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Fifty-one acylthioureas (ATUs) incorporating imidazolidine-2-thione or its upper cyclohomologue were prepared by parallel synthesis and evaluated against a high number of human cancer cell lines for antiproliferative activity. ATUs 1o (3,5-dichlorobenzoyl), 1s (2-furoyl), 3s (2-furoyl) and 1t (2-thenoyl) displayed activity against leukemia, melanoma LOX IMVI, non-small cell lung NCI-H522, renal 786-0, CAKI-1, SN12C, UO-31 and breast MCF7, MDA-MB-435, T-47D cancer cell lines in the 0.3-9.7 microM concentration range. Compound 14s exhibited selectivity for melanoma SK-MEL-5 (GI(50)<5 nM); 1s for leukemia MOLT-4 (GI(50): 300 nM); 1q, 3b and 3q for renal cancer UO-31 (GI(50): 70-200 nM); 8s, 9s for non-small cell lung cancer EKVX (GI(50): 300, 10 nM) and 3j for HOP-92 (GI(50): 700 nM) cell line.

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F(2)-NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C(2)-position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C(2)-amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C(5)-position and the other at the benzylic carbon), being thymine, alpha-methyluracil or alpha-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-alpha-methylthymine derivatives 4d, 5h'-n' showed the highest potency and selectivity among tested compounds, both a properly sized C(2)-NH side chain and the presence of two methyl groups (at C(5) and benzylic positions) being crucial for high antiviral action.

The synthesis of base modified L-nucleosides is described with pyrrolo[2,3-d]pyrimidines, pyrazolo[3,4-d]pyrimidines, benzimidazoles, and imidazo[1,2-a]-s-triazines as nucleobases. The conformation of the nucleosides is studied and the antiviral activity is evaluated.

5-Alkyl-2-(alkylthio)-6-(2,6-difluorobenzyl)-3,4-dihydropyrimidin-4(3H)-ones (S-DABOs, 2) have been recently described as a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) active at nanomolar concentrations (Mai, A. et al. J. Med. Chem. 1999, 42, 619-627). In pursuing our lead optimization efforts, we designed novel conformationally restricted S-DABOs, 3, featuring a methyl at the benzylic carbon (Y = Me) and at the pyrimidine 5-position (R = Me). Conformational analyses and docking simulations suggested that the presence of both methyls would significantly reduce conformational flexibility without compromising, in the R enantiomers, the capability of fitting into the RT non-nucleoside binding pocket. To develop structure-activity relationships, we prepared several congeners of type 3 belonging to the thymine (R = Me) and uracil (R = H) series, featuring various 2-alkylthio side chains (X = Me, i-Pr, n-Bu, i-Bu, s-Bu, c-pentyl, and c-hexyl) and aryl moieties different from the 2,6-difluorophenyl (Ar = phenyl, 2,6-dichlorophenyl, 1-naphthyl). Moreover, alpha-ethyl derivatives (Y = Et) were included in the synthetic project in addition to alpha-methyl derivatives (Y = Me). All of the new compounds were evaluated for their cytotoxicity and anti-HIV-1 activity in MT-4 cells, and some of them were assayed against highly purified recombinant wild-type HIV-1 RT using homopolymeric template primers. The results were expressed as CC(50) (cytotoxicity), EC(50) (anti-HIV-1 activity), SI (selectivity, given by the CC(50)/EC(50) ratio), and IC(50) (RT inhibitory activity) values. In the 2,6-difluorobenzylthymine (R = Me) series, methylation of the benzylic carbon improved anti-HIV-1 and RT inhibitory activities together with selectivity. Compound 3w (Ar = 2,6-F(2)-Ph, R = Y = Me, X = c-pentyl) turned out the most potent and selective among the S-DABOs reported to date (CC(50) > 200 microM, EC(50) = 6 nM, IC(50) = 5 nM, and SI > 33 333). Assays performed on the pure enantiomer (+)-3w, much more active than (-)-3w, yielded the following results: CC(50) > 200 microM, EC(50) = 2 nM, IC(50) = 8 nM, and SI > 100 000, under conditions wherein MKC-442 was less active and selective (CC(50) > 200 microM, EC(50) = 30 nM, IC(50) = 40 nM, SI > 6666). The 2,6-difluorophenylethylthymines (R = Me) were generally endowed with higher potency compared with the uracil counterparts (R = H). In the 2,6-difluorophenyl series the best and the least performant 2-alkylthio side chains were the 2-c-pentylthio and the 2-methylthio, respectively. When the methyl at the benzylic carbon was replaced by an ethyl, activity was retained or decreased slightly, thus suggesting that the dimensions of the cavity within the RT hosting this substituent would not be compatible with groups larger than ethyl. Aryl moieties different from the 2,6-difluorophenyl (phenyl, 1-naphthyl, 2,6-dichlorophenyl) were generally detrimental to activity, consistent with a favorable electronic effect exerted by the 2,6-fluorines on a putative charge-transfer interaction between the aromatic moieties of the inhibitor and Tyr188.

Worldwide, the heterosexual route is the prevalent mode of transmission of AIDS; therefore, demands have been raised for measures that block sexual spreading of the HIV infection. Development of microbicides for topical use may represent an efficacious alternative to condoms. Several approaches are being investigated. Besides surfactants, which directly act on the virus particle, and measures that enhance natural defence mechanisms, promising new candidates appear to be drugs that block the early steps of HIV multiplication. We describe herein a long-term assay which enables the establishment of whether the above drugs reversibly (virustatic action) or irreversibly (virucidal action) inhibit HIV-1 multiplication, thus allowing screening for effective and potent microbicides. We validated our assay with nucleoside (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Following a chronic treatment, the NRTIs tested (didanosine, zalcitabine, stavudine and lamivudine) simply delayed the viral breakthrough with respect to infected, untreated controls. Under the same experimental conditions, non-nucleoside reveres transcriptase inhibitors (NNRTIs), such as MKC-442, alphaAPA, nevirapine, efavirenz and 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs) MC 1047 and MC 1220 suppressed HIV-1 replication for the entire experimental period (40 days). When cell culture samples were evaluated for the presence of infectious virus, p24 antigen and viral DNA sequences, none of them was detected up to day 40 post-infection (p.i.). Identical results were obtained after a treatment with the above NNRTIs limited to the first 4 days p.i. Under more selective experimental conditions, that is drug treatments limited to the first 4 h p.i., nevirapine and efavirenz proved to be virustatic; in fact, viral breakthrough ensued shortly after their removal from the culture medium. Conversely, DABO MC 1220 was endowed with potent virucidal activity; in fact, at 3.5 microM it was able to suppress HIV-1 multiplication in cultures acutely infected with a very high multiplicity of infection (5 CCID50/cell), thus allowing exponential cell multiplication as in uninfected cultures for the next 40 days.

Molecular modeling analysis of compounds belonging to the recently published series of dihydro-alkoxy-benzyl-oxopyrimidines (DABOs), such as S-DABOs and DATNOs, gave support to the design of new 2, 6-disubstituted benzyl-DABO derivatives as highly potent and specific inhibitors of the HIV-1 reverse transcriptase (RT). To follow up on the novel DABO derivatives, we decided to investigate the effect of electron-withdrawing substituents in the benzyl unit of the S-DABO skeleton versus their anti-HIV-1 activity. Such chemical modifications impacted the inhibitory activity, especially when two halogen units were introduced at positions 2 and 6 in the phenyl portion of the benzyl group bound to C-6 of the pyrimidine ring. Various 5-alkyl-2-(alkyl(or cycloalkyl)thio)-6-(2, 6-dichloro(or 2,6-difluoro)phenylmethyl)-3, 4-dihydropyrimidin-4(3H)-ones were then synthesized and tested as anti-HIV-1 agents in both cell-based and enzyme (recombinant reverse transcriptase, rRT) assays. Among the various mono- and disubstituted phenyl derivatives, the most potent were those containing a 6-(2,6-difluorophenylmethyl) substituent (F-DABOs), which showed EC50's ranging between 40 and 90 nM and selectivity indexes up to >/=5000. An excellent correlation was found between EC50 and IC50 values which confirmed that these compounds act as inhibitors of the HIV-1 RT. The structure-activity relationships of the newly synthesized pyrimidinones are presented herein.

Novel compounds related to 2-(cyclohexylthio)-3,4-dihydro-5-methyl-6-(3-methylbenzyl)-4-ox opyrimidine (3c, MC 639) have been synthesized and tested as inhibitors of human immunodeficiency virus type-1 (HIV-1). Reaction of thiourea with ethyl arylmethylacetoacetates furnished 5-alkyl-6-(arylmethyl)-3,4-dihydro-2-mercapto-4-oxopyrimidines which were then alkylated at the sulfur atom to afford the required 2-alkylthio or 2-cycloalkylthio derivatives (S-DABOs). Chemical modifications at N-3, C-4, and C-6 of the pyrimidine ring were attempted with the aim of improving antiretroviral activity. In particular, replacement of the benzyl group with the 1-naphthylmethyl moiety enhanced the activity of S-DABOs, whereas N-3 alkylation and C=O transformation into C=S at position 4 of the pyrimidine ring led to compounds devoid of anti-HIV-1 activity. Lower activity was generally observed when 1-naphthylmethyl was replaced by the isomeric 2-naphthylmethyl moiety. The most active compounds showed activity in the low micromolar range with EC50 values comparable to that of nevirapine.

Various thio analogues of dihydroalkoxybenzyloxopyrimidines (DABOs), a new class of non-nucleoside reverse transcriptase inhibitors, were found to selectively inhibit the HIV-1 multiplication in vitro. Among the C-5 H-substituted 6-benzyl-3,4-dihydro-4-oxopyrimidines, the introduction of alkylthio or cycloalkylthio substituents at C-2 of the pyrimidine ring led to derivatives (S-DABOs) which were up to 10-fold more potent than the alkyloxy or cycloalkyloxy counterparts. The further introduction of a methyl group at the 3'-position of the benzyl portion of 2-(alkylthio)-6-benzyluracils reduced the cytotoxicity leading to more selective compounds. Among C-5 methyl-substituted S-DABOs, numerous derivatives showed EC50 values as low as 0.6 microM and lacked cytotoxicity at doses as high as 300 microM. In the C-5 double methyl-substituted series, a more pronounced cytotoxicity was observed and the further introduction of a methyl at the 3'-position in the benzylidene group resulted in total loss of antiviral activity. S-DABOs, namely 2-(alkylthio)-6-benzyl-3,4-dihydro-4-oxopyrimidines, were synthesized by reacting proper methyl (phenylacetyl)acetates or their 2-methyl compounds with thiourea to afford 6-benzyl-4-oxo-1,2,3,4-tetrahydro-2-thiaoxopyrimidines or the related 5-methyl derivatives. Treatment of the latter derivatives with alkyl or cycloalkyl halides in alkaline medium gave the required title compounds.

Novel 3,4-dihydro-6-benzyl-4-oxopyrimidines (DABOs), variously substituted at both the C-2 and C-5 positions of the pyrimidine ring, proved to be specific inhibitors of the human immunodeficiency virus type 1 (HIV-1) in vitro. Some compounds showed potency at micromolar doses, no cytotoxicity at the maximum testable doses and selectivity indexes comparable to that of 2'-3'-dideoxyinosine (ddI). Mode of action studies suggested that DABOs interfered with a step of the virus multiplication cycle following adsorption and preceding integration. Enzyme assays indicated that DABOs targeted HIV-1 reverse transcriptase: they inhibited the RNA-dependent DNA polymerase activity in a template-dependent manner and, to a lesser extent, the DNA-dependent DNA polymerase activity. No inhibition of the RNase-H associated activity was observed. When DABOs were assayed in combination with 3'-azido-3'-dideoxythymidine (AZT) or ddI against HIV-1 in cell cultures, a slightly synergistic inhibitory effect was observed. The combination of DABO 546 and AZTTP in enzyme assays showed that the two compounds were kinetically mutually exclusive.

4,6-dimethyl-2-amino-3,4,5-trimethoxyphenyl-pyrimidine (B-31) is a pyrimidine derivative previously reported to arrest the mitotic cycle of mammalian cells in metaphase. In the present study it is shown that B-31 also acts as a specific inhibitor of the cellular uptake of nucleosides. The uptake of purinic and pyrimidinic nucleosides is inhibited by 80-90% at concentrations in the range 5-20 micrograms/ml, whereas those of nucleobases, leucine, choline and glucose are unaltered at the maximum nontoxic dose of B-31 (25 micrograms/ml). Various mammalian (human, monkey and murine) and avian cell are equally sensitive to the inhibition of nucleoside transport. The antimitotic effect of B-31, by contrast, is species-specific: human cells are the most sensitive whereas monkey and chicken fibroblasts appear resistant to this effect. Both effects can be reversed by removal of B-31 from the medium; inhibition of nucleoside transport can also be reversed by high doses of the nucleosides themselves.