Ing-Jun Chen

The new generation dihydropyridine type calcium blockers, bearing 4-phenyl oxypropanolamine, display α-/β-Adrenoceptor antagonist and long-Acting antihypertensive activities

A new series of dihydropyridine derivatives, bearing oxypropanolamine moiety on phenyl ring at the 4-position of the dihydropyridine base, were prepared. Oxypropanolamine was synthesized by replacing the phenolic OH of vanillin or other compounds, having a phenyl aldehyde group, with epichlorohydrin, followed by cleavaging the obtained epoxide compounds with tert-butylamine, n-butylamine or 2-methoxy-1-oxyethylamino benzene (guaiacoxyethylamine), respectively. Obtained various oxypropanolamine compounds, still remaining a phenyl aldehyde moiety, were then performed by Hantzsch condensation reaction with methylacetoacetate or ethylacetoacetate, respectively, to give our new series of dihydropyridine linked with the 4-phenyl ring. These compounds were evaluated for inotropic, chronotropic, and aorta contractility that associated with calcium channel and adrenoceptor antagonist activities. Dihydropyridine derivatives that with oxypropanolamine side chain on their 4-phenyl ring associated α-/β-adrenoceptor blocking activities created a new family of calcium entry and the third generation β-adrenoceptor blockers. Optimizing this research to obtain more potent α-/β-adrenoceptor blocking and long-acting antihypertensive oxypropanolamine on the 4-phenyl ring of dihydropyridine series compounds was thus accomplished and classified as third generation dihydropyridine type calcium channel blockers, in comparison with previous short-acting type nifedipine and long-acting type amlodipine. We concluded that compounds 1a, 1b and 1g showed not only markedly high calcium-antagonistic activity but also the highest antihypertensive effect; compounds 1b, 1c, 1f, 1g, 1i and 1j induced sustained antihypertensive effects are major and attributed to their calcium entry and α-adrenoceptor blocking activities in the blood vessel due to their introduction of 2-methoxy, 1-oxyethylamino benzene moiety in the side chain on the 4-phenyl ring of dihydropyridine. Bradycardiac effects of all the compounds 1a–1j resulted from calcium entry and β-adrenoceptor blocking, which attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 1b as candidate compound for further pharmacological and pre-clinical evaluation studies.

Antihypertensive and vasorelaxing activities of synthetic xanthone derivatives.

A series of xanthones and xanthonoxypropanolamines have been synthesized. The activity of compounds on cardiovascular system was evaluated. All the compounds tested exhibited effective hypotensive activity in anesthetized rats. An oxypropanolamine side chain substituted at the C-3 position of the xanthone nucleus significantly enhanced the hypotensive activity. In rat thoracic aorta, all the compounds tested significantly depressed the contractions induced by Ca(2+) (1.9mM) in high K+(80mM) medium and the phasic and tonic contractions caused by norepinephrine (3 microM). In the rat thoracic aorta, the phenylephrine- and high K+ -induced 45Ca(2+) influx were both inhibited by a selective xanthone derivative, 13. In addition to the previously reported result of 13, evaluated as beta adrenoceptor blocker, the depressor and bradycardia effects of 9 are independent of the parasympathetic passway. These results suggest that 13 showed inhibitory effects on the contractile response caused by high K+ and norepinephrine in rat thoracic aorta are mainly due to inhibition of Ca(2+) influx through both voltage-dependent and receptor-operated Ca(2+) channels. The vasodilating properties of 13 is due to its calcium channel and beta adrenergic blocking effects.

A xanthine-based KMUP-1 with cyclic GMP enhancing and K+ channels opening activities in rat aortic smooth muscle

KMUP‐1 (1, 3, 5 mg kg−1, i.v.), a xanthine derivative, produced dose‐dependent sustained hypotensive and short‐acting bradycardiac effects in anaesthetized rats. This hypotensive effect was inhibited by pretreatment with glibenclamide (5 mg kg−1, i.v.). In endothelium‐intact or denuded aortic rings preconstricted with phenylephrine, KMUP‐1 caused a concentration‐dependent relaxation. This relaxation was reduced by endothelium removal, the presence of NOS inhibitor L‐NAME (100 μM) and sGC inhibitors methylene blue (10 μM) and ODQ (1 μM). The vasorelaxant effects of KMUP‐1 was attenuated by pretreatment with various K+ channel blockers TEA (10 mM), glibenclamide (1 μM), 4‐AP (100 μM), apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). Increased extracellular potassium levels (30 – 80 mM) caused a concentration‐related reduction of KMUP‐1‐induced vasorelaxations. Preincubation with KMUP‐1 (1, 10, 100 nM) increased the ACh‐induced maximal vasorelaxations mediated by endogenous NO release, and enhanced the potency of exogenous NO‐donor SNP. The vasorelaxant responses of KMUP‐1 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor IBMX (0.5 μM) had an additive action. Additionally, KMUP‐1 (100 μM) affected cyclic GMP metabolism since it inhibited the activity of PDE in human platelets. KMUP‐1 induced a dose‐related increase in intracellular cyclic GMP levels in rat A10 vascular smooth muscle (VSM) cells, but not cyclic AMP. The increase in cyclic GMP content of KMUP‐1 (0.1 – 100 μM) was almost completely abolished in the presence of methylene blue (10 μM), ODQ (10 μM), and L‐NAME (100 μM). In conclusion, these results indicate that KMUP‐1 possesses the following merits: (1) stimulation of NO/sGC/cyclic GMP pathway and subsequent elevation of cyclic GMP, (2) K+ channels opening, and (3) inhibition of PDE or cyclic GMP breakdown. Increased cyclic GMP display a prominent role in KMUP‐1‐induced VSM relaxations.

KMUP-1, a xanthine derivative, induces relaxation of guinea-pig isolated trachea: the role of the epithelium, cyclic nucleotides and K+ channels.

7‐[2‐[4‐(2‐chlorophenyl)piperazinyl]ethyl]‐1,3‐dimethylxanthine (KMUP‐1) produces tracheal relaxation, intracellular accumulation of cyclic nucleotides, inhibition of phosphodiesterases (PDEs) and activation of K+ channels. KMUP‐1 (0.01–100 μM) induced concentration‐dependent relaxation responses in guinea‐pig epithelium‐intact trachea precontracted with carbachol. Relaxation responses were also elicited by the PDE inhibitors theophylline, 3‐isobutyl‐1‐methylxanthine (IBMX), milrinone, rolipram and zaprinast (100 μM), and a KATP channel opener, levcromakalim. Tracheal relaxation induced by KMUP‐1 was attenuated by epithelium removal and by pretreatment with inhibitors of soluble guanylate cyclase (sGC) (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), 1 μM), nitric oxide synthase (Nω‐nitro‐L‐arginine methyl ester, 100 μM), K+ channels (tetraethylammonium, 10 mM), KATP channels (glibenclamide, 1 μM), voltage‐dependent K+ channels (4‐aminopyridine, 100 μM) and Ca2+‐dependent K+ channels (charybdotoxin, 0.1 μM or apamin, 1 μM). Both KMUP‐1 (10 μM) and theophylline nonselectively and slightly inhibited the enzyme activity of PDE3, 4 and 5, suggesting that they are able to inhibit the metabolism of adenosine 3′,5′‐cyclic monophosphate (cyclic AMP) and guanosine 3′,5′‐cyclic monophosphate (cyclic GMP). Likewise, the effects of IBMX were also measured and its IC50 values for PDE3, 4 and 5 were 6.5±1.2, 26.3±3.9 and 31.7±5.3 μM, respectively. KMUP‐1 (0.01–10 μM) augmented intracellular cyclic AMP and cyclic GMP levels in guinea‐pig cultured tracheal smooth muscle cells. These increases in cyclic AMP and cyclic GMP were abolished in the presence of an adenylate cyclase inhibitor SQ 22536 (100 μM) and an sGC inhibitor ODQ (10 μM), respectively. KMUP‐1 (10 μM) increased the expression of protein kinase A (PKARI) and protein kinase G (PKG1α1β) in a time‐dependent manner, but this was only significant for PKG after 9 h. Intratracheal administration of tumour necrosis factor‐α (TNF‐α, 0.01 mg kg−1) induced bronchoconstriction and exhibited a time‐dependent increase in lung resistance (RL) and decrease in dynamic lung compliance (Cdyn). KMUP‐1 (1.0 mg kg−1), injected intravenously for 10 min before the intratracheal TNF‐α, reversed these changes in RL and Cdyn. These data indicate that KMUP‐1 activates sGC, produces relaxation that was partly dependent on an intact epithelium, inhibits PDEs and increases intracellular cyclic AMP and cyclic GMP, which then increases PKA and PKG, leading to the opening of K+ channels and resulting tracheal relaxation.

KMUP‐1 relaxes rabbit corpus cavernosum smooth muscle in vitro and in vivo: involvement of cyclic GMP and K+ channels

In isolated endothelium‐intact or denuded rabbit corpus cavernosum preconstricted with phenylephrine, KMUP‐1 (0.001 – 10 μM) caused a concentration‐dependent relaxation. This relaxation of KMUP‐1 was attenuated by endothelium removed, high K+ and pretreatments with a soluble guanylyl cyclase (sGC) inhibitor ODQ (1 μM), a NOS inhibitor L‐NAME (100 μM), a K+ channel blocker TEA (10 mM), a KATP channel blocker glibenclamide (1 μM), a voltage‐dependent K+ channel blocker 4‐AP (100 μM) and Ca2+‐dependent K+ channel blockers apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). The relaxant responses of KMUP‐1 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor IBMX (0.5 μM) had additive actions on rabbit corpus cavernosum smooth muscle (CCSM). KMUP‐1 (0.01 – 10 μM) induced increase of intracellular cyclic GMP level in the primary cell culture of rabbit CCSM. This increase in cyclic GMP content was abolished in the presence of ODQ (10 μM). Both KMUP‐1 and sildenafil at 0.2, 0.4, 0.6 mg kg−1 caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose‐dependent manner. These in vivo activities of ICP for sildenafil and KMUP‐1 are consistent with those of in vitro effects of cyclic GMP. KMUP‐1 has the following merits: (1) inhibition of PDE or cyclic GMP breakdown, (2) stimulation of NO/sGC/cyclic GMP pathway, and (3) subsequent stimulation of K+ channels, in rabbit CCSM. We suggest that these merits play prominent roles in KMUP‐1‐induced CCSM relaxation‐associated increases of ICP and penile erection.

The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase

Background and purpose:  KMUP‐1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP‐1 on acute and chronic pulmonary artery hypertension (PAH) in rats.

Capsinolol: the first β‐adrenoceptor blocker with an associated calcitonin gene‐related peptide releasing activity in the heart

1 The β‐adrenoceptor blocking and calcitonin gene‐related peptide (CGRP)‐releasing properties of capsinolol (N‐[4‐(2‐hydroxy‐3 (isopropylamino) propoxy)‐3‐methoxybenzyl] ‐nonanamide), derived from nonivamide, were investigated under in vivo and in vitro conditions. 2 Capsinolol (0.1, 0.5, 1.0 mg kg−1, i.v.), as well as (±)‐propranolol, produced a dose‐dependent bradycardia response and a temporary pressor action in urethane‐anaesthetized normotensive Wistar rats. These cardiovascular effects were different from the vagus reflex and parasympathetic efferent effects shown by capsaicin (0.1 mg kg−1, i.v.) in the rat. 3 Capsinolol (1.0 mg kg−1) inhibited the tachycardia effects induced by (−)−isoprenaline, but had no blocking effect on the arterial pressor responses induced by (−)−phenylephrine. The findings suggest that capsinolol possesses β‐adrenoceptor blocking activity, but it has no α‐adrenoceptor blocking activity. 4 In guinea‐pig isolated tissues, capsinolol (10−8 to 10−6 m) antagonized (−)−isoprenaline‐induced positive chronotropic and inotropic effects of the atria and tracheal relaxation responses in a concentration‐dependent manner. The parallel shift to the right of the concentration‐response curve of (−)−isoprenaline suggests capsinolol is a β‐adrenoceptor competitive antagonist. 5 Capsinolol (10−5 to 10−4 m) exhibited a positive cardiotonic effect that was not inhibited by (±)‐propranolol and reserpine, but was inhibited by capsazepine (10−6 m) and CGRP8–37 (10−6 m). This effect was independent of intrinsic sympathomimetic effects. 6 An immunoassay of released CGRP from guinea‐pig isolated perfused heart indicated that capsinolol increases the release of CGRP and thus produces positive cardiotonic effects. 7 In conclusion, capsinolol is a non‐selective β‐adrenoceptor antagonist with capsaicin‐like cardiotonic properties unrelated to traditional intrinsic sympathomimetic effects. It is suggested that capsinolol causes CGRP release from cardiac sensory neurones via a non‐adrenergic mechanism and then activates CGRP receptors on cardiac muscle.

Inhibition of Proinflammatory Tumor Necrosis Factor-α-Induced Inducible Nitric-Oxide Synthase by Xanthine-Based 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-Nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) in Rat Trachea: The Involvement of Soluble Guanylate Cyclase and Protein Kinase G

In the study of anti-proinflammation by 7-[2-[4-(2-chlorobenzene)piperazinyl] ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3), exposure of rat tracheal smooth muscle cells (TSMCs) to tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase α1 (sGCα1), soluble guanylate cyclase β1 (sGCβ1), protein kinase G (PKG), and the release of cGMP in TSMCs. The cell-permeable cGMP analog 8-Br-cGMP, xanthine-based KMUP-1 and KMUP-3, and the phosphodiesterase 5 inhibitor zaprinast all inhibited TNF-α-induced increases of iNOS expression and NO levels and reversed TNF-α-induced decreases of sGCα1, sGCβ1, and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-α also increased protein kinase A (PKA) expression and cAMP levels, cyclooxygenase-2 (COX-2) expression, and activated productions of prostaglandin (PG) E2 and 6-keto-PGF1α (stable PGI2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-α-induced expression of COX-2 and activated productions PGE2 and PGI2. However, KMUP-1 and KMUP-3 did not affect COX-2 activities and did not further enhance cAMP levels in the presence of TNF-α. It is suggested that TNF-α-induced increases of PKA expression and cAMP levels are mediated by releasing PGE2 and PGI2, the activation products of COX-2. In conclusion, xanthine-based KMUP-1 and KMUP-3 inhibit TNF-α-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.

Antioxidant eugenosedin-A protects against lipopolysaccharide-induced hypotension, hyperglycaemia and cytokine immunoreactivity in rats and mice

Eugenosedin‐A has been demonstrated to possess α/β‐adrenoceptor and serotonergic receptor blocking activities. We have investigated by what mechanisms eugenosedin‐A prevents lipopolysaccharide (LPS)‐induced hypotension, vascular hyporeactivity, hyperglycaemia, oxidative injury or inflammatory cytokines formation in rats. Intravenous administration of eugenosedin‐A, trazodone, yohimbine (1 mg kg−1), aminoguanidine or ascorbic acid (15 mg kg−1) normalized LPS (10 mg kg−1)‐induced hypotension. Pretreatment with eugenosedin‐A or the other agents 30 min before LPS injection reduced aortic hyporeactivity. LPS‐induced increases in plasma interleukin‐1β (IL‐β), IL‐6, interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and blood glucose levels were significantly inhibited by eugenosedin‐A (1 mg kg−1, i.v.). The same dose of trazodone, a chloropiperazinylbenzene‐type antidepressant, and yohimbine, an α2‐adrenoceptor antagonist, reduced IL‐1β and TNF‐α, but it could not inhibit hyperglycaemia. Aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, and ascorbic acid, an antioxidant, decreased IL‐1β, TNF‐α contents and hyperglycaemia. Eugenosedin‐A and the other agents inhibited Fe2+‐ascorbic acid‐induced peroxidation in rat cortex, indicating that those agents had antioxidant effects, with the exception of aminoguanidine. In free radical scavenged experiments, eugenosedin‐A and ascorbic acid eliminated peroxyl radicals. All test agents inhibited the LPS‐induced increase of malondialdehyde (MDA) content in rat brain homogenates. When mice were administered an intraperitoneal injection of LPS alone, mortality occurred from 4 to 16 h, after which time all were dead. However, eugenosedin‐A significantly prolonged the survival time after LPS injection, suggesting that eugenosedin‐A protected against LPS‐induced cardiovascular dysfunction, hyperglycaemia, tissue injury and inflammatory cytokine production. This was attributable mainly to the antioxidant and peroxyl radical scavenged effects of eugenosedin‐A, and which may be, at least in part, due to its blockade on α/β‐adrenergic and serotonergic receptors.

Baicalin, a flavonoid from Scutellaria baicalensis Georgi, activates large-conductance Ca2+-activated K+ channels via cyclic nucleotide-dependent protein kinases in mesenteric artery.

Baicalin isolated from Scutellaria baicalensis is a traditional Chinese herbal medicine used for cardiovascular dysfunction. The ionic mechanism of the vasorelaxant effects of baicalin remains unclear. We investigated whether baicalin relaxes mesenteric arteries (MAs) via large-conductance Ca2+-activated K+ (BK(Ca)) channel activation and voltage-dependent Ca2+ channel (VDCC) inhibition. The contractility of MA was determined by dual wire myograph. BK(Ca) channels and VDCCs were measured using whole-cell recordings in single myocytes, enzymatically dispersed from rat MAs. Baicalin (10-100 microM) attenuated 80 mM KCl-contracted MA in a concentration-related manner. L-NAME (30 microM) and indomethacin (10 microM) little affected baicalin (100 microM)-induced vasorelaxations. Contractions induced by iberiotoxin (IbTX, 0.1 microM), Bay K8644 (0.1 microM) or PMA (10 microM) were abolished by baicalin 100 microM. In MA myocytes, baicalin (0.3-30 microM) enhanced BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) currents were abolished by IbTX (0.1 microM). Baicalin-mediated (30 microM) BK(Ca) current activation was significantly attenuated by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Perfusate with PMA (0.1 microM) abolished baicalin-enhanced BK(Ca) currents. Additionally, baicalin (0.3-30 microM) reduced the amplitude of VDCC currents in a concentration-dependent manner and abolished VDCC activator Bay K8644-enhanced (0.1 microM) currents. Baicalin produced MA relaxation by activating BK(Ca) and inhibiting VDCC channels by endothelium-independent mechanisms and by stimulating the cGMP/PKG and cAMP/PKA pathways.