Derivatives of oxazaphosphorines that are pre-activated, use and method of preparation

Abstract

The present invention relates to novel derivatives of oxazaphosphorines that are pre-activated, to the methods for preparing same, to the pharmaceutical compositions containing same and to the therapeutic use thereof, in particular for treating cancer.

Claims

1. A compound of formula (I) ##STR00020## wherein: X is O; R1 and R3 are both (CH.sub.2).sub.2CI; R2 is H; and R4 is a linear or branched, saturated, hydrocarbon chain of 5 to 30 carbon atoms; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein R4 comprises a linear saturated hydrocarbon chain having from 5 to 30 carbon atoms.

3. The compound of claim 1, wherein R1 and R3 are both (CH.sub.2).sub.2Cl and R2 is H.

4. A nanoparticle formed by a compound of claim 1 or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising a nanoparticle of claim 4.

7. A method for preparing a derivative of oxazaphosphorine of formula (I) wherein: ##STR00021## X is O; R1 and R3 are both (CH.sub.2).sub.2CI; R2 is H; and R4 is a linear or branched, saturated, hydrocarbon chain of 5 to 30 carbon atoms; said method comprising: providing a compound of formula (II) ##STR00022## wherein R1 and R3 are both (CH.sub.2).sub.2CI; R2 is H; and reacting the compound of formula (II) with R4-XH, wherein X is O and R4 is a linear or branched, saturated, hydrocarbon chain of 5 to 30 carbon atoms, in the presence of a Lewis acid.

Description

EXAMPLE 1: PREPARATION OF 4-SUBSTITUTED ANALOGUES OF IFO (IFOSFAMIDE)

(1) The anodic oxidation of ifosfamide in methanol provides access to a chemical entity (4-MeO-IFO), whose cytotoxic potency is equivalent to that of the active metabolite of IFO. However, this 4-MeO-IFO is quite unstable from a chemical point of view, releasing isophosphoramide mustard rapidly. In order to modulate the release kinetics and facilitate intracellular penetration of this very polar small molecule, the inventors considered inserting longer aliphatic chains at this position 4. This concept is the first step toward modifying ifosfamide to insert it into a lipophilic vectorized form, such as a liposome or nanoparticle system.

(2) Substitution by Direct Route

(3) Oxidation of ifosfamide generates an iminium. This is trapped by the nucleophile present in the medium, leading to a 4-substituted oxazaphosphorinane. The purpose of this description is to study the feasibility of this synthetic route and to evaluate the substrates and the minimum amount compatible with this direct route.

(4) Electrochemical Oxidation of Ifosfamide in the Presence of Pentan-1-ol:

(5) In a first step, the inventors used primary amyl alcohol to study the feasibility of this reaction in the presence of a long chain aliphatic alcohol.

(6) To define a procedure as efficient as possible, the inventors varied the experimental reaction conditions described in Scheme 1.

(7) ##STR00007##

(8) Electrolysis of IFO was carried out in the presence of pentan-1-ol in acetonitrile, at constant current through the graphite electrode. Tetraethylammonium tetrafluoroborate (TEABF.sub.4) was the supporting electrolyte that was used. The reaction was followed by thin layer chromatography (TLC) and stopped when all the IFO was consumed. After stopping the reaction, sodium bicarbonate was added to neutralize the electrogenerated acidity. The obtained products, which could be isolated, were then purified by column chromatography.

(9) IFO is a racemic mixture due to the chirality conferred by the phosphorus. As for methoxylation, pentoxylation generated four diastereomers, pairwise enantiomers.

(10) By nature, the enantiomers could not be physically differentiated. On the other hand, the .sup.31P-NMR chemical shifts for the two pairs of diastereomers were distinct.

(11) The proportions of the various products were therefore determined by .sup.31P-NMR.

(12) By analogy with methoxylation, it was possible to assign the .sup.1H-NMR signals to one or the other diastereomeric forms; in fact, addition of an alkoxy group on the carbon of the nitrogen results in a group with 3 signals corresponding to the hydrogens carried by C4 and C6, and which differ from one diastereomer to the other.

(13) Influence of the Amount of Alcohol

(14) The electrolysis was carried out by varying the amount of alcohol introduced. For a given quantity of supporting electrolyte (1 Eq.), the results are summarized in Table 1.

(15) TABLE-US-00001 TABLE 1 Influence of the amount of nucleophile Eq. of Yield Test Pentanol Current (mA) Reaction stopped (%) A 9 20 3.75 F/mol <15 B 15 20 3.60 F/mol 34 C 30 20 3.75 F/mol 41

(16) Initially, at least 15 equivalents of alcohol appeared to be necessary to obtain a satisfactory yield (calculated with reference to IFO). On the other hand, the gain obtained when increasing from 15 to 30 equivalents was low.

(17) Anodic Oxidation of IFO in the Presence of Different Alcohols

(18) The aim here, using the conditions developed with pentan-1-ol, was to study the compatibility of different functional groups on the alcohol with the electrochemical process.

(19) The general reaction is shown in Scheme 2.

(20) ##STR00008##

(21) IFO therefore was electrochemically oxidized in acetonitrile in the presence of various alcohols with 5 carbon atoms. The reaction was stopped when all the starting material appeared to be consumed as monitored by TLC. At the end of the reaction, sodium bicarbonate (1 Eq.) was added to the medium to neutralize electrogenerated hydrons. The proportions of the various diastereomers were determined on the crude reaction mixture by .sup.31P-NMR before being isolated by flash chromatography.

(22) The results of these operations are described in Table 2.

(23) TABLE-US-00002 TABLE 2 Summary of various alkoxylation tests Results Stop Overall yield Alcohol (F/mol) Proportions of diastereomers (%) Pentan-1-ol 3.6 80/20 34 Pentan-3-ol 2.7 55/45 0* Pentan-1,2-diol 2.5 52/48 22 Pentan-1,4-diol 3.5 53/47 0* 4-Penten-1-ol 2.9 ND 19 Heptan-1,7-diol 2.7 80/20 27 *products not isolated.

(24) At first, it was observed that a secondary alcohol seems more difficult than a primary alcohol to attach on IFO. Indeed, the NMR spectra of the crude electrolysis showed only traces of alkoxylation. Alkoxylated products formed in small quantities could not be isolated. Steric hindrance would prevent the alcohol from accessing the iminium of IFO.

(25) In addition, in the case of competition between a primary and secondary alcohol, the fixation was almost exclusively carried out by the primary alcohol, the rest of the chain remaining unchanged. During the tests using diols, the NMR spectra of the crude reaction mixtures revealed the presence of products compatible with the expected adducts. This was confirmed in the case of pentan-1,2-diol. However, despite a presence in the crude reaction, the products could not be purified in the case of pentan-1,4-diol.

(26) The use of a compound having two primary alcohol functions makes it possible to reduce the number equivalents to 10 Eq. (compared to 15 used for other alcohols), while maintaining an equivalent yield. Under the conditions used, the forms attached with two alcohols were not characterized.

(27) These results allow the possibility of selective fixation of polyols by a primary alcohol function. This was a first step toward the fixation of an ose on IFO, in the context of vectorization.

(28) The presence of a CC double bond did not prevent the fixation of the primary alcohol to IFO and it was not modified during the electrochemical reaction.

(29) Compatibility of Different Functions with the Direct Oxidation Protocol

(30) Besides using a large amount of nucleophile, the direct oxidation protocol requires the use of compounds resistant to oxidizing conditions.

(31) Amines

(32) Amines can be oxidized under the conditions used. Thus, in these conditions, and in the presence of ifosfamide, there is concomitant oxidation of the two compounds (IFO and amine).

(33) Thiol

(34) Under oxidizing conditions, thiols dimerize by forming a disulfide bridge (Scheme 3).

(35) ##STR00009##

(36) The nucleophilic center (sulfur atom) is no longer available for substitution on the iminium.

(37) Conclusions and Prospects

(38) The anodic oxidation protocol of IFO in the presence of 15 equivalents of alcohol is possible. It allows simple access to novel compounds, never described. This protocol is also applicable to other oxazaphosphorines such as cyclophosphamide and trofosfamide. To optimize this reaction, it is necessary to use an anhydrous reaction medium, in order to eliminate a competitor nucleophile.

(39) However, the disadvantages of this direct route are manifold.

(40) The need to use 15 Eq. of nucleophile poses several problems: this method is limited to inexpensive reagents, and this excess reagent impedes the purification of sensitive products. Lastly, the use of nucleophilic compounds with oxidizable functions is impossible.

(41) Substitution by Indirect Route

(42) Iminium Trapped then Regenerated

(43) This protocol consists of 3 stages: anodic methoxylation, and amidoalkylation, broken down into: regeneration of the iminium, then in situ reaction with the nucleophile.

(44) This 3-stage reaction sequence is carried out in 2 reaction media (Scheme 4).

(45) ##STR00010##

(46) In practice, ifosfamide undergoes electrolysis in methanol, then the 4-MeO-IFO derivative is brought into contact with a Lewis acid in a non-nucleophilic solvent to regenerate the iminium with which the nucleophile reacts.

(47) This technique allows the use of nucleophiles incompatible with anodic oxidation. Compared with the direct route, the other advantage of this technique is the use of a smaller number of nucleophile equivalents.

(48) The iminium was electrogenerated by anodic oxidation in methanol and therefore trapped in the form of 4-MeO-IFO.

(49) Once ifosfamide was totally consumed (passage of more than 2.5-3.0 F/mol), the solvent was evaporated in the presence of a base, sodium carbonate (Na.sub.2CO.sub.3), to prevent acidification of the medium. The residue was taken up in diethyl ether followed by simple filtration to give access to the methoxylated products. When a bit of starting material remains, or if the separation of the diastereomers is desired, a chromatographic column is required.

(50) Finally, many methoxylations on different amounts of IFO, ranging from a few dozen milligrams to a few grams, were carried out. In a 15 mL electrochemical cell, oxidation of 2 grams of IFO was carried out, for example. Methoxylated derivatives were obtained with a yield of 82% (1.8 g), after passage of 4.0 F. mol.sup.1. Thus this electrochemical reaction seems appropriate to use for synthesis; it is fairly simple to implement even though the purification requires a certain know-how. Moreover, the products obtained are relatively stable and can be stored for several weeks in a freezer under an inert atmosphere.

(51) Under the action of a Lewis acid, the methoxyl group can regenerate iminium which will be trapped by a variety of nucleophiles to give access to various structures. Amidoalkylation is the reaction consisting of regenerating the iminium ion from the methoxylated derivative, then carrying out a nucleophilic addition on this iminium.

(52) Regeneration of the iminium intermediate from the methoxylated derivative occurs through the use of a Lewis acid. The most conventionally used Lewis acids include BF.sub.3.OEt.sub.2, TiCl.sub.4, Yb(OTf).sub.2, and TMSOTf.

(53) The Lewis acid is merely involved in the regeneration of the iminium from the methoxylated derivative and not in the diastereoselectivity of the addition.

(54) ElectrolysisMethoxylation by Anodic Oxidation

(55) Angelo Paci and Thierry Martens described the anodic oxidation of IFO and CPM in a single-compartment cell (Paci et al., 2001b) (Scheme 5).

(56) ##STR00011##

(57) To obtain a known end product, the study focused on the following sequence using pentan-1-ol as nucleophile (Scheme 6).

(58) ##STR00012##

(59) 4-MeO-IFO was contacted with a Lewis acid at low temperature (78 C.) for 1 hour (arbitrarily). The nucleophile (1 Eq. of amyl alcohol) was then added to the mixture which was placed at 0 C. for 15 minutes. As the pentoxy-IFO have already been identified, a simple .sup.31P-NMR spectrum allowed rapid determination of reaction efficiency, as summarized in Table 3.

(60) TABLE-US-00003 TABLE 3 Influence of the Lewis acid used in the amidoalkylation Lewis acid Results Quantity Conversion Proportion dias Nature (Eq) (%) (9.6/9.3 ppm) TMSOTf 0.1 30 (+degradation) 85/15 0.5 Degradation ND 1 Degradation ND Ti(OEt).sub.4 0.1 0% Remaining 4- 0.5 0% MeO-IFO & 1 0% degradation products BF.sub.3OEt.sub.2 0.1 59 82/18 0.5 48 80/20 1 Degradation ND

(61) It appears that the Ti (OEt).sub.4 is not reactive enough to regenerate iminium from 4-MeO-IFO. Furthermore, TMSOTf appears to induce degradation of 4-MeO-IFO, except when used in catalytic amounts (10 mol %), which leads to the formation of diastereomers with a poor yield. The Lewis acid used does not seem to affect the diastereoselectivity of the reaction, but rather influences the yield and stability of the intermediate iminium generated, contributing to the purity of the crude reaction. In this study, it was found that the boron trifluoride etherate used in catalytic amounts (BF.sub.3OEt.sub.2) was the most effective Lewis acid tested.

(62) The conditions (0.1 Eq. BF.sub.3OEt.sub.2, 1 hour at 78 C. in CH.sub.2Cl.sub.2) identified above were subsequently used to evaluate the influence of the amount of pentan-1-ol on the conversion. The results are summarized in Table 4.

(63) TABLE-US-00004 TABLE 4 Influence of reaction time of the iminium Pentan-1-ol Results Quantity Time Conversion Proportion dias (Eq.) (min) (%) (9.6/9.3 ppm) 1 15 59 82/18 2 15 60 81/19 2 30 64 85.4/14.6 2 45 62 83.5/16.5 2 120 61 82/18

(64) The amount of pentan-1-ol does not seem to influence the conversion rate or the proportion of diastereomers formed. Despite comparable levels of conversion, it seems that the optimum reaction time is half an hour, with a conversion of 64% and a diastereomer ratio of 85.4/14.6.

(65) In some experiments, the inventors verified that the use of THF in place of dichloromethane did not modify the results observed.

(66) Competitor nucleophiles are methanol generated at the same time as the iminium and residual water present in the medium. The inventors have attempted to add activated 4 A molecular sieves to sequester residual water and released methanol. This addition did not appear to significantly improve the observed yields.

(67) Conjugation of other substrates such as amines, polyfunctionalized compounds or lipids is possible, and has been envisioned to allow the formation of pre-activated IFO analogues targeted and vectorized to tumors by attaching fatty acids or other lipids, a peptide or a sugar.

(68) For example, the most recent and most promising application concerns the attachment of a squalene residue by this method by adding squalenol or squalene-thiol in the presence of BF.sub.3ET.sub.2O on the phosphoryl-iminium produced from 4-methoxy-MFI (Schemes 7 and 8). The compounds formed, SQ-4-O-MFI and SQ-4-S-MFI, have two interesting properties. The first is that they are pre-activated forms of MFI due to oxidation in position 4 that can release, in a slightly acidic aqueous medium, the alkylating mustard directly without metabolic activation. The second is that they are capable of self-assembly in an aqueous medium in the form of nanoparticles of a size of about 160 nm.

(69) ##STR00013##

(70) ##STR00014##

(71) The same type of reaction was considered for cyclophosphamide or trofosfamide which can also be oxidized at position 4 in the 4-methoxy form to lead to the phosphoryl imine form.

(72) This two-stage method produces 4-substituted analogues of ifosfamide. Compared to direct oxidation, the main advantage of this technique is the use of small amounts of nucleophiles. This will be a major advantage when using expensive nucleophiles. Moreover, this technique is compatible with a wider variety of nucleophiles. Finally, the production of 4-MeO-IFO is feasible on large amounts of IFO.

Structural Analysis

(2-chloro-ethyl)-[3-(2-chloro-ethyl)-2-oxo-4-pentyloxy-25-[1,3,2]oxazaphosphinan-2-yl]-amine or 4-pentoxy-IFO

(73) ##STR00015##

(74) Protocol: In an 8 mL electrochemical cell, 110 mg IFO (0.4 mmol) were dissolved in 4.0 mL of anhydrous acetonitrile. One equivalent of a supporting electrolyte (95 mg tetraethylammonium tetrafluoroborate), then fifteen equivalents (7.1 mmol, 0.77 mL) of pentan-1-ol were added. The mixture was degassed by bubbling nitrogen and placed in an ice bath. Agitation was provided by a magnetic stirrer. Two electrodes were introduced into the cell.

(75) After the passage of 3.6 F/mol at 20 mA, the reaction was stopped by turning off the electric current. One equivalent of sodium bicarbonate was added to the medium to neutralize hydrons released during the reaction, which may degrade the product.

(76) The solvent was evaporated under reduced pressure, and the residue was taken up twice in 8 mL of acetonitrile to eliminate the maximum of alcohol. 15 mL of ethyl ether were then added to insolubilize the supporting electrolyte. After filtration of the ether phase, it was evaporated under reduced pressure.

(77) The resulting products were purified by chromatography (eluent ethyl ether/methanol; 95/5). Diastereomers 36a and 36b were obtained with 90% separation.

(78) Organoleptic character: slightly yellow oil.

(79) Rf=0.24 and 0.39 for X1 and X2 (Et.sub.2O).

(80) Molecular formula: C.sub.12H.sub.25Cl.sub.2N.sub.2O.sub.3P.

(81) NMR in CDCl.sub.3.

(82) .sup.31P (ppm): 9.6 (diastereomer 36a), 9.3 (diastereomer 36b).

(83) .sup.13C (ppm): 13.9 (C.sub.15); 22.4 (C.sub.14); 28.4 (C.sub.13); 29.5 (C.sub.5); 42.7 (C.sub.7); 44.0 (C.sub.8); 46.6 (C.sub.10); 49.5 (C.sub.9); 62.5 (C.sub.11); 68.5 (C.sub.6); 89.3 (C.sub.4).

(84) .sup.1H (ppm): 0.92 (t, 3H, H.sub.15); 1.40 (m, 4H, H.sub.13, H.sub.14); 1.62 (m, 2H, H.sub.12); 2.10 (m, 1H, H.sub.5 Eq); 2.26 (td, 1H, H.sub.5 Ax); 3.33 (m, 2H, H.sub.9); 3.45 (m, 2H, H.sub.7); 3.56 (m, 2H, H.sub.11); 3.73 (m, 4H, H.sub.10, H.sub.8); 4.15 (m, 1H, H.sub.6 Eq); 4.45 (m, 1H, H.sub.6 Ax); 4.60 (m, 1H, H.sub.4).

1-[3-(2-chloro-ethyl)-2-(2-chloro-ethylamino)-2-oxo-25-[1,3,2]oxazaphosphinan-4-yloxy]-pentan-2-ol or 4-(20H)-pentoxy-IFO

(85) ##STR00016##

(86) Protocol similar to that used for 4-pentoxy-IFO.

(87) The resulting products were purified by chromatography (eluent ethyl ether/methanol; 90/10), 2 successive columns.

(88) Organoleptic character: slightly yellow oil.

(89) Rf=0.32 and 0.50 for X1 and X2 respectively (hardly distinguishable spots) (Et.sub.2O/MeOH 90/10).

(90) Molecular formula: C.sub.12H.sub.25Cl.sub.2N.sub.2O.sub.4.

(91) NMR in CDCl.sub.3.

(92) .sup.31P (ppm): 9.78 (diastereomer 38a), 9.88 (diastereomer 38b).

(93) .sup.1H (ppm): 0.95 (t, 3H, H.sub.15); 1.30 (bs, 1H, OH); 1.40 (m, 4H, 2H.sub.13, 2H.sub.14); 1.92 (m, 2H, H.sub.5); 2.25 (bs, 1H, NH); 3.30 (m, 4H, 2H.sub.7, 2H.sub.9); 3.40 (m, 3H, 2H.sub.11, H.sub.12); 3.70 (m, 4H, 2H.sub.8, 2H.sub.10); 4.15 (m, 1H, H.sub.6Eq); 4.50 (m, 1H, H.sub.6Ax); 4.70 (m, 1H, H.sub.4).

(2-chloro-ethyl)-[3-(2-chloro-ethyl)-2-oxo-4-pent-4-enyloxy-25-[1,3,2]oxazaphosphinan-2-yl]-amine or 4-pentenoxy-IFO

(94) ##STR00017##

(95) Protocol similar to that used for 4-pentoxy-IFO.

(96) The resulting products were purified by chromatography (eluent ethyl ether/methanol; 97/3).

(97) Organoleptic character: slightly yellow oil.

(98) Rf=0.68 and 0.78 for X1 and X2 respectively (hardly distinguishable spots) (Et.sub.2O/MeOH 95/5).

(99) Molecular formula: C.sub.12H.sub.23Cl.sub.2N.sub.2O.sub.3.

(100) IR (film); (cm.sup.1): 2924 (CH), 2361, 1640 (CC), 1434 (PN), 1257 (PO), 1065-1113 (POC).

(101) NMR in CDCl.sub.3.

(102) .sup.31P (ppm): 9.51 (diastereomer 40a), 9.23 (diastereomer 40b).

(103) .sup.13C (ppm): 28.9 (C.sub.5); 35.6 (C.sub.12); 36.9 (C.sub.13); 42.6 (C.sub.9); 44.5 (C.sub.7); 47.3 (C.sub.8); 50.0 (C.sub.10); 62.5 (C.sub.6); 67.8 (C.sub.ii); 89.5 (C.sub.4); 115.9 (C.sub.15), 138.0 (C.sub.14).

(104) .sup.1H (ppm): 0.95 (bs, 1H, NH), 1.30 (m, 2H, 2H.sub.13); 1.48 (s, 1H, 1H.sub.11); 1.75 (q, 2H, H.sub.12), 1.90 (d, 1H.sub.11); 2.25 (m, 2H, 2H.sub.5); 3.20 (m, 2H, 2H.sub.9); 3.35 (m, 2H, 2H.sub.7); 3.65 (m, 2H, 2H.sub.8); 3.75 (m, 2H, 2H.sub.10); 4.25 (m, 1H, H.sub.6Eq), 4.50 (m, 1H, H.sub.6Ax), 4.65 (m, 1H, H.sub.4); 5.10 (m, 2H, 2H.sub.15), 5.80 (m, 1H, H.sub.14).

(2-chloro-ethyl)-[3-(2-chloro-ethyl)-2-oxo-4-squalenyl-[1,3,2]oxazaphosphinan-2-yl]-amine or 4-SQ-IFO

(105) ##STR00018##

(106) Protocol: From 4-methoxy-ifosfamide, under similar conditions to the previous products (78 C., CH.sub.2Cl.sub.2, BF.sub.3, EtO.sub.2, 30 min), 1 equivalent of tris-nor-squalenol was added to form the desired compound with a yield of 53%.

(107) The resulting products were purified by chromatography.

(108) Organoleptic characters: slightly yellow oil.

(109) Molecular formula: C.sub.34H.sub.61Cl.sub.2N.sub.2O.sub.3P.

(110) IR (film); (cm.sup.1): 2956 (CH), 2352, 1638 (CC), 1432 (PN), 1258 (PO), 1063-1109 (POC).

(111) NMR in CDCl.sub.3.

(112) .sup.13C (ppm): 33.4 (C.sub.5), 35.6 (C.sub.u), 36.9 (C.sub.13), 42.6 (C.sub.9); 44.5 (C.sub.7); 47.3 (C.sub.8); 50.0 (C.sub.10); 63.5 (C.sub.6); 67.8 (C.sub.11), 89.5 (C.sub.4); 125.9 (C.sub.ethylen); 132.0 (C.sub.quat).

(113) .sup.1H (ppm): 0.95 (bs, 1H, NH), 1.30 (m, 2H, 2H.sub.13); 1.55 (s, 18H); 1.95 (m, 20H); 3.20 (m, 2H, 2H.sub.9); 3.35 (m, 2H, 2H.sub.7); 3.55 (m, 2H, 2H.sub.8); 3.65 (m, 2H, 2H.sub.10); 4.25 (m, 1H, H.sub.6Eq), 4.50 (m, 1H, H.sub.6Ax), 4.65 (m, 1H, H.sub.4); 5.05 (m, 5H, H.sub.ethylen).

(2-chloro-ethyl)-[3-(2-chloro-ethyl)-2-oxo-4-mercaptoethylamidosqualen-[1,3,2]oxazaphosphinan-2-yl]-amine or 4-thio SQ-IFO

(114) ##STR00019##

(115) Protocol: From 4-methoxy-ifosfamide, under similar conditions to the previous products (78 C., CH.sub.2Cl.sub.2, BF.sub.3, EtO.sub.2, 30 min), 1 equivalent of cysteamido-squalenic acid was added to form the desired compound with a yield of 60%.

(116) The resulting products were purified by chromatography.

(117) Organoleptic characters: slightly yellow oil.

(118) Molecular formula: C.sub.36H.sub.64Cl.sub.2N.sub.3O.sub.3PS.

(119) IR (film); (cm.sup.1): 2956 (CH), 2352, 1746, (CO), 1638 (CC), 1432 (PN), 1258 (PO), 1063-1109 (POC).

(120) NMR in CDCl.sub.3.

(121) .sup.13C (ppm): 33.4 (C.sub.5); 35.6 (C.sub.u); 36.9 (C.sub.13); 42.6 (C.sub.9); 44.5 (C.sub.7); 47.3 (C.sub.8); 50.0 (C.sub.10); 63.5 (C.sub.6); 67.8 (C.sub.11); 89.5 (C.sub.4); 125.9 (C.sub.ethylen.); 132.0 (C.sub.quat).

(122) .sup.1H (ppm): 0.95 (bs, 1H, NH); 1.30 (m, 2H, 2H.sub.13); 1.55 (s, 18H); 1.95 (m, 20H); 3.20 (m, 2H, 2H.sub.9); 3.35 (m, 2H, 2H.sub.7); 3.55 (m, 2H, 2H.sub.8); 3.65 (m, 2H, 2H.sub.10); 4.25 (m, 1H, H.sub.6Eq.); 4.50 (m, 1H, H.sub.6Ax.); 4.65 (m, 1H, H.sub.4); 5.05 (m, 5H, H.sub.ethylen), 7.35 (bs, 1H, NH).

(123) Note: cysteamido-squalene acid and tris-nor squalenol can be obtained from squalene aldehyde by conventional synthetic methods. The synthesis of aldehyde derivatives of squalene is described in Ceruti et al., J. Chem. Soc, Perkin Trans. 1, 2002, 1477-1486 (see Figure 2, p. 1479).

EXAMPLE 2: BIOLOGICAL EVALUATION OF COMPOUNDS 4-THIO SQ-IFO AND 4-SQ-IFO

(124) I. Evaluation of In Vitro Cytotoxicity

(125) I.1. Materials and Methods:

(126) Cell Culture and Culture Conditions

(127) Cytotoxicity of SQ-IFO and SQ-thio-IFO nanoparticles was studied in several cell lines:

(128) A549: human alveolar basal epithelial cell adenocarcinoma MCF-7: human breast carcinoma MCF-7 MDR: multidrug resistant human breast carcinoma B16F10: mouse melanoma KB 3.1: human epidermoid carcinoma M109: mouse lung tumor cells MiaPaCa-2: human pancreatic carcinoma UW-479: human pediatric glioma IGR OV1: human ovarian cancer SK-N-MC: neuroblastoma, reclassified as Ewing (expression of EWS/Flip-1 oncogene)

(129) The cell lines were maintained in DMEM or RPMI medium supplemented with 10% fetal calf serum and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) in an incubator at 37 C. in a humid 5% carbon dioxide atmosphere.

(130) The cells were seeded in 96-well plates (TPP). Seeding was optimized for each line for an incubation time of 72 hours.

(131) A549, MCF-7, B16F10, M109, MiaPaCa-2 cells were seeded at 5.Math.10.sup.3 cells/well. KB 1.3 cells were seeded at 2.Math.10.sup.3 cells/well, IGR-OV1 cells at 10.sup.4 cells/well and UW479 and SK-N-MC cells at 5.Math.10.sup.4 cells/well.

(132) Preparation of Nanoparticle Suspensions

(133) The compounds SQ-IFO and SQ-thio-IFO were synthesized according to the protocol described above. For each compound, an aqueous suspension of nanoparticles was prepared using the technique described by Fessi, Int. J. Pharm, 1989, 55, R1-R4. The nanoparticles obtained are spherical and have an average size of 182 nm.

(134) For each compound, different concentrations between 0.1 and 100 M were tested by serial dilution of a 2 mg/mL stock solution for SQ-IFO and a 10 mg/mL stock solution for SQ-thio-IFO in culture medium.

(135) Cell Viability Test

(136) After 72 hours of incubation, cell viability was determined by observing the reduction of MTS reagent to formazan (CellTiter 96 AQ.sub.ueous One Solution kit, Promega). 20 L of a 5 mg/mL solution of MTS in PBS were added per well. The incubation time of the MTS was optimized for each cell line; the absorbance at 490 nm was then read using a plate reader (EL808, Biotek).

(137) The results are expressed as percentage of untreated cells. The data were treated with Prism 4 software (Graph Pad Software, San Diego). IC50 values were thus calculated for each line. Ifosfamide and squalenol were used as controls.

(138) I.2. Results

(139) Table 5 below shows, for each compound tested, the IC50 obtained for each cell line. It is clear that the compounds IFO-SQ and IFO-thio-SQ exhibit high in vitro cytotoxicity; these compounds are thus capable of releasing the alkylating mustard without prior activation by cytochrome P450.

(140) Remarkably, a different activity profile was observed for IFO-thio- and IFO-SQ depending on the cancer cell line. IFO-thio-SQ had high cytotoxic activity on M109 cell lines, SK-N-MC (Ewing), UW 479 (Glioma) and IGR-OV1 (Ovary) cells. Such activity was not observed for the compound IFO-SQ.

(141) Squalenol and IFO did not exhibit significant cytotoxicity at the concentrations tested.

(142) TABLE-US-00005 TABLE 5 IC50 of IFO, IFO-SQ and IFO-thio-SQ on each cell line tested. IC50 (M) Ifosfamide Ifosfamide SQ Ifosfamide-thio-SQ A549 >100 32.5 5.3 MCF-7 >100 43.6 10.9 MCF-7 MDR >100 78.8 80 B16F10 >100 73 16.9 KB 3.1 >100 50 2.96 M109 >100 >100 9.2 MiaPaCa-2 >100 32.5 4.4 SK-N-MC (Ewing) >100 >100 19 UW 479 (Glioma) >100 >100 65 IGR-OV1 (Ovary) >100 >100 81
II. Evaluation of In Vivo Efficacy

(143) In vivo cytotoxic efficacy was evaluated first on a model of human rhabdomyosarcoma xenografted in nude mice. The model used is the RD human pediatric rhabdomyosarcoma model. The cells were first amplified in culture and counted. 10.Math.10.sup.6 cells were injected subcutaneously in both flanks of each mouse. After graft uptake, when tumor volume reached >80 mm.sup.3, the mice were treated with IFO-SQ or placebo. Preliminary results of this study showed a significant decrease in tumor volume in mice treated with IFO-SQ compared to mice treated with placebo. Similar results are expected for the compound IFO-thio-SQ.