Phd inhibitor compounds, compositions, and methods of use

Abstract

Part of this invention provides novel small molecule PHD inhibitors having structures according to formula (A) and its derivatives: or pharmaceutically acceptable salts thereof. The compounds provided herein may be used to treat diseases including: heart diseases (e.g., ischemic heart disease, congestive heart failure, and valvular heart disease), lung diseases (e.g., acute lung injury, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease), liver diseases (e.g., acute liver failure, liver fibrosis, and cirrhosis), and kidney diseases (e.g., acute kidney injury and chronic kidney disease).

Description

[Previous Technology]

[0001] Hypoxia is a condition or state in which the oxygen supply is insufficient to maintain normal life functions, such as low arterial oxygen supply. Hypoxia can lead to functional and structural damage to cells. Activation of cellular defense mechanisms during hypoxia is mediated by HIF (hypoxia-inducible factor) proteins. In response to hypoxia, HIFα levels are increased in most cells due to decreased HIFα prolyl hydroxylation. HIFα prolyl hydroxylation is achieved by a family of proteins with different names, namely proteins containing prolyl hydroxylase domains (PHD1, 2, and 3) (also known as HIF prolyl hydroxylases (HPH-3, 2, and 1) or EGLN-2, 1, and 3). These PHD proteins act as oxygen sensors and regulate HIF stability in an oxygen-dependent manner. These three PHD isoforms act in different ways in regulating HIF and may have other non-HIF-related regulatory roles.

[0002] In fact, many studies have shown that stabilizing HIF can reduce tissue inflammation and promote its repair. Therefore, compounds that can inhibit the activity of PHD proteins may be particularly beneficial in new therapies (Lee et al. (2019) Exp. Mol. Med. 51:68).

[0003] This article describes novel small molecule PHD inhibitors that are effective in treating diseases including heart diseases (e.g., ischemic heart disease, congestive heart failure, and valvular heart disease), lung diseases (e.g., acute lung injury, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease), liver diseases (e.g., acute liver failure, liver fibrosis, and cirrhosis), and kidney diseases (e.g., acute kidney injury and chronic kidney disease). [Summary of the Invention]

[0004] The present invention particularly provides novel small molecule PHD inhibitors that are effective in treating diseases including (but not limited to) heart diseases (e.g., ischemic heart disease, congestive heart failure, and valvular heart disease), lung diseases (e.g., acute lung injury, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease), liver diseases (e.g., acute liver failure, liver fibrosis, and cirrhosis), and kidney diseases (e.g., acute kidney injury and chronic kidney disease).

[0005] In one embodiment, this document provides a compound having the structure according to formula (A), (A) or a pharmaceutically acceptable salt thereof, wherein: Ar1 is a phenyl or a six-membered nitrogen-containing heteroaryl group, wherein the phenyl or heteroaryl group is substituted with a halogen group, CN, OH, a C1-3 alkyl group substituted with one or more halogen groups, or a C1-3 alkoxy group; R2 is H or a C1-3 alkyl group; Ar2 is a six-membered nitrogen-containing heteroaryl group substituted with a halogen group, OH, amine or C1-3 alkyl group, wherein R4 is hydrogen or a C1-4 alkyl group; and formula (A) excludes the following compounds: ...

[0006] In the embodiment, R2 is H.

[0007] In the examples, R2 is a C1-3 alkyl group.

[0008] In the embodiments, Ar1 is, wherein X, Y and Z are independently CH or N, wherein N is oxidized as appropriate; each R1 system is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl, and C1-3 alkoxy, which are substituted with one or more halogen groups as appropriate; and m is 1, 2, 3 or 4.

[0009] In the examples, Ar1 is a substituted phenyl group. In the examples, Ar1 is substituted with at least one R1, wherein R1 is a CN or a halide group.

[0010] In the embodiments, Ar1 is substituted with one or two R1 groups, which are independently selected from C1-3 alkyl, halogen, CN or OH groups substituted with one or more halogen groups as appropriate.

[0011] In the embodiments, Ar1 is a pyridinyl N-oxide or, where appropriate, a pyridinyl group substituted with at least one R1, wherein R1 is a C1-3 alkoxy or a halogen.

[0012] In the embodiments, each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl groups substituted with one or more halogen groups as appropriate, and C1-3 alkoxy groups.

[0013] In the embodiments, Ar2 is, wherein A and B are independently CH or N, wherein N is oxidized as appropriate; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl; and n is 0, 1 or 2.

[0014] In the embodiments, Ar2 is a pyridyl or pyrazinyl group, and said group is unsubstituted or contains a halogen, C1-3 alkyl or OH substituent.

[0015] In the embodiments, each R3 group is independently selected from the group consisting of hydrogen, halogen, OH, amine and C1-3 alkyl.

[0016] In the examples, R4 is H. In the examples, R4 is a C1-4 alkyl group.

[0017] In the embodiments, the compound of formula (A) is not,,,,,,,, or.

[0018] In the embodiments, the compounds of formula (A) exclude the following compounds,,,,,,, and.

[0019] In the embodiments, the compound of formula (A) has the following structure, (I), or a pharmaceutically acceptable salt thereof.

[0020] In the embodiments, X, Y, Z, A and B are independently CH or N, wherein N is oxidized as appropriate; m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0021] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (Ia), or a pharmaceutically acceptable salt thereof.

[0022] In the embodiments, X, Y and Z are independently CH or N, wherein N is oxidized as appropriate; m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0023] In the embodiments, the compound of formula (I) is not,,,,,,,, or.

[0024] In the embodiments, the compounds of formula (I) exclude the following compounds,,,,、、、 and.

[0025] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (Ib), or a pharmaceutically acceptable salt thereof.

[0026] In the embodiments, A and B are independently CH or N, wherein N is oxidized as appropriate; m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0027] In the embodiments, the compound of formula (Ib) is not,,,,,,,, or.

[0028] In the embodiments, the compounds of formula (Ib) exclude the following compounds,,,,,,, and.

[0029] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (Ic), or a pharmaceutically acceptable salt thereof.

[0030] In the embodiments, X, Y, Z, A and B are independently CH or N, wherein N is oxidized as appropriate; m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0031] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (II), or a pharmaceutically acceptable salt thereof.

[0032] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0033] In the embodiments, the compound of formula (II) is not,,,,,,,, or.

[0034] In the embodiments, the compounds of formula (II) exclude the following compounds,,,,,,, and.

[0035] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IIa), or a pharmaceutically acceptable salt thereof.

[0036] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; and each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group.

[0037] In the embodiments, the compound of formula (IIb) is not,,,,,,,, or.

[0038] In the embodiments, compounds of formula (IIa) exclude the following compounds,,,,,,,, and.

[0039] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IIb), or a pharmaceutically acceptable salt thereof.

[0040] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0041] In the embodiments, the compound of formula (IIb) is not , , , , , or.

[0042] In the embodiments, the compounds of formula (IIb) exclude the following compounds: , , , , , or.

[0043] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IIc), or a pharmaceutically acceptable salt thereof.

[0044] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; and R2 is hydrogen or C1-3 alkyl group.

[0045] In the embodiments, the compound of formula (IIc) is not , , , , , or.

[0046] In the embodiments, the compounds of formula (IIc) exclude the following compounds: , , , , , or.

[0047] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IId), or a pharmaceutically acceptable salt thereof.

[0048] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; and each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group.

[0049] In the embodiments, the compound of formula (IId) is not,,,,,,,, or.

[0050] In the embodiments, the compounds of formula (IId) exclude the following compounds,,,,,,, and.

[0051] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IIe), or a pharmaceutically acceptable salt thereof.

[0052] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0053] In the embodiments, the compound of formula (IIe) is not , , , , , or.

[0054] In the embodiments, the compounds of formula (IIe) exclude the following compounds: , , , , , or.

[0055] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IIf), or a pharmaceutically acceptable salt thereof.

[0056] In the embodiments, m is 1, 2, 3 or 4; and each R1 group is independently selected from the group consisting of hydrogen, halogen, CN, OH, C1-3 alkyl groups substituted with one or more halogen groups as appropriate, and C1-3 alkoxy groups.

[0057] In the embodiments, the compound of formula (IIf) is not , , , , , or.

[0058] In the embodiments, the compound of formula (IIf) excludes the following compounds: , , , , , or.

[0059] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (IIg), or a pharmaceutically acceptable salt thereof.

[0060] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R4 is hydrogen or C1-4 alkyl group.

[0061] In the embodiments, the compound of formula (A), formula (I) or formula (II) has the following structure, (III), or a pharmaceutically acceptable salt thereof.

[0062] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; R4 is hydrogen or C1-4 alkyl group; and R5 is CN or halogen group.

[0063] In the embodiments, the compound of formula (III) is not,,,,,,,, or.

[0064] In the embodiments, the compounds of formula (III) exclude the following compounds,,,,、、、 and.

[0065] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIa), or a pharmaceutically acceptable salt thereof.

[0066] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R5 is CN or halogen group.

[0067] In the embodiments, the compound of formula (IIIa) is not,,,,,,,, or.

[0068] In the embodiments, the compounds of formula (IIIa) exclude the following compounds,,,,,,, and.

[0069] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIb), or a pharmaceutically acceptable salt thereof.

[0070] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl substituted with one or more halogen groups as appropriate, and C1-3 alkoxy; R2 is hydrogen or C1-3 alkyl; R4 is hydrogen or C1-4 alkyl; and R5 is CN or halogen.

[0071] In the embodiments, the compound of formula (IIIb) is not , , , , , or.

[0072] In the embodiments, the compounds of formula (IIIb) exclude the following compounds: , , , , , and .

[0073] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIc), or a pharmaceutically acceptable salt thereof.

[0074] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R2 is hydrogen or C1-3 alkyl group; and R5 is CN or halogen group.

[0075] In the embodiments, the compound of formula (IIIc) is not , , , , , or.

[0076] In the embodiments, the compounds of formula (IIIc) exclude the following compounds: , , , , and .

[0077] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIId), or a pharmaceutically acceptable salt thereof.

[0078] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; and R5 is CN or halogen.

[0079] In the embodiments, the compound of formula (IIId) is not,,,,,,,, or.

[0080] In the embodiments, the compounds of formula (IIId) exclude the following compounds,,,,,,,, and.

[0081] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIe), or a pharmaceutically acceptable salt thereof.

[0082] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; R4 is hydrogen or C1-4 alkyl group; and R5 is CN or halogen group.

[0083] In the embodiments, the compound of formula (IIIe) is not , , , , , or.

[0084] In the embodiments, the compounds of formula (IIIe) exclude the following compounds: , , , , and .

[0085] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIf), or a pharmaceutically acceptable salt thereof.

[0086] In the embodiments, m is 1, 2, 3 or 4; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; and R5 is CN or halogen.

[0087] In the embodiments, the compound of formula (IIIf) is not , , , , , or.

[0088] In the embodiments, the compounds of formula (IIIf) exclude the following compounds: , , , , and .

[0089] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIg), or a pharmaceutically acceptable salt thereof.

[0090] In the embodiments, m is 1, 2, 3 or 4; n is 0, 1 or 2; each R1 group is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl group substituted with one or more halogen groups as appropriate, and C1-3 alkoxy group; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl group; R4 is hydrogen or C1-4 alkyl group and R5 is CN or halogen group.

[0091] In the embodiments, X is CH. In the embodiments, X is N, wherein N is oxidized as appropriate.

[0092] In this embodiment, Y is CH. In this embodiment, Y is N.

[0093] In this embodiment, Z is CH. In this embodiment, Z is N.

[0094] In this embodiment, A is CH. In this embodiment, A is N.

[0095] In this embodiment, B is CH. In this embodiment, B is N.

[0096] In this embodiment, m is 1. In this embodiment, m is 2. In this embodiment, m is 3. In this embodiment, m is 4.

[0097] In this embodiment, n is 0. In this embodiment, n is 1. In this embodiment, n is 2.

[0098] In the embodiments, R1 is hydrogen. In the embodiments, R1 is CN. In the embodiments, R1 is OH.

[0099] In an embodiment, R1 is a halogen group. In an embodiment, R1 is F. In an embodiment, R1 is Cl. In an embodiment, R1 is Br.

[0100] In the embodiments, R1 is a C1-3 alkyl group substituted with one or more halogen groups, as appropriate. In the embodiments, R1 is a C1-3 alkyl group. In the embodiments, R1 is methyl. In the embodiments, R1 is ethyl. In the embodiments, R1 is CF3.

[0101] In the examples, R1 is a C1-3 alkoxy group. In the examples, R1 is a methoxy group.

[0102] In the embodiments, R2 is hydrogen.

[0103] In the examples, R2 is a C1-3 alkyl group. In the examples, R2 is a methyl group.

[0104] In the embodiments, R3 is hydrogen.

[0105] In this embodiment, R3 is a halogen group. In this embodiment, R3 is F.

[0106] In the embodiments, R3 is OH.

[0107] In the examples, R3 is an amine. In the examples, R3 is NH2.

[0108] In the examples, R3 is a C1-3 alkyl group. In the examples, R3 is a methyl group.

[0109] In the embodiments, R4 is hydrogen.

[0110] In the examples, R4 is a C1-4 alkyl group. In the examples, R4 is methyl. In the examples, R4 is ethyl. In the examples, R4 is isopropyl. In the examples, R4 is tributyl.

[0111] In this embodiment, R5 is F. In this embodiment, R5 is Cl. In this embodiment, R5 is Br.

[0112] In the embodiment, R5 is CN.

[0113] In some embodiments, the compound is any one of compounds 1-44, or a pharmaceutically acceptable salt thereof: compound serial number structure compound serial number structure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

[0114] In the embodiments, in the compounds of formula (A) and (I)–(III), such as any of compounds 1–44 or their pharmaceutically acceptable salts, at least one hydrogen atom is replaced by a deuterium atom.

[0115] In another embodiment, the invention is characterized as a pharmaceutical composition comprising any of the compounds described herein (e.g., compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0116] In another embodiment, the invention is characterized by a method for treating diseases mediated by PHD activity, comprising administering to an individual any of the compounds described herein (e.g., compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) or a medically acceptable salt thereof.

[0117] In the embodiments, the disease mediated by PHD activity is ischemic reperfusion injury (e.g., stroke, myocardial infarction or acute kidney injury).

[0118] In the embodiments, the disease mediated by PHD activity is an inflammatory bowel disease (e.g., ulcerative colitis or Crohn's disease).

[0119] In the embodiments, the disease mediated by PHD activity is cancer (e.g., colorectal cancer).

[0120] In the embodiment, the disease mediated by PHD activity is a liver disease.

[0121] In the embodiment, the disease mediated by PHD activity is atherosclerosis.

[0122] In the embodiments, the disease mediated by PHD activity is a cardiovascular disease.

[0123] In the embodiments, the disease mediated by PHD activity is an eye disease or condition (e.g., radiation retinopathy, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, and ocular ischemia).

[0124] In the embodiments, the disease mediated by PHD activity is anemia (e.g., anemia associated with chronic kidney disease).

[0125] In the embodiments, the disease mediated by PHD activity is related to hyperoxia.

[0126] In the embodiment, the disease mediated by PHD activity is retinopathy of prematurity.

[0127] In the embodiment, the disease mediated by PHD activity is bronchodystrophy (BPD).

[0128] In the embodiments, the diseases mediated by PHD activity are ischemic heart disease, valvular heart disease, congestive heart failure, acute lung injury, pulmonary fibrosis, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), acute liver failure, liver fibrosis, and cirrhosis.

Implementation Method

[0129] [Cross-reference to related applications]

[0130] This application claims priority to U.S. Provisional Patent Application No. 62 / 992,585, filed March 20, 2020, which is incorporated herein by reference in its entirety. Definitions

[0131] To facilitate understanding of the present invention, certain terms are defined below. Additional definitions of accompanying and other terms are provided throughout this specification. Publications and other references that describe the background of the invention and provide additional details about its practice are incorporated herein by reference.

[0132] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any stage of development. In some embodiments, "animal" means a non-human animal at any stage of development. In some embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be genetically modified animals, genetically engineered animals, and / or purebreds.

[0133] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest means a value similar to the stated reference value. In some specific instances, unless otherwise stated or apparent from the context, the term "approximately" or "about" means a series of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in any direction (except where such values ​​would exceed 100% of the probability value).

[0134] When used in the description and the appended claims, unless the context clearly requires otherwise, the singular forms "a," "an," and "this" include a plurality of references. Thus, for example, a reference to "composition" includes a mixture of two or more such compositions.

[0135] In this specification and the following claims, several terms will be used, which shall be defined to have the following meanings: In various places in the description of this specification and the claims, the term "comprising" and other forms of the term, such as "comprising" and "containing", means to include without limitation and not to exclude, for example, other additives, components, integers, or steps.

[0136] "Whether" or "as the case may" means that the event or circumstances described below may or may not occur, and the description includes examples of the event or circumstances occurring as well as examples of the event or circumstances not occurring.

[0137] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the commencement of the treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A “control individual” is an individual who suffers from the same form of disease as the treated individual and is approximately the same age as the treated individual.

[0138] In vitro: As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, or in a cell culture, rather than in a multicellular organism.

[0139] In vivo: As used herein, the term "in vivo" refers to an event occurring within a multicellular organism such as a human or non-human animal. In the case of cell-based systems, the term may be used to refer to an event occurring within living cells (as opposed to, for example, in vitro systems).

[0140] Patient: As used herein, the terms "patient" or "individual" mean any organism that may be administered the provided composition for (e.g.) experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include both prenatal and postnatal forms.

[0141] Pharmaceutical acceptable: As used herein, “pharmaceutical acceptable” means a substance that is suitable for use in human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.

[0142] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or an organic acid (such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods used in this art (such as ion exchange). Other medically acceptable salts include adipic acid salts, alginate salts, ascorbate salts, aspartate salts, benzenesulfonate salts, benzoate salts, hydrogen sulfate salts, borate salts, butyrate salts, camphorate salts, camphor sulfonate salts, citrate salts, cyclopentanepropionate salts, digluconate salts, dodecyl sulfate salts, ethanesulfonate salts, formate salts, transbutenedioic acid salts, gluconate salts, glycerol phosphate salts, gluconate salts, hemisulfate salts, heptaate salts, hexanoate salts, hydroiodate salts, 2-hydroxyethanesulfonate salts, and lactobionate salts. Lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, papoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and their analogues. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N+(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and their analogues. Other pharmaceutically acceptable salts appropriately include non-toxic ammonium, quaternary ammonium, and amine cations formed using balancing ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include those formed from the quaternization of amines using appropriate electrophiles (e.g., alkyl halides) to form quaternized alkylamine salts.

[0143] Individual: As used herein, the term "individual" means human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes both prenatal and postnatal forms. In many embodiments, an individual is a human. An individual can be a patient, which refers to a human who goes to a healthcare provider for diagnosis or treatment of a disease. The term "individual" may be used interchangeably with "person" or "patient" herein. An individual may have or be susceptible to a disease or condition, but may or may not show symptoms of that disease or condition.

[0144] Substantively: As used herein, the term "substantively" refers to a qualitative condition that exhibits the full or near full range or degree of the feature or property of interest. Those of ordinary skill in the field of biology will understand that biological and chemical phenomena rarely (if any) reach completion and / or proceed to full or achieve or avoid absolute results. Therefore, the term "substantively" is used herein to encompass the potential lack of completeness inherent in many biological and chemical phenomena.

[0145] Therapeutic effective amount: As used herein, the term "therapeutic effective amount" means, when administered to an individual who suffers from or is susceptible to a disease, condition, and / or symptom, sufficient to treat, diagnose, prevent, and / or delay the onset of such disease, condition, and / or symptom. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0146] Treatment: As used herein, the term "treatment" means any method used to partially or completely reduce, improve, alleviate, suppress, prevent, or repair a particular disease, condition, and / or symptom, delay the onset of one or more symptoms or features, reduce the severity of one or more symptoms or features, and / or reduce the incidence of one or more symptoms or features. Treatment may be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease in order to reduce the risk of developing disease-related symptoms.

[0147] Aliphatic: As used herein, the term aliphatic refers to C1–C40 hydrocarbons and includes both saturated and unsaturated hydrocarbons. Aliphatic groups can be straight-chain, branched, or cyclic. For example, C1–C20 aliphatic groups can include C1–C20 alkyl groups (e.g., straight-chain or branched C1–C20 saturated alkyl groups), C2–C20 alkenyl groups (e.g., straight-chain or branched C4–C20 dienyl, straight-chain or branched C6–C20 trienyl, etc.), and C2–C20 alkynyl groups (e.g., straight-chain or branched C2–C20 alkynyl). C1–C20 aliphatic groups can include C3–C20 cyclic aliphatic groups (e.g., C3–C20 cycloalkyl, C4–C20 cycloalkenyl, or C8–C20 cycloalkynyl). In some embodiments, the aliphatic group may comprise one or more cyclic aliphatic groups and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur), and may be substituted with one or more substituents (such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). The aliphatic group is either unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group may be substituted with one or more of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R' is independently a C1–C20 aliphatic group (e.g., C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted alkyl group (e.g., an unsubstituted C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted C1–C3 alkyl group. In some embodiments, the aliphatic group is unsubstituted. In some embodiments, the aliphatic group does not contain any heteroatoms.

[0148] Alkyl: As used herein, the term "alkyl" refers to an acyclic, straight-chain or branched hydrocarbon group, for example, "C1–C20 alkyl" refers to an alkyl group having 1 to 20 carbon atoms. Alkyl groups can be straight-chain or branched. Examples of alkyl groups include (but are not limited to) methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, etc. The term "lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms, whether straight-chain or branched. Other alkyl groups will readily become apparent to those skilled in the art in view of the benefits of this invention. Alkyl groups can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group may be substituted with one or more of the following: halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R' is independently a C1–C20 aliphatic group (e.g., C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted alkyl group (e.g., an unsubstituted C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted C1–C3 alkyl group. In some embodiments, the alkyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein). In some embodiments, the alkyl group is substituted with a –OH group and may also be referred to herein as a “hydroxyalkyl” group, wherein the prefix indicates a –OH group and “alkyl” is as described herein. In some embodiments, the alkyl group is substituted with a -OR' group and may also be referred to herein as an “alkoxy” group.

[0149] Adding the suffix "-ene" to a group indicates that the group is a divalent part. For example, arylene is a divalent part of aryl, while heteroarylene is a divalent part of heteroaryl.

[0150] Alkylene: As used herein, the term "alkylene" means a saturated divalent straight-chain or branched hydrocarbon group, and exemplified by methylene, ethylene, isopropylene, etc. Similarly, as used herein, the term "alkenylene" means an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon double bonds, which may be present at any stable point along the chain, and the term "alkynylene" herein means an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon triple bonds, which may be present at any stable point along the chain. In some embodiments, alkylene, alkenylene, or alkynylene may comprise one or more cyclic aliphatic groups and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur), and may be substituted with one or more substituents (such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). For example, the alkylene, alkenyl, or ynylene group may be substituted with one or more of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R' is independently a C1–C20 aliphatic group (e.g., C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted alkyl group (e.g., an unsubstituted C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted C1–C3 alkyl group. In some embodiments, the alkylene, alkenyl, or ynylene group is unsubstituted. In some embodiments, the alkylene, alkenylene, or ynylene groups do not include any heteroatoms.

[0151] Alkenyl: As used herein, "alkenyl" means any straight-chain or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds, which may be present at any stable point along the chain. For example, "C2-C20 alkenyl" means an alkenyl having 2-20 carbons. For example, alkenyl includes prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In some embodiments, the alkenyl comprises 1, 2, or 3 carbon-carbon double bonds. In some embodiments, the alkenyl comprises a single carbon-carbon double bond. In some embodiments, the multiple double bonds (e.g., 2 or 3) are conjugated. The alkenyl may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkenyl group may be substituted with one or more of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R' is independently a C1–C20 aliphatic group (e.g., C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted alkyl group (e.g., an unsubstituted C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted C1–C3 alkyl. In some embodiments, the alkenyl group is unsubstituted. In some embodiments, the alkenyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein). In some embodiments, the alkenyl group is substituted with a –OH group and may also be referred to herein as a “hydroxyalkenyl” group, wherein the prefix indicates a –OH group and “alkenyl” is as described herein.

[0152] Alynyl group: As used herein, "alkynyl group" means any hydrocarbon chain, whether straight-chain or branched, having one or more carbon-carbon triple bonds at any stable point along the chain. For example, "C2-C20 alkynyl group" means an alkynyl group having 2-20 carbons. Examples of alkynyl groups include prop-2-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-2-alkynyl, 3-methylpent-4-alkynyl, hex-2-alkynyl, hex-5-alkynyl, and so on. In some embodiments, the alkynyl group comprises a single carbon-carbon triple bond. The alkynyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group can be substituted with one or more of halogen, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', or -SO2R' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R' is independently a C1–C20 aliphatic group (e.g., C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted alkyl group (e.g., an unsubstituted C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted C1–C3 alkyl. In some embodiments, the alkynyl group is unsubstituted. In some embodiments, the alkynyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5 or 6 substituents as described herein).

[0153] Aryl: The term "aryl" when used alone or as part of a larger portion of "arylene" refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein the ring system has a single connection point to the rest of the molecule, at least one ring in the ring system is aromatic, and each ring in the ring system contains four to seven ring members. In some embodiments, an aryl has six ring carbon atoms ("C6 aryl", e.g., phenyl). In some embodiments, an aryl has ten ring carbon atoms ("C10 aryl", e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl has fourteen ring carbon atoms ("C14 aryl", e.g., anthracene). "Aryl" also includes ring systems in which an aryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment group or attachment point is on the aryl ring, and in such cases, the number of carbon atoms continues to indicate the number of carbon atoms in the aryl ring system. Exemplary aromatic rings include phenyl, naphthyl, and anthracene.

[0154] Arylene: As used herein, “aryl” means a divalent aryl group (i.e., a group with two attachment sites to the molecule). Examples of arylene groups include phenylene (e.g., unsubstituted or substituted phenylene).

[0155] Halogen or halogen group: As used herein, the term “halogen” or “halogen group” refers to fluorine, chlorine, bromine or iodine.

[0156] Acetamine: The term "acetamine" or "acetamine group" refers to a chemical moiety having the formula C(O)N(R')2, -C(O)N(R')-, -NR'C(O)R', -NR'C(O)N(R')2- or -NR'C(O)-, wherein each R' is independently selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl (chain-carbon bonded), cycloalkyl, aryl, aralkyl, heteroaryl (cycle-carbon bonded), heteroarylalkyl or heterocycloalkyl (cycle-carbon bonded), unless otherwise specified in this specification, each moiety may be substituted as described herein, or the two R' may combine with a nitrogen atom to form a 3-, 4-, 5-, 6- or 7-membered ring.

[0157] Amino group: The term "amino group" or "amine" refers to a -N(R')2 group, wherein each R' is independently selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl (chain-carbon bonded), cycloalkyl, aryl, aralkyl, heteroaryl (cycle-carbon bonded), heteroarylalkyl, heterocycloalkyl (cycle-carbon bonded), sulfonyluyl, or carbonyl groups. Unless otherwise specified herein, each part may be substituted as described herein, or the two R's may combine with a nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. In the examples, the amino group is –NHR', wherein R' is aryl ("arylamino"), heteroaryl ("heteroarylamino"), or alkyl ("alkylamino").

[0158] Sulfoyl: The term "sulfonyl" refers to a -S(=O)2R' or -S(=O)2- group, wherein R' is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl (chain-carbon bonded), cycloalkyl, aryl, aralkyl, heteroaryl (cycle-carbon bonded), heteroarylalkyl, heterocycloalkyl (cycle-carbon bonded), unless otherwise specified in this specification, each part may be substituted as described herein.

[0159] Sulphinyl: The term "sulfenyl" refers to a chemical moiety having the formula -S(=O)R', -S(=O)- or -S(=O)(=NR')-, wherein R' is selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl (chain-carbon bonded), cycloalkyl, aryl, aralkyl, heteroaryl (cycle-carbon bonded), heteroarylalkyl, heterocycloalkyl (cycle-carbon bonded), unless otherwise specified in this specification, each moiety may be substituted as described herein.

[0160] Carbonyl: The term "carbonyl" refers to a -C(=O)R' or -C(=O)- group, wherein R' is selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl (chain-carbon bonded), cycloalkyl, aryl, aralkyl, heteroaryl (cycle-carbon bonded), heteroarylalkyl, heterocycloalkyl (cycle-carbon bonded), unless otherwise specified in this specification, each part may be substituted as described herein.

[0161] Phosphooxy group: The term "phosphooxy group" refers to a -P(=O)(R')2 or -P(=O)(R')- group, wherein R' is selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl (linked by carbon atom or heteroatom), cycloalkyl, aryl, aralkyl, heteroaryl (linked by carbon atom), heteroarylalkyl or heterocycloalkyl (linked by carbon atom), unless otherwise specified in this specification, each part may be substituted as described herein, or the two R' may combine with a nitrogen atom to form a 3-membered, 4-membered, 5-membered, 6-membered or 7-membered ring.

[0162] Heteroalkyl: The term "heteroalkyl" refers to a branched or straight-chain alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms, and also having 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyl includes tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphate diesters, phosphonates, sulfonamides, and disulfides. Heteroalkyl may include monocyclic, bicyclic, or tricyclic rings, wherein each ring is preferably 3 to 6 members. Examples of heteroalkyl include polyethers, such as methoxymethyl and ethoxyethyl.

[0163] Heteroalkylene: As used herein, the term “heteroalkylene” refers to the divalent form of the heteroalkylene as described herein.

[0164] Heteroaryl: As used herein, “heteroaryl” refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein the ring system has a single connection point to the rest of the molecule, wherein at least one ring in the ring system is aromatic, wherein each ring in the ring system contains four to seven ring members, and wherein at least one ring atom is a heteroatom, such as (but not limited to) nitrogen and oxygen.

[0165] Heterocyclic alkyl: As used herein, "heterocyclic alkyl" means a non-aromatic ring in which at least one atom is a heteroatom, such as (but not limited to) nitrogen, oxygen, sulfur or phosphorus, and the remaining atoms are carbon. Heterocyclic alkyl can be substituted or unsubstituted.

[0166] Deuterium: The term "deuterium" ("D" or "2H") is also known as heavy hydrogen. Deuterium is an isotope of hydrogen with an atomic nucleus consisting of one proton and one neutron, and its mass is twice that of a normal hydrogen nucleus (one proton).

[0167] Isotope: The term "isotope" refers to variants of a particular chemical element that have different numbers of protons and therefore different numbers of nucleons. All isotopes of a given element have the same number of protons in each atom, but different numbers of neutrons.

[0168] The term "substituted" means that the specified group or part has one or more substituents. The term "unsubstituted" means that the specified group does not have substituents. The term "substituted as appropriate" means that the specified group is unsubstituted or substituted with one or more substituents. When the term "substituted" is used to describe a structural system, substitution is intended to occur at any position on the system where the valence allows, for example, substitution resulting in a stable compound (e.g., a compound that does not spontaneously undergo transformations (such as by rearrangement, cyclization, elimination, or other reactions)). Where no specified substituent is explicitly indicated as being substituted or replaced as appropriate for a specified part or group, it should be understood that such part or group is intended to be unsubstituted.

[0169] When a ring system (e.g., cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) is substituted with substituents varying within a well-defined range, the total number of substituents shall not exceed the normally available valences under existing conditions. It should also be understood that hydrogen atoms are assumed to be present to fill the remaining valences of the ring system. Substituted groups only encompass combinations of substituents and variables that result in stable or chemically viable compounds. A stable or chemically viable compound is one that, among other things, possesses stability sufficient to allow its preparation and detection.

[0170] A variety of substituents are well known, and methods for their formation and the introduction of various parental groups are also well known. Representative substituents include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, alkylaryl, aryl, arylalkoxy, arylamino, heteroarylamino, heteroaryl, heteroarylalkoxy, heterocycloalkyl, hydroxyalkyl, aminoalkyl, haloalkyl, thioalkyl, alkylthioalkyl, carboxylalkyl, imidazolyl, indolealkyl, monohaloalkyl, dihaloalkyl and trihaloalkyl, monohaloalkoxy, dihaloalkoxy and trihaloalkoxy, amino, alkylamino, dialkylamino, amide, cyano, alkoxy, hydroxy, sulfonylmethane, halogen (e.g., -Cl and -Br), nitro, hydroxyimino, -COOR50, -COR50, -SO0-2R50, -SO2NR50R51, NR52SO 2R50, ═C(R50R51), ═N—OR50, ═N—CN, ═C(halo)2, ═S, ═O, —CON(R50R51), —OCOR50, —OCON(R50R51), —N(R52)CO(R50), —N(R52)COOR50 and —N(R52)CON(R50(R51), wherein R50, R51 and R52 may be independently selected from the following: hydrogen atom and branched or straight chain, C1–6-alkyl, C3–6-cycloalkyl, C4–6-heterocycloalkyl, heteroaryl and aryl groups, and may or may not have substituents. Where permissible, R50 and R51 may be linked together to form a carbocyclic or heterocyclic ring system.

[0171] In a preferred embodiment, the substituent system is selected from halogens, -COR', -CO2H, -CO2R', -CN, -OH, -OR', -OCOR', -OCO2R', -NH2, -NHR', -N(R')2, -SR', and -SO2R', wherein each example of R' is independently a C1–C20 aliphatic group (e.g., C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). In some embodiments, R' is independently an unsubstituted alkyl group (e.g., unsubstituted C1–C20 alkyl, C1–C15 alkyl, C1–C10 alkyl, or C1–C3 alkyl). Preferably, R' is independently an unsubstituted C1–C3 alkyl group.

[0172] Any formula given herein is intended to represent a compound having the structure described by the structural formula and a particular variant or form. Specifically, a compound of any formula given herein may have an asymmetric center and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the general formula compound, and mixtures thereof, are considered to fall within the scope of this formula. Therefore, any formula given herein is intended to represent racemic compounds, one or more enantiomeric forms, one or more diastereomeric forms, one or more hysteretic isomeric forms, and mixtures thereof. Furthermore, certain structures may exist in geometric isomeric form (i.e., cis and trans isomers), tautomeric form, or hysteretic isomeric form. Additionally, any formula given herein is intended to include hydrates, solvates, and allotropes of such compounds, and mixtures thereof. Compounds of the Invention

[0173] This article discloses potent inhibitors of PHD. In some embodiments, the half-maximal inhibitory concentration (IC50) of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 100 µM. In some embodiments, the IC50 of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 50 µM. In some embodiments, the IC50 of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 25 µM. In some embodiments, the IC50 of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 20 µM. In some embodiments, the IC50 of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 15 µM. In some embodiments, the IC50 of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 10 µM. In some embodiments, the IC50 of the compounds of the present invention for any of PHD1, PHD2, and PHD3 is less than 5 µM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is less than 1 µM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is about 3 nM to about 5 nM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is about 5 nM to about 10 nM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is about 10 nM to about 20 nM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is about 20 nM to about 50 nM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is about 50 nM to about 100 nM. In some embodiments, the IC50 value of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 is about 100 nM to about 200 nM. In some embodiments, the IC50 values ​​of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 are from about 200 nM to about 500 nM. In some embodiments, the IC50 values ​​of the compounds of the present invention for any one of PHD1, PHD2, and PHD3 are from about 500 nM to about 1000 nM.

[0174] Representative examples of this class show inhibitory activity against PHD1, PHD2 and PHD3 in vitro.

[0175] This document describes exemplary compounds. In particular, these selective inhibitors are characterized by a pyrazole moiety connecting two aromatic moieties (e.g., a 5-hydroxy-substituted pyrazole). Compounds of formulas (A) and (I)–(III)

[0176] In one state, this document provides a compound having the structure according to formula (A): (A) or a pharmaceutically acceptable salt thereof, wherein: Ar1 is a phenyl or a six-membered nitrogen-containing heteroaryl group, wherein the phenyl or heteroaryl group is substituted with a halogen group, CN, OH, a C1-3 alkyl group substituted with one or more halogen groups, or a C1-3 alkoxy group; R2 is H or a C1-3 alkyl group; Ar2 is a six-membered nitrogen-containing heteroaryl group substituted with a halogen group, OH, amine, or C1-3 alkyl group, wherein the halogen group, OH, amine, or C1-3 alkyl group is substituted with a halogen group; and R4 is hydrogen or a C1-4 alkyl group.

[0177] In the examples of the chemical formula (e.g., formula (A)) described herein, the compound (e.g., compound of formula (A)) is not,,,,,,,, or.

[0178] In the examples of the chemical formula (e.g., formula (A)) described herein, the compounds (e.g., compound (A)) exclude the following compounds,,,,,,,, and.

[0179] In the embodiment, R2 is H.

[0180] In the examples, R2 is a C1-3 alkyl group. In the examples, R2 is CH3. In the examples, R2 is CH2CH3. In the examples, R2 is CH2CH2CH3. In the examples, R2 is CH(CH3)2.

[0181] In the embodiment, R4 is H.

[0182] In the examples, R4 is a C1-4 alkyl group. In the examples, R4 is CH3. In the examples, R4 is CH2CH3. In the examples, R4 is CH2CH2CH3. In the examples, R4 is CH(CH3)2. In the examples, R4 is CH2CH2CH2CH3. In the examples, R4 is CH(CH3)(CH2CH3). In the examples, R4 is C(CH3)3.

[0183] In the examples, Ar1 is an unsubstituted aryl group. In the examples, Ar1 is a substituted aryl group. In the examples, Ar1 is an unsubstituted phenyl group. In the examples, Ar1 is a substituted phenyl group.

[0184] In the embodiments, Ar1 is an unsubstituted 6-membered nitrogen-containing heteroaryl group. In the embodiments, Ar1 is a substituted 6-membered nitrogen-containing heteroaryl group.

[0185] In the embodiments, Ar1 is substituted with one or more groups selected from halogen groups, CN, OH, C1-3 alkyl groups substituted with one or more halogen groups as appropriate, and C1-3 alkoxy groups. In some embodiments, Ar1 is substituted with one substituent. In some embodiments, Ar1 is substituted with two substituents. In some embodiments, Ar1 is substituted with three substituents. In some embodiments, Ar1 is substituted with four substituents.

[0186] In the embodiments, Ar1 comprises one or more R1 groups, wherein each R1 is independently selected from halogen, halogen, CN, OH, C1-3 alkyl groups substituted with one or more halogen groups as appropriate, and C1-3 alkoxy groups. In the embodiments, Ar1 comprises some R1 groups represented by m, wherein m is 1, 2, 3, or 4. When R1 is present, R1 may substitute for hydrogen in the parent molecule structure. In the embodiments, when R1 is present and is a non-hydrogen moiety, R1 represents a substituent group. In the embodiments, R1 is independently selected from halogen, CN, OH, C1-3 alkyl groups substituted with CN or one or more halogen groups as appropriate, and C1-3 alkoxy groups.

[0187] Therefore, it should also be understood that for any value of m described herein, the hydrogen system is suitably present to fulfill the valence requirement of the Ar1 component atoms, such that the molecule is a stable molecule (e.g., a compound that does not spontaneously undergo transformations (such as by rearrangement, cyclization, elimination, or other reactions)). Exemplary examples of Ar1, R1, and m are described herein.

[0188] In the embodiments, Ar1 is, wherein X, Y and Z are independently CH or N, wherein N is oxidized as appropriate; each R1 system is independently selected from the group consisting of: hydrogen, halogen, CN, OH, C1-3 alkyl substituted with one or more halogen groups as appropriate, and C1-3 alkoxy; and m is 1, 2, 3 or 4.

[0189] In the embodiments, R1 is not hydrogen. In the embodiments, when R1 is present and is a non-hydrogen moiety, R1 represents a substituent.

[0190] In the embodiments, the value of m is based on the number of nitrogen atoms present in the ring. In the embodiments, m is 1, 2, or 3 when two and only two of X, Y, and Z are N. In the embodiments, m is 1 or 2 when each of X, Y, and Z is N.

[0191] In this embodiment, m is 1. In this embodiment, m is 2. In this embodiment, m is 3. In this embodiment, m is 4.

[0192] In the embodiments, X, Y, and Z are all N, and m is 1 or 2. In the embodiments, m is 1, and any remaining unsubstituted carbon ring atoms are assumed to be bonded to hydrogen to fill the valence. In the embodiments, m is 2.

[0193] In the embodiments, one of X, Y, and Z is CH, and the rest are N, wherein N is oxidized as appropriate, and m is 1, 2, or 3. In the embodiments, m is 1, and any remaining unsubstituted carbon ring atoms are assumed to be hydrogen-bonded to fill the valence. In the embodiments, m is 2, and any remaining unsubstituted carbon ring atoms are assumed to be hydrogen-bonded to fill the valence. In the embodiments, m is 3.

[0194] In the embodiments, two of X, Y, and Z are CH and the rest are N, wherein N is oxidized as appropriate, and m is 1, 2, 3, or 4. In the embodiments, m is 1, and any remaining unsubstituted carbon ring atoms are assumed to be hydrogen-bonded to fill the valence. In the embodiments, m is 2, and any remaining unsubstituted carbon ring atoms are assumed to be hydrogen-bonded to fill the valence. In the embodiments, m is 3, and any remaining unsubstituted carbon ring atoms are assumed to be hydrogen-bonded to fill the valence. In the embodiments, m is 4.

[0195] In the embodiments, the N atoms in Ar1 were not oxidized.

[0196] In the embodiments, the N atoms in Ar1 are oxidized.

[0197] In the examples, Ar1 is a substituted phenyl group. In the examples, Ar1 is substituted with at least one R1, wherein R1 is a CN or a halogroup.

[0198] In the embodiments, Ar1 is substituted with one or two R1 groups, which are independently selected from C1-3 alkyl, halogen, CN or OH groups substituted with one or more halogen groups as appropriate.

[0199] In the embodiments, Ar1 is a pyridinyl N-oxide or, where appropriate, a pyridinyl group substituted with at least one R1, wherein R1 is a C1-3 alkoxy or a halogen.

[0200] In the embodiments, R1 is hydrogen each time it is used.

[0201] In the embodiment, R1 is CN each time it is used.

[0202] In the embodiment, R1 is OH each time it is used.

[0203] In an embodiment, R1 is a halogen group each time it is used. In an embodiment, the halogen group is Cl. In an embodiment, the halogen group is Br. In an embodiment, the halogen group is I.

[0204] In the embodiments, R1 is a C1-3 alkyl group each time it is used.

[0205] In the embodiments, R1 is an unsubstituted C1-3 alkyl group each time it is used. In the embodiments, R1 is CH3 each time it is used. In the embodiments, R1 is CH2CH3 each time it is used.

[0206] In the embodiments, R1 is a substituted C1-3 alkyl group each time it is used. In the embodiments, R1 is a C1-3 alkyl group substituted with one or more halogen groups each time it is used. In the embodiments, the halogen group is F. In the embodiments, the halogen group is Cl. In the embodiments, the halogen group is Br. In the embodiments, the halogen group is I.

[0207] In the embodiment, R1 is CF3 each time it is used.

[0208] In the embodiments, R1 is C1-3 alkoxy in each application. In the embodiments, R1 is OMe in each application.

[0209] In the embodiments, Ar1 is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , and.

[0210] In the embodiments, Ar1 is selected from: , , , and.

[0211] In the embodiments, Ar1 is selected from: , , , , , , , , , , and.

[0212] In the embodiments, Ar1 is selected from: , , , and.

[0213] In the embodiments, Ar1 is selected from: , , , and.

[0214] In the embodiments, Ar1 is selected from: and.

[0215] In the embodiments, Ar2 is an unsubstituted six-membered nitrogen-containing heteroaryl group.

[0216] In the examples, Ar2 is a six-membered nitrogen-containing heteroaryl group substituted with a halogen, OH, amine, or C1-3 alkyl group. In the examples, Ar2 is substituted with one substituent as described herein. In the examples, Ar2 is substituted with two substituents as described herein.

[0217] In the embodiments, Ar2 is, wherein A and B are independently CH or N, wherein N is oxidized as appropriate; each R3 group is independently selected from the group consisting of: hydrogen, halogen, OH, amine and C1-3 alkyl; and n is 0, 1 or 2.

[0218] In the embodiments, the value of n is based on the number of nitrogen atoms present in the ring. In the embodiments, when one and only one of A and B is N, n is 0, 1, or 2. In the embodiments, when both A and B are N, n is 0 or 1.

[0219] In this embodiment, n is 0. In this embodiment, n is 1. In this embodiment, n is 2.

[0220] In the embodiments, both A and B are N, wherein N is oxidized as appropriate, and n is 0 or 1. In the embodiments, n is 0. In the embodiments, n is 1.

[0221] In the embodiments, one of A and B is CH and the other is N, wherein N is oxidized as appropriate, and n is 0, 1, or 2. In the embodiments, n is 0. In the embodiments, n is 1. In the embodiments, n is 2.

[0222] In the embodiments, both A and B are CH, and n is 0, 1, or 2. In the embodiments, n is 0. In the embodiments, n is 1. In the embodiments, n is 2.

[0223] In the embodiments, the N atoms in Ar2 were not oxidized.

[0224] In the embodiments, the N atoms in Ar2 are oxidized.

[0225] In the embodiments, R3 is hydrogen each time it is used.

[0226] In the embodiments, R3 is OH each time it is used.

[0227] In an embodiment, R3 is a halogen group each time it is used. In an embodiment, the halogen group is F. In an embodiment, the halogen group is Cl. In an embodiment, the halogen group is Br. In an embodiment, the halogen group is I.

[0228] In the examples, R3 is an amine each time it is used. In the examples, R3 is NH2 each time it is used.

[0229] In the embodiments, R3 is a C1-3 alkyl group each time it is used.

[0230] In the embodiments, R3 is an unsubstituted C1-3 alkyl group each time it is used. In the embodiments, R3 is CH3 each time it is used.

[0231] In the embodiments, Ar2 is selected from the group consisting of: , , , , and.

[0232] In the embodiments, the compound of formula (A) has the following structure, (I), or a pharmaceutically acceptable salt thereof, wherein A, B, X, Y, Z, R1, R2, R3 and R4 are as defined anywhere herein.

[0233] In the examples of chemical formulas (e.g., formula (I)) described herein, the compound (e.g., the compound of formula (I)) is not,,,,,,,, or.

[0234] In the examples of the chemical formula (e.g., formula (I)) described herein, the compounds (e.g., compounds of formula (I)) exclude the following compounds,,,,,,,, and.

[0235] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (Ia), or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R1, R2, R3 and R4 are as defined anywhere herein.

[0236] In the examples of chemical formulas (e.g., formula (Ia)) described herein, the compound (e.g., compound of formula (Ia)) is not,,,,,,,, or.

[0237] In the examples of the chemical formula (e.g., formula (Ia)) described herein, the compounds (e.g., compounds of formula (Ia)) exclude the following compounds,,,,,,,, and.

[0238] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (Ib), or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 are as defined anywhere herein.

[0239] In the examples of chemical formulas (e.g., formula (Ib)) described herein, the compound (e.g., compound (Ib)) is not,,,,,,,, or.

[0240] In the examples of the chemical formula (e.g., formula (Ib)) described herein, the compounds (e.g., compounds of formula (Ib)) exclude the following compounds,,,,,,,, and.

[0241] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (Ic), or a pharmaceutically acceptable salt thereof, wherein A, B, X, Y, Z, R1, R3 and R4 are as defined anywhere herein.

[0242] In the embodiments, the compound of formula (A) or formula (I) has the following structure, (II), or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 are as defined anywhere herein.

[0243] In the examples of the chemical formula (e.g., formula (II)) described herein, the compound (e.g., compound (II)) is not,,,,,,,, or.

[0244] In the examples of the chemical formula (e.g., formula (II)) described herein, the compounds (e.g., compounds of formula (II)) exclude the following compounds,,,,,,,, and.

[0245] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IIa), or a pharmaceutically acceptable salt thereof, wherein R1, R2 and R3 are as defined anywhere herein.

[0246] In the examples of the chemical formula (e.g., formula (IIa)) described herein, the compound (e.g., compound of formula (IIa)) is not,,,,,,,, or.

[0247] In the examples of the chemical formula (e.g., formula (IIa)) described herein, the compounds (e.g., compounds of formula (IIa)) exclude the following compounds,,,,,,,, and.

[0248] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IIb), or a pharmaceutically acceptable salt thereof, wherein R1, R2 and R4 are as defined anywhere herein.

[0249] In the examples of the chemical formula (e.g., formula (IIb)) described herein, the compound (e.g., the compound of formula (IIb)) is not, , , , or.

[0250] In the examples of the chemical formula (e.g., formula (IIb)) described herein, the compounds (e.g., compounds of formula (IIb)) exclude the following compounds: , , , , or .

[0251] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IIc), or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are as defined anywhere herein.

[0252] In the examples of the chemical formula (e.g., formula (IIc)) described herein, the compound (e.g., the compound of formula (IIc)) is not, , , , or.

[0253] In the examples of the chemical formula (e.g., formula (IIc)) described herein, the compounds (e.g., compounds of formula (IIc)) exclude the following compounds, , , , or.

[0254] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IId), or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are as defined anywhere herein.

[0255] In the examples of chemical formulas (e.g., formula (IId)) described herein, the compound (e.g., formula (IId) compound) is not,,,,,,,, or.

[0256] In the examples of chemical formulas (e.g., formula (IId)) described herein, the compounds (e.g., compounds of formula (IId)) exclude the following compounds,,,,,,,, and.

[0257] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IIe), or a pharmaceutically acceptable salt thereof, wherein R1 and R4 are as defined anywhere herein.

[0258] In the examples of chemical formulas (e.g., formula (IIe)) described herein, the compound (e.g., the compound of formula (IIe)) is not, , , , or.

[0259] In the examples of the chemical formula (e.g., formula (IIe)) described herein, the compounds (e.g., compounds of formula (IIe)) exclude the following compounds: , , , , or.

[0260] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IIf), or a pharmaceutically acceptable salt thereof, wherein R1 is as defined anywhere herein.

[0261] In the examples of chemical formulas (e.g., formula (IIf)) described herein, the compound (e.g., formula (IIf) compound) is not, , , , or.

[0262] In the chemical formula (e.g., formula (IIf)) examples described herein, the compounds (e.g., formula (IIf) compounds) exclude the following compounds, , , , or.

[0263] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (IIg), or a pharmaceutically acceptable salt thereof, wherein R1, R3 and R4 are as defined anywhere herein.

[0264] In the embodiments, the compounds of formula (A), formula (I) or formula (II) have the following structure, (III), or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 are as defined anywhere herein, and wherein R5 is a CN or a halogen group.

[0265] In the embodiment, R5 is CN.

[0266] In an embodiment, R5 is a halogen group. In an embodiment, the halogen group is F. In an embodiment, the halogen group is Cl. In an embodiment, the halogen group is Br. In an embodiment, the halogen group is I.

[0267] In the examples of chemical formulas (e.g., formula (III)) described herein, the compound (e.g., compound (III)) is not,,,,,,,, or.

[0268] In the examples of chemical formulas (e.g., formula (III)) described herein, the compounds (e.g., compounds of formula (III)) exclude the following compounds,,,,,,,, and.

[0269] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIa), or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4 and R5 are as defined anywhere herein.

[0270] In the examples of chemical formulas (e.g., formula (IIIa)) described herein, the compound (e.g., compound (IIIa)) is not,,,,,,,, or.

[0271] In the examples of the chemical formula (e.g., formula (IIIa)) described herein, the compounds (e.g., compounds of formula (IIIa)) exclude the following compounds,,,,,,,, and.

[0272] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIb), or a pharmaceutically acceptable salt thereof, wherein R1, R2, R4 and R5 are as defined anywhere herein.

[0273] In the examples of chemical formulas (e.g., formula (IIIb)) described herein, the compound (e.g., the compound of formula (IIIb)) is not, , , , or.

[0274] In the examples of the chemical formula (e.g., formula (IIIb)) described herein, the compounds (e.g., compounds of formula (IIIb)) exclude the following compounds: , , , , and .

[0275] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIc), or a pharmaceutically acceptable salt thereof, wherein R1, R2 and R5 are as defined anywhere herein.

[0276] In the examples of the chemical formula (e.g., formula (IIIc)) described herein, the compound (e.g., the compound of formula (IIIc)) is not, , , , or.

[0277] In the examples of the chemical formula (e.g., formula (IIIc)) described herein, the compounds (e.g., compounds of formula (IIIc)) exclude the following compounds: , , , , and .

[0278] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIId), or a pharmaceutically acceptable salt thereof, wherein R1, R3 and R5 are as defined anywhere herein.

[0279] In the examples of chemical formulas (e.g., formula (IIId)) described herein, the compound (e.g., formula (IIId) compound) is not,,,,,,,, or.

[0280] In the examples of chemical formulas (e.g., formula (IIId)) described herein, compounds (e.g., compounds of formula (IIId)) exclude the following compounds,,,,,,,, and.

[0281] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIe), or a pharmaceutically acceptable salt thereof, wherein R1, R4 and R5 are as defined anywhere herein.

[0282] In the examples of the chemical formula (e.g., formula (IIIe)) described herein, the compound (e.g., the compound of formula (IIIe)) is not, , , , or.

[0283] In the examples of the chemical formula (e.g., formula (IIIe)) described herein, the compounds (e.g., compounds of formula (IIIe)) exclude the following compounds: , , , , and .

[0284] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIf), or a pharmaceutically acceptable salt thereof, wherein R1 and R5 are as defined anywhere herein.

[0285] In the examples of chemical formulas (e.g., formula (IIIf)) described herein, the compound (e.g., the compound of formula (IIIf)) is not, , , , or.

[0286] In the examples of the chemical formula (e.g., formula (IIIf)) described herein, the compounds (e.g., compounds of formula (IIIf)) exclude the following compounds, , , , and .

[0287] In the embodiments, formula (A), formula (I), formula (II) or formula (III) has the following structure, (IIIg), or a pharmaceutically acceptable salt thereof, wherein R1, R3, R4 and R5 are as defined anywhere herein. Exemplary Compounds

[0288] In some embodiments, the PHD inhibitor compound is any one of compounds 1-44 or a pharmaceutically acceptable salt thereof. compound serial number structure compound serial number structure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Isotope molecules

[0289] It should be understood that in the compounds described herein (e.g., any of formulas (A) and (I)–(III), such as any of compounds 1–44), atoms may exhibit their natural isotopic abundance, or one or more atoms may be artificially enriched with a specific isotope, the isotope having the same number of atoms as the dominant naturally occurring element, but with a different atomic mass or mass number than the dominant naturally occurring element. This invention is intended to include all suitable isotopic variations of compounds described herein (e.g., any of formulas (A) and (I)–(III), such as any of compounds 1–44). For example, different isotopic forms of hydrogen (H) include protium (¹H), deuterium (²H), and tritium (³H). Protium is the dominant hydrogen isotope found in nature.

[0290] In some embodiments, one or more hydrogen series in the compounds described herein (e.g., any of the compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) are replaced by deuterium. Concentration of deuterium can produce certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or providing compounds suitable for characterizing biological samples. In some embodiments, one or more hydrogen series in the compounds described herein (e.g., any of the compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) are replaced by tritium. Tritium is radioactive and thus provides compounds that can be radiolabeled and used as tracers in metabolic or kinetic studies.

[0291] The isotope-enriched compounds disclosed herein (e.g., any of the compounds in formulas (A) and (I)–(III), such as any of compounds 1–44) can be produced without excessive experimentation using appropriate isotope-enriching reagents and / or intermediates, either by conventional techniques known to those skilled in the art or by methods similar to those described in the procedures and examples herein.

[0292] The term "isotope-like molecule" refers to a substance having the same chemical structure and formula as the specific compound provided herein, except for the position of isotopic substitution at one or more positions and / or the degree of isotopic enrichment. For example, hydrogen and deuterium. Therefore, as used herein, the term "compound" encompasses a collection of molecules having the same chemical structure, but with isotopic variations among the constituent atoms of such molecules. Thus, it will be apparent to those skilled in the art that a compound containing a specified deuterium atom, represented by a specific chemical structure, also contains a smaller amount of isotope-like molecules having hydrogen atoms at one or more specified deuterium positions in that structure. The relative amounts of such isotope-like molecules in the provided compounds depend on a variety of factors, including (but not limited to) the isotopic purity of the deuterating reagent used to prepare the compound and the efficiency of deuterium incorporation in the various synthetic steps used to prepare the compound.

[0293] When a position is specifically designated as “H” or “hydrogen”, the position shall be considered to have hydrogen with a naturally occurring abundance isotopic composition. When a position is specifically designated as “D” or “deuterium”, the position shall be considered to have deuterium with an abundance of at least 3340 times greater than the naturally occurring deuterium abundance (which is 0.015%) (that is, the terms “D” or “deuterium” refer to the inclusion of at least 50.1% deuterium).

[0294] In the embodiments, the compounds provided herein have isotopic enrichment factors for each deuterium present at sites designated as potential deuteration sites on the compounds of at least 3500 (incorporating 52.5% deuterium), at least 4000 (incorporating 60% deuterium), at least 4500 (incorporating 67.5% deuterium), at least 5000 (75% deuterium), at least 5500 (incorporating 82.5% deuterium), at least 6000 (incorporating 90% deuterium), at least 6333.3 (incorporating 95% deuterium), at least 6466.7 (incorporating 97% deuterium), at least 6600 (incorporating 99% deuterium), or at least 6633.3 (incorporating 99.5% deuterium). Synthesis of the compounds of the present invention

[0295] The compounds described herein (e.g., any of the compounds of formulas A and I–III, such as any of compounds 1–44) may be prepared according to methods known in the art, including exemplary synthesis of the examples provided herein, such as the synthesis shown in process A.

[0296] The purity of the compound and its synthetic intermediates was determined by reverse-phase HPLC using any of the methods described below:

[0297] Method A: Mobile phase: A: Water (0.01% TFA) B: Acetonitrile (0.01% TFA); Gradient phase: Increased from 5% B to 95% B in 1.4 min, maintained at 95% B for 1.6 min (total run time: 3 min); Flow rate: 2.3 mL / min. Column: SunFire C18, 4.6*50 mm, 3.5 µm; Column temperature: 50ºC. Detectors: ADC ELSD, DAD (214 nm and 254 nm), ES-API.

[0298] Method B: Mobile phase: A: Water (10 mM NH4HCO3) B: Acetonitrile; Gradient phase: Increased from 5% B to 95% B over 1.5 min, maintained at 95% B for 1.5 min (total run time: 3 min); Flow rate: 2.0 mL / min; Column: XBridge C18, 4.6*50 mm, 3.5 μm; Column temperature: 40 ºC. Detectors: ADC ELSD, DAD (214 nm and 254 nm), MSD (ES-API). Abbreviations and acronyms used herein include the following: the term acronym 4-Dimethylaminopyridine DMAP Acetyl Ac Aqueous solution aq. benzyl Bn Tert-butoxycarbonyl Boc Surface width single peak brs dichloromethane DCM dimethyl sulfoxide DMSO Twin Peaks d Electrospraying ionization method ESI equivalent eq Ethyl acetate EtOAc gram g hexane Hex High performance liquid chromatography HPLC Hour hr Isopropyl i-Pr Liquid Chromatography Mass Spectrometer LCMS megahertz MHz m-chloroperoxybenzyl acid m-CPBA methanol MeOH mg mg milliliters mL minute min Multiplets m N,N-Diisopropylethylamine DIPEA N,N-Dimethylformamide DMF N,N-Dimethylformamide dimethyl acetal DMF-DMA normal N Nuclear magnetic resonance NMR Palladium on carbon Pd / C Five Peaks p petroleum ether PE Phenyl Ph Four Peaks q room temperature RT Single peak s Tetrahydrofuran THF Thin-layer chromatography TLC Triethylamine TEA Trifluoroacetic acid TFA Triple Peak t Process A

[0299] Compound (IId) was prepared using commercially available materials according to process A. The ester of formula (B) reacts with N,N-dimethylformamide dimethyl acetal to produce the enamine compound of formula (C). The cyclization reaction of (C) with acehydrazine (D) provides the pyrazole compound of formula (IId). Composition and Method

[0300] This invention provides the use of any compound of formulas (A) and (I)–(III) in the manufacture of a medicament for treating the various conditions or ailments described herein. In one embodiment, a pharmaceutical composition is provided comprising at least one compound of any one of formulas (A) and (I)–(III) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. In various embodiments, the medicament or pharmaceutical composition may further comprise, or be used in combination with, at least one additional therapeutic agent.

[0301] The compounds of the present invention, or pharmaceuticals or compositions comprising such compounds, can be used to inhibit PHD activity. Inhibition of PHD may be particularly beneficial for treating the following diseases: including heart diseases (e.g., ischemic heart disease, congestive heart failure, and valvular heart disease), lung diseases (e.g., acute lung injury, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease), liver diseases (e.g., acute liver failure, liver fibrosis, and cirrhosis), and kidney diseases (e.g., acute kidney injury and chronic kidney disease). In one embodiment, the method of the present invention comprises administering to a patient in need a therapeutically effective amount of any compound of formula (A) and (I)–(III) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds of formula (A) and (I)–(III).

[0302] The present invention also relates to a method for inhibiting PHD activity. In one embodiment, the method comprises exposing the PHD to an effective amount of one or more compounds selected from the group comprising any one of the compounds of formulas (A) and (I)–(III), or a pharmaceutically acceptable salt thereof.

[0303] In other embodiments, the compounds disclosed herein (e.g., any of the compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) or their pharmaceutically acceptable salts are used to treat or prevent anemia, including treating anemia associated with: chronic kidney disease, polycystic kidney disease, aplastic anemia, autoimmune hemolytic anemia, bone marrow transplant anemia, Churg-Strauss syndrome, and congenital pure red cell aplastic anemia (Diamond Blackfan syndrome). Anemia, Fanconi's anemia, Felty's syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, hemolytic uremic syndrome, myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria, myelofibrosis, pancytopenia, pure red cell aplasia, Schoenlein-Henoch purpura, refractory anemia with polyblastic blasts, rheumatoid arthritis, Shwachman syndrome, sickle cell disease, severe thalassemia. Anemia, mild thalassemia, thrombocytopenic purpura, anemia or non-anemia in patients undergoing surgery, trauma-related or secondary anemia, sideroblastic anemia, and other treatments for secondary anemia, including: reverse transcriptase inhibitors for HIV treatment, corticosteroids, chemotherapy drugs containing or without cisplatin, vinca alkaloids, mitotic inhibitors, topoisomerase II inhibitors, anthracyclines, tranexamic acid, alkylating agents, especially anemia secondary to inflammation, aging, and / or chronic diseases. PHD1 inhibition can also be used to treat symptoms of anemia, including chronic fatigue, pallor, and dizziness.

[0304] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat or prevent metabolic disorders, including but not limited to diabetes and obesity.

[0305] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat or prevent vascular diseases. These diseases include, but are not limited to, diseases related to hypoxia or wound healing that require angiogenesis mediators for angiogenesis, vascularization, and arterial regeneration.

[0306] The compounds disclosed herein (such as compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat or prevent local ischemia-reperfusion. These conditions include, but are not limited to, stroke, myocardial infarction, and acute kidney injury.

[0307] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat inflammatory bowel diseases. These diseases include, but are not limited to, ulcerative colitis and Crohn's disease.

[0308] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat cancers, such as colorectal cancer.

[0309] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat atherosclerosis.

[0310] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat cardiovascular diseases.

[0311] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat eye diseases or conditions. These diseases include, but are not limited to, radiation retinopathy, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, and ocular ischemia.

[0312] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat diseases associated with hyperoxia.

[0313] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat bronchodystrophy (BPD).

[0314] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat heart conditions. These conditions include, but are not limited to, myocardial ischemia after pancreatic surgery, myocardial injury after percutaneous coronary intervention (PCI), myocardial injury after non-cardiac surgery, myocardial ischemia throughout the procedure of elective abdominal aortic aneurysm surgery, myocardial injury after PCI, myocardial injury in patients undergoing coronary artery bypass grafting (CABG), minimally invasive mitral valve (MIMV) repair or replacement, adult patients undergoing open-heart surgery, chronic heart failure, NYHA class II–IV.

[0315] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat lung diseases. These conditions include, but are not limited to, lung injury during selective lobectomy, lung injury during CABG surgery, and lung transplantation.

[0316] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat liver diseases. These conditions include, but are not limited to, non-alcoholic hepatitis (NASH).

[0317] In other embodiments, the compounds disclosed herein (e.g., compounds of formulas (A) and (I)–(III), such as any one of compounds 1–44) or their pharmaceutically acceptable salts are used to treat kidney diseases. These conditions include, but are not limited to, contrast agent-induced acute kidney injury, stage III–IV chronic kidney disease undergoing planned coronary angiography, acute kidney injury in patients undergoing heart valve surgery, non-dialysis-dependent chronic kidney disease, patients with chronic kidney disease initiating dialysis, and non-dialysis-dependent chronic kidney disease.

[0318] Furthermore, the compounds disclosed herein (e.g., any of the compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) or their pharmaceutically acceptable salts may be used in combination with additional active ingredients to treat the aforementioned symptoms. These additional compounds may be co-administered separately from the compounds disclosed herein (e.g., any of the compounds of formulas (A) and (I)–(III), such as any of compounds 1–44) or their pharmaceutically acceptable salts, or may be included together with the additional active ingredient in a pharmaceutical composition according to the invention. In one exemplary embodiment, the additional active ingredient is an active ingredient known or found to be effective in treating symptoms, conditions, or diseases mediated by PHD enzymes, or an active ingredient active against another target associated with that particular symptom, condition, or disease, such as an alternative PHD modulator. This combination may help improve efficacy (e.g., by including compounds in the composition that enhance the efficacy or effectiveness of the compounds of the invention), reduce one or more side effects, or reduce the required dosage of the compounds of the invention.

[0319] The compounds of the present invention are used alone or in combination with one or more other active ingredients to formulate pharmaceutical compositions of the present invention. Pharmaceutical compositions of the present invention comprise: (a) an effective amount of the compounds disclosed herein (e.g., any compound of formula (A) and (I)–(III), such as any one of compounds 1–44) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable prodrug thereof, or a pharmaceutically active metabolite thereof; and (b) a pharmaceutically acceptable excipient.

[0320] "Pharmaceutical-acceptable excipients" refers to substances that are nontoxic, biologically tolerable, or otherwise biologically suitable for administration to a subject, such as inert substances, which are added to pharmaceutical compositions or otherwise used as carriers, diluents, or loads to facilitate drug administration, and are compatible with the drug. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. Suitable excipients may also include antioxidants. These antioxidants can be used in pharmaceutical compositions or in storage media to extend the shelf life of the pharmaceutical product. Pharmaceutical formulations and routes of administration

[0321] As is known in the art, the compounds and compositions of the present invention can be delivered directly or as pharmaceutical compositions or drugs together with suitable carriers or excipients. Treatment methods of the present invention may include administering an effective amount of the compounds of the present invention to an individual in need. In a preferred embodiment, the individual is a mammalian individual; in a most preferred embodiment, the individual is a human individual.

[0322] The effective amount of the compound, composition, or drug can be readily determined by routine experiments, as can the most effective and convenient route of administration and the most suitable formulation. Various formulations and drug delivery systems exist in the art. See, for example, Gennaro, AR ed. (1995), Remington's Pharmaceutical Sciences, above.

[0323] For example, suitable routes of administration may include oral, rectal, local, nasal, pulmonary, ocular, intestinal, and non-enteric administration. Primary routes of administration for non-enteric administration include intravenous, intramuscular, and subcutaneous administration. Secondary routes of administration include intraperitoneal, intra-articular, intra-articular, intracardiac, intracisional, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intracardiac administration. The indication to be treated and the physical, chemical, and biological properties of the drug determine the type of formulation and the route of administration used, as well as whether local or systemic administration will be preferred.

[0324] The pharmaceutical dosage forms of the compounds of the present invention can be provided in the form of rapid-release, controlled-release, sustained-release, or targeted drug delivery systems. Common dosage forms include solutions and suspensions, (micro)emulsions, ointments, gels and patches, liposomes, tablets, sugar-coated pills, soft-shell or hard-shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols, and lyophilized formulations. Depending on the route of administration used, special devices may be required to administer or deliver the drug, such as syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks. Pharmaceutical dosage forms often consist of a drug, excipients, and a container / sealing system. One or more excipients (also known as inactive ingredients) may be added to the compounds of the present invention to improve or facilitate the manufacture, stability, administration, and safety of the drug, and to provide a method for obtaining the desired drug release profile. Therefore, the type of excipient added to a drug can be determined by various factors, such as the physical and chemical properties of the drug, the route of administration, and the preparation steps. Pharmaceutical excipients exist in this field and include those listed in various pharmacopoeias. See, for example, the United States Pharmacopeia (USP), the Japanese Pharmacopoeia (JP), the European Pharmacopoeia (EP), and the British Pharmacopoeia (BP); the U.S. Food and Drug Administration.

[0325] Publications from the Centre for Drug Evaluation and Research (CEDR) of the Federal Regulatory Commission (www.fda.gov), such as the Guide to Inactive Ingredients (1996); the Handbook of Drug Additives, edited by Ash and Ash (2002), Synapse Information Resources, Endicott NY, et al.

[0149] Pharmaceutical dosage forms of the compounds of the present invention may be manufactured by any method known in the art, such as by conventional mixing, sieving, dissolving, melting, granulation, manufacturing of sugar-coated pills, tableting, suspension, extrusion, spray drying, grinding, emulsification, (nano / micron) encapsulation, packaging, or lyophilization processes. As described above, the components of the present invention may include one or more physiologically acceptable inactive ingredients that facilitate the processing of active molecules into formulations for pharmaceutical use.

[0326] The appropriate formulation depends on the desired route of administration. For example, for intravenous administration, the composition may be formulated in an aqueous solution, using physiologically compatible buffers, including, for example, phosphates, histidines, or citrates for adjusting the pH of the formulation, and permeabilizers, such as sodium chloride or dextran. For transmucosal or nasal administration, semi-solid, liquid, or patch formulations may be preferred, possibly containing permeation enhancers. These permeabilizers are generally known in the art. For oral administration, the compound may be formulated in liquid or solid dosage forms and as a rapid-release or controlled-release / sustained-release formulation. Suitable dosage forms for individual oral intake include tablets, pills, sugar-coated pills, hard-shell and soft-shell capsules, liquids, gels, syrups, ointments, suspensions, and emulsions. The compound may also be formulated in rectal components, such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

[0327] Solid oral dosage forms can be obtained using excipients, which include fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, flow aids, anti-adhesion agents, cationic exchange resins, humectants, antioxidants, preservatives, colorants, and flavoring agents. These excipients may be of synthetic or natural origin. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gums, magnesium carbonate, magnesium lauryl sulfate / sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salts, sugars (i.e., dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, hydrogenated vegetable oils, and waxes. Ethanol and water can be used as granulation aids. In some cases, it is necessary to coat the tablets with, for example, a taste-masking film, an acid-resistant film, or a delayed-release film. Natural and synthetic polymers are often combined with colorants, sugars, and organic solvents or water to coat tablets, resulting in sugar-coated pills. When capsules are preferred over tablets, their drug powders, suspensions, or solutions can be delivered in compatible hard-shell or soft-shell capsule forms.

[0328] In one embodiment, the compounds of the present invention can be administered topically, for example via skin patches, semi-solid or liquid formulations such as gels, (micro)emulsions, ointments, solutions, (nano / micro) suspensions, or foams. Skin and sub-tissue penetration of the drug can be modulated, for example, by using penetration enhancers; by using appropriate selections and combinations of lipophilic, hydrophilic, and bipolar excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, and emulsifiers; by adjusting the pH value; and by using chelating agents. Other techniques, such as iontophoresis, can also be used to modulate the skin penetration of the compounds of the present invention. For example, transdermal or local administration is preferred in cases where local administration with minimal systemic exposure is required.

[0329] For administration by inhalation or nasal administration, the compounds used according to the invention are conveniently administered from a pressurized pack or nebulizer in the form of a solution, suspension, emulsion, or semi-solid aerosol, typically by means of a propellant, such as carbon halide derived from methane and ethane, carbon dioxide, or any other suitable gas. For localized aerosols, hydrocarbons such as butane, isobutene, and pentane are suitable. In the case of pressurized aerosols, appropriate dosage units can be determined by providing a valve to deliver the metering amount. Capsules and cartridges containing, for example, gelatin can be formulated for use in inhalers or blowpipes. These typically contain a powder mixture of the compound with a suitable powder matrix (such as lactose or starch).

[0330] Compounds and compositions formulated for non-enteral administration by injection are generally sterile and can be provided in unit dosage forms, such as ampoules, syringes, injection pens, or multi-dose containers, which typically contain preservatives. Compositions may be in the form of suspensions, solutions, or emulsions in oily or aqueous media and may contain formulation agents such as buffers, penetrants, viscosity enhancers, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents, and preservatives. Depending on the injection site, the media may contain water, synthetic or vegetable oils, and / or organic co-solvents. In some cases, such as for lyophilized products or concentrates, the non-enteral formulation may be reconstituted or diluted prior to administration. Reservoir formulations providing controlled-release or sustained-release of the compounds of the present invention may comprise nano / micron-sized particles, or injectable suspensions of nano / micron-sized or non-micronized crystals. Polymers such as poly(lactic acid), poly(glycolic acid), or copolymers thereof can be used as controlled-release / sustain-release matrices, as can other well-known matrices in this technology. Other reservoir-type drug delivery systems can be provided in the form of implants requiring incisions and pumps.

[0331] Suitable carriers for intravenous injection of the compounds of the present invention are well known in the art and include aqueous solutions containing a base (such as sodium hydroxide) for forming ionic compounds, sucrose or sodium chloride as a permeabilizing agent, and buffers containing phosphates or histidine. Cosolvents such as polyethylene glycol may be added. These aqueous systems effectively dissolve the compounds of the present invention and produce low toxicity after systemic administration. The proportions of the components in the solution system can be significantly altered without compromising solubility and toxicity characteristics. Furthermore, the nature of the components can be modified. For example, low-toxicity surfactants such as polysorbates or poloxamer may be used, as may polyethylene glycol or other cosolvents, biocompatible polymers such as polyvinylpyrrolidone may be added, and other sugars and polyols may be used to replace dextrose.

[0332] The effective therapeutic dose can first be estimated using various techniques well known in the art. The initial dose for animal studies can be based on the effective concentration established in cell culture assays. A suitable dose range for human individuals can be determined, for example, using data obtained from animal studies and cell culture assays. In some embodiments, the compounds of the present invention are formulated for oral administration. In pharmaceutical formulations for oral administration, an exemplary dose of the compounds of the present invention is about 0.5 to about 10 mg / kg body weight. In some embodiments, the pharmaceutical formulation contains about 0.7 to about 5.0 mg / kg body weight, or about 1.0 to about 2.5 mg / kg body weight. A typical dosing regimen for oral administration would be to administer the oral pharmaceutical formulation three times a week, twice a week, once a week, or daily.

[0333] The effective amount or therapeutically effective amount or dose of a pharmaceutical agent (such as the compounds of the present invention) refers to the amount of the agent or compound that causes improvement in individual symptoms or prolongs survival. The toxicity and therapeutic efficacy of the molecule can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures, for example, by determining the LD50 (the dose that is 50% lethal to a population) and ED50 (the dose that is 50% therapeutically effective to a population). The dose ratio of toxicity to therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Pharmaceutical agents exhibiting a high therapeutic index are preferred.

[0334] An effective or therapeutically effective amount is the amount of a compound or pharmaceutical ingredient that will elicit a biological or medical response in a tissue, system, animal, or human that is being sought by researchers, veterinarians, physicians, or other clinicians. The dosage is preferably within a cyclic concentration range including the ED50, which has minimal or no toxicity. The dosage may vary within this range depending on the dosage form and / or route of administration. The precise formulation, route of administration, dosage, and dosing interval should be selected based on methods known in this art, taking into account the specificities of individual circumstances.

[0335] Dosage and intervals can be individually adjusted to provide a sufficient plasma concentration of the active fraction to achieve the desired effect; i.e., minimum effective concentration (MEC). The MEC will vary for each compound but can be estimated, for example, from in vitro data and animal studies. The dose necessary to obtain the MEC will depend on individual characteristics and route of administration. In cases of local administration or selective absorption, the effective local concentration of the drug may be independent of plasma concentration.

[0336] The amount of the drug or composition administered may be determined by various factors, including the sex, age and weight of the individual being treated, the severity of the illness, the method of administration and the judgment of the prescribing physician.

[0337] When necessary, the compounds and compositions of the present invention can be provided using packaging or dispensing devices containing one or more unit dosage forms (containing the active ingredient). For example, the packaging or device may comprise metal or plastic foil (such as foam packaging) or glass and rubber stoppers, such as in vials. The packaging or dispensing device may be accompanied by instructions for use. Compositions of the compounds of the present invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in a suitable container, and labeled for the treatment of a specified condition.

[0338] In view of the disclosure herein, these and other embodiments of the present invention will readily conceive of by those skilled in the art and are expressly covered by the present invention. Example of illustrative compound synthesis: Example 1: Preparation of compound 1

[0339] 6-Chloronicotinic acid tert-butyl ester

[0340] To a solution of 6-chloronicotinic acid (5.0 g, 6.37 mmol) and 4-dimethylaminopyridine (0.39 g, 0.64 mmol) in tetrahydrofuran (50.0 mL), ditert-butyl dicarbonate (10.41 g, 47.77 mmol) was added. The reaction mixture was stirred under reflux for 4 hours and concentrated. The residue was purified by rapid chromatography (petroleum ether / ethyl acetate = 10 / 1) to give 6-chloronicotinic acid tributyl ester (5.5 g, yield 81.12%) as a yellow solid. LC-MS: m / z = 214.0 (M+H)+, residence time 1.83 min (Method A).

[0341] 6-Hydroxynicotinic acid tert-butyl ester

[0342] To a solution of 6-chloronicotinic acid tributyl ester (5.5 g, 25.82 mmol) in ethanol (25.0 mL), hydrazine hydrate (6.46 g, 129.11 mmol, 85% aqueous solution) was added. The mixture was stirred at 100 °C for 2 hours and concentrated to dryness. The residue was partitioned between ethyl acetate and water. The organic phase was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was ground together with petroleum ether and filtered to give 6-hydrazinotinic acid tributyl ester (5.0 g, 92.76% yield) as a yellow solid. LC-MS: m / z = 210.0 (M+H)+, residence time 1.19 min (Method A).

[0343] (E)-3-(dimethylamino)-2-(p-tolyl)ethyl acrylate

[0344] In a solution of 1.00 g (5.61 mmol) of ethyl 2-(p-tolyl)acetate in N,N-dimethylformamide (10.0 mL), N,N-dimethylformamide diethyl acetal (3.34 g, 28.05 mmol, 3.73 mL) was added. The reaction mixture was stirred at 100 °C for 3 hours and cooled to room temperature. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give ethyl (E)-3-(dimethylamino)-2-(p-tolyl)acrylate (1.1 g, 4.71 mmol, 84% yield). LC-MS: m / z = 234.0 [M+H]+, residence time 2.064 min (Method B). The product was of sufficient purity and was used directly in the next step.

[0345] 6-(5-hydroxy-4-(p-tolyl)-1H-pyrazole-1-yl) tert-butyl nicotinate

[0346] In an ethanol solution (10.0 mL) of (E)-3-(dimethylamino)-2-(p-tolyl)acrylate (50.00 mg, 2.14 mmol) and 6-hydrazinoic acid tributyl ester (448.4 mg, 2.14 mmol), p-toluenesulfonic acid monohydrate (40 mg, 0.21 mmol) was added. The reaction mixture was stirred at 90 °C for 16 hours and cooled to form a precipitate. The crude solid was purified by rapid chromatography (dichloromethane / ethyl acetate = 10 / 1) to give 6-(5-hydroxy-4-(p-tolyl)-1H-pyrazol-1-yl)nicotinic acid tributyl ester (393 mg, 1.11 mmol, yield 52%) as a yellow solid. LC-MS: m / z = 352.0 [M+H]+, residence time 6.78 min (Method A). ¹H NMR (400 MHz, DMSO-d⁶) δ 12.86 (s, ¹H), 8.92 (s, ¹H), 8.80–8.15 (m, ³H), 7.78 (s, 2H), 7.17 (d, J = 8.0 Hz, 2H), 2.29 (s, 3H), 1.58 (s, 9H). Example 2: Preparation of Compound 2

[0347] (E)-2-(4-bromophenyl)-3-(dimethylamino)acrylate ethyl ester

[0348] In a solution of ethyl 2-(4-bromophenyl)acetate (1.01 g, 4.15 mmol) and N,N-dimethylformamide (20.0 mL), N,N-dimethylformamide diethyl acetal (2.47 g, 20.7 mmol) was added. The mixture was stirred at 100 °C for 16 hours and cooled to room temperature. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give ethyl (E)-2-(4-bromophenyl)-3-(dimethylamino)acrylate (980 mg, 3.32 mmol, 80% yield) as a yellow oil. LC-MS: m / z = 298.0 [M+H]+, residence time 1.710 min (Method B). The product was of sufficient purity and was used directly in the next step.

[0349] 6-(4-(4-bromophenyl)-5-hydroxy-1H-pyrazole-1-yl) tert-butyl nicotinate

[0350] To an ethanolic solution (10.0 mL) of (E)-2-(4-bromophenyl)-3-(dimethylamino)acrylate (300.0 mg, 1.01 mmol) and 6-hydrazinoic acid tributyl ester (210.5 mg, 1.01 mmol), p-toluenesulfonic acid monohydrate (38 mg, 0.2 mmol) was added. The mixture was stirred at 80 °C for 16 hours and cooled to form a precipitate. The crude product was purified by rapid chromatography (dichloromethane / methanol = 100 / 3) to give a yellow solid, 6-(4-(4-bromophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (286 mg, 0.69 mmol, yield 68%). LC-MS: m / z = 416.0 (M+H)+, retention time 6.76 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.13 (br, 1H), 8.91 (s, 1H), 8.80–8.25 (m, 3H), 7.89 (br, 2H), 7.53 (d, J = 8.0 Hz, 2H), 1.58 (s, 9H). Example 3: Preparation of Compound 3

[0351] 6-(4-(4-bromophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0352] Trifluoroacetic acid (0.5 mL) was added to a 10.0 mL solution of 6-(4-(4-bromophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (80.00 mg, 0.19 mmol). The mixture was stirred overnight at room temperature and concentrated. The residue was ground together with ethyl acetate and filtered to give 6-(4-(4-bromophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid (67 mg, 0.18 mmol, 97% yield) as a yellow solid. LC-MS: m / z = 360.0 (M+H)+, residence time 4.995 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.29 (br, 2H), 8.96 (s, 1H), 8.51–8.44 (m, 3H), 7.89 (d, J = 7.5 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H). Example 4: Preparation of compound 4

[0353] (E)-3-(dimethylamino)-2-(4-chlorophenyl)acrylate ethyl ester

[0354] In a solution of 2-(4-chlorophenyl)ethyl acetate (5.0 g, 25.25 mmol) in N,N-dimethylformamide (25.0 mL), N,N-dimethylformamide diethyl acetal (12.0 g, 101.01 mmol) was added. The mixture was stirred overnight at 100 °C and cooled to room temperature. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give ethyl (E)-3-(dimethylamino)-2-(4-chlorophenyl)acrylate (3.5 g, 14.04 mmol, yield 55.60%) as a colorless oil. LC-MS: m / z = 254.1 [M+H]+, residence time 2.030 min (Method A). The product was of sufficient purity and was used directly in the next step.

[0355] 6-(4-(4-chlorophenyl)-5-hydroxy-1H-pyrazole-1-yl) tert-butyl nicotinate

[0356] To an ethanol solution (10.0 mL) of (E)-3-(dimethylamino)-2-(4-chlorophenyl)acrylate (0.83 g, 3.95 mmol) and 6-hydrazinoic acid tributyl ester (1.00 g, 3.95 mmol), p-toluenesulfonic acid monohydrate (150 mg, 0.79 mmol) was added. The mixture was stirred under reflux for 12 hours and cooled to form a precipitate. The solid was filtered, washed with ethanol, and dried to give a white solid of 6-(4-(4-chlorophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (800.0 mg, 2.16 mmol, yield 54.79%). LC-MS: m / z = 372.1 (M+H)+, residence time 6.645 min (Method A). ¹H NMR (400 MHz, DMSO-d⁶) δ 9.04 – 8.86 (m, 1H), 8.62 – 8.33 (m, 3H), 7.95 (d, J = 7.2 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 1.58 (s, 9H). Example 5: Preparation of compound 5

[0357] 6-(4-(4-chlorophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0358] Trifluoroacetic acid (5.0 mL) was added to a 10.0 mL solution of 6-(4-(4-chlorophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (200.00 mg, 0.54 mmol) in dichloromethane. The mixture was stirred at 40 °C for 2 hours and concentrated. The residue was ground together with ethyl acetate and filtered to give 6-(4-(4-chlorophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid (74.0 mg, 0.24 mmol, yield 43.52%) as a white solid. LC-MS: m / z = 316.0 (M+H)+, residence time 4.718 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.23 (s, ¹H), 8.97 (s, ¹H), 8.67–8.47 (m, ³H), 8.04–7.97 (m, 2H), 7.41 (d, J = 3.8 Hz, 2H). Example 6: Preparation of compound 6

[0359] (E)-3-(dimethylamino)-2-(4-fluorophenyl)acrylate ethyl ester

[0360] In a solution of 2-(4-fluorophenyl)ethyl acetate (2.0 g, 10.98 mmol) in N,N-dimethylformamide (5.0 mL), N,N-dimethylformamide diethyl acetal (8.07 g, 54.8 mmol) was added. The mixture was stirred overnight at 100 °C and cooled to room temperature. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give ethyl (E)-3-(dimethylamino)-2-(4-fluorophenyl)acrylate (2.0 g, 8.45 mmol, 77% yield) as a colorless oil. LC-MS: m / z = 238.0 [M+H]+, residence time 1.89 min (Method B). The product was of sufficient purity and was used directly in the next step.

[0361] 6-(4-(4-fluorophenyl)-5-hydroxy-1H-pyrazole-1-yl) tert-butyl nicotinate

[0362] To an ethanol solution (5.0 mL) of (E)-3-(dimethylamino)-2-(4-fluorophenyl)acrylate (567.0 mg, 2.39 mmol) and 6-hydrazinoic acid tributyl ester (500.0 mg, 2.39 mmol), p-toluenesulfonic acid monohydrate (91.2 mg, 0.48 mmol) was added. The mixture was stirred under reflux for 12 hours and cooled to form a precipitate. The solid was filtered, washed with ethanol, and dried to give a white solid of 6-(4-(4-fluorophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (350.0 mg, 0.98 mmol, yield 41%). LC-MS: m / z = 356.0 (M+H)+, residence time 6.23 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 8.41 (s, 3H), 7.93 (s, 2H), 7.19 (s, 2H), 1.57 (s, 9H). Example 7: Preparation of compound 7

[0363] 6-(4-(4-fluorophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0364] Trifluoroacetic acid (2.0 mL) was added to a 5.0 mL solution of 6-(4-(4-fluorophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (150.0 mg, 0.42 mmol) in dichloromethane. The mixture was stirred at 40 °C for 2 hours and concentrated. The residue was ground together with ethyl acetate and filtered to give 6-(4-(4-fluorophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid (103.2 mg, 0.34 mmol, yield 81%) as a white solid. LC-MS: m / z = 300.0 (M+H)+, residence time 4.23 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 8.95 (s, ¹H), 8.45 (s, ³H), 7.94 (s, ²H), 7.20–7.17 (m, ²H). Example 8: Preparation of compound 8

[0365] Ethyl 2-(4-cyano-2-methylphenyl)acetate

[0366] A mixture of 4-bromo-3-methylbenzonitrile (5.0 g, 25.6 mmol), diethyl malonate (27 g, 168 mmol), tris(dibenzylacetone)palladium(0) (0.24 g, 0.26 mmol), tris(tert-butylphosphine)tetrafluoroborate (0.08 g, 0.26 mmol), potassium carbonate (5.3 g, 38.4 mmol), and potassium bicarbonate (3.84 g, 38.4 mmol) was stirred at 160 °C for 12 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and water. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was purified by rapid chromatography (petroleum ether / ethyl acetate = 1 / 1) to give ethyl 2-(4-cyano-2-methylphenyl)acetate (2.0 g, 31.7% yield) as a yellow oil. LC-MS: m / z 204.1 (M+H)+.

[0367] (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate ethyl ester

[0368] In a solution of ethyl 2-(4-cyano-2-methylphenyl)acetate (1.0 g, 5.0 mmol) in N,N-dimethylformamide (10.0 mL), N,N-dimethylformamide diethyl acetal (2.9 g, 25.0 mmol) was added. The mixture was stirred overnight at 100 °C and cooled to room temperature. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was purified by rapid chromatography (dichloromethane / methanol = 98 / 2) to give ethyl (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate (600 mg, yield 47.2%) as a yellow oil. LC-MS: m / z 259.0 (M+H)+.

[0369] 6-Chloronicotinic acid tert-butyl ester

[0370] In a THF (50.0 mL) solution of 6-chloronicotinic acid (5.0 g, 6.37 mmol) and 4-dimethylaminopyridine (0.39 g, 0.64 mmol), ditert-butyl dicarbonate (10.41 g, 47.77 mmol) was added. The reaction mixture was refluxed for 4 hours and concentrated. The residue was purified by rapid chromatography (petroleum ether / ethyl acetate = 10 / 1) to give 6-chloronicotinic acid tributyl ester (5.5 g, yield 81.12%) as a yellow solid. LC-MS: m / z 214.0 (M+H)+.

[0371] 6-Hydroxynicotinic acid tert-butyl ester

[0372] To an ethanol solution (25.0 mL) of 6-chloronicotinic acid tributyl ester (5.5 g, 25.82 mmol), hydrazine hydrate (6.46 g, 129.11 mmol, 85% aqueous solution) was added. The mixture was stirred at 100 °C for 2 hours and concentrated. The residue was partitioned between ethyl acetate and water. The organic phase was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was ground together with petroleum ether and filtered to give 6-hydrazinotinic acid tributyl ester (5.0 g, 92.76% yield) as a yellow solid. LC-MS: m / z 210.0 (M+H)+.

[0373] 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazole-1-yl)tert-butyl nicotinate

[0374] To a 10.0 mL ethanol solution of (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate (260 mg, 1.0 mmol) and 6-hydrazinoic acid tributyl ester (200 mg, 1.0 mmol), 4-methylbenzenesulfonic acid (34.4 mg, 0.2 mmol) was added. The mixture was stirred under reflux for 12 hours and cooled to form a precipitate. The solid was filtered, washed with ethanol, and dried to give a white solid of 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (190 mg, 50% yield). LC-MS: m / z 377.0 (M+H)+. Example 9: Preparation of Compound 9

[0375] Ethyl 3-ethoxy-2-(4-cyanophenyl)but-2-enoate

[0376] Under N2 conditions, LHMDS (28.00 mL, 27.75 mmol) was added dropwise to a mixture of ethyl 2-(4-cyanophenyl)acetate (3.50 g, 18.50 mmol) in anhydrous THF (100 mL) over 20 minutes at -30°C. The reaction mixture was heated to 0°C and stirred at 0°C for 30 minutes. The reaction mixture was then cooled to -30°C, and an acetyl chloride (2.18 g, 27.75 mmol) in THF solution (8 mL) was added dropwise over 15 minutes. The reaction mixture was slowly heated to room temperature and stirred for 4 hours. After the reaction was completed by TLC analysis, the mixture was quenched with cold NH4Cl aqueous solution (50 mL) and extracted with EtOAc (3 x 80 mL). The combined organic phases were dried over anhydrous Na2SO4 (30 g), filtered, and concentrated under vacuum. The residue was purified by silicone column chromatography (EA:hexane = 1:50 to 1:5) to give the desired product (1.9 g) as a yellow oil. ¹H NMR δ ppm (300 MHz, CDCl₃) 7.68 (dd, J = 6.6, 1.8 Hz, 2H), 7.41 (dd, J = 6.6, 1.8 Hz, 2H), 4.11–4.18 (q, J = 6.9 Hz, 2H), 2.25 (s, 3H), 1.88 (s, 3H), 1.20 (t, J = 6.9 Hz, 3H).

[0377] 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl)nicotinic acid

[0378] 4-Hydroxybenzoic acid (1.06 g, 6.91 mmol) was added to a mixture of ethyl 2-(4-cyanophenyl)-3-sideoxybutyrate (0.30 g, 1.38 mmol) and HOAc (24 ml). The mixture was stirred overnight at 100 °C. After the reaction was completed by TLC analysis, the reactants were quenched with water (20 mL) and a large amount of solid precipitated. After filtration, the solid was dissolved in TEA (5 eq), water (20 mL), and MeOH (20 mL). The solution was extracted with EtOAc (3 x 10 mL), and the pH of the aqueous phase was adjusted to 3 with dilute HCl solution. A large amount of solid precipitated. The solid was collected by filtration to give the desired product (128 mg) as a yellow solid. LC-MS (ESI+): m / z 321 (M+H)+; HPLC purity 97.3%; 1H NMR (300 MHz, DMSO-d6) δ ppm 13.25 (brs, 2H), 8.95 (d, J = 1.5 Hz, 1H), 8.56 (d, J = 8.7 Hz, 1H), 8.41 (dd, J = 8.7 Hz, 1H), 7.89 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 8.1 Hz, 2H), 2.51 (s, 3H). Example 10: Preparation of compound 10

[0379] 6-Hydroxynicotinic acid tert-butyl ester

[0380] Hydrazine (0.35 g, 5.62 mmol) was added once to a 10 mL solution of 6-chloronicotinic acid tributyl ester (0.20 g, 0.94 mmol) in 1,4-dioxane. The mixture was stirred overnight at 80 °C. After the reaction was completed by TLC analysis, the mixture was concentrated under vacuum. The residue was purified by preparative HPLC to give 180 mg of the title compound as an oil. 1H NMR (300 MHz, CDCl3) δ ppm 8.71 (d, J = 1.8 Hz, 1H), 8.01 (dd, J = 8.1, 1.8 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 6.24 (brs, 1H), 3.90 (brs, 2H), 1.60 (s, 9H).

[0381] 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl) tert-butyl nicotinate

[0382] This compound was synthesized using 6-hydrazinoic acid tributyl ester according to the preparation procedure of 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI-): m / z 375 (MH)-; HPLC purity 96.3%; 1H NMR (300 MHz, CDCl3) δ ppm 13.10 (brs, 1H), 8.88 (d, J = 2.1 Hz, 1H), 8.42 (dd, J = 8.7, 2.1 Hz, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.69 (s, 4H), 2.46 (s, 3H), 1.63 (s, 9H). Example 11: Preparation of compound 11

[0383] Methyl 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl)nicotinate

[0384] This compound was synthesized using methyl 6-hydrazinoic acid according to the preparation procedure of 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS: m / z = 335 (M+H)+; 1H NMR (300 MHz, DMSO-d6) δ ppm 13.11 (s, 1H), 8.95 (d, J = 1.6 Hz, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.43 (dd, J = 7.0, 2.3 Hz, 1H), 7.90 (dd, J = 17.6, 8.3 Hz, 2H), 7.79 (dd, J = 14.5, 8.4 Hz, 2H), 3.90 (s, 3H), 2.48 (s, 3H). Example 12: Preparation of Compound 12

[0385] Di-tert-butyl 2-(4-cyano-2,5-difluorophenyl)malonate

[0386] Under nitrogen atmosphere, di-tert-butyl malonate (8.26 g, 31.19 mmol) was added dropwise to 100 mL of anhydrous DMF solution of sodium hydride (60% dispersion in mineral oil) (5.09 g, 127.31 mmol) at 0°C for 15 minutes. After stirring the reaction mixture at 0°C for 15 minutes, 2,4,5-trifluorobenzonitrile (5.00 g, 31.83 mmol) was added to the reaction mixture in one batch. The mixture was stirred at 0°C for 15 minutes, followed by stirring at 60°C overnight. After the reaction was indicated by TLC analysis, the mixture was quenched with saturated aqueous NH4Cl solution (800 mL) and extracted with EtOAc (200 mL x 3). The combined organic phases were washed with water (100 mL x 2), dried over anhydrous Na2SO4 (50 g), filtered, and concentrated under vacuum. 12.60 g of crude title compound as a yellow oil was obtained and used in the next step without further purification. LC-MS (ESI+): m / z 239 (M-(t-Bu)2)+.

[0387] 2-(4-cyano-2,5-difluorophenyl)acetic acid

[0388] TFA (35 mL) was added in a single addition to a DCM solution (35 mL) of di-tert-butyl 2-(4-cyano-2,5-difluorophenyl)malonate (12.30 g, 34.80 mmol). The reaction mixture was stirred overnight at room temperature. After the reaction was indicated by TLC analysis, the mixture was concentrated under vacuum. Toluene (50 mL) was added to the residue, stirred, and concentrated to dryness. 6.07 g of crude title compound as a white solid was given and used in the next step without further purification. LC-MS (ESI+): m / z 216 (M+H+H2O)+.

[0389] Methyl 2-(4-cyano-2,5-difluorophenyl)acetate

[0390] SOCl2 (4.0 mL) was added to a MeOH solution of 2-(4-cyano-2,5-difluorophenyl)acetic acid (4.00 g, 20.29 mmol) in 160 mL at room temperature for 5 minutes. The reaction mixture was stirred at 70 °C for 3 hours. After the reaction was completed by TLC analysis, 80% of the reaction solvent was evaporated and removed. The residue was quenched with ice water (180 mL) and extracted with EtOAc (50 mL x 3). The combined organic phases were dried over anhydrous Na2SO4 (50 g), filtered, and concentrated under vacuum. 4.30 g of crude title compound as a yellow oil was given and used in the next step without further purification. LC-MS (ESI+): m / z 230 (M+H+H2O)+.

[0391] (E)-2-(4-cyano-2,5-difluorophenyl)-3-(dimethylamino)methyl acrylate

[0392] This compound was synthesized using methyl 2-(4-cyano-2,5-difluorophenyl)acetate according to the preparation procedure for ethyl (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 267 (M+H)+.

[0393] 6-(4-(4-cyano-2,5-difluorophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0394] This compound was synthesized using (E)-2-(4-cyano-2,5-difluorophenyl)-3-(dimethylamino)acrylate according to the preparation procedure of 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI-): m / z 341 (MH)-, HPLC purity 95.4%, 1H NMR (300 MHz, DMSO-d6) δppm 13.41 (brs, 1H), 8.96 (d, J = 2.1 Hz, 1H), 8.58 – 8.42 (m, 3H), 8.31 (d, J = 3.0 Hz, 1H), 7.93 (dd, J = 10.8, 5.4 Hz, 1H). Example 13: Preparation of Compound 13

[0395] Methyl 2-(3-fluoro-4-hydroxyphenyl)acetate

[0396] Concentrated H₂SO₄ (0.2 mL) was added to a mixture of 2-(3-fluoro-4-hydroxyphenyl)acetic acid (2.0 g, 11.76 mmol) in MeOH (20 mL). The reaction mixture was incubated at 65 °C for 2 hours. After the reaction was completed by TLC analysis, the mixture was quenched with water (40 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried, filtered, and concentrated directly to give the title product (2.49 g) as an oil. ¹H NMR (300 MHz, CDCl₃) δ ppm 7.03 (d, J = 1.5 Hz, 1H), 6.91 (dd, J = 3.9, 1.5 Hz, 2H), 3.70 (s, 3H), 3.54 (s, 2H).

[0397] Methyl 2-(3-fluoro-4-(((trifluoromethyl)sulfonyl)oxy)phenyl)acetate

[0398] Under N2 atmosphere, trifluoromethanesulfonic anhydride (5.71 g, 20.25 mmol) was added dropwise to a mixture of methyl 2-(3-fluoro-4-hydroxyphenyl)acetate (2.49 g, 13.50 mmol) in DCM (52 mL) over 15 minutes at 0°C. TEA (4.10 g, 40.50 mmol) was added dropwise to the reactant over 10 minutes. The reactants were stirred at 0°C for 4 hours. After TLC analysis, the mixture was quenched with an aqueous solution of NaHCO3 (30 mL) and extracted with DCM (3 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4 (50 g), filtered, and concentrated under vacuum. The residue was purified by silicone column chromatography (EA: n-hexane = 1: 20) to give the desired product (2.18 g) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ ppm7.30 (d,J= 8.1 Hz, 1H), 7.24 (dd,J= 3.3, 1.11 Hz, 1H), 7.13 (d,J= 8.1 Hz, 1H), 3.73 (s, 3H), 3.65 (s, 2H).

[0399] Methyl 2-(4-cyano-3-fluorophenyl)acetate

[0400] Under N2 atmosphere, Zn(CN)2 (0.49 g, 4.14 mmol) and Pd(PPh3)4 (0.80 g, 0.69 mmol) were added to a mixture of methyl 2-(3-fluoro-4-(((trifluoromethyl)sulfonyl)oxy)phenyl)acetate (2.18 g, 6.90 mmol) in anhydrous DMF (40 mL). The reaction mixture was stirred at 80 °C for 4 hours. After the reaction was completed by TLC analysis, the mixture was quenched with water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with water (2 x 20 mL), dried over anhydrous Na2SO4 (50 g), filtered, and concentrated under vacuum. The residue was purified by silicone column chromatography (EtOAc:hexane = 1:40 to 1:8) to give the desired product (1.27 g) as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ ppm 7.59 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 8.7 Hz, 2H), 3.73 (s, 3H), 3.70 (s, 2H).

[0401] (E)-2-(4-cyano-3-fluorophenyl)-3-(dimethylamino)methyl acrylate

[0402] This compound was synthesized using methyl 2-(4-cyano-3-fluorophenyl)acetate according to the preparation procedure for ethyl (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 249 (M+H)+.

[0403] 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0404] This compound was synthesized using (E)-2-(4-cyano-3-fluorophenyl)-3-(dimethylamino)acrylate according to the preparation procedure of 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI-): m / z 323 (MH)-; 1H-NMR (300 MHz, DMSO-d6) δ ppm 13.32 (brs, 1H), 8.77 (d, J = 1.2 Hz, 1H), 8.55 (s, 1H), 8.34 (d, J = 8.4 Hz, 1H), 8.26 (dd, J = 8.4, 2.1 Hz, 1H), 7.88 (d, J = 12.3 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H). Example 14: Preparation of Compound 14

[0405] 5-Fluoro-6-hydrazinoic acid

[0406] N₂H₄·H₂O (0.81 g, 12.95 mmol) was added to a THF solution (22 mL) of 6-chloro-5-fluoronicotinic acid (0.46 g, 2.59 mmol) for 1 minute. After stirring the mixture overnight at 65°C, a large amount of solid precipitated out. The suspension was filtered and the solid was slurried in MeOH (3 mL) for 1 hour. After filtration, 440 mg of the desired product as a white solid was obtained. LC-MS (ESI+): m / z 172 (M+H)⁺.

[0407] 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazole-1-yl)-5-fluoronicotinic acid

[0408] This compound was synthesized using 5-fluoro-6-hydrazinoic acid and (E)-2-(4-cyanophenyl)-3-(dimethylamino)acrylate, according to the preparation procedure for 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 325 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ ppm 8.78 (s, 1H), 8.08 (d, J = 9.9 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.12 (brs, 2H). Example 15: Preparation of Compound 15

[0409] Methyl 2-(4-chlorophenyl)-3-t-oxybutyrate

[0410] Under nitrogen atmosphere, LHMDS (8.1 mL, 8.11 mmol) was added dropwise to an anhydrous THF solution (50 mL) of methyl 2-(4-chlorophenyl)acetate (1.00 g, 5.41 mmol) at -40 °C for 10 minutes. After stirring the resulting mixture at -40 °C for 1 hour, 1-(1H-imidazol-1-yl)ethyl-1-one (0.89 g, 8.11 mmol) was added fractionally over 10 minutes. After the addition, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was indicated by TLC analysis, the residue was diluted with water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic phases were dried over Na2SO4 (30 g), filtered, and concentrated to dryness to give the desired product (1.6 g) as an oil. LC-MS (ESI+): m / z 249 (M+Na)+.

[0411] 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0412] This compound was synthesized using methyl 2-(4-chlorophenyl)-3-sideoxybutyrate according to the preparation procedure of 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 330 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ ppm 12.77–13.27 (m, 2H), 8.95 (d, J = 1.5 Hz, 1H), 8.55 (brs, 1H), 8.41 (dd, J = 9.0 Hz, J = 2.1 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 2.42 (s, 3H). Example 16: Preparation of Compound 16

[0413] Methyl 2-(4-(benzooxy)-3-methylphenyl)acetate

[0414] Bromotoluene (292 mg, 1.70 mmol) and Cs₂CO₃ (1.66 g, 5.10 mmol) were added to a DMF solution (10 mL) of methyl 2-(4-hydroxy-3-methylphenyl)acetate (300 mg, 1.70 mmol). The mixture was stirred at room temperature for 1 hour. After the reaction was analyzed by TLC, the mixture was quenched with water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic phases were dried over anhydrous Na₂SO₄ (30 g), filtered, and concentrated under vacuum. The residue was purified by silicone column chromatography (PE:EtOAc = 100:1 ~ 50:1) to give 498 mg of the title compound. 1H NMR (300 MHz, CDCl3) δ ppm 7.29-7.45 (m, 5H), 7.03-7.08 (m, 2H), 6.82 (d, J = 8.1 Hz, 1H), 5.06 (s, 2H), 3.68 (s, 3H), 3.54 (s, 2H), 2.27 (s, 3H).

[0415] (Z)-2-(4-(benzooxy)-3-methylphenyl)-3-(dimethylamino)acrylate methyl acrylate

[0416] This compound was synthesized using methyl 2-(4-(benzoxy)-3-methylphenyl)acetate according to the preparation procedure for ethyl (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 326 (M+H+).

[0417] 6-(4-(4-(benzooxy)-3-methylphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0418] This compound was synthesized using (Z)-2-(4-(benzoxy)-3-methylphenyl)-3-(dimethylamino)acrylate according to the preparation procedure for 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 402 (M+H+).

[0419] 6-(5-hydroxy-4-(4-hydroxy-3-methylphenyl)-1H-pyrazol-1-yl)nicotinic acid

[0420] 6-(4-(4-benzoxy-3-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid (60 mg, 0.12 mmol) was stirred with a MeOH solution of Pd / C (10 mg) for 7 hours under a hydrogen atmosphere from a balloon. After the reaction was indicated by TLC, the suspension was filtered through a Celite kit and the filter cake was washed with MeOH (5 mL). The combined filtrates were concentrated and purified by preparative HPLC to give 9 mg of the title compound as a solid. LC-MS (ESI-): m / z 310 (MH-), 1H NMR (300 MHz, CD3OD) δ ppm 8.94 (s, 1H), 8.45 (d, J = 8.7 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.90 (s, 1H), 7.48 (s, 1H), 7.41 (d, J = 8.7 Hz, 1H), 6.75 (s, 1H), 6.65 (d, J = 6.9 Hz, 1H), 2.22 (s, 3H). Example 17: Preparation of Compound 17

[0421] (Z)-2-(6-chloropyridin-3-yl)-3-(dimethylamino)ethyl acrylate

[0422] This compound was synthesized using ethyl 2-(6-chloropyridin-3-yl)ethyl acetate according to the preparation procedure for (E)-2-(4-cyano-2-methylphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 241 (M+H+), 1H NMR (300 MHz, CDCl3) δ ppm 8.18 (d,J= 2.4 Hz, 1H), 7.66 (s, 1H), 7.51 (dd,J= 2.4, 8.1 Hz, 1H), 7.26 (d,J= 8.4 Hz, 1H), 3.64 (s, 3H), 2.75 (s, 6H).

[0423] 6-(4-(6-chloropyridin-3-yl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid

[0424] This compound was synthesized using (Z)-2-(6-chloropyridin-3-yl)-3-(dimethylamino)acrylate according to the preparation procedure of 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 317 (M+H+), 1H NMR (300 MHz, DMSO-d6) δ13.44 (brs, 2H), 8.94 (d,J= 16.8 Hz, 2H), 8.67 (s, 1H), 8.55 (s, 1H), 8.44-8.48 (m, 1H), 8.38 (d,J= 7.8 Hz, 1H), 7.51 (d,J= 8.4 Hz, 1H). Example 18: Preparation of Compound 18

[0425] 6-Hydroxy-4-methylnicotinic acid

[0426] Hydrazine (2.00 g, 32.26 mmol) was added to a THF solution (50 mL) of 6-fluoro-4-methylnicotinic acid (0.50 g, 3.23 mmol). The reaction mixture was stirred at 66 °C for 2 hours. After the reaction was indicated by TLC, the mixture was diluted with ethanol (6 mL) and a large amount of solid precipitated out. After filtration, 725 mg of the desired product was given. LC-MS (ESI+): m / z 168 (M+H+), 1H NMR (300 MHz, DMSO-d6) δ 8.44 (s, 1H), 7.56 (brs, 1H), 6.43 (s, 1H), 5.90 (brs, 2H), 2.44 (s, 3H).

[0427] 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl)-4-methylnicotinic acid

[0428] Ethyl 2-(4-cyanophenyl)-3-sideoxybutyrate (0.43 g, 1.98 mmol) was added to a 20 ml solution of acetic acid containing 0.30 g (1.80 mmol) of hydrazino-4-methylnicotinic acid. The reaction mixture was stirred overnight at 100 °C. After the reaction was indicated by LC-MS analysis, the mixture was cooled to room temperature and a large amount of solid precipitated out. After filtration, the filter cake was further purified by preparative HPLC to give 36 mg of the desired product. LC-MS (ESI+): m / z 335 (M+H+), 1H-NMR (300 MHz, DMSO-d6) δ 12.97–13.21 (brs, 2H), 8.87 (s, 1H), 8.45 (brs, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 2.78 (s, 3H), 2.52 (s, 3H). Example 19: Preparation of Compound 19

[0429] 6-Hydroxy-2-methylnicotinic acid

[0430] This compound was synthesized using 6-fluoro-2-methylnicotinic acid according to the preparation procedure of 6-hydrazino-4-methylnicotinic acid. ¹H-NMR (300 MHz, D₂O) δ 7.63 (d, J = 8.7 Hz, ¹H), 6.55 (d, J = 8.7 Hz, ¹H), 2.37 (s, ³H).

[0431] 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)-2-methylnicotinic acid

[0432] This compound was synthesized using 6-hydrazino-2-methylnicotinic acid according to the preparation procedure for 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)-4-methylnicotinic acid. LC-MS (ESI+): m / z 335 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 12.95 (brs, 1H), 8.28-8.35 (m, 2H), 7.89 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 2.80 (s, 3H), 2.58 (s, 3H). Example 20: Preparation of Compound 20

[0433] Ethyl 2-(5-chloropyridin-2-yl)-3-sideoxybutyrate

[0434] Under N2 conditions, LHMDS (5.0 mL, 5.0 mmol) was added dropwise over 20 minutes at -55°C to a mixture of ethyl 2-(5-chloropyridin-2-yl)acetate (500 mg, 2.5 mmol) in anhydrous THF (15 mL). After stirring the reaction mixture at 0°C for 1 hour, a THF solution (15 mL) of 1-(1H-imidazol-1-yl)ethyl-1-one (412 mg, 3.75 mmol) was added dropwise over 15 minutes at -55°C. The reaction mixture was slowly heated to room temperature and stirred at room temperature for 1 hour. After the reaction was indicated by TLC analysis, the mixture was quenched with an aqueous solution of NH4Cl (50 mL) and extracted with EtOAc (3 x 20 mL). The combined organic phases were dried over anhydrous Na2SO4 (20 g), filtered, and concentrated under vacuum. The residue was purified by silicone column chromatography (EtOAc:hexane = 1:100) to give the title compound as a solid (152 mg). LC-MS (ESI+): m / z 242 (M+H)+.

[0435] 6-(4-(5-chloropyridin-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0436] This compound was synthesized using ethyl 2-(5-chloropyridin-2-yl)-3-sideoxybutyrate according to the preparation procedure of 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 331 (M+H)+; 1H NMR (300 MHz, DMSO-d6) δ 13.20 (brs, 2H), 8.95 (d, J = 1.8 Hz, 1H), 8.53–8.61 (m, 1H), 8.52 (s, 1H), 8.41 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H), 8.32 (d, J = 5.1 Hz, 1H), 7.87 (dd, J = 8.7 Hz, J = 2.7 Hz, 1H), 2.62 (s, 3H). Example 21: Preparation of compound 21

[0437] Methyl 2-(4-bromo-2-methoxyphenyl)acetate

[0438] SOCl2 (6 mL, 82.71 mmol) was added dropwise to a MeOH solution (35 mL) of 2-(4-bromo-2-methoxyphenyl)acetic acid (2.00 g, 8.16 mmol) at room temperature over 5 minutes. After the addition, the mixture was stirred overnight at 55 °C. After the reaction was indicated by TLC analysis, the reaction mixture was concentrated directly to dryness. The residue was diluted with an aqueous solution of NaHCO3 (50 mL) and extracted with EtOAc (100 mL × 3). The combined organic phases were dried over Na2SO4, filtered, and concentrated to give the desired product (2.05 g) as a yellow oil. LC-MS (ESI+): m / z 281 (M+Na)+; 1H-NMR (300 MHz, CDCl3) δ 7.02-7.08 (m, 2H), 7.00 (s, 1H), 3.93 (s, 3H), 3.81 (s, 3H), 3.57 (s, 2H).

[0439] Methyl 2-(4-cyano-2-methoxyphenyl)acetate

[0440] Under nitrogen protection, Zn(CN)₂ (2.74 g, 23.32 mmol), Pd₂(dba)₃ (0.11 g, 0.12 mmol), and S-Phos (0.48 g, 1.17 mmol) were added to a DMF solution (20 mL) of methyl 2-(4-bromo-2-methoxyphenyl)acetate (3.02 g, 11.66 mmol). The reaction mixture was stirred at 110 °C for 4 hours. After the reaction was indicated by TLC analysis, the mixture was concentrated to remove undissolved solids. The filtrate was directly concentrated to dryness. The residue was purified by rapid silica gel chromatography (EtOAc / PE = 1 / 50 to 1 / 10) to give the desired product (2.04 g) as a white solid. LC-MS (ESI+): m / z 206 (M+H)+; 1H-NMR (300 MHz, CDCl3) δ 7.23-7.30 (m, 3H), 3.86 (s, 3H), 3.71 (s, 3H), 3.67 (s, 2H).

[0441] (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate methyl acrylate

[0442] A solution of methyl 2-(4-cyano-2-methoxyphenyl)acetate (0.94 g, 4.57 mmol) in DMF-DMA (27.4 g) was stirred overnight at 100 °C. After the reaction was indicated by TLC analysis, the mixture was diluted with ice water (80 mL) and extracted with EtOAc (20 mL x 3). The combined organic phases were dried over Na2SO4, filtered, and concentrated to give the desired product (1.23 g) as a brown oil. LC-MS (ESI+): m / z 261 (M+H)+.

[0443] 6-(4-(4-cyano-2-methoxyphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0444] 6-Hydroxynicotinic acid (0.19 g, 1.24 mmol) and HCl (1.25 mL, 1 M, 1.24 mmol) were added to a suspension of (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate (0.32 g, 1.24 mmol) in i-PrOH (6 mL). After stirring the reaction mixture at room temperature for 6 hours, a large amount of solid precipitated out. The suspension was filtered. The filter cake was dissolved in i-PrOH (6 mL), water (1 mL), and DIEA (320 mg). The resulting mixture was stirred overnight at 50 °C. A dilute HCl solution (1 M, 5 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for another 20 minutes. A large amount of solid precipitated out. After filtration and drying, the filter cake was slurried in methanol (5 mL) overnight. After filtration and drying, 185 mg of the title compound was obtained. LC-MS (ESI+): m / z 335 (MH)-;1H-NMR (300 MHz, DMSO-d6) δ 13.5 (brs, 1H), 8.97 (s, 1H), 8.56-8.68 (brs, 2H), 8.45-8.47 (m, 2H), 7.44-7.49 (m, 2H), 3.96 (s, 3H).

[0445] 6-(4-(4-cyano-2-hydroxyphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0446] Under nitrogen protection, EtSNa (0.11 g, 1.34 mmol) was added once to 8 mL of a DMF solution of 6-(4-(4-cyano-2-hydroxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid (0.15 g, 0.45 mmol). The reaction mixture was stirred at 150 °C for 48 hours. After the reaction was indicated by TLC analysis, the mixture was diluted with water (80 mL) and the pH was adjusted to 2 with dilute HCl solution. A large amount of solid precipitated out. After filtration, the solid was purified by preparative HPLC to give 20 mg of the title compound as a yellow solid. LC-MS (ESI+): m / z 321 (MH)-; 1H-NMR (300 MHz, CD3OD) δ 9.10 (s, 1H), 8.30–8.50 (m, 2H), 8.20 (m, 1H), 7.82 (m, 1H), 7.12 (m, 1H), 7.02 (s, 1H). Example 22: Preparation of compound 22

[0447] 1-Chloro-2-methoxy-4-methylbenzene

[0448] K₂CO₃ (2.4 g, 17.48 mmol) and MeI (1.04 g, 7.34 mmol) were added to a DMF solution (4 ml) of 2-chloro-5-cresol (1 g, 6.99 mmol). The reaction mixture was stirred at room temperature for about 6 hours. After the reaction was indicated by TLC analysis, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic phases were washed with water (10 mL), dried over Na₂SO₄ and concentrated to dryness to give 1.05 g of crude product. GC-MS (EI+): 156;1H-NMR (300 MHz, CDCl3) δ 7.22 (d,J= 8.1Hz, 1H), 6.69-6.74 (m, 2H), 3.88 (s, 3H), 2.33 (s, 3H).

[0449] 4-(bromomethyl)-1-chloro-2-methoxybenzene

[0450] NBS (26.24 g, 0.15 mol) and BPO (1.62 g, 6.7 mmol) were added to a CCl4 solution of 1-chloro-2-methoxy-4-toluene (20.98 g, 0.13 mol) (200 mL). The resulting mixture was stirred overnight at 80 °C. After the reaction was completed by TLC analysis, the mixture was filtered, and the filtrate was diluted with dilute HCl solution (20 mL, 1N) and extracted with DCM (200 mL x 3). The combined organic phases were washed with saturated NaHCO3 solution (40 mL), dried over Na2SO4 (60 g), filtered, and concentrated to give 41.44 g of crude product. The crude product was further purified by silicone column chromatography (PE / EtOAc = 100 / :1 to 60 / 1) to give 32.6 g of the title compound. 1H-NMR (300 MHz, CDCl3) δ 7.32 (d,J= 8.1Hz, 1H), 6.90-6.96 (m, 2H), 4.54 (s, 2H), 3.93 (s, 3H).

[0451] 2-(4-chloro-3-methoxyphenyl)acetonitrile

[0452] NaCN (3.14 g, 64.05 mmol) was added once to a solution of 4-(bromomethyl)-1-chloro-2-methoxybenzene (10 g, 42.7 mmol) in EtOH (50 mL) and H2O (10 mL). The resulting mixture was stirred overnight at 80 °C. After the reaction was completed by TLC analysis, the reactants were diluted with water (100 mL) and extracted with DCM (200 mL x 2). The combined organic phases were washed with saturated NaHCO3 solution (50 mL) and concentrated brine (50 mL), dried, and concentrated to give 7.58 g of crude product. The crude product was purified by column chromatography (PE / EtOAc = 60 / 1 to 15 / 1) to give 5.58 g of the title compound. 1H-NMR (300 MHz, CDCl3) δ 7.34 (d,J= 8.1Hz, 1H), 6.83-6.88 (m, 2H), 3.91 (s, 3H), 3.73 (s, 2H).

[0453] 2-(4-chloro-3-methoxyphenyl)acetic acid

[0454] KOH (8.68 g, 155 mol) was added in a single addition to a solution of 2-(4-chloro-3-methoxyphenyl)acetonitrile (5.58 g, 31 mmol) in ethanol (120 mL) and water (40 mL). The reaction mixture was stirred under reflux for about 3 hours. After the reaction was indicated by TLC analysis, the mixture was concentrated to remove most of the ethanol. The residue was adjusted to pH 3 with dilute HCl solution and extracted with EtOAc (200 mL x 3). The combined organic phases were washed with water (30 mL), dried, and concentrated to dryness to give 7.29 g of crude product. 1H-NMR (300 MHz, DMSO-d6) δ 12.40 (brs, 1H), 7.34 (d,J= 7.8 Hz, 1H), 7.05 (d,J= 1.5 Hz, 1H), 6.84 (d,J= 7.8, 1.5 Hz, 1H), 3.92 (s, 3H), 3.72 (s, 2H).

[0455] Methyl 2-(4-chloro-3-methoxyphenyl)acetate

[0456] Concentrated H₂SO₄ (3.578 g, 36.45 mmol) was added once to a methanol (100 mL) solution of 2-(4-chloro-3-methoxyphenyl)acetic acid (7.29 g, 36.45 mmol). The reaction mixture was stirred at 70 °C for 2 hours. After the reaction was indicated by TLC analysis, the mixture was cooled to room temperature and concentrated to remove most of the methanol. The residue was diluted with water (20 mL) and extracted with EtOAc (50 mL x 2). The combined organic phases were dried and concentrated. The residue was purified by column chromatography (PE / EtOAc = 60 / 1 to 20 / 1) to give 4.68 g of the title compound. 1H-NMR (300 MHz, CDCl3) δ 7.29 (d,J= 8.1 Hz, 1H), 6.86 (d,J= 1.5 Hz, 1H), 6.80 (d,J= 7.8, 1.5 Hz, 1H), 3.90 (s, 3H), 3.72 (s, 3H), 3.60 (s, 2H).

[0457] (Z)-2-(4-chloro-3-methoxyphenyl)-3-(dimethylamino)acrylate methyl acrylate

[0458] This compound was synthesized using methyl 2-(4-chloro-3-methoxyphenyl)acetate according to the preparation procedure for (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 270 (M+H)+.

[0459] 6-(4-(4-chloro-3-methoxyphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0460] This compound was synthesized using (Z)-2-(4-chloro-3-methoxyphenyl)-3-(dimethylamino)acrylate methyl acrylate according to the preparation procedure for 6-(4-(4-cyano-2-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 346 (M+H)+.

[0461] 6-(4-(4-chloro-3-hydroxyphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0462] This compound was synthesized using 6-(4-(4-chloro-3-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid, following the preparation procedure for 6-(4-(4-cyano-2-hydroxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 332 (M+H)+; 1H-NMR (300 MHz, CDCl3) δ 13.30 (brs, 1H), 10.07 (s, 1H), 8.95 (s, 1H), 8.42-8.52 (m, 2H), 8.33 (s, 1H), 7.70 (s, 1H), 7.25 (s, 2H). Example 23: Preparation of Compound 23

[0463] Methyl 2-(4-bromo-3-fluorophenyl)acetate

[0464] This compound was synthesized using 2-(4-bromo-3-fluoro)acetic acid according to the preparation procedure for methyl 2-(4-bromo-2-methoxyphenyl)acetate. ¹H-NMR (300 MHz, CDCl₃) δ 7.49 (t, J = 8.1 Hz, 1H), 7.08 (d, J = 9.0 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 3.71 (s, 3H), 3.59 (s, 2H).

[0465] Methyl 2-(4-cyano-3-fluorophenyl)acetate

[0466] This compound was synthesized using methyl 2-(4-bromo-3-fluorophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-2-methoxyphenyl)acetate. ¹H-NMR (300 MHz, CDCl₃) δ 7.59 (t, J = 8.1 Hz, 1H), 7.19 (d, J = 8.7 Hz, 2H), 3.73 (s, 3H), 3.70 (s, 2H).

[0467] Methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate

[0468] Under nitrogen protection, LiHMDS (2.9 mmol, 2.9 mL) was added dropwise to a THF solution (15 mL) of methyl 2-(4-cyano-3-fluorophenyl)acetate (363 mg, 1.88 mmol) at -78°C over 10 minutes. After stirring the resulting mixture at -78°C for 30 minutes, a THF solution (5 mL) of 1-acetylimidazole (249 mg, 2.25 mmol) was added dropwise to the reactant over 10 minutes. The mixture was stirred at -78°C for 1 hour. After the reaction was indicated by TLC analysis, the reactants were quenched with saturated NH4Cl solution (20 mL) and extracted with EtOAc (30 mL x 2). The combined organic phases were dried and concentrated to dryness to give 410 mg of crude title compound, which was used in the next step without further purification. LC-MS (ESI-): m / z 234 (MH)–.

[0469] 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0470] This compound was synthesized using methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate according to the preparation procedure for 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)-4-methylnicotinic acid. LC-MS (ESI+): m / z 337 (M+H+), 1H NMR (300 MHz, DMSO-d6) δ 8.99 (d, J = 1.5 Hz, 1H), 8.34 (m, 1H), 8.26 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 11.7 Hz, 1H), 7.68 (m, 1H), 7.57 (m, 1H), 2.44 (s, 3H). Example 24: Preparation of Compound 24

[0471] 3-Methyl-4-vinylbenzonitrile

[0472] Under nitrogen protection, Cs₂CO₃ (33.3 g, 102.04 mmol) and Pd(dppf)Cl₂ (1.8 g, 2.55 mmol) were added to a THF / water solution of 4-bromo-3-methylbenzonitrile (5 g, 25.51 mmol) and potassium vinyltrifluoroborate (6.84 g, 51.02 mmol) (250 mL / 25 mL). The resulting mixture was stirred overnight at 70 °C. After the reaction was indicated by TLC analysis, the reactants were quenched with water (100 mL) and extracted with EtOAc (50 mL x 2). The combined organic phases were dried and concentrated. The residue was purified by column chromatography (PE:EtOAc = 20 / 1 to 10 / 1) to give 3.2 g of the title compound as a pale yellow oil. GC-MS (EI+): m / z 143 (M+).

[0473] 2-(4-cyano-2-methylphenyl)acetic acid

[0474] I2 (0.57 g, 2.24 mmol) was added once to a solution of 3-methyl-4-vinylbenzonitrile (3.2 g, 22.38 mmol) in DME (200 mL) and water (48 mL). Potassium persulfate (27.52 g, 44.76 mmol) was added fractionally to the resulting mixture over 10 minutes at room temperature. After the addition, the reaction mixture was stirred overnight at room temperature. Once the reaction was indicated by TLC analysis, the suspension was filtered to remove undissolved solids, and the filtrate was concentrated under vacuum to remove most of the organic solvent. The residue was diluted with saturated Na2S2O3 (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic phases were dried and concentrated. The residue was slurried with n-hexane (30 mL) for 30 minutes and filtered. After repeating the n-hexane slurry and filtration purification twice, 3.56 g of crude product was obtained, which was used in the next step without further purification.

[0475] Methyl 2-(4-cyano-2-methylphenyl)acetate

[0476] This compound was synthesized using 2-(4-bromo-2-methyl)acetic acid according to the preparation procedure for methyl 2-(4-bromo-2-methoxyphenyl)acetate. ¹H NMR (300 MHz, CDCl₃) δ 7.45–7.48 (m, 2H), 7.30 (d, J = 8.1 Hz, 1H), 3.71 (s, 3H), 3.69 (s, 2H), 2.34 (s, 3H).

[0477] Methyl 2-(4-cyano-2-methylphenyl)-3-sideoxybutyrate

[0478] This compound was synthesized using methyl 2-(4-cyano-2-methylphenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate. LC-MS (ESI+): m / z 232 (M+H)+.

[0479] 6-(4-(4-cyano-2-methylphenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0480] This compound was synthesized using methyl 2-(4-cyano-2-methylphenyl)-3-sideoxybutyrate according to the preparation procedure for 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 332 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 12.91 (brs, 2H), 8.95 (s, 1H), 8.52 (d, J = 8.7 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 8.23 ​​(s, 1H), 7.80 (s, 1H), 6.78 (s, 1H). Example 25: Preparation of Compound 25

[0481] Methyl 2-(4-bromo-2-fluorophenyl)acetate

[0482] This compound was synthesized using 2-(4-bromo-2-fluorophenyl)acetic acid according to the preparation procedure for methyl 2-(4-chloro-3-methoxyphenyl)acetate. ¹H NMR (300 MHz, CDCl₃) δ 7.24–7.27 (m, 2H), 7.14 (t, J = 8.4 Hz, 1H), 3.71 (s, 3H), 3.63 (s, 2H).

[0483] Methyl 2-(4-cyano-2-fluorophenyl)acetate

[0484] This compound was synthesized using methyl 2-(4-bromo-2-fluorophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-3-fluorophenyl)acetate. ¹H NMR (300 MHz, CDCl₃) δ 7.86 (dd, J = 8.7, 1.2 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 3.87 (s, 2H), 3.64 (s, 3H).

[0485] Methyl 2-(4-cyano-2-fluorophenyl)-3-sideoxybutyrate

[0486] This compound was synthesized using methyl 2-(4-cyano-2-fluorophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate. LC-MS (ESI+): m / z 258 (M+Na)+.

[0487] 6-(4-(4-cyano-2-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0488] This compound was synthesized using methyl 2-(4-cyano-2-fluorophenyl)-3-sideoxybutyrate according to the preparation procedure for 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 339 (M+H+), 1H NMR (300 MHz, DMSO-d6) δ 13.22 (brs, 1H), 8.94 (d, J = 1.8 Hz, 1H), 8.59 (d, J = 9.3 Hz, 1H), 8.38 (d, J = 9.3 Hz, 1H), 7.69–7.94 (m, 3H), 2.23 (s, 3H). Example 26: Preparation of Compound 26

[0489] Methyl 2-(4-chloro-3-fluorophenyl)acetate

[0490] This compound was synthesized using 2-(4-chloro-3-fluorophenyl)acetic acid according to the preparation procedure for methyl 2-(4-chloro-3-methoxyphenyl)acetate. ¹H NMR (300 MHz, CDCl₃) δ 7.34 (t, J = 7.8 Hz, 1H), 7.10 (dd, J = 8.1, 1.8 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 3.71 (s, 3H), 3.6 (s, 2H).

[0491] Methyl 2-(4-chloro-3-fluorophenyl)-3-sideoxybutyrate

[0492] This compound was synthesized using methyl 2-(4-chloro-3-fluorophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-2-fluorophenyl)-3-sideoxybutyrate. LC-MS (ESI+): m / z 267 (M+Na)+.

[0493] 6-(4-(4-chloro-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0494] This compound was synthesized using methyl 2-(4-chloro-3-fluorophenyl)-3-sideoxybutyrate according to the preparation procedure of 6-(4-(4-cyano-2-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 348 (M+Na)+; 1H-NMR (300 MHz, DMSO-d6) δ 13.30 (brs, 1H), 13.01 (brs, 1H), 8.95 (s, 1H), 8.56 (brs, 1H), 8.41 (dd, J = 9.0, 2.1 Hz, 1H), 7.74 (d, J = 11.7 Hz, 1H), 7.55–7.58 (m, 2H), 2.47 (s, 3H). Example 27: Preparation of compound 27

[0495] 4-Amino-6-hydrazinoic acid

[0496] This compound was synthesized using 4-amino-6-fluoronicotinic acid, following the preparation procedure for 6-hydrazino-4-methylnicotinic acid. LC-MS (ESI+): m / z 169 (M+H)+.

[0497] 4-Amino-6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0498] This compound was synthesized using 4-amino-6-hydrazinoic acid according to the preparation procedure for 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 336 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.08 (s, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.37 (brs, 1H), 2.39 (s, 3H). Example 28: Preparation of compound 28

[0499] 6-(4-(4-chloro-2-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0500] This compound was synthesized according to the preparation procedure of 6-(4-(4-chloro-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 348 (M+Na)+; 1H-NMR (300 MHz, DMSO-d6) δ 13.36 (brs, 1H), 12.82 (brs, 1H), 8.95 (d, J= 1.5 Hz, 1H), 8.58 (brs, 1H), 8.40 (m, 1H), 7.48-7.56 (m, 2H), 7.33 (m, 1H), 2.21 (s, 3H). Example 29: Preparation of compound 29

[0501] 2-Methyl-4-vinylbenzonitrile

[0502] This compound was synthesized using 4-bromo-2-methylbenzonitrile according to the preparation procedure for 3-methyl-4-vinylbenzonitrile. ¹H NMR (300 MHz, CDCl₃) δ 7.55 (d, J = 7.8 Hz, 1H), 7.26–7.32 (m, 2H), 6.70 (m, 1H), 5.85 (d, J = 17.4 Hz, 1H), 5.42 (d, J = 10.8 Hz, 1), 2.54 (s, 3H).

[0503] Methyl 2-(4-cyano-3-methylphenyl)acetate

[0504] This compound was synthesized using 2-methyl-4-vinylbenzonitrile according to the preparation procedure of methyl 2-(4-cyano-2-methylphenyl)acetate. ¹H NMR (300 MHz, CDCl₃) δ 7.56 (d, J = 7.8 Hz, 1H), 7.18–7.25 (m, 2H), 3.71 (s, 3H), 3.64 (s, 2H), 2.54 (s, 3H).

[0505] Methyl 2-(4-cyano-3-methylphenyl)-3-sideoxybutyrate

[0506] This compound was synthesized using methyl 2-(4-cyano-3-methylphenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate. LC-MS (ESI-): m / z 230 (MH)-.

[0507] 6-(4-(4-cyano-3-methylphenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl)nicotinic acid

[0508] This compound was synthesized using methyl 2-(4-cyano-3-methylphenyl)-3-sideoxybutyrate according to the preparation procedure for 6-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)-4-methylnicotinic acid. LC-MS (ESI+): m / z 333 (MH)–; 1H-NMR (300 MHz, CD3OD) δ 8.96 (s, 1H), 8.40 (d, J = 8.7, 2.1 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 7.71 (s, 1H), 7.58–7.66 (m, 2H), 2.55 (s, 3H), 2.45 (s, 3H). Example 30: Preparation of Compound 30

[0509] Methyl 2-(4-bromo-2-chlorophenyl)acetate

[0510] This compound was synthesized using 2-(4-bromo-2-fluorophenyl)acetic acid according to the preparation procedure for methyl 2-(4-bromo-2-fluorophenyl)acetic acid. ¹H-NMR (300 MHz, CDCl₃) δ 7.55 (d, J = 1.8 Hz, 1H), 7.37 (dd, J = 8.1, 1.8 Hz, 1H), 7.16 (d, J = 1.8 Hz, 1H), 3.71 (s, 3H), 3.67 (s, 2H).

[0511] Methyl 2-(2-chloro-4-cyanophenyl)acetate

[0512] This compound was synthesized using methyl 2-(4-bromo-2-chlorophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-2-fluorophenyl)acetate. GC-MS (EI+): m / z 209.

[0513] (Z)-2-(2-chloro-4-cyanophenyl)-3-(dimethylamino)methyl acrylate

[0514] This compound was synthesized using methyl 2-(2-chloro-4-cyanophenyl)acetate according to the preparation procedure for (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 265 (M+H)+.

[0515] 6-(4-(2-chloro-4-cyanophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0516] This compound was synthesized using (Z)-2-(2-chloro-4-cyanophenyl)-3-(dimethylamino)acrylate according to the preparation procedure of 6-(4-(4-cyano-2-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 341 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 13.42 (brs, 2H), 8.97 (s, 1H), 8.44-8.60 (m, 3H), 8.28 (d, J = 8.1 Hz, 1H), 8.06 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H). Example 31: Preparation of Compound 31

[0517] 3,5-Dimethyl-4-vinylbenzonitrile

[0518] This compound was synthesized using 4-bromo-3,5-dimethylbenzonitrile according to the preparation procedure for 3-methyl-4-vinylbenzonitrile. ¹H-NMR (300 MHz, CDCl₃) δ 7.32 (s, 2H), 6.64 (m, 1H), 5.54 (dd, J = 11.7, 1.5 Hz, 1H), 5.32 (dd, J = 17.7, 1.5 Hz, 1H), 2.32 (s, 6H).

[0519] Methyl 2-(4-cyano-2,6-dimethylphenyl)acetate

[0520] This compound was synthesized using 3,5-dimethyl-4-vinylbenzonitrile according to the preparation procedure for methyl 2-(4-cyano-2-methylphenyl)acetate. ¹H-NMR (300 MHz, CDCl₃) δ 7.33 (s, 2H), 3.72 (s, 2H), 3.70 (s, 3H), 2.35 (s, 6H).

[0521] (Z)-2-(4-cyano-2,6-dimethylphenyl)-3-(dimethylamino)methyl acrylate

[0522] This compound was synthesized using methyl 2-(4-cyano-2,6-dimethylphenyl)acetate according to the preparation procedure of (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate. ¹H-NMR (300 MHz, CDCl₃) δ 7.62 (s, 1H), 7.28 (s, 2H), 3.59 (s, 3H), 2.64 (s, 6H), 2.20 (s, 6H).

[0523] 6-(4-(4-cyano-2,6-dimethylphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid

[0524] This compound was synthesized using (Z)-2-(4-cyano-2,6-dimethylphenyl)-3-(dimethylamino)acrylate according to the preparation procedure for 6-(4-(4-cyano-2-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 335 (M+H)+; 1H-NMR (300 MHz, CD3OD) δ 8.03 (s, 1H), 8.52 (m, 2H), 7.75 (s, 1H), 7.40-7.47 (m, 2H), 2.30 (s, 6H). Example 32: Preparation of Compound 32

[0525] Methyl 2-(4-methoxy-3-methylphenyl)acetate

[0526] This compound was synthesized using 2-(4-methoxy-3-methylphenyl)acetic acid according to the preparation procedure for methyl 2-(4-bromo-2-methoxyphenyl)acetic acid. ¹H-NMR (300 MHz, CD3OD) δ 7.05–7.07 (m, 2H), 6.77 (d, J = 7.8 Hz, 1H), 3.81 (1, 3H), 3.76 (s, 3H), 3.54 (s, 2H), 2.20 (s, 3H).

[0527] Methyl 2-(4-methoxy-3-methylphenyl)-3-t-oxybutyrate

[0528] This compound was synthesized using methyl 2-(4-methoxy-3-methylphenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate. LC-MS (ESI+): m / z 259 (M+Na)+.

[0529] 6-(5-hydroxy-4-(4-methoxy-3-methylphenyl)-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0530] This compound was synthesized using methyl 2-(4-methoxy-3-methylphenyl)-3-sideoxybutyrate according to the preparation procedure of 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 340 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 13.30 (brs, 1H), 12.35 (brs, 1H), 8.93 (s, 1H), 8.58 (s, 1H), 8.39 (d, J = 6.9 Hz, 1H), 7.34–7.36 (m, 2H), 6.96 (d, J = 9.0 Hz, 1H), 3.80 (s, 3H), 2.35 (s, 3H), 2.18 (s, 3H). Example 33: Preparation of compound 33

[0531] Diethyl 2-(2-bromo-4-cyanophenyl)malonate

[0532] Under nitrogen protection, NaH (600 mg, 15 mmol) was added sequentially to a DMF solution of diethyl malonate (2 g, 12.5 mmol) at 0°C for 5 minutes. After stirring the reaction mixture at 0°C for 30 minutes, 3-bromo-4-fluorobenzonitrile (2.08 g, 10 mmol) was added to the reaction mixture in one go, and the mixture was stirred at 80°C for 1.5 hours. After the reaction was indicated by TLC analysis, the reaction mixture was quenched with water (20 mL) and the pH was adjusted to 5 with dilute HCl solution. The resulting mixture was then extracted with EtOAc (50 mL x 2). The combined organic phases were washed with water (20 mL) and concentrated brine (20 mL), dried, and concentrated to dryness. The residue was purified by column chromatography (PE / EtOAc = 20 / 1 to 10 / 1) to give 1.8 g of the title compound. LC-MS (ESI+): m / z 340, 342 (M+H)+. 1H-NMR (300 MHz, CDCl3) δ 7.90 (s, 2H), 7.64 (s, 2H), 5.24 (s, 1H), 4.21-4.32 (m, 4H), 1.29 (t,J =7.2 Hz, 6H).

[0533] Ethyl 2-(2-bromo-4-cyanophenyl)

[0534] LiCl (339 mg, 8 mmol) was added once to a solution of diethyl 2-(2-bromo-4-cyanophenyl)malonate (1.8 g, 5.3 mmol) in DMSO (20 mL) and water (96 mg, 5.3 mmol). The resulting mixture was stirred overnight at 120 °C. After the reaction was indicated by TLC analysis, the reactants were quenched with water (20 mL), the pH was adjusted to 6 with dilute HCl, and the mixture was extracted with EtOAc (20 mL x 2). The combined organic phases were washed with water (20 mL) and concentrated brine (20 mL), dried, and concentrated. The residue was purified by column chromatography (PE / EtOAc = 20 / 1 to 10 / 1) to give 780 mg of the title compound. 1H-NMR (300 MHz, CDCl3) δ 7.87 (s, 1H), 7.59 (d,J =7.8 Hz, 1H), 7.42 (d,J =7.8 Hz, 1H), 4.20 (q,J =6.9 Hz, 2H), 3.84 (s, 2H), 1.27 (t,J =6.9 Hz, 3H).

[0535] Ethyl 2-(4-cyano-2-vinylphenyl)acetate

[0536] This compound was synthesized using ethyl 2-(2-bromo-4-cyanophenyl)acetate according to the preparation procedure for 3-methyl-4-vinylbenzonitrile. ¹H NMR (300 MHz, CDCl₃) δ 7.7 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 6.89 (m, 1H), 5.70 (d, J = 17.4 Hz, 1H), 5.42 (d, J = 10.8 Hz, 1), 4.15 (q, J = 7.2 Hz, 1H), 3.73 (s, 3H), 1.24 (q, J = 7.2 Hz, 1H).

[0537] Ethyl 2-(4-cyano-2-ethylphenyl)

[0538] Pd / C (56 mg) was added to a methanol solution (10 mL) of methyl 2-(4-cyano-2-vinylphenyl)acetate (560 mg, 2.6 mmol). The suspension was stirred under hydrogen from a balloon for about 1 hour. After the reaction was indicated by TLC analysis, the suspension was filtered through a Celite kit and the filter cake was washed with methanol (10 mL). The filtrate was concentrated to dryness to give 540 mg of crude title compound. 1H-NMR (300 MHz, CDCl3) δ 7.61 (s, 1H), 7.49 (d,J =8.1 Hz, 1H), 7.31 (d,J =8.1 Hz, 1H), 4.16 (q,J =7.2 Hz, 2H), 3.69 (s, 2H), 2.68 (q,J =7.5 Hz, 2H), 1.21-1.28 (m, 6H).

[0539] (Z)-2-(4-cyano-2-ethylphenyl)-3-(dimethylamino)acrylate ethyl ester

[0540] This compound was synthesized using ethyl (4-cyano-2-ethylphenyl)acetate according to the preparation procedure of (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate. ¹H-NMR (300 MHz, CDCl₃) δ 7.60 (s, 1H), 7.49 (s, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.23 (m, 1H), 4.12 (q, J = 6.9 Hz, 2H), 3.60 (s, 3H), 2.67 (s, 6H), 1.14 (t, J = 6.9 Hz, 3H).

[0541] 6-(4-(4-cyano-2-ethylphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0542] This compound was synthesized using (Z)-2-(4-cyano-2-ethylphenyl)-3-(dimethylamino)acrylate according to the preparation procedure for 6-(4-(4-cyano-2-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 335 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.52 (m, 2H), 8.12 (s, 1H), 7.75 (s, 1H), 7.60-7.69 (m, 2H), 2.78 (q, J = 7.5 Hz, 2H), 1.17 (q, J = 7.5 Hz, 3H). Example 34: Preparation of compound 34

[0543] 5-Hydroxypyrazine-2-carboxylic acid

[0544] This compound was synthesized using 5-fluoropyrazine-2-carboxylic acid according to the preparation procedure for 6-hydrazino-4-methylnicotinic acid. LC-MS (ESI+): m / z 155 (M+H)+.

[0545] 5-(4-(4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl)pyrazine-2-carboxylic acid

[0546] This compound was synthesized using 5-hydrazinopyrazine-2-carboxylic acid according to the preparation procedure of 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 322 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.04 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 2.51 (s, 3H). Example 35: Preparation of compound 35

[0547] Ethyl 2-(2-methoxypyridin-4-yl)ethyl acetate

[0548] Under nitrogen atmosphere, lithium diisopropylamine (16.0 mL, 32.0 mmol, 2.0 M in n-heptane) was added to anhydrous tetrahydrofuran (50.0 mL) of 2-methoxy-4-methylpyridine (2.0 g, 16.2 mmol) at -78 °C. The mixture was stirred at -78 °C for 10 min and diethyl carbonate (3.78 g, 32.0 mmol) was added. The mixture was heated to room temperature and stirred for 2.0 h. The reaction mixture was quenched with water and extracted twice with ethyl acetate. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by rapid chromatography (petroleum ether / ethyl acetate = 10 / 1) to give ethyl 2-(2-methoxypyridine-4-yl)acetate (2.0 g, 10.2 mmol, yield 63.4%) as a yellow oil. LC-MS: m / z = 196.1 (M+H)+, residence time 1.97 minutes (Method A).

[0549] (E)-3-(dimethylamino)-2-(2-methoxypyridin-4-yl)ethyl acrylate

[0550] In a solution of ethyl 2-(2-methoxypyridin-4-yl)acetate (1.95 g, 10 mmol) and N,N-dimethylformamide (3.0 mL), N,N-dimethylformamide diethyl acetal (5.95 g, 50 mmol) was added. The mixture was stirred at 100 °C for 12.0 h and cooled. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by rapid chromatography (dichloromethane / methane = 98 / 2) to give (E)-3-(dimethylamino)-2-(2-methoxypyridin-4-yl)ethyl acrylate (1.1 g, 4.4 mmol, yield 44%) as a colorless oil. LC-MS: m / z = 251.0 [M+H]+, residence time 1.68 minutes (Method B).

[0551] 6-(5-hydroxy-4-(2-methoxypyridin-4-yl)-1H-pyrazole-1-yl) tert-butyl nicotinate

[0552] In an ethanol solution (5.0 mL) of (E)-3-(dimethylamino)-2-(2-methoxypyridin-4-yl)acrylate (0.25 g, 1.0 mmol) and 6-hydrazinoic acid tributyl ester (0.21 g, 1.0 mmol), p-toluenesulfonic acid monohydrate (19 mg, 0.1 mmol) was added. The mixture was stirred at reflux for 12.0 h and cooled to form a precipitate. The solid was filtered, washed with ethanol and dried to give a white solid of 6-(5-hydroxy-4-(2-methoxypyridin-4-yl)-1H-pyrazol-1-yl)nicotinic acid tributyl ester (210 mg, 0.57 mmol, yield 57%). LC-MS: m / z = 369.0 (M+H)+, residence time 4.38 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.53 (m, 1H), 8.91 (s, 1H), 8.39–8.67 (m, 2H), 8.04–8.06 (d, J = 5.0 Hz, 1H), 7.39–7.50 (m, 2H), 3.86 (m, 3H), 1.58 (s, 9H). Example 36: Preparation of compound 36

[0553] Ethyl 6-hydrazinoic acid ester

[0554] A solution of ethyl 6-chloronicotinate (1.0 g, 5.40 mmol) in tetrahydrofuran (6.0 mL) was added to hydrazine hydrate (300 mg, 5.94 mmol, 85% aqueous solution). The mixture was stirred under reflux overnight. The mixture was cooled and concentrated to dryness. The residue was partitioned between ethyl acetate and water. The organic phase was washed with concentrated brine, dried over sodium sulfate, and concentrated. A crude product as a yellow oil (600 mg, 3.31 mmol, yield 61.4%) was given. LC-MS: m / z = 182.1 (M+H)+, residence time 0.37 min (Method A). The product was of sufficient purity and was used directly in the next step.

[0555] Ethyl 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinate

[0556] In an ethanol solution (5.0 mL) of (E)-2-(4-cyanophenyl)-3-(dimethylamino)acrylate (300 mg, 1.30 mmol) and ethyl 6-hydrazinoic acid (235 mg, 1.30 mmol), p-toluenesulfonic acid monohydrate (25 mg, 0.13 mmol) was added. The reaction mixture was stirred overnight at 90 °C and cooled to form a precipitate. The solid was filtered, washed with ethanol, and dried to give ethyl 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid (51 mg, 0.15 mmol, yield 11.7%) as a yellow solid. LC-MS: m / z = 335.1 [M+H]+, residence time 5.05 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.58 (s, ¹H), 8.97 (s, ¹H), 8.70–8.47 (m, ³H), 8.15 (s, 2H), 7.79–7.77 (m, 2H), 4.39–4.34 (m, 2H), 1.37–1.22 (m, 3H). Example 37: Preparation of compound 37

[0557] Isopropyl 6-chloronicotinate

[0558] Carbonyl diimidazole (3.42 g, 21.1 mmol) was added to a 50.0 mL solution of 6-chloronicotinic acid (3.0 g, 19.2 mmol) in dichloromethane at room temperature. The mixture was stirred for 1.0 h and isopropanol (3.78 g, 32.0 mmol) was added. Dichloromethane was removed under vacuum. A catalytic amount of sodium isopropoxy (164 mg, 2.0 mmol) was added and the mixture was heated at 90 °C for 1.0 h. The solution was concentrated under vacuum. The resulting residue was slurried with water and extracted with ethyl acetate. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give isopropyl 6-chloronicotinic acid as a yellow solid (3.4 g, 17.1 mmol, yield 89%). LC-MS: m / z = 200.0 [M+H]+, residence time 2.04 min (Method A). The product has sufficient purity and can be used directly in the next step.

[0559] Isopropyl 6-Hydroxynicotinate

[0560] A hydrazine hydrate (1.0 g, 20.1 mmol, 85% aqueous solution) was added to an ethanolic solution (10.0 mL) of 6-chloronicotinic acid isopropyl ester (1.0 g, 5.03 mmol). The mixture was stirred overnight at 80 °C. The mixture was cooled and concentrated to dryness. The residue was partitioned between ethyl acetate and water. The organic phase was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was ground with petroleum ether and filtered to give 6-hydrazinotinic acid isopropyl ester as a yellow solid (800 mg, 4.1 mmol, yield 81.6%). LC-MS: m / z = 196.0 (M+H)+, residence time 0.39 min (Method A).

[0561] 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazole-1-yl)isopropyl nicotinate

[0562] To an ethanolic solution (5.0 mL) of (E)-2-(4-cyanophenyl)-3-(dimethylamino)acrylate (300 mg, 1.30 mmol) and 6-hydrazinoic acid isopropyl ester (254 mg, 1.30 mmol), p-toluenesulfonic acid monohydrate (25 mg, 0.13 mmol) was added. The reaction mixture was stirred overnight at 90 °C and cooled to form a precipitate. The solid was filtered, washed with ethanol, and dried to give a yellow solid of 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid isopropyl ester (53 mg, 0.15 mmol, yield 11.7%). LC-MS: m / z = 349.0 [M+H]+, residence time 5.44 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.55 (s, 1H), 8.95 (s, 1H), 8.69 (s, 1H), 8.53–8.44 (m, 2H), 8.14–8.12 (m, 2H), 8.79–8.77 (m, 2H), 5.21–5.15 (m, 1H), 1.36–1.34 (m, 6H). Example 38: Preparation of compound 38

[0563] 6-Chloronicotinic acid tert-butyl ester

[0564] In a tetrahydrofuran solution (50.0 mL) of 6-chloronicotinic acid (5.0 g, 6.37 mmol) and 4-dimethylaminopyridine (0.39 g, 0.64 mmol), ditert-butyl dicarbonate (10.41 g, 47.77 mmol) was added. The reaction mixture was refluxed for 4.0 h and concentrated. The residue was purified by rapid chromatography (petroleum ether / ethyl acetate = 10 / 1) to give 6-chloronicotinic acid tributyl ester (5.5 g, yield 81.12%) as a yellow solid. LC-MS: m / z = 214.0 (M+H)+, retention time 1.83 min (Method A).

[0565] 6-Hydroxynicotinic acid tert-butyl ester

[0566] A solution of 6-chloronicotinic acid tributyl ester (5.5 g, 25.82 mmol) in ethanol (25.0 mL) was mixed with hydrazine hydrate (6.46 g, 129.11 mmol, 85% aqueous solution). The mixture was stirred at 100 °C for 2.0 h. The mixture was cooled and concentrated to dryness. The residue was partitioned between ethyl acetate and water. The organic phase was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was ground together with petroleum ether and filtered to give 6-hydrazinotinic acid tributyl ester (5.0 g, 92.76% yield) as a yellow solid. LC-MS: m / z = 210.0 (M+H)+, residence time 1.19 min (Method A).

[0567] Methyl 2-(4-cyanophenyl)acetate

[0568] A methanol solution of hydrochloric acid (20.0 mL, 3.0 M) was added to a mixture of 2-(4-cyanophenyl)acetic acid (5.0 g, 31.0 mmol) in methanol (10.0 mL) at 0 °C. The mixture was stirred at 70 °C for 3.0 h and cooled to precipitate a solid. The solid was filtered, washed with methanol and dried to give methyl 2-(4-cyanophenyl)acetate (5.0 g, 28.4 mmol, yield 92%) as a yellow solid. LC-MS: m / z = 176.0 [M+H]+, residence time 1.54 min (Method A).

[0569] (E)-2-(4-cyanophenyl)-3-(dimethylamino)methyl acrylate

[0570] In a solution of methyl 2-(4-cyanophenyl)acetate (5.0 g, 28.5 mmol) in N,N-dimethylformamide (25.0 mL), N,N-dimethylformamide diethyl acetal (14.0 g, 114.16 mmol) was added. The mixture was stirred at 100 °C for 16.0 h and cooled. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give (E)-2-(4-cyanophenyl)-3-(dimethylamino)acrylate as a yellow solid (5.20 g, 25.4 mmol, yield 89%). LC-MS: m / z = 231.0 [M+H]+, residence time 1.70 min (Method A). The product was of sufficient purity and was used directly in the next step.

[0571] 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazole-1-yl) tert-butyl nicotinate

[0572] To an ethanol solution (20.0 mL) of (E)-2-(4-cyanophenyl)-3-(dimethylamino)acrylate (2.0 g, 8.70 mmol) and 6-hydrazinoic acid tributyl ester (1.82 g, 8.70 mmol), p-toluenesulfonic acid monohydrate (171 mg, 0.9 mmol) was added. The reaction mixture was stirred at 90 °C for 16.0 h and cooled to form a precipitate. The solid was filtered, washed with ethanol, and dried to give a yellow solid of 6-(4-(4-cyanophenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid tributyl ester (2.3 g, 6.35 mmol, yield 73%). LC-MS: m / z = 363.1 (M+H)+, residence time 5.82 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.58 (s, 1H), 8.92 (s, 1H), 8.69 (s, 1H), 8.51 (s, 1H), 8.44–8.41 (m, 1H), 8.15–8.13 (m, 2H), 7.81–7.78 (m, 2H), 1.58 (s, 9H). Example 39: Preparation of compound 39

[0573] Methyl 2-(4-chloro-2-methoxyphenyl)acetate

[0574] This compound was synthesized using 2-(4-chloro-2-methoxyphenyl)acetic acid according to the preparation procedure for methyl 2-(4-bromo-2-methoxyphenyl)acetate. LC-MS (ESI+): m / z 237 (M+Na)+; 1H-NMR (300 MHz, CDCl3) δ 7.10 (d, J = 8.1 Hz, 1H), 6.85-6.92 (m, 2H), 3.86 (s, 3H), 3.71 (s, 3H), 3.67 (s, 2H).

[0575] (Z)-2-(4-chloro-2-methoxyphenyl)-3-(dimethylamino)acrylate methyl acrylate

[0576] This compound was synthesized using methyl 2-(4-chloro-2-methoxyphenyl)acetate according to the preparation procedure for (Z)-2-(4-cyano-2-methoxyphenyl)-3-(dimethylamino)acrylate. LC-MS (ESI+): m / z 270 (M+H)+.

[0577] 6-(4-(4-chloro-2-methoxyphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0578] This compound was synthesized using (Z)-2-(4-chloro-2-methoxyphenyl)-3-(dimethylamino)acrylate according to the preparation procedure for 6-(4-(4-cyano-2-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 346 (M+H)+.

[0579] 6-(4-(4-chloro-2-hydroxyphenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0580] This compound was synthesized using 6-(4-(4-chloro-2-methoxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid, following the preparation procedure for 6-(4-(4-cyano-2-hydroxyphenyl)-5-hydroxy-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 332 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 12.91 (brs, 2H), 8.95 (s, 1H), 8.52 (d, J = 8.7 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 8.23 ​​(s, 1H), 7.80 (s, 1H), 6.78 (s, 2H). Example 40: Preparation of Compound 40

[0581] Diethyl 2-(4-cyano-2-(trifluoromethyl)phenyl)malonate

[0582] Under nitrogen protection, Cs₂CO₃ (45.50 g, 139.70 mmol) was added to a DMF solution (100 mL) of ethyl 3-(ethylperoxy)-3-sideoxypropionate (10.20 g, 63.50 mmol). After stirring the reaction mixture at 70 °C for 10 min, 4-fluoro-3-(trifluoromethyl)benzonitrile (12.00 g, 63.50 mmol) was added to the reaction mixture. The resulting mixture was stirred at 70 °C for 2 h. After the reaction was indicated by TLC analysis, the reaction mixture was quenched with water (300 mL) and extracted with EtOAc (500 mL x 2). The combined organic phases were washed with water (50 mL), dried over Na₂SO₄, filtered, and concentrated to give the desired product (20.8 g) as an oil. 1H-NMR (300 MHz, CDCl3) δ 7.99 (s, 1H), 7.86-7.92 (m, 2H), 5.11 (s, 1H), 4.20-4.33 (m, 4H), 1.28 (t,J= 7.2 Hz, 6H).

[0583] Ethyl 2-(4-cyano-2-(trifluoromethyl)phenyl)acetate

[0584] LiCl (2.90 g, 68.40 mmol) and water (0.80 mL, 45.60 mmol) were added to a DMSO solution (150 mL) of diethyl 2-(4-cyano-2-(trifluoromethyl)phenyl)malonate (15.00 g, 45.60 mmol). The mixture was stirred overnight at 120 °C. After the reaction was indicated by TLC analysis, the reactants were quenched with water (300 mL) and extracted with EtOAc (150 mL x 3). The combined organic phases were washed with water (100 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (EA / PE = 1 / 25) to give the desired product (8.28 g) as a white solid. 1H-NMR (300 MHz, CDCl3) δ 7.97 (d,J= 1.8 Hz, 1H), 7.83 (d,J= 7.8 Hz, 1H), 7.57 (d,J= 7.8 Hz, 1H), 4.18 (q,J= 7.2 Hz, 2H), 3.88 (s, 2H), 1.26 (t,J= 7.2 Hz, 3H).

[0585] (Z)-2-(4-cyano-2-(trifluoromethyl)phenyl)-3-(dimethylamino)acrylate ethyl ester

[0586] Ethyl 2-(4-cyano-2-(trifluoromethyl)phenyl)acetate (4.00 g, 15.56 mmol) and DME-DMA (15 mL) solution were stirred overnight at 150 °C in a sealed tube. After the reaction was indicated by TLC analysis, the mixture was diluted with water (100 mL) and extracted with EtOAc (150 mL × 3). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (EA / PE = 1 / 9) to give the desired product (3.2 g) as a brown oil. 1H-NMR (300 MHz, CDCl3) δ 7.94 (s, 1H), 7.75 (d,J= 7.8 Hz, 1H), 7.59 (s, 1H), 7.46 (d,J= 7.8 Hz, 1H), 3.95-4.08 (m, 2H), 2.66 (brs, 6H), 1.10 (t,J= 7.2 Hz, 3H).

[0587] 6-(4-(4-cyano-2-(trifluoromethyl)phenyl)-5-hydroxy-1H-pyrazole-1-yl)nicotinic acid

[0588] 6-hydrazinoic acid (0.59 g, 0.38 mmol) and dilute HCl solution (38 mL, 1 M) were added to i-PrOH (38 mL) of (Z)-2-(4-cyano-2-(trifluoromethyl)phenyl)-3-(dimethylamino)acrylate (1.00 g, 3.20 mmol). The reaction mixture was stirred at room temperature for 24 hours. After the reaction was indicated by LCMS analysis, DIEA (15 eq) was added and the mixture was stirred at room temperature for 2 days. After adjusting the pH of the mixture to 3, a large amount of solid precipitated out. After filtration and slurry purification, 90 mg of the desired product as a solid was obtained. The HPLC purity was 93.1%. LC-MS (ESI+): m / z 375 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.47–8.57 (m, 2H), 8.34 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.97–8.02 (m, 2H). Example 41: Preparation of compound 41

[0589] Ethyl 3-O-2-(pyridin-4-yl)butyrate

[0590] This compound was synthesized using ethyl 3-sideoxy-2-(pyridin-4-yl)butyrate according to the preparation procedure for methyl 2-(4-cyano-2-fluorophenyl)-3-sideoxybutyrate. LC-MS (ESI+): m / z 208 (M+H)+.

[0591] 6-(5-hydroxy-3-methyl-4-(pyrazol-4-yl)-1H-pyrazol-1-yl)nicotinic acid

[0592] This compound was synthesized using ethyl 3-sideoxy-2-(pyridin-4-yl)butyrate according to the preparation procedure of 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 297 (M+H)+; 1H-NMR (300 MHz, CD3OD) δ 9.00 (s, 1H), 8.24-8.33 (m, 4H), 7.84 (d,J =6.0 Hz, 2H), 2.44 (s, 3H).

[0593] 4-(1-(5-carboxypyridin-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)pyridine-1-oxide

[0594] m-CPBA (166 mg, 0.96 mmol) was added once to a DMF solution (190 mg, 0.64 mmol) of 6-(5-hydroxy-3-methyl-4-(pyridin-4-yl)-1H-pyrazol-1-yl)nicotinic acid (190 mg, 0.64 mmol) in 10 mL at room temperature. The resulting mixture was stirred at room temperature for about 2 hours. After the reaction was indicated by HPLC analysis, the reaction mixture was purified directly by preparative HPLC to give 50 mg of the title compound. LC-MS (ESI+): m / z 313 (M+H)+; 1H-NMR (300 MHz, CD3OD) δ 9.03 (d, J = 2.1 Hz, 1H), 8.61 (d, J = 6.3 Hz, 1H), 8.45 (m, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 6.3 Hz, 1H), 2.07 (s, 3H). Example 42: Preparation of compound 42

[0595] Methyl 2-(3-bromo-4-chlorophenyl)acetate

[0596] This compound was synthesized using 2-(3-bromo-4-chlorophenyl)acetic acid according to the preparation procedure for methyl 2-(4-bromo-2-methoxyphenyl)acetate. ¹H-NMR (300 MHz, CDCl₃) δ 7.55 (d, J = 1.8 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.17 (dd, J = 8.1, 1.8 Hz, 1H), 3.71 (s, 3H), 3.58 (s, 2H).

[0597] Methyl 2-(4-chloro-3-cyanophenyl)acetate

[0598] This compound was synthesized using methyl 2-(3-bromo-4-chlorophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-2-fluorophenyl)acetate. ¹H-NMR (300 MHz, CDCl₃) δ 7.61 (s, 1H), 7.48 (s, 2H), 3.73 (s, 3H), 3.64 (s, 2H).

[0599] Methyl 2-(4-chloro-3-cyanophenyl)-3-sideoxybutyrate

[0600] This compound was synthesized using methyl 2-(4-chloro-3-cyanophenyl)acetate according to the preparation procedure for methyl 2-(4-cyano-3-fluorophenyl)-3-sideoxybutyrate. The crude product was used directly in the next step without further purification.

[0601] 6-(4-(4-chloro-3-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid

[0602] This compound was synthesized using methyl 2-(4-chloro-3-cyanophenyl)-3-sideoxybutyrate according to the preparation procedure for 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 355 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.57 (d, J = 6.9 Hz, 1H), 8.43 (m, 1H), 8.22 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 1.91 (s, 3H). Example 43: Preparation of compound 43

[0603] 2-(6-Synephro-1,6-dihydropyridin-3-yl)acetic acid:

[0604] A mixture of 2-(6-chloropyridin-3-yl)acetic acid (1.0 g, 6.4 mmol) in glacial acetic acid (13.0 mL) and water (3.0 mL) was stirred in a sealed tube at 150 °C for 3.0 days. The solution was cooled and evaporated to give a residue. The residue was redeposited from toluene to give 2-(6-sidek-1,6-dihydropyridin-3-yl)acetic acid (1.1 g, crude) as a brown solid. LC-MS: m / z = 154.1 [M+H]+, residence time 1.63 min (Method A). The crude product was used for the next step.

[0605] Methyl 2-(6-Symoxy-1,6-dihydropyridin-3-yl)acetate:

[0606] Concentrated sulfuric acid (0.5 mL) was added to a methanol solution (20.0 mL) of 1.1 g crude 2-(6-sideoxy-1,6-dihydropyridin-3-yl)acetic acid. The mixture was stirred at room temperature for 15 hours and concentrated. The residue was partitioned between ethyl acetate and a saturated sodium bicarbonate solution. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated under reduced pressure to give methyl 2-(6-sideoxy-1,6-dihydropyridin-3-yl)acetate as a white solid (800 mg, 4.79 mmol, yield 74.8%). LC-MS: m / z = 168.1 [M+H]+, residence time 1.35 min (Method A).

[0607] Methyl 2-(6-methoxypyridin-3-yl)acetate

[0608] Iodimethane (580 mg, 4.20 mmol) was added to an anhydrous tetrahydrofuran solution (20.0 mL) of methyl 2-(6-sideoxy-1,6-dihydropyridin-3-yl)acetate (450 mg, 2.69 mmol) and cesium carbonate (1.05 g, 3.23 mmol). The mixture was stirred at room temperature for 18 hours. The reaction solution was diluted with ethyl acetate and water. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated. The residue was purified by rapid chromatography (petroleum ether / ethyl acetate = 3 / 1) to give a mixture of methyl 2-(1-methyl-6-sideoxy-1,6-dihydropyridin-3-yl)acetate and methyl 2-(6-methoxypyridin-3-yl)acetate as a yellow oil (400 mg, 2.21 mmol, yield 82.1%). LC-MS: m / z = 182.1 (M+H)+, residence time 1.33 minutes (Method A).

[0609] (E)-3-(dimethylamino)-2-(6-methoxypyridin-3-yl)methyl acrylate

[0610] To a solution of methyl 2-(1-methyl-6-sideoxy-1,6-dihydropyridin-3-yl)acetate and methyl 2-(6-methoxypyridin-3-yl)acetate (400 mg, 2.21 mmol) in N,N-dimethylformamide (5.0 mL), N,N-dimethylformamide diethyl acetal (1.63 g, 11.1 mmol) was added. The mixture was stirred overnight at 100 °C and cooled. Ethyl acetate and water were added to the solution, and the layers were separated. The organic layer was washed with concentrated brine, dried over sodium sulfate, and concentrated to give methyl (E)-3-(dimethylamino)-2-(1-methyl-6-sideoxy-1,6-dihydropyridin-3-yl)acrylate and methyl (E)-3-(dimethylamino)-2-(6-methoxypyridin-3-yl)acrylate as a yellow oil (400 mg, 1.69 mmol, yield 76.7%). LC-MS: m / z = 237.1 [M+H]+, residence times 1.08 min, 1.51 min (Method B). This mixture was used in the next step.

[0611] 6-(5-hydroxy-4-(6-methoxypyridin-3-yl)-1H-pyrazole-1-yl) tert-butyl nicotinate

[0612] The mixture of (E)-3-(dimethylamino)-2-(1-methyl-6-sideoxy-1,6-dihydropyridin-3-yl)acrylate and (E)-3-(dimethylamino)-2-(6-methoxypyridin-3-yl)acrylate (350 mg, 1.48 mmol) in ethanol (5.0 mL) was stirred in a sealed tube at 90 °C for 16.0 h and cooled. The insoluble solids were filtered and the filtrate was concentrated to dryness. The two isomers were separated by preparative HPLC and were white solids. 6-(5-hydroxy-4-(6-methoxypyridin-3-yl)-1H-pyrazol-1-yl)nicotinic acid tributyl ester (76 mg, 0.21 mmol, yield 27.9%). LC-MS: m / z = 369.0 [M+H]+, residence time 2.29 min (Method A). 1HNMR (500 MHz, DMSO-d6)δ12.97 (br, 1H), 8.91 (d,J =1.5 Hz, 1H), 8.70 (s, 1H), 8.47-8.39 (m, 2H), 8.19-8.14 (m, 1H), 6.85-6.83 (m, 1H), 3.85 (s, 3H), 1.58 (s, 9H).

[0613] 6-(5-hydroxy-4-(6-methoxypyridin-3-yl)-1H-pyrazol-1-yl)nicotinic acid

[0614] A solution (6.0 mL) of 6-(5-hydroxy-4-(6-methoxypyridin-3-yl)-1H-pyrazol-1-yl)nicotinic acid tributyl ester (76 mg, 0.21 mmol) in dichloromethane was added to trifluoroacetic acid (3.0 mL). The mixture was stirred overnight at room temperature and concentrated. The residue was ground together with ethyl acetate and filtered to give 6-(5-hydroxy-4-(6-methoxypyridin-3-yl)-1H-pyrazol-1-yl)nicotinic acid (5.4 mg, 0.017 mmol, yield 8.2%) as a white solid. LC-MS: m / z = 313.0 [M+H]+, residence time 3.73 min (Method A). ¹H NMR (400 MHz, DMSO-d6) δ 13.47 (br, ¹H), 13.06 (br, ¹H), 8.96 (s, ¹H), 8.73–8.44 (m, 4H), 8.21–8.18 (m, ¹H), 6.84 (d, J = 8.8 Hz, ¹H), 3.85 (s, 3H). Example 44: Preparation of compound 44

[0615] Diethyl 2-(3-chloro-4-cyanophenyl)malonate

[0616] This compound was synthesized using 2-chloro-4-fluorobenzonitrile according to the preparation procedure for diethyl 2-(2-bromo-4-cyanophenyl)malonate. ¹H-NMR (300 MHz, CDCl₃) δ 7.68 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 1.2 Hz, 1H), 7.45 (dd, J = 8.1, 1.2 Hz, 1H), 4.63 (s, 1H), 4.30 (q, J = 6.9 Hz, 4H), 1.26 (t, J = 6.9 Hz, 6H).

[0617] Ethyl 2-(3-chloro-4-cyanophenyl)

[0618] This compound was synthesized using diethyl 2-(3-chloro-4-cyanophenyl)malonate according to the preparation procedure for ethyl 2-(2-bromo-4-cyanophenyl)acetate. ¹H-NMR (300 MHz, CDCl₃) δ 7.63 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 1.2 Hz, 1H), 7.27 (dd, J = 8.1, 1.2 Hz, 1H), 4.22 (q, J = 6.9 Hz, 2H), 3.66 (s, 2H), 1.26 (t, J = 6.9 Hz, 3H).

[0619] (E)-3-acetoxy-2-(3-chloro-4-cyanophenyl)but-2-enoic acid ethyl ester

[0620] This compound was synthesized using ethyl 2-(3-chloro-4-cyanophenyl)acetate according to the preparation procedure for 6-(4-(4-cyano-2-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 330 (M+Na)+.

[0621] 6-(4-(3-chloro-4-cyanophenyl)-5-hydroxy-3-methyl-1H-pyrazole-1-yl)nicotinic acid

[0622] This compound was synthesized using ethyl (E)-3-acetoxy-2-(3-chloro-4-cyanophenyl)but-2-enoate according to the preparation procedure of 6-(4-(4-cyano-3-fluorophenyl)-5-hydroxy-3-methyl-1H-pyrazol-1-yl)nicotinic acid. LC-MS (ESI+): m / z 355 (M+H)+; 1H-NMR (300 MHz, DMSO-d6) δ 13.35 (brs, 2H), 8.95 (s, 1H), 8.57 (d,J =9.0 Hz, 1H), 8.42 (d,J =9.0, 1.8 Hz, 1H), 8.14 (s, 1H), 7.92 (d,J =8.4 Hz, 1H), 7.82 (d,J =8.4 Hz, 1H), 2.53 (s, 3H). Example 45: In vitro assays demonstrate PHD inhibition.

[0623] Determine the maximum half-inhibitory concentration (IC50) of the enzyme of the compound selected in this invention.

[0624] The time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the half-maximal inhibitory concentration (IC50) of PHD inhibitors for the full-length human prolyl-4-hydroxylase domain (PHD) enzymes PHD1, PHD2, and PHD3. The TR-FRET assay was developed based on the specific binding of the hydroxylated HIF-1α peptide to a complex (VBC) formed by VHL, EloB, and EloC to generate a fluorescence signal. The Tb-donor (monoclobutrazol anti-6His-Tb-caecin Gold) and the D2-receptor (streptavidin [SA]-D2) of the TR-FRET are linked to the VBC complex and the HIF-1α peptide, respectively. Upon hydroxylation, the VBC complex specifically binds to the HIF-1α peptide, allowing energy to be transferred from the TR-FRET donor to the receptor (Figure 1). Materials and Methods

[0625] Unless otherwise stated, all chemicals and materials are standard laboratory grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Reagents: TR-FRET reagents

[0626] The monoclonal antibody against 6His-Tb-caecin Gold (catalog number 61HI2TLA) and streptavidin [SA]-D2 (catalog number 610SADLA) were purchased from CisBio International (Bedford, MA, USA).

[0627] The N-terminal biotinylated HIF-1α C35 synthetic peptide, representing amino acids 547 to 581 and containing the proline 564 PHD2 hydroxylation position, was purchased from California Peptide Research (Salt Lake City, UT, USA). Recombinant protein VBC complex

[0628] The His-tagged recombinant VHL protein, the EloB and EloC complex (His-VBC), was supplied by Axxam (Milan, Italy). Recombinant human (NCBI accession number NP_00542.1) contains a His tag at the C-terminus of amino acids 55 to 213 and is designated VHL-His. VHL-His in E. coli is expressed together with full-length human EloB (NCBI accession number Q15370.1) and full-length human EloC (NCBI accession number Q15369.1) and was purified as the His-VBC complex by affinity chromatography on a Ni-NTA column. Purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (~80%). PHD1

[0629] Recombinant human PHD1 protein (catalog number 81064, batch number 24717001) was purchased from Active Motif (Carlsbad, CA, USA). PHD1 was expressed as the full-length protein with an N-terminal FLAG tag (molecular weight 44.9 kDa) in a baculovirus expression system (NCBI accession number NP_542770.2). Purity was assessed by SDS-PAGE (>90%). PHD2

[0630] The full-length human PHD2 enzyme system was produced by Beryllium (Bedford, MA, USA) using a baculovirus-infected insect cell (BIIC) expression system. This PHD2 construct contains amino acids 1 to 426 of PHD2 (UniProt Knowledgebase [UniProtKB] / Swiss-Prot accession number Q9GZT9.1), a His tag, and a Tobacco Etch Virus (TEV) protease cleavage site at the N-terminus. This construct was expressed in Sf9 insect cells, purified using a Ni-NTA column, and the His tag was removed by TEV protease cleavage. The purity of the final cleaved protein was assessed by SDS-PAGE and found to be >94%. PHD3

[0631] Recombinant human PHD3 protein (molecular weight 31.1 kDa) was purchased from Active Motif (Carlsbad, CA, USA). It was expressed as the full-length protein in E. coli with an N-terminal 6-His tag (catalog number 81033, batch number 24417001) (NCBI accession number NP_071356.1). Purity was assessed by SDS-PAGE and found to be >75%. PHD inhibitor.

[0632] The synthesis and identification of small molecule PHD inhibitors are as described in this paper. TR-FRET assay procedure.

[0633] The PHD inhibitor compound was pre-incubated with PHD enzyme in a white 384-well Optiplate microplate (catalog number 6007290, Perkin Elmer, Waltham, MA, USA) at a reaction volume of 10 μL. For this purpose, 5 μL of PHD inhibitor was serially diluted with dilution buffer (50 mM HEPES [4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid] pH 7.5, 50 mM sodium chloride [NaCl], 0.01% Tween-20, 0.01% purified bovine serum albumin [BSA]) and mixed with 5 μL of PHD enzyme (prepared as a 4X concentrate in dilution buffer containing PHD enzyme (60 nM PHD1, 20 nM PHD2, 140 nM PHD3), 40 µM ferrous ammonium sulfate (FAS), and 4 mM sodium ascorbate (Na)). The microplate was incubated at room temperature for 30 minutes without rotation.

[0634] Next, add 5 μL of VBC / anti-6His-Tb-caecin compound Gold mixture (prepared as a 4X concentrate in dilution buffer containing 20 nM His-VBC and 1.32 nM monoclonal antibody anti-6His-Tb-caecin compound Gold). Immediately after this step, add 5 μL of HIF-1α C35 matrix mixture (prepared as a 4X concentrate in dilution buffer containing 120 nM biotinylated HIF-1α C35, 132 nM SA-D2, and 4 μM 2-OG glutarate) to reach a final concentration of 20 μL.

[0635] The final assay results contained 50 mM HEPES, pH 7.5, 50 mM NaCl, 1 μM 2-OG, 10 μM FAS, 1 mM sodium ascorbate, 0.01% Tween-20, 0.01% purified BSA, 30 nM biotinylated HIF-1α C35, 5 nM His-VBC, 0.33 nM monoclonal antibody anti-6His-Tb-crystallization compound Gold, 33 nM SA-D2, and PHD enzymes (15 nM PHD1, 5 nM PHD2, or 35 nM PHD3) and dilution compounds.

[0636] Regarding the IC50 measurement of PHD inhibitor compounds, the reactants were incubated at room temperature for 10 minutes, followed by readings on a Perkin Elmer EnVision (Waltham, MA, USA) at an excitation wavelength of 340 nm and emission wavelengths of 615 nm and 665 nm. Data representing signal intensity coefficients at 615 nm and 665 nm were automatically calculated using Envision Manager software (Perkin Elmer, Waltham, MA, USA). IC50 values ​​(mean, standard deviation, standard error of mean, geometric mean, and 95% confidence interval) were determined using four-parameter curve fitting with GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA), presenting the compound concentration as plotted against the calculated ratios at 665 nm and 615 nm. Each concentration of compound was measured using the TR-FRET method in triplicate, with each measurement independently repeated three times.

[0637] Calculate Ki from IC50 based on the Cheng-Prussoff equation: Ki = IC50 / (1 + [2-OG] / Km)

[0638] The final concentration of 2-OG in both the PHD1 and PHD2 assays was 1 μM. The Km system determination of 2-OG was 12.7 nM in the PHD1 assay, while it was 22.6 nM in the PHD2 assay. Exemplary compounds Compound numbering structure PHD1 IC50 (nM) PHD2 IC50(nM) PHD3 IC50(nM) 1 B C -- 2 B D -- 3 A A B 4 B C -- 5 A A B 6 A C -- 7 A A C 8 A A A 9 A A A 10 A A D 11 A A B 12 A A B 13 A A A 14 C C -- 15 A A -- 16 B B C 17 A A A 18 A A A 19 C C -- 20 A B -- 21 A A B 22 A A -- 23 A A -- 24 A A A 25 A A -- 26 A A B 27 A A -- 28 A B B 29 A A -- 30 A A -- 31 A B -- 32 B B -- 33 A A -- 34 A A -- 35 A A A 36 A A A 37 A A A 38 A A C 39 B A C 40 A A -- 41 D D -- 42 A A -- 43 A A -- 44 A A -- Symbol explanation: A = IC50 < 100 nM B = 100 nM ≤ IC50 < 1,000 nM C = 1,000 nM ≤ IC50 < 10,000 nM D = IC50 ≥ 10,000 nM

[0639] Those skilled in the art can easily identify the essential characteristics of the invention from the description provided, and various changes and modifications can be made to adapt the invention to various uses and conditions without departing from its spirit and scope.

[0640] All U.S. or foreign references, patents or patent applications mentioned in this application are incorporated herein by reference in their entirety as written herein. In the event of any discrepancy, the information disclosed herein shall prevail. [Simplified Explanation of the Diagram]

[0641] Figure 1 is an illustrative schematic diagram showing the principle of the TR-FRET assay for PHD enzymes (PHD1, PHD2, and PHD3). In the presence of 2-side-oxyglutarate and O2, the PHD enzyme hydroxylates the proline 564 of the biotin-labeled HIF-1α peptide, resulting in the generation of biotin-labeled HIF-1α-hydroxyproline, succinate, and CO2. The proximity of the donor luciferase complex monoclonal antibody against 6His-tib(Tb)-caecinoid Gold, which binds to the His-labeled VHL protein, EloB, and EloC complex (His-VBC), to the acceptor luciferase SA-D2 complex, which binds to HIF-1α-hydroxyproline, leads to the detection and quantification of the fluorescence resonance energy transfer signal.

Claims

1. A compound of formula III or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, (III), wherein: m is 1, 2, 3, or 4; n is 0, 1, or 2; R1 is independently selected from the group consisting of hydrogen, halogen, OH, and C1-3 alkyl groups substituted with one or more halogen groups, as appropriate; R2 is hydrogen or C1-3 alkyl; R3 is independently selected from the group consisting of hydrogen, halogen, OH, NH2, and C1-3 alkyl, as appropriate; R4 is hydrogen or C1-4 alkyl; R5 is CN or halogen; and formula III excludes the following compounds: , , , , and.

2. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIIa): (IIIa).

3. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIIb): (IIIb).

4. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIIc): (IIIc).

5. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIId): (IIId).

6. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIIe): (IIIe).

7. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIIf): (IIIf).

8. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound has the structure of formula (IIIg): III(g).

9. A compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, wherein m is 1, 2, or 3.

10. A compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, wherein R1 is hydrogen.

11. A compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein R1 is a halogen, OH or, where appropriate, a C1-3 alkyl group substituted with one or more halogen groups.

12. A compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein R1 is F, Cl, Br, OH, methyl, ethyl or CF3.

13. A compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, wherein R2 is hydrogen or methyl.

14. A compound of any one of claims 1, 2, 5 and 8, or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein R3 is hydrogen, F, NH2 or methyl.

15. A compound of any one of claims 1, 3, 6 and 8, or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein R4 is methyl, ethyl, isopropyl or tert-butyl.

16. A compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein R5 is F, Cl, Br or CN.

17. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is selected from the group consisting of:

18. A compound of claim 17 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is selected from the group consisting of:

19. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (8).

20. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (9).

21. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (13).

22. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (18).

23. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (24).

24. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (36).

25. The compound of claim 18 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is: (37).

26. A compound of claim 17 or a pharmaceutically acceptable salt, stereoisomer or hydrate thereof, wherein the compound is selected from the group consisting of:

27. A compound of any one of claims 1 to 26, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, wherein at least one hydrogen atom is replaced by a deuterium atom.

28. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 27, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, and a pharmaceutically acceptable excipient.

29. Use of any compound of any one of claims 1 to 27, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, in the preparation of a medicament for treating diseases mediated by PHD activity.

30. As claimed in claim 29, wherein the diseases mediated by the PHD activity are: ischemia-reperfusion injury; inflammatory bowel disease; cancer; liver disease; atherosclerosis; cardiovascular disease; eye diseases or conditions; anemia; chronic kidney disease; and diseases related to hyperoxia.

31. As claimed in claim 30, wherein the ischemic reperfusion injury is selected from stroke, myocardial infarction and acute kidney injury.

32. As claimed in claim 30, wherein the inflammatory bowel disease is selected from ulcerative colitis or Crohn's disease.

33. As claimed in claim 30, wherein the cancer is colorectal cancer.

34. As claimed in claim 30, wherein the eye disease or condition is selected from radiation retinopathy, retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, and ocular ischemia.

35. As requested in claim 30, wherein the anemia is anemia associated with chronic kidney disease.

36. For the purposes of claim 30, wherein the disease associated with hyperoxia is retinopathy of prematurity or bronchodystrophy (BPD).

37. Use of a compound of any one of claims 1 to 27, or a pharmaceutically acceptable salt, stereoisomer, or hydrate thereof, in the preparation of a medicament for treating diseases mediated by PHD activity, wherein the diseases mediated by PHD activity are selected from ischemic heart disease, valvular heart disease, congestive heart failure, acute lung injury, pulmonary fibrosis, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), acute liver failure, liver fibrosis, and cirrhosis.

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