DERIVATIVES OF PORPHYRINS, THEIR PROCESS OF PREPARATION AND THEIR USE FOR TREATING VIRAL INFECTIONS

Abstract

The present application provides metallated porphyrin derivatives as ligands of G-quadruplex and their novel use as anti-viral agents. More specifically, this application is related to the use of porphyrin derivatives as G-quadruplex ligands to inhibit viral infections, such as HIV, more particularly to inhibit HIV-1 replication cycle.

Claims

1. A compound of formula (IA): ##STR00023## wherein: in Formula (IA) - - - represents an optionally present single bond; M represents H.sub.2, Cobalt (Co), Manganese (Mn) or a Nickel (Ni) atom ; A.sup.−represents a counter anion; and ##STR00024## wherein - - - represents the attachment of X to the phenyl groups in Formula (IA).

2. The compound according to claim 1 wherein X represents ##STR00025## and M represents a Manganese atom.

3. The compound of formula (IA) according to claim 1, wherein A.sup.−represents a halogen ion.

4. The compound according to claim 1, wherein A.sup.−is Cl.sup.−.

5. A pharmaceutical composition comprising the compound according to claim 1, and at least one pharmaceutically acceptable excipient.

6. A process for the preparation of the compound according to claim 1 comprising reacting a compound of formula (II) ##STR00026## wherein X is defined as in claim 1, and wherein B.sup.−represents a counter anion; with a gold-containing complex.

7. The process according to claim 6 wherein the counter anion is trifluoroacetate.

8. The process according to claim 6 wherein the gold-containing complex is a complex of formula (III):
KAu.sup.IIIA.sub.4  (III) wherein A represents a counter anion.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0082] FIGS. 1 to 4 illustrate effect of the G4 ligands on HIV-1 replication.

[0083] FIG. 1: Effect of G4 ligands on G4 stability: The FRET melting experiments were performed in the presence of 0.5 μM of G4 ligand and using 0.2 μM of HIV-PRO sequence labelled with fluorophores.

[0084] FIG. 2: Effects of G4 ligands compared with AZT on HIV-1 infectivity using the HeLa P4 cell system for studying the replication of HIV-1. The HeLa P4 cells contain a gene lacZ encoding beta-galactosidase under the control of the viral LTR and whose transcription is activated by the viral protein Tat. The cells are incubated with the ligand then infected with HIV-1. After 24 hours the activity of beta-galactosidase, which is proportional to the infectivity of the virus is then measured on a fluorescence plate reader.

[0085] FIG. 3: Correlation between the apparent affinity of the ligands for the G4 (expressed in stabilisation of the G4 in ΔTm ° C.) and their inhibition activity on HIV.

[0086] FIG. 4: Measurement of the cell proliferation of HeLa cells as a function of time in the presence of increasing the concentrations of Au-MA G4 ligand.

[0087] FIG. 5 represents the structure of the compounds of the invention that are tested and illustrated in FIG. 3. X=H2 refers to the case where no metal is in the center (M is absent).

[0088] FIG. 6 represents the structure of comparative prior art compounds that are tested and illustrated in FIG. 3. X=H2 refers to the case where no metal is in the center (M is absent).

[0089] The above mentioned features of the invention are given for illustration of the invention and not intended to be limiting thereof.

[0090] The following examples describe the synthesis of some compounds according to the invention. These examples are not intended to be limitative and only illustrate the present invention.

EXAMPLES

1. Synthesis of Compounds of Formula (I) or (IA)

1.1. [Preparation of meso-tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl) porphyrinatogold(III) pentachloride] (Au-MA)

[0091] ##STR00017##

[0092] The synthesis of the non-metallated porphyrin was previously described in Sabater et al. Dalton Trans. 2015, 44, 3701.

Step 1: Synthesis of 5,10,15,20-tetrakis(4-(pyrid-2-yl)phenyl)porphyrin (1)

[0093] ##STR00018##

4-(Pyrid-2-yl)benzaldehyde (2.8 g, 15.3 mmol) was dissolved in propionic acid (72 mL), pyrrole (1 g, 15.6 mmol) was added and the mixture was refluxed for 1 h in the dark. The solvent was evaporated and the residue dried under vacuum. The crude product was taken in dimethyl formamide (50 mL) and filtered. The product was washed with dimethyl formamide (50 mL) and diethyl ether (2×50 mL) and dried under vacuum. Yield: 0.78 g (0.84 mmol, 22%) purple solid. .sup.1H NMR (250 MHz, CDCl.sub.3) δ=8.99 (s, 8H, pyrrole), 8.90 (d, J=5 Hz, 4H, pyridine), 8.44 (d, J=8 Hz, 8H, phenyl), 8.38 (d, J=8 Hz, 8H, phenyl), 8.10 (d, J=8 Hz, 4H, pyridine), 7.95 (ddd, J=8, 8, 1 Hz, 4H, pyridine), 7.40 (dd, J=8, 5 Hz, 4H, pyridine), −2.66 (s, 2H, NH). TLC Rf≈0.20 (SiO.sub.2, CH.sub.3CN/H.sub.2O/KNO.sub.3 sat. 8/1/1).

Step 2: Synthesis of 5,10,15,20-tetrakis(4-(N-methyl-pyridinium-2y1)phenyl)porphyrin tetrakis(trifluoroacetate) (2)

[0094] ##STR00019##

5,10,15,20-Tetrakis(4-(pyrid-2-yl)phenyl)porphyrin (1) (200 mg, 0.22 mmol) was dissolved in dimethylformamide (20 mL) and excess iodomethane (4 mL) was added. The mixture was heated at 155° C. for 3 h and acetone (100 mL) was added. The resulting purple precipitate was filtered off, washed with acetone, chloroform and diethyl ether. The product was purified on reverse phase C18 column (20 g), elution water with 0.1% TFA then water/acetonitrile, 80/20, v/v with 0.1% TFA. Yield: 210 mg (0,14 mmol, 66%) purple solid. .sup.1H NMR (300 MHz, d.sup.6-DMSO) δ=9.33 (d, J=6 Hz, 4H, pyridine), 9.04 (s, 8H, pyrrole), 8.84 (dd, J=8, 8 Hz, 4H, pyridine), 8.55 (d, J=8 Hz, 8H, phenyl), 8.48 (dd, J=8, 1 Hz, 4H, pyridine), 8.33 (ddd, J=8, 6, 1 Hz, 4H, pyridine), 8.19 (d, J=8 Hz, 8H, phenyl), 4.53 (s, 12H, CH.sub.3-N), -2.83 (s, 2H, NH). UV-Vis (H.sub.2O), λ max nm (ε M.sup.−1 cm.sup.−1) 416 (410×10.sup.3), 516 (15×10.sup.3), 552 (7×10.sup.3), 580 (5×10.sup.3), 634 (3×10.sup.3). HRES.sup.+-MS m/z: calculated for [C.sub.68H.sub.54N.sub.8].sup.4+=245.6112, found: 245.6106. TLC Rf ≈0.15-0.20 (SiO.sub.2, CH.sub.3CN/H.sub.2O/KNO.sub.3 sat. 8/1/1).

Step 3: 5,10,15,20-tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)porphyrinatogold(III) pentachloride (Au-MA)

[0095] Tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)porphyrin tetrakis(trifluoroacetate) (2) (30.5 mg, 0.021 mmol) was dissolved in water (10 mL). NaOH 1 M, 0.1 mL was added. KAu.sup.IIICl.sub.4 (11.4 mg, 0.030 mmol, 1.4 mol. eq.) was dissolved in water (1 mL) and added to the porphyrin solution. The mixture was refluxed for 24 h. The reaction was monitored by UV-visible spectroscopy and was stopped when the Soret band shift was complete (from 438 to 406 nm, in water, acidic pH). The reaction medium was cooled to room temperature. Desalting of the porphyrin was performed by reverse phase chromatography on a C18 Sep-Pak cartridge (5 g, Waters) by elution with water (200 mL) followed by methanol (20 mL). The collected fractions were evaporated to dryness and product taken in methanol/water, 50:50, v/v (20 mL). Anion exchange was performed on a DOWEX 1×8-200 resin column (chloride form, 1 g). The product solution was evaporated to dryness. The product was dissolved in methanol and precipitated by the addition of diethyl ether. After centrifugation the pellet was dried. Yield: 25.3 mg (0.0185 mmol, 88%) red solid. .sup.1H NMR (400 MHz, CD.sub.3OD) δ=9.61 (s, 8H, pyrrole), 9.26 (d, J=6 Hz, 4H, pyridine), 8.86 (dd, J=8, 8 Hz, 4H, pyridine), 8.66 (d, J=8 Hz, 8H, phenyl), 8.51 (d, J=8 Hz, 4H, pyridine), 8.32-8.27 (m, 12H, pyridine and phenyl), 4.66 (s, 12 H, CH.sub.3—N). UV-Vis (H.sub.2O), λ max nm (ε M.sup.−1 cm.sup.−1) 406 (400×10.sup.3), 520 (21×10.sup.3). ES.sup.+-MS m/z=235.49 [M-5 Cl].sup.5+, 303.10 [M-4Cl].sup.4+, 415.80 [M-3Cl].sup.3+. TLC Rf ≈0.24 (SiO.sub.2, CH.sub.3CN/H.sub.2O/KNO.sub.3 sat. 6/1/1).

1.2. [Preparation of 5,10,15,20-tetrakis(4-phenylguanidinium)porphyrinatogold(III) pentachloride] (Au—PG)

[0096] ##STR00020##

[0097] The synthesis of the non-metallated porphyrin was previously described in Sabater et al. J. Biol. Inorg. Chem. 2015, 20, 729.

Step 1: Synthesis of 5,10,15,20-tetrakis(4-(N,N′-ditertbutoxycarbonylphenylcarboxamidine)porphyrin (3)

[0098] ##STR00021##

Porphyrin 5,10,15,20-(tetra-4-aminophenyl)porphyrin (502 mg, 0.74 mmol) and N, N′-bis(tertbutyloxycarbonyl)pyrazole-1-carboxamidine (1.2 g, 3.8 mmol) were dissolved in 15 mL of dry chloroform and the reaction mixture was stirred at room temperature, under argon, for 5-7 days while being monitored by TLC (SiO.sub.2, diethyl ether). After removal of the solvent under reduced pressure, the product was purified by silica gel chromatography. The column was eluted with 250 mL of hexane/dichloromethane, 7/3, v/v with 1% triethylamine, followed by 500 mL hexane/dichloromethane, 6/4, v/v with 1% triethylamine, and 500 mL hexane/dichloromethane, 50/50, v/v, 1% triethylamine. The fraction of interest was dried under reduced pressure. The solid residue was washed with diethyl ether to ensure elimination of contaminating pyrazole derivative. Pure compound 3 was obtained as a purple solid (790 mg, 65%): Rf =0.7 (SiO.sub.2, hexane/ethyl acetate, 7/3); .sup.1H NMR (300 MHz, CDCl.sub.3): δ=11.83 (s, 4H, N—H), 10.80 (s, 4H, N—H), 8.95 (s, 8H, pyrrole), 8.22 (d, J=8 Hz, 8H, phenyl), 8.09 (d, J=8 Hz, 8H, phenyl), 1.65 (s, 36H, CH.sub.3), 1.61 (s, 36H, CH.sub.3), −2.75 (s, 2H, N—H porphyrin); .sup.13C NMR (75 MHz, CDCl.sub.3): δ=163.67, 153.69, 153.50, 138.50, 136.75, 135.06, 131.23, 120.18, 119.60, 83.93, 79.88, 28.26, 28.18 ppm; HRMS-ES+ m/z [M+H]+ calcd for C.sub.88H.sub.107N.sub.16O.sub.16: 1643.8051 (95%), 1644.8081 (100%), found: 1643.8074 (95%), 1644.8086 (100%). TLC Rf ≈0.7 (SiO.sub.2, hexane/Et.sub.2O 1/1).

Step 2: Synthesis of 5,10,15,20-tetrakis(4-phenylguanidinium)porphyrin tetratrifluoroacetate (4)

[0099] ##STR00022##

Porphyrin meso-5,10,15,20-tetrakis(4-(N,N′-ditertbutoxycarbonylphenylcarboxamidine)porphyrin (3) (800 mg, 0.49 mmol) was dissolved in 80 mL of dichlotomethane and reacted with 20 mL of trifluoroacetic acid under stirring for 3-4 h and the reaction mixture was evaporated under reduced pressure. The product was purified by dissolution in methanol followed by precipitation with diethyl ether. Precipitation procedure was repeated 4 times. The precipitate was filtered on a fritted glass, washed with diethyl ether to provide 4 as a microcrystalline purple solid (592 mg, 93%); .sup.1H NMR (250 MHz, d.sup.6-DMSO): δ=10.33 (s, 4H, N—H), 9.01 (s, 8H, pyrrole), 8.28 (d, J=8 Hz, 8H, phenyl), 7.87 (brs, 16H, N—H.sub.2), 7.72 (d, J=8 Hz, 8H, phenyl), −2.90 (s, 2H, N—H porphyrin); HRMS-ES+ m/z [M-4(CF.sub.3CO.sub.2)—3H].sup.+calcd for C.sub.48H.sub.43N.sub.16: 843.3857, found: 843.3873.

Step 3: 5,10,15,20-tetrakis(4-phenylguanidinium)porphyrinatogold(III) pentachioride (Au—PG)

[0100] Porphyrin meso-5,10,15,20-tetrakis(4-phenylguanidinium)porphyrin tetratrifluoroacetate (4) (50.2 mg, 0.039 mmol) was dissolved in acetic acid 10 mL. KAuCl.sub.4 (53.0 mg, 0.014 mmol) was dissolved in water 2 mL and added to the porphyrin solution. The mixture was heated at 110° C. for 24 h. The reaction was monitored by UV-visible spectroscopy and was stopped when the Soret band shift was complete (Soret band of the the metallated derivative at 408 nm in acidic water). After evaporation to dryness the product was taken in water/methanol, 80/20, v/v and the medium was centrifuged. The supernatant was loaded on a reverse phase C18 Sep-Pak cartridge (5 g, Waters). Elution with water (200 mL) was followed by methanol (20 mL) and then methanol containing 0.5% trifluoroacetic acid. The collected fractions were evaporated to dryness and product taken in methanol/water, 50/50, v/v (20 mL). Anion exchange was performed on a DOWEX 1×8-200 resin column (chloride form, 1 g). The product solution was evaporated to dryness. The product was dissolved in methanol (5 mL) and precipitated by the addition of diethyl ether (20 mL). After centrifugation the pellet was washed with diethyl ether and dried. Yield: 28.6 mg (0.023 mmol, 60%) red solid. .sup.1H NMR (400 MHz, CD.sub.3OD): δ=9.54 (s, 8H, pyrrole), 8.41 (d, J=8 Hz, 8H, phenyl), 7.87 (d, J=8 Hz 8H, phenyl). UV/vis (H.sub.2O): λ max nm (ε M.sup.−1 cm.sup.−1) 408 (285×10.sup.3). ES.sup.+-MS m/z=346.9 [M—2H—5Cl].sup.3+, 260.2 [M—H—5Cl].sup.4+.

2. Biological Activity

2.1. Methods

[0101] A. Preparation of the oligonucleotides:

Fluorescent oligonucleotides were purchased from Eurogentec (Seraing, Belgium) with “Reverse-Phase Cartridge Gold purification”. Concentrations were determined by ultraviolet (UV) absorption using the extinction coefficients provided by the manufacturer. All oligonucleotides were dissolved in 20 mM potassium phosphate buffer pH7 containing 70 mM KCI.

[0102] B. FRET melting experiments:

The HIV-PRO sequence (5′TGGCCTGGGCGGGACTGGG3′) (SEQ ID N° 1) was labelled with fluorescein at the 5′end and TAMRA at 3′end. The transfer of fluorescence energy between fluorescein and tetramethylrhodamine is only possible when the two fluorophores are close in the folded state at low temperature. In the unfolded state at high temperature, the FRET is reduced. The fluorescence melting profiles were recorded on a Stratagene quantitative PCR device (De Cian A, et al. Fluorescence-based melting assays for studying quadruplex ligands. Methods. 2007 Jun; 42(2):183-95.).

[0103] C. Cell lines and viruses: HeLa P4 cells encoding a Tat-inducible β-galactosidase were maintained in DMEM medium (Invitrogen) supplemented with 10% inactivated FCS, 1 mg/ml geneticin (G418, Gibco-BRL), gentamycin. MT4 and H9Laï cells were grown in RPMI 1640 glutamax medium (Invitrogen) supplemented with 10% inactivated FCS. HIV-1 viruses were obtained after 48 h co-culture of MT4 cells (0,5×106 /ml) and H9Laï cells (1×106 /ml), chronically infected by HIV-1Laï isolate, in RPMI 1640 glutamax medium supplemented with 10% inactivated FCS, at 37° C. under humidified atmosphere and 5% CO2. The culture was then centrifuged and the supernatant was clarified by filtration on a 0.45 μm membrane before freezing at −80° C.

[0104] D. Viral infectivity test: The G4 ligands are incubated in presence of the HelaP4 cells 20 minutes before infection. The infectivity was assayed on HeLa P4 cells expressing CD4 receptor and the β-galactosidase gene under the control of the HIV-1 LTR. HeLa P4 were plated using 200 μl of DMEM medium supplemented with 10% inactivated FCS in 96-multi-well plates at 10 000 cells per well. After overnight incubation at 37° C., under humidified atmosphere and 5% CO2, the supernatant was discarded and 200 μl of viral preparation were added in serial dilutions. After 24 h of infection, the supernatant was discarded and the wells were washed 3 times with 200 μl of PBS. Each well was refilled with 200 μl of a reaction buffer containing 50 mM Tris-HCl pH 8,5, 100 mM β-mercaptoethanol, 0,05% Triton X-100 and 5 mM 4-methylumbelliferyl-B-D-galactopyranoside (4-MUG) (Sigma). After 24 h, the reaction was measured in a fluorescence microplate reader (Cytofluor II, Applied Biosystems) at 360/460 nm Ex/Em.

[0105] E. Cytotoxicity study of the G4 ligands: HeLa, human epithelial carcinoma cell line and Wi38, normal human fibroblast cell line were used as experimental model to asses cellular proliferation in the presence of G4 ligands. Cells have been seeded in 384-well plates. All measures have been performed in duplicates in one experiment. The cell proliferation was measured as the surface of the well occupied by the cells. A proliferation index was then calculated by making a ratio on the surface occupied at the time-point preceding the treatment. The cell count is also assessed at the last time-point of the kinetics. Cytolysis Lyzed cells are stained with a nuclear marker, able to enter only cells with compromised membranes. The number of lyzed cells per well is reported all along the kinetics. For the last time-point of the kinetics, a percentage of cytolysis is also computed after getting the maximum cytolysis for each well by permeabilizing cells.

2.2. Results: Specific G4 Ligands Inhibits HIV-1 Replication

2.2.1. G-Quadruplex Ligands

[0106] As part of the anti-cancer strategies described above, a wide variety of quadruplex ligands have been developed. These molecules are very good G4 specific structural probes. They bind very little to double-stranded and single-stranded DNA but recognize very well all types of G4 with dissociation constants in the order of tens to hundreds of nano-molar.

[0107] The ability of some ligands (for instance XM14 and Br-360A) to bind to HIV-PRO3 sequence was evaluated using a stabilization test by FRET (FIG. 1). 3 different families of G4 ligands were used: Salfens, Bisquinoliniums and porphyrins. This technique allows to select molecules which bind to HIVPRO labelled with fluorophores. In this test, thermal denaturation experiments were performed followed by fluorescence and measure the fluorescence emission of fluorescein. Ligand binding to the fluorescent oligonucleotide stabilizes the structure and these results in an increase in the temperature of half-dissociation of the latter (FIG. 1). The more the T.sub.m increases, the larger is the affinity of the ligand for the target. Thus, in the example shown, the ligand 360A further stabilizes the G4 as compared to XM14 compound; its affinity for the G4 is higher. These experiments were also performed in the presence of a duplex competitor composed of 26 base pairs (data not shown). Thus, the binding selectivity of the ligand for the G4 in competition with the duplex can be evaluated. If the stabilization is maintained in the presence of 50 molar equivalents of non-fluorescent competitor duplex (600 equivalents when comparing the number of base pairs to the number of G4 tetrads), the ligand is considered to be selective, but if the stabilization disappears, the ligand is considered not very selective, and this is the case for three porphyrins (H2PYMA, AUT and Tmpyp4). 43 ligands were tested, 23 were selected from the three different chemical families that have interesting affinity and specificity features to study their inhibitory effects on HIV.

2.2.2. Inhibition of HIV

[0108] The next step was therefore to test the effect of these ligands on the viral replication (FIG. 2) and see if there is a correlation between the ability to stabilize the G4 in vitro (ΔT.sub.m) and the effect on viral infectivity in cellulo (FIG. 2). By testing these 23 ligands, it was shown that there is a significant correlation between the affinity of a ligand for the G4, whatever its chemical or structural nature (Bisquinolinium, porphyrins and Salen) and its inhibitory potential (FIG. 2). An increase of G4 stabilization by 7° C. directly translates in an IC.sub.50 which is divided by 10. Compounds in the LA, MALA, T, PYLA and PYMA series are porphyrins derivatives disclosed in the literature and are tested as a comparative examples. The best compounds are porphyrin derivatives according to the invention of the Ma and PG series having an IC.sub.50 of around 100 nM. Under the same conditions, AZT has an inhibitory effect just below with an IC.sub.50 of 40 nM. For H2PYMA, AUT and Tmpyp4 G4 ligands, the competition FRET experiments demonstrated that they were much less specific to the G4 structures. These compounds also bind to duplex and single-stranded DNA. They turned out to be weak inhibitors of viral replication. The low efficiency was interpreted by the “dilution” on non-specific targets. Cytotoxicity test on KB cells, A549, MCF7, MRC5, HCT116 and HeLa P4 showed no cytotoxic effect of these ligands on a period of 92 h and a concentration of up to 30 μM (FIG. 4). This correlation between stabilization in vitro and in vivo inhibition (FIG. 3), associated to the high specificity of these ligands, and the absence of cytotoxicity on human cells, suggest that the observed inhibition is due to the recognition of quadruplex structures of the virus in the viral RNA or DNA. What is claimed is: