The present invention relates to new titanocene com¬ pounds. More particularly the invention relates to new titanocene complexes and methods for their preparation, which possess chemotherapeutic activity being useful for the treatment of human tumors.

BACKGROUND OF THE INVENTION There are known metallocene complexes containing titan¬ ium, vanadium, niobium and molibdenum as a metal ion, which are active against a variety of tumor cell lines such as B16 melanoma, colon 38 carcinoma, Lewis lung car¬ cinoma, etc. It has been shown that the activity of vana¬ dium complexes related to the formula Cp ₂ VC1 ₂ where C is cyclopentadiene, against human epidermoid (HEP-2) tumor cells in vitro and against mouse tumor cells, is similar to that of cis-platin (Murthy M.S. et al. Proc .

Am. Assoc. Cancer Res. 1986, 27, 279). A study which was carried out with a corresponding molibdenum compound, supports the possibility that these complexes are binding 5\'-phosphate terminated polynucleotides , thus inhibiting DNA replication, by a mechanism which is different from that of cis-platinum complexes (Kon.L.Y. et al. J.A . Chem.Soc. 1991, 113, 9027).

Titanocene dichloride, one of the first metallocene compounds which was tested, was found to be indeed a very reactive anti-tumor reagent. Due to its rapid hydrolysis

to the corresponding dihydroxy derivative, it is quite reasonable to assume that this dihydroxy titanocene is the actual drug. Accordingly, many references can be found describing titanocene compounds which were tested in an attempt to possess an improved cytotoxity. Examples of such compounds include halides, pseudohal ides , carbo- xylates, and phenolates. However, no significant improve¬ ment over titanocene dichloride in the antitumor activity has been achieved. The metallocene diacido complexes, having the general formula (C ₅ H ₅ ) ₂ MX ₂ are characterized by the following structural features:

- The geometry of the complexes is that of a distorted tetrahedron . - The complexes contain two uninegative acido ligands X coordinated to the central metal atom and arranged in adjacent "cis-like" position.

- The sites of the other two ligands are occupied by two anionic cyclopentadieny1 rings. Attempts to modify the cyclopentadienide rings lead to a decreased biological activity.

In a very recent U.S. Patent No. 5,002,969 there are described cytostatic pharmaceutical compositions based on titanocene complexes. A group which is present in all these complexes is an amino or substituted amino bound to

the titancceno moiety. These compounds are obtained by a reaction between a titanocene dihalogenide and an amino phenol, lithiurr a inophenolate, or lithiurr amino thiophe- nolate. There is mentioned that the compounds have a better solubility in water than titanocene dichloride, fact which improves their application and dosing. Other titanocene complexes which were described, differ by their ionic character from the neutral titanocene compounds. Most of them correspond to the general formula [(C ₅ H ₅ ) ₂ TiXL] ⁺ Y ^" where X and Y are anions and L is a neutral donor molecule. These ionic titanocene complexes are characterized by their improved water solubility compared with the neutral titanocene compounds. It is an object of the present invention to provide novel titanocene derivatives. It is another object of the present invention to provide novel titanocene derivatives which possess a superior cytotoxic activity than the cis- platinu complexes.

BRIEF DESCRIPTION OF THE INVENTION. The invention relates to novel titanocene derivatives which comprise two cyclopentadiene rings linked tc titan¬ ium as a central atom, which are bound covalently to two phencxy groups which possess a substituent R which is selected from the group consisting of: C00CH ₃ , C00C ₂ H ₅ , H, C00CH ₂ CH ₂ 0CH ₂ CH ₂ 0CH ₃ being free from amino groups,

nitro group, chloride and fluoride. The above novel titanocene derivatives represent a compromise between the two main properties required for an antitumor agent: electrophi 1 icity and stability. DETAILED DESCRIPTION OF THE INVENTION.

It is a generally accepted assumption that titanocenes, as well as other antitumor agents, do react with DNA in a similar manner. Therefore, the two main properties requi¬ red for the drugs, in addition to the water solubility, are electrophil icity and stability in order to survive the aqueous biological medium during the time required to reach the target. The inventor\'s approach was to synthe¬ size the new compounds which should possess these two main properties. Accordingly, the titanocene compounds envisaged should contain groups such as phenolates, having the role cf moderate leaving groups, and appropri¬ ate substituents on the phenyl rings which impart stabi¬ lity to these compounds. Thus, considering the electro- philic role played by the metallocene drug in binding to the nucleophilic sites of polynucleotides , it may be concluded that an optimal biological activity would be achieved when the titanocene compounds, according to the present invention, will contain leaving groups of modera¬ te reactivity, such as phenols substituted at their 4-position with C0OCH ₃ , C0 ₂ CH ₂ CH ₂ CH ₂ 0CH _? CH ₂ 0CH ₃ , CH ₂ -CH ₃

C0CH ₃ , H and of course possessing a satisfactory hydrcly- tic stability.

Typical examples of the novel titanocene derivatives are as follows:

5

1. Bis(4-cyanophenolato)bis(n -cyclopentadienyl )titanium

(IV), hereinafter referred to as TC .

5

2. Bis(4-methoxycarbonylphenolato)bis(n -cyclopentadi¬ enyl )titanium( IV) , hereinafter referred to as TPE

5

3. Bis(4-ethoxycarbonylphenolato)bis (n -cyclopentadi¬ enyl) hereinafter referred to as TEE1.

4. Bis[4- (2-methoxyethoxy )ethoxy]carbonylphenolato]bis-

5 (n -cyclopentadienyl )titanium( IV) .hereinafter referred to as TEG. 5. Bis[4-(methoxy)ethoxycarbonylphenolato]bis (n cyclo¬ pentadienyl )titanium( IV) , hereinafter referred to as

TKEM.

6. Bis[4-(2-dimethylamino )ethoxycarbonylphenolatojbis-

5 (n -cyclopentadienyl )titanium( IV) , hereinafter refer- red to as TCA. 7. Bis[4- (2-trimethylammono)ethoxycarbonylphenolato]bis-

(n cyclopentadienyl )titanium( IV) , hereinafter referred to as TCE. Cytotoxicity measurements carried out with the above compounds show significant growth inhibition properties of these compounds expressed in terms cf IC ₅₀ [ιX] values.

In the following Table 1 are presented the results which show that these compound*; are much superior than the known titanocene dichloride (TDC) under the same conditi- ons. The value of the ratio Ti/Pt represents the relative activity of TPE as compared with that of cls-platlnura. The first four entries represent data of normal cell lines and the other ten entries represent the experiments with tumor cell lines.

TABLE 1. Cytotoxic data of titanocene derivatives.

Cell Cell type TPE TDC cisPt JJ_ line Pt

CHO Chinese Hamstead 1.3x10 -5 10 -3 3.1x10 ^"5 2

Ovary HMEC Normal Human 3.1x10 ^"5 1.3x10~ ⁴ 6.3x10 ^"5 20

Mammary NHDF Normal Human 1.6x10 ^"6 10 ^"3 3.1x10 ^"5 20

Skin

NHEK Normal Keratino 3.1x10 ^~6 10 ^"3 6.3x10 ⁴ 200

Epithelial

Capan 1 Pancreas 3.9x10 ^"7 5.0x10~ ⁴ 3.9x10 ^"6 10 Carcinoma HT-29 Colon Carcinoma 3.9x10 ^-4 5.0x10~ ⁴ 1.3x10 ^"4 200

SK-Mel-28 Melanoma 1.6x10~ ⁶ 10~ ³ 1.6x10 ^"5 10

H-322 Lung Carcinoma 6.3x10 ^"7 10 ^"3 6.3x10 ^"5 100

UCLA-P3 Lung Carcinoma 3. x10 ^"6 10~ ³ 6.3x10 ^"5 20

MCF-7 Breast Cancer 2.0x10 ^-6 10~ ³ 1.3x10 ^"4 100 HL-60 B-cell Leukemia 3.1x10 ^"6 10 ^~3 7.δx10 ^"6 2

Molt-4 T-cell Leukemia -10 ^"6 10~ ³ -10 ^"δ 10

Ovcar-3 Ovarian 6.3x10 ^"6 10 ^~3 6.3x10 ^"5 10

Carcinoma

P-388 Mouse Leukemia 3.9x10~ ⁶ 5.0χ10 ^"4 9.8x10 ^"7 025

The cytotoxicity results with a number of titanocene derivatives, expressed in concentrations (M) are present¬ ed in the attached Table 2 for a number of solid tumors. For combating solid tumors, the titanocene derivatives according to the present invention may be employed as such or as pharmaceutical compositions containing at least one titanocene complex as described above in addi¬ tion to pharmaceutically acceptable excipients, diluents and/or auxiliary agents. The excipient can serve as an agent for promoting absorption of the medicament by the body or as formulation auxiliary, sweetener, flavouring agent, colourant or preservative. The pharmaceutical formulations of the active compounds are preferably in the form of unit doses matched to the particular mode of administration. The amount of the active compound is chosen so that one or more units are usually sufficient for an individual therapeutic administration. In addition to that, the medicaments with the active compound, may contain also one or more other pharmacologically active constituents, such as: alkylating agents, antimetabol ites antibiotics, vitamins, enzymes and heavy metal compounds. The novel titanocene derivatives, according to the pre¬ sent invention, can be prepared from common chemical rea¬ gents using standard equipment. It should be realized, that the Examples for their preparations presented

hereinafter are only for illustration and many other routes may be conceived for their syntheses.

EXAMPLE 1. Preparation of Bis(4-cyanophenolato)bis

5 (n -cyclopentadienyl )titanium( IV) TCN. An amount of 238 mg(2mmol) of 4-cyanophenol was dissolved in 10 ml of benzene and 200 mg of sodium hydride 80% in mineral oil (6.67mmol) was added and stirred at room temperature for about 10 minutes. To this mixture an amount of 249 mg (1 mol) of titanocene dichloride was added and the mixture refluxed for 8 hours, cooled to room temperature and placed on a short column containing silica gel (pre-washed with acetone). The elution with methylene chloride followed by removal of the solvent under reduced pressure, yielded crude TCN. By purifying the crude TCN on a chro atographic column (silica gel, ethyl acetate-hexane), an amount of 290 mg of pure TCN (70% yield) was obtained in the form of a yellow solid. The analysis of the product on H NMR (CDC *, was as fol lows : 7.52 (d,J=8.6Hz,4H), 6.64 (d , =8.6Hz , 4Hz), 6.31 (s, 10H).

EXAMPLE 2. Preparation of Bis(methoxycarbonylphenolato)

5 bis(n -cyclopentadienyl )titanium( IV) TPE.

In the same manner as in Example 1, an amount of 273mg (2 mmol) of methyl 4-hydroxybenzoate was reacted with 249 mg

(1mmol) of titanocene dichloride. An amount of 364 mg of TPE (81% yield) in the form of a yellow solid was obtained . The analysis of the product on H NMR (CDC1 ₃ ) was as fol lows :

7.91 (d,J=8.7 Hz, 4H),6.64 (d,J=8.7Hz, 4H), 6.33 (s,10H),

2.56 (s, 6H).

EXAMPLE 3. Preparation of Bis(4-ethoxycarbonyIphenolato)-

5 bis(n -eyelopentadienyl)ti anium(IV) TEE1.

In the same manner as in Example 1, an amount of 332mg

(2mmol) of ethyl 4-hydroxybenzoate was reacted with 249mg

(1 mmol) of titanocene dichloride. An amount of 417 mg of

TEE1 (81% yield) was obtained.

The analysis of the product on ¹ H NMR (CDC1 ₃ ) was as follows:

7.90 (d, J=8.6 Hz, 4H), 6.61 (d, J=8.6Hz, 4H) ,

6.31 (s, 10H), 2.94 (q, J=7.3Hz, 4H) ,

1.21 (t, J=7.3 Hz, 6H).

EXAMPLE 4. Preparation of Bis[4-(2-(2-methoxyethoxy)

5 ethoxy]carbonylphenolato]bis(n -cyclopenta¬ dienyl )titanium(IV) TEG

(a) In a first step, an amount of 1g (43 mmol) of sodium was dissolved in 25 ml of 2-(2-methoxyethoxy)ethanol . To the resulted solution an amount of 3 g (22mmol) of methyl 4-hydroxybenzoate was added and the mixture was heated tc

130°C for 24 hours; after cooling to room temperature, it was acidified with a hydrochloric acid solution (3N) and extracted with ethyl acetate. The removal of the solvent under reduced pressure and column chromatography of the residue (silica gel, hexane:ethyl acetate 3:1) afforded 2-(2-methoxyethoxy)ethyl 4-hydroxybenzoate , in the fcrm of a colourless oil in essentially quantitative yield ^, (b) In the second step, an amount of 480 mg (2mmol) of the product obtained in step (a), was reacted with 249 mg (1mmol) of titanocene dichloride, as described in Example 1. An amount of 355 mg of TEG (54% yield) was obtained. The analysis of the product on ¹ H NMR (CDC1 ₃ ) was as fol lows :

7.95 (d, 0=8.6 Hz, 4H), 6.60 (d, J=8.6 Hz, 4H) 6.31 (s, 10H), 4.45 (t, J=5.0Hz, 4H),

3.83 (t,J=5.0Hz, 4H), 3,70 (t, J=4.6 Hz, 4H), 3.57 (t, J=4.6 Hz, 4H), 3.38(s, 6H).

EXAMPLE 5 Preparation of Bi s [4- (2-methoxy )ethoxycarbonyl -

5 phenolato]bis(n -cyclopentadienyl )titanium(IV) TMEM.

(a) In the first step (as in the Example 4) 1 g (43 mmol) of sodium was dissolved in 25 ml of 2-methoxyethanol . An amount of 3.0 g (22 mmol) of methyl 4-hydroxy-benzoate was added, producing 2-methoxyethyl 4-hydroxybenzoate, as a colourless oil, in essentially quantitative yield.

(b) In the second step, an amount of 392 mg (2mmol) of the product obtained in step (a) was reacted with 249 mg (1.1 mmol) of titanocene dichloride as described above in

Example 1, affording 330 mg of TMEM (58% yield).

*] The analysis of the product on H NMR (CDC1 ₃ ) was as fol lows :

7.96 (d, J=8.6 Hz, 4H), 6.60 (d, J=8.6 Hz, 4H),

4H), 6.29 (s, 10H), 4.42 (t, J=4.8 Hz, 4H),

3.70 (t, J=4.8 Hz, 4H), 3.70 (t, J=4.8 Hz, 4H),

10 3.40 (s, 6H).

EXAMPLE 6: Preparation of Bis[4-(2-dimethylamino)ethoxy- carbonylphenolato]bis(n ^" cyclopentadienyl ) titanium(IV) TCA

(a) In a first step, 1 g (43mmol) of sodium was dissolved l ₅ in 20 ml of 2-(dimethylamino)ethanol . An amount of 3.0 g

(22 mmol) of methyl 4-hydroxybenzoate was added and heat¬ ed to 110°C for 24 hours and then cooled to room tempera¬ ture. The solvent was removed under reduced pressure and using a column chromatography (silica gel, chloroform 2o methanol), a white solid of 2-(dimethylamino ) -ethyl 4- hydroxybenzoate was obtained.

(b) In the second step, an amount of 418 mg (2 mmol) of the product obtained in step (a) was reacted with 249 mg (Immol) of titanocene dichloride, as described in Example

25 1, affording 330 mg of TCA (58% yield).

1 The analysis of the product on H NMR (CDC1***,) was as fol lows :

7.94 (d,J=8.6 Hz, 4H), 6.59 (d, J=8.6Hz, 4H),

6.30 (s, 10H), 4.39 (t, J=7.2 Hz, 4H), 2.71 (t, J=7.2Hz, 4H), 2.34 (s, 12H).

EXAMPLE 7: Preparation of Bis[4-(2-trimethylamino)etho- xy-carbonylphenolato]bis(n -cyclopentadienyl ) titaniu (IV) .

The TCA product as obtained in the previous Example 6, was treated with an excess of methyl iodide (10 equiv) in benzene for about 6 hours. A yellow solid of TCE is formed, collected by filtration, washed by benzene and ether and dried.

^• 1

The analysis of the product on H NMR (DMSO) was as follows:

7.87 (d, J=8.6 Hz, 4H), 6.66 (d, J=8.6 Hz, 4H),

6.44 (s, 10H), 4.64 (m, 4H), 3.77 ( , 4H), 3.18 (s, 18H).

TABLE 2; DATA ON CYTOTOXICITY (log).