and mixture m.p. with authentic sample, 257') and or-ethylaminobutyric acid m.p. 248-250" dec. in good yields. n - [ 2 - (P-Hydroxyethyl)hydrazino]butyric Acid (XVI).----eBroniobntyric acid (33.4 g., 0.2 mole) was added gr:ttluiilly ivith cooling to a solution of 60.8 g. (0.8 mole) of p-hydros>.ethylhydrazine, h.p. 100-105" ( 1 mm.) in 100 nd. I I u~ x t c r , :ind t h r niisture alloived to stand a t rooni tenipernture for 5 d solution was passed through a Duolite C 20 column and eluted as usual. The eluate was concentrated t o 200 nil. and passed through an Amberlite I R C 50 column, the eiAuent evaporated to dryness in vacuo and the reFidue crystallized f r o m 300 ~ n l .of %-propanol,giving 4.4 g. (13.6%) of XYI, m.p. 133-134" dec. Reductive Cleavage with Raney Nickel. In Ethanol.--.$ suspension of 1.9 g. of XF'I in 50 inl. of ethmol :tnd 10 g. of Raney nickel catalyst was refluxed for 5 hr. in it reaction flask equipped with an ethanolic HC1 trap. The catalyst \vas filtered: the alcoholic liquors distilled into the ethanolic HCI, and thr latter evaporated t o dryness. The product (0.8 g.) was crystallized from a mixture of ethanol-ether ( 1 : 1 ), giving cthylaminciethanol hydrochloride, m.p. 225-230". A r ~ a l . Calcd. for CaHl&lNO: K, 11.21; CI, 28.51. Found: S , 11.50; C1, 28.20. The Raney nickel catalyst was combined with the alcohol residue, boiled with 150 ml. of water, and filtered. The green solution was diluted witti A00 rnl. l ; f water :rnd passed
tlirough an Amberlite IItC 50 column, the efnuent eva,porated to dryness :md the residue cryst:tllized from methanol, giving 0.4 g. of S-etti?-l:tniinobiityrii~ acid, n1.p. 24S-252' clev. (sub1iin:Ltiiin !. . I nnl. ( ' : r l ( ~ I . ioi. ( '61115S ) 2( : N , tO.08. I:ound: N,1O.W. I n Water. - - S\'I i 1 .(ig.) in 100 1111. ( i f water W:LS hydrogrnnt~etl i i i t h c ~presenc.r i l l ;i g. rif Raney nirkel cntttlyst at, atrriosphr~ric pwssiire. The fil(rat.e \vas n e u t d i z e d 11-itti8 ml. of lo(, Iiydroto dryness, the residue dissolved i n 50 id passed through an Amberlite IltC 50 colunin. The efiiuent, wis passed through a Duolite C 20 column :rnd eluted with 400 nil. of I, :tmnionia. ';, The eluate mas evitpor:itcd to dryness :ind the residue crystallized from IO0 ml. of q i i e o u s ethaxiol, giving 0.4 g. of ly-:briiixiobut,pric acid, III,[J. 298-300" dev. (sul,lixiiation). .\naZ. ( U d for C4HsN02: X, 13.58. Found: S , 13.25.
Acknowledgments.-The :iuthors wish to thank the Sloaii IG.ttcritig Iiistitute for C h c e r Itcsearch, S w 'L'orli. S. I-..Cor :L g1*:ilIt, :t11(1 ASS1:I Chemical I L L ~ or:itoric.s 1,ttl.. Israel. for m:tliiiig th&r 1al)or:ttorica :i\.ailnl)lc for this work. 'I'haiiks are also clur: to 1)I.. 1.:. uitl 111.. S . ( ' P I Y I ~ P Z for their hclp.
Phosphorylated Alkylating Agents Related to DL-Phenylalanine'
Life Sciences Reaetrrch, Stanford Reserrrcli Insfzfirte, . I f e n h I'cri b. ('trlifoi n z ( i
A group of alkylating agents has been prepared which use 3-(p-:~ininnphen~l)propionic acid antl 3-( n ~ -and paniinophenyl)-oI,-alanine as the carrier moiety for a numher of phosphorylated antl thiophosphorylated ethylenimines and bis(2-chloroetli~l)amines. llethods were devised for removing 1he blo~kinggroups used in these syntheses so that the alkylating groups remained int:ic.t. Severd of the thiophosphorylntccl rthylenimines were active antitumor agents acrording to testing results \\-it11\Tnllter 256 ~:ir(,iii[)s~r~iiiii:i.
A number of phosphorylated alkylating agent's, among which are 2- [bis(2-chloroethyl)-amiiio]-2111,3,2-0xazaphosphorinane2-oxide2 (Cytoxan) (I), tris(1-aziridiny1)phosphine oxide3 (TEPA) (II), and tris(1-aziridinyl) phosphine sulfide4 (thio-TEPA) (111) have been synthesized and used ~linically5~-" against cancer. It has been suggested5"zethat I and its analogs have latent activity until hydrolyzed by the phosphamidases, present in high concentrations in tumor tissue, to release the cytotoxic nitrogen mustard. A number of modifications of I, 11, and 111 have been synthesized with the phosphorylated alkylating groups attached to various carrier moieties in an attempt to obtain greater selectivity of antiturnor action. Because of the importance of nitrogen mustards of the phenylalkanoic acids (e.g., and of phenylI
(1) This work was carried o u t under the auspices of tlie Cancer Chernotherapy Xational Service Center, National Chnrer Institute. National Institutes of Health, Public Health Seri ice, Contract No. SA-43-pli-1892. The opinions expressed in this paper are those of the authors and not necessarily those of the Canrer Chemotherapy Xational Service Center. ( 2 ) H. .Irnold, F. I3ourseaux, and N. Brock, .Vature, 181, 931 (1958). (3) H. Bestian. Ann., 666, 210 (1950). (4) If. K u h and D. R. Seeper, P. S. Patent 2,670,347 (1954); Chem. Abstr., 49, 2481 (19%). (5) (a) T. B. Haddy, J. A . Wtiitaker, T. J . Yietti, and H. D. Riley, Jr., Cancer Chemofhernpy R e p t . , 26, 81 (1962); (b) L. R. Durall, i b i d . , 8, 156 Nasr,ibid., 13, 185 (1962): (d) 0. N. Friedman, E. 1'. Roper. Grnbliauskas, and H. Somer, J . M e d . Cliem.. 6 , 50 (1963); ( e ) I i . Arnold, I;. Honrseaiix, a n d M. Brock, il,.znei?nittP!-Forsch.. 11, 143 (1961). (6) J. I.. Everrtt, J. J. Roberts, and W . C. .I. Ross, J . C h e n . Soc.. 238ti (1953).
oJ ' 'N(CH~CH~CI), II, R = O
I
111, R = S
VI
alanine ( e . g . . sarcolysin),' it was considered of interest to use these aromatic moieties as carriers for the phosphorylated alkylating groups. This article describes the preparat'ioii and antitumor evaluation of a numbcv of such compounds typified by IT', T', and VI. Kiiunyants, r'i al..Rhave prepared examples of these cornpounds based 011 phenylacetic acid and phenylalaniiie in which cert'aiii of the functional groups ryere left in t'lw ])locked state. SYe we1.e interested in preparing compounds which possessed the free carboxyl and free arniiio acid fiinctional groups. ( i )I,. 1;. I,arionov, A. S. lilioklilov, E. Pi. Jhkodinskaia, 0. 9. Yasina V. I. Trrisheikina, and A . X. Novikova. L n n r e l . 269, 169 (1955). (8) I. L. Kniinywnts, 0. V. Kil'disherva, N. I:. Golubeva, and 8. Zurabyan, Uoki. ilkad. Xauk S S S H , 142, 370 (1962); Chem. Abstr., 66, 15604 11962).
JIarch, 1961
PHOSPHORYLATED
ALKYLATISG AGENTS RELATED TO
225
DL-PHENYL-4LANINE
CHARTI The phosphorylated ethyleniniine derivatives of phenylpropionic acid were first examined. The methyl and benzyl esters of p-aminophenylpropionic acid (VII and VII) (see Chart I for formulas) were treated successively with phosphoryl chloride and excess ethylenA = NHP(0) (N3)zB = NHP(S) (NJ), imine to yield the phosphorylated ethyleniniine esters, methyl and benzyl 3-{p- [bis(asiridinyl)phosphinylOC6H6 I aniinolphenyl ]propionate (IX and X). The experiC = NHqK(CHzCHzC1)2 L> = KHP(O)(SHCH2CHzCl)z mental modification of replacing a tertiary amine with ($ excess ethylenimine as hydrogen acceptor gave increased Phth = phthaloyl yields and purity in our preparation of IX and X, and Compound Ra also in the preparation of the known P,P-bis(1-aziri1-11 P-NK diny1)-E-(m-nitrophenyl)phosphinic amide (XI), TI11 p-NH2 which was used as a model. IX P-A It was anticipated and found that hydrogenolysis of x P-A the benzyl group in X simultaneously opened the ethylXI1 P-A enimine rings; indeed, the pure acid XI1 could not be XI11 P-A obtained in this manner. The sodium salt (XIII) XV P-D was prepared in high yield and purity by room temXT-I P-D perature hydrolysis of the methyl ester (IX) with one XVII P-B equivalent of base and a small amount of water in alXTIII P-B SIX m-NHz cohol. This salt, unlike the acid, appeared to be stable. XS m-A There is evidence that ethylenimines substituted on IT m-A the nitrogen atom with a phosphorus link are quite XXIII m-B sensitive to ring cleavage by acids, as are the unsub1m-B stituted or the S-sulfonyl substituted ethylenimines. lo XXIV p-SO2 Gaseous hydrogen chloride bubbled through a solution XXV p-xo2 of the phosphorylated ethylenimines a t room temperaXXTI p-NOz ture gave the corresponding ring-opened products, XXVII P-”Z r\’,E’-bis(2-~hloroethyl)-N”- (m-nitropheny1)phosphorXXT’III P-A amide (XIT.‘) and benzyl 3-{p - [bis(ZchloroethylXXIX P-C XXX amino)phosphinylamino ]phenyl )propionate (XY) . P-C XXXI The latter was not isolated, but was immediately hyP-C YXXIT P-C drogenolyzed to acid XVI. Grechkinll obtained similar VI P-C ring cleavage products upon reaction of equivalent amounts of dialkyl phosphorochloridates and ethylenimine with no added acid acceptor. The cleavage of the N-phosphorylated ethylenimines by acid may XI, R = A; XIV, R = D; X X X I I I , R C explain why some attempts on our part to prepaye and react P,P-bis(1-aziridiny1)phosphinic chloride in situ in alcohol containing some water were unsuccessful; with aromatic amines were not successful; some success the product obtained was the sodium phthalamate has been attained with aliphatic amines by Friedman, (XXI). Under somewhat more drastic conditions, the et a1.12 ethylenimine rings mere opened to give XXII. HowThe methods found suitable for the phenylpropionic acid derivatives were used to prepare the phosphorylated ethylenimine derivatives of DL-phenylalanine (XX and XXVIII). The respective starting materials were methyl 3-(m-aminophenyl)-N-phthaloyl-~~-ala0 nate (XIX), available from previous 1v0rk,13 and benzyl 3-(p-aminophenyl)-S-benzyloxycarbonyl-~~-alanate XXI, R = N 3 ; XXII, R = NHCH2CH,0H (XXVII). The latter was prepared particularly for ever, the use of 1 mole of hydrazine1* followed by the synthesis of the phosphorylated bis(2-chloroethy1)treatment with 1 mole of base, all a t room temperature, amino mustards that are discussed below. gave the sodium phenylalanate (IV). Attempts to remove completely the S-phthaloyl By replacing the phosphoryl chloride with thiophosand methyl ester groups from XX with 3 moles of base phoryl chloride, benzyl 3-(p-aminophenyl)propionate (1’111) was converted to the thiophosphoramidic (9) A. A . Kropacheva and V. A . Parshina, Z h . Obshch. Khzm., 29, 566 dichloride, which reacted with ethylenimine to give (1959); Chem. Abstr., 54, 472 (1960). (10) (a) J. S. Fruton, “Heterocyclic Compounds,” Vol. I, R. C. ElderXVII. When the methyl ester (T’II) was used, no field, Ed., John Wiley a n d Sons, Inc., New York, N. Y., 1950; (b) hoaever, crystalline product could be isolated. The benzyl Y. Tsuchiki, H. Wada. and Y. Suzuki [Japan Patent 918 (1960); Chem. Abstr., 64, 19539 (1960)l claimed the preparation of Z-(l-aziridinyImethy1)-3ester (XVII) could be hydrolyzed, under the same carboxycyclohexanone b y a Mannich reaction. conditions used for the oxygen analog, to the sodium (11) N. P. Grechkin. Izu. Akad. Nauk S S S R , Otdel. Khim. Nauk, 538 salt (XVIII). Attempts to acidify XVIII carefully to (1956). :
(12) 0. M. Friedman, R. S. Levi, Z. U. Papanastassiou, and W. hl. Whaley, J . Med. Chem., 6 , 449 (1963). (13) H. F. Gram, C. W. Mosher, and B. R. Baker, J . A m . Chem. Sac., 81, 3103 (1959).
(14) F. >I. Callahan. G. W. Anderson, R. Paul, and J. E. Zimmerman. ?bid., 86, 201 (1963): E . Taschner, C. Wasielewski, and J. F. Biernat. Ann. Chem., 646, 119 (1961).
226
\'01, i
hntituliior sc,reeniiig \V:LS perforiiieti under the auspiws of the Canc.rr C:lic~iiiotIier:il,~~ S:rticirial Serviw (:iintiBr :ic~c~rirding i(iits protooutlined in Cancer C'hernotherapg R e p l . , 25, 1 (1962). Froin this source are t,:iken the i*odetfesign:itiiins for ttic tumor syetc,ins: TV.4 for TValker 256 (subcutaneous): SA, Sarcoma 180; C.4, Adenocarcinonua 7%; LI$ ikenii:i 1'12 I O . ( + iridic-atcs g 111 ttw :tt)civc priitrrciil. ', hI1.11the cwnpound is a confirmed active ((3-4, SA, LE), or has therapeutic index (T.1.)2 4 ( product , 1~ec~ryst:rllizrd frmi ing points and yields are for analytical samples. Corresponding values in parenthesi water-ethanol. rl nnl. Found: CI, 11.6;N, 8.60. e Recrj-stallized from niet,hylene ~ ~ l i l o r i t l e - - d k e l l y13. s ~ ~ l ~13ccrystallized ~~~ frcitil absolute ethanol. Recrystallized from benzene. h Recrystallized from Skellysoiv . t u u / , Flllllld: s,1 1 . 1 . Isi~rlrilizeti f r o i i i ct 1ic.r :mtl :iir-ilriwI. I)r>.ing liztrd from benzene-ether; product of m.p. 164-165" was analyzed: Found: S , 10.1. iti m r i t o , either :it r w n i teinperature or :it)ove, gave :in oil. Recry iol. 'I' Ihlilitig p o i l i t . T h c 3 li'i valur~s :ire given for the appropriate solvent sj-steins listed in ref. 20. Iktc (*id
Q
form tlie free acid immediately gave a \vliitc polymer, iiisolu1)le in all organic solvents. In a similar way, treatmelit of methyl 3-(m-ainiiiopheiiyl)-~-phthaloylI)i,-alaiiate with thiophosphoryl chloride arid ethyleiiiniiiie gave XXIII. This was separable hy crystalliaatioii from the large amount of polymer formed iii the i.eactioii.15 Because the yields of XXIII were poorer than those obtained for any of the other phosphoryIated or thiophosphorylated ethylenimines, the reaction coiiditions for prepariiig X X I I I were carefully examiiicd. The. thiophosphorylatioii step was found to he romplete after 40 min. at reflux temperature and t q u i r e d a iiitrogeri atmosphere. The reaction with c%hylenimiiie proceeded best at about 20'; higher and lower temperatures were less satisfactory. Similar to the preparation of ITT, room temperature reaction of XXIIl with hydrazine, then with base, gave the sodium salt V.
111prepariiig pliospliorylatecl iiitiogeii mustards \vitli ~~~-pIienylalaiiiiie as a carrier, the required intermediate 11 as Ijeiiayl :~-(p-aminopl~enyl)-S-(t~erizyloz;ycarl~onyl)i)L-alaiiate (sx\'II). It contained thc free aiomatk niiiiiio aiid tjlockiiig groups tliat are reiiiova\)le after tlie alkylatiiig gioup is attached. Ifi 111 thc ayntlie& of tlie blocked amine (XXYII), the key step was the iurcrssful chemical reductioii of the hlocked nitro precursor (XXJ7) Itacernic p-iiiti oplieiiylalaiiiiieli was esterifid \\ i t l i heiieyl alcohol 1'. to give beiiayl ester XXIT'. 'l'liiz \\ acoiivcnrted ( a i \ cwtioiiallp t o bciizyl N-t)cliqlosp\.c.allmiiyl-8-(p-iiitropheii yl) -1,L-alatiate (XXV). 1The1I t I ie ~~eiiaylosycarboiiyl acid (XXVI) was prepared first stiicl tlieii esterified. thc over-all yields of XXI' were pourer. Refluxing nitro estcr XXV with ziiic dust and am-
PHOSPHORYLATED ALKYLATING A G E X TRELATED ~ TO DL-PHESYLALASISE
March. 1964
monium chloride in aqueous methanol gave the amino ester (XXVII) in good yield. The selective reduction of the nitro group in XXV makes an interesting comparison with 2-amino-4-benzyloxyamino-5-nitro-6methyl~yrimidine,'~ where the nitro group could not be reduced without concomitant loss of the benzyloxy group. Both the methyl and the benzyl esters (T'II and VIII), and the benzyl alanate (XXVII) reacted with phenyl N,?;-bis(2-chloroethyl)phosphoramidic chloridela (XXXVII) to give blocked mustards XXIX, XXX, and XXXII, respectively. Preparation of other phosphorylated mustards attached to phenylalanine was considered. Two intermediates, r\l',S-bis(2-chloroethyl)phenylphosphonamidic chloride (XXXIV) and K,K-bis(2-chloroethyl)phenylthiophosphonamidic chloride (XXXV) were prepared. However, both of these gave unattractive oils when condensed with esters of 3-(paminophenyl)propionic acid. Oils were also obtained in similar experiments with methyl S,?;-bis(2-~hloroethyl)phosRi
I (CICH2CHl)JPCl
X X X V I I R;
=
OCsHA, Ri = 0
phoramidic chloride (XXXTrI), which was prepared and used in situ. Antitumor screening of the phosphorylated alkylating agents were performed under the auspices of the C.C.N.S.C. The results are summarized in Table I. The phosphorothioamides V, XVII, and XVIIT were active against Kalker 256, but the corresponding oxygen analogs were not. Of these, V was the most active and had a therapeutic index of over 20.
Experimental20 Benzyl 3 4 pAminopheny1)propionate (VIII) Hydrochloride.By the method of Shields, et d , ' *the reaction of 33.0 g. (0.20 mole) of 3-(p-aminophenyl)propionic acid, 38.0 g. (0.21 mole) of p-toluenesulfonic acid monohydrate, and 150 ml. of benzyl alcohol in 500 ml. of carbon tetrachloride for 18 hr. gave the toluenesulfonate salt of the ester which was converted in 869% yield to the benzyl ester hydrochloride, m.p. 130-132', of purity suitable for use in the next step. An analytical sample of the benzyl ester hydrochloride, obtained from an earlier run, had m.p. 132-134'; ':::i 3.85, 5.10 ( N H $ + ) ,and5.71 ( C = O ) p ; R f 0 . l i i n s o l v e n t A, Rr0.85inB. Anal. Calcd. for ClsH17N02~HCl: C, 6.59; H, 6.23; S, 4.81. Found: C,6.61: H,6.52; n',4.90. Benzyl 3-{ p-[Bis( 1-aziridinyl)phosphinylamino]phenyl )propionate (X).-A mixture of 9.60 g. (32.9 mmoles) of benzyl 2-(paminopheny1)propionate (1.111) hydrochloride and 50 ml. of phosphoryl chloride was heated a t reflux for 30 min. The reW.Barton,J. O r y . Cheni. 24, 127 ( l Y 5 Y ) . ( 2 0 ) hleeting points, corrected, were determined with the Fisher-Jolins apparatus. .inhydrous magnesium sulfate was used a s the drying agent. Celite is a T h e solvent Ykellysolve 13 is essentially hexane, b.1,. 60-68'. diatomaceous e a r t h product of Johns-,llanville. Paper chroriiatoyrams were run by the descending technique on ivliatnian N o . 1 paper and the s p o t s were detected yisually under ultrayiolet light unless otheraise indicated. T h e sol\ent systerns ufied were: .i,benzene-methanol-water ( 2 :6:1) on acetylated paper (Schleiclier a n d Sclluell No. 2496) ; 13, l-hutanolwater (saturated); C , solvent .I with \Thatman No. 1 paper; D. 5% disodium liydroneri pliosphate, pH 8.9; E , I-butanol-acetic acid-water ( 5 : 2 : 3 ) ; F, 2-propanol-2 .\' Iiydrocliloric acid (65:35). (19) E. C . Taylor a n d J.
227
sultant solution was evaporated to dryness a t 40' ( 1 rnni.). The residue was taken up in 150 ml. of methylene chloride and added dropwise over 60 min. t,o a cooled (-5 t o O"), stirred solution of 50 nil. (0.96 mole) of ethylenimine in 200 nil. of methylene chloride. After the addition was completed, the bath was removed, and the reaction mixture was allowed t o stir an additional 60 min. I t was washed successively with three 200-ml. portions of water, 200 nil. of 0.01 S hydrochloric acid, 100 ml. of saturated aqueous sodium bicarbonate, and 100 ml. of water, and finally dried. Evaporation of the solvent in V U C U O left 12.2 g. (967;) of white, crystalline X, m.p. 109-11O0. Recrystallization from absolute ethanol gave a n analytical sample of X, n1.p. 112.5-113.0°; i p ' :' 3.19 ( S - H ) , 5.81 (C=O), 7.95 (P-O), 10.32, and 10.72 (aziridinyl) p ; for paper chroniatography and ana tical results see Table I. CAUTION: 1-igorous acid-catal" ed polymerization of the ethylenimine is possible and the temperature must be carefully watched. For larger scale runs, it is recommended that a tertiary amine such as triethylamine be added as an acid accept,or. Compounds I X , X I , 9 XX, and XY\7II were prepared similarly. For the preparation of XTTI and X X I I I , thiophosphoryl chloride was used instead of phosphoryl chloride. Sodium 3-(p-[Bis( 1-aziridinyl)phosphinylamino]phenyl)propionate (XIII).--A solution of 0.93 g. (3.0 mmoles) of the methyl ester ( I X ) , 3.0 ml. of N sodium methoxide in methanol, 0.10 ml. (5.6 mmoles) of water, and 10 nil. of methanol was kept a t room temperature for 3 days and then evaporated t o dryness in CUCZLO below 35' (0.1 mm.); with the amino acid derivatives, this temperature is critical. The residue was triturated with hot methylene chloride (in some cases, acetone) and dried in zucuo a t 56" for 3 hr. to afford 0.91 g. (96%) of XI11 (.0.5HzO) as a white solid, which did not melt below 300";' : : ;A 3.01, 3.18 (X-H, H20); 6.30, 7.12 (CO2-); 7.95,8.32 (P=O); 12.05 p . The paper chromatography and ana1,ytical results are given in Table I . Bn aqueous solution of XI11 was treated with a slight excess of acetic acid and the solution was extracted with ethyl acetate to give an oil which had the infrared spectrum expected of acid X I I . This oil did not crystallize; after a few days, it was no longer soluble in ethyl acetate, suggesting that polymerization had taken place. I n a similar way, X\?II was prepared from X \ X , and an attempt was made to convert the salt to the acid. I n this case, acidification immediately gave a white precipitate which was insoluble in wat,er, ethyl acetate, alcohol, and other solvents. From the ethj-1 acetate, a small amount of solid with a broad melting range was isolated. I n a few days, this solid was no longer soluble in ethyl acetate or any other solvent. N,N'-Bis( 2-chloroethyl)-N"-( m-nitropheny1)phosphoramide (XIV).-.4 solution of 2.00 g. (7.52 mmoles) of bisaziridine X I in 25 nil. of methylene chloride was saturated with anhydrous hydrogen chloride, diluted u-ith 25 ml. of benzene, evaporated in v a c ~ uto ~ cn. 20 nil., and chilled. The yellow crystals were collected and dried to afford 1.10 g. (4251) of XI\-, m.p. 89.590.0". ,4portion was recrystallized from benzene to afford X I 7 , m.p. 8'3.5-90.0";' : : , :A 2.95, 3.10, 3.19 ( X H ) ; 8.40, 8.52, and 8.67 ( P - 4 ) p ; see Table I for analytical data. 3- { p - IBis(2-chloroethylamino)phosphinylamino] phenyl )propionic Acid XV1.-A solution of 2.60 g. of the bisaziridinyl benzyl ester ( X ) was treated with hydrogen chloride, by the procedure used to prepare XIT- to obtain 3.20 g. (1027;) of a yellow oil which could not be crystallized. This oil was dissolved in 50 ml. of ethanol and hydrogenat,ed over 1.0 g. of 5%; palladium-oncharcoal a t 3.3 kg./cm.Z for 4 hr., then worked up in the usual way t o give 2.30 g. (92%) of acid 91-1, n1.p. 155-157'. One recrystallization from ethanol-water (1: 1) gave an analytiral sample as white needles, n1.p. 161.5-162'; Ai:' 2.98, 3.05 ( N H ) , 3.7-4.2 and 10.4 (COOH), 5.86 (C=O), 7.70, and 7.90 (P=O and C02H) p : for analytical data see Table I. Sodium 3-(m- [Bis(1-aziridinyl)phosphinylamino]phenyl ]-DLalanate (IV).-A solution of 4.54 g. (10 mmoles) of methyl 3iridinyl )phospliinylaniino]phenyl ) - S-phthaloyl- DL), 0.51 g. (10 mnioles) of hydrazine hydrate, and 20 nil. of absolute methanol was stirred a t room temperature for 4 days, then evaporated t o dryness in zacuo. The residue was extracted with 50 nil. of methylene chloride and filtered to remove 1.50 g. (937:,) of the hydrazide. The filtrate was evaporated t o dryness to leave 2.87 g. (89';;) o f the methyl :%lamiteas a pale yell(iw oil. This was dissolved in 25 ml. of methanol, treated with 9.0 rnl. (9.0 mmoles) of methanolir ,\'*sodium methoxide and 0.2 ml. of wat,er, allowed to stand for 20 hr. a t room temperature,
:inti ev:rporated t o dryness in ~'aciio. Tlie w h i t e residue m s triturated with 100 ml. of h i l i n g acetone and dried in 2 to leave 2.52 g. (837:) O f analytically pure I\-, See Table
:tnalyt,ical details. I n a similar way, \' was prepared f r ~ i l lS i SIII. Disodium N-( m-( [Bis(1-aziridinyl)phosphinyl]amino)-a-carboxyphenethylJphthalamate (XXl).-A solution of 9.1 g. (20 rnmoles) of the phthaliniido ester ( X S ) , 45.0 nil. of .\- sodium Inethoside in methanol (45 mmoles), 1 nil. of water, and 200 n i l . of nietha,nol was stirred at room teniperature for 4 days and evaporated to dryness in 2'aciio. 'The residue was triturated successively with 100 nil. of 95C; ethanol arid 200 nil. of boiling rsthancrl and dried in Z ' U C I I O a t 100" (0.5 nini.) t o :tH'ortl 9.50 ( (i.6lj, 7.42 (SOn), S.30, : !J.40 (ItS03C) p.
-
Treatment of the tc~1uenesulfon:ites d t with sodiuni hydroxide XSIT-, horiiogenous Irn p:iper, IZr 0.14 in A , 0.70 . . in 13, and 0.!)4 in 1,'. 3-1ii-l\iitroDhenvlbN-benzvloxvcarbonvI-uL-alanine ( XXVI ). . --The 'reaction of 2.0 g. (9.6 nimoles) of 3-(]~-nitrophenyl)-uL:rlaniiie with 1.71 g. (10.0 nimoles) of Irenz~lc~r;yi~arbc,n!.l chloritle t ) y the usu:tl rnethod gave, after c~rystallization frirni ct'her-g . (14' ) of white, rryst:tlline product, ni.p. 3.04 ( - S H ) , 3.6.5-4.20 f COOH), 5.90 (C:==C) 1. , NOP),and 7.41 ( X O y ) p ;li'r 0.31 in 13, 0.53 iii I ) . .l?//[l. C':tI(.d. for CI~HlJ'gO~: ( ' , 3!J.i;; H,4.6h: S , S.1;;. Found: C, 59.:3: H, 4.80; S , 8.22. Benzyl 3-(p-Nitrophenyl)-N-benzyloxycarbonyl-o-a~anate (,XXV).-A solution of 30.1 g. (.177 mole) of benzylo. cahloride in 80 nil. of ether was added dropwise over 30 min. to :I c ~ ) l d(10--15') stirred solution of 30.0 g. (0.100 mole) of the benzyl :tlan:ite SNI\.:ind 38 nil. (0.47 mole) of pyridine in 1.1 I. of ether. 'Ttic, re:tcation inist~lrewas stirred :it r o i i n i teniperature for :in :ctidition:11 min., then t:strac,ted siic,c.(Gvel>.H it11 800-1111. 1)ortions o f water, 3' liydroc~hloric~:ic*itl, s:itnruted :rqueous i)ic,:irhirinte, :tnd watrr. Thc txt her l:i!.c.r \\-:is dried and ev:ip~ > r : i t ( di n 7 ~ ~ 1 ~ 1 t1o0 le:ivc. :t yel11)w 11i1irhich crystallized. This n : i s tritur:cted with 50 nil. of I : 1 etlier-pctroleuiii ether, t 3 . p . : N - ( i o o , to I f % W 30.0 g. (ssc; ) of g:Lve free base
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-
I
:,
'
3.00 (XTI), 5 . i f i .05 in A , :ind ().!)I in 13. (')
(i(i.3;
H , 5.11: N, (j.l(i.
Benzyl 3-(p-Aminopheny1)-N-benzyloxycarbonyl-o~-alanine [XiVII).-A
SS\-,2.40
niisture of 9.30 g. (21.4 mmoles) of the p-nitro ester g. of ariinionirrrn chloride, 18.5 nil. of witer, 190 wil.
1iiet1i:inol~;tnd 14.0 g. (0.214 ~nole)of zinc d u e t , adiletl in this order, 1r:is stirred and heated at reflux on ~1 steam bath for 70 miri.
of
