[CANCER RESEARCH 40, 1482-1485, May 1980] 0008-5472/80/0040-0000$02.00 Pyrazofurin Inhibition of Furine Biosynthesis via 5-Aminoimidazole-4- carboxamide-1 -/J-o-ribofuranosyl 5'-Monophosphate Formyltransferase1

John F. Worzalla2 and Martin J. Sweeney

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

ABSTRACT showed that PF-PO4, formed through the phosphorylation of PF by adenosine kinase (9, 18), inhibited orotidylate decarbox- Pyrazofurin (PF), a C-nucleoside which inhibits pyrimidine ylase (8). The inhibition of orotidylate decarboxylase by PF- biosynthesis, is being tested clinically as an anticancer agent. PO4 resulted in decreased levels of uracil and cytosine nucleo- Pyrazofurin 5'-monophosphate (PF-PO4), the active metabolite sides and nucleotides (2, 4, 25). This ability of PF-PO4 to of PF, has a structural resemblance to 5-aminoimidazole-4- decrease the levels of uracil and cytosine compounds can be carboxamide-1 -/S-D-ribofuranosyl 5'-monophosphate (AICAR), exploited by using PF in combination chemotherapy to enhance a nucleotide intermediate in the biosynthesis of purines. Be the activation of other antitumor compounds such as 5-azacy- cause of this structural similarity, the effects of PF and PF-PO4 tidine and 1-ß-D-arabinofuranosylcytosine (5, 6, 16, 26). on AICAR formyltransferase and purine synthesis were studied. Clinical studies show that some patients receiving PF have PF-PO4 inhibited AICAR formyltransferase in rat liver super- decreased serum uric acid levels and increased excretion of natants 46, 69, and 89% at concentrations of 0.2, 0.4, and 1 uric acid (15, 24). This suggests a possible interference of mw, respectively. The Ki for AICAR formyltransferase by PF- purine metabolism caused by PF or its phosphate. PF-PO4 has PO4 was 3 x 10~6 M. AICAR formyltransferase was inhibited a structural resemblance not only to orotidine 5'-monophos- 32% by 2 HIM PF when ATP (20 mw) and MgCI2 (20 mW) were phate (12) but also to AICAR, an intermediate in the de novo present, but 2 mvi PF alone did not inhibit AICAR formyltrans biosynthesis of IMP (Chart 1). This report describes the inhi ferase. bition of AICAR formyltransferase (EC 2.1.2.3) in vitro by PF- The inhibition of AICAR formyltransferase in vivo should PO4 and the inhibition of de novo purine synthesis in rats given result in a buildup of AICAR and a subsequent increase in the PF. urinary excretion of 5-aminoimidazole-4-carboxamide (AIO, which is the normal urinary degradation product of AICAR. Male Sprague-Dawley rats given single i.p. doses of PF at 7.5, Materials and Methods 10, or 30 mg/kg showed increased urinary excretions of AIC PF and PF-PO4 are products of Eli Lilly and Company, as the dose of PF was increased. A single dose of 10 mg/kg Indianapolis, Ind. AIC, 5-aminoimidazole-4-carboxamide ribo- resulted in a 64% increase in the amount of urinary AIC (41 side, and brewer's bottom yeast were purchased from Sigma tig/day for the treated rats versus 25 jtig/day for the untreated Chemical Company, St. Louis, Mo. Leucovorin calcium salt and rats). A higher dose of 30 mg/kg resulted in a 233% increase methotrexate were purchased from Lederle Laboratories, Pearl in the urinary AIC (70 /¿g/day for the treated rats versus 21 River, N. Y. /ig/day for the untreated rats). AICAR was synthesized from 5-aminoimidazole-4-carbox- PF has a unique ability to inhibit the de novo biosynthesis of amide riboside with a brewer's yeast preparation, purified by both purine and pyrimidine nucleotides. ion-exchange chromatography, and crystallized (1 7). AICAR was characterized by UV spectrophotometry, mass spectrom- INTRODUCTION etry, and elemental analysis. Later supplies of AICAR were purchased from Sigma. Samples of aqueous AICAR solution PF3 is a nucleoside antibiotic isolated from a fermentation were stored at 4°. broth of a strain of Streptomyces candidus (15). PF shows A/'°-Formyltetrahydrofolate was synthesized from leucovorin activity against some viruses (30), fungi (33), and tumors in (27). Oxidation of N'°-formyltetrahydrofolate solutions was re rodents (31 ). PF has been tested clinically for cancer (3, 7,13, tarded by mercaptoethanol (0.004%). These tetrahydrofolate 14, 21, 23, 28, 32) and has shown activity against multiple solutions were frozen and stored for periods of up to 4 weeks myeloma.4 before use. The antiviral effect of PF was reversed by uridine, suggesting The assay of Flaks and Lukens (11) for AICAR formyltrans that PF inhibited pyrimidine synthesis (30). Subsequent studies ferase activity was run at 37° in a total volume of 1.0 ml containing 100 rriM Tris-HCI buffer (pH 7.4), 10 MIM KCI, 0.8 ' Presented in part at the 69th Annual Meeting of the American Association HIM AICAR, 1.6 HIM A/10-formyltetrahydrofolate, and 0.2 ml of for Cancer Research in Washington. D. C.. April 1978 (34). 2 To whom requests for reprints should be addressed, at Lilly Research a 25,000 x g supernatant fraction prepared from 20% (w/v) Laboratories, MC305. Eli Lilly and Company. 307 East McCarty Street. Indian rat liver homogenates. The loss of AICAR was determined by apolis. Ind. 46285. analyzing a 0.3-ml sample of the incubation mixture at zero 3 The abbreviations used are: PF. pyrazofurin; PF-PCv pyrazofurin 5'-mono- phosphate; AICAR, 5-aminoimidazole-4-carboxamide-1-/?-D-ribofuranosyl 5'- time and after 20 min. The reaction was stopped by adding the monophosphate; AIC, S-aminoimidazole^-carboxamide. 0.3-ml sample to 0.2 ml of 10% (w/v) cold trichloroacetic acid. 4 R. C. Dyke. Lilly Laboratories for Clinical Research and the Department of After centrifugation for 10 min at 1800 rpm, 0.3 ml of the clear Medicine. Indiana University School of Medicine, Indianapolis, Ind., personal communication. supernatants was treated with 0.1 ml of acetic anhydride for Received May 10, 1979: accepted January 31. 1980. 20 min, and then 2 ml of 0.2 N HjSO* were added. The samples

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inhibition of AICAR formyltransferase (Table 1), but 2 mw PF together with 20 mM ATP and 20 mw MgCI2 showed 32% inhibition. Presumably, this inhibition was caused by PF-PO4, the active metabolite of PF (9, 35). The inhibition of AICAR formyltransferase in vitro by PF-PO4 but not by PF is similar to HO-POCH, o. the inhibition seen for the pyrimidine-biosynthetic enzyme, orotidylate decarboxylase, which also was inhibited by PF-PO4 OH |

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Table 2 Daily urinary excretion of AIC by rats of an amount of of con imals34488881112888Av.AIC in urine(/ig)20.6 (ftest)a>0.05>0.05<0.001>0.05<0.001<0.001<0.05<0.02%trol11786163108192331159145 urines0-2424-hr control ±5.0b24.0 mg/kg)24-48hr after PF (7.5 ±11.617.8 mg/kg)24-hrhr after PF (7.5 ±11.025.2

urines0-24control ±5.241. mg/kg)24-48hr after PF (10 0±8.227.1 mg/kg)0-24hr after PF (10 ±4.948.4 onDayhr after second dose of PF (10 mg/kg) ±13.721. 424-hr

urines0-24control 2 ±4.370.2 mg/kg)24-48hr after PF (30 ±25.233.8 mg/kg)24-hrhr after PF (30 ±14.121.

urines0-24control 8 ±5.831 hr after methotrexate (3 mg/kg)No. .7 ± 6.7p a Mean of treated versus mean of respective control group. Statistical significance determined using a 2- tailed Student f test. b Mean ±S.D.

DeNovoSynthesis conversion of PF to PF-PO4, however, results in a nucleotide that in vitro is not only an inhibitor of orotidylate decarboxylase in the de novo synthesis of pyrimidines but is also an inhibitor of the purine biosynthetic enzyme, AICAR formyltransferase

AIC—¿AICRibos* —¿AICARAICAR A(1-- Drob*»-— Orotic (Chart 2). Evidence that both these enzymes are also inhibited 1T— »Inhibition » in vivo is provided by the fact that elevated urinary levels of 1PFPOT |—^UMPPvnmiiimeNudeoMes^r^Increased ~~ AIC, orotidine, and orotic acid are seen after PF is given. Many FAICARPurim anticancer drugs are active due to their inhibition of purine or 1tunarv Excretion pyrimidine biosynthesis, but PF has the unique ability to inhibit In Man And AnimalsAfter PF the de novo biosynthesis of both pathways (Chart 2).

udeotidesOMP Acknowledgments

We thank M. R. Gleissner for synthesizing the PF-PCv

RNAandDNA References Chart 2. Inhibition of purine and pyrimidine biosynthesis by PF-PCv FAICAR, formyl-AICAR; OMP, orotidine 5'-monophosphate. 1. Black. M. J., Black. S. L.. Solan. V. C., Robins. R. K., and Mangum, J. H. Inhibition studies of glycinamide ribotide transformylase and aminoimidazole carboxamide ribotide transformylase involving antifolates and aminoimida a 17% increase in the amount of AIC excreted, but the differ zole carboxamide ribotide analogues. Fed. Proc., 37: 1545. 1978. 2. Brockman, R. W.. Shaddix. S. C., and Rose. L. M. Biochemical aspects of ence was not statistically significant (p > 0.05). chemotherapy of mouse colon carcinoma. Cancer (Phila.) 40.2681-2691, Because the normal excretion of AIC is constant, these 1977. 3. Budman. D., Currie, V., and Wittes. R. Phase II trial of pyrazofurin in increases in the excretion of AIC indicate that PF inhibits AICAR malignant melanoma. Cancer Treat. Rep., 6) 1733-1734, 1977. formyltransferase and de novo purine biosynthesis in rats. PF- 4. Cadman, E. C., Dix, D. E., and Handschumacher, R. E. Clinical, biological, PO4 might be a useful biochemical tool for the study of purine and biochemical effects of pyrazofurin. Cancer Res., 38. 682-688, 1978. 5. Cadman, E., Eiferman, F., Heimer, R., and Davis, L. Pyrazofurin enhance biosynthesis, especially since there are few known inhibitors ment of 5-azacytidine antitumor activity in L5178Y and human leukemia of the latter steps in the biosynthesis of IMP, although the 5'- cells. Cancer Res.. 38. 4610-4617. 1978. phosphate of virazole has also been shown to inhibit AICAR 6. Chiuten, D. F., Johnson, R. K., and Muggia, F. M. Antitumor activity of pyrazofurin in combination with 5-azacytidine in mice. Proc. Am. Assoc. formyltransferase (1 ). Cancer Res.. 19: 230, 1978. The biological effects of PF have been previously attributed 7. Creagan, E. T., Rubin, J., Moertel, C. G.. Schutt, A. J., O'Connell. M. J.. to the ability of its monophosphate metabolite, PF-PCu, to Hahn. R. G.. Reitemeier. R. J.. and Frytak. S. Phase II study of pyrazofurin in advanced colorectal carcinoma. Cancer Treat. Rep., 6/. 491-493, 1977. inhibit the synthesis of UMP through its inhibition of orotidylate 8. Delong, D. C., Baker. L. A., Gerzon, K.. Gutowski, G. E., Williams, R. H., decarboxylase (15). Although PF-PO4 is a more potent inhibitor and Hamill, R. L. Inhibition of Friend leukemia virus-induced splenomegaly in vitro of the pyrimidine enzyme orotidylate decarboxylase (K¡ by pyrazomycin. In: Proceedings of the Seventh International Congress of = 5 x 10~9 M) (9) than of the purine enzyme AICAR formyl Chemotherapy, Prague, Czechoslovakia. 1970 1, A-5/35, Bloomfield, N. J.: Schering Corp.. 1971. transferase (K¡= 3 x 10^5 M), this report shows that PF had 9. Dix, D. S., Lehman, C. P., Jakubowski, A., Moyer. J. D.. and Handschu- macher, R. E. Pyrazofurin metabolism, enzyme inhibition and resistance in a significant effect on de novo purine metabolism in rats. L5178Y cells. Cancer Res., 39. 4485-4490, 1979. PF is a known inhibitor of pyrimidine biosynthesis, and the 10. Dixon, M. The determination of enzyme inhibitor constants. Biochem. J., 55: cytotoxic effect of PF can be completely overcome by undine 170-171, 1953. 11. Flaks, J. G., and Lukens, L. N. The enzymes of purine nucleotide synthesis (25), suggesting that inhibition of pyrimidine nucleotide synthe de novo. Methods Enzymol. 6. 52-95, 1963. sis is the primary reason for the action of this compound. The 12. Gerzon, K., DeLong, D. C., and Cline, J. C. C-nucleosides: aspects of

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John F. Worzalla and Martin J. Sweeney

Cancer Res 1980;40:1482-1485.

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