WO2023213728A1

WO2023213728A1 - Substituted aryl esters of coumarin-3-carboxylic acid and their use as host cell proteases inhibitors

Info

Publication number: WO2023213728A1
Application number: PCT/EP2023/061344
Authority: WO
Inventors: Bernard Pirotte; Pierre Francotte; Chahrazade El Amri; Michèle REBOUD-RAVAUX; Nancy CHAAYA; Cindy LESENFANTS
Original assignee: Universite de Liege; Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Sorbonne Universite
Current assignee: Universite de Liege; Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Sorbonne Universite
Priority date: 2022-05-03
Filing date: 2023-04-28
Publication date: 2023-11-09
Anticipated expiration: 2024-11-03

Abstract

Compound of Formula (I) wherein R¹ is a substituted linear or branched alkyl chain of 1 to 6 carbon atoms; R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being optionally preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms and R² being in the ortho, meta or para position of the ester group on the aryl ring; R³ is a halogen with x being an integer between 0 and 4; R⁴ is a halogen with y being an integer between 0 and 3; or a prodrug thereof, and/or a salt thereof, for its use in the treatment of respiratory diseases and compounds of formula I wherein R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms, preferably of 1 to 2 carbon atoms for use as a medicament.

Description

Title: SUBSTITUTED ARYL ESTERS OF COUMARIN-3-CARBOXYLIC ACID AND THEIR USE AS HOST CELL PROTEASES INHIBITORS TECHNICAL FIELD OF THE INVENTION The present invention relates to substituted aryl esters of coumarin-3- carboxylic acid and their use for the treatment of respiratory diseases, in particular as transmembrane serine protease 2 inhibitors. The present invention also relates to pharmaceutical compositions comprising substituted aryl esters of coumarin-3- carboxylic acid. BACKGROUND OF THE INVENTION There is an urgent need to identify new therapies to prevent acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and improve the outcome of COVID-19 patients. SARS-CoV-2 utilizes the membrane ectopeptidase angiotensin-converting enzyme 2 (ACE2) as its receptor to initiate binding and entry in host cells (Hoffmann et al., Cell 2020, 181, 271). The virus binds to ACE2 through the receptor binding domain of its spike glycoprotein (S protein). To facilitate fusion with host cell membranes, the S protein undergoes proteolytic cleavage events. In respiratory epithelia, the predominant mode of viral entry appears to be membrane fusion at the cell surface, where a cell surface-expressed serine protease, transmembrane serine protease 2 (TMPRSS2), executes the appropriate cleavage of the viral S protein. TMPRSS2 is also known to be implicated in the activation of influenza A, influenza B, and other coronaviruses to drive efficient infection of the lungs (Matsuyama et al., J. Virol.2010, 84, 12658 and Limburg et al., J. Virol.2019, 93, 1). As a result, TMPRSS2 is an attractive target for new antiviral agents, as inhibiting its proteolytic activity blocks efficiently viral entry (Hoffmann et al., Cell 2020, 181, 271, Singh et al., Eur. J. Pharm. Sci. 2020, 153, 105495). It should be specified that TMPRSS2 alone does not mediate viral infection. However, co- expression of TMPRSS2 with ACE2 results in enhanced infectivity, inducing S protein cleavage and exposing the fusion peptide for efficient viral entry (Chavez- Medina et al., Arch. Microbiol. 2022, 204, 77). According to recent literature, TMPRSS2 appears to be a druggable anti- SARS-CoV-2 host protein target for the following reasons. The host protein is not subject to mutation. Moreover, TMPRSS2 being used by other viruses (i.e. SARS- CoV, MERS-CoV and influenza viruses) for the activation of surface glycoproteins, TMPRSS2 inhibitors may be used to treat a wide class of diseases caused by different pathogens (Cannalire et al., Int. J. Mol. Sci. 2020, 21, 5707), including SARS-CoV-2 variants. Lastly, in TMPRSS2-knockout mice, TMPRSS2 appeared to be useless for normal development and organ function (Stopsack et al., Cancer Discov. 2020, 10, 779). As a result, TMPRSS2 inhibition may have few on-target side effects (Huang et al., Int. J. Mol. Sci. 2021, 22, 7060). Since TMPRSS2 constitutes an attractive therapeutic target, many recent works were dedicated to the evaluation of existing TMPRSS2 inhibitors as possible antiviral agents. The irreversible serine protease inhibitors camostat and nafamostat, currently on the Japanese pharmaceutical market for therapeutic indications other than antivirals, were found to be effective at preventing host cell entry and replication of SARS-CoV-2 in Calu-3 cells through a TMPRSS2-dependent mechanism (Hoffmann et al., Cell 2020, 181, 271, Hoffmann et al., Antimicrob. Agents Chermother. 2020, 64, e00754-20). A recent publication also reports the discovery of small-molecule ketobenzothiazole TMPRSS2 inhibitors with improved activity over the existing known inhibitors (Mahoney et al., Proc. Natl. Acad. Sci. 2021, 118, 43 e2108728118). A clinical trial is ongoing to assess the efficacity of nafamostat as a novel treatment for SARS-CoV-2 infection (Zhuravel et al., E Clinical Medicine 2021, 41, 101169). However, this drug lacks high selectivity for TMPRSS2 and presents poor bioavailability (rapid metabolization after parenteral administration) (Quinn et al, medRxiv 2021, 10.06.21264648). There is thus still a need to develop potent and highly selective TMPRSS2 inhibitors. There is also a need to develop potent and highly selective inhibitors against other serine protease involved in respiratory diseases such as human airways trypsin-like protease TMPRSS11D (HAT), cathepsin B/L or furin protease. An inhibitor being able to target both TMPRSS2 and HAT can have an effect both on infection and inflammation. A combined inhibition may thus be of interest in the treatment and/or the prevention of respiratory diseases. In previous work, some coumarin derivatives were described with their activity as protease inhibitors (Reboud-Ravaux et al. WO98/55472). However, while some coumarin derivatives were found to be potent and selective inhibitors of α- chymotrypsin (Pochet et al., J. Med. Chem. 1996, 39, 2579), human leukocyte elastase (Doucet et al., J. Med. Chem. 1999, 424161 and Pochet et al., Bioorg. Med. Chem. 2000, 8, 1489), thrombin (Frederick et al., J. Med. Chem. 2005, 48, 7592) or disease-related kallikreins (WO2013/010963, Tan et al., J. Med. Chem.2015, 58, 598; Hanke et al., J. Med. Chem. 2020, 63, 5723), no activity was yet reported towards the inhibition of TMPRSS2. It is an aim of the present invention to provide compounds for use as selective inhibitors of host cell proteases such as TMPRSS2, HAT, cathepsin B/L, furin and/or other enzymes involved in respiratory diseases. It is also an aim of the present invention to provide compounds that heal or relief from pulmonary infections, in particular viral infections. It is also an aim of the present invention to provide new substituted aryl esters of coumarin-3-carboxylic acid. SUMMARY OF THE INVENTION The invention pertains to compounds of Formula I as presented below:

wherein R¹ is a substituted linear or branched alkyl chain of 1 to 6 carbon atoms; R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being optionally preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms and R² being in the ortho, meta or para position of the ester group on the aryl ring; R³ is a halogen with x being an integer between 0 and 4; R⁴ is a halogen with y being an integer between 0 and 3; or a prodrug thereof, and/or a salt thereof, for its use in the treatment of respiratory diseases. Such compounds of Formula I present an inhibitory activity against enzymes such as host cell proteases. Host cell proteases may be transmembrane serine protease 2, generally referred to as TMPRSS2, human airways trypsin-like protease TMPRSS11D (HAT), another major serine protease involved in respiratory diseases, cathepsin B/L or furin protease. By inhibitory activity, one means a decrease of the activity of the enzyme. A test to show the inhibitory activity consists of incubating the enzyme, optionally a buffer, and a compound as recited above and then determining the activity of the enzyme by comparison of the initial rates to those obtained in control experiments without the tested compounds. The IC50 values (inhibitor concentration giving 50% inhibition) express the inhibitory activity. Advantageously, the coumarin derivatives are designed to present a basic group (R²), which bears a positive charge at physiological pH. By bearing a positive charge is also meant having a cationic group at physiological pH. A physiological pH is a pH of about 7 to about 8. Examples of such basic group are an amine, an amidine or a guanidine group, providing an aminium, an amidinium or a guanidinium group at physiological pH. These may be directly or not directly linked to the aromatic ring and introduced in the ortho, meta or para position of the ester group on the aryl group. When not directly linked, these groups are linked through a short linear or branched alkyl chain such a linear or branched alkyl chain with 1 to 6 carbon atoms. Such alkyl chain is preferably a linear alkyl chain. This cationic group enables to target the anionic carboxylate function of Asp435 of the enzymatic catalytic site of TMPRSS2 and to promote the establishment of a salt bridge between the enzyme and its inhibitor. The basic group may advantageously be chosen among a relatively strong basic group such as amidine or guanidine or a mild basic group such as a primary amine. Advantageously, the coumarine derivative are low molecular weight non peptidic molecules. Advantageously, their modular structure allows structural variations to obtain selective inhibitors of TMPRSS2 or other host cell proteases of interest (furin, cathepsin B/L, TMPRSS11D) to combat respiratory diseases. This may also lead to either dual inhibitors or use of a combination of inhibitors, both strategies being susceptible to increase antiviral potency. Advantageously they may have a dual inhibition against TMPRSS2 and HAT. Alternatively, compounds may be designed as prodrugs. By prodrug is meant a compound that, after administration, is metabolized into a pharmacologically active drug. Hence, by prodrug of Formula I is meant any compound that can be metabolized into a compound of Formula I. A prodrug form can be advantageous to improve the oral bioavailability. As an illustration, when the basic group of Formula I is an amidine or a guanidine-bearing group, it might exhibit poor oral bioavailability due to the permanent cationic form of these groups in biological media. It is for example known that the antithrombotic agent dabigatran bearing an amidine function is used as a prodrug (dabigatran etexilate) suitable for oral administration. In some embodiments, the precursor of the basic group may bear a carbamate function or a pseudocarbamate function. A carbamate function refers to - NH-COORwhile a pseudocarbamate function may refer to -(=NH)NH-COORor -NH- C(=NH)NH-COOR, or -NH-C(=NH)NH-COOR. Such a (pseudo)carbamate function may be metabolized into a basic group after administration to a subject. Other precursors of amine function or other precursors of other basic functions are also possible. In some embodiments, R² is chosen from the group consisting of -(CH₂)_n- NH₂, -(CH₂)_n-C(=NH)NH₂, -(CH₂)_n-NH-C(=NH)NH₂ or -(CH₂)_n-NH-COOR⁵ , -(CH₂)_n- C(=NH)NH-COOR^{5 ,} -(CH²)ⁿ-NH-C(=NH)NH-COOR⁵ with R⁵ being a linear or branched alkyl chain with 1 to 6 carbon atoms and with n being an integer between 0 and 6. Advantageously, n may be 1 or 2 to facilitate the target of the anionic carboxylate function of Asp435 of the enzymatic catalytic site of TMPRSS2 and to promote the establishment of a salt bridge between the enzyme and its inhibitor. Advantageously, the aryl ring linked to the ester function of the coumarin derivative is substituted by one or more halogen atoms (R³). A halogen atom may be fluorine, chlorine, bromine or iodine. Preferably a halogen atom is chlorine or bromine. Preferably the aryl ring bears one or two or three halogen atoms, to favour the inhibitory activity on the enzyme. It will be easily understood by a person skilled in the art that the aryl ring bears a hydrogen in each position where no halogen or other substituent is present. Alternatively, the aryl ring linked to the ester function of the coumarin derivative does not bear a halogen atom (R³). In other words, x in Formula I may be equal to 0. Advantageously the coumarin core also bears one or more halogen atoms (R⁴), preferably 1 or 2. It will also be easily understood by a person skilled in the art that the coumarin bears a hydrogen in each position where no halogen or other substituent is present. Alternatively, the coumarin core does not bear a halogen atom (R⁴). In other words, y in Formula I may be equal to 0. An alkyl chain of 1 to 6 carbon atom may be a methyl, an ethyl, a propyl, a butyl, a pentyl, a hexyl, a tert-butyl. A substituent on the R¹ linear or branched alkyl chain of 1 to 6 carbon atoms may be one or more suitable substituent(s) to favour interaction with the host cell protease. Suitable substituents may be a halogen. Suitable substituents may be chosen among chloride, bromide, iodide, mesylate, triflate, tosylate, acetate or trifluoroacetate. The one or more substituent(s) may be positioned on any carbon atom of the said R¹ alkyl chain. Advantageously, the substituent may be a leaving group. By leaving group is meant an atom or a group of atoms that is able to depart upon heterolysis taking with it the bonding electrons. Chloride and bromide are preferred leaving groups. Preferably a leaving group is linked to the aryl ring with a short linear or branched alkyl chain, such as an alkyl chain with 1 to 6 carbon atoms, such as a linear or branched alkyl chain of 1 to 6 carbon atoms e.g. a methyl. In preferred embodiments, R¹ is -CH₂Cl or -CH₂Br. A salt of the compound of Formula I may have as counterion for example chloride, bromide, iodide or trifluoroacetate. A compound of Formula I may also be in the form of a conjugate acid base, such as in the form of a hydrochloride for example. By respiratory disease one means a pathological condition affecting the organs, and/or the tissues and/or the cells thereof, that enable to breathe, making such breathing difficult. Such organs or tissues include the lungs, the trachea, the bronchi, the bronchioles, the alveoli, the pleura and pleural cavity, the nerves and muscles of respiration. Respiratory diseases include but are not limited to the cold, influenza, pharyngitis, bronchitis, pneumonia, tuberculosis, asthma, lung cancer, Covid-19 or other severe acute respiratory syndromes (SARS). Targeted cells may be cells expressing transmembrane serine protease 2 such as for example lung epithelial cells. Treatment may also include a reduction of the inflammation observed in such cells. The respiratory disease may be a viral infection or another type of infection affecting the respiratory system. A compound of Formula I or a prodrug thereof and/or a salt thereof, may also be used in the prevention of respiratory diseases. The invention also pertains to a compound of Formula I or a prodrug thereof and/or a salt thereof, wherein R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms, preferably of 1 to 2 carbon atoms. Examples of a basic group are an amine, an amidine or a guanidine group, providing an aminium, an amidinium or a guanidinium group at physiological pH. An example of precursor of a basic group is a carbamate function or a pseudocarbamate function. The invention also pertains to a compound of Formula I wherein R² is - (CH₂)_n-NH-COOR⁵ or -(CH₂)_n-C(=NH)NH-COOR⁵ or -(CH₂)_n-NH-C(=NH)NH-COOR⁵ with R⁵ being a linear or branched alkyl chain with 1 to 6 carbon atoms and with n being an integer between 0 and 6, preferably between 1 and 6. The invention also pertains to a compound of Formula I or a prodrug thereof and/or a salt thereof, wherein R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms, preferably of 1 to 2 carbon atoms for use as a medicament. Such compounds present an inhibitory activity against serine proteases, as shown in the examples. The invention also pertains to a pharmaceutical composition comprising a compound of Formula I or a prodrug thereof and/or salt thereof, wherein R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms, preferably of 1 to 2 carbon atoms and a pharmaceutically acceptable carrier. The invention particularly pertains to a compound of Formula I wherein R¹ is a methyl substituted by a chloride, R² is an amine or a guanidine or an amidine preceded by a methyl or an ethyl, R² being in ortho, meta or para position of the ester group on the aryl ring and wherein x and y are both 0, or a prodrug thereof and/or a salt thereof. By a prodrug is particularly meant that the amine, guanidine or amidine may be present as a carbamate or a pseudocarbamate function bearing a methyl, ethyl, propyl, butyl, pentyl, hexyl or tertbutyl function. By a salt is particularly meant that the amine, guanidine or amidine may be conjugated with a hydrochloride. The invention also particularly pertains to pharmaceutical compositions of those compounds and their use as a medicament. Examples of compounds according to the invention are:

Compound a

Compound b

Compound c

Compound d

Compound e

Compound f The invention also pertains to the preparation of such compounds. A general method of synthesis may imply the following steps: - Preparation of a phenol intermediate bearing the desired R² group in ortho, meta or para position and optionally one or more R³ groups. The basic group comprised in R² is advantageously protected by a tert-butyloxycarbonyl group or any other suitable protecting group - Preparation of a 2-oxo-2H-1-benzopyran-3-carbonyl chloride bearing the desired R¹ group and optionally one or more R⁴ groups from a salicylic aldehyde according to the process described in the literature (Pochet et al. J. Med. Chem. 1996, 39, 2579-252585) - Adding the obtained 2-oxo-2H-1-benzopyran-3-carbonyl chloride to the phenol intermediate bearing the desired protected R² group in ortho, meta or para position and optionally one or more R³ groups. - Optionally deprotecting the protected basic group of the R² group in acidic conditions. The invention also pertains to the preparation of such pharmaceutical composition. The term “pharmaceutically acceptable carrier” is intended to mean a medium that is compatible with the respiratory system, such as for example the pulmonary system. The pharmaceutical composition according to the invention can be administered through any type of administration, such as an oral administration, a cutaneous administration, an injection, an intravenous administration, a subcutaneous administration, a systemic administration, a parenteral, digestive, rectal or transcutaneous administration, a transmucosal administration, a pulmonary, nasal or sublingual administration, an inhalation or a nebulization or an aerosol administration. Preferably, the composition is administered orally, by injection or by an aerosol. A pulmonary or aerosol administration can be through a gaseous composition, a volatile composition or through a composition that is a suspension in a gaseous vector. Advantageously, the pharmaceutical composition may be in a form suitable for an aerosol administration. Compounds as recited above are also referred to as coumarin derivatives. Possible routes of synthesis of the coumarin derivatives are described in the examples. It will however be understood that such synthetic routes do not limit the present invention. EXAMPLES Example 1: Organic synthesis of the aryl esters of coumarin-3-carboxylic acid with amidine or guanidine function. One of the possible pathways to access the compounds of formula I bearing an amidine or a guanidine function is reported in schemes 1-3. It should be understood that present invention is not limited to the below synthesis strategies.

Scheme 1 Reagents are for step i: EtOH, HCl, for step ii: NH₃, MeOH, for step iii: di-tert-butyl dicarbonate (Boc)₂O, NaOH, dioxane/H₂O, Boc is tert-butyloxycarbonyl protecting group.

Scheme 2 Reagents are for step i: S-methyl isothiouronium salt (CH₃S- C(=NH)NH₂.HX), CH₃CN; for step ii: (Boc)₂O, NaOH, dioxane/H₂O; for step iii: S- methyl bis(tert-butoxycarbonyl)thiopseudourea (CH₃S-C(=N-Boc)NH-Boc), CH₃CN.

Scheme 3 Reagents are for step i: diethyl malonate, piperidine, HOAc, EtOH; for step ii: HOH, H⁺, EtOH; for step iii: SOCl₂ or SOBr₂; for step iv: intermediate 9, 12 or 13, pyridine, dioxane; for step v: HX, methylene chloride. The phenol intermediates bearing the tert-butoxycarbonyl (Boc)- protected amidines (9) or guanidines (12,13) are prepared according to schemes 1 and 2. Intermediate 17 obtained according to a well-known classical synthetic process (Pochet et al., J. Med. Chem. 1996, 39, 2579) reacts with the protected intermediates 9, 12 or 13 to provide intermediates 18. In the last step, deprotection of the tert-butoxycarbonyl group in acidic conditions provides the final compounds. According to the literature, the phenol hydroxyl group does not need to be protected before reaction with (Boc)₂O (Frederick et al., Bioorg. Med. Chem. Lett. 2006, 16, 2017). Example 2a: Organic synthesis of the aryl esters of coumarin-3-carboxylic acid with amine function Synthetic pathway to intermediates 3b is first shown in scheme 4.

Scheme 4 To obtain the aminoalkylphenol 2b, the appropriate methoxyphenylalkylamine 1b (6.6 mmol) was dissolved in acetic acid (10 mL) and concentrated HBr (11 mL) and heated to reflux for 4 hours under stirring. The reaction mixture was cooled to room temperature and the solvent and HBr were removed by distillation under reduced pressure. The title compound 2b as the hydrobromide was obtained as a solid in good yields and used in the next step without further purification. To obtain the tert-butyl hydroxyphenylalkylcarbamate 3b, the solution of compound 2b (6.6 mmol) in DMF (2 mL) and dioxane (20 mL) was supplemented with triethylamine (6.6 mmol) and stirred for 15 minutes. Di-tert-butyl dicarbonate (6.6 mmol) was added and the reaction mixture was stirred at room temperature for 18 hours. The solvents were removed by distillation under reduced pressure and the resulting residue was taken up in water (20 mL) and extracted trice with ethyl acetate (3 x 25 mL). The organic layers were combined, dried over MgSO₄, and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel chromatography to afford the title compound 3b as a white solid. Synthetic pathway to intermediate 8b is shown in scheme 5.

Scheme 5 6-Chloromethyl-2-oxo-2H-1-benzopyran-3-carbonyl chloride (intermediate 8b) was obtained as described in the literature (Pochet et al. J. Med. Chem. 1996, 39, 2579-2585). Synthetic pathway to final compounds 10b is shown in scheme 6.

Scheme 6 (Tert-butoxycarbonyl)aminoalkylphenyl 6-(chloromethyl)-2-oxo-2H-1- benzopyran-3-carboxylate 9b was obtained as follows. Compound 8b (4.5 mmol) was dissolved in anhydrous dioxane (3 mL) and added with stirring to a solution of the appropriate compound 3b (5.0 mmol) and of anhydrous pyridine (5.0 mmol) in anhydrous dioxane (7 mL). After 2 hours at room temperature, the solvent was removed by evaporation under reduced pressure. The residue was purified by silica gel chromatography to afford the title compound 9b as a white solid. Aminoalkylphenyl 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3- carboxylate hydrochloride 10b was obtained as follows. Compound 9b (4 mmol) was dissolved in anhydrous diethyl ether (10 mL) or another appropriate solvent (10 mL) and gaseous HCl was bubbled for 10 minutes. The reaction mixture was stirred for 1 hour and then the solvent was removed by distillation under reduced pressure. The residue of the final compound 10b as the hydrochloride was recrystallized in the appropriate solvent. Example 2b: alternative organic synthesis of the aryl esters of coumarin-3-carboxylic acid with amine, amidine or guanidine function yielding final products 16 of scheme 9 through intermediates 2 or 6 of scheme 7 or intermediate 12 of scheme 8. An alternative synthesis route is hereafter described with reference to schemes 7, 8 and 9. Each obtained compound was analysed by ¹H NMR and ¹³C NMR with main peaks listed. Melting temperatures of intermediates and of final products are also presented. Table 1 summarizes the obtained compounds with their formulae and molecular weight (Mw) expressed in g/mol. The formulae also include an indication of the potential presence of crystallisation water molecules. This was deduced by elemental analysis. Compounds 15a-15j, 19a bearing carbamate or pseudocarbamate groups can be considered as prodrugs. Compounds 16a-16j, 20a are positively charged at physiological pH and are here presented as examples in the form of hydrochloride salts.

Scheme 7 Reagents of scheme 7 are for step i: (Boc)₂O, NaHCO3, MeOH; for step ii: benzyl bromide, K2CO3, CH₃CN; for step iii: HCl 12N, MeOH; for step iv: S-methyl bis(tert-butoxycarbonyl)thiopseudourea (CH₃S-C(=N-Boc)NH- Boc), CH₂Cl₂; for step v: H₂, Pd/C, EtOH.

Scheme 8 Reagents of scheme 8 are for step: i: benzyl bromide, K₂CO₃, CH₃CN; for step ii: HCl, EtOH; for step iii: NH₄OH, EtOH; for step iv: (Boc)₂O, pyridine, DMAP, CH₂Cl₂; for step v: H₂, Pd/C, EtOH.

Scheme 9 Reagents of scheme 9 are in step i: -LG = -Cl: SOCl₂; -LG = -OCOR, 1: (RCO)2O, 2: SOCl₂; -LG = -OSO2R, 1: RSO2Cl, 2: SOCl₂;, in step ii: intermediate 2, 6 or 12, pyridine, dioxane; in step iii: HX, CH₃CN.

Scheme 10 Reagents of scheme 10 are in step i: (Boc)₂O, NaHCO₃, MeOH; in step ii: intermediate 14, pyridine, dioxane; in step iii: HX, CH₃CN Synthesis of intermediates 2 A mixture of compound 1 of scheme 7 (7.29 mmol), di-tert-butyl dicarbonate (1.75 g, 8.02 mmol) and sodium bicarbonate (1.29 g, 15.31 mmol) in methanol (25 mL) was refluxed for 16 h. After cooling, the reaction mixture was evaporated under reduced pressure. The residue was taken up in water (25 mL) and the resulting suspension was extracted thrice with dichloromethane (3 x 15 mL). The organic phases were combined, washed twice with distilled water (3 x 10 mL), dried over MgSO4 and filtered. The filtrate was evaporated to dryness and the resulting residue (compound 2 of scheme 7) was used in the next step without further purification. Tert-butyl (4-hydroxyphenethyl)carbamate (2a) White solid (m.p.: 73.7-75.1 °C). ¹H NMR (500 MHz, DMSO-d₆) δ 3(+. #N& +7& ;H), 6.96 (d, J = 8.4 Hz, 2H, 2-H/6-H), 6.81 (t, J = 5.7 Hz, 1H, NH), 6.66 (d, J = 8.4 Hz, 2H, 3-H/5-H), 3.05 (q, J = 15.2, 5.8 Hz, 2H, CH₂NH), 2.59 – 2.52 (m, 2H, CH₂), 1.36 (s, 9H, C(CH₃)₃).¹³C NMR (DMSO-d₆) δ 155.6, 155.5, 129.4, 129.4, 115.0, 77.4, 41.9, 34.7, 28.3. Tert-butyl (3-hydroxyphenethyl)carbamate (2b) Liquid. ¹H NMR (DMSO-d₆) δ 1.37 (s, 9H, C(CH₃)₃), 2.58 (t, J=7.6 Hz, 2H, PhCH₂), 3.08 (dt, J=8.2 Hz/6.1 Hz, 2H, CH₂N), 6.58 (m, 3H, 2-H/4-H/6-H), 6.84 (t, J=5.7 Hz, 1H, NH), 7.05 (t, J=7.3 Hz, 1H, 5-H), 9.25 (s, 1H, OH). ¹³C NMR (DMSO-d₆) δ ,2(-& 35.6, 41.6, 77.5, 113.0, 115.5, 119.2, 129.2, 140.8, 155.5, 157.3. Tert-butyl (4-hydroxybenzyl)carbamate (2c) Liquid.¹H NMR (DMSO-d₆) δ 1.38 (s, 9H, C(CH₃)₃), 3.99 (d, J=6.2 Hz, 2H, CH₂), 6.68 (d, J=8.2 Hz, 2H, 3-H/5-H), 7.02 (d, J=8.1 Hz, 2H, 2-H/6-H), 7.23 (t, J=6.2 Hz, 1H, NH), 9.23 (s, 1H, OH). ¹³C NMR (DMSO-d₆) δ 28.2, 42.8, 77.5, 114.8, 128.2, 130.3, 155.6, 156.0. Tert-butyl (3-hydroxybenzyl)carbamate (2d) Liquid.¹H NMR (DMSO-d₆) δ 1.39 (s, 9H, C(CH₃)₃), 4.03 (d, J=6.2 Hz, 2H, CH₂), 6.58- 6.64 (m, 3H, 2-H/4-H/6-H), 7.08 (t, J=8.0 Hz, 1H, 5-H), 7.31 (t, J=6.3 Hz, 1H, NH), 9.30 (s, 1H, OH). ¹³C NMR (DMSO-d₆) δ 28.8, 43.7, 78.2, 114.0, 114.2, 117.9, 129.6, 142.1, 156.3, 157.8. Synthesis of intermediates 3 A mixture of intermediate 2 of scheme 7 (24.65 mmol), benzyl bromide (3 mL, 25.26 mmol) and potassium carbonate (1.50 g, 35.24 mmol) in acetone (100 mL) was refluxed for 20 h. After cooling, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then supplemented with hexane. The resulting precipitate of intermediate 3 of scheme 7 was collected by filtration, washed with hexane and dried. Tert-butyl (4-(benzyloxy)phenethyl)carbamate (3a) White solid (m.p.: 80.7-82.8 °C).¹H NMR (500 MHz, DMSO-d₆) δ 7.43 (d, J = 6.7 Hz, 2H, 2’-H, 6’-H), 7.38 (t, J = 7.6 Hz, 2H, 3’-H/5’-H), 7.32 (t, J = 7.2 Hz, 1H, 4’-H), 7.09 (d, J = 8.6 Hz, 2H, 2-H/6-H), 6.92 (d, J = 8.6 Hz, 2H, 3-H/5-H), 6.83 (t, J = 5.7 Hz, 1H, NH), 5.06 (s, 2H, CH₂O), 3.08 (q, 2H, CH₂NH), 2.61 (t, 2H, CH₂), 1.36 (s, 9H, C(CH₃)₃).¹³C NMR (DMSO-d₆) δ 156.7, 155.5, 137.3, 131.6, 129.6, 128.4, 127.7, 127.6, 114.6, 77.4, 69.1, 41.7, 34.6, 28.3. Tert-butyl (4-(benzyloxy)benzyl)carbamate (3b) White solid. ¹H NMR (500 MHz, DMSO-d₆) δ 3(,, #N& +7& :H), 7.44 (d, J = 7.3 Hz, 2H, arom. H), 7.39 (t, J = 7.5 Hz, 1H, arom. H), 7.32 – 7.28 (m, 2H, arom. H), 7.15 (d, J = 8.4 Hz, 2H, arom. H), 6.95 (d, J = 8.5 Hz, 2H, arom. H), 5.08 (s, 2H, CH2O), 4.04 (d, J = 6.1 Hz, 2H, CH2), 1.39 (s, 9H, C(CH3)3). Synthesis of intermediates 4 To a solution of intermediate 3 of scheme 7 (11.54 mmol) in methanol (40 mL) was added a concentrated aqueous solution of HCl (30 mL) under stirring and on an ice bath. After 5 h, the reaction mixture was evaporated under reduced pressure. The residue was taken up in an aqueous solution of sodium hydroxide 10% m/v (25 mL) to obtain an aqueous solution at pH 12-14. The resulting suspension was extracted thrice with ethyl acetate (3 x 20 mL). The organic phases were combined, washed twice with distilled water (2 x 15 mL) dried over MgSO4, and filtered. The filtrate was evaporated to dryness and the resulting residue of 4 of scheme 7 was used in the next step without further purification. In some cases, intermediate 4 was obtained as the hydrochloride after treatment of intermediate 3 in methanol with concentrated HCl. The resulting precipitate was collected by filtration, washed with methanol and dried (ex: 4b). 2-(4-(Benzyloxy)phenyl)ethan-1-amine (4a) White solid (m.p.: 122.2-124.5 °C). ¹H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 7.5 Hz, 2H, 2’-H/6’-H), 7.38 (t, J = 7.5 Hz, 2H, 3’-H/5’-H), 7.32 (t, J = 7.3 Hz, 1H, 4’-H), 7.12 (d, J = 8.0 Hz, 2H, 2-H/6-H), 6.92 (d, J = 8.0 Hz, 2H, 3-H/5-H), 5.05 (s, 2H, CH2O), 2.96 (t, J = 6.9 Hz, 2H, CH2NH2), 2.72 (t, J = 6.9 Hz, 2H, CH2), 2.06 (s, 2H, NH2). ¹³C NMR (CDCl3) δ 157.38 .57.1 +-+(1& +,3(2& +,2(0& +,1(3& +,1(/& ++/(*& 1*(+& .-(,& -2(/( (4-(Benzyloxy)phenyl)methanamine hydrochloride (4b) White solid (m.p.: 240.4-241.7 °C). ¹H NMR (500 MHz, DMSO-d₆) δ 2(*0 #N& -7& NH3⁺), 7.44 (d, J = 7.2 Hz, 2H, 2-H/6-H), 7.42 – 7.37 (m, 4H, 2’-H/3’-H/5’-H/6’-H), 7.35 – 7.31 (m, 1H, 4’-H), 7.07 – 7.04 (m, 2H, 3-H/5-H), 5.14 (s, 2H, CH2O), 3.94 (s, 2H, CH2). ¹³C NMR (DMSO-d₆) δ +/2(+& +-0(1& +-*(,& +,2(,& +,1(0& +,1(-& +,0(*& ++.(1& 68.9, 41.6. Synthesis of intermediates 5 A mixture of intermediate 4 of scheme 7 as the base (7.70 mmol) and 1,3- bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (2.19 g, 7.54 mmol) in dichloromethane (35 mL) was stirred at room temperature for 28 h. The reaction mixture was then evaporated under reduced pressure. The residue (intermediate 5 of scheme 7) was purified by flash column chromatography using hexane/ethyl acetate. N1-(4-(Benzyloxy)phenethyl)-N2,N3-di(tert-butoxycarbonyl)guanidine (5a) White solid (m.p.: 99.2-101.5 °C).¹H NMR (500 MHz, CDCl3) δ ++(.1 #N& +7& :HCO), 8.36 (t, J = 5.2 Hz, 1H, CH₂NH), 7.43 (d, J = 6.8 Hz, 2H, 2’-H/6’-H), 7.38 (t, J = 7.4 Hz, 2H, 3’-H/5’-H), 7.32 (t, J = 7.2 Hz, 1H, 4’-H), 7.13 (d, J = 8.6 Hz, 2H, 2-H/5-H), 6.91 (d, J = 8.6 Hz, 2H, 3-H/5-H), 5.04 (s, 2H, CH₂O), 3.66 – 3.61 (m, 2H, CH₂NH), 2.81 (t, J = 7.3 Hz, 2H, CH₂), 1.50 (s, 9H, C(CH₃)₃), 1.48 (s, 9H, C(CH₃)₃). ¹³C NMR (CDCl3) δ +0-(0& +/1(/& +/0(+& +/-(,& +-1(+& +-*(3& +,3(2& +,2(0& +,1(3& +,1(/& ++/(*& 83.0, 79,2, 70.1, 42.5, 34.4, 28.3, 28,1. N1-(4-(Benzyloxy)benzylamino)-N2,N3-di(tert-butoxycarbonyl)guanidine (5b) White solid (m.p.: 113.6-114.7 °C). ¹H NMR (500 MHz, DMSO-d₆) δ ++(/* #N& +7& NHCO), 8.55 (t, J = 5.5 Hz, 1H, NHCH₂), 7.44 (d, J = 7.4 Hz, 2H, 2-H/6-H), 7.39 (t, J = 7.5 Hz, 2H, 2’-H/6’-H), 7.32 (t, J = 7.2 Hz, 1H, 4’-H), 7.24 (d, J = 8.5 Hz, 2H, 3’-H/5’- H), 6.98 (d, J = 8.6 Hz, 2H, 3-H/5-H), 5.09 (s, 2H, CH₂O), 4.42 (d, J = 5.6 Hz, 2H, CH₂NH), 1.43 (s + s, 18H, C(CH₃)₃).¹³C NMR (DMSO-d₆) δ +0.(/& +/2(3& +/0(0& +/-(.& 138.5, 131.7, 130.2, 129.8, 129.2, 129.0, 116.2, 84.4, 79.6, 70.6, 44.5, 29.4, 29.0. Synthesis of intermediates 6 Intermediate 5 of scheme 7 (4.17 mmol) dissolved in ethanol (40 mL) was introduced in a hydrogenator in the presence of 10% wt Pd/C (0.2 g, 1.88 mmol). After removing the air inside the reactor, the reaction mixture was pressurized to 10 bar H₂ and stirred for 48 h. The catalyst was then removed by filtration and the filtrate was evaporated to dryness under reduced pressure. The residue (intermediate 6 of scheme 7) was used in the next step without further purification. N1-(4-Hydroxyphenethyl)-N2,N3-di(tert-butoxycarbonyl)guanidine (6a) White solid (m.p.: 175.8 °C (dec.)).¹H NMR (500 MHz, DMSO-d₆) δ ++(.2 #N& +7& ;H), 9.19 (s, 1H, NH), 8.28 (t, J = 5.6 Hz, 1H, CH₂NH), 7.01 (d, J = 8.4 Hz, 2H, 2-H/6-H), 6.68 (d, J = 8.4 Hz, 2H, 3-H/5-H), 3.49-3.41 (m, 2H, CH₂NH), 2.69 (t, J = 7.4 Hz, 2H, CH₂), 1.46 (s, 9H, C(CH₃)₃), 1.40 (s, 9H, C(CH₃)₃).¹³C NMR (DMSO-d₆) δ +0-(*& +//(1& 155.1, 151.9, 129.4, 128.7, 115.1, 82.8, 78.0, 41.9, 33.5, 27.9, 27.5. Synthesis of intermediates 8 A mixture of intermediate 7 of scheme 8 (7.66 mmol), benzyl bromide (0.9 ml, 7.58 mmol) and potassium carbonate (1.50 g, 10.85 mmol) in acetone (40 mL) was refluxed for 12 h. After cooling, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and then supplemented with hexane under stirring. The resulting precipitate of intermediate 8 of scheme 8 was collected by filtration, washed with hexane and dried. 2-(4-(Benzyloxy)phenyl)acetonitrile (8a) White solid (m.p.: 67.8-69.1 °C).¹H NMR (500 MHz, DMSO-d⁶) δ 1(.. #A& J = 6.8 Hz, 2H, 2’-H/6’-H), 7.39 (t, J = 7.4 Hz, 2H, 3’-H/5’-H), 7.33 (t, J = 7.3 Hz, 1H, 4’-H), 7.26 (d, J = 8.7 Hz, 2H, 2-H/6-H), 7.03 (d, J = 8.7 Hz, 2H, 3-H/5-H), 5.10 (s, 2H, CH₂O), 3.93 (s, 2H, CH₂CN). ¹³C NMR (DMSO-d₆) δ +/1(1& +-1(*& +,3(-& +,2(.& +,1(2& +,1(1& 123.3, 119.5, 115.3, 69.2, 21.5. 4-(Benzyloxy)benzonitrile (8b) White solid (m.p.: 93.7-95.1 °C). ¹H NMR (500 MHz, DMSO-d₆) δ 1(11 #A& ,7& ,'H/6- H), 7.45 (d, J = 7.1 Hz, 2H, 2’-H/6’-H), 7.40 (t, 2H, 3’-H/5’-H), 7.37 – 7.32 (m, 1H, 4’- H), 7.18 (d, 2H, 3-H/5-H), 5.20 (s, 2H, CH₂O). ¹³C NMR (DMSO-d₆) δ +0*(*& +-.(.& 132.5, 126.8, 126.4, 126.1, 117.3, 114.1, 101.2, 67.9. Synthesis of intermediates 9 HCl gas was bubbled during 30 minutes under stirring and argon atmosphere in a solution of intermediate 8 of scheme 8 (13.39 mmol) in ethanol (30 mL) cooled on an ice bath. The reaction mixture was then stirred at room temperature for 16 h. The solvent was partly removed under reduced pressure and diethyl ether (20 mL) was added. The resulting solid (intermediate 9 of scheme 8) in suspension was collected by filtration, washed with diethyl ether and dried. Ethyl 2-(4-(benzyloxy)phenyl)acetimidate hydrochloride (9a) White solid (m.p.: 194.8-196.6 °C). ¹H NMR (500 MHz, DMSO-d₆) δ 1(.. #A& J = 6.9 Hz, 2H, 2’-H,6’-H), 7.39 (t, J = 7.4 Hz, 2H, 3’-H/5’-H), 7.33 (t, J = 7.2 Hz, 1H, 4’-H), 7.28 (d, J = 8.7 Hz, 2H, 2-H/6-H), 7.02 (d, J = 8.7 Hz, 2H, 3-H/5-H), 5.10 (s, 2H, CH₂O), 4.38 (q, J = 7.0 Hz, 2H, CH₂CH₃), 3.89 (s, 2H, CH₂), 1.28 (t, J = 7.0 Hz, 3H, CH₂CH₃). ¹³C NMR (DMSO-d₆) δ +11(0& +/2(*& +-0(3& +-*(.& +,2(/& +,1(3& +,1(1& 124.2, 115.2, 69.2, 68.9, 37.7, 13.3. Ethyl 4-(benzyloxy)benzimidate hydrochloride (9b) White solid (m.p. : 185.5-187.2 °C). ¹H NMR (500 MHz, DMSO-d₆) δ ++(*/ #N& ,7& NH₂ ⁺), 8.07 (d, 2H, 2-H/6-H), 7.46 (d, 1H, 2’-H/6’-H), 7.40 (t, 2H, 3’-H/5’-H), 7.37 – 7.33 (m, 1H, 4’-H), 7.26 (d, 2H, 3-H/5-H), 5.26 (s, 2H, CH₂O), 4.56 (q, J = 7.0 Hz, 2H, CH₂CH₃), 1.47 (t, J = 7.0 Hz, 3H, CH₂CH₃). ¹³C NMR (126 MHz, DMSO-d₆) δ +1*(+& 163.9, 136.1, 131.4, 129.3, 128.5, 128.5, 128.2, 127.9, 127.8, 117.8, 115.4, 114.2, 69.8, 68.9, 13.5. Synthesis of intermediates 10 To a solution of intermediate 9 of scheme 8 (3.34 mmol) in ethanol (20 mL) was added a concentrated aqueous solution of ammonia (7 mL) under stirring and on an ice/acetone bath. After addition, the reaction mixture was stirred at room temperature for 5 h. The reaction mixture was evaporated under reduced pressure. The residue was taken up by ethanol (10 mL) and the suspension was heated to boiling point. The hot suspension was filtered, and the filtrate was evaporated under reduced pressure. The residue of intermediate 10 of scheme 8 was used in the next step without further purification. 2-(4-(Benzyloxy)phenyl)acetimidamide hydrochloride (10a) White solid (m.p.: 130.2-132.5 °C). ¹H NMR (500 MHz, DMSO-d6) δ 3(+/ #N& ,7& NH₂ ⁺), 8.67 (s, 2H, NH₂), 7.43 (d, J = 7.3 Hz, 2H, 2’-H/6’-H), 7.41 – 7.31 (m, 5H, 3’- H/5’-H/4’-H/2-H/6-H), 7.01 (d, J = 8.2 Hz, 2H, 3-H/5-H), 5.10 (s, 2H, CH₂O), 3.62 (s, 2H, CH₂).¹³C NMR (DMSO-d₆) δ +03(0& +/2(*& +-1(,& +-*(-& +,2(0& +,2(*& +,1(2& +,0(-& 115.2, 69.4, 37.0. 4-(Benzyloxy)benzimidamide hydrochloride (10b) White solid (m.p.: 191.9-193.6 °C). ¹H NMR (500 MHz, DMSO-d₆) δ 3(** #N& .7& C(NH₂)NH₂ ⁺), 7.83 (d, J = 8.9 Hz, 2H, 2-H/6-H), 7.46 (t, J = 9.1 Hz, 2H, 2’-H/6’-H), 7.41 (t, J = 7.4 Hz, 2H, 3’-H/5’-H), 7.35 (t, J = 7.2 Hz, 1H, 4’-H), 7.24 (d, J = 8.9 Hz, 2H, 3-H/5-H), 5.25 (s, 2H, CH₂O). ¹³C NMR (DMSO-d₆) δ +0-(3& +0+(2& +-/(/& +,3(.& 127.7, 127.3, 127.0, 118.9, 114.4, 68.8. Synthesis of intermediates 11 A mixture of intermediate 10 of scheme 8 (3.25 mmol), di-tertbutyl dicarbonate (2.00 g, 9.16 mmol), 4-dimethylaminopyridine (1.03 g, 8.43 mmol), pyridine (10 mL) and dichloromethane (20 mL) was refluxed under stirring for 20 h. The reaction mixture was then evaporated under reduced pressure. The residue was taken up in an aqueous solution of NaHCO3 10 % m/v (25 mL) and the resulting suspension was extracted with dichloromethane (3 x 15 mL). The organic phases were combined, washed twice with an aqueous solution of NaHCO310% m/v (2 x 15 mL), dried over MgSO4, and filtered. The filtrate was evaporated to dryness and the residue (intermediate 11 of scheme 8) was purified by flash column chromatography using hexane/ethyl acetate. Tert-butyl (2-(4-(benzyloxy)phenyl)-1-iminoethyl)carbamate (11a) White solid (m.p.: 82.5-84.4 °C). ¹H NMR (500 MHz, DMSO-d₆) δ +*(-1 #N& +7& :H), 7.43 (d, J = 6.9 Hz, 2H, 2’-H/6’-H), 7.38 (t, J = 7.6 Hz, 2H, 3’-H/5’-H), 7.32 (t, J = 7.3 Hz, 1H, 4’-H), 7.15 (d, J = 8.7 Hz, 2H, 2-H/6-H), 6.94 (d, J = 8.6 Hz, 2H, 3-H/5-H), 5.08 (s, 2H, CH₂O), 3.60 (s, 2H, CH₂), 1.43 (s, 9H, C(CH₃)₃). ¹³C NMR (DMSO- d6) δ 170.9, 157.2, 150.2, 137.2, 130.4, 128.4, 127.8, 127.6, 127.0, 114.6, 80.6, 69.1, 41.8, 27.7. Tert-butyl ((4-(benzyloxy)phenyl)(imino)methyl)carbamate (11b) White solid. ¹H NMR (500 MHz, DMSO-d₆) δ 3(-* #N& ,7& :H), 7.91 (d, J = 8.6 Hz, 2H, 2-H/6-H), 7.46 (d, J = 7.3 Hz, 2H, 2’-H/6’-H), 7.40 (t, J = 7.4 Hz, 2H, 3’-H/5’-H), 7.34 (t, J = 7.1 Hz, 1H, 4’-H), 7.10 (d, J = 8.7 Hz, 2H, 3-H/5-H), 5.19 (s, 2H, CH₂O), 1.45 (s, 9H, C(CH₃)₃). Synthesis of intermediates 12 In a hydrogenator were introduced intermediate 11 of scheme 8 (1.38 mmol), 10% wt Pd/C (0.047 g, 0.44 mmol) and ethanol (20 mL). After removing the inside air, the reactor was pressurized to 10 bar H₂ and the reaction mixture was stirred for 24 h. The catalyst was then removed by filtration and the filtrate was evaporated to dryness under reduced pressure. The residue (intermediate 12 of scheme 8) was used in the next step without further purification. Tert-butyl (2-(4-hydroxyphenyl)-1-iminoethyl)carbamate (12a) White solid (m.p.: 134.2 °C (dec.)). ¹H NMR (500 MHz, CDCl3) δ 1(+/ #A& J = 8.4 Hz, 2H, 2-H/6-H), 6.79 (d, J = 8.5 Hz, 2H, 3-H/5-H), 5.30 (s, 1H, OH), 3.96 (s, 2H, CH₂), 1.49 (s, 9H, C(CH₃)₃). ¹³C NMR (DMSO-d₆) δ +1+(*& +/0(+& +/*(,& +-*(-& +,/(*& ++/(*& 80.5, 41.8, 27.7. Synthesis of intermediates 15 A mixture of 6-(hydroxymethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid 13 (0.50 g, 2.27 mmol) (obtained according to Pochet et al. J. Med. Chem. 1996, 39, 2579–2585) and thionyl chloride (5 mL, 68.92 mmol) was refluxed for 3 h. The reaction mixture was cooled and evaporated under vacuum. After adding anhydrous toluene (5 mL) to the residue, the solvent was removed under reduced pressure. This last operation was carried out three times. Without further purification, the acyl chloride 14 of scheme 9 (-LG = -Cl) was stirred for 18 h at room temperature under argon atmosphere with intermediate 2 of scheme 7 (or 6 of scheme 7 or compound 12 of scheme 9) (2.27 mmol) and anhydrous pyridine (0.3 mL, 3.72 mmol) in anhydrous dioxane (7 mL). The reaction mixture was then evaporated under reduced pressure. The residue was purified by flash column chromatography using dichloromethane/methanol. Starting from compound 13 of scheme 9, intermediate 14 of scheme 9 with -LG = -OCOCH₃ was obtained according to the literature (Doucet et al. J. Med. Chem. 1999, 42, 4161-4171). Then, the above-mentioned protocol was followed to obtain the corresponding 6-acetoxymethyl-substituted intermediates 15 of scheme 9. 3-(2-((Tert-butoxycarbonyl)amino)ethyl)phenyl 6-(chloromethyl)-2-oxo- 2H-chromene-3-carboxylate (15a) White solid (m.p.: 145-157 °C). ¹H NMR (DMSO-d₆) δ +(-1 #N& 37& C(CH₃)₃), 2.75 (t, J=7.4 Hz, 2H, PhCH₂), 3.17 (dt, J=7.9 Hz/6.1 Hz, 2H, CH₂N), 4.88 (s, 2H, CH₂Cl), 6.92 (t, J=5.7 Hz, 1H, NH), 7.11 (m, 2H, 2’-H/4’-H), 7.16 (d, J=7.6 Hz, 1H, 6’-H), 7.40 (t, J=8.1 Hz, 1H, 5’-H), 7.52 (d, J=8.6 Hz, 1H, 8-H), 7.85 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 8.05 (d, J=2.2 Hz, 1H, 5-H), 9.05 (s, 1H, 4-H).¹³C NMR (DMSO- d6) δ ,2(-& -/(+& .+(,& ./(*& 11(0& ++0(2& ++1(+& ++1(2& ++3(.& +,+(2& +,0(/& +,3(.& +-*(/& 134.6, 135.5, 141.4, 149.9, 150.3, 154.5, 155.5, 155.8, 161.0. 4-(((Tert-butoxycarbonyl)amino)methyl)phenyl 6-(chloromethyl)-2-oxo- 2H-chromene-3-carboxylate (15b) White solid (m.p.: > 260 °C). ¹H NMR (DMSO-d₆) δ +(.* #N& 37& 6#6H₃)₃), 4.16 (d, J=6.2 Hz, 2H, CH₂N), 4.88 (s, 2H, CH₂Cl), 7.21 (d, J=8.2 Hz, 2H, 2’-H/6’-H), 7.33 (d, J=8.2 Hz, 2H, 3’-H/5’-H), 7.44 (t, J=6.3 Hz, 1H, NH), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.85 (dd, J=8.7 Hz/2.2 Hz, 1H, 7-H), 8.05 (d, J=2.1 Hz, 1H, 5-H), 9.06 (s, 1H, 4- H). ¹³C NMR (DMSO-d₆) δ ,2(.& .-(*& ./(+& 12(*& ++0(3& ++2(*& +,+(1& +,2(,& +-*(1& 134.7, 135.6, 138.3, 149.1, 150.0, 154.6, 155.9, 155.9, 161.2. 4-(2-((Tert-butoxycarbonyl)amino)ethyl)phenyl 6-(chloromethyl)-2-oxo- 2H-chromene-3-carboxylate (15c) White solid (m.p.: 184 °C (dec)). ¹H NMR (DMSO-d₆) δ +(-1 #N& 37& C(CH₃)₃), 2.73 (t, J=7.4 Hz, 2H, PhCH₂), 3.17 (q, J=6.8 Hz, 2H, CH₂N), 4.88 (s, 2H, CH₂Cl), 6.91 (t, J=5.7 Hz, 1H, NH), 7.18 (d, J=8.1 Hz, 2H, 2’-H/6’-H), 7.29 (d, J=8.4 Hz, 1H, 3’-H/5’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.85 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 8.05 (d, J=2.1 Hz, 1H, 5-H), 9.04 (s, 1H, 4-H).¹³C NMR (DMSO-d₆) δ ,2(-& -.(2& .+(.& 45.0, 77.5, 116.8, 117.1, 117.8, 121.5, 129.8, 130.5, 134.5, 135.4, 137.4, 148.6, 149.8, 155.0, 155.5, 155.7, 161.1. 3-(((Tert-butoxycarbonyl)amino)methyl)phenyl 6-(chloromethyl)-2-oxo- 2H-chromene-3-carboxylate (15d) White solid (m.p.: > 260 °C). ¹H NMR (DMSO-d₆) δ +(.* #N& 37& 6#6H₃)₃), 4.18 (d, J=6.2 Hz, 2H, CH₂N), 4.88 (s, 2H, CH₂Cl), 7.13 (m, 2H, 2’-H/4’-H), 7.19 (d, J=7.7 Hz, 1H, 6’-H), 7.43 (t, J=7.8 Hz, 1H, 5’-H), 7.46 (t, J=6.3 Hz, 1H, NH), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.85 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 8.05 (d, J=2.2 Hz, 1H, 5-H), 9.05 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ ,2(,& .*(-& ./(*& 11(3& ++0(2& ++1(+& ++1(2& 120.0, 124.7, 129.5, 130.5, 134.5, 135.5, 142.3, 149.9, 150.3, 154.5, 155.8, 155.8, 161.1. 4-(2-(2,3-Bis(tert-butoxycarbonyl)guanidino)ethyl)phenyl 6- (chloromethyl)-2-oxo-2H-chromene-3-carboxylate (15e) White solid (m.p.: > 250 °C).¹H NMR (500 MHz, DMSO-d₆) δ ++(/* #N& +7& NH), 9.05 (s, 1H, 4-H), 8.37 (t, J = 5.6 Hz, 1H, CH₂NH), 8.05 (d, J = 2.1 Hz, 1H, 5- H), 7.85 (dd, J = 8.6, 2.2 Hz, 1H, 7-H), 7.51 (d, J = 8.6 Hz, 1H, 8-H), 7.34 (d, J = 8.3 Hz, 2H, 3’-H/5’-H), 7.21 (d, J = 8.4 Hz, 2H, 2’-H/6’-H), 4.88 (s, 2H, CH₂Cl), 3.56 (q, J = 6.3 Hz, 2H, CH₂NH), 2.87 (t, J = 7.4 Hz, 2H, CH₂), 1.47 (s, 9H, C(CH₃)₃), 1.41 (s, 9H, C(CH₃)₃). ¹³C NMR (DMSO-d₆) δ +0-(*& +0*(3& +//(1& +//(+& +/.(.& +/+(3& +.3(2& 148.6, 136.8, 135.4, 134.5, 130.4, 129.7, 121.6, 117.7, 117.0, 116.7, 82.9, 78.1, 44.9, 41.5, 33.7, 27.9, 27.5. 4-(2-((Tert-butoxycarbonyl)amino)ethyl)phenyl 6-(acetoxymethyl)-2-oxo- 2H-chromene-3-carboxylate (15f) White solid (m.p.: 181-184 °C). ¹H NMR (DMSO-d₆) δ +(-1 #N& 37& C(CH₃)₃), 2.10 (s, 3H, OCOCH₃), 2.73 (t, J=7.4 Hz, 2H, PhCH₂), 3.17 (m, 2H, CH₂N), 5.17 (s, 2H, CH₂O), 6.91 (t, J=5.7 Hz, 1H, NH), 7.18 (d, J=8.4 Hz, 2H, 3’-H/5’-H), 7.29 (d, J=8.6 Hz, 2H, 2’-H/6’-H), 7.50 (d, J=8.6 Hz, 1H, 8-H), 7.78 (dd, J=8.6 Hz/2.1 Hz, 1H, 7-H), 7.98 (d, J=2.1 Hz, 1H, 5-H), 9.05 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ ,*(1& 28.3, 32.8, 41.4, 64.3, 77.5, 116.5, 116.9, 117.7, 121.5, 129.6, 129.7, 133.1, 134.6, 137.4, 148.6, 150.0, 154.4, 155.5, 155.8, 161.1, 170.2. 3-(2-((Tert-butoxycarbonyl)amino)ethyl)phenyl 6-(acetoxymethyl)-2-oxo- 2H-chromene-3-carboxylate (15g) White solid (m.p.: 159-161.5°C). ¹H NMR (DMSO-d⁶) δ +(-0 #N& 37& C(CH₃)₃), 2.10 (s, 3H, OCOCH₃), 2.75 (t, J=7.4 Hz, 2H, PhCH₂), 3.17 (dt, J=8.0 Hz/6.1 Hz, 2H, CH₂N), 5.17 (s, 2H, CH₂O), 6.92 (t, J=5.6 Hz, 1H, NH), 7.11 (m, 2H, 2’-H/4’- H), 7.15 (d, J=7.8 Hz, 1H, 6’-H), 7.39 (t, J=8.2 Hz, 1H, 5’-H), 7.50 (d, J=8.6 Hz, 1H, 8- H), 7.78 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 7.98 (d, J=2.2 Hz, 1H, 5-H), 9.05 (s, 1H, 4- H).¹³C NMR (DMSO-d₆) δ ,*(1& ,2(,& -/(+& .+(,& 0.(-& 11(0& ++0(/& ++0(3& ++1(1& ++3(.& 121.8, 126.5, 129.4, 129.6, 133.2, 134.6, 141.4, 150.1, 150.3, 154.4, 155.5, 155.8, 161.0, 170.2. 4-(((Tert-butoxycarbonyl)amino)methyl)phenyl 6-(acetoxymethyl)-2-oxo- 2H-chromene-3-carboxylate (15h) White solid (m.p.: 164-167°C).¹H NMR (DMSO-d₆) δ +(.* #N& 37& 6#6H₃)₃), 2.10 (s, 3H, OCOCH₃), 4.16 (d, J=6.2 Hz, 2H, CH₂N), 5.17 (s, 2H, CH₂Cl), 7.21 (d, J=8.5 Hz, 2H, 3’-H/5’-H), 7.33 (d, J=8.5 Hz, 2H, 2’-H/6’-H), 7.44 (t, J=6.3 Hz, 1H, NH), 7.50 (d, J=8.6 Hz, 1H, 8-H), 7.78 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 7.98 (d, J=2.1 Hz, 1H, 5-H), 9.06 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ ,*(1& ,2(-& .,(2& 0.(-& 11(3& ++0(/& 116.8, 117.7, 121.6, 128.1, 129.6, 133.1, 134.6, 138.2, 149.0, 150.1, 154.4, 155.8, 161.1, 170.2. 3-(((Tert-butoxycarbonyl)amino)methyl)phenyl 6-(acetoxymethyl)-2-oxo- 2H-chromene-3-carboxylate (15i) White solid (m.p.: 122.5-126°C). ¹H NMR (DMSO-d₆) δ +(.* #N& 37& C(CH₃)₃), 2.10 (s, 3H, OCOCH₃), 4.18 (d, J=6.2 Hz, 2H, CH₂N), 5.17 (s, 2H, CH₂Cl), 7.14 (m, 2H, 2’-H/4’-H), 7.19 (d, J=7.6 Hz, 1H, 6’-H), 7.43 (t, J=7.8 Hz, 1H, 5’-H), 7.46 (t, J=6.1 Hz, 1H, NH), 7.50 (d, J=8.6 Hz, 1H, 8-H), 7.78 (dd, J=8.6 Hz/2.2 Hz, 1H, 7- H), 7.98 (d, J=2.1 Hz, 1H, 5-H), 9.06 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ ,*(1& ,2(,& 43.0, 64.3, 77.9, 116.5, 116.9, 117.7, 120.0, 124.7, 129.5, 129.6, 133.1, 134.6, 142.3, 150.1, 150.3, 154.4, 155.8, 155.8, 161.1, 170.2. 4-(2-(2,3-Bis(tert-butoxycarbonyl)guanidino)ethyl)phenyl 6- (acetoxymethyl)-2-oxo-2H-chromene-3-carboxylate (15j) White solid (m.p.: 222.4 °C (dec)). ¹H NMR (500 MHz, DMSO-d₆) δ ++(/* (s, 1H, NH), 9.06 (s, 1H, 4-H), 8.37 (t, J = 5.7 Hz, 1H, CH₂NH), 7.98 (d, J = 2.1 Hz, 1H, 5-H), 7.78 (dd, J = 8.6, 2.1 Hz, 1H, 7-H), 7.50 (d, J = 8.6 Hz, 1H, 8-H), 7.34 (d, J = 8.6 Hz, 2H, 3’-H/5’-H), 7.21 (d, J = 8.5 Hz, 2H, 2’-H/6’-H), 5.17 (s, 2H, CH₂OCO), 3.56 (q, J = 7.8, 7.1 Hz, 2H, CH₂NH), 2.87 (t, J = 7.3 Hz, 2H, CH₂), 2.10 (s, 3H, CH₃), 1.47 (s, 9H, C(CH³)³), 1.41 (s, 9H, C(CH³)³). ¹³C NMR (DMSO-d⁶) δ +0-(*& +0+(*& +//(1& 155.1, 154.3, 151.9, 150.0, 148.6, 136.8, 134.5, 133.1, 129.7, 129.5, 121.6, 117.6, 116.8, 116.4, 82.9, 78.1, 64.2, 41.5, 33.7, 27.9, 27.5, 20.6. Synthesis of the target compounds 16 Intermediate 15 of scheme 9 (1.12 mmol) was dissolved in acetonitrile saturated with HCl gas (10 mL) (HX = HCl). The reaction mixture was stirred for 16-96 h at room temperature. The formed precipitate of compound 16 of scheme 9 as the hydrochloride was collected by filtration, washed with acetonitrile and dried. Alternatively, trifluoroacetic acid can be used instead of HCl (HX = CF3COOH) to obtain the target compounds 16 of scheme 9 as the trifluoroacetate salts. 3-(2-Aminoethyl)phenyl 6-(chloromethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride (16a) White solid (m.p.: 220 °C (dec)). ¹H NMR (DMSO-d₆) δ ,(3. #H& ,7& PhCH₂), 3.08 (m, 2H, CH₂N), 4.89 (s, 2H, CH₂Cl), 7.19 (d, J=7.8 Hz, 1H, 4’-H), 7.21 (t, J=1.7 Hz, 1H, 2’-H), 7.24 (d, J=7.9 Hz, 1H, 6’-H), 7.46 (t, J=7.8 Hz, 1H, 5’-H), 7.53 (d, J=8.6 Hz, 1H, 8-H), 7.86 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 7.92 (bs, 3H, NH₃ ⁺), 8.06 (d, J=2.2 Hz, 1H, 5-H), 9.05 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ -,(0& ..(3& ++0(1& 116.9, 117.7, 120.1, 121.8, 126.5, 129.7, 130.4, 134.5, 135.5, 139.1, 149.9, 150.3, 154.4, 155.7, 160.9. 4-(Aminomethyl)phenyl 6-(chloromethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride (16b) White solid (m.p.: 250 °C (dec)).¹H NMR (DMSO-d₆) δ .(*1 #N& ,7& 6H₂N), 4.89 (s, 2H, CH₂Cl), 7.34 (d, J=8.6 Hz, 2H, 2’-H/6’-H), 7.52 (d, J=8.6 Hz, 1H, 8-H), 7.58 (d, J=8.5 Hz, 2H, 3’-H/5’-H), 7.86 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 8.06 (d, J=2.2 Hz, 1H, 5-H), 8.15 (bs, 3H, NH₃ ⁺), 9.08 (s, 1H, 4-H).¹³C NMR (DMSO-d₆) δ .+(0& ..(3& 116.7, 116.7, 117.7, 122.0, 130.3, 130.5, 134.5, 135.5, 150.1, 150.2, 154.4, 155.6, 160.9. 4-(2-(Aminoethyl)phenyl 6-(chloromethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride (16c) White solid (m.p.: 253 °C (dec)). ¹H NMR (DMSO-d₆) δ ,(3, #H& ,7& PhCH₂), 3.08 (m, 2H, CH₂N), 4.88 (s, 2H, CH₂Cl), 7.25 (d, J=8.5 Hz, 2H, 2’-H/6’-H), 7.38 (d, J=8.5 Hz, 2H, 3’-H/5’-H), 7.52 (d, J=8.6 Hz, 1H, 8-H), 7.86 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 7.92 (bs, 3H, NH₃ ⁺), 8.06 (d, J=2.2 Hz, 1H, 5-H), 9.05 (s, 1H, 4-H). ¹³C NMR (DMSO-d⁶) δ -,(.& ./(*& ++0(2& ++1(*& ++1(2& +,+(3& +,3(3& +-*(/& +-.(0& +-/(-& 135.5, 149.1, 150.0, 154.5, 155.8, 161.1. 3-(Aminomethyl)phenyl 6-(chloromethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride (16d) White solid (m.p.: 217 °C (dec)).¹H NMR (DMSO-d₆) δ .(*2 #N& ,7& 6H₂N), 4.88 (s, 2H, CH₂Cl), 7.31 (d, J=8.1 Hz, 1H, 4’-H), 7.43 (t, J=2.0 Hz, 1H, 2’-H), 7.47 (d, J=8.1 Hz, 1H, 6’-H), 7.53 (m, 2H, 5’-H/8-H), 7.86 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 8.07 (d, J=2.2 Hz, 1H, 5-H), 8.50 (bs, 3H, NH₃ ⁺), 9.06 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ 41.8, 45.0, 116.8, 116.9, 117.8, 122.0, 122.2, 126.7, 130.0, 130.6, 134.6, 135.6, 135.9, 150.1, 150.3, 154.6, 155.8, 161.1. 1-(4-((6-(Chloromethyl)-2-oxo-2H-chromene-3- carbonyl)oxy)phenethyl)guanidinium chloride dihydrate (16e) White solid (m.p.: 208 °C (dec)). ¹H NMR (500 MHz, DMSO-d₆) δ 3(*/ #N& 1H, 4-H), 8.05 (d, J = 2.2 Hz, 1H, 5-H), 7.86 (dd, J = 8.6, 2.2 Hz, 1H, 7-H), 7.55 – 7.50 (m, 2H, 8-H/NH), 7.39 (d, J = 8.5 Hz, 2H, 3’-H/5’-H), 7.23 (d, J = 8.5 Hz, 2H, 2’-H/6’- H), 4.88 (s, 2H, CH₂Cl), 3.44 – 3.36 (m, 2H, CH₂NH), 2.83 (t, J = 7.4 Hz, 2H, CH₂). ¹³C NMR (DMSO-d₆) δ +0+(+& +/0(1& +//(2& +/.(/& +.3(3& +.2(3& +-0(-& +-/(/& +-.(0& 130.5, 130.0, 121.7, 117.8, 117.0, 116.8, 45.0, 41.9, 33.8. 4-(2-(Aminoethyl)phenyl 6-(acetoxymethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride hemihydrate (16f) White solid (m.p.: 229-232 °C). ¹H NMR (DMSO-d₆) δ ,(+* #N& -7& 6H₃), 2.91 (dd, J=9.3 Hz/6.6 Hz, 2H, PhCH₂), 3.08 (dd, J=9.3 Hz/6.6 Hz, 2H, CH₂N), 5.17 (s, 2H, CH₂O), 7.24 (d, J=8.5 Hz, 2H, 2’-H/6’-H), 7.38 (d, J=8.5 Hz, 2H, 3’-H/5’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.79 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 7.88 (bs, 3H, NH₃ ⁺), 7.98 (d, J=2.1 Hz, 1H, 5-H), 9.06 (s, 1H, 4-H).¹³C NMR (DMSO-d₆) δ ,*(1& -,(.& -3(2& 64.3, 116.5, 116.8, 117.7, 121.9, 129.6, 129.9, 133.2, 134.7, 135.3, 149.1, 150.2, 154.4, 155.8, 161.1, 170.2. 3-(2-(Aminoethyl)phenyl 6-(acetoxymethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride hemihydrate (16g) White solid (m.p.: 198.5-200.5 °C). ¹H NMR (DMSO-d₆) δ ,(+* #N& -7& OCOCH₃), 2.95 (dd, J=9.6 Hz/6.3 Hz, 2H, PhCH₂), 3.07 (dd, J=9.4 Hz/6.5 Hz, 2H, CH₂N), 5.17 (s, 2H, CH₂Cl), 7.18 (dd, J=7.9 Hz/1.8 Hz, 1H, 4’-H), 7.20 (t, J=2.0 Hz, 2’-H), 7.23 (dd, J=7.7 Hz/1.4 Hz, 1H, 6’-H), 7.45 (t, J=7.8 Hz, 1H, 5’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.79 (dd, J=8.6 Hz/2.1 Hz, 1H, 7-H), 7.99 (d, J=2.1 Hz, 1H, 5-H), 8.05 (bs, 3H, NH₃ ⁺), 9.06 (s, 1H, 4-H).¹³C NMR (DMSO-d₆) δ ,*(1& -,(0& -3(0& 0.(-& ++0(/& 116.8, 117.7, 120.2, 121.9, 126.6, 129.6, 129.8, 133.2, 134.7, 139.3, 150.2, 150.4, 154.4, 155.8, 161.0, 170.2. 4-(Aminomethyl)phenyl 6-(acetoxymethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride hemihydrate (16h) White solid (m.p.: 243 °C (dec)). ¹H NMR (DMSO-d₆) δ ,(+* #N& -7& OCOCH₃), 4.07 (s, 2H, CH₂N), 5.17 (s, 2H, CH₂Cl), 7.34 (d, J=8.5 Hz, 2H, 3’-H/5’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.59 (d, J=8.6 Hz, 2H, 2’-H/6’-H), 7.79 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 7.99 (d, J=2.1 Hz, 1H, 5-H), 8.32 (bs, 3H, NH₃ ⁺), 9.09 (s, 1H, 4-H). ¹³C NMR (DMSO-d₆) δ ,*(1& .+(1& 0.(-& ++0(/& ++0(0& ++1(1& +,,(+& +,3(0& +-*(/& +-,(*& 133.2, 134.8, 150.3, 150.4, 154.4, 155.8, 161.0, 170.2. 3-(Aminomethyl)phenyl 6-(acetoxymethyl)-2-oxo-2H-chromene-3- carboxylate hydrochloride hemihydrate (16i) White solid (m.p.: 228 °C). ¹H NMR (DMSO-d₆) δ ,(+* #N& -7& ;6;6H₃), 4.09 (s, 2H, CH₂N), 5.17 (s, 2H, CH₂Cl), 7.32 (ddd, J=8.2 Hz/2.4 Hz/1.0 Hz, 1H, 4’-H), 7.41 (t, J=2.0 Hz, 2’-H), 7.44 (dd, J=7.8 Hz/1.4 Hz, 1H, 6’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.55 (t, J=7.9 Hz, 1H, 5’-H), 7.80 (dd, J=8.6 Hz/2.1 Hz, 1H, 7-H), 7.99 (d, J=2.1 Hz, 1H, 5-H), 8.29 (bs, 3H, NH₃ ⁺), 9.07 (s, 1H, 4-H).¹³C NMR (DMSO-d₆) δ ,*(1& .+(2& 64.3, 116.5, 116.7, 117.7, 122.0, 122.2, 126.7, 129.7, 130.0, 133.2, 134.8, 135.9, 150.3, 150.3, 154.4, 155.8, 161.1, 170.2. 1-(4-((6-(Acetoxymethyl)-2-oxo-2H-chromene-3- carbonyl)oxy)phenethyl)guanidinium chloride hemihydrate (16j) White solid (m.p.: 205.9-206.9 °C). ¹H NMR (500 MHz, DMSO-d₆) δ 3(*0 (s, 1H, 4-H), 7.98 (d, J = 2.0 Hz, 1H, 5-H), 7.79 (dd, J = 8.6, 2.1 Hz, 1H, 7-H), 7.57 (t, J = 5.8 Hz, 1H, CH₂NH), 7.51 (d, J = 8.6 Hz, 1H, 8-H), 7.39 (d, J = 8.3 Hz, 2H, 3’- H/5’-H), 7.23 (d, J = 8.2 Hz, 2H, 2’-H/6’-H), 5.17 (s, 2H, CH₂OCO), 3.40 (q, J = 6.4 Hz, 2H, CH₂NH), 2.83 (t, J = 7.4 Hz, 2H, CH₂), 2.10 (s, 3H, CH₃). ¹³C NMR (DMSO-d₆) δ 170.2, 161.1, 156.7, 155.8, 154.4, 150.1, 148.9, 136.3, 134.7, 133.2, 130.0, 129.6, 121.7, 117.7, 116.8, 116.5, 64.3, 41.9, 33.8, 20.7. Synthesis of target compounds 20 of scheme 10 Target compounds 20 of scheme 10 are compounds with R2 being a cyclic compound. The synthesis enables to obtain piperidinyl compounds (Y in compound 20 of scheme 10 = CH) and piperazinyl compounds (Y in compound 20 of scheme 10 = N). The position of R2 may be in ortho, meta or para. An example is illustrated as 20a in Table 1. The appropriate piperidinyl- or piperazinyl-substituted phenol 17 of scheme 10 was converted into the N-tert- butoxycarbonyl-substituted intermediate 18 of scheme 10 according to the conditions described for the synthesis of intermediates 2 of scheme 7. Subsequently, intermediate 19 of scheme 10 was obtained by following the conditions described for the synthesis of intermediates 15 of scheme 9. Deprotection of the tert- butoxycarbonyl group of 19 of scheme 10 was finally performed in acidic conditions as described for the synthesis of the target compounds 16 of scheme 9. tert-butyl 4-(4-((6-(chloromethyl)-2-oxo-2H-chromene-3- carbonyl)oxy)phenyl)piperazine-1-carboxylate 19a White solid (m.p.: 171.5-173.5°C). ¹H NMR (DMSO-d₆) δ +(.- #N& 37& C(CH₃)₃), 3.12 (t, J=5.2 Hz, 4H, 2’’-H₂/6’’-H₂), 3.48 (t, J=5.1 Hz, 4H, 3’’-H₂/5’’-H₂), 4.88 (s, 2H, CH₂Cl), 7.04 (d, J=9.0 Hz, 2H, 3’-H/5’-H), 7.14 (d, J=9.0 Hz, 2H, 2’-H/6’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.85 (dd, J=8.6 Hz/2.2 Hz, 1H, 7-H), 8.05 (d, J=2.1 Hz, 1H, 5-H), 9.03 (s, 1H, 4-H).¹³C NMR (DMSO-d₆) δ ,2(/& ./(/& .3(+& 13(/& ++1(,& ++1(-& 117.7, 118.3, 122.5, 131.0, 135.0, 135.9, 143.5, 149.5, 150.1, 154.3, 154.9, 156.3, 161.9. 4-(piperazin-1-yl)phenyl 6-(chloromethyl)-2-oxo-2H-chromene-3- carboxylate dichlorhydrate dihydrate 20a White solid (m.p.: 60-62 °C).¹H NMR (DMSO-d₆) δ -(,- #H& .7& ,TT'H₂/6’’- H₂), 3.38 (m, 4H, 3’’-H₂/5’’-H₂), 4.88 (s, 2H, CH₂Cl), 7.08 (m, 2H, 3’-H/5’-H), 7.17 (m, 2H, 2’-H/6’-H), 7.51 (d, J=8.6 Hz, 1H, 8-H), 7.85 (dd, J=8.7 Hz/2.2 Hz, 1H, 7-H), 8.05 (d, J=2.2 Hz, 1H, 5-H), 9.04 (s, 1H, 4-H), 9.15 (bs, 2H, NH₂ ⁺). ¹³C NMR (DMSO-d₆) δ 42.5, 44.9, 45.6, 116.7, 116.8, 117.0, 117.7, 122.1, 130.4, 134.4, 135.3, 143.4, 148.0, 149.6, 154.4, 155.7, 161.2.

Table 1: Compounds obtained in example 2b Example 3: In vitro enzymatic assays and inhibition mechanistic studies The compounds were evaluated in vitro for their inhibitory potential on the human recombinant TMPRSS2 and HAT (TMPRSS11D. The enzyme activities are measured at 25°C in the presence of appropriate commercially available fluorogenic peptide substrates, Boc-QAR-AMC for TMPRSS2 and, Boc-VPR-AMC for HAT using a fluorescence microplate reader (BMG Fluostar Optima). The experimental conditions are summarized in Table 2.

Table 2. Experimental conditions used for inhibition studies of TMPRSS2 and HAT. Compounds (5-100 µM) were tested in triplicate for each inhibitor to detect its inhibitory potential. The tested compounds were previously incubated with TMPRSS2 or HAT in the appropriate buffer for 15 min at 25°C. The enzymatic reactions were triggered by the addition of the fluorogenic substrate and the kinetics were monitored for 30 min. Initial rates (V0) determined in control experiments (no inhibitor) were considered to be 100% of the proteinase activity; initial rates Vi that were below 100% in the presence of a tested compound were considered to be inhibitions. The same percentage of co-solvent was used in both cases. The inhibitory activity of compounds was expressed as IC50 (inhibitor concentrations giving 50% inhibition). The values of IC50 were calculated by fitting the experimental data to BLP?OFJI4 " 8IEF@FOFJI 5 +**#+ U <F)<*) 5 +**=8>*)#86/* % =8>*)( 6JHKJPIAN AFNKG?SFID ?I 86/* Q?GPB @BGJR +* W9 RBMB MBO?FIBA CJM structure-activity analysis (Table 3).

Table 3. Inhibition of TMPRSS2 and HAT by compounds 16a-d, 16j and 20a at 25°C. Several compounds displayed a noticeable inhibitory activity against TPMRSS2 such as 16a, 16b, 16c, 16d, 16j and 20a. Compounds 16a, 16b, 16c and 16d also inhibited HAT. Compound 16b was more active against HAT than TMPRSS2 (factor 5.6). A similar inhibitory activity on the two enzymes was observed for compound 16c. The alkyl chain length or its position (4’or 5’) was not discriminative (16a versus 16d). The inhibition of TMPRSS2 and HAT was favored by the presence of a positive charge (factor of 74 for TMPRSS2 and of 10 for HAT) by comparing compounds 16c and 15c. The charged compounds 16f, 16e and 16i displayed about 100% inhibition à 10 µM inhibitor concentration. The nature of the charged group R² (-(CH₂)_n-NH₂ or –(CH₂)2-C(=NH)NH₂) for example) had a poor influence on the inhibitory activity whereas the cyclic charged group of 20a was unfavorable. The selectivity spectrum was studied using a panel of representative proteases including several trypsin-like kallikrein-related peptidases (KLK5, KLK6, KLK8, KLK13), plasma-blood serine proteases (plasmin, thrombin, tPA) transmembrane serine proteases (matriptase) (Table 4).

Table 4. Effect of selected compounds on a panel of serine proteases showing the percentage of inhibition at 10 µM (at 30 min and 37°C). NI: < 20% inhibition. Each inhibitor was tested in triplicate. From results summarized in Table 4, it can be observed that compound 16c has a good selectivity for TMPRSS2 and HAT in comparison with all other tested proteases. Compound 16a inhibited in a quite similar way TMPRSS2, HAT and plasmine. Compound 16b inhibited in a quite similar way TMPRSS2, HAT and tPA whereas compound 16d was less selective.

Claims

CLAIMS 1. Compound of Formula I:

wherein R¹ is a substituted linear or branched alkyl chain of 1 to 6 carbon atoms; R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being optionally preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms and R² being in the ortho, meta or para position of the ester group on the aryl ring; R³ is a halogen with x being an integer between 0 and 4; R⁴ is a halogen with y being an integer between 0 and 3; or a prodrug thereof, and/or a salt thereof, for its use in the treatment of respiratory diseases.

2. Compound for use according to claim 1 wherein the basic group bears an amine function, an amidine function or a guanidine function.

3. Compound for use according to claim 1 or 2 wherein the precursor of the basic group bears a (pseudo)carbamate function.

4. Compound for use according to claim 2 wherein R² is chosen from the group consisting of -(CH₂)_n-NH₂, -(CH₂)_n-C(=NH)NH₂, -(CH₂)_n-NH-C(=NH)NH₂, -(CH₂)_n- NH-COOR⁵ , -(CH₂)_n-C(=NH)NH-COOR⁵ , -(CH₂)_n-NH-C(=NH)NH-COOR⁵ with R⁵ being a linear or branched alkyl chain with 1 to 6 carbon atoms and with n being an integer between 0 and 6.

5. Compound for use according to claim 4 wherein n is 1 or 2.

6. Compound for use according to any of previous claims wherein x is 1 or 2.

7. Compound for use according to any of previous claims wherein y is 0, 1 or 2.

8. Compound for use according to any of previous claims wherein R¹ is -CH₂Cl or –CH₂Br.

9. Compound for use according to any of previous claims wherein said respiratory disease is a viral infection.

10. Compound of Formula I or a prodrug thereof, and/or a salt thereof for its use in the prevention of respiratory diseases.

11. Compound of Formula I or a prodrug thereof, and/or a salt thereof wherein R² is a basic group bearing a positive charge at physiological pH or a precursor thereof, such basic group or precursor thereof being preceded by a linear or branched alkyl chain of 1 to 6 carbon atoms, preferably of 1 to 2 carbon atoms.

12. Compound according to claim 11 wherein R² is -(CH₂)_n-NH-COOR⁵, -(CH₂)_n- C(=NH)NH-COOR⁵ , -(CH₂)_n-NH-C(=NH)NH-COOR⁵ with R⁵ being a linear or branched alkyl chain with 1 to 6 carbon atoms and with n being an integer between 1 and 6.

13. Compound according to any of claims 11 to 12 for use as a medicament.

14. A pharmaceutical composition comprising a compound as defined in any of claim 11 or 12 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition according to claim 14 in a form suitable for an aerosol administration.