Synthesis, structure-activity relationship of novel substituted 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates as potential anti-mycobacterial and anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2011.03.079

Series of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 7a-7zb, 8a-8d and 9a-9d were synthesized and screened for their in vitro anti-mycobacterial activity against Mycobacterium tuberculosis H₃₇Rv (MTB) and cytotoxicity against three human cancer cell lines including A549, SK-N-SH and HeLa. The results indicate that six compounds are more potent and 7za is most effective anti-mycobacterial derivative compared to the standard drugs Ethambutol and Ciprofloxacin. However, 12 compounds exhibited cytotoxicity against human neuroblastoma cell line; amongst them the compound 7v is most effective compared to the standard drug Doxorubicin. This is the first report assigning in vitro anti-mycobacterial, anticancer and structure-activity relationship for this new class of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5- carboxylates.

Full text of DOI:10.1016/j.bmcl.2011.03.079

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2855–2859
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, structure–activity relationship of novel substituted
4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates
as potential anti-mycobacterial and anticancer agents
B. China Raju a, , R. Nageswara Rao a, P. Suman a, P. Yogeeswari b, D. Sriram b,
,
⇑
⇑
Thokhir Basha Shaik c, Shasi Vardhan Kalivendi c
a Organic Chemistry Division-1, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Birla Institute of Technology & Science-Pilani, Hyderabad 500 078, India
c Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 7a–7zb, 8a–8d and 9a–9d
were synthesized and screened for their in vitro anti-mycobacterial activity against Mycobacterium tuber-
culosis H37Rv (MTB) and cytotoxicity against three human cancer cell lines including A549, SK-N-SH and
HeLa. The results indicate that six compounds are more potent and 7za is most effective anti-mycobac-
terial derivative compared to the standard drugs Ethambutol and Ciprofloxacin. However, 12 compounds
exhibited cytotoxicity against human neuroblastoma cell line; amongst them the compound 7v is most
effective compared to the standard drug Doxorubicin. This is the first report assigning in vitro anti-myco-
bacterial, anticancer and structure–activity relationship for this new class of 4H-chromen-1,2,3,4-tetra-
hydropyrimidine-5-carboxylates.
Received 3 November 2010
Revised 9 March 2011
Accepted 23 March 2011
Available online 30 March 2011
Keywords:
Substituted 4H-chromen-1,2,3,4-
tetrahydropyrimidine-5-carboxylates
Biginelli reaction
Ó 2011 Elsevier Ltd. All rights reserved.
Vilsmeier reaction
Suzuki coupling
4-Oxo-4H-chromene-3-carbaldehydes
Anti-mycobacterial activity
Anticancer activity
Chromone derivatives1 are an important class of natural, syn-
thetic compounds and pharmacologically active substances dis-
playing a broad range of biological activities including anticancer,
monoamine oxidase B (MAO-B) inhibitors, anti-mycobacterial,
antimicrobial, human-immunodeficiency virus (HIV-1) and mush-
room tyrosinase inhibition.2 These derivatives are an important
intermediates in the manufacture of agrochemicals, pharmaceuti-
cals, and dyestuff industries.3 4-Oxo-4H-chromen-3-carbaldehyde
(3-formylchromone) a useful precursor for the synthesis of several
biological active compounds4 owing to the presence of an unsatu-
rated keto function, a conjugated second carbonyl group at C-3,
and an electrophilic centre at C-2. However, there is no report for
the synthesis of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate derivatives and their biological activities. Mycobacterium
tuberculosis (MTB), single infectious causative agent of tuberculosis
(TB)5 resulted in the highest number of human death worldwide
and the threat factor in current TB problem is the prevalence of
multi-drug resistant (MDR) strains and co-infections with AIDS
has been responsible for this serious situation. Similarly cancer6
the uncontrolled, rapid and proliferation of abnormal cells is the
leading cause of human death, despite considerable progress of
its biology and pharmacology, cancer remains a serious problem,
therefore, safe, potent, and selective search for new chemothera-
peutic agents are required for tuberculosis and cancer.
Our interest in design and synthesis of diverse range of biolog-
ically active heterocyclic compounds with potential activities,7 and
on anti-mycobacterial agents,8 in this Letter, we report the synthe-
sis, anti-mycobacterial and anticancer activity of new substituted
4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylate deriva-
tives. Scheme 1 illustrates the synthesis of 6-substituted 4-oxo-
4H-chromene-3-carbaldehydes 4a–4f. Substituted phenols 1a–1e
were acetylated using acetyl chloride and on subsequent Fries
rearrangement with AlCl3 gave 3a–3e. The 1-(2-hydroxy-3-nitro-
phenyl)ethanone 3f was prepared according to the literature pro-
cedure.9 The compound 3a was subjected for Vilsmeier
conditions10 to give 4a using various reagents such as POCl3/
DMF, oxalyl chloride/DMF and triphosgene/DMF, among these oxa-
lyl chloride/DMF was found to be the better reagent in terms of
yields. Under similar conditions 4b–4f were synthesized in very
good yields (Scheme 1). The reaction of 5-bromoacetophenone
⇑
Corresponding authors. Tel.: +91 40 27191725; fax: +91 40 27160512 (B.C.R.).
E-mail addresses: chinarajuiict@yahoo.co.in (B. China Raju), drdsriram@yahoo.
com (D. Sriram).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.079

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B. China Raju et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2855–2859
R1
R2
O
O
R1
R1
O
R1
R
R
R
R
H
AlCl3
120 0
DMF/POCl3
or
AcCl
Py, DCM
C
O
OAc
OH
OH
DMF/Oxalyl chloride
or
R2
2a-e
R2
3a-f
R2
1a-e
4a-f
DMF/Triphosgene
4a R = R1 = R2 = H;
4b R = Br, R1 = R2 = H;
4c R = CH3, R1 = R2 = H;
4d R = NO2, R1 = R2 = H;
4e R1 = CH3, R = R2 = H;
4f R2 = NO2, R = R1 = H.
Scheme 1.
O
O
O
O
O
R
Br
R
H
R-B(OH)2
PdCl2(PPh3)2
DMF
Oxalyl chloride
OH
OH
4g R = Phenyl
4h R = Furyl
3g-h
2b
Scheme 2.
O
O
O
MeO
O
Br
OMe
Amberlyst-15
Toluene
+
MeO
O
K2CO3
DMF, 90 0C
OH
OH
O
O
OH
HO
OH
3k
3l
OMe
3i
3j
DMF
DMF
Oxalyl chloride
Oxalyl chloride
O
O
CHO
CHO
O
O
O
O
4i
4j
Scheme 3.
X
R1
R2
O
O
HN
NH
R3
OR4
R1
R2
O
O
X
O
O
R
R
p-TsOH
C2H5OH
H
+
R3
OR4
H2N
NH2
+
O
O
7a-zb
4a-h
6a-b
5a-e
Scheme 4.
O
O
O
HN
NH
R3
OR4
CHO
O
O
O
p-TsOH
C2H5OH
+
+
R3
OR4
H2N
NH2
O
O
O
O
O
8a-d
5a-d
4i
6a
Scheme 5.
2b subjected with phenylboronic acid and furylboronic acid in
presence of PdCl2(PPh3)2 (20 mol %) in 22–24% aq isopropanol (Su-
zuki coupling),7a11 under reflux conditions gave 3g, 3h and subse-
quent Vilsmeier reaction with oxalyl chloride/DMF afforded 4g, 4h
in good yields (Scheme 2).
Resacetophenone 3i was reacted with bromoacetaldehyde
dimethyl acetal in presence of potassium carbonate in anhy-
drous DMF yielded 1-(4-(2,2-dimethoxyethoxy)-2-hydroxyphenyl)
ethanone 3j, which upon cyclization using acidic Amberlyst
15 in toluene under reflux conditions yielded mixture of

B. China Raju et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2855–2859
2857
O
O
O
O
O
HN
NH
R3
OR4
CHO
+
O
O
O
p-TsOH
C2H5OH
+
R3
OR4
H2N
NH2
O
O
9a-d
O
4j
6a
5a-d
Scheme 6.
1-(6-hydroxybenzofuran-5-yl)-ethanone 3k (61%), 1-(4-hydrox-
ybenzofuran-5-yl)ethanone 3l (39%, Scheme 3).12 These com-
pounds 3k, 3l were separated by column chromatography and
subsequent Vilsmeier reaction afforded 5-oxo-5H-furo[3,2-g]chro-
mene-6-carbaldehyde 4i and 4-oxo-4H-furo[2,3-h]chromene-
3-carbaldehyde 4j (Scheme 3). Thus synthesized 4-oxo-4H-
chromene-3-carbaldehydes 4a–4j were reacted with various
b-ketoesters 5a–5e and urea 6a–6b in presence of p-TsOH in etha-
nol under reflux conditions (Biginelli reaction)13 afforded series of
pounds were screened in the present study, MIC’s ranging from
3.39–76.21 M and eight compounds inhibited MTB with less than
10 M. Six compounds 7t, 7u, 7v, 7za, 8d and 9d shown excellent
activity with MIC 3.39–7.38 M and were more potent than stan-
dard drugs Ethambutol (MIC of 7.63 M), Ciprofloxacin (MIC of
9.44 M). Two compounds 7d and 7s with MIC ranging from
7.86–7.90 M are potent than Ciprofloxacin and 7za found to be
most active with MIC 3.39 M. Five compounds 7n, 7q, 7zb, 8b
and 9b with MIC’s 14.53–16.62 M shown moderate activity in
l
l
l
l
l
l
l
l
new
4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates
present series. With respect to structure-MTB activity, among
2-oxo-4-(4-oxo-4H-chromen-3-yl-1,2,3,4-tetrahydropyrimidine-
5-carboxylates, it is interesting to note that in general the
7a–7zb, 8a–8d and 9a–9d in very good yields (Scheme 4–6,
Table 1). The synthesized compounds were well characterized by
spectral data and documented in Supplementary data.14
The compounds 7a–7zb, 8a–8d, and 9a–9d were screened for
their in vitro anti-mycobacterial activity against M. tuberculosis
H
MIC and values of the synthesized compounds along with the stan-
dard drugs for comparison are presented in Table 2. All the 36 com-
Table 2
Anti-mycobacterial and anticancer activities of compounds 7a–7zb, 8a–8d and 9a–9d
37Rv (MTB)15 by agar dilution method for the determination of
Compds
MTBb (MICa in
lM)
Cytotoxicityc (%)
A549
SK-N-SH
HeLa
7a
7b
7c
7d
76.21
64.10
34.53
7.90
18.1 0.8
15.4 4.4
30.7 3.1
––d
19 2.5
15 3.9
18.6 1.9 23.3 3.8
23.4 1.9 29.2 0.8
16.6 2.5
5.3 1.60
Table 1
Synthesis of substituted 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates
(7a–7zb, 8a–8d and 9a–9d)
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
7x
7y
7z
7za
7zb
8a
8b
8c
69.83
72.67
61.57
66.84
30.78
28.66
29.69
36.54
30.94
16.62
34.15
34.91
14.88
32.21
7.86
23.5 0.1
26.3 5.5
9.4 3.9
––d
23.9
3
38.5 1.8
32.7 0.3 49.3 7.4
1.9 3.8
––d
Compds
R
R1
R2
R3
R4
X
Yielda (%)
33 1.9
––d
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
CH3
C6H5
CH2Cl
CF3
Et
Et
Et
Et
O
O
O
O
O
S
97
92
89
51.4 0.04 31.1 4.7 32.8 3.8
29.9 4.5
43.5 0.2
24.8 3.8
18.6 4.4
38.4
1
40.2 2.4
41.1 0.7
16.6 0.6
29.8 4.5
b
57
20 2.5
1.7 0.2
CH3
CH3
C6H5
CH3
CH3
CH3
CH3
CH3
C6H5
CH2Cl
CH3
CH3
C6H5
CH3
CF3
C2H4OMe
Et
Et
C2H4OMe
Et
C2H4OMe
Et
Et
Et
Et
C2H4OMe
Et
Et
C2H4OMe
Et
Et
Et
Et
Et
Et
C2H4OMe
Et
Et
Et
Et
Et
C2H4OMe
Et
Et
Et
C2H4OMe
Et
92
88
80
85
72
87
92
96
94
93
90
90
89
92
73b
62b
70b
53b
85
87
82
82
40b
57
82
80
86
43b
82
82
87
42b
d
––
S
S
46.2
1
55.3 0.9 81.4 1.7
35.6 2.6 40.2 0.9
28.4 5.3
18.3 2.3
27.2 0.3
21.2 2.8
37.4 2.6
22 1.5
Br
Br
Br
O
O
S
O
O
O
O
S
7
2.4
43.2 5.4
31.5 0.007
12.1
4
22.8 1.2 17.4 1.8
45.5 0.1 33.5 5.3
35.9 1.7 61.3 5.7
28.7 2.1 54.4 3.3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
3.91
4.18
4.18
––d
47.6 1.5
56 0.9
60.6 1.6
45.3 0.9
––d
30.71
25.93
57.60
59.52
3.39
15.86
33.96
14.53
31.40
7.38
33.96
14.53
31.40
3.67
0.66
7.63
7.2
1
––d
S
S
––d
––d
34.4 0.5
37.4 2.6
31.3 2.7
34.5 2.5
29.6 0.1
13.1 3.5
34.7 1.7
13.4 0.5 33.5
25.4 2.1 40.9 0.3
10.7 1.4 28.6
4
7s
7t
O
O
O
O
O
O
O
S
O
O
O
O
O
O
O
O
O
O
CF3
3
7u
7v
7w
7x
7y
7z
7za
7zb
8a
8b
8c
8d
9a
9b
9c
9d
NO2
H
CH3
CH3
CH3
C6H5
CH3
CH3
CF3
CH3
CH3
Ph
CH3
CF3
CH3
Ph
CH3
CF3
46.4 2.4 35.4 2.6
37.5 2.2 51.5 2.7
d
C6H5
C6H5
C6H5
C6H5
C6H5
Furyl
––
––
––
––
––
––
59.1 3.6
34.7 5.7 37.6 2.4
8d
9a
9b
9c
21.8
3
6.9 5.1
40.9 0.4
42.4 2.7
d
H
H
H
H
H
H
33.8 3.1
33.3 1.8
4.5 0.97
27.5 1.7
––
39.4 0.7 43.3 1.7
8.4 0.04 61.2 5.7
34.3 2.5 32.8 0.04
––
––
––
––
––
––
––
––
––
––
––
––
––
––
––
––
9d
Isoniazid
Ethambutol
Ciprofloxacin 9.44
Doxorubicin
55 3.5
31.8 2.7 86.5 0.09
––
––
––
a
b
c
Minimum inhibitory concentration.
Mycobacterium tuberculosis H37Rv.
Cytotoxicity against three cancer cell lines [lung, neuroblastoma, and cervical
a
Isolated yields.
cancer].
b
d
AcOH was used for the cyclization reaction in stead p-TsOH.
No activity.

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B. China Raju et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2855–2859
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compounds having the trifluoromethyl group at 6th position on
1,2,3,4-tetrahydropyrimidine-5-carboxylate has the potential im-
pact in improving the activity compared to methyl, phenyl, chloro-
methyl substitutions. Nitro substitution on 4H-chromene at 6th
position 7u and 8th position 7v play an important role in enhanc-
ing the activity compared to bromo 7i, methyl 7s, and phenyl/furyl
7w/7zb. Further methyl at 5th position 7t on 4H-chromen im-
proved the activity over 6th position 7s.
The in vitro results of anti-mycobacterial activity encouraged us
to evaluate its anticancer effects against a panel of three human
cancer cell lines including lung (A549), CNS (SK-N-SH), and cervical
(HeLa) by using MTT assay16 for compounds 7a–7zb, 8a–8d and
9a–9d, and the results are presented in Table 2 along with the
standard drug Doxorubicin for comparison. Compound 7i dis-
played significant, 7k, 7n, and 7v shown moderate anticancer
activity against lung cancer cell line (A549). Compounds 7n, 7s,
7v and 7zb are more potent compared to standard drug Doxorubi-
cin, where as eight compounds 7f, 7j, 7o, 7t, 8a, 9b and 9d dis-
played potent activity against CNS cancer cell line (SK-N-SH).
Compound 7n is potent; six compounds 7t, 7u, 7v, 8a, 8b, and 9c
are significant against cervical cancer cell line (HeLa). In general
the compounds 7n and 7v have shown both anti-mycobacterial
and anticancer activity on tested cancer cell lines, where as 7s,
7t, 7v, and 9d shown anti-mycobacterial and selectively on SK-N-
SH cancer cell line.
In conclusion, compounds 7a–7zb, 8a–8d and 9a–9d were syn-
thesized applying different synthetic protocols. Suzuki coupling for
the synthesis of compounds 7w–7zb has been applied for the first
time in synthesizing substituted 4-oxo-4H-chromenyl-1,2,3,4-tet-
rahydropyrimidine-5-carboxylate derivatives. These compounds
are not known so far in the literature along with the synthetic
methodology applied, and the assignment of the in vitro anti-
mycobacterial and anticancer activity to all the 4-oxo-4H-chrome-
nyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives in this
report is new aspect. Compounds 7za, 7t, 7u, 7v, and 9d are potent
in present series in comparison with standard drugs Ethambutol
and Ciprofloxacin. Compounds 7n and 7v are the most potent
against SK-N-SH, cell line. This study further presents substituted
4-oxo-4H-chromenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate
derivatives as new class of anti-mycobacterial as well as anticancer
and it may serve as a model compounds for design and develop-
ment of therapeutic based anti-mycobacterial, anticancer agents.
The synthesis and biological activity of other heterocycles is cur-
rently under investigation.
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14. Ethyl-6-(chloromethyl)-2-oxo-4-(4-oxo-4H-chromen-3-yl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (7c). Yield: 89%; color less solid; mp 240–
242 °C. IR (KBr): 3297, 3099, 2939, 1711, 1639, 1231, 1094, 751 cmÀ1 1H NMR
.
(300 MHz, CDCl3 + DMSO-d6): d 9.50 (br s, 1H, NH), 8.20 (d, 1H, J = 7.9 Hz,
aromatic), 7.84 (s, 1H, CH), 7.88–7.69 (m, IH, aromatic), 7.54–7.40 (m, 2H,
aromatic), 6.78 (s, 1H, NH), 5.58 (d, 1H, J = 3.1 Hz, CH), 4.85–4.73 (q, 2H, CH2Cl),
4.22–4.04 (q, 2H, OCH2), 1.22 (t, 3H, J = 6.9 Hz, CH3). 13C NMR (CDCl3 + DMSO-
d6): d 175.6, 163.1, 155.2, 152.5, 151.8, 152.6, 147.1, 133.0, 124.4, 122.8, 122.5,
117.2, 97.0, 59.4, 46.5, 46.3. Mass (ESI-MS): m/z 363, 365 [M++1], 385, 387
[M++Na]. Ethyl 2-oxo-4-(4-oxo-4H-chromen-3-yl)-6-(trifluoromethyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (7d). Yield: 57%; pale cream solid; mp
255–257 °C. IR (KBr): 3233, 3100, 1611, 1569, 1438, 1363, 1241, 1093;
Acknowledgements
The authors thank Dr. J.S. Yadav, Director, and Dr. J. Madhusudana
Rao, Head, Organic Chemistry Division-1, IICT for their constant
encouragement and support of this work. R.N.R. and P.S. thank CSIR,
New Delhi for financial support.
. d 8.30–8.15 (m, 1H,
957 cmÀ1 1H NMR (300 MHz, CDCl3 + DMSO-d6):
aromatic), 8.08 (s, 1H, aromatic), 7.82–7.74 (m, 1H, aromatic), 7.66 (s, 1H,
aromatic), 7.56 (d, 2H J = 8.1, Hz, aromatic), 7.52–7.46 (m, 1H, aromatic), 5.30
(d, 1H, J = 4.1 Hz, CH), 3.98–3.75 (m, 2H, OCH2), 0.88 (t, 3H, CH3). 13C NMR
(CDCl3 + DMSO-d6): d 175.5, 174.2, 155.0, 152.4, 149.3, 132.7, 127.0, 124.1,
124.0, 112.6, 94.1, 70.8, 58.4, 46.5, 17.2, 17.0. Mass (ESI-MS): m/z 383
[M+ +H]. Ethyl 4-(6-methyl-4-oxo-4H-chromen-3-yl)-6-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (7q). Yield: 89%; color less solid; mp 220–
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.079.
222 °C. IR (KBr): 3389, 3203, 1662, 1564, 1452, 1199, 1056, 924 cmÀ1 1H NMR
.
(300 MHz, CDCl3 + DMSO-d6): d 9.32 (br s, 1H, NH), 8.48 (s, 1H, aromatic), 8.02
(s, 1H, aromatic), 7.94 (s, 1H, aromatic), 7.56–7.38 (m, 7H, aromatic), 5.66 (d,
1H, J = 3.3 Hz, CH), 3.98–3.86 (q, 2H, OCH2), 2.48 (s, 3H, CH3), 0.90 (t, 3H,
J = 7.2 Hz, CH3). 13C NMR (CDCl3 + DMSO-d6): d 175.9, 174.0, 164.0, 153.8,
153.2, 146.5, 134.6, 133.4, 128.9, 127.6, 127.3, 122.9, 122.0, 117.3, 97.6, 59.4,
48.0, 20.2. Mass (ESI-m/z 421 [M++H]. Ethyl 6-methyl-4-(6-nitro-4-oxo-4H-
chromen-3-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (7u). Yield:
71%; brown solid; mp 296–298 °C. IR (KBr): 3226, 3102, 1706, 1645, 1221,
References and notes
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1092, 965, 812 cmÀ1 1H NMR (300 MHz, CDCl3 + DMSO-d6): d 9.18 (br s, 1H,
.
NH), 8.98 (d, 1H, J = 2.8 Hz, aromatic), 8.54 (dd, 1H, J = 2.6, 9.0 Hz, aromatic),
7.90 (s, 1H, aromatic), 7.74 (d, 1H, J = 9.2 Hz, aromatic), 6.78 (br s, 1H, NH), 5.50
(d,1H, J = 2.8 Hz, CH), 4.14–4.04 (q, 2H, OCH2), 2.36 (s, 3H, CH3), 1.18 (t, 3H,
J = 6.8 Hz, CH3), 13C NMR (CDCl3 + DMSO-d6): d 174.2, 164.1, 157.8, 153.2,

B. China Raju et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2855–2859
2859
151.5, 149.6, 143.3, 126.9, 123.7, 122.7, 120.8, 119.3, 93.8, 58.3, 46.5, 17.1,
16.9; Mass (ESI-MS): m/z 374 [M++H].
plated in 96-well plates at a density of 2.0 Â 104 in 100
l
L of medium per
well of 96-well plate. Cultures were incubated with the test compounds
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metabolic activity of viable cells. Metabolically active cells reduce pale yellow
tetrazolium salt (MTT) to a dark blue water-insoluble formazan, which can be
directly quantified after solubilisation with DMSO. The absorbance of the
formazan directly correlates with the number of viable cells. The cells were
(10
containing
tetrazolium bromide (MTT) for 2–3 h. The supernatant was aspirated and
MTT-formazan crystals were dissolved in 100 L DMSO; OD measured at k
l
M) and incubated for 48 h. The medium was replaced with fresh medium
100 g/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
l
l
540 nm (reference wavelength, k 620 nm) on ELISA reader cell cytotoxicity%
was calculated by comparing the absorbance of treated versus untreated cells.
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