Synthesis and?studies on?some?new fluorine containing triazolothiadiazines as?possible antibacterial, antifungal and?anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2006.02.001

Synthesis of a series of 7-arylidene-6-(2,4-dichlorophenyl)-3-aryloxymethyl/anilinomethyl-1,2,4-t riazolo[3,4-b]-1,3,4-thiadiazines (3) by the condensation of 3-aryl-1-(2,4-dichloro-5-fluorophenyl)-2-bromo-propen-1-one (1) and 4-amino-5-mercapto-3-aryloxy

Full text of DOI:10.1016/j.ejmech.2006.02.001

European Journal of Medicinal Chemistry 41 (2006) 657–663
http://france.elsevier.com/direct/ejmech
Short communication
Synthesis and studies on some new fluorine containing
triazolothiadiazines as possible antibacterial,
antifungal and anticancer agents
B. Shivarama Holla a,*, B. Sooryanarayana Rao b, B.K. Sarojini c, P.M. Akberali b,
N. Suchetha Kumari d
a Department of Post-Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangothri 574 199, India
b Apotex Pharmachem India Pvt. Ltd., Plot No. 1A, Bommasandra Industrial Area, 4th Phase, Bangalore 562 158, India
c Department of Chemistry, P.A. College of Engineering, Nadupadavu, Kairangala Mangalore 574153, India
d Department of Biochemistry, K.S. Hegde Medical Academy, Deralakatte 574 160, India
Received 27 September 2005; received in revised form 18 December 2005; accepted 22 February 2006
Available online 17 April 2006
Abstract
Synthesis of a series of 7-arylidene-6-(2,4-dichlorophenyl)-3-aryloxymethyl/anilinomethyl-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (3) by the
condensation of 3-aryl-1-(2,4-dichloro-5-fluorophenyl)-2-bromo-propen-1-one (1) and 4-amino-5-mercapto-3-aryloxymethyl/anilinomethyl-1,2,4-
triazoles (2) is described. The newly synthesized compounds were characterized by elemental analysis IR, 1H NMR and mass spectral data. These
compounds were tested for their antimicrobial activities against Escherichia coli, Staphylococcus aureus (Smith), Psuedomonas aeruginosa
(Gessard), Bacillus subtilis and Candida albicans. Some of the newly synthesized compounds were also screened for their anticancer activity.
Among them compounds 3m, 3o, 3q showed in vitro anticancer activity.
© 2006 Elsevier SAS. All rights reserved.
Keywords: 1,2,4-Triazoles; 1,3,4-Thiadiazines; Antibacterial agents; Antifungal agents; Anticancer screening
1. Introduction
properties of previously reported 3-akyl/aryl-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazines [8], would benefit from the incorporation
of aryloxy and anilinomethyl group. A similar strategy on the
triazolothiadiazine template to produce antibacterials compar-
able or superior to nitrofurazone; actually, we found that repla-
cement of alkyl and aryl group by aryloxy and anilinomethyl
substituents was not advantageous. In the case antitumor activ-
ity in these template three compounds with 2-chlorophenoxy-
methyl, 4-chloro-3-methyl-phenoxymethyl and 4-chloro-anili-
nomethyl as substituent at C3 found to be active. Moreover,
screening of these triazolothiadiazines was expanded to include
antifungal activity.
1,2,4-Triazoles and their derivatives play important role in
medicinal, agricultural and industrial fields [1–4]. N-bridged
heterocyclic derivatives derived from 1,2,4-triazoles show var-
ied biological activities [5]. Recently, fluorinated heterocycles
have attracted attention due to the ability of fluorine to act as
polar hydrogen or hydroxyl mimic. Therefore substitution of
hydrogen by fluorine has been a strategy in designing mole-
cules for biological activity studies [6,7]. In continuation of
our search on the synthesis of biologically active fluorine con-
taining N-bridged heterocycles [8,9] and in an attempt to sig-
nificantly improve antibacterial and anticancer activities of tria-
zlothiadiazine (general structure 1) we considered substitution
at C3 by aryloxymethyl and anilinomethyl moieties. It was
hoped that these compounds in addition to retaining anticancer
2. Results and discussion
2.1. Chemistry
The synthesis of reported triazolothiadiazines 3a–3b1 was
carried out by the condensation of 3-aryl-1-(2,4-dichloro-5-
fluorophenyl)-2-bromo-2-propen-1-one (1) with 4-amono-5-
* Corresponding author. Fax: +91 8242 287 367
E-mail address: bksaroj@yahoo.com (B. Shivarama Holla).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.02.001

658
B. Shivarama Holla et al. / European Journal of Medicinal Chemistry 41 (2006) 657–663
mercapto-5-aryloxymethyl/anilinomethyl-1,2,4-triazoles (2) in
presence of alcoholic potassium hydroxide (Scheme 1). The
structure of newly synthesized compounds was confirmed by
sponding to exocyclic vinylic proton. The signals due to six
protons of 3,4-dimethoxy group appeared as two closely
packed singlets at 3.91 and 3.94, respectively. The six aromatic
protons of 3,4-dimethoxyphenyl and 3-chloro 4-fluoro phenyl
ring resonated as multiplets in the region δ 6.64–7.08. The aro-
matic protons of the 2,4-dichloro-5-fluorophenyl ring resonated
as two doublets at δ 7.33 (J = 8.5 Hz, ortho H–F coupling) and
δ 7.66 (J = 6 Hz, meta H–F coupling) integrating for one pro-
ton, respectively, this data confirmed the formation of com-
1
recording the IR, H NMR and mass spectra. Characterization
data of triazolothiadiazines are given in Table 1. IR spectrum
of 3i showed absorption bands at 3034, 2834, 1585, 1081 and
728 cm–1 due to CH, C=N, C–F, and C–Cl groups, respec-
tively. The absence of the absorption bands corresponding to
–NH2, –SH and C=O stretching frequency of the reactants
clearly confirmed the formation of triazolothiadiazines 3a–
3b1. The IR spectra of other compounds of the series showed
similar absorption bands and the data are listed in Table 1. The
1H NMR spectrum of 3n showed a singlet at δ 6.65 corre-
1
pound 3n. The H NMR spectra of some of the compounds
were recorded and their spectral data are given in Table 1.
Mass spectral data of the newly synthesized compounds were
recorded are also given in Table 1. Almost all the compounds
Scheme 1. Preparation of 7-arylidene-6-(2,4-dichloro5-fluorophenyl)-3-aryloxymethyl/anilinomethyl-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (3).

B. Shivarama Holla et al. / European Journal of Medicinal Chemistry 41 (2006) 657–663
659
Table 1
Characterization data of 7-arylidene-6-(4-dichloro-5-fluorophenyl)-3-substituted-1,2,4-triazolo [3,4-b]-1,3,4-thiadiazine (3)
Compound
numbers
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
R
R1
X
M.p. (°C)
Yield
(%)
74
78
71
68
76
73
81
76
74
78
71
73
79
75
68
77
71
76
80
82
76
70
77
78
81
80
82
76
Molecular formula
Analysis (%) Found (Calculated)
C
H
N
2-Cl
4-Cl
4-Cl, 3-CH3
2,4-Cl2
H
4-Cl
3-Cl 4-F
2-Cl
3,4-(OCH2O)
3,4-(OCH2O)
3,4-(OCH2O)
3,4-(OCH2O)
3,4-(OCH2O)
3,4-(OCH2O)
3,4-(OCH2O)
3,4-(OCH3)2
3,4-(OCH3)2
3,4-(OCH3)2
3,4-(OCH3)2
3,4-(OCH3)2
3,4-(OCH3)2
3,4-(OCH3)2
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
2,4-Cl2
2,4-Cl2
O
O
O
O
NH
NH
NH
O
O
O
246–248
296–298
168–169
185–187
180–181
215–117
158–160
198–200
160–162
165–167
184–186
173–175
201–203
174–176
168–170
178–180
180–182
168–170
182–184
226–228
196–198
196–198
175–175
96–98
C25H14Cl3FN4O3S
C25H14Cl3FN4O3S
C26H16Cl3FN4O3S
C25H13Cl4FN4O3S
C25H16Cl2FN5O2S
C25H15Cl3FN5O2S
C25H14Cl3F2N5O2S
C26H18Cl3FN4O3S
C26H18Cl3FN4O3S
C27H20Cl3FN4O3S
C26H17Cl4FN4O3S
C26H20Cl2FN5O2S
C26H19Cl3FN5O2S
C26H18Cl2F2N5O2S
C24H13Cl4FN4OS
C24H13Cl4FN4OS
C25H15Cl4FN4OS
C24H12Cl5FN4OS
52.011 (52.12) 2.52 (2.43)
9.61 (9.73)
9.82 (9.73)
9.64 (9.49)
9.02 (9.18)
12.64 (12.96)
12.06 (12.18)
11.64 (11.8)
9.23 (9.46)
9.28 (9.46)
7.74 (7.92)
8.78 (8.94)
12.34 (12.59)
11.62 (11.85)
11.36 (11.50)
9.71 (9.89)
9.74 (9.89)
9.48 (9.65)
9.18 (9.32)
13.04 (13.19)
12.28 (12.39)
12.09 (12.00)
9.24 (9.32)
9.16 (9.11)
9.16 (9.11)
8.73 (8.81)
13.04 (13.19)
12.28 (12.39)
12.09 (12.00)
51.94 (52.12)
52.71 (52.96)
48.91 (49.18)
55.21 (55.55)
52.06 (52.21)
50.34 (50.63)
52.56 (52.74)
52.42 (52.74)
53.24 (53.51)
49.67 (49.84)
55.88 (56.11)
52.57 (52.83)
51.04 (51.27)
50.61 (50.88)
50.58 (50.88)
51.48 (51.72)
47.54 (47.96)
54.07 (54.11)
50.63 (50.97)
49.08 (49.39)
47.62 (47.96)
47.71 (47.96)
48.51 (48.82)
45.12 (45.35)
54.07 (54.11)
50.63 (50.97)
49.08 (49.39)
2.30 (2.43)
2.69 (2.71)
2.04 (2.13)
2.84 (2.86)
2.58 (2.61)
2.28 (2.36)
2.95 (3.04)
2.91 (3.04)
3.23 (3.30)
2.59 (2.71)
3.61 (3.59)
3.16 (3.21)
2.90 (2.95)
2.26 (2.29)
2.24 (2.29)
2.61 (2.58)
1.91 1.99
2.80 (2.82)
2.41 (2.47)
2.26 (2.23)
1.95 (1.99)
1.93 (1.99)
2.22 (2.29)
1.71 (1.73)
2.80 (2.82)
2.41 (2.47)
2.26 (2.23)
4-Cl
4-Cl, 3-CH3
2,4-Cl2
H
4-Cl
3-Cl, 4-F
2-Cl
O
NH
NH
NH
O
O
O
4-Cl
4-Cl, 3-CH3
2,4-Cl2
H
4-Cl
3-Cl, 4-F
2-Cl
O
NH
NH
NH
O
O
O
C
24H15Cl3FN5S
C24H14Cl4FN5S
24H13Cl5F2N5S
C
C24H13Cl5FN4OS
C24H12Cl5FN4OS
C25H14Cl5FN4OS
C24H11Cl6FN4OS
C24H14Cl4FN5S
C24H14Cl4FN5S
C24H13Cl5F2N5S
4-Cl
4-Cl, 3-CH3
2,4-Cl2
H
4-Cl
3-Cl, 4-F
2,4-Cl2
2,4-Cl2
2,4-Cl2
2,4-Cl2
O
166–168
178–180
198–200
93–95
3z
NH
NH
NH
3a1
3b1
2,4-Cl2
3i: IR (KBr disc) γmax (cm−1): 3034 (C–H), 2834 (C–O), 1590 (C=N), 1081 (C–F), 728 (C–Cl); 3j: IR (KBr disc) γmax (cm−1): 2965 (C–H), 1597 (C–N), 1514
(C=C), 1086 (C–F), 731 (C–Cl); 3k: IR (KBr disc) γmax (cm−1): 3945 (C–H), 1605 (C–N), 1512 (C=C), 1076 (C–F), 731 (C–Cl); 3c: 1H NMR 400 MHz
(CDCl3): δ, 7.29 (d, 1H, Ar–H, JH–F ortho = 8.1 Hz), 7.49 (m, 2H, Ar–H), 7.61 (d, 1H, Ar–H, JH–F meta = 6.3 Hz), 7.73 (s, 1H, =CH–), 7.9 (m, 2H, Ar–H); 3d:
1H NMR 400 MHz (CDCl3): δ, 7.27 (d, 1H, Ar–H, J = 8 Hz), 7.37 (dd, 1H, Ar–H, J = 2 Hz, J = 2 Hz), 7.48 (d, 1H, Ar–H, J = 2.1 Hz), 7.57 (d, 1H, Ar–H,
JH–F meta = 6.3 Hz), 7.86, (s, 1H, CH, 8.02 (d, 1H, Ar–H, JH–F ortho = 8.5 Hz); 3f: 1H NMR 400 MHz (CDCl3): δ, 3.92 (s, 1H, NH), 4.6 (s, 2H, –CH2–), 6.67
(s, 2H, –OCH2O–), 6.71 (s, 1H, –CH=), 7.11–7.14 (m, 7H, Ar–H), 7.36 (d, 1H, Ar–H, JH–F ortho = 8.3 Hz), 7.63 (d, 1H, Ar–H, JH–F meta = 6.5 Hz); 3o: 1H
NMR 400 MHz (CDCl3): δ, 5.43 (s, 2H, –OCH2–), 6.96 (s, 1H, –CH=), 7.59 (d, 1H Ar–H, JH–F meta = 6.5 Hz), 6.93–7.53 (m, 9H, Ar–H); 3n: 1H NMR
300 MHz (CDCl3 DMSO-d6): δ, 3.91 (s, 1H, OCH3), 3.94 (s, 3H, –OCH3), 4.4 (t, 1H, NH), 4.56 (d, 2H, –CH2–), 6.65 (s, 1H, –CH=), 7.32 (d, 1H, Ar–H,
JH–F ortho = 8.5 Hz), 7.65 (d, 1H, Ar–H, JH–F meta = 6.5 Hz), 6.57–7.08 (m, 6H, Ar–H); 3t: 1H NMR 300 MHz (CDCl3): δ, 4.36 (s, 1H, –NH–), 4.56 (s, 2H,
–CH2–), 6.69 (s, 1H, –CH=). 6.56–7.44 (m, 2H, Ar–H), 7.66 (d, 1H, Ar–H JH–F meta = 6.5 Hz); 3w: 1H NMR 300 MHz (CDCl3): δ, 5.29 (s, 2H, –OCH2–),
1
6.85 (s, 1H, –CH=), 6.97–7.53 (m, 8H, Ar–H), 7.63 (d, 1H, Ar–H, JH–F meta = 8.6 Hz); 3x: H NMR 300 MHz (CDCl3): δ, 2.32 (s, 3H, –CH3), 5.26 (s, 2H,
–OCH2–), 6.71 (s, 1H, –CH=), 6.79–7.71 (m, 8H, Ar–H); 3y: 1H NMR 300 MHz (CDCl3): δ, 5.30 (s, 2H, –OCH2–), 6.87 (s, 1H, –CH=), 6.99–7.62 (m, 7H,
Ar–H), 7.87 (d, 1H, Ar–H, JH–F ortho = 8.6 Hz); 3a: m/z 574 (80%, M+), 407 (80%, M+-2-chloroaryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluor-
obenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 121 (60% 3,4-methylenedioxyphenyl cation); 3b: m/z 574 (70%, M+), 407 (80%, M+-4-chloroaryloxy
methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 121(50% 3,4-methylenedioxyphenyl cation);
3c: m/z 588 (90%, M+), 407 (80%, M+-4-chloro-3-methyl-aryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (80%, 2,4-dichloro-
5-flurophenyl cation) 121 (60% 3,4-methylenedioxyphenyl cation); 3d: m/z 608 (50%, M+), 407 (80%, M+-2,4-dichloroaryloxy methyl nitrile radical), 189
(100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (44%, 2,4-dichloro-5-flurophenyl cation) 121 (50% 3,4-methylenedioxyphenyl cation); 3e: m/z 539 (20%, M+),
407 (90%, M+-anilinomethyl nitrile radical), 189 (10%, 2,4-dichloro-5-fluorobenzonitrile), 163 (100%, 2,4-dichloro-5-flurophenyl cation) 121 (60% 3,4-methyle-
nedioxyphenyl cation); 3f: m/z 573 (30%, M+), 407 (80%, M+-4-chloroanilinomethyl nitrile radical), 189 (70%, 2,4-dichloro-5-fluorobenzonitrile), 163 (100%,
2,4-dichloro-5-flurophenyl cation) 121 (60% 3,4-methylenedioxyphenyl cation); 3g: m/z 591 (30%, M+), 407 (90%, M+-3-chloro-4-fluoro-anilinomethyl nitrile
radical), 189 (25%, 2,4-dichloro-5-fluorobenzonitrile), 163 (100%, 2,4-dichloro-5-flurophenyl cation) 121 (60% 3,4-methylenedioxyphenyl cation); 3h: m/z 590
(30%, M+), 423 (80%, M+-2-chloroaryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (14%, 2,4-dichloro-5-flurophenyl cation)
137 (60% 3,4-dimethoxyphenyl cation); 3i: m/z 590 (40%, M+), 423 (80%, M+-4-chloroaryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzoni-
trile), 163 (24%, 2,4-dichloro-5-flurophenyl cation) 137 (60% 3,4-dimethoxyphenyl cation); 3j: m/z 604 (50%, M+), 423 (60%, M+-4-chloro-3-methyl-aryloxy-
methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 137 (80% 3,4-dimethoxyphenyl cation); 3k:
m/z 628 (70%, M+), 423 (80%, M+-2,4-dichloroaryloxymethyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophe-
nyl cation) 137 (50% 3,4-dimethoxyphenyl cation); 3l: m/z 555 (20%, M+), 423 (90%, M+-anilinomethyl nitrile radical), 189 (10%, 2,4-dichloro-5-fluorobenzo-
nitrile), 163 (100%, 2,4-dichloro-5-flurophenyl cation) 137 (60% 3,4-dimethoxyphenyl cation); 3m: m/z 589 (50%, M+), 423 (70%, M+-4-chloro-anilinomethyl
nitrile radical), 189 (30%, 2,4-dichloro-5-fluorobenzonitrile), 163 (100%, 2,4-dichloro-5-flurophenyl cation) 137 (60% 3,4-dimethoxyphenyl cation); 3n: m/z 607
(100%, M+), 423 (80%, M+-3-chloro-4-fluoro anilino radical), 444 (56%, M+-2,4-dichloro-5-fluorophenyl radical), 189 (6%, 2,4-dichloro-5-fluorobenzonitrile),
163 (4%, 2,4-dichloro-5-flurophenyl cation); 3o: m/z 564 (80%, M+), 397 (80%, M+-2-chloroaryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluoro-
benzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 111 (60% 4-chlorophenyl cation); 3p: m/z 564 (80%, M+), 397 (50%, M+-4-chloroaryloxy methyl ni-
trile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 111 (60% 4-chlorophenyl cation); 3q: m/z 578 (80%,
M+), 397 (80%, M+-M+-4-chloro-3-methyl-aryloxymethyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl ca-

660
B. Shivarama Holla et al. / European Journal of Medicinal Chemistry 41 (2006) 657–663
tion) 111 (70% 4-chlorophenyl cation); 3r: m/z (M+ is not observed), 397 (8%, M+-2,4-Cl2C6H3OCH2CN+) 189 (14%, 2,4-dichloro-5-fluorobenzonitrile), 163
(22%, 2,4-dichloro-5-flurophenyl cation) 111 (100% 4-chlorophenyl cation); 3s: m/z 510 (20%, M+), 397 (80%, M+-anilinomethyl nitrile radical), 189 (100%,
2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 111 (60% 4-chlorophenyl cation); 3t: m/z 544 (20%, M+), 397 (80%, M+-4-chlor-
oanilinomethyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 111 (60% 4-chlorophenyl cation) 3u:
m/z 562 (5%, M+), 397 (70%, M+-3-chloro-4-fluoro-anilinomethyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-fluro-
phenyl cation) 111 (60% 4-chlorophenyl cation) 3v: m/z 599 (5%, M+), 432 (90%, M+-2-chloroaryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluoro-
benzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 145 (60% 2,4-dichlorphenyl cation); 3w: m/z 599 (5%, M+), 432 (90%, M+-4-chloroaryloxy methyl ni-
trile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (24%, 2,4-dichloro-5-flurophenyl cation) 145 (60% 2,4-dichlorphenyll cation); 3x: m/z 613 (5%,
M+), 432 (90%, M+-4-chloro-3-methyl-aryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (34%, 2,4-dichloro-5-flurophenyl ca-
tion) 145 (60% 2,4-dichlorphenyll cation); 3y: m/z 633 (5%, M+), 432 (90%, M+-2,4-dichloro-aryloxy methyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluoro-
benzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 145 (60% 2,4-dichlorphenyll cation); 3z: m/z 563 (20%, M+), 432 (80%, M+-anilinomethyl nitrile radi-
cal), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 145 (50% 2,4-dichlorphenyll cation); 3a1: m/z 597 (20%, M+),
432 (80%, M+-4-chloro-anilinomethyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile), 163 (4%, 2,4-dichloro-5-flurophenyl cation) 145 (60% 2,4-
dichlorphenyl cation); 3b1: m/z 615 (20%, M+), 432 (80%, M+-3-chloro-4-fluoro-anilinomethyl nitrile radical), 189 (100%, 2,4-dichloro-5-fluorobenzonitrile),
163 (4%, 2,4-dichloro-5-flurophenyl cation) 145 (80% 2,4-dichlorphenyl cation).
showed molecular ion peaks revealing the stability of the com-
pounds. The major peaks in the spectra appeared were due to
the loss of aryloxy/anilinomethyl nitrile radical from the mole-
cular ion, formation of 2,4-dichloro-5-fluoro benzonitrile radi-
cal ion and aryl cations.
GI50 value = 0.336 μM against leukemia MOLT-4 cell line
among active compounds. Compound 3m [4-chloro-anilino-
methyl at C3 and 3,4-dimethoxy benzylidene at C7 substitu-
tion] appears to be least active with GI50 value > 100 μM for
some of the tested cell lines. In the alkyl/aryl substituted tria-
zolothiadiazine series compounds with methyl group at C3 and
3,4-methylenedioxy benzylidene at C7, methyl at C3 and 4-
chloro benzylidene at C7, methyl at C3 and 3,4-dimethoxy
benzylidene at C7 and phenyl group at C3 and 3,4-diomethoxy
benzylidene at C7 were found to be active. With this informa-
tion it is immature to comment on structure activity relation-
ship. However, it appears to be the presence of 4-chloro ben-
zylidene and 3,4-dimethoxy benzylidene at C7 may also
contribute to their activity.
2.2. Anticancer activity
In the present anticancer screening program, compounds
3m, 3o, 3q possessed growth percentage to less than 32%
against all the tested cell lines and were regarded as active
compounds (Table 2). These three compounds 3m, 3o and 3q
were then passed on for evaluation in the full panel of 60 cell
lines derived from seven cancer types namely, lung, colon,
melanoma, renal, ovarian, CNS and leukemia. These com-
pounds showed antiproliferative activity on the whole cell pa-
nel, although they did not prove cytotoxic or cytostatic at the
maximum tested concentration (100 μM). The screening data
are presented in Table 3. Compound 3o [2-chloroaryloxy
methyl at C3 and 4-chloro benzylidene at C7 substitution]
showed highest activity with GI50 value < 10 μM against all
tested 60 cell lines. Whereas compound 3q [4-chloro-3-
methyl-aryloxy methyl at C3 and 4-chloro benzylidene at C7
substitution] showed moderate activity GI50 value < 50 μM
against all tested 60 cell lines though it showed highest activity
2.3. Antibacterial activity
The newly synthesized thiadiazines 3 were screened for
their antibacterial activity against Escherichia coli, Staphylo-
coccus aureus (Smith), Psuedomonas aeruginosa (Gessard)
and Bacillus subtilis bacterial strains by serial dilution method
[11]. Almost all compounds exhibited comparable activity with
standard nitrofurazone, but did not prove to be more active
than the previously reported compounds in this template. Com-
pound 3i, 7-(3,4-dimethoxy benzylidene)-6-(2,4-dichloro-5-
Table 2
Preliminary in vitro anticancer screening a data of triazolothiadiazines
Compound
numbers
NSC number
Sample
Growth percentageb
Activityc
concentration × 10−4
(M)a
Lung NCI-H 460
Breast MCF 7
CNS SF-268
3a
3b
3f
3h
3i
713313
713314
713319
713315
713316
713317
713320
713310
713311
713312
713318
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
58
83
44
47
61
66
16
–30
70
–30
97
65
89
24
49
66
58
10
0
89
44
59
73
88
79
48
14
93
–14
32
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
Active
Inactive
Active
3j
3m
3o
3p
3q
3t
67
–10
73
Inactive
a
Fixed concentration assay (100 μM; standard NCI protocol).
Percent cell growth reduction following 48-h incubation with test compounds (optical density, sulforhodamine procedure).
Active when growth percentage is < 32% for any of the three cell lines.
b
c

B. Shivarama Holla et al. / European Journal of Medicinal Chemistry 41 (2006) 657–663
661
fluorophenyl)-3-(4-chloroaryloxy methyl)-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine showed highest activity (MIC 6 μg ml–1)
against all the tested bacteria. The compound 7-(3,4-dimethoxy
benzylidene)-6-(2,4-dichloro-5-fluorophenyl)-3-ethyl-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine, which was emerged as most
active compound in the earlier template exhibited MIC value 0.
312 μg ml–1 against E. coli, S. aureus (Smith), P. aeruginosa
(Gessard) and against B. subtilis 0.625 μg ml–1. The results of
this study is given in Table 4. With this result we can not arrive
at any conclusion regarding SAR but for the same substituent
3,4-dimethoxy benzylidene at C7. This moiety may be contri-
buting to their enhanced activity.
Table 3
Sixty cell line in vitro anticancer screening (GI50, μM) triazolothiadiazines 3m,
3o and 3q
Panel/cell line
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
GI50 (μM)
3o
3m
3q
4.97
45.7
19.0
10.9
7.58
21.0
9.03
4.48
3.76
5.98
3.08
4.38
2.95
1.91
8.37
0.336
11.7
7.96
Non-small cell lung cancer
EKVX
HOP-62
> 100
> 100
17.0
> 100
28.1
> 100
53.6
27.7
10.1
15.8
1.36
20.6
1.96
3.42
3.38
5.49
39.4
43.1
18.1
46.0
27.4
38.7
25.9
14.7
HOP-92
NCI-H226
NCI-H23
NCIH322M
NCI-H460
NCI-H 522
Colon cancer
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
2.4. Antifungal activity
Newly prepared compounds were screened for their antifun-
gal activity against Candida albicans (NCIM no. 3100) in
DMSO by serial plate dilution method [12,13]. Activity of
the compound was compared with amphotericin B as standard
drug. The minimum inhibitory concentration (MIC) for the am-
photericin B in DMSO is 1 μg ml–1 against the C. albicans.
The antifungal study revealed that the tested compound 3
showed moderate to good antifungal activity with MIC value
6.0 μg ml–1 for most of the tested compounds. The results of
this study is given in Table 5. Hence these series of compounds
may emerge as good antifungal agents.
31.1
14.8
15.9
28.6
83.8
15.8
3.60
2.74
3.71
3.18
3.21
3.54
19.6
12.8
19.7
16.7
22.9
17.4
20.6
2.83
4.28
5.59
3.20
22.7
29.9
44.9
18.3
SF-295
SF-539
U251
> 100
> 100
> 100
Melanoma
LOX IMVI
M14
SK MEL-2
SK MEL-28
SK MEL-5
UACC-257
Ovarian cancer
IGRVOI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-O
A498
ACHN
CAKI-1
RXF-393
TK-10
UO-31
Prostate cancer
PC-3
Du-145
Breast cancer
MCF7
Table 4
29.3
59.8
> 100
> 100
> 100
> 100
3.92
3.75
13.3
3.54
3.97
21.8
21.9
25.0
29.0
19.8
18.0
38.5
Antibacterial activities of 7-arylidene-6-(2,4-dichloro-5-fluorophenyl)-3-(sub-
stituted)-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (3)
Compound
numbers
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
MIC (μg ml–1
P. aeruginosa B. subtilis
)
E. coli
6.0
6.0
S. aureus
12.5
12.5
12.5
6.0
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
6.0
6.0
6.0
6.0
12.5
12.5
12.5
12.5
12.5
6.0
6.0
> 100
48.1
22.5
89.0
24.7
3.80
17.1
9.85
3.96
6.02
19.3
21.0
30.5
19.5
24.1
18.8
50.9
12.5
12.5
6.0
12.5
12.5
6.0
6.0
6.0
12.5
6.0
6.0
12.5
6.0
6.0
12.5
12.5
6.0
6.0
12.5
12.5
6.0
6.0
12.5
6.0
12.5
6.0
12.5
6.0
25.0
12.5
6.0
12.5
12.5
12.5
12.5
25.0
12.5
12.5
6.0
> 100
12.5
6.0
12.5
6.0
24.5
> 100
48.4
23.5
15.8
86.7
56.9
4.23
14.2
4.65
8.02
9.88
11.6
6.40
28.0
24.2
33.5
18.6
57.0
32.6
27.5
12.5
6.0
12.5
6.0
6.0
6.0
12.5
12.5
6.0
12.5
12.5
6.0
12.5
12.5
12.5
12.5
6.0
12.5
6.0
12.5
12.5
6.0
67.2
71.6
12.3
5.97
21.5
17.4
6.0
6.0
23.9
17.9
> 100
51.1
27.2
24.0
44.3
5.15
4.24
3.72
11.8
2.26
2.24
13.6
23.3
33.2
29.7
15.3
24.2
32.4
20.4
NCI/ADR-RES
MDA-MB-231/ATCC
HS-578T
MDA-MB-435
MDA-N
3w
3x
3y
12.5
25.0
12.5
12.5
6.0
12.5
12.5
12.5
6.0
12.5
6.0
12.5
6.0
3z
3a1
12.5
6.0
12.5
12.5
12.5
–
6.0
12.5
12.5
T-47D
3b1
12.5
6.0
The other two standard parameters, TGI and LC50 were above 100 μM (max-
imum tested concentration).
Nitrofurazone

662
B. Shivarama Holla et al. / European Journal of Medicinal Chemistry 41 (2006) 657–663
Table 5
1b: 3-(3,4-Dimethoxy phenyl)-1-(2,4-chloro-5-fluorophe-
nyl)-2-bromo-2-propen-1-one, m.p.: 95–97 °C; Yield, 75%;
yellow needles Analysis: Found (calculated): C: 47.35
(47.00), H: 2.68 (2.76).
Antifungal activities of 7-arylidene-6-(2,4-dichloro-5-fluorophenyl)-3-(substi-
tuted)-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (3)
Compound numbers
MIC in μg ml–1
Zone of inhibition (in mm)
C. albicans
6.0 (15–20)
6.0 (15–20)
6.0 (15–20)
6.0 (15–20)
12.5 (< 10)
6.0 (15–20)
12.5 (< 8)
1c:
3-(4-Chlorophenyl)-1-(2,4-chloro-5-fluorophenyl)-2-
bromo-2-propen-1-one, m.p.: 115–18 °C; Yield, 75%; pale yel-
low needles Analysis: Found (calculated): C: 41.55 (42.00), H:
1.80 (1.86).
1d: 3-(4-Dichlorophenyl)-1-(2,4-chloro-5-fluorophenyl)-2-
bromo-2-propen-1-one, m.p.: 76–78 °C; Yield, 74%; pale yel-
low needles Analysis: Found (calculated): C: 40.46 (40.63), H:
1.29 (1.35).
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
12.5 (< 8)
6.0 (15–20)
12.5 (10–12)
6.0 (15–20)
12.5 (< 8)
6.0 (15–20)
6.0 (15–20)
12.5 (< 8)
6.0 (15–20)
12.5 (< 8)
6.0 (15–20)
12.5 (< 8)
3.1.2. General procedure for the synthesis of 4-amino-5-
mercapto-3-aryloxymethyl/amilinomethyl-1,2,4-triazoles (2)
A mixture of carboxylic acid (0.01 mol) and thiocarbohy-
drazide (0.01 mol) contained in round-bottomed flask was
heated on a mantle until the contents melted. The mixture
was maintained at this temperature for 15–20 min. The product
obtained on cooling was treated with sodium bicarbonate solu-
tion to dissolve the unreacted carboxylic acid if any. It was
then washed with water and collected by filtration. Then the
product was recrystallized from a mixture of dioxane and etha-
nol to afford the title compounds.
2a: 3-(4-Chlorophenoxymethyl)-4-amino-5-mercapto-1,2,4-
triazole, m.p. 183 °C (Ref. [14] m.p. 183–185 °C).
2b: 3-(2-Chlorophenoxymethyl)-4-amino-5-mercapto-1,2,4-
triazole, m.p. 162 °C (Ref. [14] m.p. 162–165 °C).
2c: 3-(4-Chloro-3-methyl-phenoxymethyl)-4-amino-5-mer-
capto-1,2,4-triazole, m.p. 167 °C (Ref. [14] m.p. 167–169 °C).
2d: 3-(2,4-Dichloro-phenoxymethyl)-4-amino-5-mercapto-
1,2,4-triazole, m.p. 186 °C (Ref. [14] m.p. 185–187 °C).
2e: 3-Anilinomethyl-4-amino-5-mercapto-1,2,4-triazole, m.
p. 216 °C (Ref. [15] m.p. 215–217 °C).
3t
6.0 (15–20)
6.0 (15–20)
12.5 (< 8)
3u
3v
3w
3x
3y
3z
3a1
3b1
12.5 (< 8)
12.5 (10–12)
6.0 (15–20)
12.5 (< 8)
6.0 (15–20)
12.5 (< 10)
3. Experimental
3.1. Chemistry
2f: 3-(4-Chloroanilinomethyl)-4-amino-5-mercapto-1,2,4-
triazole, m.p. 204 °C (Ref. [15] m.p. 204–206 °C).
2g: 3-(3-Chloro-4-fluoro-anilinomethyl)-4-amino-5-mercap-
to-1,2,4-triazole, m.p. 189–191 °C; Elemental analysis, Found
(calculated): C: 41.39 (41.61), H: 3.39 (3.46) and N: 21.50
(21.57).
Melting points were taken in open capillary tubes and are
uncorrected. IR spectra in KBr pellets were recorded on JAS-
CO FT-IR 5300 Infrared Spectrophotometer. H NMR spectra
were recorded in DMSO-d6 on a Varian (300 MHz) spectro-
meter using TMS as an internal standard and the mass spectra
were recorded on aVG-s-70 micro mass, mass spectrometer
operating at 70 eV. The purity of the compounds were checked
by TLC using ethyl acetate/methanol [8:2] solvent system. Io-
dine was used as visualizing agent.
1
3.1.3. Synthesis of 7-arylidene-6-(2,4-dichloro-5-
fluorophenyl)-3-substitued 1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazines (3a–3b1)
An equimolar mixture of 2-bromo-2-propen-1-one (1) and
3-substituted 4-amino-5-mercapto-1,2,4-triazole (2) and solu-
tion of potassium hydroxide (10%, 2.5 ml) in ethanol (25 ml)
was kept under reflux on a water bath for about 5 h. The reac-
tion mixture was cooled and the precipitated solid was filtered,
washed with water, dried and recrystallized from ethanol and
dioxane. The analytical data are given in Table 1.
3.1.1. General procedure for the synthesis of 3-aryl-1-(2,4-
chloro5-fluorophenyl)-2-bromo-2-propen-1-one (1)
A mixture of dibromopropanone (0.01 mol) and triethyla-
mine (0.05 mol) in dry toluene (50 ml) was stirred for 24 hours.
The precipitated triethylammoniumhydrobromide was filtered.
The filtrate was concentrated under reduced pressure. The pre-
cipitated solid was filtered, dried and recrystallized from
chloroform.
3.2. Pharmacology
1a: 3-(3,4-Methylenedioxy phenyl)-1-(2,4-chloro-5-fluoro-
phenyl)-2-bromo-2-propen-1-one, m.p.: 110–12 °C; Yield,
78%; yellow needles Analysis: Found (calculated): C: 45.81
(45.93), H: 1.95 (1.91).
3.2.1. Anticancer activity
Eleven of the newly synthesized compounds 3 were
screened for their antitumor activities at NIH, Bethesda, MD,

B. Shivarama Holla et al. / European Journal of Medicinal Chemistry 41 (2006) 657–663
663
USA under the Drug Discovery Programme of NCI according
to the procedure suggested by Boyd and Paull [10] in a primary
three cell line-one dose anticancer assay against NCI-H 460
(lung), MCF 7(breast) and SF 268 (CNS). In the current pro-
tocol each cell line is inoculated on an incubated microtiter-
plate. The test agents are added at a single concentration and
the culture is incubated for 48 h. Endpoint determinations are
made with sulforhodamine B, a protein binding dye. Results
for each test agents are reported as the percent growth of the
treated cells when compared with the untreated control cells.
Compounds which reduce the growth of any one of the cell
lines to 2% or less (negative numbers indicate cell kill) are
passed on for evaluation in the full panel of 60 cell lines over
a 5-log dose range.
benzylidene at C7 and phenyl group at C3 and 3,4-dimethoxy
benzylidene at C7 were found to be active. Compound 3o
[2-chloroaryloxy methyl at C3 and 4-chloro benzylidene at
C7 substitution] showed highest activity with GI50
value < 10 μM against all tested 60 cell lines. Whereas com-
pound 3q [4-chloro-3-methyl-aryloxy methyl at C3 and 4-
chloro benzylidene at C7 substitution] showed moderate activ-
ity GI50 value < 50 μM against all tested 60 cell lines though it
showed highest activity GI50 value = 0.336 μM against leuke-
mia MOLT-4 cell line among active compounds. Compound
3m [4-chloro-anilinomethyl at C3 and 3,4-dimethoxy benzyli-
dene at C7 substitution] appears to be least active with GI50
value > 100 μM for some of the tested cell lines. As the com-
pounds for the anticancer studies were selected randomly, we
are unable to comment on SAR, because it is a preliminary
study. However, it is hoped that triazolothiadiazines may
emerge as potential compounds with varied biological activ-
ities in future.
3.3. Antibacterial activity
The newly synthesized thiadiazines 3 were screened for
their antibacterial activity against E. coli, S. aureus (Smith),
P aeruginosa (Gessard) and B. subtilis bacterial strains by
serial dilution method [11].
Acknowledgements
The authors are grateful to Head, R.S.I.C., C.D.R.I., Luck-
now and The Director, R.S.I.C., Punjab University, Chandi-
garh, for providing microanalysis, IR, 1H NMR and mass spec-
tral data. The authors are grateful to Dr. V.L. Narayanan,
National Institutes of Health (NIH), Bethesda, MD, USA, for
the antitumor activity screening studies reported in this paper.
3.4. Antifungal activity
Sabourands agar media was prepared by dissolving peptone
(1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 ml)
and adjusted the pH to 5.7. Normal saline was use to make a
suspension of spores of fungal strain for lawning. A loopful of
particular fungal strain was transferred to 3 ml saline to get a
suspension of corresponding species. Agar media of 20 ml was
poured in to each Petri dishes. Excess of suspension was dec-
anted and the plates were dried by placing in an incubator at
37 °C for 1 h. Using an agar punch wells were made on these
seeded agar plates and 5–100 μg ml–1 of the test compounds in
DMSO were added in to each well labeled. A control was also
prepared for the plates in the same way using solvent DMSO.
The Petri dishes were prepared in triplicate and maintained at
37 °C for 3–4 days. Diameter of the inhibition zone and MIC
were noted. The results of such studies are given in Table 5.
References
[1] G. Martin R.A. Lahti, A.D. Rudzik, D.J. Duchamp, C. Chidester, T. Sca-
hill, J. Med. Chem. 21 (1978) 542, Chem. Abstr. 88 (1978) 192783e.
[2] C.F.H. Allen, H. Bell, J. Org. Chem. 26 (1946) 11.
[3] B.A. Shimanski, Tr. Vees. Ged. Obsh. Akad. Nauk. USSR, 14 (1963)
1474, Chem. Abstr. 62 (1965) 8836a.
[4] M.B. Weed, Chem. Abstr. 62 (1965) 4544a (US 3,152,880).
[5] B.S. Holla, B. Kalluraya, K.R. Sridhar, E. Drake, L.M. Thomas, K.K.
Bhandary, M.J. Levine, Eur. J. Med. Chem. 29 (1994) 301 (and refer-
ences cited therein).
[6] O.H. David, S.R. Henry, Chem. Commun. 45 (1997) 645.
[7] R. Filler, Y. Kabayashi, Biomedicinal Aspects of Fluorine Chemistry,
Elsevier, Amsterdam, 1992.
4. Conclusions
[8] B.S. Holla, B.K. Sarojini, B.S. Rao, P.M. Akberali, N.S. Kumari, V.
Shetty, Il Farmaco 56 (2001) 565 (and references cited therein).
[9] B.S. Holla, B.S. Rao, B.K. Sarojini, P.M. Akberali, Eur. J. Med. Chem.
39 (2004) 777.
[10] M.R. Boyd, K.D. Paull, Some practical considerations and application of
the National Cancer Institute in vitro anticancer drug discovery screen,
Drug Dev. Res. 34 (1995) 91.
[11] E.J. Stokes, G.L. Ridgway, in Clinical Bacteriology, Fifth ed., Churchill
Livingstone, Edward Arnold, London and New York, 1980 (226 pp.)
[12] R. Cruichshank, J.P. Dugid, B. Marmion, R.H.A. Swain, Medical Micro-
biology, 11, Churchill Livingstone, London, 1975 (190 pp.)
[13] A.H. Collins, Microbiological methods, second ed, Butterworth, London,
1976.
In a hope to improve antibacterial and anticancer activities
of 3-alkyl/aryl-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines 3, we
considered substitution at C3 by aryloxymethyl and anilino-
methyl group for alkyl/aryl groups which was reported in our
earlier work. But actually, we found that replacement of alkyl
and aryl group by aryloxymethyl and anilinomethyl substitu-
ents was not advantageous in the case of antibacterial activity.
In the case of antitumor activity only 3o, 3m and 3q were
emerged as active compounds whereas in the alkyl/aryl substi-
tuted triazolothiadiazine series, compounds with methyl at C3
and 3,4-methylenedioxy benzylidene at C7, methyl at C3 and
4-chloro benzylidene at C7, methyl at C3 and 3,4-dimethoxy
[14] B.S. Holla, B. Kalluraya, Ind. J. Chem. 27B (1988) 683.
[15] B.S. Holla, K.V. Udupa, Il Farmco 47 (1992) 305.