Synthesis and evaluation of 3-(benzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol- 4-amines as Bcl-2 inhibitory anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2013.02.029

A series of substituted 3-(benzylthio)-5-(1H-indol-3-yl)-4H-1,2,4-triazol- 4-amines has been synthesised and tested in vitro as potential pro-apoptotic Bcl-2-inhibitory anticancer agents. Synthesis of the target compounds was readily accomplished in good yields through a cyclisation reaction between indole-3-carboxylic acid hydrazide and carbon disulfide under basic conditions, followed by S-benzylation. Active compounds, such as the nitrobenzyl analogue 6c, were found to exhibit sub-micromolar IC₅₀ values in Bcl-2 expressing human cancer cell lines. Molecular modelling and ELISA studies further implicated anti-apoptotic Bcl-2 as a candidate molecular target underpinning anticancer activity.

Full text of DOI:10.1016/j.bmcl.2013.02.029

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2391–2394
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 3-(benzylthio)-5-(1H-indol-3-yl)-
1,2,4-triazol-4-amines as Bcl-2 inhibitory anticancer agents
Rania Hamdy a,b, Noha Ziedan a,b, Samia Ali a,c, Mohamed El-Sadek b, Elsaid Lashin b, Andrea Brancale a,
Arwyn T. Jones a, Andrew D. Westwell a,
⇑
a School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB Wales, UK
b Faculty of Pharmacy, Zagazig University, Egypt
c National Research Centre, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted 3-(benzylthio)-5-(1H-indol-3-yl)-4H-1,2,4-triazol-4-amines has been synthesised
and tested in vitro as potential pro-apoptotic Bcl-2-inhibitory anticancer agents. Synthesis of the target
compounds was readily accomplished in good yields through a cyclisation reaction between indole-3-
carboxylic acid hydrazide and carbon disulfide under basic conditions, followed by S-benzylation. Active
compounds, such as the nitrobenzyl analogue 6c, were found to exhibit sub-micromolar IC50 values in
Bcl-2 expressing human cancer cell lines. Molecular modelling and ELISA studies further implicated
anti-apoptotic Bcl-2 as a candidate molecular target underpinning anticancer activity.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 29 November 2012
Revised 1 February 2013
Accepted 7 February 2013
Available online 14 February 2013
Keywords:
Anti-apoptotic Bcl-2
Anticancer
Indoles
Triazole-amines
The intracellular balance between pro- and anti-apoptotic Bcl-2
family proteins is critical for regulation between programmed cell
death and cell survival respectively.1 For example in cancer cells,
where resistance to apoptotic cell death is a well documented hall-
mark,2 over-expression of anti-apoptotic family members such as
the prototypical Bcl-2 protein is well known.3 Hence inhibition of
anti-apoptotic Bcl-2 family members has emerged in recent years
as an exciting anticancer drug discovery strategy, since Bcl-2 inhi-
bition should lead to a selective pro-apoptotic cascade from which
cancer cells cannot easily recover.
rently in Phase I/II clinical trials,7 suggests that structurally simple
drug-like molecules may have the necessary attributes as candi-
date Bcl-2 family inhibitors. Our own previous work in the design
of indole-based pro-apoptotic heterocycles8 has included the re-
cent identification of antitumour indolyl-oxadiazoles9 and -isoxaz-
oles.10 In this Letter we extend our previous studies on pro-
apoptotic indole-based heterocycles to the synthesis and antitu-
mour evaluation of a series of novel 3-(benzylthio)-5-(1H-indol-
3-yl)-4H-1,2,4-triazol-4-amines as Bcl-2 inhibitory anticancer
agents.
Cancer drug design strategies towards the Bcl-2 family have
tended to focus on sites of protein–protein interaction between
anti-apoptotic Bcl-2 members (e.g., Bcl-2, Bcl-xL, Mcl-1) and their
pro-apoptotic binding partners (e.g., Bax, Bak, Bid).4 Pioneering
Bcl-2 inhibitory drug candidates within this class include the po-
tent structural analogues ABT-737 (1) and ABT-263 (2, Navitoclax)
from Abbott Laboratories that have progressed to Phase I/II clinical
evaluation in cancer (Fig. 1).5 The relatively large size of leading
Bcl-2 inhibitors as drug candidates (Mr = 975 in the case of ABT-
263), arising from the requirement to make interactions across a
protein–protein interface,6 has driven efforts to develop selective
Bcl-2 inhibitors of lower molecular weight in recent years. The evo-
lution of small molecules such as the pan-Bcl-2 inhibitory indole-
bipyrrole drug candidate Obatoclax mesylate (3, GX15-070), cur-
The synthesis of 3-(benzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol-
4-amines was accomplished in two steps from commercially avail-
able indole 3-carboxylic acid hydrazide (4) according to literature
precedent.11 Briefly, commercially available compound 4 was cyc-
lised by reaction with carbon disulfide under basic conditions, fol-
lowed by hydrazine hydrate under reflux. This one-pot reaction
produced the required 4-amino-5-(1H-indol-3-yl)-4H-[1,2,4]tria-
zole-3-thiol (5) in 75% yield following recrystallisation from etha-
nol.12 Reaction of intermediate compound 5 with a series of
substituted benzyl bromides under basic conditions afforded the
3-(benzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol-4-amine products
in good yields (69–95%) following recrystallisation from ethanol
(Scheme 1).13
Evaluation of newly synthesized 3-(benzylthio)-5-(1H-indol-
3-yl)-1,2,4-triazol-4-amine products14 was carried out in the
established human MDA-MB-231 (breast) and HeLa (cervical) can-
cer cell lines using the colorimetric MTT cell proliferation assay to
⇑
Corresponding author. Tel.: +44 (0) 2920 875800; fax: +44 (0) 2920 8754149.
E-mail address: WestwellA@cf.ac.uk (A.D. Westwell).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.029

2392
R. Hamdy et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2391–2394
O
CF3
S
O
NO2
H
H
N
N
S
S
H
H
O
N
O
N
S
S
O
O
O
O
N
O
N
N
N
2 (ABT-263)
N
N
1 (ABT-737)
N
.CH3SO3H
HN
OMe
N
H
3 (Obatoclax mesylate)
Cl
Cl
Figure 1. Examples of small molecule Bcl-2 inhibitors in development.
H
O
H
N
N
NHNH2
(i), (ii)
NH2
NH2
(iii)
N
N
S
R
SH
N
N
N
N
N
H
4
5
6a-i
6
6
R
R
aReagents and conditions: (i) CS2, KOH, EtOH, room temp., 16h;
(ii) N2H4.H2O, reflux, 3h; (iii) subs. benzyl bromide, KOH, EtOH, room temp., 16h
a
b
f
g
H
4-Br
3-OMe
2-NO2
c
d
e
3-NO2
4-NO2
4-F
h
4-OMe
4-CH3
i
Scheme 1. Synthesis of 3-(benzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol-4-amines. aReagents and conditions: (i) CS2, KOH, EtOH, room temp, 16 h; (ii) N2H4ꢀH2O, reflux, 3 h;
(iii) subs. benzyl bromide, KOH, EtOH, room temp, 16 h.
assess viability, as previously described (see Supplementary
data).15 The MDA-MB-231 and HeLa human cancer cell lines are
well established in our laboratory for the initial screening for
growth inhibitory activity of newly synthesized compounds. The
MDA-MB-231 cell line is known to express Bcl-2 and previous
studies have shown down-regulation of Bcl-2 in these cells by
Western blot analysis following inhibitor treatment.16 Stable
expression of Bcl-2 in the HeLa cell line is also well known.17 The
results, expressed as mean values following testing on at least
three separate occasions, are presented in Table 1.
leukaemia) and Jurkat (T-cell leukaemia). The Jurkat cell line has
previously been characterized as Bcl-2 negative, by our group18
and others,19 unlike the Bcl-2-expressing KG1a cell line.20 For these
studies we used the CellTiter-BlueÒ cell viability assay (see Supple-
mentary data), appropriate for endpoint determinations in these
non-adherent cell lines. The results, expressed as mean values fol-
lowing testing on at least three separate occasions, are presented
in Table 2.
The results presented in Tables 1 and 2 indicate some clear
structure–activity relationship trends. In general the HeLa cell line
was the most sensitive to the effects of test compounds with IC50
values in the sub- to low-micromolar range (0.25–8.0 lM). The
MDA-MB-231 breast cancer cell line also gave sub- to low-micro-
To test the effect of differing Bcl-2 status within the human can-
cer cell environment, further evaluation of new compounds was
carried out in the leukaemic cell lines KG1a (acute myelogenous
Table 1
Table 2
Growth inhibitory activity (IC50
,
l
M) values for 3-(benzylthio)-5-(1H-indol-3-yl)-
Growth inhibitory activity (IC50, lM) values for 3-(benzylthio)-5-(1H-indol-3-yl)-
1,2,4-triazol-4-amine products 6a–i in human cancer cell lines MDA-MB-231 (breast)
and HeLa (cervical), using the MTT assay. Results are expressed as triplicate mean
values (SEM)
1,2,4-triazol-4-amine products 6a–i in human leukaemia KG1a and Jurkat cell lines,
using the CellTitre-BlueÒ assay as readout for anti-proliferative activity. Results are
expressed as triplicate mean values (SEM)
Compound
MDA-MB-231
HeLa
Compound
KG1a
Jurkat
6a
6b
6c
6d
6e
6f
6g
6h
6i
26.5 (0.3)
2.30 (0.07)
2.52 (0.19)
0.40 (0.07)
0.25 (0.09)
0.29 (0.02)
8.00 (0.06)
1.73 (0.07)
0.75 (0.02)
5.38 (0.04)
6a
6b
6c
6d
6e
6f
6g
6h
6i
17.2 (0.23)
20.82 (0.69)
0.65 (0.21)
6.90 (0.18)
13.2 (0.07)
55.80 (0.68)
12.86 (0.14)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9.52 (0.13)
0.31 (0.03)
0.74 (0.03)
1.35 (0.09)
71.4 (0.68)
15.56 (0.36)
1.70 (0.07)
10.49 (0.59)
>100

R. Hamdy et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2391–2394
2393
molar IC50 values for the majority of test compounds. Although the
Bcl-2-expressing KG1a acute myelogenous leukaemia cell line was
generally less responsive to test agents, IC50 values in the low
micromolar range were observed, particularly for compounds 6a–
e and 6g. Particularly noteworthy (Table 2) was that anti-prolifer-
ative activity was concentrated within the Bcl-2-expressing KG1a
leukaemic cell line, whereas all compounds were found to be inac-
tive in the Bcl-2 negative human T-cell leukaemia Jurkat cells. The
data from Table 2 suggests that Bcl-2 may play a role in mediating
the observed anticancer activity.
The most active compound overall was the 3-nitrobenzyl deriv-
ative 6c that shows potent (sub-micromolar IC50) values across the
three Bcl-2-expressing cancer cell lines (MDA-MB-231, HeLa and
KG1a). Also active was the isomeric 4-nitrobenzyl analogue 6d,
however a marked drop in activity was observed for the 2-nitro-
benzyl derivative 6b. A further notable compound showing sub-
to low-micromolar activity across the three Bcl-2 expressing cell
lines was the 4-fluorobenzyl analogue 6e.
Further evidence that Bcl-2 might be responsible for the ob-
served anticancer activity of the new triazole-amines such as the
3-nitrobenzylthio derivative 6c was gained through molecular
modelling studies. A comparison of interactions between com-
pound 6c and several less active analogues (e.g., 6b, 6d, 6f, 6g
and 6i) with a published ligand-bound Bcl-2 crystal structure21
(PDB code: 4AQ3; BH3 domain)22 were studied using MOE23 and
the Leadit molecular docking software.24 The BH3 domain of the
Bcl-2 model consists of one hydrophobic and one hydrophilic
groove connected by hydrophobic amino acid residues. Compound
6c was found to bind within the pro-apoptotic BH3-only peptide
hydrophobic groove of Bcl-2, making a number of key binding
interactions particularly between the Bcl-2 side chain residue
Tyr-67 and the 4-amino and the sulfur atoms of 6c. The 2-nitroben-
zyl substituted compound 6b (and other less active analogues)
adopted a less favourable binding conformation in the Bcl-2 active
site. Compound 6b docked further away from the active site and
was unable to make key binding interactions with important active
site residues such as Tyr-67. Figure 2 shows the comparative bind-
ing interactions between compounds 6c and 6b within the Bcl-2
active site.
The ability of compound 6c to compete with the pro-apoptotic
BH3 (Bcl-2 homology domain 3) Bim peptide for binding to Bcl-2
was evaluated using an ELISA assay, adapted from a previously
published protocol (see Supplementary data for details).25 In brief,
biotinylated Bim peptide was attached to streptavidin-coated
microtitre plates. Mixtures of test compound 6c at varying concen-
trations and His-tagged Bcl-2 were then added to the plates, allow-
ing competitive binding between 6c and Bim peptide for His-
tagged Bcl-2. Following further washing, anti-His secondary anti-
body conjugated to the enzyme horse-radish peroxidase (HRP)
was added. Addition of o-phenylenediamine, a chromogenic sub-
strate that produces a soluble coloured end-product (2,3-diamino-
phenazine) in the presence of peroxidase enzyme, was followed by
spectrophotometric reading at 450 nm. Reduction of the signal at
450 nm by competitive binding of compound 6c to Bcl-2, is then
plotted against concentration to derive an IC50 value for 6c. Tripli-
cate measurement of the ELISA IC50 gave a mean value of 1.43
lM
(SEM = 0.19), compared with an IC50 of 2.11 M for the standard
l
Bcl-2 inhibitor gossypol, a natural product-based agent currently
in clinical trials against cancer.26
In summary, the design and synthesis of a series 3-(benzylthio)-
5-(1H-indol-3-yl)-4H-1,2,4-triazol-4-amines as potential antican-
cer agents targeting Bcl-2 has lead to the discovery of a number
of compounds with sub-micromolar IC50 activity in Bcl-2 express-
ing human cancer cell lines. The 3-nitrobenzyl derivative 6c was
identified as the most active compound of the series. Compound
6c was further found to bind within the BH3 domain of Bcl-2,
according to molecular modelling and ELISA studies, providing
support for the prototypical anti-apoptotic protein Bcl-2 as a po-
tential mechanistic target underpinning in vitro anticancer
activity.
Acknowledgments
The authors are grateful to the Higher Education Ministry of the
Arab Republic of Egypt for the award of a PhD studentship (to S.A.)
and a Channel Scholarship (to R.H.). We thank the EPSRC National
Mass Spectrometry Centre (Swansea, U.K.) for provision of accurate
mass spectrometric analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
029.
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Figure 2. 3-(3-Nitrobenzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol-4-amine (6c, blue)
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disulfide (12 mL) was added dropwise to a mixture of indole-3-carboxylic
acid hydrazide (10 mmol) and potassium hydroxide (15 mmol) in absolute
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reaction mixture heated under reflux for 3 h. After cooling, the mixture was
diluted with water (10 mL) and neutralized using 1 M HCl (aq). The crude
product precipitate was collected by vacuum filtration and washed with cold
water. Recrystallisation from ethanol gave the required 4-amino-5-(1H-indol-
3-yl)-4H-[1,2,4]triazole-3-thiol product in 75% yield, which was used
immediately in the next step.
13. General method for S-benzylation of 4-amino-5-(1H-indol-3-yl)-4H-
[1,2,4]triazole-3-thiol. A solution of substituted benzyl bromide (10 mmol) in
absolute ethanol (10 mL) was added to a solution of intermediate thiol (5,
10 mmol) and potassium hydroxide (1 mmol) in ethanol (10 mL). The reaction
mixture was stirred for 16 h at room temperature, then the resulting
precipitate was collected by vacuum filtration and recrystallised from
ethanol to give the required 3-(benzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol-4-
amines (6a–i) in 69–95% yield.
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1,2,4-triazol-4-amine (6a): (76% yield). Mp 235–237 °C. 1H NMR (DMSO-d6) d
4.43 (2H, s, CH2), 6.06 (2H, s, NH2), 7.16 (1H, t, J 8.0, ArH), 7.20 (1H, t, J 8.0, ArH),
7.27 (1H, t, J 8.0, ArH), 7.34 (2H, m, ArH), 7.48 (3H, m, ArH), 8.26 (2H, d, J 7.1,
ArH), 11.65 (1H, s, NH indole). 13C NMR (DMSO-d6) d 35.13 (CH2), 101.71 (C-3),
111.62 (ArCH), 120.08 (ArCH), 121.33 (ArCH), 122.12 (ArCH), 125.35 (ArC),
125.63 (ArCH), 127.26 (ArCH), 128.38 (ArCH), 129.00 (ArCH), 135.63 (ArC),
137.71 (ArC), 150.71 (ArC), 151.77 (ArC). MS (ESI+) 322 (M++1). Anal. Calcd for
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(1H-Indol-3-yl)-3-(3-nitrobenzylthio)-4H-1,2,4-triazol-4-amine (6c) (85% yield).