N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

Modified norcantharidins; synthesis, protein phosphatases 1 and 2A inhibition, and anticancer activity

Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC(50)=13+/-5 microM; PP2A IC(50)=7+/-3 microM) and 16 (PP1 IC(50)=18+/-8 microM; PP2A IC(50)=3.2+/-0.4 microM). Overall, only analogues possessing at least one acidic residue at the former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma), G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine leukemia).     

Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues

A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1-14) in which the sugar moiety was replaced by the conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all evaluated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC(50)=4.3microM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC(50)=18microM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.     

Design of N-acetyl-6-sulfo-beta-d-glucosaminide-based inhibitors of influenza virus sialidase

Biological activity of N-acetyl-6-sulfo-beta-d-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetylneuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC(50)=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH(3)(+) 1b without 2-NHAc showed little activity (IC(50) >50 mM). The activity was enhanced nearly 100-fold when the pNP group of 1a was converted to p-acetamidophenyl one 5 (IC(50)=30 microM) or replaced with 1-naphthyl 6 (IC(50)=10 microM) or n-propyl one 8 (IC(50)=11 microM).     

Synthesis and biological evaluation of aroylguanidines related to amiloride as inhibitors of the human platelet Na(+)/H(+) exchanger

Pyridine and benzene bioisosteres of amiloride were synthesized and evaluated for their inhibitory potency against the sodium-hydrogen exchanger (NHE) involved in intracellular pH regulation. The inhibition of NHE was determined by using the platelet swelling assay (PSA) in which the swelling of human platelets was induced by their incubation in an acid buffer (pH 6.7). Additionally, the inhibitory potency of the most active compounds was assessed by measuring the inhibition of the EIPA-sensitive (22)Na(+) uptake (UIA) by human platelets after intracellular acidosis. The results indicated that several benzene derivatives and compounds bearing an carbonylguanidine moiety in the meta position of the pyridine nitrogen were much more potent than amiloride (PSA:IC(50)=43.5 microM; UIA:IC(50)=100.1 microM), but less than EIPA, a pyrazine NHE inhibitor (PSA:IC(50)=0.08 microM; UIA:IC(50)=0.5 microM). In both biological assays (2-amino-5-bromo-pyridine-3-carbonyl)guanidine (32) was the most active molecule (PSA: IC(50)=0.8 microM, UIA : IC(50)=0.8 microM). Our investigations demonstrated that the replacement of the pyrazine ring of amiloride by a pyridine or a phenyl ring improved the NHE inhibitory potency (phenyl >pyridine >pyrazine).     

Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.     

Non-nucleoside HIV-1 reverse-transcriptase inhibitors. Part 10. Synthesis and anti-HIV activity of 5-alkyl-6-(1-naphthylmethyl)pyrimidin-4(3H)-ones with a mono- or disubstituted 2-amino function as novel 'dihydro-alkoxy-benzyl-oxopyrimidine' (DABO) analogues

A series of structurally related 2,5-disubstituted 6-(1-naphthylmethyl)-pyrimidin-4(3H)-ones, compounds 6a-6r, were synthesized and evaluated for their in vitro anti-HIV activities in MT-4 cells. Most of the new compounds investigated showed moderate-to-good activities against wild-type HIV-1, with IC(50) values in the range 5.64-0.21 microM. Compound 6d was the most potent congener (IC(50)=0.21 microM, SI=724) in inhibiting HIV-1 replication, which is ca. 25 times more effective than the reference compound 2',3'-dideoxyinosine (DDI). Preliminary structure-activity relationship (SAR) studies revealed that both modulation of the amino function at C(2) and of the alkyl group at C(5) of the pyrimidine ring are crucial for high anti-HIV-1 activity.     

Indole derivatives as potent inhibitors of 5-lipoxygenase: design, synthesis, biological evaluation, and molecular modeling

A series of novel indole derivatives was designed, synthesized and evaluated by cell-based assays for their inhibitory activities against 5-LOX in rat peritoneal leukocytes. Most of them (30 out of 35) showed an inhibitory potency higher than the initial screening hit 1a (IC(50)=74 microM). Selected compounds for concentration-response studies showed prominent inhibitory activities with IC(50) values ranging from 0.74 microM to 3.17 microM. Four compounds (1m, 1s, 4a, and 6a) exhibited the most potent inhibitory activity compared to that of the reference drug (Zileuton), with IC(50) values less than 1 microM. Molecular modeling studies for compounds 1a, 3a, 4a, and 6a were also presented. The excellent in vitro activities of this class of compounds may possess potential for the treatment of LT-related diseases.     

Investigations on the influence of terminal groups at the C2-propyl side chain of 1,1-bis(4-hydroxyphenyl)-2-phenylpent-1-ene and 1,1,2-tris(4-hydroxyphenyl)pent-1-ene on the estrogen receptor binding and the estrogenic/anti-estrogenic properties

1,1-bis(4-Hydroxyphenyl)-2-phenylpent-1-ene (5) and 1,1,2-tris(4-hydroxyphenyl)pent-1-ene (6) derivatives with terminal CN (5a, 6a), NH(2) (5b, 6b), NHCOCH(3) (5c, 6c), NHCOC(2)H(5) (5d, 6d) groups at the C2-propyl chain were synthesized and assayed in vitro for estrogen receptor (ER) binding affinity (RBA) in a competition experiment with [3H]estradiol and for estrogenic and anti-estrogenic properties in a luciferase assay with ER-positive MCF-7-2a cells, stably transfected with the plasmid ERE(wtc)luc. The CN as well as the NH(2) group reduced the RBA-values (5: 2.09%; 5a: 1.50%; 5b: 0.07%; 6: 4.03%; 6a: 0.67%; 6b: 0.20%) and the antagonistic potency (5: IC(50)=0.05 microM; 5a: IC(50)=0.43 microM; 5b: IC(50)=1.50 microM; 6: IC(50)=0.07 microM; 6a: IC(50)=0.60 microM; 6b: IC(50)=2.00 microM). Derivatization of the amino function with acetic anhydride and propionic anhydride did not change the RBA-value but altered the antagonistic profile (5c: IC(50)=2.50 microM; 5d: IC(50)=not detectable; 6c: IC(50)=0.65 microM; 6d: IC(50)=1.00 microM). Agonistic effects were only detected for the amine 6b (34.2% activation of the luciferase expression). These data document that estrogen receptor binding and the antagonistic effects can be modified by terminal groups at the C2-propyl chain of the pure antagonists 5 and 6. The mode of action is unclear. However, it can be assumed that the elongation of the side chain causes a reorientation in the LBD in order to locate the side chain in a side pocket near the ligand binding domain.     

Synthesis and biological evaluation of novel 5(H)-phenanthridin-6-ones, 5(H)-phenanthridin-6-one diketo acid, and polycyclic aromatic diketo acid analogs as new HIV-1 integrase inhibitors

A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new beta-diketo acid analogs with the phenanthridinone scaffold replaced by phenanthrene, anthracene or pyrene exhibited the highest IN inhibitory potency. There is a general selectivity against the integrase strand transfer step. The most potent IN was 2,4-dioxo-4-phenanthren-9-yl-butyric acid (27f) with an IC(50) of 0.38microM against integrase strand transfer. The phenanthrene diketo acids 27d-f were more potent (IC(50)=2.7-0.38microM) than the corresponding phenanthridinone diketo acid 16 (IC(50)=65microM), suggesting that the polar amide bridge in the phenanthridinone system decreases inhibitory activity relative to the more lipophilic phenanthrene system. This might have to do with the possible binding of the aryl group of the compounds binding to a lipophilic pocket at the integrase active site as suggested by the docking simulations. Molecular modeling also suggested that effectiveness of chelation of the active site Mg(2+) contributes to IN inhibitory potency. Finally, some of the potent compounds inhibited HIV-1 replication in human peripheral blood mononuclear cells (PBMC) with EC(50) down to 8microM for phenanthrene-3-(2,4-dioxo)butyric acid (27d), with a selectivity index of 10 against PBMCs.     

2,6-Bis(3,4,5-trihydroxybenzylydene) derivatives of cyclohexanone: novel potent HIV-1 integrase inhibitors that prevent HIV-1 multiplication in cell-based assays

A number of 2,6-bisbenzylidenecyclohexane-1-one derivatives have been synthesized and tested as HIV-1 integrase (IN) inhibitors with the aim of obtaining compounds capable to elicit antiviral activity at non-cytotoxic concentrations in cell-based assays. 3,5-Bis(3,4,5-trihydroxybenzylidene)-4-oxocyclohexaneacetic acid (20d) resulted one of the most potent and selective derivatives in acutely infected MT-4 cells (EC(50) and CC(50) values of 2 and 40 microM, respectively). In enzyme assays with recombinant HIV-1 integrase (rIN), this compound proved able to inhibit both 3'-processing and disintegration with IC(50) values of 0.2 and 0.5 microM, respectively. In order to develop a model capable to predict the anti HIV-IN activity and useful to design novel derivatives, we performed a comparative molecular field analysis (CoMFA) like 3-D-QSAR. In our model the ligands were described quantitatively in the GRID program, and the model was optimized by selecting only the most informative variables in the GOLPE program. We found the predictive ability of the model to increase significantly when the number of variables was reduced from 20,925 to 1327. A Q(2) of 0.73 was obtained with the final model, confirming the predictive ability of the model. By studying the PLS coefficients in informative 3-D contour plots, ideas for the synthesis of new compounds could be generated.     

Resveratrol analogues as selective cyclooxygenase-2 inhibitors: synthesis and structure-activity relationship

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is found in grapes and various medical plants. Among cytotoxic, antifungal, antibacterial cardioprotective activity resveratrol also demonstrates non-selective cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibition. In order to find more selective COX-2 inhibitors a series of methoxylated and hydroxylated resveratrol derivatives were synthesized and evaluated for their ability to inhibit both enzymes using in vitro inhibition assays for COX-1 and COX-2 by measuring PGE(2) production. Hydroxylated but not methoxylated resveratrol derivatives showed a high rate of inhibition. The most potent resveratrol compounds were 3,3',4',5-tetra-trans-hydroxystilbene (COX-1: IC(50)=4.713, COX-2: IC(50)=0.0113 microM, selectivity index=417.08) and 3,3',4,4',5,5'-hexa-hydroxy-trans-stilbene (COX-1: IC(50)=0.748, COX-2: IC(50)=0.00104 microM, selectivity index=719.23). Their selectivity index was in part higher than celecoxib, a selective COX-2 inhibitor already established on the market (COX-1: IC(50)=19.026, COX-2: IC(50)=0.03482 microM, selectivity index=546.41). Effect of structural parameters on COX-2 inhibition was evaluated by quantitative structure-activity relationship (QSAR) analysis and a high correlation was found with the topological surface area TPSA (r=0.93). Docking studies on both COX-1 and COX-2 protein structures also revealed that hydroxylated but not methoxylated resveratrol analogues are able to bind to the previously identified binding sites of the enzymes. Hydroxylated resveratrol analogues therefore represent a novel class of highly selective COX-2 inhibitors and promising candidates for in vivo studies.     

Conformationally restricted analogs of deoxynegamycin

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.     

Cantharimides: a new class of modified cantharidin analogues inhibiting protein phosphatases 1 and 2A.

Cantharidin and its analogues have been of considerable interest as potent inhibitors of the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A). However, limited modifications to the parent compounds is tolerated. As part of an on-going study we have developed a new series of cantharidin analogues, the cantharimides. Inhibition studies indicate that cantharimides possessing a D- or L-histidine, are more potent inhibitors of PP1 and PP2A (PP1 IC(50)=3.22+/-0.7 microM; PP2A IC(50)=0.81+/-0.1 microM and PP1 IC(50)=2.82+/-0.6 microM; PP2A IC(50)=1.35+/-0.3 microM, respectively) than norcantharidin (PP1 IC(50)=5.31+/-0.76 microM; PP2A IC(50)=2.9+/-1.04 microM) and essentially equipotent with cantharidin (PP1 IC(50)=3.6+/-0.42 microM; PP2A IC(50)=0.36+/-0.08 microM). Cantharimides with non-polar or acidic amino acid residues are only poor inhibitors of PP1 and PP2A.     

Tetraketones: a new class of tyrosinase inhibitors

Twenty-eight tetraketones (1-28) with variable substituents at C-7 were synthesized and evaluated as tyrosinase inhibitors. Remarkably compounds 25 (IC(50)=2.06 microM), 11 (IC(50)=2.09 microM), 15 (IC(50)=2.61 microM), and 27 (IC(50)=3.19 microM) were found to be the most active compounds of the series, even better than both standards kojic acid (IC(50)=16.67 microM) and L-mimosine (IC(50)=3.68 microM). This study may lead to the discovery of therapeutically potent agents against clinically very important dermatological disorders including hyperpigmentation as well as skin melanoma.