Complexes Of CU(I) As Antitumor Agents
20240239814 ยท 2024-07-18
Inventors
- Valentina Gandin (Tarzo, IT)
- Cristina Marzano (Padova, IT)
- Marina Porchia (Padova, IT)
- Francesco Tisato (Padova, IT)
Cpc classification
C07F9/6536
CHEMISTRY; METALLURGY
C07F9/5045
CHEMISTRY; METALLURGY
C07F9/650952
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a Cu(1) complex of Formula (I), wherein L is a ligand of Formula (II), wherein n1, n2, n3 are independently to each other, an integer from 0 to 1, A1, A2 and A3 are independently from each other,a phenyl optionally substituted with (C.sub.1-C.sub.3)alkoxy, (C.sub.1-C.sub.3)alkyl, F, formyl, carboxyl, sulphonyl hydroxyl, hydroxyl, methoxy(C.sub.1-C.sub.3)alkoxy oran heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2 residue, with the proviso that when A1, A2 or A3 is optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1, for use in the treatment of tumours.
Claims
1. A method for the treatment of tumours comprising the step of administering to a subject in need thereof a Cu(I) complex of Formula (I) ##STR00061## Wherein L is a ligand of Formula (II) ##STR00062## wherein X is a monovalent anion n1, n2, n3 are independently to each other, an integer from 0 to 1 A1, A2 and A3 are independently from each other, a phenyl optionally substituted with (C.sub.1-C.sub.3)alkoxy, (C.sub.1-C.sub.3)alkyl, F, formyl, carboxyl, sulphonyl hydroxyl, hydroxyl, methoxy(C.sub.1-C.sub.3)alkoxy or an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2 residue, with the proviso that when A1, A2 or A3 is an optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1.
2. The method according to claim 1, wherein A1, A2, A3 are, independently from each other, phenyl optionally substituted with a substituent selected from methyl, methoxy, F, formyl, sulphonyl hydroxyl, hydroxyl, methoxymethoxy and carboxyl, wherein at least one of A1, A2 and A3 is phenyl substituted with a substituent selected from methyl, methoxy, F, formyl, sulphonyl hydroxyl, hydroxyl, methoxymethoxy and carboxyl.
3. The method according to claim 2, wherein at least one of of A1, A2, A3, is phenyl substituted with methyl, F, or methoxy in para position.
4. The method according to claim 2, wherein at least one of of A1, A2, A3, is, phenyl substituted with formyl, sulphonyl hydroxyl, hydroxyl, methoxymethoxy or carboxyl in ortho position.
5. The method according to claim 1, wherein A1, A2, A3 are, independently from each other, an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2 residue.
6. The method according to claim 5, A1, A2, A3 are, independently from each other, substituted with methyl or ethyl.
7. The method according to claim 5, A1, A2, A3 are, independently from each other, piperazinyl, preferably substituted with methyl or ethyl.
8. The method according to claim 5, A1, A2, A3 are, independently from each other, morpholinyl or thiomorpholynyl, preferably unsubstituted.
9. The according to claim 1, wherein A1, A2, A3 are equal.
10. The method according to claim 1, wherein L comprises at least two of A1, A2, A3 equal, preferably optionally substituted, phenyl.
11. The method according to claim 1, wherein the legand L is selected from the group consisting of TABLE-US-00009 L L Formula (II) Name
12. The according to claim 1, wherein the Cu(I) is selected from the group consisting of TABLE-US-00010 Compound L Formula (II) CuL.sub.2NO.sub.3 PPh.sub.3 Bis[tris(phenylphosphine)] copper(I) nitrate 1
13. A pharmaceutical composition comprising a Cu(I) complex of Formula (I) ##STR00091## wherein L is a ligand of Formula (II) ##STR00092## wherein X.sup.? is a monovalent anion n1, n2, n3 are independently to each other, an integer from 0 to 1 A1, A2 and A3 are independently from each other, a phenyl optionally substituted with (C.sub.1-C.sub.3)alkoxy, (C.sub.1-C.sub.3)alkyl, F, formyl, carboxyl, sulphonyl hydroxyl, hydroxyl, methoxy(C.sub.1-C.sub.3)alkoxy or an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2 with the proviso that when A1, A2 or A3 is optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1, and pharmaceutically acceptable carrier.
14. A Cu(I) complex of Formula (I) ##STR00093## wherein L is a ligand of Formula (II) ##STR00094## wherein X.sup.? is a monovalent anion n1, n2, n3 are independently to each other, an integer from 0 to 1 A1, A2 and A3 are independently from each other, a phenyl optionally substituted with a substituent selected from the group consisting of carboxyl, sulphonyl hydroxyl, hydroxyl, and methoxy(C.sub.1-C.sub.3)alkoxy or an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2 residue, with the proviso that when A1, A2 or A3 is optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and at least one of A1, A2 and A3 is a phenyl substituted with a substituent selected from the group consisting of carboxyl, sulphonyl hydroxyl, hydroxyl, and methoxy(C.sub.1-C.sub.3)alkoxy. when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1.
15. The Cu(I) complex according to claim 14, wherein A1, A2, A3 are, independently from each other, a phenyl optionally substituted with F, sulphonyl hydroxyl, hydroxyl, methoxymethoxy or carboxyl and at least one of A1, A2 and A3 is a phenyl substituted with sulphonyl hydroxyl, hydroxyl, methoxymethoxy or carboxyl.
16. The Cu(I) complex according to claim 14, wherein A1, A2, A3 are heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom atom to CH.sub.2 residue.
17. The Cu(I) complex according to claim 16, wherein A1, A2, A3 are independently from each other heterocyclic ring substituted with methyl or ethyl.
18. The Cu(I) complex according to claim 17, wherein A1, A2, A3 are, independently from each other, piperazinyl, preferably substituted with methyl or ethyl.
19. The Cu(I) complex according to claim 16, A1, A2, A3 are, independently from each other morpholinyl or thiomorpholynyl, preferably unsubstituted.
20. The Cu(I) complex according to claim 14, wherein A1, A2, A3 are equal.
21. The Cu(I) complex according to claim 14, wherein L comprises at least two of A1, A2, A3 equal, preferably optionally substituted phenyl.
22. The Cu(I) complex according to claim 14, wherein the legand L is selected from the group consisting of TABLE-US-00011 L Formula (II) L
23. The Cu(I) complex according to claim 14, wherein the Cu(I) complex is selected from the group consisting of TABLE-US-00012 Compound L Formula (II) CuL.sub.2BF.sub.4 PMepip 5
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0051]
DETAILED DESCRIPTION OF THE INVENTION
[0052] Therefore the invention concerns a Cu(I) complex of Formula (I)
##STR00007##
wherein L is a ligand of Formula (II)
##STR00008##
wherein [0053] X.sup.? is a monovalent anion [0054] n1, n2, n3 are independently to each other, an integer from 0 to 1 [0055] A1, A2 and A3 are independently from each other, [0056] a phenyl optionally substituted with (C.sub.1-C.sub.3)alkoxy, (C.sub.1-C.sub.3)alkyl, F, formyl, carboxyl, sulphonyl hydroxyl, hydroxyl, methoxy(C.sub.1-C.sub.3)alkoxy or [0057] an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2-, [0058] with the proviso that [0059] when A1, A2 or A3 is optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and [0060] when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1 [0061] for use in the treatment of tumours.
[0062] The invention hence concerns complexes of Cu(I) of Formula (I) with two ligands L of Formula (II).
[0063] Each Ligand L comprises A1, A2, A3 equal or different from each other. A1, A2, A3 can be a phenyl optionally substituted with a substituent selected from the group consisting of (C.sub.1-C.sub.3)alkoxy, (C.sub.1-C.sub.3)alkyl, F, formyl, carboxyl, sulphonyl hydroxyl, hydroxyl, methoxy(C.sub.1-C.sub.3)alkoxy.
[0064] According to the invention, when A1, A2 or A3 is an optionally substituted phenyl, then n1, n2 or n3, respectively, is 0. Therefore when a phenyl is present it is directly linked to P atom.
[0065] Preferably at least one of A1, A2 and A3 is independently a phenyl substituted with a substituent selected from methyl, methoxy, F, formyl, sulphonyl hydroxyl, hydroxyl, metoxymetoxy and carboxyl.
[0066] In a more preferred embodiment the phenyl of A1, A2, A3, is substituted with methyl, F, or methoxy in para position.
[0067] In another preferred embodiment the phenyl of least one of A1, A2 and A3, is substituted with formyl, methoxy, sulphonyl hydroxyl, hydroxyl, methoxymethoxy or carboxyl in ortho position.
[0068] In a preferred embodiment A1, A2, A3 are independently from each other phenyl unsubstituted or substituted with methyl, methoxy or ethyl, more preferably in para position.
[0069] In another preferred embodiment one of A1, A2, A3 is phenyl substituted with carboxyl or sulphonyl hydroxyl or hydroxyl substituted in ortho position. A1, A2, A3 can be an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2.
[0070] According to the invention, when A1, A2 or A3 is an optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1. According to the invention when the ligand contains a heterocyclic group, the latter is directly linked to CH.sub.2 residue.
[0071] In a still another preferred embodiment A1, A2, A3 are independently from each other piperazinyl, more preferably substituted with methyl or ethyl.
[0072] In another preferred embodiment A1, A2, A3 are independently from each other morpholinyl or thiomorpholynyl, more preferably unsubstituted.
[0073] In a preferred embodiment A1, A2, A3 are equal.
[0074] In another preferred embodiment L comprises at least two of A1, A2, A3 equal. In this embodiment A1, A2, A3 are preferably optionally substituted phenyl.
[0075] In Formula (II) n1, n2 and n3 can be equal or different and they mean an integer from 0 to 1 dependently on the meaning of A1,A2 and A3, respectively.
[0076] In a preferred embodiment n1, n2, n3 are all 0, and A1, A2 and A3 are optionally substituted phenyl.
[0077] In another preferred embodiment n1, n2 and n3 are all 1, and A1, A2 and A3 are, independently from each other, optionally substituted pyridizinyl, optionally substituted morpholinyl or optionally substituted thiomorpholynyl.
[0078] X.sup.? is a monovalent anion.
[0079] Preferably X.sup.? is selected from the group consisting of BH.sub.4.sup.?, NO.sub.3.sup.?, PF.sub.6.sup.? and BF.sub.4.sup.?.
[0080] In a still more preferred aspect the ligand L of Formula (II) is selected from the group consisting of:
TABLE-US-00001 L L Formula (II) Name
[0081] The compound of Formula (I) is preferably selected from the group consisting of
TABLE-US-00002 Compound L Formula (II) CuL.sub.2NO.sub.3 PPh.sub.3 Bis[tris(phenylphosphine)] copper(I) nitrate 1
[0082] The inventors surprisingly found out that the compounds of Cu(I) were antitumor agent.
[0083] Therefore, the invention concerns a Cu(I) complex of Formula (I)
##STR00037##
wherein L is a ligand of Formula (II)
##STR00038##
wherein [0084] X.sup.? is a monovalent anion [0085] n1, n2, n3 are independently to each other, an integer from 0 to 1 [0086] A1, A2 and A3 are independently from each other, [0087] a phenyl optionally substituted with (C.sub.1-C.sub.3)alkoxy, (C.sub.1-C.sub.3)alkyl, F, formyl, carboxyl, sulphonyl hydroxyl, hydroxyl, methoxy(C.sub.1-C.sub.3)alkoxy or [0088] an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to CH.sub.2 [0089] with the proviso that [0090] when A1, A2 or A3 is optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and [0091] when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1 [0092] for use as a medicament.
[0093] Preferred embodiments for A1, A2, A3 above indicated for the compounds for use in the treatment of tumours are here recalled for the Cu(I) complex of Formula (I) for use as a medicament.
[0094] In another aspect, the invention concerns a Cu(I) complex of Formula (I)
##STR00039##
Wherein L is a ligand of Formula (II)
##STR00040##
wherein [0095] X.sup.? is a monovalent anion [0096] n1, n2, n3 are independently to each other, an integer from 0 to 1 [0097] A1, A2 and A3 are independently from each other, [0098] a phenyl optionally substituted with a substituent selected from the group consisting of carboxyl, sulphonyl hydroxyl, hydroxyl, and methoxy(C.sub.1-C.sub.3)alkoxy or [0099] an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to the residue CH.sub.2, [0100] with the proviso that [0101] when A1, A2 or A3 is an optionally substituted phenyl, then n1, n2 or n3, respectively, is 0, and at least one of A1, A2 and A3 is a phenyl substituted with a substituent selected from the group consisting of carboxyl, sulphonyl hydroxyl, hydroxyl, and methoxy(C.sub.1-C.sub.3)alkoxy. and [0102] when A1, A2 or A3 is an optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1.
[0103] The invention hence concerns new complexes of Cu(l) of Formula (I) with two ligands L of Formula (II).
[0104] Each Legand L comprises A1, A2, A3, equal or different from each other. A1, A2, A3 can be a phenyl optionally substituted with sulphonyl hydroxyl, hydroxyl or carboxyl. When A1, A2 or A3 is optionally substituted phenyl, then n1, n2 or n3, respectively, is 0 and at least one of A1, A2 and A3 is a phenyl substituted with a substituent selected from the group consisting of carboxyl, sulphonyl hydroxyl, hydroxyl, and methoxy(C.sub.1-C.sub.3)alkoxy.
[0105] In a preferred embodiment at least one of A1, A2, A3 is, phenyl substituted with hydroxyl, sulphonyl hydroxyl, metoxymetoxy or carboxyl in ortho position.
[0106] A1, A2, A3 can be an heterocyclic ring selected from piperazinyl, morpholynyl, thiomorpholynyl, optionally substituted with (C.sub.1-C.sub.3)alkyl, where the heterocyclic ring is linked with the nitrogen atom to the residue CH.sub.2 . According to the invention, when A1, A2 or A3 is optionally substituted heterocyclic ring, then n1, n2 or n3, respectively, is 1.
[0107] In another preferred embodiment one of A1, A2, A3 is phenyl substituted with carboxyl, methoxy, sulphonyl hydroxyl, methoxymethoxy or hydroxyl, more preferably substituted in ortho position.
[0108] In a still another preferred embodiment A1, A2, A3 are independently from each other piperazinyl, more preferably substituted with methyl or ethyl.
[0109] In another preferred embodiment A1, A2, A3 are independently from each other morpholinyl or thiomorpholynyl, more preferably unsubstituted.
[0110] In a preferred embodiment A1, A2, A3 are equal.
[0111] In another preferred embodiment L comprises at least two of A1, A2, A3 equal. In this embodiment A1, A2, A3 are preferably optionally substituted phenyl. In a still another preferred embodiment one of A1, A2, A3 is a substituted phenyl.
[0112] In a preferred embodiment n1, n2, n3 are all 0, and at least one of A1, A2 and A3 is a substituted phenyl.
[0113] In another preferred embodiment n1, n2 and n3 are all 1, and A1, A2 and A3 are, independently from each other, optionally substituted pyridizinyl, optionally substituted morpholinyl or optionally substituted thiomorpholynyl.
[0114] X.sup.? is a monovalent anion.
[0115] Preferably X.sup.? is selected from the group consisting of BH.sub.4.sup.?, NO.sub.3.sup.?, PF.sub.6.sup.? and BF.sub.4.sup.?. In a still more preferred aspect the ligand L of Formula (II) is selected from the group consisting of:
TABLE-US-00003 L Formula (II) L
[0116] The compound of Formula (I) is preferably selected from the group consisting of:
TABLE-US-00004 Compound L Formula (II) CuL.sub.2BF.sub.4 PMepip 5
[0117] The present invention is also directed to a pharmaceutical composition comprising Cu(I) complex of Formula (I) and a pharmaceutically acceptable carrier.
[0118] The invention relates also to a Cu(I) complex of Formula (I) as above indicated for use as a medicament.
[0119] The Cu(I) complexes of the invention may be administered by any suitable route of administration, including both systemic administration and topical administration.
[0120] Systemic administration includes oral administration, parenteral administration, trans-dermal administration, rectal administration, and administration by inhalation. The Cu(I) complexes of the invention may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for a compound of the invention depend on the pharmacokinetic properties of that Cu(I) complex, such as absorption, distribution, and half-life, which can be determined by the skilled artisan. In addition, suitable dosing regimens, including the duration such regimens are administered, for a compound of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan.
[0121] The Cu(I) complexes of the invention will be normally, but not necessarily, formulated into a pharmaceutical composition prior to administration to a patient.
[0122] The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art.
[0123] The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein an effective amount of a Cu(I) complex of the invention can be extracted and then given to the patient such as with powders, syrups, and solutions for injection. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form. A dose of the pharmaceutical composition contains at least a therapeutically effective amount of the Cu(I) complex of this invention. When prepared in unit dosage form, the pharmaceutical compositions may contain from 1 mg to 1000 mg of the Cu(I) complex of this invention.
[0124] Conventional dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) trans-dermal administration such as trans-dermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols and solutions; and (6) topical administration such as creams, ointments, lotions, pastes, sprays and gels.
[0125] Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, granulating agents, coating agents, wetting agents, suspending agents, emulsifiers, sweeteners, flavour masking agents, colouring agents, anti-caking agents, humectants, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch, cellulose, calcium sulphate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch, gelatine, sodium alginate, alginic acid, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc. Suitable carriers for oral dosage forms include but are not limited to magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, methylcellulose, sodium carboxymethyl cellulose, and the like. Techniques used to prepare oral formulations are the conventional mixing, granulation and compression or capsules filling.
[0126] The compounds of the present invention may be also formulated for parenteral administration with suitable carriers including aqueous vehicles solutions (i.e.: saline, dextrose) or and/or oily emulsions.
[0127] In a still further aspect the invention relates to a Cu(I) complex of the invention for use in the treatment of a tumour.
[0128] Among the tumours the following can be cited: [0129] Primary brain tumors including, but not limited to, astrocytic and oligodendroglia tumors such as astrocytomas, and glioblastomas; [0130] Nerve tissue tumors of infancy such as neuroblastomas; [0131] Metastatic brain tumors including, but not limited to, lung cancer, breast cancer, melanoma metastases, colon/colorectal metastases, kidney/renal metastases; [0132] Solid tumors including, but not limited to, primary, recurrent or metastatic, progressive thyroid, breast, liver, bladder and kidney cancers.
[0133] Preferably the tumours are selected from the group consisting of cervical tumor, colon tumor, lung tumor, pancreatic tumor, ovarian tumor and breast tumor.
[0134] A therapeutically effective amount is intended to mean that amount of a compound that, when administered to a patient in need of such treatment, is sufficient to effect treatment, as defined herein. Thus, e.g., a therapeutically effective amount of a Cu(I) complex of the invention is a quantity of an inventive agent that, when administered to a human in need thereof, is sufficient so as to treat a tumour. The amount of a given compound that will correspond to such an amount will vary depending upon factors such as the particular compound (e.g., the potency (IC.sub.50), efficacy (EC.sub.50), and the biological half-life of the particular compound), disease condition and its severity, the identity (e.g., age, size and weight) of the patient in need of treatment, but can nevertheless be routinely determined by one skilled in the art. Likewise, the duration of treatment and the time period of administration (time period between dosages and the timing of the dosages, e.g., before/with/after meals) of the compound will vary according to the identity of the human in need of treatment (e.g., weight), disease or condition and its severity and the specific composition and method being used, but can nevertheless be determined by one of skill in the art.
EXPERIMENTAL PART
Example 1
General Procedure for the Preparation of Compounds 1-15
[0135] Two different synthetic procedures were used for the synthesis of [CuP.sub.2]X complexes
i) With a Copper(I) Precursor (Compounds 4-14):
[0136] ##STR00059##
[0137] [CuL.sub.2]BF.sub.4 copper complexes 4-14 were prepared by an exchange reaction with the starting labile compound Cu(CH.sub.3CN).sub.4BF.sub.4 in acetonitrile using a stoichiometric amount of the pertinent legand L. The reaction mixture was kept at room temperature under nitrogen for 1-6 h, then the solvent was removed and the residue recovered and dried under vacuum overnight.
[0138] Specific work-up procedures were employed depending on the nature of L. In the case of L=PMepip, PEtpip, Pmorf, Pthiomorf, DPMPP, POH and 2-(Diphenylphosphino)(OCH.sub.2OCH.sub.3) after solvent removal a waxy white residue was obtained which was treated with diethylether. The products (compounds 5-8 and 11-13) as white solid were recovered by filtration and dried under vacuum.
[0139] Here, as an example, the synthesis of compound 5 was detailed:
[0140] To an acetonitrile solution (20 ml) of [Cu(CH.sub.3CN).sub.4][BF.sub.4] (100 mg, 0.32 mmol) PMepip (243 mg, 0.66 mmol) was added at room temperature under nitrogen. The colourless reaction mixture was stirred at room temperature for 1 h 30 min and then the solvent was removed under a gentle stream of dinitrogen leaving an oily residue.
[0141] The residue was treated with diethylether, filtered and the obtained white solid dried under vacuum for 2 days.
ii) Reduction of Nitrate Cu(II) Salt With Phosphine Oxide Formation, Followed in Case by Metathetical Reaction of Anion Exchange (Compounds 1-3) (ref. Gysling, H., & Kubas, G. (1979). Coordination Complexes of Copper(I) Nitrate Inorg. Synth. Vol. 19, pp. 92-97)
##STR00060##
[0142] This procedure was used for the synthesis of compounds 1-3. The reaction took place in hot methanol without any precautions for the exclusion of air. The phosphine was dissolved in methanol under stirring and then the Cu(Il) salt was added in small portions. The salt dissolved immediately with the formation of a clear colorless solution, but near the end of the addition a heavy white precipitate forms. After addition of all the copper salt the reaction suspension was heated at reflux for about 10 minutes and then cooled to ambient temperature. The white precipitate was filtered and washed with ethanol and diethyl ether to eliminate the phosphinoxide formed as a side product. The Cu(I) derivative was dried under vacuum and recrystallized from ethanol or methanol.
Example 2
Characterization of [CuL.SUB.2.]X Compounds
[0143] Compound CuL.sub.2NO.sub.34 L=P(p-FC.sub.6H.sub.4).sub.3
[0144] By following the procedure ii), compound Cu(P(p-FC.sub.6H.sub.4).sub.3).sub.2 NO.sub.3 was obtained as white solid.
[0145] MW: 758
[0146] Anal. Calcd. For CuP.sub.2C.sub.36F.sub.6H.sub.24NO.sub.3: C 57.04, H 3.19, N 1.85%. Found: C 60.18, H 3.42, N 1.20%.
[0147] .sup.31P-NMR in CDCl.sub.3: ? (ppm) ?3.67 (s)
[0148] .sup.1H-NMR in CDCl.sub.3: ? (ppm) 7.04 (t, [Cu[P[C(CHCH).sub.2CF].sub.3].sub.2][NO.sub.3]); 7.22-7.30 (m, [Cu[P[C(CHCH).sub.2CF].sub.3].sub.2][NO.sub.3]).
Compound CuL.sub.2 BF.sub.45 L=P(PMepip).sub.3
[0149] By following the procedure above reported compound Cu(P(PMepip).sub.3).sub.2 BF.sub.4 was obtained as white solid.
[0150] MW: 891.4 White solid.
[0151] Anal. Calcd. for CuP.sub.2N.sub.12C.sub.36H.sub.78BF.sub.4: C 48.51, H 8.82, N 18.86%
[0152] .sup.31P NMR (CDCl.sub.3): ?(ppm) ?30.42 (s)
[0153] .sup.1H NMR (CDCl.sub.3): ?(ppm) 2.29 (s, [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2)NCH.sub.3].sub.3].sub.2); 2.45 (s, [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2)NCH.sub.3].sub.3].sub.2][BF.sub.4]); 2.66 (s, [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2)NCH.sub.3].sub.3].sub.2); 2.92 (s, [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2)NCH.sub.3].sub.3].sub.2).
[0154] ESI (+)-full-MS in MeOH (m/z assignment, intensity %): 803 ([Cu[P[CH.sub.2N(CH.sub.2CH.sub.2)NCH.sub.3].sub.3].sub.2].sup.+, 100); 433 ([CuP[CH.sub.2N(CH.sub.2CH.sub.2)NCH.sub.3].sub.3].sup.+, 45).
Compound CuL.sub.2 BF.sub.4 6 L=P(PEtpip).sub.3
[0155] By following the procedure above reported compound Cu(P(PEtpip).sub.3).sub.2 BF.sub.4 was obtained as white powder.
[0156] MW 975.5
[0157] Anal. Calcd. for CuP.sub.2N.sub.12C.sub.42H.sub.90BF.sub.4 C 51.71, H 9.30, N 17.22%. Found: C 50.21, H 9.98, N 18.65%.
[0158] .sup.31P NMR (D.sub.2O): ? (ppm) ?35.13 (bs)
[0159] .sup.1H NMR (D.sub.2O): ? (ppm) 2.92 (bs, 2H, [Cu[P[CH.sub.2N.sub.2(CH.sub.2CH.sub.2).sub.2CH.sub.2CH.sub.3].sub.3].sub.2); 2.62 (dd, [Cu[P[CH.sub.2N.sub.2(CH.sub.2CH.sub.2).sub.2CH.sub.2CH.sub.3].sub.3].sub.2); 2.37 (q, [Cu[P[CH.sub.2N.sub.2(CH.sub.2CH.sub.2).sub.2CH.sub.2CH.sub.3].sub.3].sub.2); 0.96 (t, 3H, [Cu[P[CH.sub.2N.sub.2(CH.sub.2CH.sub.2).sub.2CH.sub.2CH.sub.3].sub.3].sub.2).
[0160] ESI-MS(+) in MeOH (m/z assignment, % intensity): 887 ([Cu[P(CH.sub.2Pip-Et).sub.3].sub.2].sup.+, 100); 475 ([CuP(CH.sub.2Pip-Et).sub.3].sup.+, 83); 511 ([Cu[P(CH.sub.2Pip-Et).sub.3](H.sub.2O).sub.2].sup.+, 10).
Compound CuL.sub.2 BF.sub.47 L=P(Pmorf).sub.3
[0161] By following the procedure above reported compound Cu(P(Pmorf).sub.3).sub.2 BF.sub.4 was obtained as white powder.
[0162] MW 813
[0163] Anal. Calcd. for CuP.sub.2N.sub.6O.sub.6C.sub.30H.sub.60BF.sub.4 C 44.31, H 7.44, N 10.34%. Found: C 44.51, H 7.23, N 10.04%
[0164] .sup.31P NMR (D.sub.2O): ? (ppm) ?44.5 (bs)
[0165] .sup.1H NMR (D.sub.2O): ? (ppm) 3.68 (t, 4H, Cu[P[CH.sub.2N(CH.sub.2CH.sub.2).sub.2O].sub.3].sub.2); 2.63 (s, 2H, Cu[P[CH.sub.2N(CH.sub.2CH.sub.2).sub.2O].sub.3].sub.2]); 2.92 (bt, 4H, Cu[P[CH.sub.2N(CH.sub.2CH.sub.2).sub.2O].sub.3].sub.2])
[0166] ESI-MS(+) in MeOH (m/z assignment, intensity %): 725 ([Cu[P(CH.sub.2morph).sub.3].sub.2].sup.+, 100); 332 ([P(CH.sub.2morph).sub.3+H].sup.+, 55); 394 ([CuP(CH.sub.2morph).sub.3].sup.+, 18)
Compound CuL.sub.2 BF.sub.48 L=P(Pthiomorf).sub.3
[0167] By following the procedure above reported compound Cu(P(Pthiomorf).sub.3).sub.2 BF.sub.4 was obtained as a beige solid.
[0168] MW: 909.5
[0169] Anal. Calcd. for CuP.sub.2N.sub.6S.sub.6C.sub.30H.sub.60BF.sub.4 C 39.62, H 6.65, N 9.24, S 21.15%.
[0170] .sup.31P NMR (DMSO): ?(ppm) ?36.65 (bs)
[0171] .sup.1H NMR (DMSO): ?(ppm) (s, 2.61 [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2).sub.2S].sub.3].sub.2][BF.sub.4]); 2.75 (s, [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2).sub.2S].sub.3].sub.2][BF.sub.4]); 2.80 (s, [Cu[P[CH.sub.2N(CH.sub.2CH.sub.2).sub.2S].sub.3].sub.2][BF.sub.4]).
Compound CuL.sub.2 BF.sub.49 L=P(DPBAL).sub.3
[0172] By following the procedure above reported compound Cu(P(DPBAL).sub.3).sub.2 BF.sub.4 was obtained as a yellow-orange solid.
[0173] MW: 731
[0174] Anal. Calcd. for CuP.sub.2C.sub.38O.sub.2H.sub.30BF.sub.4: C 62.44, H 4.14% Found: C 58.22, H 4.11%
[0175] .sup.31P NMR (CDCl.sub.3): ?(ppm) 1.95 (s).
[0176] .sup.1H NMR (CDCl.sub.3): ?(ppm) 9.83 (s, [Cu[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]); 7.65 (m, [Cu[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]; 7.42, 7.27 (m, [Cu[P[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]); 6.99 (m, [Cu[P[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]); 7.99 (m, [Cu[P[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2])
[0177] .sup.13C NMR (CDCl.sub.3): ?(ppm) 192.49 (s, [Cu[P[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.r)(C.sub.6H.sub.5).sub.2].sub.3].sub.2].
[0178] ESI (+)-full-MS in MeOH (m/z assignment, intensity %): 643 ([Cu[P[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2].sup.+, 100); 291 ([PH[(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sup.+, 51); 321 ([P(OCH.sub.3) [(C.sub.aC.sub.bCHO(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sup.+, 42).
Compound CuL.sub.2 BF.sub.4 10 L=P(DPBA).sub.3
[0179] By following the procedure above reported compound Cu(P(DPBA).sub.3).sub.2 BF.sub.4 was obtained as a pale yellow solid.
[0180] MW 763
[0181] Anal. Calcd. for CuP.sub.2C.sub.38O.sub.4H.sub.30BF.sub.4: C 59.82, H 3.96% Found: C 56.47, H 4.10%.
[0182] .sup.1H NMR (CDCl.sub.3): ?(ppm) 6.46 (s, [Cu[P[C.sub.aC.sub.bCOOH(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]); 6.96 (d, [Cu[P[C.sub.aC.sub.bCOOH(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2); 8.29 (d, [Cu[C.sub.aC.sub.bCOOH(CH.sub.cCH.sub.aCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]); 7.55 (m, [Cu[C.sub.aC.sub.bCOOH(CH.sub.cCH.sub.dCH.sub.eCH.sub.f)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]); 7.25 (m, [Cu[P[Ca-Cb-COOH(CHc-CHd-CHe-CHf)(C.sub.6H.sub.5).sub.2].sub.3].sub.2]).
[0183] .sup.31P NMR (CDC.sub.3): ? (ppm) 1.84 (s).
[0184] ESI-MS(+) in MeOH (m/z assignment, intensity %): 675 ([Cu(DPBA).sub.2].sup.+, 100).
Compound CuL.sub.2 BF.sub.4 11 L=P(DPMPP).sub.3
[0185] By following the procedure above reported compound Cu(P(DPMPP).sub.3).sub.2 BF.sub.4 was obtained as a white solid.
[0186] MW 734
[0187] Anal. Calcd. for CuP.sub.2C.sub.38O.sub.2H.sub.34BF.sub.4: C 62.10, H 4.66%. Found: C 61.45, H 4.62%. ESI(+)-MS (MeOH) (m/z assignment, intensity %): 551 ([Cu(DPMPP).sub.2].sup.+, 100).
Compound CuL.sub.2 BF.sub.4 12 L=P(POH).sub.3
[0188] By following the procedure above reported compound Cu(P(POH).sub.3).sub.2 BF.sub.4 was obtained as a white solid.
[0189] MW 706
[0190] Anal. Calcd. for CuP.sub.2C.sub.36O.sub.2H.sub.30BF.sub.4: C 61.16, H 4.28%. Found: C 60.35, H 4.22.
Compound CuL.sub.2 BF.sub.4 13 L=P(TPPME).sub.3
[0191] By following the procedure above reported compound Cu(P(TPPME).sub.3).sub.2 BF.sub.4 was obtained as a white solid.
[0192] MW 795
[0193] Anal. Calcd. for CuP.sub.2C.sub.40O.sub.4H.sub.38BF.sub.4: C 60.43, H 4.82%. Found: C 58.11, H 4.81%.
Compound CuL.sub.2 BF.sub.4 14 L=P(TPPMS).sub.3
[0194] By following the procedure above reported compound Cu(P(TPPMS).sub.3).sub.2 BF.sub.4 was obtained as a white solid.
[0195] MW 835
[0196] Anal. Calcd. for CuP.sub.2C.sub.36O.sub.6H.sub.30BF.sub.4: C 51.78, H 3.62%. Found: C 50.91, H 3.60.
Example 3
Evaluation of the Activity of the Compounds of the Invention
Products and Methods
Cell Cultures
[0197] Human breast (MCF-7), lung (H157), pancreatic (PSN-1) and colon (HCT-15 and LoVo) carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A431 are human cervical carcinoma cells kindly provided by Prof. F. Zunino (Molecular Pharmacology Unit, Experimental Oncology and Molecular Medicine, Istituto Nazionale dei Tumori, Milan, Italy). 2008 human ovarian cancer cells were kindly provided by Prof. G. Marverti (Dept. of Biomedical Science, University of Modena, Italy). The LoVo-OXP cells were derived using a standard protocol in which LoVo cells were grown in increasing concentrations of oxaliplatin and resistant clones were selected over a period of nine months (Gandin, V., et al., J. Cell. Mol. Med. 2012, 16, 142-151). Cell lines were maintained in the logarithmic phase at 37? C. in a 5% carbon dioxide atmosphere using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units mL.sup.?1 penicillin and 50 mg mL.sup.?1 streptomycin), and 2 mM L-glutamine in: i) RPMI-1640 medium (Euroclone) for MCF-7, HCT-15, A431, 2008, PSN-1 and H157 cells; ii) F-12 HAM'S (Sigma Chemical Co.) for LoVo, LoVo MDR, and LoVo-OXP cells.
Cytotoxicity Assay
[0198] The growth inhibitory effect towards tumor cell lines was evaluated by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Alley, M. C., et al., Cancer Res. 1988, 48, 589-601). Briefly, depending upon the growth characteristics of the cell line, 3-8?10.sup.3 cells well.sup.?1, were seeded in 96-well microplates in growth medium (100 ?L) and then incubated at 37? C. in a 5% carbon dioxide atmosphere. After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied, at the appropriate concentration, dissolved in 0.9% sodium chloride solution just before use. Triplicate cultures were established for each treatment. After 72 h, each well was treated with 10 ?L of a 5 mg mL.sup.?1 MTT saline solution and, after 5 h of incubation, 100 ?L of a sodium dodecylsulfate (SDS) solution in 0.01 M HCl were added. After an overnight incubation, the inhibition of cell growth induced by the tested complexes was determined by measuring the absorbance of each well at 570 nm using a Bio-Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as percentage of the control and plotted vs. drug concentration. Dose-response curves were fitted and IC.sub.50 values were calculated with four parameter logistic model (4PL). IC.sub.50 values represent the drug concentrations that reduce the mean absorbance at 570 nm to 50% of those in the untreated control wells.
Example 3a
Cell Studies
[0199] Compounds 1-10 were evaluated for their cytotoxic activity towards a panel of human tumor cell lines including cervical (A431), colon (HCT-15), lung (H157), pancreatic (PSN-1), ovarian (2008) and breast (MCF-7) cancers as well as compounds 11-14 were tested on human ovarian cancer cells (2008). The cytotoxicity was evaluated by means of the MTT test for 72 h treatment with increasing concentrations of the tested compounds. For comparison purposes, the cytotoxicity of cisplatin, the most widely used anticancer metallo-drug, and oxaliplatin, key drug in FOLFOX (Folinic acid, 5-Fluorouracil & Oxaliplatin) regimens for the treatment of colorectal cancers, were evaluated in the same experimental conditions. IC.sub.50 values (?M), calculated from dose-survival curves, are shown in Table 1.
TABLE-US-00005 TABLE 1 In vitro antitumor activity of compounds 1-10. A431 HCT-15 H157 PSN-1 2008 MCF-7 1 0.2 ? 0.01 1.8 ? 0.8 1.6 ? 0.1 4.5 ? 1.8 2.2 ? 0.1 1.9 ? 0.7 2 3.0 ? 1.4 0.5 ? 0.2 3.4 ? 0.2 0.3 ? 0.08 1.3 ? 0.3 3.1 ? 0.6 3 3.1 ? 1.1 0.3 ? 0.2 1.2 ? 0.01 0.5 ? 0.2 0.5 ? 0.1 1.3 ? 0.4 4 0.8 ? 0.6 1.1 ? 0.5 0.9 ? 0.1 6.2 ? 0.9 1.9 ? 0.5 1.4 ? 0.3 5 8.0 ? 2.6 4.3 ? 1.2 7.3 ? 1.4 5.2 ? 1.7 6 7.7 ? 0.5 2.5 ? 0.1 3.8 ? 0.3 13.3 ? 3.2 14.5 ? 1.2 3.1 ? 0.9 7 15.0 ? 2.2 6.4 ? 0.5 8.4 ? 1.8 12.3 ? 1.5 9.2 ? 1.0 6.1 ? 1.5 8 10.2 ? 0.1 5.3 ? 1.2 7.7 ? 1.3 11.9 ? 0.1 9 4.7 ? 0.6 0.8 ? 0.2 1.5 ? 0.2 1.1 ? 0.5 0.6 ? 0.1 1.3 ? 0.3 10 3.8 ? 1.1 1.8 ? 0.6 0.9 ? 0.1 0.6 ? 0.01 0.3 ? 0.1 1.1 ? 0.2 cisplatin 2.1 ? 0.8 15.3 ? 2.6 2.1 ? 0.9 18.3 ? 3.1 2.2 ? 0.5 7.6 ? 0.2 oxaliplatin 3.7 ? 1.0 1.3 ? 0.3 11.8 ? 3.2 16.1 ? 2.5 1.7 ? 1.0 3.4 ? 1.2
[0200] Cytotoxic activity was examined against a panel of human tumor cell lines. For comparison purposes, the cytotoxicity of cisplatin and oxaliplatin were assessed under the same experimental conditions. The growth inhibitory effect was evaluated by means of MTT test. Cells (5-8?10.sup.3 mL.sup.?1, depending on the growth characteristics of the cell line) were treated for 72 h with increasing concentrations of tested compounds. IC.sub.50 values were calculated from the dose-survival curves by four parameter logistic model (P<0.05). SD=standard deviation, -=not detected. Compounds 1 and 4 were found, on average, 3.9 times more effective than cisplatin, and 3.1 time more effective than oxaliplatin. Similarly, compound 2 elicited an average cytotoxic activity 4 and 3.3 times higher than cisplatin and oxaliplatin, respectively. Compounds 3 and 10 were on average the most effective derivatives, showing an in vitro antitumor potential significantly higher than those of cisplatin (up to 6.8 times) and oxaliplatin (up to 5.5 times). Compound 9 proved to possess an average cytotoxic potency 4.8-and 3.8-fold higher compared to that of cisplatin and oxaliplatin, respectively. Compound 5 elicited average IC.sub.50 values comparable to that of oxaliplatin but slightly lower than cisplatin, whereas compound 6 proved to be as effective as cisplatin but slightly less cytotoxic than oxaliplatin. Compounds 7 and 8 were found to possess, on average, a cytotoxic activity only slightly lower compared to the reference metal-based drugs.
[0201] Compounds 12-14 were tested on human ovarian cancer cells (2008). The cytotoxicity was evaluated by means of the MTT test for 72 h treatment with increasing concentrations of the tested compounds. For comparison purposes, the cytotoxicity of cisplatin and oxaliplatin were evaluated in the same experimental conditions. IC.sub.50 values (?M), calculated from dose-survival curves, are shown in Table 2.
TABLE-US-00006 TABLE 2 In vitro antitumor activity of compounds 11-14. 2008 11 0.4 ? 0.3 12 1.1 ? 0.3 13 1.5 ? 0.4 14 0.8 ? 0.2 Cisplatin 2.2 ? 0.5 oxaliplatin 1.7 ? 1.0
[0202] Cytotoxic activity was examined against a panel of human tumor cell lines. For comparison purposes, the cytotoxicity of cisplatin and oxaliplatin were assessed under the same experimental conditions. The growth inhibitory effect was evaluated by means of MTT test. Cells (5-8?10.sup.3 mL.sup.?1, depending on the growth characteristics of the cell line) were treated for 72 h with increasing concentrations of tested compounds. IC.sub.50 values were calculated from the dose-survival curves by four parameter logistic model (P<0.05). SD=standard deviation, -=not detected. Against human ovarian cancer cells, compounds 11-14 were much more effective than both cisplatin and oxaliplatin, with compound 11 being the most cytotoxic derivative.
[0203] Compounds 1-4, 6-7 and 9-10 have been also additionally tested for their in vitro antitumor activity in a pair of human cell lines which has been selected for their resistance to oxaliplatin (colon cancer cells LoVo/LoVo-OXP). A LoVo cell line retaining a multidrug resistance phenotype was also considered (LoVo MDR). Cross-resistance profiles were evaluated by means of the resistance factor (RF), which is defined as the ratio between the IC.sub.50 value for the resistant cells and that arising from the sensitive cells (Table 3).
TABLE-US-00007 TABLE 3 Cross-resistance profiles LoVo LoVo-OXP LoVo MDR 1 0.4 ? 0.03 0.3 ? 0.1 (0.8) 0.5 ? 0.2 (1.1) 2 1.4 ? 0.1 1.3 ? 0.5 (0.9) 0.9 ? 0.6 (0.6) 3 0.5 ? 0.2 0.5 ? 0.1 (0.9) 0.9 ? 0.2 (1.8) 4 0.9 ? 0.3 0.7 ? 0.1 (0.7) 1.0 ? 0.1 (1.1) 6 0.7 ? 0.3 1.3 ? 0.5 (1.8) 1.2 ? 0.9 (1.8) 7 0.6 ? 0.3 0.4 ? 0.2 (0.7) 0.4 ? 0.3 (0.7) 9 0.6 ? 0.2 1.1 ? 0.6 (1.7) 0.8 ? 0.5 (1.3) 10 1.5 ? 0.4 0.09 ? 0.03 (0.1) 0.9 ? 0.4 (0.6) oxaliplatin 3.1 ? 0.3 17.5 ? 1.8 (17.0) 1.4 ? 0.8 (1.3) doxorubicin 1.1 ? 0.5 19.4 ? 2.2 (17)
[0204] Cells (5?10.sup.3 mL-1) were treated for 72 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test. IC.sub.50 values (?M) were calculated by four parameter logistic model (p<0.05). -=not detected, S.D.=standard deviation. Resistant Factor (in brackets) is defined as IC.sub.50 resistant/parent line.
[0205] The molecular mechanism involved in oxaliplatin resistance appeared to depend upon the decreased cellular accumulation (which is thought to be related to a greater activity of the ATP7B exporter rather than to the activity of P-glycoprotein (P-gp) and multi-drug resistance protein 1 (MRP1)) and a more efficient repair of oxaliplatin-induced DNA-damage by NER (Nucleotide Exci-sion Repair) (P. Noordhuis et al., Biochem. Pharmacol., 2008, 76, 53-61). LoVo OXP cells (derived from LoVo cells grown in the presence of increased concentration of oxaliplatin) were about 17-fold more resistant to oxaliplatin than parental cells. The data reported in Table 3 clearly indicate that all compounds possess resistance factors much lower than that of oxaliplatin, thus confirming their ability to overcome the oxaliplatin-resistance.
[0206] In Table 3 are also reported the results obtained in a multidrug resistant (MDR) colon carcinoma subline, LoVo MDR, in which the resistance to doxorubicin, a drug belonging to the MDR spectrum, is associated with an overexpression of multi-specific drug transporters, such as the 170 kDa P-glycoprotein (P-gp) (Wersinger, C., et al., Amino Acids 2000, 19, 667-685). It is well known that acquired MDR, whereby cells become refractory to multiple drugs, poses most important challenge to the success of anticancer chemotherapy. All copper derivatives tested against this cell line showed a similar response as for the parental subline, thus suggesting that they are not P-gp substrates and clearly underlining their ability to overcome MDR occurrence.
Example 3b
In Vivo Anticancer Activity Toward Lewis Lung Carcinoma
[0207] All studies involving animal testing were carried out in accordance with the ethical guidelines for animal research adopted by the University of Padua, acknowledging the Italian regulation and European Directive 2010/63/UE as to the animal welfare and protection and the related codes of practice. The mice were purchased from Charles River, Italy, housed in steel cages under controlled environmental conditions (constant temperature, humidity, and 12 h dark/light cycle), and alimented with commercial standard feed and tap water ad libitum. Animals were observed daily, and body weight and food intake recorded. The Lewis lung carcinoma (LLC) cell line was purchased from ECACC, UK. The LLC cell line was maintained in D-MEM (Euroclone) supplemented with 10% heat-inactivated FBS (Euroclone), 10 mM L-glutamine, 100 U mL.sup.?1 penicillin, and 100 ?g mL.sup.?1 streptomycin in a 5% CO.sub.2 air incubator at 37? C. The Lewis lung carcinoma (LLC) was implanted i.m. as a 2?10.sup.6 cell inoculum into the right hind leg of 8-week old male and female C57BL mice (24?3 g body weight). After 24 h from tumor implantation, mice were randomly divided into five groups (8 animals per group, 10 controls) and treated with a daily i.p. injection of compound 6 (5 mg kg.sup.?1 in 0.9% NaCl solution) and 9 (3 mg kg.sup.?1 dissolved in a vehicle solution composed of 0.5% DMSO (v/v) and 99.5% of saline solution (v/v)), cisplatin (1.5 mg kg.sup.?1 in 0.9% NaCl solution), or the vehicle solution (0.5% DMSO (v/v) and 99.5% of saline solution (v/v)) from day 9 after tumor inoculation (palpable tumor). Compounds 6 and 9 are better tolerated than cisplatin and could be administered also at a greater dose (5 and 3 mg kg.sup.?1, respectively, which represents ? of the MTD). At day 15, animals were sacrificed, the legs were amputated at the proximal end of the femur, and the inhibition of tumor growth was determined according to the difference in weight of the tumor-bearing leg and the healthy leg of the animals expressed as % referred to the control animals. Body weight was measured every two days and was taken as a parameter for systemic toxicity. All the values are the means?S.D. of not less than three measurements. Multiple comparisons were made by Tukey-Kramer test (** p<0.01; * p or ?p<0.05).
Example 3c
In Vivo Anticancer Activity Toward Lewis Lung Carcinoma
[0208] The in vivo antitumor activity of compounds 6 and 9 were evaluated in a model of solid tumor, the syngeneic murine Lewis lung carcinoma (LLC). Tumor growth inhibition induced by compounds 6 and 9 was compared with that promoted by the reference metallodrug cisplatin. From day 9 after tumor inoculation, when tumors became palpable, tumor-bearing mice received daily doses of compound 6 (5 mg kg.sup.?1 in 0.9% NaCl solution) and 9 (3 mg kg.sup.?1 dissolved in a vehicle solution composed of 0.5% DMSO (v/v) and 99.5% of saline solution (v/v)), cisplatin (1.5 mg kg.sup.?1 in 0.9% NaCl solution), or the vehicle solution (0.5% DMSO (v/v) and 99.5% of saline solution (v/v)). Tumor growth was estimated at day 15, and the results are summarized in Table 4. For the assessment of the adverse side effects, changes in body weights of tumor-bearing mice were monitored daily (
TABLE-US-00008 TABLE 4 In vivo anticancer activity toward murine Lewis lung carcinoma (LLC). Daily dose i.p. Average tumor weight Inhibition of (mg kg.sup.?1) (mean ? SD, g) tumor growth (%) Control.sup.a 0.589 ? 0.05 6 5 0.142 ? 0.09** 75.9 9 3 0.098 ? 0.06** 83.4 cisplatin 1.5 0.119 ? 0.10** 79.8 .sup.aVehicle (0.5% DMSO (v/v) and 99.5% of saline solution (v/v)).
[0209] Starting from day 9 after tumor implantation, tested compounds were daily administered i.p. At day 15, mice were sacrified and tumor growth was detected as described above. Tukey-Kramer test ** p<0.01.
[0210] The tumor growth inhibition induced by compound 6 was compared to that promoted by the reference metallodrug cisplatin. Noteworthy, administration of compound 9 induced a 83% reduction of the tumor mass. Remarkably, the time course of body weight changes indicated that treatment with 6 and 9 did not induce significant body weight loss (<20%,