HU212105B - Process for producing titanium compounds of antitumor activiti and pharmaceutical compositions containing them - Google Patents

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

The present invention relates to novel organic titanium compounds of the formula I and to pharmaceutical compositions containing them.

In the formula (I), when (R) ₂ Ti (X) ₂ (I), when R is Q or Z, then X is the same as chlorine or ethoxy, while if R is W,

A further difference between the two complex types is that while in the bis-β-diketonate-Ti (rV) complex only the β-diketone component was altered and the other two substituents, Cl and ethoxy, remained, in the titanocene derivatives two cyclopentadiene groups a number of very different substituents have been tried, primarily to increase the water solubility of the compounds [Köpf-Maier et al., Anticancer Sl. 6, 33 (1986)].

Since literature references indicate that β-diketonate Ti (IV) complexes are more potent [Kepper et al., Arzneim. Frosch. Drug Slit. 39, 706 (1989)], the invention relates to structural variants to enhance the efficiency and solubility of these types of titanium compounds.

It is an object of the present invention to provide titanium compounds which exhibit more favorable and / or selective biological activity than known compounds.

The present invention relates to novel organo-titanium compounds of formula (I) wherein: (R) ₂ Ti (X) ₂ (1)

When R is Q or Z, then X is the same as chlorine or ethoxy, while when R is W, X is, in the combined form, 2,3-L-ascorbate.

The formula of the substituents is as follows:

then both X represent 2,3-L-ascorbate. The formula of the substituents is as follows:

Metal complexes are a significant group of anticancer compounds. Of these, the group of platinum-containing antitumor compounds is well known. The so-called. cisplatin (cis-diaminedichloroplatinium (IV)) has been successfully used in the chemotherapeutic treatment of tumors (Giraldi et al., Chem. Bioi. Internet. 22: 231, 1978). However, cisplantin develops resistance rapidly and very frequently. Therefore, drug research has focused on the development of metal complexes that can successfully treat resistant tumors. Of these new types of metal complexes having antitumor activity, titanium-containing compounds have recently become the focus of interest. There are two major classes of titanium compounds that inhibit tumor growth. The so-called. titanocene compounds [bis / n ⁵ -cyclopentadianyltitanium (IV)] and bis-β-diketonate Ti (IV)] derivatives [Köpf-Maier, Canter Chemother. Pharmacol 23, 225 (1989); Arepim of Keppler et al. Forsch. Drug Slit. 39, 706 (1989)].

From a complex chemical point of view, there is a significant difference between the two Ti (IV) complex types, since the coordination number of the titanium in the bis-dicetonate Ti (IV) derivatives is 6. whereas in the titanocene compounds only 4. The biological consistency is probably due to the because of the different coordination geometries of the metal complexes, their interaction with the DNA double helix is also different (Kovacic et al., AntiCancer Drug Design, 1988, 3, 205).

Preferred novel compounds of formula I are:

dichloro-bis-salicylanilidate-Ti (IV) (Ti 35) diethoxy-bis-salicylanilidate-Ti (IV) (Ti 96) diethoxy-bis (1-phenyl-3-methyl-4-acetylpyrazol-5-one) - Ti (Ti 45) Biobenzene 1-Acetonate Ti (IV) - L-Ascorbate (Ti 83)

A process for the preparation of organic Ti (IV) compounds of formula (I) - wherein R is Q or Z and each substituent is as defined above, then X is chlorine or ethoxy.

HU 212 105 Β

R is W and W is the former, then X may be taken together as L-2,3-ascorbate - characterized in that

a) salicylanilide of formula QH or formula ZH

-phenyl-3-methyl-4-acetylpyrazol-5-one is reacted with titanium (IV) tetrachloride in an aprotic organic solvent to give the compound of formula I (wherein R - Q, X = Cl and R = Z, X = Cl) and, if desired, the resulting compound, wherein R = Q, X = Cl, or R = Z, X = Cl, is reacted with ethyl alcohol in the presence of an acid scavenger and the resulting compound of formula I R = Q, X = OC ₂ H ₅ or R = Z, X = OC ₂ H ₅ ), or

b) Salicylanilide of formula QH or 1-phenyl-3-methyl-4-acylpyrazol-5-one of formula ZH in ethyl alcohol solvent with titanium (IV) -tetraalkoxide - wherein the alkoxide has from 1 to 4 carbon atoms - reacting and isolating the resultant compound of formula (I) - wherein the substituents represent the final product of process (a), or

c) reacting diethoxy-bis-benzoylacetonate Ti (IV) [WO ₂ TI (OC ₂ H ₅ ) ₂ ] in ethyl alcohol solvent with L-ascorbic acid and obtaining the compound of formula I (wherein R = W is as defined above); while (X) _{2 is} collectively L-ascorbate) by evaporation of the solvent.

The reaction is carried out in all the processes in anhydrous solvent at 25-110 ° C.

In the process (a), the same hydrocarbon or aliphatic chlorinated hydrocarbon - preferably toluene - chloroform solvent is used and the reaction is carried out at a boiling point of the solvent using a 2: 1 molar ratio of QH, ZH: TiCl ₄ .

In process b), ethyl alcohol is used as solvent and reagent. at least 2 molar equivalents of organic base, preferably triethylamine, are used as the acid scavenger.

In process (c), the titanium (IV) -tetra-alkoxide is titanium (IV) -tetra-ethoxide or preferably titanium (IV) -tetrahisopropoxide in a 2: 1 molar ratio of QOH, ZOH: Ti (OR) ₄ .

The anti-tumor growth inhibitory pharmaceutical compositions of the present invention may be prepared by adding to a compound of formula (I) a conventional carrier or excipient in a pharmaceutical composition.

The invention is based on the discovery that bis (n ⁵ -cyclopentadienyl) dichlorotitanum (IV) (titanocene dichloride) and diethoxy bis (1-phenylbutane-1,3-dionato) titanum (IV) are known in the art. In addition to budotitan, the salicylanediyl titanium complexes of formula (I) diethoxybis (1-phenyl-3-methyl-4-acetylpyrazol-5-oneate) Ti (IV) titanium compounds also exhibit significant tumor growth inhibitory activity.

The main advantages of the new compounds as active ingredients are:

- they are effective in the treatment of tumors resistant to other antineoplastic agents;

- the toxic side effects are more favorable than those of the titanium derivatives known in the art;

unlike other antineoplastic compounds, the novel titanium compounds produced by the process do not induce immunosuppression;

- are particularly well-suited for the treatment of colon cancer and melanoma.

In order to carry out the process according to the invention, the following examples are given. The stoichiometry of the complex compounds was determined by elemental analysis and 1 H-NMR spectroscopy.

Example 1

Preparation of dichloro-bis-salicylanilidate Ti (IV)

C ₂₆ H ₂₀ N ₂ O ₄ Cl ₂ Ti

M, 544.25

6.4 g (0.03 mol) of salicylanilide in 80 ml of abs. A solution of titanium tetrachloride (1.8 mL, 0.016 mol) in toluene was added dropwise with stirring, and the reaction mixture was refluxed for 1 h with the exclusion of moisture. The hydrochloric acid gas remaining in the solution is purged with dry nitrogen, the solid formed is filtered, washed with toluene and dried in a vacuum desiccator.

Yield: 7.8 g (95%) m.p. 228-230 'C (dec.)

El am Analysis:

Calculated: C 57.38%, H 3.70%, N5.14%;

Found: C, 58.75%, H, 3.85%, N5.16%.

1 H-NMR (DMSO-d ₆ ) δ: arH 6.7-8.0 m

Example 2

Preparation of diethoxy-bis-salicylanilidate (TiIV)

C ₃ O ₃ O N ₂ 0 ₆ TI

M: 562.46 Triethylamine ^a ) C ₂₆ H ₂₀ N ₂ O ₄ Cl ₂ Ti -► C ₃₀ H ₃₀ N ₂ O ₆ Ti + c ₂ h ₅ oh ⁺ 2Et ₃ NH ⁺ .Cl ⁺

0.54 g (0.001 mole) of the dichlorobissalicylanilidate TI (IV) prepared in Example 1 were treated with 5 ml of abs. ethanol was stirred with 0.3 ml triethylamine at room temperature for 3 hours. The precipitate is filtered off, washed with alcohol and dried.

Yield: 0.35 g (62%) m.p.

b)

CONH- @, li (0iPr) ₄ ^C - ^ C _3o y ₂ O ₆ Ti ♦ 2iPrOH (Öj-OH

6.4 g (0.03 mol) of salicylanilide in 50 ml of abs. in ethanol 4.5 ml (0.015 mol) of titanium (IV) -tetrahisopropoxide are refluxed for 2 hours

HU 212 105 B became. After cooling, the precipitated solid is filtered off and washed with alcohol.

Yield: 6.75 g (80%) Mp: 264-266 'C Elemental analysis:

Calculated: C, 64.06; H, 5.38; N, 4.98;

Found: C 65.08%, H5.18%, N 5.07%.

1 H-NMR (DMSO-d ₆ ) δ: CH ₃ 1.03 t: CH ₂ 3.9 q: ArH

6.4-8.0 m

This reaction with titanium (IV) tetraethoxide, as described above, affords a product of similar yield and purity as titanium (IV) tetraisopropoxide.

Example 3

Preparation of diethoxybis (1-phenyl-3-methyl-4-acetylpyrazole-5-nato) -Ti (IV)

C ₂₈ H ₃₂ N ₄ O ₆ Ti

M: 568.47

3a)

C 1 H OH c

8.64 g (0.04 mol) of 1-phenyl-3-methyl-4-acetylpyrazol-5-one in 80 ml of abs. 6 ml (0.02 mol) of titanium (IV) -tetra-isopropoxide was added to a solution of ethanol and refluxed for 2 hours. The product formed was isolated as in Example 2b. [Ti (IV) tetraethoxide can also be used instead of isopropoxide.]

Yield: 11.0 g (96%) M.p .: 196-198 'C Elemental analysis:

Found: C, 59.16%. H 5.67%, N 9.86%;

Found: C 59.81%, H 5.67%, N 10.03%.

1 H-NMR (CDCl ₃ ): CH ₃ CH ₂ O 1.30 t (6): 4.50 q (4): CH ₃ 2.35 s (6): CH ₃ CO 2.50 s (6) : ArH 7.25-8.0 m (10)

3b) 1.

rtOH, ® ©> ^C 28 ^H 32 ^N 4 ° 6 ^Ti diethoxy-bis (1-phenyl-3-methyl-4-acetylpyrazole-5onato-Ti (IV))

3b) 2. 2.16 g (0.01 mol) of 1-phenyl-3-methyl-4-acetylpyrazol-5-one in 2 ml of abs. 0.6 ml (0.0055 mole) of titanium tetrachloride was added dropwise to a toluene solution and proceeded as in Example 1 below.

Yield: 1.8 g (65%) of dichloro-bis (1-phenyl-3-methyl-4-acetyl) -pyrazole-5-oneato-Ti (IV) m.p. 254-255 ° C (dec.)

3b) 3. Dichloro-bis (1-phenyl-3-methyl-4-acetyl) -pyrazol-5-oneate (IV) (1.8 g, 0.003 mol) prepared according to Example 3b (1). 20 ml abs. ethanol was stirred with 1 ml triethylamine at room temperature for 6 hours. The precipitated solid was filtered off, washed with ethanol and dried.

Yield: 1.3 g (70%) m.p. 194-196 ° C

The quality of the product is the same as that obtained by method (a).

Example 4

Preparation of bis-benzoylacetonate-J ([Vf-L-ascorbate

Formula: C ₂₆ H ₂₄ O ₁₀ Ti

M: 544.36

budotitan or diethoxy bis-benzoylacetonato-Ti (IV)

ascorbic acid

- * C ₂₆ H ₂₄ O ₁₀ Ti c ₂ h ₅ oh bis-benzoylacetonato-Ti (IV) -L-ascorbate

6.5 g (0.0 mol) of benzoylacetone in 40 ml of abs. 6 ml (0.02 mol) of titanium (IV) tetraiisopropoxide was refluxed for 2 hours, after cooling, the precipitated solid was filtered off and washed with ethanol.

Yield: 6.8 g (74%) of budotitan; mp: 126-128'C

6.8 g (0.015 mol) of budotitan in 40 ml of abs. ethanol

2.6 g (0.015 mol) of L-ascorbic acid are heated under reflux for 3 hours. The solution is evaporated to dryness on a rotary evaporator and the residue is abs. triturate with ether, filter and dry.

Yield: 7.0 g (86%) m.p. 190-200 ° C (dec.)

Anal:

Calculated: C, 57.36; H, 4.44; Found: C, 58.04; H, 4.48.

General remarks:

Among the starting materials, salicylanilide and benzoylacetone commercial product, 1-phenyl-3-methyl-4-acetylpyrazol-5-one can be prepared according to the literature [B.

S. Jensen: ActaChem. Scand. 13, 1668 (1959)].

The biological activity of the biological compounds of the present invention is illustrated by the following examples:

Example 5

Effect of dichlorobis-salicylanilidate Ti (fV) (Ti-35) treatment on human melanoma and colon tumor cultures Cells were cultured in RPMI 1640 (Bibco) medium containing 10% bovine whey. His compound4

To evaluate the antitumor effect of TEKs, the ratio of living and dead cells was determined using a hematocytometer (MOM). The therapeutic efficacy of the compounds was characterized by inhibition of growth expressed as a percentage of control. 5

The inhibitory effect of dichlorobis-salicylanilidate Ti (IV) on cell proliferation is already present at a concentration of 10.0 pg / ml. HCMB in human melanoma cultures (Bowes human melanoma culture, Prof. O. 10) is reduced by 2430% within this dose range.

Collen, Leuven, Belgium) and COLO 205 colon tumor cultures (ATCC CCL 222) (Table 1). In contrast to this compound, C26 mouse colon tumor [C26 Colon 26 carcinoma, transplantable mouse; tumor origin: SRI, Birmingham, Alabama (USA) host Balb / C inbred mouse, transplantation method: S. C. tumor tissue implantation] proved to be the most sensitive as 73% growth inhibition was observed at 50.0 pg / ml.

Table 1 (Ti-35) Effect of bis-salicylanilidate Ti (IV) treatment on human melanoma and colon tumor cultures

Manage cell lines cell Number Growth inhibition Managed / Control (%) 24 hours 48 hours 24 hours 48 hours HCMB melanoma control 5.4 x 10 ⁵ 13.2 x 10 ⁵ - - Ti-35 10.0 pg / ml 3.8 X 10 ⁵ 7.5 X 10 ⁵ 70.4 75.75 50.0 pg / ml 2.6 X 10 ⁵ 2.9 X 10 ⁵ 48.1 21.9 100.0 pg / ml 1.8 x 10 ⁵ 0.8 x 10 ⁵ 33.3 6.1 Colo 205 colon tumor control 6.3 x 10 ⁵ 9.8 X 10 ⁵ - - Ti-35 10.0 pg / ml 4.8 X 10 ⁵ 7.9 X 10 ⁵ 76.1 80.6 50.0 pg / ml 3.4 X 10 ⁵ 4.8 X 10 ⁵ 53.9 48.9 100.0 pg / ml 2.8 x 10 ⁵ 2.2 X 10 ⁵ 44.4 22.4 Colo 320 colon tumor [ATCC CCL-220] control 11.7 X 10 ⁵ 22.6 X 10 ⁵ - - Ti-35 10.0 pg / ml 9.8 X 10 ⁵ 18.7 X 10 ⁵ 83.8 82.7 50.0 pg / ml 6.7 x 10 ⁵ 9.6 X 10 ⁵ 57.2 42.5 100.0 pg / ml 5.2 x 10 ⁵ 4.6 X 10 ⁵ 44.4 20.3 C 26 mouse colon tumor control 8.8 X 10 ⁵ 15.4 X 10 ⁵ - - Ti-35 10.0 pg / ml 7.9 X 10 ⁵ 10.6 X 10 ⁵ 89.7 68.8 50.0 pg / ml 2.4 X 10 ⁵ 1.8 X 10 ⁵ 27.3 11.6 100.0 pg / ml 1.6 X 10 ⁵ 0.8 x 10 ⁵ 18.2 5.2

Example 6

Antitumor effect of diethoxy-bis-salicylanilidate (TillV) (Ti-96)

The effect of the Ti-96 derivative on human melanoma and colon tumor cultures is shown in Figure 2. are summarized in Table. HCMB human melanoma cultures are more sensitive to Ti-96 treatment than human colon tumor cultures. In fact, a 50% reduction in proliferation can already be induced in melanoma cultures with a dose of 50.0 pg / ml Ti-96. The same degree of inhibition of proliferation of proli55 in colon tumor cultures can be achieved only at twice the dose (100.0 pg / ml) (Table 2).

Example 7

Antitumor Activity of Diethoxybis (1-Phenyl-3-methyl-4-acetylpyrazole-5onato) -Ti (IV) (Ti-45) The cellular proliferation inhibitory activity is shown in Table 3. Ti-45 is most effective in COLO 205 tumor cultures as it is used at doses of 10.0 pg / ml.

Also, it produced a 42% inhibition of growth 48 hours after treatment. 97.5% of the cells were killed by 100.0 pg / ml Ti-45 treatment. The tumor spectrum of Ti-45 differs from the titanium derivatives studied so far, as it is less effective against other titanium compounds in HCMB melanoma cultures than in colon tumor cultures.

Table 2 (Ti-96) Effect of diethoxy-bis-salicylanilidate Ti (IV) treatment in human melanoma and colon tumor cultures

Manage cell lines cell Number Growth inhibition Managed / Control (%) 24 hours 48 hours 24 hours 48 hours HCMB melanoma control 2.2 x 10 ⁵ 4.1 x 10 ⁵ - - Ti-96 10.0 µg / ml l 9x 10 ⁵ 4.0 X 10 ⁵ 86.4 97.5 25.0 pg / ml 1.6 X 10 ⁵ 2.8 X 10 ⁵ 74.5 68.3 50.0 pg / ml 1.0 X 10 ⁵ 1.8 x 10 ⁵ 45.4 43.9 100.0 pg / ml 0.8 x 10 ⁵ 0.7 X 10 ⁵ 35.4 17.0 COLO 205 control 6.2 x 10 ⁵ 9.8 x 10 ⁵ - - Ti-96 10.0 pg / ml 5.5 x 10 ⁵ 7.7 X 10 ⁵ 87.1 78.5 25.0 pg / ml 4.8 x 10 ⁵ 5.2 x 10 ⁵ 77.4 53.1 50.0 pg / ml 4.1 X 10 ⁵ 4.6 X 10 ⁵ 66.1 46.9 100.0 pg / ml 2.9 X 10 ⁵ 1.4 x 10 ⁵ 46.7 14.2 COLO 320 control 11.8 x 10 ^? 18.2X 10 ⁵ - - Ti-96 10.0 pg / ml 11.4 X 10 ⁵ 15.1 X 10 ⁵ 87.5 82.9 25.0 pg / ml 11.2 x 10 ⁵ 13.9 x 10 ⁵ 74.6 76.4 50.0 pg / inl 8.1 x 10 ⁵ 9.6 X 10 ⁵ 68.6 52.7 100.0 pg / ml 6.1 X 10 ⁵ 7.7 X 10 ⁵ 51.7 42.3 SW 6101 ATCC CCL 277] control 2.4 X 10 ⁵ 5.0 x 10 ⁵ - - Ti-96 10.0 pg / ml 2.3 X 10 ⁵ 4.7 X 10 ⁵ 95.8 92.1 25.0 pg / ml 2.1 X 10 ⁵ 3.9 X 10 ⁵ 87.5 78.0 50.0 µg / ml 1.7 X 10 ⁵ 2.3 X 10 ⁵ 70.8 45.1 100.0 pg / ml 1.6 X 10 ⁵ 1.7 X 10 ⁵ 66.6 33.3

Table 3: Effect of (Ti-45) Diethoxybis - (] - phenyl-3-methyl-4-acetylpyrazol-5-one) -Ti (TV) on human melanoma and colon tumors

Manage cell lines cell Number Growth inhibition Managed / Control (%) 24 hours 48 hours 24 hours 48 hours HCMB melanoma control 7.2 X 10 ⁵ 14.8 X 10 ⁵ - - Ti-45

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Manage cell lines cell Number Growth inhibition Managed / Control (%) 24 hours 48 hours 24 hours 48 hours 10.0 pg / ml 6.7 X 10 ⁵ 14.1 X 10 ⁵ 93.0 95.3 50.0 pg / ml 4.4 X 10 ⁵ 7.8 X 10 ⁵ 61.6 52.7 100.0 pg / ml 2.9 X 10 ⁵ 3.6 X 10 ⁵ 40.3 24.3 COLO 205 colon tumor control 6.4 x 10 ⁵ 11.8 X 10 ⁵ - - Ti-45 10.0 pg / ml 5.6 X 10 ⁵ 6.9 X 10 ⁵ 87.5 58.5 50.0 pg / ml 4.1 X 10 ⁵ 3.8 x 10 ⁵ 64.1 32.2 100.0 pg / ml 3.7 X 10 ⁵ 0.3 X 10 ⁵ 57.8 2.5 COLO 320 colon tumor control 13.2 X 10 ⁵ 24.2 X 10 ⁵ - - Ti-45 10.0 pg / ml 12.3 x 10 ⁵ 20.8 X 10 ⁵ 93.2 85.9 50.0 pg / ml 10,6x 10 ⁵ 11,3X 10 ⁵ 80.3 46.7 100.0 pg / ml 6.1 x 10 ⁵ 6.4 X 10 ⁵ 46.2 26.4 C26 mouse colon tumor control 7.8 x 10 ⁵ 13.1 X 10 ⁵ - - Ti-45 10.0 pg / ml 6.1 x 10 ⁵ 10.3 X 10 ⁵ 78.2 78.6 25.0 pg / ml 4.6 X 10 ⁵ 4.8 X 10 ⁵ 58.9 36.6 100.0 pg / ml 3.2 X 10 ⁵ 2.1 X 10 ⁵ 41.0 16.0

Table 4 (Ti-83) Effect of bis-benzoylacetonato-Ti (IV) ascorbate treatment on human melanoma and colon tumor cultures

! Manage cell lines cell Number Growth inhibition Managed / Control (%) 24 hours 48 hours 24 hours 48 hours HCMB melanoma control 5.7 x 10 ⁵ 12.5 X 10 ⁵ - - Ti-83 10.0 pg / ml 5.4 X 10 ⁵ 10.8 X 10 ' 94.7 86.4 50.0 pg / ml 2.9 x 10 ⁵ 3.4 X 10 ⁵ 50.8 27.2 100.0 pg / ml 1.5 X 10 ⁵ 0.4 X 10 ⁵ 26.3 3.2 Colo 205 colon tumor control 11.2Χ 10 ⁵ 19.2 X 10 ⁵ - - Ti-83 10.0 pg / ml 9.8 x 10 ⁵ 15.8 x 10 ⁵ 87.5 82.3 50.0 pg / ml 7.4 x ¹⁰ 9.6 X 10 ⁵ 66.1 30.2 100.0 pg / ml 6.2 x 10 ⁵ 5.8 x 10 ⁵ 55.4 30.2 Colo 320 colon tumor control 8.4 X 10 ⁵ 16.3 x 10 ⁵ - - Ti-83 10.0 pg / ml 7.6 x 10 ⁵ 12,8x 10 ⁵ 90.5 78.5 50.0 pg / ml 6.5 X 10 ⁵ 9.4 X 10 ⁵ 77.4 57.6 100.0 pg / ml 5.8 X 10 ⁵ 6.5 x 10 ⁵ 69.1 39.9

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Treatment of cell lines cell Number Growth inhibition Managed / Control (%) 24 hours 48 hours 24 hours 48 hours C 26 mouse colon tumor control 6.8 x 10 ⁵ 15.8 x 10 ⁵ - - Ti-83 10.0 pg / ml 6.1 x 10 ⁵ 10.9 x 10 ⁵ 89.7 68.9 50.0 pg / ml 3.2 X 10 ⁵ 5.7 X 10 ⁵ 47.1 36.1 100.0 pg / ml 1.9 x 10 ⁵ 0.4 X 10 ⁵ 27.9 2.5

Example 8

Tumor growth inhibitory effect of bis-benzoylletonato-Ti (IV) -L-ascorbate (Ti-83) The cellular proliferation inhibitory effect of Ti-83 is shown in Table 4.

50% proliferation inhibition can be induced at 50.0 pg / ml in both HCMB human melanoma cultures and C26 colon tumor cultures. The efficacy of the compound is indicated by the fact that when 100.0 pg / ml Ti-83 is administered, tumor cultures contain only 2.5-3.2% live cells after 48 hours.

The biological activity of the compounds of the invention is illustrated below in transplantable mouse tumors.

Example 9

Antitumour Effect of Dichlorobis-Salicylanilidate Ti (1V) (Ti-35) on S 180 Mouse Sarcoman The effect of the treatments on tumor growth was evaluated on the basis of the change in lifetime relative to the tumor control groups and by controlling the size of the tumor. Tumor growth was controlled by caliper on the basis of the relationship between the volume of the tumor and the square of its longest diameter. With this in mind, tumor masculinity was determined using the following formula:

V cm ³ = L x D ²

6.cm where V = volume; L = longest diameter; D = shorter diameter perpendicular to L.

Following the treatments, the animals were weighed daily for the first 10 days and subsequently every 2 days until death. The approx. In fact, weight loss of more than 10% usually refers to a toxic dose of the compound used, which may interfere with a realistic evaluation of the anti-tumor effect.

The (Ti-35) S 180 mouse exhibited significant antitumor activity at all tested doses of sarcomas. Table 5 shows that the most effective dose of Ti-35 was 200 mg / kg. At 50% survival in this dose group, the surviving animals became tumor free.

In addition to the survival data, the efficacy of the compound is confirmed by the data in Figure 1, which illustrates the effect of (Ti35) administered at different doses and rates of treatment on S180 tumor growth.

Ti-35 at 3x100 and 200mg / kg, respectively, delayed the development of measurable tumors by 3 days and 10 days by 5x100 mg / kg. At 5 days post-transplantation, 5-fold treatment resulted in a marked increase in ca. 50% growth inhibition.

Table 5

Effect of dichloro-bis-salicylanilidato-Ti (rV) (Ti-35) on the life span of S 180 sarcomas

Dose i.p. mg / kg Treatments' pace Survival (days) Average life (days) X Surviving / all animals standard deviation 50 2xq2d 17, 18, 19, 20, 22, 22, 25,27,33, 1 mouse> 110 - 1/10 100 2 X q2d 15, 15, 20, 20, 23.32, 38, 66, 2 mice> 110 - 2/10 200 2 x q2d 15, 17, 19, 20, 38.5 mice> 110 - 5/10 control 20,21,23,23,36,31, 32, 32, 32, 33 27.3, 6.3 0/10 100 3 x 2qd 27, 30, 34. 34, 34. 35, 35, 2 mice> 60 - 2/10 100 5 X 2qd 14, 14, 29,33,33,35, 35, 35,2 mice> 60- - 2/10 control 20, 20, 20, 24, 24, 27, 27, 29, 37, 37 26.5 6.3

x treatments started 2 days after tumor transplantation q? d = 2 daily repetitions

HU 212 105 Β

Example 70

Effect of diethoxybis (1-phenyl-3-methyl-4-acetylpyrazol-5-oneate) -Ti (fV) (71-45) on B 16 murine melanoma AB 16 melanoma transplantable mouse tumor [Karolinska Inst., Stockholm , host animal: BDF, hybrid mouse, the first generation from the crossing of C 57 Black female and DBA male mice; transplantation with subcutaneous tumor tissue] increased the life span of the tumor animals by increasing the dose of Ti-45 (Table 6). The 80 mg / kg dose of Ti-45 induced a nearly 60% increase in life span.

Example 77

Antitumor activity of bis-benzoylacetonato-Ti (rV) -L-ascorbate (71-83) on sarcoman in S 180 mice S 180 sarcomas showed only sensitivity to the higher (200 mg / kg) dose of Ti-83 (Table 7) . The efficacy of this dose is indicated by the fact that the treatment started to kill the animals one week later and in 20% of the treated animals the life expectancy exceeded four times the mean of the control group.

Table 6

Effect of diethoxybis (1-phenyl-3-methyl-4-acetylpyrazol-5-oneato) -Ti (IV) (Ti-45) on the survival of B 16 melanoma mice

Dose i.p. mg / kg ¹¹ treatments of treatments Number of animals Average life (days) Operator t / control (%) lifetime X S.D. 40 3 x q2d 7 19.4 7.9 120 60 3 Xq2d 7 20.4 6.8 135 80 3 X q2d 7 23.7 10.2 157 control 7 15.1 5.9 -

x Treatments Repeat q2d = 2 days 2 days after tumor transplantation

Animals that survived over 100 days became tumor free. The inhibitory effect on tumor growth at the low dose (Ti-83) was only seen in the early stages of tumor growth, but the higher dose increasing survival at the later stage of tumor growth reduced the tumor size by 40-50% compared to the control. illustrated.

Example 72

Antitumor effect of diethoxy-bis-salicylanilylated Ti (7V) (71-96) on S 180 mouse sarcoman The effect of (Ti-96) S 180 on sarcoma mice is illustrated in Table 8.

Table 7

Effect of bis-benzoylacetonato-Ti (N) -L-ascorbate (Ti-83) on survival of S 180 sarcomas mice

Dose i.p. mg / kg Rate of treatments * Survival (days) Average life (days) Surviving / all animals X S.D. 100 3 X q3d 17, 17,20,20, 20,20,20,21, 26,27 20.8 3.3 0/10 200 3 X q3d 22, 22, 22, 23, 23, 23, 34, 34, 42, 2 mice> 100 - - 2/10 control 15, 15, 18, 18, 18, 22, 25, 28, 28, 39 22.6 7.5 0/10

x Treatments began 2 days after tumor transplantation with q3d = 3 daily repetitions

Table 8

Effect of diethoxy-bis-salicylanilidato-Ti (IV) (Ti 96) on survival of S 180 sarcomas

Dose i.p. mg / kg Rate of treatments * Survival (days) Average life (days) Surviving / all animals X + S.D. 100 4 X qd 18.8 + 2.6 0/10 100 3 x q2d 16,16,21,23,23,24, 25,34, 2 mice <80 - 2/10 100 5 x q2d 20, 20, 20, 20, 20, 20, 25, 26, 29, 1 mouse <80 - 1/10 200 3 X q2d 23, 23, 27, 29, 37, 38.4 mice <80 - 4/10 control - 16, 16, 17, 20, 20, 20, 20, 20,21,21 19.1 + 1.9 0/10

x Treatments started 2 days after tumor transplantation.

HU 212 105 B

There was little difference in survival between the two doses. Survival above 80 days and tumor regression in surviving animals were observed in 40% and 20% of treated groups, respectively.

The effect of (Ti-96) on S 180 tumor growth retardation and growth inhibition is shown in Figure 3. At both doses (100 and 200 mg / kg), inhibition of growth was approximately 50% relative to control. A dose of 4 x 100 mg / kg, repeated daily, delayed the appearance of a measurable tumor by 4 days.

Example 13

Effects of titanium compounds on the immune system

The adverse side effect of antitumor cytostatic agents on the immune system, ummunsuppression, is well known. Based on this, we investigated the effect of BDF on the humoral immune response of mice to antigen, which is effective for the inhibition of cancer by salicyl-containing dichlorbis-salicylanilidate Ti (IV), (Ti-35) and diethoxybissalicylanilidate Ti (IV), (Ti-96).

B-lymphocyte antibody production was assayed by immuno-plaque assay.

Spleen cells that produce hemolytic antibodies. i.e., the number of plaque-forming cells was determined 4 days after immunization using a modified version of the direct plaque method developed by Jeme and Nordin. [Literature: Jeme N.K., Nordin A.A .: Plaque formadon in agar, by single antibodyproducing cells, Science 140, 405-406 (1963); Gaál, D. and Nowotny, A .: Immune enhancement by chemotherapeutic drugs and edotoxins. Cancer Immunize, and Immunother. 1979, 6, 9-15.

The so-called. active spleen cell suspension was used at two or three concentrations per test group. The spleens of 3-3 treated animals per sample were pooled.

Plaque counts were performed on an electric plaque counter (Titriplaque, Labor MIM). In each sample, plaques were counted in triplicate per dilution in each case.

Red blood cells from sheep blood three times washed with sterile physiological saline (Phylaxia, Budapest) were used as antigens. Sheep erythrocyte (BVVS) antigen was administered in a 1 x 8 dose, 0.2 ml volume, intraperitoneally. The complement was 1: 8 diluted guinea pig whey (a lyophilized preparation of HUMAN).

The results of intraperitoneal (i.p.) treatments at two different doses and at two different times of immunization are shown in Table 9. Treatments (Ti35) and (Ti-96) administered 24 hours prior to immunization did not affect the production of B lymphocytes against antigen at any dose. It was also ineffective when given with (Ti-35) antigen. In contrast, when administered simultaneously with (Ti-96) immunization, both doses significantly stimulated the humoral immune response of BDF mice.

The stimulatory effect of the (Ti-96) immune response when co-immunized is believed to be due to its inhibitory effect on T-suppressor cells, which are first activated by cells involved in the regulation of the immune response.

In the case of (Ti-35) and (Ti-96), it is considered significant that, contrary to known cytostatics used in other clinics, no immunosuppressive side effects detrimental to therapeutic efficacy are to be expected.

In addition, the immunostimulatory effect experienced with (Ti-96) may be advantageous in combination therapy for compensating for immunosuppression caused by other antitumor treatments (cytostatic, ionizing radiation).

Table 9

Influence of dichloro-bis-salicylanilidato-TifIV (Ti-35) and Dioxy-bis-salicylanilidato-Ti (IV) (Ti-96) on the humoral immune response of BDF, mice

Titanium compound of the invention Treatment time Dose (mg / kg) immune Response ** PFC / 10 ⁶ X spleen cell S.D. Percentage of control (%) (Ti-35) - 24h 100 40.0 ± 3.3 97.3 200 37.8 ± 2.8 92.0 immunization 100 40.6 ± 5.6 98.8 at the same time 200 43.3 ± 4.4 105.3 (Ti-96) - 24 hours 100 36.7 ± 3.0 89.3 200 44.4 ± 4.7 10,8,0 immunization 100 65.6 ± 3.0 159.6 at the same time 200 73.3 ± 4.2 178.3 * Control - - 41.1 ± 3.1 100.0

* Sheep red blood cell (BVVS) antigen-only (immunized) group ** PFC = plaque-forming cell assay

Claims (6)

A process for the preparation of organic Ti (IV) compounds of formula (I): wherein: (R) ₂ Ti (X) ₂ (I) When R is Q or Z, X is identical to chlorine or ethoxy; whereas when R is W, both X are 2,3-L-ascorbate. The substituents have the following meanings: characterized in that a) Salicylanilide of formula QH or 1-phenyl-3-methyl-4-acetylpyrazol-5-one of formula ZH is reacted with titanium (IV) tetrachloride in an aprotic organic solvent to give the compound of formula I (where R = Q, X = Cl, or R = Z. X-Cl), and if desired, reacting the resulting compound wherein R = Q, X = Cl, or R = Z, X = Cl with ethyl alcohol. in the presence of an acid scavenger and the resulting compound of formula I which is isolated by filtration (R = Q, X = OC ₂ H ₅ or R = Z, X = OC ₂ H ₅ ), or b) reacting the salicylanilide of formula QH or the 1-phenyl-3-methyl-4-acylpyrazol-5-one of formula ZH in ethyl alcohol with titanium (IV) tetraalkoxide, wherein the alkoxide is carbon atom, and Isolating the compound of formula (I) wherein the substituents represent the final product of process (a), or c) reacting diethoxy-bis-benzoylacetonate Ti (IV) [W ₂ ΊΊ (Ο ^ Η ₅ ) ₂ ] in ethyl alcohol solvent with L-ascorbic acid and obtaining the compound of formula I (wherein R = W and (X)) ₂ together (L-ascorbate) by evaporation of the solvent. Process (a) according to Claim 1, characterized in that the aprotic organic solvent is a hydrocarbon or an aliphatic chlorinated hydrocarbon, preferably toluene, chloroform, and the reaction is carried out at a boiling point of the reaction mixture in a ratio of 2: 1 QH, ZH: TiCl ₄ . finally. Process (a) according to claim 1, characterized in that at least 2 molar equivalents of the organic base, preferably triethylamine, are used as the acid scavenger. Process b) according to claim 1, characterized in that the titanium (IV) tetraalkoxide is titanium (IV) tetraethoxide or preferably titanium (IV) tetraisopropoxide QH, ZH: Ti (OR) ₄ 2 : 1 molar ratio. Process c) according to claim 1, characterized in that the reaction is carried out at a molar ratio of (W) ₂ Ti (OC ₂ H 5) ₂ : L-ascorbic acid = 1: 1. 6. A process for the manufacture of a medicament for the reduction of tumor growth inhibiting immunosuppression, comprising the preparation of a compound of formula I according to claim 1, wherein R and X are as defined in claim 1, with a conventional carrier or excipient. mixing it into a pharmaceutical preparation.