March 1967
265
NOTES
TABLEI STEROID A L ?-La CTO N E H % CoinlJd
Crystii solvent
yield
I
69
I1
Beiixeiiehexatie Acetone-benzene Benzene-hexane AIethanol-water Ether CHCI,-ether
RIIb
X*,m,* mlr
oca
285-238 232-234 217-219 248-253 252-253 260-263
249
Calcd, '/o H
[m.]o,C
deg
f
12,300
+1450
C
Forluula
C2JIa4O4 ,0.5H20 73,68 8.66 C~~H~~O~.O.~C 74.50 ~ H B 8O. 7 2 C~3H3003 77.92 8 . 5 3 C23133203 77.49 9.05 C2~H3404.0.5HjO 73.68 8.66 C2jH3203.H& 75.34 8.60
15,000 221 -3P 10,600 - 133 220 8,650 -129 219 I 223 13,600 +102 v 248f In rnethanol. c At 34-26' aiid 1'; in CHCI, except as indicated. Capillary tube, corrected. E. Childs (Anii Arbor) and by N r . F. H. Oliver ( H o ~ i n s l o ~ v )e. Lit.? nip 257-259'. J Inflection. CIlC13. 49 100 66 40
I11 \T
11
CUllIlxl
I
----~-------Infrared, OH Lactone Ketone
cm-1 Azo
A4
Ester
-
l ' o u m l , ~1% C H
73.94 74.61 77.46 77.48 73.93 75.60
8 . 61 8.46 8.49 9.21 8.44 8.34
Microanalxses are by 3Ir. C. E. 0.727; in acetotie. O.75$,; it1
Q
Cyclopropyl
18-Me
1Y-hIe
21-Ne
C-4
C-6
C-22
C-3
3440
1776 1653 1608 0.38 1.11 1 . 1 3 1 . 6 2 5 . 8 5 I1 1744 1665 1632 1618 0.95 1.19 2.15 5 . 4 4 5.87 111 3530,3480 1743 1634 0 . 9 4 1.02 2.15 538 5 . 8 7 3.50 I\' 1750 1632 1730, 1249 0.96 1.04 2 , l 5 5.43 5 . 8 8 4.54 v 1745 1658 1635 1604 0.46 1.00 1.26 2.17 5.66 5.87 'b Infrared determinations were made by 3Ir. E. Schoeb (.4llii Arbor) u h g a Beckmau 3lodel 111-9. KBr disks were used except for I1 which was run in CHCI, solution. b Nmr spectra were obtained by hIr. R. B. Scott (An11Arbor) usiiig a Varian A-60. Except for I, which was run in pyridine, solutions in CDC13 were used. Experimental Section
TABLE I11 PROPORTIONS OF REACT.LNT~ RlesSOI. Product
I
mmoles
5
NaH, mmoles
DMSO, ml
Steroid, mmoles
5 .3 5.6 5.3 4.5
90 4.75 100 5.6 100 5.3 4.5 150 4.3a \I 3 55 2.4b a ::p,l701-l)iacetoxyp~egn-5-eri-20-oiie, mp l74-177", from pyridine-acetic anhydride acetylatioii of 17a-acetoxypregiieriolG. I). Searle & Co., South African Patent 65/4327 oiie. (Feb 14, 1966).
I1 I11 IV
ones yielded the corresponding A20(22)-lactones:21701hydroxy-3-oxo-23-norchola-4,20(22)-dienicacid y-lactone (11),3@,17~t-dihydroxy-23-norchola-5,20(22)-dienic acid y-lactone (111), 3p-acetoxy-1701-hydroxy-23norchola-5,20(22)-dienic acid y-lactone (IV), and 17ahydroxy-3 -oxo-6- spirocyclopropyl-23-norchola-4,20(22)-dienic acid y-lactone (V), when run with sodium hydride in dimethyl sulfoxide. Our work has not permitted the assignment of configuration to the C-20 position. Compounds I and I1 were tested for biological activity, Compound I1 failed to prevent litters being born when fed to mice at 10 mg/kg/day. n'either compound antagonized 1 pg of aldosterone in the saltloaded rat at dose levels of 30-50 mg/kg. Seither compound showed any progestational effect in the MePhail assay in rabbits a t a dose level of 20 times progesterone by subcutaneous and 100 times norethindrone by oral administration. ( 2 ) H. G . Lehmann, Angew. Chem., 77, 808 (1965), has since reporied t h a t t h e reaction leads i o t h e unsaturated lactone (111) in t h e presence of equimolar amoiinrs of N a H while catalytic amounts of NaOH for short reaction times allou isolation of t h e hydroxylactone. Also N. H. Dyson, J. A. Edwards, a n d J. H. Fried. Tetrahedron Letters, 1841 (1966),have reported t h e conversion of 17u-acetoxypregna-4,6-diene-3,20-dionet o the corresponding unsaturated lactone by N a H in DMSO.
General Procedure.-Sodium hydride was added t o dimethyl sulfoxide (DMSO) under nitrogen aiid stirred a t room temperature for about 1 hr. A solution of trimethylsulfoxonium iodide in D l l S 0 was added and stirred for 15-30 min. The steroid was suspended in D3ISO and added in one portion. The mixture was then stirred overnight and worked up by pouring into ice-water, separating, and crystallizing. When trimethylsulfoxonium iodide wa5 not used, the remainder of the procedure was unchanged. I n two of the preparations, I1 and V, the reaction mixture wa< acidified with 3 S HC1 after being qiienched in ice-water. The reactant proportions are given in Table 111.
Acknowledgment.-The authors are indebted to Dr. 0. D. Bird, Dr. L. Blouin, and Dr. 11. R. Callantine for the pharmacological results presented.
The Synthesis of Some Aryl Nitrogen Mustard Derivatives of Estrogens' C. 11. WALK,^ T . C. CHOU,A N D HSI Hu L1x3 Department of Chemistry, Saint Joseph's College, Philadelphia, Pennsylvania Received August 3, 1966
The synthesis of several new steroidal compounds has been acconiplished from the corresponding intermediates 4-aminoestrone 3-methyl ether (I) and 2-aminoestrone 3-methyl ether (11). These mustards were prepared because the literature describes no previous attempts to study aryl nitrogen mustards of steroids and also because of their potential as anticancer agents. (1) This investigation was supported by t h e fund from the National Cancer Institute, National Institutes of Health, U. 9. Public Health Service, Bethesda, X l d . (Grant Ca-06492-03). (2) T h e d a t a published here are taken from a thesis submitted by Charles R. Walk in partial fulfillment for t h e degree of Master of Science. (3) T o whom inquiries should be addressed.
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.
