof. In some embodiments, it is
preferred for D to be a moiety of the formula:

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

15 In some embodiments, it is preferred for D to be a moiety of the formula:

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

10
 WO 2019/246349 PCT/US2019/038155

In some embodiments it is preferred for D to be a moiety of the Formula:

wherein A and B are as defined herein.

In some embodiments, it is preferred for D to be a moiety of the formula:

5
wherein A and B are as defined herein.
In some embodiments, it is preferred for D to be a moiety of the formula:

wherein A and B are as defined herein.

10 In some embodiments, it is preferred for D to be a moiety of the formula:

wherein A and B are as defined herein.

In some embodiments, it is preferred for D to be a moiety of the formula:
A

15 wherein A and B are as defined herein.

11
 WO 2019/246349 PCT/US2019/038155

In some embodiments, it is preferred for D to be a moiety of the formula:

wherein A and B are as defined herein.

In some embodiments wherein R1 and R2 are joined together, along with the peptide
5 ring to which they are attached forming thereby a cyclic structure, it is preferred for R1 and
R2 to form a moiety of the structure:

In one embodiment the present invention provides pharmaceutical compositions

comprising a compound of the invention, for example, a compound of Formula I, and at
10 least one pharmaceutical excipient, preferably a composition directed to oral administration.
In one aspect the present invention provides a method of antagonizing PCSK9 in the
provision of therapy for disease states related to PCSK9 activity, for example,
atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute
coronary syndrome, or related cardiovascular disease and cardiometabolic conditions, by
15 administering to a subject in need thereof a therapeutically effective amount of a compound
of Formula I, or a salt thereof, preferably in the form of a pharmaceutical composition.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows conventional structural representation is employed and
20 includes conventional stereochemical notation for certain asymmetric carbon centers.

12
 WO 2019/246349 PCT/US2019/038155

Thus, structural representation of compounds of the invention includes conventional

stereochemical notation for some asymmetric carbon centers shown in the example
compounds. Accordingly, in such instances, solid black “wedge” bonds represent bonds

projecting from the plane of the reproduction medium, “hashed wedge” bonds representing

5 descending bonds into the plane of the reproduction medium, and a “wavy” line appended

to a carbon bearing a double bond indicates both possible cis and trans orientations are
included. As is conventional, plain solid lines represent all spatial configurations for the
depicted bonding. Accordingly, where no specific stereochemical notation is supplied, the
representation contemplates all stereochemical and spatial orientations of the structural
10 features.
As is shown in the examples of the invention, and mentioned above, particular
asymmetric carbon centers are structurally represented using conventional “Solid Wedge”

and “Hash Wedge” bonding representation. For the most part, absolute configuration has
not been determined for the example compounds, but has been assigned by analogy to
15 specific example compounds of known stereochemical configurations (determined by X-ray
crystallography) prepared using the same or analogous reaction conditions and starting
reagents and isolated under the same chromatographic conditions. Accordingly, specific
assignment of the configurations structurally represented herein is meant to identify the
specific compounds prepared has having an excess of one particular stereoisomer and is
20 not put forth herein necessarily as being a statement of the absolute determination of the
stereochemical structure of said compound unless otherwise noted in the data presented.
It will be appreciated that where isomeric mixtures are obtained, the preparation of
individual stereoisomers in significant percentages of enantiomeric excess can be carried
out, if desired, by separation of the mixture using customary methods, for example by
25 chromatography or crystallization, or by the use of stereochemically uniform starting

13
 WO 2019/246349 PCT/US2019/038155

materials for the synthesis described, or by stereoselective synthesis. Optionally a

dehvatization can be carried out before a separation of stereoisomers. The separation of a
mixture of stereoisomers can be carried out at an intermediate step during the synthesis of
a compound of Formula I or it can be done on a final racemic product.
5 Where indicated herein, absolute stereochemistry is determined by X-ray
crystallography of crystalline products or crystalline intermediates which are derivatized, if
necessary, with a reagent containing a stereogenic center of known configuration. Unless a
particular isomer, salt, solvate (including hydrates) or solvated salt of such racemate,
enantiomer, or diastereomer is indicated, the present invention includes all such isomers,
10 as well as salts, solvates (including hydrates) and solvated salts of such racemates,
enantiomers, diastereomers and mixtures thereof.
The present invention also embraces isotopically-labeled compounds of the present
invention which are structurally identical to those recited herein, but for the fact that a
statistically significant percentage of one or more atoms in that form of the compound are
15 replaced by an atom having an atomic mass or mass number different from the atomic
mass or mass number of the most abundant isotope usually found in nature, thus altering
the naturally occurring abundance of that isotope present in a compound of the invention.
The present invention is meant to include all suitable isotopic variations of the compounds
of Formula I.
20 Examples of isotopes that can be preferentially incorporated into compounds of the
invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine,
fluorine and chlorine, for example, but not limited to: 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O,
18O, 31P, 32P, 35S, 18F, and 36CI, 123l, and 125l. It will be appreciated that other isotopes may
be incorporated by known means also.

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 WO 2019/246349 PCT/US2019/038155

In particular, certain isotopically-labeled compounds of the invention (e.g., those

labeled with 3H, 11C, and 14C) are recognized as being particularly useful in compound
and/or substrate tissue distribution assays using a variety of known techniques.
Additionally, compounds of the invention contemplate isotopic substitution include different
5 isotopic forms of hydrogen (H), including protium (1H) and deuterium (2H or D). Protium is
the predominant hydrogen isotope found in nature. Enriching for deuterium may afford
certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage
requirements, or may provide a compound useful as a standard for characterization of
biological samples. Isotopically-enriched compounds within Formula I can be prepared
10 without undue experimentation by conventional techniques well known to those skilled in
the art or by processes analogous to those described in the Schemes and Examples herein
using appropriate isotopically-enriched reagents and/or intermediates.

Where a wavy line terminates a conventional bond (as opposed to connecting two
atoms within a structure) it indicates a point of bonding to a structure, e.g.:

ch2—|

15

indicates a the secondary-butyl moiety is bonded via the methylene group via the bond
terminated with the wavy line. Where an alphabetical notation is used to depict a
substituent moiety, a dash is employed to indicate the point of bonding to the indicated
substrate, e.g.: -CH2-C(O)-CH2CI indicates the acetyl chloride moiety is bonded via the
20 methylene portion of the moiety.

When any variable (e.g., n, Ra, Rb, etc.) occurs more than one time in any
constituent or in Formula I, its definition on each occurrence is independent of its definition
at every other occurrence unless otherwise specified at the point of definition. One of
ordinary skill in the art will recognize that choice of combinations of the various substituents
15
 WO 2019/246349 PCT/US2019/038155

defined in a structural representation, i.e. R1, RA, etc., are to be chosen in conformity with
well-known principles of chemical structure connectivity and stability, and combinations of
substituents and/or variables are permissible only if such combinations result in stable
compounds.
5 A "stable" compound is a compound which can be prepared and isolated and whose
structure and properties remain or can be caused to remain essentially unchanged for a
period of time sufficient to allow use of the compound for the purposes described herein
(e.g., therapeutic administration to a subject). The compounds of the present invention are
limited to stable compounds embraced by Formula I.
10 Where any variable or moiety is expressed in the form of a range, e.g. (-CH2-)i-4,
both of the extremes of the specified range are included (i.e. 1 and 4 in the example) as
well as all of the whole number values in between (i.e. 2 and 3 in the example).

The term "halogen" includes fluorine, chlorine, bromine and iodine unless specified
otherwise at the point of use.

15 As the term is used herein, “subjects” (alternatively “patients”) refers to an animal,

preferably a mammal, and in particular a human or a non-human animal including livestock

animals and domestic animals including, but not limited to, cattle, horses, sheep, swine,
goats, rabbits, cats, dogs, and other mammals in need of treatment. In some embodiments
the subject is preferably a human. As used herein, the term "administration" and variants
20 thereof (e.g., "administering" a compound) in reference to a compound of Formula I means
providing the compound, or a pharmaceutically acceptable salt thereof, to a subject in need
of treatment.

16
 WO 2019/246349 PCT/US2019/038155

As mentioned above, in one aspect the present invention includes the provision of
compounds of Formula I, or a pharmaceutically acceptable salt thereof, which have
properties that antagonize PCSK9 function.
In an embodiment, the compounds of Formula I have the structure of Formula IA:

Formula IA,
wherein:
R1 is selected from:
(a) -H; or
10 (b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
(i) -H;
(ii) -NH2;
(iii) -N+H3;
(iv) -N+(H3C)3;
15 (v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vi) -NH-C(O)-[(CH2)yi2-O-]i-4-(CH2)yi3R14B, preferably -NH-C(O)-[(CH2)yi2-
O-]2-(CH2)yi3R14B wherein:

17
 WO 2019/246349 PCT/US2019/038155

y12 and y13 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C
is -O-(CH2)za-N+(CH3)3, wherein za is 3 or 4; and
5 (viii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
(ai) -O-(CH2)2-N+(CH3)3;
(aii) -N+(CH3)3; or
(aiii) a moiety of the formula:

10 R2 is selected from:
(a) -H; and
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is selected from:
(i) -H;
(ii) -NH2;
15 (iii) -N+H3;
(iv) -N+(H3C)3;
(v) -NH-C(O)-[(CH2)2-O-]i-4-(CH2)2R14B, preferably -NH-C(O)-[(CH2)2-O-]2-
(CH2)2R14B wherein R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vi) -NH-C(O)-[(CH2)yi2-O-]2-(CH2)yi3R14B wherein:
20 y12 and y13 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C
is -O-(CH2)zb-N+(CH3)3, wherein zb is 3 or 4; and

18
 WO 2019/246349 PCT/US2019/038155

-NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:

-O-(CH2)2-N+(CH3)3;
-N+(CH3)2R14ca, wherein R14ca is -CH3 or-(CH2)i-4-OCH3;
a moiety of the formula:

5 ; or
a moiety of the formula:
o
,N+(CH3)3
14Cc

where Y14Cb and Y14Ccare 1 to 4; or

R1 and R2 may be bonded together to form a moiety of the formula:

h2c. .ch2
N------- G1----- N
RG1b
io RG1a

wherein:
G1 RG1a anc| RG1b are defjnec| as follows:
G1 is a linker moiety of the formula:

15 wherein nq1 is 1 to 6, mq1 is 0, 1 or 2 and together the value of nq1 and mq1 are
selected such that the length of the linker moiety they define does not exceed
a total of 8 carbon and/or oxygen atoms comprising the chain including the
carbon atom in the chain that forms the carbonyl moiety;
19
 WO 2019/246349 PCT/US2019/038155

RG1a is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms; and
RG1b is selected from:
a moiety of the formula:

N
0
2-10
; and
5 a moiety of the formula:
O

; or
(b) G1 is a linker moiety of the formula:

wherein nq2 is 0, 1 or 2, mq2 is 1 to 6, and together the value of nq2 and mq2
10 are selected such that the length of the linker moiety they define does not
exceed a total of 8 carbon and/or oxygen atoms comprising the chain
including the carbon atom in the chain that forms the carbonyl moiety;
RG1a is selected from:
a moiety of the formula:

N
o
2-10
15 ; and
O

a moiety of the formula: 1-8 ; and

RG1b is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms;

20
 WO 2019/246349 PCT/US2019/038155

R8 is -CH3 or a moiety of the formula:

wherein R8a is -H, or a linear, branched or cyclic alkyl of up to four carbon atoms;
A is selected from:
(a) a moiety of the formula:

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

Ab1
(c) a moiety of the formula: , wherein Ab1 is:
(i) a moiety of the formula:

10

wherein x is 1 to 6; or
(ii) a moiety of the formula:

wherein y is 1 to 5;
15 (d) a moiety of the formula: -CH2-(CH2)m-O-(CH2)n- wherein m = 1 to 5, and n= 0 or
1 to 4;
B is:
(a) a bond;
21
 WO 2019/246349 PCT/US2019/038155

(b) —(CH2)i-4; or
(c) a moiety of the formula:

D is:
5 a moiety of the Formula:

B N

wherein E is -CH2- or -(CH2)2-4-O-, and A and B are as defined above;

(b) a moiety of the formula:

B
N
10 H

wherein A and B are as defined above;

(c) a moiety of the formula:

wherein na is 1,2, or 3, ma is 2, 3, or 4, and na + ma is at least 3, and wherein A and B are

15 as defined above;
(d) a moiety of the formula:

22
 WO 2019/246349 PCT/US2019/038155

wherein, R34b is -H or a liner, branched or cyclic alkyl of up to four carbon atoms,

and A and B are as defined above,
or a pharmaceutically acceptable salt of any thereof.
5 In an embodiment of the compounds of Formula IA, R1 is -(CH2)z-R14A, wherein: z is
1-6, and R14A is:
(i) -H;
(ii) -NH2;
(iii) -N+H3; or
10 (iv) -N+(H3C)3;
R2 is -(CH2)z-R14A, wherein: z is 1-6, and R14A is selected from:
(i) -H;
(ii) -NH2;
(iii) -NH-C(O)-[(CH2)2-O-]i-4-(CH2)2R14B, preferably -NH-C(O)-[(CH2)2-O-]2-
15 (CH2)2R14B wherein R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(iv) -NH-C(O)-[(CH2)yi2-O-]2-(CH2)yi3R14B wherein:
y12 and y13 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(v) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C
20 is -O-(CH2)zb-N+(CH3)3, wherein zb is 3 or 4; and
(vi) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
(ai) -O-(CH2)2-N+(CH3)3;
(aii) -N+(CH3)2R14ca, wherein R14ca is -CH3 or-(CH2)i-4-OCH3;

23
 WO 2019/246349 PCT/US2019/038155

(aiii) a moiety of the formula:

(aiv) a moiety of the formula:

o. N+(CH3)3
14Cb 14Cc

5 where Y14Cb and Y14Ccare 1 to 4; or

R8 is -CHs or a moiety of the formula:

wherein R8a is -H, or a linear, branched or cyclic alkyl of up to four carbon atoms;
A is selected from:
(a) a moiety of the formula:

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

Ab1 κ
(c) a moiety of the formula: , wherein Ab1 is:
(i) a moiety of the formula:
i-CH2X-^

' x
15

24
 WO 2019/246349 PCT/US2019/038155

wherein x is 1 to 6; or
a moiety of the formula:

ch2

wherein y is 1 to 5; and
5 (d) a moiety of the formula: -CH2-(CH2)m-O-(CH2)n- wherein m = 1 to 5, and n= 0 or
1 to 4;
B is:
—(CH2)i-4; or
(b) a moiety of the formula:

10

D is:
a moiety of the Formula:

B N

15 wherein E is -CH2- or -(CH2)2-4-O-, and A and B are as defined above;

(b) a moiety of the formula:

B
N
H

wherein A and B are as defined above; or

(c) a moiety of the formula:
25
 WO 2019/246349 PCT/US2019/038155

wherein, R34b is -H or a liner, branched or cyclic alkyl of up to four carbon atoms,

and A and B are as defined above,
or a pharmaceutically acceptable salt of any thereof.
5 In an embodiment of the compound of Formula IA, D is a moiety of the formula:

wherein, E is -CH2- or-(CH2)2-O-, and A and B are as defined above in Formula IA.
In an embodiment of the compound of Formula IA, A is:
(a) -(CH2)6;
10 (b) a moiety of the formula:

wherein x is 1 to 3; or
(c) a moiety of the formula:

15 In another embodiment of the compound of Formula IA, R2 is:

(a) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
(a) -H;

26
 WO 2019/246349 PCT/US2019/038155

(b) -CH3;
(c) -NH2;
(d) -N+H3;
(e) -N+(H3C)3;
5 (f) -NH-C(O)-[(CH2)2-4-O-]2-4-(CH2)2-4R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(g) -NH-C(O)-[(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
(ai) -O-(CH2)2-4-N+(CH3)3;
(aii) -N+(CH3)3; or
10 (aiii) a moiety of the formula:

(b) a moiety of the formula

(H3C)3N

In a further embodiment of the compound of Formula IA, R1 is selected from:

15 -H;
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
-H;
-N+H3; or
-NH-C(O)-[(CH2)2-O-]2-(CH2)2-N+(CH3)3.

27
 WO 2019/246349 PCT/US2019/038155

In yet another embodiment of the compound of Formula IA, A is -CH2-(CH2)y-CH2-,

wherein y is 3-5. In a further embodiment, A is -(CH2)e.
In another embodiment of the compound of Formula ΙΑ, B is a moiety of the formula:

5 In another embodiment of the compound of Formula IA, R1 is -(CH2)z-R14A, wherein:

z is 1-6, and R14A is -H. In another embodiment of the compound of Formula IA, R1 is -
(CH2)z-R14A, wherein: z is 1, and R14A is -H.
In another embodiment of the compound of Formula IA, R2 is -(CH2)z-R14A, wherein:
z is 1-6, and R14A is -NH-C(O)-(CH2)yR14C, wherein y= 1 to 6 and R14C is -N+(CH3)2R14ca,
10 wherein R14ca is -CHs.
In another embodiment of the compound of Formula IA, R8 is a moiety of the
formula:
R8a

ch3

wherein R8a is -H or linear alkyl of up to four carbon atoms. In a further embodiment, R8 is

15 a moiety of the formula:
R8b

ch3

wherein R8b is -H, -CHs, or-C(CHs)s.

28
 WO 2019/246349 PCT/US2019/038155

In some embodiments, it is preferred for the compounds of Formula I to have the

structure of Formula II or Formula HA, or a pharmaceutically acceptable salt thereof:

Formula II,

Formula HA,
wherein, A, R1 and R2 are as defined above in Formula IA and B1 is -(CH2)o-2, and D1 is
selected from:

29
 WO 2019/246349 PCT/US2019/038155

5 In some embodiments of Formula II or Formula HA, it is preferred for D1 to be a

moiety of the formula: In some embodiments Formula II or Formula HA, it is

preferred for D1 to be a moiety of the formula: In some embodiments of

Formula II or Formula HA, it is preferred for D1 to be a moiety of the formula:

In some embodiments of Formula II or Formula HA, it is preferred for D1

10 to be a moiety of the formula:

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

30
 WO 2019/246349 PCT/US2019/038155

wherein, A, R1 and R2 are as defined above in Formula IA and D2 is a moiety of the

formula:

In some embodiments of Formula III, it is preferred for D2 to be a moiety of the

formula: . In some embodiments of Formula III, it is preferred for D2 to be a

moiety of the formula: In some embodiments of Formula III, it is preferred

for D2 to be a moiety of the formula:

31
 WO 2019/246349 PCT/US2019/038155

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

Formula IV:

Formula IV,
5 wherein, A, R1 and R2 are as defined above in Formula IA.
In some embodiments the compound of Formula I is a compound of Formula V:

32
 WO 2019/246349 PCT/US2019/038155

Formula V,

wherein
A, B, R1 and R2 are as defined above in Formula IA; and
5 D2 is:

(a) a moiety of the formula:

(b) a moiety of the formula:

10 (c) a moiety of the formula:

33
 WO 2019/246349 PCT/US2019/038155

; or

(d) a moiety of the formula:

In some embodiments of Formula I, Formula IA, Formula II, Formula HA, Formula
5 III, Formula IV, or Formula V, it is preferred for A to be a moiety of the formula: -(CH2)ya,
wherein ya is 4 to 6. In some embodiments of Formula I, Formula IA, Formula II, or
Formula HA, it is preferred for A to be a moiety of the formula: -CH2-(CH2)ma-O-(CH2)na-,
wherein ma is 2 or 3 and na is 0 or 1. In some embodiments of Formula HI, Formula IV,
or Formula V, it is preferred for A to be a moiety of the formula: -CH2-(CH2)ma-O-(CH2)na-,
10 wherein ma is 2 or 4 and na is 0, 1, or 2. In some embodiments of Formula I, Formula IA,
Formula II, Formula HA, Formula HI, Formula IV, or Formula V, it is preferred for A to be
a moiety of the formula:

wherein yb is 1 to 3. In some embodiments of Formula I, Formula IA, Formula II,

15 Formula HA, Formula HI, Formula IV, or Formula V, it is preferred for A to be a moiety of
the formula:

1,2

34
 WO 2019/246349 PCT/US2019/038155

Also provided herein as compounds of Formula I are compounds Ex-1, Ex-2, Ex-3,
Ex-4, Ex-5, Ex-6, Ex-7, Ex-8, Ex-9, Ex-10, Ex-11, Ex-12, Ex-13, Ex-14, Ex-15, Ex-16, Ex-
17, Ex-18, Ex-19, Ex-20, Ex-21, Ex-22, Ex-23, Ex-24, Ex-25, Ex-26, Ex-27, Ex-28, Ex-29,
Ex-31, Ex-35, Ex-36, Ex-38, Ex-39, Ex-40, Ex-41, Ex-44, Ex-47, Ex-48, Ex-49, Ex-50, Ex-
5 51, Ex-52, Ex-53, Ex-54, Ex-55, Ex-56, Ex-57, Ex-58, Ex-59, Ex-60, and Ex-61, or any
pharmaceutically acceptable salt thereof. These compounds, which are disclosed in Table
1, are also referred to herein as “compounds of the invention.”

Table 1

35
WO 2019/246349 PCT/US2019/038155

36
 WO 2019/246349 PCT/US2019/038155

10

37
 WO 2019/246349 PCT/US2019/038155

Ex Structure Ex Structure
No No
0
Ex- Ex-
19 20

?
JX ίoA
*
HN- >■/
3-Ανη° \ ° ηHN Λ
: , ■ '

Aft#
A

O-ι O

X' A"
1s
o 0
Ex- Ex-
21 22

Unh ο ΐ 'o HNf "“·■■( N-\

Vnh"<
ζ Uγν W
H JyP
$Γν<Ρ °

0
a” 0 A
0 1
Ex- Ex-
23 24
° A s s
u U //

HN<n fSv6
\/
U Nh^xx
hn~y^ r I·
/
1
HN-- υ^ξ“ΟΗ
° o
θζΤ] A '—'
HCT OH

10

38
 WO 2019/246349 PCT/US2019/038155

10

39
 WO 2019/246349 PCT/US2019/038155

Ex Structure Ex Structure
No No
0 0
Ex- Ex- pX
35 36 „ „ XXX

°χχχχ HNX° ογ\ T k x HN^\o

7X>
// O=/
XXNH η Γ)

'Λ A~ ^γΧ

0 0
0 0
Ex- xCX Ex-
38 39
. s u Xr „ 0
°Χχ °X J° HN V
2 O=/NH F-^~y~Nx__
f / N\ '
\ J/ Q=HNS.
0 J
/ °x
S'
0
A A_
Λ A XSox 0 ]|--- NH
0

0 0
Ex- Ex-
40 41
„ 0
0 a, fa S < -1 "“-^o
/ »< „X> ? O=f ΧΧη H y>
Z Xv ■ 7- fa/fa"

0 NH —

10

15

40
 WO 2019/246349 PCT/US2019/038155

Ex Structure Ex Structure
No No
0 0
Ex- Ex- qYk
44 .. Ayy 47 γ o V^ nh

- 11
CX H Π 9x 7-I NH H 1 OH
Y F-fyl HN^n
> NH )==/ > 1 |l J
< o=/h n h
VJ
1 0=/
HN ... 0 J
J ο^Ν-Λ
Me"AZ
0Μθ

A' o+^-[Link]™ °

0
0 0

yCy
Ex- cY>° Ex-
48 49 γ 0 ν' ν’ NH
%A. AJA ’>Cz,
ο Η Y^aXho^T
ΗχΑ/τα

r° °A. / tA J °A.. A 0<Y"\ 0Me

jA HN 0 J "jC 0 o> Me"'P

A *' ” »' w ° ' A HN 0

H LAY
A Γ°\ 0 0
Ex- ,„.cw> AyY ΗΎ"0 «.o, Ex-
> h3c^oh
50 51 A" L H JLHy) hnXLo /=¾
%N V/rX VY HN-<j/
νγγ
l ΎΑ Ϋ/
Αλ NH /TVA H
I

Y-pA ’ “

10

41
 WO 2019/246349 PCT/US2019/038155

Ex Structure Ex Structure
No No
0
qX .
Ex- PjOo H3Voh Ex-
52 53 °< 1
VW X0
A" n H I
L/PY
.•HjrrzA>H S0 iκ yJ 1
a yNMLh ° n γ ”r
HNW °H
\ NH \=/ 1== -^/ CH3 τ' f—/ y-p Ύιι i
f 0=/ > /A. 0 \ > n—Λ
' T1
°η*~° HsC ) nW Λ—Λ / **
< /
LΛ H k__ P
—\ 0 ----- X"N\ 7x>-—x /n_Ao
_ N*(CH 3)3 S o ---- XZ
A
0 0
Ex- Ex- pX .
W□ o
54 ■A'A, 55
o .> f a S.
Λρ-XrM X
*Η3ΓΓ H 0 it
HNx<o rO °H hiL
X fhCH ΓΓ k X FO> ΓΠ
\-i "T 0/i NH\
A- C A\ v u
O^ jAiS^'OxX
W NW

y\ / V--\ /N"S> "ύ- Γφ-ν4'·

0 —p/
| 0 ''""Ο θ
Ex- X px Ex- V σΥχ
56 57 "ς νχύρ
VxkkAVn
Vanfh<r ρα.
p(X
0 Ρο ο Vw> 0
0 nA, „ V cr "P
in °
HpO K
0 h/° O hW°
VO uo

10

42
 WO 2019/246349 PCT/US2019/038155

Ex Structure Ex Structure
No No
0 . A 0
Ex- v cnA Ex-
58 Nt, γ O Vy NH 59
\ oAAr % T ° A NH
Vw-s PX
S rs Τ Pv°
o < O^N-\ u vT<T TO,
XPaP
o
p
HN^° ° A o ytn
Taj O tt hnT 0
uu
0 A
Ex-
T M UK
Ex- I, O
60 61 c aCa
γ o yv nh
η >A J
ΓL nH .CT/NH
J H A/ A
AA PCU

P^NP "p 1 F λ/A y-N, I I 1

0 HJ°
o ~o JT (χτ-χ
i > °
Taj hnyTAn-J "A
o Av, HN^°
LAJ
wherein A’ is a pharmaceutically acceptable anion.
The term “salt(s)”, and its use in the phrase “pharmaceutically acceptable salts”

employed herein, includes any of the following: acidic salts formed with inorganic and/or
5 organic acids, basic salts formed with inorganic and/or organic bases, zwitterionic and
quaternary ammonium complexes. Salts of compounds of the invention may be formed 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 one in which the salt precipitates or in aqueous medium followed by lyophilization.
10 Compounds of the invention contain th-coordinate nitrogen atoms, for example,
primary, secondary or tertiary amino moieties, wherein, as is known, the lone pair of
electrons residing on the nitrogen atom may be protonated with an appropriate acid or
alkylated with an appropriate reagent, for example, alkyl bromide, under the appropriate

43
 WO 2019/246349 PCT/US2019/038155

reaction conditions to provide tetracoordinate charged nitrogen stabilized by an anion

generated in the process, for example, a halogen ion or conjugate base. Accordingly,
compounds of the invention may be prepared in the form of a free-base or isolated in the
form of a quaternary complex or a salt complex. In some instances where there is an
5 appropriate acidic proton proximal to a basic nitrogen formation of a zwitterionic complex is
possible. As the term is employed herein, salts of the inventive compounds, whether acidic
salts formed with inorganic and/or organic acids, basic salts formed with inorganic and/or
organic bases, salts formed which include zwitterionic character, for example, where a
compound contains both a basic moiety, for example, but not limited to, a nitrogen atom, for
10 example, an amine, pyridine or imidazole, and an acidic moiety, for example, but not limited
to a carboxylic acid, and quaternary ammonium complexes are included in the scope of the
inventive compounds described herein.
Accordingly, structural representation of compounds of the invention, whether in a
free-base form, a salt form, a zwitterionic form or a quaternary ammonium form, also
15 include all other forms of such compounds discussed above. Thus, one aspect of the
invention is the provision of 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 those instances in which the compounds of the invention may form
such complexes, including where a tetracoordinate nitrogen can be quaternized or
20 protonated and the charged nitrogen form stabilized by an associated anion. The term
"pharmaceutically acceptable salt" refers to a salt (including a quaternary ammonium
complex and an inner salt such as a zwitterion complex) which possesses effectiveness
similar to or greater than a free-base form of the compound and which is not biologically or
otherwise undesirable (e.g., is neither toxic nor otherwise deleterious to the recipient
25 thereof).

44
 WO 2019/246349 PCT/US2019/038155

The formation of pharmaceutically useful salts from basic (or acidic) pharmaceutical
compounds are discussed, for example, by S. Berge et al., Journal of Pharmaceutical
Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217;
Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; in
5 The Orange Book (Food & Drug Administration, Washington, D.C. on their website); and P.
Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts: Properties,
Selection, and Use, (2002) Int'l. Union of Pure and Applied Chemistry, pp. 330-331. These
disclosures are incorporated herein by reference.
The present invention contemplates both freebase forms of the compounds of the
10 invention and all available salts, including salts which are generally recognized as safe for
use in preparing pharmaceutical formulations and those which may be formed presently
within the ordinary skill in the art and are later classified as being “generally recognized as

safe” for use in the preparation of pharmaceutical formulations, termed herein as

“pharmaceutically acceptable salts.” As will be appreciated, freebase compounds may be

15 prepared by controlling the conditions of isolation of the compound during synthesis or by
neutralization and ion exchange from salt forms of compounds of the invention.
Examples of pharmaceutically acceptable acid salts include, but are not limited to,
acetates, including thfluoroacetate salts, adipates, alginates, ascorbates, aspartates,
benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates,
20 camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates,
ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates,
heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-
hydroxyethanesulfonates, lactates, maleates, methanesulfonates, methyl sulfates, 2-
naphthalenesulfonates, nicotinates, nitrates, oxalates, pamoates, pectinates, persulfates, 3-
25 phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates,

45
 WO 2019/246349 PCT/US2019/038155

sulfates, sulfonates (such as those mentioned herein), tartarates, thiocyanates,

toluenesulfonates (also known as tosylates,) undecanoates, and the like.
Examples of pharmaceutically acceptable basic salts include, but are not limited to,
ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline
5 earth metal salts such as calcium and magnesium salts, aluminum salts, zinc salts, salts
with organic bases (for example, organic amines) such as benzathines, diethylamine,
dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine),
N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, piperazine,
phenylcyclohexyl-amine, choline, tromethamine, and salts with amino acids such as
10 arginine, lysine and the like. Basic nitrogen-containing groups may be converted to an
ammonium ion or quaternized with agents such as lower alkyl halides (e.g. methyl, ethyl,
propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl,
dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl and stearyl
chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and
15 others.
The term “pharmaceutically acceptable anion" refers to an anion suitable for forming
a pharmaceutically acceptable salt.
Further examples of pharmaceutically acceptable salts that may be used with the
instant invention include, but are not limited to, fluoride, chloride, bromide and iodide.
20 In general, salts of compounds are intended to be pharmaceutically acceptable salts
within the scope of the invention.
The term “purified”, “in purified form” or “in isolated and purified form” for a

compound refers to the physical state of said compound after being isolated from a
synthetic process or natural source or combination thereof. Thus, the term “purified”, “in

25 purified form” or “in isolated and purified form” for a compound refers to the physical state of

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said compound after being obtained from a purification process or processes described
herein orwell known to the skilled artisan, and in sufficient purity to be characterized by
standard analytical techniques described herein or well known to the skilled artisan.
Compounds of the invention include any form of the compound including in situ in a
5 reaction mixture as well as in isolated and purified form obtained by routine techniques.
Also included are polymorphic forms of the compounds of the invention and solvates and
prodrugs thereof.
Certain compounds of the invention may exist in different tautomeric forms, for
example, but are not limited to, ketone/enol tautomeric forms, imine-enamine tautomeric
10 forms, and for example heteroaromatic forms such as the following moieties:

In the same manner, unless indicated otherwise, presenting a structural representation of

any tautomeric form of a compound which exhibits tautomerism is meant to include all such
tautomeric forms of the compound. Accordingly, where compounds of the invention, their
15 salts, and solvates and prodrugs thereof, may exist in different tautomeric forms or in
equilibrium among such forms, all such forms of the compound are embraced by, and
included within the scope of the invention.
In another aspect, the present invention provides pharmaceutical compositions
comprising one or more compounds of the invention. As used herein, the term
20 “pharmaceutical composition” comprises at least one pharmaceutically active compound

and at least one excipient, and is intended to encompass both the combination of the
specified ingredients in the specified amounts, and any product which results, directly or
indirectly, from combination of the specified ingredients in the specified amounts.
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As will be appreciated by the ordinarily skilled artisan, excipients are any constituent
which adapts the composition to a particular route of administration or aids the processing
of a composition into a dosage form without itself exerting an active pharmaceutical effect.
In general compositions comprise more than one excipient depending upon the route of
5 administration and the characteristics of the active being administered. Examples of
excipients which impart to the composition properties which make it easier to handle or
process include, but are not limited to, lubricants or pressing aids in powdered
medicaments intended to be tableted, and emulsion stabilizers in compositions in which the
active is present in the form of an emulsion. Examples of excipients which adapt a
10 composition to a desired route of administration are, for example, but not limited to, for oral
administration, absorption enhancers promoting absorption from the gastrointestinal tract,
for transdermal or transmucosal administration, penetration enhancers, for example, those
employed in adhesive skin "patch" or compositions for buccal administration.
Notwithstanding the function excipients perform in a composition, excipients are
15 collectively termed herein "a carrier." Typically, formulations may comprise up to about 95
percent active ingredient and the balance carrier, although formulations with different ratios
may be prepared. In general, acceptable pharmaceutical compositions contain a suitable
concentration of the active that an effective amount of the PCSK9 antagonist can be
provided in an individual dosage form of acceptable volume based upon the route of
20 administration such that it can provide a therapeutic serum level of the active for an
acceptable period of time in a subject to whom the composition is administered and the
composition will retain biological activity during storage within an acceptable temperature
range for an acceptable period of time.

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Pharmaceutical composition, as used herein, refers both to a bulk composition, that

is, formulated material that has not yet been formed into individual dosage units for
administration, and the composition contained within individual dosage units.
While compositions of the invention may be employed in bulk form, it will be
5 appreciated that for most applications compositions will be incorporated into a dosage form
providing individual units suitable for administration to a patient, each dosage form
comprising an amount of the selected composition which contains an effective amount of
said one or more compounds of Formula I. Examples of suitable dosage forms include, but
are not limited to, dosage forms adapted for: (i) oral administration, e.g., a liquid, gel,
10 powder, solid or semi-solid pharmaceutical composition which is loaded into a capsule or
pressed into a tablet and may comprise additionally one or more coatings which modify its
release properties, for example, coatings which impart delayed release or formulations
which have extended release properties; (ii) a dosage form adapted for administration
through tissues of the oral cavity, for example, a rapidly dissolving tablet, a lozenge, a
15 solution, a gel, a sachet or a needle array suitable for providing intramucosal administration;
(iii) a dosage form adapted for administration via the mucosa of the nasal or upper
respiratory cavity, for example a solution, suspension or emulsion formulation for dispersion
in the nose or airway; (iv) a dosage form adapted for transdermal administration, for
example, a patch, cream or gel; (v) a dosage form adapted for intradermal administration,
20 for example, a microneedle array; (vi) a dosage form adapted for intravenous (IV) infusion,
for example, over a prolonged period using an I.V. infusion pump; (vii) a dosage form
adapted for intramuscular administration (IM), for example, an injectable solution or
suspension, and which may be adapted to form a depot having extended release
properties; (viii) a dosage form adapted for drip intravenous administration (IV), for
25 example, a solution or suspension, for example, as an IV solution or a concentrate to be

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injected into a saline IV bag; (ix) a dosage form adapted for subcutaneous administration,
including administration over an extended time period by implanting a rod or other device
which diffuses the compound into the surround tissue and thereby provides a continuous
serum therapeutic level; or (x) a dosage form adapted for delivery via rectal or vaginal
5 mucosa, for example, a suppository.
Pharmaceutical compositions can be solid, semi-solid or liquid. Solid, semi-solid and
liquid form preparations can be adapted to a variety of modes of administration, examples
of which include, but are not limited to, powders, dispersible granules, mini-tablets, beads,
which can be used, for example, for tableting, encapsulation, or direct administration. In
10 addition, liquid form preparations include, but are not limited to, solutions, suspensions and
emulsions which for example, but not exclusively, can be employed in the preparation of
formulations intended for ingestion, inhalation or intravenous administration (IV), for
example, but not limited to, administration via drip IV or infusion pump, intramuscular
injection (IM), for example, of a bolus which is released over an extended duration, direct IV
15 injection, or adapted to subcutaneous routes of administration.
Other routes of administration which may be contemplated include intranasal
administration, or for administration to some other mucosal membrane. Formulations
prepared for administration to various mucosal membranes may also include additional
components adapting them for such administration, for example, viscosity modifiers.
20 Although in some embodiments, compositions suitable for use in a solid oral dosage
form, for example, a tablet or quick-melt mouth-dissolving formulation are preferable routes
of administration for a compound of the invention or a salt thereof, a composition of the
invention may be formulated for administration via other routes mentioned above.
Examples include aerosol preparations, for example, suitable for administration via
25 inhalation or via nasal mucosa, may include solutions and solids in powder form, which may

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be in combination with a pharmaceutically acceptable propellant, for example, an inert

compressed gas, e.g. nitrogen. Also included are solid form preparations which are
intended to be converted, shortly before use, to a suspension or a solution, for example, for
oral or parenteral administration. Examples of such solid forms include, but are not limited
5 to, freeze dried formulations and liquid formulations adsorbed into a solid absorbent
medium.
For example, the compounds of the invention may also be deliverable transdermally
or transmucosally, for example, from a liquid, suppository, cream, foam, gel, or rapidly
dissolving solid form. It will be appreciated that transdermal compositions can take also the
10 form of creams, lotions, aerosols and/or emulsions and can be provided in a unit dosage
form which includes a transdermal patch of any know in the art, for example, a patch which
incorporates either a matrix comprising the pharmaceutically active compound or a
reservoir which comprises a solid or liquid form of the pharmaceutically active compound.
Examples of pharmaceutically acceptable carriers and methods of manufacture for
15 various compositions mentioned above may 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 addressing formulation issues may be
found in: Pharmaceutical compositions may be formulated by any number of strategies
known in the art, see, e.g., McGoff and Scher, 2000 Solution Formulation of
20 Proteins/Peptides: In - McNally, E.J., ed. Protein Formulation and Delivery. New York, NY:
Marcel Dekker; pp. 139-158; Akers & Defilippis, 2000, Peptides and Proteins as Parenteral

Solutions. In - Pharmaceutical Formulation Development of Peptides and Proteins.

Philadelphia, PA: Taylor and Francis; pp. 145-177; Akers et al., 2002, Pharm. Biotechnol.

14:47-127.

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In another aspect the present invention provides methods of employing PCSK9-

specific antagonist compounds described herein for antagonizing PCSK9 function; said
methods of which are further described below. Use of the term “antagonizing” throughout

the present application refers to providing to the affected tissue(s) a substance which
5 opposes the action of, inhibits, counteracts, neutralizes or curtails one or more functions of
PCSK9 in the affected tissues. Inhibition or antagonism of one or more of PCSK9-
associated functional properties can be readily determined according to methodologies
known to the art (see, e.g., 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
10 369:158-167) as well as those described herein. Inhibition or antagonism will effectuate a
decrease in PCSK9 activity relative to that seen in the absence of the antagonist or, for
example, that seen relative to the activity observed when a control antagonist of irrelevant
specificity is present. Preferably, a PCSK9-specific antagonist in accordance with the
present invention antagonizes PCSK9 functioning to the point that there is a decrease of at
15 least 10%, of the measured parameter including but not limited to the activities disclosed
herein, and more preferably, a decrease of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% and 95% of the measured parameter. Such inhibition/antagonism of PCSK9
functioning is particularly effective in those instances where PCSK9 functioning is
contributing at least in part to a particular phenotype, disease, disorder or condition which is
20 negatively impacting the subject.
In one aspect, the present invention provides a method for antagonizing the activity
of PCSK9, which comprises contacting a cell, population of cells or tissue sample capable
of being affected by PCSK9 (i.e., which expresses and/or comprises LDL receptors) with a
PCSK9-specific antagonist disclosed herein under conditions that allow said antagonist to
25 bind to PCSK9 when present and inhibit PCSK9's inhibition of cellular LDL uptake. In some

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embodiments of the present invention include such methods wherein the cell is a human
cell. Additional embodiments of the present invention include such methods wherein the
cell is a murine cell.
In one aspect, the present invention provides a method for antagonizing the activity
5 of PCSK9 in a subject, which comprises administering to the subject a therapeutically
effective amount of a PCSK9-specific antagonist of the present invention. In some
embodiments, the methods for antagonizing PCSK9 function are for the treatment, as
defined herein, of a PCSK9-associated disease, disorder or condition or, alternatively, for
providing therapy in a disease, disorder or condition that could benefit from the effects of a
10 PCSK9 antagonist.
The present invention, thus, contemplates the use of PCSK9-specific antagonists
described herein in various methods of treatment where antagonizing PCSK9 function is
desirable. As used herein, the term “method of treatment" relates to a course of action
resulting in a change in at least one symptom of a disease state which can be prophylactic
15 or therapeutic in nature. In some embodiments, the present invention relates to a method
of treatment for a condition associated with and/or attributed to PCSK9 activity, or a
condition where the functioning of PCSK9 is contraindicated for a particular subject, the
method comprising administering to the subject a therapeutically effective amount of a
PCSK9- antagonist compound of Formula I, or pharmaceutically acceptable salt thereof. In
20 some embodiments, the condition may be atherosclerosis, hypercholesterolemia, coronary
heart disease, metabolic syndrome, acute coronary syndrome or related cardiovascular
disease and cardiometabolic conditions, or may be a disease state or condition in which
PCSK9 activity is contraindicated.
Methods of treatment in accordance with the present invention comprise
25 administering to an individual a therapeutically (or prophylactically) effective amount of a

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PCSK9-specific antagonist of the present invention. Use of the terms “therapeutically

effective” or “prophylactically effective” in reference to an amount refers to the amount

necessary at the intended dosage to achieve the desired therapeutic and/or prophylactic
effect for the period of time desired. The desired effect may be, for example, the alleviation,
5 amelioration, reduction or cessation of at least one symptom associated with the treated
condition. These amounts will vary, as the skilled artisan will appreciate, according to
various factors, including but not limited to the disease state, age, sex, and weight of the
individual, and the ability of the PCSK9-specific antagonist to elicit the desired effect in the
individual. The response may be documented by in vitro assay, in vivo non-human animal
10 studies, and/or further supported from clinical trials.
In some embodiments it is preferred to administer a PCSK9 antagonist compound of
the invention in the form of a pharmaceutical composition as described herein.
Dosing of antagonist therapeutics is well within the realm of the skilled artisan, see,

e.g., Lederman et al., 1991 Int. J. Cancer47:659-664; Bagshawe etal., 1991 Antibody,

15 Immunoconjugates and Radiopharmaceuticals 4:915-922, and will vary based on a number

of factors, for example, but not limited to, those mentioned above, including the condition of
the patient, the area being treated, the route of administration, and the treatment desired,
for example, prophylaxis or acute treatment and the like. A physician or veterinarian of
ordinary skill can readily determine and prescribe the effective therapeutic amount of the
20 antagonist.
The subject may be in need of, or desire, treatment for an existing disease or
medical condition. As used herein, the subject "in need" of treatment of an existing
condition encompasses both a determination of need by a medical professional as well as
the desire of the subject for such treatment. When a compound or a salt thereof is provided
25 in combination with one or more other active agents, "administration" and its variants are

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each understood to include provision of the compound or its salt and the other agents
contemporaneously or simultaneously or over a course of separate administrations over a
period of time. When the agents of a combination are administered at the same time, they
can be administered together in a single composition or they can be administered
5 separately. It is understood that a "combination" of active agents can be a single
composition containing all of the active agents or multiple compositions each containing
one or more of the active agents. In the case of two active agents a combination can be
either a single composition comprising both agents or two separate compositions each
comprising one of the agents; in the case of three active agents a combination can be
10 either a single composition comprising all three agents, three separate compositions each
comprising one of the agents, or two compositions one of which comprises two of the
agents and the other comprises the third agent; and so forth.
The compositions and combinations of the present invention are suitably
administered in effective amounts. The term “effective amount” means the amount of active

15 compound sufficient to antagonize PCSK9 and thereby elicit the response being sought
(/.e., induce a therapeutic response in the treatment or management of conditions
associated with or impacted by PCSK9 function, including, but not limited to
atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute
coronary syndrome, and related cardiovascular disease and cardiometabolic conditions in
20 an animal or human).
The actual dosage employed may be varied depending upon the requirements of the
patient and the severity of the condition being treated. Determination of the proper dosage
regimen for a particular situation is within the skill in the art, for example, as described in the
standard literature, for example, as described in the “Physicians’ Desk Reference” (PDR),

25 e.g., 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), the

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Physician’s Desk Reference, 56th Edition, 2002 (published by Medical Economics company,
Inc. Montvale, NJ 07645-1742), or the Physician’s Desk Reference, 57th Edition, 2003
(published by Thompson PDR, Montvale, NJ 07645-1742); the disclosures of which is
incorporated herein by reference thereto. For convenience, the total daily dosage may be
5 divided and administered in portions during the day as required or delivered continuously.
The PCSK9-specific antagonist may be administered to an individual by any route of
administration appreciated in the art, including but not limited to oral administration,
administration by injection (specific embodiments of which include intravenous,
subcutaneous, intraperitoneal or intramuscular injection), or administration by inhalation,
10 intranasal, or topical administration, either alone or in combination with other agents
designed to assist in the treatment of the individual. The PCSK9-specific antagonist may
also be administered by injection devices, injector pens, needleless devices; and
subcutaneous patch delivery systems. The route of administration should be determined
based on a number of considerations appreciated by the skilled artisan including, but not
15 limited to, the desired physiochemical characteristics of the treatment.
One or more additional pharmacologically active agents may be administered in
combination with a compound of Formula I. An additional active agent (or agents) is
intended to mean a pharmaceutically active agent (or agents) that is active in the body,
including pro-drugs that convert to pharmaceutically active form after administration, which
20 are different from the compound of Formula I, and also includes free-acid, free-base and
pharmaceutically acceptable salts of said additional active agents. Generally, any suitable
additional active agent or agents, including but not limited to anti-hypertensive agents, anti-
atherosclerotic agents such as a lipid modifying compound, anti-diabetic agents and/or anti­
obesity agents may be used in any combination with the compound of Formula I in a single
25 dosage formulation (a fixed dose drug combination), or may be administered to the subject

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in one or more separate dosage formulations which allows for concurrent or sequential
administration of the active agents (co-administration of the separate active agents).
Examples of additional active agents which may be employed include but are not
limited to angiotensin converting enzyme inhibitors (e.g., alacepril, benazepril, captopril,
5 ceronaphl, cilazapril, delapril, enalapril, enalaprilat, fosinophl, imidapril, lisinopril, moveltipril,
pehndopril, quinapril, ramipril, spirapril, temocapril, or trandolapril), angiotensin II receptor
antagonists (e.g., losartan i.e., COZAAR®, valsartan, candesartan, olmesartan, telmesartan
and any of these drugs used in combination with hydrochlorothiazide such as HYZAAR®);
neutral endopeptidase inhibitors (e.g., thiorphan and phosphoramidon), aldosterone
10 antagonists, aldosterone synthase inhibitors, renin inhibitors (e.g. urea derivatives of di- and
tri-peptides (See U.S. Pat. No. 5,116,835), amino acids and derivatives (U.S. Patents
5,095,119 and 5,104,869), amino acid chains linked by non-peptidic bonds (U.S. Patent
5,114,937), di- and tri-peptide derivatives, peptidyl amino diols and peptidyl beta-aminoacyl
aminodiol carbamates, and small molecule renin inhibitors (including diol sulfonamides and
15 sulfinyls), N-morpholino derivatives, N-heterocyclic alcohols and pyrolimidazolones; also,
pepstatin derivatives and fluoro- and chloro-derivatives of statone-containing peptides,
enalkrein, RO 42-5892, A 65317, CP 80794, ES 1005, ES 8891, SQ 34017, aliskiren
(2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-
[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamid hemifumarate) SPP600, SPP630 and
20 SPP635), endothelin receptor antagonists, phosphodiesterase-5 inhibitors (e.g. sildenafil,
tadalfil and vardenafil), vasodilators, calcium channel blockers (e.g., amlodipine, nifedipine,
veraparmil, diltiazem, gallopamil, niludipine, nimodipins, nicardipine), potassium channel
activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam), diuretics
(e.g., hydrochlorothiazide), sympatholitics, beta-adrenergic blocking drugs (e.g.,
25 propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartate), alpha

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adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha methyldopa) central alpha
adrenergic agonists, peripheral vasodilators (e.g. hydralazine); lipid lowering agents e.g.,
HMG-CoA reductase inhibitors such as simvastatin and lovastatin which are marketed as
ZOCOR® and MEVACOR® in lactone pro-drug form and function as inhibitors after
5 administration, and pharmaceutically acceptable salts of dihydroxy open ring acid HMG-
CoA reductase inhibitors such as atorvastatin (particularly the calcium salt sold in
LIPITOR®), rosuvastatin (particularly the calcium salt sold in CRESTOR®), pravastatin
(particularly the sodium salt sold in PRAVACHOL®), fluvastatin (particularly the sodium salt
sold in LESCOL®), chvastatin, and pitavastatin; a cholesterol absorption inhibitor such as
10 ezetimibe (ZETIA®) and ezetimibe in combination with any other lipid lowering agents such
as the HMG-CoA reductase inhibitors noted above and particularly with simvastatin
(VYTORIN®) or with 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 acifran, as well as niacin receptor partial agonists; metabolic altering agents
15 including insulin and insulin mimetics (e.g., insulin degludec, insulin glargine, insulin lispro),
dipeptidyl peptidase-IV (DPP-4) inhibitors (e.g., sitagliptin, alogliptin, omarigliptin, linagliptin,
vildagliptin); insulin sensitizers, including (i) PPARy agonists, such as the glitazones (e.g.

pioglitazone, AMG 131, MBX2044, mitoglitazone, lobeglitazone, IDR-105, rosiglitazone,

and balaglitazone), and other PPAR ligands, including (1) PPARa/γ dual agonists (e.g.,

20 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’s), (e.g., such as those disclosed in WO 02/060388, WO 02/08188, WO

2004/019869, WO 2004/020409, WO 2004/020408, and WO 2004/066963); and (4) PPARy

25 partial agonists; (ii) biguanides, such as metformin and its pharmaceutically acceptable

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salts, in particular, metformin hydrochloride, and extended-release formulations thereof,

such as Glumetza™, Fortamet™, and GlucophageXR™; and (iii) protein tyrosine

phosphatase-1 B (PTP-1B) inhibitors (e.g., ISIS-113715 and TTP814); insulin or insulin

analogs (e.g., insulin detemir, insulin glulisine, insulin degludec, insulin glargine, insulin
5 lispro and inhalable formulations of each); leptin and leptin derivatives and agonists; amylin
and amylin analogs (e.g., pramlintide); sulfonylurea and non-sulfonylurea insulin
secretagogues (e.g., tolbutamide, glyburide, glipizide, glimepihde, mitiglinide, meglitinides,
nateglinide and repaglinide); α-glucosidase inhibitors (e.g., acarbose, voglibose and
miglitol); glucagon receptor antagonists (e.g., MK-3577, MK-0893, LY-2409021 and KT6-
10 971); incretin mimetics, such as GLP-1, GLP-1 analogs, derivatives, and mimetics; and
GLP-1 receptor agonists (e.g., dulaglutide, semaglutide, albiglutide, exenatide, liraglutide,
lixisenatide, taspoglutide, CJC-1131, and BIM-51077, including intranasal, transdermal, and
once-weekly formulations thereof); bile acid sequestering agents (e.g., colestilan,
colestimide, colesevalam hydrochloride, colestipol, cholestyramine, and dialkylaminoalkyl
15 derivatives of a cross-linked dextran), acyl CoA:cholesterol acyltransferase inhibitors, (e.g.,
avasimibe); antiobesity 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 (GKAs) (e.g.,
AZD6370); inhibitors of 11 β-hydroxysteroid dehydrogenase type 1, (e.g., such as those
20 disclosed in U.S. Patent No. 6,730,690, and LY-2523199); CETP inhibitors (e.g.,
anacetrapib, torcetrapib, and evacetrapib); inhibitors of fructose 1,6-bisphosphatase, (e.g.,
such as those disclosed in U.S. Patent Nos. 6,054,587; 6,110,903; 6,284,748; 6,399,782;
and 6,489,476); inhibitors of acetyl CoA carboxylase-1 or 2 (ACC1 or ACC2); AMP-
activated Protein Kinase (AMPK) activators; other agonists of the G-protein-coupled
25 receptors: (i) GPR-109, (ii) GPR-119 (e.g., MBX2982 and PSN821), and (iii) GPR-40 (e.g.,

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TAK875); SSTR3 antagonists (e.g., such as those disclosed in WO 2009/001836);

neuromedin II receptor agonists (e.g., such as those disclosed in WO 2009/042053,
including, but not limited to, neuromedin S (NMS)); SCD modulators; GPR-105 antagonists
(e.g., such as those disclosed in WO 2009/000087); SGLT inhibitors (e.g., ASP1941,
5 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); inhibitors of fatty acid
synthase; inhibitors of acyl coenzyme A:monoacylglycerol acyltransferase 1 and 2 (MGAT-1
and MGAT-2); agonists of the TGR5 receptor (also known as GPBAR1, BG37, GPCR19,
10 GPR131, and M-BAR); ileal bile acid transporter inhibitors; PACAP, PACAP mimetics, and
PACAP receptor 3 agonists; PPAR agonists; protein tyrosine phosphatase-1 B (PTP-1B)
inhibitors; IL-1 b antibodies, (e.g., XOMA052 and canakinumab); and bromocriptine
mesylate and rapid-release formulations thereof; or with other drugs beneficial for the
treatment of the above-mentioned conditions or disorders including the free-acid, free-base,
15 and pharmaceutically acceptable salt forms of the above active agents where chemically
possible.
The compounds of the present invention can be readily prepared according to the
following reaction schemes and examples, or modifications thereof, using readily available
starting materials, reagents and conventional synthesis procedures. In these reactions, it is
20 also possible to make use of known variants. For purification of the compounds using
reverse phase chromatography (either HPLC or MPLC, as noted below), a C18 column was
used. Other methods for preparing compounds of the invention will be readily apparent to
the person of ordinary skill in the art in light of the following reaction schemes and
examples. Abbreviations listed below may used in the exemplary schemes and/or
25 examples herein.

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ACN is acetonitrile
AcOH is acetic acid
AcO’NhU is ammonium acetate
BOC2O is di-tert-butyl dicarbonate
5 Bn is benzyl
BnBr is benzyl bromide
BzCI is benzoyl chloride
CBr4 is perbromomethane
Cbz-CI is benzyl chloroformate
10 DBU is 1,8-Diazabicyclo[5.4.0]undec-7-ene
DCC is dicyclohexylcarbodiimide
DCE is 1,2-dichloroethane
DCM is dichloromethane
DEA is N,N-diethylamine
15 DIAD is (E)-diisopropyl diazene-1,2-dicarboxylate
DIEA or DIPEA is A/,/\/-diisopropylethylamine
DMAP is 4-dimethylaminopyridine
DMF is A/,A/-dimethylformamide
DMSO is dimethyl sulfoxide
20 EA or EtOAc is ethyl acetate
EtOH is ethanol
Et20 is diethyl ether
Fmoc is fluorenylmethyloxycarbonyl protecting group
Fmoc-CI is (9/-/-fluoren-9-yl)methyl carbonochlohdate
25 Fmoc-D-Dap(Boc)-OH is N-alpha-(9-Fluorenylmethyloxycarbonyl)-N-beta-t-
butyloxycarbonyl-D-2,3-diaminopropionic acid

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Fmoc-Osu is Fmoc N-hydroxysuccinimide ester

HATII is 1-[bis(dimethylamino)methylene]-1 H-1,2,3-thazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate
HPLC is High Performance Liquid Chromatography
5 IPA is isopropyl alcohol
LiOH is lithium hydroxide
LC/MS is Liquid chromatography-mass spectrometry
Me3N is trimethyl amine
MeOH is methanol
10 MPLC is Medium pressure liquid chromatography
MsCI is methanesulfonyl chloride
NaBH(OAc)3 is sodium triacetoxyborohydride
NMR is Nuclear Magnetic Resonance
NsCI is 4-nitrobenzene-1 -sulfonyl chloride
15 PE is petroleum ether
Pd2(dba)3(HCCl3) is ths(dibenzylideneacetone)dipalladium(0)-chloroform adduct
PPh3 is triphenylphosphine
PdCl2(dppf) or Pd(ii)(dppf)Cl2 is dichloro[1 ,T-bis(diphenylphosphino)ferrocene]palladium(ll)
Pd(dppf)Cl2CH2Cl2 is dichloro[1,T-bis(diphenylphosphino)ferrocene]palladium(ll)
20 dichloromethane adduct
Pd(PPh3)4 is tetrakis(thphenylphosphine)palladium
PPTs is pyridinium p-toluenesulfonate
[Rh(OAc)2]2 is rhodium(ll) acetate dimer
RT or r.t. or rt is room temperature
25 tBuOAc is tert-butyl acetate
TEA is triethylamine
TFA is trifluoroacetic acid
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TFE is tetrafluoroethylene
THF is tetrahydrofuran
Tf2O is trifluoromethanesulfonic anhydride
Teoc-OSu is 2,5-dioxopyrrolidin-1 -yl (2-(trimethylsilyl)ethyl) carbonate

5 TBAF is tetrabutylammonium fluoride

TMS is tetramethylsilane
Zhan’s catalyst 1B is dichloro(1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)((5-
((dimethylamino)sulfonyl)-2-(1-methylethoxy-O)phenyl)methylene-C)ruthenium(ll)
[also described as 1,3-Bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene[2-(i-
10 propoxy)-5-(N,N-dimethylaminosulfonyl)phenyl[methyleneruthenium (II) dichloride]

EXAMPLE 1 Preparation of Ex-01 and Ex-51

o

o
Ex-01, (free base illustrated)
15

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Ex-51, (free base illustrated)

Salt forms of compounds Ex-01 and Ex-51 were prepared from intermediates 76 and
86 (preparation following) in accordance with the following scheme:

5 -O

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Step I
TFA/DCM
Ex-51, formate salt
HCI/DCM
Reverse phase
formic acid

Step H
TFA/DCM
95 Ex-01, formate salt
HCI/DCM
Reverse phase
formic acid

Step A: Preparation of intermediate 89

To a solution of 76 (1.56 g, 1.917 mmol, preparation following) and 86 (1.506 g, 1.936

mmol, preparation following) in DMF (40 ml) was added HATLI (0.802 g, 2.108 mmol) and
5 DIEA (0.670 ml, 3.83 mmol). The resulting solution was stirred at rt for 50 min, then
partitioned between EtOAc (300 mL) and brine (100 mL). The organic phase was washed
with brine (2x100 mL), dried over Na2SO4, concentrated and the residue was purified on
silica gel column using MeOH/DCM as eluting solvents to give 89. LC/MS: (M+1)+: 1573.8.
Step B: Preparation of intermediate 90

10 A solution of 89 (0.68 g, 0.432 mmol) in DCM (500 ml) and acetic acid (40 mL) was bubbled
with N2 for 20 min followed by addition of Zhan catalyst-1 b (0.222 g, 0.302 mmol). The
resulting mixture was further bubbled with N2 for 20 min, then heated at 55 °C for 5 h. After
cooling to rt the mixture was filtered through celite, the filtrate was concentrated, and the
residue was purified on silica gel column using MeOH/DCM as eluting solvents to give 90
15 (cis/trans mixture). LC/MS: (M+1)+: 1545.7.
Step 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 rt for 1 h, then
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concentrated and the residue was dissolved in acetonitrile (4 mL) and concentrated again.
The residue was further dried under high vacuum for 30 min to give 91 (cis and trans
mixture). LC/MS: (M+1)+: 1324.0.
Step D: Preparation of intermediate 92

5 To a solution of 91 (cis/trans mixture) (548 mg, 0.414 mmol) and 88 (227 mg, 0.455 mmol,
preparation following) in DMF (10 ml) at 0 °C was added HATLI (181 mg, 0.476 mmol) and
DIEA (0.166 ml, 0.952 mmol). The resulting solution was stirred at 0 °C for 1 h, and the
solution was purified by reverse phase MPLC over C18 column using acetonitrile
(0.05%TFA)/water (0.05%TFA) as eluting solvents to give 92 (cis/trans mixture). LC/MS:
10 (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 was added 1N aqueous LiOH (3.11 ml, 3.11 mmol) dropwise, and the resulting
solution was stirred at 0 °C for 23 h. The solution was neutralized by addition of 1N HCI to
15 pH 7-8, the volatile was evaporated, the aqueous phase was acidified to pH 5, and the
mixture was purified by reverse phase MPLC over C18 column using acetonitrile
(0.05%TFA)/water (0.05%TFA) as eluting solvents to give 93 as TFA salt. To a solution of
93 TFA salt (427 mg, 0.257 mmol)) in water (70 mL) and acetonitrile (70 ml) at 0 °C was
added 0.1 N HCI (13.5 ml, 1.350 mmol) dropwise, the resulting solution was stirred at 0 °C
20 for 5 min, then lyophilized to give 93 as HCI salt. LC/MS: (M+1)+: 1567.1.
Step F: Preparation of intermediate 94

To a solution of 93 as HCI 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 rt for 30 min, then diluted with
DCM (400 mL) followed by addition of DIEA (0.065 mL, 0.374 mmol). The resulting solution
25 was stirred at rt for 1 h, the volatile was evaporated on rotary evaporator, and the resulting

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DMF solution was purified by reverse phase MPLC using acetonitrile (0.05%TFA)/water
(0.05%TFA) as eluting 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 pmol) in MeOH (20 ml) was added 10% Pd/C (1.924 mg,
5 1.808 pmol) and the resulting mixture was subjected to hydrogenation at rt via H2 balloon
for 1 h. The mixture was filtered through celite and the filtrate was concentrated to give
intermediate compound 95. LC/MS: (M+1)+: 1550.9.
Step /-/: Preparation of Ex-01

Intermediate compound 95 prepared in the previous step (26 mg, 0.017 mmol) was
10 dissolved in DCM (2 ml). To this solution was added TFA (6 mL, 78 mmol) and the
resulting solution was stirred at rt for 30 min, then concentrated and the residue was
dissolved in DCM (3 mL) and treated with 4N HCI in dioxane (0.042 mL, 0.168 mmol), and
concentrated again to give Ex-01 as a crude product. The crude Ex-01 was purified by
reverse phase HPLC using acetonitrile (0.1%formic acid)/water (0.1%formic acid) as eluting
15 solvents to provide the formate salt form of Ex-01. LC/MS: (M+1)+: 1394.4.
Step I: Preparation of Ex-51

To a solution of intermediate compound 94 (30 mg, 0.019 mmol) in DCM (2 ml) was added
TFA (4 ml, 51.9 mmol), and the reaction mixture was stirred at ambient temperature for 30
minutes, then concentrated. The residue was dissolved in DCM (2 mL), treated with HCI
20 (4N in dioxane) (0.048 ml, 0.194 mmol), and concentrated to yield Ex-51 as a HCI salt. The
compound was purified by reverse phase HPLC using acetonitrile (0.1%formic acid)/water
(0.1%formic acid) as mobile phase to provide the formate salt form of Ex-51. LC/MS:
(M+1)-: 1392.0.
The following schemes and procedures were used to prepare intermediates 76, 86
25 and 88 used in the procedures described above.

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Preparation of Intermediate 68 used in the preparation of Intermediate 76

Step A: Preparation of Intermediate compound 65

To a suspension of (2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid (5.32 g, 40.6 mmol) in

5 dioxane (100 ml) at 0°C was added sodium hydroxide (122 ml, 122 mmol), followed by
addition of benzyl chloroformate (6.50 ml, 44.6 mmol) dropwise. The resulting suspension
was stirred at 0 °C for 5h. After removing the volatile, the aqueous phase was acidified to
pH 3, then partitioned between 30%IPA/DCM(200 mL) and brine (50 mL), the aqueous
phase was further extracted with 30%IPA/DCM(2x100mL). Combined organic phases were
10 dried over Na2SO4 and concentrated to give (2S,3S)-1-((benzyloxy)carbonyl)-3-
hydroxypyrrolidine-2-carboxylic acid (65). LC/MS: (M+1)+: 266.1.
Step B: Preparation of Intermediate compound 66

To a solution of 65 (7.48 g, 28.2 mmol) in MeOH (80 ml) was added TMS-Diazomethane
(70.5 ml, 141 mmol) dropwise, and the resulting solution was stirred at rtfor 10 min then
15 quenched by addition of acetic acid (ca. 400 uL) dropwise. The solution was concentrated,
and the residue was purified on silica gel column using EtOAc/hexane as eluting solvents to
give 66. LC/MS: (M+1)+: 280.1.
Step C: Preparation of Intermediate compound 67

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A solution of 66 (4.81 g, 17.22 mmol) in DCM (200 mL) was bubbled with N2 for 30 min,
followed by addition of rhodium(ii) acetate dimer (0.761 g, 1.722 mmol). The mixture was
cooled in a ice-water bath, and tert-butyl diazoacetate (3.58 mL, 25.8 mmol) were added at
0 °C dropwise. The resulting mixture was stirred at 0 °C for 1,5h. The reaction was
5 quenched by addition of 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
eluting solvents. The fraction containing the product was concentrated and the aqueous
phase was extracted with DCM (2x100mL). The combined organic phase was dried over
10 Na2SO4 and concentrated to give 67. LC/MS: (M+1)+: 394.2.
Step D: Preparation of Intermediate compound 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 subjected to hydrogenation via H2 balloon at
ambient temperature for 2 hours, then filtered through celite. The filtrate was concentrated
15 to give 68. LC/MS: (M+1)+: 259.9.

Preparation of intermediate compound 76

Step B

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Step D

θχ zP
HOZ CH3

tBuOAc/DCM
Step E

Step H

1. LiOH/THF/
MeOH/water
Fmoc^
Fmoc-OSu/
2.
Na2CO3/acetone

Step A: Preparation of Intermediate 69

5 To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1 H-indol-3-

yl)propanoic acid (3 g, 6.75 mmol) in THF (20 ml), MeOH (10 mL), and water (20.00 ml) at
0 °C was added NaOH (20.25 ml, 20.25 mmol) and the resulting solution was stirred at
ambient temperature for 4 hours, then the volatile was evaporated. To the aqueous mixture
was added dioxane (50 ml) and water (20 mL), the resulting solution was cooled to 0 °C
10 and Boc2O (1.881 ml, 8.10 mmol) was added to the above solution. The resulting solution
was stirred at 0 °C for 3 hours, the volatile was removed and the aqueous phase was

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extracted with Et2<D (3x40 mL), acidified to pH 3, then extracted with DCM (3x100mL),
followed by 30%IPA/DCM (2x80 mL). The combined organic phase was dried over Na2SO4
and concentrated to give 69. LC/MS: (M+1)+: 322.9.
Step B: Preparation of Intermediate 70

5 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
followed by addition of allyl bromide (1.172 mL, 13.54 mmol) dropwise. The resulting
solution was stirred at 0 °C for 1.5h, then quenched by addition of 1N HCI (ca. 3.68 mL).
The solution was then partitioned between EtOAc (200 mL) and water (100 mL), the
10 organic phase was washed with brine (2x100 mL), dried over Na2SO4, concentrated and
the residue was purified on silica gel column using MeOH/DCM as eluting solvents to give
70. LC/MS: (M+1)+: 363.0.
Step C: Preparation of Intermediate 71

To a solution of 70 (2.239 g, 6.18 mmol) and 68 (1.842 g, 7.11 mmol) in DMF (30 ml) was
15 added HATU (2.82 g, 7.41 mmol) and DIEA (2.59 ml, 14.83 mmol), and the resulting
solution was stirred at ambient temperature for 1 hour. The mixture was partitioned
between EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine
(3x100 mL), dried over Na2SO4, concentrated and the residue was purified on silica gel
column using EtOAc/hexane as eluting solvents to give 71. LC/MS: (M+1)+: 604.2.
20 Step D: Preparation of Intermediate 72

To a solution of 71 (2.83 g, 4.69 mmol) in CH2CI2 (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 h, then ambient temperature for 2.5 h. The solution (72) was directly used in
the next step. LC/MS: (M+1)+: 504.2.
25 Step E: Preparation of Intermediate 73

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To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(((tert-
butoxycarbonyl)amino)methyl)phenyl)propanoic acid (2.66 g, 5.16 mmol) in DMF (10 ml)
was added HATLI (1.961 g, 5.16 mmol) and DIEA (5.32 ml, 30.5 mmol), and the resulting
solution was stirred at rt for 30 min then added to an ice-cold bath of the above prepared 72
5 solution. The resulting solution was stirred at ambient temperature for 1 hour. Volatiles
were evaporated on rotary evaporator, and the residue was purified by reverse phase
MPLC using acetonitrile (0.05%TFA)/water (0.05%TFA) as eluting solvents. Collected
fractions were concentrated on rotary evaporator to give 73. LC/MS: (M+1)+: 1002.1.
Step F: Preparation of Intermediate 74

10 To a solution of 73 (3.235 g, 3.23 mmol) in DCM (4 ml) was added TFA (7.46 ml, 97 mmol),
and the resulting solution was stirred at ambient temperature for 1 hour, then concentrated.
The residue was dissolved in DCM (10 mL), treated with 4N HCI 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 provide 74. LC/MS: (M+1)+: 846.1.
15 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) followed by addition of DIEA (1.692 ml, 9.69 mmol) dropwise.
The resulting solution was stirred at ambient temperature for 1 hour. The solution was
20 concentrated, and the residue was purified by reverse phase MPLC over C18 column using
acetonitrile (0.05%TFA) I water (0.05%TFA) as eluting solvents. The fractions containing
the product were concentrated and the aqueous layer was partitioned between DCM (200
mL) and sat. NaHCOs (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.
25 LC/MS: (M+1)+: 828.1.

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Step H: Preparation of Intermediate compound 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 1N aqueous LiOH (9.9 ml, 9.90 mmol) dropwise, and the resulting
solution was stirred at 0 °C for 16h then quenched by addition of HCI (1N, 9.9 mL). The
5 volatile was evaporated on rotary evaporator and to the solution above at 0 °C was added
acetone (60 ml), sodium carbonate (0.371 g, 3.50 mmol), and Fmoc-Osu (0.802 g, 2.378
mmol). The resulting solution was stirred at 0 °C for 6h, the volatile was evaporated on
rotary evaporator, the aqueous phase was acidified to pH 4, then extracted with
30%IPA/DCM (3x100 mL). The combined organic phase was dried over Na2SO4,
10 concentrated and the residue was purified on silica gel column using MeOH/DCM as eluting
solvents to give 76. LC/MS: (M+1)+: 814.2.

Alternative preparation of intermediate compound 75b and intermediate compound

76B there from:

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The general procedure described above for the preparation of intermediate

compound 75 was generally followed, except that Step B was eliminated and the final step
G also included replacing the Fmoc protecting group with a Boc protecting group, thus
5 providing the intermediate compound 75a. The procedures for 75a and 76B are described
below.
Step C: Preparation of intermediate compound 71a

To a solution of 69 (5.00 g, 15.5 mmol) in DMF (40.0 mL) at -50 °C were added 68,
HATU (5.90 g, 15.5 mmol) and DIEA (4.01 g, 31.0 mmol) and the reaction mixture was
10 stirred at -50 °C for 3 h. The final solution was quenched with water (5 mL), concentrated
under reduced pressure and the residue was purified by reverse phase column
chromatography over C18 (eluting with a gradient of acetonithle/water + 0.01% ammonium
bicarbonate) to provide 71a. LCMS (ESI) calc’d for C28H38FN3O8 [M + H]+: 564.3, found
564.2.
15 Step D: Preparation of intermediate compound 72a

To a solution of 2 N HCI in dioxane (100 mL) and THF (100 mL) at rt was added 71a
(8.40 g, 14.9 mmol) and the reaction mixture was stirred for 5 h. The final solution was
concentrated under reduced pressure to afford 72a. LCMS (ESI) calc’d for C23H31CIFN3O6
[M - HCI + H]+: 464.2, found 464.3.
20 Step E: Preparation of intermediate compound 73a

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To a solution of 72a (800 mg, 1.60 mmol) in DMF (10.0 mL) at -50 °C were added
(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(((tert-
butoxycarbonyl)amino)methyl)phenyl)propanoic acid (827 mg, 1.60 mmol), HATU (608 mg,
1.60 mmol) and DIEA (620 mg, 4.80 mmol) and the mixture was stirred at -50 °C for 3 h.
5 The resulting solution was diluted with water (50 mL) and the aqueous layer was extracted
with EtOAc (3 x 100 mL). The combined organic layer was washed with brine, dried over
anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and
the residue was purified by silica gel column chromatography (eluting with a gradient 1%-
60% of EtOAc in PE) to give 73a. LCMS (ESI) calc’d for C53H60FN5O11 [M + H]+: 962.4,
10 found 962.6.
Step F: Preparation of intermediate compound 74a

To a solution of 73a (3.00 g, 3.12 mmol) in DCM (15.0 mL) at rt was added TFA
(15.0 mL) and the reaction mixture was stirred for 1 h at room temperature. The resulting
solution was concentrated under reduced pressure, co-evaporated with toluene and DCM
15 to give 74a. LCMS (ESI) calc’d for C46H45F4N5O11 [M - TFA + H]+: 806.3, found 806.7.
Step G: Preparation of intermediate compound 75a

To a solution of 74a (4.00 g, 4.35 mmol) in DMF (150 mL) at rt was added HATU
(1.65 g, 4.35 mmol) 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) then stirred at rt for 3 h. The resulting
20 solution was quenched with water (5 mL), concentrated under reduced pressure and the
residue was purified by reverse phase column chromatography over C18 (eluting with a
gradient of acetonithle/water + 0.05%TFA) to provide a Fmoc-protected intermediate.
LCMS (ESI) calc’d for C44H42FN5O8 [M + H]+: 788.3, found 788.9.
To a solution of the Fmoc-protected intermediate just above (200 mg, 0.250 mmol) in
25 DCM (5.00 mL) at rt was added piperidine (1.25 mL) and the reaction solution was stirred

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for 1 h. The final solution was concentrated under reduced pressure and the residue was
purified by silica gel column chromatography (eluting with a gradient 0%-5% of MeOH in
DCM) to afford an amine intermediate. LCMS (ESI) calc’d for C29H32FN5O6 [M + H]+:
566.2, found 566.3.
5 To a solution of the amine intermediate just above (2.86 g, 5.06 mmol) in THF (30.0
mL) and water (30.0 mL) at rtwere added BOC2O (2.21 g, 10.1 mmol) and sodium
bicarbonate (1.70 g, 20.2 mmol) and the reaction was stirred for 3 h. The final solution was
diluted with water (50 mL) and extracted with EtOAc (3 x 100 mL). The combined organic
layer was washed with brine (3 x 100 mL), dried over anhydrous Na2SO4 and filtered. The
10 filtrate was concentrated under reduced pressure and the residue was purified by silica gel
column chromatography (eluting with a gradient 0%-5% MeOH in DCM) to provide 75a.
LCMS (ESI) calc’d for C34H40FN5O8 [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,
15 9H). Step H: Preparation of intermediate compound 75b
To a solution of 75a (0.665 g, 0.99 mmol) in DMF (0.5 mL) was added CS2CO3 (1.11
g, 3.40 mmol) and 3-bromoprop-1-ene (0.43 g, 3.55 mmol) at 0 °C. The reaction mixture
was stirred at room temperature for 16 hours and poured into 5 mL of 50% sat. bhne/10%
citric acid solution, then extracted with ethyl acetate (2 x 20 mL). The organic layer was
20 washed with brine (3 x 20 mL), dried over anhydrous MgSCH and filtered. The filtrate was
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography, eluting with a 1%-5% gradient of MeOH in DCM. The fractions containing
intermediate compound 75b were combined and concentrated to afford the title compound.
LCMS (ESI) calc’d for C37H44FN5O8 [M + H]+: 706.3, found 706.3.
25 Step I: Preparation of intermediate compound 76B

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Hydrolysis of 75b with LiOH followed conditions similar to the ones described in the
preparation of intermediate 93 to provide 76B.
Preparation of Intermediate 77B

BocHN

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-(2-aminoethyl)benzylcarbamate hydrochloride
10 (5.03 g, 17.54 mmol) in DMF (140 ml) at 0°C were added HATLI (8.00 g, 21.05 mmol) and
DIPEA (9.16 ml, 52.6 mmol) then the reaction was allowed to warm to r.t. and stirred for 2
h. The final mixture was diluted with water, extracted with EtOAc, washed with brine, dried
over MgSO4, and filtered. The filtrate was concentrated and the residue was purified by
column chromatography over silica gel (eluting with a gradient of 0-60% of EtOAc in
15 hexane) to give 77A. MS (ESI): m/z (M+H)+ 584.5.
Step B: Preparation of intermediate 77B

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

5 Preparation of Intermediate 86

o 83 0 84

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Step A: Preparation of intermediate 77

To a solution of (S)-(9H-fluoren-9-yl)methyl 2-((4-(aminomethyl)phenethyl)carbamoyl)-2-

methylpyrrolidine-1 -carboxylate hydrochloride (77B) (5.87 g, 11.29 mmol) in DCM (140 ml)
5 at 0 °C was added DIEA (5.91 ml, 33.9 mmol) and CBZ-CI (1.726 ml, 11.85 mmol)
dropwise, and the resulting solution was stirred at 0 °C for 4h. The reaction solution was
partitioned between water (200 mL) and DCM (200 mL), the water phase was extracted
with DCM (100 mL), the combined organic phase was dried over Na2SO4, and the residue
was purified on silica gel column using EtOAc/hexane as eluting solvents to give 77.
10 LC/MS: (M+1)+: 618.3.
Step 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 1N aqueous NaOH (45.0 ml, 45.0 mmol), and the resulting solution was stirred
at rt for 2h. The volatile was evaporated, and to the aqueous solution was added dioxane
15 (200 ml), and BOC2O (2.508 ml, 10.80 mmol) in dioxane (20 mL). The resulting mixture was
stirred from 0 °C to rt overnight. The volatile was evaporated on rotary evaporator, and the
aqueous phase was extracted with DCM (3x150 mL). The combined organic phase was
dried over Na2SO4, concentrated and the residue was purified on silica gel column using
EtOAc/hexane as eluting solvents to give 78. LC/MS: (M+1)+: 496.2.
20 Step C: Preparation of intermediate 79

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To a solution of 78 (4.04 g, 8.15 mmol) in MeOH (100 ml) was added 10% Pd/C (0.867 g,
0.815 mmol), and the resulting mixture was subjected to hydrogenation via H2 balloon at rt
for 1,5h. The mixture was filtered through Celite, and the filtrate was concentrated to give
79. LC/MS: (M+1)+: 362.2.
5 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 4-
methylbenzenesulfonate (0.858 g, 3.57 mmol) and K2CO3 (1.973 g, 14.27 mmol), and the
resulting mixture was heated at 80 °C for 6h. After cooling down to rt, the mixture was
filtered and the filtrate was purified by reverse phase MPLC using acetonitrile (0.05%TFA)/
10 water (0.05%TFA) as eluting solvents to give the product as TFA salt, which was further
partitioned between DCM (100 mL) and 1N aqueous NaOH (50 mL). The aqueous phase
was further extracted with DCM (2x50mL), the combined organic phase was dried over
Na2SO4, and concentrated to give 80. LC/MS: (M+1)+: 430.3.
Step E: Preparation of intermediate 81

15 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), HATLI (1.009 g, 2.65 mmol), and DIEA (0.927 ml,
5.31 mmol), and the resulting solution was stirred at rt for 1h. The solution was partitioned
between EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine
(2x100mL), dried over Na2SO4, concentrated and the residue was purified on silica gel
20 column using EtOAc/hexane as eluting solvents to give 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 HCI (4N in dioxane)
(5.36 ml, 21.43 mmol). The resulting solution was stirred at rt for 3h, and the mixture was
concentrated to give 82. LC/MS: (M+1)+: 444.2.
25 Step G: Preparation of intermediate 83

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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 rt for 50 min. The solution was partitioned between
EtOAc (200 mL) and brine (100 mL), the organic phase was washed with brine (2x100 mL),
5 the combined organic phase was dried over Na2SO4, concentrated and the residue was
purified on silica gel column using EtOAc/hexane as eluting solvents to give 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
10 ml, 5.80 mmol), and the resulting solution was stirred at rtfor 1 h, then concentrated. The
residue was resuspended in acetonitrile (20 mL) and concentrated again, the cycle was
repeated once, and the residue was further dried under high vacuum to give 84. LC/MS:
(M+1)+: 621.3.
Step I: Preparation of intermediate 85

15 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 rt for 1h. The solution was partitioned between EtOAc
(200 mL) and brine (100 mL), the organic phase was washed with brine (2x100 mL), dried
over Na2SO4, concentrated and the residue was purified on silica gel column using
20 EtOAc/hexane as eluting solvents to give 85. LC/MS: (M+1)+: 1000.2.
Step J: Preparation of intermediate 86

To a solution of 85 prepared in the previous step (1.94 g, 1.940 mmol) in acetonitrile (20 ml)
was added piperidine (0.960 ml, 9.70 mmol), and the resulting solution was stirred at rt for
30min, then concentrated. The residue was re-dissolved in DCM/acetonitrile (1:1, 20 mL),

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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
o o
OH
NHFmoc

Cl Step A

OH O
o
'·■■/ OH
HATU O'
ΗΝ^>θ
then AcOH, TFE in DCM rsA
Step B 88 /^NHFmoc
5

Step A - Synthesis of Intermediate 87

To 2-ch loro-2 -ch lorotrityl resin 1-1.5 mmol/g (7.0 g, 1-1.5 mmol/g) was added dry DCM (45
ml). The resin was shaken 20 min followed by addition of half of DIPEA 0.17 N in DCM
(3.67 ml, 21.00 mmol), Fmoc-D-Dap(Boc)-OH (3.28 g, 7.70 mmol) then the remainder of
10 DIPEA 0.17 N 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 h then filtered, rinsing with DCM (3x), DMF (3x)
and DCM (3x) then dried under vacuum to give resin 87 which was used as is in the next
step.
15 Step B - Synthesis of Intermediate 88
Resin 87 (4.5 g, 2.475 mmol) was manually deprotected with 5% piperidine in DMF (30 ml)
for 30 min, filtered, retreated with 5% piperidine in DMF (30 ml) for another 30 min, filtered,
then rinsed with DMF and DCM and dried. The resin was then manually coupled with

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Fmoc-Ala-OH (1.541 g, 4.95 mmol), HATU (1.694 g, 4.46 mmol) and DIPEA (1.729 ml, 9.90
mmol) in DMF (30 ml) for 2 h then filtered, rinsing with DMF and DCM, then dried. The
resin was then treated with 10% AcOH and TFE in DCM (60 ml) for 90 min, filtered and the
filtrate was concentrated to provide 88. LC/MS: [2M+H]+ = 995.01.
5 Example 1A - Alternative Synthesis of Ex-01 and preparation of Ex-25 therefrom:
Compound Ex-01, presented above, may alternatively be prepared, and compound
Ex-25 may be prepared from Ex-01, in accordance with the following Scheme:

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85
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86
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Step A - Synthesis of Intermediate Int-cd1

To a solution of lnt-3c (synthesis from intermediate 107 described below) (7.09 g, 10.33
5 mmol) in DMF (45 ml) at 0 °C was added lnt-2d (3.63 g, 9.84 mmol, preparation described

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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 1h. The mixture
was quenched at 0 °C with brine and extracted with EtOAc. The combined organic
fractions were washed with brine, dried over MgS04, filtered and concentrated in vacuo.
5 The residue was purified by column chromatography over silica gel (eluting with a gradient
of 1% to 80% ethyl acetate in petroleum ether) to give Int-cd1. LC/MS: [M+1 ]+ = 1000.5.
Step B - Synthesis of Intermediate 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 rt for 3h. The mixture was
10 concentrated, the residue was re-dissolved in DCM/acetonithle (1:1,20 mL), concentrated
again and the residue was dried under vacuum to give Int-cd2 as a crude product. LC/MS:
(M+1 )+=778.5.
Step C - Synthesis of Intermediate Int-cd3

To a solution of 76 (preparation shown in Example 1, above) (2.45 g, 3.01 mmol) and Int-
15 cd2 (2.69 g, 3.46 mmol) in DMF (70 ml) at 0 °C was added HATU (1.37 g, 3.61 mmol)
followed by DIEA (1.05 ml, 6.02 mmol). The resulting solution was stirred at rt for 50 min,
then partitioned between EtOAc (500 mL) and brine (200 mL). The organic phase was
washed with brine (2x200 mL), dried over Na2SO4, concentrated and the residue was
purified by column chromatography over silica gel (eluting with a gradient of 1 % - 5%
20 MeOH in DCM) to give Int-cd3. LC/MS: (M+1 )+=1574.7.
Step D - Synthesis of Intermediate Int-cd4

A room temperature solution of Int-cd3 (1.91 g, 1.21 mmol) in DCM (1500 ml) and acetic
acid (30 mL) was bubbled with N2 for 30 min followed by addition of Zhan’s catalyst-1 B
(0.445 g, 0.607 mmol). The resulting mixture was further bubbled at room temperature with
25 N2 for 30 min, then heated at 55 °C for 5h. After cooling to rt the mixture was filtered over

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Celite, the filtrate was concentrated, and the residue was purified by column
chromatography over silica gel (eluting with a gradient of 1 % - 5% MeOH in DCM) to give
Int-cd4 (as mixture of cis and trans olefins). LC/MS: (M+1 )+=1546.8.
Step E - Synthesis of Intermediate Int-cd5

5 To a solution of Int-cd4 (mixture of cis and trans olefins) (5.49 g, 3.55 mmol) in DCM (20
ml) and acetonitrile (50 ml) was added piperidine (1.76 ml, 17.8 mmol). The resulting
solution was stirred at rt for 2h, then concentrated and the residue was suspended in
acetonitrile (20 ml) and concentrated again. The residue was then dried under vacuum to
give Int-cd5 (as mixture of cis and trans olefins) as a crude mixture. LC/MS:
10 (M+1 )+=1323.8.
Step F - Synthesis of Intermediate Int-cd6

To a solution of Int-cd5 (mixture of cis and trans olefins) (4.70 g, 3.55 mmol) and lnt-1d
(2.21 g, 4.44 mmol, preparation described below) in DMF (70 ml) at 0 °C was added HATU
(1.76 g, 4.62 mmol) and DIEA (1.55 ml, 8.88 mmol). The resulting solution was warmed to
15 room temperature and stirred for 1 h, then partitioned between EtOAc (300 mL) and brine
(200 mL). The aqueous phase was extracted with EtOAc (200 mL), the EtOAc phase was
combined and washed with brine (3x200 mL), dried over Na2SO4, concentrated and the
residue was purified by column chromatography over silica gel (eluting with a gradient of
1 % - 5% MeOH in DCM) to give Int-cd6 (as mixture of cis and trans olefins). LC/MS:
20 (M+1)+= 1802.8
Step G - Synthesis of Intermediate Int-cd7

To a solution of Int-cd6 (as 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 was added 1N aqueous LiOH (24.0 ml,
24.0 mmol) dropwise, and the resulting solution was stirred at 0 °C for 3h. The mixture was
25 neutralized at 0 °C by addition of 1N HCI, the volatile was evaporated, and the aqueous

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layer was neutralized to pH 5 by 1N HCI. The mixture was then frozen and lyophilized, and
the residue was purified by column chromatography over C18 (eluting with a gradient of
acetonitrile (0.05%TFA)/water (0.05%TFA)) to give Int-cd7 (as mixture of cis and trans
olefins) as a TFA salt. To the Int-cd7 TFA salt thus obtained, (as mixture of cis and trans
5 olefins) in acetonitrile (750 mL) and water (450 mL) was added at 0 °C 0.1 N aqueous HCI
(150 ml, 15.00 mmol) dropwise, then the resulting solution was stirred at 0 °C for 5 min,
frozen and lyophilized to give Int-cd7 as a HCI salt (as mixture of cis and trans olefins).
LC/MS: (M+1)+=1566.6.
Step H - Synthesis of Intermediate Int-cd8

10 To a solution of Int-cd7 HCI salt obtained from the previous step (1.01 g, 0.630 mmol) in
DMF (50 ml) and DCM (1300 ml) was added DIEA (0.330 ml, 1.890 mmol) and HATU
(0.287 g, 0.756 mmol). The resulting solution was stirred at rt for 2h, the volatile was
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
15 washed with brine (3x100 mL), dried over Na2SO4, concentrated and the residue was
purified by column chromatography over silica gel (eluting with a gradient of 1% - 10%
MeOH in DCM) to give Int-cd8 (as mixture of cis and trans olefins). LC/MS:
(M+1)+=1548.8.
Step I - Synthesis of Intermediate Int-cd9

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

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Step J - Synthesis of compound Ex-01 as the -HCI 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
5 (20 mL). The resulting mixture was concentrated, and the residue was re-dissolved in DCM
(20 mL) and treated with HCI (4N in dioxane) (0.919 ml, 3.68 mmol). The resulting mixture
was concentrated to give the product as solid. This solid product was re-dissolved in
acetonitrile (200 mL) and water (100 mL), and to the above solution at 0 °C was added 1N
aqueous HCI (3.68 ml, 3.68 mmol) dropwise. The resulting solution was stirred at 0 °C for 2
10 min, then frozen and lyophilized to give Ex-01 as a -HCI salt. LC/MS: (M+1)+=1394.7.
Step K - Synthesis of Example Ex-25 as the TFA salt

To a solution of Ex-01 HCI salt (870 mg, 0.608 mmol) and lnt-4b (170 mg, 0.669 mmol,
preparation described below) in DMF (1.2 ml) and water (0.6 ml) was added HATU (254
mg, 0.669 mmol) and DIEA (425 pl, 2.433 mmol). The resulting solution was stirred at rt for
15 1 h then quenched by addition of 1.2 mL water. The mixture was filtered, and the filtrate
was purified by column chromatography over C18 (eluting with a gradient of acetonitrile
(0.05%TFA)/water (0.05%TFA)) to give Ex-25 as a TFA salt. LC/MS: M+= 1550.6.
Step L - Preparation of Example Ex-25 as the Cl salt

Into two columns was packed 73.6 g of AG MP-1 ion exchange resin chloride form (cat#
20 141-1841 BIO-RAD) for a total of 36.8 g resin in each column. Each column was washed
with water (2x80 ml), followed by 20% acetonitrile in water (2x100ml). A solution of Ex-25
TFA salt prepared in the previous step (737 mg, 0.443 mmol) in 20% acetonitrile in water
(100 mL) was loaded evenly onto the two resin columns, then each column was eluted with
20% acetonitrile in water (130 ml). The eluents were combined, frozen and lyophilized to
25 give Ex-25 as the chloride salt. LC/MS: M+ = 1550.6.

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There follows the description of a number of intermediates which are usefully

employed in the synthesis of Ex-01 and Ex-25 described immediately preceding.

Preparation of Intermediate lnt-1d

Intermediate compound lnt-1d was prepared from starting materials in accordance with the
5 following scheme:

Step A. - Synthesis of lnt-1 da

To a solution of D-Dap(Boc)-OMe HCI 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
10 (7.03 ml, 40.2 mmol) and the mixture was stirred at 0 °C for 2 h then kept in the
refrigeratorovernight. The mixture was quenched at room temperature with water and
extracted with EtOAc. The combined organic fractions were washed with half brine, dried
over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column
chromatography over silica gel (eluting with a gradient of Hexanes/EtOAc) to give lnt-1 da.
15 LC/MS: [M+H]+= 512.3.
Step B - Synthesis of lnt-1 d

To a solution of lnt-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 was added solid
sodium hydroxide (0.691 g, 17.27 mmol). The mixture was stirred at room temperature
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overnight. The mixture was concentrated, acidified with 0.5 N to pH ~2 (~40 mL), extracted
trice with EtOAc, washed with brine, dried over Na2S04 and concentrated. The residue
was purified by column chromatography over C18 (eluting with a gradient of
acetonitrile/water + 0.1% TFA) to give lnt-1d. LC/MS: [M+H]+ = 498.25.
5 The preparation of intermediate lnt-1d is described above for use in the preparation
of Ex-01 and Ex-25. This portion of the molecule may be described as a “linker” which

cyclizes the lower peptide ring to the higher peptide ring. Other similar “linkers” may be

used in place of lnt-1d by varying the spacer used in the synthesis, including but not limited
to, using Dap and D-Ala.

10 Preparation of intermediate lnt-2d

Intermediate lnt-2d, useful as a “linker” in the preparation of compounds of the

invention, was prepared in accordance with the following scheme:

sodium
triacetoxy­
hydroborate

pd(ii)(dppf)CI2 Step B

cesium carbonate
Step A

15 Step A - Synthesis of lnt-2da

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A solution of 4-bromobenzaldehyde (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 ,T-bis(diphenylphosphino)ferrocene-palladium(ii)dichloride dichloromethane
complex (Pd(ll)(dppf)Cl2, 1.99 g, 2.43 mmol) in degassed toluene (250 ml) and water (85
5 ml) was warmed to 76 °C and stirred overnight. The mixture was quenched at room
temperature with half-saturated aqueous ammonium chloride and extracted with EtOAc.
The combined organic fractions were washed with brine, dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography over silica gel
(eluting with a gradient of DCM/EtOAc) to give lnt-2da. LC/MS: (M-56+1)+= 193.0.
10 Step B - Synthesis of lnt-2db

To a solution of lnt-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 in a water bath was added sodium
thacetoxyhydroborate (32.9 g, 155 mmol) portion wise and the mixture was stirred for 30
min. The reaction was slowly quenched at 0 °C with 3 ml of water, poured into 1 N NaOH
15 (500 ml), stirred for 15 min then extracted with DCM, dried over Na2SO4, and concentrated.
The residue was purified by column chromatography over silica gel (eluting with a gradient
of DCM/MeOH) to give lnt-2db. LC/MS: (M+1)+= 319.2.
Step C - Synthesis of lnt-2dc

To a solution of lnt-2db (8.48 g, 20.77 mmol) and 4-methoxy-4-oxobutanoic acid (3.02 g,

20 22.85 mmol) in DMF (40 ml) was added HATU (9.48 g, 24.92 mmol) and DIPEA (8.71 ml,
49.8 mmol). The resulting solution was stirred at room temperature for 1hour, then
quenched with aqueous saturated NaHCOs (10 mL). The mixture was partitioned between
EtOAc (500 mL) and aqueous saturated NaHCOs (200 mL), the organic phase was washed
with brine (3x200 mL), dried over Na2SO4, concentrated and the residue was purified on

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silica gel column (eluting with a gradient of Hexanes/EtOAc) to give lnt-2dc. LC/MS:
(M+1)+= 433.4.
Step D - Synthesis of lnt-2d

To a solution of lnt-2dc (2.9 g, 6.70 mmol) in DCM (15 mL) was added 4 M HCI in Dioxane
5 (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 h.
The mixture was concentrated under reduced pressure to afford methyl 4-((4-(2-
aminoethyl)benzyl)(pent-4-en-1-yl)amino)-4-oxobutanoate hydrochloride (lnt-2d). LC/MS
[M-HCI + H]+ = 333.3.
Preparation of lnt-3c from 107 used in the synthesis of Ex-01 and Ex-25 described above
10 Intermediate lnt-3c was prepared in accordance with the following scheme:

Step A - Synthesis of lnt-3ca

Into a solution of 107 (the preparation of which is presented in the synthesis of 109 and
15 later used for 116, which intermediate compound is 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, was added 2 N lithium hydroxide monohydrate (43.3 ml, 87 mmol) and the
mixture was warmed to 45 °C and stirred overnight to give lnt-3ca as crude solution.
LC/MS: (M+1 )+=464.3. The reaction mixture was cooled to 0 °C and treated with 1 M HCI
20 (40 mL). The mixture was directly used for the next step.

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Step B - Synthesis of lnt-3c

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

Preparation of intermediate lnt-4b

10 Intermediate lnt-4b was prepared in accordance with the following scheme:

i Br- ’ Br-

Step A- Synthesis of lnt-4ba from tert-butyl-3-(2-hydroxyethoxy)proponate

To a solution of te/t-butyl 3-(2-hydroxyethoxy)propanoate (500.0 mg, 2.63 mmol) in

DCM (2 mL) were added CBh (1395 mg, 4.21 mmol) and PPhs (965 mg, 3.68 mmol) at 0
15 °C. The mixture was stirred at room temperature for 2 h. The resulting mixture was
concentrated under reduced pressure and the residue was purified by silica gel column
chromatography, eluting with a gradient 1% -15% of ethyl acetate in petroleum ether. The
fractions containing the desired product were combined and concentrated to afford tert­
butyl 6-bromohexanoate. Thus prepared, a solution of tert-butyl 6-bromohexanoate (5 g,
20 19.91 mmol) in acetonitrile (10 ml) was treated with trimethylamine (13.56 ml, 59.7 mmol)
and the resulting solution was heated at 50 °C overnight. The solution was concentrated to
give lnt-4ba. LC/MS: M+= 230.3.
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Step B - Synthesis of lnt-4b

To a solution of lnt-4ba (6.8 g, 21.92 mmol) in DCM (6 ml) was added 4N HCI in dioxane
(27.4 ml, 110 mmol), and the resulting solution was stirred at rt for 3h. The mixture was
then concentrated to give lnt-4b. LC/MS: M+= 174.3.
5 The preparation of intermediate lnt-2d is described above for use in the preparation of Ex-
01 and Ex-25. This portion of the molecule may be described as a “linker” which cyclizes

the lower peptide ring bearing the R1, R2 and R8 substituents. Other similar “linkers” may

be used in place of lnt-2d. Following is a description of other “linkers” which may be used

to prepare examples of the invention described herein.

10 Preparation of intermediate lnt-2e

Intermediate lnt-2e, useful as a “linker” in the preparation of compounds of the

invention, was prepared in accordance with the following scheme:

15 Step A - Synthesis of Intermediate lnt-2ea

To a solution of tert-butyl (2-(3-oxoisoindolin-5-yl)ethyl)carbamate (1.60 g, 5.79 mmol) in
DCE (20 mL) were added NsCI (1.93 g, 8.69 mmol), thethylamine (1.76 g, 17.4 mmol) and
DMAP (0.141 g, 1.16 mmol). The reaction mixture was stirred for 14 h at 40 °C. The
reaction was cooled to room temperature and concentrated under reduced pressure. The
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residue was purified by column chromatography over silica gel (eluting with a 1%-40%
gradient of EtOAc in PE) to give lnt-2ea. LC/MS: (M+Na)+:= 484.4.
Step B - Synthesis of Intermediate lnt-2eb
To a solution of lnt-2ea (11.3 g, 24.5 mmol) in THF (100 mL) and water (100 mL) was
5 added LiOH (1.76 g, 73.5 mmol). The reaction mixture was stirred for 5 h at 25 °C then the
resulting solution was adjusted to pH 4~5 with HCI (1M). The solution was extracted with
EtOAc and the combined organic layers were washed with brine, dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and the
residue was purified by column chromatography over silica gel (eluting with a 1%-6%
10 gradient of MeOH in DCM) to afford lnt-2eb. LC/MS: (M+Na)+:= 502.2.
Step C - Synthesis of Intermediate lnt-2ec
To a solution of lnt-2eb (1.70 g, 3.55 mmol) in THF (8 mL) was added borane (0.147 g,
10.6 mmol) at 0 °C. The reaction mixture was stirred for 14 h at 25 °C then the resulting
solution was concentrated under reduced pressure. The residue was purified by column
15 chromatography over silica gel (eluting with a 1%-50% gradient of EtOAc in PE) to give Int-
2ec. LC/MS: (M+NH4]+= 483.2.
Step D - Synthesis of Intermediate lnt-2ed
To a solution of lnt-2ec (4.50 g, 9.67 mmol) in DMF (150 mL) was added K2CO3 (2.01 g,
14.5 mmol) and 3-bromoprop-1-ene (1.41 g, 11.6 mmol). The reaction mixture was stirred
20 for 5 h at room temperature then diluted with water and extracted with EtOAc. The
combined organic layers were washed with brine, dried over anhydrous Na2SO4 and
filtered. The filtrate was concentrated under reduced pressure and the residue was purified
by column chromatography over silica gel (eluting with a 1%-50% gradient of EtOAc in PE)
to afford lnt-2ed. LC/MS: (M+H)+:= 506.2.
25 Step E - Synthesis of Intermediate lnt-2e

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To a solution of lnt-2ed (4.50 g, 8.90 mmol) in DMF (35 mL) was added DBU (1.35 g, 8.90
mmol) and 2-mercaptoethanol (2.08 g, 26.7 mmol). The reaction mixture was stirred for 14
h at room temperature then purified by column chromatography over C18 (Column: 330 g;
Mobile Phase A: water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 85 mL/min; Gradient:
5 10% B to 20% B in 15 min, 20% B to 45% B in 15 min Detector: UV 210 nm; Rt=20 min) to
afford lnt-2e. LC/MS: (M+H)+:= 321.2. 1H NMR (300 MHz, CDCI3) 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 lnt-2f-1

10 Intermediate lnt-2f-1, useful as a “linker” in the preparation of compounds of the
invention, was prepared in accordance with the following scheme:

PPTs MgSO4, DCM

lnt-2fa StepB
Step A

lnt-2fb-racemic

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PdCl2(dppf)2, Cs2CO3
toluene/H2O
Step E

Step Z\ - Synthesis of Intermediate lnt-2fa

To a solution of 4-bromobenzaldehyde (20.0 g, 108 mmol), (S)-2-methylpropane-2-

sulfinamide (12.5 g, 103 mmol), MgSO4 (130 g, 1081 mmol) in DCM (225 mL) was added
5 pyridine 4-methylbenzenesulfonate (1.35 g, 5.40 mmol) under nitrogen protection. This
mixture was stirred at 25 °C for 72 h then the resulting solution was filtered, and the filtrate
was concentrated under reduced pressure. The resulting residue was purified by column
chromatography over silica gel (eluting with a 1 %-15% gradient of EtOAc in PE) to give Int-
2fa. LC/MS: (M+H)+:= 287.9, 289.9.
10 Step B - Synthesis of Intermediate lnt-2fb (racemate) and separation into enantiomers Int-

2fb-1 and lnt-2fb-2

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

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To a solution of HCI (100 mL, 4 N in 1,4-dioxane) at room temperature was added lnt-2fb-1
(17.0 g, 46.9 mmol). The reaction solution was stirred for 1 h then concentrated under
reduced pressure to afford lnt-2fc-1. LC/MS: (M+H-HCI)+:= 240.0, 242.0.
Step D - Synthesis of Intermediate lnt-2fd-1

5 To a solution of lnt-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
column chromatography over silica gel (eluting with a 1 %-10% gradient of EtOAc in PE) to
10 give lnt-2fd-1. LC/MS: (M+Na+CH3CN)+:= 447.3, 449.3.
Step E- Synthesis of Intermediate lnt-2fe-1

To a solution of lnt-2fd-1 (15.1 g, 37.3 mmol), potassium (2-((tert-

butoxycarbonyl)amino)ethyl) trifluoroborate (18.7 g, 74.6 mmol), CS2CO3 (36.5 g, 112
mmol) in toluene (285 mL) and water (95 mL) was added PdCl2(dppf) (1.37 g, 1.87 mmol)
15 under nitrogen protection. The mixture was stirred at 80 °C for 40 h. The resulting solution
was quenched with water and extracted with EtOAc. The combined organic layers were
dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and
the residue was purified by column chromatography over silica gel (eluting with a 1%-40%
gradient of EtOAc in PE) to afford lnt-2fe-1. LC/MS: (M+Na)+:= 471.4.
20 Step F- Synthesis of Intermediate lnt-2f-1

To a solution of Int-2fe1 (10.6 g, 22.4 mmol) in THF (100 mL) was added 1N TBAF in THF
(44.9 mL, 44.9 mmol). This mixture was stirred at room temperature for 16 h then
quenched with water and extracted with EtOAc. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under
25 reduced pressure and the residue was purified by column chromatography over silica gel

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(eluting with a 1%-70% gradient of EtOAc in PE) then by column chromatography over C18
(Column: 330 g; Mobile Phase A: water (10 mm NH4HCO3), Mobile Phase B: ACN; Flow
rate: 80 mL/min; Gradient: 10% B to 10% B in 10 min, 20% B to 45% B in 10 min, 45% B to
70% B in 20 min Detector: UV 210 nm; Rt= 25 min) to provide lnt-2f-1. LC/MS: (M+H)+: =
5 305.1. 1H NMR (300 MHz, CDsOD) 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

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

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Br MeCN, 50 °C

O
0
O'
HCI OH
N+
I Br- DCM
lnt-32 A lnt-32

Step A: Preparation of intermediate lnt-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
5 the resulting solution was heated at 50 °C overnight. The solution was concentrated to give
2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)-N,N,N-thmethylethanaminium bromide (Int 32A).
LC/MS: (M)+: 276.5.
Step B: Preparation of intermediate lnt-32

To a solution of 2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)-N,N,N-trimethylethanaminium
10 bromide (lnt-32A) (5.99 g, 16.81 mmol) in DCM (20 ml) was added HCI (4N in dioxane)
(21.01 ml, 84 mmol), and the resulting solution was stirred at rt overnight. The solution was
concentrated to give 2-(2-(2-carboxyethoxy)ethoxy)-N,N,N-trimethylethanaminium bromide
(lnt-32). LC/MS: (M)+: 220.1.
Preparation of example compound Ex-50

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To a solution of Ex-01 (crude) (17.4 mg, 0.012 mmol) and 2-(2-(2-carboxyethoxy)ethoxy)-

Ν,Ν,Ν-trimethylethanaminium bromide (lnt-32) (4.49 mg, 0.015 mmol) in DMF (2 ml) was
added HATLI (5.69 mg, 0.015 mmol) and DIEA (6.54 μΙ, 0.037 mmol), and the resulting
5 solution was stirred at rt for 50 min, then purified by reverse phase HPLC using
acetonitrile(0.1%formic acid)/water(0.1%formic acid) as mobile phase to give Ex-50.
LC/MS: M+ = 1596.3.
Preparation of example compound Ex-52
Compound Ex-52 was prepared in an analogous manner to the preparation of compound
10 Ex-50 but using Ex-51 instead of Ex-01. Ex-52 was purified using reverse phase HPLC, in
accordance with the methods described herein. LC/MS: M+ = 1593.8.
104
 WO 2019/246349 PCT/US2019/038155

EXAMPLE 3 Preparation of Ex-53, Ex-54 and Ex-55

105
 WO 2019/246349 PCT/US2019/038155

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

Ex-54 -------------------------- - Ex-55

Step C
5 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) followed by HATU (18.50 mg, 0.049
mmol) and the mixture was stirred for 30 min. The mixture was concentrated in vacuo and
10 directly purified by column chromatography over C18 (30 g, eluting with acetonithle+0.05%
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TFA/water+0.05% TFA 90:10 to 40:60) to provide 116 as a mixture of E and Z isomers as

well as 116 as pure fractions of E or Z isomer. LC/ MS (major isomer) LC/MS: M+ =
1481.19; LC/MS (minor isomer): LC/MS: M+= 1480.
Step B - Synthesis of Compound Ex-54
5 A solution of 116 (25.1 mg, 0.017 mmol) and Pd-C 10% (3.61 mg, 3.39 pmol) in MeOH (10
ml) was hydrogenated at 1 atm for 1 h. The reaction was filtered over Celite and
concentrated. The residue was treated with DCM/TFA 1:1 for 30 min then concentrated,
treated with 4N HCI in dioxane (100 uL) then concentrated to provide Ex-54 in the form of
the HCI salt. LC/MS: M+= 1383.44.
10 Step C - Synthesis of Compound Ex-55
Example compound Ex-55 was prepared in the form of the formate salt 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.
Step D - Synthesis of Compound Ex-53
15 Example compound Ex-53 was prepared in the form of the HCI salt from intermediate
compound 116 in a manner identical to that described in Example 1 for the synthesis of
compound Ex-51. LC/MS: M+ = 1381.33.
Preparation of the intermediates needed to provide intermediate compound 115 from
which compounds Ex-53, Ex-54, and Ex-55 are ultimately prepared is described in the
20 schemes and synthesis below beginning with the preparation of intermediate compound
103

107
 WO 2019/246349 PCT/US2019/038155

Step A - Synthesis of Intermediate 100

A solution of 4-bromo-2-hydroxybenzaldehyde (3.00 g, 14.92 mmol), potassium tert-butyl N-

[2-trifluoroboraniudyl)ethyl] carbamate (3.82 g, 15.22 mmol), cesium carbonate (17.02 g,
5 52.2 mmol) and 1,1'-bis(diphenylphosphino)ferrocene-palladium(ii)dichloride
dichloromethane complex (0.611 g, 0.746 mmol) in degassed toluene (45 ml) and water (15
ml) was warmed to 75 °C and stirred overnight. The mixture was quenched at room
temperature with half-saturated aqueous sodium bicarbonate and extracted with EtOAc.
The combined organic fractions were washed with brine, dried over Na2SO4, filtered and
10 concentrated in vacuo. The residue was purified by column chromatography over silica gel
(eluting with Hexanes/EtOAc 99:1 to 60:40) 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 was added potassium carbonate (1.328 g, 9.61 mmol) and the
15 mixture was warmed to 50 °C and stirred for 1 h. The mixture was quenched at room
temperature with half-saturated aqueous sodium bicarbonate and extracted with EtOAc.
The combined organic fractions were washed with brine, dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography over silica gel
(eluting with Hexanes/EtOAc 99:1 to 70:30) to give 101. LC/MS: (M-55)+ = 250.29.
20 Step C - Synthesis of Intermediate 102

108
 WO 2019/246349 PCT/US2019/038155

To a solution of 101 prepared in the previous step (1.76 g, 5.76 mmol), 4 A molecular
sieves (2 g) and ammonium acetate (4.44 g, 57.6 mmol) in MeOH (100 ml) at room
temperature was added sodium cyanoborohydride (0.380 g, 6.05 mmol) and the mixture
was shaken overnight. The mixture was concentrated, quenched at room temperature with
5 water and extracted with DCM. The combined organic fractions were dried over Na2SO4,
filtered and concentrated in vacuo. The residue was purified by column chromatography
over silica gel (eluting with DCM/MeOH 99:1 to 30:70) to provide 102. LC/MS: (2M+H)+ =
613.56.
Step D - Synthesis of Intermediate 103

10 To a solution of 102 (220 mg, 0.718 mmol) and succinic acid mono-methyl ester (114 mg,
0.862 mmol) in DMF (4 ml) at room temperature was added HATLI (300 mg, 0.790 mmol)
and DIPEA (0.314 ml, 1.795 mmol) and the mixture was stirred for 30 min. 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
15 Na2SO4, filtered and concentrated in vacuo. The residue was purified by column
chromatography over silica gel (eluting with Hexanes/EtOAc 99:1 to 30:70) to give an
intermediate that was treated with 20% TFA in DCM for 1h. The reaction was concentrated
then treated with 4N HCI (1.5 mL) and concentrated to provide 103. LC/MS: (M+H)+ =
321.29.
20 Preparation of intermediate 109

109
 WO 2019/246349 PCT/US2019/038155

1 NH HCI
*
Me
''CO 2Me
HATU HCI

Step A Step
Me B

HATU Pd/C H2 Boc2O

Step C Step D Step E

108

LiOH
SfepF

109

Step A - Synthesis of Intermediate 104

To a stirred solution of methyl (S)-2-methylpyrrolidine-2-carboxylate hydrochloride (7.00 g,

5 39 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxyphenyl)propanoic acid
(12.08 g, 40.9 mmol) in DMF (100 ml) at 0 °C was added DIPEA (17.01 ml, 97.0 mmol)
followed by HATU (19.26 g, 50.7 mmol). The resulting mixture was allowed to warm to
room temperature and stirred overnight. The reaction was quenched with a 10% aqueous
LiCI solution and extracted with EtOAc. The organic extract was washed with 10%
10 aqueous LiCI and dried over MgSO4. The solvent was removed under reduced pressure
and the residue purified by column chromatography over silica gel (eluting with
Hexanes/EtOAc 80:20 to 40:60) to provide 104.
Step B - Synthesis of Intermediate 105

no
 WO 2019/246349 PCT/US2019/038155

To a solution of 104 (16.4 g, 39.0 mmol) in EtOAc (100 ml) was added 4 N HCI in dioxane
(48.8 ml, 195 mmol). The resulting mixture was stirred at room temperature for 18 h and
was concentrated under reduced pressure to give 105 that was used in the next step
without further purification.
5 Step C - Synthesis of Intermediate 106

To a solution of 105 (13.2 g, 37.0 mmol) and N-((benzyloxy)carbonyl)-O-(tert-butyl)-L-

threonine (18.15 g, 37.0 mmol) in DMF at 0 °C was added DIPEA (16.15 ml, 92 mmol)
followed by HATLI (18.28 g, 48.1 mmol). The resulting mixture was allowed to warm to
room temperature and stirred overnight. The reaction was quenched with 10% aqueous
10 LiCI solution and extracted with EtOAc. The organic extract was washed with 10%
aqueous LiCI and dried over MgSO4. The solvent was removed under reduced pressure
and the residue purified by column chromatography over silica gel (eluting with
Hexanes/EtOAc 80:20 to 40:60) to provide 106.
Step D - Synthesis of Intermediate 107

15 To a solution of 106 (16.5 g, 27.0 mmol) in MeOH was added a slurry of 10% Pd/C and the
mixture was hydrogenated at 20 psi for 4 h. The reaction mixture was filtered over Celite
and concentrated under reduced pressure. The crude product was then re-dissolved in
DCM and the solution was filtered through a 2 μΓη filter and concentrated to give 107.
Step E - Synthesis of Intermediate 108

20 To a solution of 107 (3.2 g, 6.70 mmol) in DCM was added DIPEA (1.52 ml, 8.71 mmol)
followed by di-tert-butyl dicarbonate (1.90 g, 8.71 mmol). The resulting mixture was stirred
at room temperature for 2 h then concentrated under reduced pressure. The residue was
purified by column chromatography over silica gel (eluting with Hexanes/EtOAc 100:0 to
40:60) to afford 108.
25 Step F - Synthesis of Intermediate 109

111
 WO 2019/246349 PCT/US2019/038155

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

Preparation of Intermediate Compounds 110 to 115

Step A - Synthesis of Intermediate 110

A solution of 109 (217 mg, 0.385 mmol), HATU (133 mg, 0.350 mmol) and DIPEA (0.245
ml, 1.400 mmol) in DMF (2.5 ml) was treated with 103 (217 mg, 0.385 mmol) at 0 °C and
the mixture was 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
15 EtOAc. The combined organic fractions were washed with brine, dried over Na2SO4,
filtered and concentrated in vacuo. The residue was purified by column chromatography
over silica gel (eluting with Hexanes/EtOAc-EtOH 3:1 99:1 to EtOAc-EtOH 3:1) to give 110.
LC/MS: (M+H)+= 866.21.
Steps B and C - Synthesis of Intermediate Compounds 111 and 112

112
 WO 2019/246349 PCT/US2019/038155

To a solution of 110 (249 mg, 0.288 mmol) in DCM (1 ml) at room temperature was
added HCI 4 N in dioxane (0.359 ml, 1.438 mmol) and the mixture was stirred for 6 h then
concentrated to provide 111. LC/MS: (M+H)+ = 710.19.
5 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 min. The mixture was quenched at 0
°C with brine and extracted with EtOAc. The combined organic fractions were dried over
Na2SO4, filtered and concentrated in vacuo. The residue was purified by column
10 chromatography over silica gel (eluting with Hexanes/EtOAc-EtOH 3-1 99:1 to 30:70 then
DCM/MeOH 99:1 to 70:30) to afford 112. LC/MS: (M+H)+ = 1506.11.
Step D - Synthesis of Intermediate 113

Fmoc

112
Step D

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To a solution of 112 (173 mg, 0.115 mmol) in DCM (180 ml) and AcOH (15 ml) degassed
with nitrogen for 30 min was added Zhan’s Catalyst (59.0 mg, 0.080 mmol) and the mixture

was warmed to 50 °C and stirred for 3 h. The mixture was filtered over Celite, washing with
DCM then concentrated in vacuo. The residue was purified by column chromatography
5 over silica gel (eluting with DCM/MeOH 99:1 to 80:20) to afford 113 as a mixture of E and Z
isomers. LC/MS (major isomer): (M)+ = 1477.80; LC/MS (minor isomer): (M)+ = 1478.28.
Step E- Synthesis of Intermediate 114

To a solution of 113 (141 mg, 0.095 mmol)) in acetonitrile (2 ml) was added piperidine
10 (0.066 ml, 0.668 mmol) and the mixture was stirred for 45 min. The mixture was
concentrated in vacuo, co-evaporated with acetonitrile trice to give a crude. To a slurry of
this crude (119 mg, 0.095 mmol) and intermediate compound 88 (52.0 mg, 0.105 mmol) in
DMF (2 ml) and water (0.1 ml) at 0 °C was added HATLI (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
15 column chromatography over C18 (eluting with acetonitrile+0.05% TFA/water+0.05% TFA
90:10 to 30:70) to give 114 as a mixture of E and Z isomers. LC/MS major isomer: (M)+ =
1735.28; LC/MS (minor isomer): (M)+ = 1735.25.
Step F- Synthesis of intermediate compound 115

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 WO 2019/246349 PCT/US2019/038155

To a solution of 114 (134 mg, 0.077 mmol) in THF (1.5 ml) and MeOH (1.5 ml) at 0 °C was
added 1 N aqueous LiOH (0.386 ml, 0.386 mmol) dropwise and the mixture was stirred for
2 h. The reaction was treated at 0 °C dropwise with 0.5 N HCI until pH~7, concentrated
5 from organic solvents, then the slurry was dissolved with ~1 mL of DMF and directly purified
by column chromatography over C18 (eluting with acetonitrile+0.05% TFA/water+0.05%
TFA 90:10 to 50:50) to give 115 as a mixture of E and Z isomers. LC/MS major isomer:
(M)+ = 1498.71; LC/MS (minor isomer): (M)+ = 1499.48.
As described above in the preparation of Ex-50 from Ex-01, and Ex-55 from Ex-54
10 by reaction of the R2 amide thereof in the respective starting compounds with an acidic
substituent precursor, the following intermediate compounds may be employed in
analogous reactions to provide useful compounds of the invention.
Synthesis of R1/R2 substituent precursors:
Preparation of 5-carboxv-/V-(3-methoxvpropyl)-/V,/V-dimethvlpentan-1 -aminium
15 chloride (Intermediate Z-1a)

Step A: preparation of intermediate Z-1

115
 WO 2019/246349 PCT/US2019/038155

To a stirred solution of tert-butyl 6-(dimethylamino)hexanoate (300 mg, 1.393 mmol) in

acetonitrile (1 mL) was added 1-bromo-3-methoxypropane (853 mg, 5.57 mmol). The
reaction mixture was stirred at 50 °C for 16 h. The resulting mixture was concentrated
under reduced pressure to afford Z-1 LC/MS: (M-Br)+ = 288.4. 1H NMR (300 MHz, CDCh):
5 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 compound Z-1 a

To a stirred solution of Z-1 (460 mg, 1.249 mmol) in DCM (0.5 mL) was added 4 M HCI in
10 dioxane (2 mL) at room temperature. The reaction mixture was stirred at room temperature
for 4 h and concentrated under reduced pressure. The residue was re-dissolved in DCM (5
mL) and concentrated under reduced pressure to afford intermediate compound Z-1a.
LC/MS: (M-CI)+ = 232.3.
Preparation of Intermediate Z-2b
15 Step A: preparation of intermediate Z-2

To a stirred solution of tert-butyl 6-bromohexanoate (1.0 g, 3.98 mmol) in THF (10 mL) was
added dimethylamine (2 M in THF) (7.96 mL, 15.93 mmol). The reaction mixture was stirred
at room temperature for 16 h. The resulting mixture was concentrated under reduced
20 pressure and the residue was purified by silica gel column chromatography, eluting with a
gradient 1% -15% MeOH in DCM. The fractions containing the desired product were
combined and concentrated to afford Z-2. LC/MS: (M+H)+ = 216.2. 1H NMR (300 MHz,

116
 WO 2019/246349 PCT/US2019/038155

CDCIs): 5 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

0.
N
0
Br
Z-2a
5 To a stirred solution of Z-2 (250 mg, 1.161 mmol) inACN(1 mL) was added 1-bromo-2-
methoxyethane (645 mg, 4.64 mmol). The reaction mixture was stirred at 50 °C for 16 h.
The resulting mixture was concentrated under reduced pressure to afford Z-2a. LC/MS: (M-
Br)+ = 274.3. 1H NMR (300 MHz, CDCI3): 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).
10 Step C: preparation of intermediate Z-2b

ci
0.
N"
OH Z-2b
To a stirred solution of Z-2a (450 mg, 1.270 mmol) in DCM (0.5 mL) was added 4 M HCI in
dioxane (2 mL) at room temperature. The reaction mixture was stirred at room temperature
for 4 h and concentrated under reduced pressure. The residue was re-dissolved in DCM (5
15 mL) and concentrated under reduced pressure to afford Z-2b. LC/MS: (M-CI)+ = 218.3.
Preparation of Intermediate Z-3b
Step A: preparation of intermediate Z-3

.0
Br
O Z-3
To a solution of te/t-butyl 3-(2-hydroxyethoxy)propanoate (500.0 mg, 2.63 mmol) in DCM (2
20 mL) were added CBr4 (1395 mg, 4.21 mmol) and PPh3 (965 mg, 3.68 mmol) at 0 °C. The
mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated
under reduced pressure and the residue was purified by silica gel column chromatography,
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 WO 2019/246349 PCT/US2019/038155

eluted with gradient 1% -15% EA in PE. The fractions containing desired product were
combined and concentrated to afford Z-3. 1H NMR (400 MHz, CDCIs): 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

.0
N
O
5 Br Z-3a
To a stirred solution of tert-butyl 3-(2-bromoethoxy)propanoate Z-3 (450 mg, 1.778 mmol) in
ACN (2 mL) was added trimethylamine (955 mg, 5.33 mmol) (33%Wt, in EtOH). The
reaction mixture was stirred at 50 °C for 16 h. The resulting mixture was concentrated
under reduced pressure to afford Z-3a. LC/MS: (M-Br)+ = 232.3. 1H NMR (400 MHz,
10 CDCI3): 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

.OH
N
0
Cl Z-3b
To a solution of Z-3a (550 mg, 1.761 mmol) in DCM (0.6 mL) was added 4 M HCI in
15 dioxane (2.5 mL) at room temperature. The mixture was stirred at room temperature for 4 h.
The resulting mixture was concentrated under reduced pressure and the residue was re­
dissolved in DCM (3 mL) and toluene (3 mL). The mixture was then concentrated under
reduced pressure to afford Z-3b. LC/MS: (M-CI)+ = 176.2.
Preparation of Intermediate Z-4b
20 Step A: preparation of intermediate Z-4

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To a solution of DIAD (1.755 mL, 9.03 mmol) in THF (30 mL) was added PhsP (2.368 g,
9.03 mmol). The mixture was stirred at room temperature for 10 min, then methyl 2-(3-
hydroxyphenyl)acetate (1.0 g, 6.02 mmol) and 3-(dimethylamino)propan-1-ol (0.931 g, 9.03
mmol) were added to the solution. The mixture was stirred at 50 °C for 1 h. The resulting
5 solution was concentrated under reduced pressure and the residue was purified by silica
gel column chromatography, eluting with a gradient 1% -10% MeOH in DCM. The fractions
containing the desired product were combined and concentrated to afford Z-4. LC/MS:
(M+H)+ = 252.2. 1H NMR (300 MHz, CDCIs): 5 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),
10 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 Mel (1.288 g, 9.07
mmol). The mixture was stirred at room temperature for 1 h. The resulting solution was
15 concentrated under reduced pressure to afford -Z-4a. LC/MS: (M-l)+ = 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 h. The pH value of the
20 solution was adjusted to 4 with HCI (1 M) and the solution was concentrated under reduced
pressure. The crude product was purified by reverse phase chromatography over C18
(Mobile Phase A: water, Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 1% B to
25% B in 25 min; 25% B to 95% B in 15 min; 95% B to 95% B in 10 min) to afford Z-4b.
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LC/MS: (M-CI)+ = 252.2. 1H NMR (300 MHz, CDsOD): 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).
EXAMPLE 4 Preparation of Ex-23

AcO
AcO HBr/AcOH NaN3
O ---- »
OAc DCM, 0 °C DMF
AcO
OAc Step A Step B

AcO
AcO
H2,Pd-C
O
N3
AcO Step C 2 DMAP
OAc
Step D
S-1c

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 a solvent of 48% HBr (7.25 mL, 64.0 mmol) in AcOH and
DCM (40 mL) at 0°C and the mixture was stirred at 0°C for 1 hour. The reaction mixture
10 was poured into aqueous saturated sodium hydrogen carbonate cooled on ice and the
mixture was extracted with DCM (3 x 60mL). The organic layer was washed with brine,

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dried over Na2SO4, filtered and concentrated then the crude product was purified by flash
chromatography over silica gel (eluting with 0~30% EtOAc/PE) to 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
5 (0.854 g, 13.13 mmol) and the reaction was stirred at 18 °C for 30 min. The reaction
mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 80 mL). The organic
layer was dried over Na2SO4 and evaporated to dryness. The crude product was purified
by flash chromatography over silica gel (eluting with 0~30% EtOAc/PE) to give S-1c.

Step C: Preparation of intermediate S-1d

10 To a solution of S-1c (3.15 g, 8.44 mmol) in EtOH (60 mL) were added 10% Pd-C (0.898 g,
0.844 mmol). The reaction vessel was purged from air and filled with H2 under 50 psi. The
reaction was stirred at 18 °C for 5 h. The reaction mixture was diluted with EtOAc, filtered
through Celite, and concentrated, to give S-1d which was used for the next step.
Step D: Preparation of intermediate S-1e

15 To a solution of S-1d (2.34 g, 6.74 mmol) in anhydrous THF (20 mL) was added
dihydrofuran-2,5-dione (0.742 g, 7.41 mmol) and Et3N (0.939 mL, 6.74 mmol). The
reaction was stirred for 3 h until complete consumption of the starting material then
evaporated. The resulting crude product was purified by flash chromatography over silica
gel (eluting with 0-10% DCM/MeOH) to give S-1e. MS (ESI): m/z (M+H)+ 448.1. 1HNMR
20 (400 MHz, CDCI3) 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 (q, 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).
Step E: Preparation of Ex-23

To a solution of Ex-01 (300 mg, 0.215 mmol) and S-1e (115 mg, 0.258 mmol) in DMF (8 ml)
25 and water (0.4 ml) was added DIEA (0.150 ml, 0.860 mmol) and HATU (98 mg, 0.258
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mmol) and the resulting solution was stirred at rt for 1h. The reaction was quenched with 1N
LiOH (2.58 ml, 2.58 mmol) dropwise, the resulting solution was stirred at rt for 2h then
filtered and the filtrate was purified on reverse phase HPLC C18 column using a 29-34%
gradient of acetonitrile (0.05%TFA) in water (0.05%TFA) to give Ex-23. LC/MS: [M+1 ]+ =
5 1657.1.
EXAMPLE 5 Preparation of Ex-14

Compound Ex-14 was prepared in a manner analogous to procedures described in

io Example 1 but using different “linkers” and alternate synthetic steps. Synthesis of these
“linkers”, alternative steps and main assembly is described below.

Preparation of intermediate lnt-2q

Intermediate lnt-2g, useful as a “linker” in the preparation of compounds of the invention,
was prepared in accordance with the following scheme:

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sodium
triacetoxy­
hydroborate

Step A

Step A - Synthesis of lnt-2gb

To a solution of lnt-2da (0.5 g, 2 mmol) and 2-azidoethanamine, HCI (0.246 g, 2mmol) in

5 THF (16 ml) at room temperature in a water bath was added sodium thacetoxyhydroborate
(1.06 g, 5 mmol) portion wise and the mixture was stirred for 2h. The reaction was slowly
quenched with aqueous saturated NaHCOs solution, then extracted with DCM and washed
with brine. The combined organic layers were dried over MgS04 and concentrated to give
lnt-2gb. LC/MS: (M+1 )+= 320.3.
10 Step B - Synthesis of lnt-2gc

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

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Step C - Synthesis of lnt-2g

To a solution of lnt-2gc (0.52 g, 1.2 mmol) in DCM (9 mL) was added TFA (3 mL, 38.9
mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h.
The mixture was concentrated under reduced pressure to afford lnt-2g. LC/MS: (M+1)+=
5 334.3.

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
10 (0.141 g, 5.56 mmol), and the resulting solution was stirred at 0 °C for 20 min followed by
addition of 3-bromoprop-1-yne (80% in toluene) (0.596 ml, 5.35 mmol) dropwise. To the
resulting solution was added aqueous lithium hydroxide (2M) (3345 pl, 6.69 mmol)
dropwise. The reaction was stirred at room temperature for 2h, filtered and purified by
reverse phase HPLC (eluting with acetonithle/water + 0.1 % TFA) to give 70B. LC/MS:
15 (M+1)+: 361.0, (M+Na)+: 383.0.
Step B - Synthesis of intermediate 76C

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Conversion of 70B to intermediate 76C proceeded according to procedures analogous to

those described in the preparation of intermediate 76 Steps C to H. LC/MS: [M+1]+ =
812.16.

Assembly into Example Ex-14:

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Step A. - Synthesis of intermediate 117

Intermediate 117 was prepared from intermediates lnt-2g and 76C according to procedures
analogous to those described in Example 1 and 1A. More specifically lnt-2g was
5 functionalized following reagents and procedures for the preparation of intermediate 77B
Steps A to B, further elaborated following procedures for the preparation of intermediate 86
Steps G to J, then finally coupled with intermediate 76C following procedures for the
preparation of Example 1A Steps B to C, to provide 117. LC/MS: (M+1)+: 1573.36.
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Step B - Synthesis of intermediate 118

Tetrakis(acetonitrile)copper(l) hexafluorophosphate (51.6 mg, 0.138 mmol), tris[(1-benzyl-

1h-1,2,3-triazol-4-yl)methyl]amine (73.4 mg, 0.138 mmol) and sodium ascorbate (137 mg,
0.692 mmol) in BuOH (185.00 mL)/water (93 mL) were bubbled with nitrogen and then
5 heated at 50 °C. Intermediate 117 (435.3 mg, 0.277 mmol) was added into the reaction as
a solid. After 1 h, the reaction was treated with aqueous pH 4 buffer and extracted with
EtOAc. The combined organic layers were evaporated, and the residue was purified by
reverse phase chromatography (eluting with a gradient of acetonithle/water +0.1% formic
acid) to provide intermediate 118. LC/MS: (M+1)+: 1573.2.
10 Step C - Synthesis of Ex-14

Synthesis of Example Ex-14 proceeded from intermediate 118 according to procedures

analogous to those described in Example 1, including the use of alternate spacers for the
assembly. LC/MS: [M+1]+ = 1621.01.
Using the synthetic schemes described above, and as will be appreciated, in some
15 instances with appropriate substitution of certain intermediates including the use of
alternate spacers apparent to those skilled in the art, the preparation of which may be
described above, the following compounds of the invention, listed in Table 2 below, were
prepared. Additionally, alternate salt forms of compounds of the instant invention may also
be described in this application:
20

25

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Table 2

Ex- No/ Structure LC/MS: (Μ)+

Ex-01
ACOH salt 1394.4
0
-χ ΗΝ-Λ H.C
»ΟΗ
h3c
A Η ? $

+H3N NH 0

NH r ^y-N 0=
0=/ F~\ ^(CH2)6
\__ NH \ CH3
_ ΝΗ
Η3σ
ο "UP
o'

Ex-02 TFA ο
salt 1622.12
ΗΝ'"'^ ΟΗ
ΰΚ
cJ c -ΝΗ
οζ
ΗΝ—1.

Ο '
----- ΝΗ θ -- τ
ο. °Α
π ΝΗ
LmN / \
y /7 Ν -Ν
ΗΝ^η A0 F ο
1 * ΗΝ-----
----- Ν
-----0
'ο--\ / Ό F
"X~F
■\ ο" F

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Ex- No/ Structure LC/MS: (M)+

Ex-03 TFA
0 1622.06
salt
hn^S y°H
\ Ok. *K
HN H .
q * ΥΛ·

Lhn (Λ οΛν
' O X N-- 1 \ /
y-NH \ / \ X/ /

-4 !h <«
H\t°hnF v“> /N J /
v_° /=/

0-\ \ / °x F
NH

Ex-04 TFA
0 1595.04
salt
HN—
\ / ' / X^>0H

/ °5 J /=\ /
tVivi xc^

y— NH O \ 0 N=o
/III"./ /^, \
NH F /1 H I \
°=< \ / η
u \ H ' 1—>'
'
θ \ r~ hfl =

°—\ \ / S\^'N'^/
'—0 Ν< Ό F JT
\___ / N~(-f o
OZ F

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Ex- No/ Structure LC/MS: (M)+

Ex-05 TFA
salt
o 1594.54
HN—

( H JL Η'ύΗ/Ν’^Ύ>°υΓΎ'°/
gr'M +T

nA
NH // A m Hl/
n__z F—(' x>— N-^ N. A—/
Λ H \=/ 1__ / Ύ i
/—' °

H'NV° Vx i CJ

IT

/ Ό F
-W o^V

Ex-06 TFA
0 1549.44
salt

)=/ 0 XT
* OH
( h j? h
0<V^n^\N °

f O=/H FHfy^ HN Jp

\ jJ o

^n+ Ό F \ Λ/)

1 o^v

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Ex- No/ Structure LC/MS: (M)+

Ex-07 TFA
salt
0 1692.66
_HN----
\γ
*
ΟΗ
/=\ /

1 η
ΛAHN
*" Cr°
LT
HN^ HN^\.
0 N 1
nO</hn—//,, r''Ί3 0 H
J HN Ny / '/
A—N
\^O
0
0
3
N N----
H
°~\
0=\
'hr
0
Ό F Π
N+- r?—^~F 0
/ \ 0 F

Ex-08 TFA o
salt 1608.46
/—\ /N~
\—/
i o
°\ Η 1
0 I H 0
"y
TY"•OH
HN^
H 0 X ° A.
O NH
HN
,NX
=0 Xj
a r-<^\ /=\ )
HN· N V o
0 ° /
Ό -o'
1 F '''ΉΗ V.
U F

Ex-09 Cl 0
salt 1608.30
HN-^^
YrfOH
J 0 H„J
X HN
w
/ ° 0
^■"NH
L ,0
H /
N—' Φ N^\
O 'N---- 1
0 Ko y
NH
ALY
HN RN
7 \
II
U
OCH3 7
°=T
J
;O
oz F
0 o
\— > --NH
o
N"\
Η X- Y>4^'n/
H X
'— N < cr

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Ex- No/ Structure LC/MS: (M)+

Ex-IOTFA o
salt 1608.40
HN—

j? "I ϊ <γ° 1

TO—γ%, TO)n

1 O^NH i ] / TO .
k \ N / .=/ TOTO
YA < J Q °0 -TO

1 TOl <" < /

TO VTOnTOto /
toto toqto

Ex-11 TFA 0
salt fyTO)
1608.84

\ h 9 H-Y H 1

hnTOT y vY Ύ
fc L ° \__ , ° q<tonh
T 0 Aa rCn

ΎΝΗ XjO Λ
οΔ jdV7

\ to sAOZ

TO^-TO toV
Ex-12
Ό 1579.54
TFA salt £y?TO

/7 °\
0 θ 1 H
UrTOTOTO" /
9 Η* Η 1

TO hn TO. 0 ,nh
S ok TOY Y>to
v \

Ό F \ TO TOroro /
□TOF NTOTOV

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Ex- No/ Structure LC/MS: (M)+

EX-13TFA 0
salt 1621.74
<--- HN------------

1 Α»

' // / Ύ

I A 0 o^nh
O. .NH / 'N „1 !
Y A/\ / ana
r’NH
HN^° F rA r \ ° 77"
Γ^Ν \A 0 .NH
[ Z 0 X "'N /
i II / o. /
°\^X \AnZ \
O~~\—Ntf /
' Αχ^ A
■O F || I (
o^f LAJ

Ex-14 TFA 0
salt 1622.28
r-----HN-----------------

0 \ H H H L
ΑΛΆτ A

hn' έ
-
Η Π
ο
i
T
v-^ II
Ω
1
=
HN .0
\
X----- a υ Λ<Υ
0 NH
I

i \ aJYA
i V A Μ

A AJ < A

' \ 7 L /x /

0 pF XX/

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Ex- No/ Structure LC/MS: (M)+

EX-15TFA 0
salt Pl
1636.54

Ο^,ΝΗ
yΛ FU
P /
pip

o F

Ex-16TFA ,o
salt 1592.52
_hNp
)=/ <£

0 1
/
Η
θ
Π
% /1
Ζ^^Ν·<Ρ,ΠΗ
Η I

/ NX || i U 1
H b \ 0 0<>^NH

A-γ FXJX-o^°xO

0
/ U
A
' "
N—N
o=f\ X
1 \__ O __ J r----- -NH

R (-F
ϋ~ν
EX-17TFA
0 1582.68
salt

ύ»
{ H 0 HZz^ HN-P^O Γ~\-0
h yW A AZP
\ 0 NH Zp 7 Hl)
°λ o< hf<t\ p/H

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 WO 2019/246349 PCT/US2019/038155

Ex- No/ Structure LC/MS: (Μ)+

Ex-21 Cl
salt 0 1566.91

\=/ / V0H

0 YrVr
knh o < HNn

Γ X JLJ X? ^γΡ

Λ xl+cr Ji Ο
θ/___Ν+-

Ο
Ex-22 TFA
0 1596.66
salt ΛΑ21Ν Λ
\=/ 1 χ
°
* Η
(η 9 ™~Co Τ\Α
VnUI Ν^Ο ΗΝ-Τφ-7^

.... ( Ν-Λ
Λ ΝΗ F ^Vn Η ϊ >
0=/ h \ / \Ζ \
)---- ΝΗ — / Γ |[ i
< /—/=Υ ο
Τ°
ο^θνΛ

ο
Γ Ι+ Ό F
(TV

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Ex- No/ Structure LC/MS: (M)+

Ex-23 0 1656.57
HN--

“S H k0„
( U / Uv A—NH
\_/NY 0 1

St
h/ \ / T /NH
\ HN '0 / 1
X=o o
C /° A J

τ r
HN-- OH
θζΤ]
HO OH

Ex-24 TFA 1
salt 1810.06

NEC nN (half mass

848.64)

> Ey^NyE- po
Co i
! 5

N
h\ ov-n« 0 "Χνί
*
AS
A

ff

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Ex- No/ Structure LC/MS: (Μ)+

Ex-25 TFA
salt 1550.6
s

Π>Η3λθ κ

ο 1 ο /=\ C θ
ΑΛ ΧΧ^,ζ)
Ο=ζ ΝΗ

\_1 Λ_Ζ °
Ν+- ^-<-F
\ ° F
Ex-25 Cl
Salt
Ο 1550.6

\_/ / \>.ΟΗ
/ °\ Τ 7=\ /

Ο Ν VnU ""'C;
Ος^-Ν V—ΝΗ Οχ ο γ=Ο
1 —ΖΛΝΗ F—ΧΧ-Ν^
r>0= \ / 1
Η
> \ Η Χ=/ 1__ k Ξ
J / Ν\ ° ’
/Ν\ cr 1

||
0
Ex-26 0 1599.19

| 0 ΝΗ
ν^Ν^">|
1 Η = ι
°%^ΝΗ Βη 1
η ζηΝχ Λχ F V ')—Ν. 1 II 1
w \ΝΗ \_/ 1 [1
\ ^Χχ fl 0
1 [ ^° 0 1 1 7
1 II 1111...r\z
Ο^Ν 1
nSz-oh Hr/ °
η°Λ\^οη LJLJ
ΟΗ

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Ex- No/ Structure LC/MS: (M)+

Ex-27
OH 1628.74
hoXXoh
0 OH
HN Ο Y X. Π H

n ' -----V \
ΟγΝΗ Η XNH η X
/X H J
*^NH F
\ VX
Γ nM/HNKroH
\_ X
\X? X

>o r J/ U/
ζ \ Ο=ς
olurTP

Ex-28 TFA
0 1568.63
salt

\=/ / \>0H

wfη(9 χην ( / ’™χχ

ύ*οχΓΎ'°

C° nh _X^ h 73

0} / rC°‘

^Ntx zFr 'V \

zl (/vF )<N—

0
Ex-29 TFA o
salt 1566.60
<τν^ΝΛ
\=/ / X
* 0H
( H 2 HXl η/ν^ολΑ>ο/
H v

/N x / V χΖ~ΝΆ
\ ο NH XXii HN^/X'
0—. / 1 \ / \ J qt -
) 0=\ / 0

X X ( \J
N+~ 0
/] Ό /,p W
>—<-T 0
0 F

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Ex- No/ Structure LC/MS: (M)+

Ex-31 Cl 0
salt 1552.85
. /xX-w-

°v\ k A Ν"\>

χί·

0
Ex-35 Cl
0 1533.40
salt

/ „ <nh aX h IT
Λ;·. >

0
Ex-36 Cl
0 1520.60
salt

h °v
Up
/-NH
- Τύ
Ok 0
Λ,
hI-Ca
VO

/i ·<κ

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 WO 2019/246349 PCT/US2019/038155

Ex- No/ Structure LC/MS: (M)+

Ex-38 TFA
0 1536.21
salt

T 0 NH
η T N^''Z/i
1
U H 1 θ\
O^N XNH /—/S °Hum
Y χ"··< f-ZAJ /Υί
> nh ^=/ > l L JL /
J J
\ J HN 0 J ...
N+ JJ [ jL
\ O’
Ffort ° Η
A
/Π
i_ikJ < 0
f o

Ex-39 TFA
salt 0 1551.21
HN—
\_ / / / \>DH
/ °\ T /=\ /

1 t! aa hn T^°/---Xr#0
Ηχ VNU < Nn HN γΓ

1 / ' /NH
0== Λ- < / V-ON'"\

r / N\ \ ην^ξ
; \ —°L\ )
n+ p n. \ __ / \ /
7 \ \ P
F /—4 0 11—NH
r~\'—//
F 0 0

Ex-40 Cl
Ρ 1538.60
salt
npN Λ
\—/
/ °\
\>»
/T
oh
/=\ /
( ti aH/2 ° hn<HN 0^4T#<°
y'V

\ ' N\ °Cx ) ° '

X Cr
0 Ibx N-7/
0
H

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Ex- No/ Structure LC/MS: (M)+

Ex-41 Cl
salt 0 1566.40

\_ / / /
/ °\ T /=\ /

fafafafa
1 HN'γΛ
Ufa# 0

( <x f<5x hJ o
\ ' N\ J /=<0 °

^N+ d /v N v v

Ex-44 TFA
salt 0 1549.49

\—/
H/7N~\/ * 0H
\>
/ °\ i /=\ /

Bs
( t! a HN
> AΎ rX
~"-C„
Γ0

T < .V)

\ / S 0 =

On+ F. 0' X. \ A /)

F ° OX
0
Ex-47 TFA
0 1534.27
salt

]
Ο XX NH
_ Y^N^'^iMe^J·--^0
Η °A | Η 1 : \
O.x^-N
γ '■■··< Mfyl XnH
/A OH HN1 YYY

i
fa 0=(
nh x/ >
fa
I II JL
Ο^ΝΛ 0Me

\ J1 HNf 0 J e 11··!^
Me Γ"7/
XN+ F \ J | jL
X°- 0 X HN

0 fafafafa

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Ex- No/ Structure LC/MS: (M)+

Ex-48 TFA
0 1582.20
salt

T 0 V" NH
0 |^N

°<^N VNH °HHN ,

J x'"< f -ZXJ hnY%A
f NH \=/ > I [I
°=^.... J

CT HN 0 J ""'K/
/ F. θ' V__ U I L
-£ 7 vJj 0

Ex-49 TFA
0 1551.21
salt

γ 0 V" yS NH
Me j-,,^θ

VnH %V° 1
Y XY f YU hnYA
> NH X/ \ 1 [1 Ί
J °K 7
\ / HN?.. _ o> Mel"'P

/N\ F\ °' J- \ A 1
P-H. oZ X Ηϊ
f ° H [1 1

Ex-50
ACOH salt 1596.3
O^O \ \ 0
A < λ λ HN—\ — H3C^OH
(H3C)3N+ 1 \_/-J / -Y

V-/ 1 H 9 M / HN^O-^
/ 0 /YNUkNXJJ ]
HNK Vnh o *x '-J % J
Hm·,,../ CF N^\
\ F-Ay-N HN__ /

V
o^A^^nh ?N k
\ 'A
0 ^h3
Η3θ
o // X/"^
0

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 WO 2019/246349 PCT/US2019/038155

Ex- No/ Structure LC/MS: (M)+

Ex-51
ACOH salt 0 1392.0
HN—
\_ / ' / h3cx^oh
n / °< I H3C
( h ΗΝ<ν°,ΓΥθ

+h3n 'Illi../Vnh 0 \ 'o HN |

NH |TVN-) /X
An
H
°=<h
η’Γν Γ
s
//

\^N V
0
Ex-52
0 1593.8
ACOH salt
/T/J''__0 H’cyoH

h /
y
* 0 Αλ
Ois/ \iu'\ F—/ /Ν--Ί N\/V^
\ NH \=/ l== / CH3
1 °/ / /=^\ 0
0—1 4---- NH \ 4 /)
*-o h c 3 1 N/ V-7
1—1 0 -------
O^O N+(CH3)3 5

Ex-53 Cl 0
salt 1381.33

1 H X η·^-^^νη
cr Λ 1
+h3n^ ■<
Ηύ
0 γ
°H J,
ύ
Jl F \ / N> III Ί
tjh\ 4
N---- \

0
ZZ~—A
V h
/
°x
'’""""-L /
/ V^\ 7^0
0 —

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Ex- No/ Structure LC/MS: (M)+

Ex-54 Cl 0
salt 1383.44
Ο25 2 θ

cr γΧγΡή JNH

V°
HNX FON>
°H hiTO
L l
\ > CT N—\ U
/ [ /
0 < Η 0. P
r\ / F—\/o
*
° —. z

Ex-55
0 1583.69
ACOH salt
2 o

Arj η 1
0 -γΥΐΊΓH iA A

ς
o
X
\
f<Jy >
PCl
\
\
A—-,
0 'v H
/
°\
'"""“XL"//
0 —ya
Ex-56 Cl . _ o
salt 'V cnX>K 1628.95

> T° Y NH

°ΥΊ τΓΊ X‘r°

τ H
I_
Y
n. VF hλ /-+M
NH )=/
>-n. hhi^ >
ill
1
i /
0 po o >°<D °

o XAs HN^°
VO

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Ex- No/ Structure LC/MS: (M)+

Ex-57 Cl ""Ό 0
salt i Cl JL o 1608.95
S CO

L / cubist1 \
NV T 0 A A NH

k O^NH H 1 0H 1
kJ, AF \ UN\ lY 1

0 P° 0 Va) 0

o hn^°
uu

Ex-58 Cl . o
salt 1594.93
°1 z
L / r._ 0. ,N I X
NfoCI T 0 V" A NH
γ^Ν^·'''ι X/L

k O^NH "A 0H 1

Un. Xf~~\ Un. Ill Ί

° a° ° S°Jd 0
hn^A^n^1 A
O HN^°
uo

Ex-59 Cl , _ o
salt ζ” UA« 1593.7

O O^NH U/L 0HHf

I Η I f__A~Y / HN^^^
v 1 II 1
y yh uz > u
° Ao ο > <γτ
*

0 U^- hn^°

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Ex- No/ Structure LC/MS: (M)+

Ex-60 Cl 0
N^ n 1607.4
salt _'x'N.
m+ Cl
c LU
H
CK
0 NH
Z/ T°
H
NH As OH
0WH
V F— V-N. T
NH
kJ
0 OW-"\
X) 0 //,,, 1 >
HN, N^^V HN^0
O
•0
Ex-61 Cl iIVcr 0
salt 1592.4
n^ P
IL u°
[Iil P
O ’ NH

J O. ,NH H
'Xi °hhn
I H i F—· Nx T 'Ί
"NH \--- / [i p
'Np °Z
0 X 0
L cr
HN N"~"
Ά
0 X HNZ

ACTIVITY DETERMINATION
Selected compounds of the invention were subjected to one or more of the following
procedures to assay their activity for antagonism of PCSK9 activity.
The following is a description of the assays used to determine activity of compounds
5 of the invention, and any comparator compounds reported, toward PCSK9 antagonism.
Biotinylated PCSK9 was obtained commercially.
LDLR TR-FRET

The PCSK9 TR-FRET assay measures the interaction between PCSK9 and LDLR. A
solution containing 40 nM biotinylated PCSK9 + 10 nM Lance ULight Streptavidin is made in
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50 mM HEPES pH 7.4, 0.15 M NaCI, 5 mM CaCI2, 0.01% BSA, and 0.01% Surfactant P20.
A separate solution containing 40 nM rhLDLR-6xHis + 10 nM Eu-W1024 anti-6xHis is made
in the same buffer system. An Echo is used to transfer 0.750ul of compound to an assay
plate followed by the addition of 15uI of PCSK9+Ulight and 15uI of LDLR+Eu. The final assay
5 volume is 30.750ul containing 20nM PCSK9, 5nM lllight, 20nM LDLR, and 5nM Eu. The
reaction is incubated at room temperature for at least two hours prior to fluorescence
measurements using an Envision Multilabel Reader. IC50 values are determined by fitting
data to a sigmoidal dose-response curve using nonlinear regression. Counts (B-counts) of
the europium-labeled LDLR are followed to observe if compounds are adversely affecting
10 LDLR. A fall off of the B-counts is likely indicates a false positive of inhibition.
Alexa FRET Standard TR-FRET

The PCSK9 Alexa FRET Standard assay measures the interaction between PCSK9
and an AlexaFluor647 (AF) tagged cyclic peptide, Reagent A (Kd = 83nM). A solution
containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) is
15 made in 50 mM HEPES pH 7.4, 0.15 M NaCI, 5 mM CaCI2, 0.01% BSA, and 0.01%
Surfactant P20. A separate solution containing 40 nM of the AlexaFluor tagged cyclic peptide
is made in the same buffer system. An Echo is used to transfer 0.750ul of compound to an
assay plate followed by the addition of 15ul of PCSK9+Stept-Eu and 15ul of AF peptide. The
final assay volume is 30.750ul containing 0.5nM PCSK9, 1.25nM Strep-Eu, and 20nM AF
20 cyclic peptide. The reaction is incubated at room temperature for at least two hours prior to
fluorescence measurements using an Envision Multilabel Reader. IC50 values are
determined by fitting data to a sigmoidal dose-response curve using nonlinear regression. Ki
is then calculated from the IC50 and the Kd of AF cyclic peptide. Counts (B-counts) of the
europium-labeled PCSK9 are followed to observe if compounds are adversely PCSK9. A fall

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off of the B-counts likely indicates a false positive of inhibition. Data from this procedure is
reported as “A = ‘numerical value’ (nanomolar)”

Reagent A was prepared in accordance with the following method:

Step A - Synthesis of Intermediate Compound Int-A

The peptide was synthesized on a 0.250 mmol scale on CEM Liberty Blue, Microwave
synthesizer using Fmoc/tBu chemistry on PS Rink-Amide MBHA resin, 0.32 mmol g-1. The
assembly was performed using single-couplings using 4eq of Fmoc protected amino acid
10 0.2M in DMF, 4eq of 0.5M HATU in DMF, 4eq of 2M DIPEA (double coupling for Tyr). Fmoc
deprotection cycles were performed using 20% (V/V) piperidine in DMF.
The sequence of Fmoc protected amino acids and building blocks used are:

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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
5 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-1 H-indol-3-yl)propanoic
acid
7. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1 H-indol-3-yl)propanoic
10 acid
8. (((9H-fluoren-9-yl)methoxy)carbonyl)glycine
9. N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine
10.3-(tritylthio)propanoic acid
At the end of the assembly, the resin was washed with DMF, MeOH, DCM, Et20. The
15 peptide was cleaved from solid support using 50 ml of TFA solution (v/v) (91 % TFA, 5%
H2O, 4% TIPS) for approximately 1.5 hours, at room temperature. The resin was filtered,
washed with TFA and solution concentrated to dryness and lyophilized. Lyophilization
afforded Intermediate Compound Int. A (399mg), which was used as crude in the next step.
LCMS anal, calcd. C61H75F2N15O13S2: 1328.48, found: 1328.2 (M+1)+
20 Step B - Synthesis of Intermediate Compound Int-B: as described for reagent B
Purified by RP-HPLC (Waters Deltapak C4, double cartridge, 40x100 mm, 15um, 300A; 15%
to 35% ACN/water + 0.1% TFA modifier over 20 min). Collected fractions lyophilized to afford
35mg of Intermediate Compound Int-B. LCMS anal, calcd. for C69H81F2N15O13S2:
1430.62; found: 1430.9 (M+1)+
25 Step C - Synthesis of Compound ReagentA: as described for reagent B

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LCMS anal, calcd. for C105H122F2N17O26S63-: 2268.58; 1135.8 (M+2)2+

Alexa FRET Plus TR-FRET

The PCSK9 Alexa FRET Plus assay measures the interaction between PCSK9 and
an AlexaFluor647 (AF) tagged cyclic peptide, Reagent B (Kd = 35nM). A solution
5 containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) is
made in 50 mM HEPES pH 7.4, 0.15 M NaCI, 5 mM CaCI2, 0.01% BSA, and 0.01%
Surfactant P20. A separate solution containing 1920 nM of the AlexaFluor tagged cyclic
peptide is made in the same buffer system. An Echo is used to transfer 0.075ul of
compound plus 0.675ul of DMSO to each well of an assay plate followed by the addition of
10 15uI of PCSK9+Stept-Eu and 15uI of AF peptide. The final assay volume is 30.750ul
containing 0.5nM PCSK9, 1.25nM Strep-Eu, and 960nM AF cyclic peptide. The reaction is
incubated at room temperature for at least two hours prior to fluorescence measurements
using an Envision Multilabel Reader. IC50 values are determined by fitting data to a
sigmoidal dose-response curve using nonlinear regression. Ki is then calculated from the
15 IC50 and the Kd of AF cyclic peptide. Counts (B-counts) of the europium-labeled PCSK9
are followed to observe if compounds are adversely affecting PCSK9. A fall off of the B-
counts is likely indicates a false positive of inhibition. Data from this procedure is reported
as “P= ‘numerical value’ (nanomolar)”

Reagent B was prepared by the following procedure.

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Reagent B

Step A - Synthesis of Intermediate Compound Int-A

The peptide was synthesized on a 0.250 mmol scale on CEM Liberty Blue, Microwave
5 synthesizer using Fmoc/fBu chemistry on PS Rink-Amide MBHA resin, 0.32 mmol g·1. The
assembly was performed using single-couplings using 4eq of Fmoc protected amino acid
0.2M in DMF, 4eq of 1M Oxyme in DMF, 4eq of 0.5M Λ/,ΛΖ-di isopropylcarbodi imide (DIC)
(double coupling for Y01). Fmoc deprotection cycles were performed using 20% (V/V)
piperidine in DMF.
10 The sequence of Fmoc protected amino acids and building blocks used are:
1. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-tntyl-L-cysteine
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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)propanoic
acid
5 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-1 H-indol-3-
yl)propanoic acid
7. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1 H-indol-3-
10 yl)propanoic acid
8. (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanine
9. N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine
10.3-(tritylthio)propanoic acid
At the end of the assembly, the resin was washed with DMF, MeOH, DCM, Et20. The
15 peptide was cleaved from solid support using 50 ml of TFA solution (v/v) (91 % TFA, 5%
H2O, 4% TIPS) for approximately 1.5 hours, at room temperature. The resin was filtered,
washed with TFA and solution concentrated to dryness and lyophilized. Lyophilization
afforded Intermediate Compound Int. A (300mg), which was used as crude in the next step.
LCMS anal, calcd. C63H79F2N15O13S2: 1356.53, found: 1356.9 (M+1)+
20 Step B - Synthesis of Intermediate Compound Int-B

Crude Int-A (0.22 mmol) was redissolved in 24ml of DMF. 6ml of 1M aqueous solution of
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) were added dropwise. Reaction was left under
stirring at room temperature for 20 min, quenched with TFA (pH to 3-4) and then
25 concentrated in vacuo to provide crude Int-B, which was purified by RP-HPLC (Waters

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XBridge, C18, 50x150 mm, 5μιτι, 130A; 25% to 40% ACN/water + 0.1% TFA modifier over
20 min). Collected fractions were lyophilized to afford 35mg of Intermediate Compound Int-
B. LCMS anal, calcd. for C71H85F2N15O13S2: 1458.67; found: 1458.8 (M+1)+
Step C - Synthesis of Compound Reagent B

5 Intermediate Compound Int-B (15mg) was dissolved in 0.2ml of dry DMSO. Then 15mg of
ALEXAFLUOR 647NHS Ester (A37566, Life technology) dissolved in 1,5ml of dry DMSO
were added. 20uL of dry DIPEA were added. Reaction was left under stirring at room
temperature for 12h under Nitrogen atmosphere in the dark. Quenched with TFA (pH to 3-
4) and purified by RP-HPLC (Dr Maish, Reprosil Gold C18, 250x20 mm, 120 A, 10pm; 20%
10 to 35% of 0.1% TFA in ACN/0.1% TFA in H2O, over 20min, then 35% to 40% over 5min at
20 mL/min flow rate). Collected fractions were lyophilized to afford 16.1 mg of Compound
Reagent B. LCMS anal, for C107H126F2N17O26S63-:2296.64; found: 1150.6 (M+2)2+
Activity data obtained by one or both of the above-described procedures is reported
for selected example compounds of the invention in the following format:
15 Example No.: A (standard TR Fret) = ‘numerical value’; P (Alexa Fret plus standard TR

Fret) = ‘numerical value’ /, note that all values reported are nanomolar.
Alexa FRET Ultra TR-FRET

The PCSK9 Alexa FRET Ultra assay measures the interaction between PCSK9 and
an AlexaFluor647 (AF) tagged cyclic peptide, Reagent B (Kd = 0.99nM). A solution
20 containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) is
made in 50 mM HEPES pH 7.4, 0.15 M NaCI, 5 mM CaCI2, 0.01% BSA, and 0.01%
Surfactant P20. A separate solution containing 1920 nM of the AlexaFluor tagged cyclic
peptide is made in the same buffer system. An Echo is used to transfer 0.015 ul of
compound plus 0.735 ul of DMSO to each well of an assay plate followed by the addition of
25 15 ul of PCSK9+Stept-Eu and 15 ul of AF peptide. The final assay volume is 30.750 ul
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containing 0.5 nM PCSK9, 1.25 nM Strep-Eu, and 960 nM AF cyclic peptide. The reaction
is incubated at room temperature for at least two hours prior to fluorescence measurements
using an Envision Multilabel Reader. IC50 values are determined by fitting data to a
sigmoidal dose-response curve using nonlinear regression. Ki is then calculated from the
5 IC50 and the Kd of AF cyclic peptide. Counts (B-counts) of the europium-labeled PCSK9
are followed to observe if compounds are adversely affecting PCSK9. A fall off of the B-
counts is likely indicates a false positive of inhibition. Data from this procedure is reported
as “Ki Ultra = ‘numerical value’ (data reported is nanomolar)”

The following compounds were assessed, as shown in Table 2, using the protocol
10 described above with the results shown:

Ex-01 Ki Plus = < 0.00558, Ki Ultra = 0.0046 I 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 =
15 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
20 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 Ultra = 0.01519/ Ex-23 Ki Plus = 0.00938, Ki Ultra =
25 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/
30 Ex-36 Ki Plus = 0.00762, Ki Ultra = 0.00507/ Ex-38 Ki Plus = 0.00904, Ki
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 WO 2019/246349 PCT/US2019/038155

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
5 = 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 = 0.00359/
Ex-53 Ki Standard <1.26, Ki Plus = 0.09629, Ki Ultra = 0.21500/ Ex-54 Ki
Standard <1.26, Ki Plus = 0.36720, Ki Ultra = 0.48390/ Ex-55 Ki Standard
10 <1.26, Ki Plus = 0.07240, Ki Ultra = 0.23800/ Ex-56 Ki Standard <1.257, Ki
Plus = 0.02237, Ki Ultra = 0.00481/ Ex-57 Ki Standard <1.257, Ki Plus <
0.00558, Ki Ultra = 0.00162/ Ex-58 Ki Standard <1.257, Ki Plus = 0.00773,
Ki Ultra = 0.00196/ Ex-59 Ki Plus = 0.00788, Ki Ultra = 0.004959/ Ex-60 Ki
Plus = 0.006515, Ki Ultra = 0.005312 I Ex-61 Ki Plus = 0.00747, Ki Ultra =
15 0.006543.

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What is Claimed is:

1) A compound of Formula I:

Formula I,
wherein:
X is H F, Cl or Br;
R1 is selected from:
(a) -H; or
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
(i) -H;
(ii) -NH2;
(iii) -N+H3;
(iv) -N+(H3C)3;
(v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(vi) -NH-C(O)-[(CH2)yi2-O-]2-(CH2)yi3R14B wherein:
yi2 and yi3 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;

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(vii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C

is -O-(CH2)3-4-N+(CH3)3; and
(viii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
(ai) -O-(CH2)2-N+(CH3)3;
(aii) -N+(CH3)3; or
(aiii) a moiety of the formula:

R2 is selected from:
(a) -H; and
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is selected from:
(i) -H;
(ii) -NH2;
(iii) -N+H3;
(iv) -N+(H3C)3;
(v) -NH-C(O)-[(CH2)2-O-]2-(CH2)2R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; ογ-Ν+(ΟΗ3)3;

(vi) -NH-C(O)-[(CH2)yi2-O-]2-(CH2)yi3R14B wherein:

yi2 and yi3 are not both 2 and are independently 2 to 4; and
R14B is: -NH2; -N+H3; -N(CH3)2; ογ-Ν+(ΟΗ3)3;
(vii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C
is -O-(CH2)3-4-N+(CH3)3; and
(viii) -NH-C(O)-(CH2)yR14C, wherein, y= 1 to 6 and R14C is:
(ai) -O-(CH2)2-N+(CH3)3;

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-N+(CH3)2R14ca, wherein R14ca is -CH3 or-(CH2)i-4-OCH3;

a moiety of the formula:

; or
a moiety of the formula:
o
o. .n+(CH3)3
yl4Cb 14Cc

where R14Cb and R14Ccare 1 to 4; or

R1 and R2 may be bonded together to form a moiety of the formula:

h2c /CH2
N G1----- N
I I
oG1b .
RG1a K , wherein:
G1 RGia anc| RGw are defjnec| as follows:
G1 is a linker moiety of the formula:

wherein nq1 is 1 to 6, mq1 is 0, 1 or 2 and together the value of nq1 and mq1 are
selected such that the length of the linker moiety they define does not exceed a total
of 8 carbon and/or oxygen atoms comprising the chain including the carbon atom in
the chain that forms the carbonyl moiety;
RG1a is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms; and
RG1b is selected from:
(i) a moiety of the formula:

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(ii) a moiety of the formula:

(b) G1 is a linker moiety of the formula:

wherein nq2 is 0, 1 or 2, mq2 is 1 to 6, and together the value of nq2 and mq2
are selected such that the length of the linker moiety they define does not exceed a
total of 8 carbon and/or oxygen atoms comprising the chain including the carbon
atom in the chain that forms the carbonyl moiety;
RG1a is selected from:
(i) a moiety of the formula:

(ii) a moiety of the formula:

' '1-0 ; and

RG1b is selected from: (i) -H; and (ii) alkyl of up to 4 carbon atoms;
R8 is -CHs or a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

wherein R8a is -H, or a linear, branched or cyclic alkyl of up to four carbon

atoms;
A is selected from:
(a) a moiety of the formula:

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

(c) a moiety of the formula:

wherein Ab1 is:
(i) a moiety of the formula:
i-CH2X-£

' ' x

wherein x is 1 to 6; or
(ii) a moiety of the formula:

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

B is:

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a bond;
(b) —(CH2)i-2- ; or
(c) a moiety of the formula:

D is:
a moiety of the Formula:

wherein E is -CH2- or-(CH2)2-4-O-, and A and B are as defined above;

(b) a moiety of the formula:

B
N
H

wherein A and B are as defined above;

(c) a moiety of the formula:

wherein na is 1,2, or 3, ma is 2, 3, or 4, and na + ma is > 3, and wherein A and

B are as defined above;
(d) a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

wherein, R34b is -H or a liner, branched or cyclic alkyl of up to four carbon

atoms, and A and B are as defined above,
or a pharmaceutically acceptable salt of any thereof.

2. A compound according to Claim 1 of Formula I, wherein X is F, or a

pharmaceutically acceptable salt of any thereof.

3. A compound of Claim 2 wherein D is a moiety of the formula:

wherein, E is -CH2- or-(CH2)2-O-, or a pharmaceutically acceptable salt

thereof.

4. A compound of Claim 2 wherein D is a moiety of the formula:

wherein, E is -CH2- or-(CH2)2-O-, or a pharmaceutically acceptable salt

thereof.

5. A compound of Claim 2 wherein D is a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

or a pharmaceutically acceptable salt thereof.

6. A compound of Claim 2 wherein D is a moiety of the formula:

or a pharmaceutically acceptable salt thereof.

7. A compound of Claim 2 wherein D is a moiety of the formula:

or a pharmaceutically acceptable salt thereof.

8. A compound of Claim 2 wherein D is a moiety of the formula:

O A

or a pharmaceutically acceptable salt thereof.

9. A compound of Claim 2 wherein D is a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

or a pharmaceutically acceptable salt thereof.

10. A compound of Claim 2 wherein D is a moiety of the formula:

or a pharmaceutically acceptable salt thereof.

11. A compound of Claim 2 wherein R1 and R2 are joined together with a moiety
of the formula:

such that together with the cyclopeptide to which R1 and R2 are attached they
form acyclic structure,
or a pharmaceutically acceptable salt thereof.

12. A compound of Claim 2 having the structure of Formula IIB, or a

pharmaceutically acceptable salt thereof:

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 WO 2019/246349 PCT/US2019/038155

Formula IIB,
B1 is -((CH2)o-2)-; and
D1 is:
(a) a moiety of the formula:

(c) a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

13. A compound of Claim 2 having the structure of Formula IIC, or a

pharmaceutically acceptable salt thereof:

Formula IIC,

wherein
D2 is:

(a) a moiety of the formula:

(b) a moiety of the formula:

(c) a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

(d) a moiety of the formula:

14. A Compound of Claim 2 having the structure of Formula HD, or a

pharmaceutically acceptable salt thereof:

15. A compound of Claim 2 having the structure of Formula HE, or a

pharmaceutically acceptable salt thereof:

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 WO 2019/246349 PCT/US2019/038155

Formula HE.

16. A compound of Claim 2 having the structure of Formula HF, or a

pharmaceutically acceptable salt thereof:

Formula HF.
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 WO 2019/246349 PCT/US2019/038155

17. A compound of any of Claims 1 to 16, or a pharmaceutically acceptable salt

thereof, wherein A is:
(a) -(CH2)6 ;
(b) a moiety of the formula:

wherein x is 1 to 3; or
(c) a moiety of the formula:

18. A compound of any of Claims 1 to 17, or a pharmaceutically acceptable salt

thereof, wherein R2 is:
(a) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
(a) -H;
(b) -CH3;
(c) -NH2;
(d) -N+H3;
(e) -N+(H3C)3;
(f) -NH-C(O)-[(CH2)2-4-O-]2-4-(CH2)2-4R14B wherein R14B
is: -NH2; -N+H3; -N(CH3)2; or-N+(CH3)3;
(g) -NH-C(O)-[(CH2)yR14C, wherein, y= 1 to 6 and R14C is:

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 WO 2019/246349 PCT/US2019/038155

(ai) -O-(CH2)2-4-N+(CH3)3;
(aii) -N+(CH3)3; or
(aiii) a moiety of the formula:

; or
(b) a moiety of the formula

(H3C)3N

19. A compound of any of Claims 1 to 18, or a pharmaceutically acceptable salt

thereof, wherein R1 is selected from:
(a) -H;
(b) -(CH2)z-R14A, wherein: z is 1-6, and R14A is:
(i) -H;
(ii) -N+Hs; or
(iii) -NH-C(O)-[(CH2)2-O-]2-(CH2)2-N+(CH3)3.

20. A compound of any of Claims 1 to 19, or a pharmaceutically acceptable salt

thereof, wherein R8 is a moiety of the formula:

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 WO 2019/246349 PCT/US2019/038155

wherein R8b is -H, -CH3, or-C(CH3)3.

21 A compound of claim 1, which is selected from the group consisting of:

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174
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175
WO 2019/246349 PCT/US2019/038155

176
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or any pharmaceutically acceptable salt form thereof.

22. A composition comprising at least one compound of any of claims 1 to 21, or

a pharmaceutically acceptable salt thereof orfreebase 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 a composition of
Claim 22.

24. A method of treating hypercholesterolemia, comprising administering to a

patient in need thereof a therapeutically effective amount of a compound of
any of Claims 1 to 21.

25. A compound according to any of Claims 1 to 21, or a pharmaceutically

acceptable salt thereof, for use in therapy.

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 WO 2019/246349 PCT/US2019/038155

26. A compound according to any of Claims 1 to 21, or a pharmaceutically

acceptable salt thereof, for treating hypercholesterolemia.

180
 INTERNATIONAL SEARCH REPORT
International application No

PCT/US2019/038155
A. CLASSIFICATION OF SUBJECT MATTER
INV. C07K7/06 A61K38/00 C12N9/64
ADD.

According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
C07K A61K C12N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

EPO-Internal, BIOSIS, EMBASE, WPI Data

C. DOCUMENTS CONSIDERED TO BE RELEVANT

*
Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

A NI-YA HE ET AL: "Lowering serum lipids 1-26

via PCSK9-targeting drugs: current
advances and future perspectives",
ACTA PHARMACOLOGICA SINICA,
vol. 38, no. 3,
23 January 2017 (2017-01-23), pages
301-311, XP055484458,
GB
ISSN: 1671-4083, DOI: 10.1038/aps.2016.134
the whole document

A RAHUL CHAUDHARY ET AL: "PCSK9 inhibitors: 1-26

A new era of lipid lowering therapy",
WORLD JOURNAL OF CARDIOLOGY,
vol. 9, no. 2, 1 January 2017 (2017-01-01)
, page 76, XP055422913,
ISSN: 1949-8462, DOI: 10.4330/wjc.v9.Ϊ2.76
the whole document

Further documents are listed in the continuation of Box C. See patent family annex.

* Special categories of cited documents :

"T" later document published after the international filing date or priority
date and not in conflict with the application but cited to understand
"A" document defining the general state of the art which is not considered the principle or theory underlying the invention
to be of particular relevance
Έ" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be
filing date considered novel or cannot be considered to involve an inventive
"L" document which may throw doubts on priority claim(s) orwhich is step when the document is taken alone
cited to establish the publication date of another citation or other Ύ" document of particular relevance; the claimed invention cannot be
special reason (as specified) considered to involve an inventive step when the document is
Ό" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination
means being obvious to a person skilled in the art
"P" document published prior to the international filing date but later than
the priority date claimed document member of the same patent family
Date of the actual completion of the international search Date of mailing of the international search report

21 October 2019 05/11/2019

Name and mailing address of the ISA/ Authorized officer
European Patent Office, P.B. 5818 Patentlaan 2
NL-2280 HV Rijswijk
Tel. (+31-70) 340-2040,
Fax: (+31-70) 340-3016 Lopez Garcia, F
Form PCT/ISA/210 (second sheet) (April 2005)
 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

CORRECTED VERSION
(19) World Intellectual Property
Organization lllllllllllllllllllllllllllllllllllllllllll^
International Bureau (10) International Publication Number
(43) International Publication Date WO 2019/246349 A8
26 December 2019 (26.12.2019) WIPO I PCT

(51) International Patent Classification: NJ 07033 (US). TONG, Ling; c/o Merck Sharp & Dohme
C07K 7/06 (2006.01) C12N9/64 (2006.01) Corp, 770 Sumneytown Pike, West Point, PA 19486 (US).
A61K38/00 (2006.01) WALJI, Abbas, M.; c/o Merck Sharp & Dohme Corp,
770 Sumneytown Pike, WestPoint, PA 19486 (US). NAIR,
(21) International Application Number:
Anilkumar, G.; c/o Merck Sharp & Dohme Corp, 2000
PCT/US2019/038155
Galloping Hill Road, Kenilworth, NJ 07033 (US). DING,
(22) International Filing Date: Fa-Xiang; c/o Merck Sharp & Dohme Corp., 2000 Gal­
20 June 2019 (20.06.2019) loping Hill Road, Kenilworth, NJ 07033 (US). BIANCHI,
Elisabetta; c/o IRBM Science Park S.p.A., Via Pontina
(25) Filing Language: English
Km 30, 600-0040 Pomezia (IT). BRANCA, Danila; c/o
(26) Publication Language: English IRBM Science Park S.p.A., Via Pontina Km 30, 600-0040
Pomezia (IT). WU, Chengwei; c/o Merck Sharp & Dohme
(30) Priority Data: Corp., 770 SumneytownPike, WestPoint, PA 19486 (US).
62/687,913 21 June 2018 (21.06.2018) US XIONG, Yusheng; c/o Merck Sharp & Dohme Corp., 2000
(71) Applicant: MERCK SHARP & DOHME CORP. Galloping Hill Road, Kenilworth, NJ 07033 (US). HA,
[US/US]; 126 East Lincoln Avenue, Rahway, NJ Sookhee, Nicole; c/o Merck Sharp & Dohme Corp., 2000
07065-0907 (US). Galloping Hill Road, Kenilworth, NJ 07033 (US). LIU,
Jian; c/o Merck Sharp & Dohme Corp., 2000 Galloping
(72) Inventors: WOOD, Harold, B.; c/o Merck Sharp & Hill Road, Kenilworth, NJ 07033 (US). BOGA, Sobhana,
Dohme Corp, 2000 Galloping Hill Road, Kenilworth, NJ Babu; c/o Merck Sharp & Dohme Corp., 2000 Galloping
07033 (US). JOSIEN, Hubert, B.; c/o Merck Sharp & Hill Road, Kenilworth, NJ 07033 (US).
Dohme Corp, 2000 Galloping Hill Road, Kenilworth, NJ
07033 (US). TUCKER, Thomas, Joseph; c/o Merck Sharp (74) Agent: TRINQUE, Brian, C. et al.; Lathrop Gage LLP, 28
& Dohme Corp, 770 Sumneytown Pike, West Point, PA State Street, 7th Floor, Boston, MA 02109 (US).
19486 (US). KEREKES, Angela, Dawn; c/o Merck Sharp (81) Designated States (unless otherwise indicated, for every
& Dohme Corp., 2000 Galloping Hill Road, Kenilworth, kind of national protection available)'. AE, AG, AL, AM,

(54) Title: PCSK9 ANTAGONIST COMPOUNDS

2019/246349 as llllllllllllllllllllllllllllllllllllllll^

(I)

(57) Abstract: Disclosed are compounds of Formula I, or a salt thereof: where A, B, D, X, RI, R2 and R8 are as defined herein, which
compounds have properties for antagonizing PCSK9. Also described are pharmaceutical formulations comprising the compounds of
Formula I or their salts, and methods of treating cardiovascular disease and conditions related to PCSK9 activity, e.g. atherosclerosis,
hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, or related cardiovascular disease and
cardiometabolic conditions.
wo

[Continued on next page]

 WO 2019/246349 A8 llllllllllllllllllllllllllll^
AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,
KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
KM, ML, MR, NE, SN, TD, TG).

Published:
— with international search report (Art. 21(3))
(48) Date of publication of this corrected version:
04 February 2021 (04.02.2021)
(15) Information about Correction:
see Notice of 04 February 2021 (04.02.2021)