Patent Application
20120277230
The present invention is generally dedicated to the use of compounds for the manufacture of compositions useful to treat cancer.
In most of the cancers, mortality is not due to the primary tumor but rather to the derived metastases. This malignant progression which leads to tumor invasion and is clinically defined by the appearance of metastases is the final outcome of the primary loss of cell adhesion and increase of cell motility which together allow invasive cell to leave the initial tumor site and colonize various target tissues.
Metastases are considered as a recurrent feature of uncontrolled malignant progression of cancer. During this process, tumor cells complete their malignant transformation by increasing their migratory capacity. Cancer cells can then disseminate and establish tumor foci in far away sites. Spreading of cancer cells in the organism is the outcome of a series of events called <<metastatic cascade>>: invasion of the tissues around the tumor, venous or lymphatic intravasation, migration and establishment in a distant place of a new colony that escapes from all the defence mechanisms of the organism.
Metastatic invasion, against which there is no efficient therapeutic option available at this time, is by far the major cause of death. Due to the frequency of cancers diagnosed at the metastatic stage and to the therapeutic impasse they represent, the development of molecules that specifically target metastatic invasion is thus a crucial requirement for a major breakthrough in cancer treatments.
The present invention is in keeping with the evidence as published during the last twenty years of a link between changes in RNA alternative splicing and metastatic invasion which has opened to new therapeutic strategies.
It has now been found that derivatives of formula (I) as defined in formula (I) hereinafter are able to correct defects of alternative splicing, as illustrated in the experimental data hereinafter, a mechanism closely associated with the invasive progression of metastatic cancers, and on the basis of such activity, the compounds are useful in the treatment of cancer.
The present invention therefore relates to compounds of formula (I) as defined below for use as agents for preventing, inhibiting or treating cancer.
The present invention moreover relates to a method of preventing, inhibiting or treating cancer, which comprises at least one step consisting in administering to a patient suffering therefrom an effective amount of a compound as defined in formula (I) below or one of its pharmaceutically acceptable salts.
The present invention further relates to some particular derivatives as such, as defined below.
The present invention also provides pharmaceutical compositions comprising at least one of said particular compounds.
According to a first aspect, a subject-matter of the present invention relates to a compound of formula (I)
means an aromatic ring wherein V is C or N and when V is N, V is in ortho, meta or para of Z, i.e. forms respectively a pyridazine, a pyrimidine or a pyrazine group,
R independently represent a hydrogen atom, a halogen atom or a group chosen among a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a —NR1R2 group, a (C1-C4)alkoxy group, a phenoxy group and a (C1-C3)alkyl group, said alkyl being optionally mono-substituted by a hydroxyl group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
n is 1, 2 or 3,
n′ is 1 or 2,
R′ is a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a halogen atom, a hydroxyl group, a —COOR1 group, a —NO2 group, a —NR1R2 group, a morpholinyl or a morpholino group, a N-methylpiperazinyl group, a (C1-C3)fluoroalkyl group, a (C1-C4)alkoxy group and a —CN group,
R″ is a hydrogen atom or a (C1-C4)alkyl group,
Z is N or C,
Y is N or C,
X is N or C,
W is N or C,
T is N or C,
U is N or C,
and wherein at most four of the groups V, T, U, Z, Y, X and W are N,
and at least one of the groups T, U, Y, X and W is N,
or anyone of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to one aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is C, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is C, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is C, X is C, T is C, U is C and W is N, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is N and is in para of Z, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is N and is in para of Z, Y is C, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is N and is in meta of Z and is in para of the bond linked to NR″, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is N and is in meta of Z and is in para of the bond linked to NR″, Y is C, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is C, Y is C, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is C, Y is N, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is N and is in meta of Z and in ortho of the bond linked to NR″, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is N and is in para of Z, Y is C, X is C, T is C, U is C and W is N, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is N and is in para of Z, Y is C, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is N, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is N and is in meta of Z and is in ortho of the bond linked to NR″, Y is N, X is N, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is C, Y is C, X is C, T is N, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is C, X is C, T is N, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is C, X is C, T is C, U is N and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to one preferred aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another preferred aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is N and is in para of Z, Y is N, X is C, T is C, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another preferred aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is C, V is C, Y is C, X is C, T is N, U is C and W is C, for use as an agent for preventing, inhibiting or treating cancer.
According to another preferred aspect, the present invention relates to a compound of formula (I) as defined above, wherein Z is N, V is C, Y is C, X is C, T is C, U is N and W is C, for use as an agent for preventing, inhibiting or treating cancer.
The compounds of the invention may exist in the form of free bases or of addition salts with pharmaceutically acceptable acids.
Suitable physiologically acceptable acid addition salts of compounds of formula (I) include hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate and fumarate.
The compounds of formula (I) and or salts thereof may form solvates (e.g. hydrates) and the invention includes all such solvates.
In the context of the present invention, the term:
“halogen” is understood to mean chlorine, fluorine, bromine, or iodine, and in particular denotes chlorine, fluorine or bromine,
“(C1-C3)alkyl” as used herein respectively refers to C1-C3 normal, secondary or tertiary saturated hydrocarbon. Examples are, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl,
“(C1-C3)alkoxy” as used herein respectively refers to O—(C1-C3)alkyl moiety, wherein alkyl is as defined above. Examples are, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy,
“fluoroalkyl group” and “fluoroalkoxy group” refers respectively to alkyl group and alkoxy group as above-defined, said groups being substituted by at least one fluorine atom. Examples are perfluoroalkyl groups, such as trifluoromethyl or perfluoropropyl, and
“patient” may extend to humans or mammals, such as cats or dogs.
According to a particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ia)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a —NO2 group, a —NR1R2 group and a (C1-C3)alkoxy group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —NO2 group, a (C1-C3)alkoxy group and a —NR1R2 group,
R1 and R2 are a hydrogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ib)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —NR1R2 group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a phenoxy group and a (C1-C4)alkoxy group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is preferably 1 or 2,
n′ is as defined above and is preferably 1,
R′ is a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group and a (C1-C4)alkoxy group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ic)
wherein:
R independently represent a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a (C1-C3)fluoroalkyl group, a —NR1R2 group, a —COOR1 group, a —NO2 group and a (C1-C3)alkoxy group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Id)
wherein:
R independently represent a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a (C1-C3)fluoroalkyl group and a (C1-C3)alkoxy group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ie)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group and a (C1-C3)alkoxy group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (If)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ig)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom or a halogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ii)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ii)
wherein:
R independently represent a hydrogen atom or a group chosen among a (C1-C3)fluoroalkoxy group and a (C1-C3)alkoxy group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ij)
wherein:
R independently represent a hydrogen atom or a group chosen among a (C1-C3)fluoroalkoxy group and a (C1-C3)alkyl group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ik)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom, a halogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Il)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Im)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Io)
wherein:
R independently represent a hydrogen atom or a halogen atom or a group chosen among, a —NO2 group, a —CN group and a (C1-C3)alkyl group, said alkyl being optionally mono-substituted by a hydroxyl group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom, a halogen atom or a (C1-C3)fluoroalkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ip)
wherein:
R represents a hydrogen atom,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Iq)
wherein:
R independently represent a hydrogen atom, a (C1-C3)alkoxy group or a (C1-C3)fluoroalkoxy group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom or a group chosen among a —NR1R2 group, a N-methylpiperazinyl group, a (C1-C3)alkoxy group and a morpholino group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Ir)
wherein:
R independently represent a hydrogen atom or a (C1-C3)alkyl group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 1,
R′ is a hydrogen atom or a group chosen among a —NR1R2 group, a morpholino group and a (C1-C3)alkoxy group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, an additional subject-matter of the present invention is a compound of formula (Iee)
wherein:
R independently represent a hydrogen atom, a (C1-C3)alkyl group or a (C1-C3)fluoroalkyl group,
R″ is as defined above and is advantageously a hydrogen atom,
n is as defined above and is advantageously 1,
n′ is as defined above and is advantageously 2,
R′ is a hydrogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
Among the previous defined families of compounds of formulae (Ia) to (Iee), some are more particularly preferred for their use as an agent for preventing, inhibiting or treating cancer. These preferred compounds particularly belong to formulae (Ia), (Ie), (Iq) and (Iee), as defined above or one of its pharmaceutically acceptable salts.
Accordingly the present invention further relates to a compound chosen among compounds of formulae (Ia), (Ie), (Iq) and (Iee), and their pharmaceutically acceptable salts for use as an agent for preventing, inhibiting or treating cancer.
According to a particular embodiment, the present invention more particularly focuses on a compound of formula (Ia)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a —COOR1 group and a (C1-C3)fluoroalkyl group,
R″ is as defined above and more preferably is a hydrogen atom,
R1 is as defined above,
n is as defined above,
n′ is as defined above,
R′ is a halogen atom, a (C1-C4)alkyl group, a (C1-C4)alkoxy group or a —NO2 group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, the present invention more particularly focuses on a compound of formula (Ie)
wherein:
R represents a hydrogen atom or a (C1-C4)alkyl group,
R″ is as defined above and more preferably is a hydrogen atom,
n is as defined above,
n′ is as defined above,
R′ is a halogen atom,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, the present invention more particularly focuses on a compound of formula (Iq)
wherein:
R′, R″, n and n′ are as defined in formula (I), and
R is a (C1-C3)fluoroalkoxy group,
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
According to another particular embodiment, the present invention more particularly focuses on a compound of formula (Iee)
wherein:
R is independently a hydrogen atom or a (C1-C4)alkyl group,
R′, R″, n and n′ are as defined in formula (I),
or one of its pharmaceutically acceptable salt,
for use as an agent for preventing, inhibiting or treating cancer.
In a particular embodiment, the present invention relates to a compound of formula (Ia) or (Ie) as defined above or one of its pharmaceutically acceptable salts, for use as an agent for preventing, inhibiting or treating cancer.
According to a preferred embodiment of the present invention, the compound for use as an agent for preventing, inhibiting or treating cancer, is chosen from:
Among said compounds, compounds (6), (18), (30), (35), (36), (37), (45), (48), (51), (52), (53), (55), (56), (58), (61), (63), (64), (109), (110), (112), (143), (144) and (148) are of particular interest.
The present invention therefore extends to compounds (6), (18), (30), (35), (36), (37), (45), (48), (51), (52), (53), (55), (56), (58), (61), (63), (64), (109), (110), (112), (143), (144) and (148) or one of its pharmaceutically acceptable salts for use as an agent for preventing, inhibiting or treating cancer.
Some of said preceding compounds are new and form part of the present invention: (6), (18), (30), (35), (36), (37), (48), (51), (52), (53), (55), (56), (58), (61), (63), (64), (109), (110), (112), (143) and (144).
The compounds of formulae (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (Io), (Ip), (Iq), (Ir) and (Iee) can comprise one or more asymmetric carbon atoms. They can thus exist in the form of enantiomers or of diastereoisomers. These enantiomers, diastereoisomers and their mixtures, including the racemic mixtures, are encompassed within the scope of the present invention.
Among the compounds of formula (I), some of them are new and form part of the invention, as well as their pharmaceutically acceptable salts, such as hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate and fumarate.
According to a particular embodiment, the present invention encompasses compounds of formula (Ig)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a —NR1R2 group, and a (C1-C3)alkoxy group,
n is 1 or 2,
n′ is 1 or 2,
R′ is a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a halogen atom, a hydroxyl group, a —COOR1 group, a —NO2 group, a —NR1R2 group, a (C1-C3)alkoxy group and a —CN group,
R″ is a hydrogen atom or a (C1-C4)alkyl group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
with the proviso that R and R′ are not simultaneously a hydrogen atom,
and when n and n′ are 1 and R is a hydrogen atom then R′ is not a —COOH group,
or anyone of its pharmaceutically acceptable salt.
According to another particular embodiment, the present invention encompasses compounds of formula (If)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a —NR1R2 group, and a (C1-C3)alkoxy group,
n is 1 or 2,
n′ is 1 or 2,
R′ is a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a halogen atom, a hydroxyl group, a —COOR1 group, a —NO2 group, a —NR1R2 group, a (C1-C3)alkoxy group and a —CN group,
R″ is a hydrogen atom or a (C1-C4)alkyl group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
or anyone of its pharmaceutically acceptable salt.
According to another particular embodiment, the present invention encompasses compounds of formula (Ih)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a —NR1R2 group, and a (C1-C3)alkoxy group,
n is 1 or 2,
n′ is 1 or 2,
R′ is a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a halogen atom, a hydroxyl group, a —COOR1 group, a —NO2 group, a —NR1R2 group, a (C1-C3)alkoxy group and a —CN group,
R″ is a hydrogen atom or a (C1-C4)alkyl group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
or anyone of its pharmaceutically acceptable salt.
According to another particular embodiment, the present invention encompasses compounds of formula (II)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a —NR1R2 group, and a (C1-C3)alkoxy group,
n is 1 or 2,
n′ is 1 or 2,
R′ is a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a halogen atom, a hydroxyl group, a —COOR1 group, a —NO2 group, a —NR1R2 group, a (C1-C3)alkoxy group and a —CN group,
R″ is a hydrogen atom or a (C1-C4)alkyl group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
with the proviso that R and R′ are not simultaneously a hydrogen atom,
or anyone of its pharmaceutically acceptable salt.
According to another particular embodiment, the present invention encompasses compounds of formula (Im)
wherein:
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a (C1-C3)fluoroalkoxy group, a —NO2 group, a —NR1R2 group, and a (C1-C3)alkoxy group,
n is 1 or 2,
n′ is 1 or 2,
R′ is a hydrogen atom or a group chosen among a (C1-C3)alkyl group, a halogen atom, a hydroxyl group, a —COOR1 group, a —NO2 group, a —NR1R2 group, a (C1-C3)alkoxy group and a —CN group,
R″ is a hydrogen atom or a (C1-C4)alkyl group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
with the proviso that when n and n′ are 1 and R is a hydrogen atom, R′ is not a chlorine atom,
or anyone of its pharmaceutically acceptable salt.
For a sake of simplification, the following compounds and their corresponding definitions are called “new compounds”.
According to another particular embodiment, the present invention encompasses compounds of formula (Ia), as such,
wherein:
R″ and n are as defined in formula (Ia),
n′ is 1,
R independently represent a hydrogen atom, a halogen atom or a group chosen among a (C1-C3)alkyl group, a —CN group, a hydroxyl group, a —COOR1 group, a (C1-C3)fluoroalkyl group, a —NO2 group, a (C1-C3)fluoroalkoxy group and a (C1-C3)alkoxy group,
R′ is a hydrogen atom or a halogen atom or a group chosen among a (C1-C3)alkyl group, a —COOR1 group, and a —CN group,
R1 is a hydrogen atom or a (C1-C3)alkyl group:
with the proviso that
when R and R′ are not simultaneously a hydrogen atom,
when n is 1, R is not a methyl group in ortho or para position with respect to Z, Z being N,
when R′ is a hydrogen atom, R is not a bromine atom or a chlorine atom,
when R is a hydrogen atom, R′ is not a methyl or ethyl group, a —COOH group, a COOC2H5 group or a bromine atom, said bromine atom being in ortho position of the bond linked to NR″,
or one of its pharmaceutically acceptable salt.
Still according to this particular embodiment, the present invention more particularly focuses on compounds of formula (Ia), as such, wherein,
R independently represent a hydrogen atom or a (C1-C3)alkyl group,
R″ is as defined in formula (Ia),
R′ is a hydrogen atom, a halogen atom, a (C1-C3)alkoxy group or a —NO2 group,
n′ is 1,
n is 1,
with the proviso that
when n is 1, R is not a methyl group in ortho or para position with respect to Z, Z being N,
or one of its pharmaceutically acceptable salt.
Still according to this particular embodiment, the present invention more preferably focuses on compounds of formula (Ia′), as such,
wherein,
R independently represent a hydrogen atom, a (C1-C3)alkyl group, a (C1-C3)fluoroalkyl group, a halogen atom or a hydroxyl group,
R″ is as defined in formula (Ia),
n is 1 or 2,
or one of its pharmaceutically acceptable salt.
According to another particular embodiment, the present invention encompasses compounds of formula (Ie)
wherein:
R, R′, R″ n and n′ are as defined in formula (I),
with the proviso that
when R is a hydrogen atom, R′ is not a bromine atom,
or one of its pharmaceutically acceptable salt.
The present invention further relates to a compound of formula (Iq) as defined above, as such
wherein:
R, R′, R″ and n′ are as defined in formula (I),
n is 1 or 2,
with the proviso that
R′ and R are not simultaneously a hydrogen atom,
when R′ is a hydrogen atom, R is not a —NO2 group or a —NH2 group,
when n is 2 and R′ is a hydrogen atom, R is not a COOC2H5 group or a chlorine atom,
or one of its pharmaceutically acceptable salt.
Still according to this particular embodiment, the present invention more particularly focuses on compounds of formula (Iq), as such, wherein
R′, R″, n and n′ are as defined in formula (I), and
R is a (C1-C3)fluoroalkoxy group,
or one of its pharmaceutically acceptable salt.
Still according to this particular embodiment, the present invention more particularly focuses on compounds of formula (Iq), as such, wherein
R, R″, n and n′ are as defined in formula (I), and
R′ is a —NR1R2 group,
R1 and R2 are independently a hydrogen atom or a (C1-C3)alkyl group,
or one of its pharmaceutically acceptable salt.
Still according to this particular embodiment, the present invention more particularly focuses on compounds of formula (Iq), as such, wherein
R, R″, n and n′ are as defined in formula (I), and
R′ is a morpholinyl group, a morpholino group or a N-methylpiperazinyl group,
or one of its pharmaceutically acceptable salt.
The present invention further relates to a compound of formula (Iee) as defined above, as such
wherein:
R, R′, R″, n and n′ are as defined in formula (I),
or one of its pharmaceutically acceptable salt,
with the exclusion of the following compound
and with the exclusion of compounds wherein R is a —NO2 group or a —NH2 group when R′ is a hydrogen or a methyl group.
Still according to this particular embodiment, the present invention more particularly focuses on compounds of formula (Iee), as such, wherein
R′, R″, n and n′ are as defined in formula (I), and
R is a (C1-C3)fluoroalkyl group,
or one of its pharmaceutically acceptable salt.
Among said compounds as such, compounds (1), (2), (5)-(8), (10)-(16), (18), (21)-(44), (46)-(75), (77)-(84), (86)-(119), (121), (124)-(130), (132), (135)-(141), (143)-(147), (149)-(168) and their pharmaceutically acceptable salts are of particular interest.
The present invention therefore extends to compounds (1), (2), (5)-(8), (10)-(16), (18), (21)-(44), (46)-(75), (77)-(84), (86)-(119), (121), (124)-(130), (132), (135)-(141), (143)-(147), (149)-(168) and their pharmaceutically acceptable salts, as such.
More preferably, compounds (143), (144), (149), (166), (167) and their pharmaceutically acceptable salts are of particular interest.
The present invention therefore extends to compounds (143), (144), (149), (166), (167) and their pharmaceutically acceptable salts, such as hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate and fumarate.
Still more preferably, the present invention extends to compounds (143), (144) and their pharmaceutically acceptable salts, such as hydrobromide, tartrate, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate and fumarate.
The new compounds of the present invention, i.e. compounds of formulae (Ia), (Ie), (Iq) and (Iee) and the specific compounds as listed above, are not only useful as agent for inhibiting, preventing or treating cancer but can also be useful for inhibiting, preventing or treating premature aging or progeria and for inhibiting, preventing or treating AIDS.
According to an aspect of the invention, said compounds may be useful to inhibit, prevent and/or treat diseases with premature aging and that are likely related to an aberrant splicing of the nuclear lamin A gene. Among all, said disease may include Hutchinson Guilford Progeria Syndrome (HGPS), progeria, premature aging associated with HIV infection, muscular dystrophy, Charcot-Marie-Tooth disorder, Werner syndrome, but also atherosclerosis, insulin resistant type II diabetes, cataracts, osteoporosis and aging of the skin such as restrictive dermopathy.
The compounds of the present invention can be prepared by conventional methods of organic synthesis practiced by those skilled in the art. The general reaction sequences outlined below represent a general method useful for preparing the compounds of the present invention and are not meant to be limiting in scope or utility.
The compounds of general formula (I) can be prepared according to scheme 1 below.
As appears in said scheme two routes are available for recovering a compound of formula (I) according to the present invention.
The synthesis is based on a coupling reaction alternatively starting from a halogeno-bicycle of formula (III), wherein X, Y, W, T, U, n′, R′ and R″ are as defined above and X′ is a chlorine atom or a bromine atom or from a chloro-monocycle of formula (V), wherein Z, V, n and R are as defined above and X′ is a chlorine atom or a bromine atom.
According to route (A), the compound of formula (III) is placed in a protic solvent such as tert-butanol. The compound of formula (IV) is then added in a molar ratio ranging from 1 to 1.5 with respect to the compound of formula (III) in presence of an inorganic base, such as Cs2CO3 or K2CO3 in a molar ratio ranging from 1 and 2, in the presence of a diphosphine, such as Xantphos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene) or X-Phos (2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl) in an amount ranging from 2 mol % to 10 mol % relative to the total amount of compound of formula (III), and in the presence of a catalyst, such as Pd(OAc)2 or Pd2dba3 in an amount ranging from 2 mol % to 10 mol % relative to the total amount of compound of formula (III). The reaction mixture can then be heated at a temperature ranging from 80 to 120° C., for example at 90° C. and stirred for a time ranging form 15 to 25 hours, for example during 20 hours under inert gas and for example argon. The reaction mixture can be concentrated under reduced pressure.
According to route (B) the compound of formula (V) is placed in a protic solvent such as tert-butanol. The compound of formula (VI) is then added in a molar ratio ranging from 1 to 1.5 with respect to the compound of formula (V) in presence of an inorganic base, such as Cs2CO3 or K2CO3 in a molar ratio ranging from 1 to 2, in the presence of a diphosphine, such as Xantphos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene) or X-Phos (2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl) in an amount ranging from 2 mol % to 10 mol % relative to the total amount of compound of formula (V), and in the presence of a catalyst, such as Pd(OAc)2 or Pd2 dba3 in an amount ranging from 2 mol % to 10 mol % relative to the total amount of compound of formula (V). The reaction mixture can then be heated at a temperature ranging from 80 to 120° C., for example at 90° C. and stirred for a time ranging form 15 to 25 hours, for example during 20 hours under inert gas and for example argon. The reaction mixture can be concentrated under reduced pressure.
The starting compounds of formula (III), (IV), (V) and (VI) are commercially available or can be prepared according to methods known to the person skilled in the art.
The chemical structures and spectroscopic data of some compounds of formula (I) of the invention are illustrated respectively in the following Table I and Table II.
1H NMR (300 MHz, D2O) δ 8.31 (d, J = 5.1, 1H), 8.21 (d, J = 9.3, 1H), 7.60 (d, J =
1H NMR (300 MHz, DMSO) δ 10.23 (s, 1H), 8.96 (s, 1H), 8.18 (d, J = 8.8, 2H),
13C NMR (75 MHz, DMSO) δ 153.63, 153.61, 148.37, 147.32, 142.65, 137.52,
1H NMR (300 MHz, DMSO) δ 10.71 (s, 1H), 8.71 (d, J = 1.4, 1H), 8.62 (d, J = 8.9,
13C NMR (75 MHz, DMSO) δ 156.09, 152.40, 152.11, 146.24, 141.07, 137.83,
1H NMR (300 MHz, CDCl3) δ 7.92 (d, J = 8.9, 1H), 7.79 (d, J = 8.4, 1H), 7.65 (t, J =
13C NMR (75 MHz, CDCl3) δ 153.88, 147.62, 144.35, 139.26, 138.11, 130.13,
1H NMR (300 MHz, CDCl3) δ 9.10 (d, J = 2.5, 1H), 8.83 (d, J = 2.6, 1H), 8.02 (d, J =
1H NMR (300 MHz, CDCl3) δ 9.18 (d, J = 2.7, 1H), 8.86 (d, J = 2.5, 1H), 8.56 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.83 (d, J = 2.6, 1H), 8.37 (d, J = 2.3, 1H), 8.00 (d, J =
13C NMR (75 MHz, CDCl3) δ 157.13, 154.59, 145.81, 144.43, 138.78, 134.54,
1H NMR (300 MHz, CDCl3) δ 8.77 (dd, J = 1.5, 4.2, 1H), 8.04 (dd, J = 4.7, 8.7,
1H NMR (300 MHz, CDCl3) δ 8.53 (d, J = 59.9, 2H), 7.76 (d, J = 8.6, 1H), 7.58 (t,
1H NMR (300 MHz, CDCl3) δ 8.78 (s, 1H), 8.13 (d, J = 5.1, 1H), 7.89 (d, J = 8.3,
1H NMR (300 MHz, CDCl3) δ 8.95 (d, J = 8.4, 1H), 8.28 (d, J = 5.7, 1H), 7.87 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.64 (d, J = 8.4, 1H), 8.55 (d, J = 2.1, 1H), 8.03 (s,
1H NMR (300 MHz, CDCl3) δ 9.09 (d, J = 8.9, 1H), 8.53 (d, J = 1.7, 1H), 7.94 (dd,
1H NMR (300 MHz, CDCl3) δ 8.16 (d, J = 5.2, 1H), 8.10 (s, 1H), 7.90 (d, J = 8.8,
1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 8.3, 1H), 7.70 (d, J = 9.0, 1H), 7.64 (d, J =
1H NMR (300 MHz, CDCl3) δ 9.17 (d, J = 2.5, 1H), 8.71 (s, 1H), 8.49 (dd, J = 2.6,
1H NMR (300 MHz, CDCl3) δ 8.64 − 8.51 (m, 3H), 8.18 (d, J = 9.0, 1H), 7.93 (d, J =
1H NMR (300 MHz, CDCl3) δ 10.77 (s, 1H), 8.60 (s, 3H), 8.19 (d, J = 8.2, 1H),
1H NMR (300 MHz, CDCl3) δ 8.46 (dd, J = 1.9, 5.0, 1H), 7.87 (dd, J = 2.0, 7.6,
1H NMR (300 MHz, CDCl3) δ 8.44 (d, J = 9.1, 1H), 8.17 (d, J = 4.8, 1H), 8.03 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 8.5, 1H), 8.10 (s, 1H), 7.91 (d, J = 8.9,
1H NMR (300 MHz, CDCl3) δ 8.87 (s, 1H), 8.32 (d, J = 5.0, 1H), 7.95 (d, J = 8.8,
1H NMR (300 MHz, CDCl3) δ 8.52 (s, 1H), 8.45 (d, J = 8.6, 1H), 8.01 (d, J = 8.8,
1H NMR (300 MHz, CDCl3) δ 8.32 (d, J = 9.1, 1H), 8.07 (d, J = 4.8, 1H), 7.93 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.70 (d, J = 7.2, 1H), 8.01 (s, 1H), 7.82 (d, J = 8.9,
1H NMR (300 MHz, CDCl3) δ 9.08 (d, J = 8.5, 1H), 8.55 (s, 1H), 8.36 (s, 1H), 8.02
1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 9.1, 1H), 7.97 (d, J = 9.1, 1H), 7.80 −
1H NMR (300 MHz, CDCl3) δ 8.57 (d, J = 29.4, 1H), 7.80 (d, J = 8.8, 1H), 7.66 (t,
1H NMR (300 MHz, DMSO) δ 9.75 (s, 1H), 9.12 (d, J = 2.3, 1H), 8.50 (d, J = 2.2,
1H NMR (300 MHz, CDCl3) δ 8.52 (dd, J = 2.8, 8.6, 1H), 8.35 (s, 1H), 8.15 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.55 (d, J = 6.8, 1H), 8.01 (d, J = 8.9, 2H), 7.82 (dd,
1H NMR (300 MHz, DMSO) δ 10.41 (s, 1H), 9.08 (dd, J = 4.1, 9.3, 1H), 8.31 (d, J =
13C NMR (75 MHz, DMSO) δ 156.30, 153.32, 153.04, 150.17, 142.55, 137.73,
1H NMR (300 MHz, CDCl3) δ 11.09 (s, 1H), 8.78 (d, J = 9.0, 1H), 8.42 (dd, J =
1H NMR (300 MHz, CDCl3) δ 8.59 (d, J = 8.3, 1H), 7.73 (d, J = 8.3, 1H), 7.57 (s,
1H NMR (300 MHz, MeOD) δ 8.99 (s, 1H), 8.76 (d, J = 9.2, 1H), 8.32 (d, J = 8.7,
1H NMR (300 MHz, MeOD) δ 8.48 (d, J = 9.1, 1H), 8.40 (d, J = 6.7, 1H), 7.94 (d,
1H NMR (300 MHz, CDCl3) δ 9.34 (s, 1H), 8.95 (s, 1H), 8.21 (d, J = 5.1, 1H), 7.87
1H NMR (300 MHz, CDCl3) δ 8.57 (d, J = 29.4, 1H), 7.80 (d, J = 8.8, 1H), 7.66 (t,
1H NMR (300 MHz, CDCl3) δ 8.64 (s, 1H), 8.06 (s, 1H), 7.89 (d, J = 8.7, 1H), 7.71
13C NMR (75 MHz, CDCl3) δ 156.15, 153.17, 152.82, 150.16, 143.70, 137.92,
1H NMR (300 MHz, CDCl3) δ 8.89 (d, J = 8.8, 1H), 8.05 (d, J = 8.8, 1H), 8.01 (s,
1H NMR (300 MHz, CDCl3) δ 9.27 (s, 1H), 8.33 (d, J = 5.7, 1H), 8.13 (d, J = 5.2,
1H NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 7.90 (d, J = 9.0, 1H), 7.63 (d, J = 7.5,
1H NMR (300 MHz, CDCl3) δ 8.20 (s, 1H), 7.90 (d, J = 9.0, 1H), 7.63 (d, J = 7.5,
1H NMR (300 MHz, CDCl3) δ 8.91 (d, J = 1.7, 1H), 8.46 (d, J = 8.8, 1H), 8.28 (dd,
1H NMR (300 MHz, CDCl3) δ 8.94 (d, J = 8.9, 1H), 8.91 (d, J = 1.8, 1H), 8.37 (dd,
1H NMR (300 MHz, CDCl3) δ 8.96 (d, J = 8.8, 1H), 8.85 (d, J = 1.3, 1H), 8.28 (d, J =
1H NMR (300 MHz, CDCl3) δ 11.02 (s, 1H), 8.75 (d, J = 9.2, 1H), 8.44 (d, J = 3.7,
1H NMR (300 MHz, DMSO) δ 10.38 (s, 1H), 8.56 (s, 1H), 8.28 (d, J = 9.1, 1H),
1H NMR (300 MHz, DMSO) δ 10.51 (s, 1H), 8.83 (d, J = 2.3, 1H), 8.62 (d, J = 9.3,
1H NMR (300 MHz, CDCl3) δ 9.57 (s, 1H), 8.44 (d, J = 4.8, 1H), 8.05 (d, J = 8.8,
1H NMR (300 MHz, CDCl3) δ 9.12 (s, 1H), 7.94 (d, J = 8.6, 1H), 7.71 (d, J = 7.5,
1H NMR (300 MHz, CDCl3) δ 9.07 (d, J = 8.5, 1H), 7.97 (d, J = 8.8, 1H), 7.90 (t, J =
1H NMR (300 MHz, CDCl3) δ 8.67 (d, J = 7.9, 1H), 7.83 (d, J = 8.3, 1H), 7.71 (s,
1H NMR (300 MHz, CDCl3) δ 8.91 (dd, J = 3.8, 9.0, 1H), 8.11 (d, J = 2.9, 1H),
1H NMR (300 MHz, CDCl3) δ 8.96 (d, J = 8.3, 1H), 8.49 (s, 1H), 7.89 (dd, J = 1.9,
1H NMR (300 MHz, CDCl3) δ 7.83 (d, J = 9.0, 1H), 7.69 (dd, J = 1.3, 7.6, 1H),
1H NMR (300 MHz, CDCl3) δ 7.83 (d, J = 8.8, 1H), 7.70 (d, J = 7.6, 1H), 7.59 (d, J =
13C NMR (75 MHz, CDCl3) δ 156.40, 155.54, 144.29, 138.09, 132.96, 130.44,
1H NMR (300 MHz, CDCl3) δ 7.80 (t, J = 7.6, 2H), 7.64 (d, J = 8.9, 2H), 7.61 −
1H NMR (300 MHz, CDCl3) δ 7.78 (d, J = 8.4, 1H), 7.76 − 7.71 (m, 2H), 7.69 (s,
1H NMR (300 MHz, CDCl3) δ 7.92 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 8.3 Hz, 1H),
13C NMR (75 MHz, CDCl3) δ 152.46, 146.25, 143.86, 139.33, 136.83, 128.93,
1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 8.3, 1H), 7.66 (d, J = 8.5, 3H), 7.55 (d, J =
1H NMR (300 MHz, CDCl3) δ 7.92 (d, J = 8.9, 1H), 7.82 − 7.70 (m, 2H), 7.66 (d, J =
1H NMR (300 MHz, CDCl3) δ 7.93 − 7.86 (m, 1H), 7.85 (s, 1H), 7.82 (d, J = 8.4,
1H NMR (300 MHz, CDCl3) δ 8.67 (d, J = 8.1, 1H), 7.92 (d, J = 8.9, 1H), 7.85 (d, J =
1H NMR (300 MHz, CDCl3) δ 7.93 (d, J = 8.9, 1H), 7.83 (d, J = 8.3, 1H), 7.70 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.27 (s, 1H), 7.76 (d, J = 8.9, 1H), 7.67 (d, J = 7.5,
1H NMR (300 MHz, CDCl3) δ 8.11 (dt, J = 2.1, 12.1, 1H), 7.76 (d, J = 8.9, 1H),
1H NMR (300 MHz, DMSO) δ 11.38 (s, 1H), 8.41 (d, J = 9.1, 1H), 7.93 (d, J = 7.8,
1H NMR (300 MHz, CDCl3) δ 7.84 (d, J = 9.1, 2H), 7.79 (d, J = 8.9, 1H), 7.67 (dd,
13C NMR (75 MHz, CDCl3) δ 153.88, 144.30, 143.91, 139.00, 138.25, 131.13,
1H NMR (300 MHz, CDCl3) δ 8.74 (s, 1H), 8.54 (s, 1H), 8.46 (d, J = 8.8, 1H), 7.91
1H NMR (300 MHz, CDCl3) δ 8.67 (d, J = 7.9, 1H), 7.83 (d, J = 8.3, 1H), 7.71 (s,
1H NMR (300 MHz, CDCl3) δ 9.21 (dd, J = 1.5, 8.4, 1H), 7.85 (d, J = 8.4, 1H),
1H NMR (300 MHz, CDCl3) δ 8.16 (d, J = 8.7, 1H), 7.83 (d, J = 8.9, 1H), 7.63 (d, J =
1H NMR (300 MHz, MeOD) δ 8.42 (s, 1H), 7.94 (d, J = 7.9, 1H), 7.83 (d, J = 8.1,
1H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.9, 1H), 7.70 (d, J = 8.9, 1H), 7.64 (d, J =
1H NMR (300 MHz, MeOD) δ 8.11 (d, J = 8.4, 1H), 7.81 (s, 2H), 7.62 (d, J = 8.7,
1H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 8.9, 1H), 7.70 − 7.63 (m, 1H), 7.51 (dd,
1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 8.9, 1H), 7.76 (d, J = 8.5, 1H), 7.63 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.49 (d, J = 2.5, 1H), 7.89 (d, J = 8.8, 1H), 7.72 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.85 (d, J = 2.6, 1H), 8.45 (d, J = 2.3, 1H), 8.01 (d, J =
13C NMR (75 MHz, CDCl3) δ 157.18, 154.80, 145.42, 143.80, 138.17, 135.04,
1H NMR (300 MHz, DMSO) δ 10.24 (s, 1H), 9.06 (d, J = 2.3, 1H), 8.65 (d, J = 1.8,
1H NMR (300 MHz, CDCl3) δ 8.77 (dd, J = 1.5, 4.3, 1H), 8.06 (dd, J = 10.8, 18.4,
1H NMR (300 MHz, CDCl3) δ 8.72 (dd, J = 1.6, 4.2, 1H), 8.61 (d, J = 2.4, 1H),
1H NMR (300 MHz, CDCl3) δ 9.68 (s, 1H), 8.21 (s, 2H), 7.94 (d, J = 8.9, 1H), 7.79
1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1H), 8.33 − 8.21 (m, 2H), 8.05 (d, J = 8.9,
1H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 8.9, 1H), 7.70 − 7.63 (m, 1H), 7.51 (dd,
1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 8.9, 1H), 7.76 (d, J = 8.5, 1H), 7.63 (d, J =
13C NMR (75 MHz, DMSO) δ 152.94, 150.19, 142.48, 142.18, 138.20, 137.55,
1H NMR (300 MHz, CDCl3) δ 9.74 (s, 1H), 8.20 (s, 2H), 8.03 (d, J = 8.6, 1H), 7.87
1H NMR (300 MHz, CDCl3) δ 9.67 (s, 1H), 8.34 − 8.12 (m, 2H), 7.84 (d, J = 8.0,
1H NMR (300 MHz, CDCl3) δ 10.15 (s, 1H), 8.24 − 8.12 (m, 2H), 7.79 (s, 1H),
1H NMR (300 MHz, CDCl3) δ 8.42 (s, 1H), 7.95 (dd, J = 1.3, 8.2, 1H), 7.87 − 7.78
1H NMR (300 MHz, CDCl3) δ 8.42 (s, 1H), 8.03 (d, J = 9.5, 1H), 7.92 (d, J = 8.2,
1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 8.03 (s, 1H), 7.94 (d, J = 8.2, 1H), 7.84
1H NMR (300 MHz, CDCl3) δ 8.85 (dd, J = 1.0, 8.3, 1H), 8.47 (s, 1H), 7.96 (d, J =
1H NMR (300 MHz, CDCl3) δ 9.17 (s, 1H), 8.68 (d, J = 9.1, 1H), 8.64 (d, J = 4.8,
13C NMR (75 MHz, CDCl3) δ 158.34, 138.07, 129.85, 127.63, 127.31, 124.34,
1H NMR (300 MHz, CDCl3) δ 9.14 (s, 1H), 8.73 (d, J = 21.2, 3H), 8.17 (s, 1H),
1H NMR (300 MHz, CDCl3) δ 9.05 (s, 1H), 8.64 (d, J = 4.8, 2H), 8.52 (s, 1H), 7.89
1H NMR (300 MHz, CDCl3) δ 8.86 (d, J = 2.6, 1H), 8.70 (d, J = 2.5, 1H), 8.32 (d, J =
1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 8.71 (s, 1H), 8.54 (d, J = 8.4, 1H), 8.37
1H NMR (300 MHz, CDCl3) δ 9.02 (s, 1H), 8.70 (s, 1H), 8.30 (s, 1H), 8.20 (d, J =
13C NMR (75 MHz, CDCl3) δ 153.28, 150.20, 148.55, 147.40, 140.93, 139.83,
1H NMR (300 MHz, DMSO) δ 11.10 (s, 1H), 9.03 (s, 1H), 8.82 − 8.75 (m, 1H),
1H NMR (300 MHz, CDCl3) δ 8.72 (s, 1H), 8.53 (s, 1H), 8.20 (d, J = 8.3, 1H), 7.93
13C NMR (75 MHz, CDCl3) δ 156.86, 152.27, 148.40, 140.92, 139.70, 139.00,
1H NMR (300 MHz, CDCl3) δ 8.53 (s, 1H), 8.20 (d, J = 4.8, 1H), 8.04 (d, J = 8.3,
1H NMR (300 MHz, CDCl3) δ 9.93 (s, 1H), 8.19 (s, 1H), 8.05 (d, J = 8.1, 1H), 7.99
1H NMR (300 MHz, CDCl3) δ 9.72 (s, 1H), 9.35 (s, 1H), 8.30 (d, J = 5.0, 1H), 8.05
1H NMR (300 MHz, DMSO) δ 8.85 (s, 1H), 8.42 (d, J = 5.3, 1H), 7.96 (d, J = 9.1,
13C NMR (75 MHz, DMSO) δ 156.82, 150.25, 149.69, 143.79, 141.71, 125.95,
1H NMR (300 MHz, DMSO) δ 9.34 (s, 1H), 8.59 (d, J = 5.2, 1H), 8.53 (s, 1H),
13C NMR (75 MHz, DMSO) δ 161.31, 155.67, 151.63, 150.25, 147.77, 147.01,
1H NMR (300 MHz, CDCl3) δ 8.35 (s, 1H), 8.04 (d, J = 8.3, 1H), 7.82 (d, J = 8.9,
1H NMR (300 MHz, CDCl3) δ 8.30 (d, J = 8.5, 1H), 8.08 (s, 1H), 7.90 (d, J = 9.0,
1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.54 (s, 1H), 8.46 (d, J = 8.8, 1H), 7.91
1H NMR (300 MHz, DMSO) δ 9.08 (s, 1H), 8.12 (d, J = 8.4, 1H), 7.73 (d, J = 8.2,
1H NMR (300 MHz, CDCl3) δ 7.68 (d, J = 8.3, 1H), 7.61 (s, 1H), 7.56 (d, J = 11.5,
1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 9.1, 1H), 7.62 (d, J = 8.9, 1H), 7.58 (d, J =
1H NMR (300 MHz, CDCl3) δ 7.98 (d, J = 2.6, 1H), 7.89 (d, J = 8.9, 1H), 7.72 (d, J =
1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 9.0, 1H), 7.70 (dd, J = 1.2, 7.5, 1H),
1H NMR (300 MHz, CDCl3) δ 8.80 (d, J = 2.6, 1H), 8.37 (d, J = 2.6, 1H), 8.01 (d, J =
1H NMR (300 MHz, CDCl3) δ 9.68 − 8.90 (m, 1H), 8.77 (s, 1H), 8.35 (s, 1H), 8.14
1H NMR (300 MHz, CDCl3) δ 9.98 (s, 1H), 8.70 (s, 1H), 8.45 (s, 1H), 8.27 (d, J =
1H NMR (300 MHz, CDCl3) δ 8.73 (s, 1H), 8.70 − 8.60 (m, 1H), 8.48 (s, 1H), 8.31
1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.68 (s, 1H), 8.01 (s, 1H), 7.95 (d, J =
1H NMR (300 MHz, DMSO) δ 10.46 (s, 1H), 9.00 (s, 1H), 8.41 (s, 1H), 8.24 (d, J =
1H NMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 8.79 (s, 1H), 8.51 (s, 1H), 8.18 (s, 1H),
1H NMR (300 MHz, CDCl3) δ 8.49 (d, J = 5.0, 1H), 7.77 (d, J = 9.0, 1H), 7.32 (d, J =
The following examples illustrate in detail the preparation of compounds (51), (64), (110), (143) and (148) according to the invention. The structures of the products obtained have been confirmed at least by NMR spectra.
According to route (A), the compound of formula (III) is placed in a protic solvent such as tert-butanol. The compound of formula (IV) is then added in a 1.1 molar ratio with respect to the compound of formula (III) in presence of an inorganic base, such as Cs2CO3 or K2CO3, in a 2.8 molar ratio, in the presence of a diphosphine, such as Xantphos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene), or X-Phos 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl in a 2 mol % amount relative to the total amount of compound of formula (III), and in the presence of a catalyst, such as Pd(OAc)2 or Pd2 dba3 in a 2 mol % amount relative to the total amount of compound of formula (III). The reaction mixture is then heated at 90° C., and stirred during 20 hours, under argon. The reaction mixture is concentrated under reduced pressure and the resulting residue is diluted with ethyl acetate. The organic phase is then washed twice with water, dried on magnesium sulphate, filtered and concentrated under reduced pressure. The residue could then be purified by column chromatography on silica gel to yield pure compounds (51), (64), (110), and (143).
According to route (B), the compound of formula (V) is placed in a protic solvent such as tert-butanol. The compound of formula (VI) is then added in a 1.1 molar ratio with respect to the compound of formula (V) in presence of Cs2CO3 in a 2.8 molar ratio, in the presence of Xantphos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene) in a 2 mol % amount relative to the total amount of compound of formula (V), and in the presence of a Pd(OAc)2, in a 2 mol % amount relative to the total amount of compound of formula (V). The reaction mixture is then heated at 90° C., and stirred during 20 hours, under argon. The reaction mixture is concentrated under reduced pressure and the resulting residue is diluted with ethyl acetate. The organic phase is then washed twice with water, dried on magnesium sulphate, filtered and concentrated under reduced pressure. The residue could then be purified by column chromatography on silica gel to yield pure compound (148).
According to route (A), a mixture of 2,8-dichloroquinoline (98.5 mg) and 2-amino-4,6-dimethylpyridine (67.1 mg), Pd(OAc)2 (2.2 mg), XantPhos (5.8 mg) and Cs2CO3 (456 mg) in 2 mL of t-BuOH gave compound (51) (99.7 mg).
According to route (A), a mixture of 2-chloro-5-nitroquinoline (100.0 mg) and 2-amino-4-methylpyridine (57.6 mg), Pd2 dba3 (20 mg), XantPhos (30 mg) and K2CO3 (270 mg) in 3 mL of t-BuOH gave compound (64) (14.0 mg).
The preparation of 2-chloro-5-nitroquinoline is described in Patent application WO2009/23844.
According to route (A), a mixture of 8-bromo-2-chloroquinoline (500 mg) and aminopyrazine (216 mg), Pd2 dba3 (95 mg), XantPhos (120 mg) and K2CO3 (1.15 g) in 12 mL of t-BuOH gave compound (110) (245 mg).
The preparation of 8-bromo-2-chloroquinoline is described in Cottet, F. et al. Eur. J. Org. Chem. 2003, 8, 1559.
According to route (A), a mixture of 7-chloro-4-(N,N-dimethylamino)quinoline (500 mg), 4-trifluoromethoxyaniline (0.257 mL), Pd2dba3 (110 mg), XPhos (115 mg) and K2CO3 (1g) in 10 mL of t-BuOH gave compound (143) (410 mg).
The preparation of 7-chloro-4-(N,N-dimethylamino)quinoline is described in Sanchez-Martin, R. et al. J. Med. Chem. 2005, 48, 3354.
According to route (B), a mixture of 5,8-dimethylisoquinolin-6-amine (59 mg) and 2-bromo-5-methylpyridine (86 mg), Pd(OAc)2 (2.2 mg), XantPhos (5.8 mg) and Cs2CO3 (456 mg) in 2 mL of t-BuOH gave compound (148) (48 mg).
The preparation of 5,8-dimethylisoquinolin-6-amine is described in Australian Journal of Chemistry 1969, 22, 2489.
1H NMR (300 MHz, CDCl3) δ 9.32 (s, 1H), 8.52 (d, J=6.0, 1H), 8.07 (s, 1H), 7.72 (d, J=6.0, 1H), 7.51 (s, 1H), 7.36 (dd, J=2.1, 8.4, 1H), 6.69 (d, J=8.3, 2H), 2.72 (s, 3H), 2.48 (s, 3H), 2.26 (s, 3H)
MS (ESI) [M+H]+=264
Standard operating procedure:
Effect of drug compounds on invasion
of MDA-MB231-D3H2LN cells into collagen
Background:
A key step in the generation of tumor metastasis is the invasion of tumor cells into the extracellular matrix, a major component of which is collagen. Therefore, the invasion of tumor cells into collagen in vitro may be indicative of the generation of metastasis in vivo. E. g., MDA-MB231-luc-D3H2LN mouse breast cancer cells display indeed both higher invasion into collagen in vitro and a higher metastatic potential in vivo as compared to MDA-MB231 cells (from which they were derived). Using these MDA-MB231-luc-D3H2LN cells as a model, the aim of the experiment described here is to identify drug compounds that inhibit the invasion of tumor cells into collagen in vitro, therefore potentially inhibiting also the generation of tumor metastasis in vivo.
Assay Principle:
Step 1: Preparation of cells at the bottom of a collagen gel: Cells are suspended in a liquid collagen solution (4° C.), distributed into BSA-coated wells, and then collected at the bottom of the wells by centrifugation. The collagen is then solidified by incubation at 37° C. The BSA coating improves the adhesion of the collagen gel.
Step 2: Pre-treatment with the compounds to be tested: Concentrated drug solutions are then added on top of the collagen, and cells are pre-incubated for 24 h with the drugs at low serum conditions (0.025% FBS).
Step 3: Stimulation of invasion: Medium with 5% FBS is then added in order to stimulate invasion of the cells into the collagen gel.
Step 4: Fixation and staining: Following another 24 h incubation, cells are fixed and nuclei are stained.
Step 5: Analysis: Finally, plates are analyzed using an automated microscope. Fluorescent beads that have been included into the BSA coating serve to detect the bottom of the wells. Pictures of the stained nuclei are taken at the same level (0 μm) as well as 25 μm and 50 μm above.
Note:
In order to detect possible toxic effects, all compounds are tested in parallel in a viability assay. The viability assay is performed in parallel on serum-starved cells (as in the invasion assay) vs. cells under normal culture conditions (10% FBS).
Materials:
General equipment: Freezer (−20° C.), refrigerator (4° C.), ice machine, water bath (37° C.), incubator (37° C./5% CO2), cell culture hood, vortex, vacuum pump, microscope, Malassez cell, Pipet aid, micropipettes (for pipetting 1-1000 μl), multichannel pipettes (for pipetting 20-200 μl), standard cell culture centrifuge, refrigerated centrifuge for 96 well plates
General consumables: Sterile 96 well cell culture plates (for the viability assay), sterile tubes (1.5/15/50 ml), sterile pipettes (5/10/25 ml), sterile micropipette tips (for pipetting 1-1000 μl), sterile Pasteur pipettes, sterile reagent reservoirs
General products: Sterile PBS, sterile Milli-Q water, DMSO, decomplemented FBS (frozen aliquots), 0.1 N NaOH, 1 M Hepes, MEM without serum (not older than 1 month), 2.5×MEM without serum (not older than 1 month), MEM with 10% FBS (not older than one month), 0.25% trypsin/1 mM EDTA solution, 37% formaldehyde solution
Specific Equipment:
plate reader: Tecan Infinite F200
automated microscope: Cellomics ArrayScan VTI HCS Reader
Specific Consumables:
sterile black 96 well plates (for the invasion assay): Perkin Elmer ViewPlate-96 F TC, ref. 6005225
sterile 96 deep well polypropylene plates (for drug preparation): Starlab, ref S1896-5110
Specific Products:
rat tail collagen, type 1: BD Biosciences, ref. 354236 (note: each new lot has to be validated)
red fluorescent beads (1 μm diameter): Invitrogen, ref. F13083
Y-27632 (5 mM aqueous solution): Calbiochem, ref. 688001 (in solution) or 688000 (dry powder)
BSA without fatty acids (sterile-filtered 4% aqueous solution): Sigma, ref A8806 (dry powder)
Hoechst 33342 nuclear stain (10 mg/ml): Invitrogen, ref. H3570
MTS reagent: Promega CellTiter CellTiter 96® AQueous One Solution Reagent, ref. G3581
drug compounds to be tested: generally 25 or 50 mM in 100% DMSO (aliquots stored at −20° C., then at 4° C. for max. 3 months)
MDA-MB231-luc-D3H2LN cells:
Limits for the cell cultures to be used in the assays:
total passage number: max. 30
last passage: between 2 and 4 days before, between 1:3 and 1:20
cell density: between 50 and 90% (optimally 70%) (between 1 and 2×106 cells per 100 mm dish)
Experimental Procedures:
General considerations: Controls and plate maps:
Invasion assay: Negative control: No drug (just DMSO at equivalent concentration). Positive control: 10 μM Y-27632. To avoid edge effects, only the 60 central wells B2-G11 are used; lines A and H as well as columns 1 and 12 remain free. Each drug is tested at least in triplicate. The positive and negative controls should be tested in double triplicates at different positions on each plate. Typical plate map (−=negative control, +=positive control, 1-16=16 different drug compounds):
Viability assays: No additional controls. The MTS viability assay is based on colorimetric detection of a product generated by the mitochondrial activity of the cells. Each drug is tested at least in duplicate. To detect potential direct interactions with the assay substrate, each drug is also tested in absence of cells (background signals). Typical plate map (controls and drug compounds as in the invasion assay, lines A-B and E-F: with cells, lines C-D and G-H: without cells; each 1 plate with 10% vs. 0.025% FBS):
The volumes or other quantities indicated in the following are required for testing 16 drug compounds per 96 wells-plate at 5 μM each (+controls) in an invasion assay and each one viability assay on serum-starved cells vs. cells under normal culture conditions according to the plate maps above. According to the number of tested compounds, the volumes and other quantities should be adapted for testing more or less compounds or different concentrations.
Day 1: Preparation and treatment of the cells (all steps are performed under a cell culture hood):
Preparation of 100× concentrated drug solutions in 10% DMSO:
prepare 10% DMSO in sterile PBS: 1.8 ml sterile PBS+0.2 ml DMSO
prepare 100 μl/well 10% DMSO in PBS in 16 wells of a sterile 96 well polypropylene plate
add each 1 or 2 μl of the 50 or 25 mM compound stock solutions, respectively
mix by pipetting up and down
Preparation of 4× concentrated drug and control solutions in 0.4% DMSO in MEM+0.1% FBS:
prepare MEM+0.1% FBS: 12 ml MEM without serum+12 μl FBS (freshly thawed aliquot)
prepare 480 μl/well MEM+0.1% FBS in 20 wells of a sterile 96 deep well polypropylene plate
negative controls (no drug): add each 20 μl 110% DMSO in sterile PBS
positive controls (Y-27632): add each 14 μl sterile PBS+2 μl DMSO+4 μl 5 mM Y-27632 (freshly thawed aliquot)
drug compounds: add each 20 μl of the 100× concentrated drug solutions in 10% DMSO
mix by pipetting up and down
store at RT until use
Coating of the Plates for the Invasion Assay:
mix 9.5 ml MEM without serum+0.5 ml 4% BSA without fatty acids+1 μl vortexed fluorescent beads (i.e. dilute 1:10000), vortex, distribute 100 μl/well into the plate for the invasion assay
centrifuge 30′ with 1800×g at 4° C. (e.g. 3000 rpm in a Jouan GR412 centrifuge)
remove supernatants by aspiration
Preparation of a 10×106 Cells/ml Cell Suspension (During the Centrifugation of the Plates):
remove medium, wash cells with ˜10 ml/dish PBS, add 1 ml/dish 0.25% trypsin/1 mM EDTA
incubate 30-60 s at 37° C.
add 5-10 ml/dish pre-warmed MEM+10% FBS
homogenize by pipetting up and down using a 10 ml pipette, pool all
count cells using a Malassez cell
centrifuge 2×106 (or more) cells for 5′ with 150×g at RT (850 rpm in a std. cell culture centrifuge)
remove supernatant, resuspend cell pellet in 0.2 ml (or more, respectively) MEM without serum, yielding 10×106 cells/ml
Preparation of the Invasion Assay (on Ice; Start During the Centrifugation of the Cells):
mix on ice in a pre-chilled tube: example for a 3.4 mg/ml collagen stock solution; volumes of collagen and water to be adapted according to the stock concentration of each collagen lot:
2.8 ml 2.5×MEM
441 μl water
140 μl M Hepes
49 μl 1 N NaOH
3.5 ml 3.4 mg/ml collagen stock solution (yielding 1.7 mg/ml collagen in 7 ml)
homogenize by pipetting gently up and down (keep on ice)
add 70 μl of the 10×106 cells/ml cell suspension, homogenize by pipetting gently up and down (yields 0.1×106 cells/ml in 1.7 mg/ml collagen in 7 ml 1×MEM+20 μM Hepes) (keep on ice)
distribute 100 μl/well (i.e. 10000 cells/well) into the coated wells of the plate for the invasion assay (all on ice)
centrifuge 5′ with 200×g at 4° C. (e.g. 1000 rpm in a Jouan GR412 centrifuge)
add 200 μl/well PBS to all free wells
incubate 30′ at 37° C./5% CO2 (solidification of the collagen)
Preparation of the Viability Assay on Serum-Starved Cells:
add 50 μl of the 10×106 cells/ml cell suspension to 5 ml MEM without serum (yields 0.1×106 cells/ml)
distribute 100 μl/well of this suspension (i.e. 10000 cells/well) or MEM without serum without cells, respectively, into a standard 96 well tissue culture plate, according to the plate map above
add 200 μl/well PBS to all free wells
incubate 30′ at 37° C./5% CO2
Preparation of the Viability Assay on Cells Under Normal Culture Conditions:
add 30 μl of the 10×106 cells/ml cell suspension to 5 ml MEM+10% FBS (yields 0.06×106 cells/ml)
distribute 100 μl/well of this suspension (i.e. 6000 cells/well) or MEM+10% FBS without cells, respectively, into a standard 96 well tissue culture plate, according to the plate map above
add 200 μl/well PBS to all free wells
incubate 30′ at 37° C./5% CO2
Treatment with the Drugs:
add each 33 μl/well of the 4× concentrated drug solutions in MEM+0.1% FBS to the corresponding wells in all three plates, according to the plate maps above
incubate 24 h at 37° C./5% CO2
Day 2: Addition of FBS to stimulate the invasion:
Microscopic observation after 24 h of treatment:
examine the cells of the viability assays
Addition of FBS (under a cell culture hood):
prepare MEM+5% FBS: 7.2 ml MEM without serum+0.8 ml FBS (freshly thawed aliquot or rest of the aliquot thawed the day before if kept at 4° C.)
add 33 μl/well to all wells of invasion and viability assays
incubate 24 h at 37° C./5% CO2
Day 3: Stop:
Microscopic observation after 48 h of treatment:
examine the cells of the viability assays
Viability assays: MTS assay:
add each 33 μl/well of the MTS reagent, incubate 2.5 h at 37° C./5% CO2
shake and read absorbance at 490 nm (proportional to the viability)
calculate the background-corrected signals by subtracting the means of the background signals in absence of cells from the corresponding signals in presence of cells
normalize the background-corrected signals with respect to the mean signal of the negative controls (no drug) (viabilities are thus expressed in “% of control”)
Invasion assays: fixation and staining (formaldehyde must be manipulated under a fume cupboard):
freshly prepare 1 μg/ml Hoechst 33342 in 18.5% formaldehyde: 5 ml PBS (not necessarily sterile)+5 ml 37% formaldehyde+1 μl 10 mg/ml Hoechst 33342 (note: for one plate, a smaller volume would be sufficient, but the minimal pipetted volume should not be below 1 μl)
add 50 μl/well to all wells of the invasion assay (yields 4.3% formaldehyde final)
seal with black film (provided with the plates)
incubate at least 7 h at RT
Day 3: 17 (min. 7 h/max. 2 weeks after fixation and staining): Analysis of the invasion assay:
Lecture using the Cellomics ArrayScan VTI HCS Reader:
BioApplication: SpotDetector.V3
Plate type: Perkin Elmer 96 well
Parameters of the Assay Protocol:
objective: 10×(NA 0.45)
apotome: yes (resulting optical slice: 11.7 μM)
fields per well: 8
autofocus in each field
autofocus channel: 1
channel 1 (autofocus on, and photo of the fluorescent beads at the bottom of the wells): filter: XF93-TRITC; exposure time: usually between 0.002 and 0.01 s
channel 2 (photo of the stained cells at the same level as the fluorescent beads): filter: XF100-Hoechst; exposure time: usually between 0.02 and 0.1 s; z offset: 0 μM
channel 3 (photo of the stained cells 25 μM above the fluorescent beads): filter: XF100-Hoechst; exposure time: usually between 0.02 and 0.1 s; z offset: −25 μM
channel 4 (photo of the fluorescent cells 50 μM above the fluorescent beads): filter: XF100-Hoechst; exposure time: usually between 0.02 and 0.1 s; z offset: −50 μM
object identification: method: fixed threshold: 100-32767
Analysis of the results of the scan using vHCS Viewer:
export the results: for each well:
number of valid fields
number of objects in each valid field in each of the channels 2, 3 and 4 (“field details”)
mean numbers of objects per valid field for each well, in each of the channels 2, 3 and 4
exclude wells with less than 6 valid fields per well from further analysis
visually check all photos for any apparent problems, such as bad focusing or obviously inhomogeneous collagen structure (“bubbles”, . . . ), . . . ; in case of apparent problems: document, then exclude the corresponding wells from further analysis
Further analysis of the results of the invasion assay (using e.g. Excel):
for each well, calculate the mean invasion distance of the counted cells: (25 μm×number of cells at 25 μm+50 μm×number cells at 50 μm)/sum of cells at 0, 25 and 50 μm
for all four parameters (number of cells at 0 μm, number of cells at 25 μm, number of cells at 50 μm, mean invasion distance of the counted cells), calculate means, SD and CV of the replicates (n=6 for the controls; n=3 for the samples)
invalidate any replicate with a CV≧50% (compound to be re-tested, or assay to be repeated if CV≧50% for the untreated negative control or the compound Y-27632-treated positive control). Y27632 is a selective inhibitor of the Rho-associated protein kinase p160ROCK of the following formula
validate the assay only if the mean invasion distance of the cells treated with 10 μM Y-27632 (positive control) is decreased by ≧40% as compared to the untreated negative control
plot graphs of all four parameters (number of cells at 0 μm, number of cells at 25 μm, number of cells at 50 μm, mean invasion distance of the counted cells)
Results
Anti-invasive effect at 5 μM on MDA-MB231 breast cancer cells (fold effect compared to 10 μM Y-27632 ref compound)
The compounds according to the present invention demonstrate an anti-invasive effect predictive for their activity against cancer.
Therefore, the result of the tests carried out on the compounds disclosed in the present invention show that said compounds may be useful to inhibit, prevent and/or treat cancer. The following type of cancer may more particularly be treated by the compounds according to the present invention: colorectal cancer, pancreatic cancer, lung cancer including non-small cell lung cancer, breast cancer, bladder cancer, gall bladder cancer, thyroid cancer, melanoma, liver cancer, uterine/cervical cancer, oesophageal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, and stomach cancer, etc.
For this purpose an effective amount of a said compound may be administered to a patient suffering from cancer.
The present invention is also related to the use of at least a compound chosen among a compound of anyone of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (Io), (Ip), (Iq), (Ir) or (Iee) as defined above, and compounds (I) to (168) as defined above, or one of its pharmaceutically acceptable salts according to the present invention for the manufacture of a pharmaceutical composition intended for the treatment of cancer.
The present invention also encompasses pharmaceutical compositions comprising at least a compound chosen among new compounds of formula (Iq) or (Iee) as defined above and compounds (143), (144), (149), (166) and (167) as defined above or any pharmaceutically acceptable salt thereof.
Thus, these pharmaceutical compositions contain an effective amount of said compound, and one or more pharmaceutical excipients.
The aforementioned excipients are selected according to the dosage form and the desired mode of administration.
In this context they can be present in any pharmaceutical form which is suitable for enteral or parenteral administration, in association with appropriate excipients, for example in the form of plain or coated tablets, hard gelatine, soft shell capsules and other capsules, suppositories, or drinkable, such as suspensions, syrups, or injectable solutions or suspensions, in doses which enable the daily administration of from 0.1 to 1000 mg of active substance.
The present invention is also related to the use of a compound of anyone of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (Io), (Ip), (Iq), (Ir) or (Iee) as defined above, and compounds (I) to (168) as defined above, or one of its pharmaceutically acceptable salts according to the present invention for the manufacture of a pharmaceutical composition intended for inhibiting, preventing and/or treating cancer.
The present invention further relates to a method of treatment of patients suffering form cancer, which comprises at least a step of administration to a patient suffering thereof of an effective amount of a compound of anyone of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (Io), (Ip), (Iq), (Ir) or (Iee) as defined above and (1) to (168) or one of its pharmaceutically acceptable salts.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09162630.9 | Jun 2009 | EP | regional |
| 09305540.8 | Jun 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB10/52650 | 6/14/2010 | WO | 00 | 7/5/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61186552 | Jun 2009 | US | |
| 61186544 | Jun 2009 | US |
