WO2003033525A1

WO2003033525A1 - Amino-substituted camptothecin polymer derivatives and use of the same for the manufacture of a medicament

Info

Publication number: WO2003033525A1
Application number: PCT/IB2001/001912
Authority: WO
Original assignee: Debio Recherche Pharmacuetique S.A.
Priority date: 2001-10-12
Filing date: 2001-10-12
Publication date: 2003-04-24
Also published as: WO2003031467A3; WO2003031467A2

Abstract

The present invention relates to a pharmacologically active amino substituted 7-ethylcamptothecin polymer derivative, which has anti-tumor activity and is water-soluble.

Description

Amino-substituted camptothecin polymer derivatives and use of the same for the manufacture of a medicament

The present invention relates to new amino-substituted camptothecin polymer derivatives useful as medicaments. It relates also to the use of said derivatives for-the manufacture of a medicament.

Camptothecin, an alkaloid which was first isolated from the Chinese tree Camptotheca acuminata, has attracted much attention because of its significant anti-tumour activity in animals and has initiated medicinal chemistry studies aiming to provide with analogues having improved pharmaceutical profile.

Yaegashi et al. described, in Chem. Pharnn. Bull., 1994, 42, 2518-25, the synthesis, among others, of 9-, 10-, and 11-amino-A-ring- substituted 7-ethylcamptothecin derivatives, and those derivatives demonstrated in in-vitro assay some promising anti-tumour activity. However, due to their very poor solubility in the physiological medium and despite the presence of an amino group, their administration for in-vivo assays and their targeting to the tumour cells remain hard to conceive.

One oM ie aims of the present invention is to eliminate the above mentioned drawback by providing with new 9-, 10-, and 11-amino-A-ring- substituted 7-ethylcamptothecin derivatives, allowing the 9-, 10-, and 11-amino-A-ring-substituted 7-ethylcamptothecin pharmacophore to be administrated into the body with an effective plasma half-life and then targeted and accumulated to and into the tumour cells to be treated with a reduce efflux, in particular in multidrug-resistant cells.

To that effect,. the object of the present invention relates to amino- substituted camptothecin polymer derivatives having the following general formula (I):

in which n is an integer between 10 and 1000;

-X¹ and -X², independently, represent a residue having the following general formula (II)

wherein -S- represents a cleavable spacer arm residue of a peptide selected among the following peptides: GlyLeuPheGly, GlyPheLeuGly, GiyPhePheAla, GlyPhePheLeu, GlyPheTyrAla, AlaGlyValPhe, GlyPheTyrAla, GlyLeuAla, GlyLeuGly, GlyPheGIy, GlyPheAla, DAIaPheLys, DValLeuLys, and LysGlyLeuPheGly with at least one of any of the alpha- and epsilon-amino groups of lysine being linked to the corresponding remaining part of formula (I); or -X¹ represents -a linear or a branched CrC₆-alkyl group.

The polymeric part of the amino-substituted camptothecin polymer derivatives of the invention correspond to a polyethylene fragment and it has been selected for its hydrophilic properties. The size of this polyethylene fragment, represented by the integer n, or its molecular weight, is judiciously chosen in order to obtain an appropriate targeting and accumulation of the derivative of the invention to and into the tumour cells to be treated. The preferred derivatives of the invention of general formula (I) are those in which n is an integer between 20 and 400. i.e. those in which the polyethylene fragment exhibits a molecular weight comprised between about 880 and about 17600. More preferably, the polyethylene fragment exhibits a molecular weight of about 10000, with n being equal about 250.

In the amino-substituted camptothecin polymer derivatives of the invention, the camptothecin pharmacophore is attached to at least one of the two extremities of the polyethylene fragment through the cleavable peptidic spacer arm residue -S-. The peptidic residues are selected for their sensitiveness to lysosomal enzymatic system or to other tumour- related enzymes, such as plasmine, and their capability to be cleaved by f such enzymatic system. Preferably, the cleavable spacer arm residue

-S- is selected among the following peptides: GlyLeuPheGly,

GlyPheLeuGly, and LysGlyLeuPheGly.

The peptidic spacer arm residue is attached to the extremity of the polyethylene fragment through a carbamate group formed between the amino end group of the peptide and the end oxygen atom of the polyethylene.

When lysine is the amino end terminal amino acid of the peptidic spacer arm, at least one of any of its alpha- and epsilon-amino groups can carry the polyethylene fragment through a carbamate bond. Preferably, both of the alpha- and epsilon-amino groups carry the polyethylene fragment through a carbamaie bond in order to form a branched polymer derivative.

Preferably, the amino group carried on the A-ring of the camptothecin framework of the camptothecin pharmacophore is on position 10. It forms an amide group with the carboxyl end group of the cleavable peptidic spacer arm residue.

When the camptothecin pharmacophore is attached to only one of the two extremities of the polyethylene fragment, the second extremity is preferably terminated, through the oxygen atom, by methyl group. The process for the preparation of the derivatives of the invention is based on the linkage of the peptidic spacer arm -S- to the hydroxyl function of monoalkoxypolyethylene glycol through a carbamate linkage which involves the NH₂ group of the said peptidic arm. This reaction is followed by the activation of the COOH function of said peptidic arm to an activated ester, which, thus, becomes reactive towards the amino group of the camptothecin pharmacophore.

More specifically, when the camptothecin pharmacophore is attached to only one of the two extremities of the polyethylene fragment the process consists of: a) reacting a mono-alkoxy-polyethylene glycol derivative of formula (III)

RO-(CH₂-CH₂-0)_n-H (III)

wherein R is a linear or a branched d-Cβ-alkyl group and n have the definition provided above, with benzotriazolchloroformate, 2,4,5- trichlorphenylchloroformate or 4-nitrophenylchloroformate to obtain the corresponding carbonate; b) reacting the carbonate thus obtained with an amino acid the peptide of formula (IV)

H-S-OH (IV)

wherein -S- is defined above fo obtain a compound of formula (V)

RO-(CH₂-CH₂-0)_n-(C=O)-S-OH (V)

c) converting the compound of formula (V) thus obtained into the corresponding activated ester, and d) finally, reacting the said activated ester with the respective amino-A- ring-substituted 7-ethylcamptothecin. Steps a) through d) of the above-described method do not necessitate special reaction conditions and can be carried out according to the usual techniques. Furthermore, some of the activated carbonate polymers are commercially available. Details of each of the above reaction steps are provided in the Examples illustrating the invention.

A similar approach is applied for the preparation of the amino- substituted camptothecin polymer derivatives when both extremities of the polyethylene fragment carry respectively a camptothecin pharmacophore.

Another of the objects of the present invention relates to the use of the amino-substituted camptothecin polymer derivatives of the invention for the manufacture of a medicament for the treatment of tumour cells.

By means of the introduction of such a polymer, an improved administration and targeting of the pharmacophore is achieved. By the introduction of such a peptidic spacer arm, it is believed that a site-specific cleavage of the derivative by specific cellular enzymes takes place, releasing the pharmacophore into the tumour cells.

Some of these interesting properties are illustrated in the following Examples, which are not limitative.

In the said Examples the term "m-PEG-OH" defines the monomethoxypolyethylene glycol having a molecular weight mw of about 10000 and the amino acids or peptides are described by means of the terms usual in the art. m-PEG-benzotriazolyl carbonate (m-PEG-BTC) and H-GlyLeuPheGly-OH were commercially available. Example 1

A. Preparation of m-PEGnn m-0(C=0)-NH-GlvLeuPheGlv-OH (1

1 g (0.101 mmol) of m-PEG₍i_0kD)-BTC (mw = 10000) were added portionwise over 30 minutes to a solution of 0.24g (0.606 mmol, 6 eq.) of tetrapeptide H-GlyLeuPheGly-OH in 3 ml of borate buffer 1 M, pH 8. The resulting mixture was adjusted to pH 8 using NaOH 1 N and stirred at room temperature for 24 hours.

The reaction mixture was acidified with citric acid to pH 3, and extracted with chloroform (3 x 50 ml). The combined organic solutions were dried over sodium sulphate and concentrated to a small volume at reduced pressure. The resulting slurry was added dropwise to 200 ml of vigorously stirred diethyl ether. The white precipitate, which formed, was filtered and dried at reduced pressure, affording 0.96 g of crude product which was applied to a QAE Sephadex A-50 ion exchange column. Elution with mQ grade H₂0 afforded 0.125 g of starting material (m-PEG-OH); with increase of ionic strength (0.01 N NaCl) the desired compound eluted together with NaCl (0.896g combined). The combined fractions containing the m-PEG-tetrapeptide were freeze-dried and the residue was suspended in chloroform to remove the salts. Recristallisation with diethyl ether afforded 0.77 g (77%) of the title compound.

¹H NMR (CDCI₃) ppm: 0.91 (t, J=5.6Hz, 6H); 1.44 (m, 1 H); 3.01-3.20 (dd, J=22.8Hz; J=6.6Hz, 2H); 3.39 (s, 3H); 3.40-3.88 (m); 4.15 (m, 1 H); 4.19 (m, 2H); 4.56 (m, 2H); 6.26 (bs, 1 H); 6.90 (bs, 1 H); 7.12 (bs, 1 H); 7.24 (t, J=4.8Hz, 1 H); 7.26-7.31 (m, 5H). B. Preparation of m-PEGhn_k -O(C=O -NH-GlvLeuPheGlv-10-amino- 7-ethyl-camptothecin (2)

0.6 g (0.06 eq.)of the compound as obtained above and 45 mg

(0.12 mmol, 2 eq) of 10-amino-7-ethylcamptothecin were dissolved in 30 ml of toluene and the mixture was azeotropically distilled with removal of 10 ml of toluene. The mixture was evaporated to dryness at reduced pressure, and the residue was suspended in 30 ml of dry chloroform. pyridine (0.24 ml, 3 mmol) and phenyl dichlorophosphate (0.35 ml,

2.28 mmol) were added and the mixture was stirred at room temperature for 12 hours, adjusting the pH with DPEA.

At the end the yellow^' mixture was extracted with 1 N HCI (20 ml). The organic phase was concentrated at reduced pressure and the residue, diluted with 20 ml of 2-propanol, was recristallised. The pale yellow crystalline precipitate was washed with diethyl ether affording 0.45g of pure product (2).

¹H NMR (CDCI3) ppm: 0.83-0.91 (m, 6H); 1.03 (t, J=7.2Hz, 3H); 1.26 (m, 1 H); 1.42 (t, J=7.4Hz, 3H); 1.89 (m, 2H); 3.19 (m, 2H); 3.38 (s, 3H); 3.39- 3.89 (m); 4.17 (m); 4.26 (m, 2H); 5.24 (s, 2H); 5.29 (d, J=16.7Hz); 5.72 (d, J=16.7Hz); 6.5 (bs, 1 H); 7.24-7.30 (m, 7H); 7.7 (s, 1 H); 8.18 (s, 1 H); 8.90 (bs, 1 H).

The amount of 10-amino-7-ethylcamptothecin (2) determined in the obtained derivative was 16.f mg, corresponding to a w/w % of 3.72 % (The theoretical 100% of loading is, for this conjugate, 3.72% according to UV absorption). C. In-vivo assay

The derivative (2) was tested against murine leukemias P388 and P388 resistant to adriamycipf (P388/ADM). Female CDF1 mice were inoculated intraperitoneally with P388 or P388/ADM at a dose of 1x10⁶ cells/mouse on day 0, and injected intravenously with the derivative on days 1 , 5, and 9 at total doses of 5, 10, 20 or 25 mg/kg, then monitored survival times for 40 days. Due to poor water-solubility, the pharmacophore 10-amino-7-ethylcamptothecin was unable to be tested in comparison. However, one of its water soluble analogues, namely CPT-11 was used. The survival rate (T/C%) is calculated using the following formula:

T/C (%) = (Mean survival days of treated group / mean survival days of control group) x 100.

Table 1 reports the anti-tumour activity of the derivative against P388, while Table 2 reports the same against P388/ADM.

Table 1

Table 2

Example 2

A. Preparation of PEGn_nk -rθ-(C=0 -NH-GlvLeuPheGlv-OHl_? (3)

1g (0.101 mmol) of the iol HO-PEG-OH (mw = 10000) was dissolved in 30 ml of toluene and refluxed in a Dean-Stark apparatus to azeotropically remove water. The solution was concentrated to 5-6 ml, and then diluted with dry dichloromethane (5 ml). 0.2 g (10 eq.) of p-nitro- phenyl chloroformate and 0.14 ml (10 eq.) of triethylamine were added and the resulting mixture was stirred at room temperature for 12 hours. At the end the mixture was added dropwise to 200 ml of diethyl ether under vigorous stirring. The resulting white precipitate was filtered and dried, affording 1 g of PEG-di(p-nitrophenyl carbonate).

The activated PEG diol was added portionwise over 30 minutes to a solution of 0.24g (0.606 mmol, 6 eq.) of tetrapeptide

H-GlyLeuPheGly-OH in 3 ml of borate buffer 1 M, pH 8. The resulting mixture was adjusted to pH 8 u-sing NaOH 1 N and stirred at room temperature for 24 hours. The reaction mixture was then acidified with citric acid to pH 3, and extracted with chloroform (3 x 50 ml). The combined organic solutions were dried over sodium sulphate and concentrated to a small volume at reduced pressure. The resulting slurry was added dropwise to 200 ml of vigorously stirred diethyl ether. The white precipitate which formed was filtered and dried at reduced pressure, affording 0.96 g of crude product which was applied to a column packed with QAE Sephadex A-50 ion exchange resin. Elution with mQ grade H₂0 afforded 0.095 g of starting material (PEG-OH). The appropriate combined fractions were freeze-dried and the residue was suspended in chloroform to remove the salts. Recristallisation afforded 0.86 g (79%) of title compound.

Titration of -COOH groups: 98% ¹H NMR (CDCIa) ppm: 0.91 (t, J=5.6Hz, 12H); 1.44 (m, 2H); 3.01-3.20 (dd, J=22.8Hz; J=6.6Hz, 4H); 3.40-3.88 (m); 4.15 (m, 2H); 4.19 (m, 4H); 4.56 (m, 4H); 6.26 (bs, 2H); 6.90 (bs, 2H); 7.12 (bs, 2H); 7.24 (t, J=4.8Hz, 2H); 7.26-7.31 (m, 10H).

B. Preparation of PEG ι<m-rθ-(C=OVNH-GlvLeuPheGlv -10-amino-7- ethyl-camptothecinl? (4)

(4)

0.6 g (0.06 mmol)of PEG₁₀kD-(GlyLeuPheGlyOH)₂ (3) and 90 mg (0.24 mmoJ, 4 eq) of 10-amino-7-ethylcamptothecin were dissolved in 30 ml of toluene and the mixture was azeotropically distilled with removal of 10 ml of toluene. The mixture was evaporated to dryness at reduced pressure, and the residue was suspended in 30 ml of dry chloroform. Pyridine (0.48 ml, 6 mmol) and phenyl dichlorophosphate (0.7 ml, 4.56 mmol) were added and the mixture was stirred at room temperature for 12 hours, adjusting the pH with DPEA.

At the end the yellow mixture was extracted with 1 N HCI (20 ml). The organic phase was concentrated at reduced pressure and the residue, diluted with 20 ml of 2-propanol, was recristallised. The pale yellow crystalline precipitate was washed with diethyl ether affording 0.55g of product (4). Loading (w/w % of aminocamptothecin) = 6.1 % (theoretical = 6.78%) according to UV absorption.

¹H NMR (CDCI₃) ppm: 0.83-0.91 (m, 12H); 1.03 (t, J=7.2Hz, 6H); 1.26 (m, 2H); 1.42 (t, J=7.4Hz, 6H); 1.89 (m, 4H); 3.19 (m, 4H); 3.40-3.89 (m); 4.17 (m, 4H); 4.26 (m, 4H); 5.24 (s, 4H); 5.29 (d, J=16.7Hz, 2H); 5.72 (d, J=16.7Hz, 2H); 6.5 (bs, 2H); 7.24-7.30 (m, 14H); 7.7 (s, 2H); 8.18 (s, 2H); 8.90 (bs, 2H).

Derivative (4) was tested against P388 and P388/ADM in a similar method as described m Example 1 and demonstrated to have anti-cancer activities.

Example 3

A. Synthesis of rmPEGn_nk l9-0(C=Q -NH-LvsGlvLeuPheGlv-OH (5)

2g (0.1 mmol) of [mPEG₍ιokD)]₂-0(C=0)-NH-Lys-OSu (mw = 20000) were added portionwise over 30 minutes to a stirred solution of H- GlyLeuPheGly-OH (39 mg, 10 eq.) and Et₃N (0.14 ml, 10 eq.) in 20 ml of anhydrous dichloromethane. The resulting mixture was stirred at room temperature for 24 hours, then was extracted with HC1 1 N (2 x 20 ml) to remove the excess of tetrapeptide.

The combined organic solutions were dried over sodium sulphate and concentrated to a small volume at reduced pressure. The resulting slurry was added dropwise to 200 ml of vigorously stirred diethyl ether. The white precipitate which formed was filtered and dried at reduced pressure, affording 1.92 g (93%) of crude product which was used without further purification.

H NMR (CDC ) ppm: 0.91 (t, J=5.6Hz, 6H); 1.37-1.44 (m, 3H); 1.6-1.8 (m, 4H); 2.9 (m, 2H); 3.01-3.20 (dd, J=22.8Hz; J=6.6Hz); 3.39 (s, 6H); 3.40-3.88 (m); 4.17 (m, 1 H); 4.21 (m, 2H); 4.26 (m,. 1 H); 4.57 (m, 2H); 6.26 (bs, 1 H); 6.95 (bs, 1 H); 7.12 (bs, 1 H); 7.24 (t, J=4.8Hz, 1 H); 7.26- 7.30 (m, 5H); 7.83 (d, J=4.6Hz, 1 H).

B. Preparation of rmPEGπokm1?-0(C=0 -NH-LvsGlvLeuPheGlv-1 Q-amino- 7-ethyl-camptothecin (6)

(6)

1.23 g (0.06 eq.) of [mPEG_(10kD)]₂-O(C=O)NH-LysGlyLeuPheGlyOH (5) and 45 mg (0.12 mmol, 2 eq) of 10-amino-7-ethylcamptothecin were dissolved in 30 ml of toluene and the mixture was azeotropically distilled with removal of 10 ml of toluene. The mixture was evaporated to dryness at reduced pressure, and the residue was suspended in 30 ml of dry chloroform. Pyridine (0.24 ml, 3 mmol) and phenyl dichlorophosphate (0.35 ml, 2.28 mmol) were added and the mixture was stirred at room temperature for 12 hours, adjusting the pH with DPEA.

At the end the yellow mixture was extracted with 1 N HCI (20 ml).

The organic phase was concentrated at reduced pressure and the residue, diluted with 20 ml of 2-propanol, was recristallised. The pale yellow crystalline precipitate was washed with diethyl ether affording 0.95g (76%) of title compound (6). Loading (w/w % of amino- camptothecin) = 1.82 % (theoretical = 1.87%) according to UV absorption.

¹H NMR (CDCI₃) ppm: 0.91 (t, J=5.6Hz, 6H); 1.03 (t, J=7.2Hz, 3H); 1.37- 1.44 (m, 6H); 1.6-1.8 (m, 4H); 1.89 (m, 2H); 2.9 (m, 2H); 3.01-3.15 (dd, J=22.8Hz; J=6.6Hz); 3.19 (m, 2H); 3.39 (s, 6H); 3.40-3.88 (m); 4.15 (m, 1 H); 4.19 (m, 2H); 4.28 (m,. 1 H); 4.54 (m, 2H); 5.24 (s, 2H); 5.31 (d,

J=16.7Hz); 5.72 (d, J=16.7Hz, 1 H); 6.26 (bs, 1 H); 6.90 (bs, 1 H); 7.12 (bs, 1 H); 7.23 (t, J=4.8Hz, 1 H); 7.26-7.31 (m, 7H); 7.7 (s, 1 H); 7.81 (d, J=4.6Hz, 1 H); 8.90 (bs, 1 H).

Derivative (6) was tested against P388 and P388/ADM in a similar method as described in Example 1 a^d demonstrated to have anti-cancer activities.

Claims

1. An amino-substituted camptothecin polymer derivative having the following general formula (I):

in which n is an integer between 10 and 1000;

wherein -S- represents a cleavable spacer arm residue of a peptide selected among the following peptides: GlyLeuPheGly, GlyPheLeuGly, GlyPhePheAla, GlyPhePheLeu, GlyPheTyrAla, AlaGlyValPhe, GlyPheTyrAla, GlyLeuAla, GlyLeuGly, GlyPheGly, GlyPheAla, DAIaPheLys, DValLeuLys, and LysGlyLeuPheGly with at least one of any of the alpha- and epsilon-amino grpups of lysine being linked to the corresponding remaining part of formula (I); or -X¹ represents a linear or a branched Cι-C₆-alkyi group.

2. The amino-substituted camptothecin polymer derivative according to claim 1 having the general formula (I) in which n is an integer between 20 and 400.

3. The amino-substituted camptothecin polymer derivative according to claim 1 having the general formula (I) in which n is equal about 250.

4. The amino-substituted camptothecin polymer derivative according to any of the preceding claims having the general formula (I) in which the cleavable spacer arm residue -S- is selected among the following peptides: GlyLeuPheGly, GlyPheLeuGly, and LysGlyLeuPheGly.

5. The amino-substituted camptothecin polymer derivative according to any of the preceding claims having the general formula (l) in which, when X¹ represents an alkyl group, it is a methyl group.

6. The amino-substituted camptothecin polymer derivative according to any of the preceding claims having the general formula (I) in which the amino group, linking the camptothecin framework to the cleavable spacer arm residue -S-, is attached on the carbon atom in position 10 of the camptothecin framework.

7. The use of the amino-substituted camptothecin polymer derivative of any of the preceding claims for the manufacture of a medicament for the treatment Df tumour cells.