The invention relates to an aziri±Lno derivative of a tetrameric cyclochlorophosphazene compound.

The (NPCLj )-tetramer having the formula N P.C and the com¬ pound N.P'Az derived therefrom, ^' in which Az is aziridino, are known from the article by V.A. Chernov, V.B. ytkina, S.I. SergievsJay , A.A. ropacheva, V.A. Parshina and L.E. Sventsitskaya, Farmakol. Toksikol. (Moscow) 22, 365 (1959). Of the compound N P Az it is indicated that it has an anti-tumor activity with respect to S-45 sarcoma in rats. Moreover, Inorg. Chem. 3 _^ (1964) 757-761 discloses that the compound N.P.Az can be prepared by complete aminolysis of the tetrameric N.P.C1-, by means of aziridine or a homologue thereof in an aromatic hydrocarbon as reaction medium and triethylamine as acid acceptor. It is an object of the invention to provide an aziridino deri¬ vative of a tetrameric cyclochlorophosphazene compound which may serve as starting-material in the synthesis of tetrameric cyclophόsphazene compounds to be derived therefrom and containing one or more aziridino groups by substitution of the chlorine atoms by a properly selected substituent, of which latter compounds it may be expected that they also have an anti-tumor activity.

For this purpose the invention provides a compound of the type defined in the opening paragraph, characterized by the formula N P C1 Az , in which n » 1,2,3,4,5,6 or 7.

Although the preparation of the compounds according to the invention proceeds rather easily with good precautions, the isolation of different, mostly isomeric products is not easy. E.g. the reaction of (NPC .) . with aziridine gives at a molar ratio of 1:3.5, mainly the 6 products

N,P.C1_AZ 44 7 ea-N ₄ P ₄ Cl ₆ Az ₂

^' l,3-cis-N ₄ P ₄ Cl ₆ Az ₂

1,5-cis-N4. 4.Cl6,Az2_

1,3-trans-N4.P4.Cl6.Az2-

1,5-trans-N-P.Cl-Az-, in addition to a number of products N.P.Cl _^ Az, 44 6 2 * 44 5 3. A schematic representation of the structuralformulae of these compounds, in which the ring-N-atoms and the Cl-atoms have been omitted, is given by formulae 1-6 of the sheet of formulae.

In accordance with what has been stated in the preceding para¬ graph the invention therefore also relates to a process for preparing an aziridino derivative according to the invention by aminolysis in a reaction solution of a cyclopolychlorophosphazene compound and working up of the reaction mixture,which process is characterized in that in a compound having the formula N 4.P•.Clo.—nAzn, in which n = 0,1,2,3,4,5 or

6, 1-7 chlorine atoms are substituted by an aziridino group and that the resulting aziridino derivative are recovered from the product obtained after working up of the reaction mixture by means of HPLC ("high perfor¬ mance liquid chromatography") .

In the process according to the invention the selection of column material and eluent depends, within the scope of application of the HPLC technique, on the reaction mixture to be analyzed.

As will be elucidated hereinafter, the ratio of mono-aziri- dino to polyaziridino substitution is, e.g. in the case of starting from (NPCL ) ,the ratio in the reaction product of ono-aziridino to di-aziridino substitution, to be varied by affecting the molar ratio of the reaction components and, if required, the reaction time.

A suitable solvent in which the process according to the inven- tion can be carried out is dry diethyl-ether but also benzene, pentane, hexane and THF (tetrahydrofuran) are suitable for having reactions carried out therein.

The aziridino derivative of the tetrameric cyclochlorophosphazene compounds according to the invention are suitable starting materials for preparing compounds therefrom, the chlorine atoms being replaced by properly selected other substituents. In view of the teaching from later published Dutch patent application no. 83.00573 it may be expected that such compounds have an anti-tumor activity.

Consequently, the invention also relates to an aziridinoderivative of a tetrameric substituted cyclophosphazene compound having an anti- tumor activity, characterized by the formula N.P.R„ Az , in which

² . 4 48-n n n = 1,2,3,4,5,6 or 7 and R represents the same or different substituents.

Preferably, R is an electron donating group of low sensitivity to hydrolysis.

The invention will be illustrated by the example given herein below. Examole I

Preparation of

( PC _) . (Otsuka Chem.) was recrystaϊ3ized from hexane before use. Aziridine was distilled from KOH pills under dry nitrogen just before use. Solvents were purified and dried in the conventional manner. Reactions were carried out under a dry nitrogen atmosphere. . P and H NMR spectra were measured with a Nicolet 283A FT spectrometer equipped with an NTCFT- 1180 data system, in 10 mm tubes at 25°C. The deuterium resonance of the solvent (CDC1-) was used as "field-frequency lock". HPLC separations were carried out by using two Waters 6000A liquid pumps (each having a capacity of 20 cm /min.) and a Waters R401 refractometer. Lichrosorb Si 60/10 served as column material.

A. Reaction of (NPCL_) „ with aziridine in the molar ratio of 1:2.5.

2 4 A solution of 1.4 cm of aziridine (27.1 αmol) in 150 cm of dry diethyletherwas added dropwise to a solution of 5.0 g of (NPC1- (10,8 mmol) in 300 cm of dry diethyl etherfor 30-45 min., while vigorously stirring and cooling to - 20°C. After the reaction mixture was warmed up slowly to room temperature and after a reaction time of 18 hours filtration of the polyaeric amino-HCL salt and evapo¬ ration of the filtrate gave 5.1 g of a white waxy oil which turned out to be slightly sensitive to hydrolysis (Product A) .

B. Reaction of (NPCL ) . with aziridine inthe molar ratio of 1:3.5.

A solution of 3.9 cm of aziridine (77.8 mmol) in 100 cm of dry diethyletherwas added dropwise to a solution of 10.0 g (NPC1.,) .

24

(21.6 mmol) in 400 cm of dry diethylether for 30-45 min. , while vigorously stirring and cooling to - 0°C. ^ _{reaction ffiixture}

was warmed up slowly to room temperature and stirred further until a total reaction time of 7 hours. The working up procedure as set forth below A. gave 10.5 g of a turbid oil sensitive to hydrolysis (Product B

C. Analysis of the products.

31 Analysis of P NMR andmass spectra as well as HPLC diagrams

(Fig. 1 and Fig. 2) showed that products A and B had the same composi¬ tion in principle. A especially contained N P AzCl_ while B, in addition to this component, especially contained N P Az.C (namely 5 isomers) . The ratio of mono/disubstitution was to be affected by varying the molar ratio and the reaction time. It turned out that a reaction mixture such as product B was also to be obtained starting from N.P.AzCl-, in a 1:2 reaction with aziridine in dry diethyl ether.

D. Separation methods

It turned out that both product A and product B could be separate with HPLC by using a 25% diethyl ether/75% hexane eluent. Product A give

N P AzCl as the largest fraction (Fig 1,fraction 1). In total, 2.56 g were obtained (yield 50%). Recrystalization from entane gave 1.9 g of analytically pure material; melting point 68.5-70.0°C.

Under corresponding conditions product B gave seven fractions (Fig. 2) : Fraction no. : (1) _^ P _^ AzCl. 1.54 g

4 4 7

(2) N ₄ P ₄ Az ₂ Cl _g 2.12 g

(3) " 1,26 g

Y different isomers

(4) " 0,65 g

(5) N ₄ P ₄ Az ₂ Cl ₆ 1.63 g

"BUR£4]

Fraction no. : (6) N .P .Az.Cl 0.57 g . .. __ . .

4 4 3 s ' / different isomers

(7) 0.38 g

^N 4 ^P 4^3 ^C1 5

Total 8.15 g } ^* - ^*** 77.8% on product B.

It turned out that fraction 5 consisted of 2 components which were once again separated afterwards with the same eluent (Fig. 3) .

Yield. ,

Fraction no.: 5 : N-P.Az-.Cl_ 0.20 g II 44 2 6 5 ^± : N ₄ P ₄ Az ₂ Cl ₆ 1.02 g

Total 1.22 g = 75%, calculated on fraction 5

(1.63 g). E. Characterization

Mass spectra

Themass s ectra of both N.p.AzCl. and N.P _^ z.Cl- showed diffe-

44 7 44 2 6 rent chlorine isotope peaks in addition to parent peaks of respective¬ ly M ⁺ - 467 (for ³⁵ C1) and M ⁺ = 474 (for ³⁵ C1) . The spectra of the different isomeric forms of N.P.Az.Cl.were not distinguishable.

4 4 2 6

Infrared spectra

N P.AzCl_ gave a ring frequency at 1316 (broad) or 1279 cm (sharp) ; the "aziridino" band lay at 965 cm (sharp) . The IR spectra of the isomeric compounds N P Az Cl. were clearly distinguishable. Ring frequencies varied from 1310-1334 cm (broad) or from 1275-1279 cm (sharp) . Aziridino bands were visible from 963 to 976 cm (sharp) .

0ΛPI •Λ, WiK) fr

NMR spectra Substance HPLC 31 P, spec- δP. δP δ ^{: L} H , Isom fraction tru M. δP., 2 2

AM MX (form) (Hz) (HZ) (HZ)

N .P .AzCl. 1 8 .57 -4,68 -7.17 27.6 30.6 2, .35 22 4 4 7 ^AM 2 ^X

^N ₄ ^P Λ ^C 1 ₆ 2 A ₂ X ₂ 8 .37 -1,92 27.9 2, .32 22 (1 ,5

3 ₂ X ₂ 8 .71 -2.61 28.4 2. .32 22

^N 4 ^P 4 ^AZ 2 ^C1 6 (1 . 5 ^N 4 ^P 4 ^A2 2 ^C1 6 AA'XX' 11 .88 -4.67 2. .30 22 ( 1 .3

.II AA'XX' 10 .38 -4.85 2. .29 22 ( 1 ,3

^N 4 ^P 4 ^AZ 2 ^C1 6 N ₄ P ₄ Z ₂ C1 ₆ AM ₂ X 18 .80 -6,2(multiplet) 2.26 16 _* 5 gem

31 P "chemical shifts" in ppm relative to H.PO. 85%; H "chemical shifts"

3 4 in ppm with TMS as reference.

Elemental analysis and melting points

HPLC- Substance mpt.(°C) C{%) H(%) N(«) ?{%) C} {%) fractlon

1 N ₄ P ₄ AzCl ₇ 68,5- 70 5,07(5.11) 0,84(0,86) 14,85(14.90) 26,31(26.35) 52,60(52,78)

2 W ^Z 2 ^C1 6 ^• 103 -104 10,11(10.08) 1.60(1,69) 17,56(17.63) 26.17(25,99) 44,63(44.62)

3 ^N 4 ^P 4 ^Az 2 ^C1 6 1 12222,,55--112233,510.08(10,08) 1,61(1,69) 17.66(17.63) 25,84(25,99) 44,64(44,62)

4 4 ^P 4 ^Az 2 ^C1 6 91 - 92 10,21(10,08) 1.68(1,69) 17.73(17,63) 25.98(25,99) 44,29(44,62)

5 ^{11 N} 4 ^P 4 ^Az 2 ^C1 6 74 - 75 10.43(10,08) 1.66(1,69) 17,47(17,63) 25,92(25,99) 44,53(44,62)

5 ¹ N _{4 4} Az ₂ Cl ₆

Fractiqn 0 is solvent.

The calculated values are mentioned in brackets.

N ₄ P ₄ AzCl ₇ was recrystallized from pentane; all the other substances mentioned above, apart from fraction

' 1

5 , were crystallized from a mixture of diethyl ether and pentane.

Example II

Preparation of a number of aziridinoderivative having the

_* formula N4.P4.R8--nAzn.

In de preparation of the abovementioned aziridino derivative the resulting reaction mixture was worked up according to procedure (a) mentioned herein below:

Procedure (a)

Most reactions afforded considerable amounts of hydrochloride salts, either precipitated or in solution. The use of aziridine as a hydrochloride scavenger resulted in the aziridino chloride salt which is rather unstable andsubsequently polymerized.

Precipitated (polymeric) salts are removed by filtration and , after washing thoroughly with solvent, the combined filtrates contain¬ ing, the P-N ring compounds are evaporated in vacuo. If acetonitrile or THF is used as solvent, the complete reaction mixture is evaporated in vacuo. Extraction with diethyl ether or benzene yields solutions of the salt-free crude products.

All crude products are purified by recrys allization from an appropriate solvent. Mixtures are separated by HPLC and the resulting fractions are subsequently recrystallized.

Preparation of N.P.AzAm. and N.P„Az_Aπι_ (Am « NHme, NMe„, 44 7 4 4 2 6 2 wherein me = methyl: compounds nos. 11-22): the compounds having

II ^ - formulae 1-5 of the sheet of formulae were used as starting compounds.

N _Λ P _Λ Az(NHMe) _ and N .P .Az. (NHMe) _ 4 4 7 4 4 2 6.

To a stirred solution of 0,5 g (ca. 1 mmol) of the ring compounds in 15 cm of chloroform, cooled at 0°C, were slowly added 15cm of a

1 M solution of methylamine in benzene. After warming up to room te pe- rature and a reaction time of 18 h application of procedure (a) afforded the crude products. There was obtained a white solid when the compound having formula 2 of the sheet of formulae was used as starting compound. In all other cases the products consisted of resinous oils. All compounds were reerystaϊ-ti.zed several times from mixtures of diethyl ether and aiethylene chloride. When the compound having formula 5 of the sheet of formulae was used as starting material, a contaminated oil was obtained. Mass and NMR spectra indicated the presence of the completely a inol .zed product. Further data are listed in Table I given herein below.

TABLE I Data on the preparation of compounds nos.11-16

Starting compounds (formul a-no. of sheet of formulae) Product (compound no. ) Yield {%) mmpt(°C)

1 N ₄ P ₄ Az(NH e) ₇ 11 36 96-98

2 1. trans-5 N ₄ P ₄ Az ₂ (NHMe) ₆ 12 56 124-126

3 1. cis-5 N ₄ P ₄ Az ₂ (NHMe) ₆ 13 52 135-137

5 ¹ gem. N ₄ P ₄ Az ₂ (NH e) ₆ 14 75 136-138

4 I., trans-3 N ₄ P ₄ Az ₂ (NHMe) ₆ 15 19 104-106.5

■I I If cis-3 N ₄ P ₄ Az ₂ (NH e) ₆ 16 75 ^a -

N ₄ P ₄ z(M e ₂ ) ₇ and N^-Az,, HMe^g

To a stirred solution of 0.5 g (ca. 1 mmol) of the ring com-

3 ^* pound in 25 cm diethyl ether, cooled at 0°C, was added dropwise

15 cm of a 3 M dimethylamine solution in diethyl ether. After warming up to room temperature and a reaction time of 18 h, the working up by using procedure (a) yielded 0.57 g of an oily material. This was dissolved in 25 cm of diethyl ether -and refluxed

3 ^' overnight after adding 10 cm of a 3 M dimethylamine solution in diethyl ether. Subsequently, rocedure (a) was once again used, yielding 0.54 g of a white solid (if the starting material is the compound having formula 1 or formula 2 of the sheet of formulae) or a viscous oil (if the starting material is the compound having formula 3 or formula 5 of the sheet of formulae) . The solid was easily crystallized from hexane, whereas the oil required severalrecrystallizations from small amounts of hexane at -70°C. The product obtained by starting from the compound having formula 2 of the sheet of formulae remained an oil of -unsatis¬ factory purity. Mass and NMR spectra were in agreement with the completely aminoIYzed compound no. 22. Further data are listed in table II given herei below.

TABLE II

Data on the preparation of compounds nos. 17-22

Startingf compounds (formula-no. of sheet of formulae) Product (compound no.) Yield {%) m.p.(°C)

1 N ₄ P ₄ Az(N e ₂ ) _? 17 34 206-208 2 I - trans-5 N ₄ P ₄ Az ₂ (NMe ₂ ) ₆ 18 68 198-200

3 I f cis-5 N ₄ P ₄ Az ₂ (NMe ₂ ) ₆ 19 32 192-195

5 ¹ gem. N ₄ P ₄ Az ₂ (NMe ₂ ) ₆ 20 33 dec. >200

4 I f trans-3 N ₄ P ₄ Az ₂ (NMe ₂ ) ₆ 21 24 deo. > 200

II I f cis-3 N ₄ P ₄ Az ₂ (NMe ₂ ) ₆ 22 100 ²

1 Characterization data

TABLE III

³¹ P NMR data' of the compounds nos. 6 - 22

- - - • δ P(ppm) ² JPP(Hz) ⁴ JPP (Hz)

Com¬ δP(l) δP(3) δP(5] δP(7)

^J 13 ^J 35 ^J 57 ^J 17 pound no.

6 18,5 -3,4 6,9 -3,4 13,9 26,4 26,4 13,9

7 12,1 14,9 12,1 -2,5 27,0 27,0 26,5 26,5

8 10,3 13,7 11,7 -1,8 28,9 27,6 24,7 26,9

9 19,6 11,3 -4,4 -6,8 22,8 25,6 27,9 12,0

10 10,3 - 12,2 10,3 -3,5 29,4 29,4 27,8 27,8

11 13,8 9,6 9,7 9,6 32,6 44,6 44,6. 32,6

12 13,6 9,5 13,6 9,5 32,3 32,3 32,3 32,3

13 .13,9 9,6 13,9 9,6 32,9 32,9 32,9 32,9

14 19,1 9,5 9,4 9,5 30,5 42,7 42,7- 30,5

15 13,8 13,8 9,6 9,6 27,2 33,0 39,8 33,0 0

16 13,5 13,5 9,5 9,5 27,2 33,1 39,5 33,1 -0,2

17 13,3 9,6 8,6 9,6 36,2 49,2 49,2 36,2

18 12,8 9,6 12,8 9,6 38,3 38,3 38,3 38,3

19 13,9 9,6 13,9 9,6 39,8 39,8 39,8 39,8

20 19,2 10,3 8,5 10,3 29,5 41,4 41,4 29,5

21 14,0 14,0 8,6 8,6 31,7 38,9 43,6 38,9 -0,4

22 12,5 12,5 • 8,3 8,3 33,0 39,9 43,5 39,9 -0,1

a- "Chemical Shifts" relative to 85 % H ₃ PC

>4

^βURt.

TABLE IV

Elemental analysis data of compounds Nos. 6 - 22

Compound C(%) H(%) N(%) Cl(%) No.

6 14,95(14,91) 2,49(2,50) 20,35(20,28) 36,86(36,67)

7 14,75(14,91) 2,43(2,50) 20,36(20,28) 36,44(36,67)

8 14,72(14,91) 2,57(2,50) 20,41(20,28) 36,96(36,67)

9 14,89(14,91) 2,51(2,50) 20,37(20,28) 36,72(36,67)

10

11 24,87(25,00) 7,46(7,46) 38,35(38,88)

12 26,90(27,03) 7,26(7,26) 37,43(37,83)

13 26,94(27,03) 7,37(7,26) 37,79(37,83)

14 27,24(27,03) 7,31(7,26) 36,89(37,83)

15 26,92(27,03) 7,32(7,26) 37,12(37,83)

17 36,34(36,22) 8,78(8,74) 31,64(31,68)

18 36,35(36,36) 8,36(8,39) 31,43(31,80)

19 36,48(36,36) 8,41(8,39) 32,25(31,80)

20 36,23(36,36) 8,35(8,39) 31,20(31,80)

21 36,53(36,36) 8,61(8,39) 32,21(31,80)

a - the calculated values are mentioned in brackets

In vitro" physiological activity

-

Compound no. LAD (urn) ID ₅₀ μm)

11 150 56,9

12 0.6 4,6

13 0.6 4,6

14 18 6,5

15 2,5 1,8

16 (not tested) 17 62 12,0

18 1,0 7,5

19 4 5,5

20 2 running test

21 2

22 — - (not tested)

Compounds nos. 12 and 18 are now measured "in vivo": ^-0..- values are compound no. 12 : 165 mg/kg; 18 : 200 mg/kg (mice) . Testing compound no. 12 for L 1210 leukemia in mice gives the following picture.

Doses: 100 mg/kg T/C (= "Treated /Control") %

^ 300

(3 mice out of 5 alive)

120 mgAg T/C

225 140 mg/kg T/C

225 (one mouse alive) 160 mg/kg T/C

250 (2 mice alive) (tests conducted with mice taken in groups of 5) .