pressitre. r1, he residue was treated with lo';( aqueous KaHCOI aiid then eswacted with ether. The organic layer was washed with lO!, HCl arid the aqiieous layer was then made basic with 10c>hTaOH. The oil t>hat separated was again extracted with et,her. The organic layer was washed wit,h water and dried (ru'a&Od). I-pon removal of the solvent a white solid was obtaitied, which was recrystallized from ether t,o yield 600 mg of 111. Procedure B. 4-[Bis(2-hydroxyethyl)amino] -3-methoxy-1,35-estratrien-17p-01 (V).--k solution of 4-[bis( 2-hydroxyethy1)ai~iitio]-~3-methox~-l,~~,S-eilratrien-li-one (111) (1.4 g) in meth:mol (5U ml) was treated u-ith excess iYaBH4 (2.0 g), stirred for 1 hr at room temperatiire, aiid poured into aqueous NaHC03. The soliition was extracted with ether and the organic layer was washed and dried (Na2S04). The solveiit' was removed and t'he solid residue was rec tallized from methanol t,o yield 700 mg of
hydroxy acid (I, It = X = OH) but the hydroxy acid was recovered unchanged after prolonged heating with concentrated HCI. When heated with thionyl chloride the hydroxy acid gave an unstable product, presumably the hydrochloride of the acid chloride (I, R = X = Cl), which afforded 4-(2-chloroethylthio)nicotinic acid (I, R = OH; X = Cl), methyl +(2-~hloroethylthio)nicotinate (I, R = O N e ; X = Cl), and 4-(2-chloroethylthio)nicotinamide (I, R = KHz; X = C1) on treatment with HCl, methanol, and niethaiiolic ammonia, respectively.
v.
Procedure C. 4-[Bis(2-chloroethyl)amino]-3-methoxy-l,3,5estratrien-17-one (IV).-A mixture of Ill (500 mg) and POCll (10 i n l ) W : I ~ heated oil a stram bath for 2 hr. The excess POCIS \vas t'ctii~JVeduttder i,etluced pressure atld t,he remainiiig oil was tlirsolvrtl i t i ethcr-hetizeiie. The solutioii was washed wii'li tlilrit,c: I I C 1 aiid theti water. The orgaiiic lager was dried (Sa.SO,), atid t h e solvent was removed. The residue was dissolved i i i Iwtieciie (5 1111) arid absorbed on a coliimii of silica gel [; (50 g ) . rlfter removal of ail oilj- side product by eluting with benzene, I\- was eliited with ether-benzene (1: 1) as an oil (300 mg), which would iiot crystallize. This reaction failed with SOCI,. Treatmeiit of the triol (IS) wit,h POClp gave a compound that coiild iiot be ident,ified. l c i d shifted the ultraviolet absorption from 290 to 282-288 mM. KO hydroxyl abrorption appeared in the itifrared spectrrim. The nmr spectrum showed the aromatic prototi:: RZ, singlets 7 2.3 aiid 3.17. The 3-methoxy and 13methyl proton> appeared where they did in the starting material. S o other protons could be identified. Anal. Found: C, 43.23; I i , 6.66: N, 2.28; C1, 17.60; P, 7.6. I t is conceivable t,hat this material could be a dimer or polymer.
Acknowledgments.-We are indebted t'o H. Cheng for his hell) in the preparation of t'he starting iiiat'erials m t l to the Cancer Cheniothera,py Sational Service Center, Satioiial Institut'es of Health, U. S. Public Health Service, Bet,lwsclu, JId., for t'he biological data.
Somc Alka lating Derivalives of NicoLiuic Acid. S?ritlicsis a r i d Antineoplastic Activities
4-Substituted tiicotinztmides might act as stereospecific inhibitors of the glycolytic process by which many cancer cells derive an appreciable proportion of More effective and potheir energy tentially irreversible antagonists may be produced by iricorporating a cheniically reactive grouping into the 4 substituent. The preparation of such derivatives c~)titainingalkylating groups is now described. Tlic prepar:tt,ion of 4-(2-bromoethylthio)nicotinic :wid (I, It = OH; X = Br), isolat'ed as its hydroliruiiiide, has already been described.* The corresponding amide (I, R = XH2; X = Br) has now been prepared by the action of ammonia on the mixed anhydride formed from the acid (I, R = OH; X = Br) : ~ n disobutyl chloroformate. 'Tht. :wid (I, I< = OH; X = Rr) TWS re:idily obt:iinetl liy tlie action of concentrated hydrobromic acid on the RCJSS, : l i ~ n . H a p t . Brii. B m p m Cniwer Compn., 43, 81 (1984). Rosa, J . Chcm. Sou., Sect. C, 1816 (1966).
1
Iv
I11
Many difunct'ional alkylating agents have greater carcinostatic activity than the corresponding tnonofunct'ional analog^.^ The acid (I, R = H ; X = C1) could be converted into a novel t'ype of difurictioiial alkylating agent, having two diff erent alkylating groups, by the preparation of its ethyleniniide (I, R = S=(CHZ)t; X = CI). As a model for this synthesis the preparation of Snicotinoylethylenimine (11) was examined. The action of nicotirioyl chloride on ethyleniniine has been reported to give 114 but the product has not been adcquately characterized. When this preparation was repeated, only 2-(3-pyridyl)-2-oxazolitie4 (111), foimctl by internal alkylation of the iiiitial product, was 01)tairied. Ethyleriiniidcs car1 clinierizc to give pilwimazincs but the preparation of ~,S'-diriicoti~ioyli,iI,er.uzine b y an unambiguous synthesis showed that t'liis 1i:d iiot occurred. Xcotinoylethylenimine (11) was prepued by ('ondcrising nicotinic acid with ethyleniniine i n the prescnce of dicyclohexylcarbodiimide, and X-4-(k h l o r o ethy1thio)nicotinoylethyleniniine (I, R = S=(CH2)2; X = C1) was similarly obtained from the acid (I, R = OH; X = Cl). Biological Data.-The results of screer:ing tests against the Walker 256 tumorb and the lymphoid leukemia L12106 are given in Tables I and 11. Only moderate activity (ea. 50% inhibition) at tolerated doses against, the Walker tumor was shown by the hydrobromide of the acid (I, R = OH; X = Br) and nicotinoylethyleniniirie (11). Sjgiiificaiit :wtivity against the L1210 leulieniia was sliomi only by the chloro acid (I, R = OH; X = C1) at the niltxiniuni tolerated dose. (3) W. C. J. Ross, "Biological Alkylating Agents," Butterworth and Co. (Publishers) Ltd., London, 1962. (4) G. I. Hraa and V. A . Skorodiimor, % / i . Obsiiih. Kkim.,36, i i 0 (19.561; Chtim. Absir., 60, l l i l l (1Y56j. ( 5 ) Tlie protovol for tliid ran~illostatir assay i* xi\.eii by 1'. .i. ('uiiiims, 13. C', V. l l i t v l d e y , V , A l . Kohellarirr, alld \ \ . ( ' , . I . I < o s r , Bio(.Iirtu. /'/irlrm., 13, 395 (1984). (6) T h e protocol for t h i s assay ic esentially t h a t given in Cnnrer Chemot h e r u p y R e p i . , 1, 42 (lY59); tile Cs7/L)BA2 hybrid strain of inouae n a a used as host.
Experimental Section'
4-(2-Chloroethylthio)nicotinic Acid.--S-(2-IIyd1~11s~~eil1~llhiojiiicotiiiic acid2 (10 p) :tiid SOCI, (40 m i ) were heated wider refliis for '2 lrr. Addition of dry I)etizeric 1200 nil) arid then petroiciini ether h p 40-60°, '200 ml) to the cooled ,mlntioit caused l h e \cpiir:~tii>liof fine ricedles iproduc.t A ) which was c.ollected by riipid filtration, xvaahed with petroleiim ether, and stored in :I v:icii1irn
desiccator.
I'rodiic~t A \vas added in ~ n i a l lportiu~i,:t o s i ' ; HC1 130 nil. ri I.19) kept at 0" aiid the bdiitiori was evaporated to drytiess under rdticetl preiiiire :it 30-4O0. \{-:iter (60 nil) was added arid the d i d , nip 171-178", Tvhich proved t i l lie the hydrochloride of 4-(~--c~hlor1it~thyttI1i~i)rii1~1~i i l i i c . :ii,itl. \v:ib filtered off.
Noms by Bra2 and Skorodumov4 by the action of alkali on chloroethylnicotinamide (IT-). N-Nicotinoylethylenimine.-Dicyclohexylcarbodiimide (20.6 g) ill T H F (100 ml) was added to a solution of nicotinic acid (12.3 g) and ethylenimine (5.1 ml) in T H F (200 ml). After keeping for 1.5 hr at 25" the solution was filtered and evaporated under reduced pressure a t 25'. The residue was extracted with petroleum ether (bp 30-40", l l.), On chilling the extract in a Dry Ice-acetone bath, plates of mp 36" separated: yield 10 g. Anal. Calcd for C,H,X\;,O: C, 64.9; H, 5.4; N, 18.9. Found: C , 64.9; H, 5 . 5 : N, 18.8. The compound could be stored for several days a t 0" without decomposition but a t room temperature i n an evacuated vessel it liquefied within a few hours; this behavior probably accounts for the previously reported "hygroscopic" nat,ure of the product. A solution of the compoiind (166.4 mg) in 50YGaqueous acetone (50 ml) containing sodium thiosulfate (1 g ) rapidly developed alkalinity (phenolphthalein indicator) which was continuously titrated with 0.1 S HC1.8 The final titer was 10.2 ml, indicating that the materhl contained 97% of nicotinoylethplenimine. N-442-Chloroethylthio)nicotinoylethylenimine.-4-( 2-Chloroethylthio)nicot,inic acid was similarly converted into it's ethylenimide which formed plates, mp 66-68', from benzene-petroleum ether (bp 40-60"); yield 40%. The product was unstable a t room temperature and was stored a t -30". Anal. Calcd for Cl~H11ClN20S:C, 49.5; H, 4.6; C1, 14.6; N, 11.5; aziridine E,5.77. Found: C, 49.2; H, 4.3; C1, 14.6; IG,11.3; aziridine N (thiosulfate titrattion), 5.6. N,N'-Dinicotinoy1piperszine.-Isobutyl chloroformate (2.6 g) in T H F (10 ml) was added to a stirred, cooled (0") solution of nicotinic acid (2.46 g) and triethylamine (2.8 g) in T H F (40 ml). After allowing to stand for 30 min a t O", a solution of piperazine (0.86 g) in THF (20 ml) was added and the mixture was allowed to warm up to room temperature during 1.5 hr. The filtered solution was washed successively with aqueous 2 -17 Na2C03,aqueous 2 HCl and wat.er, and t,hen dried (Na2SOa). The product obtained on evaporation formed prisms, mp 201", from ethanol-ether (1:2). Anal. Calcd for C16H16?i402: C, 64.9: H, 5.4; K, 18.9. Found: C, 64.4; H, 5.6; S , 18.9. A\-
Acknowledgments.-Carcinostatic assays were carried out by lIr. B. C. V. llitchley and toxicity assays by l I r . 11. Jones. This investigation has been supported by grant's to the Chester Beatky Research Institute (Institute of Cancer Research, Royal Cancer Hospital) from the Medical Research Council and the British Empire Cancer Campaign and by Public Health Service Grant No. CA-03188-10 from the S a tional Cancer Institut'e, U. S. Public Health Service. (8) W. C . J. Ross, J . Chem. Sac., 2257 (1950).
Alkylating Activity of 1,3-Bis(2-chloroethyl)-l-nitrosourea and Related Compounds' GLYSN P. FVHEELER .IKD SUECHUMLEY K c l l e r i n g - X e y e r Laboratory, Southern Research Institute,
Birmingham, Alabama Received October 26, 1966
1,3-Bis(2-chloroethy1)-l-nitro~ourea~ (I) is an active compound against lymphocytic choriomeningiti~,~ L1210 leukemia and other experimental neoplasm~,~-4 (1) This work was supported h y a grant from T h e John A. Hartford Foundation, Inc. (2) T. P. Johnston, G. 9. IlcCaleb, a n d J. -1.Montgomery, J . Med. Chem.. 6 , 669 (1963). (3) R. \V. didwell, G. J. Dixon, S. AI. Sellers, and F. hl. Schabel, Jr., A p p l . .Cficrobiol.. 13, 5 i B (1965).
1.00
0.900
0.000
0.700
,0.600
.-
c
c
Ln
0
n
- 0.50C 0
U .c
~0.40c
0.30C
0.20c
0.IOC
I
2
3
4
5
6
7
)moles
8
9
1011
121314
Figure 1. Measurement of the alkylating activities of several compounds. With the exception of the tests with diazomethane, the reaction mixtures were heated in a boiling-water bath for 20 min. The reaction with diazomethane occurred at room temperature in the course of approximately 3 min. The numbers on the abscissa indicate the total micromoles of compound present in the reaction mixture. A, 1,3-bis(2-chloroethyl)-lnitrosourea; B, 1-(2-chloroethyl)-l-nitrosourea; C, 1-(2-chloroethyl)-3-cyclohexyl- 1-nit rosourea ; D, 1,3-bis(2-fluoroethy1)- 1nitrosourea; E, 2-fluoroethylamine hydrochloride; F, diazomethane; G, 1-methyl-1-nitrosourea.
and a number of neoplasms in humans.j Because a hamster plasmacytoma that was resistant to cyclophosphamide was also resistant to this compound, it was suggested that it might function as an alkylating agent.4a Cross resistance with alkylating agents mas also observed with microorganisms,6 and some of the biochemical effects of the agent are similar to those of several alkylating It is known that treatment of X-nitroso-S-methylurethanJ7 l-methyl-l-nitrosourea,* and 1-alkyl-l-nitros0-3-nitroguanidines~with alkali yields diazoalkanes, and it has been suggested that the biological effects of these agents might be due to such d i a z ~ a l k a n e s , ~which ~'~ might function as alkylating agents. The results presented below indi(4) (a) F. >I. Schabel, Jr., T. P. Johnston, G. 9. hIcCaleb, J. A. I l o n t gomery, It-. R. Laster, a n d H. E. Skipper, Cancer Res., 2 3 , 725 (1963); (b) .I. Goldin, J. >I. Venditti, J. A. R. hIead, and J. P. Glynn, Cancer Chemotherapy Rept., 40, 57 (1964): ( c j G . P. Wheeler and B. J. Bowdon, Cancer Res., 26, 1770 (1965). ( 5 ) (a) D. P. Rall, M. Ben, a n d D. hl. hIcCarthy, Proc. A m . Assoc. Cancer Res., 4, 5.5 (1963); (b) V. DeVita, P. P. Carbone, A H. Owen, Jr., G . L. Gold, >I. J. Krant, a n d J. Edmonson, Cancer Res., 26, 1876 (1965). (6) R. F. Pittillo, A. J. Narkates, and J. Burns, Cancer Res., 24, 1222 (1964). (7) 13. A. Kihlman, Ezptl. Cell Res.. 20, 657 (1960). (8) F. Arndt, O r g . S y n . 16, 4 (1935). (9) -1.F. McKay, Chem. Reu., 61, 301 (1952). (10) (a) \V. A . Skinner, H. F. Gram, hl. 0. Greene, J. Greenberg. and B. R. Baker, J . Med. Pharm. Chem., 2 , 299 (1960): ( b j L. Pasternak, Acta Bioi. 3 f e d . Ger., 10, 436 (1963); ( e ) H. Druckrey, S.Ivankovic, and R. Preussmann, 2.KreCqforsch.. 66, 389 (1B65).
