Synthesis, ADMET Properties, and Biological Evaluation of Benzothiazole Compounds Targeting Chemokine Receptor 2 (CXCR2)

Article abstract of DOI:10.1002/cmdc.201700229

Herein we describe the synthesis and biological evaluation of a series of novel benzothiazoles based on a diaryl urea scaffold previously reported in some allosteric chemokine receptor 2 (CXCR2) inhibitors. From a library of 41 new compounds, 17 showed significant inhibition of CXCR2, with IC₅₀ values less than 10 μm and selectivity over CXCR4. Our ADMET simulations suggest favorable drug-like properties for the active compounds. Importantly, we developed a predictive model that can distinguish active from inactive compounds; this will serve as a valuable tool to guide the design of optimized compounds to be evaluated in preclinical models.

Full text of DOI:10.1002/cmdc.201700229

Accepted Article
Title: Synthesis, ADMET properties, and biological evaluation of
benzothiazole compounds targeting CXCR2
Authors: Wesam E. Mehanna, Tiangong Lu, Bikash Debnath, Deena
S. Lasheen, Rabah A. T. Serya, Khaled A. Abouzid, and Nouri
Neamati
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Table of Contents entry
Synthesis, ADMET properties, and biological evaluation of
benzothiazole compounds targeting CXCR2
We combined benzothiazoles and diaryl urea scaffolds to design novel CXCR2 inhibitors that
display selectivity over CXCR4. Our molecular modeling studies support a robust approach to
further optimize these inhibitors to select highly potent compound for in vivo studies.
Wesam E. Mehanna, Tiangong Lu, Bikash Debnath, Deena S. Lasheen, Rabah A. T. Serya,
Khaled A. Abouzid, and Nouri Neamati
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Synthesis, ADMET properties, and biological evaluation of
benzothiazole compounds targeting CXCR2
Wesam E. Mehanna,[a,b] Tiangong Lu,[a] Bikash Debnath,[a] Deena S. Lasheen,[b]
Rabah A. T. Serya,[b] Khaled A. Abouzid,[b] and Nouri Neamati*[a]
[a] Department of Medicinal Chemistry, College of Pharmacy, and Translational Oncology Program, University of
Michigan, North Campus Research Complex, 1600 Huron Parkway, Ann Arbor, Michigan 48109, United States
[b] Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566,
Egypt
Abstract
Herein, we describe the synthesis and biological evaluation of a series of novel benzothiazoles
based on a diaryl urea scaffold previously reported in some allosteric CXCR2 inhibitors. From a
library of 41 new compounds, 17 showed significant inhibition of CXCR2 with IC50 less than 10
M and selectivity over CXCR4. Our ADMET simulations suggest favorable drug-like
properties for the active compounds. Importantly, we developed a predictive model that can
distinguish active from inactive compounds and will serve as a valuable tool to guide the design
of optimized compounds to be evaluated in preclinical models.
Keywords
CXCR2, CXCR4, benzothiazole, diaryl urea, ADMET
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Introduction
Cytokines are small proteins secreted by a variety of cell types. One class of cytokines, called
chemokines, target a family of G-protein coupled receptors (GPCRs) known as chemokine
receptors.[1] The chemokine receptor family is comprised of CXC, CC, C and CX3C receptors.[2]
There are seven members of the CXC subfamily (CXCR1-7). Two members of this family,
CXCR2 and CXCR4, have been shown to be implicated in several pathological processes.
CXCL8 (Interleukin 8), the ligand for CXCR2, stimulates a variety of signaling pathways
important in inflammatory diseases as well as cancer.[3] The chemokine ligand 12 (CXCL12),
also called stromal-derived-factor-1 (SDF-1), interacts with CXCR4 and is responsible for
initiating a cascade of events important for cell migration and invasion.[4] Both CXCR2 and
CXCR4 are validated therapeutic targets and intense efforts are underway to develop selective
antagonists to these GPCRs. Plerixafor (AMD3100) is a selective CXCR4 inhibitor that is
approved by the FDA to treat non-Hodgkin’s lymphoma and multiple myeloma. Currently, there
are no FDA-approved small-molecule drugs targeting CXCR2. Therefore, there is a need to
develop new selective CXCR2 inhibitors for various diseases.[5]
The availability of robust high throughput screening platforms has led to the discovery of diverse
small molecule inhibitors against both targets.[6] Among the earlier CXCR2 antagonists, the
diaryl ureas showed promising activities.[7] In addition, thiazole based compounds were found to
be active against CXCR2.[8] A biphenyl urea compound, TPD7, was also found to inhibit
CXCR4 function in breast cancer cells (Figure 1)[9]. Given that these similar scaffolds showed
activity against both receptors, we were interested in synthesizing new benzothiazole analogs
based on the diaryl urea scaffold to evaluate their selectivity for CXCR2 over CXCR4 receptors.
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Herein, we present the synthesis and biological evaluation of novel CXCR2 inhibitors. To
understand the selectivity of our compounds, we developed a pharmacophore model that
successfully distinguishes active from inactive compounds. Moreover, ADMET properties were
calculated using ADMET Predictor 8 to assess their drug-like properties. The synthesized
compounds display selectivity for CXCR2 over CXCR4 and possess desirable drug-like
properties warranting further optimization.
Results and Discussion
Synthesis
The diaryl-based target compounds 4a-p (Table 1) were prepared according to a method
described in Scheme 1. 2-amino-6-nitrobenzothiazole (1) was acetylated using acetic anhydride
in a mixture of pyridine and acetone to yield the intermediate (2).[10] Compound (2) was reduced
using sodium dithionite in ammonia to give the amine (3),[11] which subsequently reacted with
the appropriate aryl isocyanate to give the target urea 4a-p. Compounds 6a-y (Table 2) were
synthesized as outlined in Scheme 2.[12]
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Scheme 1. Synthesis of compounds 4a-p. Reagents and conditions: (a) acetic anhydride, pyridine/acetone (1:4),
reflux, 8 h (92%); (b) sodium dithionite, NH4OH, reflux, 2 h (72%); (c) aryl isocyanate, THF, pyridine, room
temperature, 12 h (40−80%).
Table 1. RefTverskyCombo Score of compounds 4a-p.
RefTverskyCombo
Compounds
R1
H
R2
H
R3
H
R4
H
Score
1.307
1.311
1.199
1.346
1.313
1.360
1.308
1.309
1.307
1.305
1.321
1.314
1.354
1.327
1.308
1.309
4a
4b
4c
4d
4e
4f
H
H
Cl
-OCF3
H
H
H
H
H
H
-CF3
H
H
H
-CF3
Cl
Cl
H
H
H
-CF3
Cl
H
H
4g
4h
4i
H
H
F
H
H
F
H
H
4j
H
Cl
Cl
Cl
H
H
H
4k
4l
H
H
Cl
H
F
H
4m
4n
4o
4p
Cl
-CF3
Cl
H
H
-CF3
H
H
Cl
H
H
H
H
F
H
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Scheme 2. Synthesis of compounds 6a-y. Reagents and conditions: (a) aryl isocyanate, THF, room temperature, 8 h
(52−84%).
Table 2. RefTverskyCombo Score of compounds 6a-y.
RefTverskyCombo
Compounds
R1
-OC2H5
-OCH3
F
R2
H
H
H
H
H
H
H
H
R3
H
R4
-OCF3
-OCF3
-OCF3
H
R5
H
H
H
H
H
H
H
H
Score
1.268
1.306
1.312
1.375
1.383
1.313
1.377
1.281
6a
6b
6c
6d
6e
6f
H
H
-OC2H5
F
-CF3
-CF3
H
H
Cl
-OCF3
H
6g
6h
-OCH3
-OC2H5
-CF3
-CF3
Cl
Cl
6i
H
Cl
Cl
H
1.328
6j
6k
6l
-CF3
F
F
F
Cl
Cl
Cl
H
H
H
H
H
H
1.343
1.354
1.356
-OC2H5
-OCH3
-OC2H5
6m
H
-CF3
H
-CF3
1.462
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-OCH3
6n
H
-CF3
H
-CF3
1.464
6o
6p
6q
6r
Cl
-OCH3
-OC2H5
Cl
H
Cl
-CF3
H
H
H
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
H
1.473
1.385
1.384
1.392
1.392
1.387
1.338
1.338
1.346
1.346
1.340
Cl
H
H
Cl
H
H
6s
F
Cl
H
H
6t
-CF3
-OCH3
-OC2H5
Cl
Cl
H
H
6u
6v
6w
6x
6y
-CF3
-CF3
-CF3
-CF3
-CF3
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
F
H
H
-CF3
H
H
In vitro characterization and structure activity relationship analysis
To assess selectivity of the compounds for CXCR2 over CXCR4, a Tango assay in U2OS cells
was performed under identical experimental conditions. The Tango assay measures ligand-
induced activation of the receptor CXCR2 or CXCR4 in a stable cell line expressing the
recombinant human CXCR2 or CXCR4 linked to a TEV protease site and a GAL4-VP16
transcription factor. Ligand binding to the receptor results in the recruitment of β-arrestin
proteins (tagged with protease) to the receptor and triggers the release of a tethered transcription
factor. The transcription factor enters the nucleus and activates the transcription of the reporter
gene (β-lactamase). The ability of a compound to inhibit CXCR2 or CXCR4 activation is
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indirectly assessed by measuring the reporter gene activity. SB265610 and AMD3100
(Plerixafor), are potent and selective CXCR2 and CXCR4 chemokine receptor antagonists,
respectively, and were included as positive controls (Figure 2). As summarized in Tables 3 and
4, most of the active compounds are selective CXCR2 inhibitors with IC50 <10 M. Interestingly,
a distinct SAR was noticeable by varying the substituents on the phenyl ring for compounds 4a-p
where the CXCR2 inhibitory activity significantly changes relative to steric and electronic
properties of these substituents. It was clear that having a bulky group with electron-withdrawing
effect increased the inhibitory activities. For example, compounds 4d, 4f and 4k showed IC50
values around 2 M whereas 4k, the dichloro derivative in the meta positions, showed the best
inhibitory activity. Shifting the orientation of the trifluoromethyl group to the para position
decreased the IC50 to 7.4 M while having an electron-donating group significantly reduces the
activity. On the other hand, compounds 6a-y produced good inhibitory activity by having bulky
electron- withdrawing groups on both the benzothiazole and the phenyl moiety. The
trifluoromethyl group showed superior activity on the phenyl ring over the chlorine atom. Most
of the compounds did not show cytotoxicity when assessed in MTT assays in the CXCR2-U2OS
cells as well as in an ovarian cancer cell line, OVCAR-8 (Table 3).
Table 3: Inhibition of CXCR2 and CXCR4, and cytotoxicity evaluation of compounds 4a-p.
Cytotoxicity (IC50, M)
CXCR2 Inhibition CXCR4 Inhibition
Compound code
(IC50, M)
(IC50, M)
OVCAR-8
>30
CXCR2-U2OS
SB265610
0.13 ± 0.04
>10
>30
0.001± 0.0002
>30
>30
NT
AMD3100
NT
4a
4b
4c
4d
4e
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.2 ± 3.1
7.4 ± 0.9
>10
>10
>10
>10
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4f
4g
4h
4i
2.5 ± 0.2
>10
>10
>10
>10
>10
>30
>10
>10
>10
>10
>10
>10
>10
>30
>10
>10
5.7
>10
>10
>10
>10
>30
>10
>10
>10
>10
>10
>10
>10
>10
4j
4.9
4k
4l
2.1 ± 1.0
>10
>10
>30
>10
>10
>30
>10
4m
4n
4o
4p
>10
>10
>10
>10
Table 4: Inhibition of CXCR2 and CXCR4, and cytotoxicity evaluation of compounds 6a-y.
Cytotoxicity (IC50, M)
CXCR2 Inhibition
CXCR4 Inhibition
Compound code
(IC50, M)
(IC50, M)
OVCAR-8
CXCR2-U2OS
SB265610
AMD3100
0.13 ± 0.04
>10
>30
0.001± 0.0002
>30
NT
>30
NT
6a
6b
6c
6d
6e
6f
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>30
>10
>30
>10
4.9
>10
9.4
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>30
2.9
7.5 ± 1.6
>10
>10
>10
2.2 ± 1.4
5.5 ± 3.6
>10
>10
6g
6h
6i
>10
>10
>10
1.0± 0.2
>10
6j
12.6 ± 1.4
>10
6k
6l
>10
>10
>10
6m
6n
6o
6p
6q
6r
2.2± 1.1
3.6± 1.1
0.3 ± 0.1
>10
5.1 ± 1.9
11.8 ± 3.1
1.4 ± 0.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.6 ± 0.4
>10
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6s
2.4 ± 1.1
2.0 ± 0.5
>10
>10
>10
>10
>10
>30
>30
>30
>10
>10
>10
>10
3.57
>10
>10
>30
>10
6t
6u
6v
6w
6x
6y
>10
>10
>10
2.2 ± 1.3
2.1 ± 0.3
2.7 ± 0.9
13.6 ± 1.2
14.0 ± 2.1
>10
Molecular modeling
A shape-based pharmacophore model was generated from the docking conformation of CX25
(Figure 3A) in the allosteric binding pocket of CXCR2.[13] We replaced an anionic feature to a
hydrogen bond donor on the sulfonamide nitrogen to better represent protein-ligand interactions
(Figure 3B). The pharmacophore model was mapped onto compounds 6r and 4f, as they showed
high RefTverskyCombo scores (Figures 3C and 3D) and were active in the CXCR2 Tango
assay, but not in the CXCR4 Tango assay. The model successfully distinguished active from
inactive analogs as shown in the ROC score plot (Figure 4). We observed an AUC value of 0.83,
which is higher than the random model (AUC = 0.5). When compounds from this work were
mixed with an independent set of inactive compounds (from public sources) as a validation
dataset, [13] the pharmacophore was able to distinguish our active compounds with an even higher
AUC value of 0.96. The pharmacophore model shows high AUC values in the validation set and
is selective for the active compounds. As a result, compounds with high RefTverskyCombo
scores are predicted to be active. The calculated RefTverskyCombo scores are shown in the
Tables 1 and 2. Urea analogs have been reported as allosteric CXCR2 inhibitors.[14] The
compounds in the present study are well mapped by the pharmacophore model derived from
CX25, which was originally discovered from ligand-based modeling of CXCR2 allosteric
inhibitors. Therefore, we predict that these compounds are also allosteric inhibitors. To
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investigate the possible binding modes of three of our active compounds, 6o, 6r and 6s,
molecular docking was performed in the allosteric site of CXCR2. We propose that 6o, 6r and
6s bind CXCR2 in a similar fashion and form two hydrogen bond interactions with D40 and
K320, as well as a pi-pi interaction with Y314. There are several other important residues, such
as S81, T83, A249, Q319, V72 and I73 that form van der Waals interactions with these active
compounds (Figure 5).
ADMET properties
All active compounds were evaluated for their ADMET properties using ADMET Predictor 8[15]
.
We calculated various risks descriptors, such as ADMET_Risk, absorption risk (Absn_Risk),
Lipinski’s rules violations (RuleOf5), Risk associated with CYP P450 enzymes (CYP_Risk),
mutational risk (MUT_Risk), risk associated with various toxicity (TOX_Risk) along with LogP
(MlogP) and polar surface area (T_PSA) (Table 5). Star plots of these risks model are shown for
all active compounds (Figure 6). From this data it is clear that most of the active compounds are
predicted to lack toxicity, have good ADMET properties, and are suitable for further
optimization.
Table 5. ADMET properties of active compounds calculated using ADMET Predictor 8.0
ADMET_
Absn_Riska
< 3.5
CYP_Riskb
< 2.5
MUT_Riskc TOX_Riskd
Riske
< 7.5
RuleOf5f
<= 1
MlogPg
< 4.3
T_PSAh
< 140
Desired
valuei
< 1
< 3.3
2.0
1.9
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
0.0
0.0
0.0
0.0
1.0
0.2
0.5
1.1
1.0
1.8
0
0
0
0
0
0
0
0
0
0
2.0
2.0
2.0
1.0
2.0
2.2
2.0
3.0
3.0
3.0
4.6
4.6
5.0
3.1
5.7
5.4
5.1
8.1
8.0
8.8
0
0
0
0
0
0
0
0
0
1
2.8
2.8
3.0
2.7
3.3
3.0
3.0
3.8
3.6
4.4
83.12
83.12
83.12
83.12
63.25
63.25
63.25
63.25
63.25
54.02
4d
4e
4f
4k
6d
6f
6g
6m
6n
6o
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2.0
2.0
2.0
2.0
2.0
2.0
1.3
1.4
1.1
1.1
1.5
1.2
0
0
0
0
0
0
2.8
2.6
3.4
1.4
1.0
3.7
7.2
6.9
8.6
5.5
5.4
9.0
0
0
1
0
0
1
4.0
54.02
54.02
54.02
54.02
54.02
54.02
6r
6s
3.9
4.4
4.0
3.9
4.4
6t
6w
6x
6y
aAbsorption risk model: eight rules based on descriptors and predicted properties which directly contribute to the
b
absorption of drugs; CYP risk model: eight rules based on predicted enzymatic clearances and CYP inhibitions;
d
cMutational risk model: qualitative estimate of overall mutagenicity; Toxicity risk model: of seven rules based on
calculated toxicities including mutational risk model; eADMET risk model: all of the risk models combined and two
f
others such as low unbound fraction and high steady state volume of distribution; Lipinski’s rule of five violations;
h
i
gCalculated LogP, Topological polar surface area; Desired values are reported in the tutorial of the ADMET
Predictor.[3] Red colored values exceed the desired values.
Conclusions
The diaryl urea is an important feature in a previously disclosed class of allosteric CXCR2
inhibitors. In this study, we synthesized a new series of diaryl urea compounds based on a
benzothiazole scaffold and identified compounds with reasonable selectivity against CXCR2
over CXCR4. Out of 41 compounds, 17 showed significant inhibition of CXCR2 with IC50 less
than 10 M. Synthesized compounds have reasonable calculated ADMET properties and are
suitable candidates for further optimization. Our predictive model will be a valuable tool to guide
the design of more potent and selective compounds that will be evaluated in preclinical models.
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Experimental Section
Chemistry
All reagents and solvents were obtained from commercial sources and used as received. 1H NMR
spectra were recorded on Bruker Ascend 400 and Bruker UltraShield 300 NMR spectrometers in
the indicated solvents using TMS as internal reference with values expressed in δ ppm. Thin-
layer chromatography (TLC, for monitoring reaction progress) was performed on pre-coated
aluminum plates (Merck, Silica gel 60 F254). Product spots were visualized using UV irradiation
at λ 254 nm. Column purifications were carried out automatically using a Biotage Isolera One.
Purity was assessed using a Shimadzu LC/MS workstation with a LC-2030C pump system, PDA
detector, and an LC/MS-2020 single quadrupole mass spectrometer. A Shimadzu C18 3 µm
column (50 x 4.6 mm) was used for analysis. Compound purities were calculated as the
percentage peak area of the analyzed compound by UV detection at 254 nm.
N-(6-nitrobenzo[d]thiazol-2-yl)acetamide (2)[16]
To a solution of 2-amino-6-nitrobenzothiazole (1) (5 g, 25 mmol) in a mixture of pyridine and
acetone (1:4, 20 ml), acetic anhydride (20 ml) was added dropwise. The mixture was heated
under reflux for 8 h. After cooling, the mixture was added to acidified cold water. The formed
solid was filtered, washed with water, then dried under vacuum to afford the product as a cotton-
like white solid (5.6 g, 92%); 1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H), 9.04 (s, 1H), 8.27
(d, J = 9.0, 2.1 Hz, 1H), 7.88 (dd, J = 8.9, 1.7 Hz, 1H), 2.26 (s, 3H).
N-(6-aminobenzo[d]thiazol-2-yl)acetamide (3)[16]
To a solution of compound 2 (2.37 g, 10 mmol) in NH4OH (25 ml, 30%), a solution of sodium
dithionite (8.7 g, 50 mmol) in water (40 ml) was quickly added, the reaction mixture was
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10.1002/cmdc.201700229
ChemMedChem
refluxed for 2 h. After cooling, the crude product was filtered off, washed and dried under
1
vacuum to afford 3 as pale buff solid (1.25 g, 72%); H NMR (400 MHz, DMSO-d6) δ 11.77 (s,
1H), 7.39 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.70 (dd, J = 8.6, 2.3 Hz, 1H), 5.13 (s,
2H), 2.15 (s, 3H).
General procedure for preparation of compounds (4a-p)
To a solution of compound 3 (1 equiv) in THF (10 ml) and pyridine (1 ml), isocyanate (1.5
equiv) was added and the mixture was stirred for 12 h at room temperature. The reaction was
quenched by the addition of water then diluted with ethyl acetate. The organic layer was washed
with brine and dried over MgSO4. After filtration and concentration, the crude product was
purified as indicated in each case to afford the desired urea in 40 to 80% yield.
N-(6-(3-phenylureido)benzo[d]thiazol-2-yl)acetamide (4a)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
(78%); 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.79 (s, 1H), 8.69 (s, 1H), 8.12 (s, 1H),
7.64 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 9.1 Hz, 1H), 7.29 (t, J = 7.8 Hz,
2H), 6.98 (t, J = 7.3 Hz, 1H), 2.19 (s, 3H); LC/MS: tR=6.59 min, purity 97.9%, [M+H]+ 326.85.
N-(6-(3-(4-chlorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4b)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(65%); H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.83 (d, J = 4.9 Hz, 2H), 8.10 (s, 1H),
7.64 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.41 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.4 Hz,
2H), 2.19 (s, 3H); LC/MS: tR=3.03 min, purity 97.4%, [M−H]- 358.35.
N-(6-(3-(4-(trifluoromethoxy)phenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4c)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford an off-white solid
(74%); 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.92 (s, 1H), 8.86 (s, 1H), 8.11 (s, 1H),
7.65 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 8.4 Hz,
2H), 2.19 (s, 3H); LC/MS: tR=6.98 min, purity 97%, [M+H]+ 410.85.
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N-(6-(3-(3-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4d)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford an off-white solid
1
(54%); H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 9.09 (s, 1H), 8.93 (s, 1H), 8.14 (d, J =
2.1 Hz, 1H), 8.05 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.59 (dd, J = 8.1, 2.0 Hz, 1H), 7.52 (t, J = 7.9
Hz, 1H), 7.46 – 7.40 (m, 1H), 7.32 (d, J = 7.6 Hz, 1H), 2.20 (s, 3H); LC/MS: tR=5.64 min, purity
>99%, [M+H]+ 394.95.
N-(6-(3-(4-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4e)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford an off-white solid
1
(58%); H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.92 (s, 1H), 8.85 (s, 1H), 8.11 (d, J =
2.1 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 9.1 Hz, 2H), 7.43 (dd, J = 8.7, 2.2 Hz, 1H),
7.33 – 7.26 (m, 2H), 2.19 (s, 3H); LC/MS: tR=3.08 min, purity >99%, [M−H]- 392.60.
N-(6-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4f)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a buff solid (50%);
1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 9.20 (s, 1H), 8.97 (s, 1H), 8.58 (s, 1H), 8.13
(dd, J = 7.3, 2.3 Hz, 2H), 7.70 – 7.58 (m, 2H), 7.47 – 7.35 (m, 1H), 2.20 (s, 3H); LC/MS: tR=5.87
min, purity 98%, [M+H]+ 428.95.
N-(6-(3-(3,4-dichlorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4g)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford an off-white solid
1
(74%); H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.99 (s, 1H), 8.90 (s, 1H), 8.09 (d, J =
2.0 Hz, 1H), 7.89 (d, J = 2.5 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.40 (dd,
J = 8.7, 2.2 Hz, 1H), 7.32 (dd, J = 8.9, 2.5 Hz, 1H), 2.17 (s, 3H); LC/MS: tR=3.13 min, purity
96.2%, [M+H]+ 392.35.
N-(6-(3-(2-fluorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4h)
The crude product was purified by Isolera One (EtOAc/Hexane 1:3) to afford an off-white solid
1
(62%); H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 9.19 (s, 1H), 8.56 (d, J = 2.7 Hz, 1H),
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8.22 – 8.06 (m, 2H), 7.63 (d, J = 8.7 Hz, 1H), 7.39 (dd, J = 8.8, 2.2 Hz, 1H), 7.22 (ddd, J = 11.6,
8.2, 1.5 Hz, 1H), 7.16 – 7.08 (m, 1H), 6.99 (tdd, J = 7.8, 5.1, 1.7 Hz, 1H), 2.17 (s, 3H); LC/MS:
tR=6.69 min, purity 98%, [M+H]+ 344.85.
N-(6-(3-(3-fluorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4i)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a buff solid (80%);
1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.92 (s, 1H), 8.85 (s, 1H), 8.09 (d, J = 2.1 Hz,
1H), 7.63 (d, J = 8.7 Hz, 1H), 7.49 (dt, J = 12.2, 2.3 Hz, 1H), 7.40 (dd, J = 8.7, 2.2 Hz, 1H), 7.29
(q, J = 7.8 Hz, 1H), 7.15 – 7.08 (m, 1H), 6.76 (td, J = 8.5, 2.6 Hz, 1H), 2.17 (s, 3H); LC/MS:
tR=2.99 min, purity >99%, [M+H]+ 344.50.
N-(6-(3-(3-chlorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4j)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford a buff solid (65%);
1H NMR (300 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.89 (d, J = 12.3 Hz, 2H), 8.11 (d, J = 2.1 Hz,
1H), 7.73 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 8.5, 2.2 Hz, 1H), 7.30 (d, J = 7.2 Hz,
2H), 7.10 – 6.95 (m, 1H), 2.19 (s, 3H); LC/MS: tR=3.04 min, purity 95.7%, [M+H]+ 360.85.
N-(6-(3-(3,5-dichlorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4k)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a buff solid (77%);
1H NMR (300 MHz, DMSO-d6) δ 12.25 (s, 1H), 9.09 (s, 1H), 9.01 (s, 1H), 8.11 (d, J = 2.1 Hz,
1H), 7.65 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 1.9 Hz, 2H), 7.43 (dd, J = 8.8, 2.1 Hz, 1H), 7.19 – 7.13
(m, 1H), 2.19 (s, 3H); LC/MS: tR=7.26 min, purity 96.3%, [M−H]- 392.85.
N-(6-(3-(3-chloro-2-fluorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4l)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a buff solid (51%);
1H NMR (300 MHz, DMSO-d6) δ 12.26 (s, 1H), 9.59 (s, 1H), 8.91 (s, 1H), 8.25 – 8.00 (m, 2H),
7.66 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 8.9, 2.2 Hz, 1H), 7.30 – 7.04 (m, 2H), 2.19 (s, 3H);
LC/MS: tR=6.99 min, purity 98%, [M−H]- 376.80.
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N-(6-(3-(2-chloro-5-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4m)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a yellow solid
(42%); 1H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 9.71 (s, 1H), 8.86 – 8.57 (m, 2H), 8.19
(d, J = 14.2 Hz, 1H), 7.70 (dd, J = 24.0, 8.4 Hz, 2H), 7.56 – 7.31 (m, 2H), 2.20 (s, 3H); LC/MS:
tR=3.17 min, purity 97.6%, [M−H]- 426.35.
N-(6-(3-(4-chloro-2-(trifluoromethyl)phenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4n)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a buff solid (47%);
1H NMR (300 MHz, DMSO-d6) δ 12.24 (s, 1H), 9.54 (s, 1H), 8.15 (d, J = 19.2 Hz, 2H), 8.03 (d,
J = 8.5 Hz, 1H), 7.77 – 7.62 (m, 3H), 7.42 (dd, J = 8.8, 2.2 Hz, 1H), 2.19 (s, 3H); LC/MS:
tR=3.16 min, purity 98.5%, [M+H]+ 428.55.
N-(6-(3-(2-chlorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4o)
The crude product was purified by Isolera One (EtOAc/Hexane 1:3) to afford a white solid
1
(56%); H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 9.20 (s, 1H), 8.58 (d, J = 2.7 Hz, 1H),
8.15 (dd, J = 18.7, 1.9 Hz, 2H), 7.66 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 8.8, 2.2 Hz, 1H), 7.28 –
7.21 (m, 1H), 7.18 – 7.12 (m, 1H), 7.05 – 6.98 (m, 1H), 2.20 (s, 3H); LC/MS: tR=3.03 min, purity
96.9%, [M−H]- 358.40.
N-(6-(3-(4-fluorophenyl)ureido)benzo[d]thiazol-2-yl)acetamide (4p)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a buff solid (45%);
1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.83 (d, J = 4.8 Hz, 2H), 8.11 (d, J = 2.1 Hz,
1H), 7.65 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.42 (dd, J = 8.7, 2.2 Hz, 1H), 7.36 – 7.31
(m, 2H), 2.19 (s, 3H); LC/MS: tR=2.96 min, purity 96.8%, [M−H]- 342.45.
General procedure for preparation of compounds (6a-y)
To a solution of the amine (1 equiv) in THF (10 ml), the corresponding isocyanate (1.5 equiv)
was added and the mixture was stirred for 8 h at room temperature. The reaction mixture was
quenched by the addition of water then diluted with ethyl acetate. The organic layer was washed
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with brine and dried over MgSO4. After filtration and concentration, the crude product was
purified as indicated in each case to afford the desired urea.
1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(4-(trifluoromethoxy)phenyl)urea (6a)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford a white solid
1
(82%); H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.36 (s, 1H), 7.63 (d, J = 9.0 Hz, 2H),
7.58 – 7.48 (m, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.98 (dd, J = 8.8, 2.6 Hz, 1H), 4.06 (q, J = 6.9 Hz,
2H), 1.35 (t, J = 6.9 Hz, 3H).); LC/MS: tR=3.47 min, purity 98.6%, [M+H]+ 397.50.
1-(6-methoxybenzo[d]thiazol-2-yl)-3-(4-(trifluoromethoxy)phenyl)urea (6b)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(79%); H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 9.34 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H),
7.54 (dd, J = 14.3, 6.1 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 6.99 (dd, J = 8.7, 2.7 Hz, 1H), 3.80 (s,
3H); LC/MS: tR=3.34 min, purity >99%, [M−H]- 381.35.
1-(6-fluorobenzo[d]thiazol-2-yl)-3-(4-(trifluoromethoxy)phenyl)urea (6c)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(76%); H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 9.35 (s, 1H), 7.84 (d, J = 8.7 Hz, 1H),
7.63 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.25 (td, J = 9.1, 2.8 Hz, 2H); LC/MS: tR=3.38
min, purity 98.8%, [M−H]- 369.80.
1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (6d)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
(84%); 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 9.53 (s, 1H), 8.04 (s, 1H), 7.70 (s, 1H),
7.62 – 7.48 (m, 3H), 7.39 (d, J = 7.7 Hz, 1H), 6.98 (dd, J = 8.7, 2.6 Hz, 1H), 4.07 (q, J = 6.9 Hz,
2H), 1.35 (t, J = 6.9 Hz, 3H); LC/MS: tR=6.35 min, purity 98.8%, [M+H]+ 382.00.
1-(6-fluorobenzo[d]thiazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (6e)
The crude product was purified by Isolera One (EtOAc/Hexane 1:3) to afford a white solid
(82%); 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.52 (s, 1H), 8.03 (s, 1H), 7.85 (dd, J =
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8.8, 2.7 Hz, 1H), 7.75 – 7.62 (m, 2H), 7.58 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.25 (td,
J = 9.1, 2.7 Hz, 1H); LC/MS: tR=7.73 min, purity 96.6%, [M−H]- 353.80.
1-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-(trifluoromethoxy)phenyl)urea (6f)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(72%); H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.36 (s, 1H), 8.04 (s, 1H), 7.61 (d, J =
8.7 Hz, 3H), 7.40 (dd, J = 8.6, 2.2 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H); LC/MS: tR=3.57 min, purity
98.3%, [M−H]- 385.30.
1-(6-methoxybenzo[d]thiazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (6g)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(68%); H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.48 (s, 1H), 8.02 (s, 1H), 7.68 (d, J =
8.2 Hz, 1H), 7.58 – 7.48 (m, 3H), 7.37 (d, J = 8.0 Hz, 1H), 6.98 (dd, J = 8.8, 2.6 Hz, 1H), 3.78 (s,
3H); LC/MS: tR=7.57 min, purity 95.4%, [M+H]+ 367.85.
1-(3,4-dichlorophenyl)-3-(6-ethoxybenzo[d]thiazol-2-yl)urea (6h)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(71%); H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 9.45 (s, 1H), 7.90 (d, J = 2.5 Hz, 1H),
7.58 – 7.39 (m, 4H), 6.96 (dd, J = 8.8, 2.6 Hz, 1H), 4.04 (q, J = 6.9 Hz, 2H), 1.32 (t, J = 6.9 Hz,
3H); LC/MS: tR=3.60 min, purity 98%, [M−H]- 379.50.
1-(3,4-dichlorophenyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea (6i)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(62%); H NMR (300 MHz, DMSO-d6) δ 11.41 (s, 1H), 9.55 (s, 1H), 8.42 (s, 1H), 7.96 – 7.90
(m, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 9.3 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.53 – 7.44
(m, 1H); LC/MS: tR=3.70 min, purity 97.4%, [M−H]- 403.25.
1-(3-chloro-2-fluorophenyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea (6j)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
(57%); 1H NMR (300 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.45 (s, 1H), 8.07 (t, J = 7.6 Hz, 1H), 7.85
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(d, J = 8.5 Hz, 1H), 7.76 – 7.65 (m, 1H), 7.26 (dt, J = 16.4, 7.6 Hz, 2H); LC/MS: tR=3.56 min,
purity 97%, [M−H]- 387.
1-(3-chloro-2-fluorophenyl)-3-(6-ethoxybenzo[d]thiazol-2-yl)urea (6k)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford a pale yellow solid
(55%); 1H NMR (300 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.08 (td, J = 7.6, 2.0 Hz, 1H), 7.61 – 7.49
(m, 2H), 7.32 – 7.16 (m, 2H), 6.98 (dd, J = 8.8, 2.6 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 1.35 (t, J =
6.9 Hz, 3H); LC/MS: tR=8.22 min, purity >99%, [M−H]- 363.75.
1-(3-chloro-2-fluorophenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (6l)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford a yellow solid
(58%); 1H NMR (300 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.09 (ddd, J = 9.1, 7.2, 2.0 Hz, 1H), 7.64
– 7.51 (m, 2H), 7.33 – 7.16 (m, 2H), 7.00 (dd, J = 8.8, 2.6 Hz, 1H), 3.80 (s, 3H); LC/MS: tR=3.39
min, purity 95.6%, [M+H]+ 351.50.
1-(3,5-bis(trifluoromethyl)phenyl)-3-(6-ethoxybenzo[d]thiazol-2-yl)urea (6m)
The crude product was purified by Isolera One (EtOAc/Hexane 1:3) to afford a white solid
(55%); 1H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.25 (s, 2H), 7.70 (s, 1H), 7.50 (d, J = 8.9
Hz, 2H), 6.99 (d, J = 8.8 Hz, 1H), 4.06 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 6.8 Hz, 3H); LC/MS:
tR=3.62 min, purity 96.5%, [M−H]- 447.35.
1-(3,5-bis(trifluoromethyl)phenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (6n)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
(54%); 1H NMR (300 MHz, DMSO-d6) δ 8.26 (s, 2H), 7.69 (d, J = 2.3 Hz, 1H), 7.51 (d, J = 7.7
Hz, 2H), 6.99 (d, J = 9.1 Hz, 1H), 3.80 (s, 3H); LC/MS: tR=3.49 min, purity 95.2%, [M−H]-
433.40.
1-(3,5-bis(trifluoromethyl)phenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea (6o)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(52%); H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.25 (s, 2H), 8.05 (s, 1H), 7.73 (s, 1H),
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7.61 (d, J = 8.4 Hz, 1H), 7.44 (dd, J = 8.9, 2.1 Hz, 1H); LC/MS: tR=3.80 min, purity 95.9%,
[M−H]- 437.80.
1-(2-chloro-5-(trifluoromethyl)phenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (6p)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford a white solid
1
(59%); H NMR (300 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.43 (s, 1H), 8.62 (d, J = 2.6 Hz, 1H),
7.77 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 2.5 Hz, 1H), 7.47 (dd, J = 8.5, 2.2
Hz, 1H), 7.01 (dd, J = 8.9, 2.6 Hz, 1H), 3.81 (s, 3H); LC/MS: tR=4.36 min, purity 95.6%,
[M+H]+ 401.60.
1-(2-chloro-5-(trifluoromethyl)phenyl)-3-(6-ethoxybenzo[d]thiazol-2-yl)urea (6q)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(62%); H NMR (300 MHz, DMSO-d6) δ 11.43 (s, 1H), 9.45 (s, 1H), 8.61 (d, J = 1.2 Hz, 0H),
7.78 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 7.47 (dd, J = 8.0, 2.2
Hz, 1H), 7.00 (dd, J = 8.8, 2.5 Hz, 1H), 4.06 (q, J = 6.9 Hz, 2H), 1.35 (t, J = 6.9 Hz, 3H);
LC/MS: tR=2.64 min, purity 96.6%, [M−H]- 413.35.
1-(2-chloro-5-(trifluoromethyl)phenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea (6r)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
(53%);1H NMR (300 MHz, DMSO-d6) δ 11.66 (s, 1H), 9.33 (s, 1H), 8.60 (d, J = 2.2 Hz, 1H),
8.09 (d, J = 2.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.46 (ddd, J = 15.3,
8.9, 2.2 Hz, 2H); LC/MS: tR=3.86 min, purity 97%, [M−H]- 403.30.
1-(2-chloro-5-(trifluoromethyl)phenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (6s)
The crude product was purified by Isolera One (EtOAc/Hexane 1:1) to afford a white solid
1
(55%); H NMR (300 MHz, DMSO-d6) δ 11.55 (s, 1H), 9.35 (s, 1H), 8.60 (d, J = 2.6 Hz, 1H),
7.87 (dd, J = 8.7, 2.7 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.71 (dd, J = 8.9, 4.8 Hz, 1H), 7.48 (dd, J
= 8.5, 2.2 Hz, 1H), 7.26 (td, J = 9.1, 2.7 Hz, 1H); LC/MS: tR =3.55 min, purity 97.2%, [M−H]-
387.35.
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1-(2-chloro-5-(trifluoromethyl)phenyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea (6t)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(44%); H NMR (300 MHz, DMSO-d6) δ 11.81 (s, 1H), 9.31 (s, 1H), 8.59 (d, J = 2.7 Hz, 1H),
8.46 (d, J = 2.0 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.5, 2.2
Hz, 1H), 7.50 (dd, J = 8.4, 2.2 Hz, 1H); LC/MS: tR =3.74 min, purity 95.3%, [M−H]- 437.30.
1-(4-chloro-2-(trifluoromethyl)phenyl)-3-(6-methoxybenzo[d]thiazol-2-yl)urea (6u)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
(56%);1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.12 (s, 1H), 8.06 (d, J = 8.7 Hz, 1H),
7.83 – 7.75 (m, 2H), 7.62 – 7.51 (m, 2H), 7.01 (dd, J = 8.9, 2.7 Hz, 1H), 3.80 (s, 3H); LC/MS:
tR=3.53 min, purity 98.4%, [M−H]- 399.35.
1-(4-chloro-2-(trifluoromethyl)phenyl)-3-(6-ethoxybenzo[d]thiazol-2-yl)urea (6v)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(67%); H NMR (300 MHz, DMSO-d6) δ 11.32 (s, 1H), 9.13 (s, 1H), 8.05 (d, J = 8.6 Hz, 1H),
7.78 (d, J = 9.3 Hz, 2H), 7.60 – 7.49 (m, 2H), 6.99 (dd, J = 8.8, 2.5 Hz, 1H), 4.06 (q, J = 7.0 Hz,
2H), 1.34 (t, J = 6.9 Hz, 3H); LC/MS: tR=3.69 min, purity 98%, [M−H]- 413.35.
1-(4-chloro-2-(trifluoromethyl)phenyl)-3-(6-chlorobenzo[d]thiazol-2-yl)urea (6w)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(48%); H NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H), 8.97 (s, 1H), 8.09 (d, J = 2.2 Hz, 1H),
8.03 (d, J = 8.6 Hz, 1H), 7.84 – 7.77 (m, 2H), 7.67 (d, J = 8.6 Hz, 1H), 7.43 (dd, J = 8.6, 2.3 Hz,
1H); LC/MS: tR=3.80 min, purity 98%, [M−H]- 403.30.
1-(4-chloro-2-(trifluoromethyl)phenyl)-3-(6-fluorobenzo[d]thiazol-2-yl)urea (6x)
The crude product was purified by Isolera One (EtOAc/Hexane 1:3) to afford a white solid
1
(50%); H NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H), 9.35 (s, 1H), 8.61 (d, J = 2.3 Hz, 1H),
7.87 (dd, J = 8.7, 2.7 Hz, 1H), 7.78 (dd, J = 8.4, 1.0 Hz, 1H), 7.71 (dd, J = 8.9, 4.8 Hz, 1H), 7.48
(dd, J = 8.5, 2.2 Hz, 1H), 7.27 (td, J = 9.1, 2.7 Hz, 1H); LC/MS: tR=3.54 min, purity 95.4%,
[M−H]- 387.30.
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10.1002/cmdc.201700229
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1-(4-chloro-2-(trifluoromethyl)phenyl)-3-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)urea (6y)
The crude product was purified by Isolera One (EtOAc/Hexane 1:2) to afford a white solid
1
(53%); H NMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H), 8.95 (s, 1H), 8.45 (s, 1H), 8.03 (d, J =
8.6 Hz, 1H), 7.88 – 7.78 (m, 3H), 7.72 (dd, J = 8.6, 2.0 Hz, 1H); LC/MS: tR=3.73 min, purity
>99%, [M−H]- 437.30.
Molecular modeling:
Generation of the shape-based pharmacophore. The shape-based pharmacophore was generated
using vROCS v3.2.0.4 (OpenEye Scientific)[17]. ROCS performs shape-based overlays of
conformers from a candidate database to a query molecule with one or more conformations[18]
.
Here, we have used combination of shape overlap and color overlap by aligning groups with
similar properties in the color force field. The score, referred to as RefTverskyCombo was
calculated as the sum of RefTversky, which is the shape score, and RefColorTversky score. The
nominal value of the Combo score is in between 0 and 2, although based on the nature of the
shape matching, the score can be higher than 2. Tversky scores include a weighting factor to deal
with size differences between query and target molecules, particularly when the query is small
(see ROCS documentation)[19]. In this case, the query molecule CX25 is smaller than the
compounds reported here.
Validation of the shape-based pharmacophore. The pharmacophore was validated for predictive
power using compounds reported in this study (active and inactive) and 99 inactive compounds
used as a validation set which can be found in our previous work [13]. ROC curves were plotted
for the prediction of these compounds and compared against a random model that has an AUC
value of 0.5. A ROC curve plots % or fraction of actives found on Y-axis vs % decoys or
inactives on the X-axis as the ROCS scores decrease. The top scoring compounds are plotted
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10.1002/cmdc.201700229
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closest to the origin. The curve gives an indication of how the actives and inactives are ranked as
a result of the ROCS run. An ideal ROC plot for a perfectly selective query would show all of
the actives being identified first because they score most highly.[17]
Molecular docking. Molecular docking studies were performed using the GOLD (Genetic
Optimization for Ligand Docking) software package, version 5.2 in the allosteric site of CXCR2
homology model.[13] Prior to docking, 10 different conformations were generated for each ligand
using Omega (OpenEye Scientific, Santa Fe, NM), a systematic, knowledge-based conformer
generator.[20] GOLD uses a genetic algorithm to explore the conformational space of a compound
inside the binding site of a protein.[21] Docking studies were performed using the standard default
settings with 100 genetic algorithm (GA) runs on each molecule with side chain flexibility of
important allosteric site residues allowed within their rotamer library.
ADMET properties calculation. ADMET properties and associated risk values were calculated
using ADMET Predictor 8 from Simulation Plus, Inc.[15] The absorption risk model includes
eight rules, each contributes a value of 1, based on descriptors and predicted properties that
directly contributes to the absorption of drugs, such as molecular weight, hydrogen bond donor,
hydrogen bond acceptor, polar surface area, number of rotatable bonds, etc. The CYP risk model
is comprised of eight rules, each contributes a value of 1, based on predicted enzymatic
clearances and CYP inhibitions. The mutational risk model yields a qualitative estimate of
overall mutagenicity. The toxicity risk model is comprised of seven rules, each contributes a
value of 1, based on calculated toxicities, such as hERG toxicity, acute toxicity in rats including
the mutational risk model. The ADMET risk model is comprised of a total of 23 rules, each
contributes a value of 1, which include all of these risk models and two others, such as low
unbound fraction and high steady state volume of distribution. Lipinski’s Rule of five violations
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10.1002/cmdc.201700229
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is another risk model for drug-likeliness. LogP and topological polar surface area were also
calculated.
Biology:
Cell Culture
OVCAR-8 cells (National Cancer Institute, Developmental Therapeutics Program, Bethesda,
MD) were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS (Gemini
Bio-products). CXCR2-bla and CXCR4-bla U2OS Tango cells were purchased from Invitrogen
and grown in McCoy’s 5A supplemented with 10% dialyzed FBS, zeocin (200 μg/mL),
hygromycin (50 μg/mL), Geneticin (100 μg/mL), 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids (NEAA), and 25 mM HEPES. All cells were grown at 37 °C in a humidified
atmosphere of 5% CO2. To remove adherent cells from the flask for subculture and counting,
cells were washed with PBS without calcium or magnesium, incubated with a small volume of
0.25% trypsin–EDTA solution (Mediatech, Inc.) for 5 minutes, resuspended with culture
medium, and centrifuged. All cell lines used were maintained in culture under 30 passages and
tested regularly for mycoplasma contamination using Plasmo Test (InvivoGen, San Diego, CA).
Compounds and Reagents
Compounds were prepared as 10 mM stock solutions in dimethyl sulfoxide (DMSO) and stored
at −20 °C. SB265610 was purchased from Tocris Bioscience (Ellisville, MI). Human CXCL8
cDNA was inserted into the expression vector pET32a at the EcoR1 and XhoI restriction sites to
generate His-tagged CXCL8. The sequence of the clone was confirmed by DNA sequencing.
SDF-1α expression vector was kindly provided by Dr. Ghalib Alkhatib. Recombinant CXCL8
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and SDF-1α were expressed in BL21-Gold (DE3) pLysS (Stratagene, La Jolla, CA) and purified
using a previously reported protocol.[22]
Cell Proliferation
Cell proliferation was assessed by MTT assay as previously described.[23] In brief, OVCAR-8 or
CXCR2-bla U2OS cells were seeded in 96-well microtiter plates (3000−5000 cells/well) and
were allowed to attach overnight. The cells were continuously treated with compounds for 72 h.
At the end of treatment, the cells were incubated with MTT solution (at a final concentration of
0.5 mg/mL) for 3 h at 37 °C. The supernatant was removed, and the formazan was dissolved in
150 μL of DMSO. The absorbance was read at 570 nm on a microplate reader (Molecular
Devices, Sunnyvale, CA). All of the experiments were performed in triplicate. IC50 values were
determined for each compound from a plot of log (drug concentration) versus percentage of cells
killed. The data reported with standard deviations are representative of at least three independent
experiments.
CXCR2 and CXCR4 Tango Assay
CXCR2-bla U2OS and CXCR4-bla U2OS cells were genetically modified to stably over-express
CXCR2 or CXCR4 linked to a tobacco etch virus (TEV) protease site and a GAL4-VP16
transcription factor. These cells also stably express a β-arrestin/TEV protease fusion protein and
a β-lactamase reporter gene. Upon CXCR2 activation in the presence of IL-8 or CXCR4
activation in the presence of SDF-1α, the β-arrestin/TEV fusion protein is recruited to the
receptor and cleaves the peptide linker that links CXCR2 to the GAL4-VP16 transcription factor.
Subsequently, GAL4-VP16 enters the nucleus and promotes the transcription of the β-lactamase
gene. β-Lactamase activity is detected using a fluorescence resonance energy transfer (FRET)-
based fluorescence assay with CCF4-AM (Invitrogen, Thermo Fisher). CCF4-AM is a β-
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10.1002/cmdc.201700229
ChemMedChem
lactamase FRET substrate and is cleaved in the presence of β-lactamase. The uncleaved substrate
excites at 409 nm and emits at 535 nm, whereas the cleaved substrate excites at 409 nm and
emits at 460 nm. Thus, IL-8 activation of CXCR2 or SDF-1α activated CXCR4 is directly
correlated with the amount of cleaved β-lactamase substrate.
In each assay, CXCR2- or CXCR4-bla U2OS cells were seeded at 1.1×104/well in 384-well
tissue culture plates (BD Falcon) for 24 h in DMEM supplemented with 1% FBS. Cells were
pretreated with various concentrations of inhibitors for 30 min prior to the addition of IL-8 (15
nM) or SDF-1α (30 nM) and incubated for 5 h at 37 °C. β-Lactamase substrate was loaded
according to the manufacturer’s protocol for 2 h, and plates were read on an CLARIOstar
microplate reader (BMGlabtech) with excitation at 409 nm and emission at 460 or 530 nm.
Percent inhibition was calculated using the following formulas:
Acknowledgments
This study was supported by the Department of Defense Lung Cancer Research Program, Grant
No. LC090363 (N. N.). WEM was supported by a joint supervision fellowship from the Egyptian
government (E.M.E.D).
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Figure Legends:
Figure 1. Select CXCR2 inhibitors based on thiazole and diaryl urea moieties.
Figure 2. A. 4d, 4e, 4f and 4k inhibit IL8-mediated CXCR2 β-arrestin-2 recruitment. B. 6d, 6g
and 6f inhibit IL8-mediated CXCR2 β-arrestin-2 recruitment. CXCR2-bla U2OS Tango cells
were pretreated with various concentrations of compounds or SB265610 for 30 min. Cells were
stimulated with 15 nM of IL8 for an additional 5 h.
Figure 3. Pharmacophore model generation and screening. A. Chemical structure of CXCR2
inhibitor, CX25. B. ROCS pharmacophore with features (four rings, one each of HBA and HBD)
generated based on CX25. C & D. Active compounds 6o and 4f were mapped onto the ROCS
pharmacophore with RefTverskyCombo score value of 1.473 and 1.36, respectively.
Figure 4. ROC score plot for the pharmacophore model screening. Red: ROC curve for the
screening of actives vs. inactives reported in this work (AUC value = 0.832); Green: ROC curve
for the screening of actives from this work vs. inactives from this work and various other sources
(AUC value = 0.967); Blue: ROC curve for the random model (AUC value = 0.5).
Figure 5. Molecular docking of compounds 6o, 6r and 6s in the allosteric site of CXCR2. A, C,
E. 3D binding poses of compounds 6o, 6r and 6s, respectively, were generated using PyMol.
Predicted binding pose of compounds (orange) in the allosteric site of CXCR2 (binding site
surface is grey, important residues are in cyan). B, D, F. 2D protein-ligand interactions of
compounds 6o, 6r and 6s, respectively, were generated using Biovia Discovery Studio 2016
client and redrawn in ChemBioDraw Ultra 14.0. Compounds are predicted to form two H-bond
interactions with D84 and K320, a pi-pi interaction with Y314, and van der Waals interactions
with A249, Q319, S81, T83, V72, I73 and Y314.
Figure 6. Star plots generated within ADMET predictor 8.0 for calculated risks for all active
compounds. Red-Toxicity risks; Green-ADMET risks; Blue-Absorption risks; Pink-CYP risks;
Yellow-Mutation risks.
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