WO2007006543A1

WO2007006543A1 - Camptothecin analogues, process for their preparation, use and formulations containing them

Info

Publication number: WO2007006543A1
Application number: PCT/EP2006/006760
Authority: WO
Inventors: Ezio Bombardelli; Gabriele Fontana
Original assignee: Indena S.P.A.
Priority date: 2005-07-14
Filing date: 2006-07-11
Publication date: 2007-01-18
Also published as: ITMI20051348A1

Abstract

The present invention relates to novel camptothecin derivatives of general formula (I) in which R1 is a straight or branched polyamino residue, optionally functionalized by acylation or alkylation of the amino residues with more than 2 nitrogen atoms; R2 is H, -OR3; R3 is H or a straight or branched C1-C24 saturated or unsaturated acyl residue; R4 is H, straight or branched C1-C4 alkyl, hydroxyalkyl, aminoalkyl or cyano; R5 and R6 can be H, OH or OCH3. The compounds of the present invention can be conveniently administered for the therapy of tumors, in the form of suitable pharmaceutical formulations.

Description

CAMPTOTHECIN ANALOGUES, PROCESS FOR THEIR PREPARATION, USE AND FORMULATIONS CONTAINING THEM

Disclosure of the invention

The present invention relates to novel camptothecin analogues modified in the E ring through lactone-opening with polyamines to give ω-hydroxy amides. The ω-hydroxy function, i.e. the hydroxy group at the 17- position, can be removed or esterified with CpC₂₄ straight, branched, saturated or unsaturated acyl residues.

The compounds of the present invention have general formula (I)

(I) wherein

R₁ is a straight or branched polyamino residue, optionally functionalized by acylation or alkylation of the amino groups, with more than 2 nitrogen atoms;

R₂ is H, -OR₃;

R₃ is H or a Cj-C₂₄ straight or branched, saturated or unsaturated acyl residue;

R₄ is H, straight or branched Cj-C₄ alkyl, hydroxyalkyl, aminoalkyl or cyano;

R₅ and R₆ can be H, -OH or -OCH₃.

Among the polyamines indicated for the R₁ function, spermine and spermidine are preferred. If the polyamine nitrogen atoms are not acylated, the product is salified with suitable organic or inorganic acids, preferably trifluoroacetic, oxalic, tartaric or hydrochloric acid.

The 17- position is preferably esterified with activated derivatives of acetic, ximeninic, c/s-4,7, 10, 13, 16, 19- docosahexenoic or c/s-5,8, 1 1, 14- eicosapentaenoic acid.

The products of the invention are usually synthesized from camptothecin, or from analogues thereof suitably functionalized at the 7-, 9- and 10- positions by reaction with a suitably protected polyamine. The reaction is usually performed in polar aprotic solvents, in the presence of a base. Upon completion of the reaction the mixture is neutralized with acids and extracted with an organic solvent. The crude obtained after evaporation of the solvent can be purified or used as such for subsequent functionalization at the 17- position. The polyamine is preferably protected with a t-butoxycarbonyl (Boc) or a carbobenzyloxy (CBz) group. The protected polyamine is prepared as described in literature.

The esterification of the 17- position is preferably performed on the crude precursor by acylation with the selected acyl chloride in pyridine. The removal of the 17-hydroxy group is instead accomplished by hydrogenolysis with Pd(C) in methanol under the conditions described in greater detail in the examples.

A simplified procedure for the preparation of the salts consists in the use of CBz protected polyamines, and deprotection through hydrogenation in the presence of stoichiometric amounts of the selected acids. Although the integrity of the E-ring is commonly deemed crucial for the cytotoxic activity of camptothecin analogues, the compounds of the present invention maintain an interesting cytotoxic in vitro activity and still inhibit topoisomerase I. As far as in vivo activity is concerned, the products of the invention show activity against the H460 lung tumor model comparable to substances are already used in clinic, such as topotecan, and other active principles currently under clinical investigation. However, the advantage of the compounds of the invention consists in lower toxicity compared with known products and in higher tolerability, which allow a broader therapeutic window.

The products can be incorporated into the most common pharmaceutical forms, either injectable or oral. The examples reported hereinbelow illustrate the invention.

Example I - Preparation of compound A

N²,N³-Di-t-butoxycarbonylspermidine (2.8 g, 8.1 mmol), prepared according to the procedures reported in literature, is added to a suspension of camptothecin (700 mg, 2.01 mmol) in dry pyridine (50 ml), under nitrogen atmosphere. The reaction mixture is heated to 8O⁰C to obtain a clear yellow mixture. After 3 days at 8O⁰C the mixture is cooled and washed with 5% HCl, extracted with CHCl₃ and dried over Na₂SO₄. After evaporation of the solvent, the crude product is purified through flash chromatography (silica gel, CHCl₃: CH₃OH 96: 4) to give the desired compound as a pale yellow solid (730 mg, 52%). ¹H-NMR (CDCl₃): δ 1.15 (t, 3H), 1.25 (4H), 1.45 (18H), 1.65 (2H), 2.39 (m, 2H), 3.1 (4H), 3.25 (m, 4H), 4.35 (d, IH, J=13.2), 5.0 (d, IH, J= 13.2, 5.15 (2H), 7.5-8.2 (6H).

Example II - Preparation of compound B

The product of example I (570 mg, 0.82 mmol) is reacted with acetic anhydride (1.54 ml) in 1.65 ml of dry pyridine to give the desired compound as a yellow solid (488 mg, 81%).

¹H-NMR (CDCl₃): δ 1.15 (t, 3H, J-6.9), 1.2 (6H), 1.45 (18H), 2.05 (s, 3H), 2.35 (m, 2H), 3.05 (4H), 3.2 (4H), 5.25 (s, 2H), 5.4 (2H), 7.5-8.4 (6H).

¹³C-NMR (CDCl₃): δ 7.87 (q), 20.97 (q), 25.66 (t), 2x27.39 (t), 6x28.37

(q), 32.93 (t), 40.15 (t), 3x46.58 (t), 50.2 (t), 58.98 (t), 78.74 (s), 79.64 (s),

100.6 (d), 125.0 (s). 127.72 (d), 127.89 (d), 2x128.35 (s), 129.32 (d), 130.19 (d), 130.61 (d), 2x144.47 (s), 2x148.45 (s), 2x155.97 (s), 161.58 (s), 2x171.15

(S).

HPLC (RP 8; 75% H₂O, 25% CH₃CN): 18.47 min. Example III - Preparation of compound C

The product of example II (488 mg, 0.664 mmol) is dissolved in anhydrous CH₂Cl₂ and deprotected by adding, drop by drop, trifluoroacetic acid (0.8 ml) at 0⁰C under nitrogen atmosphere. The reaction mixture is stirred at 0⁰C for 1.5 h, then the solvent is evaporated off under reduced pressure. The residue is dissolved in CHCl₃ and precipitated from «-hexane. The desired compound is obtained as a yellow solid (505 mg, 98%). ¹H NMR (DMSO-d₆/D₂O) δ 8.63 (s, 1 H, arom), 8.12 (d, 1 H, J = 8.4

Hz arom), 8.05 (d, 1 H, J = 8.4 Hz arom), 7.86-7.81 (m, 1 H, arom), 7.70-7.65 (m, 1 H, arom), 7.50 (s, 1 H, arom), 5.29 (d, 1 H, CH₂-O, J = 11.2 Hz), 5.24 (d, 1 H, CH2-O, J - 1 1.2 Hz), 5.21 (s, 2 H, CH₂-N), 3.20-3.00 (m, 2 H), 2.88-2.80 (m, 4 H), 2.80-2.72 (m, 2 H), 2.18-2.28 (m, 1 H), 2.06-2.18 (m, 1 H), 1.95 (s, 3 H, Me, OAc), 1.76-1.66 (m, 2 H), 1.60- 1.48 (m, 4 H), 0.88 (t, 3 H, J = 7.8 Hz, Me).

¹³C NMR (DMSO-d₆) δ 173.9, 171.0, 161.3, 158.0 (q, CO₂H of TFA), 156.5, 153.4, 148.7, 144.9, 132.3, 131.1, 130.6, 129.7, 1292, 128.7, 128.3, 124.0, 99.5, 79.4, 59.5, 51.0, 46.8, 45.3, 38.9, 36.7, 32.3, 26.5, 24.8, 23.3, 21.4, 8.6.

Example IV - Preparation of compound D

A suspension of 7-ethylcamptothecin (3 g, 7.9 mmol) in dry pyridine is added with N²,N³-dibenzyloxycarbonylspermidine (6.5 g, 15 mmol). The mixture is sonicated at 55°C for 12 hrs. Pyridine is then removed by distillation under reduced pressure at room temperature and the residue is dissolved in dichloromethane (20 ml), washed with a 5% citric acid solution (2x50 ml) and then with water (20 ml). After drying over Na₂SO₄ and filtering, the mixture is evaporated to obtain crude compound D (7.5 g), which is purified through flash-chromatography (silica gel, CHCl₃ :CH₃OH 96: 4) or is used as such for the subsequent reaction.

Example V - Preparation of compound E

The crude product obtained from example IV (7.5 g, 9.6 mmol) is acetylated with acetic anhydride (9.12 g) in dry pyridine (8.6 ml). The reaction is left under stirring overnight at room temperature and under nitrogen atmosphere. The mixture is then poured into ice-water (40 ml) and extracted with methylene chloride (2x30 ml). After evaporation of the solvent, the product is purified through flash chromatography using 98:2 ethyl acetate and methanol. 3.5 g of a yellow solid product are obtained.

Example VI - Preparation of compound F

A solution of compound E (166 mg, 0.2 mmol) in acetonitrile (20 ml) is added with 5% Pd/C (100 mg) and L-(+)-tartaric acid (60 mg, 0.4 mmol). The mixture is hydrogenated at 4 atmospheres for 3 hours. Upon completion of the reaction the mixture is filtered through syntherized glass. The filtrate is concentrated to give compound F (140 mg, 98.5%).

¹H NMR (DMSO-d₆/D₂O) δ 8.26 (d, 1 H, J = 8.4 Hz, arom), 8.17 (d, 1 H, J - 8.4 Hz, arom), 8.10-8.05 (m, 1 H, NHC=O), 7.83 (m, 1 H, arom), 7.71 (m, 1 H, arom), 7.48 (s, 1 H, arom), 5.38 (d, 1 H, CH₂O, J - 1 1.0 Hz), 5.32 (d, 1 H, CH₂O, J = 1 1.0 Hz), 5.27 (s, 2 H, CH₂N), 3.97 (s, 4 H of tartaric acid, 2 CH₂), 3.24- 3.10 (m, 4 H, CH₂ Of C₇-Et and CH₂ Of CH₂N-C=O), 2.90-2.70 (m, 6 H, 3 CH₂), 2.30-2.00 (m, 2 H, CH₂ of C₁₉-H₂), 1.97 (s, 3 H, Me, OAc), 1.80- 1.68 (m, 2 H, CH₂), 1.68- 1.50 (m, 4 H, 2 CH₂), 1.30 (t, 3 H, Me of C₇-Et), 0.87 (t, 3 H, Me at C₁₈).

Example VII - Preparation of compounds G and H

Product E (500 mg, 0.6 mmol) is hydrogenated in the presence of succinic acid (142 mg, 1.2 mmol) in acetonitrile (20 ml) over 5% Pd/C (165 mg), using the same procedure as in example VI. Compounds G and H are obtained as 1 : 1 mixture with 91% overall yield. The products are separated by flash chromatography.

Compound G

¹H NMR (DMSO-d₆/D₂O) δ 8.19 (d, 1 H, J = 8.5 Hz, arom), 8.09 (d, 1 H, J = 8.5 Hz, arom), 7.82-7.77 (m, 1 H, arom), 7.69-7.64 (m, 1 H, arom),

7.45 (s, 1 H, arom), 5.18 (s, 2 H, CH₂N), 3.10-3.2 (m, 4 H, CH₂ Of C₇-Et and

CH₂-NHC=O), 2.80-2.70 (m, 6 H, 3 CH₂), 2.28 (s, 4 H, 2 CH₂ of succinic acid), 2.23 (s, 3 H, Me), 2.30-2.10 (m, 2 H, CH₂ of C₂O-Et), 1.70- 1.50 (m,

6 H, 3 CH₂), 1.27 (t, 3 H, Me of C₇-Et), 0.87 (t, 3 H, Me at C₁₈). ¹³C NMR (DMSO) δ 175.8 (C of succinic acid), 173.7 (C), 161.8 (C),

153.4 (C), 152.1 (C), 149.2 (C), 146.0 (C), 142.1 (C), 130.5 (CH), 128.4 (CH),

128.0 (C), 127.8 (2 CH), 127.0 (C), 124.7 (CH), 99.9 (CH), 78.8 (C), 50.0

(CH₂N), 48.0 (CH₂), 46.3 (CH₂), 38.8 (CH₂) 36.6 (CH₂), 32.1 (CH₂, succinic acid), 31.5 (CH₂), 26.8 (CH₂), 24.8 (CH₂), 23.7 (CH₂), 22.9 (CH₂), 20.3 (Me), 14.6 (Me), 8.6 (Me)

Compound H

¹H NMR (DMSO-d₆/D₂O) δ 8.26 (d, 1 H, J = 8.4 Hz, arom), 8.17 (d, 1 H, J = 8.4 Hz, arom), 8.10-8.05 (m, 1 H, NHC=O), 7.83 (m, 1 H, arom), 7.71 (m, 1 H, arom), 7.48 (s, 1 H, arom), 5.38 (d, 1 H, CH₂O, J = 1 1.0 Hz), 5.32 (d, 1 H, CH₂O, J - 1 1.0 Hz), 5.27 (s, 2 H, CH₂N), 3.97 (s, 4 H of tartaric acid, 2 CH₂), 3.24- 3.10 (m, 4 H, CH₂ Of C₇-Et and CH₂ Of CH₂N-C=O), 2.90-2.70 (m, 6 H, 3 CH₂), 2.30-2.00 (m, 2 H, CH₂ of C₁₉-H₂), 1.97 (s, 3 H, Me, OAc), 1.80- 1.68 (m, 2 H, CH₂), 1.68- 1.50 (m, 4 H, 2 CH₂), 1.30 (t, 3 H, Me of C₇-Et), 0.87 (t, 3 H, Me at C₁₈).

Example VIII - Preparation of compound I

Product E (400 mg, 0.48 mmol) is hydrogenated over 5% Pd/C (5%) (100 mg) in the presence of oxalic acid (121 mg, 0.96 mmol) in acetonitrile (20 mL) using the same procedure as in example VI. 234 mg (74.7%) of product I are obtained.

¹H NMR (DMSO-d₆/D₂O) δ 8.26 (d, 1 H, J = 8.4 Hz, arom), 8.16 (d, 1 H, J = 8.4 Hz, arom), 8.10-8.00 (m, 1 H, NHC=O), 7.83 (m, 1 H, arom), 7.71 (m, 1 H, arom), 7.47 (s, 1 H, arom), 5.36 (d, 1 H, CH₂O, J = 11.0 Hz), 5.31 (d, 1 H, CH₂O, J = 11.0 Hz), 5.27 (s, 2 H, CH₂N), 3.24- 3.14 (m, 4 H, CH₂ of C₇-Et and CH₂ Of CH₂N-C=O), 2.90-2.70 (m, 6 H, 3 CH₂), 2.30-2.00 (m, 2 H, CH₂ of C₁₉-H₂), 1.97 (s, 3 H, Me, OAc), 1.82- 1.68 (m, 2 H, CH₂), 1.68-1.50 (m, 4 H, 2 CH₂), 1.31 (t, 3 H, Me of 7-Et), 0.87 (t, 3 H, Me at C₁₈). Example IX - Preparation of compound J Product D (570 mg, 0.82 mmol) is esterified with cis-4,7, 10, 13, 16, 19- docosahexenoyl chloride in dry pyridine (2.65 ml). After purification through flash chromatography product J is obtained as a yellow solid (550 mg).

¹H NMR (CD₃OD, 400 MHz) δ 8.54 (s, 1 H, Ar), 8.10 (d, 1 H, J = 8.4 Hz, Ar), 7.99 (d, 1 H, J = 8.4 Hz, Ar), 7.84-7.79 (m, 1 H, Ar), 7.78 (s, 1 H, H- 14), 7.69-7.63 (m, 1 H, Ar), 5.50 (d, 1 H, J = 1 1.2 Hz, H- 17), 5.43 (d, 1 H, J = 1 1.2 Hz, H- 17), 5.41-5.24 (m, 12 H), 5.20 (s, 2 H, H-5), 3.14-2.92 (m, 8 H, 4 CH₂), 2.86-2.72 (m, 10 H, 5 CH₂), 2.50-2.38 (m, 4 H, 2 CH₂), 2.38-2.24 (m, 1 H, H), 2.10-2.00 (m, 3 H, CH₂+H), 1.98- 1.89 (m, 2 H, CH₂), 1.86-1.69 (m, 4 H, 2 CH₂), 1.06 (t, 3 H, J = 7.2 Hz, CH₃), 0.93 (t, 3 H, J = 7.2 Hz, CH₃). ¹³C NMR (CD₃OD, 100 MHz) δ 175.5 (CO), 173.9 (CO), 162.1 , 161.8,

157.1 (NCO), 152.5, 148.4, 144.8, 132.1 , 131.6, 130.7, 129.6, 129.0, 128.7, 128.6, 128.5, 128.3, 128.1 , 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.0, 124.4, 100.9 (C- 14), 79.1 (C), 59.2 (C- 17), 50.5, (C-5), 47.1, 45.4, 38.8, 36.7, 34.0, 32.1, 26.4, 25.4, 25.3, 25.2, 24.4, 24.3, 24.2, 23.1 , 23.0, 22.7, 20.3, 13.5 (Me), 7.2 (Me).

Example X - Preparation of compound L

The product of example II (488 mg, 0.664 mmol) is dissolved in methylene chloride and deprotected with formic acid (0.8 ml) at O⁰C under nitrogen atmosphere. The reaction mixture is stirred at O⁰C for 8 h, then the solvent is evaporated off under reduced pressure. The residue is dissolved in CHCl₃ and precipitated from «-hexane. The desired compound is obtained as a yellow solid (90%). ¹H NMR (DMSO) δ 8.64 (s, 1 H, arom), 8.20-8.40 (s, 1 H arom),

8.02-8.20 (m, 2 H, arom), 7.84 (m, 1 H, arom), 7.68 (m, 1 H, arom), 7.50 (s, 1 H, arom), 5.37 (d, 1 H, CH₂-O, J = 8.0 Hz), 5.31 (d, 1 H, CH₂-O, J = 8.0 Hz), 5.24 (s, 2 H, CH₂-N), 3.24-3.12 (m, 1 H), 3.10-3.00 (m, 1 H), 2.80-2.60 (m, 6 H), 2.20-2.12 (m, 1 H), 2.12-2.04 (m, 1 H), 1.96 (s, 3 H, Me), 1.70- 1.60 (m, 2 H), 1.60- 1.44 (m, 4 H), 0.87 (t, 3 H, J = 7.5 Hz, Me).

¹³C NMR (DMSO) δ 173.7, 171.0. 166.4 (C-O of HCO₂H), 161.3, 156.6, 153.4, 148.7, 144.7, 132.2, 131.0, 130.6, 129.7, 129.2, 128.6, 128.2, 124.0, 99.6, 79.6, 59.5, 50.9, 47.6, 46.0, 39.0, 37.3, 32.3, 27.8, 25.8, 25.0, 21.4, 8.6.

Claims

1. Compounds of the general formula (I)

(I) in which

R₁ is a straight or branched polyamino residue, optionally functionalized by acylation or alkylation of the amino residues, with more than 2 nitrogen atoms;

R₂ is H, -OR₃;

R₃ is H or a Ci -C₂₄ straight or branched, saturated or unsaturated acyl residue;

R₄ is H, straight or branched Ci -C₄ alkyl, hydroxyalkyl, aminoalkyl, cyano;

R₅ and R₆ are H, -OH or -OCH₃.

2. Compounds as claimed in claim 1 wherein the polyamine is spermidine, protected spermidine or salified spermidine.

3. Compounds as claimed in claim 2 in which the polyamine is

Boc-spermidine.

4. Compounds as claimed in claim 2 in which the polyamine is N₂,N₃-Di- Cbz-spermine.

5. Compounds as claimed in claim 2 in which the polyamine is salified spermine.

6. Compounds as claimed in any one of claims 1 to 5 in which the R₃ residue is acetyl.

7. Compounds as claimed in any one of claims 1 to 5 in which the R₃ residue is cw-4,7,10,13,16,19-docosahexenoyl. 8. Compounds as claimed in any one of claims 1 to 5 in which the R₃ residue is cz5-5,

8,l l, 14-eicosapentaenoyl.

9. Compounds as claimed in any one of claims 1 to 5 in which the R₃ residue is ximeninoyl.

10. Pharmaceutical compositions comprising as active ingredient a compound of any one of claims 1-9 in admixture with suitable carriers and excipients.

11. The use of the compounds of any one of claims 1-9 for the preparation of anti-tumour medicaments.

12. Compounds of claims 1-9 as medicaments.

13. Compounds of claims 1-9 as anti-tumour agents.