Schiff bases of tryptamine as potent inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases): Structure-activity relationship

Article abstract of DOI:10.1016/j.bioorg.2018.10.046

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including

Full text of DOI:10.1016/j.bioorg.2018.10.046

Accepted Manuscript
Schiff Bases of Tryptamine as Potent Inhibitors of Nucleoside Triphosphate
Diphosphohydrolases (NTPDases): Structure-Activity Relationship
Kanwal, Khalid Mohammed Khan, Uzma Salar, Saira Afzal, Abdul Wadood,
Muhammad Taha, Shahnaz Perveen, Huma Khan, Joanna Lecka, Jean Sévigny,
Jamshed Iqbal
PII:
S0045-2068(18)30826-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.046
YBIOO 2580
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
6 August 2018
18 October 2018
23 October 2018
Please cite this article as: Kanwal, K. Mohammed Khan, U. Salar, S. Afzal, A. Wadood, M. Taha, S. Perveen, H.
Khan, J. Lecka, J. Sévigny, J. Iqbal, Schiff Bases of Tryptamine as Potent Inhibitors of Nucleoside Triphosphate
Diphosphohydrolases (NTPDases): Structure-Activity Relationship, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.10.046
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Schiff Bases of Tryptamine as Potent Inhibitors of Nucleoside Triphosphate
Diphosphohydrolases (NTPDases): Structure-Activity Relationship
Kanwal,a Khalid Mohammed Khan,a,h Uzma Salar,a Saira Afzal,b Abdul Wadood,c Muhammad
Taha,d Shahnaz Perveen,e Huma Khan,c Joanna Lecka,f,g Jean Sévigny,f,g Jamshed Iqbal*b
aH. E. J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan
bCentre for Advanced Drug Research (CADR), Department of Pharmacy COMSATS Institute of
Information Technology, Abbottabad, Pakistan
cDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University Mardan, Pakistan
dDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam Saudi Arabia
ePCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
fDépartement de Microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada
gCentre de Recherche du CHU de Québec-Université Laval, Québec, QC, G1V 4G2, Canada
hDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
Abstract: Overexpression of NTPDases leads to a number of pathological situations such as
thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological
situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and
their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and
polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless,
a range of side effects are also associated with it. Reactive blue 2 also had non-specific side
effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors
are high molecular weight compounds, always required tedious chemical steps to synthesize.
Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent
NTPDase inhibitors.
Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen
heterocycles, Schiff bases of tryptamine, 1-26, were synthesized and characterized by
1
spectroscopic techniques such as EI-MS, HREI-MS, H-, and 13C-NMR. All the synthetic
compounds were evaluated for the inhibitory avidity against activities of three major isoforms of
*Corresponding Author: khalid.khan@iccs.edu, drkhalidhej@gmail.com, Tel.: 0092-21-34824910; Fax: 0092-21-
34819018 / J. Iqbal, drjamshed@ciit.net.pk, jamshediqb@googlemail.com; Tel. 00923365101651, 0092-0992-
383591-5; Fax. 0092-0992-383441
1

NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were
found to show potent inhibition (Ki = 0.0200-0.350 μM) of NTPDase-1, twelve (Ki = 0.071-
1.060 μM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700-4.03 μM) NTPDase-8
activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ± 0.007, 6.39 ±
0.89 and 1.180 ± 0.002 μM, respectively. Kinetic studies were performed on lead compounds (6,
5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed
that they were all competitive inhibitors. In silico study was conducted on compound 6 that
showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the
active site of the enzyme.
Keywords: Schiff bases; tryptamine; nucleoside triphosphate diphosphohydrolases; in vitro;
structure-activity relationship
Introduction
Nucleoside triphosphate diphosphohydrolases (NTPDases) comprises of two families
ectoenzymes and endoenzymes located at various sites in the body. They exist in eight different
isoforms designated as NTPDase 1-8, out of which NTPDase-1 to -3 and NTPDase-8 are located
on the cell surface. These enzymes can hydrolyze extracellular nucleotides ATP and ADP to
AMP [1]. The catalytic activity of these enzymes can be stimulated through milli-molar
concentrations of either Ca2+ or Mg2+ ions. NTPDases may exist in monomeric, dimeric or
trimeric forms. NTPDase-1 hydrolyze the ATP and ADP equally, while NTPDases-2, -3, and -8
preferentially hydrolyze ATP. NTPDases express themselves in different tissues such as
NTPDase-1 is found to be expressed on natural killer cells, subsets of activated T cells,
monocytes, dendritic cells, and on vascular endothelium. NTPDase-1 enzyme controls the
cellular immune response by modulating purinergic signaling. It hydrolyzes prothrombotic ADP,
maintains vascular fluidity, modulates thrombosis, vascular inflammation, and plays a major role
in cerebro- as well as cardio-protections [2]. NTPDase-1 also controls vaso-relaxation that is
endothelial P2Y2 receptor-dependent [3]. NTPDase-2 hydrolyzes nucleoside triphosphates and
affords ADP and UDP as the main products [4]. NTPDase-3 is mainly expressed in subsets of
neurons in the brain, in various epithelial cells including kidney, airways, digestive, reproductive
systems, and in islet of langerhans [5]. The last NTPDase member identified, NTPDase-8, is
mainly expressed in the liver, intestine, and kidney. The adenine nucleotides such as ATP and
2

ADP that are released by the cells mainly regulate the vascular response to endothelial injuries.
Adenosine acts as an inhibitor of platelet aggregation, however, ADP promotes it and acts on
specific receptors. The ectonucleotidases interfere with purinergic signaling pathways and are
recognized as pharmaceutical agents. The expression of E-NTPDases at the cell surface is
associated with the development of certain pathologies, particularly proliferation of cancer cells.
E-NTPDases expression is associated to the virulence of certain parasites and bacteria by
protecting these pathological agents from the immune response of the host. The over-expression
of E-NTPDases is found to be associated with human differentiated melanoma and myeloid
leukocytes cells. The over expression of E-NTPDases at the cell surface reduced the interaction
with T-lymphocytes and thus escaping reconnaissance mechanism of the immune system. These
results suggest that E-NTPDases could be implicated into a cell-to-cell recognition. Inhibition or
modulation of E-NTPDases may prolong the effect of nucleotides at their respective receptors or
modulate cell-to-cell interaction [6]. NTPDase inhibitors are found to be important tools as
potential therapeutics against pain, cancer, and autoimmune disorders [7-12]. The good
NTPDase inhibitors should be specific and should not be P2 receptor antagonist. Some specific
inhibitors for NTPDase-1 have been reported i.e. ticlopidine an antiplatelet drug.
Schiff bases are condensed products and formed via the nucleophilic attack of nitrogen on
carbonyl carbon (aldehyde/ketone) thus forming an imine or azomethine bond. They can behave
as precursors for the synthesis of various natural products. They are easy to synthesized and due
to having special feature C=N group, are considered as good chelating agents [13-15]. Its
chelating ability increases if present just adjacent to electron donating substituents like OH and
SH on the aryl part. They are amongst the most widely used organic compounds and usually
utilized as pigments, dyes, catalysts, polymer stabilizers, and intermediates in organic synthesis
[15]. Schiff bases have also been shown a broad range of biological activities including
antifungal, antibacterial, anti-malarial, antiproliferative, antiinflammatory, antiviral, and
antipyretic activities [16-23]. Our research group had also extensively reported the different
biological aspects of Schiff bases e.g. antibacterial/anti-biofilm, antioxidant, anticancer, α-
glucosidase, ß-glucuronidase, and carbonic anhydrase inhibitory activities [24].
Efficacious inhibitors of NTPDases are always required to reduce responses induced by
nucleotides in a number of pathological situations such as thrombosis, inflammation, and cancer.
Three different classes of NTPDase inhibitors are reported so far including nucleotides and their
3

derivatives (8-Bus ATP), sulfonated dyes such as reactive blue 2, suramin and its derivatives,
and polyoxomatalates (POMs) (Figure-1) [1, 7-12, 25, 26].
Figure-1: Inhibitors of NTPDases
Despite being a potent inhibitor, a range of side effects are associated with suramin such as
abdominal pain, vomiting, and diarrhoea. Furthermore, various sensations in the skin including
tingling sensations, numbness of arm, hands, and legs, tenderness of palms and soles as well as
swelling and skin rash are also included. In addition, during usage, it also make the urine cloudy
[27, 28]. Furthermore, most commonly known NTPDase inhibitors are high molecular weight
compounds, containing multiple acidic groups such as sulfonic acid in suramin. It also requires
tedious steps to synthesize these high molecular weight compounds.
Literature revealed that some of the NTPDase inhibitors possess imine (Schiff base) moiety and
many inhibitors including nucleotides contain nitrogen heterocycle in the structure [26, 29].
These nucleotides specially, purines have fused ring structure with nitrogen atoms. Hence, due to
structural resemblance of tryptamine with purine, we intended to install Schiff base functionality
4

on tryptamine to evaluate as inhibitors of NTPDases (Figure-2). Furthermore, it is worth-
mentioning that our compounds are easy to synthesize and requires only a single step
transformation of inexpensive chemicals without any harsh conditions, thus can be called as
green synthesis. Herein, we report the synthesis, structural characterization, and evaluation of
Schiff bases of tryptamine as NTPDase inhibitors.
Figure-2: Rationale of the current study
Results and Discussion
Chemistry
Schiff bases 1-26 were synthesized by reacting tryptamine with different benzaldehyde
derivatives in methanol. The change in the color of reaction mixture indicated the progress of
reaction, however, the completion of reaction was monitored via TLC. The solvent was
evaporated under vacuum resulting in the formation of a crude product which was purified by
crystallization from methanol. The synthetic compounds 1-26 were characterized by different
spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C-NMR. Compounds 1-3, 6-13,
15-17, 19-20, 22-24, and 26 are new compounds while compounds 5, 14, 18, 21 and 25 have
already been reported [30,31].
5

Scheme-1: Synthesis of Schiff bases of tryptamine 1-26
Inhibition of NTPDase-1, -3, and -8
All synthetic compounds 1-26 were evaluated as inhibitors of three major isoforms of NTPDase
enzymes (Table-1). All compounds showed good inhibitory properties. Suramin was used as a
known inhibitor (Ki values of 1.260 ± 0.007, 6.39 ± 0.89 and 1.180 ± 0.002 μM) against
NTPDase-1, -3, and -8, respectively). A limited SAR suggested that the NTPDase inhibition by
this class of compounds mainly depends upon all persistent structural features such as indole
ring, imine group and bridging alky moiety, however, the varying features such as the “R” group
with different substitution pattern in all compounds were also responsible for the inhibitory
avidity of the synthetic compounds (Figure-3).
Figure-3: General structural features of synthetic compounds
6

Table-1: NTPDase-1, -3 and -8 inhibitory activities of the Schiff bases of tryptamine 1-26
NTPDase-1
NTPDase-3
NTPDase-8
NTPDase-1
NTPDase-3
Ki (µM)
NTPDase-8
Comp.
1
Aryl part
IC50 ± SEMa (µM)
b
b
b
b
0.43 ± 0.05
↓
↓
0.03 ± 0.04
↓
↓
b
b
0.53 ± 0.09
0.79 ± 0.08
1.01 ± 0.07
↓
4.55 ± 0.86
14.0 ± 2.01
0.04 ± 0.08
0.06 ± 0.07
0.080 ± 0.001
0.13 ± 0.01
↓
1.31 ± 0.82
4.03 ± 0.02
2
3
4
0.51 ± 0.04
0.13± 0.03
b
b
b
b
↓
↓
↓
↓
1.64 ± 0.17
0.27 ± 0.03
0.36 ± 0.01
0.26 ± 0.02
0.39 ± 0.04
3.89 ± 0.76
0.42 ± 0.03
0.79 ± 0.07
1.57 ± 0.09
0.071 ± 0.002
0.12 ± 0.01
5
6
7
0.021 ± 0.001 0.112 ± 0.002 0.220 ± 0.005
0.030 ± 0.003 1.060 ± 0.002 0.450 ± 0.005
b
b
0.65 ± 0.05
1.91 ± 0.56
↓
1.82 ± 0.11
8.50 ± 1.01
0.050 ± 0.003
↓
0.520 ± 0.002
2.45 ± 0.01
8
9
1.01 ± 0.06
0.105 ± 0.001 0.270 ± 0.002
b
b
b
b
b
b
↓
↓
↓
↓
↓
↓
10
7

0.41 ± 0.03
1.56 ± 0.14
1.06 ± 0.07
3.31 ± 0.56
0.030 ± 0.001 0.420 ± 0.006 0.301 ± 0.001
11
12
b
b
b
b
↓
↓
↓
↓
0.950 ± 0.002
0.280 ± 0.004
b
b
b
b
↓
↓
0.97 ± 0.05
2.48 ± 0.14
↓
↓
13
14
2.38 ± 0.56
0.61 ± 0.03
0.190 ± 0.005 0.170 ± 0.002 0.710 ± 0.007
b
b
b
b
↓
↓
1.32 ± 0.02
0.71 ± 0.02
7.20 ± 1.01
2.12 ± 0.13
7.85 ± 0.97
↓
↓
0.380 ± 0.002
0.201 ± 0.001
2.070 ± 0.002
0.610 ± 0.003
2.26 ± 0.01
15
16
17
18
19
b
b
4.51 ± 0.77
↓
0.350 ± 0.001
↓
b
b
b
b
↓
↓
↓
↓
b
b
0.76 ± 0.06
0.43 ± 0.02
↓
0.060 ± 0.002
↓
0.75 ± 0.06
0.35 ± 0.03
0.030 ± 0.001 0.201 ± 0.001
0.0200 ±
0.0005
0.0900 ±
0.0006
0.28 ± 0.01
1.07 ± 0.05
0.310 ± 0.002
20
8

0.0300 ±
0.0001
0.0700 ±
0.0001
0.43 ± 0.04
3.88 ± 0.99
0.26 ± 0.01
1.71 ± 0.04
1.060 ± 0.001
21
22
b
b
b
b
↓
↓
↓
↓
0.490 ± 0.002
b
b
b
b
b
b
↓
↓
↓
↓
↓
↓
23
24
0.0500 ±
0.0003
0.0700 ±
0.0002
0.64 ± 0.04
0.92 ± 0.06
4.49 ± 0.89
1.290 ± 0.003
b
b
b
b
b
b
↓
↓
↓
↓
↓
↓
25
4.31 ±0.98
16.1 ± 1.02
0.49 ± 0.04
24.1 ± 3.01
0.69 ± 0.07
4.31 ± 0.41
0.340 ± 0.003 0.130 ± 0.001 0.190 ± 0.004
1.260 ± 0.007 6.39 ± 0.89 1.180 ± 0.002
26
Suramin
Std
SEMa (Standard error of the mean), ↓b (Not active), Suraminc (Standard inhibitor for NTPDase activity)
Structure-activity relationship (SAR)
NTPDase-1 inhibitory activity
The structure-activity relationship of the synthetic derivatives is established on the basis of Ki
values. Amongst the hydroxy substituted derivatives, compound 1 with an ortho hydroxy group
on aryl part inhibited selectively NTPDase-1 (Ki = 0.03 ± 0.04 µM) more potently than suramin
(Ki = 1.260 ± 0.007µM). However, the dihydroxy analog 2 with two hydroxyl groups meta to
each other was found to have less NTPDase-1 inhibitory activity (Ki = 0.04 ± 0.08 µM) as
compared to compound 1. Furthermore, the selectivity was also lost by the compound 2 with an
additional hydroxy group as it inhibited also NTPDase-8. The inhibitory activity and selectivity
was further decreased when two hydroxy groups were present at ortho to each other, as observed
in the case of compound 3 (Ki = 0.06 ± 0.07 µM). Inhibitors selectivity is inversely related to the
number of hydroxy groups. It means that compounds having dihydroxy substitutions can interact
in various ways to the active site of enzymes and decrease their selectivity (Figure-4).
9

Figure-4: Structure-activity relationship (SAR) of compounds 1-3
Compounds 4-6 with hydroxy and chloro groups are also potent inhibitors of NTPDase-1.
Amongst them, compound 4 (Ki = 0.080 ± 0.001 µM) with chloro and hydroxyl groups ortho to
each other was found to be selective inhibitor for NTPDase-1. However, in case of its positional
isomer 5 (Ki = 0.13 ± 0.01 µM), with chloro and hydroxyl group at para each other, lost it
selectivity. It showed that compound 4 attained a conformation which only fitted well into the
active site of the NTPDase-1 enzyme. Similarly, compound 6 (Ki = 0.021 ± 0.001 µM), which
has an extra chloro substitution, displayed stronger inhibitory effect of NTPDase-1 than
compounds 4 and 5, however, selectivity was also lost in case of compound 6. It indicates that
extra chloro substitution is playing an important role in inhibition of NTPDase-1. It is worth
mentioning here that compounds 4-6 were stronger inhibitors than suramin (Figure-5).
Figure-5: Structure-activity relationship (SAR) of compounds 4-6
Compounds 8 and 9 with bromo and hydroxy substitutions were potent but non-selective
inhibitors. Amongst them, compound 8 (Ki = 0.050 ± 0.003 µM) with bromo and hydroxy
substitutions at ortho to each other, was potent inhibitor of NTPDase-1. Compound 8 with bromo
together with chloro group was more potent than compound 4. It indicated that bromo group
adjacent to hydroxy is more efficiently participating in the activity as compared to chloro moiety.
10

Its positional isomer 9 (Ki = 0.105 ± 0.001 µM), with bromo substitution at para to hydroxy
group, displayed decreased NTPDase-1 inhibitory activity as compared to compound 8 (Figure-
6).
Figure-6: Structure-activity relationship (SAR) of compounds 7-9
Compound 16 (Ki = 0.350 ± 0.001 µM) having hyroxy and methoxy groups para to each other
showed stronger inhibition of NTPDase-1 activities. This inhibitory pattern showed that the
hydroxy substitution at ortho position might be actively participating in the process, however,
causes loss of the selectivity. Compound 18 (Ki = 0.060 ± 0.02 µM) bearing two methoxy group
reveals that the replacement of OH with OCH3 leads to increase in inhibitory effect against
NTPDase-1. Compound 20 (Ki = 0.0200 ± 0.0005 µM) having an additional methoxy group
along with hydroxy and methoxy showed increased inhibition against NTPDase-1 as compared
to compound 16. It indicates that additional methoxy groupa are playing important role in the
inhibition by creating some important interaction with the active site (Figure-7).
Figure-7: Structure-activity relationship (SAR) of compounds 16-18 and 20
Inhibitory activity of compound 20 can be compared with its structurally similar analog 10,
which has bromo group substitution instead of one methoxy group that makes it completely
11

inactive. Compound 10 might attain the conformation, which is not well fitted into the active
sites of all three isozymes. Moreover, by changing the positions of the substituent as in
compound 11 (Ki = 0.030 ± 0.001 µM), inhibitory effects were restored (Table-1). Inhibitory
effect of compound 10 can also be compared with another structurally similar compound 19 (Ki
= 0.030 ± 0.001 µM), with iodo instead of bromo substitution, that demonstrates potent
inhibition. However, compounds 11 and 19 showed comparable activity to each other (Figure-8).
Figure-8: Structure-activity relationship (SAR) of compounds 10-12, and 19
Compound 24 (Ki = 0.0500 ± 0.0003 µM) with a hydroxy substituted naphthyl ring showed
potent inhibitory effect against NTPDase-1. It can be assumed that the hydroxyl group is actively
contributing in the inhibitory effect. Similarly, compound 26 (Ki = 0.340 ± 0.003 µM) with
anthracene ring have good inhibitory activity (Figure-9). The addition of another phenyl group,
however, resulted in decreased activity.
Figure-9: Structure-activity relationship (SAR) of compounds 23-26
12

NTPDase-3 inhibitory activity
Twelve compounds showed good inhibitory potential as compared to the standard suramin (Ki =
6.39 ± 0.89 µM). Among them, compound 5 (Ki = 0.071 ± 0.002 µM) having chloro and
hydroxy groups at meta and ortho positions, respectively, showed good inhibitory potential as
compared to standard suramin. The addition of another chloro group on the aryl ring resulted in
decreased activity in compound 6 (Ki = 0.112 ± 0.002 µM), might be the addition of chloro
group resulted in increased steric factor. Compound 24 (Ki = 0.071 ± 0.002 µM) having naphthol
ring also showed comparable activity to compound 5. However, compound 25 having
unsubstituted naphthyl ring was inactive, it showed that the presence of hydroxy group in
compounds 5 and 24 might be responsible for binding interactions within the active site of
enzyme (Figure-10).
Figure-10: Structure-activity relationship of compounds 5, 6, and 24.
The second most active compound was 20 (Ki = 0.0900 ± 0.0006 µM) having two methoxy and
one hydroxy group which showed that the addition of methoxy resulted in slight decreased
activity. Replacement of one methoxy with iodo resulted in further decreased in activity as in
compound 19 (Ki = 0.201 ± 0.001 µM), might be due to increased steric factor of iodo group.
Compound 3 (Ki = 0.13 ± 0.03 µM) having two hydroxy groups ortho to each other also
exhibited good inhibitory activity as compared to standard suramin. Compound 14 (Ki = 0.0900
± 0.0006 µM) bearing hydroxy and nitro groups showed good inhibition against NTPDase-3,
however, this compound was less active as compared to compound 5 which showed that the
replacement of nitro group with chloro resulted in decreased activity which might be due to the
electron withdrawing effect of nitro group resulting in deactivation of the ring (Figure-11).
13

Figure-11: Structure-activity relationship of compounds 3, 14, 19, and 20.
NTPDase-8 inhibitory activity:
Except few derivatives, all synthetic compounds showed good inhibitory potential against
NTPDase-8 enzyme. Five compounds 12, 13, 15, 17, and 22 selectively inhibited NTPDase-8
enzyme. Among them, compound 13 (Ki = 0.280 ± 0.004 µM) with flouro and hydroxy
substituents at meta and ortho positions, respectively, was most active. The selectivity was lost
when flouro group was replaced with nitro group in compound 14 (Ki = 0.710 ± 0.007 µM), the
activity of compound was also decreased. Compound 15 (Ki = 0.380 ± 0.002 µM) having flouro
and methoxy groups para to each other was found to have good inhibitory potential and showed
selectivity. Interestingly the selectivity of compound was lost and activity was increased when
flouro group was replaced with hydroxy in compound 16 (Ki = 0.201 ± 0.001 µM). The
selectivity of compounds 13 and 15 was might be due to the presence of flouro group which may
binds well only with the active site of NTPDase-8 enzyme (Figure-12).
14

Figure-12: Structure-activity relationship of compounds 13-16.
Compound 22 (Ki = 0.280 ± 0.004 µM) bearing thiomethyl group was also found to be selective
inhibitor of NTPDase-8 might be due to god interaction of thiomethyl group with enzyme’s
active site. Compound 12 (Ki = 0.950 ± 0.002 µM) having one bromo and two methoxy groups
was also found to be selective and potential inhibitor of NTPDase-8. Shifting the position of
bromo and replacement of one methoxy from hydroxy resulted in loss of activity in compound
11 (Ki = 0.301 ± 0.001 µM), however, the activity was enhanced as compared to compound 12
might be due to better binding interactions of hydroxy group to the active site of enzyme.
Compound 17 (Ki = 2.070 ± 0.002 µM) bearing hydroxy and methoxy groups ortho to each
other selectively inhibited NTPDase-8, nevertheless, the compound was less active as compared
to standard suramin (Ki = 1.180 ± 0.002 µM). The enhanced activity was observed when
hydroxy was replaced with methoxy in compound 18 (Ki = 0.610 ± 0.003 µM), however, the
selectivity was lost (Figure-13).
15

Figure-13: Structure-activity relationship of compounds 11, 12, 17, 18, and 22.
Mechanism of Inhibition
In order to determine the type of inhibition, the inhibitor 6 was selected for human NTPDase-1,5
was selected for human NTPDase-3 and 21 was selected for human NTPDase-8. The obtained
results showed that compounds 6, 5, and 21 exhibited competitive mode of inhibition against
human NTPDase-1, -3 and -8 (Figure-13). Moreover, Michaelis-Menten kinetic parameters (Km
and Vmax) of human NTPDase-1, -3 and -8 inhibition were also determined in the presence and
absence of inhibitor 6, 5 and 21. In these cases, the maximal velocity (Vmax) found almost
constant even by using different concentrations of respective inhibitors, however the Michaelis
constant (Km) was found in increasing order as depicted in Figure-14 (A-C).
16

A
B
17

C
Figure-14: Lineweaver-Burk Plot of lead compounds (A) compound 6, (B) compound 5, and
(C) compound 21, agianst NTPDase-1, -3 and -8, respectively. S: is the concentration of
substrate used (0, 250, 500, 1000 and 1500 μM); concentration of compounds (A) Compound 6,
(B) Compound 5, & (C) Compound 21 black circle (0 μM); black triangle (0.5 μM for NTPDase-
1 and -3, while 0.12 μM for NTPDase-8); black square (1 μM for NTPDase-1 and -3, while 0.25
μM for NTPDase-8); and black diamond (2 μM for NTPDase-1 and -3, while 0.50 μM for
NTPDase-8).
Molecular docking
In order to investigate the binding mode of these newly synthesized compounds in the active site
of human NTPDase-1, the most active compound 6 was docked in the developed homology
model of human NTPDase-1 (Figure-15A). Nucleotide-binding site (i.e. active site), which is
comprised of amino acid residues Asp54, Thr131, Ala132, Glu174, Asp213, Gln220, Ser361,
Phe360, Phe365 and Trp450, was proposed as the docking area. The lowest energy minimized
docked conformation (i.e. -43.30 kcal/mol with docking score -6.62) was used to analyze the
interactions. It was observed that the compound 6 interacts with the active site of human
NTPDase-1 via hydrogen bonds and hydrophobic interactions (Figure-15B). Hydrogen bonds are
created with Asp213 (side chain; 2.2 and 2.4Å), Gly215 and Gly216 (main chain; 2.6Å and 2.1
Å), whereas, some weak arene-hydrogen interactions were observed with Trp450 and Phe365.
Whereas Ca++ also shows interaction with the oxygen atom of dichloro methylphenol moiety,
which is Schiff bases of the compound. The compound 6 also revealed main hydrophobic
18

interactions with Glu174, Phe360, Ser361 and Leu214. The good interaction of compound 6 may
be due to the presence of hydroxyl group and chlorine atoms at ortho and meta positions of
benzene ring. Cumulatively, docking results showed important interactions between compound 6
and the active site of human NTPDase-1, which correlates with its competitive inhibitory effect.
19

Figure-15: (A) The 3D structure homology model and active site of human NTPDase-1. Note:
The enzyme has two extracellular domains (EC) and two transmembrane (TM) helices. (B)
Schematic illustration of the compound 6 docking in the active site of human NTPDase-1. The
dashed lines show the hydrogen bonds between the active site residues (blue) and compound 6
(pink).
In-silico toxicity evaluation
In order to determine toxicity profile of the compounds (1-26), in-silico evaluation has been
carried out with some physiochemical parameters (OSIRIS) [32]. The results are shown in Table-
2. Polar surface area (PSA) is sum of all the polar atoms in compound. It gives us the estimated
ability of synthesize derivatives to cross the blood brain barrier (BBB). The value of PSA less
than 140 is ideal and our all compounds showing the values less than 100. Another parameter,
cLogP shows the octanol water partition coefficient (ratio of compound concentration in octanol
to water concentration). The parameter cLogP is responsible to calculate the compound ability to
20

cross the membrane. Similarly, cLogS is the calculation of water solubility at 25 ˚C. The number
of hydrogen bond donors (HD) and hydrogen bond acceptors (HA) must be less than 5 and 10,
respectively. All the compounds were estimated to be none mutagenic and tumorigenic.
Table-2: In silico calculation of some ADME parameters /toxicity profile for compounds (1-26)
Compounds
PSA
48.38
68.61
68.61
48.38
48.38
48.38
48.38
48.38
48.38
57.61
57.61
46.61
48.38
94.20
37.38
57.61
57.61
46.61
57.61
66.84
48.38
28.15
41.29
48.38
28.15
28.15
cLogP
3.651
3.380
3.341
4.280
4.280
4.907
5.113
4.486
4.486
4.477
4.401
4.492
3.841
3.623
4.105
3.644
3.644
3.659
4.869
3.635
6.721
4.553
2.676
4.871
5.181
6.438
cLogS
-3.2431
-2.8811
-2.8811
-3.9774
-3.9774
-4.7117
-5.0678
-4.3335
-4.3335
-4.3839
-4.3839
-4.7962
-3.5381
-4.0333
-3.9504
-3.2935
-3.2935
-3.7058
-4.2850
-3.3438
-6.9688
-4.6264
-3.3566
-5.1210
-5.4829
-7.3608
HD
6
4
4
4
4
4
4
4
4
5
5
4
4
4
3
5
5
4
5
6
4
2
2
4
2
2
HA
3
2
2
2
2
2
2
2
2
3
3
3
2
2
2
3
3
3
3
4
2
1
1
2
1
1
Mutagenic Tumorigenic
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Conclusions
Schiff bases of tryptamine 1-26 were synthesized and evaluated as inhibitors of human
NTPDase-1, -3, and -8. Many compounds showed selective and strong inhibitory effects on
activities of three major NTPDases, compared with the commonly used inhibitor suramin.
Kinetic studies confirm that the strongest compounds competitively inhibit the enzyme activities.
Also the binding and interactions between enzyme and the ligand were performed in-silico with
the most potent inhibitor compound 6. All synthetic compounds were found to be non-cytotoxic.
21

In conclusion, newly identified NTPDase inhibitors may serve as lead candidates for further
research in order to get powerful anti-NTPDase agents for various biomedical applications.
Materials and Methods
13
1H- and C-NMR spectra were recorded in deuterated solvents on Bruker 300 and 400 MHz
spectrometers. Mass experiments were carried out on a Finnigan MAT-311A mass spectrometer
(Germany). Thin layer chromatography (TLC) was monitored on pre-coated silica gel aluminum
plates (Kieselgel 60, 254, E. Merck, Germany). Visualization of TLC chromatograms was
performed at wavelengths of 254 and 365 nm or iodine vapors. Methanol of analytical grade was
used as received from RCI Labscan Limited, Thailand. All benzaldehyde derivatives of
analytical grades were used as received from the supplier Wako, Japan, TCI, USA. Tryptamine
was purchased from Merck, Germany.
General procedure for the synthesis of Schiff bases of tryptamine (1-26)
Tryptamine (0.16 g, 1 mmol) was taken in a 50 mL round-bottom flask in methanol (10 mL).
Corresponding benzaldehydes (1 mmol) were added in the reaction mixture and stirred for 24 h.
The completion of reaction was monitored via TLC analysis. After completion of the reaction,
solvent was dried under vacuum. The residue was then washed with hexane. Pure products were
obtained after crystallization using methanol.
Spectral Data for the Synthesized Compound
2-((2-(1H-Indol-3-yl)ethylimino)methyl)phenol (1)
º
1
Yield: 76%, M.p.: 103-105 C; H-NMR (300 MHz, DMSO-d6):  13.65 (s, 1H, OH), 10.79 (s,
1H, NH), 8.48 (s, 1H, N=CH), 7.56 (d, 1H, J7,6 = 7.8 Hz, H-4), 7.31 (overlapping multiplet, 3H,
H-7, H-4′, H-6′), 7.13 (d, 1H, J = 2.1 Hz, H-2), 7.06 (t, 1H, J5(4,6) = 7.8 Hz, H-5), 6.98 (t, 1H,
J6(5,7) = 7.8 Hz, H-6), 6.84 (overlapping multiplet, 2H, H-3′, H-5′), 3.88 (t, 2H, J = 6.6 Hz, CH2),
13
3.30 (t, 2H, J = 6.9 Hz, CH2); C-NMR (75 MHz, DMSO-d6):  165.8, 163.4, 136.2, 131.5,
130.8, 125.4, 123.9, 123.4, 121.2, 120.7, 118.6, 118.1, 116.8, 111.2, 110.8, 54.5, 24.9; EI-MS
m/z (% rel. abund.): 264 (M+, 15), 130 (100.0); HREI-MS m/z: Calcd for C17H16N2O [264.1269],
Found [264.1263].
22

4-((2-(1H-Indol-3-yl)ethylimino)methyl)benzene-1,3-diol (2)
º
1
Yield: 82%, M.p.: 136-138 C; H-NMR (300 MHz, DMSO-d6):  13.93 (s, 1H, OH), 10.79 (s,
1H, NH), 8.26 (s, 1H, N=CH), 7.56 (d, 1H, J7,6 = 7.0 Hz, H-4), 7.31 (d, 1H, J4,5 = 7.8 Hz, H-7),
7.10 (overlapping multiplet, 3H, H-6′, H-2, H-5), 6.95 (t, 1H, J6(5,7) = 7.0 Hz, H-6), 6.20 (dd, 1H,
J5′,6′ = 8.7 Hz, J5′,3′ = 2.4 Hz, H-5′), 6.10 (d, 1H, J3′,5′ = 2.1 Hz, H-3′), 3.78 (t, 2H, J = 6.9 Hz,
CH2), 3.0 (t, 2H, J = 6.9 Hz, CH2); 13C-NMR (75 MHz, DMSO-d6):  164.9, 163.7, 163.4, 135.9,
132.6, 126.8, 122.8, 121.4, 118.4, 118.1, 116.7, 111.1, 110.4, 109.1, 103.4, 52.7, 26.4; EI-MS
m/z (% rel. abund.): 280 (M+, 11), 151 (23), 130 (100); HREI-MS m/z: Calcd for C17H16N2O2
[280.1206], Found [280.1212].
3-((2-(1H-Indol-3-yl)ethylimino)methyl)benzene-1,2-diol (3)
º
1
Yield: 66%, M.p.: 216-219 C; H-NMR (300 MHz, DMSO-d6):  10.03 (s, 1H, NH), 8.33 (s,
1H, N=CH), 7.63 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.35 (d, 1H, J7,6 = 8.1 Hz, H-7), 7.17 (s, 1H, H-2),
7.09 (overlapping multiplet, 1H, H-5), 7.03 (overlapping multiplet, 1H, H-6), 6.81 (dd, 1H, J6′,5′
= 7.8 Hz, J6′,4′ = 1.8 Hz, H-6′), 6.74 (dd, 1H, J4′,5′ = 7.8 Hz, J4′,6′ = 1.5 Hz, H-4′), 6.55 (t, 1H,
J5′(6′,4′) = 7.8 Hz, H-5′), 3.97 (t, 2H, J = 6.9 Hz, CH2), 3.18 (t, 2H, J = 7.2 Hz, CH2); 13C-NMR (75
MHz, DMSO-d6):  160.6, 153.4, 149.7, 135.8, 127.4, 125.6, 123.9, 123.2, 122.5, 121.3, 119.6,
118.6, 118.0, 111.1, 110.6, 54.7, 24.6; EI-MS m/z (% rel. abund.): 280 (M+, 62), 184 (20), 130
(100); HREI-MS m/z: Calcd for C17H16N2O2 [280.1212], Found [280.1212].
4-((2-(1H-indol-3-yl)ethylimino)methyl)-2-chlorophenol (4)
º
1
Yield: 72%, M.p.: 140-142 C; H-NMR (300 MHz, DMSO-d6):  10.77 (s, 1H, NH), 8.11 (s,
1H, N=CH), 7.68 (s, 1H, H-2′), 7.55 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.50 (overlapping multiplet, 1H,
H-6′), 7.32 (d, 1H, J7,6 = 8.1 Hz, H-7), 7.12 (s, 1H, H-2), 7.06 (t, 1H, J5(4,6) = 7.5 Hz, H-5), 6.96
(overlapping multiplet, 2H, H-6, H-5′), 3.80 (t, 2H, J = 7.2 Hz, CH2), 3.00 (t, 2H, J = 7.2 Hz,
CH2); 13C-NMR (75 MHz, DMSO-d6):  163.6, 158.6, 137.1, 136.4, 132.3, 129.4, 127.0, 125.4,
123.2, 121.4, 118.7, 118.2, 116.8, 111.1, 110.7, 53.6, 24.1; EI MS m/z (% rel. abund.): 298 (M+,
10), 300 (M+2, 3), 130 (100); HREI-MS m/z: Calcd for C17H15ClN2O [298.0870], Found
[298.0868].
23

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-chlorophenol (5)
º
1
Yield: 77%, M.p.: 136-138 C; H-NMR (300 MHz, DMSO-d6):  13.79 (s, 1H, OH), 10.81 (s,
1H, NH), 8.46 (s, 1H, N=CH), 7.56 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.45 (d, 1H, J6′,3′ = 2.7 Hz, H-6′),
7.31 (overlapping multiplet, 2H, H-7, H-4′), 7.13 (d, 1H, J = 1.8 Hz, H-2), 7.06 (t, 1H, J5(4,6)
=
7.8 Hz, H-5), 6.95 (t, 1H, J6(5,7) = 7.8 Hz, H-6), 6.86 (d, 1H, J3′,4′ = 8.7 Hz, H-3′), 3.88 (t, 2H, J =
13
6.6 Hz, CH2), 3.05 (t, 2H, J = 6.9 Hz, CH2); C-NMR (75 MHz, DMSO-d6): 164.6, 160.2,
136.1, 131.8, 130.4, 127.0, 122.9, 121.3, 120.9, 119.4, 118.7, 118.2, 118.2, 111.4, 111.3, 58.5,
26.3; EI-MS m/z (% rel. abund.): 298 (M+, 44), 300 (M+2, 28), 143 (34), 130 (100); HREI-MS
m/z: Calcd for C17H15ClN2O [298.0866], Found [298.0873].
2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4,6-dichlorophenol (6)
º
1
Yield: 75%, M.p.: 160-162 C; H-NMR (300 MHz, DMSO-d6):  14.61 (s, 1H, OH), 10.87 (s,
1H, NH), 8.45 (s, 1H, N=CH), 7.58 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.54 (d, 1H, J4′,6′ = 2.7 Hz, H-4′),
7.31 (overlapping multiplet, 2H, H-7, H-2), 7.16 (br. s, 1H, H-6′), 7.06 (t, 1H, J5(4,6) = 7.8 Hz, H-
5), 6.96 (t, 1H, J6(5,7) = 7.8 Hz, H-6), 3.91 (t, 2H, J = 6.6 Hz, CH2), 3.09 (t, 2H, J = 6.9 Hz, CH2);
13C-NMR (75 MHz, DMSO-d6):  169.1, 155.0, 136.0, 132.5, 127.2, 123.7, 120.9, 120.5, 118.3,
118.2, 111.3, 108.6, 55.1, 33.6; EI-MS m/z (% rel. abund.): 332 (M+, 16), 334 (M+2, 9), 130
(100); HREI-MS m/z: Calcd for C17H14Cl2N2O [332.0474], Found [332.0483].
2-((2-(1H-Indol-3-yl)ethylimino)methyl)-6-bromo-4-chlorophenol (7)
Yield: 66%, M.p.: 94-96 ºC; 1H-NMR (300 MHz, DMSO-d6):  14.65 (s, 1H, OH), 10.86 (s, 1H,
NH), 8.43 (s, 1H, N=CH), 7.66 (d, 1H, J4′,6′ = 2.7 Hz, H-6′), 7.58 (d, 1H, J4,5 = 7.3 Hz, H-4), 7.34
(overlapping multiplet, 2H, H-7, H-2), 7.15 (d, 1H, J6′,4′ = 2.7 Hz, H-6′), 7.06 (t, 1H, J5(4,6) = 7.3
Hz, H-5), 6.96 (t, 1H, J6(5,7) = 7.3 Hz, H-6), 3.91 (t, 2H, J = 6.6 Hz, CH2), 3.0 (t, 2H, J = 6.9 Hz,
CH2); 13C-NMR (100 MHz, DMSO-d6): 165.2, 164.6, 136.2, 135.8, 130.9, 126.9, 123.2, 121.0,
118.3, 118.3, 117.0, 116.2, 115.4, 111.4, 110.5, 55.6, 26.3; EI-MS m/z (% rel. abund.): 378 (M+,
19), 380 (M+2, 4), 130 (100); HREI-MS m/z: Calcd for C17H14BrClN2O [377.9881], Found
[377.9896].
24

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-bromophenol (8)
º
1
Yield: 68%, M.p.: 118-120 C; H-NMR (300 MHz, DMSO-d6):  10.77 (s, 1H, NH), 8.10 (s,
1H, N=CH), 7.84 (s, 1H, H-2′), 7.54 (d, 2H, J4,5/6′,5′ = 8.1 Hz, H-4, H-6′), 7.30 (d, 1H, J7,6 = 8.1
Hz, H-7), 7.12 (s, 1H, H-2), 7.01 (t, 2H J5(4,7)/5,6/6,5 = 8.1 Hz, H-5, H-6), 6.97 (d, 1H, J5′,6′ = 7.2
Hz, H-5′), 3.75 (t, 2H, J = 7.2 Hz, CH2), 3.01 (m, 2H, CH2); 13C-NMR (75 MHz, DMSO-d6): 
164.6, 160.7, 136.7, 135.3, 131.4, 130.6, 126.9, 123.1, 120.8, 118.8, 118.1, 117.5, 114.5, 111.1,
110.9, 54.2, 23.9; EI-MS m/z (% rel. abund.): 342 (M+, 27), 344 (M+2, 26), 130 (100); HREI-
MS m/z: Calcd for C17H15BrN2O [342.0361], Found [342.0368].
2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-bromophenol (9)
º
1
Yield: 73%, M.p.: 144-146 C; H-NMR (400 MHz, DMSO-d6):  13.79 (s, 1H, OH), 10.80 (s,
1H, NH), 8.45 (s, 1H, N=CH), 7.57 (overlapping multiplet, 2H, H-4, H-6′), 7.43 (dd, 1H, J4′,3′
=
6.4 Hz, J3′,6′ = 2.8 Hz, H-4′), 7.33 (d, 1H, J7,6 = 7.6 Hz, H-7), 7.12 (d, 1H, J = 1.2 Hz, H-2), 7.07
(t, 1H, J5(4,6) = 7.6 Hz, H-5), 6.97 (t, 1H, J6(5,7) = 7.6 Hz, H-6), 6.81 (d, 1H, J3′,4′ = 8.8 Hz, H-3′),
13
3.90 (t, 2H, J = 7.2 Hz, CH2), 3.07 (t, 2H, J = 7.2 Hz, CH2); C-NMR (75 MHz, DMSO-d6):
164.5, 160.7, 136.1, 134.6, 133.3, 127.0, 122.9, 120.8, 120.0, 119.2, 118.2, 118.2, 111.4, 111.3,
108.5, 58.4, 26.3; EI-MS m/z (% rel. abund.): 342 (M+, 33), 344 (M+2, 35), 130 (100); HREI-MS
m/z: Calcd for C17H15BrN2O [342.0367], Found [342.0368].
4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-bromo-6-methoxyphenol (10)
º
1
Yield: 69%, M.p.: 234-237 C; H-NMR (300 MHz, DMSO-d6):  10.78 (s, 1H, OH), 9.90 (s,
1H, NH), 8.10 (s, 1H, N=CH), 7.56 (overlapping multiplet, 2H, H-2′, H-6′), 7.32 (d, 2H, J7(6,5)
=
7.8 Hz, H-7, H-4), 7.13 (s, 1H, H-2), 7.04 (t, 1H, J5(4,6) = 7.8 Hz, H-5), 6.96 (t, 1H, J6(5,7) = 7.8
13
Hz, H-6), 3.77 (s, 5H, CH2, OCH3), 3.0 (s, 2H, CH2); C-NMR (75 MHz, DMSO-d6):  164.9,
162.4, 155.7, 135.8, 134.6, 126.7, 123.1, 121.3, 118.9, 118.2, 115.2, 111.2, 110.5, 109.7, 55.8,
53.7, 25.2; EI-MS m/z (% rel. abund.): 372 (M+, 7), 374 (M+2, 7), 163 (34), 130 (100); HREI-
MS m/z : Calcd for C18H17BrN2O2 [372.0457], Found [372.0473].
25

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-3-bromo-6-methoxyphenol (11)
º
1
Yield: 69%, M.p.: 138-140 C; H-NMR (300 MHz, DMSO-d6):  14.90 (s, 1H, OH), 10.85 (s,
1H, NH), 8.49 (d, 1H, J = 5.7 Hz, N=CH), 7.59 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.32 (d, 1H, J7,6 = 7.5
Hz, H-7), 7.15 (s, 1H, H-2), 7.06 (t, 1H, J5(4,6) = 7.5 Hz, H-5), 6.96 (t, 1H, J6(5,7) = 7.5 Hz, H-6),
6.72 (overlapping multiplet, 2H, H-4′, H-5′), 3.96 (overlapping multiplet, 2H, CH2), 3.15 (s, 3H,
13
OCH3), 3.07 (t, 2H, J = 6.9 Hz, CH2); C-NMR (75 MHz, DMSO-d6):  165.4, 157.9, 152.3,
136.2, 131.6, 129.7, 127.5, 126.1, 123.0, 121.2, 118.7, 118.0, 116.4, 111.3, 110.6, 56.7, 54.5,
25.7; EI-MS m/z (% rel. abund.): 372 (M+, 2), 374 (M+2, 2), 130 (100); HREI-MS m/z: Calcd for
C18H17BrN2O2 [372.0497], Found [372.0473].
N-(2-Bromo-4,5-dimethoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (12)
Yield: 71%, M.p.: 220-222 ºC, 1H-NMR (300 MHz, DMSO-d6):  7.89 (d, 2H, J4(5,6) = 7.6 Hz, H-
4, H-7), 7.48 (s, 2H, H-2, H-3′), 7.33 (s, 1H, H-6′), 7.27 (overlapping multiplet, 2H, H-5, H-6),
13
3.85 (d, 10H, J = 6.9 Hz, CH2, CH2, OCH3, OCH3); C-NMR (75 MHz, DMSO-d6):  164.5,
153.7, 150.2, 136.0, 135.1, 126.3, 123.4, 121.5, 119.6, 118.9, 118.3, 117.1, 115.8, 111.1, 110.4,
54.6, 26.2; EI-MS m/z (% rel. abund.): 387 (M+, 4), 389 (M+2, 4), 130 (9); HREI-MS m/z: Calcd
for C19H19BrN2O2 [386.1604], Found [387.2704].
2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-fluorophenol (13)
º
1
Yield: 76%, M.p.: 154-157 C; H-NMR (300 MHz, DMSO-d6):  13.38 (s, 1H, OH), 10.80 (s,
1H, NH), 8.45 (s, 1H, N=CH), 7.56 (d, 1H, J4,5 = 7.0 Hz, H-4), 7.33 (d, 1H, J7,6 = 7.0 Hz, H-7),
7.26 (overlapping multiplet, 1H, H-6′), 7.18 (overlapping multiplet, 2H, H-4′, H-2), 7.05 (t, 1H,
J5(4,6) = 7.0 Hz, H-5), 6.95 (t, 1H J6(5,7) = 7.0 Hz, H-6), 6.88 (overlapping multiplet, 1H, H-3′),
3.88 (t, 2H, J = 6.9 Hz, CH2), 3.05 (t, 2H, J = 6.9 Hz, CH2); 13C-NMR (75 MHz, DMSO-d6): 
164.9, 158.7, 156.2, 136.3, 127.3, 126.8, 122.7, 121.4, 118.9, 118.1, 117.8, 116.1, 111.3, 110.6,
53.7, 25.2; EI-MS m/z (% rel. abund.): 282 (M+, 24), 130 (100); HREI-MS m/z: Calcd for
C17H15FN2O [282.1156], Found [282.1168].
26

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-nitrophenol (14)
º
1
Yield: 65%, M.p.: 183-185 C; H-NMR (300 MHz, DMSO-d6):  14.13 (s, 1H, OH), 10.88 (s,
1H, NH), 8.63 (s, 1H, N=CH), 8.32 (d, 1H, J6′,4′ = 2.4 Hz, H-6′), 8.01 (dd, 1H, J4′,3′ = 5.1 Hz, J4′,6′
= 2.4 Hz, H-4′), 7.60 (d, 1H, J4,5 = 6.0 Hz, H-4), 7.34 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.17 (d, 1H, J =
1.5 Hz, H-2), 7.08 (t, 1H J5(4,6) = 6.0 Hz, H-5), 6.98 (t, 1H, J6(5,7) = 6.0 Hz, H-6), 6.57 (d, 1H, J3′,4′
= 7.2 Hz, H-3′), 3.94 (t, 2H, J = 5.1 Hz, CH2), 3.14 (t, 2H, J = 5.1 Hz, CH2); 13C-NMR (75 MHz,
DMSO-d6): 177.8, 166.9, 136.2, 133.5, 132.5, 129.2, 126.8, 123.3, 122.7, 121.1, 118.3, 118.2,
113.3, 111.4, 110.0; EI-MS m/z (% rel. abund.): 309 (M+, 13), 130 (100); HREI-MS m/z: Calcd
for C17H15N3O3 [309.1111], Found [309.1113].
N-(2-Fluoro-4-methoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (15)
º
1
Yield: 70%, M.p.: 118-120 C; H-NMR (300 MHz, DMSO-d6):  10.77 (s, 1H, NH), 8.36 (s,
1H, N=CH), 7.87 (d, 1H, J6′,5′ = 8.4 Hz, H-6′), 7.56 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.32 (d, 1H, J7,6 =
7.5 Hz, H-7), 7.12 (s, 1H, H-2), 7.06 (t, 1H, J5(4,6) = 7.5 Hz, H-5), 6.96 (t, 1H, J6(5,7) = 7.5 Hz, H-
6), 6.85 (overlapping multiplet, 2H, H-3′, H-5′), 3.84 (overlapping multiplet, 5H, CH2, OCH3),
13
3.06 (t, 2H, J = 7.2 Hz, CH2); C-NMR (75 MHz, DMSO-d6):  164.9, 163.7, 161.3, 135.7,
132.3, 126.4, 122.8, 120.9, 118.7, 118.3, 114.3, 111.4, 110.7, 109.4, 103.6, 56.3, 52.7, 24.9; EI-
MS m/z (% rel. abund.): 296 (M+, 22), 166 (44), 130 (100); HREI-MS m/z: Calcd for
C18H17FN2O [296.1333], Found [296.1313].
2-((2-(1H-Indol-3-yl)ethylimino)methyl)-5-methoxyphenol (16)
º
1
Yield: 71%, M.p.: 109-111 C; H-NMR (300 MHz, DMSO-d6):  12.97 (s, 1H, OH), 10.79 (s,
1H, NH), 8.45 (s, 1H, N=CH), 7.57 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.33 (d, 1H, J7,6 = 7.8 Hz, H-7),
7.12 (d, 1H, J = 2.1 Hz, H-2), 7.07 (t, 1H, J5(4,6) = 7.8 Hz, H-5), 6.97 (overlapping multiplet, 3H,
H-6, H-6′, H-3′), 6.79 (d, 1H, J5′,6′ = 8.7 Hz, H-5′), 3.89 (t, 2H, J = 7.2 Hz, CH2), 3.68 (s, 3H,
13
OCH3), 3.07 (t, 2H, J = 7.2 Hz, CH2); C-NMR (75 MHz, DMSO-d6):  165.7, 162.2, 160.6,
136.1, 134.3, 127.1, 123.1, 121.5, 118.7, 118.2, 116.3, 111.1, 110.7, 106.8, 101.7, 53.6, 24.1; EI-
MS m/z (% rel. abund.): 294 (M+, 54), 130 (100); HREI-MS m/z: Calcd for C18H18N2O2
[294.1369], Found [294.1368].
27

5-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-methoxyphenol (17)
º
1
Yield: 67%, M.p.: 147-149 C; H-NMR (300 MHz, DMSO-d6):  10.75 (s, 1H, NH), 8.10 (s,
1H, N=CH), 7.54 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.31 (d, 1H, J7,6 = 7.8 Hz, H-7), 7.22 (d, 1H, J = 1.8
Hz, H-2′), 7.10 (s, 1H, H-2), 7.03 (overlapping multiplet, 2H, H-5, H-6′), 6.94 (overlapping
multiplet, 2H, H-6, H-5′), 3.78 (overlapping multiplet, 5H, CH2, OCH3), 2.97 (t, 2H, J = 7.5 Hz,
CH2); 13C-NMR (75 MHz, DMSO-d6):  164.7, 159.8, 151.6, 135.6, 134.6, 126.9, 123.4, 122.6,
121.1, 118.5, 118.0, 116.4, 114.3, 111.2, 109.6, 56.7, 53.8, 23.8; EI-MS m/z (% rel. abund.): 294
(M+, 31), 164 (44), 130 (100); HREI-MS m/z: Calcd for C18H18N2O2 [294.1379], Found
[294.1368].
N-(3,4-Dimethoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (18)
º
1
Yield: 73%, M.p.: 100-102 C; H-NMR (300 MHz, DMSO-d6):  10.76 (s, 1H, NH), 8.19 (s,
1H, N=CH), 7.56 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.36 (s, 1H, H-2′), 7.30 (d, 1H, J7,6 = 8.1 Hz, H-7),
7.19 (d, 1H, J6′,5′ = 6.9 Hz, H-6′), 7.13 (s, 1H, H-2), 7.01 (overlapping multiplet, 3H, H-5, H-6,
13
H-5′), 3.78 (overlapping multiplet, 8H, CH2, OCH3, OCH3), 3.00 (t, 2H, J = 7.2 Hz, CH2); C-
NMR (75 MHz, DMSO-d6):  164.4, 153.7, 150.6, 135.8, 132.8, 127.2, 123.6, 123.2, 121.2,
118.9, 118.5, 112.3, 111.4, 110.1, 108.7, 55.8, 52.4, 26.2; EI-MS m/z (% rel. abund.): 308 (M+,
88), 179 (95), 130 (100); HREI-MS m/z: Calcd for C19H20N2O2 [308.1507], Found [308.1525].
4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-iodo-6-methoxyphenol (19)
º
1
Yield: 70%, M.p.: 225-228 C; H-NMR (300 MHz, DMSO-d6):  10.79 (s, 1H, NH), 8.13 (s,
1H, N=CH), 7.55 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.31 (d, 1H, J7,6 = 8.1 Hz, H-7), 7.13 (d, 1H, J = 1.8
Hz, H-2), 7.04 (m, 1H, H-5), 6.99 (overlapping multiplet, 2H, H-2′, H-6′), 6.95 (m, 1H, H-6),
3.81 (overlapping multiplet, 5H, CH2, OCH3), 2.99 (t, 2H, J = 6.9 Hz, CH2); 13C-NMR (75 MHz,
DMSO-d6):  164.6, 160.2, 158.7, 137.2, 136.4, 131.3, 126.8, 123.1, 121.1, 118.6, 118.0, 111.4,
110.6, 109.8, 89.6, 56.4, 54.2, 26.3; EI-MS m/z (% rel. abund.): 420 (M+, 41), 163 (30), 130
(100); HREI-MS m/z: Calcd for C18H17IN2O2 [420.0342], Found [420.0335].
28

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2,6-dimethoxyphenol (20)
º
1
Yield: 68%, M.p.: 119-121 C; H-NMR (300 MHz, DMSO-d6):  10.77 (s, 1H, NH), 8.14 (s,
1H, N=CH), 7.57 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.31 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.12 (s, 1H, H-2),
7.06 (overlapping multiplet, 4H, H-5, H-2′, H-6′, H-6), 3.77(overlapping multiplet, 8H, CH2 ,
13
OCH3, OCH3), 3.01 (t, 2H, J = 7.2 Hz, CH2); C-NMR (75 MHz, DMSO-d6):  164.7, 150.2,
138.7, 136.5, 133.6, 127.1, 122.8, 120.7, 118.6, 118.0, 111.4, 110.7, 105.2, 56.8, 53.9, 24.7; EI-
MS m/z (% rel. abund.): 324 (M+, 25), 130 (100); HREI-MS m/z: Calcd for C19H20N2O3
[324.1482], Found [324.1474].
2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4,6-di-tert-butylphenol (21)
º
1
Yield: 66%, M.p.: 113-115 C; H-NMR (300 MHz, DMSO-d6):  14.22 (s, 1H, OH), 10.82 (s,
1H, NH), 8.51 (s, 1H, N=CH), 7.58 (d, 1H, J7,6 = 7.8 Hz, H-4), 7.32 (d, 1H, J4,5 = 7.8 Hz, H-7),
7.28 (d, 1H, J6′,4′ = 2.4 Hz, H-6′), 7.20 (d, 1H, J4′,6′ = 2.1 Hz, H-4′), 7.15 (d, 1H, J = 2.1 Hz, H-2),
7.06 (t, 1H, J5(4,6) = 7.8 Hz, H-5), 6.96 (t, 1H, J6(5,7) = 7.8 Hz, H-6), 3.86 (t, 2H, J = 7.2 Hz, CH2),
13
3.06 (t, 2H J = 7.2 Hz, CH2), 1.37 (s, 9H, tBu), 1.24 (s, 9H, tBu); C-NMR (75 MHz, DMSO-
d6):  165.2, 157.6, 139.7, 138.4, 136.5, 129.6, 126.9, 124.7, 124.1, 123.2, 120.6, 118.7, 118.4,
111.0, 110.4, 54.5, 34.6, 33.9, 31.7, 31.2, 26.3; EI-MS m/z (% rel. abund.): 376 (M+, 89), 361
(41), 130 (100); HREI-MS m/z: Calcd for C25H32N2O [376.2509], Found [376.2515].
2-(1H-Indol-3-yl)-N-(4-(methylthio)benzylidene)ethanamine (22)
º
1
Yield: 72%, M.p.: 167-169 C; H-NMR (300 MHz, DMSO-d6):  10.76 (s, 1H, NH), 8.22 (s,
1H, N=CH), 7.65 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.56 (d, 1H, J2′,3′/6′,5′ = 7.8 Hz, H-2′, H-6′), 7.29
(overlapping multiplet, 3H, H-3′, H-5′,H-7), 7.12 (d, 1H J = 1.5 Hz, H-2), 7.06 (t, 1H, J5(4,6) = 7.5
Hz, H-5), 6.96 (t, 1H, J6(5,7) = 7.5 Hz, H-6), 3.82 (t, 2H, J = 7.2 Hz, CH2), 3.02 (t, 2H, J = 7.2 Hz,
CH2); 13C-NMR (75 MHz, DMSO-d6):  164.7, 145.4, 136.2, 133.6, 131.2, 127.6, 127.4, 123.3,
121.5, 118.8, 118.1, 111.4, , 110.1, 53.7, 24.9, 15.3; EI-MS m/z (% rel. abund.): 294 (M+, 36),
164 (73), 130 (100); HREI-MS m/z: Calcd for C18H18N2S [294.1199], Found [294.1191].
29