Identification and structure–activity relationship (SAR) studies of carvacrol derivatives as potential anti-malarial against Plasmodium falciparum falcipain-2 protease

Article abstract of DOI:10.1016/j.bioorg.2020.104142

In an effort to develop a potent anti-malarial agent against Plasmodium falciparum, a structure-guided virtual screening using an in-house library comprising 652 compounds was performed. By docking studies, we identified two compounds (JMI-105 and JMI-346

Full text of DOI:10.1016/j.bioorg.2020.104142

Journal Pre-proofs
Identification and structure-activity relationship (SAR) studies of carvacrol
derivatives as potential anti-malarial against Plasmodium falciparum Falci-
pain-2 protease
Amad Uddin, Vigyasa Singh, Iram Irfan, Taj Mohammad, Rahul Singh Hada,
Md Imtaiyaz Hassan, Mohammad Abid, Shailja Singh
PII:
S0045-2068(20)31439-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104142
YBIOO 104142
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
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30 June 2020
23 July 2020
Please cite this article as: A. Uddin, V. Singh, I. Irfan, T. Mohammad, R. Singh Hada, M. Imtaiyaz Hassan, M.
Abid, S. Singh, Identification and structure-activity relationship (SAR) studies of carvacrol derivatives as
potential anti-malarial against Plasmodium falciparum Falcipain-2 protease, Bioorganic Chemistry (2020), doi:
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Identification and structure-activity relationship (SAR) studies of
carvacrol derivatives as potential anti-malarial against
Plasmodium falciparum Falcipain-2 protease
Amad Uddin1, 2‡, Vigyasa Singh , Iram Irfan , Taj Mohammad , Rahul Singh Hada ,
2‡
1
3
4
3
1*
2*
Md Imtaiyaz Hassan , Mohammad Abid and Shailja Singh
1Medicinal Chemistry Laboratory, Department of Biosciences, Jamia Millia Islamia, Jamia
Nagar, New Delhi, 110025, India
2
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, 110067,
India
3
Center for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar,
New Delhi-110025, India
4
Department of Life Sciences, Shiv Nadar University, Gautam Buddha Nagar UP, 201314,
India
✉
To whom correspondence should be addressed: mabid@jmi.ac.in (M. Abid), shailja.jnu@gmail.com (S.
Singh).
‡These authors contributed equally to this work.
1

Abstract: In an effort to develop a potent anti-malarial agent against Plasmodium
falciparum, a structure-guided virtual screening using an in-house library comprising 652
compounds was performed. By docking studies, we identified two compounds (JMI-105 and
JMI-346) which formed significant non-covalent interactions and fit well in the binding pocket
of PfFP-2. We affirmed this observation by MD simulation studies. As evident by the
biochemical analysis, such as enzyme inhibition assay, Surface Plasmon Resonance (SPR),
live-cell imaging and hemozoin inhibition, JMI-105 and JMI-346 at 25µM concentration
showed an inhibitory effect on purified PfFP-2. JMI-105 and JMI-346 inhibited the growth of
S
R
CQ (3D7; IC = 8.8 and 13µM) and CQ (RKL-9; IC = 14.3 and 33µM) strains of P.
5
0
50
falciparum. Treatment with compounds resulted in defect in parasite growth and development.
No significant hemolysis or cytotoxicity towards human cells was observed suggesting that
these molecules are non-toxic. We pursued, structural optimization on JMI-105 and in the
process, SAR oriented derivatives (5a-5l) were synthesized and evaluated for growth inhibition
potential. JMI-105 significantly decreased parasitemia and prolonged host survival in a murine
model with P. berghei ANKA infection. The compounds (JMI-105 and JMI-346) against
PfFP-2 have the potential to be used as an anti-malarial agent.
Keywords: Plasmodium falciparum; Malaria; Falcipain-2; Virtual screening; SAR studies.
2

1
. Introduction
Malaria is one of the most endemic tropical parasitic diseases, caused by blood born
Apicoplexan parasite Plasmodium falciparum (P. falciparum) around the world [1, 2]. It is
responsible for about one million deaths annually [3, 4]. Five species of Plasmodium out of
2
00 known ones are able to infect human, which includes P. falciparum, P. vivax, P. ovale, P.
knowelsi and P. malariae. P. falciparum is the most lethal one and responsible for over 95%
cases worldwide [5]. Irrespective of the efforts made to control malaria under the eradication
programs, in 2018, the World Health Organization (WHO) estimated 228 million cases
worldwide, and 4,05,000 death [6]. The emergence and spread of the resistance to the
commonly available and inexpensive drugs limited the efficiency of anti-malarial therapies
resulting in an increase of the mortality rate [7]. To combat spread of multidrug-resistance in
P. falciparum, the WHO recommended Artemisinin based Combination Therapies (ACTs).
However, there have been reports on decreased susceptibility of the parasite to artemisinins in
the Greater Mekong Subregion (GMS), followed by ACTs’ failure leading to a worrisome
situation. [8]. Therefore, it becomes imperative to explore newer strategies for the control and
eradication of malaria.
Proteases of P. falciparum act are the attractive target for most of the drugs playing
crucial role in growth, and development of the parasite during its life cycle [9]. A complete
genome of P. falciparum consists of 33 open reading frames that encode cysteine proteases and
a family of four cathepsin L-like papain proteases collectively known as falcipains [10].
Falcipain-2 (PfFP2) and falcipain-3 (PfFP3) are key papain-family (C1) Clan CA trophozoite
cysteine proteases which lies in the digestive food vacuole that cleaves host hemoglobin (native
and denature) and also responsible for erythrocyte rupture. The inflation of malarial parasite
inside the infected red blood cell is proportionate to rate of its food assimilation [11]. Cysteine
proteases of Plasmodium are implicated with several biological processes like, rupture of
membranes, degradation of Hb, protein trafficking and host cell invasion [12, 13]. Thus, the
growth of malarial parasites is measured by the interference of key indicator protein PfFP2
[14]. Among falcipains, the most expressed and well-studied one is PfFP2 which is an attractive
target for the discovery of antimalarial drug [15].
Virtual screening methods have helped in identifying new leads for drug development
[16-18]. Structure-based virtual screening (SBVS) is commonly used for the identification of
3

potential compounds preferentially binding to a drug target [19]. SBVS involves computational
methods, including molecular docking and dynamic simulations of receptor-ligand and their
complexes [20]. First, the compounds were screened based on their binding affinities and
predicted interactions with the target and those with the higher scores (hits) were selected for
experimental activity assays. The availability of the X-ray crystal structure of PfFP2 in
complex with its known inhibitor E64 (PDB ID: 3BPF) opened newer avenue to discover its
inhibitors through structure-based virtual screening of natural/synthetic libraries as potent anti-
malarials. In the present study, an in-house library of 652 compounds was screened through
DruLiTo for their likelihood of drug molecules based on their physicochemical and ADMET
properties. The selected compounds were evaluated for their ADME/T properties. Further, we
used AutoDock Vina to identify high-affinity binding partners of PfFP2 by utilizing molecular
docking approach. The compounds showing high binding affinity towards PfFP2 were selected
further for interaction analysis and molecular dynamics (MD) simulations. We identified two
compounds (JMI-105 and JMI-346) as potential PfFP2 inhibitors and subjected them to
various biochemical studies. This included growth inhibition, live-cell imaging and
cytotoxicity assays and in vivo studies for their evaluation as potential anti-malarials.
2
. Results and Discussion
2
.1. In silico studies
In silico studies were conducted in search of antimalarial compounds by using different
bioinformatics tools encompassing AutoDock Vina [21], PyRX and Discovery Studio [22],
PyMOL [23], online resources such as RCSB Protein Data Bank, SwissADME [24],
CarcinoPred-EL [25], MGL Tools [26], VMD (Visual Molecular Dynamics), QtGrace [27] and
GROMACS [28]. These were used for data visualization, data retrieval, evaluation, and
molecular dynamics (MD) simulations.
2
.1.1. Drug-likeliness assessment of In-house library
The Lipinski's criteria generally referred to as the ''Rule of Five'' (Ro5), which has been used
as a part of the assessment of presumable oral accessibility of the selected compounds. Thus,
the drug-potential of selected compounds was assessed by calculating the Lipinski’s Ro5
descriptors. Out of 652 compounds, a total of 46 fulfilled all the Ro5 criteria showing well
defined physicochemical properties of which 17 were selected. The physicochemical properties
4

of the selected compounds are listed in (Supplementary data), while two of the selected
compounds JMI-105 and JMI-346 and their physicochemical properties are shown in Table
1
. Both compounds are showing drug-like properties following the Lipinski's rule of five.
Table 1: Physicochemical properties of JMI-105 and JMI-346 compounds
Compound
Mol.
Rotatable
Lipinski
HBD
HBA
Log P
ID
Weight
bond
Violation
JMI-105
337.18
0
1
6
5
6
3.69
2.15
0
JMI-346
336.16
7
0
On the basis of ADMET predictions, selected compounds were further filtered out. Out of 17
compounds, 12 were showing favorable ADMET properties (Supplementary data). All these
compounds were found to be non-carcinogenic as predicted through Carcinopred-EL. ADMET
properties and carcinogenicity of compounds JMI-105 and JMI-346 mentioned in (Table 2).
Table 2: ADMET properties of JMI-105 and JMI-346 compounds
Ratio
Compound
ID
Rigid
Bond
Solubilit
y (mg/l)
C
logD
logS
F
Carcinogen
Atoms
H/C
0.25
0.32
JMI-105
JMI-346
4.93
3.11
-4.51
-3.33
17
18
0.26
0.28
3698.60
12031.2
20
19
NC
NC
2
.1.2. Molecular docking
Molecular docking of 12 compounds showed discernible binding affinity with PfFP-2 than
with PfFP-3. The possible confirmations of 12 selected compounds were generated using Vina
Split program. A total of 108 docked confirmations of 12 selected compounds were subjected
to interaction analysis towards PfFP-2 and PfFP-3 (Supplementary data). Out of 12
compounds, JMI-105 and JMI-346 showed binding affinity with PfFP-2, calculated as -7.5
kcal/mol and -7.3 kcal/mol, respectively. These compounds showed binding with the ligand-
binding site of FP-2 interacting with several important residues of PfFP-2. The interactions of
JMI-105 and JMI-346 compounds along with E-64 (Co-crystallized ligand) are shown in
5

(Table 3). The interaction analysis suggests that both compounds occupy the same position of
FP-2 binding site mimicking the binding pose of each other (Fig. 1A). The PfFP-2-JMI-105
and PfFP-2-JMI-346 complexes stabilized by 3-4 hydrogen bonds showing several other
significant interactions (Fig. 1B, 1C).
Table 3. Molecular interaction of JMI-105, JMI-346 and E-64 (Co-crystallized ligand) with
PfFP-2 binding site
Protein-ligand Interaction
Affini
ty
Hydrogen Bond
Comp
ID
Other
Compound structure
Distance
Å)
Interacting
Residues
(kcal/
Residues
(
mol)
Ala157, Trp210,
Ser205, Lys37,
Asn173, Gln36,
Val152, Asn38,
His174
Lys37
Asp35
Trp206
3.08,
2.85,
2.93
JMI-105
O
-7.5
O
N
N
N
Gly209, Lys37,
Asp35, His174,
Cys42, Asn173,
Gly82, Asn81,
Ala157
3
.09,
3.25,
.02
O
Gln36
JMI-346
E-64
O
-7.3
-5.1
N
N
Trp206
NH2
N
3
HN
NH2
Gln36, Ala157,
Phe158, Trp210,
Asn173
HOOC
H
H
O
H
His174
Trp206
2.92,
2.82
N
NH
O
N
H
O
6

Figure 1: Structural organization of PfFP-2 with JMI-105 and JMI346. (A) Cartoon
representation of PfFP-2 showing the docked JMI-105 and JMI-346. Zoomed view of PfFP-
2
binding site with JMI-105 and JMI-346 (right panel). (B) 2-D representation PfFP-2 residues
interacting to JMI-105 and (C) JMI-346.
2
.1.3. MD simulations
MD simulations technique has been widely used to assess the internal motion, physical
arrangements and environmental-induced structural changes in proteins and their interactions
with other chemical molecules [47]. MD simulations can describe the dynamics of the binding
mechanism of a small molecule to a protein under an explicit solvent surroundings [48]. Here,
7

we performed all-atom MD simulations of free PfFP-2 and JMI-105 and JMI-346 docked
complexes of PfFP-2 for 30 ns to evaluate the conformational changes, stability and interaction
mechanism. Average potential energies of free PfFP-2, PfFP-2-JMI-105 and PfFP-2-JMI-346
complexes were calculated to ascertain the equilibration and stability of the systems prior to
MD analysis. The potential energy of free PfFP-2, PfFP-2-JMI-105 and PfFP-2-JMI-346
systems was calculated as -9,10,842 kJ/mol, -9,10,083 kJ/mol and -9,10,066 kJ/mol,
respectively. The volume, density, kinetic energy, enthalpy, and total energy of the systems
were also calculated over the simulations (Table 4).
Table 4: Calculated parameters for all three systems obtained after 30 ns MD simulations.
Rg
RMSD RMSF
SASA Kinetic
(nm2)
energy
Volume Density
(nm3)
(g/l)
System
Enthalpy
(nm)
(nm)
(nm)
PfFP-2
0.13
0.19
0.24
0.08
0.09
0.12
1.72 129.30 1,43,282 -763570
1.67 122.85 1,42,810 -762364
1.73 113.48 1,46,460 -762182
595.17 1,028.44
588.91 1,029.51
593.54 1,021.21
PfFP-2-JMI-105
PfFP-2-JMI-346
In the globular structure of a protein, the large conformational changes can occurs due to
binding of a small molecule [49, 50]. The stability and evaluation of structural deviation can
be determined by studying the Root mean square deviation (RMSD) of the structures [51]. The
averages RMSD of free PfFP-2, PfFP-2-JMI-105 and PfFP-2-JMI-346 complexes were found
to be 0.13 nm, 0.19 nm and 0.24 nm, respectively (Table 4). The RMSD of all three systems
do not show any significant change suggesting the stabilized binding of compounds with PfFP-
2
having fewer conformational changes (Fig. 2A). In RMSD plot, upon binding of compounds
the random variation can be seen up to 10 ns, which may be due to their initial orientation in
the binding pocket of PfFP-2. Afterward, PfFP-2 in the presence of JMI-105 and JMI-346
showed equilibration in RMSD throughout the simulation trajectory (Fig. 2A). The RMSD plot
suggested enough stability of the protein-ligand complexes. The RMSD showed the PfFP-2-
JMI-105 complex more compact and stable as compared to the PfFP-2-JMI-346 complex.
We plotted the root-mean-square fluctuation (RMSF) value of each residues to assess the
residual flexibility present in free PfFP-2 and PfFP-2 in complex with JMI-105 and JMI-346
(Fig. 2B). The residual fluctuations were shown in RMSF plot at variable regions of the PfFP-2
8

structure and stabilizations throughout the simulation. The average RMSF of free PfFP-2 and
in complex with JMI-105 and JMI-346 was obtained as 0.08 nm, 0.09 nm and 0.12 nm,
respectively. Still, many increasing residual fluctuations can be seen after the binding of JMI-
3
46 with PfFP-2, which might be due to the systematic vibrations in the protein during
simulation. Fewer increased fluctuations in the PfFP-2 after JMI-346 binding was noticed
which might be due to ligand adjustment in the binding pocket of the protein. The RMSF
analysis also showed that the PfFP-2-JMI-105 complex is more compact and stable compared
with PfFP-2-JMI-346.
The structural parameters of a protein, such as tertiary volume and overall globular state can
be signified by the radius of gyration (R ) which is broadly used to analyze the protein’s
g
stability under biological environment [2]. The average values of R for free PfFP-2, PfFP-2-
g
JMI-105 and PfFP-2-JMI-346 complexes were calculated as 1.72 nm, 1.67 nm and 1.73 nm,
respectively. R plot showed slight changes but no significant structural switching was
g
observed in binding of PfFP-2 packing with JMI-105 and JMI-346 compounds. PfFP-2 in
presence of JMI-105 and JMI-346 attained a higher R as compared with free PfFP-2, which
g
is equilibrated throughout the simulations (Fig. 2C). The results showed a fewer structural
deviation in PfFP-2 upon binding with JMI-105 and JMI-346 compounds as no
conformational shift was observed in the R plot. While comparing both the complex systems,
g
PfFP-2-JMI-105 is more compact and stable than PfFP-2-JMI-346.
The surface area of a protein that is accessible to its surrounding solvent is termed as Solvent
accessible surface area (SASA) which is precisely associated to the R of a protein [51]. The
g
SASA for free-PfFP-2, PfFP-2-JMI-105 and PfFP-2-JMI-346 complex over the simulations
was calculated. An average of SASA for PfFP-2 protein alone and in complex with JMI-105
2
2
2
and JMI-346 were found to be 129.30 nm , 122.85 nm and 113.48 nm , respectively. Change
in SASA suggested few conformational adjustments in PfFP-2 upon binding with JMI-105
and JMI-346 compounds (Fig. 2D). However, the SASA of PfFP-2-JMI-105 system attained
a stable equilibrium without any switching during the entire simulation suggesting structural
stability of PfFP-2 in the presence of JMI-105. A decrease in SASA was observed in case of
PfFP-2-JMI-346 possibly due to increased surface area of PfFP-2 in presence of JMI-346
where some outer residues might be buried to the inner core of the protein.
The protein stability is fundamentally determined by intramolecular hydrogen bonding. The
stability of polar contact between a protein and ligand can also be evaluated by studying the
9

dynamics of Hydrogen bonds that can provide a direction, and specificity of protein-ligand
interaction [52]. To further evaluate the stability of free-PfFP2, PfFP2-JMI-105 and PfFP2-
JMI-346 complex, hydrogen bonds paired within 0.35 nm were calculated over the
simulations. An average number of intra-protein hydrogen bonds within PfFP-2 before and
after binding with JMI-105 and JMI-346 were found to be 160, 159 and 157, respectively
(Fig. 2E). A little decrement in hydrogen bonding within PfFP2 itself might be due to the
occupancy of some intramolecular space by the compounds. The analysis of hydrogen bond
dynamics indicates that both complexes are quite stable with minimal change. While analyzing
the dynamics of Hydrogen bonding between the compounds and the protein, an average of
three hydrogen bonds were found between PfFP-2-JMI-105 and PfFP-2-JMI-346 throughout
the simulations. It was observed that JMI-105 and JMI-346 bind in the binding site of PfFP-2
with 5-6 conventional hydrogen bonds with higher fluctuations, and 3-4 hydrogen bonds with
least fluctuations, which were consistent with our molecular docking findings (Fig. 2 F-G).
1
0

Figure 2: Structural dynamics of PfFP-2 upon binding with JMI-105 and JMI-346
compounds. (A) The RMSD plot of PfFP-2 and its docked complexes (B) Residual fluctuations
(RMSF) plot of PfFP-2 upon binding with JMI-105 and JMI-346 (C) Time evolution of radius
of gyration (R ) (D) SASA plot of PfFP2 as a function of time. The values were obtained from
g
3
2
0 ns MD simulations time scale. Black, red and green represent values obtained for free PfFP-
, PfFP-2-JMI-105 and PfFP-2-JMI-346 complex, respectively. (E) Time evolution and
stability of hydrogen bonds formed intra-protein within PfFP2, and (F) Hydrogen bonds
formed between JMI-105 and PfFP2 (G) and Hydrogen bonds formed between JMI-346 and
PfFP2. The values were obtained from 30 ns MD simulations time scale. Black, red and green
represent values obtained for free PfFP-2, PfFP-2-JMI-105 and PfFP-2-JMI-346 complex,
respectively.
1
1

The dynamics of the secondary structure content in PfFP-2 upon binding with JMI-105 and
JMI-346 were investigated to assess the changes in PfFP-2. Structural components such as α-
helix, -sheet and turn in PfFP-2 were broken into distinct amino acid residues for each time
step, and subsequently, average residues were calculated. The structural elements in free-PfFP-
2
remain continual with the least fluctuations, which were equilibrated throughout the entire
simulations (Fig. 3). The changes in free-PfFP-2 remain continual with fewer fluctuations and
equilibrated throughout the simulations (Fig. 4). Overall residues participating in secondary
structure elements of PfFP-2 upon JMI-105 and JMI-346 binding were found to be decreased
to some extent due to bend formation in the case of PfFP-2-JMI-105, and coil formation in
PfFP-2-JMI-346 complex as compared to free PfFP-2 (Fig. 4 and Table 5). Overall, we did
not observe any significant change in the secondary structure content of PfFP-2 upon JMI-105
and JMI-346 binding that showed good stability of the complexes.
Table 5: Percent residues participating in the secondary structure formation of PfFP2.
Residues participating in the secondary structure formation (%)
System
Structure* Coil
β-sheet β-bridge Bend Turn α-helix Other#
PfFP2
52
51
49
32
31
33
18
19
17
3
3
2
13
15
17
11
9
20
20
22
3
3
1
PfFP-2-JMI-105
PfFP-2-JMI-346
8
#
*
Structure = α-helix + β-sheet + β-bridge + Turn; Other = 5-Helix + 3-Helix
Figure 3: Secondary structure content of (i) Free PfFP2 and (ii) PfFP2 upon JMI-105 binding
and (iii) PfFP2 upon JMI-346 binding. *Structure = α-helix + β-sheet + β-bridge + Turn.
2
.2. Chemistry
1
2

Based on the virtual screening of our in house library of 652 compounds, JMI-105 and JMI-
46 were selected (Supplementary data) as mentioned earlier. JMI-105 is a semi-synthetic
triazole derivative of a natural alcohol carvacrol and has been reported previously by our lab
33]. Briefly, carvacrol was propargylated in the presence of propargyl bromide and K CO to
3
[
2
3
yield alkyne. In another set of reaction, aniline bearing p-OCH group was converted into
3
corresponding azide. Together, azide & alkyne underwent Cu(I)- catalyzed [3+2] cycloaddition
reaction in the presence of CuSO .5H O in catalytic amount & sodium ascorbate in THF/H O
4
2
2
(
1:2) mixture to yield JMI-105. Another compound JMI-346 is chemically a triazole ester of
phenylalanine bearing p-methyl substitution on the phenyl ring was also reported previously
34]. Briefly, boc-protected L- phenylalanine was coupled with propargyl bromide in the
presence of K CO using DMF as solvent to give alkyne in good yield. In another reaction,
[
2
3
diazotization of p-toluidine with NaNO and concentrated HCl in the presence of NaN led to
2
3
corresponding azide. Later, both alkyne and azide were reacted to obtain the boc-protected
triazole derivative via Huisgen 1, 3-diapolar cycloadditon reaction in the presence of
CuSO .5H O and sodium ascorbate in THF: H O (1:2) mixture. Finally, deprotection of boc
4
2
2
group was carried out using p-toulene sulfonic acid (PTSA) in CH Cl to get JMI-346.
2
2
2
.3. Biochemical inhibition of PfFP-2
2
.3.1. Enzyme kinetics
Plasmodium cysteine proteases are responsible for different biological progressions. This
includes protein trafficking, rupture of membranes, Hb degradation, host cell invasion, and
egress from host erythrocytes [12, 53-56]. Among the probable targets for drugs leads
falcipain-2 (PfFP-2), known cysteine proteases hydrolyze Hb to provide amino acids for
parasite protein synthesis. It is important to find inhibitors of falcipain-2 with suitable
properties for novel antimalarial drugs [57-60].
Recombinant PfFP-2 was prepared as described earlier [36]. Enzymatic activity of
recombinant PfFP-2 was measured by spectrofluorometry; incubated with substrate Z-Phe-
Arg-AMC, a known substrate for cysteine proteases. Cleavage and release of fluorogenic AMC
was observed at Em=355 nm and Ex= 460 nm. The result showed the percentage inhibition of
PfFP-2 in a dose-determined mode by JMI-105 and JMI-346 at different concentrations (1, 5,
7
.5, 10, 12.5, 17.5, 20, 22.5 and 25μM) (Fig. 4A). The IC for JMI-105 and JMI-346 against
50
recombinant PfFP-2 was found to be 20.56μM and 25.79μM respectively. PfFP-2 was found
1
3

to be responsible for cleavage of peptide substrate and release of fluorogenic AMC. Reduction
of hydrolysis of Z-Phe-Arg-AMC was observed in JMI-105 and JMI-346 samples as
compared with untreated samples in a dose dependent manner. Therefore, expansion in
percentage inhibition of PfFP-2 with JMI-105 and JMI-346 treated samples showed their
function to block the PfFP-2.
2
.3.2. Hemoglobin degradation assay
Malaria parasites follow proteolytic catabolic pathway for degradation of Hb for their own
growth and maturation. Catabolic pathway results in generation of peptides and amino acids in
food vacuoles of the parasite [38, 54, 61, 62]. Inhibition of hemoglobinase activity of PfFP-2
by JMI-105 and JMI-346 was observed with SDS PAGE and spectrophotometer [36]. JMI-
1
05 and JMI-346 at 1, 5, 12.5 and 25µM concentrations were used for incubation with Hb and
recombinant PfFP-2 in sodium acetate buffer (pH 5.5) [38]. SDS PAGE analysis confirmed
inhibition of degradation of Hb by JMI-105 at the 12.5µM concentration and JMI-346 at
2
5µM concentration (Fig. 4B). The percent decrease in Hb hydrolysis was observed by
spectrophotometer in dose dependent manner with the treatment of JMI-105 and JMI-346 as
compared to untreated control confirming that they block the PfFP-2 activity. Use of 12.5 and
2
4
4
1
5µM concentration of JMI-105 and JMI-346, showed percentage of Hb hydrolysis to be
4.63 ± 5.16% and 34.05 ± 2.63% and 59.88 ± 2.95% and 45.97 ± 4.63% respectively (Fig.
C). Thus, JMI-105 and JMI-346 effectively blocks the activity of PfFP-2. However, JMI-
05 was observed to be more effective as compared to JMI-346 with respect to PfFP-2
inhibition activity.
2
.3.3. Surface Plasmon Resonance (SPR)
To confirm the interaction of PfFP-2 with JMI-105 and JMI-346, we studied its strength
between the protein-drug using surface plasmon resonance (SPR). Different concentrations of
JMI-105 and JMI-346 were injected over immobilized recombinant PfFP-2. E-64 was used as
positive control. We found that JMI-105 and JMI-346 at increasing concentration of 1, 10, 25,
5
0, 75µM interacted with recombinant PfFP-2. The dissociation equilibration constants (Kd)
for the compounds JMI-105 and JMI-346 were found to be 56.7μM and 74.0μM indicating
direct binding to the recombinant PfFP-2 (Fig. 4D).
1
4

A.
JMI−105
JMI−346
6
4
2
0
0
0
0
50
40
30
20
10
0
1
uM
5uM
7.5uM
10uM
12.5uM
17.5uM
20uM
22.5uM
25uM
1uM
5uM
7.5uM
10uM
12.5uM
17.5uM
20uM
22.5uM
25uM
(Concentration)
(Concentration)
C.
100
B.
80
JMI-105
JMI-346
60
2
-
4
6
b
-
P
F
H
E
40
1
6 KDa
20
6
.5 KDa
0
2
P
-64
)
)
)
)
)
)
)
)
M
M
5
M
M
M
M
M
M
F
E
µ
(5µ
.5µ
25µ
(1µ
(5µ
.5µ
25µ
(
1
5
-105
12
(
-346
-346
12
(
6
0
(
5
(
I-1
-10
6
-34
I
I
I-10
I
I
4
I
I-3
M
M
M
M
J
J
M
J
J
M
J
M
J
M
J
J
D.
2
40
10
80
50
E-64
JMI-105
JMI-346
1
60
40
400
350
300
250
200
150
100
2
1
1
1
120
100
80
1µM
1
1
2
µM
0µM
5µM
1µM
2.5µM
5µM
10µM
20µM
1
0µM
25µM
0µM
75µM
120
5
50µM
75µM
9
0
0
60
6
40
5
0
20
30
0
0
0
1
51 101 151 201 251 301 351 401 451 501
1
51 101 151 201 251 301 351 401 451 501
1
51 101 151 201 251 301 351 401 451 501
-50
-20
-30
Time (s)
Time (s)
Time (s)
Figure 4: (A) Shows PfFP-2 inhibition activity by JMI-105 and JMI-346. Hydrolysis of
substrate Z-Phe-Arg-AMC was calculated. Recombinant PfFP-2 was treated with JMI-105 and
JMI-346 at different concentrations. Z-Phe-Arg-AMC was added to the samples and incubated
to obtain fluorescence intensity. The graph showed percentage increase inhibition of PfFP-2,
indicating attenuation of hydrolysis of substrate peptide. (B) Hemoglobin hydrolysis by PfFP-
2
and its inhibition by JMI-105 and JMI-346 were checked by SDS PAGE. The dose
dependent manner attenuation in hemoglobin hydrolysis was observed in treated samples. (C)
Inhibition of hemoglobin hydrolysis by JMI-105 and JMI-346 was observed using
spectrophotometer by taking absorbance at 410 nm. Native Hb was treated with recombinant
PfFP-2 and JMI-105 and JMI-346 at 1, 5, 12.5, 25µM concentrations. Data was expressed as
1
5

mean values ± SD. Experiments were carried out in duplicate. (D) shows Surface plasmon
resonance analysis. Recombinant PfFP-2 was immobilized on surface of nickel charged NTA
SPR chip. Experiment was performed with increasing concentrations of JMI-105 and JMI-
3
46 (1, 10, 25, 50, 75µM) at association and dissociation time of 300 and 150 s. E-64 was used
as positive control. Hemoglobin degradation activity by PfFP-2 occurs during an early
trophozoite stage. PfFP-2 also works beside membrane skeletal ankyrin and protein 4.1 during
the late trophozoite and schizont stages of growth of parasite [60, 61].
2
.3.4. Live-cell imaging
Live-cell imaging was done to observe the effect of JMI-105 and JMI-346 in the intracellular
PfFP-2. Parasites from the synchronized trophozoite stage were incubated with JMI-105 and
JMI-346 to observe the intracellular hydrolysis of substrate Z-Phe-Arg-AMC. PfFP-2
undergoes proteolytic cleavage to the Z-Phe-Arg-AMC products and release fluorogenic AMC
as observed with Olympus fluoview 3000 confocal microscope. CellSens analysis software
was used to analyse the figures. Around thirty parasites of each sample were monitored but
only representative figures are shown here (Fig. 5A). Less intensity corresponds to the
hindrance of cleavage of peptide substrate Z-Phe-Arg-AMC. JMI-105 and JMI-346 treated
samples showed attenuation in fluorescence as compared to control untreated sample. Mean
gray intensity values of control and JMI-105 and JMI-346 treated samples were obtained by
spectrofluorometer. The compound treated parasites showed less intensity as compared to
untreated control in a dose dependent manner. In untreated sample, the mean intensity was
found to be 734.33 ± 133.16 whereas at 20µM concentration of JMI-105 and JMI-346, the
mean intensity was 434 ± 86.01 and 517.33 ± 62.40, respectively (Fig. 5B). JMI-105 treated
pRBCs showed significant decrease in mean (gray intensity value) as compared to JMI-346
treated pRBCs and untreated control pRBCs. This represents inhibition effect on PfFP-2 by the
hindrance of cleavage of fluorogenic peptide substrate Z-Phe-Arg-AMC [37].
2
.3.5. Hemozoin Assay
Hb degradation produces, ferric heme which is a byproduct and proves to be lethal to both the
malaria parasites and the host cells [56, 63-65]. Consequently, malaria parasite for its
protection resort to crystallization into hemozoin, converting toxic heme into nontoxic
metabolites. Hemozoin is a malaria pigment, which is insoluble in water [65, 66].
Spectrophotometric analysis was performed to examine free monomeric heme acquired from
1
6

hemozoin. For the quantification of monomeric heme, parasites having ring stage
synchronized) were incubated with JMI-105 and JMI-346 and followed up to schizont stage.
(
Parasites treated with JMI-105 and JMI-346 showed dose dependent decreased percentage of
free monomeric heme as compared to untreated control. At 50µM concentration, JMI-105
showed 29.7% whereas at JMI-346 showed 34.7% free heme (Fig. 5C). From parasite’s
hemozoin, free monomeric heme was quantified at 405/750 nm absorbance which later
correlated with the reduction in Hb hydrolysis. Crystallization of hemozoin is vital for
persistence of malaria parasite that creates an interest towards the development of novel
antimalarial drugs [64, 67].
A.
DIC
Z-Phe-Arg-AMC
MERGE
B.
900
800
700
600
500
400
300
200
100
0
l
)
M
)
M
)
)
4
6
o
r
M
M
-
t
n
0µ
0µ
0µ
0µ
2
(
E
o
1
2
1
C
(
(
(
5
5
6
6
4
0
0
4
1
1
3
3
-
-
-
-
I
I
I
I
M
M
M
M
J
J
J
J
C.
1
20
00
1
µM
5µM
12.5µM
25µM
50µM
1
80
60
40
20
0
JMI-105
JMI-346
E-64
Control
Figure 5: (A) Live cell imaging analysis of intracellular hydrolysis of substrate Z-Phe-Arg-
AMC showed PfFP-2 inhibitory effect of JMI-105 and JMI-346. Synchronized trophozoites
were treated with JMI-105 and JMI-346 at 10 and 20µM concentrations. Images showed the
1
7

inhibition of PfFP-2 by JMI-105 and JMI-346 conferred by the attenuation of proteolytic
cleavage of fluorogenic AMC. (B) The mean intensity values measured by CellSens analysis
software. Parasitized red blood cells (pRBCs) treated with JMI-105 and JMI-346; showed a
decrease in mean intensity values of JMI-105 and JMI-346 treated pRBCs. (C) Graph showed
hemozoin inhibition in JMI-105 and JMI-346 treated parasites. Synchronized ring-stage
parasites exposed to JMI-105 and JMI-346 at 1, 5, 12.5, 25 and 50µM concentrations and
Percent control free heme was estimated. Experiment was done in triplicate and presented as
mean values ± SD, **P < 0.01 vs. control: Dunnett’s test.
2
.3.6. Antimalarial activity by SYBR green I based fluorescence assay
Growth inhibition of JMI-105 and JMI-346 on 3D7 P. falciparum was observed at different
concentrations ranging from 1-100µM. One complete intraerythrocytic growth cycle of P.
falciparum was examined after the treatment. DNA-specific dye SYBR green I assay and
Giemsa staining were performed to observe the growth inhibition. Both JMI-105 and JMI-
3
46 were found to be effective against the tested strain and showed inhibitory effects in a
concentration dependent manner as compared to untreated control (Fig. 6A). Furthermore,
JMI-105 and JMI-346 were also tested against PfRKL-9 strain. Half-maximal inhibition
concentration (IC ) of JMI-105 and JMI-346 against 3D7 and RKL-9 were calculated (Graph
5
0
Pad Prism 8 software). IC values of JMI-105 and JMI-346 against 3D7 were found to be
5
0
8
.8µM and 14.3µM whereas 13µM and 33.8µM were observed against RKL-9 P. falciparum
strain, respectively. Growth progression analysis was also done with JMI-105 and JMI-346
against P. falciparum 3D7 strain. Experiment was performed for one complete growth cycle of
parasite. Synchronized parasite culture of ring stage was treated with JMI-105 and JMI-346
at their IC concentration. Parasites (no treatment) were taken as control. Thin Giemsa stained
5
0
blood smears were prepared at different time intervals of 20, 40 and 56-hour post treatment
HPT). Growth progression defect was analyzed by counting ~2000 cells per Giemsa stained
(
slide by light microscope. Morphological analysis was observed in the presence and absence
of compounds. It was observed that after 56 HPT, most of the JMI-105 and JMI-346 treated
parasites were stuck at trophozoite stage showing altered morphology. In untreated control,
growth of the cells progressed normally. As compared to untreated control, reduction in
percentage of parasitemia was detected. However, in case of JMI-105, parasites were found to
1
8

be pyknotic whereas in case of JMI-346 treated parasites, some population of ring-stage
parasite was seen indicating delayed progression (Fig. 6B).
A.
i.
ii.
CQ sensitive P. falciparum 3D7; IC50= 8.8uM CQ resistant P. falciparum RKL−9; IC50= 13uM
CQ sensitive P. falciparum 3D7; IC50= 14.3uM CQ resistant P. falciparum RKL−9; IC50= 33.8uM
6
4
2
0
0
0
6
0
0
0
4
2
OCH3
O
O
N
N
N
O
N
NH2
N
N
2
.5uM
5uM
12.5uM
25uM
8uM
10uM
12.5uM
15uM
2.5uM
5uM
12.5uM
25uM
25uM
30uM
35uM
40uM
JMI−105 (Concentration)
JMI−346 (Concentration)
20 HPT
40 HPT
56 HPT
B.
0
HPT
1.08%
3.93%
0.94%
1.20%
4.21%
0.90%
0.90%
1.37%
0
.98%
0.95%
Rings
Trophozoites
Schizonts
Figure 6: (A) In vitro growth inhibition of JMI-105 and JMI-346 at different concentrations
1-50µM) against 3D7 and RKL-9 strains of P. falciparum. Box plot graph shows the
antimalarial activity of JMI-105 and JMI-346. Inset of box plot graph show structure of JMI-
05 and JMI-346 and half-maximal inhibition concentration (IC ) of JMI-105 and JMI-346
(
1
5
0
as mentioned against 3D7 and RKL-9 strains of P. falciparum. Experiment was performed in
triplicate and data was expressed as mean values ± SD. (B) Growth progression analysis was
done with 3D7 P. falciparum after treatment with JMI-105 and JMI-346. Giemsa staining was
done to observe morphological changes for one complete growth cycle of parasites at time
intervals of 20, 40 and 56 HPT. Figures and pie graphs both showed delayed growth of the
parasites.
2
.3.7. Hemolytic activity
1
9

Spectrophotometric analysis was done to see the effect of JMI-105 and JMI-346 on human
red blood cells (hRBCs) and HEK293 cells. Up to 12.5µM concentration, no significant lysis
in RBCs was observed by the JMI-105 and JMI-346 whereas 1% Triton-X 100 treated RBCs
showed 100% hemolysis (Fig. 7A).
2
.3.8. Cytotoxicity assay
Compound JMI-105 and JMI-346 did not affect the viability of HEK293 cells (Fig. 7B)
because these compounds are non-cytotoxic to human cells in the concentration range tested.
2
.3.9. Structure-Activity Relationship (SAR) studies
The synthetic pathway to achieve SAR derivatives (5a-5l) is outlined in (Scheme 1). In the
reaction sequences, the coupling of (1) with propargyl bromide using K CO in anhyd. DMF
2
3
gives the corresponding alkyne (2) an excellent yield. In another pot, aryl azides (4a-4l) were
prepared from the substituted anilines (3a-3l) by diazotization of aniline with NaNO and HCl
2
followed by the reaction with NaN all in the single reaction vessel. Finally, both alkyne and
3
azide were subjected to Huisgen 1,3 dipolar cycloaddition reaction in the presence of
CuSO .5H O and sodium ascorbate in THF:H O (1:2) mixture. SAR derivatives (5a-5l) gave
4
2
2
1
13
rise moderate to good yields (46-88%). Using FT-IR, H, C NMR and mass spectrometry, the
structures and purity was found to be ≥94% which was confirmed by UPLC. In the IR spectra,
the disappearance of the characteristic peak of alkyne C=C and ≡CH stretching and appearance
1
of characteristic peak of =CH (triazole) stretching in the region 3017-3274 cm- provided the
1
evidence for the formation of triazole ring. In the H NMR spectra, appearance of characteristic
singlet due to =CH (triazole) in the region 7.69-8.14 ppm confirmed the formation of triazole
ring. The methylene (-CH -) proton linking the natural precursors to the triazole scaffold
2
1
3
appeared in the region 5.19-5.33 ppm. In the C NMR spectra, peak at 145.71 and 123.22-
24.51 ppm, is due C4 and C5 carbon atoms of the triazole ring. All the protons and carbons
1
appeared at the expected chemical shift values. Mass spectra of all the compounds were found
to be in agreement with their molecular formula thus confirming the formation of the desired
SAR derivatives. Details of all the spectra are given in the supporting information.
Initial active molecule JMI-105 [52.73% inhibition of PfFP-2; IC = 8.8μM (3D7); 13.0
5
0
(RKL-9)] as the lead inhibitor in cell-free and cell-based assays was further optimized through
SAR. Compounds (JMI-99 to JMI-106) belonging to the series of JMI-105 were included in
2
0

the SAR studies. SAR optimized compounds (5a-5l) along with (JMI-99 to JMI-106) were
subjected to growth inhibition studies to see their effects on the parasite growth.
NH2
OH
R
R
o-OCH3
3
a
b
3a-l
1
3
m-OCH3
Br
a
3c
o-CH3
b
3
d
m-CH3
o-F
3
3
3
3
e
N3
O
m-F
f
R
g
h
o-NO2
m-NO2
o-Cl
2
4a-l
3
3
3
3
i
m-Cl
j
k
l
p-COOCH3
p-Br
O
N
N
R
N
5a-l
Scheme 1: Synthesis of 1,2,3-triazole derivatives, Reagents and conditions: (a) DMF, K CO ,
2
3
0
°C r.t , 18 h; (b) conc. HCl, NaNO , NaN , H O, EtOAc, 0 °C r.t , 2.5 h ; (c) CuSO .5H O,
2 3 2 4 2
sodium ascorbate, THF/H O (1:2), r.t, 20-24 h.
2
JMI-105 showed up to (59.25% inhibition at 10µM) as expected, but unfortunately, none of
the SAR derivatives showed significant growth inhibition upto 10µM concentration except 5a
and 5b (Supplementary data). It was interesting to note that replacement of p-OCH group as
3
present in JMI-105 (59.25% inhibition) to ortho-position (5a, 43% inhibition) or meta-position
(
5b, 41% inhibition) at 10µM conc. which gradually diminishes the activity (Fig. 7C). To
validate our screening strategy, we conducted growth inhibition studies with JMI-99 to JMI-
06, which showed maximum of 23% inhibition (JMI-103) at 10µM concentration (data given
1
2
1

in supplementary information). This proves that our strategy for the identification of potent and
selective PfFP-2 inhibitors is valid and justified. Further, JMI-105, which is a semi-synthetic
triazole derivative of carvacrol with p-OCH substitution is indeed a potent anti-malarial
3
compound acting via PfFP-2 inhibition and can be carried forward for further biological
evaluation.
Figure 7: (A) Effect of JMI-105 and JMI-346 on human RBCs. Different concentrations 1,
5
, 12.5, 25, 50µM of JMI-105 and JMI-346 were used to treat the RBCs suspension [10%
(v/v)] and incubated for 1h. Non-significant effect was observed in the treated samples upto
2
5µM concentration. (B) Colorimetric MTT assay was used to observe the cytotoxicity of JMI-
05 and JMI-346 against human embryonic kidney (HEK293) cells at 5-200µM
1
concentrations. The graph showed percent cell viability of HEK293 and the experiment was
performed in triplicate. Data was expressed as mean values ± SD. (C) Replacement of -OCH3
group from para position (JMI-105) to ortho (5a) or meta (5b) position led to the decrease in
the inhibition of the malaria parasite.
2
.3.10. In vivo antimalarial activity
In vivo antimalarial efficacy of JMI-105 was examined in P. berghei ANKA infected BALB/c
mice model. Infected mice were administered intraperitoneally with JMI-105 at 30 and
1
00mg/Kg doses. Thin blood smears were made from P. berghei infected mice to check the
2
2

parasitemia for upto eight consecutive days [68]. Significant reduction in the percentage
parasitemia and mortality rate was observed in JMI-105 treated group of mice as compared to
untreated control group on day 8 post infection. Percentage parasitemia of JMI-105 treated
group was observed to be 14.67% and 11.21% in 30 and 100mg/Kg as compared to the
untreated control wherein percent parasitemia was 43.34% on day 8 (Fig. 8Ai). Survival of
group of mice was observed upto 18 days post infection. Mean survival time (MST) showed
improvement in mortality rate in case of JMI-105 30mg/Kg (MST= 9.26 days) and 100mg/Kg
(MST= 10.72 days) as compared to control (MST=7 days). Graph was plotted as percentage
mean survival time (Fig. 8ii). The decrease in parasite load was found to be more effective in
case of JMI-105 100mg/Kg as compared to 30mg/Kg dose concentration for eight consecutive
days post infection.
A.
i.
ii.
7
0
0
0
0
0
0
0
0
120
00
Control
JMI-105 (30mg/Kg)
JMI-105 (100mg/Kg)
Control
6
5
4
3
2
1
1
JMI-105 (30mg/Kg)
JMI-105 (100mg/Kg)
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
-
10
Post Infection (Days)
Figure 8: (A) Evaluation of in vivo antimalarial activity of JMI-105 on P. berghei ANKA
infected Balb/c mice. JMI-105 (30 mg/Kg and 100 mg/Kg body weight; n=4) was administered
intra-peritoneally in P. berghei ANKA infected Balb/c mice for eight consecutive days.
Untreated group of mice were taken as control. Thin blood smears were made upto day 8 post
infection. (Ai) Graph showed percentage increase in parasitemia; (Aii) Survival of mice was
observed up to 18 days post infection. Graph showed percentage survival of mice.
3
. Materials and Methods
Through the combination of various bioinformatic techniques such as filtering tools, AutoDock
Vina, PyMOL, Discovery Studio Visualizer and in vitro, in vivo biological evaluation of the
hit compounds as well as their SAR derivatives, this study paved the way to develop semi-
synthetic derivative of carvacrol (JMI-105) as a potential PfFP-2 inhibitor with appreciable in
vitro and in vivo antimalarial efficacy.
2
3

3
3
.1. In silico studies
.1.1 Drug-likeness assessment of In-house library
Initially, an in-house library of 652 compounds was filtered out based on their drug-like
properties using the DruLiTo tool which is an open-source software developed by NIPER
http://www.niper.gov.in/pi_dev_tools/DruLiToWeb/DruLiTo_index.html [29]. This consists
of various drug-likeness filters based on Lipinski’s rule, Veber rule, Ghose rule, CMC-50,
Quantitative Estimate of Drug-likeness (QED) properties, etc. We applied all these filters with
default parameters to obtain those compounds which show resemblance to drug molecules.
Further, the selected compounds were filtered out based on their ADMET properties using
SwissADME webserver. These compounds were also checked for any PAINS and carcinogenic
pattern to select safer compounds. The PAINS filter was applied to sidestep those compounds
possessing PAINS patterns thus may have a higher tendency of binding towards multiple
targets [2].
3
.1.2 Pre-processing of FP-2 structure and compounds
The three-dimensional coordinates of PfFP-2 and PfFP-3 crystal structures were taken from
the Protein Data Bank (PDB ID: 3BPF and 3BPM, respectively), and all the bound crystallized
water molecules and co-crystallized ligands were deleted from the original structures [30]. The
structures were remodeled while using MODELLER 9.21 to fill-up the break due to some
missing residues. The final structures were prepared using MGL tools for screening. An in-
house library of compounds was used to process three-dimensional status. Compounds were
converted into PDBQT file format using the Open Babel module of PyRx to perform structure-
based virtual screening. All the selected compound structures were drawn in ChemDraw and
processed in MGL tools [31].
3
.1.3 Molecular docking
We have performed the structure-based molecular docking to further screen the compounds on
the basis of their binding affinity towards PfFP-2 and PfFP-3. This screening was performed
while using AutoDock Vina to find out compounds based on their affinity and interaction with
PfFP-2 and PfFP-3. The docking was structurally blind for all the compounds with
exhaustiveness 8 where they were free to move and search their preferential binding sites in
2
4

the proteins. AutoDock Vina was used for docking purpose to generate the detailed ligand-
receptor interactions. PyMOL [30] and Discovery Studio were used in visualization procedure
to analyze the bound conformation stabilized by various interactions between the selected
compounds and the protein.
3
.1.4 MD simulations
All-atoms MD simulations studies were performed on DELL® server with Intel® Xeon® CPU
E5-2609 v3 @ 1.90 GHz processor. MD simulation was performed on free FP-2 and in-
presence of JMI-105 and JMI-346 at 300 K of molecular mechanics level for 30 ns. The
GROMOS96 43a1 force-field was utilized in GROMACS 5.1.2 to perform simulations. The
ligands topologies were generated from the PRODRG server and merged into the parent
coordinates of FP-2 for systems preparation. A cubic box of 10 Å dimension was used for
solvating the systems in the SPC216 model using gmx solvate module. A run of 1500 steps of
steepest descent was carried out for energy minimization [27]. The final MD run was set to
3
0,000 ps for all three systems, and the generated trajectories were examined while utilizing
the inbuilt GROMACS utilities and visualized in VMD and QtGrace tools as described in our
earlier communication [32].
3
.2 Chemical synthesis
All the required chemicals were purchased from Merck, Sigma-Aldrich and were used without
further purification. TLC was performed using precoated Merck Silica gel 60 F254 TLC
aluminium sheets and UV light (254 nm) I vapor staining was used for visualization of spots.
2
Column chromatography was performed with Merck 230-400 mesh silica gel. Digital Buchi
melting point apparatus (M-560) was used for measurement of melting points. IR spectra were
-1
recorded and only major peaks are reported in cm by Agilent Cary 630 FT-IR spectrometer.
CDCl (as solvent) with TMS as an internal standard on Bruker Spectrospin DPX-300
3
1
13
spectrometer at 300 MHz and 75 MHz were used for H and C NMR spectra. s (singlet), d
(doublet), t (triplet), m (multiplet) or brs (broad) were labelled as splitting pattern. The chemical
1
shift values of H NMR were reported in parts per million (ppm) relative to residual solvent
1
3
13
(
CDCl , δ 7.26) and C NMR chemical shifts (δ) of C NMR were reported in ppm relative
3
to (CDCl , δ 77.16) and Hertz (Hz) is the unit of coupling constants (J). Agilent Quadrupole-
3
6
150 LC/MS spectrometer was used for recording of mass spectra. Chemical synthesis and
2
5

spectral characterization of JMI-105 [33] and JMI-346 [34] was reported earlier. Synthesis
protocols and spectral characterization of SAR derivatives (5a-5l) is discussed below:
3
.2.1 General procedure for the synthesis of alkyne (2)
Briefly, the natural alcohol carvacrol (1) (1.0 mM) was dissolved in anhyd. DMF and 2.0 mmol
o
of potassium carbonate was added and allowed to stir for 15 min at 0 tort. 1.2 mM of propargyl
bromide was added to reaction flask and stirred for overnight. Upon completion of the reaction
as evident by TLC, addition of water to quenched the reaction and extracted with ethyl acetate
(30 mL×2). The organic layer was washed with brine, dried over anhyd. sodium sulphate and
concentrated under vacuo. Crude obtained was purified by column chromatography (CC) by
use of ethyl acetate:hexane (3:7) to get the pure alkyne product [33].
3
.2.2 General procedure for the synthesis of azides (4a-l)
To the solution of substituted aniline (3a-l) (3.22 mmol) in ethyl acetate (6 mL) kept at 0 °C
was added conc. HCl (1.29 mL) followed by addition of a solution of NaNO (3.87 mmol) in
2
water (4.03 mL) with constant stirring. After stirring for 1 h at 0 °C, solution of NaN (3.87
3
mmol) in water (4.03 mL) was added to the mixture and allowed to stir at rt till the reaction
completes. Reaction mixture was poured in water and extracted with ethyl acetate then dried
over anhyd. Na SO . Further, concentrated under vacuo to give azide which was used without
2
4
purification [33].
3
.2.3 Synthesis of 1,2,3-triazole derivatives of carvacrol (5a-5l)
Finally, a solution of azide (3a-l) (1.06 mmol) and alkyne (2) (1.06 mmol) in THF/H O (1:2, 9
2
ml) was added to sodium ascorbate (0.55 mmol) and CuSO .5H O (0.18 mmol) and stirred
4
2
overnight at rt. The obtained solid was filtered and washed with water then purified by CC
using a solution of ethyl acetate:hexane (1:9) to yield pure triazole derivatives of carvacrol
[35].
3
.2.3.1
4-((5-isopropyl-2-methylphenoxy)methyl)-1-(2-methoxyphenyl)-1H-1,2,3-triazole
(5a):
1
Light cream liquid, yield: 63%, R = 0.65 (ethyl acetate:hexane = 30:70); H NMR (300 MHz,
f
CDCl ) (δ, ppm): 8.14 (s, 1H, triazole), 7.80 (d, J = 7.5 Hz, 1H, Ar-H), 7.42 (d, J = 7.5 Hz, 1H,
3
2
6

Ar-H), 7.09 (d, J = 7.2 Hz, 1H, Ar-H),7.09 (d, J = 7.2 Hz, 1H, Ar-H), 7.07 (t, J = 7.2 Hz, 1H,
Ar-H), 6.89 (s, 1H, Ar-H), 6.77 (d, J = 7.2 Hz, 1H, Ar-H), 5.32 (s, 2H, CH ), 3.87( s, 3H,
2
1
3
OCH ), 2.93-2.84 (m, 1H, CH), 2.21 (s, 3H, CH ), 1.25(d, J = 6.6 Hz, 6H, isopropyl); C NMR
3
3
(
75 MHz, CDCl ) (δ, ppm): 156.46, 151.17, 148.04, 144.21, 130.56, 126.33, 124.74, 121,24,
3
1
18.73, 112.26, 110.28, 77.46, 77.04, 76.89, 76.61, 62.51, 55.96, 34.10, 24.13, 15.91; ESI-MS
+
-1
(
m/z) calcd. for C H N O : 337.18; Found: 338.13 [M+H] ; IR (neat): ν (cm ) 3017 (=CH
20 23 3 2
str., triazole).
3
.2.3.2
4-((5-isopropyl-2-methylphenoxy)methyl)-1-(3-methoxyphenyl)-1H-1,2,3-triazole
(5b):
1
White powder, yield: 69%, mp: 66.2 °C, R = 0.82 (ethyl acetate:hexane = 30:70); H NMR
f
(
300 MHz, CDCl ) (δ, ppm): 8.01 (s, 1H, triazole), 7.41 (t, J = 8.1 Hz, 1H, Ar-H), 7.34 (s, 1H,
3
Ar-H), 7.26 (d, J = 6.9 Hz, 1H, Ar-H), 7.08 (d, J = 7.5 Hz, 1H, Ar-H), 6.96 (d, J = 7.5 Hz, 1H,
Ar-H),6.79 (s,1H, Ar-H), 6.78 (d, J = 6.6 Hz, 1H, Ar-H), 5.31 (s, 2H, CH ), 3.88( s, 3H, OCH ),
2
3
1
3
2
.92-2.83 (m, 1H, CH), 2.22 (s, 3H, CH ), 1.24(d, J = 6.6 Hz, 6H, isopropyl);. C NMR (75
3
MHz, CDCl ) (δ, ppm): 160.62, 156.30, 148.11, 145.60, 130.65, 130.54, 124.27, 114.70,
3
1
10.12, 106.46, 77.59, 77.47, 77.04, 76.74, 76.62, 62.35, 55.65, 34.09, 24.12, 15.92; ESI-MS
+
-1
(
m/z) calcd. for C H N O : 337.18; Found: 338.28 [M+H] ; IR (neat): ν (cm ) 3147 (=CH
20 23 3 2
str., triazole).
3
.2.3.3 4-((5-isopropyl-2-methylphenoxy)methyl)methyl)-1-o-tolyl-1H-1,2,3-triazole (5c):
1
Light yellow liquid, yield: 84%, R = 0.65 (ethyl acetate:hexane = 30:70); H NMR (300 MHz,
f
CDCl ) (δ, ppm): 7.69 (s, 1H, triazole), 7.28 (d, J = 8.4 Hz, 2H, Ar-H), 6.99 (d, J = 7.5 Hz, 2H,
3
Ar-H), 6.79 (s, 1H, Ar-H), 6.71 (t, J = 7.8 Hz, 2H, Ar-H), 5.27 (s, 2H, CH ), 2.82-2.78 (m, 1H,
2
1
3
CH), 2.17 (s, 3H, CH ), 2.13( S, 3H, CH ), 1.16 (d, J = 6.6 Hz, 6H, isopropyl); C NMR (75
3
3
MHz, CDCl ) (δ, ppm): 156.30, 147.82, 144.86, 133.75, 131.50, 130.64, 129.95, 119.09,
3
1
18.88, 110.25, 77.47, 76.62, 75.14, 62.53, 56.00, 34.10, 29.72, 24.08, 17.85, 15.81; ESI-MS
-
-1
(
m/z) calcd. for C H N O: 321.18; Found: 320.28 [M-H] ; IR (neat): ν (cm ) 3274(=CH str.,
20 23 3
triazole).
3
.2.3.4 4-((5-isopropyl-2-methylphenoxy)methyl)-1-m-totlyl-1H-1,2,3-triazole (5d):
2
7

1
Light orange powder, yield: 46%, R = 0.68 (ethyl acetate:hexane = 30:70); H NMR (300
f
MHz, CDCl ) (δ, ppm): 7.93 (s, 1H, triazole), 7.50 (s, 1H, Ar-H), 7.31 (t, J = 7.8 Hz, 1H, Ar-
3
H), 7.17 (d, J = 7.8 Hz, 1H, Ar-H), 7.00 (d, J = 7.2 Hz, 1H, Ar-H),6.70 (d, J = 7.5 Hz, 1H, Ar-
H),6.79 (s,1H, Ar-H), 6.60 (d, J = 7.8 Hz, 1H, Ar-H), 5.27 (s, 2H, CH ), 2.82-2.80 (m, 1H,
2
1
3
CH), 2.42 (s, 3H, CH ), 1.17(d, J = 4.8 Hz, 6H, isopropyl); C NMR (75 MHz, CDCl ) (δ,
3
3
ppm): 156.32, 148.12, 145.55, 140.04, 130.64, 129.61, 124.267, 121.31, 120.65, 118.82,
17.70, 110.10, 77.47, 76.62, 62.37, 34.10, 29.72, 24.13, 15.95, 1.041; ESI-MS (m/z) calcd.
1
+
-1
for C H N O: 321.18; Found: 322.30 [M+H] ; IR (neat): ν (cm ) 3064(=CH str., triazole).
2
0
23
3
3
.2.3.5 1-(2-fluorophenyl)-4-((5-isopropyl-2-methylphenoxy)methyl)-1H-1,2,3-triazole (5e):
1
Off white powder, yield: 79%, mp:62.3°C, R = 0.63 (ethyl acetate:hexane = 30:70); H NMR
f
(
300 MHz, CDCl ) (δ, ppm): 8.14 (s, 1H, triazole), 7.97 (t, J = 9.8 Hz, 1H, Ar-H), 7.44 (d, J =
3
7
.5 Hz, 1H, Ar-H), 7.33 (t, J = 7.8 Hz, 1H, Ar-H), 7.07 (d, J = 7.5 Hz, 1H, Ar-H), 7.25 (s, 1H,
Ar-H), 6.78 (d, J = 7.5 Hz, 1H, Ar-H), 5.33 (s, 2H, CH ), 2.91-2.86 (m, 1H, CH), 2.21 (s, 3H,
2
1
3
CH ), 1.25 (d, J = 6.6 Hz, 6H, isopropyl). C NMR (75 MHz, CDCl ) (δ, ppm): 156.31, 151.72,
3
3
1
48.07, 145.31, 130.64, 130.24 (d, J = 7.72 Hz), 125.29, 125.24, 124.35, 123.76 (d, J = 8.025
Hz), 118.87, 117.18, 116.91, 110.16, 77.46, 76.61, 62.28, 34.10, 24.11, 15.88; ESI-MS (m/z)
+
-1
calcd. for C H FN O: 325.16; Found: 326.28 [M+H] ; IR (neat): ν (cm ) 3132 (=CH str.,
1
9
20
3
triazole).
3
.2.3.6 1-(3-fluorophenyl)-4-((5-isopropyl-2-methylphenoxy)methyl)-1H-1,2,3-triazole (5f):
1
White powder, yield: 80%, mp:100.1 °C, R = 0.68 (ethyl acetate:hexane = 30:70); H NMR
f
(
300 MHz, CDCl ) (δ, ppm): 8.03 (s, 1H, triazole), 7.64 (d, J = 5.4 Hz, 2H, Ar-H), 7.46 (s, 1H,
3
Ar-H), 7.16 (d, J = 7.2 Hz, 1H, Ar-H), 7.10 (t, J = 7.5 Hz, 1H, Ar-H), 6.86(s,1H, Ar-H), 6.78
d, J = 7.5 Hz, 1H, Ar-H), 5.31 (s, 2H, CH ), 2.93-2.83 (m, 1H, CH), 2.22 (s, 3H, CH ), 1.24(d,
(
2
3
1
3
J = 6.9 Hz, 6H, isopropyl); C NMR (75 MHz, CDCl ) (δ, ppm): 164.75, 161.45, 156.23,
3
1
48.14, 145.98, 138.24, 131.23 (d, J = 8.85 Hz), 130.69, 124.25, 120.48, 118.93, 115.75 (d, J
21.75 Hz), 110.07, 108.36 (d, J = 25.8 Hz), 77.47, 76.62, 62.28, 34.10, 15.93, ESI-MS (m/z)
=
+
-1
calcd. for C H FN O: 325.16; Found: 326.11 [M+H] ; IR (neat): ν (cm ) 3131(=CH str.,
1
9
20
3
triazole).
.2.3.7 4-((5-isopropyl-2-methyphenoxy)methyl)-1-(2-nitrophenyl)-1H-1,2,3-triazole (5g):
3
2
8

1
Dark yellow liquid, yield: 88%, R = 0.33 (ethyl acetate:hexane = 30:70); H NMR (300 MHz,
f
CDCl ) (δ, ppm): 7.90 (s, 1H, triazole), 8.10 (d, J = 7.8 Hz, 1H, Ar-H), 7.69 (t, J = 7.5 Hz, 1H,
3
Ar-H), 7.66 (d, J = 7.2 Hz, 1H, Ar-H), 7.63 (t, J = 6.3 Hz, 1H, Ar-H), 7.07 (d, J = 7.5 Hz, 1H,
Ar-H), ), 6.86 (s, 1H, Ar-H), 6.78 (d,1H, J=7.5Hz, Ar-H), 5.19 (s, 2H, CH ), 2.93-2.84 (m, 1H,
2
1
3
CH), 2.25 (s, 3H, CH ), 1.24 (d, J = 6.9 Hz, 6H, isopropyl); C NMR (75 MHz, CDCl ) (δ,
3
3
ppm): 156.23, 148.17, 145.66, 144.53, 133.82, 130.83, 127.99, 125.60, 124.28, 123.97, 118.96,
1
3
10.18, 77.45, 77.03, 76.61, 62.32, 34.09, 24.11, 15.89; ESI-MS (m/z) calcd. for C H N O :
19 20 4 3
+
-1
52.15; Found: 353.28 [M+H] ; IR (neat): ν (cm ) 3144 (=CH str., triazole).
3
.2.3.8 4-((5-isopropyl-2-methyphenoxy)methyl)-1-(3-nitrophenyl)-1H-1,2,3-triazole (5h):
1
Light yellow powder, yield: 83%, mp:122.8 °C, R = 0.52 (ethyl acetate:hexane = 30:70); H
f
NMR (300 MHz, CDCl3) (δ, ppm): 8.63 (s, 1H, triazole), 8.31 (d, J = 8.1 Hz, 1H, Ar-H), 8.15
(s, 1H, Ar-H), 8.20 (d, J = 8.1 Hz, 1H, Ar-H), 7.76 (t, J = 8.1 Hz, 1H, Ar-H), 7.09 (d, J = 7.8
Hz, 1H, Ar-H), 6.81(s,1H, Ar-H), 6.79 (d, J = 7.8 Hz, 1H, Ar-H), 5.34 (s, 2H, CH ), 2.91-2.84
2
1
3
(
m, 1H, CH), 2.24 (s, 3H, CH ), 1.25(d, J = 6.9 Hz, 6H, isopropyl); C NMR (75 MHz, CDCl )
3
3
(δ, ppm): 156.14, 148.96,146.57, 137.74, 131.01, 130.74, 126.00, 124.25, 123.27, 120.42,
1
19.03, 115.33, 110.02, 77.45, 76.60, 62.18, 34.09, 24.11, 15.94; ESI-MS (m/z) calcd. for
+
-1
C H N O : 352.15; Found: 353.28 [M+H] ; IR (neat): ν (cm ) 3132(=CH str., triazole).
1
9
20
4
3
3
.2.3.9 1-(2-chlorophenyl)-4-((5-isopropyl-2-methyphenoxy)methyl)-1H-1,2,3-triazole (5i):
1
Dark yellow liquid, yield: 66%, R = 0.61 (ethyl acetate:hexane = 30:70); H NMR (300 MHz,
f
CDCl ) (δ, ppm): 8.02 (s, 1H, triazole), 7.64 (t, J = 7.2 Hz, 1H, Ar-H), 7.58 (t, J = 7.2 Hz, 1H,
3
Ar-H), 7.07 (d, J = 7.8 Hz, 1H, Ar-H), 6.87 (s, 1H, Ar-H), 6.78 (d,1H, J=7.8Hz, Ar-H), 5.34
(
s, 2H, CH ), 2.90-2.86 (m, 1H, CH), 2.21 (s, 3H, CH ), 1.24 (d, J = 6.9 Hz, 6H, isopropyl);
2
3
1
3
C NMR (75 MHz, CDCl ) (δ, ppm): 156.31, 148.09, 144.77, 144.53, 134.92, 130.80, 128.68,
3
1
27.94, 127.82, 124.52, 124.39, 118.89, 110.24, 77.47, 76.62, 62.43, 34.10, 24.13, 15.90; ESI-
+
+
MS (m/z) calcd. for C H ClN O: 341.13; Found: 342.10 [M+H] , 344.09 [M+H+2] ; IR
1
9
20
3
-1
(
neat): ν (cm ) 3144 (=CH str., triazole).
3
.2.3.10 1-(3-chlorophenyl)-4-((5-isopropyl-2-methyphenoxy)methyl)-1H-1,2,3-triazole (5j):
1
White powder, yield: 80%, mp:93.9 °C, R = 0.71 (ethyl acetate:hexane = 30:70); H NMR
f
(
300 MHz, CDCl ) (δ, ppm): 8.02 (s, 1H, triazole), 7.80 (s, 1H, Ar-H), 7.65 (d, J = 7.8 Hz, 1H,
3
2
9