Insulin-releasing activity of a series of phenylalanine derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.11.018

A series of phenylalanine derivatives were synthesized and their biological activities were evaluated. Compounds (S)-3 and (R)-3 exhibited more potent insulin-releasing activity than that of nateglinide, compound (S)-3 also showed insulin-sensitizing activity in vitro. Both compounds were tested for hypoglycemia effect in vivo.

Full text of DOI:10.1016/j.ejmech.2007.11.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1997e2003
http://www.elsevier.com/locate/ejmech
Short communication
Insulin-releasing activity of a series of phenylalanine derivatives
a
Lei Tang a, Juanhong Yu a, Haoshu Wu b, Houxing Fan a, Yushe Yang a, , Ruyun Ji
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
b Department of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Received 8 October 2007; received in revised form 14 November 2007; accepted 22 November 2007
Available online 5 December 2007
Abstract
A series of phenylalanine derivatives were synthesized and their biological activities were evaluated. Compounds (S )-3 and (R)-3 exhibited
more potent insulin-releasing activity than that of nateglinide, compound (S )-3 also showed insulin-sensitizing activity in vitro. Both compounds
were tested for hypoglycemia effect in vivo.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Insulin-releasing activity; Insulin-sensitizing activity; Hypoglycemia effect; Phenylalanine
1. Introduction
end of the structure. Another compound gliquidone, the third
generation of sulfonylurea insulin secretagogue, also contains
an acidic sulfonylurea group on the right of its structure, and
on the left part of gliquidone’s structure, there is a lipophilic
aromatic alkyl group. Another type of antidiabetic agent, gli-
tazars insulin-sensitizers, also has the structural features men-
tioned above [5]. So in our work searching for compounds
with more potent insulin-releasing activity, we designed
some nateglinide analogues by adding lipophilic substituents
onto the phenyl ring of nateglinide (Fig. 1). Meanwhile,
considering the configurational difference of a-carbon atom
between nateglinide and insulin-sensitizing agent farglitazar,
we decided to synthesize both enatiomers of all designed
compounds to observe the steric effect of a-carbon atom on
biological activity (Table 1).
Type 2 diabetes is a multifaceted disease characterized by
insulin resistance and/or abnormal insulin secretion. This
metabolic disorder accounts for more than 90% of all diabetes,
its worldwide frequency is expected to grow by 6% annually,
reaching a potential total of 200e300 million cases in 2010
[1]. Hyperglycemia often leads to several complications such
as neuropathy, retinopathy, nephropathy and premature
atherosclerosis [2]. Therefore, it is important to maintain an
appropriate blood glucose level, especially during the early
stage of the disease. The therapy for type 2 diabetes is often
by a combination of diet, exercise, or with pharmacological
agents. Current pharmacological agents available for type 2 di-
abetes include mainly insulin secretagogues, insulin formula-
tions, a-glucosidase inhibitors and insulin-sensitizers [3].
Insulin secretagogues, due to their direct and fast hypoglyce-
mic effect through stimulating the releasing of insulin from
pancreatic b-cells, have been used widely in the treatment of
type 2 diabetes.
2. Chemistry
Compound 1 is the key intermediate in the synthesis of de-
signed target compounds. Optically active compound 1 was
synthesized by condensation of L- or D-tyrosine methyl ester
with trans-4-isopropylcyclohexylcarboxylic acid in the exis-
tence of N-hydroxysuccimide (HOSu) and N,N0-dicyclohexyl-
carbodiimide (DCC). Compound 1 and the corresponding
heterocycloalkyl alcohol or 4-trifluoromethylbenzyl alcohol
Nateglinide is an insulin-releasing stimulator marketed in
1999 [4]. Nateglinide contains a carboxylic group on the right
* Corresponding author. Tel./fax: þ86 21 50806786.
E-mail address: ysyang@mail.shcnc.ac.cn (Y. Yang).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.11.018

1998
L. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 1997e2003
were used in a Mitsunobu condensation, followed by hydroly-
sis to give compounds 2e4 (Scheme 1).
Compounds with strong insulin-releasing activity were re-
tested, their EC50s (effective concentration of causing 50%
increase of insulin-releasing in HIT-T15 cells) were calculated
and listed in Table 3.
Compound 1 condensed with tert-butoxycarbonyl protected
2-methylaminoethanol giving 2-[N-(trans-4-isopropylcyclo-
hexylcarbonyl)amino]-3-[4-(N-methyl-N-(tert-butoxycarbony-
l)aminoethoxy)phenyl]propionic acid methyl ester, which was
treated with trifluoroacetic acid to remove the protective group
affording 2-[N-(trans-4-isopropylcyclohexylcarbonyl)amino]-
3-[4-(2-methylaminoethoxy)phenyl]propionic acid methyl
ester (compound 5). Compounds 6 and 7 were obtained by
condensation of compound 5 with 2-fluoropyridine and 2-
chlorobenzoxazole, respectively, and then followed by hydro-
lysis (Scheme 2).
Compounds 8e10 were afforded by saponification of the
condensed products of compound 1 with corresponding alkyl
halide. Compound 11 was obtained directly by hydrolysis of
compound 1. Compound 12, the S-antipode of nateglinide,
was synthesized by using L-phenylalanine as starting material
following the method of Ref. [4].
For nateglinide derivatives, it was reported that R-
configuration was necessary for insulin-releasing activity
[4,7]. However, in our study we find that when certain substit-
uents were added onto the phenyl ring of nateglinide, the
compounds with S-configuration also showed potent insulin-
releasing activity. Comparing the activities of different config-
urations of compounds 8 and 9 (Table 3) it could be seen that
compounds with S-configuration even possessed a higher insu-
lin-releasing activity than that of R-configuration. The maxi-
mum insulin-releasing activity was achieved by compound
(R)-3, which is 63-fold more potent than the positive nategli-
nide in vitro judged from their EC50s.
Compound (S )-3 also exhibited a high insulin-releasing
activity in vitro. Considering the structural similarity between
(S )-3 and farglitazar, the insulin-sensitizing activity of (S )-3
was tested on 3T3-L1 preadipocyte cells following the litera-
ture’s method by measuring the triglyceride accumulation gen-
erating from insulin-regulated differentiation of preadipocyte
versus adipocyte [8]. The triglyceride accumulation increase
caused by (S )-3 at concentrations of 1 mM and 10 mM were
38.14% and 46.01%, respectively; for rosiglitazone at concen-
trations of 1 mM and 10 mM, the triglyceride accumulation
increased by 38.81% and 35.87%, respectively. The results
3. Pharmacological evaluation and discussion
All synthesized compounds were screened for insulin-
releasing activity in HIT-T15 cells by measuring glucose-
induced insulin-releasing [6]. Nateglinide and mitiglinide
were selected as positive controls. The insulin-releasing activ-
ities of all compounds were given in Table 2.
COOH
HN
O
Nateglinide
COOH
O
O
S
O
NH
R
HN
O
O
O
O
N
H3C
HN
Gliquidone
COOH
O
N
HN
O
O
Farglitazar
Fig. 1. Design of phenylalanine derivatives.

L. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 1997e2003
1999
Table 1
Structure of phenylalanine derivatives
which was nearly equal to the control drug mitiglinide. How-
ever, the plasma-glucose level after that gradually got back to
the level of negative control group, and was maintained till the
end of test. Meanwhile, mitiglinide exhibited a continuing
lowering action on plasma-glucose throughout the test. Com-
pound (S )-3 showed no lowering action at all time points.
The biological activity in vivo exhibits a poor correlation
with the results obtained in vitro, the underlying reasons
may include inferior bioavailability and unstable metabolic
properties, which need further exploration.
In the study of antidiabetic agents, designing of compounds
with both insulin-sensitizing activity and insulin-releasing ac-
tivity is also an important research field. Although the synthe-
sized compounds in our work didn’t show satisfactory activity
in vivo, it is no doubt that the type of compounds could offer
us a hint to design and synthesize more compounds with
higher insulin-releasing activity or even with higher insulin-
sensitizing activity.
COOH
O
R
O
HN
Compoundsa
R
Compoundsa
R
2
8
N
CH3
O
3
4
6
9
n-Bu
N
10
11
Et
4. Conclusions
F3C
N
In summary, we found that the S-configuration derivatives
of nateglinide possess potent insulin-releasing activity in vitro,
and compound (S )-3 even exhibited insulin-sensitizing
activity in vitro. Among all synthesized compounds, both com-
pounds (S )-3 and (R)-3 possessed more potent insulin-
releasing activity than that of nateglinide and mitiglinide in
HIT-T15 cells, but both compounds showed a weak in vivo
activity in rat model. Further structureeactivity relationship
exploration of this type of compounds is still undergoing.
H
N
CH3
O
N
CH3
7
N
a
Both (S ) and (R) configurations of a-carbon atom of all compounds were
synthesized for activity-screening.
5. Experimental protocols
indicated that (S )-3 showed good insulin-sensitizing activity
almost equivalent to the positive control rosiglitazone.
5.1. General chemistry
To test in vivo activities of (S )-3 and (R)-3, both com-
pounds were administered by oral gavage to glucose-loading
normal SD rats at a dose of 3 mg/kg body weight. The se-
rum-glucose levels were measured at several time points after
dosing, and the results were given as decreasing percentage of
plasma-glucose level compared to control (Table 4). In this
test, compound (R)-3 showed impressive glucose reduction
at the time point of 30 min and 60 min after dosing. The
reduction percentage amounts to 36% at the point of 30 min,
All melting points were determined on MEL-TEMP melt-
ing point apparatus and are uncorrected. 1H NMR spectra
were recorded on a Varian Mercury 400 NMR spectrometer
with TMS as internal standard, chemical shifts are expressed
as d (ppm). Mass spectra were determined using Finnigan
MAT 95, EI: 70 eV. High resolution mass spectra (HRMS)
were performed on a Kratos MS80 instrument. Elemental
analyses (C, H, N) were performed on a Leco CHN-2000
COOCH3
COOH
O
COOCH3
NH2
HN
HN
HO
b
c
O
R
O
a
HO
D- or L-
2- 4 (R and S)
1
Scheme 1. (a) trans-4-Isopropylcyclohexylcarboxylic acid, HOSu, DCC; (b) Ph3P, diethyl azodicarboxylate, ROH, THF and (c) 1 N LiOH, THF/MeOH (3:1).

2000
L. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 1997e2003
COOCH3
COOCH3
CH3
BocN
HN
HN
a
HO
b
O
O
O
1
COOCH3
COOH
O
CH3
HN
CH3
HN
N
N
HN
O
c
d
O
O
5
6
Scheme 2. (a) 2-(N-Boc-N-methlyamino) ethanol, Ph3P, diethyl azodicarboxylate, THF; (b) trifluoroacetic acid, CH2Cl2; (c) 2-fluoropyridine, reflux and (d) 1 N
LiOH, THF/MeOH (3:1).
elemental analyzer and all compounds are within ꢀ0.4% of
theory. Flash chromatography was carried out on silica gel
(200e300 mesh), and chromatographic solvent proportions
are expressed on a volume:volume basis. Optical rotations
were measured with a PerkineElmer 241 polarimeter at
room temperature (20 ꢁC). IR spectrums were recorded on
a Bruker Tensor 27 FTIR spectrometer. Synthetic procedures
of S-configuration compounds are listed below; the synthetic
procedures of R-configuration compounds are the same with
their corresponding antipodes. All analytic data of R-
configuration compounds are nearly identical with that of S-
configuration except for the optical rotations, which exhibited
opposite rotation but with a nearly identical value compared to
the corresponding S-configuration compounds.
Table 2
Insulin-releasing activities of the compounds (mean ꢀ sd, n ¼ 3)
Compounds
5.2. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-(4-hydroxyphenyl)propionic acid
methyl ester ((S )-1)
Insulin-releasing activitya
at 1 mM
at 10 mM
at 100 mM
(S )-2
35.7 ꢀ 3.3
37.8 ꢀ 3.8
68.5 ꢀ 6.0
105.2 ꢀ 7.1
47.9 ꢀ 5.1
29.8 ꢀ 2.7
26.0 ꢀ 2.0
24.4 ꢀ 6.4
35.5 ꢀ 4.1
24.8 ꢀ 3.3
53.1 ꢀ 2.0
38.9 ꢀ 3.8
73.4 ꢀ 5.6
58.9 ꢀ 9.3
55.8 ꢀ 3.6
76.2 ꢀ 4.6
17.5 ꢀ 1.6
34.2 ꢀ 8.8
26.4 ꢀ 7.2
36.3 ꢀ 5.8
52.5 ꢀ 1.62
74.9 ꢀ 0.8
58.8 ꢀ 4.1
146.6 ꢀ 6.8
111.7 ꢀ 17.2
329.2 ꢀ 12.6
487.4 ꢀ 44.6
156.9 ꢀ 6.5
152.1 ꢀ 33.8
83.7 ꢀ 20.3
59.4 ꢀ 7.6
To a solution of 0.7 g (4.1 mmol) of trans-4-isopropylcy-
clohexylcarboxylic acid and 0.53 g (4.6 mmol) of HOSu in
14 mL of chloroform was added 0.95 g (4.6 mmol) of DCC
portionwise. The resulting solution was stirred at room tem-
perature for 3 h. The precipitate produced was filtered off.
Acetic acid (0.4 mL) was added into the filtrate and stirred
at room temperature for 2 h. Saturated NaHCO3 (10 mL)
was added into the mixture and stirred together, then the water
layer was removed. The chloroform layer was washed with
5 mL of H2O and brine, then dried over Mg2SO4 and filtered.
L-Tyrosine methyl ester (0.80 g, 4.1 mmol) was added, and
stirred at room temperature for 24 h. The solution was washed
with 1 N HCl, water, then dried on Mg2SO4 and filtered. After
removal of the solvent in vacuo, the residue was dissolved in
methanol, kept at ꢂ20 ꢁC, a white solid was separated out.
The mother liquor was concentrated and then another portion
of solid was collected. Title compound (0.53 g) was afforded
together. Yield: 37.8%. M.p. 123e126 ꢁC. [a]D25 54.3
(R)-2
(S )-3
(R)-3
131.2 ꢀ 6.2
200.9 ꢀ 21.7
76.7 ꢀ 16.2
85.6 ꢀ 12.4
41.5 ꢀ 12.5
36.2 ꢀ 6.7
(S )-4
(R)-4
(S )-6
(R)-6
(S )-7
72.8 ꢀ 11.3
81.7 ꢀ 12.6
147.5 ꢀ 12.5
94.4 ꢀ 10.5
136.7 ꢀ 12.9
107.1 ꢀ 4.4
99.9 ꢀ 12.8
107.3 ꢀ 11.7
35.4 ꢀ 2.4
126.9 ꢀ 5.5
145.1 ꢀ 23.6
225.9 ꢀ 49.9
171.1 ꢀ 25.7
326.2 ꢀ 13.0
261.6 ꢀ 19.5
151.3 ꢀ 3.8
183.8 ꢀ 8.7
64.8 ꢀ 8.6
(R)-7
(S )-8
(R)-8
(S )-9
(R)-9
(S )-10
(R)-10
(S )-11
(R)-11
(S )-Nateglinide (12)
Nateglinide
Mitiglinide
a
69.6 ꢀ 9.3
122.2 ꢀ 40.2
61.1 ꢀ 14.2
124.3 ꢀ 6.9
120 ꢀ 8.16
35.3 ꢀ 11.7
66.8 ꢀ 3.7
66.0 ꢀ 1.55
Insulin-releasing activities were given as the increasing percentage of
1
insulin-releasing from HIT-T15 cells than that of the negative group (DMSO).
(c 0.50, CHCl3). H NMR (CDCl3): d ¼ 0.82 (d, J ¼ 6.9 Hz,

L. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 1997e2003
2001
Table 3
EC50s of the compounds (n ¼ 3)
Compounds
(S )-3
(R)-3
(S )-8
(R)-8
(S )-9
(R)-9
(S )-10
(R)-10
Nateglinide
7.59
Mitiglinide
0.708
EC50 (mM)
0.54
0.12
1.05
2.82
0.55
0.65
0.96
0.26
6H), 0.89e1.12 (m, 3H), 1.30e1.43 (m, 3H), 1.70e1.90
(m, 4H), 2.05 (m, 1H), 2.96 (dd, J ¼ 6.5 Hz, 14.2 Hz, 1H),
3.09 (dd, J ¼ 5.5 Hz, 14.7 Hz, 1H), 3.72 (s, 3H), 4.85 (m,
1H), 6.00 (d, J ¼ 8.1 Hz, 1H), 6.70 (d, J ¼ 8.4 Hz, 2H), 6.90
(d, J ¼ 8.2 Hz, 2H). IR (KBr, cmꢂ1): 3408.8 (OeH), 3325.7
(NeH), 1622.1 (C]O); MS (EI) m/e (%): 347 (19, Mþ),
170 (100). Anal. Calcd for C20H29NO4 (%): C, 69.14; H,
8.41; N, 4.03. Found: C, 69.25; H, 8.75; N, 3.88.
cmꢂ1): 3305.4 (NeH), 1712.5 (C]O), 1650.8 (C]O); MS
(EI) m/e (%): 476 (11, Mþ), 144 (100); HRMS: 476.2670
(C29H36N2O4).
5.4. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazole)
ethoxy]phenyl]propionic acid ((S )-3)
Using (S )-1 and 2-(5-methyl-2-phenyl-4-oxazole)ethanol
as starting material, the title compound was prepared follow-
ing the procedure in 5.3. Yield: 77.2%. M.p. 151e153 ꢁC
(dec). [a]2D5 81.1 (c 0.545, CHCl3). 1H NMR (DMSO-d6):
d ¼ 0.80 (d, J ¼ 7.6 Hz, 6H), 0.85e1.0 (m, 3H), 1.10e1.40
(m, 3H), 1.50e1.72 (m, 4H), 2.00 (m, 1H), 2.35 (s, 3H),
2.75 (dd, J ¼ 14 Hz, 9.9 Hz, 1H), 2.90 (t, J ¼ 6.6 Hz, 2H),
2.96 (dd, J ¼ 14 Hz, 4.7 Hz, 1H), 4.17 (t, J ¼ 6.6 Hz, 2H),
4.21 (m, 1H), 6.81 (d, J ¼ 8.8 Hz, 2H), 7.13 (d, J ¼ 8.8 Hz,
2H), 7.50 (m, 3H), 7.92 (m, 3H), 12.6 (s, 1H); IR (KBr,
cmꢂ1): 3282.3 (NeH), 1710.6 (C]O), 1631.5(C]O); MS
(EI) m/e (%): 518 (1, Mþ), 186 (100); Anal. Calcd for
C31H38N2O5 (%): C, 71.81; H, 7.34; N, 5.41. Found: C,
71.49; H, 7.24; N, 5.36.
5.3. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-[4-[2-(1-indolyl)ethoxy]phenyl]propionic
acid ((S )-2)
Triphenylphosphine (0.61 g, 2.31 mmol) was added into
a stirred solution of 0.24 g (1.54 mmol) of 2-(1-indolyl)etha-
nol and 0.54 g (1.54 mmol) of (S )-1 in 30 mL of anhydrous
tetrahydrofuran, then 0.37 mL (2.31 mmol) of diethyl azodi-
carboxylate was added dropwise at 0 ꢁC. The resulting solu-
tion was stirred at room temperature for 24 h. After solvent
was removed under reduced pressure, the residue was diluted
with ether and a solid separated out, the solid was filtered out
and recrystallized from methanol to give a white solid. The
solid was dissolved in 1 mL of mixed solvent of tetrahydrofur-
anemethanol (3:1), then 1 mL of 1 N solution of LiOH in H2O
was added, the resulting solution was stirred at room temper-
ature for 24 h. The pH of the solution was adjusted to 5 with
1 N HCl and 5 mL of H2O was added and then this mixture
was stirred, filtered, and the filter cake was recrystallized
from methanol to give 0.29 g of the title compound. Yield:
39.6%. M.p. 165e167 ꢁC (dec). [a]D25 26.7 (c 0.545, CHCl3).
1H NMR (CDCl3): d ¼ 0.81 (d, J ¼ 6.9 Hz, 6H), 0.80e1.10
(m, 3H), 1.30e1.45 (m, 3H), 1.70e1.90 (m, 4H), 1.99 (m,
1H), 3.07 (dd, J ¼ 14.3 Hz, 6.2 Hz, 1H), 3.15 (dd,
J ¼ 14.3 Hz, 5.2 Hz, 1H), 4.23 (t, J ¼ 5.5 Hz, 2H), 4.50 (t,
J ¼ 5.5 Hz, 2H), 4.75 (m, 1H), 5.89 (d, J ¼ 6.6 Hz, 1H), 6.50
(d, J ¼ 3.9 Hz, 1H), 6.76 (d, J ¼ 8.4 Hz, 2H), 7.00 (d,
J ¼ 8.4 Hz, 2H), 7.11 (t, J ¼ 7.3 Hz, 1H), 7.24 (m, 2H), 7.40
(d, J ¼ 8.1 Hz, 1H), 7.62 (d, J ¼ 7.7 Hz, 1H); IR (KBr,
5.5. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-[4-(4-trifluoromethylphenylmethoxy)phenyl]
propionic acid ((S )-4)
Using (S )-1 and 4-trifluoromethylbenzyl alcohol as starting
materials, ethyl ether was used as solvent, the title compound
was prepared following the procedure in 5.3. Yield: 60.6%.
1
M.p. 171e172 ꢁC. [a]D25 53.9 (c 0.285 in CHCl3). H NMR
(CDCl3): d ¼ 0.81 (d, J ¼ 6.7 Hz, 6H), 1.0 (m, 3H), 1.40 (m,
3H), 1.70e1.90 (m, 4H), 2.05 (m, 1H), 3.08 (dd, J ¼ 5.5 Hz,
14.1 Hz, 1H), 3.19 (dd, J ¼ 5.5 Hz, 14.3 Hz, 1H), 4.80 (m,
1H), 5.08 (s, 2H), 5.98 (d, J ¼ 6.4 Hz, 1H), 6.88 (d,
J ¼ 8.2 Hz, 2H), 7.08 (d, J ¼ 8.2 Hz, 2H), 7.52 (d,
J ¼ 8.0 Hz, 2H), 7.62 (d, J ¼ 8.2 Hz, 2H). IR (KBr, cmꢂ1):
3328.6 (NeH), 1731.8 (C]O), 1612.2 (C]O); MS (EI) m/e
(%): 491 (3, Mþ), 159 (100). Anal. Calcd for
C27H32F3NO4$1/2H2O: C, 64.80; H, 6.60; N, 2.80. Found:
C, 65.20; H, 6.43; N, 3.01.
Table 4
Decreasing percentage of plasma-glucose level after administration of com-
5.6. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-[4-[2-[N-methyl-N-(2-pyridyl)amino]
ethoxy]phenyl]propionic acid ((S )-6)
pound (S )-3 and (R)-3a
Group
Decreasing percentage of serum-glucose level compared to
control group (%)
0 min
15 min
30 min
60 min
120 min 180 min
A stirred solution of 0.19 g (1.1 mmol) of 2-[(N-tert-butox-
ycarbonyl)-methylamino]ethanol and 0.35 g (1 mmol) of (S )-1
in 15 mL of anhydrous tetrahydrofuran was treated with 0.38 g
(1.5 mmol) of triphenylphosphine, then 240 mL (1.5 mmol) of
diethyl azodicarboxylate was added dropwise at 0 ꢁC. The
(S )-3
ꢂ8.37
ꢂ4.73
ꢂ8.51
ꢂ36.4
ꢂ36.2
ꢂ4.47 ꢂ11.3
ꢂ2.07
ꢂ11.4
ꢂ70.3
(R)-3
Mitiglinide
ꢂ0.853 ꢂ21.2
ꢂ33.7
ꢂ17.3
ꢂ87.3
8.72
ꢂ16.5
ꢂ61.7
a
Dosage: 3 mg/kg body weight, n ¼ 8e10.

2002
L. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 1997e2003
resulting solution was stirred at room temperature for 24 h.
The solvent was removed in vacuo, and the residue was chro-
matographed over silica gel using petroleum/ethyl acetate
(2:1) as eluent to afford 0.21 g of compound (S )-5 as a color-
less syrupy. The colorless syrupy was dissolved in 4.8 mL of
dichloromethane, then 4.8 mL of trifluoroacetic acid was
added. The resulting solution was stirred at room temperature
for 1 h. Then parts of the solvent were removed in vacuo at
room temperature. The residual solution was neutralized
with saturated NaHCO3 and extracted with dichloromethane
(10 mL ꢃ 2), The combined organics were washed with
H2O, dried on Mg2SO4, filtered and concentrated in vacuo.
The residue was refluxed with 2 mL of 2-fluoropyridine for
24 h. Excessive 2-fluoropyridine was removed under reduced
pressure, the residue was dissolved in small amount of ace-
tone, chromatographed over silica gel using acetone/petroleum
(1:2) as eluent afforded 0.081 g of white solid. Hydrolysis of
the white solid following the same procedure in Section 5.3
provided 0.06 g of the title compound. Yield: 76.8%. M.p.
148 (100). Anal. Calcd for C29H37N3O5: C, 68.64; H, 7.30;
N, 8.28. Found: C, 68.45; H, 7.49; N, 8.21.
5.8. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-(4-phenylmethoxyphenyl)propionic
acid ((S )-8)
A solution of 137 mL (1.14 mmol) of benzyl bromide and
0.13 g (0.38 mmol) of (S )-1 in 1 mL of DMF was treated
with 0.16 g (1.14 mmol) of powered K2CO3. The resulting
mixture was stirred at 70 ꢁC for 12 h. After cooling to 0 ꢁC,
5 mL of H2O was added, the resulting solution was extracted
with EtOAc (5 mL ꢃ 2). The combined extract was washed
with H2O, dried on anhydrous Na2SO4. Solvent was removed
under reduced pressure, the residue mixed with ether, and
a white solid separated. The solid was hydrolyzed with
LiOH to give 0.10 g of the title compound. Yield: 62.5%.
M.p. 140e142 ꢁC (dec). [a]D25 70.9 (c 0.83, CHCl3). 1H
NMR (CDCl3): d ¼ 0.81 (d, J ¼ 6.9 Hz, 6H), 1.0 (m, 3H),
1.40 (m, 3H), 1.70e1.90 (m, 4H), 2.05 (m, 1H), 3.04 (dd,
J ¼ 5.5 Hz, 13.9 Hz, 1H), 3.19 (dd, J ¼ 5.5 Hz, 13.9 Hz, 1H),
4.81 (m, 1H), 5.01 (s, 2H), 6.12 (d, J ¼ 7.3 Hz, 1H), 6.91 (d,
J ¼ 8.4 Hz, 2H), 7.08 (d, J ¼ 8.4 Hz, 2H), 7.39 (m, 5H); IR
(KBr, cmꢂ1): 3328.6 (NeH), 1731.8 (C]O), 1648.9
(C]O); MS (EI) m/e (%): 423 (4, Mþ), 91 (100). Anal. Calcd
for C26H33NO4$1/2H2O: C, 72.22; H, 7.87; N, 3.24. Found: C,
72.34; H, 7.71; N, 3.47.
1
158e160 ꢁC. [a]D25 30.0 (c 0.26, CHCl3). H NMR (CDCl3):
d ¼ 0.80 (d, J ¼ 7.6 Hz, 6H), 0.80e1.00 (m, 3H), 1.13e1.40
(m, 3H), 1.62e1.82 (m, 4H), 1.96 (m, 1H), 3.03 (d,
J ¼ 4.4 Hz, 2H), 3.10 (s, 3H), 3.88 (t, J ¼ 6.5 Hz, 2H), 3.98
(t, J ¼ 6.6 Hz, 2H), 4.62 (m, 1H), 5.40 (s, 1H), 6.21 (d,
J ¼ 6.9 Hz, 1H), 6.55 (m, 1H), 6.75 (d, J ¼ 8.5 Hz, 2H), 6.98
(d, J ¼ 8.5 Hz, 2H), 7.48 (m, 1H), 8.03 (m, 1H). IR (KBr,
cmꢂ1): 3299.7 (NeH), 1720.2 (C]O), 1637.3 (C]O); MS
(EI) m/e (%): 467 (2, Mþ), 135 (100). Anal. Calcd for
C27H37N3O4$1/2H2O: C, 68.07; H, 7.98; N, 8.82. Found: C,
67.86; H, 7.75; N, 8.79.
5.9. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-(4-butoxyphenyl)propionic acid ((S )-9)
5.7. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-[4-[2-[N-methyl-N-(2-benzooxazolyl)amino]
ethoxy]phenyl]propionic acid ((S )-7)
Taking n-butyl bromide and (S )-1 as starting material, the
title compound was prepared following the procedure as in
Section 5.8. Yield: 70.3%. M.p. 120e121 ꢁC. [a]D25 87.9 (c
1
1.145, CHCl3). H NMR (CDCl3): d ¼ 0.82 (d, J ¼ 6.9 Hz,
To a solution of 36 mg (0.21 mmol) of compound (S )-5 in
2 mL of tetrahydrofuran were added 240 mL (0.51 mmol) of
triethylamine and 40 mg (0.26 mmol) of 2-chlorobenzoxazole.
The resulting solution was stirred at room temperature for
24 h. Tetrahydrofuran was removed under reduced pressure,
the residue was mixed with 4 mL of ethyl acetate, then added
4 mL of saturated solution of NaHCO3 in H2O and stirred, the
organic layer was separated, dried over anhydrous Na2SO4 and
concentrated, the residue was mixed with a mixed solvent of
petrolether/ethyl acetate (1:1), a white solid separated. The
solid was hydrolyzed with LiOH to give 42 mg of the title
compound. Yield: 39.6%. M.p. 179e180 ꢁC (dec). [a]D25
6H), 0.96 (m, 6H), 1.38 (m, 3H), 1.50 (m, 2H), 1.75e1.90
(m, 6H), 2.05 (m, 1H), 3.04 (dd, J ¼ 5.8 Hz, 14.3 Hz, 1H),
3.19 (dd, J ¼ 5.5 Hz, 14.2 Hz, 1H), 3.91 (t, J ¼ 6.6 Hz, 2H),
4.80 (m, 1H), 5.99 (d, J ¼ 7.3 Hz, 1H), 6.81 (d, J ¼ 8.4 Hz,
2H), 7.07 (d, J ¼ 8.4 Hz, 2H). IR (KBr, cmꢂ1): 3305.4 (Ne
H), 1716.4 (C]O), 1646.9 (C]O); MS (EI) m/e (%): 389
(7, Mþ), 163 (100). Anal. Calcd for C23H35NO4$1/3H2O: C,
69.87; H, 9.03; N, 3.54. Found: C, 69.60; H, 8.88; N, 3.64.
5.10. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-(4-ethoxyphenyl)propionic acid ((S )-10)
1
ꢂ81.1 (c 0.535, CHCl3). H NMR (DMSO-d6): d ¼ 0.81 (d,
Taking ethyl bromide and compound (S )-1 as starting
material, the title compound was prepared following the pro-
cedure as in Section 5.8. Yield: 75.0%. M.p. 168e170 ꢁC.
J ¼ 6.9 Hz, 6H), 0.80e1.0 (m, 3H), 1.15e1.40 (m, 3H), 1.60
(m, 4H), 1.95 (m, 1H), 2.78 (dd, J ¼ 13.5 Hz, 7.3 Hz, 1H),
2.96 (dd, J ¼ 13.5 Hz, 4.8 Hz, 1H), 3.20 (s, 3H), 3.82 (t,
J ¼ 5.5 Hz, 2H), 4.10 (m, 1H), 4.20 (t, J ¼ 5.5 Hz, 2H), 6.78
(d, J ¼ 8.4 Hz, 2H), 6.95 (t, J ¼ 7.7 Hz, 1H), 7.00 (d,
J ¼ 8.4 Hz, 2H), 7.10 (t, J ¼ 7.0 Hz, 1H), 7.28 (d,
J ¼ 8.3 Hz, 1H), 7.35 (d, J ¼ 8.0 Hz, 1H), 7.40 (d,
J ¼ 7.3 Hz, 1H). IR (KBr, cmꢂ1): 3322.8 (NeH), 1731.8
(C]O), 1648.9 (C]O); MS (EI) m/e (%): 507 (12, Mþ),
1
[a]2D5 93.2 (c 1.13, CHCl3). H NMR (CDCl3): d ¼ 0.81 (d,
J ¼ 6.9 Hz, 4H), 1.0 (m, 3H), 1.40 (m, 5H), 1.75e1.90 (m,
4H), 2.05 (m, 1H), 3.04 (dd, J ¼ 5.5 Hz, 14.0 Hz, 1H), 3.17
(dd, J ¼ 5.1 Hz, 14.3 Hz, 1H), 4.00 (m, 2H), 4.80 (m, 1H),
5.97 (d, J ¼ 7.6 Hz, 1H), 6.81 (d, J ¼ 8.4 Hz, 2H), 7.03 (d,
J ¼ 8.3 Hz, 2H). IR (KBr, cmꢂ1): 3303.5 (NeH), 1724.1
(C]O), 1643.1 (C]O); MS (EI) m/e (%): 361 (16, Mþ),

L. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 1997e2003
2003
192 (100). Anal. Calcd for C21H31NO4$1/3H2O: C, 68.66; H,
8.63; N, 3.81. Found: C, 68.93; H, 8.43; N, 3.86.
After an additional 2 days of incubation in DMEM supple-
mented with 10% FBS and 5 mg/mL insulin, the medium
was changed every other day with DMEM supplemented
with 10% FBS. Cells were challenged during the first 4 days
of differentiation with different compounds at 1 and
10 mmol/L, respectively, with rosiglitazone as positive control
and 0.1% DMSO as negative control. The addition of com-
pounds to the medium was accomplished by dissolving the
drug in DMSO and diluting the drug 1000-fold with medium.
Seven days after the induction of 3T3-L1 cells, oil red O stain-
ing was used to detect triglyceride accumulation in 3T3-L1
cells. The precipitation of oil red O in 3T3-L1 adipocytes
was dissolved with isopropyl alcohol, and OD value at
510 nm was determined by ELISA spectrometry. The results
were based on three or four independent experiments.
5.11. (2S )-2-[N-(trans-4-isopropylcyclohexylcarbonyl)
amino]-3-(4-hydroxyphenyl)propionic acid ((S )-11)
The title compound was prepared by hydrolysis of com-
pound (S )-1 with LiOH. Yield: 81.1%. M.p. 156e157 ꢁC.
[a]2D5 76.4 (c 0.845, CHCl3). H NMR (DMCO-d6): d ¼ 0.81
1
(d, J ¼ 7.0 Hz, 6H), 1.0 (m, 3H), 1.40 (m, 3H), 1.75e1.90
(m, 4H), 2.15 (m, 1H), 2.90 (dd, J ¼ 8.1 Hz, 13.9 Hz, 1H),
3.10 (dd, J ¼ 5.1 Hz, 13.9 Hz, 1H), 4.82 (m, 1H), 6.72 (d,
J ¼ 8.4 Hz, 2H), 7.05 (d, J ¼ 8.4 Hz, 2H). IR (KBr, cmꢂ1):
3303.5 (NeH), 1619.9 (C]O); MS (EI) m/e (%): 333 (10,
Mþ), 170 (100). Anal. Calcd for C19H27NO4$2H2O: C,
61.79; H, 8.40; N, 3.79. Found: C, 61.94; H, 8.17; N, 3.84.
5.14. In vivo screening in glucose-loading normal rats
5.12. Insulin-releasing screening in HIT-T15 cells
Normal SD rats weighting 180e220 g were fasted over-
night and divided into six groups with 8e10 rats each. Follow-
ing oral administration of 3 mg/kg of one of the agents being
tested, mitiglinide (positive control) or 0.5% of carboxyl-
methyl-cellulose sodium (CMC-Na, vehicle control), the rats
were immediately administered oral glucose 2.5 g/kg. Blood
samples were drawn before glucose and drug administration
and at 15, 30, 60, 120 and 180 min after administration from
the caudal vein, and then serum-glucose levels were measured
by the glucose oxidase method.
HIT-T15 cells from American Type Culture Collection
(ATCC, USA) were seeded into a 96-well plate at a density
of 5 ꢃ 104 cells/well, and cultured with Ham’s F12 medium
(supplemented with 10% fetal bovine serum, 100 unit/mL
penicillin, and 100 mg/mL streptomycin) for 2 days at 37 ꢁC
in 5% CO2e95% air. After being washed twice with
KrebseRinger bicarbonateeHEPES buffer (KRBeHEPES
buffer, containing 129 mM NaCl, 4.7 mM KCl, 5 mM
NaHCO3, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4,
2.8 mM glucose, 10 mM HEPES, pH 7.4) supplemented
with 0.1% bovine serum albumin, the subconfluent cells
were pre-incubated with KRBeHEPES buffer for 1 h, then in-
cubated for another 1 h with KRBeHEPES buffer containing
one of the agents being tested, nateglinide or mitiglinide at
a range of concentrations (both incubations being at 37 ꢁC
in 5% CO2e95% air, all agents being dissolved in DMSO
and the same final concentration of the solvent being added
to the control medium). Aliquots of the culture media were
collected for measurement of the insulin concentration using
an insulin radioimmunoassay kit (Atomic Energy Research
Institute, Beijing, China).
Acknowledgements
The authors are thankful for the financial support from the
National Natural Science Special Foundation of China (Project
30623008).
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