Design, synthesis, and cytotoxicity of 5-fluoro-2-methyl-6-(4-aryl-piperazin-1-yl) benzoxazoles

Article abstract of DOI:10.3390/molecules21101290

To design new compounds suitable as starting points for anticancer drug development, we have synthesized a novel series of benzoxazoles with pharmaceutically advantageous piperazine and fluorine moieties attached to them. The newly synthesized benzoxazoles and their corresponding precursors were evaluated for cytotoxicity on human A-549 lung carcinoma cells and non-cancer HepaRG hepatocyes. Some of these new benzoxazoles show potential anticancer activity, while two of the intermediates show lung cancer selective properties at low concentrations where healthy cells are unaffected, indicating a selectivity window for anticancer compounds.

Full text of DOI:10.3390/molecules21101290

molecules
Article
Design, Synthesis, and Cytotoxicity of
5-Fluoro-2-methyl-6-(4-aryl-piperazin-1-yl) Benzoxazoles
Thuraya Al-Harthy 1, Wajdi Michel Zoghaib 1, Maren Pflüger 2, Miriam Schöpel 3, Kamil Önder 4,
Maria Reitsammer 4, Harald Hundsberger 2, Raphael Stoll 3 and Raid Abdel-Jalil 1,
*
1
Chemistry Department, College of Science, Sultan Qaboos University, Muscat 123, Oman;
thurayalharthi@gmail.com (T.A.-H.); zoghaibw@squ.edu.om (W.M.Z.)
2
IMC Fachhochschule Krems University of Applied Sciences Krems, Piaristengasse 1, Krems A-3500, Austria;
maren.pflueger@fh-krems.ac.at (M.P.); harald.hundsberger@fh-krems.ac.at (H.H.)
Biomolecular NMR, Ruhr University of Bochum, Bochum 44780, Germany;
miriam.schoepel@ruhr-uni-bochum.de (M.S.); raphael.stoll@ruhr-uni-bochum.de (R.S.)
Research Program for Rational Drug Design in Dermatology and Rheumatolog, Department of Dermatology,
General Hospital of Salzburg, Paracelsus Medical, University of Salzburg, Müllner Hauptstraße 48,
Salzburg A-5020, Austria; oender@procomcure.com (K.Ö.); m.reitsammer@salk.at (M.R.)
Correspondence: jalil@squ.edu.om; Tel.: +968-2414-1483
3
4
*
Academic Editor: Derek J. McPhee
Received: 5 August 2016; Accepted: 22 September 2016; Published: 27 September 2016
Abstract: To design new compounds suitable as starting points for anticancer drug development,
we have synthesized a novel series of benzoxazoles with pharmaceutically advantageous piperazine
and fluorine moieties attached to them. The newly synthesized benzoxazoles and their corresponding
precursors were evaluated for cytotoxicity on human A-549 lung carcinoma cells and non-cancer
HepaRG hepatocyes. Some of these new benzoxazoles show potential anticancer activity, while two
of the intermediates show lung cancer selective properties at low concentrations where healthy cells
are unaffected, indicating a selectivity window for anticancer compounds.
Keywords: benzoxazole; piperazine; fluorine Cytotoxicity; A-549 lung carcinoma cells;
HepaRG hepatocytes
1. Introduction
Despite all the progress in biomedical research, cancer remains a leading cause of death worldwide.
Therefore, many efforts have been made to find new therapeutic agents that could potentially improve
cancer therapy by using more selective and less toxic drugs.
Heterocyclic compounds are considered to be the most interesting drug-based structures in
medicinal chemistry due to their widespread presence in lead compounds. Specifically, benzoxazoles
have received much attention in recent years because of their broad spectrum of biological activities.
Recent studies show that several benzoxazole analogues exhibit a number of biological activities
including anticancer [1], antifungal [2], antituberculosis [3], mPGES-1 inhibitor [4], 5-HT receptor
antagonist [5], CETP inhibitor [6], and antiplasmodial [7] activities.
Broad therapeutic spectrum compounds intrinsically possess a certain amount of cytotoxicity.
Structural modification on lead anticancer compounds may eliminate or reduce cytotoxicity to
a minimum level. Usually, a plethora of analogues are synthesized to decipher the underlying
structure activity relationship (SAR) and to identify more selective, less toxic, and ADME-improved
leads. Often, substitutions found to be bioactively advantageous on a certain active scaffold can be
introduced to other scaffolds leading to the enhancement of bioactivity [8,9]. Fluorine and piperazine
are common appendages and substituents in medicinal chemistry due to their immense utilities in
drug design and their unique bioactivities [10,11].
Molecules 2016, 21, 1290; doi:10.3390/molecules21101290
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1290
2 of 11
The introduction of fluorine and piperazine appendages played a vital role in enhancing
the antimicrobial activities of the quinolone drugs, e.g., ciprofloxacin®
) (Figure 1), the most
active antibiotic drug on the market [12]. Similarly, we have successfully synthesized a variety
of heterocyclic systems incorporating fluorine and piperazine moieties, such as quinoxaline [13] ( ),
benzimidazole [14] ( ), quinazolinone [15] ( ), and recently benzothiazole [16] ( ). Some derivatives of
(
1
2
3
4
5
our systems showed very promising biological activities, which were attributed to the presence of both
appendages. In this study, we combined the benzoxazole scaffold with both piperazine and fluorine
moieties and evaluated their potential in anticancer activity.
O
O
F
N
N
R
R
F
N
F
OH
R2
N
N
H
N
N
N
N
N
HN
3
1
2
Ar
Ar
N
O
N
N
N
N
H
N
F
N
N
S
N
OH
NO2
N
F
NH
Ar
O
N
Ar
N
CH3
F
F
4
5
6
7
Figure 1. Examples of heterocyclic systems that contain fluorine and piperazine.
Based on that, and as a part of our ongoing research in developing new potentially bioactive
heterocyclic compounds, this study describes the synthesis of new 4-fluoro-5-(substituted
phenyl-piperazin-1-yl)-2-nitro-phenoles
(6) and their corresponding 5-fluoro-6-(substituted
phenyl-piperazin-1-yl)-benzoxazoles (7) functionalized with both piperazine and fluorine moieties.
2. Results and Discussion
2.1. Chemistry
A new series of benzoxazole derivatives was synthesized using a multi-step protocol that involved
regioselective nitration and piperazinylation, followed by a one-pot in-situ reductive cyclization step
as depicted in Scheme 1.
To start with, the commercially available 3-chloro-4-fluorophenol (8) was nitrated using mild
nitration conditions. The choice of a mild nitration step was effective and led to a shorter synthetic
procedure compared to the use of strong acidic conditions. In fact, nitration yielded 5-chloro-4-fluoro-
2-nitrophenol (9) and its 3-chloro-4-fluoro-2,6-dintirophenol (10) counterpart. Carefully controlling
the reaction temperature, we were able to isolate
column chromatography.
9 in a 60% yield after purification using flash
Subsequently, the piperazinylation of
the key intermediates 6a . Changing the solvent from dimethylsulfoxide to toluene simplified the
work and considerably improved the yield up to 83% for 6a 6b 6d 6e 6g 6i, and 6j as shown in
Table 1. For the preparation of 6c 6f, and 6h chlorobenzene was the solvent of choice, due to poor
9 using excess substituted phenyl N-piperazine yielded
–
j
,
,
,
,
,
,
solubility of the corresponding aryl-piperazines in toluene nonetheless using chlorobenzene required
a longer reaction time. Purification using column chromatography was required to remove unreacted
N-piperazines.

Molecules 2016, 21, 1290
3 of 11
NO2
10
Cl
F
OH
Cl
OH
Cl
F
OH
(i)
F
NO2
NO2
8
9
(ii)
R1
N
N
OH
F
NO2
6a-j
(iii)
R1
N
N
O
CH3
N
F
7a-j
Scheme 1. Synthesis of 5-fluoro-6-(substituted phenyl-piperazin-1-yl)-benzoxazoles; reagents and
conditions: (i) 15% HNO3/AcOH, 0 ◦C, (ii) aryl piperazine, refluxing toluene or chlorobenzene
(iii) In AcOH, benzene, trimethyl orthoacetate.
Table 1. Data for the synthesis of benzoxazoles and their precursors.
Compound
R
Time (h)
Yield * %
Solvent **
Compound
Time (h)
Yield * %
Solvent **
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
p-CH3
p-OH
p-F
p-OCH3
o-OH
o-F
m-OH
m-OCH3
3,4-Cl
5.5
2
24
2
1.5
3
2.5
24
2
72
73
37
73
27
71
83
28
73
33
Toluene
Toluene
Chlorobenzene
Toluene
Toluene
Chlorobenzene
Toluene
7a
7b
7c
7d
7e
7f
7g
7h
7i
4
3
4
2.5
1
1.5
2
2
1
1
71
53
63
51
70
55
74
74
77
75
Benzene
Benzene
Benzene
Benzene
Benzene
Benzene
Benzene
Benzene
Benzene
Benzene
Chlorobenzene
Toluene
6j
5
Toluene
7j
*
The yield was calculated after purification by crystallization or column chromatography. ** Solvent at
reflux temperature.
The cyclization of intermediates (6a–j) was achieved by an indium-mediated reaction using
indium/acetic acid followed by addition of trimethyl orthoaceate to form the corresponding
benzoxazole derivatives (7a ). This one-pot, reductive cyclization simplified and shortened
–
j
the synthetic procedure and provided yields ranging from good to very good (53%–75%) after
recrystallization as shown in Table 1. All the newly synthesized compounds were characterized
using spectroscopic techniques. High-resolution mass spectrometry shows the correct molecular ion
peaks. The formation of 3-chloro-4-fluoro-2,6-dinitrophenol (10) as a byproduct during the nitration
step simultaneously with
were evident in the spectra of 10 and
the methyl proton signals appeared as singlets around
9
was confirmed by 1H-NMR in which one and two types of 1H signals
, respectively. In the 1H-NMR spectra of compounds (7a
),
2.54–2.60 ppm. The H-4 protons were the
9
–j
δ

Molecules 2016, 21, 1290
4 of 11
most deshielded protons, showing a large ortho coupling with the adjacent fluorine JH-F = 11.2–12.0 Hz
compared with H-7 meta coupling JH-F = 5.0–8.0 Hz. Piperazinyl protons resonated as broad singlets
around
around
of compounds (7a
δ
3.10–3.36 ppm. The disappearance of the exchangeable signals in intermediates (6a
10.40–10.80 ppm confirmed the successful cyclization to benzoxazoles. In 13C-NMR spectra
), the most deshielded carbon was C-2, resonating around 163.0–164.6 ppm.
13.5–14.6 ppm and the piperazine methylene
–j)
δ
–
j
δ
The methyl carbons appear in the upfield region around
δ
carbons resonated around δ 48.0–52.0 ppm.
2.2. Cytotoxicity
The cytotoxicity of prepared benzoxazoles (7a–j) and intermediates (6a–j and 9) were measured by
applying the CellTiter-Glo® luminescent cell proliferation assay (G755A, Promega, Madison, WI, USA).
This method permits the determination of viable cells numbers in the culture based on quantitation
of the ATP present, which denotes the presence of metabolically active cells [17]. In vitro cytotoxicity
analysis was overall performed following the protocol as described in “Guidance Document on Using
in Vitro Data to Estimate in Vivo Starting Doses for Acute Toxicity Based on Recommendations” [18].
Human A-549 cells are human lung cancer epithelial cells, which were chosen as the tumor model,
whereas the HepaRG
human hepatocytes. HepaRG
detect a dose-dependent impact for each of the twelve test compounds, concentrations of 0, 0.1, 1.0,
10, 50, 100, 250, and 500 M were analyzed with both cell lines, and doxorubicine (IC50 = 5.1 M for
HepaRG™ cells) was used as standard.
As shown in Table 2, we observed visible solubility problems for some compounds at
concentrations of 50 M, 100 M, and 250 M, while 6d and 7f were completely soluble at all
concentrations. Therefore, a CT50 value of ~50 M could only be obtained for 6d, which shows
™
cell line is a classic hepatic cell line that retains many characteristics of primary
™
cells are terminally differentiated. In order to obtain CT50 values and
µ
µ
µ
µ
µ
µ
equal toxicity to both cell lines, indicating a non-differential behavior.
Since the majority of test compound solutions with concentrations above 50
µ
M precipitated
M.
, 7i, and 7j, it was not possible to draw any conclusion because
in cell-culture media, we performed cytotoxicity assays for healthy and cancer cells at 50
For compounds 6i 7a 7d 7g 7h
of the precipitation problem.
Compared to healthy control cells, compounds 6a 6b, 6g, and 6j showed significant increased
µ
,
,
,
,
,
toxicity in lung cancer A-549 cells >30%–40%. Interestingly, two compounds were more toxic to healthy
cells compared with lung cancer cells 7b and 7j.
Even at concentrations as low as 10
compounds were non-toxic to healthy cells. At a 10-
compounds was toxic to healthy hepatocytes, but at least four compounds (6a
were highly toxic to lung cancer cells, killing 30%–40% of all cancer cells at this low concentration.
Concentrations below 10 M were not toxic at all, indicating a small activity window, which has to be
further extended through improving efficacy in order to obtain useful compounds.
µ
M, 6a and 6g killed ~40% of lung cancer cells, whereas all
M concentration, nearly none of the tested
6b 6g, and 6j
µ
,
,
)
µ

Molecules 2016, 21, 1290
5 of 11
Table 2. Cell viability shown in % for benzoxazole derivatives and their intermediates.
Concentration (C) µM
Liver Cells, Hepatocytes
Lung Cancer Cells
Compound
0
0.1
1
10
50
100
250 500
0
0.1
1
10
50
100 250 500
6a
6b
6c
6d
6e
6f
6g
6h
6i
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
86
107
95
97
123
101
87
110
104
109
109
95
115
97
99
107
94
108
97
112
120
110
101
104
110
112
108
99
81
94
91
101
85
91
86
87
99
91
85
90
97
101
57
74
76
87
83
76
96
89
62
79
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
97
99
104
103
101
97
96
106
95
98
101
92
92
98
93
103
96
100
105
98
104
100
103
111
104
100
96
106
93
94
96
94
97
98
92
99
92
93
64
68
102
78
96
93
62
105
96
65
90
92
101
87
75
91
92
97
88
81
61
65
94
58
71
60
51
87
69
62
60
87
90
87
74
91
73
95
76
70
58
94
44
13
54
46
34
107
105
110
117
79
119
101
105
98
46
42
58
108
60
6j
102
7a
7b
7c
7d
7e
7f
7g
7h
7i
63
93
75
83
82
68
95
63
90
75
47
91
107
106
96
100
100
100
71
59
60
27
53
7
54
7
83
28
63
106
102
102
90
102
94
7j
65

Molecules 2016, 21, 1290
6 of 11
3. Materials and Methods
3.1. Chemistry
Reagents were obtained from commercial sources and used without further purification.
All solvents were purchased from Sigma Aldrich (Munich, Germany) except for benzene and
chlorobenzene, which were purchased from BDH. The staring materials, 3-chloro-4-fluorophenol
and trimethyl orthoacetate, were purchased from Alfa Aesar (Munich, Germany).
The newly synthesized compounds were characterized using IR, 1H-NMR, 13C-NMR, HRMS,
and a melting point. IR spectra (4000–400 cm−1) were recorded as KBr discs on a Perkin–Elmer
Spectrum BX FTIR spectrophotometer (Akron, OH, USA). The 1H-NMR spectra were measured on
Varian (Palo Alto, CA, USA) 400 MHz, and 13C-NMR spectra were recorded at 100 MHz using TMS
as an internal standard assigned as zero ppm, and chemical shifts
δ were given in part per million
(ppm) downfield from TMS. The EI and FAB high-resolution mass spectra were recorded on a Finnigan
MAT 312 mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) Melting points were
determined on an SMP 10 Stuart Scientific apparatus (Keison Products, Essex, UK). The reactions
were monitored by thin layer chromatography (TLC) using silica gel plates, and the components were
visualized by UV light. Flash column chromatography was carried out using silica gel 60 (70–230 mesh)
(Aldrich, Munich, Germany) as the stationary phase.
5-chloro-4-fluoro-2-nitrophenol (
9
) and 3-chloro-4-◦fluoro-2,6-dintirophenol (10): for the nitrating
solution, 15% HNO3/AcOH was prepared and kept at 0 C. 3-chloro-4-fluorophenol (8) (5.0 g, 34 mmol)
was added in small portions to the solution and stirred for 1 h at 0 ◦C. The reaction mixture poured
into an ice-water bath, filtered by suction, washed thoroughly with water, and then dried and purified
by column chromatography 10% MeOH/CH2Cl2.
◦
5-Chloro-4-fluoro-2-nitro-phenol (
9
) (yield 60%). m.p.: 82–83 C. FTIR (KBr): 3471, 1629, 1530, 1330, 1247,
1
1150 cm−1. H-NMR (400 MHz, CDCl3)
δ
(ppm): 10.40 (bs, 1H, O-H), 7.91 (d, J = 8.4 Hz, H-6), 7.26 (d,
(ppm): 152.2(C), 151.7 (C), 150.0 (C), 132.5 (C), 121.8
J = 12.0 Hz, H-3). 13C-NMR (100 MHz, CDCl3)
δ
(CH), 112.1 (CH). MS (ESI) [M] m/z calcd for C6H3ClFNO3: 190.98; found: 189.75.
1
3-Chloro-4-fluoro-2,6-dintirophenol (10) (yield 20%). H-NMR (400 MHz, CDCl3)
δ (ppm): 10.60 (bs, 1H,
O-H), 8.11 (d, J = 9.2 Hz, H-3). MS (ESI) [M] m/z calcd for C6H2ClFN2O5: 235.96; found: 234.79.
3.1.1. General Procedure for Synthesis of 4-Fluoro-5-(substitutedphenyl-piperazin-1-yl)-2-nitro-phenol
(6a, 6b, 6d, 6e, 6g, 6i, 6j)
5-Chloro-4-fluoro-2-nitro-phenol (
corresponding aryl piperazine (13.1 mmol) was then added and refluxed for 2–6 h. Afterwards, water
was added and extracted with dichloromethane (3 30 mL), and the organic layer was concentrated,
dried over sodium sulfate, and purified by flash column chromatography.
9) (0.500 g, 2.61 mmol) was dissolved in toluene (5 mL), and the
×
◦
4-Fluoro-2-nitro-5-(4-phenyl-piperazin-1-yl)-phenol (6a) (yield 72%, 40% hexane/CH2Cl2). m.p.: 197–198 C.
1
FTIR (KBr): 3377, 1634, 1508, 1388, 1225 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 10.90 (bs, 1H,
O-H), 7.74 (d, J = 13.2 Hz, 1H, H-3), 6.46 (d, J = 7.6 Hz, 1H, H-6), 3.52 (s, 2H, piperazine), 3.34 (s,
2H, piperazine). 13C-NMR (100 MHz, CDCl3)
(ppm): 153.9 (C), 150.6 (C), 148.3 (C), 145.9 (C), 129.3
δ
(CH), 120.7 (C), 116.5 (CH), 112.0 (CH), 111.8 (CH), 105.2 (CH), 49.31 (CH2), 49.26 (CH2). HRMS (EI)
m/z calcd for C16H16FN3O3: 317.1176; found: 317.1163.
◦
4-Fluoro-2-nitro-5-(4-p-tolyl-piperazin-1-yl)-phenol (6b) (yield 73%, CH2Cl2). m.p.: 178–179 C. FTIR
1
(KBr): 3354, 1631, 1511, 1390, 1220 cm−1. H-NMR (400 MHz, CDCl3)
δ
(ppm): 10.84 (bs, 1H, O-H), 7.68
(d, J = 13.2 Hz, 1H, H-3), 6.83 (d, J = 7.2 Hz, 1H, H-6), 3.46 (s, 2H, piperazine), 3.22 (s, 2H, piperazine),
2.23 (s, 1H, CH3-Ph). 13C-NMR (100 MHz, CDCl3)
(ppm): 154.0 (C), 148.4 (C), 148.3 (C), 145.9 (C),
δ
129.9 (CH), 124.8 (C), 124.7 (C), 117.0 (CH), 112.0 (CH), 105.3 (CH), 49.8 (CH2), 49.2 (CH2), 20.5 (CH3).
HRMS (ESI) [M + H+] m/z calcd for C17H18FN3O3: 332.1405; found: 332.1407.

Molecules 2016, 21, 1290
7 of 11
4-Fluoro-5-[4-(4-fluoro-phenyl)-piperazin-1-yl]-2-nitro-phenol (6d) (yield 73%, CH2Cl2). m.p.: 151–152 ◦C.
1
FTIR (KBr): 3309, 3103, 1637, 1535, 1244 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 10.88 (bs, 1H,
O-H), 7.74 (d, J = 13.6 Hz, 1H, H-3), 6.47 (d, J = 7.60 Hz, 1H, H-6), 3.51 (s, 2H, piperazine), 3.25 (s, 2H,
piperazine). 13C-NMR (100 MHz CDCl3)
(ppm): 158.9 (C), 156.5 (C), 153.9 (C), 148.3 (C), 146.0 (C),
δ
124.9 (C), 118.5 (CH), 115.7 (CH), 111.8 (CH), 105.3 (CH), 50.2 (CH2), 49.3 (CH2). HRMS (EI) m/z calcd
for C16H15F2N3O3: 335.1081; found: 335.1066.
◦
4-Fluoro-5-[4-(4-methoxy-phenyl)-piperazin-1-yl]-2-nitro-phenol (6e) (yield 27%, CH2Cl2). m.p.: 202–204 C.
1
FTIR (KBr): 3448, 2933, 1638, 1515, 1225 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 10.84 (bs, 1H,
O-H), 7.68 (d, J = 13.2 Hz, 1H, H-3), 6.41 (d, J = 7.6 Hz, 1H, H-6), 3.72 (s, 1H, OCH3), 3.47 (s, 2H,
piperazine), 3.17 (s, 2H, piperazine). 13C-NMR (100 MHz, CDCl3)
(ppm): 153.9 (C), 148.3 (C), 145.9
δ
(C), 122.5 (C), 121.4 (C), 119.2 (C), 114.7 (CH), 112.0 (CH), 111.8 (CH), 105.5 (CH), 55.6 (CH3), 51.4
(CH2), 49.0 (CH2). HRMS (EI) m/z calcd for C17H18FN3O4: 347.1281; found: 347.1264.
4-Fluoro-5-[4-(2-fluoro-phenyl)-piperazin-1-yl]-2-nitro-phenol (6g) (yield 83%, CH2Cl2). m.p.: 180–181 ◦C.
1
FTIR (KBr): 3457, 2928, 1636, 1508, 1265 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 10.82 (bs, 1H,
O-H), 7.69 (d, J = 13.2 Hz, 1H, H-3), 6.42 (d, J = 7.6 Hz, 1H, H-6), 3.51 (s, 2H, piperazine), 3.25 (s, 2H,
piperazine). 13C-NMR (100 MHz, CDCl3)
(ppm): 157.0 (C), 154.5 (C), 154.0 (C), 148.5 (C), 145.9 (C),
δ
139.3 (C), 124.6 (CH), 123.3 (CH), 119.1 (CH), 116.2 (CH), 111.8 (CH), 105.4 (CH), 50.2 (CH2), 49.5 (CH2).
HRMS (EI) m/z calcd for C16H15F2N3O3: 335.1081; found: 335.1070.
◦
4-Fluoro-5-[4-(3-methoxy-phenyl)-piperazin-1-yl]-2-nitro-phenol (6i) (yield 73%, CH2Cl2). m.p.: 148–149 C.
1
FTIR (KBr): 3426, 2937, 1629, 1504, 1209 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 10.83 (bs, 1H,
O-H), 7.68 (d, J = 13.6 Hz, 1H, H-3), 6.52 (d, J =8.0 Hz, 1H, H-6), 3.74 (s, 1H, OCH3), 3.45 (s, 2H,
piperazine), 3.28 (s, 2H, piperazine). 13C-NMR (100 MHz, CDCl3)
(ppm): 160.6 (C), 153.9 (C), 151.9
δ
(C), 148.3 (C), 145.9 (C), 130.0 (CH), 124.8 (C), 112.0 (CH), 111.8 (CH), 109.1 (CH), 105.2 (CH), 103.0 (CH),
55.2 (CH3), 49.2 (CH2), 49.1 (CH2). HRMS (EI) m/z calcd for C17H18FN3O4: 347.1281; found: 347.1297.
5-[4-(3,4-Dichloro-phenyl)-piperazin-1-yl]-4-fluoro-2-nitro-phenol (6j) (yield 33%, CH2Cl2).
m.p.:
◦
1
211–213 C. FTIR (KBr): 3448, 2924, 1634, 1513, 1207 cm−1. H-NMR (400 MHz, CDCl3)
δ
(ppm): 10.80
(bs, 1H, O-H), 7.70 (d, J = 13.6 Hz, 1H, H-3), 6.42 (d, J = 7.6 Hz, 1H, H-6), 3.49 (s, 2H, piperazine), 3.30
(s, 2H, piperazine). 13C-NMR (100 MHz, CDCl3)
(ppm): 153.9 (C), 149.8 (C), 148.3 (C), 148.0 (C), 145.9
δ
(C), 133.0 (C), 130.7 (CH), 120.1 (C), 117.9 (CH), 115.9 (CH), 112.1 (CH), 105.4 (CH), 49.0 (CH2), 48.8
(CH2). HRMS (EI) m/z calcd for C16H14Cl2FN3O3: 385.0396; found: 385.0374.
3.1.2. General Procedure for Synthesis 5-[4-(o/m/p-Hydroxy-phenyl)-piperazin-1-yl]-4-fluoro-
2-nitro-phenol (6c, 6f, 6h)
5-Chloro-4-fluoro-2-nitro-phenol (9) (0.250 g, 1.31 mmol) was dissolved in chlorobenzene (5 mL),
substituted phenyl-piperazine (6.53 mmol) was added and refluxed overnight. The reaction mixture
was extracted with water and dichloromethane, and the organic layer was concentrated and purified
by flash column chromatography.
4-Fluoro-5-[4-(4-hydroxy-phenyl)-piperazin-1-yl]-2-nitro-phenol (6c) (yield 37%, 10% EtOAc/CH2Cl2).
1
m.p.: 218–219 ◦C. FTIR (KBr): 3475, 3117, 1631, 1515, 1242, cm−1. H-NMR (400 MHz, CDCl3 with
2 drops of CD3OD)
1H, H-6), 3.46 (s, 2H, piperazine), 3.13 (s, 2H, piperazine). 13C-NMR (100 MHz, CDCl3 with 2 drops
of CD3OD) (ppm): 152.8 (C), 147.5 (C), 147.4 (C), 147.3 (C), 144.9 (C), 123.8 (C), 118.5 (CH), 115.0
δ (ppm): 10.81 (bs, 1H, O-H), 7.67 (d, J = 13.2 Hz, 1H, H-3), 6.42 (d, J = 7.6 Hz,
δ
(CH), 110.7 (CH), 104.4 (CH), 50.4 (CH2), 50.4 (CH2). HRMS (EI) m/z calcd for C16H16FN3O4: 333.1125;
found: 331.1141.
◦
4-Fluoro-5-[4-(2-hydroxy-phenyl)-piperazin-1-yl]-2-nitro-phenol (6f) (yield 71%, CH2Cl2). m.p.: 176–178 C.
1
FTIR (KBr): 3452, 2923, 1636, 1520, 1227 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 10.81 (bs, 1H,
O-H), 7.70 (d, J = 13.2 Hz, 1H, H-3), 6.43 (d, J =8.0 Hz, 1H, H-6), 3.45 (s, 2H, piperazine), 2.99 (s, 2H,
piperazine). 13C-NMR (100 MHz, DMSO)
(ppm): 152.9 (C), 150.6 (C), 147.5 (C), 145.2 (C), 139.7 (C),
δ

Molecules 2016, 21, 1290
8 of 11
126.0 (C), 123.7 (CH), 116.1 (CH), 119.9 (CH), 119.3 (CH), 112.6 (CH), 106.5 (CH), 50.3 (CH2), 49.7 (CH2).
HRMS (EI) m/z calcd for C16H16FN3O4: 333.1125; found: 333.1111.
4-Fluoro-5-[4-(◦3-hydroxy-phenyl)-piperazin-1-yl]-2-nitro-phenol (6h) (yield 28%, 25% EtOAc/CH2Cl2).
1
m.p.: 207–208 C. FTIR (KBr): 3448, 2924, 1636, 1506, 1260 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm):
10.83 (bs, 1H, O-H), 7.69 (d, J = 13.2 Hz, 1H, H-3), 7.11 (m, 1H, H-6), 3.46 (s, 2H, piperazine), 3.28 (s,
2H, piperazine). 13C-NMR (100 MHz, DMSO)
(ppm): 158.5 (C), 152.7 (C), 147.5 (C), 147.3 (C), 145.1
δ
(C), 130.1 (CH), 126.1 (C), 112.9 (CH), 107.2 (CH), 107.0 (CH), 106.4 (CH), 103.0 (CH), 49.4 (CH2), 48.4
(CH2). HRMS (EI) m/z calcd for C16H16FN3O4: 333.1125; found: 333.1131.
3.1.3. General Procedure for Synthesis of 5-Fluoro-6-(substitutedphenyl-piperazin-1-yl)-
benzoxazoles (7a–j)
Fluoro-5-(substituted phenyl-piperazin-1-yl)-2-nitro-phenol (6a–j) (1 mmol) was added to a mixture of
indium powder (8 mmol) and acetic acid (20 mmol) in benzene (2 mL). Trimethyl orthoacetate (8 mmol)
in benzene (2 mL) was then added to the reaction mixture, stirred, and refluxed for 1–4 h. The reaction
mixture was cooled to room temperature, washed with saturated sodium bicarbonate, extracted
with dichloromethane (3
×
10 mL), and washed with water followed by saturated sodium chloride
solution (brine). The organic extracts were dried over sodium sulfate anhydrous and concentrated
using a rotary evaporator. The residue was then recrystallized from absolute ethanol to yield the
corresponding benzoxazole.
5-Fluoro-2-methyl-6-(4-phenylpiperazin-1-yl)-benzo[d]oxazole (7a) (yield 71%). m.p.: 153–154 ◦C. FTIR
1
(KBr): 2848, 1638, 1242 cm−1. H-NMR (400 MHz, CDCl3)
δ
(ppm): 7.27 (m, 1H, H-4), 7.06 (d, J = 6.8 Hz,
1H, H-7), 3.35 (s, 2H, piperazine), 3.22 (s, 2H, piperazine), 2.54 (s, 3H, CH3). 13C-NMR (100 MHz,
CDCl3) (ppm): 163.1 (C), 153.9 (C), 151.5 (C), 150.0 (C), 137.2 (C), 134.9 (C), 128.2 (CH), 119.3 (CH),
δ
115.4 (CH), 105.4 (CH), 99.5 (CH), 50.2 (CH2), 48.6 (CH2), 13.5 (CH3). HRMS (EI) m/z calcd for
C18H18FN3O: 311.1434; found: 311.1423.
◦
5-Fluoro-2-methyl-6-(4-p-tolyl-piperazin-1-yl)-benzooxazole (7b) (yield 53%). m.p.: 168–170 C. FTIR (KBr)
1
1636, 1265 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.27 (d, J =11.6 Hz, 1H, H-4), 7.06 (m, 1H, H-7),
3.28 (s, 2H, piperazine), 3.19 (s, 2H, piperazine), 2.54 (s, 3H, CH3), 2.23 (s, 3H, CH3-Ph). 13C-NMR
(100 MHz, CDCl3) (ppm): 164.1 (C), 152.5 (C), 149.0 (C), 147.5 (C), 138.2 (C), 135.9 (C), 135.8 (C), 129.7
δ
(CH), 116.8 (CH), 106.2 (CH), 100.5 (CH), 51.2 (CH2), 50.1(CH2), 20.5 (CH3), 14.6 (CH3). HRMS (EI)
m/z calcd for C19H20FN3O: 325.1590; found: 325.1577.
4-[4-(5-Fluoro-2-methyl-benzooxazol-6-yl)-piperazin-1-yl]-phenol (7c) (yield 63%). m.p.: 188–191 ◦C. FTIR
1
(KBr): 3466, 2937, 1640, 1267 cm−1. H-NMR (400 MHz, DMSO)
δ
(ppm): 8.90 (bs, 1H, O-H), 7.48 (d,
J = 12.0 Hz, 1H, H-4), 7.38 (d, J = 7.2 Hz, 1H, H-7), 3.36 (s, 2H, piperazine), 3.10 (s, 2H, piperazine), 2.54
(s, 3H, CH3). 13C-NMR (100 MHz, DMSO)
(ppm): 164.6 (C), 154.6 (C), 152.2 (C), 147.6 (C), 144.5 (C),
δ
138.4 (C), 135.5 (C), 118.5 (CH), 115.9 (CH), 106.5 (CH), 101.6 (CH), 51.3 (CH2), 50.6 (CH2), 14.6 (CH3).
HRMS (EI) m/z calcd for C18H18FN3O2: 327.1383; found: 327.1391.
5-Fluoro-6-[4-(4-fluoro-phenyl)-piperazin-1-yl]-2-methyl-benzooxazole (7d) (yield 51%). m.p.: 148–150 ◦C.
1
FTIR (KBr): 2928, 1640, 1267 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.27 (d, J = 11.6 Hz, 1H,
H-4), 7.06 (d, J = 7.2 Hz, 1H, H-7), 3.20 (s, 2H, piperazine), 2.25 (s, 2H, piperazine), 2.54 (s, 3H, CH3).
13C-NMR (100 MHz, CDCl3)
δ
(ppm): 164.3 (C), 154.9 (C), 152.2 (C), 147.6 (C), 144.5 (C), 138.4 (C), 135.5
(C), 118.5 (CH), 115.9 (CH), 106.5 (CH), 101.6 (CH), 51.6 (CH2), 51.0 (CH2), 14.6 (CH3). HRMS (EI) m/z
calcd for C18H17F2N3O: 329.1340; found: 329.1352.
◦
5-Fluoro-6-[4-(4-methoxy-phenyl)-piperazin-1-yl]-2-methyl-benzooxazole (7e): (yield 70%). m.p.: 156–157 C.
1
FTIR (KBr): 2946, 1636, 1236 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.27 (d, J = 11.6 Hz, 1H, H-4),
7.06 (d, J = 7.2 Hz, 1H, H-7), 3.72 (s, 3H, OCH3), 3.21 (s, 2H, piperazine), 3.20 (s, 2H, piperazine), 2.54
(s, 3H, CH3). 13C-NMR (100 MHz, CDCl3) δ (ppm): 164.3 (C), 153.9 (C), 151.5 (C), 146.5 (C), 144.2 (C),

Molecules 2016, 21, 1290
9 of 11
137.2 (C), 134.8 (C), 117.6 (CH), 113.5 (CH), 105.2 (CH), 99.5 (CH), 54.5 (CH3), 50.2 (CH2), 50.0 (CH2),
13.5 (CH3). HRMS (EI) m/z calcd for C19H20FN3O2: 341.1540; found: 341.1548.
2-[4-(5-Fluoro-2-methyl-benzooxazol-6-yl)-piperazin-1-yl]-phenol (7f) (yield 55%). m.p.: 172–173 ◦C. FTIR
1
(KBr): 3453, 2928, 1636, 1265 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.28 (d, J = 11.2 Hz, 1H,
H-4), 6.94 (d, J = 5.0 Hz, 1H, H-7), 3.24 (s, 2H, piperazine), 3.10 (s, 2H, piperazine), 2.55 (s, 3H, CH3).
13C-NMR (100 MHz, CDCl3)
δ
(ppm): 163.2 (C), 153.9 (C), 151.5 (C), 150.4 (C), 146.5 (C), 137.0 (C), 134.9
(C), 125.7 (CH), 120.5 (CH), 119.2 (CH), 113.2 (CH), 105.5 (CH), 99.6 (CH), 51.6 (CH2), 50.8 (CH2), 13.6
(CH3). HRMS (EI) m/z calcd for C18H18FN3O2: 327.1383; found: 327.1369.
5-Fluoro-6-(4-(2-fluorophenyl)-piperazin-1-yl)-2-methylbenzo[d]oxazole (7g) (yield 74%). m.p.: 157–158 ◦C.
1
FTIR (KBr): 2834, 1634, 1271 cm−1. H-NMR (400 MHz, DMSO)
δ (ppm): 7.49 (d, J = 12.0 Hz, 1H,
H-4), 7.42 (d, J = 7.6 Hz, 1H, H-7), 3.36 (s, 2H, piperazine), 3.15 (s, 2H, piperazine), 2.54 (s, 3H, CH3).
13C-NMR (100 MHz, DMSO)
δ
(ppm): 164.7 (C), 156.7 (C), 154.4 (C), 147.6 (C), 140.1 (C), 138.4 (C), 135.7
(C), 125.4 (CH), 123.0 (CH), 119.8 (CH), 116.4 (CH), 106.4 (CH), 101.8 (CH), 51.3 (CH2), 50.6 (CH2), 14.6
(CH3). HRMS (EI) m/z calcd for C18H17F2N3O: 329.1340; found: 329.1353.
3-[4-(5-Fluoro-2-methyl-benzooxazol-6-yl)-piperazin-1-yl]-phenol (7h) (yield 74%). m.p.: 211–213 ◦C. FTIR
1
(KBr): 3448, 2941, 1622, 1278 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.28 (d, J = 11.6 Hz, 1H,
H-4), 7.12 (d, J = 8.0 Hz, 1H, H-7), 3.35 (s, 2H, piperazine), 3.22 (s, 2H, piperazine), 2.55 (s, 3H, CH3).
13C-NMR (100 MHz, DMSO)
δ
(ppm): 164.7 (C), 158.5 (C), 154.6 (C), 152.7 (C), 147.6 (C), 138.4 (C),
135.6 (C), 130.1 (CH), 107.2 (CH), 106.9 (CH), 106.2 (CH), 103.0 (CH), 101.8 (CH), 51.1 (CH2), 48.9 (CH2),
14.5 (CH3). HRMS (EI) m/z calcd for C18H18FN3O2: 327.1383; found: 327.1368.
5-Fluoro-6-[4-(3-methoxy-phenyl)-piperazin-1-yl]-2-methyl-benzooxazole (7i) (yield 77%). m.p.: 124–125 ◦C.
1
FTIR (KBr): 2942, 1636, 1280 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.33 (d, J = 11.2 Hz, 1H, H-4),
7.11 (d, J = 7.2 Hz, 1H, H-7), 3.80 (s, 3H, OCH3), 3.38 (s, 2H, piperazine), 3.24 (s, 2H, piperazine), 2.60
(s, 3H, CH3). 13C-NMR (100 MHz, CDCl3) δ (ppm): 164.2 (C), 160.6 (C), 154.9 (C), 152.5 (C), 147.5 (C),
138.2 (C), 135.8 (C), 129.9 (CH), 109.1 (CH), 106.2 (CH), 104.8 (CH), 102.8 (CH), 100.5 (CH), 55.2 (CH3),
51.2 (CH2), 49.4 (CH2), 14.5 (CH3). HRMS (EI) m/z calcd for C19H20FN3O2: 341.1540; found: 341.1528.
◦
6-[4-(3,4-Dichloro-phenyl)-piperazin-1-yl]-5-fluoro-2-methyl-benzooxazole (7j) (yield 75%). m.p.: 147–148 C.
1
FTIR (KBr): 2928, 1638, 1272 cm−1. H-NMR (400 MHz, CDCl3)
δ (ppm): 7.28 (d, J = 11.6 Hz, 1H,
H-4), 7.06 (d, J = 7.2 Hz, 1H, H-7), 3.31 (s, 2H, piperazine), 3.17 (s, 2H, piperazine), 2.55 (s, 3H, CH3).
13C-NMR (100 MHz, CDCl3)
δ
(ppm): 163.3 (C), 153.9 (C), 151.5 (C), 149.3 (C), 146.5 (C), 136.7 (C), 135.2
(C), 131.9 (C), 129.5 (CH), 116.7 (CH), 114.7 (CH), 105.3 (CH), 99.6 (CH), 49.8 (CH2), 48.1 (CH2), 13.6
(CH3). HRMS (ESI) [M + H+] m/z calcd for C18H17Cl2FN3O: 380.0727; found: 380.0729.
3.2. Cytotoxicity
Human cell cultures: Hepatocytes (HepaRG
and were cultivated in William’s E Medium (Gibco, Life Technologies, Waltham, MA, USA) and
supplemented with 1x GlutaMAX plus 1x HepaRG General Purpose Medium Supplement in
™ cells) were obtained from Life Technologies,
™
™
25-cm2 or 75-cm2 flasks (TPP, Trasadingen, Switzerland) at 37 ◦C and 5% CO2. Lung cancer epithelial
cells A-549 were propagated in Epilife Medium (Gibco, Life Technologies; MEPICF) and combined with
CaCl2 and 1x-growth-supplement containing penicillin and streptomycin (Gibco, Life Technologies).
For sub-culturing, cells were split at a ratio of 1:4 to 1:8. For cryopreservation, cells were frozen in 10%
DMSO in liquid nitrogen.
Human cytotoxicity assay: Human hepatocytes or human lung carcinoma cells were seeded into
sterile black clear-bottomed polystyrene Corning® CellBind® 96-well plates (Munich, Germany) at
a density of 20,000 cells/well after calculating cell numbers from trypsinized samples in a classical
Ne◦ubauer Improved hemocytometer (VWR, Munich, Germany). The cells were grown for 24 h at
0
37 C in a 5% CO2 atmosphere in Epilife medium (keratinocytes) or William s E medium (hepatocytes).
After a 24-h recovery period, media were removed from the wells by aspiration. The cells were then

Molecules 2016, 21, 1290
10 of 11
immediately exposed to the respective test compounds or reference antibiotics (prepared in advance in
100 µL of the same media) for 24 h 37 ◦C with 5% CO2. The concentration tested ranged from 0.1 µM
to 500
µ
M (0.1
µ
M, 1
µ
M, 10
µ
M, 50
µ
M, 100
µ
M, 250
µ
M, and 500
µ
M) and was formulated as a 100
×
concentrated stock each, resulting in a final constant DMSO concentration of 1% per 100-
µL sample
volume. Test series were each performed in triplicate, and additionally included a vehicle reference
(DMSO only) plus another internal control for evaluating overall assay performance. Additionally, cells
were visually examined under a light microscope for any morphological abnormalities relative to the
untreated cells. Subsequently, cell viability, as an indicator of potential cytotoxicity, was measured by
applying the CellTiter-Glo® luminescent cell proliferation assay (G755A, Promega, Madison, WI, USA).
This method permits the determination of the number of viable cells in culture based on quantitation
of the ATP present, which denotes the presence of metabolically active cells. To this end, 100 µL
(equal to the volume of culture medium) of the kit-provided CellTiter-Glo® reagent (reconstituted by
transferring the thawed CellTiter-Glo® buffer to the lyophilized enzyme and substrate mixture) was
directly added to each well with a repetitive dispensing pipette. The contents were then mixed for
2 min at room temperature on an orbital minishaker (IKA® MS3 digital, Staufen, Germany) at 450 rpm
to induce efficient cell lysis, and then further incubated for 10 min at room temperature for stabilization
of the resultant luminescent signal. Luminescence was recorded using a compatible multimode reader
(GloMax® Multi Detection Platform, Promega Corporation, Madison, WI, USA) with its embedded,
preset CellTiter-Glo® parameters (integration time of 0.5 s). Output values were expressed as percent
cell viability compared with the vehicle (DMSO) control, and EC50 values were calculated from the
dose-response curves via nonlinear regression (version 7, Graphpad Prism Software Inc., La Jolla,
CA, USA).
4. Conclusions
A novel series of 5-fluoro-2-methyl-6-(4-arylpiperazin-1-yl)benzoxazoles (7a
both fluorine and piperazine were prepared in good yields. Initial cytotoxicity analysis of some
of the intermediates—4-fluoro-5-(substituted phenylpiperazin-1-yl)-2-nitrophenols (6a )—showed
–j) incorporating
–j
promising activities and cell type-dependent cytotoxicity. Although the best activity achieved is
still too low, the lung cancer sensitive pattern is a crucial property underlining a therapeutically
interesting window, which can be further investigated. The poor solubility of some benzoxazoles could
be improved by replacing the aryl-piperazine with N-methylpiperazine at position 6 and replacing
the methyl moiety at position 2 with a carbamate functional group. Manipulating the benzoxazole
structure is now under intensive investigation in our laboratory, and the results will be published
in due course. Finally, at least two compounds (6d and 6g) can be used as starting points for the
development of selective anti-cancer compounds in the future.
Acknowledgments: Financial support from Sultan Qaboos University, Oman, is gratefully acknowledged.
Author Contributions: Raid Abdel-Jalil and Wajdi Michel Zoghaib conceived and designed the experiments;
Thuraya Al-Harthy performed the experiments and characterization of new compounds; Raphael Stoll’s group
contributed to the NMR measurements and characterization of the compounds. Kamil Önder, Harald Hundsberger,
Maren Pflüger and Maria Reitsammer performed the Cytotoxicity measurements and the discussion of biological
data. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Chanda, K.; Maiti, B.; Yellol, G.S.; Chien, M.-H.; Kuoc, M.-L.; Sun, C.-M. Polymer supported synthesis of
novel benzoxazole linked benzimidazoles under microwave conditions: In vitro evaluation of VEGFR-3
kinase inhibition activity. Org. Biomol. Chem. 2011, 9, 1917–1926. [CrossRef] [PubMed]
Sheng, C.; Che, X.; Wang, W.; Wang, S.; Cao, Y.; Yao, J.; Miao, Z.; Zhang, W. Design and synthesis of antifungal
benzoheterocyclic derivatives by scaffold hopping. Eur. J. Med. Chem. 2011, 46, 1706–1712. [CrossRef]
[PubMed]
2.

Molecules 2016, 21, 1290
11 of 11
3.
4.
Klimešováa, V.; Kocˇía, J.; Waissera, K.; Kaustováb, J.; Möllmannc, U. Preparation and in vitro evaluation of
benzylsulfanyl benzoxazole derivatives as potential antituberculosis agents. Eur. J. Med. Chem. 2009, 44,
2286–2293. [CrossRef] [PubMed]
Arhancet, G.B.; Walker, D.P.; Metz, S.; Fobian, Y.M.; Heasley, S.E.; Carter, J.S.; Springer, J.R.; Jones, D.E.;
Hayes, M.J.; Shaffer, A.F.; et al. Discovery and SAR of PF-4693627, a potent, selective and orally bioavailable
mPGES-1 inhibitor for the potential treatment of inflammation. Bioorg. Med. Chem. Lett. 2013, 23, 1114–1119.
[CrossRef] [PubMed]
5.
6.
7.
8.
9.
Liu, K.G.; Lo, J.R.; Comery, T.A.; Zhang, G.M.; Zhang, J.Y.; Kowal, D.M.; Smith, D.L.; Di, L.; Kerns, E.H.;
Schechter, L.E.; et al. Identification of a series of benzoxazoles as potent 5-HT6 ligands. Bioorg. Med.
Chem. Lett. 2009, 19, 1115–1117. [CrossRef] [PubMed]
Kallashi, F.; Kim, D.; Kowalchick, J.; Park, Y.J.; Hunt, J.A.; Ali, A.; Smith, C.J.; Hammond, M.L.; Pivnichny, J.V.;
Tong, X.; et al. 2-Arylbenzoxazoles as CETP inhibitors: Raising HDL-C in cynoCETP transgenic mice.
Bioorg. Med. Chem. Lett. 2011, 21, 558–561. [CrossRef] [PubMed]
Ongarora, D.S.B.; Gut, J.; Rosenthal, P.J.; Masimirembwa, C.M.; Chibale, K. Benzoheterocyclic amodiaquine
analogues with potent antiplasmodial activity: Synthesis and pharmacological evaluation. Bioorg. Med.
Chem. Lett. 2012, 22, 5046–5050. [CrossRef] [PubMed]
Murty, M.S.R.; Rao, B.R.; Katiki, M.R.; Nath, L.R.; Anto, R.J. Synthesis of piperazinyl
benzothiazole/benzoxazole derivatives coupled with 1,3,4-oxadiazole-2-thiol: Novel hybrid heterocycles as
anticancer agents. Med. Chem. Res. 2013, 22, 4980–4991. [CrossRef]
Shao, K.-P.; Zhang, X.-Y.; Chen, P.-J.; Xue, D.-Q.; He, P.; Ma, L.-Y.; Zheng, J.-X.; Zhang, Q.-R.; Liu, H.-M.
Synthesis and biological evaluation of novel pyrimidine-benzimidazol hybrids as potential anticancer agents.
Bioorg. Med. Chem. Lett. 2014, 24, 3877–3881. [CrossRef] [PubMed]
10. Shaquiquzzaman, M.; Verma, G.; Marella, A.; Akhter, M.; Akhtar, W.; Khan, M.F.; Tasneem, S.; Alam, M.M.
Piperazine scaffold: A remarkable tool in generation of diverse pharmacological agents. Eur. J. Org. Chem.
2015, 102, 487–529. [CrossRef] [PubMed]
11. Filler, R.; Saha, R. Fluorine in medicinal chemistry: a century of progress and a 60-year retrospective of
selected highlights. Future Med. Chem. 2009, 1, 777–791. [CrossRef] [PubMed]
12. Sanchez, J.P.; Domagala, J.M.; Hagen, S.E.; Heifetz, C.L.; Hutt, M.P.; Nichols, J.B.; Trehan, A.K. Quinolone
Antibacterial Agents. Synthesis and Structure-Activity Relationships of 8-Substituted Quinoline-3-carboxylic
Acids and 1,8-Naphthyridine-3-carboxylic Acids. J. Med. Chem. 1988, 31, 983–991. [CrossRef] [PubMed]
13. Abdel-jalil, R.J.; Al-Qawasemeh, R.A.; Voleter, W.; Heeg, P.; El-Abadelah, M.M.; Sabri, S.S. Synthesis and
properties of some 2,3-disubstituted 6-fluoro-7-(4-methyl-1-piperazinyl)quinoxalines. J. Heterocycl. Chem.
2009, 37, 1273–1275. [CrossRef]
14. Abu-Elteen, K.H.; Abdel-Jalil, R.J.; Hamad, M.A.; Ghaleb, M.; Khan, K.M.; Voelter, W. fungicidal effects of
some derivatives of 2-ferrocenyl-benzimidazoles: A possible template for antifungal drug design. J. Med. Sci.
2008, 8, 673–681. [CrossRef]
15. Abdel-Jalil, R.J.; Aldoqum, H.M.; Ayoub, M.T.; Voelter, W. Synthesis and antitumor activity of
2-aryl-6-fluoro-6-(4-methyl-1-piperazinyl)-4-(3H)-Quinazolinones.
Heterocycles 2005, 65, 2061–2070.
[CrossRef]
16. Al-Harthy, T.; Abdel-Jalil, R.; Zoghaib, W.; Pflüger, M.; Hofmann, E.; Hundsberger, H. Design and Synthesis
of Benzothiazole Schiff Bases of Potential Antitumor Activitiy. Heterocycles 2016, 92, 1282–1292.
17. Crouch, S.; Kozlowski, R.; Slater, K.; Fletcher, J. The use of ATP bioluminescence as a measure of cell
proliferation and cytotoxicity. J. Immunol. Methods. 1993, 160, 81–88. [CrossRef]
18. Guidance Document on Using in Vitro Data to Estimate in Vivo Starting Doses for Acute Toxicity. Available
online: http://www.epa.gov/hpv/pubs/general/nih2001b.pdf (accessed on 5 November 2014).
Sample Availability: Samples of the compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).