Elmer J. Reist

Antiviral activities of nucleosides and nucleotides against wild-type and drug-resistant strains of murine cytomegalovirus

Resistance of human cytomegalovirus to approved antiviral drugs is becoming a problem of increasing concern. In order to further study drug resistance in a related virus, strains of murine cytomegalovirus (MCMV) have been prepared in vitro by extensive adaptation of the virus to increasingly higher concentrations of either ganciclovir, foscarnet, or (S)-9-(3-hydroxy-2-[phosphonylmethoxy]propyl)cytosine (HPMPC). Plaque reduction 50% effective concentrations (EC50) for the above inhibitors increased 9-, 7-, and 23-fold, respectively (against the corresponding virus), compared to wild-type MCMV. Each virus was then evaluated against other known anti-MCMV agents to determine cross-resistance patterns. These compounds included 3-hydroxy-phosphonylmethoxypropyl derivatives of adenine (HPMPA) and guanine (HPMPG), 2-phosphonylmethoxyethyl derivatives of adenine (PMEA) and 2,6-diaminopurine (PMEDAP), cyclobutylguanine, acyclovir, and the methylene phosphonate derivatives of acyclovir (SR3722) and ganciclovir (SR3773). The ganciclovir-resistant MCMV was cross-resistant to foscarnet, HPMPA, HPMPC, HPMPG, SR3722, and SR3773. The foscarnet-resistant virus was also resistant to acyclovir, PMEA, PMEDAP, SR3722, and SR3773. The HPMPC-resistant MCMV was cross-resistant to HPMPA, HPMPG, and SR3773. Changes in susceptibility were from 3- to 22-fold relative to the wild-type virus. Virus yield reduction data correlated with the plaque assay results. Only cyclobutylguanine was approximately equally active against wild-type and the three drug-resistant MCMVs. The patterns of cross-resistance correlated with resistance seen in human cytomegalovirus strains expressing altered DNA polymerase function. The GCV-resistant and HPMPC-resistant viruses were markedly attenuated in their ability to kill severe combined immunodeficient mice.

The synthesis of methyl 4-O-(4-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-β-d-glucopyranoside: The methyl β-glycoside of the trisaccharide related to Fabry's disease

Abstract As part of a program to synthesize the ceramide trisaccharide ( 1 ) related to Fabrys disease, methyl 4- O -(4- O -α- d -galactopyranosyl-β- d -galactopyranosyl)-β- d -glucopyranoside ( 12 ) was prepared. Methyl β-lactoside ( 2 ) was converted into methyl 4- O -(4,6- O -benzylidene-β- d -galactopyranosyl)-β- d -glucopyranoside ( 4 ). Methyl 2,3,6-tri- O -benzoyl-4- O -(2,3,6-tri- O -benzoyl-β- d -galactopyranosyl)-β- d -glucopyranoside ( 7 ) was synthesized from 4 through the intermediates methyl 2,3,6-tri- O -benzoyl-4- O -(4,6- O -benzylidene-2,3-di- O -benzoyl-β- d -galactopyranosyl)-β- d -glucopyranoside ( 5 ) and methyl 2,3,6-tri- O -benzoyl-4- O -(2,3-di- O -benzoyl-β- d -galactopyranosyl)-β- d -glucopyranoside ( 6 ). The halide-catalyzed condensation of 7 with 2,3,4,6-tetra- O -benzyl- d -galactopyranosyl bromide ( 8 ) gave methyl 2,3,6-tri- O -benzoyl-4- O -[2,3,6-tri- O -benzoyl-4- O -(2,3,4,6-tetra- O -benzyl-α- d -galactopyranosyl)- β- d -galactopyranosyl]-β- d -glucopyranoside ( 10 ). Stepwise deprotection of 10 led to 12 , the methyl β-glycoside of the trisaccharide related to Fabrys disease.

Mutagenic activities of fecapentaene derivatives in the Ames/Salmonella test system

The direct mutagenic activities of a pair of naturally-occurring and several synthetic fecapentaenes were measured in the Ames/Salmonella test system. We found that strain TA100 with preincubation was the most sensitive procedure for the naturally-occurring fecapentaene-12 (FP-12). Its natural analog, FP-14, and the synthetic isomer, cis-FP-12, yielded similar mutagenic activities to FP-12 in the range of 1000-2000 TA100 revertants per microgram of compound. The synthetic analogs of FP-12 and FP-14, MFP-12 and MFP-14, wherein the glycerol moiety was replaced by methoxy, exhibited consistently higher mutagenic activities than their parent fecapentaenes (MFP-12 was about 20 times more potent than FP-12; MFP-14 was about twice the potency of FP-14). The standard rat liver metabolizing system (S9) reduced the activities of all the fecapentaenes in a dose-related manner.

A 13c-n.m.r. study of a di- and a tri-saccharide containing the α-d-galactopyranosyl group

Abstract The 13 C-n.m.r. spectra of methyl 4- O -α- d -galactopyranosyl-α- d -galactopyranoside ( 1 ) and methyl 4- O -[4- O -(α- d -galactopyranosyl)-β- d -galactopyranosyl]-β- d -glucopyranoside ( 2 ) in D 2 O were recorded. Comparison of these spectra with the spectra of methyl α- d -galactopyranoside ( 4 ) and methyl β-lactoside (5) provided substantial confirmation of the structures of 1 and 2 .

Structure-Activity Relationship of Phosphonic Acid Analogs of Acyclovir or Ganciclovir Against Human Cytomegalovirus in MRC-5 Cells

Abstract In vitro anti-HCMV activity of 22 phosphonic acid analogs of acyclovir or ganciclovir (DHPG) was determined in MRC-5 cells to establish structure-activity relationships.

A new synthesis of 4-O-α-d-galactopyranosyl-d-galactopyranose

Abstract The bromide-catalyzed condensation of 2,3,4,6-tetra- O -benzyl- d -galactopyranosyl bromide ( 11 ) with methyl 2,3,6-tri- O -benzoyl-α- d -galactopyranoside ( 3 ) gave methyl 2,3,6-tri- O -benzoyl-4- O -(2,3,4,6-tetra- O -benzyl-α- d -galactopyranosyl)-α- d -galactopyranoside ( 12 ) in 83% yield. The yield of this glycosidation reaction was high, despite the axial orientation of the 4-hydroxyl group of 3 . Stepwise deprotection of 12 afforded methyl 4- O -α- d -galactopyranosyl-α- d -galactopyranoside ( 15 ). Acetylation of 15 , followed by acetolysis, gave the known α-octaacetate 17 . This scheme constituted a total synthesis of 4- O -α- d -galactopyranosyl- d -galactopyranose ( 2 ) in 25% yield from 3 . The disaccharide 2 is the terminal disaccharide of the ceramide trisaccharide related to Fabrys disease.

Mapping cyclic AMP binding sites on type I and type II cyclic AMP-dependent protein kinases using 2-substituted derivatives of cyclic AMP

Abstract Twenty-one derivatives of cyclic AMP with substituents or modification in the 2-position were examined for their ability to activate rabbit skeletal muscle type I cyclic AMP-dependent protein kinase (PK I) and bovine heart type II cyclic AMP- dependent protein kinase (PK II). PK I had stricter steric requirements than did PK II for the binding locale on the protein kinases adjacent to the 2 position of cyclic AMP. Derivatives with substituents that caused electron withdrawal from the purine ring were better than cyclic AMP as activators of PK I, but were less active than cyclic AMP as activators of PK II.

Synthesis of 6,6′-disubstituted sucrose derivatives from 1′,6,6′-tri-O-tripsylsucrose

Abstract An improved synthesis of 6,6′-disubstituted derivatives of sucrose is described that uses 1′,6,6′-tri- O -tripsylsucrose as the starting material. The 6- and 6′-tripsyloxy groups have been selectively displaced, leaving the (more sterically hindered) 1′-tripsyloxy group untouched. Subsequent displacement of the 1′-tripsyloxy group by sodium benzoate, under forcing conditions, effected sulfonate displacement and gave sucrose derivatives substituted at C-6 and C-6′.

Synthesis of acyclonucleoside phosphonates as antiviral agents against cytomegalovirus

Abstract Phosphonic acid analogs of ACV and DHPG that are isosteric with ACV phosphate and DHPG phosphate have been synthesized and evaluated for antiviral activity against human, murine, and guinea pig strains of cytomegalovirus. The phosphonates showed high activity against all of the strains. They were also evaluated against a DHPG resistant strain of human cytomegalovirus. Although the activity dropped considerably, significant antiviral activity was still evident.

EFFECT OF PHOSPHONIC ACID ANALOGS OF ACYCLOVIR AND GANCICLOVIR ON IN VITRO CYTOMEGALOVIRUS INFECTIONS

Abstract Eight phosphonic acid analogs of acyclovir (ACV) or ganciclovir (DHPG) inhibited human cytomegalovirus in vitro. Therapeutic indices were: phosphonate diacid of DHPG: 500; DHPG: 500; phosphonate monoethylester of DHPG: 258; phosphonate monoethylester of ACV: 94; cyclic phosphonate of DHPG: 64; ACV: 60; phosphonate monobutylester of ACV: 1.5; phosphonate monoethylester of deoxy DHPG: 4.6; 8-bromo ACV phosphonate monoethylester: >2; phosphonate monoethylester heptyl of ACV: 1. Types 1 and 2 herpesvirus (HSV-1, HSV-2) and varicella zoster virus (VZV) were poorly inhibited by these new compounds, suggesting highly specific anti-HCMV activity. None exhibited significant cytotoxic effects as measured by uptake of [3H]thymidine, [3H]uridine and [3H]leucine.