Taracad K. Venkatachalam

Rational design and synthesis of phenethyl-5-bromopyridyl thiourea derivatives as potent non-nucleoside inhibitors of HIV reverse transcriptase

Abstract A series of novel phenethylthiazolylthiourea (PETT) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of HIV reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket. The structure-based design and synthesis of these new PETT derivatives were complemented by biological assays of their anti-HIV activity. Modeling studies for rational drug design included the construction of a composite NNI binding pocket from nine RT-NNI crystal structures, the analyses of surface complementarity between NNI and RT, and application of K i calculations combined with a docking procedure involving the novel PETT derivatives. The use of the composite NNI binding pocket allowed the identification and structure-based design of three promising PETT derivatives with ortho -F ( 2 ), ortho -Cl ( 3 ), and meta -F ( 5 ) substituents on the phenyl ring. These novel PETT derivatives were more active than AZT or trovirdine and showed potent anti-HIV activity with IC 50 [p24] values of 100,000.

Novel Thiourea Compounds as Dual-Function Microbicides

Abstract Sexually active women represent the fastest growing human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we previously reported the synthesis of novel nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase with sperm-immobilizing activity (SIA). To gain further insight into the structure-function relationship controlling these two properties of NNIs, we have rationally designed and synthesized 30 novel thiourea compounds and examined them for dual-function, anti-HIV and spermicidal activity. Twelve of the 30 thiourea compounds exhibited potent anti-HIV activity in the nanomolar range (IC50 = <1–9 nM). Nine of the 30 thiourea derivatives exhibited both anti-HIV and spermicidal activity. Among the phenyl ring-containing thioureas, the 2-fluoro (HI-240) -substituted and 2-chloro (HI-253) -substituted derivatives exhibited potent anti-HIV activity (IC50 = <1 nM) with SIA (EC50 = 70 μM and 147 μM). Among the alicyclic ring-containing thioureas, the 5-bromo (HI-346) and 5-chloro (HI-445) functionalized cyclohexenyl ring-substituted thioureas were the most potent dual-function spermicides (EC50 = 42 and 57 μM), with anti-HIV activity at nanomolar range (IC50 = 3 nM). Unlike nonoxynol-9 (N-9), none of the potent dual-function thiourea compounds were cytotoxic to normal human vaginal, ectocervical, and endocervical epithelial cells at spermicidal concentrations. We conclude that as potent anti-HIV agents with SIA and reduced cytotoxicity when compared with N-9, the phenyl-substituted and cyclohexenyl-substituted thiourea derivatives, especially compounds HI-253 (N-[2-(2-chlorophenethyl)]-N′-[2-(5-bromopyridyl)-thiourea), HI-346 (N-[2-(5-bromopyridinyl)]-N′-[2-(1-cyclohexenyl)ethyl-thiourea), and HI-445 (N-[2-(5-chloropyridinyl)]-N′-[2-(1-cyclohexenyl)ethyl-thiourea) show unique clinical potential to become the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Structure-based drug design of non-nucleoside inhibitors for wild-type and drug-resistant HIV reverse transcriptase

The generation of anti-HIV agents using structure-based drug design methods has yielded a number of promising non-nucleoside inhibitors (NNIs) of HIV reverse transcriptase (RT). Recent successes in identifying potent NNIs are reviewed with an emphasis on the recent trend of utilizing a computer model of HIV RT to identify space in the NNI binding pocket that can be exploited by carefully chosen functional groups predicted to interact favorably with binding pocket residues. The NNI binding pocket model was used to design potent NNIs against both wild-type RT and drug-resistant RT mutants. Molecular modeling and score functions were used to analyze how drug-resistant mutations would change the RT binding pocket shape, volume, and chemical make-up, and how these changes could affect inhibitor binding. Modeling studies revealed that for an NNI of HIV RT to be active against RT mutants such as the especially problematic Y181C RT mutant, the following features are required: (a) the inhibitor should be highly potent against wild-type RT and therefore capable of tolerating a considerable activity loss against RT mutants (i.e. a picomolar-level inhibitor against wild-type RT may still be effective against RT mutants at nanomolar concentrations), (b) the inhibitor should maximize the occupancy in the Wing 2 region of the NNI binding site of RT, and (c) the inhibitor should contain functional groups that provide favorable chemical interactions with Wing 2 residues of wild-type as well as mutant RT. Our rationally designed NNI compounds HI-236, HI-240, HI-244, HI-253, HI-443, and HI-445 combine these three features and outperform other anti-HIV agents examined.

5-Alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-ones as potent non-nucleoside reverse transcriptase inhibitors of S-DABO series

Novel dihydroalkoxybenzyloxopyrimidine (S-DABO) derivatives targeting the non-nucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been synthesized using a novel computer model for the NNI binding pocket and tested for their RT inhibitory activity in cell-free assays using purified recombinant HIV RT as well as for their anti-HIV activity in HTL VIIIB-infected peripheral blood mononuclear cells. Our computational approach allowed the identification of several ligand derivatization sites for the generation of more potent S-DABO derivatives. Our lead S-DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one (compound 3), elicited potent anti-HIV activity with an IC50 value of less than 1nM for inhibition of HIV replication without any evidence of cytotoxicity and an unprecedented selectivity index of > 100,000.

Stereochemistry of halopyridyl and thiazolyl thiourea compounds is a major determinant of their potency as nonnucleoside inhibitors of HIV-1 reverse transcriptase.

Chiral derivatives of two cyclohexylethyl halopyridyl thiourea compounds (HI-509 and HI-510), two alpha-methyl benzyl halopyridyl compounds (HI-511 and HI-512), and a cyclohexyl ethyl thiazolyl thiourea compound (HI-513) were synthesized as nonnucleoside inhibitors (NNI) of human immunodeficiency virus (HIV) reverse transcriptase (RT). The R stereoisomers of all five compounds inhibited the recombinant RT in vitro with 100-fold lower IC50 values. HI-509R, HI-510R, HI-511R, HI-512R and HI-513R were active anti-HIV agents and inhibited HIV-1 replication in human peripheral blood mononuclear cells at nanomolar concentrations, whereas their enantiomers were inactive. Each of these five compounds was also active against NNI-resistant HIV-1 strains, with HI-511R being the most active agent. When tested against the NNI-resistant HIV-1 strain A17 with a Y181C mutation in RT, HI-511R was found to be 10,000-times more active than nevirapine, 5000-times more active than delavirdine, and 50-times more active than trovirdine. HI-511 R inhibited the HIV-strain A17 variant, containing RT mutations Y181C plus K103N, with an IC50 value of 2.7 microM, whereas the IC50 values of nevirapine, delavirdine, and trovirdine against this highly NNI-resistant HIV-1 strain were >100 microM.

In Vivo Antiretroviral Activity of Stampidine in Chronically Feline Immunodeficiency Virus-Infected Cats

ABSTRACT Here we report the antiretroviral activity of the experimental nucleoside reverse transcriptase inhibitor (NRTI) compound stampidine in cats chronically infected with feline immunodeficiency virus (FIV). Notably, a single oral bolus dose of 50 or 100 mg of stampidine per kg resulted in a transient ≥1-log decrease in the FIV load of circulating peripheral blood mononuclear cells in five of six FIV-infected cats and no side effects. A 4-week stampidine treatment course with twice-daily administration of hard gelatin capsules containing 25 to 100 mg of stampidine per kg was also very well tolerated by cats at cumulative dose levels as high as 8.4 g/kg and exhibited a dose-dependent antiretroviral effect. One of three cats treated at the 25-mg/kg dose level, three of three cats treated at the 50-mg/kg dose level, and three of three cats treated at the 100-mg/kg dose level (but none of three control cats treated with placebo pills) showed a therapeutic response, as evidenced by a ≥1-log reduction in the FIV load in peripheral blood mononuclear cells within 2 weeks. The previously documented in vitro and in vivo antiretroviral activity of stampidine against primary clinical human immunodeficiency virus type 1 isolates with genotypic and/or phenotypic NRTI resistance, together with its favorable animal toxicity profile, pharmacokinetics, and in vivo antiretroviral activity in FIV-infected cats, warrants further development of this promising new NRTI compound.

Structure-based design of non-nucleoside reverse transcriptase inhibitors of drug-resistant human immunodeficiency virus.

A computer model of reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to design thiourea compounds that were predicted to inhibit RT. The RT model was used to approximate how changes in binding pocket shape, volume and chemical properties resulting from residue mutations would affect inhibitor binding. Our lead compound, N-[2-(2,5-dime-thoxyphenylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (HI-236) was tested against clinically observed non-nucleoside inhibitor (NNI)-resistant mutated strains of HIV. HI-236 was more potent than trovirdine, MKC-442 and zidovudine against the drug-sensitive HIV-1 strain IIIB, 50–100 times more effective than delavirdine or nevirapine and twice as effective as our recently reported lead compound N-[2-(2-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (HI-240) against the NNI-resistant Y181C mutant HIV-1 strain A17. HI-236 was highly effective against the multidrug-resistant HIV-1 strain RT-MDR containing multiple mutations involving the RT residues 74V, 41L, 106A and 215Y. In general, thiourea compounds such as HI-236 and HI-240 showed better inhibition of drug-resistant strains of HIV-1 than thioalkylbenzyl-pyrimidine compounds such as HI-280 and HI-281. The improved activity of thioureas against RT mutants is consistent with a structural analysis of the NNI binding pocket model of RT. The activity of HI-236 against RT-MDR was superior to that of other anti-HIV agents tested, in the following order, from high to low activity; HI-236 (IC50 5 nM), HI-240 (IC50 6 nM), trovirdine (IC50 20 nM), zidovudine (IC50 150 nM), MKC-442 (IC50 300 nM), delavirdine (IC50 400 nM) and nevirapine (IC50 5 µM).

Stampidine Is a Potent Inhibitor of Zidovudine- and Nucleoside Analog Reverse Transcriptase Inhibitor-Resistant Primary Clinical Human Immunodeficiency Virus Type 1 Isolates with Thymidine Analog Mutations

ABSTRACT We report the antiretroviral activity of stavudine-5′-(p-bromophenyl methoxyalaninyl phosphate) (stampidine [STAMP]), a novel aryl phosphate derivative of stavudine, against primary clinical human immunodeficiency virus type 1 (HIV-1) isolates. STAMP inhibited each one of nine clinical HIV-1 isolates of non-B envelope subtype and 20 genotypically and phenotypically nucleoside analog reverse transcriptase inhibitor-resistant HIV-1 isolates at subnanomolar to low-nanomolar concentrations.

In Vivo Toxicity, Pharmacokinetics, and Anti-Human Immunodeficiency Virus Activity of Stavudine-5′-(p-Bromophenyl Methoxyalaninyl Phosphate) (Stampidine) in Mice

ABSTRACT We have evaluated the clinical potential of stavudine-5′-(p-bromophenyl methoxyalaninyl phosphate(stampidine [STAMP]), a novel aryl phosphate derivative of stavudine, as a new anti-human immunodeficiency virus (anti-HIV) agent, by examining its acute, subacute, and chronic toxicity profile in mice as well as by testing its antiviral activity in a surrogate human peripheral blood lymphocyte (Hu-PBL)-SCID mouse model of human AIDS. STAMP was very well tolerated in BALB/c and CD-1 mice, without any detectable acute or subacute toxicity at single intraperitoneal or oral bolus doses as high as 500 mg/kg of body weight. Notably, daily administration of STAMP intraperitoneally or orally for up to 8 consecutive weeks was not associated with any detectable toxicity at cumulative dose levels as high as 6.4 g/kg. Micromolar concentrations of the active STAMP metabolite in plasma were rapidly achieved and maintained for more than 4 h after parenteral as well as oral administration of a nontoxic 100-mg/kg bolus dose of STAMP. In accordance with its favorable pharmacokinetic profile and in vitro potency, STAMP exhibited dose-dependent and potent in vivo anti-HIV activity in Hu-PBL-SCID mice against a genotypically and phenotypically nucleoside analog reverse transcriptase inhibitor (NRTI)-resistant clinical HIV type 1 (HIV-1) isolate (BR/92/019; D67N, L214F, T215D, K219Q) at nontoxic dose levels. The remarkable in vivo safety and potency of STAMP warrants the further development of this promising new antiretroviral agent for possible clinical use in patients harboring NRTI-resistant HIV-1.

Regiospecific synthesis, X-ray crystal structure and biological activities of 5-bromothiophenethyl thioureas

Abstract The regiospecific synthesis of 5-bromothiophenethyl thioureas was accomplished in four steps with an overall yield of 40–60%. The requisite regioselectivity for bromination of the thiophene ring was achieved using bromine in acetic acid at low temperatures. The resulting 5-bromothiophenethylamine hydrobromide is an important precursor for the preparation of substituted thioureas. The X-ray crystal structure demonstrates that the bromine atom is indeed located at the 5-position of the thiophene ring.