GB2303627A - Anti-tumour platinum complex - Google Patents

Abstract

A novel platinum complex of the * small Greek beta *-carboline alkaloid (harmine) having the formula: ```wherein DMSO is dimethyl sulphoxide, coordinated to platinum via sulphur, is useful for treating malignant tumours in animals.

Description

COMPLEX This invention relates to a novel platinum complex of the ss-carboline alkaloid (canine) having the formula:

wherein DMSO is dimethyl sulphoxide, coordinated to platinum via sulphur. The invention also relates to methods of preparation. characterization and method for treating malignant tumours in animals comprising parenterally administering to an animals affected with a malignant tumour.

FIELD OF THE INVENTION This invention is in the field of coordination metal ion complexes and relates specifically to methods for treatment of neoplastic diseases BACKGROUND OF THE INVENTION Since the discovery of Rosenberg. that cisplalin has a potenl activity against tumour cells lB. Rosenberg et al., Nature (London),222. 385-386 (1969)j, there have been a large number of platinum complexes screened thenafter against certain types of tumour cell lines.

Some of these complexes were already drugs, e.g. carboplatin [K. R. Harrap, Cancer Treat.

Rev., 12 (A). 21-33 (1985)1. others were under filing. e.g.. oxaliplatin according to Debiotech., Switzerland, 1993j, and others were under various stages of pre-clinical and clinical trials [R. B. Weiss and M.C. Christien. Druas 46. 360-377 (1993); L. R. Kelland, Crit.

Rev. oncol. Hematol., 15. 191-219 (1993)j.

In almost all platinum complexes studied against tumours. ammonia or tert. amine was used as a ligand in the coordination with platinum, and only very rare were nalurally occurring ligands, e.g., sucrose jN.D. Sachinvala et al., J. Med. Chem.. 36. 1791-1795 (1993)1. We, the inventors. developed a new class of cis-plalinum complexes. in which the naturally occurring p-carboline alkaloids (isolated from the Deqanum harmala seeds) were used in the coordination with the platinum metal ion l T.A.K. Al-Allaf et al., J. Intra.

Biochem., 38 47-56 (1990)j. Some struclural modifications on these complexes were made later on according to several analytical means [T.A.K. Al-Allaf et at.. Dirasat (Jordan), 21B.

128-136 (1994)j. The present invention relates to a new method of preparalion leading to a single (non-mixture) and pure platinum complex of harmine. as well as relates to its antitumour activity against Leukemia P-388 and L-1210 in Swiss mice. The complex of the present invention is also useful for the treatment of various other tumours such as malignant lymphoma, breast cancer and the like. The platinum complex ot the present invention can be used for pharmaceutical use in the form of a pharmaceutical preparalion suilable for either oral or parenteral administration preferably lor parenteral administration. II rnay be used in conjunction or admixture with pharmaceutical excipients.Suitable excipients include, e.g., gelatin, lactose, glucose, sodium chloride and so forth. Further, it can also be used in the form of. injection. For injection. it is used in the form of an isotonic solution, which is prepared by admixing the complex with an isotonic agents such as manitol, sodium chloride. glucose or the like.

The dose of the complex for pharmaceutical use depends on the route of administration, age, weight. condition....etc. In general. il may be used for pharmaceutical use at a dose of ca.10 mg daily for oral roule and al a dose of 20-100 mg!kg per day parenterally.

SUMMARY OF THE INVENTION This invention is consistent with the development of a new method for preparing a novel, single (non-mixture) and pure platinum-harmine complex of the formula cis- [Pt(DMSO)(harmine)Cl2]. The complex obtained has been fully characterised physio chemically and spectroscopically.

The in vivo antitumour activity indicated that the complex of the invention is antitumour agent against P-388 and L-1210 Leukemia in mice.

CHARACTERISATION The structure of platinum-harmine complex has been characterised by CHN elemental analysis. NMR, IR. U.vNis. and mass spectral data. Its purity was checked by HPLC tecchnique, which is as follows: Status : Powder Colour : Pale Yellow Solubility: Practically insoluble in water, sparingly soluble in chloroform, slightly soluble in alcohol and acetone, and freely soluble in DMSO and DMF.

M. P. 216-2220 C (dec.) Analysis: Found, %C, 32.09; %H, 3.29; %N. 4.99 calculated for C,,H,,N,CI,OR., %C. 32.37; %H, 324; %N, 5.04.

IR spectrum: u(NH), 3300; o(C=C), 1628; u(S=O), 1160; (Csl discs) o(Pt-Cl), 320,345; u(Pt-S), 440 cm-1 1H NMR spectrum: a) In CDCl3 : 5(CH3), 3.3s; ô(CH30), 4.0s; 6(CH31DMSO)=3.5s, 3J (195Pt-S-CH), 21; 8(NH). 8.5s, b; #(aromatic),6.9-7.3 and 8.0-8.3m.

b) In DMSO-d6: 5(CH3). 3.4s; 6(CH30), 3.9s; 6(NH), 12s,b; 6(aromatic) 7.0 - 7.1 and 8.0 - 8.3 m.

U.vA(is.spectrum: (2.5mg% in methanol) #max = 312.5, 246.5 and 210 nm.

Mass spectrum : FAB technique using 3-nitrobenzyl alcohol as a solvent with cold inlet The characteristic ions formed from the complex [Pt(DMSO)(harmine) CI2] (molecular ion M/Z = 556) were of M/Z = 557. 521, 486. 460, 405, 154.

HPLC Technique: Column: Spheresorb, 5 ODS. 5 microns. 25 x 4.6 cm.

Solvent: Methanol Mobile phase: Melhanol Wave length: 254 nm Flow rate: 1 mllmin.

Rel. time: 3.1 It is obvious from the analytical data that the new complex possesses the formula cis-f,Pt(DMSO) (harmine) Cl2l, in which its molecular ion (MIZ = 556) obtained from the mass spectral data (FAB technique with 3-NBA as a solvent) is in accordance with the theorelical value (M.W. = 556). The 1H NMR signal with the chemical shift of 3.5ppm is for CH3 resonance of DMSO and the 3J (195Pt-S-CH) value (21 Hz) is in a good agreemenl with that of cis- [Pt (DMSO)2C12i (i.e., 23 Hz).The IR band appeared in the region 1160 cm-1 attributed to u(S=O) is for coordinated DMSO via sulpher and the two bands appeared in the region 320 and 345 cm-1 altributed to u(Pt-CI) are for cis-Pt Cl2 species.

This is another support for which the complex obtained possesses the cis.configuration.

The new complex is non-ionic since ils DMSO solution showed no conductivity.

METHODS OF PREPARATION The platinum-harmine complex was prepared as summarised in the following Scheme:

0 d e cisIPt(DMSO)(ha ra'ine) Cl:I cis-lpt(DOtso)(harmine) Cl & IP((harmint) Clllz \ D:vXSO / acidic solution The working starting material cis-[Pt(DMSO)2Cl2] was prepared either from K2PtCI4 (Price et al., Inora. Chem., 11. 1280-1284 (1972)l or from PtCI2. However, il can be used either as isolated solid or as in silu in DMSO solution as described in the following examples:: EXAMPLE 1 The complex cis-lPt(DMSO)2Cl2l (2.119, 0.5 mmol) was suspended in ethanol (20ml) and harmine (1.06g. 0.5 mmol) was added at once followed by the addition ot further quantity of ethanol (20ml). The reaction mixture was refluxed under stirring for ca.3h. It was tuned clear pale yellow solution soon after the addition of harmine and only for few minutes then became turbid. The reaction mixture was cooled lo room temperature and the solid thus formed was filtered, washed with small quanlities of ethanol and ether and dried under vacuum. Yield: 2.429 (87%).

Analytical data showed Ihal the product obtained is a mixture of the monomer cis (Pt(DMS0)(harmine)CI2] and the dimer it (harmine)Cl2]2 in ca. 1:4 proportions. This was redissolved in DMSO (10ml) with gentle healing and the resulting solution was filtered and cooled to room temperature. An cold acidic solution (PH = 2.5 - 5.5) was added until complete precipitation. The solid was isolated by fillralion, washed several times with the acidic solution. distilled water, cold ethanol and ether and dried. The product obtained is pure enough for further purposes, nevertheless. it can be crystallised from acetone or alcohol as a fine pale yellow cryslals.

EXAMPLE 2 Similar procedure to that of Example 1 was repeated using chloroform instead of ethanol. The yield of the complex obtained is almost certainly similar to that when ethanol was used.

EXAMPLE 3 Similar procedure to that of Example 1 or 2 was repealed using acetone instead of ethanol or chloroform. The yield of the complex obtained is almost certainly similar to that when ethanol or chloroform was used.

EXAMPLE 4 The complex cis-lPt (DMSO),CI,] (2.15g. 0.51 mmol) was dissolved in DMSO (20ml) with gentle heating and stirring. Harmine (1.09g, 0.51 mmol) was added gradually to the hot solution, and the reaction mixture was allowed to stand at ca. 800 C for ca.2h, then cooled to room temperature. An cold acidic solution (PH = 3.5 - 5.5) was added until complete precipitation. The solid obtained (2.7g. 95%) was treated as mentioned in Example 1.

EXAMPLE 5 The salt PtCI, (2g, 0.75 mmol) was added gradually to a wann DMSO (lOmi) with stining until complete dissolution. The olive- green solution formed was filtered and the filter paper was washed with wann DMSO (semi) and the washings were combined with the filtrate.

Harmine (1.5g, 0.7 mmol) was added to the solution and the reaction mixture was allowed to stand at ca.800 C for ca.2h, then cooled to room temperature. The resulting solution thus obtained was treated as mentioned in Example 4. The solid obtained (3.59, 90% based on harmine used) was treated as mentioned in Example 1.

BIOLOGICAL EVALUATION The complex of the invention was tested for anti-tumour activity in vivo in mouse models following standard protocols for this testing as set by the National Cancer Institute lancer Chemother. ReP.* 3 (1972)].

TEST EXAMPLE 1 To CD2F1 male mice, P-388 mouse leukemia were inoculated at 1 x 106 into the abdominal cavity into each mouse used in the test. The test complex was dissolved in a 10% DMSO.

The test complex was intraperitoneally introduced once (e.g., after 24h of implantation) and continued for 9 days. The results obtained on this tumour system are shown in Table 1.

Table i : The effects of the complex on life span of mice implanted with leukemia P-388.

Dose (mglkg) e/Ò increase in the life span ILS 1 65 10 130 ILS (increase in the life span) = T-C x 100 C T = the medium survival time of the treated mice.

C = the medium survival lime of the untrealed mice (control group of mice ) The results described in Table 1 indicaled that the complex of the invention is anlitumour active against the P-388 leukemia in Swiss mice. Confirmalory tests of antilumour activity against the L-1210 tumour in mice was conducted. The initial results are shown in Table 2 and confirm the activity of the complex of the invention against other tumour system TEST EXAMPLE 2 To CDF1 male mice, the L-1210 mouse leukemia were inoculaled at 1 x 106 into the abdominal cavity of each mouse used in the test. The test complex was dissolved in 10% DMSO. The test complex was intraperitoneally administered once (e.g., after 24h of implanlalion) and continued for 9 days. The results obtained on this tumour system are shown in Table 2. This confirms the activity of the complex of the invention.

Table 2. The effects of the complex on life span of mice implanted with leukemia L-1210

Dose (mg/kg) % increase in the life span (ILS') 1 58 10 127 ILS* (increase in the life span) = T-C x 100 C T = the mean survival time of the treated mice.

C = the mean survival time of the untreated mice (conlrol group)

Claims (6)

1. Claims 1. A platinum-harmine coordination complex of the formula:

   wherein DMSO is dimethylsulphoxide coordinated with platinum via sulphur.
2. 2. A process for preparing the platinum complex of the formula given in claim 1, comprising the reaction between the starting complex of the formula :cis [Pt(DMSO)2C12] and the 5-carboline alkaloid (harmine) of the formula:

   in a 1:1 molar ratio.
3. 3. A process according to claim 2 in which the reaction solvent is chloroform, alcohol, acetone or DMSO and an acidic solution of pH=2.5-5.5 is used for the precipitation of the product.
4. 4. A platinum complex of the formula given in claim 1 for use as anti-tumour agent.
5. 5. A method for treating malignant tumours in nonhuman animals which comprises administering to the animal a platinum coordination complex of claim 1.
6. 6. The use of a complex according to claim 1 in the manufacture of a medicament for the treatment of malignant tumours.

   6. A pharmaceutical composition for parenteral or oral administration to an animal affected with a malignant tumour comprising a platinum coordination complex as claimed in claim 1 and an acceptable carrier therefore.

   7. The use of a complex according to claim 1 in the manufacture of a medicament for the treatment of malignant tumours - Amendments to the claims have been filed as follows 5. A pharmaceutical composition for parental or oral administration in the treatment of tumours comprising a platinum coordination complex as claimed in claim 1 and an acceptable carrier therefore.