Inhibition of human deoxyuridine triphosphatase (dUTPase) has been identified as a promising approach to enhance the efficacy of 5-fluorouracil (5-FU)-based chemotherapy. This study describes the development of a novel class of dUTPase inhibitors based on the structure-activity relationship (SAR) studies of uracil derivatives. Starting from the weak inhibitor 7 (IC(50) = 100 μM), we developed compound 26, which is the most potent human dUTPase inhibitor (IC(50) = 0.021 μM) reported to date. Not only does compound 26 significantly enhance the growth inhibition activity of 5-fluoro-2'-deoxyuridine (FdUrd) against HeLa S3 cells in vitro (EC(50) = 0.075 μM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound 26 is a promising candidate for clinical development.

5-Fluorouracil (5-FU) is an antimetabolite and exerts antitumor activity via intracellularly and physiologically complicated metabolic pathways. In this study, we designed a novel small molecule inhibitor, TAS-114, which targets the intercellular metabolism of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addition, TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addition of TAS-114 was retained in the presence of a potent DPD inhibitor containing oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment. .

Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as a 5-fluorouracil-based combination chemotherapy. We describe the design and synthesis of a novel class of human dUTPase inhibitors, 1,2,3-triazole-containing uracil derivatives. Compound 45a, which possesses 1,5-disubstituted 1,2,3-triazole moiety that mimics the amide bond of tert-amide-containing inhibitor 6b locked in a cis conformation showed potent inhibitory activity, and its structure–activity relationship studies led us to the discovery of highly potent inhibitors 48c and 50c (IC50 = ∼0.029 μM). These derivatives dramatically enhanced the growth inhibition activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells in vitro (EC50 = ∼0.05 μM). In addition, compound 50c exhibited a markedly improved pharmacokinetic profile as a result of the introduction of a benzylic hydroxy group and significantly enhanced the antitumor activity of 5-fluorouracil against human breast cancer MX-1 xenograft model in mice. These data indicate that 50c is a promising candidate for combination cancer chemotherapies with TS inhibitors.

Recently, deoxyuridine triphosphatase (dUT-Pase) has emerged as a potential target for drug development as part of a new strategy of 5-fluorouracil-based combination chemotherapy. We have initiated a program to develop potent drug-like dUTPase inhibitors based on structure−activity relationship (SAR) studies of uracil derivatives. N-Carbonylpyrrolidine-and N-sulfonylpyrrolidine-containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (12k) having excellent potencies (IC 50 = 0.15 μM). The X-ray structure of a complex of 16a and human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a hydrophobic region, respectively, and are stacked on each other. Compounds 12a and 16a markedly enhanced the growth inhibition activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells in vitro (EC 50 = 0.27−0.30 μM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemotherapeutic strategies when used in combination with TS inhibitors.

Human deoxyuridine triphosphatase (dUTPase) inhibition is a promising approach to enhance the efficacy of thymidylate synthase (TS) inhibitor based chemotherapy. In this study, we describe the discovery of a novel class of human dUTPase inhibitors based on the conformation restriction strategy. On the basis of the X-ray cocrystal structure of dUTPase and its inhibitor compound 7, we designed and synthesized two conformation restricted analogues, i.e., compounds 8 and 9. These compounds exhibited increased in vitro potency compared with the parent compound 7. Further structure-activity relationship (SAR) studies identified a compound 43 with the highest in vitro potency (IC(50) = 39 nM, EC(50) = 66 nM). Furthermore, compound 43 had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1 breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage.

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Background: Deoxyuridine 5'-triphosphate nucleotidehydrolase (dUTPase) is a pyrophosphatase, which selectively catalyzes FdUTP and dUTP hydrolysis, and tightly restricts 5-fluorouracil (5-FU) and uracil misincorporation into DNA. TAS-114 is a novel potent inhibitor of dUTPase, and is under clinical development as a fluoropyrimidine enhancer. TAS-114 potentiates the antitumor activity of fluoropyrimidines through increasing misincorporation of 5-FU and uracil into DNA, however, precise mechanisms of cytotoxicity after misincorporation of the aberrant base is unknown. Here, we report relationships between the DNA damage repair function and the efficacy of TAS-114 on the anticancer activity of fluoropyrimidine. Materials and Methods: The antitumor activities of 5-FU or 5-FU/TAS-114 were evaluated in 9 human tumor xenograft models. Intratumor dUTPase protein was measured by Western blotting, and phosphorylation of histone H2AX was detected by immunohistochemistry. Tissue concentrations of FdUMP and dUMP were measured by HPLC and a thymidylate synthase (TS) binding assay, respectively. Suppression of DNA damage repair proteins were performed by RNA interference technology in HeLa cells, and then, the cells were used to assess an antiproliferative activity of FdUrd/TAS-114. Results: Oral administration of TAS-114 inhibited dUTPase and synergistically increased antitumor activity of 5-FU in various human tumor xenograft models. The magnitude of dUTPase inhibition was estimated by the change of dUTPase enzymatic products (FdUMP and dUMP) and it appeared to correlate with dUTPase expression and synergistic effect. These data suggest that 5-FU and uracil misincorporation is a primary mechanism of TAS-114 efficacy. In addition, TAS-114 combination with 5-FU increased phosphorylation of histone H2AX, suggesting a TAS-114 activated DNA damage response pathway. Suppression of DNA damage repair proteins involved in base excision repair (BER) and homologous recombination repair (HR) revealed that these DNA repair pathways were important in TAS-114’s synergistic increase of 5-FU cytotoxicity. Conclusion: Expression levels of DNA damage repair proteins in BER and HR affect TAS-114 sensitivity. DNA repair response appears to be a determinant of TAS-114 efficacy as well as dUTPase expression. These factors will be measured inTAS-114/fluoropyrimidines combination clinical trial. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B89. Citation Format: Sayaka Tsukioka, Wakako Yano, Tatsushi Yokogawa, Takeshi Wakasa, Akio Fujioka, Keisuke Yamamura, Satoko Itoh, Masayoshi Fukuoka, Kenichi Matsuo, Kazuharu Noguchi, Teruhiro Utsugi. Expression of DNA damage repair enzymes determine the efficacy of a novel dUTPase inhibitor, TAS-114. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B89.