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Edward E. Knaus - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of Alkyl 6-Methyl-4- (2-trifluoromethylphenyl) -1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylates Possessing a N-3 Nitro Substituent to Determine Calcium Channel Modulation Structure—Activity Relationships.
    ChemInform, 2007
    Co-Authors: K. J. Kaur, Edward E. Knaus
    Abstract:

    The Bigenelli acid catalyzed condensation of 2-trifluoromethylbenzaldehyde (1), urea (2) and an alkyl acetoacetate (3) afforded the respective alkyl (Me, Et, i-Pr, i-Bu) 6-methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylate (4-7). Subsequent N3-nitration of the alkyl esters (4-7) using Cu(NO3)2 3H2O and Ac2O furnished the target alkyl 6-methyl-3-nitro-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylates (8-11). The N3-nitro compounds (8-11) were less potent Calcium Channel antagonists (IC50 values in the 1.9 × 10−7 to 3.9 × 10−6 M range) on guinea pig ileal longitudinal smooth muscle than the reference drug nifedipine (Adalat®, IC50 = 1.4 × 10−8 M). In vitro Calcium Channel Modulation studies on guinea pig left atrium (GPLA) showed that the methyl and ethyl esters (8-9) induced a weak-to-modest positive inotropic (agonist) effect, and that the inactive isopropyl (10) and isobutyl (11) esters did not alter the cardiac contractile force of GPLA.

  • Synthesis of alkyl 6‐methyl‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates possessing a N‐3 nitro substituent to determine Calcium Channel Modulation structure‐activity relationships
    Journal of Heterocyclic Chemistry, 2007
    Co-Authors: K. J. Kaur, Edward E. Knaus
    Abstract:

    The Bigenelli acid catalyzed condensation of 2-trifluoromethylbenzaldehyde (1), urea (2) and an alkyl acetoacetate (3) afforded the respective alkyl (Me, Et, i-Pr, i-Bu) 6-methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylate (4-7). Subsequent N3-nitration of the alkyl esters (4-7) using Cu(NO3)2 3H2O and Ac2O furnished the target alkyl 6-methyl-3-nitro-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylates (8-11). The N3-nitro compounds (8-11) were less potent Calcium Channel antagonists (IC50 values in the 1.9 × 10−7 to 3.9 × 10−6 M range) on guinea pig ileal longitudinal smooth muscle than the reference drug nifedipine (Adalat®, IC50 = 1.4 × 10−8 M). In vitro Calcium Channel Modulation studies on guinea pig left atrium (GPLA) showed that the methyl and ethyl esters (8-9) induced a weak-to-modest positive inotropic (agonist) effect, and that the inactive isopropyl (10) and isobutyl (11) esters did not alter the cardiac contractile force of GPLA.

  • Synthesis of Dialkyl 1,4‐Dihydro‐2,6‐dimethylpyridine‐3,5‐dicarboxylates and Alkyl 1,4‐Dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates Possessing a C‐4 2,4‐Dioxo‐1,2,3,4‐tetrahydropyrimidin‐5‐yl (Uracil) Substituent to Determine Calcium Channel M
    ChemInform, 2004
    Co-Authors: Afshin Fassihi, Carlos A. Velázquez, Edward E. Knaus
    Abstract:

    The Hantzsch condensation of 5-formyluracil (1) with methyl, isopropyl or isobutyl acetoacetate (2a-c) in the presence of ammonium hydroxide afforded the respective dialkyl 1,4-dihydro-2,6-dimethyl-4-(2,4-dloxo-1,2,3,4,-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3a-c). A group of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylates (6a-c) were also prepared using a modified Hantzsch reaction that involved the condensation of 5-formyluracil with nitroacetone and either methyl, isopropyl or isobutyl 3-aminocrotonate (5a-c). A C-4 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl substituent is not a suitable bioisostere for the traditional C-4 aryl or heteroaryl substituents present in 1,4-dihydropyridine Calcium Channel modulators since diisopropyl 1,4-dihydro-2,6-dimemyl-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3b) and isobutyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dloxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylate (6c) did not exhibit any in vitro Calcium Channel antagonist activity using a guinea pig smooth muscle Calcium Channel antagonist assay, or a guinea pig left atrium Calcium Channel agonist (positive inotropic) assay.

  • synthesis of dialkyl 1 4 dihydro 2 6 dimethylpyridine 3 5 dicarboxylates and alkyl 1 4 dihydro 2 6 dimethyl 3 nitropyridine 5 carboxylates possessing a c 4 2 4 dioxo 1 2 3 4 tetrahydropyrimidin 5 yl uracil substituent to determine Calcium Channel mod
    Journal of Heterocyclic Chemistry, 2004
    Co-Authors: Afshin Fassihi, Carlos A. Velázquez, Edward E. Knaus
    Abstract:

    The Hantzsch condensation of 5-formyluracil (1) with methyl, isopropyl or isobutyl acetoacetate (2a-c) in the presence of ammonium hydroxide afforded the respective dialkyl 1,4-dihydro-2,6-dimethyl-4-(2,4-dloxo-1,2,3,4,-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3a-c). A group of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylates (6a-c) were also prepared using a modified Hantzsch reaction that involved the condensation of 5-formyluracil with nitroacetone and either methyl, isopropyl or isobutyl 3-aminocrotonate (5a-c). A C-4 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl substituent is not a suitable bioisostere for the traditional C-4 aryl or heteroaryl substituents present in 1,4-dihydropyridine Calcium Channel modulators since diisopropyl 1,4-dihydro-2,6-dimemyl-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3b) and isobutyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dloxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylate (6c) did not exhibit any in vitro Calcium Channel antagonist activity using a guinea pig smooth muscle Calcium Channel antagonist assay, or a guinea pig left atrium Calcium Channel agonist (positive inotropic) assay.

  • Syntheses, Calcium Channel Modulation effects, and nitric oxide release studies of O2-alkyl-1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylates
    Drug Development Research, 2003
    Co-Authors: Carlos A. Velázquez, Edward E. Knaus
    Abstract:

    A group of racemic 4-aryl(heteroary)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxy- lates possessing a potential nitric oxide donor C-5 O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate ester (alkyl ¼ (CH2)n ,n ¼ 1-4) substituent were synthesized using a modified Hantzsch reaction. Compounds having a C-4 2-trifluoromethylphenyl (16), 2-pyridyl (17), or benzofurazan-4-yl (20) substituent generally exhibited more potent smooth-muscle Calcium Channel antagonist activity (IC50 values in the 0.55 to 38.6 mM range) than related analogs having a C-4 3-pyridyl (18), or 4-pyridyl (19) substituent with IC50 values 4 29.91 mM, relative to the reference drug nifedipine (IC50 ¼ 0.0143 mM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2-pyridyl 4 3-pyridyl and 4-pyridyl. Subgroups of compounds 16a-d, 17a-d, and 20a-d having alkyl spacer groups of variable chain length (-CO2(CH2)nO-, n ¼ 1-4) exhibited small differences in Calcium Channel antagonist potency. Replacement of the ester ''methyl'' moiety of Bay K 8644 by an O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate group provided the Bay K 8644 group of analogs 16a-d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O 2 -ethyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-tri- fluoromethylphenyl)pyridine-5-carboxylate (16b ,E C 50 ¼ 0.096 mM) is about eightfold more potent positive inotrope (cardiac Calcium Channel agonist) than the reference compound Bay K 8644 (EC50 ¼ 0.77 mM). A similar replacement of the ester ''isopropyl'' group in the C-4 benzofurazan-4-yl group of compounds by an O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate ester substituent provided compounds 20 (n ¼ 1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202-791 (3 ,E C 50 ¼ 9.40 mM). The O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2- diolate ester moiety present in 1,4-dihydropyridine Calcium Channel modulating compounds 16-20 is not a suitableNO donor moiety because the percent nitric oxide released upon in vitro incubation with either L-cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204-216, 2003. � c 2003 Wiley-Liss, Inc.

Rudong Shan - One of the best experts on this subject based on the ideXlab platform.

Carol‐anne Mcewen - One of the best experts on this subject based on the ideXlab platform.

  • 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‐py
    Drug Development Research, 2002
    Co-Authors: Jeffrey‐tri Nguyen, Carol‐anne Mcewen, Rudong Shan, Edward E. Knaus
    Abstract:

    A 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 modulating effects of modified Hantzsch nitrooxyalkyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(pyridinyl or 2‐trifluoromethylphenyl)‐5‐pyridinecarboxylates
    Drug Development Research, 2000
    Co-Authors: Ramin Miri, Carol‐anne Mcewen, Edward E. Knaus
    Abstract:

    A 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 (IC 50 = 1.43 x 10 -8 M). Compounds 8 possessing a C-4 R 1 = 2-pyridyl substituent were always more potent than the approximately equiactive analogs having an R 1 = 3-pyridyl, 4-pyridyl or 2-CF3-C6H4-substituent, within each subgroup of nitrooxyalkyl compounds [R 2 = -(CH 2 ) n ONO 2 (n = 2, 3, 4) or -CH(CH 2 ONO 2 ) 2 ]. Although the length of the R 2 = -(CH 2 ) n ONO 2 substituent (n = 2-4) was not a determinant of smooth muscle Calcium antagonist activity when the C-4 R 1 -substituent was 2-pyridyl, when R 1 was a 3-pyridyl, 4-pyridyl, or 2-CF3-C6H4-substituent, the relative potency order with respect to the R 2 = -(CH 2 ) n ONO 2 substituent was n = 3 and 4 > n = 2. Replacement of the isopropyl substituent of the ester moiety of the Calcium antagonist (±)-2-pyridyl 3a by a -(CH 2 ) n ONO 2 (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 R 2 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 -CH 2 CH 2 ONO 2 moiety resulted in retention of the cardiac positive inotropic effect where the relative potency order with respect to the C-4 substituent was 2-CF 3 -C 6 H 6 - (8d) > 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.

  • Synthesis and Calcium Channel Modulation effects of isopropyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-phenylpyridine-5-carboxylates possessing ortho-, meta-, and para-CH2S(O)nMe and –S(O)nMe (n = 0–2) phenyl substituents
    Drug Development Research, 2000
    Co-Authors: Nadeem Iqbal, Carol‐anne Mcewen, Edward E. Knaus
    Abstract:

    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.

Carlos A. Velázquez - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of Dialkyl 1,4‐Dihydro‐2,6‐dimethylpyridine‐3,5‐dicarboxylates and Alkyl 1,4‐Dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates Possessing a C‐4 2,4‐Dioxo‐1,2,3,4‐tetrahydropyrimidin‐5‐yl (Uracil) Substituent to Determine Calcium Channel M
    ChemInform, 2004
    Co-Authors: Afshin Fassihi, Carlos A. Velázquez, Edward E. Knaus
    Abstract:

    The Hantzsch condensation of 5-formyluracil (1) with methyl, isopropyl or isobutyl acetoacetate (2a-c) in the presence of ammonium hydroxide afforded the respective dialkyl 1,4-dihydro-2,6-dimethyl-4-(2,4-dloxo-1,2,3,4,-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3a-c). A group of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylates (6a-c) were also prepared using a modified Hantzsch reaction that involved the condensation of 5-formyluracil with nitroacetone and either methyl, isopropyl or isobutyl 3-aminocrotonate (5a-c). A C-4 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl substituent is not a suitable bioisostere for the traditional C-4 aryl or heteroaryl substituents present in 1,4-dihydropyridine Calcium Channel modulators since diisopropyl 1,4-dihydro-2,6-dimemyl-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3b) and isobutyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dloxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylate (6c) did not exhibit any in vitro Calcium Channel antagonist activity using a guinea pig smooth muscle Calcium Channel antagonist assay, or a guinea pig left atrium Calcium Channel agonist (positive inotropic) assay.

  • synthesis of dialkyl 1 4 dihydro 2 6 dimethylpyridine 3 5 dicarboxylates and alkyl 1 4 dihydro 2 6 dimethyl 3 nitropyridine 5 carboxylates possessing a c 4 2 4 dioxo 1 2 3 4 tetrahydropyrimidin 5 yl uracil substituent to determine Calcium Channel mod
    Journal of Heterocyclic Chemistry, 2004
    Co-Authors: Afshin Fassihi, Carlos A. Velázquez, Edward E. Knaus
    Abstract:

    The Hantzsch condensation of 5-formyluracil (1) with methyl, isopropyl or isobutyl acetoacetate (2a-c) in the presence of ammonium hydroxide afforded the respective dialkyl 1,4-dihydro-2,6-dimethyl-4-(2,4-dloxo-1,2,3,4,-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3a-c). A group of alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylates (6a-c) were also prepared using a modified Hantzsch reaction that involved the condensation of 5-formyluracil with nitroacetone and either methyl, isopropyl or isobutyl 3-aminocrotonate (5a-c). A C-4 2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl substituent is not a suitable bioisostere for the traditional C-4 aryl or heteroaryl substituents present in 1,4-dihydropyridine Calcium Channel modulators since diisopropyl 1,4-dihydro-2,6-dimemyl-4-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-3,5-dicarboxylate (3b) and isobutyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2,4-dloxo-1,2,3,4-tetrahydropyrimidin-5-yl)pyridine-5-carboxylate (6c) did not exhibit any in vitro Calcium Channel antagonist activity using a guinea pig smooth muscle Calcium Channel antagonist assay, or a guinea pig left atrium Calcium Channel agonist (positive inotropic) assay.

  • Syntheses, Calcium Channel Modulation effects, and nitric oxide release studies of O2-alkyl-1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylates
    Drug Development Research, 2003
    Co-Authors: Carlos A. Velázquez, Edward E. Knaus
    Abstract:

    A group of racemic 4-aryl(heteroary)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxy- lates possessing a potential nitric oxide donor C-5 O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate ester (alkyl ¼ (CH2)n ,n ¼ 1-4) substituent were synthesized using a modified Hantzsch reaction. Compounds having a C-4 2-trifluoromethylphenyl (16), 2-pyridyl (17), or benzofurazan-4-yl (20) substituent generally exhibited more potent smooth-muscle Calcium Channel antagonist activity (IC50 values in the 0.55 to 38.6 mM range) than related analogs having a C-4 3-pyridyl (18), or 4-pyridyl (19) substituent with IC50 values 4 29.91 mM, relative to the reference drug nifedipine (IC50 ¼ 0.0143 mM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2-pyridyl 4 3-pyridyl and 4-pyridyl. Subgroups of compounds 16a-d, 17a-d, and 20a-d having alkyl spacer groups of variable chain length (-CO2(CH2)nO-, n ¼ 1-4) exhibited small differences in Calcium Channel antagonist potency. Replacement of the ester ''methyl'' moiety of Bay K 8644 by an O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate group provided the Bay K 8644 group of analogs 16a-d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O 2 -ethyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-tri- fluoromethylphenyl)pyridine-5-carboxylate (16b ,E C 50 ¼ 0.096 mM) is about eightfold more potent positive inotrope (cardiac Calcium Channel agonist) than the reference compound Bay K 8644 (EC50 ¼ 0.77 mM). A similar replacement of the ester ''isopropyl'' group in the C-4 benzofurazan-4-yl group of compounds by an O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate ester substituent provided compounds 20 (n ¼ 1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202-791 (3 ,E C 50 ¼ 9.40 mM). The O 2 -alkyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2- diolate ester moiety present in 1,4-dihydropyridine Calcium Channel modulating compounds 16-20 is not a suitableNO donor moiety because the percent nitric oxide released upon in vitro incubation with either L-cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204-216, 2003. � c 2003 Wiley-Liss, Inc.

Stephen R. Ikeda - One of the best experts on this subject based on the ideXlab platform.

  • fluorophore assisted light inactivation produces both targeted and collateral effects on n type Calcium Channel Modulation in rat sympathetic neurons
    The Journal of Physiology, 2006
    Co-Authors: Juan Guo, Huanmian Chen, Henry L Puhl, Stephen R. Ikeda
    Abstract:

    Fluorophore-assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein-conjugated polypeptide, α-bungarotoxin (BTX) and a 13 amino acid BTX-binding site engineered into the N-terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G-protein-coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of Calcium currents (ICa) using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labelled neurons, mGluR8a-mediated ICa inhibition was greatly attenuated whereas holding current and basal ICa, measures of non-specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, α2-adrenergic receptor-mediated ICa Modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium Channel β2 subunits or ionotropic 5-HT3 receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR-independent ICa Modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore-tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.

  • Fluorophore‐assisted light inactivation produces both targeted and collateral effects on N‐type Calcium Channel Modulation in rat sympathetic neurons
    The Journal of Physiology, 2006
    Co-Authors: Juan Guo, Huanmian Chen, Henry L Puhl, Stephen R. Ikeda
    Abstract:

    Fluorophore-assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein-conjugated polypeptide, α-bungarotoxin (BTX) and a 13 amino acid BTX-binding site engineered into the N-terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G-protein-coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of Calcium currents (ICa) using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labelled neurons, mGluR8a-mediated ICa inhibition was greatly attenuated whereas holding current and basal ICa, measures of non-specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, α2-adrenergic receptor-mediated ICa Modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium Channel β2 subunits or ionotropic 5-HT3 receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR-independent ICa Modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore-tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.

  • Fluorophore-assisted light inactivation produces both targeted and collateral effects on N-type Calcium Channel Modulation in rat sympathetic neurons.
    The Journal of physiology, 2006
    Co-Authors: Juan Guo, Huanmian Chen, Henry L Puhl, Stephen R. Ikeda
    Abstract:

    Fluorophore-assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein-conjugated polypeptide, alpha-bungarotoxin (BTX) and a 13 amino acid BTX-binding site engineered into the N-terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G-protein-coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of Calcium currents (I(Ca)) using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labelled neurons, mGluR8a-mediated I(Ca) inhibition was greatly attenuated whereas holding current and basal I(Ca), measures of non-specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, alpha(2)-adrenergic receptor-mediated I(Ca) Modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium Channel beta2 subunits or ionotropic 5-HT(3) receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR-independent I(Ca) Modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore-tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.

  • Endogenous regulator of G-protein signaling proteins modify N-type Calcium Channel Modulation in rat sympathetic neurons.
    The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
    Co-Authors: Seong-woo Jeong, Stephen R. Ikeda
    Abstract:

    Experiments using heterologous overexpression indicate that regulator of G-protein signaling (RGS) proteins play important roles in Gbetagamma-mediated ion Channel Modulation. However, the roles subserved by endogenous RGS proteins have not been extensively examined because tools for functionally inhibiting natively expressed RGS proteins are lacking. To address this void, we used a strategy in which Galpha(oA) was rendered insensitive to pertussis toxin (PTX) and RGS proteins by site-directed mutagenesis. Either PTX-insensitive (PTX-i) or both PTX- and RGS-insensitive (PTX/RGS-i) mutants of Galpha(oA) were expressed along with Gbeta(1) and Ggamma(2) subunits in rat sympathetic neurons. After overnight treatment with PTX to suppress natively expressed Galpha subunits, voltage-dependent Ca(2+) current inhibition by norepinephrine (NE) (10 microm) was reconstituted in neurons expressing either PTX-i or PTX/RGS-i Galpha(oA). When compared with neurons expressing PTX-i Galpha(oA), the steady-state concentration-response relationships for NE-induced Ca(2+) current inhibition were shifted to lower concentrations in neurons expressing PTX/RGS-i Galpha(oA). In addition to an increase in agonist potency, the expression of PTX/RGS-i Galpha(oA) dramatically retarded the current recovery after agonist removal. Interestingly, the alteration in current recovery was accompanied by a slowing in the onset of current inhibition. Together, our data suggest that endogenous RGS proteins contribute to membrane-delimited Ca(2+) Channel Modulation by regulating agonist potency and kinetics of G-protein-mediated signaling in neuronal cells.

  • Heterologous expression of a green fluorescent protein-pertussis toxin S1 subunit fusion construct disrupts Calcium Channel Modulation in rat superior cervical ganglion neurons.
    Neuroscience letters, 1999
    Co-Authors: Stephen R. Ikeda, Seong-woo Jeong, Paul J. Kammermeier, Victor Ruiz-velasco, Marina M. King
    Abstract:

    Abstract The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1–100 ng/μl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca2+ Channel Modulation. The heterologously expressed toxin retained specificity for Gαi/o-dependent pathways as VIP-mediated Modulation of N-type Ca2+ Channels and muscarine-mediated inhibition of M-type K+ Channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.