Design and synthesis of 5-[(2-chloro-6-fluorophenyl)acetylamino]-3-(4- fluorophenyl)-4-(4-pyrimidinyl)isoxazole (AKP-001), a novel inhibitor of p38 MAP kinase with reduced side effects based on the antedrug concept

Article abstract of DOI:10.1016/j.bmc.2014.05.045

Inhibitors of p38 mitogen-activated protein (MAP) kinase, which are closely involved in the production of inflammatory cytokines, are considered promising curative drugs for chronic inflammatory disorders. However, there is also a growing concern regarding its systemic side effects. To reduce the occurrence of side effects, we have identified a novel p38 MAP kinase inhibitor that shows properties of an antedrug, which imparts its effect solely on the inflammatory site and is metabolically inactivated right after. We have designed isoxazole derivatives through the addition of a fresh interacting fourth site to the structure of the prototypical p38 MAP kinase inhibitor that harbors three point interactive sites. The derivative 26d (AKP-001) shows excellent p38 MAP kinase inhibitory activity and a high selectivity for various kinases. Its rapid metabolism has been confirmed in rats. Moreover, 26d has been shown to be effective in animal models of inflammatory bowel disease.

Full text of DOI:10.1016/j.bmc.2014.05.045

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 5-[(2-chloro-6-fluorophenyl)acetylamino]-
3-(4-fluorophenyl)-4-(4-pyrimidinyl)isoxazole (AKP-001), a novel
inhibitor of p38 MAP kinase with reduced side effects based
on the antedrug concept
Koichi Hasumi a, Shuichiro Sato a, Takahisa Saito a, Jun-ya Kato a, Kazuhiko Shirota b, Jun Sato c,
Hiroyuki Suzuki c, Shuji Ohta d,
⇑
a Synthetic Research Department, ASKA Pharmaceutical Co., Ltd, 5-36-1, Shimosakunobe, Takatsu-ku, Kawasaki-shi, Kanagawa 213-8522, Japan
b Pharmacokinetics Research Department, ASKA Pharmaceutical Co., Ltd, 5-36-1, Shimosakunobe, Takatsu-ku, Kawasaki-shi, Kanagawa 213-8522, Japan
c Pharmacological Research Department, ASKA Pharmaceutical Co., Ltd, 5-36-1, Shimosakunobe, Takatsu-ku, Kawasaki-shi, Kanagawa 213-8522, Japan
d Synthetic Research Department, ASKA Pharmaceutical Co., Ltd, 4-3, 3-Chome, Sakae-cho, Hamura-shi, Tokyo 205-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of p38 mitogen-activated protein (MAP) kinase, which are closely involved in the production of
inflammatory cytokines, are considered promising curative drugs for chronic inflammatory disorders.
However, there is also a growing concern regarding its systemic side effects. To reduce the occurrence
of side effects, we have identified a novel p38 MAP kinase inhibitor that shows properties of an antedrug,
which imparts its effect solely on the inflammatory site and is metabolically inactivated right after. We
have designed isoxazole derivatives through the addition of a fresh interacting fourth site to the structure
of the prototypical p38 MAP kinase inhibitor that harbors three point interactive sites. The derivative 26d
(AKP-001) shows excellent p38 MAP kinase inhibitory activity and a high selectivity for various kinases.
Its rapid metabolism has been confirmed in rats. Moreover, 26d has been shown to be effective in animal
models of inflammatory bowel disease.
Received 11 April 2014
Revised 20 May 2014
Accepted 21 May 2014
Available online xxxx
Keywords:
Antedrug
p38 MAP kinase
IBD
Isoxazole
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors such as infliximab and adalimumab have been clinically
used and have shown excellent results,2,3 although these are rela-
Inflammatory bowel disease (IBD) is a collective name for intes-
tinal disorders that include Crohn’s disease (CD) and ulcerative
colitis (UC). These intestinal inflammatory and intractable diseases
of unknown etiology are characterized by repetitive periods of
activity and remission. The incidence of IBD has steadily increased
in recent years, predominantly affecting young adults, thus result-
ing in the deterioration of their quality of life (QOL) during the
acute period and forcing diet regulations even during remission.
Treatment regimens for IBD include 5-aminosalicylic acid
(5-ASA) formulations such as mesalazine,1 which is used mainly
during the remission period, and steroids such as prednisolone,
which is generally administered during the active period. In case
of the former, however, a high dose is required to observe an effect,
whereas the latter is associated with various side effects or may
tively costly and their route of administration has been described
as not very ideal. Based on these conditions, there is a need to
develop a small molecule drug of novel function that shows excel-
lent efficacy and reduced side effects.
Mitogen-activated protein kinases (MAPKs) represent a class of
serine/threonine kinases that are widely distributed across different
cell types and play an important role in various cell functions. Of
these, p38 MAP kinase is involved in the signaling pathway that reg-
ulates cellular responses to cytokines or stress. Its activation is
responsible for the formation of inflammatory cytokines such as
TNF-a or interleukin 1b (IL-1b). Accordingly, the p38 MAPK
inhibitor acts as a therapeutic drug for the treatment of chronic
inflammatory diseases such as rheumatic arthritis (RA) by regulat-
ing the secretion of cytokines. Many inhibitors have been reported
in literature. Based on its structure and interaction with p38 MAP
kinase, these inhibitors can be divided into 3 types (Fig. 1).4 Com-
pounds that competitively interact with the ATP-binding site are
classified as prototypes (teardrop binders) such as SB203580,5 and
result in drug dependency. Tumor necrosis factor-
a (TNF-a)
⇑
Corresponding author. Tel.: +81 42 578 3917; fax: +81 42 578 3925.
E-mail address: ohta-s@aska-pharma.co.jp (S. Ohta).
http://dx.doi.org/10.1016/j.bmc.2014.05.045
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. Typical inhibitors of p38 MAP kinase.
linear binders such as VX-745,6,7 and PH-797804.8–10 Teardrop bind-
ers carry a 5-membered heterocyclic ring as the nucleus and charac-
teristically forms hydrogen bonds with Lys53. Extended binders,
represented by BIRB796,11 undergo allosteric binding with a p38
MAP kinase that results in an inactive DFG-out conformation. p38
MAP kinase is involved not only in the production of inflammatory
cytokines, but also in cell differentiation and proliferation, as well
as cell-cycle regulation or apoptosis. It is ubiquitously expressed in
various systems of the body and thus, long-term exposure to inhib-
itors may result in toxicity.12,13 Although not all cases are class
effects, adverse events such as hepatotoxicity, cardiac toxicity, and
disorders involving the central nervous system have been reported,
prompting authorities to terminate its development.14 Leftheris
et al. have previously reported that p38 MAP kinase inhibitors con-
tain a triazine structure that has a short duration of action, which
suggests that its adverse effects can possibly be reduced.15 Recently,
Pfizer released the p38 MAPKs inhalant drug, PF-03715455, for the
treatment of respiratory diseases.16 Although the site of drug appli-
cation was different, we agree with the study that minimizing the
exposure of the rest of the body by using a drug with high clearance
and low absorption is extremely imperative. Most anti-inflamma-
tory drugs that have been designed with reduced side effects by lim-
iting its site or period of action are steroids that are commonly used
in the area of dermatology. After imparting its effect on the disease
site, steroids are rapidly inactivated during metabolism. This reac-
tion has served as the foundation of the concept of an antedrug.17
We believe that anti-inflammatory drugs showing the properties
of an antedrug may be applicable to not only the skin or specific
respiratory regions, but also to inflammatory bowel disease. There-
fore, we have attempted to design p38 MAP kinase inhibitors that
exclusively follows a topical action.
region II) formed by Met109, Ala111, Asp112, Ala157, and Leu167
also exist relatively close to the phosphate-binding ribbon with
which the 1-position side chain interacts.20–22 Based on this infor-
mation, by considering the presence of amino acid residues that
have the capability of interacting and a definite spatial tolerance
at a position corresponding to the direction of the 1-substitution
position of imidazole of these compounds in p38 MAP kinase,
new compounds were designed to further increase the inhibition
and adapted for application as antedrugs. Thus, compound A was
designed on the basis of the pharmacophore obtained by adding
one fresh point-of-interaction site to the prototypical structure of
p38 MAP kinase that has a 5-membered ring as its core and repre-
sented by SB203580, which possesses 3-point interaction sites
(presumed interaction sites: Met109, Lys53, and hydrophobic region
I) (Fig. 3). The concept of this compound A is as follows: (1) once the
drug reaches the inflammation site through an enteric-coated prep-
aration, an excellent topical anti-inflammatory effect showing high
inhibitory activity is exercised by a strong interaction mainly at
the 4 points, with p38 MAP kinase produced in the intestines; and
(2) once the drug is absorbed and circulates through the blood or
reaches the liver, the part of the structure containing the fourth
interaction site undergoes cleavage and releases metabolite B,
resulting in the suppression of the side effects originating from
p38 MAP kinase because of a considerable reduction in its inhibitory
activity. By using pyrazole and isoxazole as basic structures, a
substituent group was introduced into the site that was equivalent
to the imidazole 1-position of the compound previously mentioned
or into the 5-position of the individual structures.
3. Materials and methods
3.1. Chemistry
2. Design concept
Melting points were determined on a micro hot plate melting
point apparatus of Mettler-Toledo (Central Processor: Mettler
FP80HT, Hot Stage: Mettler FP82HT) and were uncorrected. Proton
NMR (1H NMR) spectra were recorded on a JEOL JNM-ECP 400.
Chemical shifts (d) are expressed in parts per million using tetra-
methylsilane as the internal standard. Mass spectra (MS) were
obtained on a Shimadzu GC/MS QP-5000 mass spectrometer sys-
tem, with electrospray ionization methodology. Infrared spectra
were recorded on JASCO FT/IR-470. High-resolution mass spectra
(ESI-HRMS) were obtained on Waters XevoQ-TOF. For normal pres-
sure and flash column chromatography, WakogelÒ C-200 (100–200
mesh) was used. The purity of the final compounds was deter-
mined by HPLC on Shimadzu HPLC system (Liquid chromatograph:
LC-10AD, System Controller: SCL-10A, Chromatopac: C-R7Aplus,
UV Spectrophotometric detector: SPD-10A, Autoinjector: SIL-10A).
The X-ray crystal structure of SB203580, which is a representa-
tive of the prototypical p38 MAP kinase inhibitor, has been
previously reported (Fig. 2).18 SB203580 interacts with the ATP-
binding site of p38 MAP kinase, and a hydrogen bond is formed
between the nitrogen of the imidazole 3-position and Lys53. Its
p-fluorophenyl group interacts with a lipophilic pocket (hydropho-
bic region I) consisting of Thr106 (gatekeeper residual group),
Leu75, Leu86, and Leu104 through the formation of van der Waals
bonds. In addition, the nitrogen atom from the pyridyl group inter-
acts with the main chain of Met109 through the formation of
hydrogen bonds. According to the same report, SB218655 has a
cyclopropylmethyl group at the 1-position of the imidazole struc-
ture, and the cyclopropylmethyl group interacts with pockets
formed by Val30, Tyr35, and Val38, and that is the cavity of the
phosphate-binding ribbon. SB220025, which carries a piperidine
ring, also interacts with Tyr35 of the phosphate-binding ribbon
and Asp168 at the C terminal position. Moreover, Wilson et al.
has also shown that VK-19911 interacts with p38 MAP kinase in
a similar manner to them.19 Relatively larger pockets (hydrophobic
3.1.1. 3-(4-Fluorophenyl)-4-(4-pyridyl)pyrazole (2)
To a solution of 3-dimethylamino-1-(4-fluorophenyl)-2-(4-pyr-
idyl)-2-propen-1-on (1) (3.21 g, 11.9 mmol) in ethanol (60 mL)
was added hydrazine monohydrate (2.99 g, 2.9 mL, 59.7 mmol)
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Figure 2. Important interactions between the prototypical p38 MAP kinase inhibitor and the ATP-binding site of p38: SB203580, SB218655, SB220025 and VK-19911.
Figure 3. Strategy for antedrugs of prototypical p38 MAP kinase inhibitors.
and the mixture was heated to reflux for 2 h. Then cooled to room
temperature, the solvent was removed in vacuo, the residue was
added water and extracted with ethyl acetate. The organic layer
was washed with brine, dried over MgSO4 and evaporated. The
crude product was recrystallized from ethyl acetate to give the title
compound 2 (2.48 g, 86%). Mp: 208.5–209 °C; 1H NMR (CDCl3) d:
8.51 (dd, J = 1.5 and 4.5 Hz, 2H), 7.82 (s, 1H), 7.5–6.9 (m, 6H); IR
(KBr) 2840, 1606, 1518, 1222, 834, 814 cmꢀ1; MS (EI): m/z 239
(M+).
removed in vacuo, the residue was added water and extracted with
ethyl acetate. The organic layer was washed with water, followed
by brine and then dried over MgSO4. The solvent was removed
under vacuum to give the title compound 3 (2.17 g, 97%). 1H
NMR (CDCl3) d: 8.49 (dd, J = 1.6 and 4.5 Hz, 2H), 7.70 (s, 1H),
7.90–7.60 (m, 6H), 5.08 (s, 2H), 2.90–2.50 (m, 4H), 2.0–1.6 (m,
4H); IR (KBr) 1602, 1222, 1142 cmꢀ1; MS (EI): m/z 239 (M+ꢀ83);
HRMS (ESI) calcd for C14H11FN3 [M+H]+ 240.0937, found 240.0948.
3.1.3. 3-(4-Fluorophenyl)-5-(1-hydroxy-3-phenylpropyl)-4-(4-
pyridyl)pyrazole (4)
3.1.2. 3-(4-Fluorophenyl)-4-(4-pyridyl)-1-(1-pyrrolidinomethyl)
pyrazole (3)
3-(4-Fluorophenyl)-4-(4-pyridyl)-1-(1-pyrrolidinomethyl)pyr-
azole (3) (3.89 g, 12.1 mmol) was dissolved in dry THF (100 mL). To
stirred the solution, 1.6 M n-butyllithium in hexane (8.3 mL,
13.3 mmol) was added dropwise while keeping the temperature
at ꢀ70 °C or lower. The solution was stirred for 30 min followed
by addition 3-phenylpropionaldehyde (1.77 g, 13.2 mmol) in THF
To a solution of 3-(4-fluorophenyl)-4-(4-pyridyl)pyrazole (2)
(1.66 g, 6.94 mmol) and 37% formaldehyde solution (0.42 g,
1.05 mL, 14.0 mmol) in ethanol (20 mL) was added pyrrolidine
(1.00 g, 1.17 mL, 14.0 mmol), the resulting mixture was heated to
reflux for 5 h. Then cooled to room temperature, the solvent was
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(15 mL) at the same temperature, then the resulting solution was
stirred while rising to room temperature gradually. After 1 h, 2 M
HCl was added to the solution and stirred for 10 min, then satu-
rated NaHCO3 aqueous solution was added until alkaline and sep-
arated the organic layer. After the aqueous layer was extracted
with ethyl acetate, those organic layers were combined and
washed with brine, dried over MgSO4, then evaporated. The result-
ing residue was purified by column chromatography (eluent: ethyl
acetate) to afford the title compound 4 (2.27 g, 51%). 1H NMR
(CDCl3) d: 8.43 (dd, J = 1.5 and 4.6 Hz, 2H), 7.4–6.8 (m, 11H), 5.0–
4.7 (m, 1H), 5.0–4.0 (br s, 1H), 2.69 (t, J = 7.3 Hz, 2H), 2.2–1.9 (m,
2H); IR (KBr) 3160, 2880, 1602, 1518, 1220, 968, 834 cmꢀ1; MS
(EI): m/z 373 (M+); HRMS (ESI) calcd for C23H21FN3O [M+H]+
374.1669, found 374.1666.
The reaction solution was filtered off by Celite and the filtrate
was evaporated in vacuo. The residue was added water and neu-
tralized with aqueous solution of HCl. The formed precipitate
was collected by filtration and washed with water, followed by
diethyl ether and dried in a circulation dryer to afford the title
compound 11 (16.3 g, 84%). Mp: 220.0–223.0 °C; 1H NMR (CD3OD)
d: 8.23–7.97 (m, 4H), 7.80–7.61 (m, 2H), 7.12 (t, J = 8.9 Hz, 2H); IR
(KBr) 2180, 1636, 1608, 1542, 1492, 1408, 1374, 1342, 1222, 1202,
1156, 830 cmꢀ1; MS (EI): m/z 240 (M+).
3.1.8. 5-Amino-3-(4-fluorophenyl)-4-(4-pyridyl)pyrazole (12)
A mixture of 3-(4-fluorophenyl)-3-oxo-2-(4-pyridyl)propioni-
trile (11) (11.24 g, 46.8 mmol) and phosphorus oxychloride
(181 g, 110 mL, 1180 mmol) was stirred at 100 °C for 1 h. After
excess phosphorus oxychloride was removed in vacuo, toluene
was added to the residue, and evaporated. Then the residue was
dissolved in ethanol (80 mL). To the solution was added hydrazine
monohydrate (7.2 g, 7.0 mL, 144 mmol) and the mixture was
heated to reflux. After 3.5 h, the mixture was evaporated in vacuo,
and sat. NaHCO3 was added to the residue. The solution was
extracted with a mixture of chloroform and methanol. The organic
layer was dried over MgSO4, and evaporated. The resulting residue
was washed with ethyl acetate to give the title compound 12
(6.40 g, 54%). Mp: 267.0–272.0 °C; 1H NMR (DMSO-d6) d: 8.41 (d,
J = 5.8 Hz, 2H), 7.37–7.03 (m, 6H), 5.26 (br s, 1H), 4.73 (br s, 1H);
MS (EI): m/z 254 (M+); HRMS (ESI) calcd for C14H12FN4 [M+H]+
255.1046, found 255.1049.
3.1.4. 3-(4-Fluorophenyl)-5-(3-phenyl-1-propenyl)-4-(4-pyridyl)
pyrazole (5)
A
mixture of 3-(4-fluorophenyl)-5-(1-hydroxy-3-phenylpro-
pyl)-4-(4-pyridyl)pyrazole (4) (0.373 g, 1.00 mmol), 4-toluenesul-
fonic acid monohydrate (0.399 g, 2.10 mmol) and toluene (10 mL)
was heated to reflux for 24 h. After cooling, a saturated NaHCO3
aqueous solution was added until alkaline and extracted with a
mixture of chloroform and methanol (9:1). The organic layer was
washed with brine, dried over MgSO4, then evaporated. The result-
ing residue was purified by column chromatography (eluent: chlo-
roform), then recrystallized from ethyl acetate to give the title
compound 5 (0.160 g, 45%). Mp: 204–205.5 °C; 1H NMR (CDCl3)
d: 8.55 (dd, J = 1.6, 4.5 Hz, 2H), 7.5–6.7 (m, 11H), 6.4–6.1 (m, 2H),
3.52 (d, J = 5.1 Hz, 2H); IR (KBr) 3220, 1600, 1516, 1442, 1220,
974, 838, 828 cmꢀ1; MS (EI): m/z 355 (M+); HRMS (ESI) calcd for
3.1.9. 3-(4-Fluorophenyl)-5-phenylacetylamino-4-(4-
pyridyl)pyrazole (13)
C
23H21FN3 [M+H]+ 356.1563, found 356.1556.
To a suspension of 5-amino-3-(4-fluorophenyl)-4-(4-pyri-
dyl)pyrazole (12) (0.254 g, 1.0 mmol) in THF (20 mL) was added
triethylamine (0.306 g, 3.0 mmol). After phenylacetyl chloride
(0.464 g, 3.0 mmol) in THF (5 mL) was added dropwise to the
suspension, the mixture was stirred at room temperature for 3 h.
Then water was added to the mixture and the aqueous solution
was extracted with ethyl acetate. The organic layer was washed
with water, dried over MgSO4, then evaporated. After the resulting
residue was dissolved in a mixed solution 2 M NaOH aqueous
solution (2 mL) and methanol (20 mL), the mixture was stirred at
room temperature for 1 h. The formed precipitate was collected
by filtration and suspended in a mixed solution 2 M NaOH aqueous
solution (2 mL) and methanol (20 mL) once again, the mixture was
heated at reflux for 5 h. After cooling, the reaction mixture was
evaporated under reduce pressure. The residue was purified by
column chromatography (eluent: chloroform/methanol = 30:1) to
give the title compound 13 (0.080 g, 22%). Mp: 289.2–290.4 °C;
1H NMR (DMSO-d6) d: 13.25 (br s, 1H), 9.93 (br s, 1H), 8.34 (d,
J = 5.9 Hz, 2H), 7.50–7.06 (m, 9H), 7.01 (d, J = 5.9 Hz, 2H), 3.56 (s,
2H); IR (KBr) 1694, 1600, 1586 cmꢀ1; MS (EI): m/z 372 (M+); HRMS
(ESI) calcd for C22H18FN4O [M+H]+ 373.1465, found 373.1458.
3.1.5. 3-(4-Fluorophenyl)-5-(3-phenylpropyl)-4-(4-pyridyl)
pyrazole (6)
To a solution of 3-(4-fluorophenyl)-5-(3-phenyl-1-propenyl)-4-
(4-pyridyl)pyrazole (5) (0.100 g, 0.28 mmol) in ethanol (30 mL)
was added 5% palladium on carbon (50 mg). The mixture was stir-
red under hydrogen atmosphere at 1 atm and room temperature
for 15 h. The catalyst was removed by filtration through Celite
and the filtrate was evaporated in vacuo. To the resulting residue
was added diethyl ether, the formed precipitate was collected by
filtration to afford the title compound 6 (60 mg, 60%). Mp:
155.5–156.5 °C; 1H NMR (CDCl3) d: 8.52 (dd, J = 1.5, 4.4 Hz, 2H),
7.4–6.8 (m, 11H), 2.9–2.5 (m, 4H), 2.2–1.7 (m, 2H); IR (KBr) 2920,
1602, 1510, 1226, 830 cmꢀ1; MS (EI): m/z 357 (M+); HRMS (ESI)
calcd for C23H21FN3 [M+H]+ 358.1720, found 358.1728.
3.1.6. tert-Butyl 1-methylhydrazinecarboxylate (8)
Methylhydrazine (7) (26 g, 564 mmol) and sodium hydroxide
(23 g, 575 mmol) was dissolved in methanol (500 mL). To the solu-
tion, di-tert-butyl dicarbonate (123 g, 564 mmol) in methanol
(500 mL) was added dropwise over a period of 2 h at 0 °C. Then
the reaction solution was stirred at room temperature for 2 h,
the deposit was removed by filtration through Celite and the fil-
trate was evaporated in vacuo. The oily residue was added water
and neutralized with aqueous solution of ammonium chloride.
The solution was extracted with dichloromethane, dried over
MgSO4, then evaporated to give the title compound 8 (79 g, 97%).
1H NMR (CDCl3) d: 4.06 (br, 2H), 3.05 (s, 3H), 1.47 (s, 9H); IR (neat)
3.1.10. 5-Amino-3-(4-fluorophenyl)-1-methyl-4-(4-
pyridyl)pyrazole (14)
A mixture of 3-(4-fluorophenyl)-3-oxo-2-(4-pyridyl)propioni-
trile (11) (2.0 g, 8.33 mmol) and phosphorus oxychloride (16.45 g,
10 mL, 107 mmol) was stirred at 100 °C for 1 h. After excess
phosphorus oxychloride was removed in vacuo, the residue was
dissolved in ethanol (150 mL). To the solution was added tert-butyl
1-methylhydrazinecarboxylate (8) (3.5 g, 20.5 mmol) and the
mixture was heated to reflux. After 1.5 h, the reaction solution
was added trifluoroacetic acid (4.44 g, 3 mL, 38.9 mmol) and the
mixture was stirred at 100 °C. After cooling, the mixture was evap-
orated in vacuo, then water was added to the residue and neutral-
ized with NaHCO3. The solution was extracted with chloroform and
dried over MgSO4, then evaporated. The resulting residue was
1694, 1365, 1154 cmꢀ1
.
3.1.7. 3-(4-Fluorophenyl)-3-oxo-2-(4-pyridyl)propionitrile (11)
To a solution of 4-pyridylacetonitrile (9) (9.6 g, 81.3 mmol) and
2,5-dioxopyrrolidinyl 4-fluorobenzoate (10) (19.3 g, 81.4 mmol) in
dimethylformamide (200 mL) was added potassium carbonate
(11.2 g, 81.0 mmol), then stirred at room temperature for 24 h.
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
purified by column chromatography (eluent: chloroform/metha-
nol = 50:1–20:1) to give the title compound 14 (1.5 g, 62%). Mp:
155.2–157.9 °C; 1H NMR (CDCl3) d: 8.53 (dd, J = 1.5, 4.4 Hz, 2H),
7.50–6.83 (m, 4H), 7.08 (dd, J = 1.5, 4.4 Hz, 2H), 3.77 (br s, 2H),
3.77 (s, 3H); IR (KBr) 1598, 1212 cmꢀ1; MS (EI): m/z 268 (M+);
HRMS (ESI) calcd for C15H14FN4 [M+H]+ 269.1202, found 269.1199.
2H); IR (KBr) 3460, 1644, 1606 cmꢀ1; MS (EI): m/z 255 (M+); HRMS
(ESI) calcd for C14H11FN3O [M+H]+ 256.0886, found 256.0877.
3.1.15. 3-(4-Fluorophenyl)-4-(4-pyridyl)isoxazole (20)
Compound 20 was prepared from 19 according to the Laufer’s
method.23
3.1.11. 5-(4-Fluorophenyl)-1-methyl-3-phenylacetylamino-4-
(4-pyridyl)pyrazole (15)
3.1.16. Typical procedure for the preparation of 5-acylamino-3-
(4-fluorophenyl)-4-(4-pyridyl)isoxazole (21)
To a solution of 5-amino-3-(4-fluorophenyl)-1-methyl-4-(4-
pyridyl)pyrazole (14) (0.398 g, 1.48 mmol) in THF (20 mL) was
added triethylamine (0.165 g, 1.63 mmol). After the mixture was
added dropwise phenylacetyl chloride (0.240 g, 1.55 mmol), the
solution was stirred at room temperature overnight. Then water
was added to the mixture, and extracted with chloroform. The
organic layer was washed with water and dried over MgSO4, then
evaporated. The resulting residue was purified by column chroma-
tography (eluent: chloroform/methanol = 40:1) and recrystallized
from n-hexane/ethyl acetate to afford the title compound 15
(0.200 g, 35%). Mp: 164.9–166.5 °C; 1H NMR (CDCl3) d: 8.40 (d,
J = 5.9 Hz, 2H), 7.50–6.66 (m, 12H), 3.74 (s, 2H), 3.74 (s, 3H); IR
(KBr) 1602, 1220 cmꢀ1; MS (EI): m/z 386 (M+); HRMS (ESI) calcd
for C23H20FN4O [M+H]+ 387.1621, found 387.1619.
3.1.16.1. 5-Acetylamino-3-(4-fluorophenyl)-4-(4-pyridyl)isoxaz-
ole (21a).
To a solution of 5-amino-3-(4-fluorophenyl)-4-(4-
pyridyl)isoxazole (19) (0.15 g, 0.59 mmol) and triethylamine
(0.143 g, 0.197 mL, 1.41 mmol) in dichloromethane (10 mL) was
added acetyl chloride (0.102 g, 0.092 mL, 1.30 mmol) at 0 °C, the
mixture was stirred at 0 °C for 2 h. Then water was added to the
solution and extracted with chloroform, dried over MgSO4, then
evaporated. After the resulting residue was dissolved in methanol
(10 mL), 2 N NaOH aqueous solution (1 mL, 2.0 mmol) was added
to the solution and stirred at room temperature for 30 min. The
mixture was evaporated in vacuo, the residue was added water
and neutralized with 2 N HCl aqueous solution. Then the formed
precipitate was collected by filtration, washed with water, and
dried under reduced pressure to afford the title compound 21a
(0.109 g, 63%). Mp: 189.5–194.0 °C; 1H NMR (DMSO-d6) d: 11.00
(br s, 1H), 8.54 (d, J = 5.9 Hz, 2H), 7.54–7.00 (m, 6H), 2.05 (s, 3H);
IR (KBr) 1722, 1630, 1440, 1225 cmꢀ1; MS (EI): m/z 297 (M+);
3.1.12. 4-Fluorobenzaldoxime (17)
To a solution of 4-fluorobenzaldehyde (16) (25.0 g, 201 mmol)
and hydroxylamine hydrochloride (15.4 g, 222 mmol) in ethanol
(47 mL) was added ice water (137 mL). Then 50% NaOH aqueous
solution (42.8 mL, 1621 mmol) was added dropwise to the solution
while keeping the internal temperature at 25–30 °C and the mix-
ture was stirred at room temperature for 1 h. After the reaction
solution was washed with diethyl ether, the aqueous layer was
neutralized with concd HCl and extracted with chloroform. The
organic extracts was washed with brine and dried over MgSO4,
then evaporated. The resulting residue was recrystallized from n-
hexane/diethyl ether to afford the title compound 17 (24.3 g,
87%). Mp: 86.6–88.0 °C; 1H NMR (CDCl3) d: 8.11 (s, 1H), 7.75 (s,
1H), 7.70–7.40 (m, 2H), 7.23–6.90 (m, 2H); MS (EI): m/z 139 (M+).
HRMS (ESI) calcd for
298.0990.
C
16H13FN3O2 [M+H]+ 298.0992, found
3.1.16.2. Compounds 21b–21e (except for 21c).
Those title
compounds were prepared according to the same method as
described above for 21a using each the corresponding reagent.
3.1.16.3. 5-Benzoylamino-3-(4-fluorophenylamino)-4-(4-pyri-
dyl)isoxazole (21b).
Yield: 76%; mp:178.5–180.5 °C; 1H
NMR (CDCl3) d: 8.54 (dd, J = 1.5, 4.4 Hz, 2H), 8.38 (br s, 1H),
7.90–7.73 (m, 2H), 7.70–7.25 (m, 5H), 7.25–6.90 (m, 4H); IR (KBr)
1708, 1276 cmꢀ1; MS (EI): 359 (M+); HRMS (ESI) calcd for C21H15-
FN3O2 [M+H]+ 360.1148, found 360.1139.
3.1.13. 4-Fluoro-N-hydroxybenzimidoyl chloride (18a)
To a solution of 4-fluorobenzaldoxime (17) (19.4 g, 139 mmol)
in DMF (140 mL) was added portion wise N-chlorosuccinimide
(NCS) (18.9 g, 142 mmol) while keeping the internal temperature
at 40 °C or lower. After the mixture was stirred at room tempera-
ture for 1.5 h, the reaction solution was poured into ice water,
and extracted with diethyl ether. The extracts was washed with
water and dried over MgSO4, then evaporated. The resulting resi-
due was recrystallized from n-hexane/diethyl ether to afford the
title compound 18a (23.4 g, 97%). Mp: 67.4–70.1 °C; 1H NMR
(CDCl3) d: 8.08 (s, 1H), 7.87 (dd, J = 2.2, 6.8 Hz, 1H), 7.14 (dd,
J = 2.2, 6.8 Hz, 1H), 7.04 (dd, J = 2.2, 6.8 Hz, 1H); MS (EI): m/z 175
(M++2).
3.1.16.4. 3-(4-Fluorophenyl)-5-(3-phenylpropionylamino)-4-(4-
pyridyl)isoxazole (21d).
NMR (CDCl3) d: 8.50 (dd, J = 1.5, 4.4 Hz, 2H), 7.60 (br s, 1H),
7.50–6.85 (m, 11H), 3.15–2.90 (m, 2H), 2.85–2.60 (m, 2H); IR
(KBr) 1722, 1505, 1224 cmꢀ1; MS (EI): m/z 387 (M+); HRMS (ESI)
calcd for C23H19FN3O2 388.1461, found 388.1468.
Yield: 25%; mp: 184.0–187.0 °C; 1H
3.1.16.5. 3-(4-Fluorophenyl)-5-(4-phenylbutanoylamino)-4-(4-
pyridyl)isoxazole (21e).
Yield: 57%; mp: 132.7–135.1 °C; 1H
NMR (CDCl3) d: 8.56 (dd, J = 1.5, 4.4 Hz, 2H), 7.63 (br s, 1H),
7.46–6.95 (m, 11H), 2.68 (t, J = 7.5 Hz, 2H), 2.50–2.34 (m, 2H),
2.19–1.91 (m, 2H); IR (KBr) 1722, 1628, 1610, 1442 cmꢀ1; MS
(EI): m/z 401 (M+); HRMS (ESI) calcd for C24H21FN3O2 [M+H]+
402.1618, found 402.1615.
3.1.14. 5-Amino-3-(4-fluorophenyl)-4-(4-pyridyl)isoxazole (19)
Sodium (2.92 g, 127 mmol) was dissolved in anhydrous ethanol
(200 mL). To stirred the solution, 4-pyridylacetonitrile (9) (15 g,
127 mmol) in THF (200 mL) was added dropwise, then a solution
of 4-fluoro-N-hydroxybenzimidoyl chloride (18a) (22.06 g,
127 mmol) in ethanol (200 mL) was added dropwise at 0 °C. The
mixture was stirred at room temperature for 1 h. After the reaction
solution was concentrated under reduced pressure, water was
added to the residue, the formed precipitate was collected by filtra-
tion and dried under reduced pressure. The precipitate was washed
with diethyl ether to yield the title compound 19 (31.86 g, 98%).
Mp: 192.5–194.5 °C; 1H NMR (CDCl3) d: 8.55 (dd, J = 1.8, 4.4 Hz,
2H), 7.50–6.90 (m, 4H), 7.05 (dd, J = 1.8, 4.4 Hz, 2H), 4.83 (br s,
3.1.16.6.
dyl)isoxazole (21c).
3-(4-Fluorophenyl)-5-phenylacetylamino-4-(4-pyri-
Imidazole (1.36 g, 20.0 mmol) and DBU
(3.04 g, 20.0 mmol) were dissolved in THF (50 mL). To stirred the
solution, phenylacetyl chloride (3.09 g, 20.0 mmol) in THF
(10 mL) was added dropwise at 0 °C, then 5-amino-3-(4-fluoro-
phenyl)-4-(4-pyridyl)isoxazole (19) (2.55 g, 10.0 mmol) and DBU
(3.04 g, 20.0 mmol) in THF (20 mL) was added dropwise. The
mixture was stirred at room temperature for 5 h. Then water was
added to the reaction solution and extracted with ethyl acetate.
The organic extracts was washed with water and dried over
MgSO4, then evaporated. The resulting residue was purified by
Please cite this article in press as: Hasumi, K.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.045

6
K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
column chromatography (eluent: chloroform/methanol = 40:1) to
give the title compound 21c (3.06 g, 82%). Mp: 164.5–165.5 °C;
1H NMR (CDCl3) d: 8.48 (dd, J = 1.7, 4.5 Hz, 2H), 7.69 (br s, 1H),
7.50–6.97 (m, 9H), 6.89 (dd, J = 1.7, 4.5 Hz, 2H), 3.75 (s, 2H); IR
(KBr) 1712, 1630, 1602, 1436 cmꢀ1; MS (EI): 373 (M+); HRMS
(ESI) calcd for C22H17FN3O2 [M+H]+ 374.1305, found 374.1301.
d: 9.02 (d, J = 1.2 Hz, 1H), 8.35 (d, J = 5.4 Hz, 1H), 7.51 (dt, J = 6.6,
8.5 Hz, 1H), 7.08–6.97 (m, 2H), 6.92 (br s, 2H), 6.60 (td, J = 1.2,
5.4 Hz, 1H); MS (EI): m/z 274 (M+); HRMS (ESI) calcd for C13H9F2N4
O [M+H]+ 275.0744, found 275.0744.
3.1.19.2.4. 5-Amino-3-(2,6-difluorophenyl)-4-(4-pyrimidinyl)isox-
azole (25e). Yield: 88%; mp:170.0–176.5 °C; 1H NMR (CDCl3)
d: 9.02 (d, J = 1.5 Hz, 1H), 8.34 (d, J = 5.4 Hz, 1H), 7.58–7.50 (m,
1H), 7.11–7.07 (m, 2H), 6.94 (br s, 2H), 6.57–6.54 (m, 1H); MS
(EI): m/z 274 (M+); HRMS (ESI) calcd for C13H9F2N4O [M+H]+
275.0744, found 275.0746.
3.1.19.2.5. 5-Amino-3-(2,6-dichlorophenyl)-4-(4-pyrimidinyl)isox-
azole (25f). Yield: 56%; mp:202.5–207.0 °C; 1H NMR (CDCl3) d:
9.00 (d, J = 1.4 Hz, 1H), 8.30 (d, J = 5.6 Hz, 1H), 7.50–7.43 (m, 3H),
7.00 (br s, 2H), 6.29 (dd, J = 1.4, 5.6 Hz, 1H); MS (EI): m/z 306
(M+); HRMS (ESI) calcd for C13H9Cl2N4O [M+H]+ 307.0153, found
307.0160.
3.1.17. (E)-N,N-Dimethyl-2-(4-pyrimidinyl)ethenamine (23)
A mixture of 4-methylpyrimidine (22) (10 g, 106 mmol), N,N-
dimethylformamide dimethylacetal (DMFDMA) (38 g, 319 mmol)
and DMF (46.6 g, 637 mmol) was stirred in a sealed-tube at
140 °C for 24 h. After cooling, the solution was evaporated in vacuo
to yield the title compound 23 (15.08 g, 95%). 1H NMR (CDCl3) d:
8.73 (br s, 1H), 8.22 (d, J = 5.5 Hz, 1H), 7.77 (d, J = 12.9 Hz, 1H),
6.72 (dd, J = 5.5, 12.9 Hz, 1H), 5.00 (d, J = 12.9 Hz, 1H), 2.96 (s, 6H).
3.1.18. 4-Pyrimidinylacetonitrile (24)
3.1.19.2.6. 5-Amino-3-(3-methylphenyl)-4-(4-pyrimidinyl)isoxaz-
ole (25g). Yield: 18%; mp:164.5–166.5 °C; 1H NMR (CDCl3) d:
9.00 (d, J = 1.6 Hz, 1H), 8.28 (d, J = 5.6 Hz, 1H), 7.40–7.28 (m, 4H),
6.86 (br s, 2H), 6.73 (dd, J = 1.6, 5.6 Hz, 1H), 2.41 (s, 3H); MS (EI):
m/z 252 (M+); HRMS (ESI) calcd for C14H13N4O [M+H]+ 253.1089,
found 253.1080.
3.1.19.2.7. 5-Amino-3-(4-fluoro-3-methyl)-4-(4-pyrimidinyl)isox-
azole (25h). Yield: 43%; mp:161.0–165.0 °C; 1H NMR (CDCl3) d:
9.02 (d, J = 1.5 Hz, 1H), 8.32 (d, J = 5.4 Hz, 1H), 7.37 (dd, J = 1.6,
7.3 Hz, 1H), 7.32–7.28 (m, 1H), 7.13 (t, J = 8.5 Hz, 1H), 6.89 (br s,
2H), 6.73 (dd, J = 1.2, 5.4 Hz, 1H), 2.33 (d, J = 1.5 Hz, 3H); MS (EI):
m/z 270 (M+); HRMS (ESI) calcd for C14H12FN4O [M+H]+
271.0995, found 271.0997.
To a solution of (E)-N,N-dimethyl-2-(4-pyrimidinyl)ethenamine
(23) (5 g, 33.5 mmol) in water (70 mL) was added hydroxylamine-
O-sulfonic acid (9.48 g, 83.8 mmol), the mixture was stirred at
50 °C for 30 min. Then a saturated NaHCO3 aqueous solution was
added until alkaline at 0 °C and extracted ethyl acetate. The organic
extracts was dried over MgSO4 and evaporated in vacuo. The
resulting residue was purified by column chromatography (eluent:
chloroform/methanol = 30:1) to give the title compound 24 (1.56 g,
39%). 1H NMR (CDCl3) d: 9.21 (d, J = 1.2 Hz, 1H), 8.80 (d, J = 5.2 Hz,
1H), 7.51 (dd, J = 1.2, 5.2 Hz, 1H), 3.93 (s, 2H).
3.1.19. Typical procedure for the preparation of 5-amino-3-aryl-
4-(4-pyrimidinyl)isoxazole 25
3.1.19.1. 5-Amino-3-(4-fluorophenyl)-4-(4-pyrimidinyl)isoxaz-
3.1.20. Typical procedure for the preparation of 3-aryl-5-
phenylacetylamino-4-(4-pyrimidinyl)isoxazole 26
ole (25a).
To a solution of sodium methoxide (2.50 g,
46.3 mmol) in methanol (50 mL) was added dropwise 4-pyrimidi-
nylacetonitrile (24) (5.0 g, 42.0 mmol) in THF (50 mL) and the mix-
ture was stirred at room temperature for 30 min. Then a solution of
4-fluoro-N-hydroxybenzimidoyl chloride (18a) (7.29 g, 42.0 mmol)
in methanol (50 mL) was added dropwise to the mixture and
stirred at room temperature for 7 h. The reaction solution was
concentrated under reduced pressure, then water was added to
the residue. The formed precipitate was collected by filtration
and washed with water, then dried under reduced pressure. The
resulting residue was purified by column chromatography (eluent:
chloroform/methanol = 50:1–30:1) and washed with diethyl ether
to give the title compound 25a (7.86 g, 73%). Mp:196.5–198.5 °C;
1H NMR (CDCl3) d: 9.03 (d, J = 1.4 Hz, 1H), 8.32 (d, J = 5.6 Hz, 1H),
7.54–7.49 (m, 2H), 7.24–7.18 (m, 2H), 6.88 (br s, 2H), 6.70 (dd,
J = 1.4, 5.6 Hz, 1H); MS (EI): m/z 256 (M+); HRMS (ESI) calcd for
3.1.20.1.
midinyl)isoxazole (26a).
3-(4-Fluorophenyl)-5-phenylacetylamino-4-(4-pyri-
Imidazole (0.43 g, 6.32 mmol) and
DBU (1.90 g, 12.5 mmol) were dissolved in THF (40 mL). To stirred
the solution, phenylacetyl chloride (0.97 g, 6.27 mmol) in THF
(10 mL) was added dropwise at 0 °C, then 5-amino-3-(4-fluoro-
phenyl)-4-(-4-pyrimidinyl)isoxazole (25a) (0.80 g, 3.12 mmol) in
THF (40 mL) was added dropwise. The mixture was stirred at room
temperature for 6 h. Then the reaction solvent was evaporated in
vacuo, water was added to the residue and extracted with ethyl
acetate. The organic extracts was dried over MgSO4 and evapo-
rated. The resulting residue was purified by column chromatogra-
phy (eluent: chloroform/methanol = 100:1) and washed with
diethyl ether to give the title compound 26a (0.77 g, 82%). Mp:
159.5–162.0 °C; 1H NMR (CDCl3) d: 11.39 (s, 1H), 8.49 (s, 1H),
8.36 (d, J = 5.6 Hz, 1H), 7.50–7.38 (m, 7H), 7.20 (t, J = 8.5 Hz, 2H),
6.73 (dd, J = 1.3, 5.6 Hz, 1H), 3.94 (s, 2H); MS (EI): m/z 374 (M+);
C
13H10FN4O [M+H]+ 257.0839, found 257.0835.
HRMS (ESI) calcd for
375.1269.
C
21H16FN4O2 [M+H]+ 375.1257, found
3.1.19.2. Compounds 25b–25g. Those title compounds were
prepared according to the same method as described above for
25a using each the corresponding reagent.
3.1.20.2. Compounds 26f, 26i and 26k–26l.
Those title com-
3.1.19.2.1. 5-Amino-3-(2-chlorophenyl)-4-(4-pyrimidinyl)isoxaz-
ole (25b). Yield: 56%; mp:181.0–184.0 °C; 1H NMR (CDCl3) d:
9.00 (d, J = 1.3 Hz, 1H), 8.29 (d, J = 5.6 Hz, 1H), 7.58–7.40 (m, 4H),
6.92 (br s, 2H), 6.41 (dd, J = 1.3, 5.6 Hz, 1H); MS (EI): m/z 272
(M+); HRMS (ESI) calcd for C13H10ClN4O [M+H]+ 273.0543, found
273.0536.
pounds were prepared according to the same method as described
above for 26a using each the corresponding reagent.
3.1.20.2.1.
3-(2-Chlorophenyl)-5-phenylacetylamino-4-(4-pyri-
midinyl)isoxazole (26f). Yield: 47%; mp: 149.5–153.0 °C; 1H
NMR (CDCl3) d: 11.50 (s, 1H), 8.47 (br s, 1H), 8.33 (d, J = 5.7 Hz,
1H), 7.55–7.40 (m, 9H), 6.46 (dd, J = 1.3, 5.7 Hz, 1H), 3.95 (s, 2H);
MS (EI): m/z 390 (M+); HRMS (ESI) calcd for C21H16ClN4O2
[M+H]+ 391.0962, found 391.0974.
3.1.19.2.2. 5-Amino-3-(4-chlorophenyl)-4-(4-pyrimidinyl)isoxaz-
ole (25c). Yield: 58%; mp:203.0–206.5 °C; 1H NMR (CDCl3) d:
9.02 (d, J = 1.2 Hz, 1H), 8.32 (d, J = 5.4 Hz, 1H), 7.51–7.45 (m, 4H),
6.88 (s, 2H), 6.70 (dd, J = 1.2, 5.4 Hz, 1H); MS (EI): m/z 272 (M+);
3.1.20.2.2. 3-(2,6-Difluorophenyl)-5-phenylacetylamino-4-(4-pyri-
midinyl)isoxazole (26i). Yield: 77%; mp: 191.0–193.5 °C; 1H NMR
(CDCl3) d: 11.48 (s, 1H), 8.48 (d, J = 1.2 Hz, 1H), 8.38 (d, J = 5.4 Hz,
1H), 7.58–7.41 (m, 6H), 7.11–7.06 (m, 2H), 6.60 (dd, J = 1.2,
5.4 Hz, 1H), 3.95 (s, 2H); MS (EI): m/z 392 (M+); HRMS (ESI) calcd
for C21H15F2N4O2 [M+H]+ 393.1163, found 393.1180.
HRMS (ESI) calcd for
273.0542.
C
13H10ClN4O [M+H]+ 273.0543, found
3.1.19.2.3. 5-Amino-3-(2,4-difluorophenyl)-4-(4-pyrimidinyl)isox-
azole (25d). Yield: 65%; mp:185.5–190.0 °C; 1H NMR (CDCl3)
Please cite this article in press as: Hasumi, K.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.045

K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
3.1.20.2.3.
3-(2,6-Dichlorophenyl)-5-phenylacetylamino-4-(4-
1H NMR (CDCl3) d: 8.52 (s, 1H), 8.34 (d, J = 5.4 Hz, 1H), 7.54–7.20
(m, 8H), 6.78 (dd, J = 1.5, 5.4 Hz, 1H), 4.07 (s, 2H), 2.39 (s, 3H);
MS (EI): m/z 404 (M+); HRMS (ESI) calcd for C22H18ClN4O2
[M+H]+ 405.1118, found 405.1109.
3.1.20.4.7. 3-(4-Fluoro-3-methylphenyl)-5-[(2-methylphenyl)ace-
tylamino]-4-(4-pyrimidinyl)isoxazole (26l). Yield: 82%; mp:193.5–
195.0 °C; 1H NMR (CDCl3) d: 11.35 (br s, 1H), 8.41 (d, J = 1.4 Hz,
1H), 8.34 (d, J = 5.4 Hz, 1H), 7.41–7.30 (m, 5H), 7.24–7.20 (m, 1H),
7.11 (t, J = 9.3 Hz, 1H), 6.74 (dd, J = 1.4, 5.4 Hz, 1H), 3.92 (s, 2H),
2.35 (s, 3H), 2.30 (d, J = 1.9 Hz, 3H); MS (EI): m/z 402 (M+); HRMS
(ESI) calcd for C23H20FN4O2 [M+H]+ 403.1570, found 403.1547.
pyrimidinyl)isoxazole (26j). Yield: 50%; mp:186.0–191.0 °C; 1H
NMR (CDCl3) d: 11.50 (s, 1H), 8.47 (s, 1H), 8.35 (d, J = 5.6 Hz, 1H),
7.52–7.42 (m, 8H), 6.34 (dd, J = 1.5, 5.6 Hz, 1H), 3.96 (s, 2H); MS
(EI): m/z 424 (M+); HRMS (ESI) calcd for C21H15Cl2N4O2 [M+H]+
425.0572, found 425.0566.
3.1.20.3. Typical procedure for the preparation of 3-aryl-4-
(4-pyrimidinyl)-5-substituted phenylacetylaminoisoxazole
26. 3.1.20.3.1.
3-(4-Fluorophenyl)-5-[(2-fluorophenyl)acetyl-
amino]-4-(4-pyrimidinyl)isoxazole (26b). To a solution of 20-fluor-
ophenylacetic acid (0.12 g, 0.78 mmol) in THF (5 mL) was added
1,10-carbonyldiimidazole (CDI) (0.126 g, 0.78 mmol) and the mix-
ture was stirred at room temperature for 1 h. Then a solution of
DBU (0.237 g, 1.56 mmol) and 5-amino-3-(4-fluorophenyl)-4-(4-
pyrimidinyl)isoxazole (25a) (0.100 g, 0.39 mmol) in THF (5 mL)
was added to the mixture and stirred at room temperature for
11 h. After the reaction solvent was evaporated in vacuo, water
was added to the residue and extracted with ethyl acetate. The
organic extracts was dried over MgSO4 and evaporated. The result-
ing residue was purified by column chromatography (eluent: chlo-
roform/methanol = 100:1) and washed with n-hexane/diethyl
ether to afford the title compound 26b (0.090 g, 59%). Mp:160.0–
163.0 °C; 1H NMR (CDCl3) d: 11.57 (s, 1H), 8.62 (s, 1H), 8.39 (d,
J = 5.7 Hz, 1H), 7.50–7.40 (m, 4H), 7.30–7.17 (m, 4H), 6.76 (dd,
J = 1.6, 5.7 Hz, 1H), 3.97 (s, 2H); MS (EI): m/z 392 (M+); HRMS
(ESI) calcd for C21H15F2N4O2 [M+H]+ 393.1163, found 393.1172.
3.2. Docking studies
All docking studies were performed on Discovery Studio Ver. 3.5
system (Accelrys Software Inc.). Three crystal structures of p38
a
MAP kinase (PDB ID: 1bl7, 2ewa, 1ouk) were selected from the Pro-
tein Data Bank (PDB) as docking templates by ligand similarity to
26d. Each crystal structure was modified using the Automatic
Preparation on default configuration. Compound 26d was docked
into each crystal structure using the docking program CDOCKER
on default configuration (Top Hits: 10, Random Conformations:
10, Orientations to Refine: 10). Because the best docking poses of
each crystal structure was relatively similar in the interaction
between 26d and p38a, the docking result for 1bl7 was only pre-
sented in context.
3.3. Biological methods
3.1.20.4. Compounds 26c–26e, 26g–26h and 26k–26l.
Those
title compounds were prepared according to the same method as
described above for 26b using each the corresponding reagent.
3.1.20.4.1. 5-[(2-Chlorophenyl)acetylamino]-3-(4-fluorophenyl)-4-
(4-pyrimidinyl)isoxazole (26c). Yield: 48%; mp:171.0–173.5 °C; 1H
NMR (CDCl3) d: 11.45 (br s, 1H), 8.54 (s, 1H), 8.38 (d, J = 5.7 Hz, 1H),
7.55–7.38 (m, 6H), 7.20 (t, J = 8.7 Hz, 2H), 6.75 (dd, J = 1.3, 5.7 Hz,
1H), 4.06 (s, 2H); MS (EI): m/z 408 (M+); HRMS (ESI) calcd for
3.3.1. Animals
All animal studies were approved by the Animal Research Com-
mittee of ASKA Pharmaceutical Co., Ltd and were conducted in
compliance with the Law Concerning the Protection and Control
of Animals (Japanese Law 105, October 1, 1973; revised on June
22, 2005). Female BALB/c mice and male Sprague-Dawley (SD) rats
were purchased from Japan SLC, Inc. (Shizuoka, Japan). All animals
were housed in temperature- and humidity-controlled rooms,
allowed to take standard rodent chow pellets and tap water ad libi-
tum., and were acclimatized for at least 7 days.
C
21H15F2N4O2 [M+H]+ 409.0868, 409.0859.
3.1.20.4.2. 5-[(2-Chloro-6-fluorophenyl)acetylamino]-3-(4-fluoro-
phenyl)-4-(4-pyrimidinyl)isoxazole (26d). Yield: 79%; mp:181.0–
182.0 °C; 1H NMR (CDCl3) d: 11.55 (s, 1H), 8.64 (s, 1H), 8.40 (d,
J = 5.7 Hz, 1H), 7.51–7.45 (m, 2H), 7.43–7.34 (m, 2H), 7.26–7.13
(m, 3H), 6.78 (dd, J = 1.3, 5.7 Hz, 1H), 4.14 (s, 2H); MS (EI): m/z
426 (M+); HRMS (ESI) calcd for C21H14ClF2N4O2 [M+H]+ 427.0773,
found 427.0770.
3.3.2. Inhibition of p38
selectivity assays
This study was conducted by Carna Biosciences, Inc. (Kobe,
Japan).
a MAP kinase and kinase isoform
3.1.20.4.3.
5-[(2,5-Dimethylphenyl)acetylamino]-3-(4-fluoro-
For details, refer to the URL; (http://www.carnabio.com/output/
pdf/ProfilingProfilingBook_en.pdf).
phenyl)-4-(4-pyrimidinyl)isoxazole (26e). Yield: 95%; mp:180.5–
182.5 °C; 1H NMR (CDCl3) d: 11.32 (s, 1H), 8.39 (d, J = 1.4 Hz, 1H),
8.35 (d, J = 5.4 Hz, 1H), 7.49–7.43 (m, 2H), 7.23–7.14 (m, 5H),
6.72 (dd, J = 1.4, 5.4 Hz, 1H), 3.88 (s, 2H), 2.39 (s, 3H), 2.31 (s,
3H); MS (EI): m/z 402 (M+); HRMS (ESI) calcd for C23H20FN4O2
[M+H]+ 403.1570, found 403.1562.
Inhibition of p38
Mobility Shift Assay (MSA). The enzyme activities of various
kinases except for p38 of 26d and 25a were measured by opti-
a MAP kinase were measured by Off-chip
a
mized ELISA or IMAP™ assay methods. IC50 values of test com-
pounds against each isoform of p38 and JNK were estimated.
3.1.20.4.4. 3-(4-Chlorophenyl)-5-[(2-chlorophenyl)acetylamino]-
4-(4-pyrimidinyl)isoxazole (26g). Yield: 49%; mp:209.0–211.5 °C;
1H NMR (CDCl3) d: 11.44 (br s, 1H), 8.53 (s, 1H), 8.39 (d,
J = 5.6 Hz, 1H), 7.54–7.39 (m, 8H), 6.76 (dd, J = 1.5, 5.6 Hz, 1H),
4.06 (s, 2H); MS (EI): m/z 424 (M+); HRMS (ESI) calcd for C21H15Cl2
N4O2 [M+H]+ 425.0572, found 425.0536.
Protein kinases evaluated 26d were; p38b, p38
JNK2, JNK3, CSK, LCK, AKT2, CRIK, ROCK1, PKAC
RSK2, CaMK4, CaMK2 , CHK1, DAPK1, MAPKAPK2, PIM1, CHK2,
CDK2/cyclinA, GSK3b, SRPK1, AurA, IKKb, NEK2, TTK, IRAK4,
PHKG1, CK1d, PKD2, Erk1, Erk2, Erk5, MAP3K5 and RAF1. Protein
c
, p38d, JNK1,
a, PDK1, PKC
a,
a
kinases evaluated 25a were; p38b, p38c, p38d, JNK1, JNK2, JNK3.
3.1.20.4.5.
5-[(2-Chlorophenyl)acetylamino]-3-(2,4-difluoro-
phenyl)-4-(4-pyrimidinyl)isoxazole (26h). Yield: 49%; mp:172.5–
175.5 °C; 1H NMR (CDCl3) d: 11.49 (br s, 1H), 8.53 (s, 1H), 8.41
(d, J = 5.4 Hz, 1H), 7.54–7.40 (m, 5H), 7.09–7.04 (m, 1H), 6.98 (dt,
J = 2.3, 8.5 Hz, 1H), 6.67 (td, J = 1.5, 5.4 Hz, 1H), 4.07 (s, 2H); MS
(EI): m/z 426 (M+); HRMS (ESI) calcd for C21H14ClF2N4O2 [M+H]+
427.0773, found 427.0788.
3.3.3. Inhibition of TNF- production in THP-1 cells
a
This study was conducted by SB Drug Discovery (Scotland). Cell
from the human monocytic cell line, THP-1, were cultured in RPMI
media supplemented with 10% fetal calf serum (FCS). Cell were
seeded out in 96-well cell culture plates at a density of 100,000
cells per well. Test compounds were diluted in 100% DMSO to yield
working stock concentrations of 1 mM. Subsequent dilutions
were carried out in cell culture media/10% DMSO. The final
3.1.20.4.6. 5-[(2-Chlorophenyl)acetylamino]-3-(3-methylphenyl)-
4-(4-pyrimidinyl)isoxazole (26k). Yield: 73%; mp:143.5–145.5 °C;
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
concentration of DMSO across the plate did not exceed 1%. Unstim-
ulated and Stimulated Control wells received an identical volume
of cell culture media/DMSO at this point. Test compounds were
added to the cells and 30 min later the cells were stimulated with
LPS (Escherichia coli 055 B55, Sigma, 1 g/mL). Two hours after addi-
tion of LPS, plates were centrifuged to pellet the cells and the con-
ethanol/saline) was instilled into the lumen of the colon. From
2 days after the colitis induction, vehicle, 26d (3, 10, 30 and
100 mg/kg) or prednisolone acetate (10 mg/kg) were orally
administrated to 8–10 rats per group twice daily for 6 days. Rats
were sacrificed 7 days after the induction of colitis, and the colonic
damage was scored according to the Wallace Criteria.24
centration of TNF-a in the supernatant was determined by ELISA
(human TNF- ELISA kits, BD Bioscience).
a
4. Results and discussion
4.1. Chemistry
3.3.4. Metabolic stability in human liver S9
Test compounds were spiked at a final concentration of 1 lM to
Although not fulfilling the conditions for the antedrug concept
(Fig. 3), first, 5-phenylpropylpyrazole 6 was synthesized by using
the procedure presented below to confirm the effect of the new
fourth interaction site (Scheme 1). By the reaction of enamine 1
and hydrazine monohydrate, the pyrazole framework was con-
structed to generate 2,25 and then 3 was obtained through the
reaction of pyrrolidine and formalin. After deprotonation of the
5-position using n-butyl lithium and introduction of the side chain
through a reaction with phenylpropionaldehyde, 4 was obtained. A
dehydration reaction resulted in compound 5 and finally, target
compound 6 was synthesized by catalytic hydrogen reduction of
compound 5.
50 mM phosphate buffer (pH 7.4), containing 3.3 mM MgCl2,
1.0 mM b-NADP+, 2.5 mM glucose 6-phosphate, and 2 U/mL glu-
cose 6-phosphate dehydrogenase, and to 0.5 mg protein/mL liver
S9. Metabolic reaction was terminated after incubation for 5 and
15 min at 37 °C, by addition of a four-fold volume of ice-cold ace-
tonitrile including 0.1 lM internal standard. The samples were
centrifuged at 2000ꢁg for 10 min, and the supernatant was sub-
jected to LC–MS/MS. Intrinsic clearance of test compounds in
human liver S9 was calculated according to a following equation:
CLint = Ke ꢁ V/proteinS9, where Ke is the elimination rate constant
of test compound, V is the initial incubation volume, and proteinS9
represents the protein concentration in the incubation mixture.
Pyrazoles 13 and 15, which contain an amide structure as side
chains at the 5-position, were synthesized using the procedure
shown in Scheme 2. Pyridylacetonitrile 9 and activated ester 10
were condensed in the presence of a base to produce cyanoketone
11, which upon chlorination of a carbonyl group, followed by a
reaction with hydrazine monohydrate, produced 5-aminopyrazole
12.26 Condensation with phenylacetylchloride in the presence of a
base produces 5-acylaminopyrazole 13. Meanwhile, 1-methyl
derivative 15 was synthesized by employing almost the same route
as previously described by using tert-butoxycarbonyl (Boc) com-
pound 8 derived from methylhydrazine 7.
3.3.5. Pharmacokinetic studies
Compound 26d was suspended in 0.5% Tween 80 (w/v) to pre-
pare oral dosing formulations. For intravenous dosing, Compound
26d was dissolved in a mixed solution of DMSO, polyethylene gly-
col-400, and saline (5:65:30). Three male SD rats received doses by
oral gavage or intravenous injection via the femoral vein, respec-
tively. Blood samples were collected via the tail vein over 24 h after
administration. Plasma was separated by centrifugation (12,000ꢁg,
3 min at 4 °C), and was stored at ꢀ20 °C until analysis.
The synthesis of 4-(4-pyridyl)isoxazole derivatives is shown in
Scheme 3. The key intermediate 5-aminoisoxazole 19 was synthe-
sized using the method of Miller et al.27 Aldehyde 16 was reacted
with hydroxylamine to form oxime 17 and then hydroxamic acid
chloride 18a was obtained by using N-chlorosuccinimide (NCS).
Through the reaction between 18a and commercially available 4-
pyridylacetonitrile 9 under basic conditions, an isoxazole frame-
work was built, which produced 5-amino isoxazole 19. Isoxazole
20, which contained an unsubstituted 5-position, was synthesized
by using the method of Laufer et al. and by deamination through
the diazotization reaction of 19.23 Introduction of the side chain
to the amino group at 5-position of 19 by condensation with a car-
boxylic acid could not proceed by using condensation reagents
such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
Therefore, this reaction was conducted through the diacyl form
obtained from the reaction using two or more equivalent quanti-
ties of carboxylic acid chloride and then, under alkaline conditions,
produced the monoacyl derivatives (21a, 21b, 21d, and 21e). To
simplify the reaction procedure and improve yield, condensation
with imidazoleamide in the presence of 1,8-diazabicyclo-[5.4.0]-
undec-7-ene (DBU) was performed and deemed optimal and was
therefore employed for the synthesis of 21c.28
4-(4-Pyrimidinyl)isoxazole derivative 26 was also synthesized
by using Scheme 4, which was basically similar to that for the
generation of the 4-(4-pyridyl)isoxazole derivative. By reacting
4-methylpyrimidine 22 and N,N-dimethylformamide dimethyl
acetal (DMFDMA), enamine 23 was obtained, and then 4-pyri-
midinylacetonitrile 24 was obtained by using hydroxylamine-
O-sulfonic acid. The target compound 26 was obtained via
5-aminoisoxazole 25 using a method similar to that previously
described.
3.3.6. LC–MS/MS analysis
The LC–MS/MS system consisted of an Alliance 2795 separation
module (Waters, Milford, MA) and a Quattro Ultima Pt triple quad-
rupole mass spectrometer (Micromass, Manchester, UK) with an
electron spray ionization interface. Aliquots of plasma samples
(50 lL) were added and vortex-mixed, followed by addition of
the internal standard solution, 0.5 mL of 0.5 M sodium hydrogen
carbonate solution, and 2 mL of diethyl ether. After vigorously
shaking for 10 min and centrifugation (2000ꢁg, 5 min), the organic
layer was evaporated under a nitrogen gas stream and were recon-
stituted with 100
lL of acetonitrile. Analytes (10
l
L) were sepa-
rated on a Discovery HSF5 column (2.1 mm I.D. ꢁ 50 mm, 3
lm)
at 40 °C with a linear gradient program consisting of 10 mM
ammonium formate (pH 3) and acetonitrile. Intra- and interday
precision and accuracy for these compounds were within 15%.
3.3.7. In vivo assays
3.3.7.1. DSS-induced colitis in mice.
Female BALB/c mice
were used in this study. Colitis was induced by allowing the mice
free access to 5% DSS (Sodium Dextran Sulfate) in their drinking
water for 7 days. Vehicle or 26d (3, 10, 30 and 100 mg/kg) were
orally administrated to 8 mice per group twice daily during DSS-
treatment. SASP (100 mg/kg) was administrated once daily to 8
mice during DSS-treatment. Mice were sacrificed 7 days after the
start of DSS-treatment and the colon length was measured.
3.3.7.2. TNBS-induced colitis in rats.
Male SD rats, fed an ele-
mental liquid diet (ElentalÒ; Ajinomoto Co., Inc.) for 7 days
to remove the gastrointestinal contents, were used in this study.
To induce colitis, 1 mL of TNBS solution (30 mg/mL in 40%
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
Scheme 1. Synthesis of 5-aralkylpyrazole.
Scheme 2. Synthesis of 5-acylaminopyrazole.
4.2. Biological evaluation and discussion
IC50 = 0.950 nM; TNF-
a: IC50 = 3.8 nM) and from this it was pre-
dicted that in the case of the freshly added fourth interaction site,
the substitution by a lipophilic group was favorable. The 5-pheny-
lacetylamino groups from 13 and 15 were not as active as the
phenylpropyl group of 6. Nevertheless, an increase in activity of
about one order of magnitude from each of 12 and 14 (before con-
densation) was observed. However, pyrazoles 13 and 15 were
stable in human liver S9, (individual percentage of unchanged
residue after 5 min: 90.4%, 84.4%, for 13 and 15, respectively) and
this metabolic stability is a desirable property for a drug to
manifest its efficacy through a systemic action. However, for the
antedrug, which is the goal of our present research, this may not
be suitable.
For primary screening, measurements of the inhibitory activity
of p38
matory cytokines in four isoforms (
TNF- inhibitory activity in THP-1 cells was also measured as an
a
MAP kinase, which is involved in the production of inflam-
a
, b, , and d), was performed.
c
a
index for inflammation. For the evaluation of its applicability as
an antedrug, a metabolic stability test using human liver S9 was
also carried out. Table 1 shows the results of biological activity of
a series of pyrazole derivatives and a metabolic stability test. First,
a comparison of a simple 3-(4-fluorophenyl)-4-(4-pyridyl)pyrazole
(2)25 not having any substituent, with 5-amino compound 12 and
5-amino-1-methyl compound (14) showed that all of these have
p38
a
inhibitory activities and TNF-
a
inhibitory activities of almost
Table 2 shows the activity and metabolic stability of isoxazole
derivatives. Comparison of activity of the 5-amino compound 19
that does not possess a fourth interaction site and an unsubstituted
the same magnitude despite the difference in these substituents. A
comparison between the compounds having a side chain at the 5-
position showed that the activity of the compounds from 6, 13, and
15 with side chains exceeds that of 2, 12, and 14 (i.e., prior to con-
densation). This finding indicates that according to our objective,
the side chain at the 5-position effectively interacts with p38
Derivative 6, in which the phenylpropyl group was introduced
at the 5-position, showed a particularly high activity (p38
compound 20 with pyrazole 12 showed that 19 has a p38a inhib-
itory activity of the same order as that of 12 (105 nM vs 97.4 nM)
and the activity of 20 was considerably low (871 nM) (in the report
by Laufer et al., the inhibitory activities of 19 and 20 were almost of
the same order23). This result differs from pyrazole, in which
the unsubstituted compound 2 and 5-amino compound 12 have
a.
a:
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10
K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Scheme 3. Synthesis of pyridylisoxazole derivatives.
Scheme 4. Synthesis of pyrimidinylisoxazole derivatives.
activities of almost the same order. In the case of isoxazole, the
5-amino group from 19 contributed to an increase in its activity
and thus is due to the interaction of the 5-amino group itself.
However, it supports the possibility of an increase in the affinity
of the nitrogen atom at the 3-position with Lys53 because of an
electron donation effect of the 5-amino group. In fact, 21a, in which
the electron-donating property has been reduced by acetylation of
the 5-amino group, showed a significantly lower activity than 19,
but it is almost of the same order as that of the unsubstituted
derivative 20. Compounds 21b–21e were synthesized to optimize
the length of the linker portion of the 5-position side chain,
which contains a benzene ring that can potentially become the
fourth interaction site. As a result, phenylacetylamide 21c and
phenylpropionylamide 21d had most excellent p38
activities, with values approaching that of pyrazole 13. 21c and
21d had almost equal p38 inhibitory activities (23.0 nM vs
23.4 nM), whereas 21c showed a higher TNF- inhibitory activity,
which was at least 2-fold (IC50: 54.7 nM vs 126.0 nM). Thus, the
optimum 5-position substituent group is phenylacetylamide from
21c in terms of the activity.
Next, in terms of metabolism, the series of isoxazole derivatives
showed an increasing tendency of clearance in human liver S9
compared to the pyrazole derivatives and thus, it is suitable as
an antedrug. However, 21c, which showed the highest activity,
was stable in isoxazole (CLint: 91.0 mL/min/mg) and thus needs
further enhancement in metabolism.
a inhibitory
a
a
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
Table 1
Biological activity and metabolic stability of pirazole derivatives
N
R1
N
R2
N
F
Compound
Structure
p38
a
MAP kinase
TNF-
a
Metabolic stability in human liver S9 (% of remaining)
Clint (mL/min/mg)
R1
H
R2
IC50 (nM)
IC50 (nM)
5 min
15 min
2
6
12
13
14
15
H
H
H
H
CH3
CH3
67.0
0.950
97.4
16.5
70.4
5.72
316.7
3.8
195.6
22.9
304.3
11.9
NAa
81.5
NAa
90.4
NAa
84.4
NAa
50.3
NAa
86.2
NAa
65.0
NAa
81.8
NAa
40.6
NAa
68.1
C6H5(CH2)3
NH2
C6H5CH2CONH
NH2
C6H5CH2CONH
a
NA: not applicable. The data of metabolic stability of compounds without a side chain at the 5-position was not applicable, since it is not so important from a perspective
of the concept of an antedrug.
Table 2
Biological activity and metabolic stability of isoxazole derivatives
N
A
R
O
N
F
Compound
Structure
p38a
MAP kinase
TNF-a
Metabolic stability in human liver S9 (% of remaining)
CLint
R
A
IC50 (nM)
IC50 (nM)
5 min
15 min
(mL/min/mg)
19
20
NH2
H
CH3CONH
CH
CH
CH
CH
CH
CH
CH
N
105
871
490.0
NAa
NAa
92.4
39.6
79.7
32.1
50.7
NAa
5.6
NAa
NAa
82.5
10.9
64.3
27.9
28.5
NAa
NTb
NAa
NAa
31.6
371
91.0
455
272
NAa
NTb
4617.0
4058.0
2771.0
54.7
126.0
762.5
>10,000
241.8
21a
21b
21c
21d
21e
25a
26a
1310
1210
23.0
23.4
271
C6H5CONH
C6H5CH2CONH
C6H5(CH2)2CONH
C6H5(CH2)3CONH
NH2
792
45.6
C6H5CH2CONH
N
a
NA: not applicable. The data of metabolic stability of compounds without a side chain at the 5-position was not applicable, since it is not so important from a perspective
of the concept of an antedrug.
b
NT: not tested.
Compound 25a was obtained by changing the 4-position of 19
from the 4-pyridyl group to the 4-pyrimidinyl group. Through this
The results of optimization using 26a as the lead compound
are presented in Table 3. First, analysis of the inhibitory effect
of individual substituents revealed that the 4-fluoro group
(26a–26e), 2,4-difluoro group (26h), 4-chloro group (26g), 3-
methyl group (26k), and 4-fluoro-3-methyl group (26l) were
preferable in the case of the isoxazole 3-position benzene ring
(R1). On the other hand, compounds with a halogen introduced
only at the ortho position (26f, 26i, and 26j) showed a consider-
ably low activity. This 3-position benzene ring was expected to
interact with the hydrophobic region I, these substituent effects
showed a similar tendency with that corresponding to the same
site in the other inhibitors. On the other hand, the introduction
of a chloro group (26c, 26d, 26g, 26h, and 26k) of the ortho posi-
tion or methyl group (26e, 26l) as substituent group (R2) of the
isoxazole 5-position side chain benzene ring was effective, show-
ing higher activities than the unsubstituted compound 26a. Of
these, 26k and 26l, which carried a 3-methyl group at R1,
change, p38
a
inhibitory activity decreased to about 1/8 (IC50
:
105 nM vs 792 nM) and TNF-
a
inhibitory activity to at least 1/20
(IC50: 490.0 nM vs >10,000 nM). The compound 26a, which was
obtained by condensing the phenylacetyl group with compound
25a, showed a significantly increased level of p38
activity, as well as TNF- inhibitory activity. In the pyrimidinylis-
oxazole form also, the 5-position substituent group effectively
showed affinity toward p38 . However, its activity was reduced
relative to that of pyridylisoxazole 21c. Thus, its p38 inhibitory
activity was reduced to about 1/2 (IC50: 45.6 nM vs 23.0 nM) and
TNF- inhibitory activity to about 1/5 (IC50 241.8 nM vs
a inhibitory
a
a
a
a
:
54.7 nM). However, surprisingly, in the metabolic stability test,
pyrimidinylisoxazole 26a showed a considerably improved meta-
bolic profile with an unchanged drug residual percentage of 5.6%
and because the activity of its anticipated metabolite 25a was
low, it was confirmed to be extremely suitable as an antedrug.
To further increase its activity, optimization was carried out using
26a as the lead compound.
showed very high TNF-
ual IC50 values exceeding p38
investigations have not been carried out, but this may be attributed
a
inhibition activities, with their individ-
a
inhibitory activities. Detailed
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12
K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 3
Biological activity and metabolic stability of 4-pyrimidinylisoxazole derivatives
O
N
N
HN
R2
O
N
R1
Compound
Structure
p38
a
MAP kinase
TNF-
a
Metabolic stability in human liver S9
(5 min, % of remaining)
R1
R2
IC50 (nM)
IC50 (nM)
26a
26b
26c
26d
26e
26f
26g
26h
26i
4-F
4-F
4-F
4-F
4-F
2-Cl
4-Cl
2,4-F
2,6-F
2,6-Cl
3-CH3
3-CH3, 4-F
H
2-F
45.6
33.2
6.88
10.9
8.43
141
10.8
2.73
294
241.8
100.6
15.5
49.1
93.9
522.8
33.6
22.9
1293.0
4242.0
0.78
5.6
10.0
5.8
2-Cl
2-F, 6-Cl
2,5-CH3
H
2-Cl
2-Cl
H
H
2-Cl
2-CH3
16.1
53.9
NTa
96.6
20.4
NTa
NTa
21.2
52.6
26j
26k
26l
1010
3.62
5.48
0.89
a
NT: not tested.
Table 4
to the lower selectivity of these compounds for other kinases [off
target, such as c-Jun N-terminal kinase (JNK)] that regulate the
production of inflammatory cytokines. From the perspective of
metabolic stability, a comparison of compounds having high
activity, obtained by introducing an ortho position substituent
at R2 (26c–26e, 26g, 26h, 26k, and 26l), showed a tendency of
undergoing stable metabolism as compared to the unsubstituted
compound (26a). All compounds, except 26g (percentage of
unchanged residues after 5 min: 96.6%), were metabolized to
about half or even more in 5 min in human liver S9 and
therefore, each of these compounds is at a level of fulfilling
the concept of an antedrug. Among these, 26c, 26d, 26h, and
Inhibitory effects of 26d and the principal metabolite 25a on p38 MAP kinase and JNK
isoforms
Compound
Kinase isoform IC50 (nM)
p38
10.9
792
a
p38b
p38c
p38d
JNK1
JNK2
JNK3
26d
25a
326
9714
NEa
NEa
NEa
NEa
NEa
NEa
198
1476
572
2199
To evaluate the selectivity profiles of 26d against protein kinases, the inhibitory
effects of 26d against 32 distinct kinases were studied other than Table 4. As those
kinases were; CSK, LCK, AKT2, CRIK, ROCK1, PKAC
a, PDK1, PKCa, RSK2, CaMK4,
CaMK2 , CHK1, DAPK1, MAPMAPK2, PIM1, CHK2, CDK2/cyclinA, GSK3b, SRPK1,
a
AurA, IKKb, NEK2, TTK, IRAK4, PHKG1, CK1d, PDK2, Erk1, Erk2, Erk5, MAP3K5 and
RAF1, 26d were inactive against them.
26k not only showed particularly excellent TNF-
a inhibition
a
NE: no effect.
(IC50: <50 nM), but also excellent metabolic stability (percentage
of unchanged residue after 5 min: <30%). Furthermore, after a
comprehensive investigation of CYP inhibition (unpublished data),
genotoxicity (negative Ames test), and drug efficacy, 26d (AKP-
001) was selected as the candidate for further development.29
To reduce the side effects of a p38 MAP kinase inhibitor, it is
essential to eliminate the class effect by optimizing the compound
to a profile that was suitable for an antedrug. At the same time, it is
necessary to increase selectivity for various kinds of kinases other
4.3. Docking studies
Compound 26d was docked to the ATP-binding site in the X-ray
crystal structure (PDB ID: 1bl7) of p38a MAP kinase obtained from
Protein Data Bank (PDB) by using the docking tool (CDOCKER) of
Discovery Studio (Fig. 4). In the resulting binding structure, a 4-
fluorophenyl site of 26d was detected in hydrophobic region I,
which consisted of Leu75, Leu86, Leu104, Val105, and Thr106.
Moreover, the 4-position nitrogen atom of pyrimidine became
the hydrogen bond acceptor of the Met109 principal chain N–H,
and the nitrogen atom of the isoxazole ring interacted with the ter-
minal amino group of Lys53 via hydrogen bonding. In the case of
the most interesting 5-position substituent, the 2-fluoro-6-chloro-
phenyl terminal site was situated in the hydrophobic region II,
which consisted of Met109, Ala111, Asp112, Ala157, and Leu167
and seem to be interacting with them by van der Waals forces.
These results were in accordance to the initial concept. The com-
pound 2-acylaminoimidazole derivative 27, reported by Bracht
et al.,29 presented a structure that was relatively similar to that
of 26d. In their docking model, the acyl site of 27 interacts with
than p38a and prevent the off-target drug effects. Accordingly, the
inhibitory effect of 26d and the presumed metabolite 25a against
various isoforms of p38 MAP kinase and against various tyrosine/
threonine kinases and serine/threonine kinases was evaluated
(Table 4). Analysis of the inhibitory effect of 26d against various
isoforms of p38 MAP kinase showed that the activity of p38
was the highest (IC50: 10.9 nM), followed by p38b, with about
1/30 of the activity, then p38 and p38d, which were inactive. In
a
c
the case of JNK, which is a kinase involved in inflammation in a
manner similar to that of p38 MAP kinase, an intermediate inhib-
itory activity was observed in JNK2 and JNK3. In addition, all were
inactive against the 32 types of kinases and 26d was confirmed as
the compound showing relatively higher p38a selectivity. Further-
more, although selectivity of the anticipated metabolite 25a for
each of the isoforms of p38 and JNK showed an overall tendency
that resembled 26d, in general, the inhibitory activity was low
compared to that of 26d.
the hydrophobic region II, and p38
after the introduction of this acyl site. However, the suitability of
27 for antedrug was not tested.
a inhibition increased 10-fold
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K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
13
Figure 4. Docking results for 26d in the p38
a
MAP kinase. X-ray structure 1bl7 is from the PDB database.
Table 5
PK parameters of 26d and its metabolites 25a in plasma after a single oral (30 mg/kg)
or intravenous (3 mg/kg) administration of 26d to male rats
Route
p.o.
25a
0.88 0.08 136 16.6
iv
Compound
26d
26d
25a
Cmax (ng/mL)
Tmax (h)
363 89a
744 111
0.5 0.0
3.33 1.15 3.33 1.15 0.08 0.0
t1/2 (h)
18.7 17.9 0.98 0.09 0.474 0.0028 0.428 0.037
AUC0ꢀ1 (ng h/
mL)
23.7 17.4 760 40.6
505 37.9
924 98.5
BAb (%)
<1
NAc
NAc
NAc
Each value represents the mean SD of three rats.
a
Plasma concentration at 5 min.
Oral bioavailability of 26d.
NA: not applicable.
b
c
value (Cmax: 136 ng/mL), but with a short half-life (t1/2) of 1 h or
less, which indicates that 25a is also rapidly metabolized. The
bioavailability of 26d after oral administration was 1% or less
and its systemic exposure has been estimated to be extremely
low. This confirms that 26d considerably reduces the undesired
Figure 5. Plasma concentrations of 26d and its metabolites 25a after a single oral
administration of 26d (30 mg/kg) to male rats.
side effects originating from p38
suits the concept of an antedrug.
a inhibition and this compound
4.4. Pharmacokinetics of 26d
The pharmacokinetics (PK) of an antedrug is particularly impor-
tant because it is directly linked to the onset of side effects. Figure 5
shows the plasma concentrations of 26d and its metabolites 25a
after a single oral administration of 26d (30 mg/kg) to male rats.
Table 5 shows the important PK parameters at that time, along
with the data for intravenous administration. After oral adminis-
tration, the maximum concentration of unchanged 26d in plasma
(Cmax) was 0.88 ng/mL. This was extremely low and confirms that
according to the strategy, it metabolizes rapidly into 25a after
absorption by cleavage of the 5-position amide bond. On the other
hand, the plasma concentration of 25a showed a relatively high
4.5. Drug efficacy evaluation of 26d
Compound 26d was evaluated by using animal models of two
types of IBD. In the dextran sulfate sodium (DSS)-induced colitis
mouse model, the shortening of the colon, which is a marker of
inflammation, was significantly inhibited at dose of 10 mg/kg or
more of 26d (Fig. 6). In a 2,4,6-trinitrobenzene sulfonic acid
sodium salt (TNBS)-induced colitis rat model, a dose of 10 mg/kg
or more of 26d significantly reduced the damage score (Fig. 7).
These results clearly show that 26d has a curative effect on colitis
in animal models of IBD.
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14
K. Hasumi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
efficacy was reduced and thus the so-called redundancy of MAP
kinase pathway was indicated30,31 On the other hand, although
the reason remains unclear, PH-797804 did not show any reduc-
tion in anti-inflammatory action even after being administered
over a long period and is currently undergoing phase III tests.16
Moreover, losmapimod and dilmapimod also have completed
phase II tests. The common factor among the 3 drugs is that
chronic obstructive pulmonary disease (COPD) is their target
illness, and the possibility of persistence of their anti-inflammatory
effect was influenced by either the disease or by the treated body
organs. In the case of BIRB796, a reduction in the anti-inflammatory
effect against IBD or digestive organs was observed, but because of
the lack of clinical cases as well as several unclear points, we are
only including the clinical results of 26d. We expect 26d to be
considered as a new and excellent anti-inflammatory drug with
reduced side effects and can be used in various organs (including
the respiratory organs).
Acknowledgments
Figure 6. Inhibitory effects of 26d and SASP on DSS-induced colitis in mice.
We thank Katsuya Ueno (Cosmo Bio Co., Ltd) as a mediator of SB
Drug Discovery in the biological testing. We are also grateful to
Hiroshi Uchida and Makoto Okada for the assistance in the docking
studies, Katsuyuki Keino and Akira Asagarasu for a comprehensive
support of writing this paper (each of ASKA Pharmaceutical Co.,
Ltd).
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