Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
• • 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
  • C07K5/0815—Tripeptides with the first amino acid being basic
  • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg

Definitions

• anticancer therapy is mainly based on the use of cytotoxic drugs displaying low therapeutic index.
• cytotoxic drugs displaying low therapeutic index.
• taxol Proclitaxel, Taxol ®
• Taxol ® Taxol
• Taxol ® is able to inhibit tumour proliferation through a mechanism interfering with the dynamics of cell microtubules and their irreversible stabilization, and today it is recognized as one of the most efficient cytotoxic agents for the treatment of different types of cancer, among which mammalian carcinoma, Kaposi's sarcoma and, in association with cisplatin, advanced ovarian carcinoma and non-small-cell lung carcinoma.
• tumour cells also other quickly dividing cells, such as white blood cells and piliferous cells, are attacked by the action of most cytotoxic drugs and consequently their administration is associated with a series of serious side effects, such as immune system suppression (neutropenia), a remarkable reduction of the sensory nervous function (peripheral neuropathy), hair loss (alopecia), as well as phenomena of blood and cardiac toxicity.
• a recent approach to anticancer therapy involves targeting cytotoxic drugs at receptors involved in tumour angiogenesis.
• AlfaV integrin receptors are overexpressed on activated endothelial cells and tumour cells, but not on resting endothelial cells and most healthy organs, and thus they represent potential targets for targeted cytotoxic interventions.
• An object of the present invention is to provide anticancer compounds that are efficient in vitro and in vivo.
• an object of the present invention is to provide anticancer compounds displaying high affinity for alfaV integrin receptors overexpressed in some types of solid neoplasias.
• an object of the invention is to provide anticancer compounds displaying high selectivity for tumour cells.
• Another object of the invention is to provide new compounds selective for tumour cells, having a sufficiently long half-life to exert their anticancer action and reduced systemic toxicity.
• a further object of the invention is to provide new compounds having the abovementioned features, that are obtainable by simple synthetic procedures and with good yields.
• a further object of the invention is to provide a method for the treatment of tumours, in particular of solid tumours, including the use of the compounds of the invention.
• the invention has as an object a compound of formula (I).
• R 2 are independently selected from hydrogen, CrC 6 -alkyl, Ci-C 6 -alkoxy, benzyl and a group of formula (II)
• - L and Z are independently selected from the following groups:
• n and m are, each independently, an integer from 0 to 10, p is an integer from 1 to 10 and Ar is an aromatic or heteroaromatic group;
• - X is a roup selected from the following
• - Y is a group selected from the following
• - Ct is a cytotoxic agent
• Ri and R 2 is a group of formula (II);
• Ri and R2 are both a sterically hindered group, for example one is a group of formula (II) and the other one is a benzyl group, compounds of formula (I) can exist as a mixture of two divisible different conformers due to the hindered rotation of one ring about the other.
• alkyl and alkoxy groups are meant to be linear or branched, saturated or unsaturated.
• aromatic or heteroaromatic group includes 1 ,2- phenylene, 1 ,3-phenylene, naphtylene, phenantrylene, anthracylene, indenylene, furylene, pyranylene, pyrrolylene, imidazolylene, pyridylene, pyrazylene, pyrimidinylene, indolylene, quinolynene, etc.
• Variables m, n and p are preferably integers comprised between 1 and 5, for example 1 , 2 or 3; advantageously 1 or 2.
• cytotoxic agent herein is meant to represent a compound having cytotoxic activity, suitable to be administered to humans and animals.
• the cytotoxic agent "Ct” is selected from taxol, daunorubicin, doxorubicin, carminomycin, 4-epiadriamycin, 4- demethoxy-daunomycin, 11-deoxy-daunorubicin, 13-deoxy-daunorubicin, adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14- naphtaleneacetate, vinblastine, vincristine, mitomycin C, N-methyl-mitomycin C, bleomycin A2, dideaza-tetrahydrofolic acid, aminopteridine, methotrexate, camptothecin, colchicine and cisplatin.
• the cytotoxic agent "Ct” is taxol.
• taxol is linked to the Y group through the hydroxyl group at position 2'.
• Ri and R 2 is a group of formula (II), even more preferably one of Ri and R 2 is a group of formula (II) and the other one is selected from hydrogen and a benzyl group, more particularly it is selected to be hydrogen.
• the L group is the
• the X group is the
• the Z roup is the
• the Y group is the
• a peptidomimetic cyclic unit RGD consisting of one suitably functionalized diketopiperazine scaffold and of the amino acid sequence Arg-Gly-Asp, and displaying high affinity for alfaV integrin receptors,
• alkyl-chained dicarboxylic acid for example succinic acid, or other acids able to improve the pharmacological properties of the final molecule relating to stability and/or solubility
• cytotoxic agent that is a molecule having cytotoxic activity, then able to inhibit and fight the development of tumours, for example taxol.
• the cyclic peptidomimetic scaffold RGD comprising a diketopiperazine
• RGD can be prepared essentially modifying what described in the abovementioned paper by A. Ressurreigao, et a!., where two unfunctionalized cyclic peptidomimetic compounds RGD were synthesized through a synthetic strategy displaying significant differences.
• Specific examples of the new synthetic pathways are provided in the experimental section of the present description, where the preparation of some representative "-L-X-Z-Y-" structures linking the cytotoxic agent to said scaffold is also described.
• the observed datum is of double interest, both because the activity of the conjugates according to the present invention is higher than that of taxol used alone as a reference compound, and also, and mainly, because said found higher activity is observed, in the case of compounds of formula (I) of the invention, with an amount of taxol halved compared with the amount of taxol used as a control.
• this aspect represents a great advantage, in that the possibility of obtaining interesting results of anticancer activity with half the dose compared with what it would be obtained with the control compound, allows to provide a class of products according to the invention, able to dramatically reduce side effects connected to the dosages of known compounds, required to reach the desired anticancer activity. This result is markedly unexpected and demonstrates that compounds of formula (I) represent a remarkable technical progress in the field of anticancer therapy.
• compounds of formula (I) have shown to be particularly efficient in vitro and in vivo, and some representative compounds containing taxol have shown even higher anticancer activity, at a clearly lower dosage, than that exerted by taxol when administered alone, and i.e. not conjugated to form the compounds of formula (I).
• compounds of formula (I) are structurally characterized by the presence of a cytotoxic agent with anticancer pharmacological action and one cyclic peptidomimetic scaffold RGD comprising the diketopiperazine, and are generally known with the term "conjugates", according to the present invention.
• the suitably functionalized scaffold described above in association with the amino acid sequence Arg-Gly-Asp, acts as a vehicle for the cytotoxic agent and is able to recognize integrin receptors overexpressed in some types of solid neoplasias.
• the resulting conjugate is surprisingly able to selectively deliver the cytotoxic agent to tumour cells, reducing its systemic toxicity and improving its efficacy.
• Binding data referred to some cyclic peptidomimetic RGD scaffolds (not conjugated) alfa v beta3 alfa v beta5
• the compound can exist as a mixture of two divisible different conformers (diastereomers) due to the hindered rotation of one ring about the other (the benzylic group of the DKP cannot pass through the macrolactamic ring).
• the benzylic group of the DKP cannot pass through the macrolactamic ring.
• Cyclo (RGDfV) is a commercially available reference compound (H-2574 Bachem AG, Switzerland).
• ST1646 is a reference compound, synthesized as described in L. Belvisi et al. (Bioorg. Med. Chem. 2006, 14, 169-180).
• IGROV- and IGROV-1/Pt1 cell lines were obtained as disclosed in Perego P., Romanelli S., Carenini N., Magnani I., Leone R., Bonetti A., Paolicchi A., Zunino F. Ovarian cancer cisplatin-resistant cell lines: Multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9:1-8, 1998 and in Perego P., Giarola M., Righetti, S. C, Supino R., Caserini C, Delia D., Pierotti M. A., Miyashita T., Reed J. C, Zunino F.
• an analysis of the anticancer activity of the conjugate Cyclo [DKP-/3-RGD]-PTX (administration every 4 days, 4 times) can be performed, showing its ability to significantly inhibit growth of the IGROV- 1/Pt1 tumour. Inhibition is dose-dependent as demonstrated by comparing the effect obtained administering the conjugate Cyclo [DKP- 3-RGD]-PTX at the doses of 30 mg/kg and 15 mg/kg.
• Conjugates of formula (I) of the invention display the further advantage of being obtainable by synthetic chemical reactions able to give high yields of the final product and display the further advantage of being able to be differently and readily functionalized on the diketopiperazine scaffold. Additionally, they display the advantage of being stable in saline, retaining purity higher than 99.5% after 4 days.
• the invention has as an object the use of compounds of formula (I) in therapy, in particular for the treatment of tumours in humans and animals, in particular in mammals, for example of solid tumours, such as mammalian carcinoma, ovarian carcinoma, Kaposi's sarcoma and non-small-cell lung carcinoma, whether metastatic or not, if needed in association with other active ingredients, for example other cytotoxic agents such as, by means of illustration, cisplatin.
• solid tumours such as mammalian carcinoma, ovarian carcinoma, Kaposi's sarcoma and non-small-cell lung carcinoma, whether metastatic or not, if needed in association with other active ingredients, for example other cytotoxic agents such as, by means of illustration, cisplatin.
• compounds of formula (I) can be administered in the form of single unit doses.
• Said unit doses are preferably formulated in pharmaceutical compositions wherein the compound of formula (I) is admixed with one or more pharmaceutical excipients.
• compositions comprising, as an active compound, a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or hydrate thereof, optionally in association with further active ingredients, for example one or more cytotoxic agents.
• compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, transdermal, local or rectal administration active ingredients can be administered in the form of unit doses, in admixture with classical pharmaceutical supports, to animals or humans.
• Suitable single administration forms comprise forms for administration via oral route such as tablets, capsules, powders, granules and oral solutions or suspensions, forms for sublingual and buccal administration, aerosols, forms for topical administration, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.
• compositions of the present invention can be prepared according to methods well known to one of ordinary skill in the art.
• Combinations of more active ingredients comprising compounds of formula (I) as well as kits comprising such combinations represent a further object of the present invention.
• the invention has as an object a method for the treatment of tumours including administering to a human or animal an efficient amount of a compound of formula (I) as described herein or a pharmaceutically acceptable salt, solvate and/or hydrate thereof, optionally in association with further active ingredients, for example other cytotoxic agents.
• Figure 1 shows the effects of the conjugate Cyclo [DKP- 3-RGD]-PTX administered intravenously to nude mice subcutaneously grafted with the ovarian carcinoma IGROV-1/Pt1 using taxol as a reference drug.
• Figure 2 shows solubility and stability of Cyclo [DKP-/3-RGDJ-PTX assessed by HPLC in time at +25°C.
• the L group is the L
• the Z group is the
• PXT is taxol and Bn is a benzyl group.
• DKP 2 and DKP 3 4-((4- ethoxy-2,3,6-trirnethyl-phenyl-sulfonyl)arninomethyI)benzyl alcohol
• the mixture was then diluted with AcOEt (250 ml) and washed with 1 M KHSO 4 (2 x 50 ml), aqueous NaHC0 3 solution (2 x 50 ml) and brine (2 x 50 ml); it was then dried over Na 2 S0 4 and the solvent was removed on the rotary evaporator, thus obtaining the raw product.
• the latter was purified by flash chromatography on silica gel (7:3 to 5:5 Hex/AcOEt) thus obtaining the desired product in the form of a transparent oil (3.2 g, 82% yield).
• butoxy-carbonylamino)succinate (3.0 g, 4.3 mmol, 1 eq) was deprotected according to general procedure A.
• the corresponding trifluoroacetate salt was dissolved in AcOEt (200 ml) and then an aqueous NaHC0 3 solution (100 ml) was added.
• the mixture was extracted with AcOEt (2 x 100 ml).
• the organic phases were combined and the solvent was removed on the rotary evaporator thus obtaining a transparent and viscous oil.
• Such compound was dissolved in / ' -PrOH (250 ml) and the mixture was allowed to stir for 18 h at room temperature.
• the toluene solution of HN 3 was titrated diluting 1 ml of the solution of HN 3 in 50 ml of distilled water, adding few drops of phenolphtalein as an indicator and using a 0.1 M NaOH solution as the titrant.
• H-Asp(OtBu)-DKP/3-Arg(Mtr)-Gly-OH 580 mg, 0.52 mmol, 1 eq
• DMF 440 mi
• HATU 900 mg, 2.36 mmol, 4.5 eq
• HOAt 323 mg, 2.36 mmol, 4.5 eq
• DIPEA 0.630 ml, 3.70 mmol, 7 eq
• the flask was placed under an atmosphere of hydrogen and allowed to stir, at room temperature, for 4 h. Then the mixture was filtered on a pad of Celite, washing it carefully with THF. The filtrate was concentrated on the rotary evaporator, thus obtaining the desired product in the form of a white spongy solid (280 mg, 99% yield), that was used without further purifications.
• H-DKP 4-OMe or H-DKP 6-OMe 280 mg, 0.52 mmol, 1 eq
• DIPEA 0.17 ml, 1.0 mmol, 2 eq
• Boc 2 0 132 mg, 0.6 mmol, 1.2 eq
• the reaction was kept under stirring for 6 h at room temperature, then the solvent was removed on the rotary evaporator and the residue was purified by flash chromatography on a small pad of silica gel (100% EtOAc), thus obtaining the desired product in the form of a white spongy solid (320 mg, 96% yield).
• Boc-DKP 4-OMe or Boc-DKP B-OMe (320 mg, 0.5 mmol, 1 eq) was dissolved in THF (20 ml) and a solution of ⁇ _ ⁇ 2 0 (52 mg, 1.24 mmol, 2.5 eq) in H 2 0 (10 ml) was slowly added at 0°C. The resulting solution was allowed to react for 1 h at 0°C; then the mixture was acidified, at 0°C, to pH 1-2 with 1 M KHSO 4 . The solution was extracted with CH 2 CI 2 (4x), the organic phases were combined and dried over Na 2 S0 4 . The solvent was removed on the rotary evaporator, thus obtaining the desired product in the form of a white spongy solid (310 mg, 100%) that was used without further purifications.
• the compound cyclo [Asp(OtBu)-DKP/4-Arg(Mtr)-Gly] (90 mg, 0.081 mmol) was dissolved in TFA (10 ml) in the presence of ion scavengers: thioanisole (1.5 ml), ethanedithiol (0.75 ml), phenol (150 mg). The mixture was cooled to 0°C and placed under a flow of nitrogen. Then, trimethyl silyl bromide (2 ml) was added. The reaction was allowed to stir until it reached room temperature and subsequently allowed to stir for 2 h.
• H-Asp(OtBu)-DKP 6-Arg(Mtr)-Gly-OH 260 mg, 0.23 mmol, 1 eq
• DMF 165 ml
• HATU 350 mg, 0.92 mmol, 4 eq
• HOAt 125 mg, 0.92 mmol, 4 eq
• DIPEA 0.236 ml, 1.38 mmol, 6 eq
• the compound cyclo [Asp(OtBu)-DKP/4-Arg(Mtr)-Gly] (90 mg, 0.081 mmol) was dissolved in TFA (10 ml) in the presence of ion scavengers: thioanisole (1.5 ml), ethanedithiol (0.75 ml), phenol (150 mg). The mixture was cooled to 0°C and placed under a flow of nitrogen. Then, trimethyl silyl bromide (2 ml) was added. The reaction was allowed to stir until it reached room temperature and subsequently allowed to stir for 2 h.
• the TFA was then removed on the rotary evaporator, and the raw product was dissolved in a 1 :1 mixture of water and diisopropyl ether (50 ml). Then, the aqueous phase was washed several times with diisopropyl ether and subsequently concentrated on the rotary evaporator, thus obtaining the raw product, which was eventually purified by semi-preparative HPLC (Water's Atlantis column 21 mm x 10 cm, gradient: 100% H 2 0 + 0.1 % TFA to 70% H 2 0 + 0.1 % TFA/30% acetonitrile), thus obtaining the desired product (as the trifluoroacetate salt) in the form of a white solid (50 mg, 71 % yield).
• the raw product was purified by semi-preparative HPLC (Xbridge column 19 mm x 10 cm, gradient: 90% H 2 0 + 0.1 % formic acid/10% acetonitrile to 30% H 2 0 + 0.1 % formic acid/70% acetonitrile), thus obtaining the desired product (after freeze-drying) in the form of a white solid (60% yield).
• Binding assays on isolated receptors purified ⁇ ⁇ ⁇ 3 and ⁇ ⁇ ⁇ ⁇ receptors (Chemicon International, Inc., Temecula, California, USA) were diluted to the concentration of 0.5 pg/ml in a coating buffer containing 20mM Tris-HCI (pH 7.4), 150mM NaCI, 1mM MnCI 2 , 2mM CaCI 2 and 1mM MgCI 2 . An aliquot of diluted receptors (100 ⁇ /well) was added in microtitration 96-well plates (NUNC MW 96F MEDISORP STRAIGHT) and incubated overnight at 4°C.
• the plates were then incubated with a blocking solution (solution of coating buffer added with 1% bovine serum albumine) for another 2 h at room temperature, so that non-specific binding was blocked, followed by 3 h of incubation at room temperature with different concentrations (10 "12 - 10 "5 M) of test compounds, in the presence of 1 pg/ml of biotinylated vitronectin.
• Biotinilation was carried out using the EZ-Link Sulfo-NHS-Biotynilation kit (Pierce, Rockford, Illinois).
• Cell sensitivity to drugs was assayed using the growth inhibition assay based on cell count. Briefly, cells were seeded in duplicate in 12-well plates (Corning Costar, Euroclone) and exposed to drugs after 24 h. Taxol and test drugs were dissolved in dimethylsulfoxide and then added to the culture medium at different concentrations starting from 200x solutions. In some wells, vehicle alone was added, which was not toxic at that concentration (0.005%). After 72 h of incubation with the drugs, cells were harvested and counted with an automated counter. IC50 is defined as the concentration of drug that reduces cell growth by 50%.
• Integrin expression was assayed by indirect immunofluorescence employing a cytofluorometer, after suitable optimization of antibody concentration. Exponentially growing cells were harvested and incubated 30 min at 4°C with antibodies targeted at human integrins ⁇ ⁇ ⁇ 3 , ⁇ (Millipore, Temecula, California), ⁇ ⁇ ⁇ ⁇ (Chemicon International) or with isotype controls. Cells were washed in buffered saline and samples were immediately analysed by cytofluorometry (FACScan, Becton-Dickinson). Integrin expression was expressed as the ratio between mean fluorescence intensity obtained in isolated cells incubated with anti-integrin antibodies, divided by that of cells incubated with isotype controls.
• the tumour line was maintained in vivo by serial steps in animals.
• tumour fragments were implanted subcutaneously into both sides of the animals.
• Each experimental group consisted of 4 animals.
• Three days after grafting, when the tumour mass was not measurable yet, animals were randomized for treatment, the administration of compounds intravenously (i.v.) every 4 days, 4 times.
• Ovarian carcinoma cell lines (IGROV-1 , IGROV-1/Pt1 and SKOV3) displayed high levels of ⁇ 5 ⁇ Evaluation of cell sensitivity to conjugates, assayed by the inhibition of proliferation test, indicated that conjugates displayed high cytotoxic potency, comparable to that of taxol (Table 2). The inhibition of observed proliferation was not to be attributed to the ligand (Table 2) that did not inhibit proliferation.
• TVI Tumour Volume Inhibition
• the amount of taxol administered to the animals treated with 30 mg/kg conjugate is equal to 16.35 mg of taxol, the treatment with the conjugate displays an evident therapeutic advantage.
• higher activity in comparison with taxol was observed with about half (54.5%) the active compound.
• the therapeutic advantage is also supported by the lack of regrowth of two tumours out of eight at the end of the experiment.
• MIA-PaCa2 1.2 ⁇ 0.1 5.6 ⁇ 0.9 4.8 ⁇ 0.2
• Perego P. Romanelli S., Carenini N., Magnani I., Leone R., Bonetti A., Paolicchi A., Zunino F. Ovarian cancer cisplatin-resistant cell lines: Multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9:1-8, 1998.
• Perego P. Giarola M., Righetti, S. C, Supino R., Caserini C, Delia D., Pierotti M. A., Miyashita T., Reed J. C, Zunino F. Association between cispfatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems.. Cancer Res., 56, 556-562, 1996.

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