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

Article abstract of DOI:10.1016/j.bmcl.2016.12.061

A series of 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines 8a–j has been designed, synthesized and tested in vitro as potential pro-apoptotic Bcl-2-inhibitory anticancer agents based on our previous lead compound 8a. Synthesis of the target compounds was readily accomplished through a cyclisation reaction between indole-3-carboxylic acid hydrazide (5) and substituted isothiocyanates 6a–j, followed by oxidative cyclodesulfurization of the corresponding thiosemicarbazide 7a–j using 1,3-dibromo-5,5-dimethylhydantoin. Active compounds of the series 8a–j were found to have sub-micromolar IC₅₀ values selectively in Bcl-2 expressing human cancer cell lines; notably the 2-nitrophenyl analogue 8a was found to exhibit potent activity, and compounds 8a and 8e possessed comparable Bcl-2 binding affinity (ELISA assay) to the established natural product-based Bcl-2 inhibitor, gossypol. Molecular modeling studies helped to further rationalise anti-apoptotic Bcl-2 binding, and identified compounds 8a and 8e as candidates for further development as Bcl-2 inhibitory anticancer agents.

Full text of DOI:10.1016/j.bmcl.2016.12.061

Accepted Manuscript
Synthesis and evaluation of 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines
as Bcl-2 inhibitory anticancer agents
Rania Hamdy, Noha Ziedan, Samia Ali, Cinzia Bordoni, Mohamed El-Sadek,
Elsaid Lashin, Andrea Brancale, Arwyn T. Jones, Andrew D. Westwell
PII:
S0960-894X(16)31341-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.061
BMCL 24550
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
1 November 2016
22 December 2016
24 December 2016
Please cite this article as: Hamdy, R., Ziedan, N., Ali, S., Bordoni, C., El-Sadek, M., Lashin, E., Brancale, A., Jones,
A.T., Westwell, A.D., Synthesis and evaluation of 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines as Bcl-2
inhibitory anticancer agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.12.061
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Bioorganic & Medicinal Chemistry Letters
Synthesis and evaluation of 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines as
Bcl-2 inhibitory anticancer agents
Rania Hamdya,b , Noha Ziedana,b , Samia Alia,c, Cinzia Bordonia, Mohamed El-Sadekb , Elsaid Lashinb ,
Andrea Brancalea , Arwyn T. Jonesa and Andrew D. Westwella
aSchool of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, U.K.
bFaculty of Pharmacy, Zagazig University, Egypt
cNational Research Centre, Cairo, Egypt
ARTICLE INFO
ABSTRACT
A series of 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines 8a-j has been designed,
synthesized and tested in vitro as potential pro-apoptotic Bcl-2-inhibitory anticancer agents
based on our previous lead compound 8a. Synthesis of the target compounds was readily
accomplished through a cyclisation reaction between indole-3-carboxylic acid hydrazide (5)
and substituted isothiocyanates 6a-j, followed by oxidative cyclodesulfurization of the
corresponding thiosemicarbazide 7a-j using 1,3-dibromo-5,5-dimethylhydantoin. Active
compounds of the series 8a-j were found to have sub-micromolar IC50 values selectively in
Bcl-2 expressing human cancer cell lines; notably the 2-nitrophenyl analogue 8a was found
to exhibit potent activity, and compounds 8a and 8e possessed comparable Bcl-2 binding
affinity (ELISA assay) to the established natural product-based Bcl-2 inhibitor, gossypol.
Molecular modeling studies helped to further rationalise anti-apoptotic Bcl-2 binding, and
identified compounds 8a and 8e as candidates for further development as Bcl-2 inhibitory
anticancer agents.
Article history:
Received
Revised
Accepted
Available online
Keywords:
Anti-apoptotic Bcl-2
Indoles
Oxadiazoles
Anticancer
Drug discovery
2009 Elsevier Ltd. All rights reserved.
Resistance to apoptosis is well documented as one of the
hallmarks of cancer,1 and the Bcl-2 family of proteins play a
critical role in regulating the intrinsic apoptosis process in
response to apoptotic stimuli. The structurally related Bcl-2
protein family is structurally and functionally classified as either
anti-apoptotic (e.g. Bcl-2, Bcl-xL, Bcl-w, Mcl-1); pro-apoptotic
(e.g. Bak, Bax, Bcl-rambo); or as pro-apoptotic BH3 domain
only proteins (e.g. Bad, BID, Bim, Noxa) that bind and regulate
anti-apoptotic Bcl-2 proteins. The carefully regulated balance
between pro- and anti-apoptotic protein family members is
crucial in dictating cellular survival or commitment to apoptosis.2
between anti-apoptotic Bcl-2 family proteins and pro-apoptotic
partners. Amongst the most advanced and well-documented
examples of Bcl-2 inhibitors that have progressed to clinical
evaluation are the orally bioavailable navitoclax (ABT-263, 1),
obatoclax (2) and the polyphenolic natural product-based
experimental drug gossypol (3), shown in Figure 1. Perhaps most
significantly, the Bcl-2 inhibitor venetoclax (ABT-199, 4) has
been granted breakthrough designation by the FDA for treatment
of patients with relapsed or refractory chronic lymphocytic
leukaemia (CLL) who have the 17p chromosomal deletion.6
One notable feature of the first generation of anti-apoptotic
Bcl-2 family inhibitors is their large size (Mr = 975 in the case of
navitoclax) and chemical complexity, in part arising from the
perceived requirement to make multiple drug binding interactions
across a substantial protein-protein interaction interface. The
development of smaller, less complex molecules that retain
selective Bcl-2/Bcl-XL inhibition and antitumour activity is an
ongoing challenge. The pro-apoptotic Bcl-2 inhibitory compound
obatoclax mesylate7 suggests that structurally simple heterocylic
compounds may possess the necessary characteristics for
selective Bcl-2 inhibitory antitumour properties. Previous work
within our group has identified pro-apoptotic Bcl-2 inhibitory
compounds of the indolyl-oxadiazole,8 isoxazole9 and indolyl-
triazole10 classes; in this paper we report the discovery and early
In terms of development of new cancer therapeutics, small
molecule anti-apoptotic Bcl-2 inhibitors offer exciting prospects.3
Following Bcl-2 inhibition, the resulting cellular mitochondrial
membrane permeabilisation and release of pro-apoptotic factors
commits the cell to apoptosis and subsequent removal.
Resistance to apoptotic stimuli is commonly induced by over-
expression of anti-apoptotic Bcl-2 proteins that are also known to
contribute to therapy failure and subsequent generation of a more
resistant cancer phenotype.2
The exciting potential of small molecule anti-apoptotic Bcl-2
inhibitors in cancer therapy has led to a number of advances in
this area,3,4 focusing on the design of new cell permeable small
molecule inhibitors that block sites of protein-protein interaction5

evaluation of a new series of Bcl-2 inhibitory 5-(1H-indol-3-yl)-
N-aryl-1,3,4-oxadiazol-2-amines.
of 0.3 – 19 M (with the exception of inactive compound 8g).
The MDA-MB-231 breast cancer cell line also gave sub- to low-
micromolar IC50 values for the majority of new compounds. The
observation of sub- to low-micromolar inhibitory activity for the
majority of compounds in the Bcl-2 expressing KG1a leukaemic
cell line, but not in the Bcl-2 negative T-cell leukaemia Jurkat
cells, suggests that Bcl-2 may play a role in mediating the
observed anticancer activity.
The most active compound overall was the 2-nitrophenyl
derivative 8a that shows potent (sub-micromolar) IC50 values
across the three Bcl2-expressing human cancer cell lines (MDA-
MB-231, HeLa and KG1a) with less potent inhibitory activity in
the Bcl-2 negative Jurkat cells. The most active and selective
compounds from the cell line assay (8a, 8c and 8e), along with
moderately active compounds 8h and 8i were chosen for more
detailed mechanistic study using the enzyme-linked
immunosorbent (ELISA) sandwich binding assay along with
gossypol as positive control.
Figure 1. Examples of small molecule Bcl-2 inhibitors.
The lead compound for this study (8a) was obtained as a
virtual screening hit of the ZINC database against a Bcl-2
pharmacophore model, using methodology previously
described.11 Structure activity relationships were studied by
variation of the phenyl/benzyl group substituents that bind within
a hydrophobic groove of our model, as shown in Scheme 1.
The ELISA assay was used to determine the ability of the test
compounds to compete with immobilized Bim peptide for
binding to His-tagged Bcl-2 protein, according to our previously
described protocol10,19 (see Supplementary Information). The
ELISA binding results (Table 2) displayed some consistency with
the predicted activity of the killing pattern, in that the potently
active compound 8e showed similar binding affinity to Bcl-2
compared with reference standard gossypol. Compound 8e was
found to be two-fold more active than our initial lead compound
8a.
Compound number
ELISA IC50, M
1.24 ± 0.18
8a
Scheme 1: Synthesis of indolyl-N-aryl-oxadiazolamines.
8c
3.83 ± 0.05
Synthesis of the target compounds was readily achieved in
two synthetic steps, through adaptation of a literature route.12,13
The first step involved heating indole-3-carbonylhydrazide (5)
with substituted phenyl/benzyl isothiocyanates (6a-j) in refluxing
ethanol to produce the intermediate thiosemicarbazide (7a-j) that
was used in the next step without further purification.14
8e
0.66 ± 0.06
8h
8i
21.12 ± 0.18
29.05 ± 0.17
0.60 ± 0.09
Gossypol (3)
Treatment
of
the
indolyl-3-carbonyl-N-phenyl/benzyl
thiosemicarbazide (7a-j) with KI and 1,3-dibromo-5,5-
dimethylhydantoin in basic solution afforded the product 5-(1H-
indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines (8a-j) in high yield
(58-86%) following recrystallisation from ethanol.15,16
Table 2. ELISA binding activity values (IC50) for selected compounds.
Results are expressed as triplicate testing mean values.
Further evidence that Bcl-2 might be responsible for the
observed anticancer activity of the new indolyl-N-aryl-
oxadiazolamines was obtained through molecular modeling
studies. Interactions between the most active compounds 8a and
8e, and a published Bcl-2 crystal structure (PDB code 4AQ320),
were studied within MOE21 making use of the LeadIt software22
for analyzing molecular interactions. Docking of compound 8a at
the Bim peptide-binding site revealed that 8a mimics residues
Leu95 and Ile97 of Bim, making important interactions between
Leu96 and the extracellular NH group, and between Arg105 and
the oxadiazole ring nitrogen (3-position), within the Bcl-2
hydrophobic groove. Compound 8e occupies a similar Bcl-2
binding site mimicking the Phe101, Leu95, and Ile97 residues of
Bim peptide, similarly making interactions between Leu96 and
the extracellular NH group, and between Arg105 and the
oxadiazole ring nitrogen (3-position). For compound 8e, an
additional interaction between key Bcl-2 hydrophobic groove
residue Tyr67 and the indole ring of 8a is apparent.
Representations of the binding models of 8a (A) and 8e (B)
within the Bcl-2 hydrophobic pocket are shown in Figure 2.
Evaluation of the newly synthesized indolyl-oxadiazolamines
was carried out across four human cancer cell lines. Viability in
the established triple-negative breast cancer cell line MDA-MB-
231 and cervical cancer (HeLa) cells (both Bcl-2 expressing) was
assessed using the MTT endpoint assay. To test the effect of
differing Bcl-2 status, further evaluation of new compounds was
carried out in the human cancer cell lines KG1a (acute
myelogenous leukaemia) and Jurkat (T cell leukaemia). The
Jurkat cell line has previously been characterized as Bcl-2
negative by our group17 and others,18 unlike the Bcl-2 expressing
KG1a cell line.19 For studies on the leukaemic cells we used the
CellTiter-Blue® viability assay, appropriate for endpoint
determinations in these non-adherent cells. The results, expressed
as mean values following testing on at least three separate
occasions, are presented in Table 1.
The results shown in Table 1 indicated that 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

Table 1. Growth inhibitory activity (IC50, M) values for 5-(1H-indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines 8a-j in human cancer cell lines MDA-MB-231
(breast), HeLa (cervical), KG1a (acute myelogenous leukaemia) and Jurkat (T cell leukaemia). Results are expressed as triplicate testing mean values.
Compound
MDA-MB-231
0.90 ± 0.02
34.6 ± 0.64
3.54 ± 0.03
9.77 ± 0.82
8.89 ± 0.05
31.08 ± 0.6
>100
HeLa
KG1a
Jurkat
30.2 ± 1.5
>100
8a
8b
8c
8d
8e
8f
0.30 ± 0.04
15.12 ± 0.32
1.76 ± 0.02
3.80 ± 0.20
5.60 ± 0.09
19.29 ± 1.09
>100
0.85 ± 0.08
25.5 ± 0.65
0.89 ± 0.04
3.16 ± 0.19
32.7 ± 0.68
45.3 ± 0.34
21.1 ± 1.6
45.0 ± 1.3
30.2 ± 1.5
>100
>100
8g
8h
8i
>100
27.25 ± 0.98
10.08 ± 0.83
6.47 ± 0.52
15.79 ± 0.96
5.82 ± 0.07
7.81 ± 0.87
4.00 ± 0.13
0.82 ± 0.05
>100 ± 0.03
>100
>100
8j
>100
further development against this well-established anti-apoptotic
target.
A
Acknowledgments
We thank the Egyptian Government for the award of a PhD
studentship (to NIZ) and the award of a Channel Scholarship (to
RH and SA). We acknowledge the support of Cancer Research
Wales for a PhD studentship (to CB). We are also grateful to the
EPSRC UK National Mass Spectrometry Facility at Swansea
University (U.K.) for provision of compound accurate mass
determinations.
References and notes
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14. General method for synthesis of indolyl-3-carbonyl-N-
phenyl/benzyl thiosemicarbazide (7a-j). To a solution of indole-3-
carbonylhydrazide (5, 1.0 mmol) in absolute ethanol (100 mL)
was added a solution of substituted phenyl/benzyl isothiocyanate
(6a-j) in ethanol (50 mL) with continuous stirring. The reaction
mixture was heated under reflux for 1h. After cooling to room
temperature, the precipitate formed was collected by filtration, and
washed with ice-cold ethanol (30 mL) to give the corresponding
Figure 2. Binding of indolyl-N-aryl-oxadiazolamine hit compounds 8a (A)
and 8e (B) within the Bcl-2 hydrophobic binding pocket. Only the Bcl-2
residues involved in hydrogen bonding (Leu-96, Arg-105) or in π-H
interaction (Tyr-67) with compounds 8a and 8e are represented.
In conclusion, we have identified the 5-(1H-indol-3-yl)-N-aryl-
1,3,4-oxadiazol-2-amines 8e and 8a as new Bcl-2 inhibitory
small molecules with moderately potent anti-proliferative activity
against Bcl-2 expressing human cancer cell lines and Bcl-2
protein. These new compounds are useful lead compounds for

thiosemicarbazide (7a-j), which was used in the next step without
further purification.
calcd. for C16H11N5O3: C, 59.81; H, 3.45; N, 21.80. Found: C,
59.46; H, 3.35; N, 21.47.
15. General method for synthesis of 5-(1H-indol-3-yl)-N-aryl-1,3,4-
oxadiazol-2-amines (8a-j). To a suspension of the indolyl-3-
carbonyl-N-phenyl/benzyl thiosemicarbazide (7a-j, 1.5 mmol) in
isopropanol (10 mL) was added a solution of potassium iodide
(1.5 mmol) in water (2 mL) and 5N NaOH (1.5 mmol) at 5 °C,
and the mixture was stirred until a clear solution was formed. A
solution of 1,3-dibromo-5,5-dimethyl hydantoin (3.8 mmol) in
acetonitrile (10 mL) was the added over 1h whilst maintaining the
temperature below 10 °C. The mixture was stirred for a further 1h
at 10 °C and then the reaction mixture was quenched with aqueous
NaHSO3 and the crude product collected by filtration.
Recrystallisation from ethanol gave the corresponding pure 5-(1H-
indol-3-yl)-N-aryl-1,3,4-oxadiazol-2-amines (8a-j).
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K.; Tian, X. F.; Town, J.; Cao, X. H.; Lin, F.; Zhai, D. Y.; Kitada,
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20. Research Collaboration for Structural Bioinformatics (RCSB)
Protein Data Bank (PDB); www.rcsb.org/pdb
21. Molecular Operating Environment (MOE), Chemical Computing
Group Inc., Montreal, Canada; www.chemcomp.com
16. Representative characterization data: 5-(1H-Indol-3-yl)-N-(4-
22. BioSolveIT GmbH; www.biosolveit.de/LeadIT
1
nitrophenyl)-1,3,4-oxadiazol-2-amine (8c): (85% yield). H NMR
(DMSO-d6)  7.26 (2H, m, ArH), 7.55 (1H, d, J 7.4 Hz, ArH),
7.84 (2H, d, J 9.1 Hz, H-3’, H-5’), 8.03 (1H, d, J 2.7 Hz, H-2),
8.11 (1H, d, J 7.0 Hz, ArH), 8.30 (2H, d, J 9.1 Hz, H-2’, H-6’),
11.40 (1H, bs, NH), 11.91 (1H, bs, NH). 13C NMR (DMSO-d6) 
99.48 (C-3), 112.38 (ArCH), 116.65 (ArCH), 120.18 (ArCH),
120.96 (ArCH), 122.78 (ArCH), 123.99 (ArC), 125.48 (ArCH),
126.93 (ArCH), 136.38 (ArC), 140.80 (ArC), 145.31 (ArC),
156.54 (ArC), 157.49 (ArC). MS (ESI+) 322.1 (M++1). Analysis
Supplementary Material
Chemical analytical and spectroscopic data (NMR, mass
spectrometry and % C,H,N elemental analysis) are detailed in
Supplementary Material, along with detailed protocols for cell
culture and endpoint assays, plus the Bcl-2 ELISA assay and
molecular modelling studies.

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1,3,4-oxadiazol-2-amines as Bcl-2 inhibitory anticancer agents
Rania Hamdy, Noha Ziedan, Samia Ali, Cinzia Bordoni, Mohamed El-Sadek, Elsaid Lashin, Andrea Brancale,
Arwyn T. Jones and Andrew D. Westwell