U.S. patent application number 14/661936 was filed with the patent office on 2015-07-09 for transition metal complexes for inhibiting resistance in the treatment of cancer and metastasis.
The applicant listed for this patent is Ecole Polytechnique Federal de Lausanne (EPFL). Invention is credited to Wee Han Ang, Paul Joseph Dyson.
Application Number | 20150190362 14/661936 |
Document ID | / |
Family ID | 37770371 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150190362 |
Kind Code |
A1 |
Dyson; Paul Joseph ; et
al. |
July 9, 2015 |
TRANSITION METAL COMPLEXES FOR INHIBITING RESISTANCE IN THE
TREATMENT OF CANCER AND METASTASIS
Abstract
The present invention relates to organometallic compounds useful
in the treatment of metastasis. The organometallic compounds
comprise a ligand that is convalently bound to a bioactive
compound, which is an inhibitor of a resistance pathway or a
derivative thereof. Preferably, the organometallic compounds are
half-sandwich ("piano-stool") compounds. The compounds of the
present invention offer a high variability with respect to the
bioactive compound and to the nature of the ligand bound to a
central transition metal.
Inventors: |
Dyson; Paul Joseph;
(Ecublens, CH) ; Ang; Wee Han; (Ecublens,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ecole Polytechnique Federal de Lausanne (EPFL) |
Lausanne |
|
CH |
|
|
Family ID: |
37770371 |
Appl. No.: |
14/661936 |
Filed: |
March 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12227210 |
Apr 2, 2009 |
9018199 |
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PCT/CH2007/000234 |
May 9, 2007 |
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14661936 |
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60799433 |
May 9, 2006 |
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Current U.S.
Class: |
514/184 ;
514/492 |
Current CPC
Class: |
C07F 15/0033 20130101;
A61P 35/00 20180101; C07F 15/0073 20130101; A61K 45/06 20130101;
C07F 15/0046 20130101; C07F 15/002 20130101; A61K 31/555 20130101;
A61K 31/28 20130101 |
International
Class: |
A61K 31/28 20060101
A61K031/28; A61K 45/06 20060101 A61K045/06; A61K 31/555 20060101
A61K031/555 |
Claims
1-13. (canceled)
14. A method for treating cancer or metastatic cancer comprising
administering to an individual an effective amount of an
organometallic compound of formula (XIV) ##STR00011## in which, A
is a monocyclic arene selected from the group of benzene,
methylbenzene, cymene; R.sub.18, R.sub.19, R.sub.20 are ligands of
the central ruthenium atom which are, independently of each other,
selected from the group of halogens consisting of F.sup.-,
Cl.sup.-, Br.sup.- and I.sup.-, from the group consisting of N-, P-
and O-donor ligands or from a bioactive organic compound; R.sub.17
is an optional residue selected from alkyl, alkenyl, alkynyl, aryl,
or from a bioactive organic compound; whereby at least one
bioactive organic compound is at least one residue or ligand
selected from R.sub.17, R.sub.18, R.sub.19, R.sub.20 or is
covalently linked to any of R.sub.17, R.sub.18, R.sub.19, R.sub.20
via a covalent bond selected from the group consisting of a
carbon-carbon alkyl bond, alkenyl bond and alkynyl bond, or
selected from the group of carbon-heteronuclear bonds consisting of
amide bond (--CONH--), ester bond (--CO.sub.2--), ether bond
(--CH.sub.2O--), thioether (--CH.sub.2S--), amine bond
(--CH.sub.2N--), imine bond (--CH.dbd.N--), and phosphorous bond
(--CH.sub.2P--), with the proviso that the residue or ligand
covalently linked to the bioactive organic molecule is not halogen;
and wherein the bioactive organic compound is selected from: an
inhibitor of Glutathione S-transferase selected from ethacrynic
acid, peptidomimetics of gluthatione, p-chlorophenoxyisobutyrate,
Gossypol, indomethacin, non-steroidal anti-inflammatory compounds
of ibuprofen and of ketoprofen, misonidazole, Piriprost,
Sulfasalazine; an inhibitor of .gamma.-Glutamyl Cysteine Synthetase
selected from sulfoxime-based compounds group consisting of
buthinone sulfoxime and methinone sulfoxime, S-sulfocysteine,
S-sulfohomocysteine, cystamine; an inhibitor of the multidrug
resistance protein selected from the group of quinidine,
vinblastine, terfernadine, tamoxifen, verapamil, cyclosporin,
amitriptyline, progesterone; or an inhibitor of a cell signaling
pathway selected from the group of pleurotin, azelaic acid,
bischloroethylnitrosourea, palmarumycin; and said N-donor ligand is
an imidazole of formula (IV) or of formula (V), which is covalently
linked to the bioactive compound ##STR00012## and wherein the
"bioactive compound" moiety of formula (IV) and formula (V)
represents a bioactive organic compound as described above and
wherein the bioactive organic compound is linked to the --NH-- of
formula (IV) or the --O-- of formula (V) via a carboxy group; n is
1-10, and M is the transition metal Ru of the organometallic
compound of formula (XIV); said P-donor ligand is selected from the
group consisting of PTA, MePTA and DAPTA, wherein PTA is
1,3,5-triaza-7-phosphadamantane, MePTA is
3-methyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane and DAPTA is
3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane; and said
O-donor ligand is selected from the group consisting of carbonate
CO.sub.3.sup.-, carboxylate ligands R.sup.CCO.sub.2.sup.-, oxalate
C.sub.2O.sub.4.sup.2-, nitrate NO.sub.3.sup.-, sulfate
SO.sub.4.sup.2- and sulphonate R.sup.S1O.sub.3.sup.-, wherein
R.sup.C and R.sup.S1 is selected from alkyl, alkenyl, alkynyl,
aryl.
15. The method according to claim 14, wherein: R.sub.20 and
R.sub.19 of formula (XIV) are independently selected from the group
of halogens consisting of F, Cl.sup.-, Br.sup.- and I.sup.-; or
from the group consisting of said P- and said O-donor ligands;
R.sub.17 is present and A-R.sub.17 of formula (XIV) is represented
by formula (XI) or in formula (XII) below: ##STR00013## wherein m
is 1-10, the monocyclic arene A of the organometallic compound of
formula (XIV) is the structure ##STR00014## and the "bioactive
compound" of formula (XI) and formula (XII) represents the
bioactive organic compound; and wherein the bioactive organic
compound is linked to the O-- of formula (XI) or the --NH-- of
formula (XII) via a carboxy group; and R.sub.18 of formula (XIV) is
selected from the group of halogens consisting of F.sup.-,
Cl.sup.-, Br.sup.- and I.sup.- or from the group consisting of said
P- and said O-donor ligands.
16. The method according to claim 14, wherein A of formula (XIV) is
benzene or cymene.
17. The method according to claim 14, wherein, said bioactive
organic compound is ethacrynic acid.
18. The method according to claim 14, wherein halogen is Cl.sup.-;
said P-donor ligand is PTA; said O-donor ligand is selected from
carboxylate ligands R.sup.CCO.sup.2- or oxalate
C.sub.2O.sub.4.sup.2-, wherein R.sup.C is selected from alkyl,
alkenyl, alkynyl, aryl; and said bioactive organic compound is
ethacrynic acid.
19. The method according to claim 14, wherein R.sub.20 and R.sub.19
of formula (XIV) are independently selected from the group of
halogens consisting of F.sup.-, Cl.sup.-, Br.sup.- and I.sup.-;
R.sub.18 is selected from the group consisting of said P-donor
ligands.
20. The method according to claim 14, wherein R.sub.20 and R.sub.19
a of formula (XIV) are independently selected from the group of
halogens consisting of F.sup.-, Cl.sup.-, Br.sup.- and I.sup.- or
from the group consisting of said P-donor ligands; R.sub.18 of
formula (XIV) is selected from said N-donor ligands of formula (IV)
or of formula (V); and R.sub.17 is absent.
21. The method according to claim 14, wherein the organometallic
compound of formula (XIV) is selected from the group consisting of
(Ethacrynic-.eta..sup.6:phenylmethylamide)Ru(PTA)Cl.sub.2,
(Ethacrynic-.eta..sup.6:phenylethanoate)Ru(PTA)Cl.sub.2,
(.eta..sup.6-cymene)RuCl.sub.2(ethacrynic-propylamide-imidazole),
and
[(.eta..sup.6-cymene)RuCl(PTA)(ethacrynic-propylamide-imidazole)]BF4.sup.-
-, wherein PTA is 1,3,5-triaza-7-phosphadamantane.
22. The method according to claim 14, wherein: R.sub.20 and
R.sub.19 of formula (XIV) are independently selected from the group
of halogens consisting of F.sup.-, Cl.sup.-, Br.sup.- and I.sup.-;
or from the group consisting of said P-- and said O-donor ligands;
R.sub.18 of formula (XIV) is selected from said N-donor ligands of
formula (IV) or of formula (V) as defined in claim 14 to which a
bioactive organic molecule is covalently bound, R.sub.17 of formula
(XIV) is present and selected from alkyl, alkenyl, alkynyl,
aryl.
23. The method according to claim 14, wherein: R.sub.20 and
R.sub.19 of formula (XIV) are independently selected from the group
of halogens consisting of F.sup.-, Cl.sup.-, Br.sup.- and I.sup.-;
or from the group consisting of said P- and said O-donor ligands;
R.sub.18 of formula (XIV) is selected from said N-donor ligand of
formula (IV) or of formula (V); and R.sub.17 of formula (XIV) is
absent.
24. The method according to claim 14, wherein R.sub.20 and R.sub.19
of formula (XIV) are independently selected from the group of
halogens consisting of F.sup.-, Cl.sup.-, Br.sup.- and I.sup.- or
from the group consisting of said P-donor ligands; R.sub.18 of
formula (XIV) is selected from said N-donor ligands of formula (IV)
or of formula (V); and R.sub.17 of formula (XIV) is present and is
selected from alkyl, alkenyl, alkynyl, aryl.
25. The method according to claim 14, wherein R.sub.20 and R.sub.19
of formula (XIV) are selected from the group of said O-donor
ligands consisting of carboxylate ligands R.sup.CCO.sup.2- or
oxalate C.sub.2O.sub.4.sup.2-, wherein R.sup.C is selected from
alkyl, alkenyl, alkynyl, aryl; R.sub.18 of formula (XIV) is
selected from said N-donor ligands of formula (IV) or of formula
(V); R.sub.17 of formula (XIV) is selected from alkyl, alkenyl,
alkynyl, aryl.
26. The method according to claim 14, wherein R.sub.19 of formula
(XIV) is selected from the group of halogens consisting of F.sup.-,
Cl.sup.-, Br.sup.- and I.sup.- and R.sub.20 of formula (XIV) is
selected or from the group consisting of said P-donor ligands;
R.sub.18 of formula (XIV) is selected from N-donor ligands of
formula (IV) or of formula (V); and R.sub.17 of formula (XIV) is
selected from alkyl, alkenyl, alkynyl, aryl.
27. The method according to claim 14, wherein R.sub.19 of formula
(XIV) is selected from the group of halogens consisting of F.sup.-,
Cl.sup.-, Br.sup.- and I.sup.- and R.sub.20 of formula (XIV) is
selected or from the group consisting of said P-donor ligands;
R.sub.18 of formula (XIV) is selected from said N-donor ligands of
formula (IV) or of formula (V); and R.sub.17 is absent.
28. The method according to claim 14, wherein R.sub.20 and R.sub.19
of formula (XIV) are Cl.sup.-; and R.sub.18 of formula (XIV) is
selected from the group consisting of said P-donor ligands.
29. The method according to claim 14, wherein: A of formula (XIV)
is cymene; R.sub.20 and R.sub.19 of formula (XIV) are independently
selected from the group of halogens consisting of F.sup.-,
Cl.sup.-, Br.sup.- and I.sup.-; or from the group consisting of
said P- and O-donor ligands; R.sub.18 of formula (XIV) is selected
from said N-donor ligands of formula (IV) or of formula (V); and
R.sub.17 is absent.
30. The method according to claim 14, wherein R.sub.20 and R.sub.19
of formula (XIV) are independently selected from the group of
halogens consisting of F.sup.-, Cl.sup.-, Br.sup.- and I.sup.-; and
R.sub.18 is selected from the P-donor ligand PTA.
31. The method according to claim 14 comprising administering to
the individual an effective amount of said organometallic compound
of formula (XIV) and an effective amount of an anti-cancer
drug.
32. A method for reducing resistance of cancers or metastatic
cancer comprising administering to an individual an effective
amount of an organometallic compound of formula (XIV) ##STR00015##
in which, A is a monocyclic arene selected from the group of
benzene, methylbenzene, cymene; R.sub.18, R.sub.19, R.sub.20 are
ligands of the central ruthenium atom which are, independently of
each other, selected from the group of halogens consisting of
F.sup.-, Cl.sup.-, Br.sup.- and I.sup.-, from the group consisting
of N-, P- and O-donor ligands or from a bioactive organic compound;
R.sub.17 is an optional residue selected from alkyl, alkenyl,
alkynyl, aryl, or from a bioactive organic compound; whereby at
least one bioactive organic compound is at least one residue or
ligand selected from R.sub.17, R.sub.18, R.sub.19, R.sub.20 or is
covalently linked to any of R.sub.17, R.sub.18, R.sub.19, R.sub.20
via a covalent bond selected from the group consisting of a
carbon-carbon alkyl bond, alkenyl bond and alkynyl bond, or
selected from the group of carbon-heteronuclear bonds consisting of
amide bond (--CONH--), ester bond (--CO.sub.2--), ether bond
(--CH.sub.2O--), thioether (--CH.sub.2S--), amine bond
(--CH.sub.2N--), imine bond (--CH.dbd.N--), and phosphorous bond
(--CH.sub.2P--), with the proviso that the residue or ligand
covalently linked to the bioactive organic molecule is not halogen;
and wherein the bioactive organic compound is selected from: an
inhibitor of Glutathione S-transferase selected from ethacrynic
acid, peptidomimetics of gluthatione, p-chlorophenoxyisobutyrate,
Gossypol, indomethacin, non-steroidal anti-inflammatory compounds
of ibuprofen and of ketoprofen, misonidazole, Piriprost,
Sulfasalazine; an inhibitor of .gamma.-Glutamyl Cysteine Synthetase
selected from sulfoxime-based compounds group consisting of
buthinone sulfoxime and methinone sulfoxime, S-sulfocysteine,
S-sulfohomocysteine, cystamine; an inhibitor of the multidrug
resistance protein selected from the group of quinidine,
vinblastine, terfernadine, tamoxifen, verapamil, cyclosporin,
amitriptyline, progesterone; or an inhibitor of a cell signaling
pathway selected from the group of pleurotin, azelaic acid,
bischloroethylnitrosourea, palmarumycin; and said N-donor ligand is
an imidazole of formula (IV) or of formula (V), which is covalently
linked to the bioactive compound ##STR00016## and wherein the
"bioactive compound" moiety of formula (IV) and formula (V)
represents a bioactive organic compound as described above and
wherein the bioactive organic compound is linked to the --NH-- of
formula (IV) or the --O-- of formula (V) via a carboxy group; n is
1-10, and M is the transition metal Ru of the organometallic
compound of formula (XIV); said P-donor ligand is selected from the
group consisting of PTA, MePTA and DAPTA, wherein PTA is
1,3,5-triaza-7-phosphadamantane, MePTA is
3-methyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane and DAPTA is
3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane; and said
O-donor ligand is selected from the group consisting of carbonate
CO.sub.3.sup.-, carboxylate ligands R.sup.CCO.sub.2.sup.-, oxalate
C.sub.2O.sub.4.sup.2-, nitrate NO3.sup.-, sulfate SO.sub.4.sup.2-
and sulphonate R.sup.S1O.sub.3.sup.-, wherein R.sup.C and R.sup.S1
is selected from alkyl, alkenyl, alkynyl, aryl.
Description
[0001] This invention relates to organometallic compounds of the
metals Ru, Os, Rh, and Ir, to their use in medicine, particularly
for the treatment and/or prevention of cancer, and more
particularly in the treatment of metastasis. This new class of
compounds contains, covalently bound to any of the ligands of the
metal, or directly to the metal, a bioactive organic compound which
is an inhibitor of resistance pathway.
BACKGROUND AND OBJECTIVES OF THE INVENTION
[0002] Resistance against bioactive principles that directly target
the origin of a disease are widespread, and one or more of a number
of known resistance pathways are at their origin. For example, the
Glutathione-S-Transferase (GST) is a superfamily class of isozymes
that constitute the main cellular defense against xenobiotics,
including bioactive principle designed for the treatment of a
disease. They catalyse the conjugation of endogenous glutathione
(GSH) with the electrophilic groups of substrates, the first step
in the mercapturic acid pathway that leads to elimination of toxic
compounds. The overexpression of several subclasses of GST, namely
GST-.pi. and GST-.alpha., has been linked to the multidrug
resistance phenomenon of certain anticancer drugs, such as
cisplatin and adriamycin. More recently, GSTP1-1 (GST-.pi.
subclass) was found to mediate the c-Jun N-Terminal Kinase (JNK)
signal transduction pathway, an important control of cell survival.
It was found to have a significant affinity for the C terminus of
JNK and therefore could potentially interfere with and suppress
downstream induction of cellular apoptosis. Clearly, GST is a
potential target for chemotherapeutic drug design, in order to
inhibit resistance against anti-cancer drugs.
[0003] Ruthenium-based compounds have shown some potential as
anticancer drugs. For example, U.S. Pat. No. 4,980,473 discloses
1,10-phenanthroline complexes of ruthenium(II) and cobalt(II) which
are said to be useful for the treatment of tumour cells in a
subject.
[0004] Some other ruthenium(II) and ruthenium(III) complexes which
have been shown to exhibit antitumour activity are mentioned in Guo
et al, Inorganica Chimica Acta, 273 (1998), 1-7, specifically
trans-[RuCl.sub.2(DMSO).sub.4], trans-[RuCl.sub.4(imidazole).sub.2]
and trans-[RuCl.sub.4(indazole).sub.2]. Clarke et al have reviewed
the anticancer, and in particular the antimetastatic, activity of
ruthenium complexes: Chem. Rev., 1999, 99, 251-253. Also, Sava has
reviewed the antimetastatic activity in "Metal Compounds in Cancer
Therapy" Ed by S P Fricker, Chapman and Hall, London 1994, p.
65-91.
[0005] Dale et al, Anti-Cancer Drug Design, (1992), 7, 3-14,
describes a metronidazole complex of ruthenium(II) ie,
[(C.sub.6H.sub.6)RuCl.sub.2(metronidazole)] and its effect on DNA
and on E. coli growth rates. Metronidazole sensitises hypoxic
tumour cells to radiation and appears to be an essential element of
the complexes of Dale et al. There is no indication that the
complexes would be at all effective in the absence of the
metronidazole ligand.
[0006] Kramer et al, Chem Eur J., 1996, 2, No. 12, p. 1518-1526
discloses half sandwich complexes of ruthenium with amino esters.
Bennett et al, Canadian Journal of Chemistry, (2001), 79, 655-669
discloses certain ruthenium(I1) complexes with acetylacetonate
ligands. Oro et al, J Chem Soc, Dalton Trans, (1990), 1463
describes ruthenium(II) complexes containing -p-cymene and
acetylacetonate ligands. WO 01/130790 discloses ruthenium(II)
compounds and their use as anticancer agents. The compounds have
neutral N-donor ligands and the resulting ruthenium complex is
generally positively charged.
[0007] WO 02/102572 also discloses ruthenium(II) compounds that
have activity against cancer cell lines. Again, the complexes are
generally positively charged. Complexes are disclosed containing a
bidentate ligand which is a neutral diamine ligand.
[0008] Chen et al, J. Am. Chem. Soc., volume 124, no 12, 3064,
(2002), describes the mechanism of binding of ruthenium complexes
to guanine bases. The binding model requires NH bonds from a
diamino ligand to be present in the complex for hydrogen bonding to
the guanine base. Similarly, Morris et al, J. Med. Chem., volume
44, 3616-3621, (2001), describes the selectivity of ruthenium(II)
complexes for binding to guanine bases.
[0009] Further references concerned with Ruthenium complexes for
treatment of cancer are WO 06/018649, US 2006/0058270, US
2005/0239765.
[0010] Very few, if any, of the compounds and complexes of the
prior art cited above have resulted in clinical phase studies, not
to mention actual therapies. The reason for the poor performance of
these principles are manifold and may be linked to toxicity
problems or un-sufficient efficiency in treatment.
[0011] It is thus an objective of the present invention to explore
new ways for treating cancer, for example based on work done in the
area of complexes of transition metals.
[0012] It is a further aspect of the present invention to increase
the activity and/or efficiency of therapies against diseases and in
particular cancer. More particularly, it is an objective to reduce
the resistance intrinsic to or developed against therapies, and in
particular against resistance in cancer chemotherapies. By reducing
resistance to bioactive principles against diseases an in
particular cancer, it is hoped to increase the overall efficiency
of the therapy. With increased efficiency, lower levels of the
bioactive principle needs to be administered, which may further
reduce side effects linked to the treatment.
[0013] Tumors of various kinds can be removed surgically, the most
relevant problem of these cancers being the development of
metastasis. It is thus an objective of the present invention to
prevent and/or treat metastasis. In particular, it is an objective
of the invention to assist the treatment for prevention and/or
treatment of metastasis.
SUMMARY OF THE INVENTION
[0014] The present invention relates to complexes of transition
metals comprising ligands in any form and of any nature, with the
proviso that at least one of the ligands comprises at least one
bioactive organic compound selected from inhibitors of resistance
pathways and/or pharmaceutically acceptable derivatives thereof.
Inhibitors may be directly attached to the transition metal as a
ligand, or it may be covalently bound to a ligand of the transition
metal.
[0015] Remarkably, the present inventors observed low toxicity and
high inhibition of resistance pathways when administering the
organometallic compounds of the present invention.
[0016] Surprisingly, high proliferation inhibition of the
organometallic compounds of the present invention on carcinoma
cells was observed. Moreover, toxicity against healthy, normal
cells remained very low.
[0017] Accordingly, in a first aspect, the present invention
provides an organometallic compound of the general formula (I),
[MR.sub.1R.sub.2R.sub.3R.sub.4R.sub.5R.sub.6] (I),
which may be charged or neutral, and which may be present in the
form of a salt and/or an optically resolved enantiomer, in which,
[0018] M is a transition metal selected from the group of Ru, Os,
Rh, and Ir; [0019] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6 are neutral or charged ligands of the transition metal,
whereby two, three or more of the ligands R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6 may be present in the form of
one or more single compounds, the single compound being [0020] a
bi-, tri- or polydentate compound, and/or, [0021] an alkene,
alkyne, cyclopentadienyl and/or an arene, the alkene, alkyne,
cyclopentadienyl and/or an arene being optionally substituted and
optionally comprising one or more heteroatoms; in which at least
one bioactive organic compound selected from inhibitors of
resistance pathways, related compounds, and/or derivatives of any
of the fore-mentioned, is present in the organometallic compound,
whereby the bioactive organic compound is directly attached to the
metal, thus constituting at least one of the ligands selected from
R.sub.1-R.sub.6, and/or is covalently bound to any of the ligands
selected from R.sub.1-R.sub.6.
[0022] In a second aspect, the present invention provides the
organometallic compounds of the invention for use as a
medicament.
[0023] In a third aspect, the present invention provides the
organometallic compounds of the invention in the preparation of a
medicament for the inhibition of resistance pathways and/or for
treating cancer, and in particular metastasis.
[0024] In a fourth aspect, the present invention provides a method
for treating and/or preventing metastasis, the method comprising
the step of administering to an individual an effective amount of
the organometallic compound according to the invention.
[0025] In a fifth aspect, the present invention provides a method
for treating and/or preventing metastasis the method comprising the
step of administering to an individual an effective amount of an
anti-cancer drug and, in parallel, an effective amount the
organometallic compound according to the invention.
[0026] In the figures,
[0027] FIG. 1 shows a single crystal X-ray diffraction structure of
compound (3), which is an example of an organometallic compound of
the invention.
[0028] FIG. 2 shows a single crystal X-ray diffraction structure of
compound (6), which is an example of an organometallic compound of
the invention.
[0029] FIG. 3 shows a single crystal X-ray diffraction structure of
compound (8), which is an organometallic compound having, amongst
others, oxalate as a bivalent ligand.
[0030] FIG. 4 illustrates the process for preparing (3), an example
of an organometallic compound of the invention.
[0031] FIG. 5 illustrates the process for preparing (4), an example
of an organometallic compound of the invention.
[0032] FIG. 6 illustrates the process for preparing (6), an example
of an organometallic compound of the invention.
[0033] FIG. 7 illustrates the process for preparing (7), an example
of an charged organometallic compound of the invention, present as
its tetrafluoroborate salt.
[0034] FIG. 8 illustrates the process for preparing (8) and (9),
organometallic compounds that are used as controls for evaluating
the activity of organometallic compounds of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0035] The present invention relates to organometallic compounds
and their use as medicaments. The organometallic compound comprises
a transition metal M, and, linked to the transition metal, a number
of ligands. Ligands include donors of electron pairs and/or donors
of m-orbitals from unsaturated bonds in organic molecules, for
example.
[0036] The term "comprise" or "comprising", for the purpose of the
present invention is intended to mean "including amongst other". It
is not intended to mean, "consisting only of".
[0037] The transition metal M is selected from the group of Ru, Os,
Rh, and Ir. Preferably, M is Ruthenium. The transition metal may be
present in any oxidation state known with respect to the specific
transition metal. For example, the oxidation state may be II or
III. Preferably, M is Ruthenium (II).
[0038] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 are
neutral or charged ligands attached to the transition metal.
Suitable ligands include halogen ions, such as F.sup.-, Cl.sup.-,
Br.sup.- and I.sup.-, preferably chloride. One, two or more ligands
selected from R.sub.1-R.sub.6 may be provided by halogens.
[0039] The term "selected from" a group indicated by
"R.sub.1-R.sub.6", for example, or "any of R.sup.N1-R.sup.N3" as in
the above paragraph or indicated below, respectively, refers to the
fact that one or more selected from all the individuals of the
group may be selected independently of each other. Accordingly, if
one specimen of R.sub.1-R.sub.6 is to be selected, any one of
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 can be
selected.
[0040] One, two, three or more of the ligands R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6 may be selected from ligands
providing electron pairs. For example, the ligands may be selected
from N-, P-, O- or S-donor ligands. Preferably, at least one ligand
of the organometallic compound of the invention is selected from
N-, P-, O- or S-donor ligands. For example, at least one N-donor
and/or at least one P-donor ligand is present.
[0041] Examples of N-donor ligands are nitrile ligands
(N.ident.C--R); azo ligands (N.dbd.N--R); aromatic N-donor ligands;
amine ligands (NR.sup.N1R.sup.N2R.sup.N3); azide (N.sub.3.sup.-);
cyanide (N.ident.C); isothiocyanate (NCS.sup.-).
[0042] In both nitrile and azo ligands, R may be selected from
alkyl, alkenyl, alkynyl, and aryl, optionally substituted and
optionally comprising one or more heteroatoms.
[0043] Aromatic N-donor ligands include optionally substituted
pyridine, pyridazine, pyrimidine, purine and pyrazine, for example.
Substituents may be selected from alkyl, alkenyl, alkynyl, and
aryl, optionally substituted and optionally comprising one or more
heteroatoms.
[0044] R.sup.N1, R.sup.N2, and R.sup.N3 may, independently of each
other, be selected from H, alkyl, alkenyl, alkynyl, aryl,
optionally substituted and optionally comprising one or more
heteroatoms.
[0045] Preferably, two or all three of R.sup.N1-R.sup.N3 may be
fused to form a cyclic N-donor compound. Furthermore, any of
R.sup.N1-R.sup.N3 may be linked covalently to any other ligand of
M, to provide bi-, tridenate or other polyvalent ligands.
Preferably, R.sup.N1, R.sup.N2, and R.sup.N3 are, independently of
each other, selected from H and C.sub.1-8alkyls, optionally
substituted.
[0046] According to a preferred embodiment, at least one of the
ligands selected from R.sub.1-R.sub.6 is an N-donor ligand
comprising the structure of formula (II) below,
##STR00001##
in which, M is the transition metal; the circle represents a mono-
or polycyclic system, comprising at least one N-heteroatom,
indicated as N, which provides an electron pair enabling attachment
to M; B is an optional linking group, which may be selected from
alkyl, alkenyl, alkynyl and aryl, which is optionally substituted,
which optionally comprises one or more heteroatoms and has 0-15,
preferably 1-8 carbon atoms; X is a functional group of B or the
cyclic system, through which the bioactive compound is bound to B
or to the cyclic system.
[0047] The cyclic system may further be substituted.
[0048] Preferably, in formula (II), the circle is a heterocyclic
ring, for example a heterocyclic arene.
[0049] Preferably, it is a 5- or 6-membered heterocyclic ring, for
example arene. Preferably, A is a C.sub.1--Cl.sub.6, more
preferably a C.sub.2-C.sub.4 alkylene.
[0050] Preferably, X is selected from --O-- and from --NH--.
Accordingly, X preferably represents an ether, ester or peptide
group, of which the carbonyl part, if applicable, may be part of
the "bioactive compound" or of B.
[0051] According to a particularly preferred embodiment, any one of
the ligands selected from R.sub.1-R.sub.6 is a N-donor ligand
comprising the structure of formula (III) below, to which the
bioactive compound is linked by means of an amide bond:
##STR00002##
in which, the dashed line represents bonding, as a monodentate
ligand, to the transition metal; the circle represents a mono- or
polycyclic system comprising at least two N-heteroatoms; n is 1-10,
preferably 2-6, most preferably 3-4; X is as defined above for
(III).
[0052] The cyclic system may, for example, be selected from
imidazole, purine, pyrazine, pyrimidine, 1,8-naphthydrin,
chinoxaline, chinazoline, pteridine. Preferably, the cyclic system
is imidazole, resulting in N-donor ligands comprising structures as
illustrated in formula (IV) and (V) below.
##STR00003##
in which n is as indicated for (III) above.
[0053] The bioactive compound comprises, in its released and/or
original, active form, a carboxy-group, which is linked to the
N-atom or O-atom indicated in (IV) or (V), upon formation of a
amide bond.
[0054] Examples of P-donor ligands are PR.sup.P1R.sup.P2R.sup.P3,
in which R.sup.P1, R.sup.P2, and R.sup.P3 are defined as R.sup.N1,
R.sup.N2, and R.sup.N3 above, wherein a fused P-donor ligand may
arise if two or all three of R.sup.N1-R.sup.N3 are fused.
[0055] A preferred example of a P-donor ligand is PTA
(1,3,5-triaza-7-phospha-adamantane).
[0056] S-donor ligands are ligands which bind to M via a sulphur
atom. Examples include thiosulfate (S.sub.2O.sub.3.sup.2-);
isothiocyanate (NCS.sup.-); sulfoxide ligands (R.sup.S1R.sup.S2SO);
thioether ligands (R.sup.S1R.sup.S2S); thiolate ligands
(R.sup.S1S.sup.-); sulfinate ligands (R.sup.S1SO.sup.2-); and
sulfenate ligands (R.sup.S1SO.sup.-), wherein R.sup.S1 and R.sup.S2
are independently selected from alkyls, alkenyls, alkynyls, aryls,
optionally substituted and optionally comprising one or more
heteroatoms.
[0057] O-donor ligands are ligands which bind to M via an oxygen
atom. Examples include carbonate (CO.sup.3-); carboxylate ligands
(R.sup.CCO.sup.2-); nitrate (NO3.sup.-); sulfate (SO4.sup.2-) and
sulphonate (R.sup.S1O3), wherein R.sup.C is selected from alkyls,
alkenyls, alkynyls, aryls, optionally substituted and optionally
comprising one or more heteroatoms.
[0058] According to the organometallic complex of the invention,
two, three or more of the ligands R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6 may be present in the form of one or more
single compounds, the single compound being [0059] a bi-, tri- or
polydentate compound, and/or, [0060] an alkene, alkyne,
cyclopentadienyl and/or an arene, the alkene, alkyne,
cyclopentadienyl and/or an arene being optionally substituted and
optionally comprising one or more heteroatoms.
[0061] Bi-, tri- or polydentate ligands generally comprise at least
two donor ligands, such as N-, P-, O- or S-donor ligands as defined
above, for example. A bi-, tri- or polydentate ligand may,
furthermore, comprise different donor ligands within the same
compound.
[0062] An example of a bidentate N-donor ligand is 2,2'-bipyridine,
optionally substituted. An example of a bidentate O-donor ligand is
oxalate. A well known example of a polydentate ligand is EDTA
(ethylene diamine tetraacetic acid), which comprise 6 donor
locations. In this case, all residues R.sub.1-R.sub.6 would be
provided by one single polydentate compound.
[0063] Two or more substituents of R.sub.1-R.sub.6 may be present
in the form of one or more single compounds being a alkene, alkyne,
cyclopentadienyl, and/or arene.
[0064] In these cases, double bonds of the ligands may play a role
in the formation of the bond with the central M, thus giving rise,
if the ligands are formed by a cyclic compound, to sandwich or
half-sandwich ("piano-stool") configurations, for example.
[0065] Accordingly, in a preferred embodiment, three ligands of the
organometallic compound of the present invention selected from
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6, are
present in the form of an alkene, alkyne, cyclopentadienyl and/or
arene, optionally substituted and optionally comprising one or more
heteroatoms, optionally bound covalently to the bioactive
compound.
[0066] Examples of linear alkenes that function as suitable ligands
to M include alkene, propene, 1,3-butadiene.
[0067] Examples of cyclic alkenes that function as suitable ligands
to M include cyclohexa-1,4-diene and cycloocta-1,5-diene.
[0068] Preferably, three ligands selected from R.sub.1-R.sub.6 are
formed by an organic molecule such as an arene. Preferably, the
selected ligands result in a pseudo-octahedral arrangement around a
central M, although other geometries are also possible, e.g.
pentagonal bipyraimd, square pyramid, tetrahedral and square planar
or intermediate structures thereof.
[0069] The compound of the invention may thus be a half-sandwich
compound. Preferably, three ligands selected from R.sub.1-R.sub.6
are formed by a cyclic alkene, cyclopentadienyl and/or by an arene,
optionally substituted, and optionally comprising one or more
heteroatoms. The at least one cyclic "tridentate" ligand may be a
mono-, bi-, tri- or polycyclic compound. Preferably, it is
monocyclic. Preferably, it is an arene. Arenes are aromatic
hydrocarbons. They may be substituted and comprise one or more
heteroatoms.
[0070] Examples of mono-cyclic arenes are benzene and
cyclopentadienyl (C.sub.5H.sub.5.sup.-). The later is considered by
the present inventors being an arene, but is often mentioned
explicitly for the sake of avoiding doubts. Examples of monocyclic
arenes comprising at least one N-heteroatom are pyridine, pyrazine,
pyrimidine, pyridazine, for example. Of course, other heteroatoms
may be present in the arene besides or instead of N, such as those
mentioned above.
[0071] Accordingly, the arene may be polycyclic. Examples of
polycyclic arenes are pentalene, indene, naphthalene, azulene, and
so forth. Examples of polycyclic arenes comprising N-heteroatoms
are indolizine, +H-indole, 2H-isoindole, 3H-indole, 1H-indazole,
/H-purine, indoline, isoindoline, 4H-quinolizine, quinoline,
isoquinoline, pteridine, phtalizine, naphthydrine, quinazoline,
cinnoline. Of course, other heteroatoms may be present in the
polycyclic arene besides or instead of N, such as those mentioned
above.
[0072] Most preferably, the organometallic compound of the present
invention comprises, as at least one ligand, at least one are
selected, independently, from benzene and cyclopentadienyl, which
are optionally substituted by alkyl, alkenyl, alkynyl or aryl
residues, optionally further substituted and comprising one or more
heteroatoms.
[0073] According to a preferred embodiment of the present
invention, three adjacent ligands of the organometallic compound of
the invention, selected from R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6 are formed by an arene of formula (VI)
##STR00004##
in which R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13
may, independently of each other, be the same or different, are
each hydrogen, alkyl, alkenyl, alkynyl, or aryl, which are, if
applicable, optionally substituted and which optionally comprise
one or more heteroatoms, and in which two or more residues selected
from R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13 may
be covalently linked with each other thus forming a bi- or tri-, or
polycyclic system, and in which any of the residues
R.sub.8-R.sub.13 is optionally bound to the bioactive compound.
[0074] Monocyclic examples of arenes according to the compound of
formula (VI) without heteroatoms are benzene, methylbenzene,
cymene, for example.
[0075] Monocyclic examples of arenes according to the compound of
formula (VI) comprising any one or more residues selected from
R.sub.8-R.sub.13 being an alkyl, alkenyl or alkynyl that is
substituted and/or comprising at least one heteroatom, the
remaining residues being hydrogens are benzyl alcohol,
2-phenylethanol, 3-phenylpropanol, 4-phenylbutanol, benzylamine,
2-phenylethanamine, 3-phenylpropanamine, 4-phenylbutanamine,
2-phenylethanaminium, N,N,-dimethyl-2-phenylethanamine, all of
which may be substituted to be bound to the bioactive organic
compound.
[0076] The residues R.sub.8-R.sub.13 may thus comprise charges, but
are preferably non-charged.
[0077] One or more residues selected from R.sub.8-R.sub.13 may
comprise heteroatoms with electron pairs or double bonds that are
linked to the central M, thus forming one of the ligands
R.sub.1-R.sub.6, which is not yet occupied. An example of a
compound falling in this category is reproduced by formula (VII)
below:
##STR00005##
in which the bioactive organic compound may be linked to any of
residues R.sub.5 or R.sub.6 or to an optional further residue of
the benzene ring.
[0078] Examples of bicyclic variants of compound (VI), in which two
residues, R.sub.12 and are R.sub.13 are covalently linked forming a
bicyclic systems are indicated with formulae (VIII) and (IX)
below
##STR00006##
in which R.sub.8-R.sub.11 have the same meaning as above in
formulae (VI).
[0079] Further substituents may be present on the cycle formed by
R.sub.12 and R.sub.13, which are not shown here.
[0080] According to a particularly preferred embodiment of the
present invention, the compound of formula (VI) comprises the
structure of formula (IX):
##STR00007##
in which, A is a optionally substituted, benzyl or
cyclopentadienyl, B is an optional linking group, which may be
selected from alkyl, alkenyl, alkynyl and aryl, which is optionally
substituted, which optionally comprises one or more heteroatoms and
has 0-15, preferably 1-8 carbon atoms; X is a functional group of B
or the cyclic system, through which the bioactive compound is bound
to B or to the cyclic system.
[0081] Preferably, in (X), A is benzene.
[0082] Preferably, in (X), B is a C.sub.1--Cl.sub.6, more
preferably a C.sub.2-C.sub.4 alkyl. Preferably the alkyl only
comprises X as a substituent and is otherwise unsubstituted.
[0083] Preferably, X is selected from --O-- and from --NH--.
Accordingly, X preferably represents ether or part of an ester or
amide group, of which the carbonyl part, if applicable, may be part
of the "bioactive compound" or of the alkylene. Preferably, the
bioactive compound, in its released and/or original, active form,
comprises a carboxy-group and is linked to the N- or O-heteroatom
indicated as X in formula (X) by means of a amide bond or ester
bond, respectively.
[0084] Accordingly, in case of an ester bond, a hydroxy group of B
may be esterified with a carboxy group of the bioactive compound,
or, vice versa, a hydroxy group of the bioactive compound may be
esterified with a carboxy-group optionally present on B. Both
alternatives allow for hydrolytic cleavage and release of the
bioactive compound.
[0085] In the case of an amide bond, an amino group of B may be
linked to a carboxy group of the bioactive compound, or, vice
versa, an amine group of the bioactive compound may be esterified
with a carboxy-group optionally present on B. Both alternatives
allow for hydrolytic cleavage and release of the bioactive
compound.
[0086] According to further, particularly preferred embodiments,
the compound of formula (VI) comprises the structures of formula
(XI) and (XII):
##STR00008##
in which, m is 1-10, preferably 2-8.
[0087] Preferably, --O-- and --NH-- represent ester and peptide
bonds, respectively, as defined for X in formula (X) above. More
preferably, the bioactive compound, in its released and/or
original, active form, comprises a carboxy-group and is linked to
the O- or the N-atom indicated in (XI) and (XII), respectively, by
means of an ester and peptide bond, respectively.
[0088] Most preferably, m in formula (XI) is 1 and in (XII) is
2.
[0089] According to a preferred embodiment, at least one of
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 of the
organometallic compound of the present invention is a residue
suitable of increasing the solubility of the complex of formula (I)
in water (H.sub.2O). This residue is preferably selected
specifically for this purpose. The bioactive organic compound may
be linked to this residue. Generally, hydrocarbons having a high
heteroatom:carbon ratio are suitable to increase the
solubility.
[0090] Accordingly, at least one of the residues R.sub.1-R.sub.6
has the purpose of increasing the solubility of the organometallic
compound of the invention in water. Preferably, the at least one
ligand has a hydrophilic group. Hydrophilic groups include (--OH),
(.dbd.O), (--COOH), (--NH.sub.2), (--NHR--), (--O--), (--SH.sub.2),
(--S--), (--SO.sub.3--), for example, with R being optionally
substituted alkyl, alkenyl or aryl.
[0091] For example, the at least one residue selected from
R.sub.1-R.sub.6 for increasing solubility in water may be a
hydrocarbon comprising one or several groups capable of engaging in
hydrogen bonding with water, such as, for example, hydroxy
groups.
[0092] In a preferred embodiment of the organometallic compound of
the present invention, at least one organometallic compound of
claim 1, in which any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6 has the formula (XIII),
PR.sub.14R.sub.15R.sub.16 (XIII),
in which, R.sub.14, R.sub.15, R.sub.16 may be the same or
different, are each C.sub.1-C.sub.6 alkyl, aryl or substituted
aryl, or R.sub.14, R.sub.15, R.sub.16 may together with the
phosphorous atom form a cycloalkyl group, such group being
optionally heterocyclic. Preferably, R.sub.14, R.sub.15, R.sub.16
together form a fused ring system comprising 1-5 heteroatoms,
preferably N. This residue, if present, preferably increases the
solubility of the organometallic compound of the present invention
in water.
[0093] An example of a residue capable of increasing solubility is
1,3,5-triaza-7-phospha-adamantane (PTA),
3-methyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (MePTA),
3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (DAPTA),
which may be linked via the P-atom to the central M in the compound
of the present invention. An example of a bivalent ligand capable
of increasing solubility of the compound of the present invention
in water is oxalate.
[0094] In a preferred embedment the organometallic compound of the
present invention comprises the structure (XIV) below,
##STR00009##
in which, A is an arene, optionally substituted and optionally
comprising one or more heteroatoms; R.sub.18, R.sub.19, R.sub.20,
are ligands of the central Ruthenium atom which are, independently
of each other, selected from halogens and/or N--, 0-, S--, or P--
donor ligands; R.sub.17 is an optional residue selected from an
alkyl, alkenyl, alkynyl, aryl, optionally substituted and
optionally comprising one or more heteroatoms; whereby the at least
one bioactive organic compound constitutes at least one selected
from R.sub.17-R.sub.20, or is covalently linked to any of the
R.sub.17-R.sub.20, with the proviso that residues selected from
R.sub.17-R.sub.20, which constitute the bioactive organic compound,
or which are covalently bound the bioactive organic compound, are
not halogens.
[0095] Preferred A and residues R.sub.17-R.sub.20 are as indicated
above. Accordingly, the grouping of A-R.sub.17 may be selected from
formulae (X)-(XII), and the residues R.sub.18-R.sub.20, may be
selected from halogens mentioned above and from N-, O-, S-, or
P-donor ligands mentioned above, for example. If the bioactive
compound is bound to one or more of R.sub.18-R.sub.20, this/these
residue(s) may be selected from formulae (III)-(V), for
example.
[0096] According to a still preferred embodiment, the
organometallic compounds of the present invention comprise the
structures (XV) below:
##STR00010##
in which, X is a halogen; Y is a halogen or N--, P--, O- or S-donor
ligand as defined above; and, in which, if the bioactive compound
is bound to R.sub.17, R.sub.17 is corresponds to "B-X--" as defined
in formula (X) above, and R.sub.18 is a N-, P-, O- or S-donor
ligand as defined above; and/or, if the bioactive compound is bound
to R.sub.18, or constitutes R.sub.18, R.sub.18 is, respectively, a
N-, P-, O- or S-donor ligand as defined above to which the
bioactive compound is covalently bound; or R.sub.18 constitutes the
bioactive compound, the bioactive compound comprising itself N-,
P-, O- or S-atoms functioning as donors suitable to attach it to
the central Ru.
[0097] Preferably, Y in (XV) is a halogen.
[0098] Preferably, R.sub.18 is P R.sub.14R.sub.15 R.sub.16 as
defined above. Preferably, it is PTA.
[0099] Preferably, in (XV), the bioactive compound is linked to
R.sub.17 by a amide or ester bond. For example, the benzene to
which R.sub.17 is bound, R.sub.17 and the bioactive compound
together may correspond to the structure illustrated in formulae
(XI) and (XII), for example.
[0100] If the bioactive compound is linked to R.sub.18, R.sub.18
and the bioactive compound preferably correspond to any of formula
(II)-(V) above.
[0101] If the bioactive compound is linked to R.sub.18, R.sub.18 is
preferably a N-, P-, O-, or S-donor ligand, to which the bioactive
compound is linked. Accordingly, R.sub.18 preferably carries a
functional group to which the bioactive compound can be linked,
such as amide and ester bonds, as mentioned above.
[0102] In this alternative case, with R.sub.18 being covalently
bound to the bioactive compound, R.sub.17 may represent one or more
residues such as R.sub.8-R.sub.13 in the compound of formula (VI)
above.
[0103] The present invention also envisages the possibility that
the organometallic compound comprises more than one bioactive
compound covalently bound to a ligand selected from
R.sub.1-R.sub.6. Accordingly, in the above example of formula (XV),
both, R.sub.17 and R.sub.18 may be covalently bound to a bioactive
organic compound, whereby R.sub.18 may, instead constitute the
bioactive compound. The bioactive compounds may then be the same or
different. It they are different, the second bioactive compound may
have a biological activity different from the bioactive compound
generally used in the context of the present invention. Preferably,
however, if more than one bioactive compounds are present, all are
useful in the treatment of cancer in general and/or metastasis in
particular.
[0104] Many residues, and in particular ligands attached to M
detailed above are indicated to be substituted. For the purpose of
the present invention, substituents are preferably selected,
independently from each other if there are more than one
substituents, from alkyls, alkenyls, alkynyls, aryls, the alkyls,
alkenyls, alkynyls, aryls, optionally comprising one or more
heteroatoms, and functional groups such as, for example,
imine-groups (.dbd.NH), amino groups (--NH.sub.2), hydroxy groups
(--OH), thiol groups (--SH), carbonyl groups (.dbd.O), thio groups
(.dbd.S), carboxyl groups (--COOH), nitrile groups (--C.ident.N),
nitro groups (--NO.sub.2), any other functional group and, if
applicable, charged derivatives (for example, if pH dependent) and
salts of the functional groups, for example. The substituent
comprising a functional group may be substituted at the functional
group.
[0105] Substituents may be branched and/or be further
substituted.
[0106] The present invention, for example in claim 1, refers to an
alkene, alkyne, cyclopentadienyl, and/or arene. The alkene or
alkyne is considered to be an unsaturated C.sub.2-C.sub.30, more
preferably a C.sub.3-C.sub.15 and most preferably C.sub.4-C.sub.8
hydrocarbon. An alkene comprises at least one (C.dbd.C)-double
bound, whereas the alkyne comprises at least one (C.ident.C)-triple
bond. The arene is preferably an aromatic C.sub.5-C.sub.35, more
preferably C.sub.6-C.sub.20, most preferably C.sub.6-C.sub.15
hydrocarbon. The alkene or alkyne may also be cyclic, if there are
.gtoreq.3 carbons. The alkene or alkyne may be branched, if there
are .gtoreq.4 or .gtoreq.5 carbons, respectively. The alkene,
alkyne, cyclopentadienyl and/or arene (or aryl radical) may further
be substituted, as indicated above, and optionally comprising one
or more heteroatoms.
[0107] Alkenyl, alkynyl and/or aryl residues or substituents, as
mentioned, for example, with respect to nitrile and azo ligands,
aromatic N-donor ligands, amine ligands, P-donor ligands, S-donor
ligands, O-donor ligands, substituents of benzene or
cyclopentadienyl ligands, substituents selected from
R.sub.8-R.sub.13, R.sub.17 in formula (III), substituents of the
alkene, alkyne, cyclopentadienyl and/or arene mentioned in the
above paragraph, substituents of an alkyl or with respect to
substituents in general are radicals of the alkenes, alkynes,
cyclopentadienyls and/or arenes as defined in the paragraph
above.
[0108] Alkyl substituents, as mentioned, for example, with respect
to substituents in general as defined above, N-donor ligands, in
particular nitrile and azo ligands, aromatic N-donor ligands, amine
ligands, substituents of B in formula (II), P-donor ligands,
S-donor ligands, O-donor ligands, substituents of the alkene,
alkyne and/or arene of claim 1, substituents of benzene and/or
cyclopentadienyl, substituents R.sub.8-R.sub.13 of formulae (VI),
substituents of B in formula (IX), substituents selected from
R.sub.14-R.sub.15, R.sub.17 in formula (III), are preferably
C.sub.1-C.sub.30 alkyls, more preferably C.sub.2-C.sub.25 alkyls,
even more preferably C.sub.4-C.sub.10 alkyls. If the alkyl
comprises more than 3 carbons, it may be cyclic or branched. Alkyls
that are cyclic and branched are also encompassed, if the comprise
more than 6 carbons. Alkyls may generally be further
substituted.
[0109] A heteroatom, for the purpose of the present invention may
be any heteroatom, but is preferably selected from B, Si, N, P, As,
O, S, Se, T, and halogens. If several heteroms are present, they
may be the same or different. More preferably, heteroatoms are
selected from N, O, P, S, and halogens. If the heteroatom is
present in a substituent, alkene, alkyne, cyclopentadienyl or
arene, it may change the chemical class of the compound. For
example, an O present in a linear alkyl results in an ether. For
the purpose of the present invention, this example would be
considered to be an alkyl comprising one O heteroatom.
[0110] The present invention provides an organometallic compound
having a bioactive agent linked to at least one of the ligands of
the central M.
[0111] Preferably, the bioactive organic compound is selected from
inhibitors of resistance pathways and/or a pharmaceutically
acceptable derivatives thereof. Inhibiting activity may, if it is
not yet described, be assessed by specific, commercially obtainable
or described assays. The skilled person is capable of using
meaningful concentrations of an presumed inhibitor in such an
assay. An example of an assay for testing Glutathione S-transferase
inhibiting activity is mentioned in Example 6.
[0112] According to a preferred embodiment, the bioactive organic
compound is an inhibitor of resistance selected from Glutathione
S-transferase (GST) inhibitors, .gamma.-Glutamyl Cysteine
Synthetase (.gamma.-GCS) inhibitors, multidrug resistance protein
(MRP)/P-glycoproteins (PgP) inhibitors, inhibitors of cell
signalling pathways.
[0113] Examples of inhibitors of Glutathione S-transferase
inhibitors comprise, but not limited to, ethacrynic acid,
peptidomimetics based on gluthatione, p-chlorophenoxyisobutyrate,
Gossypol, indomethacin, non-steroidal anti-inflammatory compounds
based on ibuprofen and ketoprofen, misonidazole, Piriprost,
Sulfasalazine, and their derivatives.
[0114] Examples of inhibitors of .gamma.-Glutamyl Cysteine
Synthetase comprise, but not limited to, sulfoxime-based compounds
such as buthinone sulfoxime and methinone sulfoxime,
S-sulfocysteine, S-sulfohomocysteine, cystamine, and their
derivatives.
[0115] Examples of inhibitors of the multidrug resistance protein
comprise, but not limited to, quinidine, vinblastine, terfernadine,
tamoxifen, verapamil, cyclosporin, amitriptyline, progesterone, and
their derivatives.
[0116] Examples of inhibitors of cell signaling pathways comprise,
but not limited to, pleurotin, azelaic acid,
bischloroethylnitrosourea, palmarumycin, and their derivatives.
[0117] Optically pure enantiomers, mixtures of enantiomers such as
racemates, diastereomers, mixtures of diastereomers, diastereomeric
racemates, mixtures of diastereomeric racemates, and the meso-form,
as well as pharmaceutically acceptable salts, solvent complexes and
morphological forms of the bioactive organic compounds are also
encompassed by the present invention.
[0118] The expression pharmaceutical acceptable derivatives and
derivatives also encompasses, but is not limited to, salts.
[0119] Salts comprise, for example, salts with inorganic acids or
organic acids like hydrochloric or hydrobromic acid, sulphuric
acid, phosphoric acid, citric acid, formic acid, acetic acid,
maleic acid, tartaric acid, benzoic acid, methanesulfonic acid,
p-toluenesulfonic acid, and the like that are non toxic to living
organisms or in case the compound (I) is acidic in nature with an
inorganic base like an alkali or earth alkali base, e.g. sodium
hydroxide, potassium hydroxide, calcium hydroxide and the like.
[0120] According to the present invention, the bioactive compound
constitutes a ligand of the central M selected from
R.sub.1-R.sub.6, or is covalently bound to a ligand to the central
M of the organometallic compound of the invention. If the bioactive
compound is covalently bound to the ligand, the covalent bond can
be a carbon-carbon alkyl, alkenyl or alkynyl bonds, or
carbon-heteronuclear bonds such as amide (--CONH--), ester
(--CO.sub.2--), ether (--CH.sub.2O--), thioether (--CH.sub.2S--),
amine (--CH.sub.2N--), imine (--CH.dbd.N--), phosphorous
(--CH.sub.2P--). Preferably, the covalent bond is a carbon
heteronuclear bond.
[0121] According to a preferred embodiment of the present
invention, the organometallic compounds of the present invention
are used as medicaments.
[0122] More particularly, according to a preferred embodiment, the
organometallic compound according to any of the preceding claims in
the treatment and/or prevention of cancer and/or metastasis. The
effectiveness of the present compounds for the treatment of
metastasis is remarkable. With many cancers, tumors may be removed
surgically, with the occurrence of metastasis remaining the
principle problem to which so far no convincing remedy has been
found. Surprisingly, the compounds of the present invention are
effective in overcoming the resistance of many cancer cells in
metastasis to anti-cancer drugs. Thanks to the compounds of the
present invention, anti-cancer drugs that are inefficient due to
the onset of resistance against it, these very drugs may again be
effectively employed. In addition, due to the increase of
efficiency against cancer drugs, lower doses of the later may be
applied, thus reducing the occurrence and severity of side
effects.
[0123] Accordingly, according to a preferred embodiment, the
organometallic compound according to the invention are useful for
reducing resistance of cancers and/or metastasis against
anti-cancer drugs.
[0124] According to a further embodiment, the present invention
comprises a composition comprising an anti-cancer drug and the
organometallic compound of any of the preceding claims for treating
and/or preventing metastasis. The organometallic compound of the
invention is most effective if administered together with an
anti-cancer drug which may be conventional, and to which cancer
cells have developed resistance, or a capable of developing
resistance.
[0125] As becomes clear from the above, the principle of the
present invention does not only encompass a single, specific
compound but is more general. In particular, with respect to the
different ligands bound to the central M, and to one of which the
bioactive compound is covalently bound, a large variability is
provided. The bioactive compound may also be directly attached to
the central atom, increasing the variability. If the bioactive
compound is bound to a ligand, it may be linked to ligands of
different general structure and to ligands that are linked to the
central M in different ways, for example as
free-electron-pair-donors or as donors of .pi.-bonds that are
capable of complexing or associating to a transition metal.
Preferably, however, the central M is Ruthenium (Ru), and the
overall compound has a sandwich or half-sandwich structure.
[0126] Therefore, a high variability exists with respect to the
ligands. For examples, the arene-part shown in exemplary ligands of
formula (VII) and (VIII) may be selected from a large number of
possible arenes that can be used in sandwich- or half-sandwich
compounds. These arenes may, of course, be substituted, for example
for improving the physico-chemical properties of the overall
compound, such as solubility, or for improving effectiveness and
reducing toxicity to normal cells. Such substituents are not shown
here in all the detail, they may be selected at the discretion of
the skilled person and are not essential for the general principle
described herein.
[0127] The following examples illustrate the principle of the
present invention on the basis of ethacrynic acid, which functions
as the bioactive compound and which is shown to be effective in the
treatment of metastasis. The examples also illustrate the
structural variably of connecting the bioactive compound to ligands
of different structures and being linked to the central M in
different ways.
A. Examples 1-5
Synthesis of the Organometallic Compounds of the Invention
Example 1
Ethacrynic-(cyclohexa-1,4-dienyl)methylamide (1)
[0128] Ethacrynic acid (500 mg, 1.65 mmol) was refluxed in oxalyl
chloride (5 ml) for 30 mins. Unreacted oxalyl chloride was removed
in vacuo and dichloromethane (10 ml) was added to redissolve the
residual colourless oil. (cyclohexa-1,4-dienyl)methamine (109 mg,
1.00 mmol) and triethylamine (1 ml) was added sequentially and the
reaction mixture was refluxed for a further 6 h. On completion, a
dark brown solution was obtained. The reaction mixture was washed
with 5% NaHCO.sub.3 (50 ml), brine (2.times.50 ml) and dried in
vacuo. The residual oil was separated on silica using 5:95
MeOH:dichloromethane as the eluent to yield a colourless oil which
crystallizes on standing (yield: 250 mg, 63.3%). .sup.1H NMR
(CDCl.sub.3, 400.13 MHz) 7.19 (d, 1H, Ar.sub.EA--H,
.sup.3J.sub.HH=8.4 Hz), 6.87 (d, 1H, Ar.sub.EA--H,
.sup.3J.sub.HH=8.4 Hz), 6.81 (b, 1H, --CH.sub.2NH--), 5.95 (s, 1H,
.dbd.CH.sub.2), 5.64-5.71 (m, 3H, C.dbd.CH--C), 5.58 (s, 1H,
.dbd.CH.sub.2), 4.60 (s, 2H, --OCH.sub.2CO.sub.2--), 3.91 (d, 2H,
--CH.sub.2NH--, .sup.3J.sub.HH=6.0 Hz), 2.83 (t, 2H,
--CH.sub.2NH--, .sup.3J.sub.HH=6.0 Hz), 2.61-2.72 (m, 4H,
.dbd.C--CH.sub.2--C.dbd.), 2.47 (q, 2H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz), 1.15 (t, 3H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz).
[(Ethacrynic-.eta..sup.6:benzylamide)RuCl(.mu.-Cl)].sub.2(2)
[0129] (1) (0.43 g, 3.32 mmol) was refluxed with
RuCl.sub.3.3H.sub.2O (50 mg, 0.192 mmol) in degassed EtOH (25 ml)
for 6 hours and left to stand at -4.degree. C. for 12 h, during
which a reddish-orange precipitate separates from the dark green
solution. The precipitate was filtered, dissolved in
dichloromethane and precipitated using diethyl ether to yield a
light orange precipitate. The product was dried in vacuo (yield:
76.0 mg, 70.4%). .sup.1H NMR (CDCl.sub.3, 400.13 MHz) 8.11 (t, 1H,
--CH.sub.2NH--), 7.16 (d, 1H, Ar.sub.EA--H, .sup.3J.sub.HH=8.4 Hz),
6.90 (d, 1H, Ar.sub.EA--H, .sup.3J.sub.HH=8.4 Hz), 5.97 (s, 1H,
.dbd.CH.sub.2), 5.77 (t, 1H, p-Ar.sub.Ph--H, .sup.3J.sub.HH=6.0
Hz), 5.63 (s, 1H, .dbd.CH.sub.2), 5.56 (dd, 2H, m-Ar.sub.Ph--H,
.sup.3J.sub.HH=6.0, 6.0 Hz), 5.43 (d, 2H, o-Ar.sub.Ph--H,
.sup.3J.sub.HH=6.0 Hz), 4.80 (s, 2H, --OCH.sub.2CO.sub.2--), 4.58
(d, 2H, --CH.sub.2NH--, .sup.3J.sub.HH=6.0 Hz), 2.47 (q, 2H,
--CH.sub.2CH.sub.3, .sup.3J.sub.HH=7.6 Hz), 1.15 (t, 3H,
--CH.sub.2CH.sub.3, .sup.3J.sub.HH=7.6 Hz). Anal.
(C.sub.40H.sub.38Cl.sub.8N.sub.2O.sub.6Ru.sub.2) C, 42.57; H, 3.39;
N, 2.48. Found C, 42.48, H, 3.52,N, 2.70.
(Ethacrynic-.eta..sup.6:benzylamide)Ru(PTA)Cl.sub.2 (3)
[0130] (2) (28.4 mg, 0.025 mmol) was refluxed with PTA (9.3 mg,
0.059 mmol) in 1:1 MeOH/dichloromethane (15 ml) for 2 hours. The
solvent was removed and the residual was recrystallised from
dichloromethane/diethyl ether to yield and an orange precipitate
(yield: 31 mg, 85.3%). .sup.1H NMR (CDCl.sub.3, 400.13 MHz) 7.87
(t, 1H, --CH.sub.2NH--), 7.18 (d, 1H, Ar.sub.EA--H,
.sup.3J.sub.HH=8.4 Hz), 6.90 (d, 1H, Ar.sub.EA--H,
.sup.3J.sub.HH=8.4 Hz), 5.96 (s, 1H, .dbd.CH.sub.2), 5.07 (t, 1H,
p-Ar.sub.Ph--H, .sup.3J.sub.HH=5.6 Hz), 5.60 (s, 1H,
.dbd.CH.sub.2), 5.70 (m, 2H, m-Ar.sub.Ph--H), 5.52 (d, 2H,
o-Ar.sub.Ph--H, .sup.3J.sub.HH=6.0 Hz), 4.69 (s, 2H,
--OCH.sub.2CO.sub.2--), 4.56 (d, 2H, --CH.sub.2NH--,
.sup.3J.sub.HH=6.0 Hz), 4.51 (s, 6H, PTA-N--CH.sub.2--N), 4.32 (s,
6H, PTA-P--CH.sub.2--N), 2.47 (q, 2H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz), 1.15 (t, 3H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz). Anal.
(C.sub.26H.sub.31Cl.sub.4N.sub.4O.sub.3PRu) C, 42.23; H, 4.50; N,
7.58. Found C, 42.54, H, 4.40, N, 7.74.
Example 2
(Ethacrynic-.eta..sup.6:phenylethanoate)Ru(PTA)Cl.sub.2 (4)
[0131] Ethacrynic acid (200 mg, 0.66 mmol),
N,N-dicyclohexylcarbodiimide (200 mg, 0.98 mmol),
N,N-dimethylaminopyridine (120 mg, 1.10 mmol) and
(.eta..sup.6:phenylethanol)Ru(PTA)Cl.sub.2) (90 mg, 0.20 mmol) was
dissolved in dichloromethane (50 ml) and stirred for 96 h. The
reaction mixture was filtered through celite to remove the urea
precipitate and separated on silica gel using acetone. The product
was triturated in diethyl ether and recrystallised from
dichloromethane/diethyl ether to yield a brown precipitate (yield:
50 mg, 34.0%). .sup.1H NMR (CDCl.sub.3, 400.13 MHz) 7.07 (d, 1H,
Ar.sub.EA--H, .sup.3J.sub.HH=8.4 Hz), 6.72 (d, 1H, Ar.sub.EA--H,
.sup.3J.sub.HH=8.4 Hz), 6.00 (s, 1H, .dbd.CH.sub.2), 5.62 (s, 1H,
.dbd.CH.sub.2), 5.48 (m, 2H, m-Ar.sub.Ph--H), 5.22 (d, 2H,
o-Ar.sub.Ph--H, .sup.3J.sub.HH=5.6 Hz), 5.13 (t, 1H,
p-Ar.sub.Ph--H, .sup.3J.sub.HH=5.2 Hz), 4.81 (s, 2H,
--OCH.sub.2CO.sub.2--), 4.55 (s, 6H, PTA-N--CH.sub.2--N), 4.50 (t,
2H, --CO.sub.2CH.sub.2CH.sub.2--, .sup.3J.sub.HH=6.0 Hz), 4.33 (s,
6H, PTA-P--CH.sub.2--N), 2.83 (t, 2H, --CO.sub.2CH.sub.2CH.sub.2--,
.sup.3J.sub.HH=6.0 Hz), 2.47 (q, 2H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz), 1.16 (t, 3H,
--CH.sub.2CH.sub.3,.sup.3J.sub.HH=7.6 Hz). Anal.
(C.sub.27H.sub.32Cl.sub.4N.sub.3O.sub.4PRu) C, 44.03; H, 4.38; N,
5.71. Found C, 43.94; H, 4.40; N, 5.72.
Example 3
N-[2-(1H-imidazol-1-yl)propyl]ethacrynic-amide (5)
[0132] Ethacrynic acid (349 mg, 1.16 mmol) was refluxed in
dichloromethane (20 ml) with an excess of oxalyl chloride (2 ml)
for 1 h. Unreacted oxalyl chloride was removed under vacuum and the
reaction mixture concentrated to yield ethacrynic acid chloride as
a colourless oil. The acid chloride was taken up in dichloromethane
(20 ml) and N-(aminopropyl)imidazole (500 mg, 4.00 mmol) was added.
The reaction mixture was then refluxed for 2 h. 5% NaHCO.sub.3
solution (25 ml) was added to quench the reaction and the aqueous
phase was extracted with dichloromethane (3.times.25 ml). The
organic phases were combined and washed with brine (2.times.25 ml)
and dried over Na.sub.2SO.sub.4. The solvent was removed and the
product was separated on silica using 20% EtOH/80% CHCl.sub.3. The
solvent was removed to yield a colourless oil (yield: 378 mg,
79.5%). .sup.1H NMR (CDCl.sub.3, 400.13 MHz) 7.52 7.07 6.96 (s, 3H,
Ar.sub.imdazole--H), 7.20 6.86 (d, 2H, Ar.sub.EA--H,
.sup.3J.sub.HH=8.4 Hz), 6.82 (m, 1H, NH), 5.96 5.59 (s, 2H,
.dbd.CH.sub.2), 4.57 (s, 2H, --OCH.sub.2CO.sub.2--), 4.04 (t, 2H,
Im-CH.sub.2--CH.sub.2), 3.42 (dt, 2H, CH.sub.2--CH.sub.2--NH), 2.47
(q, 2H, --CH.sub.2CH.sub.3, .sup.3J.sub.HH=7.6 Hz), 2.10 (tt, 2H,
CH.sub.2--CH.sub.2--CH.sub.2), 1.16 (t, 3H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz).
(.eta..sup.6-cymene)RuCl.sub.2(ethacrynic-propylamide-imidazole)
(6)
[0133] [(.eta..sup.6-cymene)RuCl(.mu.-Cl)].sub.2 (68 mg, 0.11 mmol)
and (5) (94 mg, 0.23 mmol) was dissolved in dichloromethane (25 ml)
and stirred for 12 h. The reaction mixture was concentrated and
pentane was added to precipitate the product. The product was
recyrstallised from dichloromethane/pentane to yield an orange
precipitate (yield: 144 mg, 90.3%). .sup.1H NMR (CDCl.sub.3, 400.13
MHz) 7.95 7.30 6.91 (s, 3H, imidazole-H), 7.19 6.90 (d, 2H,
Ar.sub.EA--H, .sup.3J.sub.HH=8.4 Hz), 6.94 (t, 1H, N--H), 5.96 5.61
(s, 2H, .dbd.CH.sub.2), 5.45 5.26 (dd, 4H, Ar.sub.Ph--H,
.sup.3J.sub.HH=6.0 Hz), 4.60 (s, 2H, --OCH.sub.2CO.sub.2--), 3.89
(t, 2H, --CH.sub.2CH.sub.2-imidazole, .sup.3J.sub.HH=7.2 Hz) 3.36
(q, 2H, NHCH.sub.2CH.sub.2--, .sup.3J.sub.HH=7.2 Hz), 2.96 (septet,
1H, --CH.sub.3CHCH.sub.3, .sup.3J.sub.HH=8.0 Hz), 2.47 (q, 2H,
--CH.sub.2CH.sub.3, .sup.3J.sub.HH=7.6 Hz), 2.16 (s, 3H,
--ArCH.sub.3), 1.93 (m, 2H, NHCH.sub.2CH.sub.2--), 1.26 (d, 6H,
--CH(CH.sub.3).sub.2, .sup.3J.sub.HH=8.0 Hz), 1.15 (t, 3H,
--CH.sub.2CH.sub.3, .sup.3J.sub.HH=7.6 Hz). Anal.
(C.sub.29H.sub.35Cl.sub.4N.sub.3O.sub.3Ru) C, 48.67, H, 4.93, N,
5.88. Found C, 48.75; H, 4.95; N, 5.79.
Example 4
[(.eta..sup.6-cymene)RuCl(PTA)(imidazolium-ethacrynic
amide)]BF.sub.4.sup.- (7)
[0134] (.eta..sup.6-cymene)Ru(PTA)Cl.sub.2 (96 mg, 0.21 mmol),
NaBF.sub.4 (92 mg, 0.84 mmol) and (5) (124 mg, 0.30 mmol) was
suspended in methanol (15 ml) and refluxed for 2 h, during which a
yellow solution was obtained. The reaction mixture was cooled to
room temperature and the solvent removed. The residue was extracted
with dichloromethane (2.times.25 ml) and filtered through celite.
The dichloromethane extracts was concentrated and diethyl ether was
added to precipitate the product. The product was recrystallised
from dichloromethane/diethyl ether to yield a yellow precipitate
(yield: 150 mg, 77.2%). .sup.1H NMR (CDCl.sub.3, 400.13 MHz) 8.28
7.42 7.04 (s, 3H, imidazole-H), 7.22 (t, 1H, N--H), 7.17 6.99 (d,
2H, Ar.sub.EA--H, .sup.3J.sub.HH=8.4 Hz), 5.96 5.58 (s, 2H,
.dbd.CH.sub.2), 5.81 5.61 (dd, 4H, Ar.sub.Ph--H), 4.72 (m, 2H,
--OCH.sub.2CO.sub.2--), 4.41 (m, 6H, PTA-N--CH.sub.2--N), 4.10 (m,
6H, PTA-P--CH.sub.2--N), 3.89 (t, 2H, --CH.sub.2CH.sub.2-imidazole,
.sup.3J.sub.HH=7.2 Hz) 3.36 (q, 2H, NHCH.sub.2CH.sub.2--,
.sup.3J.sub.HH=7.2 Hz), 2.96 (septet, 1H, --CH.sub.3CHCH.sub.3,
.sup.3J.sub.HH=8.0 Hz), 2.47 (q, 2H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz), 2.16 (s, 3H, --ArCH.sub.3), 1.93 (m, 2H,
NHCH.sub.2CH.sub.2--), 1.26 (d, 6H, --CH(CH.sub.3).sub.2,
.sup.3J.sub.HH=8.0 Hz), 1.15 (t, 3H, --CH.sub.2CH.sub.3,
.sup.3J.sub.HH=7.6 Hz). ESI-MS (CH.sub.2Cl.sub.2, +ve mode) m/z 840
[M].sup.+. Anal.
(C.sub.35H.sub.47BCl.sub.3F.sub.4N.sub.6O.sub.3PRu) C, 45.45; H,
5.12; N, 9.09. Found C, 45.60, H, 5.15, N, 9.07.
Example 5
(.eta..sup.6-cymene)Ru(PTA)(C.sub.2O.sub.4) (8)
[0135] [(.eta..sup.6-cymene)RuCl(.mu.-Cl)].sub.2 (196.8 mg, 0.322
mmol) and silver oxalate (240 mg, 0.797 mmol) were stirred in water
for 12 h. The mixture was then filtered through celite to remove
the AgCl precipitate. The solvent was removed under vacuum and the
residue was redissolved in methanol (25 ml). PTA (120 mg, 0.764
mmol) was added and the reaction was stirred for 2 h. The solvent
was reduced to ca. 5% of its original volume and diethyl ether (25
ml) was added. The slurry was cooled to 4.degree. C. for 12 h to
complete precipitation of the product. The precipitate was filtered
and recrystallised from methanol-diethyl ether to yield a light
orange precipitate (yield: 285 mg, 89%). .sup.1H NMR (D.sub.2O,
400.13 MHz) 5.98 5.89 (dd, 4H, Ar--H), 4.57 (s, 6H,
PTA-N--CH.sub.2--N), 4.15 (s, 6H, PTA-P--CH.sub.2--N), 2.61
(septet, 1H, --CH(CH.sub.3).sub.2), 2.05 (s, 3H, --CH.sub.3), 1.22
(d, --CH(CH.sub.3).sub.2). .sup.13C-{.sup.1H} NMR (D.sub.2O, 100.63
MHz) 166.2 (--CO.sub.2), 105.0 97.7 87.3 86.8 (Ar--C), 70.7
(N--CH.sub.2--N), 48.7 (P--CH.sub.2--N), 30.8 (--ArCH.sub.3), 21.5
(--CH(CH.sub.3).sub.2), 17.3 (--CH(CH.sub.3).sub.2).
.sup.31P-{.sup.1H} NMR (D.sub.2O, 400.13 MHz)-33.39. Anal.
(C.sub.18H.sub.26N.sub.3O.sub.4PRu.0.5H.sub.2O)C, 44.08, H, 5.55,
N, 8.57. Found C, 44.24; H, 5.58; N, 8.69.
(.eta..sup.6-cymene)Ru(PTA)(C.sub.6H.sub.6O.sub.4) (9)
[0136] [(.eta..sup.6-cymene)RuCl(.mu.-Cl)].sub.2 (228 mg, 0.373
mmol) and silver 1,1-cyclobutanedicarboxylate (300 mg, 0.838 mmol)
were stirred in acetonitrile (50 ml) for 12 h, during which a
yellow precipitate was formed. The solvent was removed and the
residue was redissolved in methanol (25 ml). The mixture was
filtered through celite to remove the AgCl precipitate. PTA (130
mg, 0.828 mmol) was added to the filtrate and the solution was
stirred for 2 hours. The solvent was reduced to ca. 5% of its
original volume and diethyl ether (25 ml) was added. The slurry was
cooled to 4.degree. C. for 4 hours to complete precipitation of the
product. The precipitate was filtered and recrystallised from
dichloromethane-diethyl ether to yield an orange precipitate
(yield: 288 mg, 72.2%). .sup.1H NMR (CDCl.sub.3, 400.13 MHz) 5.54
5.43 (dd, 4H, Ar--H), 4.49 (s, 6H, PTA-N--CH.sub.2--N), 4.15 (s,
6H, PTA-P--CH.sub.2--N), 2.76 2.66 (t, 4H,
--CH.sub.2(CH.sub.2).sub.2), 2.58 (septet, 1H,
--CH(CH.sub.3).sub.2), 2.02 (s, 3H, --CH.sub.3), 1.94 (quintet, 2H,
--CH.sub.2(CH.sub.2).sub.2), 1.24 (d, --CH(CH.sub.3).sub.2).
.sup.13C-{.sup.1H} NMR (CDCl.sub.3, 100.63 MHz) 178.7 (--CO.sub.2),
102.5 96.1 87.9 85.3 (Ar--C), 72.9 (N--CH.sub.2--N), 50.8
(P--CH.sub.2--N), 30.9 (--(O.sub.2C)C(CH.sub.2).sub.2), 30.9
(C(CH.sub.2).sub.topCH.sub.2), 30.9
(C(CH.sub.2).sub.bottomCH.sub.2), 30.9
((--CH.sub.2).sub.2CH.sub.2), 30.9 (--ArCH.sub.3), 22.5
(--CH(CH.sub.3).sub.2), 17.3 (--CH(CH.sub.3).sub.2).
.sup.31P--{.sup.1H} NMR (CDCl.sub.3, 400.13 MHz) -30.16. Anal.
(C.sub.22H.sub.32N.sub.3O.sub.4PRu.H.sub.2O)C, 47.73; H, 6.19; N,
7.59. Found C, 47.99; H, 6.45; N, 7.72.
B. Examples 6-9
In vitro and in vivo Biological Data
Example 6
Determination of Inhibition of Glutathione-S-Transferase
Activity
[0137] Human A549 lung carcinoma cells, known to express high
levels of Glutathione-S-Transferase, were routinely grown in flasks
with DMEM medium containing 4.5 g/l glucose, 10% foetal calf serum
(FCS) and antibiotics at 37.degree. C. and 6% CO.sub.2. When the
cells are confluent, they are trypsinised and concentrated in a
centrifuge at 4.degree. C. The cells were then diluted in
phosphate-buffer saline (PBS) containing protease inhibitor
cocktail (final concentration of 1 .mu.g/ml) and homogenised by
repeatedly freezing in liquid nitrogen and thawing (4 cycles). The
homogenised cell samples were centrifuged at 4.degree. C. and the
supernatant, which is the cell lysates, was separated. The cell
lysates are stored at -56.degree. C.
[0138] The Ru compounds are weighed, and dissolved in DMSO to 100
mM. They are diluted in water to 100 .mu.M, such that the DMSO
concentration did not exceed 0.1%. The cell lysates were exposed to
the drug solutions for 90 mins. Control, containing the cell
lysates with water/0.1% DMSO was also prepared.
[0139] The GST activity in the treated cell lysates was determined
using the glutathione-CDNB (1-chloro-2,4-dinitrobenzene) assay.
Glutathione and CDNB were dissolved in deionised water and ethanol
respectively to make up a 100 mM solutions. A developing solution,
containing 50 mM phosphate buffer solution (pH 6.5), was prepared
and was added 1% v/v, the 100 mM glutathione and 100 mM CDNB
solutions such that their final concentrations are both 1 mM. The
developing solution was added to a 96-well plate at 200 .mu.l per
well, followed by the treated cell lysates at 2 .mu.l per well. The
absorbance at 340 nm was monitored continuously for 5 mins at 15 s
intervals. The average slope of the change in absorbance was
determined as a fraction of the control as the percentage of
residual GST activity.
[0140] Results:
[0141] Cells lysates treated with complexes with good
GST-inhibition ability should exhibit low residual GST activity.
The summary of the results are shown in Table 1 below:
TABLE-US-00001 TABLE 1 GST activity in lung carcinoma cells
Residual GST activity Teatment (% control) Ethacrynic acid 86.32
.+-. 13.5 (Ethacrynic-.eta..sup.6:phenylmethyl- 63.57 .+-. 2.0
amide)Ru(PTA)Cl.sub.2 (3) (Ethacrynic-.eta..sup.6:phenylethanol
42.77 .+-. 13.9 ester)Ru(PTA)Cl.sub.2 (4)
(.eta..sup.6-cymene)RuCl.sub.2(ethacrynic- 23.70 .+-. 10.7
propylamide-imidazole) (6)
[(.eta..sup.6-cymene)RuCl(PTA)(ethacrynic- 62.58 .+-. 8.8
propylamide-imidazole)]BF.sub.4.sup.- (7)
Example 7
Determination of Cell Growth Proliferation Inhibition
[0142] The cells were routinely grown in flasks with DMEM medium
containing 4.5 g/l glucose, 10% foetal calf serum (FCS) and
antibiotics at 37.degree. C. and 6% CO.sub.2. When the cell are
confluent, they are trypsinised and seeded in 48-well plates as
monolayers for 24 h.
[0143] For (3), (4), (5) and (7), the Ru compounds are weighed, and
dissolved in DMSO to 100 mM. They are diluted in excess medium and
diluted to 100 .mu.M, such that the DMSO concentration did not
exceed 0.12%. The 100 .mu.M Ru complex solution was then serially
diluted to make up 50 .mu.M, 25 .mu.M, 12.5 .mu.M, 6.3 .mu.M, 3.1
.mu.M, 1.6 .mu.M solutions. The media is removed from the cell
plates are the drug solutions applied in triplicate. A set of
control cells, with media containing 1.0% DMSO, was left on each
plate. The cells were exposed to the drugs for 72 hours.
[0144] For RAPTA-C, (8) and (9), the Ru compounds are weighed, and
dissolved directly in medium to 1600 .mu.M, then serially diluted
to make up 800 .mu.M, 400 .mu.M, 200 .mu.M, 100 .mu.M, 50 .mu.M, 25
.mu.M solutions. The media is removed from the cell plates are the
drug solutions applied in triplicate. A set of control cells, with
media containing 1.0% DMSO, was left on each plate. The cells were
exposed to the drugs for 72 hours.
[0145] Cell viability was determined using the standard MTT assay
protocol, which allows the quantification of the mitochondrial
activity in metabolically active cells. MTT (final concentration
0.2 mg/ml) was added to the cells for 2 h, then the culture medium
was aspirated and the violet formazan precipitate dissolved in 0.1
N HCl in 2-propanol. The optical density, which is directly
proportional to number of surviving cells, was quantified at 540 nm
using a multiwell plate reader and the fraction of surviving cells
was calculated from the absorbance of untreated control cells.
[0146] Results:
[0147] Using the above protocol, the compounds were tested against
human T47D and MCF-7 breast carcinoma, A549 lung carcinoma and
HT-29 colon carcinoma cell lines (see FIG. 7).
TABLE-US-00002 TABLE 2 Comparison of activity between RAPTA-C and
Ru-ethacrynic acid derivatives IC.sub.50 (.mu.M) Complexes HT29
A549 T47D MCF7 (.eta..sup.6-cymene)Ru(PTA)Cl.sub.2 436 1029 1063
>1600 Ethacrynic acid 73.5 50.7 7.73 66.0
(Ethacrynic-.eta..sup.6:phenyl- 50.7 32.3 2.91 19.9
methylamide)Ru(PTA)Cl.sub.2 (3 ) (Ethacrynic-.eta..sup.6:phenyl-
105.5 66.7 6.28 104.7 ethanoate)Ru(PTA)Cl.sub.2 (4)
(.eta..sup.6-cymene)RuCl.sub.2(ethacrynic- 39.1 34.0 4.80 10.7
propylamide-imidazole) (6) [(.eta..sup.6-cymene)RuCl(PTA)(eth- 64.2
65.1 5.97 19.9 acrynic-propylamide- imidazole)]BF.sub.4.sup.-
(7)
TABLE-US-00003 TABLE 3 Comparison of activity between RAPTA-C and
RAPTA-C carboxylate derivatives IC.sub.50 (.mu.M) Complex HT29 A549
T47D MCF7 (.eta..sup.6-cymene)Ru(PTA)Cl.sub.2 436 1029 1063
>1600 (.eta..sup.6-cymene)Ru(PTA)(C.sub.2O.sub.4) (8) 267 1130
1174 >1600 (.eta..sup.6-cymene)Ru(PTA)(C.sub.6H.sub.6O.sub.4)
(9) 265 1567 1088 >1600
Example 8
IC.sub.50 Values of Compounds Tested on Tumor Cell Lines
[0148] Tumor cell lines derived from human and mouse tumours listed
below were stabilised for in vitro propagation.
TABLE-US-00004 TABLE 4 Tumor cell lines for in vitro study.
Identification Tumor histotype HT-29 .sup.1 Human colorectal
carcinoma MIA-PaCa-2 .sup.2 Human pancreatic cancer H460M .sup.3
Human lung carcinoma/metastatic A2780 Human ovarian carcinoma
A2780/CDDP .sup.4 Human ovarian carcinoma resistant to Cistplatin
TLX5 .sup.5 Mouse lymphona .sup.1 Obtained from CRO, Aviano, Italy
(Dr. P. Spessotto); .sup.2 Obtained from Istituto Nazionale per lo
Studio e la Cura dei Tumori, Milan (kindly supplied by Dr. D.
Coradini); .sup.3 , as .sup.2 , but kindly supplied by Dr. G.
Pratesi; .sup.4 A2780cis is the variant of the parental A2780
resistant to Cisplatin (ECACC No. 93112517); .sup.5 Originally
obtained from Chester Beatty Institute, London, UK.
[0149] IC.sub.50 values are determined as follows: Tumour cells are
incubated in the appropriate complete medium at 37.degree. C. and
under controlled atmosphere (5% CO.sub.2). Test compounds are
tested at doses in the range of 100 nM and 100 .mu.M. Cytotoxicity
is determined by the MTT test, by measuring cell viability as the
cell metabolic capacity to transform the tetrazolium salt of MTT in
the blue formazan, by mitochondrial dehydrogenases; the blue colour
is read at 570 nm with a spectrophotometer (Alley M C, Scudiero D
A, Monks A, Hursey M L, Czerwinski M J, Fine D L, Abbott B J, Mayo
J G, Shoemaker R H, Boyd M R. Feasibility of drug screening with
panels of human tumor cell lines using a microculture tetrazolium
assay. Cancer Res 48: 598-601, 1988. Mosmann T Rapid colorimetric
assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63,
1983). Briefly, on day 0, in 96-well plates, 2-4000 cells/well/100
.mu.l complete medium are plated. On day 1, test compounds are
added at the test concentrations of 10.sup.-7 M; 3*10.sup.-7M;
10.sup.-6 M; 3*10.sup.-6M; 10.sup.-5 M; 3*10.sup.-5M; 10.sup.-4 M.
Each concentration is studied in triplicate. Cell viability, as
determined by the MTT test is done on Day 4, after 3 days
treatment. Each well is added with 10 .mu.l/100 .mu.l MTT (prepared
previously by dissolving 5 mg/ml MTT in PBS, sterilized with
filters at a cut-off of 0.22 nm and stored at 4.degree. C.). Plates
are put in an incubator at 37.degree. C. for 4 hrs, then medium is
eliminated and each well is added with 200 .mu.l DMSO (Sigma
Chemical Co., USA). Plates are read with a spectrophotometer
(Spectra count Packard Bell, Meriden, Conn., USA) at 570 nm wave
length. IC50 is calculated with the GraphPad Prism4.
[0150] The table below shows the IC.sub.50 values for the tested
compounds. The reported values are the most representative of two
separate experiments. Test concentrations have been done by serial
dilutions of each compound starting form a mother solution of 10-2
M prepared by dissolving Compound 3, Compound 4, Ethacrynic acid
and cisplatin in dimethylsulfoxide, and RAPTA-T (the moiety of the
test compounds without the Ethacrynic acid moiety) in sterile
apirogen water.
TABLE-US-00005 TABLE 5 IC.sub.50 values (.mu.M) A2780/ MIA A2780
CDDP H460M HT-29 PaCa-2 TLX5 Comp. 3 7.1 / 9.5 4.6 / 5.3 2.5 / 3.8
2.3 / 3.8 14.7 / 20.1 0.8 / 3.0 Comp. 4 ~30 36.7 / 38.7 29.1 / 31.5
30.0 / 32.6 81.5 / 92.7 3.6 / 4.3 Ethacrynic 91.1 / 94.8 84.3 /
97.6 58.1 / 63.3 35.2 / 44.0 >100 1.3 / 1.8 acid Cisplatin 10.5
/ 11.8 >100 1.3 / 3.0 4.9 / 32.6 10.6 / 12.9 0.02 / 0.2 RAPTA-T
>100 >100 >100 >100 >100 64.1 / 92.9
[0151] It can be seen from the table above that both compounds 3
and 4 of the present invention are active, in particular also
against the Cisaplatin-resistant cell line A2780/CDDP.
Example 9
In-Vivo Study
[0152] Tumour cells (TLX5, see above in Example 8) were derived
from mouse tumours and stabilised for in vivo propagation. For the
animal study, TLX5 lymphona cells were injected in the peritoneal
cavity of CBA brown-grey inbred mice (Harlan-Nossan, San Giovanni
al Natisone, Udine, Italy) at day 0. Test compounds were applied at
day 3 at various doses, also by the intraperitoneal route, with
each drug (test compound or reference drug) diluted in appropriate
physiological solution and injected in a volume of 0.1 ml/10 g body
weight.
[0153] Results from the in vivo study are shown in the table
below.
TABLE-US-00006 TABLE 6 Survival time in an in vivo TXL5 lymphoma
model Mean survival time % increase vs (days .+-. SD) Controls
Controls 10.0 .+-. 0.82 -- Compound 3 at 200 mg/kg 12.2 .+-. 1.79**
22 Cisplatin 8 mg/kg 11.8 .+-. 1.79* 18
[0154] As can be seen from the table above, control mice die 10
after tumor inplant. The treatment with compound 3 raises the mean
survival time to 12.2 days, corresponding to +22% versus control, a
result statistically significant (**p=0.0089, Logrank test).
Cisplatin, known to have severe side effects, was also effective at
the dose indicated, although to a lesser extent (*p=0.0182).
[0155] While these results indicate that Compound 3 of the present
invention is active in vivo, it is to be noted that the compound
was administered as a suspension of the compound in
carboxymethylcellulose. This fact has most probably reduced the
bioavalability of the compound 3.
* * * * *