Corroles for treating poxvirus infection

Abstract

Disclosed is a family of corroles for their use in the treatment of an infection by poxvirus.

Claims

1. A method for treatment of an infection of a poxvirus in human or non-human animals, comprising providing a corrole of type A3 or A2B of formula (I): ##STR00017## wherein: Y.sub.1 and Y.sub.2 are identical or different and each independently chosen from, —H, —SO.sub.3H, —SO.sub.3.sup.−, —NO.sub.2, —CHO, —NH.sub.2, —NH.sub.3.sup.+, —COOH, —COO.sup.− M represents 3H or a metal chosen from the group of Cu, Mn, Fe, Co, V, Cr, Ti, Ag, Rh, Ru, Mo, Zr, Au, Pt, Ir, Re, W, Hf, Li, Al, Ga, Ge, Sn, As, Sb, Pb, Bi, La, Gd, Tb, or Th A.sub.1 and A.sub.2 are identical or different and each represents a phenyl group of formula (II), ##STR00018## or A pyridinium group of formula (III) ##STR00019## or a five membered heterocycle of formula (IV) ##STR00020## with each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 of formula (II); each of R.sub.1′, R.sub.2′, R.sub.4′ and R.sub.5′ of formula (III); each of R.sub.1″, R.sub.2″, and R.sub.3″ of formula (IV) being chosen from: (a). —H, —CN, —NO.sub.2, —CHO, —SO.sub.3H, —OH, —SH, —C≡CH, —NH.sub.2, —COOH, —CONH.sub.2, (b). a halogen atom, selected from a chlorine atom, a fluorine atom, a bromine atom, or an iodine atom, (c). a (C.sub.1-C.sub.8) alkyl chain, (d). a PEG chain of formula —(CH.sub.2—CH.sub.2—O).sub.n—H or of formula —(CH.sub.2—CH.sub.2—O).sub.n—CH.sub.3, wherein n is an integer chosen from 1, 2, 3, 4 or 5, (e). —CX.sub.3, X being a halogen atom selected from a chlorine atom, a fluorine atom, a bromine atom, or an iodine atom, (f). —OR.sub.a, —NR.sub.aR.sub.b, —NHR.sub.a, —COOR.sub.a, —CONHR.sub.a, —CONR.sub.aR.sub.b, —SO.sub.2R.sub.a, —COR.sub.a, —SR.sub.a, —C≡CR.sub.a, R.sub.a and R.sub.b being independently chosen from —(CH.sub.2—CH.sub.2—SO.sub.3H), a (C.sub.1-C.sub.8) alkyl chain, a PEG chain of formula —(CH.sub.2—CH.sub.2—O).sub.n—H, or of formula —(CH.sub.2—CH.sub.2—O).sub.n—CH.sub.3, wherein n is an integer chosen from 1, 2, 3, 4 or 5 (g). a group of formula ##STR00021## wherein R.sub.c is —COOH, or —SO.sub.3H, (h). a pyridinium group of formula ##STR00022## wherein R.sub.d is a (C.sub.1-C.sub.8) alkyl chain, or —(CH.sub.2).sub.nSO.sub.3.sup.−, n being an integer chosen from 1, 2, 3, 4 or 5, R.sub.3′ of formula (III) being chosen from —H, —CONH.sub.2, a (C.sub.1-C.sub.8)alkyl a PEG chain of formula —(CH.sub.2—CH.sub.2—O).sub.n—H or of formula —(CH.sub.2—CH.sub.2—O).sub.n—CH.sub.3, wherein n is an integer chosen from 1, 2, 3, 4 or 5, a group —(CH.sub.2).sub.nSO.sub.3.sup.−, n being an integer chosen from 1, 2, 3, 4 or 5 —OR.sub.a, —NR.sub.aR.sub.b, —NHR.sub.a, —COOR.sub.a, —CONHR.sub.a, —CONR.sub.aR.sub.b, —COR.sub.a, R.sub.a and R.sub.b being independently chosen from —(CH.sub.2—CH.sub.2—SO.sub.3H), a (C.sub.1-C.sub.8) alkyl chain, a PEG chain of formula —(CH.sub.2—CH.sub.2—O).sub.n—H, or of formula —(CH.sub.2—CH.sub.2—O).sub.n—CH.sub.3 wherein n is an integer chosen from 1, 2, 3, 4 or 5, E of formula (IV) is chosen from —O—, —S—, —Se—, —NH—, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, and administering an effective amount of the corrole to the human or non-human animals.

2. The method of claim 1, wherein the poxvirus is belonging to the subfamily Chordopoxvirinae or subfamily Entomopoxvirinae.

3. The method of claim 1, for wherein the infection of a poxvirus is myxomatosis, cowpox, smallpox, sheeppox, orf, vaccinia, monkeypox, LSDV, goatpox or an infection by any other members of the poxvirus family.

4. The method of claim 1, wherein the corrole is of formula (Ia) ##STR00023## Wherein Y.sub.1, Y.sub.2, A.sub.1 and A.sub.2 are defined as in formula (I).

5. The method of claim 4, wherein A1 and/or A2 are represented by the formula (IIa), formula (IIb), formula (IIc), formula (IId), formula (IIIa), formula (IIIb), or formula (IIIc) ##STR00024## wherein R′, R″, R.sub.1, R.sub.3 and R.sub.5 are independently chosen from: —CN, —NO.sub.2, —CHO, —SO.sub.3H, —OH, —NH.sub.2, —COOH, —CONH.sub.2, —SH, —C≡CH, a halogen atom, selected from a chlorine atom, a fluorine atom, a bromine atom, or an iodine atom, a (C.sub.1-C.sub.8) alkyl chain, a PEG chain of formula —(CH.sub.2—CH.sub.2—O).sub.n—H or of formula —(CH.sub.2—CH.sub.2—O).sub.n—CH.sub.3, wherein n is an integer chosen from 1, 2, 3, 4 or 5, —CX.sub.3, X being a halogen selected from a chlorine atom, a fluorine atom, a bromine atom, or an iodine atom, —OR.sub.a, —NR.sub.aR.sub.b, —COOR.sub.a, —NHR.sub.a, —CONR.sub.aR.sub.b, —CONHR.sub.a, —SO.sub.3R.sub.a, —SO.sub.2NHR.sub.a, —COR.sub.a, —SR.sub.a, —C≡CR.sub.a, R.sub.a and R.sub.b being independently chosen from —(CH.sub.2—CH.sub.2—SO.sub.3H), a (C.sub.1-C.sub.8) alkyl chain, a PEG chain of formula —(CH.sub.2—CH.sub.2—O).sub.n—H or of formula —(CH.sub.2—CH.sub.2—O).sub.n—CH.sub.3, wherein n is an integer chosen from 1, 2, 3, 4 or 5, a group of formula ##STR00025## wherein R.sub.c is —COOH, or —SO.sub.3H, a pyridinium group of formula ##STR00026## wherein R.sub.d is a (C.sub.1-C.sub.8) alkyl chain, or —(CH.sub.2).sub.nSO.sub.3.sup.−, n being an integer chosen from 1, 2, 3, 4 or 5.

6. The method of claim 5, wherein A.sub.1 and A.sub.2 are different and respectively represented by one formula chosen from the formula (IIa), (IIb), (IIc), (IId), (IIIa), (IIIb), (IIIc), (IIId) or (IVa).

7. The method of claim 5, wherein A.sub.1 and A.sub.2 are identical and represented by one formula chosen from the formula (IIa), (IIb), (IIc), (IId), (IIIa), (IIIb), (IIIc), (IIId) or (IVa).

8. The method of claim 1, wherein the corrole is chosen from following compounds ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

9. The method of claim 2, wherein the corrole is of formula (Ia) ##STR00032## Wherein Y.sub.1, Y.sub.2, A.sub.1 and A.sub.2 are defined as in formula (I).

10. The method of claim 3, wherein the corrole is of formula (Ia) ##STR00033## Wherein Y.sub.1, Y.sub.2, A.sub.1 and A.sub.2 are defined as in formula (I).

11. The method of claim 2, wherein the corrole is chosen from following compounds ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

12. The method of claim 3, wherein the corrole is chosen from following compounds ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

13. The method of claim 4, wherein the corrole is chosen from following compounds ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

14. The method of claim 5, wherein the corrole is chosen from following compounds ##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053## or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

15. The method of claim 6, wherein the corrole is chosen from following compounds ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058## or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the toxicity of compounds FNO2Cor, TpNO2Cor, TPFCor at different concentrations (0.1, 0.25, 0.5, 0.75, 1.5, 3.1, 6.25, 12.5, 25, 50 μM) on RK13 cells (rabbit epithelial kidney) seeded on a plate of 96 well. The Y axe shows normalized numbers of survived cells per well.

(2) FIG. 2 shows normalized infection rate of MYXV in RK13 cells at multiplicity of infection (MOI) 0.5 after 48 h post-infection by compounds FNO2Cor, TpNO2Cor, or TPFCor, at different concentrations (0.08, 0.18, 0.37, 0.75, 1.5, 3.1, 6.25 μM).

(3) FIG. 3 shows normalized infection rate of MYXV in RK13 cells at multiplicity of infection (MOI) 0.01 after 6 days post-infection by compounds FNO2Cor, TpNO2Cor, or TPFCor, at different concentrations (0.08, 0.18, 0.37, 0.75, 1.5, 3.1, 6.25 μM).

(4) FIG. 4 shows lysis plaques in RK13 infected with MYXV 6d PI observed by microscope.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Examples

(5) 1. Materials and Methods

(6) 1.1 Preparation of Corroles of the Invention

(7) All the chemical and solvents were of analytical grade and used without any further purification. Silica gel 60 (70-230 and 230-400 mesh, Sigma Aldrich) were used for column chromatography. Reactions were monitored by thin layer chromatography, UV-Vis spectroscopy and mass spectrophotometry. Chromatographic purification on column was performed on silica gel 60 (70-230 mesh, Sigma Aldrich. .sup.1H NMR spectra were recorded on a Bruker AV300 spectrometer (300 MHz). CDCl.sub.3 was used as solvent (except when indicated) and TMS as internal reference; the chemical shifts (6) are given in ppm relative to residual CHCl.sub.3 (7.26 ppm). All data were processed with TopSpin. MALDI/TOF mass spectra were recorded on Bruker Ultraflex Extreme MALDI Tandem TOF Mass Spectrometer. UV-vis spectra were measured on a Cary 50 spectrophotometer using CH.sub.2Cl.sub.2, CHCl.sub.3 or THF as solvent.

(8) General procedure #1 according to a modified Paolesse's method (Paolesse et al., J. Org. Chem. 2001, 66 (2), 550-556.).

(9) Aldehyde (40.4 mmol) and distilled pyrrole (121 mmol) were dissolved in AcOH (500 mL) and the reaction was stirred at reflux for 3 h. The reaction mixture was cooled at room temperature and AcOH was evaporated under vacuum. The crude product was filtered over a chromatography column (silica, CH.sub.2Cl.sub.2). All fractions containing corrole (green fraction) were combined and evaporated to dryness. Purification details for each compound are described below.

Preparation of 5,10,15-Tris(4-nitrophenyl)corrole (Designed as TpNO2Cor)

(10) This corrole corresponds to the above described compound H.

(11) This corrole was prepared as described for general procedure 1 starting from 4-nitrobenzaldehyde and pyrrole. The residue was purified by chromatography column (alumina, CH.sub.2Cl.sub.2/heptane, 1/1, v/v) to give pure corrole (492 mg, 5.5%). UV-Vis (DCM): λ.sub.max (nm) (ε×10.sup.−3 L mol.sup.−1 cm.sup.−1)=447 (53.4), 598 (18.7). .sup.1H NMR (300 MHz, 300 K, DMSO-d6) δ (ppm): 8.41 (m, 2H), 8.58-8.71 (m, 14H), 8.87 (m, 2H), 9.14 (m, 2H). MS (MALDI-TOF) m/z=661.92 [M+H].sup.+, 661.17 calcd for C.sub.37H.sub.23N.sub.7O.sub.5. MS (ESI) m/z=660.15 [M−H].sup.−, 662.14 [M+H].sup.+, 661.17 calcd for C.sub.37H.sub.23N.sub.7O.sub.5.

Preparation of 5,10,15-Tris(2-fluoro-5-nitrophenyl)corrole (Designed as FNO2Cor)

(12) This corrole corresponds to the above described compound B.

(13) This corrole was prepared as described for general procedure 1 starting from 2-fluoro-5-nitrobenzaldehyde and pyrrole. The residue was crystallized from CHCl.sub.3/Heptane (3/1, v/v), separating the solution, containing corrole, from the porphyrin precipitate. Solvent was removed under vacuum and the crude was crystallized from THF/heptane 1:2 v/v to give pure dark green corrole crystals (76.1 mg, 3.2% yield). UV-Vis (THF): λ.sub.max, (nm) (ε×10.sup.−3 L mol.sup.−1 cm.sup.−1) 418 (103.2), 572, 610, 645. .sup.1H NMR (300 MHz, CDCl.sub.3), δ (ppm): 9.11-9.00 (m, 5H), 8.73-8.61 (m, 5H), 8.50-8.47 (m, 4H), 7.71-7.62 (m, 3H), −2.83 (brs, 3H). MS (MALDI/TOF): m/z 715.007 [M].sup.+; 715.14 calcd for C.sub.37H.sub.20F.sub.3N.sub.7O.sub.6.

(14) General procedure #2 according to a modified Gryko's method (Gryko, D. T.; Koszarna, B. Org. Biomol. Chem., 2003, 1(2), 350-357).

(15) Aldehyde (20.0 mmol) was dissolved in distilled pyrrole (30.0 mmol) at room temperature and then a solution of TFA in CH.sub.2Cl.sub.2 (18 μL in 2.0 mL) was added and vigorously stirred. After 10 min, 800 mL of CH.sub.2Cl.sub.2 was added and stirred for further 1 h. DDQ (24.0 mmol) was added and stirred for another 1 h and solvent was removed under vacuum. The crude product was filtered over a chromatography column (silica, CH.sub.2Cl.sub.2). Purification details for each compound are described below.

Preparation of 5,10,15-Tris(pentafluorophenyl)corrole (Designed as TPFCor)

(16) This corrole correspond to the above described compound of formula A.

(17) This corrole was prepared as described for general procedure 2 starting from pentafluorobenzaldehyde and pyrrole. The residue was purified by a second column chromatography (silica, toluene/heptane, 8/2, v/v) to give pure dark green corrole crystals (228 mg, 4.3% yield). UV-Vis (THF): λ.sub.max, (nm) (ε×10.sup.−3 L mol.sup.−1 cm.sup.−1) 407 (150.1), 562 (23.9), 604 (12.4). .sup.1H NMR (300 MHz, CDCl.sub.3), δ (ppm): 9.10 (d, J=4.2 Hz, 2H), 8.79 (d, J=4.8 Hz, 2H), 8.60 (4H), 2.88 (brs, 3H). .sup.19F NMR (282 MHz, CDCl.sub.3) 137.2 (2F), 137.7 (4F), 152.2 (2F), 152.8 (1F), 161.5 (4F), 161.9 (2F). MS (MALDI/TOF): m/z 796.91 [M+H].sup.+, 796.07 calcd for C.sub.37H.sub.11F.sub.15N.sub.4.

(18) 1.2 MYXV Infection

(19) RK13 (rabbit epitelial kidney) cells were used for MYXV infection assay.

(20) Cells were grown in 200 μL Dulbecco's Modified Eagle's medium (DMEM) without phenol red (Sigma-Aldrich), 10% SVF, Pen-strep, 1× sodium pyruvate, 1× Glutamax.

(21) Infection Protocol

(22) MYXV-GFP infection was carried out on RK13 cells seeded in 96 wells plate, in triplicate at two multiplicity of infection (MOI) and 7 concentrations of FNO2Cor, TPFCor, TpNO2Cor. D0: RK13 cells seeded at 6 k per well in Corning Glass Bottom 96 well plates in 200 μL of DMEM without phenol red. D1: cells are treated with FNO2Cor, TpNO2Cor, TPFCor (from 50 to 0.1 μM by two fold dilution) in duplicate for the toxicity study.

(23) In parallel: cells are treated with FNO2Cor, TpNO2Cor, TPFCor (from 6.25 to 0.08 μM by two fold dilution) and are infected at different multiplicity of infection (MOI) in triplicate: MYXV at MOI 0.5 or 0.01.

(24) 1.3 Toxicity Assessment

(25) The toxicity of compounds FNO2Cor, TpNO2Cor or TPFCor on ARPE-19 cells was evaluated after 6 days of treatment. 10 concentrations (50, 25, 12.5, 6.25, 3.1, 1.5, 0.75, 0.5, 0.25, 0.1 μM) of FNO2Cor, TpNO2Cor and TPFCor were studied.

(26) 1.4 Microscopy

(27) RK13 cells at MOI 0.5 48 h post-infection (PI) are fixed with Formalin 10 min at room temperature. Wash with 200 μL PBS and 100 μL PBS/Hoechst 33342 (1/1000) per well. 96 well plates are kept at 4° C. in the dark until data acquisition. Image acquisition and analysis for high content quantification is performed on a Thermo Cellomics Arrayscan VTI microscope using a modified compartmental analysis algorithm.

(28) RK13 cells for the toxicity study and RK13 cells at MOI 0.01 6 days PI are fixed and image acquisition and analysis are performed as previously described.

(29) On 48 h and 6d post-infection, data acquisition was done by high content microscopy to calculate infection level according to compound concentration.

(30) 2. Results

(31) 2.1 Cytotoxicity

(32) Cytotoxicity of compounds FNO2Cor, TPFCor and TpNO2Cor were assessed at different concentration on RK13 cells according to the method described on section 1.3. CC.sub.50 values of these three compounds on RK13 cells are given in table I below.

(33) TABLE-US-00001 TABLE I FNO2Cor TpNO2Cor TPFCor CC.sub.50 RK13 3.74 4.36 6.68

(34) FIG. 1 and Table I show that three tested compounds are safe for rabbit epithelial kidney cells at low concentrations.

(35) 2.2 Antiviral Activity

(36) Compounds FNO2Cor, TPFCor, and TpNO2Cor were assessed at 7 different concentrations in RK13 cell culture on 48 h post-infection (FIG. 2) or on 6 days post-infection (FIG. 3) to evaluate their activity for inhibiting MYXV infection. Table II displays IC.sub.50 values of these three compounds on RK13 cells on 48 h or 6d PI. The selectivity index of these compounds are displayed in table III.

(37) TABLE-US-00002 TABLE II FNO2Cor TpNO2Cor TPFCor IC50 1.33 0.43 0.27 (48 h PI) IC50 0.50 0.20 0.12 (6 d PI)

(38) TABLE-US-00003 TABLE III FNO2Cor TpNO2Cor TPFCor SI (48 h PI) 2.81 10.25 24.40 SI (6 d PI) 7.52 21.45 55.31

(39) These results indicate that all three tested compounds can inhibit MYXV infection rate in RK13 cells. TPFCor and TpNO2Cor have better in vitro anti-MYXV effect than FNO2Cor.

(40) 2.3 Lysis Plaques Analysis

(41) Lysis plaque analysis was carried out in RK13 infected with MYXV 6d PI. Lysis plaques area and number of lysis plaques were assessed directly on microscope pictures (FIG. 4). The results are also displayed in table IV.

(42) TABLE-US-00004 TABLE IV Area in cm.sup.2 of lysis plaques Number of lysis plaques Concen- 0 3.1 1.5 0.75 0.37 0 3.1 1.5 0.75 0.37 tration μM Not treated 7.6 19 FNO2Cor nd nd 3.4 8 0 1.3 6 5 TpNO2Cor nd nd 3.4 9 0 2.7 6.7 4 TPFCor 6 4 3.3 6 6.7

(43) These results show that three tested compounds can prevent cell to cell propagation of MYXV infection.