ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of the thromboxane receptor antagonist and thromboxane synthase inhibitor BM-531

BM-531 (N-tert-butyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA2 receptors labeled with [3H]SQ-29548 (IC50 = 0.0078 microM) is higher than sulotroban (IC50 = 0.93 microM) and SQ-29548 (IC50 = 0.021 microM). Moreover, BM-531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet-rich plasma BM-531 prevents platelet aggregation induced by arachidonic acid (600 microM) (ED100 = 0.125 microM), U-46619, a stable TXA2 agonist (1 microM) (ED50 = 0.482 microM) or collagen (1 microgram/mL) (percentage of inhibition: 42.9% at 10 microM) and inhibits the second wave of ADP (2 microM)-induced aggregation. On the other hand, when BM-531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100) is significantly prolonged. In addition, at the concentrations of 10 and 1 microM, BM-531 totally prevents the production of TXB2 by human platelets activated by arachidonic acid. Finally, at 10 microM, BM-531 significantly prevents rat fundus contractions induced by U-46619 but not by prostacyclin. These results suggest that BM-531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.     

Protective effects of G 619, a dual thromboxane synthase inhibitor and thromboxane A2 receptor antagonist, in splanchnic artery occlusion shock.

Splanchnic artery occlusion (SAO) shock was induced in anesthetized rats by clamping the celiac trunk and the superior mesenteric artery for 45 min. The arteries were then released and survival rate, mean survival time, mean arterial blood pressure (MAP), plasma levels of thromboxane B2 (TxB2) and 6-keto-PGF1 alpha, and the phagocytotic activity of peritoneal macrophages were evaluated. Shocked animals died within 89 +/- 10 min, while all sham-shocked rats survived greater than 3 h. SAO shock produced relevant changes in MAP, significantly increased plasma levels of TxB2 and 6-keto-PFG1 alpha, and decreased peritoneal macrophage phagocytotic activity. The administration of G 619, a dual thromboxane synthase inhibitor/thromboxane A2 receptor antagonist (50 mg/kg, 15 min before SAO shock) significantly increased survival time (190 +/- 13 min) and survival rate, reduced plasma levels of TxB2, and partially restored the impairment in peritoneal macrophage phagocytosis. Finally, the administration of G 619 had beneficial effects on changes in MAP-induced bay SAO shock. These data further confirm the involvement of TxA2 in SAO shock and suggest that G 619 may have positive effects in low-flow states.     

Pharmacological characterization of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor.

1. The pharmacological effects of cinnamophilin, a new lignan, isolated from Cinnamomum philippinense, was determined in vitro in human platelet, rat isolated aorta and guinea-pig isolated trachea and in vivo in mice and guinea-pigs. 2. Cinnamophilin inhibited dose-dependently human platelet-rich plasma (PRP) aggregation induced by arachidonic acid (AA), collagen and U-46619 with IC50 of 5.0 +/- 0.4, 5.6 +/- 0.6 and 3.0 +/- 0.4 microM, respectively. The second wave of ADP- or adrenaline-induced platelet aggregation was inhibited by cinnamophilin, while the first wave was only slightly inhibited by cinnamophilin above 30 microM. 3. Cinnamophilin was found to be a thromboxane A2 (TXA2) receptor blocking agent in human platelet, rat aorta and guinea-pig trachea as revealed by its competitive antagonism of U-46619-induced aggregation of human-PRP, contraction of rat aortic rings and guinea-pig tracheal rings with pA2 values of 7.3 +/- 0.2, 6.3 +/- 0.1 and 5.2 +/- 0.2, respectively. 4. [3H]-inositol monophosphate formation and the rise of intracellular Ca2+ caused by U-46619 in human platelet was suppressed by cinnamophilin (10 microM). 5. Cinnamophilin induced a dose-dependent inhibition of thromboxane B2 (TXB2) formation, while the prostaglandin E2 (PGE2) formation was increased. Cinnamophilin did not affect unstimulated platelet adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. When the platelets were challenged with AA, a dose-dependent rise in cyclic AMP was observed. Dazoxiben (a pure TX synthase inhibitor) and SQ 29548 (a pure TXA2 receptor antagonist) did not affect cyclic AMP levels in AA-treated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thromboxane A2/prostaglandin endoperoxide receptor antagonist activity of CV-4151, a thromboxane A2 synthetase inhibitor

The thromboxane A2/prostaglandin endoperoxide (TXA2/PGH2) receptor antagonist activity of CV-4151, a potent TXA2 synthetase inhibitor, was examined. CV-4151 inhibited guinea pig and human platelet aggregation induced by U-44069 with IC50 values of 1.2 +/- 0.3 X 10(-5) and 1.9 +/- 0.4 X 10(-5) M, respectively, and inhibited the specific binding of [3H]U-46619 to washed guinea pig and human platelets with IC50 values of 1.2 +/- 0.3 X 10(-6) and 5.1 +/- 1.0 X 10(-6) M, respectively. CV-4151 competitively inhibited the contraction of rabbit aortic strips induced by U-44069 with a pA2 value of 5.90. In experiments in mice in vivo, CV-4151 (1 and 10 mg/kg i.v.) significantly inhibited the thrombocytopenia induced by U-44069 in a dose-dependent manner. These results show that CV-4151 has a distinct TXA2/PGH2 receptor antagonist effect, and that this effect together with its inhibition of TXA2 synthetase could be important for the pharmacological action of this compound.     

Pharmacological profile and therapeutic potential of BM-573, a combined thromboxane receptor antagonist and synthase inhibitor

BM-573 (N-terbutyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl]urea), a torsemide derivative, is a novel non-carboxylic dual TXA2 synthase inhibitor and receptor antagonist. The pharmacological profile of the drug is characterized by a higher affinity for the thromboxane receptor than that of SQ-29548, one of the most powerful antagonists described to date, by a complete prevention of human platelet aggregation induced by arachidonic acid at a lower dose than either torsemide or sulotroban, and by a significantly prolonged closure time measured by the platelet function analyser (PFA-100). Moreover, at the concentrations of 1 and 10 microM, BM-573 completely prevented production of TXB2 by human platelets activated by 0.6 mM of arachidonic acid. BM-573 prevents rat fundus contraction induced by U-46619 but not by prostacyclin or other prostaglandins. Despite possessing a chemical structure very similar to that of a diuretic torsemide, BM-573 has no diuretic activity. BM-573 does not prolong bleeding time and, unlike some of the other sulfonylureas, has no effect on blood glucose levels. In vivo, BM-573 appears to have antiplatelet and antithrombotic activities since it reduced thrombus weight and prolonged the time to abdominal aorta occlusion induced by ferric chloride. BM-573 also relaxed rat aorta and guinea pig trachea precontracted with U-46619. In pigs, BM-573 completely antagonized pulmonary hypertensive effects of U-46619 and reduced the early phase of pulmonary hypertension in models of endotoxic shock and pulmonary embolism. Finally, BM-573 protected pigs from myocardial infarction induced by coronary thrombosis. These results suggest that BM-573 should be viewed as a promising therapeutic agent in the treatment of pulmonary hypertension and syndromes associated with platelet activation.     

Ridogrel, a dual thromboxane synthase inhibitor and receptor antagonist: anti-inflammatory profile in inflammatory bowel disease

BACKGROUND: Thromboxanes, prostaglandins, reactive oxygen metabolites and pro-inflammatory cytokines are produced in excess in inflammatory bowel disease. Preliminary reports suggest that ridogrel, a thromboxane synthesis inhibitor and receptor blocker, may have therapeutic benefits in ulcerative colitis. AIMS: To investigate the anti-inflammatory profile of ridogrel. METHODS: The effects of ridogrel on the production of eicosanoids, reactive oxygen metabolites and cytokines by cultured inflamed colorectal mucosal biopsies were made using ELISA and chemiluminescence, reactive oxygen metabolite generation in a cell-free system, and platelet activation using flow cytometry. The effects of oral ridogrel on mucosal release of eicosanoids in two patients with active ulcerative colitis were assessed using rectal dialysis. RESULTS: Ridogrel significantly reduced the release of thromboxane B2, but not prostaglandin E2 or tumour necrosis factor-alpha, from biopsies (P < 0.01 for 10 microM ridogrel). Ridogrel showed no direct antioxidant activity but significantly reduced reactive oxygen metabolite production from cultured biopsies (P < 0.01 for 10 microM ridogrel). Platelet activation in vitro was inhibited by ridogrel (P /= 10 microM ridogrel). Mean rectal mucosal thromboxane B2 release was reduced to 86% of pre-treatment levels in two patients treated with oral ridogrel. CONCLUSIONS: Its inhibition of mucosal production of thromboxane B2, reactive oxygen metabolites, and of platelet activation, suggests that ridogrel could have a therapeutic role in inflammatory bowel disease.     

A novel thromboxane receptor antagonist and synthase inhibitor, BM-573, reduces development and progression of atherosclerosis in LDL receptor deficient mice

Atherosclerosis is a chronic inflammatory disease of the vasculature influenced by a variety of mediators. Among them, prostanoids, which include prostacyclin and thromboxane (Tx) A(2), have recently received a lot of attention. Previous studies demonstrated that antagonism or deletion of the receptor for TxA(2) retards early atherogenesis in apolipoprotein E-deficient mice, but no data are available in low-density lipoprotein (LDL) receptor deficient mice. In our study, we tested the effect of a novel TxA(2) receptor (TP) antagonist and synthase inhibitor, BM-573, on atherosclerosis development and progression in LDL receptor deficient mice. To this end, the effect of 12 weeks treatment with BM-573 on early or established aortic atherosclerotic lesions of these mice was assessed. In both treatments, while BM-573 did not affect body weight, systolic blood pressure, total plasma cholesterol or triglycerides levels, it partially reduced TxA(2) but did not affect prostacyclin biosynthesis. Moreover, BM-573 significantly decreased early atherogenesis and prevented progression of established atherosclerotic lesions. These results show for the first time that this dual Tx inhibitor is effective in reducing atherogenesis in the LDL receptor deficient mice. They also demonstrate the novel concept that this therapeutic approach halts the progression of the disease and influences the cellular composition of the atherosclerotic plaques.     

Pharmacokinetics and pharmacodynamics of terbogrel, a combined thromboxane A2 receptor and synthase inhibitor, in healthy subjects

AIMS: To characterize the pharmacokinetics of terbogrel, a new combined thromboxane A2 (TxA2) receptor and synthase inhibitor, in healthy human subjects after single or multiple oral administration. METHODS: Forty-eight healthy male subjects received a single oral dose (10, 25, 50, 100, 150 or 200 mg) of terbogrel or placebo and 32 different subjects received one of the following treatments twice daily for 7 days: 50, 100 or 150 mg terbogrel, placebo, or once-a-day 330 mg acetylsalicylic acid. RESULTS: Terbogrel was well tolerated without obvious adverse effects following either a single oral dose or administration over 7 days. Plasma drug concentrations were dose-linear and there was no accumulation over 7 days. There was a dose-dependent blockade of TxA2 receptors and of inhibition of thromboxane synthase activity with values for IC50 of 12 ng ml(-1) and 6.7 ng ml(-1), respectively. At the highest dose tested (150 mg) there was almost complete inhibition of thomboxane synthase and thromboxane receptor occupancy. Even at trough concentrations, receptor occupancy remained above 80% and thromboxane synthase was still completely inhibited. These two activities were associated with a dose-dependent inhibition of platelet aggregation (>80% at the 150 mg dose of terbogrel) and enhanced prostacyclin production. CONCLUSIONS: Terbogrel is a potent agent having two distinct, complimentary pharmacodynamic actions, namely inhibition of thromboxane synthase and antagonism of the TxA2 receptor. The antithrombotic effect of terbogrel was dose-dependent and was associated with enhanced prostacyclin production. Terbogrel is an attractive candidate for long-term antithrombotic therapy.     

Development of dual-acting benzofurans for thromboxane A2 receptor antagonist and prostacyclin receptor agonist: synthesis, structure-activity relationship, and evaluation of benzofuran derivatives

Prostacyclin (PGI(2)) is an unstable, powerful endogenous inhibitor of platelet aggregation, and thromboxane A(2) (TXA(2)) is an unstable endogenous arachidonic acid metabolite that plays a pivotal role in platelet aggregation and vasoconstriction. The balance between TXA(2) and PGI(2) greatly affects maintenance of the homeostasis of the circulatory system. A novel series of benzofuran-7-yloxyacetic acid derivatives was discovered as potent dual-acting agents to block the thromboxane A(2) receptor and to activate the prostacyclin receptor. Synthesis, structure-activity relationship, and in vitro and ex vivo pharmacology of this series of compounds are described. The most potent in the series was {3-[2-(1,1-diphenylethylsulfanyl)ethyl]-2-hydroxymethylbenzofuran-7-yloxy}acetic acid diethanolamine salt (7) with K(i) of 4.5 nM for thromboxane receptor antagonism and K(i) of 530 nM for prostacyclin receptor agonism. Remarkably, compound 7 is a promising candidate for novel treatment as an antithrombotic agent with other cardiovascular actions to avoid hypotensive side effects.     

Guanidine derivatives as combined thromboxane A2 receptor antagonists and synthase inhibitors

A new series of omega-disubstituted alkenoic acid derivatives derived from samixogrel 5 were designed and synthesized as combined thromboxane A2 receptor antagonists/thromboxane A2 synthase inhibitors with improved solubility and reduced protein binding compared to 5. Hexenoic acid derivatives with a 3-pyridyl group and 3-(2-cyano-3-alkyl-guanidino)phenyl substituent were found to be optimal with regard to this dual mode of action. The most potent compound, E-6-(3-(2-cyano-3-tert-butyl-guanidino)phenyl)-6-(3-pyridyl)hex-5-eno ic acid, "terbogrel" 32 inhibits the thromboxane A2 synthase in human gel-filtered platelets with an IC50 value of 4.0 +/- 0.5 nM (n = 4). Radioligand binding studies with 3H-SQ 29,548 revealed that 32 blocks the thromboxane A2/endoperoxide receptor on washed human platelets with an IC50 of 11 +/- 6 nM (n = 2) and with an IC50 of 38 +/- 1 nM (n = 15) in platelet-rich plasma. Terbogrel inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an IC50 of 310 +/- 18 nM (n = 8) and 52 +/- 20 nM (n = 6), respectively. This was shown to translate into a potent antithrombotic effect in vivo as demonstrated in studies using a model of arterial thrombosis in rabbits (ED50 = 0.19 +/- 0.07 mg/kg; n = 20). Thus, terbogrel is the first compound with a guanidino moiety demonstrating both a potent TXA2 synthase inhibition and a potent TXA2 receptor antagonism and has been selected for further clinical development.     

Design, synthesis, and biological evaluation of benzofuran derivatives as ET receptor antagonists

A series of novel benzofuran carboxylic acid derivatives have been designed and synthesized, with their antagonism effect screened on ET-1-induced contraction in the rat thoracic aortic ring. Some target compounds demonstrated significant inhibitory activity, especially benzo[c]thiadiazole and benzo[c]oxadiazole compounds 29 and 30 showed potent inhibition percentage higher than the contrast compound BQ123. Further affinity and selectivity for ET binding assay showed that 29 demonstrated a dual ET_A/ET_B antagonism activity in nanomole level. Moreover, 30 was effective in relieving hypoxia-induced pulmonary arterial hypertension.     

Design,synthesis, and biological evaluation of fluoroneplanocin A as the novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase

Fluoroneplanocin A (12) was designed as a novel mechanism-based inhibitor of S-adenosylhomocysteine hydrolase (SAH) and efficiently synthesized via an electrophilic vinyl fluorination reaction (n-BuLi, N-fluorobenzenesulfonimide at -78 degrees C). Fluoroneplanocin A exhibited 2-fold more potent SAH inhibitory activity than the parent neplanocin A. A new mechanism of irreversible inhibition discovered in this work might provide new alternatives in the design of a different class of antiviral agents operating via SAH inhibition.     

ICI 180080, a novel selective thromboxane receptor antagonist: synthesis and relative activity

The preparation of a novel potent thromboxane receptor antagonist ICI 180080, 5(Z)-7-[2,2-dimethyl-4-(2-hydroxyphenyl)-1,3-dioxan-cis-5-yl] heptenoic acid, is described together with its methyl ether and methyl ester. Thromboxane antagonist pA2 data against U 46619 is presented for rabbit thoracic aorta in-vitro (ICI 180080, pA2 = 7.5). The relative antagonist pA2 values obtained are discussed in terms of the chemical structure of the molecules. The potent activity of ICI 180080 is attributed to a specific orientation of the phenolic oxygen, due to an intramolecular hydrogen bond.     

Basic pharmacophore of some antithrombotic agents with combined thromboxane receptor antagonist (TxRA)/thromboxane synthase inhibitor (TxSI) activity

Platelet activation, causing formation of platelet aggregates, plays a major role in occlusive thrombus formation which may induce ischemic cardiovascular diseases. Thromboxane A₂ (TXA₂) is an extremely potent vasoconstricting and platelet-aggregating agent so that it is important to search for a new type of thromboxane synthase inhibitor combined with thromboxane receptor antagonism (TxRA/TxSI) dual blocker with high potency and less side effects. On the basis of the structures of some known TxRA/TxSI dual blockers, we used 3D molecular modelling to search for pharmacophores for the dual blockers. Drug Dev. Res. 39:197–200. © 1997 Wiley-Liss, Inc.     

A thromboxane A2 synthase inhibitor, DP-1904, prevents rat renal injury

The effects of DP-1904, a thromboxane (TX) A2 synthase inhibitor, on renal function were investigated by analysis of prostanoid metabolism in hydronephrotic and ischemic rat kidney models, and in isolated perfused normal and hydronephrotic rat kidneys. The increase in production of TXB2 in hydronephrotic or ischemic kidneys was significantly suppressed by intraperitoneal DP-1904 (10 mg/kg), with the 6-keto-prostaglandin F1 alpha to TXB2 ratio being significant increased. Urine volume, glomerular filtration rate and renal plasma flow were all improved. DP-1904 (0.3 micrograms/min) blocked the effects of infused arachidonic acid on isolated perfused normal rat kidneys thus reducing TXB2 levels and perfusion pressure but the pressor response to norepinephrine or angiotensin II remained unchanged. In isolated perfused hydronephrotic rat kidneys, DP-1904 suppressed the increase in perfusion pressure and TXB2 production caused by platelet-activating factor. These findings suggested that DP-1904 improved renal failure by specifically inhibiting TXA2 production.     

Characterization of platelet thromboxane A2/prostaglandin H2 receptor by a novel thromboxane receptor antagonist, [3H]S-145

The specific binding sites for S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonist with weak partial agonistic activity, were studied in human platelet membranes. [3H]S-145 displayed high affinity and specificity, as well as saturable and displaceable binding, to a single class of recognition sites with the same maximum number of sites (2100 fmol/mg protein) as the other two TXA2/PGH2 receptor antagonists, [3H]SQ29,548 and [3H]ONO3708. Binding of S-145 to the platelet membranes was enhanced by divalent cations (Mg2+ and Ca2+), and the binding affinity in the presence of 20 mM MgCl2 was 0.75 nM, a value which was smaller than those of SQ29,548 (8.7 nM) and ONO3708 (3.7 nM). The rank order of potency (Ki) for a series of TXA2/PGH2 receptor antagonists to displace [3H]S-145 binding to the membranes was correlated with those determined from [3H]SQ29,548 or [3H]ONO3708 binding to the same preparations. Kinetic analysis for the binding of the above radiolabeled antagonist to the crude platelet membranes revealed that the dissociation rate constant (K-1) for S-145 was much smaller than that for other ligands in human, rat and rabbit platelets. The extremely slow dissociation of S-145 from the receptors may explain the long-lasting characteristic of this compound in vivo as well as the abolishment of partial agonistic activity.     

Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist

The first druglike selective angiotensin II AT(2) receptor agonist (21) with a K(i) value of 0.4 nM for the AT(2) receptor and a K(i) > 10 microM for the AT(1) receptor is reported. Compound 21, with a bioavailability of 20-30% after oral administration and a half-life estimated to 4 h in rat, induces outgrowth of neurite cells, stimulates p42/p44(mapk), enhances in vivo duodenal alkaline secretion in Sprague-Dawley rats, and lowers the mean arterial blood pressure in anesthetized, spontaneously hypertensive rats. Thus, the peptidomimetic 21 exerts a similar biological response as the endogenous peptide angiotensin II after selective activation of the AT(2) receptor. Compound 21, derived from the prototype nonselective AT(1)/AT(2) receptor agonist L-162,313 will serve as a valuable research tool, enabling studies of the function of the AT(2) receptor in more detail.     

Preparation and biological evaluation of a potential photoaffinity label for the prostaglandin H2/thromboxane A2 receptor

Two aromatic azides (24 and 26) were prepared as potential photoaffinity probes for the PGH2/TXA2 receptor. The compounds are based on the well-characterized PGH2/TXA2 receptor antagonist 13-azaprostanoic acid, with the terminus of its lower side chain replaced with phenoxy (24) or benzyl (26) azide functionality. The two compounds were shown to irreversibly inhibit platelet function after photolysis and resuspension. However, of the two aromatic azides, only the benzyl derivative 26 appeared to be selective for the prostaglandin pathway. The latter compound was also prepared as the aromatic 125I (29) derivative, which may ultimately prove useful as a labeled probe for the identification and isolation of the putative TXA2/PGH2 receptor.     

Design, synthesis, and biological evaluation of phenylpropanamides as novel transient receptor potential valnilloid 1 antagonists

Synthesis and structure-activity relationship of N-benzyl-3-phenylpropanamides as transient receptor potential vanilloid 1 (TRPV1) antagonists are described. A variety of substituents such as halide, ester, nitro, and alkyl groups at 2 or 3-position of 4-(methylsulfonylamino) benzyl unit were examined. These compounds exhibited potent ⁴⁵Ca²⁺ uptake inhibition in rat DRG neuron via TRPV1 blockade. Especially compound 28c, has been identified as a potent antagonist with IC₅₀ of 38 nM.