Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F420H2-dependent reduction.

Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC50 (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC50, concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F420 Thus, we hypothesized that TA-C may be metabolized by Mtb F420-dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F420-dependent resistance phenotype, suggesting that F420H2-dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC50 against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active "prodrug," into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity.

Comparative assessment of competency frameworks for pharmacists in the Western Pacific region including the potential for a regional framework.

OBJECTIVES: To assess the potential for a regional competency framework for pharmacists in the Western Pacific using the Global Competency Framework (GbCF) as a reference. METHODS: Mixed-methods approach used a self-administered survey and semi-structured interviews of 13 countries to evaluate the perceived benefits, existence and content of competency frameworks. KEY FINDINGS: Variations in structure, components and emphasis of the four frameworks that do exist indicate significant tailoring to local requirements. Based on these four and the GbCF, 32 competencies allocated into four themes has been proposed. CONCLUSION: Varying national requirements mitigate against a single regional competency framework.

Using a collaborative decision-making approach to identify, prioritise and respond to issues influencing Good Pharmacy Practice across countries of the Western Pacific Region.

OBJECTIVES: The Western Pacific Pharmaceutical Forum aims to foster Good Pharmacy Practice (GPP) in the Western Pacific Region (WPR). Our objective in this work was to develop and implement a process that equitably and constructively engaged pharmacy associations from the diverse cultures, languages and models of pharmacy practice in the WPR to identify common issues influencing the implementation of GPP. METHODS: The World Café concept was incorporated into a modified Tuckman's model for group development to conduct a workshop of leaders from the major pharmacy associations of the WPR. Facilitated discussion groups and collaborative decision-making were used to gather opinions and achieve agreement between the regional leaders. KEY FINDINGS: Participants reported the method to be open, systematic and enabled full contribution. Two major issues were identified in relation to each of the four key pharmacist roles associated with GPP. The regional pharmacy leaders agreed the common priority issue for implementing GPP as: 'National competency standards should be established to enable the formulation of professional development framework that lead to enhanced pharmacy practice and patient care' and developed a strategy and work plan to address this issue. CONCLUSIONS: Identification of GPP issues and the common major priority occurred with shared knowledge of the current state of practice across the WPR. The adopted methodology overcame cultural and practice differences to ensure full and equal participation of all delegates. The approach ensured ownership by all participants of the strategy and work plan developed to address competency and professional development in the WPR.

The INN global nomenclature of biological medicines: A continuous challenge.

Medicines are assigned International Nonproprietary Names (INN) by the World Health Organization (WHO), pursuing the aim to increase patient safety. Following scientific developments in drug discovery and biotechnology, the number of biological medicines is constantly growing and a surge in INN applications for them has been observed. Pharmacologically active biological substances have a complex structure and mechanism of action posing new challenges in selecting names that appropriately reflect such properties. As a consequence, existing nomenclature naming schemes may need to be revised and new ones developed. This review reports on the recently implemented policies for naming fusion proteins, monoclonal antibodies, advanced therapy substances that cover gene and cell therapy, virus-based therapies as well as vaccines and vaccine-like substances. Different approaches, based on the use of a one-word versus a two-word naming scheme, have been developed for different categories of biological substances highlighting a major and still not completely resolved issue, i.e. how to assign a name that is both informative, short and euphonic.

Fused Heterocyclic Systems with an s-Triazine Ring. 34. Development of a Practical Approach for the Synthesis of 5-Aza-isoguanines.

Purine isosteres present excellent opportunities in drug design and development. Using isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were explored to establish a robust general protocol for the preparation of these compounds. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents.

Novel dual-targeting anti-proliferative dihydrotriazine-chalcone derivatives display suppression of cancer cell invasion and inflammation by inhibiting the NF-κB signaling pathway.

Chalcones present in edible plants possess anti-cancer and anti-inflammatory properties, with the Michael acceptor moiety reported to be responsible for their biological activities. In this study, two novel dihydrotriazine-chalcone compounds previously identified to exert anti-proliferative effects through dual-targeting of dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR), were evaluated for their anti-invasive and anti-inflammatory abilities. At non-lethal concentrations, the compounds suppressed in vitro migration of MDA-MB-231 breast carcinoma cells, which was correlated with a dose-dependent downregulation of phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) expression and secretion. At similar concentrations, these chalcone-based compounds suppressed expression of inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-stimulated murine macrophage-like RAW 264.7 cells, as well as tumor necrosis factor alpha (TNF-α) in LPS-stimulated human monocytes isolated from healthy donors. Mechanistically, inhibition of cancer cell invasion and inflammation by the compounds were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway, which corroborated with the reported mechanism of action of chalcones. Their abilities to target multiple biological mediators relevant to multi-step carcinogenesis and with bioactivities stronger than those of the parent chalcone scaffold have warranted dihydrotriazine-chalcone compounds as promising candidates for use in pharmacological intervention of aggressive cancers.

1,3,5-triazaspiro[5.5]undeca-2,4-dienes as selective Mycobacterium tuberculosis dihydrofolate reductase inhibitors with potent whole cell activity.

The emergence of multi- and extensively-drug resistant tubercular (MDR- and XDR-TB) strains of mycobacteria has limited the use of existing therapies, therefore new drugs are needed. Dihydrofolate reductase (DHFR) has recently attracted much attention as a target for the development of anti-TB agents. This study aimed to develop selective M. tuberculosis DHFR inhibitors using rationale scaffolding design and synthesis, phenotype-oriented screening, enzymatic inhibitory study, whole cell on-target validation, molecular modeling, and in vitro DMPK determination to derive new anti-TB agents. 2,4-diamino-1-phenyl-1,3,5-triazaspiro[5.5]undeca-2,4-dienes 20b and 20c were identified as selective M. tuberculosis DHFR inhibitors, showing promising antimycobacterial activities (MIC50: 0.01 µM and MIC90: 0.025 µM on M. tuberculosis H37Rv). This study provided compelling evidence that compound 20b and 20c exerted whole cell antimycobacterial activity through DHFR inhibition. In addition, these two compounds exhibited low cytotoxicity and low hemolytic activity. The in vitro DMPK and physiochemical properties suggested their potential in vivo efficacy.

Design, Synthesis, and Biological Evaluation of Coupled Bioactive Scaffolds as Potential Anticancer Agents for Dual Targeting of Dihydrofolate Reductase and Thioredoxin Reductase.

The dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) enzymes are involved in the process of tumor cell growth and survival. The 4,6-diamino-1,2-dihydro-1,3,5-triazine scaffold is well-established as a useful scaffold for DHFR inhibition, while chalcones have been reported to be inhibitors of TrxR. In this study, 15 novel compounds designed by the structural combination of the 4,6-diamino-1,2-dihydro-1,3,5-triazine and chalcone scaffolds via a diether linker were successfully synthesized and characterized. All of the compounds demonstrated dual inhibition against DHFR and TrxR when they were assessed by in vitro enzyme assays. The compounds also exhibited antiproliferative activity against the MCF-7 and HCT116 cells. The more potent analogs 14 and 15 were found to inhibit cellular DHFR and TrxR activities in HCT116 cells. Therefore, this study provided compelling evidence that 14 and 15 could exert their anticancer property via multitarget inhibition at the cellular level.

Discovery of novel dual VEGFR2 and Src inhibitors using a multistep virtual screening approach.

AIM: Simultaneous inhibition of VEGFR2 and Src may enhance the efficacy of VEGFR2-targeted cancer therapeutics. Hence, development of dual inhibitors on VEGFR2 and Src can be a useful strategy for such treatments. MATERIALS & METHODS: A multistep virtual screening protocol, comprising ligand-based support vector machines method, drug-likeness rules filter and structure-based molecular docking, was developed and employed to identify dual inhibitors of VEGFR2 and Src from a large commercial chemical library. Kinase inhibitory assays and cell viability assays were then used for experimental validation. RESULTS: A set of compounds belonging to six different molecular scaffolds was identified and sent for biological evaluation. Compound 3c belonging to the 2-amino-3-cyanopyridine scaffold exhibited good antiproliferative effect and dual-target activities against VEGFR2 and Src. CONCLUSION: This study demonstrated the ability of the multistep virtual screening approach to identify novel multitarget agents.

Design, synthesis, docking studies and biological evaluation of novel dihydro-1,3,5-triazines as human DHFR inhibitors.

A novel series of dihydro-1,3,5-triazine derivatives bearing a heteroatom spiro-ring were designed and synthesized on the basis of molecular flexible docking work, and their biological activities were evaluated. Compounds A2, A5, B1 and B3 showed potent human dihydrofolate reductase (hDHFR) inhibitory activity with IC50 values of 7.46 nM, 3.72 nM, 6.46 nM, 4.08 nM, versus reference drug methotrexate (MTX). From the molecular docking result we concluded that the conformation space generated by deformation of the flexible residue Phe31 is favorable for the binding of the spiro-ring, and inserting heteroatom into spiro ring might increase the binding affinity. There were 24 compounds with broadspectrum antiproliferative activity against several tumor cell lines (HCT116, A549, HL-60, HepG2 and MDA-MB-231) with IC50 values ranging from 0.79 to 0.001 µM. The antitumor activity in vivo of compound A2 was determined in a human alveolar basal epithelial cell line A549 xenograft model. This study offered novel anticancer agents with high inhibitory activity that target hDHFR and have a binding mode of the novel molecular scaffold with hDHFR. This provides potent support for further development of novel hDHFR inhibitors.

Towards cheminformatics-based estimation of drug therapeutic index: Predicting the protective index of anticonvulsants using a new quantitative structure-index relationship approach.

The overall efficacy and safety profile of a new drug is partially evaluated by the therapeutic index in clinical studies and by the protective index (PI) in preclinical studies. In-silico predictive methods may facilitate the assessment of these indicators. Although QSAR and QSTR models can be used for predicting PI, their predictive capability has not been evaluated. To test this capability, we developed QSAR and QSTR models for predicting the activity and toxicity of anticonvulsants at accuracy levels above the literature-reported threshold (LT) of good QSAR models as tested by both the internal 5-fold cross validation and external validation method. These models showed significantly compromised PI predictive capability due to the cumulative errors of the QSAR and QSTR models. Therefore, in this investigation a new quantitative structure-index relationship (QSIR) model was devised and it showed improved PI predictive capability that superseded the LT of good QSAR models. The QSAR, QSTR and QSIR models were developed using support vector regression (SVR) method with the parameters optimized by using the greedy search method. The molecular descriptors relevant to the prediction of anticonvulsant activities, toxicities and PIs were analyzed by a recursive feature elimination method. The selected molecular descriptors are primarily associated with the drug-like, pharmacological and toxicological features and those used in the published anticonvulsant QSAR and QSTR models. This study suggested that QSIR is useful for estimating the therapeutic index of drug candidates.

Applying the designed multiple ligands approach to inhibit dihydrofolate reductase and thioredoxin reductase for anti-proliferative activity.

The development of multi-targeting drugs is currently being explored as an attractive alternative to combination therapy, especially for the treatment of complex diseases such as cancer. Dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) are enzymes belonging to two unrelated cellular pathways that are known to contribute towards cancer cell growth and survival. In order to evaluate whether simultaneous inhibition of DHFR and TrxR by dihydrotriazines (DHFR-targeting) and chalcones (TrxR-targeting) may be beneficial, breast MCF-7 and colorectal HCT116 carcinoma cells were treated with combinations of selected dihydrotriazines and chalcones at a 1:1 M ratio. Two combinations demonstrated synergy at low-to-moderate concentrations. Based on this result, four merged dihydrotriazine-chalcone compounds were designed and synthesized. Two compounds, 11a [DHFR IC50 = 56.4 µM, TrxR IC50 (60 min) = 12.6 µM] and 11b [DHFR IC50 = 2.4 µM, TrxR IC50 (60 min) = 10.1 µM], demonstrated in vitro inhibition of DHFR and TrxR. The compounds showed growth inhibitory activity against MCF-7 and HCT116 cells, but lacked cytotoxicity. Molecular docking experiments showed 11b to possess rational binding modes to both the enzymes. In conclusion, this study has not only identified the dihydrotriazine and chalcone scaffolds as good starting points for the development of dual inhibitors of DHFR and TrxR, but also demonstrated the synthetic feasibility of producing a chemical entity that could result in simultaneous inhibition of DHFR and TrxR. Future efforts to improve the antiproliferative profiles of such compounds will look at alternative ways of integrating the two pharmacophoric scaffolds.

Sulforaphane and its methylcarbonyl analogs inhibit the LPS-stimulated inflammatory response in human monocytes through modulating cytokine production, suppressing chemotactic migration and phagocytosis in a NF-κB- and MAPK-dependent manner.

Sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)-butane], an aliphatic isothiocyanate (ITC) naturally derived from cruciferous vegetables and largely known for its chemopreventive potential also appears to possess anti-inflammatory potential. In this study, structural analogs of SF {compound 1 [1-isothiocyanato-4-(methylcarbonyl)-butane] and 2 [1-isothiocyanato-3-(methylcarbonyl)-propane]} containing a carbonyl group in place of the sulfinyl group in SF, were evaluated for their anti-inflammatory activities. In RAW 264.7 cells, the ITCs at non-toxic concentrations caused an inhibition of NO and prostaglandin E2 (PGE2) release through suppressing expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as a reduction in matrix metalloproteinase-9 (MMP-9) expression, secretion and gelatinolytic activity. Further work performed on human monocytes isolated from blood of healthy donors revealed that the ITCs not only suppressed the expression and release of pro-inflammatory mediators IL-1ß, IL-6, TNF-α and MMP-9, but also suppressed their antibody-independent phagocytic and chemotactic migratory abilities. These anti-inflammatory activities were mediated through suppression of the NF-κB and MAPK signaling pathways. In addition, the ITCs were revealed to interact with the cysteines in inhibitor of nuclear factor-κB kinase ß subunit (IKKß), which could contribute at least partly to the suppression of NF-κB signaling. In conclusion, results obtained in this study provide deeper insights into the anti-inflammatory properties of SF and its methylcarbonyl analogs and the underlying mechanisms. These compounds thus serve as promising candidates for clinical applications in controlling inflammatory conditions.

Discovery of mixed type thymidine phosphorylase inhibitors endowed with antiangiogenic properties: synthesis, pharmacological evaluation and molecular docking study of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones. Part II.

In our drug discovery program, a series of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones were designed, synthesized and evaluated for their TP inhibitory potential. All the synthesized analogues conferred a varying degree of TP inhibitory activity, comparable or better than positive control, 7-deazaxanthine (7-DX, 2) (IC50 value = 42.63 µM). A systematic approach to the lead optimization identified compounds 3c and 4a as the most promising TP inhibitors, exhibiting mixed mode of enzyme inhibition. Moreover, selected compounds demonstrated the ability to attenuate the expression of the angiogenic markers (viz. MMP-9 and VEGF) in MDA-MB-231 cells at sublethal concentrations. In addition, molecular docking studies revealed the plausible binding orientation of these inhibitors towards TP, which was in accordance with the experimental results. Taken as a whole, these compounds would constitute a new direction for the design of novel TP inhibitors with promising antiangiogenic properties.

Fragment-based approach to the design of 5-chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines as thymidine phosphorylase inhibitors.

5-Chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines were designed as new thymidine phosphorylase inhibitors based on the fragment based drug design approach. Multiple-step convergent synthetic schemes were devised to generate the target compounds. The intermediate 5-chloro-6-chloromethyluracil was synthesized by a 4-step reaction. A series of the second bicyclic intermediates, namely pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one, was obtained from various substituted 3-aminopyrazoles. These two intermediates were coupled finally in the presence of sodium ethoxide and methanol to yield the desirable target compounds. The methylthio coupling spacer was found to be suitable in enabling the interaction of the two fragments at the active site and allosteric site of the enzyme. The best coupled compound (9q) inhibited the thymidine phosphorylase with an IC50 value as low as 0.36 ± 0.1 µM. In addition, 9q demonstrated a mixed-type of enzyme inhibition kinetics, thus suggesting that it might indeed potentially bind at two different sites on the enzyme.

Synthesis, anti-thymidine phosphorylase activity and molecular docking of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones.

In our lead finding program, a series of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones and their 5-thio-alkyl derivatives were designed and synthesized which contained different substituents at ortho-position of 2-phenyl ring attached to the fused ring structure. The preliminary pharmacological evaluation demonstrated that the synthesized compounds exhibited a varying degree of inhibitory activity towards thymidine phosphorylase (TP), comparable to reference compound, 7-Deazaxanthine (7-DX, 2) (IC50 value=42.63 µM). The study also inferred that the ortho-substituted group at the phenyl ring and 5-thio-alkyl moiety imparted steric hindrance effects in the binding site of the enzyme, leading to a reduced inhibitory response. In addition, compound 3a was identified as a mixed-type inhibitor of TP. Moreover, computational docking study was performed to illustrate the important structural information on the plausible ligand-enzyme binding interactions.

A novel shogaol analog suppresses cancer cell invasion and inflammation, and displays cytoprotective effects through modulation of NF-κB and Nrf2-Keap1 signaling pathways.

Natural compounds containing vanilloid and Michael acceptor moieties appear to possess anti-cancer and chemopreventive properties. The ginger constituent shogaol represents one such compound. In this study, the anti-cancer potential of a synthetic novel shogaol analog 3-phenyl-3-shogaol (3-Ph-3-SG) was assessed by evaluating its effects on signaling pathways. At non-toxic concentrations, 3-Ph-3-SG suppressed cancer cell invasion in MDA-MB-231 and MCF-7 breast carcinoma cells through inhibition of PMA-activated MMP-9 expression. At similar concentrations, 3-Ph-3-SG reduced expression of the inflammatory mediators nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and prostanglandin-E2 (PGE2) in RAW 264.7 macrophage-like cells. Inhibition of cancer cell invasion and inflammation by 3-Ph-3-SG were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway. The 3-Ph-3-SG also demonstrated cytoprotective effects by inducing the antioxidant response element (ARE)-driven genes NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1). Cytoprotection by 3-Ph-3-SG was achieved at least partly through modification of cysteine residues in the E3 ubiquitin ligase substrate adaptor Kelch-like ECH-associated protein 1 (Keap1), which resulted in accumulation of transcription factor NF-E2 p45-related factor 2 (Nrf2). The activities of 3-Ph-3-SG were comparable to those of 6-shogaol, the most abundant naturally-occurring shogaol, and stronger than those of 4-hydroxyl-null deshydroxy-3-phenyl-3-shogaol, which attested the importance of the 4-hydroxy substituent in the vanilloid moiety for bioactivity. In summary, 3-Ph-3-SG is shown to possess activities that modulate stress-associated pathways relevant to multiple steps in carcinogenesis. Therefore, it warrants further investigation of this compound as a promising candidate for use in chemotherapeutic and chemopreventive strategies.

A structure-activity relationship study of 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione and its 5-thioxo analogues on anti-thymidine phosphorylase and associated anti-angiogenic activities.

Thirty-three 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione and its 5-thioxo analogues were designed and synthesized which contained different substituents at meta- and/or para-positions of 2-phenyl or 2-benzyl ring attached to the fused ring structure. The preliminary pharmacological evaluation demonstrated that the 5-thioxo analogues of 1,2,4-triazolo[1,5-a][1,3,5]triazine exhibited a varying degree of inhibitory activity towards thymidine phosphorylase, comparable or better than reference compound, 7-Deazaxanthine (7-DX, 2) (IC50 value = 42.63 µM). Moreover, compounds 5q and 6i displayed a mixed-type of inhibitory mechanism in the presence of variable concentrations of thymidine (dThd). In addition, selected compounds were found to have a noticeable inhibitory effect on the expression of angiogenesis markers, including VEGF and MMP-9 in MDA-MB-231 breast cancer cells.

Synthesis and in vitro evaluation of 1,2,4-triazolo[1,5-a][1,3,5]triazine derivatives as thymidine phosphorylase inhibitors.

In our lead finding program, a series of 1,2,4-triazolo[1,5-a][1,3,5]triazine derivatives were synthesized, and their in vitro thymidine phosphorylase inhibitory potential was explored. Among the different derivatives, compounds having keto group (C = O) at C7 and thioketo group (C = S) at C5 positions showed varying degrees of TP inhibitory activity comparable with positive control, 7-deazaxanthine (7-DX, 2) (IC50 value = 42.63 µm). Enzyme inhibition kinetics study suggested that compound IVn behaved as a mixed-type inhibitor of the enzyme with respect to thymidine (dThd) as a variable substrate. Compound IVn was also found to inhibit PMA-induced MMP-9 expression in MDA-MB-231 cells at sublethal concentrations. Computational docking study was performed to illustrate the enzyme inhibition kinetics and to explore the ligand-enzyme interactions.

Synthesis of pyrazolo[1,5-a][1,3,5]triazine derivatives as inhibitors of thymidine phosphorylase.

Thymidine phosphorylase (TP) is an enzyme that promotes tumor growth and metastasis and therefore is an attractive druggable target. Using a reported TP inhibitor, 7-deazaxanthine (7DX), as the lead compound; this study was set up to evaluate whether pyrazolo[1,5-a][1,3,5]triazin-2,4-diones and pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones would exhibit TP inhibitory activity. The pyrazolo[1,5-a][1,3,5]triazine nucleus was constructed using a reaction that annulated the 1,3,5-triazine ring onto a pyrazole scaffold. Among the 52 compounds synthesized and tested, it was found that 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones exhibited various extent of inhibitory activity against TP. The best compound 17p, which bears a para-substituted pentafluorosulfur group, showed an IC50 value of 0.04 µM, which was around 800 times more potent than the 7DX (IC50 = 32 µM) under the same bioassay conditions. The results of the study suggested that a substituent with +σ and +π properties inserted at position 4 of a phenyl ring that is attached to position 8 of the pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one scaffold would give excellent TP inhibitory action. In addition, 17p was found to be a non-competitive inhibitor thus suggested that it might interact with TP at a position different from the substrate binding site.