Anti-tubercular activities of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues endowed with high activity toward non-replicative Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2016.09.012

Thirty three derivatives of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine analogues were synthesized by molecular modification of a reported antimycobacterial molecule (GSK163574A). Compounds were evaluated in vitro against actively replicative and nutrient starved non-replicative Mycobacterium tuberculosis (MTB), enzymatic screening and cytotoxicity against RAW 264.7 cell line. Among the compounds, 2-ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (5c) was found to be the most active compound against non-replicative MTB with 2.7 log reduction of bacteria at 10?μg/mL and was more potent than isoniazid (1.2 log reduction) and rifampicin (2.0 log reduction) at same dose level. Compound 5c also showed activity against MTB alanine dehydrogenase enzyme with IC₅₀of 1.82?±?0.42?μM and showed 25% cytotoxicity against RAW 264.7 cell line at 50?μg/mL.

Full text of DOI:10.1016/j.bmc.2016.09.012

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-tubercular activities of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-amine analogues endowed with high activity toward
non-replicative Mycobacterium tuberculosis
Ganesh Samala, Parthiban Brindha Devi, Shalini Saxena, Saritha Gunda, Perumal Yogeeswari,
⇑
Dharmarajan Sriram
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawaharnagar, Hyderabad 500078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty three derivatives of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine
analogues were synthesized by molecular modification of a reported antimycobacterial molecule
(GSK163574A). Compounds were evaluated in vitro against actively replicative and nutrient starved
non-replicative Mycobacterium tuberculosis (MTB), enzymatic screening and cytotoxicity against RAW
264.7 cell line. Among the compounds, 2-ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (5c) was found to be the most active compound against non-replicative MTB with
Received 1 July 2016
Revised 31 August 2016
Accepted 7 September 2016
Available online xxxx
Keywords:
Tuberculosis
Dormant tuberculosis
2.7 log reduction of bacteria at 10
lg/mL and was more potent than isoniazid (1.2 log reduction) and
rifampicin (2.0 log reduction) at same dose level. Compound 5c also showed activity against MTB alanine
dehydrogenase enzyme with IC50 of 1.82 0.42
line at 50 g/mL.
lM and showed 25% cytotoxicity against RAW 264.7 cell
L
-Alanine dehydrogenase
l
Tetrahydrobenzo[4,5]thieno[2,3-d]
Ó 2016 Elsevier Ltd. All rights reserved.
pyrimidin-4-amine analogues
1. Introduction
to antibacterial discovery have kept the output of novel antibacte-
rial drug classes to extraordinarily low levels over the past
25 years, even though discovery programs have been in place at
large and small pharmaceutical companies as well as academic
laboratories over this period.4 Target-based approaches are widely
used in drug discovery; questions have been raised about the effi-
ciency of this approach given the very high attrition rates that
these projects have historically shown in the anti-infective field.5
Compounds identified in whole-cell screens provide suitable
chemical and biological starting points. The Tuberculosis Antimi-
crobial Acquisition and Coordinating Facility (TAACF) was estab-
lished by the National Institute of Allergy and Infectious Diseases
(NIAID) in 1994 to allow researchers access to high quality screen-
ing services in order to encourage anti-TB drug discovery research.
TAACF reported anti-TB high-throughput results of large libraries
of drug-like small molecules.6–8 Similarly GSK reported 177 small
molecules leads against TB,9 one among them was the molecule
GSK163574A (5-methyl-2-(6-methylpyridin-2-yl)-N-(pyrimidin-
2-yl)thieno[2,3-d]pyrimidin-4-amine) that showed activity against
MTBH37Rv with minimum inhibitory concentration (MIC) of
Mycobacterium tuberculosis (MTB), the causative agent of tuber-
culosis (TB), is one of the most lethal infectious agents affecting
mankind.1 In 2014, 9.6 million people fell ill with TB and 1.5 mil-
lion died from the disease. About one-third of the world’s popula-
tion has latent TB, which means people have been infected by TB
bacteria but without any signs and symptoms of the disease.2
MTB infection is difficult to treat, requiring 6–9 months of
chemotherapy with a cocktail of four antibiotics isoniazid, rifampi-
cin, pyrazinamide and ethambutol. In addition to toxic side effects,
the lengthy treatment regime results in poor patient compliance
and thus drug resistant strains are beginning to emerge.3 The
World Health Organization estimates that globally in 2014, an esti-
mated 480,000 people developed multidrug-resistant TB (MDR-
TB).2 This number continues to grow as 300,000 new MDR-TB cases
are diagnosed each year with 79 percent of individuals showing
resistance to three or more frontline drugs.2 Taken together, the
growing problem of MDR-TB and the lack of drugs that effectively
target latent MTB, there seem to be an urgent need for identifica-
tion of new antimicrobial targets and inhibitors. The challenges
0.76 lM (Fig. 1).
We took GSK163574A as the starting point to designed novel
series of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine by modifying A, B, and C rings of lead followed
by synthesis and biological evaluation (Fig. 1).
⇑
Corresponding author. Tel.: +91 4066303506; fax: +91 4066303998.
E-mail address: dsriram@hyderabad.bits-pilani.ac.in (D. Sriram).
http://dx.doi.org/10.1016/j.bmc.2016.09.012
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
C
Structural
N
N
N
N
1. Activity against log-
phase and nutrient
starved culture of M .
tuberculosis H37Rv
2. Enzymatic
R2 Biological
evaluation
modifications
A
at rings A, B and C
NH
N
NH
N
B
N
S
R1
S
screening
R1 = phenyl, cyclopropyl and ethyl
R2 = various substituted aryl groups
3. Cytotoxicity
screening
GSK 163574A
MTB MIC 0.76 µM
Figure 1. Design strategy and proposed plan of work.
moderate activity with MIC ranging from 2.02 to 74.08
lM and
2. Results and discussion
2.1. Chemistry
only five compounds showed MIC of <10 M. None of the com-
l
pounds were more active than lead GSK163574A, isoniazid, rifam-
picin and moxifloxacin. Two compounds 7a and 7e showed better
activity than standard first line anti-TB drug ethambutol. These
A library of thirty three molecules has been synthesised by fol-
lowing four step synthetic protocol as shown in Figure 2. In first
step, commercially available cyclohexanone (1) was converted to
2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (2)
using cyanoacetamide and elemental sulfur following Gewald
reaction. In next step, N-acylation was carried out under basic con-
ditions using benzoyl chloride, propionyl chloride and cyclopropy-
lcarbonyl chloride to get three different N-acylated compounds
(3a–c). In third step, cyclization was achieved under basic, reflux
conditions by treating with aqueous NaOH, to yield tricyclic com-
pound (4a–c). In final step, compounds (4a–c) were treated with
eleven different substituted primary aryl amines under microwave
conditions at high temperature to afford compounds (5a–k, 6a–k
and 7a–k) (Table 1). All the compounds are stable to atmospheric
air, stable on long standing in open atmosphere and they were
stable to buffers whose pH range is ꢀ1 to 12.
results indicate that modification on A,
B and C rings of
GSK163574A reduces the actively replicative MTB. Compounds
4a–c does not show any MTB activity.
In structure–MTB activity, we prepared three kinds of modifica-
tion at 2nd position and among them in general phenyl ring
showed better activity (7a–k; MIC 2.02–64.85
cyclopropyl ring (6a–k; MIC 8.78–71.53 M) and then ethyl group
(5a–k; MIC 18.18–77.29 M). Within 2-phenyl substituted com-
lM) followed by
l
l
pounds (7a–k) we have prepared different 4th substituted com-
pounds, among them simple phenyl (7a) showed MTB MIC of
4.36
groups makes less active with MIC 33.65 and 64.85
tively, whereas introduction of 2,5-dimethyl group (7e) makes
twice potent (MIC 2.02 M) but 2,4 and 2,6-dimethyl makes less
l
M. Extension of phenyl to benzyl (7b) and phenylethyl (7c)
lM respec-
l
potent than phenyl amino derivative.
Next stage we tested these compounds in nutrient starved MTB
model, in this model MTB was grown in PBS for 6 weeks, using
nutrient starvation, Betts et al.,10 established a model in which
MTB arrests growth, decreases its respiration rate and is resistant
to isoniazid, rifampicin and metronidazole. After 6 weeks we trea-
ted nutrient starved non-replicative MTB with of synthesized com-
2.2. Biological evaluation
In the preliminary screening all the thirty three synthesized
compounds were subjected to screening for their biological inhibi-
tion studies in actively replicating MTB using microplate alamar
blue assay (MABA) assay method. Isoniazid, rifampicin, moxi-
floxacin, and ethambutol were used as positive controls and for
comparison. The minimum inhibitory concentration (MIC) was
determined for each compound which was measured as the mini-
mum concentration of compound required to completely inhibit
the bacterial growth (Table 1). The tested compounds showed
pounds at 10 lg/mL for 1 week along with standard compounds
isoniazid, rifampicin and moxifloxacin for comparison. The treated
MTB cells were diluted 10-fold up to 10À6 using Middlebrook 7H9
medium supplemented with OADC and were plated in 48 well
plates in triplicates. The plates were incubated at 37 °C for 4 weeks
and the wells with visible bacterial growth were counted as
H2N
O
HO
O
O
NH2
NH2
N
a
b
R1
c
H
N
N
O
S
S
S
R1
1
2
3
4 (a-c)
R2
HN
d
N
R1
N
S
5 (a-k);
6 (a-k);
7 (a-k):
Reagents and conditions: a: Cyanoacetamide, S8, morpholine, ethanol, rt, 6 h; b: R1COCl, Et3N,
THF, 0 °C to rt, 6 h; c: 1N NaOH, CH3OH/H2O, 90 °C, 12 h; d: R2NH2, PTSA, MW, 135 °C, 45 min.
Figure 2. Synthetic protocol of the designed compounds.
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G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
Biological activities of the synthesized compounds
R
R
R
HN
HN
HN
N
N
N
CH3
N
N
N
S
S
S
5a-k
6a-k
7a-k
Compd
R
Yield (%)
Mp (°C)
MTB MIC in
l
M
MTB ADH IC50 in
lM
Cytotoxicity (RAW 264.7 cells)
at 50 lg/mL % inhibition
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
Phenyl
Benzyl
Phenethyl
79
79
91
69
76
84
87
74
78
83
84
91
85
78
92
87
80
93
91
71
78
80
84
72
70
79
81
64
72
68
88
79
90
170–171
180–181
191–192
141–142
196–197
201–202
182–183
243–244
290–291
279–280
264–265
190–191
198–199
204–205
213–214
222–223
116–117
218–219
209–210
234–235
225–226
236–237
174–175
184–185
190–191
196–197
156–157
150–151
190–191
180–181
210–211
214–215
222–223
20.20
38.64
37.04
74.08
74.08
18.52
77.29
73.65
32.19
18.18
38.18
9.72
18.63
35.77
71.53
35.77
17.88
74.52
17.78
15.61
8.78
15.10 1.80
6.03 0.97
1.82 0.42
7.41 0.87
>25
5.02 0.34
3.26 0.61
4.68 0.70
6.54 1.12
4.94 0.48
6.05 0.68
>25
4.23 0.96
17.03 3.40
9.00 1.60
2.07 0.66
7.55 1.23
13.43 3.41
>25
8.93 1.26
11.12 3.46
8.91 1.66
11.37 4.51
9.68 0.96
5.60 0.75
9.96 1.06
5.82 0.78
8.82 0.67
7.97 0.98
8.72 1.76
8.70 1.52
2.72 0.42
12.18 0.87
>25
7.82
7.05
25.23
9.65
2,4-Dimethylphenyl
2,5-Dimethylphenyl
2,6-Dimethylphenyl
4-Tolyl
4-Methoxyphenyl
4-Bromophenyl
4-Chlorophenyl
4-Flurophenyl
Phenyl
29.89
26.54
15.62
15.18
12.56
15.62
13.98
8.80
23.05
17.45
23.88
16.34
13.51
16.25
34.40
33.39
15.49
4.82
Benzyl
Phenethyl
2,4-Dimethylphenyl
2,5-Dimethylphenyl
2,6-Dimethylphenyl
4-Tolyl
4-Methoxyphenyl
4-Bromophenyl
4-Chlorophenyl
4-Flurophenyl
Phenyl
36.83
4.36
33.65
64.85
16.21
2.02
7.15
Benzyl
Phenethyl
26.52
24.58
7.97
2,4-Dimethylphenyl
2,5-Dimethylphenyl
2,6-Dimethylphenyl
4-Tolyl
4-Methoxyphenyl
4-Bromophenyl
4-Chlorophenyl
4-Flurophenyl
3.23
8.11
25.46
41.14
28.19
20.35
20.80
33.41
33.65
16.13
28.64
63.79
33.29
0.66
0.23
1.26
7.64
7k
Isoniazid
Rifampicin
Moxifloxacin
Ethambutol
12.16*
9.56*
ND
>25
>25
>25
ND
*
At 10 lg/mL; ND indicates not tested.
positive and MPN values were calculated using standard statistical
methods.11 At 10
g/ml concentrations, standard first line anti-TB
As our synthesized compounds showed good activity against
nutrient starved MTB, we decided to screen these molecules
against few of the enzymes like ADH and LAT. In the case of LAT
none of the molecules showed more than 50% inhibition at
l
drugs isoniazid and rifampicin reduced ꢀ1.2 and 2.0 log bacterial
reductions respectively whereas fluoroquinolone antibacterial
moxifloxacin showed high activity with ꢀ2.7 log bacterial reduc-
25 l
M. In MTB ADH screening,12 thirty compounds showed 50%
tion. Minimum inhibitory concentration (MIC) of INH: 0.66
lM,
inhibition at 25
lM and IC50 of synthesized compounds reported
RIF: 0.23 lM and moxifloxacin: 1.26 lM against actively replica-
in Table 1.
tive MTB. Among the thirty three tested compounds, twenty seven
compounds showed activity with more than 1 log reduction of bac-
teria at 10 lg/ml (Fig. 3). Twenty compounds showed more bacte-
Twenty four compounds inhibited ADH with IC50 < 10 lM and
compound 5c (2-ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine) emerged as more potent with
rial log reduction than isoniazid, ten compounds showed activity
better than rifampicin. One compound (5c) was equally active
(2.7 log bacterial reduction) as moxifloxacin. Betts et al., also used
microarray and proteome analysis to investigate the response of
MTB to nutrient starvation. Proteome analysis of 6-week-starved
cultures revealed the induction of several proteins which includes
fumarate reductase flavoprotein subunit, pyruvate dehydrogenase
IC50 of 1.82 0.42 lM which showed 2.7 log reduction in nutrient
starved MTB also. We found very good correlation between ADH
enzyme inhibition studies and bacterial log reduction nutrient
starved MTB model. This enzyme ADH is 6.04 times over expressed
in nutrient starved model. Increased levels of this enzyme has been
linked to the generation of alanine for peptidoglycan biosynthe-
sis13 and the maintenance of the NAD+ pool under conditions when
the terminal electron acceptor oxygen become limiting.14 NAD(H)-
E1 component subunit,
L-alanine dehydrogenase (ADH), polyketide
synthase, transcriptional regulator (ArsR family), sulfate transport
dependent
L-AlaDH catalyze the oxidative deamination of L-alanine
system permease protein and lysine-
enzymes over expressed >5 times.
e-aminotransferase (LAT)
to pyruvate and ammonia (catabolic reaction) or, in the reverse
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G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 3. In vitro anti-TB activity against nutrient starved MTB at 10 lg/mL.
direction, the reductive amination of pyruvate to
thetic reaction).
L
-alanine (biosyn-
with proteins.16 DSF is being increasingly used as a robust method
for initial screening of proteins for interacting small molecules,
either for identifying physiological partners or for hit discovery.
This technique has the advantage that it requires only a PCR
machine suitable for quantitative PCR. In this method, ADH was
incubated with a fluorescent dye (Sypro Orange dye), which alters
its fluorescence upon binding to the hydrophobic regions of the
proteins. The protein–dye sample is then heated, and the fluores-
cence monitored as the heat rises. The unfolding of the protein,
and exposure of hydrophobic parts of the protein, gives rise to a
characteristic pattern in the fluorescence as a function of temper-
ature (Fig. 5). A higher or positive shift of melting temperature
(Tm) of protein–ligand complex compared to the native protein
Tm signify a better stabilization of the protein–ligand complex,
which in turn would reflect on the inhibitor binding. The most
potent compound 5c display positive shifts towards the MTB
ADH protein (1.4 °C). The native protein melting temperature is
46.20 °C; whereas the protein complexed with inhibitor showed
melting temperature of 47.60 °C proves the stabilization of the
We also tried to dock these molecules with crystal structure of
the mycobacterial MTB L-ADH in complex with N6-methyl adeno-
sine (PDB ID: 4LMP)15 having resolution of 1.95 Å and crystal struc-
ture of the protein ADH bound with co-factor NAD+ and substrate
pyruvate (PDB ID: 2VHW)15 using Glide XP docking (Glide, version
5.7, Schrçdinger, LLC, New York, NY, 2011). The inhibitors are not
docked properly on both crystal structures indicates that they
may bind in allosteric site. To prove this concept, here we repre-
sent the active molecules from each representative core (5c, 6e
and 7j). Compounds were showing the docking score in the range
of À3.49 to À4.80 in the ADH protein complex with N6-methyl
adenosine (PDB ID: 4LMP). Binding analysis of these compounds
revealed that all the three active compounds were mainly involved
in hydrophobic interactions with the ADH protein (Fig. 4). All the
compounds were fitted in the hydrophobic pocket within the vicin-
ity of Ile199, Pro247, Leu255, Leu249, Leu197, Ile174, Ala126,
Leu127, Ala179, Leu130, Ala176 and few polar amino acid residues
Gln121, Asp198, Thr178, Ser220 respectively. Though these com-
pounds were able to fit in to active site but failed to interact with
active site residues through hydrogen bonding. This is the reason
these molecules were showing low docking score.
ligand towards the desired protein MTB L-AlaDH.
The safety profile of all the synthesized compounds was evalu-
ated by testing their in vitro cytotoxicity in mouse macrophage cell
line (RAW 264.7) cells at 50 lg/mL concentration using (4,5-
To confirm the interaction between ADH and inhibitors we used
differential scanning fluorimetry (DSF) which is a widely used
method for screening libraries of small molecules for interactions
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.17 Since MTB reside inside the macrophage, the
monocyte macrophage cell lines (RAW 264.7) were mostly used
Figure 4. Binding analysis of active compounds from the series.
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G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
and washed the organic layer with H2O (2 Â 30 mL). The separated
organic layer was dried over anhydrous Na2SO4, evaporated and
purified by column chromatography to get Compound 2 (5.40 g,
90%) as an light yellow solid. ESI-MS found 197 [M+H]+.
4.1.2. General procedure for the preparation of 3a–c
To the stirred solution of compound 2 (1.0 equiv) in CH2Cl2 at
0 °C was added Et3N (2.0 equiv) followed by R1COCl (1.2 equiv)
and allowed to stir at room temperature for 6 h. The reaction mix-
ture was diluted with CH2Cl2 and washed with satd NaHCO3, H2O
and dried over anhyd Na2SO4 and evaporated under vacuo to get
compound 3 as an off-white solid.
4.1.3. General procedure for the preparation of 4a–c
To a solution of compound 3 in MeOH (2.0 vol) was added a
solution of 1 N NaOH (10.0 vol) and the mixture was refluxed for
3 h. then the mixture was poured into water and neutralised with
a concentrated solution of HCl to give a precipitate which was fil-
tered and washed with water and dried to obtain desired
compound.
Figure 5. DSF experiment for compound 5c (protein–ligand complex, green)
showing an increase in the thermal shift of 1.4 °C when compared to the native
ADH protein (red).
4.1.4. General procedure for the synthesis of 5a–k, 6a–k and 7a–k
The mixture of compound 4a (for 5a–k)/4b (for 6a–k)/4c (for
7a–k) (1.0 equiv), substituted primary amine (R2NH2) (1.1 equiv)
and p-toluenesulfonic acid (catalytic) were taken in methanol (3
volumes) and subjected to microwave irradiation (temperature
135 °C, pressure and power automatic) for 45 min. Ice water was
added to the reaction mixture and the obtained solids were fil-
tered, washed with water, cold ethanol and hexanes to get com-
pounds (5a–k, 6a–k and 7a–k) respectively.
for tuberculosis research; in order to check whether the screened
compounds were not toxic towards macrophages but toxic to the
bacteria. All the compounds showed less than 50% inhibition with
good safety profile with low enzyme/MTB inhibitory potential.
3. Conclusion
In the present study we tried to modify reported MTB inhibitor
GSK163574A, but none of the molecules showed promising in
actively replicative MTB. Most of the molecules showed good
potency in nutrient starved non-replicative MTB. Most of the mole-
cules also showed MTB ADH enzyme which was 6.04 times over
expressed in the nutrient starved MTB. We also supported the
enzyme target with DSF studies. This kind of molecules might be
starting point for developing new drugs for dormant TB for which
currently available anti-Tb drugs were in effective.
4.1.5. 2-Ethyl-N-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-amine (5a)
To the mixture of 2-ethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-ol (4a) (0.3 g, 1.28 mmol), aniline (0.13 mL,
1.41 mmol) and PTSA (catalytic) was added methanol (0.9 mL)
and subjected to microwave irradiation (temperature 135 °C, pres-
sure and power automatic) for 45 min. Ice water was added to the
reaction mixture and the obtained solids were filtered, washed
with water, cold ethanol and hexanes to get title compound
(0.31 g, 79%) as an Off-white solid. MS(ESI) m/z 310 [M+H]+. 1H
NMR (400 MHz, DMSO-d6): d 9.24 (s, 1H), 7.92 (d, J = 7.6 Hz, 2H),
7.81–7.74 (m, 3H), 3.11 (q, J = 7.2 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H),
2.83 (t, J = 7.2 Hz, 2H), 1.86 (t, J = 6.8 Hz, 4H), 1.29 (t, J = 7.2 Hz,
3H); 13C NMR (100 MHz, DMSO-d6) d 174.8, 158.4, 146.1, 140.3,
139.8, 133.3(2C), 129.0, 128.4, 124.3(2C), 118.4, 33.4, 25.2, 24.7,
24.1(2C), 12.5. Anal. Calcd for C18H19N3S: C, 69.87; H, 6.19; N,
13.58. Found C, 69.98; H, 6.29; N, 13.66.
4. Experimental section
4.1. Chemistry
All commercially available chemicals and solvents were used
without further purification. TLC experiments were performed on
alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Ger-
many). The homogeneity of the compounds was monitored by thin
layer chromatography (TLC) on silica gel 40 F254 coated on alu-
minum plates, visualized by UV light and KMnO4 treatment. All
1H and 13C NMR spectra were recorded on
a Bruker AM
4.1.6. N-Benzyl-2-ethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidin-4-amine (5b)
(400 MHz, 100 MHz) NMR spectrometer, Bruker BioSpin Corp, Ger-
many. Molecular weights of the synthesized compounds were
checked by LCMS 6100B series Agilent Technology with electro-
spray ionization (ESI+). Chemical shifts are reported in ppm (d)
with reference to the internal standard TMS. The signals are desig-
nated as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; m, multiplet. Elemental analyses were carried out using
CHN Analyzer (Vario MICRO cube in CHN mode).
MS(ESI) m/z 324 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.66
(s, 1H), 7.56–7.48 (m, 5H), 4.41 (s, 2H), 3.13 (q, J = 7.2 Hz, 2H),
3.03 (t, J = 6.8 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 1.88 (t, J = 6.8 Hz,
4H), 1.25 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d
172.9, 160.4, 146.5, 141.2, 138.6, 133.0, 130.3(2C), 128.4, 126.6
(2C), 119.6, 49.3, 33.1, 24.8, 24.3, 23.4(2C), 12.3. Anal. Calcd for
C19H21N3S: C, 70.55; H, 6.54; N, 12.99. Found C, 70.68; H, 6.69;
N, 13.06.
4.1.1. Preparation of 2-amino-4,5,6,7-tetrahydrobenzo[b]
thiophene-3-carboxamide (2)
4.1.7. 2-Ethyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (5c)
To the stirred solution of Compound 1 (3.0 g, 30.56 mmol), 2-
cyanoacetamide (2.56 g, 30.56 mmol), sulfur powder (0.97 g,
30.56 mmol) in ethanol (40 mL) was added morpholine (5.31 mL,
61.11 mmol) and stirred the reaction mixture at room temperature
for 6 h. The reaction mixture was concentrated, diluted with EtOAc
MS(ESI) m/z 338 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.31
(s, 1H), 7.60–7.49 (m, 5H), 3.49 (s, 2H), 3.11–2.96 (m, 4H), 2.89–
2.74 (m, 4H), 1.83 (t, J = 6.8 Hz, 4H), 1.23 (t, J = 6.8 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6) d 170.8, 160.1, 144.9, 139.3, 136.4,
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G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
128.5(2C), 127.3, 125.3(2C), 123.4, 118.2, 48.1, 36.3, 34.6, 25.2,
24.3, 23.8(2C), 13.2. Anal. Calcd for C20H23N3S: C, 71.18; H, 6.87;
N, 12.45. Found C, 71.28; H, 6.99; N, 12.58.
24.6, 13.2. Anal. Calcd for C18H18BrN3S: C, 55.67; H, 4.67; N,
10.82. Found C, 55.74; H, 4.73; N, 10.94.
4.1.14. N-(4-Chlorophenyl)-2-ethyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (5j)
4.1.8. N-(2,4-Dimethylphenyl)-2-ethyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (5d)
MS(ESI) m/z 344 [M+H]+, 346 [MH+2]+. 1H NMR (400 MHz,
MS(ESI) m/z 338 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.34
(s, 1H), 7.63 (s, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 7.2 Hz,
1H), 3.09–2.94 (m, 4H), 2.81 (t, J = 6.8 Hz, 2H), 2.43 (s, 3H), 2.36
(s, 3H), 1.88 (t, J = 6.8 Hz, 4H), 1.24 (t, J = 7.2 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) d 173.6, 157.3, 146.8, 138.3, 137.5, 136.4,
133.3, 129.3, 127.4, 124.3, 119.5, 117.3, 34.2, 24.9, 24.3, 23.8(2C),
22.2, 20.9, 12.6. Anal. Calcd for C20H23N3S: C, 71.18; H, 6.87; N,
12.45. Found C, 71.28; H, 6.99; N, 12.56.
DMSO-d6): d 8.32 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.49 (d,
J = 8.0 Hz, 2H), 3.12 (t, J = 6.8 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H), 2.77
(t, J = 6.8 Hz, 2H), 1.89 (t, J = 7.2 Hz, 4H), 1.32 (t, J = 6.8 Hz, 3H);
13C NMR (100 MHz, DMSO-d6) d 176.4, 155.4, 147.6, 141.3, 139.0,
136.2, 131.2(2C), 127.1(2C), 124.3, 120.9, 34.6, 27.3, 26.0(2C),
24.9, 13.9. Anal. Calcd for C18H18ClN3S: C, 62.87; H, 5.28; N,
12.22. Found C, 62.94; H, 5.33; N, 12.34.
4.1.15. 2-Ethyl-N-(4-fluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (5k)
4.1.9. N-(2,5-Dimethylphenyl)-2-ethyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (5e)
MS(ESI) m/z 328 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.63
(s, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 3.16 (t,
J = 7.2 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H), 1.93
(t, J = 7.2 Hz, 4H), 1.33 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) d 177.3, 156.7, 146.3, 142.0, 138.4, 137.1, 132.6(2C),
128.3(2C), 125.7, 121.4, 35.3, 28.1, 26.6(2C), 24.3, 13.3. Anal. Calcd
for C18H18FN3S: C, 66.03; H, 5.54; N, 12.83. Found C, 66.14; H, 5.63;
N, 12.94.
MS(ESI) m/z 338 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 7.63–
7.54 (m, 2H), 7.24 (s, 1H), 7.18 (d, J = 6.8 Hz, 1H), 3.14 (t, J = 6.8 Hz,
2H), 2.87–2.80 (m, 4H), 2.41 (s, 3H), 2.32 (s, 3H), 1.89 (t, J = 6.8 Hz,
4H), 1.26 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d 172.4,
156.1, 145.9, 137.8, 136.5, 136.2, 133.1, 127.1, 125.3, 123.9, 120.4,
118.6, 34.4, 25.1, 24.6, 24.2(2C), 23.4, 20.7, 13.0. Anal. Calcd for
C20H23N3S: C, 71.18; H, 6.87; N, 12.45. Found C, 71.26; H, 6.94;
N, 12.61.
4.1.16. 2-Cyclopropyl-N-phenyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (6a)
4.1.10. N-(2,6-Dimethylphenyl)-2-ethyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (5f)
MS(ESI) m/z 322 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.64
(s, 1H), 7.82 (d, J = 7.6 Hz, 2H), 7.61–7.54 (m, 3H), 3.06 (t,
J = 7.2 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H), 1.84 (t, J = 6.8 Hz, 4H),
1.56–1.51 (m, 1H), 1.29–1.11 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) d 170.8, 156.2, 144.0, 139.3, 136.4, 128.9(2C), 128.1,
126.5, 123.6(2C), 119.4, 25.4, 24.9, 22.4(2C), 13.3, 10.2(2C). Anal.
Calcd for C19H19N3S: C, 70.99; H, 5.96; N, 13.07. Found C, 71.08;
H, 6.04; N, 13.16.
MS(ESI) m/z 338 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.21
(s, 1H), 7.31 (d, J = 6.8 Hz, 2H), 7.09 (t, J = 6.4 Hz, 1H), 3.08 (t,
J = 6.8 Hz, 2H), 2.90–2.82 (m, 4H), 2.31 (s, 6H), 1.86 (t, J = 6.8 Hz,
4H), 1.28 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d
173.0, 152.4, 144.9, 136.9, 137.5(2C), 133.8(2C), 131.1, 129.4,
124.4, 119.3, 34.6, 25.3, 24.9(2C), 24.5(2C), 21.2, 12.7. Anal. Calcd
for C20H23N3S: C, 71.18; H, 6.87; N, 12.45. Found C, 71.24; H,
6.90; N, 12.64.
4.1.17. N-Benzyl-2-cyclopropyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (6b)
4.1.11. 2-Ethyl-N-(p-tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (5g)
MS(ESI) m/z 336 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.53
(s, 1H), 7.58–7.49 (m, 5H), 4.43 (s, 2H), 3.04 (t, J = 7.2 Hz, 2H),
2.86 (t, J = 7.2 Hz, 2H), 1.85 (t, J = 6.8 Hz, 4H), 1.54–1.49 (s, 1H),
1.27–1.14 (m, 4H); 13C NMR (100 MHz, DMSO-d6) d 170.9, 159.4,
146.3, 139.4, 137.9, 136.3, 128.3(2C), 126.1, 125.8(2C), 117.9,
47.8, 24.6, 24.0, 23.6(2C), 12.9. 9.99 (2C). Anal. Calcd for
MS(ESI) m/z 325 [M+H]+. 1H NMR (400 MHz, CDCl3): d 8.91 (s,
1H), 7.46 (d, J = 7.6 Hz, 2H), 7.36 (d, J = 7.6 Hz, 2H), 3.12 (t,
J = 7.2 Hz, 2H), 2.96–2.87 (m, 4H), 2.46 (s, 3H), 1.87 (t, J = 6.8 Hz,
4H), 1.29 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 174.2,
156.1, 147.3, 137.6, 136.8, 133.9, 129.5(2C), 126.8, 122.6(2C),
120.3, 34.5, 26.0, 24.5(2C), 23.2, 20.5, 13.2. Anal. Calcd for
C20H21N3S: C, 71.61; H, 6.31; N, 12.53. Found C, 71.68; H, 6.39;
C19H21N3S: C, 70.55; H, 6.54; N, 12.99. Found C, 70.64; H, 6.60;
N, 12.66.
N, 13.04.
4.1.18. 2-Cyclopropyl-N-phenethyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (6c)
4.1.12. 2-Ethyl-N-(4-methoxyphenyl)-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (5h)
MS(ESI) m/z 350 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.31
(s, 1H), 7.74 (t, J = 7.2 Hz, 2H), 7.62–7.54 (m, 3H), 3.47 (s, 2H),
3.13–2.97 (m, 4H), 2.83 (t, J = 6.8 Hz, 2H), 1.86 (t, J = 6.8 Hz, 4H),
1.56–1.50 (m, 1H), 1.26–1.16 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) d 170.4, 159.6, 144.7, 138.6, 137.2, 129.2(2C), 128.1,
126.3(2C), 125.3, 117.4, 47.3, 36.9, 25.4, 24.7, 24.2(2C), 13.0, 10.3
(2C). Anal. Calcd for C21H23N3S: C, 72.17; H, 6.63; N, 12.02. Found
C, 72.28; H, 6.69; N, 12.08.
MS(ESI) m/z 340 [M+H]+. 1H NMR (400 MHz, CDCl3): d 8.46 (s,
1H), 7.54 (d, J = 7.6 Hz, 2H), 7.42 (d, J = 7.6 Hz, 2H), 3.96 (s, 3H),
3.08 (t, J = 6.8 Hz, 2H), 2.99–2.88 (m, 4H), 1.91 (t, J = 6.8 Hz, 4H),
1.30 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 173.9, 158.0,
142.4, 139.1, 134.6, 132.6, 131.4, 127.3(2C), 124.9(2C), 119.9,
61.2, 33.3, 25.6, 24.8(2C), 23.8, 12.7. Anal. Calcd for C19H21N3OS:
C, 67.23; H, 6.24; N, 12.38. Found C, 67.34; H, 6.33; N, 12.44.
4.1.13. N-(4-Bromophenyl)-2-ethyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (5i)
4.1.19. 2-Cyclopropyl-N-(2,4-dimethylphenyl)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (6d)
MS(ESI) m/z 388 [M+H]+, 390 [MH+2]+. 1H NMR (400 MHz,
MS(ESI) m/z 350 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.36
(s, 1H), 7.49 (s, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz,
1H), 3.09 (d, J = 7.2 Hz, 1H), 2.94 (d, J = 7.2 Hz, 1H), 2.41 (s, 3H),
2.37 (s, 3H), 1.85 (t, J = 6.8 Hz, 4H), 1.52–1.48 (m, 1H), 1.25–1.11
(m, 4H); 13C NMR (100 MHz, DMSO-d6) d 172.6, 154.3, 144.3,
139.4, 138.1, 136.3, 132.8, 129.3, 128.2, 126.6, 125.4, 119.6, 25.8,
DMSO-d6):
d 8.36 (s, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.48 (d,
J = 8.0 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 2.74
(t, J = 6.8 Hz, 2H), 1.86 (t, J = 7.2 Hz, 4H), 1.28 (t, J = 6.8 Hz, 3H);
13C NMR (100 MHz, DMSO-d6) d 174.4, 156.7, 146.3, 140.1, 138.3,
136.5, 134.3, 130.4(2C), 126.3(2C), 120.1, 34.2, 26.1, 25.3(2C),
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G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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25.0, 24.6(2C), 24.3, 23.8, 12.6, 9.3(2C). Anal. Calcd for C21H23N3S:
C, 72.17; H, 6.63; N, 12.02. Found C, 72.28; H, 6.79; N, 12.06.
(2C). Anal. Calcd for C19H18ClN3S: C, 64.12; H, 5.10; N, 11.81. Found
C, 64.24; H, 5.23; N, 11.94.
4.1.20. 2-Cyclopropyl-N-(2,5-dimethylphenyl)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (6e)
4.1.26. 2-Cyclopropyl-N-(4-fluorophenyl)-5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidin-4-amine (6k)
MS(ESI) m/z 350 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.46
(s, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.39–7.31 (m, 2H), 3.11 (d,
J = 7.2 Hz, 1H), 2.97 (d, J = 7.2 Hz, 1H), 2.44 (s, 3H), 2.39 (s, 3H),
1.88 (t, J = 7.2 Hz, 4H), 1.53–1.47 (m, 1H), 1.26–1.14 (m, 4H); 13C
NMR (100 MHz, DMSO-d6) d 172.9, 155.2, 145.1, 138.8, 138.1,
135.4, 133.2, 129.2, 127.6, 126.2, 126.0, 120.0, 25.6, 25.3, 24.9
(2C), 24.6, 22.2, 11.8, 9.1(2C). Anal. Calcd for C21H23N3S: C, 72.17;
H, 6.63; N, 12.02. Found C, 72.26; H, 6.72; N, 12.09.
MS(ESI) m/z 340 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.35
(s, 1H), 7.56 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 7.6 Hz, 2H), 3.10 (t,
J = 7.2 Hz, 2H), 2.92 (t, J = 7.2 Hz, 2H), 1.88 (t, J = 7.2 Hz, 4H),
1.49–1.45 (m, 1H), 1.23–1.10 (m, 4H); 13C NMR (100 MHz,
DMSO-d6) d 173.4, 158.4, 149.6, 144.2, 137.2, 136.7, 128.3, 126.1
(2C), 124.7(2C), 121.3, 25.4, 24.3, 24.0(2C), 11.6, 10.3(2C). Anal.
Calcd for C19H18FN3S: C, 67.23; H, 5.35; N, 12.38. Found C, 67.34;
H, 5.63; N, 12.44.
4.1.21. 2-Cyclopropyl-N-(2,6-dimethylphenyl)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (6f)
4.1.27. N,2-Diphenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4-amine (7a)
MS(ESI) m/z 350 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 7.42
(d, J = 7.6 Hz, 2H), 7.27–7.19 (m, 2H), 3.04 (d, J = 7.2 Hz, 1H), 2.94
(d, J = 7.2 Hz, 1H), 2.33 (s, 6H), 1.91 (t, J = 7.2 Hz, 4H), 1.51–1.46
(m, 1H), 1.24–1.12 (m, 4H); 13C NMR (100 MHz, DMSO-d6) d
171.9, 153.6, 146.3, 138.4, 136.3, 133.1, 130.2(2C), 129.5(2C),
126.2, 120.4, 25.2, 24.7, 23.8(2C), 21.4(2C), 11.6, 9.0(2C). Anal.
Calcd for C21H23N3S: C, 72.17; H, 6.63; N, 12.02. Found C, 72.26;
H, 6.72; N, 12.09.
MS(ESI) m/z 358 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.37
(d, J = 7.6 Hz, 2H), 7.82–7.69 (m, 6H), 7.54–7.42 (m, 3H), 3.03 (t,
J = 7.6 Hz, 2H), 2.85 (t, J = 7.2 Hz, 2H), 1.87 (t, J = 7.2 Hz, 4H); 13C
NMR (100 MHz, DMSO-d6) d 166.4, 155.2, 147.5, 142.7, 139.2,
137.4, 133.2, 128.1(2C), 127.3(2C), 126.0, 125.2, 124.6(2C), 120.8
(2C), 117.2, 25.6, 24.3, 23.6(2C). Anal. Calcd for C22H19N3S: C,
73.92; H, 5.36; N, 11.75. Found C, 73.99; H, 5.63; N, 12.04.
4.1.28. N-Benzyl-2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (7b)
4.1.22. 2-Cyclopropyl-N-(p-tolyl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (6g)
MS(ESI) m/z 358 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.36
(s, 1H), 7.81 (d, J = 7.2 Hz, 2H), 7.67–7.56 (m, 3H), 7.48–7.32 (m,
5H), 4.42 (s, 2H), 3.08 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H),
1.84 (t, J = 7.2 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) d 164.9,
156.6, 146.3, 141.3, 138.8, 137.2, 133.6, 127.7(2C), 127.1(2C),
125.8, 125.3, 124.3(2C), 121.2(2C), 118.4, 45.2, 24.9, 24.1, 23.2
(2C). Anal. Calcd for C23H21N3S: C, 74.36; H, 5.70; N, 11.31. Found
C, 74.49; H, 5.83; N, 11.44.
MS(ESI) m/z 325 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.36
(s, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 3.08 (t,
J = 7.6 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H), 2.41 (s, 3H), 1.84 (t,
J = 7.2 Hz, 4H), 1.52–1.47 (m, 1H), 1.23–1.08 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 173.5, 154.3, 146.3, 138.4, 137.3, 131.8,
128.7(2C), 127.3, 123.6(2C), 116.1, 26.1, 25.3(2C), 24.6, 22.3, 12.3,
9.3(2C). Anal. Calcd for C20H21N3S: C, 71.61; H, 6.31; N, 12.53.
Found C, 71.66; H, 6.40; N, 12.64.
4.1.29. N-Phenethyl-2-phenyl-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-4-amine (7c)
4.1.23. 2-Cyclopropyl-N-(4-methoxyphenyl)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (6h)
MS(ESI) m/z 352 [M+H]+. 1H NMR (400 MHz, CDCl3): d 8.39 (s,
1H), 7.63 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 7.6 Hz, 2H), 4.06 (s, 3H),
3.06 (t, J = 7.2 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 1.87 (t, J = 6.8 Hz,
4H), 1.48–1.43 (m, 1H), 1.26–1.05 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 170.8, 156.3, 155.4, 144.3, 138.3, 135.2, 128.2, 126.4
(2C), 124.2(2C), 123.2, 61.9, 25.2, 24.4(2C), 23.4, 12.4, 9.7(2C). Anal.
Calcd for C20H21N3OS: C, 68.35; H, 6.02; N, 11.96. Found C, 68.39;
H, 6.13; N, 12.04.
MS(ESI) m/z 386 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.43
(s, 1H), 8.11 (d, J = 7.2 Hz, 2H), 7.77–7.68 (m, 4H), 7.62–7.48 (m,
4H), 3.46 (t, J = 6.8 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H), 2.89–2.65 (m,
4H), 1.87 (t, J = 6.8 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) d
166.2, 158.3, 144.6, 140.3, 139.2, 137.6, 134.2, 127.9(2C), 127.2
(2C), 126.2, 125.5, 124.1(2C), 122.1(2C), 119.3, 46.1, 36.9, 24.7,
23.8, 23.4(2C). Anal. Calcd for C24H23N3S: C, 74.77; H, 6.01; N,
10.90. Found C, 74.89; H, 6.13; N, 11.04.
4.1.30. N-(2,4-Dimethylphenyl)-2-phenyl-5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7d)
4.1.24. N-(4-Bromophenyl)-2-cyclopropyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (6i)
MS(ESI) m/z 386 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.16
(s, 1H), 7.84 (d, J = 7.2 Hz, 2H), 7.72–7.66 (m, 3H), 7.56 (s, 1H),
7.49–7.41 (m, 2H), 3.03 (d, J = 6.8 Hz, 2H), 2.91 (d, J = 6.8 Hz, 2H),
2.43 (s, 3H), 2.34 (s, 3H), 1.82 (t, J = 6.8 Hz, 4H); 13C NMR
(100 MHz, DMSO-d6) d 174.9, 155.3, 145.7, 138.6, 137.4, 136.7,
133.9, 130.3, 129.6, 128.6, 127.3, 126.6(2C), 125.9, 125.1, 123.5
(2C), 118.4, 25.3, 24.7, 24.2(2C), 23.1, 22.2. Anal. Calcd for
MS(ESI) m/z 400 [M+H]+, 402 [MH+2]+. 1H NMR (400 MHz,
DMSO-d6):
d 9.42 (s, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.54 (d,
J = 7.6 Hz, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H), 1.89
(t, J = 7.2 Hz, 4H), 1.53–1.44 (m, 1H), 1.27–1.08 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 172.6, 154.6, 145.9, 140.3, 136.9, 134.6
(2C), 132.0, 124.3(2C), 120.4, 118.6, 25.5, 24.7(2C), 24.1, 11.6,
10.1(2C). Anal. Calcd for C19H18BrN3S: C, 57.00; H, 4.53; N, 10.50.
Found C, 57.04; H, 4.63; N, 10.64.
C24H23N3S: C, 74.77; H, 6.01; N, 10.90. Found C, 74.88; H, 6.09;
N, 11.06.
4.1.25. N-(4-Chlorophenyl)-2-cyclopropyl-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (6j)
MS(ESI) m/z 356 [M+H]+, 358 [MH+2]+. 1H NMR (400 MHz,
4.1.31. 2-Cyclopropyl-N-(2,5-dimethylphenyl)-5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7e)
MS(ESI) m/z 386 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.37
(s, 1H), 7.85 (d, J = 7.2 Hz, 2H), 7.68–7.58 (m, 3H), 7.53–7.47 (m,
3H), 3.04 (d, J = 6.8 Hz, 2H), 2.92 (d, J = 6.8 Hz, 2H), 2.41 (s, 3H),
2.33 (s, 3H), 1.82 (t, J = 6.8 Hz, 4H); 13C NMR (100 MHz, DMSO-
d6) d 168.7, 156.6, 146.3, 139.5, 138.7, 136.8, 134.8, 133.9, 133.0,
130.6, 128.6(2C), 127.9(2C), 125.7, 123.1, 120.4, 119.2, 25.9, 24.8,
DMSO-d6):
d 9.63 (s, 1H), 7.77 (d, J = 7.6 Hz, 2H), 7.56 (d,
J = 7.6 Hz, 2H), 3.05 (t, J = 7.2 Hz, 2H), 2.88 (t, J = 7.2 Hz, 2H), 1.87
(t, J = 7.2 Hz, 4H), 1.52–1.46 (m, 1H), 1.25–1.06 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) d 171.7, 155.5, 146.4, 139.4, 135.3, 132.3
(2C), 130.1, 128.5, 126.7(2C), 119.3, 24.9, 24.3(2C), 23.5, 11.7, 9.4
Please cite this article in press as: Samala, G.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.012

8
G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
24.2(2C), 23.1, 21.4. Anal. Calcd for C24H23N3S: C, 74.77; H, 6.01; N,
10.90. Found C, 74.86; H, 6.12; N, 10.99.
for C22H18FN3S: C, 70.38; H, 4.83; N, 11.19. Found C, 70.49; H, 4.99;
N, 11.24.
4.1.32. N-(2,6-Dimethylphenyl)-2-phenyl-5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidin-4-amine (7f)
4.2. Biological screening
MS(ESI) m/z 386 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 8.37
(d, J = 7.2 Hz, 2H), 7.87–7.69 (m, 3H), 7.63 (d, J = 7.2 Hz, 2H),
7.58–7.53 (m, 2H), 3.06 (d, J = 6.8 Hz, 2H), 2.91 (d, J = 6.8 Hz, 2H),
2.31 (s, 6H), 1.86 (t, J = 6.8 Hz, 4H); 13C NMR (100 MHz, DMSO-
d6) d 172.4, 154.3, 145.8, 139.0, 137.1, 135.8, 133.6(2C), 132.6,
130.6(2C), 129.3(2C), 128.7(2C), 128.2, 126.7, 121.3, 24.7, 24.3,
23.4(2C), 19.9(2C). Anal. Calcd for C24H23N3S: C, 74.77; H, 6.01;
N, 10.90. Found C, 74.86; H, 6.12; N, 11.01.
4.2.1. MTB MABA assay
The compounds were screened for their in vitro antimycobacte-
rial activity against Mycobacterium tuberculosis H37Rv by micro-
plate Alamar blue assay method. Briefly, the inoculum was
prepared from fresh LJ medium, re-suspended in 7H9-S medium
(7H9 broth, 0.1% casitone, 0.5% glycerol, supplemented oleic acid,
albumin, dextrose, and catalase [OADC]), adjusted to a McFarland
tube No. 1, and diluted 1:20; 100 ll was used as inoculum. Each
drug stock solution was thawed and diluted in 7H9-S at four-fold
the final highest concentration tested. Serial two-fold dilutions of
each drug were prepared directly in a sterile 96-well microtiter
4.1.33. 2-Phenyl-N-(p-tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-4-amine (7g)
MS(ESI) m/z 372 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.43
(s, 1H), 8.26 (d, J = 7.2 Hz, 2H), 7.82–7.74 (m, 3H), 7.67–7.56 (m,
4H), 3.03 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.40 (s, 3H),
1.83 (t, J = 6.8 Hz, 4H), 1.52–1.47 (m, 1H), 1.23–1.08 (m, 4H); 13C
NMR (100 MHz, DMSO-d6) d 170.7, 155.4, 144.8, 139.2, 138.0,
136.3, 134.2, 130.4(2C), 129.3(2C), 127.8(2C), 127.1, 126.3(2C),
120.4, 117.9, 25.4, 24.7(2C), 23.8, 22.5. Anal. Calcd for C23H21N3S:
C, 74.36; H, 5.70; N, 11.31. Found C, 74.46; H, 5.80; N, 11.44.
plate using 100 ll 7H9-S. A growth control containing no antibiotic
and a sterile control were also prepared on each plate. Sterile water
was added to all perimetre wells to avoid evaporation during the
incubation. The plate was covered, sealed in plastic bags and incu-
bated at 37 °C in normal atmosphere. After 7 days of incubation,
30 ml of alamar blue solution was added to each well, and the plate
was re-incubated overnight. A change in color from blue (oxidized
state) to pink (reduced) indicated the growth of bacteria, and the
MIC was defined as the lowest concentration of drug that pre-
vented this change in color.
4.1.34. N-(4-Methoxyphenyl)-2-phenyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (7h)
MS(ESI) m/z 388 [M+H]+. 1H NMR (400 MHz, CDCl3): d 8.19 (d,
J = 7.6 Hz, 2H), 7.83 (d, J = 7.6 Hz, 2H), 7.68–7.60 (m, 6H), 3.96 (s,
3H), 3.12 (t, J = 7.2 Hz, 2H), 2.96 (t, J = 7.2 Hz, 2H), 1.89 (t,
J = 6.8 Hz, 4H); 13C NMR (100 MHz, CDCl3) d 169.9, 158.5, 156.5,
146.1, 139.6, 138.3, 136.2, 134.7, 130.6(2C), 129.1(2C), 127.8,
127.2(2C), 124.7(2C), 121.1, 60.3, 25.1, 24.3(2C), 22.8. Anal. Calcd
for C23H21N3OS: C, 71.29; H, 5.46; N, 10.84. Found C, 71.38; H,
5.62; N, 10.94.
4.2.2. In vitro dormant MTB model
Culture of Mycobacterium tuberculosis H37RV were grown in
Middlebrook 7H9 medium supplemented with OADC (nutrient rich
medium) was pelleted and washed twice with PBS (Phosphate Buf-
fer Saline, HiMedia Laboratories). The pellet was re-suspended in
PBS in sealed bottles and is incubated at 37 °C for 6 weeks. Six
week starved cultures were treated with standard drugs like isoni-
azid, rifampicin and moxifloxacin along with synthesized drugs for
7 days at a concentration of 10ug/ml. The treated cell suspensions
were diluted 10-fold up to 10À6 using Middlebrook 7H9 medium
4.1.35. N-(4-Bromophenyl)-2-phenyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (7i)
supplemented with OADC and 100
ll of each dilutions were plated
MS(ESI) m/z 436 [M+H]+, 438 [MH+2]+. 1H NMR (400 MHz,
in 48 well plates in triplicates along with 900
ll of Middlebrook
DMSO-d6):
d
8.91 (s, 1H), 8.31 (d, J = 6.8 Hz, 2H), 7.87 (d,
7H9 medium (HiMedia Laboratories) supplemented with OADC
(HiMedia Laboratories). The microplates were incubated at 37 °C
for 28 days without agitation. Wells with visible bacterial growth
were counted as positive, and MPN values were calculated using
standard statistical methods.
J = 6.8 Hz, 2H), 7.62–7.56 (m, 5H), 3.14 (t, J = 6.4 Hz, 2H), 2.87 (t,
J = 6.4 Hz, 2H), 1.83 (t, J = 6.4 Hz, 4H); 13C NMR (100 MHz, DMSO-
d6) d 165.3, 157.2, 152.8, 144.6, 139.7, 136.7, 135.1, 129.2(2C),
127.8(2C), 127.0, 126.1(2C), 125.3, 124.2(2C), 118.0, 25.1, 24.4,
23.8(2C). Anal. Calcd for C22H18BrN3S: C, 60.55; H, 4.16; N, 9.63.
Found C, 60.69; H, 4.29; N, 9.74.
4.2.3. In vitro MTB ADH enzyme inhibition assay
The enzyme inhibition studies were performed in microtitre
plate containing desired substrate and enzyme concentration as
explained above. The spectrophotometric determination of the
reaction product NADH that accompanies the conversion of
4.1.36. N-(4-Chlorophenyl)-2-phenyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (7j)
MS(ESI) m/z 392 [M+H]+, 394 [MH+2]+. 1H NMR (400 MHz,
DMSO-d6): d 8.33–8.28 (m, 3H), 7.81 (d, J = 7.2 Hz, 2H), 7.53–7.48
(m, 5H), 3.13 (t, J = 6.8 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H), 1.87 (t,
J = 6.8 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) d 165.3, 156.1,
146.4, 141.3, 138.9, 137.8, 134.0, 129.2(2C), 128.3(2C), 127.3,
126.6(2C), 125.3, 124.2(2C), 118.7, 25.3, 24.8, 24.1(2C). Anal. Calcd
for C22H18ClN3S: C, 67.42; H, 4.63; N, 10.72. Found C, 67.49; H,
4.69; N, 10.84.
L-alanine into pyruvate in the oxidative deamination was mea-
sured at 340 nm in heat controlled microplate reader PerkinElmer
Victor X3 instrument. The synthesized compounds were added to
the plate with different concentrations from 25 lM to 0.5 lM in
order to determine IC50 values for the all the synthesized com-
pounds. The reaction mixture except MTB ADH was added and
background reactions were measured. Reactions were carried out
at 37 °C in a heat-controlled PerkinElmer Victor X3 Spectropho-
tometer. Further the IC50 values were calculated using GraphPad
Prism analysis software.
4.1.37. N-(4-Fluorophenyl)-2-phenyl-5,6,7,8-tetrahydrobenzo
[4,5]thieno[2,3-d]pyrimidin-4-amine (7k)
MS(ESI) m/z 376 [M+H]+. 1H NMR (400 MHz, DMSO-d6): d 9.21
(s, 1H), 8.30 (d, J = 6.8 Hz, 2H), 7.85 (d, J = 6.8 Hz, 2H), 7.60–7.54
(m, 5H), 3.10 (t, J = 6.4 Hz, 2H), 2.85 (t, J = 6.4 Hz, 2H), 1.84 (t,
J = 6.4 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) d 164.7, 156.6,
151.4, 144.2, 138.4, 137.2, 134.4, 129.4(2C), 127.1(2C), 126.5,
125.2(2C), 124.1, 123.4(2C), 118.4, 25.6, 24.7, 24.3(2C). Anal. Calcd
4.2.4. Differential scanning fluorimetry
A thermal shift assay, also called differential scanning fluorime-
try (DSF) is a thermal-denaturation assay that measures the ther-
mal stability of a target protein and a subsequent increase in
protein melting temperature upon binding of a ligand to the pro-
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G. Samala et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
tein. We used Sypro Orange dye (Sigma–aldrich) in a Real time PCR
Acknowledgements
instrument (Bio-Rad iCycle5), which binds to exposed core resi-
dues of a denatured protein and results in an increased fluores-
cence signal. The instrument was programmed to equilibrate the
samples at 25 °C for 3 min and further rise in temperature till
95 °C, with every 0.1 °C rice using a LED/Photodiode set matched
with dye excitation and emission wavelengths. Required concen-
tration of MTB L-AlaDH and dye was determined by varying differ-
ent concentrations of protein and dye in analysis buffer containing
100 mM HEPES–NaOH, pH-7.8. The selected top active compounds
D.S. is thankful to Department of Biotechnology, Government of
India for the Tata innovation fellowship.
References and notes
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were diluted in <10% DMSO. Later, in a 96 well PCR plate, 20
reaction volume containing 10 l of MTB -AlaDH (100 g/ml) in
analysis buffer, 6
l of 15Â dye (Diluted from 5000Â stock with
ll of
l
L
l
l
sub-stock of 50Â in DMSO). Based on their dilutions, the com-
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assay. Mouse macrophages were grown in RPMI medium supple-
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and 10 mg streptomycin per ml in T25 flasks to attain 80–90% con-
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for 24 h prior to addition of experimental drugs. The test com-
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pounds at 50
bated at 37 °C for 48 h; later 10
l
M concentration were then added to cells and incu-
lL of 0.5 mg/ml concentration of
MTT was added and incubated for 3 h at 37 °C and the final product
Formazon crystals were measured at 595 nm.
Please cite this article in press as: Samala, G.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.012