Design, synthesis and evaluation of new bioactive oxadiazole derivatives as anticancer agents targeting bcl-2

Article abstract of DOI:10.3390/ijms21238980

A series of 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles, 4a–m, were designed, synthesized and tested in vitro as potential pro-apoptotic Bcl-2 inhibitory anticancer agents based on our previously reported hit compounds. Synthesis of the target 1,3,4-oxadiazoles was readily accomplished through a cyclization reaction of indole carboxylic acid hydrazide 2 with substituted carboxylic acid derivatives 3a–m in the presence of phosphorus oxychloride. New compounds 4a–m showed a range of IC₅₀ values concentrated in the low micromolar range selectively in Bcl-2 positive human cancer cell lines. The most potent candidate 4-trifluoromethyl substituted analogue 4j showed selective IC₅₀ values of 0.52–0.88 μM against Bcl-2 expressing cell lines with no inhibitory effects in the Bcl-2 negative cell line. Moreover, 4j showed binding that was two-fold more potent than the positive control gossypol in the Bcl-2 ELISA binding affinity assay. Molecular modeling studies helped to further rationalize anti-apoptotic Bcl-2 binding and identified compound 4j as a candidate with drug-like properties for further investigation as a selective Bcl-2 inhibitory anticancer agent.

Full text of DOI:10.3390/ijms21238980

International Journal of
Molecular Sciences
Article
Design, Synthesis and Evaluation of New Bioactive
Oxadiazole Derivatives as Anticancer Agents
Targeting Bcl-2
Rania Hamdy 1,2,3,*, Samia A. Elseginy 1,4,5 , Noha I. Ziedan 1,6, Mohamed El-Sadek 2
,
Elsaid Lashin 2, Arwyn T. Jones 1 and Andrew D. Westwell 1,
*
1
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building,
King Edward VII Avenue, Cardiff CF10 3NB, Wales, UK; samiaaliph@gmail.com (S.A.E.);
n.ziedan@chester.ac.uk (N.I.Z.); JonesAT@cardiff.ac.uk (A.T.J.)
Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt; m.elhussenyelsadek@yahoo.com (M.E.-S.);
slashine@yahoo.com (E.L.)
Research Institute for Medical and Health Sciences, University of Sharjah, P.O. Box 27272, Sharjah, UAE
Green Chemistry Department, Chemical Industries Research Division, National Research Center,
Cairo, Egypt
School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK
Department of Mathematical and Physical Sciences, University of Chester, Chester CH2 4NU, UK
Correspondence: Rania_hamdy2000@yahoo.com (R.H.); WestwellA@cardiff.ac.uk (A.D.W.)
2
3
4
5
6
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 23 October 2020; Accepted: 21 November 2020; Published: 26 November 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles, 4a–m, were designed, synthesized
and tested in vitro as potential pro-apoptotic Bcl-2 inhibitory anticancer agents based on our previously
reported hit compounds. Synthesis of the target 1,3,4-oxadiazoles was readily accomplished through a
cyclization reaction of indole carboxylic acid hydrazide 2 with substituted carboxylic acid derivatives
3a–m in the presence of phosphorus oxychloride. New compounds 4a–m showed a range of IC50 values
concentrated in the low micromolar range selectively in Bcl-2 positive human cancer cell lines. The most
potent candidate 4-trifluoromethyl substituted analogue 4j showed selective IC50 values of 0.52–0.88 µM
against Bcl-2 expressing cell lines with no inhibitory effects in the Bcl-2 negative cell line. Moreover,
4j showed binding that was two-fold more potent than the positive control gossypol in the Bcl-2 ELISA
binding affinity assay. Molecular modeling studies helped to further rationalize anti-apoptotic Bcl-2
binding and identified compound 4j as a candidate with drug-like properties for further investigation as
a selective Bcl-2 inhibitory anticancer agent.
Keywords: oxadiazole; indole; aromatic heterocycle; anticancer; Bcl-2 inhibitor; chemical synthesis;
molecular modeling; apoptosis
1. Introduction
The archetypal member of the Bcl-2 anti-apoptotic gatekeeper protein family is a well-studied drug
target [1], validated in recent years by the clinical approval of Venetoclax (ABT-199) for the treatment
of chronic lymphocytic leukemia, small lymphocytic lymphoma and acute myeloid leukemia [
2]. As a
Bcl-2 homology domain (BH3) mimetic, Venetoclax represented an important development within the
area of protein–protein interaction (PPI) inhibitors. As a target class, PPI inhibitors had previously been
thought to be rather intractable drug targets [3]; however, Venetoclax represents an early example of a
PPI inhibitor targeting a hot-spot domain that was validated in clinical trials. Other Bcl-2 inhibitory
agents that have been studied in cancer clinical trials include obatoclax mesylate [4], an indole-based
Bcl-2 inhibitor that received orphan drug designation in the US, in 2004, for the treatment of chronic
Int. J. Mol. Sci. 2020, 21, 8980; doi:10.3390/ijms21238980
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 8980
2 of 11
lymphocytic leukemia (Figure 1). The relatively simple heterocyclic structure of obatoclax has provided
inspiration for our own studies on a range of indole-azole-linked Bcl-2 inhibitory anticancer agents.
Previously we reported a series of indole-based oxadiazole amines [
amines [ ] as Bcl-2 inhibitors (Figure 1). Within this series, we found that the oxadiazoles were more
active than the corresponding triazoles [ ], with sub-micromolar activity found for the most active
indole-oxadiazole amine series members in the Bcl-2 ELISA binding assay. In addition, we discovered
new quinolinyl-based oxadiazoles and triazoles as potent Bcl-2 inhibitory anticancer compounds [ ].
5,6] and indole-based triazole
7
7
8
In order to discover more potent and selective anticancer agents for further development and as a
continuation of our studies, we aimed to investigate the structure activity relationship and carry out
optimization of new indolyl-oxadiazoles.
H
N
S
N
N
R
R
N
N
N
O
N
HN
OCH3
NH2
N
H
CH3SO3H
N
N
H
H
Hamdy et al. Bioorg. Med.
Chem. Lett 2017
Hamdy et al. Bioorg. Med.
Chem. Lett 2013
Obatoclax mesylate
N
N
X
O
N
R
H
4(a-m)
Figure 1. Rational design of novel indole-based oxadiazoles 4a–m.
In this paper, we further modified the connector ( ) between the oxadiazole ring and an aromatic
X
ring, to further investigate the role of the connecting group in the structural optimization with respect
to fitting into the active site of the Bcl-2 protein (Figure 1).
2. Results
2.1. Synthesis of Target Compounds
Reaction of commercially available ethyl indol-3-yl carboxylate (
under reflux gave the corresponding hydrazide ( ) in 80% isolated yield, according to the
previously reported method [ ) was then reacted with aryl-linked
1) with hydrazine hydrate
2
5–7]. The intermediate hydrazide (2
carboxylic acids (3a–m) in the presence of phosphorus oxychloride (50 ◦C), to provide the product
2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles (4a–m) in 46–66% yield (Scheme 1).

Int. J. Mol. Sci. 2020, 21, 8980
3 of 11
Scheme 1. Synthesis of 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles 4a–m.
2.2. In Vitro Antiproliferative Activity
Evaluation of anti-proliferative activity for the new 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles
(
4a–m) was carried out in Bcl-2-expressing human metastatic breast (MDA-MB-231) [ ] and cervical
(HeLa) [10] cancer cell lines, using the MTT endpoint assay, following 72 h incubation of test compound,
according to previously established protocols [ ]. Further analysis within the non-adherent leukemic
cell lines KG1a (acute myelogenous leukemia, Bcl-2 +ve) [11] and Jurkat (T-cell leukemia, Bcl-2 ve,) [12
was carried out by using the CellTiter-Blue® assay according to published precedent [
]. The results
9
5–8
−
]
5–8
(IC50 values in mM, mean values from three independent experiments) are presented in Table 1.
Table 1. In vitro anti-proliferative activity of 2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles 4a–m in
the MDA-MB-231 (metastatic breast), HeLa (cervical), KG1a (AML) and Jurkat (T-cell leukemia) human
cancer cell lines.
Compound
X
R
IC50 (mM) 1
MDA-MB-2312 HeLa 2 KG1a 3
Jurkat 3
4a
4b
4c
4d
4e
4f
OCH2
OCH2
OCH2
OCH2
CH2
CH2
4-F-C6H4
4-CH3-C6H4
4-tBu-C6H4
C6H5
3-OMe-C6H4
C6H5
36.11 ± 3.4
31.53 ± 4.8
3.50 ± 0.16
1.63 ± 0.08
6.28 ± 0.20
>100
12.86 ± 1.33
13.31 ± 0.32
0.9 ± 0.03
0.57 ± 0.04
3.14 ± 0.14
>100
>100
>100
3.45 ± 0.8
2.56 ± 0.07
3.66 ± 0.15
>100
>100
>100
>100
>100
>100
>100
4g
4h
4i
4j
4k
4l
-
-
-
-
-
-
-
4-NO2-C6H4
4-OMe-C6H4
3,4,5-triOMe- C6H2
4-CF3-C6H4
6.95 ± 0.5
6.02 ± 0.12
1.19 ± 0.1
0.52 ± 0.15
0.92 ± 0.19
2.97 ± 0.06
1.79 ± 0.07
1.60 ± 0.12
0.92 ± 0.08
1.22 ± 0.9
0.88 ± 0.16
1.28 ± 0.35
1.52 ± 0.03
1.19 ± 0.4
>100
>100
2.21 ± 0.1
0.975 ± 0.02
0.73 ± 0.09
1.47 ± 0.13
4.5 ± 0.31
1.87 ± 0.14
60.59 ± 0.12
12.25 ± 1.13
>100
4-CH3-C6H4
6(1-piperidinyl)-pyridin-3-yl
5-OMe-indol-3-yl
>100
61.4 ± 1.4
4m
57.45 ± 0.58
Mean values of three independent experiments (±SEM); 2 MTT assay; 3 CellTitre-Blue® assay.
1
With the exception of compounds 4a, 4b and 4f, the test compounds were found to inhibit the
growth of the Bcl-2 expressing cell lines (MDA-MB-231, HeLa and KG1a) with IC50 values < 10 mM.
In general, the Bcl-2 negative cell line (Jurkat) was found to be unresponsive. Considering the different
linker groups, the methylene linker (X = CH2) gave low potency values for compound 4e and the
unresponsive compound 4f. The more active group of compounds appeared to be those where the
oxadiazole ring was directly connected to the aryl group (no linker), with compounds 4j (R = CF3Ph)

Int. J. Mol. Sci. 2020, 21, 8980
4 of 11
and 4k (R = 4-CH3-Ph) being particularly potent. A selection of these most active compounds plus
compounds of intermediate activity were taken forward to study for competitive Bcl-2 binding in the
ELISA assay.
2.3. Evaluation of Bcl-2 Binding (ELISA Assay)
The most active compounds from the cell line assay (4j and 4k), along with the moderately
active methyleneoxy linker compounds (4c and 4d), the methylene linker compound 4e and other
active compounds with no linker (4i and 4m), plus the inactive compound 4b, were further studied.
Compounds were evaluated according to their ability to compete with the pro-apoptotic BH3 domain
Bim peptide for binding to His-tagged Bcl-2 protein, with a readout from anti-His secondary antibody
conjugated to HRP (horseradish peroxidase) enzyme that produces a colorimetric readout in the
presence of o-phenylenediamine (competitive ELISA assay). The protocol followed our previously
published work on Bcl-2 inhibitors [5–8], and the results are shown in Table 2. The clinical trial stage
Bcl-2 inhibitory natural product (-)-gossypol [13] was used as a positive control.
Table 2. ELISA binding activity IC50 values for selected 2-(1H-indol-3-yl)-5-substituted-1, and 3,4-
oxadiazoles compared to positive control (-)-gossypol.
Compound
ELISA IC50 (µM) 1
4b
>100
4c
4d
4e
4i
4j
1.17 ± 0.13
1.41 ± 0.01
2.72 ± 0.2
21.05 ± 1.09
0.33 ± 0.05
0.43 ± 0.05
0.84 ± 0.05
0.60 ± 0.09
4k
4m
Gossypol
1
Mean of three independent experiments, with range quoted. Results are expressed as mean values
three independent experiments.
±
SEM across
ELISA assay results largely mirrored the data obtained from Bcl-2 positive cancer cell lines.
Again compounds 4j and 4k stood out as the most active at preventing Bim peptide binding, and notably
were more potent than the standard positive control Bcl-2 inhibitor ( )-gossypol. Compounds with
−
low-level cellular activity, such as 4b, were inactive in the ELISA assay; however, compound 4i, one
of the most active in the cell line assay, had only moderate Bcl-2 binding activity according to the
ELISA assay.
2.4. Molecular Modeling of Compound 4j within the Bcl-2 Binding Pocket
A molecular modeling study was performed to better understand and rationalize the potent
cellular anticancer activity of the new indolyl-aryl-oxadiazole derivative 4j, mediated by Bcl-2 inhibition.
Docking studies were carried within MOE [14], using the published Bcl-2 crystal structure (PDB: 4AQ3) [15].
Docking of our previous lead compound 5-(1H-indol-3-yl)-N-(3-chlorophenyl)-1,3,4-oxadiazol-2-amine [5]
and the most active compound 4j of the new series of indolyl-oxadiazoles was carried out at the Bim
peptide binding site. Results showed that the new derivative 4j, like the previous hit compound, fits well
within the hydrophobic pocket (Figure 2). Compound 4j showed a p-cation interaction between the
trifluorophenyl group and the key residue Arg105, and a hydrophobic interaction between the Bcl-2
hydrophobic groove residue Tyr67 and the oxadiazole moiety. Additional hydrophobic interactions were
observed with Leu96 and Ala108 key residues. Compound 4j showed a comparatively tighter binding
energy score in comparison with our previous lead (
the good fit of compound 4j within the binding pocket. Notably, the orientation of the indole ring
of compound 4j in Figure 2 is inverted compared to the corresponding indole ring of the previously
−
5.64 and 4.81 kcal, respectively), indicative of
−
reported lead compound [5]. Orientation of the indole ring of 4j within the deep hydrophobic pocket

Int. J. Mol. Sci. 2020, 21, 8980
5 of 11
allows additional interactions between the indole NH and the neighboring Asp70 carboxylate residue
that are not present in the orientation of 5-(1H-indol-3-yl)-N-(3-chlorophenyl)-1,3,4-oxadiazol-2-amine [ ],
5
which might help explain the lower relative binding energy score for compound 4j. Representations of
the binding interactions of 4j within the Bcl-2 hydrophobic pocket are shown in Figure 3.
Figure 2. Compound 4j (pink stick) showed a similar binding mode to our previous lead compound
5-(1H-indol-3-yl)-N-(3-chlorophenyl)-1,3,4-oxadiazol-2-amine [5] (red stick). Both compounds showed
a good fit within the hydrophobic pocket of the Bcl-2 protein.
Figure 3. Compound 4j showed hydrophobic interactions with key residues Tyr67, Phe71, Leu96 and
Ala108, in addition to a p-cation interaction between the trifluorophenyl group and the key
residue Arg105.
2.5. Physicochemical Properties and Assessment of Hit Optimization Potential
Assessment of key drug-like properties was undertaken for the most active compounds (4d
,
4e and 4j) from the three synthesized mini-series, representing each of the linker functions (Table 3).
Calculated physicochemical data are also presented for the structurally related clinical drug candidate
obatoclax. The key parameters of molecular weight (MW), calculated logP, topological polar surface
area (TPSA), number of hydrogen bond acceptors (nON) and number of hydrogen bond donors
(nOHNH) were calculated by using the freely available Molinspiration® software [16].

Int. J. Mol. Sci. 2020, 21, 8980
6 of 11
Table 3. Analysis of physicochemical properties of hit compounds exhibiting Bcl-2 inhibitory activity
(calculated by using software available at www.molinspiration.com).
Compound
X
R
MW
clogP
nON
nOHNH
TPSA/Å
4d
4e
4j
OCH2
CH2
C6H5
3-OMe-C6H4
4-CF3-C6H4
-
291.31
305.34
329.28
317.39
3.16
3.50
4.77
4.08
63.95
63.95
54.72
53.71
5
5
4
4
1
1
1
2
-
-
Obatoclax
Analysis of the drug-like properties of hit compounds reveals that 4d, 4e and 4j all fall within the
expected parameters for further development. Of particular note are the properties of the most potent
analogue 4j. Compound 4j is rather lipophilic, with a calculated logP value of 4.77, just within the
recommended Lipinski rule of 5 for prediction of oral bioavailability, and similar to the free base of the
related clinical drug candidate obatoclax. This preliminary physicochemical data gives confidence
for further development of compound 4j, noting the possibility that a mesylate (or other) salt form
may be required for optimization of solubility whilst maintaining potent cellular activity, as is the case
for obatoclax.
3. Discussion
The results presented here report further evidence of the ability of synthetically accessible
heterocycles based on the indolyl-oxadiazole scaffold to act as potent Bcl-2 inhibitory anticancer
agents. This early drug discovery work, inspired by clinical stage Bcl-2 inhibitors such as obatoclax
mesylate, has led to a new series of anticancer compounds obtained in two chemical steps from
commercially available starting materials. New compounds were designed to test the optimal linker
function between the 5-position of oxadiazole and a substituted aryl group, resulting in the discovery
of 2-(1H-indol-3-yl)-5-(4-trifluoromethylphenyl)-1,3,4-oxadiazole (4j) as the most potent compound of
the series. Compound 4j (and several other examples) possessed low- to sub-micromolar IC50 values
against Bcl-2 expressing human cancer cell lines such as MDA-MB-231 (invasive breast), HeLa (cervical)
and KG1a (acute myelogenous leukemia). In addition, compound 4j was able to compete with the
pro-apoptotic BH3 domain Bim peptide for binding to His-tagged Bcl-2 protein in a competitive ELISA
assay. Low micromolar IC50 values were obtained, but they were more potent than the positive control
clinical stage compound ( )-gossypol. The Bcl-2 binding activity at the Bim peptide binding site was
−
rationalized by computational modeling (MOE environment), offering a rational basis for further
optimization to a lead Bcl-2 inhibitory anticancer agent. Finally, preliminary physicochemical properties
of 4j were compatible with the possibility of further development of this compound, most likely in salt
form, to overcome the relatively high lipophilicity of this particular heterocycle.
4. Materials and Methods
4.1. General Experimental Details—Chemistry
All starting materials and solvents were used without further purification. Melting points were
measured on a Griffin apparatus and are uncorrected. Nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Avance 500 MHz instrument, and chemical shifts (δ values) are given
in ppm relative to Me4Si, with coupling constants (J values) in Hz. Mass spectrometry was run in
electrospray ionization (ESI) mode (Bruker MicroTof instrument, Coventry, UK). Elemental analysis
(% CHN) was run by combustion analysis through an outsourced service (Medac Ltd., Surrey, UK).

Int. J. Mol. Sci. 2020, 21, 8980
7 of 11
4.2. Chemical Synthesis
4.2.1. Synthesis of 3-Indolylcarboxlic Acid Hydrazide (2)
Excess hydrazine monohydrate (5 mL) was added to a solution of methyl indolyl-3-carboxylate
(1.0 g, 10 mmol) in ethanol (10 mL). The reaction mixture was heated under reflux for 7 h and then left to
cool to room temperature. The resulting precipitate was collected by filtration and washed with water,
followed by cold ethanol, to remove excess hydrazine. After drying under vacuum, the corresponding
acid hydrazide was obtained, and it was used without further purification. Yield 80%, m.p. 205–207 ◦C.
1H NMR (DMSO-d6)
δ 4.30 (s, 2H, NH2), 7.107–7.14 (m, 2H, ArCH), 7.43 (d, 1H, J = 8.10, ArCH),
7.96 (s, 1H, ArCH), 8.14 (d, 1H, J = 8.14, ArCH), 9.13 (s, 1H, NH), 11.49 (s, 1H, NH indole).
4.2.2. General Method for Synthesis of 2-(1H-Indol-3-Yl)-5-Substituted-1,3,4-Oxadiazoles (4a–m)
A mixture of the acid hydrazide
2 (10 mmol), carboxylic acid derivatives 3a–m (10 mmol) and
phosphorus oxychloride (5 mL) was heated for 4 h, at 50 ◦C, and then left to cool to room temperature.
Ice was added, and the reaction mixture was stirred overnight; then the mixture was carefully
neutralized with saturated sodium bicarbonate solution. The precipitate formed was filtered, dried
and crystallized from ethanol, to give the corresponding oxadiazole product 4a–m.
◦
2-(1H-Indol-3-yl)-5-(4-fluorophenoxymethyl)-1,3,4-oxadiazole (4a). Yield 60%, m.p. 210–212 C.
1H NMR (DMSO-d6):
δ 5.45 (s, 2H, CH2), 7.107–7.16 (m, 4H, ArCH), 7.197–7.30 (m, 2H, ArCH) 7.53
(d, 1H, J = 7.5, ArCH-2, indole), 8.12 (d, 1H, J = 7.5, ArCH), 8.30 (d, 1H, J = 1.5, ArCH), 12.14 (s, 1H, NH
indole). 13C-NMR (DMSO-d6): δ 60.18 (CH2), 99.01 (C-3), 112.36 (ArCH), 115.92 (ArCH), 121.26 (ArCH),
122.90 (ArCH), 124.04 (ArCH), 127.74 (ArCH), 128.52 (ArCH), 136.43 (ArC), 153.70 (ArC), 156.20 (ArC),
158.09 (ArC), 160.17 (CO), 162.68 (CO). Anal. (C17H12FN3O): % CHN required: C 66.02, H 3.91, N 13.59;
found: C 65.62, H 3.85, N 13.58. MS (C17H12FN3O): calcd mass 309.09, found (m/z, ES+): 310.10.
2-(1H-Indol-3-yl)-5-(4-methylphenoxymethyl)-1,3,4-oxadiazole (4b). Yield 56%, m.p. 201–203 ◦C.
1H-NMR (DMSO-d6):
δ 2.67 (s, 3H, CH3) 5.83 (s, 2H, CH2), 7.42 (s, 1H, ArCH), 7.55 (d, 2H, J = 7.0, ArCH),
7.66.7.69 (m, 2H, ArCH), 7.94 (d, 1H, J = 7, ArCH), 8.47 (d, 1H, J = 7, ArCH), 8.70 (s, 1H, ArCH), 12.50
(s, 1H, NH indole). 13C NMR (DMSO-d6): 20.05 (CH3), 59.64 (CH2), 99.06 (C-3), 112.45 (ArCH), 114.84
(ArCH), 120.11 (ArCH), 121.24 (ArCH), 122.87 (ArCH), 124.06 (ArCH), 128.49 (ArCH), 129.93 (ArC),
130.53 (ArC), 136.44 (ArC), 155.31 (ArC), 160.38 (CO), 162.64 (CO). Anal. (C18H15N3O2) % CHN required:
C 70.81, H 4.95, N 13.76; found: C 70.62, H 4.87, N 13.85. MS (C18H15N3O2): calcd mass 305.33, found (m/z,
ES+): 306.12.
2-(1H-Indol-3-yl)-5-(4-tert-butylphenoxymethyl)-1,3,4-oxadiazole (4c). Yield 51%, m.p. 218–220
◦C. 1H NMR (DMSO-d6):
δ
1.26 (s, 9H, t-butyl), 5.40 (s, 2H, CH2), 7.04 (d, 2H, J = 9, ArCH), 7.24–7.26
(m, 2H, ArCH), 7.34 (d, 2H, J = 9, ArCH), 7.54 (d, 1H, J = 8, ArCH), 8.08 (d, 1H, J = 8, ArCH), 8.20
(d, 1H, J = 3, ArCH), 12.96 (s, 1H, NH indole). 13C NMR (DMSO-d6):
31.24 (CH3), 33.81 (ArC),
δ
59.57 (CH2), 99.05 (C3), 112.46 (ArCH), 114.43 (ArCH), 120.10 (ArCH), 121.24 (ArCH), 122.88 (ArCH),
124.06 (ArC), 126.23 (ArCH), 128.49 (ArCH), 136.44 (ArC), 143.96 (ArC), 155.16 (ArC), 160.41 (CO),
162.65(CO). Anal. (C21H21N3O2) % CHN required: C 72.60, H 6.09, N 12.10; found: C 72.67, H 6.03,
N 12.05. MS (C21H21N3O2): calcd mass 347.41, found (m/z, EI+): 347.16.
2-(1H-Indol-3-yl)-5-(phenoxymethyl)-1,3,4-oxadiazole (4d). Yield 46%, m.p. 195–197 ◦C. 1H NMR
(DMSO-d6):
ArCH), 7.52 (d, 1H, J = 8, ArCH), 8.06 (d, 1H, J = 8, ArCH), 8.10 (d, 1H, J = 2.5, ArCH), 12.96 (s, 1H,
NH indole). 13C NMR (DMSO-d6):
58.70 (CH2), 99.40 (C3), 112.37 (ArCH), 120.10 (ArCH), 121.09
δ 4.30 (s, 2H, CH2), 7.237–7.25 (m, 2H, ArCH), 7.307–7.33 (m, 1H, ArCH), 7.377–7.39 (m, 4H,
δ
(ArCH), 122.76 (ArCH), 124.04 (ArC), 127.12 (ArCH), 127.89 (ArCH), 128.72 (ArCH), 128.79 (ArCH),
134.87 (ArC), 136.39 (ArC), 162.05 (CO), 163.08 (CO). Anal. (C17H13N3O2): % CHN required: C 70.09,
H 4.50, N 14.42, found: C 69.62, H 4.39, N 14.31.
2-(1H-Indol-3-yl)-5-(3-methoxybenzyl)-1,3,4-oxadiazole (4e). Yield 56%, m.p. 208–210 ◦C. 1H NMR
(DMSO-d6):
δ 3.84 (s, 3H, OCH3) 4.52 (s, 2H, CH2) 6.90–6.94 (m, 1H, ArCH), 7.15 (d, 1H, J = 7.7, ArCH),
7.22–7.26 (m, 2H, ArCH), 7.33–7.36 (m, 2H, ArCH), 7.55 (d, 1H, J = 8.5, ArCH), 8.12 (d, 1H, J = 6.5,

Int. J. Mol. Sci. 2020, 21, 8980
8 of 11
ArCH), 8.17 (d, 1H, J = 2.3, ArCH), 11.98 (s, 1H, NH indole). 13C NMR (DMSO-d6): 25.54 (CH2), 55.51
(OCH3), 99.50 (C3), 111.10 (ArCH), 112.36 (ArCH), 120.09 (ArCH), 120.49 (ArCH), 121.04 (ArCH),
122.73 (ArCH), 124.06 (ArC), 127.72 (ArC), 128.81 (ArCH), 130.23 (ArCH), 136.38 (ArC), 157.03 (ArC),
161.79 (CO), 162.99 (CO). Anal. (C18H15N3O2) % CHN required: C 70.81, H 4.95, N 13.76, found:
C 70.84, H 4.85, N 13.76. MS (C18H15N3O2): calcd mass 305.33, found (m/z, ES+): 306.12.
2-(1H-Indol-3-yl)-5-benzyl-1,3,4-oxadiazole (4f). Yield 47%, m.p. 187–189 ◦C. 1H NMR (DMSO-d6):
δ
4.30 (s, 2H, CH2), 7.227–7.26 (m, 2H, ArCH), 7.31 (t, 1H, J = 7, ArCH), 7.367–7.39 (d, 4H, J = 8, ArCH),
7.52 (d, 1H, J = 8, ArCH), 8.06 (d, 1H, J = 7.60, ArCH), 8.11 (s, 1H, ArCH), 11.97 (s, 1H, NH indole).
13C NMR (DMSO-d6):
21.09 (CH2), 99.46 (C3), 112.44 (ArCH), 120.22 (ArCH), 120.98 (ArCH), 121.23
δ
(ArCH), 122.83 (ArCH), 124.11 (ArCH), 126.28 (ArCH), 128.41 (ArCH), 129.89 (ArC), 136.46 (ArC),
141.58 (ArC), 161.72 (CO), 161.92 (CO). Anal. (C17H13N3O) % CHN required: C 74.17, H 4.76, N 15.26,
found: C 74.16, H 4.70, N 15.24. MS (C17H13N3O): calcd mass 275.30, found (m/z, EI+) 275.10.
2-(1H-Indol-3-yl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (4g). Yield 64%, m.p. 215–217 ◦C. 1H NMR
(DMSO-d6)
8.348–8.37 (m, 3H, ArCH), 8.53 (d, 2H, J = 9, ArCH), 12.18 (s, 1H, NH indole). 13C NMR (DMSO-d6):
99.06 (C3), 120.33 (ArCH), 122.95 (ArCH), 123.67 (ArCH), 124.07 (ArCH), 127.48 (ArCH), 127.71
δ 7.277–7.30 (m, 2H, ArCH), 7.57 (d, 1H, J = 8.5, ArCH), 8.17 (d, 1H, J = 8.5, ArCH),
δ
(ArCH), 129.14 (ArCH), 129.23 (ArC), 130.65 (ArC), 148.76 (ArC), 159.72 (ArC), 160.56 (CO), 162.87
(CO). Anal. (C16H10N4O3) % CHN required: C 62.74, H 3.29, N 18.29, found: C 62.75, H 3.25, N 17.93.
MS (C16H10N4O3): calcd mass 306.28, found (m/z, EI+): 306.
2-(1H-Indol-3-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (4h). Yield 64%, m.p. 210–212 ◦C. 1H
NMR (DMSO-d6):
J = 7, ArCH), 7.31 (s, 1H, ArCH), 7.51 (d, 1H, J = 8.5, ArCH), 7.70 (d, 1H, J = 8, ArCH), 8.08 (d, 2H, J = 9,
ArCH), 12.30 (s, 1H, NH indole). 13C NMR (DMSO-d6):
55.51 (OCH3), 99.50 (C3), 104.81 (ArCH),
δ 3.89 (s, 3H, OCH3) 7.12 (t, 1H, J = 6.5, ArCH), 7.21 (d, 2H, J = 9, ArCH), 7.28 (t, 1H,
δ
112.21 (ArCH), 114.90 (ArCH), 115.57 (ArCH), 120.30 (ArCH), 121.19 (ArCH) 121.40 (ArC), 124.13
(ArCH), 127.72 (ArC), 137.76 (ArC), 158.90 (ArC), 162.10 (CO), 163.30 (CO). Anal. (C17H13N3O2) %
CHN required: C 70.09, H 4.50, N 14.42, found: C 69.82, H 4.40, N 14.25. MS (C17H13N3O2): calcd mass
291.30, found (m/z, EI+): 291.00.
2-(1H-Indol-3-yl)-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazole (4i). Yield 60%, m.p. 221–222 ◦C.
1H NMR (DMSO-d6):
δ
3.83 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.90 (s, 1H, ArCH),
7.27–7.30 (m, 2H, ArCH), 7.49 (s, 1H, ArCH), 7.56 (t, 1H, J = 7.5, ArCH), 8.18 (t, 1H, J = 6, ArCH), 8.23
(d, 1H, J = 3, ArCH), 12.01 (s, 1H, NH indole). 13C NMR (DMSO-d6):
55.88 (OCH3), 56.21 (OCH3),
δ
56.82 (OCH3), 98.72 (ArCH), 98.86 (C3), 103.52 (ArCH), 112.41 (ArCH), 120.16 (ArCH), 121.12 (ArCH),
122.74 (ArCH), 124.18 (ArC), 127.90 (ArC), 136.45 (ArC), 142.85 (ArC), 152.67 (ArC), 160.75 (CO), 161.34
(CO). Anal. (C19H17N3O4) % CHN required: C 64.95, H 4.88, N 11.96, found: C 64.94, H 4.75, N 12.00.
MS (C19H17N3O4): calcd mass 351.36, found (m/z, EI+): 351.11.
2-(1H-Indol-3-yl)-5-(4-trifluoromethylphenyl)-1,3,4-oxadiazole (4j). Yield 63%, m.p. 224–226 ◦C.
1H NMR (DMSO-d6):
δ
7.267–7.30 (m, 2H, ArCH), 7.58 (d, 1H, J = 8.5, ArCH), 8.0 (d, 2H, J = 8.5, ArCH),
8.18 (d, 1H, J = 7, ArCH), 8.34 (d, 2H, J = 8, CH aromatic), 8.37 (d, 1H, J = 3, ArCH), 12.06 (s, 1H, NH
indole). 13C NMR (DMSO-d6):
99.15 (C3), 112.52 (ArCH), 121.38 (ArCH), 122.95 (ArCH), 126.35
δ
(ArCH), 126.38 (ArCH), 127.13 (ArC), 127.48 (ArCH), 128.99 (ArCH), 130.97 (ArC), 131.23 (ArC), 136.49
(ArC), 160.88 (CO), 162.58 (CO). Anal. (C17H10F3N3O) % CHN required: C 62.01, H 3.06, N 12.76,
found: C 62.10, H 3.13, N 12.75. MS (C17H10F3N3O): calcd mass 329.00, found (m/z, EI+): 329.08.
2-(1H-Indol-3-yl)-5-(4-methylphenyl)-1,3,4-oxadiazole (4k). Yield 66%, m.p. 198–200 ◦C. 1H NMR
(DMSO-d6):
J = 6.5, ArCH), 8.02 (d, 2H, J = 8, ArCH), 8.18 (d, 1H, J = 6.5, ArCH), 8.30 (s, 1H, ArCH), 12.06 (s, 1H, NH
indole). 13C NMR (DMSO-d6):
21.10 (CH3), 99.45 (C3), 112.44 (ArCH), 120.22 (ArCH), 120.99 (ArCH),
δ 2.43 (s, 3H, CH3), 7.29 (dd, 2H, J = 6.3, 2.6, ArCH), 7.45 (d, 2H, J = 7, ArCH), 7.56 (d, 1H,
δ
121.23 (ArCH), 122.83 (ArCH), 124.11 (ArCH), 126.29 (ArCH), 128.43 (ArC), 129.90 (ArC), 136.46 (ArC),
141.59 (ArC), 161.72 (CO), 161.92 (CO). Anal. (C17H13N3O) % CHN required: C 74.17, H 4.76, N 15.26,
found: C 73.97, H 4.59, N 15.25. MS (C17H13N3O): calcd mass 275.30, found (m/z, ES−): 274.10.

Int. J. Mol. Sci. 2020, 21, 8980
9 of 11
2-(1H-Indol-3-yl)-5-(6-(piperidin-1-yl)-pyridin-3-yl)-1,3,4-oxadiazole (4l). Yield: 53%, m.p. 240–242
◦C. 1H NMR (DMSO-d6):
1.56 (d, 4H, J = 4, CH aliphatic), 1.66 (d, 2H, J = 5, CH aliphatic), 3.663–3.69
δ
(m, 4H, CH aliphatic), 7.01 (d, 1H, J = 9, ArCH), 7.247–7.30 (m, 2H, ArCH), 7.55 (d, 1H, J = 8, ArCH),
8.10 (dd, 1H, J = 9, ArCH), 8.16 (d, 1H, J = 6.5, ArCH), 8.26 (d, 1H, J = 3, ArCH), 8.80 (d, 1H, J = 2.5,
ArCH), 12.02 (s, 1H, NH indole). 13C NMR (DMSO-d6):
δ 24.18 (CH2), 25.09 (CH2), 45.23 (CH2), 99.60 (C3),
106.42 (ArCH), 107.81 (ArCH), 112.38 (ArCH), 120.26 (ArCH), 121.14 (ArCH), 122.78 (ArCH), 124.08 (ArC),
128.11 (ArCH), 135.09 (ArC), 136.44 (ArC), 146.62 (ArC), 159.35 (ArC), 160.88 (CO), 160.95 (CO). Anal.
(C20H19N5O) % CHN required: C 69.55, H 5.54, N 20.28, found: C 69.26, H 5.37, N 20.18. MS (C20H19N5O):
calcd mass 345.40, found (m/z, EI+): 345.16.
2-(1H-Indol-3-yl)-5-(5-methoxy-1H-indol-2-yl)-1,3,4-oxadiazole (4m). Yield 63%, m.p. 231–233
◦C. 1H NMR (DMSO-d6):
δ
3.80 (s, 3H, OCH3), 6.93 (dd, 1H, J = 8, ArCH), 7.177–7.19 (m, 2H, J = 2.5,
ArCH), 7.267–7.32 (m, 2H, ArCH), 7.41 (d, 1H, J = 9, ArCH), 7.55-7.58 (m, 1H, ArCH), 8.21-8.23 (m, 1H,
ArCH), 8.27 (s, 1H, ArCH), 12.09 (s, 2H, NH indole). 13C NMR (DMSO-d6):
55.28 (OCH3), 99.30
δ
(C3), 101.90 (C2), 104.02 (ArCH), 112.48 (ArCH), 112.97 (ArCH), 115.03 (ArCH), 120.28 (ArCH), 121.27
(ArCH), 121.75 (ArCH), 122.90 (ArCH), 124.08 (ArC), 127.80 (ArC), 128.37 (ArC), 132.96 (ArC), 136.46
(ArC), 154.09 (ArC), 157.30 (CO), 161.26 (CO). Anal. (C19H14N4O2) % CHN required: C 69.08, H 4.27, N
16.96, found: C 68.89, H 4.14, N 16.90. MS (C19H14N4O2): calcd mass 330.34, found (m/z, EI+): 330.10.
4.3. Cell Viability—MTT Assay (MDA-MB-231 and HeLa Cells)
Human breast cancer (MDA-MB-231) and cervical cancer (HeLa) cells, obtained from the ATCC
(Manassas, VA, USA), were cultured and maintained as previously described [5–8]. For each experiment,
3000 cells in 0.2 mL of medium were seeded into each well of a clear flat-bottomed 96-well plate and
allowed to adhere for 24 h. The cells were then incubated with the test compound (from 10 mM
stock in DMSO), over a 10-fold dilution series, with concentrations ranging from 0.00001 to 100 mM
and each diluent being performed in triplicate. Cells were incubated with the test compound for
72 h, followed by treatment with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
solution and reading of absorbance (automated microplate) according to our previous protocol [5–8].
Mean IC50 values were obtained by using GraphPad Prism 5 software (San Diego, CA, USA). For each
concentration, three independent repeat experiments were carried out to establish reproducibility.
4.4. Cell Viability—CellTiter-Blue® Assay (KG1a and Jurkat Cells)
Human acute myeloid leukemia KG1a and acute T-cell lymphocytic Jurkat cells were cultured
and maintained as previously described [5–8]. For each experiment 15,000 cells in 0.1 mL of medium
were seeded into each well of a solid-black fluorescence 96-well plate and incubated for 24 h. The cells
were then incubated with test compound (from 10 mM stock in DMSO), over a 10-fold dilution series,
with concentrations ranging from 0.00001 to 100 mM and each diluent being performed in triplicate.
Cells were incubated with test compound for 24 h, followed by treatment with CellTiter-Blue® solution
and reading of fluorescence according to our previous protocol [5–8]. Mean IC50 values were obtained
by using GraphPad Prism 5 software (San Diego, CA, USA). For each concentration, three independent
repeat experiments were carried out, to establish reproducibility.
4.5. Enzyme-Linked Immunosorbent Assay (ELISA)
The ELISA assay to determine the ability of test compounds (10-fold dilution series) to compete with
the interaction between biotinylated Bim peptide and His-tagged Bcl-2 protein was carried out according
to our previously published protocol [5–8]. The addition of anti-His antibody containing horseradish
peroxidase enzyme, followed by the addition of o-phenylenediamine and hydrogen peroxide, allowed
for the determination of optical density, using a plate reader (450 nm). The experiments were carried
out on three separate occasions, including both negative and positive controls. A plot of log mM
concentration for each inhibitor against the percentage reduction in affinity for the Bim peptide was

Int. J. Mol. Sci. 2020, 21, 8980
10 of 11
created, using non-linear regression curve analysis (GraphPad Prism 5, San Diego, CA, USA) and the
software used to generate the IC50 value for the Bcl-2 inhibitor.
4.6. Molecular Modeling and Docking
Bcl-2 protein (PDB:4AQ3) was prepared by using the MOE program (Chemical Computing Group,
Cambridge, UK), ligands were built and the system was energy minimized by using the MMFF94x
force field until a RMSD gradient of 0.05 Kcal mol−1 Å−1 was reached [14
,15]. Triangle Matcher was
chosen as the replacement methodology, and London dG was applied as the scoring function.
5. Conclusions
The work reported herein describes the design and synthesis of a new series of
2-(1H-indol-3-yl)-5-substituted-1,3,4-oxadiazoles (4a–m) as inhibitors of Bcl-2. Target compounds were
readily synthesized through a cyclization reaction of indole carboxylic acid hydrazide 2 with substituted
carboxylic acid derivatives 3a–m in the presence of phosphorus oxychloride. Compound 4j, containing
a 4-trifluoromethylphenyl group directly attached to the oxadiazole ring, possessed the most potent
anticancer activity across the Bcl-2 expressing human cancer cell lines (MDA-MB-231, HeLa, KG1a;
sub-micromolar IC50 values). Compound 4j displayed similarly potent activity in a Bcl-2 ELISA binding
assay. Results were rationalized by computational docking within the Bcl-2 binding pocket, and compound
4j (and other hit compounds) possessed satisfactory calculated early physicochemical (ADME) data for
further development as a lead compound.
Author Contributions: Conceptualization, R.H. and A.D.W.; methodology, R.H., S.A.E. and N.I.Z.; investigation,
R.H. and S.A.E.; resources, A.T.J. and A.D.W.; writing, R.H. and A.D.W.; supervision, M.E.-S., E.L., A.T.J. and
A.D.W.; funding acquisition, R.H. and A.D.W. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Egyptian Government and Cardiff University, through a Channel
Scholarship (to R.H. and S.A.E.) and a PhD studentship (to N.I.Z.). The APC was funded by Cardiff University.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Ashkenazi, A.; Fairbrother, A.A.W.J.; Leverson, J.D.; Souers, J.D.L.A.J. From basic apoptosis discoveries to
advanced selective BCL-2 family inhibitors. Nat. Rev. Drug Discov. 2017, 16, 273–284. [CrossRef] [PubMed]
Valentin, R.; Grabow, S.; Davids, M.S. The rise of apoptosis: Targeting apoptosis in hematologic malignancies.
Blood 2018, 132, 1248–1264. [CrossRef] [PubMed]
White, A.; Westwell, A.D.; Brahemi, G. Protein–protein interactions as targets for small-molecule therapeutics
in cancer. Expert Rev. Mol. Med. 2008, 10, e8. [CrossRef] [PubMed]
Schimmer, A.D.; O’Brien, S.; Kantarjian, H.; Brandwein, J.; Cheson, B.D.; Minden, M.D.; Yee, K.; Ravandi, F.;
Giles, F.J.; Schuh, A.; et al. A Phase I Study of the Pan Bcl-2 Family Inhibitor Obatoclax Mesylate in Patients
with Advanced Hematologic Malignancies. Clin. Cancer Res. 2008, 14, 8295–8301. [CrossRef] [PubMed]
Hamdy, R.; Ziedan, N.I.; 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 Med. Chem. Lett. 2017, 27, 1037–1040. [CrossRef] [PubMed]
Ziedan, N.I.; Hamdy, R.; Cavaliere, A.; Kourti, M.; Prencipe, F.; Brancale, A.; Jones, A.T.; Westwell, A.D.
Virtual screening, SAR, and discovery of 5-(indole-3-yl)-2-[(2-nitrophenyl)amino] [1,3,4]-oxadiazole as a
novel Bcl-2 inhibitor. Chem. Biol. Drug Des. 2017, 90, 147–155. [CrossRef] [PubMed]
5.
6.
7.
8.
Hamdy, R.I.; Ziedan, N.; Ali, S.; El-Sadek, M.; Lashin, E.; Brancale, A.; Jones, A.T.; Westwell, A.D. Synthesis
and evaluation of 3-(benzylthio)-5-(1H-indol-3-yl)-1,2,4-triazol-4-amines as Bcl-2 inhibitory anticancer agents.
Bioorganic Med. Chem. Lett. 2013, 23, 2391–2394. [CrossRef] [PubMed]
Hamdy, R.; Elseginy, S.A.; Ziedan, N.I.; Jones, A.T.; Westwell, A.D. New Quinoline-Based Heterocycles as
Anticancer Agents Targeting Bcl-2. Molecules 2019, 24, 1274. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2020, 21, 8980
11 of 11
9.
Arisan, E.D.; Kutuk, O.; Tezil, T.; Bodur, C.; Telci, D.; Basaga, S.H. Small inhibitor of Bcl-2, HA14-1, selectively
enhanced the apoptotic effect of cisplatin by modulating Bcl-2 family members in MDA-MB-231 breast
cancer cells. Breast Cancer Res. Treat. 2010, 119, 271–281. [CrossRef] [PubMed]
10. Meredith, M.J.; Cusick, C.L.; Soltaninassab, S.; Sekhar, K.S.; Lu, S.; Freeman, M.L. Expression of Bcl-2 Increases
Intracellular Glutathione by Inhibiting Methionine-Dependent GSH Efflux. Biochem. Biophys. Res. Commun. 1998
,
248, 458–463. [CrossRef] [PubMed]
11. Watkins, C.L.; Sayers, E.J.; Allender, C.J.; Barrow, D.; Fegan, C.; Brennan, P.; Jones, A.T. Co-operative
Membrane Disruption Between Cell-penetrating Peptide and Cargo: Implications for the Therapeutic Use of
the Bcl-2 Converter Peptide D-NuBCP-9-r8. Mol. Ther. 2011, 19, 2124–2132. [CrossRef] [PubMed]
12. Rao, J.; Xu, D.; Zheng, F.; Long, Z.-J.; Huang, S.-S.; Wu, X.; Zhou, W.-H.; Huang, R.-W.; Liu, Q. Curcumin
reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia
cell lines and primary sorted CD34+ acute myeloid leukemia cells. J. Transl. Med. 2011, 9, 71. [CrossRef]
[PubMed]
13. Meng, Y.; Tang, W.; Dai, Y.; Wu, X.; Liu, M.; Ji, Q.; Ji, M.; Pienta, K.; Lawrence, T.; Xu, L. Natural BH3 mimetic
(-)-gossypol chemosensitizes human prostate cancer via Bcl-xL inhibition accompanied by increase of Puma
and Noxa. Mol. Cancer Ther. 2008, 7, 2192–2202. [CrossRef] [PubMed]
14. Cozza, G.; Moro, S. Medicinal Chemistry and the Molecular Operating Environment (MOE): Application of
QSAR and Molecular Docking to Drug Discovery. Curr. Top. Med. Chem. 2008, 8, 1555–1572. [CrossRef]
15. Research Collaboration for Structural Bioinformatics (RCSB) Protein Data Bank. Available online: www.rcsb.
org/pdb (accessed on 13 August 2020).
16. Molinspiration®Software. Available online: www.molinspiration.com (accessed on 12 October 2020).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
affiliations.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).