CAPACITATIVE CALCIUM ENTRY INHIBITORS

Abstract

The invention relates to Orai1-dependent selective capacitative calcium entry inhibitors as well as to the therapeutic use thereof.

Claims

1-11. (canceled)

12. A method for inducing immunosuppression in a subject in need thereof, comprising administering to said subject a compound of formula (I) or a pharmaceutically acceptable salt thereof: ##STR00002## wherein R1, R2, R3, R4, R5 and R6 are groups, which may be identical or different, selected from a hydrogen atom, a halogen atom, a C.sub.1-C.sub.6 alkyl group, a C.sub.1-C.sub.6 alkenyl group, a C.sub.1-C.sub.6 alkyloxy group, a C.sub.1-C.sub.6 alkylthio group, a C.sub.6-C.sub.14 aryl group and a heteroaryl group comprising 5 to 14 ring members; with the proviso that, when two of the R1, R2 and R3 groups represent a hydrogen, then the group from R1, R2 and R3 which is not a hydrogen is selected from a C.sub.3-C.sub.6 alkyl group, a C.sub.3-C.sub.6 alkenyl group, a C.sub.3-C.sub.6 alkyloxy group, a C.sub.3-C.sub.6 alkylthio group, a C.sub.6-C.sub.14 aryl group and a heteroaryl group comprising 5 to 14 ring members; with the proviso that, when two of the R4, R5 and R6 groups represent a hydrogen, then the group from R4, R5 and R6 which is not a hydrogen is selected from a C.sub.3-C.sub.6 alkyl group, a C.sub.3-C.sub.6 alkenyl group, a C.sub.3-C.sub.6 alkyloxy group, a C.sub.3-C.sub.6 alkylthio group, a C.sub.6-C.sub.14 aryl group and a heteroaryl group comprising 5 to 14 ring members; it being possible for the R1 and R2 groups to form, with the carbon atoms to which they are bound, a ring comprising 5 to 7 ring members; it being possible for the R4 and R5 groups to form, with the carbon atoms to which they are bound, a ring comprising 5 to 7 ring members; and said aryl or heteroaryl groups being optionally substituted with one or more substituents selected from halogen atoms, C.sub.1-C.sub.6 alkyl groups, C.sub.2-C.sub.6 alkenyl groups, C.sub.1-C.sub.6 alkyloxy groups and C.sub.1-C.sub.6 alkylthio groups, C.sub.2-C.sub.6 ketone groups, C.sub.1-C.sub.6 ester groups, C.sub.1-C.sub.6 aldehyde groups, —NH.sub.2, −CN, —CF.sub.3, C.sub.1-C.sub.6 amine groups and C.sub.1-C.sub.6 amine groups mono- or disubstituted with a C.sub.1-C.sub.6 alkyl group.

13. The method according to claim 12, wherein R1, R3, R4 and R6 represent a hydrogen atom and R2 and R5, which are identical, represent a C.sub.3-C.sub.6 alkyl group, in particular an n-butyl, isobutyl, sec-butyl or tert-butyl, more particularly n-butyl, group, or a C.sub.6-C.sub.14 aryl group, more particularly a C.sub.6-C.sub.8 aryl group.

14. The method according to claim 13, wherein R2 and R5 represent a substituted or unsubstituted phenyl group.

15. The method according to claim 12, said compound being 2-(dibiphenyl-4-ylboryloxy)ethamine (compound P11).

16. The method according to claim 12, said compound being 2-(bis(4-(tert-butyl)phenyl)boryl)oxy)ethamine (compound P9).

17. The method according to claim 12, wherein: R3 is a hydrogen atom and R1 and R2, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group; and/or R6 is a hydrogen atom and R4 and R5, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group.

18. The method according to claim 12, wherein: R2 represents a hydrogen atom, and R1 and R3, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group; and/or R5 is a hydrogen atom and R4 and R6, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group.

19. The method according to claim 12, for inhibiting cytokine production by an immune cell, in particular for inhibiting IL-2 production from T lymphocytes.

20. A method for treating an inflammatory disorder, an immune disorder, an allergy or a cancer, in a subject in need thereof, comprising administering to said subject a compound of formula (I) or a pharmaceutically acceptable salt thereof: ##STR00003## wherein R1, R2, R3, R4, R5 and R6 are groups, which may be identical or different, selected from a hydrogen atom, a halogen atom, a C.sub.1-C.sub.6 alkyl group, a C.sub.1-C.sub.6 alkenyl group, a C.sub.1-C.sub.6 alkyloxy group, a C.sub.1-C.sub.6 alkylthio group, a C.sub.6-C.sub.14 aryl group and a heteroaryl group comprising 5 to 14 ring members; with the proviso that, when two of the R1, R2 and R3 groups represent a hydrogen, then the group from R1, R2 and R3 which is not a hydrogen is selected from a C.sub.3-C.sub.6 alkyl group, a C.sub.3-C.sub.6 alkenyl group, a C.sub.3-C.sub.6 alkyloxy group, a C.sub.3-C.sub.6 alkylthio group, a C.sub.6-C.sub.14 aryl group and a heteroaryl group comprising 5 to 14 ring members; with the proviso that, when two of the R4, R5 and R6 groups represent a hydrogen, then the group from R4, R5 and R6 which is not a hydrogen is selected from a C.sub.3-C.sub.6 alkyl group, a C.sub.3-C.sub.6 alkenyl group, a C.sub.3-C.sub.6 alkyloxy group, a C.sub.3-C.sub.6 alkylthio group, a C.sub.6-C.sub.14 aryl group and a heteroaryl group comprising 5 to 14 ring members; it being possible for the R1 and R2 groups to form, with the carbon atoms to which they are bound, a ring comprising 5 to 7 ring members; it being possible for the R4 and R5 groups to form, with the carbon atoms to which they are bound, a ring comprising 5 to 7 ring members; and said aryl or heteroaryl groups being optionally substituted with one or more substituents selected from halogen atoms, C.sub.1-C.sub.6 alkyl groups, C.sub.2-C.sub.6 alkenyl groups, C.sub.1-C.sub.6 alkyloxy groups and C.sub.1-C.sub.6 alkylthio groups, C.sub.2-C.sub.6 ketone groups, C.sub.1-C.sub.6 ester groups, C.sub.1-C.sub.6 aldehyde groups, —NH.sub.2, —CN, —CF.sub.3, C.sub.1-C.sub.6 amine groups and C.sub.1-C.sub.6 amine groups mono- or disubstituted with a C.sub.1-C.sub.6 alkyl group.

21. The method according to claim 20, wherein R1, R3, R4 and R6 represent a hydrogen atom and R2 and R5, which are identical, represent a C.sub.3-C.sub.6 alkyl group, in particular an n-butyl, isobutyl, sec-butyl or tert-butyl, more particularly n-butyl, group, or a C.sub.6-C.sub.14 aryl group, more particularly a C.sub.6-C.sub.8 aryl group.

22. The method according to claim 21, wherein R2 and R5 represent a substituted or unsubstituted phenyl group.

23. The method according to claim 20, said compound being 2-(dibiphenyl-4-ylboryloxy)ethamine (compound P11).

24. The method according to claim 20, said compound being 2-(bis(4-(tert-butyl)phenyl)boryl)oxy)ethamine (compound P9).

25. The method according to claim 20, wherein: R3 is a hydrogen atom and R1 and R2, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group; and/or R6 is a hydrogen atom and R4 and R5, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group.

26. The method according to claim 20, wherein: R2 represents a hydrogen atom, and R1 and R3, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group; and/or R5 is a hydrogen atom and R4 and R6, which may be identical or different, represent a halogen atom or a C.sub.1-C.sub.6 alkyl group.

27. The method according to claim 20, for treating breast cancer in a subject in need thereof.

28. The method according to claim 20, for treating a hormone-independent cancer.

29. The method according to claim 20, for the treating a chronic inflammatory disorder, leukemia, lymphoma, pulmonary arterial hypertension or graft rejection.

Description

FIGURE LEGENDS

[0049] FIG. 1 represents the structure of the 2-APB compound (compound P1) and of the 2-APB analogs disclosed in the present application.

[0050] FIG. 2 shows that 2-APB has a dual effect on the SOCE of Jurkat T cells, whereas the other 2-APB analogs have only an inhibitory effect.

[0051] FIG. 3 is a graph showing that compound P11 inhibits Mn.sup.2+ influx via the SOCCs and SOCE of other cell types.

[0052] FIG. 4 is a graph showing that compounds P9 and P11 preferentially inhibit SOCE compared to loading via SERCA and compared to calcium release from the ER by IP3R (“IICR”).

[0053] FIG. 5 is a graph showing that compound P11 increases cell death and decreases interleukin-2 synthesis by PHA-stimulated Jurkat cells.

[0054] FIG. 6 is a graph showing that compound P11 increases the caspase-3 activity of PHA-stimulated Jurkat cells.

[0055] FIG. 7 is a graph showing that compound P11 increases DNA fragmentation in PHA-stimulated Jurkat cells.

EXAMPLES

[0056] 1. Materials and Methods

[0057] The Jurkat T lymphocyte line was mainly used for characterizing the effect of the molecules on SOCE. The Indo-1 ratiometric probe was used to visualize cytosolic calcium variations. For this, the cells in culture were centrifuged and then resuspended and incubated in PBS (Phosphate Buffered Saline) medium supplemented with 1 mg/ml of albumin and 4 μM of Indo-1-AM for 45 mim at ambient temperature and in the dark. The cells were then again centrifuged and resuspended in the HBS (Hepes-Buffered Saline) recording medium having the composition (in mM): 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl.sub.2, 11.6 mM Hepes, 11.5 mM glucose, pH adjusted to 7.3 with NaOH. 0.5 to 1 million cells are then placed in the cuvette of a Varian Cary Eclipse spectrofluorimeter and the Indo-1 probe is excited at 360 nm, with emission of fluorescence at 405 and 480 nm. The ratio of fluorescence at 405 and 480 nm is directly proportional to the [Ca.sup.2+]cyt.

[0058] The lines of monocyte origin U937, B lymphocyte origin (DG75) and breast cancer origin (MDA-MB231) were also used with the same protocol.

[0059] To test the selectivity of certain molecules on SOCE compared to IP3R and SERCA, the L15 line stably expressing type 1 IP3R was used. Briefly, the cells were permeabilized with saponin and incubated in the presence of .sup.45Ca.sup.2+ in a medium containing ATP allowing SERCA activity and Ca' ion entry into the ER. The addition of TG makes it possible to induce release from the ER and therefore to measure the amount of Ca.sup.2+ ions that have left the ER, which is itself directly linked to the SERCA activity. The addition of IP3 allows direct measurement of the amount of Ca' ions leaving the ER by IP3R opening.

[0060] In order to measure the interleukin-2 (IL-2) synthesis in 24 h after stimulation with 10 μg/ml of phytohemagglutinin (PHA) in the presence or absence of certain molecules, the Jurkat cells are centrifuged and the supernatant containing the IL-2 is collected. The 11-2 concentration is measured by Elisa (R&D systems kit).

[0061] The caspase-3 activity is measured with a fluorescent kit using the Ac-DEVD-AFC substrate. The Jurkat cells are incubated for 24 h with PHA in the presence or absence of certain molecules. The cells are then lyzed with RIPA medium, and the amounts of proteins are then measured with a commercial kit (Biorad). The cell lysates are then diluted in the following reaction medium: 200 mM Hepes, pH 7.4, 1 mM EDTA, 20% sucrose and 20 mM dithiothreitol, then incubated with 20 μM of the fluorogenic substrate for 45 min at 7 h at 37° C. The appearance of the fluorescent AFC product is measured by spectrofluorimetry (excitation at 405 nm, emission at 505 nm).

[0062] The Roche TUNEL kit was used to observe the nuclear DNA fragmentation and therefore cell apoptosis.

[0063] 2. Results

[0064] 2.1. Effects of the Analogs on Cytosolic Calcium Concentration

[0065] New analogs of 2-APB were synthesized in order to identify strong SOCE inhibitors. The molecules tested and their synthesis route are presented in FIG. 1.

[0066] The SOCE phenomenon was evaluated conventionally. Jurkat cells were placed in calcium-free HBS medium and stimulated with 1 μM of thapsigargin (TG, arrow in FIG. 2A) for 600 s in order to allow calcium release from the ER and SOCC channel opening(=CRAC). The addition of 1 mM of CaCl.sub.2 to the medium allows massive calcium ion entry through the SOCCs and an increase in the concentration of cytosolic calcium ([Ca.sup.2+].sub.cyt).

[0067] The synthesis of the new compounds was validated by evaluating the effect of 2-APB synthesized under the same conditions. FIG. 2A shows that 2-APB synthesized in this way clearly has its conventional properties: high potentiation of calcium entry at 5 μM and virtually total inhibition at 50 μM.

[0068] The dose-response curves were established after addition of 2-APB or of an analog thereof 30 seconds before the reintroduction of calcium. The results show that the 2-APB analogs do not exhibit the property of increasing cytosolic calcium concentration. The increase in the size of the molecules at the level of the phenyl groups therefore has a negative effect on the potentiation activity. The analogs tested are therefore SOCE inhibitors without the effect of potentiation of the increase in cytosolic calcium normally observed at low dose with 2-APB (cf. table 1).

TABLE-US-00001 TABLE 1 Compound Ki (nM) P11  75 ± 21 P6 275 ± 17 P15 294 ± 74 P8 350 ± 40 P12 374 ± 96 Dibenzothienyl-APB 405 ± 23 (Djillani 2014) P10  484 ± 116 P9  641 ± 103 P7 751 ± 97 Compound Ki (μM) P13 1.84 ± 0.1  P5 2.1 ± 0.6 P16 3.1 ± 0.6 P3 3.5 ± 0.6 P2  3.5 ± 0.65 P14 5.4 ± 0.3 P4 75 ± 21 P17 >>1 μM

[0069] Compounds P3 and P5 comprise a methyl group on each of the phenyl groups of the molecule (in the para and meta positions, respectively). P7 and P10 correspond to compounds P3 and P5 to which a second methyl group has been added on each of the phenyl groups (a group in the meta-position and a group in the para-position of each of the phenyls). These modifications result in a significant increase in the efficiency of inhibition of the molecules. However, compound P10 is the most active of the two, with a Ki of approximately 500 nM and a total inhibition obtained at 3 μM.

[0070] Other analogs were generated with a methoxy group on each of the phenyl groups of the 2-APB (compounds P2 and P4). It is observed that the placement of the methoxy group in the para-position makes it possible to obtain a compound (compound P2) having an activity equivalent to that of the compound comprising a methyl group at this position (compound P3). On the other hand, the placement of this methoxy group in the meta-position (compound P4) has a drastic negative impact on the inhibition constant, which rises to 75±21 μM, with a maximum inhibition of 80% only. Compound P16 combines a methyl group in the para-position of one of the phenyls, and a methoxy group on the other phenyl. This compound has an inhibitory activity similar to compounds P2 and P3 (3.1±0.6 μM).

[0071] Halogenated analogs were also prepared. These are compounds P13, P14 and P15. Compounds P13 and P14, wherein a chlorine and a fluorine are located in the para-position of each of the phenyl groups, respectively, have an activity equivalent to compound P3. Surprisingly, compound P15, which is similar to compound P7 in which a chlorine atom would have been replaced with methyl groups in the meta-position of each of the phenyl groups, has an improved inhibitory activity with a Ki of approximately 300 nM.

[0072] Compounds P8 and P9 are analogs comprising, in the para-position of the phenyl groups, substituents that are bulkier than the methyl group of compound P3, with respectively a tert-butyl and n-butyl group. This modification leads to a significant increase in the inhibitory activity of the analogs, with a Ki of 350±40 nM and 641±103 nM, respectively. It is therefore demonstrated that the addition of bulkier groups leads to an increase in the inhibitory activity of the compounds.

[0073] Moreover, analogs comprising a naphthyl group (compound P6) or benzothienyl group (compound P12) in place of each phenyl group of 2-APB were synthesized. Compounds P6 and P12 exhibit a significant inhibitory activity, with a Ki of 275±17 nM and 374±96 nM, respectively.

[0074] However, the best inhibitor identified is compound P11, which comprises a phenyl group bonded in the para-position of each of the phenyl groups of 2-APB, and has a Ki of 75±21 nM on Jurkat cells. Entirely surprisingly, the imposition of a constraint between the two phenyl groups by means of the introduction of a gem-dimethyl (compound P17) leads to a total absence of activity of the compound, resulting in a Ki of greater than 3 μM. Free rotation of the two phenyl groups of compound P11 therefore appears to be an important parameter for maintaining the very high efficiency of said compound.

[0075] 2.2. Specific Inhibition of SOCE without Impact on Calcium Efflux Mechanisms

[0076] In order to determine whether compound P11 has an action on SOCE and not on calcium efflux mechanisms, a quenching experiment with Mn.sup.2+ was carried out by means of the indo-1 molecule. The Mn.sup.2+ ions enter the cells via the SOCCs, but cannot be pumped into the extracellular membrane by the plasma membrane calcium-dependent ATPases (plasma membrane Ca.sup.2+ ATPases, or PMCA) or the sodium/calcium exchanger (Na.sup.+/Ca.sup.2+ exchanger, or NCX) or sent back into the lumen of the ER by the sarcoplasmic-endoplasmic reticulum Ca.sup.2+ ATPases (or SERCAs). Once in the cell, Mn.sup.2+ binds to indo-1 and quenches its fluorescence measured at 430 nm. An increase in the amplitude of the Mn.sup.2+ influx is therefore associated with an increase in the indo-1 quenching rate, and vice versa.

[0077] After 10 minutes of treatment with TG in order to open the SOCCs, 100 μM of MnCl.sub.2 were added. A degree of quenching of −0.89±0.05% Fo/s was observed (FIG. 3A). In the presence of increasing concentrations of P11 added 30 seconds before the addition of Mn.sup.2+ ions, the degree of quenching decreased and reached a blocking of approximately 90% at 1 μM (−0.09±0.01, FIG. 3A).

[0078] Compound P11 therefore clearly targets SOCE.

[0079] Moreover, FIG. 3B shows that P11 is also capable of blocking SOCE in other cell types, with an efficiency significantly greater than that observed in Jurkat cells. Thus, Ki values of 32±2 nM, 40±5 nM and 50±5 nM were calculated in DG75 cells (B lymphocytes), U937 cells (monocytes) and MDA-MB231 cells (hormone-independent breast cancer cells) respectively, compared with 75±21 nM for Jurkat cells (FIG. 3B). FIG. 3B also shows that total inhibition is achieved at 100 nM.

[0080] 2.3. Selectivity of the Compounds of the Invention

[0081] The selectivity of compound P11 and of the close compound P9 was evaluated.

[0082] The .sup.45Ca.sup.2+ loading and IP3-dependent release measurements were carried out on permeabilized cells under one-way conditions.

[0083] FIG. 4 shows the results obtained with P9 and P11.

[0084] P9 did not significantly inhibit the activity of IP3R and is only a partial inhibitor of SERCA activity, with an inhibition of −52±3% at 100 μM and a Ki of 6±0.3 μM. Below 3 μM, compound P9 inhibits only SOCE.

[0085] P11 inhibits IP3R only very slightly, with a value of 29±9% at 100 μM, the calculation of the Ki with respect to this receptor having moreover proved to be impossible because of the very high concentrations that would have been required in order to obtain this value. SERCA is 89% inhibited by P11, at a concentration greater than 30 μM. However, this activity is quite unlike that observed on SOCE, since the Ki for SERCA is 7.4 μM, whereas that for SOCE is 75 nM, that is to say 100 times less for the latter. As a result of this, P11 is a more selective inhibitor of SOCE at concentrations of less than 1 μM, without any effect on SERCA and IP3R.

[0086] 2.4. The Compounds of the Invention Prevent Jurkat Cell Activation

[0087] T lymphocyte activation requires an increase in cytosolic calcium concentration for several minutes in order to allow interleukin 2 (IL-2) synthesis and immune function activation. Consequently, inhibition of the increase in cytosolic calcium concentration due to SOCE prevents the activation of T lymphocytes and proliferation thereof.

[0088] The concentration of neosynthesized IL-2 was measured in the culture medium of phytohemagglutinin (PHA)-stimulated Jurkat cells after 24 h, in the presence of increasing concentrations of compound P11. In the absence of stimulation by PHA, Jurkat cells do not produce IL-2. Conversely, stimulation with PHA allows the detection of approximately 100 pg/ml of IL-2 (103 ±4 pg/ml, FIG. 5A). The addition of increasing concentrations of P11 at the same time as the stimulation by PHA leads to a gradual decrease in IL-2 synthesis to 26±6 pg/ml for 1 μM of P11 (-74%, FIG. 5B).

[0089] PHA alone or P11 alone did not exhibit any toxic effect on the cells. However, the combination of the treatment of the cells with PHA and increasing concentrations of P11 significantly increases the percentage of dead cells (which goes from approximately 5% without treatment, to 33.2±1.3% at 1 μM of P11, FIG. 5B). However, the calculation of the amount of IL-2 synthesized by the remaining living cells shows that increasing amounts of P11 decrease the capacity of synthesis of this cytokine by the T lymphocytes by approximately 63%, going from 107.6±4.1 pg/ml to 39.2±8.6 pg/ml (FIG. 5C).

[0090] P11 therefore has a double effect on activated Jurkat cells: a reduction in IL-2 synthesis and an induction of cell death.

[0091] 2.5. Induction of Apoptosis of the Activated Jurkat Cells

[0092] The caspase-3 activity was then evaluated by means of an Ac-DEVD-AFC fluorogenic substrate, and apoptosis was evaluated by means of a TUNEL assay.

[0093] FIG. 6 shows that compound P11 alone does not induce an increase in caspase-3 activity, including at a concentration of 1 μM. The caspase-3 activity increases by a factor of 2 after stimulation with PHA for 24 h (1836±132 arbitrary units (AU) compared with 1037±100 AU). The supplementary addition of P11, this being from 10 nM, leads to a maximum activation effect with an increase by a factor of 3 (3332±113 AU at 1 μM). Similar results were obtained by means of the TUNEL assay, which shows that, at concentrations greater than 10 nM, compound P11 induces a 4.5-fold increase in the number of cells containing fragmented DNA (17.9±1.2%), in cells stimulated with PHA (FIG. 7).

[0094] These results therefore show that compound P11 does not exhibit a toxic effect on the non-activated cells, but that it has an impact on Jurkat cell activation through an induction of apoptosis of said cells.