Synthesis and antimycobacterial activity of 3,5-disubstituted thiadiazine thiones

Article abstract of DOI:10.1016/S0968-0896(03)00480-2

A series of 3,5-disubstituted thiadiazine thiones (4-24) have been synthesized by reaction of primary amines with carbon disulphide followed by cyclocondensation of the resulting intermediate with formaldehyde and primary amines or amino acids. The compounds were screened for antitubercular activity in vitro against Mycobacterium tuberculosis H37Rv. Three compounds 4, 12 and 18 showed antimycobacterial activity with MIC 12.5 μg/mL. Compound 4, was tested in vitro against five multidrug resistant (MDR) strains of M. tuberculosis and was found to be active. Compound 4 also exhibited activity in vivo. While all the mice died in the untreated group, the mean survival time (MST) of the compound treated mice was enhanced, 33% mice were surviving in treated group and the load of bacilli in the lung was considerably less in the compound treated group than in the untreated control group.

Full text of DOI:10.1016/S0968-0896(03)00480-2

Bioorganic & Medicinal Chemistry 11 (2003) 4369–4375
Synthesis and Antimycobacterial Activity of 3,5-Disubstituted
Thiadiazine Thiones§
D. Katiyar,a V.K. Tiwari,a R. P. Tripathi,a,* A. Srivastava,b V. Chaturvedi,b
R. Srivastavab and B. S. Srivastavab
aDivision of Medicinal Chemistry, Central Drug Research Institute, Lucknow-226001, India
bDivision of Microbiology, Central Drug Research Institute, Lucknow-226001, India
Received 12 May 2003; accepted 14 July 2003
Abstract—A series of 3,5-disubstituted thiadiazine thiones (4–24) have been synthesized by reaction of primary amines with carbon
disulphide followed by cyclocondensation of the resulting intermediate with formaldehyde and primary amines or amino acids. The
compounds were screened for antitubercular activity in vitro against Mycobacterium tuberculosis H37Rv. Three compounds 4, 12
and 18 showed antimycobacterial activity with MIC 12.5 mg/mL. Compound 4, was tested in vitro against five multidrug resistant
(MDR) strains of M. tuberculosis and was found to be active. Compound 4 also exhibited activity in vivo. While all the mice died in
the untreated group, the mean survival time (MST) of the compound treated mice was enhanced, 33% mice were surviving in treated
group and the load of bacilli in the lung was considerably less in the compound treated group than in the untreated control group.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
new mode of action and at the same time possessing
anti-HIV, antimycotic and immunomodulatory activ-
ities.
Tuberculosis, caused by single infectious agent Myco-
bacterium tuberculosis, remains on the top of the killing
infectious diseases in spite of intensive funding and
research, and is a priority area globally.1,2 As per esti-
mate of W.H.O there were 8.4 million new cases of
tuberculosis in 1999 and the annual global rate of
increase of TB incidence was 3%. Assuming this rate of
increase in TB incidence, there will be over 10 million
new cases of TB in 2005. Currently, one third of world’s
population is infected with this bacillus.3 Development
of drug resistance against the frontline drugs and
synergy of this disease with HIV and mycotic infections
in immunocompromised patients have aggravated the
problem.1ꢀ4 A number of antitubercular agents belong-
ing to different class of molecules are known today but
these are associated with one or more drawbacks.5 In
spite of enormous amount of work done in genetic
engineering and structural biology of this bacterium no
new chemical entity has appeared in clinics since the last
40 years. Hence, in view of the above there is an emer-
gent need to develop new antitubercular agents having a
Thiadiazine thione nucleus has been reported for differ-
ent biological activities6 including antituberculostatic,
antibacterial, antiviral, antifungal and anthelmenthic,
where it has been postulated that biological activity in
these molecules is dependent on isothiocyanates and
dithiocarbamic acid species generated in the biosystem
on hydrolysis.7,8 Dithiocarbamates themselves are asso-
ciated with antifungal, antiviral, immunomodulatory
and many other bioactivities9ꢀ11 which could be an
added advantage in developing new drugs against
tuberculosis. Diethyl dithio carbamic acid sodium salt
(DEDTC 1), a dithiocarbamic acid derivative, a well
known immunopotentiator12 has been a safe clinical
candidate for the treatment of HIV infections.13,14
Encouraged by the above and in continuation of our
synthetic work on dithiocarbamates15 and thiadiazine
thiones,16 we were prompted to develop thiadiazine
thiones as a new class of antitubercular agents. Further,
this study was undertaken keeping in mind our recent
finding that amino acid derivatives17 have anti-
tubercular activity. The potential of this class of mole-
cule to block mycobacterial cell wall biosynthesis has
been documented.18,19 It is envisaged if amino acids or
§CDRI communication No. 6405.
*Corresponding author. Tel.: +91-522-212411-18x4382; fax: +91-
522-223405 or 223938; e-mail: rpt_56@yahoo.com
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00480-2

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D. Katiyar et al. / Bioorg. Med. Chem. 11 (2003) 4369–4375
their isosteres are coupled with appropriate thiadiazine
thione skeleton, a new class of molecules would emerge
active against mycobacteria. The immunopotentiating,
antimycotic and anti-HIV activities associated with
thiocarbamic acid moiety would be beneficial in treating
this disease in immunocompromised patients.
Scheme 1.
separately resulted in the formation of respective inter-
mediate dithiocarbamate potassium salts 3 (which were
not isolated). Further reaction of the intermediates (3)
with formaldehyde and selected amino acids, amines
and amino ester, including b-alanine, glycine, g-amino
butyric acid, phenylalanine, 6-amino caproic acid,
cyclohexyl amine, cyclopropyl amine, hexadecyl amine
and 3-amino ethyl propionate, afforded respective 3,5-
disubstituted thiadiazine-2-thiones (4–24) in fair to
good yields (Scheme 1). Structures of all the compounds
were established on the basis of spectroscopic data and
microanalysis. IR spectroscopy of the compounds
showed absorption bands ranging from 3060 to 3450
cmꢀ1 for (OH and NH), 1690 to 1720 cmꢀ1 for (C¼O)
In this context, we were interested to synthesize a com-
binatorial library of thiadiazine thiones having amino
acid and hetero aromatic acid components. Our attempt
to synthesize a small library of 48 compounds on solid
phase in a parallel format manner was unsuccessful as
the products obtained after cleavage from resin were a
complex mixture of several compounds including 3,5-
disubstituted thiadiazine thiones (molecular ion peaks
as evidenced by FAB MS) and none of them could be
isolated in pure form. However, these compounds as
such were screened against M. tuberculosis and few of
them showed mild in vitro activity (unpublished result).
We then decided to synthesize some of these compounds
with thiadiazine thione skeleton in conventional manner
and evaluate them for antitubercular activity.
1
and from 1445 to 1508 cmꢀ1 for (C¼S). In H NM R
spectra, H-4 and H-6 protons of tetrahydro-2H-1,3,5-
thiadiazine-2-thione ring appeared as two separate
singlets around d 4.50 and 4.40 respectively besides
other usual signals of the substituents.
In 13C NMR spectrum of compound 4, characteristic
signals for C=S, C-4 and C-6 appeared at d 195.1, 71.8
and 57.3, respectively. The signals corresponding to
these carbons appear at d 195, 71.6, 57.3; 195.2, 71.6,
57.3; 195.3, 71.7, 57.3; 195.2, 71.3, 53.0; 193.4, 68.4,
58.6; 195.4, 67.4, 59.6; 193.4, 68.4, 58.6; 193.6, 67.4,
56.7; 190.9, 65.1, 57.1; 191.5, 70.6, 58.2; 191.5, 70.6,
58.2; 191.5, 70.6, 58.2; 193.9, 66.7, 56.2; 193, 65.9, 56.1;
193.5, 68.3, 59.1; 195.1, 71.8, 57.5; 195.8, 69.0, 55.4 and
195.7, 71.5, 58.1 in compounds 5, 6, 7, 8, 9, 11, 12, 13,
14, 16, 17, 18, 19, 20, 21, 22, 23 and 24, respectively. All
Results and Discussion
Chemistry
The compounds 4–24 (Table 1) were prepared by a
slight modification of the earlier reported method.6
Thus reaction of the appropriate primary amines of the
general formula 2, including cyclopropyl amine, furfuryl
amine, benzyl amine, cyclohexyl amine, butyl amine,
octyl amine and dodecyl amine, with carbon disulphide
Table 1. In vitro activity of compounds 4–24 against M. tuberculosis H37Rv
Compd
R1
R2
ClogP
CMR
Water solubilities
MIC (mg/mL)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Furfuryl
Furfuryl
Furfuryl
Benzyl
Benzyl
Benzyl
Cyclohexyl
Cyclohexyl
Butyl
–CH2CH2COOH
–CH2COOH
ꢀ1.29
ꢀ0.73
ꢀ1.03
ꢀ0.46
ꢀ0.68
ꢀ0.41
2.29
0.14
0.41
3.12
0.37
6.66
6.19
7.12
8.05
7.59
8.05
10.01
8.38
8.84
10.89
8.01
9.63
7.26
9.12
10.97
7.72
9.40
14.22
7.58
7.68
6.33
3.257
5.859
Soluble
Soluble
12.5
50
25
25
50
50
50
50
12.5
50
100
50
25
25
12.5
50
–CH2CH2CH2COOH
–CH2(CH2)4COOH
–CH2CH2COOH
–CH2CH2CH2COOH
CH(CH2C6H5)COOH
–CH2CH2COOH
–CH2CH2CH2COOH
CH(CH2C6H5)COOH
–CH2CH2COOH
–(CH2C6H5)COOH
–CH2CH2COOH
–CH2CH2COOH
–CH2CH2COOH
Cyclohexyl
Partially soluble
Partially soluble
Partially soluble
Partially soluble
Insoluble
Partially soluble
Insoluble
Insoluble
Partially soluble
Insoluble
Partially soluble
Insoluble
Insoluble
3.39
ꢀ0.05
Octyl
2.06
4.18
Dodecyl
Furfuryl
Benzyl
3.54
4.91
10.48
1.96
2.93
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Soluble
Soluble
Cyclohexyl
Hexadecyl
–CH2CH2COOEt
Cyclohexyl
Cyclopropyl
50
50
12.5
100
25
Benzyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
—
23
24
1.25
ꢀ0.668
Isoniazid
Ethambutol
—
—
0.75
3.25
—
0.1188

D. Katiyar et al. / Bioorg. Med. Chem. 11 (2003) 4369–4375
4371
other signals of the above compounds are given in the
experimental section.
ethyl substituent at N-5 also exhibited antitubercular
activity with MIC of 12.5 mg/mL. In the N-3-benzyl
series of compounds only one compound with carboxy-
propyl substituent at N-5 (12) was found to be potent
with an MIC of 12.5 mg/mL. All other compounds were
inactive as they have MIC values >25 mg/mL.
Biology
A search for new antitubercular compounds based on
inhibition in the biosynthesis of cell wall components is
an ideal approach and presents an attractive target as it
is involved in the transportation of nutrients and drugs,
forms a permeability barrier and protects the organism
from the hostile environment of the host’s immune sys-
tem.20,21 Compounds having thiourea (isoxyl) function-
ality are known to inhibit biosynthesis of mycolic acid,22
an important constituent of the cell wall. Several amino
acid analogues18 are known to inhibit an enzyme d-ala-
nine racemase19 required in one of the steps during
peptidoglycan biosynthesis. Therefore, it was thought to
synthesize compounds with thioureidyl and amino acid
functionalities, wherein the thione moiety may serve as
a site for nucleophilic attack by the enzymes of M.
tuberculosis and unnatural amino acids or its isosteres
will inhibit the peptidoglycan biosynthesis by inhibiting
the enzyme d-alanine racemase. The compounds have
been designed keeping in mind the fact that in thiadi-
azine thiones N-3- and N-5-substitutions with hydro-
phobic and hydrophilic components respectively leads
better activity and low toxicity to the host.23
The in vitro high MIC values in this series of com-
pounds did not discourage us as it is known that in vitro
activity often displays poor correlation with activity in
vivo and the reason for this failure is most often con-
flicting balances between bioavailability and activity.24
The bioavailability depends mainly on solubility, per-
meability and metabolic half-life. Compounds with
thiocarbonyl and sulphide moiety functionalities are
known to be activated in vivo by oxidation of S to
sulphoxides and sulphones. MIC of the sulphides are
much less in vitro in comparison to sulphone and there
is no gain in giving the oxidized product to in vivo
system as it will only increase the cost of the drug with
no net gain in activity profile. An estimate of lipophili-
city can easily be predicted by simply counting the NH
and OH bonds and N and O atoms. Looking into the
Log P and molar refractivities values, determined using
chem office 6.0 programme, and water solubilities of
these compounds, compound 4 was selected for further
study.
The activity of the compounds 4–24 synthesized and
reported here was determined against M. tuberculosis
H37Rv. The in vitro activity of the compounds is shown
in Table 1, and expressed as the minimal inhibitory
concentration (MIC). The colony forming ability of M.
tuberculosis H37Rv was completely inhibited at the
reported MIC’s. As evident out of 21 compounds syn-
thesized, compounds 4, 6, 7, 12, 16, 17, 18, 22 and 24
showed activity in vitro at one or the other concentra-
tions. Careful examination of biological activity data
reveals that compounds with N-3 cyclopropyl and N-5
b-alanyl (4) or its congener having 2-carbethoxy ethyl
substituent at N-5 (22) showed in vitro activity at 12.5
mg/mL. Other compounds of this series having cyclo-
propyl substituent at N-3 and cyclopropyl, cyclohexyl
or other substituent at N-5 always resulted in lower
activity with higher MIC’s. However, compounds with
N-3 furfuryl substituent having any substituent at N-5
always resulted in poor activity as all the compounds of
this series have MIC’s 50 or 100 mg/mL. Compound 18
having a 12 carbon substituent at N-3 and 3-carboxy
The in vitro activity of compound 4 was determined
against some selected multi-drug resistant (MDR)
strains isolated from tuberculosis patients. Activity was
tested against only one concentration (50 mg/mL) of
compound 4 present in the media and was found to
inhibit the growth of MDR strains (Table 2).
As compound 4 showed in vitro activity in MDR strains
it was screened in vivo too in mouse model. Mice infec-
ted with M. tuberculosis H37Rv strain were treated with
the compound 4 and it was found to be promising as
compared to the untreated control group. Enhancement
of mean survival time, reduction in the load of tubercule
bacilli in the lungs and marginal increased survival of
mice were observed in the treated group (Table 3).
Compound 4 proved to be promising as the survival of
treated mice was enhanced marginally and the burden
of bacilli was reduced at least 10-fold as compared to
untreated control.
Table 3. Activity of compound 4 against M. tuberculosis H37Rv in
mice model of tuberculosis
Table 2. In vitro activity of compound 4 (50 mg/mL) against MDR
strains of M. tuberculosis isolated from TB patients
Group
MST
(Days)
% Survival on day
35 post infection
Load of bacilli
in lung (CFU/g)
Compd or drug
Growth of MDR strains after 6 weeks
BC-248 BC-243 VA-101 BC-426 BC-437
(1)
Compd 4 treated
Untreated control
Sparfloxacin
21.3
19
>35
33
00
100
2.1Â105
5Â106
(1)
(2)
(3)
(3)
Compd 4
Sparfloxacin
No drug control
ꢀ
+
++
ꢀ
ꢀ
+
++
++
ꢀ
4.8Â102
++
++
++
++
++
++
MST, Mean survival time=mortality rate each day/total no. of mice.
Results are mean values of two experiments. All the groups were
infected with 107 CFU of M. tuberculosis H37Rv/mouse intrave-
nously. Animals were kept under observation for 35 days post infec-
tion. Compound 4 (100 mg/kg)/Sparfloxacin 25 mg/kg was given
intraperitoneally for 10 days consecutively.
ꢀ, no growth; +, 1–20 colonies; ++, heavy growth.
(1) Strains resistant to rifampicin, isoniazid, ofloxicin and etham-
butol; (2) strains resistant to rifampicin, isoniazid and ethambutol; (3)
strains resistant to rifampicin and isoniazid.

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D. Katiyar et al. / Bioorg. Med. Chem. 11 (2003) 4369–4375
Experimental
5 - (4 - Carboxypropyl) - 3 - (cyclopropyl) - tetrahydro -2H -
1,3,5, thiadiazine-2-thione (6). This was obtained by
cyclopropyl amine (1.39 mL, 20 mmol) and g-amino
butyric acid (2.06 g, 20 mmol) as described above and
isolated as colourless solid, yield 65%, mp 112–115 ꢁC.
IR (KBr): 3439, 1699, 1475; MS (FAB): 261 (M+H)+;
1H NMR (CDCl3): d 4.36 (s, 2H, H-4), 4.30 (s, 2H, H-
6), 3.08 (m, 1H, NCH), 2.81 (t, 2H, J=6.9 Hz, NCH2),
2.46 (t, 2H, J=7.1 Hz, CH2COOH), 1.88 (m, 2H,
NCH2CH2), 1.04–0.87 (m, 4H, cyclopropyl ring pro-
tons); 13C NMR (CDCl3): d 195.2 (C¼S), 176.3 (C¼O),
71.6 (C-4), 57.3 (C-6), 49.8 (NCH2), 36.3 (NCH), 31.7
(CH2COOH), 22.8 (NCH2CH2), 9.3 (cyclopropyl ring
carbons). Anal. calcd for C10H16N2O2S2: C, 46.15; H,
6.15; N, 10.76. Found: C, 45.96; H, 6.15; N, 10.72.
Chemistry
Melting points were determined on a Buchi 510 appa-
ratus and are uncorrected. Elemental analysis for all
new compounds were performed on a Carlo Erba
Model EA-1108 elemental analyzer and data of C, H,
and N is withinÆ0.4% of calculated values. Thin layer
chromatography was used to monitor the reactions.
IR(KBr) spectra were recorded using Perkin-Elmer 881
spectrophotometer and the values are expressed as nmax
cmꢀ1. Mass spectral data were recorded on a Jeol
(Japan) SX 102/DA-6000 Mass Spectrometer/Data sys-
tem. The 1H NMR and 13C NMR spectra were recorded
on Brucker Spectrospin spectrometer at 200 and
50 MHz, respectively, using TMS as internal standard.
The chemical shift values are on d scale and the cou-
pling constants (J) are in Hz.
5-(6-Carboxypentyl)-3-cyclopropyl-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (7). This was obtained by cyclopro-
pyl amine (1.39 mL, 20 mmol) and 6-amino caproic acid
(2.62 g, 20 mmol) as described above and isolated as
yellow oil, yield 56%; IR (KBr): 2939, 1711, 1461; MS
(FAB): 289 (M+H)+; 1H NMR (CDCl3): d 4.37 (s, 2H,
H-4), 4.32 (s, 2H, H-6), 3.09–3.07 (m, 1H, NCH), 2.73
(t, 2H, J=7.1 Hz, NCH2), 2.37 (t, 2H, J=7.2,
CH2COOH), 1.67–1.41 (m, 6H, [CH2(CH2)3CH2], 1.03–
0.83 (m, 4H, cyclopropyl ring protons); 13C NM R
(CDCl3): d 195.3 (C¼S), 179.8 (C¼O), 71.7 (C-4), 57.3
(C-6), 50.3 (NCH2), 36.3 (NCH), 34.3 (CH2COOH),
27.6, 26.8, 24.8 (CH2’s), 9.2 (cyclopropyl ring carbons).
Anal. calcd for C12H20N2O2S2: C, 50.0; H, 6.9; N, 9.72.
Found: C, 49.9; H, 7.02; N, 9.72.
General procedure for the synthesis of compounds 4–18
5-(3-Carboxyethyl)-3-cyclopropyl-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (4). To a magnetically stirred solu-
tion of cyclopropyl amine (1.39 mL, 20 mmol) in 50 mL
of H2O, KOH (1.12 g, as 20% aq solution, 20 mmol)
and CS2 (1.20 mL, 20 mmol) were added at 30 ꢁC. The
reaction mixture was stirred for 4 h more followed by
addition of 37% formaldehyde solution (3.01 mL, 40
mmol) and stirring continued for 1 h. The reaction
mixture was filtered and resulting filtrate was added
drop wise to a suspension of b-alanine (1.78 g, 20 mmol)
in 20 mL of phosphate buffer solution (pH 7.7) and
stirred additional for 1 h. The reaction mixture filtered
off and the filtrate extracted with diethyl ether. The
aqueous solution was cooled in an ice bath and acidified
with 15% HCl (pH 2.0). The precipitated solid filtered
off and washed with cold water, dried and crystallized
from ethanol to give the title compound 4 as colourless
granules. Yield 80%, mp 92–95 ꢁC. IR (KBr): 3422,
1703, 1458; MS (FAB): 247 (M+H)+; 1H NM R
(CDCl3): d 4.41 (s, 2H, H-4), 4.34 (s, 2H, H-6), 3.09–
3.04 (m, 3H, NCH2 and NCH), 2.54 (t, 2H, J=6.4 Hz,
CH2COOH), 1.04–0.87 (m, 4H, cyclopropyl ring pro-
tons); 13C NMR (CDCl3): d 195.1 (C¼S), 176.7 (C¼O),
71.8 (C-4), 57.3 (C-6), 46.3 (NCH2), 36.2 (NCH), 33.3
(CH2COOH), 9.3 (cyclopropyl ring carbons). Anal.
calcd for C9H14N2O2S2: C, 43.90; H, 5.69; N, 11.38.
Found: C, 43.90; H, 5.70; N, 11.29.
5-(3-carboxyethyl)-3-(20 -furfuryl)-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (8). This was obtained by furfuryl
amine (1.76 mL, 20 mmol) and b-alanine (1.78 g, 20
mmol) as described above and isolated as colourless
solid, yield 71%, mp 128–30 ꢁC (lit.25 mp 131–132 ꢁC).
IR (KBr): 3122, 1699, 1493; MS (FAB): 287 (M+H)+;
1H NMR (CDCl3): d 7.54 (d, 1H, J=1.7 Hz, furfuryl H-
50), 6.50 (d, 1H, J=3.2 Hz, furfuryl H-30), 6.44 (dd, 1H,
J=3.2 and 1.7 Hz, furfuryl H-40), 5.41(s, 2H, furfuryl
CH2), 4.63 (s, 2H, H-4), 4.57 (s, 2H, H-6), 3.01 (t, 2H,
J=6.8 Hz, NCH2), 2.45 (t, 2H, J=6.7, CH2COOH);
13C NMR (CDCl3): d 195.2 (C¼S), 177.9 (C¼O), 152
(C-20), 144.9 (C-50), 115.0 (C-40), 114.1 (C-30), 71.3 (C-
4), 61.8 (furfuryl CH2), 53.0 (C-6), 51.0 (NCH2), 34.6
(CH2COOH). Anal. calcd for C11H14N2O3S2: C, 46.15;
H, 4.89; N, 9.79. Found: C, 46.16; H, 4.89; N, 9.78.
5-(2-Carboxymethyl)-3-cyclopropyl-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (5). This was obtained by cyclo-
propyl amine (1.39 mL, 20 mmol) and glycine (1.5 g,
20 mmol) as described above and isolated as colour-
less solid, yield 62%, mp 123–125 ꢁC. IR (KBr): 3439,
1711, 1480; MS (FAB): 233 (M+H)+; 1H NM R
(CDCl3+DMSO-d6): d 4.48 (s, 2H, H-4), 4.41 (s, 2H,
H-6), 3.58 (s, 2H, NCH2COOH), 3.09 (m, 1H, NCH),
1.04–0.79 (m, 4H, cyclopropyl ring protons); 13C
NMR (CDCl3): d 195 (C¼S), 175.3 (C¼O), 71.6 (C-
4), 57.3 (C-6), 54.8 (NCH2), 36.3 (NCH), 9.3 (cyclo-
propyl ring carbons). Anal. calcd for C8H12N2O2S2:
C, 41.38; H, 5.17; N, 12.06. Found: C, 41.36; H, 5.17;
N, 12.06.
5-(4-Carboxypropyl)-3-(20-furfuryl)-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (9). This was obtained by furfuryl
amine (1.76 mL, 20 mmol) and g-amino butyric acid
(2.06 g, 20 mmol) as described above and isolated as
colourless solid, yield 62%, mp 115–118 ꢁC. IR (KBr):
1
3101, 1709, 1480; MS (FAB): 301(M+H)+; H NM R
(CDCl3): d 7.37 (d, 1H, J=1.8 Hz, furfuryl H-50), 6.47
(d, 1H, J=3.0 Hz, furfuryl H-30), 6.37 (dd, 1H, J=3.0
and 1.8 Hz, furfuryl H-40), 5.30 (s, 2H, furfuryl CH2),
4.56 (s, 2H, H-4), 4.36 (s, 2H, H-6), 2.70 (t, 2H, J=6.9
Hz, NCH2), 2.37 (t, 2H, J=7.1 Hz, CH2COOH), 1.80–
1.66 (m, 2H, NCH2CH2); 13CNMR (CDCl3): d 197.3
(C=S), 179.9 (C¼O), 152.9 (C-20), 146.9 (C-50), 115.0
(C-40), 114.7 (C-30), 73.3 (C-4), 62.8 (furfuryl CH2), 53.7

D. Katiyar et al. / Bioorg. Med. Chem. 11 (2003) 4369–4375
4373
(C-6), 51.3 (NCH2), 35.6 (CH2COOH), 26.6 (NCH2CH2).
Anal. calcd for C12H16N2O3S2: C, 48.0; H, 5.33; N,
9.33. Found: C, 47.96; H, 5.33; N, 9.33.
(CDCl3+DMSO-d6): d 193.6 (C¼S), 173.6(C¼O), 136.5
and 135.4 (Ar-C), 129.4, 128.8 and 128.6 (ArCH), 67.4
(C-4), 64.2 (NCH), 56.7, 54.8, and 36.7 (CH2’s). Anal.
calcd for C19H20N2O2S2: C, 61.29; H, 5.37; N, 7.52.
Found: C, 61.30; H, 5.4; N, 7.52.
5-[ꢀ-(Benzyl)carboxymethyl]-3-(furfuryl)-tetrahydro-2H-
1,3,5, thiadiazine-2-thione (10). This was obtained by
furfuryl amine (1.76 mL, 20mmol) and dl-phenyl ala-
nine (3.3 g, 20 mmol) as described above as colourless
oil and treated witꢁh cool ether to precipitate it, yield
50%, mp 167–169 C (lit.25 mp 166–168 ꢁC). IR (KBr):
5-(3-Carboxyethyl)-3-cyclohexyl-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (14). This was obtained by cyclo-
hexyl amine (2.28 mL, 20 mmol) and b-alanine (2.06 g,
20 mmol) as described above and isolated as colourless
solid, yield 67%, mp 148–50 ꢁC (lit.25 mp 145–146). IR
1
3062, 1719, 1597, 1490; MS (FAB): 363 (M+H)+; H
NMR (DMSO-d6): d 7.37 (m, 6H, furfuryl H-50 and
ArH), 6.47 (d, 1H, J=3.0 Hz, furfuryl H-30), 6.37 (dd,
1H, J=3.0 and 1.8 Hz, furfuryl H-40), 5.32 and 5.10
(each d, each 1H, J=9.3 Hz, furfuryl CHA and CHB),
4.56 (s, 2H, H-4), 4.36 (s, 2H, H-6), 3.83–3.76 (m, 1H,
NCHCOOH), 3.32–3.28 (m, 2H, CH2C6H5). Anal.
calcd for C17H18N2O3S2: C, 56.35; H, 4.97; N, 7.73.
Found: C, 56.36; H, 4.82; N, 7.78.
(KBr): 3405, 1710, 1461; MS (FAB): 289(M+H)+; H
1
NMR (CDCl3): d 4.34 (s, 2H, H-4), 4.32 (s, 2H, H-6),
3.07 (t, 2H, J=6.5 Hz, NCH2), 2.53 (t, 2H, J=6.4,
CH2COOH), 1.87–1.36 (m, 10H, cyclohexyl ring pro-
tons); 13C NMR (CDCl3): d 190.9 (C¼S), 173.5 (C¼O),
65.1 (C-4), 57.9 (N–CH), 57.1 (C-6), 45.9, 33.3, 28.8,
25.6, 25.3 (CH2’s). Anal. calcd for C12H20N2O2S2: C,
50.0; H, 6.94; N, 9.72. Found: C, 49.98; H, 7.0; N, 9.72.
5 - (3 - Carboxyethyl) - 3 - (phenylmethyl) - tetrahydro - 2H-
1,3,5, thiadiazine-2-thione (11). This was obtained by
benzyl amine (2.18 mL, 20 mmol) and b-alanine (1.78 g,
20 mmol) as described above and isolated as colourless
solid, yield 72%, mp 134–136 ꢁC. IR (KBr): 3386, 1699,
5-[ꢀ-(Benzyl)carboxymethyl]-3-cyclohexyl-tetrahydro-
2H-1,3,5, thiadiazine-2-thione (15). This was obtained
by cyclohexyl amine (2.28 mL, 20 mmol) and dl-phenyl
alanine (3.3 g, 20 mmol) as described above and isolated
as colourless solid, yield 52%, mp 136–138 ꢁC (lit.25 mp
134–135 ꢁC). IR (KBr): 3071, 2952, 1589, 1503; MS
1
1489; MS (FAB): 297 (M+H)+; H NMR (CDCl3): d
1
7.41–7.26 (m, 5H, Ar-H), 5.35 (s, 2H, C6H5CH2), 4.39
(s, 2H, H-4), 4.29 (s, 2H, H-6), 2.92 (t, 2H, J=6.5 Hz,
NCH2), 2.19 (t, 2H, J=6.5, CH2COOH). 13C NM R
(CDCl3): d 195.4 (C¼S), 175.2 (C¼O), 135.2 (Ar-C),
129.2, 128.9 and 128.6 (ArCH), 67.4 (C-4), 59.6 (C-6),
53.2 (CH2C6H5), 48.9 (NCH2), 32.5 (CH2COOH). Anal.
calcd for C13H16N2O2S2: C, 52.70; H, 5.4; N, 9.45.
Found: C, 52.72; H, 5.4; N, 9.45.
(FAB): 365 (M+H)+; H NMR (DMSO-d6): d 7.34–
7.17 (m, 5H, ArH), 5.76 (m, 1H, NCH), 4.49 (s, 2H, H-
4), 4.43 (s, 2H, H-6), 3.85–3.78 (m, 1H, NCHCOOH),
3.16 (d, 2H, J=6.5 Hz, CH2C6H5), 1.92–1.11 (m, 10H,
cyclohexyl
ring
protons).
Anal.
calcd
for
C18H24N2O2S2: C, 59.34; H, 6.59; N, 7.69. Found: C,
59.34; H, 6.61; N, 7.69.
5-(3-Carboxyethyl)-3-butyl-tetrahydro-2H-1,3,5, thiadi-
azine-2-thione (16). This was obtained by butyl amine
(1.98 mL, 20 mmol) and b-alanine (1.78 g, 20 mmol) as
described above and isolated as colourless solid, yield
75%, mp 125–127 ꢁC; IR (KBr): 2956, 1718, 1507; MS
5-(4-Carboxypropyl)-3-(phenylmethyl)-tetrahydro-2H-
1,3,5, thiadiazine-2-thione (12). This was obtained by
benzyl amine (2.18 mL, 20 mmol) and g-amino butyric
acid (2.06 g, 20 mmol) as described above and isolated
as colourless solid, yield 67%, mp 138–140 ꢁC. IR
1
(FAB): 263 (M+H)+; H NMR (200 MHz, CDCl3): d
1
(KBr): 3424, 1703, 1485; MS (FAB): 311(M+H)+; H
4.38 (s, 2H, H-4), 4.37 (s, 2H, H-6), 3.99 (t, 2H, J=7.7
Hz, NCH2CH2COOH), 3.15 (t, 2H, J=6.5 Hz,
NCH2CH2), 2.61 (t, 2H, J=6.4 Hz, CH2COOH), 1.71–
1.60 (m, 2H, CH2CH2CH3), 1.44–1.29 (m, 2H,
CH2CH3), 0.96 (t, 3H, J=7.2 Hz, CH2CH3); 13C NM R
(CDCl3): d 191.5 (C¼S), 177.6 (C¼O), 70.6 (C-4), 58.2
(C-6), 52.6 (NCH2CH2COOH), 46.4 (NCH2CH2), 33.4
(CH2COOH), 29.1 and 20.4 (CH2’s), 14.2 (CH2CH3).
Anal. calcd for C10H18N2O2S2: C, 45.80; H, 6.87; N,
10.68. Found: C, 45.80; H, 6.79; N, 10.87.
NMR (CDCl3): d 7.49–7.32 (m, 5H, Ar-H), 5.36 (s, 2H,
C6H5CH2), 4.40 (s, 2H, H-4), 4.29 (s, 2H, H-6), 2.64 (t,
2H, J=6.9 Hz, NCH2), 2.20 (t, 2H, J=7.1 Hz,
CH2COOH), 1.57–1.43 (m, 2H, NCH2CH2); 13C
NMR(CDCl3): d 193.4 (C¼S), 176.2 (C¼O), 135.6 (Ar-
C), 129.2, 128.9 and 128.6 (ArCH), 68.4 (C-4), 58.6 (C-
6), 54.2 (CH2C6H5), 49.6 (NCH2), 31.5 (CH2COOH),
22.5 (NCH2CH2). Anal. calcd for C14H18N2O2S2: C,
54.19; H, 5.80; N, 9.03. Found: C, 54.24; H, 5.8; N,
8.98.
5-(3-Carboxyethyl)-3-octyl-tetrahydro-2H-1,3,5, thiadi-
azine-2-thione (17). It was obtained by octyl amine
(3.30 mL, 20 mmol) and b-alanine (1.78 g, 20 mmol) as
described above and isolated as colourless solid, yield
72%, mp 140–142 ꢁC. IR (KBr): 3446, 1707, 1502; MS
(FAB): 319 (M+H)+; 1H NMR (CDCl3): d 4.38 (s, 2H,
H-4), 4.37 (s, 2H, H-6), 3.97 (t, 2H, J=7.6 Hz,
NCH2CH2COOH), 3.15 (t, 2H, J=6.4, NCH2CH2),
2.61 (t, 2H, J=6.4 Hz, CH2COOH), 1.30–127 (m, 12H,
CH2’s), 0.88 (t, 3H, J=7.1 Hz, CH2CH3); 13C NM R
(CDCl3): d 191.5 (C¼S), 177.5 (C¼O), 70.6 (C-4), 58.2
R (C-6), 52.8, 46.4, 33.4, 32.1, 29.6, 29.5, 27.2, 27.0, 22.9
5-[ꢀ-(Benzyl)carboxymethyl]-3-(2-phenylmethyl)-tetra-
hydro-2H-1,3,5, thiadiazine-2-thione (13). This was
obtained by using benzyl amine (2.18 mL, 20 mmol) and
dl-phenyl alanine (3.3 g, 20 mmol) as described above
and isolated as colourless solid, yield 72%, mp 150–
152 ꢁC. IR (KBr): 3062, 1719, 1597, 1490; MS (FAB):
1
373 (M+H)+; H NMR (CDCl3+DMSO-d6): d 7.40–
7.06 (m, 10H, Ar-H), 5.43 and 5.05 (each d, each 1H,
J=14.7 Hz, C6H5CHA and CHB), 4.52 (s, 2H, H-4),
4.44 (s, 2H, H-6), 3.82 (t, 1H, J=7.2 Hz, NCH), 2.90 (d,
2H,
J=7.2
Hz,
CHCH2C6H5).
13C
NM

4374
D. Katiyar et al. / Bioorg. Med. Chem. 11 (2003) 4369–4375
(CH2’s), 14.4 (CH2CH3). Anal. calcd for C14H26N2O2S2:
C, 52.83; H, 8.18; N, 8.80. Found: C, 52.85; H, 8.14; N,
8.61.
for C16H22N2S2: C, 62.75; H, 7.19; N, 9.09. Found: C,
62.73; H, 7.19; N, 8.98.
5-Hexadecyl-3-(phenylmethyl)-tetrahydro-2H-1,3,5, thia-
diazine-2-thione (21). This was obtained by benzyl
amine (2.18 mL, 20 mmol) and hexadecyl amine (4.82 g,
20 mmol) as described above and isolated as colourless
solid, yield 64%, mp 78–81 ꢁC. IR (KBr): 2918, 1595,
5-(3-Carboxyethyl)-3-dodecyl-tetrahydro-2H-1,3,5, thia-
diazine-2-thione (18). This was obtained by dodecyl
amine (4.62 mL, 20 mmol) and b-alanine (1.78 g, 20
mmol) as described above and isolated as colourless
solid, yield 76%, mp 139–142 ꢁC. IR (KBr): 3428, 1707,
1
1489; MS (FAB): 449 (M+H)+; H NMR (CDCl3): d
1
1502; MS (FAB): 375 (M+H)+; H NMR (CDCl3): d
7.39–7.31 (m, 5H, Ar-H), 5.34 (s, 2H, C6H5CH2), 4.40
(s, 2H, H-4), 4.27(s, 2H, H-6), 2.55 (t, 2H, J=7.6 Hz,
NCH2CH2), 1.26–1.13 (m, 28H, CH2’s), 0.88 (t, 3H,
J=6.6 Hz, CH2CH3); 13C NMR (CDCl3): d 193.5
(C¼S), 135.7 (Ar-C), 129.2, 128.9 and 128.5 (Ar-CH),
68.3 (C-4), 59.1 (C-6), 54.1 (CH2C6H5), 50.7, 32.3, 30.1,
29.9, 29.8, 29.7, 29.6, 27.5, 27.2, 23.1 (CH2’s), 14.5
(CH2CH3). Anal. calcd for C26H44N2S2: C, 69.64; H,
9.82; N, 6.25. Found: C, 69.71, H, 9.82, N, 6.25.
4.38 (s, 2H, H-4), 4.36(s, 2H, H-6), 3.97 (t, 2H, J=7.7
Hz, NCH2CH2COOH), 3.15 (t, 2H, J=6.4 Hz,
NCH2CH2), 2.61 (t, 2H, J=6.5 Hz, CH2COOH), 130–
1.26 (m, 20H, CH2’s), 0.88 (t, 3H, J=6.7 Hz, CH2CH3);
13C NMR (CDCl3): d 191.5 (C¼S), 177 (C¼O), 70.6 (C-
4), 58.2 (C-6), 52.8, 46.4, 33.4, 32.3, 30.0, 29.9, 29.7,
29.6, 27.2, 27.0, 23.1 (CH2’s), 14.5 (CH2CH3). Anal.
calcd for C18H34N2O2S2: C, 57.75; H, 9.09; N, 7.48.
Found: C, 57.76; H, 9.09; N, 7.42.
5-Carbethoxyethyl-3-(cyclopropyl)-tetrahydro-2H-1,3,5,
thiadiazine-2-thione (22). This was obtained by cyclo-
propyl amine (1.39 mL, 20 mmol) and 2-amino ethyl
propionate (2.34 g, 20 mmol) as described above and
isolated as colourless solid, yield 55%, mp 80–81 ꢁC. IR
General procedure for the synthesis of compounds 19–24
5-Cyclohexyl-3-(20-furfuryl)-tetrahydro-2H-1,3,5, thiadi-
azine-2-thione (19). To a magnetically stirred solution
of furfuryl amine (1.76 mL, 20 mmol) in 50 mL of H2O,
KOH (1.12 g, as 20% aq solution, 20 mmol) and CS2
(1.20 mL, 20 mmol) were added at 30 ꢁC. The reaction
mixture was stirred for 4 h more followed by addition of
37% formaldehyde solution (3.1 mL, 40 mmol) and
stirring continued for 1 h. The reaction mixture was fil-
tered and resulting filtrate was added drop wise to a
suspension of cyclohexyl amine (2.28 mL, 20 mmol) in
60 mL of phosphate buffer (pH 7.7)/toluene (1:2 mix-
ture) and stirred again for 1 h. The reaction mixture fil-
tered off and organic layer of the filtrate was separated,
dried over sodium sulphate, concentrated and the pro-
duct was allowed to solidify overnight which was fur-
ther purified by crystallization with ethanol as
colourless granules, yield 52%, mp 131–133 ꢁC. IR
1
(KBr): 2970, 1731, 1470; MS (FAB): 275 (M+H)+; H
NMR (CDCl3): d 4.38 (s, 2H, H-4), 4.30 (s, 2H, H-6),
4.17 (q, 2H, J=7.1 Hz, OCH2CH3), 3.13–3.04 (m, 3H,
NCH2 and NCH), 2.54 (t, 2H, J=6.49 Hz,
CH2COOH), 1.27 (t, 3H, OCH2CH3), 1.08–0.81 (m, 4H,
cyclopropyl ring protons); 13C NMR (CDCl3): d 195.1
(C¼S), 171.9 (C¼O), 71.8 (C-4), 61.2 (OCH2), 57.5 (C-
6), 46.5 (NCH2), 36.2 (NCH), 33.7 (CH2COOEt), 14.6
(OCH2CH3), 9.3 (cyclopropyl ring carbons). Anal. calcd
for C11H18N2O2S2: C, 48.18; H, 6.56; N, 10.24. Found:
C, 48.22; H, 6.52; N, 10.24.
5-Cyclohexyl-3-(cyclopropyl)-tetrahydro-2H-1,3,5, thia-
diazine-2-thione (23). This was obtained by cyclopropyl
amine (1.39 mL, 20mmol) and cyclohexyl amine (2.28
mL, 20 mmol) as described above and isolated as yellow
oil, yield 42%. IR (Neat): 2934, 1450; MS (FAB): 257
1
(KBr): 2928, 1595, 1487; MS (FAB): 297 (M+H)+; H
NMR (CDCl3): d 7.37 (d, 1H, J=1.8 Hz, furfuryl H-50),
6.47 (d, 1H, J=3.2 Hz, furfuryl H-30), 6.44 (dd, 1H,
J=3.1 and 1.8 Hz, furfuryl H-40), 5.31 (s, 2H, furfuryl
CH2), 4.55 (s, 2H, H-4), 4.49 (s, 2H, H-6), 2.69 (m, 1H,
NCH), 1.80–1.04 (m, 10H, cyclohexyl ring protons); 13C
NMR (CDCl3): d 193.9 (C¼S), 149.2 (C-20), 142.8 (C-
50), 111.2 (C-40), 110.8 (C-30), 66.7(C-4), 56.2(C-6), 55.6
(NCH), 47.0, 30.8, 25.9, 25.1 (CH2’s). Anal. calcd for
C14H20N2OS2: C, 56.75; H, 6.75; N, 9.46. Found: C,
56.32; H, 6.79; N, 9.62.
1
(M+H)+; H NMR (CDCl3): d 4.46 (s, 2H, H-4), 4.42
(s, 2H, H-6), 3.12–3.06 (m, 1H, cyclohexyl NCH), 2.89
(m, 1H, cyclopropyl NCH), 1.97–1.27 (m, 10H, cyclo-
hexyl ring protons), 1.02–0.85 (m, 4H, cyclopropyl ring
protons); 13C NMR (CDCl3): d 195.8 (C¼S), 69.0 (C-4),
55.6 (NCH), 55.4 (C-6), 36.2 (NCH), 33.3, 31.5, 30.4,
25.9, 25.1, 9.5, 8.9 (CH2’s). Anal. calcd for C12H20N2S2:
C, 56.25; H, 7.81; N, 10.93. Found: C, 56.26; H, 7.92; N,
11.01.
5 - Cyclohexyl - 3 - (phenylmethyl) - tetrahydro - 2H - 1,3,5,
thiadiazine-2-thione (20). This was obtained by benzyl
amine (2.18 mL, 20 mmol) and cyclohexyl amine (2.28
mL, 20 mmol) as described above and isolated as col-
ourless solid, yield 51%, mp 180–81 ꢁC. IR (KBr): 2930,
1492, 1447; MS (FAB): 307 (M+H)+; 1H NM R
(CDCl3): d 7.45–7.26 (m, 5H, Ar-H), 5.34 (s, 2H,
C6H5CH2), 4.50 (s, 2H, H-4), 4.39 (s, 2H, H-6), 2.61–
2.58 (m, 1H, NCH), 1.58–0.93 (m, 10H, cyclohexyl ring
protons); 13C NMR (CDCl3): d 193 (C¼S), 136 (Ar-C),
129.2, 129.1 and 128.5 (Ar-CH), 65.9 (C-4), 56.1 (C-6),
54.9 (NCH), 54.1, 30.7, 25.8, 25.0, (CH2’s). Anal. calcd
5-Cyclopropyl-3-(cyclopropyl)-tetrahydro-2H-1,3,5, thia-
diazine-2-thione (24). It was obtained by cyclopropyl
amine (1.39 mL, 20 mmol) and cyclopropyl amine (1.39
mL, 20 mmol) as described above and isolated as col-
ourless solid, yield 45%, mp 92–93 ꢁC; IR(KBr): 2999,
1
1449; MS (FAB): 215 (M+H)+; H NMR (CDCl3): d
4.40 (s, 2H, H-4), 4.35 (s, 2H, H-6), 3.15–3.10 (m, 1H,
NCH), 2.46–2.40 (m, 1H, NCH), 1.05–0.89 (m, 4H,
cyclopropyl ring protons), 0.66–0.58 (m, 4H, cyclopro-
pyl ring protons); 13C NMR (CDCl3): d 195.7 (C¼S),
71.5 (C-4), 58.1 (C-6), 36.1 (NCH), 32.2 (NCH), 9.5
(cyclopropyl ring carbons), 7.4 (cyclopropyl ring

D. Katiyar et al. / Bioorg. Med. Chem. 11 (2003) 4369–4375
4375
carbons). Anal. calcd for C9H14N2S2: C, 50.46; H, 6.54;
N, 13.08. Found: C, 50.42; H, 6.51; N, 12.92.
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The authors gratefully acknowledge financial support
from ICMR New Delhi, VW Foundation Germany and
Department of Ocean Development. We also thank
RSIC staff for spectral data and microanalysis.
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References and Notes
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