The synthesis and biological evaluation of alkyl and benzyl naphthyridinium analogs of eupolauridine as potential antimicrobial and cytotoxic agents.

Eupolauridine, an indenonaphthyridine alkaloid, has been previously reported by us to exhibit antifungal activity. This study describes the synthesis of new alkyl and benzyl naphthyridinium/pyridinium analogs of eupolauridine as potential antifungal agents. A majority of the analogs exhibited antifungal activity against opportunistic pathogens such as Candida albicans and Cryptococcus neoformans. Several of them were also effective against bacteria (Staphylococcus aureus, MRS, Pseudomonas and Mycobacterium) and the malaria parasite (Plasmodium falciparum) to variable extents. A number of analogs were also cytotoxic to human cancer cell lines.

6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases.

6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(ß-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-ß-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-ß-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-ß-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the ß-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

Novel substituted pyrimidines as HCV replication (replicase) inhibitors.

Compound 1 was identified as a HCV replication inhibitor from screening/early SAR triage. Potency improvement was achieved via modulation of substituent on the 5-azo linkage. Due to potential toxicological concern, the 5-azo linkage was replaced with 5-alkenyl or 5-alkynyl moiety. Analogs containing the 5-alkynyl linkage were found to be potent inhibitors of HCV replication. Further evaluation identified compounds 53 and 63 with good overall profile, in terms of replicon potency, selectivity and in vivo characteristics. Initial target engagement studies suggest that these novel carbanucleoside-like derivatives may inhibit the HCV replication complex (replicase).

Pyridofuran substituted pyrimidine derivatives as HCV replication (replicase) inhibitors.

Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC(50)) and PK parameters in all species studied, while the selectivity in the thymidine incorporation assay (DNA·CC(50)) was low.

Synthesis and SAR of geminal substitutions at the C5' carbosugar position of pyrimidine-derived HCV inhibitors.

The installation of geminal substitution at the C5' position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5' position led to the installation of the dimethyl group as the optimal functionality. An improved route was subsequently designed to access these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These compounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in rodent as well as in higher species.

5-Benzothiazole substituted pyrimidine derivatives as HCV replication (replicase) inhibitors.

Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.

Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV replication inhibitors.

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.

The Alabama Drug Discovery Alliance: a collaborative partnership to facilitate academic drug discovery.

The Alabama Drug Discovery Alliance is a collaboration between the University of Alabama at Birmingham and Southern Research Institute that aims to support the discovery and development of therapeutic molecules that address an unmet medical need. The alliance builds on the expertise present at both institutions and has the dedicated commitment of their respective technology transfer and intellectual property offices to guide any commercial opportunities that may arise from the supported efforts. Although most projects involve high throughput screening, projects at any stage in the drug discovery and development pathway are eligible for support. Irrespective of the target and stage of any project, well-functioning interdisciplinary teams are crucial to a project's progress. These teams consist of investigators with a wide variety of expertise from both institutions to contribute to the program's success.

Alteration of the carbohydrate for deoxyguanosine analogs markedly changes DNA replication fidelity, cell cycle progression and cytotoxicity.

Nucleoside analogs are efficacious cancer chemotherapeutics due to their incorporation into tumor cell DNA. However, they exhibit vastly different antitumor efficacies, suggesting that incorporation produces divergent effects on DNA replication. Here we have evaluated the consequences of incorporation on DNA replication and its fidelity for three structurally related deoxyguanosine analogs: ganciclovir (GCV), currently in clinical trials in a suicide gene therapy approach for cancer, D-carbocyclic 2'-deoxyguanosine (CdG) and penciclovir (PCV). GCV and CdG elicited similar cytotoxicity at low concentrations, whereas PCV was 10-100-fold less cytotoxic in human tumor cells. DNA replication fidelity was evaluated using a supF plasmid-based mutation assay. Only GCV induced a dose-dependent increase in mutation frequency, predominantly GC-->TA transversions, which contributed to cytotoxicity and implicated the ether oxygen in mutagenicity. Activation of mismatch repair with hydroxyurea decreased mutations but failed to repair the GC-->TA transversions. GCV slowed S-phase progression and CdG also induced a G2/M block, but both drugs allowed completion of one cell cycle after drug treatment followed by cell death in the second cell cycle. In contrast, PCV induced a lengthy early S-phase block due to profound suppression of DNA synthesis, with cell death in the first cell cycle after drug treatment. These data suggest that GCV and CdG elicit superior cytotoxicity due to their effects in template DNA, whereas strong inhibition of nascent strand synthesis by PCV may protect against cytotoxicity. Nucleoside analogs based on the carbohydrate structures of GCV and CdG is a promising area for antitumor drug development.

Synthesis of 9-(6-Deoxy-α-L-Talofuranosyl)-6-Methylpurine and 9-(6-Deoxy-ß-D-Allofuranosyl)-6-Methylpurine Nucleosides.

6-Methylpurine (MeP) is a cytotoxic adenine analog that does not exhibit selectivity when administered systemically and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli purine nucleoside phosphorylase (PNP). 9-(6-Deoxy-ß-D-allofuranosyl)-6-methylpurine [methyl(allo)-MePR, 18] and 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine [methyl(talo)-MePR, 21] were synthesized as potential prodrugs for MeP in the E. coli PNP/prodrug cancer gene therapy approach. The detailed syntheses of [methyl(allo)-MePR] and [methyl(talo)-MePR] are described. The glycosyl donors, 1,2-di-O-acetyl-3,5-di-O-benzyl-α-D-allofuranose (12) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-α-L-talofuranose (16) were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (4) in nine and eleven steps, respectively. Vorbrüggen coupling of the latter glycosyl donors with 6-methylpurine (3), followed by deprotection of the sugar hydroxyl groups, gave the title compounds in good overall yields. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of 6-methylpurine Basic Protocol 2: Preparation of the D-allofuranose derivative (12) Basic Protocol 3: Preparation of 6-deoxy-α-L-talofuranoside Basic Protocol 4: Preparation of methyl(allo)-MePR (18) Basic Protocol 5: Preparation of methyl(talo)-MePR (21).

Activities of certain 5-substituted 4'-thiopyrimidine nucleosides against orthopoxvirus infections.

As part of a program to identify new compounds that have activity against orthopoxviruses, a number of 4'-thionucleosides were synthesized and evaluated for their efficacies against vaccinia and cowpox viruses. Seven compounds that were active at about 1 microM against both viruses in human cells but that did not have significant toxicity were identified. The 5-iodo analog, 1-(2-deoxy-4-thio-beta-d-ribofuranosyl)-5-iodouracil (4'-thioIDU), was selected as a representative molecule; and this compound also inhibited viral DNA synthesis at less than 1 microM but only partially inhibited the replication of a recombinant vaccinia virus that lacked a thymidine kinase. This compound retained complete activity against cidofovir- and ST-246-resistant mutants. To determine if this analog had activity in an animal model, mice were infected intranasally with vaccinia or cowpox virus and treatment with 4'-thioIDU was given intraperitoneally or orally twice daily at 50, 15, 5, or 1.5 mg/kg of body weight beginning at 24 to 120 h postinfection and was continued for 5 days. Almost complete protection (87%) was observed when treatment with 1.5 mg/kg was begun at 72 h postinfection, and significant protection (73%) was still obtained when treatment with 5 mg/kg was initiated at 96 h. Virus titers in the liver, spleen, and kidney were reduced by about 4 log(10) units and about 2 log(10) units in mice infected with vaccinia virus and cowpox virus, respectively. These results indicate that 4'-thioIDU is a potent, nontoxic inhibitor of orthopoxvirus replication in cell culture and experimental animal infections and suggest that it may have potential for use in the treatment of orthopoxvirus infections in animals and humans.

Inhibition of herpesvirus replication by 5-substituted 4'-thiopyrimidine nucleosides.

A series of 4'-thionucleosides were synthesized and evaluated for activities against orthopoxviruses and herpesviruses. We reported previously that one analog, 5-iodo-4'-thio-2'-deoxyuridine (4'-thioIDU), exhibits good activity both in vitro and in vivo against two orthopoxviruses. This compound also has good activity in cell culture against many of the herpesviruses. It inhibited the replication of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus with 50% effective concentrations (EC(50)s) of 0.1, 0.5, and 2 microM, respectively. It also inhibited the replication of human cytomegalovirus (HCMV) with an EC(50) of 5.9 microM but did not selectively inhibit Epstein-Barr virus, human herpesvirus 6, or human herpesvirus 8. While acyclovir-resistant strains of HSV-1 and HSV-2 were comparatively resistant to 4'-thioIDU, it retained modest activity (EC(50)s of 4 to 12 microM) against these strains. Some ganciclovir-resistant strains of HCMV also exhibited reduced susceptibilities to the compound, which appeared to be related to the specific mutations in the DNA polymerase, consistent with the observed incorporation of the compound into viral DNA. The activity of 4'-thioIDU was also evaluated using mice infected intranasally with the MS strain of HSV-2. Although there was no decrease in final mortality rates, the mean length of survival after inoculation increased significantly (P < 0.05) for all animals receiving 4'-thioIDU. The findings from the studies presented here suggest that 4'-thioIDU is a good inhibitor of some herpesviruses, as well as orthopoxviruses, and this class of compounds warrants further study as a therapy for infections with these viruses.

Discovery and development of clofarabine: a nucleoside analogue for treating cancer.

The treatment of acute leukaemias, which are the most common paediatric cancers, has improved considerably in recent decades, with complete response rates approaching approximately 90% in some cases. However, there remains a major need for treatments for patients who do not achieve or maintain complete remission, for whom the prognosis is very poor. In this article, we describe the challenges involved in the discovery and development of clofarabine, a second-generation nucleoside analogue that received accelerated approval from the US FDA at the end of 2004 for the treatment of paediatric patients 1-21 years old with relapsed or refractory acute lymphoblastic leukaemia after at least two prior regimens. It is the first such drug to be approved for paediatric leukaemia in more than a decade, and the first to receive approval for paediatric use before adult use.

Clofarabine acts as radiosensitizer in vitro and in vivo by interfering with DNA damage response.

PURPOSE: Combination treatment with radiotherapy and chemotherapy has emerged as the dominant form of cancer adjuvant regimens in recent years. Clofarabine, a newly approved drug for pediatric leukemia, is a second-generation purine nucleoside analogue that can block DNA synthesis and inhibit DNA repair. Therefore, we hypothesized that clofarabine could work synergistically with radiotherapy to increase the tumor cell response. METHODS AND MATERIALS: The effects of clofarabine on radiosensitivity have been established in several tumor cell lines in vitro and in vivo using colony-forming assays and tumor xenografts. The effect of clofarabine on the DNA damage response was also studied in vitro by measuring gamma-H2AX focus formation. RESULTS: Clonogenic survival was significantly reduced in irradiated cells treated with clofarabine, demonstrating the strong radiosensitizing effect of clofarabine. Furthermore, clofarabine displayed a radiosensitizing effect that was greater than gemcitabine or 5-fluorouracil. We also found that low doses of clofarabine can prolong the presence of radiation-induced gamma-H2AX nuclear focus formation, and high doses of clofarabine can induce DNA double-strand breaks, suggesting that clofarabine can interfere with DNA damage response pathways. In addition, clofarabine-induced radiosensitization was also established in vivo using a colorectal cancer model, DLD-1, in athymic nude mice. When combined with fractionated radiotherapy, a moderate dose of clofarabine led to a significant increase in tumor growth inhibition. CONCLUSION: Clofarabine acts as a powerful radiosensitizer both in vitro and in vivo by interfering with the DNA damage response.

Antiangiogenic activity of 4'-thio-beta-D-arabinofuranosylcytosine.

4'-Thio-beta-D-arabinofuranosylcytosine (T-araC), a new-generation deoxycytidine nucleoside analogue, showed significant efficacy against numerous solid tumors in preclinical studies and entered clinical development for cancer therapy. It is a structural analogue of cytarabine (araC), a clinically used drug in the treatment of acute myelogenous leukemia, which has no or very limited efficacy against solid tumors. In comparison with araC, the excellent in vivo activity of T-araC against solid tumors suggests that, in addition to inhibition of DNA synthesis, T-araC may target cellular signaling pathways, such as angiogenesis, in solid tumors. We studied T-araC and araC for their antiangiogenic activities in vitro and in vivo. Both compounds inhibited human endothelial cell proliferation with similar IC50s. However, only T-araC inhibited endothelial cell migration and differentiation into capillary tubules. T-araC also abrogated endothelial cell extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, a key signaling molecule involved in cellular processes of angiogenesis. Results from chick chorioallantoic membrane angiogenesis assays revealed that T-araC significantly inhibited the development of new blood vessels in vivo, whereas araC showed much less effect. The findings of this study show a role of T-araC in antiangiogenesis and suggest that T-araC combines antiproliferative and antiangiogenic activity in one molecule for a dual mechanism of drug action to achieve the excellent in vivo efficacy against several solid tumors. This study also provides important information for optimizing dosage and sequence of T-araC administration in clinical investigations by considering T-araC as both an antiproliferative and an antiangiogenic agent.

Recent development of therapeutics for chronic HCV infection.

The global prevalence of hepatitis C virus (HCV) infection and serious health consequences associated with chronic state of the disease have become a significant health problem worldwide. Currently, there is no vaccine to prevent the disease and no specific antiviral drug directed against HCV infection. The current standard of care, interferon-based therapies, both alone or in combination with ribavirin, has demonstrated limited success and is associated with undesirable side effects. Thus, the treatment of the chronic HCV infection represents an unmet medical need. With advances in the understanding of HCV replication and the crystal structures of the virally encoded enzymes, the HCV NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase have emerged as ideal targets toward the control of the disease and the development of new anti-HCV agents. In this review, we will summarize the current treatment options, and outline the approaches toward discovery of small molecule antivirals against the virally encoded enzymes. The current clinical studies of promising lead compounds are also reviewed.

Excellent in vivo bystander activity of fludarabine phosphate against human glioma xenografts that express the escherichia coli purine nucleoside phosphorylase gene.

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

6-Methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase.

Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-ß-d-ribofuranosyl)-6-methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

Antibiotic-mediated chemoprotection enhances adaptation of E. coli PNP for herpes simplex virus-based glioma therapy.

The E. coli PNP suicide gene sensitizes solid tumors to nucleoside prodrugs, such as 6-methylpurine-2'-deoxyriboside (MeP-dR). In this study using lentiviral, MuLv, and HSV-based gene transfer, we quantified thresholds for inhibition of tumor growth and bystander killing by E. coli PNP and tested the role of intestinal flora in this process. Regressions of human glioma tumors following retroviral transduction exhibited dose dependence on both the level of PNP expression and the dose of MeP-dR administered, including strong tumor inhibition when 90-99% bystander cells comprised the tumor mass. A replication competent, non-neurovirulent herpes simplex virus (HSV) deficient in both copies of the gamma-1 34.5 gene was next engineered to express E. coli PNP under the egr-1 promoter (HSV-PNP). HSV-PNP injected intratumorally (17 million pfu/0.05 ml) in nude mice bearing 300 mg human glioma flank tumors produced a delay in tumor growth (approximately 24 days delay to one doubling). MeP-dR treatment after antibiotic therapy (to eliminate enteric flora encoding PNP enzymes) resulted in antitumor enhancement, with arrest of tumor growth (delay to doubling >50 days). Bystander killing of the magnitude described here has been difficult to accomplish with other suicide genes, such as HSV-tk or cytosine deaminase. The results establish a model for applying E. coli PNP to HSV treatment of glioma.