Raman and Infrared Studies on New Anticancer Inorganic Ring Systems 1-Hexaziridinocyclotriphosphazene N3P3(NC2H&

Michel Manfait? and Alain J. P. Alix Laboratoire de Recherches Optiques, Facultt des Sciences, B.P. 347, 5 1 062 Reims Cedex, France

Jean-Franqois Labarre and Francois Sournies Laboratoire Structure et Vie, Universite Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex, France

The Raman spectra of the new anticancer agent N3P3(NC2H& (MYKO 63), in the solid state and in aqueous solution, and the infrared spectra of the molecule in the solid state, are reported for the first time. AU the assignments are discussed in terms of the following vibrations framework (N3P3); ligand (-NCzHJ; and framework-Iigand couplings (Ne,,doZPNexof). The concept of group frequencies associated with local sym- metrical valence coordinates is used extensively.

INTRODUCTION

In relation to the powerful antitumour activity of the cyclophosphazene class of compounds,132 several impor- tant questions arise: (i) on what targets does the effectiveness depend, in vitro and/or in vivo? (ii) what are the geometrical and conformational molecular structures of these drugs in the solid state and in sol- ution? (iii) is it possible to establish some clear relation- ships between these structures and the antitumour activities in order to design still better drugs?

In an attempt to answer these questions, several lines of investigation have recently been pursued. The nature and the intensity of the interactions in vitro of hexaziridinocyclotriphosphazene (MYKO 63') with DNA has been studied by combined application of the Scatchard technique3 and of Raman spectro~copy.~ Studies of the x-ray crystal structure of MYKO 63, have shown that the molecular geometry (especially the spatial orientation of its aziridinyl 'wings') depends drastically on the solvent from which the drug is crystall- ized. When MYKO 63 is crystallized from saturated solutions of either CCl, or C6H6, the unit cell of the corresponding single crystals retains several solvent molecules. The actual composition of these two struc- tures may be described as (N3P3AZ6 * 3Ccl4)' and (2N3P3Az6 - C6H6)6'7 (Az = NC2H4) respectively, and the molecular patterns of MYKO 63 in these two cases (Figs l(a) and (b)) look quite different. However when MYKO 63 is crystallized from saturated solutions in rn-xylene, carbon disulphide or water, although the single crystals so obtained belong to various space groups, the geometry of the drug molecule itself appears to be the same in the three cases, and no clathration of solvent molecules in the unit cell is observedn2 This molecular geometry (Fig. l(c)) differs from the geometries shown in Figs l(a) and (b) mainly in the orientation of aziridinyl wings.

5- Author to whom correspondence should be addressed.

Thus the molecular structure of MYKO 63 appears to be versatile and flexible, its symmetry point group varying sharply from one structure to the other in the solid state. It is noteworthy that the genuine structure of MYKO 63 as represented in Fig. l(c) has no ternary symmetry and probably no symmetry element at all (C, point group), the expected three-fold axis appearing only in the structures shown in Figs l(a) and (b), i.e. when the molecule is stressed by a D6h (C6H6) or Td (CCl,) solvent field. Thus, it seems that the real geometry of MYKO 63 in aqueous solution would be the one of Fig. l(c). The present study was undertaken to establish if this is the case. Raman spectra of aqueous solutions of MYKO 63 and Raman and IR spectra of powdered solid MYKO 63 were recorded and compared.

EXPERIMENTAL

Synthesis and purity of MYKO 63

MYKO 63 was prepared according to the Ratz pro- cedure' as improved by Labarre and Sournies.' Its purity was checked by mass spectrometry" and was found to be higher than 99.5%. Such a pure sample was found to be highly soluble in water, about 100 g1-l. 31 P NMR spectroscopy gives an unique signal at -37.2 ppm with 85% H3P04 as a standard. The melting point of pure MYKO 63 is 150 OC.'

Instrumentation

Raman spectra of the solid (powder crystallized from m-xylene solutions) and of aqueous solutions (-3 x

M) were recorded on a Coderg T800 and a PHO spectrometer respectively, both equipped with a Coher- ent Radiation Model 52B Ar' laser using 400- 1200 mW of power from the 488.0 nm line and a cooled EM1 9558 QB photomultiplier for detection. A small two-prism monochromator (Anaspec 300 S) was used

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RAMAN AND INFRARED ANTICANCER N ~ P ~ A G

out on the stored spectra," especially the subtraction of one spectrum from another. Thus, the spectrum of MYKO 63 was obtained by subtracting the water spec- trum from the MYKO aqueous solution spectrum in the range 250-1850 cm-'. The scaling factor for the subtraction of these spectra is determined from consideration of the band in the water spectrum at 1640 cm-' ($,(HOH)). This difference technique gives a horizontal base-line in the spectral area around 1850cm-' and also reveals the low frequencies of MYKO 63 (range 250-500 cm-') in solution, in contrast to normal aqueous solution spectra which have a strong background in this range arising from the solvent.

Figure 1. Projection views of the N3P3AzB molecule.23 (a) (N3P3Az.J (3CCl41 anticlathrate structure; (b) (2N3P3Az6) +

(C&G) clathrate structure; (c) free MYKO 63 molecule.

to remove background plasma lines. The PHO-spec- trometer was driven by a computer (Alcyane, MBC, France) which automatically coordinates the scanning (steps of 2 cm-' with maximum counting time of 2 s per step) and the storage of the spectral information.

The spectra were recorded with slit widths of 4 ind 6 cm-' for the solid and the solutions respectively. The Raman frequencies reported here are accurate to *2 cm-'. The polarization data were obtained with a polarizer and a scrambler.

IR spectra (on KBr disks) were recorded at room temperature on a Perkin Elmer 521 spectrometer (range 4000-250 cm-', calibration with polystyrene lines).

Analysis method

Data accumulation was used to improve the signal-to- noise ratio. Several types of operation may be carried

RESULTS AND DISCUSSION

Figure 2 present the polarized Raman spectra in aqueous solution ((a) = 41, (b) = I,) and in the solid state (c) together with the IR spectrum (d) of MYKO 63. The frequencies and their assignments are reported in Table 1.

Following the normal coordinate analysis of Alix et al., the discussion will be conducted in terms of local symmetries and the assignments will be made in terms of local symmetrical valence coordinates. As the whole molecule has no symmetry whatsoever, all the vibrations ( n = 3N - 6 = 138) belong to the A species, and thus none of them are depolarized (p # 3/4).

We have used data from the literature and data from our own work as follows.

12

Data from literature. Framework vibrations N3P3) :

N3p3B1-6; ligand vibrations ( -NC2H4) : HNC2H4. 17-19

Our own data. N ~ P ~ A Z S ; ' ~ (NPAz&NSOF, (NPAz2) (NSOF)Z;~' (NPAz&(NPClZ);21 ( N P A z ~ ) ~ N S O A Z . ~ ~

The group frequency approach provided useful infor- mation about assignments, especially for the f ramework-ligand couplings.

N3P3C16;z-'6 N3P3F6;13 mixed N3P3ClnF6-n 1 3 and

N3P3 ring vibrations

As expected from the theory," twelve lines have to be assigned. Six of them correspond to local stretch defor- mations u(PN). The ring breathing u,(PN) appears at 786 cm-' in aqueous solution (Figs 2(a) and (b)). The two very intense symmetrical IR vibrations around 880 cm-' are typical of the trimer cyclophosphazenes N3P3X6 these shift to -900cm-' for the tetramers N4P4XtJ.

The anti-symmetrical u,,(PN) (a: mode, inactive in molecules of D3h symmetry) was calculated at 1042 cm-':16 we assigned it to the very weak band at 1037cm-' (Fig. 2c). The twp other u,,(PN) modes appear in the 1150-1220 cm- range. A further three of them correspond to local deformation modes G(NPN). The symmetrical deformation vibration G,(NPN) is very intense, [706 (Figs 2(a) and (b)) and 695 cm-' (Fig. 2(c))]. The two other anti-symmetrical modes appear at 369 and 471 cm-' in aquebus solution. The remaining three lines correspond to out-of-plane ring deforma-

~ 6 . 2 0 . 2 1

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M. MANFAIT, A . J. P. ALIX, J.-F. LABARRE A N D F. SOURNIES h W

M. MANFAIT, A . J. P. ALIX, J.-F. LABARRE A N D F. SOURNIES

6, r- W

W N

200 400 600 800 I000 I200 1400

, * 200 400 600 800 1000 1200 1400

ern-'

1 L 1 I I - 200 400 600 800 1000 1200 I400

c rn-'

Fipure 2. Rarnan spectra of N3P3Az6 in aqueous solution with polarization (/,I (a), I , (b)) and in solid state ( c ) ; IR spectra: (d)

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RAMAN AND INFRARED ANTICANCER N ~ P ~ A z ~

Table 1. Vibrational frequencies (em-') of N3P3A% in aqueous solution (Raman) and in the solid state (Raman and IR)"

Raman

in HZO Solid 40 48 61 77 88 s

103 118 148 s 161 s 184 m 254 m 295 m

2 9 7 ~ ( 0 . 1 1 ) ~ 323vs 328 vw (0.34) 344 vw (0.68)

369 w (0.70) 361 m 353m,sh

392 381 433 m

471"w 455 m 487 s (0.04) 479 vs

501 m

631 652

692" v w 706 vs (0.02) 695 vs

708 w 786 v w (0.27) 790 m

804 81 8

828 m (0.55) 838 vs 844 m (0.52) 850 m

IR Assignments (approximate description)

Solid Lattice modes

265 v w 292 v w

325 v w 350 v w

385 v w 425 v w

477 v w 492 m

630 s 652 m

705 s 780 v w 796 w

Ring deformation IP, S., (NPN)

Ring deformation IP,

Ring breathing, v,(PN) SJNPN 1

Raman IR Assignments lapproximate description)

in H20 Solid Solid

881 873s } u,(PN) 889 884m 890 v w

937 v w (0.50) 926 978 w (0.13) 932

956 974 m

1036 v w 1037

109lCvw 1081 l l 0 0 m (<0.01) 1101 m 1131 v w 1132 1158m (0.23) 1151 m

1158 1172sh

1203

1267 w 1276vs (0.04) 1273vs

1452 w (0.56) 1445 m 1451 1462 sh

1475 w (0.34) 1474 w 2896 2938 w 2992 vs 2999 vs 3025 3052 3065 s 3077 3100

930 vs pW,,(CHz) 945 sh

va,(PN 1 923 I 1074 m

1088 m 1125vw

1155w

1 2 0 8 ~ ~ ~,,(PN)+P,,~(CH~) l l 9Ovs

1220 vs

1260 s uaS(NCz)

i Ring breathing Az,

vANC2) 1446 vw Sas(CH1)

1480 vw S.(CHz)

"Al l observed frequencies are in cm-'. Abbreviations used are: s, strong; m, medium: w, weak; v, very; sh, shoulder: IP, in local plane; OP, out-of local plane: pT =twist; pw = wag; pr= rock: 8 =scissor; T = torsion; Az = aziridino (NCZH,).

Depolarization ratio App= 10%. Data from the aaueous solution Raman spectrum II (Fig. 2(b)).

tions (torsional and/or puckFring modes) and are obser- ved in the range 60-90 cm- .

Ligand -NC2H4 vibrations

According to our preliminary theoretical study," the vibrations of each ligand can be classified as belonging to one of three characteristic groups of frequencies.

Three ring NC2 vibrations. The ring breathing us(NC2) is observed at 1276cm-' (Figs 2(a) and (b)). The sym- metrical deformation mode S,(NC2) appears at 844 cm-' (Figs 2(a) and (b)); the anti-s mmetrical ring stretch u,,(NC2) is pointed at 1260 cm- (Fig. 2(d)). T

Twelve (CH,) vibrations. The assignment of the four (CH) stretch and the eight (CH2) deformations of scissor-type (S), twist (pT), wag(pw) and rock (p,) modes is straightfor- ward (see Table 1).

Two -NC, vibrations. The two PN,,, deformation modes (in-plane and out-of-plane, with respect to the local

plane of symmetry of one aziridinyl wing) correspond to the twist and the wag of the NC2 ring with respect to the PN,,, bond.

Since, as for aziridine, the NH deformations are found at 109P8cm-' (in local plane) and 904 cm-' (out of locat plane) (Fig. 2(c)) and the twist of NC2, to 433 cm- .

It is assumed that the seventeen frequencies described above are nearly the same for each of the six individual aziridinyl wings and thus they would generate 6 x 17 = 102 frequencies. Indeed many of the observed bands show one or more shoulders, i.e. a non-symmetric profile, especially in the Raman spectrum of the solid (Fig. 2(c)).

we attributed the wag of NC2, ty 501 cm-

Framework-ligand coupling vibrations

Following the principles defined in Ref. 12, the twenty- four coupling vibrations belong to three characteristic groups of framework frequencies.

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M. MANFAIT, A. J. P. ALIX, J.-F. LABARRE AND F. SOURNIES

(i) Six PN,,, vibrations describing the motions of the three pairs of aziridino (NC2H4) groups along the PN,,, bonds. They combine into 3vS(PAz2) and 3v,,(PAz21 bands. We observed the lowest v,(PAz2) at 297 cm- (Figs 2(a) and (b)) and the two highest in the 450- 480 cm-' range; the three va,(PAz2) are observed (intense in IR) in the 630-655 cm-' range (Fig. 2(d)).

(ii) Twelve PNeXo2 vibrations describing deformations of scissor, twist, wag and rock types. In these cases the proposed assignments have been made taking into account the calculations and complete assignments of Huvenne et for the hexachlorocyclotriphosphazene N3P3C16 (see Table 1).

(iii) Finally, the last six frequencies are three combi- nations of local symmetrical and anti-symmetrical tor- sions of the three pairs of aziridino groups around their PN,,, bonds (i.e. the atoms involved in one torsion coordinate are Ne,o-P-Nexo-C; see Ref. (12) for details).

As for the aziridinyl ligands, the aziridino groups may be considered as independent of each other and then one would observe only the two characteristic frequen- cies 7,(Az2) and T,,(Az~), which are tentatively assigned to bands in the 100-120 cm-' range (see Table 1).

The results obtained here have been used to study the interactions in vitro between the drug and DNA.4

REFERENCES

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Acta PartA 23,195 (1967). 19. J. Le Brumant, C.R. Acad. Sci. Ser. B 268, 1424 (1969). 20. M. Manfait, unpublished work. 21. G. Guerch, J.-F. Labarre, F. Sournies, M. Manfait, F. Spreafico

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Trans. 22, 2503 (1972).

Acta PartA 29,821 (1973).

63, 47 (1980).

Received 11 May 1981

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