Pannexin-1 modulator and method for treating diseases in which pannexin-1 is involved

Abstract

Disclosed herein are small molecule modulators of pannexins. Also disclosed herein are methods of treating disorders associated with exacerbated activation of pannexin-mediated ATP-induced signaling pathways, such as pain and opioid addiction, by administering the small molecule modulators of pannexins. The present invention provides purine, indole, piperidine, pyrido(1,2-a)benzimidazole, naphthyridine, imidazoquinoline, imidazo-triazonaphthalene, and isoquinoline derivatives, salts, hydrates, solvates, and / or polymorphs thereof, which are PANX1 modulators.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 402,336, filed August 30, 2022, the entire teachings of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to small molecule modulators of pannexins and correlated channels, and to the treatment of diseases associated with exacerbated activation of ATP-induced signaling pathways and correlated channels mediated by pannexins, such as pain and opioid addiction. [Background technology]

[0003] Cell-cell and cell-matrix interactions are fundamental properties of multicellular organisms. Gap junctions allow the direct passage of ions and small molecules (<2,000 Da) between cells. Vertebrate homologs of innexins, called "pannexins," form primarily hemichannels, or pannexons, due to the high level of glycosylation in their extracellular domains. Pannexins contain a cytoplasmic N-terminal domain, four transmembrane domains with two extracellular loops, and a cytoplasmic C-terminal domain (1).

[0004] The pannexin (PANX) family consists of three proteins, PANX1, PANX2, and PANX3, all of which have been shown to form single-membrane channels. Panx1 is ubiquitously expressed in almost all cell types, including the nervous system, immune system, eye, muscle, olfactory epithelium, blood vessels, exocrine glands (e.g., lacrimal and salivary glands), thyroid, prostate, kidney, and liver. Panx1 protein is primarily localized to the plasma membrane.

[0005] Pannexins are ATP-release channels activated by caspase cleavage of their pore-associated C-terminal tails, and an autoregulatory region controls channel permeability. Regulated ATP (nucleotide) release via pannexin HCs is involved in many normal physiological functions and also plays a role in responses to stressors and pathological conditions in cells and tissues. Pannexin functions include cell differentiation and migration, tissue development and regeneration, inflammation, wound healing, and regulation of cell death. PANX1 has been shown to exhibit distinct structures with different conductance and permeability properties depending on the type of stimulus that triggers channel activation. The pore-associated C-terminus effectively inhibits PANX1 channel function. Panx1 has a low-voltage channel opening for Cl and a high-voltage channel opening for molecules such as ATP. Opening occurs gradually and can be permanent (caspase cleavage) or transient (1).

[0006] In the resting state, PANX1 is closed. Panx1 has various activation modes. The permeability and conductivity of the pannexon vary depending on the activation mode. Pannexons are activated by various physiological stimuli and extracellular [K + When activated by increasing the membrane potential, the channel becomes highly conductive and permeable to ATP. Activating pannexons only by increasing the membrane potential to positive values ​​reduces the channel conductance and allows Cl - This results in selective permeability to

[0007] Panx1 is expressed in most cell types and tissues of the somatic and nervous systems, particularly in the heart, skeletal muscle, skin, ovary, testis, placenta, prostate, thymus, lung, liver, small intestine, pancreas, spleen, colon, blood epithelium, and erythrocytes. In the central nervous system, Panx1 is present in the cerebellum, cortical lens, retina, cone cells, hippocampus, amygdala, substantia nigra, olfactory bulb, neurons, and glial cells. Therefore, there are numerous pathologies associated with PANX1. PANX1 channels are essential components of the P2X / P2Y purinergic signaling pathway and contribute significantly to pathophysiological ATP release. For example, PANX1 channels, along with ATP, purinergic receptors, and ectonucleotidases, contribute to several feedback loops during inflammatory responses (2).

[0008] Three major important processes in which extracellular ATP, and therefore Panx1, participate are:

[0009] Extracellular ATP is a key signaling molecule throughout the inflammatory cascade, functioning as a danger signal that triggers inflammasome activation, promoting immune cell infiltration, and fine-tuning several signaling cascades, including those important for inflammation resolution. Panx1-mediated ATP release is involved in inflammasome activation, neutrophil / macrophage chemotaxis, and T cell activation. The crucial role of Panx1 in inducing and propagating inflammation has been demonstrated in various organs, including the lungs and the central and peripheral nervous systems.

[0010] Furthermore, extracellular ATP can be degraded by ectonucleotidases to ADP, AMP, and adenosine, which are important for the resolution of inflammation. Thus, PANX1 contributes to an important feedback loop during the inflammatory response, making it a promising candidate for novel therapeutics (2).

[0011] In the extracellular space within the nervous system, ATP functions as a signaling molecule that can fulfill a variety of roles. ATP acts as a fast neurotransmitter, a trophic factor that promotes growth and development, and a damage-associated molecular pattern (DAMP, any molecule capable of inducing a non-infectious inflammatory response) that controls communication with phagocytes, such as acting as a microglial activator in the injured cortex. Panx1 is expressed in both neurons and glia, and it is thought to mediate crosstalk between these cells. Pathological activity of Panx1 is thought to significantly contribute to several disease processes, including seizures, stroke, migraine, and chronic pain. Emerging evidence also reveals that Panx1 has physiological functions regulating neural stem cell survival, neuronal maturation, and synaptic plasticity, suggesting its relevance to normal cognitive function (3)(4).

[0012] In the tumor microenvironment, high levels of extracellular ATP (released by Panx1) induce cell death. In cancer cells, high ATP levels are associated with enhanced cancer cell survival, proliferation, and metastasis (5). Summary of the Invention [Means for solving the problem]

[0013] The present invention relates to compounds that have PANX1 modulating activity. In some embodiments, the compound is a compound of Formula I or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0014] During the ceremony,

[0015] X and Y are each C, CH or N, or preferably Y is C or CH and X is N;

[0016] R 1 , R 2 and R 3 are independently hydrogen, lower alkyl optionally substituted with hydroxy, lower cycloalkyl, lower alkenyl, lower alkoxy, lower alkynyl, phenyl, halogenated phenyl, halogen, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably diazine, pyrazole, diazole, triazole, or alkyl derivatives thereof, 2-hydroxyisopropyl, or have one of the following structures: [ka]

[0017] During the ceremony,

[0018] X' can be C, CH, CH2, N, S, or O, or preferably CH2, N, or O;

[0019] X″ is independently C, CH, CH2, N, S, or O, preferably CH2, N, or O;

[0020] X''' is NH, N-lower alkyl, or O;

[0021] R1' to R5' are independently absent, hydrogen, lower alkyl, lower cycloalkyl, halogen, OH, NH2, hydroxy-lower alkyl, or NH-lower alkyl, O-lower alkyl, O-PO-OR6', O-lower alkyl or alkyloxy-O-PO-O-R6', or O-PO-O-lower alkyl-O-PO-R6', preferably OH, NH2, NH-lower alkyl, and adjacent R1' and R5' can form a 5- to 7-membered ring moiety;

[0022] R6' is H or O-lower alkyl;

[0023] R1" to R4" are independently absent, hydrogen, lower alkyl, lower cycloalkyl, OH, NH2, hydroxy-lower alkyl, NH-lower alkyl, O-lower alkyl, O-PO-OR6', O-lower alkyl or alkyloxy-O-PO-O-R6', or O-PO-O-lower alkyl-O-PO-R6', preferably OH, NH2, NH-lower alkyl, or halogen;

[0024] Y''' is a halogen, and

[0025] The bond between adjacent ring substituents X' or adjacent ring substituents X'' may comprise a single bond or a double bond.

[0026] In certain embodiments, the compound of formula I is [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0027] In another embodiment, the present invention relates to a compound of formula I' or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0028] During the ceremony,

[0029] X a or X b is C, CH or N, or preferably X a is C or CH, and X b is N, Y is CH, CH, C-lower alkyl, CH-lower alkyl, C-halogen, CH-halogen or C-dihalogen, Z is CH or N, preferably CH, and the bond between adjacent ring substituents Y may constitute a single or double bond;

[0030] R 1 , R 2 , R 3 and R4 are independently absent, hydrogen, lower alkyl, lower cycloalkyl, lower alkenyl, lower alkoxy, lower alkynyl, phenyl, halogenated phenyl, halogen, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole, or diazine, 2-hydroxyisopropyl, or have one of the following structures: [ka]

[0031] During the ceremony,

[0032] X' is C, CH, CH2, N, S, or O, preferably CH2, N, or O;

[0033] X″ is C, CH, CH2, N, S, or O, preferably CH2, N, or O;

[0034] X''' is NH, N-lower alkyl, or O;

[0035] R1' to R5' are independently absent, H, lower alkyl, lower cycloalkyl, OH, NH2, NH-lower alkyl, halogen, O-lower alkyl, O-PO-OR6', O-lower alkyl or alkyloxy-O-PO-O-R6', or O-PO-O-lower alkyl-O-PO-R6', preferably OH, NH2, NH-lower alkyl, and adjacent R1' and R5' can form a 5- to 7-membered ring moiety;

[0036] R6' is H or O-lower alkyl;

[0037] R1" to R4" are independently absent, hydrogen, lower alkyl, lower cycloalkyl, or halogen;

[0038] Y''' is a halogen, and

[0039] The bond between adjacent ring substituents X' or adjacent ring substituents X'' may comprise a single or double bond, thereby forming a non-aromatic or aromatic ring.

[0040] In certain embodiments, the compound of formula I' is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0041] In other specific embodiments, the present invention relates to a compound of formula II or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0042] During the ceremony,

[0043] X is N, Y is C, and Z is N, C, or CH;

[0044] R 1 ~R 4 does not exist independently when Z is N, or

[0045] R 1 ~R 4 or R 1 '~R 4 one or more of ' are low to medium chain oxo or keto fatty acids, optionally substituted with oxo or lower alkyl, H, halogen, lower alkyl, lower cycloalkyl, or COOH, or

[0046] R 1 ~R 4 or R 1 '~R 4 ' includes a substituent of formula X: [ka]

[0047] During the ceremony,

[0048] X' is CH, CO, N or O, preferably X' contains up to two N or O;

[0049] R 1’’ ~R 5’’is independently absent, H, lower alkyl, lower cycloalkyl, halogen, NH, NH-lower alkyl, hydroxy, oxo, COOH, SONH, SONH-lower alkyl, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or comprises a substituent of formula XI, [ka]

[0050] During the ceremony,

[0051] X iv is CH, CH2, CO, N or O, preferably X iv contains at most two N or O,

[0052] R 1 '''~R 5 "'' is independently absent, H, halogen, NH, lower alkyl, lower cycloalkyl, NH-lower alkyl, hydroxy, COOH, SONH, SONH-lower alkyl, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or preferably R 1 '''~R 5 and only one of the groups contains an unsubstituted aromatic or non-aromatic heterocycle; or

[0053] R 1 ~R 4 One or more of the following may comprise a substituent of formula XII: [ka]

[0054] During the ceremony,

[0055] Y' is absent or can each independently be CH2, CH2(CH3), CO, SO, SO2, CHOH or NH, and Z' is COOH, SO2NH2, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, forming a C3-C8 straight or branched chain; or

[0056] R 1 ~R 4 One or more of the following may comprise a substituent of formula XIII: [ka]

[0057] During the ceremony,

[0058] Y'' is COOH, SO2NH2, or tetrazole, and X''' is N or O.

[0059] In certain embodiments, the compound of formula II is [ka] [ka] [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0060] In another aspect, the present invention relates to a compound of formula III or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0061] During the ceremony,

[0062] each Z is independently selected from CH or C; each X is independently selected from CH, C, CH, O, N, S, SO, or SO; each Y is independently selected from N, C, CH, CH, CO, SO, S, or N-alkyl; and the bond between adjacent ring substituents X, Z, and / or Y comprises a single or double bond;

[0063] Each R 1 is absent or is H, halogen, O, NH, N-lower cycloalkyl, phenyl, alkyl (optionally substituted with one or both of oxo and carboxy), OH, SO, N(CH), C(O)N(CH), a 5-membered heterocycle (preferably tetrazole), -CO-(CH) n -COOH, where n is 0-4, and R1, together with optionally adjacent substituents, can form an aromatic or aliphatic 5-, 6-, or 7-membered ring system, which may contain heteroatoms (optionally substituted with one or more COOH, carboxamido, alkoxy, oxo, halogen, or triazole groups), or R1 optionally contains a substituent of formula XIV: [ka]

[0064] During the ceremony,

[0065] X' is absent or is O, N, or N-lower cycloalkyl; A is CH, or if X' is absent, may be a direct bond; Y' is O, N, N-lower cycloalkyl, CH, CH-lower alkyl, or CH-OH, or preferably X' is absent or O; the Y' ring contains one or two N or O atoms; and the bonds between adjacent substituents Y' and other atoms in the ring constitute single or double bonds, thereby forming a non-aromatic or aromatic ring; and

[0066] R 2 is absent or is lower alkyl optionally substituted with halogen, H, carboxy or oxo, hydroxy, alkoxy, COOH, SO2NH2, SO2NH-O-CH3, SO2NH-C(O)-CH3, SO2N(CH3)2, SO 2- lower alkyl, C(O)N(CH), a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole or tetrazole, or containing a substituent of formula XV: [ka]

[0067] During the ceremony,

[0068] B' is a bond, B' can include a linker or a direct bond, the linker is -N-, and R 3 are independently H, COOH, SO2NH2, SO2NH-lower alkyl, halogen, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole or tetrazole.

[0069] In certain embodiments of the present invention, the compound of formula III may be selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0070] In another aspect, the present invention relates to a compound of formula IV or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0071] During the ceremony,

[0072] each X is independently selected from CH or C;

[0073] X' is C,

[0074] each Y is independently selected from N, CH, C, SO, or CO;

[0075] R 1 and R 2 are each independently H, COOH, O—CH3, lower cycloalkyl, or joined together to form an optionally substituted 5- or 6-membered heterocycle;

[0076] R 3 ~R 7 are each independently H, COOH, O—CH, lower cycloalkyl, or contain a substituent of formula XVI: [ka]

[0077] During the ceremony,

[0078] Z is -N-CO-N or lower alkyl-CO-N;

[0079] X" is lower alkyl or partially halogenated lower alkyl, and

[0080] R1'-R5' are independently H, CHF2, COOH, SO2NH2, NH-CH2-COOH, lower alkyl, lower alkyl-COOH, lower alkyl-CO-, lower alkyl-COOH, -O-CO-CH3, OCH3, N-CO-NO-lower alkyl, N-CO-NO-CF2, -SO2-N(lower alkyl)2, lower alkyl-O-lower alkyl-COOH, SO2-N-lower alkyl-phenylcarboxylic acid, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, which may optionally be attached to the bicyclic ring of Formula IV by a lower alkyl linker.

[0081] In certain embodiments, the compound of formula IV is: [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0082] In a further aspect, the present invention relates to a compound of formula V or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0083] During the ceremony, X is either CH2, C, or CH; X' is C or CH; Y is C or CH; Y' is C; Z is independently N, O, or N-lower alkyl; Z' is O; R2 is H, halogen, lower alkyl, ketobutyric acid, cyclohexanonecarboxylic acid, or hydroxymethyl (cyclohexenone); the bond between adjacent ring substituents X, X', and / or Z is a single or double bond, and the bond between adjacent Y and / or Y' is a single or double bond; Z" can be CH or N, or lower alkyl, lower cycloalkyl, alkyl ether, or cycloalkyl; and R1 is H, lower alkyl, halogen, O-lower alkyl, COOH, N-CO-NO-lower alkyl or cycloalkyl, partially halogenated lower alkyl or cycloalkyl, -CO-CH3, -lower alkyl-O-phenylcarboxylic acid having a lower alkyl substituent;

[0084] [ka] It can be,

[0085] During the ceremony,

[0086] Z''' is lower alkyl or cycloalkyl, each R3 is independently H, COOH, NH-CO-NH-OCH3 or partially halogenated NH-CO-NH-CH3, O-lower alkyl, CO-CH3 or lower alkyl, and R4 is lower alkyl.

[0087] In certain embodiments, the compound of formula V is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0088] In a further aspect, the present invention is directed to a compound of formula VI or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0089] During the ceremony,

[0090] Independently, X is N or CH, and R 1 is NH, NH-lower alkyl, OH, or O-lower cycloalkyl, and R 2 and R 3 may be H, lower alkyl, SO2NH2, or may contain a substituent of formula XVIII: [ka]

[0091] During the ceremony,

[0092] X' is a direct bond, NH, N-CH3 or CH2, Y' is H or lower alkyl, and Z' is H, lower alkyl, COOH, SO2NH2, SO2NH-CH3, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole; when Y' and Z' are both H, X' is N-CH3.

[0093] In some embodiments, the compound of formula VI is [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0094] In a further embodiment, the present invention is directed to a compound of formula VII or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0095] During the ceremony,

[0096] X is N or C, X' is CH or N, X'' is N or CH, Y is CH or C, Z is CH2- or C=O, and the bond between adjacent ring substituents Y is a single bond or a double bond;

[0097] R 1 contains a substituent of formula XIX: [ka]

[0098] During the ceremony,

[0099] R1' to R3' can independently be halogen, carboxylic acid, SO2NH2, SO2-NH-lower alkyl, COOH, or NO2, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole; or

[0100] R1 may include a substituent of formula XX: [ka]

[0101] During the ceremony,

[0102] Y' is N, CH, or C; R'' is independently H, halogen, dihalogen, or lower alkyl;

[0103] R 2 is H, lower cycloalkyl, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably 1,3-diazolidine;

[0104] R 3 is a halogen, a carboxylic acid, SO2NH2, or SO2-NH-lower alkyl, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole;

[0105] R 4is H, a halogen, or a lower alkyl keto acid, preferably ketopropionic acid;

[0106] If X is N or CH, then R 5 is absent and when X is C, R5 is a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably pyrrolidine, piperidine, pyrazine, or a bicyclic structure of formula XXI: [ka]

[0107] During the ceremony,

[0108] Y'' is N,N-lower alkyl, or COOH.

[0109] In certain embodiments, the compound of formula VII is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0110] In another aspect, the present invention is directed to a compound of Formula VIII, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0111] During the ceremony,

[0112] X is SO2, NR4 or O, Z is C or N, Y is N, CR3 or CH, and R1 to R4 are each independently H, oxo, halogen, -(CH2) n -COOH (n=0-6), -NH-C(O)-NH-CH, -NH-C(O)-NH-CHF, alkyl or alkoxy (optionally substituted with COOH or oxo), or one or more of R1-R4 comprises a substituent of formula XXII:

[0113] [ka]

[0114] During the ceremony,

[0115] A is a linking site, and the substituent of formula XXII is attached to the compound of formula VIII directly or through a linker L, where L is alkyl, —CH—C(O)—NH—, or —NH—C(O)—NH—;

[0116] X' is H, O, N, or N-lower cycloalkyl;

[0117] Y' is O, N, N-lower cycloalkyl, CH2, CR5, CHR5, R5 is alkyl, hydroxy, alkoxy, halogen, alkyl is optionally substituted with one or more of COOH, oxo, alkoxy, halogen, or hydroxylamine, adjacent Y's are bonded by a single or double bond, and two R5s bonded to two adjacent Y's are optionally bonded to form an aromatic or non-aromatic 5- or 6-membered ring, preferably X' is O, and the Y' ring contains one or two N or O atoms.

[0118]

[0119] In certain embodiments, the compound of formula VIII is: [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0120] In another aspect, the invention includes a pharmaceutical composition comprising a compound of Formula I, I', II, III, IV, V, VI, VII, or VIII, or a salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

[0121] In another aspect, the present invention includes a method of treating a disease or disorder associated with aberrant PANX1 signaling, comprising administering to a subject in need thereof a compound of Formula I, I', II, III, IV, V, VI, VII, or VIII, or a salt or solvate thereof.

[0122] In certain embodiments, the disease or disorder is selected from the group comprising chronic pain, chemotherapy-related pain, addiction, particularly opioid addiction, epilepsy, Parkinson's disease, Alzheimer's disease, multiple sclerosis, traumatic brain injury, migraine, stroke, cancer, particularly melanoma, hepatocellular carcinoma, breast cancer, colon cancer, pancreatic cancer, and leukemia, cardiovascular diseases, particularly cardiac arrhythmias, vascular inflammation, hypertension, and pulmonary arterial hypertension, inflammatory diseases, particularly arthritis and wound healing inflammatory diseases, lung diseases, particularly Covid-19, asthma, and primary and secondary ciliary dyskinesia, fibrosis, diabetes, eye diseases, and skin diseases.

[0123] In a preferred embodiment, the disease or disorder is chronic pain. In another preferred embodiment, the disease or disorder is opioid addiction. [Brief explanation of the drawings]

[0124] [Figure 1] 1 shows the expression of β-catenin protein in MDA-MB-231 breast cancer cells treated with or without a PANX1 blocking agent of the present invention.

[0125] [Figure 2] 1 shows E-cadherin protein expression in MDA-MB-231 breast cancer cells treated with or without a PANX1 blocking agent of the present invention.

[0126] [Figure 3] 1 shows matrix metalloproteinase 2 (MMP2) protein expression in MDA-MB-231 breast cancer cells treated or not with a PANX1 blocking agent of the present invention.

[0127] [Figure 4] 1 shows matrix metalloproteinase 9 (MMP9) protein expression in MDA-MB-231 breast cancer cells treated or not with a PANX1 blocking agent of the present invention.

[0128] [Figure 5-1] 1 shows the tissue invasive ability of MDA-MB-231 cells cultured in Matrigel treated or not with a PANX1 blocking agent of the present invention. [Figure 5-2] 1 shows the tissue invasive ability of MDA-MB-231 cells cultured in Matrigel treated or not with a PANX1 blocking agent of the present invention. [Figure 5-3] 1 shows the tissue invasive ability of MDA-MB-231 cells cultured in Matrigel treated or not with a PANX1 blocking agent of the present invention. [Figure 5-4] 1 shows the tissue invasive ability of MDA-MB-231 cells cultured in Matrigel treated or not with a PANX1 blocking agent of the present invention.

[0129] [Figure 6] Dissection of the rat biceps femoris showing the exposed sciatic nerve (1) and its further division into the peroneal nerve (4), common peroneal nerve (2), and tibial nerve (3).

[0130] [Figure 7] 1 shows the effect of intraspinal administration of Compound 004, carbenoxolone (Cbx), or saline on the withdrawal threshold in neuropathic rats.

[0131] [Figure 8] 1 shows the effect of intraspinal administration of Compound 011, Cbx, or saline on the withdrawal threshold in neuropathic rats.

[0132] [Figure 9] 1 shows the effect of intraspinal administration of Compound 054, Cbx, or saline on the withdrawal threshold in neuropathic rats.

[0133] [Figure 10] 1 shows the effect of intraspinal administration of Compound 055, Cbx, or saline on the withdrawal threshold in neuropathic rats.

[0134] [Figure 11] 1 shows the effect of intraspinal administration of Compound 019, Cbx, or saline on the withdrawal threshold in neuropathic rats.

[0135] [Figure 12] 1 shows the effect of intraspinal administration of Compound 027, Cbx, or saline on the withdrawal threshold in neuropathic rats.

[0136] [Figure 13] 1 shows the effect of intraspinal administration of Compound 043, Cbx, or saline on the withdrawal threshold in neuropathic rats.

[0137] [Figure 14] 1 shows the effect of Compound 004 or Compound 011 on withdrawal scores in rats in an opioid-induced addiction model.

[0138] [Figure 15] ATP release by astrocytes (A) and microglial cells (B) in vitro upon stimulation with lipopolysaccharide in the presence or absence of a PANX1 blocker of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0139] The present invention provides purine, indole, piperidine, pyrido(1,2-a)benzimidazole, naphthyridine, imidazoquinoline, imidazo-triazonaphthalene, and isoquinoline derivatives, as well as their salts, hydrates, solvates, and / or polymorphs, which are PANX1 modulators. Pharmaceutical compositions containing the compounds or their salts, hydrates, solvates, and / or polymorphs, and a pharmaceutically acceptable excipient, are also provided. The salts, hydrates, solvates, and / or polymorphs of the compounds can be used to treat diseases associated with PANX-1 overactivity. Accordingly, the present invention also provides methods for treating patients suffering from diseases associated with PANX-1 overactivity, comprising administering a compound of the present invention or one of its salts, hydrates, solvates, and / or polymorphs to a patient in need thereof. Finally, the present invention also provides the use of a panx-1 modulator described herein, or a salt, hydrate, solvate, and / or polymorph thereof, for the preparation of a medicament for preventing or treating a disease associated with PANX1 overactivity.

[0140] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0141] It will be understood that the same thing can be expressed in more than one way. Accordingly, alternative language or synonyms may be used for one or more of the terms described herein. Whether a term is detailed or discussed herein is not intended to imply any special significance. Synonyms for certain terms are provided. The listing of one or more synonyms does not exclude the use of other synonyms. The use of any examples herein (including examples of terms discussed herein) is for illustrative purposes only and is not intended to further limit the scope and meaning of the disclosure or the scope and meaning of the exemplified term. Similarly, the disclosure is not limited to the various embodiments described herein.

[0142] As used herein, a PANX1 modulator refers to a small molecule that binds to PANX1 and blocks its activity. Modulators are classified as antagonists or agonists depending on their effect on PANX1, and further classified as full, partial, or inverse agonists. An agonist is a modulator that binds to a receptor and changes the receptor's state, resulting in a biological response. If the induced response is maximal, the modulator is said to be a full agonist. If the response is less than maximal at the highest agonist concentration, the modulator is a partial agonist. If a modulator binds to PANX1 and reduces its proportion in the active conformation, the ligand becomes an inverse agonist. Finally, an antagonist is a modulator that prevents a receptor agonist from binding to the receptor or, upon binding, from inducing the receptor's conformational changes that lead to signal transduction, thereby inhibiting signal transduction.

[0143] In the context of the present invention, the PANX1 modulator can prevent ATP efflux from cells upon activation of PANX1. The prevention of ATP efflux through the PANX1 channel promoted by the compounds of the present invention is also referred to as blocking. Therefore, the PANX-1 modulator of the present invention can be a PANX-1 blocker.

[0144] As used herein, the term "alkyl" has been given its normal meaning in the art, and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, alkyl groups can be lower alkyl groups, where lower alkyl groups have 10 or fewer carbon atoms in their backbone. Likewise, lower cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6, or 7 carbon atoms in the ring structure.

[0145] As used herein, the term "alkenyl" is given its ordinary meaning in the art and includes monounsaturated or polyunsaturated aliphatic groups containing one or more carbon-carbon double bonds, including straight-chain alkenyl groups, branched-chain alkenyl groups, which may be unsubstituted or substituted with alkyl, alkenyl, alkynyl, hydroxy, carboxy, alkoxy, heteroalkyl, heterocyclic, aryl, heteroaryl, oxo, and amino groups. In some embodiments, the carbon-carbon double chain may be internal or terminal. In some embodiments, an alkenyl group may be a lower alkenyl group, which has 10 or fewer carbon atoms, 6 or fewer carbon atoms, or 5 or fewer carbon atoms in its backbone.

[0146] As used herein, the term "alkynyl" is given its ordinary meaning in the art and includes unsaturated aliphatic groups containing one or more carbon-carbon triple bonds, which can be unsubstituted or substituted with alkyl, alkenyl, alkynyl, hydroxy, carboxy, alkoxy, heteroalkyl, heterocyclic, aryl, heteroaryl, oxo, and amino groups. In some embodiments, the carbon-carbon triple bond can be internal or terminal. In some embodiments, an alkynyl group can be a lower alkynyl group having 10 or fewer carbon atoms, 6 or fewer carbon atoms, or 5 or fewer carbon atoms in its backbone.

[0147] As used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to an alkyl group, as described herein, in which one or more atoms is a heteroatom (e.g., oxygen, nitrogen, sulfur, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0148] In the context of this invention, the term "aryl" refers to an optionally substituted aromatic group having a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused rings, at least one of which is aromatic (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl). That is, at least one ring has a conjugated pi-electron system, and other adjacent rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl. Aryl groups may be optionally substituted as described herein.

[0149] The term "carbocyclic aryl group" refers to an aryl group in which the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic or fused compounds (e.g., two or more adjacent ring atoms are common to two adjacent rings) such as naphthyl groups. In some cases, aryl groups include monocyclic carbocyclic aryl groups and polycyclic or fused compounds (e.g., two or more adjacent ring atoms are common to two adjacent rings) such as naphthyl groups. Non-limiting examples of aryl groups include phenyl, quinolinyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, pyrido(1,2-a)benzimidazole, imidazoquinoline, and the like.

[0150] The term "heteroaryl" is given its ordinary meaning in the art and refers to aryl groups described herein in which one or more atoms is an optionally substituted heteroatom (e.g., oxygen, nitrogen, sulfur, etc.). Examples of aryl and heteroaryl groups include, but are not limited to, phenyl, pyrrolyl, furanyl, thiophenyl, benzothiazolyl, imidazolyl, benzimidazolyl, imidazoquinolinyl, naphthyridinyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, and the like.

[0151] The atoms constituting the compounds of the present disclosure are intended to include all isotopic forms of such atoms. As used herein, "isotopes" include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 114, 115, 120, 121, 122, 12 13 C and 14 C. It is further contemplated that one or more carbon atoms of the compound may be replaced by a silicon atom. It is further contemplated that one or more oxygen atoms of the compound may be replaced by a sulfur atom or a selenium atom.

[0152] Compounds having formulas represented by dashed bonds are intended to include formulas that optionally have zero, one, or more double bonds. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom.

[0153] The term "pharmaceutically acceptable" means that the materials of a pharmaceutical composition must be compatible with each other and not deleterious to the recipient thereof. In this sense, a "pharmaceutically acceptable salt" refers to a salt that possesses the effectiveness of the parent compound and that is not biologically or otherwise undesirable (e.g., not toxic or otherwise harmful to the recipient).

[0154] As used herein, "disease associated with PANX1" refers to a disease or disorder characterized by inappropriate PANX1 activity. For example, inappropriate PANX1 activity refers to an increase or decrease in PANX1 activity measured by a cell assay compared to the activity in healthy cells or subjects. Inappropriate activity may also be due to overexpression of PANX1 in diseased tissue compared to adjacent healthy tissue.

[0155] As used herein, the term "therapeutic activity" refers to a demonstrated or potential biological activity whose effect is consistent with a desired therapeutic outcome in humans or is consistent with a desired effect in non-human mammals or other species or organisms. While a particular PANX1 modulator may have one or more therapeutic activities, the term "therapeutic activity" as used herein may refer to a single therapeutic activity or multiple therapeutic activities. "Therapeutic activity" includes the ability to elicit a desired response and may be measured in vivo or in vitro. For example, a desired effect may be measured in cell culture, isolated tissues, animal models, clinical evaluation, EC 50 Assay, IC 50 Therapeutic activity can be assessed by assay or dose-response curve. The term therapeutic activity includes prevention or treatment of a disease, disorder, or condition. Treatment of a disease, disorder, or condition includes any degree of amelioration of the disease, disorder, or condition, including elimination of the disease, disorder, or condition.

[0156] The acronyms IC and EC used herein stand for "inhibitory concentration" and "effective concentration," respectively, and the notation IC 50 and EC 50 represents the half-maximal inhibition or activation of a particular biological phenomenon (e.g., ATP release) promoted by a compound in an in vitro assay.

[0157] As used herein, the term "therapeutically effective" will vary depending on the condition of the subject and the particular compound being administered. This term refers to an amount effective to achieve a desired clinical effect. The therapeutically effective amount will vary depending on the nature of the condition being treated, the length of time the effect is desired, and the age and condition of the patient, and is ultimately determined by the attending physician.

[0158] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing, or reversing the progression of a disease or disorder, substantially ameliorating the clinical symptoms of a disease or disorder, or substantially preventing the appearance of clinical symptoms of a disease or disorder.

[0159] Pannexin-1 Modulators The PANX1 modulators of the present invention can be compounds of Formula I, II, III, IV, V, VI, or VII, which are purine, indole, naphthyridine, piperidine, pyrido(1,2-a)benzimidazole, quinoline, and isoquinoline derivatives, respectively.

[0160] The present invention relates to compounds that have PANX1 modulating activity. In some embodiments, the compound is a compound of Formula I or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0161] During the ceremony,

[0162] X and Y are each C, CH or N, or preferably Y is C or CH and X is N;

[0163] R 1 , R 2 and R 3 are independently hydrogen, lower alkyl optionally substituted with hydroxy, lower cycloalkyl, lower alkenyl, lower alkoxy, lower alkynyl, phenyl, halogenated phenyl, halogen, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably diazine, pyrazole, diazole, triazole, or alkyl derivatives thereof, 2-hydroxyisopropyl, or have one of the following structures: [ka]

[0164] During the ceremony,

[0165] X' can be C, CH, CH2, N, S, or O, or preferably CH2, N, or O;

[0166] X″ is independently C, CH, CH2, N, S, or O, preferably CH2, N, or O;

[0167] X''' is NH, N-lower alkyl, or O;

[0168] R1' to R5' are independently absent, hydrogen, lower alkyl, lower cycloalkyl, halogen, OH, NH2, hydroxy-lower alkyl, or NH-lower alkyl, O-lower alkyl, O-PO-OR6', O-lower alkyl or alkyloxy-O-PO-O-R6', or O-PO-O-lower alkyl-O-PO-R6', preferably OH, NH2, NH-lower alkyl, and adjacent R1' and R5' can form a 5- to 7-membered ring moiety;

[0169] R6' is H or O-lower alkyl;

[0170] R1" to R4" are independently absent, hydrogen, lower alkyl, lower cycloalkyl, OH, NH2, hydroxy-lower alkyl, NH-lower alkyl, O-lower alkyl, O-PO-OR6', O-lower alkyl or alkyloxy-O-PO-O-R6', or O-PO-O-lower alkyl-O-PO-R6', preferably OH, NH2, NH-lower alkyl, or halogen;

[0171] Y''' is a halogen, and

[0172] The bond between adjacent ring substituents X' or adjacent ring substituents X'' may comprise a single bond or a double bond.

[0173] In certain embodiments, the compound of formula I is [ka] [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0174] In some embodiments of the present invention, a subclass of the aforementioned class of compounds may further be represented by Formula I', or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0175] During the ceremony,

[0176] X a or X b is C, CH or N, or preferably X a is C or CH, and X b is N, Y is CH, CH, C-lower alkyl, CH-lower alkyl, C-halogen, CH-halogen or C-dihalogen, Z is CH or N, preferably CH, and the bond between adjacent ring substituents Y may constitute a single or double bond;

[0177] R 1 , R 2 , R 3 , and R 4 is independently absent, hydrogen, lower alkyl, lower cycloalkyl, lower alkenyl, lower alkoxy, lower alkynyl, phenyl, halogenated phenyl, halogen, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole, or diazine, 2-hydroxyisopropyl, or has one of the following structures: [ka]

[0178] During the ceremony,

[0179] X' is C, CH, CH2, N, S, or O, preferably CH2, N, or O;

[0180] X″ is C, CH, CH2, N, S, or O, preferably CH2, N, or O;

[0181] X''' is NH, N-lower alkyl, or O;

[0182] R1' to R5' are independently absent, H, lower alkyl, lower cycloalkyl, OH, NH2, NH-lower alkyl, halogen, O-lower alkyl, O-PO-OR6', O-lower alkyl or alkyloxy-O-PO-O-R6', or O-PO-O-lower alkyl-O-PO-R6', preferably OH, NH2, NH-lower alkyl, and adjacent R1' and R5' can form a 5- to 7-membered ring moiety;

[0183] R6' is H or O-lower alkyl;

[0184] R1" to R4" are independently absent, hydrogen, lower alkyl, lower cycloalkyl, or halogen;

[0185] Y''' is a halogen, and

[0186] The bond between adjacent ring substituents X' or adjacent ring substituents X'' may comprise a single or double bond, thereby forming a non-aromatic or aromatic ring.

[0187] In certain embodiments, the compound of formula I' is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0188] Another embodiment of the present invention includes a compound of Formula II or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0189] During the ceremony,

[0190] X is N, Y is C, and Z is N, C, or CH;

[0191] R 1 ~R 4 does not exist independently when Z is N, or

[0192] R 1 ~R 4 or R 1 '~R 4 one or more of ' are low to medium chain oxo or keto fatty acids, optionally substituted with oxo or lower alkyl, H, halogen, lower alkyl, lower cycloalkyl, or COOH, or

[0193] R 1 ~R 4 or R 1 '~R 4 ' includes a substituent of formula X: [ka]

[0194] During the ceremony,

[0195] X' is CH, CO, N or O, preferably X' contains up to two N or O, and R 1 ''~R 5'' is independently absent, H, lower alkyl, lower cycloalkyl, halogen, NH, NH-lower alkyl, hydroxy, oxo, COOH, SONH, SONH-lower alkyl, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or contains a substituent of formula XI: [ka]

[0196] During the ceremony,

[0197] X iv is CH, CH2, CO, N or O, or preferably, X iv contains at most two N or O and R 1 '''~R 5 "'' is independently absent, H, halogen, NH, lower alkyl, lower cycloalkyl, NH-lower alkyl, hydroxy, COOH, SONH, SONH-lower alkyl, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or preferably R 1 '''~R 5 Only one of the groups contains an unsubstituted aromatic or non-aromatic heterocycle and the adjacent ring substituent X iv The bond between may constitute a single or double bond, thereby forming a non-aromatic or aromatic ring, or

[0198] R 1 ~R 4 One or more of the following may comprise a substituent of formula XII: [ka]

[0199] During the ceremony,

[0200] Y' is absent or can each independently be CH2, CH2(CH3), CO, SO, SO2, CHOH or NH, and Z' is COOH, SO2NH2, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, forming a C3-C8 linear or branched chain; or

[0201] One or more of R1-R4 comprises a substituent of formula XIII: [ka]

[0202] During the ceremony,

[0203] Y'' is COOH, SO2NH2, or tetrazole, and X''' is N or O.

[0204] In certain embodiments, the compound of formula II is: [ka] [ka] [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0205] In a further embodiment, the present invention comprises a compound of formula III or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0206] During the ceremony,

[0207] Each Z is independently selected from CH or C, and each X is independently CH, C, CH 2、 each Y is independently selected from N, C, CH, CH, CO, SO, S, or N-alkyl, and the bond between adjacent ring substituents X, Z, and / or Y comprises a single or double bond;

[0208] Each R 1 is absent or is H, halogen, O, NH, N-lower cycloalkyl, phenyl, alkyl (optionally substituted with one or both of oxo and carboxy), OH, SO, N(CH), C(O)N(CH), a 5-membered heterocycle (preferably tetrazole), -CO-(CH) n -COOH, where n is 0-4, and R1, together with optionally adjacent substituents, can form an aromatic or aliphatic 5-, 6-, or 7-membered ring system, which may contain heteroatoms (optionally substituted with one or more COOH, carboxamido, alkoxy, oxo, halogen, or triazole groups), or R1 optionally contains a substituent of formula XIV: [ka]

[0209] During the ceremony,

[0210] X' is absent or is O, N, or N-lower cycloalkyl; A is CH, or if X' is absent, may be a direct bond; Y' is O, N, N-lower cycloalkyl, CH, CH-lower alkyl, or CH-OH, or preferably X' is absent or O; the Y' ring contains one or two N or O atoms, and the bonds between adjacent substituents Y' and other atoms in the ring constitute single or double bonds, thereby forming a non-aromatic or aromatic ring; and

[0211] R 2 is absent or is lower alkyl optionally substituted with halogen, H, carboxy or oxo, hydroxy, alkoxy, COOH, SO2NH2, SO2NH-O-CH3, SO2NH-C(O)-CH3, SO2N(CH3)2, SO 2- lower alkyl, C(O)N(CH), a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole or tetrazole, or containing a substituent of formula XV: [ka]

[0212] During the ceremony,

[0213] B' is a bond, B' can include a linker or a direct bond, the linker is -N-, and R 3 are independently H, COOH, SO2NH2, SO2NH-lower alkyl, halogen, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole or tetrazole.

[0214] In a particular embodiment of the invention, the compound of formula III is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0215] In a further embodiment, the invention relates to a compound of formula IV or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0216] During the ceremony,

[0217] each X is independently selected from CH or C;

[0218] X' is C,

[0219] each Y is independently selected from N, CH, C, SO, or CO;

[0220] R 1 and R 2 are each independently H, COOH, O—CH3, lower cycloalkyl, or joined together to form an optionally substituted 5- or 6-membered heterocycle;

[0221] R 3 ~R 7 are each independently H, COOH, O—CH, lower cycloalkyl, or contain a substituent of formula XVI: [ka]

[0222] During the ceremony,

[0223] Z is -N-CO-N or lower alkyl-CO-N;

[0224] X" is lower alkyl or partially halogenated lower alkyl, and

[0225] R1'-R5' are independently H, CHF2, COOH, SO2NH2, NH-CH2-COOH, lower alkyl, lower alkyl-COOH, lower alkyl-CO-, lower alkyl-COOH, -O-CO-CH3, OCH3, N-CO-NO-lower alkyl, N-CO-NO-CF2, -SO2-N(lower alkyl)2, lower alkyl-O-lower alkyl-COOH, SO2-N-lower alkyl-phenylcarboxylic acid, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, which may optionally be attached to the bicyclic ring of formula IV by a lower alkyl linker.

[0226] In certain embodiments, the compound of formula IV is: [ka] [ka] [ka] and pharmaceutically acceptable salts, hydrates, solvates and polymorphs thereof.

[0227] In other embodiments, the present invention comprises a compound of formula V or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0228] X is CH2, C, or CH, X' is either C or CH, Y is C or CH, Y' is C, Z is independently N, O, or N-lower alkyl, Z' is O, R2 is H, halogen, lower alkyl, ketobutyric acid, cyclohexanonecarboxylic acid, or hydroxymethyl (cyclohexenone), the bond between adjacent ring substituents X, X', and / or Z is a single or double bond, the bond between adjacent Y and / or Y' is a single or double bond, Z" can be CH or N, or lower alkyl, lower cycloalkyl, alkyl ether, or cycloalkyl, R1 is H, lower alkyl, halogen, O-lower alkyl, COOH, N-CO-NO-lower alkyl or cycloalkyl, partially halogenated lower alkyl or cycloalkyl, -CO-CH3, -lower alkyl-O-phenylcarboxylic acid having a lower alkyl substituent, or

[0229] [ka] It can be,

[0230] During the ceremony,

[0231] Z''' is lower alkyl or cycloalkyl, each R3 is independently H, COOH, NH-CO-NH-OCH3 or partially halogenated NH-CO-NH-CH3, O-lower alkyl, CO-CH3 or lower alkyl, and R4 is lower alkyl.

[0232] In certain embodiments, the compound of formula V is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0233] In other embodiments, the present invention comprises a compound of formula VI or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0234] During the ceremony,

[0235] Independently, X is N or CH, and R 1 is NH, NH-lower alkyl, OH, or O-lower cycloalkyl, and R 2 and R 3 may be H, lower alkyl, SO2NH2, or may contain a substituent of formula XVIII: [ka]

[0236] During the ceremony,

[0237] X' is a direct bond, NH, N-CH3 or CH2, Y' is H or lower alkyl, and Z' is H, lower alkyl, COOH, SO2NH2, SO2NH-CH3, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole; when Y' and Z' are both H, X' is N-CH3.

[0238] In certain embodiments, the compound of formula VI is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0239] In a further embodiment, the present invention comprises a compound of Formula VII or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0240] During the ceremony,

[0241] X is N or C, X' is CH or N, X'' is N or CH, Y is CH or C, Z is CH2- or C=O, and the bond between adjacent ring substituents Y is a single bond or a double bond;

[0242] R 1 contains a substituent of formula XIX: [ka]

[0243] During the ceremony,

[0244] R1' to R3' can independently be halogen, carboxylic acid, SO2NH2, SO2-NH-lower alkyl, COOH, or NO2, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole; or

[0245] R1 may include a substituent of formula XX: [ka]

[0246] During the ceremony,

[0247] Y' is N, CH, or C; R'' is independently H, halogen, dihalogen, or lower alkyl;

[0248] R 2 is H, lower cycloalkyl, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably 1,3-diazolidine;

[0249] R 3 is a halogen, a carboxylic acid, SO2NH2, SO2-NH-lower alkyl, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole;

[0250] R 4 is H, a halogen, or a lower alkyl keto acid, preferably ketopropionic acid;

[0251] If X is N or CH, then R 5 is absent and when X is C, R5 is a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably pyrrolidine, piperidine, pyrazine, or a bicyclic structure of formula XXI: [ka]

[0252] During the ceremony,

[0253] Y'' is N,N-lower alkyl, or COOH.

[0254] In certain embodiments, the compound of formula VII is: [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0255] In other embodiments, the present invention comprises a compound of Formula VIII or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: [ka]

[0256] During the ceremony,

[0257] X is SO2, NR4 or O, Z is C or N, Y is N, CR3 or CH, and R1 to R4 are each independently H, oxo, halogen, -(CH2) n-COOH (n=0-6), -NH-C(O)-NH-CH, -NH-C(O)-NH-CHF, alkyl or alkoxy (optionally substituted with COOH or oxo), or one or more of R1-R4 comprises a substituent of formula XXII:

[0258] [ka]

[0259] During the ceremony,

[0260] A is a linking site, and the substituent of formula XXII is attached to the compound of formula VIII directly or through a linker L, where L is alkyl, —CH—C(O)—NH—, or —NH—C(O)—NH—;

[0261] X' is H, O, N, or N-lower cycloalkyl;

[0262] Y' is O, N, N-lower cycloalkyl, CH2, CR5, CHR5, R5 is alkyl, hydroxy, alkoxy, halogen, alkyl is optionally substituted with one or more of COOH, oxo, alkoxy, halogen, or hydroxylamine, adjacent Y's are bonded by a single or double bond, and two R5s bonded to two adjacent Y's are optionally bonded to form an aromatic or non-aromatic 5- or 6-membered ring, preferably X' is O, and the Y' ring contains one or two N or O atoms.

[0263] In certain embodiments, the compound of formula VIII is [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.

[0264] The compounds of the present invention may be in the form of pharmaceutically acceptable salts. Suitable salts include, for example, acid addition salts that can be formed by mixing a solution of the compound of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, or benzoic acid. Acceptable salts also include salts formed with suitable organic ligands, such as alkali metal salts (e.g., sodium salts or potassium salts), alkaline earth metal salts (e.g., calcium salts or magnesium salts), and quaternary ammonium salts. In addition, when an acid (-COOH) or alcohol group is present, pharmaceutically acceptable esters can be used to modify the solubility or hydrolysis characteristics of the compound. Biological activities and therapeutic applications of PANX-1 modulators

[0265] In some embodiments, the PANX1 modulators of the present invention are applicable to the treatment of diseases. The PANX1-modulating activity of a particular compound can be measured using various in vitro models known to skilled practitioners. For example, models are available for assessing ATP release by human embryonic kidney (HEK)-293 cells and Xenopus oocytes genetically engineered to overexpress PANX1 hemichannels (6, 7). Models based on cells that naturally express PANX1 are also known. For example, skilled practitioners will be aware that human subcutaneous fibroblasts release ATP via PANX1 upon mechanical or histamine stimulation (8, 9).

[0266] It is also possible to investigate the potential preventive or therapeutic effects of PANX1 modulators against several diseases using both in vitro and in vivo models. For example, the antitumor effects of PANX1 blockers against colon cancer, breast cancer, and melanoma can be evaluated using corresponding cell lines (10-12). Animal models have also been reported to evaluate the ability of PANX1 modulators to prevent or manage the progression of symptoms of Alzheimer's disease and multiple sclerosis (13)(14).

[0267] In some embodiments of the present invention, a PANX1 modulator or a pharmaceutically acceptable salt, hydrate, solvate, and / or polymorph thereof can be used to treat a disease in which PANX1 overactivity is involved.

[0268] In another aspect, the present invention provides a method for treating a patient suffering from a disease associated with PANX1 overactivity, comprising administering to a patient in need thereof a compound of the present invention or one of its salts, hydrates, solvates, and / or polymorphs.

[0269] In another embodiment, the present invention provides the use of a PANX-1 modulator described herein, or a salt, hydrate, solvate, and / or polymorph thereof, for the preparation of a medicament for preventing or treating a disease associated with panx-1 overactivity.

[0270] In some embodiments, the therapeutic methods and uses of PANX1 modulators described herein relate to the treatment of diseases that would benefit from modulation of pannexin 1 activity, including but not limited to chronic pain, opioid addiction, epilepsy, Parkinson's disease (PD), multiple sclerosis (MS), Alzheimer's disease (AD), traumatic brain injury, migraine, stroke, neoplastic diseases and / or conditions, cardiovascular diseases, inflammatory or autoimmune diseases, and pulmonary diseases.

[0271] In some embodiments of the present invention, the neoplastic disease and / or condition treatable with the PANX1 modulators of the present invention may be selected from hepatocellular carcinoma (HCC), breast cancer, colon cancer, pancreatic cancer, leukemia, chemotherapy-related pain, and others known to those skilled in the art.

[0272] In another embodiment of the present invention, the PANX1 modulators are applicable to the treatment of cardiovascular diseases, which may be selected from arrhythmia, vascular inflammation, pulmonary arterial hypertension (PAH), hypertension, and other diseases known to those skilled in the art.

[0273] In another embodiment of the present invention, the PANX1 modulators are applicable for the treatment of inflammatory or autoimmune diseases, which may be selected from arthritis, wound healing disorders, and other diseases known to those skilled in the art.

[0274] In another embodiment of the present invention, the PANX1 modulators may be applied in the treatment of pulmonary diseases, which may be selected from asthma, COPD, primary and secondary ciliary dyskinesia (PCD and SCD), coronavirus-mediated pulmonary diseases (such as Covid-19), and other diseases known to those skilled in the art.

[0275] As already pointed out, PANX1 is involved in numerous pathologies in various organs. Designing and refining compounds from several different chemical classes with distinct physicochemical properties will allow us to assign PANX1 blockers to specific organs and their respective underlying pathologies. Below, we outline the most investigated pathologies and their relationship / role in various pathologies.

[0276] chronic pain Neuropathic pain is a disabling consequence of cancer treatment with cytotoxic chemotherapy agents such as paclitaxel. The economic cost of chronic pain, of which neuropathic pain is a significant component, is estimated to exceed $500 billion in the United States alone. Mice lacking PANX1 developed acute mechanical hypersensitivity after the first dose of paclitaxel, but unlike wild-type mice, the neuropathic pain did not persist and resolved after the second dose of paclitaxel. (16-18)

[0277] PANX1 in hematopoietic cells is required for pain-like responses after nerve injury in mice and is a potential therapeutic target. - / - ) protected against hypersensitivity in two models of sciatic nerve injury. Bone marrow transplant studies have shown that functional PANX1 expression in hematopoietic cells is required for mechanical hypersensitivity after nerve injury (16-18).

[0278] Opioid addiction, general addiction Studies in mice have shown that PANX1 is activated during opioid withdrawal. Therefore, the P2X7-Panx1 signaling cascade functions as a feed-forward loop, amplifying cellular responses to withdrawal. Mice lacking PANX1 on microglia exhibited reduced withdrawal behavior compared to controls, but morphine analgesia was unaffected. Administering the PANX1 inhibitors probenecid and mefloquine to mice before the opioid antagonist naloxone significantly reduced withdrawal behavior (19).

[0279] Ethanol-induced Cx43 hemichannel and Panx1 channel activity correlated with increased levels of the inflammatory messengers IL-1β, TNF-α, and IL-6 in the hippocampus, as well as significant changes in astrocytic dendritic complexity. Thus, unregulated opening of astrocytic hemichannels and pannexons may contribute to the development of alcohol use disorders in adulthood (20).

[0280] epilepsy PANX1 expression is elevated in several animal seizure models and resected human epileptic brain tissue, suggesting its relevance to epilepsy. In animal models of epilepsy, PANX1 knockout and application of PANX1 channel modulators suppressed seizure activity. After electrical stimulation of the hippocampal CA3 region, PANX1 knockouts exhibited significantly shorter evoked afterdischarges and resistance to kindling. Activation or inhibition of Panx channels has been shown to regulate the release of adenosine triphosphate (ATP) and other signals, which is crucial for the development and control of neurological disorders, including epilepsy. Postoperative human tissue samples from epilepsy patients showed that activation of pannexin 1 channels promotes seizure initiation and maintenance through adenosine triphosphate signaling via purinergic receptors. Pharmacological inhibition of PANX1 channels with probenecid or mefloquine, respectively, blocked seizure discharges in human cortical brain tissue slices. Genetic deletion of PANX1 channels in mice showed anticonvulsant effects when the mice were exposed to kainic acid, a model of temporal lobe epilepsy, suggesting an epileptogenic role for PANX1 channels in chronic epilepsy in human patients and suggesting that pannexin-1 channel inhibition may be an alternative therapeutic strategy for treating lesional and drug-resistant epilepsy (20-26).

[0281] Parkinson's disease Nod-like receptor pyrin domain-containing 3 plays a key role in the pathogenesis of Parkinson's disease, and therefore, PANX1 channels may play an important role in the inflammatory cascade underlying this neurodegenerative disease.

[0282] α-Synuclein has been found to promote the opening of connexin 43 (Cx43) hemichannels and PANX1 channels in mouse cortical astrocytes, and it has been proposed that α-synuclein-mediated opening of Cx43 hemichannels and Panx1 channels in astroglial cells may constitute a novel mechanism involved in the onset and progression of α-synucleinopathies (27)(28).

[0283] Multiple sclerosis Probenecid (a PANX1 inhibitor) reduced clinical symptoms (disease score) in a mouse experimental autoimmune encephalomyelitis (MS) model by reducing inflammation, the number of T lymphocytes infiltrating the spinal cord, and the loss of oligodendroglial cells (14)(29)(30).

[0284] Alzheimer's disease The age-dependent increase in PANX1 expression, along with the deterioration of PANX1 activity both basally and in response to glutamate receptor activation, correlates with increased Aβ levels in the hippocampal tissue of Tg mice. Acute inhibition of Panx1 activity with the drug probenecid (a Panx1 inhibitor) significantly attenuated excitatory synaptic dysfunction in AD models by normalizing long-term potentiation (LTP) and inhibition and improving dendritic branching and spine density in hippocampal neurons of Tg mice. This strongly suggests that Panx1 contributes significantly to the early mechanisms leading to synaptopathy in AD (31).

[0285] traumatic brain injury A murine controlled cortical impact (CCI) model using myeloid-specific PANX1 conditional knockout (Cx3cr1-Cre::Panx1fl / fl) mice demonstrated that bone marrow PANX1 mediates neuroinflammation and brain injury. CCI-related outcomes correlate well with PANX1 channel function in myeloid cells, indicating that activation of PANX1 channels in myeloid cells is a major cause of acute brain inflammation after TBI (32).

[0286] Migraine Cortical spreading depression (CSD) is a putative cause of migraine aura and headache. CSD triggers the opening of neuronal Pannexin 1 (Panx1) megachannels and activation of caspase-1, which subsequently releases high-mobility group box 1 (HMGB1) from neurons and activates nuclear factor-κB in astrocytes. Inhibition of this cascade abolished CSD-induced trigeminovascular activation, dural mast cell degranulation, and headache. Therefore, inhibition of Panx1 appears to be a logical step for ameliorating this disease (33)(34).

[0287] stroke Wild-type and Panx1KO mice were subjected to permanent middle cerebral artery (MCA) occlusion, and infarct size and astrocytic and microglial activation were assessed. Sexual dimorphism of Panx1 deficiency was also investigated and tested by analyzing the effect of probenecid on stroke volume. Panx1KO females had significantly smaller infarct volumes (approximately 50% reduction) compared with wild-type mice, whereas no such KO effect was observed in males (35).

[0288] cancer Adenosine triphosphate (ATP) is one of the major biochemical components of the tumor microenvironment (TME) and can promote tumor progression or tumor suppression depending on its concentration and specific ectonucleotidases and receptors expressed by immune cells and cancer cells. (36) Most literature refers to the microtumor environment and highlights the overexpression of Panx1 in some cancers.

[0289] melanoma Panx1 forms large pore channels that allow the passage of ions and metabolites, such as ATP, for cell-to-cell communication. Panx1 has been implicated in many diseases, including breast cancer and melanoma, and inhibiting or deleting PANX1 reduces the tumorigenic and metastatic properties of cancer cells. Potential mechanism: Direct interaction between the C-terminal region of PANX1 and the N-terminal portion of β-catenin, a key transcription factor in the Wnt pathway (11).

[0290] Hepatocellular carcinoma (HCC) The expression of Panx1 was analyzed by immunohistochemistry (IHC) in 126 cases of HCC. The effect of Panx1 on HCC cell metastasis and invasion was observed by examining the expression levels of Panx1 and epithelial-mesenchymal transition (EMT)-related proteins in HCC cells and tissues. The tumor metastatic ability of PANX1 knockout mice was compared with that of nude mice.

[0291] High expression of PANX1 in HCC was positively correlated with tumor lymph node metastasis. In conclusion, overexpression of Panx1 appears to promote HCC cell invasion and migration through regulating EMT in vitro and in vivo ( 37 ).

[0292] breast cancer Overexpression of PANX1 in breast cancer is associated with a transition to an EMT phenotype in silico and in vitro, resulting in tumor-promoting effects and worsening clinical outcomes in breast cancer patients. This association provides a novel target for breast cancer therapy (10).

[0293] colorectal cancer The nuclear factor kappa B (NF-κB) signaling pathway is activated in many colorectal cancer (CRC) cells and the tumor microenvironment. Mefloquine is an NF-κB inhibitor and has been shown to induce growth arrest and apoptosis in CRC cells with phosphorylated p65 in culture and in mice. Therefore, mefloquine may exert its antitumor effects by inhibiting the NF-κB signaling pathway (12).

[0294] Pancreatic cancer Analysis of PANX1 expression in all human cancers in the Oncomine and GEPIA2.0 databases (Kaplan-Meier plotter and OncoLnc tools) showed that PANX1 was overexpressed in most cancers compared with normal tissues. High expression of PANX1 was associated with poor prognosis in multiple tumors, especially pancreatic adenocarcinoma (PAAD) (38).

[0295] leukemia Antitumor immune responses are thought to be associated with the controlled release of ATP from apoptotic cancer cells, which activates the P2 purinergic receptor signaling cascade in nearby leukocytes. Comparison of PANX1 levels showed significantly higher expression in leukemic T lymphocytes than in normal, non-mutated T lymphoblasts, suggesting that the signaling role of PANX1 may be amplified in leukemic leukocytes (39).

[0296] Chemotherapy and Pain Neuropathic pain is a debilitating consequence of cancer treatment with cytotoxic chemotherapy agents such as paclitaxel. The economic cost of chronic pain, of which neuropathic pain is a significant component, is estimated to exceed $500 billion in the United States alone (40).

[0297] cardiovascular disease arrhythmia Crosstalk between GJC and HC / PANX1 channels may be crucial for the development of arrhythmogenic substrates, including fibrosis. Current evidence indicates that activation of HC and PANX1 channels may increase the risk of arrhythmias. This field may contribute to the development of novel therapeutic strategies for patients prone to atrial and ventricular fibrillation (41)(42).

[0298] Inflammation of blood vessels Ischemia-reperfusion (I / R) injury (IRI) poses a serious threat to graft and recipient survival and leads to increased morbidity and mortality in patients undergoing lung transplantation.

[0299] Pharmacological antagonism of PANX1 attenuated pulmonary IRI in wild-type (WT) mice. Endothelial-specific Panx1 inducible knockout mice exhibited a protective phenotype with reduced endothelial permeability, edema, and inflammation after I / R. The mechanism of Panx1-mediated protection involves endothelial cell release of ATP, thereby identifying a potentially effective therapeutic target for preventing pulmonary I / R injury.

[0300] PANX1 channels on endothelial cells mediate vascular inflammation during pulmonary ischemia-reperfusion injury ( 43 ).

[0301] PAH Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to well-ventilated lung regions, optimizing ventilation-perfusion matching. PANX1-mediated ATP release and subsequent signaling through purinergic P2Y receptors have been identified as regulators of systemic arteriolar vasoconstriction. Pharmacological inhibition and genetic deletion of the hemichannel PANX1 in pulmonary arterial smooth muscle cells attenuates the physiological HPV response (44).

[0302] blood pressure Spironolactone prevents α1AR (α1-adrenergic receptor)-mediated vasoconstriction in resistance vessels, acutely reducing blood pressure in mice. These effects require expression of the PANX1 channel in vascular smooth muscle cells but are independent of spironolactone's traditional target, the mineralocorticoid receptor (MR). It has been proposed that PANX1 is a novel target of spironolactone and, together with MR-dependent actions, may contribute to the beneficial blood pressure lowering effects of spironolactone, particularly relevant for the treatment of patients with resistant hypertension (45)(46).

[0303] inflammatory disease arthritis All joint tissues express one or more connexins and pannexins, and their expression is altered in some pathologies, such as osteoarthritis (OA) and rheumatoid arthritis (RA), suggesting that they may be involved in the onset and progression of these conditions. The aging of the global population, along with increases in obesity and metabolic dysfunction, is associated with an increased incidence of joint diseases and the associated costs and burden. Modulation of connexins and pannexins represents an attractive therapeutic target in joint diseases (47).

[0304] wound healing inflammatory disease Following dorsal skin punch biopsies from Panx1 knockout (KO) mice, these mutant mice exhibited significantly delayed wound healing. Scratch wound and proliferation assays revealed that keratinocytes cultured from KO mice were more migratory, while dermal fibroblasts were more proliferative compared to controls. Furthermore, collagen gels filled with fibroblasts from KO mice exhibited significantly reduced contraction, comparable to that of WT fibroblasts treated with the Panx1 modulator probenecid. KO fibroblasts did not increase α-smooth muscle actin expression in response to TGF-β, as did WT fibroblasts that differentiate during wound contraction. PANX1 is thought to control the cellular properties of keratinocytes and dermal fibroblasts during early skin development and regulate wound repair upon injury (48).

[0305] Pulmonary disease COVID19 PANX1 channels regulate inflammation and host responses to several pathogens, including viruses. Several lines of evidence demonstrate that the opening of PANX1 channels (and the release of ATP) enhances inflammatory responses, including in systemic endothelium (pulmonary microvasculature), pulmonary epithelium, olfactory epithelium, and the parenchyma of several tissues throughout the body. Targeting the early-stage hyperinflammation and cytokine storm, which occur especially in severe cases of COVID-19, could be an important application of PANX1 inhibitors (49).

[0306] Primary and secondary ciliary dyskinesia (PCD and SCD) Pannexin 1 contributes to the release of ATP, a key paracrine regulator of mucociliary function in the airway epithelium. Consideration of pannexin regulation may have important implications for ATP availability in airway surface fluid in airway homeostasis and disease (50).

[0307] asthma Stressed or damaged cells release ATP into the extracellular environment through the PANX1 channel, which is the underlying cause of inflammation in various pathologies, including allergic pneumonia. Blockade of PANX1 significantly reduced goblet cell hyperplasia and inflammatory cell infiltration in the lungs of OVA-sensitized mice. Inhibition of PANX1 also reduced total and eosinophil cell counts in bronchoalveolar lavage fluid (BALF) and reduced the expression of CCL11 and CCL2 in mouse lung tissue (51).

[0308] Other disorders fibrosis Liver fibrosis is the final common pathway in nearly all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components due to the transdifferentiation of primarily quiescent hepatic stellate cells into myofibroblast-like cells, which is triggered by cell death and inflammation. Gene expression profiling revealed downregulated fibrosis and immune responses in pannexin-1 knockout mice treated with carbon tetrachloride, whereas bile duct-ligated pannexin-1-deficient animals displayed a pronounced inflammatory profile (52).

[0309] Diabetes and related diseases Insulin has been identified as a novel activator of the PANX1 channel. In obese humans, PANX1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. PANX1 also appears to be involved in β-cell regulation. PANX1 is further involved in glucose resistance; impaired glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, both hallmarks of type 2 diabetes.

[0310] Adipocytes expressed functional Panx1 channels that could be activated to release ATP. Pharmacological inhibition or selective genetic deletion of PANX1 from adipocytes reduced insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice.

[0311] Fructose exposure reduced intracellular ATP levels and promoted ATP release from β cells upon acute glucose stimulation. This resulted in increased extracellular ATP via the PANX1 channel, which activated calcium mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in β cells.

[0312] Type 1 diabetes (T1D) causes various skeletal problems, including decreased bone mineral density and increased fracture risk. High blood glucose levels in T1D alter the expression and function of purinergic receptors (P2Rs) and PANX1 channels, inhibiting ATP signaling, which is essential for proper bone response to mechanical loading and maintaining skeletal integrity (53-54).

[0313] eye disease Potential changes in corneal nerve terminals in moderately severe non-insulin-dependent diabetes mellitus (NIDDM) were investigated in mice. Dissected corneas were subjected to an ultracentrifugation protocol to obtain synaptosomes from sensory nerve terminals. Two major mechanisms were investigated in these nerve terminals: changes in the mechanosensitive channel pannexin 1 and ATP release upon stimulation of these terminals. Thus, changes in the intracellular location and function of the PANX1 channel may contribute to altered corneal mechanosensitivity, which in turn may affect corneal wound healing and primary visual function (55-56).

[0314] skin diseases psoriasis Psoriasis is a chronic inflammatory disease of the skin accompanied by systemic and joint symptoms and associated complications, such as increased risk of metabolic syndrome and cardiovascular disease. Because psoriasis is likely triggered by skin-damaging events or trauma, it is highly likely that intracellular ATP released from damaged cells plays a role in activating the inflammasome, thereby triggering the inflammatory response that underlies the pathogenesis of this disease. Purinergic signaling in the skin may therefore represent a novel early stage of psoriasis, opening the door to developing new therapeutic approaches for psoriasis by targeting single molecular factors in the purinome. Therefore, preventing excessive ATP release may prevent or ameliorate this disease (57)(58).

[0315] Pharmaceutical Compositions The pharmaceutical compositions of the present invention can be prepared and formulated according to conventional methods, for example, as disclosed in the British Pharmacopoeia, the European Pharmacopoeia, the United States Pharmacopoeia (59) (60) (61), Remington's Pharmaceutical Sciences (62), Martindale: The Extra Pharmacopoeia (63), and Harry's Cosmeticology (64).

[0316] Pharmaceutical formulations may contain, for example, water, buffers (e.g., sodium bicarbonate, buffered neutral saline such as phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethyl sulfoxide, carbohydrates (e.g., lactose, sorbitol, trehalose, glucose, mannose, sucrose, amides, glycerol, mannitol, or dextran), proteins, adjuvants (e.g., stabilizers such as polymers and cyclodextrins), polypeptides or amino acids (e.g., His, Gly, Lys, Asp, Glu, and Arg), antioxidants (e.g., ascorbic acid, α-tocopherol, sulfites, B It may contain one or more of HA (butylhydroxyanisole), BHT (butylhydroxytoluene), surfactants (e.g., non-ionic detergents - Triton® X-100, Polysorbate 20, Polysorbate 80, Pluronic® F68, Pluronic® F88, Pluronic® F127, Bridge 35), chelating agents (e.g., EDTA and / or glutathione), and / or preservatives (e.g., parabens, sorbic acid, imidazole urea, quaternary ammonium compounds, hydantoins, phenolic derivatives, acidic derivatives, halogenated compounds).

[0317] Pharmaceutical forms can be formulated for any route of administration, including, for example, topical, oral, nasal, rectal, or parenteral administration. As used herein, the term parenteral includes subcutaneous injection, intradermal injection, intravascular injection (e.g., intravenous), intramuscular injection, spinal injection, intracranial injection, intrathecal injection, and intraperitoneal injection, as well as similar injection or infusion techniques. In certain modes, compositions for oral use are preferred. Such compositions include, for example, pills, tablets, solutions, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Among other modes, pharmaceutical compositions can be formulated as lyophilized powders.

[0318] Pharmaceutical forms intended for oral use may further contain other ingredients such as sweetening, flavoring, coloring, and / or preservatives in order to provide an attractive and palatable preparation.

[0319] Tablets contain the active ingredient mixed with physiologically compatible excipients that are suitable for the manufacture of tablets. These excipients include, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate), granulating and disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch, gelatin, or acacia), and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). Tablets can be formed using standard techniques, such as dry granulation, direct compression, or wet granulation. Tablets can be uncoated or coated using known techniques.

[0320] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, kaolin, talcum, lactose monohydrate, colloidal silicon dioxide, microcrystalline cellulose, sodium lauryl sulfate, sodium glycolate) or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid petrolatum, or olive oil).

[0321] Aqueous suspensions contain the active substance(s) in admixture with one or more suitable excipients, such as suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), and dispersing or wetting agents (e.g., naturally occurring phospholipids such as lecithin, polyoxyethylene stearate, etc.). stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol, such as heptadeca-ethyleneoxy-cetanol, a condensation product of ethylene oxide with a partial ester derived from a fatty acid and one hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as polyethylene sorbitan monooleate. The aqueous suspension also contains one or more preservatives, for example, ethyl p-hydroxybenzoate or n-propyl, one or more coloring agents, one or more flavoring agents, and / or one or more sweetening agents, such as sucrose or saccharin.

[0322] Oily suspensions can be formulated by suspending the active ingredient(s) in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil such as liquid paraffin. Oily suspensions may also contain thickening agents such as beeswax, hard paraffin, or cetyl alcohol. Sweetening agents, such as those set forth above, and / or flavoring agents may be added to provide a palatable oral preparation. These suspensions may be preserved by the addition of an antioxidant, such as ascorbic acid.

[0323] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in a mixture with a dispersing or wetting agent, a dispersing agent, and one or more preservatives. Suitable dispersing or wetting agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavoring agents, and coloring agents, may also be present.

[0324] Pharmaceutical formulations may be formulated as water-in-oil emulsions. The oil phase may be vegetable oil (e.g., coconut oil, almond oil, grapeseed oil, olive oil, or peanut oil), mineral oil (e.g., liquid petrolatum), or a mixture thereof. Suitable emulsifiers include natural gums (e.g., gum acacia or gum tragacanth), natural phospholipids (e.g., phosphatidylserine), anhydrides (e.g., sorbitan monooleate), and condensation products of partial esters derived from fatty acids and hexitols with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). Emulsions may also contain one or more sweeteners and / or flavoring agents.

[0325] Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose, and such formulations may also contain one or more preservatives, flavorings and / or coloring agents.

[0326] Preparations for topical administration usually contain a topical vehicle with or without additional optional ingredients, combined with the active agent(s).Suitable additional ingredients and topical vehicles are well known in the art, and the choice of vehicle will obviously depend on the physical form and method of administration.Topical vehicles include water, organic solvents such as alcohols (e.g., ethanol or isopropyl alcohol) or glycerin, glycols (e.g., butylene, isoprene, or propylene glycol), fatty alcohols (e.g., lanolin), mixtures of water and organic solvents, and mixtures of organic solvents such as glycerin alcohol, lipid-based materials such as fatty acids, acylglycerols (including oils such as mineral oil and animal or synthetic fats), phosphoglycerides, sphingolipids, and waxes, protein-based materials such as collagen and gelatin, silicone-based materials (volatile and non-volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

[0327] The composition may further comprise one or more ingredients suitable for improving the stability or effectiveness of the applied formulation, such as stabilizers, suspending agents, emulsifiers, viscosity modifiers, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers, and sustained-release materials. Examples of such ingredients are described in the prior art (65-70). The formulation may include microcapsules, such as hydroxymethylcellulose or gelatin microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules.

[0328] Topical formulations can be prepared in any of a variety of physical forms, such as, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, and emulsions. The physical appearance and viscosity of such pharmaceutically acceptable forms depend on the presence and amount of emulsifier(s) and viscosity modifier(s) present in the formulation.

[0329] Solids are generally hard and non-pourable and are commonly formulated in bar, club, or granular form. Solids can be opaque or transparent and may optionally contain solvents, emulsifiers, humectants, emollients, fragrances, colorants / dyes, preservatives, and other active ingredients that enhance or enhance the effectiveness of the final product.

[0330] Creams and lotions are often similar, differing primarily in viscosity. Lotions and creams can be opaque, translucent, or clear and often contain emulsifiers, solvents, viscosity modifiers, moisturizers, emollients, fragrances, colorants / dyes, preservatives, and other active ingredients that enhance the effectiveness of the final product.

[0331] Gels can be prepared in a range of viscosities, from thick, highly viscous gels to thin, less viscous gels. Similar to lotion and cream formulations, these formulations may also contain solvents, emulsifiers, humectants, emollients, fragrances, colorants / dyes, preservatives, and other active ingredients that enhance or increase the effectiveness of the final product.

[0332] Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, humectants, emollients, fragrances, colorants / dyes, preservatives, and other active ingredients that enhance or enhance the effectiveness of the final product.

[0333] Emulsifiers suitable for use in topical formulations include, but are not limited to, ionic emulsifiers, nonionic emulsifiers such as cetearyl alcohol, polyoxyethylene oleyl ether, cetearyl alcohols such as PEG-40 stearate, ceteareth-12, ceteareth-20, and ceteareth-30, PEG-100 stearate, and glyceryl stearate. Suitable viscosity-adjusting agents include, but are not limited to, protective colloids of hydroxyethyl cellulose, xanthan gum, aluminum magnesium silicate, silica, microcrystalline wax, beeswax, paraffin, and nonionic gums such as cetyl palmitate. Gel compositions can be formed by adding a gelling agent such as chitosan, methylcellulose, ethylcellulose, polyvinyl alcohol, polyquaternium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, carbomer, or glycyrrhizic acid together with ammonia. Suitable surfactants include, but are not limited to, nonionic surfactants, amphoteric surfactants, ionic surfactants, and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG diammonium chloride, and ammonium laureth sulfate can be used in topical formulations. Suitable preservatives include, but are not limited to, antibacterial agents such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, ascorbic acid, and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin derivatives, petrolatum, isostearyl neopentanoate, and mineral oil. Suitable flavors and colors include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5.Other suitable additional ingredients that can be used topically include, but are not limited to, abrasives, absorbents, anti-foaming agents, anti-static agents, astringents (e.g., witch hazel, alcohol, and herbal extracts such as chamomile extract), binders / excipients, buffers, chelating agents, film formers, conditioning agents, propellants, opacifiers, pH adjusters, and protectants.

[0334] Topical formulations also include skin penetration enhancers, which facilitate the release of compounds through the stratum corneum to the skin surface in transdermal systems. The main penetration enhancers used to promote drug release include alcohols such as ethanol, propylene glycol, ethoxydiglycol, 1-decanol, and 2-(2-ethoxyethoxy)ethanol; glycols and glycerides; fatty acids and esters such as palmitic, capric, oleic, myristic, or lauric acid; (71-73) sulfoxides such as dimethyl sulfoxide and dimethylformamide; (74) phospholipids such as phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine; cyclodextrins (α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin); dodecyl-N,N-dimethylaminoacetate (DDAA); and the polymers previously cited herein.

[0335] Other routes of penetration enhancers include physical methods such as iontophoresis (75), electroporation (76), and phonophoresis (77).

[0336] Common methods of administration of external topical compositions include direct application of the product by gloved hands or indirect application using a physical applicator such as a spatula, dosing syringe, dosing ruler, glue or stick, spraying (including mist spray, aerosol, or foam), using 1 ml single-dose sachets, application using a drop counter, dispersing and rinsing, etc. Another form of indication for topical use is inhalation, or application to various tissues other than the skin (such as eye drops applied to conjunctival tissue or otic solutions applied to the ear).

[0337] Exemplary forms of these inhalants include gaseous forms in aerosols (using conventional propellants, such as dichlorofluoromethane or trichlorofluoromethane), or spray-dried forms of particles, and liquid emulsions, solutions, or suspensions that are inhaled by nebulization. Further examples of drug forms for the ocular or conjunctival route include cold cream, reconstituted isotonic or sterile suspension eye drops administered with an eye dropper, and drug forms for the otic route include cold cream or liquid isotonic drug forms similarly administered with an eye dropper.

[0338] Pharmaceutical forms can be prepared as sterile injectable aqueous or oily suspensions. The compound(s) provided herein can be suspended or dissolved in such compositions, depending on the vehicle and concentration used, and can be formulated according to known techniques using appropriate dispersing, wetting, and / or suspending agents, as described above. Acceptable vehicles and solvents that can be used include water, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution, particulates that may contain adjuvants such as sodium citrate and inclusion complexes with cyclodextrins, or release systems such as nanoemulsions, nanosuspensions, microemulsions, polymeric micelles, liposomes, niosomes, transfersomes, and ethosomes (78-80).

[0339] Furthermore, sterile fixed oils can also be used as a solvent or suspending medium. For this purpose, any soft fixed oil can be used, including synthetic monoglycerides and diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injectable compositions and adjuvants, such as local anesthetics, preservatives and / or buffers, and can be dissolved in the vehicle.

[0340] The drug form can also be formulated as a suppository (e.g., for rectal administration). These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug.

[0341] Drug forms may be formulated to release at a predetermined rate. Immediate release can be obtained, for example, by sublingual administration (i.e., administration by mouth in such a way that the active ingredient(s) are rapidly absorbed through the blood vessels of the hypoglossal plexus).

[0342] Controlled-release formulations (i.e., formulations such as capsules, tablets, or coated tablets that reduce and / or delay the release of an active ingredient(s) after administration) can be administered, for example, orally, rectally, or subcutaneously, or by implantation at a target site. Generally, controlled-release formulations can be obtained by combining the active ingredient(s) with a matrix material that alters the release rate itself and / or by using a controlled-release coating that delays disintegration and absorption in the intestinal tract (or at the site of implantation), thereby providing a delayed or sustained action over a longer period of time. One type of controlled-release formulation is a sustained-release formulation. In a sustained-release formulation, at least one active ingredient is continuously released at a constant rate over a period of time. Preferably, the therapeutic agent is released at a rate that maintains blood (e.g., plasma) concentrations within a therapeutic range but below toxic levels over a period of at least 4 hours, preferably at least 8 hours, and more preferably at least 12 hours. Such formulations can generally be prepared using well-known techniques. The vehicle used within such formulations is biocompatible and may also be biodegradable. Preferably, the formulation provides a constant level of release of the modulator. The amount of modulator contained in a sustained release formulation will vary depending, for example, on the location of the implant, the expected rate and duration of release, and the nature of the condition to be treated or prevented.

[0343] The release rate can be varied using methods known in the art, including (a) varying the thickness or composition of the coating, (b) changing the amount or manner of adding plasticizer to the coating, (c) including additional components such as release-modifying drugs, (d) changing the composition, particle size, or form of the particles in the matrix, and (e) providing one or more passageways through the coating. The amount of modulator included in a sustained release formulation will vary depending, for example, on the method of administration (e.g., location of implant), the expected rate and duration of release, and the nature of the condition to be treated or prevented.

[0344] The matrix material may or may not itself perform a controlled-release function, but is generally any material that supports the active ingredient(s). For example, materials such as glyceryl monostearate or glyceryl distearate may be used. The active ingredient(s) can be combined with the matrix material before forming a dosage form (e.g., a tablet). Alternatively, or in addition, the active ingredient(s) may be coated onto the surface of the particles, granules, spheres, microspheres, globules, or pellets that make up the matrix material. Such coatings can be obtained by conventional means, such as dissolving the active ingredient(s) in another suitable solvent and spraying. If necessary, additional ingredients are added before coating (e.g., to aid in binding the active ingredient(s) to the matrix material). The matrix can then be coated with a barrier agent before applying the controlled-release coating. If desired, multiple coated matrix units can be encapsulated to produce the final dosage form.

[0345] Controlled-release coatings may be continuous, uniform, non-toxic, inert, and non-adherent films that can support pigments and other additives. Coatings that control the release of the modulator include pH-independent or pH-dependent coatings that can be used to release the modulator in the stomach, and enteric coatings (which allow the formulation to pass through the stomach intact and dissolve in the small intestine, allowing the contents to be absorbed into the body). pH-dependent coatings include, for example, shellac, cellulose acetate phthalate, polyvinyl acetate phthalate, cellulose methylhydroxypropyl phthalate, and copolymers of methacrylate and zein.

[0346] In certain embodiments, the coating is preferably a hydrophobic material used in an amount effective to reduce hydration of the gelling agent after administration. Suitable hydrophobic materials include alkylcelluloses (e.g., ethylcellulose or carboxymethylcellulose ether), cellulose ethers, cellulose esters, acrylic polymers (e.g., (poly)acrylic acid, (poly)methacrylic acid, copolymers of acrylic acid and methacrylic acid, copolymers of methyl methacrylate, ethoxyethyl methacrylate, copolymers of alkamide / methacrylic acid, (poly)methyl methacrylate, polyacrylamide, ammonium methacrylate copolymers, aminoalkyl methacrylate copolymers, (poly)methacrylic anhydride and glycidyl methacrylate copolymers), and mixtures thereof.

[0347] Representative aqueous dispersions of ethylcellulose include, for example, AQUACOAT® (FMC Corp., Philadelphia, Pa.) and SURELEASE® (Colorcon, Inc., West Point, Pa.), both of which can be applied to the substrate according to the manufacturer's instructions. Representative acrylic polymers include, for example, EUDRAGIT® (Rohm America, Piscataway, NJ), and various other polymers, which can be used alone or in combination depending on the desired release profile.

[0348] The physical properties of coatings containing aqueous dispersions of hydrophobic materials can be improved by adding one or more plasticizers. Suitable plasticizers for alkyl celluloses include, for example, dibutyl sebacate, diethyl phthalate, triethyl citrate, tributyl citrate, and triacetin. Suitable plasticizers for acrylic polymers include, for example, citric acid esters such as triethyl citrate and tributyl citrate, dibutyl phthalate, polyethylene glycol, propylene glycol, diethyl phthalate, castor bean, and triacetin.

[0349] Controlled-release coatings are typically applied using conventional techniques, such as spraying in the form of an aqueous dispersion. Optionally, the coating may be provided with pores or channels to facilitate the release of the active ingredient. Pores and channels can be created using well-known methods, including the addition of organic or inorganic materials that dissolve, extract, or are released from the coating in the environment of use. Some such pore-forming materials include hydrophilic polymers such as hydroxyalkyl cellulose (e.g., hydroxypropylmethylcellulose), cellulose ethers, water-soluble synthetic polymers (e.g., polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyethylene oxide), water-soluble polydextrose, sugars and polysaccharides, and alkali metal salts.

[0350] The amount of active ingredient that can be combined with the vehicle materials to produce a unit dose varies depending, for example, on the patient being treated, the particular method of administration, and other coadministered medications. Typically, a unit dosage form contains about 5 pg to about 2 g of active ingredient. Optimal dosages can be established using testing and routine procedures well known in the art.

[0351] In one aspect of the present invention, the compositions may comprise, in addition to one or more panx-1 modulators of the present invention, one or more additional active ingredients, including, but not limited to, analgesics, anti-inflammatory agents, antiarrhythmic agents, anticoagulants, thrombolytic agents, diuretics, antidepressants, antidiabetics, antiepileptic agents, antihistamines, antihypertensive agents, antimuscarinics, antituberculous agents, antitumor agents, immunomodulators, antivirals, anxiolytics-sedatives (hypnotics and neuroleptics), beta-adrenergic receptor blockers, cardiac inotropes, cardiac agents, corticosteroids, diuretics, dopamine agonists (antiparkinsonian agents), immunological agents, muscle relaxants, parasympathomimetics, prostaglandins, bronchodilators, antiallergic agents, sympathomimetics, antiemetics, chemotherapeutic agents, and xanthines.

[0352] Accordingly, the present invention will now be illustrated by examples which further illustrate the invention, but such examples are not intended to limit the scope of the invention. [Example]

[0353] Example 1 - In silico docking An initial virtual screening study (docking analysis performed with AUTODOCK against the identified "CBX binding site" in the Panx-1 channel (PDB code 7DWG)) using known inhibitors (difloxacin, trovafloxacin, brilliant blue G-FCF, levofloxacin, compound 14c (9), compound 5b (9), imiquimod var1, compound 15b (9), baicalein, compound 6I (9), compound 6e (9), with mefloquine, carbenoxolone, quinine, without mefloquine, probenecid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, cholesterol) and some of our recommended Panx1 blockers.

[0354] The latter approach was performed using the 3Dsim module of the software Stardrop 7.1 (https: / / optibrium.com / stardrop / ). In this assay, candidate compounds were compared to the structure of carbenoxolone, a known PANX1 inhibitor with a well-described binding site. The software calculates a similarity index of the candidate compound relative to the comparator (e.g., carbenoxolone) based on the steric and Coulombic configurations, the number of hydrogen bond donors, and the number of hydrogen bond acceptors. Other known PANX1 inhibitors were also introduced in this study as standard comparators. Specifically, these were quinine, mefloquine plus minus, imiquimod var1, baicalein, levofloxacin, trovafloxacin, difloxacin, probenecid, and compounds 14c and 6e from Crocetti et al. (2021).

[0355] This approach largely followed the method published in reference (1). For comparison, the 3D structure of human Panx1 containing the inhibitor carbenoxolone was used. The structures were downloaded from the PDB database as cryo-EM structures 6WBI and 6WBL, which represent the human Panxin1 channel in complex with CBX, with and without deletion of the N-terminal helix and C-terminal tail (Ruan et al., 2020).

[0356] The following molecules were designed and their binding energies calculated as outlined in Autodock (PDB database: 7DWB). As a reference, the calculated binding energy of a specific conformation of carbenoxolone was used.

[0357] The binding energies of known PANX1 ligands are shown in Table 1, and the results for the compounds of the present invention are listed in Table 2. A skilled artisan will appreciate that the in silico docking scores can predict the in vitro and in vivo affinity of the compounds of the present invention for PANX1. [Table 1] [Table 2]

[0358] Example 2 - Preparation of Compound 004 General procedure for the preparation of intermediate 3 [ka] Intermediate 1 (2.00 g, 12.9 mmol, 1.00 equiv), MeOH (30.0 mL), and Intermediate 2 (2.91 g, 15.6 mmol, 1.21 equiv) were charged into a 100 mL flask 1 at 20 °C.

[0359] Flask 1 was charged with TEA (1.58 g, 15.6 mmol, 2.17 mL, 1.21 equiv) at 20°C.

[0360] The mixture was stirred at 70°C for 1 hour.

[0361] HPLC (EC3602-25-P1A3) showed that intermediate 1 was completely consumed and a new major peak (Rt=1.920 min) was formed.

[0362] The reaction mixture was cooled to 20°C.

[0363] The mixture was poured into H2O (150 mL), stirred at 20 °C for 0.50 h, and the filter cake was filtered and dried under vacuum to give Intermediate 3 (3.45 g, 11.3 mmol, 87.6% yield, 100% purity) as a grey solid, as determined by LCMS and 1 Confirmed by 1 H NMR.

[0364] HPLC: Rt = 1.920 min, purity 23.2% at 220 nm

[0365] LCMS: Rt=0.406 min, m / z=305.2(M+H) +

[0366] 1H-NMR (400MHz, DMSO-d6): δ13.07(s, 1H), 8.23(s, 1H), 8.15(s, 1H), 4.20(s, 4H), 3.45(t, J=4.8Hz, 4H), 1.43(s, 9H).

[0367] General procedure for the preparation of intermediate 4 [ka] A 250 mL three-neck flask was equipped with an overhead stirrer, an addition funnel, and a thermometer.

[0368] The flask was charged with DMF (100 mL).

[0369] Intermediate 3 (3.40 g, 11.1 mmol, 1.00 equivalents) was charged into a flask at 20-25°C.

[0370] K2CO3 (1.73 g, 12.5 mmol, 1.12 equiv.) was charged into the flask at 5–10 °C.

[0371] MeI (2.38 g, 16.7 mmol, 1.04 mL, 1.50 equiv) was added dropwise to the flask at 5-10 °C.

[0372] The mixture was stirred at 20 to 25°C for 10 hours.

[0373] A sample was taken for LCMS (EC378-385-P1E1). Intermediate 3 was completely consumed and one main peak with the target mass (Rt=0.421 min) was detected.

[0374] The mixture was poured into H2O (350 mL) and stirred at 20-25°C.

[0375] To the mixture was added AcOK (10.0 g).

[0376] The mixture was extracted with ethyl acetate (100 mL*3).

[0377] The organic phase was washed with brine (100 mL*1) and separated.

[0378] The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0379] The residue was triturated with petroleum ether / ethyl acetate = 3 / 1 (10.0 mL) at 15 °C for 1 hour to give intermediate 4 (2.70 g, 8.48 mmol, 75.9% yield) as a yellow solid. 1 Confirmed by 1 H NMR and HSQC.

[0380] LCMS: Rt=0.421 min, m / z=319.1(M+H) + .

[0381] 1 H NMR (400MHz, CDCl3): δ8.40(s, 1H), 7.74(s, 1H), 4.31(s, 4H), 3.84(s, 3H), 3.59(t, J=5.2Hz, 4H), 1.50(s, 9H). [ka]

[0382] Mix intermediate 4 (1.20 g, 3.77 mmol, 1.00 equiv) with trifluoroacetic acid (TFA) (5.00 mL) in dichloromethane (DCM) (20.0 mL) and place in R1 (100 mL three-neck flask) at 20 °C.

[0383] The mixture was stirred at 20° C. for 2 hours.

[0384] A sample was taken for HPLC (EC4923-3-P1A2). Intermediate 4 was completely consumed and one main peak with the target mass (Rt=0.922 min) was detected.

[0385] The reaction mixture was poured into NaOH solution (1 mmol / mL), and NaOH solution (1 mmol / mL) was added until pH=13.

[0386] The mixture was extracted with DCM / MeOH=5 / 1 (60.0 mL*5), and the combined organic layer was dried over Na2SO4, filtered and concentrated at 45 °C.

[0387] The residue was triturated with solvent (ethyl acetate, 5 mL) at 20° C. for 2 hours and filtered to give compound 004 (572 mg, 2.49 mmol, 66.1% yield, 98.9% purity) as a yellow solid. 1 Confirmed by 1 H NMR, HPLC and LCMS.

[0388] HPLC: Rt = 0.922 min, purity 97.3% at 220 nm.

[0389] LCMS: Rt=0.227 min, m / z=219.0(M+H) + .

[0390] HPLC: Rt = 0.932 min, purity 98.9% at 220 nm.

[0391] 1 H NMR (400MHz, MeOD): δ8.25 (s, 1H), 7.99 (s, 1H), 4.26 (s, 4H), 3.80 (s, 3H), 2.94 (t, J=5.2Hz, 4H).

[0392] Example 3 - Preparation of Compound 010 General procedure for the preparation of intermediate 6 [ka] Intermediate 5 (10.0 g, 87.6 mmol, 9.35 mL, 1.00 equiv), TsCl (25.0 g, 131 mmol, 1.50 equiv), TEA (13.3 g, 131 mmol, 18.2 mL, 1.50 equiv), DMAP (1.07 g, 8.76 mmol, 0.10 equiv) in DCM (100 mL) were charged into R1 (500 mL three-neck flask) at 0 °C.

[0393] The mixture was stirred at 20° C. for 3 hours.

[0394] TLC (petroleum ether / ethyl acetate = 3 / 1) confirmed that intermediate 5 (R f =0.40) is completely consumed and one new spot (R f =0.65) was formed.

[0395] The reaction mixture was poured into H2O (300 mL) and extracted with DCM (200 mL*2).

[0396] The reaction ether phase was washed with brine (200 mL*3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo at 45°C.

[0397] The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 1 / 1, TLC: petroleum ether / ethyl acetate = 3 / 1, R f =0.65) to give Intermediate 6 (21.4 g, 79.0 mmol, 90.2% yield, 99.1% purity) as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[0398] LCMS: Rt=0.530 min, m / z=269.0(M+H) + .

[0399] HPLC: Rt = 2.345 min, purity 99.1% at 220 nm.

[0400] 1 H NMR (400MHz, DMSO-d6): δ7.79(d, J=8.4Hz, 2H), 7.48(d, J=8.0Hz, 2H), 6.29(d, J=6.0Hz, 1H), 4.67-4.65(m, 1H) ), 4.15-4.11(m, 1H), 4.08-4.04(m, 1H), 3.98-3.94(m, 1H), 2.43(s, 3H), 1.98-1.87(m, 3H), 1.53-1.45(m, 1H).

[0401] General procedure for the preparation of intermediate 7 [ka] To a solution of compound 6 (70.0 g, 260 mmol, 1.00 equiv.) in dry EtO (600 mL), LAH (19.8 g, 521 mmol, 2.00 equiv.) was then added portionwise (10 times) to the mixture at 0° C. under N. The reaction mixture was stirred at 0° C. for 30 min under N, and then the mixture was stirred at 25° C. for 2 h.

[0402] 1 A sample was taken for H NMR (EC378-428-P1D2) and intermediate 6 was completely consumed and the target peak was detected.

[0403] The mixture was cooled to -5°C, and 100 g of Na2SO4·10H2O was added portionwise (30 times) to the mixture at -5 to 0°C. The mixture was warmed to 20°C and stirred for 15 minutes. The mixture was filtered, and the filter cake was washed with Et2O (250 mL). The organic phase was washed with brine (300 mL * 2) and separated. The organic phase was dried over Na2SO4 and filtered. The filtrate was vacuum distilled (35°C, 0.05 MPa) to remove Et2O and unknown impurities.

[0404] The filtrate was vacuum distilled (35 °C, 0.05 MPa) to remove EtO and unknown impurities to give intermediate 7 (17.6 g, 179 mmol, 68.7% yield) as a colorless oil. 1 Confirmed by 1 H NMR.

[0405] 1 H NMR (400MHz, CDCl3): δ6.36(d, J=6.0Hz, 1H), 4.67-4.65(m, 1H), 3.95-3.91(m, 1H), 2.09-2 .04(m, 1H), 1.98-1.97(m, 1H), 1.84-1.82(m, 1H), 1.60-1.56(m, 1H), 1.27(d, J=6.4Hz, 3H).

[0406] General procedure for the preparation of intermediate 9 [ka] A mixture of intermediate 7 (686 mg, 6.99 mmol, 3.00 equiv), intermediate 8 (350 mg, 2.33 mmol, 1.00 equiv) and TsOH (602 mg, 3.50 mmol, 1.50 equiv) in THF (5.00 mL) was stirred at 100 °C for 10 h.

[0407] TLC analysis (dichloromethane / methanol = 5 / 1) revealed that intermediate 7 (R f =0.10) was consumed to produce intermediate 9 (R f =0.30) was detected.

[0408] The six batches were combined for workup. The mixture was poured into 40.0 mL of H2O and extracted with DCM (50.0 mL*3). The organic phase was washed with brine (100 mL*1) and separated. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0409] The residue was purified by MPLC (SiO2, R f = 0.30, dichloromethane / methanol = 5 / 1, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give intermediate 9 (1.30 g, 5.03 mmol, yield 35.9%, purity 96.0%) as a yellow solid. 1 Confirmed by 1 H NMR.

[0410] LCMS: Rt=0.364 min, m / z=497.3(2M+H) + .

[0411] HPLC: Rt = 1.126 min, purity 96.0% at 220 nm.

[0412] 1 H NMR (400MHz, MeOD): δ8.15(s, 1H), 5.75-5.71(m, 1H), 5.49(s, 1H), 3.87-3.82(m, 1H), 2.4 8(s, 3H), 2.06-2.00(m, 3H), 1.73-1.69(m, 2H), 1.38-1.37(m, 1H), 1.23(d, J=6.0Hz, 3H).

[0413] General Procedure for the Preparation of Compound 010 [ka] Compound 9 (200 mg, 805 μmol, 1.00 equivalents) and Toluene (5.00 mL) were added to R1 (40.0 mL flask) at 25 °C.

[0414] POCl3 (185 mg, 1.21 mmol, 112 μL, 1.50 equiv) was charged into R1 at 25 °C.

[0415] TEA (122 mg, 1.21 mmol, 168 μL, 1.50 equiv) was added dropwise to R1 at 25 °C.

[0416] R1 was stirred at 100° C. for 1 hour.

[0417] A sample was taken for TLC (dichloromethane / methanol = 5 / 1). Compound 9 (R f =0.30) is consumed and new points (R f =0.90) was detected.

[0418] R1 was cooled to 25°C.

[0419] Combine the five batches and work them up.

[0420] The reaction mixture was poured into warm water (30.0 mL, 30-40° C.).

[0421] Then, extraction was performed with ethyl acetate (50.0 mL*2).

[0422] The combined organic layers were washed with brine (10.0 mL*1), dried over Na2SO4, filtered, and the filtrate was concentrated to give the crude product.

[0423] The crude product was purified by preparative TLC (petroleum ether / ethyl acetate = 1 / 1, R f=0.30) to give intermediate 9 (243 mg, 856 μmol, 21.2% yield, 95.2% purity) as a yellow solid. 1 Confirmed by 1 H NMR.

[0424] LCMS: Rt=0.364 min, m / z=497.3(2M+H) + .

[0425] HPLC: Rt = 1.126 min, purity 96.0% at 220 nm.

[0426] 1 H NMR (400MHz, MeOD): δ8.63(s, 1H), 5.89-5.86(m, 1H), 3.91-3.86(m, 1H), 2.74(s, 3 H), 2.15-2.05(m, 3H), 1.85-1.72(m, 2H), 1.42-1.25(m, 1H), 1.24(d, J=6.4Hz, 3H).

[0427] Example 4 - Preparation of Compound 011 General procedure for the preparation of intermediate 11 [ka] A mixture of intermediate 10 (2.00 g, 13.3 mmol, 1.00 equiv) in N,N-dimethylaniline (3.30 mL).

[0428] To the mixture was added POCl3 (15.0 mL).

[0429] The reaction mixture was stirred at 110° C. for 1 hour.

[0430] A sample was taken for TLC (petroleum ether / ethyl acetate = 0 / 1). f =0.20) is consumed and one spot (R f =0.90) was detected.

[0431] R1 was cooled to 25°C.

[0432] The reaction mixture was poured onto finely crushed ice (100 mL).

[0433] Then, it was extracted with ethyl acetate (100 mL*2).

[0434] The combined organic layers were washed with cold water (50.0 mL*3), dried over Na2SO4, filtered, and the filtrate was concentrated to give Intermediate 11 (2.00 g, crude) as a yellow solid.

[0435] General procedure for the preparation of intermediate 12 - [ka] A 100 mL three-neck flask was equipped with an overhead stirrer, an addition funnel, and a thermometer.

[0436] The flask was charged with DMF (20.0 mL).

[0437] Intermediate 11 (2.00 g, 11.8 mmol, 1.00 equivalents) was charged into a flask at 20-25°C.

[0438] K2CO3 (4.92 g, 35.5 mmol, 3.00 equiv.) was charged into the flask at 5–10 °C.

[0439] MeI (3.37 g, 23.7 mmol, 1.48 mL, 2.00 equiv.) was added dropwise to the flask at 5-10 °C.

[0440] The mixture was stirred at 20 to 25°C for 12 hours.

[0441] A sample was taken for LCMS (EC378-408-P1D1). Intermediate 11 was completely consumed and one main peak with the target mass (Rt=1.431 min) was detected.

[0442] The mixture was poured into H2O (100 mL) and stirred at 20-25 °C.

[0443] To the mixture was added AcOK (10.0 g).

[0444] The mixture was extracted with ethyl acetate (30.0 mL*3).

[0445] The organic phase was washed with brine (30.0 mL*1) and separated.

[0446] The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0447] The residue was purified by MPLC (SiO 2、 R f =0.20, petroleum ether / ethyl acetate = 1 / 1, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to give intermediate 12 (1.00 g, 4.61 mmol, yield 38.8%, purity 84.2%) as a yellow solid, which was confirmed by LCMS.

[0448] LCMS: Rt=1.431 min, m / z=183.1(M+H) + .

[0449] LCMS: Rt=1.378 min, m / z=183.2(M+H) + .

[0450] General Procedure for the Preparation of Compound 011 [ka] To a mixture of Intermediate 3a (1.10 g, 10.9 mmol, 1.21 mL, 2.00 equiv), Intermediate 12 (1.00 g, 5.48 mmol, 1.00 equiv) and TEA (1.11 g, 10.9 mmol, 1.52 mL, 2.00 equiv) in MeOH (10.0 mL) was added. The mixture was stirred at 70 °C for 1 h.

[0451] A sample was taken for LCMS (EC378-409-P1A1) and intermediate 12 was completely consumed with one main peak having the target mass (Rt=1.351 min).

[0452] The mixture was poured into 20.0 mL of H2O and extracted with DCM (50.0 mL*3), and the organic phase was washed with brine (100 mL*1) and separated. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0453] The residue was purified by MPLC (SiO2, R f = 0.20, petroleum ether / ethyl acetate = 1 / 1, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to give intermediate 10 (588 mg, 2.37 mmol, 43.2% yield, 99.3% purity) as a blue solid, which was analyzed by LCMS, HPLC, and 1 Confirmed by 1 H NMR.

[0454] LCMS: Rt=1.351 min, m / z=247.1(M+H) + .

[0455] LCMS: Rt=0.507 min, m / z=247.1(M+H) + .

[0456] HPLC: Rt = 1.684 min, purity 99.3% at 220 nm.

[0457] 1 H NMR (400MHz, MeOD): δ7.92(s, 1H), 4.29(s, 4H), 3.78(s, 3H), 2.58-2.52(m, 7H), 2.35(s, 3H).

[0458] Example 5 - Preparation of Compound 017 General procedure for the preparation of intermediate 14 - [ka] Intermediate 13 (1.50 g, 9.77 mmol, 1.00 equiv), intermediate 3a (3.91 g, 39.0 mmol, 4.33 mL, 4.00 equiv), and DIEA (6.31 g, 48.8 mmol, 8.51 mL, 5.00 equiv) in EtOH (15.0 mL) were charged into a bottle at 20 °C.

[0459] The mixture was stirred at 100° C. for 16 hours.

[0460] A sample was taken for TLC (dichloromethane / methanol = 10 / 1). f =0.60) is consumed, and one main spot (R f =0.40) was detected.

[0461] The reaction mixture was concentrated under vacuum at 50° C. to give a residue.

[0462] The residue was purified by column chromatography (SiO2, dichloromethane / methanol = 50 / 1 to 10 / 1, TLC: dichloromethane / methanol = 10 / 1, R f =0.40) to give intermediate 14 (1.80 g, 8.28 mmol, 84.8% yield, 100% purity) as a yellow solid, which was analyzed by LCMS, HPLC, and 1 Confirmed by 1 H NMR.

[0463] LCMS: Rt=1.136 min, m / z=218.1(M+H) + .

[0464] HPLC: Rt = 0.862 min, 100% purity at 220 nm

[0465] 1 H NMR(400MHz,DMSO-d6):δ12.71(s,1H),8.06(s,1H),7.40(d,J=5.6Hz,1H),6.47(d,J=5.6Hz,1H),3.86(s,4H),2.46(s,4H),2.22(s,3H)

[0466] General procedure for the preparation of compound 017 [ka] A mixture of intermediate 7 (135 mg, 1.38 mmol, 3.00 equiv.), intermediate 14 (100 mg, 460 μmol, 1.00 equiv.) and TsOH (103 mg, 598 μmol, 1.30 equiv.) in Toluene (3.00 mL) was stirred at 50° C. for 5 h.

[0467] A sample was taken for LCMS (EC378-440-P1W9): Intermediate 14 was consumed and one peak with the target mass (Rt=1.556 min) was detected.

[0468] The eight batches were combined for workup. The mixture was poured into 20.0 mL of H2O and extracted with DCM (50.0 mL*3). The organic phase was washed with brine (100 mL*1) and separated. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0469] The residue was purified by MPLC (SiO2, R f = 0.20, dichloromethane / methanol = 5 / 1, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1), and the product was separated by SFC (column: DAICEL CHIRALCEL OD (250 mm * 30 mm, 10 μm, mobile phase: [0.1% NH3H2O ​​MeOH], B%: 15% to 15%, 8.0, 160 min) to obtain intermediate 16 (647 mg, 1.27 mmol, yield 34.4%, purity 100%) as a yellow oil. 1 Confirmed by 1 H NMR.

[0470] LCMS: Rt=1.556 min, m / z=316.2(M+H) + .

[0471] LCMS: Rt=0.482 min, m / z=316.1(M+H) + .

[0472] HPLC: Rt=1.251 min, 100% purity at 220 nm.

[0473] 1H NMR (400MHz, MeOD): δ8.26(s, 1H), 8.05(d, J=6.0Hz, 1H), 6.65(d, J=6.0Hz, 1H), 5.83-5.80(m, 1H), 3.98(s, 4H), 3.91-3.88 (m, 1H), 2.83(t, J=4.8Hz, 4H), 2.50(s, 3H), 1.85-1.82(m, 2H), 1.74-1.71(m, 2H), 1.40-1.37(m, 1H), 1.23(d, J=6.0Hz, 3H)

[0474] Example 6 - Preparation of Compound 027 General procedure for the preparation of intermediate 2 - [ka] Intermediate 1 (100 mg, 412 μmol, 1.00 equiv) and THF (2.00 mL) were charged to a flask at 10° C. under nitrogen.

[0475] Cool to -70°C.

[0476] Under nitrogen, n-BuLi (2.50 M, 247 μL, 1.50 equiv) was added dropwise to the flask at −70° C.

[0477] The reaction mixture was stirred at -70°C for 1.5 hours.

[0478] A solution of intermediate 1b (105 mg, 494 μmol, 1.20 equiv) in THF (1.00 mL) was charged to a flask at −70° C. under nitrogen.

[0479] The mixture was stirred at -70°C for 2 hours.

[0480] TLC (petroleum ether:ethyl acetate=3:1) confirmed that intermediate 1 (R f =0.8) was completely consumed, and the main spot (R f =0.3) was detected.

[0481] LCMS showed that the target mass was detected (RT=0.575 min, 0.614 min).

[0482] After the reaction was completed, the mixture was added to saturated NH4Cl solution (20 mL) at 0°C, and the mixture was extracted with ethyl acetate (2 * 20.0 mL). The organic phase was separated and washed with brine (25.0 mL). The solution was dried over Na2SO4, filtered, and then evaporated in vacuo.

[0483] The residue was analyzed by pre-TLC (petroleum ether: ethyl acetate = 3:1, R f =0.3).

[0484] Intermediate 2 (67.0 mg, 164 μmol, 39.8% yield, 92.5% purity) was obtained as a yellow oil. 1 Confirmed by H NMR (EC361-437-P1B1) and LCMS (EC361-437-P1B1).

[0485] LCMS: RT=0.575 min, 0.614 min, m / z=376.9(M+H) + , 377.0(M+H) + .

[0486] LCMS: RT=0.572 min, 0.611 min, m / z=377.0(M+H) + , 376.9(M+H) + .

[0487] 1 H NMR (400MHz, CDCl3): δ7.98-8.07(m,2H),7.79-7.80(m,1H),7.67(d,J=2.00Hz,1H),7.33-7.64(m,1H),5.09-5.13(m,1H),4.67(d, J=138Hz,1H),4.19(s,1H),3.14(t,J=20.8Hz,1H),2.17(d,J=24.8Hz,1H),1.67-1.83(m,1H),1.41-1.53(m,9H),0.91-1.02(m,4H).

[0488] General procedure for the preparation of compound 027 - [ka] Intermediate 2 (67.0 mg, 164 μmol, 92.5% purity, 1.00 equiv.), ethyl acetate (1.00 mL), and HCl / EtOAc (4.00 M, 925 μL, 22.5 equiv.) were charged into a 100 mL flask at 20 °C.

[0489] The mixture was stirred at 20° C. for 0.5 h.

[0490] TLC (plate 1, petroleum ether:ethyl acetate = 0:1) revealed intermediate 2 (R f =0.50) is completely consumed and one new spot (R f =0.20) was detected.

[0491] The mixture was filtered and the filtrate cake was washed with ethyl acetate (5.00 mL*2).The filter cake was concentrated in vacuo.

[0492] The solid was slurried in ethyl acetate (5.00 mL) at 20° C. for 0.5 h and collected by filtration.

[0493] The product was freeze-dried.

[0494] Compound 027 (31.13 mg, 106 μmol, yield 64.5%, purity 94.3%) was obtained as a yellow solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[0495] LCMS: RT=0.403 min, 0.412 min, m / z=277.2(M+H) + , m / z=277.2(M+H) + .

[0496] HPLC: RT = 1.333 min, 1.387 min, purity 94.3% at 220 nm.

[0497] 1H NMR (400MHz, MeOD): δ8.60-8.68(m, 1H), 8.14-8.22(m, 2H), 7.93(t, J=18.8Hz, 2H), 5.32(t, 1H), 4.99-5.06(m, 1H), 3.67- 379(m, 1H), 3.46(d, J=13.6Hz, 1H), 3.15(t, J=14.4Hz, 1H), 1.82-1.93(m, 2H), 1.69-1.76(m, 2H), 1.46(d, J=23.6Hz, 1H).

[0498] Example 7 - Preparation of Compound 029 General procedure for the preparation of intermediate 4 - [ka] Intermediate 3 (100 mg, 412 μmol, 1.00 equiv.), THF (3.00 mL), and Intermediate 1b (105 mg, 494 μmol, 1.20 equiv.) were charged to a flask under nitrogen at 10°C.

[0499] Cool to -70°C.

[0500] Under nitrogen, n-BuLi (2.50 M, 247 μL, 1.50 equiv) was added dropwise to the flask at −70° C.

[0501] The mixture was stirred at -70°C for 1.5 hours.

[0502] TLC (plate 1, petroleum ether:ethyl acetate = 3:1) revealed intermediate 3 (R f =0.70) was completely consumed, and three new spots (R f =0.15, R f =0.30, R f =0.80) was detected.

[0503] After the reaction was completed, the mixture was added to saturated NH4Cl solution (20.0 mL) at 0°C and extracted with ethyl acetate (2*20.0 mL). The organic phase was separated and washed with brine (25.0 mL). The solution was dried over Na2SO4, filtered, and evaporated in vacuo.

[0504] The mixture was analyzed by pre-TLC (petroleum ether:ethyl acetate=3:1, R f =0.3).

[0505] Intermediate 4 (26.0 mg, 67.7 μmol, 16.4% yield, 98.2% purity) was obtained as a yellow solid. 1 The result was confirmed by H NMR (EC3360-132-p1) and LCMS (EC3360-132-P1C1).

[0506] LCMS: RT=0.608 min, 0.639 min, m / z=377.1(M+H) + , m / z=377.1(M+H) + .

[0507] 1 H NMR (400MHz, CDCl3): δ8.12 (d, J=8.40Hz, 1H), 7.75 (d, J=8.00Hz, 2H), 7.43-7.48 (m, 2H), 5.06 (d, J=6.00Hz, 1H), 4.03-4.28(m, 3H), 3.14-3.19(m, 2H), 1.88(d, J=13.2Hz, 2H), 1.73(t, J=23.2Hz, 3H), 1.40-1.60(m, 9H).

[0508] General procedure for the preparation of compound 029 [ka] Intermediate 4 (50.0 mg, 130 μmol, purity 98.2%, 1.00 equivalents) and ethyl acetate (1.00 mL) were placed in a flask at 10° C. under nitrogen.

[0509] HCl / EtOAc (4.00 M, 2.00 mL, 60.3 equiv) was charged to the flask at 10°C.

[0510] The mixture was stirred at 10° C. for 1 hour.

[0511] TLC (plate 1, petroleum ether:ethyl acetate = 0:1) revealed intermediate 4 (R f=0.50) is completely consumed and one new spot (R f =0.20) was detected.

[0512] The mixture was filtered and the filtrate cake was washed with ethyl acetate (5.00 mL*2).The filter cake was concentrated in vacuo.

[0513] The solid was slurried in ethyl acetate (5.00 mL) at 20° C. for 0.5 h and collected by filtration.

[0514] Compound 029 (28.79 mg, 102 μmol, 78.3% yield, 98.1% purity) was obtained as a yellow solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[0515] LCMS: RT=0.402 min, m / z=277.2(M+H) + .

[0516] HPLC: RT = 1.298 min, 1.336 min, purity 98.1% at 220 nm.

[0517] 1 H NMR (400MHz, MeOD): δ8.48-8.51(m, 1H), 7.93-7.96(m, 2H), 7.86(d, J=8.4Hz, 1H), 7.59(t, J=15.6Hz, 1H), 5.12(d, J=4.8Hz, 1 H), 4.96(d, J=4.40Hz, 1H), 3.79(d, J=4.80Hz, 1H), 3.51(d, J=15.2Hz, 1H), 3.12-3.16(m, 1H), 1.84(s, 3H), 1.69-1.74(m, 2H).

[0518] Example 8 - Preparation of Compound 038 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (300 mg, 1.59 mmol, 1.00 equiv), Intermediate 2 (501 mg, 3.17 mmol, 2.00 equiv), diacetoxycopper (577 mg, 3.17 mmol, 2.00 equiv), 4A MS (13.2 mmol), TEA (321 mg, 3.17 mmol, 442 μL, 2.00 equiv) were added to DCM (3.00 mL).

[0519] The mixture was stirred under an O2 atmosphere at 20°C for 12 hours.

[0520] LCMS showed that intermediate 1 was consumed and the target mass was obtained (RT=0.570 min).

[0521] The mixture was filtered and the filtrate was concentrated.

[0522] The crude product was purified by Pre-HPLC (column: Phenomenex luna C18 150*25mm*10μm, mobile phase: [water (HCl)-ACN], B%: 36%-66%, 10 min).

[0523] Compound 038 (20 mg, 59.07 μmol, 3.72% yield, 99.7% purity, HCl) was obtained as a white solid.

[0524] LCMS: RT=0.570 min, m / z=300.9(M+H) +

[0525] LCMS: RT=0.565 min, m / z=300.9(M+H) +

[0526] HPLC: RT=2.630 min, purity 99.7% at 220 nm.

[0527] 19 F NMR (400 MHz, CDCl3)

[0528] 1 H NMR (400MHz, CDCl3): δ8.29 (d, J=0.88Hz, 1H) 7.65-7.71 (m, 1H) 7.17 (s, 2H).

[0529] Example 9 - Preparation of Compound 043 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (5.00 g, 19.7 mmol, 1.00 equivalents) was dissolved in MeOH (50.0 mL) at 15-25°C.

[0530] The solution was degassed and purged with N2 three times.

[0531] To the above solution was added EtN (5.97 g, 59.0 mmol, 8.22 mL, 3.00 equiv.) and Pd(dppf)Cl.CHCl (3.21 g, 3.94 mmol, 0.20 equiv.) at 15-25 °C under N.

[0532] The mixture was degassed and purged with CO (50 psi) three times.

[0533] The mixture was heated to 40-50°C and stirred under CO (50 psi) at 40-50°C for 16 h.

[0534] LCMS showed that about 27.1% of the desired product was detected (RT=1.194 min, m / z=234.2).

[0535] The mixture was filtered, and the filtrate was concentrated to remove MeOH.

[0536] The residue was diluted with water (20.0 mL) and ethyl acetate (20.0 mL). The aqueous phase was extracted with ethyl acetate (10.0 mL*2). The combined organic layers were washed with brine (10.0 mL*2), dried over Na2SO4, filtered, and concentrated.

[0537] The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 1 / 1, product: petroleum ether / ethyl acetate = 2 / 1, R f =0.40).

[0538] Intermediate 2 (1.10 g, 3.36 mmol, 17.1% yield, 71.3% purity) was obtained as a yellow solid and was confirmed by LCMS (EC3718-89-P1LD, RT=1.209 min, m / z=234.2).

[0539] LCMS:EC3718-89-P1L4, RT=1.194 min, m / z(M+H + )=234.2

[0540] LCMS:EC3718-89-P1LD, RT=1.209 min, m / z(M+H + )=234.2

[0541] General procedure for the preparation of intermediate 3 - [ka] Intermediate 2 (700 mg, 3.00 mmol, 1.00 equiv.) and CsCO (1.96 g, 6.00 mmol, 2.00 equiv.) were dissolved in DMF (7.00 mL) at 15-25 °C.

[0542] 3-Bromopropylbenzene (1.79 g, 9.00 mmol, 1.36 mL, 3.00 equiv) was added to the above solution.

[0543] The mixture was heated to 65-75°C and stirred at 65-75°C for 1 hour.

[0544] LCMS (EC3718-97-P1L3) showed that intermediate 2 was consumed and a new peak with the target mass (RT=2.551 min, m / z=352.1) was detected.

[0545] The reaction mixture was poured into aqueous NH4Cl (21.0 mL) and the aqueous phase was extracted with ethyl acetate (5.00 mL*2). The combined organic layers were washed with brine (5.00 mL*2), dried over Na2SO4, filtered and concentrated.

[0546] The crude product was purified by preparative HPLC (column: Phenomenex Luna C18200*40mm*10um; mobile phase: [water (TFA)-ACN]; B%: 55%-85%, 10 min).

[0547] Intermediate 3 (522 mg, 1.47 mmol, 48.9% yield, 98.7% purity) was obtained as a yellow solid and was confirmed by LCMS (EC3718-97-P1L4, RT=1.713 min, m / z=351.9).

[0548] LCMS: RT = 2.551 min, m / z (M+H + )=352.1

[0549] LCMS: RT = 1.713 min, m / z (M+H + )=351.9

[0550] General procedure for the preparation of compound 043 [ka] Two batches were run in parallel.

[0551] Intermediate 3 (200 mg, 569 μmol, 1.00 equiv.) and LiOH.HO (71.7 mg, 1.71 mmol, 3.00 equiv.) were dissolved in MeOH (2.00 mL) at 15-25 °C.

[0552] The mixture was heated to 40-50°C and stirred at 40-50°C for 2 hours.

[0553] LCMS (EC3718-99-P1L5) showed that intermediate 3 was consumed and a new peak with the target mass (RT=0.525 min, m / z=324.3) was detected.

[0554] The reaction mixture was cooled to 15-25°C and the pH was adjusted to 3-5 with 1N HCl (aq). The mixture was filtered and washed with 1N HCl (aq, 10 mL). The filter cake was concentrated to give the desired product.

[0555] PX043 (220.06 mg, 679 μmol, 59.6% yield, 99.7% purity) was obtained as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[0556] LCMS: RT = 0.525 min, m / z (M+H + )=324.3.

[0557] LCMS: RT = 2.043 min, m / z (M+H + )=324.0.

[0558] HPLC: RT=2.637 min, purity 99.7% at 220 nm.

[0559] 1 H NMR (400MHz, DMSO-d6): δ12.45(s, 2H) 8.72(d, J=1.2Hz, 1H) 8.21(s, 1H) 7.83(dd, J=8.6, 1.6Hz, 1H) 7.62(d , J=8.8Hz, 1H)7.23-7.29(m, 2H)7.18(d, J=6.8Hz, 3H)4.31(t, J=7.0Hz, 2H)2.55-2.60(m, 2H)2.11(m, 2H).

[0560] Example 10 - Preparation of Compound 048 General procedure for the preparation of intermediate 5 [ka] Intermediate 4 (2.00 g, 9.08 mmol, 1.00 equiv.) and CsCO (5.92 g, 18.2 mmol, 2.00 equiv.) were dissolved in DMF (20.0 mL) at 15-25 °C.

[0561] The mixture was stirred at 15-25°C for 0.5 hours.

[0562] Then, 3-bromopropylbenzene (5.43 g, 27.3 mmol, 4.11 mL, 3.00 equiv.) was added to the above mixture at 15-25°C.

[0563] The mixture was heated to 70-80°C and stirred at 70-80°C for 1.5 hours.

[0564] LCMS (EC3718-96-P1L2) showed that intermediate 5 was consumed and a new peak with the target mass (RT=1.876 min, m / z=339.2) was detected.

[0565] The reaction mixture was poured into aqueous NH4Cl (60.0 mL) and the aqueous phase was extracted with ethyl acetate (10.0 mL*2). The combined organic layers were washed with brine (10.0 mL*2), dried over Na2SO4, filtered and concentrated.

[0566] The crude product was used in the next step without purification.

[0567] Intermediate 5 (3.50 g, crude) was obtained as a brown oil as confirmed by LCMS.

[0568] LCMS: RT = 1.876 min, m / z (M+H + )=339.2.

[0569] LCMS: RT = 1.928 min, m / z (M+H + )=338.9.

[0570] General procedure for the preparation of intermediate 6 [ka] Intermediate 5 (400 mg, 1.18 mmol, 1.00 equiv) was dissolved in EtOH (4.00 mL) at 15-25 °C.

[0571] To the above solution was added a solution of NH4Cl (632 mg, 11.8 mmol, 10.0 equiv) in H2O (4.00 mL).

[0572] To the above mixture, Fe (330 mg, 5.91 mmol, 5.00 equiv) was added at 15-25°C.

[0573] The mixture was stirred at 15 to 25°C for 2 hours.

[0574] LCMS showed that intermediate 5 was consumed and a new peak with the target mass (RT=0.461 min, m / z=309.2) was detected.

[0575] The reaction mixture was filtered. The filtrate was concentrated to remove EtOH. The aqueous phase was extracted with ethyl acetate (10.0 mL*2). The combined organic layers were washed with brine (10.0 mL*2), dried over Na2SO4, filtered and concentrated.

[0576] The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 2 / 1, product: petroleum ether / ethyl acetate = 1 / 1, R f =0.40).

[0577] Intermediate 6 (200 mg, 588 μmol, 49.8% yield, 90.7% purity) was obtained as a yellow solid, which was confirmed by LCMS.

[0578] LCMS: RT = 0.461 min, m / z (M+H + )=309.2.

[0579] LCMS: RT = 1.816 min, m / z (M+H + )=308.9.

[0580] General procedure for the preparation of intermediate 7 - [ka] Intermediate 6 (120 mg, 389 μmol, 1.00 equiv.) and EtN (78.8 mg, 778 μmol, 108 μL, 2.00 equiv.) were dissolved in DMF (3.00 mL) at 15-25 °C.

[0581] To the above solution, CDI (69.4 mg, 428 μmol, 1.10 equiv.) was added at 15-25°C.

[0582] The mixture was stirred at 15-25°C for 0.5 hours.

[0583] O-methylhydroxylamine (42.3 mg, 506 μmol, 1.30 equiv., HCl) was added to the above solution at 15-25°C.

[0584] The mixture was stirred at 15-25°C for 0.5 hours.

[0585] LCMS showed that intermediate 6 was consumed and a new peak with the target mass (RT=0.571 min, m / z=382.2) was detected.

[0586] The reaction mixture was poured into water (10.0 mL) and the aqueous phase was extracted with ethyl acetate (5.00 mL*2). The combined organic layers were washed with brine (5.00 mL*2), dried over Na2SO4, filtered and concentrated.

[0587] The crude product was used in the next step without purification.

[0588] Intermediate 7 (130 mg, crude) was obtained as a brown oil and was confirmed by LCMS (RT=0.567 min, m / z=382.2).

[0589] LCMS: RT = 0.571 min, m / z (M+H + )=382.2

[0590] LCMS: RT = 0.567 min, m / z (M+H + )=382.2

[0591] General procedure for the preparation of compound 048 [ka] Intermediate 7 (100 mg, 262 μmol, 1.00 equiv.) and LiOH.HO (55.0 mg, 1.31 mmol, 5.00 equiv.) were dissolved in MeOH (3.00 mL) and HO (0.30 mL) at 15-25 °C.

[0592] The mixture was heated to 40-50°C and stirred at 40-50°C for 16 hours.

[0593] LCMS showed that intermediate 7 was consumed and a new peak with the target mass (RT=0.415 min, m / z=368.1) was detected.

[0594] The reaction mixture was cooled to 15-25°C and the pH was adjusted to 3-5 with 1N HCl (aq.). The mixture was filtered and washed with 1N HCl (aq., 10.0 mL). The filter cake was concentrated to give the crude product.

[0595] The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1, product: petroleum ether / ethyl acetate = 0 / 1, R f =0.60).

[0596] Compound 048 (25.62 mg, 66.0 μmol, 25.2% yield, 94.6% purity) was obtained as a white solid. 1 Confirmed by H NMR (EC3718-108-P1N2), LCMS (EC3718-108-P1L1, RT=0.418 min, m / z=368.1) and HPLC (EC3718-108-P1H2).

[0597] LCMS: RT = 0.415 min, m / z (M+H + )=368.1

[0598] LCMS: RT = 0.418 min, m / z (M+H + )=368.1.

[0599] HPLC: 94.6% purity at 220 nm

[0600] 1H NMR (400MHz, DMSO-d6): δ11.89(s, 1H)9.35(s, 1H)8.82-8.87(m, 1H)8.19(s, 1H)8.03(s, 1H)7.40-7.46(m, 2H) 7.25-7.30 (m, 2H) 7.19 (d, J=7.0Hz, 3H) 4.24 (t, J=6.8Hz, 2H) 3.63 (s, 3H) 2.54-2.59 (m, 2H) 2.04-2.13 (m, 2H).

[0601] Example 11 - Preparation of Compound 54 General procedure for the preparation of intermediate 3 [ka] Intermediate 1 (23.0 g, 133 mmol, 1.00 equiv.), Intermediate 2 (25.5 g, 200 mmol, 1.50 equiv.), DIEA (34.5 g, 267 mmol, 46.4 mL, 2.00 equiv.), and EtOH (200 mL) were charged to a flask under nitrogen at 25° C.

[0602] The mixture was stirred at 100° C. for 30 minutes.

[0603] LCMS showed that intermediate 1 was consumed and one peak with the target mass (Rt=0.583 min) was detected.

[0604] The mixture was poured into 500 mL of H2O, and the mixture was extracted with DCM (250 mL*7), and the organic phase was washed with brine (100 mL*1) and separated. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0605] The residue was purified by MPLC (SiO2, Rf=0.60, petroleum ether:ethyl acetate=5 / 1, petroleum ether / ethyl acetate=10 / 1 to 0 / 1).

[0606] Intermediate 3 (19.0 g, 62.7 mmol, 47.1% yield) was obtained as a yellow solid and was confirmed by LCMS (EC378-470-P1O1).

[0607] General procedure for the preparation of intermediate 4 [ka] Intermediate 3 (19.0 g, 68.4 mmol, 1.00 equiv.), [phenyl-(2,2,2-trifluoroacetyl)oxy-λ-iodanyl] 2,2,2-trifluoroacetic acid salt (38.3 g, 88.9 mmol, 1.30 equiv.), and ACN (200 mL) were charged to a 500 mL flask at 20 °C.

[0608] The reaction was stirred under N2 at 25°C for 2 hours.

[0609] A sample was taken for LCMS (EC378-476-P1R1). Intermediate 3 was consumed and two peaks with the target mass were detected (Rt=0.451 and 0.494 min).

[0610] The mixture was poured into 300 mL of H2O, and the mixture was extracted with DCM (500 mL*7), and the organic phase was washed with brine (100 mL*1) and separated. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo.

[0611] The crude product was purified by preparative HPLC (0.1% NH3*H2O).

[0612] Intermediate 4 (1.60 g, 5.34 mmol, 7.80% yield) was obtained as a yellow solid, confirmed by LCMS.

[0613] General procedure for the preparation of PX054 [ka] A mixture of intermediate 4 (1.60 g, 5.80 mmol, 1.00 equiv) and NaOH (464 mg, 11.6 mmol, 2.00 equiv) and HO (5.80 mL) in THF (20.0 mL).

[0614] The mixture was stirred at 25° C. for 10 minutes.

[0615] A sample was taken for TLC (petroleum ether:ethyl acetate=0:1). f =0.50) was consumed and a new spot (Rf=0.00) was detected.

[0616] The mixture was filtered and concentrated under reduced pressure to give a residue. Then 50.0 mL of HCl-H2O (1.00 N) was added to the residue, and the aqueous layer was extracted with DCM (30.0 mL * 3). The combined organic layers were washed with H2O (10.0 mL * 2), dried over Na2SO4, filtered, and concentrated.

[0617] Compound 054 (262.8 mg, 3.62 mmol, 62.3% yield, 99.5% purity) was obtained as a yellow solid, which was purified by LCMS, HPLC, and 1 Confirmed by 1 H NMR.

[0618] LCMS: RT=0.331 min, m / z=247.0(M+H) + .

[0619] HPLC: RT=1.348 min, 99.5% purity at 254 nm.

[0620] 1 H NMR (400MHz, DMSO): δ9.76 (d, J=6.80Hz, 1H), 8.48 (d, J=8.80Hz, 2H), 8.04 (s, 1H), 7.58 (m, 1H), 7.55 (m, 1H).

[0621] Example 12: Preparation of Compound 55 General procedure for the preparation of compound 055 [ka] To a solution of intermediate 055-1 (96.0 mg, 648 μmol, 1.00 equiv.) and intermediate 055-2 (119 mg, 648 μmol, 1.00 equiv.) in DMSO (2.00 mL) was added DIEA (670 mg, 5.19 mmol, 903 μL, 8.00 equiv.) at 20 °C, and the mixture was heated to 100 °C and stirred at 100 °C for 12 h.

[0622] LCMS (EC3963-107-P1A1) showed that approximately 14.0% of intermediate 055-2 (RT=0.945 min) remained and the target mass (RT=0.994 min) was detected.

[0623] The crude product was purified by reverse-phase HPLC (0.1% FA condition, Phenomenex luna C18 150*25mm*10μm, mobile phase: [water (FA)-ACN], B%: 5%-35%, 10 min), and concentrated under vacuum to remove ACN and water to obtain a residue.

[0624] The reaction was successful, affording compound 055 (30.0 mg, 92.9 μmol, 14.3% yield, 96.8% purity) as a yellow solid. 1 The product was confirmed by H NMR (EC3963-107-P1A1), LCMS (EC3963-107-P1D), and HPLC (EC3963-107-P1D2).

[0625] LCMS: RT=0.994 min, m / z=313.0(M+H) + .

[0626] LCMS: RT=0.389 min, m / z=313.1(M+H) + .

[0627] HPLC: RT=1.768 min, purity 96.8% at 220 nm.

[0628] 1 H NMR: (400MHz, DMSO-d6): δ8.29(d, J=2.0Hz, 1H), 8.00(dd, J=2.4, 2.0Hz, 1H), 7.36(d, J=8.8Hz, 1H), 6.89(t, J= 8.0Hz, 1H), 6.52(d, J=8.0Hz, 1H), 6.38(d, J=7.2Hz, 1H), 4.24(s, 2H), 3.47(t, J=5.6Hz, 2H), 2.67-2.62(m, 2H).

[0629] Example 13: Preparation of Compound 56 General procedure for the preparation of intermediate 2 [ka] To a solution of Intermediate 1 (1.00 g, 5.74 mmol, 1.00 equiv) in AcOH (20.0 mL) at 20 °C, PtO (500 mg, 2.20 mmol) was added, and the mixture was heated to 80 °C and stirred at 80 °C under 50 Psi for 24 h. TLC (petroleum ether:ethyl acetate = 1:2) showed that Intermediate 1 (R f =0.50) is consumed and the target spot (R f =0.00) was formed. The reaction mixture was filtered, and the filter cake was washed with MeOH (100 mL), after which the filtrate was concentrated under vacuum to give a residue. The residue was combined with EW3695-141 for purification. The crude product was purified by silica gel chromatography (diameter: 100-200 mesh silica gel, petroleum ether: ethyl acetate = 10:1 to 0:1, petroleum ether: ethyl acetate = 1:2, R f =0.00) to give the product. Intermediate 2 (200 mg, 1.30 mmol, crude purity) was obtained as a yellow solid and was confirmed by H NMR.

[0630] 1 H NMR (400MHz, CDCl3): δ3.58-3.55(m, 1H), 3.40-3.35(m, 1H), 3.25-3.24(m, 1H), 3.15-3.04(m, 2H), 2.16-1.94(m, 1H), 1.71-1.44(m, 9H).

[0631] General procedure for the preparation of compound 056 [ka] To a solution of intermediate 2 (200 mg, 0.524 mmol, 1.00 equiv) and intermediate 3 (96.5 mg, 0.524 mmol, 1.00 equiv) in DMSO (2.00 mL) was added DIEA (0.338 g, 2.62 mmol, 0.456 mL, 5.00 equiv) at 20 °C, and the mixture was heated to 100 °C and stirred at 100 °C for 12 h.

[0632] LCMS showed that intermediate 2 was consumed and the target mass was detected (RT=0.693 min). The reaction mixture was cooled to 25 °C, poured into H2O (50 mL), and the aqueous phase was extracted with ethyl acetate (30 mL * 2). The combined organic phase was washed with brine (50 mL * 2), dried over Na2SO4, and concentrated to give the crude product. The residue was combined with EW3977-133 for purification. The crude product was purified by reverse-phase HPLC (0.1% HCl condition, column: Welch Xtimate C18 150 * 25 mm * 5 um, mobile phase: [water (HCl)-ACN], B%: 0%-17%, 8 min), the eluate was concentrated in vacuo to remove ACN, and the aqueous phase was lyophilized to give the product.

[0633] Compound 056 (30.0 mg, 90.37 μmol, 14.3% yield, 95.9% purity) was obtained as a white solid and was characterized by LCMS, HPLC, 2D 1 HNMR and 1 HNMR.

[0634] LCMS: RT=0.693 min, m / z=319.1(MH) + .

[0635] LCMS: RT=1.119 min, m / z=319.1(MH) + .

[0636] HPLC: RT=1.733 min, purity 95.9% at 220 nm.

[0637] 1 H NMR (400MHz, MeOD): δ8.83(s, 1H), 8.45(d, J=8Hz, 1H), 8.08(d, J=7.6Hz, 1H), 3.80-3.78(m, 2H), 3.60-3.58(m, 2H), 3. 09(s, 1H), 2.37-2.34(m, 1H), 2.16(s, 1H), 1.96-1.81(m, 4H), 1.73-1.70(m, 1H), 1.60-1.58(m, 2H), 1.29-1.23(m, 1H).

[0638] Example 14: Preparation of Compound 57 General procedure for the preparation of compound 057 [ka] Intermediate 1b (9.34 g, 48.4 mmol, 1.80 equiv) was placed in THF (90.0 mL), and i-PrMgBr (2 M, 24.2 mL, 1.80 equiv) was added at 0° C., and the mixture was stirred at 0° C. for 1 h.

[0639] The mixture was added to a solution of Intermediate 1a (5.00 g, 26.9 mmol, 1.00 equiv) in THF (50.0 mL) at 0° C., and the mixture was stirred at 0° C. for 1 hour.

[0640] A sample was taken for LCMS: Intermediate 1a was completely consumed and one main peak with the target mass (Rt=0.405 min) was detected.

[0641] The mixture was poured into saturated aqueous NH4Cl (200 mL) at 0 °C, the mixture was extracted with DCM (100 mL*3), the organic phase was washed with brine (300 mL*1) and separated, the organic phase was dried over Na2SO4 and filtered, and the filtrate was concentrated under vacuum.

[0642] The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*25mm*10μm, mobile phase: [water (HCl)-ACN], B%: 36%~69%, 10 min).

[0643] Compound 057 (30 mg, 98.87 umol, 3.15% yield, 98.9% purity) was obtained as a yellow solid and was confirmed by LCMS: EC378-531-P1E2, HPLC: EC378-532-P1E4, HNMR: EC378-532-P1H.

[0644] LCMS: RT=0.405 min, m / z=300.1(M+H) + .

[0645] LCMS: RT=0.430 min, m / z=300.1(M+H) + .

[0646] HPLC: RT=1.833 min, purity 98.9% at 220 nm.

[0647] 1 H NMR (400MHz, DMSO): δ8.56 (m, 1H), 8.08 (m, 1H), 7.58 (m, 1H), 7.43 (m, 1H), 7.15 (m, 2H), 5.89 (s, 1H).

[0648] Example 15: Preparation of Compound 58 General procedure for the preparation of intermediate 4 [ka] To a solution of compound 057 (6.00 g, 20.0 mmol, 1.00 equiv.) and TBSCl (3.62 g, 24.0 mmol, 2.94 mL, 1.20 equiv.) in DMF (15.0 mL) was added imidazole (3.40 g, 49.9 mmol, 2.50 equiv.) at 25 °C, and the mixture was stirred at 25 °C for 10 h.

[0649] A sample was taken for LCMS (EC378-451-P1Q1). Compound 057 was completely consumed and one main peak with the target mass (Rt=2.351 min) was detected.

[0650] The mixture was poured into H2O (70.0 mL) at 25 °C, and the mixture was extracted with EA (60.0 mL*3), the organic phase was washed with brine (100 mL*2) and separated, the organic phase was dried over Na2SO4 and filtered, and the filtrate was concentrated in vacuo.

[0651] The residue was purified by MPLC (SiO2, Rf = 0.80, (petroleum ether: ethyl acetate = 10 / 1), petroleum ether / ethyl acetate = 100 / 1 to 50 / 1) to give intermediate 3a (8.00 g, 19.3 mmol, 96.6% yield) as a white solid, which was confirmed by LCMS.

[0652] LCMS: RT=2.351 min, m / z=415.9(M+H)+.

[0653] General procedure for the preparation of intermediate 4a [ka] To a solution of intermediate 3a (2.00 g, 4.83 mmol, 1.00 equiv) and HMPA (1.21 g, 6.76 mmol, 1.19 mL, 1.40 equiv) in THF (30.0 mL) was added the mixture, which was cooled to -70 °C. To the mixture was added n-BuLi (2.5 M, 2.70 mL, 1.40 equiv) dropwise at -70 °C under N. The mixture was then stirred at -70 °C for 1 h. Intermediate 5b (966 mg, 9.65 mmol, 2.00 equiv) was dissolved in THF (10.0 mL) and added dropwise to the mixture at -70 °C under N. The mixture was stirred at 20 °C under N for 12 h.

[0654] A sample was taken for LCMS (EC378-468-P1E2). Intermediate 3a was completely consumed and one main peak with the target mass (Rt=0.481 min) was detected.

[0655] The reaction mixture was poured into saturated aqueous NH4Cl (50.0 mL) at 0 °C. The mixture was extracted with ethyl acetate (50.0 mL * 3). The combined organic phases were washed with brine (60.0 mL) and concentrated. The residue was purified by preparative HPLC (0.1% HCl) to give intermediate 4a (250 mg, 529 μmol, 10.9% yield) as a yellow oil, which was confirmed by LCMS.

[0656] LCMS: RT=0.481 min, m / z=436.0(M+H)+.

[0657] General procedure for the preparation of compound 058 [ka] A mixture of intermediate 4a (150 mg, 344 μmol, 1.00 equiv) in TBAF (1 M, 5.00 mL, 14.5 equiv) was stirred at 25° C. for 10 h.

[0658] A sample was taken for LCMS. Intermediate 4a was consumed and one main peak with the target mass was detected (RT=0.209 min).

[0659] The reaction mixture was poured into H2O (20.0 mL) and extracted with EA (20.0 mL*3). The combined organic phase was washed with brine (20.0 mL), dried over Na2SO4, filtered and concentrated.

[0660] The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*25mm*10μm, mobile phase: [water (HCl)-ACN], B%: 7%-37%, 10 min).

[0661] The crude product was purified by preparative HPLC (column: Phenomenex Luna C18 150*25mm*10um, mobile phase: [water (HCl)-ACN], B%: 39%-69%, 10 min) to obtain compound 058 (20 mg, 59.76 umol, yield 17.35%, purity 96.0%) as a yellow oil. 1 Confirmed by 1 H NMR.

[0662] LCMS: RT=0.209 min, m / z=322.0(M+H) + .

[0663] LCMS: RT=0.430 min, m / z=300.1(M+H) + .

[0664] HPLC: RT=1.833 min, purity 98.9% at 220 nm.

[0665] 1 H NMR (400MHz, DMSO): δ8.50(m,1H), 8.06(m,1H), 7.63(m,1H), 7.61(m,1H), 7.49(m,2H), 7.25(m,1H), 6.08(s,1H), 3.10(m,2H), 2.58(m,2H).

[0666] Example 16: Preparation of Compound 59 General procedure for the preparation of intermediate 3b [ka] A mixture of Intermediate 3a (1.00 g, 2.41 mmol, 1.00 equiv), BPD (1.23 g, 4.83 mmol, 2.00 equiv), and KOAc (474 ​​mg, 4.83 mmol, 2.00 equiv) in dioxane (10.0 mL) was then added Pd(dppf)Cl (177 mg, 241 μmol, 0.10 equiv) to the mixture at 25 °C under N. The mixture was stirred at 100 °C for 3 h under a N atmosphere.

[0667] A sample was taken for LCMS (EC378-467-P1D1). Intermediate 3a was completely consumed and one main peak with the target mass (Rt=2.328 min) was detected.

[0668] The mixture was filtered, and 20.0 mL of EtOAc was added to the filtrate, then the organic phase was washed with brine (10.0 mL*1) and separated, the organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum.

[0669] The residue was purified by MPLC (SiO2, R f =0.60, (petroleum ether:ethyl acetate=5 / 1), petroleum ether / ethyl acetate=100 / 1 to 10 / 1) to give intermediate 4a (1.05 g, 2.19 mmol, yield 90.6%) as a yellow solid, which was confirmed by LCMS.

[0670] LCMS: RT=2.328 min, m / z=380.0(M+H) + .

[0671] General procedure for the preparation of intermediate 3c [ka] The bottle was charged with dioxane (15.0 mL) and H2O (3.00 mL).

[0672] Intermediate 3b (1.50 g, 3.25 mmol, 1.00 equiv.), 5-(benzyloxymethyl)-2-iodo-cyclohex-2-en-1-one (1.11 g, 3.25 mmol, 1.00 equiv.), and Pd(dppf)Cl (265 mg, 325 μmol, 0.10 equiv.) were charged into a bottle.

[0673] K2CO3 (898 mg, 6.50 mmol, 2.00 equiv) was charged into the bottle under N2.

[0674] The mixture was stirred at 80° C. under N 2 for 8 h.

[0675] A sample was taken for TLC (petroleum ether:ethyl acetate=5:1). f = 0.50) was consumed and a new main spot (Rf = 0.30) was detected.

[0676] The mixture was poured into H2O (50.0 mL) at 25 °C and extracted with DCM (100 mL*3), the organic phase was washed with brine (100 mL*1) and separated, the organic phase was dried over Na2SO4 and filtered, and the filtrate was concentrated under vacuum.

[0677] The residue was purified by MPLC (SiO2, R f =0.30, (petroleum ether:ethyl acetate=5 / 1, petroleum ether / ethyl acetate=30 / 1 to 5 / 1) to obtain intermediate 3c (1.50 g, 2.73 mmol, yield 83.9%) as a yellow oil.

[0678] General procedure for the preparation of compound 059 [ka] A mixture of intermediate 3c (500 mg, 909 umol, 1.00 equiv) in HCl / MeOH (4 M, 2.00 mL, 8.80 equiv) and H2O (1.00 mL) was stirred at 25 °C for 10 h.

[0679] A sample was taken for LCMS (EC378-535-P1Q1), which showed that intermediate 3c was consumed and one main peak with the target mass (Rt=0.418 min) was detected.

[0680] The reaction mixture was poured into H2O (20 mL) and extracted with EA (20.0 mL*3). The combined organic phase was washed with brine (20.0 mL), dried over Na2SO4, filtered and concentrated.

[0681] The crude product was purified by preparative HPLC (column: Phenomenex Luna C18200*40mm*10um; mobile phase: [water (TFA)-ACN]; B%: 30%~60%, 10 min).

[0682] The crude product was purified by preparative HPLC (column: Phenomenex Luna C18 150*25mm*10μm, mobile phase: [water (HCl)-ACN], B%: 39%-69%, 10 min) to obtain compound 059 (120mg, 275.57μmol, yield 30.30%, purity 100%) as a yellow solid. 1 Confirmed by 1 H NMR.

[0683] LCMS: RT=0.418 min, m / z=435.9(M+H) + .

[0684] LCMS: RT=0.588 min, m / z=436.0(M+H) + .

[0685] HPLC: RT=2.608 min, 100% purity at 220 nm.

[0686] 1 H NMR (400MHz, DMSO): δ8.45(s, 1H), 7.95(m, 1H), 7.66(m, 1H), 7.36(m, 1H), 7.33(m, 6H), 7. 30(m, 1H), 7.19(m, 1H), 6.05(s, 1H), 4.50(s, 2H), 3.50(s, 2H), 2.64(m, 3H), 2.40(m, 2H).

[0687] Example 17: Preparation of Compound 62 General procedure for the preparation of compound 15 [ka] Intermediate 14 (5.00 g, 24.0 mmol, 1 equivalent) was charged into R1 (100 mL) at 15-25°C.

[0688] ACN (50.0 mL) was added to R1 at 15 to 25°C.

[0689] Intermediate 14_A (3.87 g, 25.2 mmol, 1.05 equivalents, HCl) was charged into R1 at 15-25°C.

[0690] K2CO3 (5.96 g, 43.1 mmol, 1.80 equiv.) was added to R1 at 15–25 °C.

[0691] The mixture was heated to 50°C.

[0692] The mixture was stirred at 50° C. for 20 hours.

[0693] A sample was taken for LCMS: Intermediate 14 was consumed and Intermediate 15 was detected (RT=0.361 min).

[0694] The mixture was cooled to 20-25°C.

[0695] The mixture was diluted with ethyl acetate (150 mL).

[0696] The mixture was filtered under vacuum.

[0697] The filter cake was washed with ethyl acetate (100 mL) and the filtrate was concentrated in vacuo to give Intermediate 15 (6.80 g, 20.8 mmol, 86.8% yield, 88.6% purity) as a yellow solid, which was confirmed by LCMS and HPLC.

[0698] LCMS: RT=0.361 min, m / z=290.1(M+H)+

[0699] LCMS: RT=0.352 min, m / z=260.1(M+H) +

[0700] HPLC: RT = 0.998 min, purity 88.6% at 220 nm

[0701] General procedure for the preparation of compound 16 [ka] Intermediate 15 (6.80 g, 23.5 mmol, 1.00 equiv) was charged to R1 (500 mL).

[0702] Pd / C (680 mg, 10% purity) was loaded into R1 under N2.

[0703] Na2SO4 (3.40 g, 24.0 mmol, 2.43 mL, 1.02 equiv) was charged to R1 under N2.

[0704] R1 was charged with EtOAc (210 mL) under N2.

[0705] Degas under vacuum and purge with H2 three times.

[0706] The mixture was stirred at 20-25°C under H2 (15 psi) for 4 hours.

[0707] A sample was taken for LCMS and the reaction was consumed and intermediate 16 (RT=0.339 min) was detected.

[0708] The mixture was filtered under vacuum.

[0709] The filter cake was washed with MeOH (100 mL*2) and the mixture was concentrated under vacuum to give intermediate 16 (6.00 g, 20.5 mmol, 87.1% yield, 88.5% purity) as a brown solid, which was confirmed by LCMS and HPLC.

[0710] LCMS: RT=0.339 min, m / z=260.1(M+H) + .

[0711] LCMS: RT=0.345 min, m / z=260.1(M+H) + .

[0712] HPLC: RT=0.959 min, purity 88.5% at 220 nm.

[0713] General procedure for the preparation of intermediate 17 [ka] Intermediate 16 (4.00 g, 15.4 mmol, 1.00 equiv) was charged to R1 (35 mL).

[0714] CH(OEt)3 (35.6 g, 240 mmol, 40.0 mL, 15.6 equiv.) was added to R1 at 20-25 °C.

[0715] The mixture was stirred at 100°C for 3 hours.

[0716] A sample was taken for LCMS: Intermediate 16 was consumed and Intermediate 17 (Rt=0.390 min) was detected.

[0717] The mixture was cooled to 20-25°C, and 1M HCl (100 mL) was added to R1 and stirred at 20-25°C for 40 minutes. The pH was adjusted to 8-9 with saturated NaHCO3 (200 mL), and the mixture was extracted with ethyl acetate (200 mL*3). The organic layer was washed with brine (300 mL), dried over Na2SO4, and concentrated in vacuo to give the crude product.

[0718] The crude product was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate = 2:1 to 1:3 to give Intermediate 17 (2.12 g, 6.79 mmol, 44.0% yield, 86.2% purity) as a brown oil, which was confirmed by LCMS and HPLC.

[0719] LCMS: RT=0.390 min, m / z=270.1(M+H)+ .

[0720] LCMS: RT=0.412 min, m / z=270.1(M+H) + .

[0721] HPLC: RT=1.370 min, purity 86.2% at 220 nm.

[0722] General procedure for the preparation of intermediate 18 [ka] DCM (21.0 mL) was charged to R1 (50 mL).

[0723] Intermediate 17 (2.10 g, 7.41 mmol, 95% purity, 1.00 equiv) was charged to R1.

[0724] mCPBA (2.40 g, 11.11 mmol, 80% purity, 1.50 equivalents) was added to R1 at 0-5 °C.

[0725] The mixture was stirred at 20 to 25°C for 2 hours.

[0726] A sample was taken for LCMS: Intermediate 17 (Rt=0.414 min) was not completely consumed, but Intermediate 18 (Rt=0.465 min) was detected.

[0727] Saturated Na2SO3 (60 mL) was added to R1 at 0-5 °C, and the pH of the mixture was adjusted to 8-9 with saturated NaHCO3, and the mixture was extracted with DCM (30.0 mL*3), washed the organic layer with brine (40.0 mL), dried the organic layer with Na2SO4, and concentrated under vacuum at 40 °C to give intermediate 18 (2.00 g, 5.54 mmol, 74.76% yield, 79% purity) as a brown oil without purification, which was confirmed by LCMS.

[0728] LCMS: RT=0.465 min, m / z=286.1(M+H) + .

[0729] LCMS: RT=0.478 min, 79.4% purity at 220 nm.

[0730] General procedure for the preparation of intermediate 19 [ka] Intermediate 18 (2.00 g, 7.01 mmol, 1 equiv) was charged to R1 (100 mL).

[0731] DCE (40.0 mL) was charged into R1.

[0732] NH3·H2O (15.6 g, 111 mmol, 17.1 mL, 25% purity, 15.8 equivalents) was added to R1 at 25–30 °C.

[0733] TosCl (1.60 g, 8.41 mmol, 1.2 equiv.) in DCE (10 mL) was charged to R1 at 25-30 °C.

[0734] The mixture was stirred at 25 to 30°C for 3 hours.

[0735] A sample was taken for LCMS: Intermediate 18 was consumed and Intermediate 19 (Rt=0.436 min) was detected.

[0736] The mixture was diluted with DCE (50.0 mL), the aqueous and organic layers were separated, the organic layer was washed with brine (40.0 mL), the organic layer was dried over Na2SO4, and the organic layer was concentrated in vacuo to give intermediate 19 (1.20 g, 3.51 mmol, 50.03% yield, 83.1% purity) as a brown oil, confirmed by LCMS.

[0737] LCMS: RT=0.436 min, m / z=285.1(M+H) + .

[0738] LCMS: RT=0.443 min, 83.1% purity at 220 nm.

[0739] General procedure for the preparation of compound 062 [ka] Intermediate 19 (1.20 g, 4.22 mmol, 1 equiv) was charged to R1 (100 mL).

[0740] THF (24 mL) was charged to R1.

[0741] LiOH.HO (265.67 mg, 6.33 mmol, 1.5 equiv.) in HO (2.40 mL) was charged to R1 at 25-30 °C.

[0742] The mixture was stirred at 25 to 30°C for 3 hours.

[0743] A sample was taken for LCMS: intermediate 19 was consumed and target 3 was detected (RT=0.499 min).

[0744] The mixture was diluted with DCE (50 mL), the aqueous and organic layers were separated, the organic layer was washed with brine (40 mL), the organic layer was dried over Na2SO4, and the organic layer was concentrated under vacuum to give compound 062 (0.55 g, 2.01 mmol, 47.63% yield, 98.8% purity) as a grey solid, which was identified by LCMS, HPLC and 1 Confirmed by 1 H NMR.

[0745] LCMS: RT=0.499 min, m / z=329.0(M+H) + .

[0746] LCMS: RT=0.423 min, m / z=329.0(M+H) + .

[0747] HPLC: RT=1.353 min, purity 98.8% at 220 nm.

[0748] 1H NMR: (400MHz, DMSO-d6): δ12.0-14.87(m, 1H), 8.79-8.80(m, 2H), 8.46(s, 1H), 8.22-8.24(m, 1H), 7.82-7.84(m, 1H) ), 7.70-7.73(m, 1H), 7.56-7.59(m, 1H), 4.89-4.94(m, 1H), 4.68-4.73(m, 1H), 2.98-3.04(m, 1H), 1.18(d, 7.2Hz).

[0749] Example 18: Preparation of Compound 64 General procedure for the preparation of intermediate 36 [ka] Rh / C (2.08 g, 1.01 mmol, 2.03 mL, 5% purity, 0.10 equiv.) was added to R1 (100 mL) at 20-25 °C under Ar2.

[0750] PtO2 (109 mg, 480 μmol, 0.05 equiv.) was added to R1 at 20-25 °C under Ar2.

[0751] AcOH (40.0 mL) was added to R1 at 20-25°C under Ar2.

[0752] Intermediate 35 (2.00 g, 9.60 mmol, 1.00 equiv) was added at 20-25 °C under Ar.

[0753] The suspension was degassed under vacuum and purged with H2 several times.

[0754] The mixture was stirred in a 50 mL autoclave at 1.50 MPa and 75°C for 4 hours.

[0755] A sample was taken for LCMS and intermediate 35 was completely consumed and the target peak was detected (RT=0.395 min).

[0756] Cool to 20-25°C.

[0757] The reaction mixture was filtered and concentrated in vacuo to give Intermediate 36 (3.20 g, crude) as a brown oil, confirmed by LCMS.

[0758] LCMS: RT=0.395 min, m / z=215.3(M+H) + .

[0759] General procedure for the preparation of intermediate 37 [ka] n-BuOH (24.0 mL) was added to R1 (50.0 mL) at 20 to 25°C.

[0760] Intermediate 36 (1.27 g, 5.93 mmol, 1.00 equiv.) was added to R1 at 20-25° C. Intermediate 36_a (999 g, 5.93 mmol, 1.00 equiv.) was added to R1 at 20-25° C.

[0761] The mixture was stirred at 120°C for 5 hours.

[0762] A sample was taken for LCMS and intermediate 36 was completely consumed and the target peak (RT=0.333 min) was detected.

[0763] The reaction mixture was filtered, the filter cake was washed with EtOAc (30.0 mL*2), and the filter cake was concentrated in vacuo to give intermediate 37 (1.50 g, 2.99 mmol, yield 50.4%, purity 69.0%) (HPLC: EC8946-1-P1B2) as an off-white solid, which was confirmed by LCMS and HPLC.

[0764] LCMS: RT=0.333 min, m / z=347.2(M+H) +

[0765] HPLC: RT=1.210 min, purity 69.0% at 220 nm.

[0766] General procedure for the preparation of compound 064 [ka] Intermediate 37 (1.50 g, 4.33 mmol, 1.00 equiv) was added to R1 (50.0 mL) at 20-25 °C.

[0767] To R1 was added EtOAc (7.00 mL) at 20-25°C.

[0768] HCl / EtOAc (4 M, 2.16 mL, 2.00 equiv) was added to R1 at 20-25 °C.

[0769] The mixture was stirred at 20 to 25°C for 4 hours.

[0770] A sample was taken for LCMS (EC8946-6-P1A1) and intermediate 37 was completely consumed and the target peak (RT=1.300 min) was detected.

[0771] The reaction mixture was filtered and the filter cake was washed with EtOAc (10.0 mL*2).

[0772] The filter cake was concentrated in vacuo to give the crude product.

[0773] The crude product (110 mg) from notebook page EC8836-2 was combined with EC8946-6 for further purification.

[0774] The mixture was added to R2 (50.0 mL) at 20-25°C.

[0775] MeOH (7.50 mL) in EtOAc (15.0 mL) was added to R2 at 20-25°C.

[0776] The mixture was stirred at 25 to 30°C for 0.3 hours.

[0777] The reaction mixture was filtered, the filter cake was washed with EtOAc (10.0 mL*2), and the filter cake was concentrated under vacuum to give compound 064 (570 mg, 2.31 mmol, 53.2% yield, 99.7% purity) as a white solid, which was analyzed by LCMS, HPLC, and HPLC. 1 Confirmed by 1 H NMR.

[0778] LCMS: RT=1.300 min, m / z=247.2(M+H) + .

[0779] LCMS: RT=0.315 min, m / z=246.9(M+H) + .

[0780] HPLC: RT=1.451 min, purity 99.7% at 220 nm.

[0781] 1 H NMR (400MHz, MeOD): δ8.52(s, 1H), 8.32(s, 1H), 5.50(d, J=12.8Hz, 1H), 4.94-5.10(s, 1H), 4.08(d, J=7.8Hz, 1H) , 3.94(s, 3H), 3.67-3.77(m, 2H), 2.31-2.43(m, 1H), 1.88-1.99(m, 1H), 1.72-1.86(m, 1H), 1.23(d, J=7.2Hz, 3H).

[0782] Example 19 - Preparation of Compound 65 General procedure for the preparation of compound 39_a [ka] Intermediate 39 (25.0 g, 181 mmol, 1.00 equiv) was added to R1 (500 mL) at 20-25 °C.

[0783] Ac2O (166 mL) was added to R1 at 20-25 °C.

[0784] KOAc (29.6 g, 302 mmol, 1.67 equiv) was added to R1 at 20-25 °C.

[0785] The mixture was stirred at 120°C for 16 hours.

[0786] LCMS analysis showed that intermediate 39 was completely consumed and intermediate 39_a (RT=0.127 min) was detected.

[0787] Cool to 20-25°C.

[0788] Add MTBE (250 mL) to R1 and stir at 20-25° C. for 15 minutes. The mixture was filtered, the filter cake was washed with MTBE (250 mL*2), and the organic layer was concentrated under vacuum to give intermediate 39_a as a brown oil, which was confirmed by LCMS.

[0789] LCMS: RT=0.127 min, m / z=110.4(M+H) + .

[0790] General procedure for the preparation of compound 40 [ka] To R1 (500 mL) was added H2SO4 (90.0 mL) at 20-25 °C.

[0791] Intermediate 39_a (23.0 g, 210 mmol, 1.00 equiv) was added to R1 at 20-25 °C.

[0792] The solution was warmed to 60-65°C.

[0793] HNO3 (42.1 g, 656 mmol, 30.1 mL, 98.0% purity, 3.11 equiv.) in H2SO4 (40 mL) was added to R2 (250 mL) at 20-25 °C.

[0794] The mixture was slowly added from R2 to R1 at 60-65°C.

[0795] The mixture was stirred at 60 to 65°C for 2 hours.

[0796] The mixture was stirred at 75°C for 16 hours.

[0797] A sample was taken for LCMS. Intermediate 39_a (RT=0.639 min) was not completely consumed, but the target peak was detected (RT=0.588 min).

[0798] Cool to 10-20°C.

[0799] The reaction mixture was slowly poured into H2O (550 mL) at 10-20 °C.

[0800] The pH of the mixture was adjusted to 5-6 with NH3.H2O (370 mL) at 10-20 °C.

[0801] The mixture was filtered and the filter cake was concentrated under vacuum to give intermediate 40 (3.85 g, 24.9 mmol, 11.9% yield) (HNMR: EC8946-29-P1C3) as a yellow solid, as determined by LCMS and 1 Confirmed by 1 H NMR.

[0802] LCMS: RT=0.588 min, m / z=155.1(M+H) + .

[0803] 1 H NMR (400MHz, DMSO): δ12.18(s, 1H), 8.73(s, 1H), 7.73(s, 1H), 1.92(s, 3H).

[0804] General procedure for the preparation of intermediate 41 [ka] Pd / C (0.34 g, 1.70 mmol, 10% purity) was added to R1 (250 mL) at 20-25 °C under Ar2.

[0805] MeOH (100 mL) was added to R1 at 20-25°C under Ar2.

[0806] Intermediate 40 (3.40 g, 22.06 mmol, 1.00 equiv) was added at 20-25 °C under N2.

[0807] The suspension was degassed under vacuum and purged with H2 several times.

[0808] The mixture was stirred under H2 (15 psi) at 25 C for 16 h.

[0809] LCMS analysis showed that intermediate 40 was completely consumed and intermediate 41 (RT=0.123 min) was detected.

[0810] The reaction mixture was filtered and concentrated in vacuo to give Intermediate 41 (2.66 g, 19.9 mmol, 90.3% yield, 93.0% purity) as a brown solid, which was confirmed by LCMS and HPLC.

[0811] LCMS: RT=0.123 min, m / z=125.3(M+H) + .

[0812] HPLC: RT=0.467 min, purity 96.4% at 220 nm.

[0813] General procedure for the preparation of intermediate 42 [ka] THF (19.0 mL) was added to R1 (100 mL) at 20-25°C.

[0814] Intermediate 41 (950 mg, 7.65 mmol, 1.00 equiv) was charged to R1.

[0815] The mixture was degassed with N2 three times and cooled to -70 to -60 °C.

[0816] LiHMDS (1 M, 16.8 mL, 2.20 equiv.) was charged to R1 at −70 to −60 °C under N 2 .

[0817] The mixture was stirred at -70 to -60°C for 0.5 hours.

[0818] Boc2O (2.09 g, 9.57 mmol, 2.20 mL, 1.25 equiv) in THF (4 mL) was added dropwise to R1 at -70 to -60 °C under N2.

[0819] The mixture was stirred at -70 to -60°C for 0.5 hours, and then stirred at 20 to 25°C for 16 hours.

[0820] LCMS analysis showed that intermediate 41 (RT=0.102 min) was not completely consumed, but intermediate 42 (RT=0.465 min) was detected.

[0821] The mixture was poured into HO (120 mL) at 20-25°C and extracted with EtOAc (120 mL*2). The combined organic phase was washed with brine (120 mL), and the organic phase was dried over NaSO and concentrated in vacuo to give Intermediate 42 (2.66 g, 11.8 mmol, 77.5% yield, 73.1% purity) as a yellow solid, which was confirmed by LCMS and HPLC.

[0822] LCMS: RT=0.466 min, m / z=169.0(M-55).

[0823] LCMS: RT=0.469 min, m / z=169.0(M-55).

[0824] HPLC: RT=1.672 min, purity 73.1% at 220 nm.

[0825] General procedure for the preparation of intermediate 43 [ka] PtO2 (166 mg, 731 μmol, 0.20 equiv.) was added to R1 (50.0 mL) at 20-25 °C under Ar2.

[0826] Rh (827 mg, 402 μmol, 811 μL, 5% purity, 0.11 equiv.) was added to R1 at 20-25 °C under Ar2.

[0827] AcOH (11.0 mL) was added to R1 at 20-25°C under Ar2.

[0828] Intermediate 42 (820 mg, 3.66 mmol, 1.00 equiv) was added at 20-25 °C under Ar.

[0829] The suspension was degassed under vacuum and purged with H2 several times.

[0830] The mixture was stirred in a 50 mL autoclave at 4.0 MPa and 75°C for 16 hours.

[0831] A sample was taken for LCMS. Intermediate 42 (RT=0.484 min) was not completely consumed and the target peak was detected (RT=0.352 min).

[0832] Cool to 20-25°C.

[0833] The reaction mixture was filtered and concentrated in vacuo to give intermediate 43. The crude product was purified by column chromatography (SiO, ethyl acetate:petroleum ether=20 / 1, R f =0.30) to give Intermediate 43 (600 mg, 2.50 mmol, 34.2% yield, 96.0% purity) as a yellow solid, which was confirmed by LCMS and HPLC.

[0834] LCMS: RT=0.353 min, m / z=231.2(M+H + ).

[0835] LCMS: RT=0.381 min, m / z=231.2(M+H + ).

[0836] HPLC: RT=0.758 min, purity 96.6% at 220 nm.

[0837] General procedure for the preparation of intermediate 44 [ka] n-BuOH (12.0 mL) was added to R1 (50.0 mL) at 20 to 25°C.

[0838] Intermediate 43 (470 mg, 2.04 mmol, 1.00 equiv) was added to R1 at 20-25 °C.

[0839] Intermediate 36_a (365 mg, 2.17 mmol, 1.06 equiv) was added to R1 at 20-25 °C.

[0840] The mixture was stirred at 120°C for 10 hours.

[0841] LCMS analysis showed that intermediate 43 was completely consumed and intermediate 44 (RT=0.413 min) was detected.

[0842] The reaction mixture was concentrated in vacuo to give Intermediate 44 (910 mg, crude) as a yellow solid, confirmed by LCMS.

[0843] LCMS: RT=0.413 min, m / z=363.1(M+H + ).

[0844] LCMS: RT = 2.126 min, m / z (M+H + )=363.1(M+H + ).

[0845] General procedure for the preparation of compound 065 [ka] Intermediate 44 (1.00 g, 2.76 mmol, 1.00 equiv) was added to R1 (50.0 mL) at 20-25 °C.

[0846] To R1 was added EtOAc (10.00 mL) at 20-25°C.

[0847] HCl / EtOAc (4 M, 2.76 mL, 4.00 equiv) was added to R1 at 20-25 °C.

[0848] The mixture was stirred at 35 to 40°C for 4 hours.

[0849] A sample was taken for LCMS and intermediate 44 was completely consumed and the target peak was detected (RT=0.270 min).

[0850] The reaction mixture was cooled to 20-25°C.

[0851] The reaction mixture from notebook page EC8946-43 was combined with EC8946-42 for workup.

[0852] The reaction mixture was filtered and the filter cake was washed with EtOAc (10.0 mL*2).

[0853] The filter cake was concentrated in vacuo to give the crude product.

[0854] The crude product was triturated with MeOH: EtOAc = 1:2 (10V) for 0.5 h at 20-25 °C. The mixture was filtered and concentrated in vacuo to give compound 065 (700 mg, 2.25 mmol, 66.6% yield, 95.9% purity, HCl) as a white oil, which was analyzed by LCMS, HPLC, and HPLC. 1 Confirmed by 1 H NMR.

[0855] LCMS: RT=0.270 min, m / z=263.1(M+H) + .

[0856] LCMS: RT=0.285 min, m / z=263.1(M+H) + .

[0857] LCMS: RT=6.697 min, m / z=263.3(M+H) + .

[0858] HPLC: RT = 1.031 min, purity 95.9% at 220 nm.

[0859] 1 H NMR (400MHz, MeOD): δ8.50(s, 1H), 8.32(s, 1H), 4.02(s, 1H), 3.94(s, 3H), 3.57-3.72( m, 3H), 3.33-3.49(m, 1H), 2.91-3.26(m, 1H), 2.00-2.10(m, 1H), 1.13(d, J=6.8Hz, 3H).

[0860] Example 20 - Preparation of Compound 66 General procedure for the preparation of intermediate 46 [ka] DMF (35.0 mL) was added to a 100 mL three-neck round-bottom flask (R1) at 20 to 25°C.

[0861] Intermediate 45_1 (1.44 g, 13.4 mmol, 1.37 mL, 1.00 equivalent) and intermediate 45 (3.00 g, 13.4 mmol, 1.00 equivalent) were added to R1 at 20-25 °C.

[0862] K3PO4 (2.86 g, 13.4 mmol, 1.00 equivalent) was added to R1 at 20-25 °C.

[0863] The mixture (R1) was slowly heated with stirring, and the temperature was raised to 20-25°C over 4 hours.

[0864] LC-MS showed that intermediate 45 was completely consumed and intermediate 46 was detected (RT=0.520 min).

[0865] The mixture was poured into water (100 mL) at 20-25°C. The mixture was extracted with ethyl acetate (100 mL*3) and the organic phase was collected at 20-25°C. The organic layer was washed with brine (100 mL*2). The organic layer was dried over anhydrous Na2SO4. The filtrate was concentrated in vacuo at 35-45°C.

[0866] The residue was purified by preparative HPLC (neutral conditions, 330 g flash column Welch Ultimate XB_C18 20-40 μm, 120 A, gradient B%: 10-60%, 25 min) to give intermediate 46 (1.80 g, 6.10 mmol, 45.2% yield, 86.8% purity) as a yellow oil, which was confirmed by LCMS and HPLC.

[0867] LCMS: RT=0.520 min, m / z=257.1(M+H) + .

[0868] LCMS: RT=0.512 min, m / z=257.1(M+H) + .

[0869] HPLC: RT=1.908 min, purity 86.8% at 220 nm.

[0870] General procedure for the preparation of intermediate 47 [ka] EtOH (75.0 mL) was placed in a 250 mL three-neck round-bottom flask (R1) under N2 at 20-25 °C.

[0871] Intermediate 46 (1.80 g, 6.10 mmol, 86.8% purity, 1.00 equiv.) was charged to R1 at 20-25 °C under N2.

[0872] The mixture (R1) was cooled to 0-5°C.

[0873] NaBH4 (1.09 g, 28.8 mmol, 4.72 equiv) was charged to R1 at 0-5 °C under N2.

[0874] The mixture (R1) was stirred at 20 to 25°C for 3 hours.

[0875] TLC (petroleum ether:ethyl acetate=0:1) showed that intermediate 46 was completely consumed (R f= 0.6), indicating that two new spots were formed (R f = 0.4 and R f =0.3). The reaction was clean by TLC.

[0876] The mixture was poured into saturated aqueous ammonium chloride solution (300 mL) at 0-10 °C. The mixture was extracted with EtOAc (100 mL*3) and the organic phase was collected at 20-25 °C. The organic layer was washed with brine (100 mL*2). The organic layer was separated and dried over anhydrous MgSO4. The filtrate was concentrated under vacuum at 35-45 °C to give Intermediate 47 (1.60 g, crude) as a yellow oil, which was confirmed by LCMS.

[0877] LCMS: RT=2.365, 2.683 min, m / z=259.2(M+H) + .

[0878] General procedure for the preparation of intermediate 48 [ka] DCM (20.0 mL) was added to a 100 mL three-neck round-bottom flask (R1) at 20 to 25°C.

[0879] Intermediate 47 (1.60 g, 6.19 mmol, 1.00 equiv) was charged to R1 at 20-25°C.

[0880] The mixture (R1) was cooled to 0-5°C.

[0881] TFA (21.1 g, 185 mmol, 13.7 mL, 30.0 equiv) was charged to R1 at 0-5°C.

[0882] The mixture (R1) was stirred at 20 to 25°C for 3 hours.

[0883] TLC analysis (petroleum ether:ethyl acetate=0:1) showed that intermediate 47 was completely consumed (R f= 0.4 and 0.3), intermediate 48 was produced (R f =0.25).

[0884] The filtrate was concentrated under vacuum at 35-45° C. to give Intermediate 48 (900 mg, crude) as a yellow oil.

[0885] General procedure for the preparation of compound 066 [ka] Intermediate 48 (900 mg, 5.69 mmol, 1.00 equiv) and EtOH (10.0 mL) were placed in a 100 mL round-bottom flask (R1) at 20-25 °C.

[0886] Intermediate 2 (1.01 g, 5.69 mmol, 95.3% purity, 1.00 equivalents) and DIEA (2.94 g, 22.7 mmol, 3.96 mL, 4.00 equivalents) were added to R1 at 20-25 °C.

[0887] The mixture (R1) was stirred at 80 to 85°C for 10 hours.

[0888] LC-MS showed that intermediate 48 was completely consumed and two main peaks with the target mass were detected (RT1=1.820 min and RT2=2.053 min).

[0889] The mixture was poured into water (100 mL) at 0-10 °C. The mixture was extracted with EtOAc (50.0 mL*3) and the organic phase was collected at 20-25 °C. The organic layer was washed with brine (50.0 mL*2). The organic layer was dried over anhydrous Na2SO4. The filtrate was concentrated in vacuo at 35-45 °C.

[0890] The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 8 / 1, R f = 0.5 and R f =0.4). The racemic product (1.30 g) was separated by preparative chiral SFC (column: DAICEL CHIRALCEL OJ (250 mm * 30 mm, 10 μm), mobile phase: [0.1% NH3H2O ​​IPA], B%: 15%-15%, 0 min) to give compound 066 (500 mg, 1.70 mmol, 37.9% yield, 98.4% purity) as an off-white solid. 1 Confirmed by 1 H NMR.

[0891] LCMS: RT=1.820, 2.053 min, m / z=291.0(M+H)+.

[0892] LCMS: RT=0.557 min, m / z=291.0(M+H) + .

[0893] HPLC: RT=1.943 min, purity 98.4% at 220 nm.

[0894] 1H NMR: (400MHz, DMSO-d6): δ8.24(s, 1H), 8.13(s, 1H), 4.24-4.23(m, 5H), 3.72(s, 3H), 3.54-3.51(m, 1H) , 2.67-2.66(m, 2H), 2.47-2.43(m, 2H), 2.35-2.32(m, 1H), 1.05(d, J=6.0Hz, 3H), 0.86(d, J=6.4Hz, 3H).

[0895] Example 21 - Preparation of Compound 68 General procedure for the preparation of intermediate 51 [ka] Intermediate 2 (1.50 g, 8.18 mmol, 91.8% purity, 1.00 equivalents) and DMF (15.0 mL) were placed in a 100 mL three-neck round-bottom flask (R1) at 20-25 °C.

[0896] Pd(PPh3)4 (472 mg, 408 μmol, 0.05 equiv.) was charged into R1 at 20-25 °C under N2.

[0897] Zn(CN)2 (1.18 g, 10.0 mmol, 637 μL, 1.23 equiv.) was charged to R1 at 20-25 °C under N2.

[0898] The solution was degassed and purged with N2 three times.

[0899] The reaction mixture was stirred at 130° C. for 3 hours.

[0900] LC-MS showed that intermediate 2 was completely consumed and one main peak with the target mass was detected (RT=1.194 min).

[0901] The reaction mixture was cooled to 25°C. The mixture (R1) was poured into H2O (20.0 mL) and ethyl acetate (20.0 mL) at 20-25°C. The insoluble material was removed by filtration through a Celite pad. The aqueous layer was separated and extracted with ethyl acetate (30 mL*3). The combined organic layers were washed with brine (50.0 mL), dried over Na2SO4, filtered, and concentrated.

[0902] The residue was purified by preparative HPLC (basic conditions, 80 g flash column Welch Ultimate XB_C18 20-40 μm, 120 A, gradient B%: 33%, 22 min) to give intermediate 51 (490 mg, 2.56 mmol, 31.2% yield, 83.0% purity) as a white solid, which was confirmed by LCMS and HPLC.

[0903] LCMS: RT=1.194 min, m / z=160.1(M+H) + .

[0904] LCMS: RT=1.192 min, m / z=160.1(M+H) + .

[0905] HPLC: RT=1.255 min, purity 81.8% at 220 nm.

[0906] General procedure for the preparation of intermediate 52 [ka] A 250 mL three-neck round-bottom flask (R1) was charged with EtOH (75.0 mL) under N2 at 20-25 °C.

[0907] ACN (20.0 mL) was added to a 100 mL three-neck round-bottom flask (R1) at 20 to 25°C.

[0908] Intermediate 51 (920 mg, 4.99 mmol, 86.3% purity, 1.00 equiv.) was charged to R1 at 20-25 °C under N2.

[0909] N2H4.H2O (3.12 g, 52.9 mmol, 3.03 mL, 85.0% purity, 10.6 equivalents) was charged into R1 at 20-25 °C under N2.

[0910] The solution was degassed and purged with N2 three times.

[0911] The reaction mixture was stirred at 20° C. for 3 hours.

[0912] LCMS analysis showed that intermediate 51 was completely consumed and intermediate 52 (RT=0.591 min) was detected.

[0913] The solution was filtered and washed with ACN (20.0 mL). The filter cake was concentrated under vacuum at 30-40° C. to give Intermediate 52 (800 mg, crude) as a yellow solid, confirmed by LCMS.

[0914] LCMS: RT=0.591 min, m / z=192.1(M+H) + .

[0915] LCMS: RT=0.590 min, m / z=192.1(M+H) + .

[0916] General procedure for the preparation of compound 68 [ka] HCOOH (10.0 mL) was added to a 100 mL three-neck round-bottom flask (R1) at 20-25°C.

[0917] The mixture was cooled to 0-5°C.

[0918] Intermediate 52 (750 mg, 3.92 mmol, 1.00 equiv.) was slowly added dropwise to R1 at 0-5°C under N2.

[0919] The solution was degassed and purged with N2 three times.

[0920] The reaction mixture was stirred at 110° C. for 3 hours.

[0921] LC-MS (EC8860-42-P1A6) showed that intermediate 52 was completely consumed and one main peak with the target mass was detected (RT=0.329 min).

[0922] The mixture was poured into 50% NaOH and the pH was adjusted to 7-8. It was extracted three times with ethyl acetate (20.0 mL*4) and saturated brine. The combined organic layer was dried over Na2SO4. The filtrate was concentrated in vacuo at 35-45 °C.

[0923] The residue was purified by preparative HPLC (column: YMC Triart C1870*250mm*7μm, mobile phase: [water (NH4HCO3)-ACN], B%: 0%-30%, 15 min). The residue was purified by preparative HPLC (column: Phenomenex luna C18250*50mm*15μm, mobile phase: [water (FA)-ACN], B%: 0%-18%, 20 min) to obtain compound 068 (510mg, 2.26mmol, yield 13.5%, purity 94.1%) as a white solid. 1 Confirmed by 1 H NMR.

[0924] LCMS: RT=0.329 min, m / z=202.0(M+H)+.

[0925] LCMS: RT=0.192 min, m / z=201.8(M+H) + .

[0926] HPLC: RT = 0.928 min, purity 94.1% at 220 nm.

[0927] 1 H NMR: (400MHz, DMSO-d6): δ15.2-14.4(m,1H), 9.02(s,1H), 8.81-8.65(m,2H), 3.88(s,3H).

[0928] Example 22 - Preparation of Compound 69 General procedure for the preparation of intermediate 54 [ka] A 2.0 L three-necked round-bottom flask (R1) was charged with THF (200 mL) and diisopropylamine (18.5 g, 183 mmol, 25.9 mL, 1.05 equiv.) at 25°C.

[0929] Degassing and N2 purging was performed three times.

[0930] n-BuLi (2.50 M, 73.3 mL, 1.05 equiv.) was added dropwise in an ice-water bath under N2 at 0-5°C, and the mixture was then reacted under N2 at 0-5°C for 10 min.

[0931] SnHBu3 (49.7 g, 171 mmol, 45.2 mL, 9.82 e-1 equivalent) was added dropwise under N2 at 0-5 °C in a water bath, and the reaction was continued for 20 min under N2.

[0932] The mixture was cooled to -70 to -65 °C, and compound 53 (20.0 g, 174 mmol, 15.6 mL, 1.00 equiv.) was dissolved in THF (400 mL), and the mixture was N2 The solution was slowly added dropwise to R1 at -70 to -65°C.

[0933] The mixture was stirred under N2 at -70 to -65 °C for 8 h.

[0934] As a result of LCMS analysis, 38.6% of intermediate 53 (RT=0.164 min) remained, and the target compound (RT=0.660 min) was detected.

[0935] The temperature was raised to -40°C, and an aqueous potassium fluoride solution (400 mL) was added dropwise at -40 to -35°C to quench the reaction.

[0936] The mixture was stirred at 10-20°C for 0.5 hours.

[0937] The reaction mixture was filtered, extracted with ethyl acetate (400 mL*3) at 25 °C, dried over sodium sulfate, filtered, and the filtrate was concentrated and dried under reduced pressure. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 0-15%, petroleum ether / ethyl acetate = 3 / 1, product: RF = 0.35) to give intermediate 54 (3.00 g, 7.17 mmol, yield 4.10%, purity 88.2%) as a yellow oil, which was confirmed by LCMS.

[0938] LCMS: RT=0.660 min, m / z=370.9(M+H) + .

[0939] LCMS: RT=0.660 min, m / z=370.9(M+H) + .

[0940] General procedure for the preparation of compound 069 [ka] Intermediate 54 (3.10 g, 7.41 mmol, 88.2% purity, 1.00 equiv.) and DMF (31.0 mL) were charged into a 100 mL three-neck round-bottom flask (R1) at 25 °C.

[0941] Intermediate 2 (2.87 g, 8.15 mmol, 73.8% purity, 1.10 equiv.) and KPO (4.72 g, 22.2 mmol, 3.00 equiv.) were charged to R1 at 25 °C.

[0942] Pd(OAc)2 (166 mg, 740 μmol, 0.10 equiv) was charged to R1 at 25 °C under N2.

[0943] The reaction was stirred at 95-100°C for 16 hours.

[0944] LCMS analysis showed that intermediate 54 had been consumed and the target mass was detected (RT=0.189 min).

[0945] Concentrate under reduced pressure at 45-50°C to obtain a residue.

[0946] The residue was purified by column chromatography (SiO2, DCM:MeOH=50 / 1 to 10 / 1, product: DCM:MeOH=10 / 1, product: RF=0.21) to give the crude product.

[0947] The crude product was triturated with MeOH at 20-25 °C for 20 min to give compound 069 (100 mg, 454 μmol, 6.13% yield, 96.4% purity) as a yellow solid, which was analyzed by LCMS, HPLC, and HPLC. 1Confirmed by 1 H NMR.

[0948] LCMS: RT=0.189 min, m / z=213.1(M+H) + .

[0949] LCMS: RT=0.378 min, m / z=213.0(M+H) + .

[0950] HPLC: RT=1.865 min, purity 96.4% at 220 nm.

[0951] 1 H NMR (400MHz, DMSO): δ9.79(d, J=1.2Hz, 1H), 9.10(s, 1H), 8.92-8.91(t, J=2.0Hz, 1H), 8.81(d, J=2.4Hz, 1H), 8.70(s, 1H), 3.91(s, 1H).

[0952] Example 23: Preparation of Compound 70 General procedure for the preparation of intermediate 56 [ka] Intermediate 55 (1.00 g, 4.30 mmol, 1.00 equiv) was placed in a 100 mL three-neck round-bottom flask (R1) at 20-25 °C.

[0953] Intermediate 2 (770 mg, 4.30 mmol, 94.2% purity, 1.00 equivalents) was added to R1 at 20-25 °C.

[0954] DIEA (1.67 g, 12.9 mmol, 2.25 mL, 3.00 equiv.) was added to R1 at 20–25 °C.

[0955] EtOH (10 mL) was added to R1 at 20 to 25°C.

[0956] The mixture was stirred at 70-80°C for 6 hours.

[0957] LCMS showed that intermediate 55 was consumed and intermediate 56 (RT=0.347 min) was detected.

[0958] The mixture was concentrated under vacuum at 45° C. to give compound 56 (2.40 g, crude) as a brown oil, confirmed by LCMS.

[0959] LCMS: RT=0.347 min, m / z=365.0(M+H) + .

[0960] LCMS: RT=0.345 min, m / z=365.4(M+H) + .

[0961] General procedure for the preparation of compound 70 [ka] Intermediate 56 (2.40 g, 6.59 mmol, 1.00 equiv) was charged into a 100 mL stand-up bottle (R1) at 20–25 °C.

[0962] TFA (15.0 g, 131 mmol, 9.75 mL, 20.0 equiv.) was added to R1 at 20–25 °C.

[0963] The mixture was stirred at 20-25°C for 4 hours.

[0964] LCMS showed that intermediate 56 was consumed and the target compound was detected (RT=0.610 min).

[0965] The mixture was poured into saturated NaOH (10.0 mL).

[0966] It was filtered and the filter cake was washed with H2O (30.0 mL).

[0967] The aqueous phase was washed with DCM:MeOH=10:1 (80.0 ml*4).

[0968] The organic phase was washed with saturated brine and dried over Na2SO4.

[0969] The mixture was concentrated under vacuum at 45 °C, and the crude product was purified by reverse-phase HPLC (0.1% NH3·H2O) and lyophilized.

[0970] The crude product was triturated with EtOAc (30.0 mL) at 20-25 °C for 30 min and dried on a filter cake at 45 °C under vacuum to give compound 070 (631 mg, 2.35 mmol, 35.6% yield, 98.2% purity) as a white solid, which was purified by LCMS, HPLC, and FT-IR. 1 H NMR, and 19 This was confirmed by F NMR.

[0971] LCMS: RT=0.610 min, m / z=265.4(M+H) + .

[0972] LCMS: RT=0.605 min, m / z=265.2(M+H) + .

[0973] HPLC: RT=2.829 min, purity 98.2% at 220 nm.

[0974] 19F NMR (400MHz, DMSO-d6)

[0975] 1 H NMR (400MHz, DMSO-d6): δ8.22(brs, 1H), 8.12(s, 1H), 7.65(brs, 1H), 5.75(s, 1H), 2.81-2.72(m, 2H), 2.71-2.60(m, 2H), 1.81-1.40(m, 4H).

[0976] Example 24 - Preparation of Compound 71 General procedure for the preparation of compound 071 [ka] Intermediate 2_B (1.50 g, 8.01 mmol, 90.0% purity, 1.00 equiv.) and intermediate 60 (794 mg, 8.01 mmol, 939 μL, 1.00 equiv.) were placed in a 10 mL one-neck round-bottom flask (R1) in EtOH (3 mL) at 20–25 °C.

[0977] DIEA (3.10 g, 24.0 mmol, 4.18 mL, 3.00 equivalents) was added to R1 at 20-25°C.

[0978] The mixture was heated to 80°C and stirred at 80°C for 12 hours.

[0979] LCMS showed that intermediate 2_B was completely consumed and the target product was detected (RT=1.653 and 1.669 min).

[0980] The mixture was concentrated and purified by preparative HPLC (column: Phenomenex luna C18250*50mm*15μm, mobile phase: [water (NH4HCO3)-ACN], B%: 15%-45%, 20 min), and the eluate was lyophilized to obtain compound 071 (743 mg, 3.21 mmol, yield 40.1%, purity 100%) as a yellow solid. 1 This was confirmed by 1 H NMR and Nuclear Overhauser Effect (NOE) NMR.

[0981] LCMS:RT=1.653min, 232.1(M+H) + .

[0982] LCMS:RT=0.248min, 232.1(M+H) + .

[0983] 1 H NMR / NOE (400MHz, CDCl3): δ8.63(s, 1H), 7.96(s, 1H), 4.01(s, 3H), 3.79-3.64(m, 2H), 2.94(dt, J=2.8, 12.4Hz, 1H), 2.64(dd, J=10.8, 12.4Hz, 1H), 2.10(s, 2H), 1.89-1.81(m, 2H), 1.78-1.68(m, 1H), 1.16(dq, J=4.0, 12.0Hz, 1H), 0.97(d, J=6.4Hz, 3H).

[0984] Example 25: Preparation of Compound 158 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (3.00 g, 10.5 mmol, 1.00 equiv) was charged to R1 at 20°C.

[0985] DMF (30.0 mL) was added to R1 at 20°C.

[0986] CuCN (1.99 g, 22.2 mmol, 4.86 mL, 2.10 equiv) was charged to R1 at 20 °C.

[0987] The mixture was stirred at 145° C. under N 2 for 12 h.

[0988] TLC (petroleum ether:ethyl acetate=8:1) showed that intermediate 1 was consumed (R f =0.70), and intermediate 2 was detected (R f =0.01, 0.11, 0.21, 0.31).

[0989] After cooling to 20° C., the mixture was poured into NaClO (50.0 mL).

[0990] Extraction was carried out with ethyl acetate (50.0 mL*3).

[0991] Washed with brine (50.0 mL*2), dried over Na2SO4, filtered and concentrated in vacuo.

[0992] The crude product was purified by silica gel chromatography (silica gel, petroleum ether / ethyl acetate = 8 / 1, R f =0.21) to give intermediate 2 (1.88 g, 8.20 mmol, 77.4% yield, 100% purity) as a yellow gum, which was analyzed by LCMS and 1 Confirmed by 1 H NMR.

[0993] LCMS: RT=0.442 min, m / z=230.1(M+H)+.

[0994] 1H NMR: (400MHz, DMSO): δ7.55-7.52(m, 2H), 7.33-7.31(m, 1H), 4.13-4.08(m, 2H), 3.0 6-2.78(m, 5H), 2.08-2.07(m, 1H), 1.78-1.75(m, 1H), 1.20(t, J=7.2Hz, 14.0Hz, 3H).

[0995] General procedure for the preparation of intermediate 3 [ka] THF (20.0 mL) was placed in a 100 mL stand-up flask (R1) at 20°C.

[0996] Intermediate 2 (1.88 g, 8.20 mmol, 1.00 equiv) was charged to R1 at 20°C.

[0997] Tetrabutylammonium acetate (2.47 g, 8.20 mmol, 2.50 mL, 1.00 equiv) was charged to R1 at 20°C.

[0998] TMSN3 (3.78 g, 32.8 mmol, 4.31 mL, 4.00 equiv) was charged to R1 at 20°C.

[0999] The mixture was stirred at 60° C. for 16 hours.

[1000] LCMS showed intermediate 2 remained (RT=0.444 min), but intermediate 3 was detected (RT=0.389 min).

[1001] The mixture was poured into water (80.0 mL) and extracted with ethyl acetate (80.0 mL*3). The organic phases were combined, washed with saturated brine (80.0 mL*2), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography (silica gel, dichloromethane:methanol = 10 / 1, R f =0.52) to give Intermediate 3 (479 mg, 1.75 mmol, 21.3% yield, 99.6% purity) as a white solid, which was confirmed by LCMS.

[1002] LCMS: RT=0.389 min, m / z=273.1(M+H) + .

[1003] LCMS: RT=0.389 min, m / z=273.1(M+H) + .

[1004] General procedure for the preparation of intermediate 4 [ka] Intermediate 3_1 (1.12 g, 9.63 mmol, 1.29 mL, 5.00 equiv.) was charged into a 50.0 mL stand-up flask (R1) at 20°C.

[1005] THF (5.50 mL) was charged to R1 at 20°C.

[1006] After cooling to −78°C, the mixture was degassed with N 2 three times.

[1007] LiHMDS (1 M, 9.63 mL, 5.00 equiv.) is added dropwise to R1 at -78 °C.

[1008] The mixture was stirred under N2 at -78 °C for 0.5 h.

[1009] A solution of intermediate 3 (530 mg, 1.93 mmol, 1.00 equiv) in THF (3.00 mL) was added dropwise to R1 at -78°C.

[1010] The mixture was stirred at -78°C for 2 hours.

[1011] LCMS showed that intermediate 3 was consumed and intermediate 4 was detected (RT=0.426 min).

[1012] The mixture was added dropwise to NH4Cl (30.0 mL) and extracted with ethyl acetate (15.0 mL*3), and the combined organic layers were washed with brine (30.0 mL), dried over Na2SO4, filtered and concentrated.

[1013] The crude product was purified by preparative HPLC (column: Phenomenex Luna C18200*40mm*10μm, mobile phase: [water (FA)-ACN], gradient: 33% to 63% B in 10 min) to give intermediate 4 (400 mg, 1.10 mmol, yield 57.1%, purity 94.2%) as a white solid, which was confirmed by LCMS.

[1014] LCMS: RT=0.426 min, m / z=343.2(M+H) + .

[1015] LCMS: RT=0.418 min, m / z=343.0(M+H) + .

[1016] General procedure for the preparation of PX158 [ka] Intermediate 4 (500 mg, 1.38 mmol, 1.00 equiv) was charged into R1 (10.0 mL flask) at 20 °C.

[1017] HCl / EtOAc (4 M, 5.00 mL, 14.5 equiv) was charged to R1 at 20°C.

[1018] The mixture was stirred under N2 at 20°C for 4 hours.

[1019] LCMS showed that intermediate 4 remained (RT=0.663 min) but the target mass was detected (RT=0.433 min).

[1020] The reaction mixture was concentrated under reduced pressure to give a residue, to which was added aqueous K2CO3 solution (2 mL) to form a solution.

[1021] The crude product was purified by preparative HPLC (column: Waters xbridge 150*25mm 10μm, mobile phase: [water (NH4HCO3)-ACN], gradient: 1% to 20% B in 10 min) to give compound 158 (210 mg, 708 μmol, yield 51.4%, purity 96.3%) as a white solid. 1Confirmed by 1 H NMR, LCMS, HPLC, and DSC.

[1022] LCMS: RT=0.433 min, m / z=286.9(M+H) + .

[1023] LCMS: RT=0.435 min, m / z=286.9(M+H) + .

[1024] HPLC: RT=1.075 min, purity 96.3% at 220 nm.

[1025] 1 H NMR: (400MHz, DMSO): δ7.68-7.66(m, 2H), 7.23(d, J=7.60Hz, 1H), 3.58-3.50(m, 1H), 3.03-2.80(m, 5H), 2.16-2.12(m, 1H), 1.76-1.66(m, 1H).

[1026] Example 26 - Preparation of Compound 174 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (2.00 g, 10.6 mmol, 1.00 equiv), Intermediate 1b (3.36 g, 10.6 mmol, 1.00 equiv), TsOH (182 mg, 1.06 mmol, 0.10 equiv) were added to EtOAc (20.0 mL).

[1027] The mixture was stirred in a microwave reactor at 80° C. for 1 hour.

[1028] TLC (petroleum ether:ethyl acetate=2:1) ​​confirmed that compound 1 (R f =0.10) is completely consumed, and the main spot (R f =0.50) was formed.

[1029] The mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 100:1 to 2:1, R f=0.50 (petroleum ether:ethyl acetate=2:1)) to give Intermediate 2 (1.20 g, 2.68 mmol, 25.3% yield) as a white solid.

[1030] General procedure for the preparation of PX173 [ka] Intermediate 2 (1.20 g, 2.68 mmol, 1.00 equiv) was dissolved in NH3 / MeOH (12.0 mL).

[1031] The mixture was stirred at 100°C for 24 hours.

[1032] TLC (petroleum ether:ethyl acetate=2:1) ​​revealed intermediate 2 (R f =0.50) is completely consumed, and the main spot (R f =0.10) was formed.

[1033] The mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase HPLC (0.1% HCl) to give compound 173 (557.91 mg, 1.74 mmol, 64.7% yield, 100% purity) as a white solid. 1 Confirmed by 1 H NMR.

[1034] LCMS: RT=0.428 min, m / z (M+23) + =343.0.

[1035] HPLC: RT=1.440 min, 100% purity at 220 nm.

[1036] 1H NMR: (400MHz, DMSO-d6): δ8.98(s, 1H)5.97(d, J=5.2Hz, 1H)5.59(d, J=6.0Hz, 1H)5.26(d, J=5.6Hz, 1H)5.08(t, J=5.2Hz, 1H) )4.51(dd, J=10.4, 5.2Hz, 1H)4.17(dd, J=10.0, 4.8Hz, 1H)3.98(dd, J=8.0, 4.0Hz, 1H)3.75-3.67(m, 1H)3.63-3.55(m, 1H).

[1037] Example 27 - Preparation of Compound 174 General procedure for the preparation of compound 1 [ka] Intermediate 1a (4.00 g, 25.2 mmol, 1.00 equiv) was added to Intermediate 1b (60.0 mL) and HCOOH (1.00 mL).

[1038] The mixture was stirred under N2 at 120°C for 5.5 hours.

[1039] LCMS showed that intermediate 1a was consumed and the target mass was detected (RT=0.158 min).

[1040] The mixture was filtered and the filtrate was concentrated to give Intermediate 1 (3.40 g, 20.1 mmol, 79.9% yield) as an off-white solid.

[1041] LCMS: RT=0.158 min, m / z (M+H) + =168.9.

[1042] General procedure for the preparation of compound intermediate 2 [ka] Intermediate 1 (500 mg, 2.97 mmol, 1.00 equiv) was charged into a 50 mL stand-up flask (R1) at 20 °C.

[1043] Intermediate 2 (944 mg, 2.97 mmol, 1.00 equiv) was charged to R1 at 20°C.

[1044] Dioxane (15.0 mL) was charged to R1 at 20°C.

[1045] Add SnCl4 (773 mg, 2.97 mmol, 347 μL, 1.00 equiv.) dropwise to R1 at 0 °C.

[1046] The mixture was stirred at 20° C. for 12 hours.

[1047] LCMS showed that intermediate 1 remained (RT=0.202 min), but intermediate 3 was detected (RT=0.371 min).

[1048] The mixture was poured into water (50.0 mL).

[1049] Extraction was carried out with EtOAc (50.0 mL*3).

[1050] The organic phase was washed with brine (50.0 mL*2), dried over Na2MgSO4, filtered and concentrated in vacuo. The crude product was purified by preparative TLC (ethyl acetate:petroleum ether=2:1, R f= 0.4) to give Intermediate 3 (1.4 g, 3.15 mmol, 53.09% yield, 96% purity) as a yellow gum, which was confirmed by LCMS.

[1051] LCMS: RT=0.371 min, m / z=427.1(M+H) + .

[1052] LCMS: RT=0.363 min, m / z=426.9(M+H) + .

[1053] General procedure for the preparation of compound 174 [ka] Intermediate 3 (1.20 mg, 2.70 mmol, 1.00 equiv) was charged into a 50 mL stand-up flask (R1) at 20 °C.

[1054] NH3 / MeOH (7 M, 23.0 mL, 59.8 equiv) was charged to R1 at 20 °C.

[1055] The mixture was stirred at 20° C. for 12 hours.

[1056] LCMS (EC13849-3-p1a4) showed that intermediate 3 had been consumed and compound 174 was detected (RT=0.306 min), after which it was concentrated in vacuo.

[1057] The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*25mm*10μm, mobile phase: [water (FA)-ACN], gradient: 0% to 25% B in 10 min) to give compound 174 (333 mg, 1.09 mmol, yield 40.5%, purity 98.6%) as a white solid. 1 Confirmed by 1 H NMR.

[1058] LCMS: RT=0.306 min, m / z=301.1(M+H) + .

[1059] LCMS: RT=0.294 min, m / z=301.1(M+H) + .

[1060] HPLC: RT=1.272 min, purity 98.6% at 220 nm.

[1061] 1 H NMR (400MHz, DMSO): δ8.84(s, 1H), 6.00(d, J=5.6Hz, 1H), 5.53(d, J=6.0Hz, 1H), 5.26(d, J=5.2Hz, 1H), 5. 10-5.13(m, 1H), 4.56-4.58(m, 1H), 4.17-4.19(m, 1H), 3.98-3.99(m, 1H), 3.58-3.69(m, 2H), 2.69(s, 3H).

[1062] Example 28 - Preparation of Compound 175 General procedure for the preparation of intermediate 2 [ka] A 100 mL three-neck flask was equipped with a stirrer, an additional nitrogen ball, and a thermometer.

[1063] Intermediate 1b (14.4 g, 37.0 mmol, 1.00 equiv) and EtOAc (70 mL) were charged to a flask.

[1064] The flask was warmed to 100°C.

[1065] Intermediate 1 (7.00 g, 37.0 mmol, 1.00 equiv) was charged to a flask at 100°C.

[1066] SnCl4 (192 mg, 740 μmol, 86.6 μL, 0.02 equiv.) was charged into the flask at 100 °C.

[1067] The mixture was stirred under N2 at 100°C for 2 hours.

[1068] LCMS (EC12863-2-P1B1) showed that compound 1 was consumed and the target mass was detected (RT=0.396 min).

[1069] The mixture was filtered and the filtrate was concentrated to give a residue.

[1070] The residue was triturated with petroleum ether (200 mL) and ethyl acetate (10.0 mL) for 30 min at 0° C. The crude product was purified by reverse phase (0.1% HCl) to give compound 2 (2.00 g, 3.85 mmol, 10.4% yield) as a yellow solid.

[1071] LCMS:EC12863-2-P1B1, RT=0.396 min, m / z(M+H) + =518.9

[1072] 1H NMR (400MHz, DMSO): δ9.08(s, 1H), 6.36(d, J=9.2Hz, 1H), 5.79(t, J=9.2Hz, 1H), 5.66(d, J=9.6Hz, 1H), 5.24( t, J=10.0Hz, 1H), 4.47-4.41(m, 1H), 4.11(d, J=4.0Hz, 2H), 2.04(s, 3H), 1.99(d, J=7.2Hz, 6H), 1.72(s, 3H).

[1073] General procedure for the preparation of compound 175 [ka] CD-HLE-97 (0.80 g, 200% wt / wt) in buffer (20.0 mL) (0.1 M sodium phosphate solution, pH = 7.0) was charged into a reaction flask (R1).

[1074] Intermediate 2 (400 mg, 770.28 μmol, 1.00 equiv) in DMSO (2.0 mL) was charged to R1.

[1075] The mixture was stirred at 35° C. for 12 hours.

[1076] LCMS (EC6017-222-P1A2) showed the reactants were completely consumed and product was detected (RT=0.685 min).

[1077] The mixture was poured into water (30.0 mL).

[1078] Extraction was carried out with EtOAc (30.0 mL*3).

[1079] The organic phase was washed with brine (30.0 mL*2), then dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, ethyl acetate:petroleum ether=3:1, R f =0.30) to give compound 175 (136.09 mg, 314.8 μmol, purity 70.5%) as a yellow solid, which was analyzed by LCMS, HPLC, and 1 Confirmed by 1 H NMR.

[1080] LCMS: RT=0.196 min, m / z=188.8(M+H) + .

[1081] HPLC: RT=1.193 min, purity 70.5% at 220 nm.

[1082] 1 H NMR (400MHz, DMSO): δ8.98(s, 1H), 5.52(d, J=9.6Hz, 1H), 5.36-5.42(m, 2H), 5.22(d, J= 5.6Hz, 1H) 4.60-4.62(m, 1H), 3.96-3.97(m, 1H), 3.71-3.72(m, 1H), 3.27-3.49(m, 4H).

[1083] Example 29 - Preparation of Compound 178 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (2.00 g, 11.7 mmol, 1.87 mL, 1.00 equiv), i-PrOH (7.5 mL), Intermediate 2a-1 (2.42 g, 24.6 mmol, 2.42 mL, 2.1 equiv), and NH3 / MeOH (7 M, 20.0 mL, 11.9 equiv) were placed in a 100 mL flask at 20 °C and stirred at 100 °C for 24 h. A sample was taken for TLC analysis, which showed that Intermediate 1 remained, but one major new spot with greater polarity was detected (R f= The mixture was concentrated in vacuo, and the residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 5 / 1) to give Intermediate 2 (1.80 g, 7.60 mmol, 16.1% yield, 93.4% purity) as a white solid.

[1084] General procedure for the preparation of intermediate 3 [ka] Intermediate 2 (4.00 g, 18.0 mmol, 1.0 equiv) and POCl3 (32.9 g, 214 mmol, 20.0 mL, 11.8 equiv) were placed in a 100 mL flask at 20 °C and heated to 100 °C and refluxed for 12 h. A sample was taken for LCMS analysis, which indicated that intermediate 2 had been consumed and 62.7% of intermediate 3 had been detected (RT = 0.411 min). The mixture was poured into ice water (15.0 mL) and extracted with EtOAc (15.0 mL * 3). The combined organic layers were washed with brine (15.0 mL * 3), dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) R f= 0.50 to give Intermediate 3 (1.80 g, 7.30 mmol, 40.3% yield, 97.2% purity) as a white oil, which was confirmed by LCMS.

[1085] LCMS: RT=0.412 min, m / z=240.1(M+H) + .

[1086] LCMS: RT=0.373 min, m / z=240.1(M+H) + .

[1087] General procedure for the preparation of intermediate 4 [ka] Intermediate 3 (1.5 g, 6.26 mmol, 1.00 equiv), DMF (18 mL), Intermediate 13-1 (2.55 g, 31.29 mmol, 5.0 equiv), Pd(OAc) (421 mg, 1.88 mmol, 0.3 equiv), Xantphos (1.09 g, 1.88 mmol, 0.3 equiv), and TEA (5.07 g, 50.06 mmol, 6.97 mL, 8 equiv) were charged into a 100 mL flask (R1) at 20 °C. The mixture was degassed three times with CO (175 mg, 6.26 mmol, 140 μL, 1.0 equiv) and stirred at 90 °C for 12 h. A sample was taken for LCMS analysis which showed that intermediate 3 was consumed and 11.8% of intermediate 4 was detected (RT=0.295 min).

[1088] The mixture was poured into aqueous NH4Cl (15.0 mL) and extracted with EtOAc (15.0 mL*3). The combined organic layers were washed with brine (15.0 mL*3), dried over Na2SO4, filtered, and concentrated. The residue was analyzed by preparative TLC (SiO2, petroleum ether / ethyl acetate = 0:1) R f =0.10 to give intermediate 4 (310 mg, 974 μmol, 15.5% yield, 86.9% purity) as a yellow oil, which was confirmed by LCMS.

[1089] LCMS: RT=0.295 min, m / z=277.1(M+H) + .

[1090] LCMS: RT=0.291 min, m / z=277.1(M+H) + .

[1091] General procedure for the preparation of intermediate 5 [ka] Intermediate 4-1 (546 mg, 4.70 mmol, 631 μL, 5.00 equiv), THF (2.00 mL), and LiHMDS (2.5 M, 1.88 mL, 5.00 equiv) were charged to flask (R1) at −78 °C. The mixture was stirred at −78 °C for 1 h. In a separate flask (R2), intermediate 4 (260 mg, 940 μmol, 1.00 equiv) and THF (3.00 mL) were charged at 20 °C. R2 was then charged to R1 at −78 °C, and the mixture was stirred for 2 h. A sample was taken for LCMS analysis, which showed that intermediate 4 had been consumed and 70.2% of intermediate 5 had been detected (RT = 0.351 min).

[1092] The mixture was poured into aqueous NH4Cl (10.0 mL) and extracted with EtOAc (10.0 mL*3). The combined organic phase was washed with brine (10.0 mL*3), dried over Na2SO4, filtered, and concentrated. The crude product was purified by HPLC (Welch Xtimate C18 150*25 mm*5 um, mobile phase: [water (TFA)-ACN], gradient: 25% to 45% B in 10 min) to give Intermediate 5 (250 mg, crude) as a white oil.

[1093] General procedure for the preparation of intermediate 6 [ka] Intermediate 5 (250 mg, 721 μmol, 1.0 equiv.) and THF (3 mL) were added to a 50 mL flask at 15°C.

[1094] It was purged with nitrogen three times.

[1095] NaBH4 (13.6 mg, 360 μmol, 0.5 equiv) was charged to the flask at 0 °C.

[1096] The mixture was stirred at 0° C. for 1 hour.

[1097] LCMS showed that intermediate 5 was consumed and 35.8% of intermediate 6 (RT=0.312 min) was detected.

[1098] 2.00 mL of brine was charged to the flask at 0°C.

[1099] The mixture was stirred at 0° C. for 1 h.

[1100] The mixture was poured into water (5.00 mL) and extracted with EtoAc (5.00 mL*3).

[1101] The combined organic layers were washed with brine (5.00 mL*3), dried with NaSO, filtered, and concentrated. The product was analyzed by preparative TLC (SiO, petroleum ether / ethyl acetate=0:1, R f=0.20) to give Intermediate 6 (50.0 mg, 116 μmol, 16.1% yield, 81.1% purity) as a white oil, which was confirmed by LCMS.

[1102] LCMS: RT=0.312 min, m / z=349.2(M+H) + .

[1103] LCMS: RT=0.320 min, m / z=349.2(M+H) + .

[1104] General procedure for the preparation of compound 178 [ka] Intermediate 6 (50 mg, 143 μmol, 1.00 equiv) and DCM (1.00 mL) were added to a 50.0 mL flask at 15°C.

[1105] The reaction flask was purged with nitrogen three times.

[1106] TFA (1.15 g, 10.1 mmol, 749 μL, 70.3 equiv) was charged to the flask at 20 °C.

[1107] The mixture was stirred at 20°C for 3 hours.

[1108] LCMS showed that intermediate 6 was consumed and 81.8% of the target mass was detected (RT=0.186 min).

[1109] The mixture was concentrated in vacuo.

[1110] The crude product was purified by HPLC (Welch Xtimate C18 150*25mm*5um; mobile phase: [water (TFA)-ACN], gradient: 0% to 25% B in 10 min) to give compound 178 (40 mg, 135 μmol, yield 94.6%, purity 99.3%) as a white oil. 1 Confirmed by 1 H NMR.

[1111] LCMS: RT = 0.186 min, m / z (M+H + )=293.1.

[1112] LCMS: RT = 0.281 min, m / z (M+H + )=293.2.

[1113] HPLC: Rt = 1.091 min, purity 99.3% at 220 nm.

[1114] 1 H NMR (400MHz, DMSO): δ7.68-7.63(m, 1H), 7.33-7.31(m 1H), 3.87-3.80(m, 2H), 2.98(s, 3H), 2.93(s, 3H), 2.81-2.78(m, 2H), 2.64-2.54(m , 2H), 2.54-2.50(m, 1H), 2.27-2.07(m, 1H), 1.76-1.75(m, 1H), 1.73-1.47(m, 1H).

[1115] Example 30 - Preparation of Compound 180 General procedure for the preparation of intermediate 3 [ka] Intermediate 1 (5.00 g, 22.2 mmol, 1.00 equiv) and THF (75.0 mL) were charged to R1 (500 mL flask) at 20 °C. The mixture was cooled to -78 °C and degassed with N2 three times. LiHMDS (1 M, 66.6 mL, 3.00 equiv) was charged to R1 at -78 °C under N2, and the mixture was stirred at -78 °C under N2 for 1.5 h. Intermediate 2 (6.60 g, 66.6 mmol, 6.53 mL, 3.00 equiv) in THF (7.50 mL) was added dropwise to R1 at -78 °C under N2. The mixture was stirred at 20 °C under N2 for 16 h.

[1116] LCMS (EC14042-18-p1a1) showed that intermediate 1 remained (RT=0.483 min) and intermediate 3 was detected (RT=0.516 min).

[1117] The mixture was added dropwise to NH4Cl (300 mL) and extracted with ethyl acetate (200 mL * 3). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated. The combined crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1, R f =0.58) to give intermediate 3 (5.60 g, 18.1 mmol, 81.8% yield, 96.5% purity) as a yellow oil, which was analyzed by LCMS and 1H Confirmed by NMR.

[1118] LCMS: RT=0.516 min, m / z=297.0(M+H) + .

[1119] LCMS: RT=0.562 min, m / z=296.8(M+H) + .

[1120] 1 H NMR (400MHz, DMSO): δ7.48(d, J=8.40Hz, 1H), 7.35(d, J=1.60Hz, 1H), 7.13(s, 1H), 3.90-3.82(m, 2H), 2.0 8-2.02(m, 2H), 1.87-1.80(m, 1H), 1.71-1.62(m, 1H), 1.59-1.47(m, 1H), 0.91(t, J=7.20Hz, 14.0Hz, 3H).

[1121] General procedure for the preparation of intermediate 4 [ka] Intermediate 3 (4.60 g, 15.4 mmol, 1.00 equiv), TFA (35.0 mL), and EtSiH (10.0 g, 86.3 mmol, 13.8 mL, 5.58 equiv) were charged to R1 (100 mL flask) at 20 °C, and the mixture was stirred under N at 20 °C for 16 h.

[1122] TLC analysis (plate 1, petroleum ether:ethyl acetate=10:1) revealed intermediate 3 (R f= 0.52) was consumed, and intermediate 4 (R f=0.45). The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 5:1, R f =0.65) to give Intermediate 4 (2.50 g, 8.74 mmol, 56.4% yield, 99.0% purity) as a colorless oil. 1 Confirmed by 1 H NMR.

[1123] LCMS: RT=0.482 min, m / z=283.0(M+H) + .

[1124] 1 H NMR (400MHz, DMSO): δ7.27-7.24(m, 2H), 7.06(d, J=8.00Hz, 1H), 4.13-4.13(m, 2H), 2.93-2.88( m, 1H), 2.82-2.70(m, 4H), 2.08-2.04(m, 1H), 1.73-1.68(m, 1H), 1.20(t, J=6.80Hz, 14.0Hz, 3H).

[1125] General procedure for the preparation of intermediate 5 [ka] Intermediate 4 (1.10 g, 3.85 mmol, 1.00 equiv), i-PrOH (10.0 mL), DABSO (554 mg, 2.31 mmol, 0.60 equiv), TEA (1.17 g, 11.5 mmol, 1.61 mL, 3.00 equiv), and Pd(AmPhos)Cl (272 mg, 384 μmol, 272 μL, 0.10 equiv) were charged to R1 (100 mL flask) at 20 °C, and the mixture was stirred at 75 °C under N for 23 h. LCMS analysis indicated that intermediate 4 was consumed and intermediate 5 was detected (RT = 0.417 min). The resulting product was concentrated in vacuo to give intermediate 5 (1.03 g, crude) as a yellow solid, confirmed by LCMS.

[1126] LCMS: RT=0.417 min, m / z=266.8(M+H) + .

[1127] General procedure for the preparation of intermediate 6 [ka] Intermediate 5 (1.03 g, 3.84 mmol, 1.00 equiv), i-PrOH (11.0 mL), and Intermediate 5a (626 mg, 7.68 mmol, 2.00 equiv) were charged to R1 (10.0 mL flask) at 20 °C. The mixture was cooled to 0 °C, and NCS (1.03 g, 7.68 mmol, 2.00 equiv) was added to R1. The mixture was stirred at 0–20 °C under N for 2 h.

[1128] LCMS analysis showed that intermediate 5 was consumed and intermediate 5 was detected (RT=0.436 min).

[1129] The mixture was poured into H2O (60.0 mL) and extracted with ethyl acetate (40.0 mL*3), and the combined organic layers were washed with brine (50.0 mL), dried over Na2SO4, filtered and concentrated.

[1130] The crude product was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 3:1, R f =0.30) to give intermediate 6 (879 mg, 2.33 mmol, 60.8% yield, 82.7% purity) as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[1131] LCMS: RT=0.436 min, m / z=312.1(M+H) + .

[1132] LCMS: RT=0.484 min, m / z=312.0(M+H) + .

[1133] HPLC: RT=1.946 min, purity 82.7% at 220 nm.

[1134] 1H NMR (400MHz, DMSO): δ7.45(d, J=6.00Hz, 1H), 7.39(t, J=3.20Hz, 6.41H), 4.16-4.08(m, 2H), 3.09 -2.81(m, 5H), 2.59(s, 6H), 2.14-2.10(m, 1H), 1.81-1.73(m, 1H), 1.20(t, J=7.20Hz, 14.4Hz, 3H).

[1135] General procedure for the preparation of intermediate 7 [ka] Intermediate 6a (1.36 g, 11.6 mmol, 1.57 mL, 5.00 equiv) and THF (10.0 mL) were placed in R1 (50.0 mL flask) at 20 °C. The mixture was cooled to -78 °C and degassed with N2 three times. LiHMDS (1 M, 11.6 mL, 5.00 equiv) was added to R1 at -78 °C under N2, and the mixture was stirred at -78 °C under N2 for 1 h.

[1136] A solution of Intermediate 6 (879 mg, 2.33 mmol, 1.00 equiv) in THF (3.00 mL) was added dropwise to R1 at −78° C. The mixture was stirred under N2 at −78° C. for 2 hours.

[1137] LCMS (EC14042-63-p1a1) showed that intermediate 6 was consumed and intermediate 7 was detected (RT=0.458 min).

[1138] The mixture was poured into H2O (30.0 mL) and extracted with ethyl acetate (20.0 mL*3). The combined organic layers were washed with brine (40.0 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 1:1, R f =0.74) to give compound 7 (350 mg, 872 μmol, yield 37.3%, purity 95.1%) as a white solid, which was confirmed by LCMS.

[1139] LCMS: RT=0.458 min, m / z=381.95(M+H) + .

[1140] LCMS: RT=0.462 min, m / z=382.15(M+H) + .

[1141] General procedure for the preparation of intermediate 8 [ka] Intermediate 7 (350 mg, 872 μmol, 1.00 equiv) and THF (4.00 mL) were charged to R1 (10.0 mL flask) at 20 °C, and NaBH4 (70.0 mg, 1.85 mmol, 2.12 equiv) was charged to R1 at 0-5 °C. The mixture was stirred under N2 at 0-5 °C for 3 h, and a sample was taken for LCMS analysis, which showed that intermediate 7 remained (RT = 0.502), but intermediate 8 was detected (RT = 0.482 min).

[1142] Brine (5.00 mL) was added, and the resulting suspension was stirred at room temperature for 10 minutes. The reaction mixture was diluted with ethyl acetate (10.0 mL) and distilled water (10.0 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (10.0 mL*2). The combined organic layers were washed with brine (10.0 mL) and dried over Na2SO4 to give Intermediate 8 (390 mg, crude) as a white solid, which was confirmed by LCMS.

[1143] LCMS: RT=0.482 min, m / z=384.1(M+H) + .

[1144] LCMS: RT=0.450 min, m / z=384.1(M+H) + .

[1145] General procedure for the preparation of compound 180 [ka] Intermediate 8 (390 mg, 981 μmol, 1.00 equiv) and HCl / EtOAc (2 M, 8.67 mL, 17.6 equiv) were placed in R1 (50.0 mL flask) at 20° C. The mixture was stirred at 20° C. for 2 h.

[1146] LCMS showed that intermediate 8 was consumed and compound 180 (RT=0.336 min).

[1147] The reaction mixture was concentrated under reduced pressure to give a residue.

[1148] The crude product was purified by preparative HPLC (column: Welch Ultimate C18 150*25mm*5um, mobile phase: [water (FA)-ACN], gradient: 16% to 46% B in 10 min) to give compound 180 (153 mg, 467 μmol, yield 47.6%, purity 100%) as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[1149] LCMSRT=0.336min, m / z=328.0(M+H) + .

[1150] LCMS: RT=0.332 min, m / z=328.1(M+H) + .

[1151] HPLC: RT=1.329 min, 100% purity at 220 nm.

[1152] 1 H NMR (400MHz, DMSO): δ7.42(d, J=6.40Hz, 1H), 7.34(t, J=8.4Hz, 16.4Hz, 1H), 3.87-3.72(m, 1H), 2.95- 2.62(m, 4H), 2.58(s, 6H), 2.48-2.46(m, 1H), 2.33-2.25(m, 1H), 2.03-1.70(m, 2H), 1.53-1.32(m, 1H).

[1153] Example 31 - Preparation of Compound 181 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (3.00 g, 10.5 mmol, 1.00 equiv), DMF (30.0 mL), HO (3.00 mL), Pd(OAc) (1.19 g, 5.30 mmol, 0.50 equiv), TEA (3.22 g, 31.7 mmol, 4.42 mL, 3.00 equiv), and Xantphos (3.07 g, 5.30 mmol, 0.50 equiv) were placed in a 100 mL stand-up flask (R1) at 20 °C. The mixture was degassed three times with CO (15 psi) and stirred at 90 °C for 4 h.

[1154] LCMS (EC13849-40-p1a1) showed that compound 1 was consumed and the target mass was detected (RT=0.532 min).

[1155] The mixture was filtered to obtain the liquid phase, saturated Na2CO3 (80.0 mL) was added to the liquid to adjust the pH of the solution to 9-10, and extracted with ethyl acetate (50.0 mL*3), the mixture was separated to obtain the aqueous phase, 1 M HCl (50.0 mL) was added to the aqueous phase to adjust the pH of the solution to 2-3, and extracted with ethyl acetate (50.0 mL*3), the organic phase was washed with brine (50.0 mL*2), dried over Na2SO4, filtered and concentrated in vacuo to obtain intermediate 2 (1.40 g, crude) as a yellow gum, confirmed by LCMS.

[1156] LCMS: RT=0.532 min, m / z=246.9(M+H) + .

[1157] General procedure for the preparation of intermediate 3 [ka] Intermediate 2 (1.40 g, 5.64 mmol, 1.00 equiv), DMF (15.0 mL), Intermediate 2a (1.15 g, 14.1 mmol, 2.50 equiv), DIEA (1.46 g, 11.2 mmol, 1.96 mL, 2.00 equiv), and HATU (2.57 g, 6.77 mmol, 1.20 equiv) were placed in a 100 mL flask (R1), and the mixture was stirred under N at 20 °C for 2 h.

[1158] LCMS (EC13849-43-p1a2) showed that compound 2 was consumed and the target mass was detected (RT=0.403 min).

[1159] Water (30.0 mL) was added to R1 at 20° C., extracted with ethyl acetate (30.0 mL*3), washed with brine (30.0 mL*2), dried over Na2SO4, filtered and concentrated in vacuo.

[1160] The crude product was purified by preparative TLC (dichloromethane:methanol = 10:1, R f= 0.86), followed by preparative HPLC (column: Phenomenex luna C18250*50mm*15um, mobile phase: [water (FA)-ACN], gradient: 28% to 58% B in 11 min) to give intermediate 3 (430 mg, 1.48 mmol, yield 26.2%, purity 94.9%) as a colorless oil, which was analyzed by LCMS, HPLC and 1 Confirmed by 1 H NMR.

[1161] LCMS: RT=0.403 min, m / z=276.1(M+H) +

[1162] LCMS: RT=0.398 min, m / z=276.1(M+H) +

[1163] HPLC: RT=1.824 min, purity 94.9% at 220 nm.

[1164] 1 H NMR (400MHz, DMSO): δ7.17-7.10(m, 3H), 4.14-4.07(m, 2H), 2.96-2.88(m, 8H), 2.82 -2.78(m, 3H), 2.12-2.08(m, 1H), 1.78-1.75(m, 1H), 1.20(t, J=7.2Hz, 14.0Hz, 3H).

[1165] General procedure for the preparation of intermediate 4 [ka] Intermediate 3a (1.35 g, 11.6 mmol, 1.56 mL, 5.00 equiv) and THF (7.00 mL) were placed in R1 (100 mL flask) at 20 °C. The mixture was cooled to -78 °C and degassed with N2 three times. LiHMDS (1 M, 11.6 mL, 5.00 equiv) was added to R1 at -78 °C under N2, and the mixture was stirred at -78 °C under N2 for 1 h. A solution of intermediate 3 (640 mg, 2.32 mmol, 1.00 equiv) in THF (3.00 mL) was added dropwise to R1 at -78 °C, and the mixture was stirred at -78 °C under N2 for 2 h.

[1166] LCMS (EC14042-63-p1a1) showed that intermediate 3 was consumed and intermediate 4 was detected (RT=0.428 min).

[1167] The mixture was poured into H2O (20.0 mL) and extracted with ethyl acetate (15.0 mL*3), and the combined organic layers were washed with brine (20.0 mL), dried over Na2SO4, filtered and concentrated.

[1168] The crude product was purified by column chromatography (SiO, petroleum ether: ethyl acetate = 1:0 to 1:1, R f =0.73) to give Intermediate 4 (500 mg, 1.22 mmol, 52.3% yield, 84.0% purity) as a colorless oil, which was confirmed by LCMS.

[1169] LCMS: RT=0.428 min, m / z=346.0(M+H) + .

[1170] LCMS: RT=0.436 min, m / z=346.1(M+H) + .

[1171] General procedure for the preparation of intermediate 5 [ka] Intermediate 4 (400 mg, 972 μmol, 1.00 equiv) and THF (4.00 mL) were placed in R1 (10.0 mL flask) at 20 °C. NaBH4 (30.0 mg, 793 μmol, 0.82 equiv) was added to R1 at 0-5 °C, and the mixture was stirred at 0-5 °C under N2 for 1 h.

[1172] LCMS analysis showed that intermediate 4 was consumed and intermediate 5 was detected (RT=0.438 min).

[1173] Brine (8.00 mL) was added, and the resulting suspension was stirred at room temperature for 10 minutes. The reaction mixture was diluted with ethyl acetate (5.0 mL) and distilled water (10.0 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (10.0 mL*2). The combined organic layers were washed with brine (10.0 mL) and dried over Na2SO4 to give Intermediate 5 (450 mg, crude) as a white solid, which was confirmed by LCMS.

[1174] LCMS: RT=0.438 min, m / z=348.3(M+H) + .

[1175] LCMS: RT=0.423 min, m / z=348.2(M+H) + .

[1176] General procedure for the preparation of compound 181 [ka] Intermediate 5 (450 mg, 1.25 mmol, 1.00 equiv) and HCl / EtOAc (4 M, 10.0 mL, 31.9 equiv) were placed in R1 (100 mL flask) and the mixture was stirred at 20 °C under N for 1 h.

[1177] LCMS showed that intermediate 5 remained (RT=0.439 min) and the target mass was detected (RT=0.308 min).

[1178] The reaction mixture was concentrated under reduced pressure to give a residue.

[1179] The crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*25mm*10um, mobile phase: [water (FA)-ACN], gradient: 6% to 36% B in 10 min) to give compound 181 (180 mg, 610 μmol, yield 48.7%, purity 98.4%) as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[1180] LCMS: RT=0.308 min, m / z=292.2(M+H) + .

[1181] LCMS: RT=0.309 min, m / z=292.2(M+H) + .

[1182] HPLC: RT=1.115 min, purity 98.4% at 220 nm.

[1183] 1 H NMR (400MHz, DMSO): δ7.13-7.09(m, 3H), 3.85-3.76(m, 1H), 2.92(s, 6H), 2 .83-2.59(m, 5H), 2.33-2.25(m, 1H), 2.02-1.69(m, 2H), 1.50-1.30(m, 1H).

[1184] Example 32 - Preparation of Compound 184 General procedure for the preparation of intermediate 2 [ka] Intermediate 1 (10.0 g, 37.7 mmol, 1.0 equiv) in dioxane (100 mL) was placed in a 500 mL flask (R1) at 25 °C. DIEA (9.75 g, 75.4 mmol, 13.1 mL, 2.0 equiv), Pd2(dba)3 (6.91 g, 7.54 mmol, 0.2 equiv), Xphos (8.73 g, 15.0 mmol, 0.4 equiv), and BnSH (9.84 g, 79.2 mmol, 9.30 mL, 2.1 equiv) were added to R1 at 25 °C under N2, and the mixture was stirred at 100 °C for 16 h.

[1185] As a result of TLC analysis (petroleum ether: ethyl acetate = 5 / 1), intermediate 1 was consumed and intermediate 2 was detected (R f= 0.55).

[1186] The mixture was quenched with water (100 mL), extracted with DCM (3*150 ml) and concentrated in vacuo. The crude product was purified by column chromatography (SiO, petroleum ether:ethyl acetate=5 / 1, R f= 0.55) to give intermediate 2 (10.0 g, 27.1 mmol, 71.9% yield) as a yellow solid. 1 Confirmed by 1 H NMR.

[1187] 1 H NMR (400MHz, DMSO): δ8.57(s, 1H), 8.06-8.03(m, 1H), 7.96-7.91(m, 3H), 7.56 -7.53(m, 1H), 7.46-7.44(m, 2H), 7.33-7.28(m, 3H), 4.43(s, 2H), 3.90(s, 3H).

[1188] General procedure for the preparation of intermediate 3 [ka] HO (0.90 mL) was placed in a 250 mL flask (R1) at 25 °C. AcOH (4.50 mL), DCM (45.0 mL), and Intermediate 2 (9.00 g, 24.4 mmol, 1.0 equiv) were added at 25 °C under N2. Then, SO2Cl2 (8.24 g, 61.0 mmol, 6.10 mL, 2.5 equiv) was added to R1 at 0 °C under N2, and the mixture was stirred at 0 °C under N2 for 1 h.

[1189] TLC analysis (petroleum ether:ethyl acetate=5 / 1) showed that intermediate 2 was consumed and intermediate 3 was detected (R f= 0.45).

[1190] The mixture was quenched with water (40 mL) and extracted with ethyl acetate (3*30 mL). The organic phase was separated and washed with brine (40 mL). The solution was dried over Na2SO4, filtered, and evaporated in vacuo to give intermediate 3 (11.6 g, crude) as a yellow solid.

[1191] General procedure for the preparation of intermediate 4 [ka] Intermediate 3 (11.6 g, 40.8 mmol, 1.0 equiv) was placed in a 250 mL flask (R1) at 25 °C. THF (120 mL) was added to R1 at 25 °C under N2. DIEA (15.8 g, 122 mmol, 21.3 mL, 3.0 equiv) and Me2NH HCl (7.99 g, 98.0 mmol, 2.4 equiv) were added to R1 at 0-5 °C under N2, and the mixture was stirred at 0-5 °C under N2 for 2 h.

[1192] LCMS (EC13398-54-P1A1) showed that intermediate 3 was consumed and intermediate 4 was detected (RT=0.423 min).

[1193] The mixture was quenched with water (100 mL) and extracted with ethyl acetate (3*100 mL). The organic phase was separated and washed with brine (3*100 mL). The solution was dried over Na2SO4, filtered and evaporated under vacuum. The crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 5 / 1, R f= 0.20) to give Intermediate 4 (3.00 g, 10.2 mmol, 25.0% yield) as a white solid, which was confirmed by LCMS.

[1194] LCMS: RT=0.423 min, m / z=293.9(M+H) + .

[1195] General procedure for the preparation of intermediate 5 [ka] Intermediate 4_1 (7.60 g, 65.4 mmol, 8.78 mL, 6.0 equiv) was placed in a 500 mL flask (R1) at 20 °C. THF (32 mL) was added to R1 at 20 °C under N2. LiHMDS (1.0 M, 65.4 mL, 6.0 equiv) was then added to R1 at -78 to -70 °C under N2 and stirred for 0.5 h. Intermediate 4 (3.2 g, 10.9 mmol, 1.0 equiv) was added to R1 at -78 to -70 °C under N2, and the mixture was stirred at -78 to -70 °C under N2 for 1 h.

[1196] LCMS showed that intermediate 4 was consumed and intermediate 5 was detected (RT=0.738 min).

[1197] The mixture was quenched with aqueous NH4Cl (50 mL) and extracted with ethyl acetate (3*30 mL). The organic phase was separated and washed with brine (3*30 mL). The solution was dried over Na2SO4, filtered, and evaporated in vacuo.

[1198] The crude product was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 3 / 1, R f= 0.20) to give Intermediate 5 (2.80 g, 7.42 mmol, 68.0% yield) as a yellow solid, which was confirmed by LCMS.

[1199] LCMS: RT=0.738 min, m / z=377.9(M+H) + .

[1200] General procedure for the preparation of intermediate 6 [ka] Intermediate 5 (1.00 g, 2.65 mmol, 1.0 equiv) was placed in a 100 mL flask (R1) at 25 °C. THF (10 mL) was added to R1 at 25 °C under N2. Next, NaBH4 (50.1 mg, 1.32 mmol, 0.5 equiv) was added to R1 at 0-5 °C under N2, and the mixture was stirred at 0-5 °C under N2 for 0.5 h.

[1201] LCMS (EC13398-101-P1A2) showed that intermediate 5 was consumed and intermediate 6 was detected (RT=0.436 min).

[1202] Brine (10 mL) was added and the resulting suspension was stirred at room temperature for 10 minutes. The reaction mixture was diluted with AcOEt (10 mL) and distilled water (10 mL) and extracted with ethyl acetate (2 * 8 mL). The organic phase was separated and washed with brine (10 mL). The solution was dried over Na2SO4, filtered and evaporated in vacuo.

[1203] The crude product was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 3 / 1, R f= 0.30) to give Intermediate 6 (567 mg, 1.45 mmol, 54.71% yield, 97% purity) as a white solid, which was confirmed by LCMS.

[1204] LCMS: RT=0.436 min, m / z=380.3(M+H) + .

[1205] General procedure for the preparation of compound 184 [ka] Intermediate 6 (567 mg, 1.49 mmol, 1.0 equiv), DCM (1 mL), and TFA (51.1 mg, 448 μmol, 33.30 μL, 0.3 equiv) were heated at 20 °C. N2 The mixture was stirred at 20°C for 1 hour.

[1206] LCMS (EC13398-105-P1A2) shows that intermediate 6 has been consumed and compound 184 has been detected (RT=0.320 min).

[1207] The mixture was evaporated under vacuum, and the crude product was purified by preparative HPLC (column: Welch Xtimate C18 150*25mm*5μm, mobile phase: [water (TFA)-ACN], gradient: 16% to 46% B in 10 min) to give compound 184 (375 mg, 1.16 mmol, 77.8% yield, 99.6% purity) as a white solid. 1 Confirmed by 1 H NMR.

[1208] LCMS: RT=0.320 min, m / z=323.9(M+H) + .

[1209] LCMS: RT=0.316 min, m / z=324.0(M+H) + .

[1210] HPLC: RT=1.345 min, purity 99.6% at 220 nm.

[1211] 1 H NMR: (400MHz, DMSO): δ8.40(s, 1H), 8.19-8.13(m, 2H), 8.02(s, 1H), 7.76-7.68(m, 2H), 5.17-5.13(m, 1H), 2.68-2.61(m, 8H).

[1212] Example 33 - Preparation of Compound 185 General procedure for the preparation of intermediate 1 [ka] Intermediate 1 (1.50 g, 5.30 mmol, 1.00 equiv), dioxane (15.0 mL), Pd(dba) (970 mg, 1.06 mmol, 0.20 equiv), XPhos (1.01 g, 2.12 mmol, 0.40 equiv), DIEA (1.37 g, 10.5 mmol, 1.85 mL, 2.00 equiv), and BnSH (1.58 g, 12.7 mmol, 1.49 mL, 2.40 equiv) were placed in a 100 mL stand-up flask at 20 °C and stirred at 100 °C for 16 h under N.

[1213] TLC analysis (plate 1, petroleum ether:ethyl acetate = 8:1) revealed intermediate 1 (R f= 0.75) was consumed, and intermediate 2 (R f =0.60) was formed.

[1214] The mixture was quenched with water and extracted with DCM (5.00 mL*3), and the combined organic phase was washed with brine (15.0 mL), dried over Na2SO4, filtered and concentrated.

[1215] The crude product (2.00 g) from notebook page EC14042-3 was combined with EC14042-4 for further purification. The combined crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:0 to 8:1, R f =0.60) to give compound 2 (3.50 g, crude) as a yellow oil. 1 Confirmed by 1 H NMR.

[1216] 1 H NMR (400MHz, DMSO): δ7.25-7.18(m, 5H), 7.10-7.01(m, 3H), 4.17(s, 2H), 4.13-4.05(m, 2H), 2.95-2. 87(m, 2H), 2.69-2.75(m, 3H), 2.08-2.03(m, 1H), 1.75-1.65(m, 1H), 1.19(t, J=7.20Hz, 14.4Hz, 3H).

[1217] General procedure for the preparation of intermediate 3 [ka] HO (1.20 mL) was placed in R1 (100 mL flask) at 20 °C. AcOH (5.00 mL), DCM (50.0 mL), and Intermediate 2 (2.50 g, 7.66 mmol, 1.00 equiv.) were added to R1 under N2 at 20 °C. Next, SO2Cl2 (2.58 g, 19.1 mmol, 1.91 mL, 2.50 equiv.) was added to R1 at 0-5 °C under N2, and the mixture was stirred at 0-5 °C under N2 for 1 h.

[1218] TLC analysis (plate 1, petroleum ether:ethyl acetate=8:1) revealed intermediate 2 (R f= 0.53) was consumed, and intermediate 3 (R f =0.40) was produced.

[1219] The mixture was poured into H2O (100 mL) and extracted with DCM (70.0 mL*3), and the combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated.

[1220] The crude product was purified by column chromatography (SiO, petroleum ether: ethyl acetate = 1:0 to 5:1, R f =0.55) to give intermediate 3 (860 mg, 2.84 mmol, 37.0% yield) as a yellow oil. 1 Confirmed by 1 H NMR.

[1221] 1 H NMR (400MHz, DMSO): δ7.31(d, J=6.00Hz, 2H), 7.04(d, J=8.40Hz, 1H), 4.13-4.07(m, 2H), 2.96-2.70(m, 5H), 2.10-2.06(m, 1H), 1.79-1.70(m, 1H).

[1222] General procedure for the preparation of intermediate 4 [ka] Intermediate 3 (780 mg, 2.58 mmol, 1.00 equiv), Intermediate 3a (423 mg, 3.86 mmol, 1.50 equiv), and THF (7.80 mL) were placed in a 10.0 mL flask (R1) at 20 °C. The pH of the mixture was adjusted to 7.0 with TEA (521 mg, 5.15 mmol, 717 μL, 2.00 equiv) and stirred under N2 at 20 °C for 1 h.

[1223] TLC analysis (plate 1, petroleum ether:ethyl acetate=3:1) revealed intermediate 3 (R f= 0.68) was consumed, and intermediate 4 (R f =0.55) was produced.

[1224] The mixture was poured into H2O (15.0 mL) and extracted with ethyl acetate (10.0 mL*3), and the combined organic layers were washed with brine (15.0 mL), dried over Na2SO4, filtered and concentrated.

[1225] The crude reaction mixture (100 mg) from notebook page EC14042-29 was combined with EC14042-30 and purified. The crude product was purified by preparative TLC (SiO, petroleum ether:ethyl acetate = 3:1, R f =0.55) to give compound 4 (760 mg, 2.24 mmol, 86.9% yield, 100% purity) as a yellow oil, which was confirmed by LCMS.

[1226] LCMS: RT=0.452 min, m / z=340.1(M+H) + .

[1227] General procedure for the preparation of intermediate 5 [ka] Intermediate 4a (941 mg, 8.10 mmol, 1.09 mL, 5.00 equiv) and THF (6.00 mL) were charged to R1 (100 mL flask) at 20 °C. The mixture was cooled to -78 °C and degassed with N2 three times. LiHMDS (1.00 M, 8.10 mL, 5.00 equiv) was added to R1 at -78 °C under N2, and the mixture was stirred at -78 °C under N2 for 1 h. Next, a solution of intermediate 4 (550 mg, 1.62 mmol, 1.00 equiv) in THF (2.00 mL) was added dropwise to R1 at -78 °C and stirred at -78 °C under N2 for 2 h.

[1228] LCMS analysis showed that intermediate 4 was consumed and intermediate 5 was detected (RT=0.502 min).

[1229] The mixture was poured into H2O (50.0 mL) and extracted with ethyl acetate (25.0 mL*3), and the combined organic layers were washed with brine (40.0 mL), dried over Na2SO4, filtered and concentrated.

[1230] The crude product was purified by preparative TLC (SiO2, petroleum ether:ethyl acetate = 1:1, R f =0.75) to give intermediate 5 (415 mg, 962 μmol, 59.4% yield, 94.9% purity) as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[1231] LCMS: RT=0.502 min, m / z=410.0(M+H) + .

[1232] LCMS: RT=0.557 min, m / z=410.1(M+H) + .

[1233] HPLC: RT=2.341 min, purity 94.9% at 220 nm.

[1234] 1 H NMR (400MHz, DMSO): δ7.49(d, J=7.60Hz, 2H), 7.32(d, J=7.60Hz, 1H), 3.65(s, 1H), 3.15-3.10(m, 2H) , 3.00-2.83(m, 5H), 2.14-2.11(m, 1H), 1.70-1.60(m, 1H), 1.41(s, 9H), 1.04(t, J=7.20, 14.0Hz, 6H).

[1235] General procedure for the preparation of intermediate 6 [ka] Compound 5 (300 mg, 695 μmol, 1.00 equiv.) and THF (1.00 mL) were placed in R1 (10.0 mL flask) at 20 °C. NaBH4 (13.1 mg, 347 μmol, 0.50 equiv.) was added to R1 at 0-5 °C. The mixture was stirred under N2 at 0-5 °C for 0.5 h.

[1236] LCMS (EC14042-61-P1A1) showed that intermediate 5 remained (RT=0.535 min), but intermediate 6 was detected (RT=0.519 min).

[1237] Brine (5.00 mL) was added, and the resulting suspension was stirred at room temperature for 10 minutes. The reaction mixture was diluted with ethyl acetate (10.0 mL) and distilled water (10.0 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (10.0 mL * 3 mL). The combined organic layers were washed with brine (10.0 mL) and dried over Na2SO4 to give Intermediate 6 (260 mg, crude) as a yellow oil, which was confirmed by LCMS.

[1238] LCMS: RT=0.519 min, m / z=412.1(M+H) + .

[1239] LCMS: RT=0.476 min, m / z=412.4(M+H) + .

[1240] General procedure for the preparation of compound 185 [ka] Intermediate 6 (260 mg, 617 μmol, 1.00 equiv) and HCl / EtOAc (4.00 M, 6.00 mL, 38.8 equiv) were placed in R1 (100 mL flask) and the mixture was stirred at 20° C. under N for 2 h.

[1241] LCMS (EC14042-66-p1a1) showed that intermediate 6 was consumed and compound 185 was detected (RT=0.377 min).

[1242] The reaction mixture was concentrated under reduced pressure, and the crude product was purified by preparative HPLC (column: Phenomenex luna C18 150*25mm*10um, mobile phase: [water (FA)-ACN], gradient: 23% to 53% B in 10 min) to give compound 185 (83.0 mg, 231 μmol, yield 37.4%, purity 99.0%) as a white solid. 1 Confirmed by 1 H NMR, LCMS, and HPLC.

[1243] LCMS: RT=0.377 min, m / z=356.1(M+H) + .

[1244] LCMS: RT=0.398 min, m / z=356.1(M+H) + .

[1245] HPLC: RT = 1.713 min, purity 99.0% at 220 nm.

[1246] 1 H NMR (400MHz, DMSO): δ7.47-7.45(m, 2H), 7.28(t, J=5.20Hz, 15.6Hz, 1H), 3.76-3.85(m, 1H), 3.14-3.09(m, 4H) , 2.92-2.52(m, 5H), 2.52-2.31(m, 1H), 2.02-1.69(m, 2H), 1.50-1.34(m, 1H), 1.04(t, J=7.20Hz, 14.0Hz, 6H).

[1247] Example 34 - In vitro evaluation of pannexin 1 channel blockade PANX1 blockade was assessed in HEK293 cells stably expressing human PANX1 channels, as previously described by Xu et al. (2012). Briefly, wild-type cells were seeded in 6-well plates, and after 12–16 hours, 60–70% confluent cells were transfected with a plasmid encoding hPANX1 using calcium phosphate. Forty-eight hours after transfection, cells were treated with fresh medium supplemented with 0.8 mg / ml of geneticin (G418) to select for transfected clones. The medium was changed every other day. Once confluent, cells were transferred to flasks and maintained in medium containing G418 (0.8 mg / ml) for a total of 4 weeks from the day of transfection.

[1248] To validate these cells as a tool for screening potential PANX1 inhibitors, several known PANX1 inhibitors were evaluated by whole-cell patch clamp, and voltage / current diagrams were compared with current literature. The pipette solution was 151 mM CaCl, 10 mM HEPES, 10 mM EGTA, and 3 mM MgCl. Cells were maintained in normal extracellular solution (NES: 140 mM NaCl, 5 mM KCl, 2 mM CaCl, 1 mM MgCl, 10 mM D-glucose, 10 mM HEPES, pH 7.3). One-second voltage ramps from -80 mV to +80 mV were applied with 5-second intervals. Panx1 channel opening was induced by hypotonic shock by replacing the isotonic extracellular solution (310 mOs ml / L) with a hypotonic extracellular solution (198 mOs ml / L).

[1249] The first run confirmed functional expression of Panx1 in HEK293 cells (n = 6) and also confirmed the known inhibitory effects of CBX (50 μM) and spirolactone (3, 10, or 30 μM) on PANX1 (n = 3). [Table 3-1] [Table 3-2]

[1250] Example 35 - In vitro evaluation of antitumor potential The antitumor potential of the compounds of the present invention was investigated against the proliferation and tissue invasiveness of MDA-MB-231 human epithelial breast cancer cells.

[1251] For antiproliferative activity, MDA-MB-231 cells were seeded into 384-well plates in appropriate culture medium and treated with either a compound of the present invention (final concentrations of 0.1, 1, 5, 10, 50, 100, 200, or 1000 μM) or vehicle (DMSO). Treated cell cultures were maintained in a humidified cell incubator at 37°C under a 95% O2 / 5% CO2 atmosphere for 7 days. Proliferation rates were automatically recorded by an Incucyte S3 system. Representative results are shown in Table 4. [Table 4]

[1252] To determine whether the reduction in proliferation was associated with a concomitant decrease in protein expression of tumor invasion markers, MDA-MB-231 cells were seeded into 6-well cell culture plates in appropriate culture medium and treated with the drug candidates of the present invention at a final concentration of 200 μM. Cultures were maintained for 7 days in a humidified cell incubator at 37°C under a 95% O2 / 5% CO2 atmosphere. At the end of the interval, cells were harvested and the expression levels of proteins associated with breast cancer metastasis were assessed.

[1253] The expression levels of E-cadherin, matrix metalloproteinases (MMPs) 2 and 9, N-cadherin, and β-catenin were assessed by Western blotting, and β-actin expression was used as a sample normalization standard. Several compounds were able to reduce the expression of β-catenin (Figure 1), E-cadherin (Figure 2), MMP2 (Figure 3), and MMP9 (Figure 4).

[1254] Having found that the compounds of the present invention can downregulate the protein expression of several metastasis-associated proteins, the potential of these compounds to prevent tumor metastasis was investigated in vitro in MDA-MB-231.

[1255] Cells were allowed to migrate into the artificial matrix in the presence or absence of the PANX1 blockers of the present invention. Cells were seeded into 24-well transwell sets containing Matrigel and the appropriate culture medium. Cells were treated with each test compound (final concentrations of 10 μM, 200 μM, or 1000 μM) or vehicle (DMSO 0.25%). Treated cell cultures were maintained in a humidified cell incubator at 37°C under a 95% O2 / 5% CO2 atmosphere for 7 days. The Matrigel layers were then harvested, fixed, stained, and cell counted.

[1256] All tested compounds were able to significantly prevent the invasion of Matrigel supports, indicating that they may reduce the tissue invasive ability of breast cancer cells (Figure 5).

[1257] Example 36 - In vivo evaluation of analgesic effects in a rat model of neuropathic pain Nerve damage was induced in rats using a modified neuropathic pain model performed by Decosterd and Wolf (2000), which produces very strong, long-lasting, and powerful changes in mechanical and thermal sensitivity that are closely related to various clinical features of neuropathic pain. In this model, the peroneal nerve was transected transversely. In this model, the sural nerve contains few motor fibers, allowing the generation of neuropathic pain while preserving motor activity (Payronnard and Charron, 1982).

[1258] Animals were anesthetized with 7% (w / v) chloral hydroxide at 400 mg / kg i.p. After shaving the right hind limb at the level of the pelvis (the origin of the sciatic nerve), an approximately 10 mm long skin incision was made. The subcutaneous tissue and biceps femoris muscle were dissected to expose the sciatic nerve (Figure 6, (1)). The nerve's course was traced until it bifurcated into three branches: the peroneal nerve (4), the sural nerve (2), the common nerve, and the tibial nerve (3). The peroneal nerve was transected 2 mm away from the sciatic nerve, and the overlying tissue was sutured in layers. The above nerve injury resulted in mechanical hyperalgesia in the hind limb, which persisted for at least 28 days.

[1259] Pain schedule and measurement Seven days after surgical induction of neuropathic pain, test compounds (CMPD004 (Figure 7; PX004), CMPD011 (Figure 8; PX011), CMPD19 (Figure 11; PX019), CMPD027 (Figure 12; PX027), CMPD043 (Figure 13; PX043), CMPD054 (Figure 9; PX054), and CMPD055 (Figure 10; PX055)) and, as controls, carbenoxolone (a nonspecific PANX1 blocker) and saline were injected intraspinally. Pain was measured using the paw pressure test. Pain behavior was quantified using the paw pressure test, also known as the Randall and Sellito test (Randall and Sellito, 1957). Briefly, increasing pressure was applied to the right hind paw of rats using an algometer (Ugo Basile, Italy) until paw withdrawal. The maximum pressure applied to the paw was 480 g (cutoff), which does not cause damage to the rat paw. This algometry test was performed on neuropathy rats before surgery (day 0) and 7 days after surgery to confirm the occurrence of mechanical hyperalgesia. The effects of drugs were studied 7 days after neuropathy-inducing surgery. Algometry was performed before injection of the test compound, 15 minutes after injection, and every 30 minutes after injection for 4 hours.

[1260] In summary, all compounds demonstrated significant and potent analgesic effects compared to the control in the paw pressure test. Although the potency varied, five of the tested compounds performed better than carbenoxolone, a nonspecific and potent pannexin 1 channel blocker. The other two performed equally well or worse (Figures 7-13).

[1261] Example 37 - In vivo evaluation of anti-addictive potential in a rat model of opioid addiction Three groups of male Sprague-Dawley rats were treated with different treatments (Group 1: Compound 004 (PX004), Group 2: Compound 011 (PX011), and Group 3: saline; N = 4) using Alzet (copyright) mini-osmotic pumps, model 1007D (DUR-ECT Corporation, Cupertino, CA, USA). The osmotic pumps were filled with 84 μL of 150 μM PX or saline (0.9% NaCl) and aseptically implanted into the dorsal subcutaneous tissue of anesthetized rats (3% isoflurane) through a small skin incision. The Alzet pumps released saline or test molecule solution at 0.5 ± 0.1 μL / h for 5 days. Concurrently, during these 5 days, all groups received increasing doses of morphine intraperitoneally at 8-h intervals (day 1, 10 mg / kg; day 2, 20 mg / kg; day 3, 30 mg / kg; day 4, 40 mg / kg). On day 5, rats were injected with morphine (45 mg / kg) in the morning and naloxone (2 mg / kg ip, naloxone hydrochloride dihydrate, Sigma) 2 h later to rapidly induce narcotic withdrawal behavior.

[1262] Withdrawal symptoms were recorded as described by Ferrini et al. Jumping, teeth chattering, violent trembling, head shaking, and grooming behaviors were assessed in 5-minute intervals for a total 30-minute test period, with each behavior assigned a standardized score of 0 to 3. Allodynia, piloerection, salivation, ejaculation, and trembling or convulsions were also assessed, with 1 point awarded for the presence of the behavior for each 5-minute interval. All signs were counted, and a cumulative withdrawal score was generated (Figure 14).

[1263] Example 38 - In vitro assessment of ATP release by microglia and astrocytes Cortical microglia / astrocytes mechanically isolated from decapitated postnatal P2-P4 rat pups (CD strain) were seeded into P75 flasks (one brain per flask) and cultured for 10-20 days at 37°C, 5% CO2 using DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. On day 10, microglia were harvested by washing with gentle shaking and purified by centrifugation through a 20 μm cell strainer.

[1264] Resuspended microglia were counted and seeded at a density of 80,000 cells / well in poly-D-lysine-coated 96-well culture plates. Remaining astrocytes were removed (enriched astrocytes) and seeded at a density of 50,000 cells / well in poly-D-lysine-coated 96-well culture plates. Overnight microglia and / or astrocytes were treated with the PANX-1 blocker of the present invention (5 or 50 μM), vehicle, or control (carbenoxolone, CBX), and then subjected to lipopolysaccharide stimulation. Extracellular ATP was assessed using the CellTiter-Glo (Promega) kit on the Envision multilabel plate reader (ultrasensitive luminescence).

[1265] Proinflammatory LPS stimulation induced a mild release of ATP by astrocytes (Figure 15A) and microglial cells (Figure 15B), which was blocked by compounds 19 (PX019), 43 (PX043), 53 (PX053), and 54 (PX054), confirming the biological activity of the compounds. 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J Invest Dermatol.2014 Jul;134(7):2026-2035.doi: 49.Penuela S, Kelly JJ, Churko JM, Barr KJ, Berger AC, Laird DW.Panx1 regulates cellular properties of keratinocytes and dermal fibroblasts in skin development and wound healing. 50.Luu R, Valdebenito S, Scemes E, Cibelli A, Spray DC, Rovegno M, Tichauer J, Cottignies-Calamarte A, Rosenberg A, Capron C, Belouzard S, Dubuisson J, Annane D, de la Grandmaison GL, Cramer-Borde E, Bomsel M, Eugenin E.Pannexin-1 channel opening is critical for COVID-19 pathogenesis.iScience.2021 Dec 17;24(12):1 51.Ransford GA, Fregien N, Qiu F, Dahl G, Conner GE, Salathe M.Pannexin 1 contributes to ATP release in airway epithelia.Am J Respir Cell Mol Biol.2009 Nov;41(5):525-34. 52.Khan M, Huang YA, Kuo CY, Lin T, Lu CH, Chen LC, Kuo ML.Blocking pannexin1 reduces airway inflammation in a murine model of asthma.Am J Transl Res.2020 Jul 15;12(7):4074-4083. 53.Crespo Yanguas, S., da Silva, T.C., Pereira, I.V.A.et al.Genetic ablation of pannexin1 counteracts liver fibrosis in a chemical, but not in a surgical mouse model.Arch Toxicol92,2607-2627(2018). 54.Seref-FerlengezZ, Maung S, Schaffler MB, Spray DC, Suadicani SO, Thi MM.P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes.PLoS One.2016 May 9;11(5):e0155107. 55.Adamson SE, Meher AK, Chiu YH, Sandilos JK, Oberholtzer NP, Walker NN, Hargett SR, Seaman SA, Peirce-Cottler SM, Isakson BE, McNamara CA, Keller SR, Harris TE, Bayliss DA, Leitinger N.Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes.Mol Metab.2015 Jul 3;4(9):610-8. 56.Bartley C, Brun T, Oberhauser L, Grimaldi M, Molica F, Kwak BR, Bosco D, Chanson M, Maechler P.Chronic fructose renders pancreatic β-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.Am J Physiol Endocrinol Metab.2019 Jul 1;317(1):E25-E41. 57.Tozzi M, Hansen JB, Novak I.Pannexin-1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration.Acta Physiol(Oxf).2020 Feb;228(2):e13360. 58.Yang K, Xiao Z, He X, Weng R, Zhao X, Sun T.Mechanisms of Pannexin 1(PANX1)Channel Mechanosensitivity and Its Pathological Roles.Int J Mol Sci.2022 Jan 28;23(3):1523. 59.Dvoriantchikova G, Pronin A, Kurtenbach S, Toychiev A, Chou TH, Yee CW, Prindeville B, Tayou J, Porciatti V, Sagdullaev BT, Slepak VZ, Shestopalov VI.Pannexin 1 sustains the electrophysiological responsiveness of retinal ganglion cells.Sci Rep.2018 Apr 11;8(1):5797. 60.Pronin A, Pham D, An W, Dvoriantchikova G, Reshetnikova G, Qiao J, Kozhekbaeva Z, Reiser AE, Slepak VZ, Shestopalov VI.Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury.Front Mol Neurosci.2019 Mar 13;12:36. 61.Cui H, Liu Y, Qin L, Wang L, Huang Y.Increased membrane localization of pannexin1 in human corneal synaptosomes causes enhanced stimulated ATP release in chronic diabetes mellitus.Medicine(Baltimore).2016 Dec;95(49): 62.Ferrari D, Casciano F, Secchiero P, Reali E.Purinergic Signaling and Inflammasome Activation in Psoriasis Pathogenesis.Int J Mol Sci.2021 Aug 31;22(17):9449.doi:10.3390 / ijms22179449.PMID:34502368;PMCID:PMC8430580. 63.Garcia-Vega L, O’Shaughnessy EM, Jan A, Bartholomew C, Martin PE.Connexin 26 and 43 play a role in regulating proinflammatory events in the epidermis.J Cell Physiol.2019 Feb 2.doi:10.1002 / jcp.28206.Epub ahead of print.PMID:30710344. 64. 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Transcutol®(Diethylene Glycol Monoethyl Ether. 72.A Potential Penetration Enhancer.In:Dragicevic N., Maibach H.(eds)Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement.Springer, Berlin, Heidelberg, 2015. 73.WIECHERS, J.W.AND DE ZEEUW, R.A.Transdermal drug delivery:efficacy and potential applications of the penetration enhancer Azone.Drug Des Deliv.(2):87-100.1990. 74.RAIMAN, J., KOLJONEN, M., HUIKKO, K., KOSTIANEN, R., HIRVONEN, J.Delivery and Stability of LHRH and Nafarelin in Human Skin:The Effect of Constant / Pulsed Iontophoresis.European Journal of Pharmaceutical Sciences:Official Journal of the European Federation for Pharmaceutical Sciences, 21(2-3):371-77.2004. 75.WANG, Y., TRAKUR, R., FAN, Q., MICHNIAK, B.Transdermal Iontophoresis:Combination Strategies to Improve Transdermal Iontophoretic Drug Delivery.European Journal of Pharmaceutics and Biopharmaceutics:Official Journal of Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik, 60(2):179-91.2005. 76.PARK, E.J., WERNER, J., SMITH, N.B.Ultrasound Mediated Transdermal Insulin Delivery in Pigs Using a Lightweight Transducer.Pharmaceutical Research, 24(7):1396-1401.2007. 77.MELKAMU, G., WOHLRAB, J., NEUBERT, R.H.Dermal Delivery of Desmopressin Acetate Using Colloidal Carrier Systems.The Journal of Pharmacy and Pharmacology, 57(4):423-27.2005. 78.MANOSROI, A., KHANRIN, P., LOHCHAROENKAL, W., WERNER, R.G., GOTZ, F., MANOSROI, W., MANOSROI, J.Transdermal Absorption Enhancement through Rat Skin of Gallidermin Loaded in Niosomes.International Journal of Pharmaceutics, 392(1-2):304-10.2010. 79.EL MAGHRABY, G.M., WILLIAMS, A.C., BARRY, B.W.Skin Delivery of Oestradiol from Deformable and Traditional Liposomes:Mechanistic Studies.The Journal of Pharmacy and Pharmacology, 51(10):1123-34.1999. 80.DAYAN, N., and E.TOUITOU.Carriers for Skin Delivery of Trihexyphenidyl HCl:Ethosomes vs.Liposomes.Biomaterials, 21(18):1879-85.2000. 81. 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Claims

1. A compound of Formula I or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 173】 During the ceremony, X and Y are each C, CH or N, or preferably Y is C or CH and X is N; R １ , R ２ and R ３ are independently hydrogen, lower alkyl optionally substituted with hydroxy, lower cycloalkyl, lower alkenyl, lower alkoxy, lower alkynyl, phenyl, halogenated phenyl, halogen, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably diazine, pyrazole, diazole, triazole, or alkyl derivatives thereof, 2-hydroxyisopropyl, or have one of the following structures: 【Chemical 174】 During the ceremony, X' is C, CH, CH ２ , N, S, or O, preferably CH ２ , N, or O; X'' is independently C, CH, or CH ２ , N, S, or O, preferably CH ２ , N, or O; X''' is NH, N-lower alkyl or O; R1' to R5' are independently absent, hydrogen, lower alkyl, lower cycloalkyl, halogen, OH, NH ２ , hydroxy-lower alkyl, or NH-lower alkyl, O-lower alkyl, O-PO-OR ６ ', O-lower alkyl or alkyloxy-O-PO-OR ６ ', or O-PO-O-lower alkyl-O-PO-R ６ ', preferably OH, NH ２ , NH-lower alkyl, and adjacent R1' and R5' can form a 5- to 7-membered ring moiety; R ６ ' is H or O-lower alkyl, R1'' to R4'' are independently absent, hydrogen, lower alkyl, lower cycloalkyl, OH, NH ２ , hydroxy-lower alkyl, NH-lower alkyl, O-lower alkyl, O-PO-OR ６ ', O-lower alkyl or alkyloxy-O-PO-O-R ６ ', or O-PO-O-lower alkyl-O-PO-R ６ ', preferably OH, NH ２ , NH-lower alkyl, or halogen; Y''' is a halogen, and The bond between adjacent ring substituents X' or adjacent ring substituents X'' may constitute a single bond or a double bond, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. 【Request 2】 【Chemical 175-1】 【Chemistry 175-2】 【Chemistry 175-3】 【Chemistry 175-4】 2. The compound of claim 1 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof.

3. A compound of Formula I' or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【176】 During the ceremony, X ａ or X ｂ is C, CH or N, or preferably X ａ is C or CH, and X ｂ is N and Y is CH, CH ２ , C-lower alkyl, CH-lower alkyl, C-halogen, CH-halogen or C-dihalogen, Z is CH or N, preferably CH, and the bond between adjacent ring substituents Y can be a single or double bond; R １ , R ２ , R ３ , and R ４ is independently absent, hydrogen, lower alkyl, lower cycloalkyl, lower alkenyl, lower alkoxy, lower alkynyl, phenyl, halogenated phenyl, halogen, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole, or diazine, 2-hydroxyisopropyl, or has one of the following structures: 【177】 During the ceremony, X' is C, CH, CH ２ , N, S, or O, preferably CH ２ , N, or O; X'' is C, CH, CH ２ , N, S, or O, preferably CH ２ , N, or O; X''' is NH, N-lower alkyl or O; R1' to R5' are independently absent, H, lower alkyl, lower cycloalkyl, OH, NH ２ , NH-lower alkyl, halogen, O-lower alkyl, O-PO-OR ６ ', O-lower alkyl or alkyloxy-O-PO-OR ６ ', or O-PO-O-lower alkyl-O-PO-R ６ ', preferably OH, NH ２ , NH-lower alkyl, and adjacent R1' and R5' can form a 5- to 7-membered ring moiety; R ６ ' is H or O-lower alkyl, R1" to R4" are independently absent, hydrogen, lower alkyl, lower cycloalkyl, or halogen; Y''' is a halogen, and The bond between adjacent ring substituents X' or adjacent ring substituents X'' may comprise a single bond or a double bond, thereby forming a non-aromatic ring or an aromatic ring, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

4. 【Chemical 178】 4. The compound of claim 3 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

5. 1. A compound of Formula II or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【179】 During the ceremony, X is N, Y is C, Z is N, C or CH, and the bond between adjacent ring substituents X, Y and / or Z constitutes a single or double bond; R １ ~R ４ is independently absent when Z is N, or R １ ~R ４ or R １ '~R ４ one or more of ' are low to medium chain oxo or keto fatty acids, optionally substituted with oxo or lower alkyl, H, halogen, lower alkyl, lower cycloalkyl, or COOH; or R １ ~R ４ or R １ '~R ４ ' contains a substituent of formula X: 【Chemistry 180】 During the ceremony, X' is CH, CO, N or O, preferably X' contains up to two N or O, R １ ''~R ５ " is independently absent, H, lower alkyl, lower cycloalkyl, halogen, NH ２ , NH-lower alkyl, hydroxy, oxo, COOH, SO ２ NH ２ , S.O. ２ NH-lower alkyl, a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or containing a substituent of formula XI: 【Chemistry 181】 During the ceremony, X ｉｖ is CH, CH ２ , CO, N or O, or preferably X ｉｖ contains at most two N or O, and R １ '''~R ５ ''' is independently absent, H, halogen, NH ２ , lower alkyl, lower cycloalkyl, NH-lower alkyl, hydroxy, COOH, SO ２ NH ２ , S.O. ２ NH-lower alkyl, substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or preferably R １ '''~R ５ Only one of the groups X''' contains an unsubstituted aromatic or non-aromatic heterocycle and the adjacent ring substituent X ｉｖ The bond between may constitute a single or double bond, thereby forming a non-aromatic or aromatic ring, or R １ ~R ４ One or more of the following may comprise a substituent of formula XII: 【Chemistry 182】 During the ceremony, Y' is absent or each independently represents CH ２ , C.H. ２ (CH ３ ), CO, SO, SO ２ , CHOH or NH, and Z' can be COOH, SO ２ NH ２ , a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, C ３ ~C ８ or forming a linear or branched chain of R １ ~R ４ One or more of the following may comprise a substituent of formula XIII: 【Chemistry 183】 During the ceremony, Y'' is COOH, SO ２ NH ２ or tetrazole, and X''' is N or O. 【Request 6】 【Chemical 184-1】 【Chemistry 184-2】 【Chemistry 184-3】 【Chemistry 184-4】 【Chemistry 184-5】 【Chemistry 184-6】 6. The compound of claim 5, selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

7. 1. A compound of Formula III or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 185】 During the ceremony, Each Z is independently selected from CH or C; each X is independently CH, C, CH ２、 O, N, S, SO, or SO ２ and each Y is independently selected from N, C, CH, CH ２ , CO, SO, S, or N-alkyl, and the bond between adjacent ring substituents X, Z and / or Y constitutes a single or double bond; Each R １ is absent or is H, halogen, O, NH ２ , N-lower cycloalkyl, phenyl group, alkyl group (optionally substituted with one or both of oxo and carboxy), OH, SO ２ N (CH ３ ) ２ , C(O)N(CH ３ ) ２ , a 5-membered heterocycle (preferably tetrazole), —CO—(CH ２ ) ｎ -COOH, where n is 0 to 4, and R １ may optionally be taken together with adjacent substituents to form an aromatic or aliphatic 5-, 6-, or 7-membered ring system, which may contain heteroatoms (optionally substituted with one or more COOH, carboxamido, alkoxy, oxo, halogen, or triazole groups), or R １ optionally containing a substituent of formula XIV: 【Chemistry 186】 During the ceremony, X' is absent or is O, N, or N-lower cycloalkyl; A is CH, or if X' is absent, it may be a direct bond; Y' is O, N, N-lower cycloalkyl, CH ２ , CH-lower alkyl, or CH—OH, or preferably X′ is absent or O, the Y′ ring contains one or two N or O atoms, and the bonds between adjacent substituents Y′ and other atoms in the ring constitute single or double bonds, thereby forming a non-aromatic or aromatic ring, and R ２ is absent or is lower alkyl optionally substituted with halogen, H, carboxy or oxo, hydroxy, alkoxy, COOH, SO ２ NH ２ , S.O. ２ NH-O-CH ３ , S.O. ２ NH-C(O)-CH ３ , S.O. ２ N (CH ３ ) ２ , S.O. ２－ Lower alkyl, C(O)N(CH ３ ) ２ , a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably a triazole or tetrazole, or containing a substituent of formula XV: 【187】 During the ceremony, B' is a bond, B' can include a linker or a direct bond, the linker is -N-, and R ３ are independently H, COOH, SO ２ NH ２ , S.O. ２ NH-lower alkyl, halogen, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably triazole or tetrazole, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. 【Request 8】 【Chemical 188-1】 【Chemistry 188-2】 【Chemistry 188-3】 【Chemistry 188-4】 【Chemistry 188-5】 【Chemistry 188-6】 【Hua 188-7】 【Hua 188-8】 【Chemistry 188-9】 8. The compound of claim 7 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

9. 1. A compound of formula IV or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 189】 During the ceremony, each X is independently selected from CH or C; X' is C, Each Y is N, CH, C, SO ２ or CO, R １ and R ２ are each independently H, COOH, O—CH3, lower cycloalkyl, or joined together to form an optionally substituted 5- or 6-membered heterocycle; R ３ ~R ７ are each independently H, COOH, O—CH3, lower cycloalkyl, or contain a substituent of formula XVI: 【190】 During the ceremony, Z is —N—CO—N or lower alkyl-CO—N; X″ is lower alkyl or partially halogenated lower alkyl, and R1' to R5' are independently H, CHF2, COOH, SO2NH2, NH-CH2-COOH, lower alkyl, lower alkyl-COOH, lower alkyl-CO-, lower alkyl-COOH, -O-CO-CH ３ , OCH3, N-CO-N-O-lower alkyl, N-CO-N-O-CF ２ , -SO ２ -N(lower alkyl) ２ , lower alkyl-O-lower alkyl-COOH, SO ２ -N-lower alkyl-phenyl carboxylic acid, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably a tetrazole, which may optionally be attached to the bicyclic ring of formula IV with a lower alkyl linker, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

10. 【Chemical 191-1】 【Hua 191-2】 【Chemistry 191-3】 10. The compound of claim 9 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

11. 1. A compound of formula V or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 192】 During the ceremony, X is CH ２ , C, or CH; X' is either C or CH; Y is C or CH; Y' is C; Z is independently N, O, or N-lower alkyl; Z' is O; and R ２ is H, halogen, lower alkyl, ketobutyric acid, cyclohexanonecarboxylic acid, or hydroxymethyl (cyclohexenone); the bond between adjacent ring substituents X, X′, and / or Z is a single or double bond; the bond between adjacent Y and / or Y′ is a single or double bond; Z″ can be CH or N, or lower alkyl, lower cycloalkyl, alkyl ether, or cycloalkyl; R １ is H, lower alkyl, halogen, O-lower alkyl, COOH, N-CO-NO-lower alkyl or cycloalkyl, partially halogenated lower alkyl or cycloalkyl, —CO—CH ３ , -lower alkyl-O-phenylcarboxylic acids having a lower alkyl substituent, or 【193】 It can be, During the ceremony, Z''' is lower alkyl or cycloalkyl, and R ３ are each independently H, COOH, NH—CO—NH—OCH ３ or partially halogenated NH—CO—NH—CH ３ , O-lower alkyl, CO—CH ３ or lower alkyl, and R ４ is lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

12. 【Catalog 194】 12. The compound of claim 11 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

13. 1. A compound of formula VI or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 195】 During the ceremony, Independently, X is N or CH, and R １ is NH ２ , NH-lower alkyl, OH, or O-lower cycloalkyl; R ２ and R ３ is H, lower alkyl, SO ２ NH ２ or may include a substituent of formula XVIII: 【Chemistry 196】 During the ceremony, X' is a direct bond, NH, or N-CH ３ or CH ２ wherein Y' is H or lower alkyl, and Z' is H, lower alkyl, COOH, SO ２ NH ２ , S.O. ２ NH-CH ３ or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole; when Y′ and Z′ are both H, X′ is N—CH ３ or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

14. 【Hua 197-1】 【Hua 197-2】 14. The compound of claim 13 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

15. 1. A compound of Formula VII or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 198】 During the ceremony, X is N or C, X' is CH or N, X'' is N or CH, Y is CH or C, and Z is CH ２ - or C=O, and the bond between adjacent ring substituents Y is a single bond or a double bond; R １ contains a substituent of formula XIX: 【Chemistry 199】 During the ceremony, R1' to R3' are independently halogen, carboxylic acid, SO ２ NH ２ , S.O. ２ -NH-lower alkyl, COOH, or NO ２ or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole, or R1 may include a substituent of formula XX: 【Chemistry 200】 During the ceremony, Y' is N, CH, or C; R'' is independently H, halogen, dihalogen, or lower alkyl; R ２ is H, lower cycloalkyl, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably 1,3-diazolidine; R ３ is halogen, carboxylic acid, SO ２ NH ２ , or SO ２ -NH-lower alkyl, or a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably tetrazole; R ４ is H, a halogen, or a lower alkyl keto acid, preferably ketopropionic acid; When X is N or CH, R ５ is absent, and when X is C, R5 is a substituted or unsubstituted aromatic or non-aromatic heterocycle, preferably pyrrolidine, piperidine, pyrazine, or a bicyclic structure of formula XXI: 【Chemistry 201】 During the ceremony, The compound or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, wherein Y'' is N,N-lower alkyl, or COOH.

16. [Chemical 202] 16. The compound of claim 15 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

17. 1. A compound of Formula VIII or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof: 【Chemistry 203】 During the ceremony, X is SO ２ , N.R. ４ or O, Z is C or N, and Y is N, CR ３ or CH, and R １ ~R ４ are each independently H, oxo, halogen, or -(CH ２ ) ｎ -COOH (where n = 0 to 6), -NH-C(O)-NH-CH ３ , -NH-C(O)-NH-CHF ２ , alkyl or alkoxy (optionally substituted with COOH or oxo), or R １ ~R ４ One or more of the following comprises a substituent of formula XXII: 【Chemistry 204】 During the ceremony, A is a linking point, and the substituent of formula XXII is attached to the compound of formula VIII either directly or through a linker L, where L is alkyl, —CH ２ -C(O)-NH-, or -NH-C(O)-NH-, X' is H, O, N, or N-lower cycloalkyl; Y' is O, N, N-lower cycloalkyl, CH ２ , C.R. ５ , CHR ５ and R ５ is alkyl, hydroxy, alkoxy, halogen, and the alkyl is optionally substituted with one or more of COOH, oxo, alkoxy, halogen, or hydroxylamine, adjacent Y' are bonded by a single bond or a double bond, and two R's bonded to two adjacent Y's are ５ are optionally linked to form an aromatic or non-aromatic 5- or 6-membered ring, and preferably X' is O and the Y' ring contains one or two N or O atoms, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. 【Request 18】 【Chemical 205-1】 【Hua 205-2】 【Chemistry 205-3】 【Hua 205-4】 18. The compound of claim 17 selected from the group consisting of: and pharmaceutically acceptable salts, hydrates, solvates or polymorphs thereof.

19. A pharmaceutical composition comprising a compound of Formula I, I', II, III, IV, V, VI, VII, or VIII, or a salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

20. A method for treating a disease or disorder associated with abnormal PANX1 signaling, comprising administering to a subject in need thereof a compound of Formula I, I', II, III, IV, V, VI, VII, or VIII, or a salt or solvate thereof.

21. 21. The method of claim 20, wherein the disease or disorder is selected from the group comprising chronic pain, chemotherapy-related pain, addiction, particularly opioid addiction, epilepsy, Parkinson's disease, Alzheimer's disease, multiple sclerosis, traumatic brain injury, migraine, stroke, cancer, particularly melanoma, hepatocellular carcinoma, breast cancer, colorectal cancer, pancreatic cancer, and leukemia, cardiovascular diseases, particularly cardiac arrhythmias, vascular inflammation, hypertension, and pulmonary arterial hypertension, inflammatory diseases, particularly arthritis and wound healing inflammatory diseases, lung diseases, particularly Covid-19, asthma, and primary and secondary ciliary dyskinesia, fibrosis, diabetes, eye diseases, and skin diseases.

22. 22. The method of claim 20 or 21, wherein the disease or disorder is chronic pain.

23. 22. The method of claim 20 or 21, wherein the disease or disorder is opioid addiction.

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