Publications

A series of novel 1,2,4-triazole thioacetanilide derivatives has been designed, synthesized and evaluated for their anti-HIV activities in MT-4 cells. Half of these compounds showed moderate to potent activities against wild-type HIV-1 with an EC50 ranging from 38.0 μM to 4.08 µM. Among them, 2-(4-(2-fluorobenzyl)-5-isopropyl-4H-1,2,4-triazol- 3-ylthio)-N-(2-nitrophenyl)acetamide 7d was identified as the most promising compound (EC50 = 4.26 µM, SI = 49). However, no compound was active against HIV-2. The preliminary structure-activity relationships among the newly synthesized congeners are discussed.

To investigate the role of transforming growth factor alpha (TGF alpha) in tumor development, we introduced the human TGF alpha (hTGF alpha) cDNA into cultured primary mouse epidermal cells or papilloma cells using a replication-defective retroviral vector and analyzed skin grafts constructed with such cells. Expression of the exogenous gene was confirmed by detection of hTGF alpha mRNA by northern RNA blot analysis, and the secreted hTGF alpha was measured by ELISA of culture supernatants. Tumor cells expressing hTGF alpha produced benign tumors (papillomas), which were 1.5- to 2-fold larger than tumors of parental cells when tested as skin grafts on nude mice. Grafts of normal cells that expressed hTGF alpha produced normal skin. When mixtures of parental tumor cells and normal mouse keratinocytes were grafted to nude mice, papilloma formation was suppressed and tumors that did form were small. Grafts of hTGF alpha-producing papilloma cells combined with either normal epidermal cells or hTGF alpha-producing epidermal cells yielded large tumors. Mixed grafts containing keratinocytes expressing hTGF alpha and parental papilloma cells also produced large tumors. While the tumor size was substantially increased by hTGF alpha expression, the tumors that developed in all groups were histologically benign and reached a stable size 4-5 wk after grafting. These results indicate that expression of hTGF alpha by either tumor cells (autocrine) or adjoining normal cells (paracrine) can stimulate tumor growth, particularly when tumor growth is suppressed by normal tissue. However, expression of this growth factor did not appear to influence tumor progression directly.

The configurations of metallocyclams are of interest in relation to protein recognition and anti-HIV activity. We have synthesised four novel zinc(II) complexes with hexyl-Me(2)-cyclam (HMC; 3,14-dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.0(7,12))docosane), 1, and naphthyl-hexyl-Me(2)-cyclam (NHMC; 2,13-bis(1-naphthylmethyl)-5,16-dimethyl-2,6,13,17-tetraazatricyclo(16.4.0.0(7,12))docosane), 2, as ligands. X-ray crystallographic data for Zn(II)-HMC diacetate, 3 show that zinc is six-coordinate in a distorted octahedral environment bound to four equatorial N atoms from the macrocycle and two axial acetato O atoms. The 14-membered metallo-macrocycle adopts a trans-III (RRSS) configuration with two six-membered rings in chair forms and two five-membered rings in gauche forms. In the chlorido Zn(II)-HMC complex 5, zinc appears to be 5-coordinate with square-pyramidal geometry. Interestingly, the chlorido Zn(II)-NHMC complex 6 crystallised in a trans-I configuration containing 4-coordinate tetrahedral zinc bound to three cyclam ring N atoms, a possible model for intermediates formed during the uptake and release of metals by cyclams. The ligand 1 and the zinc complex 3 were active towards viral strains HIV-1 (III(B)) (IC(50) values of 10.51 ± 0.23 and 3.50 ± 0.33 μM, respectively), and HIV-2 (ROD) (IC(50) values of 133.78 ± 14.10 and >110.67 μM, respectively). 2D [(1)H, (13)C] and [(1)H, (15)N] NMR spectroscopic studies suggested that the types of configurational isomers present in solution depend on the axial ligand.

Abstract For Abstract see ChemInform Abstract in Full Text.

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the genome of a human cell is an essential step in the viral replication cycle. Understanding of the integration process has been facilitated by the development of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking regions of approximately 200 bp that represent the intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction product resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron microscopy revealed the presence of a range of reaction products resulting from single or multiple integration events. The binding of HIV-1 integrase to mini-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that adopts the structure of a three-site synapsis that is reminiscent of the Mu phage transposase complex.

Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2'-O-allyl-1-beta-D-arabinofuranosyl-uracil and -cytosine and 2'-O-allyl-9-beta-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2'-O-Allyl-araUDP proved to be inhibitory to RNR at an IC(50) of 100 microM, whereas 2'-O-allyl-araCDP was only marginally active (IC(50) 1 mM) and 2'-O-allyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2'-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2'-O-allyl-araU and -araC monophosphates, namely 2'-O-allyl-5'-(phenylethoxy-L-alanyl phosphate)-araU and -araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.

A series of 3'-C-cyano-3'-deoxynucleosides have been synthesized and evaluated as antiviral agents. Reaction of 2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-erythro-pentofuranos- 3'-ulosyl derivatives of uracil, 4-N-acetylcytosine, and adenine with sodium cyanide gave a mixture of epimeric cyanohydrins, which after 3'-deoxygenation yielded the corresponding 3'-C-cyano-3'-deoxy-beta-D-xylo-pentofuranosyl derivatives 10. These compounds were epimerized to the corresponding beta-D-ribo-pentofuranosyl derivatives 11. Desilylation of 10 and 11 gave the deprotected 3'-C-cyano-3'-deoxy-beta-D-xylo- and -ribo-pentofuranosyl nucleosides. These derivatives of uridine, cytidine, and adenine, as well as the 3'-C-cyano-3'-deoxy-beta-D-xylo- and -ribo-pentofuranosyl, 3'-C-cyano-2',3'-dideoxy-beta-D-threo- and -erythro-pentofuranosyl, and 3'-C-cyano-2',3'-dideoxy-beta-D-glycero-pent-2'-enofuranosyl derivatives of thymine, were evaluated for their antiviral activity. None of the compounds proved active against the replication of retroviruses (human immunodeficiency virus, murine sarcoma virus) at concentrations that were not toxic to the host cells. However, the 3'-C-cyano-3'-deoxy-beta-D-xylo- (12e) and -ribo-pentofuranosyl (13e) derivatives of adenine showed activity against some DNA (i.e., vaccinia) and RNA (i.e., Sindbis, Semliki forest) viruses at concentrations well below the cytotoxicity threshold.