Adamantane or pinene derivatives for use in the treatment of chlamydiales infections

Abstract

The present invention relates to a method for treating a Chlamydiales infection comprising the administration of a therapeutically effective amount of a compound of formula (I) to a subject in need thereof:
W-L.sub.1-NH-L.sub.2-Ar (I)
Wherein W, L1, L2 and Ar are as defined in claim 1.

Claims

1. A method for treating a Chlamydiales infection comprising the administration of a therapeutically effective amount of a compound of formula (I) to a subject in need thereof
W-L.sub.1-NH-L.sub.2-Ar (I) wherein: W is ##STR00015## L.sub.1 is independently selected from a single bond, or (CH.sub.2).sub.pC(O), L.sub.2 is independently selected from (CH.sub.2).sub.q, [C(O)NH].sub.rNCH, or (C.sub.6H.sub.4)SO.sub.2NH, Ar is a C.sub.6-C.sub.10 aryl, said aryl group being substituted by one to three R groups, R being independently selected from Cl, Br, I, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, and NO.sub.2, p is 0, 1 or 2, q is 1 or 2, and r is 0 or 1, and or the stereoisomeric forms, or mixtures of stereoisomeric forms or pharmaceutically acceptable salts forms thereof.

2. The method of claim 1, wherein the Chlamydiales infection is a Chlamydia or Simkania infection.

3. The method of claim 1, wherein L.sub.1 is a single bond.

4. The method of claim 1, wherein L.sub.2 is (CH.sub.2).sub.q.

5. The method of claim 4, wherein Ar is phenyl, said phenyl being substituted by one to three R groups, wherein R is independently selected from the group consisting of Cl, Br, I, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6alkoxy and NO.sub.2.

6. The method of claim 1, wherein R is selected from Br, I, methoxy, or NO.sub.2.

7. The method of claim 1, which is selected from: (1S,2S,3S,5R)N-(5-bromo-2-methoxybenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP332), (1S,2S,3S,5R)N-(5-iodo-2-methoxybenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP386) or (1S,2S,3S,5R)N-(4-iodobenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP390).

8. A pharmaceutical composition comprising a compound of formula I:
W-L.sub.1-NH-L.sub.2-Ar (I) wherein: W is ##STR00016## L.sub.1 is independently selected from a single bond, or (CH.sub.2).sub.pC(O), L.sub.2 is independently selected from (CH.sub.2).sub.q, [C(O)NH].sub.rNCH, or (C.sub.6H.sub.4)SO.sub.2NH, Ar is a C.sub.6-C.sub.10 aryl, said aryl group being substituted by one to three R groups, R being independently selected from the group consisting of Cl, Br, I, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, and NO.sub.2, p is 0, 1 or 2, q is 1 or 2, and r is 0 or 1, or the stereoisomeric forms, or mixtures of stereoisomeric forms or pharmaceutically acceptable salts forms thereof, in admixture with one or more pharmaceutically acceptable excipients.

9. The pharmaceutical composition of claim 8, wherein L.sub.1 is a single bond.

10. The pharmaceutical composition of claim 8, wherein L.sub.2 is (CH.sub.2).sub.q.

11. The pharmaceutical composition of claim 8, wherein the compound of formula (I) is selected from: (1S,2S,3S,5R)N-(5-bromo-2-methoxybenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP332), (1S,2S,3S,5R)N-(5-iodo-2-methoxybenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP386) or (1S,2S,3S,5R)N-(4-iodobenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP390).

12. A compound of formula (I) as defined in claim 8, or the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts forms thereof.

13. The pharmaceutical composition of claim 10, wherein L.sub.2 is CH.sub.2.

14. The pharmaceutical composition of claim 8, wherein Ar is phenyl, said phenyl being substituted by one to three R groups, wherein R is independently selected from the group consisting of Cl, Br, I, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy and NO.sub.2.

15. The method of claim 1, wherein L.sub.2 is CH.sub.2.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effect of Compound 20 on the Sn bacterial load of infected HeLa cells by GroEL immunoblot and Actin was used as loading control.

(2) FIG. 2 shows the effect of Compound 20 on Sn the inclusion size during primary infection of HeLa cells.

(3) FIG. 3 shows the effect of Compound 20 on the number Sn of inclusions during progeny infection of HeLa cells.

(4) FIG. 4 shows pictures illustrating effect of Compound 20 on phenotypic variations in Sn inclusion formation in infected HeLa cells.

(5) FIG. 5 shows pictures of the subcellular structure of Sn inclusions in infected HeLa cells by transmission electron microscopy in presence of DMSO or of Compound 20. N: HeLa cell nucleus, V: vacuoles, Sn: Sn inclusions. After treatment with Compound 20, the number of Sn inclusions is reduced and the vacuole size is much reduced.

(6) FIG. 6 shows immunotblot analysis of lysed HeLa cells after Ctr primary and progeny infection in presence of Compound 20; Chlamydial growth was detected with antibodies against chlamydial HSP60 protein and Actin was used as loading control.

(7) FIG. 7 shows microscopy images of cells stained for DAPI and Chlamydia trachomatis (detected by GFP-signal) after compound #20 and compound #20 derivatives application at 75 M during Chlamydia trachomatis infection of HeLa229 cells.

EXAMPLES

(8) I. Synthesis of Compounds of Formula (I)

(9) ##STR00011##

(10) To a mixture of (+)-isopinocamphenylamine (100 mg, 0.652 mmol) in 2 mL of dry methanol was added 0.652 mmol of the appropriate aldehyde. The resulting mixture was stirred for several hours and NaBH.sub.3CN (0.979 mmol, 61.5 mg) was added in one portion immediately followed by acetic acid (1.305 mmol, 73.2 L).

(1S,2S,3S,5R)N-(5-bromo-2-methoxybenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP 332)

(11) ##STR00012##

(12) To a mixture of (+)-isopinocamphenylamine (100 mg, 0.652 mmol) in 2 mL of dry methanol was added 5-bromo-2-methoxybenzaldehyde (140 mg, 0.652 mmol). The resulting mixture was stirred for several hours and NaBH.sub.3CN (0.979 mmol, 61.5 mg) was added in one portion immediately followed by acetic acid (1.305 mmol, 73.2 L). The crude mixture was washed with a saturated solution of NaHCO.sub.3 (5 mL) and extracted with dichloromethane (320 mL). The residue was concentrated under vacuum and purified by flash chromatography (cyclohexane/ethyl acetate 1:0 to 1:1) affording 226 mg (98%) of compound as a colourless oil.

(13) .sup.1H-NMR (400 MHz, CDCl.sub.3) (ppm)=0.94 (s, 3H), 0.99 (d, J=9.6 Hz, 1H), 1.06 (d, J=7.1 Hz, 3H), 1.21 (s, 3H), 1.63-1.69 (m, 1H), 1.76-1.85 (m, 2H), 1.89-1.98 (m, 2H), 2.28-2.40 (m, 2H), 2.81-2.88 (m, 1H), 3.67 (d, J=13.4 Hz, 1H), 3.82 (d, J=13.4 Hz, 1H), 3.82 (s, 3H), 6.72 (d, J=8.6 Hz, 1H), 7.33 (dd, J=8.6 Hz, J=2.5 Hz, 1H), 7.41 (d, J=2.5 Hz, 1H).

(14) .sup.13C-NMR (100 MHz, CDCl.sub.3) (ppm)=21.4, 23.4, 27.8, 33.7, 36.3, 38.6, 41.7, 44.8, 46.9, 47.8, 55.4, 56.0, 11.8, 112.7, 130.5, 130.9, 132.3, 156.6.

(15) MS (ESI) [M+H].sup.+=352.08/354.02

(16) LC/MS (X-bridge 1004.6 mm) Gradient A: t.sub.R=18.8 min Gradient D: t.sub.R=13.50 min

(17) I.R. (neat, cm.sup.1) 2901, 1485, 1462, 1240, 1030, 801.7, 622.7

(18) HRMS m/z [(M+H).sup.+] calcd for C.sub.18H.sub.27NOBr 352.1276 found. 352.1271.

(1S,2S,3S,5R)N-(5-iodo-2-methoxybenzyl)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine (VP 386)

(19) ##STR00013##

(20) To a mixture of (+)-isopinocamphenylamine (100 mg, 0.652 mmol) in 2 mL of dry methanol was added 5-iodo-2-methoxybenzaldehyde (171 mg, 0.652 mmol). The resulting mixture was stirred for several hours and NaBH.sub.3CN (0.979 mmol, 61.5 mg) was added in one portion immediately followed by acetic acid (1.305 mmol, 73.2 L). The crude mixture was washed with a saturated solution of NaHCO.sub.3 (5 mL) and extracted with dichloromethane (320 mL). The residue was concentrated under vacuum and purified by flash chromatography (cyclohexane/ethyl acetate 1:0 to 1:1) affording 260 mg (99%) of compound as a colourless oil.

(21) .sup.1H-NMR (400 MHz, CDCl.sub.3) (ppm)=0.94 (s, 3H), 1.00 (d, J=9.6 Hz, 1H), 1.06 (d, J=7.2 Hz, 3H), 1.21 (s, 3H), 1.65-1.72 (m, 1H), 1.76-1.85 (m, 2H), 1.94-2.07 (m, 2H), 2.28-2.40 (m, 2H), 2.82-2.89 (m, 1H), 3.67 (d, J=13.4 Hz, 1H), 3.82 (d, J=13.4 Hz, 1H), 3.83 (s, 3H), 6.64 (d, J=8.5 Hz, 1H), 7.52 (dd, J=8.6 Hz, J=2.2 Hz, 1H), 7.58 (d, J=2.2 Hz, 1H).

(22) .sup.13C-NMR (100 MHz, CDCl.sub.3) (ppm)=21.3, 23.4, 27.8, 33.7, 36.0, 38.6, 41.7, 44.6, 46.7, 47.8, 55.4, 56.2, 82.9, 112.5, 130.8, 136.8, 138.2, 157.5.

(23) MS (ESI) [M+H].sup.+=400.13

(24) LC/MS (X-bridge 1004.6 mm) Gradient A: t.sub.R=19.18 min Gradient D: t.sub.R=14.07 min

(25) I.R. (neat, cm.sup.1) 2900, 1484, 1240.6, 1029, 801.2, 614

(26) HRMS m/z [(M+H).sup.+] calcd for C.sub.18H.sub.27N.sub.2OI 1400.1137 found 400.1126.

(1S,2S,3S,5R)-2,6,6-trimethyl-N-(4-iodobenzyl)bicyclo[3.1.1]heptan-3-amine (VP 390)

(27) ##STR00014##

(28) To a mixture of (+)-isopinocamphenylamine (100 mg, 0.652 mmol) in 2 mL of dry methanol was added 4-iodobenzaldehyde (151 mg, 0.652 mmol). The resulting mixture was stirred for several hours and NaBH.sub.3CN (0.979 mmol, 61.5 mg) was added in one portion immediately followed by acetic acid (1.305 mmol, 73.2 L). The crude mixture was washed with a saturated solution of NaHCO.sub.3 (5 mL) and extracted with dichloromethane (320 mL). The residue was concentrated under vacuum and purified by flash chromatography (cyclohexane/ethyl acetate 1:0 to 1:1) affording 241 mg (97%) of compound as a colourless oil.

(29) .sup.1H-NMR (400 MHz, CDCl.sub.3) (ppm)=0.93 (s, 3H), 0.97 (d, J=9.6 Hz, 1H), 1.07 (d, J=7.2 Hz, 3H), 1.21 (s, 3H), 1.48.1.67 (m, 3H), 1.76-1.85 (m, 2H), 1.91-1.97 (m, 1H), 2.28-2.40 (m, 2H), 2.81-2.89 (m, 1H), 3.68 (d, J=13.4 Hz, 1H), 3.81 (d, J=13.4 Hz, 1H), 7.12 (d, J=8.1 Hz, 2H), 7.65 (d, J=2.2 Hz, 2H).

(30) .sup.13C-NMR (100 MHz, CDCl.sub.3) (ppm)=21.5, 23.4, 27.8, 33.8, 36.6, 38.5, 41.7, 45.1, 47.8, 51.2, 55.9, 92.1, 130.2, 137.3, 140.4.

(31) MS (ESI) [M+H].sup.+=370.66

(32) LC/MS (X-bridge 1004.6 mm) Gradient A: t.sub.R=19.12 min Gradient D: t.sub.R=13.57 min

(33) I.R. (neat, cm.sup.1) 2900, 1482, 1006, 799

(34) II. Biological Activity against Chlamydiales Infections

(35) II.1. Materials and Methods

(36) Cell Lines and Bacteria

(37) HeLa229 (ATCC CCL-2.1) were grown in RPMI1640 medium (Glutamax, 10% FBS, w/o HEPES) (Invitrogen). Stable HeLa229 cell lines were established to constantly label the Golgi apparatus (B4GalT1 in a pCMV6-AC-mRFP cloning vector, OriGene) and the ER (KDEL in a pDsRed2-ER expression vector).

(38) Simkania negevensis (Sn) strain Z (ATCC VR-1471) was prepared as described previously (Mehlitz A, Karunakaran K, Herweg J A, Krohne G, van de Linde S, Rieck E, Sauer M, Rudel T. The chlamydial organism Sn forms ER vacuole contact sites and inhibits ER-stress. Cell Microbiol 2014; 16(8):1224-1243).

(39) Briefly, HeLa229 cells were grown to 50-70% confluence, were inoculated with Sn in RPMI1640 with 5% FBS, for 6 h at 35 C. in a humidified incubator at 5% CO.sub.2. Medium was replaced by infection medium (RPMI1640, Glutamax, 5% FBS, w/o HEPES) and growth was allowed for 3 days. Cells were mechanically detached and bacteria were released using 2-5 mm glass beads (Carl Roth). Low speed supernatant (600g, 4 C. and 5 min) was subjected to high-speed centrifugation (20,000g, 4 C. and 30 min) to pellet bacteria. Bacteria were washed twice with 5 ml SPG (250 mM sucrose, 50 mM sodium phosphate, 5 mM glutamate, pH 7.4), aliquoted and stored at '80 C. in SPG.

(40) Chlamydia trachomatis (Ctr). Laboratory-adapted strain L2/434/Bu (ATCC VR902B) was used in assays. Full biological and genetic information is available for this strain including complete genome sequence and defined proteome. This strain has a relatively low particle to infectivity ratio, perform efficient cell infection and has a higher viability than standard genital tract isolates with faster developmental cycle. Culture conditions have been described in (Wang Y., Kahane S., Cutcliffe L. T., Skilton R. J., Lambden P. R., Clarke I. N. Development of a transformation system for Chlamydia trachomatis: Restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathogen, 2011, 7(9):e1002258. doi: 10.1371/journal.ppat.1002258).

(41) Sn Infectivity Assays in Presence of Compound According to the Present Invention

(42) 40,000 HeLa cells were seeded in 12-well cluster plates, inhibitor-treated and infected as indicated in the respective experiment.

(43) For infectivity assays cells were either fixed and stained at indicated time points (FIGS. 2,4; inclusion formation/primary infection) or bacteria were released via one freeze thaw cycle (70 C./37 C.) followed by mechanical release through pipetting and transfer to fresh HeLa229 cells (1:25-1:50, progeny/infectivity). Cells were centrifuged for 1 h at 35 C. and medium exchanged to infection medium. Progeny was fixed at day 3 post infection and processed for staining (FIG. 3) or harvested for immunoblotting (FIG. 1). Infectivity assays were imaged on an automated fluorescence microscope Leica DMIR (FIG. 4). Numbers and average sizes of the SCV as well as host cell numbers were determined via GroEL and DAPI staining and images were analysed and quantified using FIJI (ImageJ) and Excel (Microsoft).

(44) In this progeny assay, bacteria are first grown in Hela299 cells treated with inhibitors (compound 20 at a concentration of 50 and 75 M) and the infectious particles from this primary infection are applied to fresh cells in the absence of inhibitor to measure the bacterial load (GroEL immunoblot) and inclusion formation (immunofluorescence microscopy).

(45) Chlamydia trachomatis (Ctr) Infectivity Assays in Presence of Compound According to the Present Invention

(46) Compound application during Ctr infection. HeLa229 cells were pretreated with compound 20 in concentrations of 25, 50 and 75 M for 30 min until Ctr (MOI1) were added to the cells. Compound 20 was present during infection.

(47) Cells with primary infection were lysed 24 h post infection (hpi). To obtain progeny infection compound treated cells were lysed 48 hpi and lysate was used to infect fresh HeLa229 cells. Progeny infection was lysed 24 hpi and analyzed together with primary infection samples by immunoblot. Chlamydial growth was detected with antibodies against chlamydial HSP60 protein and Actin was used as loading control (FIG. 6).

(48) II2. Results

(49) Infection by Sn

(50) Tested inhibitor of endolysosomal transport Compound 20 has had an inhibitor effect on primary and progeny infection for Sn (FIGS. 1, 2, 3, 4 and 5). This was shown by western detection of Sn GroEL (FIG. 1), relative Sn inclusion sizes in primary infection (FIG. 2), relative Sn inclusion number in progeny infection (FIG. 3), fluorescence microscopy (FIG. 4) and transmission electron microscopy (TEM; FIG. 5).

(51) The maximal inhibition of Sn replication was observed at a concentration of 75 M (FIGS. 1, 2, 3 and 4).

(52) Sn inclusions formed normally in DMSO-treated control cells. Infected cells treated with Compound 20 contained dramatically smaller and less sub-vacuoles and just few bacteria (FIG. 5).

(53) In summary, Compound 20 inhibits primary and progeny infection for Sn.

(54) Infection by Ctr

(55) Experiments performed to test the anti-chlamydial activity of Compound 20 demonstrated a slight inhibitory effect of this compound on chlamydial development (FIG. 6). This was shown by western detection of Sn Hsp60 in infected cells.

(56) However, this treatment of the primary Ctr infection also resulted in consequences on the progeny infection (FIG. 6); Compound 20 at 50 M strongly reduced amounts of Ctr in the progeny. At 75 M, Compound 20 totally blocked progeny infection.

(57) These results highlight the utility of Compound 20 as an anti-chlamydial compounds.

(58) III. Biological Activity Against Chlamydiales Infections

(59) III.1. Materials and Methods

(60) Cell Lines and Bacteria

(61) HeLa229 (ATCC CCL-2.1). Cells were grown in RPMI1640 medium (Glutamax, 10% FBS, w/o HEPES) (Invitrogen).

(62) Chlamydia trachomatis (Ctr). Laboratory-adapted strain L2/434/Bu (ATCC VR902B) was used in assays. Full biological and genetic information is available for this strain including complete genome sequence and defined proteome. This strain has a relatively low particle to infectivity ratio, perform efficient cell infection and has a higher viability than standard genital tract isolates with faster developmental cycle. Culture conditions have been described in (Wang Y., Kahane S., Cutcliffe L. T., Skilton R. J., Lambden P. R., Clarke I. N. Development of a transformation system for Chlamydia trachomatis: Restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathogen, 2011, 7(9):e1002258. doi: 10.1371/journal.ppat.1002258).

(63) Chlamydia trachomatis (Ctr) Infectivity Assays in Presence of Compounds According to the Present Invention

(64) Compound application during Chlamydia trachomatis infection. HeLa229 cells were pretreated with compound #20 and its derivatives in concentrations of 25 M and 75 M for 1 hour until Chlamydia trachomatis (MOI1) were added to the cells. Compounds were present during infection. To obtain progeny infection, compound treated cells were lysed 24 h post infection (hpi) and lysate was used to infect fresh HeLa 229 cells. Infected cells of progeny infection were fixed with 4% Paraformaldehyde 48 hpi. Cells were stained for DAPI and Chlamydia trachomatis were detected by GFP-signal. Images are representative of n=2 independent experiments. Quantification of infected cells by Chlamydia trachomatis was realized from microscopy images with Image J software (FIG. 7) and cellular protection by compounds at 25 M and 75 M was then determined (Table 1) by comparison with solvent-treated cells (control) with the following equation:

(65) $\mathrm{Cellular}\mathrm{protection}=100-\frac{\%\mathrm{of}\mathrm{infected}\mathrm{cells}\mathrm{in}\mathrm{presence}\mathrm{of}\mathrm{inhibitor}}{\%\mathrm{of}\mathrm{infected}\mathrm{cells}\mathrm{in}\mathrm{control}}100$
III.2. Results

(66) TABLE-US-00002 TABLE 1 Cellular protection at 75 M Cellular protection at 25 M (%) (%) #20 75.7 19.9 RN-2-103 100 66.9 m3 87.8 46.9 m4 81.6 22.8 m9 100 100 VP332 100 100 VP386 100 100 VP390 100 91.8

(67) This treatment of the primary Ctr infection with #20 and #20 derivatives resulted in a strong diminution of the progeny infection at 75 M (FIG. 7 and Table 1) and 25 M (Table 1) with a full protection for compounds RN-2-103, m9, VP332, VP386 and VP390 at 75 M and m9, VP332 and VP386 at 25 M.

CONCLUSION

(68) These results highlight the utility of #20 derivatives as anti-chlamydial compounds.