the genetic material is occurring with time and that badly damaged sperm are being eliminated cannot now be discarded. Sperm inactivation is another possible explanation for the decrease in frequency of mutation but it is rather difficult to show since the housefly is poly~ p e r m i c . ~However, 2 does not induce sperm inactivation in Rmcou, even at doses greater than those needed to induce dominant lethal mutations in 100% of the sperm,Y but with 11, cytological examination establi\hed that the eggs were fertilized with the sperm from treated malesS3 The data also suggest that mutagenic effectiveness is not necessarily directly related to the number of aziridinyl groups since compound\ such as 5 and 7 were nearly as effective as 2, and 8 was more active. When the nonheteroaromatic compound5 9, 10, and 11 were tested for recovery, the number of recovered dominant lethal mutations did not decrease after stor:i:;e for 7 days.
recryst,allized froni CREI,~: yield 63%,; mp 132-133' der. .inn{. (CioH13Yj) C, €I, ?;. 2,6-Bis(l-aziridinyl)pyridine (7).-The compound was prepared in the same manner as 6,method B, except that the quantity of AzLi was adjusted for the 2 equiv of Br to be replaced on 2,6-dibromopyridine. The product, was recrystallized from CsHla; yield 65%; mp 98-100". A n a l . (CsH,,SI)C, H, N. 2,6-Bis(l-aziridinyl)pyrazine (S).--AzLi and 2,6-dichloropyrazine, when allowed to react by method B, produced the desired product in good yield. Holvever, the reaction was someTrhat more exothermic than the others reported: thiis, the addition of the dichloro compound !vas slower. The pr0duc.r ~ r a h recrystallized from C6HI1; yield 7.ic : mp 57.5-59'. .4nal. (c&ioN3) C, H, S . Compounds 1, 2, 3, 4,9, 10, and 11 were prepared by method.: described in the literature'i or purchaPed from commei.icA sources. Compound 12 was prepared by t'he method of Heine, et a 1 . 1 3
Ackrow1edgment.-The ziutliors express their gratitude for the t'echiiical assistance given b\. Gerald Hol t of this laboratory. (13) H.W.Hrine, R. L. Kapur, and C. XIitch, J . 1 n e r . Chrm. Roc., 76, 1173 (1954).
Experimental S e ~ t i o n g . ~ ~ Melting points were taken witjh a Thomas-Hoover apparatus and are corrected. Pmr spectra were obtained in CDC1, wit'h (CH3),Si as a n internal reference on a Varian A-60A spectrometer. The ir spectra were measured in KBr or CsI pellets on a Cary Model 14 spectrophotomet,er. 811 spectral correlat,ions were as expected.11 Elemental analyses were performed by Huffman Laboratories, Inc., Wheatride, Colo. Aziridinyl1ithium.-The azyridinyllithium (AzLi) was generated at room temperature in The aziridine (Az) was diluted t'enfold with solvent (either C6Hs or EtzO), and drops of 1IeLil2 (about 1.6 '11 in Et*())were added rapidly. Because of the low, boiling point, of Az, a 107, molar excess was used. All work with Li derivatives was done in flame-dried glassware under NP. 2-Chloro-4,6-bis(l-aziridinyl)pyrimidine(5).6--A solution of 9 g of 2,4,6-t,richloropyrimidine in 45 ml of C6He was added dropwiae to a solution of 11 ml of Az and 14 ml of Et3S in 100 ml of C6H6 (in an ice bath). The ice bath was removed, and the solution was stirred for 4 hr. Charcoal was added to the mixtiire, and the suspension was filtered through Super Cel. The sohition was concentrated in cacuo to 30 ml, and several portions of CsH,i were added; the crystals were allowed to grow between additions; yield 7 3 ' 3 . The product was recrystallized from C6Hll; mp 97" dec. A n a l . (CeH&lX4) C, H, C1, S . 2,4,6-Tris(l-aziridinyl)pyrimidine (6).s Method A.--l)irect conversion of 2,4,6-trichloropyrimidine with AzLi yielded a mixture of partially substituted pyrimidine phis some trisaziridinyl product. Pmr data indicated the presence of both monoxziridinyldichloro isomers and 5 . Column chromatography on alumina did effect a separation, but the overall yield was poor. Compound 5 was therefore iised as a starting material to improve the yield. Method B.-AzLi (40 mmoles) was generated in 30 ml of C6Hs. 2,PBis( l-aziridinyl)-6-chloropyrimidine (7.0 g ) was added slowly (about 10 mill) as a solid. After 3 hr of stirring, 1 ml of HIO and some charcoal viere added to the mixtiire and the material was filtered. The solution was evaporated to near dryness, and 30 ml of C6HI4was added. The mixture was then triturated and the supernatant C6Hi4 was discarded. The residue was taken up in hot C6H14, charcoal was added, and the solution was allowed to cool after filtration. The compound was (8) L. E. LaChance and A. P. Leverich, A n n . Entomol. SOC.Amer., 61, 164 (1968). (9) Where analyses are indicated only by symbols of the elements or functions, analytical results obtained for these elements or functions were within &0.4% of the theoretical values. (10) Mention of a proprietary product does not constitute a recommendation or a n endorsement of this product by the U. S. Department of Agriculture. (11) The ir assignments for azirdine mere baaed on the paper by H. L. Spell, Anal. Chem., S9, 185 (1967). (121 MeLi solution from Foote Mineral Co., was used directly.
Potential Antineoplastics. V. Synthesis of Ethyl 2,S-Dioxobutyrate 2-Arplhydrazono-3- thiosemicarbazones H. G . GARGASD R. A . S H ~ R M A Department of Chemistry, University of Roorlzee, Roorkee, I n d i a Received .April 2, 197'0
Ever since the initial observation made by Brockman, el al. , I that 2-formylpyridine thiosemicarbazone pos-
sessed mild but definite antileukemic activity, chemists have tried to prepare several classes of thiosemicarb a z o n e ~ , ~but - ~ virtually none of these appeared to be a clinically useful drug. I n spite of this unpromising background we synthesized a few ethyl 2,3-dioxobutyrate 2-arylhydraxono-3-thio~emicarbazones,because the incorporation of an arylazo moiety in several cases resulted in the enhancement of the potency of candiate drugs.j Characteristics of the new ethyl 2,3dioxobutyrate 2-arylhydrazono-3-thiosemicarbazones are summarized in Table I. The synthetic route in all cases involved the coupling of aryldiazonium salts with ethyl 3-oxobutyrate 3-thio~emicarbazone.~ Biological Activity.-Shown in Table I1 are the data for antitumor activity against L-1210 lymphoid leukemia. From this primary screening it appears that the level of toxicity varied greatly in this group. Ethyl 2-(2,5dimethylphenyl)hydrazono-, 2- (3,5-dimethylpheny1)hydrazono-, 2- (2,5-dimethoxyphenyl)hydrazono-, and 2-(2-methoxyphenyl)hydrozono-2,3-dioxobutyrate 3- thiosemicarbazones are somewhat more potent than other members in this series. (1) R. W. Brockman, J. R. Thomson, hI. J. Bell. and H. E . Skipper. Cancer Res., 16, 167 (1956). (2) I3. Prescott and C. P. Li, J . Med. Chem.. 7,383 (1904). (3) A. B. Sen and R. N. I h p o o r , I n d i a n J . A p p l . Chem., 81, 171 (1968). (4) I,'6 O,%
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-411 melting points were determined using a KoRer hob-stage type apparatus and are uncorrected. Ethyl 3-Oxobutyrate 3-Thiosemicarbazone.--Thiosen~ic~rbazide.HC1 (2.56 g, 0.02 mole) in H20 (15 ml) was shaken with ethyl 3-oxobiityrate (?.ti0 g, 0.02 mole) in the presence I I S:iO.i(. ~ (,%I g ). The thioseinicarbazurle separated out after 0.5 hr, ~ : i > collected, washed well with IInO, and recrystallized (CtiIi6I : yield 1.93 g (95%), mp 93-94". Anal. (CiH131i302Sj X, S. Ethyl 2,3-Dioxobutyrate -2-(5-Chloro-2,4-dimethoxyphenyl)hydrazono-3-thiosemicarbazone.-j-Chloro-~,~-dimetho~~a1~ili I if' t 1.88 g, 0.01 Inole) wvas dissolved ill 3 AVI4C1 (3 ml) allti cooleti i o 0". SaN02 (0.7 g, 0.01 mole) dissolved i n H2O (10 nil) JWgradidly added aiid the diazoiiiiim salt, sollition so obtained JY:~,. filtered into B well-cnoled, itii,red mixtiire of S a O h c (2.5 gi : I r i l l ri hyl 3-osohiityrate :J-thio,~einic:i~b~zol~e (2.03 g, 0.01 ninle) ~ I I taiiiiiig iOl€(20 nil ). 'I'hc prodiict thus precipitatctl \v:I~( ~ ~ I l ~ ~\\-:isheti l e t i , \vvII \villi I t 2 ( ), a i d 1,ecrystallized from K t O l I g, iFi',.>) as deep rccl iic~dle*,inp 2:34-2%'. Atrial. ( C l j l l ,,,c'lh;o.,s),CI, T.
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I) 'ti -IJ X 9.6 Host, BDF1 mice: vehicle, (.arbox?.niethylcellulose, acetoue, saliiie; route, intraperitoneal; parameter, meaii siuvival time :30 days; tissue, ascitic fluid; level, 106 cells. * Xumbers correspond to those in Table I. All doses were 400 mg,'kg. Ratio of mean survival time of test aiiimals tT) t u cuntrol aiiimals (r,.
Acknowledgment. T h e authors are greatly indebt t t l t o Drs. H. B. Wood arid H. W. Bond of the Cancer Chemotherapy, Sational Service Center, for their cooperation and for mal\ing the wreening data available. We are also thnnhful to Professor W. C. Alalik, Head of the Chemistry Department, for providing thc iiecehw-y facilitiei for this work arid the C.S.I.R., Sr\\ I k l h i (India flJr :i .Timior Research Fellmvship (held b> Ii A. s.1. ) ?
