ES2908523A1 - Andrographic derivatives for use in the treatment of COVID-19 disease and pulmonary fibrosis associated with COVID-19 (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Andrographic derivatives for use in the treatment of COVID-19 disease and pulmonary fibrosis associated with COVID-19. Andrographic derivatives for use as an antiviral for the treatment of COVID-19 disease or pulmonary fibrosis associated with COVID-19. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Androqrafolide derivatives for use in the treatment of CoV¡D-19 disease and CoV¡D-19 associated pulmonary fibrosis

The present invention relates to an andrographolide derivative for use as an antiviral for the treatment of CoViD-19 disease or CoViD-19 associated pulmonary fibrosis.

BACKGROUND OF THE INVENTION

The rapid expansion of the global CoViD-19 pandemic has posed a serious threat to health systems in all countries due to its high hospitalization and mortality rates. A significant percentage of CoViD-19 patients die from the cytokine storm, generated by an exaggerated inflammatory response of the human body, against an infection for which it has no prior immunity. On the other hand, many of the patients who survive the disease after their stay of greater or lesser duration in the Intensive Care Unit (ICU), are left with serious pulmonary sequelae due to the cytokine storm.

The pharmaceutical industry has developed several monoclonal antibodies directed at inhibiting inflammatory cytokines. Thus, Tocilizumab (Roche), Sarilumab (Sanofi), Siltuximab (Eusa Pharma) are interleukin-6 (IL-6) inhibitors; Anakinra (Amgen) is an interleukin-1 (IL-1) inhibitor and Canakinumab (Novartis) is an IL-ip inhibitor. IL-1beta IL-ip controls the inflammasome and therefore its inhibition is important in the management of inflammation.

On the other hand, the pharmaceutical industry has also developed drugs capable of inhibiting intracellular molecular pathways that also have an effect on the control of inflammation. Thus, for example, Baricitinib (Olumiant) is an inhibitor of the JAK1/JAK2 pathway.

Many of the above compounds have been used in clinical trials during the CoViD-19 pandemic to try to reduce the inflammatory response against the SARS-CoV-2 virus.

However, these drugs suffer from several limitations in practice: monoclonal antibodies often have a high toxicity that is reflected in the appearance of significant side effects; they inhibit only one component of inflammation without addressing the cytokine storm altogether; they are aimed exclusively at inhibiting the inflammatory response instead of modulating it.

For centuries in Ayurveda (traditional Indian medicine) the active principle andrographolide, present in the plant of Indian origin Andrographis paniculata, has been used for the treatment of acute respiratory diseases.

The Chinese pharmaceutical industry has in the past developed an injectable drug called Xiyanping. Although in the article D. Zhang et al., The clinical benefits of Chinese patent medicines against CoViD-19 based on current evidence, Pharm. Beef.

157 (2020) 104882, it is mentioned that said drug consists mainly of andrographolide sulfonate, although in reality it is a semi-synthetic preparation that contains a mixture of two derivatives of andrographolide, 9-dehydro-17-hydroandrographolide, and 9-dehydro- 17-hydro-andrographolide-19-yl sulfate, structural derivatives of andrographolide different from those presented in this invention.

Xiyanping has also been used as an effective alternative to antibiotics in clinical practice (Q. Li, et al., Xiyanping plus azithromycin chemotherapy in pediatric patients with mycoplasma pneumoniae pneumonia : a systematic review and metaanalysis of efficacy and safety, Evid.- Based Compl Alt 2019 (2019) 2346583).

Xiyanping is also effective as an antipyretic and anti-inflammatory (QW Yang, et al., Crystal structure and anti-inflammatory and anaphylactic effects of andrographlide sulphonate E in Xiyanping, a traditional Chinese medicine injection, J. Pharm. Pharmacol. 71 (2) ( 2019) 251-259).

Likewise, Xiyanping improves respiratory symptoms, inhibits opportunistic bacterial infections, and regulates immune function, with less clinical risk, especially through some liver protection, suggesting that it can alleviate the damage caused to the liver by certain drugs during CoViD treatment. -19 in acute cases (N. Cai, et al., Theoretical basis and effect characteristics of andrographolide against CoViD-19, Chin. Tradit. Herb. Drugs 51 (5) (2020) 1159-1166).

In addition, Xiyanping activity has been reported to improve sepsis in mice by suppressing MAPK, STAT3 and NF- ^k B signaling pathways, which play an important role in lung diseases (W. Guo, et al . al., Watersoluble andrographolide sulfonate exerts anti-sepsis action in mice through downregulating p38 MAPK, STAT3 and NF-kB pathways, Int. Immunopharmacol. 14 (4) (2012) 613-619).

On the other hand, the possibility of using andrographolide and other phytochemical compounds naturally present in Andrographis paniculata as antivirals has been studied using computational methods. Thus, very recently the interaction of andrographolide has been explored by computational modeling of molecular docking with specific components of SARS-CoV-2 for the treatment of acute respiratory syndrome associated with CoViD-19. In this context, S. Alagu Lakshmi et al., Ethnomedicines of Indian origin for combating CoViD-19 infection by hampering the viral replication: using structure-based drug discovery approach. J. Biomol. Struct. Dyn. (2020), doi: 10.1080/07391102.2020.1778537) indicate the affinity of bis-andrographolide for the main protease 3CLpro of SARS-CoV-2, which would potentially allow it to inhibit this enzyme.

The andrographolide-3CLpro main protease affinity of SARS-CoV-2 is also described by SK Enmozhi et al., Andrographolide as a potential inhibitor of SARS-CoV-2 main protease: an in silico approach, J. Biomol. Struct. Dyn. (2020), doi: 10.1080/07391102.2020.1760136).

Likewise, Shi et al have shown that both andrographolide and its fluorescent derivative, andrographolide conjugated with nitrobenzoxadiazole (Andro-NBD), inhibit the activity of the main protease of SARS-CoV-2 in vitro\ Andrographolide and its fluorescent derivative inhibit the main proteases of 2019-nCoV and SARS-CoV through covalent linkage. Biochem Biophys Res Commun. 2020 Aug 25; doi: 10.1016/j.bbrc.2020.08.086.

For their part, D. Sivaraman and PS Pradeep, Scope of phytotherapeutics in targeting ACE2 mediated host-viral interface of SARS-CoV2 that causes CoViD-19, doi: 10.26434/chemrxiv.12089730.v1) describe the blockade of the ACE2 cell receptor by andrographolide.

Likewise, using computational docking and molecular dynamics methods, the use of andrographolide and three structural derivatives (14-deoxy-11,12-didehydro andrographolide, neoandrographolide and 14-deoxy andrographolide) against four targets of the SARS- CoV-2, including three nonstructural proteins (major protease 3CLpro, PLpro, and RNA-directed RNA polymerase RdRp) and one structural protein ( spike protein (S)), which are responsible for virus replication, transcription, and cellular internalization, selecting neoandrogafolide as the best inhibitor (NA Murugan et al., Computational investigation on Andrographis paniculata phytochemicals to evaluate their potency against SARS-CoV-2 in comparison to known antiviral compounds in drug trials, J. Biomol. Struct. Dyn. (2020) , doi: 10.1080/07391102.2020.1777901).

Finally, in a docking study on 27 metabolites of plant origin, analyzing their coupling with various virus targets (major protease, Nsp9 protein, spike receptor domain , spike receptor ectodomain and HR2 domain), the andrographolide as significant against SARS-CoV-2 compared to other compounds (KF Azim et al., Screening and druggability analysis of some plant metabolites against SARS-CoV-2: An integrative computational approach, Informatics in Medicine Unlocked 20 (2020) 100367 ).

In addition, andrographolide has been tested as an antiviral in a microarray device containing some of the main proteins of the SARS-CoV-2 virus, obtaining positive results (P. Chen et al., Establishment and validation of a drug-target microarray for SARS -CoV-2, doi: 10.1080/07391102.2020.1777901).

On the other hand, the use of andrographolide in combination with melatonin has been suggested as a potentially effective treatment against CoViD-19. (A. Banerjee et al., Crosstalk between endoplasmic reticulum stress and anti-viral activities: A novel therapeutic target for CoViD-19, Life Sciences 255 (2020) 117842)

For all these reasons, it is critical to develop new antiviral drugs that have low toxicity and also have high bioavailability in the respiratory tract.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a compound selected from:

for use as an antiviral in the treatment of CoViD-19 or pulmonary fibrosis associated with CoViD-19.

In another embodiment, the invention relates to the compound for the use defined above, where the compound is selected from AG3 or AG5.

In another embodiment, the invention refers to the compound for the use defined above, characterized in that it can be administered parenterally or orally.

In another embodiment, the invention refers to the compound for the use defined above, where the compound is administered at a concentration of between 0.0001 mg/(kg h) and 10 mg/(kg h) for a time between 1 h to 2000 h, and preferably at a concentration between 0.01 mg/(Kg h) and 0.25 mg/(Kg h) for a time between 24 h and 480 h.

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

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a scheme of the evaluation of the antiviral effect of drugs. The experimental scheme carried out to evaluate the antiviral effect of different drugs in Vero E6 cells is shown. d0, day of infection with SARS-CoV-2.

FIG. 2 shows the evaluation of the antiviral effect of andrographolide, chloroquine and ivermectin. 2 x 104 Vero E6 cells (upper panels) were plated and infected with SARS-CoV-2 at MOI 0.05 as shown in Figure 2 in the absence (no drug) or presence of the indicated drugs, which were added 24 h before the virus (Pre-Tr) or with the virus (without Pre-tr). The supernatant was collected 48h after infection and the viral genomes were quantified by RT-qPCR with oligonucleotides specific for the viral N gene, using a stock of SARS-CoV-2 (isolate NAVARRA-2473) that had been previously titrated by a lysis plaque formation assay. The numbers on the bars indicate the percentage of inhibition of SARS-CoV-2 production in each condition with respect to cells infected without drugs, also indicating the statistical comparison between these samples, which was performed using an unpaired T test. *, p < 0.05; **, p < 0.01; ns, not significant. For all data, the mean ± standard error of the mean is shown. Androg, andrographolide; Chlor, chloroquine; Iver, ivermectin.

FIG. 3 shows the analysis of the antiviral effect of andrographolide and its derivative AG3 in Vero E6 cells. An experiment similar to the one described in Figure 3 was performed, in which the indicated concentrations of andrographolide (AG1) and AG3 were tested by putting the drugs 24h before infection with SARS-CoV2 or without drugs as a negative control. The supernatant was collected 48 hours after infection and the viral genomes were quantified as described in the previous figure. The left Y-axis shows the percentage of inhibition of viral production for each concentration of each drug (circles). In a parallel experiment, the cells were incubated only with the drugs for 96h and toxicity was analyzed using a commercial kit. (CelITiterGloassay, Promega). On the right Y axis, toxicity is represented as a percentage of dead cells for each drug concentration (squares). For all data, the mean ± standard error of the mean is shown.

FIG. 4 shows the antiviral activity of andrographolide (AG1), the original extract of Andrographis paniculata (EAp) and its structural derivatives (bath treatment) measured as a function of the gene expression of the viral protein N in Danio rerio larvae after infection by SVCV microinjection, compared to a control group that received no treatment. **, p < 0.01.

FIG. 5 shows the antiviral activity of andrographolide (AG1), the original extract of Andrographis paniculata (EAp) and its structural derivatives (bath treatment) measured as a function of protection against SVCV infection in baths in Danio rerio larvae. dpi = days post-infection.

FIG. 6 shows the antiviral activity of andrographolide (AG1), the original extract of Andrographis paniculata (EAp) and its structural derivatives (bath treatment) measured as a function of the gene expression of the viral protein N in Danio rerio larvae after infection by SVCV microinjection, compared to a control group that received no treatment. *, p < 0.05.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which reveal the effectiveness of the product of the invention.

EXAMPLE 1: Obtaining andrographolide ( 3-[2-[decahydro-6-hydroxy-5- ( hydroxy-methyl)-5,8a-dimethyl-2-methylene-1-naphthalenyl]ethylidene]dihydro-4-hydroxy-2 ( 3H)-furanone) from Andrographis paniculata extract

5 g of the crude extract of A. paniculata is suspended in 50 ml of Milli-Q® water in a separatory funnel. 500 ml of hexane is added, the mixture is shaken vigorously and allowed to decant for 1 hour. The organic phase is separated and the process is repeated twice, discarding the n-hexane fractions. Next, 500 ml of chloroform is added. The mixture is vigorously stirred and allowed to decant for 1 hour. The organic phase is carefully separated and the process is repeated twice, joining all chloroform fractions. The solvent is removed under reduced pressure and the oil obtained is diluted with 200 ml of methanol. This solution is heated to boiling, filtered and placed in an ice bath for 1 h. Next, the cold solution is stored in a refrigerator at 4 0C until almost complete evaporation of the solvent. Colorless andrographolide crystals are washed with cold methanol and dried at room temperature. A 3% yield of andrographolide (compound AG1) of purity greater than 99% is obtained.

1H NMR (400 MHz, DMSO) 6.62 (t, J= 6.4 Hz, 1H); 5.67 (d, J =5.7Hz, 1H); 5.01 (s, 1H); 4.91 (s, 1H), 4.81 (s, 1H); 4.62 (s, 1H); 4.39 (dd, J = 8.7, 6, 4Hz, 1H);4.11 (d, J=6.5Hz, 1H); 4.03 (d, J= 9.9Hz, 1H); 3.85 (d, J =10.6Hz, 1H); 3.28-3.14 (m, 2H); 2.45 (s, 1H); 2.32 (d, J =12.6 Hz, 1H); 2.03-1.82 (m, 2H); 1.70 (dd, J=29.9, 14.3Hz, 4H); 1.35 (dd, J=23.1, 12.7 Hz, 1H); 1.20 (d, J= 118 Hz, 2H), 1.09 (s, 3H); 0.66 (s, 3H).

13C NMR (75MHz, DMSO) 5,169.89; 147.57; 146.22; 128.95; 108.18; 78.43; 74.27; 64.50; 62.61; 55.47; 54.37; 42.26; 38.56; 37.48; 36.50; 27.87; 23.93; 23.03; 14.71. HR-MS (ESI, m/z) [M+H]+ Calcd for C20H29O5351.4628; found 351.4633.

EXAMPLE 2: Synthesis of 3,14,19-triacetyndrographolide

A solution of andrographolide (AG1, 7.2 mg, 0.0205 mmol) in acetic anhydride (0.5 mL) is heated to reflux and stirred for 1 h. After this time, it is diluted with ethyl acetate (20 ml) and quenched with saturated sodium bicarbonate solution (NaHCOs). It is then washed with water and extracted with ethyl acetate (3 x 20 ml). The organic phases are combined and washed with sodium chloride solution (10 ml), dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. The residue is purified by Flash chromatography in a Biotage® Selekt equipment using a hexane:ethyl acetate mixture (80:20 v/v) as eluant. In the fraction containing the product, the solvent is evaporated under reduced pressure and 9.2 mg of a white solid are obtained (3,14,19-triacetylandrographolide, compound AG3, 94%).

1H-NMR (400 MHz, CDCI3) 7.00 (td, J=7.0, 1.5 Hz, 1H, 12H); 5.92 (sd, J=6.0, 1.0 Hz, 1H, H-14); 4.90 (brs, 1H, H-17b); 4.60 (dd, J=12.0, 4.0 Hz, 1H, H-3); 4.54 (dd, J=11.0, 6.0 Hz, 1H, H-15b); 4.52 (brs, 1H, H-17a); 4.35 (d, J=11.5 Hz, 1H, H-19b); 4.24 (dd, J = 11.0, 2.0Hz, 1H, H-15a); 4.12 (d, J= 11.5 Hz, 1H, H-19a); 2.47 (ddd, J= 16.0, 6.5, 3.0 Hz, 1H, H-9); 2.44-2.33 (m, 2H, H-11); 2.12 (s, 3H, COCH3); 2.04 (s, 6H, 2 x COCH3); 2.01-1.45 (m, 5H); 1.39-1.29 (m, 4H); 1.02 (s, 3H, H-18); 0.75 (s, 3H, H-20).

13 C-NMR (100 MHz, CDCI3) 170.9 (COCH3); 170.5 (COCH3); 170.4 (COCH3), 169.0 (C-16); 150.1 (C-12); 146.5 (C-8); 124.0 (C-13); 108.9 (C-17); 79.7 (C-3); 71.6 (C-15); 67.8 (C-19); 64.7 (C-14); 55.9 (C-9); 55.3 (C-5); 41.3 (C-18); 38.9 (C-10); 37.9 (C-7); 37.0 (C-1); 25.2 (C2); 24.6 (C-6); 24.2 (C-11); 22.7 (C-18); 21.2 (COCH3); 21.1 (COCH3); 20.7 (COCH3); 14.5 (C-20).

HR-MS (ESI, m/z) [M+Na]+ calcd for C26H3603Na 499.2308; found 499.2311.

EXAMPLE 3: Synthesis of 14-deoxy-11,12-didehydroandrographolide

3.0 g of andrographolide (AG1, 8.6 mmol) are dissolved in anhydrous pyridine (8 mL). Next, 600 mg of aluminum oxide (Al203) are added to the previous solution. The reaction mixture is kept stirring at 1150C for 12 hours. After the reaction is complete, it is filtered and the solvent is evaporated to dryness. The crude is redissolved in dichloromethane (DCM) and washed with aqueous sodium chloride solution. The organic phase is dried over anhydrous sodium sulfate and the solvent is evaporated to dryness. The residue is purified by Flash chromatography in a Biotage® Selekt equipment using a hexane:ethyl acetate mixture (60:40 v/v) as eluant. In the fraction containing the product, the solvent is evaporated under reduced pressure, and obtain compound AG4 as a white solid (93%).

1H NMR (600MHz, DMSO-d6) 7.64 (s, 1H, 14-H), 6.72 (dd, J= 15.8, 10.1 Hz, 1H, 11-H), 6.10( d, J=15.8Hz, 1H, 12-H), 4.87 (s, 2H, 15-H), 4.71 (s, 1H, 17a-H), 4.40 (s, 1H, 17 bH),4.12(d, J=10.7Hz, 1H, 19a-H), 3.83 (d, J=10.9Hz, 1H, 19b-H), 3.23-3.17 (m , 1H, 3-H), 2.35 (dd, J=9.5, 4.3 Hz, 1H, 9-H), 2.00-1.91 (m, 1H, 7-H), 1 0.76-1.66 (m, 2H, 2-H), 1.59-1.31 (m,3H, 1-H, 6a-H), 1.21-1.14 (m, 1H.5 -H), 1.11 (dd, J=13.4, 4.4 Hz, 1H, 6b-H), 1.07 (s, 3H, 18-CH3), 0.74 (s, 3H, 20 -CH3).

HR-MS (ESI, m/z) [M+H]+ Calcd for C20H29O4, 333.2060; found 333,2062.

EXAMPLE 4: Synthesis of 14-deoxy-12 ( R)-sulfo-andrographolide

1.0 g of andrographolide (AG1, 2.9 mmol) are dissolved in 15 ml of 95% ethanol, heating said solution to 500C (solution 1). To 4 ml of Na2S031M solution, 4.8 ml of 2% H2SO4 (M/M) and 8 ml of water (solution 2) are added. Solution 1 is added to solution 2 and the reaction mixture is kept stirring at reflux for 30 minutes. After the completion of the reaction, the pH of the reaction is adjusted to pH 6~7 by adding 2% sulfuric acid (H2SO4) solution, and the solvent is evaporated to dryness. The residue is dissolved in water (20 ml) and extracted with chloroform (20 ml x 3). The solvent of the organic phase is evaporated under reduced pressure. The aqueous phase residue is dissolved in methanol (10 mL) and filtered. In the fraction containing the product, the solvent is evaporated under reduced pressure and 0.6 g of compound AG5 (51%) are obtained.

1H-NMR (600 MHz, CD3OD) 7.65 ppm (t, J= 1.8 Hz, 1H); 4.95 (or, 2H); 4.87 (or, 2H); 4.66 (brs, 1H); 4.16 (dd,J=10.2 and 6.1 Hz, 1H); 4.05 (d, J =11.4Hz, 1H); 3.92 (dd, J = 12.2 and 1.8 Hz, 1H); 3.30 (t, J =9.8Hz, 1H); 3.27 (d, J =11.4 Hz, 1H); 2.36 (m, H); 2.31 (dd, J =12.6 and 11.4 Hz, 1H); 2.08 (t, J =12.6 Hz, 1H); 1.86 (m, H); 1.83 (m, H); 1.80 (m, H); 1.71 (m, 2H); 1.38 (brd, J =11.8 Hz, 1H); 1.28 (qd, J =12.6 and 4.2 Hz, 1H); 1.12 (s, 3H); 1.10 (dd, J =12.6 and 2.4 Hz, 1H); 1.02 (m, H); 0.68 (s, 3H);

13C-NMR (150 MHz, CD3OD) 177.2; 152.1; 149.1; 132.5; 109.2; 81.7; 73.3; 65.8; 57.2; 56.8; 55.3; 44.5; 40.8; 40.1; 38.9; 29.8; 28.0; 26.1; 24.2; 16.4.

HR-MS (ESI, m/z) [M-H]" calcd for C20H29O7S, 413.1639; found 413.1634.

EXAMPLE 5: Antiviral ( SARS-CoV-2) efficacy of andrographolide and its structural derivatives in cell cultures

The antiviral activity of andrographolide and its structural derivatives was established based on their ability to inhibit the production of SARS-CoV-2 in the Vero E6 cell line after infection with an isolate of this coronavirus. At the Cima Universidad de Navarra there is an isolate of SARS-CoV-2 obtained from the nasal sample of a CoViD-19 patient admitted to the Clínica Universidad de Navarra (isolate NAVARRA-2473). Vero-E6 cells were infected with this sample, observing a strong cytopathic effect at 72 h. Virus-containing supernatants were titrated by lysis plaques on Vero-E6 cells yielding a titer of 4.3x107 PFU (plaque-forming units)/ml. The identity of the virus was confirmed by titration of viral genomes by RT-qPCR using a commercial diagnostic kit for CoViD-19 (BGI, China). Subsequently, a virus neutralization assay was set up, for which 2x104 Vero E6 cells were used in 96-well plates that were infected with SARS-CoV-2 at a multiplicity of infection (MOI = number of PFUs/cell). of 0.05. For infection, the cells were incubated with 50 µl of infection medium (MEM with 0.2% BSA, 2 mM glutamine and 20 mM Hepes) containing the indicated amount of virus (and the drug to be tested) for 1 h at 37° C/5% CO2 and with agitation every 15 minutes. Subsequently, the inoculum was removed, the wells were washed with 200 μl of PBS Ca++Mg++ (Invitrogene) and incubated at 37°C/5%C02 with 200 μl of normal culture medium (MEM 5% bovine fetal serum) containing also contained the drug. The culture medium, with or without the drug, was changed every 24 h during the experiment. Subsequently, the supernatants were collected at different times, the viral RNA was extracted using nucleic acid binding magnetic spheres and the virus produced was titrated by RT-qPCR with specific oligonucleotides for the N gene of SARS-CoV2 (Fw N1- 2019-nCov2: 5'- GACCCCAAAATCAGCGAAAT-3' (SEQ ID NO: 1) and Rv-N1-2019-nCov2: 5'-TCTGGTTACTGCCAGTT-GAATCTG-3' (SEQ ID NO: 2)). These trials were carried out using two treatment regimens: in one case, treatment with the drug to be tested began 24 h before virus infection and was maintained for 48 h post-infection (pi), at which time the supernatants were collected. to determine viral load. In the second case, the drug was administered at the same time as the virus and the supernatants were collected at 48h pi. Figure 1 schematically shows the process followed for the infection and treatment of the cultures.

In a first antiviral assay, only andrographolide was tested at a concentration of 5 pM (Table 1), using chloroquine (10 pM) and ivermectin (5 pM) as positive controls. Figure 2 shows the inhibition results on virus replication obtained with these compounds when treatment was started 24 h before infection (Pre-tr) or when it was started at the time of infection (without-Pre- tr). Likewise, Table 2 describes the main parameters of this study and summarizes the results. It is observed that andrographolide presented a potent antiviral activity against SARS-CoV-2 in Vero E6 cells, similar to that of ivermectin.

Table 1. Experimental conditions in in vitro assays with andrographolide and other antiviral agents on SARS-CoV-2.

Compound 11 * Concentration (pM) Collection time (p.i.) Analyzed time (p.i.)

Andrographolide 5 Vero E6: 48 h Vero E6: 48 h Chloroquine 10 Vero E6: 48 h Vero E6: 48 h

Ivermectin 5 Vero E6: 48 h Vero E6: 48 h

Table 2. Reduction of SARS-CoV-2 viral load in Vero E6 cells with andrographolide and other antiviral agents.

Compound Viral load reduction Pre-tr Viral load reduction without Pre-tr (%) (%) Andrographolide 93.5 94.0

(5 iM)

Chloroquine

_{(10 uM)} 100 100

Ivermectin

97.7 96.2

(5 iM)

Next, the activity of andrographolide and compound AG3 to inhibit the replication of the SARS-CoV-2 virus in Vero E6 cells was compared. This study was carried out with a range of drug concentrations from 1-20 p,M, in all cases with a 24h pretreatment as shown in Figure I. In Figure 3, the results obtained are shown. It is observed that both andrographolide (AG1) and the AG3 derivative show a strong inhibition of virus replication at a concentration of 5 p.M (98% AG1, 93% AG3). At higher concentrations (10 ^M), almost complete (>99%) blockade of virus replication occurs with both drugs, but their toxicity is increased. To evaluate this last parameter, the same amounts of cells were incubated with the drugs at the same concentrations and times indicated in figure 3. The measurement of toxicity was carried out by means of a luminescent assay to determine the number of viable cells in culture based on the quantification of ATP, which is an indicator of metabolically active cells. For this, a commercial kit (CelITiter-Gloassay, Promega) was used.

EXAMPLE 6: Validation of the antiviral activity of andrographolide and its derivatives in zebrafish larvae ( Danio rerio)

The antiviral activity of the compounds was evaluated in vivo by infection with the Spring viremia of carp rhabdovirus ( SVCV ), both by microinjections into Cuvier's duct (2 ni of the pathogen solution using a glass microneedle in a micro-manipulator), and by bath infections by administering the viral pathogen into Water. Fish were treated with the different compounds and infected to monitor mortalities and assess viral load by RT-qPCR of virus N protein expression.

In a first assay, the antiviral activity of compounds AG1 (28 p.M), AG3 (10 p.M ) and EAp (10 p.g/ml) was determined by micro-injection infection (1% ethanol solution in PBS ). The treatments were carried out in a bath for 24 h in fish 1 day after fertilization (5 fish/well). The experiment was carried out in triplicate (n = 3). On the second day after fertilization, each fish (2 ni in Cuvier's duct) was injected with 5x104 TCID50/ml of SVCV. At 24 h post-infection, the expression of the viral protein in the surviving fish was determined. The results obtained are shown in Figure 4, where it is observed that compounds AG1 and AG3 reduce the expression of protein N compared to a control group that did not receive treatment.

In a second assay, the antiviral activity of compounds AG1, AG4, AG5 and EAp was measured at different concentrations (10, 20 and 50 p,M for the pure compounds, 10, 20 and 50 p,g/ml for the extract). ) by bath infection and mortality monitoring. The treatments were carried out in a bath for 24 h on fish 2 days after fertilization (10 fish/well). At 4 days post-treatment, dip infection (7x104 TCID50/ml of SVCV) was carried out, and survival of the fish was observed for 5 days. The results obtained are shown in Figure 5, where it is observed that compounds AG4 and AG5 drastically inhibit SVCV infection in larvae and present very low or no toxicity in a very wide range of concentrations (up to 50 p,M ).

In a third assay, the antiviral activity of compounds AG1 (5 jo,M), AG3 (5 jM), AG4 (10 p,M) and AG5 (10 p,M), against SVCV, was analyzed. The treatments were carried out in a bath for 24 h on fish 1 day after fertilization (10 fish/well). The experiment was carried out in quadruplicate (n = 4). On the second day after fertilization, each fish (2 ni in Cuvier's duct) was injected with 5x104 TCID50/ml of SVCV. At 24 h, RNA was extracted from the fish to determine the expression of the viral protein N by means of quantitative PCR. Although all treatments reduced the burden viral, AG1, AG4 and AG5 did so significantly (Figure 6).

Ċ

Claims (4)

1. A compound selected from:

for use as an antiviral in the treatment of CoViD-19 or pulmonary fibrosis associated with CoViD-19. 2. The compound for use according to claim 2, wherein the compound is selected from AG3 or AG5. 3. The compound for use according to any of claims 1 or 2, characterized in that it can be administered parenterally or orally. 4. The compound for use according to any of claims 1 to 3, wherein the compound is administered at a concentration between 0.0001 mg/(kg h) and 10 mg/(kg h) for a time between 1 h to 2000hrs