Novel N-substituted indole Schiff bases as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes: Synthesis, biological activities in?vitro and docking study

Article abstract of DOI:10.1016/j.ejmech.2016.08.013

Two new series of N-substituted indole derivatives 4a–l and 5a–h were synthesized. Their chemical structures were confirmed using spectroscopic tools including IR,¹H NMR,¹³C NMR mass spectroscopy and elemental analyses. The results showed no significant cytotoxic activity on either cancer or normal human cells. Anti-inflammatory activity for all target compounds was evaluated in?vitro. Compounds 5a–h were found to have better anti-inflammatory activity than 4a–l. The inhibitory activity of COX-2 and 5-LOX were tested for 5a–h. Three compounds, 5c, 5d and 5f showed excellent COX-2 inhibitory activity with IC₅₀ranging from 0.98 to 1.23?μM compared to the reference celecoxib (1.54?μM). These compounds had a reasonable selectivity index between 7.03 and 8.05. Additionally, p-methylbenzoyl derivative 5g (IC₅₀?=?5.78?μM) had superior 5-LOX inhibitory activity, higher than quercetin. 5e was close to quercetin in its LOX inhibitory activity. Compounds 5a–h were docked inside the active site of COX-2 and 5-LOX enzymes.

Full text of DOI:10.1016/j.ejmech.2016.08.013

Accepted Manuscript
Novel N-substituted indole Schiff bases as dual inhibitors of cyclooxygenase-2 and 5-
lipoxygenase enzymes: Synthesis, biological activities in vitro and docking study
Phoebe F. Lamie, Waleed Ali Mohamed Ali, Vaclav Bazgier, Lucie Rárová
PII:
S0223-5234(16)30656-0
10.1016/j.ejmech.2016.08.013
EJMECH 8809
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2016
Revised Date: 29 July 2016
Accepted Date: 8 August 2016
Please cite this article as: P.F. Lamie, W.A. Mohamed Ali, V. Bazgier, L. Rárová, Novel N-substituted
indole Schiff bases as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes: Synthesis,
biological activities in vitro and docking study, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.08.013.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel N-substituted indole Schiff bases as dual inhibitors of cyclooxygenase-2
and 5-lipoxygenase enzymes: synthesis, biological activities in vitro and docking
study
Phoebe
F.
Lamiea,*,
Waleed
Ali
mohamed
Alib
Vaclav Bazgierc,d, Lucie Rárováe*
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni Suef
University, Beni Suef, Egypt; bBioChemistry Department, Cairo General Hospital, Egypt
;
cLaboratory of Growth Regulators, Centre of the Region Haná for Biotechnological
and Agricultural Research, Institute of Experimental Botany ASCR & Palacký
University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic. dDepartment of Physical
Chemistry, Faculty of Science, Palacký University, 17. Listopadu 1192/12, 771 46
Olomouc, Czech Republic. eDepartment of Chemical Biology and Genetics, Centre of
the Region Haná for Biotechnological and Agricultural Research, Institute of
Experimental Botany ASCR & Palacký University, Šlechtitelů 27, 783 71 Olomouc,
Czech Republic.
*To whom correspondence should be addressed.
Phoebe F. Lamie,Ph.D. Department of Pharmaceutical Organic Chemistry, Faculty
of Pharmacy, Beni Suef University, Beni Suef, Egypt.
Lucie Rárová Department of Chemical Biology and Genetics, Centre of the Region
Haná for Biotechnological and Agricultural Research, Institute of Experimental
Botany ASCR & Palacký University, Olomouc, The Czech Republic
Tel.: (002)-0122-3321451.
00420585634698
E-mail address: feby.farag@yahoo.com
lucie.rarova@upol.cz
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Abstract
Two new series of N-substituted indole derivatives 4a-l and 5a-h were synthesized.
1
Their chemical structures were confirmed using spectroscopic tools including IR, H
13
NMR, C NMR mass spectroscopy and elemental analyses. The results showed no
significant cytotoxic activity on either cancer or normal human cells. Anti-inflammatory
activity for all target compounds was evaluated in-vitro. Compounds 5a-h were found
to have better anti-inflammatory activity than 4a-l. The inhibitory activity of COX-2
and 5-LOX were tested for 5a-h. Three compounds, 5c, 5d and 5f showed excellent
COX-2 inhibitory activity with IC50 ranging from 0.98 to 1.23 µM compared to the
reference celecoxib (1.54 µM). These compounds had a resonable selectivity index
between 7.03-8.05. Additionally, p-methylbenzoyl derivative 5g (IC50 = 5.78 µM) had
superior 5-LOX inhibitory activity, higher than quercetin. 5e was close to quercetin in
its LOX inhibitory activity. Compounds 5a-h were docked inside the active site of
COX-2 and 5-LOX enzymes.
Keywords: Indole derivatives; Antiproliferative activity; Anti-inflammatory activity;
Molecular docking study; Cyclooxygenase enzymes.
2

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1. Introduction
Inflammation is one of the earliest signs of many well defined diseases and
characterized by symptoms including pain, redness, heat, and swelling. Search for
new drugs that relieve inflammation with high safety profiles is thus still a major
target of drug therapy [1].
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used for
the treatment of inflammatory symptoms [2]. These act by inhibiting the
biotransformation of the polyunsaturated fatty acid, arachidonic acid (AA) which is a
membrane bound phospholipid to prostaglandins (PGs), prostacyclin (PGI2) and
thromboxane A2 (TXA2) via cyclooxygenase (COX) enzymes (COX-1, 2, 3) [3-5].
Non selective NSAIDs such as aspirin, phenazone and indomethacin and,
selective COX-2 inhibitors like celecoxib, rofecoxib and valdecoxib produce
undesirable side effects either by decreasing the cytoprotective action of the
constitutive COX-1 isoform in the gastrointestinal tract (GIT) leading to ulcerogenic,
hepatic and renal toxicity or by increasing cardiovascular disorders of selective COX-
2, the inducible isoform, due to imbalance in the COX pathway. This is caused by
decreasing PGI2 concentration which is a potent vasodilator and antithrombotic -
relative to an increase in the prothrombotic - TXA2 leading to cardiovascular side
effects which was the main cause of their withdrawal from the market [6-14].
In addition to the COX-mediated pathway, AA is also metabolized to leukotrienes
(LTs) via lipoxygenase (LOX) enzymes (5-, 8-, 12-, 15-LOX). Two of these have
been found in humans. 5-LOX is associated with inflammation, bronchoconstriction,
hypersensitivity, anaphylaxis and asthma. 15-LOX is involved in atherosclerosis
[6,15-18].
Accordingly, designing drugs with dual inhibitory activity for COX/LOX
enzymatic pathways offers new options for developing more effective anti-
inflammatory agents with improved safety profile.
A large number of studies [19-23 ] hve used the indole ring based NSAIDs as in
indomethacin, as a target to improve their COX-2 selectivity and reduce their
ulcerogenic side effects attributed to their high COX-1 selectivity and the acidic
properties of the drug. Knaus et al. [24], prepared a series of indole derivatives
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substituted at N1 and C3 positions. These compounds showed excellent COX-2
inhibitory activity.
Moreover, synthesis of compounds with the pyrazole ring and N-hydroxyamide
(tepoxalin) [25] showed 5-LOX activity. Dual activity against COX and 5-LOX was
observed in compounds containing the vinyl bridge between the phenyl and the
heterocyclic five membered ring as in drabufelone [18, 26]. Although, tenidap [3] -
COX/5LOXinhibitor- was more effective than traditional NSAIDs such as piroxicam
and diclofenac in rheumatic arthritis, it was short lived, in the Europe market due to
the liver and kidney toxicity of the reactive oxidative metabolite of thiophene moiety
[3,27,28] (Fig. 1).
[Please, insert Fig. 1 about here]
Given these findings and as a continuation of our earlier work on the development
of selective COX-2 inhibitors [29], we now report the design and synthesis of two
series of novel indole containing compounds, 4a-l and 5a-h substituted at the N-1 and
C-3 positions with: i) N-(4-substituted phenyl)-acetamide derivatives of indole N-1 in
compounds 4a-l similar to 5-LOX amide pharmacophore, ii) the chlorobenzoyl
moiety of indomethacin in position 1, which is important in anti-inflammatory activity
[22], is maintained in 5d, 5h, replaced with benzoyl in 5b, 5f or with p-tolylbenzoyl
part in 5c, 5g or modified to benzyl in 5a, 5e, iii) the small methyl group at C-2 was
omitted completely, iv) the ulcerative carboxylic acid group -CH2COOH at C-3 in
indomethacin was replaced with heteroaryl ring (pyrazole in 4a-f and 5a-d or triazole
4g-l and 5e-h) through azomethine linkage to mimic compounds with dual COX/5-
LOX activity (Fig. 2).
[Please, insert Fig. 2 about here]
We evaluated the in-vitro anti-inflammatory activity of the two series 4a-l and
5a-h. COX-1, COX-2 and 5-LOX inhibitory activities of the most active compounds
5a-h were determined. The plausible binding interactions of these compounds inside
COX-1, COX-2 and 5-LOX active sites were explored using molecular modeling
studies.
2. Results and Discussion
2.1.Chemistry
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The synthesis of the target compounds is depicted in Scheme 1. The starting
compounds, N-[(1H-Indol-3-yl)methylene]-4H-1,2,4-triazol-4-amine (3a) and 4-
{[(1H-Indol-3-yl)methylene]amino}-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(3b) were prepared via reacting indole-3-carbaldehyde (1) and the appropriate
primary amine namely, 4-amino-1,2,4-triazole and 4-amino-1,5-dimethyl-2-phenyl-
1H-pyrazol-3(2H)-one (4-aminophenazone) in absolute ethanol at reflux temperature
in 77-89% yield. The C=O group of indole-3-carbaldehyde (1) disappeared in the IR
spectra of 3a&b.
1H NMR spectra of 3a&b showed a singlet peak at δ 7.86 and 8.03 due to
N=CH proton and D2O exchangeable singlet signal of indole NH at δ 11.61 and
11.99. Additionally, one singlet signal at δ 9.07 due to two triazole protons in the 3a
spectrum, and two singlet signals at δ 2.47 and 3.10 due to CH3 and N-CH3 protons in
13
3b spectrum. Moreover, the C NMR spectra of 3a&b exhibited a peak at δ 152.83,
155.32 revealed to -N=CH, as well as C-3, C-5 of triazole ring in 3a at δ 139.27 and
three different peaks at δ 10.48, 36.42 and 160.80 due to CH3, N-CH3, C=O of
phenazone ring in 3b spectrum.
The mass spectra of compounds 3a&b showed a molecular ion peak at m/z
211 (77.09%) and 330 (27.84%) due to (M), respectively.
For preparing indolyl-N-substituted acetamide derivatives 4a-l in (28-48%)
yield, compounds 3a&b reacted with 4-substituted phenylcarbonylmethyl chlorides
that were prepared by following the same reaction conditions of reported methods
[30-32], in DMF and sodium hydride (NaH) as a base. The formations of compounds
1
4a-l were confirmed by H NMR that showed a singlet signal due to CH2 protons of
acetamide moiety at δ 5.08-5.29 and only one exchangeable singlet signal at δ 10.23-
10.85 of NH proton of acetamide and not of the parent indole compounds 3a&b. This
aside, the 13C NMR spectra of compounds 4a-l exhibited C=O group of acetamide
part at δ 165.21-167.67.
On the other hand, the N-benzyl (5a & 5e) and N-benzoyl (5b-d & 5f-h)
derivatives were prepared by reaction of 3a&b with benzyl chloride, benzoyl
chloride, 4-methylbenzoyl chloride and 4-chlorobenzoyl chloride in the presence of
NaH in DMF at room temperature for 24 h. The formation of compounds 5a-h (yield;
1
43% - 86%) was confirmed by H NMR that revealed the disappearance of the D2O
5

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exchangeable singlet signal of 3a&b and the appearance of a singlet signal at δ 5.47
13
and 5.56 corresponding to benzyl CH2 protons in 5a & 5e. C NMR spectra of 5b-d
& 5f-h showed the presence of the benzoyl C=O group at δ 167.77-168.76.
[Please, insert Scheme 1 about here]
2.2. Anti-inflammatory activity
2.2.1. In vitro anti-inflammatory assay
The in vitro anti-inflammatory properties of the indole derivatives were studied
using ELISA in pretreated Human Umbilical Vein Endothelial Cells (HUVEC) where
these compounds were able to inhibit NF-κB. E-selectin (ELAM) expression was
induced by TNFα, which is indicative of NF-κB activation. The observed reduction of
ELAM expression on treatment of HUVECs with 50 µM of the first series of indole
substances was 15% decrease by 4d and 4f; the more effective second series caused
significant decrease with 30 µM or 50 µM of indole derivative 5c (decrease of 33 %).
This correlates with data from enzyme inhibition of COX-2. Slight inhibition of
ELAM expression and also cell viability was measured for all tested substances. The
results confirmed that the NF-κB pathway was targeted by the indole derivatives (Fig.
3a, b).
[Please, insert Fig. 3 about here]
2.2.2. Inhibition of COX-1, COX-2 and 5-LOX – biological assays
The COX activity assay kit measures the peroxidase activity of
cyclooxygenase enzyme isoforms. The target of the in-vitro biological activity assay
was to explore the ability of tested compounds to inhibit both ovine COX-1 and COX-
2 using a colorimetric enzyme immunoassay (EIA) kit by monitoring the appearance
of oxidized N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. The kit
includes isozyme-specific inhibitors for distinguishing COX-2 activity from COX-1
activity. The COX assay allows the screening of a vast number of inhibitors and saves
much time. The potency of tested compounds was determined as the concentration
causing 50% enzyme inhibition (IC50). Also, the COX-2 selectivity index (SI values)
which is defined as IC50 (COX-1)/IC50 (COX-2) was calculated and compared with
that of the standard drug celecoxib. Compounds 5a-h were tested and the data are
listed in Table 1.
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The results showed that compounds 5a-h were weak inhibitors of the COX-1
enzyme. They showed (IC50 = 7.65 – 11.23 ꢀM) compared with indomethacin (IC50 =
0.63 ꢀM). Compound 5c with (IC50 = 7.89 ꢀM) similar to that of celebrex (IC50 = 7.59
ꢀM ) for COX-1 enzyme inhibition, exhibited better COX-2 enzyme inhibitory
activity (IC50 = 0.98 ꢀM) than celecoxib (IC50 = 1.54 ꢀM). Moreover, compounds 5f
and 5d are classified as excellent COX-2 inhibitors with IC50 = 1.06 and 1.23 ꢀM,
respectively. Compound 5h also showed good inhibitory activity against COX-2 and
its IC50 equals 1.74 ꢀM. The other compounds 5a, 5b, 5e and 5g had moderate COX-
2 inhibitory activity with IC50 ranging from 2.01-2.61 ꢀM. A common feature of the
three most active compounds 5c, 5f and 5d was the substitution of indole - NH with a
benzoyl moiety. Generally, when fixing benzoyl moiety, compounds bearing tirazole
nucleus at C-3 is more active than those with phenazone part . Thus, 5c > 5g and 5d >
5h. Between triazole series, N-benzyl indole 5a and N-unsubstituted benzoyl indole
5b showed the lowest IC50 (2.01 and 2.19 ꢀM, sequentially). Accordingly, the least
active compound was 5e with both phenazone part and benzyl moiety.
So, the results showed COX-2 selectivity indices in the range 4.30 to 8.05 for
5a-h. Within the synthesized compounds, compounds 5c, 5d, 5f and 5h showed a
better COX-2 selectivity index (S.I. = 5.31 – 8.05) compared to celecoxib (S.I. =
4.92). Compounds 5g and 5b showed a COX-2 selectivity index close to celecoxib
(S.I. = 4.84 and 4.75, respectively). The smallest COX-2 selectivity index was
obtained from 5a and 5e (S.I = 4.58 and 4.30, sequentially).
For in vitro lipoxygenase (LOX) inhibitor screening, the assay kit detects and
measures the hydroperoxides produced in the lipoxygenation reaction using a purified
LOX enzyme. The detection reaction is equally sensitive to hydroperoxides at various
positions within the fatty acid, and will work with fatty acids of any carbon length. It
is thus a general detection method for LOX and can be used to screen libraries of
compounds for those which inhibit LOX enzymes. 5-LOX enzyme is distinguished
from other LOXs that introduce hydroperoxidate to lineolate and arachdonate
substrates. Using the enzyme assay kit, the concentration of hydroxyperoxidase was
measured. Quercetin was used as a reference drug in this study. The data are listed in
Table 1.
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Regarding 5-LOX inhibitory activity, compound 5g was the highest 5-LOX
inhibitor with (IC50 = 5.78 ꢀM) compared with that of quercetin (IC50 = 5.96 ꢀM).
Compound 5e showed inhibitory activity against 5-LOX enzyme (IC50 = 5.98 ꢀM),
most similar to quercetin. The most active compound 5g, has a phenazone nucleus at
position C-3 in its structure. This combination favours lipoxygenase inhibitory
activity.
[Please, insert Table 1 about here]
2.2.3. In vitro cytotoxic activity
Fourteen tested indole derivatives from the first series were evaluated for their
cytotoxicity on three cancer cell lines (CEM, MCF7, HeLa) and normal human
fibroblasts. These substances were mostly inactive, except for 3a (strong cytotoxicity
on HeLa) and 4l with slight cytotoxicity on all tested cell lines (Table 2).
Eight indole derivatives from second series were tested for cytotoxicity in human
umbilical vein endothelial cells (HUVEC). After 24 h, these derivatives showed no
cytotoxicity towards HUVECs except for 5a IC50 14.9 ± 3.0 (Table 2).
[Please, insert Table 2 about here]
2.3. Docking study
In a docking study, all new designed compounds were docked using AutoDock
Vina into COX-2 and 5-LOX receptors. However in the case of the N-substituted
indole derivatives synthetized in this study, we found that these compounds showed
two distinct binding poses: (i) one group of compounds (count of heavy atom around
25) with the best binding free energy characterized by binding inside the active site of
receptor 5a – 5d and in particular, H-bond interaction between ligand and amino acid
HIS90 in the COX-2 receptor, and (ii) a second group of large compounds (count of
heavy atom more than 30) 5e – 5h. The problem here was that they had to attain the
active site of both receptors (Table 3). Docking of compound 5h inside the active site
of 5-LOX is illustrated in Fig. 4.
[Please, insert Table 3 and Fig. 4 about here]
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3. Conclusion
In summary, we synthesized new indole 3a&b and N-substituted indole derivatives
4a-l and 5a-h. These derivatives were tested for both cytotoxic and anti-inflammatory
activities. There was no significant cytotoxic activity on either cancer or normal human cells.
The first series of indole derivatives 4a-l showed slight anti-inflammatory activity in vitro. In
the second series, 5a-h, 5c was the most active substance inhibiting expression of cytokine E-
selectin. The latter compounds 5a-h were evaluated against COX-1, COX-2 and 5-LOX
enzymes. P-Methylbenzoyl derivative 5c was the most active inhibitor of COX-2 enzyme
showing IC50 = 0.98 µM and selectivity index equal to 8.05, while p-chlorobenzoyl derivative
5h was the most potent selective 5-LOX inhibitor in this series with IC50 equals to 9.11 µM.
Molecular docking showed that all new compounds bound to selected receptors. It is very
important to follow the count of atoms of each ligand as the size of ligand plays an important
role in the final conformation to active site and binding energy.
4. Experimental
4.1. Chemistry
Melting points were determined using a Griffin apparatus and were uncorrected. IR
spectra were recorded on a Shimadzu IR-435 spectrophotometer using KBr discs and
1
13
values were represented in cm-1. H NMR and C NMR (DEPT-Q) were carried out
1
using the Bruker instrument at 400 MHz for H NMR and 100 MHz for 13C NMR
spectrophotometer, (Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt),
in DMSO-d6, D2O using TMS as an internal standard and chemical shifts were
recorded in ppm on the δ scale using DMSO-d6 (2.5) as a solvent. Coupling constant
(J) values were estimated in Hertz (Hz). Splitting patterns are designated as follows:
s, singlet; d, doublet, t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. The
electron impact (EI) mass spectra were recorded on a Hewlett Packard 5988
spectrometer (Palo Alto, CA). Microanalysis was performed for C, H, N on Perkin-
Elmer 2400 at the Microanalytical center, Cairo University, Egypt and was within +
0.4% of theoretical values. Analytical thin layer chromatography (TLC): pre-coated
plastic sheets, 0.2 mm silica gel with UV indicator (Macherey-Nagel) was employed
routinely to follow the course of reactions and to check the purity of products. All
other reagents, solvents and compound 1 were purchased from the Aldrich Chemical
Company (Milwaukee, WI) and, were used without further purification.
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4.1.1 General procedure for synthesis of compounds 3a&b
A mixture of indole-3-carboxaldehyde (1) (300 mg, 1 mmol) and the appropriate
primary amine 2a or 2b (1.2 mmol) in absolute ethanol (10 mL) was refluxed for 5-7
h (monitored by TLC). The precipitate that formed on hot was filtered off, dried and
recrystallized from 95% ethanol to afford 3a&b.
4.1.1.1. N-[(1H-Indol-3-yl)methylene]-4H-1,2,4-triazol-4-amine (3a). White crystals;
89% yield; mp 291-293°C; IR (KBr) 3126 (NH), 1600 (C=N) cm-1;
1H NMR (400
MHz, DMSO-d6, δ = ppm) δ = 7.21-7.29 (m, 2H, indole H-5, H-6), 7.53 (d, J = 8.00
Hz, 1H, indole H-7), 8.03 (s, 1H, N=CH), 8.21 (d, J = 8.00 Hz, 1H, indole H-4), 9.07
(s, 2H, triazole H-3, H-5), 9.15 (s, 1H, indole H-2), 11.99 (s, 1H, NH, D2O
13
exchangeable); C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 110.36 (indole C-3),
112.87 (indole C-7), 121.87 (indole C-5 ), 122.17 (indole C-6), 123.75 (indole C-4),
124.45 (indole C-3a), 134.93 (indole C-2), 137.71 (indole C-7a), 139.27 (triazole C-3,
C-5), 155.32 (CH=N); EIMS (m/z) 212 (M+1, 13.92%), 211 (M+., 77.09%), 129
(100%). Anal. Calcd for C11H9N5: C, 62.55; H, 4.29; N, 33.16. Found: C, 62.37; H,
4.15; N, 33.11.
4.1.1.2.
4-{[(1H-Indol-3-yl)methylene]amino}-1,5-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (3b). Yellow crystals; 77% yield; mp 271-273°C; IR (KBr) 3159 (NH),
2977, 2921 (CH-aliphatic), 1623 (C=O), 1594 (C=N) cm-1; 1H NMR (400 MHz,
DMSO-d6, δ = ppm) δ = 2.47 (s, 3H, CH3), 3.10 (s, 3H, N-CH3), 7.15-7.23 (m, 2H,
indole H-4, phenyl H-4), 7.34 (t, J = 7.20 Hz, 1H, indole H-5), 7.40 (d, J = 7.60 Hz,
2H, phenyl H-2, H-6), 7.45 (d, J = 7.6 Hz, 1H, indole H-7), 7.52 (t, J = 7.6 Hz, 2H,
indole H-3, H-5), 7.86 (s, 1H, N=CH), 8.43 (d, J = 7.6 Hz, 1H, indole H-4), 9.76 (s,
1H, indole H-2), 11.61 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-
d6, δ = ppm) δ = 10.48 (CH3), 36.42 (N-CH3), 112.30 (indole C-7), 116.34 (pyrazole
C-4), 118.94 (indole C-3), 121.05 (indole C-5), 122.54 (indole C-6), 123.02 (indole
C-4), 124.37 (phenyl C-2, C-6), 125.14 (indole C-3a), 126.82 (phenyl C-4), 129.54
(phenyl C-3, C-5), 132.02 (indole C-2), 135.43 (phenyl C-1), 137.71 (indole C-7a),
151.44 (pyrazole C-5), 152.83 (CH=N), 160.80 (C=O); EIMS (m/z) 331 (M+1,
7.67%), 330 (M+., 27.84%), 56 (100%). Anal. Calcd for C20H18N4O: C, 72.71; H,
5.49; N, 16.96. Found: C, 72.57; H, 5.25; N, 16.99.
4.1.2. General procedure for synthesis of compounds 4a-l
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A solution of 3a or 3b (1 mmol) in DMF (5 mL) was added to the solution of
sodium hydride (NaH) (1.5 mmol) in DMF (5 mL) and the reaction mixture was
stirred for 30 min. at room temperature. A solution of the appropriate 2-chloro-N-
phenylacetamide derivatives (1.5 mmol) in DMF (5 mL) was added, and the reaction
mixture was stirred for 24 h at room temperature. The solution was poured onto ice-
cold water. The formed precipitate was filtered off, dried and recrystallized from 95%
ethanol to furnish 4a-l.
4.1.2.1.
2-{3-[((4H-1,2,4-Triazol-4-yl)imino)methyl]-1H-indol-1yl}-N-
phenylacetamide (4a). White powder; 45% yield; mp 275-277°C; IR (KBr) 3262
1
(NH) 1693 (C=O) cm-1; H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 5.24 (s, 2H,
CH2), 7.08 (t, J = 7.2 Hz, 1H, phenyl H-4), 7.26-7.35 (m, 4H, indole H-5, H-6 and
phenyl H-3, H-5), 7.55-7.61 (m, 3H, indole H-7 and phenyl H-2, H-6), 8.09 (s, 1H,
N=CH), 8.24 (d, J = 7.2 Hz, 1H, indole H-4), 9.10 (s, 2H, triazole H-3, H-5), 9.13 (s,
1H, indole H-2), 10.51 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-
d6, δ = ppm) δ = 49.83 (CH2), 109.90 (indole C-3), 111.25 (indole C-7), 119.61
(phenyl C-2, C-6), 122.23 (indole C-5), 122.39 (indole C-6), 123.94 (indole C-4),
124.11 (phenyl C-4), 124.94 (indole C-3a), 129.36 (phenyl C-3, C-5), 138.30 (indole
C-7a), 138.75 (indole C-2), 139.08 (phenyl C-1), 139.30 (triazole C3, C-5), 154.83
(CH=N), 166.01 (C=O); EIMS (m/z) 345 (M+1, 12.99%), 344 (M+., 18.83%), 129
(100%). Anal. Calcd for C19H16N6O: C, 66.27; H, 4.68; N, 24.40. Found: C, 66.42; H,
4.53; N, 24.51.
4.1.2.2.
2-{3-[((4H-1,2,4-triazol-4-yl)imino)methyl]-1H-indol-1yl}-N-(p-
tolyl)acetamide (4b). White powder; 47% yield; mp 272-274°C; IR (KBr) 3256 (NH)
1
1695 (C=O) cm-1; H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.25 (s, 3H, CH3),
5.22 (s, 2H, CH2), 7.13 (d, J = 8 Hz, 2H, phenyl H-3, H-5), 7.26-7.34 (m, 2H, indole
H-5, H-6), 7.49 (d, J = 8 Hz, 2H, phenyl H-2, H-6), 7.57 (d, J = 8 Hz, 1H, indole H-
7), 8.09 (s, 1 H, CH=N), 8.23 (d, J = 7.6 Hz, 1H, indole H-4), 9.10 (s, 2H, triazole C-
13
3, C-5), 9.14 (s, 1H, indole H-2), 10.48 (s, 1H, NH, D2O exchangeable); C NMR
(100 MHz, DMSO-d6, δ = ppm) δ = 20.91(CH3), 49.80 (CH2), 109.85 (indole C-3),
111.27 (indole C-7), 119.61 (phenyl C-2, C-6), 122.22 (indole C-5), 122.37 (indole C-
6), 123.92 (indole C-4), 124.92 (indole C-3a), 129.71 (phenyl C-3, C-5), 133.03
(phenyl C-1), 136.60 (indole C-7a), 138.28 (phenyl C-4), 138.77 (indole C-2), 139.30
(triazole C-3, C-5), 154.83 (CH=N), 165.75 (C=O); EIMS (m/z) 359 (M+1, 13.13%),
11

ACCEPTED MANUSCRIPT
358 (M+., 14.75%), 77 (100%). Anal. Calcd for C20H18N6O: C, 67.02; H, 5.06; N,
23.45. Found: C, 66.88; H, 5.13; N, 23.42.
4.1.2.3.
2-{3-[((4H-1,2,4-triazol-4-yl)imino)methyl]-1H-indol-1yl}-N-(p-
hydroxyphenyl) acetamide (4c). Yellow powder; 39% yield; mp 230-232°C; IR (KBr)
3323 (OH), 3257 (NH) 1700 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ
= 5.17 (s, 2H, CH2), 6.72 (d, J = 8 Hz, 2H, phenyl H-3, H-5), 7.29-7.55 (m, 6H,
indole H-5, H-6, H-7, phenyl H-2, H-6, CH=N), 8.13 (d, J = 7.6 Hz, 1 H, indole H-
4), 9.11 (s, 1H, indole H-2), 9.28 (s, 2H, triazole H-3, H-5), 9.96 (s, 1H, OH, D2O
exchangeable), 10.48 (s, 1H, NH, D2O exchangeable);13C NMR (100 MHz, DMSO-
d6, δ = ppm) δ = 49.95 (CH2), 109.82 (indole C-7), 115.67 (phenyl C-3, C-5), 117.82
(indole C-3), 121.47 (phenyl C-2, C-6), 122.39 (indole C-5), 123.93 (indole C-6),
124.11 (indole C-4), 124.97 (indole C-3a) 130.64 (phenyl C-1), 138.15 (indole C-7a),
138.78 (indole C-2), 142.76 (triazole C-3, C-5), 154.10 (phenyl C-4), 154.86 (CH=N),
165.21 (C=O); EIMS (m/z) 361 (M+1, 15.28%), 360 (M+., 21.35%), 155 (100%).
Anal. Calcd for C19H16N6O2: C, 63.32; H, 4.48; N, 23.32. Found: C, 63.58; H, 4.13;
N, 23.55.
4.1.2.4.
2-{3-[((4H-1,2,4-triazol-4-yl)imino)methyl]-1H-indol-1yl}-N-(p-
methoxyphenyl) acetamide (4d). Light violet powder; 46% yield; mp 276-278°C; IR
(KBr) 3267 (NH) 1695 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ =
3.71 (s, 3H, OCH3), 5.20 (s, 2H, CH2), 6.90 (d, J = 8.8 Hz, 2H, phenyl H-3, H-5),
7.26-7.35 (m, 2H, indole H-5, H-6), 7.52 (d, J = 8.8 Hz, 2H, phenyl H-2, H-6), 7.57
(d, J = 7.6 Hz, 1H, indole H-7), 8.09 (s, 1H, CH=N), 8.24 (d, J = 7.6 Hz, 1H, indole
H-4), 9.11(s, 2H, triazole H-3, H-5), 9.14 (s, 1H, indole H-2), 10.37 (s, 1H, NH, D2O
exchangeable); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 51.97 (CH2), 57.82
(OCH3), 112.06 (indole C-3), 113.45 (indole C-7), 116.66 (phenyl C-3, C-5), 123.37
(phenyl C-2, C-6), 124.45 (indole C-5), 124.60 (indole C-6), 126.15 (indole C-4),
127.14 (indole C-3a), 134.38 (phenyl C-1), 140.48 (indole C-7a), 140.99 (indole C-2),
141.52 (triazole C-3, C-5), 157.04 (CH=N), 158.11 (phenyl C-4), 167.67 (C=O);
EIMS (m/z) 374 (M+., 5.07%), 122 (100%). Anal. Calcd for C20H18N6O2: C, 64.16;
H, 4.85; N, 22.45. Found: C, 63.99; H, 4.73; N, 22.27.
4.1.2.5. 2-{3-[((4H-1,2,4-triazol-4-yl)imino)methyl]-1H-indol-1yl}-N-(p-acetylphenyl)
acetamide (4e). Yellow powder; 32% yield; mp 286-288°C; IR (KBr) 3231 (NH),
12

ACCEPTED MANUSCRIPT
1694, 1665 (2C=O) cm-1; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.50 (s, 3H,
CH3), 5.29 (s, 2H, CH2), 7.28-7.33 (m, 2H, indole H-5, H-6), 7.59 (d, J = 8 Hz, 1H,
indole H-7), 7.74 (d, J = 8.8 Hz, 2H, phenyl H-3, H-5), 7.96 (d, J = 8.8 Hz, 2H,
phenyl H-2, H-6), 8.10 (s, 1H, CH=N), 8.25 (d, J = 6.8 Hz, 1H, indole H-4), 9.11(s,
2H, triazole H-3, H-5), 9.13 (s, 1H, indole H-2), 10.85 (s, 1H, NH, D2O
13
exchangeable); C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 26.91 (CH3), 49.82
(CH2), 109.97 (indole C-3), 111.24 (indole C-7), 118.96 (phenyl C-2, C-6), 122.32
(indole C-5), 122.40 (indole C-6), 124.03 (indole C-4), 124.87 (indole C-3a), 130.09
(phenyl C-3, C-5), 132.49 (indole C-7a), 138.30 (phenyl C-4), 138.77 (indole C-2),
139.32 (triazole C-3, C-5), 143.31 (phenyl C-1), 154.85 (CH=N), 166.73 (NHC=O),
197.16 (C=O); EIMS (m/z) 386 (M+., 0.16%), 155 (100%). Anal. Calcd for
C21H18N6O2: C, 65.27; H, 4.70; N, 21.75. Found: C, 65.39; H, 4.70; N, 21.55.
3.1.2.6.
Ethyl
4-{2-[3-(((4H-1,2,4-triazol-4-yl)imino)methyl)-1H-indol-1yl]-
acetamido}benzoate (4f). Yellow powder; 47% yield; mp 245-247°C; IR (KBr) 3269
1
(NH) 1704, 1668 (2C=O) cm-1; H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 1.30 (t,
J = 7.2 Hz, 3H, CH2CH3), 4.28 (q, J = 7.2 Hz, 2H, CH2CH3), 5.28 (s, 2H, CH2), 7.26-
7.35 (m, 2H, indole H-5, H-6), 7.59 (d, J = 8 Hz, 1H, indole H-7), 7.74 (d, J = 8.4
Hz, 2H, phenyl H-2, H-6), 7.94 (d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 8.09 (s, 1H,
CH=N), 8.24 (d, J = 7.6 Hz, 1H, indole H-4), 9.11 (s, 2H, triazole H-3, H-5), 9.13 (s,
1H, indole H-2), 10.84 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-
d6, δ = ppm) δ = 14.65 (CH3), 49.83 (CH2), 60.99 (CH2CH3), 109.98 (indole C-3),
111.27 (indole C-7), 119.07 (phenyl C-2, C-6), 122.28 (indole C-5), 122.39 (indole C-
6), 124.00 (indole C-4), 124.88 (phenyl C-4), 125.07 (indole C-3a), 130.85 (phenyl C-
3, C-5), 138.32 (indole C-7a), 138.74 (indole C-2), 139.31 (triazole C-3, C-5), 143.35
(phenyl C-1), 154.82 (CH=N), 165.74 (C=O), 166.73 (NHC=O); EIMS (m/z) 417
(M+1, 22.60%), 416 (M+., 28.08%), 91 (100%). Anal. Calcd for C22H20N6O3: C,
63.45; H, 4.84; N, 20.18. Found: C, 63.39; H, 4.71; N, 20.25.
4.1.2.7. 2-{3-[((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-yl)imino)methyl]-1H-
indol-1yl}-N-phenylacetamide (4g). Yellow powder; 39% yield; mp 228-230°C; IR
(KBr) 3163 (NH) 1695, 1627 (2C=O) cm-1; 1H NMR (400 MHz, DMSO-d6, δ = ppm)
δ = 2.50 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 5.14 (s, 2H, CH2), 7.07 (t, J = 6.8 Hz, 1H,
anilide phenyl H-4), 7.20-7.30 (m, 2H, indole H-5, H-6), 7.32-7.37 (m, 3H, phenyl H-
2, H-4, H-6), 7.41 (d, J = 7.6 Hz, 2H, anilide phenyl H-3, H-5), 7.48-7.55 (m, 3H,
13

ACCEPTED MANUSCRIPT
phenyl H-3, H-5 and indole H-7), 7.60 (d, J = 8 Hz, 2H, anilide phenyl H-2, H-6),
7.91 (s, 1H, CH=N), 8.44 (d, J = 7.6 Hz, 1H, indole H-4), 9.75 (s, 1H, indole H-2),
10.46 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ =
10.50 (CH3), 36.37 (N-CH3), 49.72 (CH2), 110.70 (indole C-7), 115.79 (pyrazole C-
4), 118.76 (indole C-3), 119.60 (indole C-5), 121.45 (anilide phenyl C-4), 122.67
(anilide phenyl C-2, C-6), 123.24 (phenyl C-4), 124.05 (indole C-6), 124.46 (phenyl
C-2, C-6), 125.65 (indole C-3a), 126.89 (indole C-4), 129.34 (phenyl C-3, C-5),
129.54 (anilide phenyl C3, C-5), 135.38 (anilide phenyl C-1), 136.07 (indole C-2),
138.26 (phenyl C-1), 139.12 (indole C-7a), 151.53 (pyrazole C-5), 152.11 (CH=N),
160.76 (C=O), 166.31 (NHC=O); EIMS (m/z) 464 (M+1, 4.46%), 463 (M+., 13.62%),
56 (100%). Anal. Calcd for C28H25N5O2: C, 72.55; H, 5.44; N, 15.11. Found: C,
72.60; H, 5.43; N, 15.01.
4.1.2.8. 2-{3-[((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-yl)imino)methyl]-1H-
indol-1yl}-N-(p-tolyl)acetamide (4h). Buff powder; 41% yield; mp 258-260°C; IR
(KBr) 3259 (NH) 1660, 1627 (2C=O) cm-1;1H NMR (400 MHz, DMSO-d6, δ = ppm)
δ = 2.25 (s, 3H, p-CH3), 2.50 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 5.11 (s, 2H, CH2),
7.12 (d, J = 7.6 Hz, 2H, anilide phenyl H-3, H-5), 7.20-7.28 (m, 2H, indole H-5, H-6),
7.34 (t, J = 7.6 Hz, 1H, phenyl H-4), 7.41 (d, J = 8 Hz, 2H, phenyl H-2, H-6), 7.48-
7.55 (m, 5H, indole H-7, phenyl H-3, H-5 and anilide phenyl H-2, H-6), 7.90 (s, 1H,
CH=N), 8.45 (d, J = 7.6 Hz, 1H, indole H-4), 9.76 (s, 1H, indole H-2), 10.36 (s, 1H,
NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 10.49 (CH3),
20.90 (p-CH3), 36.35 (N-CH3), 49.70 (CH2), 110.70 (indole C-7), 115.78 (pyrazole C-
4), 118.77 (indole C-3), 119.64 (anilide phenyl C-2, C-6), 121.44 (indole C-5), 122.67
(phenyl C-4), 123.24 (indole C-6), 124.47 (phenyl C-2, C-6), 125.66 (indole C-3a),
126.90 (indole C-4), 129.54 (phenyl C-3, C-5), 129.71 (anilide phenyl C-3, C-5),
133.01 (anilide phenyl C-1), 135.37 (anilide phenyl C-4), 136.06 (indole C-2), 136.60
(phenyl C-1), 138.26 (indole C-7a), 151.51 (pyrazole C-5), 152.12 (CH=N), 160.76
(C=O), 166.05 (NHC=O); EIMS (m/z) 478 (M+1, 0.83%), 477 (M+., 2.25%), 56
(100%). Anal. Calcd for C29H27N5O2: C, 72.94; H, 5.70; N, 14.66. Found: C, 72.75;
H, 5.73; N, 14.56.
4.1.2.9.
2-{3-[((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-yl)imino)methyl]-
1H-indol-1yl}-N-(p-hydroxyphenyl)acetamide (4i). Grey powder; 28% yield; mp 229-
1
231°C; IR (KBr) 3430 (OH), 3258 (NH) 1681, 1617 (2C=O) cm-1; H NMR (400
14

ACCEPTED MANUSCRIPT
MHz, DMSO-d6, δ = ppm) δ = 2.49 (s, 3H, CH3), 3.11 (s, 3H, N-CH3), 5.08 (s, 2H,
CH2), 6.71-6.98 (m, 3H, anilide phenyl H-3, H-5, phenyl H-4), 7.24-7.51 (m, 9H,
indole H-5, H-6, H-7, phenyl H-2, H-3, H-5, H-6, anilide phenyl H-2, H-6), 7.90 (s,
1H, CH=N), 8.44-8.45 (m, 1H, indole H-4), 9.28 (s, 1H, OH, D2O exchangeable),
9.76 (s, 1H, indole H-2), 10.23 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz,
DMSO-d6, δ = ppm) δ = 10.44 (CH3), 36.26 (N-CH3), 49.67 (CH2), 110.66 (indole C-
7), 115.66 (anilide phenyl C-3, C-5), 115.72 (pyrazole C-4), 118.68 (indole C-3),
121.87 (anilide phenyl C-2, C-6), 122.19 (indole C-5), 122.65 (phenyl C-4), 123.27
(indole C-6), 124.57 (phenyl C-2, C-6), 125.66 (indole C-3a) 126.99 (indole C-4),
129.57 (phenyl C-3, C-5), 130.69 (anilide phenyl C-1), 135.27 (phenyl C-1), 136.01
(indole C-2), 138.18 (indole C-7a), 151.40 (pyrazole C-5), 152.13 (CH=N), 154.17
(anilide phenyl C-4), 160.72 (C=O), 165.56 (NHC=O); EIMS (m/z) 480 (M+1,
1.52%), 479 (M+., 4.26%), 56 (100%). Anal. Calcd for C28H25N5O3: C, 70.13; H, 5.25;
N, 14.60. Found: C, 70.52; H, 5.37; N, 14.48.
4.1.2.10.
2-{3-[((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-yl)imino)methyl]-
1H-indol-1yl}-N-(p-methoxyphenyl)acetamide (4j). Orange powder; 42% yield; mp
220-222°C; IR (KBr) 3275 (NH) 1669, 1627 (2C=O) cm-1;1H NMR (400 MHz,
DMSO-d6, δ = ppm) δ = 2.49 (s, 3H, CH3), 3.11 (s, 3H, N-CH3), 3.71 (s, 3H, O-CH3),
5.10 (s, 2H, CH2), 6.90 (d, J = 8.8 Hz, 2H, anilide phenyl H-3, H-5), 7.15-7.22 (m,
2H, indole H-5, H-6), 7.26 (t, J = 8.4 Hz, 1H, phenyl H-4), 7.34 (d, J = 6.8 Hz, 2H,
phenyl H-2, H-6), 7.37-7.53 (m, 5H, indole H-7, phenyl H-3, H-5 and anilide phenyl
H-2, H-6), 7.90 (s, 1H, CH=N), 8.45 (d, J = 7.6 Hz, 1H, indole H-4), 9.76 (s, 1H,
indole H-2), 10.32 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6,
δ = ppm) δ = 10.50 (CH3), 36.37 (N-CH3), 49.67 (CH2), 55.61 (OCH3), 110.71
(indole C-7), 115.77 (anilide phenyl C-3, C-5), 118.79 (pyrazole C-4), 121.16 (indole
C-3), 123.01 (anilide phenyl C-2, C-6), 123.23 (indole C-5), 124.36 (phenyl C-4),
124.54 (indole C-6), 125.67 (phenyl C-2, C-6), 126.88 (indole C-3a), 129.54 (indole
C-4), 132.24 (phenyl C-3, C-5), 135.39 (anilide phenyl C-1), 136.08 (phenyl C-1),
137.71 (indole C-2), 151.52 (indole C-7a), 152.13 (pyrazole C-5), 152.82 (CH=N),
155.86 (anilide phenyl C-4), 160.76 (C=O), 165.76 (NHC=O); EIMS (m/z) 494
(M+1, 3.34%), 493 (M+., 10.21%), 56 (100%). Anal. Calcd for C29H27N5O3: C, 70.57;
H, 5.51; N, 14.19. Found: C, 70.43; H, 5.57; N, 14.08.
15

ACCEPTED MANUSCRIPT
4.1.2.11. N-(p-Acetylphenyl)-2-{3-[((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-
yl)imino)methyl]-1H-indol-1yl}acetamide (4K). Orange powder; 47% yield; mp 246-
1
248°C; IR (KBr) 3257 (NH), 1673-1627 (3 C=O) cm-1; H NMR (400 MHz, DMSO-
d6, δ = ppm) δ = 2.37 (s, 3H, CH3), 2.49 (s, 3H, COCH3), 3.12 (s, 3H, N-CH3), 5.20
(s, 2H, CH2), 7.08-7.26 (m, 2H, indole H-5, H-6), 7.35 (t, J = 6.8 Hz, 1H, phenyl H -
4), 7.41 (d, J = 7.6 Hz, 2H, phenyl H-2, H-6), 7.46-7.54 (m, 3H, indole H-7, phenyl
H-3, H-5), 7.75 (d, J = 8.4 Hz, 2H, anilide phenyl H-2, H-6), 7.92 (s, 1H, CH=N),
7.95 (d, J = 8.4 Hz, 2H, anilide phenyl H-3, H-5), 8.46 (d, J = 7.6 Hz, 1H, indole H-
13
4), 9.77 (s, 1H, indole H-2), 10.84 (s, 1H, NH, D2O exchangeable); C NMR (100
MHz, DMSO-d6, δ = ppm) δ = 10.49 (CH3), 26.91 (COCH3), 36.44 (N-CH3), 49.74
(CH2), 110.74 (indole C-7), 115.90 (pyrazole C-4), 118.76 (indole C-3), 118.93
(anilide phenyl C-2, C-6), 121.47 (indole C-5), 123.00 (phenyl C-4), 123.28 (indole
C-6), 124.47 (phenyl C-2, C-6), 125.65 (indole C-3a), 126.89 (indole C-4), 129.54
(phenyl C-3, C-5), 130.07 (anilide phenyl C-3, C-5), 132.42 (phenyl C-1), 135.38
(anilide phenyl C-4), 136.02 (indole C-2), 138.33 (indole C-7a), 143.44 (anilide
phenyl C-1), 151.53 (pyrazole C-5), 152.09 (CH=N), 160.76 (C=O), 167.05
(NHC=O), 197.01 (CH3C=O); EIMS (m/z) 506 (M+1, 3.45%), 505 (M+., 10.48%),
56 (100%). Anal. Calcd for C30H27N5O3: C, 71.27; H, 5.38; N, 13.85. Found: C,
70.98; H, 5.39; N, 13.74.
4.1.2.12.
yl)imino)methyl)-1H-indol-1yl]acetamido}benzoate (4l). Buff powder; 48% yield; mp
246-248°C; IR (KBr) 3256 (NH), 1705-1628 (3C=O) cm-1; 1H NMR (400 MHz,
Ethyl
4-{2-[3-(((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-
DMSO-d6, δ = ppm) δ = 1.30 (t, J = 6.8 Hz, 3H, CH2CH3), 2.49 (s, 3H, CH3), 3.10 (s,
3H, N-CH3), 4.28 (d, J = 6.8 Hz, 2H, CH2CH3), 5.20 (s, 2H, CH2), 7.24-7.27 (m, 2H,
indole H-5, H-6), 7.34 (t, J = 6.4 Hz, 1H, phenyl H-4), 7.41 (d, J = 7.6 Hz, 2H,
phenyl H-2, H-6), 7.51-7.52 (m, 3H, indole H-7 and phenyl H-3, H-5), 7.77 (d, J = 8
Hz, 2H, anilide phenyl H-2, H-6), 7.92 (s, 1H, CH=N), 7.95 (d, J = 8 Hz, 2H, anilide
phenyl H-3, H-5), 8.48 (d, J = 6.8 Hz, 1H, indole H-4), 9.79 (s, 1H, indole H-2),
10.82 (s, 1H, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ =
10.46 (CH3), 14.64 (CH2CH3), 36.40 (N-CH3), 49.73 (CH2), 60.98 (CH2CH3), 110.71
(indole C-7), 115.91 (pyrazole C-4), 118.72 (indole C-3), 119.07 (anilide phenyl C-2,
C-6), 121.05 (indole C-5), 121.49 (phenyl C-4), 122.68 (indole C-6), 124.51 (phenyl
C-2, C-6), 125.02 (indole C-3a), 125.64 (anilide phenyl C-4), 126.93 (indole C-4),
16

ACCEPTED MANUSCRIPT
129.54 (phenyl C-3, C-5), 131.99 (indole C-2), 135.33 (phenyl C-1), 135.99 (anilide
phenyl C-3, C-5), 138.30 (indole C-7a), 143.42 (anilide phenyl C-1), 151.49 (pyrazole
C-5), 152.10 (CH=N), 160.74 (C=O), 165.76 (COOCH2CH3), 167.02 (NHC=O);
EIMS (m/z) 536 (M+1, 1.33%), 535 (M+., 3.83%), 56 (100%). Anal. Calcd for
C31H29N5O4: C, 69.52; H, 5.46; N, 13.08. Found: C, 69.72; H, 5.21; N, 12.99.
4.1.3. General procedure for synthesis of compounds 5a-h
A solution of 3a or 3b (1 mmol) in DMF (5 mL) was added to a solution of
sodium hydride (NaH) (1.5 mmol) in DMF (5 mL) and the reaction mixture was
stirred for 30 min. at room temperature. Either benzyl chloride (0.18 g, 1.5 mmol), or
the appropriate 4-substituted benzoyl chloride derivative (1.5 mmol), was added. The
reaction mixture was stirred for 24 h at room temperature. The solvent was removed
in vacuoand, ice-cold water was added. The formed precipitate was filtered off, dried
and recrystallized from 95% ethanol to furnish 5a-h.
4.1.3.1. N-[(1-Benzyl-1H-indol-3-yl)methylene]-4H-1,2,4-triazol-4-amine (5a). White
crystals; 86% yield; mp 195-197oC; IR (KBr) 1607 (C=N) cm-1; 1H NMR (400 MHz,
DMSO-d6, δ = ppm) δ = 5.56 (s, 2H, CH2), 7.24-7.37 (m, 7H, 5 benzyl-H and indole
H-5, H-6), 7.61 (d, J = 7.6 Hz, 1H, indole H-7), 8.18 (s, 1H, N=CH), 8.23 (d, J = 7.6
Hz, 1H, indole H-4), 9.08 (s, 2H, triazole H-3, H-5), 9.11 (s, 1H, indole H-2); 13C
NMR (100 MHz, DMSO-d6, δ = ppm) δ = 50.08 (CH2), 109.85 (indole C-3), 111.72
(indole C-7), 122.40 (indole C-5), 122.45 (indole C-6), 124.05 (indole C-4), 125.13
(indole C-3a), 127.73 (phenyl C-4), 128.28 (phenyl C-2, C-6), 129.21 (phenyl C-3, C-
5), 137.39 (indole C-7a), 137.52 (phenyl C-1), 137.63 (indole C-2), 139.29 (triazole
C-3, C-5), 155.00 (CH=N);
EIMS (m/z) 302 (M+1, 1.2%), 301 (M+., 6.2%), 255
(100%). Anal. Calcd for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found: C, 71.91; H,
7.99; N, 23.15.
4.1.3.2. N-[(1-Benzoyl-1H-indol-3yl)methylene)-4H-1,2,4-triazol-4-amine (5b). Yellow
crystals; 53% yield; mp 215-217°C; IR (KBr) 1670 (C=O), 1632 (C=N) cm-1; 1H NMR
(400 MHz, DMSO-d6, δ = ppm) δ = 7.48-7.56 (m, 2H, indole H-5, H-6), 7.66 (t, J = 8.0
Hz, 2H, benzoyl H-3, H-5), 7.76 (t, J = 7.6 Hz, 1H, benzoyl H-4), 7.86 (d, J = 8 Hz, 2H,
benzoyl H-2, H-6), 8.02 (s, 1H, N=CH), 8.34-8.38 (m, 2H, indole H-4, H-7), 9.10 (s, 2H,
triazole H-3, H-5), 9.20 (s, 1H, indole H-2); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ
= 115.53 (indole C-3), 116.53 (indole C-7), 122.82 (indole C-5), 125.50 (indole C-6),
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126.70 (indole C-3a), 126.72 (indole C-4), 129.35 (benzoyl C-3, C-5), 129.75
(benzoyl C-2, C-6), 133.06 (benzoyl C-4), 133.69 (benzoyl C-1), 136.02 (indole C-
2), 136.88 (indole C-7a), 139.19 (triazole C-3, C-5), 153.81 (CH=N), 168.69 (C=O)
;
EIMS (m/z) 316 (M+1, 11.4%), 315 (M+., 63.9%), 195 (100%). Anal. Calcd for
C18H13N5O: C, 68.56; H, 4.16; N, 22.21. Found: C, 68.47; H, 4.15; N, 22.11.
4.1.3.3. N-[(1-(p-Methylbenzoyl)-1H-indol-3yl)methylene)-4H-1,2,4-triazol-4-amine
(5c). Orange crystals; 49% yield; mp 117-119 oC; IR (KBr) 1678 (C=O), 1612 (C=N) cm-
1
;
1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.42 (s, 3H, CH3), 7.42-7.54 (m, 4H,
indole H-5, H-6, phenyl H-3, H-5), 7.74 (d, J = 8.0 Hz, 2H, p-methylbenzoyl H-2, H-6), 8.00
(d, J = 8.0 Hz,1H, indole H-7), 8.04 (s, 1H, N=CH), 8.33 (d, J = 8.0 Hz, 1H, indole H-4), 9.10
(s, 2H, triazole H-3, H-5), 9.18 (s, 1H, indole H-2); 13C NMR (100 MHz, DMSO-d6, δ =
ppm) δ = 21.55 (CH3), 115.31 (indole C-3), 116.39 (indole C-7), 122.80 (indole C-5),
125.39 (indole C-6), 126.61 (indole C-4), 128.39 (indole C-3a), 129.58 (p-
methylbenzoyl C-3, C-5), 129.87 (p-methylbenzoyl C-2, C-6), 130.65 (p-methylbenzoyl
C-1), 136.09 (indole, C-2), 136.85 (indole, C-7a), 139.21 (triazole C-3, C-5), 143.56
(phenyl C-4), 153.79 (CH=N), 168.59 (C=O); EIMS (m/z) 330 (M+1, 20.2%), 329
(M+., 100%). Anal. Calcd for C19H15N5O: C, 69.29; H, 4.59; N, 21.26. Found: C,
69.37; H, 4.55; N, 20.98.
4.1.3.4. N-[(1-(p-Chlorobenzoyl)-1H-indol-3yl)methylene)-4H-1,2,4-triazol-4-amine
(5d). Yellow crystals; 64% yield; mp 209-211oC; IR (KBr) 1686 (C=O), 1628 (C=N) cm-
1
1; H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 7.53-7.57 (m, 2H, indole H-5, H-6),
7.74 (d, J = 6.8 Hz, 2H, p-chlorobenzoyl H-3, H-5), 7.88 (d, J = 6.8 Hz, 2H, p-
chlorobenzoyl H-2, H-6), 8.03 (s, 1H, N=CH), 8.34-8.38 (m, 2H, indole H-4, H-7),
9.10 (s, 2H, triazole H-3, H-5), 9.17 (s, 1H, indole H-2); 13C NMR (100 MHz,
DMSO-d6, δ = ppm) δ = 115.69 (indole C-3), 116.55 (indole C-7), 122.83 (indole C-
5), 125.58 (indole C-6), 126.73 (indole C-3a), 126.78 (indole C-4), 129.44 (p-
chlorobenzoyl C-3, C-5), 131.71 (p-chlorobenzoyl C-2, C-6), 132.56 (p-
chlorobenzoyl C-1), 136.03 (indole, C-2), 136.85 (indole, C-7a), 137.88 (p-
chlorobenzoyl C-4), 139.19 (triazole C-3, C-5), 153.73 (CH=N), 167.78 (C=O); EIMS
(m/z) 349 (M+., 5.1%), 91 (100%). Anal. Calcd for C18H12ClN5O: C, 61.81; H, 3.46;
N, 20.02 Found: C, 62.17; H, 3.35; N, 20.11.
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4.1.3.5.
4-{[(1-Benzyl-1H-indol-3yl)methylene]amino}-1,5-dimethyl-2-phenyl-1H-
o
pyrazol-3(2H)-one (5e). Yellow crystals; 68% yield; mp 174-176 C; IR (KBr) 1644
(C=O), 1604 (C=N) cm-1; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.47 (s, 3H,
CH3), 3.11 (s, 3H, N-CH3), 5.47 (s, 2H, CH2), 7.21-7.29 (m, 2H, indole H-5, phenyl
H-4), 7.31-7.37 (m, 6H, benzyl H-3, H-4, H-5, indole H-6, phenyl H-3, H-5), 7.40 (d,
J = 7.6 Hz, 2H, benzyl H-2, H-6), 7.50-7.55 (m, 3H, indole H-7, phenyl H-2, H-6),
8.05 (s, 1H, N=CH), 8.44 (d, J = 8.8 Hz, 1H, indole H-4), 9.75 (s, 1H, indole H-2);
13C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 10.39 (CH3), 36.22 (N-CH3), 49.88
(CH2), 111.16 (indole C-7), 115.83 (pyrazole C-4), 118.62 (indole C-3), 121.49
(indole C-5), 122.71 (phenyl C-4), 123.29 (phenyl C-2, C-6), 124.66 (benzyl C-4),
125.85 (indole C-3a), 127.08 (indole C-6), 127.63 (indole C-4), 128.06 (benzyl C-2,
C-6), 129.10 (phenyl C-3, C-5), 129.23 (benzyl C-3, C-5), 129.60 (phenyl C-2, C-6),
134.91 (indole C-2), 135.25 (phenyl C-1), 137.48 (indole C-7a), 137.93 (benzyl C-1),
151.31 (pyrazole C-5), 152.13 (CH=N), 160.69 (C=O); EIMS (m/z) 421 (M+1, 3.1%),
420 (M+., 9.2%), 304 (100%). Anal. Calcd for C27H24N4O: C, 77.12; H, 5.75; N,
13.32. Found: C, 77.02; H, 5.86; N, 13.17.
4.1.3.6. 4-{[(1-Benzoyl-1H-indol-3yl)methylene]amino}-1,5-dimethyl-2-phenyl-1H-
pyrazol-3(2H)-one (5f). Orange crystals; 77% yield; mp 166-168°C; IR (KBr) 1673,
1642 (2C=O), 1602 (C=N) cm-1; 1H NMR (400 MHz, DMSO-d6, δ = ppm) δ = 2.50
(s, 3H, CH3), 3.18 (s, 3H, N-CH3), 7.37-7.39 (m, 3H, indole H-5, H-6, phenyl H-4),
7.46-7.55 (m, 4H, phenyl H-2, H-3, H-5, H-6), 7.61-7.64 (m, 2H, benzoyl H-3, H-5),
7.70 (t, J = 8.4 Hz, 1H, benzoyl H-4), 7.83 (d, J = 6.8 Hz, 2H, benzoyl H-2, H-6),
7.93 (s, 1H, N=CH), 8.32 (d, J = 9.2 Hz,1H, indole H-7), 8.60 (d, J = 9.2 Hz, 1H,
indole H-4), 9.70 (s, 1H, indole H-2); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ =
10.46 (CH3), 35.87 (N-CH3), 115.50 (indole C-3), 116.26 (indole C-7), 117.56 (pyrazole
C-4), 123.24 (indole C-5), 125.02 (phenyl C-4), 125.15 (phenyl C-2, C-6), 126.14
(indole C-6), 127.36 (indole C-4), 128.00 (indole C-3a), 129.29 (phenyl C-3, C-5),
129.64 (benzoyl C-3, C-5), 129.73 (benzoyl C-2, C-6), 132.69 (benzoyl C-1), 132.84
(indole C-2), 133.98 (phenyl C-1), 137.01 (indole C-7a), 143.26 (benzoyl C-4),
150.56 (CH=N), 153.56 (pyrazole C-5), 160.07 (C=O), 168.76 (benzoyl C=O); EIMS
(m/z) 434 (M+., 3.6%), 310 (100%). Anal. Calcd for C27H22N4O2: C, 74.64; H, 5.10;
N, 12.89. Found: C, 74.88; H, 5.36; N, 13.07.
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4.1.3.7. 1,5-Dimethyl-4-{[(1-(p-methylbenzoyl)-1H-indol-3yl)methylene]amino}-1,5-
dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (5g). Orange crystals; 48% yield; mp 154-
1
156°C; IR (KBr) 1664, 1642 (2C=O), 1603 (C=N) cm-1; H NMR (400 MHz,
DMSO-d6, δ = ppm) δ = 2.44 (s, 3H, p-methylbenzoyl CH3), 2.50 (s, 3H, CH3), 3.17
(s, 3H, N-CH3), 7.37-7.42 (m, 3H, indole H-5, H-6, phenyl H-4), 7.44-7.47 (m, 4H, p-
methylbenzoyl H-3, H-5, phenyl H-2, H-6), 7.53 (t, J = 8.4 Hz, 2H, phenyl H-3, H-
5), 7.73 (d, J = 8.00 Hz, 2H, p-methylbenzoyl H-2, H-6), 7.95 (s, 1H, N=CH), 8.28 (d,
J = 8.8 Hz, 1H, indole H-7), 8.58 (d, J = 8.8 Hz, 1H, indole H-4), 9.70 (s, 1H, indole
H-2); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 10.46 (CH3), 21.63 (p-
methylbenzoyl CH3), 35.88 (N-CH3), 116.18 (indole C-7), 117.59 (pyrazole C-4),
120.87 (indole C-3), 123.21 (indole C-5), 123.80 (phenyl C-4), 125.02 (phenyl C-2,
C-6), 126.04 (indole C-6), 127.34 (indole C-4), 127.94 (indole C-3a), 129.64 (phenyl
C-3, C-5), 129.82 (p-methylbenzoyl C-3, C-5), 129.99 (p-methylbenzoyl C-2, C-6),
131.06 (p-methylbenzoyl C-1), 132.76 (indole, C-2), 135.04 (phenyl C-1), 137.02
(indole C-7a), 143.25 (p-methylbenzoyl C-4), 150.62 (CH=N), 152.14 (pyrazole C-5),
160.09 (C=O), 168.64 (p-methylbenzoyl C=O); EIMS (m/z) 449 (M+1, 3.9%), 448
(M+., 12.6%). Anal. Calcd for C28H24N4O2: C, 74.98; H, 5.39; N, 12.49. Found: C,
75.11; H, 5.28; N, 12.62.
4.1.3.8.
4-{[(1-(p-Chlorobenzoyl)-1H-indol-3yl)methylene]amino}-1,5-dimethyl-2-
phenyl-1H-pyrazol-3(2H)-one (5h). Orange crystals; 43% yield; mp 202-204°C; IR
1
(KBr) 1685, 1644 (2C=O), 1588 (C=N) cm-1; H NMR (400 MHz, DMSO-d6, δ =
ppm) δ = 2.50 (s, 3H, CH3), 3.17 (s, 3H, N-CH3), 7.37-7.39 (m, 3H, indole H-5, H-6,
phenyl H-4), 7.46, 7.47 (dd, J = 4Hz, 2H, 7.6 Hz, phenyl H-2, H-6), 7.53 (t, 2H, J =
8.4 Hz, phenyl H-3, H-5), 7.67 (d, 2H, J = 6.4 Hz, p-chlorobenzoyl H-3, H-5), 7.85
(d, J = 6.4 Hz, 2H, p-chlorobenzoyl H-2, H-6), 7.98 (s, 1H, N=CH), 8.32, 8.34 (dd, J
= 1.2 Hz, 6.8 Hz, 1H, indole H-7), 8.59, 8.61 (dd, J = 1.2 Hz, 1H, 6.4 Hz, indole H-4),
9.70 (s, 1H, indole H-2); 13C NMR (100 MHz, DMSO-d6, δ = ppm) δ = 10.46 (CH3),
35.87 (N-CH3), 116.29 (indole C-7), 117.57 (pyrazole C-4), 121.27 (indole C-3),
123.28 (indole C-5), 125.00 (indole C-6), 125.22 (phenyl C-4), 126.17 (phenyl C-2,
C-6), 127.32 (indole C-4), 128.04 (indole C-3a), 129.37 (phenyl C-3, C-5), 129.63 (p-
chlorobenzoyl C-3, C-5), 131.69 (p-chlorobenzoyl C-2, C-6), 132.76 (indole, C-2),
132.80 (p-chlorobenzoyl C-1), 135.04 (phenyl C-1), 136.98 (indole C-7a), 137.59 (p-
chlorobenzoyl C-4), 150.53 (CH=N), 152.20 (pyrazole C-5), 160.06 (C=O), 167.77
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(p-chlorobenzoyl C=O); EIMS (m/z) 470 (M+2, 30.6%), 468 (M+., 100%). Anal.
Calcd for C27H21ClN4O2: C, 69.15; H, 4.51; N, 11.95. Found: C, 69.33; H, 4.58; N,
12.13.
4.2. Biological activity
4.2.1. In vitro anti-inflammatory activity:
ELISA of E-selectin was performed as described in a reported method [33]. Briefly, 1
× 104 HUVECs/well were seeded into 96-well plates and grown to confluence. 50 µM
of indole derivatives were added 30 min prior to application of 10 ng/ml TNFα for
another 4 h. The cells were then fixed and ELAM levels analysed by ELISA. At the
same time, the compounds were analysed by Calcein AM assay to monitor non-
specific substance toxicity.
4.2.2. In vitro cyclooxygenase (COX) inhibition assay
An enzyme immune assay (EIA) kit (Cayman Chemical, Ann Arbor, MI, USA) was
used to test the ability of the test compounds listed in Table 1 to inhibit ovine COX-1
and COX-2 (IC50 value, ꢀM) according to a reported method [34, 35].
4.2.3. In vitro 5-lipoxygenase (LOX) inhibition assay
The ability of the test compounds listed in Table 1 to inhibit 5-LOX (IC50 value, ꢀM)
was determined using an enzyme immune assay (EIA) kit (catalogue no 760709,
Cayman Chemical, Ann Arbor, MI, USA). Stock solutions were freshly prepared
before use and buffer solution used (0.1 M Tris HCl, PH, 7.4). 10 ꢀl of different
compounds were prepared in dissolved at least the amount of DMSO and diluted with
the stock solution concentrations of (0.001, 01, 1, 5, 10 ꢀM) in a final volume of 210
ꢀl. The IC50 of test compounds were determined according to the manufacturer's
instructions and according to reported methods. [34, 35]
4.2.4. In vitro cytotoxicity:
4.2.4.1. Cell culture
Stock solutions (10 mmol/L) of the tested compounds were prepared by dissolving
an appropriate quantity of each substance in dimethylsulfoxide (DMSO). Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine,
penicillin and streptomycin were purchased from Sigma (MO, USA). Calcein AM
was obtained from Molecular Probes (Life Technologies, CA, USA). The screening
cell lines (T-lymphoblastic leukemia cell line CEM, breast carcinoma cell line MCF7,
cervical carcinoma cell line HeLa and human fibroblasts BJ) were obtained from the
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American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured
in DMEM medium (Sigma, MO, USA), supplemented with 10% fetal bovine serum,
L-glutamine (2 mmol/l), penicillin (100 U) and streptomycin (100 µg/ml). The cell
lines were maintained under standard cell culture conditions at 37 ºC and 5% CO2 in a
humid environment. Cells were subcultured twice or three times a week using the
standard trypsinization procedure.
4.2.4.2. Calcein AM assay
Suspensions of tested cell lines (ca. 1.0 × 105 cells/ml) were placed in 96-well
microtiter plates and after 24 h of stabilization (time zero) the tested compounds were
added (in three 20 µl aliquots) in serially diluted concentrations in dimethylsulfoxide
(DMSO). Control cultures were treated with DMSO alone. The final concentration of
DMSO in the incubation mixtures never exceeded 0.6%. The test compounds were
typically evaluated at six 3-fold dilutions and the highest final concentration was
generally 50 µM. After 72 h incubation, Calcein AM solution (100 µl, Molecular
Probes, Life Technologies, CA, USA) was added, and incubation was continued for
another hour. The fluorescence of viable cells was then quantified using a Fluoroskan
Ascent instrument (Labsystems, Finland). The percentage of surviving cells in each
well was calculated by dividing the intensity of the fluorescence signals from the
exposed wells by the intensity of signals from control wells and multiplying by 100.
These ratios were then used to construct dose-response curves from which IC50
values, the concentrations of the respective compounds that were lethal to 50% of the
tumor cells, were calculated.
4.3. Docking study
Docking was performed to obtain prediction of conformation and energy
ranking between COX2 receptor (PDB ID: 4PH9) and 5-LOX receptor (PDBID:
3V99) and the designed set of molecules. The docking studies were carried out using
AutoDock Vina 1.05 [16]. All 3D structures of ligands were obtained with Marvin
5.10.3, software which can be used for drawing, displaying and characterization of
chemical structure, substructures and reactions. Polar hydrogens were added to all
ligands and proteins with the AutoDock Tools (ADT) [36] program prior to docking
with Autodock Vina program [37]. A grid box 25 Å in size was centered on the active
site of protein. The exhaustiveness parameter was set to 20 (default 8). After docking,
we compared the docked ligand with Ibuprofen (IBP) crystal-like poses of COX2
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nature ligand and arachidonic Acid (ACD) nature ligand of 5-LOX receptor and the
best crystal-like poses of each ligand were analyzed.
The active site of the COX-2 receptor is a bent narrow gap with two important
polar amino acids around the active site. The first amino acid is ARG121 which is
located deeper in the active site. This amino acid plays an important role in the
interaction between receptor and nature ligand IBP. The second amino acid is HIS90
located at the entrance to the active site and this amino acid is important for new
designed compounds (Fig. 5). The 5-LOX active site is a long curved tunnel with a
key polar amino acid ARG182 near the surface of receptor (Fig. 5).
[Please, insert Fig. 5 about here]
Acknowledgments
V.B. and L.R. acknowledge the support of the Student Project IGA_PrF_2016_028 of
Palacky University and Czech Ministry of Education grant from the National Program
for Sustainability I (LO1204). We thank Professor Jitka Ulrichová for the kind gift of
HUVEC cells and Olga Hustáková for excellent technical assistance.
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Tables, Figures and Schemes Legends:
Table 1: In vitro COX-1, COX-2 and 5- LOX inhibition, and selectivity index (SI)
data for 5a-h and the reference drugs celecoxib and quercetin.
Table 2: IC50 (ꢀM) values obtained from Calcein AM assays on three tested cancer
cell lines and normal human fibroblasts; means ±SD obtained from three independent
experiments performed in triplicate.
Table 3: Resulting binding free energies for best poses.
Figure 1: Chemical structures of some traditional non-selective NSAIDs, some
selective cyclooxygenase-2 (COX-2) inhibitor drugs, 5-LOX inhibitor, and dual
COX/5-LOX inhibitory agent
Figure 2: Chemical structures of the traditional NSAID indomethacin (3), dual
COX/5-LOX inhibitor darbufelone (8) and the designed indomethacin analogues 4a-l
and 5a-h.
Figure 3a, b: E-Selectin (ELAM) expression in TNFα -induced HUVECs compared
to cytotoxicity determined by Calcein AM. Curcumin (10 µM) was used as a positive
control. 3a – 4l was tested at 50 µM.
Figure 4: Compound 5h around active site of 5-LOX receptor. It is possible to
observe the binding using h-bond to Arg182.
Figure 5: Left 2D image is active site of COX2 receptor with nature ligand IBP and
collection of amino acid 4Å around. Right image is 5-LOX receptor with ACD ligand
and amino acids 4Å around.
Scheme 1: Synthesis of the target compounds 4a-l and 5a-h.
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