Toru Kontani

ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2

OBJECTIVES To evaluate and describe the anti-herpesvirus effect of ASP2151, amenamevir, a novel non-nucleoside oxadiazolylphenyl-containing herpesvirus helicase-primase complex inhibitor. METHODS The inhibitory effect of ASP2151 on enzymatic activities associated with a recombinant HSV-1 helicase-primase complex was assessed. To investigate the effect on viral DNA replication, we analysed viral DNA in cells infected with herpesviruses [herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus]. Sequencing analyses were conducted on an ASP2151-resistant VZV mutant. In vitro and in vivo antiviral activities were evaluated using a plaque reduction assay and an HSV-1-infected zosteriform-spread model in mice. RESULTS ASP2151 inhibited the single-stranded DNA-dependent ATPase, helicase and primase activities associated with the HSV-1 helicase-primase complex. Antiviral assays revealed that ASP2151, unlike other known HSV helicase-primase inhibitors, exerts equipotent activity against VZV, HSV-1 and HSV-2 through prevention of viral DNA replication. Further, the anti-VZV activity of ASP2151 (EC(50), 0.038-0.10 microM) was more potent against all strains tested than that of aciclovir (EC(50), 1.3-27 microM). ASP2151 was also active against aciclovir-resistant VZV. Amino acid substitutions were found in helicase and primase subunits of ASP2151-resistant VZV. In a mouse zosteriform-spread model, ASP2151 was orally active and inhibited disease progression more potently than valaciclovir. CONCLUSIONS ASP2151 is a novel herpes helicase-primase inhibitor that warrants further investigation for the potential treatment of both VZV and HSV infections.

Novel 7H-pyrrolo[2,3-d]pyrimidine derivatives as potent and orally active STAT6 inhibitors.

Signal transducers and activators of transcription 6 (STAT6) is an important transcription factor in interleukin (IL)-4 signaling pathway and a key regulator of the type 2 helper T (Th2) cell immune response. Therefore, STAT6 may be an excellent therapeutic target for allergic conditions, including asthma and atopic diseases. Previously, we reported 4-aminopyrimidine-5-carboxamide derivatives as STAT6 inhibitors. To search for novel STAT6 inhibitors, we synthesized fused bicyclic pyrimidine derivatives and identified a 7H-pyrrolo[2,3-d]pyrimidine derivative as a STAT6 inhibitor. Optimization of the pyrrolopyrimidine derivatives led to identification of 2-[4-(4-{[7-(3,5-difluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]amino}phenyl)piperazin-1-yl]acetamide (24, AS1810722) which showed potent STAT6 inhibition and a good CYP3A4 inhibition profile. Compound 24 also inhibited in vitro Th2 differentiation without affecting type 1 helper T (Th1) cell differentiation and eosinophil infiltration in an antigen-induced mouse asthmatic model after oral administration.

Pharmacokinetics and Pharmacodynamics of ASP2151, a Helicase-Primase Inhibitor, in a Murine Model of Herpes Simplex Virus Infection

ABSTRACT ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus 1 (HSV-1), HSV-2, and varicella zoster virus. Here, to determine and analyze the correlation between the pharmacodynamic (PD) and pharmacokinetic (PK) parameters of ASP2151, we examined the PD profile of ASP2151 using in vitro plaque reduction assay and a murine model of HSV-1 infection. ASP2151 inhibited the in vitro replication of HSV-1 with a mean 50% effective concentration (EC50) of 14 ng/ml. In the cutaneously HSV-1-infected mouse model, ASP2151 dose dependently suppressed intradermal HSV-1 growth, with the effect reaching a plateau at a dose of 30 mg/kg of body weight/day. The dose fractionation study showed that intradermal HSV-1 titers were below the detection limit in mice treated with ASP2151 at 100 mg/kg/day divided into two daily doses and at 30 or 100 mg/kg/day divided into three daily doses. The intradermal HSV-1 titer correlated with the maximum concentration of drug in serum (Cmax), the area under the concentration-time curve over 24 h (AUC24h), and the time during which the concentration of ASP2151 in plasma was above 100 ng/ml (T>100). The continuous infusion of ASP2151 effectively decreased intradermal HSV-1 titers below the limit of detection in mice in which the ASP2151 concentration in plasma reached 79 to 145 ng/ml. Our findings suggest that the antiviral efficacy of ASP2151 is most closely associated with the PK parameter T>100 in HSV-1-infected mice. Based on these results, we propose that a plasma ASP2151 concentration exceeding 100 ng/ml for 21 to 24 h per day provides the maximum efficacy in HSV-1-infected mice.

Identification, synthesis, and biological evaluation of 6-[(6R)-2-(4-fluorophenyl)-6-(hydroxymethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl)pyridazin-3(2H)-one (AS1940477), a potent p38 MAP kinase inhibitor.

Several p38 MAPK inhibitors have been shown to effectively block the production of cytokines such as IL-1β, TNFα, and IL-6. Inhibitors of p38 MAP kinase therefore have significant therapeutic potential for the treatment of autoimmune disease. Compound 2a was identified as a potent TNFα production inhibitor in vitro but suffered from poor oral bioavailability. Structural modification of 2a led to the discovery of tetrahydropyrazolopyrimidine derivatives, exemplified by compound 3, with an improved pharmacokinetic profile. We found that blocking metabolism at the methyl group of the amine and constructing the tetrahydropyrimidine core were important to obtaining compounds with good biological profiles and oral bioavailability. Pursuing the structure-activity relationships of this series led to the discovery of AS1940477 (3f), with excellent cellular activity and a favorable pharmacokinetic profile. This compound represents a highly potent inhibitor of p38 MAP kinase with regard to in vivo activity in an adjuvant-induced arthritis model.

Effect of ASP2151, a Herpesvirus Helicase-Primase Inhibitor, in a Guinea Pig Model of Genital Herpes

ASP2151 is a herpesvirus helicase-primase inhibitor with antiviral activity against varicella zoster virus and herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here, we examined the potency and efficacy of ASP2151 against HSV in vitro and in vivo. We found that ASP2151 was more potent in inhibiting the replication of HSV-1 and HSV-2 in Vero cells in the plaque reduction assay and had greater anti-HSV activity in a guinea pig model of genital herpes than did acyclovir and valacyclovir (VACV), respectively. Oral ASP2151 given from the day of infection reduced peak and overall disease scores in a dose-dependent manner, resulting in complete prevention of symptoms at the dose of 30 mg/kg. The 50% effective dose (ED50) values for ASP2151 and VACV were 0.37 and 68 mg/kg, respectively, indicating that ASP2151 was 184-fold more potent than VACV. When ASP2151 was administered after the onset of symptoms, the disease course of genital herpes was suppressed more effectively than by VACV, with a significant reduction in disease score observed one day after starting ASP2151 at 30 mg/kg, whereas the therapeutic effect of VACV was only evident three days after treatment at the highest dose tested (300 mg/kg). This indicated that ASP2151 possesses a faster onset of action and wider therapeutic time window than VACV. Further, virus shedding from the genital mucosa was significantly reduced with ASP2151 at 10 and 30 mg/kg but not with VACV, even at 300 mg/kg. Taken together, our present findings demonstrated the superior potency and efficacy of ASP2151 against HSV.

Characterization of virus strains resistant to the herpes virus helicase-primase inhibitor ASP2151 (Amenamevir).

ASP2151 is an antiherpes agent targeting the helicase-primase complex of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV). We characterized the ASP2151-resistant HSV-1 and HSV-2 variants or mutants based on findings from sequencing analysis, growth, pathogenicity, and susceptibility testing, identifying several single base-pair substitutions resulting in amino acid changes in the helicase and primase subunit of ASP2151-resistant mutants. Amino acid alterations in the helicase subunit were clustered near helicase motif IV in the UL5 helicase gene of both HSV-1 and HSV-2, while the primase subunit substitution associated with reduced susceptibility, R367H, was found in ASP2151-resistant HSV-1 mutants. However, while susceptibility in the ASP2151-resistant HSV mutants to existing antiherpes agents was equivalent to that in wild-type HSV strains, ASP2151-resistant HSV mutants showed attenuated in vitro growth capability and in vivo pathogenicity compared with the parent strains. Taken together, our present findings demonstrated that important amino acid substitutions associated with reduced susceptibilities of HSV-1 and HSV-2 to ASP2151 exist in both the helicase and primase subunits of the helicase-primase complex, and that mutations in this complex against ASP2151 might confer defects in viral replication and pathogenicity.

Susceptibility of Herpes Simplex Virus Isolated from Genital Herpes Lesions to ASP2151, a Novel Helicase-Primase Inhibitor

ABSTRACT ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. To evaluate the anti-HSV activity of ASP2151, susceptibility testing was performed on viruses isolated from patients participating in a placebo- and valacyclovir-controlled proof-of-concept phase II study for recurrent genital herpes. A total of 156 HSV strains were isolated prior to the dosing of patients, and no preexisting variants with less susceptibility to ASP2151 or acyclovir (ACV) were detected. ASP2151 inhibited HSV-1 and HSV-2 replication with mean 50% effective concentrations (EC50s) of 0.043 and 0.069 μM, whereas ACV exhibited mean EC50s of 2.1 and 3.2 μM, respectively. Notably, the susceptibilities of HSV isolates to ASP2151 and ACV were not altered after dosing with the antiviral agents. Taken together, these results demonstrate that ASP2151 inhibits the replication of HSV clinical isolates more potently than ACV, and HSV resistant to this novel helicase-primase inhibitor as well as ACV may not easily emerge in short-term treatment for recurrent genital herpes patients.

Regioselective Alkylation of Thiazolylsulfonamides: Direct and Efficient Synthesis of 3‐Alkylthiazolidene Derivatives

Abstract Various N‐3‐alkylated thiazolidenesulfonamide derivatives were efficiently prepared by the direct endo‐selective alkylation of thiazolylsulfonamides. The effects of different bases and solvents were investigated, and the NaH–THF combination was found to be the most effective at conferring high yields and endo‐selectivity.

Novel quinuclidinyl heteroarylcarbamate derivatives as muscarinic receptor antagonists.

Herein, we describe the synthesis and pharmacological profiles of novel quinuclidinyl heteroarylcarbamate derivatives. Among them, the quinuclidin-4-yl thiazolylcarbamate derivative ASP9133 was identified as a promising long-acting muscarinic antagonist (LAMA) showing more selective inhibition of bronchoconstriction against salivation and more rapid onset of action in a rat model than tiotropium bromide.