Synthesis of new thiobarbituric acid derived spiroheterobicyclic compounds and their antimicrobial activity

Article abstract of DOI:10.1002/ddr.21172

Preclinical Research A series pf novel (7S, 11R) - substituted diphenyl-2,4,8,10-tetraazaspiro [5.5] undecane-3-thio-1,5,9-triones 4a-j and (7S,11R)-substituted diphenyl-2,4,8,10-tetraazaspiro[5.5] undecane-3-thio-1,5 dione, 9-thiones 5a-j were prepared using substituted aldehydes and urea/thiourea in the presence of thiobarbituric acid under solvent free, thermal and microwave conditions catalyzed by ecofriendly K-10 clay. The antibacterial potency of these compounds in vitro on the gram negative strains Escherichia coli and Pseudomonas aeruginosa and gram positive strains Staphyllococcus aureus and Staphylococcus epidermidis was evaluated.

Full text of DOI:10.1002/ddr.21172

DRUG DEVELOPMENT RESEARCH •• : ••–•• (2014)
Research Article
Synthesis of New Thiobarbituric Acid Derived
Spiroheterobicyclic Compounds and Their
Antimicrobial Activity
Gurpreet Kaur,1 Parul Gupta,2 Kusum Harjai,2 and Vasundhara Singh1*
1Department of Applied Sciences, PEC University of Technology, Chandigarh 160012, India
2Department of Microbiology, Panjab University, Chandigarh 160014, India
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
A series pf novel (7S, 11R)—substituted diphenyl-2,4,8,10-tetraazaspiro [5.5] undecane-
3-thio-1,5,9-triones 4a-j and (7S,11R)-substituted diphenyl-2,4,8,10-tetraazaspiro[5.5] undecane-3-thio-
1,5 dione, 9-thiones 5a-j were prepared using substituted aldehydes and urea/thiourea in the presence of
thiobarbituric acid under solvent free, thermal and microwave conditions catalyzed by ecofriendly K-10
clay. The antibacterial potency of these compounds in vitro on the gram negative strains Escherichia coli
and Pseudomonas aeruginosa and gram positive strains Staphyllococcus aureus and Staphylococcus
epidermidis was evaluated. Drug Dev Res •• : ••–••, 2014.
© 2014 Wiley Periodicals, Inc.
Key words: thiobarbituric acid; spiroheterobicycles; K-10 clay; solvent-less; antibacterial activity
INTRODUCTION
dermatitis agents [Nakao et al., 2008], pesticides
[Lindell et al., 2001], antioxidants [Sarma et al., 2010]
and enzyme inhibitors [Jong-Ho et al., 2011]. A broad
spectrum of biological activity due to the presence of
thiobarbituric acid and barbituric acid as the bio-
active nucleus in several heterocyclic compounds has
been dsecribed [Behera et al., 2006; Ma et al., 2011;
Vijaya Laxmi et al., 2011; Faidallah and Khan, 2012].
Prompted by these observations, a new series of
spirohetrobicyclic compounds derived via the hybrid-
ization of biologically potent thiobarbituric acid nucleus
was designed and synthesized to generate a new series
Mutations in multidrug resistant pathogens result
in antibiotic resistance, particularly methicillin-resistant
Staphyllococcus aureus (MRSA) and vancomycin-
resistant Staphyllococcus aureus (VRSA) [Han and
Madduri, 2013; Laxminarayan et al., 2013], have led to
concerns that “Infection-related mortality rates in
developed counties might return to those of the early
20th century” [Howard et al., 2013]. New classes of
antibiotics are therefore urgently required [Shute,
2013].
Spiroheterocycles are an important class of het-
erocyclic compounds which are present in many natu-
rally occurring compounds and display complex
biological activities. They include anticonvulsants
[Kaminski et al., 2008], antimicrobials and antibacteri-
als [van der Sar et al., 2006; Thadhaney et al., 2010],
analgesics [Frank et al., 2008], anticancer agents [Chin
et al., 2008], antifungals [Hyeong Beom et al., 2007],
anti-tubercular agents [Chande et al., 2005], anti-
*Correspondence to: Vasundhara Singh, Department of
Applied Sciences, PEC University of Technology, Sector-12,
Chandigarh 160012, India.
E-mail: vasun7@yahoo.co.in
Received 27 January 2014; Accepted 17 February 2014
Published online in Wiley Online Library (wileyonlinelibrary
.com). DOI: 10.1002/ddr.21172
© 2014 Wiley Periodicals, Inc.

2
KAUR ET AL.
of spiroheterocyclic rings for evalaution as antibacterial plates (Silica gel 60 F254, Merck, KGaA, Darmstadt,
agents against both gram positive and gram negative Germany). Spots were visualized via exposur to iodine
bacterial strains.
vapor. Microwave assisted reactions were carried out in
Spiroheterobicyclic compounds have been syn- IFB-30SC1 domestic microwave oven.
thesized via a multicomponent reaction (MCR)
approach, which is a promising tool for the synthesis of
structurally diverse molecules. An important MCR that
has been well exploited is the classical Biginelli [Ladani
et al., 2009] three-component condensation reaction
for generating a wide library of therapeutically useful
molecules [Kappe, 2000]. In particular, the synthesis
of spiroheterobicyclic rings using the Biginelli reaction
in iodine [Prajapati et al., 2011] and solvent free con-
ditions at 80°C and under microwave irradiation
[Shaabani and Bazgir, 2004] have been reported giving
products in moderate yields and purity.
General procedure for synthesis of thiobarbituric
acid derived spiroheterobicyclic compounds 4-5a-j
To a dry magnetically stirred 10 ml round bottom
flask in oil bath was added requisite urea/thiourea
(1.0 mol), aromatic aldehyde (2.2 mol), thiobarbituric
acid (1.0 mol) and K-10 clay (50 mg). The mixture was
stirred with a magnetic bar in a preheated oil bath at
80°C for 2–4 h and subjected to microwave irradiation
at 70 W for 2–4 min. After completion of the reaction
as monitored by TLC (hexane : ethylacetate = 8 : 2),
methanol (5 ml) was added to dissolve the product
and the catalyst was separated by filtration on a filter
paper. The clay on the filter paper was rinsed with
additional methanol (5 × 2 ml). The solvent was recov-
ered under vacuum. To the remaining material,
crushed ice (40–50 g) was added and stirred for an
additional 10 min. The solid product obtained after fil-
tration was washed with cold water and recrystallized in
hexane : ethylacetate = 8 : 2, and finally dried to obtain
the pure compounds 4a-j and 5a-j (Fig. 1).
In the present work, we synthesized several
novel thiobarbituric acid-based symmetrical spirohe-
terobicyclic compounds using the MCR methodology
catalyzed by K-10 clay under solvent free conditions to
provide compounds in higher yields with shorter reac-
tion times. These newly synthesized compounds were
1
characterized by elemental analysis, FT-IR, H NMR,
13C NMR and mass spectrometry. This is the first report
of the biological activity against both gram positive and
gram negative bacterial strains of the synthesized com-
pounds using broth microdilution MIC (minimum
inhibitory concentration) method [NCCLS, 2002,
2007a] with promising results.
S
X
MATERIALS AND METHODS
Compound Synthesis
HN
NH
RCHO
H2N
NH2
X
R
O
O
Starting materials and reagents were purchased
from Sigma-Aldrich Chemical Co. (New Delhi, India)
and were used without further purification. Melting
points were determined using a Veego melting point
apparatus (VEEGO Instruments Corp., Mumbai,
India) and are uncorrected. Infrared (IR) spectra were
recorded on a Perkin-Elmer 882 spectrophotometer
using KBr (νmax in cm−1). 1H NMR and 13C NMR spectra
were recorded using Bruker Avance II 400 (400 MHz)
spectrometer. Chemical shift values are expressed as δ
(ppm) downfield from tetramethylsilane (TMS) used as
internal standard. Mass spectra (MS) were recorded on
a Waters micromass Q-Tof Micro spectrometer and a
Finnigan Mat, LCQ spectrometer. Anhydrous sodium
sulphate was used as the drying agent. Elemental analy-
ses were carried out using a Perkin-Elmer-2400 model
CHN analyzer. All the solvents were distilled prior to
use according to standard procedures. The samples and
reaction mixture were analyzed by thin layer chroma-
1
3
4a
4b
4c
4d
4e
4f
2
C6H5
O
O
O
O
O
O
O
O
O
O
S
p-OCH3-C6H5
m-NO2-C6H5
p-NO2-C6H5
p-Cl-C6H5
p-CH3-C6H5
p-OH-C6H5
m-OH -C6H5
o-OH-C6H5
furyl
I
4g
4h
4i
4j
5a
5b
5c
5d
5e
5f
C6H5
p-OCH3-C6H5
m-NO2-C6H5
p-NO2-C6H5
p-Cl-C6H5
p-CH3-C6H5
p-OH-C6H5
m-OH -C6H5
o-OH-C6H5
furyl
S
S
S
S
S
S
S
5g
5h
5i
S
HN
NH
HN
NH
S
S
5j
S
O
R
HN
O
R
NH
O
R
HN
O
R
NH
S
O
5a-j
4a-j
Fig. 1. Synthetic pathway for thiobarbituric acid derived spirohe-
terobicyclic compounds 4–5a-j. Reaction conditions: I = K-10 clay,
tography on TLC grade gel and on precoated silica 80°C, 4 h.
Drug Dev. Res.

SPIROHETEROBICYCLIC COMPOUNDS AS ANTIMICROBIALS
3
Compounds- Analytical Data
(DMSO-d6,400 MHz): δ 5.49 (s, 2H, -CH), 7.49–7.71
(m, 8H, Ar-H), 8.55 (bs, 2H, -NH), 12.04 (s, 1H, -NH)
and 12.33 ppm (s, 1H, -NH). 13C-NMR (DMSO-
d6,100 MHz): δ 56.8 (C7), 66.3 (C11), 79.1 (C-spiro),
128.1, 129.5, 130.6, 133.6 (Ar-C), 156.2 (C9), 161.7
(C5), 165.1 (C1) and 168.2 ppm (C3); Anal.Calcd
for C19H14O7N6S: C, 48.51; H, 2.98; N, 17.87. Found:
C, 48.50; H, 2.86; N, 17.81. MS: (M + 1) 471.2.
(7S,11R)-diphenyl-2,4,8,10-tetraazaspiro[5.5]
undecane-3-thio-1,5,9-trione (4a)
Yield: 94%; m.p. 192–194°C; Off white powder;
IRνmax(KBr): 3363, 3236 (NH str.), 1732 (ArC-H str.)
and 1691 cm−1 (C = O str.); 1H-NMR (DMSO-
d6,400 MHz): δ 5.25 (s, 2H, -CH), 7.23–7.32 (m, 10H,
Ar-H), 8.63 (s, 2H, -NH), 12.06 (s, 1H, -NH)
and 12.36 ppm (s, 1H, -NH). 13C-NMR (DMSO-
d6,100 MHz): 56.2 (C7), 61.2 (C11), 78.8 (C-spiro),
127.2, 128.8, 129.2, 133.6 (Ar-C), 155.0 (C9), 160.1
(C5), 163.1 (C1) and 167.9 ppm (C3); Anal.Calcd
for C19H16O3N4S: C, 60.0; H, 4.21; N, 14.73. Found:
C, 60.11; H, 4.19; N, 14.65. MS: (M + 1) 381.0.
(7S,11R)-bis(p-chlorophenyl)-2,4,8,10-tetraazaspiro
[5.5]undecane-3-thio-1,5,9-trione (4e)
Yield: 96%; m.p. 200–202°C; Deep yellow
powder; IRνmax(KBr): 3331, 3206 (NH str.), 1736
1
(ArC-H str.) and 1701 cm−1 (C = O str.); H-NMR
(DMSO-d6,400 MHz): δ 5.21 (s, 2H, -CH), 7.15–7.41
(m, 8H, Ar-H), 8.61 (s, 2H, -NH), 11.99 (s, 1H, -NH)
and 12.29 ppm (s, 1H, -NH). 13C-NMR (DMSO-
d6,100 MHz): δ 54.8 (C7), 62.0 (C11), 79.7 (C-spiro),
125.8, 127.4, 128.2, 134.1 (Ar-C),155.2 (C9), 161.0
(C5), 163.5 (C1) and 167.1 ppm (C3); Anal.Calcd
for C19H14O3N4SCl2: C, 50.89; H, 3.12; N, 12.50.
Found:
(7S,11R)-bis(p-methoxyphenyl)-2,4,8,10-
tetraazaspiro[5.5]undecane-3-thio-1,5,9-trione (4b)
Yield: 92%; m.p. 221–224°C; Bright yellow
powder; IRνmax(KBr): 3283, 3152 (NH str.), 1715
(ArC-H str.) and 1677 cm−1 (C = O str.); H-NMR
1
(DMSO-d6,400 MHz): δ 3.89 (s, 6H, -OCH3), 5.31 (s,
2H, -CH), 7.20–7.41 (m, 8H, Ar-H), 8.12 (s, 2H, -NH),
12.20 ppm (s, 1H, -NH), and 12.33 (s, 1H, -NH). 13C-
NMR (DMSO-d6,100 MHz): δ 55.3 (CH3), 56.7 (C7),
62.5 (C11), 78.3 (C-spiro), 126.8, 129.0, 129.8, 134.1
(Ar-C), 154.4 (C9), 158.2 (ArC-OCH3), 161.1 (C5),
163.1 (C1) and 167.9 ppm (C3); Anal.Calcd for
C21H20O5N4S: C, 57.27; H, 4.54; N, 12.72%. Found:
C, 57.25; H, 4.49; N, 12.68. MS: (M + 1) 441.6.
C, 50.78; H, 3.04; N, 12.39. MS: (M + 1) 449.2.
(7S,11R)-bis(p-methylphenyl)-2,4,8,10-tetraazaspiro
[5.5]undecane-3-thio-1,5,9-trione (4f )
Yield: 94%; m.p. 219–221°C; Faint yellow crystal-
line powder; IRνmax(KBr): 3323, 3123 (NH str.), 1730
1
(ArC-H str.) and 1681 cm−1 (C = O str.); H-NMR
(DMSO-d6,400 MHz): δ 2.35 (s, -CH3) 5.21 (s, 2H,
-CH), 7.15–7.41 (m, 8H, Ar-H), 8.62 (s, 2H, -NH),
12.11 (s, 1H, -NH) and 12.30 ppm (s, 1H, -NH). 13C-
NMR (DMSO-d6,100 MHz): δ 50.2(CH3), 56.8 (C7),
62.5 (C11), 78.6 (C-spiro), 126.3, 127.2, 129.4, 134.1
(Ar-C), 155.3 (C9), 161.3 (C5), 165.1 (C1), 168.2 (C3);
Anal.Calcd for C21H20O3N4S: C, 61.31; H, 4.86; N,
13.62. Found: C,61.29; H, 4.82; N,13.59. MS: (M + 1)
412.1.
(7S,11R-)bis(m-nitrophenyl)-2,4,8,10-tetraazaspiro
[5.5] undecane-3-thio-1,5,9trione (4c)
Yield: 90%; m.p. 212–214°C; Crystalline orange
powder; IRνmax(KBr): 3342, 3249 (NH str.), 1711
(ArC-H str.) and 1681 cm−1 (C = O str.); H-NMR
1
(DMSO-d6,400 MHz): δ 5.47 (s, 2H, -CH), 7.41–7.49
(m, 4H, Ar-H), 8.01–8,09 (m, 4H, Ar-H), 8.16 (bs, 2H,
-NH), 12.05 (s, 1H, -NH) and 12.30 ppm (s, 1H, -NH).
13C-NMR (DMSO-d6,100 MHz): δ 55.5 (C7), 62.5
(C11), 79.7 (C-spiro), 128.1, 133.7, 134.2, 135.6 (Ar-C),
154.5 (C9), 160.8 (C5), 164.0 (C1) and 169.1 ppm (C3);
Anal.Calcd for C19H14O7N6S: C, 48.51; H, 2.97;
N,17.87. Found: C, 48.49; H,2.91; N,17.78. MS:
(M + 1) 471.1.
(7S,11R-)bis(p-hydroxyphenyl)-2,4,8,10-
tetraazaspiro[5.5]undecane-3-thio-1,5,9-trione (4g)
Yield: 92%; m.p. 234–236°C; Faint brown
powder; IRνmax(KBr): 3392, 3257 (NH str.), 1742
1
(ArC-H str.) and 1688 cm−1 (C = O str.); H-NMR
(DMSO-d6, 400 MHz): δ 5.01 (s,1H,-OH), 5.33 (s, 2H,
-CH), 7.23–7.47 (m, 8H, Ar-H), 8.45 (s, 2H, -NH),
12.13 (s, 1H, -NH) and 12.34 ppm (s, 1H, -NH). 13C-
NMR (DMSO-d6,100 MHz): δ 56.3 (C7), 61.3 (C11),
78.1 (C-spiro), 115.9, 127.8, 130.3, 134.6 (Ar-C), 157.5
(C9), 160.4 (C5), 164.1 (C1) and 166.9 ppm (C3);
(7S,11R-)bis(p-nitrophenyl)-2,4,8,10-tetraazaspiro
[5.5] undecane-3-thio-1,5,9-trione (4d)
Yield: 97%; m.p. 222–224°C; Bright yellow Anal.Calcd for C19H16O5N4S: C, 55.33; H, 3.88;
powder; IRνmax(KBr): 3365, 3244 (NH str.), 1720 N,13.59. Found: C, 55.28; H, 3.80; N,13.53. MS:
(ArC-H str.) and 1694 cm−1 (C = O str.); H-NMR (M + 1) 413.1.
1
Drug Dev. Res.

4
KAUR ET AL.
(7S,11R-)bis(m-hydroxyphenyl)-2,4,8,10-
Ar-H), 8.63 (s, 2H, -NH), 12.06 (s, 1H, -NH), 12.36 (s,
tetraazaspiro[5.5]undecane-3-thio-1,5,9-trione (4h) 1H, -NH). 13C-NMR (DMSO-d6,100 MHz): δ 56.2
Yield: 92%; m.p. 237–239°C; Off white powder; (C7), 61.2 (C11), 78.8 (C-spiro), 128.1, 128.4129.3133.6
IRνmax(KBr): 3358, 3246 (NH str.), 1739 (ArC-H str.) (Ar-C), 162.9 (C5), 166.9 (C1), 167.9 (C3) and
and 1698 cm−1 (C = O str.); 1H-NMR (DMSO-d6, 172.0 ppm (C9); Anal.Calcd for C19H16O2N4S2: C,
400 MHz): δ 5.01 (s,1H, -OH), 5.33 (s, 2H, -CH), 6.83– 57.57; H, 4.04; N, 14.14. Found: C, 57.51; H, 3.99;
6.99 (m, 4H, Ar-H), 7.23–7.47 (m, 4H, Ar-H), 8.27 (s, N, 14.12. MS: (M + 1) 397.1.
2H, -NH), 12.11 (s, 1H, -NH), 12.34 (s, 1H, -NH).
13C-NMR (DMSO-d6,100 MHz): δ 56.3 (C7), 61.3
(C11), 78.1 (C-spiro), 127.4, 127.8, 130.3, 134.6 (Ar-C), (7S,11R)-bis(p-methoxyphenyl)-2,4,8,10-
155.9 (C9), 160.4 (C5), 164.1 (C1) and 166.9 ppm (C3); tetraazaspiro[5.5]undecane-3-thio-1,5dione,
Anal.Calcd for C19H16O5N4S: C, 67.96; H, 3.88; 9-thione (5b)
N,13.59. Found: C, 67.85; H, 3.86; N,13.55. MS:
(M + 1) 413.1.
Yield: 96%; m.p. 226–228°C; Deep yellow;
IRνmax(KBr): 3389, 3250 (NH str.), 1761 (ArC-H str.)
and 1676 cm−1 (C = O str.); 1H-NMR (DMSO-d6,
400 MHz): δ 3.88 (s, 6H, -OCH3), 5.25 (s, 2H, -CH),
7.21–7.44 (m, 8H, Ar-H), 8.42 (s, 2H, -NH), 12.17 (s,
(7S,11R-)bis(o-hydroxyphenyl)-2,4,8,10-
tetraazaspiro[5.5] undecane-3-thio-1,5,9-trione (4i) 1H, -NH) and 12.33 ppm (s, 1H, -NH). 13C-NMR
Yield: 70%; m.p. 221–224°C; Faint yellow (DMSO-d6,100 MHz): δ 56.2 (C7), 61.2 (C11), 78.8
powder; IRνmax(KBr): 3361, 3233 (NH str.), 1728 (C-spiro), 127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5),
1
(ArC-H str.) and 1684 cm−1 (C = O str.); H-NMR 163.1 (C1), 167.9 (C3) and 178.0 ppm (C9); Anal.Calcd
(DMSO-d6, 400 MHz): δ 5.01 (s, 1H, -OH), 5.33 (s, 2H, for C21H20O4N4S2: C, 55.26; H, 4.38; N,12.28. Found:
-CH), 7.03–7.17 (m, 8H, Ar-H), 8.05 (s, 2H, -NH), C, 55.21; H, 4.29; N,12.22. MS: (M + 1) 457.2.
12.11 (s, 1H, -NH) and 12.36 ppm (s, 1H, -NH). 13C-
NMR (DMSO-d6,100 MHz): δ 56.3 (C7), 61.3 (C11),
78.1 (C-spiro), 127.4, 127.8, 130.3, 134.6 (Ar-C), 155.9 (7S,11R-)bis(m-nitrophenyl)-2,4,8,10-tetraazaspiro
(C9), 160.4 (C5), 164.1 (C1) and 166.9 ppm (C3); [5.5]undecane-3-thio-1,5-dione,9-thione (5c)
Anal.Calcd for C19H16O5N4S: C, 67.96; H, 3.88; N,
Yield: 97%; m.p. 210–212°C; Crystalline yellow
13.59. Found: C,67.87; H, 3.85; N,13.56. MS: (M + 1) powder; IRνmax(KBr): 3363, 3236 (NH str.), 1738
1
413.1.
(ArC-H str.) and 1694 cm−1 (C = O str.); H-NMR
(DMSO-d6,400 MHz): δ 5.25 (s, 2H, -CH), 7.41–7.49
(m, 4H, Ar-H), 8.11–8,19 (m, 4H, Ar-H), 8.17 (bs, 2H,
-NH), 12.13 (s, 1H, -NH) and 12.31 ppm (s, 1H, -NH).
13C-NMR (DMSO-d6,100 MHz): δ 56.2 (C7), 61.2
(7S, 11R-)bis(furyl)-2,4,8,10-tetraazaspiro[5.5]
undecane-3-thio-1,5,9-trione (4j)
Yield: 89%; m.p. 210–212°C; Dark brown crystal- (C11), 78.8 (C-spiro), 127.2, 128.8, 129.2, 133.6 (Ar-C),
line; IRνmax(KBr): 3321, 3195 (NH str.), 1753 (ArC-H 160.1 (C5), 163.1 (C1), 167.9 (C3) and 177.0 ppm (C9);
1
str.) and 1656 cm−1 (C = O str.); H-NMR (DMSO- Anal.Calcd for C19H14O6N6S2: C, 46.91; H, 2.88; N,
d6,400 MHz): δ 1.90–1.95(m, 4H, Ar-H), 3.96–4.07 17.28. Found: C,46.82; H, 2.83; N, 17.17. MS: (M + 1)
(s, 2H, Ar-H), 5.01 (s,1H, -OH), 5.33 (s, 2H, -CH), 8.05 487.2.
(s, 2H, -NH), 12.09 (s, 1H, -NH) and 12.31 ppm (s, 1H,
-NH). 13C-NMR (DMSO-d6,100 MHz): δ 30.2, 35.6,
41.4, 48.2(furyl), 56.2 (C7), 61.2 (C11), 78.8 (C-spiro), (7S,11R-)bis(p-nitrophenyl)-2,4,8,10-tetraazaspiro
127.2, 128.8, 129.2, 133.6 (Ar-C), 155.0 (C9), 160.1 [5.5]undecane-3-thio-1,5,dione,9-thione (5d)
(C5), 163.1 (C1) and 167.9 ppm (C3); Anal.Calcd for
Yield: 97%; m.p. 215–218°C; Deep yellow;
C15H20O5N4S: C, 49.71; H, 5.43; N, 15.21. Found: C, IRνmax(KBr): 3453, 3336 (NH str.), 1742 (ArC-H str.)
49.66; H, 5.41; N, 15.19. MS: (M + 1) 369.2.
and 1682 cm−1 (C = O str.); 1H-NMR (DMSO-
d6,400 MHz): δ 5.25 (s, 2H, -CH), 7.49–7.71 (m, 8H,
Ar-H), 9.24 (bs, 2H, -NH), 12.09 (s, 1H, -NH) and
12.35 ppm (s, 1H, -NH). 13C-NMR (DMSO-
d6,100 MHz): δ 56.2 (C7), 61.2 (C11),78.8 (C-spiro),
(7S, 11R)-diphenyl-2,4,8,10-tetraazaspiro[5.5]
undecane-3-thio-1,5dione,9-thione (5a)
Yield: 94%; m.p. 226–229°C; pale yellow; 127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5), 163.1
IRνmax(KBr): 3358, 3251 (NH str.), 1744 (ArC-H str.) (C1), 167.9 (C3) and 175.0 ppm (C9); Anal.Calcd
and 1687 cm−1 (C = O str.); 1H-NMR (DMSO- for C19H14O6N6S2: C, 46.81; H, 2.88; N,17.28. Found:
d6,400 MHz): δ 5.25 (s, 2H, -CH), 7.23–7.32 (m, 10H, C, 46.75; H, 2.75; N,17.21. MS: (M + 1) 487.3.
Drug Dev. Res.

SPIROHETEROBICYCLIC COMPOUNDS AS ANTIMICROBIALS
5
(7S,11R)-bis(p-chlorophenyl)-2,4,8,10-tetraazaspiro (DMSO-d6,400 MHz): δ 5.05 (s,1H, -OH),5.33 (s, 2H,
[5.5] undecane-3-thio-1,5dione,9-thione (5e)
-CH), 6.83–6.99 (m, 4H, Ar-H), 7.23–7.47 (m, 4H,
Yield: 92%; m.p. 222–224°C; Bright yellow Ar-H), 8.27 (s, 2H, -NH), 12.06 (s, 1H, -NH) and
powder; IRνmax(KBr): 3345, 3173 (NH str.), 1752 12.31 ppm (s, 1H, -NH). 13C-NMR (DMSO-
1
(ArC-H str.) and 1675 cm−1 (C = O str.); H-NMR d6,100 MHz): δ 56.2 (C7), 61.2 (C11), 78.8 (C-spiro),
(DMSO-d6,400 MHz): δ 5.33 (s, 2H, -CH), 7.42 (m, 127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5), 163.1
8H, Ar-H), 8.09 (s, 2H, -NH), 11.98 (s, 1H, -NH) and (C1), 167.9 (C3), 176.0 ppm (C9); Anal.Calcd for
12.29 ppm (s, 1H, -NH). 13C-NMR (DMSO- C19H16O4N4S2: C, 53.27; H, 3.73; N, 13.08. Found:
d6,100 MHz): δ 56.2 (C7), 61.2 (C11), 78.8 (C-spiro), C,53.19; H, 3.69; N,12.95. MS: (M + 1) 429.3.
127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5), 163.1
(C1), 167.9 (C3) and 180.0 ppm (C9); Anal.Calcd for
C19H14O2 N4S2Cl2: C, 49.13; H, 3.01; N,12.06. Found:
C, 49.04; H, 2.92; N, 11.97. MS: (M + 1) 465.3.
(7S,11R-) bis (o-hydroxyphenyl)-2,4,8,10-
tetraazaspiro[5.5] undecane-3-thio-1,5,9-trione (5i)
Yield: 67%; m.p. 218–220°C; Faint brown powder;
IRνmax(KBr): 3391, 3250 (NH str.), 1752 (ArC-H str.)
and 1654 cm−1 (C = O str.); 1H-NMR (DMSO-d6,
400 MHz): δ 5.05 (s,1H, -OH), 5.33 (s, 2H, -CH), 7.11–
7.20 (m, 8H, Ar-H), 8.10 (s, 2H, -NH), 12.11 (s, 1H,
-NH) and 12.36 ppm (s, 1H, -NH).13C-NMR (DMSO-
d6,100 MHz): δ 56.2 (C7), 61.2 (C11), 78.8 (C-spiro),
127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5), 163.1
(C1), 167.9 (C3) and 176.0 ppm (C9); Anal.Calcd for
C19H16O4N4S2: C,53.27; H, 3.73; N, 13.08. Found:
C, 53.20; H, 3.65; N,12.98. MS: (M + 1) 429.3.
(7S, 11R-) bis(p-methylphenyl)-2,4,8,10-
tetraazaspiro [5.5] undecane-3-thio-1,5dione,
9-thione mide (5f )
Yield: 95%; m.p. 231–233°C; Faint yellow
powder; IRνmax(KBr): 3414, 3316 (NH str.), 1740
1
(ArC-H str.) and 1682 cm−1 (C = O str.); H-NMR
(DMSO-d6,400 MHz): δ 2.41 (s, -CH3) 5.22 (s, 2H,
-CH), 7.12–7.40 (m, 8H, Ar-H), 8.21 (s, 2H, -NH),
12.07 (s, 1H, -NH) and 12.36 ppm (s, 1H, -NH). 13C-
NMR (DMSO-d6,100 MHz): δ 56.2 (C7), 61.2 (C11),
78.8 (C-spiro), 127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1
(C5), 163.1 (C1), 167.9 (C3) and 179.0 ppm (C9);
Anal.Calcd for C21H20O2N4S2: C, 59.43; H, 4.71;
N,13.20. Found: C, 59.36; H, 4.66; N, 13.15. MS:
(M + 1) 425.1.
(7S, 11R-) bis(furyl)-2,4,8,10-tetraazaspiro [5.5]
undecane-3-thio-1,5,9-trione (5j)
Yield: 76%; m.p. 261–263°C; Yellow crystalline
powder; IR νmax(KBr): 3340, 3276 (NH str.), 1734
(ArC-H str.) and 1685 cm−1 (C = O str.); H-NMR
1
(DMSO-d6,400 MHz): δ 1.91–2.03(m, 4H, Ar-H), 3.96–
4.11 (s, 2H, Ar-H), 5.04 (s,1H, -OH), 5.30 (s, 2H, -CH),
8.05 (s, 2H, -NH), 12.09 (s, 1H, -NH) and 12.36 ppm (s,
1H, -NH). 13C-NMR (DMSO-d6,100 MHz): δ 30.2,
35.6, 41.4, 48.2 (furyl), 56.2 (C7), 61.2 (C11), 78.8
(C-spiro), 127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5),
163.1 (C1), 167.9 (C3), 179.0 ppm (C9); Anal.Calcd
for C15H20O4N4S2: C,51.13; H, 5.68; N,15.90. Found:
C, 51.19; H, 5.61; N, 15.82. MS: (M + 1) 385.2.
(7S,11R)-bis(p-hydroxyphenyl)-2,4,8,10-
tetraazaspiro [5.5] undecane-3-thio-1,5dione,
9-thione (5g)
Yield: 92%; m.p. 219–221°C; Faint orange
powder; IRνmax(KBr): 3293, 3166 (NH str.), 1742 (ArC-
1
H str.) and 1679 cm−1 (C = O str.); H-NMR (DMSO-
d6,400 MHz): δ 5.05 (s,1H,-OH), 5.30 (s, 2H, -CH),
7.27–7.47 (m, 8H, Ar-H), 8.25 (s, 2H, -NH), 12.05
(s, 1H, -NH) and 12.31 ppm (s, 1H, -NH).13C-NMR
(DMSO-d6,100 MHz): δ 56.2 (C7), 61.2 (C11), 78.8
(C-spiro), 127.2, 128.8, 129.2, 133.6 (Ar-C), 160.1 (C5),
163.1 (C1), 167.9 (C3) and 172.0 ppm (C9); Anal.Calcd
Biological Activity
for C19H16O4N4S2: C,53.27; H, 3.73; N, 13.08. Found: In vitro antibacterial activity
C, 53.21; H, 3.67; N, 12.93. MS: (M + 1) 429.3.
The newly synthesized compounds were evalu-
ated for their in vitro antibacterial activity against
Escherichia coli, Pseudomonas aeruginosa, Staphylo-
coccus aureus and Staphylococcus epidermidis. Tube
dilution method was followed as per NCCLS guidelines
and Minimum inhibitory concentration (MIC) was cal-
(7S,11R)-bis(m-hydroxyphenyl)-2,4,8,10-
tetraazaspiro [5.5] undecane-3-thio-1,5dione,
9-thione (5h)
Yield: 92%; m.p. 200–203°C; Light brown culated. All MIC experiments were performed in trip-
powder; IRνmax(KBr): 3379, 3233 (NH str.), 1752 licate (n = 3) for each test organism. The results are
(ArC-H str.) and 1687 cm−1 (C = O str.); H-NMR expressed as mean standard deviation (n = 3) for each
1
Drug Dev. Res.

6
KAUR ET AL.
sample. Ampicillin was used as a reference antibacte-
rial. DMSO was used as a solvent control which has no
inhibitory activity. Compound dilutions (ranging from
1 μg/ml to 10 mg/ml) were prepared in nutrition media
(Luria broth) using a double dilution protocol. Serial
double dilutions were prepared for primary and sec-
ondary screening. Each compound and drug was
diluted to a 10 mg/ml concentration as the stock. For
primary screening 1, 5, and 10 mg/ml concentrations of
the synthesized compounds were tsed. The compounds
found active at this primary screening stage were
further diluted to obtain 50, 75, 100, 150, 200, 250, 500,
800,and 1000 μg/ml concentrations for secondary
screening. To check the bactericidal activity of these
compounds a mid log phase culture was used for inocu-
lation. A 1% culture was added to the media containing
the different concentrations of the compounds. After
inoculation, incubation was continued at 37°C for 24 h.
Growth inhibition was checked by observing the tubes
for the presence or absence of turbidity due to micro-
bial growth. A set of tubes containing only medium and
solvent control were maintained under identical condi-
tions to substantiate that the solvent had no influence
on the strain growth. Further confirmation of results
was done by streaking on Luria agar plates. Dilution of
the compound showing complete inhibition (no growth)
was taken as the Minimum Inhibitory Concentration
(MIC).
TABLE 1. Synthesized Symmetric Thiobarbituric Acid Derived
Spiroheterobicyclic Compounds
Reaction times
Microwave
Isolated yields
Microwave
Compound Conventional irradiation Conventional irradiation
code
heating (h)
(min)
heating (%)
(%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4.0
3.0
3.0
2.0
4.0
4.0
4.0
4.0
3.0
2.0
3.0
4.0
2.0
2.0
3.0
4.0
4.0
3.0
3.0
2.0
4.0
3.0
3.0
2.0
4.0
4.0
4.0
4.0
3.0
2.0
3.0
4.0
2.0
2.0
3.0
4.0
4.0
3.0
2.0
2.0
94
92
90
97
96
94
92
92
70
89
94
96
97
97
92
95
92
92
67
76
69
51
61
85
69
66
64
65
51
71
61
72
74
71
65
69
68
64
53
55
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
wave irradiation under solventless conditions were
less—probably due to non-uniform mixing of the reac-
tants. The recycling of K-10 clay up to 5 cycles showeds
no appreciable decrease in activity of the catalyst. The
slight loss in activity is assigned to the mechanical loss of
RESULT AND DISCUSSION
Chemistry
The condensation of various aldehydes and urea/ catalyst while filtration.
thiourea in the presence of thiobarbituric acid under
To further explore the scope and limitations of
solvent-free conditions at 80°C catalyzed by eco- this reaction it was extended to variously substituted
friendly, inexpensive and recyclable K-10 clay was used aldehydes. Good to excellent results in terms of yield
to obtain thiobarbituric acid/ spiroheterobicyclic com- and conversion were achieved Table 1. The reaction
pounds 4a-j and 5a-j in excellent yields. As compared to proceeded with high efficiency particularly with o- and
uncatalyzed reactions, the mild Lewis acid K-10 clay p-substituted benzaldehydes. All compounds were
catalyzed reactions gave improved yields with reduced obtained in high purity as confirmed by spectral and
reaction times after optimization of reaction conditions elemental analysis.
by varying catalyst loading, time of reaction by thermal
The 1H NMR (DMSO-d6) spectrum of product 5a
and microwave irradiation. While the advantage of (Fig. 2) exhibited a significant singlet peak at δ 5.25
microwave heating was a reduction in reaction time, corresponding to two CH protons. The phenyl rings
yields were improved using conventional heating prob- showed multiplets in the aromatic region of the spec-
ably due to better mixing of the reactants on the surface trum at δ 7.23–7.32 ppm. A broad singlet at δ 8.63 was
of the catalyst K-10 clay. The yields reported are those observed for the two NH groups. The 13C spectrum of
obtained by the conventional mode of heating to yield all the compounds was in good agreement with the
the desired products. The reaction proceeded effi- structure assigned. For compound 5a the presence of
ciently with 2.2 equiv of aldehyde whereas if 1.0 equiv distinct resonances in 13C NMR (DMSO-d6) spectrum
of aldehyde resulted in the formation of undesired side agreeed with the assigned σ symmetric structure for
products. Also if the reaction times were increased to compound. The signal at δ 78.8 corresponds to the spiro
check if acid was consumed completely, decomposition carbon. The observation of three different signals for
of the initial acid was indicated. The yields using micro- two carbonyl groups at δ 162.9 (C5) and 166.9 (C1) ppm
Drug Dev. Res.

SPIROHETEROBICYCLIC COMPOUNDS AS ANTIMICROBIALS
7
Fig. 2. 1H NMR (DMSO-d6) spectrum of product 5a.
further confirmed the σ symmetric stereoisomer. The
aromatic carbons gave signals around 127.2–133.6 ppm.
The IR spectrum showed characteristic stretching
Biological Activity
All the synthesized compounds were tested for in
bands at 3358, 3251 (NH str.), 1744 (Ar C-H str.) and vitro antibacterial activity on two gram negative strains
1687 cm−1 (C = O str.). These results were further sup- like Escherichia coli and Pseudomonas aeruginosa
ported with the mass spectra which detected the and gram positive strains Staphyllococcus aureus and
expected molecular ion peak as [M + 1] at 397. The Staphylococcus epidermidis. The MIC values in μg/mL
obtained elemental analysis values are in good agree- of the compounds against both of these organisms are
ment with the theoretical data. Similarly, all the com- presented in Table 2.
pounds 4a-j and 5a-j were characterized on the basis of
An assessment of the obtained data reveals that
spectral studies. The stereochemistry has been assigned the compounds displayed moderate antibacterial
as (7S,11R) based on the configuration possible for 1, 3 activity when compared with the reference standard,
substituted six membered cyclic compounds existing ampicillin. Among the complete library 4a-j and 5a-j,
in chair form and was further confirmed with that the compounds 4c (R- m-NO2, X- O) and 5b (R-
reported in literature [Prajapati et al., 2011].
p-OCH3, X-S) had good potency for gram positive strain
Drug Dev. Res.

8
KAUR ET AL.
TABLE 2. Antimicrobial Activity of Thiobarbituric Acid Derived Spiroheterobicyclic Compounds (n = 3) MIC Value in μg/mL)
Microorganisms
Gram negative
Pseudomonas aeruginosa
Gram positive
Staphylococcus epidermidis
Compound no.
Escherichia coli
Staphyllococcus aureus
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
5e
5f
1000 0.79
75 0.64
100 0.55
150 0.91
500 0.81
75 0.65
500 0.71
50 0.37
500 0.93
500 0.70
1000 0.88
75 0.56
250 0.83
250 0.92
500 0.64
75 0.74
800 0.56
50 0.49
100 0.94
500 0.78
25 0.21
−
1000 1.11
75 0.47
800 0.14
500 0.87
500 0.69
75 0.60
1000 0.34
50 0.70
500 0.34
500 0.56
800 0.78
75 0.69
500 0.43
250 0.78
500 0.58
75 0.98
1000 0.67
50 0.57
150 0.90
150 0.68
25 0.17
−
800 0.76
250 0.56
500 0.12
500 0.74
800 0.67
250 0.84
1000 0.56
200 0.87
800 0.56
800 0.76
1000 0.38
250 0.55
500 0.67
800 0.45
800 0.98
250 0.56
800 0.87
150 0.99
500 0.45
500 0.76
125 0.14
−
800 0.75
250 0.87
500 0.39
500 0.78
800 0.90
200 0.57
800 1.16
200 0.45
250 0.78
250 0.56
800 1.03
250 0.73
500 0.99
250 0.86
100 0.75
500 0.79
800 0.55
150 0.61
500 0.73
500 0.76
75 0.23
−
5g
5h
5i
5j
Ampicillin
DMSO(control)
Staphylococcus epidermidis, while for the same class of phenyl ring had enhanced inhibition of growth. Hence,
gram positive bacteria Staphyllococcus aureus 4e (R- it can be concluded that substituents at position 4 on
p-Cl, X-O), 5b (R- p-OCH3, X-S), 5d (R- p-NO2, X-S) the diphenyl ring of triones and thiones have an effect
and 5f (R- p-CH3, X- S) showed good potency with on antibacterial potency.
respect to ampicillin (75–125 μg/mL). For the gram
In conclusion, a new series of thiobarbituric acid
negative organisms, 4b (R- p-OCH3, X-O), 4f (R- symmetric spirohetrobicyclic compounds 4a-j and 5a-j
p-CH3, X-O), 5b (R- p-OCH3, X-S), 5c (R- m-NO2, X- have been synthesized using a MCR approach under
S), 5d (R- p-NO2, X-S) showed good potency as ecofreindly reaction conditions and characterized via
antibactericidal agents for Pseudomonsa aeruginosa, elemental and spectral analysis. This synthetic strategy
while 4c (R- m- NO2, X-O), 4d (R- p-NO2, X-O), and 5d led to a library of molecules based on a thiobarbituric
(R- p-NO2, X- S) have a good potency for Escherichia acid nucleus among which some derivatives have good
coli in comparision with Ampicillin (25 μg/mL).
potency against microorganisms as compared to a ref-
The study of the structure-activity relationship erence drug. The antibacterial activity is increased in
(SAR studies) of the potencies indicates that for E. coli analogs with substituents at position 4 on the diphenyl
the trione analog having m-nitro, p-nitro groups and ring in both triones and thiones. The present study
thione analogwith p-nitro groups substituted on phenyl suggests that this new class of compounds as antibacte-
ring gave comparable results for inhibiting growth of rial agents can be pursued for further structural modi-
the organism. In the case of Pseudomonas aeruginosa fications.
the trione analog having p-methoxy, p-methyl and
thione analog with p-methoxy, m-nitro, p-nitro group
substituted on phenyl ring resulted in moderate inhibi-
ACKNOWLEDGMENTS
tion of growth. For Staphyllococcus aureus the trione
analog with p-chloro and the thione analog with
The authors are thankful to PEC University of
p-methoxy, p-nitro and p-methyl had inhibition. For Technology, Chandigarh, for financial support and
Staphylococcus epidermidis both the trione and thione Panjab University, Chandigarh, for providing the nec-
analog having an m-nitro group substituted on the essary facilities to carry out this work.
Drug Dev. Res.

SPIROHETEROBICYCLIC COMPOUNDS AS ANTIMICROBIALS
9
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