WO1992013851A1

WO1992013851A1 - Pharmaceutical product for the therapy of tumors, particularly ovarian and haemopoietic system tumors, containing quercetin compounds as active substances

Info

Publication number: WO1992013851A1
Application number: PCT/IT1992/000007
Authority: WO
Inventors: Salvatore Mancuso; Giovanni Scambia; Pier Luigi Benedetti-Pacini; Giancarlo Folchitto; Mauro Piantelli; Franco Oreste Ranelletti; Arnaldo Cappelli
Original assignee: Romeo, Francesco; Dalla Costa, Giorgio
Priority date: 1991-02-05
Filing date: 1992-01-31
Publication date: 1992-08-20
Also published as: EP0570475A1; IT1244647B; ITRM910085A1; JPH07502009A; ITRM910085A0; AU1269192A

Abstract

A quercetin compound selected from quercetin zinc complex, quercetin selenium complex, fluorinated quercetin compound, glycosidic quercetin compound, pentaacetylquercetin, trimethylquercetin, tetramethylquercetin, and pharmaceutical preparations for tumor therapy.

Description

PHARMACEUTICAL PRODUCT FOR THE THERAPY OF TUMORS, PARTICULARLY OVARIAN AND HAEMOPOIETIC SYSTEM TUMORS, CONTAINING QUERCETIN COMPOUNDS AS ACTIVE SUBSTANCES.

The present invention is generally concerned with pharmaceutical products for the therapy of tumors, particularly with those containing quercetin as the active substance . Quercetin is a natural substance belonging to the group of flavonoids, compounds of plant origin showing several biochemical and pharmacological activities. This substance has been and still is the subject of studies for its capacity of interfering with chemical carcinogenesis processes. For instance, as a result of several studies, it shows mutagenic activity in the Amnes test, in procariotes (Sugimura et al. , Proc. Jour. Acad. 53B 194, 1977), in Salmonella typhi urium (MacGregor et al. , Mutation Res. 54 297, 1978) and in eucariotes (Amacher et al. , Mutation Res. 72 447, 1980), and it had been suggested that it could act as initiator in carcinogenesis. Later studies (Saito et al., Teratog. , Carcinog. , Mutagen. 1, 213, 1980; Hirono et al. , Cancer Lett. 13, 15, 1981; Morino et al. , Carcinog. 3, 93, 1982) showed on the contrary, that quercetin has an antiproliferative activity, in particular in breast cancer (Ajit et al. , Cancer Res. 48, 5754, 1988), and therefore it mav be considered useful in tumor therapy. It was also suggested that the activity of this substance may consist in an interaction with a receptor named "type II estrogen bindinc site" (Markaverich et al., J.Steroid Biochem 30 71, 1988). The practical use of quercetin in human tumor therapy was sofar prevented, however, by the observation that it is retained in the organism into which it is introduced, both by intravenous and oral way, for a time insufficient for the display of its activity in a good degree.

The present inventors carried out studies and experiments on several cell lines derived from tumors of different origin, and they found support for the hypotesis that the antiproliferative activity of quercetin is mediated by the "type II EBS" cell receptors. Moreover, the inventors ascertained that these receptors are present also in primary human tumors, particularly in those of ovaries and of the haemopoietic system, and possess characteristics similar to those found in cultured cells. The inventors have also evaluated the dependency of the quercetin effect from time, and found that the antiproliferative activity becomes evident 24 hours after the addition of quercetin to an ovarian carcinoma cell culture. They also showed that the inhibitory effect of quercetin is not due to a non-specific cytotoxic activity. However, considering that such ascertainments and remarks have a limited practical utility due to the above mentioned unability of the human organism to retain quercetin, the inventors wanted to find a quercetin containing product showing a longer time of retention by the

_* organism in order to utilize the antiproliferative properties in a product pharmacologically active against human tumors. This purpose has been achived, according to this invention by preparing a product containing at least one of the following substances: complex of quercetin with zinco, complex of quercetin with selenium, fluorinated quercetin compound, glycosidio quercetin compound, pentaacetylquercetin, tri- and tetramethylquercetin.

The preparation of the foregoing substances was carried out by the processes described in the following examples or obvious modifications thereof not described in detail because they are well known to those skilled in the art.

Each of these substances showed an antiproliferative activity comparable to or higher than quercetin. Moreover, their chemical and pharmacocynetic stability allows their use in therapy of human tumors. In particular, it was noted that when the invention product is added in vitro to cell lines. its metabolization time is longer than the time necessary for the metabolization of quercetin. As to the dosages to be administered, therapeutically useful results may be obtained by administering a product containing these substances in such amount to convey into the organism blood levels of quercetin starting from 0,01 micromoles/ml .

The antiproliferative tests on the tumoral lines were carried out according the following method: cells were plated at 4 2 10 /cm in Falcon of 24 wells having 16 mm of diameter. After

18 hours the growth medium was replaced with fresh medium containing the compounds to be tested or the carrier alone (ethanol). This treatment was repeated after 24 hours. After additional 24 hours of culture, cells were treated with tripsine and counted in a haemocytometric chamber. A portion was tested with trypan blue for the cellular vitality. In the following examples the preparation of the claimed compounds is described.

Example 1_.

Quercetine complex with zinco

The reaction in ethanol between zinc acetate and quercetin (1 to 1 moles) yields a substance insoluble in ethanol, which precipitates in the reaction medium; insoluble in water and scarcely soluble in dimethylsulfoxide. On a diluted methanol solution of this quercetin derivative, UV readings were made using complexes with A1C1 , which on the basis of batochromic

3 and ipsochromic shifts give information on the coordination of zinc. These readings showed that the zinc complex involves the two near hydroxy groups in positions 3', 4'.

Example 2

Pentaacetylguercetina. A quercetin sample (60 mg) was treated at room temperature with a mixture of Pyridine and acetic anhyd~ide (1:1) overnight. The reaction mixture was evaporated in_. vacuo and purified by column chromatography using silica gel as fixed phase and chloroform as diluent. A pure substance was thus obtained (84 mg) , crystallized from acetone, m.p. 197-201°C.

1 13 The analysis of H and C NMR data allowed to identify this compound as 3, 5, 7,3',4' -pentaacetylquercetin. The UV spectrum in methanol, which does not undergo batochromic shift by addition of A1C1 , is in agreement with the acetylation

3 also of the hydroxyl at position 5 (OH chelate). 1 H RMN (CDC1 ), c • 2.33, 2.34, 2.43 (6+6+3H, 3s, 5X0Ac) ; 3 6.87 (IH, d, J=2 Hz, H-6); 7.33 (IH, d, J=2 Hz, H-8); 7.35 (IH, d, J-8 Hz, H-5'); 7.69(1H, d, J=2 Hz, H-2'); 7.72 (IH, dd, J«8 e 2 Hz, H-6' ) . 13

C RMN (CDC1 ), ppm: 20.5, 20.6 (x2) , 21.0, 21.1 (OCOMe.);

3 a a

108.9 (C-8); 113.8 (C-6); 114.7 (C-10); 123.8 (C-5'); 123.9

(C-2'); 126.4 (C-6'); 127.7 (C-l')_; 134.0 (C-3); 142.1 (C-3') 144.3 (C-4'); 150.3 (C-2); 153.7 (C-9); 154.2 (C-5); 156.8

(C-7); 167.72, 167.77, 167.80, 167.83, 169.22 (OCOMe); 169.99

(C-4). a interchangeable

max' Example 3.

Tri- and tetramethylquercetin:

A quercetin sample (75 mg), dissolved in a small volume of methanol, was methylated with a diethyl ether solution of diazomethane. After about 8 hours the reaction mixture was evaporated, taken up with CHCl and purified on silica gel

3 (CHCl ). Two pure substances were obtained and were

3 identified as 3, 7 , 3 ',4 '-tetramethylquercetin (16 mg) and

3, 7 , 4 '-trimethylquercetin (20 mg). 3, 7 , 3' , 4 ' -tetramethyl- o + αuercetin crystallizes from acetone, m.p. 153-156 C, M at m/z 358 (EIMS) . 1 H RMN (CDC1 ) . f -. 3.86. 3.96 (6+6H, 2ε, 4XOMe) ; 6.32 (IH, d. 3 J=2 Hz, H-6); 6.41 (IH, d, J=2 Hz, H-8); 6.97 (IH, d, J=8 Hz,

H-5') : 7.6-7 flH, d, J=2 Hz, H-2' ): 7.71 (IH, dd, J=8 e 2 H:, H-6 ' ) ; 12 . 63 ( IH , s , 5-OH ) . 13

C RMN (CDC1 ), ppm: 55.7, 55.8, 55.9 (OMe in C-7, C-3', C- 3 4'); 60.0 (OMe in C-3); 92.0 (C-8): 97.7 (C-6); 105.8 (C-10): a a

110.7 (C-5'); 111.0 (C-2'); 122.0 (C-6'); 122.7 (C-l'); 138.8 (C-3); 148.6 (C-3'); 151.2 (C-4'); 155.6 (C-2); 156.5

(C-9); 161.8 (C-5); 165.3 (C-7); 178.5 (C-4). a interchangeable

UV (MeOH).λ- nm: 351, 253; (MeOH + A1C1 ): 401, 360, 268; max' 3

(MeOH + A1C1 + HC1): unchanged. 3 3,7,4'-trimethylquercetin crystallizes from CHCl , m.p.

³ 179 C. M at m/z 344 (EIMS).

1

H RMN (acetone-d ), & : 3.89, 3.93, 3.96 (3+3+3H, 3s,

6

3XOMe); 6.32 (IH, d, J-2 Hz, H-6); 6.69 (Ih, d, J=2 Hz, H-8);

7.14 (IH, d, J-8 Hz. H-5'); 7.66 (IH. d. J=2 Hz, H-2'); 7.70 (IH, dd, J=8 e 2 Hz, H-6'); 12.74 (IH, s 5-OH). 13

C RMN (CDC1 ), ppm: 55.8, 56.0 (OMe in C-7 e C-4'); 60.1 3 (OMe in C-3); 92.1 (C-8); 97.9 (C-6); 105.4 (C-10); 110.4 (C-

5'); 114.5 (C-2'); 121.6 (C-6'); 123.7 (C-l'); 139.2 (C-3);

145.6 (C-4'); 148.8 (C-3'); 155.6 (C-2); 156.8 (C-9); 162.1 (C-5); 165.5 (C-7); 178.8 (C-4).

UV

nm: 354, 268sh, 254; (MeOH+AlCl ): 402, 360, max' 3

300. 268: (MeOH+AlCl +HC1): unchanged; (MeOH+NaOMe) : 384,

3 265; (MeOH+NaOAc) : 354, 268sh, 254.

Examples 4-6 Quercetin complex with selenium

Analogously as with zinc, quercetin is reacted with selenium acetate (1:1): three quercetin salts with selenium can be obtained. Fluorinated quercetin compound.

Quercetin is reacted with one of the following substances:

AgF . CH COOF and CsSO F, obtaining several fluorinated

2 3 4 quercetin compounds. Glycosidic quercetin compound.

Quercetin is reacted with glycosylacetobromide in the presence of sodium methoxide; various polyglycosylates are obtained.

Claims

Claims 1. A quercetin compound selected from quercetin zinc complex, quercetin selenium complex, fluorinated quercetin compound, glycosidic quercetin compound, pentaacetylquercetin, trimethylquercetin, tetramethylquercetin.

2. Quercetin zinc complex at 3' ,4 '-hydroxyls of quercetin.

3. 3, 5,7,3',4' -Pentaacetylquercetin.

4. 3,7,4' -Trimethylquercetin.

5. 3,7,3',4' -Tetramethylquercetin.

6. A pharmaceutical preparation for tumor therapy, characterized by the fact that it contains as active substance a quercetin compound selected among those of claims 1 to 5 or a mixture of two or more of these compounds.

7. Use of a pharmaceutical preparation according to claim 6, in dosages sufficient to obtain a blood concentration of quercetin of at least 0,01 micromoles per milliliter.

8. Process for the production of the compound according to claim 1, characterized in that it comprises at least one of the following steps: - production of a quercetin salt with zinc by a reaction of quercetin with zinc acetate in methanol:

- production of a quercetin salt with selenium by a reaction, of quercetin with selenium acetate;

- production of a quercetin fluorinated compound by a reaction of quercetin with one of the following substances:

AgF , CH COOF and CaSO F:

2 3 4

- production of ? σlvcosidic σuercetin compound bv = reaction of quercetin with glycosylacetobromide in the presence of sodium methoxide.