WO2004092205A1

WO2004092205A1 - Hydroxy-substituted-20-acyloxy-camptothecin polymer derivatives and use of the same for the manufacture of a medicament

Info

Publication number: WO2004092205A1
Application number: PCT/IB2003/001430
Authority: WO
Original assignee: Debio Recherche Pharmacuetique S.A.
Priority date: 2003-04-16
Filing date: 2003-04-16
Publication date: 2004-10-28

Abstract

The present invention relates to pharmacologically active hydroxz-substituted-20-acyloxy-7-ethyl-campothecin polymer derivatives, which have anti-proliferative cell activity and are water-soluble.

Description

Hydroxy-substituted-20-acyloxy-camptothecin polymer derivatives and use of the same for the manufacture of a medicament

The present invention relates to new 9-, 10-, or 11- hydroxy -A-ring- substituted 20-acyloxy 7-ethyl-camptothecin derivatives their use for the manufacture of a medicament, and a pharmaceutical composition comprising said derivatives.

Camptothecins, a family of alkaloids isolated from the Chinese tree Camptotheca acuminata, have attracted much attention because of their significant anti-tumour activity in animals and have 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, ____, 89-105.

However, due to their very poor solubility in the physiological medium and despite the presence of an hydroxy 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-, or 11- hydroxy -A-ring- substituted 20-acyloxy 7-ethyl-camptothecin derivatives, allowing the 9-, 10-, or 11- hydroxy -A-ring-substituted 20-acyloxy 7-ethyl camptothecin pharmacophores to be administrated into the body with an effective plasma half-life due to a stable linkage with a polyethylene glycol fragment and then targeted and accumulated to and into the proliferative cells to be treated with a reduced efflux, in particular in multidrug-resistant cells.

The result is a large molecule that has an hydrophylic region represented by the polyethylene glycol fragment and an hydrophobic region represented by the 9-, 10, or 11-hydroxy-A-ring-substituted 20-acyloxy 7-ethyl -camptothecin. In physiological conditions, this kind of molecules form naturally micelles in which the core is the pharmacophore with an improved protection of the lactone ring.

The present invention has also for object the use of these new 9-, 10-, or 11- hydroxy -A-ring-substituted 20-acyloxy 7-ethyl-camptothecin derivatives for the manufacture of a medicament for the treatment or the prevention of cell proliferative disorders.

Another object of the present invention concerns a pharmaceutical composition comprising said derivates.

The term "plasma half-life" as used herein means the period of time required for the concentration or amount of drug in the body to be reduced to exactly one-half of a given concentration or amount. The given concentration or amount need not be the maximum observed during the course of the experiment, or the concentration or amount present at the beginning of an experiment, since the half-life is completely independent of the concentration or amount chosen as the "starting point". Half-lives can be computed and interpreted legitimately only when concentration or amount varies with time according to the law appropriate to the kinetics of a first order reaction: the common logarithm of the concentration or amount is related linearly to time, e.g.: log C=a+bt where C is concentration at time t, a (in logarithmic units) is the intercept of the line with the ordinate, and b (which has a negative sign) is the slope of the line. The parameters of the equation can be estimated from the plot of experimental values of log C and t.

The expression "multidrug resistant cells" has used herein refers to cells or tissues having acquired drug-resistant or primary drug-resistant disease on the basis of a history of previous tumour treatment. More precisely it concerns resistance to structurally quite different drugs that develops in tumor cells and that leads to decreased intracellular accumulation of drug. The term "hydrophlic region" as used herein concerns the polar or charged part of the molecules that is soluble in water.

The term "hydrophobic region" as used herein concerns the nonpolar part of the molecules that is insoluble in water.

The term "alkyl" denotes a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 21 , preferably 1 to 10, carbon atoms. Alkyl groups may be substituted as specifically provided infra. In addition the alkyl chain may include one or more heteroatoms in lieu of one or more carbon atoms. "Lower alkyl" groups having preferably from 1 to 6, and more preferably 1 to 4 carbon atoms. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl, and the like.

The term "acyloxy" denotes a carboxylic acid residue of general structure

"X-COO-" wherein "X" can be a linear or a branched aliphatic hydrocarbon chain having 1 to 21 , preferably 1 to 11 , carbon atoms. In addition the aliphatic hydrocarbon chain may include one or more heteroatoms in lieu of one or more carbon atoms. "Lower acyloxy" groups having preferably from 1 to 6, and more preferably 1 to 4 carbon atoms. Typical acyloxy groups include methyloxy, ethyloxy, propyloxy, isopropyloxy, butyloxy, t-butyloxy, 2-butyloxy, pentyloxy, hexyloxy, and the like.

The expression "carbamate bond" concerns the group -NR-CO-O- between the nitrogen atom belonging to the peptide spacer and the alkyl group of the polyethylene glycol fragment via the oxygen atom side. It concerns also the bond between the amine of the diamine linker bound to the peptidic spacer arm and one of the oxygen atoms belonging to the camptothecin derivative.

The term "cyclic aliphatic diamine" denotes an heterocyclic compound containing two secondary amino groups bound to an aliphatic hydrocarbon chain of 1 to 15, preferably 2-8, carbon atoms. The term "lactone ring" as used therein concerns cyclic esters of hydroxy carboxylic acids, containing a 1-oxacycloalkan-2- one structure, or analogues having unsaturation or heteroatoms replacing one or more carbon atoms of the ring.

The term "pharmacophore" used in the present invention concerns the particular group or arrangement of atoms in a molecule that gives the material its medicinal activity.

"A therapeutically effective amount" of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease.

The present invention concerns hydroxy -substituted-20-acyloxy-7-ethyl 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 Y is CH ₃ -(CH₂) - in which m is an integer between 1 and 19 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 through a carbamate moiety to the corresponding remaining part of formula (I) or -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- linked with a cyclic aliphatic diamine, the petidic residue being linked through a carbamate bond to the corresponding remaining part of formula (I); or

-X¹ represents a linear or a branched Ci-Cβ-alkyl group, in this case the carbonyl group between the oxygen group and -X¹ is absent, and -X² is defined as above.

The polymeric part of the hydroxy -substituted 20-acyloxy -camptothecin polymer derivatives of the invention corresponds to a polyethylene glycol fragment and it has been selected for its hydrophilic properties. The size of this polyethylene glycol 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 polyethylene glycol fragment 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 glycol fragment exhibits a molecular weight of about 10000, with n being equal to about 250.

In the hydroxy-substituted 20-acyloxy-camptothecin polymer derivatives of the invention, the camptothecin pharmacophore is attached to at least one of the two extremities of the polyethylene glycol 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-Phe-Leu-Gly-, and -Lys-Gly-Leu-Phe-Gly-.

In another embodiment of the present invention, the camptothecin pharmacophore is attached to at least one of the two extremities of the polyethylene glycol fragment through the cleavable spacer arm residue -S- comprising a peptide, preferably the peptide -Gly-Leu-Phe-Gly-, -Gly-Phe-Leu- Gly-, or -Lys-Gly-Leu-Phe-Gly-linked with a cyclic aliphatic diamine. In a preferred embodiment the diamine is the piperazine

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

When lysine is the N-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.

When the camptothecin pharmacophore is attached to only one of the two extremities of the polyethylene glycol fragment, methyl group preferably terminates the second extremity, through the oxygen atom.

The hydroxy group carried on the A-ring of the camptothecin framework of the camptothecin pharmacophore may be on position -9, -10, or -11. Preferably, it is on position 10 and forms an ester group with the carboxyl end group of the cleavable peptidic spacer arm residue or a carbamate group with the cyclic aliphatic diamine linked with the peptidic spacer. In specific physiological conditions and in aqueous solution the hydroxy - substituted-20-acyloxy-camptothecin polymer derivatives according to the present invention may be arranged together in a multimolecular self-association of micelles due to the amphiphilicity of the macromolecules These block copolymer micelles may have mesoscopic size range of approximately 10 to 800 nm. The ability to core aggregation is expected to contribute to the stabilization of the compound in the plasma circulation.

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 peptide arm. This reaction is followed by the activation of the COOH function of said peptide arm to an activated species, which, thus, becomes reactive towards the hydroxy 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-O)_n-H (III)

wherein R is a linear or a branched Ci-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 to obtain a compound of formula (V)

RO-(CH₂-CH₂-O)_n-(C=O)-S-OH (V) c) converting the carboxylic terminal group of the compound of formula (V) thus obtained into the corresponding activated species, and d) finally, reacting the said activated species with the respective hydroxy -A-ring- substituted 20-acyloxy 7-ethyl -camptothecin.

When -S- represents a cleavable spacer arm residue of a peptide linked with a cyclic aliphatic diamine, the procedure after point c) is modified accordingly as follows: e) reacting the said activated species to a mono protected cyclic aliphatic diamine f) removing the protection group g) reacting the resulting compound with a carbonate derivative of hydroxy -A-ring- substituted 7-ethyl 20-acyloxy-camptothecin.

Steps a) through g) 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 hydroxy -substituted 20-acyloxy-7-ethyl-camptothecin polymer derivatives when both extremities of the polyethylene glycol fragment carry respectively a camptothecin pharmacophore.

In an alternative embodiment, the present invention is directed to pharmaceutical compositions comprising as an active ingredient an effective amount of_hydroxy-substituted-20-acyloxy-7-ethyl-camptothecin polymer derivative according to the present invention and a pharmaceutically acceptable carrier or excipient.

These pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. They can also be administered by parenteral route, for example, in the form of injection solutions intramuscularly, intravenously, or by bolus infusion.

The pharmaceutical preparations can also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain other therapeutically valuable substances, including additional active ingredients other than those of the present invention.

The hydroxy -substituted 20-acyloxy-7-ethyl-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, capsule and the like. The compositions may be administrated by oral or parenteral routes. Typically the hydroxy -substituted-20- acyloxy-7-ethyl-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.

Another object of the present invention concerns the use of the hydroxy- substituted-20-acyloxy-7ethyl-camptothecin derivatives of the invention for the manufacture of a medicament for the treatment or the prevention and control of cell proliferative disorders, specifically tumour disorders.

The hydroxy -substituted 20-acyloxy-7ethyl-camptothecin polymer derivatives of the invention and formulations containing said compounds may be useful for the manufacture of a medicament for the treatment or the prevention and control of cell proliferative disorders, specifically solid tumors and more specifically the cancer. These compounds are water-soluble and are expected to show enhanced anti-tumour activity and reduced toxicity in comparison with the free drug. They are useful for the manufacture of a medicament 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 the manufacture of medicament for treating leukaemia and solid tumours, such as colon, colo-rectal, ovarian, mammary, prostate, lung, kidney and also melanoma tumours.

By means of the introduction of such a polymer, an improved administration and targeting of the pharmacophore is achieved. Moreover the presence of the alkyl group on position 20 improve the formation of micelles and induce a protection of the lactone ring. By the introduction of such a peptide spacer arm which may be linked to a cyclic aliphatic diamine, it is believed that a site-specific cleavage of the derivative by specific cellular enzymes takes place, releasing the pharmacophore into the tumour cells.

A human can therefore be treated by a method comprising the administering of a therapeutically effective amount of the hydroxy -substituted camptothecin polymer derivatives of the invention. The condition of the human patient can thus be improved.

The dosage range adopted will depend on the route of administration and on the age, weight and condition of the patient being treated. The hydroxy - substituted 20-acyloxy-7ethyl-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 dose expressed as pharmacophore equivalent This range is illustrative and the practitioner will determine the optimal dosing of the hydroxy -substituted 20- acyloxy 7ethyl-camptothecin polymer derivatives based on clinical experience and the treatment indication.

The present invention will be better understood on the basis of the following examples, offered by way of illustration and not by way of limitation.

The examples below are in connection with the following figures: - Fig 1 represents a kinetic curve (square) of the conversion of the hydroxy - substituted-7ethyl-camptothecin-20-O-propionate 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 7-ethyl-10-hydroxy-20-O-propionate -camptothecin and the conversion of this product in the free drug 7-ethyl-10-hydroxy-camptothecin (circle).

- Fig 2 represents a kinetic curve (square) of the degradation of the hydroxy - substituted-7ethyl-camptothecin-20-O-undecanoate polymer derivative of example 2 by in vitro hydrolysis by Cathepsin B1 at pH 5.5

- Fig 3 represents a kinetic curve (square) of the conversion of the hydroxy - substituted-7ethyl-camptothecin-20-O-propionate polymer derivative of Example 6 by in vitro hydrolysis by Cathepsin B1 at pH 5.5 and a kinetic curve (crosses) of the release of 7-ethyl-10-O[piperazin-carbonyl] -camptothecin-20-O-propionate and the conversion of this product in the pharmacophore drug 7-ethyl- 10-hydroxy- camptothecin-20-O-propionate (circle);

- Fig 4 represents a kinetic curve (square) of the conversion of the hydroxy - substituted-7ethyl-camptothecin-20-O-propionate polymer derivative of example 6 by in vitro hydrolysis at pH 5.5 and a kinetic curve (crosses) of the release of 7- ethyl-10-O-[piperazin-carbonyl]-camptothecin-20-O-propionate and the conversion of this product in the free pharmacophore 7-ethyl-10-hydroxy-camptothecin-20-O- propionate (circle);

- Fig 5 represents a kinetic curve (square) of the conversion of the hydroxy - substituted-7ethyl-camptothecin 20-O-propionate polymer derivative of example 6 by in vitro hydrolysis at pH 7.44 and a kinetic curve (crosses) of the release of 7- ethyl-10-O[piperazin-carbonyl]-camptothecin-20-O-propionate; -Fig 6 represents a kinetic curve (circles) of the conversion of the hydroxy - substituted-7ethyl-camptothecin 20-O-propionate polymer derivative of example 6 by in vitro hydrolysis in mouse plasma and a kinetic curve (crosses) of the release 7-ethyl-10-O[piperazin-carbonyl]-camptothecin 20-O-propionate;

Examples

In the examples, the term "m-PEG-OH" defines the monomethoxypoly-ethylene glycol having a molecular weight 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), 1 -[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrocloride (EDC), 1-hydroxybenzotriazole (HOBt) and H-Gly-Leu-Phe-Gly-OH were commercially available.

Example 1

A. Preparation of m-PEG_{1nkn_rO(C=0)-NH-Glv-Leu-Phe-Glv-OH (1)

318 mg (0,81 mmol) of tetrapeptide H-Gly-Leu-Phe-Gly-OH-HCI were solubilized in 7 ml of borate buffer 1M, pH 8.

2 g (0.20 mmol) of m-PEG-BTC (mw = 9876) were added portionwise to the solution over 60 minutes. The pH was monitored and adjusted to pH 8 using NaOH 1 N and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was acidified with 1 N HCI to pH 3, and extracted with chloroform (5 x 50 ml). The organic phases were pooled and dried over Na₂S0₄ , filtered and concentrated at reduced pressure. The resulting oil was dropped to 200 ml of diethyl ether. The product was kept at 4°C for 1 hour and then filtered. The resulting white powder was dried at reduced pressure. The resulted yield is of 1.948 g (96%). The product was purified from the unreacted mPEG-OH with Sephadex

QAE A-50 ion exchange column. Elution with MQ grade H₂0 removed the undesired material (m-PEG-OH); the increase of ionic strength (0.01 N NaCl) produced the elution of the title compound (mixed with NaCl). The solution was freeze-dried to obtain 1.664 g, that were suspended again in chloroform to remove the salts. Filtration and concentration returned an oil that was chrystallised by dropwise addition to diethyl ether. Filtration yielded 1.42 g of product with a final yield 69% . ¹H NMR (CDC ) 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); 3.35-3.80 (m, PEG -CH₂); 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 7-ethyl-10-O-terf-butyloxycarbonyl-camptothecin (2)

150 mg (0.37 mmol) of 10-hydroxy-7-ethyl-camptothecin was dissolved in 3 ml of dried dimethylformamide. 0.75 ml of pyridine were added at the solution.160 mg of di-te f-butyl dicarbonate previous solubilised in 1 ml of dimethylformamide were added to the mixture, that was stirred at 25°C for 4 h. At the and the mixture was diluted with 30 ml of chloroform and washed with water (2 x 20 ml) and with 1 N Hydrochloric acid (2 x 20 ml). The acqueous phases were washed with fresh chloroform (2 x 30 ml), the organic phases were pooled , dried over Na₂S0₄ and concentrated at reduced pressure. The residue was diluted with 2 ml of chloroform and crystallised with hexane yield 157 mg (87 %)

¹H NMR (DMSO) ppm: 1.3 (t, 3H); 1.42 (t, 3H); 1.9(s,9H); 2.88 (m, 2H); 2.90 (q, 2H); 3.45 (q, 2H); 5.66 (s, 2H); 5.85 (s, 2H); 7.31 (s, 1H); 7.77 (s, 1 H); 7.80 ( s, 1 H); 8.40 (d, 1H).

C. Preparation of

7-ethyl-10-O-teAf-butyloxycarbonyl-camptothecin-20-O-propionate (3)

The product from the previous step, 7-ethyl- 10-O-te t-butyloxycarbonyl- camptothecin (2), was solubilised in anhydrous chloroform. 103 mg (0.797 mmol, 2.5 equivalents) of N,N-diisopropylethylamine and dimethylaminopyridine, in catalitic amount, were added to the solution. 83.6 mg of propionic anydride (0.638 mmol, 2 equivalents) were added to the mixture. The solution was stirred at 25°C for 4 h. The mixture was diluted with 20 ml of chloroform and washed with NaHC0₃ 5% (2 x 10 ml) and then with 0.1 N hydrochloric acid (2 x 10 ml). The acqueous phases was counter-extracted with fresh chloroform (2 x 30 ml). The organic phases were pooled and dried over Na₂S0₄ , and concentrated at reduced pressure.

The resulted yield is of 169 mg (97 %)

¹H NMR (DMSO) ppm: 1.3 (t, 3H); 1.42 (t, 3H); 1.66 (t,3H); 1.9(s,9H);2.52 (q, 2H); 2.88 (m, 2H); 2.90 (q, 2H); 3.45 (q, 2H); 5.66 (s, 2H); 5.85 (s, 2H); 7.31 (s, 1 H); 7.77 (s, 1 H); 7.80 ( s, 1 H); 8.40 (d, 1 H).

P. Preparation of 7-ethyl-10-Hvdroxy-camptothecin-20-O-propionate (4)

The product from the previous step, 7-ethyl- 10-O-te/f-butyloxycarbonyl- camptothecin-20-O-propionate (3) was solubilised in 1 ml of chloroform and added dropwise into 3 ml of trifluoracetic acid at 0°C. The solution was stirred at 25°C for 4 h. The solvents were removed at reduced pressure. The product was purified by flash chromatography (stationary phase silica gel mesh 60 (0,063-0,100 mm) , mobile phase chloroform/ethyl acetate (85:15). The fractions corresponding to the product were concentrated and crystallised with petroleum ether Yield 107 mg ( 77%)

¹H NMR (DMSO) ppm: 1.3 (t, 3H); 1.42 (t, 3H); 1.66 (t,3H); 2.52 (q, 2H); 2.88 (m, 2H); 2.90 (q, 2H); 3.45 (q, 2H); 5.66 (s, 2H); 5.85 (s, 2H); 7.31 (s, 1 H); 7.77 (s, 1 H); 7.80 ( s, 1 H); 8.40 (d, 1 H).

E. Preparation of

propionate (5)

675 mg (0.0667 mmol, 1eq.) of mPEG-Gly-Leu-Phe-Gly-OH (1) and 60 mg

(0.1334 mmol, 2 eq.) of 7-ethyl-10-Hydroxy-camptothecin-20-O-propionate (2) 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.

EDC (19.2, 0.1 mmol) and HOBt (13.5 mg, 0.1 mmol) were added and the mixture was stirred at room temperature for 15 hours.

At the end, the resulting yellow mixture was washed with 0.1 N HCI (2 x 20 ml). The acqueous phase was washed with fresh chloroform. The organic phases were unified and dried over Na₂S0₄ ,then concentrated at reduced pressure. The residue, diluted with 20 ml of 2-propanol, was recrystallised 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 resulted yield is of 90 %

¹H NMR (DMSO) ppm: 0.79-0.81 (m, 6H); 0.95 (t, 3H); 1.139 (t, 3H); 1.26 (q, 1 H);1.39(m,5H) 2.22 (m,2H); 2.53 (m,2H); 3.01 (m,2H); 3.18 (q,2H) 3.22 (t, 3H); 3.43-3.76 (m, PEG -CH₂-) 3.87 (t, 2H); 4.20( m, 4H);4.54-4.67 (m,1H); 5.27 (s, 2H); 5.45-5.73 (dd, 2H);6.02(bs,1 H); 6.38-6.56 (s,3H); 7.23-7.28 (m,6H); 7.88 (bs,1 H); 7.98 (d,1 H); 8.26 (d,1 H).

The amount of 7-ethyl-10-hydroxy-camptothecin in the final product (5) was 2.81 % w/w (the theoretical 100% of loading is, for this conjugate, 3.71% according to UV absorption).

F. In vitro hydrolysis assays

The capability of derivative (5) to release selectively the 7-ethyl- 10-hydroxy- camptothecin-20-O-propionate pharmacophore by hydrolysis was evaluated under enzymatical conditions. Then, such a release was evaluated in mouse plasma. 1. Enzymatic hydrolysis of derivative (5) in presence of Cathepsin Bιι at pH 5.5

Buffer solution (A) at pH 5.5 was prepared using KH₂P0 -2H₂0 0.15 M, 10^"3 M EDTA. Solution (B) was prepared from solution (A) by addition of 5 μM of GSH. (glutathion) Solution (C) was prepared by addition of 850 μl of solution (B) to 50 μl of Cathepsin B<ι solution, containing 0.285 mg/ml of enzyme (extracted from bovine spleen) in buffer solution (A) (1mg 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 (5) 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 (5) 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 hydrolysis of the derivative (5) with release of 7-ethyl-10-hydroxy- camptothecin-20-O-propionate and the final release of 7-ethyl- 10-hydroxy- camptothecin, were monitored following the decreasing of peak areas. The obtained value was plotted on Fig 1 (in the presence of Cathepsin B1 , at pH 5.5) .

2. Conclusions

As we can see on Fig 1 , derivative (5) was progressively converted into 7-ethyl-10-hydroxy-camptothecin-20-O-propionate in presence of Cathepsin Bi at pH 5.5.The half life of derivative (5) is 30 min., in a second time there is a slow release of the free pharmacophore.

G. In vivo pharmacological assay

Mice were inoculated with P388A CR cells (dose 1x10⁶/mouse) intraperitoneally (i.p.) on day 0, and injected intravenously with the derivative (5) on days 1 , 5, and 9 at total doses ( expressed as free drug) of 50 and 100 mg/kg. Survival times were monitored for 40 days. Due to poor water-solubility, it was 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 (5) against P388/VCR.

H. Analysis of hydrodynamic diamiters of derivative (5)

The experiment were performed with a light scattering instrument argon laser operating at 488 nm interfaced with a particle sizing autocorrelator. The measures were performed at 25°C on previously centrifuged samples. The time requared for accettable data is ca. 1-1.5 h. The solutions were prepared immediately before experiments. The sample was soluble in the two solvents used ( Millipore grade unbuffered water or PBS solution).

Table 2 reports the values of hydrodynamic diameters of derivate (5)

Example 2

A. Preparation of

7-ethyl-10-O-terf-butyloxycarbonyl-camptothecin-20-O-undecanoate (6)

10-O-terf-butyloxycarbonyl-7-ethyl-camptothecin (2), was dissolved in anydrous chloroform. 102 mg ( 0.788 mmol, 2.5 eq. ) of N,N-diisopropyl ethylamine and a catalytic amount of dimethylaminopyridine were added to the solution followed by 235 mg (0.663 mmol, 2 eq.) of undecanoic anhydride. The solution was stirred at 25°C for 4 h, then diluted with 20 ml of chloroform and extracted with NaHC0₃ 5% (2 x 10 ml) and 0.1 N HCI (2 x 10 ml ). The acqueous phases were washed with fresh chloroform (2 x 30 ml). The organic phases were unified, dried with Na₂S0₄ , and concentrated at reduced pressure.

¹H NMR (DMSO): ppm 0.70 (t, 3H); 0.83 (t, 3H); 1.11(m, 2H); 1.2 (t, 3H);1.88 (s, 9H); 2.07 (q, 2H); 2.38 (m, 2H); 3.01 (q, 2H); 3.22 (d, 14H); 5.22 (s, 2H); 5.39(s, 2H); 6.84 (s,1H); 7.30 (d, 1 H); 7.91 (d, 1 H). B. 7-ethyl-10-hydroxy-camptothecin-20-O-undecanoate (7)

The product from the previous step, 7-ethyl-10-O-terf-butyloxycarbonyl - camptothecin-20-O-undecanoate was solubilised in 1 ml of chloroform and dropped in 3 ml of trifluoracetic acid at 0°C.

The solution was stirred at 25°C for 4 h. The solvents were removed at reduced pressure. The product was purified by flash chromatography (stationary phase: silica gel mesh, mobile phase: chloroform/ethyl acetate 85:15). The fractions corresponding to the product were concentrated and crystallised with petroleum ether.

The yield is 93 mg.

¹H NMR (DMSO): ppm 0.70 (t, 3H); 0.83 (t, 3H); 1.11 (m, 2H); 1.2 (t, 3H); 2.07 (q, 2H); 2.38 (m, 2H); 3.01 (q, 2H); 3.22 (d, 14H); 5.22 (s, 2H); 5.39(s, 2H); 6.84 (s, 1 H); 7.30 (d, 1H); 7.91 (d, 1 H).

C. Preparation of

10-O-rm-PEG Mnkm-0(C=0)-NH-Glv-Leu-Phe-Glyl-7-ethyl- camptothecin-20- undecanoate (8)

150 mg (0.014 mmol, 1eq.) of mPEG-Gly-Leu-Phe-Gly-OH (1) and 17.2 mg (0.02966 mmol, 2 eq.) of 10-hydroxy -7-ethyl-camptothecin-20-O-undecanoate (7) 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.

EDC (4.3 mg, 0.022 mmol) and HOBt (3 mg, 0.022 mmol) were added and the mixture was stirred at room temperature for 15 hours.

At the end, the resulting yellow mixture was washed with 0.1 N HCI (2 x 20 ml). The aqueous phase was washed with fresh chloroform. The organic phases were unified and dried over Na₂S0₄ , then concentrated at reduced pressure. The residue, diluted with 20 ml of 2-propanol, was recrystallised 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. Yield 110 mg (69 %)

¹H NMR (CDCI3) ppm: 0.81-0.88 (m, 9H); 0.93-1.0 (t, 3H); 1.36-1.43 (t, 3H);

1.66 (m, 3H); 2.05-2.34 (m, 2H); 2.40-2.53 (m, 2H); 3.1 (m, 2H); 3.18 (q, 2H); 3.52 (t, 3H); 3.66-3.80 (m, PEG -CH₂-); 3.87 (t, 2H); 4.20 ( m, 2H);4.54-4.67 (m, 1 H); 5.27 (s, 2H); 5.45-5.73 (dd, 2H); 6.38-6.56 (3s, 3H); 7.23-7.28 (m, 6H); 7.88 (bs, 1 H); 7.98 (d, 1 H); 8.26 (d, 1 H).

The amount of 7-ethyl-10-hydroxy-camptothecin in the final product (8) was

2.67 % w/w (the theoretical 100% of loading is, for this conjugate, 4.2% according to UV absorption).

D. In vitro hydrolysis assays

The capability of derivative (8) to release selectively the 7-ethyl-camptothecin-20- O-undecanoate pharmacophore by hydrolysis was evaluated under enzymatical conditions. Then, such a release was evaluated in mouse plasma.

1. Enzymatic hydrolysis of derivative (8) in presence of Cathepsin B^ at pH 5.5.

The hydrolysis conditions of the derivative (8) in Cathepsin B1 were the same of the derivative (5)

The conversion of the derivative (8) and the release of 7-ethyl- 10-hydroxy- camptothecin-20-O-undecanoate and in a second time the release of free drug 7- ethyl-10-hydroxy-camptothecin were monitored following the decreasing of peak area and the obtained value was plotted on Fig 2 (in the presence of Cathepsin B1 at pH 5.5). 2. Conclusions

Derivative (8) was progressively converted into 7-ethyl-10- hydroxy- camptothecin-20-undecanoate in presence of Cathepsin B1 at pH 5.5 (figure 5).The half life of derivative (8) is 30 min.

Example 3

A. Preparation of m-PEGfmι_<nrO(C=0)-NH-Glv-Leu-Glv-OH (9)

114,1 mg (0,405 mmol) of tripeptide H-Gly-Leu-Gly-OH • HCI were dissolved in 5 ml of aqueous borate buffer 1M, 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 the solution was stirred at room temperature for 24 hours.

The reaction mixture was acidified with 1 N HCI to pH 3, and extracted with chloroform (5 x 50 ml). The organic phases were dried over Na₂S0 , filtered and concentrated at reduced pressure. The remaining oil was dropped in 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 94%.

The product was separated from the unreacted mPEG-OH by Sephadex QAE A-50 ion exchange column. Elution with MQ grade H₂0 removed the undesired material (m-PEG-OH), while at increased ionic strength (0.01 N NaCl) the desired compound was recovered from the column. The solution was freeze- dried to obtain 667 mg, the product was treated with chloroform to remove the salts and filtered. After concentration of the chloroform solution the remaining oil was added dropwise to diethyl ether. The final yield of (9) was 66% (w/w).

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, PEG -CH₂-); 4.15 (m, 1 H); 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).

B. Preparation of 10-O-rm-PEGrιnknrO(C=O)-NH-Glv-Leu-Glyl-7-ethyl-camptothecin 20- propionate (10)

0.2 g (0.02 mmol, 1 eq.) of the compound (9) as obtained above and 17.9 mg (0.04mmol, 2 eq) of 7-ethyl-10-hydroxy-camptothecin 20- propionate (2) 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. EDC (2.9 mg, 0.015 mmol) and HOBt (2.0 mg, 0.015 mmol) were added and the mixture was stirred at room temperature for 15 hours. At the end, the yellow mixture was washed with 0.1 N HCI (2 x 30 ml). The acqueous phase was washed with fresh chloroform. The organic phases were unified, dried over Na₂S0₄ ,and concentrated at reduced pressure. 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. Yield 183 mg (88%)

¹H NMR (DMSO) ppm: 0.79-0.81 (m, 6H); 0.95-0.81 (t, 3H); 1.139 (t, 3H); 1.26 (q, 1 H);1.39(m, 5H) 2.22 (m, 2H); 2.53 (m, 2H); 3.01(m, 2H); 3.18 (q, 2H)3.22 (t, 3H); 3.43-3.76 (m, PEG -CH₂-) 3.87 (t, 2H); 4.20( m, 2H);4.54-4.67 (m, 1 H); 5.27 (s, 2H); 5.45-5.73 (dd, 2H); 6.38-6.56 (s, 3H); 7.23-7.28 (m, 1H); 7.88 (bs, 1 H); 7.98 (d, 1 H); 8.26 (d, 1 H). Example 4

A. Preparation of PEG nk -rθ-(C=0)-NH-Glv-Leu-Phe-Gly-OHl_? (11)

1g (0.101 mmol) of the diol HO-PEG-OH (mw = 10000) was dried by Dean-

Stark apparatus in 30 ml of toluene. The solution was concentrated to 5 ml, and then diluted with dry chloroform (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 45 minutes to a solution of 0.24g (0.606 mmol, 6 eq.) of tetrapeptide H-Gly-Leu-Phe-Gly-OH in 5 ml of borate buffer 1M, pH 8. The pH of mixture was mantained to pH 8 with 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 (4 x 40 ml). The combined organic solutions were dried over Na₂S0₄ and concentrated to a small volume at reduced pressure. The resulting slurry was added dropwise to diethyl ether. The white precipitate which formed was filtered and dried at reduced pressure, affording 0.96 g of crude product which was purified from the starting product through QAE Sephadex A-50 ion exchange resin. Elution with NaCl 0.01 M afforded 0.95 g of product. The appropriate combined fractions were freeze-dried and the residue was suspended in chloroform to remove the salts. Recrystallisation afforded 0.86 g (79%) of title compound.

Titration of -COOH groups: 96%

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, PEG -CH₂-); 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_iιnkn_rrθ-(C=O)-NH-Glv-Leu-Phe-Glv -(10-O-7-ethyl- camptothecin-20-propionate) 1₂ (12)

0.6 g (0.06 mmol)of PEGι₀κ_D-(-Gly-Leu-Phe-Gly-OH)₂ (11) and 108 mg (0.24 mmol, 4 eq) of 7-ethyl-10-hydroxy-camptothecin 20-propionate (4) were dissolved in 30 ml of toluene and the mixture was azeotropically distilled with removal of 25 ml of toluene. The mixture was evaporated to dryness at reduced pressure, and the residue was suspended in 25 ml of dry chloroform EDC (17.2 mg, 0.09 mmol) and HOBt (12.2 mg, 0.09 mmol) were added and the mixture was stirred at room temperature for 15 hours. At the end, the yellow mixture was washed with 0.1 N HCI (2 x 20 ml). The acqueous phase was washed with fresh chloroform. The organic phases were unified and dried over Na₂S0₄ , concentrated at reduced pressure. 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 is of 92 % W/W

¹H NMR (CDCI₃) ppm: 0.91 (t, 12H); 1.3 (t, 6H); 1.42 (t, 6H); 1.44 (m, 2H); 1.66 (t, 6H); 2.52 (q, 4H); 2.88 (m, 4H); 2.90 (q, 4H); 3.01-3.20 (dd, J=22.8Hz; J=6.6Hz, 4H); 3.40-3.88 (m, PEG -CH₂-); 4.15 (m, 2H); 4.19 (m, 4H); 4.56 (m, 4H); 5.66 (s, 4H); 5.85 (s, 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, 14H). 7.77 (s, 2H); 7.80 ( s, 2H); 8.40 (d, 2H). Loading (w/w % of 7-ethyl-10-hydroxy- camptothecin) = 6.81 % (theoretical = 8.5%) according to UV absorption.

Example 5

A. Synthesis of α.ε-rmPEGMnk -0(C=0)l Lvs-Glv-Leu-Phe-Glv-OH (13)

2g (0.1 mmol) of α,ε-[mPEG₍ι₀kD) -0(C=0)]₂ -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 HCI 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 that 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, PEG -CH₂-); 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 a.s-fmPEGnnk -O(C=O)l?Lvs-Glv-Leu-Phe-Glv-(10-O-7- ethyl-camptothecin-20-propionate) (14)

(14)

1.23 g (0.06 mmol) of [mPEG₍ι₀kD) -0(C=0)]₂-Lys-Gly-Leu-Phe-Gly-OH (13) and 55.3 mg (0.12 mmol, 2 eq) of 7-ethyl-10- hydroxy-camptothecin-20- propionate (4) 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. EDC (17.4 mg, 0.09 mmol) and HOBt (12.3 mg, 0.09 mmol) were added and the mixture was stirred at room temperature for 18 hours.

At the end, the mixture was washed with 0.1 N HCI (2 x 30 ml). The acqueous phase was washed with fresh chloroform. The organic phases were unified, dried over Na₂S0 and concentrated at reduced pressure. The residue, diluted with 50 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.

¹H NMR (CDC ) ppm: 0.91 (t, 6H); 1.3 (t, 3H)1.37-1.44 (m, 6H); 1.56 (t, 3H); 1.6-1.8 (m, 4H); 2.52 (q, 2H); 2.88 (m, 2H);2.9 (m, 4H); 3.01-3.20 (dd, 4H); 3.39 (s, 6H); 3.45 (q, 2H); 3.40-3.88 (m, PEG -CH₂-); 4.17 (m, 1 H); 4.21 (m, 2H); 4.26 (m, 1 H); 4.57 (m, 2H); 5.66 (s, 2H); 5.85 (s, 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, 6H); 7.77 (s, 1 H); 7.80 ( s, 1 H); 7.83 (d, J=4.6Hz, 1 H);8.40 (d, 1 H)

Derivative (14) was tested against P388/VCR in a similar method as described in example 1 and demonstrated to have anti-cancer activity.

Example 6

A. Preparation of m-PEG MΩknrO(C=0)-NH-Glv-Leu-Phe-Glv-piperazine-

BOC(15)

mPEG-Gly-Leu-Phe-Gly-OH (1) (300mg, 0.02966 mmol) were dissolved in 10 ml of anydrous CHCI₃. The solution was additionated of 6.1 mg ( 0.03262 ,1.1 eq.) of terf-butyl 1-piperazinecarboxylate and cooled at 0°C. 6.3 mg of EDCI

(0.033,1.1 eq.), were added, followed, after 10 min, by 4.4 mg (0.033 ,1.1 eq.) of HOBt and after further 10 min by 4.5 μl of triethylamine. The resulting mixture was stirred at 25°C for 24 hours. The solution was then diluted with 30 ml of chloroform and washed with NaHCθ₃ 5% (2 x 25 ml) and KH₂P0 10 % (2 x 25 ml). The organic phase were dried over Na₂S0₄ and concentrated at reduced pressure. The remaining oil was added dropwise to diethyl ether. The white precipitate which formed was filtered and dried at reduced pressure, affording 267 mg (90.56%) of crude product which was purified by elution throught QAE Sephadex A-50 ion exchange resin. Elution with aqueous NaCl 0.01 M afforded 220 mg (72%) of m-PEG _(10kD)-O(C=O)-NH-Gly-Leu-Phe- Gly-piperazine-BOC (16)

¹H NMR (CDCI₃) ppm:0.72-0.85 (dd, 6H); 1.27-1.46 (m, 3H); 1.6 (s, 9H); 2.9(t, 3H); 3.55 (m, 3H); 3.60-3.78 (m, PEG -CH₂-); 3.84-3.91 (m, 3H); 3.98-4.1 (bm, 5H); 4.19-4.26 (m, 2H); 6.81-6.91(m, 1 H); 7.21(t, 2H); 7.47-7.57(m, 2H). B. Preparation of m-PEG rιnkm-0(C=0)-NH-Glv-Leu-Phe-Glv-piperazine(16)

The product from the previous step (15) was solubilized in 2 ml of chloroform and dropped in 3 ml of trifluoracetic acid cooled at 0°C The solution was stirred at 25°C for 4 hours.

The eluents was evaporated at reduced pressure and the product was utilised without further purification.

¹H NMR (CDCI₃) ppm: 0.72-0.85 (dd, 6H); 1.27-1.46 (m, 3H); 2.9 (t, 3H);3.55 (m, 3H); 3.60-3.78 (m, PEG -CH₂-); 3.84-3.91 (m, 3H); 3.98-4.1 (bm, 5H); 4.19-4.26 (m, 2H); 4.64-4.78 (m, 1 H); 6.81-6.91 (m, 1 H); 7.21 (t, 2H); 7.47-7.57 (m, 2H).

C-Preparation of

7-ethyl-10-O-p-nitrophenyloxycarbonyl camptothecin-20-O-propionate (17)

39 mg ( 0.0871 eq) of 7-ethyl-10-hydroxy-camptothecin-20-propionate (4) was dissolved with 10 ml of anydrous chloroform, p-nitrophenylchloformiate (105 mg, 6 eq.) was added and the pH of the solution was corrected to 8 with 70 μl( 6 eq.) of triethylamine. The solution was stirred at 25°C for 18 hours. The eluent was evaporated at reduced pressure. The product was purified by flash chromatography (mobile phase chloroform/ ethyl acetate 85:15). Yield 26 mg.

P. preparation of

10-O-fm-PEG nk -O(C=O)-NH-Glv-Leu-Phe-Glv-piperazinyl-carbonyll-7- ethyl-camptothecin-20-propionate (18)

(18)

250 mg (0.024 mmol, 1 eq.) of the compound (16) as obtained above and 26 mg (0.048 mmol, 2 eq) of 10-O-p-nitrophenyloxycarbonyl-7-ethyl-camptothecin- 20-O-propionate (17) were dissolved in 30 ml of toluene and the mixture was azeotropically distilled with removal of 25 ml of toluene. The mixture was diluted with 10 ml of dried chloroform and the the solution was adjusted to pH 8 with triethylamine. The solution was stirred at 25°C for 18 hours, then the solvents were evaporated at reduced pressure and the remaining oil was added droppwise to diethyl ether. The solid, dissolved into 20 ml of 2-propanol, was recrystallised 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 is of 95 %

The amount of 7-ethyl-10-hydroxy-camptothecin in the final product (18) was 2.44 % w/w (the theoretical 100% of loading is, for this conjugate, 4.19%).

¹H NMR (CDCI₃) ppm: 0.79-0.81 (m, 6H); 0.95-0.81 (t, 3H); 1.139 (t, 3H); 1.26 (q, 1 H);1.44 (t, 3H);1.55 (m, 3H); 1.78(bs, 8H); 2.22 (m, 2H); 2.53 (m, 2H); 3.1 (m,2H) 3.18 (q, 2H); 3.52 (t, 3H); 3.60-3.78 (m, PEG -CH₂-); 3.87 (t, 2H); 4.20 (m, 2H);4.54-4.67 (m, 1 H); 5.27 (s, 2H); 5.45-5.73 (dd, 2H); 6.38-6.56 (s, 3H); 7.23-7.28 (m, 6H); 7.88 (bs, 1 H); 7.98 (d, 1 H); 8.26 (d, 1 H). E. In vitro hydrolysis assays

The capability of derivative (18) to release selectively the 10-O-[piperazinyl- carbonyl]-7-ethyl-camptothecin-20-propionate pharmacophore by hydrolysis was evaluated under enzymatical and chemical conditions. Then, such a release was evaluated in mouse plasma.

1. Enzymatic hydrolysis of derivative (18) in presence of Cathepsin Bτ_ at pH 5.5 and chemical hydrolysis of the derivative at pH 5.5.

Buffer solution (A) at pH 5.5 was prepared using KH₂P0 -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 B-i solution, containing 0.285 mg/ml of enzyme (extracted from bovine spleen) in buffer solution (A) (1mg 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 (18) 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 (18) 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 hydrolysis of the derivative (18) with release of 10-O-[piperazinyl- carbonyl]-7-ethyl-camptothecin-20-propionate and the final release of 7-ethyl-10- hydroxyl-camptothecin, were monitored following the decreasing of peak areas. The obtained value was plotted respectively on Fig 3 (in the presence of Cathepsin B1 , at pH 5.5) and on Fig 4 (in the absence of Cathepsin B1 at pH 5.5).

2. Chemical hydrolysis of derivative (18) 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 NaCl was prepared. 4.098 mg of derivative (18) 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 (18) and the release of 7-ethyl-10 hydroxy camptothecin-20-propionate and then the release of free drug 7-ethyl-10-hydroxy- camptothecin were monitored and the obtained value were plotted on Fig 5.

3. Hydrolysis of derivative (18) 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 (18) expressed as hydroxy ethyl-camptothecin was added to 75μl of a solution (G) containing 1/15 M of KH₂P0₄/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 for 3' 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 (18) and the release of 7-ethyl-10- camptothecin-hydroxy-20-propionate and then the release of free pharmacophore 7-ethyl-10-hydroxy-camptothecin were monitored and the obtained value were plotted on Fig 6.

Claims

1. An hydroxy-substituted-20-acyloxy camptothecin polymer derivative having the following general formula (I):

wherein Y is CH ₃ -(CH₂)_m- in which m is an integer between 1 and 18

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 through a carbamate bond to the corresponding remaining part of formula (I) or -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- linked with a cyclic aliphatic diamine linked through a carbamate bond to the corresponding remaining part of formula (I); or

-X¹ represents a linear or a branched CrCβ-alky! group, in this case the carbonyl group between the oxygen group and -X¹ is absent, and -X² is defined as above.

2. The hydroxy-substituted-20-acyloxy-camptothecin polymer derivative according to claim 1 in which n is an integer between 20 and 400.

3. The hydroxy -substituted-20-acyloxy- camptothecin polymer derivative according to claim 1 or 2 in which n is equal about 250.

4. The hydroxy -substituted-20-acyloxy-camptothecin polymer derivative according to any one of claims 1 to 3 wherein the cyclic aliphatic diamine is a piperazine when -S- represents a cleavable spacer arm residue of a peptide linked with a cyclic aliphatic diamine.

5. The hydroxy -substituted-20-acyloxy-camptothecin polymer derivative according to any one of claims 1 to 4 in which the cleavable spacer arm residue - S- is selected among the following peptides:

-Gly-Leu-Phe-Gly-, -Gly-Phe-Leu-Gly-, and -Lys-Gly-Leu-Phe-Gly-.

6. The hydroxy-substituted-20-acyloxy-camptothecin polymer derivative according to any one of claims 1 to 5, in which, when X¹ represents a linear or a branched Ci-Cβ-alkyl group, it is a methyl group.

7. The hydroxy-substituted-20-acyloxy-camptothecin polymer derivative according to any one of claims 1 to 6, in which the hydroxy 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.

8. A pharmaceutical composition comprising as an active ingredient an effective amount of a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient.

9. Use of the hydroxy-substituted-20-acyloxy- camptothecin polymer derivative according to any one of claims 1 to 7 for the manufacture of a medicament for treating or preventing a cell proliferative disorders.

10. The use of claim 9 wherein the cell proliferative disorder is cancer.