In the Search for New Anticancer Drugs, III+ Phosphorylated Diaziridine Derivatives
George Sosnovsky* and Jan Lukszo Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, U.S.A.
Z. Naturforsch. 38b, 884-894 (1983); received August 30/December 24, 1982
Phosphorylated Diaziridines, Synthesis, Anticancer Drugs, Leukemia P 388
Several N-diethoxyphosphoryl derivatives 7 of various diaziridines, and compounds 12, 15a, lob, 18 and 20, structurally related to TEPA (la) and spin labeled Thio-TEPA (lc) were synthesized.
0 Me 0
N-P(0Et)2 HC=NN-p(<]) o / NR ' • 11 f / 1 ? R-O-P-f N —C—R R X N—R CL N "2 Me Me
1 3 1 2 3 7: R . R = alkyl 12 15a: R = R =R =Me N-0
R2=H, alkyl 15b: R' = H,R2R3=-|CH2|5- Me Me In three cases, attempts to synthesize phosphorylated diaziridine derivatives resulted in rearrangements to give the corresponding phosphorylated hydrazone derivatives 11 h, Hi and 12. Me 0 CH = NN — P(0Et)j
11 h: x= H 11 i : x = ci 13C NMR spectroscopy was shown to be a valuable tool in distinguishing between the structures of diaziridine and hydrazone derivatives. The in vivo testing of five representa- tive compounds (7e, 12, 15a, 16 and 20) against murine lymphocytic leukemia P 388 showed a lack of antitumor activity of compounds 7e, 15 a, 16 and 20, and an activity of compound 12 as evidenced by a T/C value of 194 and a % ILS of 94, at a dose of 32 mg/kg.
Introduction pounds, triethylenimine thiophosphoramide, Thio- In the past three decades, extensive studies [1-9] TEPA (lb, R = ethylenimine ring), has been used have been made in the search for new antineoplastic drugs containing the ethylenimine-(aziridine) moi- X ety. In particular, the phosphorus derivatives of P R type 1 a and 1 b containing two or three phosphorus- N-aziridine bonds, have been found to be effective alkylating anticancer agents [8, 10, 11]. It is pre- sumed [12, 13] that the alkylating reactions occur 1a : X = 0. R = or other groups at the nucleophilic centers of the heterobases of lb: X = S, R = £N, or other groups DNA causing cross linking and, thereby, the cessa- 1c: X =S, R = OR. tion of the replication of DNA. One of these com-
+ Part II: P. L. Gutierrez, M. Konieczny, and G. Sosnovsky, Z. Naturforsch. 36b, 1612 (1981). * Reprint requests to Professor Dr. G. Sosnovsky. ' Me 0340-5087/83/0700-0884/$ 01.00/0 Me G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs 885 clinically in the chemotherapy of certain cancers Results and Discussion [10]. Recently, it has been claimed [14-16] that As starting materials, several selected diaziridines nitroxyl labeled analogs of Thio-TEPA, such as 2 derived either from aldehydes or ketones were SL-O-TT (lc) [14, 15] and PAT-1 (Id) [16] have prepared by the reaction of hydroxylamine-O-sul- more desirable toxicological properties than the fonic acid with the corresponding ketone 5 or clinically used Thio-TEPA. aldimine 6 in the presence of either ammonia or the In contrast to the aziridines, the three-membered corresponding amine, following either the literature diaziridine ring system 2, containing two nitrogen procedures exactly [17, 30-33] or adapting a known atoms, was only scantily studied for possible anti- procedure [31, 34] to different starting materials, as cancer activity [17]. Although the diaziridine ring in the preparation of 2f and 2i. The N-phosphory- system was discovered only about two decades ago, the methods of preparation and the reactions of N-H diaziridines have been extensively explored [18-21]. \ H,N-0-S0,H H2N-Q-SO3H' c=o — f— <1 I . ,C=N-R' The diaziridine ring system can readily undergo R H2N-R' N—R H,N-R' C ring opening reactions either by an SN 1 mechanism 5 2 [22], involving carbonium ion intermediates (3), or 5a: R - i-Pr, R2 = R3 = Me 5b: R = R2 = R3 = Me 5c: R = H, R2R3 - —ICH2I5- H 5d: R = Me, R2R3 = —ICH2I5- NR © NR NR' 3 \ /i x \ R 6e: R = Et, R2 = Me, R = H V 6f: R = i-Pr, R2 = Et, R3 = H 2 ,2/ © \ / R NR' NH 6g: R = n-Bu, R2 - Et, R3 = H 6h: R = Me, R2 = Ph, R3 — H 6i: R = Me, R2 = p-Cl-C6H4, R3 = H 6j: R - C6HU, R2 - Ph, R3 — H by unspecified mechanisms, mediated by nucleo- philes, involving a reaction of the C-N-N group lated derivatives 7 were synthesized either by an [21], formed by ring opening of the diaziridine with adaptation of the Todd-Atherton method [35], i.e. a nucleophile. A series of such reactions, formally by the reaction of the appropriate diaziridine of the alkylation type, with reagents such as nitrones (2 a, e, f, g) with diethyl phosphite and carbon [23], enamines [24], cyclopropenone [25, 26], al- tetrachloride in the presence of triethylamine, or kynyl ketones [27], diphenyl ketene [28], and lead by the reaction of the diaziridine (2 e, g) with diethyl tetraacetate [29] have been reported. On the basis phosphorochloridate in the presence of triethyl- of such reactions, it is plausible to assume that the amine, similarly to the recently reported procedure diaziridines might be useful as alkylating antineo- [36], plastic agents. In 1967, Szantay et al. reported such a study [17]. Thus, three N-phosphorylated dia- 0 ziridine derivatives 4 were synthesized and tested (EtO)2PH/CCti/NEt3 ,N—P(0Et)2 2a,e.f,g (EtQ)2PCl/NEt3 4-R' n-Bu-N* .N-Bu-n 7a: R1 i-Pr, R2 = R3 = Me N —P—N 7e: R1 = Et, R2 = Me, R3 = H EtCH CHEt 71: R1 = i-Pr, R2 = Et, R3 = H 7g: R1 = n-Bu, R2 = Et, R3 — H
A; R=Cl. OH, HNCOEt Both reactions gave the desired products 7 in com- in vitro against the Sarcoma 180 cells. The in vitro parable yields, however, compounds 7 prepared by results seemed to be encouraging [17]. However, the Todd-Atherton method seem to be of a better the lack of in vivo data, and difficulties which were quality, as evidenced by the fact that samples of 7 experienced in our laboratory in reproducing some prepared by the alternative method using diethyl of the results reported [17], prompted us to in- phosphorochloridate appeared to darken more ra- vestigate this class of compounds in greater detail. pidly on storage at 0 °C. Attempts to achieve the 886 G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs 886
Table I. Diethyl diaziridine -1-phosphonates 7 and diethyl-N1-benzylidene-N-methylhydrazinephosphonates 11.
a 15 Com- Yield b.p. [°C/torr] Purity N25 Molecular M.S.: m/e I.R.e H NMR (CCl4)h pound [%] or m.p. [°C] [%] formula0 [M++l]d v [cm-1] ö [ppm]
f 7a 38 74—79/0.04 1.4395 C10H23N2O3P 251 960, 1020, 1050, 0.93-1.51 (m, 18H), 250 1250, 2930 2.16-2.70 (m, 1H), 3.85-4.40 (m, 4H)
7c 69 79-81/0.12 94 1.4390 C8HI9N203P 223 970, 1030, 1260, 1.18 (t, 3H), (64) 76-78/0.10 96 1.4390 222 2980 1.35 (t, 6H), 1.61 (d, 3H), 2.23-2.75 (m, 2H), 2.83-3.48 (m, 1H), 3.91-4.50 (m, 4H) 7f 67 78-80/0.05 97 1.4393 C10H23N2O3P 251 950, 1010, 1040, 0.83-1.50 (m, 15H), 250 1240, 2950 1.55-2.11 (m, 3H), 2.53-3.10 (m, 1H), 3.73-4.33 (m, 4H) 7g 68 84-85/0.05 98 1.4405 C11H25N2O3P 265 960, 1020, 1250, 0.71-2.10 (m, 18H), (57) 88-89/0.07 93 1.4416 264 2920 2.36-2.78 (m, 2H), 2.81-3.56 (m, 1H), 3.78-4.38 (m, 4H) llh 44 50-51.5 C12H19N2O3P 271 970, 1010, 1140, 1.27 (t, 6H), 270 1250, 2950 3.16 (d, 3H), 3.66-4.30 (m, 4H), 7.03-7.58 (m, 6H) lli 30 94-95 Ci2Hi8ClN203Ps 305 960, 1010, 1140, 1.27 (t, 6H), 305 307 1250, 2950 3.16 (d, 3H), 3.66-4.30 (m, 4H), 7.03-7.58 (m, 5H)
a Yields of products by Todd-Atherton method, except in ( ) which were obtained by using diethyl phosphoro- chloridate as described in the general procedure B; b by iodometric titration; c the microanalyses were in agreement with the calculated values: C ±0-36%, H ±0.21%, N ±0.40%; d chemical ionization applied; e compounds 7a, e, f and g analyzed neat, compounds 11 h and lli as KBr pellets; f no reaction with acidified solution of KI, in spite of confirmed diaziridine structure by 13CNMR; g for the chloroderivative lli, two characteristic peaks with intensity ratio of 3:1 was observed; h the appearance of dublets (3.16 ppm) for methyl protons [-N(CH3)-P] in 11 h and lli is attributed to phosphorus-hydrogen coupling.
phosphorylation by the Todd-Atherton method for diaziridines derived from aromatic aldehydes. using an excess of a diaziridine as a hydrogen The reactions of Schiff bases 6 h and 6 i with hy- chloride acceptor failed, probably because of the droxylamine-O-sulfonic acid in the presence of a low basicity of diaziridines [37]. Products 7 were 40% aqueous methylamine solution following the purified by repeated vacuum distillations to con- literature procedure [34], for the preparation of 2h, stant boiling temperature at 0.04 to 0.12 torr. How- resulted in presumed intermediates 2h and 2 i which ever, in all C3/SGSj cl slight decomposition, in part- were too unstable for isolation in pure form as icular of 7 a occurred, as evidenced by a residue in evidenced by rapid change in color and T.L.C. ana- the distillation flask, thus decreasing the overall lysis. Therefore, the presumed, diaziridines 2h and yields of products 7. Nevertheless, the structure of 2 i were immediately phosphorylated to give isolable 7 a, e, f and g wras unequivocally established by phosphorylated derivatives, presumed to be pro- microanalysis, IR and NMR spectra, and in the ducts 7h, 7i and 8. The reaction of the presumed case of 7 e, f and g by iodometric titrations [38]. The products 7h, 7 i and 8 with an acidified potassium titrations failed in the case of 7 a, h and i. This iodide solution was negative, although the *H NMR, result is in line with experiences reported in litera- mass spectra, and microanalyses were in general ture [18] for diaziridines derived from ketones, agreement with the expected structures. In order whereas no iodometric titration data are available to unequivocally determine the structures of these G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs 887
I. H2N-O-SO3H/ 40%MeNH,/H,0 CH N-P(OEt), 2.(Et0)2PH/CCI4/Et3b* 1.H2N-0-S03H/ 7 i :x=ci 40%MeNH2/H20 6H:x =H 2.PCl /Et N w'V 3 3 I 6 i : x = ci Me 3. [>H/Et N 3 8:x = ci products, as well as those of some diaziridines 2 and (7 a, e-g) are in the range of 56.5-69.2 ppm, similar phosphorylated diaziridine derivatives 7 a, e, f, g, to those reported in literature [44, 45] for unsub- the 13C NMR spectra of these compounds and of stituted diaziridines, and distinctly different from those of the corresponding hydrazones 9 a and 9 b those of the corresponding hydrazones (Table II). were investigated, since it was known [33, 39, 40] Therefore, one can assume that the reactions of 6h that hydrazones can be formed by a thermal re- and 6 i with hydroxylamine-O-sulfonic acid, fol- arrangement of diaziridines. Authentic hydrazones lowed by phosphorylation proceeded via a rearran- 9 a, 9 b and 10 were prepared according to the gement of the diaziridines 2 h and 2 i into the cor- responding hydrazones 9 a and 9 b. Subsequent phosphorylation resulted in the formation of pro- CH =NNHMe Me,C = NNHMe ducts 11 h, Hi and 12, instead of products 7h, 7 i and 8, respectively, as originally expected. 9 A: x = H 10 9 b: x = ci After the unsuccessful attempts to prepare C-3- aryl substituted phosphorylated diaziridine deriva- literature [41-43] by conventional methods. The tives 7h, 7i and 8 in aqueous media, the experi- authentic phosphorylated hydrazones 11 h, i were ments were performed in anhydrous methanol, as prepared from hydrazones 9a, b by the Todd-Atherton described for several C-3-aryl substituted diaziridine method [35]. A comparison of shift values <3 [ppm] derivatives [33]. However, the results were ana- for various diaziridines and hydrazones is shown in logous to those obtained in experiments with Table II. From Table II it is evident that ö shift aqueous media, and only the corresponding hydra- values for the diaziridine ring carbon (C-3) are zones 9a and 9b were isolated. Nevertheless, the quite different from 6 values for corresponding car- preparation of l-cyclohexyl-3-phenyldiaziridine (2j) bon in the hydrazones. Thus, the (5 values for com- [33] by the same procedure was successful. On the pounds 9 a, b and 10 are 136.9, 135.1, and 163.2 ppm, basis of these results, it appears that the type of respectively. Similarly, the <5 values for the pre- substituent at nitrogen in the Schiff base may have sumed products 7h, i and 8 are 137.4, 136.8 and a decisive influence on the type of product formed. 136.3 ppm respectively. The d values for diaziridines Besides phosphorylated derivatives 7 containing (2b, j) and for the phosphorylated diaziridines one diaziridine moiety, attempts were made to pre- Me 0 (EtO)2PH/Ca4 /= ^y-CH = NN-P(OEt)2 Et,N 6h: X = H 9a:x = H 11h:x = H 2h,il 11 i:x = ci 6 i: x = ci 9b: x = ci o 1. pa3/Et3N ^ VCH = NN-p(N3)2 2.Hh<]/Et3N Me 12: x = ct 888 G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs Table II. Comparison of 13C NMR shiftsa-b for diaziridine and hydrazone derivatives. IN compound Solvent ^)c=N-N< / \2N 6 [ppm] <5 [ppm] Me2C = NNHMe 10 163.2 /NMe Me2C\ | 2 b 56.5 \NH C6H5CH = NNHMe 9a 136.9 ?>C1-C6H4CH = NNHMe 9b CDC13 135.1 r/NC6Hn CöHSCHx 2j 58.9 and 68.4e SNH d C6H5CH = NN(Me)P03Et2 llh CC14 137.4 (d, J — 13.7 Hz) d p-Cl-C6H4CH=NN(Me)P03Et2 lli CDC13 136.3 (d, J = 13.4 Hz) i-PrN >N-P03Et2 7a 64.9 (d, J = 8.1 Hz) Me2C EtN I •N-P03Et2 7e 63.7f MeCH i-PrN I N-P03Et2 7! 68.1 (J = 7.9 Hz) EtCH w-BuN I N-P03Et2 7g 69.2 (d, J — 7.8 Hz) EtCH a Shift values measured against TMS; b for all phosphorus derivatives doublets were recorded because of carbon- phosphorus coupling; c no solvent used; d no absorption peak in this region; e two values recorded account presumably for two isomers, as reported in literature [33]; f two 0-CH2CH3 carbon atom absorptions found in the region 63-64 ppm of 7 a, 7e, 7f and 7g, interference occurred only in 7 e. pare several selected analogs of TEPA (la), Thio- ing with the nitroxyl radical 13, without isolating TEPA (lb) and SL-O-TT (lc), containing the the intermediate 14. The yield of 15a and 15b, nitroxyl radical moiety and either two diaziridine after column chromatography, was 26% and 47%, groups (15 a, b), or one aziridine ring and one respectively. Compound 16 was prepared in a 29% diaziridine ring (16), or three diaziridine rings with- yield by a three-step sequence. An attempt to pre- out the nitroxyl group (19) and (20). These attempts pare the SL-O-TT (lc) analog 17 failed. Thus, the were only partially successful. Thus, the synthesis reaction of 6 e with thiophosphoryl chloride did not of 15 a, 15 b and 16 was achieved in two steps start- proceed, and the reaction of 6 e with phosphorus tri- l Rl 2b or 2c/Et3N . fl (/'l " ROP4N I D2 2U\\ 0 II Et,N/toluene 15a: R1 =R2 = R3 = Me R0H + PC13 - — * 1 2 3 15 b: R = H, R R =-|CH2I 5" 13 1. 2f/Et,N . 9 /f^-pr-i ROP-N; v R=HXN-O- 2. H Me Me 16 G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs 889 chloride and sulfur resulted, in spite of the correct tion was performed against the lymphocytic leu- stoichiometry, in the formation of a diradical 18 in kemia P388 in mice in accordance with the protocol 11% yield. [11] of the National Cancer Institute. The results are shown in Table III. The criterion of T/C, where T represents the mean survival time of the treated S NEt 1. PCl3/Et3N 11 RO• P ,N - group, and C the mean survival time of the tumor 2. HN-NEt \ CHMe bearing control group, was used to evaluate the \l /Et*3 N ROH 6e CHMe 17 chemotherapeutic effectiveness of compounds. A value of T/C > 125 is usually considered to be the 1. pq /Et N NEt 3 3 (RO), PN, minimum requirement for a compound to be con- 2. HN—NEt 13 CHMe sidered as active [11]. The percent increase life span \l /Et3N 6e CHMe 18 (ILS %) parameter was calculated using the for- 3. S8/PhCH3 mula [(T-C)/C] x 100. Clearly, the larger the value for the ILS, the more promising is the compound as The reaction of diaziridine 2 c with thiophos- an anticancer drug. On the basis of results obtained phoryl chloride proceeded slowly with the formation with leukemia P388, it is concluded that the phos- of a mixture of unidentified products from which phorylated diaziridine derivatives 7e, 15 a, 16 and no compound 19 could be isolated. The analogous 20 have no anticancer activity, combined with low toxicity, except for compound 7e which was toxic Table III. In vivo evaluation of selected phosphorus ^Hf- (oc: ,P=S compounds for antitumor activity3. 19: R = H Compound Dose T/C ILS [%] r—\ NR mg/kg ,P = 0 0 Preparation of diethyl N^-benzylidene-N-methyl- Phosphorylation of hydrazones 9 a and 9 b. hydrazinephosphonate (11 h) and diethyl General procedure C N1-(4-chlorobenzylidene )-N-methylhydrazine- phosphonate (lli) Diethyl phosphite (3.45 g, 0.025 mole) was added Reaction of N-benzylidenemethylamine rapidly to a solution of the appropriate crude with hydroxylamine-0-sulfonic acid hydrazone either 9a or 9b (90%, 0.025 mole) and triethylamine (2.52 g, 0.025 mole) in carbon tetra- N-Benzylidenemethylamine [49] (6h, 4.8 g, chloride (30 ml). After 40 h of stirring at 20 °C, 0.040 mole) and methanol (15 ml) were added to a the reaction mixture was filtered, and the solid 40% aqueous solution of methylamine (13.5 g, material, triethylamine hydrochloride, washed with 0.174 mole) at —20 °C, followed by portionwise anhydrous ethyl ether (3 X 25 ml). The filtrate and addition of hydroxylamine-O-sulfonic acid (85%, washings were combined, and concentrated on a 5.45 g, 0.041 mole) over a period of 5 min at 0-10°C. rotating evaporator at 20°/20 torr. Recrystallization The reaction mixture was stirred for 1 h at —20 °C, of the remaining solid residue from a mixture of and 15 h at 0 °C, then ethyl ether was added (70 ml) cyclohexane and hexane (v/v6:1) gave 11 h and lli, and the layers were separated. The aqueous solution respectively (Table I). was extracted with ethyl ether (25 ml). The com- Authentic samples of diethyl N1-benzylidene-N- bined ether extracts were dried over magnesium methylhydrazine phosphonate (11 h) and diethyl sulfate, filtered, and the filtrate concentrated at N1-(4-chlorobenzyhdene)-N-methylhydrazine phos- 30°/20 torr. Distillation of the residue under reduced phonate (lli) were prepared by the procedure C pressure gave 2.4 g of a yellow, oily liquid, pre- using freshly prepared benzylidenemethylhydrazone sumably 2h, b.p. 71-73°/0.1 torr, lit. [34], b.p. 9a [41], and ^5-chlorobenzylidene methylhydrazone 72-75°/0.4 torr, IR (neat): v = 3370 cm-1 (NH), 9b [42], respectively as starting materials. The yields lit. [34], v = 3370 cm-1 (NH). This product could and melting points of pure products 11 h and lli not be titrated with an acidified solution of KI, and were 30%, m.p. 49.5-51.5 °C, and 52%, m.p. was unstable, as evidenced by rapid changes in color 65.5-66.5 °C, respectively. These products were in and T.L.C. analysis. Therefore, it was immediately, all respects identical with those synthesized from without further purification, phosphorylared by the aldimines 6h and 6i. In both cases the mixture Todd-Atherton method [35] as described in general melting points 49.5-51.5 °C for 11 h and 65-66 °C procedure C. The proposed product 2h was even- for lli were not depressed as compared to the tually identified by 13C NMR (neat) and T.L.C. melting points of the individual compounds. (ethyl ether) comparison with an authentic sample [41] as benzaldehyde N-methylhydrazone (9 a). Preparation of bis( 1-aziridinyl)- [ Ni-fp-chlorobenzylidene )N-methylhydrazin-l-yl]- Reaction of N- (4-chlorobenzylidene)methylamine phosphine oxide (12) with hydroxylamine-O-sulfonic acid A solution of crude p-chlorobenzylidene N- The reaction of N-(4-chlorobenzylidene)methyl- methylhydrazone (9b, 90%, 3.75 g, 0.020 mole) and amine [50] (2i, 21.2 g, 0.138 mole) with hydroxyl- triethylamine (2.52 g, 0.025 mole) in toluene (10 ml) amine-O-sulfonic acid was performed analogously was added dropwise, over a period of 10 min to a to the preceding experiment. The crude reaction solution of phosphoryl chloride (3.07 g, 0.020 mole) product decomposed on an attempted distillation in toluene (25 ml) at —10 °C. After stirring for 1 h under reduced pressure at 0.1 torr. Therefore, it was at —5 to 0 °C, the reaction mixture was kept for purified by recrystallization from hexane by cooling 24 h in a refrigerator at 0 °C, then cooled again to the solution to —50 °C, to yield 5.0 g (21%) of a —10 °C. To this mixture, a solution of aziridine yellow solid, m.p. 26-30 °C. This product, analo- (1.76 g, 0.041 mole) and triethylamine (4.54 g, gously to 2h, could not be titrated with an acidified 0.045 mole) in toluene (25 ml) was added over a solution of KI, and was unstable as evidenced by a period of 10 min. The reaction mixture was stirred rapid change in color and T. L. C. analysis. Therefore, for 16 h at —10 °C, then stored in a refrigerator at it was immediately phosphorylated by Todd- 0 °C for 48 h. The solid material was removed by Atherton method [35] as described in C, without filtration, and the filtrate passed through an alumina further purification. The proposed product 2i was column using a mixture of benzene: acetone (v/v 4:1) eventually identified by 13C NMR and comparison as the eluant. The eluate was concentrated on a of T.L.C. (ethyl ether) with authentic sample [42] rotating evaporator at 25 °C/20 torr. The oily residue as 2>-chlorobenzylidene-N-methylhydrazone (9b). was chromatographed on silica gel using a mixture M.S.m/e = 169(100), 171(33). of £-butyl methyl ether: acetone (v/v 1:1) as the eluant, followed by recrystallization of the resulting IR (neat): v = 840,1090,1490,1600,2900, 3370cm-1. solid from a mixture of t-butyl methyl ether: hexane XH NMR (CDCI3): 6 = 2.90 (s, 3H), 5.43 (broad s, (v/v 1:1), to afford 1.51 g (25%) of 12, m.p. 92-93°C. 1H), 7.03-7.50 ppm (m, 5H). Purity control by T.L.C. (£-BuOMe:acetone, v/v 892 G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs 892 1:1) indicated one product. M.S.: ra/e = 299(100), ether and acetone filtrates were concentrated on a 301(34). rotating evaporator at 40 °C/20 torr. The resinous IR (Nujol): v = 930, 960, 990, 1250 cm-i. residue was chromatographed on alumina using m NMR (CDCls): d = 2.23 (d, 8H, J = 15 Hz), 3.23 ethyl acetate as eluant. Concentration of the eluate (d, 3H, J = 7 Hz), 7.16-7.68 ppm (m, 5H). afforded crude 15b (2.45 g). Recrystallization of the crude material from ethyl acetate afforded a pure Ci2H16CIN4OP (298.7) 15b (2.10 g, 47%). Purity control by T.L.C. Calcd C 48.25 H 5.40 N 18.76, (benzene:acetone, v/v 1:1) indicated one product, Found C 48.08 H 5.34 N 18.46. m.p. 146-147 °C. M.S.: m/e = 442. E.P.R.: 3 lines, aN = 15.5 G. Preparation of 4-[bis- (2,3,3-trimethyldiaziridin- IR (KBr): v = 1030, 1220, 1350, 2870, 3100 cm-i. 1-yl) J -phosphinyloxy-2,2,6,6-tetramethylpiperidine- C2iH39N503P (440.5) 1-oxyl (15 a) Calcd C 57.25 H 8.92 N 15.89, A solution of 4-hydroxy-2,2,6,6-tetramethyl-l- Found C 57.33 H 8.79 N 15.54. oxopiperidine [51, 52] (13, 2.58 g, 0.015 mole) and triethylamine (1.82 g, 0.018 mole) in toluene (20 ml) Preparation of 4-f (1-aziridinyl)- was added dropwise, over a period of 10 min to a (3-ethyl-2-isopropyldiaziridin-l-yl)-phosphinyloxy] - stirred solution of phosphoryl chloride (2.30 g, 2,2,6,6-tetramethylpiperidine-l-oxyl (16) 0.015 mole) in toluene (25 ml) at —20 °C. After stirring for 3 h at —5 °C, a solution of 1,3,3-tri- A solution of 4-hydroxy-2,2,6,6-tetramethyl-l- methyldiaziridine [53] (2b, 2.75 g, 0.032 mole) and oxopiperidine [51, 52] (13, 6.90 g, 0.040 mole) and triethylamine (3.23 g, 0.032 mole) in toluene (15 ml) triethylamine (5.05 g, 0.050 mole) in toluene: ethyl was added at —5 °C. The reaction mixture was ether (v/v 1:1, 50 ml) was added over a period of stirred for 30 min at —5 °C, then kept in a freezer 30 min to a solution of phosphoryl chloride (6.14 g, for 48 h at —10 °C. The insoluble material was 0.040 mole) in toluene (25 ml) at —15 °C. The removed by filtration, and the concentration of the reaction mixture was stirred for 3 h at —5 to 0 °C. filtrate on a rotating evaporator at 30 °C/20 ton- A solution of l-isopropyl-3-ethyldiaziridine (2f, gave a crude product 15 a. Purification of 15 a by 95%, 4.80 g, 0.040 mole) and triethylamine (5.05 g, flash chromatography on silica gel, using ben- 0.050 mole) in toluene (25 ml) was then added, and zene: acetone (v/v 1:1) as eluant, gave pure 15 a, the reaction mixture was stirred for 90 min at —5 1.5 g (26%), as a red oil. The purity control by to 0 °C, and for 24 h at 20 °C. After cooling to 0 °C, T.L.C. (benzene:acetone, v/v 1:1) analysis indi- a solution of aziridine (1.72 g, 0.040 mole) and tri- cated one compound. ethylamine (4.04 g, 0.040 mole) in toluene (25 ml) was added over a period of 30 min. Following the M.S. m/e = 389. E.P.R. 3 lines, aN = 15.5 G. addition, the reaction mixture was stirred for 3 h IR (neat): v = 930, 1020, 1270, 1350, 1390, 1470, at 20 °C. Triethylamine hydrochloride (16.5 g, 100% 2925, 2960 cm"1. of theory) was filtered off, and washed with an- hydrous ethyl ether (2 x 50 ml). Concentration of CnH35N503P (388.5) the combined filtrate and washings on a rotating Calcd C 52.55 H 9.08 N 18.03, evaporator at 40 °C/20 torr gave a thick, red oil Found C 52.20 H9.14 N 17.70. which partially solidified at 0 °C. The crude product was purified by chromatography on alumina using Preparation of 4-bis[3,3-(l,5-pentanediyl)- benzene as eluant. Trituration of the resultant oily diazir idin-l-yl] phosphinyloxy-2,2,6,6-tetramethyl- product using petroleum ether gave 4.35 g (29%) piperidine-1-oxyl (15 b) of 16, m.p. 106-108 °C, M.S.: m/e = 374. Purity control by T.L.C. (benzene:acetone, v/v 3:1) A solution of 4-hydroxy-2,2,6,6-tetramethyl-l- indicated one product. E.P. R.: 3 lines, aN = 15.0 G. oxopiperidine [51, 52] (13, 1.72 g, 0.010 mole) and IR (KBr): v = 940, 1020, 1040, 1230, 1270, 1350, triethylamine (2.02 g, 0.020 mole) in toluene (25 ml) 1370, 2930 cm-1. was added dropwise to a stirred solution of phos- phoryl chloride (1.53 g, 0.010 mole) in toluene CnH34N403P (373.5) (25 ml) at —20 °C. After stirring for 3 h at 0 °C, the reaction mixture was cooled to —30 °C, and a Calcd C 54.67 H 9.17 N 15.00, solution of 2c [32] (94%, 2.55 g, 0.021 mole) and Found C 54.81 H 9.21 N 15.20. triethylamine (2.02 g, 0.020 mole) in toluene (50 ml) Preparation of bis[ 1-oxyl-2,2,6,6-tetramethyl- was added over a period of 10 min. The reaction piperidin-4-yl]-2-ethyl-3-methyldiaziridin- mixture was stirred for 1 h at —10 °C, then for 16 h at 20 °C, and filtered. The filter cake was washed 1-yl-thiophosphonate (18) with anhydrous ethyl ether (3 X15 ml), then A solution of 4-hydroxy-2,2,6,6-tetramethyl-l- triturated with warm acetone (40 °C, 3 X 25 ml), oxopiperidine [51, 52] (13, 1.72 g, 0.010 mole) and and the mixture filtered. The combined toluene, triethylamine (2.12 g, 0.021 mole) in toluene (15 ml) G. Sosnovsky-J. Lukszo • In the Search for New Anticancer Drugs 893 was added dropwise, over a period of 20 min, to a the brown colored reaction mixture still contained stirred solution of phosphorus trichloride (1.37 g, a large amount of unreacted diaziridine 2d and a 0.010 mole) in toluene (10 ml) at —25 °C. Following complex mixture of several unidentified products. the addition, the reaction mixture was stirred at No attempt was made to resolve this mixture. —5 to 0 °C for 30 min. The mixture was then cooled to —20 °C, and a solution of l-ethyl-3-methyl- Preparation of trisf 3,3-(1,5-pentanediyl)-diaziridin- diaziridine [31] (2e, 95%, 1.90 g, 0.021 mole) and 1-ylJ phosphine oxide (20, R = H) triethylamine (2.02 g, 0.020 mole) in toluene (50 ml) A solution of phosphoryl chloride (2.30 g, was added over a period of 20 min at —20 °C. After 0.015 mole) in toluene (15 ml) was added over a stirring for 2.5 h at room temperature, a solution of period of 15 min to a stirred solution of 3,3-penta- sulfur (0.35 g, 0.011 mole) in toluene (25 ml) was methylenediaziridine [32] (2 c, 94%, 5.36 g, added dropwise at —10 °C. Following the addition, 0.045 mole) and triethylamine (5.05 g, 0.050 mole) the reaction mixture was stirred for 2 h at room in toluene (50 ml) at —10 °C. After stirring for 2 h temperature, then filtered. The filtrate was con- at —5 to 0 °C, and 20 h at room temperature, the centrated on a rotating evaporator. Purification of reaction mixture was filtered. The solid residue was the crude residue by column chromatography on washed with cold water (4 x 25 ml) and dried for alumina with benzene and benzene: ethyl acetate 5 h at 20 °C/1 torr. Recrystallization from toluene (v/v 4:1) as eluants gave a red, oily product (1.10 g). gave 2.3 g (40%) of 20,'R = H, m.p. 151-153 °C. Trituration with w-hexane afforded 0.55 g (11%) of M.S.: m/e = 381. the crystalline 18, m.p. 96-97 °C. Purity control by IR (Nujol): v = 950, 1010, 1130, 1230, 3130 cm-i. the T.L.C. (benzene:ethyl ether, v/v 4:1) analysis indicated one compound. E.P.R.: 5 lines, aN = m NMR (CDCls): d = 1.21-2.28 (m, 30H), 2.63 ppm 7.5 G under conditions of low resolution [54]. (d, 3H, J= 14 Hz). M.S.: m/e = 490(100), 491(91), 492(33). C18H33N6OP (380.5) IR (KBr): v = 930-960, 1160, 1340, 2880 cm-i. Calcd C 56.81 H 8.74 N 22.09, CnHwNiOiPS (490.6) Found C 56.63 H 8.99 N 22.08. Calcd C 53.85 H 8.83 N 11.42, Found C 53.51 H 8.97 N 11.17. Attempted preparation of ethyl- [bis( 2-n-butyl-3-ethyldiaziridin-l-yl )phosphinyl] - O Attempted preparation of tris[3,3-(1,5-pentanediyl) - II diaziridin-1-ylJ phosphine sulfide (19, R = H) carbamate (4, R — NHCOEt) A solution of freshly distilled thiophosphoryl This reaction was carried out according to litera- chloride (2.54 g, 0.015 mole) in toluene (10 ml) was ture procedure [17]. Thus, anhydrous ethanol added dropwise, over a period of 10 min, to a stirred (0.92 g, 0.020 mole) was added dropwise, in 1 min, mixture of 3,3-pentamethylenediaziridine (2 c, 94%, to a stirred solution of isocyanatodichlorophosphate 5.96 g, 0.05 mole) and triethylamine (5.05 g, [5] (3.2 g, 0.020 mole) in toluene (50 ml) at —30 °C, 0.050 mole) in toluene (50 ml) at 0 °C. After the followed by 1 h stirring at 20 °C. The solution of addition was completed, the reaction mixture was l-w-butyl-3-ethyldiaziridine [17] (2g, 96%, 5.33 g, stirred at 20 °C and monitored daily by T.L.C. 0.040 mole) and triethylamine (4.04 g, 0.040 mole) (acetone:hexane, v/v 1:1). After 7 days, T.L.C. in toluene (30 ml) was then added dropwise, over a analysis still indicated the presence of a large period of 10 min at —30 °C. The reaction mixture amount of unreacted diaziridine 2 c, plus a complex was stirred for 12 days at 20 °C, then filtered. The mixture of several unidentified components. No filtrate was concentrated on a rotating evaporator attempt was made to resolve this mixture. at 30 °C/20 torr, and the oily residue was triturated with w-pentane (50 ml). The pale yellow solution Attempted preparation of tris- was decanted from resinous material and stored for [2-methyl-3,3-( 1,5-pentanediyl )-diaziridin-l-yl] 3 weeks in a freezer at —10 °C. During this period phosphine oxide (20, R = Me) no crystalline product 4 was obtained, as indicated in the original procedure [17]. T.L.C. (ethyl A solution of phosphoryl chloride (2.30 g, ether: acetone, v/v 3:1) analysis of this solution 0.015 mole) in toluene (15 ml) was added over a indicated a number of unidentified compounds. No period of 15 min to a stirred solution of 1-methyl - resolution of this mixture was attempted. 3,3-pentamethylenediaziridine [32] (2d, 92%, 6.85g, 0.050 mole) and triethylamine (5.05 g, 0.050 mole) The authors would like to thank the National in toluene (50 ml) at —10 °C. After the addition Foundation for Cancer Research for their support of was completed, the reaction mixture was stirred 2 h this work, and Dr. Peter L. Gutierrez, Laboratory at 0 °C, and 7 days at 20 °C. During this period of Clinical Biochemistry, BCRP, DCT, NCI, NIH, the reaction was monitored daily by T.L.C. Baltimore, MD, for his expert advise in biological (acetone:hexane, v/v 1:1). 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