WO2021191882A1 - Antimalarial agent, methods and uses thereof - Google Patents

Abstract

The present disclosure relates to compound of the formula I; or a pharmaceutically acceptable salt, ester, solvate thereof, for use in medicine, in particular as antimalarial agent. The present disclosure also relates to methods to obtain the compound of formula I.

Description

D E S C R I P T I O N

ANTIMALARIAL AGENT, METHODS AND USES THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to compound of the formula I; or a pharmaceutically acceptable salt, ester, solvate thereof, for use in medicine, in particular as antimalarial agent.

[0002] Furthermore, the present disclosure also relates to methods to obtain the compound of formula I.

TECHNICAL BACKGROUND

[0003] Malaria affects the whole world, with half of the global population at risk of infection.

[0004] Malaria is caused by Plasmodium parasites. Five species are capable of infecting humans: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum is the most virulent form and is responsible for majority of the death toll.

[0005] Most of the drugs currently used for treating malaria patients include artemisinins, aminoquinolines, arylaminoalcohols and anti-folate drugs.

[0006] Presently, there is a growing need for antimalarial agents, either for monotherapy or as multi-drug therapies as drug-resistant strains are emerging worldwide, in particular mefloquine and chloroquine resistant strains. Therefore, new agents able to kill the resistant malaria parasites are warranted.

[0007] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION

[0008] The present disclosure relates to compound of the formula I; or a pharmaceutically acceptable salt, ester, solvate thereof, for use in medicine, in particular as antimalarial agent.

[0009] Furthermore, the present disclosure also relates to methods to obtain the compound of formula I.

[0010] The present disclosure relates to antimalarial agents and to methods of preparing them. The advantage of the present disclosure is that the compound disclosed is highly active in different parasite stages, for both drug sensitive and multidrug resistant malaria strains. This advantage is relevant to counteract development of drug resistance mechanisms. This disclosure further describes the method of synthesizing and isolating the novel class of styrylpyridine and 3-arylacrylate-based antimalarial compounds.

[0011] The present disclosure relates to novel antimalarial agents, in particular, the present disclosure describes the unique method of synthesising new styrylpyridine and 3-arylacrylate-based compounds by combining at least two different molecules thus giving rise to compounds with an interesting associated biological profile.

[0012] Furthermore, the solution herein presented relates to the synthesis of new compounds and their antimalarial activity by in vitro screening using survival and growth in vitro assays of P. falciparum cultures using SYBR^® Green DNA staining. The activity of the compounds was explored at different levels.

[0013] The present disclosure describes new antimalarial styrylpyridine and 3- arylacrylate-based compounds and the toxicity effect of these novel compounds in non- neoplastic cell lines MCF-10A (breast) and HK2 (kidney).

[0014] In an embodiment, styrylpyridines show a multistage action at the erythrocytic P. falciparum cycle.

[0015] In an embodiment, styrylpiridines show similar action on P. falciparum 3D7 sensitive line as well as P. falciparum Dd2 multidrug resistant line. [0016] In an embodiment, styrylpiridines induce oxidative cell death.

[0017] In an embodiment, the Caenorhabditis elegans (C. elegans nematode) model was used as an early-stage drug discovery process for toxicity screening.

[0018] The compound of the present subject-matter exhibits a unique and promising antimalarialprofile.

[0019] The present disclosure describes a compound of the formula I, a pharmaceutically acceptable salt, ester, solvate, hydrate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph comprising the following formula:

wherein:

R¹, R², R³ and R⁴are independently selected from each other;

R¹ is selected from a H, alkyl, aryl, alkoxyl, acetyl, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl derivative, ketone, ester, carboxyl group, heterocyclic ring;

R² is selected from a H, alkyl, aryl, alkoxyl, acetyl, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl, ketone, ester, carboxyl group, heterocyclic ring;

R³ is selected from a H, alkyl, aryl, alkoxyl, acetyl, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl, ketone, ester, carboxyl group, heterocyclic ring;

R⁴ is selected from a H, alkyl, aryl, alkoxyl, acetyl, acyl, halogen, nitro, hydroxyl, amine, amide, carbonyl, ketone, ester, carboxyl group, heterocyclic ring.

[0020] The present disclosure describes a compound of the formula I, a pharmaceutically acceptable salt, ester, solvate, hydrate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph comprising the following formula:

wherein

R¹, R², R³ and R⁴are independently selected from each other;

R¹ is selected from a list consisting of: H, alkyl, alkoxyl, halogen, hydroxyl, carbonyl; R² is selected from a list consisting of: H, hydroxyl, carbonyl;

R³ is selected from a list consisting of: H, hydroxyl, carbonyl, cyano, carboxyl;

R⁴ is selected from a list consisting of: amine, amide.

[0021] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein R¹ is selected from a list consisting of: H, fluoro, bromo, chloro, halo-methoxy.

[0022] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein R³ is a H, CN, COOH or COO-. Preferably, wherein R³ is selected from a list consisting of: H, cyano.

[0023] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein R² is a hydroxyl.

[0024] In an embodiment, R⁴ is selected from a list consisting of: heteroarylamine, amide, preferably heteroarylamine, heteroarylamide.

[0025] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein R⁴ is a pyridine or a pyridinium salt derivative.

[0026] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein R4 is a pyridine-moiety. [0027] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein R⁴ is selected from the following list: iminiopyridin-1(4H)- yl)vinyl unit, ethyl pyridin-4(1H)-ylidene unit, iminiopyridin-1(4H)-yl unit or 4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl unit.

[0028] In an embodiment, the compound or a pharmaceutically acceptable, salt, hydrate, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer, or polymorph wherein the compound is selected from the following list:

[0029] In an embodiment, the compound disclosed is for use in medicine or veterinary. [0030] In an embodiment, the compound disclosed is for use in the prevention, therapy or treatment of P. falciparum infection.

[0031] In an embodiment, the compound disclosed is for use in the prevention, therapy or treatment of parasite infection.

[0032] In an embodiment, the compound disclosed is for the prevention, therapy or treatment of malaria diseases.

[0033] In an embodiment, the compound disclosed is for the prevention, therapy or treatment of P. falciparum infection.

[0034] In an embodiment, a pharmaceutical composition comprising the disclosed compound in a therapeutically effectively amount and a pharmaceutically acceptable excipient.

[0035] In an embodiment, a pharmaceutical composition comprising the disclosed compound wherein the pharmaceutically acceptable excipient is a carrier, adjuvant, excipient or mixtures thereof.

[0036] In embodiment, the pharmaceutical composition comprising the disclosed compound may further comprise an anti-viral compound, preferably wherein the antiviral compound is an anti-HIV compound or anti-tuberculosis compound.

[0037] In embodiment, the pharmaceutical composition comprising the disclosed compound may further comprise a second anti-malarial agent, preferably wherein the second anti-malarial agent is selected from the group consisting of: Chloroquine, Fansidar, Amodiaquine, Quinine, Halofantrine, Mefloquine, Artemether/Artesunate and Malarone.

[0038] Another aspect of the present disclosure relates to a method for preparing the disclosed compound comprising the following steps:

Adding pyridinium salt (0.03-0.19 g; 1-1.6 eq) to a solution/suspension of aldehyde (0.966-1.02 g) in aqueous base (NaHC0₃ (0.01-0.1 M; 1-3 mL) and ethanol (0.2-0.3 mL) or water (2-7 mL); Stirring at a temperature between 20 °C and 110 °C for a duration of 2 h 55 min to 87.5 hours;

Cooling to room temperature in an ice bath, filtering the precipitate and washing with water, to obtain pure 3-arylacrylate-based compound or derivatives thereof.

[0039] In an embodiment, for the preparation of the styrylpyridine derivative, the present disclosure relates to a method for obtaining the compound, that comprises the following steps:

Heating a solution of 3-(2-hydroxyphenyl)-2-pyridyl)acrylate (0.02-0.14 g), in DMSO (0.2-0.7 mL) at 130 °C for a duration from 35 minutes to 3 hours;

Adding 1 mL of water to the reaction mixture;

Filtering and washing with water to isolate the pure styrylpyridine derivatives.

[0040] Another aspect of the present disclosure, relates to a method of preparing the compound of the present disclosure comprising: adding pyridinium salt to a solution of aldehyde in aqueous base and ethanol or water in order to obtain a first mixture; stirring at room temperature to 110 °C for 2 h 55 min to 87.5 hours; cooling to the first mixture down to room temperature; filtering the cooled first mixture to obtain precipitate; washing the precipitate with water to obtain 3-arylacrylate-based compound or derivatives thereof.

[0041] In an embodiment, the method may further comprise: heating a solution of 3-(2-hydroxyphenyl)-2-pyridyl)acrylate in DMSO at 130 °C for a duration from 35 minutes to 3 hours to obtain a reaction mixture; adding 1 mL of water to the reaction mixture; filtering the reaction mixture to obtain precipitate; washing the precipitate with water to obtain pure styrylpyridine derivatives. [0042] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0043] Based on the International Union of Pure and Applied Chemistry (lUPAC) definitions, an alkyl group is defined as a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom -CnH_2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H (CH₂)_n. The groups RCH₂, R₂CH (R ≠ H), and R₃C (R ≠ H) are primary, secondary and tertiary alkyl groups respectively. An aryl group is derived from arenes (monocyclic and polycyclic aromatic hydrocarbons) by removal of a hydrogen atom from a ring carbon atom.

[0044] "Alkyl" includes "lower alkyl" and extends to cover carbon fragments having up to 30 carbon atoms. Examples of alkyl groups include octyl, nonyl, norbornyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4 - propylnonyl, 2-(cyclododecyl)ethyl, adamantyl, and the like.

[0045] "Lower alkyl" means alkyl groups with 1 to 7 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2- methylcyclopropyl, cyclopropylmethyl, and the like.

[0046] In the present disclosure, halogen refers to an element selected from the list consisting of: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine (At).

[0047] In the present disclosure, the term "heterocyclic ring" denotes a ring wherein at least one of the atoms forming the ring backbone is not a carbon. Unless otherwise indicated, a heterocyclic ring can be a saturated, partially unsaturated, or fully unsaturated ring. "Saturated heterocyclic ring" refers to a heterocyclic ring containing only single bonds between ring members. "Partially saturated heterocyclic ring" refers to a non-aromatic heterocyclic ring containing at least one double bond. The term "heteroaromatic ring" denotes a fully unsaturated aromatic ring in which at least one atom forming the ring backbone is not a carbon. Typically, a heteroaromatic ring contains no more than 4 nitrogens, no more than 1 oxygen, and no more than 1 sulfur. Unless otherwise indicated, heteroaromatic rings can be attached through any available carbon or nitrogen by replacement of a hydrogen on said carbon or nitrogen. The term "heteroaromatic bicyclic ring system" denotes a ring system consisting of two fused rings in which at least one of the two rings is a heteroaromatic ring as defined above.

[0048] The term "carbocyclic ring" denotes a ring wherein the atoms forming the ring backbone are selected only from carbon. Unless otherwise indicated, a carbocyclic ring can be a saturated, partially unsaturated, or fully unsaturated ring. When a fully unsaturated carbocyclic ring satisfies Huckel's rule, then said ring is also called an "aromatic ring". "Saturated carbocyclic ring" refers to a ring having a backbone consisting of carbon atoms linked to one another by single bonds; unless otherwise specified, the remaining carbon valences are occupied by hydrogen atoms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The following figures provide preferred embodiments for the present disclosure and should not be seen as limiting the scope of the disclosure.

[0050] Figure 1: shows the in vivo toxicity of the compounds using C. elegans model.

DETAILED DESCRIPTION

[0051] The present disclosure relates to compound of the formula I; or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof, for use in medicine, in particular as antimalarial agent. [0052] The present solution describes a new styrylpyridine and 3-arylacrylate-based compounds, the method of synthesizing these compounds by combining at least two different molecules thus giving rise to compounds with an interesting associated biological profile.

[0053] In an embodiment, 19 different styrylpyridine and 3-arylacrylate-based compounds were isolated. Table 1 shows the structure of the styrylpyridine and 3- arylacrylate-based scaffolds, as well as the 19 synthesised and isolated styrylpyridine and 3-arylacrylate-based compounds.

Table 1. Structure of the styrylpyridine and 3-arylacrylate-based compounds.

[0054] In an embodiment, antimalarial activity on P. falciparum was determined using the multidrug resistant parasite line Dd2 and susceptible parasite line 3D7. The results are described in Table 2 below.

Table 2. Antimalarial action of compounds described on Table 1.

[0055] In an embodiment, the multi-parasite stage action of styrylpyridine was determined through the analysis of IC₅₀ for the different stages on P. falciparum in Dd2 parasite line. The results show IC₅₀ values as low as 3.7 nM (Table 3). Table 3. Multi erythrocytic antimalarial action of MCA-205 compound on P. falciparum Dd2 line.

[0056] In an embodiment, IC₅₀ values of the five most active compounds were determined in non-neoplastic MCF-10A (breast) and HK2 (kidney) cell lines, as part of the evaluation of the selective cytotoxicity exhibited by the compounds. Selectivity index (SI) was calculated for each tested compound and are presented in Tables 4 and 5 below.

Table 4. IC₅₀ values (μM) for non-neoplastic cell line MCF-10A and selectivity index (SI) of the compounds for Dd2 and SD7 parasite strains.

Table 5. IC₅₀ values (μM) for non-neoplastic cell line HK2 and selectivity index (SI) of the compounds for P. falciparum Dd2 and SD7 strains.

[0057] For the non-neoplastic cell lines MCF-10A and HK2, the IC₅₀ values were all in the micromolar range and high SI values were obtained, leading to excellent SI values (higherthan 10). These values indicated a promising profile of the compounds regarding toxicity for normal cells.

[0058] In an embodiment, Plasmodium falciparum lines were obtained from MR4- Malaria Resources and maintained at ~ 4% haematocrit with human red blood cells in RPMI-1640, supplemented with 2mM L-glutamine, 200μM hypoxanthine, 0.25 μg/mL gentamycin, 25mM HEPES, 0.2% NaHCO₃, and 0.25% Albumax II. Parasite cultures were maintained at 37°C under a humidified controlled atmosphere of 5% O₂/5% CO₂/90% N₂. Parasite growth was monitored through Giemsa-stained blood smears. Parasite synchronization was performed with 5% sorbitol for 15 minutes at 37 °C. To obtain highly synchronous cultures, sorbitol treatment was applied after 6 - 8 hours.

[0059] In an embodiment, in order to determine antimalarial activities, synchronized ring-stage parasite was incubated at 37°C with 0.2% starting parasitemia and 1% haematocrit. After 72h, to determine the parasite growth, the parasites were stained with SYBR Green (Thermofisher) in PBS lx for 30 minutes and then fluorescence analysed and growth normalized to the controls with no drugs. The IC₅₀ values were calculated using nonlinear regression analysis with GraphPad prism 6 software.

[0060] In an embodiment, non-neoplastic breast cell line MCF-10A was obtained from ATCC (American Type Culture Collection) and cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco) supplemented with 5% heating inactivated FBS (Gibco), 1% antibiotic solution (Penicillin-Streptomycin, Gibco), 1% steroid hormone (Hydrocortisone, Sigma-Aldrich), 0.1% peptide hormone (Insulin, Sigma-Aldrich) and 0.01% protein complex (Cholera Toxin, Gibco). The Kidney cell line HK2 was also obtained from ATCC and cultured in RPMI 1640 medium (Biochrom^®- Merck Millipore), supplemented with 10% heat inactivated FBS (Biochrom^® - Merck Millipore) and 1% antibiotic/antimitotic mixture (Invitrogen^®). All cells were grown in a humidified incubator at 37 °C and 5% CO₂. For all assays, DMSO (Dimethyl Sulfoxide, Sigma-Aldrich) controls were used.

[0061] In an embodiment, cell viability assays were performed. MCF-10A cells were plated at 3000 cells per mL and HK2 cells were plated at 2000 cells per mL, in triplicate, using 96-multiwell culture plate. The cells were then allowed to adhere in complete medium, over a period of 18-20 hours. Cells were subsequently treated with 5 different concentrations (60, 45, 30, 10 and 5 μM) of compounds or control in fresh medium. After 72h of incubation, Sulforhodamine B assay was used according to manufacturer's instructions. The data was log-transformed and the concentration of each compound that decreased the number of viable cells to 50% (IC₅₀) relative to control was calculated using the GraphPad Prism 6 software.

[0062] In an embodiment, selectivity index (SI) value was calculated using the IC₅₀ values of the five best compounds for MCF10A and HK2 cell lines versus the IC₅₀ values calculated for P. falciparum Dd2 and 3D7, using the following mathematical formula:

SI = (IC₅₀ normal cell line - IC₅₀ malaria)/ IC₅₀ malaria For SI values >1, cytotoxicity for malaria is higher than for non-neoplastic cell lines.

[0063] In an embodiment, C. elegans drug toxicity assay was performed using the five most active compounds._C. elegans wild-type strain N2 (Bristol strain) was cultured in nematode growth medium (NGM) plates seeded with Escherichia coli OP50 strain, at 20 °C. To obtain populations of eggs, adult animals were submitted to a bleaching technique (treatment with a 20% Alkaline Hypochlorite Solution). The toxicity of compounds in vivo was determined in the wild-type N2 Bristol strain, using the food clearance assay. Briefly, C. elegans were cultured in liquid culture, using 96-well plates, in the presence of inactivated OP50 and with different concentrations of each compound (from 100 μM to 0.05 μM). The consumption of food (OD 595 nm) was measured daily for 7 days. In the assay, DMSO was used as drug vehicle at a final drug concentration of 1%. C. elegans treated with 1% and 5% DMSO were used as non-toxic and toxic concentration controls, respectively.

[0064] In an embodiment, the reactions of all the chemical compounds were monitored by thin layer chromatography (TLC) using silica gel 60 plates (Macherey-Nagel, 0.2 mm) with fluorescence indicator. For revelation, a UV chamber (CN-6 Vilber Lourmat) with a 254 nm lamp was used. A hot plate stirrer IKAMAG RCT was used with appropriate magnetic stirring and at different temperatures according to the specific procedure. Solvents were evaporated in a Buchi RE 11 rotary evaporator with vacuum and variable bath temperature. IR spectra were recorded in FT-IR Bomem MB 104 using nujol mulls and NaCI cells. Melting points were determined in a Stuart SMP3 apparatus and were not corrected. Elemental analysis was performed on a LECO CHNS-932 instrument. NMR spectra were obtained in Bruker Avance III (at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR), at 25 °C and using deuterated dimethylsulfoxide (DMSO-d₆) as solvent. Chemical shifts were recorded in parts per million (ppm) using the residual solvent peak as an internal standard.

[0065] In an embodiment, pyridinium salt 2 (0.03-0.19 g; 1-1.6 eq) was added to a solution/suspension of aldehyde 1 (0.01-0.19 g) in an aqueous solution of NaHCO₃ (0.01 or 0.1 M; 2-3 mL)/mixture of an aqueous solution of NaHCO₃ (0.01 or 0.1 M; 2-3 mL) and ethanol (0.2-0.3 mL) or in water (2-7 mL). The reaction mixture was stirred at a temperature between 20 °C and 110 °C for a duration from 55 min to 87.5 hours. After cooling to room temperature in an ice bath, the precipitated solid was filtered and washed with water leading to the pure 3-arylacrylate-based compound or MC317 (17- 98%).

[0066] In an embodiment, 3-(2-hydroxy-3-methoxyphenyl)-2-(4-iminiopyridin-1(4H)- yl)acrylate MCA-195 was synthesised. 4-Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (120 mg; 0.709 mmol; 1.2 eq.) was added to a yellow suspension of 3- methoxysalicylaldehyde 1 (90.8 mg; 0.591 mmol) in an aqueous solution of NaHCO₃ 0.01M (3 mL) leading to a yellow suspension. The reaction mixture was heated at 110 °C, leading to a brownish solid suspension after 50 minutes. After 2 h, analysis of the reaction mixture by TLC showed the absence of starting materials and the green solid suspension was filtered and washed with water, analysed and identified as the pure product 3-(2-hydroxy-3-methoxyphenyl)-2-(4-iminiopyridin-1(4H)-yl)acrylate MCA-195 (125 mg; 0.437 mmol). Beige solid; yield 74 %; mp 219-220 °C; ¹H NMR (400 MHz, DMSO- d₆) δ 13.00 (s; 1H; OH), 8.20 (d; J =7.8Hz; 4H), 7.79 (d; J =7.8Hz), 6.90 (dd; J₁ = 3.3Hz; J₂ =1.5Hz), 6.89 (s, 1H), 6.87 (d; J = 1.8Hz), 6.72 (t; J = 7.8Hz), 3.74 (s; 3H); ¹³C NMR 100 MHz, DMSO- d₆) δ 164.23, 158.71, 150.45, 147.41, 143.50, 139.32, 128.47, 123.96, 122.62, 118.06, 112.61, 108.34; IR (Nujol mull) v 3289, 3084, 1664, 1598, 1533 cm^-1; Anal. Calcd for C₁₅H₁₃N₂O₄: C, 56.00; H, 4.39; N, 8.71. Found: C, 56.05; H, 4.42; N, 8.75.

[0067] In an embodiment, 3-(5-chloro-2-hydroxyphenyl)-2-(4-iminiopyridin-1(4H)- yl)acrylate MCA-201 was synthesised. 4-Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (163 mg; 0.963 mmol; 1.2 eq.) was added to a white suspension of 5- chlorosalicylaldehyde 1 (128 mg; 0.803 mmol) in ethanol (0.2 mL) and aqueous solution of NaHCO₃ 0.01M (3 mL). The suspension was stirred at 110 °C and a homogeneous solution was obtained after 55 min. A light brown solid started to precipitate after 30 minutes. After stirring for a further 2 h 40 min, the brown solid was filtered, washed with water and identified as 3-(5-chloro-2-hydroxyphenyl)-2-(4-iminiopyridin-1(4H)- yl)acrylate MCA-201 (141 mg; 0.484 mmol). Brown solid; yield 60 %; mp 229-230 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.65 (s; 1H; OH), 8.24 (brs; 2H), 8.21 (d; J = 7.6Hz; 2H), 7.54 (d; J=2.8Hz; 1H), 7.19 (dd; J₁ = 8.8Hz; J₂ = 2.8Hz; 1H), 6.84 (s; 1H), 6.80 (s; 1H), 6.81 (d; J = 7.6Hz; 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.67, 158.81, 156.10, 143.04, 140.44, 131.00, 129.76, 125.47, 123.72, 121.00, 120.55, 108.36; IR (Nujol mull) v 3368, 3334, 1656, 1638, 1607, 1589, 1537 cm^-1; Anal. Calcd for C₁₄H₁₁N₂O₃CI : C, 57.83; H, 3.79; N, 9.64. Found: C, 57.59; H, 4.01; N, 9.42.

[0068] In an embodiment, 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-2-(4- iminiopyridin-1(4H)-yl)acrylate MCB-145 was synthesised. 4-Amino-1-

(cyanomethyl)pyridin-1-ium chloride 2a (163 mg; 0.959 mmol; 1.2 eq.) was added to a brown suspension of 5-bromo-3-methoxysalicylaldehyde 1 (190 mg; 0.799mm) in ethanol (0.2 mL) and aqueous solution of NaHCO₃ 0.01M (2mL). The reaction mixture was stirred at 110 °C and after 35 min the aldehyde was partially solubilized. An orange solid started to precipitate after 3 h and 40 min and was isolated by filtration. The brown solid was identified as 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-2-(4-iminiopyridin- 1(4H)-yl)acrylate MCB-145 (260 mg; 0.746 mmol). Brown solid; yield 90 %; mp 238-239 °C; ¹H NMR (400 MHz, DMSO- d₆) δ 13.24 (brs; 1H; OH), 8.20 (d; J = 8.00Hz; 2H), 7.18 (d; J = 2.4Hz; 1H), 7.01 (d; J = 2.4Hz; 1H), 6.85 (s; 1H), 6.80 (d; J = 8.00Hz), 3.76 (s; 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.85, 158.81, 151.33, 146.83, 143.04, 140.04, 126.48, 125.37, 123.96, 114.97, 108.81, 108.50, 55.98; IR (Nujol mull) v 3370, 3149, 1656, 1599, 1537, 1465 cm^-1; Anal. Calcd for C₁₅H₁₃N₂O₄ Br: C, 49.33; H, 3.60; N, 7.67. Found: C, 49.48;

H, 3.65; N, 7.73.

[0069] In an embodiment, 3-(5-bromo-2-hydroxyphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-122 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (92.4 mg; 0.392 mmol;

1.1 eq.) was added to a white suspension of 5-bromosalicylaldehyde 1 (59.6 mg; 0.700 mmol) in ethanol (0.2 mL) and in an aqueous solution of NaHCO₃0.01M (2 mL). The white suspension was initially stirred at room temperature for 21 h and then heated in a water bath at 40 °C, precipitating solid after 7.5 h. The starting materials were absent after 59 hours, as shown byTLC analysis. The white solid was filtered, washed with water and identified as 3-(5-bromo-2-hydroxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1- ium-1-yl)acrylate MCA-122 (122 mg; 0.301 mmol). White solid; yield 79%; mp 178-179 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.23 (brs; 1H; OH), 11.41 (brs; 1H; NH), 8.80 (d; J = 7.8Hz; 2H), 7.88 (d; J = 7.8Hz; 2H), 7.80 (s; 1H), 7.67 (d; J = 2.4Hz; 1H), 7.35 (dd; J₁ = 8.8Hz; J₂ = 2.4Hz; 1H), 7.04 (s; 1H), 6.80 (d; J = 8.8Hz; 1H), 4.26 (q; J = 7.2Hz; 2H), 1.29 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 162.84, 156.80, 152.97, 152.79, 145.43, 140.20, 134.41, 133.18, 128.13, 123.78, 121.21, 112.79, 109.43, 62.16, 14.09; IR (Nujol mull) v 3139, 3084, 1742, 1624, 1606, 1533, 1513 cm^-1; Anal. Calcd for C₁₇H₁₅N₂O₅Br: C, 50.12;

H, 3.69; N, 6.88. Found: C, 50.33; H, 3.88; N, 6.88.

[0070] In an embodiment, 3-(5-chloro-2-hydroxy-3-methoxyphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-125 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (101 mg; 0.419 mmol;

1.1 eq.) was added to a beige suspension of 5-chloro-3-methoxysalicylaldehyde 1 (73.2 mg; 3.81 mmol) in an aqueous solution of NaHCO₃0.01M (2 mL) and ethanol (0.2 mL). The suspension was stirred in a water bath at 40° C for 1 hour, leading to a beige solution. The reaction was followed by TLC and after 16 h, the aldehyde was no longer present. The solid was filtered, washed with water and identified as a mixture of MCA- 125 and the starting material 2b (112 mg; 286 mmol). Water (20 mL) was added to the solid mixture and vigorously stirred at room temperature for 2 h. The solid was filtered, washed with water and identified as 3-(5-chloro-2-hydroxy-3-methoxyphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-125 (120 mg; 0.306 mmol). White solid; yield 80%; mp 201-202 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.32 (brs; 1H, NH), 8.77 (d; J = 7.2Hz; 2H), 7.86 (d; J = 7.2Hz; 2H), 7.07 (d; J = 2.4Hz; 1H), 7.04 (s; 1H), 6,95 (d; J = 2.4Hz; 1H), 4.24 (q; J = 7.6Hz; 2H), 3.77 (s; 3H), 1.29 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.08, 153.24, 153.01, 151.20, 146.65, 145.44 (2C), 140.03, 128.79, 122.74, 122.61, 121.49, 112.86, 112.75, 62.15, 56.01, 14.13; IR (Nujol mull) v 3134, 3069, 1753, 1656, 1600, 1532 cm^-1; Anal. Calcd for C₁₈H₁₇N₂O₆CI: C, 55.04; H, 4.36; N, 7.13. Found: C, 55.12; H, 4.39; N, 7.17.

[0071] In an embodiment, 2-(4-((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)-3-(2- hydroxyphenyl)acrylate MCA-64 was synthesised. 1-(Cyanomethyl)-4- (ethoxycarbonylamino)pyridinium chloride 2b (143 mg; 0.590 mmol; 1.6 eq.) was added to a solution of salicylaldehyde 1 (45.5 mg; 39.2 μL; 0.369 mmol) in water (2.5 mL). The yellow solution was stirred at 80 °C and after 2 h and 45 min, a yellow solid precipitated from the reaction mixture. After 6 h 30 min, the reaction was complete, by TLC analysis. The beige solid was filtered and washed with water and identified as 2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)-3-(2-hydroxyphenyl)acrylate MCA-64 (119 mg; 0.361 mmol). Beige solid; yield 98%; mp 185-186 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.15 (brs; 1H; OH), 11.40 (brs; 1H; NH), 8.78 (d; J = 7.6Hz; 2H), 7.88 (d; J = 7.6Hz; 2H), 7.39 (dd; J₁ = 7.6Hz, J₂ = 1.2Hz; 1H), 7.23 (dt; J₁ = 8.4Hz; J₂ = 1.6Hz; 1H), 7.09 (s; 1H), 6.84 (t; J = 8.4Hz; 1H), 6.80 (dd; J₁ = 1.2Hz; 1H), 4.26 (q; J = 7.2Hz; 2H), 1.28 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.33, 162.17, 157.57, 152.81 (2C), 145.66 (2C), 139.24, 133.03, 131.01, 130.54, 121.67, 119.46, 118.63, 112.78 (2C), 14.12; IR (Nujol mull) v 3124, 1773, 1659, 1635, 1615, 1581, 1534, 1461 cm^-1; Anal. Calcd for C₁₇H₁₆N₂O₅: C, 62.19; H, 4.91; N, 8.53. Found: C, 62.19; H, 4.92; N, 8.53. [0072] In an embodiment, 3-(2,3-dihydroxyphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-204 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (122 mg; 0.503 mmol; 1 eq.) was added to a solution of 3-hydroxysalicylaldehyde 1 (46.4 mg; 0.336 mmol) in water (7 mL) resulting in a yellow solution. After 20 minutes under reflux, a yellow solid precipitated from the reaction mixture. After 3h and 30 min, the reaction was complete (by TLC) and the solid was filtered and washed with water. The product (61.1 mg; 0.177 mmol) was identified as 3-(2,3-dihydroxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin- 1-ium-1-yl)acrylate MCA-204. Yellow solid; yield 53%; mp 247-248 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 14.02 (brs; 1H; OH), 11.13 (brs; 1H; NH), 8.75 (d; J = 7.2Hz; 2H), 7.85 (d; J = 7.2Hz; 2H), 7.13 (s; 1H), 6.75 (dd; J₁ = 5.6Hz; J₂ = 1.6Hz; 1H), 6.72 (dd; J₁ = 6.8Hz; J₂ = 1.6Hz; 1H), 6.64 (t; J = 7.6Hz; 1H), 4.25 (q; J = 6.8Hz; 2H), 1.29 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO- d₆) δ163.53, 153.21, 153.12,148.49, 146.29, 145.82 (2C), 138.42, 132.52, 123.21, 121.69, 118.48, 115.56, 113. 17 (2C), 62.05, 14.12; IR (Nujol mull) v 3130, 3069, 1738, 1645, 1527 cm^-1; Anal. Calcd for C₁₇H₁₆N₂O₆: C, 59.30; H, 4.68; N, 8.14. Found: C, 59.41; H, 4.70; N, 8.16.

[0073] In an embodiment, 3-(5-chloro-2-hydroxyphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-103 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (98.8 mg; 0.409 mmol; 1 eq.) was added to a suspension of 5-chlorosalicylaldehyde 1 (58.2 mg; 0.372 mmol) in an aqueous solution of NaHC0₃0.01M ( 3 mL). The suspension was stirred at 110 °C and after 15 min, a yellow solid precipitated from the reaction mixture. After 3 h and 15 minutes the solid was filtered, washed with water and identified by ¹H NMR as product MCA-103 slightly contaminated (125 mg; 344 mmol). Water (20 mL) was added to the isolated solid and the suspension was vigorously stirred at room temperature for 2 h. The product was filtered, washed with water and identified as 3-(5-chloro-2- hydroxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-103 (109 mg; 0.300 mmol). Yellow solid; yield 73%; mp 190-191 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.11 (s; 1H; OH), 11.90 (s; 1H; NH), 8.81 (d; J = 7.2Hz; 2H), 7.88 (d; J = 7.2Hz; 2H), 7.56 (d; J=2.4Hz; 1H), 7.30 (dd; Ji = 8.7Hz; J₂ = 2.7Hz; 1H), 7.04 (s; 1H), 6.86 (d; J = 8.7Hz; 1H), 4.26 (q; J = 7.2Hz; 2H), 1.29 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 162.90, 156.33, 154.98, 152.81, 145.94 (2C), 140.33, 131.97, 130.40, 128.00, 123.20, 121.95, 120.70, 112.84 (2C), 62.20, 14.13; IR (Nujol mull) v 3128, 3069, 1743, 1703, 1652, 1637, 1606, 1590, 1525 cm^-1; Anal. Calcd for C₁₇H15N₂O5CI: C, 56.27; H, 4.14; N, 7.72. Found: C, 56.03; H, 4.36; N, 8.00.

[0074] In an embodiment, 3-(2-hydroxy-5-methylphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-335 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (0.14 g; 0.59 mmol; 1.6 eq.) was added to a solution of 5-methylsalicylaldehyde 1 (0.05 g; 0.38 mmol) in an aqueous solution of NaHCO₃ 0.1M (2 mL) and heated at 80 °C. After 15 minutes, a beige solid precipitated from the reaction mixture. After stirring for a further 15 minutes the reaction was complete (by TLC) and the solid was filtered and washed with water. The product (0.09 g; 0.28 mmol) was identified as 3-(2-hydroxy-5-methylphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-335. Yellow solid; yield 75%; mp 180-182 °C; ¹H NMR (400 MHz, DMSO- d₆) δ 12.93 (s, 1H, OH), 11.34 (s, 1H, NH); 8.77 (d, J = 7,6Hz, 2H), 7.87 (d, J = 7,2Hz, 2H), 7.16 (d, J = 2Hz, 1H), 7.06 (s, 1H), 7.04 (dd, J = 6Hz, J = 2Hz, 1H), 6.74 (d, J = 8Hz, 1H), 2.19 (s, 3H), 4.26 (q, J = 7,2Hz, 2H), 1.29 (t, J = 7,2Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.30, 155.28, 152.86, 145.66, 139.25, 132.94, 131.74, 130.76, 126.98, 121.43, 119.47, 112.76, 62.15, 19.96, 14.12; IR (Nujol mull) v 3131, 1737, 1653, 1636, 1526 cm^-1; Anal. Calcd for C₁₈H₁₈N₂O₅: C, 63.15; H, 5.30; N, 8.18. Found: C, 63.27; H, 5.42; N, 8.10.

[0075] In an embodiment, 3-(2-hydroxy-4-methoxylphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-360 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (0.13 g; 0.53 mmol; 1.6 eq.) was added to a solution of 4-methoxylsalicylaldehyde 1 (0.05 g; 0.33 mmol) in an aqueous solution of NaHCO₃ 0.1M (3 mL) and heated at 90 °C. After 25 minutes, a beige solid precipitated from the reaction mixture. After stirring for a further 30 minutes, the reaction was complete (by TLC) and the solid was filtered and washed with water. The product (0.05 g; 0.14 mmol) was identified as 3-(2-hydroxy-4-methoxylphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-360. Yellow solid; yield 43%; mp 202-204 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 14.15 (s, 1H, OH), 11.34 (s, 1H, NH), 8.74 (d, J = 7,2Hz, 2H), 7.85 (d, J = 7,6Hz, 2H), 7,25 (d, J = 8,8Hz, 1H), 7.05 (s, 1H), 6.42 (dd, J = 6Hz, J=2,8Hz, 1H), 6.37 (d, J = 2,8Hz, 1H), 4.26 (q, J = 7,2Hz, 2H), 3.73 (s, 3H), 1.29

(t, J = 6,8Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ163.68, 162.28, 160.27, 152.90,

152.72, 146.05, 136.68, 135.33, 132.43,114.34, 112.64, 106.03, 103.76, 62.10, 55.07, 14.11; IR (Nujol mull) v 1732, 1645, 1526 cm^-1; Anal. Calcd for C₁₈H₁₈N₂O₆: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.35; H, 5.10; N, 7.84.

[0076] In an embodiment, 3-(3,5-difluoro-2-hydroxylphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-192 was synthesised. 1- (Cyanomethyl)-4-(ethoxycarbonylamino)pyridinium chloride 2b (0.24 g; 0.97 mmol; 1.2 eq.) was added to a solution of 3,5-difluorosalicylaldehyde 1 (0.13 g; 0.81 mmol) in an aqueous solution of NaHCO₃ 0.1M (3 mL) and ethanol (0.2 mL), heated at 100 °C. After 45 minutes, a beige solid precipitated from the reaction mixture. After stirring for a further 3 h and 15 minutes the reaction was complete (by TLC) and the solid was filtered and washed with water. The product (0.22 g; 0.61 mmol) was identified as 3-(3,5- difluoro-2-hydroxylphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-192. Yellow solid; yield 76%; mp 179-180 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 14.15 (s, 1H, OH), 11.43 (s, 1H, NH), 8.82 (d, J = 7.2Hz, 2H), 7.88 (d, J = 6.8Hz, 2H), 7.16 (s, 1H), 4.26 (q, J = 7.2Hz, 2H), 1.29 (t, J = 7.2Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 162.98, 145.58 (2C), 129.51, 113.13, 112.89 (2C), 105.80, 62.14, 14.13; IR (Nujol mull) v 3211, 3127, 3066, 1753, 1640, 1613, 1596, 1533 cm^-1; Anal. Calcd for C₁₇H₁₄N₂O₅F₂: C, 56.05; H, 3.87; N, 7.69. Found: C, 56.07; H, 3.87; N, 7.71.

[0077] In an embodiment, 3-(3,5-difluoro-2-hydroxyphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-156 was synthesised. 4- Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.24 g; 0.97 mmol; 1.2 eq.) was added to a solution of 3,5-difluorosalicylaldehyde 1 (0.13 g; 0.81 mmol) in an aqueous solution of NaHCO₃0.01M (3 mL) and ethanol (0.2 mL) and heated at 110 °C. After 45 minutes, a beige solid started to precipitate from the reaction mixture. After 3h and 15 min the reaction was complete (by TLC) and the solid was filtered and washed with water. The product (0.22 g; 0.61 mmol) was identified as 3-(3,5-difluoro-2-hydroxyphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-156. Beige solid; yield 76%; mp 210-211 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 14.14 (s, 1H, OH), 8.23 (d, J = 7.2Hz, 2H), 8.19 (s, 2H)6.95 (s, 1H), 6.80 (d, J = 7.6Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.23, 158.59, 158.16 (d, J = 43.0Hz), 143.94 (d, J = 4.0Hz), 142.70, 140.12, 125.75, 111.80 (d, J = 21.0Hz), 108.10, 103.88 (d, J = 25.0Hz), Cy and Cy (not observed); IR (Nujol mull) v 3243, 3178, 3087, 1681, 1651, 1638, 1608, 1535, 1514 cm^-1; Anal. Calcd for C14H10N₂O₃F2: C, 57.54; H, 3.45; N, 9.59. Found: C, 57.55; H, 3.49; N, 9.68.

[0078] In an embodiment, 3-(2-hydroxy-5-methoxyphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-193 was synthesised. 4- Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.16 g; 0.96 mmol; 1.6 eq.) was added to a solution of 5-methoxysalicylaldehyde 1 (0.09 g; 0.60 mmol) in an aqueous solution of NaHCO₃ 0.01M (3 mL) and ethanol (0.3 mL) and heated at 110 °C. After 1 h, a green solid started to precipitate from the reaction mixture. After stirring for a further 2h, the reaction was complete (by TLC). The solid was filtered, washed with water and identified as 3-(2-hydroxy-5-methoxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1-ium-1- yl)acrylate MCB-193 (0.13 g; 0.44 mmol). Greenish solid; yield 74%; mp 206-207 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12,46 (brs; 1H; OH), 8.21 (d, J = 7.6Hz, 2H), 6.79 (d, J = 7.6Hz, 2H), 6.98 (d, J = 2.8Hz, 1H), 6.81 (dd, J = 1.6Hz, J = 1.2Hz, 1H), 6.75 (d, J = 9.2Hz, 1H), 6.84 (s; 1H), 3.66 (s; 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.95, 158.65, 151.56, 150.95, 143.40, 139.91, 127.58, 122.42, 119.95, 116.19, 115.83, 108.29, 55.35; IR (Nujol mull) v 3291, 3080, 3052, 1672, 1650, 1598, 1502 cm^-1; Anal. Calcd for C₁₅H₁₄N₂O₄: C, 62.93; H, 4.93; N, 9.79. Found: C, 62.93; H, 4.96; N, 9.77.

[0079] In an embodiment, 3-(5-bromo-2-hydroxyphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-147 was synthesised. 4- Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.16 g; 0.96 mmol; 1.2 eq.) was added to a solution of 5-bromosalicylaldehyde 1 (0.16 g; 0.80 mmol) in an aqueous solution of NaHCO₃0.01M (3 mL) and ethanol (0.2 mL) and heated at 110 °C. After 30 min, a yellow solid started to precipitate from the reaction mixture. After stirring for a further 3.5 h, the reaction was complete (by TLC). The solid was filtered, washed with water and identified as 3-(5-bromo-2-hydroxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1-ium- 1-yl)acrylate MCB-147 (0.24 g; 0.66 mmol). Green solid; yield 82%; mp 220-221 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.29 (s, 1H, OH), 8.22 (d, J = 7.6Hz, 2H), 8.16 (s, 2H), 7.61 (d, J = 2.4Hz, 1H), 7.31 (dd, J = 6.4Hz, J = 2,4Hz, 1H), 6.83 (s, 1H), 6,79 (d, J = 7.6Hz, 2H), 6.76 (s, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.66, 158.73, 156.60, 143.25 (2C), 140.34, 133.99, 133.26, 132.63, 125.74, 124.41, 121.19, 108.36 (2C); IR (Nujol mull) v 3393, 3322, 3292, 3145, 3087, 3060, 1654, 1637, 1605, 1589, 1536, 1509 cm^-1; Anal. Calcd for C₁₄H₁₁N₂O₃Br: C, 50.17; H, 3.31; N, 8.36. Found: C, 50.20; H, 3.41; N, 8.42.

[0080] In an embodiment, 3-(2-hydroxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1- ium-1-yl)acrylate MCB-314 was synthesised. 4-Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.031 g; 0.18 mmol; 1.6 eq.) was added to a solution of salicylaldehyde 1 (0.01 g; 0.12 mmol; 12 mΐ) in an aqueous solution of NaHC0₃ 0.01M (1 mL) and heated at 80 °C. After 1 h and 30 min, a beige solid started to precipitate from the reaction mixture. After stirring for a further 2.5 h, the reaction was complete (by TLC). The solid was filtered, washed with water and identified as 3-(2-hydroxyphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-314 (0.03 g; 0.11 mmol). White solid; yield 97%; mp 208-210 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.16 (s, 1H, OH), 8.19 (d, J = 7,6Hz, 2H), 8.15 (s, 2H), 6.88 (s, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 164.10, 158.67, 157.30, 143.47, 139.34, 132.54, 130.42, 128.30, 122.18, 119.34, 118.51, 108.31; IR (Nujol mull) v 1657, 1580, 1531 cm^-1; Anal. Calcd for C₁₄H₁₂N₂O₃: C, 65.62; H, 4.72; N, 10.93. Found: C, 65.65; H, 4.76; N, 10.94.

[0081] In an embodiment, 3-(2-hydroxy-3-methylphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-324 was synthesised. 4- Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.10 g; 0.59 mmol; 1.6 eq.) was added to a solution of 3-methylsalicylaldehyde 1 (0.05 g; 0.37 mmol; 45 mί) in an aqueous solution of NaHC0₃0.01M (2 mL) and heated at 80 °C. After 55 min, a green solid started to precipitate and the reaction was complete by TLC. The solid was filtered, washed with water and identified as 3-(2-hydroxy-3-methylphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-324 (0.10 g; 0.36 mmol). Green solid; yield 98%; mp 203-205 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 13.71 (s, 1H, OH), 8.18 (d, J = 7.6Hz, 2H), 8.13 (s, 2H), 6.94 (s, 1H), 6.78 (d, J = 7.6Hz, 2H), 6.70 (t, J = 7.6Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆) δ 164.30, 158.68, 155.94, 143.86, 138.60, 131.33, 131.27, 130.75, 128.08, 121.85, 118.10, 108.24, 16.94; IR (Nujol mull) v 3130, 3069, 1738, 1645, 1527 cm^-1 Anal. Calcd for C₁₅H₁₄N₂O₃: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.71; H, 5.34; N, 10.27.

[0082] In an embodiment, 3-(2-hydroxy-5-methylphenyl)-2-(4-

((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-325 was synthesised. 4- Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.10 g; 0.59 mmol; 1.6 eq.) was added to a solution of 5-methylsalicylaldehyde 1 (0.05 g; 0.37 mmol) in an aqueous solution of NaHCO₃0,01M (2 mL) and heated at 80 °C. After 3h and 50 min, a green solid started to precipitate and after 20 more minutes, the reaction was complete by TLC. The solid was filtered, washed with water and identified as 3-(2-hydroxy-5-methylphenyl)-2-(4- ((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-325 (0.08 g; 0.28 mmol). White solid; yield 76%; mp 204-206 °C; ³H NMR (400 MHz, DMSO-d₆) δ 12.88 (s, 1H, OH), 8.19 (d, J = 7.6Hz, 2H), 8.14 (s, 2H), 7.13 (d, J = 2Hz, 1H), 6.99 (dd, J = 6Hz, J = 2Hz, 1H), 6.83 (s, 1H), 6.79 (d, J = 7.6Hz, 2H), 6.71 (d, J = 8Hz, 1H), 2.17 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ164.10, 158.65, 154.98, 143.48 (2C), 139.36, 132.48, 131.12, 128.47, 126.85, 121.89, 119.27, 108.29 (2C), 19.99; IR (Nujol mull) v 3130, 3069, 1738, 1645, 1527 cm- ³; Anal. Calcd for C₁₅H₁₄N₂O₃: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.67; H, 5.26; N, 10.31.

[0083] In an embodiment, 4-(2-cyano-2-(4-iminiopyridin-1(4H)-yl)vinyl)phenolate MC- 317 was synthesised. 4-Amino-1-(cyanomethyl)pyridin-1-ium chloride 2a (0.18 g; 1.04 mmol; 1.2 eq.) was added to a solution of 4-hydroxybenzaldehyde 1 (0.11 g; 0.89 mmol) in an aqueous solution of NaHCO₃0,05M (1 mL) and propylamine (54.6 mg; 0.92 mmol; 76mί). After stirring at room temperature for 4 h, an orange solid started to precipitate and after 30 more minutes, the solid was filtered and washed with water (18.4 mg). The mother-liquor was further stirred at room temperature for 15 h and a second crop of an orange solid was filtered and washed with water (18.0 mg). The two crops were identified as 4-(2-cyano-2-(4-iminiopyridin-1(4H)-yl)vinyl)phenolate MC-317 (36.4 mg; 0.153 mmol). Orange solid; yield 17%; mp 192-194 °C; ³H NMR (400 MHz, DMSO-d₆) δ 8.30-9.50 (brs; 2H), 8.26 (d; J = 7.2Hz; 2H), 7.44 (d; J = 8.8Hz; 2H), 7.30 (s; 1H), 6.83 (d; J = 7.2Hz; 2H), 6.12 (d; J = 9.2Hz; 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 178.41, 158.77, 144.18, 143.04 (2C), 133.41 (2C), 120.78 (2C), 118.70, 109.73 (2C), 109.12, 95.47; IR (Nujol mull) v 3160, 3099, 2236, 1712, 1641, 1515 cm^-1; Anal. Calcd for C₁₄H₁₁N₃O: C, 70.87; H, 4.67; N, 17.71. Found: C, 70.91; H, 4.65; N, 17.68.

[0084] In an embodiment, a solution of 3-(2-hydroxyphenyl)-2-pyridyl)acrylate (0.02- 0.14 g), in DMSO (0.2-0.7 mL) was stirred at 100-130 °C for 1 h and 10 min to 2 h. A solid precipitated from the reaction mixture by the addition of water and the product was filtered and washed with water leading to the pure styrylpyridine derivative (60-89%).

[0085] In an embodiment, ethyl (1-(2-hydroxystyryl)pyridin-4(lH)-ylidene)carbamate MCA- 189 was synthesised. A yellow suspension of 2-(4-((ethoxycarbonyl)amino)pyridin- l-ium-1-yl)-3-(2-hydroxyphenyl)acrylate MCA-64 (96.9 mg; 0.263 mmol) in DMSO (0.2 mL) was stirred at 110 °C and a red solution was obtained 15 minutes later. The reaction was complete after 1 h and 45 min (by TLC). An orange suspension was formed after addition of water (1 mL) and the solid was filtered and washed with water. The product was identified as ethyl (1-(2-hydroxystyryl)pyridin-4(lH)-ylidene)carbamate MCA-189 (64.0 mg; 0.224 mmol). Orange solid; yield 89%; mp 176-177 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d; J = 7.8Hz; 2H),7.70 (d; J = 14.4Hz), 7.15 (d; J = 1.5Hz; 1H), 7.10 (d; J = 9.9Hz; 1H), 7.06 (d; J = 7.8Hz; 2H),6.88 (dd; J₁ = 7.8Hz; J₂ = 0.9Hz; 1H), 6.82 (dt; Ji = 7.2Hz; J₂ = 0.9Hz), 3.98 (q, J = 6.9Hz; 2H), 3.83 (s; 3H), 1.16 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 164.22, 162.47, 155.38, 137.16 (2C), 129.46, 129. 25, 127.86, 120.82, 119.28, 115.89, 115.77, 115.56 (2C), 54.53, 14.68; IR (Nujol mull) v l738, 1665, 1614, 1510 cm^-1; Anal. Calcd for C₁₆H₁₆N₂O₃: C, 67.59; H, 5.67; N, 9.85. Found: C, 67.60; H, 5.66; N, 9.95.

[0086] In an embodiment, ethyl 4-chloro-2-(2-(4-iminiopyridin-1(4H)-yl)vinyl)phenolate MCA-206 was synthesised. A suspension of 3-(5-chloro-2-hydroxyphenyl)-2-(4- iminiopyridin-1(4H)-yl)acrylate MCA-201 (88.9 mg; 0.319 mmol) in DMSO (0.2) was stirred at 130 °C. After 35 min the starting material was partially solubilized and after a further hour, the brown solid was isolated by addition of water (1.5 mL) and identified as 4-chloro-2-(2-(4-iminiopyridin-1(4H)-yl)vinyl)phenolate MCA-206 (59.1 mg; 0.256 mmol). Brown solid; yield 80%; mp 197-198 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 38.19 (d; J = 7.8Hz; 2H), 7.79 (d; J= 14.7Hz; 1H), 7.60 (d; J = 2.4Hz; 1H), 7.24 (dd; J₁ = 8.4Hz; J₂ = 2.4Hz; 1H), 7.07 (d; J = 7.8Hz; 2H), 7.02 (s; 1H), 6.80 (d; J = 8.7Hz; 1H), 4.00 (q; J = 6.9Hz; 2H), 1.17 (t; J = 7.2Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.70, 162.13, 155.25, 137.11 (2C), 131.34, 130.30, 129.51, 123.43, 118.06, 115.40 (2C), 114.08, 109,89, 59.62, 14.59; IR (Nujol mull) v3150, 3069, 1656, 1605, 1589, 1537 cm^-1; Anal. Calcd for C₁₃H₁₁N₂OCI: C, 63.04; H, 4.88; N, 11.31. Found: C, 63.09; H, 4.93; N, 11.45.

[0087] In an embodiment, ethyl (1-(5-bromo-2-hydroxystyryl)pyridin-4(1H)- ylidene)carbamate MCA-116 was synthesised. A suspension of 3-(5-bromo-2- hydroxyphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCA-122 (115 mg; 0.283 mg) in DMSO (0.2 mL) was stirred at 130 °C. The starting material was solubilized 10 minutes later and after 1 h and 50 min, TLC confirmed the absence of starting material and the addition of water (1mL) led to an orange solid. The solid was filtered, washed with water and identified as ethyl (1-(5-bromo-2-hydroxystyryl)pyridin- 4(lH)-ylidene)carbamate MCA-116 (82.8 mg; 0.228 mmol). Orange solid; yield 81%; mp 212-213 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (d; J = 7.6Hz; 2H), 7.72 (d; J = 14.4Hz; 1H), 7.15 (d; J = 2.4Hz; 1H), 7.07 (d; J = 14.4Hz; 1H), 7.04 (s; 2H), 6.95 (d; J = 2Hz; 1H), 4.00 (q; J = 7.2Hz; 2H), 3.83 (s; 3H), 1.17 (t; J = 6,8Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 163.72, 162.18, 148.78, 143.43, 137.04 (2C), 130.38, 122.67, 122.33, 117.64, 115.38 (2C), 113.27, 111.28, 59.60, 54.24, 14.57; IR (Nujol mull) vl742, 1683, 1623, 1514 cm^-1; Anal. Calcd for C₁₆H₁₅N₂O₃Br: C, 52.91; H, 4.16; N, 7.71. Found: C, 53.00; H, 4.21; N, 7.79.

[0088] In an embodiment, 4-bromo-2-(2-(4-iminiopyridin-1(4H)-yl)vinyl)-6- methoxyphenolate MCA-205 was synthesised. A yellow suspension of 3-(5-bromo-2- hydroxy-3-methoxyphenyl)-2-(4-iminiopyridin-1(4H)-yl)acrylate MCB-145 (137 mg; 0.393 mmol) in DMSO (0.7 mL) was stirred at 130 °C, leading to a red solution 20 minutes later. After 50 min the reaction was complete (by TLC). Addition of water (1 mL) led to an orange solid that was filtered and washed with water. The product was identified as 4-bromo-2-(2-(4-iminiopyridin-1(4H)-yl)vinyl)-6-methoxyphenolate MCA-205 (112 mg; 0.368 mmol). Orange solid; yield 60%; mp 200-201 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.35 (d; J = 6.6Hz; 2H), 8.18 (d; J =14.1Hz; 1H), 7.11 (d; J = 13.8Hz; 1H) 6.93 (d; J = 2.4Hz; 1H), 6.87 (d; J = 6.6Hz; 2H), 6.63 (d; J = 2.4Hz; 1H), 3.53 (s; 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 158.00, 156.51, 152.03, 149.44, 139.30 (2C), 126.06, 122.82, 121.15, 113.60, 109.65 (2C), 100.32, 55.62; IR (Nujol mull) v3269, 3053, 1645, 1533 cm^-1; Anal. Calcd for C₁₄H₁₁N₂O₂Br: C, 51.56; H, 3.40; N, 8.59. Found: C, 51.59; H, 3.40; N, 8.64.

[0089] In an embodiment, 2,4-difluoro-6-(2-(4-iminiopyridin-1(4H)-yl)vinyl)phenolate MCA-249 was synthesised. A suspension of 3-(3,5-difluoro-2-hydroxyphenyl)-2-(4- iminiopyridin-1(4H)-yl)acrylate MCB-156 (0.03 g; 0.10 mmol) in DMSO (0.2 mL) and water (6.0 mί) was stirred at 130 °C. After 1 h and 15 min the reaction was complete (by TLC). Addition of water (2 mL) led to an orange solid that was filtered and washed with water. The product was identified as 2,4-difluoro-6-(2-(4-iminiopyridin-1(4H)- yl)vinyl)phenolate MCA-249 (0.02 g; 0.07 mmol). Orange solid; yield 75%; mp 210-211 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (brs, 2H), 8.35 (brs, 2H), 8.25 (d; J = 14.0Hz; 1H), 7.17 (d; J = 14.0Hz; 1H), 6.72-6.94 (m; 2H), 6.86 (brs; 2H); ¹³C NMR (75 MHz, DMSO-d₆) d 158.65, 153.79 (dd, J = 224.0Hz, J = 22.0Hz), 152.63 (d, J = 13.0Hz), 148.21 (dd, J = 212.0Hz, J = 13.0Hz), 139.3, 127.06, 121.30, 109.65, 108.30 (dd, J = 18.0Hz, J = 2.0Hz), 103.10 (dd, J = 24.0Hz, J = 3.0Hz; IR (Nujol mull) v 1653, 1647, 1540 cm^-1; Anal. Calcd for C₁₃H₁₀N₂OF₂: C, 62.90; H, 4.06; N, 11.29. Found: C, 62.92; H, 4.04; N, 11.31.

[0090] In an embodiment, ethyl (1-(3,5-difluoro-2-hydroxystyryl)pyridin-4(lH)- ylidene)carbamate MCB-376 was synthesised. A yellow suspension of 3-(3,5-difluoro-2- hydroxylphenyl)-2-(4-((ethoxycarbonyl)amino)pyridin-1-ium-1-yl)acrylate MCB-192 (0.02 g; 0.06 mmol) in DMSO (0.2 mL) was stirred at 100 °C. After 30 min the reaction temperature was raised to 130 °C and maintained for a further hour. An orange suspension was formed after addition of water (1 mL) and the solid was filtered and washed with water, leading to ethyl (1-(3,5-difluoro-2-hydroxystyryl)pyridin-4(lH)- ylidene)carbamate MCB-376 (11.0 mg; 0.033 mmol). Orange solid; yield 53%; mp 170- 171 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.25 (d; J = 7.6Hz; 2H), 7.85 (d, J = 14.4Hz, 1H), 7.09-7.18 (m, 5H), 4.03 (q, J = 7.2Hz, 2H), 1.19 (t, J = 7.2Hz, 3H); ¹³C NMR (75 MHz, DMSO- de) d 162.57, 161.23, 150.48, 137.50, 130.91, 124.26 (d, J = 11.0Hz), 113.82, 108.06 (d, J = 26.0Hz), 103.51 (d, J = 27.0Hz), 59.94, 14.53; IR (Nujol mull) v 1733, 1682 cm^-1; Anal. Calcd for C₁₆H₁₄N₂O₃F₂: C, 60.00; H, 4.41; N, 8.75. Found: C, 60.10; H, 4.42; N, 8.71. [0091] All references recited in this document are incorporated herein in their entirety by reference, as if each and every reference had been incorporated by reference individually.

[0092] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0093] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims.

[0094] Antimalarial and other in vitro assays were funded by Fundação para a Ciencia e Tecnologia (FCT) trough projects PTDC/SAU-PAR/28066/2017, UIDB/50026/2020, Ul DP/50026/2020 and grants SFRH/BPD/76614/2011 and PD/BD/127826/2016; by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Also trough the Merieux Research Grant.

[0095] Synthesis and spectroscopic characterization of the compounds were funded by The NMR spectrometer Bruker Avance III 400, part of the National NMR Network (RNRMN) and purchased within the framework of the National Program for Scientific Reequipment, contract REDE/1517/RMN/2005 with funds from POCI2010 (FEDER) and FCT; FEDERCOMPETE through Centro de Quimica (F-COMP-01-0124-FEDER-022716, PEst-C/QUI/U 10686/2011, U I D/QU 1/00686/2013 and UID/QU 1/0686/2016); FCT trough BPD grant (SFRH/BPD/79609/2011).

[0096] The present disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0097] The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S

1. A compound of the formula (I) or a pharmaceutically acceptable salt, solvate, N- oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer or polymorph

wherein

R¹, R², R³ and R⁴are independently selected from each other;

R¹ is selected from a list consisting of: H, alkyl, alkoxyl, halogen, hydroxyl, carbonyl;

R² is selected from a list consisting of: H, hydroxyl, carbonyl;

R³ is selected from a list consisting of: H, hydroxyl, carbonyl, cyano, carboxyl;

R⁴ is selected from a list consisting of: amine, amide.

2. The compounds according to any of the previous claims wherein R¹ is selected from a list consisting of: H, hydroxyl, carbonyl, fluoro, bromo, chloro, halo-methoxy, alkyl.

3. The compounds according to any of the previous claims wherein R¹ is selected from a list consisting of: H, fluoro, bromo, chloro, halo-methoxy.

4. The compounds according to any of the previous claims wherein R² is a hydroxyl.

5. The compounds according to any of the previous claims wherein R³ is selected from a list consisting of: H, cyano.

6. The compounds according to any of the previous claims R⁴ is selected from a list consisting of: heteroarylamine, amide, preferably heteroarylamine, heteroarylamide.

7. The compounds according to any of the previous claims wherein R⁴ is a pyridine- moiety.

8. The compounds according to the previous claim wherein the compound is selected from a list consisting of:

9. The compounds according to any of the previous claims wherein the salt is a hydrochloride salt.

10. The compounds according to any of the previous claims for use in medicine or veterinary.

11. The compounds according to any of the previous claims for use in the prevention, therapy, or treatment of infection.

12. The compounds according to any of the previous claims for use in the prevention, therapy, or treatment of parasite infection, preferably P. falciparum infection.

13. The compounds according to any of any one of the previous claims for use in the prevention, therapy or treatment of malaria diseases.

14. Pharmaceutical composition comprising a compound as defined in any one of claims 1-13 in a therapeutically effectively amount and a pharmaceutically acceptable excipient.

15. Pharmaceutical composition according to the previous claim wherein the pharmaceutically acceptable excipient is a carrier, adjuvant, excipient or mixtures thereof.

16. Pharmaceutical composition according to any of the previous claims 13-15 comprising a further anti-viral agent, preferably an anti-HIV agent or anti- tuberculosis agent.

17. Pharmaceutical composition according to any of the previous claims 13-16 comprising a second anti-malarial agent, preferably wherein the second anti- malarial agent is selected from the group consisting of: Chloroquine, Fansidar, Amodiaquine, Quinine, Halofantrine, Mefloquine, Artemether/Artesunate and Malarone.