Synthesis and evaluation of chromenyl barbiturates and thiobarbiturates as potential antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2011.05.055

A novel series of barbiturate and thiobarbiturate analogs of 2-benzoyl-3-methyl-5-oxo-5H-furo[3,2-g]chromene-6-carbaldehydes (3a-g and 4a-d, respectively) and 6-methyl-4,8-dioxo-4,8-dihydropyrano[3,2-g]chromenes (7a-c), were synthesized and evaluated for their antitubercular activities against Mycobacterium tuberculosis H37RV, and cytotoxicity (CC₅₀) in the VERO cell MABA assay. The results indicate that the furanochromene series of compounds (3a-g and 4a-d) showed only weak to moderate antitubercular activity. However, the pyranochromene analog 7b showed good antitubercular activity (IC₉₀: 5.9 μg/mL) and cytotoxicity (CC₅₀: 14.27 μg/mL). The antitubercular activity of 7b was superior to the antituberculosis drug, pyrazinamide (PZA; IC₉₀: >20 μg/mL). Analog 7b was considered to be a lead compound for subsequent structural optimization.

Full text of DOI:10.1016/j.bmcl.2011.05.055

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4329–4331
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of chromenyl barbiturates and thiobarbiturates
as potential antitubercular agents
S. Vijaya Laxmi a, Y. Thirupathi Reddy b, B. Suresh Kuarm a, P. Narsimha Reddy b,
Peter A. Crooks b, B. Rajitha a,
⇑
a Department of Chemistry, National Institute of Technology, Warangal 506 004, AP, India
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of barbiturate and thiobarbiturate analogs of 2-benzoyl-3-methyl-5-oxo-5H-furo[3,2-
g]chromene-6-carbaldehydes (3a–g and 4a–d, respectively) and 6-methyl-4,8-dioxo-4,8-dihydropyr-
ano[3,2-g]chromenes (7a–c), were synthesized and evaluated for their antitubercular activities against
Mycobacterium tuberculosis H37RV, and cytotoxicity (CC50) in the VERO cell MABA assay. The results indi-
cate that the furanochromene series of compounds (3a–g and 4a–d) showed only weak to moderate anti-
Received 12 October 2010
Revised 23 April 2011
Accepted 17 May 2011
Available online 25 May 2011
tubercular activity. However, the pyranochromene analog 7b showed good antitubercular activity (IC90
:
Keywords:
Barbituric acid
Thiobarbituric acid
Knoevenagel condensation
Anti tuberculosis activity
5.9 g/mL) and cytotoxicity (CC50: 14.27 g/mL). The antitubercular activity of 7b was superior to the
l
l
antituberculosis drug, pyrazinamide (PZA; IC90: >20
pound for subsequent structural optimization.
l
g/mL). Analog 7b was considered to be a lead com-
Ó 2011 Elsevier Ltd. All rights reserved.
For over many centuries, tuberculosis (TB) remains the leading
respiratory infectious disease in the world.1 The culprit bacterium,
Mycobacterium tuberculosis causes the death of 2–3 million people
and inflicts suffering on almost 8 million people annually. Oxygen
heterocycles such as chromene-2-ones, chromene-4-ones and their
derivatives are well documented in the literature as important
pharmacophores that possess a wide range of biological activities,
such as anti-inflammatory,2 antibacterial3 and anticancer activi-
ties.4,5 Recently, Prado6–8 and Luke9 reported the synthesis and
antimycobacterial activity of some novel furochromenes (Fig. 1,
structures A and B), and in the literature, pyrimidine-2,4,6-triones
are generally reported as biologically active agents.10–12
In view of the marked biological activity of both oxygen hetero-
cycles and pyrimidine-2,4,6-triones, we have focused on the design
of novel structural entities that incorporate substituted furochrom-
ene-5-one and pyrano-chromene-2,6-dione moieties, and barbitu-
ric acid and thiobarbituric acid moieties into a single molecular
scaffold, to evaluate the potential additive effects of these hetero-
cyclic systems on biological activity, especially with regard to anti-
tubercular activity.
Figure 1. Structures of antimycobacterial furochromene derivatives A and B.
ano[3,2-g]chr-omen-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)trion-
es (7a–b) and 5-(6-methyl-4,8-dioxo-4,8-dihydropyrano [3,2-g]chro-
men-3-yl)methylene)-2-thioxodihydropyrimi-dine-4,6(1H,5H)-dione
(7c) were synthesized and evaluated for in vitro antitubercular activity
against M. tuberculosis and for cytotoxicity in the VERO cell assay.
In the synthesis of the furochromene analogs, reaction of substi-
tuted 2-benzoyl-3-methyl-5-acetyl-6-hydroxy benzofurans13 (1)
with DMF/POCl3 under Vilsmeier–Haack conditions afforded the
substituted 2-benzoyl-3-methyl-5-oxo-5H-furo[3,2-g]chromene-
6-carboxaldeh-ydes (2a–g) in 90% yield. Knoevenagel condensa-
tion of each of the resulting products with barbituric acid or
thiobarbituric acid under reflux conditions in ethanol furnished
furochromenes 3a–g, or 4a–d, respectively, in 85–90% yield
(Scheme 1).
A
series of simple and substituted 5-((2-benzoyl-3-
methyl-5-oxo-5H-furo[3,2-g]chromen-6-yl)methylene)pyrimidine-
2,4,6(1H,3H,5H)-triones (3a–g), 5-((2-benzoyl-3-methyl-5-oxo-5H-
furo[3,2-g]chromen-6-yl)methyl-ene)-2-thioxodihydropyrimidine-
4,6(1H,5H)-diones (4a–d), 5-(6-methyl-4,8-dioxo-4,8-dihydropyr-
For the synthesis of the pyranochromenes, 6-acetyl-7-hydroxy-
4-methyl/4,8-dimethylcoumarin (5a, R = H; 5b, R = CH3) was trans-
formed into the corresponding 6-pyrano[3,2-g]chromene-3
⇑
Corresponding author. Tel./fax: +91 870 2459547.
E-mail address: rajitabhargavi@yahoo.com (B. Rajitha).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.055

4330
S. Vijaya Laxmi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4329–4331
Scheme 1. Reagents and conditions; (a) DMF/POCl3, 0–5 °C; (b) barbituric acid, EtOH, reflux, 4–5 h; (c) thiobarbituric acid, EtOH, reflux, 4–5 h.
Table 2
-carbaldehyde analog (6a, R = H; 6b, R = CH3) by employing Vils-
meier–Haack reaction conditions. Knoevenagel condensation of
6a/6b with barbituric acid or thiobarbituric acid, afforded
the 5-(6-methyl-4,8-dioxo-4,8-dihydropyrano[3,2-g]chromen-3-
yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-triones 7a/7b and 5-
(6-methyl-4,8-dioxo-4,8-dihydropyran[3,2-g]chromen-3-yl)methy-
lene)-2-thioxodihydropyrim-idine-4,6(1H,5H)-dione, 7c (Scheme 2).
All the synthesized compounds were characterized by 1H NMR and
13C NMR spectral analysis.14
IC90, CC50, and selectivity index (SI) values for compound 7b
Compd
7b
MABA:H37RV IC90
5.90
(
lg/mL)
CTG:VERO CC50
14.27
(
lg/mL)
SI
2.41
Table 1
Antimycobacterial activity and IC50 and IC90 values against Mycobacterium tubercu-
losis H37RV in the MABA assay for compounds 3a–3g, 4a–4d, and 7a–7c
Analogs 3a–g, 4a–d, and 7a–c were evaluated against M. tuber-
culosis H37RV (ATCC27294) in BACTEC 12B medium using a broth
microdilution assay, the Microplate Alamar Blue Assay (MABA).15
The primary antitubercular screening was performed in accor-
dance with the protocol utilized by the Tuberculosis Antimicrobial
Acquisition and Coordinating Facility (TAACF) at Southern Re-
search Institute. Rifampin was used as control drug in these tests.
Compounds demonstrating a percent inhibition of bacterial growth
P90% in the primary screen were tested against M. tuberculosis
H37RV to determine the actual minimum inhibitory concentration
(MIC) in the MABA. The MIC was defined as the lowest concentra-
tion effecting a reduction in fluorescence of 90%, relative to control.
This value was determined from the dose–response curve as the
IC90 using a curve-fitting program. Compounds with IC90 values
Compound
IC50
(
lg/mL)
IC90 (lg/mL)
3b
3c
3d
3e
3g
4a
4c
65.07
38.52
>100
55.20
>100
42.04
>100
>100
>100
4.71
93.04
55.35
>100
84.69
>100
63.80
>100
>100
>100
5.90
4d
7a
7b
7c
>100
—
>100
>20
PZAa
a
PZA: Pyrazinamide.
<10 lg/mL were considered active antitubercular agents.
Analogs 3d, 3g, 4c, 4d, 7a, and 7c did not exhibit activity in the
MABA assay, while analogs 3b, 3c, 3e, and 4a showed some inhib-
itory activity against M. tuberculosis with MIC values (IC90) of 93.04,
Analog 7b was further evaluated for cell cytotoxicity (CC50) in
the VERO cell cytotoxicity assay (Table 2) Cytotoxicity was deter-
mined from dose–response curves as a CC50 value, using a curve-
fitting program. Concurrent with the determination of MICs, active
compounds were tested for cytotoxicity in VERO cells at a concen-
55.35, 84.69, and 63.80
fied as the most potent inhibitor against M. tuberculosis, with a MIC
value (IC90) of 5.90 g/mL in the MABA assay and its antitubercular
activity was superior to that of the antitubercular drug, pyrazina-
mide (PZA; IC90: >20 g/mL).
lg/mL, respectively. Analog 7b was identi-
l
tration of 10 lM, the MIC for M. tuberculosis H37RV (Table 1). The
selectivity index (SI) is defined as the ratio of the measured CC50 in
VERO cells to the MIC from the MABA assay (Table 2), and is a
determinant of whether an analog is suitable for subsequent
l
Scheme 2. Reagents and conditions: (a) DMF/POCl3, 0–5 °C; (b) barbituric acid or thiobarbituric acid, EtOH, reflux, 4–5 h.

S. Vijaya Laxmi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4329–4331
4331
13. Thirupathi Reddy, Y.; Narasimha Reddy, P.; Kanaka-lingeshwara Rao, M.;
Rajitha, B. Indian J. Chem. 2001, 40, 479.
in vivo evaluation in a mouse functional assay. In this respect, the
MIC and SI values for advancing compounds to in vivo evaluation
14. Analytical data for the representative compounds: (2a): M.F.: C20H12O5; yellow
solid, mp 185–187 °C; Mass (ESI): M+1 333 (100%). 1H NMR (400 MHz, DMSO-
d6): 1.42 (3H, s), 7.25 (1H, s), 7.5–8.07 (5H, m), 8.55 (1H, s), 8.71 (1H, s), 10.4
(1H, s): (3a): M.F.: C24H14N2O7; yellow solid, mp >260 °C; Mass (ESI): M+1 443
(60%); 1H NMR (400 MHz, DMSO-d6): d 2.6 (s, 3H), 7.62–8.03 (m, 5H), 8.22 (s,
1H), 8.48 (s, 1H), 8.67 (s, 1H), 9.81 (s, 1H), 11.40 (s, 1H, NH), 11.49 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6): d 9.6, 101.9, 119.3, 120.0, 120.2, 126.8, 127.9,
128.9, 129.9, 132.1, 136.2, 144.1, 149.4, 150, 150.5, 155.1, 155.9, 162.4, 162.9,
163.5, 174.7, 183.6. IR (KBr): 3410, 3077, 1744, 1711, 1686, 1648, 1617, 1476,
should be 6.25 and 10 (occasionally lower)
In the case of analog 7b, the CC50 value was 14.27
the MIC (IC90) was 5.90 g/mL, affording an SI value of 2.41. Thus,
lg/mL, respectively.
lg/mL, while
l
the pyranochromene 7b was found to be a potent anti-tubercular
agent in the MABA and VERO assays.
Interestingly, the inactivity of the structurally related analogs
7a and 7c indicates that the presence of the C8-methyl group in
7b may be critical for antitubercular activity. Thus, compound 7b
was considered to be a lead analog for subsequent optimization
in the search for novel antitubercular agents.
1346, 1041 cmÀ1
.
Compound (7b): M.F.: C19H12N2O7: brown solid, mp >260 °C; Mass (ESI): M+1
381(80%); 1H NMR (400 MHz, DMSO-d6): 1.4 (3H, s), 2.2 (3H, s), 7.25 (1H, s),
7.49 (1H, s), 8.58 (1H, s), 9.87 (1H, s), 11.43 (1H, s, NH), 11.45 (1H, s, NH). 13C
NMR (100 MHz), DMSO-d6): 12.7, 18.6, 110.3, 111.8, 112.7, 113.4, 114.2, 115.8,
126.1, 151.8, 152.2, 153, 153.7, 159.3, 160.6, 162.7, 174IR (KBr) cmÀ1: 3420,
1746, 1708, 1689, 1679, 1668, 1469, 1044 cmÀ1. (4a): M.F.: C24H13ClN2O6S:
yellow solid, mp >260 °C; Mass ESI M+1 493 (100%); 1H NMR (400 MHz, DMSO-
d6): d 2.64(s, 3H), 7.14–8.0 (m, 4H), 8.23(s, 1H) 8.5 (s, 1H), 8.6 (s, 1H), 9.8 (s,
1H), 12.45 (s, 1H, NH), 12.49 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d 9.58,
101.9, 116.3, 119.3, 119.9, 125.3, 128.1, 128.3, 129.1,129.3, 131.1, 144.9, 150.1,
154.9, 155.8, 160.2, 161.2, 163.4, 174.6, 178.4, 183, 188.2. IR (KBr): 3431, 1711,
Acknowledgments
This work was supported by MHRD (Government of India) and
CSIR Research fund Grant No. (01(2061)/06/EMR-II), New Delhi,
and is gratefully acknowledged. We also thank the Tuberculosis
Antimicrobial Acquisition & Coordinating Facility (TAACF) for pro-
viding the antitubercular activity data.
1686, 1623, 1617, 1475, 1345, 1167, 1045 cmÀ1
.
15. Microbiology Assays: All compounds were evaluated for antimycobacterial
activity against Mycobacterium tuberculosis H37RV. Antitubercular activities of
the compounds were performed by the Center of Tuberculosis Antimicrobial
Acquisition and Coordinating Facility (TAACF) at Southern Research Institute.
Compounds were tested for in vitro antitubercular activity against M.
References and notes
tuberculosis H37RV (ATCC 27294) at 6.25 lg/mL, in BACTEC 12B medium
1. World Health Organization, Global; tuberculosis control: surveillance,
planning, finance. WHO report WHO/HTM/TB/2007.376 Geneva, Switzerland:
WHO; 2007.
using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA).
Compounds exhibiting fluorescence were tested in the BACTEC 460
Radiometric System.16,17 Compounds were tested in 10 twofold dilutions,
2. Lin, C. M.; Huang, S. T.; Lee, F. W.; Sawko, H.; Lin, M. H. Bioorg. Med. Chem. 2006,
14, 4402.
typically from 100
concentration effecting
controls. This value is determined from the dose–response curve using a
g/mL is considered ‘active’ for
antitubercular activity. The Minimum Inhibitory Concentration (MIC) is
defined as the lowest concentration effecting a reduction in fluorescence of
90% relative to controls. Also the cytotoxicity (CC50) values of compounds
l
g/mL to 0.19
lg/mL. The IC90 is defined as the
a
reduction in fluorescence of 90% relative to
3. Sengupta, A. K.; Sen, S.; Srivastava, V. J. Indian Chem. Soc. 1989, 66, 710.
4. Attassi, G.; Briet, P.; Berthelon, J. J.; Collonges, F. Eur. J. Med. Chem. 1985, 20, 393.
5. Birt, D. F.; Hendrich, S.; Wang, W. Pharmacol. Ther. 2001, 90, 157.
6. Prado, S.; Ledeit, H.; Michel, S.; Koch, M.; Darbord, J. C.; Coles, S. T.; Tillequin, F.;
Brodin, P. Bioorg. Med. Chem. 2006, 14, 5423.
curve-fitting program. Any IC90 value of 610
l
7. Prado, S.; Janin, Y. L.; Saint-Joanis, B.; Brodin, P.; Michel, S.; Koch, M.; Cole, S. T.;
Tillequin, F.; Bost, P. E. Bioorg. Med. Chem. 2007, 15, 2177.
8. Prado, S.; Toum, V.; Saint-Joanis, B.; Michel, S.; Koch, M.; Cole, S. T.; Tillequin,
F.; Janin, Y. L. Synthesis 2007, 1566.
9. Luke, A.; Soizic, P.; Valerie, H.; Brigitte, S. J.; Sylvie, M.; Michel, K.; Stewart, T.;
Cole, ; Francois, T.; Yves, L. J. Bioorg. Med. Chem. 2008, 16, 8264.
10. Kwan, P.; Brodie, M. J. Epilepsia 2004, 45, 1141.
11. Halder, M. K.; Scott, M. D.; Sule, N. Bioorg. Med. Chem. 2008, 18, 2373.
12. Tomasic, T.; Zidar, N.; Muller-Premru, M.; Kikelj, D.; Masic, L. P. Eur. J. Med.
Chem. 2010, 45, 1667.
against cultured VERO cells, as well as the selectivity index (SI), defined as IC90
MIC, were determined. After 72-h exposure, viability was assessed using
Promega’s Cell Titer Glo Luminescent Cell Viability Assay, homogenous
/
a
method of determining the number of viable cells in culture based on
quantization of the ATP present. Cytotoxicity was determined from the dose–
response curve as the CC50 using a curve-fitting program. The CC50 value was
divided by the IC90 value to calculate a selectivity index (SI) value.
16. Collins, L. A.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
17. Kelly, B. P.; Furney, S. K.; Jessen, M. T.; Orme, I. M. Antimicrob. Agents Chemother.
1996, 40, 2809.