Discovery of indane propanamides as potent and selective TRPV1 antagonists

A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure–activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.     

Discovery of 2-(3,5-difluoro-4-methylsulfonaminophenyl)propanamides as potent TRPV1 antagonists

A series of A-region analogues of 2-(3-fluoro-4-methylsufonamidophenyl) propanamide 1 were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that a fluoro group at the 3- (or/and) 5-position and a methylsulfonamido group at the 4-position were optimal for antagonism of TRPV1 activation by capsaicin. The most potent antagonist 6 not only exhibited potent antagonism of activation of hTRPV1 by capsaicin, low pH and elevated temperature but also displayed highly potent antagonism of activation of rTRPV1 by capsaicin. Further studies demonstrated that antagonist 6 blocked the hypothermic effect of capsaicin in vivo, consistent with its in vitro mechanism, and it showed promising analgesic activity in the formalin animal model.     

2-Aryl substituted pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as highly potent TRPV1 antagonists

A series of 2-aryl pyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Multiple compounds showed highly potent TRPV1 antagonism toward capsaicin comparable to previous lead 7. Among them, compound 9 demonstrated anti-allodynia in a mouse neuropathic pain model and blocked capsaicin-induced hypothermia in a dose-dependent manner. Docking analysis of 9 with our hTRPV1 homology model provided insight into its specific binding mode.     

Pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists

A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The structure activity relationship indicated that the 3-chlorophenyl group at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic potency and the activity was stereospecific to the S-configuration, providing exceptionally potent antagonists 13S and 16S with K_i(CAP) = 0.1 nM. Particularly significant, 13S exhibited antagonism selective for capsaicin and NADA and not for low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that the binding modes differed somewhat, with that of 13S more closely resembling that of GRT12360.     

Design,synthesis and biological evaluation of N1-(isoquinolin-5-yl)-N2-phenylpyrrolidine-1,2-dicarboxamide derivatives as potent TRPV1 antagonists

Reported herein is the design, synthesis, and pharmacologic evaluation of a class of TRPV1 antagonists constructed on a N¹-(isoquinolin-5-yl)-N²-phenylpyrrolidine-1,2-dicarboxamide platform that evolved from a 5-aminoisoquinoline urea lead. Advancing the SAR of this series led to the eventual identification of 3b, comprising a p-Br substituted phenyl. In a TRPV1 functional assay, using cells expressing recombinant human TRPV1 channels, 3b displayed potent antagonism activated by capsaicin (IC₅₀ = 0.084 μM) and protons (IC₅₀ = 0.313 μM). In the preliminary analgesic and body temperature tests, 3b exhibited good efficacy in capsaicin-induced and heat-induced pain models and without hyperthermia side-effect. On the basis of its superior profiles, 3b could be considered as the lead candidate for the further development of antinociceptive drugs.     

2-Sulfonamidopyridine C-region analogs of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides as potent TRPV1 antagonists

A series of 2-sulfonamidopyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamide were investigated as hTRPV1 ligands. Systematic modification on the 2-sulfonamido group provided highly potent TRPV1 antagonists. The N-benzyl phenylsulfonamide derivatives 12 and 23 in particular showed higher affinities than that of lead compound 1. Compound 12 exhibited strong analgesic activity in the formalin pain model. Docking analysis of its chiral S-form 12S in our hTRPV1 homology model indicated that its high affinity might arise from additional hydrophobic interactions not present in lead compound 1S.     

2-Alkyl/alkenyl substituted pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as highly potent TRPV1 antagonists

A series of 2-alkyl/alkenyl pyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Multiple compounds showed excellent and stereospecific TRPV1 antagonism with better potency than previous lead 2. Among them, compound 15f demonstrated a strong analgesic profile in a rat neuropathic pain model and blocked capsaicin-induced hypothermia in a dose-dependent manner. Docking analysis of (S)-15f with our hTRPV1 homology model provided insight into its specific binding mode.     

Design,synthesis and biological evaluation of novel analgesic agents targeting both cyclooxygenase and TRPV1

Multitarget-directed ligands might offer certain advantages over traditional single-target drugs and/or drug combinations. In the present study, a series of novel analgesic agents targeting both cyclooxygenase and TRPV1 were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazine, ethanediamine cores. These compounds were evaluated for antagonism of hTRPV1 activation by capsaicin and the ability to inhibit Ovine COX-1 and human recombinant COX-2 in vitro. The favorable potentials of these test compounds were further characterized in preliminary analgesic and side-effects tests in vivo. On the basis of comprehensive evaluations, compound 8d which showed strong TRPV1 antagonistic activity, middle COX-2 inhibition, weak ulcerogenic action and had no hyperthermia side-effect was considered as a safe candidate for the further development of analgesic drugs.     

TRPV1 antagonist with high analgesic efficacy: 2-Thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides

A series of 2-thio pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Among them, compound 24S showed stereospecific and excellent TRPV1 antagonism of capsaicin-induced activation. Further, it demonstrated strong anti-allodynic in a rat neuropathic pain model. Consistent with its action in vitro being through TRPV1, compound 24S blocked capsaicin-induced hypothermia in mice. Docking analysis of 24S with our hTRPV1 homology model was performed to identify its binding mode.     

Asymmetric synthesis and receptor activity of chiral simplified resiniferatoxin (sRTX) analogues as transient receptor potential vanilloid 1 (TRPV1) ligands

The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthesized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested the basis for its stereospecific activity and the binding mode of 3R.     

Design,synthesis and biological evaluation of novel hydrogen sulfide releasing capsaicin derivatives

Capsaicin (CAP), the prototypical TRPV1 agonist, is the major active component in chili peppers with health-promoting benefits. However, its use is limited by the low bioavailability and irritating quality. In this study, for improving the activity of CAP and alleviating its irritating effects, a series of H₂S-releasing CAPs were designed and synthesized by combining capsaicin and dihydro capsaicin with various hydrogen sulfide donors. The resulting compounds were evaluated their TRPV1 agonist activity, analgesic activity, anticancer activities, H₂S-releasing ability, and gastric mucosa irritation. Biological evaluation indicated that the most active compound B₉, containing 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione moiety as H₂S donor, had better analgesic activity and displayed more potent cytotoxic effects on the test cell lines than the lead compound CAP. Furthermore, the preferred compound, B₉ reduced rat gastric mucosa irritation caused by CAP. Notably, the improved properties of this derivative are associated with its H₂S-releasing capability.     

Receptor activity and conformational analysis of 5'-halogenated resiniferatoxin analogs as TRPV1 ligands

A series of 5′-halogenated resiniferatoxin analogs have been investigated in order to examine the effect of halogenation in the A-region on their binding and the functional pattern of agonism/antagonism for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Halogenation at the 5-position in the A-region of RTX and of 4-amino RTX shifted the agonism of parent compounds toward antagonism. The extent of antagonism was greater as the size of the halogen increased (I > Br > Cl > F) while the binding affinities were similar, as previously observed for our potent agonists. In this series, 5-bromo-4-amino RTX (39) showed very potent antagonism with K_i (ant) = 2.81 nM, which was thus 4.5-fold more potent than 5′-iodo RTX, previously reported as a potent TRPV1 antagonist. Molecular modeling analyses with selected agonists and the corresponding halogenated antagonists revealed a striking conformational difference. The 3-methoxy of the A-region in the agonists remained free to interact with the receptor whereas in the case of the antagonists, the compounds assumed a bent conformation, permitting the 3-methoxy to instead form an internal hydrogen bond with the C4-hydroxyl of the diterpene.     

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one,a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes? and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC₅₀ values of 58 and 65?nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.     

Synthesis and SAR of novel capsazepine analogs with significant anti-cancer effects in multiple cancer types

We previously demonstrated that capsazepine (CPZ), a synthetic transient receptor potential Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-cancer effects in vivo. The purpose of this study was to develop more potent analogs based upon CPZ pharmacophore and structure–activity relationships (SAR) across analogs. We generated 30 novel compounds and screened for their anti-proliferative effects in cultured HeLa cervical cancer cells. Cell viability assays identified multiple compounds with IC_50s?<?15?μM and one compound, 29 with an IC₅₀?<?5?μM; six fold more potent than CPZ. We validated the anti-proliferative efficacy of two lead compounds, 17 and 29, in vivo using HeLa-derived xenografts in athymic nude mice. Both analogs significantly reduced tumor volumes by day 8 compared to control treated animals (p?<?0.001) with no observable adverse effects. Calcium imaging determined that compound 17 activates TRPV1 whereas 29 neither activates nor inhibits TRPV1; indicating a unique mechanism-of-action that does not involve TRPV1 signaling. Cell viability assays using a panel of additional tumor types including oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, and prostate cancer cell lines (HSC-3, H460, MDA-231, and PC-3 respectively) demonstrated that both lead compounds were efficacious against every cancer type tested. Compounds 29 displayed IC_50s of 1–2.5?μM in HSC-3and PC-3cells. Thus, we propose that these novel CPZ analogs may serve as efficacious therapeutic agents against multiple tumor types that warrant further development for clinical application.     

Pyrido[2,3-b]pyrazines,discovery of TRPV1 antagonists with reduced potential for the formation of reactive metabolites

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a non-selective cation channel that can be activated by a wide range of noxious stimuli, including capsaicin, acid, and heat. Blockade of TRPV1 activation by selective antagonists is under investigation in an attempt to identify novel agents for pain treatment. During pre-clinical development, the 1,8-naphthyridine 2 demonstrated unacceptably high levels of irreversible covalent binding. Replacement of the 1,8-naphthyridine core by a pyrido[2,3-b]pyrazine led to the discovery of compound 26 which was shown to have significantly lower potential for the formation of reactive metabolites. Compound 26 was characterized as an orally bioavailable TRPV1 antagonist with moderate brain penetration. In vivo, 26 significantly attenuated carrageenan-induced thermal hyperalgesia (CITH) and dose-dependently reduced complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain after oral administration.     

Synthesis and biological evaluation of 5-substituted and 4,5-disubstituted-2-arylamino oxazole TRPV1 antagonists

The synthesis and structure–activity relationships of a series of 5-monosubstituted and 4,5-disubstituted 2-arylaminooxazoles as novel antagonists of the transient receptor potential vanilloid 1 (TRPV1) receptor are described. The 7-hydroxy group of the tetrahydronaphthyl moiety on the 2-amino substituent of the oxazole ring was important for obtaining excellent in vitro potency at the human TRPV1 receptor, while a variety of alkyl and phenyl substituents at the 4- and 5-positions of the oxazole ring were well tolerated and yielded potent TRPV1 antagonists. Despite excellent in vitro potency, the 5-monosubstituted compounds suffered from poor pharmacokinetics. It was found that 4,5-disubstitution on the oxazole ring was critical to the improvement of the overall pharmacokinetic profile of these analogues, which led to the discovery of compound (R)-27, a novel TRPV1 antagonist with good oral activity in preclinical animal models of pain.     

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design,synthesis, and biological evaluation

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory K_i value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.     

2-Methylacrylamide as a bioisoster of thiourea group for 1,3-dibenzylthioureido TRPV1 receptor antagonists

In order to replace thiourea group with the more drug-like moiety for 1,3-dibenzylthioureas having TRPV1 antagonist activity, we introduced a set of functional groups between the two aromatic rings based on bioisosteric replacement. The synthesized bioisosteres of 1,3-dibenzylthioureas were tested for their antagonist activities on TRPV1 by ⁴⁵Ca²⁺-influx assay using neonatal rat cultured spinal sensory neurons. Among the tested 14 kinds of bioisosters, 2-methylacrylamide group was the best candidate to replace thiourea group. Compound 7c, 2-methylacrylamide analog of ATC-120, showed as potent as ATC-120 in its antagonist activity. In addition, 2-methylacrylamide analog 7e having vinyl moiety showed the most potent activity with 0.022?μM of IC₅₀ value, indicating that thiourea group of 1,3-dibenzylthioureas could be replaced to 2-methylacrylamide without loss of their potencies.     

Design,synthesis, and biological evaluation of novel biphenyl-4-carboxamide derivatives as orally available TRPV1 antagonists

A new series of transient receptor potential vanilloid type 1 (TRPV1) antagonists were designed and synthesized from N-(3-hydroxyphenyl)-2-(piperidin-1-ylmethyl)biphenyl-4-carboxamide hydrochloride (8). SAR studies identified (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide hydrochloride (ASP8370, 7), as a compound with high aqueous solubility, satisfactory stability in human liver microsomes, and reduced CYP3A4 inhibition. ASP8370 was selected as a clinical development candidate with significant ameliorative effects on neuropathic pain. SAR studies also revealed the structural mechanisms underlying the switching between TRPV1 antagonism and agonism.     

Design and synthesis of donepezil analogues as dual AChE and BACE-1 inhibitors

Multi-target-directed ligands (MTDLs) centered on β-secretase 1 (BACE-1) inhibition are emerging as innovative therapeutics in addressing the complexity of neurodegenerative diseases. A new series of donepezil analogues was designed, synthesized and evaluated as MTDLs against neurodegenerative diseases. Profiling of donepezil, a potent acetylcholinesterase (hAChE) inhibitor, into BACE-1 inhibition was achieved through introduction of backbone amide linkers to the designed compounds which are capable of hydrogen-bonding with BACE-1 catalytic site. In vitro assays and molecular modeling studies revealed the dual mode of action of compounds 4–6 against hAChE and BACE-1. Notably, compound 4 displayed potent hAChE inhibition (IC₅₀ value of 4.11 nM) and BACE-1 inhibition (IC₅₀ value of 18.3 nM) in comparison to donepezil (IC₅₀ values of 6.21 and 194 nM against hAChE and BACE-1, respectively). Moreover, 4 revealed potential metal chelating property, low toxicity on SH-SY5Y neuroblastoma cells and ability to cross the blood–brain barrier (BBB) in PAMPA-BBB assay which renders 4 a potential lead for further optimization of novel small ligands for the treatment of Alzheimer's disease.