Patent Application
20240279201
The present disclosure relates generally to compounds that dually inhibit the Wnt signaling pathway and activate the AMPK pathway and more particularly, but not exclusively, to compounds that inhibit the Wnt/β-catenin pathway and activate the AMPK pathway for the treatment of diseases that implicate the Wnt/β-catenin pathway and/or the AMPK pathway.
A number of individuals are affected each year by diseases that implicate aberrant activity in Wnt signalling, which may result in abnormal levels of β-catenin. These diseases include metabolic diseases and cancer, for example. Among the known crossing pathways, the adenosine monophosphate-activated kinase (AMPK) pathway plays a role in maintaining energy homeostasis at the cellular and whole-body levels.
There is a need in the field for new and potent therapeutics that inhibit the Wnt/β-catenin pathway and/or activate the AMPK pathway as treatments for disease. The present disclosure meets those needs.
The present disclosure meets the needs in the field by providing compounds and methods for the treatment of diseases that implicate the Wnt/β-catenin signaling pathway and/or the AMPK pathway.
In one aspect the compounds of the disclosure may include a compound of formula (I):
In some embodiments, ring A is selected from substituted or unsubstituted triazole, substituted or unsubstituted tetrazole, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrrole, and substituted or unsubstituted thiazole.
In some embodiments, the compound of formula (I) is a compound of any one of formula (10)-(15), or a pharmaceutically acceptable salt thereof.
In some embodiments, R1 is selected from substituted or unsubstituted quinoline, substituted or unsubstituted quinoxaline, substituted or unsubstituted benzothiazole, substituted or unsubstituted isoquinoline, substituted or unsubstituted pyridine, substituted or unsubstituted quinazoline, substituted or unsubstituted 1,5-naphthyridine, substituted or unsubstituted 1,8-naphthyridine, substituted or unsubstituted thiazole, and substituted or unsubstituted benzoxazole.
In some embodiments, R1 is selected from
In some embodiments, R3 is selected from substituted or unsubstituted pyridyl, unsubstituted aryl, and aryl substituted with one or more groups selected from halo, —CN, alkyl, fluroroalkyl, alkoxy, and fluoroalkoxy.
In some embodiments, R3 is selected from
In some embodiments, each R4, R5, and R6 is independently selected from H, methyl, ethyl, i-propyl, t-butyl, and —CF3.
In some embodiments, the compound of formula (I) is a compound of any one of formula (20)-(29), or a pharmaceutically acceptable salt thereof.
In some embodiments, R1 is selected from
In some embodiments, R2 is H.
In some embodiments, R3 is selected from
In some embodiments, R4 is selected from methyl and ethyl.
In some embodiments, R5 is
In some embodiments, the compound is a compound of any one of formula 1001-1126, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of any one of formula 1001, 1002, 1024, 1032-1036, 1078, 1096, 1100, 1101, 1114, 1121, 1122, or 1124, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula 1001.
In some embodiments, the pharmaceutically acceptable salt is selected from valproic acid, maleic acid, tartaric acid, oxalic acid, pamoic acid, phosphonic acid, benzoic acid, citric acid, salicylic acid, succinic acid, methanesulfonic acid, malic acid, and p-toluenesulfonic acid.
In one aspect, the disclosure provides a compound of formula (II):
In some embodiments, one or more Ra1 is fluoro. In some embodiments, each occurrence of Ra1 is fluoro. In some embodiments, Ra3 is fluoro. In some embodiments, one or more Rb is fluoro. In some embodiments, each occurrence of Rb is fluoro. In some embodiments, Rc2 is fluoro. In some embodiments, Rc5 is fluoro. In some embodiments, the compound comprises only one fluoro group. In some embodiments, the only one fluoro group is at Ra2, Ra3, Ra4, Ra5, Rc1, Rc2, Rc3, Rc4, or R5c. In some embodiments, the remaining Ra2, Ra3, Ra4, Ra5, Rc1, Rc2, Rc3, Rc4, and Rc5 are hydrogen, and each occurrence of Rai is hydrogen, and each occurrence of Rb is hydrogen. In some embodiments, the compound comprises exactly three fluoro groups, wherein the three fluoro groups are at each occurrence of Rai or at each occurrence of Rb. In some embodiments, the remaining Ra1 or Rb are at each occurrence hydrogen, and Ra2, Ra3, Ra4, Ra5, Rc1, Rc2, Rc3, Rc4, or Rc5 are each hydrogen.
In some embodiments, the compound of formula (II) is a compound of any one of formula 2001-2031, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure includes a pharmaceutical composition comprising one or more of compounds of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula (2001-2031), formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In one aspect, the disclosure provides a compound of formula (III):
In some embodiments, Ra1, Ra2, Ra3, Ra4, Ra5, Rb, Rc1, Rc2, Rc3, Rc4, and Rc5 are at each occurrence hydrogen. In some embodiments, L comprises one or more linking groups selected from optionally substituted —C1-10 alkyl-, —O—C1-10 alkyl-, —C1-10 alkenyl-, —O—C1-10 alkenyl-, —C1-10 cycloalkenyl-, —O—C1-10 cycloalkenyl-, —C1-10 alkynyl-, —O—C1-10 alkynyl-, —C1-10 aryl-, —O—C1-10—, -aryl-, —O—, —S—, —S—S—, —S(O)w—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)S—, —SC(O)—, —OC(O)O—, —N(Rb)—, —C(O)N(Rb)—, —N(Rb)C(O)—, —OC(O)N(Rb)—, —N(Rb)C(O)O—, —SC(O)N(Rb)—, —N(Rb)C(O)S—, —N(Rb)C(O)N(Rb)—, —N(Rb)C(NRb)N(Rb)—, —N(Rb)S(O)w—, —S(O)wN(Rb)—, —S(O)wO—, —OS(O)w—, —OS(O)wO—, —O(O)P(ORb)O—, (O)P(O—)3, —O(S)P(ORb)O—, and (S)P(O—)3, wherein w is 1 or 2, and Rb is independently hydrogen, optionally substituted alkyl, or optionally substituted aryl. In some embodiments, L comprises one or more linking groups selected from —C1-10 alkyl-N(Rb)—, —O—C1-10 alkyl-, —O—, —N(Rb)—, and —S—S—. In some embodiments, L comprises one or more linking group selected from and
In some embodiments, L comprises
wherein n is an integer selected from 0-5. In some embodiments, L comprises
wherein n is an integer selected from 0-5. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the targeting moiety comprises one or more moieties selected from biotin, folic acid, and biguanide.
In some embodiments, the targeting moiety is selected from:
In some embodiments, the compound of formula (III) is a compound of any one of formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure includes a pharmaceutical composition for treating or preventing a disease alleviated by inhibiting Wnt/β-catenin signaling, the pharmaceutical composition comprising one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In one aspect, the disclosure includes a pharmaceutical composition for treating or preventing a disease alleviated by activating adenosine monophosphate-activated kinase (AMPK) signaling, the pharmaceutical composition comprising one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In one aspect, the disclosure includes a pharmaceutical composition for treating or preventing a disease alleviated by both inhibiting Wnt/β-catenin signaling and activating adenosine monophosphate-activated kinase (AMPK) signaling, the pharmaceutical composition comprising one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium. In some embodiments, the disease or disorder is cancer or a metabolic disease. In some embodiments, the disease or disorder is selected from the group consisting of type 2 diabetes, obesity, hyperlipidemia, fatty liver disease, adrenocortical cancer, hepatocellular cancer, hepatoblastoma, malignant melanoma, ovarian cancer, Wilm's tumor, Barrett's esophageal cancer, prostate cancer, colon cancer, colorectal cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, gastric cancer, head & neck cancer, lung cancer, mesothelioma, cervical cancer, uterine cancer, myeloid leukemia cancer, lymphoid leukemia cancer, pilometricoma cancer, medulloblastoma cancer, glioblastoma, and familial adenomatous polyposis. In some embodiments, the breast cancer is triple negative breast cancer.
In one aspect, the disclosure includes a pharmaceutical composition for treating liver fibrosis, the pharmaceutical composition comprising one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In one aspect, the disclosure includes a pharmaceutical composition for treating colorectal cancer (CRC), the pharmaceutical composition comprising one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In one aspect, the disclosure includes a pharmaceutical composition for treating alcoholic fatty liver disease (ALD) or non-alcoholic fatty liver disease (NAFLD), the pharmaceutical composition comprising one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium. In some embodiments, the non-alcoholic fatty liver disease is selected from the group consisting of simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), and liver cirrhosis.
In some embodiments, the pharmaceutical composition includes one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is selected from the group consisting of a RAF inhibitor, an MEK inhibitor, an ERK inhibitor, a VEGFR inhibitor, EGFR inhibitor, and a combination thereof. In some embodiments, the VEGFR inhibitor is selected from the group consisting of Bevacizumab (AVASTIN), Aflibercept (ZALTRAP), Regorafenib (STIVARGA), and a combination thereof. In some embodiments, the EGFR inhibitor is selected from the group consisting of Cetuximab (ERBITUX), Panitumumab (VECTIBIX), Gefitinib, and a combination thereof. In some embodiments, the additional therapeutic agent is selected from angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, caspase inhibitors, cathepsin B inhibitors, CCR2 chemokine antagonists, CCR5 chemokine antagonists, chloride channel stimulators, cholesterol solubilizers, diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors, dipeptidyl peptidase IV (DPPIV) inhibitors, farnesoid X receptor (FXR) agonists, FXR/TGR5 dual agonists, galectin-3 inhibitors, glucagon-like peptide 1 (GLPl) agonists, glutathione precursors, hepatitis C virus NS3 protease inhibitors, HMG CoA reductase inhibitors, 1 Iβ-hydroxy steroid dehydrogenase (I Iβ-HSDl) inhibitors, IL-Iβ antagonists, IL-6 antagonists, IL-10 agonists, IL-17 antagonists, ileal sodium bile acid cotransporter inhibitors, leptin analogs, 5-lipoxygenase inhibitors, LPL gene stimulators, lysyl oxidase homolog 2 (LOXL2) inhibitors, PDE3 inhibitors, PDE4 inhibitors, phospholipase C (PLC) inhibitors, PPARa agonists, PPARy agonists, PPAR5 agonists, Rho associated protein kinase 2 (ROCK2) inhibitors, sodium glucose transporter-2 (SGLT2) inhibitors, stearoyl CoA desaturase-1 inhibitors, thyroid hormone receptor β agonists, tumor necrosis factor a (TNFa) ligand inhibitors, transglutaminase inhibitors, transglutaminase inhibitor precursors, PTPlb inhibitors, and ASK1 inhibitors. In some embodiments, the additional therapeutic agent is selected from capecitabine; cetuximab; bevacizumab; a MEK inhibitor such as N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, or a pharmaceutically acceptable salt thereof; a FOLFOX4 combination including oxaliplatin, 5-fluorouracil and leucovorin; and a FOLFIRI combination include irinotecan, 5-fluorouracil and leucovorin and the like.
In one aspect, the disclosure includes a method of treating or preventing a disease or disorder alleviated by inhibiting Wnt/β-catenin signaling in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure provides a method of treating or preventing a disease or disorder alleviated by activating adenosine monophosphate-activated kinase (AMPK) signaling in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure includes method of treating or preventing a disease or disorder alleviated by both inhibiting Wnt/β-catenin signaling and activating adenosine monophosphate-activated kinase (AMPK) signaling in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In some embodiments, the disease is cancer or a metabolic disease. In some embodiments, the disease is selected from the group consisting of type 2 diabetes, obesity, hyperlipidemia, fatty liver disease, adrenocortical cancer, hepatocellular cancer, hepatoblastoma, malignant melanoma, ovarian cancer, Wilm's tumor, Barrett's esophageal cancer, prostate cancer, colon cancer, colorectal cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, gastric cancer, head & neck cancer, lung cancer, mesothelioma, cervical cancer, uterine cancer, myeloid leukemia cancer, lymphoid leukemia cancer, pilometricoma cancer, medulloblastoma cancer, glioblastoma, and familial adenomatous polyposis. In some embodiments, the disease or disorder is type 2 diabetes. In some embodiments, the disease or disorder is colon cancer and/or colorectal cancer. In some embodiments, the disease or disorder is fatty liver disease. In some embodiments, the fatty liver disease comprises a nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In some embodiments, the breast cancer is triple negative breast cancer.
In one aspect, the disclosure includes a method of treating or preventing liver fibrosis in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure includes a method of treating or preventing fatty liver disease in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In some embodiments, the disease comprises alcoholic fatty liver disease (ALD) or a non-alcoholic fatty liver disease (NAFLD). In some embodiments, the disease is selected from the group consisting of simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), and cirrhosis. In some embodiments, the disease is non-alcoholic steatohepatitis (NASH). In some embodiments, the one or more compounds are administered orally. In some embodiments, the one or more compounds are administered in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is selected from the group consisting of a RAF inhibitor, an MEK inhibitor, an ERK inhibitor, a VEGFR inhibitor, EGFR inhibitor, and a combination thereof. In some embodiments, the VEGFR inhibitor is selected from the group consisting of Bevacizumab (AVASTIN), Aflibercept (ZALTRAP), Regorafenib (STIVARGA), and a combination thereof. In some embodiments, the EGFR inhibitor is selected from the group consisting of Cetuximab (ERBITUX), Panitumumab (VECTIBIX), Gefitinib, and a combination thereof. In some embodiments, the additional therapeutic agent is selected from angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, caspase inhibitors, cathepsin B inhibitors, CCR2 chemokine antagonists, CCR5 chemokine antagonists, chloride channel stimulators, cholesterol solubilizers, diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors, dipeptidyl peptidase IV (DPPIV) inhibitors, farnesoid X receptor (FXR) agonists, FXR/TGR5 dual agonists, galectin-3 inhibitors, glucagon-like peptide 1 (GLPl) agonists, glutathione precursors, hepatitis C virus NS3 protease inhibitors, HMG CoA reductase inhibitors, 1 Iβ-hydroxy steroid dehydrogenase (I Iβ-HSDl) inhibitors, IL-Iβ antagonists, IL-6 antagonists, IL-10 agonists, IL-17 antagonists, ileal sodium bile acid cotransporter inhibitors, leptin analogs, 5-lipoxygenase inhibitors, LPL gene stimulators, lysyl oxidase homolog 2 (LOXL2) inhibitors, PDE3 inhibitors, PDE4 inhibitors, phospholipase C (PLC) inhibitors, PPARa agonists, PPARy agonists, PPAR5 agonists, Rho associated protein kinase 2 (ROCK2) inhibitors, sodium glucose transporter-2 (SGLT2) inhibitors, stearoyl CoA desaturase-1 inhibitors, thyroid hormone receptor β agonists, tumor necrosis factor a (TNFa) ligand inhibitors, transglutaminase inhibitors, transglutaminase inhibitor precursors, PTPlb inhibitors, and ASK1 inhibitors. In some embodiments, the additional therapeutic agent is selected from capecitabine; cetuximab; bevacizumab; a MEK inhibitor such as N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, or a pharmaceutically acceptable salt thereof; a FOLFOX4 combination including oxaliplatin, 5-fluorouracil and leucovorin; and a FOLFIRI combination include irinotecan, 5-fluorouracil and leucovorin and the like.
The foregoing summary and the following detailed description of the exemplary embodiments of the present disclosure may be further understood when read in conjunction with the appended drawings, in which:
The present disclosure relates generally to compounds, and methods of using such compounds, that may inhibit the Wnt/β-catenin signaling pathway and/or activate the AMPK pathway. More specifically, the compounds of the disclosure may be used in treating diseases that implicate the Wnt/β-catenin signaling pathway and/or activate the AMPK pathway.
The Wnt/β-catenin signaling pathway plays a pivotal role in cell proliferation, differentiation, and organ development. Moreover, it regulates the transcription of its target genes through the transcriptional factor β-catenin. In the “off state” without Wnt ligands, β-catenin forms a cytoplasmic “destruction complex” with adenomatous polyposis coli (APC) and Axin, which facilitates the phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1) at the N-terminal residues Ser45, Thr41, Ser37, and Ser33. Phosphorylated β-catenin is recognized and ubiquitinated by the F-box β-transducin repeat-containing protein β-TrCP), and then degraded by the proteasome. On the other hand, the “on-state” of the pathway involves increased post-translational stability and thus accumulation of β-catenin, through Wnt-dependent degradation of Axin and inhibition of GSK3 by various mechanisms. As the β-catenin level increases, it translocates to the nucleus where it binds to LEF/TCF such as TCF7L2 and activates expression of downstream genes. Increased expression of β-catenin and malfunction of the Wnt-signaling pathway are implicated in a variety of diseases.
As an emerging research area, however, the link between Wnt/β-catenin pathway and metabolic diseases has only been appreciated recently. For example, a strong association exists between type 2 diabetes risk and single nucleotide polymorphisms (SNPs) in TCF7L2, a classic effector of Wnt/β-catenin pathway. Similar genetic evidence exists for additional modulators of Wnt signaling pathway such as WNT5B, WNT10B, and LRP6. Follow-up genetic studies have indicated that global downregulation of Wnt/β-catenin signaling activity leads to overall improved metabolic homeostasis in diabetic animal models
The metabolic disease NAFLD is the most common form of chronic liver disease and ranges in severity from relatively simple benign steatosis to NASH, which is highly prevalent in type 2 diabetes or obese patients, and is a burgeoning public health problem due to the global diabetes and obesity epidemic. Dietary control and exercise are currently the recommendation to reverse NAFLD/NASH; however, their long-term effectiveness is uncertain because many patients are unable to comply. Thus, an effective pharmacological therapeutic is highly in demand.
Reducing β-catenin expression by antisense oligonucleotides decreases expression of enzymes involved in hepatic fatty acid esterification and ameliorates diet-induced hepatic steatosis and insulin resistance. Indeed, an antisense oligonucleotide against β-catenin could totally reverse diet-induced fatty liver and obesity back to normal, improve glucose tolerance, and reduce fasting glucose levels in the blood of mice.
Nonalcoholic fatty liver disease (NAFLD) affects more than 30% of Americans. At present, there are no pharmacological options approved for NAFLD and its clinical sequelae including nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Steatosis is the most outstanding feature of NAFLD/NASH, while progressive accumulation of fibrosis is the hallmark of the disease progression. Beta-catenin and adenosine monophosphate-activated kinase (AMPK) are crucial proteins in regulation of hepatic metabolism that interact with the scaffold protein Axin. Genetic evidence supports a metabolic benefit by β-catenin downregulation on hepatic metabolism; meanwhile, AMPK has emerged as a target against metabolic disorders.
Nonalcoholic fatty liver disease (NAFLD) is defined as fat accumulation in the liver exceeding 5-10% by weight in individuals that do not drink alcohol (<2 drinks/day). NAFLD affects 34-46% of Americans. While NAFLD is a generally benign condition, NASH, the most severe form, causes liver swelling, fibrosis and scarring of the liver. In 25% NASH patients, fibrosis leads to cirrhosis. NASH affects approximately 12% of Americans. Control of lipids by diet and exercise has shown some benefits for NASH; but adherence to long-term lifestyle changes remains a large challenge for patients. There are currently no approved pharmacological therapies for the treatment of NAFLD/NASH.
An investigation into drugs with metabolic side effects indicated that many of these drugs were an activator of Wnt/β-catenin pathway. In fact, increasing evidence indicates that dysregulation of Wnt/β-catenin pathway is a causative pathological mechanism for metabolic diseases (ref). Inhibition of this pathway may hold therapeutic benefits for the treatment of metabolic diseases including NASH. In one aspect, the present disclosure provides a class of compounds that can stabilize cellular Axin levels. Axin is an important scaffold protein that interacts with β-catenin and other proteins to form a destruction complex. It facilitates degradation of/i-catenin, leading to inhibition of Wnt/β-catenin pathway. Axin has been recently found to also interact with adenosine monophosphate-activated kinase (AMPK), which is a crucial cellular energy sensor.
Globally, the prevalence of NAFLD is estimated at ˜25%. NAFLD/NASH causes a growing economic challenge, with an estimated direct annual cost of $103 billion in the US. In the US, NAFLD cases are projected to expand from 83.1 million in 2015 (˜25% of the population) to 100.9 million in 2030. An increased proportion of these cases will be NASH, rising from 20% to 27% of adults with NAFLD during this interval. NASH is the second indication for liver transplantation. To date, no evidence-based drug therapy has been approved for NASH management and NASH is classified as a medical condition with high-unmet therapeutic need.
It is estimated that the drug market for NAFLD/NASH will be worth $20 billion to $35 billion per year by 2025. According to a very recent report, the global NASH drug market will reach $84.34 billion in 2029 with a CAGR of 39% (2018-2029). The current NASH drug market is wholly occupied by off-label drugs as there are no clinically approved medicines for NASH. Non-pharmacological treatments remain the first line of management for NASH patients. However, there are over 300 on-going clinical trials investigating approximate 200 candidate drugs for NASH. Because there has been a high failure rate for late-stage drug candidates that provides a significant opportunity for new approaches, there remains a significant need for new pathways to treat NASH.
There is thus a need to provide NASH patients with a novel drug therapy. In one aspect, the compounds of the instant disclosure, such as YA6060, are useful as a novel drug therapy for NASH, and for other diseases as well.
Five candidates have entered into phase III trials. While data for the other four have not been released, the likelihood of approval for Allergan's cenicriviroc is low due to recent Part I results showing lacking efficacy. The mechanisms for these phase III candidate drugs, and others in earlier phase development include FXR agonization, PPAR-α agonization, ASK1 inhibition, CCR2/5 antagonization, and THRβ agonization.
In contrast, the compounds of the instant disclosure have a unique mechanism. In a non-limiting example, YA6060 is competitive as it has dual activities of β-catenin degradation and AMPK activation via Axin stabilization that is critical in liver metabolism but not claimed by any of on-going candidate drugs. NCE's with multiple modes of action and/or targets, such as YA6060 could prove most promising, as NASH pathogenesis involves many disease drivers. Metformin is currently the only AMPK activator (e.g., metformin). However, the efficacy of metformin in NASH patients is disputed because of widely varying efficacy outcomes in various clinical trials, suggesting AMPK activation alone may not deliver a consistent efficacy in NASH patients. Known Axin stabilizers, such as XAV939 and IWR1, are tankyrase inhibitors, to which the efficacy against NASH is unknown. Concerns have arisen for GI toxicity for these class of compounds. Tankyrase inhibitors may have limited efficacy due to their undesirable ability to stabilize the protein level of tankyrase. Instead YA6060 stabilizes Axin by binding to TAB182, a protein that binds and activates tankyrase, which provides a strategy to bypass the side effects associated with tankyrase inhibitors.
One in every 23 people is expected to be diagnosed with colorectal cancer (CRC) during her/his lifetime. As the third most common cancer, CRC is the second leading cause of cancer-related death in the United States. The 5-year survival rate for patients with metastatic CRC is less than 12%. Chemotherapies 5-FU, oxaliplatin, and regimens based on them continue to be a first-line treatment despite their significant limitations and high toxicities. Emerging biologicals targeting VEGF and EGFR do not significantly improve survival rate. These underline the urgent need to discover novel targeted therapeutics against CRC.
More than 92% of CRC are characterized by aberrant activation of Wnt/β-catenin signaling due to genetic mutations or epigenetic changes. Activated Wnt signaling represents a preferred target for the treatment of CRC. However, the genetic/epigenetic changes associated with Wnt signaling upregulation are also a major reason for high heterogeneity of CRC and pose challenges to target this pathway. Meanwhile, metabolic reprogramming has been well demonstrated as a hallmark of cancer progress and adenosine monophosphate-activated kinase (AMPK), a crucial energy sensor, has emerged as a target against cancer metabolism. Thus, simultaneously targeting the Wnt signaling and AMPK pathways represents a novel therapeutic strategy for the treatment of CRC.
The Wnt/β-catenin pathway plays a key role in cell proliferation, differentiation and growth. It regulates the expression of target genes via the transcriptional factor β-catenin that forms a cytoplasmic destruction complex with the scaffolding protein Axin and adenomatous polyposis coli (APC). This destruction complex facilitates the phosphorylation of β-catenin by casein kinase 1α (CK1α) and glycogen synthase kinase β(GSK3β), causing β-catenin degradation and therefore suppressed Wnt signaling (
CRC is characterized by aberrant Wnt signaling and metabolic dysregulation. CRC causes over 50,000 deaths yearly and may soon surpass lung cancer to become the leading cause of cancer-related death in the U.S. Metastatic CRC has a 5-year survival rate of ˜12%. First-line treatments for metastatic CRC include the thymidylate synthase inhibitor 5-FU, DNA-alkylating agent oxaliplatin, and their combinations (e.g., FOLFOX, FOLFIRI, and FOLFOXIRI) despite the limited therapeutic index. New biologicals targeting vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) (e.g., bevacizumab, cetuximab and panitumumab) do not significantly improve survival rate. Therefore, novel CRC therapeutics are urgently needed. Over 92% of CRC are characterized by aberrant activation of Wnt signaling due to genetic mutations or epigenetic silencing of Wnt antagonist genes. On the other hand, metabolic reprogramming has been well demonstrated as a hallmark of cancer progress. Cancer cells including CRC are under selection pressure to downregulate AMPK, the crucial cellular energy sensor associated with cell viability. AMPK activation can reduce cancer cell proliferation and growth, mainly via the activation of the p53 and mTOR pathways.
Therapeutic targeting of Wnt signaling and AMPK pathways is a viable strategy. While safety concern is historically present due to the severe phenotypes in genetic knockout animal models, it has not been borne out either preclinically or clinically from approximate a dozen of pharmacological inhibitors, and Wnt signaling-targeting agents have entered clinical trials. Of note, a few FDA-approved drugs, such as glucocorticoids, retinoids, and celecoxib, are also found to have strong inhibitory activities. However, oncogenic mutations or epigenetic changes associated with Wnt signaling activation can lead to high heterogeneity of CRC, which may result in limited efficacy by targeting Wnt signaling alone. On the other hand, as a crucial energy sensor, AMPK is a preferred therapeutic target against cancer metabolism. In recent years, many AMPK activators have been identified and tested in preclinical models, and a small number have entered clinical trials. Notably, metformin, a widely prescribed anti-diabetic, exhibits anti-CRC effects by acting on tumor suppressor pathways via AMPK activation. Compared to single pathway modulation, simultaneously targeting both Wnt/β-catenin and AMPK pathways may offer anti-CRC agents that have not only higher efficacy but also broader application.
Wnt inhibitors have been developed by targeting different components of the pathway. Novartis clinical candidate LGK974 and its analog GNF-6231, as well as IWP-2 are porcupine inhibitors, which functions by decreasing the secretion of Wnt ligands (
AMPK includes three subunits: the protein kinase catalytic a subunit, and non-catalytic β and γ subunits (
In one aspect, the present disclosure provides knowledge on Wnt signaling and AMPK pathways to provide novel Axin stabilizers with previously unrecognized mechanism of action, e.g. first-in-class small molecules that interfere with the binding of TNKS-binding protein1 (TAB182) to TNKS by using a combined approach of biochemistry, biology and state-of-art proteomics. In some embodiments, the compounds of the disclosure are useful as anti-CRC agents.
In one aspect, the disclosure provides the development of an improved generation of Wnt inhibitors with significantly enhanced aqueous solubility (>10,000 fold) and bioavailability (>3 fold) that maintain the inhibitory potency against the Wnt signaling pathway via Axin stabilization.
In one aspect, the compounds and methods of the disclosure support targeting the Wnt/β-catenin pathway and/or the AMPK pathway as a strategy to treat NAFLD.
In one aspect, the compounds and methods of the disclosure support targeting the Wnt/β-catenin pathway and/or the AMPK pathway as a strategy to treat colorectal cancer (CRC).
In one embodiment, the compounds of the disclosure may be used as treatments for metabolic disease in a patient in need thereof. As used herein, the term “metabolic disease” may refer to diseases that involve a disruption to a patient's metabolic homeostasis, including, but not limited to, type 2 diabetes, obesity, hyperlipidemia, and fatty liver disease. In some embodiments, the metabolic disease described in the disclosure may be type 2 diabetes. In some embodiments, fatty liver disease may include alcoholic fatty liver disease (ALD) or non-alcoholic fatty liver disease (NAFLD). In some embodiments, NAFLD may include one or more of simple fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), and liver cirrhosis. In certain embodiments, the metabolic disease may be NASH.
Research in different groups has identified an association between type 2 diabetes risk and single nucleotide polymorphisms (SNPs) in TCF7L2, an effector of the Wnt/β-catenin pathway. Similar genetic evidence has been obtained for additional modulators of the Wnt signaling pathway, including WNT5B, WNT10B, and LRP6. Therefore, the Wnt/β-catenin pathway has emerged as a novel therapeutic target for treating disease, including metabolic disorders.
With respect to cancer, the Wnt pathway may be activated in a variety of cancers including, for example, colon cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, and leukemias such as CML, CLL and T-ALL. The activation is due to constitutively active β-catenin, perhaps, without being limited to any one theory, due to its stabilization by interacting factors or inhibition of the degradation pathway. Accordingly, the compounds and compositions described herein may be used to treat these cancers in which the Wnt pathway is constitutively activated.
Folate receptor (FR)-targeted therapy. The FR family is a group of three folic acid (FA)-binding receptors including FRα, FRβ, and FRγ. Different to FRγ that is a soluble protein constitutively secreted by lymphoid, FRα and FRβ are glycosyl phosphatidylinositol (GPI)-anchored receptors that share ˜70% homology and a common mechanism of endocytosis-mediated folate uptake. FRs are the only known folate-specific transporters with a very high affinity (KD<1 nM) for FA. Both FRα and FRβ are normally expressed low in healthy tissues, while selectively overexpressed in cancers including ovarian, kidney, lung, breast, and colon cancers, to meet the high folate demand of rapid dividing cells under low folate conditions. FRα and FRβ are able to transport a variety and broad size range of chemical conjugates of FA, antifolate drugs and immunological agents, as a result, FRs has been widely used as a target for tumor-selective drug delivery employing FA as a targeting ligand.
In some embodiments, the disclosure provides methods of treating a disease or disorder implicating the Wnt/β-catenin pathway and/or the AMPK pathway. In some embodiments, the disease or disorder is cancer or a hyperoliferative disease. In some embodiments, the cancer or hyperproliferative disease may be one or more of adrenocortical cancer, hepatocellular cancer, hepatoblastoma, malignant melanoma, ovarian cancer, Wilm's tumor, Barrett's esophageal cancer, prostate cancer, colon cancer, colorectal cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, gastric cancer, head & neck cancer, lung cancer, mesothelioma, cervical cancer, uterine cancer, myeloid leukemia cancer, lymphoid leukemia cancer, pilometricoma cancer, medulloblastoma cancer, glioblastoma, and familial adenomatous polyposis. In some embodiments, the cancer or hyperproliferative disease may include colon cancer. In some embodiments, the cancer or hyperproliferative disease may include colorectal cancer. In some embodiments, the cancer or hyperproliferative disease may include triple negative breast cancer.
In one aspect, the disclosure provides the development of a first in class small molecule with the dual activities of Wnt signaling inhibition and AMPK activation, via stabilization of the scaffold protein Axin. Although Axin stabilization by TNKS inhibitors are known, they have limited efficacy and gastrointestinal toxicity.
FRs are selectively overexpressed in cancers over healthy tissues. Therefore, FR-targeted FA-based hybrids provide a novel strategy to selectively deliver compounds with dual activity of Wnt inhibition and AMPK activation.
In one aspect, the disclosure provides an advanced HT-29-luc-D6 tumor Cecal implantation mouse model of CRC invasion and metastasis used to test selected compounds, which is used in addition to a commonly used animal model of intestinal neoplasia. In an embodiment, the model is highly clinically relevant to advanced human CRC for which highly effective targeted therapies are missing.
In one aspect, the disclosure provides novel Axin stabilizers with previously unrecognized mechanism of action useful as anti-CRC agents. By using a combined approach of biochemistry, biology and state-of-art proteomics, new knowledge on first-in-class small molecules that interfere with the binding of TNKS-binding protein1 (TAB182) to TNKS Wnt signaling and AMPK pathways are identified.
In some embodiments, cancers that may be treated by the compounds, compositions and methods described herein include, but are not limited to, the following:
Cancers may be solid tumors that may or may not be metastatic. Cancers may also occur, as in leukemia, as a diffuse tissue. Thus, the term “tumor cell,” as provided herein, includes a cell afflicted by any one of the above identified disorders.
A method of treating cancer using a compound or composition as described herein may be combined with existing methods of treating cancers, for example by chemotherapy, irradiation, or surgery (e.g., oophorectomy). In some embodiments, a compound or composition can be administered before, during, or after another anticancer agent, additional therapeutic agent, or treatment.
In one aspect, the disclosure provides compounds of formula (I):
In some embodiments, the compound of formula (I) comprises one or more fluoro groups. In some embodiments, the compound of formula (I) is covalently bonded to a targeting moiety, or is conjugated to a targeting moiety by way of a linking group.
In some embodiments, ring A is selected from substituted or unsubstituted triazole, substituted or unsubstituted tetrazole, substituted or unsubstituted pyrazole, substituted or unsubstituted pyrrole, and substituted or unsubstituted thiazole.
In some embodiments, the compound of formula (I) is a compound of any one of formula (10)-(15):
In some embodiments, R1 is selected from substituted or unsubstituted quinoline, substituted or unsubstituted quinoxaline, substituted or unsubstituted benzothiazole, substituted or unsubstituted isoquinoline, substituted or unsubstituted pyridine, substituted or unsubstituted quinazoline, substituted or unsubstituted 1,5-naphthyridine, substituted or unsubstituted 1,8-naphthyridine, substituted or unsubstituted thiazole, and substituted or unsubstituted benzoxazole. In some embodiments, R1 is substituted with one or more fluoro groups. In some embodiments, R1 is covalently bonded to a targeting moiety, or is conjugated to a targeting moiety by way of a linking group.
In some embodiments, R1 is selected from
wherein L is a linking group and B is a targeting moiety. In some embodiments, the targeting moiety is selected from biotin, folic acid, and biguanide.
In some embodiments, L comprises one or more linking groups selected from optionally substituted —C1-10 alkyl-, —O—C1-10 alkyl-, —C1-10 alkenyl-, —O—C1-10 alkenyl-, —C1-10 cycloalkenyl-, —O—C1-10 cycloalkenyl-, —C1-10 alkynyl-, —O—C1-10 alkynyl-, —C1-10 aryl-, —O—C1-10—, -aryl-, —O—, —S—, —S—S—, —S(O)w—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)S—, —SC(O)—, —OC(O)O—, —N(Rb)—, —C(O)N(Rb)—, —N(Rb)C(O)—, —OC(O)N(Rb)—, —N(R)C(O)O—, —SC(O)N(Rb)—, —N(Rb)C(O)S—, —N(Rb)C(O)N(Rb)—, —N(Rb)C(NRb)N(Rb)—, —N(Rb)S(O)w—, —S(O)wN(Rb)—, —S(O)wO—, —OS(O)w—, —OS(O)wO—, —O(O)P(OR)O—, (O)P(O—)3, —O(S)P(ORb)O—, and (S)P(O—)3, wherein w is 1 or 2, and Rb is independently hydrogen, optionally substituted alkyl, or optionally substituted aryl. In some embodiments, L comprises one or more linking groups selected from —C1-10 alkyl-N(Rb)—, —O—C1-10 alkyl-, —O—, —N(Rb)—, and —S—S—. In some embodiments, L comprises one or more linking group selected from
In some embodiments, L comprises
wherein n is an integer selected from 0-5. In some embodiments, L comprises
wherein n is an integer selected from 0-5.
In some embodiments, R1 is selected from
In some embodiments, R1 is selected from
In some embodiments, R3 is selected from substituted or unsubstituted pyridyl, unsubstituted aryl, and aryl substituted with one or more groups selected from halo, —CN, alkyl, fluroroalkyl, alkoxy, and fluoroalkoxy. In some embodiments, R3 is substituted with one or more fluoro groups.
In some embodiments, R3 is selected from
In some embodiments, R3 is selected from
In some embodiments, each R4, R5, and R6 is independently selected from H, methyl, ethyl, i-propyl, t-butyl, and —CF3. In some embodiments, R4 is-CF3.
In some embodiments, the compound of formula (I) is a compound of any one of formula (20)-(29):
In some embodiments, the compound of formula (I) is a compound of any one of formula (30)-(31):
In some embodiments, R5 is substituted or unsubstituted piperazine.
In some embodiments, R1 is selected from N
In some embodiments, R1 is substituted with one or more fluoro groups. In some embodiments, R1 is covalently bonded to a targeting moiety, or is conjugated to a targeting moiety by way of a linking group. In some embodiments, R1 is selected from
wherein L is a linking group and B is a targeting moiety. In some embodiments, the targeting moiety is selected from biotin, folic acid, and biguanide. In some embodiments, R1 is selected from
In some embodiments, R2 is H.
In some embodiments, R3 is selected from
In some embodiments, R4 is C1-C6 alkyl. In some embodiments, R4 is selected from methyl and ethyl.
In some embodiments, R8 is
In some embodiments, the compound is a compound of any one of formula 1001-1126, or a pharmaceutically acceptable salt thereof:
YA70044
YA70045
YA70046
YA70047
YA6177
YA7083
YA7084
YA4117
YA7085
YA4133
YA4105
YA4061
YA4045
YA4091
YA4093
YA4101
YA4103
YA4095
YA4013
YA4071
YA4097
YA4047
YA4059
YA4069
YA4065
YA4063
YA4057
YA6029
YA4029
YA1072
YA1076
YA6027
YA4055
YA1074
YA4003
YA1050
YA1086
YA6089
YA1038
YA10146
YA1084
YA6090
YA1066
YA1042
YA1068
YA1064
YA4001
YA4005
YA6176
YA6168
YA4155
YA4165
YA6172
YA6174
YA6175
YA1124
YA1122
YA6152
YA4087
YA4033
YA7018
YA7016
YA7019
YA7015
YA6179
YA4011
YA6149
YA6143
YA6144
YA4009
YA7020
YA4007
YA4025
YA4021
YA4019
YA4017
YA4023
In some embodiments, the compound is a compound of any one of formula 1001, 1002, 1024, 1032-1036, 1078, 1096, 1100, 1101, 1114, 1121, 1122, or 1124, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula 1001.
In one aspect, the disclosure provides compounds of formula (II):
In some embodiments, one or more Ra1 is fluoro. In some embodiments, each occurrence of Ra1 is fluoro.
In some embodiments, Ra3 is fluoro.
In some embodiments, one or more Rb is fluoro. In some embodiments, each occurrence of Rb is fluoro.
In some embodiments, Rc2 is fluoro.
In some embodiments, Rc5 is fluoro.
In some embodiments, the compound comprises only one fluoro group. In some embodiments, the only one fluoro group is at Ra2, Ra3, Ra4, Ra5, Rc1, Rc2, Rc3, Rc4, or Rc5. In some embodiments, the remaining Ra2, Ra3, Ra4, Ra5, Rc1, Rc2, Rc3, Rc4, and Rc5 are hydrogen, and each occurrence of Rai is hydrogen, and each occurrence of Rb is hydrogen. In some embodiments, the compound comprises exactly three fluoro groups, wherein the three fluoro groups are at each occurrence of Rai or at each occurrence of Rb. In some embodiments, the remaining Ra1 or Rb are at each occurrence hydrogen, and Ra2, Ra3, Ra4, Ra5, Rc1, Rc2, Rc3, Rc4, or Rc5 are each hydrogen.
In some embodiments, the compound of formula (II) is a compound of any one of formula 2001-2031, or a pharmaceutically acceptable salt thereof:
In some embodiments, the compound of formula (II) is a compound of any one of formula 2001-2005, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure provides compounds of formula (III):
In some embodiments, Ra1, Ra2, Ra3, Ra4, Ra5, Rb, Rc1, Rc2, Rc3, Rc4, and Rc5 are at each occurrence hydrogen.
In some embodiments, L comprises one or more linking groups selected from optionally substituted —C1-10 alkyl-, —O—C1-10 alkyl-, —C1-10 alkenyl-, —O—C1-10 alkenyl-, —C1-10 cycloalkenyl-, —O—C1-10 cycloalkenyl-, —C1-10 alkynyl-, —O—C1-10 alkynyl-, —C1-10 aryl-, —O—C1-10—, -aryl-, —O—, —S—, —S—S—, —S(O)w—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)S—, —SC(O)—, —OC(O)O—, —N(Rb)—, —C(O)N(Rb)—, —N(Rb)C(O)—, —OC(O)N(Rb)—, —N(Rb)C(O)O—, —SC(O)N(Rb)—, —N(Rb)C(O)S—, —N(Rb)C(O)N(Rb)—, —N(Rb)C(NRb)N(Rb)—, —N(Rb)S(O)w—, —S(O)wN(Rb)—, —S(O)wO—, —OS(O)w—, —OS(O)wO—, —O(O)P(OR)O—, (O)P(O—)3, —O(S)P(ORb)O—, and (S)P(O—)3, wherein w is 1 or 2, and Rb is independently hydrogen, optionally substituted alkyl, or optionally substituted aryl. In some embodiments, L comprises one or more linking groups selected from —C1-10 alkyl-N(Rb)—, —O—C1-10 alkyl-, —O—, —N(Rb)—, and —S—S—. In some embodiments, L comprises one or more linking group selected from
In some embodiments, L comprises
wherein n is an integer selected from 0-5. In some embodiments, L comprises
wherein n is an integer selected from 0-5.
In some embodiments, L is a single bond.
In some embodiment, the linker is a cleavable linker. In a non-limiting example, a cleavable linker is a linker that can selectively release the drug moiety (e.g. an YA6060 moiety or analogue thereof) from the targeting moiety under certain conditions, such as at certain pH levels or sensitivity to proteases or esterases. In some embodiments, the linker is a non-cleavable linker. In a non-limiting example, a non-cleavable linker is a linker that does not have a drug release mechanism, so the drug moiety (e.g. an YA6060 moiety or analogue thereof) stays tethered to the targeting moiety.
In some embodiments, the targeting moiety is a moiety that binds to or is a substrate for receptors that are selectively expressed and/or overexpressed in tumor cells and/or tissue. In some embodiments, the targeting moiety targets the folate receptor (FR) and/or organic cation transports (OCTs), and/or organic anion transporting polypeptide 1B1 (OATP1B1). In some embodiments, the targeting moiety comprises one or more moieties selected from biotin, folic acid, biguanide, and carboxylic acid.
In some embodiments, the targeting moiety is selected from:
In some embodiments, the compound of formula (III) is a compound of any one of formula 3001-3018, or a pharmaceutically acceptable salt thereof:
As used herein, the term “alkyl” denotes branched or unbranched hydrocarbon chains, having about 1 to 10 carbons, such as, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, 2-methylpentyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethyl pentyl, octyl, 2,2,4-trimethylpentyl, and the like. “Substituted alkyl” includes an alkyl group optionally substituted with one or more functional groups which are attached commonly to such chains, such as, hydroxy, halogen, mercapto or thio, cyano, alkylthio, carboxy, nitro, alkoxy, or optionally substituted, alkyl, amino, alkenyl, carboxamido, carbalkoxy, alkynyl, heterocyclyl, aryl, heteroaryl, and the like to form alkyl groups such as trifluoromethyl, 3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl, phenethyl, benzyl, and the like.
The term “halogen” or “halo” as used herein alone or as part of another group refers to chloro, bromo, fluoro, or iodo.
The term “alkoxy” refers to alkyl-O—, in which alkyl is as defined above.
The term “alkylthio” refers to alkyl-S—, in which alkyl is as defined above.
The term “alkylamino” refers to —NR′R″, in which R′ and R″ each may independently represent H, alkyl, or aryl, all as defined herein.
The term “alkylcarbonyl” refers to —C(═O)-alkyl, in which alkyl is as defined above.
The term “carboxy” refers to the moiety —C(═O)OH.
The term “carbalkoxy” refers to the moiety —C(═O)—O-alkyl, in which alkyl is as defined above.
The term “carboxamido” refers to the moiety —C(═O)—NR′R″, in which R′ and R″, each may independently represent H, alkyl, or aryl, all as defined herein.
The term “alkylsulfonyl” refers to the moiety —S(═O)2-alkyl, in which alkyl is as defined above.
The term “arylsulfonyl” refers to the moiety —S(═O)2-aryl, in which aryl is as defined herein. For example, arylsulfonyl may be —S(═O)2-phenyl.
The term “arylsulfonyloxy” refers to the moiety —OS(═O)2-alkyl, wherein alkyl is as defined above.
The term “amino(monoalkylamino-, dialkylamino-)sulfinyl” refers to the moiety —S(═O)NR′R″, in which R′ and R″ each may independently represent H, alkyl, or aryl, all as defined herein.
The term “amino(monoalkylamino-, dialkylamino-)sulfonyl” refers to the moiety —S(═O)2NR′R″, in which R′ and R″ each may independently represent H, alkyl, or aryl, all as defined herein.
The term “alkylsulfonylamino” refers to the moiety —NHS(═O)2-alkyl, in which alkyl is as previously defined.
The term “hydroxysulfonyloxy” refers to the moiety —OS(═O)2OH.
The term “alkoxysulfonyloxy” refers to the moiety —OS(═O)2O-alkyl, in which alkyl is as defined above.
The term “alkylsulfonyloxy” refers to the moiety —OS(═O)2-alkyl, in which alkyl is as previously defined.
The term “hydroxysulfonyl” refers to the moiety —S(═O)2OH.
The term “alkoxysulfonyl” refers to the moiety —S(═O)2O-alkyl, wherein alkyl is as previously defined.
The term “alkylsulfonylalkyl” refers to the moiety -alkyl-S(═O)2-alkyl, wherein each alkyl may be as previously defined.
The term “amino(monoalkylamino-, dialkylamino-)sulfonylakyl” refers to the moiety -alkyl-S(═O)2—NR′R″, wherein alkyl is as previously defined, and R′ and R″ each may independently represent H, alkyl, or aryl, all as defined herein.
The term “amino(monoalkylamino-, dialkylamino-)sulfinylalkyl” refer to the moieties -alkyl-S(═O)—NR′R″, wherein alkyl is as previously defined, and R′ and R″ each may independently represent H, alkyl, or aryl, all as defined herein.
Unless otherwise indicated, the term “cycloalkyl” as employed herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or more double bonds) cyclic hydrocarbon groups containing 1 to 3 rings, including monocyclicalkyl, bicyclicalkyl and tricyclicalkyl, containing a total of 3 to 20 carbons forming the rings, preferably 3 to 10 carbons, forming the ring and which may be fused to 1 or 2 aromatic rings as described for aryl, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and cyclohexenyl. “Substituted cycloalkyl” includes a cycloalkyl group optionally substituted with 1 or more substituents such as halogen, alkyl, substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl, aryloxy, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol and/or alkylthio and/or any of the substituents included in the definition of “substituted alkyl.”
Unless otherwise indicated, the term “alkenyl” as used herein by itself or as part of another group refers to straight or branched chain of 2 to 20 carbons, preferably 2 to 12 carbons, and more preferably 2 to 8 carbons in the normal chain, which include one or more double bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and the like. “Substituted alkenyl” includes an alkenyl group optionally substituted with one or more substituents, such as the substituents included above in the definition of “substituted alkyl” and “substituted cycloalkyl.”
Unless otherwise indicated, the term “alkynyl” as used herein by itself or as part of another group refers to straight or branched chain of 2 to 20 carbons, preferably 2 to 12 carbons and more preferably 2 to 8 carbons in the normal chain, which include one or more triple bonds in the normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl, 4-dodecynyl and the like. “Substituted alkynyl” includes an alkynyl group optionally substituted with one or more substituents, such as the substituents included above in the definition of “substituted alkyl” and “substituted cycloalkyl.”
Unless otherwise indicated, the term “aryl” or “Ar” as employed herein alone or as part of another group refers to monocyclic, bicyclic, and/or polycyclic aromatic groups containing 6 to 10 carbons in the ring portion (such as phenyl or naphthyl including 1-naphthyl and 2-naphthyl) and may optionally include one to three additional rings fused to a carbocyclic ring or a heterocyclic ring, such as aryl, cycloalkyl, heteroaryl, or cycloheteroalkyl rings or substituted forms thereof.
“Substituted aryl” includes an aryl group optionally substituted with one or more functional groups, such as halo, alkyl, haloalkyl (e.g., trifluoromethyl), alkoxy, haloalkoxy (e.g., difluoromethoxy), alkenyl, alkynyl, cycloalkyl-alkyl, cycloheteroalkyl, cycloheteroalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, arylalkenyl, aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro, cyano, amino, substituted amino wherein the amino includes 1 or 2 substituents (which are optionally substituted alkyl, aryl or any of the other substituents recited herein), thiol, alkylthio, arylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylaminocarbonyl, arylaminocarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino, or arylsulfonaminocarbonyl and/or any of the alkyl substituents recited herein.
Unless otherwise indicated, the term “heteroaryl” as used herein alone or as part of another group refers to a 5- to 7-membered aromatic ring which includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygen or sulfur and such rings fused to an aryl, cycloalkyl, heteroaryl or heterocycloalkyl ring (e.g. benzothiophene, indole, quinoline, thiazole, isooxazole, benzothiazole, benzimidizole, isoquinoline, pyridine, pyrimidine, benzopyrone, oxazole, thiazole, pyrazine), and includes possible N-oxides. “Substituted heteroaryl” includes a heteroaryl group optionally substituted with 1 to 4 substituents, such as the substituents included above in the definition of “substituted alkyl” and “substituted cycloalkyl.” Substituted heteroaryl also includes fused heteroaryl groups which include, for example, quinoline, isoquinoline, indole, isoindole, carbazole, acridine, benzopyrene, benzopyrone, benzimidazole, benzofuran, isobenzofuran, phenanthroline, purine, and the like.
Moreover, the terms “heterocyclo,” “heterocycle,” or “heterocyclic ring,” as used herein, refer to an unsubstituted or substituted stable 5- to 7-membered monocyclic ring system which may be saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from N, O or S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic groups include, but are not limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, benzothiophene, chromone, benzopyrene, benzopyrone, furanyl, thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, and oxadiazolyl.
As used herein, the terms “optionally substituted” or “substituted” may indicate that a chemical moiety referred to, for example, alkyl, aryl, and heteroaryl, may be unsubstituted or substituted with one or more groups including, without limitation, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, arylalkyl, substituted arylalkyl, aryl, substituted aryl, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, hydroxyl, amino, substituted amino, alkoxy, substituted alkoxy, halogen, carboxy, nitro, carbalkoxy, substituted carbalkoxy, carboxamido, substituted carboxamido, alkylamino, substituted alkyl amino, monoalkylaminosulfinyl, substituted, monoalkylaminosulfinyl, dialkylaminosulfinyl, substituted dialkylaminosulfinyl, monoalkylaminosulfonyl, substituted monoalkylaminosulfonyl, dialkylaminosulfonyl, substituted dialkylaminosulfonyl, alkylsulfonylamino, substituted alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy, substituted alkoxysulfonyloxy, alkylsulfonyloxy, substituted alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, substituted alkoxysulfonyl, alkylsulfonylalkyl, substituted alkylsulfonylalkyl, monoalkylaminosulfonylalkyl, substituted monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl, substituted dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl, substituted monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl, substituted dialkylaminosulfinylalkyl, and the like. The chemical moieties of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, above, that may be optionally substituted include alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl, heterocycle, and heteroaryl, as described herein. For example, optionally substituted alkyl may include both propyl and 2-chloro-propyl. Additionally, “optionally substituted” is also inclusive of embodiments where the named substituent or substituents have multiple substituents rather than simply a single substituent. For example, optionally substituted aryl may include both phenyl and 3-ethyl-5-methyl-6-bromo-phenyl.
The compounds of the disclosure may be administered as salts, which are also within the scope of this disclosure. Pharmaceutically acceptable (i.e., non-toxic, physiologically compatible) salts are preferred. If the compounds of the disclosure have, for example, at least one basic center, they can form acid addition salts. These are formed, for example, with strong inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid or a hydrohalic acid, with strong organic carboxylic acids, such as alkane carboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, for example acetic acid, such as saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acid, such as hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid, such as amino acids, (for example aspartic or glutamic acid or lysine or arginine), or benzoic acid, or with organic sulfonic acids, such as (C1-C4) alkyl or arylsulfonic acids which are unsubstituted or substituted, for example by halogen, for example methyl- or paratoluene-sulfonic acid. Corresponding acid addition salts can also be formed having plural basic centers, if desired. In some embodiments, the pharmaceutically acceptable salt is selected from valproic acid, maleic acid, tartaric acid, oxalic acid, pamoic acid, phosphonic acid, benzoic acid, citric acid, salicylic acid, succinic acid, methanesulfonic acid, malic acid, and p-toluenesulfonic acid. In some embodiments, the pharmaceutically acceptable salt is selected from valproic acid, maleic acid, tartaric acid, oxalic acid, and pamoic acid.
The compounds of the disclosure having at least one acid group (e.g., carboxylic acid) can also form salts with suitable bases. Representative examples of such salts include metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium salts, or salts with ammonia or an organic amine, such as morpholine, thiomorpholine, piperidine, pyrrolidine, a mono, di or trihydroxy lower alkylamine, for example ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl or dimethyl-propylamine, or a mono, di or trihydroxy lower alkylamine, for example mono, di or triethanolamine. Corresponding internal salts may also be formed.
For example, certain salts of the compounds described herein which contain a basic group include monohydrochloride, hydrogensulfate, methanesulfonate, phosphate or nitrate. Moreover, certain salts of the compounds described herein which contain an acid group include sodium, potassium and magnesium salts and pharmaceutically acceptable organic amines.
All stereoisomers of the compounds of the disclosure, either in a mixture or in pure or substantially pure form, are considered to be within the scope of this disclosure. The compounds of the disclosure may have asymmetric centers at any of the carbon atoms including any one of the substituents. Consequently, compounds of the disclosure may exist in enantiomeric or diastereomeric forms or in mixtures thereof. Furthermore, where a stereocenter existing in a compound of the disclosure is represented as a racemate, it is understood that the stereocenter may encompass the racemic mixture of R and S isomers, the S isomers, and the R isomers. The processes for preparation of such compounds can utilize racemates, enantiomers, or diastereomers as starting materials. When diastereomeric or enantiomeric products are prepared, they can be separated by conventional methods including, chromatographic, chiral HPLC, fractional crystallization, or distillation. Some compounds of the present disclosure have groups including alkenyls, iminyls, and the like, which may exist as entgegen (E) or zusammen (Z) conformations, in which case all geometric forms thereof, both E and Z, cis and trans, and mixtures thereof, are within the scope of the present disclosure. Accordingly, when such geometric isomeric products are prepared, they can be separated by conventional methods for example, chromatographic, HPLC, distillation or crystallization.
The compounds of the invention may be used as part of a therapy or methodology in treating a variety of diseases or conditions that implicate the Wnt/β-catenin pathway and/or the AMPK pathway.
In some embodiments, the compounds of the invention are used for treating, preventing, or delaying the progression of a disorder or disease alleviated by inhibiting the Wnt/β-catenin pathway, in a patient in need of such treatment or prevention, by administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, 1001-1126, or a pharmaceutically acceptable salt thereof.
In some embodiments, the compounds of the invention are used for treating, preventing, or delaying the progression of a disorder or disease alleviated by activating adenosine monophosphate-activated kinase (AMPK) signaling, in a patient in need of such treatment or prevention, by administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In some embodiments, the compounds of the invention are used for treating, preventing, or delaying the progression of a disorder or disease alleviated by both inhibiting Wnt/β-catenin signaling and activating adenosine monophosphate-activated kinase (AMPK) signaling, in a patient in need of such treatment or prevention, by administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibiting of Wnt/β-catenin signaling and activating of adenosine monophosphate-activated kinase (AMPK) signaling is associated with and/or proceeds by the mechanism of Axin stabilization.
In some embodiments, the compounds and compositions provided herein may be used as inhibitors of one or more members of the Wnt pathway, including one or more Wnt proteins, and/or activators of the AMPK pathway, including one or more AMPK proteins, and thus can be used to treat a variety of disorders and diseases, such as cancer and other diseases associated with abnormal angiogenesis, cellular proliferation, and cell cycling. In non-limiting examples, the compounds and compositions provided herein can be used to treat cancer, to reduce or inhibit angiogenesis, to reduce or inhibit cellular proliferation and correct a genetic disorder due to mutations in Wnt signaling components.
In some embodiments, the cancer may be one or more of adrenocortical cancer, hepatocellular cancer, hepatoblastoma, malignant melanoma, ovarian cancer, Wilm's tumor, Barrett's esophageal cancer, prostate cancer, colon cancer, colorectal cancer, rectal cancer, pancreatic cancer, bladder cancer, breast cancer, gastric cancer, head & neck cancer, lung cancer, mesothelioma, cervical cancer, uterine cancer, myeloid leukemia cancer, lymphoid leukemia cancer, pilometricoma cancer, medulloblastoma cancer, glioblastoma, and familial adenomatous polyposis. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer.
Non-limiting examples of diseases which may be treated with the compounds and compositions provided herein include a variety of cancers, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, psoriasis, mycotic and viral infections, osteochondrodysplasia, Alzheimer's disease, osteoarthritis, polyposis coli, osteoporosis-pseudoglioma syndrome, familial exudative vitreoretinopathy, retinal angiogenesis, early coronary disease, tetra-amelia syndrome, MGllerian-duct regression and virilization, SERKAL syndrome, Fuhrmann syndrome, Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome, odonto-onycho-dermal dysplasia, split-hand/foot malformation, caudal duplication syndrome, tooth agenesis, Wilms tumor, skeletal dysplasia, focal dermal hypoplasia, autosomal recessive anonychia, neural tube defects, alpha-thalassemia (ATRX) syndrome, fragile X syndrome, ICF syndrome, Angelman syndrome, Prader-Willi syndrome, Beckwith-Wiedemann Syndrome and Rett syndrome.
In some embodiments, the methods of the disclosure may be used in the treatment of metabolic disease, including, without limitation, type 2 diabetes, obesity, hyperlipidemia, or fatty liver disease. In certain embodiments, the methods of the disclosure may be used in the treatment of type 2 diabetes. In some embodiments, fatty liver disease may include alcoholic fatty liver disease (ALD) or non-alcoholic fatty liver disease (NAFLD). In some embodiments, NAFLD may include one or more of simple fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), and liver cirrhosis. In certain embodiments, the methods of the disclosure may be used in the treatment of NASH.
In one aspect, the disclosure includes a method of treating or preventing a disease alleviated by inhibiting Wnt/β-catenin signaling in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure includes a method of treating or preventing a disease alleviated by activating adenosine monophosphate-activated kinase (AMPK) signaling in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
In one aspect, the disclosure includes a method of treating or preventing a disease alleviated by both inhibiting Wnt/β-catenin signaling and activating adenosine monophosphate-activated kinase (AMPK) signaling in a patient in need of said treatment or prevention, the method comprising administering a therapeutically effective amount of one or more compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof.
As used herein, the terms “administer,” “administration” or “administering” refer to (1) providing, giving, dosing, and/or prescribing by either a health practitioner or his authorized agent or under his or her direction according to the disclosure; and/or (2) putting into, taking or consuming by the mammal, according to the disclosure.
As used herein, the terms “treat,” “treatment,” and/or “treating” may refer to the management of a disease, disorder, or pathological condition (e.g., cancer or metabolic disease) with the intent to cure, ameliorate, stabilize, and/or control the disease, disorder, or pathological condition. Regarding control of the disease, disorder, or pathological condition more specifically, “control” may include the absence of disease progression, as assessed by the response to the methods recited herein, where such response may be complete (e.g., placing the disease in remission) or partial (e.g., lessening or ameliorating any symptoms associated with the disease). As used herein, the terms “prevent,” “preventing,” and/or “prevention” may refer to reducing the risk of developing a disease, disorder, or pathological condition (e.g., cancer or metabolic disorder).
In some embodiments, the methods of the disclosure may include the modulation of protein activity, regulation, and/or expression. As used herein, the terms “modulate” and “modulation” refer to a change in biological activity for a biological molecule (e.g., a protein, gene, peptide, antibody, and the like), where such change may relate to an increase in biological activity (e.g., increased activity, activation, expression, upregulation, and/or increased expression) or decrease in biological activity (e.g., decreased activity, suppression, deactivation, downregulation, and/or decreased expression) for the biological molecule.
As described herein, in some embodiments, the compounds used in the methods of the disclosure inhibit Wnt signaling, which may result in a reduction of β-catenin. In some embodiments, the compounds used in the methods of the disclosure inhibit Wnt signaling by downregulating β-catenin levels. In some embodiments, the compounds used in the methods of the disclosure inhibit Wnt signaling by downregulating β-catenin levels. In some embodiments, the compounds used in the methods of the disclosure activate phosphorylation of AMPK and its downstream target Acetyl-CoA carboxylase (ACC) is enhanced. In some embodiments, the compounds of the disclosure upregulate Axin protein expression. In some embodiments, the compounds of the disclosure downregulate c-Myc. In some embodiments, the compounds of the disclosure modulate the activity of one or more of casein kinase 1 alpha (CK1α), protein kinase B (Akt/PKB), and glycogen synthase kinase 3 (GSK3). In some embodiments, the compounds of the disclosure upregulate the activity of casein kinase 1 alpha (CK1α). In some embodiments, the compounds of the disclosure downregulate the activity of 182 kDa tankyrase-1-binding protein (TAB182). In some embodiments, the compounds of the disclosure downregulate the activity of one or more of Akt/PKB and GSK3. In some embodiments, the compounds of the disclosure suppress the expression of glucose 6-phosphatase (G6P). In some embodiments, the compounds of the disclosure increase phosphorylation of 5′ adenosine monophosphate-activated protein kinase (AMP kinase or AMPK).
In some embodiments of the disclosure, the methods described herein may include the treatment of certain symptoms of diseases that implicate the Wnt/β-catenin signaling pathway and/or the AMPK pathway.
For example, the methods of the disclosure may include treatments for symptoms of cancer or metabolic diseases. In some embodiments, the methods of the disclosure may include treatments for symptoms of type 2 diabetes. In some embodiments, the methods of the disclosure include the treatment of a fatty liver disease. In some embodiments, the fatty liver disease comprises a nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).
In some embodiments, the compounds of the disclosure may suppress glucose production. Furthermore, in some embodiments, the compounds of the disclosure may improve glucose tolerance in a patient in need thereof. In some embodiments, the compounds of the disclosure may reduce fasting glucose levels in a patient in need thereof. In some embodiments, the compounds of the disclosure may suppress gluconeogenesis in a patient in need thereof. In some embodiments, the compounds of the disclosure may reverse obesity and/or decrease weight gain in a patient in need thereof. In some embodiments, the compounds of the disclosure may increase insulin sensitivity in a patient in need thereof.
In some embodiments, the methods may include the co-administration of a compound of the disclosure with an additional therapeutic agent. The term “co-administering” as used herein means a process whereby the combination of a compound of the disclosure and at least one additional therapeutic agent is administered to the same patient. The compound of the disclosure and additional therapeutic may be administered simultaneously, at essentially the same time, or sequentially. If administration takes place sequentially, the compound of the invention may be administered before or after a given additional therapeutic agent or treatment. The compound of the disclosure and additional therapeutic agent or treatment need not be administered by means of the same vehicle or physiologically compatible carrier medium. The compound of the disclosure and the additional therapeutic agent may be administered one or more times and the number of administrations of each component of the combination may be the same or different. In addition, the compound of the disclosure and additional therapeutic agent or treatment need not be administered at the same site.
In some embodiments, the methods of the disclosure may include administering (1) a therapeutically effective amount of one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt or a prodrug thereof; and (2) a therapeutically effective amount of an additional therapeutic agent.
In some embodiments, the additional therapeutic agent may include one or more of a RAF inhibitor, an MEK inhibitor, an ERK inhibitor, a VEGFR inhibitor, and an EGFR inhibitor. In some embodiments, the VEGFR inhibitor may include one or more of Bevacizumab (AVASTIN), Aflibercept (ZALTRAP), and Regorafenib (STIVARGA). In some embodiments, the EGFR inhibitor may include one or more of Cetuximab (ERBITUX), Panitumumab (VECTIBIX), and Gefitinib. In some embodiments, the additional therapeutic agent may include pyrvinium.
In some embodiments, the methods of the disclosure include co-administration of an additional therapeutic agent useful for the treatment of fatty liver disease, including, but not limited to, a nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). In some embodiments, the additional therapeutic agent is selected from a hyperlipidemic drug, an antihypertensive, an antidiabetic, and a farnesoid X receptor (FXR) ligand. In some embodiments, the additional therapeutic agent is selected from angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, caspase inhibitors, cathepsin B inhibitors, CCR2 chemokine antagonists, CCR5 chemokine antagonists, chloride channel stimulators, cholesterol solubilizers, diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors, dipeptidyl peptidase IV (DPPIV) inhibitors, farnesoid X receptor (FXR) agonists, FXR/TGR5 dual agonists, galectin-3 inhibitors, glucagon-like peptide 1 (GLPl) agonists, glutathione precursors, hepatitis C virus NS3 protease inhibitors, HMG CoA reductase inhibitors, 1 Iβ-hydroxy steroid dehydrogenase (I Iβ-HSDl) inhibitors, IL-Iβ antagonists, IL-6 antagonists, IL-10 agonists, IL-17 antagonists, ileal sodium bile acid cotransporter inhibitors, leptin analogs, 5-lipoxygenase inhibitors, LPL gene stimulators, lysyl oxidase homolog 2 (LOXL2) inhibitors, PDE3 inhibitors, PDE4 inhibitors, phospholipase C (PLC) inhibitors, PPARa agonists, PPARy agonists, PPAR5 agonists, Rho associated protein kinase 2 (ROCK2) inhibitors, sodium glucose transporter-2 (SGLT2) inhibitors, stearoyl CoA desaturase-1 inhibitors, thyroid hormone receptor β agonists, tumor necrosis factor a (TNFa) ligand inhibitors, transglutaminase inhibitors, transglutaminase inhibitor precursors, PTPlb inhibitors, and ASK1 inhibitors.
Non-limiting examples of an additional therapeutic agent include therapeutic agents for hyperlipidemia, antihypertensives, antidiabetics, antioxidants, blood flow improving agents, and bile acid derivatives.
Non-limiting examples of therapeutic agents for hyperlipidemia include polyenephosphatidylcholine, unsaponifiable soybean oil (soy sterol), gamma-oryzanol, riboflavin butyrate, dextran sulfate sodium sulfur 18, pantethine, and elastase; statins such as pravastatin, simvastatin, atorvastatin, fluvastatin, pitavastatin, rosuvastatin, and cerivastatin; fibrates such as simfibrate, clofibrate, clinofibrate, bezafibrate, and fenofibrate; lipolytic enzyme inhibitors such as orlistat and cetilistat; resins such as colestyramine and colestimide; and ezetimibe.
Non-limiting examples of antihypertensives include angiotensin II receptor blockers such as irbesartan, olmesartan medoxomil, candesartan cilexetil, telmisartan, valsartan, and losartan potassium; angiotensin-converting enzyme inhibitors such as alacepril, imidapril hydrochloride, enalapril maleate, captopril, quinapril hydrochloride, cilazapril hydrate, temocapril hydrochloride, delapril hydrochloride, trandolapril, benazepril hydrochloride, perindopril, and lisinopril hydrate; calcium antagonists such as azelnidipine, amlodipine besylate, aranidipine, efonidipine hydrochloride, cilnidipine, nicardipine hydrochloride, nifedipine, nimodipine, nitrendipine, nilvadipine, barnidipine hydrochloride, felodipine, benidipine, and manidipine; [alpha] receptor blocker such as tolazoline, and phentolamine; [beta] receptor blockers such as atenolol, metoprolol, acebutolol, propranolol, pindolol, carvedilol, and labetalol hydrochloride; a receptors stimulant such as clonidine and methyldopa; and diuretics such as eplerenone, hydrochlorothiazide, and furosemide.
Non-limiting examples of antidiabetics include [alpha]-glucosidase inhibitors such as acarbose, voglibose, and miglitol; sulfonyl urea hypoglycemics such as gliclazide, glibenclamide, glimepiride, and tolbutamide; fast-acting insulin secretagogues such as nateglinide and mitiglinide; biguanide hypoglycemics such as metformin hydrochloride and buformin hydrochloride; dipeptidyl phosphatase 4 inhibitors such as sitagliptin, vildagliptin, alogliptin, linagliptin and saxagliptin; thiazolidine reagents such as pioglitazone hydrochloride and rosiglitazone maleate; and glucagon-like peptide 1 derivative reagents such as exenatide, lixisenatide and liraglutide.
Non-limiting examples of antioxidants include vitamins such as ascorbic acid (vitamin C), tocopherol (vitamin E), and tocopherol nicotinate, and N-acetylcysteine, probucol.
Non-limiting examples of blood flow improving agents include cilostazol, ticlopidine hydrochloride, alprostadil, limaprost, beraprost sodium, sarpogrelate hydrochloride, argatroban, naftidrofuryl, isoxsuprine hydrochloride, batroxobin, dihydroergotoxine mesilate, tolazoline hydrochloride, hepronicate, and shimotsu-to extract.
Non-limiting examples of bile acid derivatives include ursodeoxycholic acid, chenodeoxycholic acid, bile powder, deoxycholic acid, cholic acid, bile extract, bear bile, oriental bezoar, and dehydrocholic acid. Preferable examples also include biotin (vitamin B7), cyanocobalamin (vitamin B12), pantothenic acid (vitamin B5), folic acid (vitamin B9), thiamine (vitamin B1), vitamin A, vitamin D, vitamin K, tyrosine, pyridoxine (vitamin B6), branched chain amino acids such as leucine, isoleucine, and valine, calcium, iron, zinc, copper, and magnesium. Other non-limiting examples include components used in designated health foods and functional nutritional foods such as soy protein, chitosan, low molecular weight sodium alginate, dietary fiber from psyllium seed coat, soy peptide with bound phospholipids, phytosterol ester, plant stanol ester, diacylglycerol, globin digest, and tea catechin.
In some embodiments, the methods of the disclosure include co-administration of an additional therapeutic agent useful for the treatment of colorectal cancer (CRC). Non-limiting examples of additional therapeutic agent useful for the treatment of colorectal cancer (CRC) include capecitabine; cetuximab; bevacizumab; a MEK inhibitor such as N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, or a pharmaceutically acceptable salt thereof; a FOLFOX4 combination including oxaliplatin, 5-fluorouracil and leucovorin; and a FOLFIRI combination include irinotecan, 5-fluorouracil and leucovorin and the like.
Furthermore, the described methods of treatment may normally include medical follow-up to determine the therapeutic or prophylactic effect brought about in the subject undergoing treatment with the compound(s) and/or composition(s) described herein.
Molecular modeling and computer-based modeling may be used in accordance with the disclosure to both understand the protein targets of the therapeutic agents described herein or to direct drug design in the preparation of analogs. Data reflecting the effect of compounds of the disclosure on protein binding, for example, or other resulting in vitro or in vivo activity data, may be used to develop a pharmacophore and pharmacophore model. As used herein, the term “pharmacophore” refers to the ensemble of steric and electronic features that are necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger, activate, block, inhibit or modulate the biological target's biological activity, as the case may be. See, IUPAC, Pure and Applied Chemistry (1998) 70: 1129-1143.
As used herein, the term “pharmacophore model” refers to a representation of points in a defined coordinate system wherein a point corresponds to a position or other characteristic of an atom or chemical moiety in a bound conformation of a ligand and/or an interacting polypeptide, protein, or ordered water. An ordered water is an observable water in a model derived from structural determination of a polypeptide or protein. A pharmacophore model can include, for example, atoms of a bound conformation of a ligand, or portion thereof. A pharmacophore model can include both the bound conformations of a ligand, or portion thereof, and one or more atoms that interact with the ligand and are from a bound polypeptide or protein. Thus, in addition to geometric characteristics of a bound conformation of a ligand, a pharmacophore model can indicate other characteristics including, for example, charge or hydrophobicity of an atom or chemical moiety. A pharmacophore model can incorporate internal interactions within the bound conformation of a ligand or interactions between a bound conformation of a ligand and a polypeptide, protein, or other receptor including, for example, van der Waals interactions, hydrogen bonds ionic bonds, and hydrophobic interactions. A pharmacophore model can be derived from 2 or more bound conformations of a ligand.
Turning to the administration of therapeutics, the compounds of the disclosure may be administered as described herein, or in a form from which the active agent can be derived, such as a prodrug. A “prodrug” is a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxyl or carboxylic acid group of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by one or three letter symbols but also include, for example, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Prodrug esters as employed herein includes esters and carbonates formed by reacting one or more hydroxyls of compounds of the method of the disclosure with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates and the like. As further examples, free hydroxyl groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groups are also included, as are carbonate prodrugs, sulfonate prodrugs, sulfonate esters and sulfate esters of hydroxyl groups. Free amines can also be derivatized to amides, sulfonamides or phosphonamides. All of the stated prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Moreover, any compound that can be converted in vivo to provide the bioactive agent (e.g., a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof) is a prodrug within the scope of the disclosure. Various forms of prodrugs are well known in the art. A comprehensive description of pro drugs and prodrug derivatives are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991).
In general, prodrugs may be designed to improve the penetration of a drug across biological membranes in order to obtain improved drug absorption, to prolong duration of action of a drug (slow release of the parent drug from a prodrug, decreased first-pass metabolism of the drug), to target the drug action (e.g. organ or tumor-targeting, lymphocyte targeting), to modify or improve aqueous solubility of a drug (e.g., i.v. preparations and eyedrops), to improve topical drug delivery (e.g. dermal and ocular drug delivery), to improve the chemical/enzymatic stability of a drug, or to decrease off-target drug effects, and more generally in order to improve the therapeutic efficacy of the compounds utilized in the disclosure.
A compound used in practicing any method of the disclosure may be administered in an amount sufficient to induce the desired therapeutic effect in the recipient thereof. Thus the term “therapeutically effective amount” as used herein refers to an amount of a compound of the disclosure that is sufficient to treat a disease in accordance with the disclosure by administration of one or more of the compounds of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt or a prodrug thereof. Preferably, the therapeutically effective amount refers to the amount appropriate to inhibit the Wnt/β-catenin pathway. In addition, the term therapeutically effective amount may include the amount of a compound necessary, for example, to bring about a detectable therapeutic, preventative, or ameliorative effect in a patient having a disease as set forth herein. The effect may include, for example, the reduction, prevention, amelioration, or stabilization of symptoms or conditions associated with a disease as described herein.
The compound(s) described herein may also be administered at a dose in range from about 0.01 mg to about 200 mg/kg of body weight per day. A dose of from about 0.1 to 100 mg/kg, or from about 1 to 50 mg/kg per day in one or more applications per day may be effective to produce the desired result. By way of example, a suitable dose for oral administration may be in the range of 1-50 mg/kg of body weight per day, whereas a dose for intravenous administration may be in the range of 1-10 mg/kg of body weight per day.
Of course, as those skilled in the art will appreciate, the dosage actually administered will depend upon the condition being treated, the age, health and weight of the recipient, the type of concurrent treatment, if any, and the frequency of treatment. Moreover, the effective dosage amount may be determined by one skilled in the art on the basis of routine empirical activity testing to measure the bioactivity of the compound(s) in a bioassay, and thus establish the appropriate dosage to be administered.
The compounds used in certain methods of the disclosure may typically be administered from 1-4 times a day, so as to deliver the above-mentioned daily dosage. However, the exact regimen for administration of the compounds described herein will necessarily be dependent on the needs of the individual subject being treated, the type of treatment administered and the judgment of the attending medical specialist. As used herein, the term “subject” or “patient” includes both humans and animals.
In general, the compounds used in the methods of the disclosure can be administered in pure form or, as described herein, with physiologically compatible and/or acceptable carrier mediums, using any acceptable route known in the art, either alone or in combination with one or more other therapeutic agents. Thus, the compound(s) and/or composition(s) of the disclosure can be administered orally, parenterally, such as by intravenous or intraarterial infusion, intramuscular, intraperitoneal, intrathecal or subcutaneous injection, by liposome-mediated delivery, rectally, vaginally, by inhalation or insufflation, transdermally or by otic delivery. In some embodiments, the compound is administered orally.
The orally administered dosage unit may be in the form of tablets, caplets, dragees, pills, semisolids, soft or hard gelatin capsules, aqueous or oily solutions, emulsions, suspensions or syrups. Suitable dosage forms for parenteral administration include injectable solutions or suspensions, suppositories, powder formulations, such as microcrystals or aerosol spray. The active agents of the disclosure may also be incorporated into a conventional transdermal delivery system.
As used herein, the expression “physiologically compatible carrier medium” (or “physiologically acceptable carrier medium” and the like) includes any and all solvents, diluents, or other liquid vehicles, dispersions or suspension aids, surface agent agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, fillers and the like as suited for the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 20th edition, A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp. 669-1015 (Lippincott Williams & Wilkins, Baltimore, MD/Philadelphia, PA) (2000) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional pharmaceutical carrier medium is incompatible with the compounds of the present disclosure, such as by producing an undesirable biological effect or otherwise interacting in an deleterious manner with any other component(s) of a formulation comprising such compounds or agents, its use is contemplated to be within the scope of this disclosure.
For the production of solid dosage forms, including hard and soft capsules, the agents of the disclosure may be mixed with pharmaceutically inert, inorganic or organic excipients, such as lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearic acid or its salts, dried skim milk, vegetable, petroleum, animal or synthetic oils, wax, fat, polyols, and the like. For the production of liquid solutions, emulsions or suspensions or syrups one may use excipients such as water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerine, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal or synthetic oils. For suppositories one may use excipients, such as vegetable, petroleum, animal or synthetic oils, wax, fat and polyols. For aerosol formulations, one may use compressed gases suitable for this purpose, such as oxygen, nitrogen and carbon dioxide. Pharmaceutical compositions or formulations may also contain one or more additives including, without limitation, preservatives, stabilizers, e.g., UV stabilizers, emulsifiers, sweeteners, salts to adjust the osmotic pressure, buffers, coating materials and antioxidants.
The disclosure further includes controlled-release, sustained release, or extended-release therapeutic dosage forms for administration of the compounds of the disclosure, which involves incorporation of the compounds into a suitable delivery system in the formation of certain compositions. This dosage form controls release of the compound(s) in such a manner that an effective concentration of the compound(s) in the bloodstream may be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and/or minimize side effects. Additionally, a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the compound.
In pharmaceutical compositions used in practicing the methods of the disclosure more particularly, the specified compound(s) may be present in an amount of at least 0.5 and generally not more than 95% by weight, based on the total weight of the composition, including carrier medium and/or supplemental active agent(s), if any. In some embodiments, the proportion of compound(s) varies between about 30-90% by weight of the composition.
In some embodiments, the compositions of the disclosure may include (1) one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof; (2) an additional therapeutic agent; and a physiologically compatible carrier medium. In some embodiments, the additional therapeutic agent may include one or more of a RAF inhibitor, an MEK inhibitor, an ERK inhibitor, a VEGFR inhibitor, and an EGFR inhibitor. In some embodiments, the VEGFR inhibitor may include one or more of Bevacizumab (AVASTIN), Aflibercept (ZALTRAP), and Regorafenib (STIVARGA). In some embodiments, the EGFR inhibitor may include one or more of Cetuximab (ERBITUX), Panitumumab (VECTIBIX) Gefitinib. In some embodiments, the additional therapeutic agent may include pyrvinium. In some embodiments, the additional therapeutic agent may include one or more of angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, caspase inhibitors, cathepsin B inhibitors, CCR2 chemokine antagonists, CCR5 chemokine antagonists, chloride channel stimulators, cholesterol solubilizers, diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors, dipeptidyl peptidase IV (DPPIV) inhibitors, farnesoid X receptor (FXR) agonists, FXR/TGR5 dual agonists, galectin-3 inhibitors, glucagon-like peptide 1 (GLPl) agonists, glutathione precursors, hepatitis C virus NS3 protease inhibitors, HMG CoA reductase inhibitors, 1 Iβ-hydroxy steroid dehydrogenase (I Iβ-HSDl) inhibitors, IL-Iβ antagonists, IL-6 antagonists, IL-10 agonists, IL-17 antagonists, ileal sodium bile acid cotransporter inhibitors, leptin analogs, 5-lipoxygenase inhibitors, LPL gene stimulators, lysyl oxidase homolog 2 (LOXL2) inhibitors, PDE3 inhibitors, PDE4 inhibitors, phospholipase C (PLC) inhibitors, PPARa agonists, PPARy agonists, PPAR5 agonists, Rho associated protein kinase 2 (ROCK2) inhibitors, sodium glucose transporter-2 (SGLT2) inhibitors, stearoyl CoA desaturase-1 inhibitors, thyroid hormone receptor β agonists, tumor necrosis factor a (TNFa) ligand inhibitors, transglutaminase inhibitors, transglutaminase inhibitor precursors, PTPlb inhibitors, and ASK1 inhibitors. In some embodiments, the additional therapeutic agent may include one or more of capecitabine; cetuximab; bevacizumab; a MEK inhibitor such as N-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, or a pharmaceutically acceptable salt thereof; a FOLFOX4 combination including oxaliplatin, 5-fluorouracil and leucovorin; and a FOLFIRI combination include irinotecan, 5-fluorouracil and leucovorin and the like.
In some embodiments, the disclosure provides a pharmaceutical composition comprising one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In some embodiments, the disclosure provides a pharmaceutical composition for treating or preventing a disease alleviated by inhibiting Wnt/β-catenin signaling and/or activating AMPK signaling comprising one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In some embodiments, the disclosure provides a pharmaceutical composition treating liver fibrosis comprising one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In some embodiments, the disclosure provides a pharmaceutical composition treating colorectal cancer (CRC) comprising one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium.
In some embodiments, the disclosure provides a pharmaceutical composition treating alcoholic fatty liver disease (ALD) or non-alcoholic fatty liver disease (NAFLD) comprising one or more of a compound of formula (I), formula (10)-(15), formula (20)-(29), formula (31)-(32), formula 1001-1126, formula (II), formula 2001-2031, formula (III), formula 3001-3018, or a pharmaceutically acceptable salt thereof, and a physiologically compatible carrier medium. In some embodiments, the non-alcoholic fatty liver disease is selected from the group consisting of simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), and liver cirrhosis.
The following examples describe the disclosure in further detail. These examples are provided for illustrative purposes only, and should in no way be considered as limiting the disclosure.
Two potent inhibitors of the Wnt/i-catenin signaling pathway, FX1128 and FX2065 were previously identified. These Wnt signaling inhibitors exhibited single digit nM inhibitory potency in cell and intriguing efficacies in mice models of fatty liver disorder and colorectal cancer, respectively. However, compounds FX1128 and FX2065 indicated suboptimal druglike profiles highlighted by their poor aqueous solubilities, which could adversely impact the ADME and bioavailability of these compounds for their future therapeutic applications. This Example describes a class of new Wnt/β-catenin signaling pathway inhibitors with dramatically enhanced aqueous solubility. As a highlight, the hydrochloride salt form of the 1-methylpiperazine analog YA6060 demonstrated a remarkable aqueous solubility of >78 mg/mL, which accounts for an over 10,000-fold increase from both compounds FX1128 and FX2065 (
The synthesis of compounds YA10951 and YA6060 began with o-toluidine 1 (Scheme 1). Diazotization of the amino group of compound 1 using sodium nitrite (NaNO2) and aqueous HCl, followed by the treatment of the intermediate with sodium azide (NaN3) provided azides, which underwent cyclization with β-ketoester in EtONa/EtOH yielded trizaole-3-carboxylic acids 2. Next, chlorodipyrrolidinocarbenium (PyCIU)-mediated coupling of compounds 2 with 6-bromoquinolin-2-amine in dichloroethane (DCE) provided compound 3 in moderate to good yields. Further, Pd-catalyzed cross-coupling of compound 3 with potassium trifluoroborate derivatives yielded products 4 in good yields. The Boc group of 4 was then removed by trifluoroacetic acid (TFA) to yield amino-containing compound YA10951 in good yields. Alternatively, compound 4 was treated with 2 M HCl in Et2O to provide the compound YA6060 in good yields.
The syntheses of compounds YA6063 and YA6079 is shown in Scheme 2. Suzuki coupling of compound 3 with bis-(pinacolato)diboron in the presence of Pd(dppf)Cl2 and KOAc yielded the boronic ester YA6063, which was subsequently converted to the hydroxy compound YA6079 in the presence of hydrogen peroxide (H2O2).
The synthesis of compounds YA6150, YA6167, YA1126, YA6146, YA6159 and YA6160 are shown in Scheme 3. Diazotization of the amino group of o-toluidine 1 using sodium nitrite (NaNO2) and aqueous HCl, followed by the treatment of the intermediate with sodium azide (NaN3) provided azides, which underwent cyclization with β-ketoester in EtONa/EtOH yielded trizaole-3-carboxylic acids 2. Next, PyCIU-mediated coupling of compound 2 with various bromoquinolin-2-amines in dichloroethane (DCE) provided compounds YA6150, YA6167, YA1126, and YA6146 in moderate to good yields. Finally, Pd-catalyzed cross-coupling of compounds YA6150 and YA6146 with potassium trifluoroborate derivatives yielded products YA6159 and YA6160 in good yields.
The synthesis of compounds YA4182, YA4179, YA1130 and YA1128 began with o-toluidines (Scheme 4). Diazotization of the amino group of aniline 1 using sodium nitrite (NaNO2) and aqueous HCl, followed by the treatment of the intermediate with sodium azide (NaN3) provided azides, which underwent cyclization with β-ketoester in EtONa/EtOH to yield trizaole-3-carboxylic acids 2. Next, PyCIU-mediated coupling of compounds 2 with various aromatic amines in dichloroethane (DCE) provided compounds YA4182, YA4179, YA1130 and YA1128 in moderate to good yields.
Table 1 shows experimental data showing the inhibition of the Wnt/β-catenin signaling pathway by compounds disclosed herein. Compound FX1128 is included as a comparison.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from theluciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of eachcompound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
The synthesis of biotinylated compounds YA1103 and YA6023 began with compound YA10951 (Scheme 5). HATU-mediated amide bond formation between compound YA10951 and either D-biotin or biotin-dpeg(4)-CO2H in the presence of DIPEA generated compounds YA1103 and YA6023 in modest yields.
Table 2 shows experimental data showing the inhibition of the Wnt/β-catenin signaling pathway by compounds disclosed herein. Compound FX1128 is included as a comparison.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
The synthesis of tetrazole-containing compounds is detailed in Scheme 6. Condensation of ethyl 2-oxoacetate 5 and benzenesulfonohydrazide 6 generated intermediate 7, which was reacted with various aromatic diazo derivatives 9 to provide tetrazoles 10. Hydrolysis of the ethylester using LiOH generated the carboxylates 11, which was coupled to different amino compounds to provide the final products.
The synthesis of triazole-containing compounds began with o-toluidines (Scheme 6). Diazotization of the amino group of anilines 1 was carried out using sodium nitrite (NaNO2) and aqueous HCl, followed by the treatment of the intermediate with sodium azide (NaN3) to provide azides, which underwent cyclization with β-ketoester in EtONa/EtOH to yield trizaole-3-carboxylic acids 2. Next, PyCIU-mediated coupling of compounds 2 with various aromatic amines in dichloroethane (DCE) provided triazole-containing compounds in moderate to good yields.
Table 3 shows experimental data showing the inhibition of the Wnt/P3-catenin signaling pathway by compounds disclosed herein. Compound FX1128 is included as a comparison.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
Table 4 shows experimental data showing the inhibition of the Wnt/β-catenin signaling pathway by compounds disclosed herein. Compound FX1128 is included as a comparison.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
Table 5 shows experimental data showing the inhibition of the Wnt/β-catenin signaling pathway by compounds disclosed herein. Compound FX1128 is included as a comparison.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
Table 6 shows experimental data showing the inhibition of the Wnt/β-catenin signaling pathway by compounds disclosed herein. Compound FX1128 is included as a comparison.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
Table 7 shows experimental data showing the inhibition of the Wnt/β-catenin signaling pathway by compounds disclosed herein.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
The synthesis of thiazole-triazole based compounds began with o-toluidines (Scheme 7). Diazotization of the amino group of anilines 1 was carried out using sodium nitrite (NaNO2) and aqueous HCl, followed by the treatment of the intermediate with sodium azide (NaN3) to provide azides, which underwent cyclization with β-ketoester in EtONa/EtOH yielded trizaole-3-carboxylic acids 2. Next, PyCIU-mediated coupling of compounds 2 with various aromatic amines in dichloroethane (DCE) provided thiazole-triazole based compounds in moderate to good yields.
The synthesis of pyrazole compounds is detailed in Scheme 8. Condensation of ethyl acetoacetate 12 with N,N-dimethylformamide dimethyl acetal in EtOH provided compound 13 in good yields. Next, pyrazole formation using compound 13 and a substituted hydrazine in the presence of DIPEA led to the generation of the ester intermediate, which underwent hydrolysis using aqueous NaOH to afford carboxylic acids 14 in good yields. Finally, PyCIU-mediated coupling of acids 14 with various aromatic amines yielded the final products in moderate to good yields.
Table 8 shows experimental data showing the inhibition of the Wnt/P3-catenin signaling pathway by compounds disclosed herein.
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined from the luciferase reporter gene assay, were calculated, and data are expressed as mean IC50 (nM) of each compound from three independent experiments.
bNote that compounds did not present any apparent cytotoxicity during the short treatment duration used for the luciferase gene reporter assay.
All chemicals were obtained from commercial suppliers and used without further purification. Analytical thin layer chromatography was visualized by ultraviolet light at 256 nM. 1H NMR spectra were recorded on a Varian (400 MHz) spectrometer. Data are presented as follows: chemical shift (in ppm on the δ scale relative to δ=0.00 ppm for the protons in tetramethylsilane (TMS)), integration, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constant (J/Hz). 13C NMR spectra were recorded at 100 MHz, and all chemical shifts values are reported in ppm on the δ scale with an internal reference of δ 77.0 or 39.0 for CDCl3 or DMSO-d6, respectively. The purities of title compounds were determined by analytic HPLC, performed on an Agilent 1100 instrument and a reverse-phase column (Waters XTerrra RP18, 5 μM, 4.6×250 mm). All compounds were eluted with 60% acetonitrile/40 water (containing 0.1% TFA) over 20 mins with a detection at 260 nM and a flow rate at 1.0 mL/min. All tested compounds were >95% pure.
5-Methyl-N-(6-(piperazin-1-ylmethyl)quinolin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA10951). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 8.53 (d, J=9.6 Hz, 1H), 8.16 (d, J=9.6 Hz, 1H), 7.85 (d, J=9.6 Hz, 1H), 7.69-7.67 (m, 2H), 7.49 (t, J=7.2 Hz, 1H), 7.43-7.37 (m, 2H), 7.25 (t, J=8.0 Hz, 1H), 3.64 (s, 2H), 2.94 (s, 4H), 2.50 (s, 7H), 2.06 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 160.0, 150.4, 146.4, 138.8, 138.2, 137.8, 135.5, 135.1, 134.2, 131.5, 130.9, 127.7, 127.2, 126.1, 114.2, 63.3, 54.0, 45.7, 17.2, 9.3;
N-(4-bromoquinolin-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6150). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.91 (s, 1H), 8.97 (s, 1H), 8.14 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.56-7.49 (m, 2H), 7.45-7.39 (m, 2H), 7.28 (d, J=8.0 Hz, 1H), 2.54 (s, 3H), 2.09 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 150.0, 147.1, 139.0, 137.5, 135.5, 135.4, 134.2, 131.5, 130.9, 130.8, 128.2, 127.1, 126.7, 126.3, 125.9, 117.9, 17.3, 9.3;
N-(5-Bromoquinolin-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6167). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.10 (d, J=6.4 Hz, 1H), 7.58 (d, J=13.6 Hz, 1H), 7.53-7.49 (m, 2H), 7.46-7.40 (m, 2H), 7.31-7.25 (m, 2H), 5.97 (d, J=13.2 Hz, 1H), 2.48 (s, 3H), 2.09 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 147.1, 139.0, 137.7, 136.3, 135.6, 134.4, 131.7, 131.4, 131.2, 130.0, 127.4, 127.3, 127.2, 124.0, 122.9, 117.1, 105.1, 17.5, 9.4;
N-(7-Bromoquinolin-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA1126). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 8.56 (d, J=8.0 Hz, 1H), 8.16 (d, J=8.0 Hz, 1H), 8.08 (s, 1H), 7.65 (d, J=8.0 Hz, 1H), 7.54-7.38 (m, 4H), 7.27-7.25 (m, 1H), 2.51 (s, 3H), 2.07 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 160.0, 151.3, 147.6, 139.0, 138.3, 137.8, 135.5, 134.2, 131.5, 131.0, 130.2, 128.7, 128.6, 127.2, 124.9, 124.1, 114.4, 17.3, 9.3;
N-(8-Bromoquinolin-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6146). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 8.55 (d, J=8.0 Hz, 1H), 8.13 (d, J=8.0 Hz, 1H), 7.93 (d, J=6.8 Hz, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.44 (t, J=8.0, 7.2 Hz, 1H), 7.37-7.32 (m, 2H), 7.23-7.20 (m, 2H), 2.46 (s, 3H), 2.01 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 160.0, 151.3, 144.0, 139.1, 137.6, 135.5, 134.2, 133.6, 131.5, 130.9, 127.4, 127.3, 127.1, 125.5, 122.9, 115.0, 17.2, 9.3;
tert-Butyl 4-((2-(5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamido)quinolin-4-yl)methyl)piperazine-1-carboxylate (YA6159). 1H-NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 8.56 (s, 1H), 8.22 (d, J=8.0 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.68 (t, J=6.8, 8.0 Hz, 1H), 7.52-7.38 (m, 4H), 7.26 (d, J=8.0 Hz, 1H), 3.96 (s, 2H), 3.46 (s, 4H), 2.52 (s, 7H), 2.07 (s, 3H), 1.45 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ 160.0, 154.8, 150.3, 147.3, 146.0, 138.8, 137.8, 135.5, 134.3, 131.5, 130.9, 129.7, 128.3, 127.2, 127.1, 125.9, 124.9, 124.4, 114.7, 79.6, 60.3, 53.2, 44.1, 43.2, 28.4, 17.2, 9.3;
tert-Butyl 4-((2-(5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamido)quinolin-8-yl)methyl)piperazine-1-carboxylate (YA6160). 1H-NMR (400 MHz, CDCl3) δ 9.91 (s, 1H), 8.54 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.0 Hz, 1H), 7.79 (d, J=6.0 Hz, 1H), 7.70 (d, J=7.6 Hz, 1H), 7.50 (t, J=7.6 Hz, 1H), 7.46-7.38 (m, 4H), 7.27 (d, J=8.0 Hz, 1H), 4.20 (s, 2H), 3.49 (s, 4H), 2.58 (s, 4H), 2.52 (s, 3H), 2.08 (s, 3H), 1.46 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 154.8, 149.7, 145.5, 138.9, 138.7, 137.8, 135.5, 134.6, 134.2, 131.5, 130.9, 129.9, 127.2, 126.5, 126.4, 124.8, 113.9, 79.5, 56.8, 53.2, 44.2, 43.3, 28.4, 17.2, 9.3;
1-(3-Bromo-2-methylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA4182). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.91 (s, 1H), 8.54 (d, J=8.8 Hz, 1H), 8.21 (d, J=8.8 Hz, 1H), 7.90 (d, J=8.8 Hz, 1H), 7.79 (d, J=8.0 Hz, 2H), 7.68 (t, J=7.6, 8.0 Hz, 1H), 7.46 (t, J=6.8, 8.0 Hz, 1H), 7.29-7.23 (m, 2H), 2.51 (s, 3H), 2.09 (S, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 150.5, 146.9, 139.0, 138.5, 137.9, 136.1, 135.1, 130.0, 127.9, 127.8, 127.5, 126.6, 126.4, 125.2, 114.2, 18.1, 9.3;
1-(4-Bromo-2-methylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA4179). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.95 (br s, 1H), 8.55 (d, J=8.0 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.70 (t, J=7.2 Hz, 1H), 7.61 (s, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.47 (t, J=7.2, 6.8 Hz, 1H), 7.14 (d, J=7.6 Hz, 1H), 2.52 (s, 3H), 2.06 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 150.5, 138.9, 138.6, 137.7, 134.5, 133.2, 130.4, 130.1, 128.6, 127.6, 127.5, 126.3, 125.3, 125.0, 114.2, 17.2, 9.27;
1-(5-Bromo-2-methylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA1130). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 8.54 (d, J=9.2 Hz, 1H), 8.21 (d, J=9.2 Hz, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.68 (t, J=7.2, 8.4 Hz, 1H), 7.62 (d, J=7.6 Hz, 1H), 7.48-7.44 (m, 2H), 7.31 (d, J=8.4 Hz, 1H), 2.53 (s, 3H), 2.02 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 150.5, 146.9, 138.9, 138.5, 135.3, 134.7, 134.0, 132.8, 130.2, 130.0, 127.8, 127.5, 126.4, 125.2, 119.9, 114.2, 16.9, 9.3;
1-(2-Bromo-6-methylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA1128). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.94 (s, 1H), 8.55 (d, J=8.0 Hz, 1H), 8.21 (d, J=8.0 Hz, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.68 (t, J=6.8, 8.0 Hz, 1H), 7.63-7.61 (m, 1H), 7.46 (t, J=7.6, 7.2 Hz, 1H), 7.37 (d, J=6.0 Hz, 2H), 2.50 (s, 3H), 2.06 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 150.6, 147.0, 138.7, 138.4, 137.9, 133.6, 132.1, 131.2, 130.3, 130.0, 127.8, 127.5, 126.4, 125.2, 122.3, 114.2, 18.0, 8.9;
To a solution of 6-bromoquinolin-2-amine (0.5 mmol) and 5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxylic acid (0.675 mmol) in DCE (2 mL) was added PyCIU (0.775 mmol) and DIPEA (2.33 mmol). The mixture was stirred at 80° C. overnight, then cooled and concentrated. To the resulting residue was added ethyl acetate (30 mL), and the mixture was washed with brine, dried over Na2SO4 and concentrated. The crude material was purified by column chromatography (hexane/AcOEt, v/v=4/1 to 2/1) to give product YA1061 (60%).
In a conical shaped microwave vial was added YA1061 (0.169 mmol), potassium ((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)trifluoroborate (0.338 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos, 0.034 mmol), cesium carbonate (0.507 mmol), and palladium (II) acetate (0.017 mmol), THF (0.5 mL) and water (0.05 mL). The reaction mixture was sealed and stirred at room temperature for 10 min. Once a clear solution was obtained, the vial was heated to 80° C. for 15 min followed by 145° C. for 45 min. After cooling, the reaction mixture was diluted with DCM and dried over Na2SO4. The solution was filtered, concentrated and purified by column chromatography (5-15% MeOH in DCM) to give compound YA1095 (70%).
A mixture of YA1095 (2 mmol) and TFA (15 mmol) in anhydrous DCM (5 mL) was stirred at room temperature for 0.5 h. The reaction mixture was then evaporated under reduced pressure. The resulting crude product was used in the next without further purification.
To a solution of Biotin-dpeg(4)-COOH in dry DMF (5 mL), the above products (3.0 mmol), HATU (6 mmol), and DIPEA (0.3 mmol) were added, and the mixture was stirred at room temperature for 24 h. The reaction solution was diluted with water and extracted with EA. The organic layer was washed with brine and then dried with sodium sulfate, filtered, and evaporated and purified by column chromatography (10% MeOH in DCM) to give final product YA6023 (50%). 1H-NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.49 (d, J=8.0 Hz, 1H), 8.13 (d, J=8.0 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.65-7.63 (m, 2H), 7.45-7.35 (m, 3H), 7.21 (d, J=6.8 Hz, 2H), 6.90 (s, 1H), 6.42 (s, 1H), 5.46 (s, 1H), 4.43 (s, 1H), 4.25 (s, 1H), 3.74 (t, J=6.4, 7.2 Hz, 2H), 3.63-3.37 (m, 22H), 3.08-3.07 (m, 2H), 2.58 (t, J=6.4 Hz, 2H), 2.46-2.43 (m, 7H), 2.17 (t, J=7.2 Hz, 2H), 2.02 (s, 3H), 1.66-1.59 (m, 4H), 1.39-1.38 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 173.3, 169.3, 163.8, 160.0, 150.5, 146.4, 138.8, 138.2, 137.7, 135.5, 134.7, 134.2, 131.5, 131.3, 130.9, 127.8, 127.1, 126.1, 114.3, 70.4, 70.3, 70.0, 69.9, 67.3, 62.6, 61.7, 60.1, 55.5, 53.1, 52.8, 45.6, 41.5, 40.5, 39.1, 35.9, 33.4, 28.1, 28.0, 25.6, 18.6, 17.2, 9.3; HRMS (ESI): calcd. for C46H63N10O8S [M+H]+ 915.4551, found 915.4546.
N-(Isoquinolin-3-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6164). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 9.07 (s, 1H), 8.72 (s, 1H), 7.93 (d, J=8.0 Hz, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.66 (t, J=6.8, 8.0 Hz, 1H), 7.52-7.49 (m, 2H), 7.44-7.39 (m, 2H), 7.27 (d, J=7.2 Hz, 1H), 2.54 (s, 3H), 2.09 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 151.4, 146.2, 138.5, 137.9, 137.8, 135.5, 134.3, 131.5, 130.8, 130.7, 127.5, 127.2, 127.1, 126.8, 126.6, 125.8, 107.8, 17.2, 9.3;
5-Methyl-N-(quinolin-3-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6161). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.43 (s, 1H), 8.99 (s, 1H), 8.92 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.66 (t, J=6.8, 8.0 Hz, 1H), 7.57-7.50 (m, 2H), 7.45-7.39 (m, 2H), 7.27 (d, J=8.0 Hz, 1H), 2.53 (s, 3H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 145.3, 144.1, 138.6, 137.7, 135.4, 134.2, 131.5, 131.3, 131.0, 129.1, 128.3, 127.7, 127.3, 127.2, 127.1, 123.5, 17.2, 9.2;
5-Methyl-N-(quinolin-6-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6056). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.32 (s, 1H), 8.82 (s, 1H), 8.51 (s, 1H), 8.14-8.07 (m, 2H), 7.76 (d, J=8.0 Hz, 1H), 7.48-7.46 (m, 1H), 7.42-7.36 (m, 3H), 7.24 (d, J=6.4 Hz, 1H), 2.49 (s, 3H), 2.05 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.5, 149.5, 145.6, 138.5, 137.9, 135.8, 135.6, 135.4, 134.2, 131.5, 130.9, 130.4, 128.9, 127.1, 124.4, 123.2, 121.7, 115.9, 17.2, 9.2;
N-(Isoquinolin-6-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6169-2). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 9.17 (s, 1H), 8.49 (s, 2H), 7.97 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.8 Hz, 1H), 7.63 (d, J=5.2 Hz, 1H), 7.51 (t, J=7.2, 6.8 Hz, 1H), 7.44-7.38 (m, 2H), 7.25 (d, J=6.4 Hz, 1H), 2.52 (s, 3H), 2.07 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 151.8, 143.6, 139.1, 138.7, 137.8, 136.9, 135.4, 134.2, 131.5, 131.0, 128.9, 127.2, 127.1, 125.8, 120.9, 120.4, 114.2, 17.2, 9.2;
N-(Isoquinolin-7-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6169-1). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.60 (s, 1H), 8.95 (d, J=3.2 Hz, 1H), 8.46 (d, J=8.0 Hz, 1H), 8.18 (d, J=7.6 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.76 (d, J=8.0 Hz, 1H), 7.50-7.38 (m, 4H), 7.26 (d, J=8.0 Hz, 1H), 2.50 (s, 3H), 2.09 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 150.5, 148.7, 138.6, 138.0, 135.5, 134.3, 132.1, 131.5, 130.9, 129.8, 129.4, 127.2, 127.1, 127.0, 122.4, 121.1, 120.5, 17.3, 9.2;
5-Methyl-N-(quinolin-7-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6162). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.37 (s, 1H), 8.90 (d, J=4.0 Hz, 1H), 8.38 (s, 1H), 8.11 (d, J=7.6 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.49 (d, J=7.6, 7.2 Hz, 1H), 7.43-7.37 (m, 2H), 7.34-7.26 (m, 1H), 7.25 (d, J=8.0 Hz, 1H), 2.51 (s, 3H), 2.06 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.4, 151.1, 148.9, 138.7, 138.0, 135.6, 135.5, 134.3, 131.5, 130.9, 128.7, 127.1, 125.4, 120.6, 120.2, 117.6, 17.2, 9.2;
5-Methyl-N-(quinolin-8-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6055). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 11.61 (s, 1H), 8.94-8.89 (m, 2H), 8.16 (d, J=8.0 Hz, 1H), 7.57-7.53 (m, 2H), 7.47 (d, J=7.2 Hz, 2H), 7.43-7.36 (m, 2H), 7.26 (d, J=6.8 Hz, 1H), 2.53 (s, 3H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 148.7, 138.9, 138.7, 138.2, 136.1, 135.6, 134.6, 134.5, 131.4, 130.7, 128.0, 127.2, 127.1, 127.0, 121.8, 121.7, 116.4, 17.2, 9.2; HRMS (ESI): calcd. for C20H18N5O [M+H]+ 344.1511, found 344.1508.
5-Methyl-N-(quinolin-5-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6169-3). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.80 (s, 1H), 9.14 (s, 1H), 8.64 (d, J=8.8 Hz, 1H), 8.37 (d, J=7.6 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.94 (t, J=8.0 Hz, 1H), 7.70-7.56 (m, 4H), 7.44 (d, J=8.0 Hz, 1H), 2.68 (s, 3H), 2.27 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 150.5, 148.6, 138.6, 138.0, 135.4, 134.3, 132.1, 131.5, 130.9, 129.8, 129.4, 127.2, 127.1, 127.0, 122.4, 121.1, 120.6, 17.2, 9.2;
5-Methyl-N-(quinazolin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6061). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.01 (s, 1H), 9.37 (s, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.89 (t, J=7.6, 8.0 Hz, 2H), 7.58-7.49 (m, 2H), 7.45-7.39 (m, 2H), 7.29 (d, J=6.0 Hz, 1H), 2.54 (s, 3H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 162.2, 158.7, 154.2, 151.2, 139.4, 135.5, 134.6, 134.3, 131.5, 130.8, 127.7, 127.3, 127.1, 127.0, 126.2, 122.5, 17.2, 9.2;
5-Methyl-N-(quinoxalin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6045). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 9.91 (s, 1H), 8.10 (d, J=7.6 Hz, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.73 (t, J=6.8, 8.0 Hz, 1H), 7.67 (t, J=7.2 Hz, 1H), 7.50 (t, J=7.2 Hz, 1H), 7.44-7.38 (m, 2H), 7.26 (d, J=6.4 Hz, 1H), 2.53 (s, 3H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 146.3, 141.0, 140.5, 139.4, 139.2, 137.3, 135.4, 134.1, 131.5, 131.0, 130.5, 129.1, 128.1, 127.8, 127.2, 127.1, 17.2, 9.3; HRMS (ESI): calcd. for C19H17N6O [M+H]+ 345.1464, found 345.1469.
5-Methyl-N-(1,5-naphthyridin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6147). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 8.86 (d, J=4.0 Hz, 1H), 8.78 (d, J=8.0 Hz, 1H), 8.42 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.0 Hz, 1H), 7.60-7.57 (m, 1H), 7.49 (t, J=7.6, 7.2 Hz, 1H), 7.43-7.37 (m, 2H), 7.24 (d, J=8.0 Hz, 1H), 2.51 (s, 3H), 2.06 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 150.9, 149.5, 142.6, 142.3, 139.6, 139.0, 137.6, 135.6, 135.4, 134.2, 131.5, 130.9, 127.1, 124.7, 117.5, 17.2, 9.3;
5-Methyl-N-(1,8-naphthyridin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA6047). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.13 (s, 1H), 9.04 (s, 1H), 8.69 (d, J=8.0 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H), 8.15 (d, J=8.0 Hz, 1H), 7.49 (t, J=7.6 Hz, 1H), 7.42-7.36 (m, 3H), 7.24 (d, J=8.0 Hz, 1H), 2.50 (s, 3H, CH3), 2.06 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) δ 160.2, 155.1, 153.9, 153.3, 139.4, 139.0, 137.5, 136.5, 135.4, 134.2, 131.5, 130.9, 127.1, 120.8, 120.7, 115.2, 17.3, 9.3; HRMS (ESI): calcd. for C19H17N6O [M+H]+ 345.1464, found 345.1463.
N-(Quinolin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA7013-1). The title compound was synthesized according to General Procedure A (50%, a white solid):
1H-NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.54 (d, J=8.4 Hz, 1H), 8.41 (s, 1H), 8.21 (d, J=8.8 Hz, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.68 (t, J=8.0, 7.6 Hz, 1H), 7.48-7.38 (m, 5H), 2.25 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.5, 150.3, 146.9, 142.9, 138.5, 135.8, 133.6, 131.7, 130.5, 130.0, 127.8, 127.7, 127.5, 127.1, 126.5, 125.9, 125.3, 114.3, 17.9;
5-Methyl-1-phenyl-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA4114). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.91 (s, 1H), 8.54 (d, J=9.2 Hz, 1H), 8.19 (d, J=9.6 Hz, 1H), 7.90 (d, J=8.4 Hz, 1H), 7.78 (d, J=8.0 Hz, 1H), 7.67 (t, J=8.0, 7.6 Hz, 1H), 7.59-7.57 (m, 3H), 7.49-7.43 (m, 3H), 2.69 (s, 1H); 13C-NMR (100 MHz, CDCl3) δ 160.0, 150.6, 146.9, 138.4, 138.2, 137.9, 135.4, 130.1, 130.0, 129.7, 127.8, 127.5, 126.4, 125.3, 125.2, 114.2, 9.9;
5-Methyl-1-(pyridin-3-yl)-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA1144). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 8.82 (d, J=5.6 Hz, 2H), 8.52 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 2H), 7.78 (d, J=7.6 Hz, 1H), 7.67 (t, J=8.0, 7.2 Hz, 1H), 7.58-7.54 (m, 1H), 7.45 (t, J=7.6, 8.0 Hz, 1H), 2.73 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 151.2, 150.5, 146.9, 145.8, 138.6, 138.5, 138.2, 132.7, 132.3, 130.0, 127.7, 127.5, 126.4, 125.3, 124.2, 114.2, 9.9;
5-Methyl-1-(pyridin-2-yl)-N-(quinolin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA4112). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 8.59-8.54 (m, 2H), 8.20 (d, J=9.6 Hz, 1H), 8.00-7.93 (m, 2H), 7.90 (d, J=7.6 Hz, 1H), 7.78 (d, J=8.0 Hz, 1H), 7.67 (t, J=8.0, 7.6 Hz, 1H), 7.46-7.41 (m, 2H), 3.02 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 150.6, 150.2, 148.5, 146.9, 139.1, 138.9, 138.7, 138.4, 130.0, 127.7, 127.5, 126.4, 125.2, 124.2, 118.2, 114.3, 11.0;
5-Methyl-1-(pyridin-3-yl)-N-(quinoxalin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA1144-1). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 9.88 (s, 1H), 8.84-8.82 (m, 2H), 8.09 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 2H), 7.73 (t, J=8.0 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.59 (t, J=6.4 Hz, 1H), 2.75 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.4, 151.4, 146.2, 145.9, 140.9, 140.5, 139.3, 138.6, 138.1, 132.7, 132.2, 130.6, 129.1, 128.3, 127.8, 124.3, 9.9;
1-(2-Methoxyphenyl)-5-methyl-N-(quinoxalin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA6136). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 9.90 (s, 1H), 8.09 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.72 (t, J=6.8, 8.0 Hz, 1H), 7.65 (t, J=8.0, 6.8 Hz, 1H), 7.55 (t, J=8.0, 7.6 Hz, 1H), 7.39 (d, J=8.0 Hz, 1H), 7.15-7.09 (m, 2H), 3.82 (s, 3H), 2.53 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 153.9, 146.4, 141.0, 140.0, 139.4, 137.0, 132.3, 130.5, 129.1, 128.4, 128.1, 127.8, 123.9, 121.1, 112.2, 55.9, 9.3;
1-(2-Cyanophenyl)-5-methyl-N-(quinoxalin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA6137). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.99 (s, 1H), 9.89 (s, 1H), 8.12 (d, J=8.0 Hz, 1H), 7.97-7.88 (m, 3H), 7.80-7.74 (m, 2H), 7.69 (t, J=8.0, 6.8 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 2.71 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.2, 146.2, 140.9, 140.6, 139.4, 136.9, 134.3, 134.2, 131.4, 130.6, 129.1, 128.3, 127.8, 114.6, 111.4, 9.6;
1-(2-Fluorophenyl)-5-methyl-N-(quinoxalin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA6138). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 9.87 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.72 (t, J=6.8, 8.0 Hz, 1H), 7.67-7.51 (m, 3H), 7.40-7.32 (m, 2H), 2.62 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.5, 156.1 (d, J=252.1 Hz), 146.3, 140.9, 140.5, 140.2, 139.4, 137.4, 132.7 (d, J=7.7 Hz), 130.5, 129.1, 128.6, 128.2, 127.8, 125.3 (d, J=3.9 Hz), 123.1 (d, J=11.6 Hz), 117.1 (d, J=19.3 Hz), 9.2;
1-(2-Chlorophenyl)-5-methyl-N-(quinoxalin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA6139). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 9.90 (s, 1H), 8.10 (d, J=7.6 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.73 (t, J=7.2, 7.6 Hz, 1H), 7.68-7.63 (m, 2H), 7.58 (t, J=7.6, 8.0 Hz, 1H), 7.53-7.48 (m, 2H), 2.57 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 146.3, 141.0, 140.5, 140.2, 139.4, 137.2, 132.9, 132.3, 131.8, 130.8, 130.5, 129.1, 128.2, 128.1, 127.8, 9.3;
5-Methyl-1-phenyl-N-(quinoxalin-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA6141). The title compound was synthesized according to General Procedure A (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 9.89 (s, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.72 (t, J=7.2, 7.6 Hz, 1H), 7.67-7.58 (m, 4H), 7.48 (d, J=6.8 Hz, 2H), 2.71 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 146.3, 140.9, 140.5, 139.4, 138.3, 137.7, 135.3, 130.5, 130.2, 129.8, 129.1, 128.2, 127.8, 125.2, 10.0;
N-(Quinolin-2-yl)-2-(o-tolyl)-2H-tetrazole-5-carboxamide (YA70031). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.24 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.69-7.64 (m, 2H), 7.49-7.42 (m, 4H), 2.41 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.5, 154.9, 149.8, 146.6, 138.9, 135.9, 133.1, 132.1, 131.0, 130.2, 127.7, 127.6, 127.1, 126.7, 125.7, 125.3, 114.4, 18.8;
2-Phenyl-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA6171). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.74 (s, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.26-8.21 (m, 3H), 7.90 (d, J=8.0 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.69 (t, J=8.0, 6.8 Hz, 1H), 7.61-7.53 (m, 3H), 7.48 (t, J=7.2, 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 154.8, 149.8, 146.6, 139.0, 136.4, 130.7, 130.3, 129.9, 127.7, 127.6, 127.0, 125.7, 120.3, 114.4;
2-(2-Fluorophenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70032). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.25 (d, J=8.0 Hz, 1H), 7.94-7.89 (m, 2H), 7.81 (d, J=8.0 Hz, 1H), 7.69 (d, J=7.6, 8.0 Hz, 1H), 7.60 (d, J=6.4 Hz, 1H), 7.48 (d, J=7.2, 7.6 Hz, 1H), 7.43-7.39 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 155.9 (d, JCF=258.6 Hz), 154.6, 149.8, 146.6, 139.0, 132.7 (d, JCF=7.7 Hz), 130.3 127.6, 126.7, 125.8, 125.6, 125.1 (d, JCF=3.8 Hz), 124.6, 118.0 (d, JCF=19.3 Hz), 114.4;
2-(2-Chlorophenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70033). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.25 (d, J=8.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.71-7.65 (m, 3H), 7.58 (t, J=8.0 Hz, 1H), 7.53-7.47 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 154.7, 149.8, 146.6, 139.0, 134.4, 132.5, 131.3, 130.3, 129.8, 127.8, 127.7, 127.6, 126.7, 125.7, 114.4;
2-(2-Bromophenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA4184). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 8.59 (d, J=8.0 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.85-7.82 (m, 2H), 7.71 (t, J=6.8, 8.0 Hz, 1H), 7.65-7.62 (m, 1H), 7.58-7.50 (m, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 154.7, 149.8, 146.7, 139.0, 136.1, 134.4, 132.7, 130.3, 128.5, 128.0, 127.7, 127.6, 126.7, 125.8, 118.9, 114.4;
2-(2-Methoxyphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70034). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.74 (s, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H), 7.88 (d, J=7.6 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.68 (t, J=7.2, 8.0 Hz, 1H), 7.59-7.54 (m, 2H), 7.47 (t, J=7.6, 7.2 Hz, 1H), 7.12 (t, J=8.0 Hz, 2H), 3.87 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.4, 155.0, 153.5, 149.9, 146.7, 138.9, 132.8, 130.2, 127.7, 127.6, 126.9, 126.7, 125.6, 120.7, 114.4, 112.7, 56.2;
2-(2-Ethylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70035). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.76 (s, 1H), 8.60 (d, J=8.0 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.70 (t, J=7.6, 7.2 Hz, 1H), 7.60-7.42 (m, 5H), 2.71 (q, J=7.2 Hz, 2H), 1.17 (t, J=6.8 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.5, 154.9, 149.8, 146.7, 139.3, 138.9, 135.4, 131.4, 130.4, 130.3, 127.7, 127.6, 127.0, 126.7, 125.8, 125.7, 114.4, 24.8, 14.8;
N-(Quinolin-2-yl)-2-(2-(trifluoromethyl)phenyl)-2H-tetrazole-5-carboxamide (YA70036). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.74 (s, 1H), 8.54 (d, J=8.0 Hz, 1H), 8.22 (d, J=8.0 Hz, 1H), 7.92 (d, J=7.2 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.82-7.78 (m, 3H), 7.71-7.65 (m, 2H), 7.46 (t, J=7.2, 7.6 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 154.6, 149.7, 146.6, 138.9, 133.7, 133.2, 132.0, 130.3, 128.1, 128.0, 127.9, 127.7, 127.6, 126.7, 125.8, 123.6 (q, J=272.8 Hz), 114.3;
N-(quinolin-2-yl)-2-(2-(trifluoromethoxy)phenyl)-2H-tetrazole-5-carboxamide (YA70037). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.76 (s, 1H), 8.57 (d, J=8.0 Hz, 1H), 8.25 (d, J=8.0 Hz, 1H), 7.89 (t, J=8.0 Hz, 2H), 7.81 (d, J=8.0 Hz, 1H), 7.69-7.66 (m, 2H), 7.58-7.54 (m, 2H), 7.48 (t, J=7.2, 7.6 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 154.6, 149.7, 146.6, 142.1, 139.0, 132.6, 130.3, 129.3, 127.8, 127.7, 127.6, 126.8, 126.7, 125.8, 122.6, 121.5 (q, J=259.8 Hz), 114.4;
2-(2-Isopropylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70038). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.76 (s, 1H), 8.59 (d, J=8.0 Hz, 1H), 8.25 (d, J=8.0 Hz, 1H), 7.89 (d, J=7.6 Hz, 1H), 7.81 (d, J=7.6 Hz, 1H), 7.69 (t, J=8.0, 7.6 Hz, 1H), 7.61-7.54 (m, 2H), 7.50-7.47 (m, 2H), 7.39 (t, J=6.8, 7.2 Hz, 1H), 2.92-2.89 (m, 1H), 1.22 (d, J=6.8 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 154.9, 149.8, 146.7, 144.2, 138.9, 134.8, 131.7, 130.2, 127.7, 127.6, 127.2, 126.8, 126.7, 126.2, 125.7, 114.4, 28.2, 23.5;
2-(2-Fluoro-6-methylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70041). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.76 (s, 1H), 8.56 (d, J=8.0 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.67 (t, J=8.0, 7.6 Hz, 1H), 7.52-7.44 (m, 2H), 7.20-7.14 (m, 2H), 2.15 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 158.3 (d, J=254.8 Hz), 154.7, 149.8, 146.6, 138.9, 137.5, 132.8 (d, J=9.0 Hz), 130.2, 127.7 (d, J=9.0 Hz), 126.7, 126.5 (d, J=2.6 Hz), 125.7, 124.5 (d, J=12.9 Hz), 114.4 (d, J=19.2 Hz), 17.3;
2-(2-Bromo-6-methylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70042). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.78 (s, 1H), 8.59 (d, J=8.0 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H), 7.70 (t, J=7.2, 7.6 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.49 (t, J=7.6, 7.2 Hz, 1H), 7.43-7.38 (m, 2H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 154.7, 149.8, 146.7, 139.0, 138.1, 135.6, 132.7, 131.2, 130.3, 130.1, 127.7, 127.6, 126.7, 125.8, 121.1, 114.4, 17.8;
2-(2,6-Dimethylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70043). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 8.59 (d, J=8.0 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H), 7.70 (t, J=7.2, 7.6 Hz, 1H), 7.49 (t, J=6.8, 8.0 Hz, 1H), 7.41 (t, J=7.6, 7.2 Hz, 1H), 7.23 (d, J=7.6 Hz, 2H), 2.02 (s, 6H); 13C-NMR (100 MHz, CDCl3) δ 159.8, 154.9, 149.8, 146.7, 138.9, 135.7, 135.2, 131.3, 130.2, 128.7, 127.8, 127.6, 126.7, 125.7, 114.4, 17.4;
2-(2-Chloro-6-methylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70044). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 8.60 (d, J=8.0 Hz, 1H), 8.28 (d, J=8.0 Hz, 1H), 7.91 (d, J=7.6 Hz, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.72 (t, J=6.8, 8.0 Hz, 1H), 7.52-7.48 (m, 3H), 7.35 (d, J=6.4 Hz, 1H), 2.10 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.9, 154.7, 149.8, 146.6, 138.9, 137.9, 134.0, 132.4, 131.9, 130.3, 129.5, 128.0, 127.7, 127.6, 126.7, 125.7, 114.4, 17.6;
2-(2,6-Dichlorophenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70045). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H), 7.69 (t, J=7.2, 8.0 Hz, 1H), 7.56 (s, 3H), 7.49 (t, J=6.8, 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 160.0, 154.5, 149.7, 146.6, 139.0, 133.8, 133.3, 132.8, 130.3, 129.0, 127.7, 127.6, 126.7, 125.8, 114.4;
2-(2,6-Difluorophenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70046). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.73 (s, 1H), 8.57 (d, J=8.0 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.71-7.61 (m, 2H), 7.49 (t, J=6.8, 8.0 Hz, 1H), 7.24-7.20 (m, 2H); 13C-NMR (100 MHz, CDCl3) δ 160.1, 158.3 (d, J=258.6 Hz), 154.4, 149.7, 146.6, 139.0, 133.6 (d, J=9.0 Hz), 133.5 (d, J=9.0 Hz), 130.3, 127.7, 127.6, 126.7, 125.8, 114.4, 112.8 (d, J=2.6 Hz), 112.6 (d, J=2.6 Hz);
2-(2,6-Diethylphenyl)-N-(quinolin-2-yl)-2H-tetrazole-5-carboxamide (YA70047). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 8.54 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.0 Hz, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.76 (d, J=8.0 Hz, 1H), 7.64 (t, J=7.6, 8.0 Hz, 1H), 7.47-7.41 (m, 2H), 7.22 (d, J=7.6 Hz, 2H), 2.19 (q, J=8.0 Hz, 4H), 1.03 (t, J=8.0 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 154.9, 149.8, 146.7, 141.1, 138.9, 134.6, 131.8, 130.2, 127.8, 127.6, 127.0, 126.7, 125.8, 114.4, 24.3, 14.7;
2-Phenyl-N-(quinoxalin-2-yl)-2H-tetrazole-5-carboxamide (YA6177). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 9.61 (s, 1H), 8.20 (d, J=7.6 Hz, 2H), 8.09 (d, J=8.0 Hz, 1H), 7.97 (d, J=7.6 Hz, 1H), 7.87-7.77 (m, 2H), 7.74-7.66 (m, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 159.7, 156.4, 146.8, 141.0, 140.7, 140.2, 136.4, 131.4, 130.7, 129.4, 129.3, 128.1, 120.8;
5-Methyl-N-(pyridin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA7083).
1H-NMR (400 MHz, CDCl3) δ 9.67 (s, 1H), 8.35-8.31 (m, 2H), 7.72 (t, J=7.6, 8.0 Hz, 1H), 7.47 (t, J=7.2, 7.6 Hz, 1H), 7.42-7.35 (m, 2H), 7.24 (d, J=7.6 Hz, 1H), 7.05 (t, J=5.6, 6.0 Hz, 1H), 2.47 (s, 3H), 2.04 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.7, 151.2, 148.3, 138.6, 138.2, 137.8, 135.5, 134.3, 131.5, 130.8, 127.1, 127.1, 119.8, 113.9, 17.2, 9.2;
5-Methyl-N-(6-methylpyridin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA7084).
1H-NMR (400 MHz, CDCl3) δ 9.61 (s, 1H), 8.12 (d, J=8.0 Hz, 1H), 7.61 (t, J=8.0, 7.6 Hz, 1H), 7.47 (t, J=8.0, 7.6 Hz, 1H), 7.41-7.35 (m, 2H), 7.23 (d, J=6.8 Hz, 1H), 6.91 (d, J=7.6 Hz, 1H), 2.48 (s, 3H), 2.04 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6, 157.2, 150.4, 138.5, 138.4, 137.8, 135.5, 134.3, 131.5, 130.8, 127.2, 127.1, 126.9, 119.3, 110.7, 24.1, 17.2, 9.2;
5-Methyl-N-(4-phenylthiazol-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4117). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.47 (s, 1H), 7.79 (d, J=7.2 Hz, 2H), 7.39-7.22 (m, 6H), 7.15-7.09 (m, 2H), 2.40 (s, 3H), 1.95 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.7, 156.7, 150.3, 139.3, 136.5, 135.4, 134.3, 134.0, 131.5, 131.0, 128.7, 128.0, 127.2, 127.1, 126.1, 107.6, 17.2, 9.2; HRMS (ESI): calcd. for C20H18N5OS [M+H]+ 376.1232, found 376.1239.
5-Methyl-N-(5-phenylthiazol-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4085). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.79 (s, 1H), 7.54 (s, 1H), 7.30-7.28 (m, 2H), 7.20 (t, J=7.2 Hz, 1H), 7.14-7.07 (m, 4H), 7.00-6.94 (m, 2H), 2.22 (s, 3H), 1.76 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.7, 156.5, 139.4, 136.6, 135.4, 134.1, 133.9, 132.9, 131.8, 131.5, 131.0, 129.0, 127.7, 127.2, 127.1, 126.2, 17.2, 9.3; HRMS (ESI): calcd. for C20H18N5OS [M+H]+ 376.1232, found 376.1235.
N-(4,5-Diphenylthiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4133). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.54 (s, 1H), 7.56-7.25 (m, 14H), 2.53 (s, 3H), 2.09 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 153.9, 149.9, 140.0, 134.6, 131.7, 130.7, 130.1, 129.3, 127.4, 126.8, 126.2, 124.8, 124.1, 124.0, 123.6, 123.1, 123.0, 122.4, 122.4, 12.5, 4.5; HRMS (ESI): calcd. for C26H22N5OS [M+H]+ 452.1545, found 452.1555.
N-(Benzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4105). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 7.80-7.78 (m, 2H), 7.45-7.27 (m, 5H), 7.18 (d, J=6.8 Hz, 1H), 2.46 (s, 3H), 2.00 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.1, 156.8, 148.7, 139.6, 136.4, 135.4, 134.0, 132.3, 131.6, 131.0, 127.2, 127.1, 126.2, 124.0, 121.3, 17.2, 9.2; HRMS (ESI): calcd. for C18H16N5OS [M+H]+ 350.1076, found 350.1076.
N-(4-Methoxybenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4061). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.97 (s, 1H), 7.84-7.81 (m, 1H), 7.78-7.70 (m, 3H), 7.64-7.56 (m, 2H), 7.25 (d, J=8.0 Hz, 1H), 4.38 (s, 3H), 2.85 (s, 3H), 2.39 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.0, 155.6, 152.2, 139.4, 138.5, 136.3, 135.3, 134.0, 133.6, 131.5, 131.0, 127.1, 127.0, 124.9, 113.3, 106.8, 55.9, 17.2, 9.2; HRMS (ESI): calcd. for C19H18N5O2S [M+H]+ 380.1181, found 380.1191.
N-(5-Methoxybenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4045). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.49 (t, J=6.4, 7.2 Hz, 1H), 7.43-7.34 (m, 3H), 7.24 (d, J=7.6 Hz, 1H), 6.95 (d, J=8.4 Hz, 1H), 3.86 (s, 3H), 2.50 (s, 3H), 2.05 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.1, 158.9, 158.0, 149.9, 139.6, 136.4, 135.4, 134.0, 131.5, 131.0, 127.2, 127.1, 124.0, 121.6, 113.6, 104.3, 55.6, 17.2, 9.2; HRMS (ESI): calcd. for C19H18N5O2S [M+H]+ 380.1181, found 380.1183.
N-(6-Methoxybenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4091). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.67 (s, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.69 (t, J=8.0, 6.8 Hz, 1H), 7.63-7.56 (m, 2H), 7.50 (s, 1H), 7.43 (d, J=8.0 Hz, 1H), 7.24 (d, J=8.8 Hz, 1H), 4.06 (s, 3H), 2.70 (s, 3H), 2.25 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.9, 156.9, 154.7, 143.0, 139.4, 136.4, 135.4, 134.0, 133.5, 131.5, 131.0, 127.2, 127.1, 121.8, 115.2, 104.2, 55.8, 17.2, 9.2; HRMS (ESI): calcd. for C19H18N5O2S [M+H]+ 380.1181, found 380.1176.
N-(4-Fluorobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4093). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.49 (t, J=7.2 Hz, 1H), 7.42-7.38 (m, 2H), 7.24-7.22 (m, 2H), 7.13 (t, J=8.0 Hz, 1H), 2.51 (s, 3H), 2.05 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.2, 157.2, 154.8 (d, JCF=252.1 Hz), 139.7, 137.5 (d, JCF=12.9 Hz), 136.2, 135.3, 135.0, 133.9, 131.5, 131.0, 127.2 (d, JCF=14.1 Hz), 124.6 (d, JCF=7.7 Hz), 117.0 (d, JCF=2.6 Hz), 112.1 (d, JCF=18.0 Hz), 17.2, 9.2; HRMS (ESI): calcd. for C18H15FN5OS [M+H]+ 368.0981, found 368.0979.
N-(5-Fluorobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4101). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 7.78-7.74 (m, 1H), 7.53-7.50 (m, 2H), 7.45-7.38 (m, 2H), 7.24 (d, J=7.6 Hz, 1H), 7.11-7.06 (m, 1H), 2.52 (s, 3H), 2.07 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.9 (d, J=241.9 Hz), 159.1, 159.0, 149.9 (d, J=12.9 Hz) 139.7, 136.2, 135.4, 134.0, 131.5, 131.0, 127.6, 127.2 (d, J=11.5 Hz), 122.0 (d, J=9.0 Hz), 112.3 (d, J=24.5 Hz), 107.7 (d, J=23.2 Hz) 17.2, 9.2; HRMS (ESI): calcd. for C18H15FN5OS [M+H]+ 368.0981, found 368.0974.
N-(6-Fluorobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4103). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.69 (s, 1H), 7.72-7.71 (m, 1H), 7.49-7.44 (m, 2H), 7.40-7.34 (m, 2H), 7.21 (d, J=7.6 Hz, 1H), 7.11 (t, J=7.6 Hz, 1H), 2.48 (s, 3H), 2.02 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 160.8, 158.4, 157.8 (d, JCF=256.0 Hz), 145.2, 139.7, 136.2, 135.4, 134.0, 133.3 (d, JCF=10.3 Hz), 131.5, 131.0, 127.1 (d, JCF=11.6 Hz), 122.2 (d, JCF=9.1 Hz), 114.5 (d, JCF=24.4 Hz), 107.6 (d, JCF=25.7 Hz), 17.2, 9.2; HRMS (ESI): calcd. for C18H15FN5OS [M+H]+ 368.0981, found 368.0979.
N-(4-Chlorobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4095). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.71 (s, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.51-7.37 (m, 4H), 7.26-7.22 (m, 2H), 2.51 (s, 3H), 2.05 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.2, 157.5, 145.8, 139.7, 136.2, 135.3, 133.9, 133.6, 131.5, 131.0, 127.2, 127.0, 126.5, 126.0, 124.5, 119.9, 17.2, 9.2; HRMS (ESI): calcd. for C18H15ClN5OS [M+H]+ 384.0686, found 384.0694.
N-(5-Chlorobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4013). The title compound was synthesized according to General Procedure B (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.76 (d, J=8.8 Hz, 1H), 7.53-7.51 (m, 1H), 7.46-7.40 (m, 2H), 7.31 (d, J=8.4 Hz, 1H), 7.26-7.24 (m, 1H), 2.53 (s, 3H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.1, 158.3, 149.8, 139.7, 136.2, 135.4, 134.0, 132.1, 131.6, 131.1, 130.6, 127.2, 127.1, 124.3, 122.1, 121.2, 17.2, 9.2; HRMS (ESI): calcd. for C18H15ClN5OS [M+H]+ 384.0686, found 384.0683.
N-(6-Chlorobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4071). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.77 (s, 1H), 7.89 (s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.59 (t, J=8.0, 6.8 Hz, 1H), 7.53-7.46 (m, 3H), 7.33 (d, J=8.0 Hz, 1H), 2.61 (s, 3H), 2.15 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.1, 157.2, 147.3, 139.7, 136.2, 135.4, 133.9, 133.5, 131.6, 131.0, 129.4, 127.2, 127.1, 126.9, 122.1, 120.9, 17.2, 9.2; HRMS (ESI): calcd. for C18H15ClN5OS [M+H]+ 384.0686, found 384.0692.
N-(4-Bromobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4097). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.64 (d, J=7.2 Hz, 1H), 7.50 (t, J=7.2 Hz, 1H), 7.43-7.39 (m, 2H), 7.24-7.16 (m, 2H), 2.51 (s, 3H), 2.05 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.2, 157.3, 147.1, 139.8, 136.2, 135.4, 133.9, 133.1, 131.6, 131.0, 129.7, 127.2, 127.1, 124.9, 120.5, 114.7, 17.2, 9.2; HRMS (ESI): calcd. for C18H15BrN5OS [M+H]+ 428.0181, found 428.0185.
N-(5-Bromobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4047). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.61 (s, 1H), 7.99 (s, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.45-7.39 (m, 3H), 7.25 (d, J=8.0 Hz, 1H), 2.53 (s, 3H), 2.07 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.1, 158.0, 150.1, 139.7, 136.2, 135.4, 133.9, 131.6, 131.1, 127.2, 127.1, 127.0, 124.2, 122.4, 119.8, 17.2, 9.2; HRMS (ESI): calcd. for C11H15BrN5OS [M+H]+ 428.0181, found 428.0187.
N-(6-Bromobenzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4059). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.47 (s, 1H), 8.10 (s, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.68-7.63 (m, 2H), 7.58-7.52 (m, 2H), 7.38 (d, J=7.2 Hz, 1H), 2.66 (s, 3H), 2.20 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.1, 157.1, 147.7, 139.7, 136.2, 135.4, 134.0, 133.9, 131.6, 131.1, 129.6, 127.2, 127.1, 123.8, 122.5, 116.9, 17.2, 9.2; HRMS (ESI): calcd. for C11H15BrN5OS [M+H]+ 428.0181, found 428.0191.
5-Methyl-N-(6-methylbenzo[d]thiazol-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4069). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.76 (s, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.74 (s, 1H), 7.61 (t, J=7.2 Hz, 1H), 7.54-7.48 (m, 2H), 7.38-7.34 (m, 2H), 2.63 (s, 3H), 2.59 (s, 3H), 2.17 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.0, 156.0 146.7, 139.5, 136.4, 135.4, 134.0, 133.9, 132.4, 131.5, 131.0, 127.7, 127.2, 127.1, 121.1, 120.8, 21.5, 17.2, 9.2; HRMS (ESI): calcd. for C19H18N5OS [M+H]+ 364.1232, found 364.1237.
5-Methyl-1-(o-tolyl)-N-(6-(trifluoromethoxy)benzo[d]thiazol-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA4065). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.47 (s, 1H), 7.48 (d, J=8.4 Hz, 1H), 7.38 (s, 1H), 7.18 (t, J=8.0, 7.2 Hz, 1H), 7.11-7.05 (m, 2H), 6.98 (d, J=8.4 Hz, 1H), 6.92 (d, J=7.2 Hz, 1H), 2.20 (s, 3H), 1.73 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.2, 157.8, 147.4, 145.4, 139.8, 136.2, 135.4, 133.9, 133.1, 131.5, 131.0, 127.2, 127.0, 124.4 (q, J=256.0 Hz, CF3), 122.0, 120.0, 114.1, 17.2, 9.2; HRMS (ESI): calcd. for C19H15F3N5O2S [M+H]+ 434.0899, found 434.0889.
5-Methyl-1-(o-tolyl)-N-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)-1H-1,2,3-triazole-4-carboxamide (YA4063). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.59 (s, 1H), 7.90 (s, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H) 7.28 (t, J=8.0, 6.8 Hz, 1H), 7.22-7.15 (m, 2H), 7.01 (d, J=8.0 Hz, 1H), 2.30 (s, 3H), 1.84 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.3, 151.1, 139.9, 136.1, 135.4, 133.9, 132.4, 131.6, 131.1, 128.4, 127.2, 127.0, 126.3 (q, J=32.2 Hz) 123.3, 123.0 (q, J=271.5 Hz, CF3), 121.5, 119.0, 17.2, 9.2; HRMS (ESI): calcd. for C19H15F3N5O [M+H]+ 418.0949, found 418.0953.
5-Methyl-N-(6-nitrobenzo[d]thiazol-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (YA4057). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.75 (s, 1H), 8.76 (s, 1H), 8.32 (d, J=9.2 Hz, 1H), 7.87 (d, J=9.2 Hz, 1H), 7.51 (t, J=8.0, 6.8 Hz, 1H), 7.45-7.38 (m, 2H), 7.24 (s, 1H), 2.52 (s, 3H), 2.06 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.4, 159.3, 153.3, 143.9, 140.2, 135.9, 135.3, 133.8, 132.7, 131.6, 131.2, 127.3, 127.0, 122.0, 121.3, 118.1, 17.2, 9.3; HRMS (ESI): calcd. for C18H15N6O3S [M+H]+ 395.0926, found 395.0932.
N-(Benzo[d]thiazol-2-yl)-5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamide (YA6029). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.54 (s, 1H), 7.85 (d, J=8.0 Hz, 2H), 7.60-7.61 (m, 3H), 7.50-7.44 (m, 3H), 7.33 (t, J=7.6, 7.2 Hz, 1H), 2.72 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.0, 156.8, 148.7, 138.6, 136.8, 135.2, 132.2, 130.3, 129.8, 126.3, 125.2, 124.0, 121.3, 121.3, 9.9; HRMS (ESI): calcd. for C17H14N5OS [M+H]+ 336.0919, found 336.0911.
N-(benzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA4029). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 7.85 (d, J=8.4 Hz, 2H), 7.65-7.60 (m, 1H), 7.54 (t, J=7.6 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H), 7.43-7.32 (m, 3H), 2.64 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.8, 156.8, 156.0 (d, JCF=252.2 Hz), 148.6, 140.5, 136.5, 132.7 (d, JCF=7.8 Hz), 132.2, 128.5, 126.2, 125.4 (d, JCF=3.8 Hz), 124.0, 123.0 (d, JCF=12.8 Hz), 121.3, 117.1 (d, JCF=19.3 Hz), 9.2; HRMS (ESI): calcd. for C17H13FN5OS [M+H]+ 354.0825, found 354.0823.
N-(Benzo[d]thiazol-2-yl)-1-(3-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1072). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.48 (s, 1H), 7.74 (d, J=8.0 Hz, 2H), 7.51-7.46 (m, 1H), 7.36 (t, J=7.6, 8.0 Hz, 1H), 7.25-7.16 (m, 4H), 2.64 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 164.0, 161.5 (d, J=170.1 Hz), 156.7, 148.7, 138.6, 137.0, 136.3 (d, J=9.0 Hz), 132.3, 131.3 (d, J=9.0 Hz), 126.3, 124.0, 121.3, 120.8 (d, J=3.9 Hz), 117.6 (d, J=21.9 Hz), 113.1 (d, J=24.5 Hz), 9.9; HRMS (ESI): calcd. for C17H13FN5OS [M+H]+ 354.0825, found 354.0826.
N-(Benzo[d]thiazol-2-yl)-1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1076). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.82-7.76 (m, 3H), 7.56-7.46 (m, 3H), 7.35 (t, J=7.2 Hz, 1H), 2.60 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 164.3, 161.8, 160.7 (d, J=254.8 Hz), 148.7, 139.9, 137.2, 131.9, 128.5 (d, J=9.1 Hz), 127.4, 126.7, 124.2, 122.2, 121.0, 117.3 (d, J=23.1 Hz), 115.6, 10.0; HRMS (ESI): calcd. for C17H13FN5OS [M+H]+ 354.0825, found 354.0831.
N-(Benzo[d]thiazol-2-yl)-1-(2-chlorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA6027). The title compound was synthesized according to General Procedure B (50%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.61 (s, 1H), 7.83 (d, J=7.2 Hz, 2H), 7.63 (d, J=7.6 Hz, 1H), 7.57 (t, J=6.8, 8.0 Hz, 1H), 7.52-7.45 (m, 3H), 7.32 (t, J=8.0, 6.8 Hz, 1H), 2.56 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.9, 156.8, 148.7, 140.5, 136.4, 132.7, 132.4, 132.2, 131.6, 130.8, 129.1, 128.2, 126.3, 124.0, 121.3, 9.27; HRMS (ESI): calcd. for C17H13ClN5OS [M+H]+ 370.0529, found 370.0527.
N-(Benzo[d]thiazol-2-yl)-1-(3-chlorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA4055). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 7.85 (d, J=8.0 Hz, 2H), 7.57-7.54 (m, 3H), 7.47 (t, J=8.0 Hz, 1H), 7.40 (d, J=7.2 Hz, 1H), 7.34 (t, J=8.0 Hz, 1H), 2.74 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.8, 156.7, 148.6, 138.7, 137.0, 136.1, 135.6, 132.2, 130.8, 130.5, 126.3, 125.5, 124.1, 123.2, 121.3, 121.2, 9.9; HRMS (ESI): calcd. for C17H13ClN5OS [M+H]+ 370.0529, found 370.0535.
N-(Benzo[d]thiazol-2-yl)-1-(4-chlorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1074). The title compound was synthesized according to General Procedure B (%, a white solid):
1H-NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.02 (d, J=7.6 Hz, 1H), 7.81-7.75 (m, 5H), 7.47 (t, J=7.2, 7.6 Hz, 1H), 7.35 (t, J=8.0, 7.2 Hz, 1H), 2.62 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 139.9, 137.3, 135.3, 134.4, 132.0, 130.3, 127.8, 126.7, 124.2, 122.2, 121.0, 10.1; HRMS (ESI): calcd. for C17H13ClN5OS [M+H]+ 370.0529, found 370.0529.
N-(Benzo[d]thiazol-2-yl)-1-(2-bromophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA4003). The title compound was synthesized according to General Procedure B (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 10.5 (br s, 1H), 7.77-7.72 (m, 3H), 7.49-7.34 (m, 4H), 7.24 (t, J=8.0, 7.2 Hz, 1H), 2.48 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.9, 156.8, 148.7, 140.3, 136.4, 134.4, 133.9, 132.5, 132.3, 129.2, 128.8, 126.3, 124.0, 121.4, 121.3, 9.4; HRMS (ESI): calcd. for C17H13BrN5OS [M+H]+ 414.0024, found 414.0023.
N-(Benzo[d]thiazol-2-yl)-1-(3-bromophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1050). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 7.83 (d, J=8.0 Hz, 2H), 7.72-7.68 (m, 2H), 7.49-7.42 (m, 3H), 7.32 (t, J=6.8, 8.0 Hz, 1H), 2.72 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.8, 156.7, 148.7, 138.7, 137.0, 136.2, 133.5, 132.3, 131.0, 128.3, 126.3, 124.0, 123.7, 123.3, 121.3, 9.9; HRMS (ESI): calcd. for C17H13BrN5OS [M+H]+ 414.0024, found 414.0024.
N-(Benzo[d]thiazol-2-yl)-1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1086). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 12.71 (s, 1H), 8.02 (d, J=7.6 Hz, 1H), 7.89 (d, J=7.6 Hz, 2H), 7.80 (d, J=8.0 Hz, 1H), 7.67 (d, J=8.0 Hz, 2H), 7.47 (t, J=7.2, 7.6 Hz, 1H), 7.34 (t, J=8.0, 6.8 Hz, 1H), 2.62 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 134.8, 133.2, 128.0, 126.7, 124.2, 123.9, 122.2, 121.1, 10.1; HRMS (ESI): calcd. for C17H13BrN5OS [M+H]+ 414.0024, found 414.0033.
N-(Benzo[d]thiazol-2-yl)-5-methyl-1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamide (YA6089). The title compound was synthesized according to General Procedure B (60%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.56 (s, 1H), 7.94 (d, J=7.2 Hz, 1H), 7.85-7.76 (m, 4H), 7.47-7.40 (m, 2H), 7.33 (t, J=8.0 Hz, 1H), 2.52 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.8, 156.7, 148.7, 140.9, 136.2, 133.4, 132.3, 131.7, 129.7, 128.2, 127.9, 127.9, 126.3, 124.0, 123.7 (q, J=270 Hz, CF3), 121.3, 120.9, 9.2; HRMS (ESI): calcd. for C18H13F3N5OS [M+H]+ 404.0793, found 404.0792.
N-(Benzo[d]thiazol-2-yl)-5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamide (YA1038). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.93 (s, 1H), 8.07 (t, J=6.4 Hz, 2H), 7.90-7.88 (m, 1H), 7.76-7.75 (m, 3H), 7.67 (t, J=6.8, 8.0 Hz, 1H), 7.55 (t, J=7.2, 7.6 Hz, 1H), 2.82 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.9, 156.8, 148.7, 143.7, 140.5, 136.4, 132.6, 132.3, 129.1, 127.9, 127.5, 126.2, 124.0 (q, J=270.2 Hz), 121.7, 118.7, 9.2; HRMS (ESI): calcd. for C18H13F3N5O2S [M+H]+ 420.0742, found 420.0739.
N-(Benzo[d]thiazol-2-yl)-1-(2,4-difluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1046). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 7.85-7.83 (m, 2H), 7.56-7.50 (m, 1H), 7.46 (t, J=8.0, 7.6 Hz, 1H), 7.33 (t, J=8.0, 6.8 Hz, 1H), 7.14-7.10 (m, 2H), 2.62 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 165.4 (d, J=254.8 Hz), 165.3 (d, J=254.7 Hz), 158.7, 156.7, 155.2, 148.7, 140.5, 136.6, 132.3, 129.8 (d, J=10.2 Hz), 126.3, 124.1, 121.3 (d, J=26.0 Hz), 113.0 (d, J=23.2 Hz), 105.7 (t, J=25.7, 23.2 Hz), 9.1; HRMS (ESI): calcd. for C17H12F2N5OS [M+H]+ 372.0731, found 372.0728.
N-(Benzo[d]thiazol-2-yl)-1-(2,5-difluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1084). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.57 (s, 1H), 7.82 (t, J=6.4 Hz, 2H), 7.43 (t, J=8.0, 7.6 Hz, 1H), 7.33-7.27 (m, 4H), 2.63 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6 (d, J=245.7 Hz), 158.7, 156.7, 153.5 (d, J=247.0 Hz), 148.7, 140.5, 136.7, 132.3, 126.3, 124.1, 121.3, 119.6 (dd, J=7.7, 23.1 Hz), 118.3 (dd, J=21.9, 9.0 Hz), 115.8 (d, J=27.0 Hz), 9.2; HRMS (ESI): calcd. for C17H12F2N5OS [M+H]+ 372.0731, found 372.0723.
N-(Benzo[d]thiazol-2-yl)-1-(2,6-difluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA6090). The title compound was synthesized according to General Procedure B (60%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.62 (s, 1H), 7.84-7.81 (m, 2H), 7.62-7.54 (m, 1H), 7.43 (t, J=6.8, 8.0 Hz, 1H), 7.31 (t, J=7.6, 7.2 Hz, 1H), 7.18 (t, J=8.0 Hz, 2H), 2.60 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.9 (d, JCF=214.9 Hz), 158.6, 156.3, 148.7, 141.2, 136.4, 133.0 (t, JCF=9.0, 10.3 Hz), 132.3, 126.2, 124.0, 121.3, 112.8 (dd, JCF=3.8, 19.3 Hz), 8.8; HRMS (ESI): calcd. for C17H12F2N5OS [M+H]+ 372.0731, found 372.0730.
N-(Benzo[d]thiazol-2-yl)-1-(2,6-dichlorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1066). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.63 (s, 1H), 7.84-7.83 (m, 2H), 7.56-7.43 (m, 4H), 7.32 (t, J=8.0 Hz, 1H), 2.54 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.8, 156.7, 148.7, 140.7, 136.4, 134.3, 132.7, 132.3, 130.9, 129.1, 126.3, 124.0, 121.3, 8.7; HRMS (ESI): calcd. for C17H12C12N5OS [M+H]+ 404.0140, found 404.0150.
N-(Benzo[d]thiazol-2-yl)-1-(2-fluoro-6-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1042). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.63 (s, 1H), 7.83 (d, J=6.4 Hz, 2H), 7.49-7.42 (m, 2H), 7.31 (t, J=7.2, 8.0 Hz, 1H), 7.20 (d, J=7.6 Hz, 1H), 7.14 (t, J=8.0 Hz, 1H), 2.53 (s, 3H), 2.08 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.9, 158.5 (d, J=252.2 Hz), 156.8, 148.7, 140.6, 138.4, 136.4, 132.3 (d, J=9.0 Hz), 126.7, 126.3, 124.0, 122.2 (d, J=14.2 Hz), 121.3, 114.2 (d, J=19.3 Hz), 17.2, 8.8; HRMS (ESI): calcd. for C18H15FN5OS [M+H]+ 368.0981, found 368.0986.
N-(Benzo[d]thiazol-2-yl)-1-(2-chloro-6-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1068). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 7.84-7.81 (m, 2H), 7.44-7.39 (m, 3H), 7.29-7.30 (m, 2H), 2.50 (s, 3H), 2.02 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.0, 156.8, 148.7, 140.3, 138.5, 136.5, 132.3, 131.9, 131.8, 129.7, 128.0, 126.2, 124.0, 121.3, 17.7, 8.8; HRMS (ESI): calcd. for C18H15ClN5OS [M+H]+ 384.0686, found 384.0693.
N-(Benzo[d]thiazol-2-yl)-1-(2-bromo-6-methylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA1064). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.58 (s, 1H), 7.84 (d, J=8.0 Hz, 2H), 7.62 (t, J=4.4 Hz, 1H), 7.45 (t, J=8.0, 7.6 Hz, 1H), 7.37-7.31 (m, 3H), 2.51 (s, 3H), 2.05 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.0, 156.7, 148.7, 140.1, 138.6, 136.5, 133.4, 132.2, 131.3, 130.3, 126.3, 124.0, 122.1, 121.3, 18.0, 8.9; HRMS (ESI): calcd. for C11H15BrN5OS [M+H]+ 428.0181, found 428.0176.
N-(Benzo[d]thiazol-2-yl)-5-ethyl-1-(2-fluorophenyl)-1H-1,2,3-triazole-4-carboxamide (YA4001). The title compound was synthesized according to General Procedure B (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 10.61 (s, 1H), 7.83 (d, J=8.4 Hz, 1H), 7.61-7.59 (m, 1H), 7.48-7.42 (m, 2H), 7.39-7.29 (m, 3H), 3.02 (q, J=6.8, 8.0, 7.2 Hz, 2H), 1.18 (t, J=6.8, 8.0 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 158.6, 156.8, 156.5 (d, JCF=253.4 Hz), 148.7, 145.8, 136.0, 132.9 (d, JCF=7.7 Hz), 132.2, 128.8, 126.2, 125.3, 124.0, 123.2 (d, JCF=11.5 Hz), 121.3, 117.2 (d, JCF=19.3 Hz), 16.9, 12.7; HRMS (ESI): calcd. for C18H15FN5OS [M+H]+ 368.0981, found 368.0976.
N-(Benzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-isopropyl-1H-1,2,3-triazole-4-carboxamide (YA4005). The title compound was synthesized according to General Procedure B (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 10.67 (br s, 1H), 7.83 (d, J=8.0 Hz, 2H), 7.65-7.59 (m, 1H), 7.47-7.44 (m, 2H), 7.40-7.30 (m, 3H), 3.31-3.24 (m, 1H), 1.40 (d, J=5.6 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ 158.4, 156.9, 156.8 (d, JCF=253.4 Hz), 149.3, 148.7, 135.9, 133.0 (d, JCF=7.7 Hz), 132.2, 129.1, 126.3, 125.2 (d, JCF=2.5 Hz), 124.0, 121.2 (d, JCF=10.3 Hz), 117.1 (d, JCF=18.0 Hz), 25.2, 19.9; HRMS (ESI): calcd. for C19H17FN5OS [M+H]+ 382.1138, found 382.1146.
N-(Benzo[d]thiazol-2-yl)-5-(tert-butyl)-1-(2-fluorophenyl)-1H-1,2,3-triazole-4-carboxamide (YA6176). The title compound was synthesized according to General Procedure B (60%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.89 (s, 1H), 7.84 (dd, J=8.0 Hz, 2H), 7.59-7.58 (m, 1H), 7.45 (t, J=7.6, 7.2 Hz, 2H), 7.35-7.26 (m, 3H), 1.42 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ 158.8, 157.4 (d, JCF=252.2 Hz), 157.2, 151.4, 148.8, 136.6, 132.8 (d, JCF=7.8 Hz), 132.2, 129.3, 126.7 (d, JCF=14.2 Hz), 126.2, 124.8 (d, JCF=3.9 Hz), 123.9, 121.3 (d, JCF=11.6 Hz), 116.7 (d, JCF=18.0 Hz), 33.2, 29.7; HRMS (ESI): calcd. for C20H19FN5OS [M+H]+ 396.1294, found 396.1290.
N-(Benzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (YA6168). The title compound was synthesized according to General Procedure B (60%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.93 (s, 1H), 7.86-7.81 (m, 2H), 7.67-7.61 (m, 1H), 7.52 (t, J=7.2 Hz, 1H), 7.45 (t, J=6.8, 8.0 Hz, 1H), 7.40-7.32 (m, 3H); 13C-NMR (100 MHz, CDCl3) δ 156.7, 156.2 (d, JCF=254.7 Hz), 155.6, 148.4, 139.5, 133.6 (d, JCF=6.4 Hz), 132.3, 128.1, 126.4, 125.1 (d, JCF=3.9 Hz), 124.4, 123.5 (d, JCF=12.8 Hz), 121.4 (d, JCF=5.2 Hz), 118.5 (q, J=270.1 Hz, CF3), 116.9 (d, JCF=19.3 Hz); HRMS (ESI): calcd. for C17H10F4N5OS [M+H]+ 408.0542, found 408.0542.
N-(6-Fluorobenzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (YA4155). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.65-7.59 (m, 3H), 7.46 (t, J=8.0 Hz, 1H), 7.35 (t, J=6.8, 8.0 Hz, 1H), 7.14 (t, J=8.0 Hz, 1H), 2.34 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 159.2 (d, JCF=238.0 Hz), 156.2 (d, JCF=250.9 Hz), 145.5, 141.0, 136.9, 133.7 (d, JCF=7.7 Hz), 133.3 (d, JCF=11.6 Hz), 129.5, 126.2, 122.9 (d, JCF=11.6 Hz), 122.2 (d, JCF=9.0 Hz), 117.6 (d, JCF=19.3 Hz), 114.8 (d, JCF=24.5 Hz), 108.6 (d, JCF=27.0 Hz), 9.4; HRMS (ESI): calcd. for C17H12F2N5OS [M+H]+ 372.0731, found 372.0735.
5-Ethyl-N-(6-fluorobenzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-1H-1,2,3-triazole-4-carboxamide (YA4165). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.60 (s, 1H), 7.76-7.72 (m, 1H), 7.63-7.58 (m, 1H), 7.51-7.45 (m, 2H), 7.39-7.32 (m, 2H), 7.14 (t, J=8.0 Hz, 1H), 3.02 (m, 2H), 1.17 (t, J=7.2 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 159.6 (d, JCF=241.8 Hz), 158.7, 156.5 (d, JCF=253.4 Hz), 156.4, 145.8, 145.2, 135.9, 133.2 (d, JCF=10.3 Hz), 132.9 (d, JCF=7.8 Hz), 128.8, 125.3 (d, JCF=3.8 Hz), 123.1 (d, JCF=12.9 Hz), 122.1 (d, JCF=9.0 Hz), 117.2 (d, JCF=18.0 Hz), 114.5 (d, JCF=24.4 Hz), 107.5 (d, JCF=25.8 Hz), 16.9, 12.6; HRMS (ESI): calcd. for C18H14F2N5OS [M+H]+ 386.0887, found 386.0888.
N-(6-Fluorobenzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-isopropyl-1H-1,2,3-triazole-4-carboxamide (YA6172). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.71 (s, 1H), 7.76-7.73 (m, 1H), 7.64-7.59 (m, 1H), 7.51-7.43 (m, 2H), 7.39-7.32 (m, 2H), 7.18-7.13 (m, 1H), 3.29-3.22 (m, 1H), 1.39 (d, J=6.4 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ 159.7 (d, JCF=239.3 Hz), 156.8 (d, JCF=253.4 Hz), 156.6, 149.4, 145.2, 135.8, 133.2, 133.0 (d, JCF=7.7 Hz), 129.1, 125.2, 125.1, 123.7 (d, JCF=12.9 Hz), 122.1 (d, JCF=9.0 Hz), 117.1 (d, JCF=19.3 Hz), 114.6 (d, JCF=24.4 Hz), 107.6 (d, JCF=25.8 Hz), 25.2, 19.9; HRMS (ESI): calcd. for C19H16F2N5OS [M+H]+ 400.1044, found 400.1042.
5-(Tert-butyl)-N-(6-fluorobenzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-1H-1,2,3-triazole-4-carboxamide (YA6174). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.89 (s, 1H), 7.76-7.73 (m, 1H), 7.61-7.56 (m, 1H), 7.51 (dd, J=7.6 Hz, 1H), 7.45 (t, J=6.8, 7.2 Hz, 1H), 7.35-7.26 (m, 2H), 7.18-7.13 (m, 1H), 1.41 (s, 9H); 13C-NMR (100 MHz, CDCl3) δ 159.6 (d, JCF=241.8 Hz), 158.6, 157.4 (d, JCF=252.2 Hz), 156.9, 151.4, 145.3, 136.4, 133.1 (d, JCF=10.3 Hz), 132.8 (d, JCF=7.7 Hz), 129.3, 126.6 (d, JCF12.9 Hz), 124.7 (d, JCF=3.9 Hz), 122.1 (d, JCF=7.7 Hz), 116.6 (d, JCF=19.3 Hz), 114.5 (d, JCF24.4 Hz), 107.5 (d, JCF=25.7 Hz), 33.2, 29.6; HRMS (ESI): calcd. for C20H18F2N5OS [M+H]+ 414.1200, found 414.1207.
N-(6-Fluorobenzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (YA6175). The title compound was synthesized according to General Procedure B (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.73 (s, 1H), 7.79-7.76 (m, 1H), 7.69-7.63 (m, 1H), 7.55-7.52 (m, 2H), 7.42-7.34 (m, 2H), 7.19 (td, J=8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ 159.8 (d, JCF=243.1 Hz), 156.3 (d, JCF=254.7 Hz), 156.3, 155.6, 145.0, 139.4, 133.6 (d, JCF=6.4 Hz), 133.3 (d, JCF=10.3 Hz), 128.0, 125.1 (d, JCF=2.6 Hz), 123.4 (d, JCF=14.2 Hz), 122.4 (d, JCF=9.0 Hz), 119.8 (q, J=270.8 Hz, CF3), 116.9 (d, JCF=18.0 Hz), 114.8 (d, JCF=23.2 Hz), 107.7 (d, JCF=25.7 Hz); HRMS (ESI): calcd. for C17H9F5N5OS [M+H]+ 426.0448, found 426.0438.
1-(2-Bromophenyl)-5-methyl-N-(5-phenylpyridin-2-yl)-1H-pyrazole-4-carboxamide (YA1124). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 8.56-8.52 (m, 2H), 8.41 (d, J=8.0 Hz, 1H), 8.06 (s, 1H), 7.95 (d, J=8.0 Hz, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.58 (d, J=8.0 Hz, 2H), 7.50-7.36 (m, 6H), 2.49 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.7, 150.7, 146.0, 145.0, 142.5, 138.7, 137.9, 137.4, 136.9, 133.6, 132.8, 131.4, 129.6, 129.5, 129.1, 128.5, 127.8, 126.8, 122.3, 114.9, 113.9, 11.4;
N-([1,1′-Biphenyl]-4-yl)-1-(2-bromophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA1122). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.90 (s, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.68 (d, J=8.0 Hz, 2H), 7.59-7.56 (m, 4H), 7.48-7.31 (m, 6H), 2.46 (m, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.8, 144.8, 140.5, 138.4, 137.9, 137.2, 137.1, 133.6, 131.4, 129.7, 128.8, 128.5, 127.6, 127.1, 126.8, 122.3, 120.6, 115.2, 11.3;
1-(2-Bromophenyl)-5-methyl-N-(5-phenylthiazol-2-yl)-1H-pyrazole-4-carboxamide (YA6152). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 12.42 (s, 1H), 8.24 (s, 1H), 7.77 (d, J=7.6 Hz, 1H), 7.55-7.40 (m, 6H), 7.37 (t, J=7.2, 7.6 Hz, 2H), 7.28 (t, J=7.2, 7.6 Hz, 1H), 2.53 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.5, 159.1, 145.7, 139.7, 137.8, 133.7, 132.6, 131.6, 131.5, 129.6, 129.0, 128.6, 127.8, 126.3, 122.3, 113.7, 11.4;
1-(2-Bromophenyl)-5-methyl-N-(4-phenylthiazol-2-yl)-1H-pyrazole-4-carboxamide (YA4087). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.65 (s, 1H), 8.07 (s, 1H), 8.02 (d, J=7.2 Hz, 2H), 7.93 (d, J=8.0 Hz, 1H), 7.67 (t, J=8.0 Hz, 1H), 7.59-7.57 (m, 3H), 7.54-7.47 (m, 2H), 7.40 (s, 1H), 2.67 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 160.9, 158.5, 149.9, 145.6, 138.6, 137.7, 134.2, 133.6, 131.4, 129.5, 128.7, 128.4, 128.0, 126.1, 122.2, 113.0, 107.9, 11.4; HRMS (ESI): calcd. for C2-H16BrN4OS [M+H]+ 439.0228, found 439.0217.
N-(Benzo[d]thiazol-2-yl)-1-(2-bromophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4033). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.84 (d, J=6.8 Hz, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.66 (d, J=7.6 Hz, 1H), 7.49-7.32 (m, 6H), 2.53 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.7, 159.7, 147.5, 146.0, 139.5, 137.7, 133.6, 131.8, 131.4, 129.6, 128.5, 126.3, 124.0, 122.1, 121.4, 120.7, 113.2, 11.60; HRMS (ESI): calcd. for C11H14BrN4OS [M+H]+ 413.0072, found 413.0074.
5-Methyl-N-(5-phenylpyridin-2-yl)-1-(o-tolyl)-1H-pyrazole-4-carboxamide (YA7018). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 7.98 (d, J=8.0 Hz, 1H), 7.67 (s, 1H), 7.58 (d, J=7.2 Hz, 1H), 7.50-7.29 (m, 5H), 7.20 (d, J=8.0 Hz, 1H), 2.40 (s, 3H), 2.02 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 167.2, 152.9, 147.7, 146.2, 141.0, 137.3, 136.9, 136.7, 135.9, 135.5, 131.2, 129.9, 129.2, 128.3, 127.5, 127.2, 126.8, 122.0, 115.8, 17.2, 11.5;
N-([1,1′-biphenyl]-4-yl)-5-methyl-1-(o-tolyl)-1H-pyrazole-4-carboxamide (YA7016). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.94 (s, 1H), 7.82 (d, J=6.8 Hz, 2H), 7.72 (d, J=7.2 Hz, 4H), 7.58-7.43 (m, 6H), 7.35 (d, J=8.0 Hz, 1H), 2.55 (s, 3H), 2.18 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 162.0, 144.0, 140.5, 137.9, 137.6, 137.3, 137.1, 136.0, 131.2, 129.9, 128.8, 127.6, 127.1, 126.9, 126.8, 120.5, 114.9, 17.2, 11.2;
5-Methyl-N-(5-phenylthiazol-2-yl)-1-(o-tolyl)-1H-pyrazole-4-carboxamide (YA7019). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 12.50 (s, 1H), 8.44 (s, 1H), 7.76-7.75 (m, 3H), 7.66-7.47 (m, 7H), 2.71 (s, 3H), 2.31 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.6, 159.1, 145.1, 139.2, 137.4, 135.9, 132.7, 132.5, 131.6, 131.3, 130.0, 129.1, 127.8, 127.5, 126.9, 126.2, 113.3, 17.3, 11.4;
5-Methyl-N-(4-phenylthiazol-2-yl)-1-(o-tolyl)-1H-pyrazole-4-carboxamide (YA7015). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 10.48 (s, 1H), 7.42 (s, 3H), 7.00-6.89 (m, 6H), 6.81 (s, 1H), 6.75 (d, J=7.6 Hz, 1H), 2.03 (s, 3H), 1.56 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.3, 158.9, 149.8, 144.8, 138.4, 137.3, 135.9, 134.2, 131.1, 129.9, 128.7, 127.9, 127.4, 126.7, 126.1, 112.7, 107.9, 17.1, 11.3;
N-(Benzo[d]thiazol-2-yl)-5-methyl-1-(o-tolyl)-1H-pyrazole-4-carboxamide (YA6179). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 11.58 (s, 1H), 8.03 (s, 1H), 7.83 (d, J=7.2 Hz, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.40-7.26 (m, 5H), 7.17 (d, J=7.2 Hz, 1H), 2.49 (s, 3H), 2.04 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.7, 159.6, 147.8, 145.2, 139.0, 137.3, 135.8, 131.9, 131.2, 130.0, 127.5, 126.8, 126.2, 123.9, 121.4, 120.6, 112.9, 17.2, 11.5;
N-(Benzo[d]thiazol-2-yl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxamide (YA4011). The title compound was synthesized according to General Procedure E (27%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.57-7.45 (m, 4H), 7.40-7.36 (m, 3H), 7.30 (t, J=7.6 Hz, 1H), 2.69 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 162.1, 160.0, 148.0, 144.5, 139.7, 138.8, 132.2, 129.7, 129.2, 126.6, 125.8, 124.3, 121.8, 120.8, 114.3, 12.6; HRMS (ESI): calcd. for C18H15N4OS [M+H]+ 335.0967, found 335.0978.
N-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-carboxamide (YA6149). The title compound was synthesized according to General Procedure E (31%, a yellow solid): 1H-NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 9.31 (s, 1H), 8.50 (s, 1H), 8.00 (d, J=7.6 Hz, 1H), 7.88 (d, J=8.0 Hz, 2H), 7.76 (d, J=7.6 Hz, 1H), 7.55 (t, J=7.2, 6.8 Hz, 2H), 7.46-7.37 (m, 2H), 7.31 (t, J=8.0, 6.8 Hz, 1H); 13C-NMR (100 MHz, DMSO-d6) δ 160.8, 158.8, 149.0, 141.7, 139.4, 132.0, 131.2, 130.2, 127.9, 126.6, 124.0, 122.2, 120.8, 119.5, 118.6;
N-(Benzo[d]thiazol-2-yl)-5-ethyl-1-phenyl-1H-pyrazole-4-carboxamide (YA6143). The title compound was synthesized according to General Procedure E (%, a white solid): 1H-NMR (400 MHz, CDCl3) δ 11.07 (s, 1H), 8.00 (s, 1H), 7.83 (d, J=7.6 Hz, 1H), 7.57-7.47 (m, 4H), 7.40-7.35 (m, 3H), 7.31-7.28 (s, 1H), 3.10 (q, J=8.0, 7.6 Hz, 2H), 1.25 (t, J=8.0, 6.8 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.2, 159.2, 150.3, 147.9, 139.0, 138.6, 132.0, 129.4, 129.2, 126.2, 125.9, 123.9, 121.4, 120.6, 112.9, 18.8, 13.7;
N-(Benzo[d]thiazol-2-yl)-5-isopropyl-1-phenyl-1H-pyrazole-4-carboxamide (YA6144). The title compound was synthesized according to General Procedure E (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 11.43 (s, 1H), 7.99 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.52-7.27 (m, 8H), 3.43-3.39 (m, 1H), 1.48 (s, 3H), 1.46 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.5, 159.6, 153.3, 147.9, 139.9, 139.3, 131.9, 129.4, 129.3, 126.6, 126.0, 123.8, 121.4, 120.4, 113.1, 26.6, 20.6;
N-(Benzo[d]thiazol-2-yl)-1-(2-ethylphenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4009). The title compound was synthesized according to General Procedure E (27%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 7.95 (s, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.53 (d, J=7.6 Hz, 1H), 7.41-7.22 (m, 5H), 7.08 (d, J=8.0 Hz, 1H), 2.42 (s, 3H), 2.28 (q, J=7.2 Hz, 2H), 1.01 (t, J=7.2 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.5, 159.2, 147.7, 145.5, 141.7, 138.7, 136.7, 131.9, 130.2, 129.6, 127.6, 126.7, 126.2, 123.9, 121.4, 120.6, 112.7, 23.9, 14.4, 11.6; HRMS (ESI): calcd. for C20H19N4OS [M+H]+ 363.1280, found 363.1285.
N-(Benzo[d]thiazol-2-yl)-1-(2-methoxyphenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA7020). The title compound was synthesized according to General Procedure E (27%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 11.59 (s, 1H), 8.05 (s, 1H), 7.82 (d, J=7.2 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.44 (t, J=7.6, 8.0 Hz, 1H), 7.35-7.25 (m, 3H), 7.07-7.02 (m, 2H), 3.77 (s, 3H), 2.51 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.9, 159.6, 154.3, 147.8, 146.3, 142.4, 139.3, 131.9, 131.1, 128.8, 127.1, 126.1, 123.8, 121.3, 120.9, 120.6, 112.9, 112.0, 55.8, 11.5;
N-(Benzo[d]thiazol-2-yl)-1-(2-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4007). The title compound was synthesized according to General Procedure E (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.84 (d, J=7.6 Hz, 1H), 7.61 (d, J=7.6 Hz, 1H), 7.51-7.39 (m, 3H), 7.34-7.29 (m, 3H), 2.60 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.5, 159.5, 157.9 (d, J=250.9 Hz), 147.5, 146.2, 139.8, 131.8, 131.4 (d, J=6.4 Hz), 128.9, 126.2, 125.0 (d, J=3.8 Hz), 124.0, 121.4, 120.5, 116.9 (d, J=19.3 Hz), 113.6, 11.4; HRMS (ESI): calcd. for C18H14FN4OS [M+H]+ 353.0872, found 353.0879.
N-(Benzo[d]thiazol-2-yl)-1-(2-chlorophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4025). The title compound was synthesized according to General Procedure E (27%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.84 (d, J=7.6 Hz, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.51-7.39 (m, 4H), 7.33 (t, J=7.6, 6.4 Hz, 1H), 2.53 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.7, 159.7, 147.6, 146.2, 139.7, 136.1, 132.2, 131.8, 131.2, 130.5, 129.5, 127.8, 126.2, 124.0, 121.4, 120.6, 113.3, 11.5; HRMS (ESI): calcd. for C18H14ClN4OS [M+H]+ 369.0577, found 369.0579.
N-(Benzo[d]thiazol-2-yl)-1-(3-chloro-2-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4021). The title compound was synthesized according to General Procedure E (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.59-7.57 (m, 2H), 7.42-7.28 (m, 4H), 2.61 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.6, 159.8, 154.1 (d, J=253.4 Hz), 147.4, 146.3, 140.3, 131.9, 131.8, 127.6 (d, J=11.6 Hz), 127.3, 126.2, 124.9 (d, J=5.2 Hz), 124.0, 122.7 (d, J=16.7 Hz), 121.4, 120.4, 113.9, 11.4; HRMS (ESI): calcd. for C18H13ClFN4OS [M+H]+ 387.0483, found 387.0472.
N-(benzo[d]thiazol-2-yl)-1-(2,6-difluorophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4019). The title compound was synthesized according to General Procedure E (31%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.84 (d, J=7.6 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.51-7.50 (m, 1H), 7.42-7.40 (m, 1H), 7.34-7.32 (m, 1H), 7.15-7.13 (m, 2H), 2.59 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.4, 159.6 (d, J=256 Hz), 157.1 (d, J=253.4 Hz), 147.5 (d, J=19.3 Hz), 140.4, 131.7 (t, J=9.0, 10.3 Hz), 126.3, 124.0, 121.3, 120.6, 113.6, 112.5 (d, J=21.9 Hz), 11.0; HRMS (ESI): calcd. for C18H13F2N4OS [M+H]+ 371.0778, found 371.0782.
N-(Benzo[d]thiazol-2-yl)-1-(2-bromo-6-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4017). The title compound was synthesized according to General Procedure E (27%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.04 (d, J=7.6 Hz, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.65-7.58 (m, 2H), 7.54-7.45 (m, 2H), 2.75 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 161.6, 160.1 (d, J=256 Hz), 159.9, 147.5, 146.9, 140.5, 132.4 (d, J=9.0 Hz), 131.8, 129.0 (d, J=2.6 Hz), 126.5, 126.3, 124.0 (d, J=15.5 Hz), 121.3, 120.7, 116.0 (d, J=19.3 Hz), 113.5, 11.1; HRMS (ESI): calcd. for C11H13BrFN4OS [M+H]+ 430.9977, found 430.9981.
N-(Benzo[d]thiazol-2-yl)-1-(2,6-dimethylphenyl)-5-methyl-1H-pyrazole-4-carboxamide (YA4023). The title compound was synthesized according to General Procedure E (27%, a yellow solid): 1H-NMR (400 MHz, CDCl3) δ 12.00 (s, 1H), 8.06 (s, 1H), 7.83 (d, J=7.6 Hz, 1H), 7.51 (d, J=6.8 Hz, 1H), 7.28-7.27 (m, 3H), 7.16 (d, J=7.2 Hz, 2H), 2.45 (s, 3H), 1.96 (s, 6H); 13C-NMR (100 MHz, CDCl3) δ 161.9, 159.9, 147.7, 145.3, 139.6, 136.5, 136.1, 131.9, 129.8, 128.4, 126.1, 123.9, 121.4, 120.6, 112.7, 17.2, 11.0;
This Example describes the synthesis and properties of a novel class of compounds, of which, triazole YA6060 showed dual activities of β-catenin downregulation and AMPK activation via Axin stabilization. YA6060 demonstrated a highly promising drug-like profile and a remarkable efficacy preventing dimethylnitrosamine-induced hepatic fibrosis. This Example also describes the synthesis and several relevant studies including toxicity assessment and anti-NASH efficacy evaluation. Animal models of hepatic metabolic disorders are used to collect critical efficacy data in support of YA6060 development.
YA6060 (
Small molecules that induce the degradation of β-catenin and the activation of AMPK was previously identified, and some of these compounds showed efficacy in a mouse model of hepatic steatosis. Described herein are an improved second generation of compounds, including YA6060 and YA6045
YA6060 is an Axin stabilizer that exhibits the dual activities (
In some embodiments, YA6060 is prescribed by physician to NASH patients. In some embodiments, YA6060 is used by patients of other diseases such as metabolic syndromes and certain cancer.
In preliminary mice studies, YA6060 exhibited no obvious toxicity at a single dose of 500 mg/kg resulting in a >1,000-fold safety margin over an efficacy dose (0.5 mg/kg) for liver fibrosis. If the toxicity evaluation in mice indicated a much narrower therapeutic window, additional compounds of the disclosure can be evaluated and/or lower doses of YA6060 can be combined with other potential therapies. In some embodiments, toxicity and off-target side effects can be addressed with hybrid compounds of which cellular uptake is mediated by the liver-specific OCT1 transporter proteins. In a non-limiting example, extrahepatic side effects of YA6060 can be addressed by synthesizing OCT1-mediated YA6060 analogs or evaluate other related compounds.
Steatosis is the most outstanding feature of NAFLD/NASH, while fibrosis is the hallmark of its progression. Compared to earlier Wnt signaling inhibitors, YA6060 demonstrated a remarkable efficacy for liver fibrosis. In some embodiments, YA6060 is combined with other potential therapies in order to improve fibrosis by at least one stage and/or statistically reduce liver triglyceride content in mouse models. In a non-limiting example, mechanistically YA6060 and metformin could synergize to activate AMPK.
Comprehensive ADME profiling and regulatory toxicity studies. A scale up of the YA6060 synthesis is prepared. Second, although preliminary data indicates an excellent safety profile for YA6060 (
Synthesis: 200 mg of YA6060 has been synthesized and purified. YA6060 is scaled up to the 5 g level for use in further studies. Compound YA6060 (and all synthetic intermediates) are characterized using 1H-NMR, 13C-NMR, and high-resolution Mass Spectrometry. Purity (by HPLC) exceeds 95%.
Toxicity evaluation in mice: YA6060 is evaluated for tolerance and general toxicity in mice. First, the single-dose MTD in mice is determined using dose escalation studies. 5 mouse/dose administered YA6060 via oral gavage are assessed for lethal toxicity within 7-21 days. Half log dose intervals re employed. The tolerable daily X5d dosing is determined, starting with 20% of the single-dose MTD as the daily dose in a 5d regimen. The daily dose is increased or decreased, sequentially as necessary, to find the 5d repeat-dose MTD. Animals are evaluated by physical exams, microscopic examination of major organs, serum chemistries, and complete blood counts. MTD>500 mg/kg and in combination with efficacy data, to achieve a TI>1,000.
Anti-NASH efficacy in ob/ob AMLN mice: The genetically obese ob/ob mice treated with AMLN diet of which lipid composition closely reflects a prototypic fast-food diet (i.e., ob/ob AMLN mice) has been widely adopted for testing the anti-NASH efficacy of various compound classes To investigate the anti-NASH efficacy of YA6060 in ob/ob AMLN mice, 8-week old ob/ob mice are fed with AMLN diet (Research Diets, Inc.; D09100310) for 8 weeks and divided into 4 groups (n=15/group). The AMLN mice then receive saline, 0.1, 0.5, or 5 mg/kg YA6060 by oral gavage daily for 4 weeks before euthanasia. Mouse body weight, food intake, and activities are examined for general toxicity response twice per week. The methods for outcome measurement have been established previously. Briefly, liver and plasma triglyceride content is determined. To assess liver injury, fresh-frozen liver sections are prepared to detect reactive oxygen species (ROS) levels and cell death by the fluorescent probe DHE and TUNEL assay, respectively. Liver damage is assessed by serum ALT and AST enzyme levels. H&E staining is used to examine general liver histology, Sirius red staining for fibrosis, and Oil Red O for lipids. The biomarkers (fibrosis and inflammation) are examined by immunoblotting and RT-PCR. To assess general metabolic status, glucose tolerance test and measurement of fasting glucose and insulin levels is performed in subgroups of mice. The primary outcome endpoints are hepatic triglyceride content and fibrosis stage. Overall, YA6060 is expected to decrease hepatic steatosis and fibrosis, enhance hepatic insulin sensitivity, and improve metabolic homeostasis. Improvement in fibrosis by at least 1 Ishak stage with significantly reduced hepatic triglyceride content is expected.
Anti-NASH efficacy in mice administered Western diet with weekly tetrachloride: None of the present models fulfill all requirements for an ideal NASH model. Multiple NASH models should be used for the preclinical characterization of candidate anti-NASH drugs. The mouse model administered a Western diet (WD) with weekly CCl4 has been reported to closely resemble human NASH. This model is also employed to test YA6060. As described, male C57BL/6J mice are fed WD and a high sugar solution, with weekly I.P. injection of CCl4, for 12 weeks. The mice are then divided into 4 groups (n=15/group) and receive saline, 0.1, 0.5, or 5 mg/kg YA6060, respectively, by oral gavage daily for 4 weeks before euthanasia. The outcome measurement and data analysis is essentially the same as described above. Improvement in fibrosis by at least 1 stage with significantly reduced hepatic triglyceride content is expected.
YA6060 was found to have dual activities of Wnt signaling inhibition and AMPK activation (
The anti-fibrotic efficacy of YA6060 was demonstrated in LX-2 cells (
Fibrosis in the liver tissues of the mice that received YA6060 treatment was quantified (
The effect of DMNA and YA6060 treatment on hepatic histology in mice was examined (
The number of mice in Ishak fibrosis stage was examined (
The effect of YA6060 treatment on the hepatic expression of fibrotic and inflammatory markers in mice was examined (
The effect of DMNA and YA6060 treatment on body weight change (
The effect of DMNA and YA6060 treatment on liver function in mice was examined (
This Example describes a novel class of compounds, of which, triazole YW2065 showed dual activities of Wnt signaling inhibition and AMPK activation via the mechanism of Axin stabilization. Compound YW2065 selectively kills Wnt-dependent CRC cells, and indicated promising efficacy against CRC cell growth both in vitro and in mice. Based on the chemical structure of YW2065, a new analogue YA6060 was designed and synthesized. While maintaining dual activity of Wnt inhibition and AMPK activation, the new analogue YA6060 demonstrated a highly promising druglike profile highlighted by oral bioavailability (F=75%), half-life (t1/2=160 min), maximum toxic dose (MTD>500 mg/kg) and aqueous solubility (>75 mg/mL). Moreover, using biotinylated analogs and proteomic approaches, it was discovered that the tankyrase-binding protein 1 (TAB182) was the protein target of the new analogues.
Wnt signaling and AMPK pathways are validated targets for anticancer therapies. YW2065 was reported to have dual activities of Wnt inhibition and AMPK activation (
YA6060 was developed based on previous compound YW2065. Structure modification of known Wnt inhibitor Pyr provided YW2065, and further modification of YW2065 provided new compound YA6060 (
YA6060 has an excellent druglike profile. Compared to Pyr and YW2065, YA6060 showed dramatically enhanced druglike properties without any clinical toxicity. The ADMET and PK properties of YA6060 was determined. Compared to Pyr and YW2065 that have poor solubility (<0.01 mg/mL), new lead YA6060 show remarkable solubility of >75 mg/mL in water (
YW2065 and YA6060 selectively kill CRC cells. Inhibition of Wnt signaling by YW2065 was confirmed in human CRC SW480 (IC50=1.2 nM) and SW620 (IC50=0.19 nM) cells using luciferase gene reporter assays (
YW2065 functions through Axin stabilization. YW2065 inhibited the colony formation for SW480, SW620, and HT29 cells (
YW2065 suppresses CRC in vivo. The anti-CRC potential of YW2065 was tested using nude mice that received implantation of SW620 cells. YW2065 reduced both tumor growth (
The effects of FX analogs in Axin stabilization and AMPK activation were further studied by genetic manipulation of Axin. Axin overexpression led to enhanced AMPK activation (
Effects of YW2065 on mitochondrial function. AMPK can be activated by falling energy status that is a function of mitochondria. Mitochondrial can be suppressed by Axin over-expression. TNKS inhibitors XAV939 and Pyr have been reported to inhibit mitochondria. Unbiased analyses were performed for the proteomic changes by YW1128 (YW2065 analog) and XAV939 in HEK293 cells (
TAB182 as a protein target for YA6060 analogs. Previously, CK1α was reported as a direct target for Pyr; however, this conclusion was challenged. CK1α was not confirmed as the target for YA6060 analogs by either SPR binding assay, or functional validation via CK1α knockdown in the reporter assay, or pulldown experiments. Two biotinylated YA6060 analogs YA2103 and YA6023 containing different length of spacers were synthesized, while both analogs maintained inhibitory activities in the reporter assay (
Assessing the anti-CRC efficacy of selected compounds. CRC is of a major interest for targeting Wnt pathway. In part, the goal of this study is to evaluate the efficacy and safety of selected compounds with dual activities of Wnt inhibition and AMPK activation against CRC.
Effects of selected compounds on CRC cell growth and proliferation. YA6060 is further tested in CRC cell models. To represent the heterogeneity of oncogenic Wnt signaling in human CRC, six widely studied CRC cell lines with different genetic status of CTNNB1 (encoding β-catenin) and APC genes are chosen (
The assays for outcome measurement have been established (
Effects of YW2065 analogs on CRC initiation and growth in vivo. The upregulation of Wnt signaling pathway is the primary transforming event in CRC. Thus, Axin stabilization provides a sound chemo-preventive strategy for patients with high risk of colon neoplasia. The chemo-preventative potential of YA6060 is tested. Apcmin/+ mice are widely used model of gastrointestinal (GI) neoplasia, due to an upregulated Wnt signaling by truncated Apc proteins. Of note, Pyr and Axin stabilizers have been reported to inhibit intestinal polyp formation in Apcmin/+ mice. As outlined in
After 11 weeks, mice are euthanized and tumor numbers and volumes are determined by investigators blinded to treatment. Cell proliferation is determined by immunohistochemistry (IHC) with anti-Ki67 antibody; cell apoptosis in tumor tissues by IHC for cleaved caspase-3; Axin, and β-catenin, p-AMPK/AMPK, and p-ACC/ACC by immunoblotting and/or IHC; and the expression of Wnt target genes by RT-PCR. Blood biochemistry is analyzed to determine liver and kidney functions. General histology of major tissues is examined to evaluate toxic effects. The primary statistical endpoints are tumor numbers and volumes. Data is analyzed by ANOVA followed by a post-hoc test (see Statistical section). Compared to vehicle treatment, an active compound is expected to significantly attenuate both adenoma number and volume in Apcmin/+ mice without notable toxicity and that cell proliferation will be reduced in adenoma, whereas apoptosis will be increased. Moreover, it is expected that the active compounds stabilize Axin protein and inhibit Wnt signaling as measured by protein and gene expression changes in the pathway in adenoma tissues.
Effects of YA6060 on CRC invasion and metastasis in vivo. The above Apcmin/+ mouse model has limitations such as the location of polyp formation, which is limited in the small intestine, and no progression from these adenomas to metastasis. In this example, YA6060 is tested to see if the compound attenuates colon cancer progression, specifically tumor growth, invasion, and metastasis in the established murine model of CRC invasion and metastasis (
Local tumor growth and distant metastasis (i.e., liver, lungs and lymph nodes) are measured quantitatively each week in real-time by BLI (
It is expected that YA6060 shows specific efficacy against CRC cells, demonstrating its efficacy in suppression of intestinal carcinogenesis, and for the first time, a small molecule that can be an effective therapy for advanced CRC. CRC are highly heterogeneous in genetics. In a non-limiting example, additional CRC cell lines are chosen to explore a broad clinical implication for compounds. In a non-limiting example, compounds of the disclosure are used together with current CRC therapeutics to achieve synergistic effects. Although YW2065 killed CRC cells at an IC50 comparable to those of first-line CRC chemotherapy (5-FU and oxaliplatin), the in vivo anti-cancer efficacy in mice was modest (
Investigating the mechanism of action of new compound. The goal of this aim is to gain molecular insights into the pharmacological action of compounds of the disclosure with dual activities of Wnt inhibition and AMPK activation. It is hypothesized that Wnt inhibition and AMPK activation via Axin stabilization is the effective mechanism for YA6060 to treat CRC (
Role of Wnt inhibition and AMPK activation in conferring the effects of YA6060 on CRC cell proliferation and growth. It is determined if Axin stabilizers depend on Wnt inhibition, AMPK activation, or both to inhibit CRC cell proliferation and growth. The Wnt signaling activity is determined by reporter assay and by analyzing the effector protein and target gene expression with immunoblotting and RT-PCR. The protein levels of p-AMPK, total AMPK, the downstream p-ACC, total ACC are determined by immunoblotting. Wnt activity is modulated via Axin knockdown with len-shAxin (
Mechanism by which YA6060 activates AMPK in CRC cells—Axin stabilization-mediated Wnt inhibition is well known. Three hypotheses are tested to address the mechanism underlying Axin stabilization-mediated AMPK activation by YA6060 (
Confirmation of protein targets for YA6060-TAB182 and other positive hits that were identified in the preliminary target search are further examined (
It is expected that this example demonstrates the efficacy of YA6060 against CRC is, at least partially, via AMPK activation that is due to mitochondrial inhibition involved with Wnt signaling and enhanced formation of Axin-AMPK-LKB1 complex. The identified direct target of YA6060 advances the understanding of signal transduction via Axin, providing novel strategy and targets to develop CRC therapy. In a non-limiting example, if the efficacy of YA6060 was independent of AMPK activation, the preliminary data and the direct target identified through these studies can be used to formulate a new and testable hypothesis, such as mitochondrial dysfunction-induced apoptosis, or TAB182-mediated cancer development and drug sensitivity. In a non-limiting example, a traditional label-based method is used instead of SPR, which widely used in characterizing interaction of small molecules with their protein targets. In a non-limiting example, additional conditions for biotinylated chemical affinity chromatography can be explored, followed by MS protein identification. In a non-limiting example, thermal proteome profiling (TPP) is used, which combines the measurement of protein thermal stability with quantitative proteomics and has recently been used to identify drug targets.
For animal studies, the sample size is expected to provide sufficient power for statistical analyses (at the P<0.05 level; α=0.05, β=0.1), which is estimated by Russ Lenth's Power and Sample Size Calculation, with the data of tumor number, growth and size measurement from previous publications. Statistical analyses are conducted with GraphPad software. T tests and analysis of variance (ANOVA) followed by a post-hoc test will be employed in this application. A P value less than 0.05 is considered as statistically different. All chromosomal or plasmid borne assays are checked by PCR following the assay. RT-PCR is performed in triplicate on a minimum of 3 biological replicates. Purified proteins are analyzed by SDS-PAGE and checked by MALDI-TOF mass spectrometry to ensure homogeneity or removal of affinity tags. All Western blots are run on 3 separate experiments with purified protein as a marker and normalized to total protein or to RNA polymerase alpha subunit as a loading control. For proteomic studies all experiments are performed on a minimum of 5 biological replicates and 3 technical replicates per sample. Verification of instrumentation sensitivity is determined by running internal standards as a control to assess day to day variation of instrumentation. Peptide assignments are set at 1% false detection rate (FDR) and relative abundance will be considered significant >1.5 fold and p<0.05. All SMPs and inhibitors are verified by NMR and high accuracy mass spectrometry prior to use.
The limitation of compound YA6060 includes its high hygroscopicity and potential metabolic instability. It is hypothesized that simultaneous inhibition of Wnt signaling by targeting the tankyrase-binding protein TAB182 and activation of the AMPK pathway, is a novel therapeutic strategy for the treatment of CRC. This Example describes the synthesis of non-hygroscopic and metabolically stable YA6060 analogs or CRC-targeting YA6060 hybrids and testing them using established assays, to validate and characterize potent compound with dual activity of Wnt inhibition and AMPK activation. In a non-limiting example, the compounds are Wnt inhibitors with significantly enhanced aqueous solubility (>10,000 fold) and bioavailability (>3 fold) while maintaining the inhibitory potency against the Wnt signaling pathway via Axin stabilization. First, new compounds that inhibit Wnt signaling and activate AMPK are synthesized and evaluated: two classes of compounds based on YA6060 are synthesized including i) acceptably non-hygroscopic and metabolically stable YA6060 analogs and ii) folate receptor (FR)-targeted folic acid (FA)-YA6060 hybrids. Wnt inhibition of the new compounds is assayed by the TOPflash reporter assays, confirming the mechanism of action by immunoblots and RT-PCR, and FR-mediated uptake is assessed using FR-positive cells. Solubility, cytotoxicity, permeability and metabolic stability of selected compounds is determined in vitro by testing the pharmacokinetic (PK) properties and in vivo toxicity (general tolerance and organ-specific toxicity) of selected compounds using C57/BL6 mice.
The anti-CRC efficacy of selected compounds is assessed by assessing the activity of selected compounds in killing CRC cells with different genetic mutations of Wnt signaling effectors, and their effects on colony formation, cell cycle, and apoptosis of CRC cells are examined. The inhibitory activity of selected compounds for CRC initiation/growth using Apcmin/+ mice is studied. The effects of selected compounds in blocking CRC invasion and metastasis are evaluated using an advanced HT-29-luc-D6 tumor Cecal implantation mouse model for CRC invasion and metastasis.
The mechanism of action of new leads is investigated by examining the role of Wnt signaling and AMPK pathways in conferring the effects of selected compounds on CRC cell proliferation and growth using immunoblotting, immunohistochemistry (IHC) and RT-PCR. The mechanism by which selected compounds activate the AMPK pathway in CRC cells is studied, and the direct protein targets of the optimal compounds are identified and confirmed using a combination of pull-down experiments, genetic manipulation, and thermo protein profiling (TPP) strategies.
This Example provides evidence to access the therapeutic potential of simultaneously targeting Wnt signaling and AMPK pathways as novel anti-CRC therapeutic strategy. Although the YA6060 indicated an excellent activity and safety profile, it is highly hygroscopic and readily absorbs moisture from the environment, which complicated its handling process and shortened the compound's shelf life. In addition, the chemical structure of YA6060 contains multiple C—H bonds that are liable to P450-mediated oxidations in the liver. Therefore, novel YA6060 analogs with acceptable non-hygroscopicity along with excellent metabolic stability are highly desirable for its further development.
Using YA6060 as a template, new compounds are synthesized that can simultaneously inhibit Wnt signaling pathway and activate AMPK pathway, with CRC selectivity, desirable PK/Tox properties and physiochemical profiles (
New compounds that inhibit Wnt signaling and activate AMPK are synthesized and evaluated. In order to discover the metabolically stable, acceptably non-hygroscopic, and CRC-selective compounds with dual activity of Wnt signaling inhibition and AMPK activation, medicinal chemistry efforts are devoted to synthesizing new compounds of three classes: fluoro-containing P450-inert YA6060 analogs, non-hygroscopic salts of YA6060, and FR-targeting FA-YA6060 hybrids. The dual activity of synthesized analogs/hybrids for Wnt signaling and AMPK pathways is confirmed. The ADME, bioavailability, Tox and solubility of selected compounds are determined. These efforts yield three selected compounds for further evaluation.
New compound design and synthesis—Based on preliminary data of YA6060, five fluorine-containing YA6060 analogs are synthesized to block the potential P450-based oxidative metabolism. Different salts of the compound employing five different counter ions are generated. Five FA-YA6060 hybrids to target the FR-overexpressing CRC cells are synthesized.
Non-hygroscopic salts of YA6060. YA6060 is an HCl salt with high hygroscopicity. YA6060 can readily absorb moisture from the environment that limits the stability and the further therapeutic application of the compound. Non-hygroscopic salts of YA6060 are generated by screening the anionic of the neutral amino parent YA6060 with a panel of acids that has been reported to form acceptably non-hygroscopic salts. Specific acids include valproic (1), maleic (2), tartaric (3), oxalic (4) and pamoic (5) acids, to get the corresponding salts.
Fluorine-containing YA6060 analogs. Five YA6060 analogs (6-10) are synthesized by substituting the P450-labile hydrogen atoms with metabolically inert F-atoms (
CRC cell-targeting FA-YA6060 hybrids. Preliminary SAR results in the have shown that extension of the piperazine group of YA6060 is well-tolerated in maintaining the dual activity of compounds (
In vitro evaluation of drug properties for new compounds. A panel of assays to assess YA6060 analogs/hybrids was established (
Determination of physiochemical properties. The profiles of six selected analogs is estimated in silico, and selected Axin stabilizers are evaluated in in vitro assays: the aqueous solubility is determined using a multiscreen solubility filter plate (Millipore, Billerica, MA) coupled with LC-MS/MS, as performed for the already synthesized YA6060 analogs; the cell viability assays on selected analogs is measured using HEK293 cells; the intestinal permeability by determining the transport from the apical to the basolateral (A to B) in Caco-2 monolayers; the metabolic stability by incubating the compounds with mouse microsomes and liver tissue S9 fraction and analyzing by LC-MS/MS. Compounds with acceptable values according to drug property criteria (
PK/Tox. The four selected compounds from in vitro evaluation above are subjected to PK and toxicity assessment in C57/BL6 mice. As for lead YA6060 PK (
The characterization of YA6060 analogs/hybrids with dual mechanism of Wnt inhibition and AMPK activation provide a platform for the development of more druglike and CRC-selective compounds. The robust suite of tests including the Western blots, RT-PCR, and FR uptake assays will further validate compounds with IC50 values (<10 nM) (
Assessing the anti-CRC efficacy of selected compounds. CRC is of a major interest for targeting Wnt pathway. In part, the goal of this study is to evaluate the efficacy and safety of selected compounds with dual activities of Wnt inhibition and AMPK activation against CRC.
Effects of selected compounds on CRC cell growth and proliferation. YA6060 and selected analogues are further tested in CRC cell models. To represent the heterogeneity of oncogenic Wnt signaling in human CRC, six widely studied CRC cell lines with different genetic status of CTNNB1 (encoding β-catenin) and APC genes are chosen (
The assays for outcome measurement have been established (
Effects of FX01 analogs on CRC initiation and growth in vivo. The upregulation of Wnt signaling pathway is the primary transforming event in CRC. Thus, Axin stabilization provides a sound chemo-preventive strategy for patients with high risk of colon neoplasia. The chemo-preventative potential of YA6060 and selected analogues is tested. Apcmin/+ mice are widely used model of gastrointestinal (GI) neoplasia, due to an upregulated Wnt signaling by truncated Apc proteins. Of note, Pyr and Axin stabilizers have been reported to inhibit intestinal polyp formation in Apcmin/+ mice. As outlined in
After 11 weeks, mice are euthanized and tumor numbers and volumes are determined by investigators blinded to treatment. Cell proliferation is determined by immunohistochemistry (IHC) with anti-Ki67 antibody; cell apoptosis in tumor tissues by IHC for cleaved caspase-3; Axin, and β-catenin, p-AMPK/AMPK, and p-ACC/ACC by immunoblotting and/or IHC; and the expression of Wnt target genes by RT-PCR. Blood biochemistry is analyzed to determine liver and kidney functions. General histology of major tissues is examined to evaluate toxic effects. The primary statistical endpoints are tumor numbers and volumes. Data is analyzed by ANOVA followed by a post-hoc test (see Statistical section). Compared to vehicle treatment, an active compound is expected to significantly attenuate both adenoma number and volume in Apcmin/+ mice without notable toxicity and that cell proliferation will be reduced in adenoma, whereas apoptosis will be increased. Moreover, it is expected that the active compounds stabilize Axin protein and inhibit Wnt signaling as measured by protein and gene expression changes in the pathway in adenoma tissues.
Effects of YA6060 analogues on CRC invasion and metastasis in vivo. The above Apcmin/+ mouse model has limitations such as the location of polyp formation, which is limited in the small intestine, and no progression from these adenomas to metastasis. In this example, YA6060 and selected analogues are tested to see if the compound attenuates colon cancer progression, specifically tumor growth, invasion, and metastasis in the established murine model of CRC invasion and metastasis (
Local tumor growth and distant metastasis (i.e., liver, lungs and lymph nodes) are measured quantitatively each week in real-time by BLI (
It is expected that YA6060 analogues/hybrids show specific efficacy against CRC cells, demonstrating its efficacy in suppression of intestinal carcinogenesis, and for the first time, a small molecule that can be an effective therapy for advanced CRC. CRC are highly heterogeneous in genetics. In a non-limiting example, additional CRC cell lines are chosen to explore a broad clinical implication for compounds. In a non-limiting example, compounds of the disclosure are used together with current CRC therapeutics to achieve synergistic effects. Although FX01 killed CRC cells at an IC50 comparable to those of first-line CRC chemotherapy (5-FU and oxaliplatin), the in vivo anti-cancer efficacy in mice was modest (
Investigating the mechanism of action of new compound. The goal of this aim is to gain molecular insights into the pharmacological action of compounds of the disclosure with dual activities of Wnt inhibition and AMPK activation. It is hypothesized that Wnt inhibition and AMPK activation via Axin stabilization is the effective mechanism for YA6060 analogues to treat CRC (
Role of Wnt inhibition and AMPK activation in conferring the effects of YA6060 analogues/hybrids on CRC cell proliferation and growth. It is determined if Axin stabilizers depend on Wnt inhibition, AMPK activation, or both to inhibit CRC cell proliferation and growth. The Wnt signaling activity is determined by reporter assay and by analyzing the effector protein and target gene expression with immunoblotting and RT-PCR. The protein levels of p-AMPK, total AMPK, the downstream p-ACC, total ACC are determined by immunoblotting. Wnt activity is modulated via Axin knockdown with len-shAxin (
Mechanism by which YA6060 analogues/hybrids activate AMPK in CRC cells—Axin stabilization-mediated Wnt inhibition is well known. Three hypotheses are tested to address the mechanism underlying Axin stabilization-mediated AMPK activation by YA6060 (
Confirmation of protein targets for YA6060 analogues-TAB182 and other positive hits that were identified in the preliminary target search are further examined (
It is expected that this example demonstrates the efficacy of YA6060 analogues against CRC is, at least partially, via AMPK activation that is due to mitochondrial inhibition involved with Wnt signaling and enhanced formation of Axin-AMPK-LKB1 complex. The identified direct target of YA6060 analogues advances the understanding of signal transduction via Axin, providing novel strategy and targets to develop CRC therapy. In a non-limiting example, if the efficacy of YA6060 analogs was independent of AMPK activation, the preliminary data and the direct target identified through these studies can be used to formulate a new and testable hypothesis, such as mitochondrial dysfunction-induced apoptosis, or TAB182-mediated cancer development and drug sensitivity. In a non-limiting example, a traditional label-based method is used instead of SPR, which widely used in characterizing interaction of small molecules with their protein targets. In a non-limiting example, additional conditions for biotinylated chemical affinity chromatography can be explored, followed by MS protein identification. In a non-limiting example, thermal proteome profiling (TPP) is used, which combines the measurement of protein thermal stability with quantitative proteomics and has recently been used to identify drug targets.
For animal studies related to the YA6060 analogues/hybrids, the animal numbers are proposed based on preliminary studies and prior experience in conducting mouse PK and toxicity studies. For animal studies, the sample size is expected to provide sufficient power for statistical analyses (at the P<0.05 level; α=0.05, β=0.1), which is estimated by Russ Lenth's Power and Sample Size Calculation, with the data of tumor number, growth and size measurement from previous publications. Statistical analyses are conducted with GraphPad software. T tests and analysis of variance (ANOVA) followed by a post-hoc test will be employed in this application. A P value less than 0.05 is considered as statistically different. All chromosomal or plasmid borne assays are checked by PCR following the assay. RT-PCR is performed in triplicate on a minimum of 3 biological replicates. Purified proteins are analyzed by SDS-PAGE and checked by MALDI-TOF mass spectrometry to ensure homogeneity or removal of affinity tags. All Western blots are run on 3 separate experiments with purified protein as a marker and normalized to total protein or to RNA polymerase alpha subunit as a loading control. For proteomic studies all experiments are performed on a minimum of 5 biological replicates and 3 technical replicates per sample. Verification of instrumentation sensitivity is determined by running internal standards as a control to assess day to day variation of instrumentation. Peptide assignments are set at 1% false detection rate (FDR) and relative abundance will be considered significant >1.5 fold and p<0.05. All SMPs and inhibitors are verified by NMR and high accuracy mass spectrometry prior to use.
This Example demonstrates that small molecules with dual activity of Wnt inhibition and AMPK activation via the Axin stabilization are a valid strategy for the development of novel anti-cancer agents. The knowledge and novel small molecules greatly benefit research in understanding the pathophysiology of not only Wnt/β-catenin signaling but also AMPK pathways.
A number of patent and non-patent publications are cited herein in order to describe the state of the art to which this disclosure pertains. The entire disclosure of each of these publications is incorporated by reference herein.
While certain embodiments of the present disclosure have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present disclosure is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope and spirit of the appended claims.
Moreover, as used herein, the term “about” means that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.
Furthermore, the transitional terms “comprising”, “consisting essentially of” and “consisting of”, when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. All compounds, compositions, formulations, and methods described herein that embody the present disclosure can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”
This Application claims the benefit of priority to U.S. Provisional Application No. 63/195,668, filed Jun. 1, 2021, the entirety of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/031798 | 6/1/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63195668 | Jun 2021 | US |
