Field of the invention

Present patent application disclose family of aromatic sulphonamides derivatives, their method of synthesis, and use as an antiviral agent, in particular for the treatment of infection with influenza A vims.

Background of the invention

In the state of the art different groups of aromatic sulphonamides are well known and different areas of application. Supuran (2017) describes various applications of sulphonamides based on their pharmacological antibacterial, anti-carbonic anhydrase, anti-obesity, diuretic, hypoglycaemic, antithyroid, antitumor, and anti-neuropathic pain activities. British patent application GB834929A discloses the group of aromatic sulphonamides as highly active diuretics. The starting sulphonamides may be obtained by direct sulphochlorination, by reaction of alkali metal salts of the corresponding sulphonic acids with add halides, or by means of an exchange of the amino group with a sulphonic acid halide group. The starting sulphonic chalides need not be recovered in pure form but may be reacted with the ammonia, ammonium salts, or ammonia donors in the form of the reaction mixtures obtained in their production. They are also known in the art other applications of sulphonamide derivatives. For example, the European patent applicahon EP0030140A1 discloses process for preparing N-(haloheterocyclic-aminocarbonyl)aromatic sulphonamides and N-(substituted heterocyclic- aminocarbonyl)aromatic sulphonamides. These compounds are useful as herbicides and also as intermediates for herbicides.

Scozzafava et al. (2003) describe that HIV protease inhibitors possessing sulphonamide moiehes in their molecules, whereas a vast number of other derivatives are continually being synthesized and evaluated to obtain compounds with less toxicity or activity against drug-resistant viruses. Several nonnucleoside HIV reverse transcriptase or HIV integrase inhibitors containing sulphonamide groups were also reported. Most compounds with antiviral activity possessing this mechanism of action incorporate in their molecules primary sulphonamide groups. Some small molecule chemokine antagonists achng as HIV entry inhibitors also possess sulphonamide functionalities in their scaffold.

Antiviral sulphonamide moiety containing drags include protease inhibitors with activity against Human Immunodeficiency Virus Type 1 ( HIV-1) such as amprenavir, fosamprenavir, danmavir, delaviridine, tipranavir, as well as hepatitis C anhvirals, containing sulphonamide moiety: Asunaprevir (NS3/4A protease inhibitor), Beclabuvir (NS5B RNA polymerase inhibitor), Dasabuvir (NS5B RNA polymerase inhibitor), Grazoprevir (NS3/4A protease inhibitor), Paritaprevir (NS3/4A protease inhibitor) or Simeprevir (NS3/4A protease inhibitor). Amprenavir (GlaxoSmithKline) is a protease inhibitor used to treat HIV infection. Fosamprenavir is used for the treatment of HIV-1 infections, typically but not necessarily in combination with low-dose ritonavir or other antiviral drags. Tipranavir is the first antiviral heteroaromatic sulphonamide of aromatic amine, containing substituted aniline moiety that is typical for antibacterial sulphonamides. Dealaviridin belongs to alkylsulphonamides of hetroaromatic primary amine. Most of anti-HIV drags of this class (except tipranavir and delaviridine) belongs to p- aminosubstituted benzenesulphonamides of secondary aliphatic amines (contains p- aminophenylsulphonic acid moiety). None of these drags are aromatic sulphonamides of heterocyclic imines, where imino group is a fragment of an aliphatic heterocyclic ring.

It is also known from the state of the art, that some cyclic sulphonamides act as HIV-1 specific nonnucleoside reverse transcriptase inhibitors (Aranz et al, 1999) or as novel inhibitors of human immunodeficiency virus type 1 integrase and replication described (Neamati et al, 1997).

There are also known Hepatitis C antivirals containing sulphonamide moiety, e.g. Asunaprevir (NS3/4A protease inhibitor), Beclabuvir (NS5B RNA polymerase inhibitor), Dasabuvir (NS5B RNA polymerase inhibitor), Grazoprevir (NS3/4A protease inhibitor), Paritaprevir (NS3/4A protease inhibitor) or Simeprevir (NS3/4A protease inhibitor). These drags are amides of cyclopropylsulphonic acid, methylsulphonic acid or dimethylaminosulfonic acid. All these compounds are different from aromatic sulfonic acids disclosed in the present invention.

According to Luciano et al. (2017), there are currently two types of anti-influenza medicines on the market, influenza neuraminidase inhibitors, oseltamivir phosphate (Tamiflu), zanamivir (Relenza), Laninamivir and viral M2 ion channel blockers amantadine and rimantadine (Wright and Webster, 2001). Unfortunately, drag resistance that has been frequently reported for both neuraminidase and M2 channel inhibitors. Therefore, there is high medical need for novel anti-influenza therapeutics with a new mechanism of anti-influenza action.

Interfering HA-mediated fusion has become a strategy of anti-influenza drag discovery for many years. Well known are two types of fusion inhibitors that block influenza infection by either non- specifically increasing endosome pH or by direct targeting of hemagglutinin (HA) protein. Examples of the first mechanism of action include chloroquine (Ooi EE et al. , 2006) and triperiden (Ghendon Y et al. , 1986). None of these compounds are belonging to chemical class of sulphonamides.

There are also known in the state of the art antivirals against influenza virus, belonging to aromatic sulphonamides. Recently R05464466 and R05487624, two representative compounds of benzenesulphonamide, were also found to be able to inhibit fusion as well as overall replication of H1N1 influenza viruses (Zhu L et al, 2011). There are also three antiviral amino-substituted benzenesulphonamides described in the literature (Yang J et al, 2013; ShenX et al, 2013)). These experimental compounds belong to non-substituted or mono-substituted amides p- or m-amino benzene sulfonic acids (S19, R05464466 , R05487624) or derivatives of m-nitroaniline (MBX2546). It means that common structural feature of all known anti-influenza aromatic sulphonamides is the presence of aniline moiety, which is in good agreement with previously established antiviral activity of other aniline derivatives that is again different as compared with aromatic sulfonic acids disclosed in the present invention.

Until now, only one approved drag that is of sulphonamide type (MEK inhibitor refametinib) is known. It can also be used to treat influenza, but this compound does not belong to sulphonamides of aromatic sulfonic acids. None of known antiviral compounds are amides of aromatic sulfonic acid and heterocyclic imines, where imino group is included in ring of heterocycle.

As a result of research, it was unexpectedly found that the group of aromatic sulphonamides display antiviral effects, in particular towards the influenza A vims.

Detailed description of the invention

The invention concerns a new group of aromatic sulphonamides of formula (I): wherein: R is alkyl C ₁ -C ₁₆ ; cyclohexyl; and R ¹ , R ² , R ³ is lower alkyl C ₁ -C ₄ ,

Preferably, the invention concerns the following compounds: l-(4-hexylphenyl)sulfonyl-N-methyl- aziridine-2 -carboxamide or l-(4-hexylphenyl)sulfonylaziridine-2 -carboxamide.

The invention also concerns the method for preparation of said aromatic sulphonamides, wherein the method comprising reaction of the appropriate p-substituted phenylsulfonylchloride with the corresponding aziridine in presence of K ₂ CO ₃ in the mixture of CHCT and water at room. The invention concerns also a new group of aromatic sulphonamide of formula (I): 1 wherein: R is alkyl C1-C16; cyclohexyl; and R ¹ , R ² , R ³ is lower alkyl C1-C4 , for use as a medicament. Preferably, in the treatment of various viral diseases, in particular infection with influenza A vims.

Brief description of drawings

The invention has been presented in embodiments and on drawing, which are not limiting of the scope of protection, in which: fig. 1 is a chart showing cytotoxicity effect of tested compounds on MDCK (top) and A549 (bottom) cells treated, the graph shows the result of the XTT test after incubating the cells with test compounds for 48 h in 3 replications; fig. 2 is a chart showing results of RT-qPCR analysis of supernatants from MDCK (top) and A549 (bottom) cells infected with influenza A vims (IAV; TCID50 = 400) and treated with 20 mM of tested compounds or the same volume of DMSO solvent; asterisks were marked with statistically different values from the control values in the student's t-test (P <0.05); fig. 3 is a chart showing results of RT-qPCR analysis of supernatants from the apical surface of HAE cultures infected with influenza A vims (IAV; TCID ₅₀ = 400) and treated with 30 pM of a tested compound or the same volume of DMSO solvent.

Example 1. Chemical synthesis of aromatic sulphonamides derivatives

It is described below the general method for the preparation of the sulfonylaziridines. (1) wherein: R is alkyl C ₁ -C ₁₆ ; cyclohexyl; and Ri, R2, R3 is lower alkyl C1-C4 , p-Substituted phenylsulfonylchloride (1 mmol) was added with stirring to solution of the appropriated aziridine (1.1 mmol) and K ₂ CO ₃ (2 mmol) in the mixture of 1 ml CHCI ₃ + 1 ml water. The mixture was stirred for 24 h at room temperature. The product was extracted with CHCL and the solution was dried over MgSOi. The solvent was evaporated. The product was purified by chromatography (silica gel, petroleum ether/ethyl acetate 4:1=>1:2) to give appropriated sulfonylaziridine.

The characteristics and properties of synthesized compounds using the method given above are described in Table 1.

Table 1

Synthesized aromatic sulphonamides derivatives

Example 2. Effects of compounds on infection with influenza A vims

Two synthesized compounds: l-(4-hexylphenyl)sulfonyl-N-methyl-aziridine-2-carboxamide (C- 3380) and l-(4-hexylphenyl)sulfonylaziridine-2 -carboxamide (C-3389) have been tested on infection with influenza A vims. Tested compounds were dissolved in 25 mM DMSO, aliquoted and frozen. A new batch of compounds was used for each experiment. The samples were not re-frozen. In the experiment following cells and vims have been used: Madin-Derby Canine Kidney (MDCK) (ATCC CCL-34 cell line), adenocarcinomic human alveolar basal epithelial cells (A549) (ATCC CCL-185 cell line) were maintained in Dulbecco modified Eagle’s medium (DMEM, high glucose, Life Technologies, USA) supplemented with 3% heat-inactivated fetal bovine semm (FBS, Life Technologies, USA), 100 U/ml penicillin, and 100 pg/ml streptomycin (3% DMEM). Cells were cultured at 37°C in an atmosphere containing 5% CO2. Human airway (tracheobronchial) epithelial (HAE) cells were obtained from airway specimens resected from patients undergoing surgery at the Silesian Center for Heart Diseases. Primary cells were cultured on plastic to generate passage 1 cells and plated at a density of 3 ^c 10 ⁵ cells/well on permeable Transwell inserts supports. HAE cultures were generated by the provision of an air-liquid interface for 6 to 8 weeks to form fully differentiated, polarized cultures that resemble pseudostratified mucociliary epithelium. Human seasonal influenza vims type A strain H3N2 was obtained within the European Vims Archive EVA project. Vims stocks were generated by infecting MDCK cells at 90% confluency for 48 h. After that time cultures were aliquoted and stored at - 80°C. Mock samples were prepared in the same manner, using uninfected cells. Vims stocks were quantified by Reed&Muench titration.

Cell viability was evaluated using the XTT Cell Viability Assay kit (Biological Industries, USA) according to the manufacturer’s instructions. Cells were incubated with tested compounds for 48 h at 37°C in an atmosphere containing 5% C0 ₂ . After incubation, the medium was discarded and 100 mΐ of fresh medium was added to each well. Then, 50 mΐ of the activated 2,3-bis-(2-methoxy-4-nitro-5- sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) solution was added, and samples were incubated for 2 h at 37°C. The absorbance (l = 450 nm) was measured using Spectra MAX 250 spectrophotometer (Molecular Devices, USA). Data were presented as percent viability, taking the solvent treated cells as 100%.

Replication inhibition assay was performed as described below: MDCK or A549 cells were seeded on 96-well plates (TPP, Switzerland) at density lOVwell. After 24 h, cells were infected with influenza A vims at a dose of 400 50% tissue culture infectious dose (TCID ₅ o)/ml in the presence of tested compounds in a total volume of 100 mΐ in DMEM supplemented with penicillin/streptomycin and TPCK-treated trypsin 1 pg/ml (Sigma - Aldrich, Poland). The infection was carried out for 48 h. For HAE cultures, vims and tested compounds were applied on the apical surfaces of HAE cultures. Control samples were inoculated in the same manner with the same volume of mock and/or DMSO. After 2 h of incubation at 37°C, cells were rinsed thrice with PBS and fresh medium supplemented with the same tested compounds added at the same concentration. The samples were collected every 24 h. After every sample collection, fresh medium containing tested compounds was applied.

Quantitive real-time PCR. Viral RNA was isolated using the Viral DNA/RNA Isolation Kit (A&A Biotechnology, Poland) according to the manufacturer’s instructions. Then, reverse transcription was carried out with a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA) according to the manufacturer's instructions using a Veriti thermal cycler (Applied Biosystems, USA). cDNA quantity was determined by quantitative real-time PCR (qPCR). The reaction was carried out in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Reaction mixture contained 1 ^c RT HS-PCR Mix Probe (A&A Biotechnology Poland), specific probe labeled with 6-carboxyfluorescein (FAM) and Black Hole Quencher 1 (BHQ1) (sequence 5'-FAM CCG CCG AAC TGA GCA GAC ACC CGC GC BHQ1-3', 100 nM), primers (450 nM each, sense primer 5'-CAT CAC CGA CCC GGA GAG GGA C-3', antisense primer 5'-GGG CCA GGC GCT TGT TGG TGT A-3') and 2.5 mΐ of viral cDNA in 10 mΐ reaction mixture. For vims copy number quantification, standard DNA template of known copy number was serially diluted, and standard curve was determined. The lower limit of quantification under the conditions of conducting measurements is < 10 ³ copies / ml.

The results are expressed as means ± standard deviations (SD). The 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) values and were calculated using the Graph Pad Prism 6.0 function. Student t-test was used and P values < 0.05 were considered significant.

The results of quantitive real-time PCR analysis are presented as a negative decimal logarithm of the ratio of the total number of viral RNA copies in the test sample and the control sample infected with IAV vims without the addition of an inhibitor. The Log Removal Value (LRV) was calculated according to the formula:

LRV = -loglO (ci / cO)

, wherein ci - number of copies of the viral RNA in the sample with the addition of inhibitor [copies / ml], c 0 - number of copies of viral RNA in the control sample [copies / ml]. Usually, this way of presenting data is used in studies on the removal / inactivation of pathogens (bacteria, viruses or parasites) due to the simplicity of the result interpretation. Reducing the number of a given pathogen by 1 log means removing it in 90%, by 2 logs means removing 99%, by 3 logs means removing 99.9%, etc.

Fig. 1 demonstrates that tested compounds C-3380 and C-3389 did not exhibit any cytotoxicity towards MDCK cells, and they exhibit cytotoxicity to A549 cells only at a concentration of 100 mM The CC ₅ o values for the C-3389 and C-3380 toxicity in A549 cells are 51.13 and 81.21 mM, respectively.

Fig. 2 shows that compounds C-3380 and C-3389 have strong anti-influenza properties based on the analysis of real-time PCR results. Compounds C-3389 and C-3380 reduced the number of copies of viral RNA in the supernatants from both MDCK and A549 cells. C-3389 and C-3380 reduced the number of IAV RNA copies in the supernatant by -5,5 and 4,5 LRV in MDCK cells, respectively, as well as -4.06 and -4.25 LRV in A549 cells, respectively.

Fig. 3 shows that compound C-3389 inhibits IAV infection in HAE cultures. Quantitative Real-time PCR showed strong antiviral properties of Compound C-3389 (Fig. 3). This compound reduced the number of copies of viral RNA in the cell supernatant by -L, 2.0, and 2.8 LRV at 24, 48 and 72 hours after infection, respectively. These results show a strong and long-lasting inhibitory effect of Compound C-3389 in an in vitro model of human epithelial cells relevant to influenza vims infection pathophysiology.

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