Design, synthesis and anti-itch activity evaluation of aromatic amino acid derivatives as gastrin-releasing peptide receptor antagonists

Article abstract of DOI:10.2174/157340612802084342

Eight aromatic amino acid derivatives (9a-9h) as gastrin-releasing peptide receptor (GRPR) antagonists were designed and synthesized. For the design, the tertiary structure of GRPR was predicted by blast searching in the premise of 1u19 protein as the template. Eight target compounds were docked into the binding pocket to investigate their possible binding interactions. Their anti-itch activities were tested by intrathecal injection using Kunming mice as experimental animals and chloroquine phosphate as a modeling medium. Compounds 9e and 9f significantly inhibited scratching behaviors. The anti-itch activities of these compounds decreased with the following sequence: 9e > 9f = 9d > 9b > 9g > 9a > 9h > 9c predicted by computer-aided drug design (CADD) and 9e > 9f > 9b > 9h > 9g > 9d > 9a > 9c evaluated by preliminary test. They were broadly in line with activity order pretested by CADD. It showed that the predicted tertiary structure of GRPR could be used for antipruritic drug design.

Full text of DOI:10.2174/157340612802084342

Medicinal Chemistry, 2012, 8, 865-873
865
Design, Synthesis and Anti-itch Activity Evaluation of Aromatic Amino
Acid Derivatives as Gastrin-Releasing Peptide Receptor Antagonists
Ri-Sheng Yao*, Ting-Ting Li, Jun Xu, Lai-En Jiang and Ban-Feng Ruan
School of Medical Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China
Abstract: Eight aromatic amino acid derivatives (9a-9h) as gastrin-releasing peptide receptor (GRPR) antagonists were
designed and synthesized. For the design, the tertiary structure of GRPR was predicted by blast searching in the premise
of 1u19 protein as the template. Eight target compounds were docked into the binding pocket to investigate their possible
binding interactions. Their anti-itch activities were tested by intrathecal injection using Kunming mice as experimental
animals and chloroquine phosphate as a modeling medium. Compounds 9e and 9f significantly inhibited scratching be-
haviors. The anti-itch activities of these compounds decreased with the following sequence: 9e > 9f = 9d > 9b > 9g > 9a >
9h > 9c predicted by computer-aided drug design (CADD) and 9e > 9f > 9b > 9h > 9g > 9d > 9a > 9c evaluated by pre-
liminary test. They were broadly in line with activity order pretested by CADD. It showed that the predicted tertiary struc-
ture of GRPR could be used for antipruritic drug design.
Key Words: Anti-itch activity, Aromatic amino acid derivatives, Computer-aided drug design, Gastrin-releasing peptide recep-
tor, 2-(1H-imidazol-1-yl) ethanol, Molecular modeling, Pruritus, Tertiary structure.
1. INTRODUCTION
tion 26-27 GRP analogs (class 3), desMet14 or GRP27 ana-
logs (class 4), peptoids (class 5), and finally the nonpeptide
analogs, kuwanon G and kuwanon H (class 6) [10-12]. Here-
into, PD 176252 is a peptoid antagonist that has nanomolar
affinity for GRPR (Ki = 1 nM). Subsequent studies demon-
strated that PD 176252 inhibited the growth of lung cancer
cells, potentiated the growth inhibitory effects of histone
deacetylase inhibitors; inhibited GRP/Bnstimulated signaling
in lung cancer cells (Ca2+ and tyrosine phosphorylation of
p125FAK) and the stimulation of increases in c-fos mRNA
and growth, and in rats had an anxiolytic effect in vivo [13].
All classes are peptides or peptoid antagonists, except for
class 6, which are flavone derivatives, isolated from extracts
of the mulberry tree Morus bombycis [14].
Itch is an unpleasant sensation that causes the desire or
reflex to scratch. It usually caused by skin disease, biliary
disease, thyroid disease, kidney disease, cancer, AIDS and so
on [1-3]. Chronic itch is difficult to treat [4]. However, gas-
trin-releasing peptide receptor, situated in the central nervous
system, is the first gene directly related to the itch which
confirmed by Yan-Gang Sun and Zhou-Feng Chen in 2007
[5, 6]. So GRPR may be a central target for therapeutic
treatment of chronic and intractable pruritus [5].
Gastrin-releasing peptide receptor belongs to the family
of the bombesin-like peptides (BLP) [7]. The human GRPR
has 384 amino acids. Hydropathy analysis of the predicted
GRPR structure revealed seven regions of hydrophobic
amino acids consistent with a seven-transmembrane structure
typical for G protein-coupled receptors [8]. However, the
tertiary structure of GRPR had not been reported.
So far, few small molecule compounds with anti-itch ac-
tivity as GRPR antagonists have been reported. The GRPR
antagonists are mainly peptides, and they are not suitable as
molecular design reference for the high molecular weights
and complex structures. The non-peptides, kuwanon G and
kuwanon H (Fig. 1), have too many rotatable bonds and are
difficult to synthesize by chemical methods. So they also
don’t fit as molecular design reference. The peptoids, espe-
cially PD 176252 with high affinity, can be used as refer-
ence. To discover some innovative drugs for anti-itch, the
tertiary structure of GRPR was predicted with a homologous
protein, then with the reported GRPR antagonists, especially
PD 176252, eight compounds (Fig. 2) were designed, syn-
thesized and preliminarily tested the anti-itch activity.
In humans, GRPR is highly expressed in the pancreas
and is also expressed in the stomach, adrenal cortex and
brain [9]. GRPR activation has been proposed to be impor-
tant in the mediation of a number of human disorders includ-
ing disorders of lung development, various pulmonary dis-
eases, CNS disorders, and the growth/differentiation of hu-
man cancers [10].
There have been a large number of different compounds
reported to function as GRPR antagonists. They can be di-
vided into six general classes. These six classes include sub-
stituted substance P analogs (class 1), [D-Phe12] bombesin
analogs (class 2), modified position 13-14 bombesin or posi
2. RESULTS AND DISCUSSION
2.1. Chemistry
Compounds 9a-9h were composed of three fragments,
see Fig. (2). They were synthesized of L-phenylalanine or L-
tryptophan (fragment 2) as the basic skeleton by esterifica-
*Address correspondence to this author at the School of Medical Engineer-
ing, Hefei University of Technology, Hefei 230009, People’s Republic of
China; Tel: +86-0551-2901771; Fax: +86-0551-2904675;
E-mail: rishengyao@163.com
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

866 Medicinal Chemistry, 2012, Vol. 8, No. 5
Yao et al.
HO
OH
H
HO
NH
HO
OH
H
H
O
O2N
OH
O
HO
O
O
H
N
N
N
H
H
N
O
OH
Kuwanon H
O
PD 176252
Fig. (1). The Structures of PD 176252 and Kuwanon H.
R1
R2
O
O
O
R3
N
H
1
2
3
Comp. No
R1
R2
R3
9a
9b
9c
9d
9e
9f
NO2
F
phenyl
phenyl
1-imidazole
1-imidazole
1-imidazole
1-imidazole
1-imidazole
1-imidazole
4-morpholine
CH3
NO2
F
phenyl
3-indole
3-indole
3-indole
3-indole
3-indole
CH3
F
9g
9h
F
4-(1-methylpiperazine)
Fig. (2). The Structures of Compounds 9a-9h.
tion with the methanol, amidation with alphatoluic acid de-
rivatives (fragment 1), then deprotection and esterification
with ethanol derivatives (fragment 3).
the new prepared acetyl chloride derivative 6 to a solution of
4. Then, the hydrolysis of 7 was conducted in a mixed sol-
vent of MeOH and water in a (4:1, V/V) ratio and in the pres-
ence of Na2CO3 [19]. Finally, compounds 9a-9f were pre-
pared by the reaction of DCC condensation. Compounds 9g
and 9h were synthesized applying the same preparation pro-
tocols followed in Scheme 2. The only difference was the
use of 2-morpholinoethanol and 2-(4-methylpiperazin-1-yl)
ethanol instead of 2-(1H-imidazol- 1-yl) ethanol.
In Scheme 1, ethyl 2-(1H-imidazol-1-yl) acetate (1) was
prepared in 79.0% yield by refluxing imidazole in THF with
ethyl 2-chloroacetate for 10 h [15, 16]. NaBH4 was slowly
added to a solution of 1 in MeOH at 0 °C. Then heating to 50
°C, 2-(1H-imidazol-1-yl) ethanol (2) was obtained for 8 h in
a good yield [17]. While 2-morpholinoethanol and 2-(4-
methylpiperazin-1-yl) ethanol were synthesized with the
total yields of 76.6% and 72.2% applying the same prepara-
tion protocols followed in Scheme 1. The only difference
was the use of morpholine and methylpiperazine instead of
imidazole used in Scheme 1.
The structures of intermediates and target compounds
were confirmed by FT-IR and 1H NMR.
2.2. Molecular Modeling
In order to get a better insight into the binding affinity,
molecular modeling studies were performed using Molecular
Operating Environment (MOE). 2008. 10 software. Since no
crystal structure of GRPR is yet available, the tertiary struc-
ture of GRPR was predicted using the crystal structure given
in PDB code 1u19 (Fig. 3).
As shown in Scheme 2, compounds 9a-9f were synthe-
sized using L-Phenylalanine or L-Tryptophan as starting
material. Firstly, SOCl2 was added to MeOH at 0 °C for 1 h.
After the addition of 3 and refluxing for 5 h, 4 was obtained
[18]. Secondly, 7 was carried out by the dropwise addition of

Design, Synthesis and Anti-itch Activity Evaluation of New GRPR Antagonists
COOC2H5
Medicinal Chemistry, 2012, Vol. 8, No. 5 867
OH
i
N
ii
N
N
NH
N
N
1
2
Scheme 1. Reagents and Conditions: (i) Ethyl 2-chloroacetate, NEt3, THF, Reflux, 10 h, 79%; (ii) NaBH4, MeOH, 0 °C to 50 °C, 8 h, 90%.
R2
R2
O
OH
i
H2N
H2N
R1
R2
O
O
O
O
iii
3
4
O
N
H
R1
R1
7a-7f
ii
COOH
COCl
5
6
R1
R2
R1
R2
O
O
iv
v
OH
O
N
N
N
H
N
H
O
O
8a-8f
9a-9f
Scheme 2. Reagents and Conditions: (i) SOCl2, MeOH, 0 °C to Reflux, 5 h; (ii) (COCl)2, NEt3, Cat. DMF, DCM, N2, 0 °C to rt, 2 h; (iii)
NEt3, DCM, N2, 0 °C to rt, 5 h; (iv) Na2CO3, MeOH/H2O (4:1), rt, 6 h; (v) 2-(1H-imidazol-1-yl)ethanol, DCC, cat. DMAP, THF, rt, 15 h.
Fig. (3). The Tertiary Structure of GRPR.
As well as GRPR, 1u19 protein belongs to the family of
G protein coupled receptor. By blast searching, the two pro-
teins have a high degree of similarity in the structure and the
identities and positives are 22% and 42%, respectively. So
1u19 protein was chosen as the template of homology mod-
eling.
In compound 9a (Fig. 4), the benzene ring of L-
phenylalanine establishes an arene-cation interaction with
Lys-188. The left C=O forms a hydrogen bonding interaction
with the backbone NH of Lys-265 and the right C=O forms a
hydrogen bonding interaction with Val-187 through H2O.
The 4-nitro-benzene shows an arene-cation interaction with
His-186. The binding interactions of compounds 9b and 9c
(Table 1) are the same with 9a. Compounds 9d and 9f both
have one more interaction with His-207 than 9a. Compound
9e as well as 9a, interacts with Lys-265, Val-187, and His-
Compounds 9a-9h were docked over the ligand atoms in
the binding site. They occupy nearly the same space in the
binding pocket, and are overlaid well over the skeleton of the
ligand.

868 Medicinal Chemistry, 2012, Vol. 8, No. 5
Yao et al.
of L-tryptophan shows an interaction with His-207, while the
benzene ring of L-phenylalanine does not.
2.3. Anti-itch Activity
Compounds 9a-9h were tested the anti-itch activity. In
the experiments, half female and half male Kunming mice
were used as experimental animals and chloroquine phos-
phate was used as a modeling medium [5, 20]. The treatment
animals were given intrathecal injection of the solution of
target compounds (10 nmol/2.5 μL). While the control ani-
mals were given the solution not containing compounds.
After 10 min, the chloroquine phosphate solution was ap-
plied to the nape adopting subcutaneous injection [21].
It was found that various concentrations (200 μg/100 μL,
400 μg/100 μL, 600 μg/100 μL, 645 μg/100 μL, 800 μg/100
μL) of chloroquine phosphate made scratching behaviors
significantly different. When the concentrations ꢀ 600 μg/
100 μL, scratching frequency was faster and faster as the
concentration increased. After 15 min of the injection of 600
μg/100 μL, scratching behaviors appeared, and lasted for 1 h.
While the injection of 645 μg/100 μL, scratching behaviors
appeared after 2 min, and lasted for 0.5 h. When injecting
800 μg/100 μL, backwardness emerged after 2 min, and
scratching with a high frequency returned to normal after 25
min. So 645 μg/100 μL was chosen as the dose of itch mod-
eling and 0.5 h was chosen as the time of counting the num-
ber of scratching behaviors.
The result of anti-itch activity of the target compounds
was shown in Fig. (5). Compounds 9e and 9f had significant
activities and compounds 9b, 9d, 9g and 9h had obvious
activities. The concrete anti-itch activity decreased with the
following sequence: 9e > 9f > 9b > 9h > 9g > 9d > 9a > 9c.
By preliminary test, the optimum structure of fragment 1 was
2-(4-fluorophenyl) acetic acid, fragment 2 was L-tryptophan
and fragment 3 was 2-(1H-imidazol-1-yl) ethanol. It was
speculated that polycyclic aromatic structure of amino acid
(fragment 2) may increase the activities. For it was near the
active site of amino acid in space, and could enhance the
electrostatic force with the active site.
Compared with the two results by CADD and prelimi-
nary test, the activity of compound 9d was worse than 9f,
while they were the same by CADD. These two compounds
were similar in structure and the only difference between
them lied in the replacement of the nitro group of 9d with a
methyl group in 9f. This change of group R1 was associated
with a large difference in activity, which suggested a possi-
ble critical binding role for this group in the binding site of
the molecular target [22]. The activity of 9h was better than
9g, while they were on the contrary by CADD. It is to be
presumed that the 4-(1-methylpiperazine)-yl (group R3) has
two nitrogen atoms for converting into salt while 4-
morpholine-yl has one, and the water-solubility of 9h may be
greater than 9g in vivo. Further study should be emphasized
on this aspect so as to comprehend the differences clearly.
Fig. (4). 2D-presentation for the Binding Interactions of Compound
9a, 9d, 9e and 9f with Receptor.
186 respectively. The difference of the two compounds is
that the pyrrole ring of L-tryptophan establishes the arene-
cation interaction with His-207 rather than Lys-188. The
binding interactions of 9g and 9h are the same with 9e.
With the binding interactions of the eight compounds by
CADD, the anti-itch activity of them decreased with the fol-
lowing sequence: 9e > 9f = 9d > 9b > 9g > 9a > 9h > 9c.
Eight compounds mainly form interactions with His-186,
Val-187, Lys-188 and Lys-265. In addition, the pyrrole ring

Design, Synthesis and Anti-itch Activity Evaluation of New GRPR Antagonists
Medicinal Chemistry, 2012, Vol. 8, No. 5 869
Table 1. The Binding Interactions of Compounds 9a-9h by CADD
Comp. No
Structure
Binding Interactions
O2N
O
9a
Lys-188, Lys-265, Val-187, His-186
O
N
N
N
H
O
Lys-188, Lys-265, Val-187, His-186
F
O
9b
O
N
N
N
H
O
Lys-188, Lys-265, Val-187, His-186
O
9c
O
N
H
N
N
O
NH
O2N
9d
9e
9f
Lys-188, Lys-265, Val-187, His-186, His-207
O
O
N
N
N
H
O
NH
O
F
Lys-265, Val-187, His-186, His-207
O
N
N
N
H
O
NH
O
Lys-188, Lys-265, Val-187, His-186, His-207
O
N
H
N
N
O
NH
O
Lys-265, Val-187, His-186, His-207
F
O
9g
N
H
N
O
O
NH
Lys-265, Val-187, His-186, His-207
F
O
9h
O
N
N
H
O
N

870 Medicinal Chemistry, 2012, Vol. 8, No. 5
Yao et al.
Fig. (5). The Anti-itch Activity Results of Compounds 9a-9h. The First line was the Number of Scratching Behaviors of Control Animals and
the others were the Numbers of Scratching Behaviors of Treatment Animals. All the Treatments Resulted in a Significant Decrease in
Scratching Numbers Compared to the Control (p < 0.05).
3. CONCLUSION
(COCl)2 (1.84 mL, 21.6 mmol) in DCM (10 mL) was slowly
added to the mixture. After the dropwise addition, the mix-
ture was stirred at room temperature for 2 h. Solvent and
excess (COCl)2 was removed under vacuum. The brown
solid was obtained, which was directly used in the next step.
Eight compounds were designed and synthesized. Com-
pounds 9e and 9f, which had prominence activities, were
screened out as GRPR activity antagonists. The optimum
structure of fragment 1 was 2-(4-fluorophenyl) acetic acid,
fragment 2 was L-tryptophan and fragment 3 was 2-(1H-
imidazol-1-yl) ethanol. The anti-itch activity of them de-
creased with the following sequence: 9e > 9f = 9d > 9b > 9g
> 9a > 9h > 9c by CADD and 9e > 9f > 9b > 9h > 9g > 9d >
9a > 9c by preliminary test. On the whole, they were broadly
in line with activity order pretested by CADD. It showed that
the predicted GRPR tertiary structure was of a certain refer-
ence value and it can be used for antipruritic drug design.
The suspension of new prepared acetyl chloride deriva-
tive 6 in DCM (15 mL) was added to the mixture of 4 (18
mmol), TEA (5.2 mL, 36 mmol) and DCM (20 mL) at 0 °C
under the protection of N2 gas. After the dropwise addition,
the mixture was stirred at room temperature for 5 h. Com-
pounds 7a-7h were obtained by column chromatography
(silica gel, 200-300 mesh, ethyl acetate/petroleum ether, 1:2,
V/V).
4.2.1.1. (R)-methyl-2-(2-(4-nitrophenyl)acetamido)-3-
phenylpropanoate (7a)
4. EXPERIMENTAL SECTION
4.1. General
The title compound was separated by column chromatog-
1
1
raphy. Yield (5.62 g, 91.2%); yellow solid; H NMR (CD-
The H NMR spectra were recorded on a Bruker 300
Cl3) ꢀ: 6.94-8.17 (m, 9H), 4.88 (m, 1H), 3.72 (s, 3H), 3.66 (s,
2H), 3.11 (d, 2H); IR ꢁ/cm-1: 3284, 2925, 1743, 1665, 1542,
1345, 1280, 1172, 825, 701.
MHz spectrometer. The chemical shifts were expressed in
ppm using residual CDCl3 and (CD3)2SO as reference. The
FT-IR spectra were recorded in solid-state KBr dispersion on
a Perkins-Elmer FT-IR spectrophotometer. The melting
points were taken on a Buchi apparatus. All reactions were
monitored by TLC with HuanghaiGF 254 silica gel-coated
plates. Commercially obtained reagents were used without
further purification. Compounds 1, 2 and 4 were prepared
according to the literature methods as described [15-18].
4.2.1.2.
(R)-methyl-2-(2-(4-fluorophenyl)acetamido)-3-
phenylpropanoate (7b)
The title compound was separated by column chromatog-
1
raphy. Yield (5.23 g, 92.3%); cream white solid; H NMR
(CDCl3) ꢀ: 6.89-7.26 (m, 9H), 4.85 (m, 1H), 3.71 (s, 3H),
3.50 (s, 2H), 3.06 (dd, 2H); IR ꢁ/cm-1: 3285, 2953, 1755,
1669, 1546, 1512, 1364, 1222, 705.
4.2. Synthesis
4.2.1.3. (R)-methyl-3-phenyl-2-(2-p-tolylacetamido) propa-
noate (7c)
4.2.1. General Procedure for the Synthesis of Compounds
7a-7h
The title compound was separated by column chromatog-
raphy. Yield (4.89 g, 87.3%); white solid; H NMR (CDCl3)
ꢀ: 6.87-7.24 (m, 9H), 4.84 (m, 1H), 3.69 (s, 3H), 3.48 (s,
The mixture of alphatoluic acid derivatives (5) (18
mmol), DCM (30 mL), TEA (2.6 mL, 18 mmol) and DMF
(0.1 mL) was stirred at 0 °C under the protection of N2 gas.
1

Design, Synthesis and Anti-itch Activity Evaluation of New GRPR Antagonists
Medicinal Chemistry, 2012, Vol. 8, No. 5 871
2H), 3.02 (d, 2H), 2.34 (s, 3H); IR ꢀ/cm-1: 3319, 2925, 1757,
3.06 (d, 2H), 2.35 (s, 3H); IR ꢀ/cm-1: 3358, 2924, 1770,
1646, 1533, 1438, 1373, 1275, 1171, 782, 699.
1743, 1611, 1534, 1396, 1209, 1170, 784, 700.
4.2.1.4. (R)-methyl-3-(1H-indol-3-yl)-2-(2-(4-
nitrophenyl)acetamido)propanoate (7d)
4.2.2.4. (R)-3-(1H-indol-3-yl)-2-(2-(4-
nitrophenyl)acetamido)propanoic acid (8d)
The title compound was separated by column chromatog-
raphy. Yield (6.03 g, 87.8%); brown solid; 1H NMR (CDCl3)
ꢀ: 7.03-8.04 (m, 9H), 4.91 (m, 1H), 3.72 (s, 3H), 3.54 (s,
2H), 3.29 (d, 2H); IR ꢁ/cm-1: 3381, 2925, 1734, 1655, 1520,
1438, 1346, 1106, 741.
The title compound was evaporated under vacuum. Yield
(1.68 g, 91.4%); brown solid; mp 211~214 °C; H NMR
(CD3)2SO-d6) ꢁ: 10.84 (s, 1H), 6.94-8.50 (m, 9H), 4.51 (m,
1H), 3.59 (s, 2H), 3.15 (d, 2H); IR ꢀ/cm-1: 3402, 3305, 2926,
1711, 1618, 1515, 1348, 1243, 747.
1
4.2.1.5. (R)-methyl-2-(2-(4-fluorophenyl)acetamido)-3-(1H-
indol-3-yl)propanoate (7e)
4.2.2.5. (R)-2-(2-(4-fluorophenyl)acetamido)-3-(1H-indol-
3-yl)propanoic acid (8e)
The title compound was separated by column chromatog-
raphy. Yield (5.82 g, 91.2%); cream white solid; H NMR
The title compound was evaporated under vacuum. Yield
(1.62 g, 95.2%); cream white solid; mp 196~197 °C; H
1
1
(CDCl3) ꢁ: 8.07 (s, 1H), 6.74-7.44 (m, 9H), 4.90 (m, 1H),
3.68 (s, 3H), 3.45 (s, 2H), 3.25 (d, 2H); IR ꢀ/cm-1: 3407,
3291, 2923, 1733, 1662, 1547, 1513, 1451, 1361, 1220, 830,
746.
NMR (CD3)2SO-d6) ꢁ: 10.83 (s, 1H), 8.32 (s, 1H), 6.94-7.52
(m, 9H), 4.47 (m, 1H), 3.42 (s, 2H), 3.14 (dd, 2H); IR ꢀ/cm-1:
3408, 3309, 2925, 1709, 1615, 1508, 1460, 1218, 1170, 793,
746.
Compounds 7g and 7h are the same as 7e.
Compounds 8g and 8h are the same as 8e.
4.2.1.6. (R)-methyl-3-(1H-indol-3-yl)-2-(2-p-
tolylacetamido)propanoate (7f)
4.2.2.6. (R)-3-(1H-indol-3-yl)-2-(2-p-
tolylacetamido)propanoic acid (8f)
The title compound was separated by column chromatog-
raphy. Yield (5.74 g, 91.1%); white solid; H NMR (CDCl3)
ꢁ: 8.02 (s, 1H), 6.72-7.42 (m, 9H), 4.90 (m, 1H), 3.66 (s,
3H), 3.48 (s, 2H), 3.24 (d, 2H), 2.32 (s, 3H); IR ꢀ/cm-1: 3331,
2924, 2855, 1751, 1652, 1517, 1461, 1377, 1198, 753, 659.
The title compound was evaporated under vacuum. Yield
(1.63 g, 96.9%); gray oily substance; 1H NMR (CD3)2SO-d6)
ꢁ: 10.83 (s, 1H), 8.23 (s, 1H), 6.94-7.53 (m, 9H), 4.48 (m,
1H), 3.38 (s, 2H), 3.06 (d, 2H), 2.25 (s, 3H); IR ꢀ/cm-1: 3403,
2924, 1728, 1624, 1515, 1344, 1232, 1100, 743.
1
4.2.2. General Procedure for the Synthesis of Compounds
8a-8h
4.2.3. General Procedure for the Synthesis of Compounds
9a-9f
Compound 7 (5 mmol) was added to a solution of MeOH
(32 mL), H2O (8 mL) and Na2CO3 (0.79 g, 7.5 mmol) [19].
The mixture was stirred at room temperature for 6 h. MeOH
was removed under vacuum, and H2O (20 mL) was added to
the residue. pH was adjusted to 5 to 6 using 3 N HCl. The
mixture was extracted with ethyl acetate (50 mL ꢂ 3). The
organic layer was separated, dried over anhydrous Na2SO4
and then evaporated under vacuum.
A mixture of compound 8 (4 mmol), THF (10 mL), DCC
(0.99 g, 4.8 mmol) and DMAP (10 mg) was stirred at 0 °C
for 1 h. Compound 2 in THF (5 mL) was added to the solu-
tion. The mixture was stirred at room temperature for 15 h.
Target compound was obtained by column chromatography
(silica gel, 200-300 mesh, DCM/MeOH, 70:1, V/V).
4.2.3.1.(R)-2-(1H-imidazol-1-yl)ethyl-2-(2-(4-
nitrophenyl)acetamido)-3-phenylpropanoate (9a)
4.2.2.1. (R)-2-(2-(4-nitrophenyl)acetamido)-3-
phenylpropanoic acid (8a)
The title compound was separated by column chromatog-
raphy. Yield (1.52 g, 90.1%); brown oily substance; 1H
NMR (CDCl3) ꢁ: 6.92-8.15 (m, 12H), 4.78 (m, 1H), 4.36 (t,
2H), 4.15 (t, 2H), 3.61 (s, 2H), 3.01 (d, 2H); IR ꢀ/cm-1: 3206,
2941, 1746, 1641, 1532, 1350, 1283, 1178, 820, 698.
The title compound was evaporated under vacuum. Yield
1
(1.53 g, 93.2%); yellow solid; mp 165~168 °C; H NMR
(CD3)2SO-d6) ꢁ: 12.73 (s, 1H), 7.19-8.53 (m, 9H), 4.45 (m,
1H), 3.57 (s, 2H), 3.01 (d, 2H); IR ꢀ/cm-1: 3319, 2894, 1711,
1621, 1520, 1347, 1246, 1050, 831, 700.
4.2.3.2.(R)-2-(1H-imidazol-1-yl)ethyl-2-(2-(4-
fluorophenyl)acetamido)-3-phenylpropanoate (9b)
4.2.2.2. (R)-2-(2-(4-fluorophenyl)acetamido)-3-
phenylpropanoic acid (8b)
The title compound was separated by column chromatog-
raphy. Yield (1.43 g, 90.5%); yellow oily substance; 1H
NMR (CDCl3) ꢁ: 8.02 (s, 1H), 6.72-7.42 (m, 9H), 4.90 (m,
1H), 3.66 (s, 3H), 3.48 (s, 2H), 3.24 (d, 2H), 2.32 (s, 3H); IR
ꢀ/cm-1: 3201, 2932, 1747, 1658, 1543, 1509, 1355, 1222,
1159, 825, 702.
The title compound was evaporated under vacuum. Yield
(1.41 g, 94.0%); cream white solid; mp 152~154 °C; H
NMR (CDCl3) ꢁ: 6.94-7.26 (m, 9H), 4.81 (m, 1H), 3.51 (s,
2H), 3.08 (d, 2H); IR ꢀ/cm-1: 3295, 2928, 1709, 1613, 1559,
1355, 1251, 1225, 1158, 825, 700.
1
4.2.3.3.(R)-2-(1H-imidazol-1-yl)ethyl-3-phenyl-2-(2-p-
tolylacetamido)propanoate (9c)
4.2.2.3. (R)-3-phenyl-2-(2-p-tolylacetamido)propanoic acid
(8c)
The title compound was separated by column chromatog-
raphy. Yield (1.41 g, 90.0%); white oily substance; 1H NMR
(CDCl3) ꢁ: 6.81-7.35 (m, 12H), 4.75 (m, 1H), 4.21 (t, 2H),
The title compound was evaporated under vacuum. Yield
(1.34 g, 90.4%); white solid; mp 124~125 °C; H NMR
(CDCl3) ꢁ: 6.91-7.25 (m, 9H), 4.80 (m, 1H), 3.49 (s, 2H),
1

872 Medicinal Chemistry, 2012, Vol. 8, No. 5
Yao et al.
4.08 (t, 2H), 3.49 (s, 2H), 2.95 (d, 2H), 2.34 (s, 3H); IR ꢀ/cm-1:
3206, 2941, 1746, 1641, 1532, 1350, 1283, 1178, 820, 698.
800 μg/100 μL) of chloroquine phosphate solutions were
prepared, filtered by 0.22 μm water film, and loaded 1 mL in
each sample device in the sterile console.
4.2.3.4.(R)-2-(1H-imidazol-1-yl)ethyl-3-(1H-indol-3-yl)-2-
(2-(4-nitrophenyl)acetamido)propanoate (9d)
4.3.2.2. The Preparation of Eight Target Compounds Solu-
tions
The title compound was separated by column chromatog-
raphy. Yield (1.72 g, 93.2%); brown oily substance; 1H
NMR (CDCl3) ꢁ: 8.78 (s, 1H), 6.65-8.02 (m, 12H), 4.81 (m,
1H), 4.26 (t, 2H), 3.96 (t, 2H), 3.55 (s, 2H), 3.17 (dd, 2H);
IR ꢀ/cm-1: 2924, 1744, 1662, 1518, 1346, 1184, 822, 745.
With saline as a solvent, and 2 mL Tween 80 and 2.5 mL
glycerol as solubilizers, 10 nmol/2.5 μL eight target com-
pounds solutions were prepared, filtered by 0.22 μm water
film, and loaded 1 mL in each sample device in the sterile
console.
4.2.3.5.(R)-2-(1H-imidazol-1-yl)ethyl-2-(2-(4-
fluorophenyl)acetamido)-3-(1H-indol-3-yl)propanoate (9e)
4.3.3. Statistical Analysis
The title compound was separated by column chromatog-
raphy. Yield (1.63 g, 93.8%); yellow oily substance; H
NMR (CDCl3) ꢁ: 8.67 (s, 1H), 6.58-7.38 (m, 12H), 4.81 (m,
1H), 4.22 (t, 2H), 3.89 (t, 2H), 3.46 (s, 2H), 3.17 (dd, 2H);
IR ꢀ/cm-1: 3274, 1744, 1657, 1509, 1356, 1223, 825, 745.
Data were expressed as mean ± standard deviation and
they were analyzed by Student’s t-test. Values of p < 0.05
were considered significant.
1
CONFLICT OF INTEREST
4.2.3.6.(R)-2-(1H-imidazol-1-yl)ethyl-3-(1H-indol-3-yl)-2-
(2-p-tolylacetamido)propanoate (9f)
The author(s) confirm that this article content has no con-
flicts of interest.
The title compound was separated by column chromatog-
raphy. Yield (1.62 g, 94.1%); white oily substance; 1H NMR
(CDCl3) ꢁ: 8.36 (s, 1H), 6.56-7.38 (m, 12H), 4.81 (m, 1H),
4.20 (t, 2H), 3.86 (t, 2H), 3.48 (s, 2H), 3.15 (dd, 2H), 2.32 (s,
3H); IR ꢀ/cm-1: 3265, 2924, 1744, 1656, 1514, 1438, 1355,
1233, 1187, 745.
ACKNOWLEDGMENTS
This work was supported by Key Program of Natural
Science Research by Education Department of Anhui Prov-
ince (2011AJZR0919) and Condition Construction Cost of
Hefei University of Technology (407-037022).
4.2.3.7.(R)-2-morpholinoethyl-2-(2-(4-
fluorophenyl)acetamido)-3-(1H-indol-3-yl)propanoate (9g)
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Received: July 18, 2011
Revised: April 25, 2012
Accepted: April 26, 2012