Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D QSAR-assisted design

Article abstract of DOI:10.1016/j.bmcl.2019.01.038

Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro screening of 2-,4,-6-, and/or 7-substituted quinoline derivatives as hDHODH inhibitors

Full text of DOI:10.1016/j.bmcl.2019.01.038

Accepted Manuscript
Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihy-
droorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D
QSAR-assisted design
Vivek K. Vyas, Gulamnizami Qureshi, Drashti Oza, Hardik Patel, Krupali
Parmar, Palak Patel, Manjunath D. Ghate
PII:
S0960-894X(19)30059-9
https://doi.org/10.1016/j.bmcl.2019.01.038
BMCL 26275
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 August 2018
22 January 2019
30 January 2019
Please cite this article as: Vyas, V.K., Qureshi, G., Oza, D., Patel, H., Parmar, K., Patel, P., Ghate, M.D., Synthesis
of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH)
inhibitors and anticancer agents: 3D QSAR-assisted design, Bioorganic & Medicinal Chemistry Letters (2019), doi:
https://doi.org/10.1016/j.bmcl.2019.01.038
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Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as
human dihydroorotate dehydrogenase (hDHODH) inhibitors and
anticancer agents: 3D QSAR-assisted design
Vivek K. Vyasa*, Gulamnizami Qureshia, Drashti Ozaa, Hardik Patelb, Krupali
Parmara, Palak Patela, Manjunath D. Ghatea
a
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University,
Ahmedabad 382481, Gujarat, India
b Institute of Science, Nirma University, Ahmedabad 382481, Gujarat, India
ABSTRACT
Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors
as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro
evaluation of 2-,4,-6-,and/or 7-substituted quinoline derivatives as hDHODH inhibitors
and anticancer agents. We have designed 2-,4,-6-,and/or 7-substituted quinoline
derivatives and predicted their hDHODH inhibitory activity based on 3D QSAR study on
45 substituted quinoline derivatives as hDHODH inhibitors, and also predicted toxicity.
Designed compounds were docked into the binding site of hDHODH. Designed
compounds which showed good predictive activity, no toxicity, and good docking score
were selected for the synthesis, and in vitro screening as hDHODH inhibitors in an
enzyme inhibition assay, and anticancer agents in MTT assay against cancer cell lines
(HT-29 and MDA-MB-231). Synthesized compounds 7 and 14 demonstrated IC50 value
of 1.56 µM and 1.22 µM, against hDHODH, respectively, and these are our lead
compounds for the development of new hDHODH inhibitors and anticancer agents.
Keywords: Human Dihydroorotate dehydrogenase (hDHODH); Substituted quinolines;
hDHODH inhibitors; Anticancer agents; 3D QSAR
*Corresponding author at: Department of Pharmaceutical Chemistry, Institute of
Pharmacy, Nirma University, Ahmedabad 382481, Gujarat, India
E-mail addresses: vivekvyas@nirmauni.ac.in, vicky_1744@yahoo.com (V.K. Vyas)
1

Flavoenzyme dihydroorotate dehydrogenase (DHODH) [EC 1.3.99.11] catalyzes
oxidation of dihydroorotate (DHO) to orotate (ORO), and reduction of flavin
mononucleotide (FMN) to dihydroflavin mononucleotide (FMNH2)1. This is a fourth step
in de novo pyrimidine biosynthesis. Pyrimidine bases are important precursors for the
biosynthesis of DNA, RNA, glycoproteins and phospholipids and required for the cellular
metabolism and cell growth 2. For DNA and RNA biosynthesis in cells, salvage pathway
or de novo synthesis provides nucleosides. Salvage pathway does not produce enough
nucleosides for survival of rapidly proliferating cells such as cancer cells and T-
lymphocytes, thus progression of tumor, proliferation of and replication of DNA are
sensitive to de novo pyrimidine biosynthesis inhibition, and makes them ideal targets for
the development of new drug candidates against cancer, rheumatoid arthritis and multiple
5
sclerosis 3,4. Brequinar
and leflunomide (Arava ®)6 are two examples of such
compounds
(Fig.1).
Brequinar
(6-fluoro-2-(2'-fluoro-[1,1'-biphenyl]-4-yl)-3-
methylquinoline-4-carboxylic acid) is an antitumor agent and leflunomide (5- methyl-N-[4-
(trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide) is a prodrug, which converts into
active its metabolite teriflunomide (Aubagio ®/A77 1726), and used as
immunosuppressive drug. Brequinar is a quinoline carboxylic acid derivative and a potent
inhibitor of hDHODH. Brequinar was developed as a potential anticancer agent in clinical
trials, however it was not approved as anticancer drug. Leflunomide is an isoxazole-based
6
drug, and it was reported on melanoma cells proliferation . Recently many small
molecules as inhibitors of DHODH are designed, synthesized and evaluated as
anticancer agents7, antimalarial agents, antiviral agents, antibacterials, and for the
treatment of immunological disorders. Combined in silico approaches were used for the
identification of these lead compounds as hDHODH inhibitors 8–11. Following our research
for the identification of hDHODH inhibitors, in the year 2014 12, we designed and
synthesized quinoline-2-carboxamide derivatives as hDHODH inhibitors and anticancer
agents. Comprehensive in silico methods both structure- and ligand-based were used for
the design of quinoline-2-carboxamide derivatives. Very recently, in the year 2017 13, we
have designed and synthesized 1,2,5-trisubstituted benzimidazole derivatives using liquid
phase combinatorial synthesis method as hDHODH inhibitors. In continuation of work for
the generation of new lead compounds as hDHODH inhibitors and anticancer agents, in
2

this work, we performed 3D QSAR study (CoMFA and CoMSIA models) on the
substituted quinoline derivatives as a computational design strategy for the design of
hDHODH inhibitors. Contour maps generated through CoMFA and CoMSIA analysis
were used for the selection of substituents on quinoline ring. In the designed compounds,
quinoline scaffold was maintained as a core structure and substituents which were found
significant with the contour maps analysis were selected. Furthermore, 3D QSAR models
helped in the prediction of the activity of designed compounds. In silico toxicities were
also predicted for designed compounds. Prior to their selection for the synthesis and
activity, designed compounds were docked in the hDHODH active site. Selected
designed compounds (good predicted activity and docking score) were synthesized as
2,4,-6-,and/or 7-substituted quinoline derivatives. Synthesized compounds were
screened for hDHODH inhibition activity in a colorimetric assay, and as antiproliferative
agents in MTT assay. Overall, a combined strategy was employed in this work, which
resulted in lead compounds, which were targeted against hDHODH enzyme and
anticancer agents.
3

Fig. 1. Human dihydroorotate dehydrogenase (hDHODH) inhibitors
12,14
3D QSAR was performed on reported hDHODH inhibitors
(Table S1 under
supplementary data). 3D QSAR models were validated on statistical parameters
calculated using PLS analysis implemented in sybyl x software. (Fig S1 and Table S2
under supplementary data). Align 3 (Distill) was selected as a best method of alignment
based on highest value of q2, r2cv, r2ncv, r2pred, SEE and F value in CoMFA and CoMSIA
models (Table S2 under supplementary data). An external test set of 9 compounds were
used for the external validation of models in which CoMFA and CoMSIA model showed
r2pred value of 0.823 and 0.782, respectively.
CoMFA and CoMSIA contour maps are demonstrated using template compound Tr7
inside the electrostatic, steric, hydrophobic, hydrogen bond donor (HBD) and hydrogen
bond acceptor (HBA) fields. CoMFA stearic map (Fig. S2A under supplementary data)
is shown in green coloured (contributed 80%) stearically favoured regions, and yellow
coloured (contributed 20%) stearically disfavoured areas. In steric contour map, two small
green coloured contour were present; one was above the –C4 carboxylate position of
quinoline ring, and second was present near the –C6 position of quinoline ring. Both the
contour suggested presence of bulky group at these positions (expansion of functional
group) in order to design hDHODH inhibitors. Stearically disfavoured yellow coloured
contour was present on terminal phenyl ring of biphenyl ring system at –C2 position of
quinoline ring. This suggested that in the design compounds further extension of single
phenyl ring at –C2 position of quinoline ring might leads to decrease in the activity.
CoMFA electrostatic map (Fig. S2B under supplementary data) is shown in blue
coloured (contributed 80%) electrostatic favoured regions, and red coloured (contributed
20%) electrostatic disfavoured areas. A large blue coloured contour covered –C8 position
of quinoline ring, which suggested that the presence of electropositive groups at these
positions would increase the inhibitory activity of designed compounds. A large red
coloured contoured cover the terminal phenyl ring at –C2 position of quinoline ring, which
revealed that the presence of electronegative groups on terminal phenyl ring of biphenyl
ring system or extension of first phenyl ring with the electronegative groups at 4th position
would favour the inhibitory activity of the compounds. CoMSIA model calculated
4

additionally hydrophobic, HBD and HBA fields. Hydrophobic contour map of CoMSIA
analysis is shown in Fig. S3A under supplementary data, represented by yellow
(contributed 80%, hydrophobic favoured), and grey (contributed 20%, hydrophobic
disfavoured) coloured contours. Yellow coloured contours positioned same as green
coloured contour in CoMFA steric map, and suggested the extension of substitution at
this position (-C4 carboxylate) by hydrophobic groups for the design of inhibitors. Grey
coloured contour covered the –N1 of quinoline ring, which represented those regions of
the molecules where hydrophobic groups resulted in decreased activity. CoMSIA
hydrogen bond acceptor (HBA) contour map (Fig. S3B under supplementary data) is
shown in magenta (contributed 80% HBA favoured), and red (contributed 20% HBA
favoured) coloured contours. Red coloured contour covered the –N1 position of quinoline
ring, and magenta coloured contour covered the -C4 carboxylic acid functional group,
which suggested the presence of HBA group at this position. CoMSIA hydrogen bond
donor (HBD) contour map (Fig. S3C under supplementary data) is shown in cyan
(contributed 80%, HBD favoured), and purple (contributed 20%, HBD disfavoured)
coloured contours. HBD disfavoured purple coloured contour, and HBA magenta coloured
contour are placed at same position (-C4 carboxylic acid) which suggested the need of
HBA group at this position in the design of new compounds. HBD favoured cyan contour
covered the terminal phenyl ring of biphenyl ring system, which suggested the presence
of HBD group on terminal phenyl ring or at the –C4 position of first phenyl ring system for
the design of compounds.
Contour maps representation obtained by the 3D QSAR study was taken as the design
rational, and 30 substituted quinoline derivatives (1d-30d) were designed (Table S3
under supplementary data) as hDHODH inhibitors and anticancer agents. Quinoline ring
was maintained as a main scaffold in the designed compounds. CoMFA steric counter
map and CoMSIA hydrophobic map suggested the extension of functional group (bulky
and hydrophobic) at –C4 position of quinoline ring, thus phenyl, benzyl and biphenyl ring
were selected, and ester and ether spacers were introduced between quinoline and
phenyl, benzyl and biphenyl rings at –C4 position of quinoline ring system as per CoMSIA
HBA and HBD contour maps for H-bonding with the active site of hDHODH. CoMFA
electrostatic counter map suggested the presence of electropositive groups at -C8
5

position (substituted) of quinoline ring. Electron withdrawing groups –NO2, –Cl –F, -Br
and methoxy were selected at –C’4 position of first phenyl ring, which was substituted at
–C2 position of quinoline ring as per CoMFA electrostatic counter. As per the CoMFA
steric counter map –CH3, -C2H5, -CF3, -OCF3, -C3H7,–Cl, and -diCl groups were selected
at –C6 and –C7 positions of quinoline ring. Overall design rational of the molecules on
the basis of 3D QSAR contour maps is shown in Fig. 2.
Fig. 2. Design strategy for the design of 2-,4,-6-,and/or 7-substituted quinoline derivatives
using 3D QSAR generated CoMFA and CoMSIA contour maps.
3D QSAR models (CoMFA and CoMSIA) were used for the prediction of hDHODH
inhibition activity of designed compounds. Predicted activity (pIC50) of designed
compound is shown in Table S3 under supplementary data. Compound 28d
(synthesized compound number 14) was predicted with highest activity (pIC50) of 8.056
and 8.166 using CoMFA and CoMSA models, respectively, and compounds 1d, 2d, 4d,
17d, and 19d also showed good predicted activity (pIC50). Substitution of electron
6

withdrawing groups specially -Cl at R1-position of phenyl ring, along with the presence of
small alkyl group -6CH3, and -6,7-diCl at R-position of quinoline ring were found important
groups in the designed compounds with good predicted activity. Prediction of toxicity risk
(in silico) was carried out for the toxicity factors like mutagenicity, tumorigenicity, irritant
and reproductive effects. Designed compounds were predicted with no toxicity for all
these toxicity risk factors.
Human DHODH is a flavoenzymes which catalyze stereoselective oxidation of L-
dihydroorotate (DHO) to orotate (ORO). In this electron transfer reaction, flavin
mononucleotide (FMN) serve as an intermediate. Human DHODH exhibits a two-site
ping-pong mechanism with FMN. Human DHODH belongs to family-2 enzymes, which
transfer electrons to ubiquinone (CoQ) for reoxidizing FMN. Crystal structures of
hDHODH in complexes with inhibitors helped in the structure-based design of specific
inhibitors. Docking study with designed compounds was performed to further scrutinise
the designed compounds for their selection as candidates for the synthesis and hDHODH
inhibition activity. Brequinar was also docked as standard reference compound, and
binding interactions of designed compounds were compared with brequinar at the binding
site of hDHODH. In hDHODH structure a tunnel is formed from two α-helices, which
represent the active site of hDHODH, and described as the target site of hDHODH
inhibitors. Brequinar showed highest docking score of 58.92, and formed interactions with
Leu42, Pro52, Arg136, Tyr365 and Leu68 (Fig. S4 under supplementary data) at the
active site of hDHODH. Carboxyl group of brequinar formed H-bond interaction with the
side chain of Arg136. Designed compounds showed interaction with Met43, Leu46,
Gln47, Pro52, Glu53, Ala55, Thr63, Leu67, Arg136, Tyr356, Leu359, and Pro364
residues. The designed compounds also showed a water (HOH444) mediated interaction
with Thr360. Due to absence of carboxyl group in the designed compounds, they did not
showed brequinar-like binding mode. Docking results of the designed compounds are
shown in Table S3 under supplementary data. Most of the compounds showed good
docking score, compounds 4d (8) and 15d (10) showed highest docking score of 56.76
and 55.75, respectively.
7

Fig. 3. Docking pose of compound 8 (A), 10 (B), in human DHODH enzyme. Compounds
8, and 10 are in wire frame with C-atom in light green colour and other atoms by their
colour, H-bond interactions were highlighted using light green dotted lines, and distance
of H-bonding between the atoms of ligand and residues of protein in Å. The labelled
protein residues are capped in stick model with colour by atom.
8

Binding modes of compounds 4d (8) and 15d (10) are shown in Fig. 3. Compound 4d (8)
showed binding interactions at the active site with Leu42, Met43, Gln47 and Leu67 as
shown in Fig. 3(A), and compound 15d (10) showed binding interactions with Met43,
Leu46, Ala59, Gln47, Leu67, Thr356 and Pro364 as shown in Fig. 3(B). Docking
revealed a clear preference of electron withdrawing -Cl and -F groups in the designed
compounds.
Quinoline ring was synthetically derivatized at -C2, -C4 and -C6-, and/or -C7 positions to
obtain a series of 2,4,6-,and/or 7 substituted quinoline derivatives, and characterized as
target compounds. In the first step of synthesis, 2-(4-subsitutedphenyl)-6,7-substituted-
quinoline-4-carboxylic acid (4a-e) were synthesized based on Doebner reaction
using substituted anilines (1) with substituted benzyldehydes (2) and pyruvic acid (3)
(Scheme 1). In the second step of reaction, substituted-quinolin-4-ylmethanol derivatives
(5a-e) were synthesized. Substituted quinoline-4-carboxylic acid derivatives (4a-e) were
reacted with lithium aluminum hydride (LiAlH4) in the presence of tetrahydrofuran (THF).
Target compounds (6-10) (ester derivatives) were synthesized via the reaction of 4a-e
with benzylbromide in the presence of sodium hydride (NaH) and dimethylformamide
(DMF). Other target compounds (ether derivatives) (11-15) were synthesized using
Williamson ether synthesis via reaction of substituted-quinolin-4-ylmethanol (5a-e)
derivatives in DMF with benzyl bromide.
Chemical structures of all the compounds were confirmed using FTIR, 1H NMR, 13C NMR,
mass spectra and elemental analysis data (Supplementary data). Ester and ether
derivatives were prepared, and observed with significant features in FTIR spectrum.
Compounds demonstrated characteristic O-H (–COOH) stretching peaks near broad rang
3500–3205 cm-1, –C=O stretching peaks (–COOH) near 1720–1780 cm-1 whereas
compounds showed O-H stretching peaks (–CH2OH) at 3500–3200 cm-1, aliphatic C-H
stretching peaks near 2950–2870 cm-1, and –C=O stretching peaks were absent. Mass
spectrums of synthesized compounds correlated with M+1 peaks as stable base peaks
1
(Supplementary data). The significant features of H NMR spectra of the synthesized
compounds were observed as the aromatic region showed sets of aromatic protons in the
range of δ 6.8–8.4 ppm and correlated with C atoms (δ 120-138 ppm) in 13C NMR
9

spectrum, C-6 methyl group in the quinoline ring showed a resonance peak in the range
1
13
δ 2.4-2.5 ppm in H NMR spectra and correlated with C atoms (δ 21 ppm) in C NMR
spectrum (Supplementary data), signal of aliphatic ester as a linker at –C4 position of
quinoline ring observed in the region δ 5.4-5.5 ppm in 1H NMR spectra, and correlated
with C atoms (δ 67 ppm) in 13C NMR spectrum.
Scheme 1. Synthetic strategy for the synthesis of substituted quinoline derivatives via
Doebner reaction, LiAlH4 catalyzed reduction and Williamson ether synthesis reactions.
Reagents and conditions (a) ethanol, reflux 3-4 h (b) LiAlH4, THF, 0˚C (c) benzyl bromide,
DMF, NaH, 0˚C
Synthesized compounds were screened for hDHODH colorimetric enzyme inhibition
assay with recombinant hDHODH enzyme. All the synthesized compounds were also
screened for antiproliferative activity against two different human cancer cell lines,
10

colorectal adenocarcinoma cell line (HT-29) and breast cancer cell line (MDA-MB-231) to
further obtain biologically relevant information as anticancer agents.
Chromogen reduction method with 2,6-dichlorophenolindophenol (DCIP) dye was used
to perform hDHODH enzyme inhibition assay. Chromogen reduction was coupled with
the oxidation of L-dihydroorotate (L-DHO) as well as reduction of ubiqinone (CoQ).
Enzyme inhibition was resulted in loss of chromogen absorbance at 600 nm. Absorbance
of each well was measured at 600 nm and quantified in triplicate using a 96 microplate
reader (Bio-Rad-680 instrument) and the data were exported to an Excel (Microsoft)
spreadsheet for analysis. The percentage enzyme inhibition was measured from 0.01µM
to 30 10 µM concentration of synthesized compounds, along with the standard brequinar
sodium. The IC50 value (Table 1) as a practical readout of the enzyme inhibition of
synthesized compounds under comparable conditions was obtained from dose-response
plot (Fig. 4) using GraphPad. Most of the synthesized compounds showed comparative
biological activity, and the best compounds of ester series were 7 and 8, which showed
IC50 value of 1.56 µM and 1.96 µM, respectively against hDHODH enzyme, and in ether
series compound 14 showed highest hDHODH inhibition activity with an IC50 value of 1.22
µM. Presence of –Cl group at R1 position of phenyl ring, which is substituted at –C2
position of quinoline ring along with 6,7-diCl substituents in quinoline ring were found best
substitutions in the ether series (14). In the ester series of compounds presence of –CH3
at –R position (-C6) (7, 8) along with of –F group at R1 position in 7, and –OCH3 in 8
contributed to the potency of synthesized compounds against hDHODH enzyme. The
activity data revealed a clear preference of electron withdrawing –Cl and –F groups at
phenyl ring substituted at -C2 position of quinoline ring for the potency against hDHODH
enzyme. Compounds (8, 10) with –OCH3 group at R1 positions also showed better
activity. Furthermore the presence of -6,7diCl in compounds 10 and 14 have a marked
impact on activity against hDHODH enzyme. The results of hDHODH enzyme inhibition
activity are in agreement with the in silico results (predicted activity and docking score).
11

Fig. 4. Human DHODH inhibition dose-response curve of synthesized compounds. Six
different concentrations 0.01 µM, 0.1 µM, 1 µM, 10 µM, 20 µM, 30 µM of the compounds
were tasted and IC50 values were determined.
12

Table 1. Human DHODH enzyme inhibitory activity of 2-,4,-6-,and/or 7-substituted
quinoline derivatives
Compound IC50 (µM)
Compound
IC50 (µM)
6
13
12
14
15
7.37
1.56
1.96
7.48
3.34
nd
7
13.44
1.22
15.48
0.016
8
9
10
Brequinar
sodium
11
nd
IC50 values are determined as the mean ± SD of three independent
experiments performed
nd = not determined
All the synthesized compounds (6-15) along with two standards brequinar sodium and
paclitexal were evaluated for antiproliferative activity against two different cancer cell
lines, named breast cancer (MDA-MB-231) and colorectal adenocarcinoma (HT-29). The
IC50 value (cell growth inhibition) of synthesized compounds were determined from dose-
response curve (Table 2). Compounds 7 (IC50 = 15.06 µM against HT-29, and IC50 =
13.16 µM against MDA-MB-231), and 8 (IC50 = 17.29 µM against HT-29, and IC50 = 20.52
µM against MDA-MB-231) with –CH3 group at –R position (-C6) (7, 8) along with of –F
group at R1 position in 7, and –OCH3 in 8 were found the most potent compounds in MTT
assay. Compound 14 showed IC50 value of 16.78 µM against HT-29. Compound 7 and 8
exhibited good antiproliferative activity against both the cancer cell lines, which were
almost equally potent as brequinar sodium and paclitaxel.
Table 2 Antiproliferative activity of substituted quinoline derivatives against HT-29 and
MDA-MB-231 cancer cell lines with standards brequinar sodium and Paclitaxel
Colorectal adenocarcinoma cell lines
(HT-29)
Breast cancer cell lines
(MDA-MB-231)
13

Compound
IC50 (µM)
>100
Compound
IC50 (µM)
>100
6
6
15.06
17.29
48.71
42.18
>100
13.16
20.53
>100
7
7
8
8
9
9
39.16
>100
10
10
11
12
13
14
15
11
>100
12
>100
>100
>100
>100
>100
13
14
16.78
>100
15
Paclitaxel
Paclitaxel
Brequinar
Sodium
14.56
13.23
12.45
Brequinar Sodium
13.98
In the present study, we described 3D QSAR-based design, synthesis and in vitro
hDHODH inhibition and antiproliferative activity, of 2-,4,-6-,and/or 7-substituted quinoline
derivatives. We designed 30 substituted quinoline derivatives using the information
obtained from the generated CoMFA and CoMSIA contour maps. The designed
compounds showed good predicted activity using ligand-based 3D QSAR models, and
no “in silico” toxicity. In addition to ligand-based QSAR method a structure-based method
(molecular docking) was used to select designed compounds as candidates for the
synthesis and activity, based on the docking results and predicted activity. Compounds
4d (8), and 15d (10) (ester-linker), and 27d (15) and 28d (14) (ether-linker) showed very
good results in “in silico” studies. Synthesized compounds were evaluated in an in vitro
hDHODH inhibition assay, and also screened for antiproliferative activity using MTT
assay on cancer cell lines. Compounds 7, 8 and 14 exhibited good hDHODH inhibition
activity, and in vitro anticancer activity against HT-29 and MDA-MB-231 cell lines.
14

However, in comparison with standard brequinar, the synthesized compounds showed
weak hDHODH inhibition. Even the most potent compound 14 showed IC50 value of 1.22
µM, whereas the IC50 value of brequinar was 0.016 µM (Table 1). We investigated the
reason of potential differences in the hDHODH inhibitory activity and found that all the
compounds in the training set (Tr1 to Tr37 ) (Table S1 under supplementary data),
where brequinar belongs, are characterized by the presence of a carboxylic group, which
is mainly involved in a key polar interaction with hDHODH active site (Arg 136 and Gln47).
Other known more potent hDHODH inhibitors are also involved in such kind of interaction
with the active site of hDHODH, as the well structure activity relationship studies of known
hDHODH suggested the presence of an acidic moiety in the compound to retain the
activity. 3D QSAR model was not able to predict in the carboxyl area (–C4 position of
quinoline ring) any other influence different from the presence of a carboxyl group type of
interaction (HBD and HBA), this because all the training set compounds were lacking
chemiodiversity in that specific region. So the synthesized compounds did not adopted a
brequinar-like binding mode. This might be the reason of weak hDHODH inhibitory activity
of the synthesized compounds. On the other hand, the inhibitory activities of synthesized
compounds toward the proliferation of two cancer cells were comparable to those of
brequinar. Recently, Wong et al. 15 showed that DHODH inhibitors induce cell cycle arrest
in cancer cells via additional DHODH-independent pathway. Their study showed that
DHODH inhibitors does not affect the inherent abundance or the expression of DHODH
in the cancer cells although they observed significant decreased in enzymatic activity in
all cell lines. So the anticancer activity of synthesized compounds might be due cell cycle
arrest in cancer cell lines via additional DHODH-independent pathway. Comparison of in
silico computational study and in vitro study revealed that the compounds which showed
better results (predicted activity and docking score) were found as the most potent
compounds in both the screening methods. Overall, predicted activity (pIC50), docking
study, and in vitro activity data revealed a clear preference of electron withdrawing -Cl
and -F groups. Results of both the screening assay validated the computational design
of substituted quinoline derivatives. Finally, it is concluded that synthesized compounds
7 and 14 are lead compounds as hDHODH inhibitors for the development of new
anticancer agents.
15

ACKNOWLEDGEMENTS
The authors would like to thank GUJCOST Gandhinagar, Gujarat, India for Minor
Research Project grant (GUJCOST/MRP/2015-16/1177), and Nirma University,
Ahmedabad, India for providing the necessary facilities.
Supplementary data: Supplementary data associated with this article can be found,
in the online version, at…………………………..
Detail experimental procedures of 3D QSAR, Docking Study Experimental Procedures
1
regarding general procedure for the synthesis of compounds, FTIR, Mass spectra, H
NMR, 13CNMR and in vitro hDHODH inhibition assay and antiproliferative MTT assay of
synthesized compounds are given under supplementary data.
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Graphical Abstract
Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human
dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents:
3D QSAR-assisted design
Vivek K. Vyas*a, Gulamnizami Qureshia, Drashti Ozaa, Hardik Patelb,
Krupali Parmarb, Palak Patelb, Manjunath D. Ghatea
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, Ahmedabad
382481, Gujarat, India
bInstitute of Science, Nirma University, Ahmedabad 382481, Gujarat, India
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