Journal of Medicinal Chemistry, 1976, Vol. 19, No. 2 339

Notes product was collected by filtration, washed (HzO), and dried in vacuo to give 3.8 g (92% yield) of 7b as red crystah mp 14-147'. An analytical sample was obtained by recrystallization from CHCMC14 as fine red needles: mp 158160'. Anal. (ClzH1006) C, H. 2,3-Bis[[ (aminocarbonyl)oxy]methyl]naphthazarin (712). To a gently stirred solution of 7 g of 7b in 2000 ml of CHzClz was added 9.1 g of NaOCN followed by 20 ml of CF3COzH. Stirring was continued for 23 hr at room temperature followed by addition of 50 ml of HzO. The mixture was stirred for 15 min and the resulting solid collected by filtration. It was washed with CHzClz and air-dried to give 3.1 g (33% yield) of 7c as a red powder. An analytical sample was prepared by reprecipitation from MezSO-CHCL as a red powder which did not melt up to 300'. Anal. (Ci4HizNz08) C, H, N. 2,3-Bis[ [(methylsulfonyl)oxy]met hyllnaphthazarin (7d). A mixture of 5.9 g of 6b and 12.8 g of silver mesylate in 80 ml of CH3CN was stirred at room temperature for 24 hr. The CH3CN was removed in vacuo at 40'; the residue was triturated with 100 ml of CHzClz, filtered, and washed with 2 x 50 ml of CHzClz. The combined CHzClz solution was concentrated to a small volume (25 ml) in vacuo, filtered from some insoluble material, and diluted with 100 ml of C a s to give 4.9 g (76% yield) of 7d as a red powder. Depending on the rate of heating, a decomposition point of the 7d was observed at ca. 150". An analytical sample was prepared as a red powder by two recrystallizations from CsHs-CHzC1z: mp 158-160". Anal. (Ci4Hi40ioSz) C, H. Naphthazarin Diacetate (8b). To a warm (30-40') solution of 2.4 g of naphthazarin (sa)in 30 ml of AczO was added six drops of concentrated HzS04. The mixture was stirred overnight at room temperature and poured into 300 ml of ice water. The resulting yellow solid was collected by filtration, washed with HzO (2 X 10 ml), and dried to give 2.4 g (70% yield) of 8b: mp 193-195'. An analytical sample was recrystallized from Hz0-MeOH: mp 193.5-195' (lit.17 mp 195').

R. Gravatt for performing analyses and instrumental measurements. References and Notes S. K. Gupta and I. S. Mathur, Indian J . Cancer, 9,50 (1972). R. H. Thomson, "Naturally Occurring Quinones", 2nd ed, Academic Press, New York, N.Y., 1971, p 248. A. J. Lin, L. A. Cosby, C. W. Shansky, and A. C. Sartorelli, J . Med. Chem., 15, 1247 (1972). A. J. Lin and A. C. Sartorelli, J . Org. Chem., 38,813 (1973). A. J. Lin, R. S. Pardini, L. A. Cosby, B. J. L i b , C. W. Shansky, and A. C. Sartorelli, J . Med. Chem., 16, 1268 (1973). A. J. Lin, C. W. Shansky, and A. C. Sartorelli, J . Med. Chem., 17, 558 (1974). A. J. Lin, R. S. Pardini, B. J. Lillis, and A. C. Sartorelli, J . Med. Chem, 17, 668 (1974). R. K. Neogy, K. Chowdhury, and G. G. Thakurta, Biochim. Biophys. Acta, 299, 241 (1973). G. C. Das, S. Dasgupta, and N. N. Dasgupta, Biochim. Biophys. Acta, 353, 274 (1974). M. G. Brazhnikova, V. B. Zbarskii, V. I. Ponomarenko, and N. P. Potapova, J . Antibiot., 27, 254 (1974). G. F. Gause, M. G. Brazhnikova, and V. A. Shorin, Cancer Chemother. Rep., Part I , 58, 255 (1974). C. S. Contreras, Rev. R. Acad. Cienc. Exactas, Fis. Nut. Madrid, 57, 385 (1963); Chem. Abstr., 60, 9211h (1964). M. Lora-Tamayo and J. L. Leon, J. Chem. Soc., 1499 (1948). H. B. Henbest, M. Smith, and A. Thomas, J . Chem. Soc., 3293 (1958). A. N. Vereshchagin, A. P. Anastaseva, and B. A. Arbuzov, Izu. Akad. Nauk SSSR, Ser. Khim., 1420 (1971); Chem. Abstr., 75, 129266~(1971). J. R. Scheffer, K. S. Bhandari, R. E. Gayler, and R. H. Wiekenkamp, J . Am. Chem. SOC.,94, 285 (1972). C. Kuroda, Proc. Imp. Acad. (Tokyo),20, 20 (1944). V. N. Iyer and W. Szybalsky, Science, 145, 55 (1964). K. Sugiura, Cancer Res., 19, 438 (1959). S. Kinoshita, K. Uzu, K. Nakano, M. Shimizu, T. Takahashi, and M. Matsui, J . Med. Chem., 14, 103 (1971). S. Kinoshita, K. Uzu, K. Nakano, and T. Takahashi, J . Med. Chem., 14, 109 (1971).

Acknowledgment. This investigation was supported by Contract N01-CM-33743 from the Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare. The authors thank Mrs. Margaret L. Rounds and h4r.John

Inhibition of Tumor Cell Transplantability by Iron and Copper Complexes of 5- Substituted 2-Formylpyridine Thiosemicarbazones William E. Antholine, Judith M. Knight, and David H. Petering* Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201. Received May 27, 1975 The cytotoxicity of copper and iron complexes of 5-substituted 2-formylpyridine thiosemicarbazones against Ehrlich ascites tumor cells has been measured. Brief in vitro incubation of cells and drugs is followed by implantation into host mice. Subsequent degree of tumor development is a measure of cytotoxicity. A spectrum of activities for the iron complexes is observed, starting with the least active as designated by its 5-substitution: OH < OCOCH3 N(CH3)z < H < CH3 C1 CF3. The last three complexes can prevent completely tumor growth in the new host. Copper complexes of 5-H and 5-CH3 also prevent successful tumor cell transplantation.

-

- -

The possibility that metal complexes of a-N-formyl heterocyclic thiosemicarbazones may be cytotoxic to tumor cells has been raised recently in preliminary communications.12 Experimental findings together with relevant background information are reported here in support of this proposal. Thiosemicarbazones of 1-formylisoquinoline and 2formylpyridine and a multitude of their derivatives have been examined for activity against a variety of transplanted animal tumors.314 A number of studies have indicated that these compounds (L) can act a t a molecular level by inhibiting ribonucleoside diphosphate reductase, an obligatory enzyme in the pathway of synthesis of precursors of DNA.5-7 Furthermore, it has been hypothesized that

Chart I. (a) 2-Formylpyridine Thiosemicarbazone and (b) Octahedral Binding of Iron by LigandJo

N 'NH

a

b

the basis of the inhibition is the binding of iron a t the active site of the enzyme (E) in an E.Fe.L complex which prevents catalysis.89 In fact, Ablov and Belichuk have

340 Journal of Medicinal Chemistry, 1976, Vol. 19, No. 2

Notes

Table I. Cytotoxicity of Metal Complexes of 5-Substituted 2-Formylpyridine Thiosemicarbazone

i H E N "c_ hb

__._______.

-_.

~

II

i

Compound

5-x -H

FeL,+ -H

-OH

Dose, mglml

Solubility: inglml

-___

-c1

-CF, -CH, c u r -H

0.74

315

1.37

717

20 14

0.55

213 ( 2 , 18) 015

21 23

515

19 17

1.1 1.05

1.05 0.105 1.00

4.5

0.10

0.5

1.00 1.00 1.05 1.10 1.10 1.00 0.100

0.9 2.5

0.75 0.80

0.1

0.75

0.1

1.5 1.3 1.9

0.53 0.89 1.16 0.75 0.60 0.50 0.15

414 (1,3 )

616

4/4 (1,19) 313 ( 2 , 2 2 ) 314 ( 1 , 1 4 1

015 015 115

0.17 0.42 0.12

015 415

0.89

0.050

015 015

0.29 0.22 0.13 0.24 0.20

115

015 014 ( 1 , 19)

Long-term survivors: days

Dayd

0.5

0.050

-CH,

Tumor incidence"

1.0f

1.00

-OCOCH, -N(CH,)*

TICb

315 ( 4 5 )

14 20 22 22 23 23 21 23 20

515 ( 4 5 ) 515 ( 7 5 ) 215 ( 7 5 ) 515 (45) 515 ( 7 5 ) 515 (75)

21

23 22 20 20

Solubility in pH 7.5 Tris with an estimated uncertainty of +20%. (a wtlwk treated)/(A wt/wk control). Determined by analysis of weight changes with groups on day of final observation. Deaths of tumor-bearing animals occurred as shown in parentheses (number, day). Day of final observation. e Animal groups with tumors were sacrificed a t conclusion of period of observation. Others were maintained and analyzed as in footnote b for tumor development. f Drug delivered to 30-gmouse; 13 mg/kg.

prepared the iron complex of 2-formylpyridine thiosemicarbazone as well as its cobalt and nickel complexes (Chart I).lO Other workers have reported the x-ray structure of 1-formylisoquinoline thiosemicarbazonatonickel(II).11 In each of these cases a 1:2 metal to ligand stoichiometry has been found. Although there have been no detailed studies heretofore to examine the validity of the mechanism of cellular inhibition described above including ligand specificity for iron, there is ample physiological evidence that compounds with the constellation of metal binding groups shown in Chart I (a) markedly disrupt iron metabolism in animals and man, with the probable complexation of significant quantities of iron in vivo.12-14 It may be argued, therefore, that it is the iron complex, itself, which is cytotoxic to tumor cells in vivo. This is the case with 3-ethoxy-2-oxobutyraldehyde bidthiosemicarbazone), which is activated in vivo by Cu2+.15 However, as will be described elsewhere, the ligand, 2-formylpyridine thiosemicarbazone, also has a large formation constant with Cu2+.1J6 Hence the possibility is not excluded that this chelating agent may be activated by copper. Preliminary communications have outlined chemical and biochemical evidence to support the plausibility of formation and stability of iron and copper complexes of 2-formylpyridine thiosemicarbazone in vivo, the centrality of the iron(I1) complex in the inhibition of ribonucleoside diphosphate reductase, as well as the antitumor activity of iron, copper, and zinc complexes of 1-formylisoquinoline thiosemicarbazone.l,2 In the present work the effects of iron and copper complexes of selected &substituted 2formylpyridine thiosemicarbazones upon Ehrlich ascites tumor cell transplantability are examined.

Results and Discussion As an initial screen for compounds which are toxic to

tumor cells after short exposures, a combination of in vitro incubation of test material and tumor cells followed by implantation of cells into the host organism has been used. This procedure provides information on drug-induced tumor cell toxicity which is relatively uncomplicated by attendant host responses to the compounds. In this study a number of iron and copper complexes of 5-substituted 2-formylpyridine thiosemicarbazones were examined. Table I summarizes the results of several experiments. Average change of weight of treated vs. controls served as a quantitative basis of comparison of cytotoxic effects upon tumor and animal host. Experiments were repeated using many of the compounds with qualitatively similar results. Only in the case of the iron complex of 5-H was there a marked difference of results among the runs. An ordering of the relative effectiveness of the iron complexes from the least effective at 1 mg/ml in vitro incubation is OH < N(CH3)2 OCOCH3 < H < Me C1 CF3. At this concentration of drug, about 13 mg/kg is delivered to an average 30-g mouse. Neither 5-H nor 5Me, examined at 0.1 mg/ml, had activity. Strikingly, the 5-OH complex was least active, in contrast to its outstanding in vivo activity against a variety of transplanted tumors including sarcoma-180 ascites tumors.3.4 In part this may be due to its lower solubility in the aqueous solution used for incubation. However, the correlation fails with other compounds which are considerably more soluble (Table I). The ordering of complexes on this basis from least soluble is OH < OCOCH3 < CF3 C1 < CH3 < N(CH3)z < H. In general, the order of activity of these complexes differs from the patterns seen with their ligands with respect to several different tumor systems.3~4For instance, the qualitative order in L1210 leukemia is H C1 CHs < CF3 OCOCH3 < N(CH3)2