Synthesis of some new biologically active thiadiazolotriazinones - Part III

Article abstract of DOI:10.1016/S0014-827X(02)01260-0

4-Amino-6-arylmethyl/tert-butyl-3-mercapto-1,2,4-triazin-5(4H)-ones (1) were condensed with arylfuroic acids (2) to yield 7-(5-aryl-2-furyl)-3-arylmethyl/tert-butyl-4H-1,3,4-thiadiazolo[2,3-c]-1,2, 4-triazin-4-ones (3). The newly synthesized compounds exh

Full text of DOI:10.1016/S0014-827X(02)01260-0

Il Farmaco 57 (2002) 693–696
www.elsevier.com/locate/farmac
Short Communication
Synthesis of some new biologically active
thiadiazolotriazinones–Part IIIꢀ
B. Shivarama Holla a,*, B. Sooryanarayana Rao a, Richard Gonsalves a, B.K. Sarojini a,
K. Shridhara b
a Department of Post-Graduate Studies and Research in Chemistry, Mangalore Uni6ersity, Mangalagangothri 574 199, Manglore, Karnataka
State, India
b Rallis Agrochemical Research Station, Plot Nos. 21 and 22, Phase II, Peenya, Industrial Area, Bangalore 560 058, India
Received 30 April 2001; accepted 12 February 2002
Abstract
4-Amino-6-arylmethyl/tert-butyl-3-mercapto-1,2,4-triazin-5(4H)-ones (1) were condensed with arylfuroic acids (2) to yield
7-(5-aryl-2-furyl)-3-arylmethyl/tert-butyl-4H-1,3,4-thiadiazolo[2,3-c]-1,2,4-triazin-4-ones (3). The newly synthesized compounds
exhibited antibacterial activity comparable to that of nitrofurazone. In addition, two compounds displayed in vitro antitumor
´
activity with moderate growth inhibition against a panel of 60 tumor cell lines. © 2002 Editions scientifiques et me´dicales Elsevier
SAS. All rights reserved.
Keywords: Arylmethyltriazinones; Arylfuroicacid; Thiadiazolotriazinones; Antibacterial activity; Antitumor activity
1. Introduction
In continuation of our studies on the synthesis of
biologically active thiadiazolotriazinones [1,2] and in an
attempt to significantly improve antibacterial activity,
we considered substitution at C7 by furyl moieties. It
was hoped that these compounds (general structure I),
in addition to retaining good properties of previous
Moreover selection of C3 substituents was made after
7-aryl thiadiazolotriazinones, would benefit from the
previous results in this template, favoring alkyl [2],
incorporation of great part of the furaldehyde semicar-
benzyl and halo-benzyl groups. Screening of these novel
thiadiazolotriazinones was expanded to include antitu-
bazone pattern(OꢀCꢁNꢁNꢀC-furyl) characteristic of ni-
trofuran antibiotics. A similar strategy on the tria-
mor activity.
zolothiadiazole template produced antibacterials com-
parable or superior to nitrofurazone; actually, we found
that replacement of nitro by halophenyls as the 2-furyl
substituent (e.g. Ib versus IIa) was advantageous [3].
Following these results, and with a view to avoid long
term toxicity of nitrofuran drugs, halophenylfurans
were selected as C7 moieties for the present work.
2. Chemistry
For the present work, three 6-arylmethyl-4-amino-3-
mercapto-1,2,4-triazin-5(4H)-ones
(R=benzyl,
4-
chlorobenzyl and 2,4-dichlorobenzyl) were prepared by
condensing the respective azalactones with thiocarbo-
hydrazide [4]. The 6-tert-butyl-4-amino-3-mercapto-
1,2,4-triazin-5(4H)-one was obtained commercially and
used after recrystallization from ethanol. The 5-aryl-2-
furoic acids (2) were synthesized through Meerwein
reaction [5]. The triazinones 1 were then condensed
ꢀ Presented at the ‘National Seminar on New Vistas in Bio-active
Agents’ held at the Department of Chemistry, Gandhigram Rural
University, Gandhigram, Tamil Nadu, India, 21–23 December 1999.
* Corresponding author
E-mail address: hollabs@yahoo.com (B. Shivarama Holla).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(02)01260-0

694
B. Shi6arama Holla et al. / Il Farmaco 57 (2002) 693–696
Table 1
Data of 7-substituted-3-arylmethyl/tert-butyl-1,3,4-thiadiazolo-[2,3-c]-1,2,4-triazin-4-ones (3)
All the compounds were analyzed satisfactorily for their N content (90.3%). IR (KBr, kmax cm−1): 3f, 3111(CꢁH), 1699(CꢀO), 1537(CꢀN),
1519(CꢀC); 3h, 3107(CꢁH), 1698(CꢀO), 1498(CꢀC), 1082(CꢁF), 736(CꢁCl); 3j, 1694(CꢀO), 1588 (CꢀN), 1518(CꢀC), 734(CꢁCl). 1H NMR (300
MHz, CDCl3): 3a, l, 1.48 (s, 9H, C(CH3)3), 6.91 (d, 1H, furan H, J=3.7 Hz), 7.36–7.51 (m, 1H, ArꢁH), 7.53 (d, 1H, furan H, J=3.7 Hz), 7.71
(d, 1H, ArꢁH, J=8.7 Hz), 7.83 (d, 1H, ArꢁH, J=8.7 Hz); 3e, l, 4.29 (s, 2H, ꢁCH2ꢁ), 6.88 (d, 1H, furan H, J=3.7 Hz), 7.45 (d, 1H, furan H,
J=3.7 Hz), 7.21–7.54 (m, 5H, ArꢁH), 7.72 (d, 1H, ArꢁH, J=8.7 Hz), 7.83 (d, 1H, ArꢁH, J=8.7 Hz); 3f, l, 4.23(s, 2H, ꢁCH2ꢁ), 7.17 (d, 1H,
furan H, J=3.6 Hz), 7.13–7.49 (m, 6H, ArꢁH), 7.55 (d, 1H, furan H, J=3.6 Hz), 7.62(d, 1H, ArꢁH, J=7.5 Hz), 7.74 (d, 1H, ArꢁH, J=7.5 Hz),
7.99 (d, 1H, ArꢁH, J=10.2 Hz); 3g, l, 4.29 (s, 2H, ꢁCH2ꢁ), 6.88 (d, 1H, furan H, J=3.7 Hz), 7.16–7.59 (m, 5H, ArꢁH), 7.46 (d, 1H, furan H,
J=3.7 Hz), 7.7 (d, 1H, ArꢁH, J=8.5 Hz), 7.82 (d, 1H, ArꢁH, J=8.5 Hz); 3j, l, 4.43(s, 2H, ꢁCH2ꢁ), 6.9 (d, 1H, furan H, J=3.7 Hz), 7.20–7.55
(m, 4H, Ar–H), 7.51 (d, 1H, furan H, J=3.7 Hz), 7.72 (d, 1H, ArꢁH, J=8.6 Hz), 7.86 (d, 1H, ArꢁH, J=8.6 Hz); 3k, l, 4.43 (s, 2H, ꢁCH2ꢁ),
6.88 (d, 1H, furan H, J=3.7 Hz), 7.21–741 (m, 2H, ArꢁH), 7.35–4.41 (m, 2H, ArꢁH), 7.51 (d, 1H, furan H, J=3.7 Hz), 7.66 (d, 1H, ArꢁH), 7.82
(d, 1H, ArꢁH, J=8.9Hz). MS: 3a, m/z 420 (24%, M+), 392 (14%, M+ꢁCO), 237 (12%, 5-(2,4-dichlorophenyl)-2-furonitrile), 183 (100%, M+-237),
83 (10%, (CH3)3CCN+); 3e, m/z 438 (8%, M+), 217 (33%, Mꢁ5(3-chloro-4-fluorophenyl)-2-furonitrile), 167 (8%, (3-chloro-4-
fluorophenyl)cyclopropylcation), 117 (30%, C6H5CH2CN+), 91 (89%, C6H5CH+2 ); 3f, m/z 464 (100%, M+), 438 (38% MꢁCO), 266 (26%,
5-(4-bromophenyl)furo-2-thiocarbonyl cation), 91 (28%, C6H5CH+2 ); 3j, m/z 522 (5%, M+), 489 (18%, MꢁCl), 237 (32%, 5-(2,4-dichlorophenyl)-2-
furonitrile).
with 5-aryl-2-furoic acids (2) in the presence of phos-
phorus oxychloride to afford 7-(5-aryl-2-furyl)-3-aryl-
methyl/tert-butyl-4H-1,3,4-thiadiazolo[2,3-c]-1,2,4-tria-
zin-4-ones (3) in good yields. Formation of these thiadia-
zolotriazinones 3 was confirmed by elemental analysis,
pounds reported in Part I and II of the series, it is
evident that the introduction of arylfuryl moieties at C7
Table 2
Antibacterial activity a of thiadiazolotriazinones (MIC, mg ml−1
)
1
IR, H NMR and mass spectral data. The characteriza-
Comp. No.
S. aureus E. coli P. aeruginosa B. subtilis
tion data of these compounds 3 are given in Table 1.
The key spectral features are as reported by us
previously.
3a
3b
3c
3d
3e
3f
3g
3h
3I
3j
12.5
12.5
25
12.5
12.5
12.5
6
6
6
12.5
12.5
12.5
6
25
6
6
12.5
12.5
6
25
12.5
6
12.5
12.5
6
6
25
12.5
12.5
25
12.5
12.5
12.5
25
12.5
6
12.5
12.5
12.5
6
2.1. Antibacterial acti6ity
6
All the newly synthesized compounds were screened
for their in vitro antibacterial activity against Bacillus
subtilis, Pseudomonas aeruginosa, Escherichia coli and
Staphilococus aureus Smith according to the serial dilu-
tion technique [6]. Nitrofurazone was used as a stan-
dard drug. All the tested compounds (Table 2) showed
moderate activities against tested bacterial straines.
Comparing the antibacterial activities of the com-
12.5
12.5
12.5
12.5
6
6
3k
3l
12.5
12.5
12.5
Nitrofurazone 12.5
12.5
a S. aureus, Staphillococcus aureus Smith; E. coli, Escherichia coli;
P. aeruginosa, Pseudonmonas aeruginosa; B. subtilis, Bacillus subtilis.

B. Shi6arama Holla et al. / Il Farmaco 57 (2002) 693–696
695
Table 3
Table 4
Preliminary in vitro antitumor screening a data of thiadiazolotriazi-
Sixty cell line in vitro antitumor screening a (GI50, mM) thiadiazolotri-
azinones 3g and 3h
nones 3a, 3b, 3g and 3h
Comp. No. Growth percentage b
Panel/cell line
3g
3h
NCI-H 460 MCF-7
SF 268
(CNS)
Activity c
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
(Lung)
(Breast)
61.7
23.2
89.9
43.6
45.8
43.6
37.6
43.6
3a
3b
3g
3h
42
35
39
30
34
46
27
12
64
72
40
43
Inactive
Inactive
Active
MOLT-4
Non-small cell lung Cancer
A549/AATCC
EKVX
Active
80.5
29.4
43.1
38.4
66.5
31.1
59.6
52.8
51.2
24.6
a Fixed concentration assay (100 mM; standard NCI protocol).
b Percent cell growth reduction following 48-h incubation with test
HOP-62
compounds (optical density, sulforhodamine procedure) [6].
c Active when growth percentage is B32% for any of the three cell
lines.
HOP-92
NCI-H23
NCIH322M
NCI-H460
NCI-H522
58.8
54.9
62.1
did not enhance antibacterial activities noticeably.
However, among tested compounds the best (3a) and
worst compounds overall (3b) were characterized by
tert-butyl at C3, while differing for 2,4-diclorophenyl
versus 3-chloro 4-flurophenyl as the furan substituent.
Colon cancer
COLO205
HCC-2998
HCT-116
HCT-15
72.2
49.6
27.6
41.4
64.1
76.5
57.2
29.7
34.9
50.4
91.2
54.5
HT29
KM12
2.2. Antitumor acti6ity
CNS cancer
SNB-19
U251
Four newly synthesized compounds were screened
for their antitumor activities at NIH, Bethesda, Mary-
land, USA under the Drug Discovery Programme of
NCI according to the procedure suggested by Boyd and
Paull [7] in a primary three cell line–one dose anti-
cancer assay against NCI-H 460 (Lung), MCF 7
(Breast) and SF 268 (CNS). In the current protocol
each cell line is inoculated on a preincubated microtiter
plate. The test agents are added at a single concentra-
tion and the culture is incubated for 48 h. End point
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
32% 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. In the present screening pro-
gram (Table 3) compounds 3a and 3b (tert-butyl substi-
tution at C3) were found to be inactive while the
compounds 3g and 3h (4-chlorobenzyl substitution at
C3) possessed growth percentage to less than 32%
against Breast (MCF-7) cell lines and are regarded as
active compounds. These two compounds 3g and 3h
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 (Table 4). These compounds showed antipro-
liferative activity on the whole cell panel, although they
did not prove cytotoxic or cytostatic at the maximum
tested concentration (100 mM). Compound 3g showed
33.4
31.0
76.5
88.9
Melanoma
LOXIMVI
M14
SKMEL-2
UACC-62
47.1
70.5
64.5
51.4
53.2
36.7
63.1
O6arian cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
34.1
42.8
94.8
47.8
72.2
42.1
56.2
58.8
Renal cancer
786-O
A498
ACHN
CAK-I
RXF393
SN12C
UO-31
48.0
41.5
52.0
73.5
53.8
44.6
57.8
71.0
42.1
35.7
42.9
42.7
33.6
Prostate cancer
PC-3
Du-145
79.8
69.9
57.1
Breast cancer
MCF7
NCI/ADR-RES
MDA-MB-435
MDA-N
30.5
80.6
93.3
47.9
32.2
56.8
82.0
95.4
78.2
80.2
BT-549
T-47D
74.5
a The other two standard parameters, TGI and LC50 were above
100 mM (maximum tested concentration).

696
B. Shi6arama Holla et al. / Il Farmaco 57 (2002) 693–696
highest activity against Leukemia HL-60 (TB) cell line
(GI50=23 mM) while 3h showed highest activity against
non-small cell lung cancer EKVX cell line (GI50=24.6
mM).
Compound 2c: 5-(4-Bromophenyl)-2-furoic acid, m.p.
178 °C (Lit. [8] m.p. 178 °C), yield 68%. IR: (KBr disc,
kmax cm−1): 3420(OꢁH str.), 3080(CꢁH), 1668(CꢀO).
3.2. General procedure for the preparation of
7-(5-aryl-2-furyl)-3-arylmethyl/tert-butyl-4H-1,3,4-
thiadiazolo[2,3-c]-1,2,4-triazin-4-ones (3)
3. Experimental
A mixture of triazinone 1 (0.01 mol), aryl furoic acid
2 (0.01 mol) and phosphorus oxychloride (10 ml) was
refluxed on a water bath for about 5 h. Excess of
phosphorus oxychloride was removed under reduced
pressure. The reaction mixture was cooled and poured
onto crushed ice (200 g). The resulting solid product was
filtered, washed with sodium bicarbonate solution (2%),
followed by distilled water. It was dried and recrystal-
lized from a mixture of ethanol and dioxane. The yield
and characterization data of thiadiazolotriazinones (3)
prepared according to this method are given in Table 1.
Melting points were taken in open capillary tubes and
are uncorrected. The IR spectra in KBr disc were
recorded either on a Shimadzu FT IR or a JASCO FT
1
IR spectrophotometer. H NMR spectra were recorded
in CDCl3–DMSO-d6 either on a Bruker AC-300F (300
MHz) or a 400 MHz NMR spectrometer using TMS as
an internal standard. The mass spectra were recorded
either on a JEOL-JMS D-300 or on a VG 70S mass
spectrometer operating at 70 eV. Purity of the com-
pounds was checked by thin-layer chromatography
(TLC) on silica gel plates using a toluene:acetone (8:2)
solvent system. Iodine was used as the visualizing agent.
Triazinones 1 was prepared according to the method
reported by us earlier.
Acknowledgements
The authors are grateful to Head, R.S.I.C., C.D.R.I.,
Lucknow and The Director, R.S.I.C., Punjab Univer-
sity, Chandigarh, for providing microanalysis, IR, H
NMR and mass spectral data. The authors are grateful
to Dr. V.L. Narayanan, National Institutes of Health
(NIH), Bethesda, Maryland, USA, for the antitumor
activity screening studies reported in this paper. One of
the authors (B.S.R.) is grateful to Mangalore University
for the award of Senior Research Fellowship.
3.1. General procedure for the preparation of
5-aryl-2-furoic acids (2)
1
A mixture of substituted aniline (0.1 mol), dilute
hydrochloric acid (15%, 60 ml) and water (90ml) was
heated to get clear solution. The solution was cooled to
0 °C and was diazotized by the addition of sodium
nitrite solution (30%, 24 ml). After filtration, the cold
clear solution of diazonium salt was treated with furoic
acid (0.1 mol) and water (50 ml). To this, an aqueous
solution of cupric chloride (2.5 g in 10 ml of water) was
added drop wise with stirring. Stirring was continued for
4 h and the precipitated solid was collected by filtration.
The crude acid was recrystallized from a mixture of
ethanol and dioxane to yield pure 5-aryl-2-furoic acid.
The compounds synthesized using this procedure are
as follows.
Compound 2a: 5-(2,4-Dichlorophenyl)-2-furoic acid,
m.p. 199 °C, yield 72%. IR: (KBr disc, kmax cm−1):
3431(OꢁH str.), 3135(CꢁH), 1694(CꢀO), 1585(CꢀC),
726(CꢁCl). 1H NMR (300 MHz, CDCl3+DMSO-d6): l,
7.22 [d, 1H (J=3.7 Hz), furan H], 7.26[d, 1H (J=3.7
Hz), furan H], 7.39 [d, 1H, (J=9.0 Hz), ArꢁH], 7.51 [d,
1H (J=2.2 Hz), ArꢁH], 7.95 [d, 1H (J=9.0 Hz), ArꢁH].
Compound 2b: 5-(3-Chloro-4-fluorophenyl)-2-furoic
acid acid, m.p. 183 °C, yield 66%. IR: (KBr disc, kmax
cm−1): 3405(OꢁH str.), 3090(CꢁH), 1687(CꢀO),
1567(CꢀC), 1068(CꢁF), 726(CꢁCl). 1H NMR (400 MHz,
CDCl3+(CD3)2CO): l, 6.76 [d, 1H, (J=3.7 Hz), furan
H], 7.22–7.31 (m, 1H, ArꢁH), 7.38 (d, 1H (J=3.7 Hz),
furan H), 7.68 (m, 1H, ArꢁH), 7.87 (d, 1H, (J=9.0 Hz),
ArꢁH).
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