The effect of synthesized 5-R-1H-benzo[d]imidazole-2-thiol derivatives on intraocular pressure in normal and pathological conditions

Alena S. Taran¹, Olga N. Zhukovskaya², Lyudmila V. Naumenko¹, Alina M. Chebanko¹, Yuliya V. Efremova¹, Irina D. Bodrug¹, Anna A. Beloshapka¹, Svetlana B. Zaichenko², Anatoly S. Morkovnik², Alexander A. Spasov¹

1 Volgograd State Medical University, 1 Pavshikh Bortstov Sq., Volgograd 400066 Russia

2 Institute of Physical and Organic Chemistry of the Southern Federal University, 194/2 Stachki Ave., Rostov-on-Don 344090 Russia

Corresponding author: Alena S. Taran (alena-beretta-taran@mail.ru)

Abstract

Introduction: Development of new drugs that reduce intraocular pressure and do not have a resorptive effect is a topical task for the treatment of glaucoma. Eighteen derivatives of 5-R-1H-benzo[d]imidazole-2-thiols were synthesized and studied for ophthalmic hypotensive activity in animals with normal intraocular pressure and in animals with dexamethasone-induced ophthalmic hypertension.

Materials and Methods: Ophthalmohypotensive activity was studied by tonometry with the TonoVet veterinary tonometer in 90 intact and experimental sexually mature mongrel rats weighing 250-350g before and after instillation of solutions of the studied compounds. Local irritant effects were determined by performing a conjunctival test using 25 guinea pigs.

Results: The most active compound among the 5-R-1H-benzo[d]imidazole-2-thiol derivatives was compound 1a, which reduced the ophthalmotonus in normotensive animals by 31.37%, exceeding the effect of the reference drugs timolol (-26.84%) and melatonin (-30.95%), and in rats with dexamethasone-induced glaucoma – by 23.74%, similar to melatonin (-23.72%), but inferior to timolol (-29.75%). It was found that substance 1a has no local irritant effect. It was revealed that at the screening concentration (4 mg/mL), compound 1a has a systemic effect, at lower concentrations, the ophthalmohypotensive effect decreases, the latent period increases, and the severity of the resorptive effect decreases.

Conclusion: The most active compound, 1a, was found to have demonstrated in vivo IOP-lowering activity when administered as a single instillation at a concentration of 0.4% (4 mg/mL) in ophthalmonormotensive animals and animals with steroid-induced glaucoma. Compound 1a was shown to have no local irritant effect.

Graphical Abstract

Keywords: glaucoma, intraocular pressure, benzimidazole, melatonin

Introduction

Glaucoma is characterized by progressive degeneration of the optic nerve with associated visual field loss and eventually blindness if not treated appropriately (Storgaard et al. 2021; Iomdina et al. 2021). Despite the expansion of the glaucoma drug lineup with new drug classes, many barriers and challenges still exist for topical therapy drugs – non-compliance, economics, and side effects such as ocular surface damage, pain, irritation, contact dermatitis, hyperpigmentation, periorbitopathy, and systemic effects such as bronchoconstriction, heart failure, bradycardia, etc. (Shalaby et al. 2020; Wu et al. 2021; Patchinsky et al. 2022).

Ophthalmotonus (intraocular pressure, IOP) is an important indicator for controlling the normal function of the visual organs. Its increase is one of the main causes of glaucoma (Pagano et al. 2023). In all forms of glaucoma (primary, secondary, congenital), elevated intraocular pressure is recognized as a major risk factor for glaucoma development and progression, and IOP lowering is currently the only drug treatment for glaucoma (Storgaard et al. 2021). There is no doubt that IOP reduction and maintenance at the “target pressure” (target IOP) is an important goal in stopping disease progression, but practice shows that IOP compensation does not always lead to glaucoma arrest and stabilization (Katz et al. 2022). The multifactorial nature of glaucoma and the multifaceted definition of “target pressure” requires attention to many different mechanisms. In addition, knowledge of exactly how IOP level affects glaucoma development is constantly improving (Pagano et al. 2023).

Melatonin is a ubiquitous molecule found in living organisms, from bacteria to plants to mammals. It has diverse properties, partly due to its powerful antioxidant nature, but also because of its specific interaction with melatonin receptors, which are present in almost all tissues. Melatonin regulates various physiological functions and contributes to homeostasis throughout the body. The human eye also contains small amounts of melatonin, which is produced by cells in the anterior (cornea, iris, ciliary body and lens) and posterior (vitreous, retina, chorioid, optic nerve) segments. In the eye, melatonin may provide antioxidant protection as well as regulate physiologic functions of ocular tissues, including IOP. Thus, it is possible that exogenous topical administration of sufficiently large amounts of melatonin and its analogues into the eye may be useful in several instances: for the treatment of ocular pathologies such as glaucoma because of the IOP-lowering and neuroprotective effects of melatonin; for the prevention of other dysfunctions such as dry eye and refractive defects (cataract and myopia), mainly because of its antioxidant properties; in diabetic retinopathy due to metabolic effects and neuroprotective effects; in yellow spot degeneration due to antioxidant and neuroprotective properties; and in uveitis, mainly due to anti-inflammatory and immunomodulatory properties (Rusciano and Russo 2024).

Recently, the synthesis of melatonin analogues with similar pharmacological properties has attracted the interest of researchers. In general, the strategy involves changing groups in different parts of the indole ring or replacing the indole ring with an analogue. Melatonin is a heterocyclic compound based on an indole pharmacophore, and the compounds under study are benzimidazole derivatives (Fig. 1), which gives reason to consider them isosteres (Shirinzadeh et al. 2020). Isosterism is widely used in the rational modification of parent compounds to increase efficacy, selectivity, improve pharmacokinetic properties, eliminate toxicity and obtain new chemical compounds (Kumari et al. 2020).

Figure 1. Chemical structures of melatonin and the compounds studied.

Currently, nitrogen-containing heterocyclic molecules are of great interest to synthetic chemists and pharmacologists. Among heterocyclic compounds, benzimidazole frameworks significantly predominate as potential drugs. They are accessible, functional, stable, and due to the isostructural pharmacophore of natural active biomolecules, benzimidazole derivatives are of great importance as chemotherapeutic agents in various clinical conditions. Over the past decades, researchers have synthesized many benzimidazole derivatives, with a significant proportion of these compounds exhibiting high biological activity against many diseases with good bioavailability, safety, and stability (Patchinsky et al. 2022).

2-Mercaptobenzimidazole derivatives exhibit a wide range of biological activities: inhibition of cell migration and suppression of CD-44 mRNA expression (Radwan et al. 2023), inhibition of activity against COX in humans (Veerasamy et al. 2021), anthelmintic (Hajnal et al. 2021), anxiolytic activity (Spasov et al. 2020), inhibitory activity against DPP-4 (Zhukovskaya et al. 2019), inhibitory activity against α-glucosidase (Mumtaz A. et al. 2018), antimicrobial and antioxidant activity (Ouasif et al. 2017), antituberculosis activity (Anand et al. 2016), and inhibitory activity against NHE-1 (Zhang et al. 2007).

Previous studies have shown that among melatonin isosteres – benzimidazole derivatives – there are highly active compounds that lower intraocular pressure (Spasov et al. 2020; Naumenko et al. 2021; Kibalova et al. 2023). It was advisable to study the effect of new mercaptobenzimidazole derivatives on the ophthalmotonus of normotensive animals and animals with steroid glaucoma.

Materials and Methods

Experimental animals

All procedures involving animals in the study were performed in accordance with generally accepted ethical standards for animal manipulations. The animals were kept in accordance with the rules of laboratory practice for preclinical research in the Russian Federation (GOST 351.000.3-96 and 51000.4-96), the Order of the Ministry of Health and Social Development of the Russian Federation of August 23, 2010 № 708n “On Approval of the Rules of Laboratory Practice”, and also in compliance with the directives 2010/63/EU of the European Parliament and the Council of the European Union of 22.09.2010 “On the Protection of Animals Used for Scientific Purposes”. The experiments were approved by the Biomedical Ethics Commission of Volgograd State Medical University (VolSMU) IRB 00005839 IORG 0004900, OHRP, Certificate No. 2021/056 dated 15.06.2021. All sections of this study comply with the ARRIVE Guidelines for reporting animal studies (Shevchenko et al. 2023). The animals were kept in the vivarium of VolSMU of the Ministry of Health of Russia at a temperature of 24°C and relative humidity of 60% under natural light cycle with free access to food and water.

Chemical synthesis

The synthesis of salts 1-((1H-benzo[d]imidazol-2-yl)thio)propan-2-one and 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(R¹-phenyl)ethan-1-one (1-a-r), containing a phenylethanone-1-group bound to sulfur, is presented in Figure 2.

Figure 2. Synthesis of 5-R-1-benzo[d]imidazole-2-thiol derivatives. Note: 1: R=H, R₁=Me (a), X=Cl; R₁= 3,5-di-tert-butyl-4-hydroxyphenyl (b); C₆H₃(O₂CH₂)-3,4 (c); C₆H₄-C₆H₅(d); 5-bromothienyl-2 (e); R=Cl, R₁= 2-thienyl (f); 2-furyl (1g); 3,5-di-tert-butyl-4-hydroxyphenyl (h); C₆H₄(OMe)-3 (i); C₆H₃(O₂CH₂)-3,4 (j), C₆H₃(OMe)-3,4 (k); R=OMe, R₁= 3,5-di-tert-butyl-4-hydroxyphenyl (l); C₆H₄(OMe)-3 (m); C₆H₄-C₆H₅(n); C₁₀H₇ (o); C₆H₃(O₂CH₂)-3,4 (p); C₆H₄(OMe)-4 (q); 2-furyl (r), X=Br. 2: R=H (a), Cl (b), MeO (c).

In this work, a synthesis between 5R-1H-benzo[d]imidazole-2-thiol derivatives and various phenacyl bromides is presented. Products 1a–r were obtained in good yields and high purity. Both analytical and spectral data (IR, ¹H-NMR, ¹³C-NMR) of all synthesized compounds 1a–r are in complete agreement with the proposed structures. It is known that the ambidentate anion of benzimidazoline-2-thione is alkylated exclusively at the sulfur atom (Bespalov et al. 2015). The IR spectra of compounds 1a-r showed some general characteristics: 3452-3394 cm^-1 (very weak N-H absorption), 2955-3030 cm^-1 (C-H) aromatic, 1583-1513 cm^-1 (C=C) aromatic, (1626-1616) cm^-1 (C=N) imidazole, 1664–1716 (C=O) cm^-1, 593-688 cm-1 (C²-S). Vibrations of the (C=S) group with n (C=S) at 1098, 1099, 1104, 1108, characteristic of the thione structure, were absent. The NMR spectra showed the following general data: S-CH₂ - H¹ 4.84-5.36 ppm; C¹³38.9-43.48 ppm. In the ¹H NMR spectra of compounds 1a-r, the signal of the NH group proton merges with the baseline, which is explained by the rather rapid migration of the proton between two nitrogen atoms in the NMR time scale. In the ¹³C NMR spectra of compounds 1a-r, containing an alkylated sulfur atom and an aromatic benzimidazole fragment, the signal of the C² atom is located around 149-150 ppm.

IR spectra (ν/cm^-1) of the compounds obtained were recorded on a Varian Excalibur 3100 FT-IR spectrophotometer (Varian, USA), using the method of attenuated total reflection in powder; NMR-spectra were recorded on Bruker Avance 600 N (USA) (600 MHz for ¹H and 150 MHz for ¹³C) spectrometer. The chemical shifts are given relative to the residual signals of protons of the deuterated solvent (2.49 for ¹H, 39.7 ¹³C in DMSO-d6). Melting points were measured on a Fisher-Johns Melting Point Apparatus (Fisher Scientific, USA). Elemental analysis was carried out using a classical method (Gelman et al. 1987). Reaction progress and purity of synthesized compounds were monitored by TLC (plates with Al₂O₃ III degree of activity, eluent CHCl₃, visualization with iodine vapors in a moist chamber).

Benzimidazole-2-thiol, 5-chloro-, 5-methoxy-benzimidazole-2-thiols, chloroacetone used in the work were purchased from the company Alfa Aesar (Great Britain).

4-, 3-methoxy-, 3,4-dimethoxy, 3,4-methylenedioxy-phenacyl bromides are obtained by bromination of the corresponding acetophenones in alcohol according to the method (Anisimova et al. 2005); 2-bromo-1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethanone-1 was obtained by the action of bromine on the corresponding acetophenone in octane or isooctane (Volod’kin et al. 1966).

Acetylthiophene and 2-acetylfuran are brominated with bromine in an ether-dioxane solution similarly to (Shevchuk et al. 1963); 5-bromo-2-acetylthiophene in dry chloroform according to the method of (Smirnov et al. 1971).

General procedure for synthesizing

To a solution of the corresponding 1H-benzo[d]imidazole-2-thiol 2a-c (5 mmol) in ethanol at room temperature, there was added the corresponding 2-bromo-1-(4-R¹-phenyl)ethan-1-one (5 mmol). The mixture was stirred until it dissolved, after which it was kept for 8-10 hours at 20-25 °C. Salts 1a-r were filtered off and thoroughly washed with acetone. The obtained compounds 1 a-r were dried in air and crystallized from the corresponding solvents.

1-((1H-benzo[d]imidazol-2-yl)thio)propan-2-one hydrochloride (1a^.HCl)

Yield 85 %, m.p. 158.5-160º C (MeOH) (dec). IR spectrum, ν/ cm^-1: 3420 (NH) , 2941, 3002 (C-H)_Ar, 1716 (C=O). 1625, 1589, 1514 (C=C, C=N), 598, 619, 659 (C²-S). Found (%): C 49.37; H 4.68; Cl 14.48; N 11.41; S 13.07. C₁₀H₁₀N₂OS^. HCl. Calculated (%): C 49.48; H 4.57; Cl 14.61; N 11.54; S 13.21. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 2.31(с, 3H, Me); 4.85 (c, 2H, S-CH₂); 7.4-7.42 (k, 2H, H_Ar, J=3.1); 7.63-7.65 (k, 2H, H_Ar, J=4.0). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 28.69, 43.00, 112.93, 124.40, 124.40, 132.76, 150.14, 200.31

2-((1H-benzo[d]imidazol-2-yl)thio)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethan-1-one hydrogen bromide (1b .HBr)

Yield 88 %, m.p. 203-204º C (MeCN) (dec). IR spectrum, ν/ cm^-1: 3626(OH), 3425 (NH), 2948, 1659 (C=O), 1624, 1586, 1512 (C=C, C=N), 593, 619, 655. Found (%): C 57.75; H 6.24; Br 16.61; N 5.75; S 6.57. C₂₃H₂₈N₂O₂S^. HBr. Calculated (%): C 57.86; H 6.12; Br 16.74; N 5.87; S 6.71. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1,42 (c, 18H, 6 CH₃); 5.36 (c, 2H, S-CH₂); 7.45 (dd, 2H, H^5,6, J=9.24); 7.68 (dd, 2H, H^4,7, J=9.24); 7.82 (c, 2H, H_Ph); 8.01 (c, 1H, OH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 29.89, 34.45, 40.98, 113.07, 124.85, 125.79, 126.25, 132.86, 138.34, 150.55, 159.56, 190.91.

2-((1H-benzo[d]imidazol-2-yl)thio)-1-(benzo[d][1,3]dioxol-5-yl)ethan-1-one hydrogen bromide (1c ^.HBr)

Yield 82 %, m.p. 219-220º C (EtOH) (dec). IR spectrum, ν/ cm^-1: 2975, 1656 (C=O), 1623, 1616, 1597, 641, 665. Found (%): C 48.75; H 3.44; Br 20.19; N 7.08; S 8.01. C₁₆H₁₂N₂O₃S ^.HBr. Calculated (%): C 48.87; H 3.33; Br 20.32; N 7.20; S 8.15. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 5.34 (c, 2H, SCH₂); 6.17 (c, 2H, CH₂); 7.14 (d, 1H, H_Ph, J=8.2); 7.44-7.45 (m, 2H, H^5,6);7.52 (d, 1H, H_Ph, J=1,8); 7.66-7.68 (m, 2H, H^4,7), 7.72-7.74 (m, 1H, H_Ph). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 41.36, 102.27, 107.67, 108.25, 113.05, 124.88, 125.33, 129.15, 132.82, 147.89, 150.39, 152.24, 190.17.

2-((1H-benzo[d]imidazol-2-yl)thio)-1-([1,1'-biphenyl]-4-yl)ethan-1-one hydrogen bromide (1d ^.HBr)

Yield 95 %, m.p. 228-229º C (EtOH/DMF) (dec). IR spectrum, ν/cm^-1: 3392, 3127, 2975, 1667 (C=O), 1625, 1602, 1583, 593, 616. Found (%): C 59.19; H 4.15; Br 18.65; N 6.47; S 7.41. C₂₁H₁₆N₂OS HBr. Calculated (%): C 59.30; H 4.03; Br 18.79; N 6.59; S 7.54. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 5.44 (c, 2H, CH₂); 7.44-7.45 (m, 3H, H_Bif ); 7.52 (t, 2H, H^5,6, J=7.65), 7.68 (k, 2H, H^4,7, J=3.1); 7.78 (t, 2H, H_Bif, J= 3.6); 7.92 (d, 2H, H_Bif, J=8.46); 8.16 (d, 2H, H_Bif, J=8.46). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 41.44, 113.09, 124.8, 126.96, 125.98,128.54, 129.04, 129.22, 132.98, 133.48, 138.53, 145.39, 150.35, 191.68.

2-((1H-benzo[d]imidazol-2-yl)thio)-1-(5-bromothiophen-2-yl)ethan-1-one hydrogen bromide (1e ^.HBr)

Yield 85 %, m.p. 229-230º C (EtOH/DMF) (dec). IR spectrum, ν/ cm^-1: 3130, 3048, 2974, 2918, 1646, 1590, 1522, 1505, 618, 596. Found (%): C 35.85; H 2.43; Br 36.68; N 6.33; S 14.63. C₁₃H₉BrN₂OS₂HBr. Calculated (%): C 35.96; H 2.32; Br 36.81; N 6.45; S 14.77. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 5.16 (c, 2H, CH₂); 7.4 (dd, 2H, H⁵⁽⁶⁾, H³_thioph, J= 3.1, J= 3.12), 7.49 (d, 1H, H⁶⁽⁵⁾, J= 4.1); 7.64 dd, 2H, H^3,4, J= 3.2, J= 3.18); 8.02 (d, 1H, H⁴_thioph, J= 2.04).¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 39.9, 113.24, 123.04, 124.45, 132.58, 133.79, 135.55, 142.79, 149.65, 184.56.

2-((5-chloro-1H-benzo[d]imidazole-2-yl)thio)-1-(thiophen-2-yl)ethan-1-one hydrogen bromide (1f ^.HBr)

Yield 87 %, m.p. 223-225º C (EtOH) (dec). IR spectrum, ν/ cm^-1: 3130, 3048, 2974, 2918, 1646, 1590, 1522, 1505, 618, 596. Found (%): C 39.97; H 2.70; Br+Cl 29.46; N 7.08; S 16.30.C₁₃H₉ClN₂OS₂HBr. Calculated (%): C 40.07; H 2.59; Br + Cl 29.60; N 7.19; S 16.44. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 5.15 (c, 2H, CH₂); 7.31-7.33 (m, 2H, H⁶, H⁴_thioph); 7.58 (d, 1H, H⁷, J=8.64); 7.64 (d, 1H, H⁴, J=1.86); 8.10-8.11 (k, 1H, H³_{thioph, J}=5.88); 8.16-8.17 (k, 1H, H⁵_thioph_, J=4.8). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 40.0, 113.26, 114.57, 123.66, 127.87, 128.90, 134.28, 134.68, 136.0, 135.50, 141.30, 151.51.

2-((5chloro-1H-benzo[d]imidazole-2-yl)thio)-1-( furan-2-yl)ethan-1-one hydrogen bromide (1g .HBr)

Yield 86 %, m.p. 218.5-220 ºC (EtOH) (dec). IR spectrum, ν/cm^-1: 3126, 3029, 2916, 1665, 1633, 1557, 1501, 650, 621. Found (%): C 41.68; H 2.82; Br + Cl 30.74; N 7.37; S 8.44. C₁₃H₉ClN₂O₂S HBr. Calculated (%): C 41.79; H 2.70; Br + Cl 30.87; N 7.50; S 8.58. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 4.98 (c, 2H, S-CH₂); 6.78 (k, 1H, H⁴_furf,J=1.58); 7.33 (dd, 1H, H⁶, J= 1.68, J=1.74); 7.57 (d, 1H, H³_furf,J=6.42); 7.63-7.64 (m, 2H, H⁷, H⁵_furf); 8.08 (c, 1H, H⁴). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 36.9, 112.81, 113.25, 114.57, 119.91, 123.68, 127.90, 134.22, 136.44, 148.55, 150.27, 151.45, 180 59.

2-((5chloro-1H-benzo[d]imidazole-2-yl)thio)-1-(3,5-di-tert-bityl-4hydrpxyphenyl)ethan-1-one hydrogen bromide (1h .HBr)

Yield 89 %, m.p. 216-217º C (dec). IR spectrum, ν/cm^-1: 3624(OH), 3421 (NH), 1665 (C=O), 2941, 3002 (C-H)_Ar, 1716 (C=O). 1625, 1589, 1514 (C=C, C=N), 598,619, 659. Found (%): C 53.86; H 5.62; Br + Cl 22.41; N 5.34; S 6.12. C₂₃H₂₇ClN₂O₂S HBr. Calculated (%): C 53.97; H 5.51; Br + Cl 22.54; N 5.47; S 6.26. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1,42 (c, 18H, 6 CH₃); 5.35 (c, 2H, CH₂); 7.45 (dd, 1H, H⁶, J=9.24); 7.68 (dd, 2H, H^4,7, J=9.24); 7.82 (c, 2H, H_Ph); 8.01 (c, 1H, OH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 31.6, 34.40, 38.2, 115.8, 116.6. 123.3, 124.1, 128.5, 129.2, 136.0, 137.0, 140.3, 147.1, 159.0, 194.1.

2-((5-chloro-1H-benzo[d]imidazole-2-yl)thio)-1-(3-methoxyphenyl)ethan-1-one hydrogen bromide (1i .HBr)

Yield 91 %, m.p. 214-216º C (EtOH/DMF) (dec). IR spectrum, ν/cm^-1: 3003, 2959, 1681 (C=O), 1665, 1633, 1597, 1585, 1249, 1038, 621, 654, 653. Found (%): C 46.54; H 3.52; Br + Cl 27.75; Br + Cl 27.75; N 6.65; S 7.42. C₁₆H₁₃ClN₂O₂S HBr. Calculated (%): C 46.65; H 3.41; Br + Cl 27.88; N 6.77; S 7.56 . ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.75 (c, 3H, OCH₃); 5.03(c, 2H, S-CH₂); 7.23 (dd, 2H, H⁶, H_ph, J= 8.7, J= 8,28); 7.43-7.46 (m, 3H, H_ph), 7,51 (c, 1H, H⁷), 7.58 (d, 1H, H⁴, J=7.38). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 40.89, 56.16, 113.64, 114.05, 115.39, 120.85, 121.73, 124.24, 128.55, 131.02, 135.76, 136.87, 137.99, 152.29, 160.14, 193.61.

1-(benzo[d][1,3]dioxol-5-yl)-2-((5-chloro-1H-benzo[d]imidazole-2-yl)thio)ethan-1-one hydrogen bromide (1j .HBr)

Yield 93 %, m.p. 231-232 ºC (EtOH) (dec). IR spectrum, ν/cm^-1: 2975, 1656 (C=O), 1623, 1616, 1597, 641, 665. Found (%): C 44.82; H 2.94; Br + Cl 26.84; N 6.43; S 7.29. C₁₆H₁₁ClN₂O₃S HBr. Calculated (%): C 44.93; H 2.83; Br + Cl 26.97; N 6.55; S 7.43 . ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 5.18 (c, 2H, S-CH₂); 6.16 (c, 2H, O-CH₂-O); 7.11(d, 1H, H_Ph, J=8.2); 7.32-7.34 (dd, 1H, H⁶);7.51 (d, 1H, H⁷, J=1,8); 7.58 (d, 1H, H_ph, J=8.6), 7.64 (d, 1H, H_Ph, J=1,9); 7.71-7.72 (m, 1H, H⁴). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 40.53, 102.2, 107.90, 108.18, 113.19, 114.22, 123.69, 125.21, 125.84, 126.42, 127.62, 147.86, 151.85, 152.09, 190.48.

2-((5-chloro-1H-benzo[d]imidazole-2-yl)thio)-1-(3,4-dimethoxyphenyl)ethane-1-one hydrogen bromide (1k .HBr)

Yield 95 %, m.p. 229-230.5 ºC (EtOH) (dec). IR spectrum, ν/cm^-1: 3106, 3004, 2886, 1664, (C=O), 1615, 1593, 1583, 1513, 1260, 1060, 621, 589. Found (%): C 45.91; H 3.74; Br + Cl 25.87; N 6.18; S 7.08. C₁₇H₁₆ClN₂O₃S HBr. Calculated (%): C 46.02; H 3.63; Br + Cl 26.00; N 6.31; S 7.22. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.75 (c, 3H, OCH₃); 3.80 (c, 3H, OCH₃); 5.03 (c, 2H, S-CH₂); 7.04 (d, 1H, H_ph, J=8.58); 7.26 (dd, 1H, H⁶, J=2.4, J=1.92); 7.41 (d, 1H, H_ph, J= 1.98); 7.48 (d, 1H, H_ph, J= 8.64); 7.54 (d, 1H, H⁷, J= 1.92); 7.67 (dd, 1H, H⁴, J=2.04, J=1.98). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 40.71, 56.34, 56.60, 111.21, 111.77, 113.97, 115.35, 124,41, 125.50, 128.27, 128.81, 135.28, 137.47, 149.27, 152,49, 154.53, 192.08.

1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-((5-methoxy-1H-benzo[d]imidazo-2-yl)thio)ethane - 1-one hydrogen bromide (1l .HBr)

Yield 94 %, m.p. 205-207 ºC (MeCN) (dec). IR spectrum, ν/cm^-1: 3624 (OH), 3331(NH), 2948, 1666 (C=O), 1632, 1624, 1586, 1521 (C=C, C=N), 621, 655(C²-S). Found (%): C 56.69; H 6.06; Br 15.62; N 5.40; S 6.18. C₂₄H₃₀N₂O₃S HBr. Calculated (%): C 56.80; H 6.16; Br 15.75; N 5.52; S 6.32. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.33 (c, 18H, 6 CH₃); 3.75 (c, 3H, OCH₃); 5.09 (c, 2H, S-CH₂); 6.98 (m, 3H, H^4,6.7),7.48 (d, 1H, OH, J= 8.94); 7.75 (c, 2H, H_Ph). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 30.53, 35.12, 41.13, 56.57, 96.85, 114.82, 114.99, 126.66, 126.83, 128.60, 135.01, 139.19, 149.55, 158.08, 160.52, 192.39.

2-((5-methoxy-1H-benzo[d]imidazole-2-yl) thio)-1-(3- methoxyphenyl)ethan-1-one hydrogen bromide (1m .HBr)

Yield 96 %, m.p. 213-214 (EtOH/DMF) (dec). IR spectrum, ν/cm^-1: 3003, 2959, 1681 (C=O), 1665, 1633, 1597, 1585, 1249, 1038, 621, 654, 653. Found (%): C 49.78; H 4.30; Br 19.39; N 6.72; S 7.69. C₁₇H₁₆N₂O₃S HBr. Calculated (%): C 49.89; H 4.19; Br 19.52; N 6.84; S 7.83. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.83, 3.84 (c, 6H, 2OCH₃), 5.36 (c, 2H, S-CH₂), 7.05-7.07 (dd, 1H, H⁶, J= 2.34, J= 2.34); 7.12 (d, 1H, H⁴, J= 2.28), 7.30 (t, 1H, H_Ph, J=4.11), 7.52 (t, 1H, H_Ph, J=7.95), 7.54 (t, 1 H, H_Ph, J=1.89), 7.56 (d, 1H, H⁷_,J=8.8), 7.66 (d, 1H, H_Ph, J=7.95). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 41.54, 55.49, 55.83, 96.01, 113.14, 113.92, 114.34, 120.04, 120.92, 127.15, 130.05, 133.68, 136.00, 148.76, 157.43, 159.43.

1-([1,1'-biphenyl]-4-yl)-2-((5-methoxy-1H-benzo[d]imidazole-2-yl)thio)ethan-1-one hydrogen bromide (1n .HBr)

Yield 94 %, m.p. 221-222 (EtOH/DMF) (dec). IR spectrum, ν/cm^-1: 3392, 3127, 2975, 1667 (C=O), 1625, 1602, 1583, 593, 616. Found (%): C 57.92; H 4.32; Br 17.42; N 6.03; S 6.90. C₂₂H₁₈N₂O₂S HBr. Calculated (%): C 58.03; H 4.21; Br 17.55; N 6.15; S 7.04. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): .82 (c, 3H, OCH₃); 5.52 (c, 2H,S- CH₂); 7.04 (dd, 1H, H⁶, J=2.4, J=2.34);7.11 (d, 1H, H⁴, J=0.84); 7.43-7.45 (k,1H, H⁷, J= 12); 7.51 (t, 2H, H_Bif, J= 7.62); 7.56 (d, 1H, H_Bif, J= 8.94); 7,76 (c,1H, H_Bif); 7.78 (d, 1H, H_Bif, J=1.4); 7,88 (c, 1H, H_Bif); 7.89 (c, 1H, H_Bif); 8,16 (c, 1H, H_Bif); 8.17 (c, 1H, H_Bif). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 41.78, 55.75, 95.92, 113.84, 114.09, 126.92, 126.97, 128.50, 129.02, 129.22, 133.52, 133.94, 138.56, 145.33, 148.70; 157.25, 191.96.

2-((5-methoxy-1H-benzo[d]imidazole-2-yl)thio)-1-(naphthalene-1-yl)ethan-1-one hydrogen bromide (1o .HBr)

Yield 90 %, m.p. 228-229 (EtOH/DMF) (dec). IR spectrum, ν/cm^-1: 3040, 3005, 2907, 2815, 1662, 1636, 1592, 1571, 660, 641. Found (%): C 55.84; H 3.88; Br 18.48; N 6.41; S 7.33. C₂₀H₁₆N₂O₂S HBr. Calculated (%): C 55.95; H 3.99; Br 18.61; N 6.53; S 7.47. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.75 (c, 3H, OCH₃); 5.19 (c, 2H,S- CH₂ ); 6.99 (d, 1H, H⁶, J=8.8); 7.05(c, 1H, H⁴); 7.47 (d, 1H, H_Ar, J=8.9); 7.54 (t, 2H, H⁷, H_Ar, J=4.29); 7.61 (t, 1H, H_Ar, J=7.71); 7.95 (t, 1H, H_Ar, J=4.4); 8.14 (d, 1H, H_Ar, J=8.3); 8.2 (d, 1H, H_Ar, J=7.1);8.4 (c, 1H, H_Ar). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): ¹³C: 43.48, 56.58, 96.86, 11.86, 114.96, 125.56, 125.74, 127.54, 128.80, 129.15, 129.43, 130.22, 130.58, 132.83, 134.18, 134..82, 135,15, 149.18, 158.06, 196.39.

1-(benzo[d][1,3]dioxol-5-yl)-2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)ethan-1-one hydrogen bromide (1p .HBr)

Yield 89 %, m.p. 217-218 (EtOH) (dec). IR spectrum, ν/cm^-1: 2975, 1656 (C=O), 1623, 1616, 1597, 641, 665. Found (%): C 48.13; H 3.68; Br 18.75; N 6.50; S 7.43. C₁₇H₁₄N₂O₄S HBr. Calculated (%): C 48.24; H 3.57; Br 18.88; N 6.62; S 7.57. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.81 (c, 3H, OCH₃); 5.19 (c, 2H, S-CH₂); 6.17 (c, 2H, O-CH₂O); 6.99 (dd, 1H, H⁶, J= 2.4, J=2.4); 7.08 (d, 1H, H_Ph, J=2.34); 7.11(d, 1H, H⁴, J= 8.16); 7.50-7.51 (dd, 2H, H⁷, H_Ph_,J=3.66, J=3.48); 7.72 (dd, 1H, H_Ph_,J=1.8, J=1.74). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 40.83, 55.73, 96.20, 102.22, 107.65, 108.20, 113.49, 114.02, 125.24, 128.63, 129.29, 134.89, 147.87, 148.75, 152.13, 156.96, 190.48.

2-((5-methoxy-1H-benzo[d]imidazole-2-yl)thio)-1-(4-methoxyphenyl)ethan-1-one hydrogen bromide (1q .HBr)

Yield 92 %, m.p. 221.5-222 (EtOH) (dec). IR spectrum, ν/cm^-1: 3003, 2959, 1681 (C=O), 1665, 1633, 1597, 1585, 1249, 1038, 621, 654, 653. Found (%): C 49.78; H 4.28; Br 19.39; N 6.71; S 7.69. C₁₇H₁₆N₂O₃S^. HBr. Calculated (%): C 49.89; H 4.19; Br 19.52; N 6.84; S 7.83. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.83 (c, 3H, OCH₃); 3.86 (c, 3H, OCH₃); 5.31 (c, 2H, S-CH₂); 7.06 (m, 1H, H⁶); 7.12 (m, 2H, H⁴, H_ph); 7.58 (d, 1H, H⁷, J=8.94); 8.04 (d, 2H, H_ph, J= 8.9). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 41.29, 55.67, 55.82, 95.99, 113.88, 114.11, 114.34, 127.04, 127.49, 130.89, 133.59, 149.01, 157.44, 163.87, 190.49.

1-( furan-2-yl)-2-((5-methoxy-1H-benzo[d]imidazole-2-yl)thio)ethan-1-one hydrogen bromide (1r..HBr)

Yield 90 %, m.p. 208-210 (EtOH) (dec). IR spectrum, ν/cm^-1: 3126, 3029, 2916, 1665, 1633, 1557, 1501, 650, 621. Found (%): C 45.43; H 3.64; Br 21.51; N 7.47; S 8.54. C₁₄H₁₂N₂O₃S ^.HBr. Calculated (%): C 45.54; H 3.55; Br 21.64; N 7.59; S 8.68. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.75 (c, 3H, OCH₃); 4.83 (c, 2H, CH₂ ); 6.72 (m, 1H, H⁴_furf); 7.00 (dd, 1H, H⁶, J=1.62, J=2,34); 7.08 (d, 1H, H⁴, J= 2.28); 7.49 (d, 1H, H³_furf, J= 8.94); 7.55 (d, 1H, H⁷, J= 3.6); 7.94 (c, 1H, H⁵_furf). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 39.65, 56.59, 96.83, 113.88, 114.93, 115.23, 121.45, 128.59, 134.98, 148.77, 149.62, 150.61, 158.17, 181.43.

Influence of compounds on intraocular pressure

The study of the effect of compounds on IOP level was performed on adult intact rodent rats and rats with dexamethasone-induced ophthalmic hypertension by tonometry using a veterinary tonometer TonoVet (Finland) (Du Sert et al. 2020). The study of compounds was performed according to the method of Marcus et al. (Pease et al. 2006), according to which one drop of the solution of the compound under study was instillated into the right (tested) eye of the laboratory animal, and one drop of the solvent - deionized water - was instilled into the left eye. IOP was measured in both eyes. The left eye served to assess the possible resorptive effect of the tested compounds.

The investigated melatonin isosters – mercaptobenzimidazole derivatives – were instilled at a concentration of 0.4% once in a volume of 50 μL. IOP was measured at four time points (0, 1, 2, 3 h), where 0 h was the baseline value. The concentration dependence of ophthalmohypotensive activity was evaluated for the most active compounds. The presence of ophthalmohypotensive activity was evaluated by the maximum IOP reduction from the initial data. 0.5% solution of timolol (Grotex LLC, Russia) (a drug used in clinical practice) and 0.4% solution of melatonin (SigmaAldrich, USA) were used as comparison drugs. To model steroidal ophthalmic hypertension in rats, bilateral local instillation of 0.1% dexamethasone solution (Elfa Research and Production Center JSC, Russia) was performed for 1 month (Marcus et al. 2018).

Local irritant action

Local irritant action of the most active compounds was studied on guinea pigs. For this purpose, a conjunctival test was performed (Marcus et al. 2019). 50 μL of 0.4% aqueous solution was instilled into the right (test) eye of guinea pigs using an automatic pipette; the left (contralateral) eye served as a control. Experimental groups included 3 animals each. Reactions were recorded after 15, 30, 60 minutes, 24 hours and evaluated in scores.

Statistical processing

Statistical processing and graphing were performed in Prism 8.0 program (GraphPad Inc.) with automatic calculation of arithmetic mean and standard deviation. Student’s t-test was used to evaluate the change in intraocular pressure relative to the initial values.

Results and Discussions

As a result of this work, it was found that the comparison drug timolol 2 hours after instillation reduces IOP by 26.84%, and melatonin maximally lowers IOP after 3 hours by 30.95%, after which ophthalmotonus gradually begins to return to the initial values (Table 1). Both drugs decreased IOP in the contralateral eye, which indicates the presence of systemic action after absorption into the bloodstream.

Table 1.

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Effect of 0.5% solution of timolol, melatonin and new derivatives of mercaptobenzimidazole on IOP of normotensive rats at a single instillation at a concentration of 0.4%

Compound

Concentration, %

Мах Δ IOP, %

Effect on the contralateral eye

Timolol

0.5

-26.84±0.94*

Melatonin

0.4

-30.33±2.91*

0.4

-31.37±1.01*

0.4

-17.29±6.75

0.4

-13.1±2.56

0.4

-11.92±2.39

0.4

-26.87±3.85*

0.4

-19.09±5.00

0.4

-17.78±3.40*

0.4

-3.7±3.7

0.4

-15.87±4.88

0.4

-12.12±9.10

0.4

-12.73±6.39

0.4

-19.7±6.48

0.4

-29.95±6.79*

0.4

-13.15±3.52

0.4

-13.13±3.33

0.4

-17.41±8.99

0.4

-23.16±2.15*

0.4

-11.11±5.56

Note: ”-“ – absence of effect; “+” – presence of effect; * – differences are statistically significant relative to baseline values (Student’s t-test, p˂0.05).

According to the results obtained, all the tested compounds can be divided into groups: highly active (ophthalmohypotensive effect > 25%) – 1a, 1e, 1m; compounds with moderate ophthalmic hypotensive activity (15-25%) – 1b, 1h, 1i, 1f, 1l, 1o, 1p and low-activity (ophthalmohypotenchive effect ˂15%) – 1c, 1d, 1j, 1g, 1k, 1r, 1n, 1q.

Thus, the most active compounds 1a, 1m were superior to timolol in their ophthalmic hypotensive activity (-31.37%, -29.95% and -26.84%, respectively), and compound 1e was not inferior to the comparison drug timolol (-26.87% and 26.84%, respectively). After instillation of compound 1a, the maximum IOP-lowering effect developed after 60 minutes and amounted to - 31.37%, which exceeds the ophthalmic hypotensive effect of melatonin (-30.33%), but after 2 hours, IOP returned to the initial values. Similar dynamics was observed in the control eye, which indicates undesirable systemic action of the substance.

In the study of compound 1e, the maximum reduction of IOP by 26.87% was observed 2 hours after instillation and insignificantly changed by 3 hours. Reduction of ophthalmotonus in the control eye may be a manifestation of resorptive effect.

The maximum IOP decrease after instillation of compound 1m was observed after 2 hours and amounted to -29.95% of the initial values and insignificantly changed by 3 hours. The dynamics of IOP decrease in the control eye is the basis for judgment about the resorptive effect of the compound under study.

The following substances showed average ophthalmohypotensive effect. Instillation of compound 1p resulted in decrease of ophthalmotonus by 23.16% only after 180 minutes. At the same time IOP decrease was also observed in the control eye.

Substance 1l 60 minutes after instillation into the test eye reduced IOP by 13.8%, and after 120 minutes – by 19.7%. By the third hour, the index of ophthalmotonus remained at the level of -19.7% relative to the initial values. The compound had no significant effect on the control eye.

After instillation of compounds 1h and 1i, the maximum IOP-lowering effect developed after 3 hours and amounted to -19.09% and -17.78%, respectively. Compound 1o reduced IOP by 17.41% after 60 minutes, after 120 minutes the pressure increased and amounted to -10.37% relative to the initial data. When studying these compounds in the contralateral eye, a decrease in ophthalmotonus was also observed, which may indicate a systemic effect.

Compounds 1b and 1f gradually decreased the ophthalmotonus of the test eye after instillation, reaching a maximum after 3 hours (-17.29% and -15.87, respectively). No IOP changes were observed in the contralateral eye.

The other compounds showed low ophthalmohypotensive activity. The results revealed that compounds 1c, 1r and 1n reduced IOP by 13.1%, 13.15% and 13.13%, respectively. Compounds 1r and 1n, unlike 1c, had a systemic effect.

Substances under laboratory codes 1d, 1g and 1k caused a decrease in ophthalmotonus by -11.92%, -12.12% and -12.73% 120 minutes after instillation, respectively, subsequently IOP returned to the initial values. The substance under the laboratory code 1q showed a weak ophthalmohypotensive effect after 60 minutes (-11.11%); by the third hour, there was an increase in pressure to the initial values. None of four substances had any significant effect on the contralateral eye.

Instillation of compound 1j hardly caused any ophthalmohypotensive effect. The maximum IOP decrease amounted to -3.7% relative to the initial values.

According to the screening results, for the most active compounds, concentration dependence, effect on ophthalmotonus in rats with dexamethasone-induced glaucoma, evaluation of their local irritant effect, and study of the ophthalmohypotensive effect of the most promising compound were carried out.

The study of the dose-dependent effect of the test substance for compounds 1a, 1e and 1m was carried out at concentrations of 0.1%, 0.2%, 0.4%.

When substance 1a was instilled at a concentration of 0.4%, the maximum IOP reduction was observed after 60 minutes, -28.53% relative to baseline values. In lower concentrations (0.2% and 0.1%), the maximum ophthalmohypotensive effect developed only by 2 hours (-15.56 % and -16.5%, respectively). The studied compound 1a at a concentration of 0.4% caused a decrease in IOP in the contralateral eye, which was not detected when 0.2% and 0.1% solutions were instilled. Thus, the decrease in concentration leads to an increase in the latency period and a decrease in the ophthalmohypotensive effect, as well as a decrease in the manifestations of resorptive action (Fig. 3).

Figure 3. Effect of compound 1a on the intraocular pressure level of normotensive rats by a single instillation of 30 μL into the test eye at concentrations of 0.1%, 0.2%, 0.4%.

In the study of compound 1e at a concentration of 0.4%, IOP was significantly reduced by 27.39% 2 hours after instillation. At instillation of 0.2% solution after 60 minutes, the pressure decreased only by 18.06% relative to the initial values. The investigated compound in 0.1% concentration had no effect on ophthalmotonus (-6.67% relative to the initial data – not significant). The investigated compound 1e at a concentration of 0.4% caused a decrease in ophthalmotonus in the contralateral eye, and, at lower concentrations, there were no manifestations of systemic effect. Thus, a decrease in the concentration leads to a decrease in the ophthalmohypotensive effect, as well as a decrease in the manifestations of resorptive action (Fig. 4).

Figure 4. Effect of compound 1e on the intraocular pressure level of normotensive rats by a single instillation of 30 μL into the test eye at concentrations of 0.1%, 0.2%, 0.4%.

Instillation of compound 1m at a concentration of 0.4% resulted in the development of maximum IOP-lowering effect after 120 minutes (-22.46% relative to baseline values). At concentrations of 0.2% and 0.1%, the reduction in ophthalmotonus was -17.68% and -15.00%, respectively. The compound 1m in any of three concentrations had no effect on the control eye (Fig. 5).

Figure 5. Effect of compound 1m on the intraocular pressure level of normotensive rats by a single instillation of 30 μL into the test eye at concentrations of 0.1%, 0.2%, 0.4%.

The next stage was to study the local irritant effect of the compounds that showed the highest ophthalmohypotensive effect in the study on normotensive rats. Thus, during the conjunctival test in guinea pigs by instillation aqueous solutions of compounds 1a, 1e and 1m at a concentration of 0.4%, there was no reddening of the lacrimal duct, sclera and conjunctiva, which may indicate the absence of irritant effect.

At the next stage, the most active compounds were studied on the model of dexamethasone-induced glaucoma (Fig. 6). Instillation of timolol in animals with ophthalmic hypertension caused a decrease in ophthalmotonus after 3 hours by 29.76% relative to baseline values. Similar dynamics was observed in the control eye, which confirms the undesirable resorptive effect of this drug.

Instillation of melatonin to animals with dexamethasone-induced glaucoma also resulted in IOP reduction by 23.72% by 3 hours, no systemic effect was detected, as no IOP change was observed in the control eye.

After instillation of compound 1a the maximum ophthalmohypotensive effect developed after 180 minutes and amounted to -23.74%, which does not exceed the effect of the comparison drug timolol, but corresponds to the activity of melatonin. In the control eye similar dynamics was observed, which may indicate undesirable systemic action of the substance.

In the study of compound 1e on animals with ophthalmohypertension, a decrease in ophthalmotonus was observed 1 hour after instillation by 7.85%, which is not significant. The substance had no effect on the control eye.

There was almost no ophthalmohypotensive effect after instillation of substance 1m.

Figure 6. Effect of 0.5% solution of timolol and compounds 1a, 1e, 1m at a concentration of 4 mg/mL on IOP of rats with dexamethasone-induced glaucoma by a single instillation of 30 μL into the test eye.

Conclusion

Thus, according to the results of our studies, 2-mercaptobenzimidazole derivatives have shown to be a promising class for the search of new compounds that reduce IOP.

At a single instillation of 30 μL of solutions of the studied compounds in the concentration of 4mg/mL, the most active compounds were compounds 1a and 1m, which were superior to timolol in their ophthalmohypotensive activity. Compound 1e is comparable to the comparison drug in its ophthalmic hypotensive activity.

The study of the dependence of IOP-lowering effect on the concentration (0.4%, 0.2% and 0.1%) of the administered compounds showed that with decreasing concentration, there was an increase in the latent period of the effect development and a decrease in the investigated activity.

The most active compounds 1a, 1e and 1m had no local irritant effect in the conjunctival test when 30 μL of 0.4% solutions were instilled once in guinea pigs.

Compound 1a also showed high activity in a steroid-induced glaucoma model (dexamethasone), reducing IOP by 23.74%, with a single instillation of 30 μL of solution at a dose of 4 mg/mL, which is similar to melatonin (-23.72%) but inferior to the comparison drug timolol (-29.76%), making it most promising for further study.

Conflict of interests

The authors have declared that no competing interests exist.

Funding

The chemical section was financially supported by the Ministry of Science and Higher Education of the Russian Federation (state task in the field of scientific activity, Southern Federal University, grant FENW-2023-0011).

Pharmacological studies were carried out within the framework of project No. 22-15-2002 with financial support from the Russian Science Foundation and the Volgograd Region Administration.

Acknowledgments

This work was carried out using the equipment of the Common Use Center of the Southern Federal University, Rostov-on-Don, Russia.

Data availability

All of the data that support the findings of this study are available in the main text.

Additional information

Conflict of interest

The authors declare the absence of a conflict of interests.

Funding

Pharmacological studies were carried out within the framework of project No. 22-15-2002 with financial support from the Russian Science Foundation and the Volgograd Region Administration.

Data availability

All of the data that support the findings of this study are available in the main text.

Acknowledgments

This work was carried out using the equipment of the Common Use Center of the Southern Federal University, Rostov-on-Don, Russia.

Ethics Statements

The experiments were approved by the Biomedical Ethics Commission of Volgograd State Medical University (VolSMU) IRB 00005839 IORG 0004900, OHRP, Certificate No. 2021/056 dated 15.06.2021.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Funding

Pharmacological studies were carried out within the framework of project No. 22-15-2002 with financial support from the Russian Science Foundation and the Volgograd Region Administration.

Acknowledgments

This work was carried out using the equipment of the Common Use Center of the Southern Federal University, Rostov-on-Don, Russia.

Data availability

All of the data that support the findings of this study are available in the main text.

Ethics Statements

The experiments were approved by the Biomedical Ethics Commission of Volgograd State Medical University (VolSMU) IRB 00005839 IORG 0004900, OHRP, Certificate No. 2021/056 dated 15.06.2021.

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Author Contributions

§ Alena S. Taran, PhD of Medical Sciences, Associate Professor, Associate Professor of the Department of Pharmacology and Bioinformatics, Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: alena-beretta-taran@mail.ru; ORCID ID: https://orcid.org/0000-0001-8477-254X. The author designed the experimental study, participated in the experimental design, and wrote the pharmacologic part of the manuscript.

§ Olga N. Zhukovskaya, PhD in Chemical Sciences, Senior researcher at the Laboratory of Organic Synthesis, Institute of Physical and Organic Chemistry, Rostov-on-Don, Russia; e-mail: zhukowskaia.ol@yandex.ru; ORCID ID: https://orcid.org/0000-0003-2485-2139. The author proposed the design of the structures, synthesized the compounds, studied their structure, and wrote the chemical part of text.

§ Lyudmila V. Naumenko, Associate Professor, Professor of the Department of Pharmacology and Bioinformatics, Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: milanaumenko@mail.ru; ORCID ID: https://orcid.org/0000-0002-2119-4233. The author developed the design of the experimental study.

§ Alina M. Chebanko, Assistant of the Department of Pharmacology and Bioinformatics, Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: alina.chebanko@yandex.ru; ORCID ID: https://orcid.org/0009-0004-3140-5040. The author took part in conducting experimental work and analyzing the material.

§ Yuliya V. Efremova, student of Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: julia.efremova01@gmail.com. The author conducted animal studies of pharmacologic activity.

§ Irina D. Bodrug, student of Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: ira-bodrug-irina-bodrug@mail.ru. The author conducted animal studies of pharmacologic activity.

§ Anna A. Beloshapka, student of Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: ann.hlopkova2001@yandex.ru. The author conducted animal studies of pharmacologic activity.

§ Svetlana B. Zaichenko, researcher, Laboratory of Organic Synthesis, Institute of Physical and Organic Chemistry, Rostov-on-Don, Russia; e-mail: s-zaychenko@inbox.ru. The author conducted an analysis of IR-spectra.

§ Anatoly S. Morkovnik, Doctor Habil. of Chemical Sciences, Head of the Laboratory of Organic Synthesis, Institute of Physical and Organic Chemistry, Rostov-on-Don, Russia; e-mail: asmork@mail.ru; ORCID ID: https://orcid.org/0000-0002-9182-6101. The author was engaged in editing the chemical part of the manuscript.

§ Alexander A. Spasov, Doctor Habil. of Medical Sciences, Professor, Member of the Russian Academy of Sciences, Head of the Department of Pharmacology and Bioinformatics, Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: aspasov@mail.ru; ORCID ID: https://orcid.org/0000-0002-7185-4826. The author proposed the concept and design of the manuscript and approved of the final version of the manuscript.

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