Synthesis and ophthalmic hypotensive effect of new potential benzimidazole-based Rho-kinase-2 inhibitors

Olga N. Zhukovskaya¹, Alena S.Taran^2*, Lyudmila V. Naumenko², Alina M. Chebanko², Umida M. Ibragimova², Anatoly S.Morkovnik¹, Alexander A.Spasov²

¹Institute of Physical and Organic Chemistry, Southern Federal University; 194/2 Stachki Ave., Rostov-on-Don 344090 Russia

²Volgograd State Medical University; Pavshikh Bortstov Sq., Volgograd 400066 Russia

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

Abstract

Introduction: Based on the chemical structures of known Rho-kinase-2 inhibitors, 18 benzimidazole derivatives were synthesized and studied for their ophthalmic hypotensive activity in animals with normal intraocular pressure. The dependence of the pharmacological effect on the chemical structure of the compounds was analyzed. The effect of the most active compounds on Rho-kinase-2 activity was assessed.

Materials and Methods: Ophthalmic hypotensive activity was assessed by measuring intraocular pressure with a TonoVet veterinary tonometer in 120 mongrel rats (6 in each group) before and after instillation of reference drug solutions (timolol and melatonin) and 18 test substances. The effect of the test compounds on Rho-kinase activity was assessed using an in vitro enzyme-linked immunosorbent assay spectrophotometrically.

Results: The most active compounds among the new benzimidazole derivatives after a single instillation at a concentration of 0.4% were compound 7a (1-(4-fluorobenzyl)-3-(2-(pyrrolidin-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-imine hydrochloride), which reduced intraocular pressure in normotensive animals by 28.21%, exceeding the effect of the reference drug timolol (26.84%), but inferior to melatonin (30.95%), and compound 1d (1-(1-(2-(azepan-1-yl)ethyl)-1H-benzo[d]imidazol-2-yl)-3-(3-trifluoromethyl)phenyl)urea hydrochloride), which reduced ophthalmotonus by 23.96%, slightly inferior to timolol. The test compounds do not affect intraocular pressure dynamics in the contralateral eye and, therefore, do not have a systemic effect, unlike the reference drugs. It was also found that compounds 7a and 1d at a concentration of 1*10-4 mol/L inhibit Rho-kinase-2 by 26.72% and 18.11%, respectively.

Conclusion: The most active compounds, 7a and 1d, were identified as Rho kinase-2 inhibitors that exhibit ocular hypotensive effects in normotensive animals in vivo.

Graphical Abstract

Keywords: glaucoma, benzimidazole, azepan, biphenyl, intraocular pressure, Rho-kinase (ROCK)

Introduction

The synthesis and study of the biological activity of compounds spans various areas of modern medicinal chemistry. The presence of a heterocyclic fragment significantly influences the biological activity of hybrid substances. For example, the benzimidazole fragment is present in the structure of many drugs with varying biological activity. Among the widely used medications, mention should be made of dabigatran (an anticoagulant, trade name Prodaxa), dibazol (an antispasmodic), omeprazole (an antiulcer drug), droperidol and pimozide (neuroleptics), astemizole (an antihistamine), and others.

Glaucoma is currently one of the leading causes of blindness or visual impairment (Movsisyan et al. 2022). The leading symptom of this disease is increased IOP, primarily caused by impaired aqueous humor (AHF) outflow, followed by the development of typical visual defects and optic nerve atrophy (Cheng et al. 2012; Cholkar et al. 2015; Jonas et al. 2018; Efimenko et al. 2024). Thus, the main goal of glaucoma therapy is to reduce ophthalmotonus, regardless of the type and stage of glaucoma (Razhko et al. 2021).

Current glaucoma treatments primarily focus on reducing intraocular pressure but do not address pressure-independent neurodegenerative mechanisms. The endogenous indoleamine, melatonin, has attracted the attention of scientists due to its potential to regulate intraocular pressure (through activation of melatonin receptors and synergism with adrenergic and enzymatic regulators), as well as its ability to correct neurodegeneration (protecting retinal ganglion cells, mitigating oxidative stress, preventing mitochondrial dysfunction, and inhibiting apoptotic and inflammatory cascades) (Babkov et al. 2024). Benzimidazole compounds are melatonin isosteres and exhibit affinity for melatonin receptors, making this class promising not only as ophthalmic hypotensive agents but also as neuroprotective agents (Marcus et al. 2018a; Marcus et al. 2018b; Turanlı et al. 2020; Taran et al. 2025).

Recently, the emergence of a new class of antihypertensive drugs, Rho-kinase-2 inhibitors, has been discussed in the literature. They have a neuroprotective effect, affect trabecular tissue, increase the outflow of intraocular fluid, and reduce intraocular pressure (Abbhi et al. 2017; Yao et al. 2018; Wu et al. 2024; Hou and Pan 2025). Among the Rho-kinase-2 inhibitors, there are representatives of several groups depending on the chemical structure, such as: derivatives of isoquinoline, urea, indazole, aminopyrimidine, chroman-3-amine, benzimidazole, quinazolinone, indole, 7-azaindole (Cholkar et al. 2015).

Figure 1. Chemical structures of Rho kinase-2 inhibitors.

Ripasudil and netarsudil are isoquinoline derivatives, and sovesudiil is a biphenyl derivative of pyridine (Fig. 1). These compounds are approved by the US Food and Drug Administration (FDA) (Tan et al. 2025).

Figure 2. Chemical structures of benzimidazole-based Rho kinase-2 inhibitors.

The structures of Rho-kinase-2 inhibitors containing benzimidazole as a structural subunit have been reported in (Fig. 2) (Abbhi et al. 2017; Marcus et al. 2019). In this study, using the privileged structure of benzimidazole and existing data on the chemical structure of a new class of antiglaucoma agents, potential ophthalmic hypotensive agents were synthesized that exert their effect through inhibition of Rho-kinase-2.

Materials and Methods

Experimental animals

120 outbred rats took part in an experiment to study the effect on intraocular pressure.

All animal procedures in the study were conducted in accordance with generally accepted ethical standards for animal handling. Animal care complied with the rules of laboratory practice for preclinical studies in the Russian Federation (GOST 351.000.3-96 and 51000.4-96), Order No. 708n of the Ministry of Health and Social Development of the Russian Federation dated August 23, 2010, “On Approval of the Rules of Laboratory Practice”, and also complied with Directive 2010/63/EU of the European Parliament and of the Council of the European Union dated September 22, 2010, on the protection of animals used for scientific purposes. The experiments were approved by the Biomedical Ethics Committee of Volgograd State Medical University (IRB 00005839 IORG 0004900, OHRP, Certificate No. 2021/056 dated June 15, 2021). All sections of this study comply with the ARRIVE Guidelines for Reporting Animal Studies. Animals were maintained in the vivarium of Volgograd State Medical University, Ministry of Health of the Russian Federation, at a temperature of 24°C and a relative humidity of 60% under a natural light cycle with free access to food and water.

Chemical synthesis

This work included three stages. The first stage included the synthesis of 18 new benzimidazole derivatives, confirmation of the structure of all obtained compounds using IR, 1H, 13C spectroscopy, elemental analysis (C, H, Hal, N) and determination of the melting point. The second stage: studying the ophthalmic hypotensive activity of the obtained substances and analyzing the dependence of the activity on the chemical structure of the substances. The third stage: studying the inhibition of Rho-kinase-2 of the most active compounds.

The compounds under study

The synthesis of 1H-benzo[d]imidazol-2-yl)urea salts (1a-f) and 1H-benzo[d]imidazol-2-yl)-2-(4-nitrophenyl)piperazin-1-yl)acetamides (4a,b) is shown in Scheme 1. Compounds 1a-f were obtained by prolonged refluxing of a mixture of starting amines 2a-c and isocyanates in toluene. Urea derivatives 1a-f are colorless substances with high melting points. The ¹H NMR spectra of these compounds contain proton signals from two NH groups of the urea moiety. Intermediates 3a,b were synthesized by acylation of amines 2d,e with chloroacetyl chloride in absolute benzene in the presence of pyridine. Compounds 3a and 3b, 1-(4-nitrophenyl)piperazine, and triethylamine were then refluxed for 4 hours in dimethylformamide. The resulting compounds 4a and 4b are colorless substances whose ¹H NMR spectra contain characteristic signals from the NH protons of the carbamide moiety.

Scheme 1. Synthesis of 1H-benzo[d]imidazol-2-yl)urea and 1H-benzo[d]imidazol-2-yl)-2-(4-nitrophenyl)piperazin-1-yl)acetamide derivatives. Note: 1: R= (CH₂)₂N(CH₂)₄, R₁=Cl, R₂=H (a); R₁=H, R₂=CF3 (b); R₁=H, R₂=Cl (c). R= (CH₂)₂N(CH₂)₆, R₁=H, R₂=CF₃ (d); R= 4-(2’-MeOCO)C₆H₄C₆H₄CH₂, R₁=H, R₂=Cl (e); CF₃ (f). 2: R=(CH₂)₂N(CH₂)₄ (a), 4-(2’-MeOCO)C₆H₄C₆H₄CH₂(b), (CH₂)₂N(CH₂)₆(c). 3,4: R=All (a); CH₂-(4-tert-butyl)C₆H₄ (b).

Synthesis of 2-aminobenzimidazolium bromides 5 (a-f), 9-(2-azepan-1-yl)ethyl)-2-(3-methoxyphenyl)-9H-benzo[d]imidazo[1,2-a]-imidazole (6) and 2-iminobenzimidazole derivatives 7 (a-d) is shown in Scheme 2.

Scheme 2. Scheme of the synthesis of 2-aminobenzimidazolium bromides 5 (a-е), 9-(2-azepan-1-yl)ethyl)-2-(3-methoxyphenyl)-9H-benzo[d]imidazo[1,2-a]imidazole (6) and 2-iminobenzimidazole derivatives 7 (a-d). Note: 5: R= C₆H₅ (a), 3-MeOC₆H₄(b), 4-CF₃C₆H₄(c), 4- C₆H₅C₆H₄ (d), X=Br; (4-MeOC6H4)NH (e), X=Cl; 6: R= 3-MeOC6H4, X= Cl; 7: R= CH₂C₆H₄F-4, X=Cl (a), C₆H₅OCH₂CH₂(b), R₁= CH₂CH₂N(CH₂)₄; R= 4-(2´-MeOOCC₆H₄)C₆H₄, R₁= C₆H₅OCH₂CH₂ (c); R= C₆H₅CH₂, R₁= CH₂C₆H₄C₆H₅(d), X=Br. 8: R= CH₂C₆H₄F-4 (a), C₆H₅OCH₂CH₂(b, c), CH₂C₆H₅ (d).

For the synthesis of bromides 5a-e, a method based on the condensation of amine 2c with bromoketones was chosen. The quaternization reaction of amine 2c proceeds smoothly in acetone, with the yield of quaternary salts 5a-e being 74-96%. The ¹H NMR spectra of the resulting bromides 5a-e contain two-proton singlet signals (5.20-6.05), which correspond to the absorption of the methylene protons of aroylmethyl radicals. The signals of the amino group proton (2H) appear as broadened signals at 9.27-9.4 ppm. These signals are absent from the ¹H NMR spectra of tricyclic compound 6, obtained by cyclization of the imine bromide base 5c under reflux in 7.5% hydrochloric acid for 4 hours. Compounds 7a-d are obtained by quartenization of known amines with alkyl halides under reflux in nitromethane. The compounds precipitate during the refluxing of the reaction mixture. The yields of reaction products 7a-d are 50-87%. In the NMR spectra, the signals of the methylene protons at N¹ and N³ appear at 4.29-5.59 ppm.

2-Phenoxyethyl bromide, 4-fluorobenzyl chloride, 2-bromo-4-(trifluoromethyl)acetophenone, 1-(4-nitrophenyl)piperazine, isocyanates, and biphenyl-containing reagents used in this work were purchased from Alfa Aesar (Great Britain).

2-Aminobenzimidazole was obtained by alkaline hydrolysis of methyl N-benzimidazole-2-carbamate (Pozharsky et al. 1988); its N-substituted derivatives 2a (Di Braccio et al. 2013), 2b (Spasov et al. 2020), 2c (Zhao et al. 2021), 2d (Anisimova and Tolpygin 2011), 2e (Divaeva et al. 2015) were obtained according to the indicated methods, and phenacyl bromides were obtained by bromination of the corresponding acetophenones in alcohol. The reaction progress and the individuality of the compounds were monitored by TLC on Al₂O₃ plates. IR spectra (ν/cm⁻¹) of the obtained compounds were recorded on a Varian Excalibur 3100 FTIR IR spectrophotometer (Varian, USA) using the attenuated total reflectance method in powder; NMR spectra were recorded on a Bruker Avance 600 spectrometer (USA) (600 MHz for ¹H and 150 MHz for ¹³C). Chemical shifts are given relative to residual proton signals deuterated solvent (2.49 for ¹H, 39.7 for ¹³C in DMSO-d₆). Melting points were measured on a Fisher-Johns melting point apparatus (Fisher Scientific, USA). Elemental analysis was performed using the classical method (Gelman et al. 1987).

General procedure for synthesizing (scheme 1)

5 mmol of the appropriate isocyanate was added to a solution of 5 mmol of the appropriate 2-aminobenzimidazole 2a-c in 5-10 mL of toluene, and the mixture was refluxed for 8-10 hours. After refluxing, the reaction mixture was cooled in an ice bath, the precipitate was filtered, and washed with petroleum ether. The resulting compounds 1a-f were air-dried and crystallized from the appropriate solvent. Compounds 1a-f were then dissolved in a minimal amount of 2-PrOH, concentrated hydrochloric acid was added to pH 1, and the reaction mixture was maintained at room temperature for 1 hour.

The precipitated salt product was filtered, washed with acetone and ether, and air-dried.

1-(4-chlorophenyl)-3-(1-(2-pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole-2-yl)urea (1a НСl)

Yield 81 %, m.p.167-169º C. IR spectrum, ν/cm^-1: 3225, 3160 (2NH), 1702 (C=O). Found (%): C 57.04, H 5.63; Cl 16.74; N 16.54. С₂₀Н₂₂ClN₅О^.HCl. Calculated (%): C 57.15, H 5.52; Cl 16.87; N 16.66. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.94 (c, 4H, 2CH₂^endo), 3.52-3.64 (m, 6H, CH₂NCH₂+CH₂^exoN), 4.81 (c, 2H, CH₂), 7.36-7.95 (m, 8H, H_Ar), 10.54 (broad c, 1H, NH), 11.48 (c, 1H, NH), 13.25 (broad c, 1H, N⁺H). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 22.82, 38.92, 50.21, 53.12, 110.73, 113.06, 119.93, 123.79, 124.17, 126.68, 128.88, 128.95, 129.12, 137.47. M.p. of base 120-121 ºC (benzene/hexane, 1:1).

1-(1-(2-pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole-2-yl)-3-(3-(trifloromethyl) phenyl)urea (1b НСl)

Yield 66 %, m.p. 125-126º C. IR spectrum, ν/ cm^-1: 3225, 3160 (2NH), 1702 (C=O). Found (%): N 15.32. C₂₁H₂₂F₃N₅O^.HCl. Calculated (%): N 15.43. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.86 (t. 4H, 2CH₂^endo, J=5.97), 3.18 (t, 2H, CH₂^exoN, J= 5.43), 3.51-3.63 (m, 4H, CH₂NCH₂), 4.78 (c, 2H, N-CH₂^exo), 7.26-7.28 (m, 1H, H_Ar), 7.34-7.41 (m, 3H, H_Ar), 7.57-7.66 (m, 2H, H_Ar), 7.80-7.94 (m, 1H, H_Ar), 8.2 (c, 1H, H_Ar), 10.65 (b.c, 1H, NH), 11.69 (c, 1H, NH), 12.96 (c, 1H, N⁺H). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 22.82, 22.85, 38.29, 39.02, 49.76, 50.31, 52.85, 53.13, 110.37, 112.87, 119.14, 121.90, 123.66, 123.75, 124.01, 124.99, 128.97, 129.74, 130.21, 139.65, 150.1.

1-(3-chlorophenyl)-3-(1-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole-2-yl)urea (1c НСl)

Yield 65 %, m.p.166-168 º C. IR spectrum, ν/ cm^-1: 3225, 3160 (2NH), 1702 (C=O). Found (%): C 57.04; H 5.63; Cl 16.74; N 16.54. С₂₀Н₂₂ClN₅О^.HCl. Calculated (%): C 57.15; H 5.52; Cl 16.87; N 16.66. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.95 (t, 4H, 2CH₂^endo, J= 1.2), 3.24-3.27 (m, 2H, CH₂^exoN), 3.64-3.61 (k, 4H, (CH₂^endo)₂N, J=6.4), 4.78 (c, 2H, NCH₂^exo), 7.12 (d, 1H, H⁴_Ph), 7.35-7.40 (m, 4H, H_Bzm), 7.61 (d, 1H, H⁵_Ph, J=7.3), 7.81 (c, 1H, H⁶_Ph), 7.93 (b.c, 1H, H²_Ph), 10.54-10.53 (m, 1H, NHPh), 11.35 (t, 1H, C²_BzmNH, J=3.2), 13,3 (d.c, 1H, N⁺H).¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 22.80, 38.85, 50.38, 110.61, 112.91, 116.77, 117.76, 122.52, 123.68, 124.05, 128.89, 129.16, 130.68, 133.33, 140.29. M.p. of base 119-120 ºC (benzene/hexane, 1:1).

1-(1-(2-(azepan-1-yl)ethyl)-1H-benzo[d]imidazole-2-yl)-3-(3-trifloromethyl) phenyl)urea (1d НСl)

Yield 86%, m.p.218-219 ºC. IR spectrum, ν/ cm^-1: 3225, 3160 (2NH), 1734 (C=O). Found (%): 14.41 C₂₃Н₂₆F₃N₅О^.HCl. Calculated (%): N 14.53. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.54 (c, 4H, CH₂), 1.88 (c, 4H, CH₂), 3.44 (c, 4H, CH₂), 3.56 (t, 2H, CH₂, J=1.6), 4.87 (t, 2H, CH₂, J=7.23), 7.36-7.61 (m, 6H, H_Ar), 8.04-8.16 (m, 2H, H_Ar), 10.59 (c, 1H, NH), 11.34 (c, 1H, NH), 13.26 (br.c, 1G, N⁺H). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J.

Methyl-4’-((2-(3-(3-chlorophenyl)ureido)-1H-benzo[d]imidazole-1-yl)methyl)-[1,1’-biphenyl]-2-carboxylate (1e НСl)

Yield 60 %, m.p. 199-200º C. IR spectrum, ν/ cm^-1: 3225, 3160 (2NH), 1736 (COOMe), 1710 (C=O). Found (%): C 63.52, H 4.53; Cl 12.82; N 10.11. С₂₉H₂₃ClN₄O₃^.HCl. Calculated (%): C 63.63, H 4.42; Cl 12.95; N 10.23. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.52 (c, 3H, OMe), 5.7 (c, 1H, N-CH₂), 7.11, 7.12 (c, c, 1H, H_Ar), 7.25 (c, 1H, H_Ar), 7.26 (c, 1H, H_Ar), 7.31-7.41 (m, 6H, H_Ar), 7.42-7.46 (m, 4H, H_Ar), 7.47-7.51 (m, 1H, H_Ar), 7.52-7.57 (m, 1H, H_Ar), 7.59-7.72 (m, 1H, H_Ar), 7.80 (c, 1H, NH), 10.63 (b.c, 1H, NH), 13.36 (b.c, 1H, N⁺H). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 45.83, 51.79, 110.79, 112.93, 113.11, 116.81, 117.81, 122.60, 123.84, 127.35, 127.39, 127.50, 128.49, 129.00, 129.12, 129.29, 130.43, 130.56, 130.65, 131.46, 133.27, 133.31, 133.78, 140.14, 140.20, 140.72, 168.22. M.p. of base m.p. 152-153ºC.

Methyl 4’-((2-(3-(3-(trifluoromethyl)phenyl)ureido)-1H-benzo[d]imidazole-1-yl)methyl)-[1,1’-bithenyl]-2-carboxylate (1f)

Yield 88 %, m.p. 128-129º C. IR spectrum, ν/ cm^-1: 3225, 3160 (2NH), 1736 (COOMe), 1710 (C=O). Found (%): 9.55. С₃₀H₂₃F₃N₄O₃ HCl. Calculated (%): N 9.67. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.52 (c, 3H, MeO), 5.72 (c, 2H, N-CH₂), 7.25 (d, 2H, H_Ar, J=6.0), 7.28-7.35 (m, 2H, H_Ar), 7.37-7.38 (m, 2H, H_Ar), 7.44-7.49 (m, 4H, H_Ar), 7.56-7.59 (m, 2H, H_Ar), 7.63 (d, 1H, H_Ar, J=7.26), 7.67 (d, 1H, HAr, J= 8.34, 7.70-7.72 (m, 1H, H_Ar), 8.2 (c, 1H, H_Ar), 9.00 (с, 1H, NH), 10.52 (b.c, 1H, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 45.89, 51.76, 110.74, 112.90, 114,27, 118.95, 121.93, 123.58, 125.01, 126.96, 127.36, 127.48, 128.46, 128.99, 129.14, 129.27, 129.50, 129.70, 130.08, 130.40, 130.63, 131.44, 133.95, 139.80, 140.13, 140.71, 168.20, 124.19, 124.94, 126.74, 128.82, 129.03, 129.59, 129.81, 130.28, 139.37, 146.22, 153.07. M.p. of base 168-169ºC.

General procedure for synthesizing of compounds 3а,b and 4a,b

To a boiling solution of 0.015 mol of 2-aminobenzimidazole 2 d,e in 80 mL of absolute benzene, add 1-2 mL of pyridine. A solution of 0.018 mol of chloroacetyl chloride in 5 mL of absolute benzene is added dropwise over 1 hour with stirring. The mixture is refluxed for 8-10 hours. By the end of this time, a white fibrous precipitate begins to separate from the solution. The reaction mixture is cooled, the precipitate is filtered, and washed repeatedly with water until the washings test negative for ionic halide, сrystallized from 2-PrOH, DMF, MeNO₂.~~a suitable solvent.~~ Next, a solution of 0.002 mol of the above-described compounds 3a or 3b, 0.3 mL of triethylamine, and 0.002 mol of 1-(4-nitrophenyl)piperazine in 5-10 mL of dimethylformamide was refluxed for 4 hours. The reaction mixture was poured onto ice and left overnight at room temperature. The following day, the resulting precipitate was filtered, washed with water, and air-dried. The mixture was crystallized from nitromethane.

N-(1-allyl-1H-benzo[d]imidazole-2-yl)-2-chloroacetamide (3a)

Yield 85 %, m.p. 161-161.5º C (2-PrOH). IR spectrum, ν/ cm^-1: 3172 (NH), 1735 (C=O), 1625, 1585 (C=N). Found (%): C 57.65; H 4.95; Cl 14.07; N 16.71. C₁₂H₁₂ClN₃O. Calculated (%): C 57.77; H 4.84; Cl 14.20; N 16.83. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 4.25 (c, 2H, CH₂Cl), 4.76 (d, 2H, N-CH₂, J= 5.1), 5.04-5.18 (m, 2H, =CH₂), 5.87-5.96 (m, 1H, CH), 7.19-7.22 (m, 2H, H_Ar), 7.38-7.52 (m, 2H, H_Ar), 12.62 (c, 1H, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 42.7, 46.9, 110.0, 118.0, 117.2, 123.0, 123.0, 132.9, 134.2, 141.3, 152.8, 166.5.

N-(4-(tert-butyl)benzyl)-1H-benzo[d]imidazole-2-chloroacetamide (3b)

Yield 94 %, m.p. 208-210ºC (DMF). IR spectrum, ν/ cm^-1: 3210 (NH), 1732 (C=O), 1623, 1583 (C=N). Found (%): C 67.39; H 6.32; Cl 9.83; N 11.69. C₂₀H₂₂ClN₃O. Calculated (%): C 67.50; H 6.23; Cl 9.96; N 11.81. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.20 (c, 9H, C(Me)₃, 4.36 (c, 2H, CH₂Cl), 5.39 (c, 2H, N-CH₂), 7.21-7.24 (m, 2H, H_Ar), 7.31-7.39 (m, 4H, H_Ar), 4.44-7.47 (m, 1H, H_Ar), 7.52-7.55 (m, 1H, H_Ar). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 31.3, 31.3, 31.3, 34.2, 42.7, 49.7, 110.0, 118.5, 123.0, 123.0, 124.9, 124.9, 126.9, 126.9, 134.2, 134.2, 141.3, 148.3, 152.0, 166.5.

N-(1-allyl-1H-benzo[d]imidazole-2-yl)-2-(4-(4-nitrophenyl)piperazin-1-yl)acetamide (4a)

Yield 77%, m.p. 201-202ºC (MeNO₂). IR spectrum, ν/ cm^-1: 3423 (NH), 1725 (C=O), 1633, 1591 (C=N). Found (%): C 62.73; H 5.86; N 19.87. C₂₂H₂₄N₆O₃. Calculated (%): C 62.84; H 5.75; N 19.99. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 2.79 (t, 4H, CH₂NCH₂, J=4.95), 3.39-3.52 (m, 6H, CH₂NCH₂+ CH₂N ), 4.75 (c, 2H, N-CH₂), 5.14-5.26 (m, 2H, CH₂^All), 5.85-5.95 (m, 1H, CH), 6.82 (d, 2H, H_Ph, J=9.0), 7.22 (t, 3H, H_Bzm, J=4.05), 7.31 (c,1H, H_Bzm), 8.11 (d, 2H, H_Ph, J=9.3), 12.0 (b.c, 1H, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 43.32, 44.14, 50.85, 58.57, 110.55, 112.73, 113.35, 113.42, 117,51, 123.41, 123.45, 125.60, 128.80, 131.94, 137.99, 149.50, 153,74, 170.89.

N-(1-(4-(tert-butyl)benzyl)-1H-benzo[d]imidazole-2-yl)-2-(4-(4-nitrophenyl) piperazin-1-yl)acetamide (4b)

Yield 86 %, m.p. 199-200ºC (MeNO₂). IR spectrum, ν/ cm^-1: 3423 (NH), 1725 (C=O), 1633, 1591 (C=N). Found (%): C 68.31; H 6.62; N 15.84. C₃₀H₃₄N₆O₃. Calculated (%): C 68.42; H 6.51; N 15.96. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.21 (c, 9H, (Me)₃), 2.78 (c, 4H, CH₂NCH₂), 3.46 (m, 6H, CH₂NCH₂+ CH₂N), 5.28 (c, 2H, N-CH₂), 6.81 (d, 2H, H_Ph, J=9.3 ), 7.17-7.32 (m, 8H, 4H_Bzm+4H_Ph), 8.11 (d, 2H, H_Ph, J= 9.3), 12.1 (b.c, 1H, NH). ), NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 31.0, 34.17, 43.32, 44.57, 50.79, 58.79. 110.53, 112.65, 113.41, 123.35, 123.39, 125.39, 125.58, 127.05, 128.80, 128.85, 133.09, 138.02, 150.09, 150.43, 153.72, 171.51.

General procedure for the synthesis of 2-aminobenzimidazolium bromides 5 (a-f) and tricyclic compound 6 (Scheme 2)

5 mmol of the corresponding α-bromoketone was added to a hot solution of 5 mmol of amine (2c) in acetone. The mixture was stirred until the reagent dissolved, heated until the quaternary salt began to precipitate, and then kept at room temperature for 6-8 hours. The precipitate was filtered, washed thoroughly with acetone, and dried at 40-45°C. Chromatographically pure salts were obtained, which were used without further purification. ~~General procedure for synthesizing~~

2-Amino-1-(2-(azepan-1-yl)ethyl)-3-(2-oxo-2-phenylethyl)-1H-benzo[d] imidazole-3-ium bromide (5a)

Yield 86 %, m.p. 177-178ºC. IR spectrum, ν/ cm^-1: 3202, 3154 (NH₂),1710 (C=O), 1633, 1591 (C=N). Found (%): C 60.28; H 6.50; Br 17.34; N 12.13. C₂₃H₂₉BrN₄O. Calculated (%): C 60.39; H 6.39; Br 17.47; N 12.25. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.46, 1.52 (d, c, 8H, (CH₂)₄^endo, J=2.34), 2.68 (c, 4H, (CH₂)₂^endo, 2.86 (c, 2H, CH₂^exo), 4.32 (c, 2H, N_BzmCH₂), 5.99 (c, 2H, CH₂CO), 7.26-7.35 (m, 2H, H_Ar), 7.60, (d, 1H, H_Ar, J= 7.98), 7.63-7.66 (k, 3H, H_Ar, J= 5.78), 7.75-7.78 (k, 1H, H_Ar, J= 5.36), 8.09-8.11 (m, 2H, H_Ar), 9.3 (b.c, 2H, N⁺H₂). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 26.22, 27.65, 41.96, 49.85, 55.15, 55.27, 110.39, 110.62, 123.39, 123.49, 128.42, 128.81, 129.80, 130.21, 134.02, 134.22, 150.91, 191.07.

2-Amino-1-(2-(azepan-1-yl)ethyl)-3-(2-(3-methoxyphenyl)-2-oxoethyl)-1H-benzo[d]imidazole-3-ium bromide (5b)

Yield 80 %, m.p. 182-183ºC. IR spectrum, ν/ cm^-1: 3205, 3154 (NH₂), 1710 (C=O), 1633, 1591 (C=N). Found (%): C 59.03; H 6.52; Br 16.26; N 11.37. C₂₄H₃₁BrN₄O_2. Calculated (%): C 59.14; H 6.41; Br 16.39; N 11.49. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.45-1.5 (k, 8H, (CH₂)₄^endo, J=12.2), 2.67 (c, 4H, (CH₂)₂^endo, 2.87 (c, 2H, CH₂^exo), 3.86 (c, 3H, OMe), 4.31 (t, 2H, N_BzmCH₂, J=5.58), 5.98 (c, 2H, CH₂CO), 7.25-7.35 (m, 3H, H_Ar), 7.55-7.63 (m, 4H, H_Ar), 7.70 (d, 1H, H_Ar,J=74), 9.3 (b.c, 2H, N⁺H₂). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 26.24, 27.70, 42.02, 49.95, 55.18, 55.29, 55.56, 110.37, 110.64, 113.14, 120.14, 120.89, 123.39, 123.51, 129.84, 130.08, 134.18, 135.33, 150.92, 159.44, 190.98.

2-amino-3-(2-(azepan-1-yl)ethyl)-1-(2-oxo-2-(4-(trifluoromethyl)phenyl)ethyl)-1H-benzo[d]imidazole-3-ium bromide (5c)

Yield 96 %, m.p. 205-207ºC. IR spectrum, ν/ cm^-1: 3207, 3154 (NH₂),1703 (C=O), 1660, 1618, 1584 (C=N). Found (%): N 10.54. C₂₄H₂₈BrF₃N₄O. Calculated (%): 10.66. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.47 (c, 4H, (CH₂)₂^endo), 1.52 (c, 4H, (CH₂)₂^endo), 2.68 (c, 4H, (CH₂)₂^endo, 2.88 (c, 2H, CH₂^exo), 4.33 (c, 2H, N_BzmCH₂), 6.05 (c, 2H, CH₂CO), 7.28 (t, 1H, H_Ar, J=7.68), 7.34 (t, 1H, H_Ar, J=7.74), 7.65 (t, 2H, H_Ar, J=4.32), 8.04 (d, 2H, H_Ar, J=8.28), 8.30 (t, 2H, H_Ar, J=8.16), 9.27 (b.c, 2H, N⁺H₂). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 26.23, 27.66, 42.02, 50.26, 55.27, 110.48, 110.68, 120.97, 122.77, 123.43, 123.56, 124.57, 125.75, 126.38, 129.32, 129.81, 130.16, 132.99, 133.20, 133.41, 133.62, 137.28, 150.84, 190.82.

3-(2-([1,1’-biphenyl]-4-yl)-2-oxoethyl)-2-amino-1-(2-(azepan-1-yl)ethyl)-1H-benzo[d]imidazole-3-ium bromide (5d)

Yield 75 %, m.p. 185-187ºC. IR spectrum, ν/ cm^-1: 3207, 3240 (NH₂), 1687 (C=O) 1663 (C=N). Found (%): C 65.18; H 6.34; Br 14.77; N 10.38. C₂₉H₃₃BrN₄O. Calculated (%): C 65.29; H 6.23; Br 14.98; N 10.50. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.48, 1.54 (c, c, 8H, (CH₂)₂^endo), 2.69 (c, 4H, (CH₂)₂^endo, 2.90 (c, 2H, CH₂^exo), 4.34 (c, 2H, N_BzmCH₂), 6.02 (c, 2H, CH₂CO), 7.29 (t, 1H, H_Ar, J=7.7), 7.35 (t, 1H, H_Ar, J=7.71), 7.45-7.47 (m. 1H, H_Ar), 7.62 (d, 1H, H_Ar, J=7.9), 7.65 (b.d, 1H, J=6.9), 7.81 (c, 2H, H_Ar), 7.97 (d, 2H, H_Ar, J=8.2), 8.18 (d, 2H, H_Ar, J=8.22), 8.30 (t, 2H, H_Ar, J=8.16), 9.25 (b.c, 2H, N⁺H₂). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 25.23, 27.63, 41.93, 49.87, 55.24, 110.43, 110.55, 123.46, 123.54, 126.91, 127.04, 128.64, 129.14, 129.18, 129.78, 130.25, 132.86, 138.63, 145.53, 150.90, 190.65.

Аzepan-1-yl)ethyl)-1-(2-((4-methoxyphenyl)amino)-2-oxoethyl)-1H-benzo[d]imidazole-3-ium chloride (5e)

Yield 74 %, m.p. 242-243ºC. IR spectrum, ν/ cm^-1: 3332, 3240, 3207(NH+NH₂), 1657 (C=O) 1663 (C=N). Found (%): C 62.83; H 7.15; Cl 7.61; N 15.17. C₂₄H₃₂ClN₅O₂. Calculated (%): C 62.94; H 7.04; Cl 7.74; N 15.29. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.45, 1.54 (c, c, 8H, (CH₂)₂^endo), 2.68 (t, 4H, (CH₂)₂^endo, J=5.31), 2.85 (t, 2H, CH₂^exo, J=6.0), 3.70 (c, 3H, MeO), 4.30 (t, 2H, N_BzmCH₂, J=5.73), 5.20 (c, 2H, CH₂CO), 6.88 (d, 2H, H_Ar, J=9.0), 7.26-7.33 (m, 2H, H_Ar), 7.52-7.57 (m, 4H, H_Ar), 9.4 (b.c, 2H, N⁺H₂), 10.84 (c, 1H, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 26.22, 27.64, 41.83, 45.61, 55.07, 55.11, 55.22, 110.19, 110.45, 113.86, 120.53, 123.3, 123.36, 129.82, 130.14, 131.7, 151.1, 155.41, 163.04.

9-(2-(azepan-1-yl)ethyl)-2-(3-methoxyphenyl)-9H-benzo[d]imidazo[1,2-a]imidazole (6 HCl)

0.002 mol of bromide 5b is thoroughly stirred in 5 mL of 20% NaOH solution for 45 min. After cooling, the resulting imine precipitate is filtered off, washed with ice water until the washings are neutral, and then used in the cyclization reaction without additional purification and drying. Imine 5b is dissolved in 7.5 mL of 7.5% concentrated hydrochloric acid solution and refluxed for 3.5-4 hours, monitoring the reaction progress by TLC (Al₂O₃), eluent chloroform, visualization with iodine vapor in a humid chamber. Rf of the starting imine is 0.16, Rf of the reaction product is 0.27. After cooling to 10-15ºC, the reaction mixture is maintained at this temperature for 3-4 hours. The resulting precipitate of compound 6 is filtered off, washed with ethanol, then with acetone. The compound is dried in a desiccator over P₂O₅ for 10 hours.

Yield 80%, m.p. 238-240ºC. Found (%): C 67.72; H 6.99; Cl 8.21; N 12.98. C24H28N4O.HCl. Calculated (%): C 67.83; H 6.88; Cl 8.34; N 13.18. 1H NMR spectrum (600 MHz, DMSO-d6), δ, ppm, J (Hz): 1.65 (s, 4H, (CH2)2endo), 1.88 (s, 4H, (CH2)2endo), 3.38 (s, 2H, CH2exo), 3.63 (s, 2H, CH2endo), 3.67 (s, 2H, CH2endo, J=15), 3.85 (s, 3H, MeO), 5.12 (s, 2H, NBzmCH2), 6.95 (k, 1H, HAr, J=3.5), 7.42 (t, 1H, HAr, J=7.95), 7.54-7.56 (m, 2H, HAr), 7.66 (s, 1H, HAr), 7.98 (d, 1H, HAr, J=7.98), 8.1 (d, 1H, HAr, J=8.1), 8.69 (s, 1H, HAr), 11.2 (s, 1H, N+ H). 13C NMR spectrum (150 MHz, DMSO-d6), δ, ppm, J (Hz): 23.07, 25.94, 37.98, 52.85, 53.25, 53.82, 55.09, 55.47, 105.22, 110.72, 111.92, 112.19, 112.9, 117.17, 122.83, 124.15, 125.21, 130.21, 133.08, 134.22, 134.79, 159.77.

General Method for solts 2-imino-2,3-dihydro-1H-benzimidazole (7a-d)

A solution of 3 mmol of the appropriate 2-aminobenzimidazole and 3 mmol of an alkyl halide in 5–12 mL of dioxane is refluxed for 5.5 hours. During the reaction, the solution turns yellow-red, and a precipitate forms. The precipitate is filtered off, washed with dioxane and acetone until the filtrate becomes colorless. The mixture is dried and crystallized from ethanol or 2-propanol.

1-(4-flurobenzyl)-3-(2-(pyrrolidin-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazole-2-imine hydrogen chloride (7a)

Yield 87%, m.p. 239-240ºC. IR spectrum, ν/ cm-1: 3332 (NH). Found (%): N 14.83. C20H23FN4.HCl. Calculated (%): N 14.95. 1H NMR spectrum (600 MHz, DMSO-d6), δ, ppm, J (Hz): 1.66 (s, 4H, (CH2)2endo), 2.63 (s, 4H, (CH2)2endo), 3.80 (s, 2H, CH2 exo), 5.02 (s, 2H, NBzmCH2), 5.56 (s, 2H, NBzmCH2), 7.18-7.38 (m, 7H, HAr), 7.39 (d, 1H, J=6.0), 7.8 (s, 1H, NH). 13C NMR spectrum (150 MHz, DMSO-d6), δ, ppm, J (Hz): 21.38, 23.12, 44.73. 44.83, 53.50, 56.46, 56.46, 110.5, 110.6, 115.9, 115.95, 118.0, 118.0, 128.50, ~~129.50,~~ 129.50, 130.9, 131.0, 150.27, 160.8.

1-(2-phenoxyethyl)-3-(2-pyrrolidin-1-yl)ethyl)-1,3-dihedro-2H-benzo[d]imidazole-2-imine hydrogen bromide (7b)

Yield 50 %, m.p. 201-202º C (2-PrOH). IR spectrum, ν/ cm^-1: 3495, 3442 (NH₂). Found (%): C 58.36; H 6.41; Br 18.37; N 12.85 С₂₁Н₂₆N₄ОHBr. Calculated (%): C 58.47, H 6.31; Br 18.52; N 12.99. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 1.62 (c, 4H, 2CH₂^endo), 2.52 (c, 4H, CH₂NCH₂), 2.79 (c, 2H, CH₂^exoN), 4.29 (c, 4H, N¹-CH₂, N³-CH₂), 4.62 (c, 2H, CH₂-O), 6.8 (д,d, 2H, H_Ar, J=7.86), 6.91 (t,1H, H_Ar, J=6.87), 7.23 (t, 2H, H_Ar, J=7.17), 7.32 (c, 2H, H^5,6), 7,64-7.54 (m, 2H, H_Ar,). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 23.12, 42.26, 42.40, 52.74, 53.54, 64.92, 110.32, 110.82, 114,21, 120.97, 123.23, 123.36, 129.41, 129.70, 129.67, 150.90, 157.70.

Methyl 4'-((2-imino-3-(phenoxyethyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-carboxylate hydrobromide (7c)

Yield 72 %, m.p. 230-231º C (EtOH). IR spectrum, ν/ cm^-1: 3344 (NH), 1726 (COOCH₃), 1655 (C=N). Found (%): C 64.41; H 5.17; Br 14.18, N 7.39. С₃₀Н₂₇N₃О₃HBr. Calculated (%): C 64.52; H 5.05; Br 14.31, N 7.52. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 3.52 (c, 3H, OCH₃), 4.35 (t, 2H, N³-CH_2,J=5.13), 4.69 (t, 2H, O-CH_2,J=5.13), 5.54 (c, 2H, N¹-CH₂), 6.81, 6.81 (c, c, 2H, H_Ar), 6.89-6.92 (m, 1H, H_Ar),7.22-7.31 (m ,7H, H^5,6, 2H_Ar), 7.34-7.37 (m, 2H, H^4,7), 7.46-7.49 (m, 2H, H_Ar), 7.58-7.61 (m, 1H, H_Ar), 7.71-7.73 (m, 2H, H_Ar), 9.12 (c, 2H, N⁺H₂). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 42.62, 45.32, 51.73, 64.83, 110.56, 111.09, 114,25, 121.02, 123.64, 123.66, 126.72, 127.52, 128.50, 129.28, 129.33, 128.44, 129.90, 130.39, 130.56, 131.45, 133.42, 140.15, 140.56, 150.14, 157.72, 168.08.

1-([1,1’-biphenyl]-4-yl-methyl)-3-benzyl-1,3-dihydro-2H-benzo[d]imidazole-2-imine hydrobromide (7d)

Yield 84 %, m.p. 286-287ºC (EtOH/H₂O). Found (%) C 68.83; H 5.25; N 8.81. C₂₇H₂₃N₃^.HBr. Calculated (%): C 68.94; H 5.14; N 8.93. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm, J (Hz): 5.55 (c, 2H, CH₂), 5.59 (c, 2H, CH₂), 7.27-7.29 (m, 2H, H^5,6_Bzm), 7.32, 7,34 (c c, 3H, H_ph+H^4,7_Bzm), 7.35-7.55 (m, 9H, 4H_ph+ 5H_Bif), 7.62-7.64 (m, 2H, H_Bif), 7.67 (c, 1H, H_Bif), 7.69 (c, 1H, H_Bif), 9.26 (c, 2H, N⁺H₂). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm, J (Hz): 45.51, 45.80, 110.89, 123.87, 126.62, 127.01, 127.10, 127.61, 127.66, 128.03, 128.86, 128.92, 129.57, 133.67, 134.50, 139.40, 139.91, 149.99.

Effect of compounds on intraocular pressure

The ophthalmic hypotensive effect of the compounds was studied on adult outbred rats using a tonometer mounted on a TonoVet veterinary tonometer (Finland) (Du Sert et al. 2020). All animals were divided into 20 groups, with each group containing 6 rats: 2 groups of reference drugs (timolol and melatonin), and 18 experimental groups instilled with solutions of the test compounds. The compounds were tested using the method of Marcus et al. (Pease et al. 2006), according to which one drop of the test compound solution was instilled into the right (test) eye of the laboratory animal, and 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 test compounds.

The studied melatonin isosteres – benzimidazole derivatives – were instilled into the test eye at a screening concentration of 0.4% once in a volume of 30 μL. IOP measurements were performed at four time points (0, 1, 2, and 3 hours), with 0 hours being the baseline value. Ophthalmic hypotensive activity was assessed by the maximum reduction in IOP relative to baseline. A 0.5% timolol solution (a drug used in clinical practice) and a 0.4% melatonin solution (SigmaAldrich, USA) were used as comparators.

Effect of compounds on Rho-kinase-2 activity in vitro

For the study, a 15 ng/mL Rho-kinase-2 standard solution was prepared.

The test compounds were diluted to the desired concentration (1*10^-4 mol/L).

The test compound solutions were mixed with the standard solution at a ratio of 1:9 and incubated for 30 minutes at 37°C to initiate the enzyme inhibition reaction. The effect of the test compounds on Rho-kinase-2 activity was then assessed using the Rho Associated Coiled Coil Containing Protein Kinase 2 ELISA Kit (Cloud-Clone Corp., China). The enzymatic-substance reaction is assessed by the color change spectrophotometrically at a wavelength of 450 nm ± 10 nm with Infinite 200 PRO multiplate reader (Tecan, Austria).

Statistical analysis

Statistical analysis was performed in Prism 8.0 (GraphPad Inc.), with calculation of the mean and standard deviation. Student's t-test was used to assess changes in IOP relative to baseline values. The nonparametric Mann-Whitney test was used to compare groups.

Results and Discussion

The study revealed that after instillation of the comparator drugs timolol and melatonin, IOP decreased in the test eye by 26.84% and 30.95%, respectively, relative to baseline values. Similar changes were observed in the contralateral (control) eye, which may indicate an undesirable resorptive effect, likely related to the absorption of the test substance solutions into the bloodstream (Table 1).

Table 1.

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

Compound

Мах Δ IOP, %

Effect on the contralateral eye

Timolol

-26.84±0.94*

Melatonin

-30.33±2.91*

Base structure

R¹

R²

Мах Δ IOP, %

Effect on the contralateral eye

16.26±0.43*

CF₃

12.50±12.50

-22.98±7.53

CF₃

-23.96±6.78*

-11.36±2.66

CF₃

-17.17±4.84

Base structure

R¹

Мах Δ IOP, %

Effect on the contralateral eye

-CH₂-CH=CH₂

no effect

-12.93±2.46

Base structure

Мах Δ IOP, %

Effect on the contralateral eye

-14.43±8.93

3-MeOC₆H₄

no effect

4-CF₃C₆H₄

-10.74±6.43

4-PhC₆H₄

-22.71±10.84

NH(4-MeOC₆H₄)

-16.48±1.19*

Base structure

Мах Δ IOP, %

Effect on the contralateral eye

3-MeOC₆H₄

2HCl

no effect

Base structure

R¹

Мах Δ IOP, %

Effect on the contralateral eye

CH₂C₆H₄F-4

-28.21±2.56*

CH₂CH₂OC₆H₅

-14.47±10.85

4-(2´-MeOOCC₆H₄)C₆H₄

CH₂CH₂OC₆H₅

-19.10±8.56

CH₂C₆H₄C₆H₅

-17.81±9.47

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

Melatonin, known Rho-kinase-2 inhibitors, and the new compounds synthesized for the study are heterocyclic nitrogen-containing structures. When synthesizing the new benzimidazole-based compounds, the structure of Rho-kinase-2 inhibitors was taken into account. As a result, the structures of the studied compounds included certain fragments (radicals) that could theoretically be responsible for the corresponding mechanism of action and the ophthalmic hypotensive effect.

All synthesized compounds were divided into five groups.

The first group included six compounds 1a-f, which are urea derivatives where one nitrogen atom contains benzimidazole and the other a substituted phenyl. In compounds 1a-d, the N¹ nitrogen atom in the benzimidazole contains a dialkylaminoethyl substituent, and the phenyl moiety contains a p-Cl, m-Cl, or m-CF₃group. Instillation of 1-(4-chlorophenyl)-3-(1-(2-pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole-2-yl)urea hydrogen chloride (1a) resulted in a 16.26±0.43% decrease in IOP in the study animals. In the structure of compound 1c, the Cl atom is transferred to the meta position, resulting in a significant increase in ophthalmic hypotensive activity and a 22.98±7.53% decrease in IOP relative to baseline values. At the same time, replacing m-Cl with the m-CF₃ group (compound 1b) led to a decrease in ophthalmic hypotensive activity to 12.50±12.50%. However, replacement of the pyrrolidine ethyl substituent with 1-ethylazepane in compound 1d (1-(1-(2-( (azepan-1-yl)ethyl)-1H-benzo[d]imidazole-2-yl)-3-(3-trifloromethyl) phenyl)urea hydrogen chloride) significantly increased the IOP-lowering activity, and instillation of a solution of this compound decreased the ophthalmotonus by 23.96±6.78% (p≤0.05, t-test). In compounds 1e and 1f, only replacement of the diethylaminoethyl radical at the N¹ atom of benzimidazole with methyl biphenylcarboxylate negatively affected the ophthalmohypotensive effect. Thus, compound 1e, with one electronegative m-Cl atom, reduced IOP by 11.36±2.66% when instilled into normotensive animals, while compound 1f, with three more electronegative F atoms, reduced IOP by 17.17±4.84%.

Among the studied subgroup, only compound 1c demonstrated a decrease in IOP in the contralateral eye, which may indicate a systemic effect, which is an undesirable effect. The remaining compounds – 1a, 1b, 1d, 1e, and 1f – had no effect on the ophthalmotonus of the control eye.

The second group is represented by benzimidazole acetamides 4a and 4b, containing 4-nitrophenylpiperazine. Compound N-(1-allyl-1H-benzo[d]imidazole-2-yl)-2-(4-(4-nitrophenyl)piperazin-1-yl)acetamide (4a) did not affect IOP when instilled into normotensive animals. This may be due to the negative effect of the allyl radical at the N¹ nitrogen of benzimidazole on the biological activity. Instillation of compound 4b resulted in a decrease in ophthalmotonus by 12.93±2.46% relative to the initial values; similar dynamics were not observed in the contralateral eye, which may indicate the absence of the resorptive effect of the test substance.

The third group of the studied compounds are benzimidazolium halides 5-a-e, obtained by quaternization of 1-(2-(azepan-1-yl)ethylbenzimidazoleamine with haloketones. After instillation of bromide solution 5a (R=Ph), there was a decrease in IOP in the test eye by 14.43±8.93%. The inclusion of a methoxy group in the meta-position of phenyl R (compound 5b) resulted in the absence of an ophthalmohypotensive effect, thus, instillation of the test compound had no effect on IOP. At the same time, the replacement of phenyl in the R radical with biphenyl – compound 5d – on the contrary, significantly increased the IOP-lowering activity. Thus, in animals that were instilled with a solution of compound 5d in the test eye, ophthalmotonus decreased by 22.71±10.84%. Instillation of solutions of compounds 5c (R=CF₃) and 5e (R= 4-MeOC6H4NH-) resulted in a decrease in IOP by 10.74±6.43% and 16.48±1.19%, respectively. Among the compounds in this group, only substance 5d showed no effect on the contralateral eye, which may indicate the absence of a systemic effect.

Compound 6 was isolated into a separate group, since the basic structure is imidazobenzimidazole. Cyclization of the inactive benzimidazolium bromide 5b to the tricyclic structure of imidazo[1,2-a]benzimidazole 6, unfortunately, did not contribute to the appearance of ophthalmic hypotensive activity.

The fifth group is represented by derivatives of 1,3-dihydro-2H-benzo[d]imidazole-2-imine, in which the radicals R = 4-fluorobenzyl (7a), phenoxyethyl (7b), methyl biphenylcarboxylate (7c), benzyl (7d) are located at the nitrogen atom N¹, and the radical R¹ = ethylpyrrolidine (7a,b), ethylphenoxy (7c) and 1.1’-biphenylmethyl (7d) are in position N³.

After instillation of 1-(4-flurobenzyl)-3-(2-(pyrrolidin-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazole-2-imine hydrogen chloride (7a) solution, IOP in the test eye was significantly reduced by 28.21±2.56% (p≤0.05, t-test) relative to baseline, which is the highest effect among all the studied substances. Replacing the 4-fluorobenzyl substituent with ethylphenoxy decreases the IOP-lowering activity by 2-fold to 14.47±10.85% in the case of compound 7b. Replacing the pyrrolidinoethyl fragment with methyl biphenylcarboxylate, as in the structure of compound 7c, slightly increases the ophthalmic hypotensive effect to 19.10±8.56%. In compound 7d, the R and R¹radicals are represented by benzyl and 1.1'-biphenylmethyl, respectively. However, this structure has virtually no effect on the strength of the ophthalmic hypotensive effect, and instillation of a solution of this compound reduces IOP by 17.81±9.47%. No effect on the contralateral eye was observed for the studied compounds of this subgroup, which may indicate the absence of a systemic effect.

Thus, following the screening, compounds with laboratory codes 7a and 1d emerged as the most promising, as they reliably reduced IOP, slightly inferior to the reference drugs timolol and melatonin, and did not exhibit a systemic effect. For these compounds, the probable mechanism of action – impact on Rho kinase activity – was assessed (Table 2).

Table 2.

Effect of benzimidazole derivatives at a concentration of 1*10-4 mol/L on the activity of Rho-kinase-2 in the ELISA test in vitro

Test compound

% ROCK-2 inhibition

18.11

26.72

The study revealed that both compounds exhibit inhibitory activity against Rho kinase. Compound 1d inhibited this enzyme by 18.11%, while compound 7a inhibited it by 26.72%.

Conclusion

Thus, based on the results of the studies, newly synthesized benzimidazole derivatives containing fragments of known Rho-kinase-2 inhibitors have proven to be a promising class for the search for new IOP-lowering compounds. According to the results of a study on normotensive outbred rats, with a single instillation at a concentration of 0.4%, the most active compounds are compound 7a, which has higher ophthalmic hypotensive activity than the reference drug timolol, and compound 1d, which is slightly inferior to the reference drug timolol. However, unlike timolol, these compounds do not affect IOP dynamics in the contralateral eye, indicating the absence of an undesirable resorptive effect.

Based on the structural analysis, it can be suggested that the most promising compounds for the development of ophthalmic hypotensive agents are those containing dimethylbenzimidazolimine (compounds 7a-d) and benzimidazole urea (compounds 1a-f) in their structure, as all compounds exhibited IOP-lowering activity. The presence of strong electronegative atoms and heterocyclic structures – hexamethyleneimine and pyrrolidine – in the radicals (compounds 7a and 1d) also leads to increased activity.

Based on the ophthalmic hypotensive activity results and the assessment of the effects of new benzimidazole derivatives, it can be assumed that there is a relationship between these activities: the stronger the inhibitory activity against ROCK, the greater the ophthalmic hypotensive effect.

Additional Information

Conflict of interest

The authors declare no conflict of interest.

Funding

The chemical section was funded by the Ministry of Science and Higher Education of the Russian Federation (state assignment in the field of scientific activity, Southern Federal University, grant FENW-2026-0018).

Ethics statement

The experiments were approved by the Biomedical Ethics Committee of Volgograd State Medical University (extract from minutes № 2022/043 dated December 2, 2022).

Acknowledgments

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

Data Availability

All data supporting the results of this study are available in the main text.

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Author Contributions

§ Olga N. Zhukovskaya, PhD in Chemical Sciences, Senior researcher at the Laboratory of Organic Synthesis, Institute of Physical and Organic Chemistry, Southern Federal University, Rostov-on-Don, Russia; e-mail: zhukowskaia.ol@yandex.ru; ORCID ID: http://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.

§ 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; ОRCID 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.

§ Lyudmila V. Naumenko, Doctor Habil. of Medical Sciences, Associate Professor, Professor of the Department 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.

§ Umida M. Ibragimovа, Assistant of the Department of Pharmacology and Bioinformatics, Volgograd State Medical University of the Ministry of Health of Russia, Volgograd, Russia; e-mail: iumida@list.ru; ORCID ID: https://orcid.org/0000-0001-9141-4417. The author took part in conducting experimental work and analyzing the material.

§ Anatoly S. Morkovnik, Doctor Habil. of Chemical Sciences, Head of the Laboratory of Organic Synthesis, Institute of Physical and Organic Chemistry, Southern Federal University, 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, Volgograd, Russia; e-mail: aspasov@mail.ru; ORCID ID: http://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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