ES2941090A1 - Compounds derived from phenylhydrazides as antiviral agents (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Compounds derived from phenylhydrazides as antiviral agents. The present invention relates to phenylhydrazide derivatives of formula (I) and their use as antiviral agents. These compounds are therefore useful for the treatment and/or prevention of viral diseases, such as the disease caused by the Ebola virus and/or swine fever. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Compounds derived from phenylhydrazides as antiviral agents

The present invention relates to phenylhydrazide derivatives and their use for the prevention and/or treatment of viral diseases. More particularly, it refers to the use of said compounds in the treatment and/or prevention of the disease caused by the Ebola virus (EBOV) and/or African swine fever (ASF). Therefore, the invention falls within the field of medicinal chemistry.

BACKGROUND OF THE INVENTION

Currently, we are witnessing an increase in emerging and re-emerging viral diseases worldwide. In fact, infectious diseases are one of the greatest public health concerns, both human and veterinary. Such is the case with the Ebola virus disease. It is a very serious and contagious disease, causing hemorrhagic fever and with a high mortality rate in humans, which can be as high as 90%. Although a vaccine has recently been approved (Saphire, EO A vaccine against Ebola virus. Cell 2020 , 181, 6) and treatments based on monoclonal antibodies (Markham, A. REGN-EB3: first approval. Drugs 2021, 81, 175-178 ) for this disease, due to the seriousness of the infection and the unpredictability of the appearance of new outbreaks, the need to develop specific antivirals is great (Malvy, D.; McElroy, AK; de Clerck, H.; Gunther, S. .; van Griensven, J. Ebola virus disease. Lancet 2019, 393, 936-948). In the case of veterinary infections, African swine fever monopolizes the news, being also one of the animal diseases with the greatest socioeconomic repercussions. The African swine fever virus (ASFV) causes a hemorrhagic disease with high mortality in domestic pigs and wild boars (Alejo, A.; Matamoros, T.; Guerra, M.; Andrés, G. A Proteomic atlas of the African swine fever virus particle. J Viro! 2018, 92, doi:10.1128/JVI.01293-01218).

Document WO2021111022A1 describes sulfur derivatives of phenylhydrazides as antiviral agents. These compounds are therefore useful for the treatment and/or prevention of viral diseases, such as the disease caused by the Ebola virus and swine fever.

In view of the state of the art, the present invention proposes new compounds as an alternative to those known for the treatment of viral diseases, especially the disease caused by the Ebola virus (EBOV) and/or African swine fever (ASF). .

DESCRIPTION OF THE INVENTION

The present invention presents a family of compounds that possess micromolar antiviral activity, and that are useful for the treatment of viral diseases such as those caused by the Ebola virus and the African swine fever virus.

In a first aspect, the present invention relates to a compound of formula (I)

or any of its isomers or their pharmaceutically acceptable salts, where x is 0 and R is a group selected from the following:

where n is 1 or 2 and Bn represents a benzyl group,

O well,

x is 1 and R is a group selected from the following:

where n is 1 or 2 and Bn represents a benzyl group.

In a preferred embodiment, x is 0 and R is a group selected from the following:

In a more preferred embodiment of the compounds for their uses indicated above, the invention relates to the compounds of formula (I) for their use selected from the list comprising:

2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-W',W'-diphenylacetohydrazide

2-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-W',W'-diphenylacetohydrazide

3-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-W,W-diphenylpropanohydrazide

3-(4-Methylpiperazin-1-yl)-W',W'-diphenylpropanohydrazide

3-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-W,W-diphenylpropanohydrazide

W-Benzyl-2-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-W-phenylacetohydrazide

W-Benzyl-3-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-W-phenylpropanohydrazide and

W-Benzyl-3-((2-(4-benzylpiperazin-1-yl)ethyl)amino)-W-phenylpropanohydrazide.

A second aspect of the invention refers to the process for the preparation of compounds of formula (I), as defined in the first aspect of the invention. Said procedure comprises the following stages:

a) Reaction of the corresponding hydrazide (1) with the corresponding acid chloride (2) in the presence of potassium carbonate to give the compound of formula (3),

b) Reaction of the compound of formula (3) obtained in the previous step with the corresponding amine (4) in the presence of triethylamine and under reflux of acetonitrile to give the compound of formula (I)

x = 0.1

n = 1.2

(3) (I)

where the amine (4) is selected from the following:

In a preferred embodiment, step a) is carried out using an acetone/water mixture as solvent, more preferably, an acetone/water mixture in a 1:2 volume ratio.

In a preferred embodiment, for each mole of hydrazide (1) 1.5 moles of potassium carbonate and/or 1.25 moles of acid chloride (2) are used.

Preferably the reaction of step a) is carried out at a temperature between 20 -22 °C room temperature and/or for a time between 14 and 18 hours, more preferably for 16 hours.

Preferably 10 mL of acetonitrile are used for each mole of compound of formula (3) used.

The reaction of step b) is carried out for a preferred time of between 14 and 18 hours, more preferably for 16 hours.

Preferably, two moles of triethylamine are used for each mole of compound of formula (3) in step b).

In a third aspect, the present invention relates to a compound of formula (I) or any of its isomers or their pharmaceutically acceptable salts, as defined in the first aspect of the invention, for use as a medicament.

In a fourth aspect, the present invention relates to a compound of formula (I) or any of its isomers or their pharmaceutically acceptable salts, as defined in the first aspect of the invention, for use in the treatment and / or prevention of viral diseases.

The expression "treatment or prevention" as used herein, unless otherwise indicated, means to reverse, alleviate, inhibit the progress of, or prevent the disorder or condition to which it applies in such terms, one or more symptoms of such disorder or condition.

A preferred embodiment of the invention relates to the compounds of general formula (I), as described above, for use in the treatment and/or prevention of Ebola disease and/or African swine fever.

The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), any of its isomers or their pharmaceutically acceptable salts, with a pharmaceutically acceptable excipient, adjuvant and/or vehicle.

The term "excipients, adjuvants and/or vehicles" refers to molecular entities or substances with which the active ingredient is administered. Such excipients, adjuvants or pharmaceutical vehicles can be sterile liquids, such as waters and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, excipients, disintegrants, wetting agents, or diluents.

In general, the therapeutically effective amount of the compound of formula (I) to be administered will depend, among other factors, on the individual (human or animal) to be treated, the severity of the disease suffered by said individual, the manner of elected administration, etc. For this reason, the doses mentioned in this invention should only be considered as guides for those skilled in the art, and the latter should adjust the doses based on the aforementioned variables.

The compounds of the invention represented by formula (I) for use may be in crystalline form as free compounds or as solvates and both forms are intended to be within the scope of the present invention. In this sense, the term "solvate", as used herein, includes both pharmaceutically acceptable solvates, that is, solvates of the compound of formula (I) that can be used in the preparation of a medicine, and non-pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically acceptable salts or solvates. The nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. Solvates can be obtained by conventional solvation methods well known to those skilled in the art.

Compounds of formula (I) for therapeutic use are prepared in solid or aqueous suspension form, in a pharmaceutically acceptable diluent. These preparations can be administered by any appropriate route of administration, for which said preparation will be formulated in the appropriate pharmaceutical form for the chosen route of administration. In a particular embodiment, the administration of the compound of formula (I) is carried out by oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, etc.).

The compounds described for use herein, their pharmaceutically acceptable salts, solvates as well as pharmaceutical compositions containing them can be used together with additional drugs to provide combination therapy. These additional drugs can form part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for co-administration or not with that of the pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof .

Unless otherwise indicated, compounds of the invention for use also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having such a structure, except for substitution of hydrogen for deuterium or tritium, or substitution of carbon for 13C- or 14C-enriched carbon or 15N-enriched nitrogen, are within the scope of this invention.

The present invention also relates to the use of a compound of formula (I), or any of its isomers or their pharmaceutically acceptable salts, for the preparation of a medicament, preferably a medicament for use in the treatment and/or prevention of viral diseases, more preferably Ebola disease and African swine fever.

The present invention also relates to a method for the treatment of Ebola disease and African swine fever in a subject or animal comprising the administration of a compound of formula (I) or any of its isomers or their pharmaceutically acceptable salts .

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages, and features of the invention will emerge in part from the description and in part from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

Next, the invention will be illustrated by means of tests carried out by the inventors, which show the effectiveness of the compounds of the invention as antivirals.

Example 1: Synthesis of compounds of the present invention

To carry out the tests, the inventors synthesized the compounds represented in the following table, following the synthesis procedure represented in scheme 1:

n = 1.2 x = 0.1

n = 1.2 Scheme 1: Synthesis of the new compounds of the invention

To an emulsion of the corresponding hydrazide (commercially available from Sigma Aldrich or Fluorochem) (10 mmol) and potassium carbonate (15 mmol) in a mixture of acetone (10 mL) and water (20 mL) the corresponding acid chloride is added slowly. (commercially available from Sigma Aldrich or Fluorochem) (12.5 mmol) and leave stirring overnight (16 h). Next, the acetone is removed under reduced pressure and the aqueous phase is extracted with ethyl acetate (25 mL x 3). The organic phase is washed successively with a saturated sodium bicarbonate solution (15 mL x 3), water (25 mL x 3) and a saturated sodium chloride solution (15 mL x 3). The organic phase is dried, filtered and evaporated under reduced pressure, obtaining the corresponding chloride that is used in the next step without further purification.

The corresponding previously synthesized chloride (0.3 mmol) is dissolved in anhydrous acetonitrile (3 mL). Next, add the appropriate amine commercially available from Sigma Aldrich or Fluorochem (0.3 mmol) and triethylamine (85 pL, 0.6 mmol), and reflux overnight. Subsequently, it is cooled to room temperature, ethyl acetate (15 mL) is added and it is washed with a saturated sodium chloride solution (10 mL x 3). The organic phase is dried, filtered and evaporated under reduced pressure, obtaining a crude reaction that is purified by column chromatography, eluting with the C^CEM eO H solvent mixture (from 99:1 to 90:10).

2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-W,W-diphenylacetohydrazide (la)

2-Chloro-W,W-diphenylacetohydrazide (78.0 mg, 0.3 mmol) and 2-(1-benzylpiperidin-4-yl)ethane-1-amine (65.5 mg) were used to obtain after purification the compound as a brown oil (33% yield).

1H NMR (300 MHz, DMSO-da) 510.89 (s, 1H), 7.36-7.23 (m, 8H), 7.17-7.02 (m, 5H), 7.02-6 .88 (m, 2H), 3.53 (s, 3H), 3.51 (s, 3H), 2.98 (t, J = 7.3 Hz, 2H), 2.80 (m, 2H) , 2.67 (m, 2H), 2.00 (m, 2H), 1.66 - 1.53 (m, 2H), 1.43 (m, 2H), 1.26 (t, J = 7, 2 Hz, 1H), 1.26 (m, 1H), 1.19 (c, J = 7.3 Hz, 3H), 13C NMR (75 MHz, DMSO-CI ⁶ ) 5 169.2, 146.1 , 138.0, 129.5, 129.4, 128.6, 127.5, 122.6, 119.2, 62.4, 53.3, 49.6, 46.3, 45.8, 34 .8, 33.1, 31.8, 9.2. HRMS (ESI+) calculated for C ²⁸ H ³⁵ N ⁴ O [MH]+ 443.2806; found 443,2792.

2-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-W,W-diphenylacetohydrazide (Id)

2-Chloro-W,W-diphenylacetohydrazide (78.0 mg, 0.3 mmol) and 2-(1-benzylpiperazin-4-yl)ethane-1-amine (65.8 mg) were used, obtaining after purification the compound as a brown oil (30% yield).

1H NMR (300 MHz, DMSO-da) 10.69 (s, 1H), 7.35 - 7.19 (m, 11H), 7.13 - 7.04 (m, 5H), 7.02 - 6.91 (m, 2H), 3.40 (s, 2H), 3.29 (s, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.36 (dt, J = 13.6, 4.6 Hz, 11H), 13C NMR (75 MHz, DMSO-da) 5 171.5, 146.1, 138.6, 129.4, 129.2, 128.5, 127, 3, 122.4, 119.1, 62.5, 57.7, 53.1, 53.0, 51.2, 46.3. HRMS (ESI+) calculated for C ²⁷ H ³⁴ N ⁵ O [MH]+ 444.2758; found 444,2743.

3-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-W,W-diphenylpropanohydrazide (Ib)

3-Chloro-W,W-diphenylpropanohydrazide (82.4 mg, 0.3 mmol) and 2-(1-benzylpiperidin-4-yl)ethane-1-amine (65.5 mg) were used, obtaining after purification the compound as a brown oil (42% yield).

1H NMR (300 MHz, DMSO-da) 510.81 (s, 1H), 7.32 - 7.24 (m, 9H), 7.15 - 7.04 (m, 4H), 7.02 - 6.92 (m, 2H), 3.45 (s, 2H), 3.05 (t, J = 7.2 Hz, 2H), 2.87 - 2.75 (m, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.89 (d, J = 11.0 Hz, 3H), 1.68 - 1.54 (m, 2H), 1.49 (m, 2H), 1, 38 - 1.23 (m, 1H), 1.22 - 0.98 (m, 2H), 13C NMR (75 MHz, DMSO-d6) 5169.6, 146.0, 138.7, 129.7, 129.3, 129.1, 127.2, 122.5, 119.0, 62.6, 53.3, 45.4, 43.4, 33.3, 33.1, 31.9, 30, 7. HRMS (ESI+) calculated for C ²⁹ H ³⁷ N ⁴ O [MH]+: 457.2962; found 457,2953.

3-(4-Methylpiperazin-1 -yl)-W,W-diphenylpropanohydrazide (Ic)

3-Chloro-W,W-diphenylpropanohydrazide (82.4 mg, 0.3 mmol) and 1-methylpiperizine (30.5 mg) were used, the compound being obtained after purification as a brown oil (44% yield).

1H NMR (300 MHz, DMSO-^) 510.56 (s, 1H), 7.36 - 7.21 (m, 4H), 7.21 - 7.04 (m, 4H), 7.02 - 6.88 (m, 2H), 3.04 (q, J = 7.3 Hz, 1H), 2.70 - 2.53 (m, 10H), 2.36 (m, 5H), 1.19 (t, J = 7.3 Hz, 2H), 13C NMR (75 MHz, DMSO-d6) 5170.9, 146.1, 129.3, 122.4, 119.1,54.4, 53.9, 51.8, 45.8, 45.1, 31.8, 9.0. HRMS (ESI+) calculated for C ²⁰ H ²⁷ N ⁴ O [MH]+: 339.2180; found 339.2173.

3-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-W,W-diphenylpropanohydrazide (le)

3-Chloro-W,W-diphenylpropanohydrazide (82.4 mg, 0.3 mmol) and 2-(1-benzylpiperizin-4-yl)ethane-1-amine (65.8 mg) were used, obtaining after purification the compound as a brown oil (39% yield).

¹ H NMR (300 MHz, DMSO-da) 510.76 (s, 1H), 7.32-7.24 (m, 9H), 7.10 (dd, J = 7.5, 1.6 Hz, 4H), 6.98 (t, J = 7.3 Hz, 2H), 3.43 (s, 2H), 3.02 (t, J = 6.9 Hz, 3H), 2.86 (t, J = 6.0 Hz, 2H), 2.59 (t, J = 6.9 Hz, 2H), 2.50-2.30 (m, 10H), ¹³ C NMR (75 MHz, DMSO-da) 5170.3, 146.0, 138.5, 129.7, 129.3, 129.1, 128.4, 122.4, 119.0, 62.3, 55.3, 53.0, 52, 7, 44.8, 44.1, 31.5. HRMS (ESI+) calculated for C ²⁸ H ³⁶ N ⁵ O [MH]+: 458.2914; found 458.2906.

M,-Benzyl-2-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-M'-phenylacetohydrazide (If) W-benzyl-2-chloro-W-phenylacetohydrazide (82.4 mg) was used , 0.3 mmol) and 2-(1-benzylpiperidin-4-yl)ethane-1-amine (65.5 mg), the compound being obtained after purification as an orange oil (36% yield).

¹ H NMR (300 MHz, DMSO-da) 510.20 (s, 1H), 7.45 - 7.37 (m, 3H), 7.37 - 7.22 (m, 9H), 7.21 - 7.12 (m, 2H), 6.84 - 6.77 (m, 2H), 6.76 - 6.69 (m, 1H), 4.67 (s, 2H), 3.51 (s, 2H), 3.36 (d, J = 17.6 Hz, 3H), 2.81 (d, J = 11.1 Hz, 3H), 2.60 (t, J = 7.4 Hz, 2H) , 1.96 (q, J = 8.7, 6.3 Hz, 2H), 1.59 (d, J = 12.9 Hz, 2H), 1.49 - 1.22 (m, 3H), 1.17 - 1.14 (m, 2H), ¹³ C NMR (75 MHz, DMSO-d ⁶ ) 5 168.9, 149.2, 138.3, 129.4, 129.2, 128.7, 128.6, 128.0, 127.4, 118.9, 112.9, 62.5, 56.9, 53.4, 49.6, 46.2, 34.9, 33.2, 31, 9. HRMS (ESI+) calculated for C ²⁹ H ³⁷ N ⁴ O [MH]+: 457.2962; found 457,2958.

M,-Benzyl-3-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-M'-phenylpropanohydrazide (Ig) W-benzyl- 3-chloro-W-phenylpropanohydrazide (86.6 mg) was used , 0.3 mmol) and 2-(1-benzylpiperidin-4-yl)ethane-1-amine (65.5 mg), the compound being obtained after purification as a brown oil (26% yield).

¹ H NMR (300 MHz, CDCh) 59.64 (s, 1H), 7.33 - 7.03 (m, 11H), 6.95 - 6.65 (m, 4H), 4.65 (s, 2H), 3.39 (s, 2H), 2.76 (d, J = 10.9 Hz, 2H), 2.68 - 2.62 (m, 2H), 2.40 (t, J = 7 .2 Hz, 2H), 2.27 (t, J = 5.8 Hz, 2H), 1.79 (t, J = 10.4 Hz, 2H), 1.53 - 1.43 (m, 2H ), 1.27 -1.08 (m, 5H), ¹³ C NMR (75 MHz, CDCh) 5 172.3, 149.2, 137.6, 129.7, 129.6, 128.9, 128 .6, 128.5, 127.8, 127.4, 119.8, 113.3, 63.7, 56.3, 54.0, 46.9, 45.4, 36.6, 34.5 , 33.9, 32.5. HRMS (ESI+) calculated for C ³⁰ H ³⁹ N ⁴ O [MH]+: 471.3118; found 471.3114.

W-Benzyl-3-((2-(4-benzylpiperazin-1-yl)ethyl)amino)-W-phenylpropanohydrazide (Ih) W- benzyl-3-chloro-W-phenylpropanohydrazide (86.6 mg, 0 0.3 mmol) and 2-(1-benzylpipericin-4-yl)ethane-1-amine (65.8 mg), the compound being obtained after purification as a brown oil (22% yield).

1H NMR (300 MHz, CDCh) 59.64 (s, 1H), 7.32 - 7.02 (m, 12H), 6.86 - 6.80 (m, 2H), 6.79 - 6.72 (m, 1H), 4.66 (s, 2H), 3.39 (s, 2H), 2.94 - 2.59 (m, 3H), 2.61 - 2.08 (m, 14H), 13C NMR (75 MHz, CDCh) 5 171.8, 148.9, 137.9, 137.3, 129.2, 129.2, 128.5, 128.2, 127.4, 127.1, 119 .4, 113.0, 62.9, 56.8, 56.0, 53.0, 52.9, 52.9, 52.8, 45.2, 44.89, 33.9. HRMS (ESI+) calculated for C ²⁹ H ³⁷ N ⁴ O [MH]+: 472.3071; found 472,3074.

Example 2: Study of the antiviral activity of the compounds of formula (I) against the Ebola virus (EBOV)

Materials and methods

cell lines

Human embryonic kidney 293T/17 cells (ATCC-CRL-11268) and HeLa human cervical adenocarcinoma cells (ATCC-CCL-2), baby hamster kidney (BHK-21/WI-2, Kerafast #EH1011) and Kidney cells from the African green monkey Cercopithecus aethiops (VeroE6) were cultured in DMEM medium supplemented with 10% FBS, 1% L-glutamine and 25 pg/mL gentamicin in an incubator at 37 °C in the presence of 5% CO2. .

Production of recombinant viruses based on the human immunodeficiency virus (HIV), with envelopes (GP/G) of Ebola virus (ZEBOV) Mayinga or Vesicular Stomatitis Virus (VSV) and expressing luciferase after infection of susceptible cells.

Viral particles based on the HIV virus were generated, pseudotyped with the glycoprotein of the Ebola-Mayinga strain (GeneBank: U23187.1) (ZEBOV-GP) or the glycoprotein of the Vesicular Stomatitis Virus (VSV-G) (GenBank: X03633.1) expressing luciferase protein after infection of susceptible cells.

The production of these viral particles was carried out using the calcium chloride transfection method (Life Technologies, Carlsbad, CA, USA) in 293T cells by adding the plasmids: pNL4.3.Luc.RE- (Program of NIH AIDS reagents, Dr. Nathaniel Landau's Division of AIDS) and the respective glycoprotein of each virus.

Supernatants containing recombinant viruses were collected 48 h after transfection, centrifuged at 1200 rpm for 10 min at room temperature to remove cell debris, and stored frozen at -80 °C in aliquots.

Infectious titers of each viral pseudotype were estimated by performing serial dilutions (triplicate 1:5 serial dilutions) of viral supernatants and infecting 293T cells. Luciferase activity was determined 48 h after infection by luciferase assay (Luciferase Assay System, Promega, Madison, WI) using the GloMax® Navigator Microplate Luminometer (Promega) for reading.

Production of recombinant viruses based on Vesicular Stomatitis Virus (VSV), with envelopes (GP/G) of Ebola virus (ZEBOV) Mayinga or Vesicular Stomatitis Virus (VSV) and expressing luciferase after infection of susceptible cells.

For the generation of pseudotypes based on the Vesicular Stomatitis Virus, BHK-21 cells (Kerafast) were transfected with plasmids encoding the Ebola virus envelope protein (Mayinga strain) or the Vesicular Stomatitis Virus glycoprotein itself ( G) using Lipofectamine 3000 (Thermo Fisher Scientific). After 24 h, the cells are inoculated with recombinant pseudotypes of VSV-delta G-luc, deficient in their replicative capacity (MOI: 3-5) that contain the firefly luciferase gene in substitution of the virus's own glycoprotein. thus allowing the incorporation of heterologous glycoproteins into the new viral particles that are generated.

After 1 h of incubation at 37 °C, the inoculum is removed, the cells are washed several times with PBS and finally fresh medium is added.

Viral particles are collected 20–24 h post-inoculation, cell debris is removed by centrifugation (1200 rpm, 10 minutes) and stored in aliquots in a -80 °C freezer.

The calculation of the infectious titer (infectious dose per milliliter) was calculated by limiting dilution of the virus stock using Vero E6 cells as the susceptible line. Luciferase activity was determined 24 h after infection by luciferase assay (Steady-Glo® Luciferase Assay System, Promega) using the GloMax® Navigator Microplate Luminometer (Promega) for reading.

Screening for compounds inhibitory to Ebola virus infection

Screening for Ebola virus entry inhibitory compounds was performed by infecting 293T cells with ZEBOV-GP pseudotypes in 96-well plates in the presence of each compound at a final concentration of 10 pM.

293T cells (2x10 4 cells/well) were incubated at 37 °C for 1 h with each of the compounds and then exposed to 5000 TCID (culture infectious dose) of the aforementioned recombinant viruses. After 48 h of incubation, cells were washed with PBS, lysed by the addition of Steady-Glo Lyssis Buffer (Promega), and light was measured on a GloMax®-Multi detection system (Promega, Madison, WI, USA). USA) with the luciferase assay system (Promega, Madison, WI).

As a specific inhibition control, the recombinant virus VSV-G was used under the same conditions and in parallel to the infection with Ebola pseudotypes.

Inhibition values were calculated from 3 independent experiments (n=6) and are reported as the arithmetic mean.

Compounds whose inhibitory activity against ZEBOV-GP pseudotypes reduced infection by more than 80% at a final concentration of 10 pM were selected for further analysis (IC ⁵⁰ and cytotoxicity).

Calculation of CI values ^fifty

To calculate the IC ⁵⁰ value, serial concentrations of each compound from 10 pM to 10 nM were used.

Inhibition values were calculated using the mean of 3 independent experiments (n=6) using GraphPad Prism v6.0 software, with a 95% confidence interval and a setup to normalize dose-response curves.

Compound toxicity analysis

Hela cells (2x104) treated with different concentrations were used: 10, 100 and 250 pM of each compound for 48 hours in a 96-well plate. After this period, the cells were washed with culture medium and viability was measured by the colorimetric method (Cell Titer 96 AQueous non-radioactive cell proliferation assay, Promega).

Briefly, 20 pL of the combined MTS/PMS solution (reagent) was mixed with 100 pL of cells in culture medium and further incubated for 2 hours at 37 °C in a humidified atmosphere of 5% CO ² . The absorbance was recorded at 490 nm using an ELISA plate reader. Cell viability was calculated as the percent absorbance in cells with compound relative to untreated cells.

Results

aCl ⁵⁰ : Inhibitory concentration 50% or required to reduce viral infection by 50% in the pseudotypes indicated in each case.

bCC ⁵⁰ : Cytotoxic concentration 50% or required to reduce cell growth in 50%

cIS: Selectivity index (CC ⁵⁰ /CI ⁵⁰ )

Example 3: Study of the antiviral activity of the compounds of formula (i) against the African swine fever virus (ASFV)

Materials and methods

Treatment and infection

To carry out the various assays, ASFV infections were performed on Vero cell monolayers (ATCC-CCL-81) (renal fibroblasts) at a multiplicity of infection (moi) of 5 pfu/cell of the non-pathogenic ASFV Ba71V isolate. adapted to Vero cells (Enjuanes, L., Carrascosa, AL, Moreno, MA, Vinuela, E., 1976. Titration of African swine fever (ASF) virus. J Gen Virol 32, 471-477.) or with recombinant viruses fluorescents for flow cytometry as described below. Viral inocula were always added to the minimum volume necessary to cover the cell mat, after prior treatment of the cells with the compounds for one hour at 37 °C. Once the pre-treatment time had elapsed, the viral inoculum was allowed to absorb for 60 minutes at 37 °C and finally, the medium was removed together with the viral inoculum and replaced with Dulbecco's Modified Eagle culture medium (DMEM) supplemented. with 2% fetal bovine serum (SFB) in combination with the different compounds to continue with the treatment until reaching 16 hours post infection (hpi).

All compounds were initially diluted in dimethyl sulfoxide (DMSO) to a concentration of 50 mM. Next, they were diluted in 2% DMEM medium in fetal bovine serum (FBS) until reaching the required concentration in each case.

Cytotoxicity Assays

Cell viability in the presence of the different compounds at various concentrations was determined after 24 hours using the commercial kit "Cell titer 96 Aqueous Non-Radioactive Cell Proliferation Assay" (Promega) by reading the absorbance values at 490 nm, following the manufacturer's instructions. Those concentrations that allowed a cell viability equal to or greater than 80% were considered non-cytotoxic. This system is based on the colorimetric quantification of the reduction of tetrazolium to formazan by enzyme activity. metabolically active cell dehydrogenases.

Detection and quantification of viral DNA

After the infection and treatment with the different compounds, the cells were extracted and the DNA was subsequently obtained and purified with the "Dneasy blood and tissue kit" (Quiagen). The concentrations obtained were quantified in the spectrophotometer " UV-Vis Nanodrop ND-1000” (Fisher Thermo Scientific).

For the analysis of viral replication, quantitative real-time PCR (qPCR) was performed using fluorescent hybridization probes aimed at amplifying the viral p72 gene region as previously described (King, DP; Reid, SM; Hutchings, GH; Grierson, SS; Wilkinson, PJ; Dixon, LK; Bastos, AD; Drew, TW Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. J Virol Methods 2003, 107, 53-61). For this, previously purified DNA samples were analyzed, using water as a negative control, samples treated with DMSO as a positive control, and 5 serial dilutions of purified viral DNA of known concentration as a standard curve. The amplification mixture was made up of 150 ng of DNA from each of the samples, the Taqman probe (Roche), the OE3F and OE4R primers whose sequence is described by King et al. ( J Virol Methods 2003, 107, 53-61), obtained from Fisher Scientific and "Premix Ex Taq (2X)" (Takara).

The amplification and quantification process was carried out in an "ABI 7500 Fast Real-Time PCR System” thermal cycler (Applied Biosystems) with the following parameters: 1 cycle of 94 °C for 10 minutes, 45 cycles of 94 °C for 15 seconds and 58 °C for 60 seconds.

Flow cytometry

For flow cytometric analysis without antibody labeling, cells were infected with laboratory-made recombinant viruses BPP30GFP or B54GFP, described in Barrado-Gil, L.; Galindo, I.; Martinez-Alonso, D.; Viedma, S.; Alonso, C. The ubiquitin-proteasome system is required for African swine fever replication. PLoS One 2017, 12, e018974 and in Hernaez, B.; Escribano, JM; Alonso, C. Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera. Virology 2006, 350, 1-1, respectively. After 16 hpi the cells were lifted with trypsin and centrifuged at 2500 rpm for 5 minutes. The supernatants were discarded and the cells were resuspended in FACS (fluorescenceactivated cell sorter) buffer. Finally, 10,000 cells per experiment were analyzed in a "FACS Canto II" flow cytometer (BD Science) to determine the percentage of infected cells. The percentage obtained after treatment with the compounds was normalized with the values of the controls. Once the results were obtained with the optimal concentrations, the IC ^50s were calculated using different concentrations of compounds.

Statistic analysis

All data were obtained by performing the experiments in triplicate and normalizing the measurements to control values. The statistical analysis of the variance between data was performed with the GraphPad Prism 6 software, applying the Bonferroni test in multiple comparisons.

Results

aCI ⁵⁰ : 50% inhibitory concentration or required to reduce viral infection by 50%.

bCC ⁵⁰ : Cytotoxic concentration 50% or required to reduce cell growth by 50%.

cIS: Selectivity index (CC ⁵⁰ /CI ⁵⁰ )

Claims (11)

1. Compound of formula (I):

or any of its isomers or their pharmaceutically acceptable salts, where x is 0 and R is a group selected from the following:

where n is 1 or 2 and Bn represents a benzyl group, O well, x is 1 and R is a group selected from the following:

where n is 1 or 2 and Bn represents a benzyl group. 2. Compound according to claim 1, where x is 0 and R is a group selected from the following:

where n is equal to 2. 3. Compound according to claim 1, selected from the list comprising: 2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-W',W'-diphenylacetohydrazide 2-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-W',W'-diphenylacetohydrazide 3-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-W,W-diphenylpropanohydrazide 3-(4-Methylpiperazin-1-yl)-W',W'-diphenylpropanohydrazide 3-((2-(4-Benzylpiperazin-1-yl)ethyl)amino)-W,W-diphenylpropanohydrazide W-Benzyl-2-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-W-phenylacetohydrazide W-Benzyl-3-((2-(1-benzylpiperidin-4-yl)ethyl)amino)-W-phenylpropanohydrazide and W-Benzyl-3-((2-(4-benzylpiperazin-1-yl)ethyl)amino)-W-phenylpropanohydrazide. 4. Process for the preparation of a compound of formula (I), as described in any of claims 1 to 3, comprising: a) the reaction of the corresponding hydrazide (1) with the corresponding acid chloride (2) in the presence of potassium carbonate to give the compound

b) the reaction of the compound of formula (3) obtained in the previous step with the corresponding amine (4) in the presence of triethylamine and under reflux of acetonitrile to give the compound of formula (I)

x = 0.1 n = 1.2 (3) (I) where the amine (4) is selected from the following:

5. Process according to claim 4, wherein stage a) is carried out using an acetone/water mixture as solvent. 6. Process according to claim 4 or 5, wherein for each mole of hydrazide (1) 1.5 moles of potassium carbonate and/or 1.25 moles of acid chloride (2) are used. 7. Process according to any of claims 5 to 6, wherein the reaction of step a) is carried out at room temperature and/or for a time between 14 and 18 h. 8. Compound of formula (I) or any of its isomers or their pharmaceutically acceptable salts, as described in any of the preceding claims 1 to 3, for use as a medicament. 9. Compound of formula (I) or any of its isomers or their pharmaceutically acceptable salts, as described in any of the preceding claims 1 to 3, for use in the treatment and/or prevention of viral diseases. 10. Compound of formula (I) or any of its isomers or their pharmaceutically acceptable salts for use, according to claim 9, wherein the viral diseases are Ebola disease and/or African swine fever. 11. Pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), any of its isomers or their pharmaceutically acceptable salts, as defined in any of the preceding claims 1 to 3, with an excipient, adjuvant and/or or pharmaceutically acceptable vehicle.