Guinea-pig Ileum Longitudinal Muscle Research Articles

Hexamethonium-induced augmentation of the electrical twitch response in the guinea-pig ileum longitudinal muscle–myenteric plexus strip

Longitudinal muscle–myenteric plexus strips of the guinea-pig ileum were used to investigate the nature of the hexamethonium-induced augmentation of the twitch response. All preparations were set up in Tyrode solution and intermittent longitudinal twitch contractions were evoked by single pulse electrical field stimulation. Hexamethonium, a blocker of nicotinic ganglionic transmission, at 300μmol/l and 1mmol/l augmented the twitch contractions by 21% and 35%, respectively. First we tested for a possible nicotinic drive onto an inhibitory neuronal component to the longitudinal smooth muscle cells. However, guanethidine (5μmol/l), naloxone (1μmol/l), or l-NAME (300μmol/l) were without effect on the hexamethonium-induced augmentation. The P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS), 25–100μmol/l, without altering the control twitch responses, dose-dependently reduced the hexamethonium-induced augmentation; at 100μmol/l a statistically significantly inhibition was observed. Based on these functional experiments we found no evidence that blocking nicotinic transmission removed a tonic adrenergic, opioidergic or nitrergic inhibitory input to the longitudinal muscle. However, we provide evidence for a hexamethonium-induced augmentation of the P2 purinergic input to cholinergic motoneurons of the guinea-pig ileum longitudinal muscle. The P2-nicotinic receptor interaction presents a novel modulatory mechanism to cholinergic myenteric motor neurons.

Searching for new antiarrhythmic agents: Evaluation of meta-hydroxymexiletine enantiomers

Mexiletine is a very well-known class IB antiarrhythmic drug, whose enantiomers differ in both pharmacodynamic and pharmacokinetic properties, the (R)-isomer being the eutomer on experimental arrhythmias and in binding studies on cardiac voltage-gated sodium channels. meta-Hydroxymexiletine (MHM) is a minor metabolite of mexiletine, which has demonstrated to be more potent than the parent compound. Herein we report the synthesis and biological evaluation of MHM enantiomers for their potential antiarrhythmic activity. The same stereoselectivity pattern observed for mexiletine was found for MHM: the (R)-enantiomer of MHM was the eutomer on ac-arrhythmia also showing a negative inotropism higher than the one displayed by mexiletine and, at the same time, a decreased vasorelaxant activity on guinea-pig left atrium and guinea-pig ileum longitudinal smooth muscle.

The influence of peptide impurity profiles on functional tissue–organ bath response: The 11-mer peptide INSL6[151–161] case

Bioactive peptides have great pharmaceutical potential as nutraceuticals, diagnostics, and therapeutic drugs in several clinical areas. Thus, the search for novel lead peptides with a biological function has attracted renewed interest. Crude peptide material (i.e., ∼70% purity) of INSL6[151–161] (NH2-FRSLFWGNHSQ-COOH) was found to trigger a contractile response in guinea pig ileum longitudinal smooth muscle preparations using tissue–organ baths. However, the purified peptide (i.e., ⩾95% purity) had no effect on this model. Further investigation with crude materials from other suppliers, with purities ranging between 50% and 80%, indicated that the crude products gave a false-positive functional tissue–organ bath conclusion. These observations question the functionality conclusions when using crude-purity peptide materials; during the initial research or discovery phase, peptide quality is generally neglected, possibly leading to misinterpretation of biological results due to by-products from peptide synthesis and, thus, wrong fail/pass decisions. Therefore, we strongly recommend appropriate quality control testing before using any peptides for initial biomedical research or discovery purposes.

Modification of electrical field stimulation-induced contractions in the guinea-pig ileum by metoclopramide and ICS 205-930 depends on the integrity of the mucosa.

Contractions induced by electrical field stimulation of guinea-pig ileum longitudinal muscle strips were enhanced by metoclopramide and ICS 205-930 at concentrations similar to those required to antagonize at 5-hydroxytryptamine 'M' receptors. The enhancement of contraction was observed in intact ileum strips but was not recorded in the longitudinal muscle myenteric plexus preparation or from the ileum with the mucosal layer removed. It is concluded that an intact mucosal layer is required for metoclopramide and ICS 205-930 to enhance electrical field stimulation-induced contractions of the guinea-pig ileum.

Estimation of endogenous adenosine activity at adenosine receptors in guinea‐pig ileum using a new pharmacological method

Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p-sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10(-6) m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose-response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems.

Alterations in β‐arrestin expression in guinea‐pig ileum and colon following morphine tolerance

Repeated administration of morphine results in the development of tolerance to its antinociceptive effects. We have previously shown that tolerance also occurs to morphine‐induced inhibition of neurogenic contractions in the mouse ileum but not in the colon. The aim of the present study was to examine the development of morphine tolerance in the guinea‐pig ileum and colon and determine the role of G‐protein coupled receptor kinase 2 (GRK2) and β‐arrestin. Isolated guinea‐pig ileum longitudinal muscle‐myenteric plexus (LMMP) and colonic muscle strips were set‐up in the organ bath. Field stimulated contractions were dose‐dependently inhibited by morphine (10nM −10 μM) in both ileum and colon. Incubation with 10 μM morphine for 1 hr induced tolerance to subsequent administration in the ileum but not in the colon. The expression and interaction of GRK2 and β‐arrestin with the μ opioid receptor (MOR) were determined by Western blots. Immunoprecipitation of MOR with GRK2 and β‐arrestin was enhanced after morphine treatment in both tissues. The expression ratio of GRK2/GAPDH was similar, however, that of β‐arrestin/GAPDH was significantly less in the ileum (50 ± 20%) compared to the colon. Morphine treatment (10 μM for 1hr) did not alter the expression levels of GRK2 in either tissues, but markedly reduced β‐arrestin expression by approx. 30% in the ileum. In stably MOR transfected CHO cells, overexpression of GRK2 increased MOR internalization. These studies suggest that an enhanced expression of β‐arrestin in the colon may preclude development of morphine tolerance. Supported by NIH DA024009.

Tachykinin Receptor Assays

Described in this unit are methods for obtaining, preparing, and testing smooth muscle preparations bearing tachykinin receptors to study the agonist or antagonist properties of test compounds. Concentration-response curves to agonists are constructed to measure their ability to produce smooth muscle contractions and thus evaluate the potency and efficacy of the agonists. Antagonists are tested for their ability to shift the agonist concentration-response curve and to calculate their potency. Two different protocols are described for each of the three tachykinin receptors (NK(1), NK(2), and NK(3)). The NK(1) receptor assays use guinea pig ileum longitudinal muscle myenteric plexus (GPI) and rat urinary bladder (RUB), the NK(2) receptor assays use isolated endothelium-deprived rabbit pulmonary artery (RPA) and hamster trachea (HT), and the NK(3) receptor assays use GPI and rat portal vein (RPV).

Synthesis, Evaluation of Pharmacological Activities and Quantitative Structure–Activity Relationship Studies of a Novel Group of bis(4‐Nitroaryl‐1,4‐dihyropyridine)

Voltage-dependent calcium channels are crucial targets for a wide range of clinically active pharmacological agents. From these agents, 1,4-dihydropyridines constitute a group of small organic compounds are based on a core pyridine structure which can both block and enhance calcium currents. They are considered specific for L-Type calcium channels; however, other channel types, and in particular certain T-Type channels, may show sensitivity to dihydropyridine compounds. In this study, we synthesized a novel group of bis-1,4-dihydropyridines using the procedure reported by Dagnino that involved the condensation of n-alkyl diacetoacetate (n = 2-7) with methyl-3-aminocrotonate and nitrophenylaldehyde. The synthesis was run under two conditions: (i) reflux and (ii) microwave. Calcium channels antagonist activity were determined in vitro using guinea-pig ileum longitudinal smooth muscle assay. Synthesis of these compounds was confirmed with 1H-NMR, IR and mass spectrometry. Then IC(50) of them are calculated and compared with Nifedipine. Finally, the result of this pharmacological assay was used in quantitative structure-activity relationship studies utilizing multiple linear regression analysis. Most of these compounds are less active compared with Nifedipine. Decrease in activity is the result of increase in steric hindrance. The quantitative structure-activity relationship study indicates that the activity is related to the electrostatic and topological parameters and the distance between two C5-esteric groups of 1,4-dihydropyridine rings.

Synthesis, QSAR and Calcium Channel Modulator Activity of New Hexahydroquinoline Derivatives Containing Nitroimidazole

The discovery that 1,4-dihydropyridine class of calcium channel antagonists inhibit Ca2+ influx represented a major therapeutic advance in the treatment of cardiovascular disease. In contrast to the effects of known calcium channel blockers of the Nifedipine-type, the so-called calcium channel agonists, such as Bay K8644 and CGP 28392, increase calcium influx by binding at the same receptor regions. Our goal was to discover a dual cardioselective Ca2+-channel agonist/vascular selective smooth muscle Ca2+ channel antagonist third-generation 1,4-dihydropyridine drug which would have a suitable therapeutic profile for treating congestive heart failure (CHF) patients. A series of unsymmetrical alkyl, cycloalkyl and aryl ester analogues of 2-methyl-4-(1-methyl)-5-nitro-2-imidazolyl-5-oxo-1,4,5,6,7, 8-hexahydroquinolin-3-arboxylate were synthesized using modified Hantzsch reaction. All compounds show calcium antagonist activity on guinea-pig ileum longitudinal smooth muscle and some of them show agonist effect activity on guinea-pig auricle. Effect of structural parameters on the Ca2+ channel agonist/antagonist was evaluated by quantitative structure-activity relationship analysis. These compounds could be considered as a synthon for developing a suitable drug for treating CHF patients.

Synthesis, QSAR and Calcium Channel Antagonist Activity of New 1,4‐Dihydropyridine Derivatives Containing 1‐Methyl‐4,5‐dichloroimidazolyl Substituents

A group of dialkyl and diarylester analogues of nifedipine, in which the ortho-nitrophenyl group at position 4 was replaced by a 1-methyl-4,5-dichloroimidazolyl substituent, were synthesized and evaluated as calcium-channel antagonists using the high K(+)concentration of guinea-pig ileum longitudinal smooth muscle. The structure of all compounds was confirmed by IR,(1)H-NMR, and mass spectra. The calcium-channel antagonist activity of compounds 10a-f demonstrated that compound 10b was the most active and 10f the least active one. With unsymmetrical diesters 12a-k, the most active compound was the ethyl, phenethyl derivative. Structural parameters on the calcium-channel antagonist activity were evaluated by QSAR analysis and a linear correlation was found between the -log IC(50) values of these compounds and their constitutional and topological properties.

Synthesis and calcium channel antagonist activities of new derivatives of dialkyl 1,4-dihydro-2,6-dimethyl-4-(5-phenylisoxazol-3-yl)pyridine-3,5-dicarboxylates

The new analogues of nifedipine, in which 2-nitrophenyl group at position 4 is replaced by phenylisoxazolyl substituent, were synthesized. The symmetrical dialkyl 1,4-dihydro-2,6-dimethyl-4-(5-phenylisoxazol-3-yl)pyridine-3,5-dicarboxylates were prepared by classical Hantzsch condensation, and the asymmetrical analogues were synthesized using a procedure reported by Dagnino that involved the condensation of alkyl acetoacetate with alkyl 3-aminocrotonate and 5-phenylisoxazole-3-carboxaldehyde. The structure of all compounds was confirmed by IR, 1H NMR and Mass spectra. In vitro calcium channel antagonist activities were evaluated as calcium channel antagonists using the high K+ concentration of guinea-pig ileum longitudinal smooth muscle (GPILSM) assay. These compounds exhibited moderate calcium antagonist activity (IC50 = 10−7 to 10− 5 M range) relative to the reference drug nifedipine (IC50 = 1.10 ± 0.40 × 10−8 M).

MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B 2 receptor. In vitro pharmacology and molecular characterization

The pharmacological characterization of the novel nonpeptide antagonist for the B 2 receptor, namely MEN16132 (4-( S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2 H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B 2 receptor has been investigated by means of competition studies at [ 3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B 2 receptor (pK i 10.5), human lung fibroblasts (pK i 10.5), guinea pig airways (pK i 10.0), guinea pig ileum longitudinal smooth muscle (pK i 10.2), or guinea pig cultured colonic myocytes (pK i 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B 2 receptor was checked at the human bradykinin B 1 receptor (pK i < 5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pK B 10.3) and guinea pig (colonic myocytes: pK B 10.3) B 2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pK B 9.9) and guinea pig (ileum longitudinal smooth muscle: pK B 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B 2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B 2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B 2 receptor antagonist.

Evaluation of the effect of age on cannabinoid receptor functionality and expression in guinea-pig ileum longitudinal muscle–myenteric plexus preparations

Cannabinoid drugs exert a wide range of biological effects and are currently under study for their multiple potential therapeutic uses. Cannabinoids reduce gastrointestinal (GI) motility and this is mediated by the CB1 cannabinoid receptor (CB1R) present in the myenteric neurones. GI motility can also be affected by a variety of pathophysiological situations, including ageing. The purpose of this work was to study the influence of age on the functionality and expression of CB1R in the myenteric plexus. Ileal longitudinal muscle–myenteric plexus (LMMP) preparations from young, adult and old guinea-pigs were used in two sets of experiments: in vitro assessment of the inhibitory cannabinoid effect upon electrically stimulated contractions and immunohistochemical quantification of myenteric neurones expressing CB1R. LMMP preparations responded to the synthetic cannabinoid WIN 55,212-2, and the endogenous cannabinoid ligand anandamide in an age-independent manner. The total number of CB1R-immunoreactive (IR) myenteric neurones, which included at least part of the motor neurones to the longitudinal smooth muscle, decreased in proportion to the general neuronal population; however, the proportion of CB1R-IR neurones was preserved in old animals. These data may justify the preservation of the effectiveness of the cannabinoids in the isolated guinea-pig ileum. This age-related independency of CB1R expression and effect on GI motility could be of interest if cannabinoids are to be used therapeutically.

Design and synthesis of alkyl 7,7-dihalo-3-methyl-5-(nitrophenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates with calcium channel antagonist activity

A group of alkyl 7,7-dihalo-3-methyl-5-(2- or 3-nitrophenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates were prepared by reaction of dihalocarbenes (:CX 2, X = Br, Cl) with alkyl 2-methyl-4-(2- or 3-nitrophenyl)-1,4-dihydropyridine-3-carboxylates. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. The title compounds exhibited weaker CC antagonist activity (10 −5 to 10 −7 M range) than the reference drug nifedipine (1.4 × 10 −8 M). Structure–activity relationships showed that the position ( ortho or meta) of the nitro-substituent on the C-5 phenyl ring, the size (van der Waal’s radius for Br and Cl are 1.95 and 1.80 Å, respectively) and/or electronegativity (Cl > Br) of the C-7 geminal halogen atoms do not appear to have a significant effect on CC antagonist activity. In contrast, the effect of the alkyl ester substituent was more pronounced where compounds having a Me or Et alkyl ester group showed superior potency (IC 50 in the 10 −7 M range) relative to the reference drug nifedipine (IC 50 = 1.40 × 10 −8 M). Replacement of a 2-methyl-3-methoxycarbonylvinyl moiety present in nifedipine by a bioisosteric geminal-dihalocyclopropyl moiety provided a novel class of calcium channel antagonists that do not exhibit any inotropic effect on guinea pig atria.

PHARMACOLOGICAL STUDY FOR CHARACTERIZATION OF THE NON-ADRENERGIC, NON-CHOLINERGIC (NANC) NEUROTRANSMITTER IN THE BOVINE UTERUS

Pharmacological characteristics of electrical field stimulation (EFS)-induced mechani-cal responses in the bovine uterus indicate smooth muscle layer-specific variations in autonomic innervation (exclusive adrenergic innervation in the longitudinal muscle and non-adrenergic, non-cholinergic (NANC) innervation in the circular muscle).The aim of the present study was to characterize the neurotransmitter of NANC nerves in the bovine uterus using functional and biochemical approaches.EFS of uterine circular muscle caused atropine and guanethidine-resistant contraction, which was abolished by tetrodotoxin.The EFS-induced contraction was not decreased by pyrilamine, methysergide or indomethacin.In the presence of atropine and guanethidine, bioactive peptides (angiotensin II, bombesin, calcitonin-gene related peptide, enkephalins, galanin, neurokinin A, neurokinin B, substance P, neurotensin, vasoactive intestinal polypeptide), ATP and 5-hydroxytryptamine did not produce contraction of the myometrium.An acid extract of the bovine uterus (EBU) contracted both guinea-pig ileum longitudinal muscle (GPILM) and bovine uterus.Neither tetrodotoxin nor methysergide decreased the EBU-induced contraction.Atropine and pyrilamine inhibited the EBU-induced responses, but some of the contractions (5-15%) were resistant to both antagonists.On a Sephadex G50 column, smooth muscle contracting material was eluted in one peak.Active fractions were pooled and applied to a Sephadex G25 column to compare the elution volume of the active material with those of peptides of known molecular weights.The active material was eluted in one peak and the molecular weight was estimated to be 1200.The gel-filtrated fraction induced contraction of both GPILM and bovine uterus, and its contractile activity was abolished by a-chymotrypsin.In conclusion, uterotonic peptide was discovered in extract of the bovine uterus and was thought to be one of transmitter for NANC nerves.However, further studies are needed to clarify the structure of this unknown peptide and its physiological roles.

Design and synthesis of methyl 2-methyl-7,7-dihalo-5-phenyl-2-azabicyclo64.1.09hept-3-ene-4-carboxylates with calcium channel antagonist activity

Graphic A group of methyl 2-methyl-7,7-dihalo-5-(substituted-phenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates were prepared by reaction of dihalocarbenes (:CX 2 , X = Br, Cl) with methyl 1-methyl-4-(substituted-phenyl)-1,4-dihydropyridine-3-carboxylates. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. The title compounds exhibited weaker CC antagonist activity (10 −5 –10 −6 M range) than the reference drug nifedipine (1.4 × 10 −8 M). Structure–activity relationship studies showed that the position of a nitro substituent on the C-5 phenyl ring where the relative potency order was ortho > meta > para , and the size and/or electronegativity of the C-7 geminal-dihalo substituents (Br > Cl), were determinants of calcium channel antagonist activity. This class of compounds did not exhibit any inotropic effect on guinea pig left atria. A dihalocyclopropyl moiety is a potential bioisostere for the 2-methyl-3-methoxycarbonylvinyl moiety present in the calcium channel antagonist nifedipine.

Design and synthesis of methyl 2-methyl-7,7-dihalo-5-phenyl-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates with calcium channel antagonist activity

A group of methyl 2-methyl-7,7-dihalo-5-(substituted-phenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates were prepared by reaction of dihalocarbenes (:CX 2, X = Br, Cl) with methyl 1-methyl-4-(substituted-phenyl)-1,4-dihydropyridine-3-carboxylates. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. The title compounds exhibited weaker CC antagonist activity (10 −5–10 −6 M range) than the reference drug nifedipine (1.4 × 10 −8 M). Structure–activity relationship studies showed that the position of a nitro substituent on the C-5 phenyl ring where the relative potency order was ortho > meta > para, and the size and/or electronegativity of the C-7 geminal-dihalo substituents (Br > Cl), were determinants of calcium channel antagonist activity. This class of compounds did not exhibit any inotropic effect on guinea pig left atria. A dihalocyclopropyl moiety is a potential bioisostere for the 2-methyl-3-methoxycarbonylvinyl moiety present in the calcium channel antagonist nifedipine.

Syntheses, calcium channel agonist-antagonist modulation activities, and nitric oxide release studies of nitrooxyalkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate racemates, enantiomers, and diastereomers.

A novel group of hybrid calcium channel (CC) modulators was prepared where the isopropyl ester moiety of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate (PN 202-791) was replaced by a variety of nitric oxide (*NO) donor nitrooxyalkyl ester substituents. Enantiomers, or diastereomers, having the (R)-configuration at the C-4 position of the 1,4-dihydropyridine ring (1,4-DHP) exhibited more potent in vitro CC antagonist activity on guinea pig ileum longitudinal smooth muscle (GPILSM) than compounds having the (4S)-configuration. None of the nitrooxyalkyl compounds exhibited a contraindicated CC agonist effect on GPILSM that would cause smooth muscle contraction. Structure-activity studies showed the enantiomers having the (S)-configuration at the C-4 position of the 1,4-DHP ring or diastereomers having the a (4S)-configuration at the C-4 position of the 1,4-DHP ring in conjunction with a (1R-)-1-methyl-2-nitrooxyethyl ester substituent exhibited the most potent cardiac CC agonist (positive inotropic) activity on guinea pig left atrium (GPLA). This class of compounds releases *NO in vitro that is enhanced by the presence of a thiol such as N-acetylcysteamine. The novel *NO donor (-)-(S,R)-1-methyl-2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,1,3-benzoxadiazol-4-yl)pyridine-5-carboxylate [(-)-(S,R)-38], which acts as a dual cardioselective calcium channel agonist (GPLA)/smooth muscle selective calciumchannel antagonist (GPILSM), is a useful lead compound for drug discovery targeted to the treatment of congestive heart failure, and it provides a useful research probe to study the structure-function relationship of calcium channels.

Synthesis and Calcium Channel Antagonist Activities of 3‐Nitrooxyalkyl, 5‐Alkyl 1,4‐Dihydro‐2,6‐dimethyl‐4‐ (1‐methyl‐5‐nitro‐2‐imidazolyl)‐3,5‐pyridinedicarboxylates.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Syntheses, calcium channel agonist‐antagonist modulation effects, and nitric oxide release studies of [3‐(Benzenesulfonyl)furoxan‐4‐yloxy]alkyl 1,4‐Dihydro‐2,6‐dimethyl‐5‐nitro‐4‐(2‐trifluoromethylphenyl, benzofurazan‐4‐yl, 2‐, 3‐, or 4‐pyridyl)‐3‐pyridinecarboxylates

AbstractA group of racemic 1,4‐dihydro‐2,6‐dimethyl‐5‐nitro‐3‐pyridinecarboxylates possessing either a C‐4 2‐trifluoromethylphenyl (22–24), benzofurazan‐4‐yl (42–44), 2‐pyridyl (45–47), 3‐pyridyl (48–50), or 4‐pyridyl (51–53), substituent in conjunction with a nitric oxide donor C‐3 ester [3‐(benzenesulfonyl)furoxan‐4‐yloxy]alkyl substituent were synthesized using modified Hantzsch reactions. Compounds 45–53 having a C‐4 2‐, 3‐, or 4‐pyridyl substituent exhibited more potent in vitro calcium channel antagonist activity (IC50's in the 0.46 to 5.23 μM range) on guinea pig ileum longitudinal smooth muscle (GPILSM) than related analogs having a C‐4 2‐trilfuoromethylphenyl (22–24) or benzofurazan‐4‐yl (42, 44) substituent (IC50 ≥ 29.91 mM). The point of attachment of the pyridyl ring (2‐, 3‐, or 4‐), and the length of the C‐3 ester alkyl spacer [‐CH2(CH2)n, n=1–3], were not determinants of smooth muscle calcium channel antagonist activity. Replacement of the C‐3 ester methyl substituent of Bay K 8644, or the ester isopropyl substituent of the 4‐(pyridyl) isomers 4a‐c by a [3‐(benzensulfonyl)furoxan‐4‐yloxy]alkyl moiety retained the desired calcium channel agonist (positive inotropic) effect on guinea pig left atrium (GPLA). Compounds having C‐4 3‐pyridyl (48–50), or 4‐pyridyl (51–53), substituents were the most potent cardiac positive inotropes (EC50's in the 3.02 to 19.74 μM range) relative to the reference drug Bay K 8644 IC50=0.77 μM). The % nitric oxide released in vitro in the presence of L‐cysteine for this group of compounds was higher (36–74% range) than for the reference drug glycerol trinitrate (20%). A quantitative structure‐activity analysis showed an inverse correlation between a molecular weight (MW) descriptor and % nitric oxide released. Model hybrid (calcium channel modulation, nitric oxide donor) compounds that show dual cardioselective agonist (positive inotropic)/smooth muscle selective antagonist activities constitute a novel type of 1,4‐dihydropyridine calcium channel modulator that provides a potential drug design concept targeted toward the treatment of congestive heart failure, and is a useful probe to study the structure‐function relationship of calcium channels. Drug. Dev. Res. 56:1–16, 2002. © 2002 Wiley‐Liss, Inc.