A multicomponent reaction to design antimalarial pyridyl-indole derivatives: Synthesis, biological activities and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2020.103673

Using fragment-based design strategy, new pyridyl-indole hybrids 4a–y and indole intermediates 3a–e were synthesized using multicomponent one pot reaction. The synthesized compounds were subjected to screening for antimalarial activity against chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum. Several compounds exhibited antimalarial activity with IC₅₀ values in the range of 1.47–9.23 μM, and 1.16–7.66 μM, for D6 and W2 strains, respectively. Compounds 4a, 4k and 4u showed the highest selectivity index among all the tested compounds (S.I. ranged 3.8–10). Binding interactions between the active antimalarial compounds and the active site of quadruple mutant Plasmodium falciparum dihydrofolate reductase enzyme have been investigated using molecular docking analysis.

Full text of DOI:10.1016/j.bioorg.2020.103673

Journal Pre-proofs
A multicomponent reaction to design antimalarial pyridyl-indole derivatives:
Synthesis, biological activities and molecular docking
Heba A.H. Elshemy, Mohamed A. Zaki, Enas I. Mohamed, Shabana I. Khan,
Phoebe F. Lamie
PII:
S0045-2068(19)32015-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103673
YBIOO 103673
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
23 November 2019
18 January 2020
15 February 2020
Please cite this article as: H.A.H. Elshemy, M.A. Zaki, E.I. Mohamed, S.I. Khan, P.F. Lamie, A multicomponent
reaction to design antimalarial pyridyl-indole derivatives: Synthesis, biological activities and molecular docking,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103673
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A multicomponent reaction to design antimalarial pyridyl-indole derivatives: Synthesis,
biological activities and molecular docking
Heba A. H. Elshemy1, Mohamed A. Zaki2, Enas I. Mohamed2, Shabana I. Khan3, Phoebe F.
Lamie1,*
1Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Beni-Suef University,
Beni-Suef, 62514, Egypt
2Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 62514,
Egypt
3National Center for Natural Products Research, School of Pharmacy, The University of
Mississippi, University, MS, 38677, USA
* Corresponding author. E-mail addresses: feby.farag@yahoo.com
Abstract
Using fragment-based design strategy, new pyridyl-indole hybrids 4a-y and indole intermediates
3a-e were synthesized using multicomponent one pot reaction. The synthesized compounds were
subjected to screening for antimalarial activity against chloroquine sensitive (D6) and chloroquine
resistant (W2) strains of Plasmodium falciparum. Several compounds exhibited antimalarial
activity with IC50 values in the range of 1.47-9.23 μM, and 1.16-7.66 μM, for D6 and W2 strains,
respectively. Compounds 4a, 4k and 4u showed the highest selectivity index among all the tested
compounds (S.I. ranged 3.8-10). Binding interactions between the active antimalarial compounds
and the active site of quadruple mutant Plasmodium falciparum dihydrofolate reductase enzyme
have been investigated using molecular docking analysis.
Keywords: Anticancer, Antimalarial, Indole, Pyridine, Docking.
1

1. Introduction
Malaria is a fatal disease of the tropical and subtropical regions, causing death of about 0.8-1.2
million people annually [1]. Until now, approved commercial malaria vaccine is not available.
Artemisinin combination therapies (ACTs) and other available drugs are less effective due to the
resistance of parasite [2-4]. This excruciating problem of malaria treatment leads the researchers
to search for more effective and potent drugs with potential antimalarial activity.
Indole skeleton has emerged as one of the most valuable sources of synthons for therapeutic drugs
[5-8]. It is for example a vital part of the amino acid tryptophan and the neurotransmitter serotonin.
It was reported that indole nucleus possesses a wide range of biological activities such as
anticancer [9, 10], antimalarial [11], antimicrobial [12], analgesic [13], cardiovascular [14],
antihypertensive [15] antidiabetic [16], anti-inflammatory [17-19] and anti-HIV activities [20].
Furthermore, the substitution at 3-position of the indole ring with another heterocyclic ring was
found to furnish potent agents particularly with anticancer [21, 22], antimalarial [23-25], anti-
pyretic, hypoglycemic, analgesic and anti-inflammatory activities [26, 27].
On the other hand, the pyridine scaffold is the core structure of several synthetically and naturally
bioactive compounds [28-30] with wide range of biological applications such as anticancer [31]
and antimalarial [32, 33].
Recently, multi-component reactions (MCRs) have appeared as a great tool for the synthesis of
bioactive chemotherapeutics leading to interesting scaffolds that are mainly valuable for the design
of ‘drug-like’ compounds [34-37].
It was imagined that the combination of indole and pyridine moieties would produce molecular
hybrids, which might be developed as lead antimalarial agents (Figure 1). Motivated by the
aforementioned facts, herein we reported the synthesis and antimalarial activity of 4,6-diaryl-2-
(1H-indol-3-yl)-3-cyanopyridine series 4a-y. Additionally, the binding interactions of the
antimalarial compounds to quadruple mutant Plasmodium falciparum dihydrofolate reductase
enzyme were studied through molecular docking analysis.
[Please, insert Figure 1 about here]
2. Results and discussion
2.1. Chemistry
Generally, in this work, the indole nucleus was introduced at position 2 of pyridine ring with
different aryl groups at 4- and 6-positions using multi-component reactions.
2

Reaction of 3-cyanoacetyl indole (2) prepared according to the procedure described by Slatt et al
[38] with the respective aryl aldehyde gave 3a-e. The latter compounds were reacted with aromatic
ketones and ammonium acetate in glacial acetic acid and catalytic amount of I2 to afford target
compounds 4a-y in 61-83% yields, Scheme 1.
[Please, insert Scheme 1 about here]
The structures of target compounds 4a-y were confirmed by IR, 1H NMR, 13C NMR (DEPT-Q),
mass spectra and elemental analyses. IR spectra of 4a-y showed absorptions at 3128-3409 and
2202-2361 cm-1 for the indolic NH and C≡N, respectively. 1H NMR spectra for 4a-y revealed the
presence of singlet signal at δ 7.6-8.3 ppm attributed to pyridine H-5, also, indolic NH proton (D2O
exchangeable) appeared at δ 8.6-8.9 ppm and 11.8-11.8 ppm according to the solvent used (CDCl3
or DMSO, respectively). Construction of pyridine ring was confirmed by 13C NMR spectroscopy
showing signals at δ 157.8-163.2, 100.0-111.6, 150.6-157.4, 116.0-121.2 and 153.2-15 ppm due
to C-2 to C-6, sequentially.
The mass spectra exemplified for compounds 4n and 4y, displayed molecular ion peak (M+.) at
m/z = 441 (83.53%) and 539 (93.14%), respectively. Moreover, compound 4x showed molecular
ion peak at 495 as a base peak.
According to the data obtained, a suggested mechanism to explain the formation of 3-cyano-2-
(1H-indol-3-yl)pyridine derivatives 4a-y is proposed [39] (Figure 2). Firstly, 3-(cyanoacetyl)
indole (2) condensed with different aryl aldehydes to give 3a-e. The latter reacts with enol form
of the appropriate acetophenone derivative to form an intermediate A/A'. Then, ammonium acetate
reacts with the true carbonyl group in A/A' to give B/B' which by elimination of water affords the
Hantzsch 1,4-dihydropyridine derivative C. By the action of iodine – as an oxidizing agent – the
latter undergoes dehydrogenation to give the aromatized products 4a-y.
[Please, insert Figure 2 about here]
2.2. Biological activity
2.2.1. Antimalarial activity
The antimalarial activities of two series of compounds 3a-e and 4a-y were evaluated against
chloroquine sensitive (D6) and chloroquine resistant (W2) strains of P. falciparum. Both
chloroquine and artemisinin were included as reference drugs. The results for the most active
derivatives are represented in Table 1, in terms of IC50 values (μM) against D6 and W2 strains.
The compounds were also evaluated for in vitro cytotoxic activity against a mammalian kidney
3

cell line (VERO) and their IC50 values were determined. Selectivity index (S.I) of antimalarial
activity for all tested compounds was calculated from the equation, IC50 for VERO cells/IC50 for
P. falciparum. An S.I. value of >1 indicates that the antimalarial activity is stronger than the
cytotoxic activity. Cytotoxic activity of the compounds was determined in the same concentration
range as the antimalarial activity (4760 to 528.9 ng/mL).
The most active compounds against P. falciparum D6 and W2 strains showed IC50 ranged from
1.47-9.23 μM, and 1.16-7.66 μM, respectively.
In series 1, from all indole derivatives 3a-e, only trimethoxyphenyl derivative 3e showed activity
9.23 and 7.66 μM against D6 and W2, respectively.
Whereas in series 2 (compounds 4a-y), concerning D6 strain, the most potent compounds were 4a,
4k, and 4u with IC50 values equal to 1.47-1.60 μM, with high selectivity index values of 6.4-8.3.
Three other compounds 4w, 4o and 4m were found to have IC50 in the range of 5.40-6.78 μM, but
selectivity index values were lower (1.5-1.8). While, for W2 strain, 4k was the most active
derivative with IC50 of 1.16 μM having high selectivity index value of 10. Compounds 4u, 4w and
4a had nearly equal IC50 values (2.70, 2.96 and 2.99 μM, respectively) and S.I. value (3.8, 3.4 and
4.1, sequentially). The least active compounds were 4m and 4o with IC50 = 3.82 and 4.77 μM, and
S.I. 2.9 and 2, sequentially. Finally, we concluded that pyridyl-indole derivatives (series 2) had
antimalarial activity better than indole derivatives (series 1). Compounds 4a, 4k and 4u showed
antimalarial activity on both chloroquine sensitive and resistant strains D6 and W2. The common
feature in these analogues were presence of 4-flurophenyl, 3,4-difluorophenyl or 3,4,5-
trimethoxyphenyl ring at C-4 of pyridine ring together with unsubstituted phenyl ring at C-2
pyridine scaffold in their structures.
[Please, insert Table 1 about here]
2.2.2.Docking study
Dihydrofolate reductase (DHFR) is an essential enzyme for DNA formation in Plasmodium
falciparum through de novo biosynthesis of folate, a molecule which transfers carbons to enzymes
that need them for reactions. One of these enzymes is thymidylate synthase (TS) which uses these
carbons to form thymine bases [40]. In this context, the vitality of the organism is dependent on
this metabolic pathway. The effectiveness of DHFR-inhibitor drugs is now weakened due to
enzyme mutations [41]. Therefore, we thought to screen the active antimalarial compounds with
quadruple mutant Pf DHFR-TS using molecular docking analysis. The protozoan DHFR and TS
4

coexist together forming single chain bi functional enzyme; unlike prokaryotes and higher
eukaryotes where DHFR and TS exist as separate enzymes [42].
Docking of compounds 3e, 4a, 4k, 4o, 4m, and 4u to quadruple mutant Pf DHFR revealed the
ligands-protein interactions and the residues involved in these combinations.
Although the DHFR of P. falciparum shares sequence similarity to some extent with those of other
species, but there are differences that are enough for preferential inhibition [41]. The active site
region contains the following residues; ASP54, CYS15, ILE14, LEU164, ASN108, PHE58,
PRO113, ILE112 and MET55 [43]. Docking analysis of the active compounds revealed certain
interactions with amino acids in the binding pocket that playing a role in stabilizing the compounds
conformations. One of these amino acids is ASP54, a crucial residue for binding of both inhibitors
and substrates [44], where there is hydrogen bonding between one of the carbonyl side chains of
ASP54 and an aromatic hydrogen of the indole ring of the active compounds. The compounds are
oriented in such a way to avoid steric clash with ASN108 side chain which is reported to be
responsible of the resistance of P. falciparum to pyrimethamine and cycloguanil [41].
Additionally, hydrophobic interactions (π- π stackings) with PHE58 and PHE116 (4k, 4a, 4o, 4m,
4u) were observed. Moreover, formation of hydrogen bonding between NH of the indole ring and
backbone carbonyl group of LEU164 (4k, 4a, 4o, 4m) and hydrogen bonding with SER111 (4k,
4a, 4m) and ILE14 (4k, 4a, 4o, 4m, 3e), Figure 3.
It is notable that the most active antimalarial compounds 4k and 4a share all the mentioned
interactions with the active site amino acid residues, indicating strong protein-ligand binding
which may contribute to their higher antimalarial activity. Collectively, the molecular insight
presented by the docking study may explain the antimalarial activity of these compounds and
contribute to future design of more active antimalarials.
For docking scores, see Table in supporting information.
[Please, insert Figure 3 about here]
5

3. Conclusion
In summary, we have synthesized two series of indole derivatives, the intermediates 3a-e and the
newly synthesized pyridyl-indole target compounds 4a-y.
All the synthesized compounds were evaluated for antimalarial activity against chloroquine
sensitive strain (D6) and chloroquine resistant strain (W2) of P. falciparum. The most potent
antimalarial activities against chloroquine-sensitive D6 and chloroquine-resistant W2 strains were
obtained from compounds 3e, 4a, 4k, 4m, 4o, 4u and 4w, with IC50 values ranged from 1.47-9.23
μM for D6 and 1.16-7.66 μM for W2. S.I. values were found to be in the range of 1.47-8.3 and
1.7-10 for D6 and W2, respectively. Compounds 4a, 4k and 4u showed antimalarial activity on
both chloroquine sensitive and resistance strains D6 and W2. The common feature in these
analogues was presence of 4-flurophenyl, 3,4-difluorophenyl or 3,4,5-trimethoxyphenyl ring at C-
4 of pyridine ring together with unsubstituted phenyl ring at C-2 pyridine scaffold in their
structures.
Molecular docking of the active antimalarial compounds 4a, 4k, 4m and 4o into the active site of
quadruple mutant Pf DHFR-TS binding cavities indicated potential binding interactions which
could be a possible mechanism contributing to the antimalarial activity of these compounds.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined using a Griffin apparatus and are uncorrected. IR spectra were
recorded on a Shimadzu IR-435 spectrophotometer using KBr discs and values are represented in
cm-1. 1H NMR and 13C NMR were carried out on Bruker apparatus at 400 MHz for 1H NMR and
100 MHz for 13C NMR (DEPT-Q) 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 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. Mass spectra (MS) were recorded
on a Water’s Micromass ZQ 4000 mass spectrometer using the electrospray (ES) ionization mode.
6

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 and solvents were purchased from the Aldrich Chemical Company
(Milwaukee, WI) and were used without further purification.
4.1.2. General procedure for synthesis of compounds 4a-y:
A mixture of 3a-e (1 mmol), the appropriate acetophenone derivative (1 mmol), ammonium acetate
(5g, 0.064 mol) and catalytic amount of iodine in glacial acetic acid (2mL) was refluxed at 120 oC
for 10-12 h. After the completion of the reaction (monitored by TLC), it was poured onto water
and extracted with ethyl acetate. The organic layer was evaporated to dryness. The crude product
was crystallized from methanol/chloroform mixture (2:1) to give compounds 4a-y.
4.1.2.1. 4-(p-Flurophenyl)-2-(1H-indol-3-yl)-6-phenylnicotinonitrile (4a)
Yellow solid; yield 63%; mp 211-213 °C; IR (KBr) 3311 (NH), 2213 (C≡N) cm-1; 1H NMR
(DMSO-d6) δ 7.2-7.2 (m, 2H, indole H-5, H-6), 7.4 (t, 2H, J = 8.4 Hz, phenyl H-3, H-5), 7.5-7.6
(m, 4H, indole H-7, p-flurophenyl H-3, H-5, phenyl H-4), 7.9 (dd, 2H, J = 2.8 Hz, 8.4 Hz, p-
flurophenyl H-2, H-6), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 6.8 Hz, phenyl H-2, H-6), 8.3-8.4
13
(m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ 101.9
(pyridine C-3), 112.5 (indole C-7), 113.4 (indole C-3), 116.0 (p-fluorophenyl C-3, C-5), 116.2
(pyridine C-5), 119.3 (C≡N), 121.2 (indole C-5), 121.8 (indole C-4), 122.8 (indole C-6), 126.5
(indole C-3a), 127.9 (indole C-2), 129.2 (phenyl C-2, C-6), 129.5 (phenyl C-4), 130.9 (phenyl C-
3, C-5), 133.7 (p-fluorophenyl C-2, C-6), 133.7 (p-fluorophenyl C-1), 136.8 (indole C-7a), 138.0
(phenyl C-1), 154.5 (pyridine C-4), 157.8 (pyridine C-6), 158.7 (pyridine C-2), 164.7 (p-
fluorophenyl C-4); EIMS (m/z) 390 (M+1, 4.8%), 389 (M+., 9.9%), 304 (100%); Anal. Calcd for
C26H16FN3: C, 80.19; H, 4.14; N, 10.79. Found: C, 79. 97; H, 4.22; N, 10.81
4.1.2.2. 4-(p-Flurophenyl)-2-(1H-indol-3-yl)-6-(p-tolyl)nicotinonitrile (4b)
1
Yellow solid; yield 62%; mp 180-182 °C; IR (KBr) 3303 (NH), 2214 (C≡N) cm-1; H NMR
(DMSO-d6) δ 2.4 (s, 3H, CH3), 7.1-7.2 (m, 2H, indole H-5, H-6), 7.3 (d, 2H, J = 8.4 Hz, p-tolyl
H-3, H-5), 7.4 (dd, 2H, J = 2.0 Hz, 8.8 Hz, p-flurophenyl H-3, H-5), 7.5 (d, 1H, J = 7.2 Hz, indole
7

H-7), 7.8 (dd, 2H, J = 2.0 Hz, 8.8 Hz, p-flurophenyl H-2, H-6), 7.9 (s, 1H, pyridine H-5), 8.2 (d,
2H, J = 8.4 Hz, p-tolyl H-2, H-6), 8.3-8.4 (m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH, D2O
13
exchangeable); C NMR (DMSO-d6) δ 21.4 (CH3), 101.5 (pyridine C-3), 112.5 (indole C-7),
113.4 (indole C-3), 116.0 (p-fluorophenyl C-3, C-5), 116.2 (pyridine C-5), 116.4 119.40 (C≡N),
121.2 (indole C-5), 121.7 (indole C-4), 122.8 (indole C-6), 126.5 (indole C-3a), 127.8 (indole C-
2), 129.1 (p-tolyl C-2, C-6), 130.1 (p-fluorophenyl C-2, C-6), 130.8 (p-tolyl C-3, C-5), 133.7 (p-
tolyl C-4), 135.2 (p-fluorophenyl C-1), 136.8 (p-tolyl C-1), 140.9 (indole C-7a), 154.4 (pyridine
C-4), 157.7 (pyridine C-6), 158.7 (pyridine C-2), 164.7 (p-fluorophenyl C-4); EIMS (m/z) 404
(M+1, 2.8%), 403 (M+., 10.8%), 245 (100%); Anal. Calcd for C27H18FN3: C, 80.38; H, 4.50; N,
10.42. Found: C, 80.46; H, 4.53; N, 10.27
4.1.2.3. 4,6-Bis(p-flurophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4c)
1
Yellow solid; yield 61%; mp 243-245 °C; IR (KBr) 3175 (NH), 2218 (C≡N) cm-1; H NMR
(DMSO-d6) δ 7.2-7.2 (m, 2H, indole H-5, H-6), 7.4-7.5 (m, 4H, p-flurophenyl-4-yl H-3, H-5, p-
flurophenyl-6-yl H-3, H-5), 7.5 (d, 1H, J = 8.4 Hz, indole H-7), 7.9 (dd, 2H, J = 3.2 Hz, 8.4 Hz,
p-flurophenyl-4-yl H-2, H-6), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 6 Hz, p-flurophenyl-6-yl
H-2, H-6), 8.4 (dd, 1H, J = 3.2 Hz, J = 8.4 Hz, indole H-4), 8.5 (s, 1H, indole H-2), 11.8 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 101.9 (pyridine C-3), 112.6 (indole C-7), 113.3
(indole C-3), 116.3 (p-fluorophenyl-6-yl C-3, C-5), 116.3 (p-fluorophenyl-4-yl C-3, C-5), 116.6
(pyridine C-5), 119.2 (C≡N), 121.4 (indole C-5), 121.6 (indole C-4), 123.0 (indole C-6), 126.3
(indole C-3a), 129.1 (indole C-2), 130.2 (p-fluorophenyl-6-yl C-2, C-6), 131.7 (p-fluorophenyl-
4-yl C-2, C-6), 134.3 (p-fluorophenyl-6-yl C-1), 136.7 (p-fluorophenyl-4-yl C-1), 137.8 (indole
C-7a), 154.6 (pyridine C-4), 157.8 (pyridine C-6), 157.8 (p-fluorophenyl-6-yl C-4), 162.1 (p-
fluorophenyl-4-yl C-4), 163.2 (pyridine C-2); Anal. Calcd for C26H15F2N3: C, 76.65; H, 3.71; N,
10.31. Found: C, 76.55; H, 3.62; N, 10.11
4.1.2.4. 6-(p-Clorophenyl)-4-(p-fluorophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4d)
1
Pale yellow solid; yield 65%; mp 240-242 °C; IR (KBr) 3167 (NH), 2245 (C≡N) cm-1; H NMR
(CDCl3) δ 7.2-7.2 (m, 3H, p-flurophenyl H-3, H-5, indole H-6), 7.3-7.4 (m, 3H, p-flurophenyl H-
2, H-6, indole H-5), 7.5 (dd, 2H, J = 3.6 Hz, 7.6 Hz, p-chlorophenyl H-3, H-5), 8.0-8.1 (m, 3H,
p-chlorophenyl H-2, H-6, indole H-7), 8.3 (s, 1H, pyridine H-5), 8.4 (dd, 1H, J = 3.2 Hz, 8.8 Hz,
8

13
indole H-4), 8.5 (s, 1H, indole H-2), 8.7 (s, 1H, NH, D2O exchangeable); C NMR (CDCl3) δ
111.6 (pyridine C-3), 112.9 (indole C-7), 113.9 (indole C-3), 116.7 (p-fluorophenyl C-3, C-5),
116.9 (pyridine C-5), 118.0 (C≡N), 121.8 (indole C-5), 122.9 (indole C-4), 124.0 (indole C-6),
126.5 (indole C-3a), 128.8 (indole C-2), 129.6 (p-chlorophenyl C-4), 133.4 (p-chlorophenyl C-2,
C-6), 133.5 (p-chlorophenyl C-3, C-5), 134.9 (p-fluorophenyl C-1), 136.6 (p-fluorophenyl C-2, C-
6), 137.0 (p-chlorophenyl C-1), 137.1 (indole C-7a), 154.3 (pyridine C-4), 156.0 (pyridine C-6),
163.2 (pyridine C-2), 165.7 (p-fluorophenyl C-4); Anal. Calcd for C26H15ClFN3: C, 73.67; H,
3.57; N, 9.91. Found: C, 73.54; H, 3.59; N, 10.01
4.1.2.5. 6-(p-Bromophenyl)-4-(p-fluorophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4e)
Pale yellow solid; yield 63%; mp 234-236 °C; IR (KBr) 3341 (NH), 2203 (C≡N) cm-1; 1H NMR
(DMSO-d6) δ 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4 (dd, 2H, J = 2.0 Hz, 8.8 Hz, p-flurophenyl H-
3, H-5), 7.5 (d, 1H, J = 8.8 Hz, indole H-7), 7.7 (d, 2H, J = 8.8 Hz, p-bromophenyl H-3, H-5), 7.8
(dd, 2H, J = 2.0 Hz, 8.8 Hz, p-flurophenyl H-2, H-6), 7.9 (s, 1H, pyridine H-5), 8.2 (d, 2H, J = 8.8
Hz, p-bromophenyl H2, H-6), 8.3-8.4 (m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 103.6 (pyridine C-3), 110.7 (indole C-7), 110.7 (indole C-
3), 116.2 (p-fluorophenyl C-3, C-5), 119.6 (pyridine C-5), 120.0 (C≡N), 121.6 (indole C-5), 122.1
(indole C-4), 123.4 (indole C-6), 123.6 (p-fluorophenyl C-2, C-6), 126.3 (p-bromophenyl C-4),
126.4 (indoleC-3a), 129.8 (indole C-2), 130.6 (p-bromophenyl C-2, C-6), 131.2 (p-bromophenyl
C-3, C-5), 133.9 (p-fluorophenyl C-1), 134.2 (p-bromophenyl C-1), 136.2 (indole C-7a), 154.4
(pyridine C-4), 157.9 (pyridine C-6), 159.7 (pyridine C-2), 163.3 (p-fluorophenyl C-4); EIMS
(m/z) 468 (M+2, 8.7%), 467 (M+1, 37.0%), 466 (M+., 9.9%), 424 (100%); Anal. Calcd for
C26H15BrFN3: C, 66.68; H, 3.23; N, 8.97. Found: C, 66.53; H, 3.17; N, 9.01
4.1.2.6. 2-(1H-Indol-3-yl)-4-(p-methoxyphenyl)-6-phenylnicotinonitrile (4f)
1
Yellow solid; yield 65%; mp 242-244 °C; IR (KBr) 3356 (NH), 2207 (C≡N) cm-1; H NMR
(DMSO-d6) δ 3.9 (s, 3H, OCH3), 7.1 (d, 2H, J = 8.4 Hz, p-methoxyphenyl H-3, H-5), 7.2-7.3 (m,
2H, indole H-5, H-6), 7.5-7.6 (m, 4H, indole H-7, phenyl H-3, H-4, H-5), 7.8 (d, 2H, J = 8.4 Hz,
p-methoxyphenyl H-2, H-6), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 7.6 Hz, phenyl H-2, H-6),
8.3-8.4 (m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ
55.8 (OCH3), 101.7 (pyridine C-3), 112.5 (indole C-7), 113.5 (indole C-3), 114.6 (p-
9

methoxyphenyl C-3, C-5), 116.6 (pyridine C-5), 119.6 (C≡N), 121.2 (indole C-5), 121.8 (indole
C-4), 122.8 (indole C-6), 126.5 (indole C-3a), 127.9 (indole C-2), 129.1 (phenyl C-2, C-6), 129.4
(p-methoxyphenyl C-1), 129.4 (phenyl C-3, C-5), 130.8 (phenyl C-4), 130.9 (p-methoxyphenyl
C-2, C-6), 136.8 (indole C-7a), 138.1 (phenyl C-1), 155.1 (pyridine C-4), 157.9 (pyridine C-6),
158.5 (pyridine C-2), 161.0 (p-methoxyphenyl C-4); Anal. Calcd for C27H19N3O: C, 80.78; H,
4.77; N, 10.47. Found: C, 80.59; H, 4.81; N, 10.35.
4.1.2.7. 2-(1H-Indol-3-yl)-4-(p-methoxyphenyl)-6-(p-tolyl)nicotinonitrile (4g)
Pale yellow solid; yield 71%; mp 219-221 °C; IR (KBr) 3352 (NH), 2210 (C≡N) cm-1; 1H NMR
(DMSO-d6) δ 2.4 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 7.1 (d, 2H, J = 8.8 Hz, p-methoxyphenyl H-3,
H-5), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4 (d, 2H, J = 8.0 Hz, p-tolyl H-3, H-5), 7.5 (d, 1H, J =
8.4 Hz, indole H-7), 7.7 (d, 2H, J = 8.8 Hz, p-methoxyphenyl H-2, H-6), 7.8 (s, 1H, pyridine H-
5), 8.2 (d, 2H, J = 8.0 Hz, p-tolyl H-2, H-6), 8.3-8.4 (m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6) δ 21.3 (CH3), 55.8 (OCH3), 101.3 (pyridine C-3), 112.5
(indole C-7), 113.5 (indole C-3), 114.6 (p-methoxyphenyl C-3, C-5), 116.2 (pyridine C-5), 119.7
(C≡N), 121.2 (indole C-5), 121.7 (indole C-4), 122.8 (indole C-6), 126.5 (indole C-3a), 127.8 (p-
tolyl C-2, C-6), 129.0 (indole C-2), 129.4 (p-tolyl C-4), 130.1 (p-tolyl C-3, C-5), 130.9 (p-
methoxyphenyl C-2, C-6), 135.3 (p-methoxyphenyl C-1), 136.7 (p-tolyl C-1), 140.8 (indole C-7a),
155.0 (pyridine C-4), 157.9 (pyridine C-6), 158.6 (p-methoxyphenyl C-4), 160.9 (pyridine C-2);
Anal. Calcd for C28H21N3O: C, 80.94; H, 5.09; N, 10.11. Found: C, 81.07; H, 5.17; N, 10.01.
4.1.2.8. 6-(p-Flurophenyl)-2-(1H-indol-3-yl)-4-(p-methoxyphenyl)nicotinonitrile (4h)
1
Yellow solid; yield 67%; mp 206-208 °C; IR (KBr) 3325 (NH), 2218 (C≡N) cm-1; H NMR
(CDCl3) δ 3.9 (s, 3H, OCH3), 7.1 (d, 2H, J = 8.0 Hz, p-methoxyphenyl H-3, H-5), 7.2-7.3 (m, 4H,
indole H-5, H-6, p-flurophenyl H-3, H-5), 7.4 (d, 1H, J = 8.0 Hz, indole H-7), 7.6 (s, 1H, pyridine
H-5), 7.7 (d, 2H, J = 8.0 Hz, p-methoxyphenyl H-2, H-6), 8.2 (d, 2H, J = 7.6 Hz, p-flurophenyl
H-2, H-6), 8.3 (s, 1H, indole H-2), 8.4 (d, 1H, J = 7.6 Hz, indole H-4), 8.9 (s, 1H, NH, D2O
exchangeable); 13C NMR (CDCl3) δ 55.4 (OCH3), 101.8 (pyridine C-3), 110.7 (indole C-7), 114.4
(p-methoxyphenyl C-3, C-5), 114.6 (indole C-3), 116.1 (p-fluorophenyl C-3, C-5), 116.2 (pyridine
C-5), 119.7 (C≡N), 121.5 (indole C-5), 123.2 (indole C-4), 123.4 (indole C-6), 126.4 (C-3a), 127.5
(indole C-2), 129.3 (p-methoxyphenyl C-1), 129.4 (p-fluorophenyl C-2, C-6), 130.1 (p-
10

methoxyphenyl C-2, C-6), 134.3 (p-fluorophenyl C-1), 136.2 (indole C-7a), 155.3 (pyridine C-4),
157.8 (pyridine C-6), 160.9 (p-methoxyphenyl C-4), 163.0 (pyridine C-2), 165.5 (p-fluorophenyl
C-4); Anal. Calcd for C27H18FN3O: C, 77.31; H, 4.33; N, 10.02. Found: C, 77.26; H, 4.19; N,
9.92.
4.1.2.9. 6-(p-Clorophenyl)-2-(1H-indole-3-yl)-4-(p-methoxyphenyl)nicotinonitrile (4i)
1
White solid; yield 69%; mp 183-185 °C; IR (KBr) 3329 (NH), 2218 (C≡N) cm-1; H NMR
(CDCl3) δ 3.9 (s, 3H, OCH3), 7.1 (d, 2H, J = 7.2 Hz, p-methoxyphenyl H-3, H-5), 7.3-7.4 (m, 2H,
indole H-5, H-6), 7.5 (d, 2H, J = 7.2 Hz, p-methoxyphenyl H-2, H-6), 7.6 (d, 1H, J = 8.0 Hz,
indole H-7), 7.7 (s, 1H, pyridine H-5), 7.8 (d, 2H, J = 8.4 Hz, p-chlorophenyl H-3, H-5), 8.1 (d,
2H, J = 8.4 Hz, p-chlorophenyl H-2, H-6), 8.4 (s, 1H, indole H-2), 8.5 (d, 1H, J = 8.8 Hz, indole
H-4), 8.7 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 55.5 (OCH3), 103.6 (pyridine
C-3), 114.5 (indole C-7), 114.6 (p-methoxyphenyl C-3, C-5), 114.6 (indole C-3), 116.1 (C≡N),
119.8 (pyridine C-5), 121.7 (indole C-5), 123.3 (indole C-4), 123.6 (C-3a), 124.0 (indole C-6),
127.6 (indole C-2), 128.7 (p-methoxyphenyl C-2, C-6), 129.1 (p-chlorophenyl C-2, C-6), 129.4
(p-fluorophenyl C-2, C-6), 130.1 (p-methoxyphenyl C-2, C-6), 130.2 (p-chlorophenyl C-3, C-5),
130.3 (p-methoxyphenyl C-1), 133.5 (p-chlorophenyl C-4), 135.8 (indole C-7a), 137.8 (p-
chorophenyl C-1), 156.8 (pyridine C-4), 157.3 (pyridine C-6), 160.2 (p-methoxyphenyl C-4),
161.3 (pyridine C-2); EIMS (m/z) 437 (M+2, 3.4%), 435 (M+., 1.2%). Anal.Calcd for
C27H18ClN3O: C, 74.39; H, 4.16; N, 9.64. Found: C, 74.48; H, 4.23; N, 9.53.
4.1.2.10. 6-(p-Bromophenyl)-2-(1H-indole-3-yl)-4-(p-methoxyphenyl)nicotinonitrile (4j)
Pale yellow solid; yield 71%; mp 199-201 °C; IR (KBr) 3341 (NH), 2210 (C≡N) cm-1; 1H NMR
(DMSO-d6) δ 3.8 (s, 3H, OCH3), 7.1 (d, 2H, J = 8.4 Hz, p-methoxyphenyl H-3, H-5), 7.2-7.3 (m,
2H, indole H-5, H-6), 7.5 (d, 1H, J = 7.2 Hz, indole H-7), 7.7-7.8 (m, 4H, p-methoxyphenyl H-2,
H-6, p-bromophenyl H-3, H-5), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 8.4 Hz, p-bromophenyl
H-2, H-6), 8.3 (d, 1H, J = 7.6 Hz, indole H-4), 8.4 (s, 1H, indole H-2), 11.8 (s, 1H, NH, D2O
13
exchangeable); C NMR (DMSO-d6) δ 56.2 (OCH3), 102.1 (pyridine C-3), 112.1 (indole C-7),
113.1 (p-methoxyphenyl C-3, C-5), 113.3 (indole C-3), 116.7 (pyridine C-5), 119.6 (C≡N), 121.3
(indole C-5), 121.7 (indole C-4), 122.3 (indole C-6), 122.8 (indole C-2), 124.6 (indole C-3a), 126.5
(p-bromophenyl C-4), 129.2 (p-methoxyphenyl C-2, C-6), 129.3 (p-methoxyphenyl C-1), 129.9
11

(p-bromophenyl C-2, C-6), 132.4 (p-bromophenyl C-3, C-5), 136.8 (p-bromophenyl C-1), 137.3
(indole C-7a), 150.6 (pyridine C-4), 155.4 (pyridine C-6), 157.4 (p-methoxyphenyl C-4), 157.9
(pyridine C-2); Anal. Calcd for C27H18BrN3O: C, 67.51; H, 3.78; N, 8.75. Found: C, 67.42; H,
3.91; N, 8.68.
4.1.2.11. 4-(3,4-Diflurophenyl)-2-(1H-indol-3-yl)-6-phenylnicotinonitrile (4k)
Pale yellow solid; yield 65%; mp 291-293 °C; IR (KBr) 3314 (NH), 2217 (C≡N) cm-1; 1H NMR
(DMSO-d6) δ 7.2-7.3 (m, 2H, indole H-5, H-6), 7.5-7.6 (m, 4H, indole H-7, phenyl H-3, H-4, H-
5),7.7-7.8 (m, 2H,difluorophenyl H-5, H-6), 8.0 (s, 1H, difluorophenyl H-2), 8.0 (s, 1H, pyridine
H-5), 8.3-8.4 (m, 4H, indole H-2, H-4, phenyl H-2, H-6), 11.8 (s, 1H, NH, D2O exchangeable);
13C NMR (DMSO-d6) δ 101.8 (pyridine C-3), 112.6 (indole C-7), 113.0 (indole C-3), 116.8
(pyridine C-5), 118.3 (difluorophenyl C-5), 118.9 (difluorophenyl C-2), 119.1 (C≡N), 121.3
(indole C-5), 121.7 (indole C-4), 122.9 (indole C-6), 126.4 (indole C-3a), 127.4 (indole C-2), 127.9
(difluorophenyl C-6), 129.2 (phenyl C-2, C-6), 129.5 (phenyl C-4), 131.0 (phenyl C-3, C-5), 134.5
(difluorophenyl C-1), 136.8 (indole C-7a), 137.8 (phenyl C-1), 151.0 (difluorophenyl C-4), 152.0
(difluorophenyl C-3), 152.1 (pyridine C-4), 153.2 (pyridine C-6), 157.8 (pyridine C-2); Anal.
Calcd for C26H15F2N3: C, 76.65; H, 3.71; N, 10.31. Found: C, 76.47; H, 3.84; N, 10.11.
4.1.2.12. 4-(3,4-Diflurophenyl)-2-(1H-indol-3-yl)-6-(p-tolyl)nicotinonitrile (4l)
1
Yellow solid; yield 77%; mp 242-242 °C; IR (KBr) 3298 (NH), 2217 (C≡N) cm-1; H NMR
(CDCl3) δ 2.4 (s, 3H, CH3), 7.1 (s, 1H, diflurophenyl H-2), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4
(d, 2H, J = 8.4 Hz, p-methylphenyl H-3, H-5), 7.5-7.6 (m, 3H,difluorophenyl H-5, H-6, indole H-
7), 7.7 (s, 1H, pyridine H-5), 8.1 (d, 2H, J = 8.4 Hz, p-methylphenyl H-2, H-6), 8.3 (s, 1H, indole
H-2), 8.6 (dd, 1H, J = 3.6 Hz, 5.6 Hz, indole H-4), 8.7 (s, 1H, NH, D2O exchangeable); 13C NMR
(CDCl3) δ 21.4 (CH3), 101.8 (pyridine C-3), 111.4 (indole C-7), 114.5 (indole C-3), 116.0
(pyridine C-5), 117.9 (difluorophenyl C-5), 118.1 (difluorophenyl C-2), 118.1 (indole C-5), 118.8
(C≡N), 121.7 (indole C-4), 122.4 (indole C-6), 123.3 (p-tolyl C-2, C-6), 125.3 (diflurophenyl C-
6), 126.4 (indole C-3a), 127.6 (indole C-2), 129.8 (p-tolyl C-3, C-5), 131.0 (p-tolyl C-4), 134.9
(difluorophenyl C-1), 136.2 (p-tolyl C-1), 141.0 (indole C-7a), 151.0 (difluorophenyl C-4), 152.0
(difluorophenyl C-3), 152.1 (pyridine C-4), 157.7 (pyridine C-6), 159.4 (pyridine C-2); Anal.
Calcd for C27H17F2N3: C, 76.95; H, 4.07; N, 9.97. Found: C, 76.79; H, 3.91; N, 9.82.
12

4.1.2.13. 4-(3,4-Diflurophenyl)-6-(p-flurophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4m)
Buff solid; yield 74%; mp 282-284 °C; IR (KBr) 3309 (NH), 2218 (C≡N) cm-1; 1H NMR (DMSO-
d6) δ 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4 (dd, 2H, J = 3.2 Hz, J = 8.8 Hz, p-fluorophenyl H-3,
H-5), 7.5 (d, 1H, J = 7.2 Hz, indole H-7),7.6-7.7 (m, 2H,difluorophenylH-5, H-6), 8.0 (s, 1H,
difluorophenylH-2), 8.1 (s, 1H, pyridine H-5), 8.3-8.4 (m, 2H, indole H-2, H-4), 8.5 (dd, 2H, J =
3.2 Hz, J = 8.8 Hz, p-fluorophenyl H-2, H-6), 11.8 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) δ 102.0 (pyridine C-3), 112.4 (indole C-7), 114.1 (indole C-3), 116.9 (pyridine C-5),
117.8 (difluorophenyl C-5), 118.0 (difluorophenyl C-2), 118.9 (p-fluorophenyl C-3, C-5), 119.1
(indole C-5), 119.2 (C≡N), 120.6 (indole C-4), 123.4 (indole C-6), 125.3 (diflurophenyl C-6),
126.7 (indole C-3a), 128.1 (indole C-2), 130.3 (p-fluorophenyl C-2, C-6), 134.1 (p-flurophenyl C-
1), 135.8 (difluorophenyl C-1), 137.8 (indole C-7a), 151.0 (difluorophenyl C-4), 152.1
(difluorophenyl C-3), 153.2 (pyridine C-4), 157.6 (pyridine C-6), 161.0 (p-flurophenyl C-4), 162.0
(pyridine C-2); Anal. Calcd for C26H14F3N3: C, 73.41; H, 3.32; N, 9.88. Found: C, 73.42; H, 3.27;
N, 9.86.
4.1.2.14. 6-(p-Clorophenyl)-4-(3,4-diflurophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4n)
1
Yellow solid; yield 76%; mp 264-266 °C; IR (KBr) 3341 (NH), 2361 (C≡N) cm-1; H NMR
(CDCl3) δ 7.1-7.2 (m, 2H, indole H-5, H-6), 7.2-7.3 (m, 2H, difluorophenyl H-5, H-6), 7.3-7.4 (m,
2H, difluorophenyl H-2,indole H-7), 7.5 (d, 2H, J = 8.8 Hz, p-chlorophenyl H-3, H-5), 7.6 (s, 1H,
pyridine H-5), 8.1 (d, 2H, J = 8.8 Hz, p-chlorophenyl H-2, H-6), 8.4 (s, 1H, indole H-2), 8.5 (dd,
1H, J = 2.4 Hz, J = 5.6 Hz, indole H-4), 8.6 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3) δ
101.9 (pyridine C-3), 111.5 (indole C-7), 114.9 (indole C-3), 116.1 (pyridine C-5), 118.0
(difluorophenyl C-5), 118.2 (difluorophenyl C-2), 118.3 (indole C-5), 118.5 (C≡N), 121.8 (indole
C-4), 122.2 (indole C-6), 123.5 (diflurophenyl C-6), 126.2 (indole C-3a), 127.8 (indole C-2), 128.8
(p-chlorophenyl C-2, C-6), 129.3 (p-chlorophenyl C-3, C-5), 134.4 (p-chlorophenyl C-4), 136.1
(indole C-7a), 136.7 (difluorophenyl C-1), 139.0 (p-chlorophenyl C-1), 153.1 (difluorophenyl C-
4), 154.0 (difluorophenyl C-3), 155.0 (pyridine C-4), 157.9 (pyridine C-6), 158.2 (pyridine C-2);
EIMS (m/z) 443 (M+2, 30.30%), 441 (M+., 83.53%), 440 (100%). Anal.Calcd for C26H14ClF2N3:
C, 70.67; H, 3.19; N, 9.51. Found: C, 70.54; H, 3.07; N, 9.58.
4.1.2.15. 6-(p-Bromophenyl)-4-(3,4-diflurophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4o)
13

1
Yellow solid; yield 81%; mp 246-248 °C; IR (KBr) 3409 (NH), 2215 (C≡N) cm-1; H NMR
(CDCl3) δ 7.3-7.4 (m, 4H, difluorophenyl H-5, H-6, indole H-5, H-6), 7.4-7.5 (m, 2H,
difluorophenyl H-2,indole H-7), 7.6 (s, 1H, pyridine H-5), 7.7 (d, 2H, J = 8.0 Hz, p-bromophenyl
H-3, H-5), 8.1 (d, 2H, J = 8.0 Hz, p-bromophenyl H-2, H-6), 8.4 (s, 1H, indole H-2), 8.5 (dd, 1H,
13
J = 3.6 Hz, J = 5.2 Hz, indole H-4), 8.6 (s, 1H, NH, D2O exchangeable); C NMR (CDCl3) δ
101.9 (pyridine C-3), 111.5 (indole C-7), 114.4 (indole C-3), 116.0 (pyridine C-5), 118.0
(difluorophenyl C-5), 118.1 (difluorophenyl C-2), 118.5 (C≡N), 119.6 (indole C-5), 121.8 (indole
C-4), 122.2 (indole C-6), 123.4 (diflurophenyl C-6), 123.6 (p-bromophenyl C-4), 125.2 (indole C-
3a), 126.3 (indole C-2), 129.0 (p-bromophenyl C-2, C-6), 132.3 (p-bromophenyl C-3, C-5), 133.4
(diflurophenyl C-1), 136.2 (indole C-7a), 136.6 (p-bromophenyl C-1), 148.1 (difluorophenyl C-
4), 149.0 (difluorophenyl C-3), 153.5 (pyridine C-4), 157.9 (pyridine C-6), 158.2 (pyridine C-2);
EIMS (m/z) 487 (M+2, 64.25%), 485 (M+., 66.56%), 202 (100%). Anal.Calcd for C26H14BrF2N3:
C, 64.21; H, 2.90; N, 8.64. Found: C, 64.42; H, 3.02; N, 8.75.
4.1.2.16. 4-(3,4-Dimethoxyphenyl)-2-(1H-indol-3-yl)-6-phenylnicotinonitrile (4P)
1
Yellow solid; yield 71%; mp 264-266 °C; IR (KBr) 3367 (NH), 2207 (C≡N) cm-1; H NMR
(DMSO-d6) δ 3.8 (s, 3H, OCH3), 3. 9 (s, 3H, OCH3), 7.1 (d, 1H, J = 8.4 Hz, 3,4-dimethoxyphenyl
H-5), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4 (dd, 1H, J = 2.0 Hz, 8.4 Hz, 3,4-dimethoxyphenyl
H-6), 7.4 (d, 1H, J = 2.0 Hz, 3,4-dimethoxyphenyl H-2), 7.5-7.6 (m, 4H, indole H-7, phenyl H-3,
H-4, H-5), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 8.4 Hz, phenyl H-2, H-6), 8.4-8.5 (m, 2H,
indole H-2, H-4), 11.8 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 56.1 (OCH3), 56.2
(OCH3), 101.8 (pyridine C-3), 112.1 (indole C-7), 112.5 (dimethoxyphenyl C-5), 113.1
(dimethoxyphenyl C-2), 113.5 (indole C-3), 116.7 (pyridine C-5), 119.7 (C≡N), 121.2 (indole C-
5), 121.7 (indole C-4), 122.2 (indole C-6), 122.8 (dimethoxyphenyl C-6), 126.5 (C-3a), 127.9
(indole C-2), 129.1 (phenyl C-2, C-6), 129.4 (phenyl C-4), 130.8 (phenyl C-3, C-5), 134.0
(dimethoxyphenyl C-1), 136.8 (indole C-7a), 138.1 (phenyl C-1), 149.1 (dimethoxyphenyl C-4),
150.6 (dimethoxyphenyl C-3), 155.3 (pyridine C-4), 157.9 (pyridine C-6), 158.5 (pyridine C-2);
EIMS (m/z) 432 (M+1, 3.9%), 431 (M+., 7.2%), 164 (100%); Anal. Calcd for C28H21N3O2: C,
77.94; H, 4.91; N, 9.74. Found: C, 77.79; H, 5.03; N, 9.58
4.1.2.17. 4-(3,4-Dimethoxyphenyl)-2-(1H-indol-3-yl)-6-(p-tolyl)nicotinonitrile (4q)
14

Yellow solid; yield 73%; mp 195-197 °C; IR (KBr) 3341 (NH), 2202 (C≡N) cm-1; 1H NMR
(CDCl3) δ 2.4 (s, 3H, CH3), 4.0 (s, 3H, OCH3), 4.1 (s, 3H, OCH3), 7.0 (d, 1H, J = 8.4 Hz,
dimethoxyphenyl H-5), 7.1 (s, 1H, dimethoxyphenyl H-2), 7.2-7.3 (m, 3H, indole H-5, H-6,
dimethoxyphenyl H-6), 7.4 (d, 2H, J = 8.0 Hz, p-tolyl H-3, H-5), 7.5 (dd, 1H, J = 2.8 Hz, 6 Hz,
indole H-7), 7.6 (s, 1H, pyridine H-5), 8.1 (d, 2H, J = 8.0 Hz, p-tolyl H-2, H-6), 8.3 (s, 1H, indole
H-2), 8.5 (dd, 1H, J = 3.2 Hz, 6.4 Hz, indole H-4), 8.7 (s, 1H, NH, D2O exchangeable); 13C NMR
(CDCl3) δ 21.5 (CH3), 55.5 (2OCH3), 103.5 (pyridine C-3), 111.8 (indole C-7), 114.5
(dimethoxyphenyl C-5), 114.6 (dimethoxyphenyl C-2), 115.0 (indole C-3), 117.0 (pyridine C-5),
118.6 (C≡N), 121.6 (indole C-5), 121.7 (indole C-4), 123.2 (indole C-6), 126.1 (C-3a), 128.0
(dimethoxyphenyl C-6), 128.7 (indole C-2), 129.8 (p-tolyl C-4), 130.2 (p-tolyl C-2, C-6), 130.3
(p-tolyl C-3, C-5), 134.9 (dimethoxyphenyl C-1), 136.3 (indole C-7a), 141.4 (p-tolyl C-1), 150.1
(dimethoxyphenyl C-4), 153.0 (dimethoxyphenyl C-3), 156.9 (pyridine C-4), 158.2 (pyridine C-
6), 161.0 (pyridine C-2); EIMS (m/z) 446 (M+1, 11.3%), 445 (M+., 34.5%), 231 (100%); Anal.
Calcd for C29H23N3O2: C, 78.18; H, 5.20; N, 9.43. Found: C, 78.35; H, 5.17; N, 9.46
4.1.2.18. 4-(3,4-Dimethoxyphenyl)-6-(p-flurophenyl)-2-(1H-indol-3-yl)nicotinonitrile (4r)
1
Yellow solid; yield 69%; mp 205-207 °C; IR (KBr) 3345 (NH), 2203 (C≡N) cm-1; H NMR
(CDCl3) δ 4.0 (s, 3H, OCH3), 4.1 (s, 3H, OCH3), 7.0 (d, 1H, J = 8.0 Hz, dimethoxyphenyl H-5),
7.2 (s, 1H, dimethoxyphenyl H-2), 7.3-7.4 (m, 2H, indole H-5, H-6), 7.5 (d, 1H, 8 Hz,
dimethoxyphenyl H-6), 7.5-7.6 (m, 3H, p-flurophenyl H3, H-5, indole H-7), 7.7 (s, 1H, pyridine
H-5), 8.2 (d, 2H, J = 7.2 Hz, p-flurophenyl H2, H-6), 8.3 (s, 1H, indole H-2), 8.5 (dd, 1H, J = 3.6
Hz, 8.8 Hz, indole H-4), 8.7 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 55.8 (OCH3),
56.2 (OCH3), 103.4 (pyridine C-3), 109.9 (indole C-7), 111.4 (dimethoxyphenyl C-5), 111.8
(dimethoxyphenyl C-2), 112.3 (indole C-3), 116.2 (pyridine C-5), 117.8 (C≡N), 121.0 (indole C-
5), 121.7 (indole C-4), 122.2 (p-fluorophenyl C-3, C-5), 123.3 (indole C-6), 125.7 (C-3a), 127.5
(dimethoxyphenyl C-6), 128.5 (indole C-2), 129.8 (p-fluorophenyl C-2, C-6), 129.9 (p-
fluorophenyl C-1), 135.2 (dimethoxyphenyl C-1), 136.6 (indole C-7a), 149.3 (dimethoxyphenyl
C-4), 151.6 (dimethoxyphenyl C-3), 154.8 (pyridine C-4), 156.2 (pyridine C-6), 160.3 (pyridine
C-2), 169.1 (p-fluorophenyl C-4); Anal. Calcd for C28H20FN3O2: C, 74.82; H, 4.48; N, 9.35.
Found: C, 74.67; H, 4.29; N, 9.14
4.1.2.19. 6-(p-Clorophenyl)-4-(3,4-dimethoxyphenyl)-2-(1H-indol-3-yl)nicotinonitrile(4s)
15

Pale yellow solid; yield 75%; mp 245-247 °C; IR (KBr) 3128 (NH), 2210 (C≡N) cm-1; 1H NMR
(DMSO, d6) δ 3.8 (s, 3H, OCH3), 3.9 (s, 3H, OCH3), 7.1-7.2 (m, 3H, dimethoxyphenyl H-5, indole
H-5, H-6), 7.3-7.4 (m, 2H, dimethoxyphenyl H-2, H-6), 7.5 (d, 1H, J = 7.2 Hz, indole H-7), 7.6
(d, 2H, J = 8.4 Hz, p-chlorophenyl H-3, H-5), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 1H, J = 8 Hz,
indole H-4), 8.4 (d, 2H, J = 8.4 Hz, p-chlorophenyl H-2, H-6), 8.5 (s, 1H, indole H-2), 11.8 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 56.1 (OCH3), 56.25 (OCH3), 103.5 (pyridine C-
3), 112.1 (indole C-7), 112.5 (dimethoxyphenyl C-5), 113.2 (dimethoxyphenyl C-2), 113.4 (indole
C-3), 116.8 (pyridine C-5), 119.6 (C≡N), 121.2 (indole C-5), 121.7 (indole C-4), 122.3 (indole C-
6), 122.8 (dimethoxyphenyl C-6), 126.5 (C-3a), 129.2 (indole C-2), 129.3 (dimethoxy C-1), 129.5
(p-chlorophenyl C-2, C-6), 129.7 (p-chlorophenyl C-3, C-5), 135.7 (p-chlorophenyl C-4), 136.8
(indole C-7a), 137.0 (p-chlorophenyl C-1), 149.1 (dimethoxyphenyl C-4), 150.6
(dimethoxyphenyl C-3), 155.5 (pyridine C-4), 157.2 (pyridine C-6), 157.9 (pyridine C-2); Anal.
Calcd for C28H20ClN3O2: C, 72.18; H, 4.33; N, 9.02. Found: C, 71.97; H, 4.42; N, 9.15
4.1.2.20. 6-(p-Bromophenyl)-4-(3,4-dimethoxyphenyl)-2-(1H-indol-3-yl)nicotinonitrile(4t)
1
Yellow solid; yield 79%; mp 212-214 °C; IR (KBr) 3337 (NH), 2207 (C≡N) cm-1; H NMR
(DMSO-d6) δ 3.8 (s, 3H, OCH3), 3.9 (s, 3H, OCH3), 7.1 (d, 2H, J = 8.4 Hz, dimethoxyphenyl H-
5), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4 (d, 1H, J = 8.4 Hz, dimethoxyphenyl H-6),7.5 (s, 1H,
dimethoxyphenyl H-2), 7.6 (d, 1H, J = 7.2 Hz, indole H-7), 7.8 (d, 2H, J = 8.4 Hz, p-bromophenyl
H-3, H-5), 7.9 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 8.4 Hz, p-bromophenyl H-2, H-6), 8.4 (d,
13
1H, J = 7.6 Hz, indole H-4), 8.5 (s, 1H, indole H-2), 11.8 (s, 1H, NH, D2O exchangeable); C
NMR (DMSO-d6) δ 55.8 (OCH3), 56.1 (OCH3), 102.0 (pyridine C-3), 112.5 (indole C-7), 113.0
(indole C-3), 114.6 (dimethoxyphenyl C-5), 115.2 (dimethoxyphenyl C-2), 116.6 (pyridine C-5),
119.5 (C≡N), 121.3 (indole C-5), 121.7 (indole C-4), 121.8 (indole C-6), 124.6 (dimethoxyphenyl
C-6), 124.6 (p-bromophenyl C-4), 129.2 (indole C-2), 129.2 (indole C-3a), 129.9 (p-bromophenyl
C-3, C-5), 131.0 (dimethoxyphenyl C-1), 132.4 (p-bromophenyl C-2, C-6), 136.8 (indole C-7a),
137.3 (p-bromophenyl C-1), 155.3 (dimethoxyphenyl C-4), 156.0 (dimethoxyphenyl C-3), 157.4
(pyridine C-4), 158.0 (pyridine C-6), 161.0 (pyridine C-2); Anal. Calcd for C28H20BrN3O2: C,
65.89; H, 3.95; N, 8.23. Found: C, 65.99; H, 3.82; N, 8.17
4.1.2.21. 2-(1H-Indol-3-yl)-6-phenyl-4-(3,4,5-trimethoxyphenyl)nicotinonitrile (4u)
16

Buff solid; yield 68%; mp 192-194 °C; IR (KBr) 3330 (NH), 2212 (C≡N) cm-1; 1H NMR (DMSO-
d6) δ 3.7 (s, 3H, p-OCH3), 3.8 (s, 6H, 2 m-OCH3), 7.1 (s, 2H, trimethoxyphenyl H-2, H-6), 7.2-7.3
(m, 2H, indole H-5, H-6), 7.5-7.6 (m, 4H, indole H-7, phenyl H-3, H-4, H-5), 8.0 (s, 1H, pyridine
H-5), 8.3 (d, 2H, J = 7.2 Hz, phenyl H-2, H-6), 8.4-8.5 (m, 2H indole H-2, H-4), 11.8 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6) δ 56.7 (2m-OCH3), 60.6 (p-OCH3), 100.0 (pyridine C-
3), 102.0 (trimethoxyphenyl C-2, C-6), 107.2 (indole C-7), 113.4 (indole C-3), 116.8 (pyridine C-
5), 119.5 (C≡N), 121.2 (indole C-5), 122.5 (indole C-4), 123.3 (indole C-6), 126.3 (C-3a), 127.9
(phenyl C-2, C-6), 129.2 (indole C-2), 129.5 (phenyl C-3, C-5), 130.9 (phenyl C-4), 136.8
(trimethoxy C-1), 137.0 (indole C-7a), 138.1 (phenyl C-1), 139.0 (trimethoxyphenyl C-4), 153.3
(trimethoxyphenyl C-3, C-5), 155.4 (pyridine C-4), 157.8 (pyridine C-6), 158.6 (pyridine C-2);
Anal. Calcd for C29H23N3O3: C, 75.47; H, 5.02; N, 9.10. Found: C, 75.29; H, 4.88; N, 9.17
4.1.2.22. 2-(1H-Indol-3-yl)-6-(p-tolyl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile (4v)
1
White solid; yield 76%; mp 265-267 °C; IR (KBr) 3340 (NH), 2207 (C≡N) cm-1; H NMR
(DMSO-d6) δ 2.4 (s, 3H, CH3), 3.7 (s, 3H, p-OCH3), 3.9 (s, 6H, 2 m-OCH3), 7.1 (s, 2H,
trimethoxyphenyl H-2, H-6), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.4 (d, 2H, J = 8.0 Hz, p-tolyl H-
3, H-5), 7.5 (d, 1H, J = 7.6 Hz, indole H-7), 7.9 (s, 1H, pyridine H-5), 8.2 (d, 2H, J = 8 Hz, p-tolyl
13
H-2, H-6), 8.3-8.4 (m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH, D2O exchangeable); C NMR
(DMSO-d6) δ 21.4 (CH3), 56.6 (m-OCH3), 60.6 (p-OCH3), 101.6 (pyridine C-3), 107.1
(trimethoxyphenyl C-2, C-6), 112.5 (indole C-7), 113.4 (indole C-3), 116.4 (pyridine C-5), 119.6
(C≡N), 121.2 (indole C-5), 121.7 (indole C-4), 122.8 (indole C-6), 126.5 (indole C-3a), 127.9 (p-
tolyl C-2, C-6), 129.1 (indole C-2), 130.1 (p-tolyl C-3, C-5), 132.6 (p-tolyl C-4), 135.3
(trimethoxyphenyl C-1), 136.7 (indole C-7a), 139.0 (p-tolyl C-1), 140.8 (trimethoxyphenyl C-4),
153.3 (trimethoxyphenyl C-3, C-5), 155.3 (pyridine C-4), 157.8 (pyridine C-6), 158.6 (pyridine C-
2); Anal. Calcd for C30H25N3O3: C, 75.77; H, 5.30; N, 8.84. Found: C, 75.91; H, 5.47; N, 9.09
4.1.2.24. 6-(p-Flurophenyl)-2-(1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile (4w)
1
Yellow solid; yield 73%; mp 273-275 °C; IR (KBr) 3358 (NH), 2213 (C≡N) cm-1; H NMR
(CDCl3) δ 3.9 (s, 9H, 3OCH3), 6.9 (s, 2H, trimethoxyphenyl H-2, H-6), 7.2-7.3 (m, 4H, p-
flurophenyl H-3, H-5, indole H-5, H-6), 7.4 (d, 1H, J = 8.4 Hz, indole H-7), 7.6 (s, 1H, pyridine
H-5), 8.2 (d, 2H, J = 7.2 Hz, p-flurophenyl H-2, H-6), 8.4 (s, 1H, indole H-2), 8.5 (d, 1H, J = 8.4
17

13
Hz, indole H-4), 8.7 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ 56.4 (2m-OCH3),
61.0 (p-OCH3), 102.1 (pyridine C-3), 106.1 (trimethoxyphenyl C-2, C-6), 111.6 (indole C-7),
113.9 (indole C-3), 116.2 (pyridine C-5), 116.3 (p-fluorophenyl C-3, C-5), 118.9 (C≡N), 121.6
(indole C-5), 122.0 (indole C-4), 123.3 (indole C-6), 126.3 (indole C-3a), 127.9 (indole C-2), 129.7
(p-fluorophenyl C-2, C-6), 132.2 (trimethoxy C-1), 133.7 (p-fluorophenyl C-1), 136.2 (indole C-
7a), 139.5 (trimethoxyphenyl C-4), 153.5 (trimethoxyphenyl C-3, C-5), 156.0 (pyridine C-4),
157.6 (pyridine C-6), 163.1 (pyridine C-2), 165.6 (p-fluorophenyl C-4); Anal. Calcd for
C29H22FN3O3: C, 72.64; H, 4.62; N, 8.76. Found: C, 72.62; H, 4.71; N, 8.58
4.1.2.25. 6-(p-Clorophenyl)-2-(1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile (4x)
1
Off-white solid; yield 79%; mp 268-270 °C; IR (KBr) 3326 (NH), 2209 (C≡N) cm-1; H NMR
(DMSO-d6) δ 3.7 (s, 3H, p-OCH3), 3.9 (s, 6H, 2 m-OCH3), 7.1 (s, 2H, trimethoxyphenyl H-2, H-
6), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.5 (d, 1H, J = 8.0 Hz, indole H-7), 7.6 (d, 2H, J = 8.4 Hz, p-
chlorophenyl H-3, H-5), 8.0 (s, 1H, pyridine H-5), 8.3-8.4 (m, 4H, p-chlorophenyl H-2, H-6,
indole H-2, H-4), 11.8 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 56.6 (2m-OCH3),
60.6 (p-OCH3), 102.2 (pyridine C-3), 107.1 (trimethoxyphenyl C-2, C-6), 112.5 (indole C-7),
113.3 (indole C-3), 116.8 (pyridine C-5), 119.5 (C≡N), 121.4 (indole C-5), 121.6 (indole C-4),
122.9 (indole C-6), 126.4 (C-3a), 129.2 (indole C-2), 129.5 (p-chlorophenyl C-2, C-6), 129.7 (p-
chlorophenyl C-3, C-5), 132.4 (trimethoxy C-1), 135.8 (p-chlorophenyl C-4), 136.7 (p-
chlorophenyl C-1), 136.8 (indole C-7a), 139.0 (trimethoxyphenyl C-4), 153.3 (trimethoxyphenyl
C-3, C-5), 155.5 (pyridine C-4), 157.4 (pyridine C-6), 157.9 (pyridine C-2); EIMS (m/z) 497 (M+2,
39.20 %), 495 (M+., 100%). Anal.Calcd for C29H22ClN3O3: C, 70.23; H, 4.47; N, 8.47. Found: C,
70.26; H, 4.39; N, 8.52
4.1.2.26. 6-(p-Bromophenyl)-2-(1H-indol-3-yl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile (4y)
1
Off-white solid; yield 83%; mp 281-283 °C; IR (KBr) 3327 (NH), 2208 (C≡N) cm-1; H NMR
(DMSO-d6) δ 3.7 (s, 3H, p-OCH3), 3.9 (s, 6H, 2 m-OCH3), 7.1 (s, 2H, trimethoxyphenyl H-2, H-
6), 7.2-7.3 (m, 2H, indole H-5, H-6), 7.5 (d, 1H, J = 7.6 Hz, indole H-7), 7.8 (d, 2H, J = 8.4 Hz, p-
bromophenyl H-3, H-5), 8.0 (s, 1H, pyridine H-5), 8.3 (d, 2H, J = 8.4 Hz, p-bromophenyl H-2, H-
6), 8.4-8.5 (m, 2H, indole H-2, H-4), 11.8 (s, 1H, NH, D2O exchangeable);13C NMR (DMSO-d6)
δ 56.6 (2m-OCH3), 60.6 (p-OCH3), 102.3 (pyridine C-3), 107.2 (trimethoxyphenyl C-2, C-6),
18

112.5 (indole C-7), 113.3 (indole C-3), 116.8 (pyridine C-5), 119.5 (C≡N), 121.3 (indole C-5),
121.6 (indole C-4), 122.8 (indole C-6), 124.7 (p-bromophenyl C-4), 126.4 (indole C-3a), 129.2
(indole C-2), 129.9 (p-bromophenyl C-2, C-6), 132.4 (trimethoxy C-1), 132.5 (p-bromophenyl C-
3, C-5), 136.8 (indole C-7a), 137.2 (p-bromophenyl C-1), 139.1 (trimethoxyphenyl C-4), 153.3
(trimethoxyphenyl C-3, C-5), 155.6 (pyridine C-4), 157.4 (pyridine C-6), 157.9 (pyridine C-2);
EIMS (m/z) 541 (M+2, 96.55%), 539 (M+., 93.14%), 164 (100%). Anal.Calcd for C29H22BrN3O3:
C, 64.45; H, 4.10; N, 7.78. Found: C, 64.38; H, 3.97; N, 7.74.
4.2. Biological evaluation
4.2.1. In vitro antimalarial and cytoxicity assays
Two strains of Plasmodium falciparum, D6 (chloroquine sensitive) and W2 (chloroquine-
resistant), were used to determine the in vitro antimalarial activity of the prepared target
compounds. They were obtained from the Division of Experimental Therapeutics, Walter Reed
Army Institute of Research (WRAIR), Washington, DC. The assay for determining antimalarial
activity is based on plasmodial lactate dehydrogenase (LDH) activity. In brief, a suspension of red
blood cells infected with D6 strain or W2 strain of P. falciparum (200 µL, with 2% parasitemia
and 2% hematocrit in RPMI 1640 medium supplemented with 10% human serum and 60 µg/ml
amikacin) was mixed with 10 µL of serially diluted test samples inside the wells of a 96-well plate.
Using a modular incubation chamber flushed with a gas mixture of 90% N2, 5% O2, and 5% CO2,
the plate was incubated for 72 h at 37°C. The procedure of Makler and Hinrichs [45] was applied
to determine Parasite LDH activity. The incubation mixture (20 µL) is mixed with Malstat reagent
(100 µL) and incubated for 30 min. at room temperature. Nitrobluetetrazolium and
phenazinethosulfate mixture (20 µL, 1:1 mixture) (Sigma, St. Louis, MO) was then added. The
plate is further incubated in the dark for 1 h. Then, 5% acetic acid (100 µL) was added and the
plate was read at 650 nm. The percent growth was plotted against test concentrations, and IC50
values were determined from dose-response curves. Drug controls used in this study were
artemisinin and chloroquine. Dimethyl sulfoxide (DMSO) was included as a vehicle control. In
order to determine the selectivity index (SI) of the antimalarial activity for test compounds, the in
vitro cytotoxicity to mammalian cells was also measured. Vero cells (monkey kidney fibroblasts
[American Type Culture Collection, Manassas, VA]) were seeded into the wells of 96-well plates
(25,000 cells/well) and incubated for 24 h. Different concentrations of test compounds were added,
19

and the plates were incubated again for 48 h. At the end of incubation, cell viability was determined
with WST-8 dye. The IC50 values were obtained from dose-response curves. Compounds which
failed to yield an IC50 over the concentration range tested (4760-528.9 ng/mL) were considered
not active.
4.3. Docking study
The crystal structure of quadruple mutant (N51I+C59R+S108N+I164L) Plasmodium falciparum
dihydrofolate reductase-thymidylate synthase (Pf DHFR-TS) (PDB ID: 1j3k) [41] was
downloaded from the protein data bank repository [40]. Protein structures were prepared by
Protein Preparation Wizard (PrepWizard) [46, 47] of the Schrӧdinger suite for adding missing
hydrogen atoms, assigning hydrogen bonds, adjusting bond orders, and relaxing the protein −
ligand complex. Filling the missing loops and amino acid side chains were accomplished using
Prime [48-50]. The center of the docking receptor grid was determined as the coordinate of cognate
ligand. The potential of nonpolar parts of the receptor was softened by scaling the van der Waals
radius by a factor of 1.0 and partial charge cutoff of 0.25. Rotation of the receptor thiol and
hydroxyl groups was allowed for optimal hydrogen bond formation with predicted docked poses.
Ligand preparation was performed using LigPrep [51] of Schrödinger by using the OPLS3 force
field [52]. Possible ligand states at pH 7.4 were generated by Epik [53], and ligand stereogenic
centers were retained during ligand preparation. Then, the compounds were docked using Glide
[54] SP (standard precision) mode, and the best five poses were kept for analysis.
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Figures, Schemes and Table captions
Figure 1. Examples of some potent natural and synthetic indole and pyridine containing
compounds as antimalarial agents and target compounds 4a-y.
Figure 2. A plausible mechanism for the formation of 4a-y.
Figure 3. Binding modes of compound 4k (A), 4a (B), 4m (C) and 4o (D) into quadruple mutant
Pf DHFR-TS binding cavities (PDB: 1j3k). Ligands are depicted in ball and stick model. The
residues potentially interacting with the ligands are shown in labelled tube models.
Scheme 1. Synthesis of target pyridyl-indole derivatives 4a-y; Reagents and conditions: i)
CNCH2COOH, Ac2O, 50oC, 30 min.; ii) ArCHO, TEA, abs. ethanol, reflux 4-8h.; iii) Ar'COCH3,
NH4OAc, I2, gl. acetic acid, 10-12h.
Table 1. In vitro antimalarial activity, selectivity index of most active indole derivatives 3e and
pyridyl-indole derivatives 4a, k, m, o, u, w and reference drugs chloroquine and artemisinin.
26

Conflict of interest:
Authors declare that there is no conflict of interest.
27

O
O
Ar
S
H
Ar'
H
N
O
H
O
N
N
N
NC
N
H
N
NH
N
N
N
O
O
N
H
H
Target compounds 4a-y
Pyridine thiosemicarbazide
Apicidin
The natural antimalarial product
synthetic potent antimalarial agent
Figure 1.
28

Ar
CN
O
O
CN
- H2O
O
Ar-CHO
+
Ar'
-O
Ar'
N
H
N
H
2
3a-e
O
O
Ar'
Ar'
Ar
Ar
O
HO
CN
CN
N
N
H
H
A
A'
NH4OAc
H2N
H2N
Ar'
Ar
Ar'
Ar
HO
O
CN
CN
N
N
H
H
B'
B
- H2O
Ar'
N
Ar'
H
Ar
Ar
CN
N
H
CN
- 2H
I2
N
H
N
H
C
4a-y
29