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1.  Methanocarba Modification of Uracil and Adenine Nucleotides: High Potency of Northern Ring Conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but Not P2Y6 Receptors 
Journal of medicinal chemistry  2002;45(1):208-218.
The potency of nucleotide antagonists at P2Y1 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 β-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 P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5′-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC50 of 85 nM. (N)-Methanocarba-uridine 5′-diphosphate (UDP) was inactive at the hP2Y6 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 P2Y4 response (pEC50 = 6.1 ± 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 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 P2Y1, P2Y2, P2Y4, and P2Y11 receptors.
PMCID: PMC4957029  PMID: 11754592
2.  Diacylglycerol Lactones Targeting the Structural Features That Distinguish the Atypical C1 Domains of Protein Kinase C ζ and ι from Typical C1 Domains 
Journal of Medicinal Chemistry  2014;57(9):3835-3844.
To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the arginine residues that distinguish the atypical C1 domains of PKCζ and PKCι from typical C1 domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.
PMCID: PMC4310642  PMID: 24684293
3.  Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists 
Journal of medicinal chemistry  2000;43(11):2196-2203.
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 A1 and A3 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 A1, A2A, and A3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A1 or A3 receptors, respectively, were synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.
PMCID: PMC3471159  PMID: 10841798
4.  2-Substitution of Adenine Nucleotide Analogues Containing a Bicyclo[3.1.0]hexane Ring System Locked in a Northern Conformation: Enhanced Potency as P2Y1 Receptor Antagonists 
Journal of medicinal chemistry  2003;46(23):4974-4987.
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of P2Y1 receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829–842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y1 receptor and by using the radiolabeled antagonist [3H]2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y1 receptor was an (N)-methanocarba N6-methyl-2-iodo analogue 12, which displayed a Ki value in competition for binding of [3H]5 of 0.79 nM and a KB value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y1 receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y1 receptors. An enzymatic method of synthesis of the 3′,5′-bisphosphate from the corresponding 3′-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.
PMCID: PMC3408611  PMID: 14584948
5.  The Nucleoside Analog D-carba T Blocks HIV-1 Reverse Transcription 
Journal of medicinal chemistry  2009;52(17):5356-5364.
A major pathway for HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) involves reverse transcriptase (RT) mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. We analyzed novel NRTIs for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. D-carba T is a carbocyclic nucleoside that has a 3′ hydroxyl on the pseudosugar. The 3′ hydroxyl group allows RT to incorporate additional dNTPs, which should protect D-carba TMP from excision. D-carba T can be converted to the triphosphate form by host cell kinases with moderate efficiency. D-carba T-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a D-carba TMP at the primer terminus. D-carba T effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells. NRTIs based on the carbocyclic pseudosugar may offer an effective approach for the treatment of HIV-1 infections.
PMCID: PMC2756836  PMID: 19678643
HIV-1; Reverse Transcriptase; NRTI; Resistance; Kinase
6.  Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2’-deoxyzebularine 
Journal of medicinal chemistry  2008;51(23):7593-7601.
We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2’-deoxyzebularine-5’-triphosphate (dZTP). Because 2’-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine-5’-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
PMCID: PMC2659950  PMID: 19006382
7.  Conformationally Constrained Analogues of Diacylglycerol. 29. Cells Sort Diacylglycerol-Lactone Chemical Zip Codes to Produce Diverse and Selective Biological Activities 
Journal of medicinal chemistry  2008;51(17):5198-5220.
Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology produced a variety of unique biological activities. Subtle differences in chemical diversity in two areas of the molecule, the combination of which generates what we have termed “chemical zip codes”, are able to transform a relatively small chemical space into a larger universe of biological activities, as membrane-containing organelles within the cell appear to be able to decode these “chemical zip codes”. It is postulated that after binding to protein kinase C (PKC) isozymes or other non-kinase target proteins that contain diacylglycerol responsive, membrane interacting domains (C1 domains), the resulting complexes are directed to diverse intracellular sites where different sets of substrates are accessed. Multiple cellular bioassays show that DAG-lactones, which bind in vitro to PKCα to varying degrees, expand their biological repertoire into a larger domain, eliciting distinct cellular responses.
PMCID: PMC2574997  PMID: 18698758
8.  Conformationally Constrained Analogues of Diacylglycerol. 30. An Investigation of Diacylglycerol-lactones Containing Heteroaryl Groups Reveals Compounds with High Selectivity for Ras Guanyl Nucleotide-Releasing Proteins 
Journal of medicinal chemistry  2008;51(17):5371-5386.
Using a diacylglycerol-lactone (DAG-lactone) template previously developed in our laboratory as a scaffold with high binding affinity for C1 domains, we describe herein a series of novel DAG-lactones containing heterocyclic moieties (pyridines, quinolines and indoles) as α-arylidene fragments. Some of the DAG-lactones obtained show selective binding to RasGRP3 as compared to PKCα by more than two orders of magnitude and possess subnanomolar affinities. Because activated C1 domains bound to their ligands (DAG or DAG-lactones) insert into membranes, the lipid composition of membranes (cellular, nuclear, and those of internal organelles) are an important determinant for specificity. Therefore, reaching a proper hydrophilic/lipophilic balance for these molecules is critical. This was achieved by carefully selecting partnering acyl fragments for the DAG-lactones with the appropriate lipophilicity. The results clearly show that the combination of chemical and physical properties in these molecules needs to be perfectly balanced to achieve the desired specificity.
PMCID: PMC2574699  PMID: 18707088
9.  Conformationally Constrained Analogues of Diacylglycerol (DAG). 31. Modulation of the Biological Properties of Diacylgycerol Lactones (DAG-lactones) Containing Rigid-Rod Acyl Groups Separated from the Core Lactone by Spacer Units of Different Lengths 
Journal of Medicinal Chemistry  2009;52(10):3274-3283.
Diacylglycerol lactones built with a rigid 4-[(methylphenyl)ethynyl]phenyl rod that is separated from the exocyclic acylcarbonyl of the DAG-lactone core by a spacer unit of variable length were synthesized and studied. Binding affinities for a panel of classical and novel PKC isozymes in two different phospholipid environments, one corresponding to the plasma membrane of cells, were determined. The kinetics and site of translocation for the PKC isozymes α and δ upon treatment with the compounds were also studied as well as the early response of ERK phosphorylation and the late response of induction of apoptosis in the human prostatic carcinoma cell line LNCaP. Finally, the compounds were evaluated in terms of their interaction with biomimetic lipid/polydiacetylene membranes by the associated chromatic response. The different spatial disposition of the rigid structural motif on the DAG-lactones contributes to differential activity.
PMCID: PMC2708097  PMID: 19379015
10.  Human P2Y6 Receptor: Molecular Modeling Leads to the Rational Design of a Novel Agonist Based on a Unique Conformational Preference 
Journal of medicinal chemistry  2005;48(26):8108-8111.
Combining molecular dynamics (MD) in a hydrated phospholipids (DOPC) bilayer, Monte Carlo search, and synthesis of locked nucleotide analogues we discovered that the Southern conformation of the ribose is preferred for ligand recognition by the P2Y6 receptor. 2′-Deoxy-(S)-methanocarbaUDP was found to be a full agonist of the receptor and displayed a 10-fold higher potency than the corresponding flexible 2′-deoxyUDP. MD results also suggested a conformational change of the second extracellular loop consequent to agonist binding.
PMCID: PMC2583457  PMID: 16366591
11.  Conformationally Constrained Analogues of Diacylglycerol (DAG). 25. Exploration of the sn-1 and sn-2 carbonyl functionality reveals the essential role of the sn-1 carbonyl at the lipid interface in the binding of DAG-lactones to protein kinase C 
Journal of medicinal chemistry  2005;48(18):5738-5748.
A group of DAG-lactones with altered functionality (C=O → CH2 or C=O → C=S) at the sn-1 and sn-2 carbonyl pharmacophores was synthesized and used as probes to dissect the individual role of each carbonyl in binding to protein kinase C (PKC). The results suggest that the hydrated sn-1 carbonyl is engaged in very strong hydrogen bonding interactions with the charged lipid headgroups and organized water molecules at the lipid interface. Conversely, the sn-2 carbonyl has a more modest contribution to the binding process as a result of its involvement with the receptor (C1 domain) via conventional hydrogen bonding to the protein. The parent DAG-lactones, E-6 and Z-7, were designed to bind exclusively in the sn-2 binding mode to insure the correct orientation and disposition of pharmacophores at the binding site.
PMCID: PMC2563800  PMID: 16134942

Results 1-11 (11)