Manuscript CARBAZOLE-STILBENE (corrMRY05052020)

Novel Carbazole-Stilbene Hybrids as Multifunctional anti-Alzheimer agents
Dushyant V. Patel1, Nirav R. Patel1, Ashish M. Kanhed2, Divya M. Teli3, Kishan B. Patel1,
Prashant D. Joshi1, Sagar P. Patel1, Pallav M. Gandhi1, Bharat N. Chaudhary1, Navnit K.
Prajapati1, Kirti V. Patel1, Mange Ram Yadav1,4*
1
Faculty of Pharmacy, Kalabhavan Campus, The Maharaja Sayajirao University of Baroda,
Vadodara-390001, Gujarat, India
2
Shobhaben Pratapbhai Patel - School of Pharmacy & Technology Management, SVKM’s
NMIMS University, Vile Parle, Mumbai-400056, India
3
Department of Pharmaceutical Chemistry, L. M. College of Pharmacy, Navrangpura,
Ahmedabad-380009, Gujarat, India
4
Current Position: Director (R & D), Parul University, Limbda, Waghodia Road, Vadodara,
Gujarat, India
Abstract:
1
Molecules capable of engaging with multiple targets associated with pathological
condition of Alzheimer's disease have proved to be potential anti-Alzheimer's agents. In our goal
to develop multitarget-directed ligands for the treatment of Alzheimer’s disease, a novel series of
carbazole-based stilbene derivatives were designed by the fusion of carbazole ring with stilbene
scaffold. The designed compounds were synthesized and evaluated for their anti-AD activities
including cholinesterase inhibition, A aggregation inhibition, antioxidant and metal chelation
properties. Amongst them, (E)-1-(4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(pyrrolidin-
1-yl)ethyl)thiourea (50) appeared to be the best candidate with good inhibitory activities against
AChE (IC50 value of 2.64 μM) and BuChE (IC 50 value of 1.29 μM), and significant inhibition of
self-mediated A1–42 aggregation (51.29 % at 25 μM concentration). The metal chelation study
showed that compound (50) possessed specific copper ion chelating property. Additionally,
compound (50) exhibited moderate antioxidant activity. To understand the binding mode of 50,
molecular docking studies were performed, and the results indicated strong non-covalent
interactions of 50 with the enzymes in the active sites of AChE, BuChE as well as of the A1-42
peptide. Additionally, it showed promising in silico ADMET properties. Putting together, these
findings evidently showed compound (50) as a potential multitarget-directed ligand in the course
of developing novel anti-AD drugs.
Keywords: Alzheimer’s disease, MTDL, Acetylcholinesterase, Butyrylcholinesterase, Aβ
aggregation, Metal chelation, Carbazole, Stilbene
Introduction
2
Alzheimer’s disease (AD) is an age-related devastating neurodegenerative disorder,
characterized by slow and inexorable memory loss that begins many years before the symptoms
emerge [1]. It is the most common type of senile dementia with prevalence of 50 million people
worldwide. By 2050, it is estimated that the number will rise to 150 million if no preventative
means are made available [2]. The two major hallmarks of AD are the accumulation of beta-
amyloid (Aβ) plaques outside the neurons and the twisted strands of tau protein inside the
neurons in the brain. Although the etiology of AD is very complex and not fully resolved, several
factors like deficit of acetylcholine (ACh) [3], abnormal Aβ deposition and accumulation [4], tau
hyperphosphorylation [5], oxidative stress [6], and dyshomeostasis of biometals [7] are
considered to play notable roles in the pathophysiology of AD. Currently, four drugs are
approved for the treatment of AD namely: donepezil, rivastigmine, galantamine and memantine
[8]. They only give symptomatic relief to the patient while none of the drugs available today
stops the damage and destruction of neurons that make the AD fatal.
The cholinergic hypothesis reveals that the cognitive deficit associated with AD is due to
cholinergic system dysfunction, mainly due to the declining levels of ACh in the brain [3, 9].
ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in
the brain. Therefore, ChE inhibition is a useful strategy to increase ACh levels within the brain
[10]. Besides its catalytic role, AChE also plays proaggregatory role by accelerating A peptide
aggregation and deposition into the fibrils. It is reported that AChE binds through peripheral
anionic site (PAS) to non-amyloidogenic form of A, acting as a pathological chaperone
inducing conformational changes in amyloidogenic form of A with subsequent A fibril
formation [11]. In a healthy brain, AChE plays a major role in the hydrolysis of ACh. As the
disease progresses, AChE level decreases, but BuChE level increases up to 40 to 90 % in the
3
hippocampus and temporal cortex region of the brain [12]. BuChE plays several roles both in
neural and non-neural functioning. Clinical data suggested that high cortical levels of BuChE
were associated with some important AD hallmarks, such as the extracellular deposition of the
Aβ and aggregation of hyperphosphorylated tau protein [13]. This attests to the crucial role
played by both the cholinesterases and the imperative to develop multi-targeted directed ligands
(MTDLs) which could act on both of these ChEs.
Amyloid hypothesis states that Aβ plaques in the brain play a critical role in AD
pathogenesis [14, 15]. The plaques consist of aggregated oligomeric A of variable lengths
which are produced by sequential cleavage of the amyloid precursor protein (APP) by - and -
secretase. These aggregates initiate pathogenic cascade and eventually lead to neuronal loss and
dementia [16]. The A plaques generated from A1-42 are neurotoxic which continuously activate
inflammatory mediators, such as TNF- and IL-6. Furthermore, A1-42 itself can serve as an
oxygen-free radical donor that produces reactive oxygen species and directly affects the normal
physiological functions of neurocytes [17]. Hence, the inhibition of A1-42 aggregation could
serve as a rational approach for the treatment of AD.
Recent research indicates that oxidative stress is an event that precedes the appearance of
other hallmarks of AD. The subtle equilibrium between oxidants/antioxidants disturbed
by inequity between generation and scavenging of free radicals cause oxidative stress [18]. By
pathological oxidation-reduction steps, reactive oxygen species (ROS) and reactive nitrogen
species (RNS) can denature biomolecules like proteins, lipids and nucleic acids. This can induce
tissue damage through necrosis and apoptosis [19]. Thus, anti-oxidants endowed with additional
pharmacological properties are thought to offer a hope to combat this complex disease, in which
free radicals are important culprits but not the sole drivers.
4
Likewise, dyshomeostasis of metal ions such as iron, zinc, and copper, clearly occur in
AD brains. The elevated concentrations of metal ions hasten the formation of A aggregates and
neurofibrillary tangles, which activate neurotoxic pathways and promote inflammation, leading
to dysfunction and death of brain cells [20, 21]. Additionally, redox-active metal ions, Cu(I/II),
and Fe(II/III) associated with Aβ, were demonstrated to generate ROS under physiological
conditions through Fenton-like reactions [22]. The overproduction of ROS leads to oxidative
stress and eventually neuronal death in AD patients. Therefore, A aggregation and ROS
production induced by metal ions can be modulated by metal chelators, which highlights metal-
ion chelation therapy as a promising AD treatment. However non-selective metal chelators are
likely to exhibit adverse side effects which is liable to limit their long-term clinical use.
In continuation of our research for unraveling potential novel MTDLs to combat AD, a
series of novel carbazole-based stilbene derivatives as multifunctional anti-AD agents are
presented here. Herein, we report the designing, synthesis and anti-AD activities of these novel
compounds including cholinesterase inhibition, A aggregation inhibition, antioxidant and metal
chelation properties. Molecular modeling studies were also performed to know the binding mode
of these compounds with the target proteins. ADMET properties of the synthesized compounds
were also predicted using in silico methods.
Rationale of designing
The multifactorial nature of AD demands assaulting its key pathological hallmarks using
MTDLs. Although ChEIs provide only symptomatic and transient benefits to the patients, they
still remain the drugs of choice. Neurotoxic A plaques, metal ion dyshomeostasis and oxidative
stress play crucial roles in the pathogenesis of AD. However, targeting these factors all alone
might not be enough to combat such a highly complex pathological disease like AD. So,
5
cholinesterase inhibitors endowed with additional A aggregation inhibitory, and metal chelating
and antioxidant activities could prove to be the competent candidates to confront this
multifaceted disease.
Carbazole is an important nitrogen-containing heterocycle present widely in many
phytochemicals with a range of biological activities [23]. It has been reported that the carbazole
derivatives possess cholinesterase inhibitory [24], Aβ aggregation inhibitory [25], ROS
scavenging, and neuron protective activities against oxidative damage [26]. Recently, various
hybrid molecules having the carbazole scaffold and some other biologically active moieties have
been developed as potential MTDLs for AD, like carbazole-tacrine hybrid (1) [27], carbazole-
ferulic acid hybrid (2) [24] and carbazole-thiazole hybrid (5) [28] (Figure 1). A plethora of
biological activities associated with carbazole makes it an interesting privileged scaffold in the
search for new anti-AD agents. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) (6) is a naturally
occurring stilbene derivative possessing multiple anti-AD properties i.e. Aβ aggregation
inhibitory [29], neuroprotective [30] and antioxidant activities [31]. Several stilbene derivatives
with encouraging Aβ binding abilities have been developed as Aβ imaging probes and Aβ
aggregation inhibitors during the last two decades (Figure 1) [32]. In combination with other
bioactive moieties, these stilbene hybrids showed cholinesterase inhibitory, metal chelating, Aβ
aggregation inhibitory, and free radical scavenging activities [33–35]. A central lipophilic
scaffold with a terminal amine group is the salient feature of most of the reported stilbene
derivatives which could have an impact on their anti-AD properties.
6
Figure 1. Chemical structures of some previously reported (A) carbazole-based compounds (1-5)
and (B) stilbene-based compounds (6-10) as anti-AD agents.
A combination of pharmacophoric moieties having different activities offering some
novel hybrids have brought new hope for the treatment of AD. By molecular hybridization
approach, a fusion of one pharmacophore to other results into a highly integrated scaffold with
lower molecular weight. Here, a carbazole based stilbene scaffold was generated by the fusion of
carbazole ring with a stilbene scaffold. We have previously reported substituted triazinoindole
derivatives as anti-AD agents, in which pyrrolidine and piperidine side chains played an
7
indispensable role in offering cholinesterase inhibitory activity [36]. Herein, we have designed
two series of carbazole based stilbene derivatives in which the heterocyclic amines were linked
with the designed scaffold using linkers endowed with additional anti-AD property (Figure 2).
Figure 2. Molecular hybridization approach used to design carbazole-based stilbene derivatives.
8
Results and discussion
Chemistry
Based on the attachment of the heterocyclic amines through suitable linkers on either of
the two sites of the designed scaffold, these carbazole based stilbene derivatives have been
divided into two series i.e. Series-1, wherein the attachment of the linker was to the carbazole
ring and Series-2, in which the attachment was to the phenyl ring of the stilbene moiety (Figure
2). Synthesis of the designed carbazole derivatives of Series 1 has been depicted in Scheme
1 and Scheme 2. The synthetic route for the required key intermediate (E)-9-ethyl-6-styryl-9H-
carbazol-3-amine (16) from the commercially available carbazole (11) is outlined in Scheme 1.
Ethylation of carbazole (11) by ethyl bromide in the presence of aqueous NaOH solution in
DMSO afforded N-ethylcarbazole (12) in excellent yield. Mono-formylation of N-
ethylcarbazole (12) by Vilsmeier-Haack formylation using DMF and phosphorus oxychloride
generated 9-ethyl-3-formylcarbazole (13). Treatment of 13 with concentrated nitric acid gave the
nitro derivative (14), which was reacted with benzyltriphenylphosphonium bromide under Wittig
reaction condition to yield a mixture of cis- and trans-stilbenes. This isomeric mixture was
converted to a single trans isomer (15) by performing the reaction with catalytic amounts of
iodine in toluene. Reduction of the nitro group in (E)-9-ethyl-3-nitro-6-styryl-9H-carbazole (15)
by stannous chloride offered the key amine intermediate (16).
9
O
H
(i) (ii)
N N N
H
Me Me
11 12 13
(iii)
Ph Ph O
H
(v) (iv) O2N

H2N O2N
N N N
Me Me Me
16 15 14
Scheme 1. Synthesis of the key intermediate (E)-9-ethyl-6-styryl-9H-carbazol-3-amine (16).

Reagents and conditions: (i) EtBr, NaOH, DMSO, RT; (ii) POCl 3, DMF, CHCl3; (iii) conc.
HNO3, AcOH; (iv) (a) benzyltriphenylphosphonium bromide, LiOH, IPA, reflux, (b) I 2, toluene,
reflux; (v) SnCl2, THF, MeOH, reflux.
N-(9-Ethyl-3-styryl-9H-carbazole-6-yl)aminoalkylamide derivatives (20-25) have been
synthesized in two steps as shown in Scheme 2. Reaction of the amine intermediate (16) with the
respective acid chlorides in presence of K2CO3 in acetone yielded the amide intermediates (17-
19), which were further linked with alicyclic amines to offer the designed N-(9-ethyl-3-styryl-
9H-carbazole-6-yl)aminoalkylamides (20-25).
Synthetic route for the designed (E)-1-(9-ethyl-6-styryl-9H-carbazol-3-yl)aminoalkylurea
derivatives (26-31) is shown in Scheme 2. The amine intermediate (16) was reacted with p-
nitrophenyl chloroformate in the presence of triethylamine, followed by reaction with alicyclic
amines and aminoalkylamines yielded the designed (E)-1-(9-ethyl-6-styryl-9H-carbazol-3-
yl)aminoalkylurea derivatives (26-31).
10
Ph Ph Ph
(i) n H (ii) n H
H2N N R1 N
X N
N O R2 O N
N
Me Me Me
16 17-19 20-25
(iii) Compd. n X Compd. n NR1R2 Compd. n NR1R2
17 2 Cl 20 2 21 2
18 3 Cl 22 3 N 23 3 N
Ph
19 4 Br 24 4 25 4
R1
H
N A N Compd. A n NR1R2 Compd. A n NR1R2
R2
n
O N 26 - 0 27 - 0
28 NH 2 N 29 NH 2 N
Me 31
30 NH 3 NH 3
26-31
Scheme 2. Synthetic route for the synthesis of compounds (20-31). Reagents and conditions: (i)
acid chloride, K2CO3, acetone; (ii) NHR1R2, THF, reflux; (iii) (a) p-nitrophenyl chloroformate,
TEA, DCM:THF (1:1), 0 °C to RT, (b) pyrrolidine/piperidine/aminoalkylamines, RT.
Synthesis of the designed carbazole derivatives of Series 2 has been outlined in Scheme
3 and Scheme 4. Synthetic route for the required key intermediate (E)-4-(2-(9-ethyl-9H-
carbazol-3-yl)vinyl)aniline (34) from 9-ethyl-9H-carbazole-3-carbaldehyde (13) is shown
in Scheme 3. The reaction of 13 with 4-nitrophenylacetic acid (32) in the presence of piperidine
under microwave conditions afforded the trans nitro stilbene derivative (33) as the sole product,
configuration of which was confirmed by XRD. Reduction of the nitro group in compound (33)
by stannous chloride yielded the desired amine intermediate (34).
11
NO2
O
NO2
H O (i)
N HO
Me N
Me
13 32 33
(iii)
NH2
N
Me
34
Scheme 3. Synthesis of the key intermediate (E)-4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)aniline

(34). Reagents and conditions: (i) Piperidine, MW; (ii) SnCl2, THF, MeOH, reflux.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)aminoalkylamide derivatives (38-
43) have been synthesized in a way similar to compounds (20-25) in two steps as shown
in Scheme 4. Reaction of the amine intermediate (34) with the respective acid chlorides offered
the amide intermediates (35-37), which were further reacted with alicyclic amines to afford the
designed (E)-N-(4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)phenyl)aminoalkylamides (38-43).
Synthesis of the designed urea derivatives (44-49) and thiourea derivatives (50-52) was
performed as depicted in Scheme 4. Reaction of the amine intermediate (34) with p-nitrophenyl
chloroformate in the presence of triethylamine, followed by reaction with aminoalkylamines
afforded the designed urea derivatives (44-49). Similarly, reaction of the amine intermediate (34)
with thiocarbonyldiimidazole followed by reaction with the respective aminoalkylamines
afforded the designed thiourea derivatives (50-52).
12
Scheme 4. Synthetic route for the synthesis of compounds (38-52). Reagents and conditions: (i) Acid
chloride, K2CO3, acetone; (ii) NHR1R2, THF, reflux; (iii) For 44-49 (a) p-nitrophenyl chloroformate,
TEA, DCM:THF (1:1), 0 °C to RT, (b) pyrrolidine/piperidine/aminoalkylamines, RT; For 50-52 (a)
thiocarbonyldiimidazole, DCM:THF (1:1), 0 °C to RT; (b) aminoalkylamines, RT.
Biological evaluation
In vitro cholinesterase inhibition studies
The potential of the synthesized compounds to inhibit ChEs was assessed in vitro by
Ellman’s assay, as reported by our group earlier [36–38]. The obtained IC50 values of the
compounds for both AChE and BuChE enzymes and their selectivity indices (SI) are
summarized in Table 1 and Table 2. All the compounds (20-31, 38-52) offered IC50 values in the
range of 1.84-6.63 µM for AChE and 1.02-5.01 µM for BuChE.
The inhibitory potential of the compounds from Series 1 having a side chain attached to
the carbazole (ring A) has been shown in Table 1. It was observed that changing the length of
the carbon chain affected the inhibitory activity. A comparative analysis of the inhibitory
potential of compounds (21, 23 and 25) having a piperidine ring, revealed that compound (25, n
= 4) showed comparatively better AChE inhibitory activity (IC 50 value of 1.84 μM) while
13
compound (21, n = 3) and compound (23, n = 2) showed slightly poorer AChE inhibitory
activities (IC50 values of 3.54 and 4.59 μM, respectively) (Table 1). A similar activity pattern
was also observed for the compounds (20, 22 and 24) having a pyrrolidine ring. Among these
compounds, 21 (n = 2) showed better BuChE inhibition (IC 50 value of 1.40 μM) in comparison to
the other two derivatives. When the pyrrolidinyl (compound 26) and piperidinyl (compound 27)
moieties were attached directly (n = 0, Table 1) to form urea derivatives, the inhibitory activities
against both the enzymes decreased notably. Due to direct attachment, these compounds lost
their basic centers, which are seemingly indispensable for cation- interaction with the enzymes.
Compounds (26 and 27) showed inhibitory activities (AChE; IC50 = 6.63 µM and 5.99 µM,
respectively and BuChE; IC50 = 4.48 µM and 5.01 µM, respectively). There was no significant
change observed in inhibitory activities when the amide linkers (compounds 20-25) were
substituted with urea linkers (compounds 28-31) (Table 1).
Table 1. In vitro hAChE, EqBuChE and self-induced A aggregation inhibitory activities and
DPPH radical scavenging activity of compounds (20-31)
Ph
R1
H
N A N
R2
n
O N
Me
20-31
IC50 ± SEM (µM) Aβ1-42 aggregation RP of DPPHe

Compd A n 1
RRN 2
SI c Inhibition (%)d IC50 ± SEM (µM) or
hAChEa EqBuChEb at 25 µM % inhibition at 100 µM
20 - 2 3.00 ± 0.52 1.53 ± 0.31 0.51 42.72 ± 0.31 144.61 ± 2.32 (35.46 %)
21 - 2 4.69 ± 0.25 1.40 ± 0.22 0.29 52.08 ± 0.64 135.42 ± 2.05 (39.31 %)
22 - 3 2.91 ± 0.14 1.51 ± 0.35 0.52 53.92 ± 0.28 145.02 ± 3.61 (36.15 %)
14
23 - 3 3.54 ± 0.56 2.56 ± 0.29 0.72 49.88 ± 0.15 122.41 ± 3.20 (41.63 %)
24 - 4 2.63 ± 0.31 3.17 ± 0.43 1.20 46.72 ± 0.33 134.63 ± 1.29 (38.11 %)
25 - 4 1.84 ± 0.27 2.51 ± 0.19 1.36 48.09 ± 0.54 138.18 ± 1.53 (36.97 %)
26 - - 6.63 ± 0.54 4.48 ± 0.15 0.67 17.58 ± 0.42 nd
27 - - 5.99 ± 0.07 5.01 ± 1.02 0.83 21.55 ± 0.62 nd
28 -NH 2 2.65 ± 0.32 1.70 ± 0.18 0.64 52.29 ± 0.42 104.28 ± 3.87 (48.94 %)
29 -NH 2 3.79 ± 0.41 1.99 ± 0.21 0.52 50.08 ± 0.42 110.42 ± 2.62 (47.54 %)
30 -NH 3 4.54 ± 0.52 3.19 ± 0.35 0.70 54.94 ± 0.42 123.62 ± 3.53 (42.49 %)
31 -NH 3 3.57 ± 0.29 1.02 ± 0.18 0.28 54.35 ± 0.42 118.58 ± 3.20 (45.62 %)
Tacrine 0.056 ± 0.01 0.008 ± 0.00 0.14 nd > 500
Donepezil 0.023 ± 0.01 1.87 ± 0.08 81.3 nd > 500
Curcumin 20.43 ± 0.72 µM

nd nd - nd
(IC50)
Ascorbic acid nd nd - nd 13.91 ± 1.33 (98.25 %)

a
AChE from human erythrocytes, bBuChE from equine serum, IC50 = 50 % inhibitory concentration (means ± SEM of three
experiments). cSelectivity index = IC50 (BuChE)/IC50 (AChE). dAβ1-42 peptide/inhibitor 1:1 with 25 µM inhibitor
concentration. eRP of DPPH (%) = reduction percentage of DPPH.
Shifting the chain from the carbazole (ring A, Figure 2) to the phenyl ring (ring B) of
stilbene preserved the ChEs inhibitory potency (Table 2). A comparative analysis of the
inhibitory potential of compounds (38, 40 and 42) having a pyrrolidine ring, revealed that
compound (40, n = 2) showed the best profile of AChE and BuChE inhibitory activities (IC50
values of 2.36 μM and 1.46 μM, respectively) while compound (38, n = 1) and compound (42, n
= 3) showed slightly lower AChE and BuChE inhibitory activities. A similar activity pattern was
also observed for the compounds (39, 41, and 43). Among these, compound (41, n = 2) showed
15
the highest AChE and BuChE inhibitory activities (IC 50 values of 2.25 μM and 1.74 μM,
respectively).
All the urea derivatives (46-49) showed good ChEs inhibition, whereas compounds
(44 and 45) in which the heterocyclic amine was directly attached (n = 0) to form urea showed
moderate inhibitory activities (AChE; IC50 = 16.22 µM and 12.37 µM, respectively) and
(BuChE; IC50 = 11.65 µM and 8.58 µM, respectively). There was no significant change observed
in AChE inhibitory activities when the amide linkers (compounds 42 and 43) were substituted
with urea linkers (compounds 46 and 47) whereas the BuChE inhibitory activity was observed to
be increased by two fold. All the thiourea derivatives (50-52) showed the best ChEs inhibitory
activities profile amongst all of the derivatives. Among them, compound (50) was conferred with
the highest AChE and BuChE inhibitory activities (n = 2, IC50 value of 2.64 μM and 1.29 μM,
respectively).
Table 2. In vitro hAChE, EqBuChE and self-induced A aggregation inhibitory activities and
DPPH radical scavenging activity of compounds (38-52)
R1
H
N A N 2
R
n
X
Me
(38-52)
IC50 ± SEM (µM) RP of DPPHe

Aβ1-42 aggregation
Compd X A n R1R2N SIc IC50 ± SEM (µM) or
hAChEa EqBuChEb Inhibition (%)d
% inhibition at 100 µM
38 O - 1 2.98 ± 0.78 2.49 ± 0.65 0.84 51.14 ± 0.31 132.42 ± 1.17 (42.34 %)
39 O - 1 3.52 ± 0.87 2.72 ± 0.98 0.79 45.17 ± 0.64 146.28 ± 3.21 (35.46 %)
40 O - 2 2.36 ± 0.20 1.46 ± 0.42 0.62 54.27 ± 0.28 189.10 ± 2.26 (30.15 %)
16
41 O - 2 2.25 ± 0.31 1.74 ± 0.19 0.77 51.92 ± 0.15 178.51 ± 3.66 (25.81 %)
42 O - 3 4.77 ± 0.61 4.76 ± 1.03 0.99 53.08 ± 0.33 126.43 ± 2.27 (41.34 %)
43 O - 3 3.29 ± 1.09 2.11 ± 0.69 0.64 49.78 ± 0.54 118.58 ± 1.12 (42.91 %)
44 O - - 16.22 ± 1.03 11.65 ± 0.51 0.68 42.84 ± 0.82 nd
45 O - - 12.37 ± 0.87 8.58 ± 1.02 0.69 44.15 ± 0.67 nd
46 O -NH 2 4.71 ± 1.05 2.32 ± 0.97 0.49 38.90 ± 0.42 161.45 ± 1.46 (13.67 %)
47 O -NH 2 3.13 ± 0.71 1.20 ± 0.57 0.38 55.79 ± 0.42 97.36 ± 1.46 (51.19 %)
48 O -NH 3 3.04 ± 0.43 1.92 ± 0.25 0.63 53.86 ± 0.42 106.01 ± 1.39 (45.95 %)
49 O -NH 3 2.94 ± 0.98 1.98 ± 0.84 0.67 54.06 ± 0.42 134.70 ± 1.17 (41.98 %)
50 S -NH 2 2.64 ± 0.41 1.29 ± 0.10 0.49 51.29 ± 0.42 86.13 ± 1.23 (72.36 %)
51 S -NH 2 3.41 ± 0.25 1.72 ± 0.25 0.50 53.24 ± 0.42 91.72 ± 3.43 (66.36 %)
52 S -NH 3 3.19 ± 0.34 1.32 ± 0.17 0.41 53.29 ± 0.42 90.33 ± 1.20 (70.36 %)
a
AChE from human erythrocytes, bBuChE from equine serum, IC50 = 50 % inhibitory concentration (means ± SEM of three
experiments). cSelectivity index = IC50 (BuChE)/IC50 (AChE). dAβ1-42 peptide/inhibitor 1:1 with 25 µM inhibitor concentration.
e
RP of DPPH (%) = reduction percentage of DPPH.
Self-mediated A1–42 aggregation inhibition study
A Peptides present in the extracellular amyloid plaques are produced by sequential
cleavage of APP by - and -secretases. A1-40 and A1-42 are the two main isoforms of A
peptides present in the plaques. A1-40 is the predominant product in the proteolytic cleavage,
whereas A1-42 is more fibrillogenic in nature [39, 40]. So, A1-42 was chosen for the A
aggregation inhibition study. The potential of the compounds to inhibit self-mediated A1-42
aggregation was assessed using Thioflavin T (ThT) fluorescence assay. 165 Curcumin was used as
17
a positive control in this assay. Percentage inhibitions of self-mediated A1-42 aggregation of all
the tested compounds at 25 μM concentrations are listed in Tables 1 and 2. All the tested
compounds showed good A1-42 aggregation inhibition ranging from 38.9 to 55.79 %. Amongst
them, compound (47) showed the best A1-42 aggregation inhibition (55.79 %) at 25 μM
concentration.
Antioxidant Activity
The antioxidant activity of the compounds was evaluated by their ability to reduce
DPPH· radical (purple) to DPPHH (yellow) and the corresponding radical-scavenging potential
was assessed by a decrease in the absorbance at 517 nm [36]. Ascorbic acid was employed as a
positive control in this assay. All the test compounds displayed moderate free radical scavenging
activity (Tables 1 and 2). Amongst the thiourea derivatives (compounds 50 - 52) showed
moderate free radical scavenging activity (IC50 values 86.13 μM, 91.72 μM and 90.33 μM,
respectively) compared to ascorbic acid (IC50 value of 13.9 μM) whereas tacrine and donepezil
(IC50 values > 500 μM) were found to be practically devoid of significant free radical scavenging
activity at this concentration.
Metal Chelation study
High levels, and at the same time deregulation of biometal ions, such as Cu2+, Zn2+, and
Fe2+, are closely involved in the pathogenesis of AD [41]. Thus, the potential of compounds to
form chelates with these biometals present in the brain of AD patients is like adding a feather in
the cap of ideal MTDLs to treat AD patients.
The ability of the test compounds to chelate biometals was assessed using UV-
vis spectrophotometric assay [42]. 8-Hydroxyquinoline (HQ) was selected as a positive control
(Figure 3). The results demonstrated that when CuSO4 was added to a solution of compound
18
(50), its maximum absorbance at 343 nm decreased dramatically, indicating the formation of
ligand-Cu2+ complex (Figure 3). There were insignificant changes in the positions and values of
absorbance when FeSO4, FeCl3, ZnCl2 or AlCl3 were added into the solution of the test
compound, suggesting that the test compound (50) had poor chelating abilities for Fe2+, Fe3+,
Zn2+, and Al3+. The test compounds were also assessed for their binding ability to other
biologically significant metal ions, such as Mg2+ and Ca2+ wherein the compound (50) exhibited
very poor/no binding to these metal ions.
Figure 4.21. Metal chelation study of compound (50) and HQ. UV-vis spectra of (A) compound
(50) and (B) HQ (25 μM) alone and in the presence of CuSO 4 (25 μM), ZnCl2 (25 μM), FeSO4
(25 μM), FeCl3 (25 μM), AlCl3 (25 μM) MgSO4 (25 μM) and CaCl2 (25 μM) in methanol at room
temperature
The above results evinced that the target compound (50) could selectively chelate Cu2+.
This high specificity for a particular metal ion is of prime importance in the design of a metal
chelator to avoid chaotic binding to other critical biometal ions, the depletion of
which can lead to allied side effects.
Computational studies
19
Docking studies of compound (50) with target proteins
To understand the molecular interactions and binding mode of the most active compound
(50) with the ChEs, docking studies were carried out within the active sites of TcAChE (PDB
code: 2CKM) and hBuChE (PDB code: 4BDS) [43].
In the docking study of 50 with AChE, orientation of the compound along with the active
site was found to be similar to that of donepezil, extending from the active site amino acid
residue Trp84 to the peripheral site amino acid residue Tyr70. Binding to the dual sites having
such an interaction with these amino acids is crucial to display a strong affinity to the enzyme. In
the classical binding mode, the tricyclic scaffold without an amine side chain is generally
positioned near the CAS but in case of compound (50) an inverted binding mode is observed
where instead of the aromatic scaffold, the protonated tertiary amine is positioned in the CAS,
deep in the binding gorge. Pyrrolidinylethyl thiourea part of the compound (50) was observed
interacting with the CAS of the active site gorge, whereas the N-ethylcarbazole moiety of the
scaffold was found to be interacting with the receptor active site in PAS of the gorge (Figure 4).
In PAS, the aromatic carbazole moiety exhibited very strong - interactions with Tyr70 and
Tyr334 (hAChE: Tyr72 and Tyr341). The phenyl ring of compound (50) was observed to be
stabilized comfortably in the active site of the enzyme by forming hydrophobic interactions with
the aromatic amino acids Tyr116, Phe330 and Phe331 (hAChE: Tyr119, Tyr337 and Phe338).
Stability to the ligand-receptor complex in CAS is mainly observed because of hydrogen
bonding, cation- interaction and salt bridge. The -NH of thiourea group interacted with Tyr130
(hAChE: Tyr133) by forming a stable hydrogen bond. In addition to this, at physiological pH, the
protonated nitrogen of pyrrolidine moiety exhibited strong cation- interaction with Trp84
20
(hAChE: Trp86) along with a hydrogen bonding and salt bridge interactions with Glu199
(hAChE: Glu202).
Figure 4. Docking model of compound (50) with TcAChE (PDB ID: 2CKM). (A) Binding mode of 50 in
the active site of TcAChE. The ligand is shown as green balls and sticks. AChE residues are shown as
atom type colour sticks. Hydrogen bonds formed between the ligand and the receptor are indicated by red
lines. (B) Ligand interaction diagram of 50 with TcAChE.
The binding mode of 50 with the BuChE enzyme indicated that it also occupied a large
catalytic cavity of BuChE (Figure 5). Carbazole ring was found to be stabilized in the
hydrophobic pocket of nonpolar amino acid residues Ala277, Val280, Pro285 and Leu286. The
21
phenyl ring stabilized the ligand-receptor complex by forming stable - interaction with Tyr332
residue. Hydrogen bond between the -NH of thiourea and Tyr332 residue imparted stability to
the ligand-receptor complex. Further stability to this complex was also provided by the
protonated nitrogen of pyrrolidine by forming cation- interaction with Trp82 residue of the
active site.
Figure 5. Docking model of compound (50) with hBuChE (PDB ID: 4BDS). (A) Binding mode of 50 in
the active site of hBuChE. Ligand is shown as green balls and sticks. hBuChE residues are shown as atom
type colour sticks. (B) Ligand interaction diagram of 50 with hBuChE.
22
To understand the binding interaction of compound (50) with Aβ1-42, a blind docking
study was performed using the X-ray crystal structure of human Aβ 1-42 (PDB code: 1IYT) [44].
The important regions involved in A aggregation include the N-terminal region, central
hydrophobic core (Leu17-Ala21), hinge regions (Arg5-Ser8 and Glu22-Asn27) and the
hydrophobic region (Ile32-Ala42). The hydrophobic core around Leu17-Ala21 residues plays a
crucial role in the β-sheet formation. In this study, the most stable ligand-receptor complex
offered promising interactions (Figure 6). Compound (50) was observed to be aligned with the
chain of Aβ1-42. The carbazole ring showed stable interaction with Phe19 residue, whereas both
the -NH of thiourea group were observed to be interacting strongly with Asp7 residue by forming
hydrogen bonding. Further, the protonated nitrogen of pyrrolidine established stable salt bridge
interactions with Asp7 and Glu11 residues. Along with this interaction, the ligand-receptor
stability was further supported by hydrogen bonding of the nitrogen of pyrrolidine with Asp7
residue of the active site. As hydrogen bondings, salt bridges and hydrophobic interactions of
Aβ1-42 monomers represent a crucial factor governing their aggregation, binding of 50 with Aβ1-42
monomers by π-π interactions and hydrogen bondings suggests that compound (50) could inhibit
Aβ aggregation by blocking intermolecular interactions among multiple Aβ monomers. It needs
to be specified that this docking is only a predictive tool, the real binding mode and interaction of
compound (50) with A1-42 has not been verified practically.
23
Figure 6. Docking model of compound (50) with Aβ1−42 (PDB code 1IYT): (A) Binding mode of 50 with
Aβ1−42. Ligand is shown as green balls and sticks. Aβ 1−42 is shown as cartoon. Hydrogen bonds formed
between the ligand and the receptor are indicated by red lines. (B) Ligand interaction diagram of 50 with
hBuChE.
In silico physicochemical and pharmacokinetics parameters prediction
For considering any synthesized compound as a therapeutically important molecule, it
should not only be biologically active but should also be endowed with desirable
physicochemical and pharmacokinetic properties. Approximately 40 % of the drug candidates
failed in the clinical trials due to the unacceptable ADME (absorption, distribution, metabolism,
and excretion) properties [45]. The in silico prediction of the ADME properties during the drug
development process is a useful strategy to identify these physicochemical and pharmacokinetic
liabilities and to identify and reject those compounds that are suspected to withstand the rigors of
24
the later stages of drug developments. Due to the significant progress made in the field of
computational sciences in recent times, in silico prediction of ADMET parameters becomes
relatively simple and reliable. The virtual physicochemical and pharmacokinetic parameters like
Lipinski’s parameters, NRB, PSA, QPPCaco, QPPMDCK, CNS, QPlogBB, QPlogKhsa were
predicted for compound (50) with QikProp module (Table 3) [46].
Table 3. Predicted ADMET parameters of compound (50) and donepezila
Parameter Limit 50 Donepezil
MW 130-725 468.659 379.498
HBA 2-20 4.5 5.5
HBD 0-6 2 0
NRB 0-8 8 6
QPlogPo/w -2 to 6.5 7.002 4.242
PSA 7 to 200 38.55 46.234
Volume 500-2000 1571.159 1248.451
ReFG 0-2 0 0
SASA 300 to 1000 878.316 681.675
Rule of Five
0-1 1 0
(violation)
CNS - 1 1
QPPMDCK - 1284.443 589.289
QPlogBB -3 to 1.2 0.219 0.223
QPPCaco - 1235.258 1070.771
QPlogKhsa -1.5 to 1.5 1.58 0.516
QPlogS -6.5 to 0.5 -8.12 -4.059
% HOA 0-100 100 100
#star 0-5 4 0
25
a
MW: molecular weight, HBA: hydrogen-bond acceptor atoms, HBD: hydrogen-bond donor
atoms, NRB: number of rotatable bonds, QPlogP o/w: Predicted octanol/water partition coefficient,
PSA: polar surface area, #rtvFG: number of reactive functional groups; SASA: total solvent
accessible surface area, CNS: predicted central nervous system activity on a -2 (inactive) to +2
(active) scale, QPPMDCK: Predicted apparent MDCK cell permeability in nm/s, QPlogBB:
brain/blood partition coefficient, QPPCaco: Caco-2 cell permeability in nm/s, QPlogKhsa:
binding to human serum albumin, QPlogS: predicted aqueous solubility, % HOA: human oral
absorption on 0–100% scale, #star: number of parameters with values that fall outside the 95%
range of similar values for known drugs.

Lipinski’s rule-of-five states that most of the "drug-like" molecules should have
molecular weight ≤ 500, number of hydrogen bond acceptors ≤ 10, number of hydrogen bond
donors ≤ 5 and LogP ≤ 5 [47]. Poor absorption or permeation is more likely to be the case when
molecules violate more than one of these rules. Compound (50) satisfied all these parameters
offering values in the given acceptable ranges except for QPlogP o/w (value > 5).
Compounds (50) violates only one limit of the Lipinski’s rule-of-five, making it as a promising
lead as a drug candidate. The NRB and TPSA are the two key parameters introduced by Veber
[48]. NRB is a simple topological parameter that indicates molecular flexibility. It is an
important descriptor for oral bioavailability of drugs. The compound could have 0−8 rotatable
bonds or less than 7 linear chains outside rings for good oral bioavailability. TPSA is another
important descriptor that is well correlated with passive transport through membranes and
therefore, it allows the prediction of bioavailability and penetration through blood-brain barrier
(BBB) and drug absorption, including intestinal absorption [49]. The mean value of a TPSA is
40.5 Å2 (range 4.63–108 Å2) for the marketed CNS drugs. Compound (50) possesses eight
rotatable bonds and TPSA value of 38.55 Å2 [50]. QPCaco-2 value relates to the oral absorption
of a drug. It shows apparent permeability through gut-blood barrier. Values above 500 predict
26
high oral absorption which has been attained by compound (50). Good oral bioavailability of
compound (50) is also supported by the predicted human oral absorption percent (% HOA)
value. Brain/blood partition coefficient (QPlogBB), CNS, n-octanol−water partition coefficient
(QPlogPo/w), and apparent MDCK cell permeability (QPPMDCK) all predict the ability of the
compound to cross the BBB. Compound (50) is predicted to be CNS active as it possesses a CNS
value as 1 and QPlogBB value as 0.219. QPPMDCK value is predicted apparent MDCK cell
permeability in nm/s. It is recognized as a good mimic for the BBB [51]. A QPPMDCK value
higher than 25 is viewed as good, and the compound (50) has shown considerably high value.
The QPlogKhsa value predicts the binding of the compound with human serum albumin.
Compound (50) showed a slightly higher value than the recommended QPlogKhsa values. #Star
shows the number of parameters with values that fall outside the 95% range of similar values for
known drugs. A larger number of #stars suggests that the compound is less druglike than the
compound with few #stars. The value of #star for compound (50) suggests its drug-likeness.
Further, a compound having a tertiary nitrogen-containing moiety, which is a common feature in
many CNS active drugs, normally exhibits a higher degree of brain permeation [50]. As
discussed above, the compound (50) is predicted to possess a good pharmacokinetic profile,
which highlights its biological significance.
Conclusion
By molecular hybridization approach, a combination of carbazole and stilbene rings
resulted in (E)-3-styryl-9H-carbazole scaffold. Modifications by attaching alicyclic amines with
different linkers to the (E)-3-styryl-9H-carbazole scaffold resulted in a novel series of anti-AD
agents. Among the series, compound (50) having pyrrolidine moiety and thiourea linker showed
the most promising inhibitory activities against AChE (IC50 value of 2.64 μM) and BuChE (IC50
27
value of 1.29 μM). Compound (50) exhibited a significant self-mediated A1–42 aggregation
inhibition (51.29 % at 25 μM concentration). It also displayed specific metal (Cu 2+) chelating
ability and moderate antioxidant activity. Molecular modeling studies indicated significant
interactions between this most potent compound (50) with PAS as well as CAS sites of both the
enzymes as well as with A1-42 peptide. Additionally, compound (50) exhibited favorable in
silico ADMET properties. All these results suggest that compound (50) could be a leading
candidate with a high potential for further development as a novel anti-AD drug.
EXPERIMENTAL SECTION
General: All of the commercial reagents and solvents required during the synthesis of the
compounds were procured from Sigma-Aldrich, Spectrochem, S. d. fine chemicals and Avra
chemicals, and were purified by general laboratory techniques whenever needed. Reaction
monitoring was carried out by thin-layer chromatography (TLC), using silica gel precoated
plates (60F254, Merck, 0.25 mm thickness) and visualizing in ultraviolet (UV) light (λ = 254
nm) or in an iodine chamber. Compounds were purified by flash column chromatography with a
Teledyne ISCO CombiFlash Rf system using RediSep Rf columns. Yields reported here are
unoptimized. Melting points were determined in glass capillary tubes using a silicon oil-bath
type melting point apparatus (Veego) and the reported melting points are uncorrected. The IR
spectra were recorded on a Bruker ALPHA-T (Germany) FT-IR spectrophotometer for all the
reported compounds and are consistent with the assigned structures. 1H NMR and 13
C NMR
spectra were recorded on a Bruker Advance-II 400 MHz spectrometer in CDCl3 or DMSO-d6
solvents. Chemical shifts (δ) are expressed in parts per million (ppm) relative to the standard
TMS, and the peak patterns are indicated as s (singlet), d (doublet), t (triplet), m (multiplet), and
br (broad signal). Mass spectra were recorded using a Thermo Fisher mass spectrometer with an
28
ESI ion source. LCMS analyses were performed on WATERS-2690 with QDA-Mass detector
and electrospray ionization. LCMS method is described in the Supporting Information.
Elemental analyses were performed on a Thermo Fisher FLASH 2000 organic elemental
analyzer. The elemental compositions of the compounds were within ± 0.4 % range of the
calculated values.
Chemistry
9-Ethyl-9H-carbazole (12)
To a rapidly stirring solution of carbazole 1 (0.5 g, 2.99 mmole) in DMSO (10 mL), a few
drops of aqueous sodium hydroxide (0.25 g) were added and stirred for 5 min. To it ethyl iodide
(0.3 mL, 3.58 mmole) was added slowly. The reaction mixture was stirred for further 4-5 hrs to
complete the reaction. After completion of the reaction, the reaction mixture was poured in
crushed ice, and the solid precipitate so obtained was collected and washed with water to remove
the residual solvent and dried to obtain the titled compound (12) (0.54 g, 94 %), m.p. 64-66 °C
(lit [52] m.p. 67-69 °C); IR (KBr, cm−1): 3049, 2978, 2869, 1596, 1018, 753, 700; MS (m/z):
196.3 [M+H]+.
9-Ethyl-9H-carbazole-3-carbaldehyde (13)
Phosphorus oxychloride (0.47 mL, 5.12 mmol) was added, over a period of 10 min to an
ice cooled stirred solution of 9-ethyl-9H-carbazole (12) (1.0 g, 5.13 mmol) and
dimethylformamide (0.38 mL, 5.12 mmol) in 10 mL of chloroform. The resulting reaction
mixture was refluxed for overnight. The reaction mixture was then poured into crushed ice. After
warming to RT the resulting product was extracted into chloroform and the organic phase was
washed with water and brine, dried over magnesium sulphate and evaporated at reduced
pressure. The obtained residue was purified by column chromatography on silica gel using
29
petroleum ether-ethyl acetate (15 %) to obtain the titled compound (13) (0.96 g, 88 %), m.p. 84-
86 °C (lit [53] m.p. 84-86 °C); IR (KBr, cm −1): 2971, 2929, 2822, 2743, 1679, 1588, 620; 1H
NMR (DMSO-d6): δ 10.12 (s, 1H, -CHO), 8.64 (s, 1H, ArH), 8.18 (d, J = 8 Hz, 1H, ArH), 8.04
(d, J = 8 Hz, 1H, ArH), 7.37-7.58 (m, 4H, ArH), 4.43 (q, J = 7.2 Hz, 2H, -NCH2CH3), 1.49 (t, J
= 7.2 Hz, 3H, -NCH2CH3); MS (m/z): 224 [M+H]+.
9-Ethyl-6-nitro-9H-carbazole-3-carbaldehyde (14)
Nitric acid (0.44 mL) was added drop-wise to a stirring solution of 9-ethyl-9H-carbazole-
3-carbaldehyde (13) (1.0 g, 4.47 mmol) in acetic acid (10 mL) under cold conditions. After
completion of addition, the reaction mixture was further stirred for additional 1 h. The solid so
precipitated was collected by filtration and washed with acetic acid (10 mL). Excess acetic acid
was removed by washing with water. The solid so obtained was dried to obtain greenish colored
compound (14) (1.1 g, 92 %); m.p. 241-243 °C (lit [54] m.p. 247-248 °C); IR (KBr, cm−1): 3081,
2967, 2870, 2822, 2728, 1685, 1591, 1021, 752; 1H NMR (CDCl3): δ 10.16 (s, 1H, -CHO), 9.09
(d, J = 2.4 Hz, 1H, ArH), 8.69 (d, J = 1.2 Hz, 1H, ArH), 8.47 (dd, J = 2.4 Hz, 9.2 Hz, 1H, ArH),
8.15 (dd, J =1.6, 8.4 Hz, 1H, ArH), 7.61 (d, J = 8.4 Hz, 1H, ArH), 7.53 (d, J = 9.2 Hz, 1H,
ArH), 4.68 (q, J = 7.2 Hz, 2H, -NCH2CH3), 1.54 (t, J = 7.2 Hz, 3H, -NCH2CH3); MS (m/z): 269
[M+H]+.
(E)-9-Ethyl-3-nitro-6-styryl-9H-carbazole (15)
To a stirred solution of benzyltriphenylphosphonium bromide (2.4 g, 5.58 mmol) in isopropyl
alcohol (25 mL), lithium hydroxide (0.18 g, 7.44 mmol) was added. The reaction mixture was
stirred further for 30 min at room temperature. To it 9-ethyl-6-nitro-9H-carbazole-3-
carbaldehyde 14 (1.0 g, 3.72 mmol) was added in small portions over a period of a few minutes.
The resulting reaction mixture was heated to 80 °C for 5-6 h. Progress of the reaction was
30
monitored by TLC. After completion of the reaction, the solid so precipitated was collected by
filtration and washed with isopropyl alcohol (15 mL). The obtained solid contained mixture of
cis and trans isomers. This isomeric mixture was dissolved in benzene (50 mL) and catalytic
amount of iodine was added to it. The reaction mixture was heated to 70 °C for 4-5 h.
Conversion of the isomeric mixture to a single trans isomer product was confirmed by TLC.
After completion of the reaction, the organic phase was washed with aqueous sodium thiosulfate
solution (10 %) to quench the remaining quantity of iodine. The organic layer was collected,
washed with water and brine, dried over sodium sulfate, filtered and evaporated to give the titled
compound (15) as yellow solid. (Yield: 0.99 g, 78.5 %); m.p. 151-154 °C; IR (KBr, cm −1): 2986,
2939, 1592, 1482, 1315, 1088, 804, 748; 1H NMR (CDCl3): δ 9.00 (d, J = 2 Hz, 1H, ArH), 8.36-
8.39 (dd, J = 7.2 Hz, 2 Hz, 1H, ArH), 8.23 (s, 1H, ArH), 7.74-7.76 (m,1H, ArH), 7.57-7.59 (m,
2H, ArH), 7.16-7.45 (m, 5H, ArH, 2H, vinylic-H), 4.40 (q, J = 7.2 Hz, 2H, -NCH2CH3), 1.48 (t,
J = 7.2 Hz, 3H, -NCH2CH3); MS (m/z) : 343 [M+H]+.
(E)-9-Ethyl-6-styryl-9H-carbazol-3-amine (16)
To a solution of (E)-9-ethyl-3-nitro-6-styryl-9H-carbazole 15 (1.0 g, 2.92 mmol) in 1:1
THF/MeOH mixture (50 mL), stannous chloride (1.10 g, 5.84 mmol) was added in small
portions over a period of a few minutes. After completion of addition, the reaction mixture was
refluxed for 6-7 hrs. Progress of reaction was monitored by TLC. After completion of the
reaction, pH of the mixture was adjusted to eight with NaOH solution (10 %), and then extracted
with ethyl acetate (20 mL × 3). The collected organic layer was washed with water and brine,
dried over magnesium sulfate, filtered and evaporated to give crude product. It was further
purified by recrystallization with methanol to give yellowish green colored crystals of the titled
compound (16). (Yield: 0.7 g, 77 %); m.p. 132-134 °C; IR (KBr, cm −1): 3401, 3304, 3053, 3023,
31
2971, 2933, 1592, 1491, 797, 688; 1H NMR (DMSO-d6): δ 8.15 (s, 1H, ArH), 7.65-7.67 (m, 1H,
ArH), 7.57-7.59 (m, 2H, ArH), 7.23-7.48 (m, 5H, ArH, 2H, vinylic-H), 7.12-7.16 (m, 1H, ArH),
6.94-6.96 (m, 1H, ArH), 4.31-4.33 (m, 2H, -NCH2CH3), 1.42 (t, 3H, -NCH2CH3); MS (m/z) :
313.5 [M+H]+.
General method for the synthesis of (E)-N-(9-ethyl-6-styryl-9H-carbazol-3-yl)alkylamides
(17-19)
Method A: To a stirring solution of 9-ethyl-6-styryl-9H-carbazole-3-amine 16 (1.0 g, 3.20
mmol) in dry acetone (25 mL), potassium carbonate (1.12 g, 7.98 mmol) was added. To it,
respective acid chloride (4.8 mmol) was added dropwise under cold conditions. After completion
of addition, the reaction mixture was kept on stirring for additional 1 h. The progress of the
reaction was monitored by TLC. After completion of the starting material, the reaction mixture
was poured into ice-cold water and extracted with ethyl acetate (20 mL × 3). The organic layer
was washed with saturated sodium bicarbonate solution followed by water and brine, dried over
sodium sulfate, and evaporated to yield the title compound.
(E)-3-Chloro-N-(9-ethyl-3-styryl-9H-carbazol-6-yl)propanamide (17)
The title compound (17) was synthesized from compound (16) (1.0 g, 3.20 mmol) and 3-
chloropropionyl chloride (0.47 mL, 4.8 mmol) following Method A (yield: 0.96 g, 74 %).
Brown colored solid; m.p. 154-156 °C; IR (KBr, cm −1): 3297, 3024, 2972, 1652, 1595, 1554,
1231, 799, 699; MS (m/z): 403.6 [M]+, 405.6 [M+2]+.
(E)-4-Chloro-N-(9-ethyl-3-styryl-9H-carbazol-6-yl)butanamide (18)
chlorobutyryl chloride (0.53 mL, 4.8 mmol) following Method A (yield: 0.99 g, 74 %). Brown
32
colored solid; m.p. 161-163 °C; IR (KBr, cm −1): 3291, 3118, 2969, 1648, 1542, 1486, 1229, 956,
799, 692; MS (m/z): 417.3 [M]+, 419.3 [M+2]+.
(E)-5-Bromo-N-(9-ethyl-3-styryl-9H-carbazol-6-yl)pentanamide (19)
bromovaleryl chloride (0.96 g, 4.8 mmol) following Method A (yield: 0.90 g, 72 %). Brown
colored solid; m.p. 147-150 °C; IR (KBr, cm−1): 3288, 3025, 2967, 1647, 1593, 1540, 1486, 793;
MS (m/z): 476.4 [M+H]+, 477.4 [M+2]+.
General procedure for the synthesis of (E)-N-(9-ethyl-6-styryl-9H-carbazol-3-yl)
aminoalkylamide (20-25)
Method B: Pyrrolidine/piperidine (5 equiv.) was added to a solution of (E)-N-(9-ethyl-6-styryl-
9H-carbazol-3-yl)alkylamides (17-19, 0.5 g) in THF (10 mL). The reaction mixture was refluxed
under nitrogen atmosphere till completion of the reaction as judged by TLC. The reaction
mixture was then evaporated under reduced pressure and the residue was dissolved in 20 mL of
water and extracted with ethyl acetate (20 mL × 3). The combined organic phase was then dried
and evaporated to obtain the crude product, which was further purified by column
chromatography using a mixture of chloroform and methanol as eluent to obtain off-white
colored compound.
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-3-(pyrrolidin-1-yl)propanamide (20)
The title compound (20) was synthesized from compound (17) (0.5 g, 1.24 mmol) and
pyrrolidine (0.5 mL, 6.20 mmol) following Method B (yield: 0.36 g, 67 %). Brown colored
solid; m.p. 129-131 °C; IR (KBr, cm −1): 3366, 3056, 3018, 2966, 1596, 1488, 1229, 963, 806,
791; 1H NMR (DMSO-d6): δ 10.11 (s, 1H, -NHCO), 8.53 (d, 1H, ArH), 8.31 (d, 1H, ArH), 7.72-
7.74 (dd, 1H, ArH), 7.22-7.63 (m, 8H, ArH and 2H, vinylic-H), 4.41 (q, J = 7.2 Hz, 2H,
33
-NCH2CH3), 2.76 (t, 2H, -NHCOCH2), 2.45-2.54 (m, 6H, -NCH2CH2), 1.68-1.71 (m, 4H,
-NCH2CH2), 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS (m/z): 438.26 [M+H]+, Purity: nearly
100 %.
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-3-(piperidin-1-yl)propanamide (21)
piperidine (0.61 mL, 6.20 mmol) following Method B (yield: 0.38 g, 67 %). Off-white solid;
m.p. 148-151 °C; IR (KBr, cm−1): 3309, 3023, 2967, 1690, 1587, 1520, 1478, 1233, 749; 1H
NMR (DMSO-d6): δ 10.20 (s, 1H, -NHCO), 8.53 (d, 1H, ArH), 8.30 (d, 1H, ArH), 7.73 (dd, 1H,
ArH), 7.45-7.63 (m, 3H, ArH, 2H, vinylic-H), 7.36-7.41 (m, 3H, ArH), 7.24-7.30 (m, 2H, ArH),
4.40 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.64 (t, 2H, -NHCOCH2), 2.48-2.52 (m, 4H, -NCH2CH2),
2.35-2.42 (m, 2H, -NCH2CH2), 1.49-1.55 (m, 4H, -NCH2CH2), 1.39-1.40 (m, 2H, -CH2) and 1.30
(t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS (m/z): 452.16 [M+H]+, Purity: 98.58 %.
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-4-(pyrrolidin-1-yl)butanamide (22)
pyrrolidine (0.5 mL, 6.20 mmol) following Method B (yield: 0.34 g, 63 %). Off-white solid;
m.p. 127-130 °C; IR (KBr, cm−1): 3292, 3118, 3027, 2961, 2876, 2795, 1651, 1596, 1532, 1486,
1305 and 797; 1H NMR (DMSO-d6): δ 9.94 (s, 1H, -NHCO), 8.53 (d, 1H, ArH), 8.30 (s, 1H,
ArH), 7.72-7.74 (dd, 1H, ArH), 7.24-7.63 (m, 8H, ArH, 2H, vinylic-H), 4.41 (q, J = 7.2 Hz, 2H,
-NCH2CH3), 2.37-2.46 (m, 6H, -NCH2CH2, 2H, -NHCOCH2), 1.78-1.82 (m, 2H, -NCH2CH2),
1.66-1.69 (m, 4H, -NCH2CH2) and 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS (m/z): 452.26
[M+H]+, Purity: nearly 100 %.
N-(9-Ethyl-3-styryl-9H-carbazol-6-yl)-4-(piperidin-1-yl)butanamide (23)
34
m.p. 114-118 °C; IR (KBr, cm−1): 3286, 3117, 3025, 2928, 2770, 1644, 1594, 1536, 1486, 1152,
791, 691; 1H NMR (DMSO-d6): δ 9.89 (s, 1H, -NHCO), 8.35 (d, 1H, ArH), 8.15-8.18 (m, 1H,
ArH), 7.70 (dd, 1H, ArH), 7.17-7.62 (m, 8H, ArH, 2H, vinylic-H), 4.44 (q, J = 7.2 Hz, 2H,
-NCH2CH3), 2.24-2.34 (m, 6H, -NCH2CH2, 2H, -NHCOCH2), 1.69-1.77 (m, 2H, -NCH2CH2),
1.44-1.50 (m, 4H, -NCH2CH2), 1.35-1.38 (m, 3H, -NCH2CH2CH2), 1.32 (t, J = 7.2 Hz, 3H,
-NCH2CH3); LCMS (m/z): 466.4 [M+H]+, Purity: nearly 100 %.
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-5-(pyrrolidin-1-yl)pentanamide (24)
m.p. 161-164 °C; IR (KBr, cm−1): 3289, 3026, 2962, 2933, 1648, 1596, 1484, 1082, 796 and 687;
1
H NMR (DMSO-d6): δ 9.91 (s, 1H, -NHCO), 8.52 (d, 1H, ArH), 8.30 (s, 1H, ArH), 7.73 (dd,
1H, ArH), 7.24-7.63 (m, 8H, ArH, 2H, vinylic-H), 4.40 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.34-
2.42 (m, 6H, -NCH2, 2H, -NHCOCH2), 1.65-1.69 (m, 6H, -NCH2 CH2), 1.49-1.52 (m, 2H, -CH2)
and 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3; LCMS (m/z): 466.26 [M+H]+, Purity: nearly 100 %.
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-5-(piperidin-1-yl)pentanamide (25)
m.p. 159-161 °C; IR (KBr, cm−1): 3290, 3025, 2929, 2856, 1647, 1540, 1487, 1228, 799 and 689;
1
H NMR (DMSO-d6): δ 9.91 (s, 1H, -NHCO), 8.52 (d, 1H, ArH), 8.30 (d, 1H, ArH), 7.73 (dd,
1H, ArH), 7.22-7.63 (m, 8H, ArH, 2H, vinylic-H), 4.41 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.23-
2.37 (m, 6H, -NCH2CH2, 2H, -NHCOCH2), 1.61-1.65 (m, 2H, -NHCOCH2CH2), 1.45-1.49 (m,
35
6H, -NCH2CH2), 1.35-1.37 (m, 2H, -NCH2CH2 CH2) and 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3);
LCMS (m/z): 480.17 [M+H]+, Purity: 98.79 %.
General method for the synthesis of (E)-1-(9-ethyl-6-styryl-9H-carbazol-3-yl)aminoalkyl-
urea (26-31)
Method C: To a stirring solution of (E)-9-ethyl-6-styryl-9H-carbazol-3-amine 16 (1.0 g, 3.20
mmol) and triethylamine (0.5 mL, 3.52 mmol) in dry THF (20 mL), 4-nitrophenyl chloroformate
(0.71 g, 3.52 mmol) in dry THF (10 mL) was added dropwise at 0-5 °C over a period of 15 min.
The resulting reaction mixture was allowed to stir at room temperature for 2 h. Progress of the
reaction was monitored by TLC. After complete consumption of the starting material, the
respective amine (4.0 mmol) was added to the reaction mixture and the reaction was further
stirred for 30 min. After completion of the reaction, the solvent was recovered at reduced
pressure. The residues so obtained was triturated with chilled methanol (20 mL), filtered and
dried to obtain the title compound.
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)pyrrolidine-1-carboxamide (26)
The title compound was synthesized from compound (16) (1.0 g, 3.20 mmol) and
pyrrolidine (0.33 mL, 4.00 mmol) as per Method C (yield: 1.02 g, 78 %). Off-white solid; m.p.
193-195 °C; IR (KBr, cm−1): 3313, 3058, 3023, 2960, 2868, 1635, 1551, 1481, 1151, 956, 801
and 691; 1H NMR (DMSO-d6): δ 8.29-8.27 (m, 2H, ArH), 8.14 (s, 1H, -NHCO), 7.70-7.72 (m,
1H, ArH), 7.61-7.63 (m, 2H, ArH), 7.55-7.57 (m, 1H, ArH), 7.21-7.52 (m, 5H, ArH, 2H, vinylic-
H), 4.40 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.39-3.43 (m, 4H, -NCH2CH2), 1.86-1.90 (m, 4H,
-NCH2CH2) and 1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS (m/z): 410.30 [M+H]+, Purity:
nearly 100 %
(E)-N-(9-Ethyl-6-styryl-9H-carbazol-3-yl)piperidine-1-carboxamide (27)
36
piperidine (0.40 mL, 4.00 mmol) as per Method C (yield: 1.08 g, 82 %). Off-white solid; m.p.
217-219 °C; IR (KBr, cm−1): 3346, 3026, 2929, 2847, 1627, 1532, 1486, 1232, 1140, 860 and
750; 1H NMR (DMSO-d6): δ 8.47 (s, 1H, -NHCO), 8.26-8.29 (m, 2H, ArH), 7.69-7.72 (m, 1H,
ArH), 7.61-7.63 (m, 2H, ArH), 7.55-7.57 (m, 1H, ArH), 7.22-7.49 (m, 5H, ArH, 2H, vinylic-H),
4.40 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.45-3.48 (m, 4H, -NCH2CH2), 1.52-1.60 (m, 6H, -NCH2
CH2CH2) and 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS (m/z): 424.25 [M+H]+, Purity: nearly
100 %.
(E)-1-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-3-(2-(pyrrolidin-1-yl)ethyl)urea (28)
The title compound was synthesized from compound (16) (1.0 g, 3.20 mmol) and 2-
(aminoethyl)pyrrolidine (0.40 mL, 4.00 mmol) as per Method C (yield: 1.03 g, 75 %). Off-white
solid; m.p. 176-178 °C; IR (KBr, cm−1): 3322, 3022, 2961, 2872, 1630, 1562, 1486, 1132, 952,
799 and 691; 1H NMR (DMSO-d6): δ 10.23 (s, 1H, -NHCO), 8.52 (s, 1H, -NHCO) 8.31 (s, 1H,
ArH), 7.24-7.57 (m, 10H, ArH, 2H, vinylic-H), 4.41 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.62-2.67
(m, 2H, -NHCH2), 2.35-2.45 (m, 6H, -NCH2) and 1.31 (t, J = 7.2 Hz, 3H, -NCH 2CH3); LCMS
(m/z): 453.3 [M+H]+, Purity: nearly 100 %.
(E)-1-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-3-(2-(piperidin-1-yl)ethyl)urea (29)
(aminoethyl)piperidine (0.57 mL, 4.00 mmol) as per Method C (yield: 1.06 g, 71 %). Off-white
796 and 748; 1H NMR (DMSO-d6): δ 8.61 (s, 1H, -NHCO), 8.29 (s, 1H, ArH), 7.21-7.71 (m, 9H,
ArH, 2H, vinylic-H), 6.01 (bs, 1H, -NHCO), 4.39 (q, J = 7.2 Hz, 2H, -NCH2), 3.22-3.24 (m, 2H,
-NHCH2), 2.35-2.39 (m, 6H, -NCH2), 1.50-1.52 (m, 4H, -NCH2CH2), 1.40-1.39 (m, 2H,
37
-NCH2CH2CH2) and 1.29 (t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS (m/z): 467.26 [M+H]+, Purity:
99.59 %.
(E)-1-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-3-(3-(pyrrolidin-1-yl)propyl)urea (30)
(aminopropyl)pyrrolidine (0.51 g, 4.00 mmol) as per Method C (yield: 1.1 g, 75 %). Off-white
800 and 692; 1H NMR (DMSO-d6): δ 8.43 (s, 1H, -NHCO), 8.26-8.28 (m, 2H, ArH), 7.21-7.71
(m, 9H, ArH, 2H, vinylic-H), 6.18 (t, 1H, -NHCO), 4.39 (q, J = 7.2 Hz, 2H, -NCH2), 3.14-3.19
(m, 2H, -NHCH2), 2.52-2.55 (m, 6H, -NCH2), 1.62-1.74 (m, 6H, -NCH2CH2) and 1.29 (t, J = 7.2
Hz, 3H, -NCH2CH3); LCMS (m/z): 467.26 [M+H]+, Purity: nearly 100 %.
(E)-1-(9-Ethyl-6-styryl-9H-carbazol-3-yl)-3-(3-(piperidin-1-yl)propyl)urea (31)
The title compound was synthesized from compound (16) (1.0 g, 3.20 mmol) 3-
(aminopropyl)piperidine (0.57 g, 4.00 mmol) as per Method C (yield: 1.2 g, 79 %). Off-white
798 and 688; 1H NMR (DMSO-d6): δ 8.36 (s, 1H, -NHCO), 8.26-8.29 (m, 2H, ArH), 7.21-7.71
(m, 9H, ArH, 2H, vinylic-H), 6.01 (t, 1H, -NHCO), 4.39 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.11-
3.16 (m, 2H, -NHCH2), 2.27-2.33 (m, 6H, -NCH2CH2), 1.57-1.64 (m, 4H, -NCH2CH2), 1.46-1.51
(m, 4H, -NCH2CH2), 1.34-1.39 (m, 2H, -CH2) and 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3); LCMS
(m/z): 481.4 [M+H]+, Purity: nearly 100 %.
2-(4-Nitrophenyl)acetic acid (32)
A mixture of concentrated nitric acid (1.47 mL) and an equal volume of concentrated
sulphuric acid (1.47 mL) was placed in a two neck flask fitted with a thermometer and a
dropping funnel. The mixture was cooled to 10 °C in ice bath and benzyl cyanide (1.0 g, 8.53
38
mmol) was run at such a rate that the temperature was maintained around 10 °C and did not rise
above 20 °C. The solution was further stirred for 1 h at room temperature and then poured into
crushed ice. The mass was filtered under vacuum and pressed well to remove as much oil as
possible. The crude product was recrystallize from methanol to obtain 2-(4-
nitrophenyl)acetonitrile (yield:1.21 g, 88 %); m.p. 110-112 °C (lit. [55] m.p. 113-115 °C); IR
(KBr, cm−1): 3117, 3054, 2943, 2851, 2253, 1517, 1345, 1107, 732. A solution of sulphuric acid
(50 %) was prepared by adding concentrated sulphuric acid cautiously to water. Two third of the
sulphuric acid was added into a round bottom flask containing 2-(4-nitrophenyl)acetonitrile (1.0
g) and the nitrile adhering to the walls of the flask was washed down with the remaining acid.
The content was boiled under reflux for 15 min and diluted with 25 mL of ice-cold water. The
resulting pale yellow solid mass was filtered, washed, decolorized and recrystallized from hot
water to yield titled compound (32) (yield: 0.87 g, 78 %); m.p. 148-150 °C (lit [55] m.p. 151-152
°C); IR (KBr, cm−1): 3450, 3084, 2931, 2847, 1708, 1514, 1346, 951, 709.
(E)-9-Ethyl-3-(4-nitrostyryl)-9H-carbazole (33)
A mixture of 2-(4-nitrophenyl)acetic acid (32, 1.63 g, 8.96 mmol) and 9-ethyl-9H-
carbazole-3-carbaldehyde (13, 1.0 g, 4.47 mmol) in the presence of piperidine (1.33 mL, 13.44
mmol) was irradiated under microwave at 800 W in a microwave reactor. Reaction progress was
monitored by TLC. After completion of reaction, the reaction mixture was cooled and methanol
(15 mL) was added to it. The solid so obtained was filtered and dried to obtain the orange
colored crystals of the titled compound (33) [56]. (Yield:1.25 g, 82 %); m.p. 215-217 °C; IR
(KBr, cm−1): 3048, 2976, 1621, 1593, 1505, 1333 and 749; 1H NMR (DMSO-d6): δ 8.49 (s, 1H,
ArH), 8.24 (d, 2H, ArH), 8.18 (d, 1H, ArH), 7.86 (d, 2H, ArH), 7.81-7.83 (m, 1H, ArH), 7.62-
39
7.74 (m, 3H, ArH), 7.42-7.50 (m, 1H, ArH, 1H, Vinylic-H), 7.22-7.26 (m, 1H, Vinylic-H), 4.46
(q, J = 7.2 Hz, 2H, -NCH2) and 1.32 (t, J = 7.2 Hz, 3H, -NCH2CH3); MS (m/z): 343 [M+H]+.
(E)-4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)aniline (34)
To a solution of (E)-9-ethyl-3-(4-nitrostyryl)-9H-carbazole (34) (1.0 g, 2.92 mmol) in
tetrahydrofuran: methanol (1:1) mixture (50 mL), stannous chloride (1.11 g, 5.84 mmol) was
added in portion. After completion of addition, the reaction mixture was refluxed for 6-7 h.
Progress of reaction was monitored by TLC. After completion of the reaction, pH of the mixture
was adjusted to basic with aqueous NaOH solution (10 %), and then extracted with ethyl acetate
(20 mL × 3). The collected organic layer was washed with water and brine, dried and evaporated
to give crude product, which was further purified by recrystallization with methanol to give
white colored crystals of the titled compound (34). (Yield: 0.88 g, 92 %); m.p. 162-164 °C; IR
(KBr, cm−1): 3417, 3368, 3029, 2966, 2926, 1615, 1513, 958, 819 and 748; 1H NMR (DMSO-d6):
δ 8.26 (s, 1H, ArH), 8.14-8.16 (m, 1H, ArH), 7.62-7.65 (m, 1H, ArH), 7.54-7.59 (m, 2H, ArH),
7.41-7.48 (m, 1H, ArH), 7.27-7.30 (m, 2H, ArH), 7.17-7.21 (m, 1H, ArH), 7.05-7.07 (m, 2H,
vinylic-H), 6.56-6.59 (m, 2H, ArH), 5.20 (bs, 2H, -NH2), 4.42 (q, J = 7.2 Hz, 2H, -NCH2CH3)
and 1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); MS (m/z) : 313 [M+H]+.
(E)-2-Chloro-N-(4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)phenyl)acetamide (35)
chloroacetyl chloride (0.38 mL, 4.8 mmol) following Method A (yield: 0.91 g, 71 %). Brown
colored solid; m.p. 207-209 °C; IR (KBr, cm −1): 3249, 3038, 2974, 1664, 1593 and 738; MS
(m/z): 388.2 [M]+, 390.2 [M+2]+.
(E)-3-Chloro-N-(4-(2-(9-ethyl-9H-carbazol-3yl)vinyl)phenyl)propanamide (36)
40
chloropropionyl chloride (0.46 mL, 4.8 mmol) following Method A (yield: 0.87 g, 65 %).
Brown colored solid; m.p. 219-222 °C; IR (KBr, cm−1): 3273, 3036, 2970, 1644, 1594 and 744;
MS (m/z): 403.3 [M]+, 405.3 [M+H]+.
(E)-4-Chloro-N-(4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)phenyl)butanamide (37)
chlorobutyryl chloride (0.54 mL, 4.8 mmol) following Method A (yield: 0.88 g, 66 %). Brown
colored solid; m.p. 193-195 °C; IR (KBr, cm −1): 3295, 3028, 2968, 1653, 1589 and 746; MS
(m/z): 417.3 [M]+, 419.3 [M+H]+.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-2-(pyrrolidin-1-yl)acetamide (38)
m.p. 120-122 °C; IR (KBr, cm−1): 3306, 3022, 2867, 1691, 1584, 1521, 1233 and 749; 1H NMR
(DMSO-d6): δ 9.76 ( s, 1H, -NHCO), 8.37 (s, 1H, ArH), 8.17 (d, 1H, ArH), 7.67-7.72 (m, 3H,
ArH), 7.54-7.60 (m, 4H, ArH), 7.43-7.47 (m, 1H, ArH), 7.31-7.35 (m, 1H, ArH), 7.19-7.23 (m,
2H, Vinylic-H), 4.43 (q, J = 7.2 Hz, 2H, -NCH2), 3.25 (s, 2H, -NHCOCH2), 2.56-2.61 (m, 4H,
13
-NCH2), 1.69-1.81 (m, 4H, -NCH2CH2) and 1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); C NMR
(DMSO-d6): 169.00, 140.33, 139.66, 138.15, 133.32, 128.85, 128.61, 126.85, 126.34, 125.69,
125.01, 123.03, 122.71, 120.87, 120.10, 119.38, 118.78, 109.78, 109.73, 59.97, 54.20, 37.53,
23.95, 14.21; LCMS (m/z): 424.25 [M+H]+, Purity: 99.71 %.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-2-(piperidin-1-yl)acetamide (39)
41
m.p. 131-134 °C; IR (KBr, cm−1): 3326, 3016, 2938, 2795, 1693, 1582, 1516, 1231, 815 and 749;
1
H NMR (DMSO-d6): δ 9.72 (s, 1H, -NHCO), 8.37 (d, 1H, ArH), 8.17 (d, 1H, ArH), 7.55-7.70
(m, 7H, ArH), 7.43-7.47 (m, 1H, ArH), 7.31-7.35 (m, 1H, ArH), 7.19-7.23 (m, 2H, Vinylic-H),
4.43 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.07 (s, 2H, -NHCOCH2), 2.46-2.49 (m, 4H, -NCH2CH2),
1.54-1.60 (m, 4H, -NCH2CH2), 1.37-1.42 (m, 2H, -NCH2CH2CH2) and 1.32 (t, J = 7.2 Hz, 3H,
13
-NCH2CH3); C NMR (DMSO-d6): 168.96, 140.44, 139.67, 138.00, 133.39, 128.84, 128.64,
126.89, 126.34, 125.67, 125.01, 123.04, 122.72, 120.87, 120.04, 119.39, 118.80, 109.79, 109.73,
63.16, 54.57, 37.52, 25.95, 24.03 and 14.21; LCMS (m/z): 438.26 [M+H]+, Purity: 99.81 %.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(pyrrolidin1yl)propanamide (40)
m.p. 158-160 °C; IR (KBr, cm−1): 3281, 3175, 3102, 3025, 2963, 2930, 1653, 1595 and 745; 1H
NMR (DMSO-d6): δ 10.14 ( s, 1H, -NHCO), 8.36 (s, 1H, ArH), 8.17 (d, 1H, ArH), 7.69-7.72 (m,
1H, ArH), 7.53-7.63 (m, 6H, ArH), 7.43-7.47 (m, 1H, ArH), 7.29-7.33 (m, 1H, ArH), 7.18-7.23
(m, 2H, Vinylic-H) 4.43 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.71 (t, 2H, -NHCOCH2), 2.46-2.50 (m,
6H, -CH2 , -NCH2CH2), 1.66-1.69 (m, 4H, -NCH2CH2),1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C
NMR (DMSO-d6): 170.57, 140.43, 139.65, 138.77, 133.01, 128.88, 128.46, 126.94, 126.33,
125.74, 124.99, 123.04, 122.71, 120.88, 119.66, 119.38, 118.77, 109.77, 109.71, 53.88, 52.04,
37.52, 36.64, 23.65 and 14.20; LCMS (m/z): 438.21 [M+H]+, Purity: nearly 100 %.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(piperidin-1-yl)propananamide (41)
m.p. 173-176 °C; IR (KBr, cm−1): 3018, 2974, 2931, 2839, 2799, 1683, 1595, 1537, 817 and 747;
42
1
H NMR (DMSO-d6): δ 10.24 (s, 1H, -NHCO), 8.36 (s, 1H, ArH), 8.17 (d, 1H, ArH), 7.70-7.72
(m, 1H, ArH), 7.53-7.61 (m, 6H, ArH), 7.44-7.48 (m, 1H, ArH), 7.30-7.34 (m, 1H, ArH), 7.18-
7.22 (m, 2H, Vinylic-H), 4.44 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.58-2.61 (m, 2H, -NHCOCH2),
2.45-2.49 (m, 2H, -NCH2), 2.37-2.40 (m, 4H, -NCH2), 1.49-1.52 (m, 4H, -NCH2CH2), 1.36-1.41
(m, 2H, -NCH2CH2CH2) and 1.32 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 170.70,
140.43, 196.65, 138.73, 133.0, 128.8, 128.47, 126.96, 126.34, 125.73, 125.01, 123.03, 122.71,
120.88, 119.63, 119.37, 118.75, 109.78, 109.72, 54.93, 54.13, 37.52, 34.57, 26.10, 24.48 and
14.21; LCMS (m/z): 452.21 [M+H]+, Purity: nearly 100 %.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-4-(pyrrolidin-1yl)butanamide (42)
m.p. 179-181 °C; IR (KBr, cm−1): 33294, 3023, 2960, 2874, 2791, 1657, 1523, 818 and 745; 1H
NMR (DMSO-d6): δ 9.96 (s, 1H, -NHCO), 8.36 (d, 1H, ArH), 8.17 (d, 1H, ArH), 7.71 (dd, 1H,
1H, ArH), 7.18-7.23 (m, 2H, Vinylic-H), 4.44 (q, J = 7.2 Hz, 2H, -NCH2CH3), 2.34-2.42 (m, 2H,
-NHCOCH2, 6H, -NCH2CH2), 1.73-1.77 (m, 2H, -NCH2CH2), 1.65-1.68 (m, 4H, -NCH2CH2) and
1.32 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 171.59, 140.43, 139.64, 138.87,
132.90, 128.89, 128.40, 126.90, 126.33, 125.76, 124.99, 123.03, 122.71, 120.88, 119.65, 119.37,
118.75, 109.78, 109.72, 55.63, 54.00, 37.52, 35.00, 24.85, 23.60 and 14.20; LCMS (m/z): 452.21
[M+H]+, Purity: 99.05 %.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-4-(piperidin-1-yl)butanamide (43)
piperidine (0.59 mL, 6.0 mmol) following Method B (yield: 0.35 g, 63 %). Off-white solid; m.p.
43
173-176 °C; IR (KBr, cm−1): 3296, 3023, 2928, 2850, 1657, 1594, 1523, 1409, 961, 858 and 745;
1
H NMR (DMSO-d6): δ 9.93 (s, 1H, -NHCO), 8.36 (d, 1H, ArH), 8.17 (d, 1H, ArH), 7.71 (dd,
(m, 2H, Vinylic-H), 4.44 (q, J = 7.2 Hz, 2H,-NCH2CH3), 2.24-2.44 (m, 2H, -NHCOCH2, 6H,
-NCH2CH2), 1.72-1.75 (m, 2H, -NCH2CH2), 1.44-1.50 (m, 4H, -NCH2CH2), 1.33-1.36 (m, 2H,
-CH2) and 1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 171.61, 140.43, 139.64,
138.88, 132.87, 128.90, 128.39, 126.89, 126.34, 125.77, 124.99, 123.03, 122.71, 120.88, 119.65,
119.37, 118.74, 109.78, 109.72, 58.54, 54.52, 37.52, 34.99, 26.08, 24.66, 22.85 and 14.21;
LCMS (m/z): 466.21 [M+H]+, Purity: 99.88 %.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)pyrrolidine-1-carboxamide (44)
pyrrolidine (0.33 mL, 4.00 mmol) following Method C (yield: 0.76 g, 58 %). Off-white solid;
m.p. 144-146 °C; IR (KBr, cm−1): 3334, 3018, 2974, 2873, 1630, 1522, 1234, 965 and 753; 1H
NMR (DMSO-d6): δ 8.23 (d, 1H, ArH), 8.15 (d, 1H, ArH), 7.67-7.69 (dd, 1H, ArH), 7.38-7.52
(m, 7H, ArH), 7.10-7.47 (m, 1H, ArH, 2H, Vinylic-H), 6.28 (bs, 1H, -NHCO), 4.39 (q, J = 7.2
Hz, 2H, -NCH2), 3.48-3.52 (m, 4H, -NCH2), 1.98-2.02 (m, 4H, -NCH2CH2) and 1.46 (t, J = 7.2
Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 140.39, 131.58, 129.01, 127.68, 126.64, 126.37,
125.97, 124.92, 123.00, 122.66, 120.89, 120.11, 120.01, 119.38, 118.61, 109.76, 109.68, 46.17,
37.51, 25.46 and 14.16; MS (m/z): 410.3 [M+H]+.
(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)piperidine-1-carboxamide (45)
piperidine (0.40 mL, 4.00 mmol) following Method C (yield: 0.58 g, 55 %). Off-white solid;
m.p. 214-216 °C; IR (KBr, cm−1): 3305, 3026, 2972, 2931, 2854, 1633, 1589, 1515, 1234, 951
44
and 746; 1H NMR (DMSO-d6): δ 8.24 (s, 1H, ArH), 8.15 (d, 1H, ArH), 7.67-7.69 (m, 1H, ArH),
7.39-7.52 (m, 8H, ArH), 7.24-7.27 (m, 1H, ArH, 2H, Vinylic-H), 7.10-7.14 (m, 1H, ArH), 6.47
(bs, 1H, -NHCO), 4.39 (q, J = 7.2 Hz, 2H,-NCH2CH3), 3.49-3.51 (m, 4H, -NCH2), 1.64-1.71 (m,
6H, -NCH2CH2) and 1.46 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 155.26, 140.41,
140.38, 139.55, 131.51, 129.05, 127.65, 126.61, 126.32, 126.00, 124.92, 123.02, 122.70, 120.87,
120.09, 119.35, 118.59, 109.76, 109.70, 45.17, 37.51, 26.00, 24.58 and 14.20; MS (m/z): 424.3
[M+H]+.
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(pyrrolidin-1-yl)-ethyl)urea (46)
(aminoethyl) pyrrolidine (0.40 mL, 4.00 mmol) following Method C (yield: 0.88 g, 60 %). Off-
white solid; m.p. > 250 °C; IR (KBr, cm−1): 3299, 3025, 2969, 2931, 1645, 1593, 1234, 960 and
743; 1H NMR (DMSO-d6): δ 8.73 (s, 1H, -NHCONH), 8.34 (s, 1H, ArH), 8.17 (d, 1H, ArH),
7.69 (d, 1H, ArH), 7.56-7.60 (m, 2H, ArH), 7.41-7.50 (m, 5H, ArH), 7.15-7.27 (m, 1H, ArH, 2H,
Vinylic-H), 6.15 (t, 1H, -NHCONH), 4.42 (q, J = 7.2 Hz, 2H, -NCH2), 3.20-3.24 (m, 2H,
-NHCONHCH2), 2.43-2.49 (m, 6H, -NCH2), 1.66-1.72 (m, 4H, -NCH2CH2) and 1.31 (t, J = 7.2
Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 155.64, 140.43, 139.60, 139.03, 132.00, 129.10,
127.94, 127.40, 127.03, 125.86, 124.98, 123.03, 122.71, 120.89, 119.37, 118.87, 118.68, 118.14,
109.78, 55.83, 53.97, 38.06, 37.52, 23.61 and 14.02; LCMS (m/z): 453.16 [M+H] +, Purity:
nearly 100%.
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(piperidin-1-yl)-ethyl)urea (47)
(aminoethyl) piperidine (0.57 mL, 4.00 mmol) following Method C (yield: 0.89 g, 60 %). Off-
white solid; m.p. 213-216 °C; IR (KBr, cm−1): 3307, 3023, 2962, 2853, 1641, 1581,1237, 962
45
and 743; 1H NMR (DMSO-d6): δ 8.22 (d, 1H, ArH), 8.13 (d, 1H, ArH), 7.67 (dd, 1H, ArH),
7.10-7.54 (m, 10H, ArH, 2H, Vinylic-H), 6.0 (bs, 1H, -NHCONH), 4.37 (q, J = 7.2 Hz, 2H,
-NCH2CH3), 3.43-3.47 (m, 2H, -NHCONHCH2); 2.50-2.69 (m, 6H, -NCH2), 1.70-1.76 (m, 4H,
-NCH2CH2), 1.51-1.59 (m, 2H, -NCH2CH2CH2) and 1.45 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C
NMR (DMSO-d6): 155.54, 140.41, 140.19, 139.53, 131.02, 129.07, 127.41, 127.41, 126.31,
126.00, 124.90, 123.02, 122.71, 120.88, 119.34, 118.56, 118.18, 109.76, 109.70, 58.60, 54.50,
37.51, 36.92, 26.01, 24.57 and 14.02; LCMS (m/z): 467.21 [M+H]+, Purity: 99.88 %.
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(3-(pyrrolidin-1-yl)-propyl)urea (48)
(aminopropyl)pyrrolidine (0.51 g, 4.00 mmol) following Method C (yield: 0.97 g, 65 %). Off-
white solid; m.p. 215-217 °C; IR (KBr, cm −1): 3322, 3022, 2982, 2803, 1634, 1588, 1235, 959
and 745; 1H NMR (DMSO-d6): δ 8.74 (s, 1H, -NHCONH), 8.34 (d, 1H, ArH), 8.17 (d, 1H,
ArH),7.69 (dd, 1H, ArH), 7.57-7.60 (m, 2H, ArH), 7.40-7.49 (m, 5H, ArH), 7.15-7.27 (m, 1H,
ArH, 2H, Vinylic-H), 6.07 (t, 1H, -NHCONH), 4.44 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.17-3.22
(m, 2H, -NHCH2), 2.33-2.36 (m, 6H, -NCH2), 1.49-1.54 (m, 4H, -NCH2CH2), 1.36-1.42 (m, 2H,
-CH2) and 1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C NMR (DMSO-d6): 155.64, 140.41, 140.18,
139.58, 131.03, 129.07, 127.42, 127.01, 126.31, 126.00, 124.89, 123.02, 122.71, 120.87, 119.34,
118.57, 118.26, 109.75, 109.69, 54.09, 53.75, 38.06, 37.51, 29.51, 23.56 and 14.20; LCMS
(m/z): 467.21 [M+H]+, Purity: 97.38 %
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(3-(piperidin-1-yl)-propyl)urea (49)
(aminopropyl) piperidine (0.57 g, 4.00 mmol) following Method C (yield: 1.0 g, 67 %). Off-
46
white solid; m.p. 206-207 °C; IR (KBr, cm −1): 3308, 3027, 2961, 2870, 1641, 1591, 1233, 959
and 745; 1H NMR (DMSO-d6): δ 8.53 (s, 1H, -NHCONH), 8.34 (d, 1H, ArH), 8.17 (d, 1H,
ArH), 7.69 (dd, 1H, ArH), 7.58-7.60 (m, 2H, ArH), 7.41-7.49 (m, 5H, ArH), 7.15-7.27 (m, 1H,
ArH, 2H, Vinylic-H), 6.17 (t, 1H, -NHCONH), 4.43 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.08-3.13
(m, 2H, -NHCH2), 2.23-2.29 (m, 6H, -NCH2), 1.55-1.59 (m, 2H, -NHCH2CH2), 1.46-1.51 (m,
4H, -NCH2CH2), 1.35-1.40 (m, 2H, -NCH2CH2CH2) and 1.31 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C
NMR (DMSO-d6): 155.64, 140.41, 140.19, 139.52, 131.03, 129.07, 127.41, 127.01, 126.30,
126.00, 124.88, 123.03, 122.71, 120.87, 119.34, 118.58, 118.28, 109.74, 109.63, 56.64, 54.57,
38.09, 37.50, 27.52, 26.01, 24.62 and 14.19; LCMS (m/z): 481.17 [M+H]+, Purity: nearly 100%.
General method for the synthesis of (E)-1-(9-ethyl-6-styryl-9H-carbazol-3-yl)-3-
(aminoalkyl)thiourea (50-52)
Method D. To a stirring solution of 34 (1.0 g, 3.20 mmol) and triethylamine (0.5 mL, 3.52
mmol) in dry 1:1 THF/DCM mixture (20 mL), thiocarbonyldiimidazole (0.63 g, 3.52 mmol) in
1:1 THF/DCM mixture (10 mL) was added drop wise at 0-5 °C over a period of 15 min. The
resulting reaction mixture was allowed to stir at room temperature for 2 h. Progress of the
reaction was monitored by TLC. After complete consumption of the starting material, the
appropriate amine (4 mmol) was added to the reaction mixture and the reaction was further
stirred for 30 min. After the completion of the reaction, the solvent was removed at reduced
pressure. The residue so obtained was triturated with chilled methanol (20 mL). The solid was
collected by filtration, washed again with chilled methanol (10 mL) and dried to the give titled
compound [57].
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(pyrrolidin-1-yl)ethyl)thiourea
(50)
47
(aminoethyl) pyrrolidine (0.40 mL, 4.00 mmol) following Method D (yield: 1.13 g, 74 %). Light
yellow solid; m.p. 114-116 °C; IR (KBr, cm−1): 3248, 3026, 2966, 2804, 1597, 1512, 1480, 1339,
1057, 961 and 746; 1H NMR (DMSO-d6): δ 9.71 (s, 1H, -NHCSNH), 8.36 (d, 1H, ArH), 8.17 (d,
(m, 1H, ArH, 2H, Vinylic-H), 4.44 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.54-3.61 (m, 2H, -NHCH2),
2.61 (t, 2H, -NCH2CH2), 2.47-2.51 (m, 4H, -NCH2CH2), 1.74-1.67 (m, 4H, -NCH2CH2) and 1.30
(t, J = 7.2 Hz, 3H, -NCH 2CH3); 13C NMR (DMSO-d6): 139.96, 139.22, 133.43, 128.48, 128.32,
126.27, 125.90, 125.10, 124.62, 122.55, 122.23, 120.44, 118.94, 118.39, 109.35, 109.30, 53.43,
42.90, 37.06, 23.20 and 13.77; LCMS (m/z): 469.3 [M+H]+, Purity: nearly 100 %.
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(piperidin-1-yl)ethyl)thiourea
(51)
(aminoethyl) piperidine (0.57 mL, 4.00 mmol) following Method D (yield: 1.08 g, 70 %). Light
yellow solid; m.p. 183-185 °C; IR (KBr, cm −1): 3290, 3029, 2934, 2846, 2801, 1599, 1513, 1478,
1236, 961, 821 and 750; 1H NMR (DMSO-d6): δ 9.72 (s, 1H, -NHCSNH), 8.37 (d, 1H,ArH),
8.17 (d, 1H, ArH), 7.71-7.73 (m, 1H, ArH), 7.55-7.61 (m, 5H, ArH), 7.43-7.48 (m, 3H, ArH),
7.19-7.37 (m, 1H, ArH, 2H, Vinylic-H), 4.44 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.52-3.58 (m, 2H,
-NHCH2), 2.47 (t, 2H, -NCH2CH2), 2.33-2.38 (m, 4H, -NCH2CH2), 1.45-1.52 (m, 2H,
-NCH2CH2), 1.34-1.41 (m, 2H, -NCH2CH2CH2) and 1.30 (t, J = 7.2 Hz, 3H, -NCH2CH3); 13C
NMR (DMSO-d6): 139.96, 139.24, 133.61, 128.59, 128.30, 126.41, 125.91, 125.06, 124.64,
122.85, 122.55, 122.23, 120.44, 118.95, 118.42, 109.35, 109.30, 53.87, 41.20, 37.06, 25.63,
24.08 and 13.76; LCMS (m/z): 483.4 [M+H]+, Purity: nearly 100 %.
48
(E)-1-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(3-(pyrrolidin-1-yl)-propyl)thiourea
(52)
(aminopropyl)pyrrolidine (0.51 g, 4.00 mmol) following Method D (yield: 1.11 g, 72 %). Light
yellow solid; m.p. 188-190 °C; IR (KBr, cm −1): 3186, 2967, 2871, 2816, 1594, 1524, 1307, 1236,
958 and 747; 1H NMR (DMSO-d6): δ 9.55 (s, 1H, -NHCSNH), 8.37 (d, 1H, ArH), 8.17 (d, 1H,
3H, ArH, 2H, Vinylic-H), 4.44 (q, J = 7.2 Hz, 2H, -NCH2CH3), 3.51-3.55 (m, 2H, -NHCH2),
2.37-2.45 (m, 6H, -NCH2), 1.66-1.75 (m, 2H, -NCH 2CH2), 1.55-1.59 (m, 4H, -NCH2CH2) and
13
1.32 (t, J = 7.2 Hz, 3H, -NCH 2CH3); C NMR (DMSO-d6): 179.9, 139.96, 139.24, 133.69,
128.60, 128.29, 126.40, 125.04, 124.64, 123.09, 122.55, 122.23, 120.44, 118.95, 118.42, 109.35,
109.30, 53.59, 37.06, 27.34, 23.03 and 13.77; LCMS (m/z): 483.4 [M+H]+, Purity: nearly 100 %.
Biology
In vitro cholinesterase inhibition studies
The ability of the test compounds to inhibit ChEs was evaluated using Ellman’s method
as presented in our earlier report [36-38]. All the experiments were performed in 50 mM Tris-
HCl buffer (pH 8). Five different concentrations (0.001-100 μM) of each test compound were
used to determine the enzyme inhibition activity. Briefly, 10 μL of the test or standard
compounds and 50 μL of AChE (0.22 U/mL) or 50 μL of BuChE (0.06 U/mL) were incubated in
96-well plates at room temperature for 30 min. Further, 30 μL of the substrate ATCI (15 mM) or
BTCI (15 mM) was added and the solution was incubated additionally for 30 min. Finally, 160
μL of DTNB (1.5 mM) was added to it, and the absorbance at 415 nm wavelength was measured
using the microplate reader 680 XR (BIO-RAD, India). The IC 50 values were calculated from the
49
absorbance obtained for various concentrations of the test and the standard compounds. All
determinations were carried out in triplicate.
Antioxidant Activity
The antioxidant potential of the compounds was estimated using spectrophotometric
DPPH assay as described earlier [36]. In brief, 10 μL of the test compound (10, 20, 50, 100 and
200 μM in methanol) was mixed with 20 μL of DPPH (10 mM in methanol) in a 96-well plate.
Finally, the volume was adjusted to 200 μL using methanol. After a 30 s incubation at room
temperature with protection from light, the absorbance was recorded using a microplate reader
680 XR (BIORAD, India) at 520 nm wavelength. The free radical scavenging activity was
determined as the reduction percentage (RP) of DPPH using the equation RP = 100[(A 0 −
AC)/A0], where A0 is the untreated DPPH absorbance and AC is the absorbance value for the
added sample concentration C. Ascorbic acid was used as the standard antioxidant.
Self-induced A1-42 aggregation inhibition study
The potential of carbazole-based stilbene derivatives to inhibit self-mediated A1-42
aggregation was evaluated by a thioflavin T (ThT)-based fluorescence assay [58]. 1,1,1,3,3,3-
Hexafluoro-2-propanol (HFIP)-pretreated Aβ1−42 peptide (Sigma Aldrich) was resolubilized with
a 50 mM phosphate buffer (pH = 7.4) to get a stable stock solution (100 μM). The assay was
performed in Costar, clear-bottom, black-surround 96-well plates. For the experiment, the Aβ 1−42
stock solution was additionally diluted to 50 μM (by a 50 mM phosphate buffer, pH = 7.4)
before use. A mixture of the peptide (10 μL) with or without the test compounds (10 μL) at 25
μM and 50 μM final concentrations were incubated at 37 °C temperature for 48 h with frequent
shaking. Blank readings using 50 mM phosphate buffer (pH = 7.4) instead of a peptide with or
without test compounds were also taken. After incubation, the samples were diluted with 180 μL
50
of thioflavin T (5 μM, in 50 mM glycine-NaOH buffer, pH = 8). The fluorescence intensities
were measured on a SpectraMax iD3 multi-mode microplate reader with 446 nm excitation
wavelength and 490 nm emission wavelengths. Each test compound was examined in duplicate.
The fluorescence intensities were compared and the percent inhibition due to the presence of the
test compound was calculated by the following equation, % inhibition = (1-IF i/IF0) × 100, where
IFi and IF0 are the fluorescence intensities obtained for Aβ1-42 in the presence and the absence of
test compound, respectively.
Metal-chelating study
The metal chelating ability of the selected compound was assessed using UV
spectrophotometry [42]. The absorption spectra of compound (50) and 8-hydroxyquinoline (25
M, final concentration) alone or in the presence of CuCl2, FeSO4, FeCl3, or ZnCl2 (25 M, final
concentration) for 30 min were recorded at room temperature at wavelengths ranging from 200
to 600 nm.
Computational Studies
Docking studies of compound (50) with ChEs
Docking studies were carried out with Glide module of Schrodinger Suite. It offers grid-
based ligand docking and explores the promising interactions between ligand molecules and a
protein. The 3D crystallographic structures of AChE (PDB code: 2CKM, 1B41) and of BuChE
(PDB code: 4BDS) were retrieved from the RCSB Protein Data Bank and prepared for docking
by the Protein Preparation Wizard of Schrödinger. The grid was generated on the active site of
the respective protein structure. For the validation of the generated grids for docking studies, the
cocrystallized molecules in the 3D structures of TcAChE and hBuChE (PDB code: 2CKM and
4BDS, respectively) were knocked out of the binding sites. The knocked-out molecules were
51
built within Maestro using the Build module, energy minimized, and redocked into the active
sites of the grids. Very similar interactions were observed between the redocked molecules and
the enzymes as was the case with the original cocrystallized ligands. The root-mean-square
deviation (RMSD) values of the redocked ligands with those of the original orientations in
cocrystallized forms in 2CKM and 4BDS were observed to be 0.40 and 0.26 Å, respectively. The
3D structure of the ligand molecule (50) was built within Maestro using the Build module, and a
single low energy conformation search was carried out for it using the OPLS_2005 force field at
physiological pH conditions using the LigPrep module of Schrödinger, keeping all parameters at
standard values [59]. Docking calculations for the minimized 3D ligand structure were carried
out in extra precision (XP) mode within the active sites of the protein structures [60]. Docking
protocol was validated by comparing the interactions of the docked conformer of donepezil in
the active site of AChE with the reported literature.
Docking studies of compound (50) with Aβ1-42
The docking studies were carried out by using AutoDock4.2 [61, 62]. Aβ1-42 peptide
structure was obtained from RCSB (PDB Code: 1IYT) and was cleaned and prepared for
docking in AutoDock. The docking study of compound (50) was carried out by the blind docking
method. Grid was generated over the entire protein structure, and the ligand under study was
allowed to interact with the entire sequence to know the most stable/possible interactions
between the ligand and the target protein. For the ligand-receptor complex, 10 docking
experiments were carried out using the Lamarckian genetic algorithm. The maximum number of
energy evaluations of 25 million was applied for each docking experiment.
In silico prediction of physicochemical and pharmacokinetics parameters
52
In silico prediction of physicochemical and pharmacokinetic properties was carried out
using the QikProp module of Schrodinger Suite [46]. The structures of the ligand molecules built
for the docking studies were employed to determine the various physicochemical and
pharmacokinetic descriptors. The major descriptors that were analyzed in this study were
Lipinski’s rule of five, the number of rotatable bonds, polar surface area, total solvent-accessible
surface area, central nervous system permeability, apparent MDCK cell permeability, Caco-2
cell permeability, brain/blood partition coefficient, human serum albumin binding, aqueous
solubility, and percent human-oral absorption.
Corresponding Author
Mange Ram Yadav*
The Maharaja Sayajirao University of Baroda,
Vadodara-390001, Gujarat, India
Current Position: Director (R & D)
Parul University, Vadodara, Gujarat, India
E-mail address: [email protected]
Author Contributions
M.R.Y. conceived and designed the study. D.V.P. and N.R.P. performed synthesis and collected
data. A.M.K. designed and performed computational studies. D.M.T., K.B.P. and P.D.J.
contributed reagents and materials and assisted in synthesis and data collection. K.V.P. drafted
the biological evaluation studies. S.P.P., P.M.G., and B.N.C. carried out biological studies and
collected data. N.K.P. assisted in the synthesis and data interpretation. D.V.P., N.R.P. and
53
A.M.K. wrote the manuscript. All authors have given approval to the final version of the
manuscript.
Acknowledgements
Dushyant V. Patel gratefully acknowledges the DST-INSPIRE, New Delhi for awarding
Research Fellowship (IF-150660). Nirav R. Patel is thankful to the UGC, New Delhi, for
awarding Senior Research Fellowship [F. No. 25-1/2014-15(BSR)/7-129/2007/(BSR)]. The
authors thank Dr. Kapil Juvale, SPPSPTM, NMIMS University, Mumbai for providing
SpectramMax iD3 multi-mode microplate reader to perform Aβ1-42 aggregation study.
Conflicts of interest
There are no conflicts of interest to disclose.
Abbreviations used
ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; APP, amyloid
precursor protein; Aβ, beta-amyloid; BuChE, butyrylcholinesterase; 8-HQ, 8-hydroxyquinoline;
MTDLs, multi-target directed ligands; PAS, peripheral anionic site; RMSD, root-mean-square
deviation; RNS, reactive nitrogen species; ROS, reactive oxygen species; ThT, thioflavin T; XP,
extra precision.
54
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Table of Contents graphic
HIGHLIGHTS
► A series of novel carbazole-stilbene derivatives were designed and synthesized as anti-AD

agents.
► Compound (50) exhibited good inhibitory activities against AChE (IC50 value of 2.64 μM)
and BuChE (IC50 value of 1.29 μM).
► Compound (50) showed notable inhibition of self-mediated A1–42 aggregation (51.29 % at
25 μM concentration). It also possessed specific copper ion chelation property.
► It offered promising in silico ADMET properties.
61

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Novel Carbazole-Stilbene Hybrids as Multifunctional anti-Alzheimer agents

including cholinesterase inhibition, A aggregation inhibition, antioxidant and metal chelation

properties. Amongst them, (E)-1-(4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)phenyl)-3-(2-(pyrrolidin-

self-mediated A1–42 aggregation (51.29 % at 25 μM concentration). The metal chelation study

findings evidently showed compound (50) as a potential multitarget-directed ligand in the course

of developing novel anti-AD drugs.

Keywords: Alzheimer’s disease, MTDL, Acetylcholinesterase, Butyrylcholinesterase, Aβ

aggregation, Metal chelation, Carbazole, Stilbene

ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in

anionic site (PAS) to non-amyloidogenic form of A, acting as a pathological chaperone

inducing conformational changes in amyloidogenic form of A with subsequent A fibril

(MTDLs) which could act on both of these ChEs.

serve as a rational approach for the treatment of AD.

Recent research indicates that oxidative stress is an event that precedes the appearance of

other hallmarks of AD. The subtle equilibrium between oxidants/antioxidants disturbed

by inequity between generation and scavenging of free radicals cause oxidative stress [18]. By

pathological oxidation-reduction steps, reactive oxygen species (ROS) and reactive nitrogen

tissue damage through necrosis and apoptosis [19]. Thus, anti-oxidants endowed with additional

series of novel carbazole-based stilbene derivatives as multifunctional anti-AD agents are

compounds including cholinesterase inhibition, A aggregation inhibition, antioxidant and metal

were also predicted using in silico methods.

Carbazole is an important nitrogen-containing heterocycle present widely in many

derivatives possess cholinesterase inhibitory [24], Aβ aggregation inhibitory [25], ROS

search for new anti-AD agents. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) (6) is a naturally

occurring stilbene derivative possessing multiple anti-AD properties i.e. Aβ aggregation

derivatives which could have an impact on their anti-AD properties.

A combination of pharmacophoric moieties having different activities offering some

Figure 2. Molecular hybridization approach used to design carbazole-based stilbene derivatives.

carbazol-3-amine (16) from the commercially available carbazole (11) is outlined in Scheme 1.

Ethylation of carbazole (11) by ethyl bromide in the presence of aqueous NaOH solution in

DMSO afforded N-ethylcarbazole (12) in excellent yield. Mono-formylation of N-

ethylcarbazole (12) by Vilsmeier-Haack formylation using DMF and phosphorus oxychloride

generated 9-ethyl-3-formylcarbazole (13). Treatment of 13 with concentrated nitric acid gave the

reaction condition to yield a mixture of cis- and trans-stilbenes. This isomeric mixture was

converted to a single trans isomer (15) by performing the reaction with catalytic amounts of

iodine in toluene. Reduction of the nitro group in (E)-9-ethyl-3-nitro-6-styryl-9H-carbazole (15)

by stannous chloride offered the key amine intermediate (16).

(v) (iv) O2N

Scheme 1. Synthesis of the key intermediate (E)-9-ethyl-6-styryl-9H-carbazol-3-amine (16).

N-(9-Ethyl-3-styryl-9H-carbazole-6-yl)aminoalkylamide derivatives (20-25) have been

Synthetic route for the designed (E)-1-(9-ethyl-6-styryl-9H-carbazol-3-yl)aminoalkylurea

nitrophenyl chloroformate in the presence of triethylamine, followed by reaction with alicyclic

amines and aminoalkylamines yielded the designed (E)-1-(9-ethyl-6-styryl-9H-carbazol-3-

yl)aminoalkylurea derivatives (26-31).

(iii) Compd. n X Compd. n NR1R2 Compd. n NR1R2

3 and Scheme 4. Synthetic route for the required key intermediate (E)-4-(2-(9-ethyl-9H-

carbazol-3-yl)vinyl)aniline (34) from 9-ethyl-9H-carbazole-3-carbaldehyde (13) is shown

by stannous chloride yielded the desired amine intermediate (34).

Scheme 3. Synthesis of the key intermediate (E)-4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)aniline

(E)-N-(4-(2-(9-Ethyl-9H-carbazol-3-yl)vinyl)phenyl)aminoalkylamide derivatives (38-

performed as depicted in Scheme 4. Reaction of the amine intermediate (34) with p-nitrophenyl

chloroformate in the presence of triethylamine, followed by reaction with aminoalkylamines

with thiocarbonyldiimidazole followed by reaction with the respective aminoalkylamines

afforded the designed thiourea derivatives (50-52).

In vitro cholinesterase inhibition studies

range of 1.84-6.63 µM for AChE and 1.02-5.01 µM for BuChE.

potential of compounds (21, 23 and 25) having a piperidine ring, revealed that compound (25, n

substituted with urea linkers (compounds 28-31) (Table 1).

IC50 ± SEM (µM) Aβ1-42 aggregation RP of DPPHe