Synthesis, biological evaluation and docking studies of benzoxazoles derived from thymoquinone

Article abstract of DOI:10.3390/molecules23123297

Thymoquinone (TQ), a natural compound with antimicrobial and antitumor activity, was used as the starting molecule for the preparation of 3-aminothymoquinone (ATQ) from which ten novel benzoxazole derivatives were prepared and characterized by elemental analysis, IR spectroscopy, mass spectrometry and NMR (¹H,¹³C) spectroscopy in solution. The crystal structure of 4-methyl-2-phenyl-7-isopropyl-1,3-benzoxazole-5-ol (1a) has been determined by X-ray diffraction. All compounds were tested for their antibacterial, antifungal and antitumor activities. TQ and ATQ showed better antibacterial activity against tested Gram-positive and Gram-negative bacterial strains than benzoxazoles. ATQ had the most potent antifungal effect against Candida albicans, Saccharomyces cerevisiae and Aspergillus brasiliensis. Three benzoxazole derivatives and ATQ showed the highest antitumor activities. The most potent was 2-(4-fluorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1f). Western blot analyses have shown that this compound inhibited phosphorylation of protein kinase B (Akt) and Insulin-like Growth Factor-1 Receptor (IGF1R β) in HeLa and HepG2 cells. The least toxic compound against normal fibroblast cells, which maintains similar antitumor activities as TQ, was 2-(4-chlorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1e). Docking studies indicated that 1e and 1f have significant effects against selected receptors playing important roles in tumour survival.

Full text of DOI:10.3390/molecules23123297

molecules
Article
Synthesis, Biological Evaluation and Docking Studies
of Benzoxazoles Derived from Thymoquinone
3
3
Una Glamocˇlija 1,2,3,
*
, Subhash Padhye 4, Selma Špirtovic´-Halilovic´ , Amar Osmanovic´ ,
Elma Veljovic´ , Suncˇica Roca 5, Irena Novakovic´ , Boris Mandic´ , Iztok Turel 8, Jakob Kljun 8,
3
6
7
7
9
10
Snežana Trifunovic´ , Emira Kahrovic´
, Sandra Kraljevic´ Pavelic´
, Anja Harej 10
,
Marko Klobucˇar 10 and Davorka Završnik 3
1
Scientific Research Department, Bosnalijek JSC, Jukic´eva 53, 71000 Sarajevo, Bosnia and Herzegovina
School of Medicine, University of Mostar, Bijeli Brijeg bb, 88000 Mostar, Bosnia and Herzegovina
Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8, 71000 Sarajevo, Bosnia and Herzegovina;
selmaspirtovic@yahoo.com (S.Š.-H.) amar.osmanovic@ffsa.unsa.ba (A.O.); elma.veljovic@ffsa.unsa.ba (E.V.);
davorka.zavrsnik@ffsa.unsa.ba (D.Z.)
Interdisciplinary Science and Technology Research Academy, University of Pune, 2390-B,
Hidayatullah Road, 411001 Pune, India; subhashpadhye@hotmail.com
NMR Centre, Ruđer Boškovic´ Institute, Bijenicˇka cesta 54, 10000 Zagreb, Croatia; Suncica.Roca@irb.hr
ICTM, Center for Chemistry, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia;
irenan@chem.bg.ac.rs
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia;
borism@chem.bg.ac.rs (B.M.); snezanat@chem.bg.ac.rs (S.T.)
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecˇna pot 113, 1000 Ljubljana,
Slovenia; iztok.turel@fkkt.uni-lj.si (I.T.); jakob.kljun@fkkt.uni-lj.si (J.K.)
Department of Chemistry, Faculty of Science, University of Sarajevo, Zmaja od Bosne 35, 71000 Sarajevo,
Bosnia and Herzegovina; emira_kahrovic@yahoo.com
Centre for High-throughput Technologies, Department of Biotechnology, University of Rijeka,
Radmile Matejcˇic´ 2, 51000 Rijeka, Croatia; sandrakp@biotech.uniri.hr (S.K.P.); aharej@biotech.uniri.hr (A.H.);
mklobucar@biotech.uniri.hr (M.K.)
2
3
4
5
6
7
8
9
10
*
Correspondence: una.glamoclija@gmail.com; Tel.: +387-61-147-401
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 15 November 2018; Accepted: 2 December 2018; Published: 12 December 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Thymoquinone (TQ), a natural compound with antimicrobial and antitumor activity,
was used as the starting molecule for the preparation of 3-aminothymoquinone (ATQ) from
which ten novel benzoxazole derivatives were prepared and characterized by elemental analysis,
IR spectroscopy, mass spectrometry and NMR (1H, 13C) spectroscopy in solution. The crystal structure
of 4-methyl-2-phenyl-7-isopropyl-1,3-benzoxazole-5-ol (1a) has been determined by X-ray diffraction.
All compounds were tested for their antibacterial, antifungal and antitumor activities. TQ and ATQ
showed better antibacterial activity against tested Gram-positive and Gram-negative bacterial strains
than benzoxazoles. ATQ had the most potent antifungal effect against Candida albicans, Saccharomyces
cerevisiae and Aspergillus brasiliensis. Three benzoxazole derivatives and ATQ showed the highest
antitumor activities. The most potent was 2-(4-fluorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol
(
1f). Western blot analyses have shown that this compound inhibited phosphorylation of protein kinase
B (Akt) and Insulin-like Growth Factor-1 Receptor (IGF1R ) in HeLa and HepG2 cells. The least
toxic compound against normal fibroblast cells, which maintains similar antitumor activities as TQ
β
,
was 2-(4-chlorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1e). Docking studies indicated that
1e and 1f have significant effects against selected receptors playing important roles in tumour survival.
Keywords: thymoquinone; benzoxazoles; anticancer activity; antimicrobial activity; western blotting;
molecular docking
Molecules 2018, 23, 3297; doi:10.3390/molecules23123297
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3297
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1. Introduction
Thymoquinone (TQ) is a natural benzoquinone previously found in Nigella sativa L. [
and some other plant species that are used in traditional medicine for therapeutic treatment
of various diseases [ ]. TQ is a molecule with numerous biological and medicinal activities
such as antitumor [ ], antimicrobial [ 11], antiepileptic, anti-inflammatory, antioxidant,
anti-hypertensive [ ], immunomodulatory [11 12], and neuromodulatory effects [13]. The main
1]
2
3–
7
8–
1
,
disadvantage of TQ is its low aqueous solubility. There are examples of preparation of TQ derivatives
with improved solubility and biological effects [14–18].
TQ can be used as starting compound for synthesis of benzoxazoles that have aromatic chemical
properties and pronounced biological activities. Benzoxazoles can be found in natural sources and
there are many examples of synthetic compounds which contain benzoxazole rings [19
,20]. Various
studies have shown the antitumor activities of benzoxazoles [20 25]. Among the main mechanisms
–
is inhibition of topoisomerases I and II, where the benzoxazole ring is crucial for the activity [20].
Other mechanisms are inhibition of cyclooxygenase-2 (COX-2) [26], thymidylate synthase [27] and
aurora B kinase [25]. Benzoxazoles have antimicrobial activity [24,28], mainly through binding and
depleting metal ions (especially Ca2+ and Mg2+) from the microorganism cell. Nitrogen in benzoxazole
ring is very important for this activity. Moreover, benzoxazoles inhibit enzyme deformylase from
Mycobacterium tuberculosis, while some benzoxazoles inhibit activity of the human immunodeficiency
virus (HIV) [19].
There are few examples of benzoxazole compounds in clinical trials or on the market (Figure 1).
Benzoxazoles are sensitive to hydrolysis which leads to ring opening depending on the structure
of molecule, different conditions and pH values [29,30]. Since this process can take place under
physiological conditions [29] benzoxazoles can be used as pro-drugs targeted at the specific site of
activity [30]. In case of benzoxazole derivatives prepared from TQ, the products of hydrolysis would
be Schiff bases with expected high biological activity (Scheme 1).
Herein we report the synthesis, antimicrobial and cytotoxic activities and molecular docking
studies of TQ derivative 3-aminothymoquinone (ATQ) and a series of ten novel benzoxazole
derivatives produced by reaction between ATQ and aromatic aldehydes.
a
c
b
Figure 1. Benzoxazole compounds currently on the market (
utrophin modulator indicated in Duchenne muscular dystrophy. (
receptor antagonist indicated in insomnia. (
treatment of different types of cancer.
a
,
b
) or in clinical trials (
) Suvorexant is selective, dual orexin
c) Sapanisertib is an mTOR inhibitor in clinical trials for
c). (a) Ezutromid is
b

Molecules 2018, 23, 3297
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NaN3
ArCHO
HCl
CH3COO
reflux
reflux
3 h, 80°C
4 h, 75°C
TQ
ATQ
Benzoxazoles 1a 1j

30%
Schiff base intermediate
Ar =
1e
40%
1c
1d
34%
1a
30%
1b
27%
30%
1j
1i
41%
1f
22%
1g
1h
29%
29%
44%
Scheme 1. Synthesis of 3-aminothymoquinone (ATQ) and series of benzoxazoles 1a
–1j from starting
compound thymoquinone (TQ).
2. Results and Discussion
2.1. Chemistry
TQ was reacted with sodium azide in the presence of catalytic amount of acetic acid to obtain ATQ
,
as previously reported in the literature [31] with slight modifications (Scheme 1). ATQ was then reacted
with different aryl aldehydes to produce Schiff bases which underwent cyclization into corresponding
benzoxazoles (Scheme 1). The reaction itself has been well known for decades, however reports are
limited to the synthesis of benzobisoxazoles obtained by reacting two molar equivalents of aldehydes
with 2,5-diaminoquinone [32,33], a reaction which was recently employed by the research group of
2016 Nobel laureate Fraser Stoddart in the design of macrocyclic compounds pillar[n]arenes [34].
Synthesis of monocyclized products is thus far limited to substructures of extended aromatic systems
such as naphthoquinones [35,36], quinolinequinones and indoloquinones [37]. Herein we thus present
the novel synthesis of 2-aryl-5-hydroxybenzoxazoles. The structure and purity of the compounds were
determined by means of IR and NMR spectroscopy, mass spectrometry, CHN elemental analysis and
single crystal X-ray diffraction. Numbering of compounds is presented in Supplementary Scheme S1.
The crystal structure of representative compound 4-methyl-2-phenyl-7-isopropyl-1,3-benzoxazole-
5-ol (1a) was determined by single crystal X-ray structure analysis. The ORTEP diagram of compound
1a is shown in Figure 2. The crystal structure data of compound 1a are presented in Supplementary
Table S1. All analytical data confirmed the assumed structures.

Molecules 2018, 23, 3297
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Figure 2. ORTEP view of compound 1a with heteroatom labelling. Thermal ellipsoids are drawn at
50% probability level.
2.2. Antibacterial Activity
The compounds TQ, ATQ and 1a–1j were tested for antibacterial activity against four
Gram-positive and four Gram-negative bacteria. All derivatives had lower activity when compared
to TQ (which showed higher activity than reference drug amikacin) in Pseudomonas aeruginosa and
Bacillus subtilis. The activity was lowered by addition of amino group in position C-3 of TQ (ATQ)
with up to eight-fold higher minimal inhibitory concentration (MIC) values (Table 1).
Table 1. In vitro antibacterial activity represented as minimum inhibitory concentration (MIC/µM)
values of investigated compounds.
Compound
TQ
ATQ
Amikacin
Bacteria
MIC (µM)
E. coli
P. aeruginosa
P. hauseri
K. pneumoniae
S. aureus
B. subtilis
C. sporogenes
M. luteus
29.84
59.68
29.84
59.68
29.84
59.68
121.80
59.68
111.59
217.61
217.61
435.22
111.59
217.61
435.22
59.68
8.54
85.38
11.95
13.71
18.78
71.72
25.61
3.42
With the introduction of the benzoxazole core in 1a
completely lost.
–1j, antibacterial activity is partially or
2.3. Antifungal Activity
Antifungal activity of compounds TQ, ATQ and 1a–1j was tested against three fungi with nystatin
as a reference drug. Addition of an amino group in position C-3 of TQ, resulted in an improvement of
the antifungal activity. ATQ showed remarkable inhibitory activity against all tested fungi, with MIC
values significantly lower when compared to TQ and nystatin (Table 2). With addition of benzoxazole
ring, antifungal activity is completely lost and no inhibition zones were seen for compounds 1a–1j in
the disk diffusion assay.

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Table 2. In vitro antifungal activity presented as minimum inhibitory concentration (MIC/mM) values
of investigated compounds.
Compound
ATQ
Fungus
TQ
Nystatin
MIC (mM)
C. albicans
S. cerevisiae
A. braziliensis
3.81
1.90
3.81
1.75
0.22
0.88
2.70
1.35
1.35
2.4. Cytotoxicity
MTT assay was used in order to determine IC50 values (values at which 50% of cells are inhibited)
of compounds (TQ ATQ 1a 1j). Tested compounds have shown various levels of activity against
,
,
–
four carcinoma cell lines used (SW620, CFPAC-1, HepG2 and HeLa) (Table 3). Human lung fibroblasts
(WI38) were used as healthy control cell line. WI38 is widely used as a model for evaluation of the
effects of chemicals on normal cells [38]. It is highly desirable that new drug candidates show no toxic
effects in healthy cell lines.
Table 3. IC50 values (values at which 50% of cells are inhibited) (µM) of compounds TQ, ATQ,
and 1a 1j in tested cell lines. The most potent compound in each tumour cell line is bolded. The least
–
toxic compound against WI38 is bolded. SW620, CFPAC, HepG2 and HeLa are carcinoma cell lines,
WI38 is healthy control human fibroblast cell line.
Cell Line
Compound SW620 CFPAC HepG2 HeLa
WI38
IC50 (µM)
TQ
ATQ
1a
1b
1c
1d
1e
1f
39.95
25.82
>100
43.68
>100
51.53
79.88
5.82
54.93
>100
55.30
31.55
48.26
30.87
90.22
31.02
>100
34.51
49.47
33.81
>100
97.00
40.58
27.01
23.97
8.26
28.85
15.74
32.22
25.00
>100
30.10
42.67
4.13
19.83
9.41
39.34
32.27
>100
30.46
39.48
9.36
>100
>100
4.58
19.89
29.81
84.48
57.61
>100
5.54
6.21
75.89
9.32
1g
1h
1i
>100
>100
6.38
1j
7.28
10.27
9.97
Different substituents play very important role in the activity of this series of compounds.
With addition of amino group at position C-3 of TQ, antitumor activity towards all tested cell lines
was improved. Addition of benzoxazole ring to the structure, further affects cytotoxic activity, which is
depending on substituent at the position 2 of benzoxazole ring (Figure 3). Significantly improved
antitumor activity (compared to starting compound TQ) had compounds 1f, ATQ, 1i and 1j.
Various molecules and signaling pathways play important role in tumor development.
Determination of interactions between newly synthesized molecules and cancer signaling pathways is
crucial phase in identification and development of new potential drug candidates. Understanding
mechanisms of activity directs towards more efficient evaluation of new chemical entities. Western
blot technique and molecular docking studies were used for evaluation of the mechanisms involved in
the anticancer activity of the most potent compound 1f and the least toxic derivative 1e. Results were
compared to the ones of starting compound TQ.

Molecules 2018, 23, 3297
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Antitumor activity
R
=
Benzoxazole
Figure 3. Effects of various substituents on position 2 of benzoxazole ring on antitumor activity
of compound.
Among the main mechanisms of TQ antitumor activity is inhibition of protein kinase B (Akt)
phosphorylation [39–42] which can be initiated through activation of phosphatase and tensin homolog
located on chromosome 10q23 (PTEN) receptor. PTEN is one of the most usually inactivated
tumor suppressors with important role in induction of apoptosis and control of cell migration and
angiogenesis. It interacts with various signaling pathways in a cell. TQ transcriptionally up-regulates
PTEN [39,43], which is then leading to apoptosis through several signaling pathways among which
is Phosphoinositide 3-kinase (PI3K)/Akt pathway. Decreased levels of phosphorylated Akt were
measured after TQ treatment in breast [39,41], lymphoma [40], and prostate tumor cells [42].
In order to determine whether the compounds 1f and 1e retain pAkt inhibitory activity of TQ
a western blot analysis was performed. Compound 1f has shown stronger inhibitory activity towards
Akt phosphorylation in HepG2 cells where IC50 concentration caused significant effect after 24 h and
the absence of pAkt signal after 48 h of treatment (Figure 4B). In HeLa cells, significantly decreased
pAkt levels were observed only after 48 h treatment (Figure 4A). IC50 concentration of compound 1e
showed weak, time and concentration dependent inhibition of Akt phosphorylation in both cancer cell
lines (HeLa and HepG2) (Figure 4).
,
IGF1 Receptor
no significant changes in IGF1 Receptor
HeLa and HepG2 cells with compound 1e (Figure 4). In contrast, IGF1 Receptor
β
plays important role in tumor development and metastasis [44
activated form expression upon 24 and 48 h treatments of
activated form
,45]. There were
β
β
signals were not detectable upon 24 and 48 h treatments of HeLa cells with compound 1f. Moreover,
significantly decreased expression (24 h treatment) and lack of signal (48 h treatment) were observed
in compound 1f-treated HepG2 cells (Figure 4).

Molecules 2018, 23, 3297
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Figure 4. Representative membranes with IGF1R-
compounds 1e and 1f for 24 h and 48 h. Relative expression is shown on the right panel as the average
of three experiments standard error. ( ) HepG2 cells treated with compounds 1e and 1f for 24 h and
β and pAkt signals in: (A) HeLa cells treated with
±
B
48 h. Relative expression is shown on the right panel as the average of three experiments
error. Statistically significant changes are marked with an asterisk (*).
±
standard
2.5. Docking Studies
It is already known that compounds containing benzoxazole rings can have different interactions
with proteins. The oxygen and nitrogen atoms in the benzoxazole ring can act as hydrogen bond
acceptors. The benzoxazole ring has an aromatic planar nature and therefore can form
and -cation interactions. Because of lipophilic character of benzoxazole derivatives, hydrophobic
interactions between benzoxazoles and host proteins can be formed [19].
π stacking
π
Docking studies of the most potent antitumor compound 1f, a compound least toxic to normal
cells 1e and a starting compound TQ, were performed with the aim to understand the interactions
with three molecules crucial in tumorigenesis: PTEN (PDB ID: 1D5R), topoisomerase II (PDB ID: 3QX3)
and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) (PDB ID: 1K3Z) (Table 4,
Figure 5).
Table 4. Binding characteristics of TQ
, 1e and 1f to phosphatase and tensin homolog located on
chromosome 10q23 (PTEN) (PDB ID: 1D5R), topoisomerase II (PDB ID: 3QX3) and nuclear factor
kappa-light-chain-enhancer of activated B cells (NF
molecular docking study.
κB) (PDB ID: 1K3Z) receptors, as assessed by
PTEN
Topoisomerase II
NFκB
Binding
Energy
(kcal/mol)
Ki *
Value
(mM)
No. of
H
bonds
Binding
Energy
(kcal/mol)
Ki *
Value
(mM)
No. of
H
bonds
Binding
Energy
(kcal/mol)
Ki *
Value
(mM)
No. of
H
bonds
Compound
TQ
1e
1f
−4.02
−3.57
−3.88
1.12
2.41
1.43
1
0
0
−3.02
−4.32
−3.80
6.1
0.677
1.65
0
0
0
−4.75
−6.02
−5.58
0.328
0.039
0.082
1
2
1
* Ki = inhibition constant.

Molecules 2018, 23, 3297
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Figure 5. Binding mode of TQ, compounds 1e and 1f at the active site of the (
(PDB ID: 1D5R), ( ) topoisomerase II receptor (PDB ID: 3QX3), and ( ) NF B receptor (PDB ID: 1K3Z),
as assessed by molecular docking study.
A) PTEN receptor
B
C
κ
The docking studies results indicate that PTEN receptor binding of TQ is stronger when compared
to 1f and 1e compounds. On the other hand, the western blot analyses have revealed that 1f inhibits Akt
phosphorylation significantly, while compound 1e shows a weak, time and concentration dependent
inhibition. Whether those effects are mediated through PTEN remains to be determined.
Topoisomerase II is an enzyme which plays important role in carcinogenesis and it is one of
the most studied molecular targets in anticancer research [46,47]. Both TQ [48] and benzoxazole
derivatives inhibit topoisomerases I and II [20]. Based on docking studies results, and lower binding
energies of benzoxazoles, compared to starting compound TQ, it can be expected that compounds 1f
and 1e possess stronger topoisomerase II inhibitory activities than TQ.
Inhibition of NF
transcriptional factor found in cytoplasm which is stimulated by different substances including
pro-oxidants, carcinogens and growth factors. After activation, NF B enters nucleus and binds
DNA and stimulates transcription of genes involved in the regulation of apoptosis, proliferation,
angiogenesis and inflammation [50]. TQ inhibits NF B through direct interaction with the p65 subunit
of NF B and through suppression of tumor necrosis factor (TNF)-induced I B kinase (IKK) activation.
NF B activation induced by different carcinogens and inflammatory stimuli is suppressed by TQ
As a result, anti-apoptotic and proliferation regulatory proteins are suppressed [50]. According to
κB is one of the most important characteristics of TQ [4,49,50]. NFκB is a
κ
κ
κ
κ
κ
.
docking studies results, it can be expected that compounds 1f and 1e possess stronger NF
activities than TQ.
κB inhibitory

Molecules 2018, 23, 3297
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3. Materials and Methods
3.1. General Experimental Procedures
All solvents and reagents were used as received from the manufacturers. All chemicals were
purchased from Sigma Aldrich (St. Gallen, Switzerland). Melting points were determined on a
Kofler Hot Stage Microscope apparatus (Reichert, Wien, Austria) and are uncorrected. The IR
spectra were recorded on a Spectrum BX FTIR System (Perkin Elmer, Waltham, MA, USA) as KBr
pellets, in the wavelength range from 4500 to 400 cm−1. The elemental analysis was accomplished
by combustion analysis on a Vario EL III C,H,N,S/O Elemental Analyzer (Elementar, Langenselbold,
Hesse, Germany). The NMR spectra in solution were measured in DMSO-d6 on an AV600 spectrometer
1
(Bruker, Rheinstetten, Germany) at 298 K in 5 mm NMR tubes. H-NMR spectra were acquired at
600.130 MHz, while 13C APT and 13C {1H} spectra were acquired at 150.903 MHz. Digital resolutions
in 1H and 13C spectra were 0.37 and 0.60 Hz per point. Chemical shifts (
δ
/ppm) in 1H spectra were
/ppm) in 13
= 39.51 ppm. Splitting of peaks in 1H-NMR spectra
referred to the methyl protons of TMS (tetramethylsilane);
spectra were referred to the signal of DMSO-d6;
δ
= 0.0 ppm. Chemical shifts (
δ
C
δ
was assigned as: s (singlet), br s (broad singlet), d (doublet), t (triplet), hept (heptet), m (multiplet),
dd (doublet of doublets). Double resonance experiments (1H-1H COSY, 1H-13C HMQC, 1H-13C HMBC)
were performed in order to assist in signal assignment.
Mass spectral (HRESIMS) data were obtained from an Agilent Technologies 6210 Time-of-Flight
LC/MS system consisting of an HPLC instrument Agilent 1200 Series (Agilent Technologies,
Waldbronn, Germany), equipped with a degasser, a binary pump, an auto-sampler, a thermostated
column compartment and a diode array detector (DAD) and coupled with a 6210 Time-of-Flight
LC/MS system (Agilent Technologies, Santa Clara, CA, USA) via an electrospray ionization (ESI)
interface. For LC/MS analyses acetonitrile (HPLC grade) purchased from Merck KG (Darmstadt,
Germany) and Milli Q water 18.2 MΩcm, obtained from a Millipore Simplicity 185 purification system
were used. The mass spectra were recorded in ESI-Mass Spectrum mode (Agilent-6310 ion trap).
X-ray diffraction data were collected on a SuperNova diffractometer (Oxford Diffraction,
Abingdon, UK) with a Cu microfocus X-ray source with mirror optics and an Atlas detector.
The structures were solved by direct methods implemented in SIR92 [51] and refined by a full-matrix
least-squares procedure based on F2 using SHELXL-2014 [52]. All non-hydrogen atoms were
refined anisotropically. The hydrogen atoms were placed at calculated positions and treated using
appropriate riding models. The programs Platon and Mercury were used for data analysis and figure
preparation [53,54]. The crystal structure of 1a has been deposited in the CCDC database and has been
assigned the deposition number CCDC 1586630.
3.2. General Procedure for the Synthesis of 3-aminothymoquinone (ATQ)
ATQ was prepared according to the protocol previously reported in literature [31] (Scheme 1)
with slight modifications. 1 mmol of TQ and 1.3 mmol of sodium azide were dissolved in absolute
ethanol. Glacial acetic acid was added as catalyst. Mixture was refluxed for 3 h at 80 ◦C. Reaction
progression was followed by thin layer chromatography (TLC). Reaction was stopped by neutralization
with sodium bicarbonate. The obtained crude reaction mixture was filtered and purified by column
chromatography with Silicagel 60 as a stationary phase and dichloromethane:diethyl ether:n-hexane =
1:2:5 as eluent. The main impurity after synthesis of ATQ was TQ (Rf value 0.5–0.6) which was easily
separated from ATQ (Rf value 0.38).
◦
3-Aminothymoquinone (ATQ). Dark red solid (0.054 g, 30%); m.p. 44 C; IR (KBr)
ν
3462, 3330, 1650,
max
1580 cm−1; 1H-NMR (DMSO-d6)
JH,H = 6.89 Hz, H-9), 1.73 (3H, s, CH3-12), 1.05 (6H, d, JH,H = 6.89 Hz, CH3-10, CH3-11); 13C-NMR
(DMSO-d6) 184.82 (C, C-1), 183.66 (C, C-4), 148.99 (C, C-5), 145.21 (C, C-2), 132.01 (CH, C-6), 106.59 (C,
δ 6.43 (2H, s, NH2, H-13a, H-13b), 6.29 (1H, s, H-6), 2.87 (1H, hept,
δ

Molecules 2018, 23, 3297
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C-3), 25.87 (CH, C-9), 21.11 (CH3, C-10, C-11), 8.65 (CH3, C-12); MS m/z 180.10 [M + H]+ (calcd. for
C10H13NO2, 180.1025); anal. C 67.02, H 7.31, N 7.82%, calcd. for C10H13NO2, C 66.78, H 7.58, N 7.71%.
General Procedure for the Synthesis of Benzoxazole Derivatives 1a–1j
Aromatic aldehydes were added in equimolar quantities to ATQ in absolute ethanol with HCl as
catalyst and were refluxed for 4 h at 75 ◦C to produce Schiff bases which subsequently cyclized into
corresponding benzoxazoles. Mixture was poured over crushed ice, precipitate was filtered, washed
with distilled water and purified by column chromatography on Silicagel 60. Each compound required
different mobile phase for purification (Supplementary Table S2).
◦
4-Methyl-2-phenyl-7-isopropyl-1,3-benzoxazole-5-ol (1a). White solid (0.081 g, 30.3%); m.p. 164 C;
1H-NMR (DMSO-d6)
δ
9.17 (1H, s, OH), 8.20-8.15 (2H, m, H-12, H-16), 7.63–7.58 (3H, m, H-13, H-14,
H-15), 6.76 (1H, s, H-6), 3.26 (1H, hept, JH,H = 6.65 Hz, H-8), 2.34 (3H, s, CH3-17), 1.36 (6H, d, JH,H
6.95 Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
161.5 (C, C-2), 152.2 (C, C-5), 141.7 (C, C-1), 141.6 (C,
=
δ
C-3), 131.4 (CH, C-14), 129.2 (CH, C-12, C-16), 128.1 (C, C-7), 127.0 (CH, C-13, C-15), 126.9 (C, C-11),
111.6 (C, C-4), 110.6 (CH, C-6), 28.9 (CH, C-8), 22.4 (CH3, C-9, C-10), 10.1 (CH3, C-17); HRESIMS
m/z 268.13339 [M + H]+ (calcd. for C17H17NO2, 268.1338); anal. C 76.38, H 6.41, N 5.24%, calcd. for
C17H17NO2, C 76.08, H 6.54, N 5.23%.
2-(2-Hydroxyphenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1b). White solid (0.078 g, 27.5%);
◦
1
m.p. 172 C; IR (KBr)
ν
3378, 1597, 1545, 1629, 1338, 1155 cm−1; H-NMR (DMSO-d6, 600.130
max
MHz) 11.42 (1H, s, OH-12), 9.35 (1H, s, OH-5), 8.01 (1H, d, JH,H = 7.76 Hz, H-16), 7.75 (1H, t, JH,H
δ
=
8.36 Hz, H-14), 7.11 (1H, d, JH,H = 8.21 Hz, H-13), 7.08 (1H, t, JH,H = 7.63 Hz, H-15), 6.81 (1H, s, H-6),
3.28 (1H, hept, JH,H = 6.98 Hz, H-8), 2.34 (3H, s, CH3-17), 1.36 (6H, d, JH,H = 6.84 Hz, CH3-9, CH3-10);
13C-NMR (DMSO-d6)
δ 161.7 (C, C-2), 157.5 (CH, C-12), 152.6 (C, C-5), 140.0 (C, C-1), 139.5 (C, C-3),
133.5 (CH, C-14), 128.4 (C, C-7), 127.0 (CH, C-16), 119.9 (CH, C-15), 117.0 (CH, C-13), 110.92 (C, C-4),
110.87 (CH, C-6), 110.5 (C, C-11), 28.8 (CH, C-8), 22.4 (CH3, C-9, C-10), 10.0 (CH3, C-17); HRESIMS
m/z 284.1 [M + H]+ (calcd. for C17H17NO3, 284.1287); anal. C 67.76, H 6.35, N 4.65%, calcd. for
.
C17H17NO3 H2O, C 67.55, H 6.41, N 4.54%.
2-(2-Hydroxynaphtalene)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1c). White solid (0.100 g, 30%);
◦
1
m.p. 204 C; IR (KBr)
ν
3338, 1576, 1624, 1338, 1134 cm−1; H-NMR (DMSO-d6)
δ 12.64 (1H,
max
s, OH-22), 9.33 (1H, s, OH-25), 8.64 (1H, d, JH,H = 8.63 Hz, H-17), 8.07 (1H, d, JH,H = 8.63 Hz, H-14),
7.96 (1H, d, JH,H = 8.63 Hz, H-20), 7.65 (1H, t, JH,H = 7.84 Hz, H-18), 7.45 (1H, t, JH,H = 7.06 Hz, H-19),
7.35 (1H, t, JH,H = 7.84 Hz, H-13), 6.83 (1H, s, H-6), 3.32 (1H, hept, JH,H = 6.90 Hz, H-8), 2.38 (3H, s,
CH3-21), 1.40 (6H, d, JH,H = 6.95, Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
δ 162.3 (C, C-2), 158.6 (C,
C-12), 152.6 (C, C-5), 140.4 (C, C-1), 138.9 (C, C-3), 134.1 (CH, C-14), 130.9 (C, C-16), 129.0 (CH, C-20),
128.3 (CH, C-18), 128.0 (CH, C-7), 127.96 (C, C-15), 123.7 (CH, C-19), 123.6 (CH, C-17), 118.9 (CH, C-13),
110.7 (CH, C-6), 103.7 (C, C-11), 29.1 (CH, C-8), 22.3 (CH3, C-9, C-10), 10.1 (CH3, C-21), n.d. (C, C-4);
HRESIMS m/z 334.14366 [M+H]+ (calcd. for C21H19NO3, 334.1443); anal. C 75.66, H 5.74, N 4.20%,
calcd. for C21H19NO3, C 75.18, H 5.77, N 4.17%.
◦
2-(4-Bromophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1d). White solid (0.090 g, 33.7%); m.p. 190 C;
1H-NMR (DMSO-d6)
δ
9.21 (1H, s, OH), 8.10 (2H, d, JH,H = 8.70 Hz, H-13, H-15), 7.80 (2H, d, JH,H = 8.70
Hz, H-12, H-16), 6.78 (1H, s, H-6), 3.25 (1H, hept, JH,H = 5.99 Hz, H-8), 2.33 (3H, s, CH3-17), 1.34 (6H, d,
H,H = 6.67 Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
160.7 (C, C-2), 152.3 (C, C-5), 141.7 (C, C-3),
J
δ
141.6 (C, C-1), 132.3 (CH, C-13, C-15), 128.8 (CH, C-12, C-16), 128.2 (C, C-7), 126.1 (C, C-11), 125.0 (C,
C-14), 111.7 (C, C-4), 110.9 (C, C-6), 28.8 (CH, C-8), 22.4 (CH3, C-9, C-10), 10.1 (CH3, C-17); HRESIMS
m/z 346.04319 [M + H]+ (calcd. for C17H16BrNO2, 346.0443); anal. C 58.97, H 4.66, N 4.05%, calcd. for
C17H16BrNO2, C 58.36, H 4.58, N 3.99%.
2-(4-Chlo◦rophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1e).
Yellow solid (0.120 g, 39.8%);
δ 9.23 (1H, s, OH), 8.17 (2H, d, JH,H = 8.52 Hz, H-12, H-16), 7.66 (2H,
m.p. 180 C; 1H-NMR (DMSO-d6)

Molecules 2018, 23, 3297
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d, JH,H = 8.52 Hz, H-13, H-15), 6.78 (1H, s, H-6), 3.25 (1H, hept, JH,H = 6.65 Hz, H-8), 2.34 (3H, s,
CH3-17), 1.35 (6H, d, JH,H = 6.95 Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
160.6 (C, C-2), 152.3 (C,
δ
C-5), 141.7 (C, C-3), 141.6 (C, C-1), 136.1 (C, C-14), 129.4 (CH, C-12, C-16), 128.7 (CH, C-13, C-15),
128.2 (C, C-7), 125.8 (C, C-11), 111.6 (C, C-4), 110.8 (CH, C-6), 28.8 (CH, C-8), 22.4 (CH3, C-9, C-10),
10.1 (CH3, C-17); HRESIMS m/z 302.09382 [M + H]+ (calcd. for C17H16ClNO2, 302.0948); anal. C 67.66,
H 5.34, N 4.64%, calcd. for C17H16ClNO2, C 67.34, H, 5.29, N 4.59%.
◦
2-(4-Fluorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1f). White solid (0.060 g, 22.4%); m.p. 152 C;
1H-NMR (DMSO-d6)
δ
9.18 (1H, s, OH), 8.22 (2H, dd, JH,H = 8.78 Hz, JH,F = 5.50 Hz, H-12, H-16),
7.43 (2H, dd, JH,H = 8.78, Hz, JH,F = 8.78 Hz, H-13, H-15), 6.76 (1H, s, H-6), 3.25 (1H, hept, JH,H = 6.65
Hz, H-8), 2.34 (3H, s, CH3-17), 1.34 (6H, d, JH,H = 6.95 Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
163.9 (C, JC,F = 256.0 Hz, C-14), 160.7 (C, C-2), 152.2 (C, C-5), 141.68 (C, C-1), 141.66 (C, C-3), 129.5 (CH,
C,F = 9.13 Hz, C-12, C-16), 128.2 (C, C-7), 123.6 (C, C-11), 116.4 (CH, JC,F = 22.4 Hz, C-13, C-15), 111.6 (C,
δ
J
C-4), 110.6 (CH, C-6), 28.8 (CH, C-8), 22.4 (CH3, C-9, C-10), 10.1 (CH3, C-17); HRESIMS m/z 286.12359
[M + H]+ (calcd. for C17H16FNO2, 286.1243); anal. C 71.56, H 5.65, N 4.91%, calcd. for C17H16FNO2,
C 71.12, H 5.42, N 4.90%.
◦
4-Methyl-2-(4-nitrophenyl)-7-isopropyl-1,3-benzoxazole-5-ol (1g). Yellow solid (0.090 g, 28.8%); m.p. 188 C;
1H-NMR (DMSO-d6)
δ
9.29 (1H, s, OH), 8.42–8.40 (4H, m, H-12, H-13, H-15, H-16), 6.83 (1H, s, H-6),
3.28 (1H, hept, JH,H = 6.91 Hz, H-8), 2.36 (3H, s, CH3-17), 1.36 (6H, d, JH,H = 7.14 Hz, CH3-9, CH3-10);
13C-NMR (DMSO-d6)
159.6 (C, C-2), 152.5 (C, C-5), 148.7 (C, C-14), 142.1 (C, C-1), 141.7 (C, C-3),
δ
132.4 (C, C-11), 128.5 (C, C-7), 128.1 (CH, C-12, C-16), 124.4 (CH, C-13, C-15), 112.0 (C, C-4), 111.8 (CH,
C-6), 28.8 (HC, C-8), 22.4 (CH3, C-9, C-10), 10.0 (CH3, C-17); HRESIMS m/z 313.11768 [M + H]+ (calcd.
for C17H16N2O4, 313.1188); anal. C 65.38, H 5.16, N 8.97%, calcd. for C17H16N2O4, C 64.73, H 4.87,
N 8.89%.
2-(4-Methoxyphenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1h). White solid (0.130 g, 43.7%);
◦
m.p. 178 C; 1H-NMR (DMSO-d6)
δ
9.11 (1H, s, OH), 8.11 (2H, d, JH,H = 7.98 Hz, H-12, H-16), 7.14 (2H,
d, JH,H = 7.98 Hz, H-13, H-15), 6.71 (1H, s, H-6), 3.86 (3H, s, OCH3), 3.24 (1H, hept, JH,H = 6.65 Hz, H-8),
2.32 (3H, s, CH3-17), 1.34 (6H, d, JH,H = 6.95 Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
161.74 (C,
δ
C-14), 161.67 (C, C-2), 152.1 (C, C-5), 141.8 (C, C-3), 141.4 (C, C-1), 128.7 (CH, C-12, C-16), 127.9 (C, C-7),
119.4 (C, C-11), 114.6 (CH, C-13, C-15), 111.3 (C, C-4), 110.0 (CH, C-6), 55.4 (CH3, C-19), 28.8 (CH, C-8),
22.5 (CH3, C-9, C-10), 10.1 (CH3, C-17); HRESIMS m/z 298.14311 [M + H]+ (calcd. for C18H19NO3,
298.1443); anal. C 72.71, H 6.44, N 4.71%, calcd. for C18H19NO3, C 71.96, H 6.43, N 4.62%.
2-(4-Hydroxy-3-methoxyphenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1i). White solid (0.130 g, 41.5%);
m.p. 168 ◦C; 1H-NMR (DMSO-d6)
δ
9.82 (1H, s, OH-18), 9.07 (1H, s, OH-23), 7.65–7.61 (2H, m, H-12,
H-16), 6.96 (1H, d, JH,H = 8.48 Hz, H-15), 6.69 (1H, s, H-6), 3.90 (3H, s, OCH3), 3.24 (1H, hept, JH,H
6.98 Hz, H-8), 2.32 (3H, s, CH3-17), 1.33 (6H, d, JH,H = 6.89 Hz, CH3-9, CH3-10); 13C-NMR (DMSO-d6)
162.0 (C, C-2), 152.0 (C, C-5), 150.0 (C, C-14), 147.9 (C, C-13), 141.9 (C, C-1), 141.4 (C, C-3), 127.8 (C,
=
δ
C-7), 120.8 (CH, C-16), 118.1 (C, C-11), 115.9 (CH, C-15), 111.2 (C, C-4), 110.4 (CH, C-12), 109.7 (CH,
C-6), 55.7 (CH3, C-20), 28.7 (CH, C-8), 22.5 (CH3, C-9, C-10), 10.1 (CH3, C-17); HRESIMS m/z 314.13840
[M + H]+ (calcd. for C18H19NO4, 314.1392); anal. C 68.99, H 6.11, N 4.47%, calcd. for C18H19NO4,
C 68.38, H 6.08, N 4.48%.
◦
4-Methyl-7-isopropyl-2-(thiophene-2-yl)-1,3-benzoxazole-5-ol (1j). White solid (0.080 g, 29.3%); m.p. 152 C;
1
IR (KBr)
ν
3108, 1568, 1302, 1115, 856, 1036, 720 cm−1; H-NMR (DMSO-d6)
δ
9.18 (1H, s, OH),
max
7.90 (2H, d, JH,H = 4.17 Hz, H-13, H-15), 7.28 (1H, t, JH,H = 4.64 Hz, H-14), 6.74 (1H, s, H-6), 3.25 (1H,
hept, JH,H = 6.65 Hz, H-8), 2.30 (3H, s, CH3-16), 1.33 (6H, d, JH,H = 7.01, Hz, CH3-9, CH3-10); 13C-NMR
(DMSO-d6) δ 157.7 (C, C-2), 152.3 (C, C-5), 141.6 (C, C-3), 141.2 (C, C-1), 131.1 (CH, C-15), 129.6 (CH,
C-13), 129.2 (C, C-11), 128.7 (CH, C-14), 128.0 (C, C-7), 111.4 (C, C-4), 110.4 (CH, C-6), 28.7 (CH, C-8),
22.4 (CH3, C-9, C-10), 10.1 (CH3, C-16); HRESIMS m/z 274.08914 [M + H]+ (calcd. for C15H15NO2S,

Molecules 2018, 23, 3297
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274.0902); anal. C 65.91, H 5.53, N 5.12, S 11.73%, calcd. for C15H15NO2S, C 65.55, H 5.54, N 5.07,
S 11.46%.
3.3. Biological Assays
3.3.1. Antimicrobial Activity
Compounds and Microorganisms
Antimicrobial activity of compounds was evaluated in vitro on eight bacterial strains (Escherichia
coli ATCC 25922 (ATCC = American Type Culture Collection, Manassas, VA, USA), Pseudomonas
aeruginosa ATCC 9027, Proteus hauseri ATCC 13315, Klebsiella pneumoniae ATCC 10031, Staphylococcus
aureus ATCC 6538, Bacillus subtilis ATCC 6633, Clostridium sporogenes ATCC 19404 and Micrococcus
luteus ATCC 10240) and three fungi (Candida albicans ATCC 10231, Saccharomyces cerevisiae ATCC 9763
and Aspergillus brasiliensis ATCC 16404).
Diffusion and Micro-Dilution Assays
Agar well diffusion and micro-dilution assays were used for evaluation of antimicrobial activity.
To each sterile Petri dish (90 mm diameter) 22 mL of nutrient agar and 100
(106 cell per dish) or 22 mL Sabouraud dextrose agar and 100 L of fungi (105 spores per dish)
were added. A well with the diameter of 8 mm was punched using a sterile cork borer. The tested
compounds were dissolved in DMSO at the concentration 10 mg mL−1 and 100
L of this solution
was added to each well. Final concentration of tested compounds was 1 mg/well. At the same time,
amikacin and nystatin in final concentration 30 g/well were used as reference drugs for antibacterial
µL of bacterial suspension
µ
µ
µ
and antifungal testing, respectively. Petri dishes were maintained at room temperature for 4 h, and after
that they were incubated: bacterial strains 24 h at 37 ◦C, and fungi 48 h at 25 ◦C. After incubation,
inhibition zone diameters were measured by using a Readbiotic Microbiological Culture Analyzer
(International PBI SpA, Milan, Italy), with the precision of 0.1 mm. Experiments were performed in
triplicate, and average values presented.
Micro-dilution assay was used for determination of tested compounds MIC. A serial dilution
method in sterile 96-well microtitre plates was performed. Fresh Mueller–Hinton broth (for bacteria)
and Sabouraud dextrose broth (for fungi) were used. In sterile physiological solution, suspensions
−1
−1
107 CFU mL for fungi were made (according
containing 1.5
to McFarland). One hundred microliters of broth were added to each well of the plate. Stock solutions
of tested compounds were made in DMSO at concentration 10 mg mL−1, and then 100
L of the
×
108 CFU mL for bacteria and 1.5
×
µ
compound stock solution was added to each well of the first column. Double dilution was made in
whole row for one tested compound, and repeated for all tested compounds in other rows. Amikacin
served as positive control for bacteria, while nystatin served as positive control for fungi. The solvent
(DMSO) was used as the negative control. Afterwards, each well was inoculated with 10◦
µL of
◦
microorganism suspension. After incubation, 24 h at 37 C for bacteria and 48 h at 25 C for
fungi, results were read. The lowest concentration where no growth of microorganism was detected
represented MIC.
3.3.2. Cytotoxic Activity
Compounds and Cell Lines
The tested compounds were dissolved in DMSO in five, 10-fold dilutions (0.01–100 µM). The cell
lines HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic), HepG2 (hepatocellular
carcinoma), CFPAC-1 (ductal pancreatic adenocarcinoma), and WI38 (human lung fibroblast cell
line), were cultured as monolayers and maintained in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U mL−1 penicillin and
100 µg mL−1 streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦C.

Molecules 2018, 23, 3297
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MTT Assay
The MTT assay was used for evaluation of effects of tested compounds on viability of carcinoma
and control cell lines. About 5000 cells per well were seeded onto a series of standard 96-well plates
on day 0. Test agents were then added and incubated for further 72 h. Working dilutions of tested
compounds were freshly prepared in the growth medium, on the day of testing. The solvent (DMSO)
was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in the
working concentrations (DMSO concentration never exceeded 0.1%). After 72 h of incubation, the cell
growth rate was evaluated by performing the MTT assay: experimentally determined absorbance
values were transformed into a cell percentage growth (PG) using the formulas proposed by NIH
and described previously [55]. This method directly relies on control cells number at the day of assay
because it compares the growth of treated cells with the growth of untreated cells in control wells on
the same plate, the results are therefore a percentile difference from the calculated expected value.
The IC50 values for each compound were calculated from dose-response curves using linear
regression analysis by fitting the mean test concentrations that give PG values above and below the
reference value. If, however, all of the tested concentrations produce PGs exceeding the respective
reference level of effect (e.g., PG value of 50) for a given cell line, the highest tested concentration
is assigned as the default value (in the screening data report that default value is preceded by a “>”
sign). Each test point was performed in quadruplicate in three individual experiments. The results
were statistically analyzed (Analysis of variance (ANOVA), Tukey post-hoc test at p < 0.05). Finally,
the effects of the tested substances were evaluated by plotting the mean percentage growth for each
cell type in comparison to control on dose response graphs.
Western Blot
HeLa and HepG2 cells were seeded in six well plate, at 400,000 cells/well, and treated with
compounds 1e and 1f at micromolar concentrations assessed as active on the cell cycle without
immediate cell death activity, compound 1e (HeLa IC50: 42.67
µM and HepG2 IC50: 39.48 µM) and
compound 1f (HeLa IC50: 4.13 M and HepG2 IC50: 9.36 M) for 24 h and 48 h. Protein lysates were
µ
µ
lysed in the buffer 50 mM Tris HCl (pH 8), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS and protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). A total of
50
Hercules, CA, USA). Membranes were incubated with primary antibodies against phospho (Tyr 1135)
-IGF1 Receptor (1:1000, rabbit mAb, Cell Signalling Technology, Danvers, MA, USA), phospho
µg of proteins were resolved on 10% SDS polyacrylamide gels by use of Mini-protean cell (Bio-Rad,
β
(Ser 473) Akt (1:1000, rabbit mAb, Cell Signalling Technology) and
α tubulin (1:1000, mouse mAb,
◦
Sigma Aldrich) at 4 C overnight. Secondary antibody linked to anti-mouse was used at 1:1000
(Dako, Santa Clara, CA, USA) and to anti-rabbit at 1:1000 (Dako). Signals were visualized by Western
Lightening Chemiluminescence Reagent Plus Kit (Perkin Elmer, USA) on the ImageQuant LAS500 (GE
Healthcare, Chicago, IL, USA). Signal intensities of particular bands were normalized with the intensity
of the loading control
expressed as average value of three independent experiments
analysis was performed in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) by using the
α
tubulin and compared in Quantity One software (Bio-Rad). The values are
standard error of the mean. Statistical
±
ANOVA (p < 0.05).
3.4. Docking Studies
The docking studies were performed with the aim of determination of probable antitumor activity
mechanisms of the most potent 1f and the least toxic 1e compounds in comparison with starting
compound TQ. 3D coordinates for the crystal structures of PTEN (PDB ID: 1D5R), topoisomerase II
(PDB ID: 3QX3) and NFκB (PDB ID: 1K3Z) were obtained from Brookhaven protein data bank [56,57].
AutoDockTools (ADT, The Scripps Research Institute, San Diego, CA, USA) software was used
for preparation and analysis of docking simulations. Lamarckian genetic algorithm (LGA) was used

Molecules 2018, 23, 3297
14 of 17
for searching conformers which are energetically the most favorable. Up to 100 conformers of each
compound were analyzed. Coordination x, y, z network with dimensions 126 126 126 dots,
×
×
and distance between dots 0.375 Å, was prepared and centered into main catalytic part of the receptor.
Size of population was 150, with the random selection of individuals. Maximal number of energetic
calculations was 2,500,000, maximal number of generated conformations was 27,000, with number of
individuals restricted to 1, mutation speed 0.02, recombination speed 0.8, 2 Å translation, quaternion
50◦ and torsion angle 50◦. 100 LGA calculations were performed, with cluster tolerance 2 Å, external
network energy 1000, maximal initial energy 0 and maximal number of repetitions 10,000. Structures
of tested compounds were optimized in Chem3D Ultra 9.0.1. software (Perkin Elmer) with the use of
AM1 semi empirical method [58]. Docking studies were performed by using AutoDock 4.0. software
(The Scripps Research Institute). Discovery Studio Visualizer software (Biovia, San Diego, CA, USA)
was used for preparation of receptors and ligands. PyMol 1.1 (Schrödinger, LLC, New York, NY, USA)
was used for final visualization of the best conformers (tested compound-receptor).
4. Conclusions
TQ was used as a starting compound for preparation of ATQ and benzoxazoles 1a
studied for their antimicrobial and cytotoxic activities. ATQ had stronger antifungal activity than TQ
and reference drug nystatin, while benzoxazoles 1a 1j didn’t show antifungal activity. All prepared
–1j which were
–
derivatives had weaker antibacterial activity in comparison to TQ. Amination at position C-3 of
TQ, resulted in increased toxicity against four selected cancer cell lines. An additional increase
in cytotoxicity was gained through synthesis of benzoxazole derivatives with specific substituents
at the 2 position of the benzoxazole ring. The most potent antitumor compound 1f contains the
p-fluoro-phenyl group, while the derivative 1e, which is the least toxic towards human lung fibroblasts,
contains a p-chlorophenyl substituent at the 2 position of the benzoxazole ring. Moreover, benzoxazole
derivatives 1i (containing 4-hydroxy-3-methoxyphenyl group at the 2 position of the benzoxazole
ring) and 1j (containing a thiophene-2-yl group at the 2 position of the benzoxazole ring) had stronger
antitumor activities, when compared to TQ. The compound 1f inhibited phosphorylation of Akt and
IGF1 Receptor
β in HeLa and HepG2 cell lines, while the compound 1e showed weak, time and
concentration dependent inhibition of Akt phosphorylation in both cancer cell lines. According to
docking studies results, it can be expected that compounds 1f and 1e have significant effects towards
topoisomerase II, NFκB and PTEN receptors. With the aim of better understanding the mode of action
of newly synthesized compounds, their interactions with molecules involved in tumor cells signaling
need to be further evaluated.
Supplementary Materials: The following are available online. Scheme S1: Numbering scheme for
3-aminothymoquinone (ATQ) and series of benzoxazoles (1a
of compounds 1a
1c and 1j; Mass spectra for compounds ATQ and 1a
–
1j); Table S1: Mobile phases used in purification
1b
–1j.
–
1j; Table S2: The crystal structure data of compound 1a; IR spectra for compounds ATQ
,
,
–
1j; 1H- and 13C-NMR spectra for compounds ATQ and 1a
Author Contributions: Conceptualization, U.G., S.P. and D.Z.; Data curation, U.G.; Formal analysis, S.Š.-H. and
E.V.; Funding acquisition, D.Z.; Investigation, U.G., S.Š.-H., A.O., E.V., S.R., I.N., B.M., I.T., J.K., S.T., E.K., S.K.P.,
A.H. and M.K.; Methodology, U.G., S.P., S.K.P. and D.Z.; Project administration, S.Š.-H., A.O. and D.Z.; Resources,
D.Z.; Supervision, S.P. and D.Z.; Writing-original draft, U.G.; Writing- review & editing, U.G., S.P., S.Š.-H., A.O.,
E.V., S.R., I.N., B.M., I.T., J.K., S.T., E.K., S.K.P., A.H., M.K. and D.Z.
Funding: The authors are grateful to financial support from Federal Ministry of education and science in Bosnia
and Herzegovina (grant number: 05-39-3629-1/14, Most: Završnik D. “Modeliranje i doking studije novih
potentnih azometinskih derivata timokinona i njihovih organometalnih kompleksa”. Ministarstvo za obrazovanje,
nauku, kulturu i sport FBiH, 2014–2015. godine (Mostar, 22.12.2014. godine, Ugovor broj: 05-39-3629-1/14).
Acknowledgments: This paper is supported by University of Rijeka research grant “Development and research
of prodrugs with antitumour effects”. We greatly appreciate access to equipment in possession of University
of Rijeka within the project “Research Infrastructure for Campus-based Laboratories at University of Rijeka”,
financed by European Regional Development Fund (ERDF). We thank EN-FIST Centre of Excellence, Trg of 13,
1000 Ljubljana, Slovenia for using SuperNova diffractometer and Slovenian Research Agency for financial support
(P1-0175).

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Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds ATQ and 1a–1j are available from the authors.
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