Patent Application
20210369671
The present invention is related to a family of corroles for its use in the treatment of an infection by human cytomegalovirus.
Human cytomegalovirus (hCMV) is a member of the Herpesviridae family that infect 60-80% of worldwide population.
Although primo-infection is generally silent or associated with a mononucleosic syndrome in immunocompetent people, it causes severe syndromes in immunocompromised persons (e.g., seroconversion during the first quarter of pregnancy or HIV positive people, for example).
As for all herpes viruses, after primary infection, the virus persists in a latent state lifelong in monocytes and CD34+ progenitors. Latent infection reactivates in outbreaks that depend on environmental factors, immune state, and stress level. hCMV spreads from person to person through body fluids, such as blood, saliva, urine, semen, and breast milk. hCMV is a common virus that infects people at any age. Most hCMV infections are silent, which means that the majority of people who are infected with hCMV have no signs or symptoms.
However, pregnant women who are infected during the first quarter of pregnancy can transmit hCMV to their foetus, sometimes causing a congenital hCMV infection. Congenital hCMV infection can cause problems from hearing loss to severe developmental and neuronal disabilities. In the US, hCMV infection is the first cause of congenital malformation.
Nowadays many previous vaccine approaches fail to provide complete protection against hCMV infection. Current anti-hCMV therapies include treatment with nucleoside analogues such as Cidofovir and Ganciclovir that inhibit virus replication by acting as fraudulent building blocks for viral DNA synthesis.
However, these drugs have adverse effects, such as myelosuppression or nephrotoxicity, and may lead to the emergence of antiviral-resistant hCMV strains during long-term or repeated treatments. Thus, the development of non-nucleoside hCMV inhibitors with novel mechanisms of action is an important focus area of antiviral research. The strategies for the design of new antiviral drugs must aim to develop more selective compounds with a broad spectrum of antiviral activity and little or no drug resistance induction.
Gros et al. (Acs Infect. Dis. 2015, 1, 350-356) reported on 2015 potential antiviral activity of nitrocorroles bearing nitro groups as substituents at the meta- or para-position of phenyl groups on position 5, 10 or 15 of corroles. However, it was observed that a nitrocorrole of type A3 was able to reduce the proportion of infected MRC5 cells only when added before infection. When this compound is added after hCMV adsorption and entry, it does not seem to have an impact on hCMV infection.
By the way, these nitrocorroles do not display a satisfactory selectivity index, that is to say the ratio between the cytotoxicity of a compound on cells without hCMV infection and the cytotoxicity of said compound on infected cells.
Taking these shortcomings in account, there is still a need to develop a new drug with unique mechanism of action (moA), being used in hCMV infection and having better selectivity index in the treatment of hCMV infection.
Against all expectations, the Inventors of the present invention have observed that a family of corroles bearing at least one fluorine atom as the substituent of groups on meso position 5, 10 or 15 of corroles can display an anti-hCMV activity in host cells after hCMV infection and a significantly better selectivity index.
The first subject-matter of the present invention is to provide a corrole of type A3 or A2B of formula (I):
wherein
wherein Rc is —COOH, or —SO3H,
wherein Rd is a (C1-C8) alkyl chain, or —(CH2)nSO3−, n being an integer chosen from 1, 2, 3, 4 or 5,
According to an embodiment, the present invention provides a corrole of formula (I) or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, for its use in the treatment of an infection by human cytomegalovirus, such as Pneumonitis, esophagitis, gastritis, enterocolitis, retinitis, hepatitis, encephalitis causing by human CMV infection.
More particularly, the corroles of the present invention can be used in the treatment of an infection of human cytomegalovirus in an immunodeficient patient, such as HIV-infected patients, or a patient receiving organ transplant or in pregnant women.
According to in vitro trials carried out in human retinal cell culture, it is observed that a corrole of the present invention, even at low concentration, can significantly decrease hCMV infection level of cells and viral DNA accumulation level in infected cells. Moreover, the corroles of the present invention have better selectivity index than that of nitrocorroles previously described by Gros et al. (2015).
According to the invention, the term “a corrole of type A3” is meant to be a corrole wherein the A1 and the A2 are identical. In another word, a corrole of type A3 is a corrole wherein the three substituent groups on meso position 5, 10 and 15 of said corrole are identical.
According to the invention, the term “a corrole of type A2B” is meant to be a corrole wherein the A2 is different from the A1. In another word, a corrole of type A2B is a corrole wherein the substituent groups on meso position 5 and 15 are identical and they are different from the substituent group on meso position 10 of said corrole.
The term “a (C1-C8) alkyl chain” is meant to be a saturated straight or branched hydrocarbon chain containing from 1 to 8 carbons. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl.
The term “with the proviso that at least one of A1 or A2 bears at least one fluorine atom on position R1, R2, R3, R4, R5, R1′, R2′, R4′ or R5′” means that:
The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans without undue toxicity, irritation, allergic response and the like.
A pharmaceutically acceptable salt of a corrole of formula (I) of the present invention refers to salts which retain the biological effectiveness of corrole of formula (I) and are not biological undesirable for human.
According to the present invention, a pharmaceutically acceptable salt of a compound of the invention can be a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound of the invention.
A pharmaceutically acceptable salt of a corrole of formula (I) can be obtained by reacting said corrole with a variety of organic and inorganic positive counter ions well known in the art, for example sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like. A pharmaceutically acceptable salt of a corrole of formula (I) can also be obtained by reacting said corrole with pharmaceutically acceptable acids. Specific examples include inorganic acid salts such as hydrochlorides and sulfates; and organic acid salts such as formates, trifluoroacetates, acetates, tartrates, maleates, fumarates, succinates and methanesulfonates.
The term “optical isomers” refers to molecules that differ three-dimensionally by the placement of substituents around one or more atoms in a molecule.
According to an embodiment of corroles of type A2B of the present invention, A1 and A2 are both phenyl groups of formula II which are substituted differently.
In another embodiment of corroles of type A2B of the present invention, A1 and A2 are both pyridinium groups of formula II which are substituted differently.
In an embodiment of the corrole of the present invention, when A1, A2 or R3′ is a pyridinium group which does not bear a —SO3 group, said corrole comprises a counter ion such as Cl−, Br− or I−.
According to an embodiment of the corroles of the present invention, the substituent groups A1 and/or A2 are represented by one of formula (II1), (II2), (II3), (III1), (III2), (IV1) or (IV2)
or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
In said formulas, the substituents R1, R2, R3, R4, R5, R1′, R2′, R3′, R4′, R5′, R1″, R2″, R3″ and E are as defined above.
Thus in this embodiment at least one of A1 and A2 or both are selected from (II1), (II2), (II3), (III1), (III2), (IV1) or (IV2). A1 and A2 may be identical or different.
According to a more particular embodiment of the corroles of the present invention, A1 and/or A2 are represented by one formula chosen from formula (II4), formula (II5), formula (II6), formula (III3), formula (III4), formula (III5) or formula (IV3).
wherein R′ and R″ represent respectively two different substituents among R1, R2, R3, R4 and R5 or two different substituents among R1′, R2′, R4′ and R5′, R1, R2, R3, R4, R5, R1′, R2′, R3′, R4′, or R5′ being defined as before.
According to formula (II4), the fluorine atom and R′ represent two of R1, R2, R3, R4 or R5, while the remaining three other substituents among R1, R2, R3, R4 or R5 are hydrogen.
Thus for example, if F corresponds to R1 and R′ corresponds to R2, then R3, R4 and R5 are hydrogen.
According to formula (II5), the fluorine atom, R′ and R″ represent three of R1, R2, R3, R4 or R5, while the remaining two other substituents among R1, R2, R3, R4 or R5 are hydrogen.
Thus for example, if F, R′ and R″ respectively correspond to R1, R2 and R3, then R4 and R5 are hydrogen.
According to formula (III3), the fluorine atom and R′ represent two of R1′, R2′, R4′ or R5′, while the remaining two other substituents among R1′, R2′, R4′ or R5′ are hydrogen.
Thus for example, if F corresponds to R1′ and R′ corresponds to R2′, then R4′ and R5′ are hydrogen.
According to formula (III4), the fluorine atom, R′ and R″ represent three of R1′, R2′, R4′ or R5′, while the reaming forth substituent among R1′, R2′, R4′ or R5′ is hydrogen.
Thus for example, if F, R′ and R″ respectively correspond to R1′, R2′ and R4′, then R5′ is a hydrogen.
Thus in this embodiment at least one of A1 and A2 or both are selected from formula (II4), formula (II5), formula (II6), formula (III3), formula (III4), formula (III5), or formula (IV3). A1 and A2 may be identical or different.
According to an embodiment of the corroles of the present invention, at least one of A1 or A2 bears at least one substituent, among R1, R2, R3, R4, or R5 of formula (II) or among R1′, R2′, R4′ or R5′ of formula (III), or among R1″, R2″ or R3″ of formula (IV) being chosen from:
wherein Rc is —COOH, or —SO3H,
wherein Rd is a (C1-C8) alkyl chain, or —(CH2)nSO3−, n being an integer chosen from 1, 2, 3, 4 or 5.
Thanks to the presence of above-mentioned substituents which are hydrophilic, the corroles of this embodiment is also water-soluble.
In a particular embodiment, the corroles of the invention are corroles of type A3, wherein A1 and A2 on the corrole of formula (I) are represented by one formula chosen from formula (II4), formula (II5), formula (II6), formula (III3), formula (III4), formula (III5), or formula (IV3) or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
In another particular embodiment, the corroles of the invention are corroles of type A2B, wherein A1 and A2 on the corrole of formula (I) are different,
wherein R′ and R″ represent respectively 2 different substituents among R1, R2, R3, R4 and R5 or 2 different substituents among R1′, R2′, R4′ and R5′, R1, R2, R3, R′, R″ and R3′ being independently chosen from:
wherein Rc is —COOH, or —SO3H
wherein Rd is a (C1-C8) alkyl chain, or —(CH2)nSO3−, n being an integer chosen from 1, 2, 3, 4 or 5.
According to formula (II8), R′ can be any one substituent of R1, R2, R3, R4 and R5, while the other substituents of R1, R2, R3, R4 and R5 are hydrogen.
According to formula (II9), R′ and R″ represent any two substituents among R1, R2, R3, R4 and R5, while the other three substituents of R1, R2, R3, R4 and R5 are hydrogen.
According to formula (III6), R′ can be any one of R1′, R2′, R4′ and R5′, while the other three substituents of R1′, R2′, R4′ and R5′ are hydrogen.
According to formula (III7), R′ and R″ can be any two substituents of R1′, R2′, R4′ and R5′, while the other two substituents of R1′, R2′, R4′ and R5′ are hydrogen.
In another particular embodiment, the corroles of the invention are corroles of type A2B, wherein A1 and A2 on the corroles of formula (I) are different,
In a more particular embodiment of the present invention, the corroles are chosen from following compounds:
or a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
In a particular embodiment, the present invention concerns one of the above-mentioned compound A, B, C, D, E, F, G, H, I, J, K, L, M for their use in the treatment of retinitis.
Another subject-matter of the present invention concerns an association formed by:
In the present invention, the terms “medicament” and “drug” are interchangeable.
The term “active ingredient” is meant to the biologically active component of a medicament.
It is observed that an association of a commercially available anti-hCMV medicament with a corrole of the present invention can produce a synergistic therapeutic effect in treatment of hCMV infection. This synergy may be due to the fact that a corrole of the present invention reacts on hCMV by a very different mechanism of action then that of actually commercially available anti-hCMV drugs, such as Ganciclovir.
In a particular embodiment, the present invention concerns an association formed by:
According to an embodiment of said association, the corrole of the present invention is associated to another anti-hCMV active ingredient by a chemical linker. This chemical linker can be in vivo cleavable.
The present invention also relates to a combination product comprising:
The term “simultaneous use” is meant to be an administration of two active ingredients by the same route and at the same time.
The term “sequential use” is meant to be an administration sequentially on the time of two active ingredients by the same route.
The term “separate use” is meant to be an administration of 2 active ingredients at the same or substantially the same time by different routes.
Another subject-matter of the present invention concerns a pharmaceutical composition for its use in treating an infection of human herpesvirus, in particular of human cytomegalovirus, comprising a corrole of formula (I) as defined above or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or an association as defined above, or a combination product as defined above, as active ingredient, and a pharmaceutically acceptable carrier.
The term “pharmaceutically acceptable carrier” means a carrier that is useful in preparing a pharmaceutical composition or formulation that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for human pharmaceutical use. The carrier can act as a vehicle, medium, or for dilution of the active ingredient. The formulation of the pharmaceutical composition of the present invention can be determined and carried out according to well-known prior art relating to drug formulation. The carrier material can be an organic or inorganic inert carrier material, for example one that is suitable for oral administration or injection. Suitable carriers include water, gelatin, arabic gum, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene-glycols, glycerine and petroleum jelly. Additional additives such as flavoring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.
The compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
According to the formulation, the pharmaceutical composition of the present invention can be administrated by oral route or by injection.
The present invention provides also a method for treating the infections by human cytomegalovirus, such as pneumonitis, esophagitis, gastritis, enterocolitis, retinitis, hepatitis, encephalitis causing by human CMV infection, comprising the step of:
The term “pharmaceutically effective amount” means the amount of a before defined corrole or of a before defined combination product as pharmaceutical active in a pharmaceutical composition to produce the desired therapeutic effect.
The present invention is illustrated by the following figures and examples.
1. Materials and Methods
1.1 Preparation of Corroles of the Invention
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. 1H NMR spectra were recorded on a Bruker AV300 spectrometer (300 MHz). CDCl3 was used as solvent (except when indicated) and TMS as internal reference; the chemical shifts (δ) are given in ppm relative to residual CHCl3 (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 CH2Cl2, CHCl3 or THF as solvent.
General procedure #1 according to a modified Paolesse's method (Paolesse et al., J. Org. Chem. 2001, 66 (2), 550-556).
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, CH2Cl2). All fractions containing corrole (green fraction) were combined and evaporated to dryness. Purification details for each compound are described below.
This corrole was prepared as described for general procedure 1 starting from 4-nitrobenzaldehyde and pyrrole. The residue was purified by chromatography column (alumina, CH2Cl2/heptane, 1/1, v/v) to give pure corrole (492 mg, 5.5%). UV-Vis (DCM): λmax (nm) (ε×10−3 L mol−1 cm−1)=447 (53.4), 598 (18.7). 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]+, 661.17 calcd for C37H23N7O5. MS (ESI) m/z=660.15 [M−H]−, 662.14 [M+H]+, 661.17 calcd for C37H23N7O5.
This corrole corresponds to the above described compound of formula B. This corrole was prepared as described for general procedure 1 starting from 2-fluoro-5-nitrobenzaldehyde and pyrrole. The residue was crystallized from CHCl3/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): λmax, (nm) (ε×10−3 L mol−1 cm−1) 418 (103.2), 572, 610, 645. 1H NMR (300 MHz, CDCl3), 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]+⋅, 715.14 calcd for C37H20F3N7O6.
General procedure #2 according to a modified Gryko's method (Gryko, D. T.; Koszarna, B. Org. Biomol. Chem., 2003, 1(2), 350-357).
Aldehyde (20.0 mmol) was dissolved in distilled pyrrole (30.0 mmol) at room temperature and then a solution of TFA in CH2Cl2 (18 μL in 2.0 mL) was added and vigorously stirred. After 10 min, 800 mL of CH2Cl2 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, CH2Cl2). Purification details for each compound are described below.
This corrole correspond to the above described compound of formula A.
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): λmax, (nm) (ε×10−3 L mol−1 cm−1) 407 (150.1), 562 (23.9), 604 (12.4). 1H NMR (300 MHz, CDCl3), 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). 19F NMR (282 MHz, CDCl3) 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]+, 796.07 calcd for C37H11F15N4.
1.2 hCMV Infection
ARPE-19 human retinal pigmented epithelium cells were used for hCMV infection assay.
Cells were grown in DMEM without phenol red (Sigma-Aldrich) 10% SVF, Pen-strep, 1× sodium pyruvate, 1× Glutamax.
hCMV Infection Protocol
hCMV infection was carried out on ARPE-19 cells seeded in 96 wells plate, in duplicate at one or two MOI (multiplicity of infection) and 7 concentrations of FNO2Cor, TPFCor, TpNO2Cor.
On 4 d and 10 d post-infection (2 post-infection times), data acquisition per virus and cell line was done by high content microscopy to calculate infection level and DNA replication according to compound concentration.
1.3 Toxicity Assessment
The toxicity of compounds FNO2Cor 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 and TPFCor were studied. The compound TpNO2Cor was used as positive control. Cell culture without treatment was used as negative control.
2. Results
2.1 Cytotoxicity
Cytotoxicity of compounds FNO2Cor, TPFCor and TpNO2Cor were assessed at different concentrations on ARPE-19 cells according to the method described on section 1.3. TpNO2Cor worked as positive reference. CC50 values of these three compounds on ARPE-19 cells are given in table I below.
It is already known that TpNO2Cor is not cytotoxic (Gros et al., 2015).
These results indicate that FNO2Cor and TPFCor are safe for human retinal pigmented epithelium cells.
2.2 Antiviral Activity
Compounds FNO2Cor, TPFCor, TpNO2Cor and anti-hCMV drug Ganciclovir were assessed at 8 different concentrations in ARPE-19 cell culture on 4 days post-infection (
These results indicate that the compounds FNO2Cor and TPFCor already have a better selectivity index than that of TpNO2Cor only after 4 d post-infection both for inhibiting hCMV infection of healthy cells and hCMV replication in infected cells.
The results obtained on 10 d PI are displayed below in table IV. The selectivity index of these compounds on 10 d PI are displayed in table V.
On 10 d post-infection, the compounds FNO2Cor and TPFCor show significantly much better selectivity index than TpNO2Cor for inhibiting hCMV infection in cells.
2.3 Synergic Antiviral Activity
A combinatory experiment is carried out by using a combination of Ganciclovir and the compound TPFCor.
The experience design is based on Straetemans R et al. (Biometrical Journal 47 (2005) 3,299-308) derived from the Hill model (Hill 1910).
It is based on a ray design, each ray (f) being a combination of the 2 compounds in various ratio (Table VI). The calculation of IC50 of each compound alone and of the combination, allows to calculate an interaction index. The results are displayed in
According to this model an interaction index (Ir) of 1 means additivity, an Ir below 1 means synergy and above 1 antagonism of the two compounds.
The results of
| Number | Date | Country | Kind |
|---|---|---|---|
| 17306655.6 | Nov 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/082695 | 11/27/2018 | WO | 00 |
