Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine and its bromo-derivative as dual cholinesterase inhibitors

Bioorganic Chemistry 90 (2019) 103062

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Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine and its bromo-derivative as dual cholinesterase inhibitors

T

Vijay K. Nuthakkia, Ramesh Mudududdlaa,b, Ankita Sharmab,c, Ajay Kumarb,c, ⁎ Sandip B. Bharatea,b, a

Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India Academy of Scientific & Innovative Research, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India c PKPD Toxicology & Formulation Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India b

ARTICLE INFO

ABSTRACT

Keywords: Cryptolepine Acetylcholinesterase Butyrylcholinesterase Alzheimer's disease Dual cholinesterase inhibitor

Alkaloids have always been a great source of cholinesterase inhibitors. Numerous studies have shown that inhibiting acetylcholinesterase as well as butyrylcholinetserase is advantageous, and have better chances of success in preclinical/ clinical settings. With the objective to discover dual cholinesterase inhibitors, herein we report synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine (1) and its bromo-derivative 2. Our study has shown that cryptolepine (1) and its 2-bromo-derivative 2 are dual inhibitors of acetylcholinesterase and butyrylcholinesterase, the enzymes which are involved in blocking the process of neurotransmission. Cryptolepine inhibits Electrophorus electricus acetylcholinesterase, recombinant human acetylcholinesterase and equine serum butyrylcholinesterase with IC50 values of 267, 485 and 699 nM, respectively. The 2-bromo-derivative of cryptolepine also showed inhibition of these enzymes, with IC50 values of 415, 868 and 770 nM, respectively. The kinetic studies revealed that cryptolepine inhibits human acetylcholinesterase in a non-competitive manner, with ki value of 0.88 µM. Additionally, these alkaloids were also tested against two other important pathological events of Alzheimer’s disease viz. stopping the formation of toxic amyloid-β oligomers (via inhibition of BACE-1), and increasing the amyloid-β clearance (via P-gp induction). Cryptolepine displayed potent P-gp induction activity at 100 nM, in P-gp overexpressing adenocarcinoma LS-180 cells and excellent toxicity window in LS-180 as well as in human neuroblastoma SH-SY5Y cell line. The molecular modeling studies with AChE and BChE have shown that both alkaloids were tightly packed inside the active site gorge (site 1) via multiple π-π and cation-π interactions. Both inhibitors have shown interaction with the allosteric “peripheral anionic site” via hydrophobic interactions. The ADME properties including the BBB permeability were computed for these alkaloids, and were found within the acceptable range.

1. Introduction The cholinesterase (ChE) enzymes acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) catalyzes the breakdown of acetylcholine and of some other choline esters that function as neurotransmitters, resulting in hampered neurotransmission [1,2]. The drugs acting as cholinesterase inhibitors (CIs) have proved to attenuate the cholinergic deficit in Alzheimer's disease (AD) by re-establishing the level of synaptic acetylcholine (ACh) [3–5]. Restoration of cholinergic neurotransmission by CIs ameliorates the cognitive and behavioral dysfunctions associated with AD. Hence, CIs have clinically shown benefits in symptomatic treatment of AD [6–9]. Although AChE is the primary ChE responsible for the hydrolysis of ACh, the BChE was

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found to compensate for AChE particularly when AChE levels are depleted (a condition observed in AD brain with increased or unchanged levels of BChE) [10]. Therefore, drugs acting as dual inhibitors of both AChE and BChE, like rivastigmine, were recognized as more potent and promising candidates to positively improve the course of AD [11]. The physostigmine alkaloids is a class of dual CIs, and the rivastigmine, which belongs to this class is a FDA approved anti-AD drug [10]. Apart from cholinergic hypothesis, two other pathological hypotheses such as “amyloid” and “tau” hypothesis are widely accepted in AD etiology [12]. According to the general consensus, β-amyloid peptide (Aβ) is considered as the major culprit in AD, which is formed as a result of the sequential cleavage of amyloid precursor protein (APP) by β-secretase (BACE-1) and γ-secretase and its aggregation into oligomers

Corresponding author at: Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India. E-mail address: [email protected] (S.B. Bharate).

https://doi.org/10.1016/j.bioorg.2019.103062 Received 3 March 2019; Received in revised form 4 June 2019; Accepted 9 June 2019 Available online 12 June 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

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(AChE, EC 3.1.1.7, from electric eel, Type V-S, 827 U/mg solid; 1256 U/ mg of protein), recombinant human AChE (EC 3.1.1.7, recombinant, expressed in HEK 293 cells, lyophilized powder, ≥1,000 units/mg protein), butyrylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum, 246 U mg/ solid, 855 U/mg protein), 5,5-dithiobis-(2-nitrobenzoic acid) (Ellman reagent, DTNB), acetylthiocholine iodide (ATChI), S-butyrylthiocholine iodide (BTChI), and donepezil were purchased from Sigma-Aldrich. The NMR spectra was recorded on Bruker-Avance DPX FT-NMR 500 and 400 MHz instruments. The chemical shift values for proton and carbon were reported in parts per million (ppm) downfield from tetramethylsilane. ESI-MS and HRMS spectra were recorded on Agilent 1100 LC-Q-TOF and HRMS-6540-UHD machines, respectively. IR spectra were recorded on Perkin-Elmer IR spectrophotometer. Melting points were recorded on BUCHI digital melting point apparatus. Absorbance readings for Ellman assay and fluorescence readings for FRET/ rhodamine 123 assay were recorded on Molecular Devices and BioTek plate readers, respectively.

and fibrils. Hence, strategies that result in increased clearance of Aβ from AD brains were proved to be helpful in the prevention and treatment of AD [13,14]. Induction of the membrane-associated drug transporter P-glycoprotein (P-gp) has recently been emerged as a useful strategy to increase Aβ clearance from AD brains. Many natural products have been reported to possess potent P-gp induction activity, including rifampicin, hyperforin, oleocanthal [15,16], fascaplysin [17], etc. Recently, we have demonstrated that P-gp inducers (colupulone and quinoline analogues) modulates Aβ transport [18,19] across BBB, and also increases Aβ clearance in mice model [20]. Moreover, as AD is quite complex and multifaceted, to put it in a nutshell, multitargeted approaches are advantageous to combat its outrageous prevalence. The nitrogen containing heterocycles, particularly alkaloids, are a matter of great interest in search of new drug leads for AD [21,22]. In particular, several indole alkaloids viz. fascaplysin [23], serpentine [24], ungeremine [25], 19,20-dihydrotabernamine and 19,20-dihydroervahanine A [26], dehydroevodiamine [27] are reported as CIs. However, to date, there is no report on “indoloquinoline alkaloids” as CIs. Indoloquinoline alkaloids, encompassing a heterocyclic system formed by the fusion of indole and quinoline, were found in various plants used in traditional medicine. Extensive research has been published on this class of alkaloids displaying their broad spectrum of pharmacological activities [28]. One such naturally occurring alkaloid is cryptolepine (1, 5-methyl-10H-indolo[3,2-b]quinoline) which was isolated from the West-African climbing shrub, Cryptolepis sanguinolenta (Family: Apocynaceae) [29–31]. The cryptolepine scaffold has primarily been extensively investigated for its antimalarial activity, based on the traditional usage of Cryptolepis sanguinolenta plant for management of malaria in African countries. Apart from this primary biological activity, cryptolepine has also been reported to possess numerous other pharmacological activities such as anticancer, antipyretic, anti-inflammatory, antibacterial, hypotensive, antidiabetic, antithrombotic and renovascular vasodilatory activity [32–35]. The 2-bromocryptolepine (2) is a synthetic derivative of cryptolepine with better antimalarial potency, which was discovered from medicinal chemistry efforts [33]. The chemical structures of alkaloids 1–2 are shown in Fig. 1. Herein, we report the discovery of indoloquinoline alkaloid cryptolepine (1) as a multitargeted agent acting on four targets of AD viz. AChE, BChE, BACE-1 and P-gp. The enzyme kinetic study was performed with AChE to know the type of inhibition, and modeling studies were performed to rationalize the type of inhiibiton and further to understand the interaction pattern of these inhibitors with the active site gorge of AChE and BChE. In addition to dual CI property, the cryptolepine also showed potent P-gp induction activity in P-gp overexpressing adenocarcinoma LS-180 cells, and a mild BACE-1 inhibition activity. The ADME parameters were computed in order to assess the drug like properties of these lead compounds.

2.2. Synthesis of cryptolepine (1) The synthesis of cryptolepine was started from anthranilic acid (3) and was accomplished in 6-steps (see, Scheme 1), as described below. 2.2.1. Synthesis of 2-(2-bromoacetamido)-benzoic acid (4) The solution of anthranilic acid (3, 5 g) in DMF: 1,4-dioxane − 1: 1 (30 mL) was stirred at 0 °C for 10 min. To this stirring solution, 2-bromoacetyl bromide (4.0 mL, 1.2 equiv.) was added drop-wise. The resultant mixture was allowed to stir at room temperature for 10 h, after which, the water (30 mL) was added to the reaction mixture. The obtained precipitate was filtered and residue was washed with water (3–4 times). Finally, the product was dried under vacuum to get white powder (9.1 g) of 2-(2-bromoacetamido)-benzoic acid (4). m.p. 162–166 °C; 1H NMR (CD3OD, 400 MHz): δ 8.46 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.4 Hz, 1H), 7.07 (t, J = 8.4 Hz, 1H), 3.99 (s, 2H); ESI-MS: m/z 256.9 [M + H]+ [36]. 2.2.2. Synthesis of 2-(2-(phenylamino)-acetamido)benzoic acid (6) The solution of 2-(2-bromoacetamido)-benzoic acid (4, 5.0 g) and aniline (5, 3 equiv.) in DMF (10 mL) was refluxed at 120 °C for 18 h. The reaction mixture was then cooled to room temperature and poured into ice-water followed by adjusting the pH to 10. Resulting mixture was extracted with methylene chloride (3 × 100 mL) and combined CH2Cl2 layers were kept aside. The pH of aqueous layer was adjusted to 3.0 with HBr solution to get precipitate. The obtained precipitate was collected, washed with water, and dried to yield desired product 6 as a white solid (3.6 g). m.p. 194–197 °C; 1H NMR (CD3OD, 400 MHz): δ 8.60 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.8 Hz, 1H), 7.01 (t, J = 7.6 Hz, 3H), 6.54 (m, 3H), 3.78 (s, 2H); ESI-MS: m/z 270.0 [M + H]+ [36].

2. Materials and methods 2.1. General

2.2.3. Synthesis of indolo[3,2-b]quinolin-11-one (7) The mixture of 2-(2-(phenylamino)-acetamido)benzoic acid (6, 3.0 g) and polyphosphoric acid (200 g) was stirred at 130 °C for 2 h. The reaction mixture was then allowed to cool to room temperature. The crushed ice was added to the cooled reaction mixture followed by adjusting the pH to neutral using KOH solution. The resulting solution was extracted with EtOAc (2 × 250 mL) and the obtained organic layer was washed with water and then with brine solution. Solvent evaporation on rotary evaporator yielded light brown solid (1.75 g) of indolo[3,2-b] quinolin-11-one (7). m.p. > 300 °C; 1H NMR (DMSO‑d6, 400 MHz): δ 12.41 (s, 1H), 11.64 (s, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.4 Hz, 1H), 7.69 (dd, J = 7.4, 12.5 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 7.7, 12.8 Hz, 1H), 7.29 (dd, J = 7.9, 13.3 Hz, 1H), 7.20 (dd, J = 7.7, 12.5 Hz, 1H); ESI-MS: m/z 234.0 [M + H]+ [36].

All chemicals, reagents, and solvents were purchased from commercial suppliers such as Sigma-Aldrich, TCI Chemicals, or Merck, and were used as received. Electrophorus electricus acetylcholinesterase

Fig. 1. Structures of cryptolepine (1) and 2-bromocryptolepine (2). 2

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Scheme 1. Synthesis of cryptolepine (1). Reagents and conditions: (a) bromoacetyl bromide (1.2 equiv), 1,4-dioxane, DMF, 18 h, 96%; (b) DMF, 130 °C, 70%; (c) PPA, 100 °C, 2 h, 67%; (d) POCl3, 2 h, 60%; (e) H2/ Pd-C, AcOH, NaOAc, 60 psi, 2 h, 80%; (f) CH3I, ACN, 80 °C, 12 h, 70%.

Scheme 2. Synthesis of 2-bromocryptolepine (2). Reagents and conditions: (a) chloroacetic acid (1.2 equiv), 1,4-dioxane, H2O, 18 h, 80%; (b) Ac2O (1.2 equiv), DMF, 85% ; (c) Ac2O 100 °C, 2 h, 80%; (d) aq KOH, 6 h, reflux; (e) diphenyl ether, 250 °C, 4 h; (f) CH3I, ACN, 80 °C, 12 h, 70%.

2.2.4. Synthesis of 11-chloro-10H-indolo-[3,2-b]quinoline (8) The mixture of indolo[3,2-b]quinolin-11-one (7, 1.5 g) and POCl3 (20 mL) was refluxed for 2 h. Reaction mixture was then allowed to cool to the room temperature, followed by addition of crushed ice to it, and neutralizing with KOH solution. The resulting aquous solution was extracted with EtOAc (3 × 150 mL) and combined organic layers were dried over sodium sulphate, and concentrated on rotary evaporator to get brown solid of 11-chloro-10H-indolo-[3,2-b]quinoline (8, 0.95 g). m.p. 180–185 °C; 1H NMR (DMSO‑d6, 400 MHz): δ 11.82 (s, 1H), 8.28 (d, J = 7.7 Hz, 1H), 8.21 (m, 2H), 7.69 (m, 2H), 7.61 (m, 2H), 7.27 (m, 1H); ESI-MS: m/z 253.0 [M + H]+ [36].

EtOAc and combined organic layer was evaporated to dryness to get yellow solid (180 mg) of 10H-indolo [3,2-b]quinoline (9). m.p. 200–203 °C; 1H NMR (DMSO‑d6, 400 MHz): δ 11.43 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 8.29 (s, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.61 (m, 4H), 7.29 (dd, J = 7.0, 16.2 Hz, 1H); ESI-MS: m/z 219.0 [M + H]+ [36]. 2.2.6. Synthesis of cryptolepine (1) To the stirred solution of indolo [3,2-b]quinoline (9, 500 mg) in DMF (5 mL) was added methyl iodide (3 equiv.) and resulting mixture was refluxed at 100 °C for 8 h. Reaction was allowed to cool to the room temperature. To it, EtOAc (10 mL) was added, which produces orange red solid in the reaction mixture. The formed solid was filtered, and residue was washed with ethyl acetate. The obtained residue was dried to get cryptolepine iodide (1) in 70% yield. 1H NMR (DMSO‑d6, 400 MHz): δ 12.89 (s, 1H), 9.29 (s, 1H), 8.79 (dd, J = 8.4, 13.6 Hz, 2H), 8.59 (d, J = 8.0 Hz, 1H), 8.16 (t, J = 7.6 Hz, 1H), 7.94 (dd, J = 7.6, 12.0 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 5.03 (s, 3H); 13C NMR (DMSO‑d6, 100 MHz): δ 145.6, 138.0, 135.2, 133.9,

2.2.5. Synthesis of 10H-indolo [3,2-b]quinoline (9) The solution of 11-chloro-10H-indolo-[3,2-b]quinoline (8, 200 mg), sodium acetate (1.0 g, 10 equiv.) and 10% Pd/ C in acetic acid (25 mL) was hydrogenated at 60 psi for 2 h. The reaction mixture was filtered and washed with small amount of AcOH. The residual acetic acid was removed under vacuum and the mixture was neutralized with ice cold saturated sodium bicarbonate solution. The product was extracted with 3

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1048 cm−1; ESI-MS: m/z 341.2 [M + H]+ [33].

133.2, 132.4, 129.8, 127.0, 126.2, 126.1, 124.7, 121.3, 117.8, 113.8, 113.1, 54.4; IR (CHCl3): νmax 3742, 3435, 2920, 2850, 1637, 1609, 1577, 1503, 1452, 1384, 1252, 1034; ESI-MS: m/z 233.0 [M]+; HRMS: m/z 233.1071 calcd for C16H13N2 (233.1073) [36].

2.3.5. Synthesis of 2-bromo-5-methyl-10H-indolo[3,2-b]quinolin-5-ium iodide (2-bromocryptolepine, 2) The solution of 2-bromo-10H-indolo[3,2-b]quinoline-11-carboxylic acid (14, 3.4 g, 10 mmol) in 20 mL diphenyl ether was heated for 4 h at 250 °C. Reaction mixture was allowed to cool to room temperature, and then 30 mL hexane was added to it. Mixture was then filtered and obtained residue was washed with hexane, dried and crystallized from MeOH. The 500 mg of this compound (1 equiv.) was dissolved in DMF (5 mL) and kept on a magnetic stirrer to which was added methyl iodide (3 equiv.) and the resultant mixture was refluxed at 100 °C for 8 h. The reaction mixture was then cooled to room temperature, and EtOAc (10 mL) was added, which resulted in formation of an orange red solid. The solid was filtered, washed with excess of EtOAc and dried over vaccum to get 2-bromocryptolepine (2). Brown red solid; 1H NMR (DMSO-d6, 400 MHz): δ 12.98 (s, 1H), 9.23 (s, 1H), 8.91 (s, 1H), 8.84 (d, J = 6.4 Hz, 1H), 8.76 (d, J = 7.2 Hz, 1H), 8.29 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.88 (d, J = 6.4 Hz, 1H), 7.55 (s, 1H), 5.04 (s, 3H); 13C NMR (DMSO-d6, 100 MHz): δ 146.0, 138.6, 134.5, 134.4, 134.1, 133.9, 131.2, 127.4, 126.4, 123.4, 121.6, 120.2, 119.9, 113.8, 113.2, 40.5; ESI-MS: m/z 311.0 [M + H]+; HRMS: m/z 311.0176 calculated for C16H12BrN2 (311.0178) [33].

2.3. Synthesis of 2-bromocryptolepine (2) The synthesis of 2-bromocryptolepine was achieved from anthranilic acid (3) in 5-steps (see, Scheme 2), as described below. 2.3.1. Synthesis of 2-((carboxymethyl)amino)benzoic acid (10) To a stirred solution of chloroacetic acid (347 g) in 500 mL water, sodium carbonate (200 g, 6 equiv.) was added at room temperature. The resulting solution was heated to 40–50 °C and was quickly added to the mixture of anthranilic acid (3, 500 g, 3.65 mmol) in water (340 mL) and 35% aq NaOH solution (320 mL). The resulting mixture was then stirred at 45 °C for 4 days and the solid reaction mixture was treated with a NaOH solution (150 g in 4 L water). The mixture was heated to 60 °C and was immediately filtered. The solid residue was washed with 20% aqueous NaOH until the residue was dissolved. The combined filtrate was acidified with HCl to pH 3.0 and the resulting precipitate was then filtered off and was dried to yield cream colored solid (567 g) of 2-((carboxymethyl)amino)benzoic acid (10). m.p. 220–223 °C; 1H NMR (CD3OD, 400 MHz): δ 8.30 (d, J = 8.4 Hz, 1H), 7.70 (s, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.28–7.17 (m, 2H), 3.20 (s, 2H); ESI-MS: m/z 195.0 [M + H]+ [33].

2.4. In vitro AChE and BChE inhibition assay The AChE/BChE inhibitory activity of cryptolepine and 2-bromocryptolepine was determined using Ellman assay [37] with some modifications. The Ellman assay is based on the quantitative estimation of yellow coloured nitrobenzoate anion formed via AChE mediated cleavage of Ellman reagent 5,5′-dithiobis-(2-nitrobenzoic acid). Thiocholine, a byproduct of enzymatic hydrolysis of acetylthiocholine (ATChI), reacts with DTNB to produce 5-thio-2-nitrobenzoate anion which is yellow in colour and absorbs at a wavelength of 412 nm. The decrease in absorbance at this wavelength is indicator of the enzyme inhibition. The assay protocol was followed exactly, as described in our recent publication [38]. IC50 values were determined graphically from observed percentage inhibition values at different inhibitor concentrations using graph-Pad Prism 6 software and are reported as mean ± SEM (an average of three experiments). Donepezil was used as reference compound in the assay.

2.3.2. Synthesis of 2-(N-(carboxymethyl)acetamido)benzoic acid (11) To a stirred solution of sodium carbonate (8.9 g, 1 equiv. with respect to 10) in water (83.0 mL) was added (carboxymethyl amino) benzoic acid (10, 10.0 g) in small portions with continuous stirring at room temperature. Acetic anhydride (8.56 g, 1 equiv.) was then added and the reaction mixture was stirred at rt for another 30 min. The reaction mixture was then acidified with aqueous HCl, resulting in formation of a precipitate; which was collected and dried to get brown solid (11 g) of 2-(N-(carboxymethyl)acetamido)benzoic acid (11). Light brown solid; m.p. 208–210 °C; 1H NMR (CD3OD, 400 MHz): δ 7.97 (d, J = 8.4 Hz, 1H), 7.56 (m, 2H), 7.44 (m, 1H), 3.60 (d, J = 8.4, 17.6 Hz, 2H), 1.69 (s, 3H); ESI-MS: m/z 260.0 [M + Na]+ [33]. 2.3.3. Synthesis of 1-acetyl-1H-indol-3-yl acetate (12) To a stirred solution of acetic anhydride (4.6 g, 10 equiv. with respect to 11) and triethylamine (1.3 g, 1.4 mmol) was added 2-(N-(carboxymethyl)acetamido)benzoic acid (11, 1.36 g). The mixture was refluxed for 20 min and concentrated under vaccum to get oily residue (35 mL) which was refrigerated for 8–10 hrs. The solid product was filtered off and dried to get light green solid of 1-acetyl-1H-indol-3-yl acetate (12). m.p. 72–74 °C; 1H NMR (CDCl3, 400 MHz): δ 8.36 (d, J = 7.9 Hz, 1H), 7.90 (s, 1H), 7.41 (m, 3H), 2.62 (s, 3H), 2.39 (s, 3H); ESI-MS: m/z 217.0 [M + H]+ [33].

2.5. Kinetics for inhibition of rHuAChE and eqBChE Kinetic study of interaction of compounds 1 and 2 was performed with rHuAChE and eqBChE using similar protocol as mentioned above for the in vitro AChE/BChE assay using five different concentrations of the substrate (0.0625 mM to 1 mM for each concentration of test compounds) [39,40]. The enzyme kinetics experiment and determination of ki value was carried out exactly in a similar way, as described in our recent publication [38].

2.3.4. Synthesis of 2-bromo-10H-indolo[3,2-b]quinoline-11-carboxylic acid (14) The solution of 1-acetyl-1H-indol-3-yl acetate (12, 6.1 g) in water (50 mL) was stirred at room termperature. To this mixture was then added, the aqueous solution of 5-bromoisatin (13, 1 equiv.) and KOH (26 g, 2 equiv.). The resulting mixture was refluxed for 4 h, and was then allowed to cool down upto 70 °C. At this temperature, air was bubbled through the solution for about 20 min. The reaction mixture was filtered off and filtrate was acidified to pH 1.0 with conc. HCl. The obtained precipitate was collected, washed with water and dried to get brown solid of 2-bromo-10H-indolo[3,2-b]quinoline-11-carboxylic acid (14). m.p. > 300 °C; 1H NMR (DMSO‑d6, 200 MHz): δ 11.54 (s, 1H), 9.40 (s, 2H), 8.39 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.86–7.69 (m, 3H), 7.40 (t, J = 7.8 Hz, 1H); IR (CHCl3): νmax 3670, 3645, 3584, 3418, 2921, 2348, 2054, 1612, 1460, 1317, 1122,

2.6. BACE-1 inhibition The BACE-1 FRET assay kit was purchased from Sigma-Aldrich (Product No. CS0010, Saint Louis, USA) and the assay was carried out according to the protocol provided by the supplier. The assay was carried out using Fluorescence Resonance Energy Transfer (FRET) technique in which, the fluorescence energy of the donor group of the substrate (APP-based peptide) upon light excitation is significantly quenched by the acceptor present in the same substrate moiety through FRET. Enzymatic cleavage of the substrate by BACE-1 results in restoration of full fluorescence quantum yield of the donor. Subsequently, a weakly fluorescent peptide substrate becomes highly fluorescent upon enzymatic cleavage. Reduction in the fluorescence quantum yield due to inhibition of BACE-1 enzymatic activity has been considered as a 4

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measure of inhibitory activity of compounds. The assay protocol was followed exactly, as described in our recent publication [38]. The BACE-1 inhibitor IV (Calbiochem IV; CAS no. 797035-11-1) was used as a reference compound (IC50 = 17.98 ± 1.07 nM) (Lit. 15 nM) in the assay.

the base-mediated cyclization of 12 with 5-bromoisatin (13) produced 2-bromo-10H-indolo[3,2-b]quinoline-11-carboxylic acid (14). Finally, pyrolytic decarboxylation of 14 at 250 °C followed by N-methylation using methyl iodide in ACN under reflux condition, produced 2-bromocryptolepine (2). Both synthesized compounds 1 and 2 were fully characterized using spectral analysis, and the data was compared with the literature values [36,46].

2.7. P-gp induction activity in LS-180 cells As described earlier [17,19].

3.2. Cholinesterase inhibition

2.8. Cell viability studies in LS-180 and SH-SY5Y cells

From recent studies [6,10,47,48], it is evident that BChE also plays a crucial role in the progression of AD apart from AChE. In the advanced stages of AD, while the level of AChE plunges, it is surprising that the levels of BChE is maintained constant or may increase in the regions of brain which are involved in cognitive functions [48–50]. In view of these evidences, it can be presumed that inhibition of both AChE and BChE may serve as a better choice in the symptomatic treatment of AD. Structurally, the cholinesterase enzymes from different species are very similar. Because of their structural and functional conservations, AChE from the electric eel (EeAChE), and BChE from equine serum (eqBChE) are being routinely used as suitable models for the corresponding enzymes from humans [51]. Furthermore, these enzymes have better stability over their human counterparts; therefore they have became routine tool enzymes in anti-Alzheimer's drug discovery [52,53]. The EeAChE and HuAChE shares 87% sequence identity [54]. Similarly, eqBChE shares 93.4% sequence identity to that of HuBChE [55]. Therefore, cryptolepine (1) and 2-bromocryptolepine (2) were evaluated for anti-cholinesterase activity against EeAChE and eqBChE, following Ellman method [37] and donepezil as a reference standard in the assay. The preliminary screening of both compounds was performed against both enzymes at 2 µM. These compounds displayed > 50% inhibition of both the enzymes at this test concentration. The IC50 values were then determined. Cryptolepine inhibited EeAChE and eqBChE with IC50 values of 267 and 699 nM, respectively. 2-Bromocryptolepine also inhibited both the enzymes, with the IC50 values of 415 and 770 nM, respectively (Table 1). These results clearly indicated that both compounds are dual inhibitors of AChE and BChE. This type of situation was also noticed in some of the previous studies [56–59]. The IC50 values are listed in Table 1. Next, we determined their activity against recombinant human AChE (rHuAChE), wherein both of them inhibited rHuAChE with IC50 values of 485 and 868 nM, respectively (Table 1). AChE consists of two binding sites namely, catalytic anionic site (CAS) and peripheral anionic site (PAS). CAS is the primary site responsible for the hydrolysis of substrates like ACh and ATCh whereas, PAS is the site to which inhibitors bind to elicit their response. Kinetic studies were performed to deduce the type of inhibition exhibited by compounds 1 and 2 against AChE and BChE. Michaelis–Menten kinetics relates the reaction velocity to the concentration of substrate, using which Lineweaver–Burk double-reciprocal plots (Fig. 2a and c) of compounds 1 and 2 were constructed to determine the type of

As described earlier [17,19]. 2.9. Molecular modelling Docking study of compounds 1 and 2 was performed by using human AChE (PDB ID: 4EY7) [41] and human BChE (PDB ID: 6EP4) [42] crystal structures retrieved from protein data bank. These crystal structures were subjected for protein preparation using Maestro v9.0 and Impact program v5.5 (Schrodinger, Inc., New York, 2009). The chemical structures of cryptolepine and 2-bromocryptolepine were sketched, minimized and docked using GLIDE XP. The ligand-protein complexes were minimized using macromodel. For AChE docking, the ligands were docked at three known sites - site 1 (catalytic gorge, substrate binding site), site 2, and site 3. The sites 2 and 3 were proposed by Roca et al. [43] as allosteric sites. The grid for site 1 was constructed using co-crystallized ligand 'donepezil' whereas the grids for site 2 (Pro 232, Asn 233, Gly 234, Pro 235, Trp 236, Thr 238, Val 239, Gly 240, Glu 243, Arg 246, Arg 247, Leu 289, Por 290, Gln 291, Ser 293, Arg 296, Phe 297, Val 300, Thr 311, Pro 312, Glu 313, Pro 368, Gln 369, Val 370, Asp 404, His 405, Cys 409, Pro 410, Gln 413, Trp 532, Asn 533, Leu 536, Pro 537, Leu 540) and site 3 (Glu 81, Gly 82, Glu 84, Met 85, Asn 87, Asn 89, Leu 130, Asp 131, Val 132, Thr 436, Leu 437, Ser 438, Trp 439, Tyr 449, Glu 452, Ile 457, Ser 462, Arg 463, Asn 464, Tyr 465) were constructed using site residues, as proposed by Roca et al. [43]. Donepezil was used as a reference ligand for site 1 whereas rosmarinic acid [44] for site 2 and VP2.33 [43] for site 3. For BChE, only catalytic site is known and no any allosteric site is reported; therefore docking was performed only at catalytic active site gorge. AMDE parameters were also computed using Schrodinger software, and are listed in Table 5. 3. Results and discussion 3.1. Synthesis Cryptolepine (1) was synthesized starting from anthranilic acid (8) in 6-steps, as depicted in Scheme 1 [45,46]. The coupling of anthranilic acid (3) with bromoacetyl bromide yielded N-bromoacetyl anthranilic acid (4), which further on amination with aniline (5) led to the formation of 2-(2-(phenylamino)acetamido)benzoic acid (6). The cyclization of intermediate 6 using polyphosphoric acid produced indolo[3,2b]quinolin-11-one (7), which further on treatment with POCl3 resulted in replacement of carbonyl functionality with chlorine atom to yield 11chloro-10H-indolo[3,2-b]quinoline (8). The hydrogenation of chloroderivative 8 with H2/Pd in AcOH produced de-chlorinated product 10H-indolo[3,2-b]quinoline (9), which finally on N-methylation yielded cryptolepine iodide (1). 2-Bromocryptolepine (2) was also synthesized from commercially available anthranilic acid (3), however using modified synthetic route, as illustrated in Scheme 2. The reaction of anthranilic acid (3) with chloroacetic acid in the basic medium yielded 2-((carboxymethyl) amino) benzoic acid (10), which further on N-acetylation using Ac2O produced 2-(N-(carboxymethyl)acetamido) benzoic acid (11). The cyclization of 11 was done by acetic anhydride and triethyl amine to give 1-acetyl-1H-indol-3-yl acetate (12). Further,

Table 1 Inhibition of EeAChE, rHuAChE and eqBChE by cryptolepine and 2-bromocryptolepine. Entry

1 2 Donepezil a b c d

5

IC50 (nM) ± SDa EeAChEb

rHuAChEc

eqBChEd

267 ± 17 417 ± 18 49 ± 1

485 ± 67 868 ± 100 32 ± 4.2

699 ± 17 770 ± 25 5520 ± 125

The 50% inhibitory concentration of EeAChE, rHuAChE or eqBChE. EeAChE is Electrophorus electricus AChE. rHuAChE is recombinant human AChE. eqBChE is equine serum BChE.

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18

(a)

(b)

Control 0.15625 µM

16

0.9

0.3125 µM

14

1.25 µM

0.7

2.5 µM

1

10

Slope

[V]-1 min/ΔA

12

8 6

Ki 0.1

2 0 -2

0

4

16 14 12

8

12

-1.2 -0.8 -0.4 0 -0.1

16

(d)

Control 0.15625 μM

0.8

1.2

1.6

2

2.4

2.8

0.8 y = 0.211x + 0.209 R² = 0.997

0.7

0.3125 μM

0.625 μM

0.6

1.25 μM

2

2.5 μM

0.5

Slope

10 8

0.4 0.3

6

0.2

4

Ki

2 0 -6 -4 -2 0 -2

0.4

[Cryptolepine] μM

[ATCh]-1 mM

(c)

[V]-1 min/ΔA

0.5

0.3

4

-4

y = 0.276x + 0.244 R² = 0.995

0.625 µM

0.1

0

2

4

6

-1.2 -0.8 -0.4

8 10 12 14 16 18

-0.1

[ATCh] -1 mM

0

0.4

0.8

1.2

1.6

2

2.4

2.8

[2-Bromocryptolepine] μM

Fig. 2. The enzyme kinetics of rHuAChE inhibition by compounds 1 and 2. (a) The Lineweaver–Burk double reciprocal plot representing reciprocal of AChE velocity versus reciprocal of different substrate concentrations (0.0625–1 mM) at five different concentrations of cryptolepine (1). (b) Estimation of ki for cryptolepine for AChE from slope replot versus inhibitor concentration. (c). The Lineweaver–Burk double reciprocal plot representing reciprocal of AChE velocity versus reciprocal of different substrate concentrations (0.0625–1 mM) at five different concentrations of 2-bromocryptolepine (2). (d) Estimation of ki for 2-bromocryptolepine for AChE from slope replot versus inhibitor concentration.

inhibition. From the Lineweaver–Burk double-reciprocal plots, compounds 1 and 2 were found to inhibit human AChE by non-competitive mode with decreasing AChE reaction velocity at increasing inhibitor concentration. Replots of slopes of Lineweaver–Burk double-reciprocal plots against inhibitor concentrations were constructed to determine the ki values of the compounds, which were found to be 0.88 and 0.99 μM (Fig. 2b and d) respectively for compounds 1 and 2. The Dixon plots were drawn using reciprocal of AChE velocity versus increasing concentrations of 1 and 2 (0.15625 to 2.5 μM) at various concentrations of ATCh (0.0625 to 1 mM) (Fig. S1 of supporting information). The Dixon plots also supported the calculated ki values as well as the mode of inhibition determined i.e non-competitive inhibition for both the compounds.

Similarly, the type of inhibition displayed by compounds against BChE was determined by constructing Lineweaver–Burk double reciprocal plots (Fig. 3a and c) followed by replotting slopes against inhibitor concentrations (Fig. 3b and d). Compounds 1 and 2 showed ki values of 0.51 and 0.22 µM for BChE. The summary of enzyme kinetics is shown in Table 2. Dixon plots for both the compounds are presented in Fig. S2 of supporting information. The Lineweaver–Burk double reciprocal plots shown in Fig. 3 are indicative of the fact that compounds 1 and 2 display mixed- type of inhibition for BChE. The Km for the BTChI substrate was found to be 0.81 μM in the absence of inhibitor. Apparent Km and apparent Vmax values at various concentrations of inhibitor are listed in Table S1 of supporting information. The comprehensive kinetic study results indicate that compounds 1 6

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3

(a) 32 28

2.5

24

0.25µM

20

0.5 µM

16

1 µM

2

1

2 µM

12 8

0

0

28 24

[V]-1 min/ΔA

20

2

4 6 [BTCh] -1 mM

8

-1

10

-0.5

0

0.5

1

1.5

2

[Cryptolepine] μM

-0.5

(d)

0.15625 µM

2

0.3125 µM 0.625 µM

2

1.25 µM

y = 0.607x + 0.133 R² = 0.999

1.5

2.5 µM

16 12

Slope

(c)

0.5

Ki

0 -4

1.5

1

4

-2

y = 0.946x + 0.485 R² = 0.992

0.125µM

Slope

[V] -1 min/ ΔA

(b)

Control

8

0.5

Ki

4 0 -6 -4 -2 0 -4

1

0

2

4

-0.5

6 8 10 12 14 16 18 [BTCh] -1 mM

-0.5

0

0.5

1

1.5

2

2.5

3

[2-Bromocryptolepine] μM

Fig. 3. The enzyme kinetics of eqBChE inhibition by compounds 1 and 2. (a) The Lineweaver–Burk double reciprocal plot representing reciprocal of BChE velocity versus reciprocal of different substrate concentrations (0.0625–1 mM) at five different concentrations of cryptolepine (1). (b) Estimation of ki for cryptolepine for BChE from slope replot versus inhibitor concentration. (c). The Lineweaver–Burk double reciprocal plot representing reciprocal of BChE velocity versus reciprocal of different substrate concentrations (0.0625–1 mM) at five different concentrations of 2-bromocryptolepine (2). (d) Estimation of ki for 2-bromocryptolepine for BChE from slope replot versus inhibitor concentration.

interaction pattern of these inhibitors with cholinesterase enzymes, the in-silico docking studies were performed with AChE (PDB: 4EY7) and BChE (PDB: 6EP4). These studies were conducted using Glide module of the Schrodinger molecular modelling software. As the kinetic study has indicated non-competitive type of inhibition of AChE, the docking was carried out at catalytic gorge (site 1) as well as at other known allosteric sites (site 2 and 3) proposed by Marcelo et al and Roca et al. [43,44] (Fig. 4A). The active site gorge (site 1) comprises catalytic triad (CAS, Ser-Glu-His) at the base of the gorge, anionic subsite, acyl pocket, oxyanion hole, and peripheral anionic site (PAS) at the mouth of the gorge (active site is shown in Fig. 4B). The key residues of various subsites of active site gorge are tabulated in Table 3. The PAS located at the gorge of 'site 1′ is known to allosterically modulate the catalysis and therefore, it is referred as one of the allosteric site. Our docking study at site 1 has indicated that both cryptolepine and 2-bromocryptolepine does not interact with catalytic triad Ser-Glu-His of CAS; however both of them display interactions with anionic subsite (choline binding site)

Table 2 AChE and BChE inhibitory constants (ki) for cryptolepine (1) and 2-bromocryptolepine (2). Entry

1 2 a

ki (μM) ± SDa

Type of inhibition

rHuAChE

eqBChE

rHuAChE

eqBChE

0.88 ± 0.09 0.99 ± 0.12

0.51 ± 0.07 0.22 ± 0.05

Non-competitive Non-competitive

Mixed Mixed

Values are mean ± SD of at least three independent measurements.

and 2 binds equally well to the free enzyme, as well as to the bound enzyme (enzyme-substrate complex) in case of AChE by displaying noncompetitive inhibition. However, in case of BChE, compounds 1 and 2 may bind to the free-enzyme as well as to the bound enzyme via mixed type inhibition. To support the enzyme kinetic results and further to understand the 7

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Fig. 4. Molecular docking of cryptolepine (orange) and 2-bromocryptolepine (green) with site 1 of human AChE (PDB ID: 4EY7). (A) The surface view of AChE showing the three binding sites. (B). active site of human acetylcholinesterase (PDB ID: 4EY7) (site 1) indicating its various sub-sites. (C). the closer surface view of the entrance of AChE active site gorge showing entry of cryptolepine. (D) Interaction map of cryptolepine with site 1 of human AChE. (E). the closer surface view of the entrance of AChE active site gorge showing entry of 2-bromocryptolepine. (F) Interaction map of 2-bromocryptolepine with site 1 of human AChE. The π-π interactions are represented by light-blue dotted lines; whereas the green dotted lines indicate cation-π interactions. The color of displayed amino acid residues is indicative of their sub-site (dark blue: catalytic site; light green: choline binding pocket; pink: acyl binding pocket; and red: peripheral anionic site). The pink dotted lines (along with numerical value above the line) between the ligand and Trp 86 indicates the distance between ligand and Trp 86 residue, which is located at the bottom of the gorge.

which is also located at the bottom of the gorge. Both compounds showed π-π interactions with Tyr 337 and Phe 338 of anionic subsite. In addition, cryptolepine also displayed π-π interaction with Trp 86, which is the key residue of anionic subsite. The quinoline ring of both ligands was found facing towards the bottom of the gorge, whereas the indole ring towards peripheral anionic site. Although the orientation of

2-bromocryptolepine was also found similar to that of cryptolepine in the AChE active site gorge; however because of the presence of additional bulkier halogen group in 2-bromocryptolepine, it stayed away from the CAS, and thus the indole ring (ring C and D) stayed more towards the opening of active site gorge, displaying better interaction with PAS residues. Cryptolepine showed π-π interaction with Tyr 341 8

Bioorganic Chemistry 90 (2019) 103062

−1.71 (VP2.33 [43]: −6.37)

NI: no interaction with any of the key residue. Donepezil, rosmarinic acid and VP.233 were used as reference ligands for site 1, 2, and 3, respectively.

−3.64 (VP2.33 [43]: −6.37) NI Site 3

Site 2

(of PAS), whereas 2-bromocryptolepine interacted with three residues of PAS [π-π stacking with Tyr 124 and Tyr 341 and cation-π stacking with Trp 286). These interactions with PAS could be accounting for the non-competitive type inhibition of these inhibitors in kinetic study. It is noteworthy to mention that donepezil is also a non-competitive inhibitor, and it interacts with both CAS and PAS sites within the active site gorge. The overlay of cryptolepine with donepezil (Fig. 4D) revealed that both the molecules occupy the same binding site, and display similar interactions with the CAS. However because of the shorter length of cryptolepine, it does not interact with one of the important PAS residue 'Trp 286′. Furthermore, the quaternary nitrogen center of both the ligands was found closer to each other, and both showing a cation-π interaction with the CAS residue Trp 86. In addition to site 1, we also investigated the interaction pattern of these inhibitors at other two known allosteric sites of AChE (Fig. 5A–B). The site 2 (also called as site B) has been proposed by Marcelo et al. [44] for rosmarinic acid. Therefore, in our docking study at site 2, we used rosmarinic acid as a reference ligand. The key interaction of rosmarinic acid with Arg 296, at site 2 was replicated in our docking [44] (Section S4 of Supporting information). The rosmarinic acid ranked 'top' with the glide score of −9.89; whereas both these inhibitors showed poor glide score (−3.424 and −3.292, respectively), with no interaction with any of the key residue of this site (Fig. 5A), which ruled out the possibility of binding of these ligands at site 2. Recently, Roca et al proposed another allosteric site (site 3) along with site 2 [43]. We further docked these inhibitors at site 3, and again poor dock sore and absence of key interactions (Fig. 5B) ruled out the possibility of site 3 as a binding site of these inhibitors. Overall, both these ligands displayed good dock score at site 1, along with key interactions with most of the important residues. This indicates that “site 1″ is the binding site of both inhibitors; and they display non-competitive type of inhibition because of their interaction with PAS (the allosteric site) of site 1. The BChE active site gorge is wider and has ~200 Å3 large volume compared with AChE gorge [60]. The active site of BChE is displayed in Fig. 6A and the active site residues are listed in Table 4. Because of the wider opening and larger volume of the gorge, cryptolepine was found to enter deep into the active site gorge and orient in a horizontal manner (Fig. 6A–C). Inside the pocket, it primarily displayed hydrophobic interactions with two amino acid residues namely His 438 of CAS and Trp 82 of anionic subsite (Fig. 6B). The flat cryptolepine molecule was found tightly packed via π-π stacking interactions with the hydrophobic residues of BChE active site gorge. As it was seen in case of AChE, the 2-bromocryptolepine again stayed away from the CAS of BChE, and was oriented closer to the PAS (Fig. 6C). The indolic NH of 2-bromocryptolepine showed H-bonding interaction with Tyr 332 of PAS. In addition, the Tyr 332 residue interacted with ring A, B and C via π-π stacking. Thus, both these cryptolepines showed strong interactions with the active site gorge of both enzymes, resulting in inhibition of the catalytic activity of these enzymes. As both inhibitors occupy the active site gorge and interact with catalytic as well as PAS sites, the mixed type of inhibition was observed in kinetic studies.

Glu 452 (H-bond)

−3.29 (Rosmarinic acid [44]: −9.89) Tyr 124 (π-π); Tyr 341 (π-π); Trp 286 (cation-π) NI Tyr 341 (π-π)

−3.42 (Rosmarinic acid [44]: −9.89)

NI NI

NI

NI

Tyr 337 (π-π); Phe 338 (π-π)

Trp 86 (π-π); Tyr 337 (π-π, cation-π); Phe 338 (π-π) NI

–9.66 NI −11.46 (Donepezil = −18.04)

Catalytic anionic (or acylation) site (CAS) – (Ser 203, His 447, Glu 334) “anionic” subsite (choline binding pocket) – (Trp 86, Tyr 133, Tyr 337, Phe 338) Acyl binding pocket – (Phe 295, Phe 297) Oxyanion hole – (Gly121, Gly 122, Ala 204) Peripheral anionic site – (Tyr 72, Asp 74, Tyr 124, Trp 286 and Tyr 341) Arg 296, Gln 369, His 405, Gln 413, Trp 532 Glu 81, Val 132, Tyr 449, Glu 452, Arg 463 Site 1 (catalytic site)

NI

Interactions Interactions

Glide score

2-Bromocryptolepine Cryptolepine Key residues Site

Table 3 Details of binding sites of AChE and the list of key interactions observed for cryptolepine and 2-bromocryptolepine at catalytic and allosteric sites of AChE.

Glide score

V.K. Nuthakki, et al.

3.3. BACE-1 inhibition activity The role of BACE-1 in formation of Aβ plaques has been well established. Recent study has also shown that BACE-1 expressed in the BBB endothelium is upregulated in a murine model of AD [61]. Following FRET based enzymatic assay, the inhibitory potential of compounds 1 and 2 against BACE-1 (beta-secretase) was determined at 100 and 10 μM concentrations. At 100 μM compounds 1 and 2 have shown 55 and 51% inhibition, respectively, while at 10 μM, these compounds inhibited the enzyme by 14 and 10%, respectively. Despite their low inhibition activity against BACE-1; however this would be “add-on” activity of these inhibitors; and in in-vivo models, the actual level of inhibition could be ascertained in future. 9

Bioorganic Chemistry 90 (2019) 103062

V.K. Nuthakki, et al.

A.

B.

Val 132

Leu 414 Gln 413

Glu 452

Glu 313 Arg 463

Tyr 449 Trp 532

Glu 81

Gln 369

Arg 296

Fig. 5. Docking of cryptolepine (orange) and 2-bromocryptolepine (green) at site 2 (A) and 3 (B) of human AChE. (A) orientation of cryptolepine and 2-bromocryptolepine in site 2. (B) orientation of cryptolepine and 2-bromocryptolepine in site 3. The π-π interactions are represented by light-blue dotted lines; whereas the green dotted lines indicate cation-π interactions.

3.4. P-gp induction activity of cryptolepine in LS-180 cells

for a drug’s pharmacokinetics in the human body, including its ADME. Both alkaloids along with donepezil were evaluated for their ADME properties using QikProp [64]. The partition coefficient (QPlogPo/w), water solubility (QPlogS) and brain/blood partition coefficient (QPlogBB) are important parameters to know whether the compound will have adequate distribution in brain. The brain/blood partition coefficient of both alkaloids was found to be 0.344 and 0.525 which was found to be within the acceptable range. Cell permeability (QPPCaco and QPPMDCK), a key factor governing drug metabolism and its access to biological membranes, ranged from 3179 to 8389, which is within the acceptable range. None of the compound was found to violate Lipinki Rule of 5. In summary, the ADME parameters were within the acceptable range defined for human use (see Table 5 footnote), indicating their drug-likeness.

The P-gp located at BBB plays important role in clearance of Aβ from brain [62,63]. Several studies have shown that the AD brain has decreased levels of P-gp, thus resulting in accumulation of Aβ plaques in the brain. Therefore, induction of P-gp has been considered as one of the novel therapeutic approaches to combat AD. The P-gp overexpressing LS-180 cell line was used to study P-gp induction activity. Toxicity window of compounds 1 and 2 was first determined in LS-180 cell line and GI50 of both the compounds was found to be > 12.5 μM. Therefore, for P-gp induction assay, the 10-fold lower concentrations were choosen. The LS-180 cells were treated with compounds 1 and 2 at 100 nM and 1 μM. Cryptolepine (1) treatment at 100 nM and 1 μM resulted in a significant increase in the efflux of substrate rhodamine 123 dye, as determined by the respective decrease of intracellular Rh123 levels by 37 and 34%. However, compound 2 did not show any induction of P-gp at both the test concentrations. Further medicinal chemistry efforts could make out the reason behind this differential effect of two very similar compounds on P-gp induction. Rifampicin was used as a positive control in this study, which decreased the intracellular accumulation of Rh123 levels by 35% at 10 μM (37% at 1 µM).

4. Conclusion In summary, we have identified the indoloquinoline alkaloids cryptolepine and 2-bromocryptolepine as a new class of dual inhibitors of AChE and BChE. These alkaloids inhibit EeAChE as well as rHuAChE with IC50 values of 267 and 485 nM, respectively. The mechanism of the inhibition has been unraveled using kinetic studies, indicating that both the alkaloids possess non-competitive type of inhibition of human AChE. Docking studies have supported these observations. Apart from dual cholinesterase inhibition, cryptolepine also displayed mild inhibition of BACE-1 and potent P-gp induction activity. Although large number of newer approaches have emerged and extensively studied in last decade for AD treatment, so far none of them have been successful to reach the market. In such complicated scenario, it is clear that a combination or multitargeted approaches (a single drug targeting muliple targets of the disease) are more likely to succeed in the AD treatment. For such strategy, the cholinergic pathology should be better taken care by dual AChE/ BChE inhibitors rather than only AChE inhibitors. Thus, because of the multitargeted profile of cryptolepine (dual ability to inhibit AChE as well as BChE enzymes and displaying a potent P-gp induction), the further exploration of this class of natural products is warranted in preclinical animal efficacy studies.

3.5. Cell proliferation activity in human neuroblastoma SH-SY5Y cells In order to assess the toxicity profile of most active compound (cryptolepine) in neuronal cells, its in-vitro cell proliferation effect was checked on human neuroblastoma SH-SY5Y cell line. Results indicate that cryptolepine (1) has good safety window, as it shows GI50 of > 5 µM in SH-SY5Y cells, which is 5–10 fold higher than the effective concentrations for P-gp induction and cholinesterase inhibition. 3.6. ADME properties It is highly important to assess the drug-like parameters/ ADME properties of any new lead compound at early discovery stage. The first and foremost criteria is to check whether the lead compound obeys Lipinski’s rule of 5. This rule describes molecular properties important

10

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A. Trp231 Val 288 CAS

Gly 117

Anionic subsite Acyl binding pocket

Ser 198

Gly 116

Leu 286

Oxyanionhole Tyr 332

Phe 329

PAS

Glu 325 His 438

Asp 70

Tyr 128 Trp82

B.

Leu 286

C. Trp231

Ring D

Glu 325 Tyr 332

Leu 286

Trp231

Glu 325

His 438

Ring D

Tyr 332 Ring A

Ring A Asp 70 Trp82

Asp 70 Tyr 128

Trp82

Tyr 128

Fig. 6. Molecular docking of cryptolepine and 2-bromocryptolepine with human BChE. (A) active site of human butyrylcholinesterase (PDB ID: 6EP4). (B) Interaction map of cryptolepine with human BChE active site residues. (C) Interaction map of 2-bromocryptolepine with human BChE active site residues. The π-π interactions are represented by blue dotted lines; whereas the green dotted lines indicate cation-π interactions. The color of displayed amino acid residues is indicative of their sub-site (dark blue: catalytic site; light green: choline binding pocket; pink: acyl binding pocket; and red: peripheral anionic site). Table 4 Details of binding site of BChE and the list of key interactions for cryptolepine and 2-bromocryptolepine at catalytic site gorge of huBChE. Sub-sites of active site gorge

Catalytic anionic (or acylation) site (CAS) – (Ser 198, His 438, Glu 325) “anionic” subsite (choline binding pocket) – (Trp 82, Tyr 128, Phe 329) Acyl binding pocket – (Val 288, Leu 286, Trp 231) Oxyanion hole – (Gly 116, Gly 117) Peripheral anionic site – (Asp 70, Tyr 332)

Cryptolepine

2-Bromocryptolepine

Interactions

Glide score

Interactions

Glide score

His 438 (π-π)

−8.86 (Donepezil = −10.52)

NI

−4.20 (Donepezil = −10.52)

Trp 82 (π-π)

NI

NI

NI

NI

NI

NI

Tyr 332 (π-π); Tyr 332 (H-bond)

NI: no interaction with any of the key residue. Donepezil, rosmarinic acid and VP.233 were used as reference ligands for site 1, 2, and 3, respectively. 11

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Table 5 ADME properties cryptolepine, 2-bromocryptolepine and donepezil.a Entry

1 2 Donepezil

Cholinesterase inhibition (IC50) HuAChE

eqBChE

0.48 0.87 0.032

0.70 0.77 5.52

QPlogP o/wb

QPlogSc

QPP Cacod

QPP MDCKe

QPlogBBf

CNSg

Lipinski Violationsh

3.481 4.047 4.446

−3.677 −4.33 −4.678

5591 5593 892

3179 8389 484

0.344 0.525 0.114

1 1 2

0 0 0

a ADME properties were determined using QikProp module of Schrodinger 10.2 software. Range mentioned below is for 95% of known drugs (these values are obtained from QikProp User Manual). b Predicted octanol/water partition co-efficient log p (acceptable range: −2.0 to 6.5). c Predicted aqueous solubility; S in mol/L (acceptable range: −6.5 to 0.5). d Apparent Caco-2 Permeability (nm/sec) (< 25 poor, > 500 great). e Apparent MDCK Permeability (nm/sec). MDCK cells are considered to be a good mimic for the blood-brain barrier. QikProp predictions are for non-active transport (< 25 poor, > 500 great). f Predicted brain/blood partition coefficient (acceptable range: −3.0 to +1.2). g Predicted central nervous system activity on a −2 (inactive) to +2 (active) scale (acceptable range: −2.0 to +2.0). h Lipinski Rule of 5 Violations (maximum is 4).

Acknowledgements [12]

Authors thank analytical department, IIIM for analytical support. The financial support from CSIR Young Scientist Grant (P90807) is greatly appreciated.

[13] [14]

Author contributions S.B.B. designed, executed and coordinated this whole study; V.K.N. performed AChE, BChE, BACE-1 enzyme inhibition and kinetic studies. S.B.B. performed molecular modelling. R.M. performed synthesis. A.S. performed cellular assays. A.K. designed and coordinated cellular studies. V.K.N. and S.B.B. contributed to manuscript writing.

[15]

Funding

[17]

[16]

This work was supported by CSIR Young Scientist Grant (P90807), awarded to SBB.

[18]

Appendix A. Supplementary material

[19]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bioorg.2019.103062.

[20]

References

[21]

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