Gnana Ravi - Academia.edu (original) (raw)

Papers by Gnana Ravi

Research paper thumbnail of ChemInform Abstract: Structurally Related Nucleotides as Selective Agonists and Antagonists at P2Y1 Receptors

Research paper thumbnail of ChemInform Abstract: Synthesis and Purine Receptor Affinity of 6-Oxopurine Nucleosides and Nucleotides Containing (N)-Methanocarbapseudoribose Rings

Research paper thumbnail of ChemInform Abstract: Ring-Constrained (N)-Methanocarba Nucleosides as Adenosine Receptor Agonists: Independent 5′-Uronamide and 2′-Deoxy Modifications

Research paper thumbnail of ChemInform Abstract: Asymmetric Reactions of α-Ketoacid-Derived Hemiacetals: Stereoselective Synthesis of α-Hydroxy Acids

ChemInform, 1999

1999 stereochemistry stereochemistry (general, optical resolution) O 0030

Research paper thumbnail of ChemInform Abstract: Asymmetric Synthesis of α-Alkyl-α-hydroxy-γ-butyrolactones

Research paper thumbnail of Asymmetric synthesis of α-alkyl-α-hydroxy-γ-butyrolactones

Tetrahedron: Asymmetry, 1999

A new enantioselective approach to α-alkyl-α-hydroxy-γ-butyrolactones employing (1R,2S)-ephedrine... more A new enantioselective approach to α-alkyl-α-hydroxy-γ-butyrolactones employing (1R,2S)-ephedrine-derived chiral allyl morpholinones as starting materials is described.

Research paper thumbnail of Stereodivergent approach to α-hydroxy acids involving substrate directed reduction of α-keto amides

Tetrahedron Letters, 1995

Research paper thumbnail of Asymmetric reactions of α-ketoacid-derived hemiacetals: Stereoselective synthesis of α-hydroxy acids

Tetrahedron, 1998

NoAcylation of prolinol with a-ketoacid chlorides results in concomitant hemiacetalization of the... more NoAcylation of prolinol with a-ketoacid chlorides results in concomitant hemiacetalization of the a-keto amide by the prolinol hydroxyl group. (R) or (S) a-hydroxy acids are obtained with good enantiomeric excess by stereodivergent reduction of these hemiacetals. Reaction with Grignard reagents at ambient temperature furnishes (R) a-alkyl mandelic acids with good stereoselectivity.

Research paper thumbnail of Steric and Electronic Effects in Arene Formylations involving Pyrophosphoryl Chloride

Research paper thumbnail of Quantitation of the P2Y1 Receptor with a High Affinity Radiolabeled Antagonist

Molecular Pharmacology, 2002

2-Chloro-N(6)-methyl-(N )-methanocarba-2&... more 2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.

Research paper thumbnail of Induction of Novel Agonist Selectivity for the ADP-Activated P2Y1 Receptor Versus the ADP-Activated P2Y12 and P2Y13 Receptors by Conformational Constraint of an ADP Analog

Journal of Pharmacology and Experimental Therapeutics, 2004

Research paper thumbnail of Asymmetric Allylation and Reduction on an Ephedrine-Derived Template:  Stereoselective Synthesis of α-Hydroxy Acids and Derivatives

The Journal of Organic Chemistry, 1998

Research paper thumbnail of Methanocarba Modification of Uracil and Adenine Nucleotides:  High Potency of Northern Ring Conformation at P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 but Not P2Y 6 Receptors

Journal of Medicinal Chemistry, 2002

The potency of nucleotide antagonists at P2Y 1 receptors was enhanced by replacing the ribose moi... more The potency of nucleotide antagonists at P2Y 1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5′-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with -ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5′-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5′-monophosphates, which then were treated with 1,1′-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y 1 or human P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y 1 and P2Y 2 receptors, (N)-methanocarba-ATP was 138-and 41fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)methanocarba-ATP activated P2Y 11 receptors with a potency similar to ATP. (N)-Methanocarbauridine 5′-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y 2 receptors and also activated P2Y 4 receptors with an EC 50 of 85 nM. (N)-Methanocarba-uridine 5′diphosphate (UDP) was inactive at the hP2Y 6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y 4 response (pEC 50 ) 6.1 ( 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y 6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 receptors.

Research paper thumbnail of Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation:  Enhanced Stability and Potency as P2Y 1 Receptor Agonists

Journal of Medicinal Chemistry, 2002

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5&am... more Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5'-triphosphate agonists at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors, but not P2Y(6) receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y(1) or human P2Y(1) and P2Y(2) receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC(50) at the hP2Y(1) receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5'-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N(6)-Me analogue were also highly selective, full agonists at P2Y(1) receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y(1) receptors, while the corresponding ribosides were inactive. Although beta,gamma-methylene-ATP was inactive at P2Y receptors, beta,gamma-methylene-(N)-methanocarba-ATP was a potent hP2Y(1) receptor agonist with an EC(50) of 160 nM and was selective versus hP2Y(2) and hP2Y(4) receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5'-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y(1) receptors.

Research paper thumbnail of Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists

Journal of Medicinal Chemistry, 2000

Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory proper... more Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A 1 and A 3 receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A 1 , A 2A , and A 3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A 3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N 6substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A 1 or A 3 receptors, respectively, were synthesized. The N 6 -cyclopentyl derivatives were A 1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A 1 receptors, as indicated by stimulation of binding of [ 35 S]GTP-γ-S. The (N)-methanocarba-N 6 -(3-iodobenzyl)adenosine and its 2-chloro derivative had K i values of 4.1 and 2.2 nM at A 3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A 3 receptors (EC 50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y 1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.

Research paper thumbnail of Unprecedented Acceleration of Zirconium(IV)-Assisted Peptide Hydrolysis at Neutral pH

Inorganic Chemistry, 2004

4,13-Diaza-18-crown-6 substantially increases the rate of zirconium(IV) hydrolysis of unactivated... more 4,13-Diaza-18-crown-6 substantially increases the rate of zirconium(IV) hydrolysis of unactivated peptide amide bonds under nearphysiological conditions of temperature and pH. In the presence of this azacrown ether, ZrCl 4 efficiently hydrolyses both neutral and negatively charged peptides (pH 7.0−7.3, 37−60°C).

Research paper thumbnail of Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors

Il Farmaco, 2001

The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth musc... more The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth muscles prostate, ovary, and brain. A selective antagonist may be useful as an antithrombotic agent. We have analyzed the binding site of this G protein-coupled receptor using ligand design, site-directed mutagenesis, and homology modeling based on rhodopsin. We have designed and synthesized a series of deoxyadenosine 3&#39;,5&#39;-bisphosphate derivatives that act as antagonists, or, in some cases with small structural changes, as agonists or partial agonists. The 2-position accommodates Cl or thioethers, whereas the N6-position is limited to Me or Et. 2&#39;-Substitution with OH or OMe increases agonist efficacy over 2&#39;-H. Using molecular modeling of the binding site, the oxygen atoms of the ribose moiety were predicted to be non-essential, i.e. no specific H-bonds with the receptor protein appear in the model. We have, therefore, substituted this moiety with carbocylics, smaller and larger rings, conformationally constrained rings, and acyclics, with retention of affinity for the receptor. With simplified pharmacophores we are exploring the steric and electronic requirements of the receptor binding site, and the structural basis of receptor activation.

Research paper thumbnail of 2-Chloro N 6 -methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate is a selective high affinity P2Y 1 receptor antagonist

British Journal of Pharmacology, 2002

Research paper thumbnail of Ring-Constrained (N)-Methanocarba nucleosides as adenosine receptor agonists: independent 5′-Uronamide and 2′-deoxy modifications

Bioorganic & Medicinal Chemistry Letters, 2001

Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation prefer... more Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation preferred at adenosine receptors (ARs), were synthesized and tested in binding assays. The 5 0 -uronamide modification preserved [N 6 -(3-iodobenzyl)] or enhanced (N 6 -methyl) affinity at A 3 ARs, while the 2 0 -deoxy modification reduced affinity and efficacy in a functional assay. Published by Elsevier Science Ltd. 0960-894X/01/$ -see front matter Published by Elsevier Science Ltd. P I I : S 0 9 6 0 -8 9 4 X ( 0 1 ) 0 0 2 1 3 -X

Research paper thumbnail of Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings

Bioorganic & Medicinal Chemistry Letters, 2001

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopro... more 6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5 0position with an N-methyluronamide or with phosphate groups. Published by Elsevier Science Ltd. 0960-894X/01/$ -see front matter. Published by Elsevier Science Ltd. P I I : S 0 9 6 0 -8 9 4 X ( 0 1 ) 0 0 4 5 0 -4

Research paper thumbnail of ChemInform Abstract: Structurally Related Nucleotides as Selective Agonists and Antagonists at P2Y1 Receptors

Research paper thumbnail of ChemInform Abstract: Synthesis and Purine Receptor Affinity of 6-Oxopurine Nucleosides and Nucleotides Containing (N)-Methanocarbapseudoribose Rings

Research paper thumbnail of ChemInform Abstract: Ring-Constrained (N)-Methanocarba Nucleosides as Adenosine Receptor Agonists: Independent 5′-Uronamide and 2′-Deoxy Modifications

Research paper thumbnail of ChemInform Abstract: Asymmetric Reactions of α-Ketoacid-Derived Hemiacetals: Stereoselective Synthesis of α-Hydroxy Acids

ChemInform, 1999

1999 stereochemistry stereochemistry (general, optical resolution) O 0030

Research paper thumbnail of ChemInform Abstract: Asymmetric Synthesis of α-Alkyl-α-hydroxy-γ-butyrolactones

Research paper thumbnail of Asymmetric synthesis of α-alkyl-α-hydroxy-γ-butyrolactones

Tetrahedron: Asymmetry, 1999

A new enantioselective approach to α-alkyl-α-hydroxy-γ-butyrolactones employing (1R,2S)-ephedrine... more A new enantioselective approach to α-alkyl-α-hydroxy-γ-butyrolactones employing (1R,2S)-ephedrine-derived chiral allyl morpholinones as starting materials is described.

Research paper thumbnail of Stereodivergent approach to α-hydroxy acids involving substrate directed reduction of α-keto amides

Tetrahedron Letters, 1995

Research paper thumbnail of Asymmetric reactions of α-ketoacid-derived hemiacetals: Stereoselective synthesis of α-hydroxy acids

Tetrahedron, 1998

NoAcylation of prolinol with a-ketoacid chlorides results in concomitant hemiacetalization of the... more NoAcylation of prolinol with a-ketoacid chlorides results in concomitant hemiacetalization of the a-keto amide by the prolinol hydroxyl group. (R) or (S) a-hydroxy acids are obtained with good enantiomeric excess by stereodivergent reduction of these hemiacetals. Reaction with Grignard reagents at ambient temperature furnishes (R) a-alkyl mandelic acids with good stereoselectivity.

Research paper thumbnail of Steric and Electronic Effects in Arene Formylations involving Pyrophosphoryl Chloride

Research paper thumbnail of Quantitation of the P2Y1 Receptor with a High Affinity Radiolabeled Antagonist

Molecular Pharmacology, 2002

2-Chloro-N(6)-methyl-(N )-methanocarba-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;... more 2-Chloro-N(6)-methyl-(N )-methanocarba-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyadenosine-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deoxyadenosine-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;, 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.

Research paper thumbnail of Induction of Novel Agonist Selectivity for the ADP-Activated P2Y1 Receptor Versus the ADP-Activated P2Y12 and P2Y13 Receptors by Conformational Constraint of an ADP Analog

Journal of Pharmacology and Experimental Therapeutics, 2004

Research paper thumbnail of Asymmetric Allylation and Reduction on an Ephedrine-Derived Template:  Stereoselective Synthesis of α-Hydroxy Acids and Derivatives

The Journal of Organic Chemistry, 1998

Research paper thumbnail of Methanocarba Modification of Uracil and Adenine Nucleotides:  High Potency of Northern Ring Conformation at P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 but Not P2Y 6 Receptors

Journal of Medicinal Chemistry, 2002

The potency of nucleotide antagonists at P2Y 1 receptors was enhanced by replacing the ribose moi... more The potency of nucleotide antagonists at P2Y 1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5′-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with -ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5′-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5′-monophosphates, which then were treated with 1,1′-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y 1 or human P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y 1 and P2Y 2 receptors, (N)-methanocarba-ATP was 138-and 41fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)methanocarba-ATP activated P2Y 11 receptors with a potency similar to ATP. (N)-Methanocarbauridine 5′-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y 2 receptors and also activated P2Y 4 receptors with an EC 50 of 85 nM. (N)-Methanocarba-uridine 5′diphosphate (UDP) was inactive at the hP2Y 6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y 4 response (pEC 50 ) 6.1 ( 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y 6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 receptors.

Research paper thumbnail of Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation:  Enhanced Stability and Potency as P2Y 1 Receptor Agonists

Journal of Medicinal Chemistry, 2002

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5&amp;am... more Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-triphosphate agonists at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors, but not P2Y(6) receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y(1) or human P2Y(1) and P2Y(2) receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC(50) at the hP2Y(1) receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N(6)-Me analogue were also highly selective, full agonists at P2Y(1) receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y(1) receptors, while the corresponding ribosides were inactive. Although beta,gamma-methylene-ATP was inactive at P2Y receptors, beta,gamma-methylene-(N)-methanocarba-ATP was a potent hP2Y(1) receptor agonist with an EC(50) of 160 nM and was selective versus hP2Y(2) and hP2Y(4) receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y(1) receptors.

Research paper thumbnail of Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists

Journal of Medicinal Chemistry, 2000

Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory proper... more Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A 1 and A 3 receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A 1 , A 2A , and A 3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A 3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N 6substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A 1 or A 3 receptors, respectively, were synthesized. The N 6 -cyclopentyl derivatives were A 1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A 1 receptors, as indicated by stimulation of binding of [ 35 S]GTP-γ-S. The (N)-methanocarba-N 6 -(3-iodobenzyl)adenosine and its 2-chloro derivative had K i values of 4.1 and 2.2 nM at A 3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A 3 receptors (EC 50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y 1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.

Research paper thumbnail of Unprecedented Acceleration of Zirconium(IV)-Assisted Peptide Hydrolysis at Neutral pH

Inorganic Chemistry, 2004

4,13-Diaza-18-crown-6 substantially increases the rate of zirconium(IV) hydrolysis of unactivated... more 4,13-Diaza-18-crown-6 substantially increases the rate of zirconium(IV) hydrolysis of unactivated peptide amide bonds under nearphysiological conditions of temperature and pH. In the presence of this azacrown ether, ZrCl 4 efficiently hydrolyses both neutral and negatively charged peptides (pH 7.0−7.3, 37−60°C).

Research paper thumbnail of Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors

Il Farmaco, 2001

The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth musc... more The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth muscles prostate, ovary, and brain. A selective antagonist may be useful as an antithrombotic agent. We have analyzed the binding site of this G protein-coupled receptor using ligand design, site-directed mutagenesis, and homology modeling based on rhodopsin. We have designed and synthesized a series of deoxyadenosine 3&#39;,5&#39;-bisphosphate derivatives that act as antagonists, or, in some cases with small structural changes, as agonists or partial agonists. The 2-position accommodates Cl or thioethers, whereas the N6-position is limited to Me or Et. 2&#39;-Substitution with OH or OMe increases agonist efficacy over 2&#39;-H. Using molecular modeling of the binding site, the oxygen atoms of the ribose moiety were predicted to be non-essential, i.e. no specific H-bonds with the receptor protein appear in the model. We have, therefore, substituted this moiety with carbocylics, smaller and larger rings, conformationally constrained rings, and acyclics, with retention of affinity for the receptor. With simplified pharmacophores we are exploring the steric and electronic requirements of the receptor binding site, and the structural basis of receptor activation.

Research paper thumbnail of 2-Chloro N 6 -methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate is a selective high affinity P2Y 1 receptor antagonist

British Journal of Pharmacology, 2002

Research paper thumbnail of Ring-Constrained (N)-Methanocarba nucleosides as adenosine receptor agonists: independent 5′-Uronamide and 2′-deoxy modifications

Bioorganic & Medicinal Chemistry Letters, 2001

Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation prefer... more Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation preferred at adenosine receptors (ARs), were synthesized and tested in binding assays. The 5 0 -uronamide modification preserved [N 6 -(3-iodobenzyl)] or enhanced (N 6 -methyl) affinity at A 3 ARs, while the 2 0 -deoxy modification reduced affinity and efficacy in a functional assay. Published by Elsevier Science Ltd. 0960-894X/01/$ -see front matter Published by Elsevier Science Ltd. P I I : S 0 9 6 0 -8 9 4 X ( 0 1 ) 0 0 2 1 3 -X

Research paper thumbnail of Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings

Bioorganic & Medicinal Chemistry Letters, 2001

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopro... more 6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5 0position with an N-methyluronamide or with phosphate groups. Published by Elsevier Science Ltd. 0960-894X/01/$ -see front matter. Published by Elsevier Science Ltd. P I I : S 0 9 6 0 -8 9 4 X ( 0 1 ) 0 0 4 5 0 -4