Title of Invention

"ANIONIC COMPLEX OF RU(III) WITH AN AMMONIUM CATION AND PROCESS FOR PREPARATION THEREOF"

Abstract A Salt of an anionic complex of Ru(III) with an ammonium cation of formula (I): Where R1, R2 and R3, equal or different from each other, are selected from the group consisting of H, C1-C6 alkyl, linear or branched, saturated or unsaturated, C3-C7 cycloalkyl, phenyl and aryl; or NR1R2R3 is a 5-7 membered. nitrogen-containing heterocycle, saturated or unsaturated, optionally containing one or more O, S, and/or N atoms, said nitrogen atom being optionally substituted with a C1-C4 alkyl, aryl or benzyl residue; said nitrogen containing heterocyde being optionally condensed with a benzo group and/or substituted with C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkoxyl, C1-C4 alkylthio, aryl or benzyl groups; where R4 and R5, equal or different from each other, are selected from the group consisting of H, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl and aryl or R4 form, together with the S atom, a 4-7 membered heterocyde. Furthermore the process for the preparation of said salts, the pharmaceutically compositions containing the same and their use as antimetastatic and antineoplastic agents are described.
Full Text 1A
NEW SALTS OF ANIONIC COMPLEXES OF RU(III), AS ANTIMETASTATIC AND ANT1NEOPLASTIC AGENTS FIELD OF THE INVENTION
The present invention relates to new salts of anionic complexes of Ru(lll) with lammonium cations which are particularly useful as antimetastatic and "antineoplastic agents. STATE-OF-THE-ART
The discovery of the antineoplastic properties of cisplatin (cis-diammino-dichioro-platinum (II)), so far widely used both in antitumor mono- and polychemotherapy, has fostered the interest in the study of the antitumor activity of the metallo-organic complexes. As for many antitumor agents, the specificity of action of the cisplatin, is directed towards neoplasms which affect specific compartments (testis, ovary, bladder, head-neck), while other kinds of tumors, such as those localised in the lungs, and, in the breast and the colon-rectal tumor,.are almost insensitive to treatment with this drug (C.F.J. Barnard et a!., Chemistry in Britain, 1001-1004, 1986).
Thus, research in the field of the coordination complexes is aimed at the development of new drugs containing Pt and/or other transition elements which can enable both the enlargement of the spectrum of use and the achievement of toxicity levels lower than those of the antitumor agents which are known in the state-of-the-art.
The potentiality of the use of ruthenium as antitumor agent as an alternative to platinum was studied for complexes of Ru (II) like, for example, cis-Ru(ll)-tetrakis-dimethylsulfoxide (T. Giraldi et al., Cancer Res., 37, 2662, 1977) and more recently for neutral complexes of Ru (III), such as fac-[RuCI3 (NH3)3] (M.J. Clarke, Metal Ions in Biological Systems, 11(5), 231-281, 1980, Helmut Sigel Ed.). Moreover, several anionic complexes of Ru(lll) with 5-membered heterocycles have been prepared, in particular imidazolium-bis-imidazole-tetrachloro-ruthenate (111) (B.K. Keppleretal., J. Cancer Res. Clin. Oncol., 111: 166-168, 1986). More recently, some Ru(lll) complexes with DMSO have been developed; in particular, the WO 90/13553 international patent application describes some

2 Ru(lll) complexes having the formula (i):

where RxRySO is a suifoxide, preferably dimethylsulphoxide, A is a suiphoxide or
a chloride and B is a nitrogen ligand, selected from the group consisting of
ammonia, primary, secondary and tertiary amines, and heterocycies containing
nitrogen atoms.
These complexes, which can be used as antineoplastic agents, are neutral when
A is a suifoxide, while they possess a negative charge when A is a chloride; in this
latter case, the anionic complexes are isolated in the form of the corresponding
salts with alkaline and alkaline earth cations and preferably sodium.
Despite their antimetastatic activity, the above mentioned complexes exhibit some
serious inconveniences which make the administration and formulation in
adequate therapeutical compositions extremely difficult. As a matter of fact, these
anionic complexes when isolated in the form of sodium salts, always contained
two solvent.molecules of crystallisation and cannot be isolated in a pure form, that
is without these crystallisation molecules.
in particular, the [trans-RuCl4(DMSO)lm]Na 2DMS0, corresponding to formula (i)
where Rx=Ry=methyl, A=CI, B=imidazole and where the negative charge has been
neutralized by Na+, is capable of exerting good antineoplastic and antimetastatic
activities on tumor models in mice. Moreover, this salt has been characterised by
good water solubility which allows for an easier administration.
However, this compound can be isolated only with two DMSO solvation
molecules, as evidenced in the examples reported in the above mentioned"
international patent application (in particular, in the Examples 3, 4, 8 and 10)
which reduce the compound stability.
Moreover, a qualitative variability of the crystallisation molecules, which can be
DMSO, acetone and water, and more commonly two dimethylsulfoxide molecules
(DMSO) (E. Alessio et al. lnorganica Chimica Acta, 203 (1993): 205-217) can be
observed.

3
The fact that the crystallisation molecules vary in a way which is hardly controlled from one preparation to another causes problems of uncertainty in structural analysis, in the molecular weight determination and in the elemental analysis. Hence, problems related to the of the purity of the compound and its subsequent formulation may occur. As a matter of fact, the scarce structural reproducibility of these compounds implies that not always the same amount of ruthenium corresponds to the administered dose. Thus non-constant dosages and therapeutical activities that cannot easily be foreseen reduce the pharmacological interest of these compounds.
A further disadvantage of the above mentioned compound is due to pharmacological negative effects caused by DMSO, introduced in the organism in equimolar or double quantities with respect to the ruthenium complex and to its differentiating properties, as reported by Klaas Kramer et al. (Gen. Pharmac, vol. 26, n° 6, pp. 1403-1497, 1995).
Finally, since the above mentioned salts contain solvation molecules, they rapidly
*
degrade when exposed to open air at room temperature up to providing some
brown semisolid substance.
SUMMARY
The Applicant has now surprisingly found new salts of Ru(lll) anionic complexes
with ammonium cations which exhibit a remarkable antimetastatic and
where R1 R2 and R3 equal or different from each other, are selected from the
antineoplastic activity. These salts correspond to formula (I):


4
group consisting of H, C1-C6 alkyl, linear or branched, saturated or unsaturated,
C3-C7cycloalkyl, phenyl and aryl;
or NR1R2R3 is a 5-7 membered nitrogen containing heterocycle, saturated or
unsaturated, optionally containing one or more O, S and /or N atoms, said
nitrogen atom being optionally substituted with a C1-C4 alkyl, aryl or benzyl
residue; said nitrogen containing heterocycle being optionally condensed with a
benzo group and/or substituted with C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkylthio, aryl
or benzyl groups;
where R4 and R5, equal or different from each other, are selected from the group
consisting of H,C1-C6 alkyl, C3-C7cycloaikyl, phenyl and aryi or R4 and R5form,
together with the S atom , a 4-7 membered heterocycle.
A further object of the present invention is a new process for the preparation of
the above salts (1) which comprises the synthesis of [trans-RuCI4(R4R5SO)2] [
(R4R5SO)2H] (II), where R4 and R5 have the above meanings, which is obtained by
treatment of RuCI3 with R4-SO-R5 in the presence of HCI, and the following
reaction of the complex (II) with a nitrogen containing compound of formula
NR1R2R3, in the presence of an organic solvent.
Moreover, the present invention relates to the pharmaceutical compositions
containing therapeutically effective amount of at least one of the above salts of
formula (I), in combination with the proper excipients and/or diluents and their use
as antineoplastic and/or antimetastatic agents.
DESCRIPTION OF THE FIGURES
Figure A1 and Figure A2 respectively show the external connective capsule and a
necrotic area of the tumor tissue of the control mice affected by MCa mammary
carcinoma.
Figures B1 and B2 respectively show the external connective capsule and a
necrotic area of the tumor tissue in mice affected by MCa mammary carcinoma,
after treatment with [trans-RuCl4(DMSO)lm]Na 2DMS0, as described in the
above mentioned WO 90/13553 international patent application.
Finally, the C1 and C2 Figures exhibit the external connective capsule and a
necrotic area in the tumor tissue of mice affected by MCa mammary carcinoma,

5
after treatment with the salt [trans-RuCl4(DMS0) (Im)] [ImH] (Example 2) of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
The features and the advantages of the present invention will be better illustrated in the course of the following detailed description.
In the above mentioned salts of formula (I), the nitrogen atom of the nitrogen compound of formula NR1R2R3 supplies its long pair in the formation of the coordination bond with the Ru(lll) atom.
More specificafly, in the salts of the invention, R1 is preferably the same as R2 and R3 and they are H or ethyl.
When NR1R2R3 is a 5-membered nitrogen containing heterocycle, it selected from the group consisting of imidazole, N-methyl-imidazole, pyrazole, and oxazole; most preferably said nitrogen-containing heterocycie is imidazole. When NR1R2R3 is a 6-membered heterocycie, it is selected preferably in the group consisting of pyridine, pyrazine, 3,5-lutidine and 4-methylpyridine. When NR1R2R3 is a 7-membered heterocycie, it is selected from the group consisting of azepine, diazepine, and oxazepine.
Finally, when said heterocycie is condensed with a benzo group, it is selected preferably from the group consisting of indazole, isoquinoline, benzimidazole and 1,5,6-trymethyl-benzimidazole.
In the salts of the invention of formula (I), the R4-SO-R5 sulphoxide ligand is preferably dimethylsuifoxide (R4=R5=methyl), diethylsulphoxide (R4=R5=ethyl) or tetramethylensulphoxide (together with the S atom, R4 and R5 form a 5-membered ring).
These salts can unexpectedly exert an antitumor and in particular antimetastatic activity which is significantly higher than that of the corresponding sodium salts, when equimolar doses and equal treatment patterns are applied. Moreover, the salts of formula (I), with respect to those described in the state-of-the-art, are much more favourable since they are indefinitely stable in the air, while the corresponding sodium salts, being extremely hygroscopic, are easily subject to hydrolysis. In addition to that, with respect to the known complexes, the

6
salts of the invention do not exhibit the pharmacological drawbacks which are due
to the presence of DMSO crystallisation molecules.
Since the salts of formula (I) have no crystallisation molecules, they have constant
MW values as well as reproducible analytical results. This allows for formulations
containing definite amounts of active compound and consistently constant
dosages.
The salts of Ru(lll) complexes of formula (I) are obtained by means of a new
particularly easy and favourable procedure which comprises the following steps:
1) reactions of RuCI3 with R4-SO-R5 in the presence of HCl to produce the
complex of formula (Il)

where R4 and R5 have the above reported meanings.
In this reaction step, R4 and R5 are preferably methyl, that is RUCI3 undergoes
reaction with dimethyisulfoxide (DMSO) to produce [(Me2S0)2H] [trans-
RuCl4(Me2SO)2].
According to a preferred way of preparation, RUCI3 is previously dissolved in a
heated organic solvent, preferably ethanol or methanol;. R4-SO-R5 and
concentrated HCI are added to the solution obtained in this way and heated at a
temperature ranging preferably from 60 and 90°C, and even more preferably at a
temperature of about 80°C.
2) The complex (II), obtained from step (1), is reacted at room temperature with a
NR1R2R3 nitrogen containing compound, in molar ratios ranging from 1:2 to 1:6 in
one or more organic solvents, preferably acetone and dichloromethane, to provide
the salts of formula (I), according to the scheme reported hereunder:

7

where R1-R5 have the previoulsy defined meanings.
In the step (2), the intermediate (II) reacts with the NR1R2R3 nitrogen containing
compound by leading to the replacement of one of the two axial sulphoxide
groups and to the protonation of a molecule of the nitrogen compound and to the
final release of two sulphoxide crystallisation molecules. Thus the complexes (I)
are obtained in very high yields.
A further object of the present invention is the use of the above salts (I) in the
treatment of neoplasms of a various nature and in the prevention of the formation
of metastases. The above neoplasms are preferably solid spawning tumors, such
as the carcinoma of the gastro-intestinal tract, the mammary carcinoma, the lung
tumors, the metastatic carcinoma and the lung metastases of metastatic tumors.
The salts of the invention can be profitably administered by parenteral, oral,
topical or transdermal route.
Among the parenteral administration, the intravenous, intramuscular,
intraperitoneal and subcutaneous routes are preferred.
The dosage of these salts varies according to the route and ways of
administration as well as to the seriousness of the neoplasms; besides, it varies in
relation to age, to the body weight and to the general healthiness of the patient.

8
The therapeutically effective close of these salts, to be administered in single or
multiple doses, ranges preferably from 0.1 to 300 mg/kg/day and even more
preferably is 10-200 mg/kg/day, when these salts are administered parenterally,
while orally the dosages are from 3 to 10 times higher than the above reported
range.
Moreover, the above salts (I) can profitably be used in experimental protocols of
polychemotherapy in combination with other antitumor drugs of common clinical
use in the above described pathologies, such as for example cisplatin, 5-
fluorouracil, vinblastin, cyclophosphamide, bleomycin, anthracycline, taxol.
The present invention also relates to pharmaceutical compositions containing, as
an active compound, a therapeutically effective amount of at least one salt of
formula (I) in combination with suitable excipients and/or diluents.
These pharmaceutical compositions can be prepared in the form of solutions or
suspensions, both in aqueous and non-aqueous media, which are particularly
suitable for intravenous injections, infusions and intramuscular or subcutaneous
injections. These solutions can be prepared before use, by solubilizing or
suspending the lyophilized compounds of the invention in suitable solvents.
Solid or semisolid compositions in the form of inserts, gels or ointments for
topical, dermal or transdermal administration or in the form of powder, pills,
tablets, and capsules are also indicated. Furthermore, the salts of the invention
can be administered in the form of controlied-release compositions which are
known in the state-of-the-art.
The above compositions can be prepared easily according to procedures which
are known in the state-of-the-art.
The following examples are reported to illustrate the invention without limiting it:
EXPERIMENTAL PART
Example 1:
Preparation of [trans-RuCI4(Me2SO)2][ (Me2SO)2H] complex corresponding to
formula (II) where R4=R5=methyl.
1 g of RUCI3 3 H2O (0,0038 moles) was suspended in 30 ml ethanol and
underwent reflux heating for three hours to obtain a dark green solution. The

9
solution was filtered on paper to remove possible traces of undissolved solid; it was then concentrated by using a rotary evaporator up to 1/10 of the initial volume; therefore, 1 ml of aqueous concentrated HCI, 37% and 2 ml of DMSO were added and the mixture obtained in this way was kept at a temperature of 80°C for about 15 minutes, up to obtaining a bright orange solution. After cooling of the mixture to room temperature and after addition of 10 ml acetone, the product, after setting up, separated from the solution in the form of red-orange crystals; the formation of these crystals was accelerated by the addition of a few drops of ethyl ether. The crystals were then collected on a filter, washed with cold acetone (20 ml), then with ethyl ether (10 ml) and finally dried under vacuum at room temperature. 1.5 g of the final product with a 72% yield was obtained.
The physico-chemical features of [trans-RuCI4(Me2SO)2][ (Me2SO)2H] are as follows:
- physical state: a red-orange, crystalline solid
- raw formula: C8H25Cl4O4RuS4
-molecular weight: 556,40
- elemental analysis:
experimental:
C=17,33, H=4,61 Cl=25,2 S=23,2
theoretical:
C=17,27, H=4,53 Cl=25,48 S=23,04
Moreover, the IR and UV spectra and the determination of the structure by means
of X-rays turned out to be in agreement with what reported by E. Alessio et al.
(above mentioned reference).
Example 2:
Preparation of [trans-RuCI4(DMSO) (lm)][lmH] corresponding to formula (I)
where NR1R2R3 is imidazole (Im) and R4=R5=methyl.
1,0 g (0,0018 moles) of the [trans-RuCI4(Me2SO)2][ (Me2SO)2H] complex prepared
as described in Example N.1, was suspended in acetone (20 ml) at room
temperature.

10
After the addition of 0.49 g (0.0072 moles) of imidazole, the mixture was kept under stirring for 4 hours; during this time the colour of the precipitate gradually changed from orange to brick red. After being collected on a filter and washed with acetone (10 ml) and therefore with ethyi ether (10 mi), the product was dried under vacuum at room temperature or in a oven at 60°C for a few hours. Hence, 0.75 g of final product were obtained with a 92% yield. The physico-chemical properties of [trans-RuCl4(DMSO) (lm)][lmH] are as follows:
- physical state: a brick-red crystalline solid
- raw formula: C8H15N4Cl4ORuS
-molecularweight: 458,17
-elemental analysis:
experimental:
C=20,8, H=3,30 N=12,2
theoretical:
'C=20,87, H=3,30 N=12,23
- 1H-NMR spectrum in D2O (ppm vs DSS): -15,2 (Very broad, DMSO),
- 3,53 (broad, H2 Im), 7,48 (2, H3 and 4H ImH*), 8,70 (1, H2 lmH+);
- IR spectrum (selected frequencies, Nujoi, cm-1): VNH 3150 (very broad,
medium), vso 1159 (very strong), vRu-S 421 (medium), vRu-CI 342 (strong).
Example 3:
Preparation of [trans-RuCI4(DMSO) (1-Me-lm)][1-Me-lmH] corresponding to
formula (I) where NR1R2R3 is 1-methyl-imidazole and R4=R5=methyl.
1,0 g (0,0018 moles) of the [trans-RuCl4(Me2SO)2][ (Me2SO)2H] complex prepared
as described in Example N.1, was treated with 0,59 g (0,0072 moles) of 1-
methylimidazole as described in Example N.2, and 0.8 g of final product were
obtained in a 93% yield.
The physico-chemical properties of the [trans-RuCl4(DMSO)(1-Me-irn)][1-!V!e-
ImH] are as follows:
- physical state: a brick-red crystalline solid
- raw formula: C10H19N4CI4ORUS
-molecularweight: 486,23

11
- elemental analysis:
experimental:
C=24,8, H=3,83 N=11,4
theoretical:
C=24,7, H=3,94 N=11,52
- 1H-NMR spectrum in D20 (ppm vs DSS): -15,7 (Very broad, DMSO),
- 3,76 (broad, H2 1-Me-lm), -0,95 (broad, Me1, 1-Me-lm) 3,95 (3, Me1, 1-Me-
lmH+), 7,45 (2, H3 and H4 1-Me-lmH+), 8,68 (1, H2 1-Me-lmH+);
- IR spectrum (selected frequencies, Nujol, cm"1): vNH 3150 (very broad, medium),
vSO 1093 (very strong), vRu-S 424 (medium), v Ru-CI 326 (strong).
Example 4
Preparation of [trans-RuCU(DMSO) (Py)][PyH] corresponding to formula (I)
where NR1R2R3 is pyridine and R4=R5=methyl.
1,0 g (0,0018 moles) of the [trans-RuCl4(Me2SO)2][ (Me2SO)2H] complex prepared
as described in Example 1, was treated with 0,57 g (0,0072 moles) of pyridine as
described in Example 2. 0,8 g. of the final product were obtained with a 94% yield.
The physico-chemical features of the [trans-RuCU(DMSO) (Py)][PyH] are as
follows:
- physical state: a dark yellow microcrystalline solid
- raw formula: C12H17N2Cl4ORuS
- molecular weight: 480,22
- elemental analysis:
experimental:
0=31,8, H=3,77 N=5,43
theoretical:
C=30,01 H=3,57 N=5,83
- 1H-NMR spectrum in D2O (ppm vs DSS): -14,5 (Very broad, DMSO),
- 2,90 (broad, Py), 8,10, 8,65, 8,82 (PyH+);
IR spectrum (selected frequencies, Nujol, cm-1): vNH 3150 (very broad, medium), vSO 1074 (very strong), v Ru-S 432 (medium), v Ru-CI 344 (strong).

12
Example 5
Preparation of [trans-RuCU(DMSO) (NH3)][NH4] corresponding to formula (I)
where NR1R2R3 is NH3 and R4=R5=methyl.
1,0 g (0,0018 moles) of the [trans-RuCI4(Me2SO)2][ (Me2SO)2H] complex prepared
as described in Example 1, was suspended in CH2CI2 (20 ml) at room temperature
after creating vacuum by a water pump, gaseous NH3 was bubbled and the
system obtained in this way was/ kept under stirring for 4 hours, at room
temperature, under ammonia atmosphere; during this time, the colour of the
precipitate gradually passed from orange to dark red.
The product collected on a filter and washed with cold CH2CI2 and then with ethyl
ether (10 ml), was then dried under vacuum at room temperature.
The physico-chemical features of [trans-RuCI4(DMSO) (NH3)][NH4J are as
follows:
- physical state: brick red microcrystafline solid
- raw formula: C2H13N2CI4ORuS
-molecularweight: 356,08
- elemental analysis:
experimental:
C=6,75 H=3,44 N=7,63
theoretical:
C=6,75 H=3,68 N=7,87
- IR spectrum (selected frequencies, Nujol, cm-1): vNH 3304, 3174 (medium), vSO
1069 (very strong), \/Ru-N 458 (weak), vRu-S 431 (medium), vRu-CI 334, 321
(strong).
Example 6:
Preparation of [trans-RuCI4(DMSO) (Pyr)][PyrH] corresponding to formula (I)
where NR1R2R3 is pyrazine (Pyr) and R4=R5=mef/jy/.
1,0 g (0,0018 moles) of the [trans-RuCI4(Me2SO)2][ (Me2SO)2H] complex prepared
as described in Example N.1, was treated with 0,43 g (0,0053 moles) of pyrazine
as described in Example N.2, and 0.5 g of final product were obtained in a 57%
yield.

13
The physico-chemical properties of the [trans-RuCI4(DMSO) (Pyr)][PyrH] are as follows:
- physical state: a dark yellow crystalline solid
- raw formula: C10H15N4Cl4ORuS
- molecular weight: 482,20
- elemental analysis:
experimental:
C=247, H=3,07 N=11,1
theoretical:
C=24,9, H=3,13 N=11,6
-1H-NMR spectrum in D2O (ppm vs DSS): -13,8 (broad, DMSO),- 7,5 (very broad,
H2,6 Pyr), -2,1 (broad, H3,5 Pyr) 8,69 (Pyr)
- IR spectrum (selected frequencies, Nujol, cm-1): vPyrazine1605 (medium), vSO
1076 (very strong), vPyrazine 807 (strong), vProtonated pyrazine 773 (strong), v
Ru-S 435 (medium), v Ru-CI 351,326 (strong).
- UVNIS in water (nm, e (mol-1 cm-1): 400 (4200), 469 (490).
BIOLOGICAL ACTIVITY
i) In vivo activity test of the salts (I), according to the present invention, on
mice affected by Lewis Lung Carcinoma.
Example (i)-A
Materials and methods
Three groups, each made up of 10 female BD2F1 mice of 21 ±1 g, obtained from
Charles River (Calco, Como, Italy) were inoculated by intramuscular injection, with
106 Lewis lung carcinoma cells suspended in 0,05 ml of a Dulbecco's buffered
calcium and magnesium-free solution (PBS) using a sterile insulin syringe.
The tumor line was obtained by the Tumor Repository Bank, NCI, NIH, Bethesda
(USA) and maintained in liquid nitrogen.
Five days after the above inoculation, the average weight of the tumor was equal
to 0,4 ± 0,01 g. From the 5th to the 11th day, the three groups of mice were
treated intraperitoneally as follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free

14
physiological solution;
Group 2-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 44 mg of [trans-RuCU(DMSO) (Im)] Na 2DMSO, as described
in the above mentioned WO 90/13553 international patent application;
Group 3-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 35 mg of [trans-RuCI4(DMSO) (Im)] [ImH] (Example 2);
12 days after inoculation, the primary tumor was surgically removed.
21 days after inoculation, the mice were sacrificed by cervical dislocation and the
lung metastases were counted. The lungs were abducted immediately after death
of the mice and were divided in single lobes, which were then subsequently
examined by the use of a low magnification stereoscopic microscope, equipped
with a grid on the eye-piece, which allowed the detection of a and b orthogonal
axes (where a The lung metastases were then classified according to their dimensions and the
weight of the metastases for each animal was then calculated as the sum of the
weight of each single metastases, each one regarded as a solid by means of the
formula (n /6) a2xb. The experimental data obtained were then processed with the
Student- Newmann-Keuls statistical test.
The results obtained are hereunder reported in Table 1, where the number of
metastases and their weight in both groups of treated mice (2) and (3) are
reported with respect to the control Group (1). The figures are reported as the
average ± S. E. of the single values obtained for each group.

15
Table 1: Antitumor and antimetastatic activity on mice affected by Lewis lung carcinoma, of [trans-RuCU(DMSO) (lm)][lmH] of Example 2 (35 mg/kg/day) with respect to the [trans-RuCl4(DMSO) (lm)]Na 2DMS0 (44mg/kg/day).

* p The figures reported above outline the fact that the treatment with [trans-
RuCU(DMSO) (lm)][lmH] according to the present invention causes a reduction
both in the weight and in the number of metastases which is higher than the one
obtained with the reference compound known in the state of the art.
The % reduction in the number of metastases and in their weight is hereunder
reported in Table 2.
Table 2: % reduction in the number of metastases and in their weight in mice
affected by Lewis lung carcinoma and treated with the compounds according to
the present invention.


16
The advantages obtained with the compound of the invention with respect to the reference compound, as known in the state-of-the-art, are pointed out even more neatly by the figures reported above.
After the above treatments, the lung metastases isolated from the groups (1) and (3) of mice were counted and divided in three groups according to the diameter dimensions (d): metastases with a diameter d2mm (large). The results obtained are hereunder reported in Table 3.
Table 3: distribution according to diameter dimensions (d) of lung metastases in mice affected by Lewis lung carcinoma, treated with the compounds of the present invention.

These figures outline the fact that metastatic nodules of medium and large
dimensions in the group of animals treated are scarcely present (nodules of
medium dimensions) or absent (nodules of large dimension) with respect to the
controls.
Example (i)-B
Materials and methods
Three groups, each made up of 10 female BD2F1 mice of 21 ±1 g, obtained from
Charles River (Calco, Como, Italy) were inoculated by intramuscular injection, with
106 Lewis lung carcinoma cells suspended in 0,05 ml of a Dulbecco's buffered
calcium and magnesium-free solution (PBS) using a sterile insulin syringe.
The tumor line was obtained by the Tumor Repository Bank, NCI, NIH, Bethesda

17
(USA) and maintained in liquid nitrogen.
Five days after the above inoculation, the average weight of the tumor was equal
to 0,4 ± 0,01 g. From the 12th to the 17th day, the three groups of mice were
treated intraperitoneally as follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free
physiological solution;
Group 2-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 2 mg of Cisplatin
Group 3-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 35 mg of [trans-RuCl4(DMSO) (Im)] [ImH] (Example 2).
11 days after inoculation, the primary tumor was surgically removed.
25 days after inoculation, the mice were sacrificed by cervical dislocation and the
lung metastases were counted. The lungs were abducted immediately after death
of the mice and were divided in single lobes, which were then subsequently
examined by the use of a low magnification stereoscopic microscope, equipped
with a grid on the eye-piece, which allowed the detection of a and b orthogonal
axes (where a The lung metastases were then classified according to their dimensions and the
weight of the metastases for each animal was then calculated as the sum of the
weight of each single metastases, each one regarded as a solid by means of the
formula ( /6) a2xb. The experimental data obtained were then processed with the
Student- Newmann-Keuls statistical test.
The results obtained are hereunder reported in Table 4, where the number of
metastases and their weight in both groups of treated mice (2) and (3) are
reported with respect to the control Group (1). The figures are reported as the
average ± S. E. of the single values obtained for each group.

18
Table 4: Antimetastatic activity on mice affected by Lewis lung carcinoma, of [trans-RuCI4(DMSO) (lm)][lmH] of Example 2 (35 mg/kg/day) with respect to Cisplatin (2mg/kg/day).

* p The figures reported above outline the fact that the treatment with [trans-RuCl4(DMSO) (lm)][lmH] according to the present invention causes a reduction both in the weight and in the number of metastases which is higher than the one obtained with the reference compound.
ii) In vivo activity test of the compounds of the invention in mice affected by MCa mammary carcinoma EXAMPLE (ii)-A: Materials and Methods
Three groups of 7 female CBA mice of 23±2 g, obtained from a colony grown according to the procedures for kin animals. The colony obtained originally from Chester Beatthy Institute of London, was subsequently kept by means of serial couplings between kins (brother and sister) in a ratio ranging from 1:1 to 1:4. The couplings took place at the peak of the sexual maturity of the animals born in the 5th week. Animals underweight or with evident organic anomalies were discarded. The three groups were inoculated by intramuscular injection, with 106 Lewis lung carcinoma cells suspended in 0,05 ml of a Dulbecco's buffered calcium and magnesium-free physiological solution (PBS) using a sterile syringe.

19
The tumor line was obtained from Rudjer Boskovic Institute and maintained in
liquid nitrogen.
13 days after inoculation, the average tumor weight was 1.2 ± 0.2 g. From day 13
to 18, the three groups of mice were treated intraperitoneally as follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free
physiological solution;
Group 2-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 44 mg of [trans-RuCl4(DMSO) (lm)]Na . 2 DMSO, as described
in the above mentioned WO 90/13553 international patent application;
Group 3-10 mf/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 35 mg of [trans-RuCl4(DMSO) (Im)] [ImH] (Example 2).
19 days after inoculation, the primary tumor was surgically removed.
27 days after inoculation, the mice were sacrificed by cervical dislocation and the
lung metastases were then counted, as described in the test (i).
The experimental figures obtained were then processed according to Student-
Newmann-Keuls statistical test.
In order to evaluate the activity of the salts of the invention, the primary tumor was
removed surgically from the mice in the three above groups and dissected. One
tumor for each group was selected in a random way and 10 sections/tumor
obtained in such a way to evaluate the whole neoplastic mass. The tumor slices
were fixed in 10% formaline, embedded in paraffin and stained with the Cajal-
Gallego dye in order to evidence the epithelial cells, the connective matrix and the
presence of erythrocytes. The infiltration was evaluated by observing the
degranulation of the polymorphonucleates (PMN) as well as the presence of
apoptotic bodies.
Results:
The results obtained are reported in Table 5, where the number of metastases
and their weight obtained in the groups (2) and (3) with respect to the control
group (1). The figures were expressed as the average ± S.E. of the single values
obtained for each group.

20
Table 5: antitumor and antimetastatic activity of [trans-RuCl4(DMSO) (Im)] [ImH] in Example 2 (35 mg/kg/day) with respect to [trans-RuCI4(DMSO) (lm)]Na . 2 DMSO (44 mg/kg/day) on mice affected by MCa mammary carcinoma,.

* p ** p Table 6: % reduction in the number of metastases and in their weight obtained with the compounds according to the present invention.


21
The results related to the effect of the salts of the invention are hereunder reported in Table 7.
Table 7: Effects of the compounds according to the present invention on the primary tumor.

From the figures reported above it is possible to detect that the [trans-RuCl4(DMSO) (lm)][lmH] compound, with respect to the [trans-RuCI4(DMSO) (lm)]Na 2DMS0 reference compound, 44 mg/kg/day, causes profound hystologic modifications. In particular, the tumor treated with the salt according to the present invention has wide hemorrhagic and necrotic areas combined with

22
neutrophilic leukocytes, a neat degranulation of the polymorphonucleates (PMN),
the presence of a large number of apoptoptic bodies and the thicknening of the
connective capsule surrounding the tumor.
These results are even more evident in the figures where the outer connective
capsule is labelled with a C letter (figure A1, B1 and C1), while the necrotic area
is labelled with the N letter (Figure A2, B2 and C2).
Without meaning to restrict the mechanism of action with which the salts
according to the present invention exert their pharmacological effects, the above
observations contribute to explain, at least partially, the antimetastatic effect of
the above salts as the result of the prevention of the tumor cell spawning from the
primary tumor.
Example (ii)-B Materials and methods
Two groups of 12 and one group of 14 (controls) female CBA mice of 22g
obtained from a colony grown according to the procedures for kin animals were
inoculated with 106 cells of MCa mammary carcinoma, as reported in the test (ii)-
A.
From the 9th to the 14th day since the inoculation, the three groups of mice were
daily treated intraperitoneally as follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free
physiological solution;
Group 2-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution, containing 35 mg of [trans-RuCl4(DMSO) (Im)] [ImH] (Example 2);
Group 3-10 m!/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 27 mg of [trans-RuCl4(DMSO) (NH3)] NH4 (Example 5).
On day 27 from inoculation, the rats were sacrificed by cervical dislocation and
the lung metastases were then counted, as described in the test (i).
The experimental figures obtained were then processed according to the Student-
Newmann-Keuls statistical test.
In order to evaluate the activity of the salts of the invention on the primary tumor,

23
the average weight of the primary tumor was assessed at the beginning and in the
end of the above treatment.
Results:
The results obtained are reported in Table 8, where the number of metastases
and their weight obtained in the groups (2) and (3) with respect to the control
group (1) are shown. The figures are expressed as the average ± S.E. of the
single values obtained for each group.
Table 8: antitumor and antimetastatic activity of [trans-RuCl4(DMSO) (Im)] [ImH]
in Example 2 (35 mg/kg/day) and of the [trans-RuCl4(DMSO) (NH3)] NH4
described in Example 5 (27 mg/kg/day) on mice affected by MCa mammary
carcinoma,

* p
24
Table 9: % reduction of the number of metastases and of their weight, obtained with the compounds according to the present invention.

The results related to the effect of the salts of the invention on the growth of the primary tumor are reported in Table 10, where the weight of the primary tumor at the beginning and in the end of the treatment with the salts of the invention, as well as the corresponding percentage of growth.
Table 10: effect exerted by the compounds according to the present invention on the growth of the primary tumor in mice affected by MCa mammary carcinoma.

* p
25
is 44,5% in the [trans-RuCI4(DMSO) (NH3)] NH4. Example (ii)-C Materials and methods
Two groups of 7 and one group of 9 (controls) of female CBA mice of 22g
obtained from a colony grown according to the procedures for kin animals, were
inoculated by intramuscular injection with 106 cells of MCa mammary carcinoma
as reported in the test (ii)-A.
From the 9th to the 14th day, the three groups of mice were daily treated
intraperitoneally as follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free
physiological solution;
Group 2-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 44 mg of [trans-RuCl4(DMSO) (NH3)] Na 2DMSO, as
described in the above mentioned WO 90/13553 international patent application;
Group 3-10 ml/kg of body weight /day of a sterile and pyrogen-free physiological
solution containing 27 mg of [trans-RuCl4(DMSO) (NH3)] NH4 (Example 5).
In order to evaluate the activity of the salts of the invention on the primary tumor,
the average weight of the primary tumor at the beginning and in the end of the
treatment with the salts of the invention, as well as the percentage variation of the
growth were assessed.
The obtained results are reported herehelow in Table 11, showing the weight of
the primary tumor at the beginning and at the end of the treatment, as well as the
growth % variation.

26
Table 11: effect exerted by the compounds according to the present invention on the growth of the primary tumor of the mice affected by MCa mammary carcinoma.

p From the figures reported above, it is evident that the reduced growth of the primary tumor in the groups treated with the salts of Ru(lll) complexes, as normalized by the controls, is 18,7% for [trans-RuCl4(DMSO) (NH3) ]Na 2DMSO while it is 32,8% in [trans-RuCl4(DMSO) (NH3)] NH4. These figures show that the [trans-RuCl4(DMSO) (NH3)] NH4 of the present invention can exert antitumor effects on the growth of the primary tumor which are remarkably higher with respect to the known compounds in the state-of-the-art. EXAMPLE (ii)-D Materials and Methods
Three groups of 7 female CBA mice of 23±2 g, obtained from a colony grown according to the procedures for kin animals. The colony obtained originally from Chester Beatthy Institute of London, was subsequently kept by means of serial couplings between kins (brother and sister) in a ratio ranging from 1:1 to 1:4. The couplings took place at the peak of the sexual maturity of the animals born in the 5th week. Animals underweight or with evident organic anomalies were discarded.

27
The three groups were inoculated by intramuscular injection, with 106 MCa
mammary carcinoma cells suspended in 0,05 ml of a Dulbecco's buffered calcium
and magnesium-free physiological solution (PBS) using a sterile syringe.
The tumor line was obtained from Rudjer Boskovic Institute in Zagabria and
maintained in liquid nitrogen.
13 days after inoculation, the primary tumor was surgically removed.
From day 14 to 19, the three groups of mice were treated intraperitoneally as
follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free
physiological solution;
Group 2-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 2 mg of Cisplatin
Group 3-10 ml/kg of body weight/day of a sterile and pyrogen-free physiological
solution containing 35 mg of [trans-RuCl4(DMSO) (Im)] [ImH] (Example 2).
* p Table 12: Comparison of antimetastatic effect of [trans-RuCl4(DMSO) (lm)][lmH] of Example 2 (35 mg/kg/day) with respect to Cisplatin (2mglkg/day) on mice affected by MCa mammary carcinoma.


28
Example (ii)-E
In vivo activity test of the salts (I), according to the present invention, on
mice affected by TS/A mammary adenocarcinoma.
Materials and methods
Three groups, each made up of 10 female BALBc mice of 21±1 g, obtained from
Harlan Nossan (Italia) were inoculated by intramuscular injection, with 105 TS/a
mammary adenocarcinoma cells suspended in 0,05 ml of a Dulbecco's buffered
calcium and magnesium-free solution (PBS) using a sterile insulin syringe.
The tumor line was obtained from Istituto immunogenetica (Univ.Torino, IT) and
maintained in liquid nitrogen.
From the 13th to the 18th day, the three groups of mice were treated
intraperitoneally as follows:
Group 1 - Control: 10 ml/kg of body weight/day of a sterile and pyrogen-free
physiological solution;
Group 2-10 ml/kg of body weight/clay of a sterile and pyrogen-free physiological
solution containing 2 mg of Cisplatin
Group 3-10 ml/kg of body weight/clay of a sterile and pyrogen-free physiological
solution containing 35 mg of [trans-RuCI4(DMSO) (Im)] [ImH] (Example 2).
19 days after inoculation, the primary tumor was surgically removed.
33 days after inoculation, the mice were sacrificed by cervical dislocation and the
lung metastases were counted. The lungs were abducted immediately after death
of the mice and were divided in single lobes, which were then subsequently
examined by the use of a low magnification stereoscopic microscope, equipped
with a grid on the eye-piece, which allowed the detection of a and b orthogonal
axes (where a The lung metastases were then classified according to their dimensions and the
weight of the metastases for each animal was then calculated as the sum of the
weight of each single metastases, each one regarded as a solid by means of the
formula {n /6) a2xb. The experimental data obtained were then processed with the
Student- Newmann-Keuls statistical test.
The results obtained are hereunder reported in Table 13, where the number of

29
metastases and their weight in both groups of treated mice (2) and (3) are reported with respect to the control Group (1). The figures are reported as the average ± S. E. of the single values obtained for each group.
Table 13: Antimetastatic activity on mice affected byTS/A mammary adenocarcinoma, of [trans-RuCl4(DMSO) (lm)][lmH] of Example 2 (35 mg/kg/day) with respect to Cisplatin (2mg/kg/day).

* p The dosages chosen for the comparison of the effects of [trans-RuCl4(DMSO) (lm)][lmH] and cisplatin on solid metastasizing tumors are comparable in that they represent the maximum tolerated doses with the treatment adopted (1 injection /day/ for 6 consecutive days).
The comparison of the effects of the compound of the invention and cisplatin on the loss of body weight gain during tratment shows that the ruthenium compound (% loss body weight gain vs control= +1 (lewis lung car), -6 (Mca mammary car.)) is always less toxic than cisplatin (% loss body weight gain vs control= -11). The comparison of spleen weight of the treated animals (% loss spleen weight vs controi= -11) also shows that the drug treatment with the compound of the invention is much better tolerated than that of with cisplatin animals (% loss spleen weight vs control= -52).
On tumor metastasis, the compound of the invention is as effective (Mca mammary carcinoma) or even more effective (Lewis lung carcinoma and TS/A

30
adenocarcinama) than cisplatin: It reduces the number of lung metastasis either when given at early stages of tumor growth, ie prior to surgical ablation (TS/A adenocarcinoma) or when it is given to mice with lung metastases in advanced stage of growth, ie after surgical ablation of primary tumour (Lewis lung carcinoma) and the reduction of lung metastases is in agreement with a significant prolongation of the post-surgical life time expentancy of the treated mice (Mca mammary carcinoma)
The in vivo experimental models reported above are related to the treatment, with the compounds of the present invention, of two solid spawning tumors in rodents at an advanced stage of growth. A statistically significant and marked reduction both in the growth of the primary tumor and in the formation of lung metastases is observed. This reduction, as can be seen both in the model (i) of Lewis lung carcinoma and in model (ii) of MCa mammary carcinoma, is to be ascribed to a pronounced inhibition of the growth of lung metastases. In fact, with respect to the controls, the presence of medium/large dimensions in groups of animals treated is notably lower or even inexistent in as far as large nodules are concerned., In particular, from the overall comparison of the data regarding effective reduction in the metastases, the compounds of the present invention are surprisingly more active than the reference compounds known in the state-of-the-art, both in the treatment of the Lewis lung carcinoma and, in a more evident and statistically significant manner, in the treatment of the MCa mammary carcinoma.

WE CLAIM :- 31
1 1. A salt of an anionic complex of Ru(lll) with an ammonium cation of formula (I):
2
4 5 6 7 8 9 10
11 where R1, R2 and R3, equal or different from each other, are selected from the
12 group consisting of H, C1-C6 alkyl, linear or branched, saturated or unsaturated,
13 C3-C7cycloalkyl, phenyl and aryl;
14 or NR1R2R3 is a 5-7 membered nitrogen-containing heterocycle, saturated or
15 unsaturated, optionally containing one or more 0, S and /or N atoms, said
16 nitrogen atom being optionally substituted with a C1-C4 alkyl, aryl or benzyl
17 residue; said nitrogen-containing heterocycle being optionally condensed with a
18 benzo group and/or substituted with C1-C4 alkyl.C1-C4 alkoxyl, C1-C4 aikylthio, aryl
19 or benzyl groups;
20 where R4 and R51 equal or different from each other, are selected from the group
21 consisting of H, C1-C6 alkyl, C3-C7 cycloalkyl, phenyf and aryl or R4 and R5 form,
22 together with the S atom , a 4-7 membered heterocycle.

1 2. The salt, according to Claim 1, characterized by the fact that R1 R2and R3, are
2 H or ethyl.

1 3. The salt, according to Claim 1, characterized by the fact that NR1R2R3 is a 5-
2 membered nitrogen containing heterocycle selected from the group consisting of
3 imidazole, N-methyl-imidazole, pyrazole, and oxazole.

1 4. The salt, according to Claim 1, characterized by the fact that NR1R2R3 is a 6-
2 membered heterocycle selected from the group consisting of pyridine, pyrazine,
3 3,5 lutidine and 4-methylpyridine.

32
1 5. The salt, according to Claim 1, characterized by the fact that NR1R2R3 is a
2 nitrogen-containing heterocycle being condensed with a benzo group selected
3 from indazole, isoquinoline, benzirnidazole and 1,5,6-trimethyl-benzimidazole.

1 6. The salt, according to the Claims from 1 to 5, characterized by the fact that R4-
2 SO-R5 is selected from the group consisting of dimethylsulphoxide,
3 diethylsulphoxide, tetramethylensulphoxide.

1 7. The salt, according to the Claims 1, 3 and 6, characterized by .the fact that
2 NR1R2R3 is imidazole and R4-SO-R5 is dimethylsulphoxide;

1 8. The salt according to Claims 1, 2 and 6, characterized by the fact that
2 NR1R2R3 is NH3 and R4-SO-R5 is dimethylsulphoxide.
1 9. Process for the preparation of a salt of formula (I) , according to the Claims
1 from 1 to 8, comprising the following reaction steps:
3 1) RUCI3 is reacted with R4-SO-R5 in the presence of HCI to obtain the complex
4 of formula (II).
5
6
7
8
9 10 11 12 13 14
15 where R4 and R5 have the meanings reported above:
16 2) The complex (II), obtained from step (1), is reacted at room temperature with a
17 nitrogen containing compound, NR1R2R3, in one or more organic solvents, to
¦18 obtain the salt of formula (I).

1 10. The process, according to Claim 9, characterized by the fact that R4 and R5
2 are methyl or ethyl.
1 11. The process, according to Claim 9, characterized by the fact that, in step (1),

-33-
RuCl3 is dissolved in an organic solvent and the solution obtained in this way is reacted with R4-SO-R5 and concentrated HCI, at a temperature ranging from 60 to 90°C.
12. The process, according to Claim 11, characterized by the fact that RuCl3
dissolved in ethanol or methanol and the solution obtained in this way is reacted
with R4-SO-R5 and concentrated HCI, at a temperature of about 80°C.
13. The process, according to Claim 9, characterized by the fact that, in
step(2), said complex (II) and said nitrogen-containing compound, NR1R2R3, are
reacted in molar ratios ranging from 1:2 to 1:6;
14. The process, according to Claim 9, characterized by the fact that, in
step(2), said organic solvents are acetone or dichloromethane.
15. A pharmaceutical composition containing, as an active compound, a
therapeutically effective amount of at least one of the salts of formula (1), as
defined in the Claims from 1 to 8, in combination with suitable excipients and
diluents.
16. The pharmaceutical composition according to Claim 15, characterized by
the fact that it is in the form of solution or suspension.
17. the pharmaceutical composition according to Claim 15, characterized by
the fact that it is in the form of gel, ointment, powder, pill, tablet, capsule or
insert.
18. The pharmaceutical composition, according to claim 15, characterized by
the fact that said salts of formula (1) are combined with one or more antitumor
drugs.





-34-
19. The pharmaceutical composition, according to claim 18, characterized by the fact that said antitumor drugs are selected from the group consisting of cisplatin, 5-5-fluorouracil, vinblastine, cyclophosphamide, bleomycin, anthracyclin, and taxol.

A Salt of an anionic complex of Ru(III) with an ammonium cation of formula (I):

Where R1, R2 and R3, equal or different from each other, are selected from the group consisting of H, C1-C6 alkyl, linear or branched, saturated or unsaturated, C3-C7 cycloalkyl, phenyl and aryl; or NR1R2R3 is a 5-7 membered. nitrogen-containing heterocycle, saturated or unsaturated, optionally containing one or more O, S, and/or N atoms, said nitrogen atom being optionally substituted with a C1-C4 alkyl, aryl or benzyl residue; said nitrogen containing heterocyde being optionally condensed with a benzo group and/or substituted with C1-C4 alkyl, C1-C4 alkoxyl, C1-C4 alkoxyl, C1-C4 alkylthio, aryl or benzyl groups; where R4 and R5, equal or different from each other, are selected from the group consisting of H, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl and aryl or R4 form, together with the S atom, a 4-7 membered heterocyde. Furthermore the process for the preparation of said salts, the pharmaceutically compositions containing the same and their use as antimetastatic and antineoplastic agents are described.

Documents:

01263-cal-1997-abstract.pdf

01263-cal-1997-claims.pdf

01263-cal-1997-correspondence.pdf

01263-cal-1997-description(complete).pdf

01263-cal-1997-drawings.pdf

01263-cal-1997-form-1.pdf

01263-cal-1997-form-18.pdf

01263-cal-1997-form-2.pdf

01263-cal-1997-form-3.pdf

01263-cal-1997-form-5.pdf

01263-cal-1997-letters patent.pdf

01263-cal-1997-others document.pdf

01263-cal-1997-p.a.pdf

01263-cal-1997-priority document.pdf

1263-CAL-1997-CORRESPONDENCE.pdf

1263-CAL-1997-FORM 27.pdf

1263-CAL-1997-FORM-27.pdf


Patent Number 207135
Indian Patent Application Number 1263/CAL/1997
PG Journal Number 21/2007
Publication Date 25-May-2007
Grant Date 23-May-2007
Date of Filing 02-Jul-1997
Name of Patentee SIGEA S.r.I.
Applicant Address PADRICIANO 99, 34012 TRIESTE
Inventors:
# Inventor's Name Inventor's Address
1 GIOVANNI MESTRONI VICOLO DEL CASTAGNETO 27, 34127 TRIESTE
2 ENZO ALESSIO ORIANI 3, 34131 TRIESTE
3 GIANNI SAVA DE' GUARDI 2, 34143 TRIESTE
PCT International Classification Number A61K 31/28
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 961359 1996-07-02 Italy