Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Phosphorus containing other than solely as part of an...
Reexamination Certificate
1998-09-15
2001-06-05
Ambrose, Michael G. (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Phosphorus containing other than solely as part of an...
C514S114000, C514S119000, C514S129000, C514S134000, C558S173000, C562S008000, C562S015000, C562S021000, C562S023000
Reexamination Certificate
active
06242433
ABSTRACT:
The present invention relates to novel geranylgeranyl-derivatives and their pharmaceutically acceptable salts having inhibiting activity with respect to protein geranylgeranylation in eukaryotic cells.
It is known in biology that proteins bearing the aminoacidic sequence CysAAX (that is to say a cysteine molecule which is linked to two aliphatic aminoacids AA, linked to each other and, on their turn, linked to any aminoacid X) on their carboxy end group are subjected to a number of post-translational modifications in eukaryotic cells.
Said modifications consist in the formation of a covalent bond between the cysteinic residue of the protein and an isoprenoid lipid, followed by the proteolysis of the three terminal aminoacids AAX and by the &agr;-carboxy-methyl esterification of the isoprenyl-cystein (Giannakouros T., Magee A. I.,
Lipid′ modifications of proteins
(Schlesinger M J, ed.), CRC Press, Boca Raton, 1993; 136-162). The isoprenoid lipid can be farnesyl (15 carbon atoms) or geranylgeranyl (20 carbon atoms) and the isoprenylation is necessary for the acquisition of the biological properties of the protein itself (Grünler J., Ericsson J., Dallner G.,
Biochim. Biophys. Acta,
1994; 1212, 259-277). A typical example of a farnesylated protein is the p21ras, while the low-molecular weight proteins of the ras superfamily among which p21rho, p21rap1, p21rac and cdc42, are geranylgeranylated.
The proteins isoprenylation enzymology has been the object of an intense research and the following enzymes which catalyze the most important steps of the biochemical way leading to the formation of isoprenoid compounds have been characterized (Grünler J., Ericsson J., Dallner G.,
Biochim. Biophys. Acta,
1994; 1212, 259-277):
3-hydroxy-3-methylglutaryl coenzyme A reductase;
farnesyl diphosphate synthetase;
protein:farnesyl transferase;
geranylgeranyl diphosphate synthetase;
protein:geranylgeranyl transferase type I;
protein:geranylgeranyl transferase type II.
The 3-hydroxy-3-methylglutaryl coenzyme A reductase is the enzyme responsible for the synthesis of mevalonic acid from which originate, as a result of the activity of farnesyl diphosphate synthetase and geranylgeranyl diphosphate synthetase, both farnesyl diphosphate (farnesyl-FF) and geranylgeranyl diphosphate (geranylgeranyl-FF) which are the two isoprenoids used for protein isoprenylation. On the contrary the protein:farnesyl transferase and the protein:geranylgeranyl transferases (type I and II) catalyze the transfer of the group farnesyl and geranylgeranyl, respectively, to proteins.
It has been recently demonstrated the fundamental part played by geranylgeranylated proteins, among which specifically p21rho, p21rap1, rac and cdc42, in eukaryotic cells cell proliferation, where they control essential functions as the actin organization of cytoskeleton and of intercellular adhesion plaques (Olson M. F., Ashworth A., Hall A.,
Science
1995; 269; 1270-1272). The above discovery is of great importance as regards the possible implications in the therapy of pathologies characterized by an excessive cell proliferation.
The object of the present invention are the novel compounds having general formula:
wherein:
Q=
CH
2
—CH
2
, CHOH
X=ONH, ONHCO, OCH
2
CO, OCH
2
P(O)OH, CH
2
P(O)OH, NHCO, NCH
3
CO, OSO
2
, NHSO
2
;
A=R′CR″, CHR″′CH
2
, NH when X=OSO
2
, NHSO
2
;
B=OCO, O, ONHCO, NHCO, NCH
3
CO;
R=H, CH
3
, CH
2
CH
3
;
R′=H, CH
3
, CH
2
CH
3
;
R″=H, CH
3
, CH
2
CH
3
;
R″′=H, COOH;
and pharmaceutically acceptable salts thereof, with acids and bases, organic and inorganic, having inhibiting activity with respect to protein geranylgeranylation in eukaryotic cells.
Preferred compounds within the scope of the invention are the following:
It has been in fact surprisingly found that the compounds according to the present invention can prevent the post-translational geranylgeranylation and, as a consequence, the biological function of cellular proteins which perform essential functions in cellular replication mechanism. Said compounds can therefore be useful in the treatment of pathologies characterized by an excessive cell proliferation, among which are benign and malignant tumors, vascular and renal diseases on a degenerative-proliferative basis, as for example atherosclerosis and glomerulonephritis.
The ability of the compounds according to the present invention of inhibiting the protein geranylgeranylation has been evaluated by means of an in vitro test on a malignant tumoral human cell line which has been treated with the following geranylgeranyl-derivatives:
dipotassium salt of (E,E,E)-{2-oxo-2-[[(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)oxy]amino]ethyl}phosphonic acid (reported on Table 1 with the abbreviation BAL9504) whose preparation is disclosed in example 1;
monosodium salt of the monoethyl-ester of (E,E,E)-{2-oxo-2[[(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)oxy]amino]ethyl}phosphonic acid (reported on Table 1 with the abbreviation BAL9505) whose preparation is disclosed in example 2;
dipotassium salt of (E,E,E)-{3-oxo-3-[[(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)oxy]amino]propyl}phosphonic acid (reported on Table 1 with the abbreviation BAL9603) whose preparation is disclosed in example 5;
dipotassium salt of (E,E,E)-[2-oxo-2-[(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)amino]ethyl]phosphonic acid (reported on Table 1 with the abbreviation BAL9605) whose preparation is disclosed in example 7;
dipotassium salt of (E,E,E)-[3-oxo-3-[(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)amino]propyl]phosphonic acid (reported on Table 1 with the abbreviation BAL9608) whose preparation is disclosed in example 10;
tripotassium salt of (E,E,E)-[[(4,8,12,16-tetramethyl-3,7,11,15-heptadecatetraenyl)hydroxyphosphoryl]methyl]phosphonic acid (reported on Table 1 with the abbreviation BAL9609) whose preparation is disclosed in example 12;
tripotassium salt of (E,E,E)-1-methyl-1[(4,8,12,16-tetramethyl-3,7,11,15-heptadecatetraenyl)hydroxyphosphoryl]ethylphosphonic acid (reported on Table 1 with the abbreviation BAL9610) whose preparation is disclosed in example 13;
tripotassium salt of (E,E,E)-1-ethyl-1-[(4,8,12,16-tetramethyl-3,7,11,15-heptadecatetraenyl)hydroxyphosphoryl]propylphosphonic acid (reported on Table 1 with the abbreviation BAL9611) whose preparation is disclosed in example 14;
dipotassium salt of (E,E,E)-(4,8,12,16-tetramethyl-3,7,11,15-heptadecatetraenyl)phosphonic acid (reported on Table 1 with the abbreviation BAL9613) whose preparation is disclosed in example 16;
(E,E,E)-O-(3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl)-N-(aminosulfonyl)urethane (reported on Table 1 with the abbreviation BAL9503) whose preparation is disclosed in example 18;
(E,E,E)-1-[(aminosulfonyl)aminoxy]-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraene (reported on Table 1 with the abbreviation BAL9614) whose preparation is disclosed in example 19.
The cells have been treated with increasing amounts of the geranylgeranyl derivatives BAL9504, BAL9505, BAL9603, BAL9605, BAL9608, BAL9609, BAL9610, BAL9611, BAL9613, BAL9503, BAL9614, (1-75 &mgr;M) and, after 24 hours, they have been collected and solubilized in a lysis solution containing non-ionic detergents. The soluble fraction has been collected by centrifugation and proteins separated by means of acrylamide gel electrophoresis and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane.
The presence of the geranylgeranylated protein p21rap1 has been then evidenced by means of a specific polyclonal antibody followed by chemiluminescent detection by means of a secondary antibody linked to alkaline phosphatase as previously described (M. A. Mansfield,
FASEB J., S
, A1428, 1994). The presence of the protein under examination has been demonstrated by the appe
Baldacci Massimo
Balsamo Aldo
Danesi Romano
Del Tacca Mario
Macchia Bruno
Ambrose Michael G.
Laboratori Baldacci SpA
Nixon & Vanderhye
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