New quinoline-based heterocycles as anticancer agents targeting Bcl-2

Article abstract of DOI:10.3390/molecules24071274

The Bcl-2 protein has been studied as an anticancer drug target in recent years, due to its gatekeeper role in resisting programmed cancer cell death (apoptosis), and the design of BH3 domain mimetics has led to the clinical approval of Venetoclax (ABT-19

Full text of DOI:10.3390/molecules24071274

molecules
Article
New Quinoline-Based Heterocycles as Anticancer
Agents Targeting Bcl-2
Rania Hamdy 1,2,3, Samia A. Elseginy 1,4,5, Noha I. Ziedan 1,3,6, 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, Wales CF10 3NB, UK; rania_hamdy2000@yahoo.com (R.H.);
samiaaliph@hotmail.com (S.A.E.); n.ziedan@chester.ac.uk (N.I.Z.); JonesAT@cardiff.ac.uk (A.T.J.)
Sharjah Institute for Medical Research, College of Pharmacy, University of Sharjah,
P.O. Box 27272, Sharjah, UAE
Faculty of Pharmacy, Zagazig University, Zagazig 445519, Egypt
Green Chemistry Department, Chemical Industries Research Division, National Research Center,
Dokki, Giza, P.O. Box 12622, Egypt
2
3
4
5
6
School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK
Department of Natural Sciences, University of Chester, Chester CH2 4NU, UK
Correspondence: WestwellA@cardiff.ac.uk; Tel.: +44-2920-875800
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Fawaz Aldabbagh
Received: 5 March 2019; Accepted: 28 March 2019; Published: 2 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The Bcl-2 protein has been studied as an anticancer drug target in recent years, due to
its gatekeeper role in resisting programmed cancer cell death (apoptosis), and the design of BH3
domain mimetics has led to the clinical approval of Venetoclax (ABT-199) for the treatment of
chronic lymphocytic leukaemia. In this work we extend our previous studies on the discovery of
indole-based heterocycles as Bcl-2 inhibitors, to the identification of quinolin-4-yl based oxadiazole
and triazole analogues. Target compounds were readily synthesized via a common aryl-substituted
quinolin-4-carbonyl-N-arylhydrazine-1-carbothioamide (5a–b) intermediate, through simple variation
of the basic cyclisation conditions. Some of the quinoline-based oxadiazole analogues (e.g. compound
6i) were found to exhibit sub-micromolar anti-proliferative activity in Bcl-2-expressing cancer cell lines,
and sub-micromolar IC50 activity within a Bcl2-Bim peptide ELISA assay. The Bcl-2 targeted anticancer
activity of 6i was further rationalised via computational molecular modelling, offering possibilities to
extend this work into the design of further potent and selective Bcl-2 inhibitory heteroaromatics with
therapeutic potential.
Keywords: aromatic heterocycles; quinoline; oxadiazole; triazole; anticancer; Bcl-2 inhibitor; ELISA;
molecular modelling; apoptosis
1. Introduction
Resistance to apoptosis (programmed cell death) is an extensively studied cancer hallmark [
that has inspired the development of targeted therapeutic approaches towards unlocking apoptotic
signaling and selective induction of cancer cell death [ ]. Bcl-2 is the archetypal member of a
well-studied family of anti-apoptotic gatekeeper proteins, with Bcl-2 inhibition exploited in anticancer
drug design [ ]. Extensive drug development work over many years focused on Bcl-2 homology 3
1]
2
2
(BH3) mimetics has recently led to the clinical approval of Venetoclax (ABT-199, Figure 1) for the
treatment of chronic lymphocytic leukaemia in patients with a 17p deletion and at least one prior
therapy [3]. Obatoclax mesylate (Figure 1) is a further example of an indole-based pan-Bcl-2 inhibitor
that has been studied in cancer clinical trials (e.g. leukaemia and lymphoma) [4].
Molecules 2019, 24, 1274; doi:10.3390/molecules24071274
www.mdpi.com/journal/molecules

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Figure 1. Bcl-2 small molecule inhibitors.
Our own work has led to the early stage design and discovery of indole-based
heterocycles as new Bcl-2 inhibitors based on molecular modelling and virtual
a
screening approach to identify hit molecules. We have previously reported on series of
3-(benzylthio)-5-(indol-3-yl)-1,2,4-triazol-4-amines, 5-(indol-3-yl)-N-aryl-[1,3,4]-oxadiazol-2-amines
and 5-(indol-3-yl)-2-[(2-nitrophenyl)amino][1,3,4]-oxadiazole as Bcl-2 inhibitory anticancer agents
(Figure 1) [5–7]. In this study, we further explore structure-activity relationships around this
heterocyclic framework and synthesise further novel nitrogen-containing aromatic heterocycles for
characterisation and biological testing, focusing on the corresponding quinoline derivatives.
2. Results
2.1. Synthesis of Quinolinyl-N-Aryl-Oxadiazolamines and Related Triazole Derivatives as Bcl-2 Inhibitory
Anticancer Agents
The synthetic route to target compounds is outlined in Scheme 1. Commercially available
isatin compounds 1a–b were first reacted with 4-bromoacetophenone (
in refluxing ethanol to provide the intermediate 2-(4-bromophenyl)quinoline-4-carboxylic
acids 3a–b according to the standard Pfitzinger reaction protocol [ ]. Following construction
2) under basic conditions
8
of the quinoline framework, the carboxylic acid function was first esterified then
reacted with hydrazine hydrate to provide the corresponding hydrazide derivatives
4a–b. Reaction of compounds 4a–b with aryl isothiocyanates in ethanol under reflux gave
the corresponding 2-(2-(4-bromophenyl)quinoline-4-carbonyl)-N-aryl-hydrazine-1-carbothioamide
(acylthiosemicarbazide) intermediates (5a–k) in good yields (>60%) that were appropriately
functionalised to form the desired oxadiazole-2-amine and triazole-2-thione target heterocycles.
Careful control of the quinoline-4-carbonyl-N-aryl-hydrazine-1-carbothioamide basic cyclisation
conditions led selectively to alternative heterocyclic cyclisation products. Treatment of compounds
5a–k with aqueous sodium hydroxide in the presence of iodine as oxidant led to the
N-aryl-1,3,4-oxadiazole products 6a–k according to literature precedent [9], whereas refluxing in 2N
sodium hydroxide produced the N-aryl-1,3,4-triazole-2-thiones 7a–b. Intramolecular cyclodehydration
of acrylthiosemicarbazides to give product 1,3,4-triazole-2-thione derivatives under basic conditions
as is the case here, compared to product 1,3,4-thiadiazole-2-amine derivatives under acidic conditions,
follows literature precedent [10–12]. The N-aryl-1,3,4-triazine-2-thiones (7a–b) were further S-alkylated
using 2-bromoacetophenone under basic conditions to give the final products 8a–b, containing a bulky
S-substituent projected to sit within a hydrophobic pocket of the Bcl-2 binding site.

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Scheme 1.
Synthesis of substituted quinolin-4-yl-N-aryl-oxadiazol-2-amines (6a–k) and
quinolin-4-yl-benzoylmethylthiotriazole (8a–b) derivatives.
2.2. In Vitro Cancer Cell Line Testing for Anti-Proliferative Activity
Evaluation of anti-proliferative activity for the new quinolin-4-yl-N-aryloxadiazol-2-amines (6a–k
)
and quinolin-4-yl-benzoylmethylthiotriazoles (8a–b) was carried out in established MDA-MB-231
(metastatic breast) and HeLa (cervical) cancer cell lines using the MTT endpoint assay, according
to methodology previously described by our group and others [
Bcl2-expressing cancer cell lines where Bcl-2 inhibitory molecules have shown protein down-regulation
following treatment [13 14]. Additionally, to test the effect of differing cellular Bcl-2 status, compounds
5,6]. MDA-MB-231 and HeLa are
,
6a–k and 8a–b were evaluated for activity in the leukaemic cell lines KG1a (acute myelogenous
leukaemia, Bcl-2 positive, [15]) and Jurkat (T-cell leukaemia, Bcl-2 negative, [16]). As previously
described [
appropriate for testing anti-proliferative activity in these non-adherent cells.
The results presented in Table 1 indicate some potent (sub-micromolar) IC50 activity, particularly
for some of the oxadiazole-based compounds in the solid tumour cell lines MDA-MB-231 (6b–c
6f–k) and HeLa (6c 6e–h 6j). Potent (low to sub-micromolar) activity was also observed for a few
oxadiazole compounds in the Bcl-2-expressing KG1a cell line (6d–e 6i–j). All compounds were found
5,6], for the studies in the leukaemic cell lines we used the CellTitreBlue®endpoint assay,
,
,
,
,
to be essentially inactive within the Bcl-2 negative Jurkat cell line. Amongst individual compounds
with potent anti-proliferative activity across the three sensitive cell lines, compound 6j is of particular
note, having sub-micromolar IC50 values in these three cell lines. The two triazole-based analogues 8a
and 8b were found to be inactive in all cell lines tested, and were not explored further.

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Table 1. Growth inhibitory activity (IC50
6a–k) and quinolin-4-yl-benzoylmethylthiotriazoles (8a–b) in human cancer cell lines MDA-MB-231
(breast), HeLa (cervical), KG1a (AML) and Jurkat (T-cell leukaemia).
, µM) values for quinolin-4-yl-N-aryloxadiazol-2-amines
(
Compound
R
R 1
IC50 (µM) 1
MDA-MB-231
HeLa 2
KG1a 3
Jurkat 3
2
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
-
4-Cl-Ph
3,4-diCl-Ph
4-Me-Ph
4-NO2-Ph
4-OMe-Ph
4-Cl-Ph
3,4-diCl-Ph
4-Me-Ph
4-NO2-Ph
4-OMe-Ph
4-F-Ph
9.13 ± 0.82
0.25 ± 0.022
0.55 ± 0.042
7.04 ± 0.11
9.85 ± 0.82
0.55 ± 0.01
0.34 ± 0.07
0.43 ± 0.01
0.54 ± 0.02
0.44 ± 0.04
0.30 ± 0.01
>100
7.13 ± 0.63
8.60 ± 0.72
0.05 ± 0.002
6.39 ± 0.12
0.74 ± 0.04
0.84 ± 0.03
0.21 ± 0.04
0.32 ± 0.021
1.42 ± 0.78
0.77 ± 0.09
5.44 ± 0.32
>100
14.95 ± 0.54
70.52 ± 2.5
16.99 ± 0.14
1.24 ± 0.13
1.31 ± 0.1
12.4 ± 0.27
26.96 ± 1.41
12.25 ± 1.12
1.21 ± 0.044
0.36 ± 0.024
>100
44.25 ± 2.12
>100
>100
56 ± 1.24
>100
>100
>100
>100
>100
6j
65 ± 1.47
6k
8a
8b
1
>100
4-OMe-Ph
3,4-diCl-Ph
>100
>100
>100
>100
-
>100
>100
Mean of three independent experiments with range quoted; 2 MTT assay; 3 CellTitre-Blue® assay.
2.3. Evaluation of Bcl-2 Binding via Enzyme-Linked Immunosorbent Assay (ELISA)
The ability of active compounds 6c
(Bcl-2 homology domain 3)-binding Bim peptide was evaluated using an ELISA (enzyme-linked
immunosorbent assay), according to our previously described protocol [ ]. Compounds 6c 6d and
6i were chosen to represent compounds that were amongst the most potent tested across the cell lines
6c and 6i) and a compound of intermediate potency (6d) to assess whether cellular potency in Bcl-2
, 6d and 6i to compete with the pro-apoptotic BH3
5,
6
,
(
expressing cell lines correlates with Bcl-2 binding activity.
Briefly, biotinylated Bim peptide was attached to streptavidin-coated microtitre plates.
The addition of mixtures of 6c, 6d, or 6i at various concentrations and His-tagged Bcl-2 allowed
competitive binding between test compounds and Bim peptide for His-tagged Bcl-2 binding. Addition
of anti-His secondary antibody conjugated to HRP (horseradish peroxidase) enzyme was followed
by the addition of o-phenylenediamine, which produces a colourimetric readout in the presence of
peroxidase enzyme (monitored at 450 nm). Reduction of the signal by competitive binding of the test
compound to Bcl-2 is then plotted against concentration to derive an IC50 for test compound inhibition
of Bim-BH3 binding. The well-known Bcl-2 inhibitory natural product (-)-gossypol, which has been
used in cancer clinical trials [17], was used as a positive control. The results of the ELISA assay are
shown in Table 2.
Table 2. Enzyme-linked immunosorbent assay (ELISA) binding activity values (IC50) for selected
compounds compared to positive control (-)-gossypol. Results are expressed as triplicate testing
mean values.
Compound
ELISA IC50 (µM) 1
6c
6d
6i
1.93 ± 0.007
14.52 ± 1.20
0.15 ± 0.02
0.60 ± 0.09
Gossypol
1
Mean of three independent experiments with range quoted.
The results of the ELISA study shown in Table 2 provide direct evidence for the ability of the
new compounds to compete with Bim peptide for direct binding to Bcl-2. Notably compound 6d with

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moderate anti-proliferative activity in Bcl-2 expressing cell lines was the least potent compound in the
ELISA assay. In contrast, compound 6i gave an IC50 value (0.15 µM) more potent than the positive
control Bcl-2 inhibitor gossypol.
2.4. Molecular Modelling of Compound 6i Interacting within the Bcl-2 Binding Pocket
To gain insight into binding interactions at the molecular level, and to further validate our drug
design and Bcl-2 binding (ELISA) results allowing future optimization of heterocyclic Bcl-2 inhibitors,
we undertook computational molecular modeling work on hit compound 6i within the Molecular
Operating Environment (MOE) platform [18]. Our starting point was a published co-crystal structure
(Protein Data Bank (PDB) code: 4AQ3) between a sulfonamide-based dual Bcl-2/Bcl-xL antagonist
(N,N-dibutyl-4-chloro-1-[2-(3,4-dihydro-1H-isoquinolin-2-ylcarbonyl)-4-[(7-iodonaphthalene-2-yl)
sulfonylcarbamoyl]phenyl]-5-methyl-pyrazole-3-carboxamide) and the Bcl-2 binding pocket [19].
The active site of Bcl-2 is formed of two hydrophobic pockets (G1 and G2) and a shallow linker.
The interaction of the native sulfonamide ligand within the Bcl-2 binding site showed a number of
key interactions, including: (i) a H-bond between the oxygen atom of the sulfonamide SO2 group
and NH2 of Gln58; (ii) two H-bonds between the oxygen atom of sulfonamide SO2/NH and the
OH of Tyr67. The iodonaphthalene moiety showed a face–face aromatic interaction with Tyr161.
Additional hydrophobic interactions with Ala59, Phe63, Tyr161 constitute the hydrophobic site of
Bcl-2. Furthermore, the isoquinoline moiety formed hydrophobic interactions with Phe63, Phe71 and
Ala108, and the dibutylamine side chain showed hydrophobic interactions with Val92, Leu96, Phe112,
and the hydrophobic part of Glu95 and Met97. Figure 2 shows this ligand binding in more detail
(yellow stick model).
Our docking results for {5-[2-(4-bromophenyl)-6-methoxyquinolin-4-yl}-N-(4-nitrophenyl)-[1,3,4]
oxadiazol-2-amine (6i; green stick model) in the Bcl-2 binding pocket indicate a number of similarities
to the native co-crystallised sulfonamide ligand, showing five H-bonds with key Bcl-2 binding site
residues. The following interactions are notable: (i) the oxygen atom of the NO2 group was a H-bond
acceptor from the OH of Tyr161 (distance 3.4Å); (ii) the NH group bridging the oxadiazole and
nitrophenyl formed H-bonds with the NH2 of Gln58 (distance 3.4Å); (iii) similarly to the co-crystalized
ligand the oxadiazole moiety of 6i formed two H-bonds with key residue Tyr67; (iv) the nitrogen and
oxygen atoms of the oxadiazole formed two H-bonds with the OH of Tyr67 (distance 3.5Å and 3.3Å
respectively); (v) the O atom of the OCH3 group was a H-bond acceptor with the NH2 of Arg105
(distance 2.6Å). Figure 2a shows the binding interactions of 6i in more detail and binding modes
between the two ligands.
Overall the quinolin-4-yl-N-aryl-[1,3,4]oxadiazol-2-amine (6i) showed a similar binding
mode within the Bcl-2 pocket compared to the native sulfonamide ligand, noting that the
nitrophenylamino-oxadiazole component of 6i occupied the same position as the iodonaphthalene
sulfonamide moiety of the native ligand (Figure 2a). Similarly the phenyl-quinoline component of
compound 6i occupied the same position as the phenyl-isoquinoline moiety of the native ligand within
the hydrophobic pocket of Bcl-2. Figure 2b illustrates the key binding interactions for compound
6i within the Bcl-2 binding pocket in more detail, with the nitrophenylamino-oxadiazole showing
interactions with Gln58, Phe63, Tyr67 and Tyr161. Meanwhile the bromophenyl-quinoline moiety
showed hydrophobic interactions with Val92, Leu96, and Phe112 residues, with the 6-methoxy
group making an additional interaction with Arg105. Overall, it could be concluded that the
quinoline-oxadiazole based hit compound 6i makes key interactions with the Bcl-2 active site thereby
contributing to Bcl-2 inhibitory activity.

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(b)
(a) Representation of the binding mode of compound 6i (green stick) and native
(a)
Figure 2.
iodonaphthalene sulfonamide-based ligand (yellow stick) from the co-crystal structure with Bcl-2
(PDB code 4AQ3), showing the overlap in binding poses between the two ligands. ( ) representative
b
interactions of compound 6i (dark green stick) with key residues (purple stick) of the Bcl-2 binding
pocket. H-bonds represented as yellow dotted lines.
3. Discussion and Conclusions
This new series of quinoline-based heterocycles further supports our concept that rationally
designed Bcl-2 inhibitory aromatic heterocycles can effectively interfere with Bcl-2–Bim peptide
binding and exhibit potent anti-proliferative activity against Bcl-2 expressing human cancer cell lines.
Access to the target compounds was achieved via a straightforward and efficient synthetic route.
In particular, our research demonstrated complete regiochemical control of cyclisation from a common
2-(2-(4-aryl)quinoline-4-carbonyl)-N-aryl-hydrazine-1-carbothioamide intermediate (5a–k) to form
either quinolin-4-yl-N-aryl-oxadiazol-2-amines (6a–k) or quinolin-4-yl-benzylthiotriazoles (8a–b) by
simple variation of the basic reaction conditions. From the compounds active in Bcl-2 expressing
cancer cell lines and selected for further study, compound 6i exhibited potent inhibitory activity in
the Bcl-2-Bim peptide ELISA assay. On the other hand, compound 6c, overall one of the most potent
compounds against Bcl-2 expressing cell lines, gave a moderate ELISA IC50 value. This mismatch
between cellular and target-based activity is likely indicative of additional molecular targets for these
molecules as yet undiscovered, as would be expected to be the case for small heterocyclic molecules of
this type. The quinoline-oxadiazole based compound 6i (R1 = 4-nitrophenyl) in particular was found
to exhibit potency within Bcl-2 expressing cancer cells and the ELISA assay and will be the subject of
further investigation.
4. Experimental
4.1. General Experimental Details—Chemistry
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 MHz
instrument, and chemical shifts are given in ppm relative to Me4Si with coupling constants
(J values) in Hz. Mass spectrometry was run in electrospray positive ionization mode
(Bruker MicroTof instrument, Coventry, UK). Elemental analysis (% CHN) was run by
combustion analysis through an outsourced service (Medac Ltd, Surrey, UK). Commercial
compounds were used as received; 2-(4-bromophenyl)quinoline-4-carboxylic acid (3a) [20] and
2-(4-bromophenyl)-6-methoxyquinoline-4-carboxylic acid (3b) [21] were accessed via the standard
Pfitzinger reaction protocol [8].

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4.2. Preparation of 2-(4-Bromophenyl)Quinoline-4-Carboxylic Acid Hydrazides (4a–b)
A mixture of the appropriate 2-(4-bromophenyl)quinoline-4-carboxylic acid (3a–b) (10 mmol),
absolute ethanol (20 mL) and concentrated sulfuric acid (2 mL) was heated under reflux for 12 h.
Excess ethanol was removed under reduced pressure and the resulting oil was rendered alkaline using
aqueous sodium bicarbonate. The aqueous layer was extracted with dichloromethane (2
×
50 mL) and
the combined organic extracts were dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The intermediate crude ester product was re-dissolved in ethanol (10 mL) and
98% hydrazine hydrate (10 mL) added. The solution was heated under reflux for a further 12 h and
then allowed to cool to room temperature. The precipitate that formed was collected by filtration,
washed with water (3
intermediate quinoline carboxylic hydrazides (4a–b) in 75%–76% overall yield.
×
10 mL) to remove excess hydrazine hydrate, and dried in vacuo to give the
◦
1
2-(4-Bromophenyl)quinoline-4-carboxylic acid hydrazide (4a). Yield 75%, m.p. 246–248 C. H-NMR
(DMSO-d6 ): 4.7 (s, 2H, NH2), 7.65 (t, 1H, ArCH), 7.79 (d, 2H, J = 8.9, ArCH), 7.87 (t, 1H, J = 7.5, ArCH),
8.13 (d, 2H, J = 2.9, ArCH), 8.24 (d, 1H, J = 5.7, CH aromatic), 8.27 (d, 2H, J = 8.9, ArCH), 10.02 (s, 1H,
NH).
◦
2-(4-Bromophenyl)-6-methoxyquinoline-4-carboxylic acid hydrazide (4b). Yield 76%, m.p. 266–268 C.
1H-NMR (DMSO-d6 ): 3.9 (s, 3H, OCH3), 4.7 (s, 2H, NH2), 7.5 (d, 1H, J = 7.5, ArCH), 7.68 (d, 1H, J = 2.4,
ArCH), 7.70 (d, 2H, J = 7.5, ArCH), 8.07 (d, 1H, J = 7.5, ArCH), 8.13 (s, 1H, ArCH), 8.27 (d, 2H, J = 7.5,
ArCH), 10.09 (s, 1H, NH).
4.3. Preparation of 2-(2-(4-Bromophenyl)Quinoline-4-Carbonyl)-N-Arylhydrazine-1-Carbothioamides (5a–k)
To a solution of quinoline-4-carboxylic acid hydrazide (4a–b, 1 mmol) in absolute ethanol (20 mL)
was added a solution of substituted phenylisothiocyanate (1 mmol) in ethanol (10 mL) with continuous
stirring. The reaction mixture was heated under reflux for 12 h. After cooling to room temperature, the
precipitate formed was collected by filtration, and washed with ice-cold ethanol (5 mL) to give the
corresponding quinoline-4-carbonyl-N-arylhydrazine-1-carbothioamide (5a–k) which was used in the
next step without further purification.
2-(2-(4-Bromophenyl)quinoline-4-carbonyl)-N-(4-chlorophenyl)hydrazine-1-carbothioamide (5a). Yield 74%,
◦
m.p. 247–249 C. 1H-NMR (DMSO-d6): 7.45–7.62 (m, 4H, ArCH), 7.67 (t, 1H, J = 7.5, ArCH), 7.80 (d, 1H,
J = 9.2, ArCH), 7.90 (d, 1H, J = 8.6, ArCH), 8.14 (d, 1H, J = 8.5, ArCH), 8.25 (d, 1H, J = 9.2, ArCH), 8.34
(d, 2H, J = 7.5, ArCH), 8.42 (d, 2H, J= 2.5, ArCH), 10.02 (s, 2H, NH), 10.95 (s, 1H, NH).
2-(2-(4-Bromophenyl)quinoline-4-carbonyl)-N-(3,4-dichlorophenyl)hydrazine-1-carbothioamide (5b). Yield
65%, m.p. 265–267 ◦C. 1H-NMR (DMSO-d6): 7.60 (m, 3H, ArCH), 7.77 (d, 1H, J = 8.5, ArCH), 7.85
(d, 2H, J = 8.5, ArCH), 8.09 (d, 1H, J = 7.5, ArCH), 8.19 (m, 1H, ArCH), 8.29 (m, 2H, ArCH), 8.42 (d, 2H,
J = 7.5, ArCH), 10.18 (s, 1H, NH), 10.82 (s, 1H, NH), 11.28 (s, 1H, NH).
2-(2-(4-Bromophenyl)quinoline-4-carbonyl)-N-(4-methylphenyl)hydrazine-1-carbothioamide (5c). Yield 69%,
◦
1
m.p. 245–247 C. H-NMR (DMSO-d6): 2.31 (s, 3H, CH3), 7.18 (d, 2H, J = 8.5, ArCH), 7.35 (s, 2H,
ArCH), 7.70 (m, 1H, ArCH), 7.85 (m, 3H, ArCH), 8.16 (d, 1H, J = 8.5, ArCH), 8.26 (d, 2H, J = 8.47,
ArCH), 8.41 (d, 1H, J = 8.5, ArCH), 8.46 (s, 1H, ArCH), 9.83 (s, 2H, NH), 10.86 (s, 1H, NH).
2-(2-(4-Bromophenyl)quinoline-4-carbonyl)-N-(4-nitrophenyl)hydrazine-1-carbothioamide (5d). Yield 75%,
m.p. 255–257 ◦C. 1H-NMR (DMSO-d6): 7.72 (m, 1H, ArCH), 7.75 (d, 3H, J = 8.5, ArCH), 7.86 (m, 2H,
ArCH), 8.15 (d, 1H, J = 8.5, ArCH), 8.33 (m, 4H, ArCH), 8.49 (d, 2H, J = 3.5, ArCH), 10.35 (s, 2H, NH),
11.09 (s, 1H, NH).
2-(2-(4-Bromophenyl)quinoline-4-carbonyl)-N-(4-methoxyphenyl)hydrazine-1-carbothioamide (5e). Yield 61%,
m.p. 216–218 ◦C. 1H-NMR (DMSO-d6): 3.79 (s, 3H, OCH3), 6.85 (d, 2H, J = 7.8, ArCH), 7.35 (d, 2H,
J = 2.5, ArCH), 7.70 (m, 1H, ArCH), 7.82 (d, 2H, J = 7.8, ArCH), 7.90 (d, 1H, J = 6.5, ArCH), 8.17 (d, 1H,

Molecules 2019, 24, 1274
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J = 8.5, ArCH), 8.26 (d, 2H, J = 7.8, ArCH), 8.40 (d, 2H, J = 2.5, ArCH), 9.80 (s, 2H, NH), 10.45 (s, 1H,
NH).
2-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(4-chlorophenyl)hydrazine-1-carbothioamide (5f).
Yield 75%, m.p. 186–188 ◦C. 1H-NMR (DMSO-d6): 3.9 (s, 3H, OCH3), 7.48 (m, 5H, ArCH), 7.70 (d, 3H,
J = 8.9, ArCH), 8.04 (d, 1H, J = 8.9, ArCH), 8.21 (d, 3H, J = 8.8, ArCH), 10.2 (s, 2H, NH), 10.89 (s, 1H,
NH).
2-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(3,4-dichlorophenyl)hydrazine-1-carbothioamide (5g).
◦
Yield 73%, m.p. 199–201 C. 1H-NMR (DMSO-d6): 3.9 (s, 3H, OCH3), 7.51 (m, 4H, ArCH), 7.56 (m, 2H,
ArCH), 7.75 (d, 2H, J = 8.5, ArCH), 8.09 (d, 1H, J = 7.5, ArCH), 8.19 (d, 2H, J = 8.5, ArCH), 10.13 (s, 2H,
NH), 11.28 (s, 1H, NH).
2-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(4-methylphenyl)hydrazine-1-carbothioamide (5h).
Yield 65%, m.p. 209–211 ◦C. 1H-NMR (DMSO-d6): 2.30 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 7.19 (d, 2H,
J = 8.8, ArCH), 7.35 (s, 2H, ArCH), 7.53 (d, 2H, J = 7.2, ArCH), 7.69 (d, 3H, J = 7.5, ArCH), 8.03 (d, 1H,
J = 7.5, ArCH), 8.23 (d, 2H, J = 7.5, ArCH), 9.83 (s, 2H, NH), 10.85 (s, 1H, NH).
2-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(4-nitrophenyl)hydrazine-1-carbothioamide (5i).
◦
Yield 71%, m.p. 175–177 C. 1H-NMR (DMSO-d6): 3.90 (s, 3H, OCH3), 7.51 (d, 1H, J = 7.8, ArCH), 7.82
(d, 3H, J = 6.3, ArCH), 7.95 (s, 2H, ArCH), 8.10 (d, 1H, J = 9.0, ArCH), 8.30 (m, 5H, ArCH), 10.35 (s, 2H,
ArCH), 11.09 (s, 1H, NH).
2-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(4-methoxyphenyl)hydrazine-1-carbothioamide (5j).
Yield 70%, m.p. 207–209 ◦C. 1H-NMR (DMSO-d6): 3.75 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.90 (d, 2H,
J = 7.5, ArCH), 7.37 (m, 2H, ArCH), 7.55 (d, 1H, J = 6.9, ArCH), 7.80 (d, 3H, J = 7.5, ArCH), 8.10 (d, 1H,
J = 7.5, ArCH), 8.25 (d, 2H, J = 7.5, ArCH), 8.45 (s, 1H, ArCH), 9.80 (s, 2H, NH), 10.09 (s, 1H, NH). m/z
(ES+) 537.04 (M+).
2-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(4-fluorophenyl)hydrazine-1-carbothioamide (5k).
◦
Yield 60%, m.p. 183–185 C. 1H-NMR (DMSO-d6): 3.90 (s, 3H, OCH3), 7.19 (m, 2H, ArCH), 7.45 (m, 3H,
ArCH), 7.76 (d, 3H, J = 7.9, ArCH), 8.12 (d, 1H, J = 8.6, ArCH), 8.23 (d, 2H, J = 7.6, ArCH), 8.43 (s, 1H,
ArCH), 9.98 (s, 2H, NH), 10.89 (s, 1H, NH).
4.4. Preparation of {5-[2-(4-Bromophenyl)-Quinolin-4-yl}-N-Aryl-[1,3,4]Oxadiazol-2-Amines (6a-k)
A suspension of quinoline-4-carbonyl-N-arylhydrazine-1-carbothioamide (5a–k, 1 mmol) in 2N
sodium hydroxide (10 mL) was heated under reflux for 3h. After cooling to room temperature,
the crude product precipitate was collected by filtration. Recrystallisation from ethanol gave the
corresponding pure quinolin-4-yl-N-aryl-[1,3,4]oxadiazol-2-amine (6a-k) in 40%–78% isolated yield.
{5-[2-(4-Bromophenyl)quinolin-4-yl}-N-(4-chlorophenyl)-[1,3,4]oxadiazol-2-amine (6a). Yield 76%, m.p.
◦
1
254–257 C. H-NMR (DMSO-d6): 7.49 (d, 2H, J = 6.9, ArCH), 7.72 (d, 2H, J = 7.2, ArCH), 7.83
(d, 3H, J = 7.2, ArCH), 7.95 (m, 1H, ArCH), 8.25 (m, 3H, ArCH), 8.5 (s, 1H, ArCH), 9.12 (d, 1H, J = 6.9,
ArCH), 11.20 (s, 1H, NH). m/z (ES+) 478.10 (M+). Anal. (C23H14BrClN4O): % CHN required: C 57.82,
H 2.95, N 11.73; found C 57.82, H 2.82, N 11.74.
{5-[2-(4-Bromophenyl)quinolin-4-yl}-N-(3,4-dichlorophenyl)-[1,3,4]oxadiazol-2-amine (6b). Yield 78%, m.p.
◦
264–266 C. 1H-NMR (DMSO-d6): 7.61 (dd, 1H, J = 7.5, 2.9, ArCH), 7.68 (d, 1H, J = 9.3, ArCH), 7.83 (m,
2H, ArCH), 7.88 (s, 1H, J = 2.5, ArCH), 7.94 (t, 1H, J = 7.5, ArCH), 8.04 (d, 1H, J = 2.7, ArCH), 8.22 (s,
1H, ArCH), 8.24 (d, 2H, J = 8.3, ArCH), 8.47 (s, 1H, ArCH), 9.13 (d, 1H, J = 7.8, ArCH), 11.38 (s, 1H,
NH). m/z (ES+) 512.25 (M+). Anal. (C23H13BrCl2N4O): % CHN required: C 53.93, H 2.56, N 10.94;
found C 53.59, H 2.68, N 10.67.
{5-[2-(4-Bromophenyl)quinolin-4-yl}-N-(4-methylphenyl)-[1,3,4]oxadiazol-2-amine (6c). Yield 40%, m.p.
265–267 ◦C. 1H-NMR (DMSO-d6): 2.96 (s, 3H, CH3), 7.23 (d, 2H, J = 8.2, ArCH), 7.6 (d, 2H, J = 8.2,

Molecules 2019, 24, 1274
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ArCH), 7.70 (d, 2H, J = 8.5, ArCH), 7.95 (m, 1H, ArCH), 8.21 (d, 1H, J = 8.2, ArCH), 8.31 (d, 2H, J = 8.2,
ArCH), 8.35 (d, 1H, J = 8.5, ArCH), 8.46 (s, 1H, ArCH), 9.15 (d, 1H, J = 8.5, ArCH), 10.86 (s, 1H, NH).
13C-NMR (DMSO-d6): 20.32 (CH3), 116.85, 117.36, 122.16, 123.86, 125.85, 128.37, 129.00, 129.10, 129.56,
129.99, 130.76, 131.24, 132.04, 135.81, 137.11, 148.32, 154.72, 156.20, 160.28. HRMS m/z (ES+) required
457.0586 (M++1), found 457.0589.
{5-[2-(4-Bromophenyl)quinolin-4-yl}-N-(4-nitrophenyl)-[1,3,4]oxadiazol-2-amine (6d). Yield 59%, m.p.
◦
1
237–239 C. H-NMR (DMSO-d6): 7.86 (d, 3H, J = 8.5, ArCH), 7.98 (m, 3H, ArCH), 8.27 (m, 3H,
ArCH), 8.35 (d, 2H, J = 8.5, ArCH), 8.50 (s, 1H, ArCH), 9.15 (d, 1H, J = 7.5, ArCH), 11.84 (s, 1H, NH).
m/z (ES+) 489.25 (M+). Anal. (C23H14BrN5O3): % CHN required: C 56.57, H 2.89, N 14.34; found C
56.59, H 2.79, N 14.37.
{5-[2-(4-Bromophenyl)quinolin-4-yl}-N-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-amine (6e). Yield 65%, m.p.
229–231 ◦C. 1H-NMR (DMSO-d6): 3.79 (s, 3H, OCH3), 7.00 (d, 2H, J = 8.9, ArCH), 7.56 (d, 2H, J = 8.9,
ArCH), 7.97 (m, 3H, ArCH), 7.92 (m, 1H, ArCH), 8.20 (m, 3H, ArCH), 8.50 (s, 1H, ArCH), 9.17 (d, 1H,
J = 7.5, ArCH), 10.78 (s, 1H, NH). m/z (ES+) 474.05 (M+). Anal. (C24H17BrN4O2): % CHN required: C
60.90, H 3.62, N 11.84; found C 60.69, H 3.33, N 11.72.
{5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl}-N-(4-chlorophenyl)-[1,3,4]oxadiazol-2-amine (6f). Yield 48%,
m.p. 261–263 ◦C. 1H-NMR (DMSO-d6): 3.97 (s, 3H, OCH3), 7.41 (d, 2H, J = 6.9, ArCH), 7.54 (d, 1H,
J = 7.6, ArCH), 7.63 (d, 2H, J = 6.9, ArCH), 7.75 (d, 2H, J = 7.45, ArCH), 8.15 (d, 1H, J = 8.5, ArCH),
8.24 (d, 2H, J = 8.5, ArCH), 8.40 (s, 1H, ArCH), 8.7 (d, 1H, J = 2.5, ArCH). m/z (ES+) 508.02 (M+). Anal.
(C24H16BrClN4O2): % CHN required: C 56.77, H 3.18, N 11.03; found C 56.83, H 3.12, N 11.13.
{5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl}-N-(3,4-dichlorophenyl)-[1,3,4]oxadiazol-2-amine (6g). Yield
◦
59%, m.p. 227–229 C. 1H-NMR (DMSO-d6): 3.95 (s, 3H, OCH3), 7.18 (d, 2H, J = 7.3, ArCH), 7.52 (d, 1H,
J = 6.3, ArCH), 7.78 (d, 2H, J = 8.0, ArCH), 7.89 (d, 1H, J = 2.5, ArCH), 8.09 (d, 2H, J = 8.3, ArCH),
8.29 (d, 2H, J = 7.3, ArCH), 8.31 (s, 1H, NH), 8.90 (d, 1H, J = 2.3, ArCH). m/z (ES+) 541.98 (M+). Anal.
(C24H15BrCl2N4O2): % CHN required: C 53.16, H 2.79, N 10.33; found C 52.92, H 2.86, N 10.12.
{5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl}-N-(4-methylphenyl)-[1,3,4]oxadiazol-2-amine (6h). Yield 54%,
m.p. 239–241 ◦C. 1H-NMR (DMSO-d6): 2.2 (s, 3H, CH3), 4.0 (s, 3H, OCH3), 7.20 (d, 2H, J = 8.3, ArCH),
7.62 (d, 3H, J = 8.7, ArCH), 7.75 (d, 2H, J = 9.1, ArCH), 8.09 (d, 1H, J = 9.1, ArCH), 8.2 (d, 2H, J = 8.3,
ArCH), 8.40 (s, 1H, ArCH), 8.65 (d, 1H, J = 3.0, ArCH), 10.85 (s, 1H, NH). HRMS m/z (ES+) required
487.0691 (M++1), found 487.0693.
{5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl}-N-(4-nitrophenyl)-[1,3,4]oxadiazol-2-amine (6i). Yield 45%,
m.p. 272–274 ◦C. 1H-NMR (DMSO-d6): 3.99 (s, 3H, OCH3), 7.55 (dd, 1H, J = 9.0, ArCH), 7.64 (d, 2H,
J = 9.0, ArCH), 7.78 (d, 2H, J = 9.0, ArCH), 7.96 (s, 1H, ArCH), 8.10 (d, 3H, J = 9.2, ArCH), 8.24 (d, 2H,
J = 8.6, ArCH), 8.39 (s, 1H, NH), 8.79 (d, 1H, J = 3.1, ArCH). HRMS (C24H16BrN5O4) m/z (ES+) required
517.0380 (M++1), found 517.0428. Anal. (C24H16BrN5O4): % CHN required: C 56.10, H 3.27; found C
55.90, H 3.11.
{5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl}-N-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-amine (6j). Yield
◦
57%, m.p. 232–234 C. 1H-NMR (DMSO-d6): 3.69 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.90 (d, 2H, J = 7.3,
ArCH), 7.58 (m, 3H, ArCH), 7.80 (d, 2H, J = 7.9, ArCH), 8.10 (d, 1H, J = 7.3, ArCH), 8.19 (d, 2H, J = 7.9,
ArCH), 8.40 (s, 1H, ArCH), 8.54 (d, 1H, J = 2.5, ArCH). m/z (ES+) 505.07 (M+). Anal. (C25H19BrN4O3):
% CHN required: C 59.65, H 3.80, N 11.13; found C 59.35, H 3.68, N 10.91.
{5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl}-N-(4-fluorophenyl)-[1,3,4]oxadiazol-2-amine (6k). Yield 47%,
m.p. 230–232 ◦C. 1H-NMR (DMSO-d6): 3.98 (s, 3H, OCH3), 7.12 (m, 2H, ArCH), 7.54 (d, 1H, J = 8.5,
ArCH), 7.62 (m, 2H, ArCH), 7.78 (d, 2H, J = 8.5, ArCH), 8.08 (d, 1H, J = 8.5, ArCH), 8.19 (d, 2H, J = 7.8,
ArCH), 8.31 (s, 1H, ArCH), 8.75 (d, 1H, J = 2.9, ArCH). m/z (ES+) 492.07 (M+). Anal. (C24H16BrFN4O2):
% CHN required: C 59.01, H 3.68, N 10.90; found C 58.67, H 3.28, N 11.20.

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4.5. Preparation of 5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-aryl-4H-[1,2,4]triazole-3-thiols (7a–b).
A solution of 2-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-N-(4-aryl)hydrazine-1-
carbothioamide (10 mmol) in 2N NaOH was heated under reflux for 3 h. After cooling to room
temperature, water was added and the mixture carefully neutralized with 0.1M HCl. The precipitate
formed was filtered, dried and recrystallized from ethanol to give the corresponding triazole thiol
product (7a–b).
5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-(4-methoxyphenyl)-4H-[
1,
2,
4]triazole-3-thiol
(7a).
Yield 58%, m.p. 199–201 ◦C. 1H-NMR (DMSO-d6): 3.70 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.86 (d, 2H,
J = 8.9, ArCH), 7.14 (d, 2H, J = 8.9, ArCH), 7.41 (dd, 1H, J = 9.3, ArCH), 7.51 (s, 1H, ArCH), 7.65 (d, 2H,
J = 8.5, ArCH), 7.83 (m, 3H, ArCH), 7.95 (d, 1H, J = 9.2, ArCH).
5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-(3,4-dichlorophenyl)-4H-[1,2,4]triazole-3-thiol (7b).
Yield 64%, m.p. 170–172 ◦C. 1H-NMR (DMSO-d6): 3.86 (s, 3H, OCH3), 7.08 (dd, 1H, J = 8.3, ArCH),
7.42 (dd, 2H, J = 9.2, ArCH), 7.49 (d, 1H, J = 8.3, ArCH), 7.61 (d, 1H, J = 2.6, ArCH), 7.67 (d, 2H, J = 8.8,
ArCH), 7.82 (d, 1H, J = 2.4, ArCH), 7.93 (d, 2H, J = 8.8, ArCH), 7.97 (d, 1H, J = 9.4, ArCH).
4.6. Preparation of
5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-aryl-4H-[1,2,4]triazole-3-ylsulfanyl]-1-phenylethanone
(8a–b).
2-Bromoacetophenone (5 mmol) was added to a solution of 5-[2-(4-bromophenyl)-6-methoxyquinolin-
4-yl]-4-aryl-4H-[1,2,4]triazine-3-thiol (7a–b, 5 mmol) in ethanol (70%) containing KOH (5 mmol).
The reaction mixture was left to stir at room temperature for 16 h. Water was then added and the
precipitate formed was filtered, dried and recrystallized from ethanol to give the corresponding S-alkyl
triazole thiol product (8a–b).
5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-(4-methoxyphenyl)-4H-[
1,2,
4
]triazole-3-ylsulfanyl]-1-phenylethanone (8a). Yield 63%, m.p. 240–242 ◦C. 1H-NMR (DMSO-d6): 3.70
(s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.42 (s, 2H, CH2), 6.86 (d, 2H, J = 8.9, ArCH), 7.14 (d, 1H, J = 8.9,
ArCH), 7.46 (m, 5H, ArCH), 7.51 (m, 2H, ArCH), 7.62 (d, 2H, J = 9.5, ArCH), 7.83 (m, 2H, ArCH), 8.15
(m, 3H, ArCH). m/z (ES+) 639.08 (M+).
5-[2-(4-Bromophenyl)-6-methoxyquinolin-4-yl]-4-(3,4-dichlorophenyl)-4H-[
1,2,
4
]triazole-3-ylsulfanyl]-1-phenylethanone (8b). Yield 63%, m.p. 247–249 ◦C. 1H-NMR (DMSO-d6): 3.70
(s, 3H, OCH3), 5.12 (s, 2H, CH2), 7.32 (d, 1H, J = 2.3, ArCH), 7.54 (m, 2H, ArCH), 7.62 (m, 2H, ArCH),
7.76 (m, 4H, ArCH), 8.10 (m, 6H, ArCH), 8.16 (s, 1H, ArCH). Anal. (C32H21BrCl2N4O2S): % CHN
required: C 56.82, H 3.13, N 8.28; found C 57.31, H 3.03, N 8.43.
4.7. Cell Viability—MTT Assay (MDA-MB-231 and HeLa Cells)
Human breast cancer MDA-MB-231 cells were cultured in RPMI 1640 medium (Life Technologies,
Paisley, UK) supplemented with 10% foetal bovine serum, 100 IU/mL pencillin, and 100
µg/mL
streptomycin (Life Technologies). Human cervical cancer HeLa cells were cultured in D-MEM (Life
Technologies) supplemented with 10% foetal bovine serum, 100 IU/mL pencillin, and 100
µ
g/mL
◦
streptomycin (Life Technologies). Cells were passaged routinely and maintained at 37 C and 5%
CO2. 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 test
compound (from 10 mM stock in DMSO) over a 10 fold dilution series with concentrations ranging
from 0.00001 mM to 100
conducted using DMSO vehicle control with volumes equivalent to test compound concentrations.
Cells were incubated with test compound for 72 h., followed by treatment of wells with 20 L of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) solution (from a freshly made
5.5 g/mL stock in PBS, Sigma-Aldrich, Gillingham, Dorset, UK). After 4 hours the solutions from
µM, each diluent being performed in triplicate. Control experiments were
µ
µ
the wells were removed and the resulting formazan crystals were dissolved in DMSO (200
incubated for 30 minutes at 37 C. Absorbance was measured at 550 nm using an automated microplate
µ
L) and
◦

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reader. Mean IC50 values were obtained from plots of absorbance versus test compound concentration
using GraphPad Prism 5 software (San Diego, CA, USA). For each concentration, three independent
repeat experiments were carried out to establish reproducibility.
4.8. Cell Viability—CellTiter-Blue®Assay (KG1a and Jurkat Cells)
Human acute myeloid leukaemia KG1a cells and acute T-cell lymphocytic Jurkat cells were
cultured in RPMI 1640 medium (Life Technologies, Paisley, UK) supplemented with 10% foetal bovine
serum, 100 IU/mL pencillin, and 100
µ
g/mL streptomycin (Life Technologies). Cells were passaged
◦
routinely and maintained at 37 C and 5% CO2. 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 mM to 100 µM, each diluent being performed in triplicate.
Control experiments were conducted using DMSO vehicle control with volumes equivalent to test
compound concentrations. Cells were incubated with test compound for 24 h, followed by treatment
with 20 µL of CellTiter-Blue®(Promega, Southampton, UK). Fluorescence was measured using 544 nm
excitation and 590 nm emission filters on a fluorescent plate reader. Mean IC50 values were obtained
from plots of absorbance versus test compound concentration using GraphPad Prism 5 software
(San Diego, CA, USA). For each concentration, three independent repeat experiments were carried out
to establish reproducibility.
4.9. Enzyme-Linked Immunosorbent Assay (ELISA)
A streptavidin-coated 96-well microtitre plate (Thermo Scientific, Rockford, IL) was washed
three times with PBS containing 0.05% Tween-20. Biotinylated Bim peptide (residues 81–106) was
diluted to 0.09
µg/mL (20 nM) in Superblock blocking buffer in PBS (Thermo Scientific), and aliquots
(200 L) were transferred to each well. Following incubation (1.5 h) at room temperature to allow
µ
immobilization of the Bim peptide on to the solid surface (via streptavidin-biotin interaction), the
plate was washed three times with 0.5% BSA in PBS containing 0.05% Tween-20. Test compounds
were dissolved in DMSO to obtain a 50 mM stock solution, and different concentrations of tested
compound (10 fold dilution series from stock solution) were incubated with 20 nM His-tagged Bcl-2
protein (Abcam, Cambridge, UK) in PBS for one hour. The inhibitor and protein mixture (100 µL)
were then transferred to the wells containing bound Bim peptide and incubated at room temperature
for two hours. Following further washing of the plate in triplicate (0.5% BSA in PBS containing
0.05% Tween-20), anti-His antibody containing horseradish peroxidase enzyme (Qiagen, Crawley,
UK) was diluted in 0.5% BSA in PBS (1:1000), and aliquots of 100
incubation (one hour) at room temperature, the plate was washed three times as previously to remove
any unbound antibody. A 200 M solution of o-phenylenediamine (Sigma-Aldrich, UK) was freshly
prepared at pH5 using phosphate-citrate buffer (Sigma-Aldrich), and hydrogen peroxide was added to
the solution to give a final concentration of 0.004 %. Aliquots (200 L) of the prepared solution were
µL were added to each well. After
µ
µ
added to the wells and the plate incubated in the dark for 30 minutes at room temperature. The optical
density was then read using a plate reader at 450 nm. The experiments were carried on three separate
occasions, including both negative and positive controls, where the negative control contains all the
components except the Bcl-2 protein, whereas the positive control contains all the components except
the inhibitors.
The reduction of affinity of the Bim peptide for Bcl-2 was calculated as:
% Reduction in Bim affinity = l450 of treated wells (with inhibitor)/l450 of positive control × 100
A plot of log µM concentration for each inhibitor against the percentage reduction in affinity for
the Bim peptide was created using non-linear regression curve analysis (GraphPad Prism 5), and the
software used to generate the IC50 value for the Bcl-2 inhibition.

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4.10. Molecular Modeling and Docking
All molecular modeling studies were performed on a RM Innovator Pentium IV (2.4 GHz)
running Linux Fedora Core 3. The protein crystal structure of Bcl-2 was downloaded from (http:
//www.rcsb.org/pdb code: 4AQ3). Hydrogen atoms were added to the protein, using the protonate
3D option in MOE (Molecular Operating Environment). Ligand structures were built within MOE
and energy minimized using the MMFF94x force field until a RMSD gradient of 0.05 Kcal mol-1 A◦-1
was reached. The defined pocket was taken as the active site; Alpha triangle was chosen as the
replacement methodology, London dG as the scoring function and ten conformations were retained
for each compound.
Author Contributions: Conceptualization, R.H., N.I.Z. and A.D.W.; methodology, R.H. and S.A.E.; writing, R.H.
and A.D.W.; supervision, A.T.J. and A.D.W.; funding acquisition, R.H., S.A.E., N.I.Z. and A.D.W.
Funding: This research was funded by the Egyptian Government and Cardiff University, through a Channel
Scholarship award (to RH and SAE) and a PhD studentship (to NIZ).
Acknowledgments: We thank the National Mass Spectrometry Facility at Swansea University (Wales, UK) for
provision of accurate mass data as a service.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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