KR20070022290A - 7-t-butoxyiminomethylcamptothecin conjugated in position 20 with integrin antagonists - Google Patents

Abstract

A compound of Formula (I) is disclosed which comprises a condensation of a cyclopeptide containing an RGD sequence with 7-t-butoxyiminomethylcamptothecin at the position 20 as defined in the specification and at position 20. The compound has both high affinity for integrin receptors α _v ß ₃ and α _v ß ₅ and selective cytotoxic activity against human tumor cell lines at micromolar concentrations.

[Formula I]

Cyclopeptide, 7-t-butoxyiminomethylcamptothecin, condensation, integrin receptor

Description

7-T-BUTOXYIMINOMETHYLCAMPTOTHECIN CONJUGATED IN POSITION 20 WITH INTEGRIN ANTAGONISTS} Conjugated with an integrin antagonist at position 20

FIELD OF THE INVENTION The present invention relates to compounds with cytotoxic activity, consisting of cyclopeptides containing RGD sequences and derivatives of camptothecin, methods for their preparation, their use as medicaments and compositions containing them.

In particular, the compounds disclosed herein have both high affinity for integrins α _v ß ₃ and α _v ß ₅ and selective cytotoxic activity against human cell lines at micromolar concentrations.

Chemotherapy anticancer agents are drugs with the most limited therapeutic window. Indeed, since the cytotoxic activity of the agent is non-selective, the agent can indiscriminately damage all the cells of the body that come into contact with the agent.

There is currently a problem regarding the directing of cytotoxic agents to tumor cells, such that the agent does not damage surrounding healthy tissue cells or exerts its activity, at least as much as possible, limiting the damage.

The literature describes the use of selective cyclopeptides, which are reference compounds considered as cyclopentapeptides c (Arg-Gly-Asp-D-Phe-Val) (JACS 1997, 119, 1328-35; International Patent Application WO 97 / 06791), or blocking the integrins α _v ß ₃ and α _v ß ₅ by the use of monoclonal antibodies (Cell, 1994, 79, 1157-64) has been reported to result in blocking angiogenesis and decreasing tumor growth. It is. In addition, metastasis inhibitory effects were also observed (J. Clin. Invest., 1995, 96, 1815). Brooks et al., Science 1994, 264, 569-71 report that endothelial cells of the tumor vasculature and the tumor cells themselves express integrin α _v ß ₃ preferentially over silent cells of normal tissues. Among the compounds in the stage of clinical development, c (Arg-Gly-Asp-D-Phe-MeVal) or EMD 12197 or silicided.

Ruoslatati and co-workers have shown that RGD homologues that bind tumor endothelial cells, once conjugated with the cytotoxic doxorubicin, form more efficient and less toxic compounds than doxorubicin alone (Current Opinion in Oncology, 1998). , 10, 560-5). The authors also demonstrated that, without any reasonable doubt, the effect can be attributed to conjugation to RGD as long as the binding is antagonized by the free peptide itself (Arap, Pasqualini and Ruoslati, Science, 1998, 279, 377). -380). Later, the same authors performed other experiments chemically binding the apoptotic precursor peptide sequence to the RGD homologue, demonstrating that the novel compounds are selectively toxic to angiogenic endothelial cells and have anticancer activity in mice. (Rouslati, Nature Medicine, 1999, 5, 1032-8).

Marcus et al., In international patent application WO 01/17563, describe cytotoxic agents conjugated to non-peptide inhibitor antagonists of integrins α _v ß ₃ and α _v ß ₅ by spacers consisting of one or more amino acids, for example. For example, the specific anticancer activity of camptothecins.

Aoki et al., Cancer Gene Therapy, 2001, 8, 783-787, disclose the specific anticancer activity of histidylated oligolysine conjugated to the RGD sequence, which demonstrates the homing effect on tumors in mice. Indicates.

The concept of binding at the cell surface mediated by integrins has been proposed for gene transport (Hart, et al., J. Biol. Chem., 1994, 269, 12468-12474).

7-tert-butoxyiminomethylcamptothecin (or CPT184 or ST 1481 or Gimatecan) is an orally active derivative of camptothecin and is disclosed in European patent EP 1 044 977.

According to the present invention, 7-t-butoxyiminomethylcamptothecin conjugated at position 20 to a cyclopeptide derivative containing an RGD sequence may be advantageously used for the preparation of a medicament for the treatment of tumors by a presumably suitable spacer. Has been found to produce compounds endowed with high selective anticancer activity.

The compounds according to the invention provide drugs with fewer side effects and less severe thanks to their selective cytotoxic activity against tumor cells.

An object of the present invention is a derivative of 7-t-butoxyiminomethylcamptothecin conjugated to a cyclopeptide derivative containing an RGD sequence. The resulting molecule has both unchanged cytotoxic properties of the original camptothecins and integrin binding properties with affinity comparable to the affinity observed for non-conjugated cyclopeptides. The result of this combination is to promote the enrichment (reduction) of cytotoxic agents in cells that express the most of the integrins of the α _v ß ₃ and α _v ß ₅ types. The cytotoxic agent is conjugated through enzymatic or hydrolytic action and / or exerts its intracellular activity in free form.

Accordingly, the main object of the present invention is a compound of the formula (I), N ₁ -oxide, racemic mixture thereof, single enantiomer, single diastereoisomer, form E and Z, mixtures thereof, pharmaceutically acceptable salts:

Where

R ₁ is a UXY group, where:

U is absent or is one of the following groups -COCHR ₁₀ NH- and CON [(CH ₂ ) _n2 NHR ₇ ] -CH _2- , wherein R ₁₀ is H, or optionally C ₆ -C ₁₄ aryl Substituted linear or branched C ₁ -C ₄ alkyl and amino-alkyl C ₁ -C ₄ ; R ₇ is H or linear or branched C ₁ -C ₄ alkyl; n ₂ is an integer from 2 to 6;

X is either not present or H or a _{-COCHR 3 NH-, -COCHR 6 (CH} 2) n3 R 4 -, -R 4 -CH 2 (OCH 2 CH 2) n4 OCH 2 R 4 -, -R 4 (Q) R _4- , -R ₅ [Arg-NH (CH ₂ ) _n5 CO] _n6 R _5- , -R _5- [N-guanidinopropyl-Gly] _n6 R _5- n ₃ is an integer from 0 to 5, n ₄ is an integer from 0 to 50, n ₅ is an integer from 2 to 6, n ₆ is an integer from 2 to 7;

R ₃ is H or linear or branched C ₁ -C ₄ alkyl optionally substituted by —COOH, —CONH ₂ , —NH ₂ or —OH;

R ₄ is selected from the group consisting of -NH-, -CO-, -CONH-, -NHCO-;

R ₅ is absent or is a group -R ₄ (Q) R _4- ;

R ₆ is H or NH ₂ ;

Q is linear or branched C ₁ -C ₆ alkylene; Linear or branched C ₃ -C ₁₀ cycloalkylene; Linear or branched C ₂ -C ₆ alkenylene; Linear or branched C ₃ -C ₁₀ cyclo-alkenylene; C ₆ -C ₁₄ arylene; Arylene (C ₆ -C ₁₄ ) -alkylene; (C ₁ -C ₆ ), alkylene (C ₁ -C ₆ ) -arylene (C ₆ -C ₁₄ ); An aromatic or non-aromatic heterocyclyl (C ₃ -C ₁₄ ) containing one or more heteroatoms selected from the group consisting of O, N, S;

Y is absent or H, or is the group c (Arg-Gly-Asp-AA ₁ -AA ₂ ), wherein:

c means cyclic;

AA ₁ is (D) -Phe, (D) -Trp, (D) -Tyr, (D) -2-naphthylAla, (D) -4-terbutyl-Phe, (D) -4,4 ′ -Biphenyl-Ala, (D) -4-CF ₃ -Phe, (D) -4-acetylamino-Phe;

AA ₂ is NW-CH [(CH ₂ ) _n7 -CO] -CO, NW-CH [(CH ₂ ) _n7 -NH] -CO, NW- [4- (CH ₂ ) _n7 -CO] -Phe, NW -[4- (CH ₂ ) _{n 7} -NH] -Phe, [NW] -Gly, NW-Val, wherein W is H, linear or branched C ₁ -C ₆ alkyl,-(CH ₂ ) _n7 -COOH where n ₇ is an integer from 0 to 5, 4-carboxybenzyl, 4-aminomethylbenzyl; only

It is not possible for both X and Y to be present.

The present invention encompasses the use of the above-mentioned compounds of formula I as active ingredients of medicaments useful as isomerase I inhibitors. Parasitic or viral infections are among the therapeutic indications derived from isomerase I inhibition.

The compounds of formula (I) provided their specific pharmacological properties, but are also useful in the manufacture of a medicament for the treatment of tumors and their metastases.

The present invention also encompasses pharmaceutical compositions containing as an active ingredient a compound of formula (I) in admixture with one or more pharmaceutically acceptable vehicles and / or excipients.

Compounds according to the invention are the result of condensation of cyclopeptides containing the Arg-Gly-Asp (RGD) sequence with 7-t-butoxyiminomethylcamptothecin functionalized at position 20. This structural combination has the advantage of facilitating the enrichment of cytotoxic agents in cells that express the most integrins of the α _v ß ₃ and α _v ß ₅ types. The cytotoxic agent is conjugated through enzymatic or hydrolytic action and / or exerts its activity in free form.

The definitions of the various functional groups and moieties as well as the pharmaceutically acceptable salts shown in formula (I) above are common sense to any professional chemist and require no special definition. However, reference may be made to technical and patent documents such as the international patent applications WO 00/53607, WO 03/101995 and WO 03/101996 for such groups.

One initial group of preferred compounds consists of compounds of formula I in which U and / or X are present.

Preferred compounds according to the invention are as follows:

Compounds of formula (I) may be prepared by the process described below in the present invention and exemplified by the preferred compounds according to the present invention. The method constitutes a further object of the present invention.

Essentially, 7-t-butoxyiminomethylcamptothecin ("7-t-but"), functionalized via putatively suitable crosslinking (represented as "U ₁ -X ₁ "), is a compound of formula (I) which is the object of the present invention. -CP ") and cyclopeptide derivatives (represented as" Y ₁ ").

The condensation reactions can be carried out according to one of the following schemes:

7-t-but-CP + U ₁ -X ₁ -Y ₁ or

7-t-but-CP-U ₁ + X ₁ -Y ₁ or

7-t-but-CP-U ₁ -X ₁ + Y ₁ or

7-t-but-CP + U ₁ + X ₁ + Y ₁ or

7-t-but-CP-U ₁ + X ₁ + Y ₁

In the above,

7-t-but-CP represents 7-t-butoxyiminomethylcamptothecin,

U ₁ , X ₁ and Y ₁ each represent groups U, X and Y as defined in formula (I), and are finally functionalized and / or protected appropriately so that the conjugated compounds of formula (I) are obtained.

Such reactions can be carried out in conventional manners, for example in Journal of Controlled Release 2003, 91, 61-73; S.S. Dharap et al .; Journal of Medicinal Chem. 2003, 46, 190-3, R. Bhatt.

Cyclopeptide Y ₁ may be prepared according to conventional peptide synthesis techniques, as described in Examples 1-6. The peptide synthesis can be carried out in solid phase or in solution.

Once the desired cyclopeptide has been obtained, it will be used in the condensation reaction in protected form, and only after the final compound is obtained the protective group will be removed. The deprotection is carried out using acidic conditions using known methods, for example by the use of pure trifluoroacetic acid or in the presence of chlorinated organic solvents.

The compounds disclosed herein are topical isomerase I inhibitors and are therefore useful as medicaments, in particular as medicaments for the treatment of diseases in which the inhibition of said isomerases is beneficial. In particular, the compounds according to the invention exhibit anti-proliferative activity, and therefore their therapeutic properties are utilized and they have physicochemical properties which make them suitable for the formulation of pharmaceutical compositions.

The pharmaceutical composition contains, as active ingredient, one or more compounds of formula (I), for example in an amount that produces a significant therapeutic effect. The compositions included in the present invention are obtained using methods that are wholly conventional and commonly practiced in the pharmaceutical industry. Depending on the route of administration chosen, the composition will be in solid or liquid form and will be suitable for oral, nasal or intravenous administration. The composition according to the invention contains one or more pharmaceutically acceptable vehicles or excipients together with the active ingredient. Formulation aids such as solubilizers, dispersants, suspending agents or emulsifiers may be particularly useful.

The compounds of formula (I) can also be used in both separate and single dosage forms with other active ingredients, such as anticancer agents or other drugs with parasitic or viral inhibitory activity.

The compounds according to the invention are for example non-microcytoma and small cell lung cancer, or colorectal or prostate cancer, glioblastoma and neuroblastoma, cervical cancer, ovarian cancer, gastrointestinal carcinoma, liver carcinoma, Kaposi's sarcoma, renal carcinoma, sarcoma And agents having anticancer activity in osteosarcoma, testicular carcinoma, breast carcinoma, pancreatic carcinoma, melanoma, urinary bladder and head and neck carcinoma. One of the advantages provided by the compounds according to the invention is the combination of the local isomerase inhibitory activity inherent in the camptothecin portion of the molecule with the integrin inhibitory activity provided by the cyclopeptide portion of the molecule. The result is a possible complex action of the compounds according to the invention, which will be favorably accepted in the field by experts in the field of oncology. Indeed, the cyclopeptide moiety containing the Arg-Gly-Asp sequence not only directs the molecule to tumors expressing integrins, but also once the target has been reached, a number of actions ranging from internalization of the cytotoxic moiety of the molecule to integrin inhibitory activity. , Resulting in advantages, particularly with regard to tumor angiogenesis inhibition. The cyclopeptide moiety, once isolated from the camptothecin moiety, can also exert its action away from the tumor site, thus demonstrating that the compounds according to the invention are also useful for the prevention or treatment of metastatic forms.

Agents which are an object of the present invention can also be used for the treatment of parasitic diseases.

The following examples further illustrate the invention.

Abbreviations used are as follows:

Aad (aminoadipic acid);

Amb (aminomethylbenzyl);

Amp (aminomethylphenylalanine);

Boc (tert-butoxycarbonyl);

CSA (camphorsulfonic acid);

CTH (catalyst transfer hydrogenation);

DCC (dicyclohexylcarbodiimide);

DCM (dichloromethane);

DIEA (diisopropylethylamine);

DMF (dimethylformamide);

Dy (OTf) ₃ (diprosium triflate);

Fmoc (9-fluorenylmethyloxycarbonyl);

HOBT (hydroxybenzotriazole);

NMP (N-methyl-pyrrolidone);

Pht (phthaloyl);

Pmc (pentamethylchroman-6-sulfonyl);

ST1481 (7-t-butoxyiminomethylcamptothecin, also named Jimatecan);

TBTU (tetrafluoroborate-O-benzotriazol-1-yl-tetramethyluronium);

TFA (trifluoroacetic acid).

Example  One

c ( Arg ( Pmc )- Gly - Asp ( OtBu ) -D- Phe - Amp ) (Protected ST2581 ) Synthesis

1.587 moles of Fmoc-Gly-Res (Res = Sasrin Resin®, Bachem) were suspended in 75 ml of DMF under stirring for 30 minutes, after which 18 ml of piperidine was added and stirring continued for another 30 minutes. . The resin washed with filtration and DMF was suspended in 50 ml of NMP (N-methyl-pyrrolidone) for 15 minutes, after which Fmoc-Arg (Pmc) -OH, HOBT, TBTU and DIEA were added (3.174 mmol each); After stirring for 2 hours, the suspension was filtered and washed with DMF. After deprotection with piperidine, the coupling was described above each time using a different amino acid: Fmoc-Amp (Cbz) -OH, Fmoc-D-Phe-OH, and Fmoc-Asp (OtBu) -OH in turn. Repeated as described. After the final deprotection of the Fmoc-N-terminus, linear pentapeptide was released from the resin by 45 ml of 1% TFA in DCM. It is dissolved in approximately 1 L of CH ₃ CN and 4.761 mmol of HOBT and TBTU, and 10 ml of DIEA are added; The solution was kept stirring for 30 minutes, the solvent was evaporated to a small volume and the water was used to complete the precipitation of the product. The filtered crude product was dissolved in 27 ml of a 1: 1 mixture of MeOH and DMF, 5 mmol of ammonium formate and 0.55 g of 10% Pd / C were added and left under stirring for 30 minutes at room temperature. The suspension was filtered over celite and dried. The residue was purified by preparative RP-HPLC (column: Alltima® C-18, Alltech; mobile phase: 50% CH ₃ CN + 0.1% TFA in water; retention time (Rt) = 9.13 minutes). 483 mg of white powder was obtained.

Maldi-Tof: 973

Example  2

c ( Arg ( Pmc )- Gly - Asp ( OtBu ) -D- Phe - Aad ) (Protected ST2650 ) Synthesis

0.69 mmol of Fmoc-Gly-Res was treated as described in Example 1 except that in this case the third and fourth amino acids were added in the form of the dipeptide Fmoc-D-Phe-Aad (OBzl) -OH. After deprotection by CTH and the crude product was purified by preparative RP-HPLC (mobile phase: 66% CH ₃ CN + 0.1% TFA in water; Rt = 17.29 min), 187 mg of pure peptide was obtained.

Molecular Mass (Maldi-Tof): 940

Example  3

c ( Arg ( Pmc )- Gly - Asp ( OtBu ) -D- Phe -N- Me - Amp ) (Protected ST2700 ) Synthesis

Two equivalents of CSA and 20 equivalents of paraformaldehyde were added to a suspension of Fmoc-Phe (4-Pht-N-CH ₂ ) -COOH in refluxed anhydrous toluene and divided into four aliquots at 15 minute intervals. The mixture was cooled, diluted with 120 mL of toluene and washed with 5% NaHCO ₃ and water. After evaporation of the solvent, the residue was dissolved in 15 mL of CHCl ₃ + 15 mL of TFA + 700 μl of Et ₃ SiH; The mixture was left stirring in the dark for 42 hours. After evaporation of the solvent, the residue was purified by filtration on silica gel. Overall yield: 90%.

The linear peptide was synthesized in the solid phase as described in Example 1 by inserting Fmoc-N-Me-Phe- (4-Pht-N-CH ₂ ) -COOH as the third amino acid, prepared as described above. In this case, deprotection of the N-Fmoc-terminus on the dendrite was carried out with 30% diisopropylamine (30 equiv) in solution in DMF (due to the presence of phthalimide). After cyclization, 500 mg of peptide was dissolved in 10 ml of absolute EtOH at high temperature, and 0.9 ml of a 1M NH ₂ -NH ₂ · H ₂ O solution in ethanol was added thereto. After heating under reflux for 2 hours, the solvent was evaporated and the residue was dissolved in vigorous shaking in a solution of 10 ml DCM + 10 ml Na ₂ CO ₃ . Crude final product was recovered from the organic phase after evaporation and purified by preparative RP-HPLC (mobile phase: 52% CH ₃ CN + 0.1% TFA in water; Rt = 10 min).

Maldi-Tof: 987

Example  4

c [ Arg ( Pmc )- Gly - Asp ( OtBu ) -D- Phe -Amp ( CO -( CH ₂ ) ₂ - COOH )] (Protected ST2649 ) Synthesis

120 mg of cyclopeptide c [Arg (Pmc) -Gly-Asp (OtBu) -D-Phe-Amp] .TFA (prepared as disclosed in Example 1) was added in 3.6 ml of a mixture of DCM-DMF 2: 1. It was dissolved with a theoretical amount of TEA and succinic anhydride. After 1 hour the reaction mixture was diluted with 30 mL of DCM and washed with water. The organic phase was dried and concentrated to give a residue of 100 mg of pure product.

Analytical RP-HPLC: Column: Purosphere STAR®, Merck; Mobile phase: 45% CH ₃ CN + 0.1% TFA in water; Rt = 13.17 min.

Molecular Mass (Maldi-Tof): 1073

Example  5

c ( Arg ( Pmc )- Gly - Asp ( OtBu ) -D- Phe -N- Amb - Gly ) (Protected ST2701 ) Synthesis

1.83 mmol of TEA was added to a 1-22 mmol solution of Boc-ylprotected p-xylylenediamine in 6 mL of THF, and a solution of 1.22 mmol of benzyl bromoacetate in 2 mL of THF was added dropwise. The mixture was left under stirring overnight, after which the solvent was evaporated and the residue was purified on flash column (CHCl ₃ -EtOAc, 9: 1). 0.69 mmol of N- (4-Boc-NH-CH ₂ -benzyl) -glycine benzylester was obtained.

250 mg of Fmoc-D-Phe-OH was dissolved in 27 ml of DCM and 40 µl of diphosgene and 230 µl of sym-collidine were added; After 15 minutes 190 mg of the previously prepared ester was added and dissolved in 3 ml of DCM. After 3 hours, 80 μl of N-Me-piperazine was added to the reaction mixture and stirred for 10 minutes, after which the mixture was diluted with 10 mL of DCM and extracted with water, 0.5N HCl, water, 5% NaHCO ₃ and water. Was performed. After evaporating the solvent, the residue was purified by flash chromatography on silica gel (DCM-EtOAc, 9: 1). Yield 80%.

To 100 mg of the product obtained above, dissolved in 6 ml of MeOH, 76 μl of AcOH and 42 mg of HCOONH ₄ were added, the mixture was cooled to 0 ° C. and 10 mg Pd / C 50 mg. After 30 minutes, the reaction mixture was filtered over celite. The filtrate was dried and purified on flash column (CHCl ₃ -MeOH 9: 1). Yield: 90%.

190 mg of the product thus obtained was dissolved in 1.2 ml of TFA and dried (deprotection of Boc); The residue was re-dissolved in 9 ml of 10% Na ₂ CO ₃ + 6 ml of dioxane cooled to 0 ° C. and a solution of 120 μl of benzyloxycarbonyl chloride diluted with 3 ml of dioxane was added dropwise. After stirring for 1 hour at room temperature, evaporation was performed in vacuo in small volumes, after which the mixture was diluted with water, the pH was reduced to 1 with HCl and extraction with EtOAc. After evaporation of the solvent, the residue was purified by filtration on silica gel, washing with CHCl ₃ -MeOH (8: 2). Pure Dipeptide Yield: 82%.

0.69 mmol of Fmoc-Gly-Res was treated as described in Example 1. After Arg, the previously prepared dipeptide Fmoc-D-Phe-N (4-Cbz-NH-CH ₂ -benzyl) -G1 was added sequentially. After deprotection of Cbz by CTH, crude product c (Arg (Pmc) -Gly-Asp (OtBu) -D-Phe-N-Amp-Gly) was added to preparative RP-HPLC (mobile phase: 50% CH in water). ₃ CN + 0.1% TFA; Rt = 10.5 min).

Maldi-Tof: 973

Example  6

c ( Arg ( Pmc )- Gly - Asp ( OtBu ) -D- Phe -N- Amp ( CO - CH ₂ -( OCH ₂ CH ₂ ) _n -O- CH ₂ - COOH Synthesis of))

Substantially excess in a 200 mg solution of c (Arg (Pmc) -Gly-Asp (OtBu) -D-Phe-Amp) .TFA (obtained as described in Example 1) in 4 ml of a 3: 1 DCM-DMF mixture. Glycol diacid was added. DIEA (3 equiv) and DCC (2 equiv) were added to the same solution. The mixture was left overnight under stirring, after which it was diluted with DCM and washed with water.

The crude product is recovered by evaporation of the organic phase and purified by flash chromatography (mobile phase: CHCl ₃ -MeOH 7: 3 + 1% AcOH); Fractions containing product were collected, washed with water, dehydrated and dried to give a residue of 157 mg of pure product.

Analytical RP-HPLC: (Column: Purosphere STAR®, Merck; Mobile Phase: 50% CH ₃ CN + 0.1% TFA in Water; Rt = 10.96 min)

Molecular mass (Maldi-Tof): corresponds to different glycols of various molecular weights used.

Jimatekan  Synthesis of Derivatives

Example  7

20-O- Val - Jimatekan  - ST2678 Synthesis of

1 mmol of ST1481, 0.6 mmol of Dy (OTf) ₃ , ₃ mmol of dimethylaminopyridine and 3 mmol of Boc-Val-OH prepared as disclosed in Example 2 of patent EP 1 044 977 are suspended in 15 ml of anhydrous CH ₂ Cl _2. To -10 ° C; After 30 minutes 3.1 mmol of DCC was added and after an additional 30 minutes at −10 ° C. the reaction mixture was heated to room temperature. After 2 hours the reaction was further diluted with 20 mL of CH ₂ Cl ₂ , washed with 1N HCl, NaHCO ₃ and dried over Na ₂ SO ₄ . The crude product was purified by chromatography on SiO ₂ with CH ₂ Cl ₂ / MeOH 97: 3 to give the product in 92% yield as a yellow solid. Rf = 0.72 in CH ₂ Cl ₂ / MeOH 96: 4

Analytical RP-HPLC: (Column: Luna C18, Phenomenex®; Mobile Phase: 45% CH ₃ CN in Water; Rt = 23.0)

Molecular Mass (ESI): 646

The intermediate product ST2678 [N-Boc] is deprotected in DCM / TFA (75/25) in quantitative yield at 0 ° C. The ST2678 obtained as above can be used to directly bind the RGD derivative or as an additional intermediate, which can be used to bind a second residue (see Examples 8-9).

Example  8

20-O- Val - Asp - Jimatekan  - ST2676 [N- Boc Synthesis

1 mmol of ST2678 and 3.7 mmol of DIPEA were added in this order to a solution of 1.2 mmol, suitably protected aspartic acid, 1.8 mmol HOBt and 1.4 mmol EDC at 0 ° C. at 0 ° C. The reaction mixture was partitioned between water and dichloromethane after standing overnight at room temperature and the crude product thus obtained was purified by chromatography on SiO ₂ with CH ₂ Cl ₂ / MeOH 96: 4 to yield 66% as a yellow solid. To give the product. Rf = 0.5 in CH ₂ Cl ₂ / MeOH 96: 4

Analytical RP-HPLC: (Column: Luna C18, Phenomenex®; Mobile Phase: 45% CH ₃ CN in Water; Rt = 23.3)

Molecular Mass (ESI): 851

Carboxyl  Group Deprotection

Benzylester was hydrolyzed at 20 psi with H ₂ /10% Pd-C and purified with CH ₂ Cl ₂ / MeOH 94: 6 to yield a yield of 70%. Rf = 0.52 in CH ₂ Cl ₂ / MeOH 92: 8

Analytical RP-HPLC: (Column: Luna C18, Phenomenex®; Mobile Phase: 45% CH ₃ CN in Water; Rt = 22.9)

Molecular Mass (ESI): 761

Example  9

compound[ ST2677 Synthesis

After 1 mmol of deprotected ST2678- [N-Boc] was dissolved in 10 mL of anhydrous pyridine and the solution was brought to 0 ° C., 2.5 mmol of succinic anhydride was added: The mixture was restored to room temperature for 1 hour. Solvent was removed, the residue was dissolved in CH ₂ Cl ₂ and the organic phase was washed with 0.5N HCl. The crude product was purified by chromatography on SiO ₂ with CH ₂ Cl ₂ / MeOH 95: 5 to give the expected product in 90% yield as a yellow solid. Rf = 0.41 in CH ₂ Cl ₂ / MeOH 92: 8.

Analytical RP-HPLC: (Column: Luna C18, Phenomenex®; Mobile Phase: 45% CH ₃ CN in Water; Rt = 17.0)

Molecular Mass (ESI): 646

Conjugate  Synthesis of Derivatives

Example  10

compound ST2670 (or ST2671 ) Synthesis

The same synthetic process was used for both of these.

To a 1.2 mmol solution of ST2676 [N-Boc] (or ST2677) in anhydrous DMF cooled to 0 ° C., 2.1 mmol of HOBt and 1.4 mmol of EDC were added and the resulting mixture was stirred for 30 minutes, followed by 1 mmol of ST2581 and DIPEA. In order. After standing overnight the mixture was partitioned between water and dichloromethane, then the organic phase was dried over Na ₂ S0 ₄ and the crude product was purified by chromatography over SiO ₂ with CH ₂ Cl ₂ / MeOH 92: 8. The product protected ST2670 (or protected ST2671) was provided in a yield ranging from 55% to 65%.

Analytical RP-HPLC: (Column: Luna C18, Phenomenex®; Mobile Phase: 45% CH ₃ CN in Water; Rt = 20.1 for Protected ST2670, and Rt = 23.2 for Protected ST2671)

Molecular Mass (ESI): 1718 for protected ST2671

Molecular Mass (ESI): 1601 for protected ST2670

Conjugate  Product Deprotection

Final deprotection to obtain two compounds ST2670 and ST2671 was performed for 2 hours using CH ₂ Cl ₂ / TFA 1: 1 for both compounds, making the mixture at 0 ° C. to room temperature; This process was followed by an ion exchange resin step to provide the product as hydrochloride.

Analytical RP-HPLC: (Column: Luna C18, Phenomenex®; Mobile Phase: 35% CH ₃ CN in Water; Rt = 14.5 for ST2670, and Rt = 14.3 for ST2671)

Molecular Mass (ESI): 1294 for ST2671

Molecular Mass (ESI): 1279 for ST2670

Biological results

Integreen  α _v ß ₃  Binding to receptors

Purified α _v ß ₃ receptor (Chemicon, cat.CC1020) at a concentration of 0.5 μg / ml in buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM MnCl ₂ ) Diluted in. 100 μl aliquots were added to 96-well plates and incubated at + 4 ° C. overnight. Plates were washed once with buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM MnCl ₂ , 1% bovine serum albumin) and then incubated for an additional 2 hours at room temperature. It was. Plates were washed twice with the same buffer and incubated with 0.05 nM of radioligand [ ¹²⁵ I] equistatin (Amersham Pharmacia Biotech) in the presence of a competitive ligand for 3 hours at room temperature. At the end of the incubation, the wells were washed and radioactivity was measured using a gamma counter (Packard). Non-specific binding of the ligand was measured in the presence of excess low temperature echistatin (1 μM).

Integreen  α _v ß ₅  Binding to receptors

Purified α _v ß ₅ receptor (Chemicon, cat.CC1020) at a concentration of 1 μg / ml buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM MnCl ₂ ) Diluted in. 100 μl aliquots were added to 96-well plates and incubated at + 4 ° C. overnight. Plates were washed once with buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM MnCl ₂ , 1% bovine serum albumin) and then incubated for an additional 2 hours at room temperature. It was. Plates were washed twice with the same buffer and incubated with 0.15 nM of radioligand [ ¹²⁵ I] equistatin (Amersham Pharmacia Biotech) in the presence of competing ligands for 3 hours at room temperature. At the end of the incubation, the wells were washed and radioactivity was measured using a gamma counter (Packard). Non-specific ligand binding was measured in the presence of excess low temperature echistatin (1 μM).

IC ₅₀  Evaluation of parameters

The affinity of the product for the vitronectin receptor is expressed as an IC ₅₀ value ± SD, ie as a concentration capable of inhibiting 50% of specific radioligand-receptor binding. The IC ₅₀ parameters were calculated using "ALLFIT" software.

result

The tables below provide the results of affinity for camptothecin-RGD conjugates and RGD peptides for the Vitronectin α _v ß ₃ and α _v ß ₅ receptors. The conjugate showed strong affinity for both of the integrin receptors comparable to the affinity observed for RGD peptides.

TABLE 1

Vitronectin  α _v ß ₃  And α _v ß ₅  For receptors Camptothecin - RGD Conjugate  Affinity

TABLE 2

Vitronectin  α _v ß ₃  And α _v ß ₅  For receptors RGD  Peptide Affinity

Above for different tumor cell lines Of conjugates  Cytotoxicity

To assess the effect of the compound on living cells, the sulforhodamine B test was used. To test the effect of these compounds on cell growth, PC3 human prostate carcinoma, A498 human kidney carcinoma, A2780 human ovarian carcinoma cells and NCI-H460 non-small cell lung carcinoma were used. A2780, NCI-H460 and PC3 tumor cells were grown in RPMI 1640 containing 10% fetal bovine serum (GIBCO), while A498 tumor cells were grown in EMEM containing 10% fetal bovine serum (GIBCO).

Tumor cells were seeded and adhered to 96-well tissue culture plates (Corning) at approximately 10% confluence and recovered for at least 24 hours. Various concentrations of drug were then added to each well to calculate their IC50 values (concentrations that inhibit 50% of cell survival). The plates were recovered 72 hours at 37 ° C. or after 72 hours of incubation. At the end of the treatment, the plates were washed by removing the supernatant and adding 3 times of PBS. 200 μl of PBS and 50 μl of cold 80% TCA were added. The plates were incubated for at least 1 hour on ice. TCA was removed and the plate was immersed three times in distilled water and dried on paper at 40 ° C. for 5 minutes.

200 μl of 0.4% sulforhodamine B in 1% acetic acid was added. The plates were incubated for an additional 30 minutes at room temperature. The sulforhodamine B was removed and the plate was immersed three times in 1% acetic acid and then dried on paper at 40 ° C. for 5 minutes.

Then 200 μl of 10 mM Tris was added and the plate was kept for 20 minutes under stirring. Viable cells were measured as absorbance at 540 nm by a multiscan spectrophotometer. The amount of dead cells was calculated as the rate of reduction of sulforhodamine B binding to the control culture.

IC ₅₀ values were calculated using the "ALLFIT" program.

Conjugate ST2670 showed the most potent cytotoxic activity with an IC50 value of 0.4 μM against A2780 ovarian tumor cells. Moreover, the conjugate ST2670 exhibited cytotoxicity against tumor cells comparable to that observed for ST2677 (free camptothecin). The conjugate ST2671 also showed potent cytotoxicity against PC3 tumor cells comparable to the cytotoxicity found for free camptothecin ST2676.

TABLE 3

PC3 For A498 and A2780 tumor cells Conjugate ST2670  And ST2671 , And free camptothecin ( ST2676  And ST2677 ) Cytotoxicity (72 hours treatment)

(n.d. = not measured)

TABLE 4

H460  Non-small cell Lung carcinoma  Glass for cells Camptothecin  20-O derivative ( ST2676 , ST2677 , ST2678) cytotoxicity (2 hours treatment)

Claims (13)

Compounds of formula I, N ₁ -oxides, racemic mixtures thereof, single enantiomers, single diastereoisomers, forms E and Z, mixtures thereof, pharmaceutically acceptable salts: Formula I

Where R ₁ is a UXY group, where: U is absent or is one of the following groups -COCHR ₁₀ NH- and CON [(CH ₂ ) _n2 NHR ₇ ] -CH _2- , wherein R ₁₀ is H, or optionally C ₆ -C ₁₄ aryl Substituted linear or branched C ₁ -C ₄ alkyl and amino-alkyl C ₁ -C ₄ ; R ₇ is H or linear or branched C ₁ -C ₄ alkyl; n ₂ is an integer from 2 to 6; X is either not present or H or a _{-COCHR 3 NH-, -COCHR 6 (CH} 2) n3 R 4 -, -R 4 -CH 2 (OCH 2 CH 2) n4 OCH 2 R 4 -, -R 4 (Q) R _4- , -R ₅ [Arg-NH (CH ₂ ) _n5 CO] _n6 R _5- , -R _5- [N-guanidinopropyl-Gly] _n6 R _5- n ₃ is an integer from 0 to 5, n ₄ is an integer from 0 to 50, n ₅ is an integer from 2 to 6, n ₆ is an integer from 2 to 7; R ₃ is H or linear or branched C ₁ -C ₄ alkyl optionally substituted by —COOH, —CONH ₂ , —NH ₂ or —OH; R ₄ is selected from the group consisting of -NH-, -CO-, -CONH-, -NHCO-; R ₅ is absent or is a group -R ₄ (Q) R _4- ; R ₆ is H or NH ₂ ; Q is linear or branched C ₁ -C ₆ alkylene; Linear or branched C ₃ -C ₁₀ cycloalkylene; Linear or branched C ₂ -C ₆ alkenylene; Linear or branched C ₃ -C ₁₀ cyclo-alkenylene; C ₆ -C ₁₄ arylene; Arylene (C ₆ -C ₁₄ ) -alkylene; (C ₁ -C ₆ ), alkylene (C ₁ -C ₆ ) -arylene (C ₆ -C ₁₄ ); An aromatic or non-aromatic heterocyclyl (C ₃ -C ₁₄ ) containing one or more heteroatoms selected from the group consisting of O, N, S; Y is absent or H, or is the group c (Arg-Gly-Asp-AA ₁ -AA ₂ ), wherein: c means cyclic; AA ₁ is (D) -Phe, (D) -Trp, (D) -Tyr, (D) -2-naphthylAla, (D) -4-terbutyl-Phe, (D) -4,4 ′ -Biphenyl-Ala, (D) -4-CF ₃ -Phe, (D) -4-acetylamino-Phe; AA ₂ is NW-CH [(CH ₂ ) _n7 -CO] -CO, NW-CH [(CH ₂ ) _n7 -NH] -CO, NW- [4- (CH ₂ ) _n7 -CO] -Phe, NW -[4- (CH ₂ ) _{n 7} -NH] -Phe, [NW] -Gly, NW-Val, wherein W is H, linear or branched C ₁ -C ₆ alkyl,-(CH ₂ ) _n7 -COOH where n ₇ is an integer from 0 to 5, 4-carboxybenzyl, 4-aminomethylbenzyl; only It is not possible for both X and Y to be present. The compound of claim 1, wherein U and X are present. The compound according to claim 1, wherein the compound has the formula: N ₁ -oxide, racemic mixture thereof, single enantiomer, single diastereoisomer, form E and Z, mixtures thereof, pharmaceutically acceptable salts:

The compound according to claim 1, wherein the compound has the formula: N ₁ -oxide, racemic mixture thereof, single enantiomer, single diastereoisomer, form E and Z, mixtures thereof, pharmaceutically acceptable salts:

Process for preparing a compound according to any one of claims 1 to 4, which is carried out according to one of the following schemes: 7-t-but-CP + U ₁ -X ₁ -Y ₁ or 7-t-but-CP-U ₁ + X ₁ -Y ₁ or 7-t-but-CP-U ₁ -X ₁ + Y ₁ or 7-t-but-CP + U ₁ + X ₁ + Y ₁ or 7-t-but-CP-U ₁ + X ₁ + Y ₁ In the above, 7-t-but-CP represents 7-t-butoxyiminomethylcamptothecin, U ₁ , X ₁ and Y ₁ each represent groups U, X and Y as defined in formula (I) and are finally suitably functionalized and / or protected. A pharmaceutical composition comprising, as an active ingredient, at least one compound according to any one of claims 1 to 4 in a mixture with one or more pharmaceutically acceptable excipients and / or vehicles. Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament. Use of a compound according to any one of claims 1 to 4 for the preparation of a medicament endowed with isomerase 1 inhibitory activity. Use according to claim 8 for the manufacture of a medicament having anticancer activity. 10. The method of claim 9, wherein the agent is non-microcytoma and small cell lung cancer, or colorectal or prostate cancer, glioblastoma and neuroblastoma, cervical cancer, ovarian cancer, gastrointestinal carcinoma, liver carcinoma, Kaposi's sarcoma, renal carcinoma, sarcoma and Useful for the treatment of osteosarcoma, testicular carcinoma, breast carcinoma, pancreatic carcinoma, melanoma, urinary bladder and head and neck carcinoma. Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament useful for the prophylaxis or treatment of metastatic forms. Use according to claim 8 for the manufacture of a medicament with parasite inhibitory activity. Use according to claim 8 for the manufacture of a medicament having a viral inhibitory activity.