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The design of fluoroquinolone-based cholinesterase inhibitors: Synthesis, biological evaluation and in silico docking studies
⁎Corresponding author. nullah@kfupm.edu.sa (Nisar Ullah)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
An enhanced acetylcholinesterase (AChE) activity is a hallmark in early stages of Alzheimer's ailment that results in decreased acetylcholine (ACh) levels, which in turn leads to cholinergic dysfunction and neurodegeneration. Consequently, inhibition of both AChE and butyrylcholinesterase (BChE) is important to prolong ACh activity in synapses for the enhanced cholinergic neurotransmission. In this study, a series of new fluoroquinolone derivatives (7a-m) have synthesized and evaluated for AChE and BChE inhibitory activities. The screening results suggested that 7 g bearing ortho fluorophenyl was the most active inhibitor against both AChE and BChE, exhibiting IC50 values of 0.70 ± 0.10 µM and 2.20 ± 0.10 µM, respectively. The structure–activity relationship (SAR) revealed that compounds containing electronegative functions (F, Cl, OMe, N and O) at the ortho position of the phenyl group exhibited higher activities as compared to their meta- and/or para substituted counterparts. Molecular docking studies of synthesized compounds 7a, 7g, 7j and 7l docked into the active site of AChE and 7a-f docked into the active site of BChE revealed that these compounds exhibited conventional H-bonding along with π-π interaction with the active residues of AChE through their electronegative functions and phenyl ring, respectively. All the synthesized compounds are characterized by spectroscopic methods including FT-IR, 1H- and 13C NMR as well as elemental analysis. This is the first example of fluoroquinolone-based cholinesterase inhibitors.
Keywords
Fluoroquinolone derivatives
Acetylcholinesterase
Butyrylcholinesterase
Molecular docking
SAR
1 Introduction
Alzheimer's disease (AD) is a chronic clinical syndrome associated with senile dementia in older adults (Jagust, 2013). The progressive neurodegenerative disorder is characterized by a global impairment of higher mental function including deterioration of memory and cognitive functions (Bartus et al., 1982). The major characteristics of AD are accumulation of phosphorylated microtubule-associated proteins (Tau) and senile plaques containing aggregated amyloid beta (AB) peptides in the cholinergic neurons inside the brain (Glabe, 2005; Morris et al., 2015). In addition, an enhanced acetylcholinesterase (AChE) activity in early stages of the ailment is another hallmark that leads to decrease in acetylcholine (ACh) levels. The consequences of these features result in cholinergic dysfunction and neurodegeneration, which lead to memory and cognitive deficits (Moodie et al., 2019). Meta-analysis of datasets suggests that global dementia cases were 35.6 million in 2010, which will be increasing to 115.4 million in 2050 (Prince et al., 2013).
The main role played by cholinesterase enzymes, AChE and butyrylcholinesterase (BChE), is the hydrolysis of choline esters, including natural acetylcholine (Moodie et al., 2019). The AChE and BChE possess more than 50% sequence homology and bear a catalytic triad (Ser–Glu–His) in the bottom of a deep and narrow cavity. AChE is abundantly found in neurons and erythrocytes whereas BChE is typically located in the liver and plasma. AChE has a remarkably high specific catalytic activity; it can hydrolyze up to 5000 ACh molecules per second into acetate and choline. On the other hand, BChE can effectively hydrolyze both ACh and the larger, non-natural butyrylcholine.
Inhibition of cholinesterase impede the breaking down of ACh, which promote increase in both the level and duration of the neurotransmitter action. It is pertinent to mention that inhibition of AChE alone can lead to decline in its activity in the brain. However, decrease in the AChE activity is compensated by a considerable increase of brain BChE levels (up to 90%), which causes further ACh deficiency and deterioration of cognitive functions (Anand and Singh, 2013). Consequently, inhibition of both AChE and BChE is important in order to prolong the ACh activity in synapses for the enhancement of cholinergic neurotransmission. At present, cholinesterase inhibitors (ChEI) such as tacrine, galanthamine, donepezil and rivastigmine are the only class of compounds that have proven to be effective in alleviating cognitive and functional symptoms of AD (Fig. 1) (Francis et al., 2005).
Chemical structures of selected AChE inhibitors in pharmacotherapy of AD.
Nevertheless, short half-lives of approved ChEI have restricted their clinical effectiveness. In addition, they suffer from limitations such as activation of peripheral cholinergic systems, hepatotoxicity, inadequate activity, and gastric disorder (Farlow, 1992; Knapp, 1994; Rogers et al., 1998). To surmount the side effect of approved ChEI, search for long acting ChEI for optimum therapeutic benefits and minimal liabilities is a continued process.
The 4-quinolone-3-carboxylic acid scaffold is a privileged molecular framework frequently found in many commercial drugs (Morten et al., 2015; Bisacchi, 2015; Huse and Whiteley, 2011; Jadulco, et al., 2014) and wide range of bioactive compounds that possess antiviral, antibiotic, antitumor and antiparasitic activities (Mugnaini, et al., 2009; Baraldi, et al., 2012; Hiltensperger, et al., 2012; Ma, et al., 2009; Lucero, et al., 2006). In addition, introduction of fluorine at C-6 of the quinolone scaffold produces 6-fluoroquinolones which bear improve tissue penetration and binding to the DNA gyrase enzyme, resulting in broadening the spectrum of activity against both Gram-negative and Gram-positive pathogens (Van Bambeke and Tulkens, 2009; Wright, 2000; Gootz and Brighty 1996). For example, fluoroquinolones are known to be effective against difficult to treat infections such as prostatitis (Dalhoff and Weidner, 1988) and meningitis (Anderson, 2004). Soukup et al., have synthesized hybrid molecules as multifunctional agents for AD (Fig. 2) (Hepnarova, et al., 2018). Two pharmacologically distinct entities, tacrine and benzyl quinolone carboxylic acid, were connected by the alkyl spacer of varying lengths into one molecule. The hybrid molecules compounds 2 (hACHE IC50 = 0.0745 ± 0.0031 μM; hBCHE IC50 = 0.0833 ± 0.0050 μM) and and 3 (hACHE IC50 = 0.0419 ± 0.0011 μM; hBCHE IC50 = 3.72 ± 0.13 μM) that were linked through 2 to 3 carbon spacer were proved to be the most potent inhibitors of human AChE (hAChE) and human BChE (hBChE), exhibiting activities in the nanomolar range. On the other hand, phenylcarbamoyl moiety is considered an important entity of ACHE inhibitors phenserine and tolserine (Sharma, 2019). Moreover, tolserine that contains 2‑methyl group in its phenylcarbamoyl moiety was found to be 200‑fold more selective and more potent against hAChE as compared to phenserine, which suggests variation in the phenylcarbamoyl moiety is important. In our designed compounds, we replaced tacrine function with arylacetyl moiety that bears various substituents in the phenyl ring. The introduction of fluorine at C-6 of quinolone in known to enhance tissue penetration and volume of distribution of the drug (Gootz and Brighty, 1996) (Fig. 2).
Design of fluoroquinolones cholinesterase inhibitors based on tacrine- benzyl quinolone carboxylic acid hybrids.
Inspire by the work of Soukup et al., coupled with our earlier efforts devoted towards the synthesis of ChEI (Yar et al., 2014), we have designed fluoroquinolones cholinesterase inhibitors having a general chemical structure of 3. Through this communication, we wish to disclose the synthesis, biological evaluation and in-silico docking studies of these AChE and BChE inhibitors.
2 Experimental
2.1 Chemistry
General: Melting points were determined on a Büchi apparatus (Büchi Labortechnik AG, Switzerland) and uncorrected. Elemental analysis was carried out on a Perkin Elmer Elemental Analyzer Series 11 Model 2400 (PerkinElmer Inc. USA). IR spectra were recorded on a Perkin Elmer 16F PC FTIR spectrophotometer (PerkinElmer Inc. USA). 1H and 13C NMR spectra in CDCl3 and d6-DMSO on a Bruker AVANCE III 400 MHz spectrometer, using TMS as internal standard. Analytical TLC on silica gel 60 F254 plates (E. Merck) and column chromatography was performed on silica gel (200–400 mesh, E. Merck).
2.2 Synthesis of intermediate 6 and fluoroquinolone derivatives 7a-m
2.2.1 N-(2-Aminoethyl)-7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (6)
To a solution of fluoroquinolonic acid 4 (5 g, 17.8 mmol) in anhydrous CHCl3 (40 mL) at 0 °C was added SOCl2 (7.5 mL) dropwise in 30 min and the mixture was stirred overnight at room temperature. The volatiles were evaporated under vacuum and residues were co-concentrated with anhydrous CHCl3 and evaporated under reduced pressure to produce acid chloride (5), which was then added portionwise to a solution of ethylenediamine (10 mL, 184.80 mmol) in anhydrous CHCl3 (30 mL) at 0 °C with a continuous stirring. After being stirred overnight at room temperature, the mixture was diluted with chloroform (70 mL) and washed twice with sat. NaHCO3 (20 mL) followed by distilled water (20 mL). The organic layer was dried with anhydrous Na2SO4 and evaporated to dryness under reduced pressure to obtain the residues, which was then resolved by passing through a plug of silica, eluting with methanol:chloroform (5:95) to afford the desired compound 3 as a light yellow solid (4.31 g, 75%). M.p. 160–162 °C. IR (neat): 3370, 3263, 3084, 2940, 1657, 1598, 1539, 1471, 1342, 1253, 1183, 1030, 898, 854, 763, 700 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.11 (m, 2H, CH2-cyclopropyl), 1.29 (m, 2H, CH2-cyclopropyl), 2.69 (t, J = 5.6 Hz, 2H, N-CH2), 3.30 (t, J = 5.6 Hz, 2H, N-CH2), 3.76 (m, 1H, CH-cyclopropyl), 8.08 (d, JH-F = 9.2 Hz, 1H, H-5), 8.35 (d, JH-F = 6.0 Hz, 1H, H-8), 8.67 (s, 1H, H-2), 9.75 (t, J = 5.2 Hz, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.07, 35.84, 41.65, 111.14, 112.21 (d, J = 22.4 Hz), 120.76, 125.99 (d, J = 22.4 Hz), 127.32, 138.03, 148.08, 153.71, 164.0, 174.53. Anal. Calcd for C15H15ClFN3O2 (%): C 55.65, H 4.67, N 12.98. Found: C 55.58, H 4.70, N 12.94.
2.2.2 7-Chloro-N-(2-(2-chlorobenzamido)ethyl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (7a)
To a solution of mixture of compound 6 (0.20 g, 0.62 mmol) and 2-chlorobenzoic acid (0.15 g, 0.93 mmol) in anhydrous DMF (15 mL) was sequentially added hydroxybenzotriazole (0.21 g, 1.55 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.24 g, 1.24 mmol) and Et3N (0.18 mL, 1.85 mmol) and the reaction was stirred overnight at room temperature for 24 h. The mixture was diluted with ethyl acetate (15 mL) and the organic layer was washed twice with 1 M NaOH (10 mL), brine (10 mL) and distilled water (10 mL). The organic layer was dried with anhydrous Na2SO4 and evaporated under reduced pressure to obtain a yellowish residue, which was purified by silica column, eluting with chloroform:methanol (97.5:2.5) to afford the desired 7a as a light-yellow solid (yield 85%). M.p. 185–187 °C. IR (neat): 3256, 3073, 2934, 1655, 1597, 1546, 1475, 1319, 1253, 1037, 763, 670 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.19 (m, 2H), 1.40 (m, 2H), 3.54 (m, 1H), 3.73 (m, 4H), 7.07 (br. s, 1H, aromatic-H), 7.27–7.37 (m, 3H, aromatic-H), 7.64 (s, 1H, aromatic-H), 8.09 (s, 1H, aromatic-H), 8.21 (d, J = 3.8 Hz, 1H, aromatic-H), 8.85 (br. s, 1H, NH), 10.13 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.55, 29.71, 35.59, 40.86, 113.14 (d, J = 21.9 Hz), 119.49, 126.98, 129.90, 130.13, 130.89, 131.05, 135.47, 137.40, 148.06, 162.63, 167.08, 175.48. Anal. Calcd for C22H18Cl2FN3O3 (%): C 57.16, H 3.92, N 9.09. Found: C 57.10, H 3.93, N 9.04.
2.2.3 7-chloro-N-(2-(3-chlorobenzamido)ethyl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (7b)
Following the same procedure adopted for synthesis of 7a, the reaction of compound 6 with 3-chlorobenzoic acid afforded the desired compound 7b as a white solid (yield 83%). M.p. 174–176 °C. IR (neat): 3260, 3070, 2931, 1653, 1599, 1540, 1474, 1340, 1257, 1180, 1101, 1032, 852, 758, 697 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.22 (m, 2H), 1.43 (m, 2H), 3.49 (m, 1H), 3.68 (m, 2H), 3.75 (m, 2H) 7.27 (s, 1H, aromatic-H), 7.39 (m, 1H, aromatic-H), 7.46 (d, 1H, J = 6.8 Hz, aromatic-H), 7.80 (d, 1H, J = 6.8 Hz, aromatic-H), 7.89 (s, 1H, aromatic-H), 8.10 (s, 1H, aromatic-H), 8.19 (d, J = 9.2 Hz, 1H, aromatic-H), 8.92 (br. s, 1H, NH), 10.27 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 7.63, 31.93, 38.49, 40.81, 110.78, 112.20 (d, J = 22.8 Hz), 119.13, 125.23, 127.23, 129.30, 130.61, 133.94, 136.27, 147.28, 161.68, 165.48, 174.46. Anal. Calcd for C22H18Cl2FN3O3 (%): C 57.16, H 3.92, N 9.09. Found: C 57.11, H 3.93, N 9.05.
2.2.4 Synthesis of 7-chloro-N-(2-(4-chlorobenzamido)ethyl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (7c)
Following the same procedure adopted for synthesis of 7a, the reaction of compound 6 with 4-chlorobenzoic acid gave compound 7c as a light yellow solid (yield 81%). M.p. 199–200 °C. IR (neat): 3257, 3071, 2931, 1652, 1603, 1543, 1473, 1353, 1310, 1255, 1095, 1026, 850, 760, 606 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.22 (m, 2H), 1.43 (m, 2H), 3.48 (m, 1H), 3.65 (m, 2H), 3.72 (m, 2H), 7.39 (d, J = 8.4, 2H, aromatic-H), 7.83 (d, J = 8.4 Hz, 2H, aromatic-H), 8.02 (s, 1H, aromatic-H), 8.08 (d, J = 7.4 Hz, 1H, aromatic-H), 8.18 (d, J = 8.8 Hz, 1H, aromatic-H), 8.87 (br. s, 1H, NH), 10.26 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.50, 35.47, 39.39, 42.34, 112.89 (d, J = 22.2 Hz), 119.46, 128.65, 128.79, 132.71, 137.32, 137.42, 147.88, 153.12, 156.40, 161.40, 166.37, 175.30. Anal. Calcd for C22H18Cl2FN3O3 (%): C 57.16, H 3.92, N 9.09. Found: C 57.12, H 3.94, N 9.04.
2.2.5 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(2-methoxybenzamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7d)
Following the same procedure adopted for synthesis of 7a, the reaction of compound 6 with 2-methoxybenzoic acid afforded the desired compound 7d as a white solid (yield 80%). M.p. 173–174 °C. IR (neat): 3365, 3254, 3070, 2928, 1642, 1603, 1528, 1467, 1347, 1296, 1246, 1178, 1108, 1077, 886, 755, 699, 592 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.25 (m, 2H), 1.42 (m, 2H), 3.54 (m, 1H), 3.75 (br. s, 4H), 4.00 (s, 3H), 6.97 (m, 2H, aromatic-H), 7.08 (t, J = 7.6 Hz, 1H, aromatic-H), 7.45 (t, J = 8.4 Hz, 1H, aromatic-H), 8.15 (m, 1H, aromatic-H), 8.20 (m, 2H, aromatic-H), 9.03(br. s, 1H, NH), 10.18 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.31, 35.17, 39.10, 39.88, 55.85, 110.23, 113.0 (d, J = 22.5 Hz), 119.35, 121.07, 132.15, 132.74, 137.39, 147.72, 157.61, 161.32, 165.63, 175.26. Anal. Calcd for C23H21ClFN3O4 (%): C 60.33, H 4.62, N 9.18. Found: C 60.29, H 4.66, N 9.14.
2.2.6 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(3-methoxybenzamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7e)
Following the procedure adopted for synthesis of 7a, the reaction of compound 6 with 3-methoxybenzoic acid yielded the desired compound 7e as a white solid (yield 84%). M.p. 167–168 °C. IR (neat): 3366, 3264, 3074, 2942, 1648, 1597, 1535, 1472, 1353, 1300, 1182, 1029, 892, 759, 698 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.29 (m, 2H), 1.45 (m, 2H), 3.61 (m, 1H), 3.75–3.83 (m, 4H), 3.89 (s, 3H), 7.04 (dd, J = 3.2, 8.2 Hz, 1H, aromatic-H), 7.23 (m, 2H, aromatic-H), 7.28 (m, 1H, aromatic-H), 7.54 (m, 2H, aromatic-H), 8.24 (m, 1H, aromatic-H), 8.20 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.35, 35.37, 39.29, 41.75, 55.43, 112.18, 112.95 (d, J = 22.7 Hz), 117.80, 119.23, 119.38, 129.45, 135.78, 137.29, 147.84, 157.03, 159.67, 167.35, 175.24. Anal. Calcd for C23H21ClFN3O4 (%): C 60.33, H 4.62, N 9.18. Found: C 60.28, H 4.66, N 9.13.
2.2.7 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(4-methoxybenzamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7f)
Following the procedure adopted for synthesis of 7a, the reaction of compound 6 with 4-methoxybenzoic acid yielded the desired compound 7f as a white solid (yield 86%). M.p. 176–177 °C. IR (neat): 3317, 3252, 3082, 2937, 1649, 1544, 1469, 1350, 1300, 1298, 1250, 1182, 1027, 843, 765, 614 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.25 (m, 2H), 1.45 (m, 2H), 3.56 (m, 1H), 3.72 (br. s, 2H), 3.76 (br. s, 2H), 3.87 (s, 3H), 6.94 (d, J = 8.8 Hz, 2H, aromatic-H), 7.87 (d, J = 7.2 Hz, 2H, aromatic-H), 8.13 (s, 1H, aromatic-H), 8.20 (d, J = 8.8 Hz, 2H, aromatic-H), 8.95 (br. s, 1H, NH), 10.25 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.42, 35.43, 39.46, 41.62, 55.38, 112.98 (d, J = 22.7 Hz), 113.60, 119.39, 126.61, 129.07, 137.30, 147.87, 154.72, 157.19, 162.01, 167.15, 175.25. Anal. Calcd for C23H21ClFN3O4 (%): C 60.33, H 4.62, N 9.18. Found: C 60.28, H 4.66, N 9.15.
2.2.8 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(2-fluorobenzamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7 g)
Following the procedure adopted for synthesis of 7a, compound 7 g was obtained from the reaction of 6 with 2-fluorobenzoic acid as an off-white solid (yield 81%). M.p. 205–206 °C. IR (neat): 3243, 3062, 2928, 1643, 1543, 1466, 1350, 1310, 1249, 1177, 1104, 1026, 900, 760 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.23 (m, 2H), 1.42 (m, 2H), 3.54 (m, 1H), 3.73–3.76 (m, 4H), 7.12 (dd, J = 8.0, 3.6 Hz, 1H, aromatic-H), 7.26 (m, 2H, aromatic-H), 7.47 (m, 2H, aromatic-H), 8.12 (m, 2H, aromatic-H), 8.21 (d, J = 8.8 Hz, 1H, aromatic-H), 8.92 (br. s, 1H, NH), 10.11 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.48, 35.29, 39.15, 40.82, 113.62 (d, J = 22.9 Hz), 116.04 (d, J = 23.9 Hz), 119.31, 121.46, 124.60, 131.89, 133.00, 137.42, 147.94, 159.40, 161.86, 163.72, 175.44. Anal. Calcd for C22H18ClF2N3O3 (%): C 59.27, H 4.07, N 9.42. Found: C 59.25, H 4.10, N 9.38.
2.2.9 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(3-fluorobenzamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7 h)
Following the procedure adopted for synthesis of 7a, compound 7 h was obtained from the reaction of 6 with 3-fluorobenzoic acid as an off-white solid (yield 84%). M.p. 184–186 °C. IR (neat): 3320, 3216, 3041, 2945, 1642, 1583, 1537, 1468, 1349, 1251, 1122, 1022, 932, 798, 747, 677, 616 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.24 (m, 2H), 1.44 (m, 2H), 3.57 (m, 1H), 3.71–3.76 (m, 4H), 7.19 (t, J = 7.4 Hz, 1H, aromatic-H), 7.43 (d, J = 6.0 Hz, 1H, aromatic-H), 7.61 (d, J = 9.2 Hz, 1H, aromatic-H), 7.69 (d, J = 7.2 Hz, 1H, aromatic-H), 7.96 (br. s, 1H, aromatic-H), 8.13 (s, 1H, aromatic-H), 8.21 (d, J = 8.8 Hz, 1H, aromatic-H), 9.01 (br. s, 1H, NH), 10.33 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.61, 35.46, 39.37, 42.39, 113.06, 114.76 (d, J = 22.7 Hz), 118.16 (d, J = 21.2 Hz), 122.90, 130.03, 137.43, 148.03, 154.70, 155.52, 161.60, 166.50, 175.50. Anal. Calcd for C22H18ClF2N3O3 (%): C 59.27, H 4.07, N 9.42. Found: C 59.24, H 4.10, N 9.36.
2.2.10 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(4-fluorobenzamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7i)
Following the procedure adopted for synthesis of 7a, the compound 7i was obtained from the reaction of 6 with 4-fluorobenzoic acid as an off-white solid (yield 83%). M.p. 217–219 °C. IR (neat): 3254, 3068, 2928, 1649, 1600, 1545, 1469, 1356, 1301, 1229, 1165, 1095, 1028, 846, 764, 660 cm−1; 1H NMR (400 MHz, DMSO‑d6): δ 1.21 (m, 2H), 1.43 (m, 2H), 3.55 (m, 1H), 3.69 (br. s, 2H), 3.76 (m, 2H), 7.11 (m, 2H, aromatic-H), 7.84 (br. s, 1H, aromatic-H), 8.92 (m, 2H, aromatic-H), 8.12 (d, J = 5.6 Hz, 1H, aromatic-H), 8.19 (d, J = 8.8 Hz, 1H, aromatic-H), 8.89 (br. s, 1H, NH), 10.27 (br. s, 1H, NH); 13C NMR (100 MHz, DMSO‑d6): δ 8.08, 35.88, 38.48, 111.07, 112.42 (d, J = 22.2 Hz), 115.64 (d, J = 21.5 Hz), 120.86, 127.40, 130.26 (d, J = 8.9 Hz), 131.38, 138.36, 148.22, 153.79, 156.25, 164.35, 165.65, 174.67. Anal. Calcd for C22H18ClF2N3O3 (%): C 59.27, H 4.07, N 9.42. Found: C 59.23, H 4.12, N 9.36.
2.2.11 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(furan-2-carboxamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7j)
Following the procedure adopted for synthesis of 7a, the reaction of compound 6 with 2-furoic acid yielded the desired compound 7j as a light yellow solid (yield 82%). M.p. 215–216 °C. IR (neat): 3366, 3258, 3074, 2941, 1648, 1534, 1471, 1346, 1249, 1183, 1112, 1024, 890, 759, 700, 600 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.25 (m, 2H), 1.43 (m, 2H), 3.57 (m, 1H), 3.68 (m, 2H), 3.72 (m, 2H), 6.50 (d, J = 1.6 Hz, 1H, aromatic-H), 7.13 (s, 1H, aromatic-H), 7.48 (d, J = 6.2 Hz, 1H, aromatic-H), 8.12 (s, 1H, aromatic-H), 8.17 (d, J = 7.2 Hz, 1H, aromatic-H), 8.36 (m, 1H, aromatic-H), 8.99 (d, J = 8.8 Hz, 1H, aromatic-H), 8.93 (br. s, 1H, NH), 10.16 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.50, 35.59, 39.71, 42.61, 111.90, 113.08 (d, J = 21.8 Hz), 114.01, 118.64, 119.48, 137.36, 141.70, 144.07, 148.05, 157.19, 158.82, 161.80, 165.60, 175.39. Anal. Calcd for C20H17ClFN3O4 (%): C 57.49, H 4.10, N 10.06. Found: C 57.45, H 4.13, N 10.00.
2.2.12 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(furan-3-carboxamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7 k)
Following the procedure adopted for synthesis of 7a, the reaction of compound 6 with 3-furoic acid yielded the desired compound 7 k as a pale yellow solid (yield 80%). M.p. 197–198 °C. IR (neat): 3366, 3255, 3075, 2939, 1648, 1596, 1533, 1470, 1343, 1249, 1185, 1021, 888, 758, 696, 602 cm−1; 1H NMR (400 MHz, DMSO‑d6): δ 1.11 (m, 2H), 1.29 (m, 2H), 3.38 (m, 2H), 3.48 (m, 2H), 3.76 (m, 1H), 6.82 (d, J = 1.6 Hz, 1H, aromatic-H), 7.70 (t, J = 1.6 Hz, 1H, aromatic-H), 8.10 (s, 1H, aromatic-H), 8.14 (d, J = 7.2 Hz, 1H, aromatic-H), 8.32 (m, 1H, aromatic-H), 8.38 (d, J = 6.4 Hz, 1H, aromatic-H), 8.69 (s, 1H, aromatic-H), 9.79 (t, J = 6.0 Hz, 1H, NH); 13C NMR (100 MHz, DMSO‑d6): δ 7.65, 35.47, 39.08, 108.96, 110.66, 112.07 (d, J = 22.1 Hz), 120.48, 122.78, 125.42, 127.02, 137.76, 144.00, 145.08, 147.85, 153.36, 155.82, 161.82, 163.85, 174.25. Anal. Calcd for C20H17ClFN3O4 (%): C 57.49, H 4.10, N 10.06. Found: C 57.44, H 4.14, N 10.01.
2.2.13 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(nicotinamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7 l)
Following the procedure adopted for synthesis of 7a, the reaction of compound 6 with nicotinic acid afforded the desired compound 7 l as an off-white solid (yield 79%). M.p. 216–218 °C. IR (neat): 3355, 3257, 3072, 2940, 1648, 1535, 1470, 1345, 1250, 1182, 1024, 890, 757, 700, 608 cm−1; 1H NMR (400 MHz, CDCl3): δ 1.27 (m, 2H), 1.48 (m, 2H), 3.60 (m, 1H), 3.73 (br. s, 2H), 3.79 (br. s, 2H), 7.95 (m, 2H, aromatic-H), 8.13 (m, 2H, aromatic-H), 8.14 (d, J = 8.8 Hz, 1H, aromatic-H), 8.58 (br. s, 1H, aromatic-H), 9.01 (s, 1H, aromatic-H), 9.48 (n, 2H, aromatic-H, NH), 10.42 (br. s, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 8.42, 35.20, 38.93, 42.70, 110.98, 113.07 (d, J = 22.7 Hz), 119.27, 123.93, 127.19, 127.82, 130.66, 136.86, 137.33, 147.18, 147.79, 150.47, 164.83, 166.76, 175.17. Anal. Calcd for C21H18ClFN4O3 (%): C 58.82, H 4.23, N 13.06. Found: C 58.78, H 4.26, N 13.00.
2.2.14 Synthesis of 7-chloro-1-cyclopropyl-6-fluoro-N-(2-(isonicotinamido)ethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7 m)
Following the procedure adopted for synthesis of 7a, the reaction of compound 6 with isonicotinic acid gave the desired compound 7 m as an off-white solid (yield 84%). M.p. 219–220 °C. IR (neat): 3355, 3257, 3073, 2940, 1648, 1536, 1471, 1346, 1250, 1182, 1025, 890, 757, 698, 608 cm−1; 1H NMR (400 MHz, DMSO‑d6): δ 1.24 (m, 2H), 1.49 (m, 2H), 3.61 (m, 1H), 3.73 (m, 2H), 3.78 (m, 2H), 8.14 (d, J = 6.0 Hz, 1H, aromatic-H), 8.18 (d, J = 8.8 Hz, 1H, aromatic-H), 8.45 (d, J = 5.2 Hz, 1H, aromatic-H), 8.96 (m, 3H, aromatic-H), 9.52 (br. s, 1H, NH), 10.58 (br. s, 1H, NH); 13C NMR (100 MHz, DMSO‑d6): δ 8.09, 35.89, 38.34, 111.08, 112.46 (d, J = 22.3 Hz), 120.88, 121.68, 126.23, 127.41, 138.14, 141.84, 148.22, 150.70, 153.79, 156.24, 164.33, 165.40, 174.65. Anal. Calcd for C21H18ClFN4O3 (%): C 58.82, H 4.23, N 13.06. Found: C 58.77, H 4.25, N 13.01.
2.3 In vitro AChE and BChE inhibition assay
The AChE and BChE (source human: sigma 9000–81-1 and 9001–08-5, respectively) inhibitory studies were performed according to Ellman’s method (Ellman et al., 1961). Donepezil was used as a positive control. Solutions of tested compounds 7a-m in DMSO with five different concentrations (0.01, 0.1, 0.5, 5.0, 10.0 µM) were prepared. For enzyme inhibition assay, 20 μL of the corresponding enzyme (0.2 units/mL in 1 M phosphate-pH 8.0 containing 25% v/v glycerol) was added to a 24- well plate containing 2000 μL of PBS, 30 μL solution of the tested compound and 60 μL of 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, (0.5 mM). After 3 min of incubation, 20 μL of acetylthiocholine iodide/S-butyrylthiocholine chloride (10 mM) was added and then further incubated for at least 1 min at 25 °C. The reaction was initiated when enzyme was added and the blank reading were taken for all chemicals except the inhibitor. The absorbance of the reaction was measured, within the 5 min, at 412 nm on a microplate reader (BioTek synergy HT). The absorbance was measured using a microplate reader at 410 nm wavelength against the blank reading containing DMSO instead of test compound. The IC50 values, the concentration of inhibitor needed for 50% inhibition of the enzyme, were deduced graphically from inhibition curves of log inhibitor concentration vs. percent of inhibition. Results are reported as mean ± standard deviation for at least three different experiments.
2.4 Docking protocol
Autodock 4.2.6 molecular docking software was employed for protein and ligand preparations (Morris et al., 2009). The crystal structures of AChE in complex with donepezil (PDB ID: 4EY7) and BChE in complex with 2-(butyrylsulfanyl)-N,N,N-trimethylethanaminium were retrieved from the protein data bank (Cheung et al., 2012, Nicolet et al., 2003). Enzyme active sites were identified based on co-crystallized receptor-ligand complex structure of AChE and BChE. The re-docking of the original ligands donepezil and 2-(butyrylsulfanyl)-N,N,N-trimethylethanaminium into the active sites of AChE and BChE was well reproduced with RMSD value of 0.63 and 2.78 Å, respectively. Molecular geometries of fluoroquinolone derivatives were minimized at Merck molecular force field 94 (MMFF94) level44 and the optimized structures were saved as PDB files. Docking studies were performed using Lamarckian genetic algorithm, with 500 as total number of run for binding sites for the original ligand, the synthesized derivatives. In each run, a population of 150 individuals with 27,000 generations and 250,000 energy evaluations were settled. Operator weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. The grid box centered at (10.634, −56.163, –23.873) Å has a dimension of 61 × 61 × 61 points with the spacing of 0.375 Å. 2D and 3D binding interactions between the docked fluoroquinolone derivatives were visualized using Discovry Studio Client (Discovery Studio Client is A Product of Accelrys Inc., San Diego, CA, USA).
3 Results and discussion
3.1 Chemistry
Synthesis of the target compounds 7a-m was achieved as outlined in scheme 1. The reaction of fluoroquinolonic acid 4 with SOCl2 produced the acid chloride 5, which was then condensed with ethylenediamine to generate compound 6 in 75% yield from 4. The amidation of intermediate 6 with the appropriate acids in DMF, using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxybenzotriazole as coupling agents, provided the desired 7a-m in good (79–86%) yields (Scheme 1).
Synthesis of fluoroquinolone-based cholinesterase inhibitors: (a) SOCl2, CHCl3, RT overnight; (b) Ethylenediamine, RT overnight; (c) RCO2H, EDC hydrochloride, HOBt, Et3N, DMF, RT overnight.
3.2 Biological activities
3.2.1 In vitro AChE and BChE activity
All the synthesized compounds (7a-m) were screened for in vitro inhibition against AChE according to the Ellman method (Ellman et al., 1961). Donepezil was used as a positive control and screening results are summarized in Table 1.
Compound
Structure
AChE
IC50 (µM)BChE
IC50 (µM)
7a
2.70 ± 0.10
4.40 ± 0.20
7b
6.30 ± 0.20
12.30 ± 0.50
7c
4.40 ± 0.20
8.90 ± 0.30
7d
5.20 ± 0.20
10.20 ± 0.40
7e
12.80 ± 0.50
20.40 ± 0.60
7f
8.90 ± 0.40
15.10 ± 0.50
7 g
0.70 ± 0.10
2.20 ± 0.10
7 h
2.30 ± 0.20
3.70 ± 0.10
7i
1.30 ± 0.10
2.80 ± 0.10
7j
1.80 ± 0.10
3.30 ± 0.10
7 k
6.80 ± 0.20
9.90 ± 0.40
7 l
1.50 ± 0.10
2.40 ± 0.10
7 m
9.30 ± 0.40
16.90 ± 0.50
Donepezil (standard drug)
0.016 ± 0.12
4.5 ± 0.11
For the AChE inhibition assay, a clear trend of increased inhibitory activity for fluorophenyl based analogues was observed. In particular, placing a fluorine at the ortho position of the phenyl ring (C-2) has significantly enhanced the activity. Compound 7 g (IC50 = 0.70 ± 0.10 µM) turned out to be the most active compound in the series whose inhibition potential was closer to the standard donepezil (IC50 = 0.016 ± 0.12 µM) as compared other tested compounds. The activity was diminished to more than three-fold when fluorine was inserted at meta position to produce 7 h (2.30 ± 0.20 µM). Likewise, two-fold decline in the activity was observed when fluorine was moved to para position to generate 7i (1.30 ± 0.10 µM). The substitution of hydrogen atom with fluorine atom is a common practice in drug discovery (Gillis, et al., 2015). The introduction of fluorine into a therapeutic candidate can improve drug potency [Deng, et al., 2014; Lou, et al., 2015; Boehringer, et al., 2010] and target selectivity (Abbas et al., 2016; Meanwell et al., 2014; Gillis, et al., 2015). The enhancement in the binding affinity to a target protein can be achieved by either direct interaction with the protein or influencing the polarity of other functions of the molecule that interact with the protein. The stronger dipole of C-F bond may interact with other dipoles causing conformational bias that may result higher affinity and specificity (Mansha et al., 2016; Shah and Westwell, 2007). Moreover, the hydrogen bond acceptor ability of the fluorine permits the processing of fluorodeoxy analogues by enzymes, which would normally act on corresponding hydroxyl groups (Abeles RH, 1990). The highest activity of compound 7 g suggested that the electronegative fluorine with comparable van der Waals radius (1.47 Å) to hydrogen atom at the ortho position is important. Moreover, substituting fluorine with chlorine atom at the ortho position to provide 7a (2.70 ± 0.10 µM) resulted in almost four-fold decreased in the activity. Likewise, inserting less electronegative methoxy function at the ortho position, generating 7d (5.20 ± 0.20 µM), resulted in diminishing more than seven-fold activity. Interestingly, the activity pattern of both chloro- and methoxy analogues was similar to fluoro analogues i.e., the ortho derivatives, 7a and 7d, were found to be more active than the meta derivatives (7b and 7e), which in turn were more active than their para counterparts (7c and 7f). Furthermore, the 2-furyl analogue 7j (1.80 ± 0.10 µM) was proved to be more than eight-fold more active than its 3-furamide counterpart 7 k (6.80 ± 0.20 µM). In a similar fashion, 2-pyridyl derivative 7 l (1.50 ± 0.10 µM) showed six-fold higher activity than the 3-pyridyl analogue 7 m (9.30 ± 0.40) (Table 1). These results suggested that the electronegative function at the ortho position or closer to the amide linkage is vital for the improved AChE inhibitory activities of these compounds.
3.2.2 In vitro BChE activity
The BChE and AChE possess more than 50% sequence homology and share similar tertiary and quaternary structures. The two esterases also share matching catalytic triad (Ser–Glu–His) placed in the bottom of the active gorge. Nevertheless, the acyl pocket of BChE can accommodate larger substrates, which in turn provides wider substrate specificity (Nicolet et al., 2003). The in vitro inhibition potential of all the synthesized compounds (7a-m) against the BChE was studied. The screening results revealed that these compounds possess variable degree of inhibition, ranging between 2.20 and 16.90 μM. Compound 7 g (2.20 ± 0.10 µM) showed the highest inhibitory activity. As was the case with AChE inhibition, the BChE inhibitory activity was slightly decreased when the fluoro substituent was moved to para position (7i, 2.80 ± 0.10 µM). The activity was diminished to more than one-fold when the fluoro substituent was place to meta position (7 h, 3.70 ± 0.10 µM). In case of BChE inhibition, the structure–activity relationship exhibited almost similar as was the case with AChE inhibition. Compounds that bear electronegative function at the ortho position or closer to the amide linkage displayed better BChE inhibition as compared to their meta- and/or para counterparts. For instance, 2-pyridyl derivative 7 l (2.40 ± 0.10 µM) that exhibited comparable activity to the most active compound 7 g, displayed seven-fold higher activity than its 3-pyridyl counterpart 7 m (16.90 ± 0.50 µM). Likewise, the ortho substituted chloro- (7a, 4.40 ± 0.20 µM), methoxy- (7d, 10.20 ± 0.40 µM) and 2-furyl analogue (7j, 3.30 ± 0.10 µM) were found to be more active than their meta- and/or para-counterparts.
3.3 Molecular docking
In order to examine the possible binding modes, the synthesized compounds were docked into the active site of AChE and BChE, using Autodock package (Morris et al., 2009). Table 2 summarizes the calculated free binding energies of the stable complex (ligand-AChE and ligand-BChE), number of established intermolecular hydrogen bonding (H-bonding) between the docked ligand and active site residues of AChE and BChE, the closest residues to the docked compounds and their corresponding IC50 values.
Compound
Free binding energy (kcal/mol)
H-Bonds
Number of closest residues to the docked ligand in the active site
IC50 ± SEM
(a)
7a
−11.80
4
11
2.70 ± 0.10
7b
−12.14
3
10
6.30 ± 0.20
7c
−11.66
5
11
4.40 ± 0.20
7d
−11.18
3
8
5.20 ± 0.20
7e
−11.48
7
9
12.80 ± 0.50
7f
−11.32
5
9
8.90 ± 0.40
7 g
−11.49
3
9
0.70 ± 0.10
7 h
−11.54
3
13
2.30 ± 0.20
7i
−11.26
3
10
1.30 ± 0.10
7j
−10.62
3
8
1.80 ± 0.10
7 k
−10.50
3
9
6.80 ± 0.20
7 l
−11.13
5
10
1.50 ± 0.10
7 m
−11.21
4
11
9.30 ± 0.40
Donepezil
−11.11
1
8
0.016 ± 0.12
(b)
7a
−9.03
1
9
4.40 ± 0.20
7b
−9.21
2
9
12.30 ± 0.50
7c
−8.87
1
7
8.90 ± 0.30
7d
−8.68
3
9
10.20 ± 0.40
7e
−8.74
2
10
20.40 ± 0.60
7f
−8.54
2
10
15.10 ± 0.50
7 g
−8.52
2
10
2.20 ± 0.10
7 h
−8.44
1
9
3.70 ± 0.10
7i
−8.41
5
12
2.80 ± 0.10
7j
−7.62
3
11
3.30 ± 0.10
7 k
−8.24
3
11
9.90 ± 0.40
7 l
−8.26
3
9
2.40 ± 0.10
7 m
−8.63
4
9
16.90 ± 0.50
All the complexes between docked compounds and the active residues of AChE and BChE exhibited negative binding energies, indicating that binding of docking molecules was thermodynamically favorable process. For both AChE and BChE, the binding energies of the stable complexes formed between the docked compounds into the active sites of AChE and BChE are slightly varied, with variation less than 1.65 and 1.59 kcal mol−1 for AChE and BChE, respectively. Hence, binding energy may be considered as a weak descriptor in rationalizing the observed AChE and BChE inhibitions. Nevertheless, H-bonding between the substituents of the selected compounds and the active residues of AChE were helpful in explaining the observed AChE inhibitory activities. Since it has been experimentally observed that compounds having electronegative substituents at the ortho position (7a, 7 g, 7j and 7 l) exhibited higher AChE inhibitory activities as compared to their meta- and/or para substituted counterparts. The molecular docking study revealed that chloro moiety at the ortho position of 7a acted as H-bonding acceptor and showed two H-bonding with TYR B:337 and TYR B:341 in the active residues of AChE of distances 3.02 and 3.13 Å, respectively. The higher inhibition of 7c as compared to 7b may be associated to interactions between the chloro group at para position with GLN B:71 and TYR B:72 of distances 3.25 and 3.01 Å. Moreover, in 7a-AChE complex five hydrogen bonds were established between the functional groups of 7a and the amino acids ASP B:74, TYR B:341, TYR B:124, ARG B:296 and SER B:293 with distances of 2.27, 2.68, 2.76, 3.32 and 3.05 Å, respectively. Likewise, 7 g, 7j and 7 l exhibited H-bonding with TYR B:124 through their fluoro, 2-furyl and 2-pyridyl functions of distances 2.62, 3.27, 3.27 Å, respectively. The highest AChE inhibitory activity of compound 7 g can be explained by the shortest H-bonding (2.62 Å) and hence the strongest H-bonding between the fluoro group at the ortho position of the phenyl ring. In addition to the above, fluoroquinolone core of the docked molecules exhibited conventional H-bonding and π-π interaction with the active residues of AChE through its fluoro groups and phenyl ring, respectively (Fig. 3).
3D (right) and 2D (left) closest interactions between active site residues of acetylcholinesterase and synthesized compounds 7a, 7 g, 7j, and 7 l.
Similar to AChE inhibition, compounds having electronegative substituents at the ortho position (7a, 7 g, 7j and 7 l) exhibited higher BChE inhibitory activities as compared to their meta- and/or para substituted counterparts. Within the compounds bearing chlorophenyl moiety (7a-7c), 7a exhibited higher BChE inhibition compared to 7b and 7c, which may be linked to the higher number of interactions with the active site residues of the enzyme. Indeed, 7a that has chloro group at the ortho position in the chlorophenyl moiety exhibited three interactions with TRP A:430, ALA: 328 and TYR A:332, whereas compounds 4b and 4c that possess chloro group in the meta and para positions, respectively, showed fewer interactions with the active site residues (Fig. 4).
3D (right) and 2D (left) closest interactions between active site residues of BChE and synthesized compounds 7a, 7b, and 7c.
Likewise, in the methoxyphenyl analogues (7d-7f), compound 7d bearing methoxy function at the ortho position displayed higher BChE inhibition (IC50 = 10.20 ± 0.40 µM) as compared to the meta- (7e, (IC50 = 20.40 ± 0.60 µM) or para- (7f, IC50 = 15.10 ± 0.50 µM) counterparts. The higher inhibitory activity of 7d could be linked to higher strength of hydrogen bonding between the methoxy group and the amino acid Tyr 332. Indeed, the hydrogen bond distance between the methoxy group of 7d and TYR 332 (2.59 Å) was found to be shorter than that of between the methoxy group of 7e and 7f with TYR 440 (3.57 Å) and SER 198 (2.88 Å), respectively (Fig. 5). Finally, similar binding trends were observed for compounds 7 g-7i and 7j-7 m.
3D (right) and 2D (left) closest interactions between active site residues of BChE and synthesized compounds 7d, 7e, and 7f.
4 Conclusion
In conclusion, we synthesized a series of fluoroquinolone derivatives and evaluated them for in vitro inhibition against AChE and BChE enzymes. The screening results indicated a clear trend of increased inhibitory activity for compounds bearing electronegative functions at the ortho position of the phenyl group. Compounds 7 g that possesses ortho fluorophenyl was the most active inhibitor against both AChE (0.70 ± 0.10 µM0 and BChE (2.20 ± 0.10 µM). Molecular docking studies suggested that complexes between docked compounds and the active residues of AChE and BChE exhibited negative bending energies, suggesting binding of docking molecules was a thermodynamically favorable process. Moreover, ligands exhibited conventional H-bonding along with π-π interaction with the active residues of both AChE and BChE through their electronegative functions and phenyl ring, respectively.
Acknowledgements
The financial support from KFUPM project # SB191017 is gratefully acknowledged.
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