Clostridium difficile is a potentially lethal gut pathogen that causes nosocomial and community-acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA polymerase of Gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E [N2-(3,4-dichlorobenzyl)-7-(2-[1-morpholinyl] ethyl) guanine; MorE-DCBG] is a DNA polymerase inhibitor in preclinical development as a novel therapeutic against C. difficile infection. This synthetic purine shows preferential activity against C. difficile PolC over those of other organisms in vitro and is effective in an animal model of C. difficile infection. In this study, we have determined its efficacy against a large collection of clinical isolates. At concentrations below the MIC, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response of C. difficile to replication inhibition and observed an overrepresentation of upregulated genes near the origin of replication in the presence of PolC inhibitors, but not when cells were subjected to subinhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal and terminus-proximal gene copy number upon exposure to PolC inhibitors. Moreover, we show that certain genes differentially regulated under PolC inhibition are controlled by the originproximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition in C. difficile.

Despite the growing global crisis caused by antimicrobial drug resistance among pathogenic bacteria, the number of new antibiotics, especially new chemical class of antibiotics under development is insufficient to tackle the problem. Our review focuses on an emerging class of antibacterial therapeutic agents that holds a completely novel mechanism of action, namely, inhibition of bacterial DNA polymerase IIIC. The recent entry of this new class into human trials may herald the introduction of novel drugs whose novel molecular target precludes cross-resistance with existing antibiotic classes. This review therefore examines the evolution of DNA pol IIIC inhibitors from the discovery of 6-(p-hydroxyphenylazo) uracil ( HPUra) in the 1960s to the development of current first-in-class N7-substituted guanine drug candidate ACX-362E, now under clinical development for the treatment of Clostridioides difficile infection.

Zoonotic monkey B virus ( Macacine alphaherpesvirus 1; BV) infections are extremely serious and usually fatal. Drugs currently used for treatment were developed for the treatment of herpes simplex virus but are less effective against BV. Effective suppression of viral replication in the skin could prevent the virus from invading the nervous system. To test this hypothesis, the efficacy of topical administration of several drugs against lethal BV infection was evaluated in female BALB/c mice that were infected by scarification. Drugs were then applied to the site of inoculation. As 3% preparations, most drugs were only minimally effective or ineffective. In contrast, ganciclovir and cidofovir were very effective. The ED 50 for cidofovir was 0.007%, compared with 1.1% for ganciclovir. At 0.5%, cidofovir protected against both death and neurologic signs, whereas 5% ganciclovir only protected against death but not neurologic involvement. All genotypes of BV were equally susceptible to cidofovir and ganciclovir. For maximal effectiveness, treatment with both cidofovir and ganciclovir had to be initiated within 8 h of infection. Cidofovir was completely protective when administered only on the day of infection, whereas a minimum of 5 d of treatment was required for maximal ganciclovir efficacy. These studies showed that topical cidofovir treatment started soon after BV exposure was very effective in preventing BV from invading the nervous system, whereas ganciclovir treatment was only partially effective. In addition, cidofovir was protective against a ganciclovir-resistant BV mutant, whereas ganciclovir was not. These studies showed that topical cidofovir treatment started soon after BV exposure is more effective than ganciclovir in preventing BV from invading the CNS.

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12 ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes.

There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by phosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.

Introduction. Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor inhibitor is a mainstay of pharmacological therapy in acute coronary syndromes. GLS-409 is a novel diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12platelet ADP receptors.

Objective— Unlike currently approved adenosine diphosphate receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet adenosine diphosphate receptor P2Y1 and may, therefore, be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409. Approach and Results— We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited adenosine diphosphate- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, that is, platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a nonsignificant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake. Conclusions— Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy.

Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme.

ABCB1/Pgp and ABCG2/BCRP multidrug transporters are important in developing multidrug resistance and significantly modify ADME‐Tox parameters of several therapeutic compounds. Regulatory agencies (EMA, FDA) state in their guidelines that drug candidates be tested for in vitro inhibition of Pgp and ABCG2. Only the application of different assays can reveal complex drug interactions of these polyspecific transporters. We investigate these interactions either in a native cell membrane environment or by using purified proteins reconstituted in a liposomal platform. ABC transporters extrude drugs at the expense of ATP hydrolysis, thus drug interactions can be followed in proteoliposomes (PL) by measuring ATPase activity or transport of labeled compounds. Providing appropriate lipid environment for ABCG2 was a key step in establishing new assays. PLs of ABCG2, in contrast to those containing ABCB1, have to contain cholesterol. In addition, phosphatidylethanolamine‐containing E.coli lipids support the ABCG2 activity better than phosphatidylcholine‐based lipids. In our international collaboration we are working on new assays similar to that developed earlier for the Pgp (the Fluorosome‐trans‐pgp assay). This method involves PLs that contain a fluorescent drug sensing probe in the interior of the PL, and measures in real time changes in fluorescence which reflect the Pgp‐ or ABCG2‐mediated transport. With Fluorosome‐trans‐abcg2/pgp assay, and direct liposomal assay observing the inhibition of uptake of fluorescently labeled substrate drugs we examine transporter‐drug interactions. The results are analyzed in correlation with the ATPase activity measurements.

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P1,P4-di(adenosine-5′) tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure–activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.

Background: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P ,P-dithio and/or P,Pchloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 59,500-P,P-dithio-P,P-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca , P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral Pand P-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P,P-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors. Citation: Chang H, Yanachkov IB, Dix EJ, Yanachkova M, Li Y, et al. (2014) Antiplatelet Activity, P2Y1 and P2Y12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 59,590-P,P-dithio-P,P-chloromethylenetetraphosphate. PLoS ONE 9(4): e94780. doi:10.1371/journal.pone.0094780 Editor: Dermot Cox, Royal College of Surgeons, Ireland Received November 6, 2013; Accepted March 19, 2014; Published April 10, 2014 Copyright: 2014 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by SBIR grants HL081992 and HL088828 (to I.B. Yanachkov) from the National Heart, Lung and Blood Institute. Support for H. Chang was provided by grant CMRP G380211, provided by Chang Gung Memorial Hospital. There were no other funding sources for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: A.L. Frelinger and A.D. Michelson have been investigators on research grants to Boston Children’s Hospital from GLSynthesis and Eli Lilly. A.D. Michelson has been a member of the Data Monitoring Committee of clinical trials sponsored by Lilly. I.B. Yanachkov, M. Yanachkova and G.E. Wright are employees of GLSynthesis, Inc. E.J. Dix has been a consultant to GLSynthesis, Inc. The other authors state that they have no competing interests. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. * E-mail: Andrew.Frelinger@childrens.harvard.edu

Introduction: This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance.Methods: To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted. The interior of the Fluorosome particle (a unilamellar liposome) contains a fluorescent drug sensing probe whose fluorescence is quantitatively quenched by transporter substrates. Using an injecting fluorescence plate reader to initiate a proton gradient and to monitor subsequent fluorescence change, real time transport kinetics can be followed and transport inhibition characterized.Results: Fluorosomes containing the Escherichia coli EmrE efflux pump demonstrated transport of two known EmrE substrates, ethidium and methyl viologen upon creation of a proton gradient. For Fluorosomes containing the inactive EmrE mutant, E14Q, no transport was observed. When the gradient was fully collapsed by the addition of nigericin, full inhibition of substrate transport was observed. The IC50 for nigericin inhibition of ethidium was shown to be 0.71 mu M.Discussion: We have for the first time prepared and validated a single bacterial efflux pump assay, Fluorosome-trans- EmrE, that faithfully mimics properties of the transporter in vivo. It is faster than whole cell screens, simple to use, amenable to robotics, and reports on very specific targets. We have demonstrated proof of principle with EmrE and have created the first of an intended series of proton driven Fluorosomes. (C) 2015 Elsevier Inc. All rights reserved.

Background Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Methods Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Results Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy- mCF 3 PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N 2 -[3-(trifluoromethyl)pheny])guanine ( mCF 3 PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Conclusions Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo.

Monkey B virus (Macacine herpesvirus 1; BV) is endemic in macaques. BV (a BSL4 agent) is the primary zoonotic concern for persons working with macaques in research, and human BV infections frequently are fatal. We assessed the use of a BSL2 baboon herpesvirus (Papiine herpesvirus 1; HVP2) for predicting the drug sensitivity of BV by comparing the sensitivity of the 2 viruses to 12 antiherpetic drugs. Plaque reduction assays showed that 4 drugs (HBPG, BVdU, PFA, and BrdU) were ineffective against both viruses. Of the 8 effective drugs, both viruses were most sensitive to TFT, whereas sensitivity to the remaining 7 drugs varied between BV and HVP2 as well as between strains of HVP2. In addition, the efficacy of 5 drugs (ACV, PCV, GCV, CDV, and EDU) was tested by using a murine model. ACV and EDU were completely ineffective against both HVP2 and BV, and high doses of PCV only delayed death by a few days. GCV and CDV both protected mice against death, and CDV also prevented the development of neurologic symptoms. When the initiation of drug therapy was delayed until after virus gained access to the CNS, both GCV and CDV were ineffective. The similarity of the drug sensitivities of HVP2 and BV in both models validates the use of HVP2 as a BSL2 level model that can be used to predict drug sensitivity of BV. The greater efficacy of CDV relative to GCV suggests the potential for use of CDV in the treatment of zoonotic BV infections.

Background Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5′,5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors.

BACKGROUND:Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5',5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.OBJECTIVE:To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma.METHODS:We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma.RESULTS:The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers.CONCLUSIONS:The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors.

BACKGROUND:Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target.METHODS:Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests.RESULTS:The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino)uracil (IC50 of 16.75 μM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 μM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 μM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2 cells, with IC50 of > 160 μM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 μM.CONCLUSIONS:The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future.

Background: Diadenosine tetraphosphate (Ap(4)A), a constituent of platelet dense granules, and its P-1, P-4-dithio and/or P-2, P-3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y(1) and P2Y(12). The most active of those analogs, diadenosine 5',5''''-P-1, P-4-dithio-P-2, P-3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.Objective: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma.Methods: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y(1)-mediated changes in cytosolic Ca2+, P2Y(12)-mediated changes in VASP phosphorylation, and metabolism in human plasma.Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P-1 - and P-4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P-2,P-3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers.Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y(1) and P2Y(12) receptors.

The enantiomers of 9-hydroxyrisperidone, the major metabolite of risperidone and the active component of the antipsychotic drug Invega (R), were synthesized by efficient methods. Their optical rotation and enantiomeric purity were characterized for the first time. The absolute configurations were assigned by comparing H-1 NMR chemical shifts of Mosher esters of a key stereoisomeric intermediate. Thus, the stereoisomeric center of (+)-9-hydroxyrisperidone has been assigned as the S configuration, and its enantiomer has the configuration R.