COMPARISON OF THE ANTIVIRAL ACTIVITY OF HYDROPHOBIC AMINO ACID PHOSPHORAMIDATE MONOESTERS OF 2′, 3′-DIDEOXYADENOSINE (DDA) AND 3′-AZIDO-3′-DEOXYTHYMIDINE (AZT)

A series of hydrophobic, water soluble and non-toxic amino acid phosphoramidate monoesters of dideoxyadenosine (ddA) and 3′-azido-3′-deoxythymidine were shown to inhibit the replication of HIV-1 in human peripheral blood mononuclear cells (PBMC) from two donors. The tryptophan methyl ester phosphoramidates of AZT and ddA were equally potent (EC₅₀S = 0.3–0.4 μM), while the phenyl methyl ester of ddA was 40- to 100- fold more potent than the AZT derivatives. The alaninyl methyl ester of AZT was found to be 70- fold more potent than the ddA derivative. The methyl amide derivatives were found to be 5–20 fold less active than the methyl esters for the ddA series, while for AZT the derivatives were found to be of similar potency or 60- to 166- fold more potent than the methylesters.     

HPLC and mass spectrometry analysis of the enzymatic hydrolysis of anti-HIV pronucleotide diastereomers

In one current strategy to develop membrane-soluble pronucleotides, the phosphoramidate derivatives of the approved anti-HIV nucleosides 2',3'-didehydro-3'-deoxythymidine (d4T), 3'-azido-3'-deoxythymidine (AZT), (-)-beta-L-2',3'-dideoxy-3'- thiacytidine (3TC), and 2',3'-dideoxyadenosine (ddA) exhibit promising antiviral activity. However, the non-stereoselective synthetic route results in a mixture of diastereoisomers, which differ in the configuration of the phosphorus chiral center. Since it is believed that enzymatic ester hydrolysis is the first step in the intracellular activation of these prodrugs and that this process could be dependent on the stereochemistry at the phosphorus center, analytical methods must be developed. In the present work, in vitro evaluation of the selectivity of pig liver esterase (PLE) towards each diastereomer of d4T, AZT, 3TC, and ddA prodrugs has been investigated, applying our recently published HPLC-MS procedure using a polysaccharide-type chiral stationary phase. This method has been used to analyze the products of the PLE-catalyzed hydrolysis of the pronucleotides. It was found that both diastereomers of the four prodrugs were substrates for PLE.     

Synthesis and anti-HIV activities of symmetrical dicarboxylate esters of dinucleoside reverse transcriptase inhibitors

Three nucleoside analogues, 3'-fluoro-2',3'-dideoxythymidine (FLT), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-dideoxy-3'-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC(50) values of 0.8-1.0nM and 3-4nM against HIV-1(US/92/727) and HIV-1(IIIB) cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC(50)=3-60nM) was improved by 1.5-66 fold when compared to 3TC (EC(50)=90-200nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile.     

AIDS dementia: synthesis and properties of a derivative of 3'-azido-3'-deoxythymidine (AZT) that may become 'locked' in the central nervous system

In an attempt to provide a derivative of 3'-azido-3'-deoxythymidine (AZT) which might be sequestered in the central nervous system and release AZT, the dihydropyridine ester 5'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-3'-deoxythymidine, was synthesized in a three step sequence. This material showed potent anti-HIV-1 activity in MT-4 cells most probably by hydrolysis to the parent nucleoside, AZT. This dihydropyridine derivative of AZT could be easily oxidized to a positively charged pyridinium derivative of AZT in rat brain cytosol. In turn the pyridinium form could be hydrolyzed, non-enzymatically, to AZT.     

Cysteinyl methyl ester of AVP(4-8), a potent agonist on the maintenance of passive avoidance in rats

A series of short AVP analogs with D- or L-arginine forming the C-terminal was synthesized, and their peripheral effects, i.e., vasoconstrictor and antidiuretic activities, and behavioral effects in enhancing retention in passive avoidance in rats were evaluated. We found that: 1) AVP(4-8) and its cysteinyl methyl ester were more potent in the behavioral response than its D-Arg homologs; 2) the methyl ester derivative was the most effective analog in this behavioral test among the peptides we synthesized, with a potency 40 times as high as AVP; 3) neither the D- nor the L-Arg short derivatives of AVP showed any peripheral effects up to a dose thousands of times greater than AVP. The results support the contention that arginine in the short analogs plays an important role in their behavioral response associated with a relatively steady peptide conformation.     

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) > (+)3TC > (-)3TC > PMPA > azidothymidine (AZT) > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T > ddA (ddI) > ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.     

ANTI-HIV ACTIVITY OF NOVEL PHOSPHONATE DERIVATIVES OF AZT,d4T,AND ddA

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2′,3′-Dideoxy-2′,3′-didehydrothymidine 5′-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2′,3′-Dideoxy-3′-azidothymidine 5′-cyclohexylphosphite.     

Anti-HIV activity of novel phosphonate derivatives of AZT, d4T, and ddA

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2',3'-Dideoxy-2',3'-didehydrothymidine 5'-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2',3'-Dideoxy-3'-azidothymidine 5'-cyclohexylphosphite.     

bis(S-Acyl-2-thioethyl)esters of 2′,3′-Dideoxyadenosine 5′-Monophosphate Are Potent Anti-HIV Agents in Cell Culture

Abstract

It is shown that ddA bis(SATE)phosphotriesters have potent anti-HIV activity in cell culture. Thus, compared with the parent nucleoside, a decrease of 3 or 4 orders of magnitude was observed in the EC₅₀ values for the bis(S-acetyl-2-thioethyl)phosphotriester derivative, which makes this compound as active as AZT.     

Anti-AIDS agents. Part 47: Synthesis and anti-HIV activity of 3-substituted 3',4'-Di-O-(S)-camphanoyl-(3'R,4'R)-(+)-cis-khellactone derivatives

Six 3-substituted 3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone derivatives (3-8) were synthesized from 3-methyl DCK (2). 3-Hydroxymethyl DCK (6) exhibited potent anti-HIV activity in H9 lymphocytes with EC(50) and TI values of 1.87 x 10(-4) microM and 1.89 x 10(5), respectively. These values are similar to those of DCK and better than those of AZT in the same assay.     

Synthesis and anticancer activity of 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (AZT)

A series of novel N-alkyl 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (6-15) was synthesized by means of phosphonylation of 3'-azido-3'-deoxythymidine (4) with P-chloromethylphosphonic ditriazolide (3) followed by a reaction with the appropriate amine. The synthesized phosphonamidates 6-15 were evaluated for their cytotoxic activity in two human cancer cell lines: oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in KB human cancer cells was displayed by phosphonamidate 8 (IC(50)=5.8 μg/mL), however, this compound was less potent than the parent AZT (IC(50)=3.1 μg/mL). Phosphonamidate 10 showed only moderate activity (IC(50)=12.1 μg/mL) whereas the other phosphonamidates proved inactive. Similarly, the highest activity in MCF-7 human cancer cells was displayed by phosphonamidate 8 (IC(50)=3.7 μg/mL) but it proved somewhat less active than AZT (IC(50)=2.6 μg/mL). Some activity was also displayed by phosphonamidate 10 (IC(50)=12.8 μg/mL) but the other phosphonamidates were found inactive. Hydrolysis studies indicate that the synthesized phosphonamidates are likely to act as prodrugs of the parent nucleoside (AZT). Transport measurements showed that the most active phosphonamidates (8 and 10) were able to permeate across the intestinal epithelium in vitro. The apparent permeability coefficients determined in Caco-2 cell monolayers indicated that these compounds could be moderately absorbed in humans.     

3alpha-(4-Substituted phenyl)nortropane-2beta-carboxylic acid methyl esters show selective binding at the norepinephrine transporter

A series of 3alpha-(4-substituted)nortropane-2beta-carboxylic acid methyl esters was synthesized and evaluated for the ability to inhibit radioligand binding at the dopamine, serotonin, and norepinephrine transporters. 3alpha-(4-Methylphenyl)nortropane-2beta-carboxylic acid methyl ester (4c) was found to be selective and highly potent for the norepinephrine transporter (NET) relative to the dopamine and serotonin transporters.     

Substituted E-3-(2-Chloro-3-indolylmethylene)1,3-dihydroindol-2-ones with antitumor activity

The synthesis and antitumor activity of a new series of E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones is described. Several compounds were active on the primary test (three human cell lines) and entered the second level (60 human cell lines). All of them were potent growth inhibitors with GI(50) ranging from -5.32 to -7.27. Four are now under review by BEC (Biological Evaluation Committee of the NCI). The most potent antitumor derivatives were also evaluated as cardiotonic agents (in view of a possible coanthracyclinic activity). In order to find a possible mechanism of action their effects on cell cycle progression in an adenocarcinoma cell line (HT29) were tested, evidencing that these molecules are able to block HT29 in mitosis. The introduction of new substituents in the indolinone moiety while maintaining the same chloroindole portion generated interesting derivatives. 3-(2-Chloro-5-methoxy-6-methyl-3-indolylmethylene)5-hydroxy-1,3-dihydroindol-2-one was the most active of the whole series. It was more potent than vincristine against seven of the nine tumors considered. Moreover it was selective towards some cell lines such as MDA-MB-435 (breast), OVCAR-3 (ovarian) and SK-MEL-28 (melanoma). Even the introduction of a benzyl ring at the nitrogen of the chloroindole portion, gave rise to potent compounds.     

Methyl 2-(2-(4-formylphenoxy)acetamido)-2-substituted acetate derivatives: a new class of acetylcholinesterase inhibitors

A new class of inhibitors of acetylcholinesterase (methyl 2-(2-(4-formylphenoxy)acetamido)-2-substituted acetate derivatives) is described. Compounds 4b and 4i were found to be more potent than galanthamine in inhibiting acetylcholinesterase.     

Steric and charge factors in the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase hydrolysis.

The contribution of steric and negative charge factors to the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase was investigated. The hydrolysis rates of uridylyl(3'-5')N6-(N-threonylcarbonyl)-adenosine, its model derivatives, methyl ester and O-benzyl ester, together with unmodified uridyly (3'-5')adenosine, were studied. It was found that the contribution of both factors is of the same order. The steric inhibition of digestion is distinctly higher than that confirmed by N6-(delta2-isopentenyl)adenosine [1], which is ascribed to the rigid conformation of the threonylcarbonyladenosine side chain.     

Synthesis of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-ones and their 2-methylene derivatives as potential spermicidal and microbicidal agents

A series of twenty two derivatives of 3-(1-alkyl/aminoalkyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one and their 2-methylene derivatives were synthesized from naturally abundant cinchonine (I). Tartarate salts of these compounds were prepared and evaluated for spermicidal activity. The most active compounds (24, 27, 34, 36, and 38) showing potent spermicidal activity were further evaluated against different strains of Trichomonas vaginalis, for antimicrobial activity, in HeLa cell lines for cytotoxicity and against Lactobacillus jensenii for eco-safety. The tartarate of 3-(1-pentyl-3-vinyl-piperidin-4-yl)-1-(quinolin-4-yl)-propan-1-one (27) was found to be more active than N-9 in spermicidal activity.     

Novel P450(17alpha) inhibitors: 17-(2'-oxazolyl)- and 17-(2'-thiazolyl)-androstene derivatives

Twelve 17-(2'-oxazolyl)- and 17-(2'-thiazolyl)-androsta-5,16-diene derivatives were designed and synthesized from 3 beta-acetoxy-pregna-5,16-dien-20-one (1b) as inhibitors of 17 alpha-hydroxylase-C(17,20)-lyase (P450(17 alpha)). Potent inhibitors of this enzyme could be of value as treatment of prostate cancer. Two substituents (methyl and phenyl) were introduced either at their 4'- or 5'-position in order to investigate their structure-activity relationship. Due to the 16,17-double bond, 17-thiazoles were generally obtained in low yield. The pharmacological results showed that the compounds containing 17-(2'-oxazolyl) (14c) and 17-(2'-thiazolyl) (8c) (41.5%) demonstrated reasonable inhibition against P450(17 alpha). Their 3-acetate (13c and 7c) were less potent than their 3-OH counterparts. The introduction of a phenyl or methyl group generally decreased inhibitory activity. Surprisingly, 17-(5'-methyl-2'-thiazolyl) (12a) was the most potent compound in this series and was almost as potent as L-39, which has good antitumor activity.     

Novel adenosine A1 receptor antagonists. Synthesis and structure-activity relationships of a novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5 -a]pyridines

A novel series of 3-(2-cyclohexenyl-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazol o[1,5-a]pyridines was synthesized and evaluated for in vitro adenosine A1 and A2A receptor binding activities. Most of the cyclohexenyl derivatives (7a-e, 8a-s) were found to be potent adenosine A1 receptor antagonists. In a series of analogues of FR166124 (3a), alcohol 7c, nitrile 7e and amide derivatives (7d, 8c, 8r) were found to be more potent A1 antagonists with higher A2A/A1 selectivity than FR166124. Amongst them, 8r showed considerable water solubility (33.3 mg/mL), but lower than that of the sodium salt of FR166124 (> 200 mg/mL). Additionally, FR166124 had strong diuretic activity by both p.o. and iv administration in rats (minimum effective dose=0.1 and 0.032 mg/kg, respectively).