Investigation of 8-Aza-7-Deaza Purine Nucleoside Derivatives.

Glycosylation of 6-amino-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine and its iodo- and bromo- analogues with the protected ribofuranose and 2'-deoxyribofuranose under different conditions resulted in the synthesis of N8- and N8-glycosylated purine nucleosides. Five key intermediate nucleosides, having 6-methoxy, 7-iodo, and 2-bromo groups, were further derivatized to 23 final 8-aza-7-deazapurine nucleoside derivatives. The structures of N8- and N8-glycosylated products were assigned based on UV and NMR spectra. HMBC analysis of 2D NMR spectra and X-ray crystallographic studies of the representative compounds unambiguously verified the connection of ribose ring to N8- or N8-position of the purine ring. The anticancer activity of these new compounds was evaluated.

Urea-containing peptide boronic acids as potent proteasome inhibitors.

A novel class of urea-containing peptide boronic acids as proteasome inhibitors was designed by introducing a urea scaffold to replace an amido bond. Compounds were synthesized and their antitumor activities were evaluated. After two rounds of optimizations, the compound I-14 was found to be a potent proteasome inhibitor. Compared with Bortezomib, I-14 showed higher potency against the chymotrypsin-like activity of human 20S proteasome (IC50 < 1 pM), similar potency against four different cancer cell lines (IC50 < 10 nM), and better pharmacokinetic profile. Furthermore, I-14 significantly inhibited tumor growth in Bel7404 mouse xenograft model. The excellent proteasome inhibition by I-14 was rationalized through docking and molecular dynamics studies.

Versatile synthesis and biological evaluation of novel 3'-fluorinated purine nucleosides.

A unified synthetic strategy accessing novel 3'-fluorinated purine nucleoside derivatives and their biological evaluation were achieved. Novel 3'-fluorinated analogues were constructed from a common 3'-deoxy-3'-fluororibofuranose intermediate. Employing Suzuki and Stille cross-coupling reactions, fifteen 3'-fluororibose purine nucleosides 1-15 and eight 3'-fluororibose 2-chloro/2-aminopurine nucleosides 16-23 with various substituents at position 6 of the purine ring were efficiently synthesized. Furthermore, 3'-fluorine analogs of natural products nebularine and 6-methylpurine riboside were constructed via our convergent synthetic strategy. Synthesized nucleosides were tested against HT116 (colon cancer) and 143B (osteosarcoma cancer) tumor cell lines. We have demonstrated 3'-fluorine purine nucleoside analogues display potent tumor cell growth inhibition activity at sub- or low micromolar concentration.

Antiviral activity of FNC, 2'-deoxy-2'-ß-fluoro-4'-azidocytidine, against human and duck HBV replication.

BACKGROUND: New drugs are needed to combat HBV infection. We investigated the anti-HBV activity of the deoxycytidine analogue FNC, which has anticancer activity and has been found to inhibit HCV replication. METHODS: In this study, a human hepatoma HepG2.2.15 cell culture system and duck HBV (DHBV) infection model were used as the in vitro and in vivo models to evaluate the anti-HBV activity of FNC. RESULTS: In the cell model, FNC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner, with 50% effective concentration values of 0.037 µM for hepatitis B surface antigen and 0.044 µM for hepatitis B e antigen on day 9. Consistent with the HBV antigen reduction, FNC also reduced the HBV DNA level by 92.31% and 93.90% intracellularly and extracellularly, respectively. DHBV DNA levels were markedly reduced after treatment with the FNC at 0.5, 1.0 and 2.0 mg/kg•day dosages. The inhibition rate of FNC at the dose of 2.0 mg/kg•day reached 91.68% and 81.96%, in duck serum and liver, respectively, on day 10. Furthermore, significant liver histology restoration after FNC treatment was observed, as evaluated by the histopathological analysis. CONCLUSIONS: FNC can evidently inhibit the replication of HBV in the HepG2.2.15 cell line in vitro and inhibits DHBV replication in ducks in vivo. It could be potentially developed into a new anti-HBV drug.

Synthesis and anti-HIV-1 activity of 4-substituted-7-(2'-deoxy-2'-fluoro-4'-azido-ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine analogues.

Three novel 4-subsituted-7-(2'-deoxy-2'-fluoro-4'-azido-ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine analogues were designed, synthesized, and tested for their anti-HIV-1 activity. Initial biological studies indicated that among these pyrrolo[2,3-d]pyrimidine ribonucleoside analogues, 4-amino-7-(2'-deoxy-2'-fluoro-4'-azido-ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine 10 exhibited the most potent anti-HIV-1 activity (EC(50)=0.5±0.3 µM), while 4-hydroxy-7-(2'-deoxy-2'-fluoro-4'-azido-ß-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine 9 and 4-amino-5-fluoro-7-(2'-deoxy-2'-fluoro-4'-azido-ß-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine 11 showed moderate activity (EC(50)=13±8 and 5.4±0.3 µM, respectively). The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds with concentrations up to 25 µM.

Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of beta-tubulin at Cys 12 and suppression of microtubule dynamics.

Bis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potent anticancer activities to a broad spectrum of tumor cell lines with IC50 values from high nanomolar to low micromolar and showed equal anticancer potency between tumor cell lines overexpressing multidrug-resistant gene, MDR1 (MCF7/adr line and KBv200 line), and their parental MCF7 line and KB lines. BFBTS inhibited microtubule polymerization dynamics in MCF7 cells, at a low nanomolar concentration of 54 nmol/L, while disrupting microtubule filaments in cells at low micromolar concentration of 1 micromol/L. Tumor cells treated with BFBTS were arrested at G2-M phase, conceivably resulting from BFBTS-mediated antimicrotubule activities. Mass spectrometry studies revealed that BFBTS bound and modified beta-tubulin at residue Cys12, forming beta-tubulin-SS-fluorobenzyl. The binding site differs from known antimicrotubule agents, suggesting that BFBTS functions as a novel antimicrotubule agent. BFBTS at a dose of 25 mg/kg inhibited tumor growth with relative tumor growth rates of 19.91%, 18.5%, and 23.42% in A549 lung cancer, Bcap-37 breast cancer, and SKOV3 ovarian cancer xenografts, respectively. Notably, BFBTS was more potent against MDR1-overexpressing MCF7/adr breast cancer xenografts with a relative tumor growth rate of 12.3% than paclitaxel with a rate of 43.0%. BFBTS displays a novel antimicrotubule agent with potentials for cancer therapeutics.

Identification and simultaneous determination of p-FHA and p-FBA, two metabolites of anti-tumor agent--Fluorapacin in rat urine.

Fluorapacin, bis(4-fluorobenzyl)trisulfide, a small molecule natural product derivative of trisulfide, has revealed a broad spectrum of anti-proliferative activity and in vivo anti-tumor efficacy in human xenograft mice models with excellent safety profile. In the present study, two new metabolites, para-fluorohippuric acid (p-FHA) and para-fluorobenzoic acid (p-FBA), were identified by GC-MS and HPLC as the main metabolites in urine of rats after intravenous administration of fluorapacin. A simple HPLC-UV method for simultaneous determination of these two metabolites in urine has been developed and validated. The newly developed method demonstrated excellent specificity, accuracy, precision, and stability. This method was successfully employed to study the urinary excretion of fluorapacin in rats. The results indicated that p-FHA was the major metabolite in urine, and the total excretion recovery of p-FHA and p-FBA was 67.6+/-4.9% (mean+/-SE, n=6) of dosage after 48h of administration.

Stability studies of anticancer agent bis(4-fluorobenzyl)trisulfide and synthesis of related substances.

Bis(4-fluorobenzyl)trisulfide, fluorapacin, has been extensively developed as a promising new anticancer drug candidate. Its degradation products were identified and verified by the newly synthesized compounds bis(4-fluorobenzyl)disulfide (A) and bis(4-fluorobenzyl)tetrasulfide (B) which were resulted from the disproportionation of fluorapacin under forced conditions. A stability-indicating HPLC method was used for the stability evaluation of active pharmaceutical ingredient (API) fluorapacin and finished pharmaceutical product (FPP) under various conditions. High recovery (99.57%) of API was found after three freeze-thaw cycle processes of fluorapacin FPP. Susceptibility of fluorapacin to oxidative degradation was studied by treating fluorapacin and FPP in 30% hydrogen peroxide aqueous solution, and the result verified the oxidative stability of fluorapacin. However, treatment of this drug candidate under strong light (4500 Lx+/-500 Lx) for 10 days showed substantial effect on the recovery of fluorapacin, especially from fluorapacin FPP. Strong acid (1.0M, HCl) did not affect the recovery of fluorapacin while strong basic condition (1.0M, NaOH) accelerated the disproportionation of fluorapacin to its related substances A and B. The stability of fluorapacin in its aqueous media at a pH range of 2.0-10.0 for up to 6h was further investigated, and 4.0-8.0 was found to be the most stable pH range. Fluorapacin and FPP were exposed to the elevated temperatures of 40 and 60 degrees C for 10 days without obvious impact on their stability. The thermal stability of fluorapacin API and FPP under constant humidity with light protection was also thoroughly investigated under accelerated (40+/-2 degrees C, RH 75+/-5%, 6 months) and long-term (25+/-2 degrees C, RH 60+/-10%, 24 months) conditions. There was no significant change except minor color change of fluorapacin FPP. Therefore, fluorapacin has excellent stability as a potential drug candidate for further clinical development investigation.

Determination of anti-tumor agent bis(p-fluorobenzyl)trisulfide and its degraded compound in rat blood using reversed phase high-performance liquid chromatography.

Bis(p-fluorobenzyl)trisulfide (BFTS) demonstrated a broad spectrum of anti-proliferative activity and in vivo anti-tumor efficacy in human xenograft mice models. BFTS is rapidly degraded to its major metabolite bis(p-fluorobenzyl)disulfide (BFDS) in blood. In this study, we developed a reliable procedure for stable storage and treatment of blood samples containing BFTS. An HPLC assay method was developed and validated for the quantitative determination of BFTS and BFDS in rat blood. The developed procedure and method have been successfully utilized to study the concentration-time curves of BFTS and BFDS in the blood of rats after vein injection of 12.5 mg kg(-1) BFTS.

Crystal structure determination of new antimitotic agent bis(p-fluorobenzyl)trisulfide.

The purpose of this research was to investigate the physical characteristics and crystalline structure of bis(p-fluorobenzyl)trisulfide, a new anti-tumor agent. Methods used included X-ray single crystal diffraction, X-ray powder diffraction (XRPD), Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetric (DSC) and thermogravimetric (TG) analyses. The findings obtained with X-ray single crystal diffraction showed that a monoclinic unit cell was a = 12.266(1) A, b = 4.7757(4) A, c = 25.510(1) A, beta = 104.25(1) degrees ; cell volume = 1,448.4(2) A(3), Z = 4, and space group C2/c. The XRPD studies of the four crystalline samples, obtained by recrystallization from four different solvents, indicated that they had the same diffraction patterns. The diffraction pattern stimulated from the crystal structure data is in excellent agreement with the experimental results. In addition, the identical FT-IR spectra of the four crystalline samples revealed absorption bands corresponding to S-S and C-S stretching as well as the characteristic aromatic substitution. Five percent weight loss at 163.3 degrees C was observed when TG was used to study the decomposition process in the temperature range of 20-200 degrees C. DSC also allowed for the determination of onset temperatures at 60.4(1)-60.7(3) degrees C and peak temperatures at 62.1(3)-62.4(3) degrees C for the four crystalline samples studied. The results verified that the single crystal structure shared the same crystal form with the four crystalline samples investigated.

Quantitative determination of anti-tumor agent bis(4-fluorobenzyl)trisulfide, fluorapacin and its pharmaceutical preparation by high-performance liquid chromatography.

A simple isocratic and stability-indicating HPLC method was developed and validated for the quantitative determination of anti-tumor agent fluorapacin and its pharmaceutical preparation. A Spherisorb ODS II C(18) (250 mm x 4.6 mm, 5 microm) column was eluted with a mobile phase consisting of acetonitrile/water (85:15, v/v). The analyses were performed at 40+/-1 degrees C with a flow rate of 1.0 mL/min and UV detection at 218 nm. The calibration curve was linear over a concentration range of 160-240 microg/mL with the correlation coefficient of 0.9997. The LOD and LOQ were determined to be 1.4 and 7.0 ng/mL, respectively. Average recoveries were 98.27% and 100.40% for fluorapacin API and its drug product with corresponding relative standard deviations (R.S.D.) of 0.41% and 0.30%, respectively. Good repeatability (precision and intermediate precision), accuracy and tolerability were obtained with R.S.D. of <1.0%. This specific and reliable method has been successfully applied for quality control of fluorapacin API and drug product.

Synthesis of pyrrolo[2,3-d]pyrimidine nucleoside derivatives as potential anti-HCV agents.

Several Toyocamycin (4) analogues were examined for their ability to inhibit HCV RNA in a replicon assay. Among the compounds examined 4-methylthio (18) and 5-carboxamide oxime derivatives (23 and 27) of Toyocamycin were found to have good activity and selectivity.

Synthesis and anti-tumor evaluation of new trisulfide derivatives.

New bis-aromatic and heterocyclic trisulfide derivatives 5, 7-10 were synthesized by optimizing lead dibenzyl trisulfide natural product (4) to evaluate their anti-tumor activities. Five compounds 5-7, 9, and 10 exhibited potent anti-tumor activities against eight different tumor cell lines with low cytotoxicity against HepG2. Initial SAR was discussed, and MOA of these anti-microtubule agents was suggested based on cell kinetic response patterns observed on RT-CES system.

Solid-phase synthesis of 5'-deoxy-5'-amino-clitocine analogues.

Several 6-substituted-amino-5'-deoxy-5'-amino-clitocine analogues were synthesized in a parallel fashion in solid phase. The desired scaffold was generated by coupling 2,3-O-bis-(t-butyldimethylsilyl)-5-N-(monomethoxytrityl-polystyrene-resin)-1,5-diamino-5-deoxy-beta-D-ribofuranose and 4, 6-dichloro-5-nitropyrimidine. The scaffold was then reacted with a variety of amines to generate a small library of 14 analogues of 5'-deoxy-5'-amino-clitocine following a protocol developed earlier.

Synthesis of 9-(2-beta-C-methyl-beta-d-ribofuranosyl)-6-substituted purine derivatives as inhibitors of HCV RNA replication.

A series of 9-(2'-beta-C-methyl-beta-d-ribofuranosyl)-6-substituted purine derivatives were synthesized as potential inhibitors of HCV RNA replication. Their inhibitory activities in a cell based HCV replicon assay were reported. A prodrug approach was used to further improve the potency of these compounds by increasing the intracellular levels of 5'-monophosphate metabolites. These nucleotide prodrugs showed much improved inhibitory activities of HCV RNA replication.

Synthesis of 2'-beta-C-methyl toyocamycin and sangivamycin analogues as potential HCV inhibitors.

Coupling reaction of 2-beta-C-methyl-1,2,3,4-tetra-O-benzoyl-d-ribofuranose with 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine, followed by debromination and debenzoylation, gave the 2'-beta-C-methyl toyocamycin in high yield. Based on this result, a series of 2'-beta-C-methyl-4-substituted toyocamycin and sangivamycin analogues were synthesized for biological screening as potential inhibitors of HCV RNA replication.

Solid-phase parallel synthesis of 4-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine nucleosides.

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H, 6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.

Synthesis of new 2'-beta-C-methyl related triciribine analogues as anti-HCV agents.

Ten new beta-D-ribofuranosyl and 2'-beta-C-methyl-beta-D-ribofuranosyl triciribine derivatives 4-13 with various N4 and 6-N substituents on the tricyclic ring were synthesized from the corresponding toyocamycin and new 2'-beta-C-methyl toyocamycin derivatives. The inhibitory studies of these compounds in the HCV replicon assay reveal that some of them possess interesting anti-HCV properties with low cytotoxicity.

Synthesis of nucleoside libraries on solid support. I. N2,N6-disubstituted diaminopurine nucleosides.

Starting with 2-iodo-6-chloro-9-(beta-D-ribofuranosyl)purine, a library of more than 1,300 N2,N6-polysubstituted diaminopurine nucleosides was created. The starting material was condensed with a polystyrene monomethoxytrityl resin and a pool of primary and secondary amines was used to displace the 6-chloro atom with high regioselectivity. The 2-iodo was subsequently displaced by various primary amines. Nucleosides were cleaved from the resin with hexafluoroisopropanol solutions. A majority of compounds reached a purity of more than 80% without the need for any type of purification.

Synthesis of nucleoside libraries on solid support. II. 2,6,8-Trisubstituted purine nucleosides using 8-bromoguanosine as precursor.

A series of 2,6,8-trisubstituted purine nucleoside libraries was prepared by parallel solid-phase synthesis using 8-bromoguanosine as a common synthetic precursor. Polystyrene-methoxytrityl chloride resin was linked to the N2 or O5' position of the guanosine analogues. 8-Bromoguanosine was derivatized at the C8 position via carbon-carbon bond formation. Nucleophilic aromatic substitution at C2 and/or C6 positions with various amines produced two series of purine nucleoside libraries with very diverse substitution.