Benzodiazepine derivatives for use in the treatment of Chlamydiales infections

Abstract

The invention relates to a method for treating a Chlamidyales infection comprising the administration of a therapeutically effective amount of a compound of formula (I) to a subject in need thereof: ##STR00001##
Wherein R.sub.1, R.sub.2, R.sub.3 and Ar are as defined in claim 1.

Claims

1. A pharmaceutical composition comprising a compound of formula (A): ##STR00022## wherein: p is 1; is a single bond C—N; R.sub.1 is selected from F, Cl, Br and I; R.sub.2 is C.sub.3-C.sub.8 cycloalkyl or (5- to 10-membered)heteroaryl(C.sub.1-C.sub.6)alkyl, wherein said cycloalkyl group is optionally substituted by one to three R.sub.4; Ar is selected from C.sub.6-C.sub.10 aryl, C.sub.5-C.sub.10 heteroaryl and (5- to 10-membered)heteroaryl(C.sub.1-C.sub.6)alkyl, said aryl or heteroaryl groups being optionally substituted by one to three R.sub.5; R.sub.4 is, at each occurrence, independently selected from Cl, Br, I, F and C.sub.1-C.sub.6 alkyl; and R.sub.5 is C.sub.1-C.sub.6 alkyl, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof, in admixture with one or more pharmaceutically acceptable excipients.

2. The pharmaceutical composition of claim 1, wherein Ar is unsubstituted.

3. The pharmaceutical composition of claim 1, wherein R.sub.1 is Br and/or is located at position 7.

4. The pharmaceutical composition of claim 1, wherein the compound of formula (A) is selected from the group consisting of: 7-bromo-4-cyclopentyl-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (HA229); 7-bromo-4-(3-methylcyclopentyl)-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (HA280); and 4-((1H-imidazol-2-yl)methyl)-7-bromo-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (VP196).

5. A compound of formula (A) as defined in claim 1, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof.

6. The pharmaceutical composition of claim 1, wherein R.sub.2 is C.sub.3-C.sub.8 cycloalkyl.

7. The pharmaceutical composition of claim 6, wherein Ar is phenyl.

8. The pharmaceutical composition of claim 1, wherein R.sub.2 is (5- to 10-membered)heteroaryl(C.sub.1-C.sub.6)alkyl.

9. The pharmaceutical composition of claim 8, wherein Ar is phenyl.

10. A compound of formula (A) as defined in claim 6, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof.

11. A compound of formula (A) as defined in claim 7, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof.

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

13. A compound of formula (A) as defined in claim 9, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof.

14. A method for treating a Chlamidyales infection comprising the administration of a therapeutically effective amount of a compound of formula (I) to a subject in need thereof, ##STR00023## wherein: R.sub.1 is selected from F, Cl, Br and I; R.sub.2 is C.sub.3-C.sub.8 cycloalkyl or (5- to 10-membered)heteroaryl(C.sub.1-C.sub.6)alkyl, wherein said cycloalkyl group is optionally substituted by one to three R.sub.4; R.sub.3 is H; Ar is selected from C.sub.6-C.sub.10 aryl, C.sub.5-C.sub.10 heteroaryl and (5- to 10-membered)heteroaryl(C.sub.1-C.sub.6)alkyl, said aryl and heteroaryl groups being optionally substituted by one to three R.sub.5; R.sub.4 is, at each occurrence, independently selected from Cl, Br, I, F and C.sub.1-C.sub.6 alkyl; R.sub.5 is C.sub.1-C.sub.6 alkyl; is a single bond C—N; and p is 1, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts thereof.

15. The method of claim 14, wherein the Chlamidyales infection is a Chlamidya or Simkania infection.

16. The method of claim 14, wherein at least one of R.sub.1 is Br or Cl, and/or is located at position 7.

17. The method of claim 14, wherein Ar is phenyl or pyridyl.

18. The method of claim 14, wherein R.sub.3 is H.

19. The method of claim 14, wherein the compound of formula (I) is selected from the group consisting of: 7-bromo-4-cyclopentyl-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (HA229); 7-bromo-4-(3-methylcyclopentyl)-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (HA 280); and 4-((1H-imidazol-2-yl)methyl)-7-bromo-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (VP 196).

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effect of Retro-1 and VP-184 on the Sn bacterial load of infected HeLa cells by bacterial GroEL immunoblot and Actin was used as loading control.

(2) FIG. 2 shows the effect of Retro-1 and VP-184 on the inclusion size during primary infection of HeLa cells.

(3) FIG. 3 shows the effect of Retro-1 and VP-184 on the number of inclusions during progeny infection of HeLa cells.

(4) FIG. 4 shows pictures illustrating effect of Retro-1 and VP-184 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 Retro-1 or of VP-184.

(6) FIG. 6 shows immunoblot analysis of lysed HeLa cells after Ctr primary and progeny infection in presence of Retro-1; 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 Retro-1 and Retro-1 derivatives application at 75 μM during Chlamydia trachomatis infection of HeLa229 cells.

EXAMPLES

I. Synthesis of Compounds of Formula (I)

(8) Compounds of formula (I) are prepared according to the following general method:

(9) ##STR00016##

7-bromo-5-(2-bromophenyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one

(10) ##STR00017##

(11) To a solution of 2-amino-5-bromobenzophenone (1.014 mmol, 200 mg) in dichloromethane (10 mL) was added N-bromosuccinimide (1.014 mmol, 189 mg) at 0° C. The mixture was stirred for 1 hour at this temperature and 2 hours at room temperature. The organic layer was washed with water (20 mL), dried over Na.sub.2SO.sub.4, filtrated and concentrated under vacuum. The crude mixture was used in the next step without purification.

(12) To a solution of 3,5-dibromo-2-aminobenzophenone (1.014 mmol) in dichloromethane (100 mL) was added bromoacetyl bromide (1.216 mmol, 106 μL) followed by a 2M aqueous solution of Na.sub.2CO.sub.3 (1.521 mmol, 760 μL) at 0° C. The mixture was stirred 2 hours at this temperature. The organic layer was separated and washed with water, dried over Na.sub.2SO.sub.4, filtrated and concentrated under vacuum to give 3,5-bromo-2-bromoacetamidebenzophenone as a brown solid. At 0° C., 3,5-bromo-2-bromoacetamidebenzophenone (1.014 mmol) was dissolved in a solution of NH.sub.3 (7M in MeOH, 13 mL) and the mixture was stirred 1 hour at this temperature than allowed to warm up to room temperature overnight. The crude mixture was dried under vacuum, diluted in ethyl acetate than washed with water. The organic layer was concentrated under vacuum, dried over Na.sub.2SO.sub.4, filtrated and concentrated under vacuum. The crude mixture was purified by flash Chromatography (cyclohexane-ethyl acetate, 5-1 to 1-1). The desired compound was obtained as a yellowish solid (120 mg, 30% over 2 steps).

(13) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ (ppm)=3.89 (m, 1H), 4.49 (m, 2H), 7.35-7.40 (d, J=2.2 Hz, 1H), 7.41-7.57 (m, 5H), 8.17-8.22 (d, J=2.2 Hz, 1H), 9.98 (s, 1H)

(14) .sup.13C-NMR (150 MHz, CDCl.sub.3) δ (ppm)=26.3, 56.7, 115.5, 117.8, 128.4, 129.1, 130.6, 136.6, 137.0, 138.1, 167.5, 169.3

(15) I.R. (neat, cm.sup.−1) 3367, 3204, 3073, 1688, 1607, 1579, 1461, 1446, 1379, 1317, 1231, 1175, 1151, 1011, 858, 736

(16) HRMS m/z [(M+H).sup.+] calcd for C.sub.15H.sub.11Br.sub.2N.sub.2O, 329.9238 found 329.9233.

7-bromo-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one

(17) ##STR00018##

(18) To a solution of 7-bromo-5-(2-bromophenyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (1.58 mmol, 500 mg) in methanol (15 mL), was added NaBH.sub.3CN (2.37 mmol, 150 mg) followed by acetic acid (7.9 mmol, 440 μL) dropwise. The mixture was stirred at room temperature until complete conversion of the starting material. The mixture was then evaporated to dryness, diluted in ethyl acetate and washed with a saturated solution of NaHCO.sub.3, then water. The organic layer was concentrated under vacuum, dried over Na.sub.2SO.sub.4, filtrated and concentrated under vacuum. The crude mixture was purified by flash chromatography (cyclohexane-ethyl acetate, 1-1 to 1-2). The desired compound was obtained as a white solid (370 mg, 74%).

(19) 1H NMR (400 MHz, (CD.sub.3).sub.2SO) δ ppm: 9.96 (s, 1H), 7.44-7.26 (m, 6H), 7.06 (d, 1-H, J=8.5 Hz, 9-H), 6.83 (d, J=2.14 Hz, 1H), 5.23 (d, 1H), 3.68 (s br, 1H), 3.39 (dd, J=15.7 Hz, J=5.4 Hz, 1H), 3.26 (dd, J=15.7 Hz, J=8.0 Hz, 1H)

(20) 13C NMR (400 MHz, (CD.sub.3).sub.2SO) δ ppm: 173.6, 141.9, 136.6, 128.8, 127.8, 122.9, 115.5, 61.7, 50.8

(21) IR: 3441, 3309, 3258, 3208, 3150, 3095, 3064, 2947, 2825, 1675, 1578, 1482, 1380, 1284, 1248, 1227, 1173, 119, 1076, 1052, 1027, 948, 913, 880, 855

(22) HRMS m/z [(M+H).sup.+] calcd for C.sub.15H.sub.14BrN.sub.2O, 317.0290. found 317.0291

7-bromo-4-cyclopentyl-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (HA229)

(23) ##STR00019##

(24) To a solution of 7-bromo-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (500 mg, 1.58 mmol) in methanol (16 mL, 0.1M) was added NaBH.sub.3CN (300 mg, 4.76 mmol) and acetic acid (880 μL, 15.8 mmol). The solution was stirred at room temperature for 4 h then cyclopentanone (167 μL, 1.89 mmol) was added and the solution was stirred at 60° C. until complete consumption of starting materials. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a saturated solution of NaHCO.sub.3 (5 mL). The residue was concentrated under vacuum and purified by flash chromatography (cyclohexane/ethyl acetate 5:1 to 1:1) affording 390 mg (64%) of compound as a white solid.

(25) .sup.1H-NMR (400 MHz, (CD.sub.3).sub.2SO) δ (ppm)=1.36-1.50 (m, 4H), 1.62-1.64 (m, 2H), 1.77-1.89 (m, 2H), 2.90-2.96 (m, 1H), 5.30 (s, 1H), 7.02-7.15 (t, J=8.4 Hz, 3H), 7.21-7.31 (m, 3H), 7.43-7.48 (dd, J=8.5 Hz, J=2.2 Hz, 1H), 7.48-7.51 (d, J=2.1 Hz, 1H), 9.98 (s, 1H)

(26) .sup.13C-NMR (100 MHz, (CD.sub.3).sub.2SO) δ (ppm)=23.9, 23.1, 53.02, 63.2, 66.5, 115.05, 122.4, 127.0, 127.7, 128.3, 131.0, 132.1, 133.8, 136.8, 142.3, 172.3

(27) I.R. (neat, cm.sup.−1) 3194, 3070, 2993, 2961, 1648, 1579, 1491, 1449, 1421, 1401, 1377, 1357, 1324, 1294, 1253, 1225, 1179, 1131, 980, 870, 812

(28) HRMS m/z [(M+H).sup.+] calcd for C.sub.20H.sub.22BrN.sub.2O, 385.0916 found 385.0932.

7-bromo-4-(3-methylcyclopentyl)-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (as a mixture of diastereoisomers) (HA280)

(29) ##STR00020##

(30) To a solution of 7-bromo-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (1 g, 3.18 mmol) in methanol (25 mL, 0.12 M) was added NaBH.sub.3CN (300 mg, 4.77 mmol), acetic acid (880 μL, 15.8 mmol) and 3-methylcyclopentanone (1.02 mL, 9.54 mmol). The solution was stirred at 60° C. until complete consumption of starting materials. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a saturated solution of NaHCO.sub.3 (5 mL). The residue was concentrated under vacuum and purified by flash chromatography (cyclohexane/ethyl acetate 5:1 to 1:1) affording 1.19 g (94%) of compound as a white solid.

(31) .sup.1H-NMR (400 MHz, CDCl.sub.3) δ (ppm)=0.92-1.10 (m, 3H), 1.18-2.32 (m, 8H), 3.02-3.29 (m, 1H), 3.45-3.58 (m, 2H), 6.95-7.09 (m, 1H), 7.11-7.15 (m, 1H), 7.20-7.29 (m, 4H), 7.34-7.42 (m, 1H), 9.52-9.74 (m, 1H)

(32) .sup.13C-NMR (100 MHz, CDCl.sub.3) δ (ppm)=20.1, 20.9, 21.1, 21.4, 22.0, 22.1, 27.0, 30.5, 31.0, 31.8, 31.9, 32.1, 32.2, 32.3, 32.7, 38.7, 39.3, 40.7, 41.0, 41.4, 48.9, 49.4, 52.4, 52.6, 52.7, 61.6, 61.8, 62.3, 62.4, 67.5, 67.7, 67.8, 67.9, 116.4, 116.5, 122.2, 122.3, 122.4, 127.5, 127.6, 128.4, 128.5, 128.6, 128.7, 131.5, 134.2, 134.3, 135.8, 141.5, 141.6, 141.7, 176.0, 176.2, 176.3

(33) I.R. (neat, cm.sup.−1) 3204, 3083, 2950, 2866, 1657, 1579, 1488, 1449, 1401, 1365, 1224, 976, 907

(34) HRMS m/z [(M+H).sup.+] calcd for C.sub.21H.sub.24BrN.sub.2O, 399.1072 found 399.1071.

7-bromo-5-phenyl-4-propyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (VP184)

(35) ##STR00021##

(36) To a solution of 7-bromo-5-phenyl-1,3,4,5-tetrahydro-2H-benzo[e][1,4]diazepin-2-one (100 mg, 0.315 mmol) in methanol (1.5 mL, 0.21 M) was added NaBH.sub.3CN (29.7 mg, 0.473 mmol), acetic acid (94.6 μL, 1.576 mmol) and propionaldehyde (22 μL, 0.378 mmol). The solution was stirred at room temperature until complete consumption of starting materials. The crude mixture was evaporated, diluted in ethyl acetate (10 mL), and washed with a saturated solution of NaHCO.sub.3 (5 mL). The residue was concentrated under vacuum and purified by flash chromatography (cyclohexane/ethyl acetate 4:1 to 3:2) affording 103 mg (91%) of compound as a white solid.

(37) .sup.1H-NMR (400 MHz, DMSO) δ (ppm)=0.9 (t, J=7.3 Hz, 3H), 1.53-1.66 (m, 2H), 2.58-2.70 (m, 2H), 3.40 (d, J=16.1 Hz, 1H), 3.52 (d, J=16.1 Hz, 1H), 4.97 (s, 1H), 6.95 (d, J=8.5 Hz, 1H), 7.02 (m, 1H), 7.19-7.42 (m, 6H), 9.00 (s, 1H).

(38) .sup.13C-NMR (100 MHz, CDCl.sub.3) δ (ppm)=11.5, 20.8, 36.7, 52.8, 55.3, 68.5, 117.1, 122.0, 127.8, 128.5, 128.6, 131.3, 133.0, 133.8, 136.1, 140.5, 173.9

(39) MS (ESI) [M+H].sup.+=359.1, 361.0

(40) LC/MS (X-bridge 100×4.6 mm): t.sub.R=9.28 min, m/z: 359, 361 ([M+H.sup.+])

(41) Gradient A: t.sub.R=18.30 min

(42) Gradient B: t.sub.R=24.01 min

(43) I.R. (neat, cm.sup.−1) 3202, 3084, 2960, 2932, 2872, 1662, 1486, 1400, 1375, 732, 699

II. Biological Activity Against Chlamydiales Infections

(44) II.1. Materials and Methods

(45) Cell Lines and Bacteria

(46) 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).

(47) 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).

(48) 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 (600×g, 4° C. and 5 min) was subjected to high-speed centrifugation (20,000×g, 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.

(49) 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, performs 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).

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

(51) 40,000 Hela cells were seeded in 12-well cluster plates, inhibitor-treated and infected as indicated in the respective experiment.

(52) For infectivity assays cells were either fixed and stained at indicated time points (FIG. 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).

(53) In progeny assay, bacteria are first grown in Hela299 cells treated with inhibitors, Retro-1 and VP-184 (25 and 75 μM), 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).

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

(55) Compound application during Ctr infection. HeLa229 cells were pretreated with Retro-1 in concentrations of 25, 50 and 75 μM for 30 min until Ctr (MOI1) were added to the cells. Retro-1 was present during infection.

(56) 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).

(57) II.2. Results

(58) Infection by Sn

(59) Tested inhibitors of retrograde transport, Retro-1 and VP-184, have had an inhibitor effect on primary and progeny infection for Simkania (FIGS. 1, 2, 3 and 4). This was shown by western detection of Sn GroEL (FIG. 1), relative inclusion sizes in primary infection (FIG. 2), relative inclusion number in progeny infection (FIG. 3), fluorescence microscopy (FIG. 4) and transmission electron microscopy (TEM; FIG. 5).

(60) The maximal inhibition of Sn replication was observed at a concentration of 75 μM for Retro-1 and 25 μM for VP-184 (FIGS. 1, 2, 3 and 4). Sn inclusions formed normally in DMSO-treated control cells. The inclusions were smaller in Retro-1-treated cells and less sub-inclusions were visible; cellular effects caused by VP-184 strongly reduced Sn inclusion formation, size, numbers and Sn replication (FIG. 5).

(61) In summary, Retro-1 and VP-184 compounds inhibit primary and progeny infection for of HeLa cells by Sn.

(62) Infection by Ctr

(63) Retro-1 in concentrations ranging from 50-75 μM in the primary infections results in significant reduced infectivity in the progeny (FIG. 6). This was shown by western detection of Sn Hsp60 in infected cells.

III. Biological Activity Against Chlamydiales Infections

(64) III.1. Materials and Methods

(65) Cell Lines and Bacteria

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

(67) 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).

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

(69) Compound application during Chlamydia trachomatis infection. HeLa229 cells were pretreated with Retro-1 and Retro-1 derivatives in concentrations of 25 μM and 75 μM 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 μM and 75 μM was then determined (Table 1) by comparison with solvent-treated cells (control) with the following equation:

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

(71) TABLE-US-00002 TABLE 1 Cellular protection Cellular protection at 75 μM (%) at 25 μM (%) Retro-1 2.4 26.5 VP173 100 61.5 VP174 52.8 27.6 HA061 100 57.8 HA197 84.3 90.6 HA200 100 47.0 HA229 — 20.2 HA280 100 100

(72) This treatment of the primary Ctr infection with Retro-1 and Retro-1 derivatives resulted in a diminution of the progeny infection at 75 μM (FIG. 7 and Table 1) and 25 μM (Table 1) with a full protection for compounds VP 173, HA061, HA200 and HA280 at 75 μM and HA280 at 25 μM.

CONCLUSION

(73) These results highlight the utility of Retro-1 derivatives as anti-chlamydial compounds.