AZIRIDINO DERIVATIVE OF A CYCLOPHOSPHAZENE COMPOUND

Abstract: Aziridino derivative of a cyclophosphazene compound having the formula (NP <)3, characterized in that the derivative has the formula N3P3AznR6-n, in which n = 2, 3, 4, 5 or 6, and R represents an electron donating substituent of low sensitivity to hydrolysis, and may represent the same or different groups when n = 2, 3 or 4, except for the compounds N3P3Az5Morph and N3P3AZ6, in which formulae Az is aziridino and Morph is morpholino.

Description: Title: Aziridino derivative of a cyclophosphazene compound. The invention relates to an aziridino derivative of a " cyclophosphazene compound having the formula (NP<) . A similar compound is known from Netherlands Patent Application No. 80 07107 ,which further states that such hetero- cyclic derivatives of the cyclojtosxhazene type have an ' anti-tumor activity. It is an object of the present invention to provide novel compounds of the type defined in the opening paragraph, which have an enhanced anti-tumor activity. . According to the invention, the aziridino. derivative referred to in the opening paragraph is characterized in that it has the formula N_P_.Az R→ ,- in which n = 2,3,4,5, or 6, and R represents j 3 n o—n an electron-donating substituent of low sensitivity to hydrolysis, and may represent the same or different groups when n = 2,3 or 4, except for the compounds N_P,Az_Morph and N.P.Az. , in which → b 3 3 6 formulae Az is aziridino and Morph !is morpholino. It is true that the compound N.P.Az is known to have an - . o anti-tumor activity (Eur. J. Cancer 15, 637 (1979), as also the compound N,P_Az.Morph (See, for example, T.S. Safonova, Zh. Vses. Khim. Ob-va im. Mend. 18, 6, 657 (1973)), but in none of these cases is any attention paid to, let alone do the authors appreciate, the effect of substituents other than the Az groups on the anti- tumor activity. Experiments have now shown that, surprisingly, the anti-tumor activity of the cyclophosphazene compounds of the present type depends- less exclusively on the number of aziridino groups in the molecule; but that this activity is greatly determined by the number of activity centres in the molecule, i.e. ' the number of P atoms to which at least one aziridino group is attached and by the presence of electron donating substituents in the molecule or by the interaction between the substituents and the aziridino groups. It is on these experiments that the invention is based, according to which the aziridino derivative of the cyclophosphazene compound contains at least two p-to-Az bonds, preferably in non-geminal position, and further one or more electron donating substituents. According to a further elaboration of the invention, the electron donating group R is a different substituent from i idazolyl, ethylglycinato, amino, and chloro. In the order of increasing preference, based on sensitivity to hydrolysis, R is preferably NΞMe, NHPh, Pyr, Pip, or Morph, in which Me is methyl, Ph is phenyl, Pyr is pyrrolidino,-and Pip is piperidi as in this order the compounds according to the invention maintain their cytostatic activity for a longer period of time. The non-geminal isomers of a group of isomers of a cyclophospha- zene compound according to the invention have been found to have the most outspoken cytostatic activity, and are therefore the preferred compounds of the group of isomers. The invention is illustrated in and by the following examples. The preparation of the compounds described in the following examples was in each case carried out under dry nitrogen. When a ine compounds and tri luroethanol were used, these were purified by distillation over KOH-granules. When imidazole was used, this compound purified by sublimation. Analytically pure (NPC1 ) was used as such, while solvents were.-puri ied and dried using conventional methods. P NMR (Table B) and C NMR spectrums were taken using a Nicolet 283 A FT spectrometer with an NTCFT - 1180 data system in 5 mm tubes at 25°C and 80.9 MHz. The deuterium resonance of the solvent (CDCl ) was used as "field-frequency lock". Purification by HPLCC'high- performarEe-iiquid-chromatography") was carried out using a Spectra-Physi 740 B pump and an LDC refractometer, model 1107. Separation treatments were carried out on a Lichnosorb Si 60/10 column. Mass spectrums for routine purity checks were recorded by means of an AE M.S. 9 mass spectrometer operating at 70 eV, and using an accelleration voltage of 8 kV. Samples were supplied directly and in a conventional manner at a temperature of approximately 100°C. Exanrole I. Preparation of compounds having the generic formula N.P.Az . (A) Geminally substituted derivatives via gem-N ^ P_,Cl_R„. 3 3 3 4 2 (a) Preparation of compounds 16 and 18 (Table A) (R = NH , Ph) . OMPI ' •• Precursors N,P Cl R with R = NH and R - Ph were prepared in known manner (J. Inorg. Nucl. Chem. , 29, 2731 {1967); Chem. Commun. 407, (1968). 32 M oles of aziridine in 30 ml of diethyl ether was added slowly to a stirred solution .cooled at--70°C of .2 mmoles of precursor in 30 ml of diethyl ether. The reaction mixture was allowed to warm up slowly to room temperature and stirred for an additional period of 18 h. The precipitate was removed by filtration and washed with diethyl ether. The combined solutions were evaporated practically do dryness and the resulting crude products were crystallized from hot benzene (compound 16) and diethyl ether (compound 18), respectively, yielding 10% of compound 16, m.p. 250°C (decomp.) and 60% of compound 18, m.p. 155-156°C. (b) Preparation compound 22 (Table A) (R = NHPh) ; Gem - N P Cl (NHPh) was prepared as described by Lederle et al., Inorg. Chem. 5,1818(1966). To a slution of 2.0 mmoles of this starting material in 30 ml of benzene, cooled at 5°C, was added dropwise a solution of 32.0 mmoles,of aziridine in 30 ml of benzene. After warming up to room temperature and an additional 18 h of stirring the temperature was raised to about 50°C fo ' 3 h. Removal of the precipitate by filtration and evaporation of the filtrate practically to dryness gave the crude (solid) product. Recrystallization from THF yielded 75% of compound 22 as a 1:1 THF adduct , m.p. 177-178°C. (B) Via gem— N P Az Cl (compound- 12) . Compound 12 was prepared as .described by Kobayashi et al., Inorg. Chem. 2, 212 (1963).. (c) Preparation of compound 11 (R = OCH CF ) . A solution of 3.0 mmoles of sodium trifluoroethoxide in 10 ml of THF was added slowly to a solution of 2.0 mmoles of compound 12 in 5 ml of THF, cooled at 10°C. The reaction mixture was allowed to warm up slowly to room temperature and left stirring for an additional 48 h. Filtration of the formed salt and evaporation of the filtrate to substant dryness yielded compound 11 as a crude solid product. Recrystallization from diethyl ether gace compound 11 in 38% yield, m.p. 132 - 133°C. (d) Preparation of compound 13 (R = OPh) . A reaction of compound 12 with sodium phenoxide carried out in a similar way to that described for the preparation of compound 11 gave a crude oil, consisting of a 1 : 1 mixture of gem - N_P Az (OPh)Cl 31 and compound 13, according to P NMR. Separation of the two products by HPLC using an acetone/n-hexane mixture as eluent yielded compound 13 (20%) , ' .p. 95°C. (e) Preparation of compounds 15,17,19, 20 and 21, R = Morph, ' NHBu, Pip, Pyr, NΞMe) . To a solution of 2.0 mmoles of compound 12 in 25 ml of benzene, cooled at 5°C, was. added dropwise a solution of 12.0 mmoles of the appropriate a ine in 20 ml of benzene. After being allowed to warm up to room temperature the reaction mixture was stirred for 18 h. Then, for an additional period of 24 h, it was maintained at 55°C. Filtration of the precipitated amine hydrochloride and evaporation of the filtrate to substantial dryness in vacuo yielded the crude products. Recrystallization from diethyl ether/n-pentane mixtures gave analytically pure compounds: compound 15, yield 75%, m;o. 138 - 139.5 C C; compound 17, yield 60%, m.p. 92.5 - 94 β C; compound 19, yield 75%, m.p. 118.5 - 119°C; compound 20, yield 55%, m.p. 88 - 90°C ? compound 21, yield.60%, m.p. 119 - 121°C. (C) Non-geminally substituted derivatives. (f) Preparation of cis-/trans-N P,Az R. (compounds 23,25,27 and 28) via cis-/trans-N-P Cl R (R = Pyr, Pip, Morph) . The starting compounds were prepared as described in Inorg. Chim. Acta, 66, 85(1982). To a solution of 2.0 mmoles of N.P Cl R in 25 ml of benzene, cooled at 5°C, was added dropwise a solution of 60 mmol ofaziridine in 25 ml of benzene. The reaction mixture was warmed up slowl to 50 - 55°C. Dependingon the reactivity of the starting material this temperature was retained for 24 - 96 h. Filtration and subsequent evapora of the filtrate to substeintial dryness in vauo gave the crude product, wh was recrystallized several times from diethyl ether/n-pentane mixtures. Compound 23, yield 80%, m.o. 112.5 - 114°C; 24, yield 40%, m.p. 99 - 100 β OMPI _Λ>__ IP 25, yield 65%, m.p. 99,5 - 100.5°C 27, yield 50%, m.p. 71.5 - 73°C; 28, yield 40'*, m.p, 80 - S2°C. .. ' , (g) Preparation of trans-N P Az Cl~ (compound 26) A (1:8) reaction of NPCl ) , with aziridine, as described by Kobayashi et al. (Inorg. Chem. 2, 212 (1963) gives compound 26 as one of the products. It was isolated in 10% yield by.preparative HPLC, using diethyl ether with traces of ethanol as eluent, nd subsequent recrystallizations from diethyl ether. M.p. 102.5 - 104°C. The trans- structure was assigned due to the simplicity of the right part of the • C NMR spectrum (Fig. 2, doublet at 23.9 ppm) . Moreover, a reaction with excess of pyrrolidine gave compound 24 as the. ltimate product. Example II (A) Preparation of compounds N.P.Az R via compound 33. Compound 33 was prepared as described by Kobayashi et al., Inorg. Chem. 2, 212 (1963). (a) Preparation of compound 39 (R = Im) . To a stirred solution of 2.0 mmoles of compound 33 in 30 ml of THF at a temperature of 10°C,was added slowly a solution of 5.0 mmoles of imidazole in 50 ml of THF. ' The resulting mixture was warmed up to room temperature and stirring was continued at that temperature for 18 h. Thereafter the mixture was kept at ca 45°C for 3 h. The resulting clear solution was evaporated practically to dryneww in vacuo. Compound 19 was isolated from the remaining hygroscopic oil by fractional ..crystallization from THF under dry nitrogen. Yield: 30%. Recrystallization from diethyl ether gave the pure product, m.p.148 - 149°C. (b) Preparation of compound 31 (R = OPh) . At room temperature a solution of- 4.0 mmoles of sodium phenoxide in 15 ml of THF was added dropwise to a stirred solution of 2.0 mmoles of compound 33 in 15 ml of THF. The reaction mixture was refluxed for 18 hour After cooling the precipitated NaCl was removed by filtration. Evaporation of the solvent have an oil, from which compound 31 was isolated by preparative HPLC; using acetone as an eluent, and subsequent recrystalliza from n-pentane. Yield: 50%, m.p. 90 - 92°C. Example III (A) Preparation of compounds 7 and 8. OMPI The starting compounds trans- and cis-(NPPyrCl) were prepared simultaneously as follows: to a stirred solution of 2.0 mmoles of compound 1 in 25 ml of acetonitrile, .-cooled at -14 β C, was added slowly a solution of 12.0 mmoles of pyrrolidine in 15 ml > of acetonitrile. The reaction mixture was warmed up to room temperature and stirring was continued overnight. Evaporation of the solvent .in vacuo yielded a yellowish oil, which was washed three times with 25 ml portions of n-hexane. The combined n-hexane solutions were evaporated to 1/10 of the original volume. Trans-and cis-(NPPyrCl), were separated by HPLC,using n-hexane as an eluent and subsequent recrystallization from n-hexane. Yields: trans-(NPPyrCl) 40%, m.p. 161-163°C; cis-(NPPyrCl) 10%, m.p. 73-75°C. Both compounds were converted to their corresponding aziridino derivatives 7 and 8. For this purpose, the procedure described in Example I-C was used with a reaction time of two days for the trans-isomer and 4 days for the cis-isomer. Both yields were 30%, m. . 105°C (compound 7) and 89-91°C (compound 8). Examole IV. Cytostatic activity was investigated on the basis of the ID50 value (dose for 50% growth inhibition) in a leukaemia L1210 clonogenic assay, and on the basis of the LAD value (smallest active dose) using lymphoma L5178T cells of mice as follows. A. Determination of ID50 values for leukaemia L1210 cells. Drug activity, particularly cytostatic activity on leukaemia L1210 cells was determined using an in vitro clonogenic assay. From a suspension culture, 100 L1210 cells were plated into 35 mm culture dishes (Falcon) containing 1 kl of soft agar growth medium and the test compound in appropriate concentrations. The soft agar growth medium consisted of Dulbecco's medium supplemented with 20% horse serum, 4.75 "mgL 2-mercaptoethanol, 20 mgL L-asparagine, 75 mgL DEAE dextran g (molecular weight 2 x 10 ) and 0.3% bacto-agar (Difco) .The culture dishes were incubated at 37°C in an atmosphere of 10% CO in humidified air for 8 days. After this period of continous drug exposure colonies were counted and dose effect curves were made (Fig. 1). From these curves the drug dose causing 50% inhibition of colony formation (ID50) relative to untreated control cells was calculated (Table I) . B. Determination of LAD values for lymphoma L5178Y. The cytostatic activity of the agents on lymphoma L5178Y cells was determined using a method recently described by Lamberts et al. [28]. The cells were grown in a suspension culture, the medium consisting of RPMI 1640 enriched with 10% fetal calf serum. Penicillin and streptomycin were added as antibiotics. After filtration through a Millipore filter (φ 0.22y) 100 yl of a solution of a compound in physiological saline or, as sometimes required byreason of the slighter solubility in water, in 96% ethanol were serially diluted by a factor of 2 over the wells of a microtiter test plate (Cooke. Mikrotiter System, type U) . Addition of an equal volume of cell suspension containing 2 x 10 cells gave the final concentration range. A row of wells, only containing culture medium and cell suspension was used as control. The cells propagated during four days at 37°C in an incubator, adjusted to standard CO percentage and humidity. After this incubation the lowest growth inhibitive dose was determined by comparing the diameter of each of the precipitation spots with that of the control (growth inhibition causes a decrease in diameter.This gives the lowest active dose (LAD) values within an accuracy factor of 2, as listed in Table A. The tumour cell growth inhibition by the 38 compounds tested, expressed as ID50 and LAD values for the L1210 and L5178Y cells, respec- —6 —1 tively, ranges from 1.1 to 31.5 x 10 mol.l for the first and from —6 —1 0.25 to 250 x 10 mol.l for the latter (Table A). Compound 3 does not possess any activity, and compound 4 was found to be cytotoxic only towards L1210 cells. Direct comparison of data, obtained by such different methods, is hardly feasible. ^ However, within both series of values the same trends can be observed. The cytostatic activities of all compounds are listed together with the substituent constants α and γ (R*Az) . Obviously, there is R R a relationship between α (γ ) and the ID50 or LAD values. Hparticular, a R it is seen that in the series with formula gem-N P Az R (compounds 29-36) and N P Az R (compounds29-36) higher α (γ )values (i.e. stronger electron-releasing capacity) lead to a higher activity for both the leukaemi and the lymphoma cells. Compounds 14, 16 and 30 do not fit this rule. his can be explained from their relatively high sensitivity towards hydrolysis. In an extended study on compounds of type N_,P_R. Allcocϊ 336 _^JR£ et al. obtained the following order in hydrolysis rates for R ~~ ~ OMPI ~*( mo > GlyE > NH > NHMe > NHPh > Pyr , Pip, Morph (Inorg. Chem. 21 ,515 ( 1982) ) . under physiological circumstances aziridino derivatives will also react with water. Whether the first hydrolysis step will be opening of one of the three membered rings or nucleophilic attack on other sites of the molecule will strongly depend on the nature of other substituents. For compounds 16 and especially 14, 29 and 30 hydrolysis can be expected to overrule the biological alk lating action. In addition to the.imidazolyl, amino and ethylglycinato derivatives, compounds with chlorine ligands are easily hydrolyzed and it is experimentally found that solutions of these compounds in neutral water appreciably lose cytostatic activity within 24 hours on standing at room temperature. Ihaddition to hydrolysis effects the activity spectrum might be explained from electronic effects. An explanation might be that substituents with high α ( ) values will increase the R R electron density on the P-N system as well as on the three-membered rings. The resulting higher basicity of endocyclic and exocyclic nitrogen atoms leads to easier electrophilic attack, which might be rate-determining in the opening of the aziridino rings and promote the alkylating ability of the compounds. I.e. the trifluoroethoxy derivative 11 should process a rather low basicity compared with the corresponding amino derivatives. This might explain the relatively low cytostatic activity of the former compound. With regard to the dependency of the effect of the number of aziridino groups in the- olecule, the following can be concluded. The series of ethylglycinato derivatives 3, 5, 14 and 30 suggest some dependence, but the activerange can also be correlated to the sensitivity relative to hydrolysis (see the series of rates of hydrolysis listed hereinbefore). Moreover, the pyrrolidino derivatives, except for coiπpound 4 and 6, all exhibit approximately the same activity. This shows that an evaluation of biological activity merely in terms of the number of aziridino groups is an oversimplified representation: A change of the OMP >. number of alkylation centers from two to one may result in a greater effect. Experiments, conducted with the three isomers N P.Az.R and with N-P-AzR- (R=NHMe, NMe 23 Pyr, Morph) show the following activity sequence: Trans _> Cis x * Gem ?// Mono Example V. Preparation of N P Az Cl (Az = NC 2 H 4 ) • 6.2 ml Aziridine (120 mmoles) in 50 ml dry benzene was dropped into a stirred solution of 10.43 g (NPC1.), (30 mmoles) in 100 ml dry benzene, cooled to 6° C. After being slowly warmed up to room temperature, and a total reaction period of 18 hours, the amine HC1 salt formed was filtered off. Evaporation of the filtrate in vacuo gave 11.6 g of a white waxy product (107%, calculated as N_P,Az.Cl .) . Subsequently, to remove any remaining salt and polymeric material, the product was transferred to a silica gel column with a hexane/ether eluent (3:2). 31 Analysis by means of P NMR, mass spectrums and HPLC showed the product contained mono-, bis-, and some tris-aziridino-substituted derivatives of (NPCl.),. Separation was effected with preparative HPLC over a Si-column (30 cm; 0 22 mm) with hexane/ether (2:1) as the eluent. Fractions according to increasing retention period (see Fig. 3) : 1 solvent 2 N 3 P 3 ZC1 5 K' = 0.6 3 N 3 P 3 AZ 2 C1 4 (non-geminal) k' = 1.2 4 N 3 P 3 z 2 Cl 4 (geminal) k' = 2.0 5 N 3 P 3 AZ 2 1 4 (non-geminal) k' = 3.4 6 N 3 P 3 Z 3 C1 3 k' = 5.7 The various fractions were separatelycollected and processed by evaporation and re-crystallization: Fraction 2: 1.50 g of a white solid. Re-crystallization from hexane yielded 1.05 g white crystalline material (9.9 %) . Melting OMPI point 67-68.5° C (lit,value: 69-70° C Inorg. Chem 3,757 (1964)) . Fraction 3: 1.8-7 g colourless oil. Re-crystallization from hexane gave 1.49 g white crystalline platelets (13.7 %) . Melting point 66.5-68.0° C. Fraction ' 4: 1.66 g of a white solid. Re-crystallization from hexane/ diethyl ether gave 1.43 colourless needles (13.2 %) . Melting point 105.5- 07° C (lit. value: 104-105.5° C, Inorg. Chem. 3,757 (1964)) . Fraction 5: 1.49 g ofacolourless oil. Re-crystallization from hexane yielded 1.06 g of a white crystalline material (9.8 %) . Melting point 65.5-67.0° C. Further characterization was effected by means of I.R. , M.S., 1 31 ΞNMR, PNMR and elemental analysis. It was not possible, for that matter, on the basis of this data to assign any (cis/trans) structure with regard to fractions 3 and 5. This assignment was not possible until after conversion of the resulting products to e.g. N-P.Az.(NHMe) 13 3 3 2 4 derivatives and analysis of C spectrums. On the basis thereof, the transstructure could be assigned to fraction 3 and the cis-structure to fraction 5. A closer examination of fraction 5 shows that it consists of mainly Cis-N P Az Cl that is contaminated with ca. 10% Trans-N P_Az Cl_, However, the re-crystallized material is free of contamination. NMR data H NMR 31 P NMR 6 H (ppm) δPAz 2 (ppm) δPAzCl δPC J (Hz) J PH (ΞZ) N 3 3 ZC1 5 2.35 21.5 31.2 22.2 39.0 Trans-N,P,Az_Cl. 3 3 2 4 2.34 22.0 34.7 25.0 38.0 Cis-N 3 P 3 Z 2 C1 4 2.32 22.0 34.1 24.9 38 a .2 Gem-N 3 3 z 2 Cl 4 2.20 17.5 34.2 21.9 30.0 Ξle ental analysis (calculated values indicated in brackets) C ( % ) H ( %) N ( %) Cl ( %) N P AzCl. 6.93 ( 6.78) 1 .12 ( 1 .14) 15 .61 ( 15 .82) 49.60 ( 50.03) Trans-N P Az Cl 4 13.27 (13.31) 2.25 (2.24) 19.53 (19.41) 39.01 (39.29) Cis-N P Az Cl 13.40 (13.31) 2.11 (2.24) 19.30 (19.41) 39.53 (39.29) Gem-N P Az Cl 13.35 (13.31) 2.29 (2.24) 19.18 (19.41) 39.54 (39.29) Mass spectra The mass spectrumof. N,P,AzCl_ shows a parent peak M =354, together with the expected isotopes. The mass spectra of the bisazizi- dinyl derivatives are identical: M = 361. Preparation of N P Az and N P AzR (38-53) (R = NHMe, NME., Pyr, Morph) As reaction media served either benzene (R=NHMe, NMe„, and Morph) or diethyl ether (R = Pyr) . To a solution of 500 mg N.P.Az.Cl. or N_P_,AzCl_ (ca. 1.4 mmoles) 3 3 2 4 3 3 5 in 15 ml of dry solvent, cooled to the appropriate temperature and vigorously stirred, was .added dropwise a solution of an excessive amount of the desired amine, dissolved in 15 ml of dry solvent. After warming up to room temperature and a reaction time of 18 hours, the temperature was raised to 50° C (benzene) or to reflux temperature (ether) for an additional period. Removal of amine-HCl salts by filtration and evaporation "in vacuo"of the solvent afforded the crude products that were crystallized from an appropriate solvent. The purification of Cis-N P Az Morph , Cis- and Trans-N P Az (N e , and N P AzfNMe . required the use of HP1C because of the presence of unreactive inter¬ mediate products. Trans-N,P,Az. (NMe^). was finally purified by sublima¬ tion (60° C; 1 mm Hg) as it was too soluble in all suitable solvents. The table provides further details. Preparative Data (38-53) Product No. Molar Ratio Temp.Range Reaction Time Recryst. Yield Ring/Amine (°C) (hrs.) from (%) N,P,Az(N__Me)_ 38 1/40 5-50 24 benzene 49 Gem-N P Az (NHMe) 39 1/30 5-50 24 benzene/pentane 63 Trans-N_P_,Az,_(N__Me) Λ 3 3 2 4 40 1/30 5-50 24 benzene/pentane 73 41 1/30 5-50 48 benzene/pentane 40 N 3 P 3 Az( Me 2 ) 5 42 1/25 5-50 68 pentane Gem- 3 P 3 Az (NMe ) 43 1/20 5-50 48 pentane 50 rans-N 3 P 3 Az (NMe ) 44 1/20 5-50 48 (subl.) 36 Cis-N 3 P 3 Az 2 (NMe 2 ) 4 45 1/20 5-50 48 pentane N P Az(Horph 46 1/30 5-50/refl. 100 diethyl ether 20 Gem-N.-P-.Az (Morph) 47 1/20 5-50 48 diethyl ether 55 Trans-N P Az (Morph) 48 1/20 5-50 68 diethyl ether 41 Cis-N P Az (Morph) 49 1/20 5-50 68 diethyl ether 36 N 3 P 3 Az(Py ) 5 50 1/20 5-refl. 29 pentane 30 Gem-N P Az (Pyr) 51 1/14 5-ref1. 20 pentane 70 Trans-N .-P Az., (Pyr) , 3 3 2 4 52 1/14 5-refl. 18 pentane 32 Characterization Data on N.P-AzJR. and N-,P_AzR_ (38-53) j J _s 4 3 3 5 31 PNMR* Product m.p. δP(AzR) 6P(R). δP(Az) IPP Eli emental Anal * ; ses No. (°C) (ppm) (ppm) (ppm) (Hz) C(%) H(%) N(%) 38 154-156 30.5 21.9 40.0 39 147-148 21.8 38.2 37.9 28.47(28.32) 7.20(7.13) 36.59(37.16) 40 153-155 : 30.0 22.1 37.4 28.21(28.32) 7.18(7.13) 36.67(37.16) 41 152-153 29.7 21.9 38.2 28.27(28.32) 7.11(7.13) 36.61(37.16) 42 43 85.87.5 25.6 37.7 38.4 36.51(36.45) 8.21(8.17) 31.91(31.88) 44 60-63 31.6 25.4 37.4 36.40(36.45) 8.18(8.17) 32.05(31.88) 45 30.8 24.5 37.6 46 150-154 27.6 19.8 39.1 47 158-159. 5 20.0 37.0 37.9 42.73(42.63) 7.22(7.15) 22.34(22.37) 48 157-158. 5 28.5 19.5 38.9 42.56(42.63) 7.25(7.15) 22.59(22.37) 49 149-150 27.3 19.8 38.2 42.80(42.63) 7.19(7.15) 22.53(22'.37) 50 106-108 27.8 17.3 38.5 50.04(50.09) 8.43(8.41) 24.20(23.89) 51 97-98 16.9 37.1 37.9 48.20(48.09) 8.18(8.07) 25.59(25.24) 52 93.5-94. ,5 27.6 17.0 37.6 48.13(48.09) 8.26(8.07) 25.28(25.24) Exa ple VI Preparation of Gem-N P Az (NH ) Cl (54) Attempts to aminolyse N.P. z.Cl^ with NH, have led to the synthesis of 54. A solution of 750 mg of Gem-N_P Az.Cl. in 30 ml of CH-CN, cooled to -10° C, was saturated with NH, gas for 30 minutes. The precipitate was filtered off and washed with CH_CN. The filtrates were combined and evaporated to dryness "in vacuo". Re-crystallization of the solid white product from CH 7 Cl_/diethyl ether yielded several fractions of white crystalline needles. Total yield: 529 mg (79 %) m.p. 149-150° C. Elemental analysis: C 15.07(14.92), H3.74(3.70), N30.31 (30.45) , C122.19 (22.02) 31 p N R : AMX spin system δPAz = 35.7 ppm; PCl 2 = 26.0 ppm; δP(NH ) = 14.9 ppm 2y pp= 34.0/34.4/41.4 Hz Mass spectrum : M = 32.3 See for the compounds the accompanying sheet of formulae, which illustra¬ tes the structural formulae. The horizontal line is a diagrammatic representation of the plane of the molecule, and the points of intersec¬ tion of this line with the bond-lines to the various substituents each represent a P atom. OMPI Cytostatic Activity in vitro Lymphona L51784 Leukemia L1210 Compound No LAD ( ,UM) 1050(V,UM) N P Az(NHMe) 5 38 ' 200 > 245 Gem-N P Az (NHMe) . 39 8 27.8 Trans-N P Az (NHMe) . 40 1.5 3.3 Cis-N P AZ 2 (NHMe) 41 1.0 5.0 N 3 P 3 z(N E 2 ) 5 42 632 > 160 Gem-N P 3 Az 2 (NMe 2 ) 4 43 20 19.5 Trans-N P Az (N e 2 ) 44 0.5 2.9 Cis-N 3 P 3 z 2 (NMe 2 ) 4 45 0.5 N P Az(Pyr) 50 12 44.3 Gem-N 3 P 3 Az 2 (P r) 4 51 4 18.6 Trans-N P Az (Pyr) 52 1.0 4.8 Cis-N P z 2 (Pyr) 53 1.0 6.7 P z(Morph) 46 n.a. n.a. Gem-N P Az (Morph) 47 116 130.7 Trans-N P Az (Morph) 48 28 24.7 Cis-N P Az (Morph) 4 49 59 33.5 Gem-N 3 P 3 Z 2 (NH 2 ) 2 C1 2 54 0.3 3.2 General sequence: Trans ^ Cis y Gem Mono of activity Exa ple VII Some compounds have been tested in vivo in mice, with the following results: Toxicity Dose single ComDOund number: mg per kg -1 T/C (%) L1210 mg.kg mouse ID 50 20. Gem-(NPAz ) NPPyr 200-250 160 178 200 211 19. Gem-(NPAZ ) NPPiP 250 200 156 240 167 ° 15. Gem-(NPAz ) NPMorph 2 400-450 400 211 + • 20% cured 14. Gem-(NPAz 2 ) 2 NP(GlyE) 2 170 inactive * 25. Trans-(NPAz ) (NPPipAz) 140 90 167 110 189 36. (NPAz 2 ) NPAzNΞMe 45 40 190 + 20% cured 5 23. Trans-NPAz (NPMorphAz) 250 160 210 + 20% cured 35. (NPAz ) 2 NPPipAz 80 70 210 + 20% cured 40. Trans-N 3 P Az,(NHMe) 4 50 30 200 + 20% cured Trans-NP(NHMe) (NPAzNHMe) 35 213 0 T = average survival in days of the mice treated with the compound 5 C = average survival in days of untreated mice *) With a dose of 150, a T/C value of 89 was measured, and with a dose of 180 a value of 89. 0 The mice for the toxicity investigation were F mice, which were a crossbreed of C57 Black and CBA Black. The mice for the activity investigation were F mice, which were a crossbreed of Balb C mice with DBA/2 mice. In each instance series of 5 mice were used. OMPI A B L E A Cytostatic activity in vitro of aziridino cyclofosfazenes- b b Formula a compound leukaemia L1210 lymphoma L5178Y number R ≠ Az ID50 (10 "6 mol.L _1 ) LAD(10~ mol.L -1 ) 3 P 3 (GlyE) 6 3 5.0" 2.5" not active not active 3 P 3 Pyr 6 4 5.9 2.9 18.0 not active gem-N 3 P 3 Az 2 (GlyE) 5 5.0 d 2.5 d 31.5 250 3 P 3 z 2 Pyr 2 Cl 2e 6 5.9/0 1 2.9/0 13.6 250 trans-N P,Az Pyr, 7 5.9 2.9 4.5 2 cis- 3 P 3 A Z3 Pyr 3 8 5.9 2.9 2.1 2 gemn--NN,,PP A Azz MMoorrpphhCCl 9 5.0/0 2.5/0 25.4 16 J J 4 ggeemm--NN PP AAzz PPiippCCll 10 5.6/0 2.8/0 16.6 16 ggeemm--NN,.PP..AAZz, (OCH-CF,)_ 11 1.0 0.3 3 9.1 16 ■i 33 44 3 ^ gem-N,P_Az.Cl„ 12 0 3 3 4 2p 0 8.9 8 gem-N 3 P 3 Az 4 (Oph) 13 3.1 1.3 7.6 8 gem-N P Az (GlyE) 14 5.0 d _.5 d 7.2 16 gem-N_P_Az.Morph„ 15 5.0 2.5 5.8 4 3 3 4 2 gem-N 3 P 3 Az 4 ( H 2 ) 2 16 6.0 2.7 3.9 4 gem-N,P_Az.(NHBu) 17 5.8 3.6 3.9 2 3 3 4 2 gem-N P Az Ph 18 4.2 2.2 3.7 2 gem- 3 P 3 Az 4 Pi 2 " 19 5.6 2.8 3.6 2 gem-N-P-Az Pyr 20 5.9 2.9 2.7 1.0 Con d. Table A trans-N P,Az.M 23 0 2.5 2 3 orph„ 5. 3 4 2 4.7 trans-N,P,Az.Pyr. 24 5.9 2.9 2.2 2 3 3 4 J 2 trans-N P Az Pi 25 5.6 2.8 2.1 2 trans-N_P-.Az.Cl_ 26 3 3 4 2 0 0 1.9 2 cis-N.P Az Pyr 27 5.9 2.9 1.8 1.0 cis-N P.Az Pi 28 5.6 2.8 1.6 2 N3.P3,Az5_OPh 31 3,1 1.3 4.4 2 N,P,Az_Morph 32 J J 5 5.0 2.5 3.5 2 3 P 3 Az 5 Cl 33 0 0 3.5 2 N 3 3 Az 5 Pyr 34 5.9 2.9 1.6 1.0 N P Az Pip 35 5.6 2.8 1.5 1.0 N,P_Az_(NHMe) 36 5.8 3.1 1.2 0.25 37 - - N 3 P 3 AZ 6 2.0 1.0 a. he following abbreviations have been used: Az: (Aziridino) , GlyE (Ethylglycinato), Im (Imidazolyl), Morph (Morpholino) , Pip (Piperidino) , Pyr (Pyrrolidino) . b.Substituent constants for N P derivatives. Values taken from Rev.Inorg.Chem. 3, 1,1 (1981), J.Chem.Soc. (A) 10, (1968), J.Chem. Soc. (A) 196, (1969) ,and J.Chem. Soc. (A) 2468, (1969). c.Estimated valye: α ( γ ) _ α ( γ ) Morph d.1>l-bisaziridino-cis-3.5-dichloro-3.5-bispyrrolidino derivative. TABLE B O -T V * . P NMR and Elemental Analysis data Compound δ(P z 2 ) (PR 2 ) δ(PAzR) J. pp (Ξz) C(%) H(%) N(%) 7 27.1/27.7 34.9 45.75(45.86) 7.66(7.70) 26.93( 8 26 .7 — 45.77(45.86) 7.57(7.70) 26.90( 11 38.4 18.0 52.7 28.89(28.75) 3.99(4.02) 19.73(1 13 38.2 9.5 52.4 49.11(49.08) 5.39(5.36) 19.93( 15 36.7 20.2 37.6 39.88(40.42) 6.79(6.78) * 26.44(2 16 37.4 18.6 . 36.6 28.45(28.66) 6.08(6.01) 36.65(3 17 37.6 18.5 36.2 42.64(42.95) 8.09(8.11) 27.87(2 18 36.2 18.9 15.6 52.32(52.52) 5.72(5.73) 21.05(2 19 36.8 20.7 36.5 46.00(45.86) 7.77(7.70) 26.70(2 20 36.9 16.5 35.8 43.36(43.34) 7.44(7.27) 28.24(2 21 37.6 21.6 36.1 32.87(33.06). 6.68(6.66) 33.81(3 22.THF 38.1 7.0 38.5 51.43(51,52) 6.55(6.49) 22.50(2 23 37.1 28. .5 33.0 40.36(40.42) 6.85(6.78) 26.50(2 24 36.8 27, .4 33.8 43.36(43.34) 7.24(7.27) 28.13(2 25 37.4 28. .7 33.3 45.76(45.86) 7.70(7.70) 26.53(2 26. 37.6 40. .0 29.7 25.69(25.69) 4.28(4.31) 26.31(2 27 36.6 26. .8 33.3 43.13(43.34) 7.23(7.27) 28.25(2 28 37.1 27. .4 32.6 45.87(45.86) 7.74(7.70) 26.73(2 29 37.1 20. ,8 35.4 38.03(37.87) 5.76(5.62) 33.07(3 31 37.4 25. ,1 39.8 43.52(43.84) 5.74(5.75) 25.57(2 Chemical shifts (ppm) are relative to 85% H PO and positive in down field direction; solvent CDC1_. Calculated values in parentheses. " Deviation as a result of systematical errors.

Claims: C L I S 1. Aziridino derivative of a cyclophosphazene compound having the formula (NP £) , characterized in that the derivative has the formula N,P,Az R_ , in which n = 2, 3, 4, 5 or 6, and R represents 3 3 n 6— an electron donating substituent of low sensitivity to hydrolysis, and may represent the same or different groups when n = 2, 3 or 4, except for the compounds N,P,Az_Morph and N P,Az_, in which formulae Az is aziridino and Morph is morpholino. 2. A derivative according to claim 1, characterized in that R is a different substituent from imidazolyl, ethylglycinato, NE_ and chloro with the exception of the compound Gem-N P Az (NH_)Cl . 3. A derivative according to claim 2, characterized in that the electron donating substituent is NHMe, NHPh, Pyr, Pip or Morph, in which Me is methyl, Ph is phenyl, Pyr is pyrrolidino, and Pip is piperidino. 4. A derivative according to claims 1-3, characterized by a non- geminal isomer thereof.

Inventors: VAN DE GRAMPEL JOHAN CHRISTOPH, VAN DER HUIZEN ADRIAAN ALBERT, VAN DE GRAMPEL, JOHAN, CHRISTOPH, VAN DER HUIZEN, ADRIAAN, ALBERT
Applicants: UNIV GRONINGEN, RIJKSUNIVERSITEIT TE GRONINGEN
Application year: 1984
Main classification: C07F9
IPC codes: C07F9