Synthesis and biological evaluation of Matijing-Su derivatives as potent anti-HBV agents

Article abstract of DOI:10.1016/j.bmc.2011.08.001

A series of Matijing-Su (MTS, N-(N-benzoyl-l-phenylalanyl)-O-acetyl-l- phenylalanol) derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activity in 2.2.15 cells. The IC₅₀ of compounds 14a (0.71 μM), 13c (2.85 μM), 13b (4.37 μM), etc. and the selective index of 13g (161.01), 13c (90.45), 13a (85.09) etc. of the inhibition on the replication of HBV DNA were better than those of the positive control lamivudine (IC₅₀: 82.42 μM, SI: 41.59). Compounds 13o, 13p, and 16a also exhibited significant anti-HBV activity.

Full text of DOI:10.1016/j.bmc.2011.08.001

Bioorganic & Medicinal Chemistry 19 (2011) 5352–5360
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of Matijing-Su derivatives as potent
anti-HBV agents
Jingying Qiu a,b, Bixue Xu b, Zhengming Huang c, Weidong Pan b, Peixue Cao b, Changxiao Liu d,
Xiaojiang Hao b, Baoan Song a, Guangyi Liang b,e,
⇑
a Center for Research and Development of Fine Chemicals of Guizhou University, Huaxi Road, Guiyang 550025, PR China
b The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, 202 Shachong South Road, Guiyang 550002, PR China
c 302 Hospital of PLA, 100 Xisihuan Zhong Road, Fengtai, Beijing 100039, PR China
d Tianjin Institute of Pharmaceutical Research, 308 Anshan Xi Dao, Nankai District, Tianjin 300193, PR China
e Guiyang College of Traditional Chinese Medicine, 50 Shidong Road, Guiyang 550002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Matijing-Su (MTS, N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-phenylalanol) derivatives were
Received 20 June 2011
Revised 31 July 2011
Accepted 2 August 2011
Available online 6 August 2011
synthesized and evaluated for their anti-hepatitis B virus (HBV) activity in 2.2.15 cells. The IC50 of com-
pounds 14a (0.71 M), 13c (2.85 M), 13b (4.37 M), etc. and the selective index of 13g (161.01), 13c
(90.45), 13a (85.09) etc. of the inhibition on the replication of HBV DNA were better than those of the
l
l
l
positive control lamivudine (IC50: 82.42
nificant anti-HBV activity.
l
M, SI: 41.59). Compounds 13o, 13p, and 16a also exhibited sig-
Keywords:
MTS derivatives
Synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
Anti-hepatitis B virus activity
1. Introduction
showed the significative inhibitions on the replication of HBV
DNA and the SI and IC50 of them were better than those of the po-
sitive control lamivudine. These results greatly encouraged us to
make further research on it for discovery of more potent anti-
HBV derivatives. In this paper, the synthesis of new MTS deriva-
tives and the evaluation of their anti-HBV activity were reported.
Despite the presence of an effective vaccine, hepatitis B virus
(HBV) infection remains a global public health problem with over
400 million people chronically infected worldwide.1,2 Approxi-
mately, 80% of carriers have different levels of hepatocyte destruc-
tion, which may develop into liver cirrhosis and hepatocellular
carcinoma.3 HBV causes acute and chronic infections of the liver
and is responsible for 1.2 million deaths annually.4,5 Nowadays,
at least two different treatment options, including interferon and
nucleoside analogs, such as lamivudine, adefovir dipivoxil, and ent-
ecavir are considered as antiviral therapy for chronic hepatitis B
infection.6 Although various treatment options exist for chronic
HBV infection, none is entirely satisfactory.7–11 The development
of new anti-HBV agents is focused on discovering diverse com-
pounds with either novel chemical structures or novel anti-HBV
targets and mechanisms.12,13
2. Results and discussion
2.1. Chemistry
The synthesis of the key synthetic intermediates was described
in Scheme 1. 2-Chloroacetyl chloride was added to the solution of
4-amino-benzoic acid in 1.0 M NaOH to produce compound 2.16
Ac2O or (EtCO)2O was treated with the solution of 4-amino-benzoic
acid in pyridine to obtain compound 3 or 4. N-Bromosuccinimide
(NBS) and azobisisobutyronitrile (AIBN) were added to the solution
of methyl 4-methylbenzoate in dry CCl4 to give compound 6,17
which was dropped into the mixture of amine, K2CO3, and KI in
ethanol. The product was salified with HCl to produce compound
Matijing-Su (MTS, N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-
phenylalanol), a dipeptide derivative, was isolated from a Chinese
ethnic drug Matijing (Dichondra repens Forst.) which has been
widely used in the treatment of chronic liver diseases as folk med-
icine in China. The anti-HBV activity of MTS and its derivatives had
been reported in our previous studies.14,15 Some derivatives
7. The solution of L-phenylalanine in H2SO4 was reacted with a
mixed acid of concentrated HNO3 and concentrated H2SO4 to
obtain compound 9,18 which was treated with SOCl2 to afford
compound 10.
The procedure for synthesis of the target compounds was illus-
trated in Scheme 2. The substituted L-phenylalanine methyl ester
⇑
Corresponding author. Tel./fax: +86 851 5652109.
E-mail address: guangyi_liang@126.com (G. Liang).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.001

J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
5353
COOH
COOH
a
NHCOCH2Cl
2
NH2
1
COOH
COOH
COOH
b
NHAc
3
NH2
1
NHCOEt
4
O
O
O
c
d
e
H3CO
H3CO
Br
O
HO
R2
O
6
7
5
O
OH
g
OCH3
NH2HCl
f
OH
NH2
NH2
O2N
O2N
9
10
8
Scheme 1. Synthesis of the key intermediates. Reagents and conditions: (a) ClCH2COCl, 1.0 M NaOH, ꢀ5 °C, 2 h, then stirred overnight, rt; (b) Ac2O/(EtCO)2O, pyr., rt; (c) NBS,
AIBN, CCl4, reflux for 12 h; (d) amine, K2CO3, KI, EtOH, rt, 24 h; (e) 1.0 M HCl, reflux for 8 h; (f) HNO3/H2SO4 (V:V = 1.4:1.1), 10 °C, 2.5 h; (g) MeOH, thionyl chloride (SOCl2), rt,
2.5 h, then reflux for 30 min.
O
X
O
O
OCH3
O
OH
O
OCH3
NH2HCl
HN
HN
a
b
R1
R1
R1
R2
R2
10
12
11
X
c
O
R4
O
R4'
N
H
O
N
H
O
d
14
HN
HN
R1'
e
R1
15
g
f
16
R2'
17
R2
13
R3'
R3
X
X
Scheme 2. Synthesis of the target compounds. Reagents and conditions: (a) substituted benzoic acid or isonicotinic acid, CH2Cl2, IBCF, NMM, 0 °C; (b) NaOH, DMF, rt; (c)
substituted -phenylalanine methyl ester hydrochloride or substituted -phenylalanol, CH2Cl2, IBCF, NMM, 0 °C; (d) SOCl2, 1,4-dioxane, 70 °C; (e) Ac2O, pyr, rt; (f) K2CO3, DMF,
rt; (g) NaOH/EtOH–CHCl3, rt.
L
L
was treated with substituted benzoic acid or isonicotinic acid to
give compound 11,19 which was then hydrolyzed in the 1 M solu-
tion of sodium hydroxide to give compound 12. Compound 12 was
2.2. Biological activity test
The target compounds 13a–13p, 14a, 15a, 15b, 16a–16g, and
17a–17c were tested for their abilities to inhibit the replication of
HBV DNA and the production of HBsAg and HBeAg in HBV-infected
2.2.15 cells. The bioactivity of each compound was evaluated by
the combination of its IC50 values and SI. The results of the inhibition
on the replication of HBV DNA were summarized in Table 2.
As shown in Table 2, some MTS derivatives could be potent HBV
inhibitors with high selectivity indices. However, the inhibition on
the production of HBsAg and HBeAg for most of the tested
compounds did not be found in this test, except for compound
reacted with substituted
L-phenylalanine methyl ester hydrochlo-
ride or -phenylalanol to afford compound 13, which was followed
L
by treatment with SOCl2 in 1,4-dioxane to provide compound 14.
Compound 13 was reacted with Ac2O in pyridine at room temper-
ature to provide compound 15. Compound 13 was treated with
chlorides or iodides to obtain compound 16. The target compound
17 was synthesized by hydrolysis of compounds 16 with NaOH in
EtOHACHCl3. Thus, the structures of the target compounds are
listed in Table 1.

5354
J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
Table 1
Structures of the target compounds13a–13p, 14a, 15a, 15b, 16a–16g, and 17a–17c
O
1
R4
2'
3'
5
2
4
6
R1
7
3
N
H
A
8
4'
9
HN
1'' O
5'
9'
8'
R3
B
2''
6'
3''
7''
7'
R2
C
4''
6''
X
5''
No.
R1
R2
R3
7-OCH2CH3
7-O(CH2)3CH3
7-OH
7-NO2
H
H
H
H
H
H
7-NO2
7-OH
H
7-NO2
H
H
H
H
H
H
H
H
H
R4
X
Compound
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
7-OH
7-OH
H
7-OH
7-OH
H
H
H
H
H
H
H
H
H
H
H
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
CH2OH
CH2OH
CH2OH
CH2Cl
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OAc
CH2OAc
COONa
COONa
COONa
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
16a
16b
13a
13b
13c
16c
13d
16d
16e
13e
13f
13g
13h
13i
13j
13k
13l
5-NHAc
5-NHCOEt
5-NHCOEt
5-O(CH2)2N(CH3)2
5-NHCOCH2Cl
5-NHCOCH2N(CH3)2
5-Pyrrolidinylacetamido
H
H
H
H
H
H
H
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
5-NHAc
5-NHAc
7-OH
14a
13m
16f
7-NHCOCH2N(CH3)2
5-CH2N(CH3)2
5-O(CH2)2N(CH3)2
5-Pyrrolidin-1-ylmethyl
5-Morpholinomethyl
5-Morpholinomethyl
5-CH2N(CH3)2
H
13n
16g
13o
13p
15a
15b
17a
17b
17c
H
H
H
H
H
H
7-OCH3
7-OCH2CH3
7-O(CH2)3CH3
H
H
17c, which exhibited significant inhibitory for the inhibition on the
production of HBsAg in 2.2.15 cells.
b. The substituting groups on ring A, B, and C of the MTS deriv-
atives could affect their anti-HBV activities. The 500-acetam-
ido or 500-propionamido substituted on the benzene ring C
and no substituent on the benzene ring A was an important
feature in conferring relatively potent inhibitory activity.
When ring C was pyridine and the 700-hydroxyl was substi-
tuted on ring B, the inhibitory activities of the derivatives
were obviously strengthen. When ring C was pyridine and
the 700-nitro were substituted on ring B, the inhibitory activ-
ities of the derivatives were obviously weaken. Ring C of the
derivatives might be one of the pharmacophores. Most of the
MTS derivatives substituted with pyridine or amines on ring
C exhibited more potent inhibitory activity.
The IC50 of compound 14a was 0.71
times higher than that of the control lamivudine (IC50
82.42 M). Compound 14a, with a SI of 42.26, showed the most po-
tent in vitro anti-HBV activity within all of the tested compounds.
Compounds 13c (IC50: 2.85 M, SI: 90.45), 13b (IC50: 4.37 M, SI:
M, SI: 161.01), 13a (IC50: 9.06 M, SI:
M, SI: 58.61), etc. also exhibited signifi-
lM, which was about 116
:
l
l
l
33.53), 13g (IC50: 5.39
85.09), 16a (IC50: 13.95
l
l
l
cant inhibitory on the replication of HBV DNA in 2.2.15 cells.
3. Conclusions
c. The methyl carboxylate and hydroxymethyl substitution at
20-position were potential pharmacophores for anti-HBV
activities. Whereas both CH2OAc and COONa substitution
at 20-position, the inhibitory activities of the derivatives
were obviously weaken.
A series of novel MTS derivatives were synthesized and their
anti-HBV activities in vitro were examined with lamivudine as ref-
erence control. According to the above experiment results, conclu-
sions could be made as:
a. In general, a series of MTS derivatives exhibited significant
anti-HBV activity. The IC50 of the inhibition on the replica-
tion of HBV DNA of the thirteen MTS derivatives were lower
and the SI of the five MTS derivatives were higher than those
4. Experimental
4.1. Chemistry
of the positive control lamivudine (IC50: 82.42
41.59). Some MTS derivatives could be potent HBV inhibi-
tors, such as the compounds 14a (IC50: 0.71 M, SI: 42.26),
13c (IC50 2.85 M, SI: 90.45), 13g (IC50 5.39 M, SI:
161.01), and 13a (IC50: 9.06 M, SI: 85.09).
lM, SI:
l
Melting points were measured with a model XT-4 apparatus
and are uncorrected. 1H (400 MHz) and 13C (100 MHz) NMR spec-
tra were recorded on a Varian Unit INOVA-400 spectrometer in
:
l
:
l
l

J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
5355
Table 2
The results of the inhibition for the replication of HBV DNA of compounds 13a–13p, 14a, 15a, 15b, 16a–16g, and 17a–17c
a
No.
TC50
(lM)
DNA replication
Compounds
b
IC50
(
lM)
SIc
58.61
6.60
85.09
33.53
90.45
4.74
–
–
–
–
–
1
2
3
4
5
6
7
8
817.70
1148.73
770.59
146.61
257.87
497.10
223.26
110.51
106.33
1057.08
812.44
867.14
219.23
237.82
213.70
612.15
748.87
30.13
13.95
174.09
9.06
4.37
2.85
16a
16b
13a
13b
13c
104.86
16c
d
–
13d
16d
16e
13e
13f
13g
13h
13i
13j
13k
13l
14a
13m
16f
13n
15g
13o
13p
15a
15b
17a
17b
17c
–
–
–
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
MTS
3TC
5.39
–
–
28.92
–
–
0.71
57.83
–
15.84
27.59
14.09
13.91
–
161.01
–
–
7.39
–
–
42.26
3.69
–
11.84
11.86
9.11
11.25
–
1055.00
73.70
187.63
327.30
128.39
156.53
608.96
507.70
184.06
1010.07
979.32
120.18
3427.82
–
–
20.47
–
64.56
11.16
82.42
8.99
–
15.20
10.78
41.59
MTS
Lamivudinee
a
b
c
TC50: 50% toxic concentration in HepG2.2.15 cells.
IC50: 50% inhibitory concentration.
SI (Selectivity index) = TC50/IC50
.
d
e
Means no antiviral activity at a concentration lower than its TC50
Lamivudine: an antiviral agent used as positive control.
.
CDCl3 or DMSO-d6 with Me4Si (TMS) as an internal standard. Mass
spectra (MS) were run on a Agilent MS-5973 spectrometer.
was refluxed for 12 h under an Ar atmosphere. The solid was fil-
tered off and the filtrate was evaporated in vacuo to give methyl
4-bromomethylbenzoate (6) as milk white oil (6.58 g, 96%).
4.1.1. Synthesis of some intermediates
4.1.1.1. 4-(2-Chloroacetamido)-benzoic acid (2). 2-Chloroacetyl
chloride (12 mL, 150.0 mmol) was added to the solution of 4-ami-
no-benzoic acid (1) (13.70 g, 100.0 mmol) in 1.0 M NaOH (220 mL,
220.0 mmol) cooled in an ice-salt bath. The reaction mixture was
stirred at ꢀ5 °C in ice-salt bath for 2 h, then stirred overnightat room
temperature. The reaction was acidified to pH 1–2 with concen-
trated hydrochloric acid. The resulting precipitate was collected by
filtration, washed with water (80 mL) and dried to give 4-(2-chloro-
acetamido)-benzoic acid (2) as a white powder (15.58 g, 73%).
4.1.1.5. 4-Dimethylaminomethyl-benzoic acid hydrochloride
(7). 4-Bromomethylbenzoate ( 6) (11.50 g, 50.2 mmol) was
dropped to the mixture of amine (75.0 mmol), K2CO3 (17.25 g,
125.0 mmol), and KI (1.66 g, 10.0 mmol) in absolute ethanol
(50 mL). The reaction was stirred overnight under an Argon atmo-
sphere. The product was purified by extraction and hydrolyzed
with 1.0 M HCl to give 4-dimethylaminomethyl-benzoic acid
hydrochloride (7) as a pale white powder.
4.1.1.6. 4-Nitro-L-phenylalanine (9). To the solution of L-phenyl-
4.1.1.2. 4-Acetamido-benzoic acid (3). Ac2O (5.7 mL, 60.3 mmol)
was added to the solution of 4-amino-benzoic acid (1) (6.86 g,
50.0 mmol) in pyridine (25 mL). The reaction was stirred at room
temperature for 5 h. The solvent was removed in vacuo and the res-
idue dispersed in water (100 mL) and acidified to pH 2–3 with con-
centrated hydrochloric acid. The resulting precipitate was
collected by filtration, washed with water (30 mL) and dried to give
4-acetamido-benzoic acid (3) as a pale yellow powder (7.80 g, 99%).
alanine (8) (8.26 g, 50.0 mmol) in H2SO4 (85%, 25 mL) was added
dropwise the mixed acid of concentrated HNO3 7 mL and concen-
trated H2SO4 5.5 mL (V/V = 1.4/1.1) under stirring, which was
pre-prepared and cooled down to 10 °C. The reaction mixture
was stirred at 10 °C for 2.5 h. The reaction solution was adjusted
to pH 5–6 with ammonia solution and the resulting precipitate
was collected by filtration and dried to give 4-nitro-L-phenylala-
nine (9) as a pale yellow powder (9.86 g, 94%).
4.1.1.3. 4-Propionamido-benzoic acid (4). It was prepared with
a similar procedure to that described for the synthesis of com-
pound 3.
4.1.1.7. 4-Nitro-L-phenylalanine methyl ester hydrochloride
(10). SOCl2 (4.75 mL, 66.0 mmol) was added to methanol
(45 mL), which was cooled in an ice-salt bath. The mixture was
warmed up to room temperature. 4-Nitro-L-phenylalanine ( 9)
4.1.1.4. 4-Bromomethylbenzoate (6). NBS (6.57 g, 36.9 mmol)
and AIBN (12 mg) were added to the solution of methyl 4-meth-
ylbenzoate (4.51 g, 30.0 mmol) in dry CCl4 (60 mL). The reaction
(10.51 g, 50.0 mmol) was added and the mixture was stirred for
2.5 h at room temperature, then refluxed for 60 min. The solvent
was removed and the residue was recrystallized from methanol/

5356
J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
ether to afford
colorless needles (11.56 g, 88%).
L
-phenylalanine methyl ester hydrochloride (10) as
EI-MS m/z: 546 (M+), 354, 220, 192, 176 (100), 120, 91; TOFESMS:
calcd for C29H30N4O7Na [M+Na]+ 569.2012, found 569.2018.
4.1.2.4. N-(N- 4-Propionamido-benzoyl-L-phenylalanyl)-L-phen-
4.1.2. Synthesis of the target compounds
ylalanine methyl ester (13c). White crystal, yield 62%, mp
225–227 °C (EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 10.09 (1H,
s, Ar-NHCO), 8.52 (1H, d, J = 7.6 Hz, NHCO), 8.40 (1H, d, J = 8.4 Hz,
NHCO), 7.74 (2H, d, J = 8.4 Hz, H-300, 700), 7.64 (2H, d, J = 8.8 Hz,
H-400, 600), 7.34–7.13 (10H, m, H-5, 9, 50, 90), 4.73 (1H, m, H-2),
4.52 (1H, m, H-20), 3.59 (3H, s, OCH3), 3.08–2.91 (4H, m, H-3, 30),
2.35 (2H, q, J = 7.2 Hz, COCH2CH3), 1.08 (3H, t, J = 7.6 Hz,
COCH2CH3); 13C NMR (DMSO-d6, 100 MHz) d: 172.4, 171.82,
171.78, 165.7 (C-100), 142.1 (C-500), 138.4 (C-4), 137.0 (C-40), 129.2
(ꢁ2), 129.1 (ꢁ2), 128.3 (ꢁ4, C-300, 700, 50, 90), 128.1 (C-200), 128.0
(C-5, 9), 126.6 (C-7), 126.2 (C-70), 118.0 (C-400, 600), 54.4 (C-2),
53.7 (C-20), 51.9 (OCH3), 36.9 (C-3), 36.6 (C-30), 29.6 (COCH2CH3),
9.5 (COCH2CH3); EI-MS m/z: 501 (M+), 310, 295, 192, 176 (100),
120, 91; TOFESMS: calcd for C29H31N3O5Na [M+Na]+ 524.2161,
found 524.2166.
The synthetic procedures used in the preparation of target com-
pounds are illustrated in Scheme 2.
L-Tyrosine methyl ester hydro-
chloride or -phenylalanine methyl ester hydrochloride was
L
reacted with substituted benzoic acid or isonicotinic acid and IBCF
in the presence of NMM to give compound 11, then hydrolyzed in
2 M NaOH solution to give compound 12, which was reacted with
IBCF and substituted
L-phenylalanine methyl ester hydrochloride
or substituted -phenylalanol in the presence of NMM to afford tar-
L
get compound 13.
4.1.2.1. General procedure for the synthesis of compounds
13a–13p. IBCF (1.1 mmol) was added dropwise to the mixture of
substituted benzoic acid or isonicotinic acid (1.0 mmol),
L-tyrosine
methyl ester hydrochloride or -phenylalanine methyl ester hydro-
L
chloride (1.1 mmol) and NMM (2.3 mmol) in CH2Cl2 (50 mL) at 0 °C
within 60 min. The mixture was stirred for 30 min and the bulk of
CH2Cl2 was removed in vacuo. The residue was dissolved in ethyl
acetate and washed sequentially with water, 5% HCl, saturated
NaHCO3 solution and brine, dried with Na2SO4. Removal of the sol-
vent gave a residue which was recrystallized from ethyl acetate to
afford target compound 11. 2 M NaOH (1 mL) was added to the
solution of compound 11 (1.0 mmol) in DMF (5 mL). After stirring
at room temperature for 2 h, the mixture was acidified to pH 6–7
with concentrated hydrochloric acid, and partitioned between
ethyl acetate and water. The organic phase was separated and
washed with brine, dried over Na2SO4 and evaporated in vacuo
to give the compound 12. Compound 13 was prepared with a sim-
ilar procedure to that described for the synthesis of compound 11.
4.1.2.5. N-(N- 4-Chloroacetamido-benzoyl-L-phenylalanyl)-L-
phenylalanine methyl ester (13d). White powder, yield 55%, 1H
NMR (DMSO-d6, 400 MHz) d: 10.64 (1H, s, Ar-NHCO), 8.57 (1H, d,
J = 7.6 Hz, NHCO), 8.50 (1H, d, J = 8.4 Hz, NHCO), 7.78 (2H, d,
J = 8.4 Hz, H-300, 700), 7.65 (2H, d, J = 8.8 Hz, H-400, 600), 7.34–7.14
(10H, m, H-5, 9, 50, 90), 4.74 (1H, m, H-2), 4.51 (1H, m, H-20), 4.31
(2H, s, NHCOCH2Cl), 3.59 (3H, s, OCH3), 3.09–2.92 (4H, m, H-3,
13
30); C NMR (DMSO-d6, 100 MHz) d: 171.9, 171.8, 165.6 (C-100),
165.0 (Ar-NHCO), 141.2 (C-500), 138.4 (C-4), 137.1 (C-40), 129.2
(C-6, 8), 129.1 (C-60, 80), 129.0 (C-200), 128.4 (C-300, 700), 128.3 (C-50,
90), 128.1 (C-5, 9), 126.6 (C-70), 126.2 (C-7), 118.4 (C-400, 600), 54.5
(C-2), 53.8 (C-20), 51.9 (OCH3), 43.6 (COCH2Cl), 37.0 (C-3), 36.6
(C-30). EI-MS m/z: 521 (M+), 343, 310, 196 (100), 120, 91; TOFESMS:
calcd for C28H28N3O5NaCl [M+Na]+ 544.1615, found 544.1621.
4.1.2.2. N-(N-4-acetamido-benzoyl-L-phenylalanyl)-L-tyrosine
methyl ester (13a). White needle crystal, yield 63%, mp
216–217 °C (EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 10.23 (1H,
s, Ar-NHCO), 9.26 (1H, s, Ar-OH), 8.47–8.43 (2H, m, NHCO ꢁ 2),
7.73 (2H, d, J = 8.4 Hz, H-300, 700), 7.62 (2H, d, J = 8.4 Hz, H-400, 600),
7.32 (2H, d, J = 7.2 Hz, H-5, 9), 7.23 (2H, t, H-6, 8), 7.14 (1H, t, H-
7), 6.99 (2H, d, J = 8.4 Hz, H-50, 90), 6.62 (2H, d, J = 8.0 Hz, H-60,
80), 4.71 (1H, m, H-2), 4.40 (1H, m, H-20), 3.56 (3H, s, OCH3),
3.07–2.86 (4H, m, H-3, 30), 2.05 (3H, CH3CO); 13C NMR (DMSO-d6,
100 MHz) d: 172.0, 171.7, 168.7 (Ar-NHCO), 165.7 (C-100), 156.1
(C-70), 142.0 (C-500), 138.4 (C-4), 130.1 (C-50, 90), 129.2 (C-6, 8),
128.3 (C-300, 700), 128.2 (C-40), 128.0 (C-5, 9), 127.0 (C-200), 126.2
(C-7), 117.9 (C-400, 600), 115.1 (C-60, 80), 54.5 (C-2), 54.1 (C-20), 51.8
(OCH3), 36.9 (C-3), 35.9 (C-30), 24.1 (CH3CO); EI-MS m/z: 503
(M+), 326, 309, 281, 178, 162 (100), 120; TOFESMS: calcd for
4.1.2.6. N-(N- 4-Pyridinyl-formyl-L-phenylalanyl)-L-phenylala-
nine methyl ester (13e). White needle crystal, yield 49%, mp
174–175 °C (EtOAc), 1H NMR (CDCl3, 400 MHz) d: 8.71 (2H, d,
J = 6.0 Hz, H-400, 600), 7.52 (2H, d, J = 5.6 Hz, H-300, 700), 7.31–7.17 (8H,
m, H-5, 6, 8, 9, 50, 60, 80, 90), 7.07 (1H, br, NHCO), 6.99–6.97 (2H, m,
H-7, 70), 6.30 (1H, br, NHCO), 4.86–4.75 (2H, m, H-2, 20), 3.72 (3H, s,
OCH3), 3.20 (1H, dd, J = 6.0, 14.0 Hz, H-3a), 3.14–3.08 (2H, m, H-3b,
30a), 2.97 (1H, dd, J = 7.6, 13.6 Hz, H-30b); 13C NMR (CDCl3,
100 MHz) d: 171.2 (C-1), 170.0 (C-10), 165.0 (C-100), 150.6 (C-400, 600),
140.7 (C-200), 136.0, 135.3, 129.3 (C-60, 80), 129.0 (C-6, 8), 128.7
(C-50, 90), 128.6 (C-5, 9), 127.24, 127.19, 120.8 (C-300, 700), 54.6 (C-2),
53.5 (C-20), 52.4 (OCH3), 38.2 (C-3), 37.8 (C-30); EI-MS m/z: 431
(M+), 309, 269, 253, 225, 162, 147, 131, 106 (100), 78, 57, 43; TOF-
ESMS: calcd for C25H25N3O4Na [M+Na]+ 454.1743, found 454.1748.
C
28H29N3O6Na [M+Na]+ 526.1954, found 526.1959.
4.1.2.7.
N-(N- 4-Pyridinyl-formyl-L-phenylalanyl)-4-nitro-L-
4.1.2.3. N-(N-4-Propionamido-benzoyl-
L-phenylalanyl)-4-nitro-
phenylalanine methyl ester (13f). Pale yellow powder, yield
1
48%, H NMR (CDCl3, 400 MHz) d: 8.77 (2H, s, H-400, 600), 8.06 (2H,
L
-phenylalanine methyl ester (13b). Pale yellow powder, yield
59%, 1H NMR (DMSO-d6, 400 MHz) d: 10.14 (1H, s, Ar-NHCO),
8.61 (1H, d, J = 7.2 Hz, NHCO), 8.42 (1H, d, J = 8.4 Hz, NHCO), 9.05
(2H, d, J = 8.0 Hz, H-60, 80), 7.72 (2H, d, J = 7.6 Hz, H-300, 700), 7.63
(2H, d, J = 8.4 Hz, H-50, 90), 7.51 (2H, d, J = 8.0 Hz, H-400, 600), 7.31
(2H, d, J = 7.2 Hz, H-5, 9), 7.23 (2H, t, H-6, 8), 7.14 (1H, t,
J = 7.2 Hz, H-7), 4.68–4.59 (2H, m, H-2, 20), 3.61 (3H, s, OCH3),
3.25–2.90 (4H, m, H-3, 30), 2.34 (2H, q, J = 7.6 Hz, COCH2CH3),
1.07 (3H, t, J = 7.6 Hz, COCH2CH3); 13C NMR (DMSO-d6, 100 MHz)
d: 172.4 (Ar-NHCO), 171.8 (C-1), 171.4 (C-10), 165.7 (C-100), 146.3
(C-70), 145.5 (C-40), 142.2 (C-500), 138.3 (C-4), 130.6 (C-50, 90),
129.2 (C-6, 8), 128.3 (C-300, 700), 128.1 (C-5, 9), 128.0 (C-200), 126.3
(C-7), 123.2 (C-60, 80), 117.9 (C-400, 600), 54.5 (C-2), 52.9 (C-20), 52.1
(OCH3), 36.8 (C-3), 36.2 (C-30), 29.6 (COCH2CH3), 9.6 (COCH2CH3);
d, J = 8.0 Hz, H-60, 80), 7.54 (2H, s, H-300, 700), 7.29–7.19 (7H, m,
H-5, 9, 50, 90), 6.86 (1H, br, NHCO), 6.29 (1H, br, NHCO), 4.79 (2H,
m, H-2, 20), 3.72 (3H, s, OCH3), 3.21–3.08 (4H, m, H-3, 30); 13C
NMR (DMSO-d6, 100 MHz) d: 170.5 (C-1), 170.1 (C-10), 165.2
(C-100), 150.7 (C-400, 600), 147.1 (C-70), 143.2 (C-40), 140.4 (C-200),
135.7 (C-4), 130.1 (C-50, 90), 129.2 (C-6, 8), 128.9 (C-5, 9), 124.4
(C-7), 123.7 (C-60, 80), 120.7 (C-300, 700), 54.8 (C-20), 53.0 (C-2), 52.7
(OCH3), 38.1 (C-30), 37.1 (C-3); EI-MS m/z: 476 (M+), 385, 354,
225, 131, 106 (100), 78, 57, 43; TOFESMS: calcd for C25H24N4O6Na
[M+Na]+ 499.1594, found 499.1598.
4.1.2.8. N-(N- 4-Pyridinyl-formyl-L-phenylalanyl)-L-tyrosine
methyl ester (13g). White needle crystal, yield 46%, mp 216–

J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
5357
217 °C (MeOH/EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 9.26 (1H, s,
Ar-OH), 8.91 (1H, d, J = 8.8 Hz, NHCO), 8.70 (2H, d, J = 5.2 Hz, H-400,
600), 8.57 (1H, d, J = 7.6 Hz, NHCO), 7.66 (2H, d, J = 5.2 Hz, H-300, 700),
7.35 (2H, d, J = 7.6 Hz, H-5, 9), 7.25 (2H, t, H-6, 8), 7.16 (1H, t, H-7),
7.02 (2H, d, J = 8.0 Hz, H-50, 90), 6.64 (2H, d, J = 8.0 Hz, H-60, 80), 4.78
(1H, m, H-2), 4.44 (1H, m, H-20), 3.59 (3H, s, OCH3), 3.12–2.85 (4H,
m, H-3, 30); 13C NMR (DMSO-d6, 100 MHz) d: 172.0 (C-1), 171.3 (C-
10), 164.7 (C-100), 156.1 (C-70), 150.2 (C-400, 600), 140.9 (C-200), 138.1
(C-4), 130.1 (C-50, 90), 129.2 (C-6, 8), 128.1 (C-5, 9), 127.0 (C-40),
126.3 (C-7), 121.4 (C-300, 700), 115.1 (C-60, 80), 54.5 (C-2), 54.2 (C-
20), 51.9 (OCH3), 37.0 (C-3), 35.8 (C-30); EI-MS m/z: 447 (M+), 270
(100), 253, 225, 178, 106, 78; TOFESMS: calcd for C25H25N3O5Na
[M+Na]+ 470.1692, found 470.1696.
7.69 (2H, d, J = 5.6 Hz, H-300, 700), 7.23–7.09 (7H, m, H-5, 9, 50, 90),
6.63 (2H, d, J = 8.4 Hz, H-6, 8), 4.82 (1H, t, OH), 4.62 (1H, m, H-2),
3.90 (1H, m, H-20), 3.37–3.27 (2H, m, H-10), 2.96–2.79 (3H, m,
H-3, 30a), 2.66 (1H, dd, J = 8.0, 13.6 Hz, H-30b); 13C NMR (DMSO-
d6, 100 MHz) d: 171.7 (C-1), 164.5 (C-100), 155.7 (C-7), 150.2 (C-
400, 600), 141.1 (C-200), 139.0 (C-40), 130.1 (C-5, 9), 129.2 (C-60, 80),
128.2 (C-4), 128.1 (C-50, 90), 125.9 (C-70), 121.4 (C-300, 700), 114.9
(C-6, 8), 62.3 (C-10), 55.3 (C-2), 52.5 (C-20), 36.6, 36.4; EI-MS m/z:
419 (M+), 297, 269, 241, 206, 162, 147, 123, 106 (100), 91, 78;
TOFESMS: calcd for
442.1747.
C
24H25N3O4Na [M+Na]+ 442.1743, found
4.1.2.13. N-(N- 4-Acetamido-benzoyl-L-tyrosineyl)-L-phenylala-
nol (13l). Pale yellow powder, yield 54%, 1H NMR (DMSO-d6,
400 MHz) d: 10.17 (1H, s, Ar-NHCO), 9.14 (1H, s, Ar-OH), 8.28
(1H, d, J = 8.0 Hz, NHCO), 7.83 (1H, d, J = 8.4 Hz, NHCO), 7.76 (2H,
d, J = 8.8 Hz, H-300, 700), 7.63 (2H, d, J = 8.4 Hz, H-400, 600), 7.22–7.07
(7H, m, H-5, 9, 50, 90), 6.61 (2H, d, J = 8.0 Hz, H-6, 8), 4.81 (1H, t,
OH), 4.57 (1H, m, H-2), 3.88 (1H, m, H-20), 3.34–3.25 (2H, m,
H-10), 2.91–2.50 (4H, m, H-3, 30), 2.07 (3H, CH3CO); 13C NMR
(DMSO-d6, 100 MHz) d: 171.2 (C-1), 168.7 (Ar-NHCO), 165.5
(C-100), 155.7 (C-7), 142.0 (C-500), 139.0 (C-40), 130.1 (C-5, 9),
129.2 (60, 80), 128.4 (C-4, 200), 128.3 (C-50, 90), 128.1 (C-300, 700),
125.9 (C-70), 117.9 (C-400, 600), 114.8 (C-6, 8), 62.2 (C-10), 55.1
(C-2), 52.4 (C-20), 36.5, 36.4, 24.2 (CH3CO); EI-MS m/z: 475 (M+),
297, 206, 162 (100), 147, 120, 91; TOFESMS: calcd for C27H29N3O5-
Na [M+Na]+ 498.2005, found 498.2009.
4.1.2.9. N-(N- 4-Pyridinyl-formyl-L-tyrosineyl)-L-phenylalanine
methyl ester (13h). Pale yellow powder, yield 46%, 1H NMR
(DMSO-d6, 400 MHz) d: 9.17 (1H, s, Ar-OH), 8.82 (1H, d,
J = 8.8 Hz, NHCO), 8.70 (2H, d, J = 5.6 Hz, H-400, 600), 8.59 (1H, d,
J = 7.6 Hz, NHCO), 7.67 (2H, d, J = 5.6 Hz, H-300, 700), 7.27–7.18 (5H,
m, H-50, 90), 7.12 (2H, d, J = 8.4 Hz, H-5, 9), 6.63 (2H, d, J = 8.4 Hz,
H-6, 8), 4.68 (1H, m, H-2), 4.50 (1H, m, H-20), 3.59 (3H, s, OCH3),
3.09–2.77 (4H, m, H-3, 3); 13C NMR (DMSO-d6, 100 MHz) d:
171.8 (C-1), 171.4 (C-10), 164.6 (C-100), 155.7 (C-7), 150.2 (C-400,
600), 141.0 (C-200), 137.1 (C-40), 130.1 (C-5, 9), 129.1 (C-60, 80),
128.3 (C-50, 90), 128.1 (C-4), 126.6 (C-70), 121.4 (C-300, 700), 114.9
(C-6, 8), 54.9 (C-2), 53.8 (C-20), 51.9 (OCH3), 38.9, 36.5; EI-MS m/
z: 447 (M+), 325, 241, 180, 163, 147, 123, 106 (100), 91; TOFESMS:
calcd for C25H25N3O5Na [M+Na]+ 470.1692, found 470.1696.
4.1.2.14. N-(N- 4-Hydroxy-benzoyl-L-phenylalanyl)-L-phenylala-
4.1.2.10. N-(N- 4-Pyridinyl-formyl-
L
-tyrosineyl)-4-nitro-
L
-phen-
nol (13m). White needle crystal, yield 61%, mp 223–225 °C
(EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 9.98 (1H, s, Ar-OH),
8.22 (1H, d, J = 8.8 Hz, NHCO), 7.83 (1H, d, J = 8.4 Hz, NHCO), 7.66
(2H, d, J = 8.8 Hz, H-300, 700), 7.29–7.09 (10H, m, H-5, 9, 50, 90), 6.76
(2H, d, J = 8.4 Hz, H-400, 600), 4.80 (1H, t, J = 5.2 Hz, OH), 4.62 (1H,
m, H-2), 3.87 (1H, m, H-20), 3.34–3.22 (2H, m, H-10), 3.02–2.92
(2H, m, H-3), 2.84 (1H, dd, J = 5.6, 13.6 Hz, H-30a), 2.64 (1H, dd,
J = 8.0, 13.6, Hz, H-30b); 13C NMR (DMSO-d6, 100 MHz) d: 171.2
(C-1), 165.8 (C-100), 160.2 (C-500), 139.0 (C-40), 138.5 (C-4), 129.4
(C-300, 700), 129.23 (C-60, 80), 129.21 (C-6, 8), 128.09 (C-50, 90),
128.03 (C-5, 9), 126.2 (C-7), 125.9 (C-70), 124.7 (C-200), 114.7
(C-400, 600), 62.2 (C-10), 54.7 (C-2), 52.5 (C-20), 37.2 (C-30), 36.5
(C-3); EI-MS m/z: 418 (M+), 268, 240, 190, 121 (100), 91; TOFESMS:
calcd for C25H26N2O4Na [M+Na]+ 441.1790, found 441.1794.
ylalanine methyl ester (13i). Pale yellow powder, yield 43%, 1H
NMR (DMSO-d6, 400 MHz) d: 9.17 (1H, s, Ar-OH), 8.80 (1H, d,
J = 8.4 Hz, NHCO), 8.68 (2H, d, J = 4.8 Hz, H-400, 600), 8.63 (1H, d,
J = 8.0 Hz, NHCO), 8.06 (2H, d, J = 8.4 Hz, H-60, 80), 7.64 (2H,
d, J = 5.6 Hz, H-300, 700), 7.50 (2H, d, J = 8.4 Hz, H-50, 90), 7.09 (2H, d,
J = 8.4 Hz, H-5, 9), 6.62 (2H, d, J = 8.0 Hz, H-6, 8), 4.65–4.58 (2H, m,
H-2, 20), 3.61 (3H, s, OCH3), 3.22 (1H, dd, J = 5.6, 13.6 Hz, H-30a),
3.09 (1H, dd, J = 9.2, 13.2 Hz, H-30b), 2.93 (1H, dd, J = 4.4, 14.0 Hz,
H-3a), 2.79 (1H, m, H-3b); 13C NMR (DMSO-d6, 100 MHz) d:
171.4 (ꢁ2, C-1, 10), 164.5 (C-100), 155.8 (C-7), 150.2 (C-400, 600),
146.3 (C-70), 145.5 (C-40), 140.9 (C-200), 130.6 (C-50, 90), 130.1 (C-5,
9), 128.0 (C-4), 123.2 (C-60, 80), 121.3 (C-300, 700), 114.9 (C-6, 8),
54.9 (C-2), 52.9 (C-20), 52.1 (OCH3), 38.9, 36.2; EI-MS m/z: 492
(M+), 370, 162, 123, 107 (100), 91, 78; TOFESMS: calcd for
C
25H24N4O7Na [M+Na]+ 515.1543, found 515.1548.
4.1.2.15. N-(N-(4-Dimethylamino-methyl-benzoyl)-
L-phenylala-
nyl)- -phenylalanol (13n). White needle crystal, yield 45%, mp
L
4.1.2.11. N-(N- 4-Pyridinyl-formyl- -phenylalanyl)-
L
L
-phenylala-
197–198 °C (EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 8.46 (1H, d,
J = 8.8 Hz, NHCO), 7.87 (1H, d, J = 8.4 Hz, NHCO), 7.75 (2H, d,
J = 8.0 Hz, H-300, 700), 7.34 (2H, d, J = 8.0 Hz, H-400, 600), 7.30–7.10
(10H, m, H-50, 90, 5, 9), 4.81 (1H, t, OH), 4.67 (1H, m, H-2), 3.87
(1H, m, H-20), 3.43 (2H, s, Ar-CH2N(CH3)2), 3.34–3.25 (2H, m,
H-10), 3.02–2.93 (2H, m, H-3), 2.84 (1H, dd, J = 5.6, 13.6 Hz, H-
30a), 2.65 (1H, dd, J = 8.4, 13.6 Hz, H-30b), 1.14 (6H, s, Ar-
CH2N(CH3)2); 13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-1), 165.9
(C-100), 143.0 (C-500), 139.0 (C-40), 138.4 (C-4), 132.8 (C-200), 129.2
(ꢁ4, C-6, 8, 60, 80), 128.5 (C-400, 600), 128.1 (C-50, 90), 128.0 (C-5, 9),
127.4 (C-300, 700), 126.2 (C-7), 125.9 (C-70), 62.8 (Ar-CH2N(CH3)2),
62.2 (C-10), 54.8 (C-2), 52.5 (C-20), 44.9 (Ar-CH2N(CH3)2), 37.2,
36.4; EI-MS m/z: 459 (M+), 416, 309, 281, 162 (100), 105, 91; TOF-
ESMS: calcd for C28H33N3O3Na [M+Na]+ 482.2420, found 482.2425.
nol (13j). Yellow needle crystal, yield 40%, mp 199–201 °C
(MeOH/EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 8.83 (1H, d,
J = 8.8 Hz, NHCO), 8.69 (2H, d, J = 6.4 Hz, H-400, 600), 7.96 (1H, d,
J = 8.0 Hz, NHCO), 7.66 (2H, d, J = 6.0 Hz, H-300, 700), 7.31–7.11
(10H, m, H-5, 9, 50, 90), 4.81 (1H, t, J = 5.2 Hz, OH), 4.68 (1H, m,
H-2), 3.89 (1H, m, H-20), 3.35–3.25 (2H, m, H-10), 3.04 (1H, dd,
J = 4.4, 14.0 Hz, H-3a), 2.95–2.83 (2H, m, H-3b, 30a), 2.65 (1H, dd,
J = 7.6, 13.2 Hz, H-30b); 13C NMR (DMSO-d6, 100 MHz) d: 170.5
(C-1), 164.5 (C-100), 150.1 (C-400, 600), 141.0 (C-200), 139.0 (C-40),
138.2 (C-4), 129.2 (ꢁ4, C-6, 8, 60, 80), 128.1 (ꢁ4, C-5, 9, 50, 90),
126.3 (C-7), 125.9 (C-70), 121.4 (C-300, 700), 62.2 (C-10), 54.8 (C-2),
52.5 (C-20), 37.3 (C-3), 36.4 (C-30); EI-MS m/z: 403 (M+), 270, 253,
225, 190, 120, 106 (100), 91, 78; TOFESMS: calcd for C24H25N3O3Na
[M+Na]+ 426.1794, found 426.1798.
4.1.2.16. N-(N- (4-Pyrrolidin-1-ylmethyl-benzoyl)-L-phenylala-
4.1.2.12. N-(N- 4-Pyridinyl-formyl-
L
-tyrosineyl)-
L
-phenylalanol
nyl)-L-phenylalanol (13o). Yellow crystal, yield 43%, mp 164–
(13k). Pale yellow powder, yield 43%, 1H NMR (DMSO-d6,
400 MHz) d: 9.17 (1H, s, Ar-OH), 8.79 (1H, d, J = 8.4 Hz, NHCO),
8.71 (2H, d, J = 5.6 Hz, H-400, 600), 7.95 (1H, d, J = 8.0 Hz, NHCO),
165 °C (EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 8.44 (1H, d,
J = 8.0 Hz, NHCO), 7.87 (1H, d, J = 8.4 Hz, NHCO), 7.74 (2H, d,
J = 8.4 Hz, H-300, 700), 7.35 (2H, d, J = 8.0 Hz, H-400, 600), 7.31–7.09

5358
J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
(10H, m, H-50, 90, 5, 9), 4.81 (1H, t, OH), 4.67 (1H, m, H-2), 3.88 (1H,
m, H-20), 3.58 (2H, s, Ar-CH2N), 3.34–3.24 (2H, m, H-10), 3.04–2.91
(2H, m, H-3), 2.85 (1H, dd, J = 5.6, 13.6 Hz, H-30a), 2.65 (1H, dd,
J = 8.0, 13.6 Hz, H-30b), 2.40 (4H, s, Ar-CH2N(CH2)2), 1.67 (4H, s,
NCH2CH2CH2CH2N); 13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-1),
165.9 (C-100), 143.0 (C-500), 139.0 (C-40), 138.4 (C-4), 132.6 (C-200),
129.2 (ꢁ4, C-6, 8, 60, 80), 128.14 (C-400, 600), 128.08 (C-50, 90),
128.04 (C-5, 9), 127.4 (C-300, 700), 126.2 (C-7), 125.9 (C-70), 62.2
(C-10), 59.2 (Ar-CH2N), 54.8 (C-2), 53.5 (Ar-CH2N(CH2)2), 52.5
(C-20), 37.2, 36.4, 23.1 (NCH2CH2CH2CH2N); EI-MS m/z: 416, 335,
307, 265, 188 (100), 152, 118, 91; TOFESMS: calcd for
3.92 (1H, dd, J = 4.8, 11.2 Hz, H-10a), 3.82 (1H, dd, J = 4.0, 11.2 Hz,
H-10b), 3.71 (4H, t, J = 4.4 Hz, CH2OCH2), 3.54 (2H, s, Ar-CH2N),
3.22 (1H, dd, J = 6.0, 13.6 Hz, H-3a), 3.07 (1H, dd, J = 8.0, 13.6 Hz,
H-3b), 2.78–2.73 (2H, m, H-30), 2.44 (4H, s, Ar-CH2N(CH2)2), 2.02
(3H, s, OCOCH3); 13C NMR (CDCl3, 100 MHz) d: 171.1, 170.6,
167.2 (C-100), 142.5 (C-500), 137.0, 136.9, 132.8 (C-200), 129.6 (ꢁ2),
129.5 (ꢁ2), 129.4 (ꢁ2), 129.0 (ꢁ2), 128.8 (ꢁ2), 127.4 (C-300, 700),
127.0 (C-7, 70), 67.2 (CH2OCH2), 64.8 (C-10), 63.2 (Ar-CH2N), 55.2
(C-2), 53.9 (Ar-CH2N(CH2)2), 49.7 (C-20), 38.8, 37.7, 21.1 (OCOCH3);
EI-MS m/z: 543 (M+), 398, 351, 323, 220, 204 (100), 146, 105, 91;
TOFESMS: calcd for
566.2638.
C
32H37N3O5Na [M+Na]+ 566.2631, found
C
30H36N3O3 [M+H]+ 486.2757, found 486.2762.
4.1.2.17. N-(N- (4-Morpholinomethyl-benzoyl)-
L
-phenylalanyl)-
4.1.2.20. N-(N-(4-Dimethylamino-methyl-benzoyl)-
L-phenylala-
L
-phenylalanol (13p). White crystal, yield 46%, mp 183–185 °C
nyl)-O-acetyl- -phenylalanol (15b). White needle crystal, yield
L
(MeOH/EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 8.45 (1H, d,
J = 8.4 Hz, NHCO), 7.87 (1H, d, J = 8.0 Hz, NHCO), 7.76 (2H, d,
J = 7.6 Hz, H-300, 700), 7.37 (2H, d, J = 7.6 Hz, H-400, 600), 7.31–7.10
(10H, m, H-50, 90, 5, 9), 4.81 (1H, t, OH), 4.69 (1H, m, H-2), 3.89
(1H, m, H-20), 3.57 (4H, s, CH2OCH2), 3.48 (2H, s, Ar-CH2N), 3.34–
3.25 (2H, m, H-10), 3.05–2.92 (2H, m, H-3), 2.86 (1H, dd, J = 5.2,
13.6 Hz, H-30a), 2.66 (1H, dd, J = 8.0, 13.6 Hz, H-30b), 2.34 (4H, s,
Ar-CH2N(CH2)2); 13C NMR (DMSO-d6, 100 MHz) d: 170.9 (C-1),
165.9 (C-100), 141.4 (C-500), 139.0 (C-40), 138.4 (C-4), 132.8 (C-200),
129.19 (ꢁ2), 129.17 (ꢁ2), 128.6 (C-400, 600), 128.06 (ꢁ2), 128.01
(ꢁ2), 127.4 (C-300, 700), 126.2 (C-7), 125.9 (C-70), 66.2 (CH2OCH2),
62.2 (C-10), 61.96 (Ar-CH2N), 54.7 (C-2), 53.2 (Ar-CH2N(CH2)2),
52.5 (C-20), 37.2, 36.4; EI-MS m/z: 501 (M+), 351, 323, 204 (100),
44%, mp 196–198 °C (EtOAc), 1H NMR (CDCl3, 400 MHz) d: 7.68
(2H, d, J = 8.4 Hz, H-300, 700), 7.40 (2H, d, J = 8.0 Hz, H-400, 600), 7.30–
7.06 (10H, m, H-50, 90, 5, 9), 6.76 (1H, br, NHCO), 6.07 (1H, br,
NHCO), 4.78 (1H, m, H-2), 4.35 (1H, m, H-20), 3.92 (1H, dd, J = 4.8,
11.6 Hz, H-10a), 3.81 (1H, dd, J = 4.0, 11.6 Hz, H-10b), 3.49 (2H, s,
Ar-CH2N(CH3)2), 3.22 (1H, dd, J = 5.6, 13.6 Hz, H-3a), 3.06 (1H, dd,
J = 8.4, 13.2 Hz, H-3b), 2.76–2.73 (2H, m, H-30), 2.26 (6H, s, Ar-
CH2N(CH3)2), 2.03 (3H, s, COCH3); 13C NMR (CDCl3, 100 MHz) d:
170.8 (C-1), 170.2 (OCOCH3), 166.9 (C-100), 143.0 (C-500), 136.7,
136.6, 132.5 (C-200), 129.3 (ꢁ4, C-6, 8, 60, 80), 129.1 (C-400, 600),
128.7 (C-50, 90), 128.5 (C-5, 9), 127.1 (C-300, 700), 126.7 (C-7, 70),
64.5 (C-10), 63.8 (Ar-CH2N(CH3)2), 54.9 (C-2), 49.3 (C-20), 45.3
(Ar-CH2N(CH3)2), 38.4, 37.4, 20.8 (OCOCH3); EI-MS m/z: 501 (M+),
309, 281, 176, 162 (100), 91; TOFESMS: calcd for C30H35N3O4Na
[M+Na]+ 524.2525, found 524.2529.
146, 118, 91; TOFESMS: calcd for
524.2525, found 524.2529.
C
30H35N3O4Na [M+Na]+
4.1.2.18. N-(N-4-Acetamido-benzoyl-L-phenylalanyl)-
L-phenylal-
4.1.2.21.
N-(N-Benzoyl-L-phenylalanyl)-O-
ethyl-L-tyrosine
anol (14a). SOCl2 (1.1 mmol) was added dropwise to the mixture
of compound 13 (1.0 mmol) in 1,4-dioxane (10 mL), stirred at 70 °C
for 1 h. The reaction solution was partitioned in ethyl acetate and
water, washed sequentially with saturated NaHCO3 solution, and
brine. The solution was dried with Na2SO4 and concentrated, the
residue was recrystallized from ethyl acetate to afford target com-
pound 14a as needle crystal. Yield 56%, mp 193–195 °C (EtOAc), 1H
NMR (DMSO-d6, 400 MHz) d: 10.16 (1H, s, Ar-NHCO), 8.39 (1H, d,
J = 8.4 Hz, NHCO), 8.26 (1H, d, J = 8.4 Hz, NHCO), 7.76 (2H, d,
J = 8.4 Hz, H-300, 700), 7.62 (2H, d, J = 8.8 Hz, H-400, 600), 7.32–7.13
(10H, m, H-5, 9, 50, 90), 4.70 (1H, m, H-2), 4.17 (1H, m, H-20), 3.64
(1H, dd, J = 4.8, 11.2 Hz, H-10a), 3.52 (1H, dd, J = 7.0, 11.2 Hz, H-
10b), 3.05–2.78 (4H, m, H-3, 30), 2.06 (3H, CH3CO); 13C NMR
(DMSO-d6, 100 MHz) d: 171.4 (C-1), 168.7 (Ar-NHCO), 165.6 (C-
100), 142.0 (C-500), 138.3 (C-4), 137.8 (C-40), 129.2 (ꢁ4), 128.3 (ꢁ4),
128.2 (C-200), 128.0 (C-300, 700), 126.4 (C-7), 126.2 (C-70), 118.0 (C-
400, 600), 54.7 (C-2), 51.5 (C-10), 46.8 (C-20), 37.3 (C-3), 37.0 (C-30),
24.1 (CH3CO); EI-MS m/z: 441 (100), 350, 279, 264, 162, 120, 91;
TOFESMS: calcd for C27H28N3O3ClNa [M+Na]+ 500.1717, found
500.1722.
methyl ester (16a). CH3CH2I (1.40 g, 9.0 mmol) was added to
the mixture of compound 13 (1.34 g, 3.0 mmol) and K2CO3
(3.11 g, 22.5 mmol) in DMF (15 mL). After stirring at room temper-
ature overnight. The reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was washed with
brine, dried with Na2SO4 and evaporated in vacuo to give the com-
pound 16a as a white solid (1.29 g, 91%). Yield 56%, 1H NMR (CDCl3,
400 MHz) d: 7.69 (2H, d, J = 7.6 Hz, H-300, 700), 7.51 (1H, t, H-500), 7.42
(2H, t, H-400, 600), 7.31–7.20 (5H, m, H-5, 9), 6.86 (2H, d, J = 8.4 Hz, H-
50, 90), 6.76 (1H, br, NHCO), 6.66 (2H, d, J = 8.4 Hz, H-60, 80), 6.29
(1H, br, NHCO), 4.84 (1H, m, H-2), 4.72 (1H, m, H-20), 3.90 (2H,
m, Ar-OCH2CH3), 3.70 (3H, s, OMe), 3.21 (1H, dd, J = 6.0, 14.0 Hz,
H-30a), 3.12 (1H, dd, J = 7.6, 14.0 Hz, H-30b), 3.02 (1H, dd, J = 5.6,
13.6 Hz, H-3a), 2.90 (1H, dd, J = 6.4, 13.6 Hz, H-3b), 1.36 (3H, t,
Ar-OCH2CH3); 13C NMR (CDCl3, 100 MHz) d: 171.7 (C-1), 170.6
(C-10), 167.3 (C-100), 158.3 (C-70), 136.7 (C-4), 133.9 (C-200), 132.2
(C-500), 130.4 (C-50, 90), 129.8 (C-6, 8), 129.0 (ꢁ2), 128.9 (ꢁ2),
127.5 (C-40), 127.4 (C-7), 127.3 (C-300, 700), 114.8 (C-60, 80), 63.5
(Ar-OCH2CH3), 54.8 (C-2), 53.8 (C-20), 52.7 (COOMe), 38.4 (C-3),
37.3 (C-30), 15.1 (Ar-OCH2CH3); EI-MS m/z: 474 (M+), 224, 206
(100), 178, 135, 105, 91, 77; TOFESMS: calcd for C28H30N2O5Na
[M+Na]+ 497.2052, found 497.2056.
4.1.2.19. N-(N-(4-Morpholinomethyl-benzoyl)-L-phenylalanyl)-
O- acetyl- -phenylalanol (15a). Compound 13p (502 mg,
L
1.0 mmol) was dissolved in pyridine (5 mL), then Ac2O (1 mL)
was added. After stirred at room temperature for 5 h, the reaction
was quenched with ethanol (5 mL) and concentrated in vacuo. The
residue was dissolved in ethyl acetate and washed sequentially
with water, saturated NaHCO3 solution, and brine. The solution
was dried with Na2SO4 and concentrated, the residue was recrys-
tallized from ethyl acetate to afford target compound 15a as white
powder (516 mg, 95%). Yield 44%, 1H NMR (CDCl3, 400 MHz) d: 7.68
(2H, d, J = 8.4 Hz, H-300, 700), 7.40 (2H, d, J = 8.4 Hz, H-400, 600), 7.30–
7.05 (10H, m, H-50, 90, 5, 9), 6.84 (1H, d, J = 7.6 Hz, NHCO), 6.16
(1H, d, J = 8.4 Hz, NHCO), 4.80 (1H, m, H-2), 4.34 (1H, m, H-20),
4.1.2.22.
N-(N-Benzoyl-L-phenylalanyl)-O- butyl-L-tyrosine
methyl ester (16b). White needle crystal, yield 59%, mp 177–
1
178 °C, H NMR (CDCl3, 400 MHz) d: 7.69 (2H, d, J = 7.6 Hz, H-300,
700), 7.51 (1H, t, H-500), 7.42 (2H, t, H-400, 600), 7.31–7.23 (5H, m,
H-5, 9), 6.85 (2H, d, J = 8.4 Hz, H-50, 90), 6.76 (1H, br, NHCO), 6.66
(2H, d, J = 8.4 Hz, H-60, 80), 6.31 (1H, br, NHCO), 4.83 (1H, m, H-2),
4.72 (1H, m, H-20), 3.82 (2H, m, Ar-OCH2CH2CH2CH3), 3.70 (3H, s,
OMe), 3.21 (1H, dd, J = 5.6, 14.0 Hz, H-30a), 3.12 (1H, dd, J = 7.2,
13.6 Hz, H-30b), 3.01 (1H, dd, J = 5.6, 14.0 Hz, H-3a), 2.90 (1H, dd,
J = 6.8, 14.0 Hz, H-3b), 1.70 (2H, m, Ar-OCH2CH2CH2CH3), 1.45
(2H, m, Ar-OCH2CH2CH2CH3), 0.96 (2H, t, J = 7.6 Hz, Ar-

J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
5359
OCH2CH2CH2CH3); 13C NMR (CDCl3, 100 MHz) d: 171.4 (C-1), 170.3
(C-10), 167.0 (C-100), 158.2 (C-70), 136.4 (C-4), 133.6 (C-200), 131.8
(C-500), 130.1 (C-50, 90), 129.4 (C-6, 8), 128.7 (C-5, 9), 128.6 (C-400,
600), 127.04 (C-40, C-7), 127.01 (C-300, 700), 114.5 (C-60, 80), 63.5 (Ar-
OCH2CH2CH2CH3), 54.5 (C-2), 53.5 (C-20), 52.3 (OMe), 38.1 (C-3),
36.9 (C-30), 31.3 (Ar-OCH2CH2CH2CH3), 19.2 (Ar-OCH2CH2CH2CH3),
13.8 (Ar-OCH2CH2CH2CH3); EI-MS m/z: 502 (M+), 234 (100), 178,
105; TOFESMS: calcd for C30H34N2O5Na [M+Na]+ 525.2365, found
525.2372.
(1H, m, H-2), 3.89 (1H, m, H-20), 3.35–3.24 (2H, m, H-10), 3.09
(2H, COCH2N), 3.07–2.91 (2H, m, H-3), 2.86 (1H, dd, J = 5.6,
13.6 Hz, H-30a), 2.66 (1H, dd, J = 7.6, 13.6 Hz, H-30b), 2.28 (6H, s,
N(CH3)2); 13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-1), 169.1
(Ar-NHCO), 165.5 (C-100), 141.4 (C-500), 139.0 (C-40), 138.5 (C-4),
129.2 (ꢁ4, C-6, 8, 60, 80), 128.6 (C-200), 128.2 (C-300, 700), 128.1
(ꢁ2), 128.0 (ꢁ2), 126.2 (C-7), 125.9 (C-70), 118.5 (C-400, 600), 63.3
(COCH2N), 62.2 (C-10), 54.8 (C-2), 52.5 (C-20), 45.4 (ꢁ2, N(CH3)2),
37.3 (C-3), 36.4 (C-30); EI-MS m/z: 502 (M+), 474, 352, 205, 119,
91, 58 (100); TOFESMS: calcd for C29H35N4O4 [M+H]+503.2658,
found 503.2662.
4.1.2.23. N-(N-4-(2-(Dimethylamino)-ethoxy)-benzoyl-
L-phenyl-
alanyl)- -phenylalanine methyl ester (16c). White needle crys-
L
tal, yield 50%, mp 167–168 °C, 1H NMR (CDCl3, 400 MHz) d: 7.64
(2H, d, J = 8.8 Hz, H-300, 700), 7.30–6.95 (10H, m, H-5, 9, 50, 90), 6.92
(2H, d, J = 8.8 Hz, H-400, 600), 6.58 (1H, d, J = 7.6 Hz, NHCO), 6.35
(1H, d, J = 7.2 Hz, NHCO), 4.83–4.76 (2H, m, H-2, 20), 4.10 (2H, t,
J = 6.0 Hz, Ar-OCH2), 3.70 (3H, s, OCH3), 3.21–2.93 (4H, m, H-3,
30), 2.75 (2H, t, J = 6.0 Hz, CH2N(CH3)2), 2.35 (6H, s, N(CH3)2); 13C
NMR (CDCl3, 100 MHz) d: 171.3 (C-1), 170.5 (C-10), 166.6 (C-100),
161.7 (C-500), 136.4 (C-4), 135.5 (C-40), 129.4 (C-300, 700), 129.1 (C-
6, 8), 128.8 (C-60, 80), 128.7 (C-50, 90), 128.5 (C-5, 9), 127.0 (C-7,
70), 125.9 (C-200), 114.3 (C-400, 600), 66.1 (Ar-OCH2), 58.1
(CH2N(CH3)2), 54.4 (C-2), 53.4 (C-20), 52.3 (OCH3), 45.9 (N(CH3)2),
38.0, 37.8; EI-MS m/z: 517 (M+), 192, 176 (100), 120, 91, 58
(100); TOFESMS: calcd for C30H36N3O5 [M+H]+ 518.2655, found
518.2661.
4.1.2.27. N-(N-4-(2-(Dimethylamino)-ethoxy)-benzoyl-
L-phenyl-
alanyl)-
L
-phenylalanol (16g). Pale yellow powder, yield 53%, 1H
NMR (CDCl3, 400 MHz) d: 7.67 (2H, d, J = 8.8 Hz, H-300, 700), 7.31–
7.07 (10H, m, H-5, 9, 50, 90), 6.92 (2H, d, J = 8.8 Hz, H-400, 600), 6.78
(1H, d, J = 7.6 Hz, NHCO), 6.28 (1H, d, J = 8.0 Hz, NHCO), 4.80 (1H,
m, H-2), 4.11–4.07 (3H, m, Ar-OCH2, H-20), 3.42 (2H, m, H-10),
3.23 (1H, dd, J = 6.4, 13.6 Hz, H-3a), 3.06 (1H, dd, J = 8.4, 13.6 Hz,
H-3b), 2.80–2.66 (4H, m, H-30, CH2N(CH3)2), 2.34 (6H, s, N(CH3)2);
13C NMR (CDCl3, 100 MHz) d: 171.0 (C-1), 166.8 (C-100), 161.8
(C-500), 137.4 (C-4), 136.8 (C-40), 129.3 (C-300, 700), 129.1 (C-6, 8),
128.9 (C-60, 80), 128.8 (C-50, 90), 128.5 (C-5, 9), 127.1 (C-7), 126.5
(C-70), 125.8 (C-200), 114.4 (C-400, 600), 66.2 (Ar-OCH2), 63.4 (C-10),
58.1 (CH2N(CH3)2), 55.1 (C-2), 52.9 (C-20), 45.9 (N(CH3)2), 38.6 (C-
30), 36.8 (C-3); EI-MS m/z: 489 (M+), 418, 339, 192, 105, 91, 58
(100); TOFESMS: calcd for C29H36N3O4 [M+H]+ 490.2706, found
490.2710.
4.1.2.24.
N-(N- 4-(2-Dimethylamino-acetamido)-benzoyl-
L-
phenylalanyl)-
L
-phenylalanine methyl ester (16d). White
powder, yield 49%, 1H NMR (DMSO-d6, 400 MHz) d: 9.92 (1H, s,
Ar-NHCO), 8.52 (1H, d, J = 7.6 Hz, NHCO), 8.41 (1H, d, J = 8.4 Hz,
NHCO), 7.71 (4H, m, H-300, 700, 400, 600), 7.33–7.14 (10H, m, H-5, 9,
50, 90), 4.72 (1H, m, H-2), 4.50 (1H, m, H-20), 3.57 (3H, s, OCH3),
3.07 (2H, s, NHCOCH2N), 3.06–2.89 (4H, m, H-3, 30), 2.26 (6H, s,
N(CH3)2); 13C NMR (DMSO-d6, 100 MHz) d: 172.5, 172.4, 169.8
(Ar-NHCO), 165.3 (C-100), 142.1 (C-500), 139.0 (C-4), 137.7 (C-40),
129.83 (C-6, 8), 129.78 (C-60, 80), 129.1 (C-200), 128.9 (C-50, 90),
128.8 (C-300, 700), 128.7 (C-5, 9), 127.2 (C-70), 126.9 (C-7), 119.1
(C-400, 600), 63.9 (COCH2N), 55.1 (C-2), 54.4 (C-20), 52.5 (OCH3),
46.0 (N(CH3)2), 37.6 (C-3), 37.2 (C-30); EI-MS m/z: 530 (M+), 310,
205, 91, 58 (100); TOFESMS: calcd for C30H34N4O5Na [M+Na]+
553.2427, found 553.2433.
4.1.2.28. Sodium N-(N-benzoyl-L-phenylalanyl)-O- methyl-L-
tyrosinate (17a). 5 M NaOH (0.2 mL) was added to the solution
of compound 16 (1.0 mmol) in EtOH–CHCl3 (5 mL). After stirring
at room temperature for 2 h, the reaction was evaporated in vacuo.
The residue was recrystallised from EtOAc to afford the compound
17. White needle crystal, yield 61%, mp 271–271 °C, 1H NMR
(DMSO-d6, 400 MHz) d: 8.81 (1H, d, J = 8.4 Hz, NHCO), 7.78 (2H,
d, J = 7.2 Hz, H-300, 700), 7.48 (2H, m, H-500, NHCO), 7.42 (2H, t,
H-400, 600), 7.30 (2H, d, J = 8.4 Hz, H-5, 9), 7.22 (2H, t, H-6, 8), 7.13
(1H, t, H-7), 7.01 (2H, d, J = 8.4 Hz, H-50, 90), 6.57 (2H, d,
J = 8.4 Hz, H-60, 80), 4.57 (1H, m, H-2), 3.95 (1H, m, H-20), 3.59
(3H, s, Ar-OCH3), 3.13–2.86 (4H, m, H-3, 30); 13C NMR (DMSO-d6,
100 MHz) d: 172.7 (C-10), 169.9 (C-1), 166.2 (C-100), 157.3 (C-70),
138.9 (C-4), 134.1 (C-200), 131.3 (C-500), 131.0 (C-40), 130.7 (C-50,
90), 129.1 (C-6, 8), 128.2 (C-400, 600), 128.1 (C-5, 9), 127.4 (C-300,
700), 126.1 (C-7), 112.9 (C-60, 80), 55.6 (ꢁ2), 54.7 (Ar-OCH3), 36.7,
36.3; EI-MS m/z: 446, 309, 246, 121 (100), 105, 91, 77; TOFESMS:
calcd for C26H25N2O5Na [M+Na]+ 491.1559, found 491.1563.
4.1.2.25. N-(N- (4-Pyrrolidinylacetamido-benzoyl)-
L-phenylala-
nyl)- -phenylalanine methyl ester (16e). Pale yellow powder,
L
yield 43%, 1H NMR (DMSO-d6, 400 MHz) d: 9.93 (1H, s, Ar-NHCO),
8.53 (1H, d, J = 7.2 Hz, NHCO), 8.43 (1H, d, J = 8.8 Hz, NHCO), 7.75
(2H, d, J = 8.8 Hz, H-300, 700), 7.69 (2H, d, J = 8.8 Hz, H-400, 600),
7.34–7.15 (10H, m, H-5, 9, 50, 90), 4.74 (1H, m, H-2), 4.52 (1H, m,
H-20), 3.59 (3H, s, OCH3), 3.26 (2H, COCH2N), 3.07–2.94 (4H, m,
H-3, 30), 2.59 (4H, m, COCH2N(CH2)2), 1.74 (4H, m,
NCH2CH2CH2CH2N); 13C NMR (DMSO-d6, 100 MHz) d: 171.8,
171.7, 169.2 (Ar-NHCO), 165.6 (C-100), 141.4 (C-500), 138.3 (C-4),
137.0 (C-40), 129.2 (ꢁ2), 129.1 (ꢁ2), 128.5 (C-200), 128.3 (ꢁ2),
128.2 (ꢁ2), 128.0 (ꢁ2), 126.6 (C-70), 126.2 (C-7), 118.5 (C-400, 600),
59.5 (COCH2N), 54.4 (C-2), 53.73 (C-20), 53.69 (COCH2N(CH2)2),
51.9 (OCH3), 36.9 (C-3), 36.6 (C-30), 23.5 (NCH2CH2CH2CH2N); EI-
MS m/z: 556 (M+), 231, 119, 84 (100); TOFESMS: calcd for
4.1.2.29.
N-(N-Benzoyl-L-phenylalanyl)-O-
ethyl-L-tyrosine
(17b). White needle crystal, yield 57%, mp 263–264 °C, 1H NMR
(DMSO-d6, 400 MHz) d: 12.85 (1H, br, COOH), 8.55 (1H, d,
J = 8.4 Hz, NHCO), 8.24 (1H, d, J = 8.0 Hz, NHCO), 7.76 (2H, d,
J = 7.6 Hz, H-300, 700), 7.52 (1H, t, H-500), 7.43 (2H, t, H-400, 600),
7.33 (2H, d, J = 7.6 Hz, H-5, 9), 7.23 (2H, t, J = 7.6 Hz, H-6, 8),
7.15–7.10 (3H, m, J = 8.4 Hz, H-7, H-50, 90), 6.70 (2H, d,
J = 8.4 Hz, H-60, 80), 4.73 (1H, m, H-2), 4.41 (1H, m, H-20), 3.87
(2H, m, Ar-OCH2CH3), 3.10–2.84 (4H, m, H-30, 3), 1.26 (3H, t, Ar-
13
32H37N4O5 [M+H]+ 557.2764, found 557.2770.
OCH2CH3); C NMR (DMSO-d6, 100 MHz) d: 172.9 (C-10), 171.4
C
(C-1), 166.2 (C-100), 157.2 (C-70), 138.4 (C-4), 134.0 (C-200), 131.4
(C-500), 130.2 (C-50, 90), 129.2 (C-6, 8), 129.0 (C-40), 128.2 (C-400,
600), 128.1 (C-5, 9), 127.4 (C-300, 700), 126.2 (C-7), 114.1 (C-60, 80),
62.8 (Ar-OCH2CH3), 54.5 (C-20), 53.8 (C-2), 36.8 (C-3), 35.9 (C-
30), 14.7 (Ar-OCH2CH3); EI-MS m/z: 460, 251, 207, 135, 121, 105,
4.1.2.26. N-(N-4-(Dimethylamino)-acetamido-benzoyl-
L
-phenyl-
alanyl)-L-phenylalanol (16f). Pale yellow crystal, yield 47%, mp
162–164 °C, 1H NMR (DMSO-d6, 400 MHz) d: 9.94 (1H, s, Ar-
NHCO), 8.39 (1H, d, J = 8.8 Hz, NHCO), 7.89 (1H, d, J = 8.4 Hz,
NHCO), 7.77 (2H, d, J = 8.8 Hz, H-300, 700), 7.73 (2H, d, J = 8.8 Hz, H-
400, 600), 7.31–7.11 (10H, m, H-5, 9, 50, 90), 4.82 (1H, t, OH), 4.67
91 (100), 77; TOFESMS: calcd for
505.1715, found 505.1720.
C
27H27N2O5Na2 [M+Na]+

5360
J. Qiu et al. / Bioorg. Med. Chem. 19 (2011) 5352–5360
4.1.2.30. Sodium N-(N-benzoyl-
L
-phenylalanyl)-O- butyl-
L
-tyro-
4.2.2. Cytotoxicity assay
sinate (17c). White needle crystal, yield 55%, mp 274–276 °C, 1H
NMR (DMSO-d6, 400 MHz) d: 8.82 (1H, d, J = 8.4 Hz, NHCO), 7.78
(2H, d, J = 7.2 Hz, H-300, 700), 7.51–7.47 (2H, m, H-500, NHCO), 7.41
(2H, t, H-400, 600), 7.30 (2H, d, J = 7.6 Hz, H-5, 9), 7.22 (2H, t, H-6,
8), 7.12 (1H, t, H-7), 6.99 (2H, d, J = 8.4 Hz, H-50, 90), 6.55 (2H, d,
J = 8.0 Hz, H-60, 80), 4.57 (1H, m, H-2), 3.94 (1H, m, H-20), 3.75
(2H, t, J = 6.4 Hz, Ar-OCH2CH2CH2CH3), 3.12–2.85 (4H, m, H-3, 30),
1.61 (2H, m, Ar-OCH2CH2CH2CH3), 1.37 (2H, m, ArOCH2CH2CH2-
CH3), 0.89 (2H, t, J = 7.6 Hz, Ar-OCH2CH2CH2CH3); 13C NMR
(DMSO-d6, 100 MHz) d: 172.5 (C-10), 169.6 (C-1), 166.0 (C-100),
156.6 (C-70), 138.7 (C-4), 134.1 (C-200), 131.0 (C-500), 130.9 (C-40),
130.5 (C-50, 90), 128.9 (C-6, 8), 127.93, 127.87, 127.3 (C-300, 700),
125.9 (C-7), 113.4 (C-60, 80), 66.8 (Ar-OCH2CH2CH2CH3), 55.4,
55.3, 36.6, 36.3, 30.7 (ArOCH2CH2CH2CH3), 18.6 (Ar-OCH2CH2-
CH2CH3), 13.5 (Ar-OCH2CH2CH2CH3); EI-MS m/z: 488, 309, 163,
121, 105 (100), 91, 77; TOFESMS: calcd for C29H31N2O5Na2
[M+Na]+ 533.2028, found 533.2033.
Cytotoxicity induced by the test compounds in cultures of
2.2.15 cells was also determined. The 2.2.15 cells were grown to
confluence in 96-well flat-bottomed tissue culture plates and trea-
ted with test compound (in 0.2 mL culture medium/well) as de-
scribed above. Untreated control cultures were maintained on
each 96-well plate. The toxicity was determined by measuring
neutral red dye uptake, as determined from the absorbance at
510 nm relative to untreated cells, at 24 hours following 9 days
of treatment.
Acknowledgments
This work was supported by the Grants from the National Nat-
ural Science Foundation of China (NSFC No. 30760292), the Minis-
try of Science and Technology of China (No. 2011ZX09102-009-2),
and the Science and Technology Department of Guizhou Province
(No. QKHZYZ [2010] 5018).
The authors are grateful to the staff of the analytical group of
The Key laboratory of Chemistry for Natural Products of Guizhou
Province and Chinese Academy of Sciences for measurements of
all spectra.
4.2. Biological assay
4.2.1. In vitro anti-HBV assays
The anti-HBV activities in vitro included the ability to inhibit
the production of HBsAg and HBeAg in 2.2.15 cells, and the replica-
tion of HBV DNA in HBV-infected 2.2.15 cells. Confluent cultures of
2.2.15 cells were maintained on 96-well flat-bottomed tissue cul-
ture plates in RPMI 1640 medium with 2% fetal bovine serum for
the antiviral analyses. Cultures were treated with eight consecu-
tive daily doses of the test compounds and lamivudine (produced
by Glaxo & Welcome Co.). The Hep G2.2.15 cells were incubated
in 24-well plates at a density of 1.0 ꢁ 105 cells/mL in 1 L MEM
medium containing 10% FBS for 24 h. After attachment to plates,
the supernatants in each well were replaced carefully with 1 mL
of fresh DMEM containing different concentrations (50, 25, and
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