Shaoman Zhou

In Vitro Activity and Mechanism of Action of Methylenecyclopropane Analogs of Nucleosides against Herpesvirus Replication

ABSTRACT We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8. None of the 18 compounds had activity against HSV-1 or HSV-2, but four were active against VZV by plaque reduction (PR) assay at 50% effective concentration (EC50) levels of ≤50 μM. Six of the 18 compounds were active against HCMV by cytopathic effect or PR assays with EC50s of 0.5 to 44 μM, and all were active against MCMV by PR (0.3 to 54 μM). Four of the compounds were active against EBV by enzyme-linked immunosorbent assay (<0.3 to 4.4 μM). Four compounds with CMV activity were also active against HHV-6A and HHV-6B (0.7 to 28 μM), and three compounds were active against HHV-8 (5.5 to 16 μM). One of these, ZSM-I-62, had particularly good activity against CMV, HHV-6, and HHV-8, with EC50s of 0.7 to 8 μM. Toxicity was evaluated in adherent and nonadherent cells, and minimal cytotoxicity was observed. Mechanism of action studies with HCMV suggested that these compounds are phosphorylated by the ppUL97 phosphotransferase and are potent inhibitors of viral DNA synthesis. These results indicate that at least one of these compounds may have potential for use in treating CMV and other herpesvirus infections in humans.

Fluoroanalogues of Anti-Cytomegalovirus Agent Cyclopropavir: Synthesis and Antiviral Activity of (E)- and (Z)-9-{[2,2-Bis(Hydroxymethyl)-3-Fluorocyclopropylidene]Methyl}-Adenines and Guanines

Synthesis of fluorinated cyclopropavir analogues 13a, 13b, 14a, and 14b is described starting from alkene 15. Addition of carbene derived from dibromofluoromethane gave bromofluoro cyclopropane 16. Reduction (compound 17) followed by desilylation gave intermediate 18, which was transformed to 2-nitrophenylselenenyl derivative 19. Oxidation to selenoxide 20 was followed by β-elimination to afford methylenecyclopropane 21. Addition of bromine provided compound 22 for alkylation-elimination of adenine and 2-amino-6-chloropurine. The resultant E,Z isomeric mixtures of methylenecyclopropanes 23a + 24a and 23c + 24c were resolved and the individual isomers were deprotected to give adenine analogues 13a and 14a as well as compounds 13c and 14c. Hydrolytic dechlorination of 13c and 14c furnished guanine analogues 13b and 14b. The only significant antiviral effects were observed with analogue 13a against HCMV and 14a against VZV in cytopathic inhibition assays.

Synthesis Of 2,2,3-Tris(Hydroxymethyl)Methylenecyclopropane Analogues Of Nucleosides

Synthesis of 2,2,3-tris(hydroxymethyl)methylenecyclopropane analogues 16a, 16b, 17a, and 17b is described. Diethyl ester of Feists acid 18b was hydroxymethylated via carbanion formation using formaldehyde under simultaneous isomerization to cis diester to give intermediate 19. Reduction followed by acetylation gave triacetate 22. Addition of bromine afforded reagent 23, which was used for alkylation-elimination of adenine and 2-amino-6-chloropurine to provide Z,E-isomeric mixtures of 24a and 24b. Deacetylation and separation furnished the Z-isomers 16a, 16c and E-isomers 17a, 17c. Hydrolytic dechlorination of 16c and 17c gave guanine analogues 16b and 17b. None of the analogues exhibited a significant antiviral activity. Adenosine deaminase is refractory toward adenine analogues 16a and 17a.

Stereoselective approach to the Z-isomers of methylenecyclopropane analogues of nucleosides: a new synthesis of antiviral synguanol.

Stereoselective synthesis of antiviral synguanol (1) is described. Reaction of 6-benzyloxy-2-(dimethylaminomethyleneamino)purine (10) with ethyl (cis,trans)-2-chloro-2-(chloromethyl) cyclopropane-1-carboxylate (2c) under the conditions of alkylation-elimination gave (Z)-6- benzyloxy-2-formylamino-9-[(2-carbethoxycyclopropylidene)methyl]purine (11) but no E,N9-isomer. Minor amounts of (Z)-6-benzyloxy-2-formylamino-7-[(2-carbethoxy-cyclopropylidene)methyl]purine (13) were also obtained. Hydrolysis of compounds 11 and 13 in 80% acetic acid afforded (Z)-9-[2-(carbethoxycyclopropylidene)methyl]guanine (14) and (Z)-7-[2-(carbethoxy- cyclopropylidene)methyl]guanine (15). Reduction of 14 furnished synguanol (1). Reaction of N4-acetylcytosine (7) with ester 2c led to (Z,E)-1-(2-carbethoxycyclopropropylidenemethyl)cytosine (8, Z/E ratio 6.1:1). Basicity of purine base, lower reactivity of alkylation intermediates as well as interaction of the purine N3 or cytosine O2 atoms with the carbonyl group of ester moiety seem to be essential for the observed high stereoselectivity of the alkylation-elimination. The Z-selectivity is interpreted in terms of E1cB mechanism leading to a transitory “cyclic” cyclopropenes which undergo a cyclopropene-methylenecyclopropane rearrangement.