Acetylphenyl-Substituted Imidazolium Salts: Synthesis, Characterization, in silico Studies and Inhibitory Properties against Some Metabolic Enzymes

doi:10.21203/rs.3.rs-2161155/v1

Download PDF

Research Article

Acetylphenyl-Substituted Imidazolium Salts: Synthesis, Characterization, in silico Studies and Inhibitory Properties against Some Metabolic Enzymes

https://doi.org/10.21203/rs.3.rs-2161155/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Herein, we present how to thirteen new synthesize 1-(4-acetylphenyl)-3-alkylimidazolium salts by reacting 4-(1-H-imidazol-1-yl)acetophenone with a variety of benzyl halides that contain either electron-donating or electron-withdrawing groups. The structures of the new imidazolium salts were conformed using different spectroscopic method (¹H NMR, ¹³C NMR, ¹⁹F NMR and FTIR) and elemental analysis techniques. Furthermore, the carbonic anhydrase and acetylcholinesterase enzyme inhibition activities of these compounds were investigated. They showed highly potent inhibition effect toward acetylcholinesterase (AChE) and carbonic anhydrases (hCAs) with K_i values in the range of 8.30±1.71 to 120.77±8.61 nM for AChE, 16.97±2.04 to 84.45±13.78 nM for hCA I, and 14.09±2.99 to 69.33±17.35 nM for hCA II, respectively. Most of the synthesized imidazolium salts were appeared to be more potent than the standard inhibitor of tacrine (TAC) against AChE, and Acetazolamide (AZA) against CA. In the meantime, to prospect for potential synthesized imidazolium salt inhibitor(s) against acetylcholinesterase (AChE) and carbonic anhydrases (hCAs), molecular docking and ADMET-based approach was exerted.

Acetylcholinesterase

Acetyl group

ADMET

Carbonic anhydrase

Imidazolium salt

Molecular docking

Since the discovery of N-heterocyclic carbene (NHC) by Arduengo et al three decades ago, in the field of organic and organometallic chemistry has developed significantly due to the extraordinary uses of NHC ligands [1, 2]. NHCs could form more stable complexes with the majority of transition metals than heterocycles with P, O, and S atoms Furthermore; the metal-carbene bond is more reactive than the metal-phosphine bond in other metal complexes such as phosphine complexes [3]. Both σ-bonds with inductive effects and π-bonds with resonance effects influence the stability of NHCs. These ligands have been identified as promising candidates for biological activities and various oxidation states because they are better σ-donating ligands than known trialkyl phosphines [4, 5].

Imidazoles and benzimidazoles are important N-heterocyclic compounds because of their interesting framework. These compounds are mostly found in alkaloids and have biological activities such as antimicrobial [6, 7], antifungal [8], antiviral [9], anti-inflammatory [10], antitumor [11], and anticancer properties [12]. Several of these heterocyclic compounds, particularly those with fused tricyclic or bicyclic rings, have important medical applications [13–16]. Furthermore, these salts have found quite important application fields across a wide range of chemical reactions. They have been used as curing agents for epoxy resins [17], for example, and to obtain chiral zwitterionic carbene precursors [18]. While imidazolium salts are important biologically, they have also found widespread use in organic synthesis as ionic liquids that can be used as solvents or electrolytes in green chemistry [10] Imidazolium salts are bioactive azole-based compounds derived from imidazole. Imidazoles, as ligands, could bond with metals or form hydrogen bonds with drugs and proteins [20, 21]. Furthermore, imidazolium salts can interact electrostatically with biological molecules [22, 23].

Drug-like compounds that are successful in terms of chemical characteristics and biological activities will be in high demand in today's modern pharmaceutical industry [24, 25]. Recent research has revealed that it is critical to impart certain structural features to fluorine-containing molecules [26]. Nearly 300 fluorine-containing medications have been approved and are in use around the world as a result of the improvements [27]. Furthermore, half of the fluorine-containing drugs are blockbusters [28]. In organic chemistry, the acetyl group is a acyl group with the chemical formula CH₃CO and the symbol acyl or Ac for short (not to be confused with the element actinium) [29]. Many biologically active chemicals, such as acetylcysteine, acetyl-CoA, acetaminophen (also known as paracetamol), acetylsalicylic acid (commonly known as aspirin), acetyl-L-carnitine, and the neurotransmitter acetylcholine have an acetyl group in their structure. In organisms, acetyl groups are frequently transferred from acetyl-CoA to other organic molecules. Acetyl-CoA serves as an intermediate in both biological synthetase and the degradation of many organic molecules. Acetyl-CoA is also formed by the action of pyruvate dehydrogenase on pyruvic acid during the second step of cellular respiration, pyruvate decarboxylation. Acetylation is a common method of modifying histones and other proteins. On the DNA level, for example, histone acetylation by acetyltransferases causes chromatin architecture to expand, allowing genetic transcription to occur. However, acetyl group removal by histone deacetylases condenses DNA structure, preventing transcription [30]. Furthermore, there is some evidence that acetyl-L-carnitine, which contains the acetyl group, may be more effective than L-carnitine in some applications [31]. Resveratrol acetylation holds promise as one of the first anti-radiation drugs for human populations [32].

Carbonic anhydrases (CAs) are a superfamily of metalloenzymes and metabolize acid-base concentrations and they are also play a role fluid balance in parts of the body such as the eyes, kidneys, and stomach [33, 34]. CA isoenzymes are CA is the second most plentiful protein after hemoglobin in erythrocytes and effectively interconnect and carbon dioxide (CO₂) and water to bicarbonate (HCO₃⁻) and protons (H⁺) [35, 36]. It is well-known CO₂, HCO₃⁻ and H⁺ are essential molecules and ions in moat of crucial physiologic processes in life kingdoms. The family of human CA (hCAs) is divided into many isoforms, depending on the location of origin such as saliva, mitochondria and cytosol, etc [37–39]. The discovery and characterization of human CA isoforms, of which 16 are known today, has led to many new applications of inhibitors. They are widely used clinically as antiepileptic, anticonvulsant, antiglaucoma, and antiobesity agents, antitumor drugs in clinical development, as well as for the treatment of diseases such as idiopathic intracranial hypertension and acute mountain sickness [40–42]. Also, inhibition of CA isoenzymes has been fully elucidated. The inhibition of most classical inhibitory occurs by binding to the metal center in the active site of enzymes [43, 44]. Especially, increased levels of several isoforms of hCAs have been linked to variety of diseases such as obesity, glaucoma and cancer [45, 46]. CA inhibitors (CAIs) are used clinically for many years as antiepilepsy, antimetastatic, antiglaucoma, diuretics, and antitumor. To that end, development of new CAIs has been extremely important [47, 48].

The cholinergic hypothesis is known that the degeneration of cholinergic neurons and the consequent loss of cholinergic neurotransmission in the cerebral cortex are responsible for the deterioration of cognitive function observed in the brain of AD patients [49–51]. In the cholinergic hypothesis, acetylcholine (ACh) is a key neurotransmitter and it is decreased both in function and concentration in brains with AD [52, 53]. It should be noted that many other symptoms such as cognitive impairment, depression and psychosis occur simultaneously with AD. In the AD brains, ACh is hydrolyzed by acetylcholinesterase (AChE), which regulates the amount of ACh in the body [54, 55]. Regarding the hydrolysis of ACh to choline and acetate by AChE, the concentration of this neurotransmitter in the central nervous system is insufficient to keep the brain functioning properly [56, 57]. AChE inhibitors are a type of drug used to treat AD. These drugs, on the other hand, do not stop the disease from progressing; they just hide the symptoms. Some AChE inhibitors including Tacrine as reversible and potent AChE inhibitor have been used in the treatment of AD [58, 59]. There is evidence to suggest that tacrine has additional properties as well as an effect on the reduction of amyloid-β peptide-induced apoptosis in cortical neurons [60]. Despite its inhibitory power, it has been withdrawn from use due to its toxicity and side effects such as hepatotoxicity and gastrointestinal discomfort [61, 62]. While clinical treatment has a variety of adverse effects, these medications can effectively reduce the symptoms of AD, for example, transaminase elevation, nausea, emesis and flatulence [63, 64]. Hence, there is an urgent need for developing potent AChE inhibitors to treat AD without adverse effects.

Recently, there are few studies on the enzyme inhibition activities of imidazolium salts in the literature [65]. Our workgroup recently described the synthesis, characterization, and enzyme inhibition activities on some metabolic enzymes of various alkyl/aryl-substituted benzimidazolium salts [66, 67]. The new imidazolium salts had an acetophenyl substituent on one nitrogen atom and an alkyl (bearing trifluoromethyl)/aryl group on the other. In this study, 1-(4-acetylphenyl)-3-alkylimidazolium salts (2a-g, 3a-f) containing electron-withdrawing and electron-donating groups were synthesized and its structure was elucidated by spectroscopic methods. The inhibitory effects of 4′-(imidazol-1-yl)acetophenone (1) and new 1-(4-acetylphenyl)-3-alkylimidazolium salts (2a-g, 3a-f) against acetylcholinesterase and carbonic anhydrase as cholinergic enzymes were investigated. Also, their inhibition profiles and molecular docking were compared to the standard compounds like Tacrine and Acetazolamide as clinical inhibitors. Many computational approaches have been developed to research and develop robust for different targets; It has been widely applied in modern drug discovery, including virtual scanning, molecular docking, in silico absorption, distribution, metabolism, excretion and toxicity (ADMET) and so on. In addition, in this study, by performing computational studies on the basis of the experimental data, the potential first three synthesized imidazolium salts to provide the elucidation and support of the interaction mechanisms with the target enzymes (hCA-I, hCA II and AChE).

Synthesis

This work presents the synthesis method for unsymmetrically 1-acetyl-3-alkyl imidazolium salts. 4-(1-H-Imidazol-1-yl)acetophenone was reacted with benzyl halides containing electron donating groups such as methyl and methoxy and electron withdrawing groups such as trifluoromethyl, fluorine, chlorine in acetonitrile at 80°C for 18 hours (Scheme 1 and Scheme 2). New 1-acetyl-3-alkylimidazolium salts were obtained in yields of 56–89%. The 1-acetylimidazolium salts (2a-g and 3a-g), which are stable to air and moisture, were soluble in polar organic solvents such as ethyl alcohol, dimethylformamide, water, and dimethylsulfoxide. However, imidazolium salts were also soluble in halogenated solvents such as dichloromethane and chloroform. But, none of these salts are soluble in polar organic solvents such as diethyl ether and nonpolar organic solvents such as pentane, hexane, and toluene. The chemical structures of all synthesized compounds were confirmed by using ¹H NMR, ¹³C NMR, ¹⁹F NMR, and FTIR spectroscopic methods and elemental analysis techniques. The observation of characteristic acidic proton signals at the second carbon (2-CH) for the imidazole ring between 11.12 and 11.84 ppm in ¹H NMR spectra indicated the formation of imidazolium salts (2a-g and 3a-g). While characteristic ethylene (-CH = CH-) peaks for the imidazole ring were observed as doublet peaks in compounds 2d, 3a, and 3b, other compounds were observed as singlets. However, aromatic protons directly attached to the nitrogen atom in the imidazolium ring appeared between 6.93 and 8.13 ppm as doublets. The characteristic peaks of the benzyl group attached to the nitrogen atom of the imidazolium ring were observed between 5.62 and 5.92 ppm as singlets. Protons of the methyl and methoxy groups at the ortho, meta and para positions on benzene gave sharp singlets between 2.17 and 3.82 ppm. In addition, the methyl protons of the acetyl group gave sharp singlets between 2.51 and 2.57 ppm. When the ¹³C NMR spectra of all imidazolium salts were examined, the observation of characteristic salt peaks at the second carbon (2-CH) for the imidazole ring between 137.8 and 139.8 ppm in ¹³C NMR spectra indicated the formation of imidazolium salts. The peaks of the carbonyl carbons in the 4-acetylphenyl group were observed between 196.2 and 196.5 ppm. The peaks of benzylic carbons were observed between 49.9 and 54.1 ppm. Peaks of methyl carbon in the 4-acetylphenyl group were observed between 26.4 and 26.8 ppm. The peaks of the methyl carbon in the ortho, meta and para positions of the benzene attached to the nitrogen atom of the imidazolium ring were observed between 19.4 and 21.3 ppm, while the methoxy carbons in the 2g compound were observed between 56.7 and 60.8 ppm. The carbon peaks of the CF₃ group in 3a, 3b and 3c compounds were found at 126.6, 126.2, and 126.4 ppm, respectively. The carbon peak of the -OCF₃ group in compound 3d was observed at 150.1 ppm. In compound 3e, the benzene carbon peak to which the F atom is attached was observed at 162.1 and 164.6 ppm. In the 3f compound, the peak of the carbon to which the F atom is attached was observed at 164.6 ppm, while the peak of the carbon to which the Cl atom is attached was observed at 162.0 ppm. When the ¹⁹F NMR spectra of imidazolium salts (3a-f) containing fluorine, trifluoromethyl and trifluoromethoxy groups were examined, fluorine peaks were observed as singlet. The fluorine peaks of the 3a, 3b, 3c, and 3d compounds containing the CF₃ group in the ortho, meta and para position of the benzene ring were observed at -58.38, -60.97, -61.97, and − 56.79 ppm, respectively. Fluorine peaks of 3a and 3e compounds containing F atom in the para position of the benzene ring were observed at -112.96 and − 110.06 ppm, respectively. The FTIR spectral data of all imidazolium salts revealed a characteristic ν(C–N) band 1552, 1552, 1546, 1552, 1545, 1556, 1556, 1554, 1552, 1552, 1553, 1552, and 1555 cm − 1, respectively for 3a-f. When the elemental analysis results were examined, it was seen that the values found were very close to the calculated values. Elemental analysis results and all spectroscopic data are compatible with the literature [68–70].

Enzyme Inhibition Results

The 1-(4-acetylphenyl)imidazole (1) and acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) were researched in vitro for their ability to inhibit hCA I, hCA II and AChE enzymes. Acetazolamide (AZA) and Tacrine (TAC) were used as a positive control these enzymes. The inhibitory assay results are presented in Table 1. When the results are examined, the following structure-activity relationship can be easily conveyed:

hCA I isoenzyme is expressed in erythrocytes and physiologically linked to retina and cerebral edema. On the other hand, physiologically dominant CA II isoform is expressed in almost all tissues primarily including testis, erythrocytes, bone osteoclasts, eye, brain, lung, gastrointestinal tract and kidney [71]. However, CA inhibitors (CAIs) have been related to various diseases such as cancer, glaucoma and obesity [72]. In the current study, all the novel synthesized a series of acetylphenyl-substituted imidazolium salts (2a-g and 3a-f) effectively inhibited hCA I with IC₅₀ values ranging from 21.66 to 88.76 nM and K_i values ranging from 16.97 ± 2.04 to 84.45 ± 13.78 nM. In this group, compound 3f, 1-(4-acetylphenyl)-3-(2-chloro-4-fluorobenzyl)imidazolium chloride, showed the best inhibition (K_i: 16.97 ± 2.04 nM). In addition, the enhanced effect of halogen atoms on the CA inhibition activity was reported. It is well-known that the compound possessed bromide group demonstrated efficient CA inhibition effects [73]. The inhibition effects of studied compounds (1, 2a-g, 3a-f) against hCA I were decreased in the following order: 3f (K_i: 16.97 ± 2.04 nM) > 3c (K_i: 19.56 ± 6.55 nM) > 2c (K_i: 30.19 ± 6.66 nM) > 2g (K_i: 35.54 ± 1.56 nM) > 3a (K_i: 38.97 ± 7.23 nM) > 3d (K_i: 40.41 ± 9.58 nM) > 2e (K_i: 41.53 ± 8.32 nM) > 2a (K_i: 47.24 ± 5.02 nM) > 3e (K_i: 52.21 ± 6.38 nM) > 1 (K_i: 57.19 ± 11.62 nM) > 3b (K_i: 60.67 ± 4.59 nM) > 2f (K_i: 64.84 ± 7.35 nM) > 2d (K_i: 84.45 ± 13.78 nM) > 2b (K_i: 94.45 ± 5.47 nM) (Table 1). On the other hand, Acetazolamide, which employed as a reference CA inhibitor demonstrated Ki value of 82.58 ± 3.13 nM against cytosolic hCA I isoform. In addition, acetylphenyl-substituted imidazolium salts except of 2b (K_i: 94.45 ± 5.47 nM) and 2d (K_i: 84.45 ± 13.78 nM) outperformed the reference clinical drug AZA (K_i: 63.44 ± 5.32 nM). For hCA I, the positions of the methyl group attached to the benzyl structure caused changes in the inhibition value. The effect of the methyl attached to the benzyl group on the inhibition position is in the form of 2nd position > 4th position > 3rd > position. The fact that methoxy groups are attached to the benzyl structure caused it to exhibit more effective inhibition than methyl groups (2g: 35.54 ± 1.56 nM). The addition of trifluoro groups to the structure of the methyl groups attached to the benzyl group caused an increase in the inhibition value, except that the methyl group in the 3rd position was attached.

Some CA isoenzymes have been reported to lead to the development of some pathological conditions. For example, CA I and CA II isoforms are known to contribute to the pathogenesis and development of vision loss in patients with diabetes and glaucoma. In addition, expression of CA I and II isoforms has been reported in different types of hematological cell lines, colon and rectal malignancies [74–76]. The CA II isoform was the most susceptible to inhibition by the reported 1-(4-acetylphenyl)imidazole (1) and acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) K_is was in the range of 14.09 ± 2.99 to 69.33 ± 17.35 nM. According to inhibition data, 1-(4-acetylphenyl)imidazole (1) and acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) effectively inhibited hCA II with IC₅₀ values ranging from 18.13 to 78.54 nM. Dominant cytosolic CA II was potently inhibited in the low nanomolar range and compound 2b, 1-(4-acetylphenyl)-3-benzylimidazolium chloride showed the best inhibition (K_i: 14.09 ± 2.99 nM). In addition, acetylphenyl-substituted imidazolium salts except of 2d K_i: 69.33 ± 17.35 nM) outperformed the reference clinical drug AZA (K_i: 63.44 ± 5.32 nM). Similar to hCA I, the effect of the methyl attached to the benzyl group on the inhibition position is in the form of 2nd position > 4th position > 3rd > position. In contrast to hCA I, the 3rd position exhibited more effective inhibition of trifluoromethyl benzyl-linked compounds. In addition, the methyl group was more effective than methoxy the group (3c: K_i: 28.57 ± 4.01 nM, 3d: K_i: 42.81 ± 9.56 nM). Similar results are observed in hCA I.

AD is a progressive and chronic neurodegenerative brain disease in elderly people [77]. Today, the mechanism underlying the etiology of AD is still not fully elucidated due to its complex and multifactorial nature. However, acetylcholine (ACh) deficiency is thought to have a crucial role in the onset and progression of AD [78, 79]. Therefore, the inhibition of AChE would be beneficial way to the treatment of AD. Finally, 1-(4-acetylphenyl)imidazole (1) and the acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) were tested against cholinergic AChE enzyme. Synthesized compounds effectively inhibited AChE with IC₅₀ values ranging from 10.34 to 103.12 nM and K_i values ranging from 8.30 ± 1.71 to 120.77 ± 8.61 nM. The inhibitor effects of studied compounds (1, 2a-g, 3a-f) against AChE were decreased in the following order: 3f > (K_i: 8.30 ± 1.71 nM) 3d> (K_i: 12.11 ± 2.17 nM) 3e > (K_i: 16.27 ± 1.81 nM) 3c > (K_i: 19.50 ± 4.24 nM) 2g > (K_i: 20.09 ± 2.23 nM) 3b> (K_i: 23.22 ± 3.97 nM) 3a > (K_i: 27.13 ± 0.53 nM) 2f > (K_i: 29.75 ± 5.45 nM) 2e > (K_i: 31.96 ± 6.67 nM) 2b > (K_i: 52.81 ± 9.12 nM) 1 > (K_i: 91.13 ± 2.75 nM) 2c > (K_i: 101.84 ± 6.54 nM) 2a > (K_i: 104.94 ± 9.62 nM) 2d > (K_i: 120.77 ± 8.61 nM) (Table 1). According to our inhibition data, acetylphenyl-substituted imidazolium salts except of 2d (K_i: 120.77 ± 8.61 nM) outperformed the reference clinical standard of Tacrine (K_i: 106.45 ± 6.78 nM). The exchange of substituents demonstrated a substantial influence on the inhibitory effect of the compounds. For instance, the inhibition strength was greatly affected by the addition of the trifluoromethyl group instead of the methyl group. Looking at the position of the groups of the trifluoromethyl group, the inhibitor effects of studied compounds were decreased in the following order: 4th position > 3rd > position > 2nd position. The chlorine binding at position 2 to the 4-fluorobenzyl structure was almost twice as effective in inhibition (3f: K_i: 8.30 ± 1.71 nM).

Table 1

Inhibition data of novel synthesized acetylphenyl-substituted imidazolium salts (2a-g and 3a-f) against AChE, hCA I and hCA II

Compounds

IC₅₀ (nM)

K_i (nM)

hCA I

r²

hCA II

r²

AChE

r²

hCA I

hCA II

AChE

65.33

0.9812

69.11

0.9811

85.15

0.9813

57.19 ± 11.62

56.55 ± 7.24

91.13 ± 2.75

35.76

0.9721

40.56

0.9789

93.12

0.9809

47.24 ± 5.02

51.18 ± 12.49

104.94 ± 9.62

72.23

0.9813

21.00

0.9799

63.13

0.9878

94.45 ± 5.47

14.09 ± 2.99

52.81 ± 9.12

29.12

0.9765

23.10

0.9809

89.98

0.9878

30.19 ± 6.66

19.44 ± 4.80

101.84 ± 6.54

88.76

0.9843

78.54

0.9899

103.12

0.9898

84.45 ± 13.78

69.33 ± 17.35

120.77 ± 8.61

46.20

0.9896

57.21

0.9789

27.72

0.9943

41.53 ± 8.32

62.95 ± 9.56

31.96 ± 6.67

71.12

0.9813

28.44

0.9959

26.32

0.9911

64.84 ± 7.35

21.96 ± 5.03

29.75 ± 5.45

34.65

0.9956

40.56

0.9807

18.13

0.9865

35.54 ± 1.56

34.29 ± 7.62

20.09 ± 2.23

49.16

0.9823

60.11

0.9754

36.13

0.9855

38.97 ± 7.23

54.53 ± 14.22

27.13 ± 0.53

50.32

0.9745

18.13

0.9832

27.92

0.9911

60.67 ± 4.59

12.52 ± 2.54

23.22 ± 3.97

22.45

0.9834

32.13

0.9943

17.72

0.9856

19.56 ± 6.55

28.57 ± 4.01

19.50 ± 4.24

49.50

0.9891

40.76

0.9812

19.80

0.9799

40.41 ± 9.58

42.81 ± 9.56

12.11 ± 2.17

53.31

0.9941

23.90

0.9848

12.83

0.9767

52.21 ± 6.38

15.34 ± 4.05

16.27 ± 1.81

21.66

0.9856

30.13

0.9723

10.34

0.9911

16.97 ± 2.04

23.89 ± 5.84

8.30 ± 1.71

AZA*

70.13

0.9745

49.45

0.9818

82.58 ± 3.13

63.44 ± 5.32

TAC**

120.13

0.9932

106.45 ± 6.78

*Acetazolamide (AZA) was used as positive control for both hCA I and II isoforms.

*Tacrine (TAC) was used as positive control for acetylcholinesterase.

In Silico Studies

Our in silico study consists of two stages as molecular docking and ADMET analysis. All the computational operations performed in this section are to explain the relationship between the three compounds that exhibit the highest activity against the three target enzymes discussed as a result of in vitro analyzes, both in terms of structural and biological activity. The binding energies (Table 2) and binding types (Table S1) obtained as a result of the calculations were compared with the biological results and evaluated.

Table 2

Free energy of binding energy values for first and second group compounds by using molecular docking studies
FIRST GROUP COMPOUNDS
hCA I	Free Energy of Binding (kcal/mol)
2c	-6.83
2g	-6.72
2e	-6.65
Acetazolamide (AZA)	-6.14
hCA II	Free Energy of Binding (kcal/mol)
2b	-7.73
2c	-7.03
2f	-6.68
Acetazolamide (AZA)	-6.56
AChE	Free Energy of Binding (kcal/mol)
2g	-10.38
2f	-9.39
2e	-9.19
Tacrine(TAC)	-7.37
SECOND GROUP COMPOUNDS
hCA I	Free Energy of Binding (kcal/mol)
3f	-6.81
3c	-6.47
3a	-6.36
Acetazolamide (AZA)	-6.14
hCA II	Free Energy of Binding (kcal/mol)
3b	-6.81
3e	-6.47
3f	-6.46
Acetazolamide (AZA)	-6.56
AChE	Free Energy of Binding (kcal/mol)
3f	-9.29
3d	-8.97
3e	-8.74
Tacrine(TAC)	-7.37

First of all, compounds 2c, 2g and 2e, which show high enzyme inhibition tendency against hCA-I target, were investigated within the scope of molecular docking study. Then, the status of possible compounds showing activity of compounds 2b, 2c and 2f for hCA-II and compounds 2g, 2f and 2e against AChE enzyme, respectively, will be evaluated. Similarly, a second group (imidazolium bromide salts) of compounds with the same targets, compounds 3f, 3c and 3a for hCA-I, compounds 3b, 3e and 3f for hCA-II, and compounds 3f, 3d and 3e towards the AChE target will be examined in terms of their visual orientation and interaction.

As shown in Fig. 1, with the binding energy value of -6.83 kcal/mol, compound 2c shows the best binding affinity with the target enzyme hCA-I. Then, with the energy value of -6.72 kcal/mol, the compound 2g and finally − 6.65 kcal/mol, compound 2e tend to interact with the hCA-I. Considering these conditions at the atomic level, evaluations were made on the basis of the Acetazolamide (AZA) status of the target enzyme, which was accepted as the control compound. Compared to the hydrogen bonding (His200, Thr199, and Gln92), π-sulfur (His96) and hydrophobic interactions (His94, Ala121 and Leu198) with the current target, of the control compound in Figure S1; it is seen that 2c, 2g and 2e compounds contribute to their biological activities by forming electrostatic interactions as well as other non-bonding interactions. They also have a salt forms and their surface area is larger than the control compound.

In Fig. 2, the biological activity of the first three potential compounds 2b, 2c and 2f against hCA-II was examined by molecular docking study. According to the findings, compound 2b shows the best binding affinity with the target enzyme with a binding energy of -7.73 kcal/mol. Then, with the binding energy value of -7.03 kcal/mol, the second compound 2c and finally the compound 2f showed a tendency to bind with the target enzyme to -6.68 kcal/mol. When this situation is compared with the control compound AZA, it is seen that it is biologically active with the hCA-II enzyme in a situation where hydrophobic and hydrophobic interactions take place at certain rates compared to hydrophilic interactions.

When we computationally evaluate the first three compounds 2g, 2f and 2e, which show the best inhibition tendency against the AChE target in Fig. 3; the compound 2g creates the best biological activity with its binding energy value of -10.38 kcal/mol. target enzyme. Because the compound 2g makes hydrogen bonds with Gly122, Ser203, Ser293, His447, Glu292 and Tyr124, and hydrophobic interactions with Phe338, Phe297, Trp86, Trp236, Phe295, Phe297, Tyr337 and His447 residues of the target enzyme.

In addition, the same target protein models and 2nd group compounds were investigated by molecular docking method. The poses and three-dimensional interactions of the hCA-I, hCA-II and AChE enzymes and the potential first three 2nd group compounds in the active site of each enzyme, respectively, are shown in Figs. 4–6.

In Fig. 4, compounds showing activity against the hCA-I enzyme are 3f, 3c and 3a. According to the calculation results, the binding energy values of the related compounds were − 6.81 kcal/mol, -6.47 kcal/mol and − 6.36 kcal/mol, respectively. Compound 3f forms hydrogen bonds with Thr199, His67, Leu198, and His200; and also hydrophobic interactions with His200, Leu198, and Pro202 amino acids in target enzyme.

The orientation of 3f compound in the binding site of the target enzyme, and the presence of fluorine and chlorine atoms in the ortho and para positions of the phenyl ring in the structure of 3f compound against the control compound AZA and the other two potent compounds 3c and 3a increased its biological efficiency. This state is revealed in Fig. 4 and details were given in Table S1.

On the other hand, we evaluated the orientation and interactions in the visual active region for the first three compounds 3b, 3e and 3f, which showed the best interaction with the hCA-II enzyme among the 2nd group compounds. According to these findings, compound 3b stands out as the most effective structure by forming hydrogen bonds with Asn67, halogen bonds with Glu69, Ile91, hydrophobic interactions with Phe131, Ile91, Val121 and Leu198 as given in Fig. 5.

Finally, the most active compound against the AChE target is 3f. Then, compounds 3d and 3e, respectively, exhibit the best inhibition behavior with the respective target. Considering this situation at the atomic level, it appears that fluorine and chlorine atoms in compound 3f have a more dominant biological activity than trifluoromethoxy in compound 3d and fluorine atoms in compound 3e, as seen in Fig. 6.

As a result of molecular calculations, it is seen that the 1st group compounds show a better binding tendency with three targets than the 2nd group compounds. Especially the 1st groups and 2nd groups show the best binding with the AChE enzyme, which is supported by both their binding energies and their three-dimensional orientation and interactions.

Besides docking studies, in silico ADMET analyzes of both groups of compounds were evaluated separately, and their properties in biological systems were analyzed by estimating their pharmacokinetic properties as well as their pharmacodynamics interactions. These are given in Fig. 7 for both group compounds and details data are tabulated in Table 3 and Table 4.

Table 3

Druglikeness properties of the first group and the second group potential compounds and control compounds (Acetazolamide for hCA-I and hCA-II; Tacrine for AChE) based on the Lipinski and Veber rules
Name	MW (≤ 500 g/mol)	AlogP(≤ 5)	MPSA(≤ 140 A²)	NRB(≤ 10)	HA_Lipinski (≤ 10)	HD_Lipinski (≤ 5)	HA_Veber (≤ 12)	HD_Veber (≤ 12)
2f	382.926	3.923	64.36	4	3	0	1	0
2b	312.793	1.492	64.36	4	3	0	1	0
2c	326.82	1.978	64.36	4	3	0	1	0
2e	326.82	1.978	64.36	4	3	0	1	0
2g	402.871	1.442	92.05	7	6	0	4	0
Acetazolamide	222.245	-1.329	151.66	2	7	3	5	2
Tacrine	198.264	2.79	38.9	0	2	2	2	1

Name	MW (≤ 500 g/mol)	AlogP(≤ 5)	MPSA(≤ 140 A²)	NRB(≤ 10)	HA_Lipinski (≤ 10)	HD_Lipinski (≤ 5)	HA_Veber (≤ 12)	HD_Veber (≤ 12)
3f	411.696	2.095	76.14	4	3	0	1	0
3d	441.242	3.867	85.37	6	4	0	2	0
3c	425.242	2.690	76.14	5	3	0	1	0
3a	425.242	2.690	76.14	5	3	0	1	0
3b	425.242	2.690	76.14	5	3	0	1	0
3e	375.235	1.953	76.14	4	3	0	1	0
AZA	222.245	-1.329	151.66	2	7	3	5	2
Tacrine	198.264	2.790	38.90	0	2	2	2	1
[MW, molecular weight; ALogP, octanol/water partition coefficient, a measure for lipophilicity; MPSA, molecular polar surface area; nRB, Number of Rotatable bonds; Num_H_Acceptors_Lipinski, Number of hydrogen bond acceptors; Num_H_Donors_Lipinski, Number of hydrogen bond donors; Num_H_Acceptors, Number of hydrogen bond acceptors based on Veber; Num_H_Donors, Number of hydrogen bond donors based on Veber.]

Table 4

ADMET analysis of the first group and the second group potential compounds and control compounds (Acetazolamide for hCA-I and hCA-II; Tacrine for AChE)
Comp.	PSA_2D(< 140 Å²)	AlogP98(< 5)	HIA	Solubility	BBB	CYP2D6	Hepatotoxic	PPB
2f	27.997	5.356	0(good)	1(very low but possible)	0(very high)	false	true	true
2b	27.997	2.925	0(good)	3(good)	1(high)	false	false	true
2c	27.997	3.411	0(good)	2(low)	1(high)	false	false	true
2e	27.997	3.411	0(good)	2(low)	1(high)	false	false	true
2g	54.787	2.876	0(good)	3(good)	2(medium)	false	true	true
Acetazolamide	113.774	-0.579	0(good)	4(optimal)	4(undefined)	false	true	false
Tacrine	37.800	2.790	0(good)	2(low)	1(high)	false	true	false

Comp.	PSA_2D(< 140 Å²)	AlogP98(< 5)	HIA	Solubility	BBB	CYP2D6	Hepatotoxic	PPB
3f	27.997	3.273	0(good)	2(low)	1(high)	true	true	true
3d	36.927	5.045	0(good)	2(low)	0(very high)	false	true	true
3c	27.997	3.867	0(good)	2(low)	1(high)	true	false	true
3a	27.997	3.867	0(good)	2(low)	1(high)	true	false	true
3b	27.997	3.867	0(good)	2(low)	1(high)	true	false	true
3e	27.997	3.130	0(good)	2(low)	1(high)	false	true	true
AZA	113.774	-0.579	0(good)	4(optimal)	4(undefined)	false	true	false
Tacrine	37.800	2.790	0(good)	2(low)	1(high)	false	true	false
[PSA_2D, 2D polar surface area; AlogP98, the logarithm of the partition coefficient between n-octanol and water; HIA: Human intestinal absorption; BBB blood brain barrier; CYP2D6 cytochrome P450 2D6 binding, false: non-inhibitor; for CYP2D6; false: non-toxic for Hepatotoxic; PPB plasma protein binding, more than 90% for PPB value is true: Chemicals strongly bound. Less than 90% for PPB value is false: Chemicals weakly bound.]

As a result of the in vitro analyzes of the 1st and 2nd group compounds, the drug similarity and ADMET analysis results of the first three active compounds according to the activity results against three types of enzymes are within the desired limits and are seen as potential drug candidates for future studies.

This work includes the synthesis of two new series of acetylphenyl substituted imidazolium salts (2a-g, 3a-f). All synthesized compounds have been characterized using ¹H NMR, ¹⁹F NMR, ¹³C NMR, and FTIR spectroscopy techniques. We think that 1-(4-acetylphenyl)imidazole (1) and acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) were identified as potent AChE and both CA isoenzymes inhibitors. In this study, low nanomolar level of K_i values were observed for each acetylphenyl-substituted compounds (1, 2a-g, 3a-f) and these compounds can be selective inhibitor of both cytosolic CA isoenzymes and AChE enzyme and can be used as potent medicals for treatment of Alzheimer’s disease, glaucoma, intracranial hypertension, idiopathic epileptic seizure, altitude sickness, cystinuria, central sleep apnea, periodic paralysis, and dural ectasia after some further investigations. In molecular docking and ADMET applications, imidazolium chloride salts containing a methyl (2c) or methoxy (2g) substituted benzyl group in the 1st group compounds; and among the second group compounds, imidazolium bromide salts containing a trifluoromethyl (3b) or fluorinated and chlorinated (3f) benzyl group appear to be favored against three target enzymes.

Material and equipment

The synthesis of 1-(4-acetylphenyl)-3-alkylimidazolium salts (2a-g, 3a-f) was carried out in flame-dried glassware under an inert atmosphere using standard Schlenk techniques. Acetylcholinesterase from the electric eel, 5,5’-dithiobis-(2-nitrobenzoic) acid, acetylthiocholine iodide and tacrine were purchased from Sigma Aldrich. The solvents commercially purchased were used without exposure to any purification and drying process. All other reagents were commercially available by Merck, Alfa Aesar, Carlo Erba, and Sigma-Aldrich Chemical Co. and used without further purification. Melting points were identified in glass capillaries under air with an Electrothermal-9200 melting point apparatus. FT-IR spectra were saved in the range 400–4000 cm^− 1 on Perkin Elmer Spectrum 100 FT-IR spectrometer. Carbon (¹³C), Proton (¹H), and Fluorine (¹⁹F) NMR spectra were recorded using either a Bruker Avance III 400 MHz NMR spectrometer operating at NMR (400 MHz for ¹H, 100 MHz for ¹³C NMR, and 376 MHz for ¹⁹F NMR, respectively) in the CDCl₃ with tetramethylsilane (TMS) as an internal reference. All spectroscopic data in this study were performed by Inonu University Scientific and Technology Centre (Malatya, TÜRKİYE).

Synthesis

Synthesis of 1-(4-acetylphenyl)-3-ethylimidazolium bromide (2a)

The method described in the literature was used to synthesize compound 2a. 1-(4-acetylphenyl)imidazole (745 mg, 4 mmol) and ethyl bromide (436 mg, 4 mmol) were dissolved in acetonitrile (4 mL) and stirred for 24 hours at 80°C. The white solid that formed at the end of the reaction was filtered off, the solvent was removed, and the product was washed with diethyl ether. The crude product was crystallized from a mixture of ethyl alcohol/diethyl ether (1:3) [80]. Yield: 67% (791 mg). m.p.: 220–221°C; ν_(C=O): 1678 cm^− 1; ν_(CN): 1551 cm^− 1. Anal. calc. for C₁₃H₁₅BrN₂O: C: 52.90; H: 5.12; N: 9.49. Found: C: 52.91; H: 5.14; N: 9.51. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 1.63 (t, 3H, J: 7.3 Hz, -NCH₂CH₃); 2.57 (s, 3H, -NC₆H₄COCH₃); 4.58 (quar., 2H, J: 7.3 Hz, -NCH₂CH₃); 7.76 and 7.98 (s, 2H, imidazol-4,5-CH); 8.00 and 8.07 (d, 4H, J: 8.8 and 8.7 Hz, -NC₆H₄COCH₃); 11.12 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 15.8 and 46.0 (-NCH₂CH₃); 26.9 (-C₆H₄COCH₃); 120.7 ve 123.1 (imidazol-4,5-CH); 122.0, 130.6, 136.1 and 137.5 (Ar-C); 138.0 (NCHN); 196.5 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-benzylimidazolium Chloride (2b)

The compound 2b was synthesized by using the same way for 2a. But benzyl chloride (506 mg, 4 mmol) was used instead of ethyl bromide. Yield: 79% (988 mg). m.p.: 184–185°C; ν_(C=O): 1682 cm^− 1; ν_(CN): 1552 cm^− 1. Anal. calc. for C₁₈H₁₇ClN₂O: C: 69.12; H: 5.48; N: 8.96. Found: C: 69.10; H: 5.47; N: 8.97. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.51 (s, 3H, -C₆H₄COCH₃); 5.71 (s, 2H, -NCH₂C₆H₅); 7.65 and 8.06 (s, 2H, imidazol-4,5-CH); 7.27 (m, 3H, -NCH₂C₆H₅); 7.56 (m 2H, -NCH₂C₆H₅); 7.72–7.79 (m, 1H, -NC₅H₅); 7.89–8.03 (m, 4H, -NC₆H₄COCH₃); 11.64 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.8 (-NC₆H₄COCH₃); 53.6 (-NCH₂C₆H₅); 120.7 and 123.1 (imidazol-4,5-CH); 121.6, 123.1, 129.3, 129.4, 129.6, 130.6, 133.0, 136.6 and 137.6 (Ar-C); 137.8 (NCHN); 196.4 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-(2-methylbenzyl)imidazolium Chloride (2c)

The compound 2c was synthesized by using the same way for 2a. But 2-methylbenzyl chloride (562 mg, 4 mmol) was used instead of ethyl bromide. Yield: 84% (1.10 g). m.p.: 205–206°C; ν_(C=O): 1665 cm^− 1; ν_(CN): 1545 cm^− 1. Anal. calc. for C₁₉H₁₉ClN₂O: C: 69.83; H: 5.86; N: 8.57. Found: C: 69.81; H: 5.88; N: 8.56. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.32 (s, 3H, -NCH₂C₆H₄CH₃-2); 2.55 (s, 3H, -NC₆H₄COCH₃); 5.74 (s, 2H, -NCH₂C₆H₄CH₃-2); 7.16–7.20 (m, 4H, -NCH₂C₆H₄CH₃-2); 7.25 and 7.39 (d, 2H, J: 7.5 and 7.2 Hz, imidazol-4,5-CH); 7.97 and 8.05 (d, 4H, J: 8.3 and 8.1 Hz, -NC₆H₄COCH₃); 11.74 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 19.4 (-NCH₂C₆H₄CH₃-2); 26.8 (-NC₆H₄COCH₃); 52.1 (-NCH₂C₆H₄CH₃-2); 120.3 and 122.4 (imidazol-4,5-CH); 121.8, 122.2, 127.1, 130.1, 130.4, 130.6, 130.7, 131.4, 137.4 and 137.6 (Ar-C); 138.0 (NCHN); 196.3 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-(3-methylbenzyl)imidazolium Chloride (2d)

The compound 2d was synthesized by using the same way for 2a. But 3-methylbenzyl chloride (562 mg, 4 mmol) was used instead of ethyl bromide. Yield: 79% (1.03 g). m.p.: 174–175°C; ν_(C=O): 1682 cm^− 1; ν_(CN): 1552 cm^− 1. Anal. calc. for C₁₉H₁₉ClN₂O: C: 69.83; H: 5.86; N: 8.57. Found: C: 69.82; H: 5.87; N: 8.58. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.26 (s, 3H, -NCH₂C₆H₄CH₃-3); 2.54 (s, 3H, -C₆H₄COCH₃); 5.67 (s, 2H, -NCH₂C₆H₄CH₃-3); 7.12 (d, 2H, J: 7.1 Hz, -NCH₂C₆H₄CH₃-3); 7.31 (s, 2H, -NCH₂C₆H₄CH₃-3); 7.45 (s, 1H, imidazol-4,5-CH); 7.85 (d, 1H, J: 8.8 Hz, imidazol-4,5-CH); 7.95 (d, 2H, J: 8.4 Hz, -NC₆H₄COCH₃); 8.02 (s, 2H, -NC₆H₄COCH₃); 11.76 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 21.3 (-NCH₂C₆H₄CH₃-3); 26.8 (-NC₆H₄COCH₃); 53.9 (-NCH₂C₆H₄CH₃-3); 120.3 and 122.7 (imidazol-4,5-CH); 121.7, 126.4, 129.4, 129.9, 130.5, 130.7, 132.6, 137.1, 137.5 and 138.0 (Ar-C); 139.5 (NCHN); 196.3 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-(4-methylbenzyl)imidazolium Chloride (2e)

The compound 2e was synthesized by using the same way for 2a. But 4-methylbenzyl chloride (562 mg, 4 mmol) was used instead of ethyl bromide. Yield: 78% (1.02 g). m.p.: 187–188°C; ν_(C=O): 1665 cm^− 1; ν_(CN): 1545 cm^− 1. Anal. calc. for C₁₉H₁₉ClN₂O: C: 69.83; H: 5.86; N: 8.57. Found: C: 69.84; H: 5.84; N: 8.57. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.26 (s, 3H, -NCH₂C₆H₄CH₃-4); 2.53 (s, 3H, -NC₆H₄COCH₃); 5.66 (s, 2H, -NCH₂C₆H₄CH₃-4); 7.11 and 7.42 (d, 4H, J: 6.7 and 7.5 Hz, -NCH₂C₆H₄CH₃-4); 7.48 and 7.88 (s, 2H, imidazol-4,5-CH); 7.94 and 8.02 (d, 4H, J: 8.1 and 7.5 Hz, -NC₆H₄COCH₃); 11.72 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 21.1 (-NCH₂C₆H₄CH₃-4); 26.7 (-NC₆H₄COCH₃); 53.6 (-NCH₂C₆H₄CH₃-4); 120.3 and 122.7 (imidazol-4,5-CH); 121.7, 129.4, 129.8, 130.2, 130.6, 136.9, 137.6 and 137.9 (Ar-C); 139.8 (NCHN); 196.3 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-(2,3,4,5,6-pentamethylbenzyl)imidazolium Chloride (2f)

The compound 2f was synthesized by using the same way for 2a. But 2,3,4,5,6-pentamethylbenzyl chloride (787 mg, 4 mmol) was used instead of ethyl bromide. Yield: 81% (1.24 g). m.p.: 242–243°C; ν_(C=O): 1683 cm^− 1; ν_(CN): 1555 cm^− 1. Anal. calc. for C₂₃H₂₇ClN₂O: C: 72.14; H: 7.11; N: 7.32. Found: C: 72.12; H: 7.10; N: 7.30. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.17 and 2.21 (s, 15H, -NCH₂C₆(CH₃)₅-2,3,4,5,6); 2.54 (s, 3H, -NC₆H₄COCH₃); 5.81 (s, 2H, -NCH₂C₆(CH₃)₅-2,3,4,5,6); 6.86 and 7.82 (s, 2H, imidazol-4,5-CH); 8.00 and 8.07 (d, 4H, J: 8.4 and 8.2 Hz, -NC₆H₄COCH₃); 11.84 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 16.9 and 17.3 (-NCH₂C₆(CH₃)₅-2,3,4,5,6); 26.4 (-C₆H₄COCH₃); 49.9 (-NCH₂C₆(CH₃)₅-2,3,4,5,6); 121.6 and 125.0 (imidazol-4,5-CH); 119.8, 121.8, 130.7, 133.8, 133.9, 137.5, 137.7 and 137.4 (Ar-C); 138.0 (NCHN); 196.3 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-(3,4,5-trimethoxybenzyl)imidazolium Chloride (2g)

The compound 2g was synthesized by using the same way for 2a. But 3,4,5-trimethoxybenzyl chloride (864 mg, 4 mmol) was used instead of ethyl bromide. Yield: 75% (1.21 g). m.p.: 222–223°C; ν_(C=O): 1681 cm^− 1; ν_(CN): 1555 cm^− 1. Anal. calc. for C₂₁H₂₃ClN₂O₄: C: 62.61; H: 5.75; N: 6.95. Found: C: 62.59; H: 5.74; N: 6.93. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.54 (s, 3H, -NC₆H₄COCH₃); 3.75 and 3.82 (s, 9H, -NCH₂C₆H₂(OCH₃)₃-3,4,5); 5.62 (s, 2H, -NCH₂C₆H₄(OCH₃)₃-3,4,5); 6.93 (s, 2H, NCH₂C₆H₂(OCH₃)₃-3,4,5); 7.74 and 7.60 (s, 2H, imidazol-4,5-CH); 7.91 and 8.02 (d, 4H, J: 8.6 and 8.4 Hz, -NC₆H₄COCH₃); 11.73 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.8 (-NC₆H₄COCH₃); 54.1 (-NCH₂C₆H₄(OCH₃)₃-3,4,5); 56.7 and 60.8 (-NCH₂C₆H₄(OCH₃)₃-3,4,5); 121.3 and 122.9 (imidazol-4,5-CH); 107.0, 119.9, 121.7, 128.3, 130.5, 130.6, 137.0, 137.5, 138.0 and 153.9 (Ar-C); 138.9 (NCHN); 196.3 (-NC₆H₄COCH₃).

Synthesis Of 1-(4-acetylphenyl)-3-(2-trifluoromethylbenzyl)imidazolium Bromide (3a)

The compound 3a was synthesized by using the same way for 2a. But 2-trifluoromethylbenzyl bromide (717 mg, 3 mmol) was used instead of ethyl bromide. Yield: 77% (982 mg). m.p.: 234–235°C; ν_(C=O): 1678 cm^− 1; ν_(CN): 1554 cm^− 1. Anal. calc. for C₁₉H₁₆BrF₃N₂O: C: 53.66; H: 3.79; N: 6.59. Found: C: 53.67; H: 3.80; N: 6.58. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.55 (s, 3H, -NC₆H₄COCH₃); 5.92 (s, 2H, -NCH₂C₆H₄CF₃-2); 7.27 (s, 1H, -NCH₂C₆H₄CF₃-4); 7.49 (t, 1H, J: 7.7 Hz, -NCH₂C₆H₄CF₃-2); 7.61 (t, 1H, J: 7.6 Hz, -NCH₂C₆H₄CF₃-2); 7.80 (s, 1H, -NCH₂C₆H₄CF₃-2); 7.68 and 8.13 (d, 2H, J: 7.8 and 7.7 Hz, imidazol-4,5-CH); 7.97 and 8.06 (d, 4H, J: 8.6 and 8.6 Hz, -NC₆H₄COCH₃); 11.34 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.8 (-NC₆H₄COCH₃); 50.1 (-NCH₂C₆H₄CF₃-2); 120.5 and 122.8 (imidazol-4,5-CH); 122.1, 125.4, 126.6, 128.7, 129.0, 130.2, 130.3, 130.7, 133.4, 133.6, 136.9 and 137.4 (Ar-C and CF₃); 138.2 (NCHN); 196.2 (-NC₆H₄COCH₃). ¹⁹F NMR (376 MHz, CDCl₃, 298 K), δ (ppm): −58.38 (Ar–CF₃).

Synthesis Of 1-(4-acetylphenyl)-3-(3-trifluoromethylbenzyl)imidazolium Bromide (3b)

The compound 3b was synthesized by using the same way for 2a. But 3-trifluoromethylbenzyl bromide (717 mg, 3 mmol) was used instead of ethyl bromide. Yield: 80% (1.02 g). m.p.: 204–205°C; ν_(C=O): 1676 cm^− 1; ν_(CN): 1551 cm^− 1. Anal. calc. for C₁₉H₁₆BrF₃N₂O: C: 53.66; H: 3.79; N: 6.59. Found: C: 53.68; H: 3.78; N: 6.58. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.57 (s, 3H, -NC₆H₄COCH₃); 5.91 (s, 2H, -NCH₂C₆H₄CF₃-3); 7.43 and 7.62 (s, 2H, -NCH₂C₆H₄CF₃-3); 7.53 and 7.61 (d, 1H, J: 7.7 Hz, -NCH₂C₆H₄CF₃-3); 7.73 (s, 1H, imidazol-4,5-CH); 7.96 (d, 1H, J: 7.3 Hz, imidazol-4,5-CH); 7.90 and 8.09 (d, 4H, J: 8.4 and 8.2 Hz, -NC₆H₄COCH₃); 11.56 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.8 (-NC₆H₄COCH₃); 53.0 (-NCH₂C₆H₄CF₃-3); 120.3 and 122.7 (imidazol-4,5-CH); 111.8, 122.0, 125.8, 126.2, 130.5, 130.8, 133.7 and 137.2 (Ar-C and CF₃); 138.4 (NCHN); 196.3 (-NC₆H₄COCH₃). ¹⁹F NMR (376 MHz, CDCl₃, 298 K), δ (ppm): −60.97 (Ar–CF₃).

Synthesis Of 1-(4-acetylphenyl)-3-(4-trifluoromethylbenzyl)imidazolium Bromide (3c)

The compound 3c was synthesized by using the same way for 2a. But 4-trifluoromethylbenzyl bromide (717 mg, 3 mmol) was used instead of ethyl bromide. Yield: 78% (995 mg). m.p.: 195–196°C; ν_(C=O): 1678 cm^− 1; ν_(CN): 1551 cm^− 1. Anal. calc. for C₁₉H₁₆BrF₃N₂O: C: 53.66; H: 3.79; N: 6.59. Found: C: 53.65; H: 3.77; N: 6.60. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: ¹H NMR (400 MHz, DMSO), δ: 2.55 (s, 3H, -NC₆H₄COCH₃); 5.92 (s, 2H, -NCH₂C₆H₄CF₃-4); 7.57 and 7.78 (d, 4H, J: 8.0 and 8.2 Hz, -NCH₂C₆H₄CF₃-4); 7.58 and 7.69 (s, 2H, imidazol-4,5-CH); 7.88 and 8.04 (d, 4H, J: 8.7 and 8.6 Hz, -NC₆H₄COCH₃); 11.35 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.8 (-NC₆H₄COCH₃); 53.0 (-NCH₂C₆H₄CF₃-4); 120.4 and 123.1 (imidazol-4,5-CH); 122.0, 126.4, 126.5, 130.0, 130.7, 136.7 and 137.4 (Ar-C and CF₃); 138.3 (NCHN); 196.2 (-NC₆H₄COCH₃). ¹⁹F NMR (376 MHz, CDCl₃, 298 K), δ (ppm): −61.10 (Ar–CF₃).

Synthesis Of 1-(4-acetylphenyl)-3-(4-trifluoromethoxybenzyl)imidazolium Bromide (3d)

The compound 3d was synthesized by using the same way for 2a. But 4-trifluoromethoxybenzyl bromide (765 mg, 3 mmol) was used instead of ethyl bromide. Yield: 73% (966 mg). m.p.: 194–195°C; ν_(C=O): 1682 cm^− 1; ν_(CN): 1552 cm^− 1. Anal. calc. for C₁₉H₁₆BrF₃N₂O₂: C: 51.72; H: 3.66; N: 6.35. Found: C: 531.70; H: 3.67; N: 6.33. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.53 (s, 3H, -NC₆H₄COCH₃); 5.83 (s, 2H, -NCH₂C₆H₄OCF₃-4); 7.14 and 7.73 (d, 4H, J: 8.1 and 8.6 Hz, -NCH₂C₆H₄OCF₃-4); 7.71 and 7.80 (s, 2H, imidazol-4,5-CH); 7.89 and 8.02 (d, 4H, J: 8.7 and 8.6 Hz, -C₆H₄COCH₃); 11.23 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.7 (-NC₆H₄COCH₃); 52.7 (-NCH₂C₆H₄OCF₃-4); 120.6 and 123.3 (imidazol-4,5-CH); 119.0, 121.7, 121.9, 130.6, 131.4, 131.6, 136.2, 137.4 and 150.1 (Ar-C and OCF₃); 138.2 (NCHN); 196.2 (-C₆H₄COCH₃). ¹⁹F NMR (376 MHz, CDCl₃, 298 K), δ (ppm): −56.79 (Ar–OCF₃).

Synthesis Of 1-(4-acetylphenyl)-3-(4-fluorobenzyl)imidazolium Bromide (3e)

The compound 3e was synthesized by using the same way for 2a. But 4-fluorobenzyl bromide (752 mg, 4 mmol) was used instead of ethyl bromide. Yield: 82% (1.23 g). m.p.: 167–168°C; ν_(C=O): 1672 cm^− 1; ν_(CN): 1552 cm^− 1. Anal. calc. for C₁₈H₁₆BrFN₂O: C: 57.62; H: 4.30; N: 7.47. Found: C: 57.60; H: 4.31; N: 7.46. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.54 (s, 3H, -NC₆H₄COCH₃); 5.75 (s, 2H, -NCH₂C₆H₄F-4); 6.98 (t, 2H, J: 8.5 Hz, -NCH₂C₆H₄F-4); 7.63–7.65 (m, 2H, -NCH₂C₆H₄F-4); 7.67 and 7.83 (s, 2H, imidazol-4,5-CH); 7.90 and 8.02 (d, 4H, J: 8.6 and 9.0 Hz, -NC₆H₄COCH₃); 11.66 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.7 (-NC₆H₄COCH₃); 52.9 (-NCH₂C₆H₄F-4); 120.4 and 122.9 (imidazol-4,5-CH); 116.4, 116.6, 121.1, 121.7, 129.0, 130.4, 130.6, 131.8, 136.8, 137.5 and 137.4 (Ar-C); 138.0 (NCHN); 162.1 and 164.6 (Ar-C-F); 196.3 (-NC₆H₄COCH₃). ¹⁹F NMR (376 MHz, CDCl₃, 298 K), δ (ppm): −112.96 (Ar–F).

Synthesis Of 1-(4-acetylphenyl)-3-(2-chloro-4-fluorobenzyl)imidazolium Bromide (3f)

The compound 3f was synthesized by using the same way for 2a. But 2-chloro-4-fluorobenzyl bromide (670 mg, 3 mmol) was used instead of ethyl bromide. Yield: 74% (910 mg). m.p.: 168–169°C; ν_(C=O): 1679 cm^− 1; ν_(CN): 1554 cm^− 1. Anal. calc. for C₁₉H₁₅BrFClN₂O: C: 52.77; H: 3.69; N: 6.84. Found: C: 52.80; H: 3.70; N: 6.86. ¹H NMR (400 MHz, CDCl₃, 298 K), δ: 2.56 (s, 3H, -NC₆H₄COCH₃); 5.88 (s, 2H, -NCH₂C₆H₃ClF); 7.03 (t, 1H, J: 7.0 Hz, -NCH₂C₆H₃ClF); 7.11 (d, 1H, J: 2.0 Hz, -NCH₂C₆H₃ClF); 7.50 (d, 1H, J: 8.5 Hz, -NCH₂C₆H₃ClF); 7.54 and 7.77 (s, 2H, imidazol-4,5-CH); 7.92 and 8.06 (d, 4H, J: 8.4 and 8.3 Hz, -NC₆H₄COCH₃); 11.66 (s, 1H, NCHN). ¹³C NMR (100 MHz, CDCl₃, 298 K), δ: 26.8 (-NC₆H₄COCH₃); 50.3 (-NCH₂C₆H₄ClF); 120.0 and 122.9 (imidazol-4,5-CH); 115.9, 117.4, 120.9, 121.8, 127.0, 130.4, 130.7, 134.9 and 137.6 (Ar-C); 138.1 (NCHN); 162.0 and 164.6 (Ar-C-F); 196.2 (-NC₆H₄COCH₃). ¹⁹F NMR (376 MHz, CDCl₃, 298 K), δ (ppm): −110.06 (Ar–F).

Biochemical Studies

AChE inhibition assay

In the present work, in vitro effects of acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) and 1-(4-acetylphenyl)imidazole (1) on AChE activity evaluated by Ellman method [81] as described in prior studies [82, 83]. Results were performed spectrophotometrically at 412 nm using acetylthiocholine iodide according to previous study [84].

Hcas Inhibition Assay

Commercial sources usually isolate both CA isoenzymes from fresh human erythrocytes and typically produce these isoenzymes by recombinant technology from E. coli [85]. In this study, both hCA isoenzymes were purified via Sepharose-4B-L-Tyrosine-sulfanilamide affinity chromatography [86]. CA isoenzymes were purified by Sepharose-4B-L-Tirozyne-sulfanylamide affinity column chromatography [87]. The protein content during the purification steps was determined by Bradford method [88]. Also, bovine serum albumin was used as standard protein [89]. The purity of both hCA isoenzymes was controlled by SDS-PAGE as described in prior studies [90]. During the isoenzyme’s purification and inhibition process, esterase activity was performed [91]. Both hCA isoenzyme activities were determined by following the change in absorbance at 348 nm according to the assay by Verpoorte et al. [92] as described in detail [93].

Ache And Hcas Kinetic Assay

To investigate the in vitro inhibitory mechanisms of acetylphenyl-substituted imidazolium salts (2a-g, 3a-f) and 4-(1-H-imidazol-1-yl)acetophenone (1) kinetic studies were made with the variable compound and substrate concentrations [94, 95]. From the observed data, IC₅₀ and K_i values for these derivatives were computed, and the types of inhibition of AChE and hCAs were determined as in previous studies [96].

In Silico Studies

The 3D structures of carbonic anhydrases (hCA-I, hCA-II) and acetylcholinesterase (AChE) were obtained from the RCSB Protein Data Bank (PDB) having identifiers 1AZM (2.00 Å), 3HS4 (1.10 Å), and 4EY6 (2.40 Å), respectively. The 3D structures of proteins were evaluated in Discovery Studio 3.5 [97], and missing residue(s) and polar hydrogen atoms were added, and optimized by CHARMM force field [98] of DS 3.5 software. The data on ligand-binding sites in all three enzyme proteins were obtained from the literatures and with help of the “define and edit binding site” sub-protocol of DS software. Also the potential first three synthesized imidazolium salts as ligands were sketched and minimized at DFT/B3LYP/SDD level in Gaussian 09 (G09) [99]. Lastly, the current ligands were docked into the related ligand-binding sites of each target using AutoDock 4.2 software [100]. Intermolecular interactions among enzymes and ligands were assessed and 3D images were rendered with help of DS 3.5 software.

As control compounds, molecular docking calculations were also applied the drugs acetazolamide (AZA) and tacrine (TAC) for among carbonic anhydrases (hCA-I, hCA-II) and acetylcholinesterase (AChE), respectively. The data of the control compounds were also utilized to compare and analyze the selected ligands.

Besides these, the pharmacokinetics features of docking-based prioritized molecules as ligands ADMET were examined to define their activity within the human body via were calculated using DS 3.5 [97]. The Lipinski [101] and Veber Rules [102] were used to estimate the drug-like qualities of the ligands. These rules are generally used and important rules in the pharmaceutical industry as a first step in describing the optimal compound for a medicine. So that, the bioavailability of the compounds can be forecasted [103]. After then, ADMET descriptors [Aqueous solubility, intestinal absorption (HIA), blood brain barrier penetration (BBB), and cytochrome P450 2D6 binding (CYP2D6), hepatotoxicity and plasma protein binding (PPB)] were predicted with sub-protocol of DS 3.5 to be suitable drug properties or not of the current compounds.

Acknowledgments

The authors thank the İnönü University Faculty of Science Department of Chemistry for the spectroscopy data and characterization of compounds. The authors also thank Esin Akı Yalcin and the research group for technical assistance. This study was financially supported by Inonu University Research Fund (Project Code: FBG‐2021-2525 and FOA-2021-2320).

Conflicts Of Interests The authors declare that there are no conflicts of interests.

Wang MP, Chiu CC, Lu TJ, Lee DS (2018) Indolylbenzimidazole-based ligands catalyze the coupling of arylboronic acids with aryl halides. Appl Organomet Chem 32:e4348.
Nguyen VH, Dang TT, Nguyen HH, Huynh HV (2020) Platinium(II) 1,2,4-triazolin-5-ylidene complexes: stereoelectronic influences on their catalytic activity in hydroelementation reactions. Organometallics 39:2309–2319.
Herrmann WA, Goo βen LJ, Spiegler M (1997) Functionalized imidazoline-2-ylidene complexes of rhodium and palladium. J Organomet Chem 547:357–366.
Marion N, Nolan SP (2008) N-Heterocyclic carbenes in gold catalysis. Chem Soc Rev 37:1776–1782.
Ung G, Soleilhavoup M, Bertrand G (2013) Gold(III)- versus gold(i)-induced cyclization: synthesis of six-membered mesoionic carbene and acyclic (aryl)(heteroaryl) carbene complexes. Angew Chem Int Ed Engl 52:758–761.
El-Gohary NS, Shaaban MI (2017) Synthesis and biological evaluation of a new series of benzimidazole derivatives as antimicrobial, antiquorum-sensing and antitumor agents. Eur J Med Chem 131:255–262.
Albayrak S, Gök Y, Sari Y, Taskın Tok T, Aktaş A (2021) Benzimidazolium salts bearing 2-methyl-1,4-benzodioxane group: synthesis, characterization, computational studies, in vitro antioxidant and antimicrobial activity. Biointerface Research in Applied Chemistry 11:13333–13346.
Malinowska M, Sawicka D, Niemirowicz-Laskowska K, Wielgat P, Car H, Hauschild T, Hryniewicka A (2021) Steroid-functionalized imidazolium salts with an extended spectrum of antifungal and antibacterial activity. Int J Mol Sci 22:12180.
Song XQ, Vig BS, Lorenzi PL, Drach JC, Townsend LB, Amidon GL (2005) Amino acid ester prodrugs of the antiviral agent 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole as potential substrates of hpept1 transporter. J Med Chem 48:1274–1277.
Bukhari SNA, Lauro G, Jantan I, Chee CF, Amjad MW, Bifulco G, Sher H, Addullah I, Abd Rahman N (2016) Anti-inflammatory trends of new benzimidazole derivatives. Future Med Chem 8:1953–1967.
Fan QW, Zhong QD, Yan H (2017) Synthesis, antitumor activity, and docking study of 1,3-disubstituted imidazolium derivatives. Russian Journal of General Chemistry 87:3023–3028.
Aher SB, Muskawar PN, Thenmozhi K, Bhagat PR (2014) Recent developments of metal n-heterocyclic carbenes as anticancer agents. Eur J Med Chem 81:408–419.
Yadav G, Ganguly S (2015) Structure activity relationship (SAR) study of benzimidazole scaffold for different biological activities: A mini-review. Eur J Med Chem 97:419–443.
Kotovskaya SK, Zhumabaeva GA, Perova NM, Baskakova ZM, Charushin VN, Chupakhin ON, Belanov EF, Bormotov NI, Balakhnin SM, Serova OA (2007) Synthesis and antiviral activity of fluorinated 3-phenyl-1,2,4-benzotriazines. Pharmaceutical Chemistry Journal 41(2):62–68.
Wu L, Wang X (2014) One-pot synthesis of 1-aryl-1H,3H-thiazolo[3,4-a]benzimidazoles using magnetite-linked sulfonic acid as catalyst. Phosphorus, Sulfur, and Silicon and Relat Elem 189:1851 – 1857.
Abdel-Hafez AA (2007) Benzimidazole condensed ring systems: New synthesis and antineoplastic activity of substituted 3,4-dihydro- and 1,2,3,4-tetrahydro-benzo [4,5]imidazo[1,2-a]pyrinnidine derivatives. Arch Pharm Res 30:678–684.
Yang S, Zhang Q, Hu Y, Ding G, Wang J, Huo S, Zhang B, Cheng J (2018) Synthesis of s-triazine based tri-imidazole derivatives and their application as thermal latent curing agents for epoxy resin. Materials Letters 216:127–130.
Kühl O, Palm G (2010) Imidazolium salts from amino acids—a new route to chiral zwitterionic carbene precursors. Tetrahedron: Asymmetry 21:393–397.
Padvi SA, Dalal DS (2020) Task-specific ionic liquids as a green catalysts and solvents for organic synthesis. Curr Green Chem 7:105–119.
Bando T, Hiroshi S (2006) Synthesis and Biological Properties of Sequence-Specific DNA-Alkylating Pyrrole-Imidazole Polyamides. Acc Chem Res 39:935–944.
Bredas JL, Poskin M.P, Delhalle J, Andre JM, Chojnacki H (1984) Electronic structure of hydrogen-bonded imidazole chains. Influence of the proton position. J Phys Chem 88:5882–5887.
Ganapathi P, Ganesan K, Vijaykanth N, Arunagirinathan N (2016) Anti-bacterial screening of water soluble carbonyl diimidazolium salts and its derivatives. J Mol Liq 219:180–185.
Anderson EB, Long TE (2010) Imidazole- and imidazolium-containing polymers for biology and material science applications. Polymer 51:2447–2454.
Grygorenko OO, Volochnyuk DM, Ryabukhin SV, Judd DB (2020) The symbiotic relationship between drug discovery and organic chemistry. Chem Eur J 26:1196–1237.
Meanwell NA (2011) Improving drug candidates by design: A focus on physicochemical properties as a means of improving compound disposition and safety. Chem Res Toxicol 24:1420–1456.
Begue JP, Bonnet-Delpon D (2008) Bioorganic and Medicinal Chemistry of Fluorine. John Wiley & Sons, Hoboken p 365 https://doi.org/10.1051/978-2-7598-0266-1.
Meanwell NA (2018) Fluorine and fluorinated motifs in the design and application of bioisosteres for drug design. J Med Chem 61:5822–5880.
Han J, Remete AM, Dobson LS, Kiss L, Izawa K, Moriwaki H, Soloshonok VA, O’Hagan D (2020) Next generation organofluorine containing blockbuster drugs. J Fluorine Chem 239:109639.
Hanson JR (2001) Functional group chemistry. Cambridge, Eng: Royal Society of Chemistry p 11.
Lehninger AL, Nelson DL, Cox MM, Cox MM (2005) Lehninger principles of biochemistry. Macmillan.
Gülçin İ (2006) Antioxidant and antiradical activities of L-Carnitine. Life Sciences 78:803–811.
Gülçin İ (2010) Antioxidant properties of resveratrol: A structure-activity insight. Innovative Food Science and Emerging Technologies 11:210–218.
Bilginer S, Gul HI, Anil B, Demir Y, Gulcin I (2021) Synthesis and in silico studies of triazene-substituted sulfamerazine derivatives as acetylcholinesterase and carbonic anhydrases inhibitors. Archiv der Pharmazie 354:2000243.
Bilginer S, Anil B, Koca M, Demir Y, Gülçin İ (2021) Novel Mannich bases with strong carbonic anhydrases and acetylcholinesterase inhibition effects: 3-(aminomethyl)-6-{3-[4-(trifluoromethyl) phenyl] acryloyl}-2 (3H)-benzoxazolones. Turkish Journal of Chemistry 45:805–818.
Gümüş M, Babacan ŞN, Demir Y, Sert Y, Koca İ, Gülçin İ (2022) Discovery of sulfadrug-pyrrole conjugates as carbonic anhydrase and acetylcholinesterase inhibitors. Archiv der Pharmazie 355: e2100242.
Mahmudov I, Demir Y, Sert Y, Abdullayev Y, Sujayev A, Alwasel SH, Gulcin I (2022) Synthesis and inhibition profiles of N-benzyl- and N-allyl aniline derivatives against carbonic anhydrase and acetylcholinesterase – A molecular docking study. Arabian Journal of Chemistry 15:103645.
Aydın BO, Anil D, Demir Y (2021) Synthesis of N-alkylated pyrazolo[3,4‐d]pyrimidine analogs and evaluation of acetylcholinesterase and carbonic anhydrase inhibition properties. Archiv der Pharmazie 354:2000330.
Kaya Y, Erçağ A, Zorlu Y, Demir Y, Gülçin İ (2022) New Pd(II) complexes of the bisthiocarbohydrazones derived from isatin and disubstituted salicylaldehydes: Synthesis, characterization, crystal structures and inhibitory properties against some metabolic enzymes. Journal of biological inorganic chemistry: JBIC: a publication of the Society of Biological Inorganic Chemistry 27:271–281.
Ozbey F, Taslimi P, Gulcin İ, Maraş A, Goksu S, Supuran CT (2016) Synthesis, acetylcholinesterase, butyrilcholinesterase, carbonic anhydrase inhibitory and metal chelating properties of some novel diaryl ether. Journal of Enzyme Inhibition and Medicinal Chemistry 31:79–85.
Supuran CT (2021) Emerging role of carbonic anhydrase inhibitors. Clinical Science 135:1233–1249.
Turkan F, Cetin A, Taslimi P, Karaman M, Gülçin İ (2019) Synthesis, biological evaluation and molecular docking of novel pyrazole derivatives as potent carbonic anhydrase and acetylcholinesterase inhibitors. Bioorganic Chemistry 86:420–427.
Nar M, Çetinkaya Y, Gülçin İ, Menzek A (2013) (3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry 28:402–406.
Küçük M, Gülçin İ (2016) Purification and characterization of carbonic anhydrase enzyme from black sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on the enzyme activity. Environmental Toxicology and Pharmacology 44:134–139.
Hisar O, Beydemir Ş, Gülçin İ, Küfrevioğlu Öİ, Supuran CT (2005) Effect of low molecular weight plasma inhibitors of rainbow trout (Oncorhyncytes mykiss) on human erythrocytes carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo. Journal of Enzyme Inhibition and Medicinal Chemistry 20:35–39.
Tugrak M, Gul HI, Demir Y, Levent S, Gulcin I (2021) Synthesis and in vitro carbonic anhydrases and acetylcholinesterase inhibitory activities of novel imidazolinone-based benzenesulfonamides. Archiv der Pharmazie 354:2000375.
Yamali C, Gul HI, Cakir T, Demir Y, Gulcin I (2020) Aminoalkylated phenolic chalcones: Investigation of biological effects on acetylcholinesterase and carbonic anhydrase i and ii as potential lead enzyme inhibitors. Letters in Drug Design & Discovery 17:1283–1292.
Askin S, Tahtaci H, Türkeş C, Demir Y, Ece A, Akalın Çiftçi G, Beydemir Ş (2021) Design, synthesis, characterization, in vitro and in silico evaluation of novel imidazo[2,1-b][1,3,4]thiadiazoles as highly potent acetylcholinesterase and non-classical carbonic anhydrase inhibitors. Bioorganic Chemistry 113:105009.
Kalaycı M, Türkeş C, Arslan M, Demir Y, Beydemir Ş (2021) Novel benzoic acid derivatives: Synthesis and biological evaluation as multitarget acetylcholinesterase and carbonic anhydrase inhibitors. Archiv der Pharmazie 354:e2000282.
Ozgeris B, Göksu S, Köse Polat L, Gülçin İ, Salmas RE, Durdagi S, Tümer F, Supuran CT (2016) Acetylcholinesterase and carbonic anhydrase inhibitory properties of novel urea and sulfamide derivatives incorporating dopaminergic 2-aminotetralin scaffolds. Bioorganic and Medicinal Chemistry 24:2318–2329.
Garibov E, Taslimi P, Sujayev A, Bingöl Z, Çetinkaya S, Gulcin İ, Beydemir S, Farzaliyev V, Alwasel SH, Supuran CT (20169 Synthesis of 4,5-disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines and investigation of their acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase I/II inhibitory and antioxidant activities. Journal of Enzyme Inhibition and Medicinal Chemistry 31:1–9.
Burmaoğlu S, Yılmaz AO, Polat MF, Kaya R, Gülçin İ, Algül Ö (2019) Synthesis and biological evaluation of novel tris-chalcones as potent carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase inhibitors. Bioorganic Chemistry 85:191–197.
Erdemir F, Barut Celepci D, Aktaş A, Gök Y, Kaya R, Taslimi P, Demir Y, Gülçin İ (2019) Novel 2-aminopyridine liganded Pd(II) N-heterocyclic carbene complexes: synthesis, characterization, crystal structure and bioactivity properties. Bioorganic Chemistry 91:103134.
Bayrak Ç, Taslimi P, Kahraman HS, Gülçin İ, Menzek A (2019) The first synthesis, carbonic anhydrase inhibition and anticholinergic activities of some bromophenol derivatives with S including natural products. Bioorganic Chemistry 85:128–139.
Huseynova M, Taslimi P, Medjidov A, Farzaliyev V, Aliyeva M, Gondolova G, Şahin O, Yalçın B, Sujayev A, Orman EB, Özkaya AR, Gülçin İ (2018) Synthesis, characterization, crystal structure, electrochemical studies and biological evaluation of metal complexes with thiosemicarbazone of glyoxylic acid. Polyhedron 155:25–33.
Çetin Çakmak K, Gülçin İ (2019) Anticholinergic and antioxidant activities of usnic acid-An activity-structure insight. Toxicology Reports 6:1273–1280.
Sever B, Türkeş C, Altıntop MD, Demir Y, Akalın Çiftçi G, Beydemir Ş (2021) Novel metabolic enzyme inhibitors designed through the molecular hybridization of thiazole and pyrazoline scaffolds. Archiv der Pharmazie 354:e2100294.
Şahin İ, Bingöl Z, Onur S, Güngör SA, Köse M, Gülçin İ, Tümer F (2022) Enzyme inhibition properties and molecular docking studies of 4-sulfonate containing aryl α-hydroxyphosphonates based hybrid molecules. Chemistry & biodiversity 19:e202100787.
Gülçin İ, Scozzafava A, Supuran CT, Akıncıoğlu H, Koksal Z, Turkan F, Alwasel S (2016) The effect of caffeic acid phenethyl ester (CAPE) metabolic enzymes including acetylcholinesterase, butyrylcholinesterase, glutathione s-transferase, lactoperoxidase and carbonic anhydrase isoenzymes I, II, IX and XII. Journal of Enzyme Inhibition and Medicinal Chemistry 31:1095–1101.
Taslimi P, Caglayan C, Gulçin İ (2017) The impact of some natural phenolic compounds on carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase enzymes: an antidiabetic, anticholinergic, and antiepileptic study. Journal of Biochemical and Molecular Toxicology 31:e21995.
Takım K, Yigin A, Koyuncu İ, Kaya R, Gulçin İ (2021) Anticancer, anticholinesterase and antidiabetic activities of Tunceli garlic (Allium tuncelianum) - Determining its phytochemical content by LC-MS/MS analysis. Journal of Food Measurement and Characterization 15:3323–3335.
Akıncıoğlu A, Göksu S, Naderi A, Akıncıoğlu H, Kılınç N, Gülçin İ (2021) Cholinesterases, carbonic anhydrase inhibitory properties and in silico studies of novel substituted benzylamines derived from dihydrochalcones. Computational Biology and Chemistry 94:107565.
Riaz MT, Yaqup M, Shafiq Z, Ashraf Z, Khalid M, Taslimi P, Taş R, Tuzun B, Gulçin İ (2021) Synthesis, biological activity and docking calculations of bis-naphthoquinone derivatives from lawsone. Bioorganic Chemistry 114:105069.
Kızıltaş H, Bingol Z, Goren AC, Alwasel SH, Gülçin İ (2021) Anticholinergic, antidiabetic and antioxidant activities of Ferula orientalis L.-Analysis of its polyphenol contents by LC-HRMS. Records of Natural Products 15:513–528.
Atmaca U, Alp C, Akıncıoğlu H, Karaman HS, Gulçin İ, Çelik M (2021) Novel hypervalent iodine catalyzed synthesis of α-sulfonoxy ketones: Biological activity and molecular docking studies. Journal of Molecular Structure 1239:130492.
Karataş MO, Uslu H, Alıcı B, Gökçe B, Gencer N, Arslan O, Arslan NB, Özdemir N (2016) Functionalized imidazolium and benzimidazolium salts as paraoxonase 1 inhibitors: Synthesis, characterization and molecular docking studies. Bioorg Med Chem 24:1392–1401.
Hamide M, Gök Y, Demir Y, Yakalı G, Taskin Tok T, Aktaş A, Sevinçek R, Güzel B, Gülçin İ (2022) Pentafluorobenzyl-substituted Benzimidazolium salts: Synthesis, characterization, crystal structures, computational studies and inhibitory properties of some metabolic enzymes. Journal of Molecular Structure 1265:133266.
Tezcan B, Gök Y, Sevinçek R, Taslami P, Taskin-Tok T, Aktaş A, Güzel B, Aygün M, Gülçin İ (2022) Benzimidazolium salts bearing the trifluoromethyl group as organofluorine compounds: Synthesis, characterization, crystal structure, in silico study, and inhibitory profiles against acetylcholinesterase and α-glycosidase. Journal of Biochemical and Molecular Toxicology 36:e23001.
Kazancı A, Gök Y, Kaya R, Aktaş A, Taslimi P, Gülçin İ (2021) Synthesis, characterization and bioactivities of dative donor ligand N-heterocyclic carbene (NHC) precursors and their Ag(I)NHC coordination compounds. Polyhedron 193:114866.
Aktaş A, Noma SAA, Barut Celepci D, Erdemir F, Gök Y, Ateş B (2019) New 2-hydroxyethyl substituted N-Heterocyclic carbene precursors: Synthesis, characterization, crystal structure and inhibitory properties against carbonic anhydrase and xanthine oxidase. Journal of Molecular Structure 1184:487–494.
Dolbier Jr W R (2009) Guide to Fluorine NMR for Orgonic Chemists, Wilev. Hoboken 79.
Burmaoglu S, Yilmaz AO, Polat MF, Kaya R, Gulcin İ, Algul O (2021) Synthesis of novel tris-chalcones and determination of their inhibition profiles against some metabolic enzymes. Archives of physiology and biochemistry 127:153–161.
Tokalı FS, Taslimi P, Demircioğlu İH, Karaman M, Gültekin MS, Şendil K, Gülçin İ (2021) Design, synthesis, molecular docking, and some metabolic enzyme inhibition properties of novel quinazolinone derivatives. Archiv der Pharmazie 354:e2000455.
Erdoğan M, Polat Köse L, Eşsiz S, Gülçin İ (2021) Synthesis and biological evaluation of some 1-naphthol derivatives as antioxidants, acetylcholinesterase, and carbonic anhydrase inhibitors. Archiv der Pharmazie 354:e2100113.
Güney M, Coşkun A, Topal F, Daştan A, Gülçin İ, Supuran CT (2014) Oxidation of cyanobenzocycloheptatrienes: Synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes inhibition properties of some new benzotropone derivatives. Bioorganic and Medicinal Chemistry 22:3537–3543.
Krymov SK, Scherbakov AM, Salnikova DI, Sorokin DV, Dezhenkova LG, Ivanov IV, Vullo D, De Luca V, Capasso C, Supuran CT, Shchekotikhin AE (2022) Synthesis, biological evaluation, and in silico studies of potential activators of apoptosis and carbonic anhydrase inhibitors on isatin-5-sulfonamide scaffold. European Journal of Medicinal Chemistry 228:113997.
Supuran CT (2008) Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 7:168–181.
Bekku S, Mochizuki H, Yamamoto T, Ueno H, Takayama E, Tadakuma T (2000) Expression of carbonic anhydrase I or II and correlation to clinical aspects of colorectal cancer. Hepato-Gastroenterology 47: 998–1001.
Scozzafava A, Kalın P, Supuran CT, Gülçin İ, Alwasel S (2015) The impact of hydroquinone on acetylcholine esterase and certain human carbonic anhydrase isoenzymes (hCA I, II, IX, and XII). Journal of Enzyme Inhibition and Medicinal Chemistry 30:941–946.
Ozmen Ozgün D, Gül Hİ, Yamali C, Sakagami H, Gulçin İ, Sukuroglu M, Supuran CT (2019) Synthesis and bioactivities of pyrazoline benzensulfonamides as carbonic anhydrase and acetylcholinesterase inhibitors with low cytotoxicity. Bioorganic Chemistry 84:511–517.
Bal S, Kaya R, Gök Y, Taslimi P, Aktaş A, Karaman M, Gülçin İ (2020) Novel 2-methylimidazolium salts: Synthesis, characterization, molecular docking, and carbonic anhydrase and acetylcholinesterase inhibitory properties. Bioorganic chemistry 94:103468.
Ellman GL, Courtney KD, Andres Jr V, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7:88–95.
Koçyiğit UM, Budak Y, Gürdere MB, Ertürk F, Yencilek B, Taslimi P, Gulçin İ, Ceylan M (2018) Synthesis of chalcone-imide derivatives and investigation of their anticancer and antimicrobial activities, carbonic anhydrase and acetylcholinesterase enzymes inhibition profiles. Archives of Physiology and Biochemistry 124:61–68.
Küçükoğlu K, Gül Hİ, Taslimi P, Gülçin İ, Supuran CT (2019) Investigation of inhibitory properties of some hydrazone compounds on hCA I, hCA II and AChE enzymes. Bioorganic Chemistry 86:316–321.
Taslimi P, Türkan F, Güngördü Solğun D, Aras A, Erden Y, Celebioglu HU, Tuzun B, Ağırtaş MS, Günay S, Gulcin I (2022) Metal contained Phthalocyanines with 3,4-Dimethoxyphenethoxy substituents: their anticancer, antibacterial activities and their inhibitory effects on some metabolic enzymes with molecular docking studies. Journal of Biomolecular Structure & Dynamics 40:2991–3002.
Akıncıoğlu A, Topal M, Gülçin İ, Göksu S (2014) Novel sulfamides and sulfonamides incorporating tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors. Archiv der Pharmazie 347: 68–76.
Huseynova A, Kaya R, Taslimi P, Farzaliyev V, Mammadyarova X, Sujayev A, Tüzün B, Kocyigit UM, Alwasel S, Gulçin İ (2022) Design, synthesis, characterization, biological evaluation, and molecular docking studies of novel 1,2-aminopropanthiols substituted derivatives as selective carbonic anhydrase, acetylcholinesterase and α-glycosidase enzymes inhibitors. Journal of Biomolecular Structure & Dynamics 40:236–248.
Taslimi P, Gülçin İ, Öztaşkın N, Çetinkaya Y, Göksu S, Alwasel SH, Supuran CT (2016) The effects of some bromophenol derivatives on human carbonic anhydrase isoenzymes. Journal of Enzyme Inhibition and Medicinal Chemistry 31:603–607.
Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254.
Köksal E, Gülçin İ (2008) Purification and characterization of peroxidase from cauliflower (Brassica oleracea L.) buds. Protein and Peptide Letter 15:320–326.
Turan B, Sendil K, Sengul E, Gultekin MS, Taslimi P, Gulcin İ, Supuran CT (2016) The synthesis of some β-lactams and investigation of their metal chelating activity, carbonic anhydrase and achetylcholinesterase inhibition profiles. Journal of Enzyme Inhibition and Medicinal Chemistry 31: 79–88.
Gul HI, Mete E, Taslimi P, Gulcin I, Supuran CT (2017) Synthesis, carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines. Journal of Enzyme Inhibition and Medicinal Chemistry 32:189–192.
Verpoorte JA, Mehta S, Edsall JT (1967) Esterase activities of human carbonic anhydrases B and C. The Journal of biological chemistry 242:4221–4229.
Sujayev A, Garibov E, Taslimi P, Gülçin İ, Gojayeva S, Farzaliyev V, Alwasel SH, Supuran CT (2016) Synthesis of some tetrahydropyrimidine-5-carboxylates, determination of their metal chelating effects and inhibition profiles against acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase. Journal of Enzyme Inhibition and Medicinal Chemistry 31:1531–1539.
Caglayan C, Taslimi P, Türk C, Gulcin İ, Kandemir FM, Demir Y, Beydemir Ş (2020) Inhibition effects of some pesticides and heavy metals on carbonic anhydrase enzyme activity purified from horse mackerel (Trachurus trachurus) gill tissues. Environmental Science and Pollution Research International 27:10607–10616.
Artunc T, Menzek A, Taslimi P, Gulcin I, Kazaz C, Sahin E (2020) Synthesis and antioxidant activities of phenol derivatives from 1,6-bis(dimethoxyphenyl)hexane-1,6-dione. Bioorganic chemistry 100: 103884.
Gulçin I, Alwasel SH (2022) Metal ions, metal chelators and metal chelating assay as antioxidant method. Processes 10(1):132.
Spassov VZ, & Yan L (2013) Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0. Proteins Struct Funct Bioinforma 81:704–714.
Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: A program for macromolecular energy minimization and dynamics calculations. J Comp Chem 4:187–217.
Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, et. al (2009) Wallingford CT Gaussian 09 Revision E.01.
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791.
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Delivery Rev 23:3–25.
Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD (2002) Molecular properties that influence the oral bioavailability of drug candidates. J Med Chem 45:2615–2623.
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv. Rev 23:3–25.

Scheme 1 and 2 is available in supplementary section.

No competing interests reported.

Scheme1.png
Scheme 1 Synthesis of imidazolium chloride/bromide salts containing an ethyl group and methyl/methoxy substituted benzyl group (2a–g)
Scheme2.png
Scheme 2 Synthesis of imidazolium bromide salts containing trifluoromethyl, trifluoromethoxy, chloride, and fluorine substituted benzyl group (3a-f)
SUPPLEMENTARYDATA.docx

Download PDF

Editorial decision: Major revision
01 Nov, 2022
Reviews received at journal
24 Oct, 2022
Reviewers agreed at journal
17 Oct, 2022
Reviewers invited by journal
16 Oct, 2022
Editor assigned by journal
14 Oct, 2022
Submission checks completed at journal
13 Oct, 2022
First submitted to journal
13 Oct, 2022

You are reading this latest preprint version

Acetylphenyl-Substituted Imidazolium Salts: Synthesis, Characterization, in silico Studies and Inhibitory Properties against Some Metabolic Enzymes

Status:

Version 1

Abstract

Figures

Introduction

Results And Discussion

Synthesis

Enzyme Inhibition Results

In Silico Studies

Conclusions

Material And Methods

Material and equipment

Synthesis

Synthesis of 1-(4-acetylphenyl)-3-ethylimidazolium bromide (2a)

Synthesis Of 1-(4-acetylphenyl)-3-benzylimidazolium Chloride (2b)

Synthesis Of 1-(4-acetylphenyl)-3-(2-methylbenzyl)imidazolium Chloride (2c)

Synthesis Of 1-(4-acetylphenyl)-3-(3-methylbenzyl)imidazolium Chloride (2d)

Synthesis Of 1-(4-acetylphenyl)-3-(4-methylbenzyl)imidazolium Chloride (2e)

Synthesis Of 1-(4-acetylphenyl)-3-(2,3,4,5,6-pentamethylbenzyl)imidazolium Chloride (2f)

Synthesis Of 1-(4-acetylphenyl)-3-(3,4,5-trimethoxybenzyl)imidazolium Chloride (2g)

Synthesis Of 1-(4-acetylphenyl)-3-(2-trifluoromethylbenzyl)imidazolium Bromide (3a)

Synthesis Of 1-(4-acetylphenyl)-3-(3-trifluoromethylbenzyl)imidazolium Bromide (3b)

Synthesis Of 1-(4-acetylphenyl)-3-(4-trifluoromethylbenzyl)imidazolium Bromide (3c)

Synthesis Of 1-(4-acetylphenyl)-3-(4-trifluoromethoxybenzyl)imidazolium Bromide (3d)

Synthesis Of 1-(4-acetylphenyl)-3-(4-fluorobenzyl)imidazolium Bromide (3e)

Synthesis Of 1-(4-acetylphenyl)-3-(2-chloro-4-fluorobenzyl)imidazolium Bromide (3f)

Biochemical Studies

AChE inhibition assay

Hcas Inhibition Assay

Ache And Hcas Kinetic Assay

In Silico Studies

Declarations

References

Scheme

Additional Declarations

Supplementary Files

Status:

Version 1