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

Article abstract of DOI:10.1016/j.bioorg.2019.103062

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.

Full text of DOI:10.1016/j.bioorg.2019.103062

BioorganicChemistry90(2019)103062
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine
and its bromo-derivative as dual cholinesterase inhibitors
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
b 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
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alkaloids have always been a great source of cholinesterase inhibitors. Numerous studies have shown that in-
hibiting acetylcholinesterase as well as butyrylcholinetserase is advantageous, and have better chances of suc-
cess 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 acet-
ylcholinesterase and butyrylcholinesterase, the enzymes which are involved in blocking the process of neuro-
transmission. Cryptolepine inhibits Electrophorus electricus acetylcholinesterase, recombinant human acet-
ylcholinesterase 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 oli-
gomers (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 al-
losteric “peripheral anionic site” via hydrophobic interactions. The ADME properties including the BBB per-
meability were computed for these alkaloids, and were found within the acceptable range.
Cryptolepine
Acetylcholinesterase
Butyrylcholinesterase
Alzheimer's disease
Dual cholinesterase inhibitor
1. Introduction
found to compensate for AChE particularly when AChE levels are de-
pleted (a condition observed in AD brain with increased or unchanged
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-es-
tablishing 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
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 hy-
potheses such as “amyloid” and “tau” hypothesis are widely accepted in
AD etiology [12]. According to the general consensus, β-amyloid pep-
tide (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: sbharate@iiim.ac.in (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
Availableonline12June2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
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 pro-
ducts 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-dihy-
droervahanine 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 pub-
lished 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 pri-
marily been extensively investigated for its antimalarial activity, based
on the traditional usage of Cryptolepis sanguinolenta plant for manage-
ment 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-in-
flammatory, antibacterial, hypotensive, antidiabetic, antithrombotic
and renovascular vasodilatory activity [32–35]. The 2-bromocryptole-
pine (2) is a synthetic derivative of cryptolepine with better anti-
malarial potency, which was discovered from medicinal chemistry ef-
forts [33]. The chemical structures of alkaloids 1–2 are shown in Fig. 1.
Herein, we report the discovery of indoloquinoline alkaloid cryp-
tolepine (1) as a multitargeted agent acting on four targets of AD viz.
AChE, BChE, BACE-1 and P-gp. The enzyme kinetic study was per-
formed 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 over-
expressing 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.
(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-bu-
tyrylthiocholine 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 appa-
ratus. Absorbance readings for Ellman assay and fluorescence readings
for FRET/ rhodamine 123 assay were recorded on Molecular Devices
and BioTek plate readers, respectively.
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-bro-
moacetyl bromide (4.0 mL, 1.2 equiv.) was added drop-wise. The re-
sultant mixture was allowed to stir at room temperature for 10 h, after
which, the water (30 mL) was added to the reaction mixture. The ob-
tained 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 ad-
justing 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 com-
mercial 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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
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)
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].
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].
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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
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].
1048 cm−1; ESI-MS: m/z 341.2 [M + H]+ [33].
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 ob-
tained 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 cal-
culated for C16H12BrN2 (311.0178) [33].
2.3. Synthesis of 2-bromocryptolepine (2)
The synthesis of 2-bromocryptolepine was achieved from anthra-
nilic 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-bromo-
cryptolepine 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). Thio-
choline, 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 concentra-
tions using graph-Pad Prism 6 software and are reported as mean
SEM (an average of three experiments). Donepezil was used as re-
ference 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 re-
spect 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 re-
action mixture was then acidified with aqueous HCl, resulting in for-
mation 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 re-
spect to 11) and triethylamine (1.3 g, 1.4 mmol) was added 2-(N-(car-
boxymethyl)acetamido)benzoic acid (11, 1.36 g). The mixture was re-
fluxed 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 determina-
tion 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)
2.6. BACE-1 inhibition
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,
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 re-
storation 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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
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
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-bro-
mocryptolepine (2). Both synthesized compounds 1 and 2 were fully
characterized using spectral analysis, and the data was compared with
the literature values [36,46].
a reference compound (IC50 = 17.98
assay.
1.07 nM) (Lit. 15 nM) in the
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
As described earlier [17,19].
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 en-
zymes 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 eval-
uated for anti-cholinesterase activity against EeAChE and eqBChE,
following Ellman method [37] and donepezil as a reference standard in
the assay.
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 pro-
posed 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.
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 va-
lues 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 re-
sponsible 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
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 for-
mation of 2-(2-(phenylamino)acetamido)benzoic acid (6). The cycliza-
tion of intermediate 6 using polyphosphoric acid produced indolo[3,2-
b]quinolin-11-one (7), which further on treatment with POCl3 resulted
in replacement of carbonyl functionality with chlorine atom to yield 11-
chloro-10H-indolo[3,2-b]quinoline (8). The hydrogenation of chloro-
derivative 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-bromo-
cryptolepine.
Entry
IC50 (nM)
EeAChEb
SDa
rHuAChEc
eqBChEd
1
267
417
49
17
18
485
868
32
67
699
17
25
125
2
100
770
Donepezil
1
4.2
5520
a
b
c
The 50% inhibitory concentration of EeAChE, rHuAChE or eqBChE.
EeAChE is Electrophorus electricus AChE.
rHuAChE is recombinant human AChE.
d
eqBChE is equine serum BChE.
5

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
18
16
14
12
10
8
(a)
Control
(b)
0.15625 µM
0.3125 µM
0.625 µM
1.25 µM
0.9
0.7
0.5
0.3
0.1
y = 0.276x + 0.244
R² = 0.995
2.5 µM
1
6
4
Ki
2
0
-1.2 -0.8 -0.4
-0.1
0
0.4 0.8 1.2 1.6
2
2.4 2.8
-4
0
4
8
12
16
-2
-1
[Cryptolepine] μM
[ATCh] mM
(c)
(d)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control
16
14
12
10
8
0.15625 μM
0.3125 μM
0.625 μM
1.25 μM
y = 0.211x + 0.209
R² = 0.997
2
2.5 μM
6
4
Ki
2
0
-1.2 -0.8 -0.4
-0.1
0
0.4 0.8 1.2 1.6
2
2.4 2.8
-6 -4 -2
-2
0
2
4
6
8
10 12 14 16 18
[ATCh]-1 mM
[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, com-
pounds 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 re-
ciprocal plots (Fig. 3a and c) followed by replotting slopes against in-
hibitor 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 re-
ciprocal 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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
3
(b)
2.5
(a)
32
28
24
20
16
12
8
Control
0.125µM
0.25µM
0.5µM
1µM
y = 0.946x + 0.485
R² = 0.992
2
1.5
1
1
2µM
0.5
0
Ki
4
0
-1
-0.5
0
0.5
1
1.5
2
-2
0
2
4
6
8
10
-4
[Cryptolepine] μM
[BTCh]-1mM
-0.5
(c)
(d)
28
24
20
16
12
8
0.15625 µM
0.3125 µM
0.625 µM
1.25 µM
2
2
y = 0.607x + 0.133
R² = 0.999
1.5
1
2.5 µM
0.5
0
Ki
4
0
-0.5
0
0.5
1
1.5
2
2.5
3
-6 -4 -2
-4
0
2
4
6
8
10 12 14 16 18
[BTCh]-1 mM
[2-Bromocryptolepine] μM
-0.5
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 sub-
sites 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-bromo-
cryptolepine (2).
Entry
ki (μM)
SDa
Type of inhibition
rHuAChE
eqBChE
rHuAChE
eqBChE
1
2
0.88
0.99
0.09
0.12
0.51
0.22
0.07
0.05
Non-competitive
Non-competitive
Mixed
Mixed
a
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 non-
competitive 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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
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 addi-
tional 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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
(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 in-
hibitor, and it interacts with both CAS and PAS sites within the active
site gorge. The overlay of cryptolepine with donepezil (Fig. 4D) re-
vealed that both the molecules occupy the same binding site, and dis-
play 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 ros-
marinic 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 hydro-
phobic 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.
3.3. BACE-1 inhibition activity
The role of BACE-1 in formation of Aβ plaques has been well es-
tablished. Recent study has also shown that BACE-1 expressed in the
BBB endothelium is upregulated in a murine model of AD [61]. Fol-
lowing FRET based enzymatic assay, the inhibitory potential of com-
pounds 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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
A.
B.
Val 132
Leu 414
Glu 452
Glu 313
Gln 413
Arg 463
Tyr 449
Trp 532
Glu 81
Arg 296
Gln 369
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-bromo-
cryptolepine 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 vio-
late 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 over-
expressing 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 re-
sulted 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 in-
duction 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 in-
tracellular 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 in-
hibition of BACE-1 and potent P-gp induction activity.
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 ef-
fective concentrations for P-gp induction and cholinesterase inhibition.
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 crypto-
lepine (dual ability to inhibit AChE as well as BChE enzymes and dis-
playing a potent P-gp induction), the further exploration of this class of
natural products is warranted in preclinical animal efficacy studies.
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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
A.
Trp231
Val 288
CAS
Gly 117
Gly 116
Anionicsubsite
Acyl binding pocket
Ser 198
Leu 286
Oxyanionhole
PAS
Tyr 332
Phe 329
Glu 325
His 438
Asp 70
Tyr 128
Trp82
C.
B.
Leu 286
Trp231
Leu 286
Ring D
Trp231
Glu 325
Glu 325
Tyr 332
His 438
Ring D
Tyr 332
Ring A
Ring A
Asp 70
Asp 70
Trp82
Tyr 128
Tyr 128
Trp82
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
Cryptolepine
Interactions
His 438 (π-π)
2-Bromocryptolepine
Interactions
NI
Glide score
Glide score
Catalytic anionic (or acylation)
site (CAS) – (Ser 198, His
438, Glu 325)
−8.86 (Donepezil = −10.52)
−4.20 (Donepezil = −10.52)
“anionic” subsite (choline
binding pocket) – (Trp 82,
Tyr 128, Phe 329)
Trp 82 (π-π)
NI
Acyl binding pocket – (Val
288, Leu 286, Trp 231)
Oxyanion hole –
NI
NI
NI
NI
NI
(Gly 116, Gly 117)
Peripheral anionic site –
(Asp 70, Tyr 332)
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

V.K. Nuthakki, et al.
BioorganicChemistry90(2019)103062
Table 5
ADME properties cryptolepine, 2-bromocryptolepine and donepezil.a
Entry
Cholinesterase inhibition (IC50
)
QPlogP o/wb
QPlogSc
QPP
QPP
QPlogBBf
CNSg
Lipinski
Cacod
MDCKe
Violationsh
HuAChE
eqBChE
1
0.48
0.87
0.032
0.70
0.77
5.52
3.481
4.047
4.446
−3.677
−4.33
5591
5593
892
3179
8389
484
0.344
0.525
0.114
1
1
2
0
0
0
2
Donepezil
−4.678
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
c
d
e
Predicted octanol/water partition co-efficient log p (acceptable range: −2.0 to 6.5).
Predicted aqueous solubility; S in mol/L (acceptable range: −6.5 to 0.5).
Apparent Caco-2 Permeability (nm/sec) (< 25 poor, > 500 great).
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
g
h
Predicted brain/blood partition coefficient (acceptable range: −3.0 to +1.2).
Predicted central nervous system activity on a −2 (inactive) to +2 (active) scale (acceptable range: −2.0 to +2.0).
Lipinski Rule of 5 Violations (maximum is 4).
Acknowledgements
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Funding
This work was supported by CSIR Young Scientist Grant (P90807),
awarded to SBB.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
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