Hybrids of thienopyrimidinones and thiouracils as anti-tubercular agents: SAR and docking studies

Article abstract of DOI:10.1016/j.ejmech.2017.01.009

A number of hybrid molecules containing thienopyrimidinones and thiouracil moieties were designed, synthesized and tested against Mycobacterium tuberculosis H37Ra wherein it was observed that the compounds 11–14 exhibited antitubercular activity in?vitro

Full text of DOI:10.1016/j.ejmech.2017.01.009

Accepted Manuscript
Hybrids of thienopyrimidinones and thiouracils as anti-tubercular agents: SAR and
docking studies
Mahesh M. Pisal, Laxman U. Nawale, Manoj D. Patil, Sujit G. Bhansali, Jayant M.
Gajbhiye, Dhiman Sarkar, Subhash P. Chavan, Hanumant B. Borate
PII:
S0223-5234(17)30017-X
10.1016/j.ejmech.2017.01.009
EJMECH 9160
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 November 2016
Revised Date: 5 January 2017
Accepted Date: 7 January 2017
Please cite this article as: M.M. Pisal, L.U. Nawale, M.D. Patil, S.G. Bhansali, J.M. Gajbhiye, D.
Sarkar, S.P. Chavan, H.B. Borate, Hybrids of thienopyrimidinones and thiouracils as anti-tubercular
agents: SAR and docking studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.01.009.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Hybrids of thienopyrimidinones and thiouracils as anti-
tubercular agents: SAR and docking studies
Mahesh M. Pisal, Laxman U. Nawale, Manoj D. Patil, Sujit G. Bhansali, Jayant M. Gajbhiye,
Dhiman Sarkar, Subhash P. Chavan, Hanumant B. Borate

ACCEPTED MANUSCRIPT
Hybrids of thienopyrimidinones and thiouracils
as anti-tubercular agents: SAR and docking studies
Mahesh M. Pisala, Laxman U. Nawaleb, Manoj D. Patila, Sujit G. Bhansalib,
Jayant M. Gajbhiyea, Dhiman Sarkarb, Subhash P. Chavana, Hanumant B. Boratea*
aDivision of Organic Chemistry, CSIR-National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune 411 008, India
bCombichem Bio-resource Centre, CSIR-National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune 411 008, India
*Corresponding author
hb.borate@ncl.res.in, Tel: 91-20-25902546, Fax: 91-20-25902629
Abstract
A number of hybrid molecules containing thienopyrimidinones and thiouracil moieties were
designed, synthesized and tested against Mycobacterium tuberculosis H37Ra wherein it was
observed that the compounds 11-1514 exhibited antitubercular activity in vitro (MIC 7.6-19.1
µg/ml, 12-35 µM) against dormant stage while compound 15 exhibited antitubercular activity in
vitro against dormant (MIC 23.4 µg/ml, 41 µM) as well as active (MIC 25.4 µg/ml, 45 µM)
stage. Structural modifications of compound 15 were carried out to study the structure-activity
relationship and it was observed that compound 18 exhibited antitubercular activity comparable
to compound 15. Cytotoxicity studies revealed that these molecules were non-toxic. The docking
study of compound 15 showed that there is was binding with the active site of mycobacterial
pantothenate synthetase. Further docking studies led to the synthesis of the compounds 16 and 17
and the antitubercular activity screening results showed that these compounds have significant
antitubercular activity. The compounds 15-18 (MIC 11-29 µg/ml, 19-51 µM) can be used as
starting points for further optimization. The synthetic strategies used in the present work have
potential to prepare a large number of compounds for further refinement of structures and the
present results will be very useful in the development of a new class of antimycobacterial agents.

ACCEPTED MANUSCRIPT
Key words: Tuberculosis; Antitubercular activity; Thienopyrimidinone; Thiouracil; Docking.
1. Introduction
Tuberculosis is an infectious disease that claims a number of deaths paralleled only by those
from HIV/AIDS [1]. Development of new antitubercular drugs is very important due to
occurrence of multidrug resistant tuberculosis (MDR-TB) and emergence of extensively drug-
resistant tuberculosis (XDR-TB). However, tuberculosis is one of the neglected tropical diseases
(NTDs), a diverse group of communicable diseases that prevail in tropical and subtropical
conditions which affect populations living in poverty, without adequate sanitation and in close
contact with infectious vectors. The development of new antitubercular drugs is very slow due to
lack of adequate funding. As a result, after a gap of 40 years, the U.S. Food and Drug
Administration (FDA) approved bedaquiline [2,3] in December 2012 as part of combination
therapy in adults to treat pulmonary MDR-TB, followed by the interim guidance on the use of
delamanid [4] in 2014. New drug development is a continuous, lengthy process and it is
necessary to synthesize and screen a large number of chemical entities as a slight modification in
the structure can cause dramatic decrease/increase in the biological activity and the same is
applicable to the development of antitubercular agents [5-8]. As a part of a program to develop
new drugs, we have synthesized a number of new molecules containing thienopyrimidinone
moiety and have found that some of them exhibited promising antitubercular activity [5,69,10]
while some exhibited antifungal activity [7,811,12]. We wished to explore the potential of
hybrid molecules containing thienopyrimidinones and thiouracils (which are known to exhibit
various biological activities [9-1413-18] including anticancer, anti-inflammatory, antibacterial,
antifungal etc) as the hybrid molecules are reported to have better activity in many cases [1519].
Accordingly, various new molecules 11-15 and 18-27 were synthesized and characterized with
the help of spectral methods. Structure of the representative molecule 18 was confirmed with X-
ray crystallography. These molecules were screened against Mycobacterium tuberculosis H37Ra
(ATCC 25177) wherein it was observed that the compounds 11-15, 18, 22 and 25 exhibited
significant antitubercular activity. Based on the docking study results for compound 15,
compounds 16 and 17 were prepared and were found to be more active than the compound 15
and the results are reported herein.
2. Results and discussion
2.1. Chemistry
The synthetic route for various compounds in the present study is shown in Scheme 1. The
intermediate
4-oxo-2-thioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydropyrimidine-5-
carbonitrile (1) was prepared by reaction of 3,4,5-trimethoxybenzaldehyde, ethyl cyanoacetate
and thiourea by the known method [1317]. The thienopyrimidinones 3a-e were prepared by the
methods described in our earlier work [5-89-12] involving Gewald reaction of required
aldehyde/ketone with ethyl cyanoacetate and sulphur in DMF in the presence of triethylamine to
get the corresponding substituted ethyl 2-aminothiophene-3-carboxylate followed by reaction
with formamide in the presence of ammonium acetate. The bromides 4-10 were obtained from
thienopyrimidinones 3a-e by reacting them with corresponding dibromoalkanes in the presence
of
base.
The
reactions
of
4-oxo-2-thioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-

ACCEPTED MANUSCRIPT
tetrahydropyrimidine-5-carbonitrile (1) with bromides 4-8 in DMF in the presence of potassium
carbonate at room temperature afforded the hybrid molecules 11-15. The compound 15 was
reacted with various (un)substituted alkyl halides, propargyl bromide, (un)substituted benzyl
bromides or p-toluenesulfonyl chloride in DMF in the presence of potassium carbonate at room
temperature to obtain novel molecules 18-27.

ACCEPTED MANUSCRIPT
OMe
CHO
OMe
b
MeO
MeO
CN
O
S
a
O
+
+
NC
OEt
H2N
HN
NH2
MeO
R1
OMe
HN
NH
1
S
O
O
R1
O
R1
R1
X
Br
N
EtO
H2N
c
O
d
R2
R2
R2
R2
S
N
S
N
S
4-10
2
3
4: R1=H, R2=propyl, X=-(CH2)2-
5: R1=H, R2=pentyl, X=-(CH2)2-
6: R1=H, R2=hexyl, X=-(CH2)2-
7: R1=H, R2=heptyl, X=-(CH2)2-
8: R1,R2=-(CH2)4-, X=-(CH2)2-
9: R1=H, R2=hexyl, X=-CH2-
10: R1=H, R2=heptyl, X=-CH2-
a: R1=H, R2=propyl
b: R1=H, R2=pentyl
c: R1=H, R2=hexyl
d: R1=H, R2=heptyl
e: R1,R2=-(CH2)4-
a: R1=H, R2=propyl
b: R1=H, R2=pentyl
c: R1=H, R2=hexyl
d: R1=H, R2=heptyl
e: R1,R2=-(CH2)4-
OMe
OMe
MeO
MeO
CN
MeO
CN
O
O
X
d
4-10
1
+
d
MeO
N
NH
N
N
O
R3
X
R1
O
S
R1
N
S
N
R2
R2
N
S
11-17
18-27
N
S
11: R1=H, R2=propyl, X=-(CH2)2-
12: R1=H, R2=pentyl, X=-(CH2)2-
13: R1=H, R2=hexyl, X=-(CH2)2-
14: R1=H, R2=heptyl, X=-(CH2)2-
15: R1,R2=-(CH2)4-, X=-(CH2)2-
16: R1=H, R2=hexyl, X=-CH2-
17: R1=H, R2=heptyl, X=-CH2-
18: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=methyl
19: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=pentyl
20: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=octyl
21: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=undecyl
22: R1,R2=-(CH2)4-, X=-(CH2)2-,
R3=3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl
23: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=propargyl
24: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=benzyl
25: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=2,3,4,5,6-
pentafluorobenzyl
26: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=p-toluenesulfonyl
27: R1,R2=-(CH2)4-, X=-(CH2)2-, R3=3-(4-oxo-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidin-
3(4H)-yl)propyl)
o
Scheme 1. Reagents and conditions: a: K2CO3, DMF, 110 C, 3 h; b: Ethyl cyanoacetate, sulphur, triethyl
amine, DMF, 55 oC, 12 h; c: Ammonium acetate, formamide, 145 oC, 12 h; d: Required halide,
K2CO3, DMF, RT, 12 h.
The structure of representative molecule 18 was confirmed by X-ray crystallography [1620] and
the ORTEP diagram is shown in Figure 1.

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Figure 1. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) of compound 18 showing the atom
numbering scheme. The displacement ellipsoids are drawn at the 30% probability level and H-
atoms are shown as small spheres with arbitrary radii (CCDC No. 1504297).
When the thiouracil 1 was reacted with more than one equivalents of the bromide 8, the N,S-
dialkylated compound 27 was obtained in addition to the S-alkylated compound 15. The N,S-
dialkylated compound 27 was also obtained by reacting the S-alkylated compound 15 with the
bromide 8.
Based on results of antitubercular activity screening and docking study of the above molecules,
compounds 16 and 17 were synthesized by reactions similar to those used for the synthesis of
compound 15.
The 6-(4-hydroxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (28) and 4-
oxo-2-thioxo-6-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (29) were prepared by
procedure [17] that was used for 4-oxo-2-thioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-
tetrahydropyrimidine-5-carbonitrile (1) wherein 3,4,5-trimethoxybenzaldehyde was replaced
with 4-hydroxybenzaldehyde and 4-methylbenzaldehyde.
2.2. Evaluation of biological activity
The new molecules synthesized in the present work were screened for antitubercular
activity (Table 1) against Mycobacterium tuberculosis H37Ra (ATCC 25177) by in vitro [21]
and ex vivo methods [17-1922, 23].
Table 1. Antimycobacterial activity data for various hybrids (11 to 27) of thiouracils and
thienopyrimidinones and thiouracils 1, 28 and 29 against H37Ra
Entry Compound Intracellular (Ex Vivo)
no no
Extracellular (In Vitro)
In vitro

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M.
M.
M.
M.
Cytotoxicity
against
tuberculosis tuberculosis tuberculosis tuberculosis
H37 Ra
(Dormant
Stage)
H37 Ra
(Active
Stage)
H37 Ra
(Dormant
Stage)
H37 Ra
(Active
Stage)
THP-1
monocytes
MIC
(µg/mL)
MIC
(µg/mL)
GI50
(µg/mL)
11.9±1.80
>30
>30
>30
19.1±0.16
>30
>30
>30
>100
>100
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
9.8±0.81
7.1±0.91
6.91±0.12
26.1±1.45
18.8±0.17
13.6±0.23
21.5±1.23
>30
12.3±0.27
8.7±1.11
7.6±0.38
23.4±1.42
19.1±0.56
17.1±0.85
26.1±1.52
>30
54.3±4.78
68.2±0.39
97.6±1.84
96.4±1.55
88.1±3.61
89.7±0.93
>100
>30
>30
28.8±3.0
17.1±0.61
11.1±2.6
26.9±1.66
>30
25.4±0.65
15.6±0.59
11.1±0.15
27.9±1.82
>30
19
>30
>30
8.8±0.20
>30
>30
26.5±1.62
>30
>30
11.3±0.49
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
11.7±
>30
>30
28.3±
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>100
>100
>100
>100
>100
>100
>100
10
11
12
13
14
15
16
17
18
19
20
21
20
21
22
23
24
25
26
27
81.4±1.11
>100
93.0±1.35
88.9±1.92
1
28
29
>30
>30
>30
17.3±0.44
>30
>30
>30
>30
>30
>30
0.75±0.014 0.51±0.012 0.48±0.016 0.41±0.02 0.1374±0.12
Rifampicin
Ex vivo: Intracellular antitubercular activities of each agent in differentiated THP-1
cells.
It was found that the compounds 15, 16, 17 and 18 exhibited very good antitubercular activity
against dormant as well as active stage of M. tuberculosis H37Ra (MIC 11-29 µg/ml, 19-51 µM)
while compounds 1, 11, 12, 13, 14, 22 and 25 exhibited very good antitubercular activity against
dormant stage of M. tuberculosis H37Ra. The cytotoxicity studies [19-2124-25] against THP-1
monocytes (GI50 values >50 µg/mL) showed that these compounds were non-toxic. The
compounds synthesized in the present study were further studied for cytotoxicity against three
human cancer cell lines and the results are shown in Table 2.
Table 2. Cytotoxicity profile of various hybrids (11 to 27) of thiouracils and
thienopyrimidinones and thiouracils 1, 28 and 29 against human cancer cell lines
Entry
no
A549
(Human lung)
PANC 1
(Human pancreas)
HeLa
(Human
Comp no

ACCEPTED MANUSCRIPT
cervix)
GI50 (µg/mL)
GI50 (µg/mL)
GI50 (µg/mL)
>100
>100
>100
72.8±3.81
28.1±7.10
>100
52.8±7.04
36.5±2.60
72.6±6.48
24.8±2.53
>100
52.5±4.91
75.8±4.32
67.0±2.76
76.1±3.65
55.7±6.77
76.3±6.84
75.4±3.40
70.7±0.84
73.0±3.55
>100
>100
>100
66.3±1.83
72.1±2.12
>100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
90.2±2.73
90.5±3.54
91.4±3.54
96.8±1.40
79.7±7.32
95.7±4.18
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
51.9±4.80
>100
>100
>100
>100
64.2±3.91
89.6±2.86
63.7±4.71
90.8±1.43
67.6±3.12
0.0048±0.0012
83.1±4.42
>100
87.4±2.12
86.7±4.33
0.0035±0.0014
1
28
29
89.1±6.93
>100
0.1279±0.022
Paclitaxel
It was observed that the GI50 values for all compounds studied were >50 µg/mL indicating that
the compounds arewere non-toxic.
The results of selectivity of hybrid molecules 11 to 27 towards human cell lines against H37Ra
in terms of the selectivity index are shown in Table 3. The selectivity index reflects the
concentration of the compound at which it is active against mycobacteria but is not toxic towards
host cells. According to the study of Hartkoorn [26] on the drug susceptibility of TB,
antimycobacterial activity was considered to be specific when the selectivity index was >10.
Some of the compounds e.g. 14 and 22 showed SI >10 against dormant M. tuberculosis H37Ra,
which were found to be good inhibitors of dormant M. tuberculosis H37Ra. Although the
selectivity index values for other compounds studied in the present work were <10, it is
important to consider the significance of this study with respect to the antitubercular activity
exhibited by the compounds studied, flexibility of synthetic strategies used in the present work
and occurrence of multidrug resistant tuberculosis (MDR-TB) as well as emergence of
extensively drug-resistant tuberculosis (XDR-TB). As the synthetic strategies used in the present
work have potential to prepare a large number of compounds for further refinement of structures,
the present preliminary results will be very useful in the development of a new class of

ACCEPTED MANUSCRIPT
antimycobacterial agents with selectivity index in the desired range by suitable structural
modifications.
Table 3. Selectivity index ratio of various hybrids (11 to 27) of thiouracils and
thienopyrimidinones and thiouracils 1, 28 and 29 against three human cancer cell lines
En Co
try mp
no no
SI against dormant M. tuberculosis
SI against active M. tuberculosis
H37Ra
H37Ra
A549
PANC-1
HeLa
A549
PANC-1
HeLa
A
B
A
>8
>7
4
B
>5
>6
3
A
B
3
A
B
>3
>3
3
A
>3
>2
1
B
>3
>2
1
A
B
2
1
2
>8
>5
4
8
>3
>3
3
2
3
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1
>10 >8
6
3
3
13
13
3
10
12
4
9
8
2
2
4
14
2
13
2
11
2
10
2
3
3
3
3
3
3
5
3
4
2
2
2
2
6
5
5
2
2
4
4
6
6
2
2
4
5
7
6
5
5
4
6
4
7
7
6
7
7
7
8
4
4
1
1
3
3
4
3
1
1
3
3
9
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
2
2
>3
>3
>3
>3
>3
>3
>3
>3
3
>3
>3
>3
>3
>3
>3
>3
>3
3
>3
>3
>3
>3
>3
2
>3
>3
>3
>3
>3
2
2
2
10
11
12
13
14
15
16
17
18
19
20
21
>3
>3
>3
>3
>3
>3
>3
2
>3
>3
>3
2
>11 >8 >11 >8 >11 >8
>3
>3
>4
>3
3
>3
>3
>4
>3
3
>3
2
>3
2
2
2
2
2
2
2
>4
>3
>3
>9
3
>4
>3
>3
>6
3
>4
2
>4
2
>3
>3
>3
>3
3
>3
>3
>3
>3
3
>3
2
>3
2
3
3
3
3
>9
3
>6
3
6
4
>3
3
>3
3
2
2
3
3
3
3
28
29
Rif
3
3
>3
>3
2
2
3
3
>3
196
>3
2
2
133 208 133 208 133 208 196 244
244 196 244
A: Ex vivo; B: In vitro; Rif: Rifampicin

ACCEPTED MANUSCRIPT
The antitubercular screening results indicated following points regarding the structure-activity
relationship of the compounds studied in the present work.
1. The hybrid molecules 11 to 17 with free NH in thiouracil moiety exhibited antitubercular
activity against M. tuberculosis H37Ra.
2. The compounds 11 to 14 with three-carbon linker between thienopyrimidinone and
thiouracil moieties and having alkyl substituent on thiophene ring of thienopyrimidinone
exhibited antitubercular activity against dormant stage of M. tuberculosis H37Ra while
corresponding compound 15 with three-carbon linker between thienopyrimidinone and thiouracil
moieties and having cycloalkyl substituent on thiophene ring of thienopyrimidinone exhibitsed
antitubercular activity against dormant as well as active stage of M. tuberculosis H37Ra.
3. The compounds 16 and 17 with two-carbon linker between thienopyrimidinone and
thiouracil moieties and having alkyl substituent on thiophene ring of thienopyrimidinone also
exhibited antitubercular activity against dormant as well as active stage of M. tuberculosis
H37Ra.
4. Methyl group iswas tolerated on thiouracil moiety of compound 15 but alkylation with
longer side chains resultsed in loss of activity (compound 15 v/s compound 18 v/s compounds
19, 20 and 21).
5. The reaction of 15 with propargyl bromide, benzyl bromide, p-toluenesulfonyl chloride etc
resultsed in loss of activity indicating that free NH iswas preferred (compound 15 v/s compounds
23, 24 and 26). The compound 27 iswas also inactive supporting the above
observationindication.
6. The compounds 20 and 24 arewere inactive while the corresponding fluorinated compounds
22 and 25 arewere active against dormant stage of M. tuberculosis H37Ra indicating that
introduction of fluorine atoms helpsed to get better antitubercular activity.
The structure-activity relationship studies clearly indicated that it would be possible to get
molecules with better antitubercular activity/selectivity index by suitable structural
modifications.
2.3. Docking Studies
In silico based approaches have provided a new perspective in the development of highly
efficient chemical leads and have huge potential to impart as starting points in the development
of new chemical entities against TB. So, docking studies were performed against mycobacterial
pantothenate synthetase due to availability of limited resources availability for carrying out
enzyme-based experimental studies to find out the best possible mode of action of the
synthesized hybrids of thienopyrimidinones and thiouracil derivatives
A distinctive characteristic of M. Tuberculosis is that its cell wall is enriched with the
lipids which are essential for its intracellular endurance, pathogenicity and also it is believed that
it makes entry of antimicrobial agents in to the cells difficult [2227]. In M. Tuberculosis genome
huge numbers of genes encoding several enzymes are involved in the metabolism of fatty acids
[2328], so inhibition of this pathway can be an important target in antitubercular drug discovery.
The enzyme pantothenate synthetase (PS or PanC) is encoded by the gene panC, which is
important for the synthesis of pantothenate in bacteria [2227]. Pantothenate is required for
biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP) which are the important

ACCEPTED MANUSCRIPT
elements for fatty acid synthesis [2328]. In the in vitro studies it was found that the gene
encoding panC is essential for the optimum growth of bacteria and when it was genetically
broken down in M. tuberculosis, it made the strain auxotrophic and required supplementation of
pantothenate for its growth [24-2629-31]. Also, its pathogenicity is weakened in this strain
[2732]. In mammals, panC is not present [28,2933,34] so targeting this enzyme will have huge
potential for developing drugs which will not have any side effects in the hosts. Thus, it makes
pantothenate synthetase an important target for drug discovery against tuberculosis (TB). To
continue our ongoing endeavor for discovering new potent antitubercular agents [610], this study
providesd valuable guidance for rationally designing more potent inhibitors for treatment of TB.
The antitubercular activity exhibited by compounds 11-15 prompted us to carry out
docking studies [35-40] of compound 15 (Figure 2). From the docking studies (details given in
supporting information), it iswas clear that compound 15 iswas showing hydrogen bonding
interactions with Gln72 with the distance of 2.10A° and 2.18A° and with Gly158 with the
distance of 2.16A°. Also, it iswas observed that thiophene ring showsed weak π- π stacking
interaction with amino acid Arg198 which leadsled to weak binding of compound 15 in the
active site of PanC with the docking score of -5.863.
Figure 2. 3D view of binding of compound 15 with the active site of mycobacterial PanC
Encouraged by the antitubercular activity of compounds 11-15 and docking study of
compound 15 indicating its binding in the active site of pantothenate synthetase, compounds 16
and 17 were designed and subjected to docking studies (details given in supplementary
information). The docking scores of compounds 16 and 17 were -7.949 and -8.666 respectively.
Molecular binding interactions (3D and 2D views) of compound 17 with the active site of
mycobacterial pantothenate synthetase are shown in Figures 3 and 4.

ACCEPTED MANUSCRIPT
Figure 3. 3D view of the binding of compound 17 with the active site of mycobacterial
pantothenate synthetase

ACCEPTED MANUSCRIPT
Figure 4. 2D view of the binding of compound 17 with the active site of mycobacterial
pantothenate synthetase
From the lowest energy docking pose of compound 17, it was noticed that strong
hydrogen bonding interactions were observed between methoxy groups present on the phenyl
ring and amino acid residue Gln72 with the distance of 2.08A°. Oxo group attached to the
pyrimidine ring in compound 17 showsed the hydrogen bonding with Ser196 with the distance of
2.53A°. Oxo group attached to thienopyrimidinone showsed the hydrogen bonding interactions
with the amino acid residue Met40 with the distance of 2.59A°. Further, the heptyl group
attached to thienopyrimidinone ring of compound 17 fitsfitted into hydrophobic pocket formed
by amino acid residues Phe157, Val142, Val143 and Ile168 present within the active site of
mycobacterial pantothenate synthetase as seen in Figure 4. The strong hydrogen bonding and
hydrophobic interaction leadsled to orientation of ligands within the active site so that they
formed strong steric and electrostatic interactions with the amino acid residues present in the
active site of mycobacterial pantothenate synthetase.
The compound compound 17 showsed firm binding with the active site of mycobacterial
pantothenate synthetase with the binding energy of -54.413 kcal/mol. Many favorable van der

ACCEPTED MANUSCRIPT
Waals interactions (details given in supporting information) were observed with amino acid
residues present in the active site. Also, several strong electrostatic interactions (details given in
supporting information) were seen with amino acid residues which stabilized the compound 17
in to the active site of mycobacterial pantothenate synthetase.
Validation of docking procedure: Co-crystallized ligand FG6 present in the active site of
mycobacterial pantothenate synthetase was extracted and was docked again into the active site.
Root mean square deviation (RMSD) value was found to be below 1.5Å which validatesed our
docking studies.
Accordingly, compounds 16 and 17 were synthesized and screened for antitubercular activity
(Table 1, entries 6 and 7) wherein it was found that these compounds were more active than the
corresponding compounds 13 and 14 as well as compound 15. Thus, from the docking studies
and antitubercular activity results, it iswas clear that hybrids of thienopyrimidinones and
thiouracils havehad significant binding with the active site of mycobacterial pantothenate
synthetase. So, the mechanism of action for antitubercular activity of these compounds might be
through the inhibition of mycobacterial pantothenate synthetase.
3. Conclusions
Synthesis of hybrid molecules containing thiouracil and thienopyrimidinone moieties was
achieved. The new chemical entities thus synthesized were tested against Mycobacterium
tuberculosis H37Ra and it was observed that the compounds 11-14 exhibited antitubercular
activity against dormant stage while compound 15 exhibited antitubercular activity against
dormant as well as active stage. Structural modifications of compound 15 were carried out to
study the structure-activity relationship and it was observed that compound 18 exhibited
antitubercular activity comparable to compound 15 while compounds 22 and 25 exhibited
antitubercular activity against dormant stage. Cytotoxicity studies revealed that these molecules
were non-toxic. The docking study of compound 15 showed that there iswas binding with the
active site of mycobacterial pantothenate synthetase with the docking score of -5.863. Further,
the compounds 16 and 17 were designed based on docking studies and their docking scores were
found to be -7.949 and -8.666 respectively indicating the possibility of these compounds to be
more active. The synthesis and antitubercular activity screening of the compounds 16 and 17
was carried out and it was found that these compounds were having potent antitubercular activity
supporting the binding of these compounds with mycobacterial pantothenate synthetase. Hence,
the compounds 15-18 can be used as starting points for further optimization. The selectivity
index for the compounds studied needs to be improved but tThe synthetic strategies used in the
present work have potential to prepare a large number of compounds for further refinement of
structures and the present results will be very useful in the development of new class of
antimycobacterial agents.
4. Experimental section
4.1. Chemistry
All reagents and solvents were used as received from the manufacturers. Melting points
are recorded in capillary tubes and are uncorrected and the temperatures are in centigrade scale.
1H (200, 400 and 500 MHz) and 13C (50, 100 and 125 MHz) NMR spectra were recorded on AC

ACCEPTED MANUSCRIPT
200 MHz, AV 400 MHz or AV-500 MHz NMR spectrometers using CDCl3 or DMSO-d6 as
solvent. The chemical shifts (
with reference to chloroform,
δ ) and coupling constants (Hz) are reported in the standard fashion
1
13
δ
7.27 (for H) or the central line (77.0 δ) of CDCl3 (for C). In
the 13C NMR spectra, the natures of the carbons (C, CH, CH2, or CH3) were determined by
recording the DEPT-135 spectra. The following abbreviations were used to explain the
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. FTIR spectra were
recorded using KBr plate. The mass spectra were recorded on Orbitrap MS (LC-HRMS). The
reaction progress was monitored by the TLC analysis using thin layer plates precoated with silica
gel 60 F254 and visualized by UV light or iodine or by charring after treatment with p-
anisaldehyde.
Synthesis of molecules
The thiouracils 1, 28 and 29 were prepared by the reported procedure [1317].
4-Oxo-2-thioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1)
Yield: 82%, White solid, Melting point: 283
(s, 3H), 3.51 (s, 6H), 6.60 (s, 2H). C NMR (50 MHz, CDCl3 + DMSO-d6):
⁰
C. 1H NMR (200 MHz, CDCl3 + DMSO-d6):
55.2 (2C), 59.6,
δ 3.47
13
δ
62.4, 105.4 (2C), 113.6, 122.3, 140.2, 151.7 (2C), 157.6, 158.9, 175.3. IR (CHCl3): 1214, 1689,
2405, 3417 cm-1. HRMS (ESI) m/z calculated for [C14H13N3O4S+H]: 320.0700, found:
320.0696; [C14H13N3O4S +Na]: 342.0519, found: 342.0516.
6-(4-Hydroxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (28)
1
Yield: 75%, White solid, Melting point: >295
⁰
C. H NMR (200 MHz, CDCl3 + DMSO-d6):
δ
13
6.00 (d, J = 8 Hz, 2H), 6.94 (d, J = 8 Hz, 2H), 9.06 (bs, 1H). C NMR (50 MHz, CDCl3 +
DMSO-d6):
δ 82.8, 113.0 (2C), 117.4, 126.1, 128.6 (2C), 158.0, 161.6, 165.3, 180.3. IR (CHCl3):
1225, 1625, 2209 cm-1. HRMS (ESI) m/z calculated for [C11H7N3O2S+H]: 246.0332 found:
246.0330.
4-Oxo-2-thioxo-6-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (29)
1
Yield: 79%, White solid, Melting point: >295
⁰
C. H NMR (200 MHz, DMSO - d6):
δ
2.40 (s,
3H), 7.39 (d, J = 8 Hz, 2H), 7.60 (d, J = 8 Hz, 2H), 13.05 (bs, 1H). 13C NMR (50 MHz, DMSO-
d6):
δ 21.4, 90.6, 115.1, 126.6, 129.0 (2C), 129.2 (2C), 142.8, 158.8, 161.2, 176.4. IR (CHCl3):
1217, 1675, 2232, 3317 cm-1. HRMS (ESI) m/z calculated for [C12H9N3OS+H]: 244.0538,
found: 244.0539; [C12H9N3OS + Na]: 266.0357, found: 266.0359.
Synthesis of 6-oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (15)
Potassium carbonate (6.48 g, 0.047 mol, 1.5 eq) was taken in a 500 mL two-necked RB flask and
heated under vacuum to remove the traces of moisture and flushed with nitrogen. 4-Oxo-2-
thioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1) (10 g, 0.031
mol, 1.0 eq) was added under nitrogen followed by dry DMF (125 mL) and stirred for 10 min. 3-
(3-Bromopropyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (10.20 g, 0.031
mol, 1.0 eq) was added and the reaction mixture was stirred at RT for 12 h. It was then diluted
with water (400 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layer

ACCEPTED MANUSCRIPT
was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure.
The residue was purified by column chromatography to afford 6-oxo-2-((3-(4-oxo-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-
1,6-dihydropyrimidine-5-carbonitrile (15) as off-white solid, 12.0 g (68%). Melting point: 230
⁰
C.
1H NMR (200 MHz, CDCl3):
2.94 (t, J = 6 Hz, 2H), 3.40 (t, J = 7 Hz, 2H), 3.89 (s, 6H), 3.94 (s, 3H), 4.16 (t, J = 7 Hz, 2H),
δ
1.73-1.91 (m, 4H), 2.33 (t, J = 7 Hz, 2H), 2.75 (t, J = 6 Hz, 2H),
13
7.33 (s, 2H), 7.99 (s, 1H). C NMR (125 MHz, CDCl3):
δ 21.4, 22.0, 24.4, 24.8, 27.2, 27.9,
44.3, 55.6 (2C), 60.1, 92.1, 105.8 (2C), 115.5, 121.8, 129.4, 130.6, 133.5, 140.4, 145.0, 152.2
(2C), 157.0, 161.3, 161.6, 164.7, 166.0. IR (CHCl3): 1217, 1243, 1550, 1668, 2210, 2857, 2933,
3429 cm-1. HRMS (ESI) m/z calculated for [C27H27N5O5S2+H]: 566.1517, found: 566.1526.
The following compounds were prepared by using procedure described for compound 15:
6-Oxo-2-((3-(4-oxo-6-propylthieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (11)
1
Yield: 73%, White solid, Melting point: 185
⁰
C. H NMR (200 MHz, CDCl3):
δ
0.91 (t, J = 7
Hz, 3H), 1.40-1.70 (m, 2H), 2.19 (s, 2H), 2.70 (t, J = 6 Hz, 2H), 3.23 (s, 2H), 3.76 (s, 6H), 3.85
(s, 3H), 4.11 (s, 2H), 7.04 (s, 1H), 7.15 (s, 2H), 8.01 (s, 1H). 13C NMR (50 MHz, CDCl3):
δ
12.7,
23.4, 26.9, 28.1, 31.6, 44.6, 55.4 (2C), 59.9, 90.5, 105.4 (2C), 116.9, 117.3, 123.6, 130.2, 139.6,
143.4, 145.3, 150.2, 152.0 (2C), 156.4, 161.6, 166.1, 167.6. IR (CHCl3): 1215, 1683, 2210, 3450
cm-1. HRMS (ESI) m/z calculated for [C26H27N5O5S2+H]: 554.1526, found: 554.1526;
[C26H27N5O5S2 +Na]: 576.1344, found: 576.1346.
6-Oxo-2-((3-(4-oxo-6-pentylthieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (12)
1
Yield: 78%, White solid, Melting point: 183
⁰
C. H NMR (200 MHz, CDCl3):
δ
0.85 (t, J = 7
Hz, 3H), 1.15-1.40 (m, 4H), 1.60 (t, J = 7 Hz, 2H), 2.18 (s, 2H), 2.72 (t, J = 7 Hz, 2H), 3.22 (s,
13
2H), 3.77 (s, 6H), 3.86 (s, 3H), 4.11 (s, 2H), 7.05 (s, 1H), 7.14 (s, 2H), 8.00 (s, 1H). C NMR
(125 MHz, CDCl3):
δ 13.9, 22.3, 28.0, 28.7, 30.5, 30.7, 31.1, 45.5, 56.1 (2C), 60.8, 90.3, 106.2
(2C), 117.9, 118.3, 118.4, 124.4, 130.3, 140.8, 145.3, 145.7, 152.7 (2C), 157.4, 162.5, 167.0,
167.1. IR (CHCl3): 1257, 1651, 2215, 2932, 3415 cm-1. HRMS (ESI) m/z calculated for
[C28H31N5O5S2+H]: 582.1838, found: 582.1839.
2-((3-(6-Hexyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-6-oxo-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (13)
1
Yield: 81%, White solid, Melting point: 293
⁰
C. H NMR (200 MHz, CDCl3):
δ
0.88 (t, J = 7
Hz, 3H), 1.20-1.45 (m, 6H), 1.57-1.78 (m, 2H), 2.35 (t, J = 7 Hz, 2H), 2.82 (t, J = 7 Hz, 2H),
3.42 (t, J = 7 Hz, 2H), 3.90 (s, 6H), 3.94 (s, 3H), 4.20 (t, J = 7 Hz, 2H), 7.11 (s, 1H), 7.34 (s,
13
2H), 8.03 (s, 1H). C NMR (100 MHz, CDCl3):
δ 14.0, 22.5, 28.3, 28.4, 28.6, 30.6, 31.0, 31.4,
45.4, 56.3 (2C), 61.0, 92.5, 106.7 (2C), 115.5, 115.6, 118.0, 124.6, 129.4, 141.8, 145.4, 145.6,
145.9, 153.0 (2C), 157.4, 162.3, 167.5. IR (CHCl3): 1216, 1258, 1652, 2218, 3418 cm-1. HRMS
(ESI) m/z calculated for [C29H33N5O5S2+H]: 596.1995, found: 596.1996; [C29H33N5O5S2+Na]:
618.1813, found: 618.1815.

ACCEPTED MANUSCRIPT
2-((3-(6-Heptyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-6-oxo-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (14)
Yield: 83%, White solid, Melting point: 187 ⁰ δ 0.83 (t, J = 6
C. 1H NMR (400 MHz, DMSO-d6):
Hz, 3H), 1.16-1.33 (m, 8H), 1.59 (t, J = 7 Hz, 2H), 2.17 (t, J = 7 Hz, 2H), 2.79 (t, J = 7 Hz, 2H),
3.27 (t, J = 7 Hz, 2H), 3.76 (s, 3H), 3.83 (s, 6H), 4.10 (t, J = 7 Hz, 2H), 7.06 (s, 1H), 7.29 (s,
2H), 8.36 (s, 1H). 13C NMR (200 MHz, DMSO-d6):
δ 14.1, 22.2, 27.8, 28.47, 28.50, 28.7, 29.9,
30.8, 31.4, 44.8, 56.2 (2C), 60.4, 92.2, 106.6 (2C), 116.8, 118.4, 124.0, 130.5, 140.6, 143.7,
147.7, 152.7 (2C), 156.7, 162.1, 162.2, 166.3, 166.5. IR (CHCl3): 1255, 1655, 2214, 3418 cm-1.
HRMS (ESI) m/z calculated for [C30H35N5O5S2+H]: 610.2151, found: 610.2152; [C30H35N5O5S2
+Na]: 632.1968, found: 632.1972.
2-((3-(6-Hexyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)ethyl)thio)-6-oxo-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (16)
1
Yield: 80%, White solid, Melting point: 125
⁰
C. H NMR (200 MHz, CDCl3):
δ
0.86 (t, J = 7
Hz, 3H), 1.13-1.40 (m, 6H), 1.41-1.70 (m, 2H), 2.70 (t, J = 7 Hz, 2H), 3.64 (s, 2H), 3.87 (s, 6H),
3.93 (s, 3H), 4.39 (s, 2H), 6.89 (s, 1H), 7.16 (s, 2H), 8.15 (s, 1H). 13C NMR (125 MHz, CDCl3):
δ
14.0, 22.6, 29.0, 29.2, 29.5, 30.4, 30.9, 31.8, 56.1 (2C), 60.9, 106.4 (2C), 116.1, 117.8, 124.3,
129.0, 141.41, 141.42, 145.05, 145.08, 146.02, 146.03, 152.7 (2C), 157.0, 162.1, 167.6. IR
(CHCl3): 1223, 1672, 2213, 3403 cm-1. HRMS (ESI) m/z calculated for [C28H31N5O5S2+H]:
582.1839, found: 582.1839.
2-((2-(6-Heptyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)ethyl)thio)-6-oxo-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (17)
1
Yield: 77%, White solid, Melting point: 130
⁰
C. H NMR (200 MHz, CDCl3):
δ
0.84 (s, 3H),
1.22 (s, 8H), 1.53 (s, 2H), 2.65 (t, J = 6 Hz, 2H), 3.41 (s, 2H), 3.81 (s, 6H), 3.89 (s, 3H), 4.25 (s,
2H), 6.95 (s, 1H), 7.08 (s, 2H), 8.03 (s, 1H). 13C NMR (50 MHz, CDCl3):
δ
14.0, 22.5, 28.6,
28.9, 29.0, 30.5, 31.1, 31.7, 47.4, 56.0 (2C), 60.8, 88.5, 105.8 (2C), 117.7, 121.1, 124.1, 131.1,
139.9, 145.5, 152.7 (2C), 157.4, 162.8, 167.1, 167.2, 172.4, 174.7. IR (CHCl3): 1227, 1666,
2219, 3412 cm-1. HRMS (ESI) m/z calculated for [C29H33N5O5S2+H]: 596.1996, found:
596.1985.
Synthesis
of
1-methyl-6-oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-3(4H)-yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-
carbonitrile (18)
Potassium carbonate (243 mg, 1.76 mmol, 2.0 eq) was taken in a 100 mL two-necked RB flask
and heated under vacuum to remove the traces of moisture and flushed with nitrogen. 6-Oxo-2-
((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-4-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (15) (500 mg, 0.88 mmol, 1.0 eq) was
added under nitrogen followed by dry DMF (1.5 mL) and stirred for 10 min. Iodomethane (0.08
ml, 188 mg, 1.32 mmol, 1.5 eq) was added by microlitre syringe and the reaction mixture was
stirred at RT for 12 h. It was then diluted with water (10 mL) and extracted with ethyl acetate (3
x 10 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and
concentrated under reduced pressure. The residue was purified by column chromatography to
afford 1-methyl-6-oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (18) as off-
white solid, 435 mg (85%). Melting point: 185 ⁰C.

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3):
2.95 (t, J = 6 Hz, 2H), 3.42 (t, J = 7 Hz, 2H), 3.55 (s, 3H), 3.90 (s, 6H), 3.92 (s, 3H), 4.13 (t, J =
δ 1.75-1.91 (m, 4H), 2.22-2.41 (m, 2H), 2.76 (t, J = 6 Hz, 2H),
13
7 Hz, 2H), 7.33 (s, 2H), 7.88 (s, 1H). C NMR (100 MHz, CDCl3):
δ 22.1, 22.7, 25.1, 25.5,
28.3, 29.8, 30.9, 44.9, 56.3 (2C), 60.9, 92.1, 106.5 (2C), 115.7, 122.6, 129.5, 131.4, 134.7, 141.5,
145.0, 153.0 (2C), 157.8, 160.2, 161.9, 164.5, 165.3. IR (CHCl3): 1218, 1669, 2219, 2358, 3411
cm-1. HRMS (ESI) m/z calculated for [C28H29N5O5S2+H]: 580.1674, found: 580.1683;
[C28H29N5O5S2 +Na]: 602.1490, found: 602.1502.
The following compounds were prepared by using procedure described for compound 18
wherein the required halide was used in place of iodomethane:
6-Oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-1-pentyl-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile
(19)
Yield: 74%, White solid, Melting point: 86 ⁰ δ 0.93 (t, J = 7 Hz,
C. 1H NMR (200 MHz, CDCl3):
3H), 1.30-1.52 (m, 4H), 1.70-1.96 (m, 6H), 2.20-2.42 (m, 2H), 2.67-2.85 (m, 2H), 2.91-3.06 (m,
2H), 3.26 (t, J = 7 Hz, 2H), 3.93 (s, 9H), 4.13 (t, J = 7 Hz, 2H), 4.46 (t, J = 7 Hz, 2H ), 7.33 (s,
13
2H), 7.88 (s, 1H). C NMR (50 MHz, CDCl3):
δ 13.9, 22.1, 22.3, 22.8, 25.2, 25.6, 27.9, 28.1
(2C), 28.5, 45.3, 56.3 (2C), 60.9, 68.8, 87.6, 106.4 (2C), 114.9, 122.7, 129.8, 131.4, 134.4, 141.2,
145.2, 153.1 (2C), 157.7, 162.0, 167.8, 169.8, 174.0. IR (CHCl3): 1217, 1247, 1468, 1536, 1669,
2219, 2358, 2932 3411 cm-1. HRMS (ESI) m/z calculated for [C32H37N5O5S2+H]: 636.2297,
found: 580.1683; [C32H37N5O5S2 +Na]: 658.2114, found: 658.2128.
1-Octyl-6-oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (20)
Yield: 79%, White solid, Melting point: 78 ⁰ δ 0.89 (t, J = 7 Hz,
C. 1H NMR (200 MHz, CDCl3):
3H), 1.21-1.52 (m, 12H), 1.75-1.95 (m, 6H), 2.25-2.38 (m, 2H), 2.79 (t, J = 7 Hz, 2H), 3.00 (t, J
= 7 Hz, 2H), 3.27 (t, J = 7 Hz, 2H), 3.94 (s, 9H), 4.14 (t, J = 7 Hz, 2H), 4.47 (t, J = 7 Hz, 2H),
13
7.34 (s, 2H), 7.89 (s, 1H). C NMR (100 MHz, CDCl3):
δ 14.1, 22.2, 22.6, 22.8, 25.2, 25.6,
25.8, 28.2, 28.5 (2C), 29.16, 29.23, 31.8, 45.3, 56.3 (2C), 61.0, 68.9, 87.7, 106.3 (2C), 115.0,
122.7, 129.9, 131.5, 134.6, 141.1, 145.2, 153.1 (2C), 157.8, 162.0, 167.9, 169.9, 174.0. IR
(CHCl3): 1197, 1230, 1450, 1530, 1677, 2209, 2911, 3409 cm -1. HRMS (ESI) m/z calculated for
[C35H43N5O5S2+H]: 678.2765, found: 678.2778.
6-Oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-1-undecyl-1,6-dihydropyrimidine-5-carbonitrile
(21)
Yield: 78%, White solid, Melting point: 74 ⁰ δ 0.87 (t, J = 7 Hz,
C. 1H NMR (200 MHz, CDCl3):
3H), 1.15-1.55 (m, 16H), 1.70-1.94 (m, 6H), 2.19-2.40 (m, 2H), 2.68-2.85 (m, 2H), 2.89-3.06
(m, 2H), 3.26 (t, J = 7 Hz, 2H), 3.93 (s, 9H), 4.13 (t, J = 7 Hz, 2H), 4.45 (t, J = 7 Hz, 2H ), 7.33
(s, 2H), 7.88 (s, 1H). 13C NMR (50 MHz, CDCl3):
δ 14.0, 22.1, 22.6, 22.8, 25.1, 25.6, 25.8, 28.2,
28.45, 28.53, 29.2 (2C), 29.4, 29.5 (2C), 31.8, 45.3, 56.3 (2C), 60.9, 68.8, 87.6, 106.4 (2C),
114.9, 122.6, 129.8, 131.4, 134.4, 141.2, 145.2, 153.1 (2C), 157.7, 162.0, 167.8, 169.8, 173.9.
IR (CHCl3):1247, 1280, 1422, 1542, 1638, 2139, 2891, 3401 cm-1. HRMS (ESI) m/z calculated
for [C38H49N5O5S2+H]: 720.3248, found: 720.3242; [C38H49N5O5S2 +Na]:742.3067, found:
742.3060.

ACCEPTED MANUSCRIPT
6-Oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-1-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-4-(3,4,5-trimethoxyphenyl)-
1,6-dihydropyrimidine-5-carbonitrile (22)
Yield: 64%, White solid, Melting point: 140
1
⁰
C. H NMR (200 MHz, CDCl3):
δ
1.73-1.96 (m,
4H), 2.21-2.42 (m, 2H), 1.54-1.87 (m, 4H), 2.89-3.07 (m, 2H), 3.28 (t, J = 7 Hz, 2H), 3.94 (s,
13
9H), 4.13 (t, J = 7 Hz, 2H), 4.82 (t, J = 7 Hz, 2H), 7.35 (s, 2H), 7.88 (s, 1H). C NMR (125
MHz, CDCl3):
δ 22.2, 22.8, 25.2, 25.6, 28.3, 28.6, 29.7, 30.5 (t), 45.3, 56.3 (2C), 61.0, 87.6,
106.5 (2C), 108.0-119.7 (m, 6C), 114.4, 122.7, 129.5, 131.5, 134.6, 141.5, 145.2, 153.2 (2C),
157.8, 162.0, 168.1, 169.1, 174.1. IR (CHCl3): 760, 1250, 1301, 1432, 1492, 1608, 2149, 2931,
3411 cm-1. HRMS (ESI) m/z calculated for [C35H30N5O5F13S2+H]: 912.1553, found: 912.1554;
[C35H30N5O5F13S2+Na]: 934.1371, found: 934.1373.
6-Oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-1-(prop-2-yn-1-yl)-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-
carbonitrile (23)
Yield: 71%, White solid, Melting point: 171
1
⁰
C. H NMR (500 MHz, CDCl3):
δ
1.79-1.91 (m,
4H), 2.28-2.37 (m, 2H), 2.55 (t, J = 2 Hz, 1H), 2.78 (t, J = 6 Hz, 2H), 2.99 (t, J = 6 Hz, 2H),
3.28 (t, J = 7 Hz, 2H), 3.93 (s, 9H), 4.14 (t, J = 7 Hz, 2H), 5.12 (d, J = 2 Hz, 2H), 7.34 (s, 2H),
13
7.90 (s, 1H). C NMR (125 MHz, CDCl3):
δ 22.2, 22.8, 25.2, 25.6, 28.3, 28.7, 45.4, 55.6, 56.3
(2C), 61.0, 76.2, 76.9, 87.6, 106.5 (2C), 114.5, 122.7, 129.5, 131.5, 134.5, 141.4, 145.2, 153.2
(2C), 157.8, 162.0, 168.2, 168.8, 174.2. IR (CHCl3): 1220, 1655, 2219, 2230 2355, 3410 cm-1.
HRMS (ESI) m/z calculated for [C30H29N5O5S2+H]: 604.1675, found: 604.1683;
[C30H29N5O5S2+Na]: 626.1491, found: 626.1502.
1-Benzyl-6-oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (24)
1
Yield: 64%, White solid, Melting point: 110
⁰
C. H NMR (200 MHz, CDCl3):
δ
1.65-2.00 (m,
4H), 2.13-2.44 (m, 2H), 2.59-2.84 (m, 2H), 2.88-3.11 (m, 2H), 3.37 (t, J = 7 Hz, 2H), 3.90 (s,
6H), 3.93 (s, 3H), 4.04 (t, J = 6 Hz, 2H), 5.30 (s, 2H), 7.28-7.56 (m, 7H), 7.75 (s, 1H). 13C NMR
(50 MHz, CDCl3):
δ 22.1, 22.7, 25.2, 25.6, 28.1, 30.0, 45.0, 48.0, 56.4 (2C), 61.0, 92.7, 106.6
(2C), 115.6, 122.6, 128.1 (2C), 128.4, 128.8 (2C), 129.4, 131.4, 133.6, 134.7, 141.7, 145.0, 153.0
(2C), 157.7, 160.5, 162.0, 164.5, 165.0. IR (CHCl3): 1217, 1241, 1455, 1527, 1538, 1624, 1680,
2213, 2901, 3410 cm-1. HRMS (ESI) m/z calculated for [C34H33N5O5S2+H]: 656.1981, found:
656.1972.
6-Oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-1-((perfluorophenyl)methyl)-4-(3,4,5-trimethoxyphenyl)-1,6-
dihydropyrimidine-5-carbonitrile (25)
Yield: 76%, White solid, Melting point: 138
1
⁰
C. H NMR (200 MHz, CDCl3):
δ
1.73-2.00 (m,
4H), 2.21-2.45 (m, 2H), 2.75 (t, J = 5 Hz, 2H), 2.94 (t, J = 5 Hz, 2H), 3.31 (t, J = 7 Hz, 2H), 3.93
13
(s, 9H), 4.14 (t, J = 6 Hz, 2H), 5.60 (s, 2H), 7.32 (s, 2H), 7.90 (s, 1H). C NMR (125 MHz,
CDCl3):
δ 22.1, 22.7, 25.1, 25.5, 28.3, 28.8, 45.4, 56.3 (2C), 56.8, 60.9, 87.4, 106.5 (2C), 108.4
(dt), 114.3, 122.6, 129.4, 131.4, 134.5, 136.5-146.8 (m, 5C), 141.5, 145.2, 153.1 (2C), 157.7,
162.0, 168.2, 168.8, 174.1. IR (CHCl3):1233, 1321, 1442, 1502, 1628, 2159, 2922, 3401 cm-1.
HRMS (ESI) m/z calculated for [C34H28N5O5F5S2+Na]: 768.1334, found: 768.1344.

ACCEPTED MANUSCRIPT
6-Oxo-2-((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-
yl)propyl)thio)-1-tosyl-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile
(26)
1
Yield: 58%, White solid, Melting point: 163
⁰
C. H NMR (200 MHz, CDCl3):
δ
1.69-2.01 (m,
4H), 2.23-2.44 (m, 2H), 2.51 (s, 3H), 2.78 (t, J = 6 Hz, 2H), 3.02 (t, J = 6 Hz, 2H), 3.26 (t, J = 7
Hz, 2H), 3.93 (s, 9H), 4.10-4.28 (m, 2H), 7.32 (s, 2H), 7.47 (d, J = 8 Hz, 2H), 8.04 (d, J = 8 Hz,
13
2H), 8.18 (s, 1H). C NMR (50 MHz, CDCl3):
δ 21.8, 22.1, 22.8, 25.1, 25.6, 28.6, 29.0, 45.3,
56.3 (2C), 60.9, 88.7, 106.6 (2C), 113.3, 122.7, 128.7 (2C), 128.9, 130.1 (2C), 131.5, 132.5,
134.2, 141.9, 145.6, 146.8, 153.2 (2C), 157.7, 161.8, 165.1, 169.2, 175.1. IR (CHCl3): 1145,
1239, 1284, 1362, 1373, 1396, 1492, 1557, 1668, 2225, 2936 cm-1. HRMS (ESI) m/z calculated
for [C34H33N5O7S3+H]: 720.1609, found: 720.1615; [C34H33N5O7S3+Na]: 742.1427, found:
742.1434.
6-Oxo-1-(3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)propyl)-2-
((3-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)propyl)thio)-4-
(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile (27)
Yield: 60% (from compound 15 by reaction with bromide 8), White solid, Melting point: 129
1H NMR (200 MHz, CDCl3):
1.75-1.93 (m, 8H), 2.19-2.45 (m, 4H), 2.77 (s, 4H), 2.97 (s, 4H),
3.24 (t, J = 7 Hz, 2H), 3.94 (s, 9H), 4.12 (t, J = 7 Hz, 2H), 4.23 (t, J = 7 Hz, 2H), 4.56 (t, J = 7
⁰C.
δ
13
Hz, 2H), 7.34 (s, 2H), 7.91 (s, 1H), 8.00 (s, 1H). C NMR (50 MHz, CDCl3):
δ 22.1 (2C), 22.7
(2C), 25.1 (2C), 25.5 (2C), 27.9, 28.2, 28.6, 43.6, 45.3, 56.3 (2C), 60.9, 65.1, 87.3, 106.4 (2C),
114.7, 122.6, 122.7, 129.5, 131.4, 134.3, 134.4, 141.4, 141.5, 145.2, 145.5, 153.1 (2C), 157.7,
157.8, 162.0, 162.1, 167.8, 169.4, 174.2. IR (CHCl3): 1127, 1500, 1556, 1667, 2212, 2937 cm-1.
HRMS (ESI) m/z calculated for [C40H41N7O6S3+H]: 812.2347, found: 812.2353;
[C40H41N7O6S3+Na]: 834.2166, found: 834.2173.
4.2 Biological Screening
Antitubercular activity
The compounds synthesized in the present work were tested for their in vitro and ex vivo effects
against dormant and active stages MTB using XRMA protocol [17, 18]. inhibitory activity
against dormant (12 days incubation) and active (8 days incubation) H37Ra mycobacteria using
XRMA protocol as described by Singh et al. [21]. The absorbance of XRMA was measured at
470 nm. Ex vivo activity against dormant and active stages of MTB was estimated through nitrate
reductase (NR) assay reading absorbance at 540 nm as per the protocol described by Khan and
Sarkar [23]. MTB (ATCC No. 25177) were grown to logarithmic phase (O. D. 1.0) in a M. pheli
medium. The stock culture was maintained at -70°C and sub-cultured once in M. pheli medium
before inoculation into the experimental culture. All experiments were performed in triplicates
and IC50 and IC90 values were calculated from their dose–response curves [19].
Cytotoxicity assay
The cytotoxicity of the compounds was determined using MTT assay against three different
human cancer cell lines and THP-1 monocytes in duplicate [19, 20, 2124,25]. Leukaemia THP-1,
lung A549 adenocarcinoma, pancreatic PANC-1 adenocarcinoma and HeLa cervical carcinoma
cell lines were obtained from the European Collection of Cell Cultures (ECCC), Salisbury, UK.

ACCEPTED MANUSCRIPT
ο
Cell lines were maintained under standard cell culture conditions at 37 C and 5% CO2 in a
humidified environment.
Docking studies
Molecular docking studies were carried out to predict the probable mode of action of
antitubercular activity of the synthesized hybrids of thienopyrimidinones and thiouracil
derivatives with mycobacterium tuberculosis pantothenate synthetase (PDB id: 3IVX).
To perform docking studies Glide 7.1[3035] was used. All chemical structures were
drawn using 2D-sketcher incorporated within Maestro 10.6 [3136]. LigPrep 3.8 [3237] was used
to prepare 3D structures with corrected chiralities and each individual structure is refined with
the optimized energy and best possible conformations. Protein obtained was initially purified by
adding H-atoms wherever necessary to the amino acid residues and bond order was assigned to
find out the correct ionization and tautomeric states of them. All the water molecules present
within the active site were removed and missing side chains were added using Prime 4.4 [3338].
For further purification protein was refined by checking the protonation state of histidines,
terminal acids and of polar hydrogens. The existing steric clashes present within the protein were
relaxed using OPLS-2005 force field and was terminated once root mean square deviation
reached 0.30 Å [34, 3539,40].
Grid was prepared by selecting the co-crystallized ligand FG6 and the length of enclosing
cubic box was set to 20A° to cover the maximum area of active site around the co-crystallized
ligand.
Acknowledgements
The authors thank CSIR, New Delhi, India, for financial support as part of 12th Five Year Plan
Program under titles ACT (CSC-0301) and ORIGIN (CSC-0108). Authors also wish to thank
Mr. Shridhar Thorat for X-ray crystallography.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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ACCEPTED MANUSCRIPT
Highlights
* New hybrids of thienopyrimidinones and thiouracils synthesized.
* Some of the compounds exhibited significant antitubercular activity against MTB H37Ra.
* The docking study done to predict the probable mode of action.
* Inhibition of mycobacterial pantothenate synthetase predicted.