MXPA00003031A - Polymeric derivatives of camptothecins - Google Patents

Abstract

Water soluble polymeric conjugates of camptothecin comprise N-(2-hydroxypropyl)methacryloylamide units linked via a spacer of the formula -Gly-(CH2)n-CO-Gly wherein n=2-8 to the C-20 position of a camptothecin residue. The conjugates possess enhanced antitumor activity and decreased toxicity with respect to the free drug. A process for their preparation and pharmaceutical compositions containing them are also described.

Description

POLYMERIC DERIVATIVES OF CAMPTOOTECINS FIELD AND BACKGROUND OF THE INVENTION The invention relates to polymeric solvents of 20-O- [glycyl-a-inoethyl-glisyl] camptotesins. WO-95/10304 describes and claims samptotesin conjugates attached to a polymer through a peptidyl spaser. Currently, it has been discovered that conjugates in which the spacer is a glycyl-aminoasilyl glycillide have an exceptional value as an antitumor agent and are endowed with a remarkable antitumor activity and a reduced toxicity in comparison with the free drug.

DESCRIPTION OF THE INVENTION In particular, the present invention provides polymer conjugates of the formula (1) consisting of: (i) from 85 to 97 mol% of N- (2-hydroxypropyl) methacryloylamide units represented by the formula (3) REF .: 33054 I CH3-C-CO-NH-CH2-CHOH-CH3 O) (ii) from 3 to 15 mol% of 20-O- (N-methacryloyl-glycyl-aminoacyl-glycyl) -camptothecin units represented by the formula ().

CH2 CHj-C-CO-Gly-NH ^ CH ^ n-CC jly-tO-CPT] OD wherein n is from 2 to 8, - [O-CPT] represents the residue of a camptothecin of the formula (2) which is attached to the C-20 position and in which each of Ri, R2, R3, R4 and Rs, which are the same or different, is: hydrogen, straight or branched C1-C12 alkyl, nitro, amino , (CH2) aNR6R7 wherein a is from 0 to 4 and Re and R7 are hydrogen or one of Rβ or R7 is hydrogen and the other of Rβ or Ri is Ci-Cß alkyl or NR6R7 represents a piperazino or N ring -alkyl-piperazine optionally substituted with linear or branched Ci-Ce alkyl or a piperidino ring, (CH2) aNHCOR8, wherein a is as defined above and Rs is linear or branched Ci-Cs alkyl or a group R6R7 as above, hydroxy or O-CO-Rs, wherein Rs is as defined above or represents a 1-piperidino ring or 1,4'-bipiperidino, or R2 and R3, taken together, represent the residue of 0- (CH2) b-0, where b is 1 or 2, or R and R5 represent the residue of (CH2) m, where m is from 2 to 4, or the residue CH2-O-CH2 or CH2NHCH2 and (iii) from 0 to 12 mol% of N-methacryloyl-glycine or N- (2-hydroxy-propyl) units methacryloyl-glycinamide represented by the formula (_5) CH.

CHrC-CO-Gly-fZ] tf) wherein [Z] represents a hydroxy group or a residue of the formula -NH-CH2-CH (OH) -CH3. The polymer conjugates of formula (1) can be indicated as MAG-CPT (s) and can also be represented as follows: [(3)] x; [(4)] and; [(5)] z wherein (3), (4) and (5) are units of the formula defined above, and x is from 85 to 97 mol%, and from 3 to 15 mol% and z is from 0 to 12% mol. A preferred embodiment of the compounds of the present invention are those in which - [O-CPT] in the formula (4_) constitutes a residue of a camptothecin of the formula (2) selected from: camptothecin [2: Ri = R2 = R3 = R4 = Rs = H]; 9-aminocamptothecin [2b: Ri = R2 = R3 = Rs = H, R4 = NH2]; 9-nitrocamptothecin [2c: Ri = R2 = R3 = Rs = H, R4 = NO2]; 7-ethyl-10-hydroxycamptothecin [2d: Ri = R2 = R = H, R3 = OH, R5 = CH2CH3]; 7-eti-10- [1,4'-bipiperidinyl] carbonyloxycamptothecin [2e: Ri = R2 = R = H, R3 = OCO- [1,4'-bipiperidinyl], R5 = CH2CH3], 7-methylenedimethylamino-10- hydroxycamptothecin [2f_: Ri = R2 = R4 = H, R3 = OH, Rs = CH2N (CH3) 2] and 7- [methylene- (A '-methylpiperazino)] -9, 10-ethylenedioxisamptotesine [J2g: Ri = R = H, R2, R3 = 0- CH2CH2-O, Rs = methylene- (4'-methylpiperazino)].

Preferably, the polymer conjugates of the formula (JL) contain the N- (2-hydroxypropyl) metharyloyl amide units represented by the formula (J3) in a propionion of 90% or more; more preferably, 90%. Likewise, the conjugate can be from 3 and 15 mol% of the units represented by the formula (A), more preferably, 10 mol% of said units. Preferably, the conjugate of the formula (JL) does not contain residues of the formula (_5), ie, z is 0. The content of the active camptothecin derivative of the formula (2) in the conjugate of the formula (JL) can be from 2 to 15% (weight / weight), more preferably 10% (w / w) • The preparation of the compounds of the present invention can be carried out following a procedure (hereinafter referred to as Via I), which comprises reacting a 20-O- (aminoacyl-glycyl) camptothecin derivative of the formula 6) NH2- (CH) n-CO-Gly- [OCPT] (6) wherein n and [OCPT] are as defined above, with a polymer (B) consisting essentially: from 85 to 97 mol% of N- (2-hydroxypropyl) methacryloylamide units represented by the formula (3) as defined above, and from 3 to 15 mol% of units of an N-methacryloylglycyl derivative represented by the formula (7) CH, wherein [Y] is the residue of an active ester and, preferably, of a p-nitrophenyl ester, or a hydroxy group; and optionally, displacing the remainder of the active ester groups with an l-amino-2-propanol. The condensation of the derivative of the formula (6) with the polymer of the formula (B) is carried out under conditions capable of preserving the nature of the linkage between the camptothecin and the aminoacyl-glycyl spacer as well as that of the conjugate. The polymers of the formula (B), which consist of N- (2-hydroxypropyl) -methacryloylamide units of the formula (J3) and of N-methacryloyl-glycine units of the formula (J7), are prepared by the copolymerization of N- (2-hydroxypropyl) metasyrylamide with N-methacryloyl-glycine or active ester derivatives of N-methacryloyl-glisine, as described in Makromol. Chem. 178, 2159 (1977). The residue [Y] can represent a phenoxy group which is substituted on the phenyl ring with one or more electron uptake groups, such as nitro or halogen. Preferably, the residue [Y] represents p-nitro phenoxy. The reaction between (6) and (B) to form a conjugate of the polymeric drug of the formula (JL) of the present invention, can generally be carried out at a temperature of from 15 to 35 ° C and, preferably, at room temperature for 15 hours; then the aminolysis of the remaining active ester groups can be carried out in the presence of l-amino-2-propanol at room temperature, for 0.5 to 1 hour. Conveniently, the conjugate is precipitated with ethyl acetate, dissolved in ethanol and reprecipitated with ethyl acetate. For example, polymer (B) is treated, wherein [Y] represents the residue of an active ester, provided at a concentration of 15% (weight / volume) in dry dimethyl sulfoxide, with a derivative of 20-O-) aminoacyl-glycyl) camptothecin (< 5), 3% (w / v), at room temperature for 15 hours. Subsequently, l-amino-2-propanol, 0.1% (w / v) is added and the reaction mixture is kept at room temperature for 1 hour. It is possible to precipitate the polymeric drug conjugate, MAG-CPTs, with ethyl acetate, and collect, wash with ethyl acetate, and then dissolve with absolute ethanol at a concentration of 10% (w / v) and become to precipitate with ethyl acetate to produce the conjugates of the formula (JL) according to the invention. The content of camptothecin in the polymer conjugate of the invention is determined by HPLC or by absorption spectroscopy analysis. The compounds of the formula (JL) can also be prepared by a process (wherein the present is designated as Via II), which comprises the polymerization between N- (2-hydroxypropyl) methacrylamide of the formula (8) CH, Cll3-C-CO-NH-CH, -CHOH-CH3 (S) and derivatives of 20-O- [methacryloyl-glisyl- (aminoacyl) -glycyl] camptothecin of the formula (J9) CH, CH3-C-CO-Gly-NH- (CH2) p-CO-Gly- [OCPT] (2) wherein n and [OCPT] are as defined above, under conditions capable of preserving the nature of the linkage between the camptotesine and the glisil-aminoacyl-glycyl spaser as well as that of the conjugate. Reaction between (8_) and (J9), in general, can be carried out at a temperature of 55 to 70 ° C, preferably at 60 ° C, from 6 to 24 hours, preferably for 15 hours, in an aprotic solvent , such as dimethylsulfoxide, and in the presence of a catalyst, such as 2,2'-azobisisobutyronitrile. The conjugate is precipitated with ethyl acetate, dissolved in ethanol and reprecipitated with ethyl acetate. For example, N- (2-hydroxypropyl) methacrylamide (8_) is heated, provided at a concentration of 22% (w / v) and a derivative of 20-O- [methacryloyl-glycyl- (6-aminohexanoyl) -glycyl] camptothecin ( 9) at a concentration of 6% (w / v) in dry dimethylsulfoxide a is heated at 60 ° C under nitrogen and then 2,2'-azobisisobutyronitrile is added at a concentration of 1.3% (w / v). The mixture is maintained with stirring for 24 hours. After this, the reaction mixture is cooled to room temperature and, conveniently, the conjugate is precipitated with ethyl acetate, dissolved in ethanol and re-precipitated with ethyl acetate to produce the conjugate of the formula (JL ) according to the invention. The invention also provides 20-O-acylamino-glisil-camptothecin derivatives (6) as defined above and their salt derivatives. Aionally, the present invention provides a process for preparing 20-O- (aminoacyl-glycyl) camptothecin derivatives (6), said process comprising condensing the residue of formula (2) as defined above with an aminoacyl-glycyl derivative N-protected formula (10) Rg-NH- (CH2) n-CO-Gly- [P] (10) wherein n is as defined above, Rg represents an amino protecting group, such as Boc, FMOC, triphenylsilyl, diphenylmethylene or triphenylmethyl, and [P] is the residue of an activated ester, such as p-nitro phenoxy or N-hydroxysuccinimido, to produce a compound represented by the formula (11): Rg-NH- (CH2) n-CO-Gly- [OCPT] (11! wherein n, [OCPT] and Rg are as defined above; and removing the protection group N from the resulting compound. The preparation of the compounds of the formula (10) follows the standard synthetic procedures known from the literature. Suitable N-protected aminoacyl derivatives of the formula (10) include: p-nitrophenyl ester of 6-N- (triphenylmethyl) hexanonyl glycyl (10a), p-nitrophenyl ester of 6-N- (tert-butoxycarbonyl) hexanonyl) -glycyl (10b). In this way, for example, camptothecin (2a) can be allowed to react with a molar excess of, for example, up to five times the molar excess or more, in particular, 2 mole equivalents of an N-protected aminoacyl derivative of the formula JH) in anhydrous solvent, such as dry dimethylsulfoxide, in the presence of 4-dimethylaminopyridine. In general, the reaction can be effected from 8 to 48 hours. Typically, the reaction is carried out at a temperature comprised between 15 to 40 ° C. the removal of the temporary amino-protected group R9 with an appropriate deprotection agent to produce the 20-O- (aminoasilyl-glycyl) camptothecin of the formula (> a). Accordingly, deprotection can be obtained by treatment with acid, such as, by treatment with aqueous hydrochloric acid 1.5 N in acetic acid or aqueous 90% trifluoroacetic acid, from one to 6 hours at a temperature comprised between 10 to 30 °. C; preferably, for two hours at room temperature. Additionally, the invention provides 20-O- [methacryloyl-glycyl- (aminoacyl) -glycyl] camptothecin derivatives (J9) as defined above and a process for their preparation, which comprises condensing camptothecin derivatives of the formula (6). ), as defined above with N-methacryloyl-glycyl of the formula (1 '), where [Y '] is a leaving group. Thus, for example, 20-O- [aminoacyl-glycyl] camptothecin (6a), reacted at a concentration of 25% (w / v) in dry dimethyl sulfoxide, is reacted with N- (methacryloyl-glycyl ester p-Nitrophenyl ((1_J), [Y '] = p-nitro-phenol), 13% (w / v) in the presence of an equivalent amount of a base, such as triethylamine, for 15 at room temperature. The final derivative is isolated by precipitation and purified by chromatography. The compounds of the formula (8) and the polymer (B) are known or can be prepared by known synthetic methods. All camptothecin derivatives of the formula (2) are known, see for example Medicinal Research Reviews, Volume 17, No. 4, 367-425, 1997, or they can be prepared by well-known methods. The conjugates bound by polymers of the formula (JL) are within the molecular weight range of 5,000 to 45,000, preferably from 18,000 to 35,000. The polymerized polymer conjugates of the formula (1) are soluble in water and have a remarkable antitumor activity and a low toxicity compared to free camptothecin.

Antitumor activity The compound Al was analyzed in human colon carcinoma (HT29) transplanted in hairless mice, in comparison with the free drug (2a) via i.v. it was discovered that Al was not toxic and produced a tumor inhibition of > 95% in all the doses analyzed with a high exceptional number of animals free of the tumor at the time of the end of the experiment (90 days). Table 1. Additionally, it was discovered that compound A2, analyzed with the same model, had activity and was not toxic in comparison with free 7-ethyl-10-hydroxycamptothecin (2d) and it allowed to obtain a tumor inhibition of 98 % at the highest dose analyzed of 40 mg / kg (Table 2). Additionally, the Al compound was analyzed by i.v. in a large panel of other human tumor models: ovarian carcinoma A2780, breast carcinoma MX1, lung carcinoma A549 NSC and melanoma M14. Compound Al had a higher activity with respect to the corresponding free camptothecin (2a) and allowed obtaining a large number of cured animals. Respectively, Tables 3, 4, and 5 report the activity against breast carcinoma MX1, carcinoma of ovaries A2780 and melanoma M14, in comparison with free camptothecin. The Al compound had a high activity against these tumor models, for which a total inhibition of tumor growth was observed with a total of 7/7 and 8/8 mice cured through the repeated administration by i.v. of the drug at 15 or 20 mg / kg with the schedule of q4dx6. It was observed that compound Al also had activity against lung carcinoma NSC at 20 mg / kg (iv, q4dx6) with 94% IT and with a notable delay in tumor growth of 70 days never before observed with other useful chemotherapeutic agents (Table 6).

Table 1: Antitumor activity of Al in human colon carcinoma (HT29) compared to (2a.) • Treatment iv q4dx6.

Fragments of the sc tumor were implanted. The treatment began when the tumor was palpable. % IT (% inhibition of the tumor) was calculated on day 37.? TGD: Delay in tumor growth of treated animals - Delay in tumor growth of control animals.

Table 2: Antitumor activity of A2 in human colon carcinoma (HT29) compared to (2d).

Fragments of the sc tumor were implanted. The treatment began when the tumor was palpable. % IT (% inhibition of the tumor) was calculated on day 37.? PDD: Delayed tumor growth of treated animals - Delay in tumor growth of control animals.

Table 3: Anti-tumor activity of Al against MX1, breast carcinoma, in comparison with camptothecin (2a).

Fragments of the tumor were implanted, the treatment began when the tumor was palpable. The doses were expressed as camptothecin equivalents. % IT: Inhibition of tumor growth 1 week after the last treatment. Tox. : Number of mice that died due to the toxicity / total number of mice. • TGD: Delay in the growth of the treated tumor. Control of growth retardation of the tumor. Tumor free: Mice healed on day 90 after tumor implantation.

Table 4: Anti-tumor activity of Al against A2780, carcinoma of human ovaries, compared with camptothecin (2a).

Fragments of the tumor were implanted, the treatment began when the tumor was palpable. The doses were expressed as camptothecin equivalents. % IT: Inhibition of tumor growth 1 week after the last treatment. Tox. : Number of mice that died due to the toxicity / total number of mice. • TGD: Delay in the growth of the treated tumor. Control of growth retardation of the tumor. Tumor free: Mice healed on day 90 after tumor implantation.

Table 5: Anti-tumor activity of Al against M14, lung melanoma, in comparison are camptothecin (2a.).

Fragments of the tumor were implanted, the treatment began when the tumor was palpable. The doses were expressed as camptothecin equivalents. % IT: Inhibition of tumor growth 1 week after the last treatment. Tox. : Number of mice that died due to the toxicity / total number of mice. • TGD: Delay in the growth of the treated tumor. Control of growth retardation of the tumor. Tumor free: Mice healed on day 90 after tumor implantation.

Table. 6: Anti-tumor activity of Al against A549, human NSC lung carcinoma, compared to samptothecin (2a).

Fragments of the tumor were implanted, the treatment began when the tumor was palpable. The doses were expressed as camptotesine equivalents. % IT: Inhibition of tumor growth 1 week after the last treatment. Tox. : Number of mice that died due to the toxicity / total number of mice. • TGD: Delay in the growth of the treated tumor. Control of growth retardation of the tumor. Tumor free: Mice healed on day 90 after tumor implantation.

Accordingly, the compounds of the present invention are useful in the treatment of leukemia and solid tumors, such as tumors: colon, colo-rectal, gastric, ovarian, breast, prostate, lung, kidney. and also melanoma. Therefore, it is possible to treat a human being by following a method comprising administering thereto a therapeutically effective amount of a polymer conjugate of the invention. In this way, the condition of the human patient can be improved. The dosage range adopted will depend on the route of administration and the age, the weight and condition of the patient who is going to undergo treatment. In general, the polymer conjugates of the formula (JL) are administered parenterally, for example intramuscularly, intravenously or by bolus infusion. A suitable dosage range ranges from 1 to 1000 mg of camptothecin, equivalent, per m body surface area, for example between 10 and 100 mg / m. The polymer conjugate (JL) can be formulated in the form of a pharmaceutic composition together with a pharmaceutically acceptable carrier or diluent. It is usually formulated for parenteral administration, for example, by dissolution in water for injections or physiological saline. The Examples that follow illustrate the invention.

Example 1 Preparation of: N- (tert-butoxycarbonyl) -6-aminohexanoyl-glisyl p-nitrophenyl ester [10a: n = 5, Rg = Boc, P = p-nitrophenol] Glycine ethyl ester hydrochloride (9.55 g, 68.4 mmol), dissolved with dimethylformamide (100 ml), with triethylamine (9.5 ml, 68.4 mmol) and then with N- (tert-butyloxycarbonyl) -6-aminohexanoyl p-nitrophenyl ester (19 g, 54 mmol), was prepared according to the same procedure described in the EP No. 0673258. The reaction mixture was kept for two hours at room temperature, then the solvent was removed under reduced pressure. The residue was dissolved with ethyl acetate (300 ml) and washed in a consis- tent manner with a very cold ascorbic slurry (3 x 200 ml), water (100 ml), 5% aqueous sodium sulphate (2 x) 200 ml) and water (2 x 200 ml). The organic phase was dried over anhydrous sodium sulfate, then the solvent was removed under reduced pressure. The residue was crystallized from ethyl ether to produce ethyl N- (tert-butyloxycarbonyl) -6-aminohexanoyl-glycyl ester (15 g; TLC on Kieselgel F254 plate (Merck), ethyl ether elution system, Rf = 0.3) the sual was suspended with ethanol (150 ml) and treated with stirring with IN aqueous sodium hydroxide (48 ml, 48 mmol). After one hour, the reaction was added with IN aqueous hydrochloric acid (48 ml, 48 mmol) and distilled under reduced pressure. The residue was suspended with dry tetrahydrofuran (200 ml), p-nitrophenol (6.53 g, 47 mmol) was added, cooled to 0 ° C and then added with a solution of 1,3-dicyclohexyl-carbodiimide (9.7 g, 47 mmol) in tetrahydrofuran (100 ml). The reaction was left in the same conditions overnight, then filtered in a sintered glass funnel. The solvent was removed under reduced pressure. The residue was crystallized from ethyl ether to yield 17.5 g of the title compound (10a). TLC on plate F254 Kieselgel (Merck), elution system of methylene chloride / methanol (95/5 v / v) Ri = 0.34. ^? - NMR (200 MHz, DMSO) d: 1.34 (S, 9H, t-Bu); 1.0-1.7 [m, 6H, NH-CH2- (CH2) 3-CH-CO]; 2.15 (t, J = 7.2Hz, 2H, NH- (CH2) 4-CH2-CO); 2.85 [q, J = 6.5 Hz, 2H, NH-CH2- (CH2) 4-CO); 4.11 (d, J = 5.5 Hz, 2H, CONHCH2COO); 6.70 [bs, 1H, NH- (CH2) 5-CO]; 7.40 (m, 2H, aromatic 2,6-H); 8.30 (m, 2H, aromatic 3,5-H); 8.43 (t, J = 5.5 Hz, 1H, CONHCH2COO).

Example 2 Preparation of: 20-O- [(N-tert-Butyloxycarbonyl) -6-aminohexanoyl-glycyl] camptothecin [Ilia; n = 5, Ro = t-Boc, OCPT = (2a)] Camptothecin (2a: 3.4 g, 10 mmol), suspended with dimethylsulfoxide (50 ml), was treated with N- (ters-butyloxysarbonyl) -6-aminohexanoyl-glycyl p-nitrophenyl ester (10a: 6.3 g, 15 mmol) and 4-dimethylaminopyridine (2.4 g, 20 mmol). The reaction mixture was allowed to stand for 24 hours and then an additional aliquot of N- (tert-butyloxycarbonyl) -6-aminohexanoyl-glycyl p-nitrophenyl ester (6.3 g, 15 mmol) was added. After 48 hours, the reaction mixture was diluted with methylene chloride (500 ml) and washed with 0.2 N aqueous hydrochloric acid (2 x 250 ml) and water (2 x 250 ml). The organic phase was dried over anhydrous sodium sulfate, then the solvent was removed under reduced pressure. The residue was dissolved with methylene chloride (100 ml), added with ethyl ether (500 ml) and kept at 0 ° C overnight to yield 5 g of the title compound (lia) as a solid. TLC on plate F254 Kieselgel (Merck), elution system of methylene chloride / methanol (95/5 v / v) Rf = 0.44.

Example 3 Preparation of: 20-O- (6-aminohexanoyl-glycyl) camptothecin [6a: n = 5, OCPT = (2a.)] -O- [(N-tert-butyloxycarbonyl) -6-aminohexanoyl-glycyl] camptothecin (lia; 5g) was treated with 90% aqueous trifluoroacetic acid (40 ml) for 1 hour, then the solvent was removed under reduced pressure. . The residue was triturated with ethyl ether (300 ml) and filtered. The solid was dissolved in methanol (200 ml), reduced to a small volume (50 ml) under reduced pressure, ethyl ether (300 ml) was added. The precipitate was pelleted to yield 3.9 g of the title compound (6a). TLC on plate F25- Kieselgel (Merck), elution system of methylene chloride / methanol / acetic acid / water (80/20/7/3 v / v), Rf = 0.83.

Example 4 Preparation of: 20-O- [methacryloyl-glycyl- (6-aminohexanoyl) -glycyl] camptothecin [9a: n = 5, OCPT = (2a)] -O- (6-aminohexanoyl-glycyl] camptothecin trifluoroacetate (6a, 2.53 g, 4 mmol) was dissolved with anhydrous dimethyl sulfoxide (10 ml) and added with p-nitrophenyl methacryloyl-glycyl ester (1.32 g, 5 mmol). ), prepared as described in Makromel Chem. 178, 2159 (1977), and triethylamine (0.56 ml, 4 mmol) After allowing to stand overnight at room temperature, the solution was poured into water (100 ml). and the precipitate was collected and washed with water (2 x 100 ml) The solid material was subjected to flash chromatography on silica gel using a mixture of methylene chloride / ethanol (95/5 v / v) as the elution system. to produce 2.2 g of the title compound (J9a). TLC on plate F25 Kieselgel (Merck), methylene chloride / methanol elution system (9/1 v / v) Rf = 0.62. 1 H-NMR (200 MHz, DMSO) d: 0.90 (t, J = 7.3 Hz, 3H, CH 3 -I8); 1.1-1.6 [m, 6H, NHCH2- (CH2) 3-CH2CO]; 2.0-2.2 [m, 4H, CH2-19 + NH- (CH2) -CH2-CO]; 2.95 (q, J = 6.3 Hz, 2H, NH-CH2- (CH2) CO); 3.63 (d, J = 5.8 Hz, 2H, CONH-CH2-CONH); 3.97 (dd, J = 17.8, 5.9 Hz, 1H, CONHCHaCHbCOO); 4.15 (dd, J = 17.8, 5.9 Hz, 1H, CONHCHaCHbCOO); 5.29 (s, 2H, CH2-5); 5.33 [q, J = 1.6 Hz, 1H, CH3CH = C (Ha) (Hb)]; 5.48 (S, 2H, "CH2-17); 5.69 [m, 1H, CH3CH = C (Ha) (Hb)]; 7.15 (s, 1H, H-14); 7.6-7.9 (m, 3H, H- 10 + H-II + NH- (CH2) sCO]; 8.0 (t, J = 5.8 Hz, 1H, CO-NH-CH2CONH); 8.15 (m, 2H, H-9 + H-12); 8.32 (t , J = 5.9 Hz, 1H, CO-NH-CHaCHbCOO], 8.69 (s, 1H, H-7).

Example 5 Preparation of: 7-ethyl-10-hydroxy-20-O- (6-aminohexanoyl-glycyl) camptothecin trifluoroacetate [6b: n 5, OCPT = (2d)] 7-Ethyl-10-hydroxy-camptothecin (2d, 0.8 g, 2 mmol), N- (tert-butoxycarbonyl) -6-amino-hexanonyl-glycyl p-nitrophenyl ester (10a: 2.5 g, 6 mmol) were dissolved. and 4-dimethylaminopyridine with dry dimethylsulfoxide (30 ml) and kept at room temperature for 3 days with stirring. After that, the reaction mixture was poured into 0.1 N aqueous hydrochloric acid (500 ml) to produce a precipitate which was collected, then dissolved in methylene chloride (300 ml) and washed with water (2 x 100 ml). . The organic phase was separated, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was treated with 90% aqueous trifluoroacetic acid (40 ml) for three hours, then the solvent was removed under reduced pressure and the residue was subjected to flash chromatography on silica gel using a methylene chloride / acetic acid / mixture. methanol (100/5/20 v / v) as an elution system. The fractions which were in the title pool were collected and evaporated under reduced pressure to yield 1.02 g of (6b) as a trifluoroacetate salt derivative. TLC on plate F25- Kieselgel (Merck), elution system of methylene chloride / methanol / acetic acid / water (80/20/7/3 v / v), Rf = 0.4. ^? - NMR (200 MHz, DMSO) d: 0.89 (t, J = 7.2 Hz, 3H, CH3-CH2-20); 1.1-1.5 (m, 9H, NH2-CH2-CH2-CH2-CH2-CH2 + CH3-CH2-7); 1.80 (s, 3H, CH 3 -COOH); 2.10 (m, 4H, CH3-CH2-20 + CH? -CONH); 2.53 (t, 6.8 Hz, 2H, CH2-NH2); 3.06 (m, 2H, CH3-CH2-7); 3.98, 4.13 (two-dd, J = 17.6, 5.7 Hz, 2H, CONH-CH2-CO); 5.27 (s, 2H, CH2-5); 5.46 (s, 2H, CH -17); 7.01 (s, 1H, H-14); 7.40 (, 2H, H-9 + H-ll); 7.99 (d, J = 9.8 Hz, 1H, H-12); 8.33 (t, J = 5.7 Hz, 1H, CONH-CH2-CO).

Example 6 Preparation of: 7-ethyl-10-hydroxy-20-O- (6-aminohexanoyl-glycyl) camptothecin hydrochloride [6b: n = 5, OCPT = (2d)] 7-Ethyl-10-hydroxy-camptothecin (2d, 0.8 g, 2 mmol) was reacted with N- (tert-butoxy-carbonyl) -6-aminohexanonyl-glycyl p-nitrophenyl ester (10a, 2.5 g, 6 mmol). ) and 4-dimethylaminopyridine as described in Example 5. The crude material obtained was dissolved from the reaction mixture by extraction of methylene chloride in a mixture of hydrochloric acid and acetic acid 1.5 N (20 ml). After allowing it to stand for 1 hour with stirring at room temperature, the solution was reduced to a small volume by distillation and ethyl ether (100 ml) was added. The precipitate was collected and washed with ethyl ether (2 x 50 ml) to yield 1 g of the title compound (6b) as a free amino derivative.

Example 7 Preparation of MAG-camptothecin by I [Al] n = 5, OCPT = (2a)] The polymeric precursor B (R2 = p-nitrophenyloxy, 2.58 g, containing 1.16 x 10 ~ 3 eq. Of p-nitrophenyl ester residue) was dissolved, prepared as described in Makromol. Chem. 178, 2159 (1977), with dry dimethylsulfoxide (15 ml) and added are 20-O- (6-aminohexanoyl-glycyl) camptothecin trifluoroacetate (j> 0.63 g, 1 mmol), followed by triethylamine. (0.14 ml, 1 mmol). The reaction mixture was kept at room temperature for 22 hours with stirring, then 2-propanolamine (0.05 ml) was added and the mixture was left under stirring for an additional hour.

After this, the reaction mixture was precipitated with ethyl acetate (200 ml) and the mixture was stirred for 30 minutes. The solid material was cored in a sintered glass funnel, washed with ethyl acetate (200 ml) and ethyl ether (100 ml) and then dissolved with ethanol (30 ml). The alcoholic solution was treated with wet DOWEX-50, in sulphonic form, (1.2 g) under stirring for 30 minutes and, after this, n-hexane (200 ml) was added dropwise. The precipitate was collected in a sintered glass funnel, washed with ethyl ether and dried at a constant weight to yield 2.68 g of the title compound (Al). Weight-average molecular weight (Mw): 19,800. Polydispersion (Mw / Mn): 1.5. Camptothecin content, determined after alkaline hydrolysis, 10% w / w.

Example 8 Preparation of MAG- (7-ethyl-10-hydroxycamptothecin) by Via I [A2: n = 5, OCPT = (2d)] The polymer precursor (B) (R2 = p-nitrophenyloxy, 2.58 g, containing 1.16 x 10"eq p-nitrophenyl ester residue) was dissolved, prepared as described in Makromol Chem. 178, 2159 ( 1977), with dry dimethylsulfoxide (15 ml) and was added with 7-ethyl-10-hydroxy-20-O- (6-aminohexanoyl-glycyl) camptothecin trifluoroacetate (6b, 0.68 g, 1 mmol), followed by triethylamine ( 0.14 ml, 1 mmol) The reaction mixture was kept at room temperature for 22 hours under stirring, then 2-propanolamine (0.05 ml) was added and the mixture was left to stir for another hour. The reaction mixture was treated with ethyl acetate (200 ml) and left to stir for 30 minutes, the solid material was collected in a sintered glass funnel, washed with ethyl acetate (200 ml) and ethyl ether (100 ml). and then it was dissolved with ethanol (30 ml) The alcoholic solution was treated with wet DO EX-50, in sulphonic form ca, (1.2 g) under stirring for 30 minutes and, after this, n-hexane (200 ml) was added dropwise. The precipitate was collected in a sintered glass funnel, washed with ethyl ether and dried at a constant weight to yield 2.68 g of the title compound [A2] Weight-average molecular weight (Mw): 20,500. Polydispersity (Mw / Mn): 1.87. Content of 7-ethyl-10-hydroxy-camptothecin, determined after alkaline hydrolysis, 10% w / w.

Example 9 Preparation of MAG-samptotesin by Pathway II [Al: n = 5, OCPT = (2a)] -O- [methacryloyl-glycyl- (6-aminohexanoyl) -glycyl] samptotesine (J9a: 1.26 g, 2 mmol) was dissolved, N- (2-hydroxypropyl) methacrylamide (J3, 4.4 g, 31 mmol), prepared as described in Makromol. Chem. 178, 2159 (1977), and 2, 2'-azobisisobutyronitrile (0.26 g, 1. 6 mmol) with anhydrous dimethyl sulfoxide (20 ml) was maintained at 60 ° C under nitrogen and stirred for 24 hours. After this, the reaction mixture was cooled to room temperature and poured into ethyl acetate (500 ml). The precipitate was collected and dissolved with ethanol (50 ml) from which it was reprecipitated by the addition of ethyl acetate (500 ml). The solid was re-evaporated, washed with ethyl acetate ethyl ether (2 x 100 ml) to yield 5 g of the title compound (Al).

It is noted that in relation to this date the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above it claims as property what is contained in the following

Claims (17)

1. A polymeric conjugate characterized in that it consists of: (i) from 85 to 97 mol% of N- (2-hydroxypropyl) methacryloylamide units represented by the formula (3) CH, CH3-C-CO-NH-CH2-CHOH-CH3 Q) (ii) from 3 to 15 mol% of 20-O- (N-methacryloyl-glycyl-aminoacyl-glycyl) -camptothecin units represented by the formula (4_). CH2 I CH3-C-CO-Gly-NH- (C? 2) n-CO-Gly- [0-CPri) wherein n is from 2 to 8, - [O-CPT] represents the residue of a camptothecin of the formula (2) which is attached to the C-20 position and in which each of Ri, R2, R3, R4 and Rs, which are the same or different, is hydrogen, straight or branched C1-C12 alkyl, nitro, amino, (CH2) aNR6R7 wherein a is from 0 to 4 and Re and R7 are hydrogen or one of Re or R7 is hydrogen and the other of R6 or R is Ci-Cß alkyl or R6R7 represents a piperazino or N- ring alkyl-piperazino optionally substituted with linear or branched Ci-Cβ alkyl or a piperidino ring, (CH 2) aNHCORs, wherein a is as defined above and Re is linear or branched Ci-Cβ alkyl or a group NR6R7 as above, hydroxy or O-CO-Rs, wherein Rs is as defined above or represents a ring of 1-piperidino or 1, '-bipiperidino, or R2 and R3, taken together, represent the residue of 0 - (CH2) b-0, wherein b is 1 or 2, or R and R5 represent the residue of (CH2) m, wherein m is from 2 to 4, or the residue CH2-O-CH2 or CH2NHCH2 and (iii) from 0 to 12% mole of unit N-methacryloyl-glycine or N- (2-hydroxy-propyl) methacryloyl-glycinamide ades represented by the formula (5_) CH. CH3-C-CO-Gly-fZ] wherein [Z] represents a hydroxy group or a residue of the formula -NH-CH2-CH (OH) -CH3.

2. A polymeric conjugate, according to claim 1, characterized in that it contains the N- (2-hydroxypropyl) methacryloylamide units represented by the formula (3_) in a molar ratio of 90%.

3. A polymeric conjugate, according to claim 1, characterized in that it contains 10 mol% of those represented by the formula (4.

4. A polymer conjugate, according to claim 1, characterized in that the unit of the formula (5) is absent.

5. A polymer conjugate, according to claim 1, characterized in that - [O-CPT] in the formula (A_) is a residue of a camptothecin of the formula (2) selected from: camptothecin, 9-aminocamptothecin, 9- nitrocamptothecin, 7-ethyl-10-hydroxy-camptothecin, 7-ethyl-10- [1,4'-bipiperidinyl] carbonyloxycamptothecin, 7-methylenedimethylamino-10-hydroxycamptothecin and 7- [methylene- (4'-methylpiperazino)] -9 , 10-ethylenedioxicam.ptothecin.

6. A polymer conjugate, according to claim 1, characterized in that the content of the active camptothecin derivative of the formula (2) is 10% (w / w).

7. A process for producing a polymer conjugate according to claim 1, characterized in that said method comprises reacting a derivative of 20-O- (aminoacyl-glycyl) samptotesine of the formula (6) NH2- (CH2) n-CO-Gly- [OCPT] (6) wherein n and [OCPT] are as defined in claim 1, with a polymer (B) consisting essentially: from 85 to 97 mol% of N- (2-hydroxypropyl) methacryloylamide units represented by the formula (J3) as defined in claim 1, and from 3 to 15 mol% of units of an N-methacryloyl-glycyl derivative represented by the formula (J7) CH, CH3-C-CO-Gly- [Y] (2) wherein [Y] is the residue of an active ester or a hydroxy group; and optionally, displacing the remainder of the active ester groups with an l-amino-2-propanol.

8. A process for producing a polymer conjugate according to claim 1, characterized in that said process comprises the polymerization between N- (2-hydroxypropyl) methacrylamide of the formula (8) CH, ClI3-C-CO-NH-CH, -CHOH-CH3 (£) and derivatives of 20-O- [methacryloyl-glycyl- (aminoacyl] glycyl] camptothecin of the formula (9 ^) CH, CH3-C-CO-Gly-NH CH2) p-CO-Gly- [OCPT] ß) in which ny [OCPT] are as defined in claim 1, under conditions capable of preserving the nature of the link between camptothecin and the glycyl-aminoacyl-glycyl spacer as well as that of the conjugate.

9. A 20-O-acylamino-glycyl-samptotesine derivative of the formula (6-), of the form of claim 7, or a salt thereof.

10. A process for producing a compound of the formula (6) according to claim 7, wherein the derivative of the present invention comprises condensing a derivative of the formula (2) as defined in claim 1 with an α-inoacyl-glycyl derivative. -protected from the formula (JL_0): Rg-NH- (CH) n-CO-Gly- [P] (io; wherein n is as defined in claim 1, Rg represents an amino protecting group and [P] is a residue of an activated ester, to produce a compound represented by the formula (JL_1): Rg-NH- (CH2) n-CO-Gly- [OCPT] (11) wherein n and Rg are as defined above and [OCPT] is as defined in claim 1, and removing the protecting group N from the resulting compound.

11. A 20-O- [methacryloyl-glycyl- (aminoacyl) -glisyl] camptothecin derivative of the formula (J9) according to claim 8 or a salt thereof.

12. A process for producing a derivative of the formula (J9) according to claim 11, characterized in that said process comprises condensing a camptothecin derivative of the formula (-6), as defined in claim 7 with N-methacryloyl-glycyl of the formula (J7 _ ^ _), CH2 CHj-C-CO-Gly-rY'J (21) where [Y '] is a leaving group.

13. A pharmaceutic composition, characterized in that it comprises a pharmaceutically acceptable diluent or carrier and, as an active ingredient, a polymer conjugate as defined in any one of claims 1 to 6 or a compound of the formula (-6) or (J3) as it was defined in claims 9 and 11.

14. A polymeric conjugate according to claim 1, characterized in that it is MAG-camptothecin, in which the content of camptothecin is 10% (w / w).

15. A polymeric conjugate according to claim 1, characterized in that it is obtained by reacting a derivative of 20-O- (6-aminohexanoyl-glycyl) camptothecin of the formula (_6) NH2- (CH2) e-CO-Gly- [OCPT] (6) wherein [O-CPT] is the residue of camptothecin, with a polymer (B) consisting essentially: from 85 to 97 mol% of N- (2-hydroxypropyl) methacryloylamide units represented by the formula (J3) as defined in claim 1, and from 3 to 15 mol% of units of an N-metasyrylglycyl derivative represented by the formula (7) CH2 I CH3-C-C0-Gly- [Y] (2) wherein [Y] is a p-nitrophenoxy group; and optionally, displacing the remainder of the active ester groups with an l-amino-2-propanol.

16. A polymer conjugate according to claim 1, characterized in that it is obtained by the polymerization between N- (2-hydroxypropyl) methacrylamide of the formula (8) CH, ClI3-C-CO-NH-CH, -CHOH-CH3 (fi) and derivatives of 20-O- [methacryloyl-glycyl- (6-aminohexanoyl) -glycyl] camptothecin of the formula (J9) CH, CH3-C-CO-Gly-NH- (CH2) t-CO-Gly- [OCPTj (S > wherein [OCPT] is the residue of camptothecin.

17. A pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable diluent or carrier and, as an active ingredient, a polymer conjugate as defined in any of claims 14 to 16.