Coumarin-1,2,3-triazole-isatin hybrid molecules and their application

Abstract

Compounds of general structural formula I: Where: R is -H, -CH3, -F, -CI, -Br, -I and -NO2; n is the number of -CH2-(methylene) groups; or a pharmaceutically acceptable salt thereof, for use as a drug in a therapeutically effective concentration for the treatment of disorders involving acetylcholine deficiency in the central nervous system.

Description

[0001] COUMARIN-1,2.3-TRIAZOLE-ISATIN HYBRID MOLECULES AND THEIR APPLICATION

[0002] Field of the Invention

[0003] The present invention generally relates to the fields of biology, chemistry, and medicine. More specifically, it concerns compounds intended for the prevention and / or treatment of diseases and conditions caused by illnesses in humans and other mammals

[0004] According to the International Patent Classification (IPC), the invention is designated under the classification:

[0005] A61P 25 / 28

[0006] A61P 35 / 00

[0007] A61K 31 / 4192

[0008] C07P 249 / 04

[0009] C07D 405 / 14

[0010] Presentation of the Technical Problem for Which Patent Protection is Claimed

[0011] The technical problem for which patent protection is claimed relates to the discovery of novel compounds that act as inhibitors of enzymes responsible for the degradation of acetylcholine, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A further objective of the invention is to identify and synthesize new compounds that function as hybrid agents, or multi-target-directed ligands (MTDLs), capable of simultaneously modulating multiple pharmacological targets. These compounds are designed to exhibit polypharmacological effects, enabling a single molecule to act on different targets and pathophysiological pathways involved in a specific disease.

[0012] The compounds are additionally intended to possess a comparable or improved therapeutic profile relative to compounds known from the prior art — for example, in terms of their pharmacokinetic and pharmacodynamic properties, and / or their metabolic stability, and / or their dose-activity relationship.

[0013] The compounds described in this invention may be used in the treatment of disorders and diseases mediated by acetylcholine deficiency, including enhancement of cognitive functions in Alzheimer's disease and other disorders associated with cholinergic imbalance in the central nervous system.

[0014] The invention relates to compounds with tire following general structural formula:

[0015] Where:

[0016] R is -H, -CH3, -F, -Cl. -Br, -I and -NO2; n is the number of -CH2- (methylene) groups.

[0017] This invention further relates to compounds defined as:

[0018] -N2 group

[0019]

[0020] -N3 group

[0021]

[0022] N4-group

[0023] This invention also encompasses useful forms of the compounds described herein, such as hydrates, solvates, salts— particularly pharmaceutically acceptable salts— and / or co- precipitates.

[0024] Furthermore, the compounds of this invention may exist in the form of tautomers. This invention includes all possible tautomers of the compounds, whether as individual tautomers or as any mixture of said tautomers. in any ratio.

[0025] State of the Art

[0026] The concept of rational drug design through the incorporation of two or more distinct pharmacophores into a single ligand, forming so-called hybrid molecules, represents a modem trend in medicinal chemistry. Hybrid molecules exhibit improved pharmacological properties and demonstrate synergistic effects, along with enhanced activity and selectivity toward the target site compared to single-pharmacophore ligands. Additionally, rational design may lead to the development of hybrid agents with polypharmacological activity, enabling a single molecule to act on multiple targets and pathophysiological pathways of a specific disease (Bolognesi, 2013; Abdolmaleki and Ghasemi, 2017; Singh et al., 2022; Sampath Kumar et al, 2020).

[0027] The etiopathogenesis of Alzheimer's disease (AD) is closely associated with a deficiency in the cholinergic system, making the use of cholinesterase inhibitors an effective strategy in the development of new drugs for the treatment of neurodegenerative disorders. Donepezil is one of the most successful drug molecules currently used in the treatment of Alzheimer’s disease. It belongs to the class of acetylcholinesterase inhibitors (AChEls) and also acts as a polypharmacological agent. Structurally, donepezil is (RS)-2-[(1-benzyl-4- piperidyl)methyl]-5,6-dimethoxy-2,3-dihydro-1H-inden-1 -one. Its structure features an N- benzylpiperidine ring linked to a dimethoxyindanone moiety via a methylene bridge. Donepezil is a selective, potent, and reversible inhibitor of AChE, targeting both of the enzyme's main binding sites——the catalytic anionic site (CAS) and the peripheral anionic site (PAS). Molecular docking studies have shown that the indanone ring of donepezil is crucial for binding to the PAS of AChE, while the benzylpiperidine moiety exhibits activity toward the CAS. Additionally, donepezil possesses favorable pharmacokinetic and toxicological profiles. These features position donepezil as an ideal candidate for further modification and optimization to improve its activity, potency, and selectivity (Kareem et al., 2021).

[0028] Donepezil also inhibits butyrylcholinesterase (BChE) activity, although it has significantly lower affinity for BChE compared to AChE (Cacabelos R., 2007). Both BChE and AChE are involved in the metabolism of acetylcholine (ACh) and are thus important for cholinergic function in the brain. Most cholinesterase activity in the brain is attributed to AChE, while BChE has traditionally been considered to play a minor role. However, several studies confirm that during the progression of AD, as AChE activity declines, BChE activity progressively increases— suggesting that BChE may compensate for the loss of AChE function overtime (Ballard C.G., 2002).

[0029] Although AChE and BChE both belong to the same family of serine hydrolases and catalyze the hydrolysis of choline esters, their active sites differ structurally, resulting in distinct selectivity toward substrates and inhibitors. AChE features a narrow and deep catalytic gorge (~20 A) leading to the active site. This structural constraint is due to the presence of aromatic amino acids, which facilitate selective binding of small, planar molecules via π-π interactions and hydrophobic effects. In contrast, BChE has a broader and more accessible active-site gorge, a consequence of the substitution of Phe330 (in AChE) with Aia328 (in BChE). This substitution significantly reduces steric hindrance at the active site, enabling BChE to hydrolyze larger substrates and accommodate bulkier inhibitors compared to AChE (Masson and Lockridge. 2010).

[0030] Coumarin, isatin, and some of their derivatives are secondary plant metabolites that have been identified and isolated from various families of plants, fungi, and bacteria, It is well known that coumarin derivatives exert their effect on acetylcholinesterase (AChE), a protease responsible for hydrolyzing acetylcholine (ACh), which is predominantly present at neuromuscular junctions and Cholinergic synapses in the brain.

[0031] Research has highlighted the ability of isatin and its derivatives to inhibit AChE enzymatic activity through interactions with its catalytic active site (CAS) (Davis and Eckroat, 2021). Compounds containing coumarin or isatin rings exhibit a broad spectrum of pharmacological activities, including antibacterial, anticoagulant, anti-HIV, antioxidant, antihypertensive, anticonvulsant, antifungal, antitubercular, antihyperglycemic, and anticancer properties (Bhagat et al., 2021).

[0032] Extensive data in the literature support the significant AChE inhibitory activity of both coumarin and isatin derivatives (Zha et al., 2016; Leng et al., 2016; Shi et al, 2020; Wang et al., 2018; Raza et al.. 2012; Lan et al., 2017).

[0033] In the literature, various N-alkyl isatins, isatin dimers, 3-indolyl-3-hydroxy-2roxindoles, and isatin-thiosemicarbazones have been reported to exhibit inhibitory activity against BuChE (Zafar et al, 2023; Wager et al, 2019; Singh et al., 2023). Among coumarin derivatives with similar activity are hybrid molecules such as coumarin-Schiff bases, tacrine- coumarins, and halogenated coumarin-chalcones (Ahmed et al.. 2023; Xie et al., 2021 ; Singh et al., 2022).

[0034] Triazole is an N-heterocyclic compound known for its diverse biologteal activities, including antiproliferative, anticonvulsant, antimicrobial, antineoplastte. antiviral, analgesic, and anti-inflammatory effects (Kaushik and Pahwa, 2018; Al-Masoudi et al., 2002; Sarigol et al., 2015). Due to the unique structure and electronic characteristics of the triazole ring, its incorporation into potential drug candidates can be particularly advantageous for achieving the desired potency or selectivity. Additionally, triazoles can be readily synthesized via click chemistry reactions and demonstrate stability toward oxidation, reduction, and hydrolysis. As a result, they are often used to link two distinct structural motifs into a single drug molecule (Halddn et al.. 2015). Literature data further indicate that the triazole ring acts as an active pharmacophore with high potency and selectivity toward AChE (Xu et al., 2019).

[0035] The patent EP 3400938A1 refers to compounds that are derivatives of N-(1,2,3- triazolylmethyl)isatin and N-(1,2,34riazolylmethyl)-3-hydroxy-3-aryl-oxindole isatin, with reported cytotoxic and antitumor activities. Although this patent discloses isatin derivatives N-linked via a methylene bridge to a 1,2.3-triazofe ring, position 4 of the triazole does not include 4-hydroxycoumarin as a substituent, as described in the present invention.

[0036] A search of the PubChem electronic database did not reveal any compounds corresponding to the general structure (I) as disclosed in this invention. Singh et al. (2017) reported isatin-coumarin hybrids with activity against human cancer cell lines such as THP- 1, COLO-205, and HCT-116. Gulati et al. (2020) published indolindione-coumarln hybrid molecules with inhibitory activity against the enzyme xanthine oxidase.

[0037] However, the state of the art does not describe compounds of general formula (I), as defined and disclosed in the present invention, The development of new hybrid compounds remains of great interest, as their biological activity profiles vary and include a broad spectrum of potential diseases and disorders that could be treated or prevented through their use.

[0038] Essence of the Invention

[0039] The invention relates to the synthesis of novel chemical entities previously unknown to the public. These compounds are rationally designed to incorporate three distinct heterocyclic structural unite: coumarin, 1,2.3-triazole, and isatin. The structure of donepezil served as a model compound for their design— where the dimethoxyindanone moiety was replaced with 4-hydroxycoumarin, the piperidine ring with a triazole, and the benzyl group with isatin or its derivatives substituted at position 5 with fluorine, chlorine, bromine, iodine, methyl, or nitro groups.

[0040] In this manner, three series of new compounds were developed, differing by the length of the hydrocarbon linker between the coumarin and triazole units (2C, 3C, or 4C atoms). Based on literature and docking studies, the replacement of dimethoxyindanone with coumarin is expected to enhance the interaction of the molecule with the peripheral anionic site (PAS) of AChE. The carbonyl oxygen of the isatin may further strengthen interactions with the catalytic active site (CAS) via hydrogen bonding, white its aromatic ring could improve π-π interactions with the aromatic amino acids within the enzyme's active site.

[0041] Triazole is used as a linker between the coumarin and isatin moieties due to its known potency and selectivity toward AChE. as well as its ease of formation via click chemistry, which is characterized by excellent speed, regioselectivity, high yields, and operational simplicity.

[0042] The newly synthesized compounds are expected to exhibit significant inhibitory activity against both AChE and BuChE.

[0043]

[0044] Figure 1. Design of Coumarin- 1, 2, 3-Triazole-lsatin Hybrid Molecules

[0045] The compounds described in this invention, as outlined above, are usefol in the treatment of cognitive dysfunction associated with Alzheimer's disease. This utility is demonstrated by their ability to inhibit the enzyme acetylcholinesterase (AChE), thereby correcting the cholinergic deficit through the elevation of acetylcholine levels in the brain.

[0046] Detailed Description of at Least One Mode for Carrying Out the Invention

[0047] The compounds of the invention having the general formula (I) may be prepared according to Scheme 1 (Tri et al., 2021 ; Saftid et al., 2015; BrSse et al., 2010; Hamlin et al.,

[0048] 2018). The scheme and procedures described below illustrate the synthetic pathways leading to the compounds of general formula (I) of the invention and are not intended to be limiting.

[0049]

[0050] Experimental Section - General

[0051] All reagents for which synthesis is not described in the experimental section are either commercially available, known compounds, or can be prepared from known compounds by known methods by a person skilled in the art

[0052] Compounds and intermediates obtained according to the methods of the invention may require purification. The purification of organic compounds is well known to those skilled in the art, and multiple methods may exist for purifying a given compound. In some cases, purification may not be necessary. In other cases, the compounds may be purified by crystallization or chromatography, most commonly by column chromatography.

[0053] Reaction monitoring and purity checks can be performed using thin-layer chromatography (TLC). Silica gel 60 F254 plates (Merck. Germany) may be used as the stationary phase, and mixtures of solvents in various ratios (V / V) may be used as the mobile phase: ethyl acetate and n-hexane (10:90, 20:8G, 30:70, 50:50, 70:30, 90:10) and / or ethyl acetate and toluene (20:80, 30:70, 50:50, 90:10). Visualization of the spots can be performed under UV light (254 nm). For column chromatography, silica gel with a particle size of 0.063- 0.200 mm (Merck) can be used as the stationary phase, and eluents may include various solvent ratios (V / V) of ethyl acetate and n-hexane (10:90, 30:70).

[0054] 1H and13C NMR spectra were recorded on a BRUKER AVANCE III 400 MHz NMR spectrometer (Broker, Biospin GmbH, Rheinstetten, Germany). DMSO-d6and CDCl3were used as solvents, and chemical shifts are reported in δ ppm relative to tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are given in Hz. The signals are designated as follows: s = singlet; d = doublet; t = triplet; m = multiplet. Mass spectra were recorded on a UHPLC-MS / MS system— -a liquid chromatograph coupled with a triple quadrupole mass detector (ThermoSdentific, USA, Accela 1000 TSQ Quantum Access Max). Electrospray ionization (ESI) in either positive or negative mode was used as the ionization technique.

[0055] Chemical names were generated using MarvinSketch software. In some cases, commonly accepted names of commercially available reagents were used instead of tiie names generated by MarvinSketch.

[0056] Procedure 1. General Procedure for the Synthesis of 4-(ω-Bromoalkoxy)-2H- chromen-2-ones

[0057] 1 equiv of 4-hydroxycoumarin was dissolved in anhydrous dimethylformamide (DMF), and 1.5 equiv of potassium carbonate (K2CO3) were added to the solution. The resulting suspension was stirred for 10 minutes at room temperature (RT). Then, 1.2 equiv of the appropriate dibromoalkane are added, and the reaction mixture was left stirring on a magnetic stirrer until the reaction is complete.

[0058] Upon completion, the reaction mixture was poured into a container with crushed ice. The resulting precipitate was filtered and dried at room temperature to constant weight. The crude product was purified by column chromatography using ethyl acetate / n-hexane as the eluent.

[0059] Procedure 2. General Procedure for the Synthesis of 4-(ω-Azidoalkoxy)-2H- chromen-2-ones

[0060] 1 equiv of 4-(ω-bromoalkoxy)-2H-chromen-2-one was dissolved in anhydrous DMF, and 1.1 equivalents of sodium azide (NaN3) were added to the solution. The reaction mixture was stirred until the reaction is complete.

[0061] After completion, tiie reaction mixture was poured into a container with crushed ice. The resulting precipitate was filtered and dried at room temperature to constant weight. The crude product was then purified by column chromatography using ethyl acetate / n-hexane as eluent.

[0062] The following intermediates were synthesized using Procedures 1 and 2.

[0063] Procedure 3. General Procedure for the Synthesis of 1-(Prop-2-ynyl)indolin-2, 3-one and Its Substituted Derivatives

[0064] 1 equiv of isatin or the appropriate isatin derivative was dissolved in anhydrous DMF. and 1.5 equivalents of potassium carbonate (K2CO3) were added to the solution. The resulting suspension was stirred for 10 minutes at room temperature (RT). Then, 1.5 equivalents of propargyl bromide were added, and the reaction mixture was left stirring on a magnetic stirrer until the reaction was complete.

[0065] Upon completion, the reaction mixture was poured into a container with crushed ice. The resulting precipitate was filtered and dried at room temperature to constant weight The crude product was then purified by column chromatography using ethyl acetate / n-hexane as the eluent.

[0066] The foliowing intermediates were synthesized using Procedure 3.

[0067] Procedure 4. General Procedure for the Synthesis of Coumarin-1,2,3-Triazole- Isatin Hybrid Molecules

[0068] To 1 equiv at 4-(ω-azidoalkoxy)-2H-chromen-2-one and 1 equiv of 1-(prop-2- ynyl)indolin-2.3-one or its derivative previously dissolved in anhydrous DMF, an aqueous solution (10% V / V relative to DMF) of copper(ll) sulfate (CuSO4, 10 mol%) and sodium ascorbate (20 mol%) was added. The reaction mixture was stirred at room temperature until tee reaction was complete.

[0069] After completion, the reaction mixture was poured into a container with crushed ice. The resulting precipitate was filtered and dried at room temperature to constant weight. The crude product was then purified by column chromatography using ethyl acetate / n-hexane as the eluent

[0070] Identification of the Obtained Products

[0071] 1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]ethyl}-1H-1,2,3-triazol-4-yl)methyl]-2,3-dihydro- 1H-indole-2, 3-d lone (N2-1)

[0072] Light orange powder (64% yield).1H NMR (DMSO-d6, 400 MHz) δ: 4.57 (1. 2H. CH2), 4.86 (t, 2H, CH2), 4.96 (s, 2H, CH2), 5.92 (s, 1H, CH), 7.04-7.11 (m, 2H, Ar). 7.26 (t, 1H, Ar), 7.36 (d. 1H, Ar), 7.45-7.59 (m, 2H, Ar). 7.63-7.72 (m, 2H, Ar). 8.33 (s, 1H, Ar).18C NMR (DMSO- d6, 100 MHz) δ: 35.49, 48.89, 68.18, 91.52, 111.52, 115.30, 116.86, 117.98, 123.16, 124.65, 124.90, 124,93, 13327, 138.39, 142.00. 150.58, 153.14, 158.21, 161.84, 164.63, 183.52. HRMS (ESI+) calcd. mass for C22H17N4O5[M+H]+417.1199, found:417.1191.

[0073] 5-methyl-1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]ethyl)-1H-1,2,3-triazol-4-yl)methyl]-2,3- dihydro-1 H-indole-2, 3-dione (N2-2)

[0074] Light orange powder (59% yield).1H NMR (DMSO-d6, 400 MHz) δ: 2.20 (s, 3H, CH3), 4.56 (t, 2H. CH2), 4.86 (t, 2H. CH2), 4.95 (s, 2H, CH2), 5.90 (s, 1H, CH). 6.94 (d. 1H, Ar). 7.23 ft, 2H, Ar), 7.33-7.41 (m, 2H, Ar), 7.58-7.65 (m, 2H, Ar), , 8.34 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 20.45. 35.49, 48.89, 68.20, 91.47, 111.35, 115.27, 116.81, 117.91, 123.15,

[0075] 124.62, 124,90, 125.10, 133.13, 133.23, 138.64. 142.04. 148.40, 153.12. 158.23, 161.83,

[0076] 164.62, 183.74. HRMS (ESI+) caled. mass for C23H19N4O5[M+H]+431.1355, found:431.1351.

[0077] 5-fiuoro-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]ethyl}-1 H-1 ,2,3-triazol-4-yl)methyl]-2,3- dihydro-1H-indole-2, 3-dione (N2-3) Light orange powder (61% yield).1H NMR (DMSO-d6, 400 MHz) δ: 4.57 (t, 2H, CH2), 4.86 (t, 2H, CH2). 4.96 (s, 2H. CH2), 5.91 (s, 1H. CH), 7.07-7.11 (m, 1H, Ar), 7.29-7.43 (m, 3H, Ar), 7.47 (t, 1H. Ar), 7.62-7.65 (m, 2H. Ar), 8.33 (s, 1H, Ar).18C NMR (DMSO-d6, 100 MHz) δ: 35.57, 48.92, 68.21, 91.49, 111.78, 112.02, 112.87, 112.94, 115.27, 116.83, 118.87, 118.94, 123.17, 124.14. 124.38, 124.61, 124.96. 133.25, 141.91, 146.75, 153.12, 157,71, 158.23, 161.84, 164.63, 182.91. HRMS (ESI+) calcd. mass for C22H19FN4O5[M+H]+435.1105, found:435<1111.

[0078] 5-chloro-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]ethyl}-1 H-1 ,2,3-triazol-4-yl)methyl]-2,3- dihydro-1H-indole-2, 3-dione (N2-4)

[0079] Light orange powder (71% yield).1H NMR (DMSO-d6, 400 MHz) δ: 4.56 (t, 2H, CH2). 4.87 (t, 2H. CH2), 4.96 (s, 2H, CH2), 5.91 (s. 1H, CH), 7.07-7.10 (m. 1H, Ar), 7,30-7.44 (m, 3H, Ar), 7.48 (t, 1H. Ar), 7.64-7.68 (m. 2H, Ar), 8.34 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 35.48, 48.89, 68.18, 91.52. 11.51, 115.30, 116.86, 117.98, 123.16, 123.78, 124.65, 124.90, 124.93, 133.27, 138.39, 142.00, 150.58, 163.13, 15821, 161.84, 164.63, 183.53. HRMS (ESI+) calcd. mass for C22H18CIN4O5[M+H]+451.0809, found:451.0812.

[0080] 5-bromo-1 -[(142-[(2-oxo-2H-chromen-4-yl)oxy]ethy l}-1 H-1 ,2,3-triazol-4-y l)methyl]-2,3- dihydro-1H-indoie-2,3 -dione (N2-5)

[0081] Light orange powder (51% yield).1H NMR (DMSO-d6, 400 MHz) δ: 4.63 (t, 2H. CH2). 4,92 (t, 2H. CH2), 5.03 (s, 2H, CH2), 5.98 (s. 1H, CH), 7,10 (d. 1H, Ar), 7.31 (t, 1H, Ar), 7.43 (d, 1H, Ar), 7.65-7.74 (m, 4H, Ar), 8.34 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 35.59, 48.92, 68.21, 91.49, 113.61, 115.26, 115.57, 116.85, 119.78, 123.16, 124.62, 124.97, 127.13, 133.25, 140.02, 141.82, 149.40, 153.12. 157.81. 161.84, 164.62, 182.27. HRMS (ESI+) calcd. mass for C22H16BrN4O5[M+H]+495.0304, found :495.0310.

[0082] 5-iodo-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]ethyI}-1 H-1 ,2,3-triazol-4-y l)methyl]-2,3- dihydro-1H-indole-2, 3-dione (N2-6)

[0083] Yellow powder (39% yield).1H NMR (DMSO-d6, 400 MHz) δ: 4.56 (t. 2H, CH2), 4.86 (t, 2H, CH2), 4.95 (s, 2H, CH2), 5.91 (s, 1H, CH), 6.90 (d, 1H, Ar), 7.24 (t, 1H, Ar), 7.37 (d» 1H, Ar), 7.58-7.75 (m, 2H, Ar), 7.77-7.83 (m, 2H, Ar), 8,32 (s, 1H, Ar),13C NMR (DMSO-d6, 100 MHz) δ: 35.55, 48.91, 68.20. 86.80. 91.50, 113.91, 115.27, 116.86, 120.04. 123.16, 124.64. 124.95, 132.57, 133.26, 141.83, 145.82, 149.83. 153.12, 157.53, 161.83, 164.62. 182.18. HRMS (ESI+) calcd. mass for C22H16IN4O5[M+H]+543.0165, found:543.0159.

[0084] 5-nitro-1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]ethyl)-1H-1,2,3-triazol-4-yl)methyI]-2,3- dihydro-1H-indole-2, 3-dione (N2-7)

[0085] Light yellow powder (42% yield).1H NMR (DMSO-d6, 400 MHz) δ: 4.54 (t, 2H, CH2), 4.87 (t, 2H, CH2). 5.06 (s, 2H, CH2). 5.89 (s, 1H, CH), 7.16-7.23 (m. 2H, Ar), 7.33 (t, 1H, Ar), 7.57 (t, 2H, Ar), 8.20 (s. 1H, Ar), 8.30 (t. 1H, Ar), 8.39 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 35.54, 48.91, 68.20, 91.47, 113.61, 115.26, 116.86, 119.94, 120.06, 123.11, 124.62, 132.27, 133.24, 141.81, 145.81, 149.88. 153.12, 157.51, 161.84, 164.64, 182.27. HRMS (ESI+) calcd. mass for C22H16N5O7[M+H]+462.1050, found:462.1042.

[0086] 1-[(142-[(2-oxo-2H-chromen-4-yl)oxy]propyl}-1 H-1 ,2,3-triazol-4-yl)methyl]-2,3- dihydro-1H-indole-2,3-dione (N3-1)

[0087] Yellow-orange powder (80% yield).1H NMR (CDCI3, 400 MHz) δ: 2.53-2.59 (m. 2H, CH2), 4.17 (t, 2H, CH2), 4.61 (t, 2H, CH2). 5,01 (s, 2H, CH2), 5.62 (s, 1H, CH), 7.12 (t, 1H, Ar), 7.25- 7.32 (m, 3H, Ar), 7.53-7.61 (m, 3H. Ar). 7.73 (d, 2H, Ar).13C NMR (CDCI3, 100 MHz) δ: 29.15, 35.37, 47.11, 65.64, 90.96, 111.39, 115.32, 116.94. 117.52, 122.68, 123.36, 124.00, 124.13, 125.38, 132.65, 138.66. 150.14, 153.33, 158.00. 162.47, 165.00, 183.00. HRMS (ESI+) calcd. mass for C23H19N4O5[M+H]+431 1355, found.431.1347.

[0088] 5-methyl-1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]propyl}-1H-1,2,3-triazol-4-yl)methyl]

[0089] 2,3-dihydro-1H-indole-2,3-dione (N3-2)

[0090] Organge powder (54% yield).1H NMR (DMSO-d6, 400 MHz) δ: 2.25 (s, 3H, CH3), 2.32-3.39 (m, 2H, CH2), 4.18 (t. 2H, CH2). 4.56 (t, 2H, CH2). 4.90 (s, 2H, CH2), 5.82 (s, 1H, CH), 6.98 (d, 1H, Ar). 7.31-7.40 (m, 4H, Ar), 7.62 (t, 1H, Ar), 7.69-7.72 (m. 1H, Ar), 8.21 (s, 1H. Ar).13C NMR (DMSO-d6, 100 MHz) δ: 20.48, 29.11, 35.47, 47.14, 67.15, 91.02, 111.41, 115.51, 116.84, 117.92, 123.44, 124.32, 124.51. 125.13, 133.23, 138.75, 141.90, 148.45, 153.16, 158.16, 158.22. 162.00, 165.17. 183.74. HRMS (ESI+) calcd. mass for C24H21N4O5445.1512, found :445.1503. 5-fluoro-1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]propyI}-1 H-1 ,2,3-triazol-4-yl)methyl]-

[0091] 2, 3-dihydro-1H-indole-2, 3-dione (N3-3)

[0092] Light orange powder (71% yield).1H NMR (DMSO-d6, 400 MHz) δ: 2,33-3.39 (m, 2H, CH2). 4.20 (t 2H. CH2). 4.56 (t, 2H, CH2), 4.92 (s, 2H, CH2), 5.83 (s, 1H, CH). 7,14 (t, 1H. Ar). 7.31- 7.39 (m, 2H, Ar), 7.44-7.49 (m, 2H, Ar), 7.61-7.66 (m, 1H, Ar), 7.72 (d, 1H. Ar), 8.26 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 29.15, 35.58, 47.16, 67.18, 91.05, 111.78, 112.02, 112.87, 112.94. 115.27, 116.83, 116.89, 118.87. 123.17, 124.14, 124.61, 124.96, 133.25, 141.91, 146.75, 153.12. 157.71, 158.23, 161.84, 164.63, 182.91. HRMS (ESI+) calcd. mass for C23H18FN4O5[M+H]+449.1261. found:449.1270.

[0093] 5-chloro-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]propyl)-1 H-1 ,2,3-biazol-4-yl)methyl]-

[0094] 2, 3-dihydro-lH-indole-2, 3-dione (N3-4)

[0095] Orange powder (74% yield). NMR (DMSO-d6, 400 MHz) δ: 2.32-3.39 (m. 2H, CH2), 420 (t, 2H, CH2), 4.56 (t, 2H, CH2), 4.93 (s. 2H, CH2), 5.84 (s. 1H, CH), 7.14 (d, 1H, Ar), 7.31- 7.39 (m. 2H, Ar), 7.60-76 (m. 3H. Ar), 7.71-7.73 (d. 1H, Ar) 8.20 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 29.15, 35.58, 47.15, 67.15, 91 05, 113.28, 115.52, 116.85, 119.40, 123.44, 124.35, 124.42, 124.53, 128.06, 133.23, 137.32, 141.71, 149.10, 153.17, 157.97, 162.00. 165.16, 182.41. HRMS (ESI+) calcd. mass for C23H18CIN4O5[M+H]+465.0966. found:465.0963.

[0096] 5-bromo-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]propy l}-1 H-1,2,3-triazol-4-yl)methyl]-

[0097] 2, 3-dihydro-1H-indole-2, 3-dione (N3-5)

[0098] Orange powder (75% yield).1H NMR (CDCI3, 400 MHz) δ: 2.53-2.59 (m, 2H, CH2), 4.18 (t, 2H, CH2), 4.62 (t, 2H, CH2), 4.99 (s, 2H, CH2). 5.62 (s, 1H, CH), 7.25-7.32 (m, 3H, Ar), 7.53- 7.61 (m, 3H, Ar). 7.55 (t, 1H, Ar), 7.66-7.74 (m, 4H, Ar).13C NMR (CDCI3, 100 MHz) δ: 29.15, 35.39, 47.17, 65.65, 90.97, 113.28, 115.31, 116.96, 117.05, 118.66, 122.67, 123.41, 124.01, 128.09. 132.67, 140.86. 148.66, 153,33, 157.25, 162.47, 165.00, 181.90. HRMS (ESI+) calcd. mass for C23H18CIN4O5[M+H]+509.0461, found.509.0466.

[0099] 5-iodo-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]propyl}-1 H-1 ,2,3-triazol-4-yl)methyl]-2,3- dihydro-1H-indole-2, 3-dione (N3-6) Orange-red powder (71% yield).1H NMR (DMSO-d6, 400 MHz) δ: 2.32-3.39 (m, 2H, CH2). 4.21 (t, 2H, CH2), 4.56 (t, 2H, CH2), 4.91 (s, 2H, CH2), 5.84 (s, 1H. CH), 6.96 (d, 1H, Ar), 7.31-7.39 (m. 2H, Ar), 7.65 (1, 1H, Ar), 7.71 (d, 1H, Ar), 7.80 (d, 1H, Ar), 7.90-9.93 (m, 1H. Ar), 8.21 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 29.15, 36.53, 47.16, 67.17, 86.80, 91.06, 114.03, 115.53, 116.86, 120.05, 123.44, 124.30, 124.53. 132.59, 13323, 141.72, 145.93, 149.89, 153.18, 157.53, 162.00, 165.16. 182.18. HRMS (ESI+) calcd. mass for C23H18IN4O5[M+H]+557.0322, found.557.0320.

[0100] 5-nitro-1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]propyl}-1H-1,2,3-triazol-4-yl)methyl]-2,3- d i by dro-1H- indole- 2,3-dione (N3-7)

[0101] Yellow powder (59% yield).1H NMR (DMSO-d6, 400 MHz) δ: 2.33-3.37 (m. 2H, CH2), 4.20 (t. 2H, CH2), 4.57 (t, 2H, CH2). 5.02 (s, 2H, CH2), 5.82 (s, 1H, CH), 7.31-7.37 (m, 3H, Ar), 7.63 (t, 1H, Ar), 7.71 (d, 1H. Ar), 8.23 (d, 2H, Ar), 8.46-8.49 (m, 1H. Ar).13C NMR (DMSO- d6, 100 MHz) δ: 29.14, 35.87, 47.15, 67.12, 91.03, 111.97, 115.50, 116.85, 118.43, 119.62, 123.43, 124.51, 133.24, 141,42. 143.58. 153.15. 154.76, 158.72, 161.96. 165.14, 181.31. HRMS (ESI+) calcd. mass for C23H18N5O7[M+H]+476.1206, found:476.1209.

[0102] 1-[(1-{2-((2-oxo-2H-chromen-4-yl)oxy]butyl}-1H-1,2,3-triazol-4-yl)methyl]-2,3-dihydro- 1H-in dole-2 ,3-dione (N4-1)

[0103] Yellow powder (76% yield).1H NMR (DMSO-d6, 400 MHz) δ: 1.74-1.79 (m, 2H. CH2). 1.95- 2.03 (m, 2H, CH2), 4.20 (t, 2H, CH2), 4.42 (t, 2H. CH2), 4.95 (s, 2H, CH2), 5.86 (s, 1H, CH), 7.08-7.16 (m, 2H, Ar). 7.33-7.39 (m, 2H, Ar), 7.54-7.67 (m, 3H, Ar), 7.81 (d, 1 H. Ar), 8.20 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 25.44, 26.73.35.54, 69.19.91 .02, 111.62, 115.66, 116.91, 118.05, 123.31, 123.80. 124.02, 124.65, 124.91, 133.20, 138.50, 141.89, 150.65, 153.21, 158.24, 162.08, 165.32, 183.12. HRMS (ESI+) calcd. mass for C24H21N4O5[M+H]+445,1512, found:445.1507.

[0104] 5-methyl-1-[(1-{2-[(2-oxo-2H-chromen-4-yl)oxy]butyl}-1H-1,2,3-triazol-4-yl)methyl]-2,3- dihydro-1 H-indole-2, 3-dione (N4-2)

[0105] Orange powder (82% yield).1H NMR (DMSO-d6, 400 MHz) δ: 1.74-1.79 (m. 2H, CH2), 1.95- 2.02 (m, 2H. CH2), 2.23 (s, 3H, CH3). 4-20 (t, 2H, CH2). 4.41 (t, 2H. CH2), 4.93 (s, 2H. CH2), 5.86 (s, 1H, CH). 7.02 (d, 1H. Ar), 7.33-7.42 (m, 4H, Ar), 7.63-7.67 (m. 1H, Ar), 7.76 (t. 1H, Ar), 8.19 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 20.46, 25.44, 26.72, 35.54, 49.45, 69.18, 91.01, 111.48, 115.66, 116.90, 117.99, 123.31, 123.98, 124.65. 125.11, 133.20, 138.78, 141.93, 148.51, 153.21, 158.28, 162.08, 165,32, 183.78. HRMS (ESI+) calcd. mass for C25H23N4O5[M+H]+459.1668, found:459.1661.

[0106] 5-fluoro-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]butyl}-1 H-1 ,2,3-triazol-4-yl)methyl]-2,3- dihydro-1H-indole-2, 3-dione (N4-3)

[0107] Orange powder (71% yield). NMR (DMSO-d6, 400 MHz) δ: 1 ,74-1.79 (m, 2H, CH2). 1.95- 2.02 (m. 2H, CH2), 4.20 (t, 2H, CH2). 4.42 (t, 2H. CH2), 4.96 (s, 2H. CH2), 5.86 (s. 1H, CH), 7.17 (d, 1H, Ar), 7.33-7.40 (m. 2H, Ar), 7.60-7.76 (m, 3H, Ar), 7.82 (d, 1H, Ar), 8.21 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 25.43. 26.73, 35.62, 49.47, 69.18, 91.03, 111.78, 112.02, 112.87, 112.94, 115.27, 115.32, 116.83, 118.87. 118.92, 123.31, 123.80, 124.02,

[0108] 124.65, 133.25, 141.91, 150.75, 153.12, 157.71, 158.23, 162.08, 165.32, 183.12. HRMS (ESI+) calcd. mass for C24H20FN4O5[M+H]+463.1418, found.445.1409.

[0109] 5-chloro-1 -[(1 -{2-[(2-oxo-2H-chromen-4-y l)oxy]butyl}-1 H-1,2,3-triazol-4-y l)methy l]-2,3- dihydro-1H-indole-2, 3-dione (N4-4)

[0110] Orange powder (67% yield).1H NMR (DMSO-d6, 400 MHz) δ: 1 .72-1.79 (m, 2H, CH2), 1.95- 2.02 (m, 2H, CH2), 4.20 (t, 2H, CH2), 4.42 (t 2H, CH2), 4.97 (s, 2H, CH2), 5.87 (s, 1H, CH). 7.17 (d. 1H, Ar), 7.32-7,39 (m, 2H, Ar), 7.60-7.76 (m, 3H, Ar), 7.84 (d, 1H. Ar), 8.21 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 25.44, 26.73. 35.65, 49.47, 69.18, 91.02, 113.33,

[0111] 115.66. 116.91, 119.47, 123.30, 124.04. 124.40. 124.65, 128.04, 133.20, 137.32. 141.71, 149.13, 158.02, 162.09. 165.09, 182.44. HRMS (ESI+) calcd. mass for C24H20CIN4O5[M+H]+479.1122, found:479.1115.

[0112] 5-bromo-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yi)oxy]buty1}-1 H-1 ,2,3-triazol-4-yl)methyl]-2,3- dihydro-1H-indole-2, 3-dione (N4-5)

[0113] Orange powder (81% yield). <H NMR (DMSO-d6, 400 MHz) δ: 1.74-1.79 (m, 2H, CH2), 1.95- 2.02 (m, 2H, CH2), 4.20 (t, 2H, CH2), 4.42 (t. 2H, CH2), 4.95 (s, 2H, CH2), 5.86 (s, 1H. CH), 7.11 (d, 1H, Ar). 7.32-7.40 (m, 2H, Ar). 7.63-7.77 (m, 2H. Ar), 7.79 (t, 2H, Ar), 8.21 (s, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 25.43, 26.72, 35.65, 49.46, 69.19, 91.02, 113.32,

[0114] 115.66, 116.91, 119.47, 123.31, 124.02, 124.40, 124.65, 124.64, 133.20, 13742, 141.81. 149.58. 158.07, 162.09, 165.07. 182.47. HRMS (ESI+) calcd, mass for C24H20CIN4O5[M+H]+523.0617, found:523.0622. 5-iodo-1 -[(1 -{2-[(2-oxo-2H-chromen-4-yl)oxy]butyl}-1 H-1 ,2,3-triazol-4-y l)methy 0-2,3- dihydro-1H-indole-2, 3-dione (N4-6)

[0115] Red powder (72% yield).1H NMR (DMSO-d6, 400 MHz) δ: 1.73-1.79 (m, 2H, CH2), 1.95- 2.02 (m, 2H, CH2), 4.20 (t, 2H. CH2), 4.42 (t, 2H, CH2), 4.94 (s, 2H, CH2), 5.87 (s, 1H, CH), 7.10 (d, 1H, Ar), 7.32-7.39 (m. 2H, Ar). 7.64-7.78 (m, 2H, Ar), 7.83 (t, 2H. Ar). 8.20 (s. 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 25.44, 26.73, 35.59, 49.47, 69.18, 86.76, 91.03, 114.07, 115.67, 116.91, 120.11, 123.31, 124.00, 124.65, 132.58, 133.20, 141.71, 145.92, 149.92, 153.22, 157.57, 162.08, 165.32, 182.20. HRMS (ESI+) calcd. mass for C24H20IN4O5[M+H]+571.0478, found:571.0484.

[0116] S-nitro-1 -[(1 -{2-[(2-oxo-2H-chr<Hnen-4-yl)oxy]butyl)-1 H-1,2,3-triazol-4-yl)methyI]-2,3- dihydro-1H-indole-2, 3-dione (N4-7)

[0117] Orange powder (64% yield).1H NMR (DMSO-d6, 400 MHz) δ: 1.73-1.79 (m, 2H, CH2), 1.95- 2.02 (m, 2H, CH2), 4.20 (t, 2H, CH2), 4.42 (t, 2H, CH2). 5.05 (s, 2H. CH2), 5.85 (s, 1H, CH), 7.32-7.39 (m, 3H. Ar), 7.64 (t, 1H. Ar). 7.75 (d. 1H, Ar), 8.22 (t, 2H, Ar). 8.48 (t, 1H, Ar).13C NMR (DMSO-d6, 100 MHz) δ: 25.44, 26.74, 35.94, 49.49, 69.17, 91.01 , 112.03, 115.64, 116.91, 118.52, 119.60, 123.27, 124.15, 124.62, 133.20. 133.27, 141.45, 143.56, 153.20, 154.82, 158.80, 162.07, 165.29, 181.33. HRMS (ESI+) calcd. mass for C24H20N5O7[M+H]+490.1363, found:490.1366.

[0118] Biological Activity

[0119] The compounds of this invention were evaluated for their inhibitory activity against cholinesterases (AChE and BuChE) using a modified Ellman’s method- The samples and positive control were dissolved in dimethyl sulfoxide (DMSO) and diluted with phosphate buffer (pH 8.0) so that the final concentration of DMSO did not exceed 1% (V / V).

[0120] A 0.1 M phosphate buffer (pH 8.0) was used as the aqueous medium for the enzymatic reaction. A 0.01 M solution of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was prepared by dissolving it in phosphate buffer (pH 8.0). The substrates, acetylthiocholine iodide (AChl. Sigma Aldrich, USA) and butyrylthiocholine iodide (BuChl, Sigma Aldrich, USA), were prepared at a concentration of 0.015 M. Acetylcholinesterase (AChE) from electric eel (Sigma Aldrich, USA) and butyrylcholinesterase (BuChE) from horse serum (Sigma Aldrich, USA) were used as solutions in phosphate buffer (pH 8.0) at a concentration of 100 U / L.

[0121] The results of the inhibitory activity against acetylcholinesterase and butyrylcholinesterase for selected compounds from this invention are presented as half-maxima) inhibitory concentration (IC50) values in Table 1.

[0122] Table 1. Examples of Inhibitory Activity Against Acetylcholinesterase Expressed as IC50

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Claims

AMENDED CLAIMS received by the International Bureau on 11 November 2025 (11.11.2025)1 . A compound of general formula (I),Where:R is -H, -CH3, -F, -Cl, -Br, -I and -NO2; n is the number of -CH2- (methylene) groups. or a pharmaceutically acceptable salt thereof;2. The compound according to claim 1 , wherein the 4-hydroxycoumarin ring (4-hydroxy- 2-oxo-2H-chromen) at position 4 is linked through the oxygen atom to the 1 H-1 ,2,3- triazole-isatin ring at position 1 by a chain containing two, three, or four methylene (-CH2- ) groups, or a pharmaceutically acceptable salt thereof;3. The compound according to claim 1 , wherein the 2, 3-dihydro-1 H-indole-2, 3-dione (isatin) ring at position 5 is substituted with substituent (R) selected from the group: H, CH3, F, Cl, Br, I, or NO2, or a pharmaceutically acceptable salt thereof;4. A pharmaceutical composition comprising:(A) a compound according to any of claims 1-3, and(B) a pharmaceutically acceptable excipient.

5. A compound according to any of claims 1-3 or a pharmaceutical composition according to claim 4 for use as a medicinal product in a therapeutically effective amount, in a method for treating a disorder or disease mediated by acetylcholine deficiency, including improvement of cognitive function in Alzheimer’s disease and other disorders involving cholinergic imbalance in the central nervous system.

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