Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent. Trovirdine is currently in phase one clinical trials for potential use in the treatment of AIDS. Extension of these structure-activity relationship studies to identify additional compounds in this series with improved properties is ongoing. A part of this work is described here. Replacement of the two aromatic moieties of the PETT compounds by various substituted or unsubstituted heteroaromatic rings was investigated. In addition, the effects of multiple substitution in the phenyl ring were also studied. The antiviral activities were determined on wild-type and constructed mutants of HIV-1 RT and on wild-type HIV-1 and mutant viruses derived thereof, Ile100 and Cys181, in cell culture assays. Some selected compounds were determined on double-mutant viruses, HIV-1 (Ile 100/Asn103) and HIV-1 (Ile100/Cys181). A number of highly potent analogs were synthesized. These compounds displayed IC50's against wild-type RT between 0.6 and 5 nM. In cell culture, these agents inhibited wild-type HIV-1 with ED50's between 1 and 5 nM in MT-4 cells. In addition, these derivatives inhibited mutant HIV-1 RT (Ile 100) with IC50's between 20 and 50 nM and mutant HIV-1 RT (Cys 181) with IC50's between 4 and 10 nM, and in cell culture they inhibited mutant HIV-1 (Ile100) with ED50's between 9 and 100 nM and mutant HIV-1 (Cys181) with ED50's between 3 and 20 nM.     

Polyanion inhibitors of HIV and other viruses. 7. Polyanionic compounds and polyzwitterionic compounds derived from cyclodextrins as inhibitors of HIV transmission

New polyanionic compounds were obtained from radical addition of thiomalic acid and mercaptopropionic acid onto perallylated cyclodextrins (CDs) under UV irradiation with a catalytic amount of alpha,alpha'-azobis(isobutyronitrile). All these polyanions, bearing 18-48 carboxylate groups, inhibited human immunodeficiency virus type 1 (HIV-1) strain IIIB replication in MT-4 cells at a 50% inhibitory concentration (IC50) of 0.1-2.9 microM, while not being toxic to the host cells at concentrations up to 62 microM. These compounds were also active against a clinical HIV-1 isolate (HE) at >/=4-fold higher concentrations. Only some compounds showed activity against the two HIV-2 strains (ROD and EHO) but at higher concentrations than those required to inhibit HIV-1 (IIIB and HE) replication. In addition, these compounds were not active against the M-tropic HIV-1 strain BaL but were active against simian immunodeficiency virus [SIV (MAC251)]. These compounds were also inhibitory to the replication of human cytomegalovirus at an IC50 of 1-10 microM, but not herpes simplex virus (type 1 and type 2) or other (picorna-, toga-, reo-, orthomyxo-, paramyxo-, bunya-, rhabdo-, and poxvirus) viruses. Radical addition on perallylated CDs of a protected cysteine gave polyzwitterionic compounds. None of these last compounds proved inhibitory to the replication of HIV-1, HIV-2, or any of the other viruses tested.     

Macromolecular recognition: effect of multivalency in the inhibition of binding of yeast mannan to concanavalin A and pea lectins by mannosylated dendrimers

The synthesis and binding properties of a new family of high affinity alpha-D-mannopyranoside ligands are described. The synthesis of the new multivalent ligands is based on the scaffolding of multiantennary branches of L-lysine residues having electrophilic N-chloroacetylated end groups as core structures. An alpha-D-mannopyranoside with p-substituted aryl aglycon ending with a thiol group was prepared and covalently attached to each of the branches of the dendritic structures. The resulting glycodendrimers with 2 (12), 4 (14), 8 (16), and 16 (18) mannoside residues were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl and p-nitrophenyl alpha-D-mannopyranosides as standards. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohemagglutinin (Canavalia ensiformis, concanavalin A) and to pea lectin (Pisum sativum) were determined. Analogous mannosylated copolyacrylamides were also prepared for comparison. The IC50 values were also plotted as a function of dendrimer valencies. The inhibitions showed 16-mer 18 to be approximately 600- and 2000-fold more potent than methyl alpha-D-mannopyranoside, and 66- and 1383-fold more potent than p-nitrophenyl alpha-D-mannopyranosides with Con A and pea lectins, respectively. Even when these numbers are expressed relative to single mannopyranoside residues per dendrimers, the relative potencies against the aromatic mannoside are still 4- and 86-fold better against Con A and pea lectins. These results unequivocally indicate that the optimum inhibitory binding properties of the new mannosylated dendrimers vary with both dendrimers and lectin valencies.     

Resistance-modifying agents. 5. Synthesis and biological properties of quinazolinone inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP)

Clinical studies concerning the role of poly(ADP-ribose) polymerase (PARP) in the repair of drug- and radiation-induced DNA damage have been impeded by the poor solubility, lack of potency, and limited specificity of currently available inhibitors. A series of 2-alkyl- and 2-aryl-substituted 8-hydroxy-, 8-methoxy-, and 8-methylquinazolin-4(3H)-ones has been synthesized and evaluated for PARP inhibitory activity in permeabilized L1210 murine leukemia cells. 8-Methoxy- and 8-methylquinazolinones (14-34) were readily prepared by acylation of 3-substituted anthranilamides with the appropriate acid chloride, followed by base-catalyzed cyclization. The requisite 8-hydroxyquinazolinones (6, 35-39) were synthesized by demethylation of the corresponding 8-methoxyquinazolinones with BBr3. N-Methylation of 8-methoxy-2-methylquinazolinone (15) with MeI, followed by O-demethylation by BBr3, afforded the control N3-methylquinazolinones 42 and 43, respectively. In general, an 8-hydroxy or 8-methyl substituent enhanced inhibitory activity in comparison with an 8-methoxy group. 2-Phenylquinazolinones were marginally less potent than the corresponding 2-methylquinazolinones, but the introduction of an electron-withdrawing or electron-donating 4'-substituent on the 2-aryl ring invariably increased potency. This was particularly evident in the 8-methylquinazolinone series (IC50 values 0.13-0.27 microM), which are among the most potent PARP inhibitors reported to date. N3-Methylquinazolinones 42 and 43 were essentially devoid of activity (IC50 values > 100 microM). In studies with L1210 cells in vitro, a concentration of 200 microM 8-hydroxy-2-methylquinazolinone (6, NU1025) (IC50 value 0.40 microM) potentiated the cytotoxicity of the monomethylating agent 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide and gamma-radiation 3.5- and 1.4-fold, respectively, at the 10% survival level.     

Indole and benzimidazole derivatives as steroid 5alpha-reductase inhibitors in the rat prostate

A novel series of indole and benzimidazole derivatives were synthesized and evaluated for their inhibitory activity of rat prostatic 5alpha-reductase. Among these compounds, 4-?2-[1-(4,4'-dipropylbenzhydryl)indole-5-carboxamido]phenoxy?buty ric acid (15) and its benzimidazole analogue 25 showed potent inhibitory activities for rat prostatic 5alpha-reductase (IC50 values of 9.6+/-1.0 and 13+/-1.5 nM, respectively), with the potency very close to that of finasteride. Compound 30, in which the moiety between the benzene ring and amide bond was replaced by quinolin-4-one ring, showed almost equipotent activity (IC50= 19+/-6.2nM) with the correspondent amide derivative 13. This result was consistent with the previous observation that the coplanarity of this moiety might contribute to the potent inhibitory activity.     

Potent inhibitors of acyl-CoA:cholesterol acyltransferase. 2. Structure-activity relationships of novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amides

Novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives 1 were synthesized and tested for their ability to inhibit rabbit small intestinal ACAT (acyl-CoA:cholesterol acyltransferase) and lower serum total cholesterol in cholesterol-fed rats. Among the synthesized compounds, N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives showed potent ACAT inhibitory activity. The synthesis and structure-activity relationships of these compounds are described. A methyl group at position 6 of the 2,3-dihydrobenzofuran moiety was important for potent ACAT inhibitory activity. In the series of N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl) amides, lipophilicity of the acyl moiety was necessary for the potent ACAT inhibitory activity. The highly lipophilic acid amides N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)-2,2- dimethyldodecanamide (10) and 6-(4-chlorophenoxy)-N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-y l)-2,2-dimethyloctanamide (50) showed potent activity. Introduction of a dimethylamino group at position 5 of the 2,3-dihydrobenzofuran moiety resulted in highly potent activity. The most potent compound, N-[5-(dimethylamino)-2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl ]-2,2-dimethyldodecanamide (13, TEI-6620), showed highly potent ACAT inhibitory activity (rabbit small intestine IC50 = 0.020 microM, rabbit liver IC50 = 0.009 microM), foam cell formation inhibitory activity (rat peritoneal macrophage IC50 = 0.030 microM), extremely potent serum cholesterol-lowering activity in cholesterol-fed rats (71% at a dose of 0.3 mg/kg/day po), and good bioavailability in fed dogs (Cmax = 2.68 microg/mL at 1 h, 10 mg/kg po).     

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.     

Thieno[2,3-d]pyrimidine-3-acetic acids. A new class of nonpeptide endothelin receptor antagonists

On the basis of structural information for the cyclic hexapeptide endothelin (ET) receptor antagonist, TAK-044, a series of thieno[2,3-d]pyrimidine-2,4-dione derivatives bearing a carboxyl group and aromatic rings that were important for receptor binding were designed, synthesized, and evaluated for ET receptor binding affinities and inhibitory activities against ET-induced vasoconstriction. Optimization of each substituent in the thieno[2,3-d]pyrimidine ring led to the discovery of a novel and potent nonpeptide ET receptor antagonist, 6-(4-methoxymethoxyphenyl)-5-methylsulfonylaminomethyl-1-(2- methylthiobenzyl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]p yrimidine-3- acetic acid (32 g), which binded to human ETA and ETB receptor subtypes with affinities (IC50) of 7.6 and 100 nM, respectively. Compound 32 g effectively antagonized ET-induced vasoconstriction and the inhibitory effect mediated by the ETB receptor was more potent than that of bosentan, while the inhibitory effect mediated by the ETA receptor was slightly less potent than that of bosentan.     

Human papillomavirus and p53 oncoprotein in verrucous carcinoma of the larynx

An in vitro screening method for selective acetylcholinesterase (AChE) inhibitors was established. Inhibitory activity of AChE and butyrylcholinesterase (BuChE) was measured and the culture broths of microorganisms that showed selective inhibition against AChE were characterized. By using this method, a strain producing the novel and selective inhibitors of AChE, arisugacins A and B, was picked out among over seven thousand microorganisms tested. Arisugacins were obtained as white powders from the culture broth together with three known compounds, territrems B and C and cyclopenin that also showed selective inhibition against AChE. Arisugacins and territrems are members of the meroterpenoid compounds. They showed potent inhibitory activities against AChE with IC50 values in range of 1.0 approximately 25.8 nM. Furthermore, they showed greater than 2,000-fold more potent inhibition against AChE than BuChE.     

Fibrinogen receptor (GPIIb-IIIa) antagonists derived from 5,6-bicyclic templates. Amidinoindoles, amidinoindazoles, and amidinobenzofurans containing the N-alpha-sulfonamide carboxylic acid function as potent platelet aggregation inhibitors

A series of highly potent and specific fibrinogen receptor antagonists have been discovered and optimized through structural modification of the novel amidinoindole and benzofuran compounds, I and II. Systematic linker optimization afforded the amidinobenzofuran-containing inhibitor 29, which displayed an IC50 value of 250 nM in platelet aggregation assays. Attempts to enhance activity by modification of the beta-position of the beta-alanyl carboxylate group of 29 had only a modest effect on inhibitory activity in aggregation assays. Analogues prepared to enhance the activity by conformational restriction were also found to be equally or less potent. In contrast, modification at the alpha-position of the beta-alanyl carboxylate group resulted in the identification of extremely potent and novel amidinobenzofuran-containing derivatives 46-49. Reexamination of 5,6-bicyclic aromatic nucleus led to the further identification of amidinoindole- and amidinoindazole-containing derivatives 53-55. These analogues, 46-49 and 53-55, exhibited potent in vitro activity with IC50 values of 25-65 nM in platelet aggregation assays and an IC50 value of 2 nM in fibrinogen binding assays and demonstrated a selectivity of > 50,000-fold for GPIIb-IIIa versus the most closely related integrin, the vitronectin receptor, alpha v beta 3.     

Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of the nonpolyglutamatable dihydrofolate reductase inhibitor Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl- L-ornithine (PT523)

Six new B-ring analogues of the nonpolyglutamatable antifolate Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloy l-L-ornithine (PT523, 3) were synthesized with a view to determining the effect of modifications at the 5- and/or 8-position on dihydrofolate reductase (DHFR) binding and tumor cell growth inhibition. The 5- and 8-deaza analogues were prepared from methyl 2-L-amino-5-phthalimidopentanoate and 4-amino-4-deoxy-N10-formyl-5-deaza- and -8-deazapteroic acid, respectively. The 5,8-dideaza analogues were prepared from methyl 2-L-[(4-aminobenzoyl)amino]-5-phthalimidopentanoate and 2, 4-diaminoquinazoline-6-carbonitriles. The Ki for inhibition of human DHFR by the 5-deaza and 5-methyl-5-deaza analogues was about the same as that of 3 (0.35 pM), 11-fold lower than that of aminopterin (AMT, 1), and 15-fold lower than that of methotrexate (MTX, 2). However the Ki of the 8-deaza analogue was 27-fold lower than that of 1, and that of the 5,8-dideaza, 5-methyl-5,8-dideaza, and 5-chloro-5,8-dideaza analogues was approximately 50-fold lower. This trend was consistent with the published literature on the corresponding DHFR inhibitors with a glutamate side chain. In colony formation assays against the human head and neck squamous carcinoma cell line SCC25 after 72 h of treatment, the 5- and 8-deaza analogues were approximately as potent as 3, whereas the 5,8-dideaza analogue was 3 times more potent. 5-Methyl and 5-chloro substitution was also favorable, with the 5-methyl-5-deaza analogue being 2. 5-fold more potent than the 5-deaza analogue. However the effect of 5-methyl substitution was less pronounced in the 5,8-dideaza analogues than in the 5-deaza analogues. The 5-chloro-5,8-dideaza analogue of 3 was the most active member of the series, with an IC50 = 0.33 nM versus 1.8 nM for 3 and 15 nM for MTX. The 5-methyl-5-deaza analogue of 3 was also tested at the National Cancer Institute against a panel of 50 human tumor cell lines in culture and was consistently more potent than 3, with IC50 values in the low-nanomolar to subnanomolar range against most of the tumors. Leukemia and colorectal carcinoma cell lines were generally most sensitive, though good activity was also observed against CNS tumors and carcinomas of the breast and prostate. The results of this study demonstrate that B-ring analogues of 3 inhibit DHFR activity and tumor cell colony formation as well as, or better than, the parent compound. In view of the fact that 3 and its B-ring analogues cannot form polyglutamates, their high cytotoxicity relative to the corresponding B-ring analogues of AMT is noteworthy.     

In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine

Transition state mimetic tripeptide human immunodeficiency virus (HIV) protease inhibitors containing allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were synthesized and tested for activity against HIV in vitro. Two compounds, KNI-227 and KNI-272, which were highly potent against HIV protease with little inhibition of other aspartic proteases, showed the most potent activity against the infectivity and cytopathic effect of a wide spectrum of HIV strains. As tested in target CD4+ ATH8 cells, the 50% inhibitory concentrations of KNI-227 against HIV type 1 LAI (HIV-1LAI), HIV-1RF, HIV-1MN, and HIV-2ROD were 0.1, 0.02, 0.03, and 0.1 microM, respectively, while those of KNI-272 were 0.1, 0.02, 0.04, and 0.1 microM, respectively. Both agents completely blocked the replication of 3'-azido-2',3'-dideoxythymidine-sensitive and -insensitive clinical HIV-1 isolates at 0.08 microM as tested in target phytohemagglutinin-activated peripheral blood mononuclear cells. The ratios of 50% cytotoxic concentrations to 50% inhibitory concentrations for KNI-227 and KNI-272 were approximately 2,500 and > 4,000, respectively, as assessed in peripheral blood mononuclear cells. Both compounds blocked the posttranslational cleavage of the p55 precursor protein to generate the mature p24 Gag protein in stably HIV-1-infected cells. The n-octanol-water partition coefficients of KNI-227 and KNI-272 were high, with log Po/w values of 3.79 and 3.56, respectively. Degradation of KNI-227 and KNI-272 in the presence of pepsin (1 mg/ml, pH 2.2) at 37 degrees C for 24 h was negligible. Current data warrant further careful investigations toward possible clinical application of these two novel compounds.     

Homeless at risk of mortality: a proactive approach to vulnerable "repeaters"

Certain flavonoids having a C-2,3-double bond were reported to show an inhibitory activity against T-lymphocyte proliferation, but not against B-lymphocyte proliferation in vitro. In the course of these studies, vitexicarpin (3',5-dihydroxy-3,4',6,7-tetramethoxyflavone) isolated from the fruits of Vitex rotundifolia was found to show potent inhibition against lymphocyte proliferation. Vitexicarpin inhibited T-lymphocyte proliferation as well as B-lymphocyte proliferation at > 0.1 microM. IC50's were approximately 0.7 microM both for T- and B-cell proliferation. The inhibitory activity of vitexicarpin was reversible. Vitexicarpin also inhibited the growth of certain cancer cell lines, EL-4 and P815.9 (IC50 = 0.25-0.3 microM). These results suggest that vitexicarpin may be a potential therapeutic agent involved in inflammatory/immunoregulatory disorders such as rheumatoid arthritis and lymphomas.     

Synthesis and anti-HIV activities of urea-PETT analogs belonging to a new class of potent non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of potent specific HIV-1 RT inhibitory compounds is described. The compounds are urea analogs of PETT (PhenylEthylThiazoleThiourea) derivatives and the series includes derivatives with an ethyl linker (1-6) and conformationally restricted analogs (7-13). The antiviral activity is determined both at the RT level and in cell culture on both native and mutant forms of HIV-1. Many compounds display activity in the nM range against wt-RT.