Synthesis, antimalarial evaluation and molecular docking studies of some thiolactone derivatives

Article abstract of DOI:10.1016/j.molstruc.2016.12.095

In present study novel thiolactone derivatives were designed, synthesized and characterized by various analytical techniques such as IR, ¹H NMR, ¹³C NMR, mass spectral data and elemental analysis. All synthesized compounds were evaluated for in?vitro antimalarial activity against Dd2 and 3d7 strain of P.?falciparum. All synthesized compounds were also subjected for molecular docking study with pf KASI/II enzyme to analyze their binding orientation in the active site of the enzyme. Compounds 5d, 5e, and 5i found to be most potent with IC₅₀ in the range of 0.09–0.19?μM and 0.03–0.04?μM against the Dd2 strain and 3D7 strain respectively as well as they showed good binding affinities with the residues of the active site of pf KASI/II.

Full text of DOI:10.1016/j.molstruc.2016.12.095

Accepted Manuscript
Synthesis, antimalarial evaluation and molecular docking studies of some
thiolactone derivatives
Jitendra Sainy, Rajesh Sharma
PII:
S0022-2860(16)31414-4
10.1016/j.molstruc.2016.12.095
MOLSTR 23293
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
26 May 2016
19 December 2016
28 December 2016
Please cite this article as: Jitendra Sainy, Rajesh Sharma, Synthesis, antimalarial evaluation and
molecular docking studies of some thiolactone derivatives, Journal of Molecular Structure (2016),
doi: 10.1016/j.molstruc.2016.12.095
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ACCEPTED MANUSCRIPT
Highlights
 A new series of thiolactone derivatives have been synthesized.
 The structures of the synthesized compounds have been investigated by FT-IR, 1H-NMR,
13C NMR, mass spectral data and elemental analysis.
 The synthesized compounds were evaluated in vitro for their antimalarial activity.
 The binding mode of the synthesized compounds in the active site of pf KASI/II have
been evaluated through molecular docking study.

ACCEPTED MANUSCRIPT
Synthesis, antimalarial evaluation and molecular docking studies of some thiolactone
derivatives
Jitendra Sainy, Rajesh Sharma*
School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshashila Campus, Ring road, Indore
(M.P.)-452001, India
Sainy_007@rediffmail.com, rbsm73@yahoo.co.in
Address for Correspondence
*Rajesh Sharma
E. Mail: rbsm73@yahoo.co.in
Phone: +91-9425478418

ACCEPTED MANUSCRIPT
Abstract
In present study novel thiolactone derivatives were designed, synthesized and characterized by
1
13
various analytical techniques such as IR, H NMR, C NMR, mass spectral data and elemental
analysis. All synthesized compounds were evaluated for in vitro antimalarial activity against Dd2
and 3d7 strain of P. falciparum. All synthesized compounds were also subjected for molecular
docking study with pf KASI/II enzyme to analyze their binding orientation in the active site of
the enzyme. Compounds 5d, 5e, and 5i found to be most potent with IC50 in the range of 0.09-0.19
μM and 0.03-0.04 μM against the Dd2 strain and 3D7 strain respectively as well as they showed
good binding affinities with the residues of the active site of pf KASI/II.
Key words: Plasmodium falciparum, thiolactone, homology modeling, molecular docking,
β- keto acyl –ACP synthase I/II.

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1. Introduction
Malaria remains a significant public health problem in various countries of the World. According
to per World malaria report 2015 of World Health Organization (WHO) 214 million cases of
malaria observed worldwide in 2015 leading to 438,000 malaria deaths. More than 35% of the
global totals of predicted malaria deaths occur in countries like sub-Saharan Africa, Democratic
Republic of the Congo and Nigeria. The rates of decreasing in malaria cases and mortality are
slower in high burden countries. In India maximum number of malaria cases occurs among the
Southeast Asian countries. The whole Southeast Asian region reported more than 100 million
malaria cases out of which about 2 million malaria cases were observed in India. Globally there
were 6.2 million malaria deaths observed between 2001 to 2015, Out of which 5.9 million (95%)
children were died age less than 5 years. These deaths signify 13% of the 46 million deaths from
all causes in children of less than 5 years [1].
In spite of noteworthy improvement in decreasing malaria cases and deaths over the past 20
years malaria continues to affect vast populations in sub-tropical countries such as India and
Vietnam. Malaria is one of the major infectious diseases, caused by a protozoan parasite of the
genus Plasmodium. Among the five malaria parasite species viz. P. vivax, P. falciparum, P.
ovale and P.knowlesi, P. falciparum is the most lethal form and has contributed about 50% of the
total cases. The development of resistance in P. falciparum against antimalarial drugs causes a
serious problem to control disease [2]. Recently P. falciparum resistance to artemisinins has
identified in five countries of the Greater Mekong sub region like Cambodia, Lao People’s
Democratic Republic, Myanmar, Thailand and Viet Nam [3]. Artemisinins is the most important
component of the artemisinin-based combination therapies (ACTs).If artemisinins resistance will
spread to India or Sub-Saharan Africa the global mortality rate may increase because no other
drug is available with same level efficacy and tolerability. Therefore there is an urgent
requirement of newer drug against malaria.
Fatty acids are vital and one of the most abundant components of the malaria parasite
P.falciparum. They need an ample supply of lipids particularly fatty acids for the membrane
biosynthesis which is essential for invasive stage formation. Initially, it was assumed that
Plasmodium parasites were deficient in synthesizing their own fatty acids and thus depends on
their hosts for lipids[4].Later it was found that Plasmodium synthesizes fatty acids in its

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apicoplast (which is a plastid organelle of Plasmodium) with the help of FAS II (fatty acid
synthase II) enzyme complex similar to plant and bacteria[5-8].The synthesis of fatty acids is a
de novo pathway and it occurs in liver stage of malaria parasite life cycle in which P. falciparum
synthesize fatty acids from derivatives of acetate and malonate with the help of FAS II enzyme
complex[9-10]. However in human beings the process of fatty acid synthesis is carried out by a
single, multifunctional polypeptide type I fatty acid synthase (FAS I) which differs significantly
from FAS II enzyme complex[11].Therefore FAS II enzymes are attractive target for the
development of new antimalarial drugs. FAS II enzyme complex includes β-keto acyl –ACP
synthase I/II (pf KASI/II) (genome of P. falciparum indicates that enzymes pf KAS I and pf KAS
II are very similar at protein level so they are replaced by single enzyme pf KAS I/II ) [12], β-
keto acyl –ACP synthase III (pf KASIII) [13, 14] and enoyl-ACP reductase [15, 16].pf KAS III
catalyzes the initial step of fatty acid biosynthesis which is the condensation of malonyl-ACP
and acetyl-CoA forming acetoacetyl-ACP while pfKASI/II is engaged in further chain
elongation[17].
Thiolactomycin (TLM) is found to be a known inhibitor of dissociable FAS II enzymes [18-20].
It is a natural thiolactone antibiotic obtained from fermentation broth of Nocardia species of
actinomycetes [21, 22].As TLM and its derivatives target more than one enzyme in the FAS II
enzyme complex, the possibility of development of resistance due to point mutation in p.
falciparum parasite will be minimum[19].TLM exhibits potent in vivo activity against many
pathogenic bacteria[18,23,24,].In addition TLM and its derivatives showed antimycobacterial
(Richard, et al., 1996) and antimalarial activity [13,25,26].It was also found that TLM has
negligible toxicity towards mammals[21],hence TLM or its derivatives may be used as lead
structure for further drug discovery. Thus with the aim of obtaining novel antimalarial
compounds we have designed and synthesized a series of thiolactone derivatives and evaluated
them for antimalarial activity. Syntheses of the compounds (5a–5u and 6a) were carried out as
per scheme 1[27] and the structures of all the synthesized compounds were established on the
basis of FT-IR, 1H-NMR, 13C NMR, mass spectral data and elemental analysis.

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2. Experimental
2.1. Materials and methods
All the designed compounds (5a– 5u and 6a) were synthesized as per the scheme 1 in which
methyl acetoacetate was treated with alkyl iodide in the presence of potassium carbonate as base
to get the alkylated products. After the formation of β-ketoesters, it was treated with pyridinium
tribromide in presence of acetic acid afforded the bromides. Removal of the bromide group is
achieved by the use of thioacetic acid under basic conditions forming the thioacetates.
Cyclisation to the desired thiolactones was carried out upon treatment with aqueous potassium
hydroxide solution. Derivatives of thiolactones were prepared by the treatment of thiolactone
with R2-Ar-Br/ bromo hexane in presence of acetone and potassium carbonate.
The structures of all the synthesized compounds were established based on elemental analysis,
1
FT-IR, H-NMR, 13C NMR and mass spectral data. Melting points of all the compounds were
measured in an open capillary tube and were uncorrected. Purity of the compounds was checked
on precoated silica gel G plates (0.2 mm thickness) using iodine vapor as visualizing agent.
13
Infrared spectra were recorded on an FTIR-8400S Shimadzu IR spectrometer, 1H and C NMR
spectra were obtained on 400 MHz FT NMR spectrometer and 100 MHz FT NMR spectrometer
(model Advance-II Bruker) respectively. Mass spectra were recorded on a Waters Micromass Q-
Tof Micro mass spectrometer. Satisfactory elemental analyses of the synthesized compounds
were obtained on EURO VECTOR E 3000 elemental analyzer. All the reagents used in the
present work were of synthetic grade.

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R1
Br
R1
O
O
O
a
b
O
O
O
O
O
O
Methyl aceto acetate
IM02
c
IM 01
O
S
R1
O
O
O
S
O
IM 03
O
R1
R2
d
6a
f
S
O
S
e
O
O
R1
HO
R1
R2
IM 04
5
5a-f: R1=Methyl, 5a R2=SO2NH2, 5b R2=CH3, 5c R2= CF3 , 5d R2= Cl,5e= F,5f R2= Br
5g-l : R1=Ethyl, 5g R2=CF3, 5h R2=CH3, 5i R2= F, 5j R2=Cl, 5k R2= SO2NH2,5l R2=Br
5m-p: R1=Propyl, 5m R2=F, 5n R2= CH3, 5o R2=Cl, 5p R2=CF3
5q-u: R1=Isopropyl, 5q R2=SO2NH2, 5r R2=CH3, 5s R2=CF3, 5t R2 =Cl, 5u R2=F
6a: R1=Methyl, R2= C6H13
4a:R1= methyl
4b: R1= Ethyl
4c: R1= Propyl
4d: R1=Isopropyl
Scheme1. (a) R1I, K2CO3, THF, reflux (b) PyHBr3, acetic acid, room temp. (c) AcSH, Et3N,
EtOAc, room temp (d) KOH, ethanol/H2O (1:1),45°C (e)/(f) R2-Ar-Br/ Bromo hexane, anhyd
K2CO3, acetone, reflux.

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2.2 Synthesis
2.2.1 General synthetic procedure for IM 04(4a-4d):
In a round bottom flask equipped with reflux condenser methyl acetoacetatae (0.01mol) was
allowed to react with R1I (0.01mol) in 15mL of tetrahydrofuran in presence of potassium
carbonate (4 equiv) as a base to obtained alkylated IM 01.Treatment of IM 01 with pyridinium
tribromide in acetic acid at room temperature afforded the IM 02.Then removal of bromide
group was achieved by the reaction of thioacetic acid in presence of triethyl amine and ethyl
acetate at room temperature to get IM 03 (thioacetates). The cyclization of IM 03 is carried out
by adding potassium hydroxide (2.0 equiv) in water (10 mL) to a stirred solution of the
thioacetate (1.0 equiv) in ethanol (20 mL) at ambient temperature to get IM 04. Then the
resulting solution was allowed to warm up to 40ºC and stirred for 4h. The ethanol was
evaporated in vacuo and 10mL water was added. The aqueous layer was washed two times with
10mL of diethyl ether and acidified up to pH 1 with 10mL of 2M HCl. The aqueous layer was
extracted with diethyl ether and the organic layer was washed with brine and allowed to dry over
anhydrous magnesium sulphate and the solvent removed in vacuo. The crude residue was
recrystallized and purity of intermediates was ascertained by thin layer chromatography with a
suitable solvent. The physical properties and spectral data derived from the obtained products are
listed below.
Synthesis of 4-hydroxy-3, 5-dimethylthiophen-2(5H)-one(4a)
Yield 83 %; m.p. 125-126ºC; IR (KBr) (vmax,cm–1): 3310(O-H) ,1690 (C=O),620 (C-S) ,2870(C-
H), 1545 (C=C), 2950 (C-H); 1H NMR (400 MHz, DMSO-d6) δ 10.33(s, 1H,OH), 4.47 (q, 1H, 5-
H), 1.41 (d, 3H, 5-CH3), 1.98 (s, 3H, 3-CH3);13C NMR (100 MHz, DMSO-d6) δ 8.9, 15.7, 44.6,
107.1, 186.6, 197.5; Mass spectrum m/z: 144.02[M+]; Anal. Calcd. (%) C12H13NO4S2: C, 49.98;
H, 5.59; O, 22.19. Found: C, 49.94; H, 5.51; O, 22.14.
Synthesis of 3-ethyl-4-hydroxy-5-methylthiophen-2(5H)-one (4b)
Yield 83 %; m.p. 130-131ºC; IR (KBr) (vmax,cm–1): 3185(O-H), 1715 (C=O), 625 (C-S),2850(C-
H), 1610 (C=C), 2910 (C-H); 1H NMR (400 MHz, DMSO-d6) δ 10.22(s, 1H,OH),4.44 (q, 1H, 5-
13
H), 1.39 (d, 3H, 5-CH3), 2.11 (q,2H,1’-CH2), 1.14 (t,3H,2’-CH3); C NMR (100 MHz, DMSO-
d6) δ 11.4, 15.9,14.1,45.3, 111.1, 188.2, 196.4; Mass spectrum m/z: 158.05[M+]; Anal. Calcd.
(%) C12H13NO4S2: C, 53.14; H, 6.37; O, 20.22. Found: C, 53.10; H, 6.29; O, 20.14.

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Synthesis of 4-hydroxy-5-methyl-3-propylthiophen-2(5H)-one (4c)
Yield 76 %; m.p. 144-145ºC; IR (KBr) (vmax,cm–1): 3190(O-H),1725(C=O),628 (C-S), 2890(C-
H),1560 (C=C),2910 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H,OH),1.37-1.42 (m,
2H,2’-CH2), 4.49 (q, 1H, 5-H), 1.40 (d, 3H, 5-CH3), 0.98 (t, 3H, 3’-CH3), 2.10 (t, 2H, 1’-CH2);
13C NMR (100 MHz, DMSO-d6) δ 14.5, 21.1, 20.5, 23.1, 46.1, 110.1, 186.8, 196.6; Mass
spectrum m/z: 172.06 [M+]; Anal. Calcd. (%) C12H13NO4S2: C, 55.78; H, 7.02; O, 18.58. Found:
C, 55.70; H, 7.01; O,18.53.
Synthesis of 4-hydroxy-3-isopropyl-5-methylthiophen-2(5H)-one (4d)
Yield 79 %; m.p. 139-140ºC; IR (KBr) (vmax,cm–1): 3200(O-H),1730 (C=O),615 (C-S), 2880(C-
H), 1560 (C=C), 2910 (C-H); 1H NMR (400 MHz, DMSO-d6) δ 10.31(s, 1H,OH) 4.48 (q, 1H, 5-
H), 1.47 (d, 3H,5- CH3), 1.11-1.14 (m,3H,CH3a,CH3b), 2.56(t,1H,CH);13C NMR (100 MHz,
DMSO-d6) δ 15.6, 21.6, 21.5, 23.8, 45.8, 116.9, 187.1, 193.1; Mass spectrum m/z: 172.06 [M+];
Anal. Calcd. (%) C12H13NO4S2: C, 55.78; H, 7.02; O, 18.58. Found: C, 55.74; H, 6.99; O, 18.55.
2.2.2 General synthetic procedure for IM 04 to 5a-5u and 6a:
IM 04 (0.01mol) was dissolved in acetone and 0.01 mol of R2-Ar-Br/ bromo hexane was added
in reflux condenser. Then anhydrous potassium carbonate (4 equiv) was added to reaction
mixture and allowed to reflux for 4 hours. After completion of reaction the solution was
neutralized and the reaction mixture was poured into ice-cold water, then the aqueous layer was
extracted with diethyl ether twice, solvent was removed in vacuo and the dried product was
subjected to column chromatography.
4-(4- aminosulfonyl phenoxy)-5-methyl-3-methylthiophen-2(5H)-one (5a). Yield 73 %; m.p.
160-161ºC; IR (KBr) (vmax,cm–1):3470 (N-H),1242(C-O-C),1762(C=O),625(C-S),2890(C-H),
1610 (C=C),1355(S(=O2)asym),1140 (S(=O2)sym), 2930(C-H) ; 1H NMR (400 MHz, DMSO-d6)
δ 4.43 (q, 1H, 5-H), 1.45 (d, 3H, 5-CH3), 1.93 (s, 3H, 3-CH3), 7.01–7.76 (d, 4H, Ar-
H),3.24(s,2H,NH2); 13C NMR (100 MHz, DMSO-d6) δ 12.4, 22.5, 46.9, 114.1, 180.6, 199.5,
118.1, 128.4, 151.8, 133.9; Mass spectrum m/z: 299.03 [M+]; Anal. Calcd. (%) C12H13NO4S2: C,
48.12; H, 4.38; O, 28.38. Found: C, 48.09; H, 4.36; O, 28.35.

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4-(p-tolyloxy)-3,5-dimethylthiophen-2(5H)-one (5b). Yield 74 %; m.p. 155-156ºC; IR (KBr)
(vmax,cm–1): 1225(C-O-C),1750(C=O),615(C-S), 1610,1595,1450 (C=C ring str.),2890(C-H),
1
1625(C=C), 2950 (C-H); H NMR (400 MHz, DMSO-d6) δ 4.52 (q, 1H, 5-H), 1.47 (d, 3H, 5-
CH3), 1.93 (s,3H, 3-CH3), 6.61(d, 2H, Ar-Ha), 6.89 (d, 2H, Ar-Hb), 2.35 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6) δ 11.6, 21.9, 45.5, 113.4, 176.6, 200.10, 117.1, 129.8, 153.0, 134.6,
25.3;Mass spectrum m/z: 234.90 [M+]; Anal. Calcd. (%) C13H14O2S: C, 66.64; H, 6.02, O, 13.66.
Found: C, 66.61; H, 6.01, O, 13.63.
4-(4-(trifluoromethyl)phenoxy)-3,5-dimethylthiophen-2(5H)-one (5c). Yield 83 %; m.p. 219-
220ºC; IR (KBr) (vmax,cm–1): 1250 (C-O-C), 1760(C=O), 618(C-S), 1630(C=C), 2960(C-H)
1210(C-F), 2900(C-H), 1409,1614,1505 (C=C ring str.) ; 1H NMR (400 MHz, DMSO-d6) δ 1.50
(d, 3H, 5- CH3), 4.45 (q, 1H, 5-H), 1.90 (s, 3H, 3-CH3), 6.66 (d, 2H, Ar-Ha), 7.28(d, 2H, Ar-Hb);
13C NMR (100 MHz, DMSO-d6) δ 22.0, 44.6, 112.6, 175.5, 205.0, 117.8, 126.9, 157.3, 126.6,
124.2; Mass spectrum m/z: 287.10 [M+]; Anal. Calcd. (%) C13H14O2S: C, 54.16 H, 3.85, O,
11.10. Found: C, 66.60; H, 3.81, O, 11.09.
4-(4-chlorophenoxy)-3,5-dimethylthiophen-2(5H)-one (5d).Yield 73 %; m.p. 175-176ºC; IR
(KBr) (vmax,cm–1): 1260 (C-O-C),1735(C=O),630(C-S),1615(C=C), 2940(C-H), 1580,1630,1540
(C=C ring str.) 2910 (C-H) 760(C-Cl) ; 1H NMR (400 MHz, DMSO-d6) δ 4.46 (q,1H, 5-H), 1.43
(d, 3H, 5-CH3), 1.96 (s, 3H, 3-CH3), 6.67(d, 2H, Ar-Ha), 7.10 (d, 2H, Ar-Hb); 13C NMR (100
MHz, DMSO-d6) δ 12.0, 21.8, 43.5, 114, 174.9, 206.1, 118.9,130.1, 155.1, 128.5; Mass spectrum
m/z: 255.10 [M+1]; Anal. Calcd. (%) C12H11O2S: C, 56.58; H, 4.35, O, 12.56. Found: C, 56.53;
H, 4.31, O, 12.54.
4-(4-fluorophenoxy)-3,5-dimethylthiophen-2(5H)-one (5e). Yield 71 %; m.p. 182-183ºC; IR
(KBr) (vmax,cm–1): 1015(C-F), 1220 (C-O-C),1740(C=O),645(C-S), 2870(C-H),1605(C=C), 2900
(C-H), 1475,1620,1580 (C=C ring str.) ; 1H NMR (400 MHz, DMSO-d6) δ 1.89 (s, 3H, 3-CH3),
4.40 (q, 1H, 5-H), 1.50 (d, 3H, 5-CH3), 6.71(d, 2H, Ar-Ha), 6.80(d, 2H, Ar-Hb); 13C NMR (100
MHz, DMSO-d6) δ 11.8, 21.0, 42.9,112.9, 175.6, 203.0, 119.0, 117.0, 151.6, 158.1(ArC2); Mass

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spectrum m/z: 238.20 [M+]; Anal. Calcd. (%) C12H11FO2S: C, 60.49; H, 4.65, O, 13.43. Found:
C, 60.47; H, 4.61, O, 13.39.
4-(4-bromophenoxy)-3,5-dimethylthiophen-2(5H)-one(5f). Yield 78 %; m.p. 185-186ºC; IR
(KBr) (vmax,cm–1): 560 (C-Br), 635 (C-S),1210 (C-O-C), 2910 (C-H) 1478,1619,1585 (C=C ring
1
str.); 1750(C=O), 1615(C=C), 2875 (C-H) ; H NMR (400 MHz, DMSO-d6) δ 1.92 (s, 3H, 3-
CH3), 4.45 (q, 1H, 5-H), 1.51 (d, 3H, 5-CH3), 6.63(d, 2H, Ar-Ha), 7.28 (d, 2H, Ar-Hb); 13C NMR
(100 MHz, DMSO-d6) δ 12.5, 19.6, 43.6, 113.7, 172.7, 198.7, 121.4, 131.4, 153.6, 117.8; Mass
spectrum m/z: 299.9 [M++2]; Anal. Calcd. (%) C12H11BrO2S: C, 60.49; H, 4.65, O, 13.43. Found:
C, 60.47; H, 4.61, O, 13.39.
4-(4-(trifluoromethyl)phenoxy)-3-ethyl-5-methylthiophen-2(5H)-one(5g).Yield 72 %; m.p. 175-
176ºC; IR (KBr) (vmax,cm–1): 1190 (C-F), 1460 (C-H), 620 (C-S), 2890 (C-H), 1450,1600,1500
1
(C=C ring str.), 1240(C-O-C), 1740(C=O), 1620(C=C), 2915 (C-H) ; H NMR (400 MHz,
DMSO-d6) δ 1.43 (d, 3H, 5-CH3), 4.36 (q, 1H, 5-H), 1.89 (t,3H,2’-CH3), 2.14 (q,2H,1’-CH2),
13
6.66 (d, 2H, Ar-Ha), 7.28 (d, 2H, Ar-Hb); C NMR (100 MHz, DMSO-d6) δ 14.0, 16.3, 21.5,
42.8, 118.2, 170.6, 206.0, 118.1, 130.8, 153.0, 134.0, 125.0; Mass spectrum m/z: 324.80
[M++Na]; Anal. Calcd. (%) C14H13F3O2S: C, 55.62; H, 4.33, O, 10.58. Found: C, 55.57; H, 4.29,
O, 10.55.
4-(p-tolyloxy)-3-ethyl-5-methylthiophen-2(5H)-one (5h). Yield 82 %; m.p. 179-180ºC; IR (KBr)
(vmax,cm–1): 2870 (C-H), 640(C-S), 1250 (C-O-C), 2940 (C-H), 1755(C=O), 2910 (C-
1
H),1560,1600,1480 (C=C ring str.),1450(C-H), 1600(C=C), 2930 (C-H) ; H NMR (400 MHz,
DMSO-d6) δ 4.46 (q, 1H, 5-H), 1.44 (d, 3H, 5-CH3), 1.96 (t,3H,2’-CH3), 2.22 (q,2H, 1’-CH2),
2.35 (s,3H,CH3), 6.61 (d, 2H, Ar-Ha), 6.89 (d, 2H, Ar-Hb); 13C NMR (100 MHz, DMSO-d6) δ
14.2, 16.3, 21.8, 42.6, 119.2, 169.2, 210.0, 117.9, 129.5, 154.0, 133.6, 26.0; Mass spectrum m/z:
249.0 [M+]; Anal. Calcd. (%) C14H16O2S: C, 67.71; H, 6.49,O, 12.89. Found: C, 67.65; H, 6.46,
O, 12.85.

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4-(4-fluorophenoxy)-3-ethyl-5-methylthiophen-2(5H)-one (5i). Yield 79 %; m.p. 183-184ºC; IR
(KBr) (vmax,cm–1): 1000 (C-F), 1460 (C-H), 635 (C-S), 1260 (C-O-C),
1
2890 (C-H), 1600,1580,1465 (C=C ring str.) 1745(C=O), 1610(C=C), 2926 (C-H) ; H NMR
(400 MHz, DMSO-d6) δ 1.96 (t,3H,2’-CH3), 4.50 (q, 1H, 5-H),1.44 (d, 3H, 5-CH3), 2.15
(q,2H,2’-CH2), 6.71 (d, 2H, Ar-Ha), 6.80 (d, 2H, Ar-Hb); 13C NMR (100 MHz, DMSO-d6) δ
14.6, 16.5, 22.5, 43.0, 118.8, 169.8, 208.0, 119.1, 115.2, 150.6, 158.6; Mass spectrum m/z:
252.06 [M+]; Anal. Calcd. (%) C13H13FO2S: C, 61.89; H, 5.19, O, 12.68. Found: C, 61.85; H,
5.14, O, 12.65.
4-(4-chlorophenoxy)-3-ethyl-5-methylthiophen-2(5H)-one (5j). Yield 71 %; m.p. 185-186ºC; IR
(KBr) (vmax,cm–1): 610 (C-Cl), 1455 (C-H), 640 (C-S), 2870(C-H), 1255(C-O-C), 1750
1
(C=O),1605(C=C), 1580,1620,1475 (C=C ring str.), 2914 (C-H) ; H NMR (400 MHz, DMSO-
d6) δ 4.46 (q, 1H, 5-H), 1.95 (t,3H,2’-CH3), 1.43 (d, 3H, 5-CH3), 2.20 (q,2H,1’-CH2), 6.67 (d,
2H, Ar-Ha) 7.10 (d, 2H, Ar-Hb); 13C NMR (100 MHz, DMSO-d6) δ 14.8, 16.3, 22.6, 42.7, 118.3,
170.8, 201.0, 118.7, 130.6, 153.7, 130.1;Mass spectrum m/z: 271.03 [M++1]; Anal. Calcd. (%)
C13H13ClO2S: C, 67.71; H, 6.49, O, 12.89. Found: C, 67.65; H, 6.46, O,12.85.
4-(4- aminosulfonyl phenoxy)-3-ethyl-5-methylthiophen-2(5H)-one (5k). Yield 73 %; m.p. 197-
198ºC; IR (KBr) (vmax,cm–1):3472 (N-H), 1240(C-O-C), 1755(C=O), 620(C-S), 1480(C-H),
1630(C=C),1359(S(=O2)asym), 2890(C-H),1140(S(=O2)asym), 1460,1680,1510 (C=C ring str.)
1
2940(C-H) ; H NMR (400 MHz, DMSO-d6) δ 4.46 (q,1H, 5-H), 1.43 (d, 3H, 5-CH3), 1.93 (t,
3H, 2’-CH3), 7.01 (d, 2H, Ar-Ha) 7.76(d, 2H, Ar-Hb), 2.16 (q, 2H, 1’-CH2), 3.40 (s,2H,NH2); 13C
NMR (100 MHz, DMSO-d6) δ 14.5, 16.4, 21.5, 42.8, 120.1, 170.1, 198.9, 117.8, 129.1, 158.6,
134.1;Mass spectrum m/z: 313.04 [M+]; Anal. Calcd. (%) C13H15NO4S2: C, 49.82; H, 4.82; O,
20.42.Found: C, 49.79; H, 4.76; O, 20.41.
4-(4-bromophenoxy)-3-ethyl-5-methylthiophen-2(5H)-one (5l). Yield 76 %; m.p. 189-190ºC; IR
(KBr) (vmax,cm–1): 570 (C-Br), 630 (C-S),1240 (C-O-C), 2940 (C-H) 1490,1580,1500 (C=C ring
str.); 1485(C-H), 1755(C=O), 1645(C=C), 2890 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ 4.40
(q, 1H, 5-H), 1.49 (d, 3H, 5-CH3), 1.95 (t,3H,2’-CH3), 6.98(d, 2H, Ar-Ha), 7.33 (d, 2H, Ar-Hb),
2.10 (q, 2H, 1’-CH2) ; 13C NMR (100 MHz, DMSO-d6) δ 14.1, 16.5, 20.6, 43.0, 119.5, 170.9,

ACCEPTED MANUSCRIPT
198.6, 120.5, 134.6, 154.1, 120.5; Mass spectrum m/z:311.9[M+],313.90 [M++2]; Anal. Calcd.
(%) C13H13BrO2S: C, 49.82; H, 4.82, O, 20.42. Found: C, 49.79; H, 4.76, O, 20.41.
4-(4-fluorophenoxy)-5-methyl-3-propylthiophen-2(5H)-one (5m). Yield 72 %; m.p. 180-181ºC;
IR (KBr) (vmax,cm–1): 1012 (C-F), 1220 (C-O-C), 2885(C-H),1750(C=O), 1455(C-H), 630(C-S),
1
1460,1610,1540 (C=C ring str.), 1475 (C-H), 1580(C=C), 1455(C-H), 2951(C-H) ; H NMR
(400 MHz, DMSO-d6) δ 1.37-1.42 (m,2H,2’-CH2), 4.36 (q, 1H, 5-H), 1.50 (d, 3H, 5-CH3), 0.90
13
(t, 3H, 3’-CH3),2.50 (t, 2H, 1’-CH2), 6.71 (d, 2H, Ar-Ha), 6.80 (d, 2H, Ar-Hb); C NMR (100
MHz, DMSO-d6) δ 17.3, 25.3, 25.8, 20.5, 46.8, 120.1, 176.1, 199.6, 119.1, 120.2, 156.9,
158.1; Mass spectrum m/z: 265.0 [M+-H-], Anal. Calcd. (%) C14H15FO2S : C, 63.14; H,
5.68,O,12.01. Found: C, 63.01; H, 5.59, O, 12.00.
4-(p-tolyloxy)-5-methyl-3-propylthiophen-2(5H)-one (5n). Yield 77 %; m.p. 190-191ºC; IR
(KBr) (vmax,cm–1): 1240(C-O-C), 2855(C-H), 1745(C=O), 615(C-S), 1465(C-H), 1620 (C=C),
1
1480 (C-H), 2930(C-H) ; 1480,1620,1520 (C=C ring str.); H NMR (400 MHz, DMSO-d6) δ
4.56 (q, 1H, 5-H), 1.47 (d, 3H, 5-CH3), 1.39-1.44 (m,2H,2’-CH2), 0.97 (t, 3H, 3’-CH3), 2.50 (t,
2H, 1’-CH2), 2.36(s,3H,CH3), 6.61 (d, 2H, Ar-Ha), 6.89 (d, 2H, Ar-Hb); 13C NMR (100 MHz,
DMSO-d6) δ 18.0, 25.7, 28.6, 20., 45.4, 120.1, 175.1, 198.6, 118.5, 130.6, 152.7, 138.4, 22.1;
Mass spectrum m/z: 262.10 [M+]; Anal. Calcd. (%) C15H18O2S : C, 68.67; H, 6.92,O, 12.20.
Found: C, 68.62; H, 6.88, O, 12.19.
4-(4-chlorophenoxy)-5-methyl-3-propylthiophen-2(5H)-one (5o). Yield 85 %; m.p. 190-191ºC;
IR (KBr) (vmax,cm–1): 610 (C-Cl), 2880(C-H), 1258 (C-O-C), 1470(C-H), 1753 (C=O), 640 (C-
S), 1485 (C-H), 1460,1600,1520 (C=C ring str.), 2910 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ
1.46 (d, 3H, 5-CH3), 1.00 (t, 3H, 3’-CH3), 4.44 (q, 1H, 5-H), 1.36-1.40 (m, 2H,2’-CH2 ), 2.49(t ,
13
2H,1’-CH2) 6.61 (d, 2H, Ar-Ha), 6.89 (d, 2H, Ar-Hb); C NMR (100 MHz, DMSO-d6) δ 18.2,
23.7, 26.2, 19.8, 45.3, 119.8, 174.8, 199.6, 118.8, 129.8, 153.8, 125.6 ; Mass spectrum m/z: 284.4
[M++1], Anal. Calcd. (%) C14H15ClO2S : C, 59.46; H, 5.35,O 11.32. Found: C, 59.42; H, 5.30,
O, 11.29.

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4-(4-(trifluoromethyl)phenoxy)-5-methyl-3-propylthiophen-2(5H)-one (5p). Yield 75 %; m.p.
189-190ºC; IR (KBr) (vmax,cm–1): 1160 (C-F), 2920(C-H), 1210 (C-O-C), 1730 (C=O), 1480 (C-
1
H), 643 (C-S), 1450 (C-H), 1450,1580,1500 (C=C ring str.), 2850 (C-H) ; H NMR (400 MHz,
DMSO-d6)δ 1.43 (d, 3H, 5-CH3), 0.95 (t, 3H, CH3), 4.46 (q, 1H, 5-H), 2.50 (t, 2H, 1’-CH2),
13
1.37-1.40 (m, 2H, 2’-CH2 ), 6.66 (d, 2H, Ar-Ha), 7.28 (d, 2H, Ar-Hb); C NMR (100 MHz,
DMSO-d6) δ 17.9, 23.6, 25.7, 19.0, 45.2, 119.6, 176.8, 199.8, 117.9, 126.6, 152.7, 124.3
Mass spectrum m/z: 338.20 [M++Na]; Anal. Calcd. (%) C15H15F3O2S : C, 56.95; H, 4.78,
O,10.12. Found: C, 56.89; H, 4.73, O, 10.09.
4-(4- amino sulfonyl phenoxy)-3-isopropyl-5-methylthiophen-2(5H)-one (5q). Yield 76 %; m.p.
185-186ºC; IR (KBr) (vmax,cm–1): 1303 (S(=O2) asym), 1206 (S(=O2) asym), 1260 (C-O-C),
1735 (C=O), 2855(C-H), 640 (C-S), 1480,1600,1540 (C=C ring str.), 2910 (C-H) ; 1H NMR (400
MHz, DMSO-d6) δ (ppm) 4.46 (q, 1H, 5-H), 1.46 (d, 3H,5- CH3), 1.11-1.14 (m,3H,CH3a,CH3b,),
2.53(t,1H,CH) ,7.01 (d, 2H, Ar-Ha), 7.76 (d, 2H, Ar-Hb),3.46 (s,2H,NH2); 13C NMR (100 MHz,
DMSO-d6) δ 20.6, 20.5, 23.9, 19.2, 45.8, 122.9, 174.9, 198.7, 117.7, 128.2, 158.9, 134.1;
Mass spectrum m/z: 327.06 [M+]; Anal. Calcd. (%) C14H17 NO4S2 : C, 51.36; H, 5.23; S,19.55.
Found: C, 51.31; H, 5.19; N, 4.23, O, 19.51.
4-(p-tolyloxy)-3-isopropyl-5-methylthiophen-2(5H)-one (5r). Yield 70 %; m.p. 192-193ºC; IR
(KBr) (vmax,cm–1): 1260 (C-O-C), 1735 (C=O), 2840 (C-H), 635 (C-S), 1460,1620,1560 (C=C
ring str.), 2930 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ 2.52 (t,1H,CH) 4.46 (q, 1H, 5-H), 1.43
(d, 3H, 5-CH3), 1.11-1.15 (d,3H,CH3a,CH3b), 2.35 (d, 3H, CH3), 6.61 (d, 2H, Ar-Ha), 6.89 (d, 2H,
13
Ar-Hb); C NMR (100 MHz, DMSO-d6) δ 21.1, 21.3, 23.6, 19.1, 44.9, 122.5, 175.8, 199.4,
117.8, 130.1, 152.1, 132.1, 21.6 ; Mass spectrum m/z: 262.10 [M+]; Anal. Calcd. (%) C15H18O2S
: C, 68.67; H, 6.92,O,12.20. Found: C, 68.65; H, 6.95,S, 12.19.
4-(4-(trifluoromethyl)phenoxy)-3-isopropyl-5-methylthiophen-2(5H)-one (5s). Yield 78 %; m.p.
190-191ºC; IR (KBr) (vmax,cm–1): 1220(C-F), 1260 (C-O-C), 2860(C-H), 1765 (C=O), 615 (C-S),
1460,1600,1580 (C=C ring str.), 2950 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ 2.60 (t,1H,CH)
4.26 (q, 1H, 5-H), 1.41 (d, 3H, 5-CH3), 1.12-1.14 (d,3H,CH3a, CH3b), 6.70 (d, 2H, Ar-Ha), 7.32
13
(d, 2H, Ar-Hb); C NMR (100 MHz, DMSO-d6) δ 21.8, 21.7, 22.6, 19.0, 43.9, 123.6, 173.8,

ACCEPTED MANUSCRIPT
200.4, 116.8, 124.6, 158.1, 125.6; Mass spectrum m/z: 338.2 [M++Na]; Anal. Calcd. (%)
C15H15F3O2S : C, 56.95; H, 4.78,O, 10.16. Found: C, 56.92; H, 4.76, O, 10.14.
4-(4-chlorophenoxy)-3-isopropyl-5-methylthiophen-2(5H)-one (5t). Yield 78 %; m.p. 165-
166ºC; IR (KBr) (vmax,cm–1): 650(C-Cl), 1255 (C-O-C), 2990 (C-H),1735 (C=O), 627 (C-S),
1595,1500,1390 (C=C ring str.), 2870 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ 4.66 (q, 1H, 5-
H), 1.49 (d, 3H, 5-CH3), 1.13-1.16 (d,3H,CH3a ,CH3b), 2.52 (t, 1H,CH), 6.67 (d, 2H, Ar-Ha),
7.28 (d, 2H, Ar-Hb); 13C NMR (100 MHz, DMSO-d6) δ 21.4, 21.8, 23.6, 19.3, 44.5,122.5,
173.9, 198.8, 118.9, 130.6, 153.8, 128.6; Mass spectrum m/z: 282.80 [M+]; Anal. Calcd. (%)
C14H15ClO2S : C, 59.46; H, 5.35,O, 11.32. Found: C, 59.43; H, 5.30,O, 11.30.
4-(4-fluorophenoxy)-3-isopropyl-5-methylthiophen-2(5H)-one (5u). Yield 82 %; m.p. 185-
186ºC; IR (KBr) (vmax,cm–1): 1151 (C-F), 1735 (C=O), 1255 (C-O-C), 620 (C-S), 2890 (C-H),
1500,1575,1600 (C=C ring str.), 2930 (C-H) ; 1H NMR (400 MHz, DMSO-d6) δ 4.27 (q, 1H, 5-
H), 1.45 (d, 3H, 5-CH3), 1.13-1.14 (d,3H,CH3a, CH3b), 2.59 (t, 1H,CH) , 6.71 (d, 2H, Ar-Ha),
6.80 (d, 2H, Ar-Hb); Mass spectrum m/z: 265.20 [M++H-]; Anal. Calcd. (%) C14H15FO2S : C,
63.14; H, 5.68, O,12.01 . Found: C, 63.10; H, 5.66, O, 11.98.
4-(hexyloxy)-3,5-dimethylthiophen-2(5H)-one (6a).Yield 61 %; m.p. 150-151ºC; IR (KBr)
1
(vmax,cm–1): 1760 (C=O), 1220 (C-O-C), 630 (C-S), 1456 (C-H), 2930 (C-H), 2875(C-H) ; H
NMR (400 MHz, DMSO-d6): δ (ppm): 4.45 (q, 1H, 5-H), 0.96 (m, 3H,CH3),1.43(d,3H,CH3a),
1.93 (s, 3H, CH3b), 1.33-3.96 (m, 2H, 5×CH2), Mass spectrum m/z: 229.0 [M+]; Anal. Calcd.
(%) C12H20O2S : C, 63.12; H, 8.33, O, 14.01. Found: C, 63.10; H, 8.30, O, 13.97.
2.2.3 In vitro antimalarial activity of synthesized thiolactone derivatives
The in vitro antimalarial assay was carried out i
microassay protocol of Rieckmann and o-work
cultures of falciparum strain (Dd2 strain and 3d7 strain) were maintained in medium RPMI
1640 supplemented with 25mM HEPES,1%D-g os 0.23% sodium bicarbonate and 10% eat
acti ate human serum. The syn hronous parasites of P. falciparum were syn hronize te
5% D-sorbit treatment to obtain only the ring stage itize cells. For carrying out the
n
96 well micro titre plates according to
the
c
e
rs with minor modifications[28-32].The
P.
lu
c
e
,
h
in
v
d
a
c
c
d af r
o
l
p
a
ra
s
d

ACCEPTED MANUSCRIPT
assay, an
in
itial ring
sta
g
e
parasitaemia of 0.8 to 1.5% at 3% haematocrit in a total volume of
200µl of medium RPMI-1640
w
a
s
determined by Jaswant Singh Bhattacharya (JS
itaemia (rings) and un formly maintained with 50%
DMSO
culture medium. The diluted samples in 20 µ l
volume were added to the test wells so as to obtai final concentrations (at five fold dilutions)
ng ng between 0.4 µ g/ml to 100 µ g/ml in duplicate well containing itize cell
preparation. The culture plates were incubated at 37 C in a candle jar. After 36 to 40 h
ubation, thin ood smears from each well were prepared and tai with JSB stain. The
slides were micros opicall observed for regular development of parasite stages presence of
ff concentrations of the test compounds. TLM and Chloroquine (CQ) were used as the
B)
staining[33] to assess the percent
+
p
a
r
a
s
i
RBCs (O ). A stock solution of 5mg/ml of each of the test samples was prepared in
and subsequent dilutions were prepared with
n
ra
i
p
a
r
as
d
o
in
c
b
l
s
ned
c
y
in
di erent
reference drug.
The inhibition of parasite growth in the test groups was calculated as follows.
Percentage inhibition = Control-Test ×100
Control
Where ‘‘control’’ is the parasitemia in the non-treated group and ‘‘test’’ is the parasitemia in the
test compounds treated groups expressed as percentages. All values are expressed as percentage
growth inhibition. Dose–response curves were obtained by plotting percentage inhibition against
log concentration. The values of the compounds provided a mid-point value where parasite
growth would be 50%. Linear regression analysis was applied to the linear portion of the sigmoid
curve and IC50 values were derived for each test compound. The results of in vitro evaluation are
reported in Table 1.
Table1. In vitro antimalarial activity of synthesized compounds against CQ resistant strain (Dd2)
& CQ-sensitive strain (3D7) of P. falciparum.

ACCEPTED MANUSCRIPT
O
1
S
2
5
R1
3
4
O
O
S
R1
O
R2
R2
5a-5u
6a
Cl
N
N
H
H
S
O
N
HO
Thiolactomycin
R1
Chloroquine
Compounds
R2
Pf Dd2 strain
Pf 3d7 strain
(IC50µg/ml)
1.59
(IC50µg/ml)
5a
5b
5c
5d
5e
5f
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
SO2NH2
CH3
CF3
Cl
1.19
0.97
0.87
0.19
0.09
1.35
0.90
0.98
1.39
1.28
0.04
0.03
1.89
1.31
1.41
F
Br
5g
5h
CF3
CH3
Ethyl

ACCEPTED MANUSCRIPT
5i
0.03
1.02
1.63
1.96
0.09
1.51
1.26
1.38
1.71
1.45
1.35
1.25
0.05
1.99
2.21
0.03
Ethyl
Ethyl
F
Cl
0.16
0.56
1.25
1.56
0.34
1.03
0.85
0.96
1.29
1.02
0.92
0.82
0.28
1.79
5j
5k
5l
Ethyl
SO2NH2
Br
Ethyl
5m
5n
5o
5p
5q
5r
Propyl
Propyl
Propyl
Propyl
F
CH3
Cl
CF3
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Methyl
SO2NH2
CH3
CF3
Cl
5s
5t
5u
6a
TLM
CQ
F
Hexyl
-
-
1.82
0.20
2.2.4 Molecular docking study of synthesized compounds
All the synthesized thiolactone derivatives were subjected for molecular docking to evaluate
their binding affinities in the active site of enzyme pfKAS I/II.
2.2.4.1Building of molecules using Chem Draw ultra and Chem 3D ultra
2D Structure of all the synthesized compounds were draw with the help of Chem Draw ultra
version 8.0.3 and exported to window of Chem 3D ultra version 8.0.3. Energy of all the 3D
structures were minimized through MOPAC up to RMS gradient 0.001 and saved in MDL Mol
file (.Mol) format.

ACCEPTED MANUSCRIPT
2.2.4.2 Homology modeling of enzyme pfKAS I/II
As the crystal structure of pfKAS I/II is not available in the protein data bank (PDB), hence the
3D structure of pfKAS I/II was predicted by homology modeling. The protein sequence (FASTA
sequence) of enzyme pfKASI/II was retrieved from Uniprot/Swissprot database. The sequence
was submitted to swiss model [34] and modweb [35] web servers to find a suitable template with
sufficient query sequence coverage and sequence identity. The final model derived through
modweb web server was selected on the basis of maximum sequence similarity (45.43%) with
the template (X-ray crystal structure of β-ketoacyl-acyl carrier protein synthase II (KASII) of
Synechocystis ; PDB code: 1e5Ma; resolution 1.54 Aº)[36].Then homology model of enzyme
pfKASI/II was energy minimized using GROMOS 96 implemented with spdbv viewer v 4.1.0
using conjugate gradient method[37].
The resemblance of structure of pf KAS I/II obtained from the homology modeling with the X-
ray crystal structure of KAS I/II of Synechocystis [36] that was used as the template showed in
the Fig.1a and b. The two enzymes share the catalytic residues that are located in their respective
active sites in a similar fashion which reflected the reliability of model generated through
homology modeling.
Fig.1. Comparative view of active sites of (a) Homology-modeled structure of pfKAS I/II (b) X-
ray crystal structure of KAS II of template Synechocystis.

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2.2.4.3 Alignment of target and template amino acid sequences
The percentage identity matrix between query (pfKASI/II of P.falciparum) and template (KASII
of Synechocystis) amino acid sequences was confirmed by the alignment of query and template
amino acid sequences. The alignment of the query and template amino acid sequences was
carried out through clustal o version 1.2.1(Fig.2) of European Molecular Biology Laboratory
(EMBL) which is a part of European Bioinformatics Institute (EBI).The percentage identity
matrix between the query and template sequence was found to be 45.43%[38].

ACCEPTED MANUSCRIPT
Fig.2. Clustal o v 1.2.1 alignment of pfKAS I/II of P.falciparum amino acid sequence with the
sequence of the crystallographic structure of 1e5mA available in PDB. (*) Asterisk indicates
positions which have a single, fully conserved residue. (:) Colon indicates conservation between
groups of strongly similar properties. (.) Period indicates conservation between groups of weakly
similar properties. Red color: Small amino acids possess hydrophobic side chain. Blue color:
Amino acids possess acidic side chain. Magenta color: Amino acids possess basic side chain.
Green color: Glycine and other amino acids possess side chain having hydroxyl, sulfhydryl and
amide group.
2.2.4.4 Structure validation of modeled pf KAS I/II enzyme
Structure of modeled enzyme was validated by PROCHECK analysis. For this analysis, the PDB
format file of modeled pfKASI/II enzyme was uploaded in PDB SUM web server of EBI. The
results of PROCHECK analysis was obtained in the form of Ramachandran plot (Fig. 3) which
resolve stereo chemical aspects along with main chain and side chain parameters with
widespread analysis[39,40].The Ramachandran plot (PROCHECK) statistics are summarized in
Table 2.

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Fig. 3. Ramachandran plot of homology modeled pfKASI/II.
Table 2. Ramachandran plot (PROCHECK) Statistics of homology modeled pfKASI/II.
Regions
No. of residues
Percentage
Most favoured regions[A,B,L]
Additional allowed regions [a,b,l,p]
Generously allowed regions [~a,~b,~l,~p]
Disallowed regions[XX]
313
37
5
87.4%
10.3%
1.4%
3
0.8%
Non-glycine and non-proline residues
End-residues (excl. Gly and Pro)
Glycine residues
358
2
100.0%
39

ACCEPTED MANUSCRIPT
Proline residues
14
413
Total number of residues
2.2.4.5 Docking with Molegro Virtual Docker (MVD) version 5.0
The molecular docking studies of the designed compounds were carried out through Molegro
Virtual Docker (MVD version 5) software [41]. For the docking purpose modeled pfKASI/II
enzyme was imported in the work space area. Afterward this enzyme was prepared for the
optimal docking. Then through an automatic procedure possible binding cavities were detected.
During this process the maximum number of cavities was fixed to 5, the grid resolution was 0.80
Å, and probe size was 1.20 Å, while other parameters were set default. Then ligands (synthesized
thiolactone derivatives) were imported in work space area, necessary bonds, bond orders,
hybridizations, hydrogen atoms, charges were assigned and flexible torsions were detected in the
ligands.
From the docking wizard ligands were selected and the scoring functions used in the docking
were moldock score and rerank score. The search algorithm is taken as moldock SE and numbers
of runs were taken 10 and maximum iterations were 1500 with population size 50 and the energy
threshold for pose generation was 100.00 and similar poses were ignored. Similar poses were
clustered depending upon an RMSD threshold of 1.00A°. After docking simulation the generated
poses were sorted on the basis of their moldock scores and rerank scores. The reranking score
function is computationally more expensive than the scoring function used during the docking
simulation but it is generally better than the docking score function at determining the best pose
among several poses originating from the same ligand. Docking output of MVD as moldock
score and rerank score (Kcal/mol) are showed in Table 3.
Table.3 Synthesized thiolactone derivatives moldock scores and rerank scores.
Compounds pfKAS I/II
Mol dock
pfKAS I/II
Rerank
Score
Score
(kcal/mol)
(kcal/mol)
-120.36
5a
5b
-138.52
-148.52
-129.85

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5c
5d
5e
5f
-159.29
-181.23
-185.58
-133.76
-156.20
-145.23
-165.11
-171.35
-138.09
-131.21
-175.29
-140.19
-162.30
-151.20
-137.07
-141.57
-153.29
-166.11
-178.37
-128.18
-105.89
-141.52
-164.52
-170.23
-115.26
-139.89
-124.51
-165.81
-153.25
-119.25
-114.22
-160.21
-120.58
-143.55
-133.54
-117.20
-123.44
-135.24
-148.23
-162.35
-110.52
-85.24
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
6a
TLM
3. Results and Discussion
3.1Chemistry
Infrared spectrum of all synthesized derivatives showed similar trend of stretching frequency
modes, existence of peaks in the region 1260-1220 cm-1 explained the linkage between
thiolactone and phenyl ring in all the compounds. C-S absorption peak appeared in the region

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640-615 cm-1 in all the compounds. A typical C=O absorption band appeared in the range 1765-
1725 cm-1 in all the compounds. Aromatic C=C ring stretching found in the region
1600,1550,1400.The S(=O)2 symmetric and asymmetric stretching in sulfonamide group (at R2
position) containing compounds existed in the region 1300-1355 cm-1 and 1206-1140 cm-1
respectively. C-H stretching of the methyl group appeared in the region of 2990-2850 cm-1 while
CH2 bend appeared in the region of 1485-1450 cm-1.
In the 1H NMR spectra of compounds 5a, 5b, 5c, 5d, 5e a sharp singlet is observed ranges from δ
1.89-1.93 for CH3 group at third position of the thiolactone ring. In compounds 5f, 5g, 5h, 5i, 5j
5k and 5l due to ethyl group at third position of thiolactone ring a quartet at δ 2.00–2.50 and a
triplet is appeared between δ 1.89-1.96 for CH2 and CH3 group respectively. The NMR spectra
of compounds 5l, 5m, 5n, 5o, 5p due to propyl group at third position of thiolactone ring
displayed triplet at δ 2.49-2.50 for 1’-CH2, multiplet at δ 1.36-1.44 for 2’-CH2 and δ 0.90-1.00
for CH3 group. Compounds 5q,5r,5s,5t and 5u due to isopropyl group showed doublet at δ 1.11-
1.15 for CH3a, and CH3b group and triplet at δ 2.52-2.60 for CH group.
13C-NMR spectra of the synthesized compounds in conformity with the assigned structures and
exhibited characteristics signals observed at around δ 190-210 for carbonyl carbon peak,
aromatic carbons was appeared around δ 117-160 and aliphatic carbon at δ 14.0-26.0. Carbon in
the thiolactone ring appeared at δ 42.0-176.0.
The mass spectra of all the synthesized compounds exhibited a prominent molecular ion peak
[M+] with different intensities. In some compounds [M+1] and [M+2] peaks have appeared due to
the presence of chloro and bromo group respectively.
3.2 Antimalarial activity
All the synthesized compounds were evaluated in vitro for their antimalarial activity against CQ
sensitive (3D7) as well as CQ-resistant (Dd2) strain of P. falciparum and their order of
antimalarial potencies are 5e > 5i > 5d > 5u > 5m > 5j >5t > 5o> 5c > 5g> 5s > 5p > 5b > 5h >
5r >5n> 5a > 5k > 5q > 5f > 5l > 6a. All the synthesized compounds were found to be more
active than TLM (Table 1). Variations of the different substituents on the core thiolactone ring

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have been investigated to establish the structure-activity relationship among the synthesized
compounds.
3.2.1 Substitution of the alkyl chain on the 3rdPosition of thiolactone ring
In an attempt to improve hydrophobic interactions in the active site of the enzyme pfKASI/II,
alkyl chain was introduced at 3rd position of thiolactone ring. As the chain length increased from
methyl to propyl, the antimalarial activity was found to be decreased. However compounds
substituted with isopropyl group at 3rd position (5q-5u) was found to be more active than
compounds possessed propyl group (5m-5p) at this position (Table 1). The docking study also
revealed that as the chain length increased on the third position of the thiolactone ring, it
produced steric clashes with the active site residues (Table 3).
3.2.2 Substitution of the para substituted phenyl ring on 4th position of hydroxyl group of the
thiolactone ring
The substitution of the para substituted phenyl ring on the hydroxyl group (at 4th position of
thiolactone ring) leads to an enhanced activity against the parasite P.falciparum compared to
TLM (Table 1). The substituted phenyl ring at 4th position of most of the synthesized compounds
oriented towards a hydrophobic cavity of the enzyme and stabilized by hydrophobic interactions
with active site residues of the enzyme. Such as the fluoro substituted phenyl ring of most potent
compound 5e is stabilized by the hydrophobic residues Phe 459, Phe 287, Gly 405, Gly460 and
Gly 461(Fig.4a).These residues are supposed to play a significant role in the catalytic
mechanism, as this is the region of the active site of the enzyme pfKASI/II where ACP of the
natural substrate malonyl-ACP interacted hydrophobically with residues Phe 459 and Phe 287.
The hydrophobic substituents such as fluoro, chloro, trifluoromethyl and methyl at para position
of the phenyl ring improved the antimalarial activity of compounds, because these groups were
stabilized by the amino acid residues of the hydrophobic groove of the pf KASI/II enzyme.
However the compounds (5a, 5k and 5q) hold sulfonamide group at para position of the phenyl
ring found to be less active than compounds possessed fluoro, chloro and methyl group at this
position. Because above compounds bound differently in the active site of the enzyme. Moreover
hydrophilic sulfonamide group was not stabilized by the residues of the active site of the enzyme
results in the weak binding of the compounds in the active site of pfKASI/II. Interestingly the
compounds (5f and 5l) substituted with bromo group at para position of phenyl ring was found to

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be less potent than compounds 5a, 5k and 5q. The probable reason behind this is that compounds
5f and 5l oriented altogether in a different manner in the active site of enzyme which account for
its lower inhibitory potency. However the substitution of the hexyl chain on the hydroxyl group
made it least active among all the synthesized compounds, because the long hexyl chain
produced steric perturbation with the active site amino acid residues account for its weak binding
in the active site of the enzyme (Fig. 5a). Further compound 6a did not show any hydrogen bond
interaction with the active site residues (Fig. 5a). The standard drug TLM showed weak binding
interactions and oriented differently in the active site of the enzyme as compared to all designed
compounds (Fig. 5b).
3.3.3 Substitution of methyl group on the 5th position of the thiolactone ring
In molecular docking study of the compounds 5a-5u and 6a in the active site of the pfKASI/II, It
was found that the methyl group at 5th position of the thiolactone ring of the designed
compounds displayed favorable steric interaction with the active site residues of the enzyme
pfKASI/II. Such as the methyl group at 5th position of the thiolactone ring of the most active
compound 5e showed favorable steric interaction with active site residue His 398 of the enzyme
(Fig. 4b).
Fig. 4. Interactions of compound 5e in the docking model of active site of enzyme pfKASI/II (a)
Position of the substituted phenyl ring in hydrophobic pocket formed by residues Gly460,

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Gly461, Phe287, Phe459, and Gly 405. (b) Hydrogen bond interactions (blue dotted line) of
compound 5e with residues Cys 220 and Thr 365, while fluoro group (grey color) substituted at
para position (R2) of phenyl ring oriented toward hydrophobic groove of enzyme.
Fig.5. (a) Interactions of the compound 6a in the active site of enzyme pfKASI/II (b) Interactions
of the TLM in the active site of enzyme pfKASI/II.
4. Conclusions
A series of twenty two thiolactone derivatives was synthesized and evaluated (in vitro) against
Dd2 and 3D7 strain of P.falciparum for their antimalarial activity. Three compounds 5d, 5e and
5i showed encourageous antimalarial activity. The most potent compound 5e have methyl and
fluoro group at R1 and R2 positions respectively while compound 5d possess methyl group at R1
position and chloro group at R2 position. Compound 5i hold ethyl and fluoro group at R1 and R2
positions respectively. Docking studies also showed that compounds 5e have good binding
affinity for pf KASI/II. The structure activity relationships discussed above may provide valuable
information for further design and synthesis of compounds with optimized antimalarial activity.
Declaration of interest
The authors have no conflict of interest.

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Acknowledgment
This work was supported by the Madhya Pradesh Council of Science and Technology, Bhopal
(M.P.), India [Grant No:A/R&D(BS)/02/21].
References
1. World Health Organization, WHO 2015 World Malaria Report. Available at
http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/, Geneva,
2015.
2. World Health Organization, WHO 2013 World Malaria Report. Available at
http://www.who. Int/malaria/publications/world_malaria_report_2013/en/, Geneva, 2013.
3. World Health Organization Global Malaria Programme. Status report on artemisinin
resistance,September2014(http://www.who.int/malaria/publications/atoz/status_rep_arte
misinin_resistance_sep2014.pdf, accessed 16 November 2015).
4. H.J. Vial, M.L. Ancelin Malarial lipids. Biochem. 18 (1992) 259–306.
5. S. Kohler, C. F. Delwiche, P.W. Denny, L.G. Tilney, P. Webster, R.J. Wilson, J.D.
Palmer, D.S. Roos, Science 275(1997)1485-1489.
6. G.I. McFadden, M.E. Reith, J. Munholland, U. Lang, Nature 381 (1996) 482-482.
7. G.I. McFadden, R.F. Waller, Bio. Essays. 19 (1997) 1033-1040.
8. R.J.M. Wilson, P.W. Denny, P.R. Preiser, K. Rangachari, K. Roberts, A. Roy, A. Whyte,
M. Strath, D.J. Moore, P.W. Moore, D.H. Williamson, J. Mol. Biol. 261(1996) 155-172.
9. A.M. Vaughan, M.T. O’Neill, A.S. Tarun, N. Camargo, T.M. Phuong, A.S. Aly, A.F.
Cowman, S.H. Kappe, Cell Microbiol. 11(2009) 506-520.
10. M. Yu, T.R. Kumar, L.J. Nkrumah, A. Coppi, S. Retzlaff, C.D. Li, B.J. Kelly, P.A.
Moura, V. Lakshmanan, J.S. Freundlich, J.C. Valderramos, C. Vilcheze, M. Siedner,
J.H. Tsai, B. Falkard, A.B. Sidhu, L.A. Purcell, P. Gratraud, L. Kremer, A.P. Waters,
G. Schiehser, D.P. Jacobus, C.J. Janse, A. Ager, W.R. Jacobs, J.C. Jr Sacchettini,V.
Heussler, P. Sinnis, D.A. Fidock, Cell Host Microbe 4 (2008) 567-578.
11. J.L. Harwood, Biochim. Biophys. Acta 1301(1996) 7-56.
12. M.J. Gardner, N. Hall, E. Fung, O. White, M. Berriman, R.W. Hyman , J.M. Carlton, A.
Pain, K.E. Nelson, S. Bowman, I.T. Paulsen, K. James, J.A. Eisen, K. Rutherford, S.L.
Salzberg, A. Craig, S. Kyes, M.S. Chan, V. Nene, S.J. Shallom, B. Suh, J. Peterson, S.
Angiuoli, M. Pertea, J. Allen, J. Selengut, D. Haft, M.W. Mather, A.B. Vaidya, D.M.A.