Synthesis of N-Phosphorylated Derivatives of Nitrogen Mustards with Latent Cytotoxicity'
The phosphoramide nitrogen niiietards reported iiiclude series of phosphoro-trianiides, diainidic esters, amidic diesters, diamidic acids, ester amidic acids and sZ/t,1-i)Srophosphoro~iai~iidic diesters that were synthesized because of their possible selective action against tumors. Some of t'hese compounds already tested in experimental animals have shown significant antitumor activity.
some have produced significant inhibition of the growth of tumors.8 -2 preliminary report of the rebults obtained a t the Children's Cancer Researclh F'oundachemotherapy since compounds of this type were inition, Inc., and the CCXSC Screening Program is given tially synthesized2 because of their possible selectirity in a later section. A structurally related cyclic pho+ of action against malignant tumors. These comphoramide ester of the Same type, cyclophosphamitlc>, pounds are non-toxic precursors from which cytotoxic , ~ proven to be an extramustards would be liberated by enzymatic h y d r ~ l y s i s . ~ prepared by Arnold et ~ l . has ordinarily activo inhibitor of many experimental 13y the action of phosphamidases that are abundant in tumors in animals; and although much less dramatic neoplastic cells, for example, a greater concentration in its effects against human cancer it is a useful drug of a nitrogen mustard given in this inactive phosphorylthat has been studied very midely in the clinic. P h o ated form could he localized in the cells of a malignant phamidase has bcen presumed to be involved in the a c t tumor than would reach them by the administration vation of this compound although the exact mechanism of a tolerated dose of the parent mustard. of action still remains to be elucidated. I n :i rtwnt Recent work in this laboratory has provided evidencr study10evidence was found that indicated that ( and pyrofor the existence of phosphom~noamidase~ phosphamide undergoes activation in the liver of ratphosphamidase6 as discrete enzymes; and it is probable since extracts of liver of animals pretreated n ith thv from the results of Gomori and ~ t h e r s ~that ~ - mam~ drug are significantly cytotoxic to tumor cells groir ing malian tissues also contain an enzyme, phosphodiin culture whereas the parent drug prior to biologid amidase, specific for phosphorodiamidic acids. h activation show> no cytotoxicity to the same cells in study of the former two enzymes in rat and mouse vitro. This metabolic intermediate which is presumably tissues has been made and some preliminary informathe active form of the drug has not been identified ation is available on the enzyme content of normal yet. It map possibly be either the phosphoramidico and malignant human tissues.6 There have also lieen acid ester or the phosphorodiamidic acid XXXT'II reports of high concentrations of the phosphodiamidase if the metabolism of this drug can be represented as a These enzymes are of a in human simple one-step hydrolytic process. The synthesis of type that could possibly "activate" phosphoramide the latter, cytoxyl alcohol, is included among the commustards with the appropriate structural requirements. pounds reporbed here. Certain of the compounds, the syntheses of which arc' I n thth present papcr Tye report the syntheses of a reported here, that havr been studied in biological number of n m phosphoramide mustards, some of systcms have shown various degrees of cytotoxicity which arc deriwd from bis-(0-chloroethy1)-amineagainst tumor and bacterial cells grom-n in culture; (norHS2) and others from the more potent secondary and of those studied to date in experimental animals ,3-chloroethylaminesiia that had been developed pre(1) Supported in part by a research grant (CY-2130), a Cancrr Cheriioviously in relation to this program in cancer rhemotlirrapy National Service Centre research contract (SA-43-ph-307.5) f r o n i thcmpy. Thew tlerivatives include both aliphatic, t i l e National Cancer Institute, National Institutes of Healtli. U. 8 . Piiblic aromatic and c-yclic phosphorodiamidates, phosphoroIir~altliService; a n d a n Institutional American Cancer Society Grant, 1 S 2%(-, triamidates, phosphorodiimidic monoesters, phosphor12) 0 . h i . Friedman and .\. SI. Seligtiian, J . - t m . Chem. So,,., 76, 6.55 amidic diester\ a.: well as various phosphorodiamidic il924). acids and phosphoramidir acid monoesters. Thew . Seligman, hI. 31. S a c h l a s , I.. If. llanheiiner, 0 . 31. Frietiiunn f , Ann. Suro., 130, 333 (1949). compounds are listed in Tables I-T'II. . Rutenburg, L. Pprsky, 0. 31. Friedman and A . AI. Seligman, J . The cyclic phosphoramide mustard derivatives 11, I'harmacol. E x p t l . Therap., 3, 483 (1954). (A) To be published: see 0. Lf. Friedman a n d E. Bower, Abstr. 139th 111, IV, T'I and TI11 listed in Table I were prepared .$.C;.S. Meeting, St. Louis, N o . , hiarch 1961, pp. 26-C. by the same general method that had been used earlier.2 (6) S e e 0. M. Friedman, S. Yciiiclior and E. Boger, Pioc. A m . S o r . Canrrr The known dichlorophosphoramides were condensed I ( P Y . , 3, 320 (1962). ( 7 ) f:ij E. ~ ~ a l d s c i i m i i l t - L e i tBiocliem. ~, Z t s c h r . , 267-268, :JGO il983); with the appropriate propylenediamine, propylenediol S-Phosphorylated derivatives of nitrogen mustards
of the type reported here have been of interest in cancer
(IJ)11. Ictiiiiara, ,I. B i o c h e m . , J a p a n , 18, 87 (1983); (c) i;, Goniuri. / ' r o c . Sei,. E x p . Biol. & .\led., 69, 107 (1948); (d) .J. 3Ieyer and .I. 1'. \Teinnian, J . Histochem. & C y f o c h e m . , 5 , 364 (1957); ( e ) .J. 3Ieyer and J. 1'. Wf.inrnan, J . Hisfochem. B C y f o c h e m . , 1, 305 ( 1 9 3 ) ; and J . Denf. R e s . , 32, 669 (19x3): ( f ) P. Sepp, S t r a h l e n t i i e m p i e , 99, 393 (1956); ( 9 ) H. IIolter and Si-Oh Li, Cumpt. rend. trar. l a b . Carlsbarg Ser. Chim., 293 (1951):
( h ) C . E. Perlman, "Phosphorus Membolism." Vol. 11, ed. W. I). lIcElro? and B. Glass, T h e Johns Hopkins Press, 1952, p. 167; (i) H. Bredereck and 2. phyaiol. Chem., 264, 223 (1938): (j) E. H. Screcker, Deut. Meled. . , 74, 126R il9,;l); 1k) 1.. \ , Twitlin. Riokhinii!,n, 17, 208 (19521.
( 8 ) 1Iii'se studies tiax e k e n done and are being continued by l l r G r o r g e 1: I olev, Dr Charlotte L JIaddoch and Dr Alfred H. Handler and their associates at tlie Children s Cancer Research Foundation, l n c Bo\tori Maus Tht. biological data uiil be publ~shedin detail elsewhere % ) If 4rnold I' Bourseaux and N Brock . I a l u r e 181, 131 11958) 110) L E, Folrj B I' Ilrolet ,ind 0 SI Friedman, Cancer Hes , 21, i 7 (1961) (111 (a) 0 AI Friedman and A LI Belipman, J . A m . C h e n S o c , 7 6 , 638 (Iq5.t) (1)) 0 \I Friedman and F R o w r , obnd , 78, 4659 (1956)
January, 1963
N-PHOSPHORYLATED NITROGEN MUSTARDS
or propanolamine in the presence of two moles of base. The base generally used mas triethylamine although in the case of the triamide 11, the use of excess of propylenediamine as the base gave much better results. This series includes cyclophosphamide (IV) the preparation of which had been reported earlier,g although the details of synthesis have not been given. This compound was isolated as the monohydrate which was also the form in which the two nitrogen mustard analogs VI and VI11 were obtained. TABLEI CYCLIC PHOSPHORAMIDE MUSTARDS
so.
I1 111 IV
X
R
(ClCH&Hg):X(ClCHjCH2)2X( CICH:CH2)?X-
Y
-KH-0-XH-
-KH-
-XH-
-0-
-NH-
-0-
-0-0-
CHiCl
VI
c‘“7 CH&l
CHICl
t“i
c1
VI11
CHzCI
CHzCI
The phosphorotriamidate mustard derivatives XVIII, X I X and XX listed in Table I1 were also prepared from the dichlorophosphoramide of norHS2 by the stepwise replacement of chlorine through condensation with the appropriate amines in sequence, a procedure again analogous to that reported earlier.2 Two of the compounds, XVIII and X I X , mere prepared by way of the chloroaziridinyl intermediate XVII. TABLE I1 PHOSPHOROTRIBMIDATE hfUSTARD5
0 R’
R-P
II / R‘
xx
( ClCH?CH2)*N-
d
-NH@
The phosphorodiamidates XXI through XXXIV, XLVII, L and LI listed in Table I11 were prepared by sequence of reactions analogous to those above by way of the intermediate chlorophosphoramide esters formed by condensation of the particular dichlorophosphoramide with the appropriate carbinol or phenol. The very useful although unstable intermediate chlorobenxylphosphoramide from which compounds X X I through XXVI, L and LI were derived was obtained by condensation of the dichlorophosphoramide of norHN2 with one mole of sodium benzyloxide. The indicated products were formed by condensation of these chloroesters with the various amines used in
51
excess or with one mole of the particular amine in the presence of triethylamine. The phosphoramidic mustard diesters XXXV and XXXVI listed in Tahle I Y were prepared by sequential replacement of chlorine atoms by the groups indicated. The phosphorodiamidic acid mustards XXXYII through XLIII, LII and LIII were prepared by hydrogenolysis of either the phenyl or benzyl ester precursors. The phosphoramidic acid monoester XLV was prepared by an alternate method in which the chlorophosphoramide phenyl ester mas condensed with triethylammonium acetate to give theacetylphosphoramide fromwhich the product was obtained on treatment with cyclohexylamine. Compound XLV had been prepared previously by selective hydrogenolysis of the benzylphenyl diester with palladium.2 The symmetrical diphenyl XLT’III and dibenzyl XLIX, pyrophosphodiamide derivatives of norHX2 illustrated in Table VI1 were prepared by treatment of the intermediate chloroesters with small amounts of mater in the presence of a base such as lutidine or triethylamine. Biological Data.-The results of biological testing of some of these compounds a t the Cancer Chemotherapy National Service Centre, S I H , are summarized in Table VIII. The seven compounds listed are structurally related in the sense that they are either phosphorodiamides containing the propanolamino grouping or they are isologs of cyclophosphamide. Although the tumor system used here, L1210 leukemia in BDF, mice, is generally responsive to the more “active” alkylating agents, it is possibly significant that only cyclophosphamide (IT) and cytoxyl alcohol (XXXVII) (a primary hydrolytic product that would derive from cyclophosphamide), showed substantial activity, whereas the other compounds which are structural variants of these two were completely inactive. ,4summary of some preliminary data from the primary screening program a t the Children’s Cancer Research Foundation, Boston, on ten of these compounds shown in Table IX essentially corroborates the CCNSC results with regard to the L1210 leukemia system. These results indicate, in addition, that four of the compounds including cyclophosphamide (IT’) the phosphorotriamidate mustard XVIII and the two phosphorodiamidic acid mustards XXXVII and XXXVI11 are very active inhibitors of the other three tumor systems generally. An assessment of structure-activity relationships in this series may be possible when the results of testing of several of the other compounds now in progress become available. Cyclophosphamide, IV, already has undergone very extensive clinical study. The other three, XVIII, XXXVII and XXXVIII, are in the process of preclinical pharmacologic evaluation a t the Children’s Cancer Research Foundation, Inc. Experimentall2 The reagents and solvents used in these preparations including dioxane, benzene, triethylamine and propanolamine were dried carefully and distilled just prior t o use. 1,3,2-Diazaphosphorine, 2-[Bis(2-~hloroethyl)amino]pentahydro-, 2-Oxide (II).-A solution of 3 g. of the dichlorophosphoramide (I) in 30 ml. of dioxane was added dropwise t o a stirred (12) Microanalyses by Dr. Carol Fitz. Needham. Mass.; points are uncorrected.
all melting
TABLEI11 P H O S I ' H O R O D I A ~ ~ I D A T E?rfUSTARDS
0
R'
R--1' No.
XXI
XXII ?(XI11 SXIY SXV XXVI
- >:
A
,A'
I, T,I XXVII
XXVIII
XXIX
XXX XXXI XXXII XXXIII
XXXIV
XLVII
KO.
XXXV
XXXVI solution of 0.87 ml. of l&diaminopropane and 4.5 ml. of triethylamine in 15 ml. of dioxane. Stirring was continued for an additional hr. and the mixture was stored a t 4" overnight. After removal of the precipitated triethylamine hydrochloride, the dioxane was evaporated, leaving an oil which was taken up in acetone-ether (1:2). Filtration and evaporation of the solvent gave 1.6 g. ( 5 3 7 3 of n-hite crystalline product, m.p. 106-107". Recrystallization from acetone-petroleum ether raised the m.p. to 109-110". Anal. Calcd. for C ~ H I ~ P C ~ Z NC, & 32.31; : H, 6.15; N, 16.15; P, 11.92; C1, 27.31. Found: C, 32.65; H, 6.17; N, 16.22; P, 11.86; C1, 27.60. 2H-1,3,2-Dioxaphosphorine, 2- [Bis(2-chloroethyl)amino]trihydro-, 2-Oxide (III).-A solution of 14 g. of the dichlorophosphoramide I in 50 ml. of dioxane was added dropwise over a period of 1.5 hr. to a solution of 4.5 ml. of 1,3-dihydroxypropanc and 20.5 ml. of triethylamine in 100 ml. of dioxane. Reflux was maintained during and for 2 hr. after addition. After standing
overnight the precipitated triethylamine hydrochloride was rcmoved by filtration. The dioxane was evaporated leaving an oil which was taken up in benzene, washed 4 times with water, dried and evaporated. The residual oil on refrigeration yielded 7.8 g. (55YG)of product as a thick semi-crystalline syrup. Anal. Calcd. for C1Hl4C12N03: C, 32.06; H, 5.34; N, 5.34: P, 11.83; C1, 27.10. Found: C, 31.73; H, 5.65; S, 5.43; l', 11.75; C1, 27.0. 2H-1,3,2-0xazaphosphorine, 2- [Bis(2-~hloroethyl)amino] tetrahydro-, 2-Oxide (IV).-A solution of 0.3 ml. of propanolamine and 1.5 ml. of triethylamine in 4 ml. of dioxane was added dropwise to a stirred solution of 1 g. of the dichlorophosphornmidc ( 1 , in 10 ml. of dioxane. After standing overnight the precipitatrd triethylamine hydrochloride was removed by filtration. T l w ctioxaiie was evaporated leaving an oily residue which on triturati?n 'i\ it?i petroleum ether-benzene (3: 1) solution with which a crystal of ice \vas added yielded 0.55 g. (55.5%) of white crystalline prodnct,, m.p. 42-45'. This material was identical v i t h
January, 1963
N-PHOSPHORYLATED NITROGEN MUSTARDS
53
anhydrously over a period of 1 hr. to a solution of 25.9 g. of the dichlorophosphoramide (I)in 100 ml. of dry benzene with stirring and cooling in ice. After an additional 2 hr. of stirring, the sodium chloride was filtered off in a large Buchner funnel. This process was accelerated by the addition of 200 ml. of ether to R-P the mixture in the Biichner. The filtrate was evaporated and the ‘OH.NH, residual oil was taken up in ether and treated with 10 g. of a 507, Nuchar-Norit mixture. Evaporation of the filtrate afforded 24 NO. R R‘ g. (72%) of oil which was stored in solid carbon dioxide, ~ Z D 1.5300. XXXVII (ClCH?CH?)TX-XH( CH?)30H Anal. Calcd. for CI1Hl&l3NO2: N, 4.24; C1, 32.2. Found: XXXVIII (ClCH&H*)?X-NHz N, 4.2; C1, 32.4. XXXIX ( ClCH>CHi)INBenzyl-N,N’-di-(2-chloroethy1)-”,”-ethylene Phosphorodiu amidate (XXIII).-To a solution of 0.99 g. of the benzyl phosphoramidic chloride (X) in 15 ml. of dry benzene was added quickly XL ( C1CH~CHZ)TX0.63 ml. of triethylamine, and 0.155 ml. of ethylenimine was added dropwise to the mixture with stirring and ice cooling. The reaction was allowed to proceed t o completion by stirring for an XLI ( ClCH&H>)zXadditional hr. and then stored a t 5’ overnight. The precipitated triethylamine hydrochloride was filtered off and washed with benzene After treatment with Norit the filtrate was evaporated XLII a t reduced pressure, leaving 0.8 g. (787,) of product as a light oil, n Z 51.5305. ~ XLIII ( ClCH&H?)*N-Nnh Anal. Calcd. for C I ~ H ~ ~ C ~ Z O C, ~ N46.29; ~ P : H, 5.64; N, 8.3; v C1, 21.07. Found: C, 46.45; H, 5.76; N, 8.2; C1, 20.90. Benzyl-N,N-di-(2-chloroethyl)-N’,N’-tetramethylene Phos( ClCH&H?)?NLII phorodiamidate (XXIV).-This compound was prepared in essen0.5 tially the same manner as (XXIII). Reaction of 3.3 g. of the benzyl chlorophosphoramide (X), 1.4 ml. of triethylamine and -NnN--CH2CsHs LIII ( ClCH?CH*)&u 0.835 ml. of pyrrolidine yielded 2.27 g. (62%) of oily product. Anal. Calcd. for C15H28C12N202P: N, 7.67; C1, 19.45. Found: N, 7.60; C1, 19.6. TABLEVI Benzyl-N,N-di-(2-chloroethyl)-N’,N’-pentamethylene PhosPHOSPHORANIDIC ACID ESTERMUSTARDS phorodiamidate (XXV).-A solution of 1.98 ml. of piperidine in 30 ml. of dry benzene was added dropwise to a stirred solution of 3.3 g. of the benzyl phosphoramidic chloride (X) in 30 ml. of dry benzene. The reaction was allowed to proceed to completion by stirring for an additional 30 min. and then was stored a t 5” overnight. The precipitated piperidine hydrochloride was filtered off and washed with benzene. The combined filtrate was No. R R‘ evaporated a t reduced pressure and the residual oil dissolved in XLV (ClCHzCH?)2X-CeH5 ethyl ether and treated with 2 g. of a 507, Kuchar-Norit mixture. Evaporation of the filtrate afforded 2.7 g. (657’) of product as an oil. respect to m.p., mixture m.p., and infrared spectrum with a samAnal. Calcd. for C1eH26C12N202P: N, 7.38; C1, 18.73. Found: ple of the compound kindly supplied by the CCNSC, National x, 7.4; c1, 18.9. Institutes of Health, USPHS. Benzyl-N,N-di- (2-chloroethyl)-N’,N’- (2,2-diethyleneoxido) 2H-1,3,2-0xazaphosphorine, 2- [N-2-Chloroethyl-N-5-chloroPhosphorodiamidate (XXVI).-This compound was prepared in (1-chloromethyl)pentylamino] tetrahydro-, 2-Oxide (VI).-A soessentially the same manner as (XXV). Reaction of 1.74 ml. of lution of 4 ml. of propanolamine and 12 ml. of triethylamine in 20 morpholine and 3.3 g. of (X) yielded 2.85 g. (74%) of oily product. ml. of dry benzene and 10 ml. of chloroform was added dropwise Anal. Calcd. for C1SH23C12?J203P: N,7.35; C1, 18.63. Found: to a stirred solution of 12.6 g. of the dichlorophosphoramide (V) N, 7.3; c1, 18.80. in 50 ml. of dry benzene. Stirring was continued for an additional 1,4-Bis- [benzyl-N,N-di- (2-~hloroethyl)phosphorodiamidate] 1.5 hr. ilfter standing overnight a t room temperature the piperazine (L).-This compound was also prepared in essentially precipitated triethylamine hydrochloride was filtered off. The the same manner as (XXV). Reaction of 0.77 g. of piperazine filtrate after treatment with charcoal was washed 4 times with a and 2.97 g. of the benzyl chlorophosphoramide ( X ) yielded 2.18 total of 75 ml. of water and dried over sodium sulfate. Evapog. (367”) of an oil which was purified by treatment with Norit in ration of the solvent gave 9 g. (72Y0b)of oily product. acetone. Anal. Calcd. for CllHt2C13X402P: N, 7.95; C1, 30.3. Found: Anal. Calcd. for C26H38C14N404P2: N, 8.31; C1, 21.07. N, 7.7; C1, 29.6. 2H-1,3,2-Oxazaphosphorine, 2-(N-2-Chloroethyl-N-3,5-di- Found: N, 8.00; C1, 21.0. Benzyl-N,N-di- (2-chloroethyl)-N’,N’-diethyleneaminobenzyl chloropentylamino) tetrahydro-, 2-Oxide (VIII).-A solution of Phosphorodiamidate (LI).-This compound was prepared in 6.5 ml. of propanolamine and 19.5 ml. of triethylamine in 30 ml. essentially the same manner as (XXV). Monobenzylpiperazine of dry benzene and 10 ml. of chloroform was added dropwise to a (2.11 g.) and 1.98 g. of (X) yielded 2.10 g. (74%) of product w an stirred solution of 18.5 g. of the dichlorophosphoramide (VII) oil. in 200 ml. of dry benzene. Reflux was maintained during and for Anal. Calcd. for C22H30C12X~0ZP: N, 8.94; C1, 15.10. Found: 3 hr. after addition. After standing overnight the precipitated N, 8.90; C1, 15.0. triethylamine hydrochloride was removed by filtration. The Benzyl-N,N-di- (2-chloroethyl)-N’-cyclohexyl Phosphorodiamifiltrate was evaporated, and the residual oil treated with charcoal date (=I).-This compound was prepared in essentially the in a benzene solution. Evaporation gave 12.5 g. (67y0) of oily same manner as (XXV). Reaction of 0.96 ml. of cyclohexylamine product. and 1.32 g. of the benzyl chlorophosphoramide (X) yielded an Anal. Calcd. for CloH?oCls03N2P.H~0: N, 7.88; C1, 29.96. oily product which after dilution to cloudiness with petroleum Found: N, 7.8; C1, 30.6. ether gave on refrigeration 0.77 g. (497’7,) of white crystalline Benzvl-di-(2-chloroeth~l)-uhosphoramidic Chloride (XI.-Soproduct, m.p. 85-86’. dium hydride‘suspension 153.5%, 4.48 g.) was washed 3 times with Anal. Calcd. for C17H27C12S202P: C, 51.91; H, 6.87; N, petroleum ether to remove the mineral oil. A solution of 10.29 7.12; C1, 18.09. Found: C, 51.37; H, 7.0; N, 7.30; C1, ml. of benzyl alcohol in 25 ml. of dry benzene was added dropwise 18.00. to a stirred suspension of the pure sodium hydride in 70 ml. of Benzyl-N,N-di-(2-chloroethyl)Phosphorodiamidate (XXI).dry benzene, ice-cooled and under anhydrous conditions. The reaction mixture was allowed to stir a t 5” overnight. The soAmmonia gas was bubbled througii a solution of 1.5 g. of the benzyl phosphoramidic chloride ( X ) in 50 ml. of dry benzene dium benzylate suspension thus obtained was added dropwise and
TABLE V PHOSPHORODIAMIDIC ACID MUSTARDS
bR’
@
4”
-
cooled in ice for 2 lir. nntil the precipitation of ammonium chloride was complete. The ammonium chloride mas separated Iry filtration. The residual oil obtained after evaporation of the filtrate \vas taken up in ether and treated with Norit. The resulting clear filtrate on evaporation afforded 1.07 g. ( i 6 ( I C )of light-colored oil, n Z 41.5352. ~ .4nal. Calcd. for C11H1$12X2C)?P: C, -12.4-1; H, 5.46; X, (3.0: Cl, 22.83. Found: C, 42.77; H, 5.59; S,9.0; C1, 22.80. Cyclohexylammonium Hydrogen-N,N-di-(2-chloroethyl)-N',N'-tetramethylene Phosphorodiamidate (XLI).-Hydrogenation of 1.10 g. of the benzyl phosphorodiamidate (XXI\-) in 25 nil. of c1ios:tne iii the presence of 0.4 nil. of cyclohexylamine arid 0.25 p. of p:blladiuin-charcoal a t ice-bath temperature was complettl \sit>hin30 min. with the absorption of 67 ml. of hydrogen. After filtration, the palladium and precipitated white solid w r c extracted with ethyl alcohol. Evaporation of the alcohol filtrate gave a solid product which was suspended in acetone and filtered yiddiiig 0.38 g. (3.17;) of 15-hite crystalline product, m.p. 1 1 5 110'. . 1 m l . (hlcd. for C I ~ H & ~ Z X ~ ~x, ? P 11.23; : C1. 18.98. Found: X, 11.23; Clj 18.70. Cyclohexylammonium Hydrogen-N,N-di-(2-chloroethyl)-N',"-pentamethylene Phosphorodiamidate (XLII).-This conipoiind WLS prepared in a manner essentially similar to (XI&. l'lw Irenzylpiperidine interniedi:ite (SXV, 1.4 g.) took t i p 70 inl. of hydrogen in 25 min. The alcoholic filtrate yielded on vv:tporatmion0.54 g. (33") of ~rliitecrystallitic product. 1n.1).
1,4-Bis[cyclohexylammonium Hydrogen-N,N-di-(Z-chloroethyl) Phosphorodiamidate]-piperazine (LII).--Hydroger~:~liI 11 I of 2 g. of the hcnzyl phosphoraniidc (S1,TY j i n t h t a 1)rcw.ncv: of 0. I 1111. of cyclohesj.lamine over 0.25 g. of p:tlladiiitii -~rtim-cu:tIi i i iilcohol in thcl cold rcwilted in an iipt:tkc: of 52 nil. o f hy(Irog(ai1ill 20 niiii. The \\.hiti: solid that prccipitatcd dririiig tht' coiirs(s of the rtructioii \\-as leached from thr catalyst 1)). repcat with alcohol which on evaporation yirldctl 0. I p. (5' c.ry&illine product, iii.p. 1:18-140". A l ~ l d . C'alctl. for ( ! ~ ~ € ~ ~ ~ ( ' i , S ~S~, O 12.14: , l ' ~ : ( $ 1 , 20.>8. r ~ f ~ l i u t i : N,i i . ~ )r;i j ~ 5 . Cyclohexylammonium Hydrogen-N,N-di-(Z-chloroethyl)-N'cyclohexyl Phosphorodiamidate (XXXIX).-Hytirogenatioii of 0.5 g. of the tivnzyl phosphorodianiidstc S S I I iii 25 nil. of absoliito etliaiiol ovtlr 0.15 g. of palladium-charcoal in tlie cold \vas c o n i plrtc iii 14 min. \\itti thr absorption of 3 2 nil. (lOO'>) of hydrogen. 'The pd1:diuni \\-as filtpred off and 0.20 in]. of c~yclohesylaniiiic~ added to the filtrate. Evaporation afforded 0.24 g. ( 17' ) 111 c~rystnllirioprodiict, 1ri.p. 110-1 12'. Purification of the crutlr materid with c~harconli n a chloroforin solution raiseti t l i c x i t i . 1 ~ . t o 11T--lltiC. d i ~ ~ ( iCdc(1. /. for Cl,&4ClnS3(hl': ', 4 10.45: I'l. 17.tXj. Found: C, 47.7; H, 8 17.:30.
Cyclohexylammonium Hydrogen-N,N-di- (2-chloroethylj -N',-
N'-diethylenebenzylaminoPhosphorodiamidate (LIII).-Hydro-
i of 2.35 g. of the rnonobenzylpiperazirie interniediatr S)in nl~solutealcohol over 0.7 g. of palladiuiii-rh:tl.co;Ll A 4 r i c i l . Calcd. for C15H3&l2X3OJ': S . 10.8; C1, 18.3. Fountl: i n thr cold was complete iri 100 min. with the absorption of !):3 S , 10.6; CI,18.1. ml. of hydrogen (86"; of the amount required to re1)lacr oii(' Cyclohexylammonium Hydrogen-N,N-di-(I-chloroethyl)-N',- Iit~iizyl group ). The ],alladiurn-ch:trconl w:ts filttwd off :tnd Ni'-(2,2'-diethyleneoxido) Phosphorodiamidate (XLIII).-This 0.7 nil. of cycloliesyliLmirie added to the filtrate. E:v:tpor:itioii rompountl was prepared in a manner similar to XLI. The : I t rrtliicetl prtmure gave a yellowish solid residue u.hich \ s h i ~ t i I)c.nz?-lmorpholine intermediak ( S S V I , 1.4 g.) absorbed 05 stirred in :icetone gnvr L: white crystalline niatc:rial, 0.32 g. i 1:i' ; 1, ni.l). 12iT--lL)So. nil. of hydrogen in 15 min. The alcoholic filtrate gave on c~vapor:ii,ion at reduced pressure a white solid, which was suspended i n . l t ! ~ l .(lalctl. for G I I & : C I ~ ~ T ~ O ~X, P :1 l . W ; ('1, 1.4.8. J'oiiiitl: :~cc.toneand filtered; 0.31 g. ( 2 6 7 ) of \\.hit(, cryst,zllinca prodiict. N , 1 1 . 5 0 : ( > I , 1.5.0. ni.1). 118--11'3",was obtained. Cyclohexylammonium Hydrogen-N,N-di- (2-chloroethyl) Phos..I nal. Calcd. for C14HaoCl,KiaOsP: S , 1 O . T T ; C'l, 18.2, Foiintl: phorodiamidate (XXXVIII).-This romponnd \?-as prepared 1)y :I s,10.80; CI, 18.0. procrdiirr simil:ir t o th:tt for SSSIS. The t)enzyI intrrnitdi:i~tc.
120-1 2 2 O .
A?-PHOSPHORYLATED NITROGEN MUSTARDS
January, 1963
/OR
2(ClCHzCH2),NP
It \
P i,
+(C1CHzCHt)zNP-0I
0 c1 I X R = C6H5 X R = CHzCsHs \ /
OR OR XL\'III = C E& XLIX R = CHzCeH6 0 II/
\
lI/
N-P
"3
/
XIX
2
/* 0
N-P, NHz
N-P
/
NH-@
\OR XXXI, R = -CH3 XXXII, R = -CzH5
2
4
o
II/
\
O(CHZ)~NHCOOCHZC~H~
N-P
/N-P
xx
XVIII
N(CH?CHaCl)*
I
'
'OCH~C~H~
xxxv
1
1(
/
It
/
O(CH2)3NHCOOCH2C6H5
u
xxvlll
J
XXVII
I
/
/
XLIV
\
XXX;"
R
NH(CH2)zOH XXXVII XXI,R=
-NH~
XXII, R =
-NHO
XXIII, R = - N \ N-P, r
\
X
)),O-+N&a
XXXVIII, R = -NH2 XL, R = -NJ
XLII, R = -NJ'
XXXIX, R =
-NHO
XLI, R = - N g XLIII, R
-N
/
OCsH5 XLV
(XXI, 2.6 g.) took up essentially the theoretical amount of hydrogen. After filtration the palladium and precipitated white solid were stirred in a solution of 1.1 ml. of cyclohexylamine in 50 ml. of ethyl alcohol for 30 min. The mixture was again filtered and the filtrate evaporated to give a solid product which was suspended in ether. On filtration 1.9 g. (51.2y0) of white crystalline product, m.p. 107-108", was obtained in pure form. Anal. Calcd. for C I O H ~ ~ C ~ ~ YC,~ 37.5; O ~ P H, : 7.50; N, 13.12; C1, 22.18. Found: C, 37.06; H, 7.08; K, 13.10; C1, 22.2. Cyclohexylammonium Hydrogen-N,N-di-(2-chloroethyl)-N',"-ethylene Phosphorodiamidate (XL).-Hydrogenation of 0.8 g. of XXIII in 25 ml. of ethyl alcohol over 0.2 g. of palladiumcharcoal in the cold was complete with the absorption of 106 ml. of hydrogen in 25 min. The palladium was filtered off and 0.75 ml. of cyclohexylamine added to the filtrate. Evaporation of the filtrate gave an oily residue which was taken up in ether and stored a t 5" overnight. A precipitate, 0.26 g. (31.3y0), of crystalline product, m.p. 103-105", separated. The crude material, purified by treatment with Norit in methanol, melted a t 110-1 12O . Anal. Calcd. for C I ? & C ~ ~ N ~ ON,~ 12.14; : C1, 20.52. Found: N, 11.9; c1, 20.30.
N,N-Di-(2-Chloroethyl)-N',N'-ethylene Phosphorotriamidate (XVIII).-Ethyleneimine (0.26 ml.) was added dropwise to a mixture of 1.3 g. of the dichlorophosphoramide I and 0.7 ml. of triethylamine in 20 ml. of dry benzene with stirring and ice cooling. After refrigeration for 1 hr. the precipitated triethylamine hydrochloride was filtered off. Ammonia gas was bubbled through the cooled filtrate for 2 hr. when the precipitated ammonium chloride was separated by filtration and petroleum ether added to the oily residue obtained after evaporation. The petroleum ether was removed by evaporation leaving a solid residue which when stirred with ether gave 0.22 g. (17.6%) of crystalline material, m.p. 83-85'. Anal. Calcd. for CBH14C12N30: C, 29.27; H , 5.69; N, 17.08; C1, 28.86. Found: C, 29.24; H, 5.58; N, 16.9; C1, 28.7. Methyl-N,N-di-(2-chloroethy1)-","-ethylene Phosphorodiamidate (XXIX).-The intermediate methyl phosphoramidic chloride X I was prepared in the same manner aa the benzyl phosphoramidic chloride X. Reaction of 4.5 g. of a sodium hydride suspension (53.5y0), 4.0 ml. of methyl alcohol and 25.9 g. of the dichlorophosphoramide (I) gave 14.8 g. (62'%) of methyl-di-(2-chloroethyl)-phosphoramidicchoride (XI) aa an oil. To a stirred solution of 1.27 g. of crude X I and 0.7 ml. of tri-
0. M. FRIEDMAN, E. ROGER,T. GRUBLIAUSKAS AND H. SOMMER
56
TABLE VI11
cq(.ssc SCREENING
DATA SuMMARYa
O N ASTITUMOR
ACTIVITY
01. SOMEPHOSPHORAMIDE KITROGEN R~USTARDS ACAIXSTL1210
LEGKEMIA IK BDFi
?IfICEh
ReJponse: Test/Control ( T I C ) Animal w t . change, Survival, Per days cent R.
Daily dose, mg./kg.
100 50 25 12 100 50 i5 100 50 25 60 25
-3.3,’-0.2
1’11 11.1/9.2 1G.2/9.2 17G -0.0/-0.2 12.0/9.2 130 0.0/-0.2 1 3 , 0 / 9 . 2 141 -1.7/0.5 10.0;9.5 105 -0 , 4 / 0 , 5 102 9.7/9.5 0 .i j o . d 9-1 9 . 0/9 . 9 1 .9/0, 5 10,4,‘9.5 109 91 0.9j0.5 8.7/9.5 0.6/0.5 8,7/9.5 71 0.3/0.9 10.5/9.5 1 10 111 1.0/0.9 10.6/9.6 12 5 0 . s i 0 .I) 1 0 . 0 / 9 . 5 10.5 1 1 , 2 / 9 . 5 117 200 -0 . 3 / 0 . 9 9 . 8 / 0 . 5 103 0 . 8/0. 9 100 10’1 1. 2 / 0 . 9 9 . 7 p5 50 -2 . 5 / 0 , 7 100 13 , 8 / 8 . 9 1 % - 1. 2 / 0 . 7 1 1 . 3 / 8 . 9 126 50 11 3 p . 9 -0.1,’0.7 120 25 117 12 5 -0 :+/O.T 10.5/s,9 1000 G/G -0 9 / - 0 . 5 $I,U/8.9 101 500 G/G 0 (j:0.8 ‘3.8I9.G 102 2j0 f/G 0.4’0.8 0 8 / 9 . 6 102 Kindly supplied by Dr. Joseph 11. Leiter, CCNSC, Xational Institutes of Health, Bethesda, lid. b Administered intraperit oneally in saline. Assays for activity were performed according t o specifications established by the CCSSC (Cancer Chemothrrtrpy Reports, 1 , 4 2 (1959), Cancer Chemotherapy Sational Servire Centre, National Cancer Institute, Bethesda, 3Id.j.
- 1 ,G / - 0 . 2
1~
cthylamine in 25 ml. of dry benzene, 0.26 ml. of ethyleneimine i n 15 nil. of dry benzene was added dropwise. After standing overnight :It 5’ the triethylamine hydrochloride \vas removed tly filtration. The benzene was evaporated and purification of thP residue with charcoal in an ether solution gave 0.7 g. ( 5 0 7 ) of product as an oil. .Anal. Calcd. for CloHljCLSrO?: K, 10.73; C1, 27.2. Foi~nd: s.10.6; c1, 27.0. Ethyl-N,N-di-(2-chloroethyl)-N’N’-ethylenePhosphorodiamidate (XXX).-The intermediate ethyl phosphoramidic chloride TABLE
Yol. 6
(XII) was prepared in the same manner as the benzyl phosphoramidic chloride (X). Reaction of 4.5 g. of a sodium hyclridc suspension (53.5%), 5.8 ml. of ethyl alcohol and 25.9 g. oi t l i ~ dichlorophosphormide (I) gave 15.1 g, (6070) of ethyl-di-t2chloroethy1)-phosphoramidic chloride (XII) as an oil. To :i stirred solution of 1.34 e. of the crude Droduct (SIT) antl 0 7 n i l of triethylamine in 25 ml. of dry benzene, 0.26 nil. of et,liylerir.imine was added dropvise. After standing overnight a t 5” t h t t triethylamine hydrochloride was removed hy filtration. T t i c s Ixnzene was waporated antl purification of the residue xvitli charcoal in an ether solution gave 0.75 g. ( 5 4 ( < ) of oily protluct. ~ 2 ~ ~1.4850. 1 ) :1n,al. Calcd. for CnlI17C12NrOyP:X, 10.16; TI, 25.8. Foiiritl: s,10.0; C1, 29.5. N-Carbobenzoxvorooanolamhe (XIV).---A cooled solutio11 01 10 g. of cartjobenzh& chloride in 20 ml. of chloroform vias tzddcd dropwise to a solution of 11 inl. of propanolamine in 50 ml. of chloroform with stirring and cooling in ice. After standing overnight the mixture was washed with water, dilute hydrochloric. acid, dilute potassium carbonnte and again with TTater. Tlirb chloroform layer was dried over sodium sulfate and evaporated. The oily residuc! olitained on dilution with petroleum ether gave :I crl-stalline product, m.p. 48-51 ’. Recrystallization from chloroforrn--p~.t,roleuiri ether (1 :4) yielded 5.5 g. (45y0),m.p. 53-55”. .1md. (Ialcd. for C 1 ~ M ~ ~ ? ; O C,~ :63.16; H, 7.17; ?;, 6.69. Found: C , 63.1; H, 7.1; S , G . i . Benzyl N-carbobenzoxyaminopropyl di-(2-chloroethyl) -phosphoramidate (XXXV).-A solution of 2.09 g. of carbobenzosypr~~Imnolarnine in 40 nil. of benzene was added to 0.44 g. of sodium hydride suspension (53.5cj,\. After stirring for 2 hr., 2.59 g. of the dichlorophosphoramide (I) was added to the mixture and the stirring was coiit,inued for an additional 70 min. The precipitated sodium chloride was removed by filtration and the filtrate treated with charcoal. The benzene was removed by evaporation, leaving n n oily residue Ivhich was n-ashed three times with petroleum ether t o remove the mineral oil. The residue m-as treated wit 11 charcoal i r i a henzene solution and evaporation gave 2.95 g. (68.476) of N-carbobenzoxyaminopropyl-di-(2-chloroethyl)-phosphoramidic chloride (XV) as an oil. A solution of 22 g. of crude N V in 100 ml. of dry Leiizene wis added dropwise to a suspensioil of 6.6 g. of sodium bcnzyloxide (preparation previously recorded in S )in 100 nil. of dry benzene with stirring and cooling in iccx. The stirring was continued for an additional 2 hr. and the misture 11-33 stored at 5 ” overnight. The benzene was evaporated leaving ail oily rc!sidue which was taken up in ethw. After separation of the sodium chloride precipitate by centrifugation und filtration. the ether \vas evaporated leaving 14.3 g. (555‘; 1) of oily product.. . l t ~ c I .Calcti. for ClrH,,ClyT205T’: 5 ,5 . Z ; CI, 14.11. J+uiid: s , 5.5: CY, 14.0. 2-Chloroethyl-N,N-di-(2-chloroethyl) Phosphorodiamidate (XXXIII).-A solution of 3.32 ml. of chloroethanol in 15 ml. of dry benzene n-its added dropwise to a stirred solution of 12.95 g . of the dieIi1oroi)hosphoraiiii~c( I )and 0.99 nil. of t,rit:thylarnirir+i i i Y
~I
~I
-
-
Ix’’
CCRF RESULTS IX PRIMARY T m o R SCREEN WITH SOMEPHOSPHORAMIDE MUSTARDS~ L1310 I
Compound
Survival increase
Survival increase
Tumor inhibition
Survival increase
%
l-7
%
0-
/o
DBRB
(21498
Pl534
-_
/c
Tumor inhibition
Survival increase
n1
n
Tu mor inhibition
