Synthesis, biological evaluation and molecular docking studies of 6-Aryl-2-styrylquinazolin-4(3H)-ones

Article abstract of DOI:10.3390/molecules21010028

Suzuki-Miyaura cross-coupling of 6-bromo-2-styrylquinazolin-4(3H)-ones with arylboronic acids afforded a series of novel 6-aryl-2-styrylquinazolin-4(3H)-ones. These compounds were evaluated for potential anticancer properties against the human renal (TK-10), melanoma (UACC-62) and breast cancer (MCF-7) cell lines. Their antimicrobial properties were also evaluated against six Gram-positive and four Gram-negative bacteria, as well as two strains of fungi. Molecular docking studies (in silico) were conducted on compounds 5a, b, d and 6a, b, d-f to recognize the hypothetical binding motif of the title compounds within the active site of the dihydrofolate reductase and thymidylate synthase enzymes.

Full text of DOI:10.3390/molecules21010028

Article
Synthesis, Biological Evaluation
and Molecular Docking Studies of
6-Aryl-2-Styrylquinazolin-4(3H)-Ones
Emmanuel Ndubuisi Agbo 1, Tshepiso Jan Makhafola 2, Yee Siew Choong 3,
Malose Jack Mphahlele 1,* and Ponnadurai Ramasami 4
Received: 1 December 2015 ; Accepted: 22 December 2015 ; Published: 25 December 2015
Academic Editor: Derek J. McPhee
1
Department of Chemistry, College of Science, Engineering and Technology, University of South Africa,
P. O. Box 392, Pretoria 0003, South Africa; tagboen@unisa.ac.za
Department of Life and Consumer Sciences, College of Agriculture and Environmental Sciences,
2
University of South Africa, Private Bag X06, Florida 1710, South Africa; makhat@unisa.ac.za
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Minden,
3
Penang, Malaysia; yeesiew@usm.my
Computational Chemistry Group, Department of Chemistry, Faculty of Science, University of Mauritius,
4
Réduit 80837, Mauritius; ramchemi@intnet.mu
*
Correspondence: mphahmj@unisa.ac.za; Tel.: +27-11-670-9301; Fax: +27-12-429-8549
Abstract: Suzuki-Miyaura cross-coupling of 6-bromo-2-styrylquinazolin-4(3H)-ones with arylboronic acids
afforded a series of novel 6-aryl-2-styrylquinazolin-4(3H)-ones. These compounds were evaluated
for potential anticancer properties against the human renal (TK-10), melanoma (UACC-62) and
breast cancer (MCF-7) cell lines. Their antimicrobial properties were also evaluated against six Gram-positive
and four Gram-negative bacteria, as well as two strains of fungi. Molecular docking studies (in silico)
were conducted on compounds 5a, b, d and 6a, b, d–f to recognize the hypothetical binding
motif of the title compounds within the active site of the dihydrofolate reductase and thymidylate
synthase enzymes.
Keywords: 6-bromo-2-styrylquinazolin-4(3H)-ones; Suzuki-Miyaura cross-coupling; 6-aryl-2-
styrylquinazolin-4(3H)-ones; in vitro cytotoxicity; antimicrobial activity; docking studies
1. Introduction
2-Styrylquinazolin-4(3H)-one–based compounds continue to attract considerable attention because
of their interesting biological activity and pharmacological properties as anticancer [1], anticonvulsant [2],
sedative-hypnotic [3,4], antibacterial and antifungal agents [5,6]. A series of 6-substituted 2-styrylquinazolin-
4(3H)-ones, for example, was evaluated for biological activity as antimitotic agents and the compounds were
found to inhibit tubulin polymerization [7]. The presence of a 2-styryl moiety was also found to enhance
the antimicrobial activity of quinazolinone derivatives, viz., the 3-[5-(4-substituted)phenyl-1,3,4-thiadiazole-
2-yl]-2-styrylquinazolin-4(3H)-ones 1a (X = N) [
2-styrylquinazolin-4(3H)-ones 1b (X = CH) [ ], which exhibit significant antibacterial activity (Figure 1).
The analogous (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one ( ) is an effective
antibiotic in vivo against methicillin-resistant Staphylococcus aureus (MRSA) and it inhibits cell wall
biosynthesis by binding to DD-transpeptidases involved in cross-linking of the cell wall [ ]. Extensive
structure activity relationship studies on the 3-substituted 2-styrylquinazolin-4(3H)-ones revealed that
5,6] and the analogous 3-thiazole–substituted
8
2
9
the entire styrylquinazolin-4(3H)-one scaffold was required for inhibition of tubulin polymerization [7]
and for antimicrobial activity [6,10].
Molecules 2016, 21, 28; doi:10.3390/molecules21010028
www.mdpi.com/journal/molecules

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1: X = N (a), CH (b)
2
Figure 1. 3-substituted 2-styrylquinazolin-4(3H)-ones 1 and 2 with antimicrobial activity.
Quinazolinones produce their anticancer activity through inhibition of various enzymes, such
as epidermal growth factor receptor tyrosine kinase, dihydrofolate reductase, folate thymidylate
synthase, tyrosine kinase, aldose reductase, cyclic GMP phosphodiesterase and DNA repairing enzymes [11].
Raltitrexed (Tomudex™), for example, is a quinazolinone derivative with chemotherapeutic properties
known to inhibit dihydrofolate reductase (DHFR) and thymidylate synthase (TS) [12,13], which are
the primary targets for the development of new anticancer agents. Some of the compounds that
inhibit these enzymes have also been found to exhibit antibiotic and antimalarial properties [14].
DHFR and TS are also often used as models in molecular docking studies to predict the hypothetical
protein-ligand binding mode, which plays a significant role in structural based drug design
and structure activity relationship. Our interest in the synthesis and biological property
studies of polycarbo-substituted quinazolin-4-ones prompted us to investigate the possibility
to synthesize novel 2-styrylquinazolinon-4(3H)-ones bearing an aryl substituent at the 6-position
for further studies of anticancer and antimicrobial activity. Our approach to the requisite
6-aryl-2-styrylquinazolin-4(3H)-ones involves palladium catalysed Suzuki-Miyaura cross-coupling
of 6-bromo-2-styrylquinazolin-4(3H)-ones with arylboronic acids. These polycarbo-substituted
quinazolin-4(3H)-ones were evaluated for potential in vitro cytotoxicity against a panel of cancer cell
lines and for in vitro antimicrobial activity against both Gram-positive and Gram-negative bacteria
as well as fungal strains. Computer docking (in silico) studies of the most active compounds were
performed against active sites of DHFR and TS to predict the hypothetical protein-ligand binding
mode of these compounds as well as to compare binding affinity and conformations with the inhibitor
in the crystal structure of DHRF and TS.
2. Results and Discussion
2.1. Chemistry
Several methods have been developed to date for the synthesis of 2-styrylquinazolin-4(3H)-ones
and these include the reaction of anthranilic acid with styrylcarboxylic acids followed by amine
insertion [1], and Knoevenagel condensation of 2-methyl-3-substituted quinazolin-4(3H)-ones with
aromatic aldehydes in the presence of a base or acid [
7,15–17]. Amidation of 2-aminobenzonitrile
with 3-arylacryloyl chlorides, followed by oxidative ring closure under basic conditions also
afforded 2-styrylquinazolin-4(3H)-ones [18]. Kumar et al. recently developed a simple, convenient and
green synthetic protocol involving a one-pot tandem condensation of substituted isatoic
anhydrides, carbo-substituted amines and orthoesters under catalyst- and solvent-free conditions
to afford 3-aryl/heteroaryl-2-styrylquinazolin-4(3H)-ones [19]. We opted for the method that
involves initial acetylation of 2-amino-5-bromobenzamide with acetic anhydride to afford
2-acetamido-5-bromobenzamide (
reflux yielded 6-bromo-2-methylquinazolin-4-one (
of with benzaldehyde derivatives in the presence of acetic acid under reflux afforded the
corresponding 2-(arylethenyl)-6-bromoquinazolin-4(3H)-ones 5a . The structures of compounds
3
), which upon base-promoted cyclization in ethanol under
4) (Scheme 1). Knoevenagel condensation
4
–
d

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5a–d were assigned based on their NMR, IR and mass spectrometric data. Styrylquinazolines can
exist as either E- or Z-isomers according to the orientation of the ethylene linker, with coupling
1
constant values J > 16 Hz or J < 12 Hz for the E or Z-isomers, respectively [19]. The H-NMR
spectra of compounds 3a
or 6.99 ppm (R = Cl, OMe) and in the
J = 16.0 Hz, which indicates that the double bond exists in an E-geometry in agreement with the
literature assignment for the analogous compounds [20 23]. The analogous 3-phenyl/pyridinyl
–
d
reveal the presence of two doublets at
δ ca. 6.91 ppm (R = H, F)
δ
7.91–7.93 ppm region with coupling constant values
–
substituted trans-2-(aryl/hetearyl)vinylquinazolin-4(3H)-ones exhibited significantly reduced emission
intensities in solution at room temperature due to competitive quenching through trans-cis C=C
photo-isomerization [24]. However, these conformational changes were found to be suppressed at 77
K in a frozen glass leading to significantly enhanced emission intensity [24].
3
4 (90%)
5a–d
Reagents and conditions: (i) 5% NaOH (aq), EtOH, reflux, 1 h; (ii) RC6H4CHO, AcOH, reflux, 6 h.
Compound
R
%Yield of 5
5a
5b
5c
4-H
4-F
4-Cl
73
65
68
71
5d
3-OMe
Scheme 1. Synthesis of 6-bromo-2-styrylquinazolin-4(3H)-ones 5a–d.
The 6-bromoquinazolin-4(3H)-ones 5a–d were subjected to the Suzuki-Miyaura cross-coupling
with arylboronic acids in the presence of PdCl2(PPh3)2 as the source of active Pd(0) species and
potassium carbonate as a base in 3:1 dioxane–water (v/v) under reflux for 3 h to afford the
corresponding novel 6-aryl–substituted derivatives 6a–h (Scheme 2).
5a–d
6a–h
Reagents and conditions: (i) ArB(OH)2 (1.2 equiv.), PdCl2(PPh3)2, K2CO3 (3 equiv.), dioxane–H2O
(3:1, v/v), reflux, 3 h.
Compound
R
Ar
%Yield of 6
6a
6b
6c
6d
6e
6f
4-H
4-F
4-Cl
3-OMe
4-H
4-F
-C6H5
-C6H5
-C6H5
61
68
71
75
66
63
65
72
-C6H5
4-FC6H4-
4-FC6H4-
4-FC6H4-
4-FC6H4-
6g
6h
4-Cl
3-OMe
Scheme 2. Suzuki-Miyaura cross-coupling of 5a–d to afford 6a–h.

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The 6-carbo–substituted 2-styrylquinazolin-4(3H)-ones 6a
–h
do not dissolve in most commonly
used deuterated solvents and are only sparingly soluble in DMSO-d6 to enable acquisition of the
1H-NMR spectra within a few minutes. Some of the compounds, e.g., 6c and 6g were found to
precipitate from DMSO-d6 at room temperature and this behaviour compromised the quality of their
13C-NMR spectra. These compounds are easily distinguished from the corresponding precursors by
the increased number of proton signals in the aromatic region of their 1H-NMR spectra. Moreover,
the accurate calculated m/z values represent close fits consistent with the assigned structures. As a
guide to quinazolinone derivatives with potential biological properties, we decided to evaluate the
6-bromo-2-styrylquinazolinones 5a–d and the 6-aryl-2-styrylquinazolin-4(3H)-ones 6a–h for in vitro
cytotoxicity against a panel of cancer cell lines and for antimicrobial activity against the Gram-positive
and Gram-negative bacteria, as well as fungi.
2.2. Biological Properties
2.2.1. In vitro Cytotoxicity Studies of 5a–d and 6a–h
The 6-bromoquinazolinones 5a–d and their derivatives 6a–h were evaluated for their in vitro
cytotoxicity against the human renal (TK-10), melanoma (UACC-62) and breast cancer (MCF-7) cell
lines using the sulforhodamine B stain (SRB) assay [25] to correlate between both structural variations
and cytotoxic activity of the synthesized compounds. The IC50 values of the tested compounds
5a
–
d
and 6a
–
h
against human renal (TK-10), melanoma (UACC-62) and breast cancer (MCF-7) cell
M using parthenolide as reference drug are
lines at concentrations 0.01, 0.1, 1.0, 10.0 and 100
µ
represented in Table 1 (see Supplementary Materials for the corresponding cell viability percentages
and graphs for each compound). Increased potency against the three cell lines was observed for the
6-bromo-2-styrylquinazolin-4(3H)-ones 5a (R = 4-H), 5b (R = 4-F) and 5d (R = 3-OCH3). Moderate
activity was observed for the 2-(4-chlorostyryl) derivative 3c against human renal (IC50 = 22.48
µM)
and melanoma (IC50 = 14.96 M) cell lines with no activity against the breast cancer (MCF-7) cell line.
µ
Replacement of bromine with phenyl ring diminished activity against all the cell lines for majority
of the 6-phenyl-2-styrylquinazolin-4-ones. Compound 6a showed selectivity and moderate activity
against the melanoma (UACC-62) and breast cancer (MCF-7) cell lines with IC50 values of 22.99 µM
and 37.18
µM, respectively. Moderate activity against breast cancer (MCF-7) cell line was also observed
for the 2-(3-methoxystyryl) derivative 6d with IC50 value of 25.23
µM. Replacement of 6-Br with
4-fluorophenyl group, on the other hand, resulted in diminished activity against all the three cell
lines except for the 2-(4-fluorostyryl) derivatives 6f, which showed moderate cytotoxicity against
melanoma (UACC-62) and breast cancer (MCF-7) cell lines with IC50 value of 19.70 µM and 29.26 µM,
respectively. In general, the 6-bromo-2-styrylquinazolinon-4-ones exhibit enhanced inhibitory activity
than the corresponding 6-aryl–substituted derivatives. Literature precedents for analogous compounds
demonstrated that inhibitory activity increases with decreasing size of the hydrophobic substituents at
position 6 [1].
Compounds that have both anticancer and antimicrobial activity have promising therapeutic
potential due to their selective cytotoxicity coupled with the potential to reduce the occurrence
of bacterial and fungal infections in immune-compromised cancer patients [26]. To evaluate the
potential of compounds 5a–d and 6a–h to serve as dual anticancer and antimicrobial agents, we also
evaluated them for in vitro antimicrobial activity against ATCC bacterial and fungal strains representing
pathogenic species of different classes commonly associated with nosocomial infections [26].

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Table 1. IC50 values of the test compounds against the three cancer cell lines.
5a–d, 6a–h
Compound
X
R
IC50 (µM) TK-10
IC50 (µM) UACC-62
IC50 (µM) MCF-7
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
6g
6h
Br
Br
Br
4-H
4-F
4-Cl
3-OMe
4-H
4-F
4-Cl
3-OMe
4-H
4-F
4-Cl
7.72 ˘ 0.08
2.42 ˘ 0.07
22.48 ˘ 0.48
1.12 ˘ 0.05
100 ˘ 0.00
100 ˘ 0.00
100 ˘ 0.00
100 ˘ 0.00
100 ˘ 0.00
100 ˘ 0.00
100 ˘ 0.00
100 ˘ 0.00
1.56 ˘ 0.25
1.98 ˘ 1.89
1.03 ˘ 0.19
14.95 ˘ 2.67
0.62 ˘ 0.04
22.99 ˘ 1.03
42.44 ˘ 8.59
100 ˘ 0.00
2.12 ˘ 0.20
3.47 ˘ 0.013
100 ˘ 0.00
Br
1.80 ˘ 0.30
37.18 ˘ 3.36
48.83 ˘ 4.93
100 ˘ 0.00
-C6H5
-C6H5
-C6H5
-C6H5
4-FC6H4-
4-FC6H4-
4-FC6H4-
4-FC6H4-
44.68 ˘ 13.72
96.31 ˘ 5.22
19.70 ˘ 3.19
100 ˘ 0.00
25.23 ˘ 0.87
77.41 ˘ 26.01
29.26 ˘ 4.99
100 ˘ 0.00
3-OMe
96.31 ˘ 5.22
3.26 ˘ 0.40
77.41 ˘ 26.01
3.68 ˘ 0.01
Parthenolide
2.2.2. In vitro Antimicrobial Activity of 3a–d and 4a–h
The in vitro antimicrobial activities of compounds 3a
–d and 4a–h were tested by determining the
minimal inhibitory concentrations (MIC) using the microplate serial dilution assay [27] with slight
modifications for fungi [28]. A total of ten bacterial pathogens (nine ATCC strains and one CSIR isolate)
and two ATCC fungal pathogens were selected as test organisms in this study. The microorganisms
used in this study are ATCC strains and represent pathogenic species of different classes commonly
associated with nosocomial infections [26]. For both the antibacterial and antifungal assays, the MIC
values were determined visually after 24 h of incubation with the compounds to determine if these
compounds possess either bacteriostatic or bactericidal activity and either fungistatic or fungicidal
activity. The MIC was recorded as the lowest concentration of the compounds at which the bacterial
or fungal growth was inhibited. The test compounds have varying antimicrobial activity in both the
antibacterial and antifungal assays, and the results are presented in Tables 2 and 3.
Table 2. Minimum inhibitory concentration (MIC,
µ
g/mL) of 5a
–
d
and 6a–h against Gram-positive
and Gram-negative bacterial strains
Compound
B. c
E. h
E. f
E. g
E. c
S. a
A. c
E. c
P. a
S. t
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
15.62
7.81
62.5
31.25
15.62
15.62
125
31.25
125
15.62
15.62
62.50
31.25
15.62
15.62
31.25
31.25
31.25
62.50
62.50
62.50
1.56
31.25
31.25
31.25
31.25
15.62
15.62
62.50
125
31.25
125
62.50
>250
6.25
7.81
7.81
7.81
31.25
15.62
15.62
125
15.62
62.50
62.50
>250
31.25
31.25
125
31.25
31.25
>250
62.50
31.25
31.25
62.50
>250
31.25
>250
125
62.50
62.50
125
31.25
62.50
125
31.25
15.62
31.25
62.50
31.25
31.25
62.50
>250
62.50
125
31.25
125
125
62.50
62.50
31.25
31.25
>250
62.50
>250
>250
62.50
1.56
62.50
62.50
31.25
>250
>250
62.50
62.50
62.50
31.25
0.39
31.25
62.5
15.62
7.81
31.25
15.62
15.62
125
125
125
62.50
31.25
31.25
>250
1.56
62.50
>250
125
31.25
1.56
62.50
62.50
>250
6g
6h
31.25
125
6.25
62.50
0.78
125
0.78
Gentamicin 3.12
B. c = Bacillus cereus; E. h = Enterococcus hirae; E. f = Enterococcus faecalis; E. g = Enterococcus gallinarium;
E. c = Enterococcus casseliflavus; S. a = Staphylococcus aureus; A. c = Acinetobacter calcaoceuticals anitratus;
E. c = Escherichia coli; P. a = Pseudomonas aeruginosa; S. t = Salmonella typhi.

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Table 3. Minimum inhibitory concentration (MIC, µg/mL) of 5a–d and 6a–h against fungal strains.
Compound
Candida albicans
Cryptococcus neoformans
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
6g
6h
15.62
31.25
31.25
31.25
62.50
31.25
62.50
31.25
62.50
31.25
62.50
31.25
3.90
15.62
31.25
31.25
31.25
31.25
31.25
31.25
62.50
31.25
62.50
62.50
15.62
1.95
Amphotericin-B
Moderate growth inhibitory activity against all the Gram-positive bacteria was observed for
the 6-bromoquinazolinones 5a 5b and 5c. Substitution of bromine with phenyl or 4-fluorophenyl
group led to loss of activity for compounds 6a Less or no activity was observed against
the Gram-negative bacterial strains for these compounds in analogy with literature observation
for the 3-[5-(4-substituted)phenyl-1,3,4-thiadiazole-2-yl]-2-styrylquinazolin-4(3H)-ones activity [ ].
,
–h.
8
Gram-negative bacterial strains are surrounded by an additional outer membrane layer, which
presumably presents a barrier to penetration or entry of noxious compounds [29]. Both series of
compounds were found to exhibit moderate inhibitory activity against the fungal strains.
In the last part of this investigation, we performed computer docking (in silico) studies on the
most active compounds 5(a, b, d) and 6(a, b, d–f) against active sites of DHFR and TS using raltitrexed
as a standard to predict the hypothetical protein-ligand binding mode of these compounds.
2.2.3. Molecular Docking Studies
Control docking of the co-factor for DHFR and TS showed that docking simulations (Figures 2 and 3
Table 4) were able to reproduce the experimental data where the docked conformations have the
root mean square deviation (RMSD) from the crystal structure of only 0.5 and 1.7 Å, respectively. In
general, the 6-bromoquinazolinones 5a, 5b and 5d have the more favorable binding affinity with
DHRF compared with that of TS. A total of five hydrogen bond interactions between the inhibitor
(N-{4-[(2-amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-l-glutamic acid) with
DHFR were detected in the crystal structure of DHFR (Table 4). This has thus explained the most
favorable binding affinity of the inhibitor with DHFR compared with that of the test compounds. It is
also interesting to note hydrogen bonding was not formed between carbo-substituted derivatives
6a, 6b and 6d–f with DHRF. These carbo-substituted derivatives could interact with DHFR through other
non-bonded in4teractions such as van der Waals and electrostatic interactions, hydrophobic
contacts and aromatic-aromatic stacking interactions. The binding energy for the carbo-substituted
derivatives 6a
5a, 1-3 hydrogen bonding was formed between 5b
,
6b and 6d
–
f
were similar with that of the 6-bromoquinazolinones. Except
5d 6a 6b 6d with TS. The most favorable
,
,
,
,
–f
binding free energy of 4a with TS compared with other compounds might also be due to contribution by other
non-bonded interactions besides hydrogen bonding. Compound 5b exhibits significant binding affinity
towards DHRF and TS, respectively. Taking into consideration of both binding free energy and hydrogen
bonding, the 6-bromoquinazolinones (5a, 5b, 5d) would exhibit significant inhibitory activity against DHFR,
while their 6-aryl–substituted derivatives (6a, 6b, 6d–f) display potential to inhibit TS. The
6-bromoquinazolinones and their substituted derivatives appear to bind at the same binding cavity
with the inhibitor (N-{4-[(2-amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-
l-glutamic acid) of DHRF (Figure 2) and TS (Figure 3) as observed in the crystal structure. In
addition, from the binding affinity, the test compounds could inhibit better for DHRF than TS. It is
noticed that the benzene ring of all the compounds was pointing towards the exit of the binding

Molecules 2016, 21, 28
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cavity in DHFR. As compared with the classical antifolate in the crystal structure of DHFR, its
carboxylic tail was orientated toward the hydrophilic helix formed by Arg28-Thr40. This might
contribute to a better interaction of this antifolate with DHFR, thus explain the more favourable
binding affinity (<
aromatic-aromatic stackings (
´
13 kcal/mol) compared with the test compounds (ca.
´
10 kcal/mol). Favourable
π-
π
and interactions) were observed between the quinazolinone
π-σ
ring of the test compounds with the pyridine ring of the co-factor for DHRF. It is also noticed that
interactions of the quinazolinone ring docked at similar position with the thieno[2,3-d]pyrimidine of
the N-{4-[(2-amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-l-glutamic
acid in the crystal structure of DHFR [12].
(a)
(b)
Figure 2. The binding conformation in human dihydrofolate reductase (DHFR) for the 6-bromoquinazolinones
(a) and their 6-aryl–substituted derivatives (b).
(a)
Figure 3. Cont.

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(b)
Figure 3. The binding conformation in human thymidylate synthase (TS) for the 6-bromoquinazolinones
(a) and their 6-aryl substituted derivatives (b).
The docked conformation of the test compounds was comparable with the inhibitor (Tomudex) of
the crystal of thymidylate synthase (TS) [13]. The benzene ring of all the test compounds was pointing
towards the C-terminus of TS. While the carboxylic tail of Tomudex was pointing towards the exit
of the binding cavity in TS which might not increase the interaction with TS. Therefore, it could be
explained that the binding affinity of Tomudex is comparable with the test compounds. Favorable
aromatic-aromatic stacking (π-π and π-σ interactions) were also observed between the quinazolinone
ring of 6-bromoquinazolinones compounds with the pyrimidine ring of the cofactor (dUMP) for TS.
However, this aromatic-aromatic stacking was not observed for the 6-aryl-quinazolinone derivatives.
Table 4. Molecular docking results of test compounds against human dihydrofolate reductase (DHFR)
and thymidylate synthase (TS).
Dihydrofolate Reductase (DHFR)
Thymidylate Synthase (TS)
No. Hydrogen Bond
& Distance (Å)
Binding Free
Binding Free
Compound
5a
Hydrogen Bond
Energy (kcal/mol)
Energy (kcal/mol)
2
´10.36
´7.71
(i) H(5a)-O(Val115): 2.16
(ii) HO(5a)-O(Ile7): 1.85
0
3
3
(i) HZ1(Lys77)-O(5b): 2.16
(ii) HO(5b)-O(Leu221): 2.19
(i) H(5b)-O(Val115): 2.14
(ii) HH(Tyr121)-O(5b): 2.46
(iii) HO(5b)-O(Ile7): 1.79
´10.33
´8.08
5b
(iii) H(5b)-O(Leu221): 2.17
2
1
(i) H(5d)-O(Val115): 1.90
(ii) HH(Tyr121)-O(5d): 2.47
´10.86
´10.32
´7.66
´9.14
5d
6a
(i) H(Gly222)-O(5d): 1.79
2
(i) HD22(Asn226)-O(6a): 2.48
(ii) H(6a)-O4(UMP): 2.11
0
1
´10.24
´10.78
´9.07
´8.89
6b
6d
0
0
(i) HD22(Asn226)-O(6b): 2.48
2
(i) HD22(Asn226)-O(6d): 2.35
(ii) H(6b)-O4(UMP): 2.11
2
´10.10
´9.08
6e
0
(i) HD22(Asn226)-O(6e): 2.31
(ii) H(6e)-O4(UMP): 1.97

Molecules 2016, 21, 28
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Table 4. Cont.
Dihydrofolate Reductase (DHFR)
No. Hydrogen Bond
Thymidylate Synthase (TS)
Binding Free
Energy (kcal/mol)
Binding Free
Energy (kcal/mol)
Compound
6f
Hydrogen Bond
& Distance (Å)
2
´10.84
´13.09
´9.00
0
(i) HD22(Asn226)-O(6f): 2.35
(ii) H(6f)-O4(UMP): 2.09
3
5
(i) H(Phe80)-OE1(Ctrl): 2.25
(i) HH22(Arg70)-OAG(Ctrl): 1.69
(ii) HZ2(Lys77)- OE1(Ctrl): 2.03
Control
(Ctrl)
´9.07
(ii) HH22(Arg70)-OAD(Ctrl): 2.66
(iii) HH12(Arg70)-OAD(Ctrl): 1.83
(iv) HD21(Asn64)-OAE(Ctrl): 2.27
(v) (Try121) HH-S4(Ctrl): 3.23
(v) H1(Ctrl)-OE2(Glu): 2.03
(iii) H3(Ctrl)-OD2(Asp218): 1.80
3. Materials and Methods
3.1. General Information
Melting points were recorded on a Thermocouple digital melting point apparatus and are
uncorrected. IR spectra were recorded as powders using a Bruker VERTEX 70 FT-IR Spectrometer
(Bruker Optics, Billerica, MA, USA) with a diamond attenuated total reflectance (ATR) accessory by
using the thin-film method. For column chromatography, Kieselgel 60 (0.063–0.200 mm) (Merck KGaA,
Frankfurt, Germany) was used as stationary phase. NMR spectra were obtained as CDCl3 or DMSO-d6
solutions using Mercury 300 MHz (Varian Inc., Palo Alto, CA, USA) or Agilent 500 MHz NMR (Agilent
Technologies, Yarnton, UK) spectrometers and the chemical shifts are quoted relative to the TMS
peak. Low- and high-resolution mass spectra were recorded at an ionization potential of 70 eV using
Synapt G2 Quadrupole 6 Time-of-flight mass spectrometer (Waters Corp., Milford, MA, USA) at the
University of Stellenbosch Mass Spectrometry Unit. The synthesis and analytical data of compound
3
has been described before [30]. The 1H- and 13C-NMR spectra of compounds
4–6 are listed in the
Supplementary Materials.
3.2. Synthesis of 6-Bromo-2-Methylquinazolin-4(3H)-One (4)
A stirred mixture of 2-acetamido-5-bromobenzamide
3 (0.30 g, 1.16 mmol) and 5% NaOH (20 mL)
in ethanol (10 mL) was refluxed for 1 h and then allowed to cool to 0 ˝C. The mixture was acidified
dropwise with concentrated hydrochloric acid. The resulting precipitate was filtered on a sintered
funnel, washed sequentially with cold water and an ice-cold ethanol and then dried in an oven to
afford compo´u1nd (
4
) as a white solid (0.25 g, 89%), mp. 299–302 ˝C;
ν
(ATR) 495, 777, 898, 1302,
max
1469, 1641 cm
; δH (500 MHz, DMSO-d6) 2.33 (3H. s, -CH3), 7.51 (1H, d, J = 9.0 Hz, 8-H), 7.90 (1H, dd,
J = 2.0 and 9.0 Hz, 7-H), 8.12 (1H, d, J = 2.0 Hz, 5-H), 12.38 (1H, s, NH); δC (125 MHz, DMSO-d6) 21.9,
118.6, 122.7, 128.2, 129.5, 137.5, 148.4, 155.5, 161; m/z 239 (100, MH+); HRMS (ES): MH+ found 238.9820.
C9H8N2O79Br+ requires 238.9812.
3.3. Typical Procedure for the Synthesis of the 6-Bromo-2-(styryl)Quinazolin-4(3H)-Ones 5a–d
A stirred mixture of
4 (1 equiv.) and benzaldehyde derivative (1.2 equiv.) in acetic acid
(20 mL/mmol of ) was refluxed for 6 h. The mixture was allowed to cool and then quenched
4
with an ice-cold water. The resultant precipitate was filtered and washed with methanol to afford
compound 5. The following compounds were prepared in this fashion:
˝
(E)-6-Bromo-2-(phenylstyryl) quinazolin-4(3H)-one (5a). Solid (0.30 g, 74%), mp. 328–330 C (Lit. [7]
˝
334–338 C);
ν
(ATR) 534, 827, 967, 1308, 1463, 1667, 3174 cm´1
; δH (300 MHz, DMSO-d6) 6.98
max
(1H, d, Jtrans = 16.0 Hz, Ha), 7.40–7.48 (3H, m, ArH), 7.61 (1H, d, J = 8.7 Hz, 8-H), 7.64 (2H, dd, J = 1.8
and 8.4 Hz, ArH), 7.92 (1H, dd, J = 2.5 and 8.7 Hz, 7-H), 7.95 (1H, d, Jtrans = 16.0 Hz, Hb), 8.16 (1H, d,
J = 2.5 Hz, 5-H), 12.49 (1H, s, NH); δC (75 MHz, DMSO-d6) 119.0, 121.2, 123.2, 128.1, 128.4, 129.5, 129.9,

Molecules 2016, 21, 28
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130.4, 135.3, 137.8, 139.3, 148.5, 152.4, 161.1; m/z 329 (100, MH+); HRMS (ES): MH+, found 329.0130.
C16H12N2O79Br+ requires 329.0133.
˝
(E)-6-Bromo-2-(4-fluorostyryl)quinazolin-4(3H)-one (5b). Solid (0.34 g, 78.3%), mp. 332–334 C;
νmax (ATR)
506, 643, 817, 967, 1233, 1463, 1673, 3174 cm´1
;
δH (500 MHz, DMSO-d6) 6.91 (1H, d, Jtrans = 16.0 Hz
,
Ha), 7.28 (2H, t, J = 8.5 Hz, 31,51-H), 7.58 (1H, d, J = 8.0 Hz, 8-H), 7.71 (2H, t, J = 8.5 Hz, 21,61-H), 7.90
(1H, dd, J = 2.5 and 8.5 Hz, 7-H), 7.93 (1H, d, Jtrans = 16.0 Hz, Hb), 8.14 (1H, d, J = 2.5 Hz, 5-H), 12.48
(1H, s, NH); δC (125 MHz, DMSO-d6) 116.6 (d, 2JCF = 21.8 Hz), 119.0, 121.1, 123.1, 128.4, 129.9, 130.4 (d,
3JCF = 8.5 Hz), 131.9 (d, 4JCF = 3.1 Hz), 137.8, 138.1, 148.4, 152.4, 161.0, 163.4 (d, 1JCF = 246.5 Hz); m/z
345 (100, MH+); HRMS (ES): MH+, found 345.0036. C16H11N2O79BrF+ requires 345.0039.
˝
˝
(E)-6-Bromo-2-(4-chlorostyryl)quinazolin-4(3H)-one (5c). Solid (0.34 g, 76%) mp. > 345 C (Lit. [31] > 280 C);
ν
(ATR) 492, 624, 808, 831, 967, 1088, 1306, 1463, 1577, 16451, 1671, 3173 cm´1
; δH (500 MHz,
max
1
DMSO-d6) 6.98 (1H, d, Jtrans = 16.0 Hz, Ha), 7.51 (2H, d, J = 8.5 Hz, 3 ,5 -H), 7.60 (1H, d, J = 8.5 Hz, 8-H),
7.66, (2H, d, J = 8.5 Hz, 21,61-H), 7.91 (1H, dd, J = 2.5 and 8.5 Hz, 7-H), 7.92 (1H, d, J = 16.0 Hz, Hb),
8.16 (1H, d, J = 2.5 Hz, 5-H), 12.50 (1H, s, NH); δC (125 MHz, DMSO-d6) 119, 122.1,123.1,128.4, 129.6,
129.8, 129.9, 134.3, 134.7, 137.9, 137.8, 148.3, 152.4, 161.1; m/z 361 (100, MH+); HRMS (ES): MH+, found
360.9746. C16H11N2O35Cl79Br+ requires 360.9743.
˝
(E)-6-Bromo-2-(3-methoxystyryl)quinazolin-4(3H)-one (5d). Solid (0.32 g, 71%), mp. 287–288 C (Lit. [31
]
˝
´1
268–270 C);
ν
(ATR) 535, 775, 890, 976, 1464, 1586, 1676, 3173 cm
;
δH (500 MHz, DMSO-d6) 3.80
max
(3H, s, -OCH3), 6.98 (1H, dd, J = 2.5 and 7.5 Hz, 41-H), 6.99 (1H, d, Jtrans = 16.5 Hz, Ha), 7.20 (1H, d,
J = 2.5 Hz, 21-H), 7.22 (1H, d, J = 7.5 Hz, 61-H), 7.36 (1H, t, J = 8.5 Hz, 51-H), 7.59 (1H, d, J = 8.5 Hz
,
8-H), 7.91 (1H, d, Jtrans = 16.0 Hz, Hb), 7.93 (1H, dd, J = 2.5 and 8.5 Hz, 7-H), 8.16 (1H, d, J = 2.5 Hz,
5-H), 12.47 (1H, s, NH); δC (125 MHz, DMSO-d6) 55.6, 113.1, 116.2, 119.0, 120.5, 121.6, 128.4, 129.9,
130.6, 136.7, 137.8, 139.2, 148.4, 152.4, 160.1 (2xC), 161.0; m/z 357 (100, MH+); HRMS (ES): MH+, found
357.0235. C17H14N2O279Br+ requires 357.0239.
3.4. Typical Procedure for the Suzuki-Miyaura Cross-Coupling of 5a–d with Arylboronic Acids
(E)-6-Phenyl-2-styrylquinazolin-4(3H)-one (6a). A stirred mixture of 5a (0.20 g, 0.61 mmol), PdCl2(PPh3)2
(0.02 g, 0.03 mmol), PCy3 (0.017 g, 0.06 mmol) and K2CO3 (0.10 g, 0.73 mmol) in 3:1 dioxane–water
(v/v; 10 mL) was purged with argon gas for 30 minutes. Phenylboronic acid (0.11 g, 0.91 mmol) was
added to the mixture using a syringe. The reaction mixture was heated at 100 ˝C for 2 h and then
quenched with an ice-cold water. The product was extracted into chloroform and the combined organic
layers were washed with water, dried over Na2SO4 and filtered dry on a sintered funnel to afford 6a
as a yellow solid (0.12 g, 61%), mp. 329 ˝C;
cm´1
δH (500 MHz, DMSO-d6) 7.01 (1H, d, Jtrans = 16.0 Hz, Ha), 7.40–7.52 (6H, m, ArH), 7.66 (2H, d,
νmax (ATR) 491, 695, 753, 794, 966, 1259, 1473, 1579, 1655
;
J = 7.5 Hz, ArH), 7.76 (3H, t, J = 7.5 Hz, ArH), 7.96 (1H, d, Jtrans = 16.0 Hz, Hb), 8.13 (1H, dd, J = 2.5
and 7.5 Hz, 7-H), 8.31 (1H, d, J = 2.5 Hz, 5-H), 12.41 (1H, s, NH); δC (125 MHz, DMSO-d6) 121.5, 121.9,
123.6, 127.2 (2xC), 128.1, 128.4, 128.6, 128.7, 130.3, 133.5, 135.4, 138.3, 138.9, 139.3, 148.8, 151.9, 162.3;
m/z 325 (100, MH+); HRMS (E) (ES): MH+ found 325.1341. C22H17N2O+ requires 325.1339.
˝
(E)-2-(4-Fluorostyryl)-6-phenylquinazolin-4(3H)-one (6b). Solid (0.13 g, 66%), mp. 345 C;
ν
(ATR) 499,
max
698, 761, 824, 968, 1157, 1552, 1660 cm´1
(2H, t, J = 9.0 Hz, 21,61-H), 7.43 (1H, d, J = 7.5 Hz, 8-H), 7.52 (2H, t, J = 9.0 Hz, 31,51-H), 7.71–7.80 (5H,
m, Ph), 7.96 (1H, d, Jtrans = 16.0 Hz, Hb), 8.13 (1H, dd, J = 2.0 and 9.0 Hz, 7-H), 8.33 (1H, d, J = 2.0 Hz
5-H), 12.39 (1H, s, NH); δC (75 MHz, DMSO-d6) 116.6 (d, 2JCF = 21.9 Hz), 118,6, 119.0, 121.5, 123.1,
; δH (300 MHz, CDCl3) 7.00 (1H, d, Jtrans = 16.0 Hz, Ha), 7.31
,
3
4
128.2, 128.4, 129.5, 130.4 (d, JCF = 8.5 Hz), 132.0 (d, JCF = 3.2 Hz), 137.6, 137.8, 138.1, 148.4, 152.4,
161.1, 163.4 (d, 1JCF = 246.6 Hz); m/z 343 (100, MH+); HRMS (ES): MH+ found 343.1239. C22H16N2OF+
requires 343.1247.

Molecules 2016, 21, 28
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˝
(E)-2-(4-Chlorostyryl)-6-phenylquinazolin-4(3H)-one (6c). Solid (0.22 g, 71%), mp. 345 C;
ν
(ATR) 490,
max
697, 764, 793, 969, 1652 cm´1
;
δH (500 MHz, DMSO-d6) 7.01 (1H, d, Jtrans = 16.0 Hz, Ha), 7.40 (1H, d,
J = 7.5 Hz, 8-H), 7.48–7.520 (4H, m, ArH), 7.67 (2H, d, J = 8.0 Hz, 31,51-H), 7.74 (1H, d, J 7.5 Hz, 8-H),
7.75 (2H, d, J = 8.5 Hz, 2 ,6 -H), 7.93 (1H, d, Jtrans = 16.5 Hz, Hb), 8.12 (1H, dd, J = 2.0 and 7.5 Hz, 7-H),
8.32 (1H, d, J = 2.0 Hz, 5-H), 12.41 (1H, s, NH); m/z 359 (100, MH+); HRMS (E)(ES): MH+ found 359.094.
C22H16N2O35Cl+ requires 359.095.
1
1
(E)-2-(3-Methoxystyryl)-6-phenylquinazolin-4(3H)-one (6d). Solid (0.23 g, 75%), mp. 268 ˝C;
529, 827, 871, 1156, 1226, 1310, 1474, 1582, 1661, 3176 cm´1
δH (500 MHz, DMSO-d6) 3.80 (3H, s,
-OCH3), 6.98 (1H, dd, J = 1.5 and 7.5 Hz, 6 -H), 7.02 (1H, d, Jtrans = 16.0 Hz, Ha), 7.22 (1H, d, J = 1.5 Hz,
νmax (ATR)
;
'
1
1
2'-H), 7.23 (1H, d, J = 7.5 Hz, 4 -H), 7.36 (1H, t, J = 9.0 Hz, 5 -H), 7.41 (1H, d, J = 7.5 Hz, 8-H), 7.50 (2H, t,
J = 9.0 Hz, ArH), 7.73–7.77 (3H, m, ArH), 7.93 (1H, d, Jtrans = 16.0 Hz, Hb), 8.11 (1H, dd, J = 2.0 and
7.5 Hz, 7-H), 8.31 (1H, d, J = 2.0 Hz, 5-H), 12.38 (1H, s, NH); δC (125 MHz, DMSO-d6) 55.6, 113.1, 116.1,
120.5, 121.8, 121.9, 123.6, 127.2 (2xC), 128.3, 129.6, 130.5, 133.4, 136.9, 138.3, 138.7, 139.3, 148.7, 151.8,
160.2, 162.1; m/z 355 (100, MH+); HRMS (ES): MH+ found 355.1435. C23H19N2O2+ requires 355.1447.
˝
(E)-6-(4-Fluorophenyl)-2-styrylquinazolin-4(3H)-one (6e). Solid (0.20 g, 66%), mp. 343 C;
ν
(ATR) 490,
max
528, 696, 755, 968, 1156, 1226, 1478, 1661 cm´1
; δH (500 MHz, DMSO-d6) 7.01 (1H, d, Jtrans = 16.0 Hz,
Ha), 7.33 (2H, t, J 7.5 Hz, 31,51-H), 7.40–7.447 (3H, m, ArH), 7.65 (2H, d, J = 7.8 Hz, ArH), 7.73 (1H,
d, J = 7.8 Hz, 8-H), 7.82 (2H, t, J = 8.5 Hz, 21,61-H), 7.95 (1H, d, Jtrans = 16.0 Hz, Hb), 8.10 (1H, dd,
J = 2.5 and 7.8 Hz, 7-H), 8.28 (1H, d, J = 2.5 Hz, 5-H), 12.40 (1H, s, NH); δC (125 MHz, DMSO-d6) 116.6
2
3
(d, JCF = 21.75 Hz), 121.1, 121.4, 128.6, 128.8 (d, JCF = 8.0 Hz), 129.0, 129.7, 131.3, 131.4, 131.9 (d,
4JCF = 2.8 Hz), 132.8, 134.9, 135.3, 136.7, 138.3, 151.5, 161.7, 162.0 (d, 1JCF = 243.6 Hz); m/z 343 (100,
MH+); HRMS (ES): MH+. found 343.1247. C22H16N2OF+ requires 343.1238.
˝
(E)-6-(4-Fluorophenyl)-2-(4-fluorostyryl)quinazolin-4(3H)-one (6f). Solid (0.20 g, 63%), mp. 345 C;
(ATR) 508, 551, 823, 968, 1157, 1237, 1476, 1659, 3174 cm´1
δH (500 MHz, DMSO-d6) 6.96
(1H, d, Jtrans = 16.0 Hz, Ha), 7.29 (2H, t, J = 9.0 Hz, 31,51-H), 7.32 (2H, t, J = 9.0 Hz, 3”,5”-H), 7.72
(2H, t, J = 9.0 Hz, 2”,6”-H), 7.73 (1H, d, J 8.5 Hz, 8-H), 7.81 (2H, t, J = 9.0 Hz, 2 ,6 -H), 7.94 (1H, d,
trans = 16.0 Hz, Hb), 8.10 (1H, dd, J = 2.0 and 8.5 Hz, 7-H), 8.29 (1H, d, J = 2.0 Hz, 5-H), 12.40 (1H,
ν
;
max
'
'
J
2
br s, NH); δC (125 MHz, DMSO-d6) 116.4 (d, JCF = 21.7 Hz), 121.9, 122.3, 123.6, 128.3, 128.7, 129.3
(d, JCF = 8.5 Hz),129.6, 129.7, 133.5, 134.4, 134.6, 135.7 (d, JCF = 2.8 Hz), 137.3, 137.4, 152.6, 161.6,
163.1 (d, JCF = 243.0 Hz); m/z 361 (100, MH+); HRMS (ES): MH+ found 361.1143. C22H15N2OF2
3
4
1
+
requires 361.1152.
(E)-2-(4-Chlorophenyl)-6-(4-fluorostyryl)quinazolin-4(3H)-one (6g). Solid (0.20 g, 65%), mp. 345 ˝C;
ν
max
(ATR) 494, 523, 811, 967. 1092, 1241, 1474, 1490, 1518, 1665, 3173 cm´1
; δH (500 MHz, DMSO-d6)
7.01 (1H, d, Jtrans = 16.0 Hz, Ha), 7.32 (2H, d, J = 9.0 Hz, 3”,5”-H-), 7.51 (2H, t, J 8.5 Hz, 3,5-H), 7.68
(2H, d, J = 8.5 Hz, 2”,6”-H), 7.73 (1H, d, J = 8.5 Hz, 8-H), 7.81 (2H, d, J = 9.0 Hz, 2,6-H), 7.93 (1H, d,
J
trans = 16.0 Hz, Hb), 8.10 (1H, dd, J = 2.0 and 8.5 Hz, 7-H), 8.29 (1H, d, J = 2.0 Hz, 5-H), 12.4 (1H, s,
NH); m/z 377 (100, MH+); HRMS (ES): MH+ found 377.0852. C22H15N2O35ClF requires 377.0857.
˝
(E)-6-(4-Fluorophenyl)-2-(3-methoxystyryl)quinazolin-4(3H)-one (6h). Solid (0.22 g, 72%), mp. 315 C;
(ATR) 493, 672, 762, 841, 1249, 1433, 1669 cm
ν
max
´1
; δH (500 MHz, DMSO-d6) 3.80 (3H, s, -OCH3), 6.98 (1H,
dd, J 2.0 and 9.0 Hz, 61-H), 7.02 (1H, d, Jtrans = 16.0 Hz, Ha), 7.21 (1H, d, J = 2.0 Hz, 21-H), 7.22 (1H, d,
J = 7.5 Hz, 41-H), 7.32 (2H, t, J = 7.8 Hz, 3,5-H), 7.36 (1H, t, J = 7.5 Hz, 51-H), 7.73 (1H, d, J = 8.5 Hz,
8-H), 7.81 (2H, t, J = 7.5 Hz, 2,6-H), 7.92 (1H, d, Jtrans = 16.0 Hz, Hb), 8.10 (1H, dd, J = 2.0 and 8.5 Hz,
7-H), 8.29 (1H, d, J = 2.0 Hz, 5-H), 12.37 (1H, s, NH); δC (125 MHz, DMSO-d6) 55.6, 113.1, 116.1,
116.4 (d, 2JCF = 20.9 Hz), 120.5, 121.8, 121.9, 123.6, 128.3, 129.3 (d, 3JCF = 7.5 Hz), 130.6, 133.4, 135.3 (d,
4JCF = 2.9 Hz), 136.7, 137.3, 137.8, 148.7, 151.9, 160.1, 162.1, 162.6 (d, 1JCF = 243.7 Hz); m/z 373 (100,
MH+); HRMS (ES): MH+ found 373.1346. C23H18N2O2F+ requires 373.1352.

Molecules 2016, 21, 28
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3.5. Materials and Methods for Bioassays
Cytotoxicity studies [25]: The SRB Assay was performed at the Council for Scientific Research
(CSIR, Pretoria, South Africa) in accordance with the protocol of the Drug Evaluation Branch of
the National Cancer Institute (NCI, Rockville, MD, USA) and the assay has been adopted for this
screening. The human cell lines TK-10, UACC-62 and MCF-7 were obtained from NCI in the framework
a collaborative research program between CSIR and NCI. Cell lines were routinely maintained as
˝
monolayer cell cultures at 37 C, 5% CO2, 95% air and 100% relative humidity in RPMI containing 5%
fetal bovine serum, 2 mM L-glutamine and 50 g/mL gentamicin. For screening experiment, the cells
µ
(3–19 passages) were inoculated in a 96-well microtiter plates at plating densities of 7–10,000 cells/well
and were incubated for 24 h. After 24 h the cells were treated with the experimental drugs which were
previously dissolved in DMSO and diluted in medium to produce 5 concentrations: 100–0.01
10-fold serial dilutions). Cells without drug addition served as control and the blank contained
complete medium without cells with parthenolide as a standard drug at concentrations: 100–0.01
(5 10-fold serial dilutions). The plates were incubated for 48 h after addition of the compounds.
µM (5
ˆ
µM
ˆ
Viable cells were fixed to the bottom of each well with cold 50% trichloroacetic acid, washed, dried
and dyed by SRB. Unbound dye was removed and protein-bound dye was extracted with 10 mM Tris
base for optical density determination at the wavelength 540 nm using a multi-well spectrophotometer.
Data analysis was performed using GraphPad Prism software. 50% of cell growth inhibition (IC50)
was determined by linear regression.
In vitro antibacterial microdilution assay: The microplate serial dilution assay developed by
Eloff [27] was used to determine the minimal inhibitory concentration of the compounds against
six Gram-positive and four Gram-negative bacterial strains (Table 5). The microbial test organisms
were prepared using the method described by Pauw and Eloff [26]. The bacterial cultures were
maintained on Müller-Hilton (MH) agar at 4 ˝C and in Müller-Hilton broth at 37 ˝C prior to use for
the determination of minimal inhibitory concentrations. Three to five colonies of bacteria from fresh 24
h agar plate culture were inoculated into fresh MH broth and incubated for 14 h prior to antibacterial
testing. This experiment was performed in triplicate and repeated twice for verification. Each test
compound was dissolved in the appropriate solvent (DMSO or acetone) to a concentration of 1 mg/mL
and serially diluted two-fold in a 96-well microplate with equal volumes of water for each of the 10
bacteria used. 100 µL of freshly prepared bacterial cultures of each bacterium were added to each well.
DMSO was used as negative control and gentamicin as the positive control to confirm the sensitivity
of the system. The plates were then incubated overnight at 37 ˝C in 100% relative humidity. As an
indicator of bacterial growth, 40 µL of 0.2 mg/mL INT was added to each well after incubation and
the plates incubated further at 37 ˝C for 2 h. The MIC was determined and recorded as the lowest
concentration of the compounds at which the bacterial growth was inhibited.
Table 5. Bacterial test organisms used in this study.
Bacteria
Strain
Escherichia coli
Pseudomonas aeruginosa
Salmonella typhi
ATCC 35218, Gram-negative
ATCC 7700, Gram-negative
ATCC 14028, Gram-negative
CSIR isolate, Gram-negative
ATCC 10702, Gram-positive
ATCC 8043, Gram-positive
ATCC 19433, Gram-positive
ATCC 49573, Gram-positive
ATCC 25788, Gram-positive
ATCC 25925, Gram-positive
Acinetobacter calcaoceuticals anitratus
Bacillus cereus
Enterococcus hirae
Enterococcus faecalis
Enterococcus gallinarium
Enterococcus casseliflavus
Staphylococcus aureus
In vitro antifungal microdilution assay: For the antifungal assay, the microplate serial dilution assay
developed by Eloff [26] and modified by Masoko et al. [28] was used. The fungal strains (Table 6)
were maintained on Sabouraud dextrose agar and Sabouraud dextrose broth prior to inhibition tests.

Molecules 2016, 21, 28
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Two-fold serial dilutions of the compounds were prepared as described for the bacteria. Using sterile
cotton swabs, actively growing fungal organisms were transferred from agar to Sabouraud dextrose
broth and 100
previous section. DMSO was used as negative control and Amphotericin-B as the positive control. As
an indicator of fungal growth, 40 L of 0.2 mg/mL INT was added to each well and the plates were
µL of the cultures was then added to each well as described for the bacteria in the
µ
˝
incubated at 30 C after which the MIC was determined after 24 h incubation.
Table 6. Fungal test organisms used in this study.
Fungi
Strain
Candida albicans
Cryptococcus neoformans
ATCC 24433, yeast
ATCC 14116, yeast
3.6. Molecular Docking Studies
Protein Structure: The starting structures of human dihydrofolate reductase (DHFR) and
thymidylate synthase (TS) enzyme were obtained from PDB with id 3GHW201 and 1I00 [12],
respectively. All water molecules and inhibitors were removed except the co-factor NADPH in
DHFR and dUMP in TS remained as part of the protein. Polar hydrogen atoms, Kollman-Amber
united atom partial charges and solvation parameters were added by utilizing AutoDockTools [13].
Compound Structure: The initial coordinates for compounds 5a, b, d, and 6a, b, d–f were
generated using Hyperchem 7.0 (Hypercube Inc., Gainesville, FL, USA). The initial coordinates of
inhibitor for DHFR (N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-L-
glutamic acid) and TS (Tomudex) were obtained from the ligand of PDB id 3GHW and 100, respectively, and
used as control docking. All compounds/inhibitors were retained with only polar hydrogen atoms.
Gasteiger charges and torsional angles were added by utilizing AutoDockTools.
Molecular Docking Simulation: Grid maps of 60
ˆ
60
ˆ
60 points with 0.375 Å spacing were centered
at the ligand binding site in the crystal structures. The docking was performed employing Lamarckian
genetic algorithm with pseudo-Solis and Wets local search with population size of 150 and energy
evaluation of 2500,000; root mean square tolerance of 1.0 Å for control docking and 2.0 Å for the test
compounds; and 200 docking runs by AutoDock 4.2 [32]. The ligand conformation with lowest free
energy of binding in the most populated cluster was selected for comparison.
4. Conclusions
The 6-bromo-2-styrylquinazolin-4(3H)-ones are suitable candidates for palladium-catalyzed
Suzuki-Miyaura cross-coupling to afford 6-aryl–substituted 2-styrylquinazolinones with potential
biological activity. In general, the 6-bromo-2-styrylquinazolinon-4-ones were found to exhibit more
significant inhibitory activity than the corresponding 6-aryl–substituted derivatives. However,
the presence of a 4-chlorostyryl group at the 2-positions of the 6-bromoquinazolin-4(3H)-one
framework is not favourable for cytotoxicity. A combination of a substituted benzene ring at
the 2- and 6-position resulted in diminished activity against the cancer cell lines, as observed for
6-(4-fluorophenyl)–substituted derivatives 6g and 6h bearing 4-chlorostyryl and 3-methoxystyryl
groups, respectively. Some of the compounds with high cytotoxicity against cancer cells (i.e., 5a–d,
6a and 6b) were also found to exhibit good antibacterial and antifungal activity. Overall, the test
compounds had moderate bacteriostatic and fungistatic activity. The observed results serve as a
guide for the design and synthesis of more potent styrylquinazolinones with dual anticancer and
antimicrobial activity to inhibit bacterial and fungal infections in frequently immune compromised
cancer patients. Structure-activity relationship and molecular docking indicate that the entire
2-styrylquinazolinone framework is required for biological activity, however, activity is enhanced by a
halide or small lipophilic group at the 6-position of the scaffold.

Molecules 2016, 21, 28
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Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/1/28/s1. Copies of 1H- and 13C-NMR spectra of
a– and 4a (S1) and the % cell viability and IC50 values
of parthenolide and compounds 3a–d and 4a–h (S2).
2,
3
d
–h
Acknowledgments: The authors are grateful to the University of South Africa and the National Research
Foundation for financial assistance. We also thank N. Kolesnikova of the CSIR (SA) for in vitro cytotoxicity assays.
Author Contributions: The synthesis and spectrometric analyses were performed by E.N.A. as part of an MSc
research project under the supervision of M.J.M. who is the lead author. T.J.M. was responsible for the antimicrobial
activity screening and also contributed in the discussion of the corresponding results. Y.S.C. performed molecular
docking and together with P.R. contributed in the discussion of the corresponding data.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3–6 are available from the authors.
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2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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