Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids

(+)-1(R)-Coclaurine (1) and (-)-1(S)-norcoclaurine (3), together with quercetin 3-O-beta-D-glucuronide (4), were isolated from the leaves of Nelumbo nucifera (Nymphaceae), and identified as anti-HIV principles. Compounds 1 and 3 demonstrated potent anti-HIV activity with EC50 values of 0.8 and <0.8 microg/mL, respectively, and therapeutic index (TI) values of >125 and >25, respectively. Compound 4 was less potent (EC50 2 microg/mL). In a structure-activity relationship study, other benzylisoquinoline, aporphine, and bisbenzylisoquinoline alkaloids, including liensinine (14), negferine (15), and isoliensinine (16), which were previously isolated from the leaves and embryo of Nelumbo nucifera, were evaluated for anti-HIV activity. Compounds 14-16 showed potent anti-HIV activities with EC50 values of <0.8 microg/mL and TI values of >9.9, >8.6, and >6.5, respectively. Nuciferine (12), an aporphine alkaloid, had an EC50 value of 0.8 microg/mL and TI of 36. In addition, synthetic coclaurine analogs were also evaluated. Compounds 1, 3, 12, and 14-16 can serve as new leads for further development of anti-AIDS agents.     

Are seroepidemiological surveys for human immunodeficiency virus infection based on tests on pools of serum specimens accurate and cost-effective?

Serum specimens (n = 17668) from UK antenatal patients in the Thames Regions were tested by Wellcozyme HIV 1/2 EIA singly and in pools of 6, 12 and 24: 35 (0.2%, 1 in 505) were confirmed as anti-HIV positive. The pools of 12 were also tested for anti-HIV 1/2 by IAF Biochem, Behring and Diagnostics Pasteur EIAs. All 35 positive specimens were easily detectable after pooling in groups of 12. The false positive rate for Wellcozyme was nearly halved compared with individual testing (1 in 309 false positive compared with 1 in 174). For the other assays false positive rates on pools of 12 were: IAF Biochem 1 in 193, Behring 1 in 140, Diagnostics Pasteur 1 in 1547. Twenty-two known anti-HIV 2-positive sera were detected by all four EIAs when diluted as in pools of 6 and 12, but by only three EIAs in pools of 24 and 48. Pooling in groups of 6 did not seem to delay detection of HIV 1 seroconversion, but pooling in groups of 12, 24 and 48 might delay it by 1, 2 and 3 weeks respectively. For this study the effect of pooling in groups of 12 would have been a reagent saving of 87-91% and a labour saving of about 50%. Because of the low HIV incidence and rarity of specimens collected around seroconversion in UK, little, if any, loss of sensitivity would result from it. Pooling in groups of 12 has therefore been chosen for the screening of anonymous antenatal specimens in the UK.     

3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives as potent anti-HIV agents

3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC(50) value of 2 x 10(-5) microM and a TI value of 1.12 x 10(6). 3-O-(3',3'-Dimethylsuccinyl)- and 3-O-(3',3'-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC(50) values of 0.0017 and 0.0013 microM, respectively, and TI values of 16,160 and 19,530, respectively.     

Synthesis of 5-alkenylated D4T analogues via the Pd-catalyzed cross-coupling reaction

The target compounds 5-[N-(6-amino-hexyl)-acrylamide]-2',3'-didehydro-2',3'-dideoxy-uridine (12) and 5-[N-[5-(methoxycarbonyl)-pentyl]-acrylamide]-2',3'-didehydro-2',3'- dideoxy-uridine (15) were prepared by the palladium acetate-triphenylphosphine-catalyzed reaction of the 5'-O-acetyl-5-iodo-d4T analogue (3). These compounds 12 and 15 can be used to prepare nucleotide probes carrying fluorescent labels and were nevertheless screened for their anti-HIV activity. The biological data demonstrated that none of them were active against HIV-1.     

Compounds from Kadsura heteroclita and related anti-HIV activity

Phytochemical investigation of the stems of Kadsura heteroclita led to isolation of 16 compounds, including the triterpenoid named longipedlactone J (2), and two dibenzocyclooctadiene type lignans named heteroclitin I and J (3, 4). Compounds 8-10, 14, and 15 were weakly active as anti-HIV agents, whereas compounds 6 and 12 exhibited moderate anti-HIV activity with EC50 values of 1.6 microg/mL, and 1.4 microg/mL, therapeutic index (TI) values of 52.9, and 65.9, respectively. Their structures were established by spectroscopic methods, including application of 2D NMR techniques and CD spectra.     

3,28-Di-O-(dimethylsuccinyl)-betulin isomers as anti-HIV agents

Four isomers of 3,28-di-O-(dimethylsuccinyl)-betulin were prepared and evaluated for anti-HIV activity against HIV-1 replication in H9 lymphocyte cells. 3-O-(3',3'-Dimethylsuccinyl)-28-O-(2", 2"-dimethvlsuccinyl)-betulin (11) was the most potent anti-HIV compound with an EC5, value of 0.00087 microM and a TI value of 42,400.     

Potent anti-HIV activity of scytovirin domain 1 peptide

Scytovirin (SVN) is a novel anti-HIV protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. SVN contains two apparent domains, one comprising amino acids 1-48 and the second stretching from amino acids 49 to 95. These two domains display significant homology to each other and a similar pattern of disulfide bonds. Two DNA constructs encoding scytovirin 1-48 (Cys7Ser) (SD1) and 49-95 (Cys55Ser) (SD2) were constructed, and expressed in E. coli, with thioredoxin fused to their N-terminus. Purified recombinant products were tested for binding activities with the HIV surface envelope glycoproteins gp120 and gp41. Whole cell anti-HIV data showed that SD1 had similar anti-HIV activity to the full-length SVN, whereas SD2 had significantly less anti-HIV activity. Further deletion mutants of the SD1 domain (SVN(3-45)Cys7Ser, SVN(6-45)Cys7Ser, SVN(11-45)Cys7Ser) showed that the N-terminal residues are necessary for full anti-HIV activity of SD1 and that an eight amino acid deletion from the C-terminus (SVN(1-40)Cys7Ser) had a significant effect, decreasing the anti-HIV activity of SD1 by approximately five-fold.     

Design and synthesis of novel 2',3'-dideoxy-4'-selenonucleosides as potential antiviral agents

On the basis of potent anti-HIV activity of 2',3'-dideoxynucleosides (ddNs), their bioisosteric analogues, 2',3'-dideoxy-4'-selenonucleosides (4'-seleno-ddNs) were first synthesized from a chiral template, d-glutamic acid using stereoselective ring-closure reaction of the dimesylate with Se(2-) and Pummerer type condensation of the selenoxide with nucleobases as key steps. X-ray crystallographic analysis indicated that 4'-seleno-ddNs adopted the same C2'-endo/C3'-exo (South) conformation as anti-HIV active ddNs, but did not show anti-HIV activity, indicating that RT seems to prefer the C2'-exo/C3'-endo (North) conformation on binding with their triphosphates.     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.     

SYNTHESIS OF 5-ALKENYLATED D4T ANALOGUES VIA THE Pd-CATALYZED CROSS-COUPLING REACTION

The target compounds 5-[N-(6-amino-hexyl)-acrylamide]-2′,3′-didehydro-2′,3′-dideoxy-uridine (12) and 5-{N-[5-(methoxycarbonyl)-pentyl]-acrylamide}-2′,3′-didehydro-2′,3′-dideoxy-uridine (15) were prepared by the palladium acetate-triphenylphosphine-catalyzed reaction of the 5′-O-acetyl-5-iodo-d4T analogue (3). These compounds 12 and 15 can be used to prepare nucleotide probes carrying fluorescent labels and were nevertheless screened for their anti-HIV activity. The biological data demonstrated that none of them were active against HIV-1.     

Synthesis and anti-HIV evaluation of 3'-triazolo nucleosides

A series of hitherto unknown 3'-α-[1,2,3]-substituted triazolo-2',3'-dideoxypyrimidine nucleoside analogues of the anti-HIV 3'-azido-3'-deoxythymidine (AZT) were synthesized through catalyzed alkyne-azide 1,3-dipolar cycloaddition (Huisgen reaction). Those 3'-[1,2,3]-triazolo analogues bearing an azido alkyl chain were evaluated for their anti-HIV activity against HIV-1 in primary human lymphocytes as well as for their cytotoxicity in different cells. None of them inhibit HIV replication (EC(50) > 20 μM); two of them were converted to their triphosphate form to evaluate their HIV-RT inhibition.     

Anti-AIDS agents 73: structure-activity relationship study and asymmetric synthesis of 3-O-monomethylsuccinyl-betulinic acid derivatives

3-O-3'(or 2')-Methylsuccinyl-betulinic acid (MSB) derivatives were separated by using recycle HPLC. The structures of four isomers were assigned by NMR and asymmetric synthesis. 3-O-3'S-Methylsuccinyl-betulinic acid (3'S-MSB, 4) exhibited potent anti-HIV activity with an EC(50) value of 0.0087microM and a TI value of 6.3x10(3), which is comparable to the data for bevirimat (DSB, PA-457), a current clinical trials drug that was also derived from betulinic acid. The anti-HIV potency of 4 was slightly better than that of AZT.     

Anti-AIDS agents 72. Bioisosteres (7-carbon-DCKs) of the potent anti-HIV lead DCK

Three 9,10-di-O-(-)-camphanoyl-7,8,9,10-tetrahydro-benzo[h]chromen-2-one (7-carbon-DCK) analogs (3a-c) were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. All three new carbon bioisosteres of the anti-HIV lead DCK showed anti-HIV activity. Compound 3a had an EC(50) value of 0.068 microM, which was comparable to that of DCK in the same assay. The preliminary results indicated that 7-carbon-DCK analogs merit attention as potential HIV-1 inhibitors for further development into clinical trials candidates.     

Synthesis of analogues of flazin, in particular, flazinamide, as promising anti-HIV agents

Flazin isolated from the fruiting bodies of Suillus granulatus was found to possess weak anti-HIV activity (EC(50)=2.36 microM, TI=12.1). To establish a SAR study, 46 flazin analogues were synthesized, and their anti-HIV activities were evaluated in vitro. Among them, flazinamide (9a) showed the most potent activity with an EC(50) value of 0.38 microM and a TI value of 312.0. The results suggested that appropriate substituents at positions 3, 1', and 5' of flazin might play a crucial role in determining their anti-HIV activities, and that flazinamide can be considered as a promising, readily available anti-HIV agent.     

Metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-2',3'-dideoxyadenosine derivatives

Both 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine have been shown (Mitsuya, H., and Broder, S. (1987) Nature 325, 773-778) to have in vitro activity against the human immunodeficiency virus-1 (HIV). However, these dideoxynucleosides may be catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we have synthesized the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of 2',3'-dideoxyadenosine. The metabolism and anti-HIV activity of the 2-halo-2',3'-dideoxyadenosine derivatives and of 2',3'-dideoxyadenosine were compared. The 2-halo-2',3'-dideoxyadenosine derivatives were not deaminated significantly by cultured CEM T lymphoblasts. Experiments with 2-chloro-2',3'-dideoxyadenosine showed that the T cells converted the dideoxynucleoside to the 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 microM), the 2-halo-2',3-dideoxyadenosine derivatives inhibited the cytopathic effects of HIV toward MT-2 T lymphoblasts, and retarded viral replication in CEM T lymphoblasts. Experiments with a deoxycytidine kinase-deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-chloro-2',3'-dideoxyadenosine. In contrast, 2',3'-dideoxyadenosine was phosphorylated by the deoxycytidine kinase-deficient mutant and retained anti-HIV activity in this cell line. Thus, the 2-halo derivatives of 2',3'-dideoxyadenosine, in contrast to 2',3'-dideoxyadenosine itself, are not catabolized by T cells. Their anti-HIV and anti-proliferative activities are manifest only in cells expressing deoxycytidine kinase. The in vivo implications of these results for anti-HIV chemotherapy are discussed.     

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.     

Anti-HIV drugs: comparative toxicities in murine fetal liver and bone marrow erythroid progenitor cells

The toxicity of anti-HIV drugs, 3'-azido-3'-deoxythymidine (zidovudine, AZT), 2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine (d4T) and ribavirin was studied in vitro in murine fetal liver cells (FLC) and in bone marrow cells. These studies indicate that d4T is the least toxic drug and ribavirin is the most toxic agent in both models. However, the murine FLC system was found to be a more sensitive model for the assessment of toxicity of anti-HIV agents towards erythroid progenitor cells as indicated by the IC50 values.     

Synthesis and anti-HIV activity of 4'-cyano-2',3'-didehydro-3'-deoxythymidine

A new anti-HIV agent 4'-cyano-2',3'-didehydro-3'-deoxythymidine (9) was synthesized by allylic substitution of the 3',4'-unsaturated nucleoside 14, having a leaving group at the 2'-position, with cyanotrimethylsilane in the presence of SnCl4. Evaluation of the anti-HIV activity of 9 showed that this compound is much less potent than the recently reported 2',3'-didehydro-3'-deoxy-4'-(ethynyl)thymidine (1).     

Structure–activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers. Part II: P2/P3 region and discovery of cobicistat (GS-9350)

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.