WO2003031467A2 - Amino-substituted camptothecin polymer derivatives and use of the same for the manufacture of a medicament - Google Patents

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

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Publication number
WO2003031467A2
WO2003031467A2 PCT/CH2002/000562 CH0200562W WO03031467A2 WO 2003031467 A2 WO2003031467 A2 WO 2003031467A2 CH 0200562 W CH0200562 W CH 0200562W WO 03031467 A2 WO03031467 A2 WO 03031467A2
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gly
amino
phe
leu
camptothecin
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PCT/CH2002/000562
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French (fr)
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WO2003031467A3 (en
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Francesco Veronese
Andrea Guiotto
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Debio Recherche Pharmaceutique S.A.
Yakult Honsha Co. Ltd.
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Publication of WO2003031467A2 publication Critical patent/WO2003031467A2/en
Publication of WO2003031467A3 publication Critical patent/WO2003031467A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient

Definitions

  • 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.
  • pre-clinical and clinical data on some Camptothecin analogues that are already being used in clinical practice or are currently in clinical development have been recently reviewed by Keher et al. in Anti-Cancer Drugs, 2001, 12, 89-105.
  • One of the 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.
  • the object of the present invention relates to amino- substituted camptothecin polymer derivatives, or their pharmaceutically acceptable salts, having the following general formula (I):
  • n is an integer between 10 and 1000;
  • -S- represents a cleavable spacer arm residue of a peptide selected among the following peptides: -Gly-Leu-Phe-Gly-, -Gly-Phe-Leu-Gly-,
  • -X 1 represents a linear or a branched CrC ⁇ -alkyl group and -X 2 is defined as above.
  • the polymeric part of the amino-substituted camptothecin polymer derivatives of the invention corresponds 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.
  • 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 such enzymatic system.
  • the cleavable spacer arm residue -S- is selected among the following peptides:
  • 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 fragment.
  • At least one of any of its alpha- and epsilon-amino groups can carry the polyethylene fragment through a carbamate bond.
  • both of the alpha- and epsilon-amino groups carry the polyethylene fragment through a carbamate bond in order to form a branched polymer derivative.
  • 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.
  • methyl group preferably terminates the second extremity, through the oxygen atom.
  • 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 mono- alkoxypolyethylene glycol through a carbamate linkage which involves the NH 2 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.
  • the process consists of: a) reacting a mono-alkoxy-polyethylene glycol derivative of formula (III)
  • R is a linear or a branched CrC- 6 -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)
  • Steps a) through d) of the above-described process 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.
  • 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.
  • the amino-substituted camptothecin polymer derivatives of the invention are water-soluble and show enhanced therapeutic index and/or anti-tumour activity and reduced toxicity in comparison with the corresponding amino- substituted camptothecin or its pharmaceutically acceptable salt. They are useful for treating neoplastic disease, reducing tumour burden, preventing metastasis of neoplasms and preventing recurrences of tumour/neoplastic growths in mammals in the treatment. More specifically, they are useful for treating leukaemia and solid tumours, such as colon, colo-rectal, ovarian, mammary, prostate, lung, kidney and also melanoma tumours. Applying a method comprising administering thereto a therapeutically effective amount of the amino-substituted camptothecin polymer derivatives of the invention can therefore treat a human patient.
  • the dosage range adopted will depend on the route of administration and on the age, weight and condition of the patient being treated.
  • the amino- substituted camptothecin polymer derivatives of the invention are typically administered by parenteral route, for example intramuscularly, intravenously or by bolus infusion.
  • a suitable dose range is from 10 to 500 mg/m2. This range is illustrative and the practitioner will determine the optimal dosing of the amino- substituted camptothecin polymer derivatives based on clinical experience and the treatment indication.
  • the amino-substituted camptothecin polymer derivatives of the invention may be formulated into a pharmaceutical composition well known by the person skilled in the art.
  • the composition may be in the form of solutions, suspensions, tablets, capsules and the like.
  • the compositions may be administrated by oral or parenteral routes.
  • the amino-substituted camptothecin polymer derivatives of the invention are formulated for parenteral administration, for example by dissolution in water for injection or physiological saline.
  • Other ways for administrating the compositions may be envisaged, for instance by inhalation or through intranasal routes.
  • - Fig 1 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis by Cathepsin B1 at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino- 7-ethylcamptothecin
  • - Fig 2 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin
  • - Fig 3 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis at pH 7.44 and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin;
  • - Fig 4 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis in mice plasma and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin;
  • - Fig 5 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 2 by in-vitro hydrolysis by Cathepsin B1 at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino- 7-ethylcamptothecin;
  • - Fig 6 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 2 by in-vitro hydrolysis at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin.
  • m-PEG-OH defines a monomethoxypoly- ethylene glycol residue 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-benzo-triazolyl carbonate (m-PEG-BTC) and H-Gly-Leu-Phe-Gly-OH were commercially available.
  • 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 drop-wise 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 H 2 0 afforded 0.125 g of starting material (m-PEG-OH); with increase of ionic strength (0.01 N NaCI) the desired compound eluted together with NaCI (0.896g combined).
  • the amount of 10-amino-7-ethylcamptothecin (2) determined in the obtained derivative was 16.7 mg, corresponding to a w/w % of 3.72 % (the theoretical 100% of loading is, for this conjugate, 3.72% according to UV absorption).
  • Buffer solution (A) at pH 5.5 was prepared using KH 2 P0 4 -2H 2 0 0.15 M, 10 "3 M EDTA.
  • Solution (B) was prepared from solution (A) by addition of 5 ⁇ M of GSH.
  • Solution (C) was prepared by addition of 850 ⁇ l of solution (B) to 50 ⁇ l of Cathepsin ⁇ solution, containing 0.285 mg/ml of enzyme (extracted from bovine spleen) in buffer solution (A) (1 mg of enzyme powder contains 11.36 units). The mixture (C) was incubated for 5 min at 37 °C.
  • a PBS buffer solution (D), pH 7.44, containing 0.156 g/ml NaH 2 P0 4 . H 2 0 and 0.88 g/ml of NaCI were prepared. 4.098 mg of derivative (2) was solubilised in 3 ml of PBS solution in volumetric flask.
  • the mixture (E) was incubated at 37°C. At fixed period of time a 50 ⁇ l sample of the mixture was injected in an HPLC system (RP-C18 column).
  • derivative (2) was progressively converted into 10-amino-7-ethylcamptothecin when placed in mouse plasma. After one day, about 18% of derivative (2) were converted.
  • Derivative (2) was tested against murine leukaemia P388 and P388 resistant to adriamycin (P388/ADM).
  • Female CDF1 mice were inoculated intra- peritoneally with P388 or P388/ADM at a dose of 1x10 6 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.
  • Table 1 reports the anti-tumour activity of the derivative against P388, while Table 2 reports the same against P388/ADM.
  • Derivative (2) was tested against Meth A fibrosarcoma cells. 7 weeks old male BALB/c mice were inoculated subcutaneously with Meth A cells at a dose of 5x10 5 cells/mouse on day 0. Samples were injected intravenously with the derivative on day 5, and the tumours were weighed on day 21. Due to poor water-solubility, a first reference group was given 10-amino- 7-ethylcamptothecin as its open lactone sodium salt. A second reference group was given water-soluble analogue CPT-11. Control group was given with saline.
  • the reaction mixture was acidified with HC1 1 N to pH 3, and extracted with chloroform (5 x 50 ml). The organic phases were dried over Na 2 SO 4 , filtered and concentrated at reduced pressure. The remaining oil was dropped to 200 ml of diethyl ether. The mixture was maintained at 4°C for 1 hour, filtered and the resulting white powder was dried at reduced pressure. The crude yield was 96%.
  • the product was separated from the unreacted m-PEG-OH by Sephadex QAE A-50 ion exchange column. Elution with MQ grade H 2 O removed the undesired material (m-PEG-OH), while at increased ionic strength (0.01 N NaCI) the desired compound was recovered from the column. The solution was freeze- dried to obtain 603.7 mg, the product was treated with chloroform to remove the salts and filtered. After concentration of the chloroform solution the remaining oil was added drop-wise to diethyl ether. The final yield of (3) was 57% (w/w).
  • Derivative (4) was tested against P388 and P388/ADM in a similar method as described in Example 1 and demonstrated to have anti-cancer activities.
  • 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 drop-wise 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 H 2 0 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.
  • Derivative (8) was tested against P388 and P388/ADM in a similar method as described in Example 1 and demonstrated to have anti-cancer activities.

Abstract

The present invention relates to a pharmacologically active amino-substituted 7-ethylcamptothecin polymer derivative, which has anti-tumour 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. As an example, pre-clinical and clinical data on some Camptothecin analogues that are already being used in clinical practice or are currently in clinical development have been recently reviewed by Keher et al. in Anti-Cancer Drugs, 2001, 12, 89-105.
Yaegashi et al. described, in Chem. Pharm. 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 assays 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 of the 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, or their pharmaceutically acceptable salts, having the following general formula (I):
Figure imgf000003_0001
in which n is an integer between 10 and 1000;
-X1 and -X2, independently, represent a residue having the following general formula (II)
Figure imgf000003_0002
wherein -S- represents a cleavable spacer arm residue of a peptide selected among the following peptides: -Gly-Leu-Phe-Gly-, -Gly-Phe-Leu-Gly-,
-Gly-Phe-Phe-Ala-, -Gly-Phe-Phe-Leu-, -Gly-Phe-Tyr-Ala-, -Ala-Gly-Val-Phe-, -Gly-Phe-Tyr-Ala-, -Gly-Leu-Ala-, -Gly-Leu-Gly-, -Gly-Phe-Gly-, -Gly-Phe-Ala-, -DAIa-Phe-Lys-, -DVal-Leu-Lys-, and -Lys-Gly-Leu-Phe-Gly- 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 CrCβ-alkyl group and -X2 is defined as above.
The polymeric part of the amino-substituted camptothecin polymer derivatives of the invention corresponds 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 such enzymatic system. Preferably, the cleavable spacer arm residue -S- is selected among the following peptides:
-Gly-Leu-Phe-Gly-, -Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, and -Lys-Gly-Leu-Phe-Gly-.
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 fragment.
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 carbamate 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, methyl group preferably terminates the second extremity, through the oxygen atom.
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 mono- alkoxypolyethylene 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 CrC-6-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 to obtain a compound of formula (V)
RO-(CH2-CH2-0)n-(C=0)-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 process 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.
The amino-substituted camptothecin polymer derivatives of the invention are water-soluble and show enhanced therapeutic index and/or anti-tumour activity and reduced toxicity in comparison with the corresponding amino- substituted camptothecin or its pharmaceutically acceptable salt. They are useful for treating neoplastic disease, reducing tumour burden, preventing metastasis of neoplasms and preventing recurrences of tumour/neoplastic growths in mammals in the treatment. More specifically, they are useful for treating leukaemia and solid tumours, such as colon, colo-rectal, ovarian, mammary, prostate, lung, kidney and also melanoma tumours. Applying a method comprising administering thereto a therapeutically effective amount of the amino-substituted camptothecin polymer derivatives of the invention can therefore treat a human patient.
The dosage range adopted will depend on the route of administration and on the age, weight and condition of the patient being treated. The amino- substituted camptothecin polymer derivatives of the invention are typically administered by parenteral route, for example intramuscularly, intravenously or by bolus infusion. A suitable dose range is from 10 to 500 mg/m2. This range is illustrative and the practitioner will determine the optimal dosing of the amino- substituted camptothecin polymer derivatives based on clinical experience and the treatment indication.
The amino-substituted camptothecin polymer derivatives of the invention may be formulated into a pharmaceutical composition well known by the person skilled in the art. The composition may be in the form of solutions, suspensions, tablets, capsules and the like. The compositions may be administrated by oral or parenteral routes. Typically the amino-substituted camptothecin polymer derivatives of the invention are formulated for parenteral administration, for example by dissolution in water for injection or physiological saline. Other ways for administrating the compositions may be envisaged, for instance by inhalation or through intranasal routes.
Some of the interesting properties of the amino-substituted camptothecin polymer derivatives of the invention are illustrated in the following Examples and drawing, those Examples being not limitative.
In the drawing, - Fig 1 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis by Cathepsin B1 at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino- 7-ethylcamptothecin; - Fig 2 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin; - Fig 3 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis at pH 7.44 and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin;
- Fig 4 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 1 by in-vitro hydrolysis in mice plasma and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin;
- Fig 5 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 2 by in-vitro hydrolysis by Cathepsin B1 at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino- 7-ethylcamptothecin; and
- Fig 6 represents a kinetic curve (circles) of the conversion of the amino- substituted camptothecin polymer derivative of Example 2 by in-vitro hydrolysis at pH 5.5 and a kinetic curve (crosses) of the release of 10-amino-7-ethyl- camptothecin.
In the Examples, the term "m-PEG-OH" defines a monomethoxypoly- ethylene glycol residue 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-benzo-triazolyl carbonate (m-PEG-BTC) and H-Gly-Leu-Phe-Gly-OH were commercially available.
Example 1
1.1.1. Preparation of m-PEGπnknrO(C=OVNH-Glv-Leu-Phe-Glv-OH (1 )
1g (0.101 mmol) of m-PEG(ιokD)-BTC (mw = 10000) were added portion- wise over 30 minutes to a solution of 0.24g (0.606 mmol, 6 eq.) of tetrapeptide H-Gly-Leu-Phe-Gly-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 drop-wise 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 NaCI) the desired compound eluted together with NaCI (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).
1.1.2. Preparation of m-PEGMn_,nrO(C=OVNH-Glv-Leu-Phe-Glv-10-amino-7-ethyl- camptothecin (2)
(2)
0.6 g (0.06 eq.)of the compound (1) 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 phenyldichlorophosphate (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 16.7 mg, corresponding to a w/w % of 3.72 % (the theoretical 100% of loading is, for this conjugate, 3.72% according to UV absorption).
1.2. In-vitro hydrolysis assays
The in-vitro capability of derivative (2) to release selectively the aminoethyl- camptothecin pharmacophore by hydrolysis was evaluated under enzymatic conditions and chemical conditions. Then, such an in-vitro release was evaluated in mouse plasma.
1.2.1. Enzymatic hydrolysis of derivative (2) in presence of Cathepsin B1 at pH 5.5 and chemical hydrolysis of the derivative at pH 5.5
Buffer solution (A) at pH 5.5 was prepared using KH2P04-2H20 0.15 M, 10"3 M EDTA. Solution (B) was prepared from solution (A) by addition of 5 μM of GSH. Solution (C) was prepared by addition of 850 μl of solution (B) to 50 μl of Cathepsin ^ solution, containing 0.285 mg/ml of enzyme (extracted from bovine spleen) in buffer solution (A) (1 mg of enzyme powder contains 11.36 units). The mixture (C) was incubated for 5 min at 37 °C. At the end 100 μl of derivative (2) in buffer solution (A) (580μg/ml expressed as free drug) was added to solution (C) and this mixture was incubated at 37 °C. At the same time derivative (2) was solubilised in buffer solution at the same concentration. At fixed intervals a 50 μl sample of both drug mixtures was injected in an HPLC system (RP-C18 column).
The conversion of the derivative (2) and the release of 10-amino-7-ethyl- camptothecin were monitored following the decreasing of peak area and the obtained values were plotted respectively on Fig 1 (in the presence of Cathepsin B1 at pH 5.5) and on Fig 2 (in the absence of Cathepsin B1 at pH 5.5).
1.2.2. Chemical hydrolysis of derivative 12) at pH 7.44
A PBS buffer solution (D), pH 7.44, containing 0.156 g/ml NaH2P04 . H20 and 0.88 g/ml of NaCI were prepared. 4.098 mg of derivative (2) was solubilised in 3 ml of PBS solution in volumetric flask. The mixture (E) was incubated at 37°C. At fixed period of time a 50 μl sample of the mixture was injected in an HPLC system (RP-C18 column).
The conversion of derivative (2) and the release of 10-amino-7-ethyl- camptothecin were monitored and the obtained values were plotted on Fig 3.
1.2.3. Hydrolysis of derivative (2) in mouse plasma
1 ml of blood taken from female BALB mice was centrifuged at 12000 rpm for 1 minute obtaining 600 μl of plasma (F). 130μg of derivative (2) expressed as aminoethylcamptothecin was added to 75μl of a solution (G) containing 1/15 M of KH2PO4/Na2HP04 pH 7.4. The plasma solution was incubated at 37°C. At fixed periods of time 50 μl samples of the mixture were added to 300 μl of acetonitrile and centrifuged at 12000 rpm. The supernatant liquid was dried in "speed-vac" concentrator. The residue was solubilised with 100 μl of deionised H20. The samples were analysed by an HPLC system (RP-C18 column).
The conversion of derivative (2) and the release of 10-amino-7-ethyl- camptothecin were monitored and the obtained values were plotted on Fig 4.
1.2.4. Conclusions
As we can see on Fig 1 , derivative (2) was progressively converted into
10-amino-7-ethylcamptothecin in presence of Cathepsin B1 at pH 5.5. After one day, about 25% of derivative (2) were converted. As shown on Fig 2, acidic condition (pH 5.5) applied in this assay does not act much upon this conversion. After one day, less than 5% of derivative (2) were converted. As shown on Fig 3, similar observation can be made under physiological pH condition (pH 7.44). After one day, about 5% of derivative (2) were converted.
As we can see on Fig 4, derivative (2) was progressively converted into 10-amino-7-ethylcamptothecin when placed in mouse plasma. After one day, about 18% of derivative (2) were converted.
1.3. In-vivo pharmacological assay
Derivative (2) was tested against murine leukaemia P388 and P388 resistant to adriamycin (P388/ADM). Female CDF1 mice were inoculated intra- peritoneally 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.
Figure imgf000013_0001
Table 1
Figure imgf000013_0002
Table 2
Derivative (2) was tested against Meth A fibrosarcoma cells. 7 weeks old male BALB/c mice were inoculated subcutaneously with Meth A cells at a dose of 5x105 cells/mouse on day 0. Samples were injected intravenously with the derivative on day 5, and the tumours were weighed on day 21. Due to poor water-solubility, a first reference group was given 10-amino- 7-ethylcamptothecin as its open lactone sodium salt. A second reference group was given water-soluble analogue CPT-11. Control group was given with saline.
Obtained tumour weight value and calculated inhibition rate are collected in Table 3.
Dose (mg/kg) Tumour weight (g) Inhibition rate (%)
Group Mean ± S.D.)
Control (-) 2.45 ± 0.69 (-)
10-amino-7-ethyl- 25 0.93 ± 0.30 62.3 camptothecin; 50 0.88 ± 0.21 64.0 sodium salt 100 0.75 ± 0.09 69.3
(opened lactone)
Derivative (2) 25 1.51 ± 0.23 38.4
50 1.09 ± 0.29 55.5
100 0.51 ± 0.28 79.1
CPT-11 25 1.24 ± 0.46 49.5
50 1.31 ± 0.28 46.6
100 1.16 ± 0.37 52.7
Table 3
Pharmacokinetic parameters were determinated by i.v. administration to rats and the respective concentrations in 7-amino-10-ethylcamtothecin were measured on a 7 hours period. The obtained values and the calculated parameters are collected in Table 4. Derivative (2) 10-amino-7-ethyl 10-amino-7-ethyl
-camptothecin camptothecin; sodium salt
(opened lactone)
DMSO/10mM H3P04
Vehicle Sal ine 10mM NaOH
(10/1 , V/V)
Dose (mg/kg) 100 50 4 4
Parameters Mean (n=2) Mean (n=2) Mean (n=2) N=1
Cmax (μg/mL) 1.354 0.436 10.543 10.503
Tmax (h) 0.033 0.033 0.033 0.033
AUC0-7h (μg h/mL) 1.597 0.457 1.995 1.652
AUQnf (μg h/mL) 1.890 0.531 2.075 1.714
MRTιnf (h) 3.564 3.308 1.158 1.093
CI,ot (LVkg/h) - - 2.066 2.421
VDSS (Ukg) - - 2.369 2.646
T* (h) 2.815 2.687 2.246 2.256
Table 4
Example 2
2.1.1. Preparation of m-PEG,ιn.,nrO(C=0 -NH-Glv-Leu-Glv-OH 3^
114,1 mg (0,404 mmol) of tripeptide H-Gly-Leu-Gly-OH . HCI were solubilised in 7 ml of borate buffer 1 M, pH 8. 1g (0.101 mmol) of m-PEG-BTC (mw = 9876) was added portionwise over 60 minutes. The pH was maintained at pH 8 by NaOH 1 N and stirred at room temperature for 24 hours.
The reaction mixture was acidified with HC1 1 N to pH 3, and extracted with chloroform (5 x 50 ml). The organic phases were dried over Na2SO4, filtered and concentrated at reduced pressure. The remaining oil was dropped to 200 ml of diethyl ether. The mixture was maintained at 4°C for 1 hour, filtered and the resulting white powder was dried at reduced pressure. The crude yield was 96%.
The product was separated from the unreacted m-PEG-OH by Sephadex QAE A-50 ion exchange column. Elution with MQ grade H2O removed the undesired material (m-PEG-OH), while at increased ionic strength (0.01 N NaCI) the desired compound was recovered from the column. The solution was freeze- dried to obtain 603.7 mg, the product was treated with chloroform to remove the salts and filtered. After concentration of the chloroform solution the remaining oil was added drop-wise to diethyl ether. The final yield of (3) was 57% (w/w).
2.1.2. Preparation of m-PEGMnknrO(C=O)-NH-Glv-Leu-Glv-10-amino-7-ethyl- camptothecin (4)
Figure imgf000016_0001
(4)
0.1 g (0.01 mmol, 1 eq.) of the compound (3) as obtained above and 7.8 mg (0.02 mmol, 2 eq) of 10-amino-7-ethylcamptothecin were dissolved in 30 ml of toluene and the mixture was azeotropically distilled with removal of 25 ml of toluene. The mixture was suspended in 30 ml of dry chloroform. Pyridine (0.040 ml, 0.5 mmol) and phenyldichlorophosphate (0.06 ml, 0.4 mmol) were added and the mixture was stirred at room temperature for 15 hours, adjusting the pH with DPEA (0.075 ml).
At the end, the yellow mixture was washed with 1 N HCI (2 x 20 ml). The organic phase was concentrated at reduced pressure and the residue, diluted with 20 ml of 2-propanol, was recristallised at 4 °C. The pale yellow crystalline precipitate was washed with cold 2-propanol and then with diethyl ether affording the pure product that was dried at reduced pressure. The yield was 96% (w/w). 10-amino-7-ethylcamptothecin loading (w/w) 3.21 %, theoretical loading (w/w) 3.78 %
2.2. In-vitro hydrolysis assays
The capability of derivative (4) to release selectively the aminoethylcamptothecin pharmacophore by hydrolysis was evaluated under enzymatic conditions (Cathepsin B1 , pH 5.5) and chemical conditions (pH 5.5) by applying the experimental procedures as described in Example 1.
The obtained value were plotted respectively on Fig 5 and Fig 6. As we can see, derivative (4) was progressively converted into 10-amino-7-ethylcamptothecin in presence of Cathepsin B1 at pH 5.5, these acidic conditions being without any effect on this conversion.
2.3. In-vivo pharmacological assay
Derivative (4) was tested against P388 and P388/ADM in a similar method as described in Example 1 and demonstrated to have anti-cancer activities.
Example 3
3.1.1. Preparation of PEGMn.,m-rθ-(C=0)-NH-Glv-Leu-Phe-Glv-OHlp (5)
1g (0.101 mmol) of the diol 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 dichloro- methane (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 drop-wise 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-Gly-Leu-Phe-Gly-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 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 drop-wise 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 (CDCI3) 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).
3.1.2. Preparation of PEGMnk -rθ-(C=O)-NH-Glv-Leu-Phe-Glv-10-amino-7-ethyl- camptothecinl? (6)
Figure imgf000019_0001
(6)
0.6 g (0.06 mmol)of PEGι0kD-(-Gly-Leu-Phe-Gly-OH)2 (5) and 90 mg (0.24 mmol, 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 amino- camptothecin) = 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 (6) was tested against P388 and P388/ADM in a similar method as described in Example 1 and demonstrated to have anti-cancer activities.
Example 4
4.1.1. Synthesis of rm-PEGMnι«mVO C=0)-NH-Lvs-Glv-Leu-Phe-Glv-OH (7)
2g (0.1 mmol) of [m-PEG(ιokD)]2-0(C=0)-NH-Lys-OSu (mw = 20000) were added portionwise over 30 minutes to a stirred solution of H-Gly-Leu-Phe-Gly-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 drop-wise to 200 ml of vigorously stirred diethyl ether. The white precipitate, which was formed, was filtered and dried at reduced pressure, affording 1.92 g (93%) of crude product which was used without further purification.
1H NMR (CDCI3) 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 (mf. 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).
4.1.2. Preparation of rm-PEG nk lp-O(C=OVNH-Lvs-Glv-Leu-Phe-Glv-10-amino- 7-ethylcamptothecin (8)
Figure imgf000021_0001
(8)
1.23 g (0.06 eq.) of [m-PEG(ιokD)]2-0(C=0)-NH-Lys-Gly-Leu-Phe-Gly-OH (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, 1H); 6.26 (bs, 1H); 6.90 (bs, 1H); 7.12 (bs, 1H); 7.23 (t, J=4.8Hz, 1H); 7.26-7.31 (m, 7H); 7.7 (s, 1H); 7.81 (d, J=4.6Hz, 1H); 8.90 (bs, 1H).
Derivative (8) was tested against P388 and P388/ADM in a similar method as described in Example 1 and demonstrated to have anti-cancer activities.

Claims

Claims
1. An amino-substituted camptothecin polymer derivative, or pharmaceutically acceptable salt thereof, having the following general formula (I):
Figure imgf000023_0001
in which n is an integer between 10 and 1000; -X1 and -X2, independently, represent a residue having the following general formula (II)
Figure imgf000023_0002
wherein -S- represents a cleavable spacer arm residue of a peptide selected among the following peptides: -Gly-Leu-Phe-Gly-,
-Gly-Phe-Leu-Gly-, -Gly-Phe-Phe-Ala-, -Gly-Phe-Phe-Leu-,
-Gly-Phe-Tyr-Ala-, -Ala-Gly-Val-Phe-, -Gly-Phe-Tyr-Ala-, -Gly-Leu-Ala-,
-Gly-Leu-Gly-, -Gly-Phe-Gly-, -Gly-Phe-Ala-, -DAIa-Phe-Lys-, -DVal-Leu-Lys-, and -Lys-Gly-Leu-Phe-Gly- 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 C C6-alkyl group and -X2 is defined as above.
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:
-Gly-Leu-Phe-Gly-, -Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, and -Lys-Gly-Leu-Phe-Gly-.
5. The amino-substituted camptothecin polymer derivative according to any of the preceding claims having the general formula (I) 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 of tumour cells.
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