New bis-aminomercaptotriazoles and bis-triazolothiadiazoles as possible anticancer agents

Article abstract of DOI:10.1016/S0223-5234(02)01358-2

A series of bis-phenoxyacetic acids 2 were prepared starting from corresponding unsubstituted/substituted 1,4-quinols 1. The fusion of bis-phenoxyacetic acids 2 with thiocarbohydrazide gave the corresponding bis-[4-amino-5-mercapto-1,2,4-triazol-3-yl-methyleneoxy]phenylenes (3) in a one pot reaction. The reaction of bis-triazoles 3 with various reagents afforded N-bridged heterocycles 4-6 in good yields. The newly synthesised compounds were screened for their anticancer activity against a panel of 60 cell lines derived from seven cancer types namely, lung, colon, melanoma, renal, ovarian, CNS and leukemia. Some of the tested compounds showed promising anticancer properties.

Full text of DOI:10.1016/S0223-5234(02)01358-2

Eur. J. Med. Chem. 37 (2002) 511–517
www.elsevier.com/locate/ejmech
Short communication
New bis-aminomercaptotriazoles and bis-triazolothiadiazoles as
possible anticancer agentsꢀ
B. Shivarama Holla *, K. Narayana Poojary, B. Sooryanarayana Rao,
M.K. Shivananda
Department of Post-Graduate Studies and Research in Chemistry, Mangalore Uni6ersity, Mangalagangothri, Mangalore 574 199, India
Received 2 January 2001; received in revised form 4 March 2002; accepted 7 March 2002
Abstract
A series of bis-phenoxyacetic acids 2 were prepared starting from corresponding unsubstituted/substituted 1,4-quinols 1. The
fusion of bis-phenoxyacetic acids 2 with thiocarbohydrazide gave the corresponding bis-[4-amino-5-mercapto-1,2,4-triazol-3-yl-
methyleneoxy]phenylenes (3) in a one pot reaction. The reaction of bis-triazoles 3 with various reagents afforded N-bridged
heterocycles 4–6 in good yields. The newly synthesised compounds were screened for their anticancer activity against a panel of
60 cell lines derived from seven cancer types namely, lung, colon, melanoma, renal, ovarian, CNS and leukemia. Some of the
´
tested compounds showed promising anticancer properties. © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
Keywords: Bis-triazoles; Bis-triazolothiadiazoles; N-bridged heterocycles; Anticancer activity
1. Introduction
antiviral, analgesic, anthelmintic, herbicidal and plant
growth regulatory effects [10].
Various 3-substituted-4-amino-5-mercapto-1,2,4-tria-
zoles have been reported to possess analgesic, antibac-
terial, antifungal, antiinflammatory, antitubercular,
antiviral, herbicidal and sedative properties [1–7]. The
amino and mercapto groups of these compounds serve
as readily accessible nucleophilic centers for the prepa-
ration of N-bridged heterocycles. The 1,3,4-thiadiazoles
exhibit broad spectrum of biological activities, possibly
due to the presence of toxophoric ꢀNꢁCꢁS moiety [8].
They find applications as antibacterials, -tumour, and
-inflammatory agents, pesticides, herbicides, dyes, lubri-
cants and analytical reagents [9]. The 1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazole derivatives obtained by fusing the
bio-labile 1,2,4-triazole and 1,3,4-thiadiazole rings to-
gether, are reported to possess antibacterial, antifungal,
anti-inflammatory, CNS depressant, hypocholesteremic,
It is also observed that incorporation of ary-
loxymethyl substituent and the halogen atom into the
heterocyclic ring systems augments the biological activi-
ties considerably [11,12]. Further, it has been reported
that many biologically active natural and synthetic
products have interesting molecular symmetry [13]. Re-
cently, some bis-triazole derivatives endowed with ex-
cellent antibacterial activities have been reported from
our laboratory [14–16].
Prompted by these observations and in continuation
of our search for bio-active molecules, we designed the
synthesis of a series of novel 1,4-bis-[4-amino-5-mer-
capto-1,2,4-triazol-3-yl-methoxy]phenylenes (3) and
their triazolothiadiazole derivatives 4–6 starting from
hydroquinones 1. The synthesis, characterization and
the results of anticancer activity screening studies of the
newly synthesised compounds are presented in this
paper.
ꢀ Presented at ‘National Seminar on New Vistas in Bio-active
Agents’ held at Department of Chemistry, Gandhigram Rural Uni-
versity, Gandhigram, Tamilnadu, India, during December 21–22,
1999.
* Correspondence and reprints
E-mail address: hollabs@yahoo.com (B. Shivarama Holla).
2. Chemistry
1,4-Bis-phenoxyacetic acids (2) were synthesised by
the reaction of hydroquinones 1 with monochloroacetic
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(02)01358-2

512
B. Shi6arama Holla et al. / European Journal of Medicinal Chemistry 37 (2001) 511–517
acid in presence of sodium hydroxide. The fusion of
bis-phenoxyacetic acids 2 with thiocarbohydrazide af-
forded 1,4-bis-[4-amino-5-mercapto-1,2,4-triazol-3-yl-
methoxy]phenylenes (3) in good yields (Fig. 1). The
condensation of bis-triazoles 3 with various aromatic
carboxylic acids in presence of phosphorus oxychloride
furnished a series of 1,4-bis-(6-aryl-1,2,4-triazolo[3,4-b]-
1,3,4-thiadiazol-3-ylmethoxy)phenylenes (4). The reac-
tion of bis-triazoles 3 with formic acid in benzene
followed by treatment of the product with concentrated
sulphuric acid yielded bis-(1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazol-3-ylmethoxy)phenylenes (5). The reaction of
bis-triazoles with carbon disulphide in the presence of
potassium hydroxide in methanol gave 1,4-bis-(6-mer-
capto-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazol-3-ylmetho-
xy)phenylenes (6).
3. Results and discussion
The characterization data of bis-aminomercaptotria-
zoles 3a–e and the bis-triazolothiadiazoles 4a–w, 5a–e
and 6a–e are given in Tables 1 and 2, respectively. The
formation of bis-aminomercaptotriazoles was confir-
med by recording the IR, 1H-NMR and mass spectra of
a few selected compounds. IR spectrum of compound
t
Fig. 1. R=H, Cl, Bu; R1=H, Cl.
Table 1
Characterization data of compounds 3a–e
Compound
R
R1
M.p. (°C)
Yield (%)
Molecular formula
12H14N8O2S2
C12H13ClN8O2S2
C12H12Cl2N8O2S2
C16H22N8O2S2
Anal. %N found [calculated]
3a
3b
3c
3d
3e
H
Cl
H
H
Cl
H
Cl
226–28
215–17
228–30
164–66
175–77
70
72
68
72
76
C
30.56 [30.60]
27.94 [27.97]
25.71 [25.75]
26.57 [26.54]
24.49 [24.53]
Cl
tBu
tBu
C16H21ClN8O2S2
IR (KBr, wmax cm−1): 3a: 3225 (NH2), 3143 (NH2), 2947 (CꢁH), 1614 (CꢂN), 1583 (CꢂC); 3b: 3230 (NH2), 3146 (NH2), 3010 (ArꢁH), 2936 (CꢁH),
1609 (CꢂN), 1591 (C=C); 3c: 3228 (NH2), 3152 (NH2), 3016 (ArꢁH), 2940 (CꢁH), 1610 (CꢂN), 1585 (CꢂC); 3d: 3249 (NH2), 3148 (NH2), 2956
(CꢁH), 1618 (CꢂN), 1583 (CꢂC); 3e: 3218 (NH2), 3139 (NH2), 2951 (CꢁH), 1608 (CꢂN), 1593 (CꢂC). 1H-NMR (DMSO-d6): 3a: l 5.14 (bs, 4H,
2×NH2), 5.62 (bs, 4H, 2×OCH2), 6.92–7.12 (m, 4H, ArꢁH), 13.8 (bs, 2H, 2×SH–NH); 3b: l 5.11 (bs, 4H, 2×NH2), 5.58 (bs, 4H, 2×OCH2),
7.04–7.23 (m, 3H, ArꢁH), 13.8 (bs, 2H, 2×SH–NH); 3c: l 4.95 (s, 2H, NH2), 5.15 (s, 2H, NH2), 5.64 (bs, 4H, 2×OCH2), 6.98 (m, 1H, ArꢁH),
7.25 (m, 1H, ArꢁH), 13.82 (bs, 2H, 2×SH–NH); 3d: l 1.41 (bs, 9H, t-butyl), 5.08 (bs, 4H, 2×NH2), 5.32 (bs, 4H, 2×OCH2), 6.95–7. 28 (m, 3H,
ArꢁH), 13.61 (bs, 2H, 2×SH–NH); 3e: l 1.35 (bs, 9H, t-butyl), 5.12 (bs, 4H, 2×NH2), 5.22 (bs, 4H, 2×OCH2), 6.82–7.12 (m, 2H, ArꢁH), 13.74
(bs, 2H, 2×SH–NH). MS: 3a: m/z 366 [12%, M+]; 110 [100%, M+ of hydroquinone]; 3b: m/z 400 [12%, M+], 144 [100%, M+ of
2-chlorohydroquinone] 41 [50%, M+ of CH3CN]; 3c: m/z 434 [9%, M+]; 3d: m/z 422 [22%, M+]; 3e: m/z 455 [19%, M+], 200 [100%, M+ of
2-t-butyl-5-cholorohydroquinone], 41 [62%, M+ of CH3CN].

B. Shi6arama Holla et al. / European Journal of Medicinal Chemistry 37 (2001) 511–517
513
Table 2
Characterization data of compounds 4a–w
Compound
R
R1
R11
M.p. (°C)
Yield (%)
Molecular formula
Anal. %N found [calculated]
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
C6H5
114–16
168–70
88–90
190–92
166–68
106–08
58–60
65
71
75
66
80
69
72
78
65
75
73
69
77
82
74
78
84
81
78
76
72
80
79
C26H18N8O2S2
C26H16N10O6S2
20.79 [20.82]
22.25 [22.29]
18.41 [18.45]
17.49 [17.46]
19.49 [19.56]
18.61 [18.65]
24.32 [24.37]
21.08 [21.13]
17.52 [17.46]
16.62 [16.57]
18.38 [18.45]
17.59 [17.64]
20.14 [20.09]
16.50 [16.57]
18.80 [18.86]
20.52 [20.47]
16.81 [16.89]
15.98 [16.06]
17.75 [17.82]
17.12 [17.06]
22.12 [22.20]
21.36 [21.39]
16.02 [16.06]
4-NO2C6H4
2-ClC6H4
4-ClC6H4
C6H5
C6H5CH2
3-pyridyl
4-NO2C6H4
2-ClC6H4
4-ClC6H4
C6H5
C6H5CH2
4-NO2C6H4
2-ClC6H4
C6H5
4-NO2C6H4
2-ClC6H4
4-ClC6H4
C6H5
C26H16Cl2N8O2S2
C26H15Cl3N8O2S2
C26H17ClN8O2S2
C28H21ClN8O2S2
C24H15ClN10O2S2
C26H15ClN10O6S2
C26H15Cl3N8O2S2
C26H14Cl4N8O2S2
C26H16Cl2N8O2S2
C28H20Cl2N8O2S2
C26H14Cl2N10O6S2
C26H14Cl4N8O2S2
C30H26N8O2S2
236–38
92–94
4j
4k
4l
218–20
128–30
102–04
196–98
108–10
100–02
148–50
124–26
118–20
102–04
98–100
118–20
138–40
106–08
4m
4n
4o
4p
4q
4r
4s
4t
tBu
tBu
tBu
tBu
tBu
tBu
tBu
tBu
tBu
C30H24N10O6S2
C30H24Cl2N8O2S2
C30H23Cl3N8O2S2
C30H25ClN8O2S2
C32H29ClN8O2S2
C28H23ClN10O2S2
C30H23ClN10O2S2
C30H23Cl3N8O2S2
C6H5CH2
3-pyridyl
4-NO2C6H4
2-ClC6H4
4u
4v
4w
IR (KBr, wmax cm−1): 4a: 3125 (CꢁH), 1614 (CꢂN), 1585 (CꢂC); 4b: 3132 (CꢁH), 1610(CꢂN), 1581 (CꢂC), 1529 (NO2), 1348 (NO2), 4c: 3113
(CꢁH), 1609 (CꢂN), 1586 (CꢂC), 728 (CꢁCl); 4d: 3120 (CꢁH), 1612 (CꢂN), 1584 (CꢂC), 730 (CꢁCl); 4e: 3126 (CꢁH), 1608 (CꢂN), 1585 (C=C);
4g: 3115 (CꢁH), 1624(CꢂN), 1589 (CꢂC); 4h: 3123 (CꢁH), 1604 (CꢂN), 1589 (CꢂC), 1531 (NO2), 1352 (NO2); 4j: 3115 (CꢁH), 1606 (CꢂN), 1588
(CꢂC), 726 (CꢁCl); 4m: 3135 (CꢁH), 1614 (CꢂN), 1593 (CꢂC), 1534 (NO2), 1357 (NO2); 4q: 3128 (CꢁH), 1612 (CꢂN), 1581 (CꢂC), 731 (CꢁCl);
4v: 3142 (CꢁH), 1616 (CꢂN), 1594 (CꢂC), 1535 (NO2), 1356 (NO2); 4w: 3121 (CꢁH), 1613 (CꢂN), 1589 (CꢂC), 728 (CꢁCl). 1H-NMR
(DMSO-d6–CDCl3): 4a: l 5.48 (bs, 4H, 2×OCH2), 7.04–7.93 (m, 14H, ArꢁH); 4b: l 5.45 (bs, 4H, 2×OCH2), 7.04–7.63 (m, 12H, ArꢁH); 4c: l
5.45 (s, 2H, OCH2), 5.51 (s, 2H, OCH2), 7.04–7.13 (m, 2H, ArꢁH), 7.43–7.63 (m, 7H, ArꢁH), 7.79 (d, 1H, ArꢁH, J=7.6 Hz), 8.02–8.06 (m, 2H,
ArꢁH); 4f: l 4.26 (bs, 4H, 2×CH2), 5.44 (bs, 4H, 2×OCH2), 7.12–8.17 (m, 13H, ArꢁH); 4h: l 5.53 (s, 2H, OCH2), 5.60 (s, 2H, OCH2), 7.09 (d,
1H, ArꢁH, J=6 Hz), 7.22 (s, 1H, ArꢁH), 7.34 (d, 1H, ArꢁH, J=9.1 Hz), 7.57 (s, 1H, ArꢁH), 8.03 (s,1H, ArꢁH), 8.17–8.34 (m, 3H, ArꢁH), 8.42
(m, 3H, ArꢁH); 4k: l 5.42 (bs, 4H, 2×OCH2), 7.14–7.84 (m, 12H, ArꢁH); 4m: l 5.58 (bs, 4H, 2×OCH2), 7.09 (d, 1H, ArꢁH, J=9 Hz), 7.22 (s,
1H, ArꢁH), 7.34 (d, 1H, ArꢁH, J=9 Hz), 7.57 (s, 1H, ArꢁH), 8.17–8.34 (m, 6H, ArꢁH); 4o: l 1.32 (bs, 9H, t-butyl), 5.50 (s, 2H, OCH2), 5.54
(s, 2H, OCH2), 6.94–8.04 (m, 13H, ArꢁH,); 4q: l 1.34 (bs, 9H, t-butyl), 5.53 (s, 2H, OCH2), 5.55 (s, 2H, OCH2), 6.94–8.12 (m, 11H, ArꢁH,); 4s:
l 1.29 (bs, 9H, t-butyl), 5.53 (bs, 4H, 2×OCH2), 6.99–8.14 (m, 12H, ArꢁH); 4u: l 1.34 (bs, 9H, t-butyl), 5.51 (bs, 4H, 2×OCH2), 6.92–8.22 (m,
10H, ArꢁH,); 4w: l 1.37 (bs, 9H, t-butyl), 5.51 (bs, 4H, 2×OCH2), 7.13–8.32 (m, 10H, ArꢁH). MS: 4a: m/z 538 [8%, M+], 430 [15%,
M−hydroquinone], 190 (100%), 121 (25%, C6H5ꢁCꢂS), 77 (19%, phenyl cation); 4b: m/z 628 [12%, M+], 148 [100%, 4-NO2C6H4CN]; 4d: m/z 640
[14%, M+] 137 [100%, 4-ClC6H4CN]; 4f: m/z 600 [12%, M+], 91 (100%, C6H5CH2); 4h: m/z 662 (46%, M+); 4o: m/z 594 [9%, M+], 103 [100%,
C6H5CN]; 4r: m/z 696 [9%, M+], 137 [100%, 4-ClC6H4CN], 57 (48%, ꢁC(CH3)3); 4v: m/z 654 [14%, M+], 148 (100%, 4-NO2lC6H4CN), 57 (25%,
ꢁC(CH3)3); 4w: m/z 696 [16%, M+], 137 (100%, 2-ClC6H4CN), 57 (41%, ꢁC(CH3)3).
3a showed absorption bands at 3225, 2947,1614, 1583
and 1026 cm−1 corresponding to the NH2, CꢁH, CꢂN,
CꢂC, and CꢂS groups, respectively. The absence of
band due to carbonyl stretching frequency of the parent
bis-phenoxyacetic acid clearly indicates the formation
of bis-triazole 3a. The IR spectra of other bis-triazoles
of the series showed similar absorption bands.
Mass spectra of compounds 3a, 3b and 3d showed
molecular ion a peaks at m/z 366, 400 and 422, respec-
tively, in agreement with their molecular formulae.
These bis-aminomercaptotriazoles were converted
into bis-triazolothiadiazoles 4, 5 and 6 in good yields.
These N-bridged bis-heterocycles were characterised on
1
the basis of IR, H-NMR and mass spectral data. IR
1
The H-NMR spectrum of 3a showed a broad singlet
spectrum of compound 4a showed an absorption band
at 1614 cm−1 corresponding to the CꢂN stretching
frequency of thiadiazole ring. The absence of absorp-
tion bands due to NH2, SH and CꢂO stretching fre-
quencies of starting triazoles and carboxylic acids
clearly indicated the formation of cyclised products.
at l 5.62 corresponding to OCH2 protons. A sharp
singlet observed at l 5.14 is attributed to the NꢁNH2
protons. The aromatic protons resonated as a multiplet
in the region l 6.92–7.12 integrating for four protons.
The SH protons resonated as a broad singlet at l 13.8.
1
1
The H-NMR spectra of 3c and 3e were also recorded
The H-NMR spectrum compound 4o showed broad
and data are given in Table 1.
singlet at l 1.32 integrating for nine protons of the

514
B. Shi6arama Holla et al. / European Journal of Medicinal Chemistry 37 (2001) 511–517
t-butyl group. The signal due to the two OCH2 groups
resonated as two singlets at l 5.50 and 5.54, respec-
tively. The aromatic protons signal appeared as a multi-
plet in the region l 6.94–8.04 integrating for 13
protons.
The structures of newly synthesised bis-triazolothia-
diazoles were also confirmed by recording their mass
spectra. The mass spectra of compounds 4a and 4d
showed molecular ion peaks at m/z 538 and 640 in
conformity with the assigned molecular formulae
C26H18N8O2S2 and C26H15Cl3N8O2S2, respectively. In
both the cases, the molecular ion peaks were fairly
intense suggesting the presence of stable triazolothiadia-
zole ring system in these cyclised products.
The bis-aminomercaptotriazole 3 was converted into
compound 6 by refluxing 3 with potassium hydroxide
and carbon disulphide. The liberation of hydrogen sul-
phide gas during the reaction and absence of ꢁNH2
group frequency in the IR spectra of compounds 6
clearly indicated the formation of cyclised product. The
300 MHz 1H-NMR spectrum of 6c showed a broad
singlet at l 5.42 corresponding to the four protons of
two OCH2 groups. The signals due to two SH protons
resonated as a broad singlet at l 13.7. The aromatic
protons appeared as two singlets at l 7.36 and 7.86.
The mass spectra gave further evidence for the forma-
tion of 6. The spectral data are given in Tables 1–3.
the Drug Discovery Programme. Fourteen compounds
were submitted for the three cell line one dose primary
anticancer assay. The three cell lines used in the present
investigation are NCI-H 460 (lung), MCF 7 (breast)
and SF 268 (CNS). In this current protocol, each cell
line is pre-inoculated, pre-incubated on microtiter plate.
The test agents are then added at a single concentration
and the culture incubated for 48 h. End point determi-
nations are made with sulphorhodamine B, a protein
binding dye. Results for each test agent are reported as
the percentage of growth of the treated cells when
compared to the untreated control cells. The com-
pounds which reduce the growth of any one of the cells
to 32% or less (negative numbers indicate the cell kill)
are passed on for the evaluation in the full panel of 60
cell lines over a five-log dose range. Interestingly, six
compounds (3c, 4d, 4r, 4t, 6c and 6e) carrying chloro,
tbutyl, butyl, chloro and butyl substituents, respec-
tively were found to be active in the preliminary scree-
ning studies. The results of such studies are given in
Table 4. These compounds were further tested against a
panel of 60 cell lines derived from seven cancer types
namely, lung, colon, melanoma, renal, ovarian, CNS
and leukemia. Their GI50, TGI and LC50 values were
determined.
t
t
5. Conclusions
4. Pharmacology
Compound 3c was found to be active against 16
cancer cell lines: leukemia (5), non-small cell lung can-
cer (2), colon cancer (1), CNS cancer (1), melanoma (3),
renal cancer (1), prostate cancer (1) and breast cancer
(2). Compound 4d was found to be active against 25
The newly synthesised bis-aminomercaptotriazoles
and bis-triazolothiadiazoles were screened for their an-
ticancer activities at NIH, Bethesda, MA, USA under
Table 3
Characterization data of compounds 5a–e and 6a–e
Compound
R
R1
M.p. (°C)
Yield (%)
Molecular formula
14H10N8O2S2
Anal. %N found [calculated]
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
H
Cl
Cl
H
H
Cl
H
Cl
H
H
Cl
H
Cl
78–80
114–16
108–10
96–98
82
79
69
75
80
83
79
78
72
69
C
28.96 [29.02]
26.60 [26.63]
24.58 [24.62]
25.28 [25.34]
23.44 [23.50]
24.82 [24.89]
23.03 [23.12]
21.62 [21.58]
22.10 [22.13]
20.77 [20.72]
C14H9ClN8O2S2
C14H8Cl2N8O2S2
C18H18N8O2S2
C18H17ClN8O2S2
C14H10N8O2S4
C14H9ClN8O2S4
C14H8Cl2N8O2S4
C18H18N8O2S4
C18H17ClN8O2S4
tBu
tBu
H
88–90
244–46
200–02
178–80
78–80
Cl
Cl
tBu
tBu
104–06
IR (KBr, wmax cm−1): 5a: 3141 (CꢁH), 1614 (CꢂN); 5b: 3133 (CꢁH), 1608 (CꢂN), 732 (CꢁCl); 5c: 3139 (CꢁH), 1612 (CꢂN), 728 (CꢁCl); 5e: 3136
(CꢁH), 1610 (CꢂN), 730 (CꢁCl); 6a: 3133 (CꢁH), 1607 (CꢂN), 1588 (CꢂC); 6c: 3121 (CꢁH), 1602 (CꢂN), 1594 (CꢂC), 736 (CꢁCl); 6e: 3141 (CꢁH),
1617 (CꢂN), 1594 (CꢂC), 734 (CꢁCl). 1H-NMR (DMSO-d6–CDCl3): 5a: l 5.46 (bs, 4H, 2×OCH2), 6.99–8.48 (m, 6H, ArꢁH); 5b: l 5.43 (bs, 4H,
2×OCH2), 7.12–8.26 (m, 5H, ArꢁH); 5d: l 1.34 (s, 9H, t-butyl), 5.44 (bs, 4aH, 2×OCH2), 7.05–8.18 (m, 5H, ArꢁH); 5e: l 1.34 (s, 9H, t-butyl),
5.41 (bs, 4H, 2×OCH2), 7.15–8.56 (m, 6H, ArꢁH). 6a: l 5.46 (bs, 4H, 2×OCH2), 7.21–7.78 (m, 4H, ArꢁH), 13.5 (bs, 2H, 2×SH); 6c: l 5.42 (bs,
4H, 2×OCH2), 7.21(s, 1H, ArꢁH), 7.36 (s, 1H, ArꢁH), 7.86 (s, 1H, ArꢁH), 13.7 (bs, 2H, 2×SH); 6e: l 1.32 (s, 9H, t-butyl), 5.45 (bs, 4H,
2×OCH2), 7.18 (s, 1H, ArꢁH), 7.38 (s, 1H, ArꢁH), 13.5 (bs, 2H, 2×SH). MS: 5a: m/z 386 (23%); 110 [100%, M+of hydroquinone]; 5b: m/z 420
[34%, M+]; 144 [100%, M+of 2-chlorohydroquinone]; 5d: m/z 442 [13%, M+]; 57 (100%, C(CH3)3 cation); 6a: m/z 450 [15%, M+], 110 [100%,
M+of hydroquinone]; 6c: m/z 518 [11%, M+]; 6e: m/z 540 [8%, M+], 505 [5%, M+−Cl], 57 (100%, C(CH3)3 cation).

B. Shi6arama Holla et al. / European Journal of Medicinal Chemistry 37 (2001) 511–517
515
Table 4
Anticancer activity screening data of compounds 3, 4 and 6
Compound
Growth percentage a
NCI code no.
NCI-H 460 (lung)
MCF-7 (breast)
SF 268 (CNS)
Activity b
3b
3c
3e
4d
4f
4g
4j
4l
4r
4t
4u
6b
6c
6e
NSC 716886
NSC 716888
NSC 716887
NSC 716889
NSC 716890
NSC 716892
NSC 716897
NSC 716898
NSC 716893
NSC 716894
NSC 716896
NSC 716891
NSC 716899
NSC 716895
105
18
95
34
74
96
71
64
−20
11
80
20
69
25
59
86
44
42
−57
−66
55
74
23
104
40
93
−54
76
82
49
65
8
18
inactive
active
inactive
active
inactive
inactive
inactive
inactive
active
active
56
105
79
74
100
52
inactive
inactive
active
6
−27
0
active
Fixed concentration assay (100 mM; standard NCI protocol).
a Percent cell growth reduction following 48-h incubation with test compounds (optical density, sulphorhodamine procedure) [19].
b ‘Active’ when growth percentage is B32% for any one of the three cell lines. Negative numbers indicate the cell kill.
cancer cell lines: leukemia (5), non-small cell lung can-
cer (2), colon cancer (4), CNS cancer (3), melanoma (2),
ovarian cancer (2), renal cancer (5) and breast cancer
(2). Compound 4r was found to be active against 24
cancer cell lines: leukemia (3), non-small cell lung can-
cer (3), colon cancer (4), CNS cancer (3), melanoma (3),
ovarian cancer (1), renal cancer (2) and breast cancer
(5). Compound 4t was found to be active against 19
cancer cell lines: leukemia (4), non-small cell lung can-
cer (2), colon cancer (4), CNS cancer (2), ovarian
cancer (2), renal cancer (2) and breast cancer (3).
Compound 6c was found to be active against 10 cancer
cell lines: leukemia (2), non-small cell lung cancer (2),
colon cancer (1), melanoma (2), ovarian cancer (1),
renal cancer (1) and breast cancer (1). Compound 6e
was found to be active against 24 cancer cell lines:
leukemia (4), non-small cell lung cancer (2), colon
cancer (1), CNS cancer (2), melanoma (6), ovarian
cancer (2), renal cancer (2) and breast cancer (5).
Compound 4d possessed a LC50 value less than 50 mM
against renal cancer. Compound 4r possessed LC50
values less than 50 mM against leukemia, colon cancer,
melanoma and breast cancer. All other tested com-
pounds had LC50 values higher than 50 mM. Hence, it
is concluded that there is ample scope for further study
in developing these compounds as anticancer agents.
recorded on a Shimadzu FTIR 8700 spectrophotome-
ter. H-NMR spectra were recorded (CDCl3/CDCl3–
1
DMSO-d6 mixture) on a Bruker AC 300 F (300 MHz.)
NMR spectometer using TMS as an internal standard.
The mass spectra were recorded on a JEOL JMS 300
mass spectrometer operating at 70 eV. The purity of the
compounds was checked by thin layer chromatography
(TLC) on silica gel plates. The required chlorosubsti-
tuted hydroquinones and 1,4-bis-phenoxyacetic acids
were synthesised by adopting literature procedures
using commercially available hydroquinone [17,18].
6.1.1. General procedure for the preparation of
1,4-bis-[4-amino-5-mercapto-1,2,4-triazol-3-yl
methoxy]phenylenes (3a–e)
A mixture of bis-phenoxyacetic acids 2 (0.01 mol)
and thiocarbohydrazide (0.02 mol) contained in a flat-
bottomed flask was heated in an oilbath until the
contents melted. The mixture was maintained at this
temperature for 15–20 min (Fig. 1). The product ob-
tained on cooling was heated with dilute Na2CO3 solu-
tion to remove the unreacted bis-phenoxyacetic acid, if
any. It was then washed with water and collected by
filtration. The product was recrystallised from a mix-
ture of dimethylformamide and water to afford pure
title compounds 3a–e. Their characterization data are
given in Table 1 (Fig. 2).
6. Experimental
6.1.2. General procedure for the preparation of
1,4-bis-(6-aryl-1,2,4-triazolo[3,4-b]-1,3,4-
6.1. Chemistry
thiadiazol-3-ylmethoxy)phenylenes (4a–w)
To a mixture of aminomercapto triazole 3 (0.01 mol)
and aromatic carboxylic acid ( 0.02 mol) phosphorus
oxychloride (20 mL) was added and the contents were
M.p.s were determined by open capillary method and
are uncorrected. The IR spectra (in KBr pellets) were

516
B. Shi6arama Holla et al. / European Journal of Medicinal Chemistry 37 (2001) 511–517
heated under reflux on a waterbath for 4 h. Excess of
phosphorus oxychloride was then distilled off and the
residue was poured onto crushed ice with vigorous
stirring. The resulting solid was washed with cold wa-
ter, dilute NaHCO3 solution and then recrystallised
from dimethylformamide or from a mixture of dioxane
and EtOH. The characterization data of title com-
pounds 4a–w are given in Table 2.
6.1.4. General procedure for the preparation of
1,4-bis-(6-mercapto-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazol-3-ylmethoxy)phenylenes (6a–e)
A mixture of bis-aminomercaptotriazole 3 (0.01 mol),
KOH (1 g) and carbon disulphide (2 mL) in MeOH (30
mL) was kept under reflux on a waterbath for 4 h. The
solvent was removed. The reaction mixture was cooled
and poured on to crushed ice with vigorous stirring.
The resulting solid was washed with water, cold dilute
EtOH and recrystallised from DMF or a mixture of
dioxane and EtOH. The characterization data of these
compounds are given in Table 3.
6.1.3. General procedure for the preparation of
1,4-bis-(1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazol-3-ylmethoxy)phenylenes (5a–e)
A mixture of bis-aminomercaptotriazole 3 (0.01 mol)
and HCOOH (1 mL) in C6H6 (20 mL) was refluxed for
30 min. The solvent was removed by evaporation and
the reaction mixture was cooled. The precipitated
product was recrystallised from MeOH. The product
(0.01 mol) was then treated with concd. H2SO4 (15 mL)
in cold whereby a pasty mass was obtained. It was
poured into water when the title compound 5 precipi-
tated out. It was recrystallised from DMF or a mixture
of dioxane and EtOH. The characterization data of title
compounds 5a–e are given in Table 3.
Acknowledgements
The authors thank the Director, R.S.I.C., Punjab
University, Chandigarh and the Head, RSIC, CDRI,
Lucknow for recording H-NMR, Mass and IR spectra
and C,H&N-analyses. The authors are grateful to Dr
V.L.N., National Institute of Health (NIH), Bethesda,
MA, USA, for arranging the anticancer activity scree-
ning studies reported in this paper. One of the authors
1
t
Fig. 2. R=H, Cl, Bu; R1=H, Cl; R¦=C6H5, 4-NO2C6H4, 2-ClC6H4, 4-ClC6H4.

B. Shi6arama Holla et al. / European Journal of Medicinal Chemistry 37 (2001) 511–517
517
[7] B.S. Holla, K.N. Poojary, B. Kalluraya, P.V. Gowda, II Far-
maco 51 (1996) 793–799.
[8] A. Omar, M.E. Mohsen, O.M. Aboul Wafa, J. Heterocycl.
Chem. 23 (1986) 1339–1341.
[9] F. Kurtzer, in: A.R. Katritzky, A.J. Boulton (Eds.), Advances in
Heterocyclic Chemistry, vol. 5, Academic Press, New York,
1965, p. 165.
[10] Z.Y. Zhang, S. Xiao-Wen, Heterocycles 48 (1998) 561–584.
[11] R.R. Mohan, R. Agarwal, V.S. Misra, Indian J. Chem. 24B
(1985) 78–82.
(K.N.P.) is grateful to University Grants Commission,
New Delhi for the award of a Minor Research Project.
B.S.N.R. is grateful to Mangalore University for the
award of a Senior Research Fellowship. The financial
support received from D.S.T., New Delhi is also grate-
fully acknowledged.
[12] V.S. Dubey, V.N. Ingle, J. Indian Chem. Soc. 66 (1989) 174–
175.
[13] E.J. Ariens, in: E.J. Ariens (Ed.), Drug Design, vol. 1, Academic
Press, New York, 1971, p. 1.
[14] B.S. Holla, R. Gonsalves, Boll. Chim. Farmaceutico. 137 (1998)
467–472.
[15] B.S. Holla, R. Gonsalves, S. Shenoy, II Farmaco 53 (1998)
574–578.
[16] B.S. Holla, R. Gonsalves, S. Shenoy, Eur. J. Med. Chem. 35
(2000) 267–271.
[17] J. Cason, C.F. Allen, C. Goodwin, J. Org. Chem. 13 (1948)
403–408.
[18] B.S. Furniss, A.J. Hannaford, P.W.G. Smith, A.R. Tatchell,
Vogel’s Text Book of Practical Organic Chemistry, 5th ed.,
Longman, England, 1989, p. 1249.
References
[1] G. Thomas, D.V. Mehta, R. Tahilramani, D. Joy, P.K. Tal-
walker, J. Med. Chem. 14 (1971) 335–338.
[2] U. Srivastava, R.H. Khan, S.C. Bahel, Bokin Bobai. 7 (1979)
T414–T417.
[3] W. Rudnicka, Z. Osmialowska, Acta Pol. Pharm. 36 (1979)
411–419.
[4] K. Shu-Qing, T. Sun-Yung, T. Ki, C. Hung-Shan, Wei Sheng
Wu Hsueh Pao 20 (1980) 208–212.
[5] H. Singh, L.D.S. Yadav, B.K. Bhattacharya, J. Indian Chem.
Soc. 56 (1979) 1013–1016.
[6] G. Martin, R.A. Lahti, A.D. Rudzik, D.J. Duchamp, C.
Chidester, T. Scahill, J. Med. Chem. 21 (1978) 542–548.
[19] M.R. Boyd, Principles and Practices of Oncology 3 (1989) 1–10.