Synthesis and anti-hepatitis B virus activities of Matijing-Su derivatives

Article abstract of DOI:10.1016/j.bmc.2009.03.003

A series of derivatives of Matijing-Su (MTS, N-(N-benzoyl-l-phenylalanyl)-O-acetyl-l-phenylalanol) was synthesized and evaluated for their anti-hepatitis B virus (HBV) activities in 2.2.15 cells. The IC₅₀ of compounds 9c (1.40 μM), 9g (2.33 μM) and 9n (2.36 μM), etc. and the selective index of 9n (45.93) of the inhibition on the replication of HBV DNA were higher than those of the positive control lamivudine [41.59, (IC₅₀: 82.42 μM)]. Compounds 11d, 12a and 12e also exhibited significant anti-HBV activities.

Full text of DOI:10.1016/j.bmc.2009.03.003

Bioorganic & Medicinal Chemistry 17 (2009) 3118–3125
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-hepatitis B virus activities of Matijing-Su derivatives
Bixue Xu a, Zhengming Huang b, Changxiao Liu c, Zegui Cai a, Weidong Pan a, Peixue Cao a,
Xiaojiang Hao a, Guangyi Liang a,
*
a The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, 202 Shachong South Road, Guiyang 550002, PR China
b 302 Hospital of PLA, 100 Xisihuan Zhong Road, Fengtai, Beijing 100039, PR China
c Tianjin Institute of Pharmaceutical Research, 308 Anshan Xi Dao, Nankai District, Tianjin 300193, 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 derivatives of Matijing-Su (MTS, N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-phenylalanol) was
Received 10 January 2009
Revised 1 March 2009
Accepted 3 March 2009
Available online 9 March 2009
synthesized and evaluated for their anti-hepatitis B virus (HBV) activities in 2.2.15 cells. The IC50 of com-
pounds 9c (1.40 M), 9g (2.33 M) and 9n (2.36 M), etc. and the selective index of 9n (45.93) of the
inhibition on the replication of HBV DNA were higher than those of the positive control lamivudine
l
l
l
[41.59, (IC50: 82.42
lM)]. Compounds 11d, 12a and 12e also exhibited significant anti-HBV activities.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anti-hepatitis B virus activity
Derivatives of Matijing-Su
Synthesis
1. Introduction
which has been widely used in the treatment of chronic liver dis-
eases in China. The anti-HBV activities of MTS were found in our
Hepatitis B virus (HBV)-infected hepatitis is the most common
infectious disease all over the world. More than 400 million people
worldwide are infected by HBV.1 Previous research results showed
that HBV played a major role in occurrence and development of li-
ver fibrosis2,3 and hepatocelluar carcinoma.4–6 Important advances
in the anti-HBV agents have been made with the introduction of
non(low)-toxic, efficacious nucleotide analogues that can interfere
directly with HBV genomic DNA synthesis. Lamivudine (LMV), ade-
fovir (ADV) and entecavir (ETV) are well-tolerated and may lead to
viral suppression in the majority of treated patients while the ther-
apy is maintained.7–10 Interferon (IFN) alpha has both immuno-
stimulatory and direct antiviral properties. Although various
treatment options exist for chronic HBV infection, none is entirely
satisfactory. The clinical application of these agents, particularly
LMV, is limited by the frequent development of resistance, occur-
ring at the rates of up to 15–30% per year.11,12 For ADV, the devel-
opment of resistance is slower, however, 28% of treated patients
develop genotypic resistance by year 5.13 Interferon (IFN) alpha
is effective in only one-third of patients14 and the treatment is
greatly hampered by significant adverse effects.15
previous studies.16 Since the anti-HBV activities of the compounds
with a dipeptide framework still have not been found in the liter-
atures and patents before, these study results of MTS greatly
encouraged us to make further research on it for discovery of more
potent anti-HBV derivatives. In this paper, we report some results
of synthesis of the new derivatives of MTS and evaluation of their
anti-HBV activities.
2. Results
2.1. Synthetic approach
The synthesis of the key synthetic intermediates was described
in Scheme 1.
The procedure for synthesis of the target compounds was illus-
trated in Scheme 2. The substituted benzoic acid was treated with
thionyl chloride to give the substituted benzoyl chloride (7), which
was then reacted with the substituted
NaOH solution to give compound 8. Compound 8 was reacted with
isobutylchloroform (IBCF) and substituted -phenylalanine methyl
ester hydrochloride or substituted -phenylalanol in the presence
L-phenylalanine in a 2 M
L
On the other hand, the natural products, with their enormous
structural diversity, provide a large opportunity for screening
anti-HBV agents possessing novel basic structural backbones and
L
of N-methylmorpholine (NMM) to afford compound 9. For the
one route, hydrogenation of nitro group of compound 9 give com-
pound 10, which was followed by treatment with Ac2O in pyridine
at room temperature to provide compound 11. At this stage, the
target compound 12 was synthesized by hydrolysis of compounds
9 or 11 with NaOH in DMF/EtOH. Thus, the structures of the syn-
thesized target compounds are listed in Table 1.
mechanisms of action. Matijing-Su (MTS, N-(N-benzoyl-
L-phenylal-
anyl)-O-acetyl- -phenylalanol), a dipeptide derivative, was isolated
L
from a Chinese ethnic drug Matijing (Dichondra repens Forst.)
* Corresponding author. Tel./fax: +86 851 5652109.
E-mail address: guangyi_liang@126.com (G. Liang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.003

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
3119
O
O
a
OH
OH
O
NH2
O
NH2
O2N
2
4
1
OH
b
OCH3
NH2HCl
c
OH
NH2
NH2
R1
R1
R1
3
5
Scheme 1. Synthesis of the key intermediates. Reagents and conditions: (a) HNO3/H2SO4 (1.4: 1.1), rt.; (b) MeOH, thionyl chloride (SOCl2), rt, 2.5 h, then reflux for 30 min; (c)
NaBH4/H2O.
O
O
R4
O
OH
R2
O
Cl
OH
O
N
H
O
HN
HN
R1
R1
c
b
a
R2
R2
R2
R3
6
7
8
9
O
R4
O
R4'
N
H
O
N
H
O
d
e
f
10
11
HN
HN
R1
R1'
or
f
R2
R '
R3
R3'
2
9
12
Scheme 2. Synthesis of the target compounds. Reagents and conditions: (a) SOCl2, reflux 30 min; (b) substituted
L
-phenylalanine, 2 M NaOH (aq), ꢀ5 °C to rt, 3 h; (c)
substituted
L-phenylalanine methyl ester hydrochloride or substituted
L
-phenylalanol, CH2Cl2, IBCF, NMM, ꢀ5 °C; (d) MeOH, H2, Pd/C (10%), rt, 3 h; (e) Ac2O, pyr, rt.; (f) NaOH,
DMF/EtOH, rt.
2.2. Biological activity test
lamivudine as reference control. According to the above results,
conclusions could be made as:
Hep G2.2.15 cell contained multiple copies of the HBV genome,
which were stably integrated into the host cell genome and was
widely used as a useful ‘in vitro’ model for evaluation of novel
anti-HBV drugs. So, in the experiment, the Hep G2.2.15 cell line
as in vitro cellular model was chosen. The results indicated that
the derivatives of MTS could be potent HBV inhibitors (there were
(a) In general, a series of derivatives of MTS could be potent
HBV inhibitors (there were four derivatives which
IC50 < 4
the IC50 of compounds 9c (1.40
(2.36 M) and the selective index of 9n [45.93] of the inhibi-
tion on the replication of HBV DNA were higher than those of
the positive control lamivudine [41.59, (IC50: 82.42 M)].
l
M) with high selectivity indices (SI > 20), such as
l
M), 9g (2.33 M) and 9n
l
l
four derivatives which IC50 < 4
(SI > 20).
lM) with high selectivity indices
l
The target compounds 9a–9r, 11b–11d, 12a–12g were evalu-
ated for their cytotoxicities and anti-HBV activities, namely the
ability to inhibit the replication of HBV DNA and the production
of HBsAg and HBeAg in HBV-infected 2.2.15 cells. The results of
the inhibition for the replication of HBV DNA were summarized
in Table 2.
(b) The substitution with deferent groups on the ring A, B and C
of the derivatives of MTS could change the anti-HBV activi-
ties and cytotoxicities. The 500-halide substituted on the ben-
zoyl ring C was an important feature in conferring relatively
potent inhibitory activity; but the 300-halide substitution on
the ring C might weaken the inhibitory activity.
As shown in Table 2, compound 9c showed the most potence
in vitro anti-HBV activity within all of the tested compounds. Its
(c) Both hydroxymethyl and methyl carboxylate substituted on
the 20-position derivatives might be the pharmacophores
from the evaluation of their cytotoxicities and anti-HBV
activities with potent HBV inhabitation.
IC50 was 1.40
the control lamivudine (82.42
also exhibited significant inhibitory efficacy on the replication of
HBV DNA (IC50: 2.33, 2.36, 3.94 M).
lM, which was about 59 times higher than that of
l
M). Compounds 9g, 9n and 12a
l
4. Experimental
4.1. Chemistry
However, the inhibition for the production of HBsAg and
HBeAg of the tested compounds did not be found in the
examination.
Melting points were measured with a model XT-4 apparatus
and are uncorrected. 1H (400 MHz) and 13C (100 MHz) NMR spec-
tra were performed on a Varian Unit INOVA-400 spectrometers in
CDCl3 or DMSO-d6 with Me4Si (TMS) as an internal standard. Mass
spectra (MS) were recorded on a Agilent MS-5973 spectrometer.
3. Conclusion
A series of new MTS derivatives was synthesized and their anti-
HBV activities as well as cytotoxicities in vitro were examined with

3120
B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
Table 1
Structures of the synthesized target compounds 9a–9r, 11b–11d, 12a–12g
O
1
R4
2'
5
2
4
6
R1
7
3'
4'
3
N
H
O
A
9'
9
HN
2''
5'
R3
6'
8
B
1''
8'
7''
3''
R2
4''
7'
C
6''
5''
No
R1
R2
R3
R4
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
H
H
H
H
H
H
H
H
H
H
H
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Methyl carboxylate
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Hydroxymethyl
Carboxy
9a
9d
9o
11c
9c
9j
9i
9l
11b
9k
9n
9q
9r
11d
9b
9p
12b
9h
9g
9e
9m
9f
70-Hydroxy
70-Nitro
70-Acetamido
H
H
H
500-Fluoro
300-Chloro
400-Methyl
7-Nitro
7-Acetamido
H
7-Nitro
H
7-Nitro
7-Acetamido
H
H
H
H
H
H
H
7-Nitro
7-Acetamido
H
H
H
H
H
H
H
H
300-Chloro
70-Hydroxy
H
70-Hydroxy
500-Methyl
70-Hydroxy
500-Methyl
70-Hydroxy
500-Methyl
70-Acetoxy
H
H
H
H
70-Nitro
70-Acetamido
300-Chloro
H
H
H
H
H
H
H
500-Chloro
400-Methyl
500-Methyl
H
H
H
H
H
H
H
12a
12e
12f
12c
12d
12g
70-Hydroxy
70-Acetamido
70-Propionamido
H
Carboxy
Carboxy
Carboxy
Carboxy
7-Nitro
4.1.1. Synthesis of some intermediates17,18
4.1.1.1. 4-Nitro- -phenylalanine (2). To a solution of L-phenyl-
J = 8.4 Hz, H-5, 9), 4.37 (1H, m, H-2), 3.67 (3H, s, OMe), 3.35–3.22
(4H, m, H-3). EI-MS m/z: 223 [MꢀHClꢀ1]+, 165, 119, 88 (100), 78,
60, 43.
L
alanine (16.5 g, 100 mmol) in H2SO4 (85%, 50 mL) was added drop-
wise a mixed acid of concentrated HNO3 and concentrated H2SO4
(V/V = 1.4/1.1) under stirring, which was pre-prepared and cooled
down to room temperature. The reaction mixture was stirred for
5 h at room temperature. The pH of the reaction solution was then
adjusted to 2–3 with 40% NaOH and the resulting precipitate was
4.1.1.4. 4-Nitro-L-phenylalanol (5a). Compound 4b (2.4 g,
9.2 mmol) was dissolved in water (15 mL), then the solution was
slowly added to a stirred solution of NaBH4 (1.13 g, 30 mmol in
15 mL water) in an ice-salt-bath. The resulting mixture was
warmed up to room temperature over 2 h and kept overnight at
room temperature. The reaction mixture was extracted with EtOAc
(3 ꢂ 50 mL). The combined organic phase was successively washed
with saturated aqueous NaHCO3 (20 mL), brine (20 mL) and dried
over Na2SO4. The solvent was evaporated in vacuo to afford 4-ni-
collected by filtration and dried to give 4-nitro-
L-phenylalanine (2)
as
a
pale yellow powder (20 g, 95.2%). 1H NMR (DMSO-d6,
400 MHz) d: 8.18 (2H, d, J = 8.4 Hz, H-6, 8), 7.55 (2H, d, J = 8.0 Hz,
H-5, 9), 4.01 (1H, m, H-3), 3.5(br, NH2), 3.27-3.12, (2H, m, H-3). EI-
MS m/z: 194 [MꢀH2O]+, 184, 177, 138, 124, 110, 79 (100), 67, 52, 42.
tro-L-phenylalanol (5a) as a yellow powder (1.23 g, 68.2%). EI-MS
4.1.1.2.
L-Phenylalanine methyl ester hydrochloride (4a). SOCl2
m/z: 196 (M+), 178, 165, 88 (100), 78, 60.
(9.5 mL, 132 mmol) was added to 90 mL of MeOH, which was cooled
in an ice-salt-bath. The mixture was warmed up to room tempera-
4.1.2. Synthesis of the target compounds
ture.
L
-Phenylalanine (16.5 g, 100 mmol) was then added and the
The synthetic procedures used in the preparation of target com-
pounds are illustrated in Scheme 2. Substituted benzoic acid was
reacted with thionyl chloride to give substituted benzoyl chloride
mixture was stirred for 2.5 h at room temperature, then refluxed for
30 min. The solvent was removed and the residue was recrystallized
from methanol/ether to afford
L
-phenylalanine methyl ester hydro-
(7), then compound 7 was treated with substituted
nine in 2 M NaOH solution to give compound 8, which was reacted
with IBCF and substituted -phenylalanine methyl ester hydrochlo-
ride or substituted -phenylalanol in the presence of NMM to
L-phenylala-
chloride (4a) as colorless needles (18.67 g, 86.8%). EI-MS m/z: 179
[MꢀHCl]+, 120, 103, 91, 88 (100), 77, 65.
L
L
4.1.1.3. 4-Nitro-L-phenylalanine methyl ester hydrochloride
afford target compound 9.
(4b). It was prepared with a similar procedure to that described
for the synthesis of compound 4a. 1H NMR (DMSO-d6, 400 MHz) d:
8.7 (3H, br, –NH2ꢁHCl), 8.19 (2H, d, J = 8.4 Hz, H-6, 8), 7.55 (2H, d,
4.1.2.1. General procedure for the synthesis of compounds 9a–
9r. Benzoic acid or substituted benzoic acid (20 mmol) was

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
3121
Table 2
4.1.2.3. N-(N-Benzoyl-L-phenylalanyl)-L-phenylalanol (9b). Color-
less needle crystal, yield 58.0%, mp 168.0–171.0 °C (MeOH), 1H NMR
(CDCl3, 400 MHz) d: 7.72 (2H, d, J = 8.0 Hz, H-300, 700), 7.53 (1H, t,
J = 7.2 Hz, H-500), 7.44 (2H, t, H-400, 600), 7.35–7.07 (10H, m, H-5-9, 50-90),
6.80 (1H, d, J = 7.6 Hz, NHCO), 5.92 (1H, d, J = 7.6 Hz, NHCO), 4.77 (1H,
m, H-2), 4.10 (1H, m, H-20), 3.42 (2H, m, H-10), 3.27 (1H, dd, J = 5.6,
13.6 Hz, H-3a), 3.04 (1H, dd, J = 9.2, 13.6 Hz, H-3b), 2.77 (1H, dd,
J = 7.6, 13.6 Hz, H-30a), 2.68 (1H, dd, J = 6.8, 13.6 Hz, H-30b); 13C NMR
(CDCl3, 100 MHz) d: 170.9 (C-1), 166.1 (C-100), 139.0 (C-4), 138.4 (C-40),
134.1 (C-200), 131.3 (C-500), 129.2 (C-6, 8, 60, 80), 128.2 (C-400, 600), 128.1
(C-5, 9), 128.0 (C-50, 90), 127.4 (C-300, 700), 126.2 (C-70), 125.9 (C-7), 62.2
(C-10), 54.8 (C-2), 52.5 (C-20), 37.3 (C-3), 36.5 (C-30). EI-MS m/z: 402
(M+), 384, 311, 252, 224 (100), 92. Anal. Calcd for C25H26N2O3: C,
74.60; H, 6.51; N, 6.96. Found: C, 74.40; H, 6.90; N, 6.83.
The results of the inhibition for the replication of HBV DNA of compounds 9a–9r,
11b–11d, 12a–12g
No.
TC50
(
l
M)
DNA replication
Compound
IC50
—
(l
M)
SI
1
2
3
4
5
6
7
8
2557.75
1009.34
96.09
530.65
39.71
—
9a
9d
9o
11c
9c
9j
9i
9l
11b
9k
9n
9q
9r
11d
9b
9p
12b
9h
9g
9e
115.97
—
8.70
—
—
1.40
—
—
—
—
28.27
—
—
—
620.95
540.79
816.05
925.54
1039.72
108.69
1010.27
89.57
693.41
574.21
116.96
164.22
381.55
65.78
184.97
1863.29
119.37
93.82
313.47
1151.20
840.80
120.18
3427.82
9
122.01
—
7.59
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
27
28
MTS
3TC
2.36
—
—
45.93
—
—
4.1.2.4. N-(N-500-Fluoro-benzoyl-
L-phenylalanyl)-L-phenylalanine
methyl ester (9c). Mp 194.0–196.0 °C (EtOAc), 1H NMR (CDCl3,
400 MHz) d: 7.69–7.72 (2H, m, H-300, 700), 7.07–7.28 (10H, m, H-5-9,
50-90), 6.96–6.98 (2H, m, H-400, 600), 6.80 (1H, d, J = 7.6 Hz, NH), 6.34
(1H, d, J = 7.6 Hz, NH), 4.76–4.84 (2H, m, H-2, 20), 3.70 (3H, s, OCH3),
2.98–3.18 (4H, m, H-3, 30); 13C NMR (CDCl3, 100 MHz) d: 171.24 (s),
170.41 (s), 165.97 (2 ꢂ s), 136.28 (s), 135.44 (s), 129.47 (s), 129.38
(4 ꢂ d), 129.11 (2 ꢂ d), 128.69 (2 ꢂ d), 128.55 (2 ꢂ d), 127.11
(2 ꢂ d), 115.74 (d), 115.52 (d), 54.56 (d), 53.44 (d), 52.38 (q), 38.13
(t), 37.82 (t). EI-MS m/z: 448 (M+), 416, 389, 357, 309, 286, 270, 242,
218, 180, 162, 147, 131, 123 (100), 120, 95, 91, 77, 41, 28, 15. Anal.
Calcd for C26H25FN2O4: C, 69.63; H, 5.62; N, 6.25. Found: C, 69.43;
H, 5.98; N, 6.02.
23.45
—
29.57
—
—
—
—
—
—
—
2.33
60.48
—
28.29
3.06
—
9m
9f
—
—
3.94
25.35
—
23.82
12.36
—
12a
12e
12d
12g
MTS
Lamivudine
54.17
11.16
82.42
15.52
10.78
41.59
4.1.2.5. N-(N-Benzoyl-L-phenylalanyl)-L-tyrosine methyl ester
(9d). White crystal, mp 184.0–186.0 °C (MeOH/EtOAc); 1H NMR
(DMSO-d6, 400 MHz) d: 9.18 (1H, s, Ar-OH), 8.52 (1H, d, J = 8.4 Hz,
NHCO), 8.40 (1H, d, J = 7.6 Hz, NHCO), 7.77 (2H, d, J = 7.6 Hz, H-300,
700), 7.50 (1H, t, H-500), 7.42 (2H, t, H-400, 600), 7.33 (2H, d, J = 7.2 Hz,
H-5, 9), 7.24 (2H, t, H-6, 8), 7.16 (1H, t, H-7), 6.99 (2H, d, J = 8.4 Hz,
H-50, 90), 6.62 (2H, d, J = 8.4 Hz, H-60, 80), 4.74 (1H, m, H-2), 4.45
(1H, m, H-20), 3.60 (3H, s, OMe), 3.11–2.84 (4H, m, H-3, 30); 13C
NMR (DMSO-d6, 100 MHz) d: 171.8 (C-1), 171.5 (C-10), 166.2 (C-100),
156.0 (C-70), 138.2 (C-4), 134.0 (C-200), 131.1 (C-500), 129.9 (C-50, 90),
129.1 (C-6, 8), 128.0 (ꢂ2), 127.9 (ꢂ2), 127.3 (C-300, 700), 126.8 (C-7),
126.1 (C-40), 115.0 (C-60, 80), 54.4 (C-2), 53.9 (C-20), 51.7 (C–OMe),
36.9 (C-3), 35.9 (C-30). EI-MS m/z: 446 (M+), 269, 252, 224, 178, 105
(100), 91, 77. Anal. Calcd for C26H26N2O5: C, 69.94; H, 5.87; N, 6.27.
Found: C, 70.34; H, 5.93; N, 6.17.
dissolved in SOCl2 (7.2 mL, 100 mL). The reaction was refluxed for
5 h and evaporated in vacuo to give the intermediate of benzoyl
chloride or substituted benzoyl chloride 7. Then, compound 7
(10.0 mmol) was added, without further purification, to a solution
of L-phenylalanine or substituted L-phenylalanine (10.0 mmol) in
2 M NaOH (11 mL) in ice-salt-bath. The mixture was allowed to
warm up to room temperature within 2 h. The resulting mixture
was acidified to pH 5–6 with hydrochloric acid (1.8 mL, 21.6 mmol)
and the resulting precipitate was collected by filtration and dried
to give compound 8. IBCF (1.07 mmol) was added dropwise to a
mixture of compound 8 (1.0 mmol), substituted
methyl ester hydrochloride or substituted
L
-phenylalanine
-phenylalanol
L
(1.05 mmol) and NMM (23.0 mmol) in (50 mL) at 0 °C within 60–
70 min. The mixture was stirred for 30 min and the bulk of CH2Cl2
was removed in vacuo. The residue was dissolved in EtOAc and
washed sequentially with water, dilute HCl (40 mL), saturated
NaHCO3 solution and brine, dried over Na2SO4 and evaporated in
vacuo. The residue was recrystallized from ethyl acetate or appro-
priate solvents to afford target compound 9.
4.1.2.6. N-(N-400-Methyl-benzoyl-
L-phenylalanyl)-L-phenylalanol
(9e). Whitepowder, mp177.0–178.5 °C (MeOH);1H NMR (DMSO-
d6, 400 MHz) d: 8.44 (1H, d, J = 8.4 Hz, NHCO), 7.89 (1H, d, J = 8.4 Hz,
NHCO), 7.59–7.56 (2H, m, H-300, 700), 7.32–7.10 (12H, m, H-400, 500, H-5-
9, 50-90), 4.83 (1H, t, CH2OH), 4.67 (1H, m, H-2), 3.88 (1H, m, H-20),
3.33–3.23 (2H, m, H-1), 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), 2.33
(3H, s, Ar-CH3); 13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-1), 166.2
(C-100), 139.0 (C-40), 138.5 (C-4), 137.5 (C-400), 134.1 (C-200), 131.9 (C-
500), 129.3 (ꢂ3, C-60, 80, 600), 128.13 (ꢂ4, C-50, 90, C-6, 8), 128.07 (ꢂ2,
C-5, 9), 128.0 (C-300), 126.2 (C-70), 126.0 (C-7), 124.6 (C-700), 62.2 (C-
10), 54.8 (C-2), 52.5 (C-20), 33.3, 36.5, 21.0 (ArCH3). EI-MS m/z: 416
(M+), 398, 386, 325, 283, 266, 238, 190, 131, 119 (100), 104, 91. Anal.
Calcd for C26H28N2O3: C, 74.97; H, 6.78; N, 6.73. Found: C, 75.15; H,
6.87; N, 6.51.
4.1.2.2. N-(N-Benzoyl-L-phenylalanyl)-L-phenylalanine methyl
ester (9a). Colorless needl crystal, yield 65.0%, mp 206.0–
210.0 °C (EtOAc); 1H NMR (CDCl3, 400 MHz) d: 7.68 (2H, d,
J = 7.6 Hz, H-300, 700), 7.51 (1H, m, H-500), 7.42 (2H, t, H-400, 600),
7.31–7.13 (8H, m, H-5, 9, 6, 8, 50, 90, 60, 80), 6.97 (2H, m, H-7, 70),
6.76 (1H, d, J = 7.4 Hz, NHCO), 6.37 (1H, d, J = 7.6 Hz, NHCO), 4.85
(1H, m, H-2), 4.77 (1H, m, H-20), 3.70 (3H, s, OMe), 3.23–2.94
(4H, m, H-3, 30); 13C NMR (CDCl3, 100 MHz) d: 171.3 (C-1), 170.4
(C-10), 167.0 (C-100), 136.4 (C-4), 135.5 (C-40), 133.6 (C-200), 131.8
(C-500), 129.4 (C-6, 8), 129.1 (C-60, 80), 128.7 (C-400, 600), 128.6 (C-5,
9), 128.5 (C-50, 90), 127.1 (C-300, 700), 127.0 (C-7, 70), 54.5 (C-2),
53.4 (C-20), 52.3 (C–COMe), 38.0 (C-3), 37.8 (C-30). EI-MS m/z:
444 (M+), 384, 353, 311, 269, 252, 224, 172, 131, 105 (100), 91,
77, 60. Anal. Calcd for C26H26N2O4: C, 72.54; H, 6.09; N, 6.51.
Found: C, 72.63; H, 5.95; N, 6.36.
4.1.2.7. N-(N-Benzoyl-7-nitro-L-phenylalanyl)-L-phenylalanol
(9f). Pale yellow powder, mp 196.0–199.0 °C (MeOH); 1H NMR
(DMSO-d6, 400 MHz) d: 8.62 (1H, d, J = 8.4 Hz, NHCO), 8.12 (2H,
d, J = 8.8 Hz, H-6, 8), 8.05 (1H, d, J = 8.8 Hz, NHCO), 7.78 (2H, d,
J = 6.4 Hz, H-300, 700), 7.59 (2H, d, J = 8.4 Hz, H-5, 9), 7.51 (1H, t, H-

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B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
500), 7.43 (2H, t, H-400, 600), 7.26–7.12 (5H, m, H-50-90), 4.85 (1H, t,
OH), 4.76 (1H, m, H-2), 3.89 (1H, m, H-20), 3.32–3.26 (2H, m, H-
10), 3.19–3.07 (2H, m, H-3), 2.92–2.81 (2H, m, H-30); 13C NMR
(DMSO-d6, 100 MHz) d: 170.5 (C-1), 166.1 (C-100), 145.0 (C-7),
146.2 (C-4), 139.0 (C-40), 133.8 (C-200), 131.4 (C-500), 130.5 (C-5,
9), 129.2 (C-60, 80), 128.2 (C-400, 600), 128.1 (C-50, 90), 127.4 (C-300,
700), 125.9 (C-70), 123.2 (C-6, 8), 62.2 (C-10), 54.3 (C-20), 52.6 (C-2),
37.1 (C-30), 34.9 (C-3). EI-MS m/z: 447 (M+), 417, 356, 314, 297,
269, 134, 120, 105 (100), 91, 77, 60. Anal. Calcd for C25H25N3O5:
C, 67.10; H, 5.63; N, 9.39. Found: C, 67.53; H, 5.89; N, 9.17.
4.1.2.12. N-(N-300-Chloro-benzoyl-
L-phenylalanyl)-L-tyrosine
methyl ester (9k). Mp 164.5–167.0 °C (EtOAc), 1H NMR (DMSO-
d6, 400 MHz) d: 9.27 (1H, s, Ar-OH), 8.60 (1H, d, J = 8.4 Hz, NHCO),
8.40 (1H, d, J = 7.2 Hz, NHCO), 7.42–7.14 (9H, m, H-400-700, H-5-9),
7.03 (2H, d, J = 8.4 Hz, H-50, 90), 6.67 (2H, d, J = 8.4 Hz, H-60, 80),
4.75 (1H, m, H-2), 4.46 (1H, m, H-20), 3.08–2.79 (4H, m, H-3, 30);
13C NMR (DMSO-d6, 100 MHz) d: 171.9 (C-1), 171.1 (C-10), 166.0
(C-100), 156.1 (C-70), 137.8 (C-4), 136.4 (C-500), 130.8 (C-300), 130.1
(C-50, 90), 130.0 (C-200), 129.2 (400), 129.2 (C-6, 8), 128.9 (700), 128.0
(C-5, 9), 126.9 (C-600, C-40), 126.3 (C-7), 115.1 (C-60, 80), 54.0 (C-2),
51.8 (C-20), 37.2, 36.0. EI-MS m/z: 480 (M+), 303, 286, 258, 178,
139 (100), 107, 91, 77. Anal. Calcd for C26H25ClN2O5: C, 64.93; H,
5.24; N, 5.82. Found: C, 65.28; H, 5.66; N, 5.61.
4.1.2.8. N-(N-500-Chloro-benzoyl-
L-phenylalanyl)-L-phenylalanol
(9g). Mp 211.5–214.0 °C (MeOH), 1H NMR (DMSO-d6, 400 Mz) d:
8.63 (1H, d, J = 8.4 Hz, NHCO), 7.93 (1H, d, J = 8.0 Hz, NHCO), 7.81
(2H, d, J = 8.0 Hz, H-300, 700), 7.51 (2H, d, J = 8.0 Hz, H-400, 600), 7.12–
7.31 (10H, m, H-5-9, 50-90), 4.82 (1H, s, OH), 4.68 (H, m, H-2),
3.90 (1H, m, H-20), 3.29–3.39 (2H, m, H-10), 2.49–3.01 (4H, m, H-
4.1.2.13. N-(N-Benzoyl-7-nitro-L-phenylalanyl)-L-phenylalanine
methyl ester (9l). Mp 202.0–204.5 °C (MeOH), 1H NMR (CDCl3,
400 MHz) d: 8.09 (2H, d, J = 8.8 Hz, H-6, 8), 7.71 (2H, d, J = 7.6 Hz,
H-300, 700), 7.54 (1H, t, H-500), 7.43 (2H, t, H-400, 600), 7.38 (2H, d,
J = 8.8 Hz, H-5, 9), 7.13 (3H, m, H-60-80), 6.99 (2H, m, H-50, 90),
6.93 (1H, br, NHCO), 6.56 (1H, br, NHCO), 4.94 (1H, m, H-2), 4.80
(1H, m, H-20), 3.73 (3H, s, OCH3), 3.29 (1H, dd, J = 7.2, 13.6 Hz, H-
30a), 3.22 (1H, dd, J = 5.6, 14.0 Hz, H-30b), 3.09 (1H, dd, J = 5.6,
14.0 Hz, H-3a), 3.01 (1H, dd, J = 6.4, 14.0 Hz, H-3b); 13C NMR
(CDCl3, 100 MHz) d: 171.3 (C-1), 169.8 (C-10), 167.1 (C-100), 147.0
(C-7), 144.1 (C-4), 135.2 (C-40), 133.1 (C-200), 132.2 (C-500), 130.3
(C-5, 9), 129.0 (C-60, 80), 128.7 (C-50, 90), 128.6 (C-400, 600), 127.2
(C-70), 127.0 (C-300, 700), 123.7 (C-6, 8), 54.0 (C-2), 53.2 (C-20), 52.5
(OCH3), 38.0, 37.8. EI-MS m/z: 475 (M+), 339, 297, 269, 162, 120,
105 (100), 91, 77. Anal. Calcd for C26H25N3O6: C, 65.67; H, 5.30;
N, 8.84. Found: C, 65.87; H, 5.49; N, 8.58.
13
3, 30), C NMR (DMSO-d6, 100 MHz) d: 170.9 (C-1), 165.1 (C-100),
139.0 (C-4), 138.1 (C-40), 136.2 (C-500), 132.4 (C-200), 129.4 (ꢂ2),
129.2 (ꢂ2), 128.3 (ꢂ2), 128.09 (ꢂ2), 128.07 (ꢂ2), 126.2, 125.9,
62.2 (C-10), 54.9 (C-2), 52.5 (C-20), 37.3 (C-3), 36.4 (C-30). EI-MS
m/z: 436 (M+), 406, 345, 327, 303, 286, 258, 190, 139 (100), 120,
111, 104, 91, 73, 57, 43, 28. Anal. Calcd for C25H25ClN2O3: C,
68.72; H, 5.77; N, 6.41. Found: C, 69.21; H, 6.01; N, 6.08.
4.1.2.9. N-(N-300-Chloro-benzoyl-
L-phenylalanyl)-L-phenylalanol
(9h). 1H NMR (CDCl3, 400 MHz) d: 7.15–7.40 (14H, m, H-5-9, 5-
90, 400-700), 4.59–4.82 (1H, m, H-2), 4.07–4.10 (1H, m, H-20), 3.41–
3.42 (2H, m, 10), 2.71–3.17 (4H, m, H-3, 30), 13C NMR (CDCl3,
100 MHz) d: 172.81 (s), 169.53 (s), 139.61 (s), 138.42 (s), 137.02
(s), 132.27 (d), 131.96 (s), 130.92 (d), 130.45 (2 ꢂ d), 130.43
(2 ꢂ d), 130.01 (d), 129.46 (2 ꢂ d), 129.42 (2 ꢂ d), 127.96 (d),
127.80 (d), 127.33 (d), 63.73 (t), 56.55 (d), 54.14 (d), 38.93 (t),
37.87 (t). EI-MS m/z: 436 (M+), 418, 406, 345, 327, 303, 286, 258,
190, 139 (100), 120, 104, 91, 73, 60, 43, 28, 18. Anal. Calcd for
C25H25ClN2O3: C, 68.72; H, 5.77; N, 6.41. Found: C, 69.03; H,
5.80; N, 6.26.
4.1.2.14. N-(N-500-Methyl-benzoyl-
L-phenylalanyl)-L-phenylala-
nol (9m). Mp 200.0–202.0 °C (EtOAc), 1H NMR (DMSO-d6,
400 MHz) d: 8.39 (1H, d, J = 8.8 Hz, NHCO), 7.86 (1H, d, J = 8.8 Hz,
NHCO), 7.68 (2H, d, J = 8.0 Hz, H-300, 700), 7.29–7.11 (12H, m, H-400,
600, H-50-90, 5-9), 4.81 (1H, t, OH), 4.65 (1H, m, H-2), 3.88 (1H, m,
H-20), 3.33–3.24 (2H, m, H-10), 3.03–2.90 (2H, m, H-3), 2.84 (1H,
dd, J = 6.0, 13.6 Hz, H-30a), 2.65 (1H, dd, J = 8.0, 13.6 Hz, H-30b),
2.33 (3H, s, Ar-CH3); 13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-
1), 166.0 (C-100), 141.2 (C-500), 139.0 (C-40), 138.4 (C-4), 131.3 (C-
200), 129.2 (ꢂ4, C-6, 8, C-60, 80), 128.7 (C-400, 600), 128.1 (C-50, 90),
128.0 (C-5, 9), 127.5 (C-300, 700), 126.2 (C-7), 125.9 (C-70), 62.2 (C-
10), 54.8 (C-20), 52.5 (C-2), 37.3, 36.4, 21.0 (Ar-CH3). EI-MS m/z:
432 (M+), 414, 341, 297, 282, 254, 206, 147, 136, 119 (100), 107,
91, 77, 57. Anal. Calcd for C26H28N2O3: C, 74.97; H, 6.78; N, 6.73.
Found: C, 74.93; H, 7.14; N, 6.56.
4.1.2.10. N-(N-400-Methyl-benzoyl-
L-phenylalanyl)-L-phenylala-
nine methyl ester (9i). Mp: 190.0–192.0 °C (EtOAc), 1H NMR
(CDCl3, 400 MHz) d: 7.51 (1H, s, H-300), 7.46 (1H, d, J = 4.4 Hz, H-
700), 7.31–7.23 (6H, m, H-500, 600, H-6, 8, 60, 80), 7.16–7.11 (4H, m,
H-5, 9, H-50, 90), 6.99–6.95 (2H, m, H-7, 70), 6.68 (1H, d, J = 8.0 Hz,
NHCO), 7.89 (1H, d, J = 7.4 Hz, NHCO), 4.80 (2H, m, H-2, 20), 3.70
(3H, s, OCH3), 3.23–2.93 (4H, m, H-3, 30), 2.39 (3H, s, Ar-CH3); 13C
NMR (CDCl3, 100 MHz) d: 171.2 (C-1), 170.3 (C-10), 167.2 (C-100),
138.4 (C-400), 136.4 (C-4), 135.5 (C-40), 133.5 (C-200), 132.6 (C-500),
129.4 (ꢂ2), 129.1 (ꢂ2), 128.7 (ꢂ2), 128.5 (ꢂ2), 128.4 (C-600),
127.7 (C-300), 127.0 (ꢂ2, C-7, 70), 124.0 (C-700), 54.4 (C-2), 53.4 (C-
20), 52.4 (OCH3), 38.0, 37.8. EI-MS m/z: 444 (M+), 353, 309, 266,
238, 147, 119 (100), 104, 91. Anal. Calcd for C27H28N2O4: C,
72.95; H, 6.35; N, 6.30. Found: C, 73.16; H, 6.94; N, 6.14.
4.1.2.15. N-(N-Benzoyl-7-nitro-L-phenylalanyl)-L-tyrosine
methyl ester (9n). Mp 200.0–202.0 °C (MeOH/EtOAc), 1H NMR
(DMSO-d6, 400 MHz) d: 9.27 (1H, s, Ar-OH), 8.67 (1H, d,
J = 8.4 Hz, NHCO), 8.57 (1H, d, J = 7.2 Hz, NHCO), 8.13 (2H, d,
J = 8.4 Hz, H-6, 8), 7.76 (2H, d, J = 7.2 Hz, H-300, 700), 7.62 (2H, d,
J = 8.4 Hz, H-5, 9), 7.51 (1H, t, H-500), 7.43 (2H, t, H-400, 600), 7.00
(2H, d, J = 8.0 Hz, H-50, 90), 6.63 (2H, d, J = 8.0 Hz, H-60, 80), 4.83
(1H, m, H-2), 4.42 (1H, m, H-20), 3.57 (3H, s, OMe), 3.22–2.84
(4H, m, H-3, 30); 13C NMR (DMSO-d6, 100 MHz) d: 171.9 (C-1),
171.2 (C-10), 166.3 (C-100), 156.1 (C-70), 146.8 (C-7), 146.2 (C-4),
133.8 (C-200), 131.4 (C-500), 130.6 (C-5, 9), 130.4 (C-40), 130.1 (C-50,
90), 128.3 (C-400, 600), 127.4 (C-300, 700), 122.2 (C-6, 8), 115.1 (C-60,
80), 54.2 (C-2), 50.0 (C-20), 51.9 (C-OMe), 36.8 (C-3), 35.9 (C-30).
EI-MS m/z: 491 (M+), 385, 314, 297, 269, 178, 105 (100), 91, 77,
51. Anal. Calcd for C26H25N3O7: C, 63.54; H, 5.13; N, 8.55. Found:
C, 63.88; H, 5.34; N, 8.19.
4.1.2.11. N-(N-300-Chloro-benzoyl-
L-phenylalanyl)-L-phenylala-
nine methyl ester (9j). Mp 152.5–153.5 °C (EtOAc), 1H NMR
(CDCl3, 400 MHz) d: 7.51 (1H, d, J = 8.0 Hz, H-700), 7.35–7.34 (2H, m,
H-400, 500), 7.30–7.17 (9H, m, H-5, 6, 8, 9, 50, 60, 80, 90, 600), 6.97–6.95
(2H, m, H-7, 70), 6.75 (1H, d, J = 7.6 Hz, NHCO), 6.30 (1H, d,
J = 7.6 Hz, NH), 4.85 (1H, m, H-2), 4.78 (1H, m, H-2), 3.67 (3H, s,
OCH3), 3.20–3.00 (4H, m, H-3, 30); 13C NMR (CDCl3, 100 MHz) d:
171.2, 169.9, 166.1, 136.2, 135.5, 134.1, 131.6, 130.8, 130.3, 130.2,
129.4 (ꢂ2), 129.2 (ꢂ2), 128.7 (ꢂ2), 128.6 (ꢂ2), 127.14, 127.12,
127.0, 54.9, 53.4, 52.3, 37.9, 37.8. EI-MS m/z: 464 (M+), 432, 373,
309, 302, 286, 258, 218, 180, 162, 147, 139 (100), 131, 120, 112,
102, 97, 91, 83, 73, 60, 44, 28. Anal. Calcd for C26H25ClN2O4: C,
67.17; H, 5.42; N, 6.03. Found: C, 66.89; H, 5.19; N, 6.22.
4.1.2.16. N-(N-Benzoyl-
methyl ester (9o). Mp 220.0–221.5 °C (MeOH/EtOAc), 1H NMR
L L-phenylalanine
-phenylalanyl)-70-nitro-

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
3123
(DMSO-d6, 400 MHz) d: 8.57 (1H, d, J = 8.0 Hz, NHCO), 8.52 (1H, d,
J = 8.8 Hz, NHCO), 8.04 (2H, d, J = 8.4 Hz, H-60, 80), 7.73 (2H, d,
J = 7.6 Hz, H-300, 700), 7.49 (3H, m, H-500, H-50, 90), 7.41 (2H, t, 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, H-
7), 4.70 (1H, m, H-2), 4.62 (1H, m, H-20), 3.61 (3H, s, OCH3), 3.25–
2.90 (4H, m, H-3, 30); 13C NMR (DMSO-d6, 100 MHz) d: 171.6 (C-1),
171.4 (C-10), 166.2 (C-100), 146.3 (C-70), 145.5 (C-40), 138.3 (C-4),
133.9 (C-200), 131.4 (C-500), 130.6 (C-50, 90), 129.2 (C-6, 8), 128.2 (ꢂ2),
128.1 (ꢂ2), 127.4 (C-300, 700), 126.3 (C-70), 123.2 (C-60, 80), 54.5 (C-2),
52.9 (C-20), 52.1 (OCH3), 36.8, 36.2. EI-MS m/z: 475 (M+), 354, 252,
224, 177, 131, 105 (100), 91, 77, 51. Anal. Calcd for C26H25N3O6: C,
65.67; H, 5.30; N, 8.84. Found: C, 65.36; H, 5.16; N, 9.03.
the compound 10a (469 mg, <1.0 mmol) was dissolved in pyridine
(5 mL), then Ac2O (2 mL) was added, After stirred at room temperature
for 5 h, the reaction was quenched with EtOH (5 mL) and concentrated
in vacuo. The residue was dissolved in EtOAc (250 mL) and washed
sequentially with water, saturated NaHCO3 solution, and brine. The solu-
tion was dried and concentrated, the residue was recrystallized from
EtOAc to afford N-(N-benzoyl-7-acetamido-L-phenylalanyl)-O-acetyl-L-
phenylalanol (11a) as white powder (300 mg, 60.0%). Mp 233.5–235.5 °C
(EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 9.84 (1H, s, ArNHCO), 8.46
(1H, d, J = 8.4 Hz, NHCO), 7.89 (1H, d, J = 8.0 Hz, NHCO), 7.79 (2H, d,
J = 7.6 Hz, H-300 , 700), 7.52 (1H, t, H-500), 7.47–7.42 (4H, m, H-400, 600 , H-6,
8), 7.23–7.11 (7H, m, H-50-90, H-5, 9), 4.83 (1H, t, OH), 4.64 (1H, m, H-2),
3.90 (1H, m, H-20), 3.34–3.25 (2H, m, H-10), 3.00–2.64 (4H, m, H-3, 30),
1.99 (3H, CH3CO); 13C-NMR (DMSO, 100 MHz) d: 170.9 (C-1), 168.0
(ArNHCOCH3), 166.0 (C-100), 139.0 (C-7), 137.5 (C-40), 134.1 (C-200 ), 132.9
(C-4), 131.3 (C-500), 129.4 (C-5, 9), 129.2 (C-60, 80), 128.2 (ꢂ2), 128.1
(ꢂ2), 127.4 (C-300, 700 ), 125.9 (C-70), 118.6 (C-6, 8), 62.2 (C-10), 54.9 (C-2),
52.5 (C-20), 36.7 (C-30), 36.4 (C-3), 24.0 (NHCOCH3). EI-MS m/z: 501
(M+), 380, 309, 281, 229, 203, 188, 148, 105 (100), 91, 77. Anal. Calcd for
C29H31N3O5: C, 69.44; H, 6.23; N, 8.38. Found: C, 69.86; H, 6.45; N, 8.27.
Then, 2.0 mL NaOH (5 M) was added to a solution of compound 11a
(1.003 g, 2.0 mmol) in dry DMF (5 mL) and EtOH (10 mL). After stirring
at room temperature for overnight the reacted mixture was acidified to
pH 2–3 with concentrated hydrochloric acid and partitioned between
EtOAc (150 mL) and water (50 mL). The organic phase was combined
andwashed successivelywith dilute HCl(40 mL), brine (2 ꢂ 30 mL), dried
and removed the solvent. The residue was recrystallized from EtOAc to af-
ford target compound 12a as white needles (680 mg, 74.0%). mp 233.5–
235.5 °C (EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 9.84 (1H, s, ArNHCO),
8.46 (1H, d, J = 8.4 Hz, NHCO), 7.89 (1H, d, J = 8.0 Hz, NHCO), 7.79 (2H, d,
J = 7.6 Hz, H-300 , 700), 7.52 (1H, t, H-500), 7.47–7.42 (4H, m, H-400, 600 , H-6,
8), 7.23–7.11 (7H, m, H-50-90, H-5, 9), 4.83 (1H, t, OH), 4.64 (1H, m, H-2),
3.90 (1H, m, H-20), 3.34–3.25 (2H, m, H-10), 3.00–2.64 (4H, m, H-3, 30),
1.99 (3H, CH3CO); 13C NMR (DMSO-d6, 100 MHz) d: 170.9 (C-1), 168.0
(ArNHCOCH3), 166.0 (C-100), 139.0 (C-7), 137.5 (C-40), 134.1 (C-200 ), 132.9
(C-4), 131.3 (C-500), 129.4 (C-5, 9), 129.2 (C-60, 80), 128.2 (ꢂ2), 128.1
(ꢂ2), 127.4 (C-300 , 700), 125.9 (C-70), 118.6 (C-6, 8), 62.2 (C-10), 54.9
(C-2), 52.5 (C-20), 36.7 (C-30), 36.4 (C-3), 24.0 (NHCOCH3). EI-MS m/z:
459 (M+), 441, 338, 309, 281, 229, 204, 188, 148, 105 (100), 91, 77. Anal.
Calcd for C27H29N3O4: C, 70.57; H, 6.36; N, 9.14. Found: C, 70.08; H,
6.18; N, 8.68.
4.1.2.17. N-(N-Benzoyl-L L-phenylalanol
-phenylalanyl)-70-nitro-
(9p). Mp 192.0–195.0 °C (MeOH/EtOAc), 1H NMR (DMSO-d6,
400 MHz) d: 8.46 (1H, d, J = 8.4 Hz, NHCO), 7.97 (2H, d, J = 8.0 Hz,
H-60, 80), 7.92 (1H, d, J = 8.8 Hz, NHCO), 7.76 (2H, d, J = 8.0 Hz, H-300,
700), 7.52–7.40 (5H, m, H-400-600, H-50, 90), 7.29 (2H, d, J = 7.2 Hz, H-5,
9), 7.23 (2H, t, H-6, 8), 7.14 (1H, t, H-7), 4.89 (1H, t, OH), 4.63 (1H,
m, H-2), 4.97 (1H, m, H-20), 3.36–3.27 (2H, m, H-10), 3.05–2.72 (4H,
m, H-3, 30); 13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-1), 166.1
(C-100), 147.6 (C-70), 145.8 (C-40), 138.4 (C-4), 133.9 (C-200), 131.4
(C-500), 130.5 (C-50, 90), 129.2 (C-6, 8), 128.2 (ꢂ2), 128.0 (ꢂ2), 127.3
(C-300, 700), 126.2 (C-70), 123.0 (C-60, 80), 62.6 (C-10)54.8 (C-2), 51.9
(C-20), 37.0, 36.4. EI-MS m/z: 447 (M+), 429, 252, 224, 190, 131, 105
(100), 91, 77, 51. Anal. Calcd for C25H25N3O5: C, 67.10; H, 5.63; N,
9.39. Found: C, 67.51; H, 5.88; N, 9.02.
4.1.2.18. N-(N-500-Methyl-benzoyl-
L-phenylalanyl)-L-tyrosine
methyl ester (9q). Mp 207.0–208.5 °C (EtOAc), 1H NMR
(DMSO-d6, 400 MHz) d: 9.24 (1H, s, Ar-OH), 8.45 (2H, m,
NHCO ꢂ 2), 7.68 (2H, d, J = 8.0 Hz, H-300, 700), 7.33 (2H, d,
J = 7.2 Hz, H-5, 9), 7.22 (4H, m, H-400, 600, H-6, 8), 7.14 (1H, t, H-
7), 7.00 (2H, d, J = 8.0 Hz, H-50, 90), 6.63 (2H, d, J = 8.0 Hz, H-60,
80), 4.73 (1H, m, H-2), 4.42 (1H, m, H-20), 3.57 (3H, s, OCH3),
3.08–2.87 (4H, m, H-3, 30), 2.32 (3H, s, Ar-CH3); 13C NMR
(DMSO-d6, 100 MHz) d: 172.0, 171.7, 166.1 (C-100), 156.1 (C-70),
141.2 (C-500), 138.4 (C-4), 131.2 (C-200), 130.1 (C-50, 90), 129.2
(C-6, 8), 128.7 (C-400, 600), 128.0 (C-5, 9), 127.4 (C-300, 700), 127.0
(C-40), 126.2 (C-7), 115.1 (C-60, 80), 54.5 (C-2), 54.1 (C-20), 51.8
(OCH3), 36.9 (C-3), 35.9 (C-30), 21.0 (Ar-CH3). EI-MS m/z: 460
(M+), 283, 266, 238, 119 (100), 91. Anal. Calcd for C27H28N2O5:
C, 70.42; H, 6.13; N, 6.08. Found: C, 70.43; H, 6.76; N, 5.89.
4.1.2.21. By a similar synthetic method described above for
compound 12a (from compound9f), thecompoundsof 12b(from
9p), 12c (from 9o), 12d (from 9o) was synthesized.
4.1.2.19. N-(N-500-Methyl-benzoyl-7-nitro-
L-phenylalanyl)-L-tyrosine
methyl ester (9r). Mp 213.0–215.0 °C (MeOH/EtOAc), 1H NMR
(DMSO-d6,400 MHz)d: 9.31 (1H, s, Ar-OH), 8.61–8.57 (2H, m, NHCO ꢂ 2),
8.12 (2H, d, J = 8.8 Hz, H-6, 8), 7.67 (2H, d, J = 7.6 Hz, H-300, 700), 7.60 (2H, d,
J = 8.8 Hz,H-5,9),7.23(2H,d,J = 8.0 Hz,H-400,600), 7.00 (2H, d, J = 8.4 Hz,H-
50, 90), 6.63 (2H, d, J = 8.0 Hz, H-60, 80), 4.81 (1H, m, H-2), 4.41 (1H, m, H-20),
3.56 (3H, s, OCH3), 3.21–3.06 (2H, m, H-30), 2.94–2.84 (2H, m, H-3), 2.32
(3H, s, Ar-CH3); 13C NMR (DMSO-d6, 100 MHz) d: 172.0 (C-1), 171.3 (C-
10), 166.2 (C-100), 156.1 (C-70), 146.9 (C-7), 146.2 (C-4), 141.3 (C-500),
131.0 (C-200), 130.6 (C-5, 9), 130.1 (C-50, 90), 128.8 (C-400, 600), 127.4 (C-300,
700), 126.9 (C-40), 123.2 (C-6, 8), 115.1 (C-60, 80), 54.2, 53.9, 51.9 (OCH3),
36.8, 35.9, 21.0 (Ar-CH3). EI-MS m/z: 505 (M+), 328, 311, 174, 119 (100),
106, 91, 78, 65. Anal. Calcd for C27H27N3O7: C, 64.15; H, 5.38; N, 8.31.
Found: C, 63.96; H, 5.64; N, 8.06.
4.1.2.21.1. N-(N-Benzoyl-L L-phenylala-
-phenylalanyl)-70-acetamido-
nol (12b). Mp 235.5–238.0 °C (MeOH), 1H NMR (DMSO-d6,
400 MHz) d: 9.85 (1H, s, ArNHCO), 8.54 (1H, d, J = 8.0 Hz, NHCO),
7.95 (1H, d, J = 8.4 Hz, NHCO), 7.77 (2H, d, J = 7.6 Hz, H-300, 700), 7.50
(1H, t, H-500), 7.44–7.40 (4H, m, H-400-600, 60, 80), 7.31 (2H, d,
J = 7.6 Hz, H-5, 9), 7.23 (1H, t, H-6, 8), 7.15–7.12 (3H, m, H-7, 50, 90),
4.84 (1H, t, OH), 4.67 (1H, m, H-2), 3.86 (1H, m, H-20), 3.34–3.27
(2H, m, H-10), 3.05–2.92 (2H, m, H-3), 2.79 (1H, dd, J = 6.0, 13.2 Hz,
H-30a), 2.61 (1H, dd, J = 7.6, 13.2 Hz, H-30b), 2.00 (3H, s, COCH3);
13C NMR (DMSO-d6, 100 MHz) d: 171.0 (C-1), 168.1 (ArNHCO),
166.2 (C-100), 138.5 (C-4), 137.4 (C-70), 134.1 (C-200), 133.5 (C-40),
131.3 (C-500), 129.4 (C-50, 90), 129.2 (C-6, 8), 128.2 (C-400, 600), 128.1
(C-5, 9), 127.4 (C-300, 700), 126.2 (C-70), 118.8 (C-60, 80), 62.1 (C-10),
55.0 (C-2), 52.6 (C-20), 37.4 (C-3), 35.8 (C-30), 24.0 (COCH3). EI-MS
m/z: 459 (M+), 441, 293, 252, 224, 191, 177, 149, 106, 91, 77, 60
(100), 43. Anal. Calcd for C27H29N3O4: C, 70.57; H, 6.36; N, 9.14.
Found: C, 70.69; H, 6.58; N, 8.98.
4.1.2.20. N-(N-Benzoyl-7-acetamido-L-phenylalanyl)-L-phenylalanol
(12a). A solution of compound 9f (1.788 g, 4 mmol) in MeOH (100 mL)
was vigorously stirred overnight under an atmosphere of hydrogen in
presence of Pd/C (20%, 21 mg) at room temperature. The catalyst was re-
moved by filtration and the solvent was evaporated under reduced pres-
4.1.2.21.2. N-(N-Benzoyl-L L-phenyl-
-phenylalanyl)-70-acetamido-
alanine (12c). 1H NMR (DMSO, 400 MHz) d: 12.80 (1H, br, COOH),
9.87 (1H, s, ArNHCO), 8.55 (1H, d, J = 8.4 Hz, NHCO), 8.32 (1H, d,
J = 8.0 Hz, NHCO), 7.75 (2H, d, J = 7.2 Hz, H-300, 700), 7.51–7.13
sure to give the compound 10a, N-(N-benzoyl-7-amino-L-phenylalanyl)-L-
phenylalanol (1.876 g), as a white powder. Without further purification,

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B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
13
(12H, m, H-400-600, H-5–9, H-50, 90, 60, 80), 4.73 (1H, m, H-2), 4.44 (1H,
m, H-20), 3.10–2.88 (4H, m, H-3, 30), 2.02 (3H, s, COCH3); 13C NMR
(DMSO-d6, 100 MHz) d: 172.9 (C-10), 171.6 (C-1), 168.1 (ArNHC-
OCH3), 166.3 (C-100), 138.5 (C-4), 137.9 (C-70), 134.1 (C-200), 131.8
(C-40), 131.3 (C-500), 129.4 (C-50, 90), 129.2 (C-6, 8), 128.2 (C-400,
600), 128.1 (C-5, 9), 127.4 (C-300, 700), 126.3 (C-7), 118.8 (C-60, 80),
54.6 (C-2), 53.7 (C-20), 37.0 (C-3), 36.1 (C-30), 24.0 (COCH3). EI-MS
m/z: 473 (M+), 455, 429, 251, 225, 148, 105, 91 (100), 77, 65, 51.
Anal. Calcd for C27H27N3O5: C, 68.48; H, 5.75; N, 8.87. Found: C,
68.19; H, 5.89; N, 8.92.
OCH3); C NMR (DMSO-d6, 100 MHz) d: 171.4 (ꢂ2, C-1, 10), 169.2
(ArOCO), 168.1 (ArNHCO), 166.0 (C-100), 149.2 (C-70), 141.2 (C-500),
137.6 (C-7), 134.5 (C-40), 132.8 (C-4), 131.2 (C-200), 130.1 (C-50,
90), 129.4 (C-5, 9), 128.7 (C-400, 600), 127.4 (C-300, 700), 121.6 (C-60,
80), 118.7 (C-6, 8), 54.5 (C-2), 53.7 (C-20), 51.9 (OCH3), 36.5, 35.9,
24.0 (OCOCH3), 21.0 (ArCH3), 20.9 (NHCOCH3). EI-MS m/z: 559
(M+), 424, 382, 295, 203, 188, 119 (100), 91. Anal. Calcd for
C31H33N3O7: C, 66.53; H, 5.94; N, 7.51. Found: C, 66.75; H, 6.09;
N, 7.38.
4.1.2.21.3. N-(N-Benzoyl-
L
-phenylalanyl)-70-propionamido-
L-phenyl-
4.1.2.23. N-(N-Benzoyl-L-phenylalanyl)-L-phenylalanine (12e). 5 M
alanine (12d). 1H NMR (DMSO-d6, 400 MHz) d: 12.78 (1H, br, COOH),
9.81 (1H, s, ArNHCO), 8.56 (1H, d, J = 8.4 Hz, NHCO), 8.33 (1H, d,
J = 8.0 Hz, NHCO), 7.75 (2H, d, J = 7.2 Hz, H-300, 700), 7.50–7.48 (3H, m,
H-500, H-60, 80), 7.42 (2H, t, H-400, 600), 7.34 (2H, d, J = 7.2 Hz, H-5, 9), 7.23
(2H, t, H-6, 8), 7.17–7.12 (3H, m, H-7, H-50, 90), 4.73 (1H, m, H-2), 4.44
(1H, m, H-20), 3.10–2.86 (4H, m, H-3, 30), 2.28 (2H, q, J = 7.6 Hz, NHC-
OCH2CH3), 1.06 (3H, t, J = 7.6 Hz, NHCOCH2CH3); 13C NMR (DMSO-d6,
100 MHz) d: 172.9 (C-10), 171.9, 171.6, 166.3 (C-100), 138.5 (C-4), 138.0
(C-70), 134.1 (C-200), 131.7 (C-40), 131.3 (C-500), 129.4 (C-50, 90), 129.2 (C-
6, 8), 128.2 (C-400, 600), 128.1 (C-5, 9), 127.5 (C-300, 700), 126.3 (C-7), 118.9
(C-60, 80), 54.7 (C-2), 53.7 (C-20), 37.0, 36.1, 29.6 (NHCOCH2CH3), 9.8
(NHCOCH2CH3). EI-MS m/z: 487 (M+), 382, 252, 163, 148, 105 (100),
91, 77, 65, 43. Anal. Calcd for C28H29N3O5: C, 68.98; H, 6.00; N, 8.62.
Found: C, 68.63; H, 6.18; N, 8.35.
NaOH (1 mL) was added to a solution of compound 9a (2.0 mmol) in
dry DMF (5 mL) and EtOH (10 mL). After stirring at room temperature
for overnight the mixture was acidified to pH 2–3 with concentrated
hydrochloric acid, and partitioned between EtOAc (150 mL) and water
(50 mL). The organic phase was separated and washed with brine
(2 ꢂ 30 mL), dried and evaporated in vacuo to give the compound of
(12e). As a white powder (750 mg, 90.1%). 1H NMR (DMSO-d6,
400 MHz) d: 12.81 (1H, br, COOH), 8.53 (1H, d, J = 8.4 Hz, NHCO), 8.31
(1H, d, J = 8.0 Hz, NHCO), 7.74 (2H, d, J = 7.6 Hz, H-300 , 700 ), 7.46 (1H, t,
H-500 ), 7.40 (2H, t, H-400 , 600 ), 7.33–7.12 (10H, m, H-5–9, 50-90), 4.72 (1H,
m, H-2), 4.46 (1H, m, H-20), 3.10–2.90 (4H, m, H-3, 30); 13C NMR
00
(DMSO-d6, 100 MHz) d: 172.8 (C-10), 171.5 (C-1), 166.2 (C-1 ), 138.4 (C-
4), 137.4 (C-40), 134.0 (C-200 ), 131.3 (C-500 ), 129.2 (ꢂ4, C-6, 8, C-60, 80),
128.2 (ꢂ4, C-400 , 600 , C-50, 90), 128.1 (C-5, 9), 127.4 (C-300 , 700 ), 126.5 (C-
70), 126.2 (C-7), 54.6 (C-2), 53.6 (C-20), 36.9 (C-3), 36.7 (C-30). EI-MS m/
z: 416 (M+), 398, 251, 120, 105 (100), 91, 77. Anal. Calcd for
C25H24N2O4: C, 72.10; H, 5.81; N, 6.73. Found: C, 72.59; H, 6.16; N, 6.47.
4.1.2.22. By a similar synthetic method described above for
compound11a (from compound9f), thecompoundsof 11b (from
91), 11c (from 9o), 11d (from 9r) was synthesized.
4.1.2.22.1. N-(N-Benzoyl-7-acetamido-
L
-phenylalanyl)-
L
-phenylala-
4.1.2.24. By a similar synthetic method described above for
compound 12e (from compound 9a), the compounds of 12f
(from 9d), 12 (from 91) was synthesized.
nine methyl ester (11b). Mp 221.0–222.0 °C (EtOAc); 1H NMR
(DMSO-d6, 400 MHz) d: 9.84 (1H, s, ArNHCO), 8.52 (2H, m,
NHCO ꢂ 2), 7.76 (2H, d, J = 7.6 Hz, H-300, 700), 7.51 (1H, t, H-500),
7.46–7.42 (4H, m, H-400, 600, H-6, 8), 7.26–7.17 (7H, m, H-50-90, H-5,
9), 4.71 (1H, m, H-2), 4.51 (1H, m, H-20), 3.59 (3H, s, OCH3), 3.09–
2.85 (4H, m, H-3, 30), 1.99 (3H, CH3CO); 13C NMR (DMSO-d6,
100 MHz) d: 171.9, 171.7, 168.1 (ArNHCOCH3), 166.2 (C-100), 137.6
(C-7), 137.1 (C-40), 134.0 (C-200), 132.8 (C-4), 131.3 (C-500), 129.4 (C-
5, 9), 129.1 (C-60, 80), 128.3 (C-400, 600), 128.2 (C-50, 90), 127.4 (C-300,
700), 126.6 (C-70), 118.6 (C-6, 8), 54.6 (C-2), 53.8 (C-20), 51.9 (OCH3),
36.6, 36.4, 24.0 (COCH3). EI-MS m/z: 478 (M+), 366, 281, 204, 188,
148, 105 (100), 91, 77. Anal. Calcd for C28H29N3O5: C, 68.98; H,
6.00; N, 8.62. Found: C, 68.84; H, 5.88; N, 8.43.
4.1.2.24.1. N-(N-Benzoyl-L-phenylalanyl)-L
-tyrosine (12f). 1H NMR
(DMSO-d6, 400 MHz) d: 12.75 (COOH), 9.22 (1H, s, Ar-OH), 8.55
(1H, d, J = 8.4 Hz, NHCO), 8.23 (1H, d, J = 7.6 Hz, NHCO), 7.76 (2H,
d, J = 7.6 Hz, H-300, 700), 7.51 (1H, t, H-500), 7.43 (2H, t, H-400, 600),
7.34 (2H, d, J = 7.2 Hz, H-5, 9), 7.24 (2H, t, H-6, 8), 7.15 (1H, t, H-
7), 7.02 (2H, d, J = 8.4 Hz, H-50, 90), 6.61 (2H, d, J = 8.4 Hz, H-60,
80), 4.74 (1H, m, H-20), 4.38 (1H, m, H-2), 3.11–2.82 (4H, m, H-3,
13
30); C NMR (DMSO-d6, 100 MHz) d: 172.9 (C-10), 171.5 (C-1),
166.3 (C-100), 156.0 (C-70), 138.4 (C-4), 134.0 (C-200), 131.3 (C-500),
130.1 (C-50, 90), 129.2 (C-6, 8), 128.2 (C-400, 600), 128.0 (C-5, 9),
127.4 (C-300, 700), 127.3 (C-40), 126.2 (C-7), 115.0 (C-60, 80), 54.6 (C-
20), 53.9 (C-2), 36.9 (C-3), 35.9 (C-30). EI-MS m/z: 432 (M+), 414,
386, 269, 252, 224, 120, 105 (100), 91, 77. Anal. Calcd for
C25H24N2O5: C, 69.43; H, 5.59; N, 6.48. Found: C, 69.13; H, 5.79;
N, 6.18.
4.1.2.22.2. N-(N-Benzoyl-L L-phenyl-
-phenylalanyl)-70-acetamido-
alanine methyl ester (11c). Mp 228.0–229.5 °C (EtOAc), 1H NMR
(DMSO-d6, 400 MHz) d: 9.95 (1H, s, ArNHCO), 8.60 (1H, d,
J = 8.8 Hz, NHCO), 8.57 (1H, d, J = 7.6 Hz, NHCO), 7.77 (2H, d,
J = 6.8 Hz, H-300, 700), 7.53–7.14 (12H, m, H-400-600, H-5–9, H-50, 90,
60, 80), 4.74 (1H, m, H-2), 4.47 (1H, m, H-20), 3.59 (3H, s, OCH3),
3.09–2.91 (4H, m, H-3, 30), 2.02 (3H, s, COCH3); 13C NMR (DMSO-
d6, 100 MHz) d: 171.9, 171.7, 168.2 (ArNHCOCH3), 166.2 (C-100),
138.4 (C-4), 138.0 (C-70), 134.0 (C-200), 131.4 (C-40), 131.3 (C-500),
129.4 (C-50, 90), 129.2 (C-6, 8), 128.2 (C-400, 600), 128.1 (C-5, 9),
127.4 (C-300, 700), 126.2 (C-7), 118.8 (C-60, 80), 54.6 (C-2), 53.9 (C-
20), 51.9 (OCH3), 37.0, 36.1, 24.0 (COCH3). EI-MS m/z: 487 (M+),
396, 269, 252, 219, 177, 148, 105 (100), 91, 77, 43. Anal. Calcd
for C28H29N3O5: C, 68.98; H, 6.00; N, 8.62. Found: C, 68.58; H,
6.19; N, 8.79.
4.1.2.24.2. N-(N-Benzoyl-7-nitro-L-phenylalanyl)-L-phenylalanine
(12g). 1HNMR(DMSO-d6, 400 MHz)d:12.85(1H, COOH), 8.54(1H,
d, J = 8.8 Hz, NHCO), 8.42 (1H, d, J = 8.4 Hz, NHCO), 8.14 (2H, d,
J = 8.8 Hz, H-6, 8), 7.77 (2H, d, J = 6.8 Hz, H-300, 700), 7.63 (2H, d,
J = 8.4 Hz, H-5, 9), 7.52 (1H, t, H-500), 7.45 (2H, t, H-400, 600), 7.26–
7.18 (5H, m, H-50-90), 4.85 (1H, m, H-2), 4.49 (1H, m, H-20), 3.24–
2.94 (4H, m, H-3, 30); 13C NMR (DMSO-d6, 100 MHz) d: 172.7 (C-10),
171.0 (C-1), 166.3 (C-100), 146.8 (C-7), 146.2 (C-4), 137.4 (C-40),
133.8 (C-200), 131.4 (C-500), 130.5 (C-5, 9), 129.2 (C-60, 80), 128.2 (ꢂ4,
C-400, 600, 50, 90), 127.4 (C-300, 700), 126.5 (C-70), 123.2 (C-6, 8), 53.9
(C-20), 53.6 (C-2), 36.8, 36.6. EI-MS m/z: 461 (M+), 443, 296, 269,
148, 120, 105 (100), 91, 77, 57, 43. Anal. Calcd for C25H23N3O6: C,
65.07; H, 5.02; N, 9.11. Found: C, 64.67; H, 5.25; N, 8.96.
4.1.2.22.3. N-(N-500-Methyl-benzoyl-7-acetamido-
O-acetyl-
L-phenylalanyl)-
L-tyrosine methyl ester (11d). Mp 222.0–223.0 °C (MeOH/
EtOAc), 1H NMR (DMSO-d6, 400 MHz) d: 9.84 (1H, s, ArNHCO), 8.55
(1H, d, J = 7.2 Hz, NHCO), 8.42 (1H, d, J = 8.4 Hz, NHCO), 7.69 (2H, d,
J = 8.0 Hz, H-300, 700), 7.44 (2H, d, J = 8.4 Hz, H-6, 8), 7.27–7.23 (6H,
m, H-5, 9, 50, 90, 400, 600), 6.98 (2H, d, J = 8.4 Hz, H-60, 80), 4.71 (1H,
m, H-2), 4.52 (1H, m, H-20), 3.59 (3H, s, OCH3), 3.07–2.86 (2H, m,
H-3, 3), 2.34 (3H, s, Ar-CH3), 2.24 (3H, s, OCOCH3), 1.99 (3H, s, NHC-
4.2. Biological assay
4.2.1. In vitro anti-HBV assays
The antiviral activities of compounds 9a–9r, 12a–12 against
HBV in 2.2.15 cells were evaluated .The anti-HBV activities

B. Xu et al. / Bioorg. Med. Chem. 17 (2009) 3118–3125
3125
in vitro included the ability to inhibit the production of HBsAg and
Acknowledgment
HBeAg, and the replication of HBV DNA in HBV-infected 2.2.15
cells. For the antiviral analyses, confluent cultures of 2.2.15 cells
were maintained on 24-well flat-bottomed tissue culture plates
in RPMI 1640 medium with 2% fetal bovine serum. Cultures were
treated with eight consecutive daily doses of the test compounds
and lamivudine (Glaxo & Welcome Co.). The Hep G2.2.15 cells were
incubated in 24-well plates at a density of 1.0 ꢂ 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 very carefully
with 1 mL of fresh DMEM containing different concentrations
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.
This work was supported by the grants from the National Nat-
ural Science Foundation of PR China (NSFC No.30760292), the Sci-
ence and Technology Department of Guizhou Province (No. QKHNZ
[2006]5001, QSZHZ[2005]246), the Science and Technology Bureau
of Guiyang City (No. ZKJHTZ [2001]12) and the Ministry of Science
and Technology of the PR China (No. 2008DFB30110).
(50 lg/mL, 25 lg/mL, 12.25 lg/mL) of compounds 9a–9r, 12a–
12g and did 4 wells at each concentration. Cells grew in the pres-
ence of drugs for 9 d with changes of medium every 3 d. After 6 d
and 9 d, Supernatant was collected and performed at 20 °C. The
HBsAg and HBeAg in culture medium were simultaneously mea-
sured by EIA kits on days 6 and 9. This test was done twice under
the same conditions. Untreated cells were used as the control.
Medium was changed daily with fresh test compounds and posi-
tive control. HBV nucleic acid and protein levels were measured
eight days after the first treatment. The HBsAg and HBeAg in the
culture medium were evaluated by semiquantitative enzyme
immunoassay (EIA) methods using commercial kits (HBsAg,
HBeAg, Beijing North Institute of Biological Technology.) as previ-
ously described. Intracellular HBV DNA levels were measured by
quantitative Southern blot hybridization. The IC50 and selected in-
dex of the evaluated compounds and lamivudine were calculated
by one-way repeated-measure ANOVA and t-test for comparisons
between groups, respectively.
References and notes
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Man, R. A. Ned. Tijdschr. Geneeskd. 2002, 146, 1380.
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4.2.2. Cytotoxicity assay
Cytotoxicity induced by the test compounds in cultures of
2.2.15 cells was also determined. Briefly, 2.2.15 cells were grown
to confluence in 24-well flat-bottomed tissue culture plates and
treated with test compound (in 0.2 mL culture medium/well) as
described above. Untreated control cultures were maintained on
each 24-well plate. Toxicity was determined by measuring neu-
tral red dye uptake, as determined from the absorbance at
510 nm relative to untreated cells, at 24 h following day 9 of
treatment.
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