THIENOPYRIDINE DERIVATIVES FOR USE IN THE TREATMENT OF CORONAVIRUS INFECTION

Abstract

Coronaviridae is a family of enveloped, positive-sense, single-stranded RNA viruses. The emergence of a new beta-coronavirus SARS-CoV-2 has led to a major health-related crisis associated with a significant mortality in intensive care units, due to the pulmonary complications of COVID-19. The inventors showed that an inhibitor of EPAC1 (i.e. AM-001) is suitable for inhibiting replication of coronavirus and thus would be suitable for the treatment of infections mediated by said type of virus.

Claims

1. A method of treating a coronavirus infection in a subject in need thereof comprising administrating to the subject a therapeutically effective amount of a compound having the following formula (I) ##STR00012## wherein: R.sub.1 is selected from the group consisting of: H; (C.sub.2-C.sub.20)alkyl; (C.sub.3-C.sub.10)cycloalkyl; 3-10 membered heterocycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; wherein said alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are optionally substituted; R.sub.2 is selected from the group consisting of: H; (C.sub.1-C.sub.20)alkyl; (C.sub.3-C.sub.10)cycloalkyl; 3-10 membered heterocycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; or R.sub.2 and R.sub.4 together with the carbon atoms carrying them form a (C.sub.3-C.sub.10)cycloalkyl group; wherein said (C.sub.2-C.sub.20) alkyl and said (C.sub.1-C.sub.20) alkyl, said (C.sub.3-C.sub.10) cycloalkyl, said 3-10 membered heterocycloalkyl, said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted; R3 is selected from the group consisting of: H; (C.sub.3-C.sub.10)cycloalkyl; 3-10 membered heterocycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; wherein said (C.sub.3-C.sub.10) cycloalkyl, said 3-10 membered heterocycloalkyl, said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted; and R.sub.4 is selected from the group consisting of: H, —OH, —NRxRy and —C(O)ORz, Rx, Ry and Rz being independently of each other H or a (C.sub.1-C.sub.10)alkyl; or R.sub.2 and R.sub.4 together with the carbon atoms carrying them form a (C.sub.3-C.sub.10)cycloalkyl group; or its pharmaceutically acceptable salt, hydrate or hydrated salt or its polymorphic crystalline structure, racemate, diastereomer or enantiomer.

2. The method of claim 1 wherein in formula (I), R.sub.3 is selected from the group consisting of: (C.sub.3-C.sub.10)cycloalkyl; 3-10 membered heterocycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; and wherein said (C.sub.3-C.sub.10) cycloalkyl, said 3-10 membered heterocycloalkyl, said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted.

3. The method of claim 1, wherein, in formula (I), R.sub.1 is selected from the group consisting of: H; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; wherein said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted by one or more substituent(s) selected from the group consisting of —NR7R8, (C.sub.1-C.sub.10)alkyl and halogen atom; wherein R7 and R8 are independently of each other selected from (C.sub.1-C.sub.10)alkyl and H.

4. The method of claim 1, wherein, in formula (I), R.sub.2 is selected from the group consisting of: H; (C.sub.1-C.sub.20)alkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; or R.sub.2 and R.sub.4 together with the carbon atoms carrying them form a (C.sub.3-C.sub.10)cycloalkyl group; wherein said (C.sub.1-C.sub.20) alkyl, said (C.sub.3-C.sub.10) cycloalkyl, said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted by one or more substituent(s) selected from the group consisting of: (C.sub.1-C.sub.10)alkyl and halogen atom.

5. The method of claim 1 wherein in formula (I), R.sub.3 is a (C.sub.6-C.sub.10)aryl optionally substituted by one or more substituent(s).

6. The method of claim 1 wherein, in formula (I), R.sub.4 is H or R.sub.2 and R.sub.4 together with the carbon atoms carrying them form a (C.sub.5-C.sub.6)cycloalkyl group.

7. The method of claim 1 wherein, in formula (I), R.sub.1 is a phenyl group and/or R.sub.2 is a thienyl group, said phenyl and thienyl groups being optionally substituted.

8. The method of claim 1 wherein, in formula (I): R.sub.1 is selected from the group consisting of: (C.sub.2-C.sub.20)alkyl; (C.sub.3-C.sub.10)cycloalkyl; 3-10 membered heterocycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; wherein said (C.sub.2-C.sub.20) alkyl, said (C.sub.3-C.sub.10) cycloalkyl, said 3-10 membered heterocycloalkyl, said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted; and R2 is selected from the group consisting of: H; (C.sub.1-C.sub.20)alkyl; (C.sub.3-C.sub.10)cycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; or R.sub.2 and R.sub.4 together with the carbon atoms carrying them form a (C.sub.3-C.sub.10)cycloalkyl group; wherein said (C.sub.1-C.sub.20) alkyl, said (C.sub.3-C.sub.10) cycloalkyl, said (C.sub.6-C.sub.10) aryl and said 5-10 membered heteroaryl groups are optionally substituted.

9. The method of claim 1 wherein the compound is characterized by the following formula (II): ##STR00013## wherein Ra, Rb, Rc, Rd, Re, Rx, Ry and Rz are selected among the group consisting of: H, —OH, halogen atom, —C(O)OH, (C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkoxy, and NR5R.sub.6, wherein R.sub.5 and R.sub.6 are independently of each other selected from (C.sub.1-C.sub.10)alkyl or H; R.sub.4 is selected from the group consisting of H, —OH, —NH2 and —C(O)OH; and R.sub.3 is selected from the group consisting of: H; (C.sub.3-C.sub.10)cycloalkyl; 3-10 membered heterocycloalkyl; (C.sub.6-C.sub.10)aryl; and 5-10 membered heteroaryl; wherein said (C.sub.3-C.sub.10) cycloalkyl, said 3-10 membered heterocycloalkyl, said (C.sub.6-C.sub.10) aryl and said 3-10 membered heteroaryl groups are optionally substituted.

10. The method of claim 1 wherein the compound has one of the following formulae: ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## .

11. The method of claim 1 wherein the compound has the following formula: ##STR00021## .

12. The method of claim 1 wherein the coronavirus is the SARS-Cov-2.

13. The method of claim 5 wherein the one or more substituent(s) are (C.sub.1-C.sub.10)alkyl or halogen atom.

Description

FIGURES

[0139] FIG. 1. Inhibition of SARS-CoV-2 replication in VeroE6 treated with AM-001. VeroE6 cells pretreated for one hour with the indicated concentrations of AM-001 were infected at a multiplicity of infection of 0.001. After one hour of virus inoculation, the cell culture medium was removed and replaced with cell culture medium containing AM-001 at the indicated concentrations. 24 hours post-infection, viral RNA was extracted from the supernatants of infected cells and subjected to RT-qPCR using primers and probe specific for the E gene. The dotted line indicated 90% viral inhibition.

[0140] FIG. 2. Inhibition of SARS-CoV-2 replication in human lung epithelial Calu-3 cells pretreated with AM-001. Calu-3 cells pretreated for two hours with the indicated concentrations of AM-001 were infected at a multiplicity of infection of 0.001. After one hour of virus inoculation, the cell culture medium was removed and replaced with cell culture medium containing AM-001 at the indicated concentrations or Remdesivir at 6 .Math.M. (A) 24 hours post-infection, viral RNA was extracted from the supernatants of infected cells and subjected to RT-qPCR using primers and probe specific for the E gene. (B) 24 hours post-infection, infectious particles in the supernatant were titrated using the TCID.sub.50 method.

[0141] FIG. 3. Analysis of cellular viability in human lung epithelial Calu-3 cells treated with AM-001. Calu-3 cells treated for 24 hours with the indicated concentrations of AM-001. Cellular viability was measured with the Cell Proliferation Kit I (MTT) (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol.

[0142] FIG. 4 Evaluation of the antiviral properties of AM-001 against SARS-CoV-2 replication in human lung epithelial Calu-3 cells upon pre-treatment, treatment or post-treatment. Calu-3 cells were treated and infected as described in Material and Methods. 24 hours post-infection, infectious particles in the supernatant were titrated using the TCID.sub.50 method.

EXAMPLE 1

Material & Methods

[0143] VeroE6 cells (ATCC CRL-1586) were cultured in Dulbecco modified Eagle’s minimal essential medium (DMEM) containing 10% fecal calf serum (FCS). The SARS-CoV-2 virus used in these studies was isolated from a nasal swab kindly provided by the Toulouse hospital (CHU Toulouse Purpan, France) following two passages in VeroE6 cells. For the SARS-CoV-2 infection, the cells were plated in 6-well plates at a density of 2.10.sup.5 cells per well. The next day, the cells were pretreated for one hour with AM-001 at different concentrations. Following one hour pretreatment, the medium was removed and the cells were infected at a multiplicity of infection of 0.001 with SARS-CoV-2 in DMEM containing 2% FCS (infection medium). After one hour of virus inoculation, the cell culture medium was removed and replaced with infection medium containing AM-001 at the different concentrations. 24 hours post-infection, viral RNA was extracted from the supernatants of infected cells and subjected to RT-qPCR using primers and probe specific for the E gene. The result is depicted in FIG. 1.

EXAMPLE 2

Material & Methods

Cell Culture

[0144] Human lung epithelial cells Calu-3 (ATCC HTB-55) were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM:F12) containing 20% fecal calf serum (FCS) and GlutaMAX™ Supplement 1X (Gibco).Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco modified Eagle’s minimal essential medium (DMEM) containing 10% fecal calf serum (FCS). The SARS-CoV-2 virus used in these studies was isolated from a nasal swab kindly provided by the Toulouse hospital (CHU Toulouse Purpan, France) following two passages in Vero E6 cells. For the SARS-CoV-2 infection and evaluation of the antiviral properties of AM-001, Calu-3 cells were plated 48 hours before treatment and infection in 12-wells plates at a density of 1.10.sup.6 cells per well.

Pretreatment

[0145] Cells were pretreated for two hours with AM-001 at different concentrations or with Remdesivir at 6 .Math.M. Following pretreatment, the medium was removed and the cells were infected at a multiplicity of infection of 0.001 with SARS-CoV-2 in medium containing 2% FCS (infection medium) and AM-001 at the different concentrations. After one hour of virus inoculation, cells were washed twice with PBS and the cell culture medium was replaced with infection medium containing AM-001 at the different concentrations or with Remdesivir at 6 .Math.M. 24 hours post-infection, viral RNA was extracted from the supernatants of infected cells and subjected to RT-qPCR using primers and probe specific for the E gene to quantify viral RNA in the supernatants. 24 hours post-infection, infectious virus was also titrated from the supernatants of infected cells by the tissue culture infectious dose 50% (TCID.sub.50) method on Vero E6 cells.

Treatment

[0146] Cells were infected at a multiplicity of infection of 0.001 with SARS-CoV-2 in medium containing 2% FCS (infection medium) and AM-001 at the different concentrations or with Remdesivir at 6 .Math.M. After one hour of virus inoculation, cells were washed twice with PBS and the cell culture medium was replaced with infection medium containing AM-001 at the different concentrations. 24 hours post-infection, viral RNA was extracted from the supernatants of infected cells and subjected to RT-qPCR using primers and probe specific for the E gene to quantify viral RNA in the supernatants. 24 hours post-infection, infectious virus was also titrated from the supernatants of infected cells by the tissue culture infectious dose 50% (TCID.sub.50) method on VeroE6 cells.

Post-Treatment

[0147] Cells were infected at a multiplicity of infection of 0.001 with SARS-CoV-2 in medium containing 2% FCS (infection medium). After one hour of virus inoculation, cells were washed twice with PBS and the cell culture medium was replaced by fresh infection medium. Four hours post-infection, the cell culture medium was removed and replaced by infection medium containing AM-001 at the different concentrations or with Remdesivir at 6 .Math.M. 24 hours post-infection, viral RNA was extracted from the supernatants of infected cells and subjected to RT-qPCR using primers and probe specific for the E gene to quantify viral RNA in the supernatants. 24 hours post-infection, infectious virus was also titrated from the supernatants of infected cells by the tissue culture infectious dose 50% (TCID.sub.50) method on VeroE6 cells.

Cellular Viability Assay

[0148] Calu-3 cells were plated 48 hours before treatment in 96-wells plates at a density of 1.10.sup.5 cells per well. After 24 hours of treatment with the different concentrations of AM-001, cells viability was measured using the Cell Proliferation Kit I (MTT) (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol.

Results

[0149] AM-001 pre-treatment inhibit SARS CoV-2 replication (FIGS. 2A-2B). AM-001 has no detected cellular toxicity (FIG. 3). When used at concentration ≥ 10 .Math.M, AM-001 totally blocks infectious virus production even when given 4 h post-infection in human lung epithelial Calu3 cells (FIG. 4). The reported antiviral potency of AM-001 is similar to the anti-viral molecule Remdesivir (FIGS. 2A-2B and 4).

REFERENCES

[0150] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.