Synthesis and study of antibacterial and antifungal activities of novel 8-methyl-7,9-diaryl-1,2,4,8-tetraazaspiro[4.5]decan-3-thiones

Article abstract of DOI:10.1016/j.ejmech.2005.02.001

Some novel spiropiperidinyl-1,2,4-triazolidin-3-thiones have been synthesized and studied for their antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa and antifungal activity against Candida albicans, Candida-6, Candida-51, Aspergillus niger and Aspergillus flavus. Compounds 30-32 exhibited potent in vitro antibacterial activity against E. coli and P. aeruginosa whereas the same set of compounds exerted potent in vitro antifungal activity against Candida-6, A. niger and A. flavus.

Full text of DOI:10.1016/j.ejmech.2005.02.001

European Journal of Medicinal Chemistry 40 (2005) 694–700
www.elsevier.com/locate/ejmech
Synthesis and study of antibacterial and antifungal activities of novel
8-methyl-7,9-diaryl-1,2,4,8-tetraazaspiro[4.5]decan-3-thiones
S. Balasubramanian, C. Ramalingan, G. Aridoss, S. Kabilan *
Department of Chemistry, Annamalai University, Annamalainagar, Tamil Nadu 608 002, India
Received 1 February 2005; accepted 10 February 2005
Available online 09 March 2005
Abstract
Some novel spiropiperidinyl-1,2,4-triazolidin-3-thiones have been synthesized and studied for their antibacterial activity against Staphy-
lococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa and antifungal activity against Candida albicans, Candida-6,
Candida-51, Aspergillus niger and Aspergillus flavus. Compounds 30–32 exhibited potent in vitro antibacterial activity against E. coli and
P. aeruginosa whereas the same set of compounds exerted potent in vitro antifungal activity against Candida-6, A. niger and A. flavus.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Piperidin-4-one; 1,2,4-Triazolidin-3-thiones; Hydrogen peroxide; Antibacterial activity; Antifungal activity
1. Introduction
lanosterol 14a-demethylase (cytochrome P45014DM) and
orally active have been widely used in antifungal chemo-
therapy. Reports are available on the developments of resis-
tance to currently available antifungal azoles in Candida spp.,
as well as clinical failures in the treatment of fungal infec-
tions [4–7]. Furthermore, most of the present antifungal drugs
are not effective against invasive Aspergillosis and the only
drug of choice in such patients is the injectable amphotericin
B. Some examples of 1,2,4-triaole based antibacterial and anti-
fungal drugs are estazolam [8,9], alprazolam [10] and riza-
triptane [11].
These observations places new emphasis on the need of as
well as search for alternative new and more effective antimi-
crobial agents with a broad spectrum.
Recently, we exploited the synthesis of 2,6-diarylpiperidin-
4-one derivatives [12–20] with a view to incorporate various
other bioactive heterocyclic nucleus such as triazolidin-3-
thione, benzimidazole, benzoxazole, D2-1,3,4-thiadiazoline
intact for evaluation of associated antibacterial and antifun-
gal activities and also as a reagent for effecting functional
group interconversion.
Over the past decades, the incidence of systemic micro-
bial infections has been increasing dramatically due to an
increase in the number of immuno-compromised hosts [1].
The increasing incidence of bacterial resistance to a large
number of antibacterial agents such as glycopeptides (vanco-
mycin, inhibition cell walls synthesis), sulfonamide drugs
(inhibitors of tetrahydrofolate synthesis), b-lactam antibiot-
ics (penicillins and cephalosporins), nitroimidazoles and qui-
nolones (DNA inhibitors), tetracyclins, chloramphenicol and
macrolides (erythromycin, inhibiting protein synthesis) is
becoming a major concern [2]. For the past several years,
vancomycin has been considered the last line of defense agent
against Gram-positive infections and no alternative drugs for
treating diseases that have become resistant to vancomycin
[3].
Patients undergoing organ transplants, anticancer chemo-
therapy or long treatment with antimicrobial agents and
patients with AIDS are immuno suppressed and very suscep-
tible to life threatening systemic fungal infections like Can-
didiasis, Cryptococcosis andAspergillosis.Antifungal azoles,
fluconazole and itraconazole which are strong inhibitors of
Synthesis of molecules, which are novel still resembling
known biologically active molecules by virtue of the pres-
ence of some critical structural features, is an essential com-
ponent of the search for new leads in drug designing pro-
gramme. Certain small heterocyclic molecules act as highly
functionalized scaffolds and are known pharmacophores of a
* Corresponding author.
E-mail address: skabilan@rediffmail.com (S. Kabilan).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.02.001

S. Balasubramanian et al. / European Journal of Medicinal Chemistry 40 (2005) 694–700
695
Scheme 1
.
Table 1
number of biologically active and medicinally useful mol-
ecules. In the interest of above, we planned to synthesize a
system, which combines both bioactive piperidine and
triazolidin-3-thione components together to give a compact
structure like title compounds.
Analytical data of compounds 25–32a
Entry
25
26
27
28
R1
R
Yield (%)
M.p. (°C)
116
H
H
84
76
80
72
82
73
80
70
CH3
H
H
109-110
127-128
114-115
148
CH3
CH3
Cl
CH3
H
29
2. Chemistry
30
31
32
CH3
H
Cl
129-130
104
OCH3
OCH3
CH3
91
Triazoles and their derivatives can be conveniently synthe-
sized from aldehyde/ketone thiosemicarbazones and also from
substituted thiosemicarbazides by cyclization using suitable
reagents [21].
a The micro analysis values for C, H and N were within 4% of the theo-
retical values.
Earlier MnO2 [22], FeCl3·6H2O [23], H2O2 [24] and
m-CPBA [25] were used to effect cyclization of steroidal and
non-steroidal ketone thiosemicarbazones into corresponding
1,2,4-triazolidin-3-thiones. In our earlier communication [12],
we found and reported that the use of MnO2, FeCl3·6H2O to
effect cyclization of 2,6-diaryl piperidin-4-one thiosemicar-
bazones results in mixture of 1,2,4-triazolidin-3-thiones and
-3-ones. Use of m-CPBA was not also so successful as that
resulted in mixture of 1,2,4-triazolidin-3-thione and
N-hydroxy derivative along with other non-separable impu-
rities. But cyclization using hydrogen peroxide at 0 °C results
in exclusive formation of 1,2,4-triazolidin-3-thione in good
yields.
The schematic representation and analytical data for the
synthesized compounds 25–32 are furnished in Scheme 1 and
Table 1, respectively. Condensation of respective ketone, alde-
hyde and ammonium acetate in the ratio of 1:2:1, respec-
tively, afforded the formation of piperidin-4-ones 1–8.
N-alkylation of piperidin-4-ones 1–8 using methyl iodide in
the presence of anhydrous potassium carbonate as a base
resulted in 1-methylpiperidin-4-ones. Use of methyl iodide
over dimethyl sulfate for N-alkylation is found to be superior
in terms of yield, handling and easy workup. 1-Methyl-
piperidones are converted into their thiosemicarbazones and
are eventually cyclized into spiro-1,2,4-triazolidin-3-thiones
using H2O2 at 0 °C.
To comprehend structure activity relationship well, num-
berings of the target compound are done (Fig. 1).
Conversion of thiosemicarbazones into spiro-1,2,4-
triazolidin-3-thiones involves a free radical mechanism
(Scheme 2) and is consistent with a one already reported for
similar cyclization [25]. The mode of cyclization takes up
stereo chemical preferences.1
1 There are three stereogenic centers in the case of compounds 25, 27, 29
and 31. So normally one can expect eight stereoisomers. There are four ste-
reogenic centers in the case of compounds 26, 28, 30 and 32. Hence expec-
tation of 16 stereoisomers is quite normal. But it had been well proved that
mostly and favorably 2,6-diarylpiperidin-4-one ring adopts chair conforma-
tion with all its substituents at equatorial disposition [26]. Moreover equa-
torial disposition of phenyl group at C2 and C6 makes the chair conforma-
tion more rigid thereby preventing interconvertion of one chair from into
another. During the mode of cyclization, trigonal (sp2 carbon) carbon is
converted into tetrahedral carbon (sp3 carbon). Hence in the case of com-
pounds 25–32, only two diastereoisomers are now possible having NH.CS...
group either at axial or equatorial orientation. Here only one diastereoiso-
mer, in which NH.CS... group is cis with respect to 6aH, was selectively
obtained as evidenced by spectral data, which could be explained on the
basis that during cyclization, the heterocyclic ring was preferably closed in
such a way that NH.CS... group became equatorially attached to the C5 to
avoid steric effects due to C6–CH3 group and high-energy situation.

696
S. Balasubramanian et al. / European Journal of Medicinal Chemistry 40 (2005) 694–700
In general all the synthesized novel spiro-triazolidin-3-
thiones 25–32 exerted a wide range of modest antibacterial
activity in vitro against the tested organisms.
Compounds 25 and 26 without any substituent at para posi-
tion of the aryl moieties at C7 and C9 positions of the six
membered heterocyclic ring exhibited antibacterial activity
in vitro at 100 µg ml–1 against all the tested organisms except
E. coli. They inhibit E. coli at a MIC of 50 µg ml–1. It is
obvious from the Table 2 that substitution of methyl group
for hydrogen at C6 position in 25 (compound 26) did not
change the activity.
Fig. 1
.
Introduction of methyl groups at the para position of the
aryl moieties at C7 and C9 in 25 (compound 27) produced
twice the activity against P. aeruginosa whereas introduction
of another methyl group at C6 position in addition to the above
methyl group in 25 (compound 28) exhibited double the activ-
ity compared to 27 against B. subtilis alone. From this it is
clear that methyl group modification at both the para position
of the aryl moieties at C7, C9 and at C6 position of heterocy-
clic ring did not result in enhancing the activity against rest
of the bacterial strains S. aureus and E. coli.
Replacement of methyl group present at the para position
of the aryl moieties at C7 and C9 of 27 (compound 29) by
chloro functionalities yielded good improvement in activity
against all the tested organisms except E. coli. Substitution
of methyl group at C6 in 29 (compound 30) for hydrogen
exhibited remarkable antibacterial activity against all the
tested organisms.
Scheme 2
.
Instead of chloro functionalities, substitution of methoxy
groups in 29 (compound 31) results twofold increase in activ-
ity against B. subtilis and E. coli except P. aeruginosa whereas
against S. aureus, the antibacterial activity was reduced by
about 50%. Introduction of methyl group at C6 position of 31
(compound 32) exerted maximum activity at a MIC of
25 µg ml–1 against all the tested bacterial strains.
3. Results and discussion
Structure activity relationship results.
3.1. Antibacterial activity
All the synthesized novel spiro-piperidinyl heterocycles
25–32 were tested for their antibacterial activity in vitro
against Staphylococcus aureus (NCLM-2492), Bacillus sub-
tilis (NCLM-2439), Escherichia coli (NCLM-2345) and
Pseudomonas aeruginosa (NCLM-2035). Penicillin and
Streptomycin were used as standard drugs whose minimum
inhibitory concentration (MIC) values were provided in
Table 2.
3.2. Antifungal activity
The in vitro antifungal activity of the synthesized novel
spiro heterocyclic compounds 25–32 was studied against the
fungal strains viz., Candida albicans (NCLM-C27),
Candida-6 (NCLM-C27), Candida-51 (NCLM-C27), As-
pergillus niger (NCLM-590) and Aspergillus flavus (NCLM-
539). Amphotericin B was used as a standard drug whose
MIC values are provided in Table 3.
Table 2
In vitro antibacterial activity of compounds 25–32
Entry
MIC in µg ml–1
Generally all the synthesized compounds exerted a wide
range of modest in vitro antifungal activity against all the
tested organisms except 25, which failed to show activity
against Candida-6, Candida-51 and A. flavus and 26 against
Candida-51 even at a high concentration of 200 µg ml–1.
The compound 25 without any substituent at the para posi-
tion of the aryl groups present at C2 and C6 positions of the
six membered heterocyclic moiety did not show in vitro anti-
fungal activity even at a maximum concentration of
200 µg ml–1 against Candida-6, Candida-51 and A. flavus
while against C. albicans and A. niger registered activity at a
S. aureus B. subtilis E. coli
P. aeruginosa
25
26
27
28
29
30
31
32
100
100
100
100
25
100
100
100
50
50
50
50
50
50
25
25
25
50
12.5
100
100
50
100
25
50
25
25
25
50
25
25
25
25
25
Penicillin
Streptomycin
12.5
50
25
50
12.5
25

S. Balasubramanian et al. / European Journal of Medicinal Chemistry 40 (2005) 694–700
697
Table 3
In vitro antifungal activity of compounds 25–32
Entry
MIC in µg ml–1
Candida-51
C. albicans
Candida-6
A. niger
50
A. flavus
–
25
100
100
200
200
25
–
–
26
200
100
100
50
–
50
200
200
200
100
50
27
200
200
100
50
50
25
25
100
50
28
29
25
30
50
25
25
31
25
25
25
25
32
25
25
25
50
Amphotericin B
25
25
50
50
‘–’ No inhibition at 200 µg ml–1
.
MIC of 100 and 50 µg ml–1, respectively. Introduction of
methyl group at C6 in 25 (compound 26) did not enhance
activity against all the tested fungal strains except Candida-6
and A. flavus for which the inhibition is observed only at
200 µg ml–1. This compound also failed to show antifungal
activity against Candida-51 even at 200 µg ml–1.
4. Conclusion
A close examination of the in vitro antibacterial and anti-
fungal activity profile in differently substituted novel spiro-
triazolidinethiones against the tested bacterial strains viz. S.
aureus, B. subtilis, E. coli and P. aeruginosa and the fungal
strains viz. C. albicans, Candida-6, Candida-51, A. niger
and A. flavus, respectively, provides a better structure activ-
ity relationship correlate, which may be summarized as fol-
lows.
The compounds with chloro (or) methoxy functions at the
para position of the aryl moieties present at C7 and C9 posi-
tions of the six membered heterocyclic ring along with/
without methyl group at C6 play an important role in eliciting
its antibacterial and antifungal potency.
Furthermore, among the novel spiroheterocycles tested, the
compounds with methoxy group at the para position of the
aryl moieties at C7 and C9 positions of heterocyclic moiety
was found to be superior in inhibiting all the bacterial strains
than chloro modification at the same position. But the intro-
duction of methyl group at C6 position in the above said com-
pounds exerted an improved inhibition against all the tested
bacterial strains particularly against E. coli.
The novel spiropiperidinyl heterocycles with chloro func-
tions at the para position of the aryl moieties along with methyl
group modification at C6 position and the compounds with
methoxy functions in place of chloro functionalities along
with/without methyl modification at C6 position exhibited well
pronounced inhibition against A. niger and Candida-6 fun-
gal strains.
By the introduction of methyl groups at the para position
of the aryl moieties at C7 and C9 of 25 (compound 27), anti-
fungal activity was suppressed to half against C. albicans
and A. niger whereas the activity was improved against
Candida-6 and Candida-51. Against A. flavus compound 27
recorded no change in activity when compared to 26. Due to
the substitution of methyl group at C6 in 27 (compound 28)
the MIC’s for C. albicans, Candida-6, Candida-51 and A.
flavus were not changed when compared to compound 27
whereas against A. niger, inhibition was observed at
50 µg ml–1.
Due to the replacement of methyl groups by chloro func-
tionalities at aryl moieties in compound 27 (compound 28), a
marked improvement in activity was noticed when compared
to 27. This antifungal activity of 29 was significantly pro-
moted against C. albicans. Introduction of methyl group at
C6 position of compound 29 (compound 30) caused a further
enhancement in activity against Candida-6, Candida-51 and
A. flavus whereas against A. niger, there was no change in
activity. The antifungal activity of 30 was reduced by 50%
due to the introduction of methyl group at C6 position of the
heterocyclic moiety in 29.
Substitution of methoxy groups in place of chloro func-
tions in 29 and 30 led to compounds 31 and 32. These two
compounds exerted good antifungal activity in vitro at a MIC
of 25−50 µg ml–1 against all the tested organisms. The com-
pound 31 showed improved activity against C. albicans and
A. flavus whereas against remaining fungal strain no change
in activity was observed compared to that of compound 30.
On comparison with 31, enhanced activity was observed
by methyl group modification at C6 position in 31 (com-
pound 32) against Candida-51 while against rest of the organ-
isms no change in activity was resulted with the exception of
A. flavus, which recorded diminished antifungal activity.
From this study it is evident that compounds 30–32 are
showing promising activity when compared to penicillin
towards inhibiting E. coli and P. aeruginosa bacterial strains.
Similarly, the same set of compounds is also comparatively
better than amphotericin B against A. niger in terms of activ-
ity.
Results of this study show that the nature of substituent on
the phenyl ring is determinant, besides a methyl a group at
C6, for the nature and extent of the activity of the synthesized
compounds, which might have influences on their inhibiting
mechanism of actions. The effect of the C6 methyl group on
SAR may be due to its effect on stereo chemical preferences

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S. Balasubramanian et al. / European Journal of Medicinal Chemistry 40 (2005) 694–700
1
on the piperidine ring. The method of action of these com-
pounds is unknown. Theses observations may promote a fur-
ther development of our research in this field. Further devel-
opment of this group of compounds may lead to compounds
with better pharmacological profile than standard drugs and
serve as templates for the construction of better drugs to com-
bat bacterial and fungal infection.
rocal centimeter) alone are listed. H-NMR spectra were
recorded at 400 MHz on Bruker amx 400 MHz spectropho-
tometer using CDCl3 as solvent and TMS as internal stan-
dard. 13C-NMR spectra were recorded at 125 MHz on Brucker
amx 400 MHz spectrophotometer using CDCl3. The mass
spectra were recorded on a VG analytical 7070E instrument
equipped with VG 11-250 data acquisition system. Satisfac-
tory micro analysis were obtained on Carlo Erba 1106 and
Perkin Elmer models 240 CHN analyzer.
By adopting the literature precedent [27], 2,6-diaryl-
piperidin-4-ones 1-8 were prepared by the condensation of
appropriate ketones, aldehydes and ammonium acetate in
1:2:1 ratio.
5. Experimental
5.1. Microbiology
5.1.1. Materials
5.2.1. General method of preparation of 1-methyl-2,6-
diarylpiperidin-4-one thiosemicabazones (17–24)
A mixture of 1-methyl-2,6-diarylpiperidin-4-one (0.01 mol)
and thiosemicarbazide (0.01 mol) in methanol (45 ml) was
refluxed for 3 h in acidic medium and was concentrated to
one third of its original volume. After cooling, the mixture
was poured over crushed ice. The solid product thus obtained
was filtered off and recrystallized twice from methanol to give
1-methyl-2,6-diarylpiperidin-4-one thiosemicarbazone as
crystalline solid.
The bacterial strains E. coli (NCLM-2345), P. aeruginosa
(NCLM-2035), S. aureus (NCLM-2492), B. subtilis (NCLM-
2439) and antifungal strains C. albicans (NCLM-C27),
Candida-6 (NCLM-C27), Candida-51 (NCLM-C27), A.
niger (NCLM-590), A. flavus (NCLM-539) are procured from
National Chemical Laboratory, Pune, India.
5.1.2. In vitro antibacterial and antifungal activity
The in vitro activities of the compounds were tested in
sabourauds dextrose broth (SDB) (Hi-media, Mumbai) for
fungi and nutrient broth (NB) (Hi-media, Mumbai) for bac-
teria by the twofold serial dilution method [28]. The test com-
pounds were dissolved in dimethylsulfoxide (DMSO) to
obtain 1 mg ml–1 stock solutions. Seeded broth (broth con-
taining microbial spores) was prepared in NB from 24 h old
bacterial cultures on nutrient agar (Hi-media, Mumbai) at
37 1 °C while fungal spores from 1 to 7 days old sab-
ourauds agar (Hi-media, Mumbai) slant cultures were sus-
pended in SDB. The colony forming units (cfu) of the seeded
broth were determined by plating technique and adjusted in
the range of 102–105 cfu ml–1. The final inoculum size was
105 cfu ml–1 for antibacterial assay and 1.1–1.5 × 102 cfu ml–1
for antifungal assay. Testing was performed at pH 7.4 0.2.
0.2 ml of the solution of test compound was added to 1.8 ml
of seeded broth to form the first dilution. One milliliter of
this was diluted with a further 1 ml of the seeded broth to
give the second dilution and so on till six such dilution were
obtained.A set of assay tubes containing only inoculated broth
was kept as control and likewise solvent controls were also
sun simultaneously. The tubes were incubated in BOD incu-
bators at 37 1 °C for bacteria and 28 1 °C for fungi. The
MICs were recorded by visual observations after 24 h (for
bacteria) and 72–96 h (for fungi) of incubation. Penicillin,
streptomycin and amphotericin B were used as standards.
5.2.2. 8-Methyl-7,9-diphenyl-1,2,4,8-tetra-
azaspiro[4.5]decan-3-thiones (25)
A solution of 1-methyl-2,6-diphenylpiperidin-4-one thi-
osemicarbazone 17 (0.005 mol) in chloroform (40 ml) was
treated with excess of hydrogen peroxide (30%, 20 ml) and
stirred for 3 h at 0 °C. After the completion of reaction, the
organic layer was separated and dried over anhydrous sodium
sulfate. The solvents were removed under reduced pressure.
The crude product thus obtained was purified over silica gel
column (pet ether/ethyl acetate, 5:2) and the product 25 was
obtained as needles when it was recrystallized from metha-
nol.
IR (KBr) (cm–1): 3338, 3316 (N–H), 2361, 1609, 1490,
1452, 1346, 1258 (C=S), 1210, 1067, 1026, 938, 905, 856,
809, 752, 695, 664, 602, 593, 555, 525; Mass (m/z): 338(M+),
323, 294, 278, 263, 209, 194, 179, 159, 145, 133, 120, 105
(100%), 91, 77, 65, 51; 1H-NMR (d ppm): 3.60(dd,
3J = 12.11 Hz; 3.23 Hz, 2H, H7a, H9a); 2.20–2.40 (m, 4H,
H6a, H10a, H6e, H10e); 7.28–7.50 (m, 10H, aryl protons); 8.45
(b.s, 2H, NHCSNH); 7.13 (s, 1H, NH at 1); 1.63 (s, 3H,
NCH3); 13C-NMR (d ppm): 66.034 (C7, C9); 39.349 (C6, C10);
77.789(C5); 180.984 (C=S); 41.010 (NCH3); 124.934,
126.714, 127.263, 147.824 (aryl carbons).
The compounds 26–32 were synthesized similarly.
5.2. Chemistry
5.2.2.1. 6,8-Dimethyl-7,9-diphenyl-1,2,4,8-tetraaza-
spiro[4.5]decan-3-thione (26). IR (KBr) (cm–1): 3424, 3341,
3310 (N–H), 2358, 1567, 1490, 1452, 1376, 1350, 1265
(C=S), 1073, 842, 790, 751, 696, 665, 632, 524; Mass (m/z):
352(M+), 337, 308, 292, 277, 223, 207, 194, 179, 159, 146,
132, 118 (100%), 104, 91, 77, 65, 55, 51; 1H-NMR (d ppm):
The reactions and the purity of the products were assessed
by performing TLC. All the reported melting points were
taken in open capillaries and were uncorrected. IR spectra
were recorded in KBr (pellet forms) on a Perkin–Elmer 297 IR
spectrophotometer and note worthy absorption values (recip-

S. Balasubramanian et al. / European Journal of Medicinal Chemistry 40 (2005) 694–700
699
3.61(dd, 3J = 12.35 Hz; 3.01 Hz, 1H, H9a); 3.19(d,
3J = 11.10 Hz, 1H, H7a); 2.22–2.47 (m, 3H, H6a, H10a, H10e);
7.17–7.46 (m, 10H, aryl protons); 8.40 (b.s, 2H, NHCSNH);
6.90 (s, 1H, NH at 1); 1.59 (s, 3H, NCH3); 0.81 (d,
J = 6.53 Hz, 3H, CH3 at 6); 13C-NMR (d ppm): 72.212 (C7);
65.888 (C9); 39.561 (C6); 37.897 (C10); 78.398(C5); 181.683
(C=S); 42.621 (NCH3); 124.620, 126.131, 126.471, 127.290,
127.572, 139.976, 146.436 (aryl carbons); 12.940 (CH3 at
6).
1242 (C=S), 1090, 982, 882, 801, 710, 693, 620; Mass (m/z):
420(M+), 405, 376, 360, 291, 277, 247, 193, 180, 166, 140,
138 (100%), 111, 75, 65, 55, 50; 1H-NMR (d ppm): 3.61(dd,
3J = 12.35 Hz; 3.01 Hz, 1H, H9a); 3.21(d, 3J = 11.14 Hz, 1H,
H7a); 2.22–2.50 (m, 3H, H6a, H10a, H10e); 7.33–7.45 (m, 8H,
aryl protons); 8.45 (b.s, 2H, NHCSNH); 6.86 (s, 1H, NH at
1); 1.60 (s, 3H, NCH3); 0.81(d, J = 6.56 Hz, 3H, CH3 at 6);
13C-NMR (d ppm): 71.317 (C7); 65.892 (C9); 39.433 (C6);
37.852 (C10); 78.461(C5); 181.427 (C=S); 41.014 (NCH3);
127.287, 128.725, 128.992, 131.360, 131.987, 133.921,
138.923, 146.446 (aryl carbons); 12.931 (CH3 at 6).
5.2.2.2. 8-Methyl-7,9-bis(p-methylphenyl)-1,2,4,8-tetraaza-
spiro[4.5]decan-3-thione (27). IR (KBr) (cm–1): 3365, 3306
(N–H), 2345, 1602, 1502, 1463, 1341, 1265 (C=S), 1210,
1026, 998, 920, 895, 821, 800, 764, 692, 671, 613, 542, 524,
490; Mass (m/z): 366(M+), 351, 322, 306, 291, 237, 217, 207,
5.2.2.6. 8-Methyl-7,9-bis(p-methoxyphenyl)-1,2,4,8-tetraaza-
spiro[4.5]decan-3-thione (31). IR (KBr) (cm–1): 3361, 3304
(N–H), 2361, 1596, 1474, 1442, 1370, 1268 (C=S), 1230,
1082, 1029, 943, 914, 881, 838, 796, 678, 620, 586, 543;
Mass (m/z): 398(M+), 383, 354, 338, 323, 267, 239, 189, 163,
1
173, 147, 120, 118 (100%), 91, 65, 50; H-NMR (d ppm):
3
3.63(dd, J = 12.12 Hz; 3.24 Hz, 2H, H7a, H9a); 2.15–2.38
1
(m, 4H, H6a, H10a, H6e, H10e); 7.25, 7.35 (2d, 8H, aryl pro-
tons); 8.47 (b.s, 2H, NHCSNH); 7.13 (s, 1H, NH at 1); 2.42
(s, 6H, aryl CH3); 1.62 (s, 3H, NCH3); 13C-NMR (d ppm):
65.326 (C7, C9); 38.447 (C6, C10); 77.814(C5); 180.518 (C=S);
40.982 (NCH3); 127.848, 128.526, 136.976, 146.436 (aryl
carbons); 20.582 (aryl CH3).
134 (100%), 117, 107, 91, 75, 65, 50; H-NMR (d ppm):
3
3.62(dd, J = 12.09 Hz; 3.20 Hz, 2H, H7a, H9a); 2.14–2.38
(m, 4H, H6a, H10a, H6e, H10e); 6.89, 7.34 (2d, 8H, aryl pro-
tons); 8.44 (b.s, 2H, NHCSNH); 7.18 (s, 1H, NH at 1); 3.93
(s, 6H, aryl OCH3); 1.61 (s, 3H, NCH3); 13C-NMR (d ppm):
65.347 (C7, C9); 38.474 (C6, C10); 77.760(C5); 180.671 (C=S);
40.631 (NCH3); 115.545, 127.841, 141.900, 158.539 (aryl
carbons); 54.516 (aryl OCH3).
5.2.2.3. 6,8-Dimethyl-7,9-bis(p-methylphenyl)-1,2,4,8-
tetraazaspiro[4.5]decan-3-thione (28). IR (KBr) (cm–1):
3426, 3312 (N–H), 2346, 1581, 1498, 1472, 1420, 1381, 1320,
1268 (C=S), 1201, 1132, 1026, 963, 890, 526, 772, 720, 680,
626; Mass (m/z): 380(M+), 365, 336, 320, 305, 251, 237, 235,
173, 160, 146, 132 (100%), 118, 91, 65, 51; 1H-NMR (d ppm):
3.59(dd, 3J = 12.34 Hz; 3.02 Hz, 1H, H9a); 3.20(d,
3J = 11.09 Hz, 1H, H7a); 2.22–2.46 (m, 3H, H6a, H10a, H10e);
7.28–7.42 (m, 8H, aryl protons); 8.39 (b.s, 2H, NHCSNH);
6.91 (s, 1H, NH at 1); 2.45 (s, 6H, aryl CH3); 1.58 (s, 3H,
NCH3); 0.82(d, J = 6.53 Hz, 3H, CH3 at 6); 13C-NMR (d
ppm): 71.478 (C7); 66.104 (C9); 39.618 (C6); 38.028 (C10);
78.434(C5); 181.414 (C=S); 41.432 (NCH3); 128.314,
128.522, 129.779, 130.615, 135.679, 137.851, 138.648,
146.135 (aryl carbons); 20.995 (aryl CH3); 12.889 (CH3 at 6).
5.2.2.7. 6,8-Dimethyl-7,9-bis(p-methoxyphenyl)-1,2,4,8-
tetraazaspiro[4.5]decan-3-thione (32). IR (KBr) (cm–1):
3416, 3372, 3298 (N–H), 2367, 1580, 1491, 1463, 1390, 1340,
1262 (C=S), 1118, 1020, 941, 846, 797, 660, 618; Mass (m/z):
412(M+), 397, 368, 352, 337, 283, 267, 247, 239, 189, 162,
148 (100%), 135, 117, 107, 75, 65, 55, 50; 1H-NMR (d ppm):
3.60(dd, 3J = 12.36 Hz; 2.97 Hz, 1H, H9a); 3.20(d,
3J = 11.14 Hz, 1H, H7a); 2.20–2.47 (m, 3H, H6a, H10a, H10e);
7.35–7.37, 6.83–6.85 (m, 8H, aryl protons); 8.47 (b.s, 2H,
NHCSNH); 6.98 (s, 1H, NH at 1); 3.94 (s, 6H, aryl OCH3);
1.59 (s, 3H, NCH3); 0.80(d, J = 6.57 Hz, 3H, CH3 at 6); 13C-
NMR (d ppm): 71.476 (C7); 66.033 (C9); 39.615 (C6); 38.115
(C10); 78.450(C5); 181.468 (C=S); 41.142 (NCH3); 114.381,
115.892, 128.012, 130.952, 134.743, 141.683, 157.051,
159.236 (aryl carbons); 54.793 (aryl OCH3); 13.008 (CH3
at 6).
5.2.2.4. 8-Methyl-7,9-bis(p-chlorophenyl)-1,2,4,8-tetraaza-
spiro[4.5]decan-3-thione (29). IR (KBr) (cm–1): 3328, 3296
(N–H), 2396, 1601, 1451, 1435, 1376, 1246 (C=S), 1185,
1072, 1036, 978, 923, 891, 820, 800, 673, 606, 593, 560;
Mass (m/z): 406(M+), 391, 362, 346, 331, 277, 247, 237, 193,
Acknowledgements
1
166, 139 (100%), 111, 95, 75, 65, 50; H-NMR (d ppm):
3
3.64(dd, J = 12.12 Hz; 3.23 Hz, 2H, H7a, H9a); 2.18–2.42
Authors are thankful to Professor K. Pandiarajan, Head,
Department of Chemistry,Annamalai University for the facili-
ties provided. One of the authors S.K. is grateful to Univer-
sity Grants Commission, New Delhi, India for financial sup-
port in the form of Major Research Project. S.B. wishes to
thank Council of Scientific and Industrial Research, New
Delhi, India for the award of Senior Research Fellowship.
The authors place sincere thanks to Professor M. Vasudevan,
JSS College of Pharmacy, Ooty, India for his kind help in
conducting screening studies.
(m, 4H, H6a, H10a, H6e, H10e); 7.31, 7.37 (2d, 8H, aryl pro-
tons); 8.47 (b.s, 2H, NHCSNH); 7.08 (s, 1H, NH at 1); 1.59
(s, 3H, NCH3); 13C-NMR (d ppm): 65.195 (C7, C9); 38.251
(C6, C10); 77.771(C5); 180.622 (C=S); 40.532 (NCH3);
128.251, 128.404, 133.201, 146.744 (aryl carbons).
5.2.2.5. 6,8-Dimethyl-7,9-bis(p-chlorophenyl)-1,2,4,8-
tetraazaspiro[4.5]decan-3-thione (30). IR (KBr) (cm–1):
3433, 3327, 3293 (N–H), 2393, 1586, 1486, 1472, 1360, 1346,

700
S. Balasubramanian et al. / European Journal of Medicinal Chemistry 40 (2005) 694–700
[15] C. Ramalingan, S. Balasubramanian, S. Kabilan, Synth. Commun. 34
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