Design, synthesis, and evaluation of the anticancer activity of 2-amino-aryl-7-aryl-benzoxazole compounds

Article abstract of DOI:10.1111/cbdd.13025

A series of 2-amino-aryl-7-aryl-benzoxazole derivatives have been designed, synthesized, and evaluated as anticancer agents. Fourteen of the compounds exhibited cytotoxic effects toward human A549 lung cancer cells. We found 12l was the most potent with a

Full text of DOI:10.1111/cbdd.13025

MR MATTHEW PAUL GLEESON (Orcid ID : 0000-0001-6998-7446)
Article type : Research Article
Title
Design, Synthesis and Evaluation of the Anti-Cancer Activity of 2-
Amino-aryl-7-aryl-benzoxazole Compounds.
Authors
Patcharaporn Khajondetchairita, Oraphan Phuangsawaia,c, Praphasri Suphakunb, Siriruk
Rattanabunyongb, Kiattawee Choowongkomonb and M. Paul Gleesona,d*
Institutions
a Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c present address: Nano-Cosmeceuticals Laboratory, National Nanotechnology Center, National
Science and Technology Development Agency, Pathumthani 12120, Thailand.
d Department of Biomedical Engineering, Faculty of Engineering, King Mongkut’s Institute of
Technology Ladkrabang, Bangkok, 10520, Thailand.
Short Title: Anti-Cancer Activity benzoxazole inhibitors
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13025
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Corresponding Author: Paul Gleeson e-mail: paul.gl@kmitl.ac.th.
Key words: 2-Amino-aryl-7-aryl-benzoxazole, A549, cytotoxicity, drug design, SAR, JAK2
Abstract
A series of 2-aminoaryl-7-arylbenzoxazole derivatives have been designed, synthesized and
evaluated as anti-cancer agents. Fourteen of the compounds exhibited cytotoxic effects towards
Human A549 lung cancer cells. We found 12l was the most potent with an EC50 of 0.4 µM, equivalent
to the anti-cancer drug doxorubicin, but had low selectivity following cross screening in monkey
kidney Vero cells. Eight of the most potent or most selective compounds were further profiled in
additional cell lines (MCF7, NCI-H187 and KB) to better understand their cytotoxic activity. Only
compound 12l had a measurable EC50 in a single cell line (3.3 µM in the KB cell line). Taken together,
this data suggests the series as a whole display specific cytotoxicity towards A549 cells.
Cheminformatics searches pointed to JAK2 as a possible target. A subset of compounds assayed at
this target showed IC50s ranging from 10 µM to 0.08 µM, however no clear correlation between JAK
potency and A549 cytotoxicity was observed.
Introduction
According to the World Health Organization, lung cancer is the most common cause of cancer
death.[1] Non-small cell lung cancer (NSCLC) is the most deadly form of this disease and has a 5 year
survival rate below 15% [2]. Platinum-containing chemotherapies,[3] DNA intercalators such as
Doxorubicin and Etoposide[4] and protein kinase inhibitors such as Lapatinib and Imatinib [5] can be
used to treat NSCLC. This range of treatment options is necessary to reduce patient specific side-
effects [6] and to avoid disease resistance in certain sub-populations [7]. Nevertheless, the low
survivability associated with lung cancer is a clear indication of the need for more effective drugs,
particularly those that act on targets or pathways of greater relevance to the disease. Kinases
inhibitors have been of immense interest to the pharmaceutical industry[5] due to the critical role
these targets play in cellular signaling processes, including cell growth, differentiation and death. [8]
Specific signaling and regulatory pathways are often overexpressed in cancers leading to tumor
growth and blocking these pathways is a recognized strategy to target the disease [9]. Kinases are
now the most intensively studied class of molecular targets in drug discovery [10] with
approximately 30 new inhibitors on the market.[11] In NSCLC, the tyrosine kinases EGFR and VEGFR
[7] are established targets used to tackle this specific disease. Other kinases are currently under
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investigation including JAK inhibitors [12, 13]. The JAK/STAT pathway has been identified as a
potential intervention point [3, 12, 14-16] and has seen JAK inhibitors entering clinical trials.[12, 16]
In this study, we report the anti-cancer activity of 2-aminoaryl-7-arylbenzoxazole compounds
at an NSCLC cell line (Figure 1). Compounds 1-3 were obtained from the focused screening of
approximately 100 diverse kinase-like compounds in an A549 based cytotoxicity assay. This
manually curated set consisted of low molecular weight compounds with the typical donor-acceptor
interaction features required for kinase ATP site binding. The 2-aminoaryl-7-arylbenzoxazole series
was selected on the grounds of its good activity and low MWT (Figure S1), synthetic tractability, and
based on previous reports of the scaffolds kinase activity. Substructure and similarity searching
identified 2-amino-7-aryl-benzoxazole derivatives and related 2-Amino-[1,2,4] triazolo [1,5-
a]pyridines as JAK2 inhibitors which have been used to target leukemia (Figure 2). [17, 18] Our
observation of activity in an NSCLC cell line is consistent with reports that the JAK/STAT pathway
represents an intervention point in lung cancer [3, 12, 14-16]. RSK2 is also a possible target [19],
however the IC50s associated with this target are at the sub micromolar level compared to the nano- to
sub-nanomolar level of the former (Figure S2).
We undertook alterations at the 2- and 7- positions guided by the SAR of the initial hits (Figure 1)
and knowledge from previous literature reports. We selected aryl substituents for the 7-position (R1)
of benzoxazole scaffold that were primarily hydrophobic in nature (Figure 1). In an effort to reduce
the overall hydrophobicity of the molecules, relatively polar substituted aniline reagents were
selected for substitution at the 2-position of benzoxazole scaffold (R2). While kinase biochemical
data is available in the literature for this chemotype, very little phenotypic data has been reported.
The primary goal of this study has been to shed light on the cytotoxic properties of this scaffold, to
improve our SAR understanding the series with a view to obtaining more potent molecules. We have
assessed the cytotoxicity of the series further using the monkey kidney epithelial cell line (Vero), the
MCF7 breast cancer cell line, the SCLC NCI-H187 cell line and the KB cell line. In addition, an
assessment of the activity of a subset of compounds at JAK2 kinase have also been undertaken.
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N
O
N
O
1
NH
NH
2
7
R2
R1
H C
3
CH
3
N
A549 EC50 = 7.62
M
μ
N
O
N
2
3
NH
NH
O
CH
H C
3
3
Cl
N
N
NH
2
A549 EC50 = 6.04
M
μ
A549 EC50 = 27.89
M
μ
Figure 1: Structurally related screening hits identified from cytotoxicity screening in the
A549 cell line.
N
N
N
O
4
5
NH
NH
N
N
N
OMe
N
N
O
N
O
JAK2 IC50
= 0.008 μM
JAK2 IC50 = 0.0006 μM
Figure 2: Nanomolar active amino-7-aryl-benzoxazole kinase inhibitors reported in the
literature [17, 19].
1 Experimental Section
1.1 Compound Design
From our initial screening, compounds 1 and 2 display low μM activity towards A549, however they
also have unacceptably high clogPs of 6. 3 has lower lipophilicity, but lower A549 activity also. From
the outset, incorporation of polarity at the R2 position was therefore considered a higher priority in
the overall effort to improve physiochemical properties. As 2-amino-7-aryl-benzoxazole derivatives
and related 2-Amino-[1,2,4] triazolo [1,5-a]pyridines have are reported as JAK2 inhibitors. As such
consideration was given to how molecules would bind to the ATP binding site of the protein kinase
JAK2 given this is a probable target. Compounds were docked to JAK2 via (a) manual superposition
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onto the 1,2,4-triazolo[1,5-a]pyridin-2-amine of JAK2 (PDB 4JIA).[18, 20] Computational docking
using GOLD5.1 was also undertaken. All solvent and co-factors were removed and compounds
docked with default settings.[21]
The expansion of the SAR around our hits was achieved by designing structural modifications to
(a) lower the overall lipophilicity of the series, (b) explore the hydrophobic/hydrophilic balance
between the R1 and R2 positions guided by property considerations and structural understanding of
JAK2 binding mode (c) focusing on substituents that allow us to explore novel chemical space of the
series. Reagent selection was primarily driven by a desire to reduce the hydrophobicity at either the
R1 and R2 positions (Scheme 2), or both, in order to improve the overall molecular properties.
1.2 Chemical Synthesis
All chemicals received from suppliers were used without further purification. All reactions were
monitored by thin layer chromatography aluminum/silica gel plate with visualization of UV light.
Column chromatography was performed by irregular silica gel (60Å). Mass spectra (MS) were
obtained on an Agilent 1100 HPLC instrument coupled to a LC/MSD Trap mass spectrometer, in ESI
(+) mode or APCI mode and on a Bruker micrOTOF-Q III in ESI (+) mode. Compounds showed a purity
≥ 95% as determined from the corresponding UV visible absorbance HPLC chromatogram. 1H and 13
C
NMR spectra were recorded on a Varian instrument at 400 MHz and 101 MHz. Chemical shifts are
reported in ppm (δ) using the residual solvent line as the internal standard. Compound synthesis
followed the general routes described in Scheme 1 and Scheme 2 with full details being provided in
the supporting information.
O
N+
O-
NH2
OH
N
O
N
O
a
b
c
SH
S
CH3
OH
Br
Br
Br
Br
6
7
8
9
Scheme 1: General route to 7-bromo-2-methylsulfanyl-benzoxazole (9): (a) SnCl2, EtOH, (b)
Potassium ethylxanthogenate, EtOH, (c) K2CO3, MeI, DMF [22].
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9
10a-c
N
O
N
O
(a)
NH
S
Br
Br
R2
R2 a=CN,b=CONH2, c=morpholine
(b)
(b)
11a-b
12
N
O
N
S
NH
O
(a)
R2
R1
R1
R1 a=OMe,b=NHCOMe
Scheme 2: Synthetic approach used for the preparation of final compounds from 9.
1.2.1 Preparation of 7-bromo-2-methylsulfanyl-benzoxazole (9)
A mixture of 2-bromo-6-nitrophenol (6) (18.4 mmol) and tin (II) chloride dihydrate (91.7 mmol) in
o
EtOH (80 ml) was heated at 70 C for 1 h. The mixture was poured into ice and the pH was made
slightly basic (pH 7-8) by addition of 1N sodium hydroxide solution. The aqueous solution was
extracted with EtOAc. The extract was washed with brine, dried over magnesium sulfate and
concentrated under vacuum to afford of the title compound 2-Amino-6-bromophenol (7), 40% yield.
2-amino-6-bromo-phenol (7) (26.6 mmol) was dissolved in 20 ml EtOH and (39.9 mmol)
potassium ethylxanthogenate was added. This mixture was stirred at reflux for 6 h. After it had
cooled down to room temperature, the reaction mixtures were concentrated in vacuum and 50 ml
water was added along with the addition of acetic acid, to obtain a pH of 5. The precipitate was
filtered off, washed with water and dried to afford the title compound 8, 90% yield.
A mixture of (26.1 mmol) 7-bromo-benzoxazole-2-thiol (8), (52.2 mmol) K2CO3, (28.7 mmol) MeI in
80 ml, DMF was stirred at room temperature for 1 h. The reaction mixture was poured into water
and extracted with EtOAc. The combined organic layers were washed with water and saturated NaCl
solution, dried over MgSO4, and the filtrate was concentrated in vacuum to afford the title
compound 9[22], 30% yield.
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1.2.2 General procedure for amine substitution (Step a)
To 7-bromo-2-methylsulfanyl-benzoxazole (9) in DCM, m-CPBA was added. This mixture was
stirred at room temperature for 1 h. It was then diluted in sat. NaHCO3, water and brine. After that it
was dried by anhydrous Na2SO4, filtered and concentrated. Then aniline was added and the reaction
mixture was heated to 40°C with stirring for 20 h. The reaction mixture was concentrated in vacuum
and the crude was purified by irregular silica gel (60Å) column chromatography using a mixture of
EtOAc and hexane to give 10a-c.
1.2.3. General procedure for boronic acid coupling (Step b)
A solution of 9 and phenyl boronic acid were dissolved in dioxane. A solution of Na2CO3 (in 5 ml of
water) was added and a stream of N2 (g) was bubbled through the mixture in order to exclude
oxygen from the reaction mixture. Tetrakis (triphenylphosphine) palladium was added and the
o
reaction mixture was stirred at 100 C for 24 h. After that, the reaction mixture was poured into
water and was extracted with EtOAc. The combined organic layers were washed with water and
dried over MgSO4. It was purified by an irregular silica gel-G60 column chromatography using a
system of EtOAc/hexane or MeOH/DCM to give 11a-b.
1.2.4 General Procedure for the preparation of compound 1-3, 12a-12m
The preparation of compounds 1-3, 12h, 12l and 12m used intermediates 10a-c which were
reacted with the phenyl boronic acid using the general procedure described in section 1.2.3. The
preparation of compound 12a-12g and 12i-12k used intermediates 11a-b reacted with anilines using
the general procedure described in 1.2.2 (Scheme 2).
1.3 Biological Testing
1.3.1 Cytotoxicity at A549 and Vero cell lines
The EC50 of compounds were accessed in A549 (ATCC CCL-185) and Vero (ATCC CCL-
81) cell lines using the MTT method by Mosman et.al.,[3, 23, 24] Cells were incubated in
DMEM medium with 10% fetal bovine serum, 100 U/ml of penicillin and streptomycin at 37
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oC in a humidified atmosphere with 5% CO2. The cells were incubated in DMEM for 16-18
h, after with test compound for 72 h in triplicate. Then, to each well was added DMEM
containing 3-[4, 5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT), and
cells were incubated for a further 3 h. Subsequently, 50 μl dimethyl sulfoxide was added. The
plate was shaken, and then absorbance readings at a wavelength of 570 nm were performed
on a Sunrise microplate reader (Tecan). Doxorubicin was used as the positive control and
0.5% DMSO as the negative control. The EC50 values of compounds were derived from dose-
response-curve plotted between %cytotoxicity versus the sample concentrations fitted using
Prism 6.0 GraphPad Software Inc., (San Diego, CA, USA).
1.3.2 Cytotoxicity at MCF7 NCI-H187 KB cell lines
The cytotoxicity against the MCF7 (ATCC HTB-22), NCI-H187 (ATCC CRL-5804) and KB cell lines
(ATCC CCL-17) using the method described by Brien et.al,.[25] Cells were grown and maintained in a
o
complete medium and incubated at 37 C humidified incubator with 5% CO2. Briefly, to a 384-well
plate in triplicate, 5 μl of test compound and 45 μl of cell suspension were added. The plate was then
incubated at 37 oC in a humidified incubator with 5% CO2 for 72 h. After that, resazurin solution was
added to each well and the plate was further incubated at 37 oC for 4 h. Fluorescence was monitored
at 530 nm (excitation) and 590 nm (emission) using bottom-reading mode. Ellipticine and
Doxorubicin were used as the positive control and 0.5% DMSO as the negative control. The EC50
value was derived from dose-response-curve that is plotted between %cytotoxicity versus the
sample concentrations by using SOFTMax Pro software (Molecular Devices, USA).
1.3.3 JAK2 kinase activity
Kinase assays were performed as previously described.[24] Reactions were performed using 10
ng JAK2 enzyme, 0.1 mg/ml poly (Glu:Tyr) substrate and 0.1 mg/ml ATP. Inhibition was determined
by using Antibody BeaconTM Tyrosine Kinase Assay Kit (A-35725; Invitrogen, USA) 4-aryl-N-
phenylpyrimidin-2-amine [26] was used as the positive control and 0.5% DMSO as the negative
control. The IC50 value was derived from dose-response-curve plotted between % cytotoxicity versus
the sample concentrations by using GraphPad Software Inc. (San Diego, CA, USA).
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2 Results
2.1 Synthesis
2-amino-aryl-7arylbenzoxazole derivatives have been synthesized using the general synthetic
procedure described in Scheme 1 and Scheme 2. The hydrogenation reaction of 2-bromo-6-
nitrophenol (6) was carried out using tin (II) chloride dehydrate to give 2-amino-6-bromo-phenol (7)
(40% yield). This was followed by ring closure in the presence of potassium ethyl xanthogenate to
give 7-bromo-benzoxazole-2-thiol (90% yield) (8). Intermediate compound bromo-2-
(methylsulfanyl)-1, 3-benzoxazole (9) was obtained from the reaction of compound 8 and MeI (38%
yield).
1-3, 12h, 12l and 12m were produced in two steps, starting with the coupling of 7-bromo-2-
methylsulfanyl-benzoxazole (9) with anilines by activating the sulphur leaving group using m-CPBA to
give 10a-c. Suzuki-Miyaura cross-coupling of 10a-c with phenylboronic acid utilized Pd (PPh3)4 and
Na2CO3 under a nitrogen environment. We investigated changing the order of the substitution and
coupling reactions to overcome the lack of reactivity for certain reagent combinations. 12a-g and
12i-k were produced in two steps starting with the coupling of boronic acids with 9, leading to the
formation of 11a-b, followed by aniline substitution at the 2-position. Overall, the yields ranged from
3-30% which is in line with reports elsewhere. [19]
2.1 Biological Testing
The anti-cancer properties (EC50) of all synthesized compounds in A549 and Vero cell lines are
reported in Table 1. Also reported are computed physico-chemical properties and the selectivity
index (SI), the latter representing the selectivity compounds show for A549 over Vero cells. The
cytotoxicities of the most potent or more selective exemplars were evaluated in the MCF7, NCI-H187
and KB cell lines and these results are reported in Table 2. Finally, the IC50s associated with a sub-set
of the compounds were evaluated in a JAK2 inhibition assay and are reported in Table 2.
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Table 1: Activities and compounds 1-3, 12a-m
A549
EC50 (μM)
Vero
EC50 (μM)
SI
ID
1
R1
R2
clogPa
MWTa
CH3
CH3
6.0
339
7.6 ±3.4
6.0±1.7
11.9±0.5
8.6±2.7
1.6
1.4
N
N
H3C
6.0
339
2
H3C
Cl
4.8
4.9
4.8
361
342
342
27.9±4.1
12.4±0.2
3.6±0.8
41.4±23.9
0.6±0.06
2.3±0.04
1.5
0
N
N
3
NH2
O
12a
12b
0.6
O
N
CH3
O
O
S
3.5
4.9
3.9
409
401
332
1.2±0.5
1.8±0.3
>100
>100
3.0±0.6
>100
80.0
1.7
-
O
O
O
12c
12d
12e
NH
N
O
N
CH3
N
4.4
4.5
3.3
3.1
3.5
4.8
356
428
386
436
410
401
1.6±0.02
16.4±1.9
>100
30.4±15.9
8.9±0.3
>100
19.0
0.5
-
O
12f
12g
12h
12i
NH
NH
N
O
O
O
O
NH
NH
NH2
CH3
O
S
31.8±25.7
13.0±0.8
6.7±0.6
>100
3
O
NH
CH3
O
O
S
6.4±0.2
0.8±0.01
0.5
0.1
O
O
12j
12k
NH
O
N
O
4.9
401
0.4±0.01
0.8±0.02
2.0
N
N
O
12l
O
O
4.7
1.4
431
544
3.2±0.9
2.5±0.9
1.4 ±0.1
0.8
-
O
12m
O
0.4±0.08
Doxorubicin
a Calculated logP and MWT using JChem Version 14.9.100.707.[27]
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Table 2: Activities of compounds towards additional cell lines and JAK2 kinase.
% inhibition at 10 M / [EC50, μM]
ID
MCF7
NCI-H187
KB
JAK2 IC50 µM
n.d.a
12b
37.1 ±1.7 / [-]
0.0±0.1 / [-]
0.0±0.1 / [-]
12c
12d
12f
12g
12i
41.3±4.7 / [-]
0.0 ±0.4 / [-]
13.7 ±0.2 / [-]
43.1±4.3 / [-]
0.0±0.1 / [-]
43.8 ±3.6 / [-]
12.9±0.4 / [-]
0.0±0.9 / [-]
0.0±0.1 / [-]
1.7±0.1 / [-]
26.9±0.4 / [-]
25.9 ±2.0 / [-]
8.3±0.1 / [-]
0.0±0.1 / [-]
4.9±0.15
0.08±0.04
n.d.a
0.0±0.1 / [-]
7.4±0.1 / [-]
0.11±0.05
10±0.08
n.d.a
0.0±0.1 / [-]
12l
90.8 ±11.5 / [3.29]
12m
0.0±0.2 / [-]
20.7±0.6 / [-]
99.2± 7.4 / [3.9]
96.9±8.7 / [0.11]
n.d.a
0.0±0.1 / [-]
99.7±14.6 / [11.4]
96.3± 18.6 / [2.5]
n.d.a
n.d.a.
n.d.a
Ellipticine
-
Doxorubicin
4-aryl-N-phenyl
93.1± 6.6 / [16.5]
n.d.a
n.d.a
0.06±0.01
pyrimidin-2-amine [28]
a Activity not determined (n.d.).
4 Discussion
4.1 Cytotoxicity SAR
The A549 anti-cancer values ranged from 0.4 µM to >100 µM. 12l was the most potent of all
compounds with an EC50 of 0.4 µM. This was almost equivalent to the known NSCLC anti-cancer drug
doxorubicin (EC50 = 0.4 µM). Compounds 12c, 12d, and 12f all inhibited the A549 cell line with EC50s
below 2 µM (1.2, 1.6, and 1.8 µM respectively). Two additional compounds had EC50s between 6-7
µM, five compounds had EC50s between 7-50 μM (original hits 1-3, 12a, 12g, 12i and 12j), while the
final two compounds had EC50s > 100 μM (12e & 12h).
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At the R1, incorporation of phenyl groups with methoxy at the 3- position, 4- position, or both
was undertaken due to the previously discussed JAK2 SAR. Compound 1 and 2 showed reasonable
activity in A549 cells. The introduction of the 4-OMe phenyl at R1 (12a) results in a desirable drop in
clogP (6.02 to 4.8), however the A549 activity also drops by approximately 2 fold. Incorporation of
additional polar anilines at the R2 position was undertaken (12b-12f). The 3-CN-phenyl of 12b was
found to be 3.4 fold more potent than the equivalent molecule substituted at the 4-position (12a).
Substituting the R2 phenyl for 4- methyl-sulphonamide and 4-morpholine also results in a dramatic
improvement in potency, with EC50s values of 1.2 and 1.8 μM being observed. 1H-indazol-6-amine
(12f) resulted in a comparable EC50 of 1.6 μM while the methyl-pyridine of 12e was inactive. This
could be rationalized on the grounds that the more polar derivatives can interact more effectively at
or around the active site pocket of the postulated kinase target, as well as potentially from the
improved solubility improving the cell based response.
We next investigated the use of acetamide at R1, this having both donor and acceptor features
available for target interaction (12g-12j). With 4-Morpholine, 4-amide and 4-methylsulphonamide at
the R2, relatively poor activity was observed showing a subtle balance exists in terms of the
hydrophobic/hydrophilic balance needed to achieve activity. Significant polarity at both R groups,
while potentially beneficial in terms of overall lipophilicity, and solubility, appears detrimental to
overall activity. Compounds 12j differs from 12c in that the N-methyl-sulphonamide substitution is at
the 3- rather than the 4-position. This change results in a 10 fold loss in potency. Modification of 12d
in a similar manner results in a 3 fold loss in potency suggesting the 4-position substitution is
preferred. Two additional compounds were prepared with 3-MeO-phenyl and 2, 3 di-MeO-phenyl at
R1 and 4-substituted morpholine at R2. The former resulted in compound 12l, the most potent
observed here with an EC50 of 0.4 μM, while the latter had a potency of 3.2 μM.
Certain classes of cancer drugs can show broad cytotoxicity across a number of cell lines. [6, 29]
We therefore assessed all compounds against the monkey kidney epithelial cell line (Vero) (Table 1).
The most potent compound observed was 12a (0.6 μM). Doxorubicin, a relatively non-specific DNA
intercalator, was observed to have an EC50 of 1.4 μM. Compounds 12k and 12l have Vero cell EC50
values of 0.80 µM, while compounds 1, 2, 12b, 12d, 12g, 12j and 12m displayed EC50s in range 3.6-
11.9 µM. The SAR associated with the Vero cell-line broadly follows that for A549. However, there
are three noticeable outliers in compounds 12c, 12f and 12i, which display noticeably greater
selectivity towards A549 cells. These compounds were characterized as having generally lower
lipophilicity values (mean clogP = 3.6, N=3) than their more non-selective, A549 active counterparts
(mean clogP = 4.9, N=11).
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Eight compounds were evaluated in the three additional cell lines NCI-H187 (small cell lung
cancer), MCF7 (breast cancer), and KB (oral cavity cancer) and it was found that the majority of the
compounds showed only minimal cross-activity. Only compound 12l displayed a measurable EC50 at
the KB cell line (3.3 μM). Compounds 12c and 12g showed a degree of inhibition (~40% inhibition at
the highest concentration screened) in the MCF7 cell line. This would suggest a more specific
mechanism of activity for these compounds, which is consistent with the hypothesis that these are
functioning as kinase inhibitors.
4.2 Role of JAK2 Inhibition
The results in Table 2 show that the series shows some JAK2 activity, in two cases at the sub μM
level. Compounds were docked into a JAK2 crystal structure (4JIA) [18] for visual assessment using a
scaffold based superposition of the benzoxazole ring onto the triazolopyrimidine scaffold of the
inhibitor bound to the protein (Figure 3). The compounds appear to be classic hinge-binders, making
two H-bonds with Leu932 (Figure 2). The 7-positon phenyl ring binds in the hydrophobic back pocket
and the 2-position aniline group is directed towards the ATP pocket opening (Figure S3).
Figure 3: Configuration of JAK2 binding site (left) and the 3D docked model of 12c bound to the JAK2
X-ray crystal structure (4JIA, Purple) [17]. The hinge region of JAK2 is also shown in stick form and H-
bonds are indicated by dotted lines.
The SAR data shows that 12d showed the most significant inhibition of JAK2 with an IC50 0.08 µM.
The 4-phenyl-morpholine is a classic group incorporated by other JAK2 classes at this portion,
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including 4-aryl-2-aminoalkylpyrimidines.[28] 12g also displayed reasonable activity at 0.11 µM while
12c and 12i displayed IC50s of 4.9 and 10 µM respectively. It would be inappropriate to conclude that
JAK2 activity is the primary cause of the observed cytotoxicity [3, 12, 14-16] of these compounds in
A549 cells given the relatively limited correlation with the cell based response. Further detailed
studies will be needed to ascertain the target(s) associated with these compounds.
5 Conclusions
We have designed and synthesized sixteen compounds of 2-amino-aryl-7-arylbenoxazole
derivatives and assessed their biological activities at a range of cancer cell lines: A549, Vero, MCF7,
NCI-H187, KB cell lines. 12l displayed the best anti-cancer properties at the A549 cell line with an
EC50 value of 0.4 µM. Compound 12c displayed the best balance of A549 activity (1.2 μM) and
selectivity towards other cell lines (SI = 80). The most potent, or most selective compounds, were
further profiled in additional cancer cell lines (MCF7, NCI-H187 and KB). Only compound 12l
displayed a measurable EC50 (3.3 µM) in KB cell line confirming the series displays relatively low
general cytotoxicity overall, suggesting a more specific mode of action.
Biochemical testing at a possible target, JAK2 kinase, confirmed the chemotypes activity towards
this recognized NSCLC target.[3, 12, 14-16] 12d showed the most significant potency at JAK2 (0.08
μM). The compounds as a whole display good physicochemical properties; MWT mean of 382 with a
maximum of 436; clogP mean of 4.5, with only 2 examples with clogP values above 5 (the initial hits
1, 2). Further detailed studies are needed to better understand the anti-cancer potential of these
compounds.
Acknowledgements
PK gratefully acknowledges financial support provided by the Graduate School at Kasetsart
University. MPG would like to acknowledge funding provided the Thailand Research Fund
(RSA570068) and the Kasetsart University Research and Development Institute (KURDI).
References
[1] WHO, World Health Organisation, http://www.who.Int/mediacentre/factsheets/fs297/en/. ,
2016.
This article is protected by copyright. All rights reserved.

[2] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA
Cancer J. Clin., 61 (2011) 69-90.
[3] Y. Hu, Y. Hong, Y. Xu, P. Liu, D.-H. Guo, Y. Chen, Inhibition of the JAK/STAT pathway with
ruxolitinib overcomes cisplatin resistance in non-small-cell lung cancer NSCLC, Apoptosis, 19 (2014)
1627-1636.
[4] F. Reboul, Y. Brewer, P. Vincent, B. Chauvet, C.F. Faure, M. Taulelle, Concurrent cisplatin,
etoposide, and radiotherapy for unresectable Stage III nonsmall cell lung cancer: A Phase II study,
IJROBP, 35 (1996) 343-350.
[5] J. Zhang, P.L. Yang, N.S. Gray, Targeting cancer with small molecule kinase inhibitors, Nat Rev
Cancer, 9 (2009) 28-39.
[6] S. Jaeger, M. Duran-Frigola, P. Aloy, Drug sensitivity in cancer cell lines is not tissue-specific,
Mol. Cancer, 14 (2015) 40.
[7] P.Y. Julian R. Molina, Stephen, Cassivi, Steven E. Schild and Alex A. Adjei, Non–Small Cell Lung
Cancer: Epidemiology, Risk Factors, Treatment, and Survivorship, Mayo Clinic proceedings. Mayo
Clinic, 83 (2008) 584-594.
[8] A. Kontzias, A. Kotlyar, A. Laurence, P. Changelian, J.J. O’Shea, Jakinibs: A New Class of Kinase
Inhibitors in Cancer and Autoimmune Disease, Curr. Opin. Pharmacol., 12 (2012) 464-470.
[9] R.J. Scheff, B.J. Schneider, Non-Small-Cell Lung Cancer: Treatment of Late Stage Disease:
Chemotherapeutics and New Frontiers, Semin. Intervent. Radiol., 30 (2013) 191-198.
[10] D.P. Carbone, J.D. Minna, Chemotherapy for non-small cell lung cancer, Br. Med. J., 311
(1995) 889-890.
[11] P. Wu, T.E. Nielsen, M.H. Clausen, FDA-approved small-molecule kinase inhibitors, Trends
Pharmacol. Sci., 36 (2015) 422-439.
[12] S.J. Thomas, J.A. Snowden, M.P. Zeidler, S.J. Danson, The role of JAK/STAT signalling in the
pathogenesis, prognosis and treatment of solid tumours, Br J Cancer, 113 (2015) 365-371.
[13] A. Quintás-Cardama, S. Verstovsek, Molecular Pathways: JAK/STAT Pathway: Mutations,
Inhibitors, and Resistance, Clin. Cancer Res., 19 (2013) 1933-1940.
[14] D.W.B. Thomas J. Lynch, Raffaella Sordella, Sarada Gurubhagavatula, Ross A. Okimoto, B.S.,
Brian W. Brannigan, Patricia L. Harris, Sara M. Haserlat, Jeffrey G. Supko, Frank G. Halusk, David N.
Louis, David C. Christiani, Jeff Settleman and Daniel A. Haber, Erlotinib in Previously Treated Non–
Small-Cell Lung Cancer, N. Engl. J. Med., 353 (2005) 123-132.
[15] D.H. Brendan D. Looyenga, Irene Cherni, Chris Kingsley, Glen J. Weiss, Jeffrey P., MacKeigan,
STAT3 Is Activated by JAK2 Independent of Key Oncogenic Driver Mutations in Non-Small Cell Lung
Carcinoma, PLoS ONE, 7 (2012) e30820.
[16] M. Buchert, C.J. Burns, M. Ernst, Targeting JAK kinase in solid tumors: emerging opportunities
and challenges, Oncogene, 35 (2016) 939-951.
[17] P.F. Marc Gerspacher , Carole Pissot-Soldermann, Christoph Gaul, Philipp Holzer,, M.L. Eric
Vangrevelinghe, Dirk Erdmann, Thomas Radimerski, Catherine H. Regnier, Patrick Chene,, F.H. Alain
De Pover, Fabienne Baffert, Thomas Buhl, Reiner Aichholz, Francesca Blasco,, J.T. Ralf Endres, Peter
Drueckes, 2-Amino-aryl-7-aryl-benzoxazoles as potent, selective and orally available JAK2 inhibitors,
Bioorg. Med. Chem. Lett.
, 20 (2010) 1724-1727.
[18] M. Siu, R. Pastor, W. Liu, K. Barrett, M. Berry, W.S. Blair, C. Chang, J.Z. Chen, C. Eigenbrot, N.
Ghilardi, P. Gibbons, H. He, C.A. Hurley, J.R. Kenny, S. Cyrus Khojasteh, H. Le, L. Lee, J.P. Lyssikatos, S.
Magnuson, R. Pulk, V. Tsui, M. Ultsch, Y. Xiao, B.Y. Zhu, D. Sampath, 2-Amino-[1,2,4]triazolo[1,5-
a]pyridines as JAK2 inhibitors, Bioorg. Med. Chem. Lett 23 (2013) 5014-5021.
[19] M.M. Abran Costales, Savithri Ramurthy, Jiong Lan, Sharadha Subramanian, Rama Jain,, L.S.
Gordana Atallah, Mika Lindvall, Brent A. Appleton, Elizabeth Ornelas, Paul Feucht, Bob Warne,, S.E.B.
Laura Doyle, Ida Aronchik, Anne B. Jefferson, Cynthia M. Shafer ⇑, 2-Amino-7-substituted
benzoxazole analogs as potent RSK2 inhibitors, Bioorg. Med. Chem. Lett., 24 (2014) 1592-1596.
[20] M. Siu, R. Pastor, W. Liu, K. Barrett, M. Berry, W.S. Blair, C. Chang, J.Z. Chen, C. Eigenbrot, N.
Ghilardi, P. Gibbons, H. He, C.A. Hurley, J.R. Kenny, S. Cyrus Khojasteh, H. Le, L. Lee, J.P. Lyssikatos, S.
This article is protected by copyright. All rights reserved.

Magnuson, R. Pulk, V. Tsui, M. Ultsch, Y. Xiao, B.-y. Zhu, D. Sampath, 2-Amino-[1,2,4]triazolo[1,5-
a]pyridines as JAK2 inhibitors, Bioorg Med Chem Lett, 23 (2013) 5014-5021.
[21] GOLD5.1: https://www.ccdc.cam.ac.uk/solutions/csd-discovery/components/gold/.
[22] Benzoxazoles and oxazolopyridines being useful as Janus kinases inhibitors. WO2008031594
(A1) ― 2008-03-20, in, CIPO, 2008.
[23] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983) 55-63.
[24] O. Phuangsawai, P. Beswick, S. Ratanabunyong, L. Tabtimmai, P. Suphakun, P. Obounchoey,
P. Srisook, N. Horata, I. Chuckowree, S. Hannongbua, S.E. Ward, K. Choowongkomon, M.P. Gleeson,
Evaluation of the anti-malarial activity and cytotoxicity of 2,4-diamino-pyrimidine-based kinase
inhibitors, European Journal of Medicinal Chemistry, 124 (2016) 896-905.
[25] J. O'Brien, I. Wilson, T. Orton, F. Pognan, Investigation of the Alamar Blue (resazurin)
fluorescent dye for the assessment of mammalian cell cytotoxicity, European Journal of
Biochemistry, 267 (2000) 5421-5426.
[26] T. Forsyth, P.C. Kearney, B.G. Kim, H.W.B. Johnson, N. Aay, A. Arcalas, D.S. Brown, V. Chan, J.
Chen, H. Du, S. Epshteyn, A.A. Galan, T.P. Huynh, M.A. Ibrahim, B. Kane, E.S. Koltun, G. Mann, L.E.
Meyr, M.S. Lee, G.L. Lewis, R.T. Noguchi, M. Pack, B.H. Ridgway, X. Shi, C.S. Takeuchi, P. Zu, J.W.
Leahy, J.M. Nuss, R. Aoyama, S. Engst, S.B. Gendreau, R. Kassees, J. Li, S.-H. Lin, J.-F. Martini, T. Stout,
P. Tong, J. Woolfrey, W. Zhang, P. Yu, SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as
potent and selective Janus kinase 2 (JAK2) inhibitors, Bioorg. Med. Chem. Lett., 22 (2012) 7653-7658.
[27] ChemAxon JChem: www.chemaxon.com.
[28] T. Forsyth, P.C. Kearney, B.G. Kim, H.W.B. Johnson, N. Aay, A. Arcalas, D.S. Brown, V. Chan, J.
Chen, H. Du, S. Epshteyn, A.A. Galan, T.P. Huynh, M.A. Ibrahim, B. Kane, E.S. Koltun, G. Mann, L.E.
Meyr, M.S. Lee, G.L. Lewis, R.T. Noguchi, M. Pack, B.H. Ridgway, X. Shi, C.S. Takeuchi, P. Zu, J.W.
Leahy, J.M. Nuss, R. Aoyama, S. Engst, S.B. Gendreau, R. Kassees, J. Li, S.-H. Lin, J.-F. Martini, T. Stout,
P. Tong, J. Woolfrey, W. Zhang, P. Yu, SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as
potent and selective Janus kinase 2 (JAK2) inhibitors, Bioorg Med Chem Lett, 22 (2012) 7653-7658.
[29] P.A. Janne, N. Gray, J. Settleman, Factors underlying sensitivity of cancers to small-molecule
kinase inhibitors, Nat. Rev. Drug Discov., 8 (2009) 709-723.
Notes
The authors confirm no conflicts of interest.
Figure legends
•
Figure 2: Structurally related screening hits identified from cytotoxicity screening in
the A549 cell line.
•
•
Figure 2: Nanomolar active amino-7-aryl-benzoxazole kinase inhibitors reported in
the literature [17, 19].
Figure 3: Configuration of JAK2 binding site (left) and the 3D docked model of 12c bound to
the JAK2 X-ray crystal structure (4JIA, Purple) [17]. The hinge region of JAK2 is also shown in
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stick form and H-bonds are indicated by dotted lines.
•
•
Scheme 1: General route to 7-bromo-2-methylsulfanyl-benzoxazole (9): (a) SnCl2, EtOH, (b)
Potassium ethylxanthogenate, EtOH, (c) K2CO3, MeI, DMF [21].
Scheme 2: Synthetic approach used for the preparation of final compounds from 9.
Supplementary Material
Additional experimental details and characterization data of selected compounds have been
provided.
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