C-N bond formation under Cu-catalysis: Synthesis and in vitro evaluation of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones against chorismate mutase

Article abstract of DOI:10.1016/j.bmc.2012.07.011

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive to the presence of air or atmospheric moisture. A range of compounds were prepared by using this method and single crystal X-ray diffraction study was performed using a representative compound. In vitro pharmacological data of some of the compounds synthesized along with dose response studies using active molecules are presented. In silico interactions of these molecules with chorismate mutase are also presented.

Full text of DOI:10.1016/j.bmc.2012.07.011

Bioorganic & Medicinal Chemistry 20 (2012) 5127–5138
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C–N bond formation under Cu-catalysis: Synthesis and in vitro
evaluation of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones
against chorismate mutase
Raju Adepu a, K. Shiva Kumar a, Sandhya Sandra a, D. Rambabu a, G. Rama Krishna b, C. Malla Reddy b,
Ajit Kandale a, Parimal Misra a, Manojit Pal a,
⇑
a Institute of Life Sciences, University of Hyderabad Campus, Hyderabad 500046, India
b Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, West Bengal 741252, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as
potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using
Cu-mediated C–N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic
acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive
to the presence of air or atmospheric moisture. A range of compounds were prepared by using this
method and single crystal X-ray diffraction study was performed using a representative compound. In
vitro pharmacological data of some of the compounds synthesized along with dose response studies
using active molecules are presented. In silico interactions of these molecules with chorismate mutase
are also presented.
Received 9 June 2012
Revised 5 July 2012
Accepted 6 July 2012
Available online 16 July 2012
Keywords:
Thienopyrimidinone
Copper
Coupling
Chorismate mutase
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
people a year worldwide and chorismate mutase being considered
as a promising target for the development of anti tubercular agents
Pyrimidine nucleus fused with another heterocycle has been
found to be an integral part of many natural products, agro chemi-
cals and veterinary products.1,2 This class of compounds has also
found wide applications in the design and discovery of novel bioac-
tive molecules and drugs.3 Recently, thienopyrimidine I (Fig. 1) that
belongs to this class has attracted considerable interest because of
the generation of a library of small molecules based on the frame-
work C was undertaken.
Preparation of N-aryl substituted thieno[2,3-d]pyrimidin-
4(3H)-one derivatives usually involves (i) the construction of the
six membered pyrimidone ring by using 2-amino-N-aryl substi-
tuted thiophene-3-carboxamide via a multi-step process14a or (ii)
condensation reaction of aryl amine with substituted thieno[2,3-
d][1,3]oxazin-4-one.14b,c However, all these methods require te-
dious steps along with harsh reaction conditions and afforded
the desired product in low yield. 14 Moreover, we required a more
flexible, robust and high yielding method for the preparation of
our target molecules based on C. Copper promoted C–N bond
4
their various pharmacological properties such as antimicrobial,
antiviral, 5 anti inflammatory, 6 antidiabetic7 and anticancer activi-
ties. 8 Among thieno[2,3-d]pyrimidine derivatives, 3-amino-5,6-di-
methyl-2-[4-(1-phenyl-methyl)-1-piperazinyl]thieno[2,3-d]pyr-
imidin-4(3H)-one9 II and 4-(4-methyl-1-piperazinyl)-2-methyl
thio-6,7-dihydro-5H-cyclopenta [4,5]thieno[2,3-d]pyrimidine10 III
(Fig. 1) exhibited remarkable affinity and selectivity for the 5-HT3
receptor. Due to our continuing interest in the identification of no-
vel anti-tubercular agents, 11 we became interested in exploring the
N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-one C framework
for the design of small molecules12 as potential inhibitors of Myco-
bacterium tuberculosis H37Rv chorismate mutase (CM).13 Indeed,
the framework C was derived from the general structure of inhibi-
tors represented by A (reported by us earlier11d) via B (Fig. 2). In
view of the fact that tuberculosis (TB) kills more than two million
N
O
O
NH2
N
N
N
NH
S
N
N
S
N
S
N
SCH3
N
I
II
III
⇑
Corresponding author. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
Figure 1. Thieno[2,3-d]pyrimidine (I) and its biologically active derivatives (II and
III).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.011

5128
R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
O
Ar
O
O
N
Ar
Ar
N
N
N
N
Aryl/
Hetaryl
Aryl/
Hetaryl
N
N
S
A
C
B
Figure 2. Design of novel and potential inhibitors (C) of chorismate mutase from A.
Table 1
Effect of reaction conditions on Cu-mediated coupling of 1a with 2aa
formation between the –NH– moiety of a heteroaryl derivative and
organoboronic acids via cross coupling reactions have emerged as a
powerful synthetic tool for the preparation of N-(hetero)aryl
substituted derivatives.15 Recently we have reported11d efficient
N-arylation of 1,2,3-triazin-4-ones successfully under mild reac-
tion conditions in the presence of a copper catalyst and a base at
ambient temperature. Additionally, the reactions were carried
out without using any inert atmosphere as the methodology was
found to be not sensitive to air oxygen. We adopted a similar strat-
egy to generate the compound library based on thieno[2,3-d]pyr-
imidin-4(3H)-one framework C where Cu-mediated N-arylation
was used as a key synthetic step. Thus, aryl boronic acids (2) were
coupled with various substituted thieno[2,3-d]pyrimidin-4(3H)-
ones (1) in the presence of a Cu-salt to give the corresponding N-
arylated products (3) (Scheme 1). Herein we report the preliminary
results of this study and in vitro pharmacological evaluation of a
series of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-one (3)
as potential inhibitors of CM. Notably, only few heteroaryl based
inhibitors of CM has been reported so far11 and to the best of our
knowledge synthesis and CM inhibiting properties of this class of
compounds has not been reported earlier.
O
HO
OH
Cusalt
O
B
solvent
NH
N
Et3N
rt, air
S
N
S
N
1a
2a
3a
Entry
Cu salts
Solvent
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2
Cu(OAc)2.H2O
CuI
DCM
DCE
DMF
THF
DCE
DCE
DCE
DCE
1.0
1.0
3.0
3.0
5.0
6.0
6.0
3.0
82
90
65
55
50
35
30
75
CuBr
Cu(OTf)2
a
All the reactions were carried out using 1a (0.485 mmol), 2a (0.728 mmol), a
Cu-salt (0.485 mmol) and Et3N (0.970 mmol) in a solvent (5 mL) at room temper-
ature without using inert or anhydrous atmosphere (DCM = dichloromethane;
DCE = 1,2-dichloroethane).
b
Isolated yield.
2. Results and discussion
2.1. Chemistry
however was found to be not sensitive to the atmospheric mois-
ture. The methodology therefore does not require the use of an in-
ert or anhydrous atmosphere. Overall, the combination of
anhydrous Cu(OAc)2 and Et3N in DCE was found to be optimum
for the N-arylation of compound 1a.
Initially, the coupling of 4,5,6,7-tetrahydrobenzo[b]thieno[2,3-
d]pyrimidin-4(3H)-one (1a) with phenyl boronic acid (2a,
1.5 equiv) was chosen as a model reaction to establish the opti-
mized reaction conditions. Thus the reaction was performed in
the presence of various copper catalysts and solvents using Et3N
as a base at room temperature under anhydrous conditions (Ta-
ble 1). All the reactions were performed in the presence of air.
The desired product 3a was isolated in 82% yield when the reaction
was carried out using anhydrous Cu(OAc)2 in dichloromethane
(DCM) (Table 1, entry 1). The reaction was completed within 1 h.
A further improvement of yield was observed when 1,2-dichloro-
ethane (DCE) was used in place of DCM (Table 1, entry 2). The
use of DMF and THF not only decreased the product yield but in-
creased the reaction time from 1 to 3 h (Table 1, entry 3 and 4).
The use of other catalysts e.g. Cu(OAc)2ꢀH2O, CuI, CuBr and
Cu(OTf)2 was also examined (entries 5–8, Table 1) but found to
be inferior in terms of product yield. It is worthy to mention that
while significant decrease in product yield was observed when hy-
drated Cu(OAc)2 was used in place of anhydrous one the reaction
We then used the optimized conditions for the coupling of a
range of thieno[2,3-d]pyrimidin-4(3H)-ones (1) with a variety of
aryl boronic acids (2) and the results are summarized in Table 2.
It is evident from Table 2 that the reaction proceeded well irrespec-
tive of the nature of thieno[2,3-d]pyrimidin-4(3H)-ones (1a–d)
employed. Thus 4,5,6,7-tetrahydrobenzothieno[2,3-d] pyrimidin-
4(3H)-one16a (1a), tert-butyl-4-oxo-3,4,5,6,7,8-hexahydropyri-
do[40,30:4,5]thieno-[2,3-d]pyrimidine-7-carboxylate16b (1b), 6,7,8,
9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4(3H)-one16c
(1c) and 5-methylthieno[2,3-d]pyrimidin-4(3H)-one16a (1d) were
employed successfully to afford the desired products 3a-o in good
yields (Table 2). The substituent like F, Cl, OCH3, CH3 and CHO group
present in aryl boronic acids were well tolerated and the use of
naphthalen-2-yl boronic acid (Table 2, entries 6 and 10) was found
to be effective. It is worthy to mention that no desired product was
isolated when pyridin-2-yl boronic acid was reacted with 1a under
the optimized reaction conditions. Additionally, alkyl boronic acid
such as cyclopropyl boronic acid was also found to be ineffective
in the present C–N bond forming reaction. All the compounds syn-
thesized were well characterized by spectral (NMR, MS and IR) data.
The molecular structure of a representative compound 3m was
established unambiguously by single crystal X-ray diffraction study
(Fig. 3).17 The molecule 3m crystallized in the monoclinic space
group P21/c with one molecule in the asymmetric unit (Z = 4)
(Fig. 3). The molecule in the asymmetric unit does not possess
any conventional functional groups to form the strong hydrogen
bonding. Interestingly, the molecule in the asymmetric unit
form tetramer via C–H. . .O (C16–H16. . .O1: 2.58 Å, 134°:
O
Ar
O
Cu(OAc)2
Et3N
HO
OH
B
N
NH
+
Ar
ClCH2CH2Cl
rt, air
N
S
N
S
3
2
1
Scheme 1. Cu-mediated coupling of substituted thieno[2,3-d]pyrimidin-4(3H)-one
(1) with aryl boronic acids (2).

R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
5129
Table 2
Synthesis of N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-one derivatives (3) via Cu(OAc)2 mediated C–N bond forming reaction between 1 and 2 (Scheme 1)a
Entry
Thieno[2,3-d]pyrimidin-4(3H)-one (1)
ArB(OH)2 (2)
Products (3)
Time (h)
Yieldb (%)
O
HO
OH
B
NH
O
N
N
1
1.0
90
S
2a
1a
N
S
3a
HO
OH
F
B
O
2
1a
1.0
88
N
F
2b
N
S
3b
HO
HO
HO
OH
B
OMe
O
S
N
3
4
5
1a
1a
1a
1.2
1.0
1.5
88
85
80
OMe
2c
N
3c
OH
Cl
B
B
O
S
N
N
Cl
2d
3d
OH
CHO
O
N
N
CHO
2e
S
3e
OH
B
OH
O
N
N
6
1a
1.2
82
2f
S
3f
O
NH
O
N
N
N
7
2a
1.0
90
S
Boc
N
N
S
1b
Boc
3g
F
O
N
8
1b
2b
1.0
88
N
N
S
Boc
3h
(continued on next page)

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R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
Table 2 (continued)
Entry
Thieno[2,3-d]pyrimidin-4(3H)-one (1)
ArB(OH)2 (2)
Products (3)
Time (h)
Yieldb (%)
CHO
O
N
9
1b
2e
1.5
80
N
N
S
Boc
3i
O
N
10
1b
2f
1.2
82
N
N
S
Boc
3j
HO
OH
CH3
B
O
N
11
1b
1.5
85
CH3
N
2g
N
S
Boc
3k
O
NH
O
N
N
12
2a
1.0
88
S
N
1c
S
3l
F
O
13
1c
2b
1.0
85
N
N
S
3m
O
F
NH
N
O
S
1d
14
2b
1.0
86
N
N
S
3n
O
F
NH
N
O
S
1e
15
2b
1.0
82
N
N
S
3o
a
All the reactions were carried out using 1 (0.485 mmol), 2 (0.728 mmol), Cu(OAc)2 (0.485 mmol) and Et3N (0.970 mmol) in DCE (5 mL) at room temperature without using
inert or anhydrous atmosphere.
b
Isolated yield.

R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
5131
Figure 3. ORTEP representation of the compound 3m (Thermal ellipsoids are drawn at 50% probability level).
Figure 4. Showing the tetramer formation via C–H. . .O interactions (compound 3m).
Figure 5. Showing the packing arrangement along the ac axis (compound 3m).

5132
R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
O
O
OH
O
N
O
N
I
TFA, DCM
N
N
O
N
N
rt, 1h
85%
S
N
EDC.HCl, HOBt
DIPEA, DCM
0 ºC-rt
N
S
HN
Boc
S
I
5
74%
3g
4
Scheme 2. Structural elaboration of compound 3g.
C17–H17. . .O1: 2.42 Å, 156°: C3–H3B. . .O1: 2.38 Å, 132°) synthon
as shown in the Figure 4 which is although a very weak interaction.
These interactions propagated in a 2D zipper fashion and made the
packing as much as strong in both the directions on the ac plane as
shown in Figure 5.
as an attractive target for the identification of novel antibacterial
agents. Furthermore, the low sequence homology amongst known
CM provides a new opportunity for developing unique inhibitors
targeted to specific microorganisms. The pharmacological assay20
involved determination of activity of enzyme CM which catalyzes
the conversion of chorismate to prephenate. Thus determination
of activity of CM is based on the direct observation of conversion
In order to demonstrate the further scope of the present meth-
odology, further structure elaboration of compound 3g was carried
out using a two-step method (Scheme 2). Thus, the Boc group of
compound 3g was removed in the presence of trifluoroacetic acid
to give the secondary amine 4 in good yield (Scheme 2). The amine
obtained was then reacted with 2-iodobenzoic acid in the presence
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) hydrochlo-
ride (EDCꢀHCl), N-hydroxybenzotriazole (HOBt) and N,N-diisopro-
pylethylamine (DIPA) in dichloromethane to give the
corresponding amide 5. The iodo group of amide 5 is amenable
for further functionalization via transition metal catalyzed C–C or
of chorismic acid to prephenate spectrophotometrically at OD274
.
This reaction was performed in the presence of test compounds
to determine their CM inhibiting properties. A known inhibitor of
CM that is 4-(3,5-dimethoxyphenethylamino)-3-nitro-5 sulfamoyl
benzoic acid12a was prepared and used as a reference compound,
the IC50 value of which was found to be less than 10
lM. The com-
pounds were tested at 50 M and the results are summarized in
l
Table 3. It is evident from Table 3 that a number of compounds
showed significant inhibition (>30%) of CM. In general, a p-substi-
tuent on the N-aryl ring of 3 was unfavorable in terms of activity
(3a vs 3b, Table 3) whereas a m-substituent enhanced the activity
(3a vs 3c–e, Table 3). A N-naphthyl group though appeared as bet-
ter than p-substituent (3f vs 3b, Table 3) was found to be inferior to
a m-substituent. A trend similar to that of 3a versus 3e was ob-
served in case of compound 3g or 4 versus 3i. Notably, a six mem-
bered saturated carbocyclic/N-heterocyclic ring was fused with the
thiophene moiety in all these cases for example 3a–g and 3i and 4.
Replacing the 6-membered ring by a 7-membered one (3l–m) or its
removal (e.g. 3o) was found to be detrimental. Nevertheless, based
on the preliminary data generated compounds 3c and 3i were cho-
sen for further evaluation in vitro. In a dose–response study, com-
pounds 3c and 3i showed inhibition of CM in a dose dependent
18
C–N bond forming reactions.
A
plausible mechanism can be proposed for the present
Cu-mediated coupling reaction between thieno[2,3-d]pyrimidin-
4(3H)-ones (1) and aryl boronic acids (2) (Scheme 3).19 Thus trans-
metallation of aryl boronic acid (2) with Cu(OAc)2 provides the
Cu(II) complex E-1. Subsequent displacement of OAc moiety of
E-1 by the reactant 1 through its amide NH provides the Cu(II)
complex E-2 which undergoes reductive elimination to give the
19
product 3. In the presence of air oxygen, as reported earlier,
it
is possible that the Cu(II) species E-2 might undergo change of
its oxidation state (prior to reductive elimination) leading to the
corresponding Cu(III) species which then afford the product 3 via
reductive elimination.
manner with an IC50 value of 19.80 1.02 and 23.88 0.90 lM
2.2. Pharmacology
respectively (Fig. 6). Thus, N-aryl thieno[2,3-d]pyrimidin-4(3H)-
one has been identified as a new scaffold for the development of
novel inhibitors of chorismate mutase.
To understand the nature of interactions of these molecules
with CM protein docking studies were carried out using the com-
pounds 3c and 3i. The results showed (i) an H-bonding interaction
Having prepared a range of N-aryl substituted thieno[2,3-d]pyr-
imidin-4(3H)-ones (3) via Cu(OAc)2 mediated C–N bond forming
reaction between 1 and 2 some of the compounds synthesized
were tested for CM inhibiting properties in vitro. The CM
(EC5.4.99.5) an enzyme in the shikimate pathway is responsible
for the production of tyrosine and phenylalanine via the Claisen
rearrangement of chorismate to prephenate. Due to the absence
of this pathway in animals but not in bacteria CM is considered
Table 3
Inhibition of chorismate mutase by N-aryl pyrimidones (3) in vitro
N-Aryl thieno[2,3-d]pyrimidin-4(3H)-ones (3)
% Inhibitiona @ 50
lM
3a
3b
3c
3d
3e
3f
3g
3i
35.6
12.8
47.0
42.5
45.3
33.0
37.2
49.9
13.0
14.6
17.7
36.2
12.6
CuII(OAc)2
Et3N
NEt3
O
NEt3
OAc
Et3N
Ar
Et3N
Ar
1
II
II
Cu
Cu
2
N
HOAc
AcO-B(OH)2
E-2
(transmetallation)
E-1
N
S
3l
3m
3o
4
3
(reductive
elimination)
5
a
Data presented are average of three experiments.
Scheme 3. Proposed mechanism for the Cu-mediated coupling of 1 with 2.

R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
5133
Figure 6. Dose dependent inhibition of CM by compounds 3c and 3i.
between the pyrimidine nitrogen of 3c and arginine 49 residue of
CM and (ii) a -cation interaction between the benzene ring of
3c and arginine 134 residue (Fig. 7). In the case of compound 3i
an H-bonding interaction was observed between the carbonyl oxy-
gen of the ester moiety and the lysine 60 residue of CM (Fig. 8).
normalization method and the condition specified in each case:
column, mobile phase (range used), flow rate, detection wave-
length, and retention times.
p
4.1.2. Typical procedure for the synthesis of N-aryl substituted
thieno[2,3-d]pyrimidin-4(3H)-one derivatives (3a–o)
A mixture of the substituted thieno[2,3-d]pyrimidin-4(3H)-one
1 (0.485 mmol), aryl boronic acid 2 (0.728 mmol), anhydrous
Cu(OAc)2 (0.485 mmol) and Et3N (0.970 mmol) in 1,2-dichloroeth-
ane (5 mL) was stirred at ambient temperature for 1.0–2.0 h. Pro-
gress of the reaction was monitored by TLC. After completion of
the reaction, the mixture was filtered through celite. The filtrate
was concentrated under vacuum, and the residue was purified by
column chromatography on silica gel using hexane/ethyl acetate
to afford the desired product.
3. Conclusions
In conclusion, synthesis of novel N-aryl substituted thieno[2,3-
d]pyrimidin-4(3H)-ones was carried out for the first time by using
Cu-mediated C–N bond forming reaction between thieno[2,3-
d]pyrimidin-4(3H)-ones and aryl boronic acids. The methodology
does not require the use of inert or anhydrous atmosphere and
can be performed in an open flask. A range of compounds were
prepared in good to excellent yields by using this direct N-aryla-
tion process and the molecular structure of a representative com-
pound was established unambiguously by single crystal X-ray
diffraction. Among all the compounds synthesized two showed
promising inhibitory properties when tested against chorismate
mutase in vitro. In silico interactions of these molecules with CM
are presented. Overall, Cu-mediated direct access to N-aryl substi-
tuted thieno[2,3-d]pyrimidin-4(3H)-ones has resulted in identifi-
cation of novel small molecules as inhibitors of chorismate
mutase for the potential treatment of tuberculosis.
4.1.3. 3-Phenyl-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-d]
pyrimidin-4(3H)-one (3a)
Yield: 90% (0.12 g); Light brown solid; mp: 187–189 °C; 1H NMR
(400 MHz, CDCl3) d 7.97 (s, 1H), 7.54–7.45 (m, 3H), 7.38 (d,
J = 7.2 Hz, 2H), 3.01 (t, J = 5.8 Hz, 2H), 2.80 (t, J = 5.8 Hz, 2H),
1.90–1.82 (m, 4H); 13C NMR (100 MHz, CDCl3) d 161.7, 157.4,
145.1, 137.1, 134.5, 132.0, 129.4, 129.0 (2C), 127.1 (2C), 122.8,
25.5, 25.2, 22.8, 22.2; HPLC: 97.8%, column: Zorax XDB C-18
150 ꢁ 4.6 mm 5
l, mobile phase A: 0.1% Formic Acid in water, mo-
bile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/98, 14/98, 16/50,
18/50; flow rate: 1.0 mL/min; UV 220 nm, retention time 7.47 min;
IR (KBr) vmax 2924, 2866, 1682, 1562 cmꢂ1; m/z (CI) 282.9 (M+1).
4. Experimental section
4.1. Chemistry
4.1.4. 3-(4-Fluorophenyl)-5,6,7,8-tetrahydrobenzo[b]thieno
[2,3-d]pyrimidin-4(3H)-one (3b)
4.1.1. General methods
Unless stated otherwise, reactions were performed under nitro-
gen atmosphere using oven dried glassware. Reactions were mon-
itored by thin layer chromatography (TLC) on silica gel plates (60
F254), visualizing with ultraviolet light or iodine spray. Flash chro-
matography was performed on silica gel (230–400 mesh) using
distilled hexane, ethyl acetate, dichloromethane. 1H NMR and 13C
NMR spectra were determined in CDCl3 or DMSO-d6 solution by
using 400 or 100 MHz spectrometers, respectively. Proton chemi-
cal shifts (d) are relative to tetramethylsilane (TMS, d = 0.00) as
internal standard and expressed in ppm. Spin multiplicities are gi-
ven as s (singlet), d (doublet), dd (doublet of doublet), t (triplet)
and m (multiplet) as well as b (broad). Coupling constants (J) are
given in hertz. Infrared spectra were recorded on a FT-IR spectrom-
eter. Melting points were determined using melting point appara-
tus and are uncorrected. MS spectra were obtained on a mass
spectrometer. Chromatographic purity by HPLC (Agilent 1200
series Chem Station software) was determined by using area
Yield: 88% (0.13 g); White solid; mp: 171–173 °C; 1H NMR
(400 MHz, CDCl3) d 7.71 (s, 1H), 7.14–7.12 (m, 2H), 7.00–6.96 (m,
2H), 2.78 (br s, 2H), 2.58 (br s, 2H), 1.65–1.62 (m, 4H); 13C NMR
(100 MHz, CDCl3) d 167.6, 162.1 (C-F J = 248.1 Hz), 157.4, 144.8,
134.7, 133.0 (C-F J = 3.0 Hz), 130.8, 128.9 (C-F J = 8.3 Hz, 2C),
122.7, 116.4 (C-F J = 22.9 Hz, 2C), 25.5, 25.2, 22.8, 22.1; HPLC:
99.4%, column: Zorax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase
A: 0.1% Formic Acid in water mobile phase B: CH3CN (gradient)
T/%B: 0/50, 2/50, 9/98, 14/98, 16/50, 18/50; flow rate: 1.0 mL/
min; UV 220 nm, retention time 7.56 min; IR (KBr) vmax 2948,
2889, 1692, 1560, 1508 cmꢂ1; m/z (CI) 300.9 (M+1).
4.1.5. 3-(3-Methoxyphenyl)-5,6,7,8-tetrahydrobenzo[b]thieno
[2,3-d]pyrimidin-4(3H)-one (3c)
Yield: 88% (0.13 g); White solid; mp: 169–171 °C; 1H NMR
(400 MHz, CDCl3) d 7.96 (s, 1H), 7.71–7.69 (m, 2H), 7.54–7.51 (m,
2H), 3.83 (s, 3H), 3.03 (t, J = 5.6 Hz, 2H), 2.80 (t, J = 5.4 Hz, 2H),

5134
R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
Figure 7. Docking of compound 3c at the active site of CM.
1.90–1.82 (m, 4H); 13C NMR (100 MHz, CDCl3) d 161.7, 160.3,
157.4, 145.0, 138.1, 134.5, 132.3, 130.8, 128.7, 122.8, 115.0,
112.9, 55.4, 25.5, 25.2, 22.8, 22.2; HPLC: 98.1%, column: Zorax
water mobile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/98,
14/98, 16/50, 18/50; flow rate: 1.0 mL/min; UV 220 nm, retention
time 7.75 min; IR (KBr) vmax 2936, 2862, 1679, 1600, 1560 cmꢂ1
m/z (CI) 312.9 (M+1).
;
XDB C-18 150 ꢁ 4.6 mm 5l, mobile phase A: 0.1% Formic Acid in

R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
5135
Figure 8. Docking of compound 3i at the active site of CM.
4.1.6. 3-(3-Chlorophenyl)-5,6,7,8-
4.1.7. 3-(4-Oxo-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-
d]pyrimidin-3(4H)-yl)benzaldehyde (3e)
tetrahydrobenzo[b]thieno[2,3-d]pyrimidin-4(3H)-one (3d)
Yield: 85% (0.13 g); White solid; mp: 207–209 °C; 1H NMR
(400 MHz, CDCl3) d 7.94 (s, 1H), 7.47–7.46 (m, 2H), 7.43 (s, 1H),
7.30–7.27 (m, 1H), 3.01 (t, J = 5.8 Hz, 2H), 2.81 (t, J = 5.6 Hz, 2H),
1.91–1.82 (m, 4H); 13C NMR (100 MHz, CDCl3) d 161.7, 157.1,
144.5, 138.1, 134.9, 132.0, 130.4, 129.3, 128.7, 127.6, 125.4,
122.8, 25.5, 25.2, 22.8, 22.1; HPLC: 93.9%, column: Zorax XDB C-
Yield: 80% (0.12 g); White solid; mp: 204–206 °C; 1H NMR
(400 MHz, CDCl3) d 10.01 (s, 1H), 8.01–7.98 (m, 2H), 7.92 (s, 1H),
7.74–7.67 (m, 2H), 3.01 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H),
1.93–1.81 (m, 4H); 13C NMR (100 MHz, CDCl3) d 190.6, 161.7,
157.2, 144.4, 138.1, 137.6, 135.2, 133.0 (2C), 132.1, 130.3, 130.2,
128.0, 25.6, 25.3, 22.8, 22.2; HPLC: 99.3%, column: X Bridge C-18
18 150 ꢁ 4.6 mm 5
l
, mobile phase A: 0.1% Formic Acid in water
150 ꢁ 4.6 mm 5
l, mobile phase A: 0.1% Formic Acid in water mo-
mobile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/98, 14/98,
16/50, 18/50; flow rate: 1.0 mL/min; UV 220 nm, retention time
8.84 min; IR (KBr) vmax 2936, 2868, 1678, 1558 cmꢂ1; m/z (CI)
316.8 (M+1).
bile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/98, 14/98, 16/50,
18/50; flow rate: 1.0 mL/min; UV 245 nm, retention time 5.84 min;
IR (KBr) vmax 3049, 2932, 2845, 1705, 1669, 1556 cmꢂ1; m/z (CI)
310.8 (M+1).

5136
R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
4.1.8. 3-(Naphthalen-2-yl)-5,6,7,8-tetrahydrobenzo[b]thieno
[2,3-d]pyrimidin-4(3H)-one (3f)
154.6, 145.7, 134.5, 133.3, 133.1, 129.5 (2C), 128.1 (2C), 127.8
(2C), 127.3, 127.1, 125.8, 124.6, 80.4, 43.0, 42.9, 28.4 (3C), 25.8;
Yield: 82% (0.13 g); White solid; mp: 204–206 °C; 1H NMR
(400 MHz, CDCl3) d 8.07 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.92–7.87
(m, 2H), 7.83 (d, J = 1.6 Hz, 1H), 7.58–7.56 (m, 2H), 7.52–7.50 (m,
1H), 3.03 (t, J = 5.4 Hz, 2H), 2.82 (t, J = 5.6 Hz, 2H), 1.91–1.83 (m,
4H); 13C NMR (100 MHz, CDCl3) d 161.8, 157.7, 145.2, 134.8,
134.6, 133.2, 133.0, 132.0, 129.3, 128.1, 127.8, 127.2, 126.9,
125.8, 124.8, 122.9, 25.6, 25.2, 22.8, 22.2; HPLC: 97.0%, column:
HPLC: 97.3%, column: X Bridge C-18 150 ꢁ 4.6 mm 5
l, mobile
phase A: 5 mM NH4OAc in water mobile phase B: CH3CN (gradient)
T/%B: 0/50, 2/50, 10/95, 15/95, 18/50, 20/50; flow rate: 1.0 mL/
min; UV 220 nm, retention time 8.89 min; IR (KBr) vmax 2981,
2895, 1680, 1563 cmꢂ1; m/z (CI) 434.2 (M+1).
4.1.13. tert-Butyl-3-p-tolyl-4-oxo-3,4,5,6,7,8-hexahydropyrido
[40,30:4,5]thieno-[2,3-d]pyrimidine-7-carboxylate (3k)
Zorax XDB C-18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.1% Formic
Acid in water mobile phase B: CH3CN (gradient) T/%B: 0/50, 2/50,
9/98, 14/98, 16/50, 18/50; flow rate: 1.0 mL/min; UV 220 nm,
retention time 9.49 min; IR (KBr) vmax 3053, 2927, 2864, 1677,
1560 cmꢂ1; m/z (CI) 333.0 (M+1).
Yield: 85% (0.16 g); White solid; mp: 87–89 °C; 1H NMR
(400 MHz, CDCl3) d: 7.93 (s, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.17 (d,
J = 8.0 Hz, 2H), 4.58 (s, 2H), 3.63 (t, J = 5.2 Hz, 2H), 3.03 (br s, 2H),
2.35 (s, 3H), 1.42 (s, 9H); 13C NMR (100 MHz, CDCl3) d 162.5,
157.4, 154.6, 145.7, 139.4 (2C), 134.3, 130.1 (2C), 129.8, 126.8
(2C), 115.1, 80.3, 43.0, 42.9, 28.4 (3C), 25.8, 21.2; HPLC: 94.8%, col-
4.1.9. tert-Butyl-3-phenyl-4-Oxo-3,4,5,6,7,8-hexahydropyrido
[4’,3’:4,5]thieno-[2,3-d]pyrimidine-7-carboxylate (3g)
umn: X Bridge C-18 150 ꢁ 4.6 mm 5
l, mobile phase A: 5 mM
Yield: 90% (0.17 g); White solid; mp: 98–100 °C; 1H NMR
(400 MHz, CDCl3) d 8.00 (s, 1H), 7.56–7.48 (m, 3H), 7.38 (d,
J = 6.8 Hz, 2H), 4.66 (s, 2H), 3.71 (t, J = 5.4 Hz, 2H), 3.11 (br s, 2H),
1.49 (s, 9H); 13C NMR (100 MHz, CDCl3) d 162.6, 157.4, 145.8,
136.8 (2C), 129.6 (3C), 129.3 (2C), 127.1 (2C), 122.5, 80.4, 42.9,
NH4OAc in water mobile phase B: CH3CN (gradient) T/%B: 0/50,
2/50, 10/95, 15/95, 18/50, 20/50; flow rate: 1.0 mL/min; UV
220 nm, retention time 8.06 min; IR (KBr) vmax 2973, 2927, 1689,
1560 cmꢂ1; m/z (CI) 397.8 (M+1).
42.3, 28.4 (3C), 25.8; HPLC: 95.6%, column:
X
Bridge C-18
4.1.14. 3-Phenyl-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno
[2,3-d]pyrimidin-4(3H)-one (3l)
150 ꢁ 4.6 mm 5
l
, mobile phase A: 5 mM NH4OAc in water mobile
phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/95, 13/95, 15/50, 18/
50; flow rate: 1.0 mL/min; UV 218 nm, retention time 6.78 min; IR
(KBr) vmax 2974, 2928, 1689, 1558 cmꢂ1; m/z (CI) 384.2 (M+1).
Yield: 88% (0.13 g); White solid; mp: 91–93 °C; 1H NMR
(400 MHz, CDCl3) d 7.97 (s, 1H), 7.55–7.49 (m, 3H), 7.39–7.37 (m,
2H), 3.36–3.34 (m, 2H), 2.89–2.87 (m, 2H), 1.93–1.88 (m, 2H),
1.76–1.64 (m, 4H); 13C NMR (100 MHz, CDCl3) d 160.1, 157.9,
144.8, 138.9, 137.7, 137.3, 129.5 (2C), 129.1, 127.3 (3C), 32.6,
4.1.10. tert-Butyl-3-(4-fluorophenyl)-4-oxo-3,4,5,6,7,8-hexahy
dropyrido[4’,3’:4,5]thieno-[2,3-d]pyrimidine-7-carboxylate (3h)
Yield: 88% (0.17 g); White solid; mp: 129–131 °C; 1H NMR
(400 MHz, CDCl3) d 8.01 (s, 1H), 7.38–7.35 (m, 2H), 7.26–7.20 (m,
2H), 4.66 (s, 2H), 3.71 (t, J = 4.2 Hz, 2H), 3.10 (br s, 2H), 1.49 (s,
9H); 13C NMR (100 MHz, CDCl3) d 163.9 (C-F J = 248.6 Hz), 162.6,
157.3, 154.3, 145.3, 136.2, 132.8 (C–F J = 3.1 Hz, 2C), 130.8, 129.1
(C–F J = 8.8 Hz, 2C), 116.7 (C–F J = 23.0 Hz, 2C), 80.4, 42.9, 41.4,
30.0, 27.9, 27.7, 27.2; HPLC: 95.5%, column:
X Bridge C-18
150 ꢁ 4.6 mm 5
l
, mobile phase A: 0.1% Formic Acid in water mo-
bile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/98, 14/98, 16/50,
18/50; flow rate: 1.0 mL/min; UV 215 nm, retention time 7.93 min;
IR (KBr) vmax 3057, 2920, 2846, 1678, 1563 cmꢂ1; m/z (CI) 296.9
(M+1).
28.4 (3C), 25.8; HPLC: 96.7%, column:
X
Bridge C-18
4.1.15. 3-(4-Fluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta
[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (3m)
150 ꢁ 4.6 mm 5
l
, mobile phase A: 5 mM NH4OAc in water mobile
phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 10/95, 15/95, 18/50,
20/50; flow rate: 1.0 mL/min; UV 220 nm, retention time
7.06 min; IR (KBr) vmax 2974, 2931, 1690, 1566 cmꢂ1; m/z (CI)
402.2 (M+1).
Yield: 85% (0.13 g); White solid; mp: 144–146 °C; 1H NMR
(400 MHz, CDCl3) d 7.96 (s, 1H), 7.38–7.34 (m, 2H), 7.21 (t,
J = 8.4 Hz, 2H), 3.35–3.32 (m, 2H), 2.89–2.87 (m, 2H), 1.93–1.88
(m, 2H), 1.75–1.64 (m, 4H); 13C NMR (100 MHz, CDCl3) d 163.9
(C-F J = 248.2 Hz), 160.0, 157.9, 144.6, 144.5, 139.2, 137.7, 133.2
(C-F J = 3.3 Hz), 129.2 (C-F J = 8.8 Hz, 2C), 116.6 (C-F J = 22.9 Hz,
2C), 32.5, 29.9, 27.9, 27.7, 27.1; HPLC: 99.2%, column: X Bridge C-
4.1.11. tert-Butyl-3-(3-formylphenyl)-4-oxo-3,4,5,6,7,8-hexahy
dropyrido[40,30:4,5]thieno-[2,3-d]pyrimidine-7-carboxylate (3i)
Yield: 80% (0.16 g); White solid; mp: 94–96 °C; 1H NMR
(400 MHz, CDCl3) d 10.08 (s, 1H), 8.03–8.01 (m, 2H), 7.92 (s, 1H),
7.75–7.67 (m, 2H), 4.67 (s, 2H), 3.72 (t, J = 4.2 Hz, 2H), 3.11 (br s,
2H), 1.49 (s, 9H); 13C NMR (100 MHz, CDCl3) d 190.6, 162.5,
157.1, 154.6, 145.0, 137.8, 137.6 (2C), 132.9 (2C), 130.4 (2C),
130.3, 127.8, 80.4, 43.1, 42.9, 28.4 (3C), 25.8; HPLC: 95.0%, column:
18 150 ꢁ 4.6 mm 5
l, mobile phase A: 0.1% Formic Acid in water
mobile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/98, 14/98,
16/50, 18/50; flow rate: 1.0 mL/min; UV 220 nm, retention time
7.97 min;IR (KBr) vmax 3072, 2924, 2852, 1686, 1566 cmꢂ1; m/z
(CI) 314.8 (M+1).
X Bridge C-18 150 ꢁ 4.6 mm 5
l
, mobile phase A: 5 mM NH4OAc in
4.1.16. 3-(4-Fluorophenyl)-6,7-dihydro-5H-cyclopenta[4,5]
thieno[2,3-d]pyrimidin-4(3H)-one (3n)
water mobile phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 10/95,
15/95, 18/50, 20/50; flow rate: 1.0 mL/min; UV 220 nm, retention
Yield: 86% (0.12 g); White solid; mp: 179–181 °C; 1H NMR
(400 MHz, CDCl3) d 7.93 (s, 1H), 7.40–7.36 (m, 2H), 7.21 (t,
J = 8.4 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 2.98 (t, J = 7.2 Hz, 2H),
2.51–2.44 (m, 2H); 13C NMR (100 MHz, CDCl3) d 167.7, 162.5 (C-F
J = 248.0 Hz), 157.3, 144.6, 140.7, 133.0 (C-F J = 3.3 Hz) 132.4,
128.9 (C-F J = 8.8 Hz, 2C), 120.4, 116.4 (C-F J = 22.9 Hz, 2C), 30.3,
28.9, 23.7; HPLC: 99.7%, column: Zorax XDB C-18 150 ꢁ 4.6 mm
time 5.87 min; IR (KBr) vmax 2974, 2929, 1690, 1599, 1556 cmꢂ1
m/z (CI) 412.2 (M+1).
;
4.1.12. tert-Butyl-3-(naphthalen-2-yl)-4-oxo-3,4,5,6,7,8-hexahy
dropyrido[40,30:4,5]thieno-[2,3-d]pyrimidine-7-carboxylate (3j)
Yield: 82% (0.17 g); White solid; mp: 174–176 °C; 1H NMR
(400 MHz, CDCl3) d: 8.15 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.93–
7.88 (m, 2H), 7.13 (d, J = 1.2 Hz, 1H), 7.60–7.55 (m, 2H), 7.49 (dd,
J = 8.2, 1.8 Hz, 1H), 4.68 (s, 2H), 3.72 (t, J = 7.4 Hz, 2H), 3.13 (br s,
2H), 1.49 (s, 9H); 13C NMR (100 MHz, CDCl3) d: 162.6, 157.5,
5l, mobile phase A: 0.1% Formic Acid in water mobile phase B:
CH3CN (gradient) T/%B: 0/50, 2/50, 9/98, 14/98, 16/50, 18/50; flow
rate: 1.0 mL/min; UV 220 nm, retention time 6.23 min; IR (KBr)
vmax 2909, 2853, 1698, 1563, 1510 cmꢂ1; m/z (CI) 286.8 (M+1).

R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
5137
4.1.17. 3-(4-Fluorophenyl)-5-methylthieno[2,3-d]pyrimidin-
4(3H)-one (3o)
using a stereo zoom microscope supported by a rotatable polariz-
ing stage. The data was collected at room temperature on Bruker’s
Yield: 82% (0.10 g); White solid; mp: 181–183 °C; 1H NMR
(400 MHz, CDCl3) d 8.03 (s, 1H), 7.40–7.36 (m, 2H), 7.22 (t,
J = 8.2 Hz, 2H), 6.89 (s, 1H), 2.58 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 164.0, 162.5 (C–F J = 248.4 Hz), 158.0, 145.7, 135.3, 132.8 (C–F
J = 3.1 Hz), 129.0 (C-F J = 8.8 Hz, 2C), 122.8, 119.0, 116.5 (C-F
J = 23.0 Hz, 2C), 16.5; HPLC: 99.5%, column: Zorbax XDB C-18
KAPPA APEX II CCD Duo with graphite monochromated Mo-Ka
radiation (0.71073 Å). The crystals were glued to a thin glass fibre
using FOMBLIN immersion oil and mounted on the diffractometer.
The intensity data were processed using Broker’s suite of data pro-
cessing programs (SAINT), and absorption corrections were applied
using SADABS.22a The crystal structure was solved by direct meth-
ods using SHELXS-97 and the data was refined by full matrix least-
squares refinement on F2 with anisotropic displacement parame-
ters for non-H atoms, using SHELXL-97.22b Crystal data of (3m):
Molecular formula = C17H15FN2OS, Formula weight = 314.38, Crys-
tal system = Monoclinic, space group = P21/c, a = 13.849 (6) Å,
b = 4.433 (2) Å, c = 24.677 (10) Å, V = 1514.5 (11) Å3, T = 296(2) K,
150 ꢁ 4.6 mm 5
l, mobile phase A: 0.1% Formic Acid in water mobile
phase B: CH3CN (gradient) T/%B: 0/50, 2/50, 9/95, 14/95, 16/50, 18/
50; flow rate: 0.8 mL/min; UV 220 nm, retention time 5.93 min; IR
(KBr) v
max 2923, 2857, 1692, 1581, 1513 cmꢂ1; m/z (CI) 260.9 (M+1).
4.1.18. 3-Phenyl-3,4,5,6,7,8-hexahydropyrido[40,30:4,5]thieno
[2,3-d]pyrimidin-4(3H)-one (4)
Z = 4, Dc = 1.379 Mg m–3
measured, 3626 independent reflections, 2057 observed reflections
[I > 2.0 (I)], R1_obs = 0.037, Goodness of fit = 1.01.
, l(Mo-Ka
) = 0.23 mmꢂ1, 12508 reflections
To a mixture of compound 3g (200 mg, 0.52 mmol) in dichloro-
methane (5 mL) at 0 °C was added trifluoroacetic acid (TFA) (1 mL)
and then warmed to room temperature. The reaction mixture was
stirred for 2 h, then removed the solvent under vacuum, diluted
the reaction mixture with ethyl acetate (20 mL) and washed with
saturated NaHCO3 solution (2 ꢁ 10 mL) followed by brine solution
(10 mL). The organic layer dried over anhydrous Na2SO4 and con-
centrated under low vacuum to provide the desired compound 4
(125 mg, 85%) as a brown solid; mp: 144–146 °C; 1H NMR
(400 MHz, CDCl3) d 8.01 (s, 1H), 7.56–7.47 (m, 3H), 7.38 (d,
J = 6.8 Hz, 2H), 4.19 (s, 2H), 3.40 (br s, 1H), 3.28 (t, J = 5.8 Hz, 2H),
3.18–3.16 (m, 2H); 13C NMR (100 MHz, CDCl3) d 162.4, 157.4,
145.7, 136.9, 131.8, 130.3, 129.6 (2C), 129.3, 127.1 (2C), 122.6,
r
4.3. Pharmacology12a,21
4.3.1. Chorismate mutase activity assay
Mycobacterium tuberculosis Chorismate Mutase (MtCM) gene
was PCR amplified and cloned into expression vector pET22b.
MtCM was purified from over expressed culture of BL21 (DE3) har-
boring pET22b/MtCM by Ni-NTA affinity chromatography.
Activity of chorismate mutase enzyme is based on the direct
observation of conversion of chorismate to prephenate spectro-
photometrically at OD274. The reaction volume of 100 ll contained
44.1, 42.4, 25.9; HPLC: 92.1%, column:
X
Bridge C-18
50 mM Tris–HCl (pH 7.5), 0.5 mM EDTA, 0.1 mg/mL bovine serum
albumin, and 10 mM b-Mercaptoethanol, and chorismic acid
4 mM. The reaction was started by adding 180 pmol of purified
protein to the pre-warmed chorismic acid solution. Inhibitory
screening of the test compounds against Chorismate Mutase activ-
150 ꢁ 4.6 mm 5
l
, mobile phase A: 0.1% Formic Acid in water mo-
bile phase B: CH3CN (gradient) T/%B: 0/10, 2/10, 9/95, 14/95, 16/10,
18/10; flow rate: 1.0 mL/min; UV 220 nm, retention time 6.06 min;
IR (KBr) vmax 3304, 3060, 2944, 1677, 1555 cmꢂ1; m/z (CI) 283.8
(M+1).
ity was measured at 50
reaction was allowed to proceed at 37 °C and was terminated after
5 min with 100 l of 1 N HCl. A blank with no enzyme for every
lM concentration of the effectors. The
4.1.19. 7-(2-Iodobenzoyl)-3-phenyl-3,4,5,6,7,8-hexahydropyrido
[40,30:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (5)
l
reaction was kept as a control to account for the non enzymatic
conversion of chorismate to prephenate.
The percentage of enzyme inhibition caused by the test com-
pound is calculated by the following formula
A mixture of compound 4 (100 mg, 0.35 mmol), 2-iodo benzoic
acid (104 mg, 0.42 mmol), EDC.HCl (80 mg, 0.42 mmol), HOBT
(56 mg, 0.42 mmol) and DIPEA (0.12 ml, 0.70 mmol) in dry CH2Cl2
(5 ml) was stirred at 0 °C under a nitrogen atmosphere. Then, the
reaction mixture was allowed to stir at room temperature for
14 h. After completion of the reaction, the mixture was concen-
trated under reduced pressure, diluted with ethyl acetate (25 mL)
and washed with water (2 ꢁ 20 mL) followed by brine (20 mL).
The organic layer was collected, dried over anhydrous Na2SO4, fil-
tered and concentrated under low vacuum. The residue was puri-
fied by column chromatography to give the desired product 5
%inhibition ¼ 100 ꢂ residual activity of CM
ꢀ
ꢁ
A274ðS þ E0 þ CÞ ꢂ ðA274S þ CÞ
A274ðS þ EÞ ꢂ A274ðSÞ
Residual activity of CM ¼
ꢁ 100
S = Absorbance of the Substrate (Chorismic Acid) at 274 nm;
E0 = Absorbance of the Enzyme (Chorismate Mutase) at 274 nm with
Compound; E = Absorbance of the Enzyme (Chorismate Mutase) at
274 nm without Compound; C = Test Compound; A274 = Absor-
bance at 274
Dose–response study of the compound 3c and 3i against choris-
mate mutase activity was carried out using the concentration from
1 to 100 lM.
(132 mg, 74%) as
a
white solid; mp: 143–145 °C; 1H NMR
(400 MHz, CDCl3) d 8.03 (s, 1H), 7.88–7.84 (m, 1H), 7.56–7.49 (m,
3H), 7.44–7.37 (m, 3H), 7.23–7.21 (m, 1H), 7.14–7.10 (m, 1H),
5.22 (d, J = 17.2 Hz, 1H), 4.84 (d, J = 17.2 Hz, 1H), 3.54 (d,
J = 6.0 Hz, 2H), 3.31–3.23 (m, 2H) (extra signals due to rotamers);
13C NMR (100 MHz, CDCl3) d 169.8, 162.9, 157.4, 145.9, 142.0,
139.3, 136.8, 131.3, 130.5, 130.1, 129.9, 129.6 (2C), 129.4, 128.5,
127.1 (2C), 122.3, 92.4, 44.0, 41.3, 26.3 (Extra signals due to rota-
4.4. Docking studies
mers); HPLC: 95.4%, column: X Bridge C-18 150 ꢁ 4.6 mm 5
l, mo-
bile phase A: 0.1% Formic Acid in water mobile phase B: CH3CN
(gradient) T/%B: 0/50, 2/50, 9/98, 14/98, 16/50, 18/50; flow rate:
1.0 mL/min; UV 310 nm, retention time 5.43 min; IR (KBr) vmax
3059, 2930, 2851, 1682, 1642, 1555 cmꢂ1; m/z (CI) 513.7 (M+1).
The docking studies were carried out by using schrodinger 2012
software. The PDB ID 2F6L was used for this study. The protein was
prepared by giving preliminary treatment like adding hydrogen,
adding missing residues, refining the loop with prime and finally
minimized by using OPLS 2005 force field. The search grid was gen-
erated by picking the active site residues manually and extended
up to 20 Å. The hydroxyl groups of search area were allowed to
move. The molecules were minimized by using macromodule
4.2. Single crystal X-ray data for compound 3m
Single crystals suitable for X-ray diffraction of 3m were grown
from dimethyl formamide. The crystals were carefully chosen

5138
R. Adepu et al. / Bioorg. Med. Chem. 20 (2012) 5127–5138
Subbaiah, N. V.; Lingappa, Y.; Sandra, S.; Kapavarapu, R.; Misra, P.; Pal, M.
Bioorg. Med. Chem. Lett. 2011, 21, 6433; (c) Kumar, K. S.; Rambabu, D.; Sandra,
S.; Kapavarapu, R.; Krishna, G. R.; Rao, M. V. B.; Chatti, K.; Reddy, C. M.; Misra,
P.; Pal, M. Bioorg. Med. Chem. 2012, 20, 1711; (d) Kumar, K. S.; Adepu, R.;
Sandra, S.; Rambabu, D.; Krishna, G. R.; Reddy, C. M.; Misra, P.; Pal, M. Bioorg.
Med. Chem. Lett. 2012, 22, 1146; (e) Reddy, T. R.; Reddy, L. S.; Reddy, G. R.;
Yarbagi, K.; Lingappa, Y.; Rambabu, D.; Krishna, G. R.; Reddy, C. M.; Kumar, K.
S.; Pal, M. Green Chem. 1870, 2012, 14.
application. We used 1000 iteration for minimization using OPLS
2005 force field and charges were also added from force field only.
The PRCG (Polak-Ribier conjugate gradient) method was used for
minimization. All the molecules were docked by using glide XP
(extra precision) dock application. We performed flexible docking
by allowing sample ring conformations and sample nitrogens to
move to possible extent in docking.
12. For selected references, see (a) Agarwal, H.; Kumar, A.; Bal, N. C.; Siddiqi, M. I.;
Arora, A. Bioorg. Med. Chem. Lett. 2007, 17, 3053; (b) Hediger, M. E. Bioorg. Med.
Chem. 2004, 12, 4995; (c) Bartlett, P. A.; Nakagawa, Y.; Johnson, C. R.; Reich, S.
H.; Luis, A. J. Org. Chem. 1988, 53, 3195.
Acknowledgements
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The authors thank Professor Seyed E. Hasnain and Professor J.
Iqbal for encouragement and DBT, New Delhi, India for financial
support (Grant NO BT/01/COE/07/02). RA thanks CSIR, New Delhi,
India for a Junior Research Fellowship. KS thanks UGC, New Delhi,
India for a Dr. D. S. Kothari Post doctoral fellowship [No.F.4-2/
2006(BSR)/13-324/2010(BSR)].
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17. Crystallographic data (excluding structure factors) for 3m have been deposited
with the Cambridge Crystallographic Data Center as Supplementary
publication number CCDC 875760.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.011.
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