Synthesis and structure-activity relationships of 2-aminoacetamide derivatives as peroxisome proliferator-activated receptor α/γ dual agonists

Article abstract of DOI:10.1248/cpb.c15-00221

We describe the design, syntheses, and structure-activity relationships of novel zwitterionic compounds as nonthiazolidinedion-based peroxisome proliferator-activated receptor (PPAR) α/γ dual agonists. In our previous report, we obtained compound 1 showin

Full text of DOI:10.1248/cpb.c15-00221

Vol. 63, No. 8
Chem. Pharm. Bull. 63, 591–602 (2015)
591
Regular Article
Synthesis and Structure–Activity Relationships of 2-Aminoacetamide
Derivatives as Peroxisome Proliferator-Activated Receptor α/γ Dual
Agonists
,a
Yoshihiro Shibata,* Katsuji Kagechika,a Masahiro Ota,a Mitsuhiro Yamaguchi,a
Masaki Setoguchi,a Hideo Kubo,b Kiyoshi Chiba,a Hiromichi Takano,a Chiyuki Akiyama,a
Mayumi Ono,a Mina Nishi,a and Hiroyuki Usuia
a R&D Division, Daiichi Sankyo Co., Ltd.; 1–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan: and
b Biological Research Department, Daiichi Sankyo RD Novare Co., Ltd.; 1–16–13 Kitakasai, Edogawa-ku, Tokyo
134–8630, Japan.
Received March 10, 2015; accepted May 22, 2015
We describe the design, syntheses, and structure–activity relationships of novel zwitterionic compounds
as nonthiazolidinedion-based peroxisome proliferator-activated receptor (PPAR) α/γ dual agonists. In our
previous report, we obtained compound 1 showing potent PPARα/γ dual agonistic activities, together with
a sufficient glucose-lowering effect in db/db mice. However, this compound possessed an issue, i.e., the
1,3,4-oxadiazole ring was not stable in acidic conditions. Thus, we carried out further optimization to im-
prove the stability while maintaining the other favorable profile features including potent PPARα/γ dual ago-
nistic activity. We addressed the issue by changing the oxadiazole ring to a bioisostere amide group. These
amide derivatives were stable in acidic conditions and decreased plasma glucose and plasma triglyceride
levels significantly without marked weight gain.
Key words peroxisome proliferator-activated receptor; type 2 diabetes; bioisostere
In 2014, 4.9 million people died from diabetes.1) Diabetes is remedying effect without body weight gain at 10mg/kg in the
a serious health problem since the number of individuals with db/db mice. Moreover, the exchange of the ring system into
diabetes is 387 million at present and is estimated to be 592 this 1,3,4-oxadiazole ring reduced direct or mechanism-based
million by 2035.1) Of these cases, the populations of type 2 di- inhibitions (MBI) of CYP3A4 inducing drug–drug interaction
abetes currently account for >90% of all diabetes worldwide.2) risks, which our early furan derivatives posessed.11) Although
The type 2 diabetic people who have insulin resistance are these oxadiazole derivatives showed excellent profiles as
more susceptible to cardiovascular risk factors including dys- above, they were found to have a low stability in acidic media,
lipidemia, coagulopathy, hypertension and obesity.3) Further- which was not adequate for orally-administered agents. When
more, it has been revealed that type 2 diabetes with insulin
resistance and hyperinsulinemia is associated with the risk of
cancer in recent years.4) In fact, the prevention and the treat-
ment of type 2 diabetes becomes more and more important to
maintaining the quality of life of people all over the world.
Today, we have a wide variety of antidiabetic agents. Among
these, the use of the thiazolidinedione (TZD) insulin-sensitizer
makes sense for treating patients with type 2 diabetes since
TZD activates peroxisome proliferator-activated receptor
gamma (PPARγ) and leads to abated insulin resistance by pro-
moting hypertrophied adipocyte differentiation and improving
the balance of physiological active substances.5,6) However, its
activation also causes adverse effects including weight gain,
fluid retention and edema.7) On the other hand, the activa-
tion of another subtype PPARα has been identified to mediate
the lipid-lowering activity in studies with the fibrate class
of hypolipidemic drugs. Furthermore, some PPARα agonists
have been reported to reduce weight gain in rodents.8,9) Thus,
PPARα/γ dual agonists are expected to be a safer type 2 dia-
betic agent.
In our previous report,10) we obtained zwitterionic com-
pounds showing the PPARα/γ dual agonistic activities. In
Fig. 1. Profile of Compound 1
a) The value at pH 7.4. b) The % remaining value using human microsomes. c)
The % inhibition value at a 10µM concentration of compound 1. d) The % remain-
ing value at a 100µM concentration of compound 1 reacted with CYP3A4 probe
substrates after 30min preincubation in human liver microsomes.
particular, compound 1 provided highly potent dual activities
(α: EC50=0.55 nM, γ: EC50=18 nM, shown in Fig. 1), accompa-
nied by a pronounced glucose lowering effect and triglyceride
*To whom correspondence should be addressed. e-mail: shibata.yoshihiro.da@daiichisankyo.co.jp
© 2015 The Pharmaceutical Society of Japan

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Fig. 2. Time Course of Residual Compound 1 Contents in Various Aqueous Solutions at 37°C
Reagents and conditions: (a) Glyoxylic acid, NaBH(OAc)3, MeOH, 91%; (b) Corresponding commercially available amino reagents, EDCI, HOBt, DMF; (c) NaOH,
MeOH, 4a: 59% (2 steps), 4b: 66% (2 steps), 4c: 52% (2 steps), 4d: 36% (2 steps), 4e: 52% (2 steps), 4f: 43% (2 steps), 4g: 59% (2 steps), 4h: 30% (2 steps), 4i: 61% (2
steps).
Chart 1
the chemical stability of 1 was studied under Japanese Phar- and synthesized as depicted in Chart 1. The reductive al-
macopoeia (JP) XIV 1st fluid (JP1, pH 1.2), JP XIV 2nd fluid kylation of the secondary amine 211) with glyoxylic acid in
(JP2, pH 6.8) and Britton–Robinson buffer (pH 4.0) at 37°C, the presence of NaBH(OAc)3 in methanol gave a common
it was revealed that the 1,3,4-oxadiazole ring was decomposed key intermediate carboxylic acid, 3. The amidation reac-
in JP1 acidic solution (Fig. 2). It was considered that this sta- tion of 3 with the corresponding amine using 1-ethyl-3-(3-
bility was not tolerable for the oral administration. Thus, our dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and
medicinal strategy to address the issue consisted of a conver- N-hydroxybenzotriazole (HOBt) in N,N-dimethylformamide
sion of the 1,3,4-oxadiazole to a bioisostere, i.e., amide group, (DMF), followed by hydrolysis provided 4a–i.
which would ensure enough stability.
The syntheses of the (R)- or (S)-methyl group at the
We report herein the optimization of the side chain on the α-position introducing compounds (i.e., the alanyl amide
nitrogen atom and the lipophilic tail of our lead with an aim compounds) were depicted in Chart 2. The treatment of the
to improve the chemical stability and to obtain antidiabetic aldehyde 611) with alanine tert-butyl ester under reflux condi-
drugs without adverse effects.
tions to form the imine, followed by the reduction with NaBH4
gave the secondary amines 7a and 7b. The reductive alkyla-
tion of 7a and 7b with commercially available aldehyde using
Results and Discussion
Chemistry Firstly, in order to confirm the potential of NaBH(OAc)3 in methanol afforded 8a and 8b, respectively.
amide derivatives and to explore substituent effects on the The acidic hydrolysis of 8a and 8b, followed by the amidation
nitrogen of the amide group, compounds 4a–i were designed with methylamine under the conventional EDCI/HOBt condi-

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
593
Reagents and conditions: (a) (1) THF, reflux; (2) NaBH4, MeOH, 7a: 93%, 7b: 80%; (b) 5-Methyl-2-phenyl-1,3-oxazole-4-carbaldehyde, NaBH(OAc)3, MeOH, 8a: 58%,
8b: 73%; (c) (1) 4M HCl in dioxane, DCM; (2) methylamine, EDCI, HOBt, DMF, 9a: 78%, 9b: 27%; (d) NaOH, MeOH, 10a: 67%, 10b: 33%.
Chart 2
Reagents and conditions: (a) (1) 6, THF, reflux; (2) NaBH4, MeOH; (b) 4-(Chloromethyl)-5-methyl-2-phenyl-1,3-oxazole, K2CO3, MeCN, 87% (2 steps); (c) (1) 4M HCl in
dioxane, DCM; (2) methylamine, EDCI, HOBt, DMF, 96%; (d) NaOH, MeOH, 80%.
Chart 3
Reagents and conditions: (a) N-Boc-2-aminoacetaldehyde, NaBH(OAc)3, DCM, 71%; (b) (1) TFA, DCM; (2) AcCl, TEA; (3) NaOH, MeOH, 31% (2 steps).
Chart 4
tion gave 9a and 9b, respectively. Finally, the hydrolysis of 9a is shown in Chart 3. The treatment of the aldehyde 611) with
and 9b provided the carboxamides 10a and 10b, respectively.
commercially available 3-amino propionic acid tert-butyl ester
Synthesis of a one-carbon homologation compound, 15, 11 under reflux conditions to form the imine, followed by

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Reagents and conditions: (a) K2CO3, MeCN, 98%; (b) (1) TFA, DCM; (2) NH4Cl, EDCI, HOBt, DMF, 89%; (c) NaOH, MeOH, 46%.
Chart 5
the reduction with NaBH4 gave the secondary amine 12. The activities. Thus, we obtained encouraging results, showing
alkylation of 12 with 4-(chloromethyl)-5-methyl-2-phenyl-1,3- that the amide variants possessed the potential as PPARα/γ
oxazole in the presence of K2CO3 in acetonitrile gave 13. dual agonists.
Acidic catalyzed hydrolysis of the tert-butyl ester group of 13,
followed by amidation with methylamine provided 14. Finally, cal property by further optimization of compound 4a, whose
the hydrolysis of the ethyl ester group gave 15.
lipophilicity was slightly low to obtain a good oral exposure.
Then, we moved on to the balancing of the physicochemi-
Synthesis of reverse amide compound 17 is depicted in We introduced the methyl group at 4-position on the lipophilic
Chart 4. The reductive alkylation of the secondary amine tail as a promising substituent in the previous study.10) As a
211) with N-Boc-2-aminoacetaldehyde in the presence of result, our compound, 22 (Fig. 3), showed good activity and
NaBH(OAc)3 in dichloromethane gave 16. The deprotection of selectivity for PPARα (EC50=2.8nM, ten times stronger than
the Boc group of 16, followed by acetylation and hydrolysis EC50=26nM for PPARγ), which might be preferable to reduce
prepared 17.
the side effects of PPARγ activation, e.g., weight gain. Fur-
Compound 22 was synthesized as shown in Chart 5. This thermore, 22 showed good physicochemical properties such
compound was synthesized by using chloride 1810) and sec- as low lipophilicity, high metabolic stability and weak CYP
ondary amine 1910) in a similar manner to the synthetic ap- inhibition.
proach of compound 15.
With potent agonist 22 having a distinguished Absorption–
Biological Evaluation Novel compounds were evaluated Distribution–Metabolism–Excretion (ADME) profile, in vivo
in a cell-based transcription assay using GAL4-PPAR chi- studies were carried out; oral dosing was once daily for 10d in
meric receptors and plasmids for functional analysis-secreted db/db mice, an obese animal model of type 2 diabetes charac-
alkaline phosphatase (pFA-SEAP) as a reporter vector, and terized by severe insulin resistance and marked hypertriglyc-
the activities are reported as the EC50 value. The logD values eridemia (six per group). Rosiglitazone, which is a selective
were determined from the partition coefficient for 1-octanol/ PPARγ agonist, showed the effect of a significant decrease in
phosphate buffer saline (PBS) at pH 7.4. CYP3A4 direct plasma glucose and plasma triglyceride. However, this drug
inhibitory activity values were shown in % inhibition at a led to the adverse effects of an increase in the body weight on
10 µM concentration of the compounds for 60min incuba- account of only PPARγ activation. Compound 22, on the other
tion.12) The MBI values were shown in % remaining at a hand, showed a dose-dependent response and exhibited a good
100 µM concentration of the compounds reacted with CYP3A4 efficacy without marked weight gain at 1mg/kg as depicted
probe substrates after 30min preincubation in human liver in Fig. 4 and Table 2. Although the inhibitory activities of
microsomes.13,14) JP1 solubility was shown as a marker of acid 22 for PPARα and PPARγ in the in vitro assay were weaker
stability.
than those of 1, the physicochemical properties improved 22
The results of various amide derivatives are presented in showed good in vivo efficacy in the lower dose as compared
Table 1. We have had a lot of information of the structure– with 1, which showed the activity at 10mg/kg.10)
activity relationship (SAR) of non-substituted phenyloxazole
Furthermore, a comparative study of 22 versus Muraglitazar
derivatives such as compound 23. And we aimed to avoid (α: EC50=0.3µM, γ: EC50=0.1 µM)15) was performed in Zucker
false effects on activities from high lipophylicity. Thus, we Fatty rats, which develop type 2 diabetes with obesity, hy-
undertook SAR analysis of the amide group on the linker with perglycemia and hyperinsulinemia. The results are displayed
low lipophilicity showing a non-substituted phenyloxazole in Table 3. While both compounds showed a similar and sig-
group at the lipophilic tail. The derivatives 4a–c, which had nificant blood glucose lowering effect and promoted triglyc-
unsubstituted or lower alkyl amide groups, showed acceptable eride decreasing, compound 22, a PPARα dominant variant,
agonistic activity. However, bulky amide compounds 4d–g produced less weight gain than Muraglitazar (dosed orally at
showed weak activities. Among these, methoxyethyl amide 10mg/kg in Zucker Fatty Rat [six per group] for 13d).
4f or thiazolyl amide 4g gave low selectivity for PPARα. The
cyclic amino group introduced amide compounds 4h and i
Conclusion
also showed weak activities. The introduction of the methyl
We described the design, syntheses and evaluation of novel
group at the α-position of the amide (10a, b), a one-carbon zwitterionic compounds as PPARα/γ dual agonists. The issue
homologation of the methylene chain to the ethylene one (15) faced by the 1,3,4-oxadiazole derivatives, e.g., low stability
or the reverse of the amide group (17) largely decreased the under acidic conditions, was addressed by changing the oxa-

Vol. 63, No. 8 (2015)
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595
Table 1. In Vitro Activity and Physicochemical Properties of Compound 1 and Amide Compounds
CYP3A4
Solubilitye)
PPARα EC50
PPARγ EC50
Compd
R
LogDa)
Direct inhibitionb) MBI remainingc)
(nM)
(nM)
JP1 (µg/mL)
(%)
(%)
23f)
2.9
33
1.1
28
110
Decomposed
4a
4b
41
25
92
53
0.8
0.9
<10
98
89
>740
>760
26
4c
24
27
110
230
1.1
1.8
16
21
122
>720
>640
4d
NTd)
4e
4f
15
280
470
1.4
1.0
39
19
NT
NT
>890
>920
340
4g
4h
250
150
67
2.1
1.1
75
51
NT
NT
810
220
>690
4i
60
340
2.2
37
NT
>810

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Table 1. Continued.
Table 1. In Vitro Activity and Physicochemical Properties of Compound 1 and Amide Compounds
CYP3A4
Solubilitye)
PPARα EC50
PPARγ EC50
Compd
R
LogDa)
Direct inhibitionb) MBI remainingc)
(nM)
(nM)
JP1 (µg/mL)
(%)
(%)
10a
1500
620
180
290
2100
1400
1500
1100
1.5
22
NT
>700
>780
>860
>570
10b
15
1.6
0.8
1.0
22
41
27
86
NT
NT
17
a) The logD values were determined from the partition coefficient for 1-octanol/phosphate buffer saline (PBS) at pH 7.4. b) The CYP3A4 direct inhibition values were
shown in % inhibition at a 10µM concentration of the compounds for 60min incubation. c) The MBI values were shown in % remaining at 100µM concentration of the com-
pounds reacted with CYP3A4 probe substrates after 30min preincubation in human liver microsomes. d) Not tested. e) Water solubility was measured at JP1. 1 was decom-
posed. Amide derivatives were all stable under the acidic condition. f) Ref. 10.
ing orally-administered agents. Moreover, we are certain that
PPARα dominant dual agonistic activity is necessary in order
to reduce side effects of PPARγ activation by comparative
study of the PPARγ dominant dual agonist. Finally, we are
looking forward to contributing to improving the quality of
life of patients by this PPARα/γ dual agonist.
Experimental
Chemistry Unless otherwise noted, materials were ob-
tained from commercial suppliers and used without further
purification. 1H-NMR spectra were determined on a JEOL
JNM-EX400 spectrometer. Chemical shifts are reported in
parts per million relative to tetramethylsilane (TMS) as an
internal standard. Significant 1H-NMR data are tabulated
in the following order: number of protons, multiplicity (s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,
broad), and coupling constant(s) in hertz. Electron spray
ionization condition (ESI) mass spectra were recorded on
an Agilent 1100 and SCIEX API-150EX spectrometer. Fast
Fig. 3. Profile of Compound 22
atom bombardment ionization condition (FAB) mass spectra
a) The value at pH 7.4. b) The % remaining value using human microsomes. c)
The % inhibition value at a 10µM concentration of compound 22. d) The % remain-
impact ionization condition (EI) mass spectra were recorded
ing value at a 100µM concentration of compound 22 reacted with CYP3A4 probe
were recorded on a JEOL JMSHX110 spectrometer. Electron
on a JEOL JMS-AX505W. Column chromatography was per-
substrates after 30min preincubation in human liver microsomes.
formed with Merck silica gel 60 (particle size 0.060–0.200
diazole ring to amide groups. Amide variant 22 exhibited the or 0.040–0.063mm). Flash column chromatography was per-
effects of a significant decrease in plasma glucose and plasma formed with Biotage FLASH Si packed columns. Thin layer
triglyceride levels without weight gain. The agonistic activi- chromatography (TLC) was performed on Merck pre-coated
ties of amide derivatives are lower compared to the oxadiazole TLC glass sheets with silica gel 60 F254, and compound visu-
derivatives previously reported. However, we are convinced alization was effected with a 5% solution of phosphomolybdic
that this stable amide variant has superior quality for target- acid in ethanol, UV lamp, iodine, or Wako ninhydrin spray.

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
597
phenoxy]-2-methylpropanoate (2) (3.0g, 6.9mmol) in methanol
(30mL), glyoxylic acid monohydrate (0.84g, 8.9mmol) and
sodium triacethoxyborohydride (2.3g, 10mmol) were added.
The mixture was stirred at room temperature overnight. After
the solvent was removed in vacuo, the residue was dissolved
in ethyl acetate (EtOAc), washed with water and brine, dried
over Na2SO4 and concentrated. The crude product was puri-
fied by column chromatography on silica gel (chloroform/
methanol=19/1 as eluent, v/v) to provide 3 as a colorless solid
(3.1g, 91%): MS (ESI) m/z: 495 (M+H)+.
General Procedure for 2-{4-[({2-[(Substituted)-amino]-
2-oxoethyl}[(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]-
amino)methyl]-2,6-dimethylphenoxy}-2-methylpropanoic
Acid (4a–i) To a solution of 3 in DMF or acetonitrile
(MeCN), commercially available amines, EDCI and HOBt
were added. The mixture was stirred at room temperature
overnight. After the solvent was removed in vacuo, the residue
was dissolved in EtOAc, washed with water and brine, dried
over Na2SO4 and concentrated. The crude product was puri-
fied by column chromatography on silica gel (hexane/EtOAc
as eluent) to provide 4a–i ethyl ester form. To a mixture of
4a–i ethyl ester form and methanol was added NaOH (1M
aqueous solution) and stirred at 50°C overnight. After HCl
(1 M aqueous solution) was added to the reaction mixture, the
solvent was removed in vacuo. After EtOAc was added to
the residue, the mixture was washed with water and brine,
dried over Na2SO4 and concentrated. The crude product was
purified by column chromatography on silica gel (chloroform/
methanol). The residue was triturated with EtOAc–hexane or
freeze-dried from 1,4-dioxane to provide 4a–i.
2-[4-({(2-Amino-2-oxoethyl)[(5-methyl-2-phenyl-1,3-
oxazol-4-yl)methyl]amino}methyl)-2,6-dimethylphenoxy]-
2-methylpropanoic Acid (4a): This compound was obtained
as a colorless solid in 59% yield by using 3 (0.28mmol),
NH4Cl (0.42mmol), N-methylmorpholine (NMM) (0.42mmol),
EDCI (0.42mmol), HOBt (0.42mmol), MeCN (2.0mL) and
1
NaOH (4.5eq): H-NMR (400MHz, DMSO-d6) δ: 1.32 (6H,
s), 2.13 (6H, s), 2.24 (3H, s), 3.07 (2H, s), 3.53 (2H, s), 3.57
(2H, s), 7.15 (1H, brs), 7.31 (1H, brs), 7.49–7.53 (3H, m),
7.91–7.94 (2H, m). MS (ESI) m/z: 466 (M+H)+. Anal. Calcd
for C26H31N3O5·0.5H2O·0.2EtOAc: C, 65.40; H, 6.88; N, 8.54.
Found: C, 65.22; H, 6.78; N, 8.16.
2-[2,6-Dimethyl-4-({[2-(methylamino)-2-oxoethyl][(5-
methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}methyl)-
phenoxy]-2-methylpropanoic Acid (4b): This compound
was obtained as a colorless solid in 66% yield by using
3
(5.9mmol), methylamine (8.9mmol), EDCI (12mmol),
1
HOBt (12mmol), DMF (10mL) and NaOH (2.9eq): H-NMR
(400MHz, DMSO-d6) δ: 1.31 (6H, s), 2.12 (6H, s), 2.22 (3H,
s), 2.60 (3H, d, J=4.6Hz), 3.09 (2H, s), 3.50 (2H, s), 3.56
(2H, s), 6.99 (2H, s), 7.48–7.53 (3H, m), 7.74–7.78 (1H, m),
7.91–7.93 (2H, m). MS (ESI) m/z: 480 (M+H)+. Anal. Calcd
for C27H33N3O5: C, 67.62; H, 6.94; N, 8.76. Found: C, 67.57; H,
Fig. 4. Plasma Glucose Decrease Test in db/db Mice
Blood glucose change (A), triglyceride change (B) and body weight change (C)
were conducted with 10d of treatment.
Elemental analysis was performed using a PerkinElmer, Inc. 7.09; N, 8.56.
CHNS/O 2400II or a Yokokawa Analysis IC7000RS, and
2-[4-({[2-(Dimethylamino)-2-oxoethyl][(5-methyl-2-
analytical results were within 0.4% of the theoretical values phenyl-1,3-oxazol- 4-yl)methyl]amino}methyl)-2,6-
unless otherwise noted.
2N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)oxy]-3,5- pound was obtained as HCl salts of a colorless solid in 52%
dimethylbenzyl}-N-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)- yield by using (4.5mmol), dimethylamine (6.7mmol),
dimethylphenoxy]-2-methylpropanoic Acid (4c): This com-
3
methyl]glycine (3) To a solution of ethyl 2-[2,6-dimethyl-4- EDCI (8.9mmol), HOBt (8.9mmol), DMF (10mL) and NaOH
({[(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}methyl)- (2.9eq), and being hydrochlorinated with HCl (4M dioxane

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Chem. Pharm. Bull.
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BW change (%)
Table 2. Parameters of in Vivo Study with 22 on db/db Mice
Plasma glucosea)
Plasma triglyceridea)
(mg/dL)
Exp.
1
Compd
Dose (mg/kg)
Change (%)
Change (%)
(mg/dL)
Vehicle
22
563 7.6
227 15
223 17
251 36
67 5.4
77 6.2
1
−40b)
−40b)
−73b)
−69b)
0.0
+9.9b)
Rosiglitazone
10
a) Mean standard deviation (n=6). b) p<0.001 versus vehicle control (t-test).
Table 3. Parameters of in Vivo Study with 22 on Zucker Fatty Rat
Plasma glucosea)
Plasma triglyceridea)
(mg/dL)
Exp.
1
Compd
Dose (mg/kg)
Change (%)
Change (%)
BW change (%)
(mg/dL)
Vehicle
22
191 26
88 9.5
82 5.2
274 39
89 5.8
83 11
591 97
133 13
175 18
350 32
132 6.8
147 11
10
10
−53b)
−57b)
−77
−70
+2.3
Rosiglitazone
Vehicle
+11c)
2
Muraglitazar
Rosiglitazone
10
10
−68b)
−70b)
−62b)
−58b)
+9.7
+8.7
a) Mean standard deviation (n=6). b) p<0.01. versus vehicle control (t-test).
solution): 1H-NMR (400MHz, DMSO-d6) δ: 1.36 (6H, s), (2H, m), 3.32–3.35 (2H, m), 3.52 (2H, s), 3.57 (2H, s), 6.99
2.18 (6H, s), 2.42 (3H, s), 2.84 (3H, s), 2.89 (3H, s), 4.11–4.40 (2H, s), 7.49–7.54 (3H, m), 7.85 (1H, t, J=6.1Hz), 7.94–7.96
(6H, m), 7.31 (2H, s), 7.54–7.59 (3H, m), 7.97–7.99 (2H, m). (2H, m). MS (ESI) m/z: 524 (M+H)+. Anal. Calcd for
MS (ESI) m/z: 494 (M+H)+. Anal. Calcd for C28H35N3O5·H C29H37N3O6·0.5H2O: C, 65.40; H, 7.19; N, 7.89. Found: C,
Cl·1.5H2O·0.2dioxane: C, 60.19; H, 7.12; N, 7.31. Found: C, 65.49; H, 6.84; N, 7.73.
60.15; H, 6.74; N, 7.36.
2-[2,6-Dimethyl-4-({[(5-methyl-2-phenyl-1,3-oxazol-4-yl)-
2-[4-({[2-(Ethylamino)-2-oxoethyl][(5-methyl-2-phenyl-1,3- methyl][2-oxo-2-(1,3-thiazol-2-ylamino)ethyl]amino}methyl)-
oxazol-4-yl)methyl]amino}methyl)-2,6-dimethylphenoxy]- phenoxy]-2-methylpropanoic Acid (4g): This compound
2-methylpropanoic Acid (4d): This compound was obtained was obtained as a colorless solid in 59% yield by using
as HCl salts of a colorless solid in 36% yield by using 3
3
(0.28mmol), 1,3-thiazol-2-amine (0.42mmol), EDCI
(0.40mmol), ethylamine (0.81mmol), EDCI (0.81mmol), HOBt (0.42mmol), HOBt (0.42mmol), DMF (2.0mL) and NaOH
(0.81mmol), DMF (2.0mL) and NaOH (6.9eq), and being (7.8eq): 1H-NMR (400MHz, DMSO-d6) δ: 1.32 (6H, s),
hydrochlorinated with HCl (4M EtOAc solution): 1H-NMR 1.43–1.49 (1H, m), 1.73–1.83 (3H, m), 2.13 (6H, s), 2.25 (3H,
(400MHz, DMSO-d6) δ: 0.99 (3H, t, J=7.1Hz), 1.36 (6H, s), 3.06–3.10 (1H, m), 3.12 (2H, s), 3.16–3.24 (1H, m), 3.53 (2H,
s), 2.18 (6H, s), 2.41 (3H, s), 3.08 (3H, s), 3.79–3.90 (2H, m), s), 3.57 (2H, s), 3.60–3.64 (1H, m), 3.72–3.85 (2H, m), 6.99
4.01–4.06 (2H, m), 4.24–4.39 (4H, m), 7.27 (2H, s), 7.54–7.57 (2H, s), 7.49–7.54 (3H, m), 7.87 (1H, t, J=5.8Hz), 7.94–7.96
(3H, m), 8.00–7.96 (2H, m). MS (ESI) m/z: 494 (M+H)+. Anal. (2H, m). MS (ESI) m/z: 549 (M+H)+. Anal. Calcd for
Calcd for C28H35N3O5·HCl·H2O·0.2EtOAc·0.1hexane: C, C29H32N4O5S·0.75H2O·0.1dioxane: C, 61.84; H, 6.05; N, 9.81;
61.49 H, 7.20; N, 7.32. Found: C, 61.75; H, 7.12; N, 6.97.
2-[4-({[2-(Cyclopropylamino)-2-oxoethyl][(5-methyl-2-
S, 5.62. Found: C, 61.62; H, 5.69; N, 9.44; S, 5.51.
2-[4-({[2-(Azetidin-1-yl)-2-oxoethyl][(5-methyl-2-phenyl-1,3-
phenyl-1,3-oxazol- 4-yl)methyl]amino}methyl)-2,6- oxazol-4-yl)methyl]amino}methyl)-2,6-dimethylphenoxy]-
dimethylphenoxy]-2-methylpropanoic Acid (4e): This com- 2-methylpropanoic Acid (4h): This compound was obtained
pound was obtained as a colorless solid in 52% yield by as HCl salts of a colorless solid in 30% yield by using
using 3 (0.28mmol), cyclopropylamine (0.42mmol), EDCI
3 (0.28mmol), azetidine hydrochloric acid (0.47mmol),
(0.42mmol), HOBt (0.42mmol), DMF (2.0mL) and NaOH NMM (0.85mmol), EDCI (0.56mmol), HOBt (0.56mmol),
(9.5eq): 1H-NMR (400MHz, DMSO-d6) δ: 0.36–0.40 (2H, DMF (5.0mL) and NaOH (11eq), and being hydrochlori-
m), 0.59–0.63 (2H, m), 1.32 (6H, s), 2.11 (6H, s), 2.25 (3H, nated with HCl (4M dioxane solution): 1H-NMR (400MHz,
s), 2.66–2.68 (1H, m), 3.06 (2H, s), 3.53 (2H, s), 3.57 (2H, DMSO-d6) δ: 1.36 (6H, s), 2.16–2.21 (8H, m), 2.41 (3H, s),
s), 6.94 (2H, s), 7.50–7.55 (3H, m), 7.83 (1H, d, J=4.2Hz), 3.31–3.40 (4H, m), 3.84–3.90 (2H, m), 4.03–4.07 (2H, m),
7.92–7.95 (2H, m). MS (ESI) m/z: 506 (M+H)+. Anal. Calcd 4.20–4.32 (2H, m), 7.24 (2H, s), 7.54–7.59 (3H, m), 7.97–8.00
for C29H35N3O5·0.75H2O: C, 67.10; H, 7.09; N, 8.09. Found: C, (2H, m). MS (ESI) m/z: 506 (M+H)+. Anal. Calcd for
67.21; H, 6.69; N, 7.93.
C29H35N3O5·HCl·2H2O·0.25EtOAc: C, 60.04; H, 7.05; N, 7.00.
2-{4-[({2-[(2-Methoxyethyl)amino]-2-oxoethyl}[(5-methyl-2- Found: C, 60.08; H, 6.72; N, 6.60.
phenyl-1,3-oxazol- 4-yl)methyl]amino)methyl]-2,6-
2-[2,6-Dimethyl-4-({[(5-methyl-2-phenyl-1,3-oxazol-4-
dimethylphenoxy}-2-methylpropanoic Acid (4f): This com- yl)methyl][2-oxo-2-(piperidin-1-yl)ethyl]amino}methyl)-
pound was obtained as a colorless solid in 43% yield by phenoxy]-2-methylpropanoic Acid (4i): This compound was
using 3 (0.28mmol), 2-methoxyethylamine (0.42mmol), EDCI obtained as HCl salts of a colorless solid in 61% yield by using
(0.42mmol), HOBt (0.42mmol), DMF (2.0mL) and NaOH 3 (0.40mmol), piperidine (0.81mmol), EDCI (0.81mmol),
1
(12eq): H-NMR (400MHz, DMSO-d6) δ: 1.33 (6H, s), 2.13 HOBt (0.81mmol), DMF (2.0mL) and NaOH (5.6eq), and
(6H, s), 2.24 (3H, s), 3.11 (2H, s), 3.22 (3H, s), 3.23–3.26 being hydrochlorinated with HCl (4M dioxane solution):

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
599
1H-NMR (400MHz, DMSO-d6) δ: 1.56–1.33 (6H, m), 2.18 (6H, dichloromethane (3.0mL) and sodium triacethoxyborohydride
1
s), 2.42 (3H, s), 3.56–3.58 (4H, m), 4.17–4.42 (6H, m), 7.31 (2H, (0.76mmol): H-NMR (400MHz, CDCl3) δ: 1.31–1.36 (6H, m),
s), 7.55–7.58 (3H, m), 7.96–7.99 (2H, m). MS (ESI) m/z: 534 1.42 (6H, s), 1.51 (9H, s), 2.14 (6H, s), 2.26 (3H, s), 3.51–3.77
(M+H)+. Anal. Calcd for C31H39N3O5·HCl·2H2O·0.25EtOAc: (5H, m), 4.27 (2H, q, J=7.1Hz), 6.94 (2H, s), 7.38–7.43 (3H,
C, 61.18; H, 7.38; N, 6.69. Found: C, 61.08; H, 7.28; N, 6.35.
General Procedure for tert-Butyl N-{4-[(1-Ethoxy-
m), 7.96–7.99 (2H, m). MS (ESI) m/z: 565 (M+H)+.
General Procedure for Ethyl 2-[2,6-Dimethyl-4-({[1-
2-methyl-1-oxopropan-2-yl)oxy]-3,5-dimethylbenzyl}alani- (methylamino)-1-oxopropan-2-yl][(5-methyl-2-phenyl-1,3-
nate (7a, b) The mixture of tert-butyl alaninate 5 (a or b), oxazol-4-yl)methyl]amino}methyl)phenoxy]-2-methylpro-
ethyl 2-(4-formyl-2,6-dimethylphenoxy)-2-methylpropanoate panoate (9a, b) To a solution of 8 (a or b) in dichlorometh-
(6) and triethylamine (TEA) in tetrahydrofuran (THF) was ane, HCl (4M dioxane solution) was added and stirred at room
stirred under reflux conditions. After the insoluble matter was temperature overnight. After the solvent was removed in
removed by filtration through celite, the filtrate was concen- vacuo, the residue was dissolved in DMF. Methylamine hydro-
trated. After the residue was dissolved in methanol, sodium chloric acid, NMM, EDCI and HOBt was added to the reac-
borohydride was added to the solution and the mixture was tion solution and stirred at room temperature overnight. After
stirred at room temperature. After the solvent was removed in EtOAc was added to the residue, the mixture was washed
vacuo, the residue was dissolved in EtOAc, washed with satu- with water, 10% citric acid aqueous solution, satd. sodium bi-
rated sodium bicarbonate aqueous solution and brine, dried carbonate aqueous solution and brine, dried over Na2SO4 and
over Na2SO4 and concentrated to provide 7 (a or b).
tert-Butyl N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)- matography on silica gel (hexane/EtOAc as eluent) to provide
oxy]-3,5-dimethylbenzyl}-D-alaninate (7a): This compound 9 (a or b).
concentrated. The crude product was purified by column chro-
was obtained as a pale yellow oil in 93% yield by using
Ethyl 2-[2,6-Dimethyl-4-({[(2R)-1-(methylamino)-1-oxopro-
L-alanine tert-butyl ester hydrochloride (5a, 1.7mmol), 6 pan-2-yl][(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}-
(1.1mmol), TEA (1.7mmol), THF (10mL), sodium borohydride methyl)phenoxy]-2-methylpropanoate (9a): This compound
(1.1mmol) and methanol (5.0mL): MS (ESI) m/z: 394 (M+H)+. was obtained as a colorless oil in 78% yield by using 8a
tert-Butyl
N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)- (0.28mmol), HCl (4M dioxane solution, 2.0mL), dichlorometh-
oxy]-3,5-dimethylbenzyl}-L-alaninate (7b): This compound ane (5.0mL), DMF (5.0mL), methyl amine hydrochloric acid
was obtained as a pale yellow oil in 80% yield by using (0.43mmol), NMM (0.43mmol), EDCI (0.43mmol) and HOBt
D-alanine tert-butyl ester hydrochloride (5b, 1.7mmol), 6 (0.43mmol): MS (ESI) m/z: 522 (M+H)+.
(1.1mmol), TEA (1.7mmol), THF (10mL), sodium borohydride
Ethyl 2-[2,6-Dimethyl-4-({[(2S)-1-(methylamino)-1-oxopro-
1
(1.1mmol) and methanol (5.0mL): H-NMR (400MHz, CDCl3) pan-2-yl][(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}-
δ: 1.28 (3H, d, J=6.9Hz), 1.35 (3H, t, J=7.1Hz), 1.45 (6H, methyl)phenoxy]-2-methylpropanoate (9b): This compound
s), 1.49 (9H, s), 2.18 (6H, s), 3.28–3.22 (1H, m), 3.52 (1H, d, was obtained as a colorless oil in 27% yield by using 8b
J=12.3Hz), 3.66 (1H, d, J=12.3Hz), 4.29 (2H, q, J=7.1Hz), (0.35mmol), HCl (4M dioxane solution, 5.0mL), dichloro-
6.93 (2H, s). MS (ESI) m/z: 394 (M+H)+.
methane (5.0mL), DMF (5.0mL), methyl amine hydrochloric
General Procedure for tert-Butyl N-{4-[(1-Ethoxy- acid (0.43mmol), NMM (0.43mmol), EDCI (0.43mmol) and
2-methyl-1-oxopropan-2-yl)oxy]-3,5-dimethylbenzyl}alani- HOBt (0.43mmol): H-NMR (400MHz, CDCl3) δ: 1.31–1.35
1
nate (8a, b) To a solution of tert-butyl N-{4-[(1-ethoxy-2- (6H, m), 1.42 (6H, s), 2.11 (6H, s), 2.22 (3H, s), 2.91 (3H, d,
methyl-1-oxopropan-2-yl)oxy]-3,5-dimethylbenzyl}alaninate
7
J=4.9Hz), 3.31–3.61 (5H, m), 4.26 (2H, q, J=7.1Hz), 6.84
(a or b) and 5-methyl-2-phenyl-1,3-oxazole-4-carbaldehyde in (2H, s), 7.43–7.47 (3H, m), 7.99–8.01 (2H, m), 8.50 (1H, s). MS
dichloromethane, sodium triacethoxyborohydride was added (ESI) m/z: 522 (M+H)+.
and stirred at room temperature overnight. After EtOAc was
General Procedure for N-{4-[(1-Ethoxy-2-methyl-
added to the residue, the mixture was washed with water and 1-oxopropan-2-yl)oxy]-3,5-dimethylbenzyl}-N-[(5-meth-
brine, dried over Na2SO4 and concentrated. The crude product yl-2-phenyl-1,3-oxazol-4-yl)methyl]alanine (10a, b) To a
was purified by column chromatography on silica gel (hexane/ mixture of 9 (a or b) in methanol, NaOH (1M aqueous solu-
EtOAc as eluent) to provide 8 (a or b).
tion) was added and stirred under reflux conditions. After
tert-Butyl N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)oxy]- HCl (1M aqueous solution) was added to the reaction mixture,
3,5-dimethylbenzyl}-N-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)- the solvent was removed in vacuo. After EtOAc was added
methyl]-D-alaninate (8a): This compound was obtained to the residue, the mixture was washed with water and brine,
as a colorless oil in 58% yield by using 7a (0.51mmol), dried over Na2SO4 and concentrated. The crude product was
5-methyl-2-phenyl-1,3-oxazole-4-carbaldehyde
(0.51mmol), purified by column chromatography on silica gel (chloroform/
dichloromethane (3.0mL) and sodium triacethoxyborohydride methanol as eluent). The residue was hydrochlorinated with
(0.76mmol): 1H-NMR (400MHz, CDCl3) δ: 1.31–1.36 (6H, HCl (4M dioxane solution), and then concentrated. Recrystal-
m), 1.42 (6H, s), 1.51 (9H, s), 2.14 (6H, s), 2.26 (3H, s), 3.65 lization from EtOAc–hexane produced 10 (a or b) HCl salts.
(5H, dq, J=56.2, 14.0Hz), 4.27 (2H, q, J=7.1Hz), 6.94 (2H,
s), 7.38–7.43 (3H, m), 7.96–7.99 (2H, m). MS (ESI) m/z: 565 2-yl][(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}-
(M+H)+.
methyl)phenoxy]-2-methylpropanoic Acid (10a): This com-
2-[2,6-Dimethyl-4-({[(2R)-1-(methylamino)-1-oxopropan-
tert-Butyl N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)oxy]- pound was obtained as a colorless solid in 67% yield by using
3,5-dimethylbenzyl}-N-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)- 9a (0.21mmol), methanol (5.0mL) and NaOH (1M aqueous
methyl]-L-alaninate (8b): This compound was obtained solution, 2.0mL): 1H-NMR (400MHz, DMSO-d6) δ: 1.34
as a colorless oil in 73% yield by using 7a (0.51mmol), (6H, s), 2.14 (6H, s), 2.69 (3H, d, J=4.2Hz), 3.10–3.61 (4H,
5-methyl-2-phenyl-1,3-oxazole-4-carbaldehyde
(0.51mmol), m), 3.97–4.41 (4H, m), 7.06–7.60 (5H, m), 7.97–7.99 (2H, m),

600
Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
8.23–8.75 (1H, m). MS (ESI) m/z: 494 (M+H)+. Anal. Calcd d, J=4.9Hz), 2.74–2.83 (2H, m), 3.49 (2H, s), 3.56 (2H, s),
for C28H35N3O5·HCl·2H2O: C, 59.41; H, 7.12; N, 7.42. Found: 4.24–4.33 (2H, m), 6.90 (2H, s), 7.39–7.49 (3H, m), 7.95–8.04
C, 59.58; H, 6.91; N, 7.21.
2-[2,6-Dimethyl-4-({[(2S)-1-(methylamino)-1-oxopropan-
(2H, m), 8.26–8.37 (1H, m). MS (ESI) m/z : 522 (M+H)+.
2-[2,6-Dimethyl-4-({[3-(methylamino)-3-oxopropyl][(5-
2-yl][(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}- methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}methyl)-
methyl)phenoxy]-2-methylpropanoic Acid (10b): This com- phenoxy]-2-methylpropanoic Acid (15) To a solution of
pound was obtained as a colorless solid in 33% yield by using 14 (0.30g, 0.58mmol) in THF (5.0mL), NaOH (0.5M aqueous
9b (0.096mmol), methanol (5.0mL) and NaOH (1M aqueous solution, 5.0mL) was added and stirred under reflux condition
solution, 2.0mL): 1H-NMR (400MHz, DMSO-d6) δ: 1.34 overnight. After the reaction mixture was cooled to room tem-
(6H, s), 2.14 (6H, s), 2.69 (6H, d, J=4.7Hz), 3.03–3.73 (4H, perature, HCl (1M aqueous solution, 2.5mL) and water were
m), 3.98–4.57 (4H, m), 7.06–7.63 (5H, m), 7.96–8.00 (2H, m), added and the organics was extracted with dichloromethane.
8.41–8.54 (1H, m). MS (ESI) m/z: 494 (M+H)+. Anal. Calcd The organic layer was dried over Na2SO4 and concentrated.
for C28H35N3O5·HCl·0.75H2O·0.2EtOAc: C, 62.14; H, 6.99; N, The residue was purified by column chromatography on silica
7.55. Found: C, 62.26; H, 7.09; N, 7.22.
gel (dichloromethane/methanol=95/5 then 91/9 as eluent, v/v)
tert-Butyl N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)- to provide 15 as a colorless solid (0.24g, 80%): 1H-NMR
oxy]-3,5-dimethylbenzyl}-β-alaninate (12) To a solution of (400MHz, CDCl3) δ: 1.48 (6H, s), 2.20 (6H, s), 2.28 (3H, s),
6 (0.19g, 0.72mmol) in THF (5.0mL), 11 (0.16g, 0.86mmol) 2.50–2.61 (2H, m), 2.73 (3H, d, J=4.6Hz), 2.82–2.93 (2H,
and TEA (0.12µL, 0.86mmol) were added and stirred under m), 3.62 (2H, s), 3.69 (2H, s), 6.96 (2H, s), 7.40–7.49 (3H, m),
reflux condition for 3.5h. After the solvent was removed in 7.96–8.03 (2H, m), 8.25–8.37 (1H, m). IR attenuated total
vacuo, the residue was dissolved in methanol (5.0mL). Sodium reflectance (ATR) cm−1: 3276, 2991, 2917, 2817, 1720, 1637,
borohydride (40mg, 1.1mmol) was added to the reaction solu- 1556, 1482, 1448, 1407, 1382, 1361, 1338, 1301, 1286, 1214,
tion at 0°C and stirred at the same temperature for 40min. 1133, 1068, 1025. MS (ESI) m/z: 494 (M+H)+. Anal. Calcd
After water and satd. sodium bicarbonate aqueous solution for C28H35N3O5·0.5H2O: C, 66.91; H, 7.22; N, 8.36. Found: C,
were added to the reaction mixture, the organics were ex- 67.12; H, 7.35; N, 8.13.
tracted with dichloromethane. The organic layer was dried
Ethyl 2-{4-[({2-[(tert-Butoxycarbonyl)amino]ethyl}[(5-meth-
over Na2SO4 and concentrated to provide 12 (0.30g, crude yl-2-phenyl-1,3-oxazol-4-yl)methyl]amino)methyl]-2,6-
product). This compound was used at the next step without dimethylphenoxy}-2-methylpropanoate (16) To a solution
further purification.
of 2 (0.43g, 0.98mmol) and tert-butyl (2-oxoethyl)carbamate
tert-Butyl N-{4-[(1-Ethoxy-2-methyl-1-oxopropan-2-yl)- (0.28g, 1.7mmol) in dichloromethane (10mL), sodium triace-
oxy]-3,5-dimethylbenzyl}-N-[(5-methyl-2-phenyl-1,3-oxa- thoxyborohydride (0.49g, 0.84mmol) was added and stirred
zol-4-yl)methyl]-β-alaninate (13) To
a solution of 12 at room temperature for 16h. After the reaction mixture
(0.30g, ca. 0.72mmol) and 4-(chloromethyl)-5-methyl-2-phe- was diluted with EtOAc, the mixture was washed with satd.
nyl-1,3-oxazole (0.21g, 1.0mmol) in MeCN (6.0mL), K2CO3 sodium bicarbonate aqueous solution and brine, dried over
(0.15g, 1.1mmol) was added and stirred at 70°C for 2d. After Na2SO4 and concentrated. The residue was purified by column
the insoluble matter was removed by filtration through celite, chromatography on silica gel (hexane/EtOAc=9/1 then 2/1 as
the filtrate was concentrated. The residue was purified by eluent, v/v) to provide 16 as a colorless oil (0.40g, 71%): MS
column chromatography on silica gel (hexane/EtOAc=46/1 (ESI) m/z: 580 (M+H)+.
then 21/4 as eluent, v/v) to provide 13 as a colorless oil (0.35g,
2-[4-({[2-(Acetylamino)ethyl][(5-methyl-2-phenyl-1,3-
1
87% for 2 steps): H-NMR (400MHz, CDCl3) δ: 1.35 (3H, t, oxazol-4-yl)methyl]amino}methyl)-2,6-dimethylphenoxy]-
J=7.1Hz), 1.42 (9H, s), 1.45 (6H, s), 2.17 (6H, s), 2.23 (3H, s), 2-methylpropanoic Acid (17) To a solution of 16 (1.5g,
2.47 (2H, t, J=7.2Hz), 2.91 (2H, t, J=7.2Hz), 3.47–3.56 (4H, 4.1mmol) in dichloromethane (5.0mL), trifluoroacetic acid
m), 4.28 (2H, q, J=7.1Hz), 6.95 (2H, s), 7.36–7.45 (3H, m), (2.0mL, 26mmol) was added at 0°C and stirred at room tem-
7.96–8.03 (2H, m). MS (ESI) m/z: 565 (M+H)+.
Ethyl
perature for 20h. After the solvent was removed in vacuo, the
2-[2,6-Dimethyl-4-({[3-(methylamino)-3-oxopro- residue was used at the next step without further purification:
pyl][(5-methyl-2-phenyl-1,3-oxazol-4-yl)methyl]amino}- MS (ESI) m/z: 480 (M+H)+. To the mixture of the above
methyl)phenoxy]-2-methylpropanoate (14) To a solution of crude product (200mg, ca. 0.35mmol) in dichloromethane
13 (0.35g, 0.63mmol) in dichloromethane, HCl (4M dioxane (10mL), acetyl chloride (37µL, 0.52mmol) and TEA (0.19mL,
solution, 8.0mL) was added and stirred at room tempera- 1.4mmol) were added at 0°C and stirred at room temperature
ture overnight. After the solvent was removed in vacuo, the for 16h. After the reaction mixture was diluted with EtOAc,
residue was dissolved in DMF (6.0mL). Methylamine hydro- the mixture was washed with satd. sodium bicarbonate aque-
chloric acid (50mg, 0.75mmol), EDCI (0.18g, 0.94mmol), ous solution and brine, dried over Na2SO4 and concentrated.
HOBt (84mg, 0.63mmol) and TEA (0.24mL, 1.7mmol) were The residue was purified by column chromatography on
added to the reaction mixture and stirred at room tempera- silica gel (hexane/EtOAc=3/1 then 1/3 as eluent, v/v) to pro-
1
ture overnight. After the reaction mixture was diluted with vide acetamide intermediate as a pale yellow oil: H-NMR
dichloromethane, the mixture was washed with satd. sodium (400MHz, CDCl3) δ: 1.35 (3H, t, J=7.1Hz), 1.46 (6H, s),
bicarbonate aqueous solution, dried over Na2SO4 and concen- 1.90 (3H, s), 2.18 (6H, s), 2.24 (3H, s), 2.69 (2H, t, J=5.5Hz),
trated. The residue was purified by column chromatography 3.30–3.34 (2H, m), 3.53 (2H, s), 3.58 (2H, s), 4.28 (2H, q,
on silica gel (dichloromethane/methanol=97/3 then 93/7 as J=7.2Hz), 6.68–6.70 (1H, m), 6.93 (2H, s), 7.42–7.46 (3H, m),
eluent, v/v) to provide 14 as a pale yellow oil (0.30g, 96%): 8.01–7.98 (2H, m). MS (ESI) m/z: 522 (M+H)+. To the mixture
1H-NMR (400MHz, CDCl3) δ: 1.33–1.37 (3H, m), 1.46 (6H, of acetamide intermediate (60mg, 0.12mmol) in methanol
s), 2.18 (6H, s), 2.24 (3H, s), 2.39–2.49 (2H, m), 2.72 (3H, (5.0mL), NaOH (1M aqueous solution, 2.0mL) was added and

Vol. 63, No. 8 (2015)
Chem. Pharm. Bull.
601
stirred at room temperature for 2d. HCl (1M aqueous solution, 1.50 (6H, s), 2.20 (6H, s), 2.28 (3H, s), 2.40 (3H, s), 3.22 (2H,
2.5mL) and water were added and the organics was extracted s), 3.57 (2H, s), 3.62 (2H, s), 5.75 (1H, br), 6.95 (2H, s), 7.26
with EtOAc. The organic layer was washed with water and (2H, m), 7.75 (1H, br), 7.87 (2H, d, J=8.2Hz). IR (ATR) cm−1:
brine, dried over Na2SO4 and concentrated. The residue was 3334, 2920, 1730, 1678, 1120, 733, 700. FAB-MS m/z: 480
purified by column chromatography on silica gel (chloroform/ (M+H)+. HR-FAB-MS m/z: 480.2500 (Calcd for: C27H34O5N3;
methanol=9/1 as eluent, v/v) and triturated with EtOAc–hex- 480.2498). Anal. Calcd for C27H33O5N3·H2O: C, 65.17; H, 7.09;
1
ane to provide 17 as a colorless solid (51mg, 31%): H-NMR N, 8.44. Found: C, 64.92; H, 6.73; N, 8.20.
(400MHz, DMSO-d6) δ: 1.33 (6H, s), 1.76 (3H, s), 2.13 (6H, s),
PPAR Transactivation Assay The fusion protein (PPAR
2.27 (3H, s), 2.52–2.55 (2H, m), 3.17–3.22 (2H, m), 3.49 (2H, ligand binding domain–GAL4 DNA binding domain) expres-
s), 3.52 (2H, s), 6.97 (2H, s), 7.48–7.53 (3H, m), 7.68–7.71 (1H, sion plasmid (pFA-PPARγ/GAL4 or pFA-PPARα/GAL4) and
m), 7.93–7.91 (2H, m). MS m/z: 494 (M+H)+. Anal. Calcd for the reporter plasmid (pFA-SEAP, Stratagene) were used. HEK
C28H35N3O5·1.25H2O: C, 65.16; H, 7.32; N, 8.14. Found: C, 293T cells were grown in Dulbecco’s modified Eagle’s me-
65.43; H, 7.35; N, 7.74.
dium (DMEM) supplemented with 10% fetal bovine serum
Ethyl 2-(4-{[(2-tert-Butoxy-2-oxoethyl){[5-methyl-2-(4- (FBS) at 37°C in 5% CO2. After 24h of culture, the cells were
methylphenyl)-1,3-oxazol-4-yl]methyl}amino]methyl}-2,6- co-transfected with pFA-PPAR/GAL and pFA-SEAP using
dimethylphenoxy)-2-methylpropanoate (20) To a solution Lipofectamine (Invitrogen) and Plus Reagents (Invitrogen)
of 18 (1.0g, 4.5mmol) and 19 (2.0g, 5.2mmol) in MeCN according to the manufacturer’s protocol. After 5h of trans-
(20mL), K2CO3 (0.94g, 6.8mmol) was added and stirred under fection, the cells were treated with DMEM-FBS containing a
reflux conditions for 2h. After the solvent was removed in test compound at 37°C in 5% CO2. After 48h of incubation,
vacuo, the residue was dissolved in EtOAc, washed with water the conditioned medium was collected and the SEAP activ-
and brine, dried over Na2SO4 and concentrated. The crude ity in it was measured using the Reporter assay kit—SEAP
product was purified by column chromatography on silica gel (TOYOBO) according to the manufacturer’s protocol. Chemi-
(hexane/EtOAc=9/1 then 3/1 as eluent, v/v) to provide 20 as luminescence was read by ARVOsx, PerkinElmer, Inc. The
1
a pale yellow oil (2.5g, 98%): H-NMR (400MHz, CDCl3) δ: fold increase in chemiluminescence in the presence of a test
1.35 (3H, t, J=7.2Hz), 1.45 (6H, s), 1.47 (9H, s), 2.17 (6H, s), compound compared to that in the absence of it was calcu-
2.29 (3H, s), 2.39 (3H, s), 3.30 (2H, s), 3.71 (2H, s), 3.74 (2H, lated, and then the EC50 value was obtained.
s), 4.28 (2H, q, J=7.2Hz), 6.99 (2H, s), 7.23 (2H, d, J=8.3Hz),
Animal Animal facilities, animal care, and study pro-
grams were in accordance with the in-house guideline of
7.89 (2H, d, J=8.1Hz). MS (ESI) m/z: 565 (M+H)+.
Ethyl 2-(4-{[(2-Amino-2-oxoethyl){[5-methyl-2-(4-methylphen- the Institutional Animal Care and Use Committee of Daiichi
yl)-1,3-oxazol-4-yl]methyl}amino]methyl}-2,6-dimethylphen- Sankyo Co., Ltd. Female db/db (C57BLKS/J-m+/+Leprdb)
oxy)-2-methylpropanoate (21) To a solution of 20 (1.5g, mice were purchased at 10 weeks old from CREA Japan, Inc.
2.7mmol) in dichloromethane, trifluoroacetic acid (10mL, (Tokyo, Japan) and used as a type 2 diabetic animal model.
0.13mol) was added at 0°C and stirred at room temperature Male Zucker Fatty (Crlj:ZUC-Leprfa) rats were purchased
for 5d. After the solvent was removed in vacuo, the residue at 6 weeks old from Charles River Laboratories Japan, Inc.
(0.15g, 0.29mmol) was dissolved in DMF (5.0mL), and then (Kanagawa, Japan) and used as a type 2 diabetic animal
NH4Cl (20mg, 0.44mmol), TEA (0.16mL, 1.2mmol), EDCI model. All of the animals were housed six per cage and main-
(80mg, 0.44mmol) and HOBt (40mg, 0.29mmol) were added tained on an 8 a.m. light/8 p.m. dark schedule. Rodent chow
to the reaction solution and stirred at room temperature for and water were given ad libitum.
20h. After the reaction mixture was diluted with chloroform,
In Vivo db/db Mouse Studies Mice (six per group) re-
the mixture was washed with water, dried over Na2SO4 and ceived a once daily oral dosing of a test compound or vehicle
concentrated. The residue was purified by column chro- (0.5% methylcellulose) by oral gavage for 10d. Blood was col-
matography on silica gel (chloroform/methanol=20/1 as lected from the tail vein immediately prior to the next dosing
eluent, v/v) to provide 21 as a colorless oil (0.13g, 89%): on days 0, 5 and 10 for measurement of the plasma glucose
1H-NMR(400MHz, CDCl3) δ: 1.35 (3H, t, J=7.1Hz), 1.45 (6H, and triglyceride levels.
s), 2.16 (6H, s), 2.24 (3H, s), 2.40 (3H, s), 3.23 (2H, s), 3.53
In Vivo Zucker Fatty Rat Studies Mice (six per group)
(2H, s), 3.60 (2H, s), 4.27 (2H, q, J=7.1Hz), 5.44 (1H, br), 6.91 received a once daily oral dosing of a test compound or ve-
(2H, s), 7.25 (2H, m), 7.69 (1H, br), 7.87 (2H, d, J=8.2Hz). MS hicle (0.5% methylcellulose) by oral gavage for 13d. Blood
(ESI) m/z: 508 (M+H)+.
was collected from the tail vein immediately prior to the next
2-(4-{[(2-Amino-2-oxoethyl){[5-methyl-2-(4-methylphenyl)- dosing on days 1, 7 and 13 for measurement of the plasma
1, 3 - ox a z ol - 4 -yl]methyl}am i no]methyl}-2 , 6 - d i - glucose and triglyceride levels.
methylphenoxy)-2-methylpropanoic Acid (22) To a solu-
Distribution Coefficient The distribution coefficients
tion of 21 (0.13g, 0.26mmol) in methanol (2.0mL) and THF (logD) between 1-octanol and phosphate buffered saline (PBS)
(5.0mL) NaOH (1M aqueous solution, 1.3mL) was added and were assayed by a shaking flask method.16) Equal amounts of
stirred under reflux conditions for 15.5h. After the reaction PBS and 1-octanol were shaken and left for over 12h. The
mixture was cooled to room temperature, water was added upper layer (1-octanol) and lower layer PBS were collected
and the aqueous layer was washed with diethyl ether. HCl (1M individually. Each compound was dissolved in 1-octanol or
aqueous solution) was added to the aqueous layer and the or- PBS (200µM). The same amount of either PBS or 1-octanol
ganics were extracted with chloroform. The organic layer was was added and the mixture was shaken vigorously for 30min
dried over Na2SO4 and concentrated. The residue was purified at room temperature. Then, both phases were separated and
by crystallization from Hexane-Diethyl ether to provide 22 as assayed using LC-MS methodologies (LC-Mass spectrometer:
1
a colorless solid (56mg, 46%): H-NMR (400MHz, CDCl3) δ: 1100 Series LC/MSD, Agilent; Analytical Column: X Terra®

602
Chem. Pharm. Bull.
Vol. 63, No. 8 (2015)
MSC18 3.5µM, 3.0×30mm, Waters; Mobile Phase: 10mM chika, Masahiro Ota, Mitsuhiro Yamaguchi, Masaki Seto-
ammonium acetate buffer (pH 4.5)/0.05% acetic acid in aceto- guchi, Kiyoshi Chiba, Hiromichi Takano, Chiyuki Akiyama,
nitrile=95/5 to 10/90 v/v). The values of logD were analyzed Mayumi Ono, Mina Nishi and Hiroyuki Usui are employees
using Analyst software program (version 1.4, Applied Bio. of Daiichi Sankyo Co., Ltd. Hideo Kubo is an employee of
Systems).
CYP3A4 Direct Inhibition Assay P450 3A4 inhibition
Daiichi Sankyo RD Novare Co., Ltd.
activities were measured with a high throughput inhibitor
screening kit (Baculovirus-insect cell-expression system,
Supersomes™, and a fluorescent substrate (7-benzyloxy-tri-
fluoromethylcoumarin)) available from CORNING.12) Percent
inhibition was estimated by fluorescence at 10µM of the test
compounds.
MBI Assay Mechanism based inactivation against
CYP3A4 is estimated as the percentage of the enzymatic
activity (1′-hydroxylation of midazolam) remaining, after the
30-min preincubation of the test compounds in pooled human
liver microsomes.14)
References and Notes
1) International Diabetes Federation, IDF Diabetes Atlas, Sixth edi-
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accessed 9 March 2015.
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Solubilities The solubilities were determined by HPLC
analysis. Ten millimolars of compound solution in DMSO
(50 µL) was freeze-dried. To the residue JP XIV 1st fluid
(250 µL, pH 1.2) was added, and the mixture was stirred by
pipette operation. The mixture was saved under shading over
12h. After filtration of the mixture, the resulting filtrate was
diluted 20 times by adding aqueous DMSO solution (1:1
(v/v)) to obtain the measurement sample solution. Five micro-
molars of compound solution in aqueous DMSO solution (1:1
(v/v)) and 100µM of compound solution in aqueous DMSO so-
lution (1:1 (v/v)) were prepared to create a calibration curve.
The measurement sample solution, 5µM solution and 100µM
solution were assayed using HPLC methodologies (Analytical
Column: X Terra® MSC18 3.5µM, 3.0×30mm, Waters; Mobile
Phase: 10mM ammonium acetate buffer (pH 4.5)/0.05% acetic
acid in acetonitrile=95:5 to 10:90 v/v; Wave length: PDA
220–420nm). The solubilities were analyzed using Millenium
software (Waters).
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Bioorg. Med. Chem. Lett., 22, 7075–7079 (2012).
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13) We judged that MBI was considered sufficient below 80% remain-
ing from an examination of previously reported drugs.
14) Watanabe A., Nakamura K., Okudaira N., Okazaki O., Sudo K.,
Drug Metab. Dispos., 35, 1232–1238 (2007).
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H., Cap M., Farrelly D., Golla R., Grover G., Harrity T., Ma Z.,
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Acknowledgments We are grateful to the members in
Biological Research Laboratory for performing the biological
assays. Also, we would like to acknowledge Dr. Kiyoshi Na-
kayama for his helpful scientific guidance and support in the
preparation of this manuscript.
16) Manners C. N., Payling D. W., Smith D. A., Xenobiotica, 19, 1387–
1397 (1989).
Conflict of Interest Yoshihiro Shibata, Katsuji Kage-