Synthesis, characterization, in silico molecular docking study and biological evaluation of a 5-(phenylthio) pyrazole based polyhydroquinoline core moiety

Article abstract of DOI:10.1039/c7nj01962a

A multicomponent cyclocondensation reaction has been developed by incorporating 3-methyl-5-substituted phenylthio-1-phenyl-1H-pyrazole-4-carbaldehydes 3a-c, various enaminones 6a-c and different active methylene compounds 7a-c (malononitrile 7a/ethylcayno

Full text of DOI:10.1039/c7nj01962a

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J. Chem., 2017, DOI: 10.1039/C7NJ01962A.
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Synthesis, characterization, in silico molecular docking study and biological
evaluation of 5-(phenylthio) pyrazole based polyhydroquinoline core
moiety
Nirav H. Sapariyaa*, Beena K. Vaghasiyaa, Rahul P. Thummara, Ronak D. Kamania, Kirit H. Patela,
Parth Thakorb, Sampark S. Thakkarc, Arabinda Rayc and Dipak K. Ravala
aDepartment of Chemistry, Sardar Patel University, Vallabh Vidyanagar- 388 120, Gujarat, India
b P. G. Department of Biosciences, Sardar Patel Maidan, Bakrol-Vadtal Road, Satellite Campus, Sardar Patel
University, Vallabh Vidyanagar -388120, Gujarat, India
cAdvanced Organic Chemistry Department, P. D. Patel Institute of Applied Sciences, CHARUSAT, Changa-
388421, Gujarat, India
*Corresponding author. Tel.: +91-02692-226856 - Ext. - 211; Fax: +91-02692 236475.
E-mail: nir.sapariya@gmail..com , dipanalka@yahoo.com
Abstract
A multicomponent cyclocondensation reaction has been developed by incorporating 3-
methyl-5-substituted phenylthio-1-phenyl-1H-pyrazole-4-carbaldehydes 3a-c, various
enaminones 6a-c and different active methylene compounds 7a-c (malononitrile 7a
/ethylcaynoacetate7b /caynoacetamide7c) in presence of piperidine as basic catalyst, to afford
combinatorial library of polyhydroquinoline scaffolds i.e. 8a-p. The targeted compounds
were synthesized in good to excellent yield (71-84%). All the synthesized compounds have
been characterized by 1H NMR, 13C NMR, IR, mass spectrometric techniques and elemental
analysis. All the synthesized compounds were evaluated in vitro for their antibacterial,
antitubercular and antimalarial activities. In silico molecular docking study as well as in silico
pharmacokinetics evaluation have been carried out. Many candidates of this new class
revealed noticeable activities against first line drugs.
Key words: In silico molecular docking, In silico pharmacokinetics evaluation, 5-
(phenylthio) pyrazole, Polyhydroquinoline, Multi-component reaction, Biological screening.
1. Introduction
Malaria causes serious social and economic consequences. 300-500 million people are
affected annually and about 3 million deaths occur due to this severe global health issue [1,
2]. Amongst the four Plasmodium parasites, Plasmodium falciparum is considered

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responsible for ~95% of death [3]. Tuberculosis (TB) is caused by Mycobacterium
tuberculosis (MTB) which is a human pathogen. Nearly one-third of the global population is
infected by MTB. TB is declared as a global emergency by World Health Organization as it
is suspected that about 30 million people will suffer from TB in next two decades [4, 5]. The
second most common reason for death is microbial infections after heart attack being the
prime one. This is because of their resistance towards the existing antibiotic remedy.
Fluorine plays a crucial role [6-8] in improving pharmacodynamic and pharmacokinetic
properties [9, 10] of drugs molecules. Trifluoromethylation is the most significant strategy to
modulate physical and biological properties. High lipophilicity of trifluoromethyl group
enhances in vitro uptake and transport of the candidate [11].
Fluoro-substituted pyrazole and its derivatives are the significant class of heterocycles
enjoying a remarkable position in medicinal chemistry. Pyrazole derivatives exhibit variety of
pharmacological activities including antimicrobial [12, 13], anti-inflammatory [14, 15],
cytotoxic [16], antitumor [17-19], antiviral [20], anticancer [21], analgesic [22-24] and anti-
parkinson [25] activities.
Multicomponent condensation reaction, MCR [26, 27] is one of the leading methods to
synthesize small molecules. Several condensation reagents react in a single step by
simultaneous reactions to yield the desired products. Alteration in the reaction components of
MCR can easily lead to the molecular diversity in combinatorial libraries.
From the discovery of the drug molecules, their exposures to the various disease causative
agents make them drug-resistant. To overcome that problem, there is an immense need to
design fluoro-pyrazole combined polyhydroquinoline nucleus which combats against such
pathogens in an effective manner. In this perspective, the current communication describes
design and synthesis of polyhydroquinoline core molecules from the 5-substituted
phenylthio-1-phenyl-1H-pyrazole-4-carbaldehydes, various active methylene and enaminones
under conventional conditions. The synthesized compounds were also tested for their
diversified activities such as antimalarial, antitubercular and antimicrobial activities. Out of
all listed biological activities, synthesized compounds showed comparatively good
antimalarial activity. As an outcome of it, we decided to perform molecular docking of
synthesized compounds with the dihydrofolate reductase (DHFR) receptor.

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The antimalarial agents target the malaria enzyme, dihydrofolate reductase (DHFR). The
incorporation of wild-type and the quadruple mutant DHFR from the P. falciparum malarial
strain should be considered for development of new antimalarials [28]. The quinolone
containing cores (8a, 8c, 8d, 8l, 8m, 8o) were found active against P. falciparum strain.
Molecular docking studies of active quinolone compounds and standard drugs (chloroquine
and quinine) were performed to check the possible interactions of synthesized molecules
with molecular target wild-type Plasmodium falciparum dihydrofolate reductase-thymidylate
synthase (pfdhfr-ts) (PDB ID: 4DPD).
2. Chemistry
The synthesis of targeted polyhydroquinoline clubbed 5-phenylthioxypyrazole derivatives is
presented in Scheme 1. 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 1 was
prepared according to the reported method [29, 30].
Scheme 1. Synthesis of substituted 2-amino-4-(5-((4-substitutedphenyl)thio)-3-methyl-1- phenyl-1H-pyrazol-4-
yl)-1-(substitutedphenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8 hexahydro quinoline-3-carbonitrile 8a-p (i) DMF,
K2CO3, Reflux 2 h (ii) Methanol, Acetic acid, reflux 0.5-1 h (iii) Piperidine, Ethanol, Reflux for 1-3 hr.
5-((4-substituted-phenyl)thio)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 3a-c was
synthesized by refluxing compound 1 and thiophenols 2a-c in presence of K2CO3(anhydrous)
as a basic catalyst using DMF as a solvent. The required enaminones 6a-c were synthesized

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by the reaction of dimedone 4 with various amines 5a-c by refluxing in the presence of
catalytic amount of glacial acetic acid in methanol.
Table 1. Preliminary characterization of synthesized compounds 8a-p.
Comp.
R
R1
R2
Yielda(%)
4-Cl
4-Cl
4-F
4-F
-CN
-COOEt
-CONH2
-CN
81
79
73
84
76
72
80
79
73
75
78
81
83
71
72
77
8a
8b
8c
8d
8e
8f
4-Cl
4-F
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-Cl
4-F
4-F
-COOEt
-CN
4-CF3
4-CF3
2,4-F
2,4-F
2,4-F
2,4-F
2,4-F
4-F
-COOEt
-CN
8g
8h
8i
-COOEt
-CN
8j
4-Cl
-COOEt
-CONH2
-CN
8k
8l
4-Cl
4-F
8m
8n
8o
8p
4-F
4-F
-COOEt
-CN
4-F
4-CF3
4-CF3
4-F
-COOEt
aIsolated yield
The targeted molecules were synthesized by refluxing the mixture of 5-((4-substituted-
phenyl)thio)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 3a-c, various enaminones 6a-c
and different active methylene compounds 7a-c (malononitrile 7a, ethylcyanoacetate 7b and
caynoacetamide 7c) using piperidine in absolute ethanol (Scheme 1).
Scheme 2 represents a plausible mechanism for the reaction. The formation of quinoline
derivatives 8a-p was attempted through an initial in situ formation of heterylidenenitrile via
Knoevenagel condensation between aldehyde 3a-c and various active methylene compounds
7a-c. Finally, Michael addition of enaminones 6a-c to the heterylidene olefins gave acyclic
intermediate 8a’-p’. Nucleophilic attack of the -NH group on the cyano carbon cyclises this
intermediate which then tautomerizes to afford cyclised polyhydroquinoline derivatives 8a-p.

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Scheme 2. Mechanistic pathway for the synthesis of polyhydroquinoline derivatives 8a-p.
3. Pharmacology
3.1. In vitro antimicrobial activity
The synthesized compounds 8a-p were tested for their antimicrobial activity by broth
microdilution method according to National Committee for Clinical Laboratory Standards
[31-33]. Three Gram-positive (Bacillus subtilis MTCC 441, Clostridium tetani MTCC 449,
and Streptococcus pneumoniae MTCC 1936) and three Gram-negative (Salmonella typhi
MTCC 98, Escherichia coli MTCC 443, and Vibrio cholerae MTCC 3906) bacteria were
chosen for antibacterial screening using ampicillin, ciprofloxacin, norfloxacin and
chloramphenicol as the standard antibacterial agents. Two fungal species (Aspergillus
fumigats MTCC 3008 and Candida albicans MTCC 227) were screened employing
griseofulvin and nystatin as the standard antifungals. Mueller-Hinton broth was used to
dilute the drug suspension for the test. The desired test concentration of compounds was
achieved using DMSO as the diluents. The result of antimicrobial testing is depicted in Table
2.
3.2. In vitro antituberculosis activity
Using Lowenstein-Jensen medium as described by Rattan [34] the synthesized compounds
8a-p were screened for their in vitro antituberculosis activity. Screening was conducted at
250 ꢀg/mL of drug concentration against Mycobacterium tuberculosis H37Rv strain using
isoniazid and rifampicin as the standard drugs. The results are presented in Table 3 in form
of % inhibition.

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3.3. In vitro antimalarial activity
Quinine and chloroquine as the reference compounds were employed for in vitro
antimalarial activity of the compounds 8a-p against P. falciparum strain. The result of
antimalarial screening is expressed in Table 4.
3.4. In silico study
3.4.1. In silico molecular docking study
The mol2 file of active compounds (8a, 8c, 8d, 8l, 8m, 8o) and standard drugs (chloroquine
and quinine) were prepared using ChemBio3D Ultra 14.0. The mol2 formats of all ligands
were used in the molecular docking study. The 3d structure of wild-type Plasmodium
falciparum dihydrofolate reductase-thymidylate synthase’ (PDB ID: 4DPD) was downloaded
from RCSB website (www.rcsb.org/pdb). The protein structure was opened in CLC Drug
Discovery Workbench 4.0 software. The water molecules, intramolecular bonds, cofactors
and ligands were removed from receptors. The 23/23 binding pockets were found with set up
of 25 °A radius size of the protein.
3.4.2. In silico pharmacokinetics evaluation
The pharmacokinetic parameters of active compounds indicated earlier were obtained using
programs VlifeMDS 4.6 (Vlife Sciences, Pune, India) and ADMET software. These
programs computed pharmacokinetic parameters such as logP, molecular weight, PSA, HBA,
HBD, BBB, %HIA, Caco2 etc.
4. Results and discussion
4.2. Analytical results
The structures of the newly synthesized compounds were confirmed by mass spectrometry,
1H NMR, 13C NMR, FT-IR and elemental analysis. The IR spectrum of compounds 8a-p
exhibited characteristic absorption band in the range 781-755 cm-1 This can be assigned the
.
presence of thioether linkage. The characteristic absorption band around 1680-1652 cm-1
which may be due to the presence of carbonyl group (C5 of the polyhydroquinoline ring). All
the compounds show -C≡N stretching in the range of 2297-2180 cm-1, strong absorption band
in the range of 1373-1351 cm-1 due to -CH3 stretching and absorption band in the range 3483-
3324 cm-1 due to asymmetric & symmetric stretching of -NH2. The presence of the -CH
proton (C4-H of polyhydroquinoline ring) and –NH2 protons are confirmed by 1H NMR
spectra of compounds 8a-p as a sharp singlet around δ 5.43-4.63 ppm and a broad singlet at
δ 6.69-3.60 ppm respectively. Amide and aromatic protons are found to be resonated as

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multiplets in the range between δ 7.79-6.57 ppm. In 13C NMR spectra, the carbonyl carbon
(C5 of polyhydroquinoline ring) was displayed at δ 193.88-195.97 ppm. The chemical
structures are further confirmed by molecular ion peak (M+) corresponding to their respective
molecular weights in mass spectra.
4.3. Biological section
4.3.1. In vitro antibacterial activity
All the synthesized compounds (8a-p) showed moderate to very good antibacterial activity.
Table 2. In vitro antimicrobial activity expressed in terms of MIC, µg/mL for prepared
derivatives 8a-p
Gram-positive bacteria
Gram-negative bacteria
Fungi
S.P.
B.S.
C.T.
E.C.
S.T.
V.C.
C.A.
MTCC MTCC MTCC MTCC MTCC MTCC MTCC MTCC
A.F.
Compound
1936
500
100
200
62.5
500
500
100
100
200
500
100
500
200
500
200
500
100
10
441
500
500
200
250
250
500
100
62.5
500
500
1000
500
100
100
200
1000
250
100
50
449
250
62.5
500
125
1000
500
500
100
250
200
250
200
200
500
62.5
200
250
50
443
100
200
250
100
250
250
500
100
500
100
200
500
62.5
100
62.5
500
100
10
98
3906
250
200
200
1000
1000
62.5
500
500
100
200
100
500
500
500
100
100
100
10
227
>1000
1000
1000
500
3008
>1000
>1000
500
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
A
250
500
200
200
250
100
200
500
100
200
200
500
100
200
100
500
100
10
500
>1000
>1000
1000
>1000
100
1000
1000
100
1000
1000
500
250
n. t.
500
1000
1000
500
500
250
250
1000
1000
250
500
500
n. t.a
n. t.
B
n. t.
n. t.
n. t.
100
C
D
E
F
50
25
n. t.
n. t.
50
50
25
n. t.
n. t.
50
25
n. t.
n. t.
50
25
n. t.
n. t.
n. t.
n. t.
100
500
50
n. t.
n. t.
100
n. t.
n. t.
100
S.P.= Streptococcus pneumoniae, B.S.= Bacillus subtilis, C.T.= Clostridium tetani, E.C.= Escherichia coli
S.T.= Salmonella typhi, V.C.= Vibrio cholerae, C.A.= Candida albicans, A.F.= Aspergillus fumigatus, MTCC:
Microbial Type Culture Collection. A: Ampicillin, B: Norfloxacin, C: Chloramphenicol, D: Ciprofloxacin, E:
Nystatin, F: Griseofulvin, a n.t.: not tested.
It has been noticed that against S. pneumoniae, compound 8d (R = 4-CH3, R1 = 4-F, R2 = -
CN) was found to be more potent i.e. 62.5 µg/mL as compared to ampicillin i.e. 100 µg/mL.
The compounds 8b (R = 4-Cl, R1 = 4-F, R2 = -COOEt), 8g (R = 4-CH3, R1 = 4-CF3, R2 = -

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COOEt), 8h (R = 4-CH3, R1 = 2,4-F, R2 = -COOEt), 8k (R = 4-Cl, R1 = 2,4-F, R2 = -COOEt)
showed equivalent activity to that of ampicillin. Compound 8h (R = 4-CH3, R1 = 2,4-F, R2 =
-COOEt) showed maximum potency i.e. 62.5 µg/mL as compared to ampicillin i.e. 250
µg/mL as well as norfloxacin i.e. 100 µg/mL against B. Subtilis. Compound 8d (R = 4-CH3,
R1 = 4-F, R2 = -CN), 8e (R = 4-CH3, R1 = F, R2 = -COOEt) MIC = 250 µg/mL was found to
be equipotent to ampicillin. Compounds 8c (R = 4-Cl, R1 = 4-F, R2 = -CONH2) and 8o (R =
4-F, R1 = 4-CF3, R2 = -CN) i.e. 200 µg/mL were noticed to be highly potent as compared to
ampicillin MIC = 250 µg/mL. Compounds 8b (R = 4-Cl, R1 = 4-F, R2 = -COOEt) and 8o (R
= 4-F, R1 = 4-CF3, R2 = -CN) showed maximum potency i.e. MIC = 62.5 µg/mL as compared
to ciprofloxacin against C. tetani. Compounds 8a (R = 4-Cl, R1 = 4-F, R2 = -CN), 8i (R = 4-
CH3, R1 = 2,4-F, R2 = -COOEt), 8k (R = 4-Cl, R1 = 2,4-F, R2 = -COOEt), MIC = 250 µg/mL
exhibited same influence as compared to ampicillin. Compounds 8h (R = 4-CH3, R1 = 2,4-F,
R2 = -COOEt), MIC = 100 µg/mL and 8d (R = 4-CH3, R1 = 4-F, R2 = -CN), MIC = 125
µg/mL were found to be more potent as compared to ampicillin MIC = 250 µg/mL.
Compounds 8m (R = 4-F, R1 = 4-F, R2 = -CN), and 8o (R = 4-F, R1 = 4-CF3, R2 = -CN), MIC
= 62.5 µg/mL were found to have higher potency against E. coli as compared to ampicillin
against Gram-negative bacteria. Compound 8f (R = 4-CH3, R1 = 4-CF3, R2 = CN), MIC =
62.5 µg/mL was found to have excellent potency against V. Cholerae as compared to that of
ampicillin. Compounds 8f (R = 4-CH3, R1 = 4-CF3, R2 = -CN), 8i (R = 4-CH3, R1 = 2,4-F, R2
= -COOEt), 8k (R = 4-Cl, R1 = 2,4-F, R2 = -COOEt), 8m (R = 4-F, R1 = 4-F, R2 = -CN), 8n
(R = 4-F, R1 = 4-F, R2 = -COOEt) and compounds 8i (R = 4-CH3, R1 = 2,4-F, R2 = -COOEt)
8k (R = 4-Cl, R1 = 2,4-F, R2 = -COOEt) were noticed to be equipotent as that of ampicillin
i.e. 100 µg/mL respectively against S. typhi and V. cholerae (Table 2).
4.3.2. In vitro antifungal activity
Table 2 depicts antifungal screening data. Compound 8j (R = 4-Cl, R1 = 2,4-F, R2 = -CN)
and 8n (R = 4-F, R1 = 4-F, R2 = -COOEt) i.e. 250 µg/mL, were found to be more active than
griseofulvin against C. albicans. Also against C. albicans, compounds 8d (R = 4-CH3, R1 =
4-F, R2 = -CN), 8e (R = 4-CH3, R1 = F, R2 = -COOEt), 8i (R = 4-CH3, R1 = 2,4-F, R2 = -
COOEt), 8o (R = 4-F, R1 = 4-CF3, R2 = -CN) and 8p (R = 4-F, R1 = 4-CF3, R2 = -COOEt)
showed comparatively similar influence as that of griseofulvin. Compounds 8i (R = 4-CH3,
R1 = 2,4-F, R2 = -COOEt) and 8l (R = 4-Cl, R1 = 2,4-F, R2 = -CONH2) exhibited equivalent
potency i.e. 100 ꢀg/mL against A. fumigates. The results suggest that compound 8o may be a
potent candidate for new class of antimicrobial agent in future.

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4.3.3. In vitro antituberculosis activity
The synthesized compounds 8a–p were screened against Mycobacterium tuberculosis H37Rv
strain at 250 µg/mL concentration. Compounds 8e (R = 4-CH3, R1 = 4-F, R2 = -COOEt), 8i (R
= 4-CH3, R1 = 2,4-F, R2 = -COOEt), and 8l (R = 4-Cl, R1 = 2,4-F, R2 = -CONH2) were found
to exhibit excellent antituberculosis activity 94%, 95% and 91% respectively.
Table 3. In vitro antituberculosis activity (% inhibition) of polyhydroquinoline derivatives8a-
p against M. tuberculosis H37Rv (at concentration 250 µg/mL).
Compound
% Inhibition
Compound
% Inhibition
8a
8b
8c
8d
8e
8f
8j
65
20
30
46
94
46
50
73
95
79
89
91
67
80
65
87
98
99
8k
8l
8m
8n
8o
8g
8h
8i
8p
Rifampicin
Isoniazid
The compounds 8k(R = 4-Cl, R1 = 2,4-F, R2 = -COOEt ) and 8p (R = 4-f, R1 = 4-CF3, R2 = -
COOEt ) were noticed to be quite active. All other remaining compounds showed poor
inhibition against M. tuberculosis H37Rv growth (Table 3).
4.3.4. In vitro anti-malarial activity
The synthesized compounds 8a-p were tested for their anti-malarial screening using
chloroquine and quinine as the reference. Duplicate runs were performed for each
experiment and mean values of IC50 are summarized in Table 4.
Table 4. In vitro antimalarial activity of polyhydroquinoline derivatives 8a-p.
Compound
IC50 (µg/mL)
Compound
IC50 (µg/mL)
8j
8a
8b
8c
8d
8e
8f
0.54
1.27
0.065
0.69
8k
8l
0.085
0.076
0.79
0.083
0.097
1.32
8m
8n
8o
1.25
0.047
0.54
8g
8h
8i
8p
0.47
0.020
0.268
Chloroquine
Quinine
1.10.
1.40

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The compounds 8a (R = 4-Cl, R1 = 4-F, R2 = -CN), 8c (R = 4-Cl, R1 = 4-F, R2 = -CONH2), 8d
(R = 4-CH3, R1 = 4-F, R2 = -CN), 8l (R = 4-Cl, R1 = 2,4-F, R2 = -CONH2), 8m (R = 4-F, R1 =
4-F, R2 = -CN), 8o (R = 4-f, R1 = -CF3, R2 = -CN) exhibited IC50 in the range of 0.042 to
0.097 which is remarkable against P. falciparum as compared to quinine IC50 0.268.
4.3.4.1. In silico molecular docking study
To understand the atomistic level details of plausible protein-ligand interactions and to
identify potential ligands against target disease, molecular docking studies was performed.
Considering the in vitro results against P. falciparum strain, it was thought worthy to screen
for supportive coordination by in silico studies. Thus, molecular docking was performed
between ligands (8a, 8c, 8d, 8l, 8m, 8o, chloroquine, quinine) and wild-type P. falciparum
dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) (PDB ID: 4DPD) using CLC drug
discovery workbench 4.0. The docking scores are presented in Table 5.
Table 5. Molecular Docking Results
Hydrogen
bond
score
-6.13
Ligand
Conformation
Penalty
27.97
Steric
Compound Score
Interaction
-27.88
-28.78
-27.82
-20.92
-27.84
-27.10
-49.93
-45.82
-49.71
-48.60
-49.73
-47.18
-49.78
-53.51
-50.06
-44.21
8a
-9.32
29.14
8c
-6.07
27.98
8d
-9.28
35.54
8l
8m
-6.00
27.94
-1.72
28.13
8o
-2.00
2.14
Chloroquine
Quinine
-2.51
0.90
A potential interaction was observed between the active molecules (8a, 8c, 8d, 8l, 8m, 8o,
chloroquine, quinine) and the Pf-DHFR enzyme. The molecules interacted with the active
pockets of protein by forming H-bond. The docking scores of molecules (8a, 8c, 8d, 8l, 8m,
8o) were found to be ranging from -20.92 to -28.78 (Table 5) which were not so good as
compared to the standard drugs chloroquine and quinine. Such lower score was due to
higher ligand conformation penalty. The -C≡N group of compounds 8a (Fig. 1a), 8d (Fig.
1c) and 8m (Fig. 1e) and -C=O of amide of 8c (Fig. 1b) and 8l (Fig. 1d) interacted with the
active pockets of the enzyme forming hydrogen bonds with TYR 365 at different distances

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(Table 6). It is interesting to note that the -C=O of cyclohexane of these compounds formed a
hydrogen bond with LYS 297 also at different distances (Table 6). In case of ligand 8o (Fig.
1f), -NH2 group formed H-bond with GLU 287 at a distance of 2.559°A. The standard drug
chloroquine did not show any H-bond with the receptor 4DPD (Fig. 1g), while -OH group of
quinine molecule formed H-bond with TYR 322 (3.248 °A) (Fig. 1h).
Table 6. Hydrogen bonding of compounds (8a, 8c, 8d, 8l, 8m, 8o) and standard drugs with
the receptor wild-type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase
(PDB ID: 4DPD).
Functional group
of ligands
Amino acid
Bond
Compound
of receptor length (°A)
-C≡N
TYR 365
LYS 297
LYS 297
TYR 365
TYR 365
LYS 297
LYS 297
TYR 365
TYR 365
LYS 297
GLU 287
-
2.521
2.942
3.009
2.956
2.522
2.213
3.016
2.996
2.493
2.929
2.559
-
8a
8c
-C=O of cyclohexene
-C=O of cyclohexene
-C=O of amide
-C≡N
8d
8l
-C=O of cyclohexene
-C=O of cyclohexene
-C=O of amide
-C≡N
8m
-C=O of cyclohexene
-NH2
8o
No H-bonding
-OH
Chloroquine
Quinine
TYR 322
3.248
a
b

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c
e
g
d
f
h
Fig. 1 shows all ligands docked in best of its conformation with receptor 4DPD. (a) ligand 8a
bound to 4DPD; (b) ligand 8c bound to 4DPD; (c) ligand 8d bound to 4DPD; (d) ligand 8l
bound to 4DPD; (e) ligand 8m bound to 4DPD; (f) ligand 8o bound to 4DPD; (g) ligand
Chloroquine bound to 4DPD and (h) ligand Quinine bound to 4DPD

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4.3.4.1. In silico pharmacokinetic evaluation
The drug-likeness of the molecules synthesized was identified by predicting the
pharmacokinetic properties in silico. The potent molecules (8a, 8c, 8d, 8l, 8m, 8o) were
preliminarily screened considering the basic parameters of Lipinski’s rule of five, BBB,
Caco2, %HIA etc. The pharmacokinetic parameters were obtained by VlifeMDS 4.6 and
ADMET. The data found are presented in Table 7 and Table 8. For an orally active
compound, two violations of the Lipinski’s rule are acceptable. The molecules of the present
study were found to follow the rule with maximum violation of two, thus demonstrating their
drug-likeness properties.
Table 7. Evaluation of pharmacokinetic parameters by VlifeMDS 4.6
Mol. Wt.a
(gm/mol)
610.15
PSAf
(°A2)
66.77
83.78
66.77
83.48
66.77
66.77
Compound
HBDb
HBAc
logPd
RotBe
2
4
2
4
2
2
6
7
6
7
6
6
7.478
6.669
7.214
6.770
6.951
7.734
6
6
6
6
6
7
8a
8c
628.16
589.73
8d
8l
646.15
593.70
8m
8o
643.70
a = Molecular Weight ≤ 500 (gm/mol) [35]; b = Hydrogen Bond Acceptor ≤ 10 [35]; c =
Hydrogen Bond Donor ≤ 5 [35]; d = Rotatable Bonds ≤ 10 ; e =logP ≤ 5 [36]; f = Polar
Surface Area ≤ 140°A2 [36]
The logP value and molecular weight of these compounds violated the Rule of Five (RO5)
(Table 7). The oral bioavailability of the drug molecules are greatly influenced by the
optimum values of the descriptors like the polar surface area and rotatable bonds. The
important pharmacokinetic parameters with their permissible ranges are delineated in Table
7. The active quinolone derivatives showed optimum values of hydrogen bond donor (≤ 5)
and hydrogen bond acceptor (≤ 10) and thus owing good bioavailability.

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Table 8. Evaluation of pharmacokinetic parameters by ADMET
Compound
Caco2
(nm/sec)
41.57
MDCK
BBB
% HIA
(nm/sec)
0.0434176
0.0434176
0.0434176
0.0434158
0.0434183
0.0434160
0.0832
0.0497
0.1542
0.0426
0.0718
0.1350
97.67
96.97
97.42
96.98
97.37
97.44
8a
8c
25.63
38.54
8d
8l
25.92
39.09
8m
8o
38.31
BBB (Blood Brain Barrier) [37]: High absorption CNS >2.0, Middle absorption CNS 2.0-0.1,
Low absorption to CNS <0.1; Caco2 [38]: High permeability >70, Middle permeability 4-70,
Low permeability <4; %HIA (Human Intestinal Absorbance) [38]: Well absorbed
compounds 70-100%, Moderately absorbed compounds 20-70%, Poorly absorbed
compounds 0-20%; MDCK [39]: Higher permeability >500, Medium Permeability 25-500,
lower permeability <25.
The molecules except 8d and 8o showed moderate absorption to central nervous system.
Human intestinal absorption is one of the important factors to be studied in relation to the
absorption of the drug molecule, which was further confirmed by predicted Caco2 cell
permeability. However, the value of %HIA suggested that they had higher absorption to
intestine but moderate permeability to Caco2 cell. Moreover, these molecules had lower
MDCK permeability. The results showed that these compounds might be good candidates for
drug.
5. Conclusion
In finding out of new structural motifs looking promising as powerful antimicrobial,
antimalarial and antituberculosis agents, we have demonstrated the multicomponent synthesis
of 16 new polyhydroquinoline derivatives bearing a polyhydroquinoline nucleus. Many of
these new polyhydroquinoline heterocycles displayed noticeable antibacterial activities.
Amongst the screened compounds 8e, 8i, 8k, 8l, and 8p were found to be superior
antituberculosis agents against M. tuberculosis H37Rv.Some of the compounds 8a, 8c, 8i, 8l
and 8o demonstrated more significant antimalarial activity against strains of P. falciparum as
compared to quinine. In silico docking study demonstrated the potentiality of these
molecules to interact and inhibit the enzyme P. Falciparum dihydrofolate reductase (DHFR)
by occupying the active binding pocket with great ease. To avoid late stage failure, it is

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important to study the preliminary pharmacokinetic parameters. The results of in silico
pharmacokinetic data suggested that, molecules may be considered as a drug.
6. Experimental section
6.1. Chemistry
All the reagents were obtained commercially and used without further purification.
Solvents used were of analytical grade. Melting points (°C, uncorrected) were
determined in open capillaries on µThermoCal10 melting point apparatus (Analab
Scientific Pvt. Ltd, India). Precoated silica gel plates (silica gel 0.25 mm, 60 G F 254;
Merck, Germany) were used for thin layer chromatography. Electron impact Mass
Spectra were recorded on Shimadzu LCMS 2010 spectrometer (Shimadzu, Tokyo,
Japan) purchased under PURSE programme of DST at Sardar Patel University,
Vallabh Vidyanagar, India. The IR spectra were recorded on Shimadzu FTIR 8401
spectrophotometer using potassium bromide pellets in the range 4000–400 cm-1and
frequencies of only characteristic peaks are expressed in cm-1. The elemental analysis
was performed on Perkin-Elmer 2400 series-II elemental analyzer (Perkin-Elmer,
USA) at Sophisticated Instrumentation Centre for Applied Research & Training
(SICART), Vallabh Vidyanagar, India. All the compounds were found to be within
0.4% of their theoretical values. The reaction mixtures were irradiated by ultrasound
at room temperature in a D-compact ultrasonic cleaner with a frequency of 30 kHz and
an output power of 250 W. The reaction flask was kept at the maximum energy area in
the cleaner and the level of the reactants was kept slightly lower than the level of water
1
in the bath. H NMR spectra (in DMSO-d6) were recorded on Bruker Avance 400F
(MHz) NMR Spectrometer at 400 MHz using TMS as the internal standard.
6.1.1. General procedure for the synthesis of 3-methyl-5-substituted phenylthio-1-phenyl-
1H-pyrazole-4-carbaldehydes (3a-c)
5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 1(1 mmol), substituted thiophenols
2a–c (1 mmol) and anhydrous potassium carbonate (2.5 mmol) in dimethyl formamide (10
mL) were charged in a 100 mL round bottom flask equipped with a mechanical stirrer and a
°
condenser. The reaction mixture was heated at 90 C for 2 h and the progress of the reaction
was monitored by TLC. After the completion of reaction as confirmed by the TLC, the
reaction mixture was poured in to 100 mL ice water. The solid separated was filtered, washed
thoroughly with water, dried and recrystallized from hot ethanol (10 mL) to obtain 3a-c.

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6.1.2. General procedure for the synthesis of substituted 3-((substituted)amino)-5,5-
dimethylcyclohex-2-enone (6a-c).
1,3-Dimedone 4 (10 mmol), fluoro substituted amine 5a–c (10 mmol) and methanol (10 mL)
with catalytic amount of acetic acid were charged in a 100 mL round bottom flask equipped
with a mechanical stirrer. The reaction mixture was stirred at room temperature for 2 h. After
the completion of reaction (checked by TLC), the separated substituted enhydrazinoketones
6a–c were filtered and washed with methanol to obtain the pure solid product.
6.1.3. General procedure for the synthesis of 2-amino-4-(5-((substituted phenyl)thio)-3-
methyl-1-phenyl-1H-pyrazol-4-yl)-1-(phenylamino)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carbonitrile (8a-p)
A 50 mL round bottom flask, fitted with a reflux condenser, was charged with a mixture of 3-
methyl-5-substituted phenylthio-1-phenyl-1H-pyrazole-4-carbaldehydes (3a-c) (1 mmol),
malononitrile 7a or ethylcynoacetate 7b or cynoacetamide 7c (1 mmol), substituted
enaminones 6a-c (1 mmol), and catalytic amount of piperidine (2-3 drops) in ethanol (10
mL). The mixture was heated under reflux for 1-3 h and the progress of the reaction was
monitored by TLC. After the completion of reaction, the reaction mixture was cooled to room
temperature and stirred magnetically for further 10 min. The solid mass separated was
collected by filtration, washed well with ethanol (10 mL) and crystallized from hot
chloroform. The physicochemical and spectroscopic characterization data of the synthesized
compounds 8a-p are added in to the Supplementary Information.
Acknowledgment
We sincerely express our thanks to Head, Department of Chemistry, Sardar Patel University,
for providing necessary research facilities. We are also thankful to Dhanji P. Rajani,
Microcare Laboratory, Surat for antimicrobial, antituberculosis and antimalarial screening of
the compounds reported herein and Sophisticated Instrumentation Centre for Applied
Research and Training (SICART), Vallabh Vidyanagar for FT-IR analysis at concessional
rate. BKV, RPT are thankful to UGC meritorious fellowship and NHS specially thanks the
UGC, New Delhi, India for providing financial assistance under the UGC-JRF Scheme and
also for mass analysis at PURSE central facility at Sardar Patel university sponsored under
PURSE program grant vide sanction letter DO. No. SR/59/Z-23/2010/43 dated 16th March
2011.

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Graphical Abstract