Discovery of 4-arylamido 3-methyl isoxazole derivatives as novel FMS kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.08.031

A series of 4-arylamido 3-methyl isoxazoles were synthesized and evaluated for their antiproliferative activities against the A375P melanoma and U937 hematopoietic cell lines. Most compounds showed selective antiproliferative activity toward the U937 cell line and the activities were better than that of sorafenib, the reference standard. Derivatives were made as amide 5a-b, 6a-o and urea 7a-n, 8a-g with hydrophobic moieties, and one of the most potent inhibitor 6a, 5-methyl-N-(2-methyl-5-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)benzamido)phenyl)isoxazole-4-carboxamide was found to be very potent inhibitor of FMS kinase (GI₅₀ Combining double low line 0.016 μM, IC₅₀ Combining double low line 9.95 nM) with excellent selectivity profiles and is a promising candidate for further development in therapeutics for cancer.

Full text of DOI:10.1016/j.ejmech.2015.08.031

Accepted Manuscript
Discovery of 4-Arylamido 3-methyl isoxazole derivatives as novel FMS kinase
inhibitors
Daseul Im, Kyungjin Jung, Songyi Yang, Waqar Aman, Jung-Mi Hah
PII:
S0223-5234(15)30215-4
10.1016/j.ejmech.2015.08.031
EJMECH 8067
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2015
Revised Date: 13 August 2015
Accepted Date: 15 August 2015
Please cite this article as: D. Im, K. Jung, S. Yang, W. Aman, J. Mi Hah, Discovery of 4-Arylamido 3-
methyl isoxazole derivatives as novel FMS kinase inhibitors, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.08.031.
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ACCEPTED MANUSCRIPT
Graphical abstract
CF3
O
H
N
H
N
O
N
N
O
N
IC / FMS = 9.95 nM
5
0
GI / U937 = 16.0 nM
50

ACCEPTED MANUSCRIPT
Discovery of 4-Arylamido 3-methyl isoxazole derivatives as
novel FMS kinase inhibitors
*
Daseul Im, Kyungjin Jung, Songyi Yang, Waqar Aman, and Jung-Mi Hah
Department of Pharmacy, College of Pharmacy, & Institute of Pharmaceutical Science and Technology,
Hanyang University 55 Hanyangdaehak-ro, Sangnok-gu, Ansan Kyeonggi-do, 426-791, Korea
Abstract – A series of 4-Arylamido 3-methyl isoxazoles were synthesized and evaluated for their antiproliferative
activities against the A375P melanoma and U937 hematopoietic cell lines. Most compounds showed selective
antiproliferative activity toward the U937 cell line and the activities were better than that of sorafenib, the reference
standard. Derivatives were made as amide 5a-b, 6a-o and urea 7a-n, 8a-g with hydrophobic moieties, and one of the most
potent inhibitor 6a, 5-methyl-N-(2-methyl-5-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)benzamido)phenyl)isoxazole-
4
-carboxamide was found to be very potent inhibitor of FMS kinase (GI = 0.016 ꢀM, IC = 9.95 nM) with excellent
50 50
selectivity profiles and is
a promising candidate for further development in therapeutics for cancer.
Keywords: 4-arylamido 3-methyl isoxazoles; antiproliferative activity; hematopoietic cell line; kinase inhibitor; kinase selectivity.
Corresponding authors. Tel.: +82 31 400 5803; fax: +82 31 400 5958
*
E-mail address: jhah@hanyang.ac.kr
1
. Introduction
Since the discovery of a protein kinase in 1954 [1], the field of protein kinase drug discovery has advanced
enormously [2-5]. Early studies were focused on receptor tyrosine kinases including EGFR, VEGFR, and PDGFR,
especially as therapeutic targets for cancer. Receptor tyrosine kinases have been shown not only to be key regulators
of normal cellular processes but also to have critical roles in the development and progression of many types of
cancer [6]. The impact of RTK inhibition on cancer cells can be illustrated by the assortment of kinase drugs that
have been approved by the FDA [7].
Figure 1. FDA approved RTK inhibitors.
Among all the receptor tyrosine kinases known as drug targets, FMS kinase [8-10] has not been given a lot of
attention as a therapeutic target. FMS, first discovered as the oncogene responsible for Feline McDonough Sarcoma,
is a type III receptor tyrosine kinase that binds to macrophage or monocyte colony-stimulating factor (M-CSF or
CSF-1). Ligand stimulated FMS tyrosine phosphorylation regulates the survival, proliferation, and differentiation of
monocyte/macrophage lineages [11]. Macrophages, microglia, and osteoclasts play important roles in inflammatory
processes [12], and tumor-associated macrophages are increasingly recognized as a driving force for tumor
progression and metastasis [13] as well as resistance to chemo- and radiotherapy [14,15]. Thus, the inhibition of

ACCEPTED MANUSCRIPT
FMS kinase activity has great potential in the treatment of bone osteolysis and inflammation as well as cancers
promoted by macrophages [16-20].
Here we report the discovery of a highly selective and potent FMS inhibitor through screening of our in-house
protein kinase inhibitor library on A375P [21] and U937 cell lines [22]. During the cellular screening of protein
kinase inhibitors, several type II chemical scaffolds were identified as possessing high antiproliferative activities
toward the two cell lines, but the compound with a methyl isoxazole scaffold as the hinge binder showed potent
antiproliferative activity only for the U937 cell line, prompting us to investigate its kinase profiling. The following
experiments showed that 2-methyl-N-(5-methylisoxazol-4-yl)benzamide is a selective FMS inhibitor (IC50 = 216
nM) with negligible activities toward the other 45 kinases tested (Supplementary information). Subsequently, we
decided to synthesize more 2-methyl-N-(5-methylisoxazol-4-yl)benzamide derivatives with various aromatic tails.
Figure 2. Initial investigations of various hinge binder and its preference toward cancer cell lines
2
2
. Results and discussion
.1 Chemistry
The route that enabled the synthesis of the 4-arylamido 3-methyl isoxazoles derivatives is outlined in Scheme 1.
The synthesis of these 4-arylamido 3-methyl isoxazoles started from commercially available 5-methylisoxazle-
carboxylic acid (1). The carboxylic acid was converted to acid chloride (2) using SOCl and subsequently coupled
2
with 2-methyl-4/5-nitroaniline. Then, the nitro group of 5-methyl-N-(2-methyl-4-nitrophenyl) isoxazole-4-
carboxamide (3a and 3b) was reduced to aniline (4a and 4b) using SnCl in acidic conditions. The N-(5-amino-2-
2
methylphenyl)-5-methylisoxazole-4/5-carboxamide was then linked with various aromatic acids by using HATU in
DMF condition or aromatic carboxylic acid chloride to give amide analogues (5a-5b ,6a-6o), and coupled with
propane-1-sulfonyl chloride to yield 9a-9b. Urea analogues (7a–7n, 8a-8g) were synthesized by directly reacting
with various aromatic isocyanates.
Scheme 1.
2
2
.2. Biological evaluation
.2.1 Antiproliferation assays on cancer cell lines
All the synthesized compounds 5a~5b, 6a~6o, 7a~7g, 8a~8g, and 9a~9b were evaluated for their anticancer
activity against U937 (human histiocytic lymphoma) and A375P (human melanoma) cell lines by MTT assay. They
were also evaluated for cytotoxicity on a human normal cell line, HS27. The results are summarized in Table 1. The
results of this assay showed that all the tested compounds had selective potency against U937 rather than against
A375P (data not shown for A375P because the measured activity for all compounds was negligible). The results also
indicated that these compounds exhibited weak cytotoxic activity against normal cell line HS27.

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Among the compounds tested, amide link analogue 6a showed the most potent activity against U937, with an IC50
value of 16 nM, which was much better than that of the positive control sorafenib. Structure activity relationships
(
SARs) were inferred from the data of cell proliferative experiments reported in Table 1. First of all, amide link
analogues showed more potent activities against U937 than did the urea link analogues. Urea analogues (7a~7g,
a~8g), generally, were not as active toward U937, for both the 3- and 4-substituted patterns of the middle phenyl
ring.
Secondly, out of these amide analogues, the effects of the substitution pattern of the middle phenyl ring was also
8
very clear, and 3-amide was absolutely more potent, while 4-amide analogues (5a~5b) showed only weak inhibitory
activities. The sulfonamide analogues with a simple propyl chain were not active at all (9a~9b). The type, number,
and position of substituents on the phenyl ring linking the middle phenyl moiety also played important roles in the
antiproliferative activities. The inhibitory activities of compounds 6a~6o against U937 varied depending on the
different hydrophobic substituents included in the carboxylic acid moiety. Based on the IC50 values, compounds
could be grouped by activity from most to least potent as follows : compounds with a 1,3,5-substituted benzoic acid
tail (6a, 6m, 6n, 6o) exhibited potent inhibitory activities with IC s of 0.016, 0.019, 0.026 and 0.049 µM,
50
respectively; compounds with a 1,3,4-bulky substituents on aromatic acid tail or in similar patterns of imidazole
substituent (6b, 6j) displayed slightly weaker inhibitory activities with IC50 of 0.465 and 0.139 µM; compounds with
simpler mono-substitution on a benzene tail (6d, 6k) or compounds with a bulky aromatic tail (6e, 6f, 6g, 6h)
showed even weaker activities.
The above results indicated that the introduction of a bulky aromatic acid substituent through direct amide
connection caused dramatic decrease in activity while introduction of 3, 5-disubstituted benzoic acid resulted in high
activity, especially when one substituent was a bulky morpholine, alkylpiperazine, or alkylimidazole. Comparing the
activities of compounds 6a, 6m, 6n, and 6o against U937, we found that compound 6a with –CF and
3
methylpiperazinyl substituents at the 3 and 5 positions exhibited the most potent activity (IC = 0.016 µM).
50
Table1. Antiproliferative activities of N-(4 or 5-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide derivatives.
2
.2.2. Protein Kinase Profiling Assay
As shown in Table 2, the representative compound 6a was screened against a selected panel of 30 different
kinases at a single dose concentration of 10 µM and it was revealed that the compound has an excellent selectivity
profile. While this compound effectively inhibited the activities of FMS, V600E B-Raf, C-Raf, and Lyn, the activity
level of inhibition exerted on most of the other kinases tested was below 10 %. Furthermore, we evaluated the in
vitro enzymatic inhibition of the compounds, 6a and 8a toward FMS, V600E B-Raf, C-Raf, and Lyn. As shown in
Table 3, compound 6a demonstrated superb activity on FMS kinase (9.95 nM), but only good potency on V600E B-
Raf, C-Raf, and Lyn (5.96 µM, 668 nM, 65.9 nM, and 44.8 nM).
Table 2. Percentages of enzymatic inhibitions by compound 6a (10 µM) on selected protein kinase panel.

ACCEPTED MANUSCRIPT
Table 3. IC for enzymatic inhibitory activity of the selected compounds.
50
2
.3. Molecular docking studies
In order to better understand the interaction between the synthesized compounds and FMS kinase, molecular
docking of the potent compound 6a into the ATP binding site of FMS kinase (PDB: 3LCO) was performed using
Glide (SCHRODINGER software package Version 14.2). The binding model is depicted in Fig. 3. In this binding
model, compound 6a is nicely bound to the ATP-binding site of FMS via four hydrogen bonds, two π-cation
interactions, and an ion interaction.
Figure 3. Docking structures of designed 4-arylamido 3-methyl isoxazoles scaffold amide derivatives (thin, cyan) in FMS
(
PDB: 3LCO) [27]
The nitrogen atom of 5-methylisoxazole forms a hydrogen bond (N/HeN: 2.0Å) with the amino hydrogen atom of
Cys666 and another hydrogen bond (2.43 Å) between oxygen atom of Thr663 with N-H of amide bond, which
suggests that the isoxazole group plays an important role in the combination of the receptor and ligand. The middle
phenyl ring of 6a forms a π-cation interaction with the amino group of Lys 616 and there is another π-cation
interaction between the positively charged nitrogen atom of the piperazine ring and His776 (shown in light green).
Two more hydrogen bonds were identified at the amide bond, one between the N-H of the amide bond and the
oxygen atom of the GLU 633 residue, and the other between the carbonyl oxygen of the amide bond and hydrogen
atom (2.4 Å) of the Asp796 backbone. Furthermore, the strong ion interaction between the protonated nitrogen of
the piperazine ring (1.6 Å) and the negative charge of Asp796 might enhance the binding affinity, resulting in the
increased antiproliferative activity of this compound. The light blue color shows the hydrophobic regions around the
hydrophobic tail.
3
. Conclusions
A series of 4-arylamido 3-methyl isoxazole derivatives containing various aromatic moieties were synthesized and
verified as novel FMS inhibitors that displayed good antiproliferative activities against tumor cell lines (U937) and
weak cytotoxic activities against a human normal cell line and melanoma cell line A375P. Compound 6a exhibited
the most potent inhibitory activity against FMS with an IC50 of 9.95 nM, and also showed the greatest inhibitory
activities against the hematopoietic cancer cell line U937 with a GI50 of 16 nM. Molecular docking of the most
potent inhibitor 6a into the ATP binding site of FMS kinase was performed and the result suggested that compound
6
a could bind well with the FMS active site. The above findings provided a theoretical basis for further structural
optimization of 4-arylamido 3-methyl isoxazole derivatives as FMS inhibitors and demonstrated that compound 6a
could be a promising scaffold for new therapeutics for cancer, due to its strong kinase profiling.

ACCEPTED MANUSCRIPT
4
4
. Materials and methods
.1. General chemistry
All chemicals (reagent grade) used were purchased from Aldrich (USA). Separation of the compounds by column
chromatography was carried out with silica gel 60 (200–300 mesh ASTM, E. Merck, Germany). The quantity of
silica gel used was 50–100 times the weight charged on the column. Thin layer chromatography (TLC) was run on
the silica gel coated aluminum sheets (silica gel 60 GF254, E. Merck, Germany) and visualized in ultraviolet (UV)
1
13
light (254 nm). H NMR and C NMR spectra were recorded on Brucker model digital AVANCE III 400 MHz
o
spectrometer at 25 C, using tetramethylsilane (TMS) as the internal standard. High-resolution MS (HR/MS)
experiments were conducted with a Finnigan LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific Inc, MA,
USA) operated in positive-ion electrospray mode.
4
.1.1. Synthesis of 5-methylisoxazole-4-carbonyl chloride (2)
o
The 5-methylisoxazole-4-carboxylic acid (1) (1g, 7.86 mmol) in SOCl (3 mL) was heated at 50 C until compound 1
2
disappeared in TLC. After reaction was terminated, the mixture was cooled to ambient temperature and solvent was
evaporated under reduced pressure. 5-methylisoxazole-4-carbonyl chloride, a crude yellow oil (2) (96%) was used
1
for the next step without additional purification; H NMR (400 MHz, DMSO) δ 8.77 (1H, s), 2.64 (3H, s).
4
.1.2. General Syntheses of 5-methyl-N-(2-methyl-4-or 5-nitrophenyl) isoxazole-4-carboxamide (3a-3b)
The mixture of 5-methylisoxazole-4-carbonyl chloride (2) (1.1 g, 7.55mmol) and 2-methyl-4-nitroaniline (1.15g,
o
7
.55mmol) in THF (75 ml) was heated at 65 C until compound 2 disappeared in TLC. After completion of the
reaction, the mixture was cooled to ambient temperature and solvent was removed in vacuo. The concentrated crude
product was purified by flash column chromatography with EA/Hex (1:3) as the eluent to give product
4
.1.2.1. 5-methyl-N-(2-methyl-4-nitrophenyl)isoxazole-4-carboxamide (3a) : The title compound was isolated
1
as a yellow solid (100%); H NMR (400 MHz, DMSO) δ 9.91 (1H, s), 9.06 (1H, s), 8.19 (1H, s), 8.11 (1H, t, J =
1
3
2
1
2
1
.8 Hz), 7.79 (1H, t, J = 4.4), 2.68 (3H, s), 2.38 (3H, s); C NMR (101 MHz, DMSO) δ 173.12, 159.36, 149.29,
44.26, 142.15, 133.75, 125.56, 125.44, 121.55, 111.55, 17.89, 12.13; HRMS (ESI) calcd for C12 [M+H]+:
11 3 4
H N O
o
-1
62.2334, found 261.9872. 261.0187.; m.p. 169-170 C; IR(ATR) ν max/cm : 3437, 1698, 1586, 1543, 1499, 1336,
276, 1103.
4
.1.2.2. 5-methyl-N-(2-methyl-4-nitrophenyl)isoxazole-4-carboxamide (3b): The title compound was isolated
1
as a yellow solid (100%); H NMR (400 MHz, DMSO) δ 9.94 (1H, s), 9.05 (1H, s), 8.33 (1H, s), 8.04 (1H, t, J =
1
3
4
.0 Hz), 7.58 (1H, d, J = 8), 2.68 (3H, s), 2.37 (3H, s); C NMR (101 MHz, DMSO) δ 173.01, 159.53, 149.30,
45.70, 141.31, 136.57, 131.59, 120.46, 120.33, 111.50, 18.22, 12.16; HRMS (ESI) calcd for C12 [M+H]+:
1
11 3 4
H N O

ACCEPTED MANUSCRIPT
o
-1
2
1
62.2334, found 261.9872. 261.0187
532, 1344, 1074
;
m.p.: 173-174 C.; IR(ATR) ν /cm : 3340, 3113, 3095, 2928, 2854, 1646,
max
4
.1.3. General Synthesis of N-(4 or 5-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide (4a-4b)
5
-methyl-N-(2-methyl-4 or 5-nitrophenyl) isoxazole-4-carboxamide (3a or 3b) (460 mg, 1.76mmol) and 35% HCl
.
o
(
0.69 mL), SnCl 2H O (1.98 g, 8.80mmol) in EtOH (3.5 mL) was stirredat 80 C. Stirring was continued for 7h and
2 2
the clear solution was cooled to room temperature. Solvent was removed in vacuo and was then poured into ice.
The pH was made slightly basic (pH 7–8) by addition of saturated aqueous sodium bicarbonate before being
extracted with ethyl acetate. The organic phase was thoroughly washed with brine, and dried over sodium sulfate.
The concentrated crude product was purified by flash column chromatography with MC:MeOH (10:1) as the eluent
to yield product.
4
.1.3.1. N-(4-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide (4a): The title compound was isolated
1
as a yellow solid (93%); H NMR (400 MHz, DMSO) δ 9.42 (1H, s), 9.01 (1H, s), 6.87 (1H, d, J = 8.4 Hz), 6.44
1
3
(
1H, s), 6.40 (1H, dd, J=2.4, J=2.4), 5.06 (2H, –NH, br, s), 2.64 (3H, s), 2.04(3H, s); C NMR (101 MHz, DMSO)
δ 172.05, 159.29, 149.05, 147.16, 134.55, 127.72, 123.87, 115.19, 111.83, 111.47, 17.98, 11.97.; HRMS (ESI) calcd
o
-1
for C H N O [M+H]+: 231.2505, found 231.9919. 232.0234.; m.p.: 155-156 C.; IR(ATR) ν /cm : 3419,
12
13
3
4
max
3
337, 3248, 2925, 1656, 1613, 1506, 1485, 1239, 1228.
4
.1.3.2 N-(5-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide (4b) : The title compound was isolated
1
as a yellow solid (91%); H NMR (400 MHz, DMSO) δ 9.47 (1H, s), 9.00 (1H, s), 6.89 (1H, d, J = 8.4 Hz), 6.56
13
(
1H, d, J = 2.0 Hz), 6.40 (1H, dd, J=2.4, J=2.4), 4.94 (2H, –NH, br, s), 2.65 (3H, s), 2.03 (3H, s); C NMR (101
MHz, DMSO) δ 172.28, 158.92, 149.14, 146.36, 135.69, 130.50, 120.40, 112.51, 112.36, 111.89, 16.97, 12.03; HRMS
o
-1
(
ESI) calcd for C H N O [M+H]+: 232.0234, found 232.0549.; mp:169-170 C; IR(ATR) ν /cm : 3436, 3358,
12 13 3 4 max
3
245, 3204, 2919, 1645, 1526, 1494, 1236, 1151.
4
.1.4. General Syntheses of N-(4-Arylamido-2-methylphenyl)-5-methylisoxazole-4-carboxamide (5a-5b)
o
The aryl carboxylic acid (0.18 mmol, 1.2 eq.) in SOCl
2
(0.3 mL) was heated at 50 C until aryl carboxylic acid
disappeared in TLC. After reaction was terminated, the mixture was cooled to ambient temperature and solvent was
evaporated under reduced pressure. Aryl carboxylic acid chloride, a crude yellow oil was diluted with THF and
poured into N-(4-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide (4a) (34.6 mg, 0.15 mmol, 1eq.) in
o
THF (1.5 mL), was heated at 65 C until compound 4a disappeared in TLC. Purification of column chromatography
with MC/MeOH eluents was performed to afford compound 5.

ACCEPTED MANUSCRIPT
4
.1.4.1. 5-methyl-N-(2-methyl-4-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)benzamido)phenyl) isoxazole
1
-
4-carboxamide (5a) : The title compound was isolated as a white solid (16%); H NMR (400 MHz, DMSO) δ
1
0.35 (1H, s), 9.78 (1H, s), 9.03 (1H, s), 7.81 (1H, d, J=2.0 Hz), 7.71 (1H, s), 7.61-7.57 (2H, m), 7.37 (1H, s), 7.27
13
(
1H, d, J=8.4 Hz), 3.38-3.29 (4H, m), 2.67 (3H, s), 2.53-2.50(4H, m), 2.24-2.21 (6H, m); C NMR (101 MHz,
DMSO) δ 172.49, 164.34, 159.26, 151.26, 149.16, 137.04, 136.79, 136.58, 133.97, 131.48, 131.38, 126.82, 122.80,
22.41, 118.45, 117.33, 113.50, 111.71, 54.27, 47.31, 45.58, 18.14, 12.07.; HRMS (ESI) calcd for C H F N O
3
1
2
5
26
3
5
o
-1
[
M+H]+: 502.1988, found 502.3596. 502.1993.; m.p.: 222-230 C. IR(ATR) ν /cm : 3253, 2922, 2844, 1648,
max
1
604, 1507, 1452, 1375 1251, 1166, 1120.
4
.1.4.2.
5-methyl-N-(2-methyl-4-(3-morpholino-5-(trifluoromethyl)benzamido)phenyl)isoxazole-4-
1
carboxamide (5b) : The title compound was isolated as a white solid (69%); H NMR (400 MHz, DMSO) δ
1
0.33 (1H, s), 9.76 (1H, s), 9.05(1H, s), 8.24-8.21 (2H, m), 7.81 (1H, d, J=2.0 Hz), 7.65 (1H, d, J = 8.0 Hz), 7.58
1
3
(
1H, s), 7.25 (1H, d, J=8.4 Hz), 3.73-3.71 (4H, m), 2.95-2.93 (4H, m), 2.68 (3H,s), 2.15(3H, s); C NMR (101
MHz, DMSO) δ 172.46, 163.68, 159.23, 154.40, 153.93, 149.12, 136.88, 133.96, 132.90, 131.27, 130.61, 126.92,
26.87, 126.82, 124.46, 124.17, 123.83, 122.18, 118.25, 111.69, 66.41, 53.09, 18.12, 12.04.; HRMS (ESI) calcd for
1
o
-1
C H F N O [M+H]+: 489.1671, found 489.3348. 489.1672; m.p.: 176-180 C; IR(ATR) ν /cm : 3273, 2969,
24
23
3
4
4
max
2
856, 1645, 1611, 1500, 1298, 1237, 1114, 1060, 1039.
4
.1.5. General syntheses of 5-Methyl-isoxazole-4-carboxylic acid (2-methyl-5-aryl-phenyl)-amide (6a-6o)
o
Method (A) The aryl carboxylic acid (0.18 mmol, 1.2 eq.) in SOCl (0.3 mL) was heated at 50 C until aryl
2
carboxylic acid disappeared in TLC. After reaction was terminated, the mixture was cooled to ambient temperature
and solvent was evaporated under reduced pressure. Aryl carboxylic acid chloride, a crude yellow oil was diluted
with THF and poured into N-(4-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide (4b) (34.6 mg, 0.15
o
mmol, 1eq.) in THF (1.5 mL), was heated at 65 C until compound 4b disappeared in TLC. Purification of column
chromatography with MC/MeOH eluents was performed to afford compound 6.
Method (B) N-(5-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide (4b) (25 mg, 0.11mmol), Aryl acid (1
o
eq., 0.11mmol), and HATU (61.75 mg, 0.16mmol) in DMF (1.1 mL) was heated at 45 C overnight. The reaction
mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. The organic layer
was dried over Na SO . Purification was performed by column chromatography with MC/MeOH = 20:1 to afford
2
4
compound 6.
4
.1.5.1. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-5-[3-(4-methyl-piperazin-1-yl)-5-trifluoromethyl-
1
benzoylamino]-phenyl}-amide (6a) : Method (A) The title compound was isolated as a white solid (24%); H
NMR (400 MHz, DMSO) δ 10.36 (1H, s), 9.78 (1H, s), 9.06 (1H, s), 7.81 (1H, d, J=2.0 Hz), 7.71 (1H, s), 7.61-
7
.57 (2H, m), 7.37 (1H, s), 7.27 (1H, d, J=8.4 Hz), 3.38-3.29 (4H, m), 2.68 (3H, s), 2.53-2.50 (4H, m), 2.24-2.21
13
(
6H, m); C NMR (101 MHz, DMSO) δ 172.58, 164.31, 159.16, 151.24, 149.16, 136.85, 136.56, 135.46, 130.28,

ACCEPTED MANUSCRIPT
1
29.96, 128.99, 125.50, 122.79, 118.72, 118.51, 117.31, 113.50, 111.69, 54.26, 47.31, 45.57, 17.42, 12.06.; HRMS
o
(
ESI) calcd for C H F N O [M+H]+: 502.1988, found 502.2390. 502.1995.; m.p.: 208-210 C; IR(ATR) ν
25
26
3
5
3
-1
max/cm : 3255, 2923, 2845, 1651, 1598, 1526, 1450, 1304, 1278, 1244, 1167, 1119.
4
.1.5.2. 5-Methyl-isoxazole-4-carboxylic acid [2-methyl-5-(4-morpholin-4-yl-3-trifluoromethyl-benzoyl
1
amino)-phenyl]-amide (6b) : Method (B) The title compound was isolated as a white solid (19%); H NMR (400
MHz, DMSO) δ 10.37 (1H, s), 9.76 (1H, s), 9.05(1H, s), 8.24-8.21 (2H, m), 7.81 (1H, d, J=2.0 Hz), 7.65 (1H, d,
J=8.0 Hz), 7.58-7.55 (1H, m), 7.25 (1H, d, J=8.4 Hz), 3.73-3.71 (4H, m), 2.95-2.93 (4H, m), 2.67 (3H,s), 2.19
13
(
3H, s); C NMR (101 MHz, DMSO) δ 172.56, 163.65, 159.13, 154.38, 149.15, 136.93, 135.45, 132.90, 130.59,
1
30.28, 128.90, 126.90, 126.85, 124.44, 123.83, 118.50, 118.32, 111.67, 66.41, 53.08, 17.40, 12.05.; HRMS (ESI)
o
-1
calcd for C H F N O [M+H]+: 489.1671, found 489.2651. 489.1680.; m.p.: 205-206 C.; IR(ATR) ν /cm :
2
4
23
3
4
4
max
3
247, 3128, 2956, 2864, 1641, 1609, 1520, 1502, 1486, 1291, 1275, 1240, 1131, 1111.
4
.1.5.3. 5-Methyl-isoxazole-4-carboxylic acid [5-(4-chloro-3-trifluoromethyl-benzoylamino)-2-methyl-
1
phenyl]-amide (6c) : Method (A) The title compound was isolated as a white solid (64%); H NMR (400 MHz,
DMSO) δ 10.53 (1H, s), 9.77 (1H, s), 9.05 (1H, s), 8.39 (1H, s), 8.38-8.24 (1H, m), 7.93 (1H, d, J=8.4 Hz), 7.83
13
(
1H, d, J=2 Hz), 7.59 (1H, dd, J=2.4 Hz), 7.28 (1H, d, J = 8.4 Hz), 2.68 (3H, s), 2.21 (3H, s); C NMR (101 MHz,
DMSO) δ 173.91, 163.44, 159.64, 149.64, 137.15, 135.99, 134.53, 134.36, 133.86, 132.41, 130.85, 129.64,
1
4
1
27.47, 127.25, 126.94, 119.01, 118.84, 112.16, 17.92, 12.55.; HRMS (ESI) calcd for C H ClF N O [M+H]+:
20 15 3 3 3
o
-1
38.0754, found 438.2612; m.p.: 179-180 C.; IR(ATR) ν /cm : 3227, 3119, 3069, 1638, 1607, 1524, 1490,
max
332, 1308, 1279, 1255, 1246, 1110, 1091, 1036.
4
.1.5.4. 5-Methyl-isoxazole-4-carboxylic acid {5-[2-(2-fluoro-phenyl)-acetylamino]-2-methyl-phenyl}-amide
1
(
6d) : Method (A) The title compound was isolated as a white solid (79%); H NMR (400 MHz, DMSO) δ 10.22
(
1H, s), 9.70 (1H, s), 9.03 (1H, s), 7.67 (1H, d, J=1.6 Hz), 7.40-7.36 (2H, m), 7.34-7.28 (1H, m), 7.19-7.14 (3H,
13
m), 2.66 (3H, s), 2.16 (3H, s); C NMR (101 MHz, DMSO) δ 172.14, 167.50, 158.66, 148.73, 136.74, 135.09,
1
31.53, 131.49, 129.96, 127.71, 123.80, 123.77, 116.73, 116.54, 114.69, 114.47, 111.26, 35.86, 16.94, 11.63. ;
o
HRMS (ESI) calcd for C20
H
18FN
3
O
3
[M+H]+: 368.1332, found 368.1450. 368.1332 ; m.p.: 171-174 C.; IR (ATR)
-1
ν
max/cm : 3236, 3042, 1666, 1638, 1606, 1521, 1308, 1291, 1242, 1180, 1130.
4
.1.5.5. 5-Methyl-isoxazole-4-carboxylic acid {5-[2,2-bis-(4-chloro-phenyl)-acetylamino]-2-methyl-phenyl}-
1
amide (6e) : Method (A) The title compound was isolated as a white solid (88%); H NMR (400 MHz, DMSO) δ
1
0.47 (1H, s), 9.69 (1H, s), 9.02 (1H, s), 7.71 (1H, s), 7.41 (4H, d, J=8.4 Hz), 7.35 (5H, d, J=7.2 Hz), 7.19 (1H, d,
13
J=8.4 Hz), 5.17 (1H, s), 2.65 (3H, s), 2.16 (3H, s); C NMR (101 MHz, DMSO) δ 172.60, 169.11, 159.11, 149.18,
1
38.51, 136.84, 135.60, 131.80, 130.47, 130.35, 128.53, 128.48, 117.29, 117.14, 111.69, 55.76, 17.41, 12.08.; HRMS
o
(
ESI) calcd for C26
H
21Cl
2
N
3
O
3
[M+H]+: 494.0960, found 494.0594. 494.0989.; m.p.: 178-181 C.; IR (ATR) ν
-1
max/cm : 3230, 3063, 1639, 1607, 1521, 1489, 1241, 1092.

ACCEPTED MANUSCRIPT
4
.1.5.6. 5-Methyl-isoxazole-4-carboxylic acid {5-[(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carbonyl)-
1
amino]-2-methyl-phenyl}-amide (6f) : Method (B) The title compound was isolated as a white solid (51%); H
NMR (400 MHz, DMSO) δ 9.72 (1H, s), 9.21 (1H, s), 9.02 (1H, s), 7.67 (1H, d, J=2.0 Hz), 7.52 (1H, s), 7.36 (1H,
dd, J=8.4 Hz, J=2.4 Hz), 7.18 (1H, d, J=8.4 Hz), 2.68 (3H, s), 2.16 (6H, d, J=7.6 Hz), 2.07 (3H, s), 2.17-1.50 (4H,
13
m), 1.99 (3H, d, J=7.2Hz) 1.50 (3H, d, J=4.8 Hz); C NMR (101 MHz, DMSO) δ 172.59, 172.11, 159.11, 149.17,
1
2
4
1
46.03, 143.90, 136.30, 135.45, 130.22, 128.92, 122.79, 121.43, 120.35, 118.15, 118.08, 117.05, 111.66, 77.43, 29.35,
3.80, 20.13, 17.43, 12.81, 12.17, 12.08, 11.83.; HRMS (ESI) calcd for C26 [M+H]+: 464.2107, found
29 3 5
H N O
o
-1
64.3754. 464.2116.; m.p.: 155-156 C. ; IR (ATR) ν /cm : 3395, 3292, 2923, 2854, 1655, 1610, 1519, 1449,
max
415, 1240, 1126, 1087.
4
.1.5.7. 5-Methyl-isoxazole-4-carboxylic acid [5-(2-1H-indol-3-yl-acetylamino)-2-methyl-phenyl]-amide (6g) :
1
Method (B) The title compound was isolated as a white solid (17%); H NMR (400 MHz, DMSO) δ 10.88 (1H, s),
1
6
1
1
3
1
0.06 (1H, s), 9.67 (1H, s), 9.01 (1H, s), 7.66-7.58 (2H, m), 7.33 (2H, s), 7.24 (1H, s), 7.17 (1H, s), 7.06 (1H, s),
13
.99 (1H, d, J=7.2 Hz), 3.71 (2H, s), 2.66 (3H, s), 2.15 (3H, s); C NMR (101 MHz, DMSO) δ 172.57, 169.63,
59.07, 149.18, 137.45, 136.12, 135.48, 130.34, 128.00, 127.20, 123.86, 121.00, 118.71, 118.39, 117.12, 116.98,
11.70, 111.38, 108.59, 33.84, 17.39, 12.08.; HRMS (ESI) calcd for C H N O [M+H]+: 389.1535, found
2
2
20
4
3
o
-1
89.3299.389.1539 ; m.p.: 169-171 C.; IR(ATR) ν /cm : 3267, 2922, 1561, 1606, 1523, 1488, 1455, 1287,
max
131, 1093.
4
.1.5.8. 5-Methyl-isoxazole-4-carboxylic acid {5-[2-(2-cyano-phenylsulfanyl)-benzoylamino]-2-methyl-
1
phenyl}-amide (6h): Method (B) The title compound was isolated as white solid (52%); H NMR (400 MHz,
DMSO) δ 10.51 (1H, s), 9.80 (1H, s), 9.03 (1H, s), 7.91(1H, s), 7.81(1H, s), 7.72-7.65 (2H, m), 7.52-7.39 (5H, m),
13
7
1
1
.24 (1H, d, J=8.0 Hz), 7.18-7.15 (1H, m), 2.67 (3H, s), 2.18 (3H, s); C NMR (101 MHz, DMSO) δ 172.53,
65.69, 159.09, 149.16, 139.10, 138.48, 136.97, 135.54, 134.25, 134.03, 132.81, 132.22, 131.63, 131.12, 130.31,
29.01, 128.51, 128.40, 127.79, 117.83, 117.74, 116.85, 114.05, 111.67, 17.47, 12.05.; HRMS (ESI) calcd for
o
-1
C H N O S [M+H]+: 469.1256, found 469.0677. 469.1261.; m.p.: 218-220 C. IR(ATR) ν /cm : 3227, 3119,
26
20
4
3
max
2
220, 1643, 1633, 1607, 1595, 1519, 1319, 1242.
4
.1.5.9. 5-Methyl-isoxazole-4-carboxylic acid [5-(2-biphenyl-4-yl-acetylamino)-2-methyl-phenyl]-amide (6i) :
1
Method (B) The title compound was isolated as white solid (30%); H NMR (400 MHz, DMSO) δ 10.21 (1H, s),
9
2
1
1
.70 (1H, s), 9.03 (1H, s), 7.67-7.61(5H, m), 7.47-7.33 (6H, m), 7.19 (1H, d, J=8.0 Hz), 3.66 (2H, d, J=8.0 Hz),
1
3
.68 (3H, d, J=8.0 Hz), 2.16 (3H, d, J=7.6 Hz); C NMR (101 MHz, DMSO) δ 172.59, 168.96, 159.10, 149.19,
39.99, 138.52, 137.25, 135.53, 135.31, 130.40, 129.66, 128.94, 128.15, 127.34, 126.66, 126.59, 117.17, 117.00,
11.71, 42.98, 17.40, 12.09.; HRMS (ESI) calcd for C H N O [M+H]+: 426.1739, found 426.1245. 426.1740.;
26
23
3
3
o
-1
m.p.: 135-138 C.; IR(ATR) ν /cm : 3234, 3047, 1643, 1622, 1613, 1601, 1543, 1240, 1134.
max
4
.1.5.10. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-5-[(1-phenyl-5-trifluoromethyl-1H-pyrazole-4-
1
carbonyl)-amino]-phenyl}-amide (6j) : Method (B) The title compound was isolated as white solid (74%); H

ACCEPTED MANUSCRIPT
NMR (400 MHz, DMSO) δ 10.53 (1H, s), 9.79 (1H, s), 9.05 (1H, s), 8.31 (1H, s), 7.80 (1H, s), 7.62-7.60 (3H, m),
1
3
7
.54-7.53 (2H, m), 7.47 (1H, d, J = 8.0 Hz), 7.26 (1H, d, J = 8.4 Hz), 2.68 (3H, s), 2.19 (3H, d, J = 8.0 Hz);
NMR (101 MHz, DMSO) δ 172.61, 159.16, 158.85, 149.20, 139.75, 138.74, 136.75, 135.62, 130.45, 130.09, 129.57,
29.43, 129.15, 126.01, 121.50, 120.69, 117.83, 117.71, 111.68, 17.49, 12.09.; HRMS (ESI) calcd for C23
C
1
18 3 5 3
H F N O
o
-1
[
M+H]+: 470.1362, found 470.0538. 470.1365.; m.p.: 173-176 C.; IR(ATR) ν max/cm : 3252, 3027, 1646, 1608,
1
531, 1499, 1488, 1297, 1242, 1177, 1149, 1135
4
.1.5.11. 5-Methyl-isoxazole-4-carboxylic acid [5-(3,5-dimethyl-benzoylamino)-2-methyl-phenyl]-amide (6k) :
1
Method (B) The title compound was isolated as white solid (53%); H NMR (400 MHz, DMSO) δ 10.16 (1H, d,
J = 7.6 Hz), 9.78 (1H, s), 9.06 (1H, s), 7.83 (1H, d, J = 6.0 Hz), 7.57-7.54 (3H, m), 7.24 (2H, m), 2.68 (3H, s), 2.35
13
(
6H, d, J = 8.4 Hz), 2.19 (3H, d, J =8.0 Hz); C NMR (101 MHz, DMSO) δ 172.57, 165.61, 159.13, 149.18, 137.51,
1
37.31, 135.41, 134.91, 132.79, 130.22, 128.67, 125.33, 118.39, 118.21, 111.70, 20.86, 17.44, 12.07; HRMS (ESI)
o
-1
calcd for C H N O [M+H]+: 364.1583, found 364.1226. 364.1587.; m.p.: 215-216 C.; IR(ATR) ν /cm : 3225,
21
21
3
3
max
3
047, 1650, 1635, 1621, 1597, 1537, 1238, 1135.
4
.1.5.12. 5-Methyl-isoxazole-4-carboxylic acid {5-[(biphenyl-4-carbonyl)-amino]-2-methyl-phenyl}-amide
1
(
(
(
6l) : Method (B) The title compound was isolated as white solid (14%); H NMR (400 MHz, DMSO) δ 10.31
1H, s), 9.79 (1H, s), 9.06 (1H, s), 8.05 (2H, s), 7.85-7.83 (3H, m), 7.75 (2H, m), 7.59 (1H, s), 7.50 (2H, d), 7.42
13
1H, s), 7.24 (1H, s), 2.68 (3H, d, J = 3.2 Hz), 2.20 (3H, d, J = 2.8 Hz); C NMR (101 MHz, DMSO) δ 172.58,
1
1
4
65.01, 159.15, 149.19, 143.08, 139.11, 137.25, 135.45, 133.63, 130.27, 129.07, 128.78, 128.35, 128.15, 126.92,
26.58, 118.46, 118.30, 111.71, 17.46, 12.08.; HRMS (ESI) calcd for C H N O [M+H]+: 412.1583, found
2
5
21
3
3
o
-1
12.1126. 412.1590.; m.p.: 182-186 C.; IR(ATR) ν /cm : 3284, 1639, 1607, 1530, 1486, 1326, 1240.
max
4
.1.5.13. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-5-[3-(2-methyl-imidazol-1-yl)-5-trifluoromethyl-
1
benzoylamino]-phenyl}-amide (6m) : Method (A) The title compound was isolated as white solid (60.5%) H
NMR (400 MHz, DMSO) δ 10.82 (1H, s), 9.90 (1H, s), 9.15 (1H, s), 8.54 (2H, s), 8.31 (1H, d, s), 7.99 (1H, s),
13
7
.85 (1H, s), 7.71 (1H, s), 7.65-7.63 (1H, m), 7.28 (1H, d, J=8.4 Hz), 2.67 (3H, s), 2.55 (3H, s), 2.21 (3H, s);
NMR (101 MHz, DMSO) δ 172.65, 162.76, 159.21, 149.20, 138.56, 137.16, 136.62, 135.57, 130.69, 130.43, 129.27,
28.34, 127.68, 124.90, 124.87, 123.67, 123.63, 121.13, 118.61, 118.41, 111.71, 17.46, 13.58, 12.10.; HRMS (ESI)
C
1
o
-1
calcd for C H F N O [M+H]+: 484.1518, found 483.3688. 483.1524.; m.p.: 210-214 C.; IR(ATR) ν /cm :
2
4
20
3
5
3
max
3
292, 3118, 2928, 2853, 2811, 1674, 1651, 1601, 1336, 1314, 1287, 1243, 1171, 1164, 1042
4
.1.5.14. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-5-[3-(5-methyl-imidazol-1-yl)-5-trifluoromethyl-
1
benzoylamino]-phenyl}-amide (6n) : Method (B) The title compound was isolated as white solid (22%); H
NMR (400 MHz, DMSO) δ 10.79 (1H, s), 9.88 (1H, s), 9.46 (1H, s), 9.14 (1H, s), 8.60 (2H, d, J = 10.8 Hz), 8.41
13
(
1H, s), 7.85 (1H, s), 7.65 (2H, s), 7.29 (1H, d, J = 8.0 Hz), 2.67 (3H, s), 2.26 (3H, s) 2.21 (3H, s); C NMR (101
MHz, DMSO) δ 172.66, 162.46, 159.23, 149.32, 137.43, 136.60, 136.37, 135.60, 134.50, 130.97, 130.72, 130.39,
30.33, 130.20, 129.57, 124.56, 121.85, 118.86, 118.53, 117.30, 111.67, 17.55, 12.12, 9.26.; HRMS (ESI) calcd for
1

ACCEPTED MANUSCRIPT
o
-1
C H F N O [M+H]+: 484.1518, found 484.0913. 484.1525.; m.p.: 225-226 C.; IR(ATR) ν /cm : 3328, 3206,
24
20
3
5
3
max
3
109, 3047, 2981, 2719, 2649, 1672, 1661, 1614, 1538, 1524, 1375 1168, 1128.
4
.1.5.15. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-5-[3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-
1
benzoylamino]-phenyl}-amide (6o) : Method (B) The title compound was isolated as white solid (32%); H
NMR (400 MHz, DMSO) δ 10.88 (1H, s), 9.88 (1H, s), 9.84 (1H, s), 9.14 (1H, s), 8.77 (1H, s), 8.43 (1H, s), 8.28
13
(
2H, s), 7.89 (1H, s), 7.70 (1H, d, J = 8.0 Hz), 7.29 (1H, d, J = 8.0 Hz), 2.68 (3H, s), 2.36 (3H, s) 2.22 (3H, s);
NMR (101 MHz, DMSO) δ 172.68, 162.55, 159.24, 149.31, 137.47, 136.66, 135.76, 135.57, 134.58, 130.90, 130.84,
30.57, 130.31, 129.57, 125.23, 124.86, 124.61, 118.83, 118.54, 117.43, 111.68, 17.56, 12.13, 9.89.; HRMS (ESI)
C
1
o
-1
calcd for C H F N O [M+H]+: 484.1518, found 483.0598. 484.1517.; m.p.: 239-240 C.; IR(ATR) ν /cm :
2
4
20
3
5
3
max
3
233, 1665, 1613, 1541, 1523, 1495, 1378, 1275, 1122, 1094.
4
.1.6. General Syntheses of N-(5-(3-Aryl ureido)-2-methylphenyl)-5-methylisoxazole-4-carboxamide (7a-7g)
The mixture of compound 4b (10 mg, 0.043 mmol), and aryl isocyanate (6.7 mg, 0.043 mmol), in THF (0.4 ml) was
stirred at r.t overnight. After completion of the reaction, solvent was removed in vacuo and the concentrated crude
product was purified by flash column chromatography with MC: MeOH = 20:1 as the eluent.
4
.1.6.1. 5-Methyl-isoxazole-4-carboxylic acid {5-[3-(4-chloro-phenyl)-ureido]-2-methyl-phenyl}-amide (7a) :
1
The title compound was isolated as white solid (85%); H NMR (400 MHz, DMSO) δ 9.75 (1H, s), 9.35 (1H, s),
13
9
.11 (1H, s), 9.03 (1H, s), 8.06 (1H, s), 7.84-7.58 (5H, m), 7.23 (1H, s), 2.67 (3H, s), 2.15 (3H, s); C NMR (101
MHz, DMSO) δ 172.55, 170.38, 161.41, 159.11, 152.42, 149.22, 138.75, 137.56, 135.67, 130.48, 128.64, 126.90,
1
3
1
25.31, 119.70, 116.29, 111.77, 59.79, 20.79, 17.33, 14.11, 12.10.; HRMS (ESI) calcd for C H ClN O [M+H]+:
19 17 4 3
+
o
-1
85.0989, found 385.1133. 407.0978. (Na ); m.p.: 205-207 C.; IR(ATR) ν /cm : 3381, 1698, 1642, 1596, 1537,
max
490, 1281, 1206, 1134, 1094, 1042.
4
.1.6.2. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-5-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-
1
amide (7b) : The title compound was isolated as pale yellow solid (37%); H NMR (400 MHz, DMSO) δ 9.75
(
(
1H, s), 9.38 (1H, s), 9.11 (1H, s), 9.06 (1H, s), 8.01 (1H, s),7.94 (2H, s), 7.55 (1H, s), 7.51-7.48 (1H, m), 7.30
13
1H, d, J=7.6 Hz), 7.23-7.15 (1H, m),2.67 (3H, s), 2.15 (3H, s); C NMR (101 MHz, DMSO) δ 172.52, 159.10,
1
1
4
52.48, 149.20, 140.60, 137.36, 135.67, 130.45, 129.90, 129.67, 129.35, 127.07, 125.57, 121.78, 118.03, 116.44,
14.09, 111.75, 17.32, 12.07.; HRMS (ESI) calcd for C20 [M+H]+: 419.1253, found 419.1513.
17 3 4 3
H F N O
o
-1
19.1258.; m.p.: 204-205 C. IR(ATR) ν max/cm : 3320, 3227, 2914, 1644, 1613, 1533, 1336, 1170, 1123.
4
.1.6.3. 5-Methyl-isoxazole-4-carboxylic acid {5-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-2-methyl-
1
phenyl}-amide (7c) : The title compound was isolated as pale yellow solid (90%); H NMR (400 MHz, DMSO) δ
9
2
1
.71 (1H, s), 9.10 (1H, s), 9.03 (1H, s), 8.85 (1H, s), 8.12 (1H, s), 7.60-7.56 (3H, m), 7.19 (2H, s), 2.67 (3H, s),
13
.16 (3H, s); C NMR (101 MHz, DMSO) δ 172.14, 167.50, 158.66, 148.73, 136.74, 135.09, 131.53, 131.49,
29.96, 127.71, 123.80, 123.77, 116.73, 116.54, 114.69, 114.47, 111.26, 35.86, 16.94, 11.63.; HRMS (ESI) calcd

ACCEPTED MANUSCRIPT
o
-1
for C H ClF N O [M+H]+: 453.0863, found 453.1885. 453.0866.; m.p.: 215-216 C. IR(ATR) ν /cm : 3227,
20
16
3
4
3
max
3
110, 2930, 1678, 1656, 1610, 1598, 1540, 1231, 1180, 1131, 1112.
4
.1.6.4. 5-Methyl-isoxazole-4-carboxylic acid {5-[3-(2-fluoro-phenyl)-ureido]-2-methyl-phenyl}-amide (7d) :
1
The title compound was isolated as pale yellow solid (76%); H NMR (400 MHz, DMSO) δ 9.71 (1H, s), 9.05
13
(
1H, s), 8.93 (1H, s), 8.80 (1H, s), 7.65-7.02 (7H, m), 2.67 (3H, s), 2.16 (3H, s); C NMR (101 MHz, DMSO) δ
1
1
3
1
72.55, 159.11, 152.15, 150.76, 149.22, 137.46, 135.75, 130.55, 127.64, 127.54, 126.91, 124.54, 122.43, 122.35,
20.43, 116.00, 111.77, 17.33, 12.09.; HRMS (ESI) calcd for C H FN O [M+H]+: 369.1285, found 369.1312.
19
17
4
3
o
-1
69.1291.; m.p.: 215-216 C. IR(ATR) ν /cm : 3322, 3222, 1685, 1633, 1599, 1547, 1312, 1287, 1255, 1234,
max
185.
4
.1.6.5. 5-Methyl-isoxazole-4-carboxylic acid {5-[3-(3-chloro-phenyl)-ureido]-2-methyl-phenyl}-amide (7e) :
1
The title compound was isolated as white solid (36%); H NMR (400 MHz, DMSO) δ 9.70 (1H, s), 9.03 (1H, s),
8
6
1
.84 (1H, s), 8.76 (1H, s), 7.70 (1H, s), 7.55 (1H, s), 7.30-7.23 (3H, m), 7.18 (1H, d, J = 5.6Hz), 7.01 (1H, d, J =
13
Hz), 2.67 (3H, s), 2.15 (3H, s); C NMR (101 MHz, DMSO) δ 173.00, 159.56, 152.82, 149.67, 141.76, 139.05,
37.89, 136.13, 133.66, 130.93, 130.86, 127.46, 121.86, 117.97, 117.06, 116.80, 112.22, 17.79, 12.55.; HRMS
o
(
ESI) calcd for C H ClN O [M+H]+: 385.0989, found 385.0453. 385.0984.; m.p.: 218-221 C. IR(ATR) ν
19
17
4
3
-1
max/cm : 3298, 3223, 2922, 1638, 1586, 1557, 1281, 1228, 1173, 1130, 1103.
4
.1.6.6. 5-Methyl-isoxazole-4-carboxylic acid {5-[3-(3,5-dichloro-phenyl)-ureido]-2-methyl-phenyl}-amide
1
(
7f) : The title compound was isolated as white solid (16%); H NMR (400 MHz, DMSO) δ 9.75 (1H, s), 9.52 (1H,
s), 9.22 (1H, s), 9.07 (1H, s), 7.53-7.52 (3H, m), 7.22 (1H, dd, J = 8.3, 2.2 Hz), 7.17 (1H, d, J = 8.4 Hz), 7.14 (1H,
1
3
t, J = 1.9 Hz), 2.68 (3H, s), 2.16 (3H, s); C NMR (101 MHz, DMSO) δ 172.55, 159.10, 152.32, 149.23, 142.43,
1
37.26, 135.69, 134.10, 130.49, 127.24, 120.70, 116.43, 116.35, 116.01, 111.74, 17.35, 12.10.; HRMS (ESI) calcd for
+
o
-1
C H Cl N O [M+H]+: 419.0599, found 419.0153. 441.0591(Na ); m.p.: 212-213 C. IR(ATR) ν /cm : 3302,
19
16
2
4
3
max
3
241, 2920, 2853, 1652, 1584, 1538, 1504, 1306, 1239.
4
.1.6.7. 5-Methyl-isoxazole-4-carboxylic acid {5-[3-(3,4-dichloro-phenyl)-ureido]-2-methyl-phenyl}-amide
:
1
(
7g) The title compound was isolated as white solid (33%); H NMR (400 MHz, DMSO) δ 9.70 (1H, s), 9.13-
8
.81 (3H, m), 7.87 (1H, d, J=6.4 Hz), 7.53(2H, d, J=9.6 Hz), 7.33 (2H, d, J=8.0Hz), 7.18 (1H, d, J=2.0Hz), 2.66
13
(
3H, s), 2.15 (3H, s); C NMR (101 MHz, DMSO) δ 172.56, 159.11, 152.30, 149.21, 140.01, 137.30, 135.68, 131.04,
1
30.57, 130.49, 127.14, 123.05, 119.26, 118.33, 116.48, 116.46, 111.76, 17.34, 12.10.; HRMS (ESI) calcd for
o
-1
C
19 2 4 3
H16Cl N O [M+H]+: 419.0599, found 419.0493. 419.0595.; m.p.: 209-210 C.; IR(ATR) ν max/cm : 3366, 3273,
3
181, 3113, 2906, 1669, 1608, 1582, 1544, 1474, 1320, 1133, 1125, 1027.
4
.1.7. General Syntheses of N-(4-(3-Aryl ureido)-2-methylphenyl)-5-methylisoxazole-4-carboxamide (8a-8f)

ACCEPTED MANUSCRIPT
The mixture of compound 4a (10 mg, 0.043 mmol), aryl isocyanate (1 eq., 0.043 mmol), in THF (0.4 ml) was stirred
at r.t for overnight. After completion of the reaction, solvent was removed by vacuo and the concentrated crude
product was purified by flash column chromatography with MC: MeOH = 20:1 as the eluent.
4
.1.7.1. 5-Methyl-isoxazole-4-carboxylic acid {4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-2-methyl-
1
phenyl}-amide (8a): The title compound was isolated as white solid (99%); H NMR (400 MHz, DMSO) δ 9.63
(
(
1H, s), 9.15 (1H, s), 9.02 (1H, s), 8.81 (1H, s), 8.12 (1H, s), 7.61 (2H, s), 7.38 (1H, s), 7.29 (1H, d, J=6.0Hz), 7.20
13
1H, s), 2.66 (3H, s), 2.18 (3H, s); C NMR (101 MHz, DMSO) δ 172.44, 159.28, 152.42, 149.16, 139.40, 137.29,
1
34.38, 132.02, 129.95, 127.19, 126.56, 123.05, 122.25, 120.25, 116.76, 116.70, 116.41, 111.73, 18.14, 12.07.; HRMS
o
(
ESI) calcd for C H ClF N O [M+H]+: 453.0863, found 453.0186. 453.0865.; m.p.: 217-219 C. IR(ATR) ν
20
16
3
4
3
-1
max/cm : 3283, 3214, 3063, 1646, 1632, 1609, 1548, 1293, 1257, 1230, 1170, 1136, 1122, 1113.
4
.1.7.2. 5-Methyl-isoxazole-4-carboxylic acid {2-methyl-4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-
1
amide (8b) : The title compound was isolated as white solid (64%); H NMR (400 MHz, DMSO) δ 9.64 (1H, s),
9
7
1
1
.04 (2H, d, J=4.8 Hz), 8.77 (1H, s), 8.03 (1H, s), 7.57-7.49 (2H, m), 7.39(1H, d, J = 2.4 Hz), 7.31-7.28 (2H, m),
13
.20 (1H, d, J = 8.4Hz), 2.66 (3H, s), 2.19 (3H, s); C NMR (101 MHz, DMSO) δ 172.88, 159.74, 152.98, 149.62,
41.08, 137.91, 134.83, 130.39, 130.27, 130.15, 127.65, 123.35, 122.27, 120.56, 118.49, 116.72, 114.53, 112.20, 18.60,
o
2.53.; HRMS (ESI) calcd for C H F N O [M+H]+: 419.1253, found 419.1173. 419.1252.; m.p.: 206-208 C.
20
17
3
4
3
-1
IR(ATR) ν max/cm : 3286, 3241, 3041, 1639, 1561, 1338, 1125.
4
.1.7.3. 5-Methyl-isoxazole-4-carboxylic acid {4-[3-(3-chloro-phenyl)-ureido]-2-methyl-phenyl}-amide (8c) :
1
The title compound was isolated as white solid (66%); H NMR (400 MHz, DMSO) δ 9.63 (1H, s), 9.03 (1H, s),
8
.93 (1H, s), 8.76 (1H, s), 7.71 (1H, s), 7.37 (1H, s), 7.29 (2H, d, J = 6.4 Hz), 7.19 (2H, d, J = 4.8Hz), 7.01 (1H, d,
13
J = 4.0Hz), 2.66 (3H, s), 2.18 (3H, s); C NMR (101 MHz, DMSO) δ 172.43, 159.30, 152.52, 149.20, 141.43, 137.65,
1
34.40, 133.23, 130.42, 129.65, 127.25, 121.32, 119.82, 117.36, 116.45, 115.99, 111.76, 18.17, 12.09.; HRMS (ESI)
+
o
calcd for C H ClN O [M+H]+: 385.0989, found 385.0793. 407.0981(Na ); m.p.: 217-219 C.; IR(ATR) ν
19
17
4
3
-1
max/cm : 3313, 3270, 1638, 1622, 1587, 1548, 1305, 1221.
4
.1.7.4. 5-Methyl-isoxazole-4-carboxylic acid {4-[3-(2-fluoro-phenyl)-ureido]-2-methyl-phenyl}-amide (8d) :
1
The title compound was isolated as white solid (53%); H NMR (400 MHz, DMSO) δ 9.64 (1H, s), 9.05 (2H, d,
J=5.2 Hz), 8.55 (1H, s), 8.15 (1H, s), 7.36 (1H, s), 7.28 (1H, s), 7.23 (2H, d, J=9.2 Hz), 7.13 (1H, s), 7.00 (1H, s),
1
3
2
1
.67 (3H, s), 2.19 (3H, s); C NMR (101 MHz, DMSO) δ 172.43, 159.28, 152.19, 150.79, 149.16, 137.56, 134.46,
29.71, 127.64, 127.53, 127.29, 124.56, 122.46, 120.50, 119.70, 115.86, 115.08, 114.89, 111.75, 18.16, 12.07.; HRMS
o
(
ESI) calcd for C19
H
17FN
4
O
3
[M+H]+: 369.1285, found 369.0972. 369.1280.; m.p.: 240-245 C.; IR(ATR) ν
-1
max/cm : 3353,3281, 1651, 1638, 1619, 1596, 1565, 1460, 1308, 1233.
4
.1.7.5. 5-Methyl-isoxazole-4-carboxylic acid {4-[3-(4-chloro-phenyl)-ureido]-2-methyl-phenyl}-amide (8e) :
1
The title compound was isolated as white solid (63%); H NMR (400 MHz, DMSO) δ 9.68 (1H, s), 9.38 (1H, s),

ACCEPTED MANUSCRIPT
9
.20 (1H, s), 9.07 (1H, s), 7.49 (2H, dd, J = 2.4 Hz, J = 2.4 Hz), 7.34-7.28 (4H, m), 7.17 (1H, d, J = 8.8 Hz), 2.65
13
(
3H, s), 2.17 (3H, s); C NMR (101 MHz, DMSO) δ 172.38, 159.26, 152.43, 149.13, 138.73, 137.63, 134.33, 128.61,
27.18, 125.30, 119.91, 119.82, 119.70, 116.07, 111.73, 18.14, 12.05.; HRMS (ESI) calcd for C H ClN O
3
1
19
17
4
+
o
-1
[
M+H]+: 385.0989, found 385.1133. 407.0982(Na ); m.p.: 230-240 C.; IR(ATR) ν /cm : 3295, 2922, 2855,
max
1
637, 1559, 1520, 1489, 1305, 1221.
4
.1.7.6. 5-Methyl-isoxazole-4-carboxylic acid {4-[3-(3,4-dichloro-phenyl)-ureido]-2-methyl-phenyl}-amide
:
1
(
8f) The title compound was isolated as white solid (42%); H NMR (400 MHz, DMSO) δ 9.63 (1H, s), 9.02 (2H,
s), 8.80 (1H, s), 7.88 (1H, s), 7.51 (1H, d, J = 7.2 Hz), 7.36(1H, s), 7.32-7.27 (2H, m), 7.19 (1H,s), 2.65 (3H, s),
1
3
2
1
.18 (3H, s); C NMR (101 MHz, DMSO) δ 172.43, 159.28, 152.35, 149.16, 140.03, 137.39, 134.37, 131.05, 130.59,
29.84, 127.20, 123.03, 120.09, 119.22, 118.30, 116.24, 111.73, 18.15, 12.07.; HRMS (ESI) calcd for C H Cl N O
3
1
9
16
2
4
o
-1
[
M+H]+: 419.0599, found 419.0493. 419.0596.; m.p.: 235-237 C. IR(ATR) ν /cm : 3305, 3272, 1651, 1637,
max
1
621, 1586, 1550, 1305, 1224, 1131.
4
.1.7.7. 5-Methyl-isoxazole-4-carboxylic acid {4-[3-(3,5-dichloro-phenyl)-ureido]-2-methyl-phenyl}-amide
1
(
8g) : The title compound was isolated as white solid (38%); H NMR (400 MHz, DMSO) δ 9.87 (1H, s), 9.70
(
1H, s), 9.38 (1H, s), 9.08 (1H, s), 7.52 (2H, s), 7.35 (1H, s), 7.28 (1H, s), 7.19 (1H,s), 7.14 (1H,s), 2.66 (3H, s),
1
3
2
1
4
1
.18 (3H, s); C NMR (101 MHz, DMSO) δ 172.38, 159.24, 152.20, 149.11, 142.28, 137.20, 134.32, 134.06, 129.95,
27.14, 120.83, 120.22, 116.37, 116.27, 111.70, 18.10, 12.03.; HRMS (ESI) calcd for C H Cl N O [M+H]+:
19
16
2
4
3
o
-1
19.0599, found 419.2533. 419.0591.; m.p.: 230-231 C.; IR (ATR) ν /cm : 3300, 3266, 2922, 1638, 1584, 1543,
max
517, 1304, 1240.
4
.1.8. General Syntheses of 5-Methyl-isoxazole-4-carboxylic acid [2-methyl-5 or 4-(propane-1-sulfonylamino)-
phenyl]-amide (9a-9b)
The mixture of compound 4 (20 mg, 0.086 mmol), propane-1-sulfonyl chloride (13.85 mg, 97%, 0.095 mmol), in
MC (0.4 ml) was stirred at r.t for overnight. After the reaction was completed, solvent was removed by evaporation,
and the concentrated crude product was purified by flash column chromatography with EA/Hex (1:1) as the eluent
to produce compound 9.
4
.1.8.1. 5-Methyl-isoxazole-4-carboxylic acid [2-methyl-5-(propane-1-sulfonylamino)-phenyl]-amide (9a) :
1
The title compound was isolated as yellow solid (99%); H NMR (400 MHz, DMSO) δ 9.75 (1H, s), 9.66 (1H, s),
9
.01 (1H, s), 7.24 (1H, d, J=8.4 Hz), 7.09-7.04 (2H, m), 3.07-3.04 (2H, m), 2.65 (3H, s), 2.17 (3H, s), 1.69 (2H, d,
1
3
J=7.2Hz), 0.93 (3H, s); C NMR (101 MHz, DMSO) δ 173.09, 159.57, 149.68, 136.78, 136.58, 131.52, 129.37,
1
3
1
19 3 4
17.98, 117.94, 112.10, 52.49, 17.82, 17.28, 13.04, 12.56.; HRMS (ESI) calcd for C15H N O S [M+H]+:
o
-1
38.1096, found 338.2801. 338.1090.; m.p.: 34-36 C.; IR(ATR) ν max/cm : 3247, 2963, 2917, 2878, 1650, 1610,
525, 1484, 1383, 1320, 1240, 1132.

ACCEPTED MANUSCRIPT
4
.1.8.2. 5-Methyl-isoxazole-4-carboxylic acid [2-methyl-4-(propane-1-sulfonylamino)-phenyl]-amide (9b) :
1
The title compound was isolated as yellow solid (64%); H NMR (400 MHz, DMSO) δ 9.74 (2H, s), 9.02 (1H, s),
13
7
.20 (2H, s), 7.03 (1H, d, J = 8.0 Hz), 3.03 (2H, s), 2.66 (3H, s), 2.15 (3H, s), 1.68-1.66 (2H, m), 0.93 (3H, s);
C
NMR (101 MHz, DMSO) δ 172.48, 159.26, 149.13, 136.34, 134.88, 131.35, 127.57, 121.21, 117.29, 111.66,
5
3
1
2.29, 18.11, 16.85, 12.57, 12.04.; HRMS (ESI) calcd for C H N O S [M+H]+: 338.1096, found 338.1524.
15 19 3 4
o
-1
38.1094.; m.p.: 38-43 C.; IR(ATR) ν /cm : 3248, 2967, 2922, 1649, 1613, 1500, 1482, 1384, 1320, 1240,
max
141, 1092.
4
.2 Determinations of antiproliferation assays on cancer cell lines
A375P cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were
maintained in DMEM medium (Welgene, Daegu, Korea) supplemented with 10% FBS (Welgene) and 1%
o
penicillin/streptomycin (Welgene) in a humidified atmosphere with 5% CO at 37 C. A375P cells were taken from
2
3
the culture substrate with 0.05% trypsin-0.02% EDTA and plated at a density of 5 × 10 cells/well in 96 well plates
o
and then incubated at 37 C for 24 h in a humidified atmosphere with 5% CO prior to treatment with test
2
compounds at various concentrations (3-fold serial dilution, 12 points). The A357P cell viability was assessed by the
conventional 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays
®
were carried out with CellTiter 96 (Promega) according to the manufacturer’s instructions. The absorbance at 590
nm was recorded using EnVision 2103 (Perkin Elmer; Boston, MA, USA). The IC was calculated using GraphPad
50
Prism 4.0 software.
U937 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, US) and maintained
in RPMI 1640 medium (Welgene, Daegu, Korea) supplemented with 10% FBS (Welgene), 1%
o
penicillin/streptomycin (Welgene) and 25 mM HEPES (Welgene) in a humidified atmosphere with 5% CO at 37 C.
2
3
U937 cells were taken from the culture substrate and plated at a density of 5 × 10 cells/well in 96 well plates and
2
then incubated at 37℃ for 24 h in a humidified atmosphere with 5% CO prior to test compounds at various
concentration (3-fold serial dilution, 12 points). The U937 cell viability was assessed by the conventional 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays were carried out with
Thiazolyl Blue Tetrazolium Bromide (SIGMA) according to the manufacturer’s instructions. The absorbance at 570
nm was recorded using Multiskan EX (Thermo; Waltham, MA, USA). The IC50 was calculated using GraphPad
Prism 4.0 software
4
.3 Docking Simulations
Molecular docking of compound 6a into the 3D x-ray structure of FMS (PDB code: 3LCO) was carried out using
Glide (Schrodinger software package Version 14.1). The 3D x-ray protein structure of FMS as a complex with
ligand was obtained from the PDB and prepared using Protein Preparation Wizard of the Schrodinger Maestro
program. The structures of new designed inhibitors were drawn using Chemdraw, and their 3D conformations were
generated using the Schrodinger LigPrep program with the OPLS 2005 force field. When making an optimal grid

ACCEPTED MANUSCRIPT
file, a grid box was manually adjusted from a cube into a cuboid, constraints (hydrogen bonding and hydrophobic
cube) were added, and unwanted pockets were treated as excluded volume. Molecular docking of compound 6a into
the structure of FMS produced predictive docking pose, (1) SP (standard precision) and XP (extra precision)
docking of compound 6a, (2) revision of the docking poses through substructure energy minimization using
Schrodinger Macro Model, and (3) scoring of revised the docking pose.
4
.4 Selected Kinases Profiling
SM
We used Reaction Biology Corp. Kinase HotSpot service (www.reactionbiology.com) for screening of 6a, and
IC Profiler Express for IC measurement. Their assay protocol was as follows: in a final reaction volume of 25 ꢀL
5
0
50
FMS (h) (5-10 mU) was incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic protein, 10
33
mM magnesium acetate and [γ- P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The
reaction is initiated by the addition of the Mg-ATP mix. After incubation for 40 minutes at room temperature, the
reaction was stopped by the addition of 5 ꢀL of a 3% phosphoric acid solution. 10 ꢀL of the reaction was then
spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol
prior to drying and scintillation counting.
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) Grant (Grant No. NRF-
2
012R1A1A2A10038773: J.-M. Hah) and (Grant No. NRF-2014R1A1A3051648: J.-M. Hah).
Supplementary data
Supplementary data related to this article can be found at
References
[
[
1] G. Burnett, E.P. Kennedy The enzymatic phosphorylation of proteins J. Biol. Chem. 211(1954), 969-980.
2] C. Prével, L.Kurzawa, T. N. N. Van, M. C. Morris, Fluorescent biosensors for drug discovery new tools for old
targets - Screening for inhibitors of cyclin-dependent kinases European Journal of Medicinal Chemistry, 88
(
2014), 74-88.
[
[
3] D. Zhao, C. Chen, H. Liu, L. Zheng, Y. Tong, D. Qu, S. Han, Biological evaluation of halogenated
thiazolo[3,2-a]pyrimidin-3-one carboxylic acid derivatives targeting the YycG histidine kinase European
Journal of Medicinal Chemistry, 87 (2014), 500-507.
4] M. I. El-Gamal, M. A. Khan, M. S. Abdel-Maksoud, M. M. G. El-Din, C.-H. Oh, A new series of diarylamides
possessing quinoline nucleus: Synthesis, in vitro anticancer activities, and kinase inhibitory effect, European
Journal of Medicinal Chemistry, 87 (2014), 484-492.

ACCEPTED MANUSCRIPT
[
[
5] R. P. de la Bellacasa, G. Roué, P. Balsas, P. Pérez-Galán, J. Teixidó, D. Colomer, J. I. Borrell, 4-Amino-2-
arylamino-6-(2,6-dichlorophenyl)-pyrido[2,3-d]pyrimidin-7-(8H)-ones as BCR kinase inhibitors for B
lymphoid malignancies European Journal of Medicinal Chemistry, 86 (2014), 664-675.
6] E. Zwick, J. Bange, A. Ullrich. Receptor tyrosine kinase signalling as a target for cancer intervention strategies
Endocr. Relat. Cancer 8 (2001), 161–73.
[
[
7] http://www.brimr.org/PKI/PKIs.htm
8] M. El-Gamal, H. S. Anbar, K.H. Yoo, and C-H Oh, FMS Kinase Inhibitors: Current Status and Future
Prospects, Medicinal Research Reviews, 33 (2013), 599–636.
[
[
9] C.J. Burns, A. F. Wilks, c-FMS inhibitors: a patent review Expert Opin. Ther. Patents 21 (2011), 147-165.
10] E. P. Toy, T. Lamb, M. Azodi, W. J. Roy, H.-H. Woo, S. K. Chambers, Inhibition of the c-fms proto-oncogene
autocrine loop and tumor phenotype in glucocorticoid stimulated human breast carcinoma cells, Breast Cancer
Res Treat, 129 (2011), 411–419.
[
[
[
[
11] V. Chitu, E.R. Stanley, Colony-stimulating factor-1 in immunity and inflammation, Curr Opin Immunol. 18
(
2006), 39–48.
12] P. J. Murray, T. A. Wynn, Protective and pathogenic functions of macrophage subsets, Nat. Rev. Immunol. 11
2011), 723–737.
(
13] B. Ruffell, N. I. Affara, L. M. Coussens, Differential macrophage programming in the tumor
microenvironment, Trends Immunol. 33 (2012), 119–126.
14] G.O. Ahn, D. Tseng, C.-H. Liao, M. J. Dorie, A. Czechowicz, J. M. Browna, Inhibition of Mac-1
(
CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment. Proc. Natl. Acad.
Sci. USA. 107 (2010), 8363–8368.
[
15] D. G. DeNardo, D. J. Brennan, E. Rexhepaj, B. Ruffell, S. L. Shiao, S. F. Madden, W. M. Gallagher, N.
Wadhwani, S. D. Keil, S. A. Junaid, H. S. Rugo, E. S. Hwang, K. Jirstrom, B. L. West, L. M. Coussens
Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy.
Cancer Discov. 1 (2011), 54–67.
[
[
[
[
16] T. L. Smalley Jr, S. D. Chamberlain, W. Y. Mills, D. L. Musso, S. A. Randhawa, J. A. Ray, V. Samano, L.
Frick, Synthesis of novel anilinoquinolines as c-fms inhibitors, Bioorg. Med. Chem. Lett., 17 (2007) 6257–
6
260.
17] H. Huang, D. A. Hutta, H. Hu, R. L. DesJarlais, C. Schubert, I. P. Petrounia, M. A. Chaikin, C. L. Manthey, M.
R. Player, Design and synthesis of a pyrido[2,3-d]pyrimidin-5-one class of anti-inflammatory FMS inhibitors,
Bioorg. Med. Chem. Lett.18 (2008) 2355–2361.
18] C. J. Burns, M. F. Harte, X. Bu, E. Fantino, M. Giarrusso, M. Joffe, M. Kurek, F. S. Legge, P. Razzino, S. Su,
H. Treutlein, S. S. Wan, J. Zeng, A. F. Wilks, Discovery of 2-(a-methylbenzylamino) pyrazines as potent Type
II inhibitors of FMS, Bioorg. Med. Chem. Lett., 19 (2009) 1206–1209.
19] K. J. Wilson, C. R. Illig, J. Chen, M. J. Wall, S. K. Ballentine, R. L. DesJarlais, Y. Chen, C. Schubert, R.
Donatelli, I. Petrounia, D. S. Crysler, C. J. Molloy, M. A. Chaikin, C. L. Manthey, M. R. Player, B. E.
Tomczuk, S. K. Meegalla, Reducing ion channel activity in a series of 4-heterocyclic arylamide FMS inhibitors,
Bioorg. Med. Chem. Lett., 20 (2010) 3925–3929.

ACCEPTED MANUSCRIPT
[
[
[
[
20] M. I. El-Gamal, M.-H. Jung, C.-H. Oh, Discovery of a new potent bisamide FMS kinase inhibitor Bioorg. Med.
Chem. Lett., 20 (2010) 3216–3218.
21] P. Sriramarao, M. A. Bourdon, Melanoma Cell Invasive and Metastatic Potential Correlates with Endothelial
Cell Reorganization and Tenascin Expression, Endothelium, 4 (1996), 85–97.
22] M. H. Lehmann, Recombinant human granulocyte-macrophage colony-stimulating factor triggers interleukin-
1
0 expression in the monocytic cell line U937, Mol. Immunol. 35 (1998), 479–485.
23] K. Lackey, M. Cory, R. Davis, S. V. Frye, P. A. Harris, R. N. Hunter, D. K. Jung, O. B. McDonald, R. W.
McNutt, M. R. Peel, R. D. Rutkowske, J. M. Veal, E. R. Wood, The discovery of potent cRaf1 kinase inhibitors,
Bioorg. Med. Chem. Lett. 10 (2000), 223-226.
[
[
24] I. Vivanco, N. Palaskas N, C. Tran, S. P. Finn, G. Getz, N. J. Kennedy, Identification of the JNK signaling
pathway as a functional target of the tumor suppressor PTEN, Cancer Cell 11 (2007), 555-569.
25] B. G. Szczepankiewicz, K. Christi, L.T. J. Nelson, L. Gang, L. Bo, Z. Hongyu, M. D. Serby, X. Zhili, L. Mei, J.
G. Rebecca, D. L. Haasch, S. Wang, J.E. Clampit, E. F. Johnson, T.H. Lubben, M. A. Stashko, E. T. Olejniczak,
C. Sun, S. A. Dorwin, K. Haskins, C. Abad-Zapatero, E. H. Fry, C. W. Hutchins, H. L. Sham, C.M. Rondinone,
J. M. Trevillyan, Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal
cross-kinase activity, J. Med. Chem. 49 (2006), 3563-3580.
[
[
26] C. J. Vlahos, W. F. Matter, K.Y. Hui, R. F. Brown, A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-
morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), J. Biol. Chem. 269 (1994), 5241-5248.
27] M. J. Meyers, M. Pelc, S. Kamtekar, J. Day, G. I. Poda, M. K. Hall, M. L. Michener, B. A. Reitz, K. J. Mathis,
B. S. Pierce, M. D. Parikh, D. A. Mischke, S. A. Long, J. J. Parlow, D. R. Anderson, A. Thorarensen,
Structure-based drug design enables conversion of a DFG-in binding CSF-1R kinase inhibitor to a DFG-out
binding mode, Bioorg. Med. Chem. Lett. 20 (2010), 1543-1547.

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Table1. Antiproliferative activities of N-(4 or5-amino-2-methylphenyl)-5-methylisoxazole-4-carboxamide derivatives.
HS27
HS27
(GI50,
µM)
Substit-
ution
U937
Substit-
ution
U937
No
R
(GI50
,
No
7a
7b
7c
R
(GI , µM)
(GI , µM)
50
50
µM)
5a
5b
6a
6b
4-amide
4-amide
3-amide
3-amide
> 30
> 30
> 30
3-urea
3-urea
3-urea
3-urea
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
> 30
0.016 + 0.006
0.465 + 0.022
7d
6
c
3-amide
3-amide
> 30
> 30
> 30
> 30
7e
7f
3-urea
3-urea
> 30
> 30
> 30
> 30
6
d
6
e
3-amide
3-amide
> 30
> 30
> 30
> 30
7g
8a
3-urea
4-urea
1.89 + 0.31
16.5 + 1.44
> 30
> 30
6
f
6
6
g
3-amide
3-amide
> 30
> 30
> 30
> 30
8b
8c
4-urea
4-urea
> 30
> 30
> 30
> 30
CN
S
h
6
i
3-amide
> 30
> 30
8d
4-urea
> 30
> 30
6
j
3-amide
3-amide
0.139 + 0.019
3.1 + 0.05
> 30
> 30
8e
8f
4-urea
4-urea
> 30
> 30
> 30
> 30
6
k
6
l
3-amide
3-amide
> 30
> 30
> 30
8g
9a
9b
4-urea
> 30
> 30
> 30
> 30
3
-
6m
0.019 + 0.007
sulfone
amide
4
-sulfon
6
n
o
3-amide
3-amide
0.026 + 0.003
0.049 + 0.040
> 30
> 30
> 30
> 30
> 30
eamide
6
Sorafenib
2.74 + 0.05

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Table 2. Percentages of enzymatic inhibitions by compound 6a (10 µM) on selected Protein Kinases panel.
Staurosporine
Kinase
% Inhibition
5.73
IC (nM)
50
AKT1
4.62
< 1.00
< 1.00
14.0
ALK
0
0
Aurora A
BRAF
61.2
87.9
7.5
0
BRAF (V599E)
c-Kit
4.50
84.2
c-MET
179
CDK1/cyclin B
CDK2/cyclin E
EGFR
0
2.52
1.85
4.68
7.54
0
< 1.00
283
ERK1
6540
13.2
FAK/PTK2
FGFR2
3.60
0
1.77
FGFR3
10.3
FLT3
2.12
98.7
0
< 1.00
1.14
FMS
GSK3b
2.62
IGF1R
0
43.3
JAK3
0
< 1.00
b
JNK3
15.3
8.27
97.8
0
1100
KDR/VEGFR2
Lyn
10.7
< 1.00
12.1
MEK1
c
mTOR/FRAP1
PKA
1.14
0
9900
< 1.00
192
PLK1
0
a
RAF1
95.9
0
2.87
RON/MST1R
ROS/ROS1
SYK
326
< 1.00
< 1.00
0
3.81
a.
b.
c.
Data of GW5074 [23]
Data of JNKi VIII [24-25]
Data of LY294002 [26]
Table 3. IC for enzymatic inhibitory activity of the selected compounds.
5
0
IC50
B-Raf
B-Raf V600E
668 nM
FMS
Lyn
C-Raf
6
a
5.96 µM
9.95 nM 65.9 nM 44.8 nM

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Figure 1. FDA approved RTK inhibitors.
Antiproliferative A375P
activity
U937
+++
-
+++
+
+++
Figure 2. Initial investigations of various hinge binder and its preference toward cancer cell lines
o
Scheme 1. (i) 5-methylisoxazole-4-carboxylic acid, SOCl , 50 C; (ii) 2-methyl-4-nitroaniline, 2-methyl-5-
2
o
o
nitroaniline, THF, 65 C, (iii) SnCl , HCl, EtOH, 7h, 80 C (iv) (a) RCOOH, HATU, DMF, rt, overnight (b) RCOCl,
2
o
THF, 65 C (v) RNCO,THF, rt, overnight (vi) propane-1-sulfonyl chloride, pyridine, M,C , rt

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Figure 3. Docking structures of designed 4-arylamido 3-methyl isoxazoles scaffold amide derivatives (thin, cyan) in FMS
(
PDB: 3LCO) [27]

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We synthesized 4-arylamido 3-methyl isoxazoles as potential protein kinase inhibitor.
We found their potent antiproliferative activities on U937 hematopoietic cell line.
The most potent inhibitor was found to be a potent and selective FMS inhibitor.

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Supplementary material
Discovery of 4-Arylamido 3-methyl isoxazole derivatives as novel FMS kinase inhibitors
*
Daseul Im, Kyungjin Jung, Songyi Yang, Waqar Aman, and Jung-Mi Hah
Department of Pharmacy, College of Pharmacy, & Institute of Pharmaceutical Science and Technology, Hanyang
University 55 Hanyangdaehak-ro, Sangnok-gu, Ansan Kyeonggi-do, 426-791, Korea
[
1] The list of kinases tested for the first hit compound 2-methyl-N-(5-methylisoxazol-4-yl)benzamide analogue (10
µM) in figure 2 is ABL1, AKT1, ALK, Aurora A, BRAF, BRAF (V599E), c-Kit, c-MET, c-Src, CDK1/cyclin B,
CDK2/cyclin E, EGFR, ERK1, FAK/PTK2, FGFR1, FGFR2, FGFR3, FLT1, FLT3, FLT4, GSK3b, Hck, IGF1R,
ITK, JAK1, JAK3, JNK2, JNK3, KDR/VEGFR2, Lck, Lyn, MEK1, mTOR, PDGFRa, PDGFRb, PI3K, PKA,
PKC, PLK1, RAF1, Ret, RON/MST1R, ROS/ROS1, SYK, Tie2.
1

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1
13
[
2] H and C NMR Spectra of representative compounds
O
N
H
N
O
NO2
3
a
1
H NMR (DMSO-d , 400MHz)
6
2

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