WO1994021652A1

WO1994021652A1 - Novel titanium compounds inhibiting tumour growth, pharmaceutical compositions containing them and process for preparing same

Info

Publication number: WO1994021652A1
Application number: PCT/HU1994/000007
Authority: WO
Inventors: István Bitter; István PÁLYI; Dezso^' GAÁL; Orsolya Csuka; Mária BODNÁR; Zoltán KOLONICS; Csaba SÖPTIEI; Béla KARÁCSONYI; Erzsébet DIÓSZEGINÉ EICHHARDT
Original assignee: Nitrokémia Ipartelepek; Országos Onkológiai Intézet
Priority date: 1993-03-19
Filing date: 1994-03-18
Publication date: 1994-09-29
Also published as: HUT66457A; HU9300789D0; HU212105B

Abstract

This invention relates to novel organotitanium (IV) compounds of the general formula (R)2Ti(X)2 wherein X means chlorine or an ethoxy group when R stands for a salicylanilidato group of formula (Q) or the group of formula (Z) whereas X2 together means 2,3-L-ascorbate group when R stands for the group of formula (W). The novel substances inhibit the tumour growth, diminish the degree of immunosuppression, are useful for the treatment of resistant tumours and induce fewer adverse side effects than other organotitanium derivatives known in the art. They are particularly effective against melanoma and colonic tumours. The invention relates furthermore to the pharmaceutical compositions containing a compound of general formula (I) as active agent; as well as to the preparation of the above compounds and compositions. The compounds of general formula (I) of the invention are prepared e.g. by reacting salicylanilide (QH) or 1-phenyl-3-methyl-4-acetylpyrazolone (ZH), respectively with titanium tetrachloride in an aprotic organic solvent and separating the obtained compound of general formula (I), wherein R means (Q) or (Z), respectively and X stands for chlorine.

Description

NOVEL TITANIUM COMPOUNDS INHIBITING TUMOUR GROWTH, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND PROCESS FOR PREPARING SAME

The invention relates to novel organotitanium compounds of general formula (I)

(R)₂Ti(X)₂ (I) wherein

X means chlorine or an ethoxy group when R stands for a salicylanilidato group of formula (Q)

Salicylanilidato

or 1-phenyl-3-methyl-4-acetylpyrazol-5- -onato group of formula (Z)

whereas

X₂ together means 2,3-L-ascorbate moiety when

R stands for a benzoy lacetonato group of formula (W)

and pharmaceutical compositions containing these compounds as active agents. The invention furthermore relates to a process for preparation of the above compounds and compositions.

The compounds of general formula (I) according to the invention exert an effect diminishing immunosuppression and inhibit tumour growth. Accordingly, the invention relates also to a method of treatment which comprises administering a therapeutically effective amount of a compound of general formula (I) into the organism of a patient for diminishing immunosuppression and/or inhibiting tumour growth.

Metal complexes represent an important class of antitumour compounds. A well known group of these substances are platinum-containing compounds inhibiting the tumour growth. From these, the socalled cisplatin (chemically cis-diaminedichloro- platinum of formula (II)

Cl₂Pt(NH₃)₂ (II) is successfully used for the chemotherapeutic treatment of tumours [Giraldi et al.: Chem. Biol. Interact. 22 , page 231 (1978)]. However, the development of drug resistance to cisplatin is rather rapid and very frequent. Thus, the research was directed to the development of metal complexes which are useful for the treatment of resistant tumours. From these antitumour metal complexes, titanium-containing substances recently got to the centre of interest. Two more important groups of antitumour titanium compounds are known: the so-called ti tanocene compounds, i.e. bis(n⁵-cyclopentadienyl)- titanium(IV) compounds and bis(β-diketonato)titanium(IV) compounds. [Köpf-Maier: Cancer Chemother. Pharmacol. 23 , page 225 (1989); Keppler et al.: Arzneim. Forsch./Drug Res. 39, page 706 (1989)].

From the viewpoint of complex chemistry, there exists an important difference between the two above complex types of titanium(IV): the coordination number of titanium is 6 in the bis(β-diketo- nato)titanium(IV) compounds whereas it is k in the titanocenes. Presumbly, the biological consequence of this difference between the coordination numbers consists therein that, due to the different coordination geometry of the metal complexes, their interaction with the double helix of DNA is also divergent [Kovacic et al.: Anti-Cancer Drug Design 2, page 205 (1988)].

An additional difference between the above two types o f comp lexes l i es i n that , main l y for increasing the water-solubility of titanocenes, a number of widely varying substituents were tried beside the two cyclopentadiene groups characterizing the metalocene structure; whereas the β-diketone component was only varied in the diketonato- -titanium(IV) complexes while chlorine and ethoxy groups were retained [köpf-Maier et al.: Anti- cancer Res. 6, page 33 (1986)].

Based on literature references, β-diketonato- - t i t anium ( I V ) comp lexes proved to be mo re active [keppler et al.: Arzneim. Forsch./Drug Res. 39, page 706 (1989)] therefore, the task of this invention has been to increase the effectivity and solubility of this type of titanium compounds by developing structural variations.

Thus, the invention is aimed to prepare novel titanium compounds showing a more favourable and/or more selective biological activity in comparison to those known in the art.

The invention relates to novel organotitanium compounds of general formula (I)

(R)₂Ti(X)₂ (I) wherein

X means chlorine or an ethoxy group when R

stands for the group of formula (Q)

or the group of formula (Z)

whereas

X₂ together means 2,3-L-ascorbate group when

R stands for the group of formula (W)

The compounds listed hereinafter are mentioned as examples of the novel compounds of general formula (I) of the invention (code numbers in parentheses):

dichloro-bis(salicylanilidato)titanium(IV) (compound Ti-35),

diethoxy-bis(salicylanilidato)titanium(IV) (compound Ti-96),

diethoxy-bis(1-phenyl-3-methyl-4-acetylpyrazol- -5-onato)titanium(IV) (compound Ti-45) and

bis(benzoylacetonato)titanium(IV) L-ascorbate (compound Ti-83).

The invention relates also to a process for the preparation of titanium(IV) compounds of general formula (I) wherein X means chlorine or ethoxy group when R stands for a group of formula (Q) or a group of formula (Z), the substituents being as defined abovej or X₂ together means L-2,3-ascor- bate group when R stands for a group of formula (W), which is as defined above. This process comprises

a) reacting salicylic acid anilide (hereinafter abbreviated: salicylanilide) (QH) or 1-phenyl- -3-methyl-4-acetylpyrazol-5-one (ZH), respectively with titanium(IV) tetrachloride in an aprotic organic solvent and separating the obtained compound of general formula (I), wherein: R means (Q) and X stands for chlorine; or R means (Z) and X stands for chlorine, respectively by filtration; or

b) reacting a compound of general formula (I), wherein R means (Q) and X stands for chlorine or R means (Z) and X stands for chlorine, respectively prepared according to process variant a), with ethanol in the presence of an acid binding agent and separating the obtained compound of general formula (I), wherein: R means (Q) and X stands for an ethoxy group; or R means (Z) and X stands for an ethoxy group, respectively by filtration: or

c) reacting salicylanilide (QH) or 1-phenyl-3- -methyl-4-acetylpyrazol-5-one (ZH), respectively with a titanium(IV) tetraalkoxide in ethanol as solvent and then separating the obtained compound of general formula (I), wherein the substituents are as defined in variant b) above, by filtration; or

d) reacting diethoxy-bis ( benzoy lacetonato) titanium(IV) of formula (W)₂Ti(OC₂H₅)₂ with L-ascorbic acid in ethanol as solvent and separating the obtained compound of general formula (I), wherein R means (W) and X₂ together stands for L-ascorbate group, by evaporation of the solvent.

In process variant a) a hydrocarbon or chlorinated aliphatic hydrocarbon, preferably toluene or chloroform are used as solvents and the reaction is carried out at the boiling point of the solvent by using 2 moles of (QH) or (ZH) calculated for 1 mole of titanium tetrachloride.

In process variant b) ethanol is used as solvent and reagent; and at least 2 molar equivalents of an organic base, preferably triethy lamine are employed as acid binding agent.

In process variant c) titanium(IV) tetra- ethoxide or preferably titanium(IV) tetraiso- propoxide are used as titanium(IV) tetraalkoxide in a 2:1 molar ratio of (QH) or (ZH), respectively to titanium(IV) tetraalkoxide.

The process variant d) is accomplished by using an 1:1 molar ratio of (W)₂Ti(OC₂H₅)₂ to L- -ascorbic acid.

The pharmaceutical compositions of the invention inhibiting tumour growth and diminishing immunosuppression comprise a compound of general formula (I) together with carriers and/or auxiliaries commonly used in the drug manufacture.

The method according to the invention for inhibiting tumour growth and diminishing immunosuppression comprises administering to a patient suffering from tumour a compound of general formula (I) together with carriers and/or auxiliaries commonly used in the therapy.

The invention is based on the recognition that, in addition to titanocene dichloride [lchemicallybis(n⁵-cyclopentadienyl)-dichlorotitanium(IV)] and budotitane [chemically diethoxy-bis(1-phenyl- butane-1,3-dionato)titanium(IV)], the salicyl anilide-titanium complexes or diethoxy-bis(1-phenyl-3-methyl-4-acetylpyrazol-5-onato)titanium(IV) as titanium compounds also possess a significant tumour growth-inhibiting effect.

The main advantages of the novel compounds of general formula (I) as active agents can be summarized as follows.

- The novel titanium compounds can effectively be utilized for the treatment of tumours which are resistant to other antitumour agents.

- From the viewpoint of adverse side effects, the novel titanium compounds are more favourable than titanium derivatives known from the literature.

- In contrast to other antitumour agents, the novel titanium compounds do not induce any immunosuppression.

- The novel compounds proved to be particularly useful for the treatment of colonic tumours and melanoma.

The invention is illustrated in detail by the following non limiting Examples. It is noted that the stoichiometry of complexes was determined by elemental analysis and ¹H-NMR spectroscopy.

Example 1

Dichloro-bis(salicylanilidato)titanium(IV)

Empirical formula: C₂₆H2₀N₂O₄ Cl₂Ti

Molecular weight: 544.25

The title compund is prepared by the follow- i ng r e ac t i on :

TiCl₄ → C₂₆H₂₀N₂O₄CI₂Ti + 2HCl

After dropwise adding 1.8 ml (0.016 mol) of titanium tetrachloride to a solution of 6.4 g (0.03 mol) of salicylanilide in 80 ml of abs. toluene under stirring, the reaction mixture is refluxed under anhydrous conditions for 1 hour. Thereafter, gaseous hydrogen chloride remaining in the solution is blown off by bubbling dry nitrogen, the solid product formed is filtered, washed with toluene and dried in a desiccator under reduced pressure to yield 7.8 g (95%) of the title compound, m.p.: 228-230 °C (with decomposition). Analysis:

calculated: C 57.38; H 3.70; N 5.14%;

found: C 58.76; H 3.85; N 5.16%.

¹H-NMR (DMS0-d₆, δ ppm): ArH 6.7-8.0, m

Example 2

Diethoxy-bis(salicylanilidato)titanium(IV)

Empirical formula: C₃₀H₃₀N₂O₆Ti

Molecular weight: 562.46

The title compound is prepared by using any of the following methods. a ) C _{2 6} H _{2 0} N ₂ O ₄ Cl₂Ti C₃₀H₃₀N₂O₆Ti + 2Et

After stirring for 3 hours 0.54 g (0.001 mol) of dichloro-bis(salicylanilidato)titanium(IV) (prepared as described in Example 1) with 0.3 ml of

triethylamine in 5 ml of abs. ethanol at room temperature for 3 hours, the precipitate is filtered,

washed with ethanol and dried to give 0.35 g (62%)

of the title compound, m.p.: 260-262 °C. b)

+ Ti(0iPr)₄ HC₃₀H₃₀N₂O₆Ti + 2iPrOH

After boiling under reflux 6.4 g (0.03 mol)

of salicylanilide with 4.5 ml (0.015 mol) of titanium(IV) tetraisopropoxide in 50 ml of abs. ethanol under anhydrous conditions for 2 hours and then

cooling down the reaction mixture, the solid precipitate is filtered and washed with ethanol to

give 6.75 g (80%) of the title compound, m.p.:

264-266 °C.

Analysis:

calculated: C 64.06; H 5.38; N 4.98%;

found: C 65.08; H 5.18; N 5.07%. ¹H-NMR (DMSO-d₅, δ ppm): CH₃ 1.03 t; CH₂ 3.9 q;

ArH 6.4-8.0 m.

On carrying out the reaction as described, except using titanium(IV)-tetraethoxide instead of titanium(IV) tetraisopropoxide, the product is obtained in a similar yield and purity.

Example 3

Diethoxy-bis(1-phenyl-3-methyl-4-acetyl- pyrazol-5-onato)titanium(IV)

Empirical formula: C₂₈H₃₂N ₄ O₆ Ti

Molecular weight: 568.47

The title compound is prepared by using any of the following methods.

a)

C₂₈H₃₂N₄O₆Ti +2iPrOH

After adding 6 ml (0.02 mol) of titanium(IV) tetraisopropoxide to the solution of 8.64 g (0.04 mol) of 1-phenyl-3-methyl-4-acetylpyrazol-5- -one in 80 ml of abs. ethanol and boiling the reaction mixture under reflux for 2 hours, the precipitate formed is isolated as described in process b) of Example 2 to give the title compound in a yield of 11.0 g (96%), m.p.: 196-198 °C. [Also here, titanium(IV) tetraethoxide may be used in- stead of the isopropoxide reagent.]

Analysis:

calculated: C 59.16; H 5,67; N 9.86%;

found: C 59.81; H 5.67; N 10.03%.

1H-NMR (CDCl₃, δ ppm): CH₃CH₂O 1.30 t (6); 4.50 q (4);

2.35 s (6) ; CH₃CO 2.50 s (6);

ArH 7.25-8.0 m (10) .

TiCl₄ → C₂₄H₂₂N₄O₄Cl₂

0

→ C₂₈H₃₂N₄O₆Ti + 2Et

E

diethoxy-bis(1-phenyl-3-methyl-4-acetylpyrazol-5- -onato)titanium(IV)

b/1)

After dropwise adding 0.6 ml (0.0055 mol) of titanium tetrachloride to a solution containing 2.16 g (0.01 mole) of 1 -phenyl-3-methyl-4-acetyl- pyrazol-5-one in 20 ml of abs. toluene, the Example 1 is followed to obtain 1.8 g (65%) of dichloro-bis(1-phenyl-3-methyl-4-acetylpyrazol-5-

-onato)titanium(IV), m.p, 254-255 °C (with decomposition). b/2)

Ater stirring 1.8 g (0.003 mol) of dichloro-

-bis(1-phenyl-3-methyl-4-acetylpyrazol-5-onato)ti- tanium(IV) [prepared as described in the preceding

b/1)] with 1 ml of triethy lamine in 20 ml of abs.

ethanol at room temperature for 6 hours, the solid

precipitate is filtered, washed with ethanol and

dried to give 1.3 g (70%) of the aimed product,

m.p.: 194-196 °C.

The quality of this product is identical to

that prepared by using method a).

Example 4

bis(Benzoylacetonato)titanium(IV) L-ascorbate

Empirical formula: C₂₆H₂₄O₁₀Ti

Molecular weight: 544.36

The title compound is prepared as follows:

2 CH₃COCH₂COC₆H₅ + Ti(OiPr)₄

budotitane, chemically diethoxy-bis(benzoylacetonato)titanium(IV); otherwise diethoxy-bis(1-phenylbutane-1,3-di- onato)titatium(IV) C₂₆H₂₄O₁₀Ti

bis(benzoylacetonato)titanium(IV) L-ascorbate

After boiling under reflux 6.5 g of benzoyl- acetone with 6 ml (0.02 mol) of titaπium(IV) tetraisopropoxide in 40 ml of abs. ethanol for 2 hours and then cooling down the reaction mixture, the solid precipitate is filtered and washed with ethanol to give 6.8 g (74%) of budotitane, m.p.: 126-128 °C.

After boiling under reflux 6.8 g (0.015 mol) of budotitane with 2.6 g (0.015 mol) of L-ascorbic acid in 40 ml of abs. ethanol for 3 hours, the solution obtained is evaporated to dryness in a rotavapor equipment, the residue is thoroughly triturated with abs. ether, then filtered and dried to yield 7.0 g (86%) of the aimed title compound, m.p.: 190-200 ° C (with decomposition). Analysis:

calculated: C 57.36; H 4.44%;

found: C 58.04; H 4.48%.

Notes to the above Examples:

From the starting materials used, salicylanilide and benzoylacetone are commercially available; 1-phenyl-3-methyl-4-acetylpyrazol-5-one may be prepared as described in the literature [B. S. Jensen: Acta Chem. Scand. 13 , page 1668 (195917.

The biological effects of the compounds according to the invention are illustrated in the following non limiting Examples.

Example 5

Effect of treatment with dichloro-bis(salicylanilidato)titanium(IV) (compound Ti-35) on human melanoma and colonic tumour cultures The cells were grown (cultivated) in RPMI 1640 (Bibco) medium containing 10% of calf serum. For evaluating the antitumour effect of the compounds, the ratio of living cells to the destroyed cells was determined by using a haemo- cytometer (product of MOM). The therapeutical activity of the compounds was characterized by inhibition of the growth expressed as percentage of control.

The effect of dichloro-bis(salicylanilidato)- titanium(IV) inhibiting the cell proliferation was observed in a concentration of as low as 10 /jg/ml. In this dose range it decreases by 24 to 30% the proliferation of HCMB human melanoma cultures (Bowes human melanoma culture received from Prof. 0. Collen, Leuven, Belgium) and COLO 205 colonic tumour cultures (ATCC CCL 222) as shown in Table 1. Against this compound, C26 Colonic carcinoma of mice [C26 Colon 26 mouse-transplantable carcinoma; origin of the tumour: SRI, Birmingham, Alabama (USA); host animal: Balb/C inbred mouse; method of transplantation: subcutaneous (s.c.) implantation of tumour tissue] proved to be most sensitive since a growth inhibition of 73% was observed by using a dose of 50.0μg/.ml. Table 1

Effect of treatment with dichloro-bis(salicylanilidato)- titanium(IV) (Ti-35) on human melanoma and colonic tumour cultures

Example 6

Antitumour effect of diethoxy-bis(salicyl- anilidato)titanium(IV) (compound Ti-96) The effect of the derivative Ti-96 on human melanoma and colonic tumour cultures is summarized in Table 2. The HCMB human melanoma culture was more sensitive to the treatment with Ti-96 than human colonic tumour cultures: namely, an 50% decrease in the proliferation of melanoma cultures could be induced by 50.0 yug/ml dose of Ti-96. An. inhibition of the same degree of proliferation could be achieved only by administering a twofold dose (100.0 /ug/ml) to colonic tumour cultures (Table 2).

Table 2

Effect of treatment with diethoxy-bis(salicylanilidato)- titanium(IV) (Ti-96) on human melanoma and colonic tumour cultures

Example 7

Antitumor effect of diethoxy-bis(1-phenyl-

-3-methyl-4-acetylpyrazol-5-onato)titanium(IV)

(compound Ti-45)

The effect inhibiting cell proliferation is shown in Table 3. The compound Ti-45 was most effective on COLO 205 tumour cultures since, when used in a 10 μg/ml dose, it resulted in a growth inhibition of 42%, 48 hours after the treatment. Under effect of treatment with 100.0 μg/ml of Ti-45, 97.5% of the cells were destroyed. The tumour spectrum of Ti-45 is different from those of titanium derivatives studied till now since its effect on HCMB melanoma cultures, which are extremely sensitive to other titanium compounds, is weaker than on colonic tumour cultures.

Table 3

Effect of treatment with diethoxy-bis(1-phenyl-3-methyl-4- -acetylpyrazol-5-onato)titanium(IV) (Ti-45) on human melanoma and colonic tumour cultures

Example 8

Inhibitory effect of bis(benzoylacetonato)titanium(IV) L-ascorbate (compound Ti-83) on the tumour growth

The inhibitory effect of compound Ti-83 on the cell proliferation is summarized in Table 4.

An 50% inhibition of proliferation could be achieved by a dose of 50.0 μ g/ml either in HCMB human melanoma cultures or C26 colonic tumour cultures. The effectiveness of the compound is shown thereby that, on administering 100 μg/ml of Ti-83, the tumour cultures contained altogether not more than 2.5 to 3.2% of living cells after 48 hours.

Table 4

Effect of treatment with bis (benzoylacetonato)titanium(IV) L-ascorbate (Ti-83) on human melanoma and colonic tumour cultures

The biological activity of the compounds according to the invention is hereinafter illustrated on transplantable tumours of mice.

Example 9

Inhibitory effect of dichloro-bis(salicylanilidato)titanium(IV) (compound Ti-35) on the growth of 5180 mouse sarcoma

The inhibitory effect of treatments on the tumour growth was evaluated by the change in the life span in comparison to tumour-bearing control groups as well as by observing the size of the tumour. The tumour growth was determined by using a slide caliper on basis of the relation that the volume of tumour is in a direct ratio to the second power of its longest diameter. By considering this relation, the volume of the tumour was calculated by the means of the following formula:

V cm³ = L x D²

wherein: V is the volume;

L is the longest diameter; and

D means the shorter diameter being perpendicular to L.

Measurements of body weight were performed from the time of treatments up to the death of the animal, daily in the first 10 days and later in every two days. Namely, a decrease of higher than 10% in body weight indicates a toxic dose of the compound used, by which the correct evaluation of antitumour effect may be disturbed. The compound Ti-35 exerted a significant antitumour action on 5180 mouse sarcoma in all doses tested. According to data of Table 5 the most effective dose of Ti-35 was 200 mg/kg. Beside a survival of 50%, the surviving animals became tumour-free in this dose group.

In addition to the data of survival, the effectivity of the compound was proven by the data of Figure 1 illustrating the effect of Ti-35 used in various doses and treatment rates on 5180 tumour growth.

The development of a measurable tumour was delayed by 3 days on using Ti-35 in a dose of 3x100 or 200 mg/kg, respectively; and by 10 days on using a dose of 5x100 mg/kg. Under the effect of five treatments, about 50% inhibition of tumour growth was evident in comparison to the untreated control group even in the 25th day following the transplantation.

^*

Example 10

Effect of diethoxy-bis(1-phenyl-3-methyl-4- -acetylpyrazol-5-onato)titanium(IV) (compound Ti-45) on the B16 mouse melanoma

In the case of B16 transplantable melanoma mouse tumour [k arolinska Inst. , Stockholm; host animal: BDF₁ hybride mouse, first generation of the crossing of C57 Black female mice with DBA male mice; the transplantation was carried out by using subcutaneous tumour tissue]. The life span of tumour-bearing animals was prolonged by increasing the dose of Ti-45 (as shown in Table 6) . An increase of nearly 60% in the life span was achieved by using Ti-45 in a dose of 80 mg/kg.

Table 6

Effect of diethoxy-bis(1-phenyl-3-methyl-4-acetyl- pyrazol-5-onato)titanium(IV) (Ti-45) on the survival of mice bearing B16 melanoma

* Treatments were begun 2 days following the tumour transplantation

q2d: Repetitions in every 2 days E xampl e 1 1

The antitumour effect of bis (benzoylacetonato)titanium(IV) L-ascorbate (compound Ti-83) on the S180 mouse sarcoma

Mice bearing S180 sarcoma were sensitive only to a higher (200 mg/kg) dose of Ti-83 (see Table 7) The efficacy of this dose was shown by the fact that, under effect of the treatment, the death of animals began one week later and the life span of 20% of the treated animals surpassed the fourfold of the average life span of control group. Animals living over 100 days became tumour-free. The tumour growth-inhibiting effect exerted by a low dose of Ti-83 was observed only in the early stage of tumour growth^ whereas the higher dose prolonging the survival diminished the tumour size by 40 to 50% in comparison to the control also in a later stage of tumour growth as shown in Figure 2.

Example 12

Antitumour effect of diethoxy-bis(salicylanilidato)titanium(IV) (compound Ti-96) on the S180 mouse sarcoma

The effect of Ti-96 on the survival of mice bearing S180 sarcoma is shown by the data of

Table 8.

Table 8

Effect of diethoxy-bis(salicylanilidato)titanium(IV) (Ti-96) on the survival of mice bearing S180 sarcoma

* Treatments were begun 2 days following the tumour transplantation

q2d: Repetitions in every 2 days Concerning the survival, nearly no difference was found between the effects of both doses. A survival over 80 days and, in the cases of surviving animals, the remission of tumour were observed on 40% or 20%, respectively of the treated groups. Both doses (100 or 200 mg/kg) caused nearly 50% inhibition of the tumour growth in comparison to the control. A daily repeated dose of 4 x 100 mg/kg delayed the appearance of a tumour of measurable size by 4 days.

Example 13

Effect of titanium compounds on the immune system

The adverse side effect of antitumour cytostatics on the immune system, i.e. the immunosuppression is commonly known. Starting from this fact, the influence of the effective antitumour, salicylic-containing dichloro-bis(salicylanilidato)- titanium(IV) (compound Ti-35) and diethoxy-bis(salicylanilidato)titanium(IV) (compound Ti-96) on the humoral immune response of BDF mice to the antigen was investigated.

The antibody production of B lymphocytes was examined by using the immune-plaque method.

The count of cells producing haemolytic antibodies of the spleen, i.e. the count of plaque- -forming cells was determined 4 days following the immunization by using a modified variant of the direct plaque method developed by Jerne and Nordin [N. K. Jerne and A. A. Nordin: Plaque formation in agar by single antibody-producing cells, Science 140, pages 405 to 406 (1963); D. Gaal and A. No- wotny: Immune enhancement by chemotherapeutic drugs and endotoxins. Cancer Immunol, and Immunother. 6 , pages 9 to 15 (1979)].

The suspension of splenocytes being present in the so-called active layer was used in two or three concentrations in each test group. The spleens of 3 animals each treated in the same manner were combined for each sample. The count of plaques was determined by using an electrical plaque-counting device (Titriplaque, Labor MIM). Within each sample, the plaques were parallelly counted in all cases in 3 dishes each in each dilution.

Sheep erythrocytes washed 3 times with sterile physiological saline solution (Phylaxia, Budapest) were used as antigen. The sheep erythrocyte antigen was intraperitoneally (i.p.) administered in 8 x 1 doses, in 0.2 ml volume. Guinea pig serum (lyophilized preparation of HUMAN) diluted in 1:8 ratio was employed as complement.

The results obtained by intraperitoneal treatments used in two doses and in two different times related to the time of immunization are shown in Table 9. None of the doses of Ti-35 or Ti-96, respectively administered 24 hours before immunization influenced the antibody production of B lymphocytes against the antigen. Contrarily, when employed simultaneously with the immunization, both doses of Ti-96 significantly stimulated the humoral immune response of BDF,. mice.

The immune response-stimulating effect of Ti-96 administered simultaneously with the immuni zation has presumably to be attributed to its inhibitory action on T-suppressor cells being firstly activated on effect of an antigen stimulus among cells playing any role in the regulation of immune response.

In our opinion, it is important in the cases of Ti-35 and Ti-96 that, in opposition to other known cytostatics used in the clinical practice, no immunosuppressi ve side effect adversely affecting the therapeutical activity has to be considered.

The immunostimulant activity observed at Ti-96 may be favourable for the use in combinations for the compensation of immunosuppression induced by other antitumour treatments (e.g. with cytostatics or ionizing irradiation).

Table 9

Influence of dichloro-bis(salicylanilidato) titanium(IV) (Ti-35) and diethoxy-bis(salicylanilidato)- titanium(IV) (Ti-96) on the humoral immune response of BDF₁ mice

*

Group treated (immunized) only by sheep erythrocyte antigen

^**

PFC: plaque-forming cell test

Claims

Cl aims

1. Novel organotitanium compounds of general formula (I)

(R)₂Ti(X)₂ (I) wherein

X means chlorine or an ethoxy group when R stands for the group of formula (Q)

or the group of formula (Z)

whereas

X₂ together means 2,3-L-ascorbate group when R stands for the group of formula (W)

2. Pharmaceutical composition inhibiting tumour growth and inducing a lower degree of immunosuppression, which c o m p r i s e s as active ingredient a therapeutically effective amount of a compound of general formula (I), wherein the substituents are as defined in claim 1, in admixture with carriers and/or auxiliaries commonly used in the drug manufacture.

3. Process for the preparation of the organo- titanium(IV) compounds of general formula (I), wherein the substituents are as defined in claim 1, which c o m p r i s e s

a) reacting salicylic acid anilide (hereinafter abbreviated: salicylanilide)(QH) or 1-phenyl- -3-methyl-4-acetylpyrazol-5-one (ZH), respectively with titanium(IV) tetrachloride in an aprotic organic solvent and separating the obtained compound of general formula (I), wherein: R means (Q) and X stands for chlorine; or R means (Z) and X stands for chlorine, respectively by filtration; or

b) reacting a compound of general formula (I), wherein R means (Q) and X stands for chlorine or R means (Z) and X stands for chlorine, respectively prepared according to process variant a), with ethanol in the presence of an acid binding agent and separating the obtained compound of general formula (I), wherein: R means (Q) and X stands for an ethoxy group; or R means (Z) and X stands for an ethoxy group, respectively by filtration; or c) reacting salicylanilide (QH) or 1-phenyl-3- -methyl-4-acetylpyrazol-5-one (ZH), respectively with a titanium(IV) tetraalkoxide in ethanol as solvent and then separating the obtained compound of general formula (I), wherein the substituents are as defined in variant b) above, by filtration; or

d) reacting diethoxy-bis(benzoylacetonato)titanium(IV) of formula (W) ₂Ti(OC₂H₅)₂ with L-ascorbic acid in ethanol as solvent and separating the obtained compound of general formula (I), wherein R means (W) and X₂ together stands for L-ascorbate group, by evaporation of the solvent.

4. A process as claimed in claim 3, which c o m p r i s e s carrying out the reaction in all process variants in an anhydrous solvent at a temperature between 25 °C and 110 °C.

5. A process as claimed in claim 3 or claim 4, which c o m p r i s e s using in process variant a) the same hydrocarbon or chlorinated aliphatic hydrocarbon, preferably toluene or chloroform as solvent and carrying out the reaction at the boiling point of the solvent by using a 2:1 molar ratio of (QH or ZH) : TiCl₄.

6. A process as claimed in claim 3 or claim 4, which c o m p r i s e s using in process variant b) ethanol as solvent and reagent and at least 2 molar equivalents of an organic base, preferably triethylamine as acid binding agent.

7. A process as claimed in claim 3 or claim 4, which c o m p r i s e s using in process variant c) titanium(IV) tetraethoxide or preferably titanium(IV) tetraisopropoxide as titanium(IV) tetraalkoxide in a 2:1 molar ratio of (QH) or (ZH), respectively to titanium(IV) tetraalkoxide.

8. A process as claimed in claim 3 or claim 4, which c o m p r i s e s carrying out the reaction of process variant d) with an 1:1 molar ratio of (W)₂Ti (OC₂H₅)₂:L-ascorbic acid.

9. A process for the preparation of a pharmaceutical composition inhibiting tumour growth and inducing an immunosuppression of lower degree, which c o m p r i s e s mixing as active ingredient a therapeutically effective amount of a compound of general formula (I), wherein the substituents are as defined in claim 1, prepared by using any of the process variants a) to d) of claim 3 with carriers and/or auxiliaries commonly used in the drug manufacture.

10. Method for inhibiting tumour growth as well as diminishing immunosuppression/ c h a r a c t e r i z e d by administering to a patient to be treated (in the need of such treatment) a thera peutically effective amount of a compound of general formula (I), wherein the substituents are as defined in claim 1, alone or in the form of a pharmaceutical composition.