Pig Ileum Longitudinal Smooth Muscle Research Articles

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.

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.

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.

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.

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

A group of racemic 3-[(2-nitrooxyethyl), (3-nitrooxypropyl), (4-nitrooxybutyl) or (1,3-dinitrooxy-2-propyl)], 5-methyl (ethyl or propyl) 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicarboxylates ( 18– 29) were synthesized using modified Hantzsch reaction that involved the condensation of 2-nitrooxyethyl ( 8), 3-nitrooxypropyl ( 9), 4-nitrooxybutyl ( 10) or 1,3-dinitrooxy-2-propyl ( 13) acetoacetate with methyl ( 14), ethyl ( 15) or isopropyl ( 16) 3-aminocrotonate and 1-methyl-5-nitroimidazole-2-carboxaldehyde ( 17). In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. Compounds 18– 29 exhibited superior, or equipotent, calcium antagonist activity (IC 50=10 −11–10 −13 M range) relative to the reference drug nifedipine (IC 50=1.07±0.12×10 −11 M), which could serve as potential probes to investigate the in vivo release of nitric oxide which induces vascular muscle relaxation.

Syntheses, calcium channel agonist-antagonist modulation activities, nitric oxide release, and voltage-clamp studies of 2-nitrooxyethyl 1,4-dihydro- 2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate enantiomers.

The novel (-)-(S)-2 and (+)-(R)-3 enantiomers of 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate were synthesized for evaluation as calcium channel modulators. Determination of their in vitro calcium-channel-modulating activities using guinea pig left atria (GPLA) and ileum longitudinal smooth muscle (GPILSM) showed that the (-)-(S)-2 enantiomer acted as a dual cardioselective calcium channel agonist (GPLA)/smooth muscle selective calcium channel antagonist (GPILSM). In contrast, the (+)-(R)-3 enantiomer exhibited calcium channel antagonist activity on both GPLA and GPILSM. The 2-nitrooxyethyl racemate is a nitric oxide (.NO) donor that released 2.7% .NO, relative to the reference drug glyceryl trinitrate (5.3% .NO release/ONO(2) moiety), in the presence of N-acetylcysteamine. Whole-cell voltage-clamp studies using isolated guinea pig ventricular myocytes indicated that both enantiomers inhibit calcium current but that the (-)-(S)-2 enantiomer is a weaker antagonist than the (+)-(R)-3 enantiomer. These results indicate that replacement of the methyl ester substituent of (-)-(S)-methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate [(-)-(S)-1] by the 2-nitrooxyethyl ester .NO donor substituent present in (-)-(S)-2 provides a useful drug design concept to abolish the contraindicated calcium channel agonist effect of (-)-(S)-1 on vascular smooth muscle. The novel (-)-(S)-2 enantiomer is a useful lead compound for drug discovery targeted toward the treatment of congestive heart failure, and it provides a useful probe to study the structure-function relationships of calcium channels.

Synthesis and calcium channel modulating effects of modified Hantzsch nitrooxyalkyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(pyridinyl or 2‐trifluoromethylphenyl)‐5‐pyridinecarboxylates

AbstractA group of racemic nitrooxyalkyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(pyridinyl or 2‐trifluoromethylphenyl)‐5‐pyridinecarboxylates 8a–o were synthesized using modified Hantzsch reactions. In vitro calcium channel antagonist activities, determined using a guinea pig ileum longitudinal smooth muscle (GPILSM) assay, showed that compounds 8a–o exhibited weaker calcium antagonist activity (10–5 to 10–7 M range) than the reference drug nifedipine (IC50 = 1.43 × 10–8 M). Compounds 8 possessing a C‐4 R1 = 2‐pyridyl substituent were always more potent than the approximately equiactive analogs having an R1 = 3‐pyridyl, 4‐pyridyl or 2‐CF3‐C6H4‐substituent, within each subgroup of nitrooxyalkyl compounds [R2 = – (CH2)nONO2 (n = 2, 3, 4) or –CH(CH2ONO2)2]. Although the length of the R2 = –(CH2)nONO2 substituent (n = 2–4) was not a determinant of smooth muscle calcium antagonist activity when the C‐4 R1‐substituent was 2‐pyridyl, when R1 was a 3‐pyridyl, 4‐pyridyl, or 2‐CF3‐C6H4‐substituent, the relative potency order with respect to the R2 = –(CH2)nONO2 substituent was n = 3 and 4 &gt; n = 2. Replacement of the isopropyl substituent of the ester moiety of the calcium antagonist (±)‐2‐pyridyl 3a by a –(CH2)nONO2 (n = 2–4) moiety increased calcium antagonist activity on GPILSM by 8‐fold. In contrast, replacement of the isopropyl substituent of the ester moiety of the calcium agonists (±)‐3‐pyridyl 3b, (±)‐4‐pyridyl 3c or the methyl substituent of the ester moiety of Bay K8644 by a R2 nitrooxyalkyl substituent resulted in abolition of their calcium agonist effects on GPILSM that is replaced by a smooth muscle calcium antagonist effect. These calcium antagonist data support the concept that incorporation of a nitrooxyalkyl ester substituent constitutes a valuable drug design strategy to enhance Hantzsch 1,4‐dihydropyridine calcium antagonist and/or abolish calcium agonist effects on smooth muscle. Replacement of the isopropyl (8b–c), or the methyl (8d) group by a –CH2CH2ONO2 moiety resulted in retention of the cardiac positive inotropic effect where the relative potency order with respect to the C‐4 substituent was 2‐CF3‐C6H6‐ (8d) &gt; 3‐pyridyl (8b) ≈ 4‐pyridyl (8c). Model hybrid (calcium channel modulation, ·NO release) compounds, that exhibit dual cardioselective agonist / smooth muscle selective antagonist activities, represent a novel type of 1,4‐dihydropyridine CC modulator that offers a potential approach to drug discovery targeted toward the treatment of congestive heart failure and for use as probes to study the structure–function relationship of calcium channels. Drug Dev. Res. 51:225–232, 2000. © 2001 Wiley‐Liss, Inc.

Synthesis and calcium channel modulation effects of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-phenylpyridine-5-carboxylates possessingortho-,meta-, andpara-CH2S(O)nMe and -S(O)nMe (n = 0-2) phenyl substituents

A group of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-phenylpyridine-5-carboxylates (13–15) possessing ortho-, meta-, and para-CH2S(O)nMe and –S(O)nMe (n = 0–2) phenyl substituents were synthesized using a modified Hantzsch reaction. Calcium channel (CC) modulating activities were determined using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) in vitro assays. This class of –CH2S(O)nMe and –S(O)nMe (n = 0–2) compounds (13–15a–f) exhibited weaker CC antagonist activity on GPILSM (IC50 = > 1.1 × 10–5 to 4.1 × 10–6 M range) than the reference drug nifedipine (IC50 = 1.4 × 10–8 M). The oxidation state of the sulfur atom was a determinant of smooth muscle CC antagonist activity where the relative activity profile was generally thio (13, -CH2SMe, -SMe) and sulfonyl (15, -CH2SO2Me, -SO2Me) > sulfinyl (14, -CH2SOMe, -SOMe). The point of attachment of the phenyl substituent was a determinant of activity for the –CH2SMe (13a–c), -CH2SOMe (14a–c) and SOMe (14d–f) isomers where the relative potency order was meta and para > ortho. Compounds in this group (13–15), unlike Bay K 8644 (EC50 = 2.3 × 10–7 M on GPILSM), did not exhibit an agonist effect on GPILSM. The meta-CH2SMe (13b), ortho-CH2SMe (13c), meta-SMe (13e), and ortho-CH2SO2Me (15c) C-4 phenyl derivatives exhibited respectable in vitro cardiac positive inotropic activities (EC50 = 1.00 × 10–6 to 7.57 × 10–6 M range) relative to the reference drug Bay K 8644 (EC50 = 7.70 × 10–7 M) in the GPLA assay. In contrast to Bay K 8644, which acts as an undesirable calcium channel agonist on smooth muscle (GPILSM), compounds 13b (IC50 = 4.11 × 10–6 M), 13c (IC50 = 2.29 × 10–5 M), 13e (IC50 = > 1.20 × 10–5 M) and 15c (IC50 = 6.22 × 10–6 M) exhibited a desirable simultaneous calcium channel antagonist effect on smooth muscle at a similar (13b, 15c), or lower (13c, 13e), concentration relative to its cardiac agonist EC50 value. Model compounds such as 13b, 13c, 13e, and 15c, that exhibit dual cardioselective agonist / smooth muscle selective antagonist activities, represent a novel type of 1,4-dihydropyridine CC modulators that offer a potential approach to drug discovery targeted toward the treatment of congestive heart failure and for use as probes to study the structure–function relationship of calcium channels. Drug Dev. Res. 51:177–186, 2000. © 2001 Wiley-Liss, Inc.

A new class of pseudopeptide antagonists of the kinin B1 receptor containing alkyl spacers.

Four previously reported kinin receptor peptide antagonists, including the B1 receptor-selective peptides desArg10-HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg10-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-Arg-Arg-NH-(CH2)10-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA2 = 7.1) as a kinin B1 receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B1 receptor (rat ileum) and antagonist activity at the kinin B2 receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg10-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B1 receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on Calpha-tetrasubstituted alpha-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc).

Synthesis, rotamer orientation, and calcium channel modulation activities of alkyl and 2-phenethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(3- or 6-substituted-2-pyridyl)-5-pyridinecarboxylates.

A group of racemic alkyl and 2-phenethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(3- or 6-substituted-2-pyridyl)-5-pyridinecarboxylates (13a-q) was prepared using a modified Hantzsch reaction that involved the condensation of a 3- or 6-substituted-2-pyridinecarboxaldehyde (7a-j) with an alkyl or 2-phenethyl 3-aminocrotonate (11a-d) and nitroacetone (12). Nuclear Overhauser (NOE) studies indicated there is a significant rotamer fraction in solution where the pyridyl nitrogen is oriented above the 1,4-dihydropyridine ring, irrespective of whether a substituent is located at the 3- or 6-position. A potential H-bonding interaction between the pyridyl nitrogen free electron pair and the suitably positioned 1,4-dihydropyridine NH moiety may stablize this rotamer orientation. In vitro calcium channel antagonist and agonist activities were determined using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) assays, respectively. Compounds having an i-Pr ester substituent acted as dual cardioselective calcium channel agonists (GPLA)/smooth muscle-selective calcium channel antagonists (GPILSM), except for the C-4 3-nitro-2-pyridyl compound which exhibited an antagonist effect on both GPLA and GPILSM. In contrast, the compounds with a phenethyl ester group, which exhibited antagonist activity (IC50 = 10(-5)-10(-7) M range) on GPILSM, were devoid of cardiac agonist activity on GPLA. Structure-activity relationships showing the effect of a substituent (Me, CF3, Cl, NO2, Ph) at the 3- or 6-position of a C-4 2-pyridyl moiety and a variety of ester substituents (Me, Et, i-Pr, PhCH2CH2-) upon calcium channel modulation are described. Compounds possessing a 3- or 6-substituted-2-pyridyl moiety, in conjuction with an i-Pr ester substituent, are novel 1,4-dihydropyridine calcium channel modulators that offer a new drug design approach directed to the treatment of congestive heart failure and may also be useful as probes to study the structure-function relationships of calcium channels.

Synthesis and Calcium Channel-Modulating Effects of Alkyl (or Cycloalkyl) 1,4-Dihydro-2,6-dimethyl-3-nitro-4-pyridyl-5-pyridinecarboxylate Racemates and Enantiomers

A group of racemic alkyl (or cycloalkyl) 1,4-dihydro-2,6- dimethyl-3-nitro-4-(2-, 3-, or 4-pyridyl)-5-pyridinecarboxylate isomers (6-14) were prepared using a modified Hantzsch reaction that involved the condensation of nitroacetone with an alkyl (or cycloalkyl) 3-aminocrotonate and 2-, 3-, or 4-pyridinecarboxaldehyde. Determination of their in vitro calcium channel-modulating activities using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) assays showed that the 2-pyridyl isomers acted as dual cardioselective calcium channel agonists (GPLA)/smooth muscle selective calcium channel antagonists (GPILSM). In contrast, the 3-pyridyl and 4-pyridyl isomers acted as calcium channel agonists on both GPLA and GPILSM. In the C-4 2-pyridyl group of compounds, the size of the C-5 alkyl (or cycloalkyl) ester substituent was a determinant of GPILSM antagonist activity where the relative activity profile was cyclopentyl and cyclohexyl > t-Bu, i-Bu, and Et > MeOCH2CH2 > Me. The point of attachment of the C-4 pyridyl substituent was a determinant of GPLA agonist activity where the potency order was generally 4- and 3-pyridyl > 2-pyridyl. (+)-Cyclohexyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-pyridyl)-5- pyridinecarboxylate [(+)-14a] was a less potent calcium antagonist (IC50 = 5.27 x 10(-6) M) than the (-)-enantiomer (IC50 = 7.48 x 10(-8) M) on GPILSM. In the GPLA assay, (+)-14a exhibited a much more potent agonist effect (EC50 = 8.45 x 10(-6) M) relative to the marginal agonist effect produced by (-)-14a. The C-4 2-pyridyl compounds (enantiomers) constitute a novel type of 1,4-dihydropyridine calcium channel modulator that could provide a new drug design concept directed toward the treatment of congestive heart failure, and for use as probes to study the structure-function relationships of calcium channels.

Tachykinin-induced contractions of the guinea pig ileum longitudinal smooth muscle: tonic and phasic muscular activities

In vitro tachykinin-induced contractions of guinea pig ileum longitudinal smooth muscle were investigated under isometric conditions by using selective agonists ([Sar9,Met(O2)11]substance P, [Nle10]neurokinin A-(4-10), senktide) and antagonists (SR 140333, SR 48968, SR 142801), respectively, for the tachykinin NK1, NK2, and NK3 receptors. [Sar9,Met(O2)11]Substance P (10 nM) induced a tonic contraction with superimposed phasic contractions. Both tonic and phasic muscular activities were completely abolished by SR 140333 (10 nM) and were not modified by SR 142801 (10 nM). SR 48968 (10 nM) and atropine (0.001 mM) did not modify the tonic muscular activity but inhibited the phasic muscular activity. [Nle10]Neurokinin A-(4-10) (10 nM) only caused a phasic contractile response that was inhibited by SR 48968. Atropine, SR 140333, and SR 142801 were without effect. Senktide (1 nM) induced combined tonic and phasic contractile responses. SR 142801 blocked the phasic and tonic muscular activities, whereas SR 48968 and SR 140333 were inactive. After addition of atropine, only tonic contractile response was abolished. These results showed fundamental differences in isometric tonic and phasic contractile responses of guinea pig ileum longitudinal smooth muscle to tachykinins.

Tachykinin-induced contractions of the guinea pig ileum longitudinal smooth muscle: tonic and phasic muscular activities

Tachykinin-induced contractions of the guinea pig ileum longitudinal smooth muscle: tonic and phasic muscular activities

Syntheses, calcium channel antagonist and anticonvulsant activities of substituted 1,4-dihydro-3,5-pyridinedicarboxylates containing various 3-alkyl ester substituents.

A group of 3-alkyl 5-isopropyl 4-aryl-1,4-dihydro-2,6-dimethyl- 3,5-pyridinedicarboxylates 10-20 containing an amine, quaternary ammonium, aryl (heteroaryl)alkenyl, 4-(4-fluorophenyl)- piperazin-1-yl or methoxy moiety in the C-3 alkyl ester R-substituent in combination with a C-4 phenyl ring bearing a 2,3-Cl2, 3-NO2, 3-NMe2, 4-NMe2 or 3,4,5-(OMe)3 X-substituent were prepared using the Hantzsch 1,4-dihydropyridine reaction. In vitro calcium channel antagonist activity (CCA) was determined using a guinea pig ileum longitudinal smooth muscle assay. In the C-4 3-nitrophenyl series of compounds, the C-3 ester R-substituent was a determinant of CCA activity where the relative potency order was -CH2CH2CH=C-(2-methylphenyl)2 > or = -CH2CH2NMe2.HCI> -CH2CH2CH=C-(3-methyl-2-thienyl)2 > -CH2CH2+NMe3I -. The position and nature of the C-4 phenyl X-substituent, were also determinants of CCA activity where the relative activity order was 3-NMe2 > 4-NMe2 > 3,4.5-(OMe)3. Anticonvulsant activities were determined in mice using the subcutaneous metrazol (scMet) and maximal electroshock (MES) screens. The compounds investigated were generally not effective for protecting against scMet induced seizures, except for 10 (X = 2,3-Cl2, R = 2-[4-(4- fluorophenyl)piperazin-l-yl]ethyl] and 14a (X = 3-NMe2.HCl, R = CH2CH2OMe), which exhibited modest activity. Compound 11a (X = 3-NO2, R = -CH2CH2NMe2.HCl) was the most effective agent in the MES screen. All of the compounds investigated, except for 11b (X = 3-NO2, R = -CH2CH2+NMe3 I-, Kp = 0.15), are lipophilic with n-octanol/aqueous phosphate buffer (pH = 7.4) partition coefficients (Kp) in the 121-424 range relative to the reference drug nimodipine (Kp = 187). The structure-activity relationships acquired reinforce the concept that calcium is only one of several factors that are involved in seizure generation.

Synthesis and calcium channel modulating effects of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(thienyl)-5-pyridinecarboxylates.

A group of racemic isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(thienyl)-5-pyridinecarboxylates++ + 7a-f were prepared using a modified Hantzsch reaction that involved the condensation of a thienylcarboxaldehyde 4a-f with isopropyl 3-aminocrotonate 5 and nitroacetone 6. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle (GPILSM) assay. Compounds 7a-f exhibited weaker calcium channel antagonist activity (IC50 = 10(-5) to 10(-7) M range) than the reference drug nifedipine (IC50 = 1.43 x 10(-8) M). The point of attachment of the C-4 thienyl ring system was a determinant of antagonist activity [3-thienyl (7b) > 2-thienyl (7a)]. A 5-substituent in the 2-thienyl moiety influenced antagonist activity where the potency order was 5-bromo-2-thienyl 7f > or = 5-methyl-2-thienyl 7c > 2-thienyl 7a. Although the 5-methyl-2-thienyl 7c and 3-methyl-2-thienyl 7d isomers are equipotent antagonists, the 5-bromo-2-thienyl compound 7f appears to be marginally more active than the 4-bromo-2-thienyl isomer 7e. The 2-thienyl compound 7a, unlike the 3-thienyl isomer 7b, exhibited an agonist effect on GPILSM in the absence of the muscarinic agonist carbachol. Effects of the 2-thienyl 7a and 3-thienyl 7b isomers on the magnitude of calcium current were determined in guinea pig ventricular myocytes with voltage clamp techniques. Results showed that 2-thienyl 7a inhibited calcium current (antagonist) when voltage steps were made from a potential of -40 mV. However, when voltage steps were made from -60 mV, 7a enhanced calcium current (agonist). The 3-thienyl isomer 7b had little, if any, effect on calcium current.

Synthesis and smooth muscle calcium channel effects of dialkyl 1,4-dihydro-2,6-dimethyl-4-aryl-3,5-pyridinedicarboxylates containing a nitrone moiety in the 4-aryl substituent.

A group of dialkyl 1,4-dihydro-2,6-dimethyl-4-¿3-(or 4-)[[(Z)-N- oxo-N-[4-substituted-phenylmethylene (or vinylmethylene)]-lambda 5- azanyl]phenyl¿-3,5-pyridinedicarboxylates 7a-n were synthesized. Reaction of the C-4 nitrophenyl compounds 6a-d with an aryl Grignard reagent afforded the corresponding nitrone derivatives 7a-e. Alternatively, reaction of the aryl hydroxylamine compounds 8a-b prepared by reduction of the nitrophenyl compounds 6c-d with Zn/NH4Cl, or the aryl hydroxylamine compounds 8c-d prepared by reduction of the nitrophenyl compounds 6e-f with 5% rhodium-on-charcoal and 65% hydrazine hydrate, with a 4-substituted-benzaldehyde, benzaldehyde or acrolein afforded the respective nitrone compounds 7f-n. In vitro calcium channel (CC) antagonist activities were determined using the guinea pig ileum longitudinal smooth muscle assay. This class of compounds containing a nitrone moiety on the 1,4-dihydropyridine C-4 phenyl ring exhibited CC antagonist activities (10(-5) to 10(-9) M range) relative to the reference drug nifedipine (IC50 = 1.43 x 10(-8) M). Structure-activity relationships showed that the position of the nitrone moiety on the C-4 phenyl ring was a determinant of CC antagonist activity where the potency order was always meta-nitrone > para-nitrone. The effect of the ester alkyl substituent was variable depending upon whether the nitrone substituent was at the meta or para-position (meta-nitrone, Et > i-Pr approximately Me; para-nitrone, i-Pr > Me approximately Et). In the diethyl ester series of compounds having a meta-nitrone moiety, the difference in potency for the various R2-nitrone substituents varied by a factor of 15-fold (IC50 = 1.51 x 10(-7) to 9.84 x 10(-9) M range) (4-Cl-C6H4- > or = 4-Me-C6H4- approximately C6H5- > or = 4-O2N-C6H4- 4-F3C-C6H4- > > CH2 = CH-). Whole-cell voltage-clamp studies using isolated guinea pig ventricular myocytes indicated that the 4-¿3-[(Z)-N-oxo-N-(phenylmethylene)-lambda 5-azanyl]-phenyl¿ compound 7c (10 microM) is a calcium channel antagonist which decreased the calcium current (ICa).