Synthesis and Biological Evaluation of Matijin-Su Derivatives as Potential Antihepatitis B Virus and Anticancer Agents

Article abstract of DOI:10.1002/cbdv.201600113

A series of Matijin-Su (MTS, (2S)-2-{[(2S)-2-benzamido-3-phenylpropanoyl]amino}-3-phenylpropyl acetate) derivatives were synthesized and evaluated for their anti-HBV and cytotoxic activities in?vitro. Six compounds (4g, 4j, 5c, 5g, 5h and 5i) showed signi

Full text of DOI:10.1002/cbdv.201600113

1584
Synthesis and Biological Evaluation of Matijin-Su Derivatives as
Potential Antihepatitis B Virus and Anticancer Agents
Jingying Qiu,a,1 Bixue Xu,b,1 Qineng Gong,a Weidong Pan,b Changxiao Liu,c Zhengming Huang,d
Xiaoke Gu,*a and Guangyi Liang*b
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College, 209 Tongshan Road,
Xuzhou 221004, P. R. China, e-mail: gu_xk@163.com
bThe Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, 202
Shachong South Road, Guiyang 550002, P. R. China, e-mail: guangyi_liang@126.com
cTianjin Institute of Pharmaceutical Research, 308 Anshan Xi Dao, Nankai District, Tianjin 300193, P. R. China
d302 Hospital of PLA, 100 Xisihuan Zhong Road, Fengtai Beijing 100039, P. R. China
A
series of Matijin-Su (MTS, (2S)-2-{[(2S)-2-benzamido-3-phenylpropanoyl]amino}-3-phenylpropyl acetate)
derivatives were synthesized and evaluated for their anti-HBV and cytotoxic activities in vitro. Six compounds (4g, 4j,
5c, 5g, 5h and 5i) showed significant inhibition against HBV DNA replication with the IC50 values in range of
2.18 – 8.55 lM, which were much lower than that of positive control lamivudine (IC50 82.42 lM). In particular,
compounds 5h (IC50 2.18 lM; SI 151.59) and 5j (IC50 5.65 lM; SI 51.16) displayed relatively low cytotoxicities, resulting
in high SI values. Notably, besides the anti-HBV DNA replication activity, compound 4j also exhibited more potent
in vitro cytotoxic activity than 5-fluorouracil in two hepatocellular carcinoma cell (HCC) lines (QGY-7701 and SMMC-
7721), indicating that 4j may be a promising lead for the exploration of drugs with dual therapeutic effects on HBV
infection and HBV-induced HCC.
Keywords: MTS Derivatives, Antihepatitis B virus activity, Cytotoxic activities, Synthesis, Structure-Activity Relationships.
dipeptide derivative, was isolated from a Chinese eth-
nic drug Matijin (Dichondra repens FORST.) widely used
Introduction
Hepatitis B virus (HBV)-infected hepatitis is one of the in treatment of chronic liver diseases in China. In pre-
most common infectious disease worldwide.[1 – 5] vious studies, we have described that the derivatives
More than 400 million people worldwide are infected of MTS could significantly inhibit HBV DNA replication,
by HBV. These individuals face a high risk for cirrhosis and displayed more potent anti-HBV activity than the
or hepatocellular carcinoma (HCC).[6 – 8] HBV Infection positive control lamivudine.[12 – 14] Notably, several
has become the most important etiologic factor of derivatives exhibited potential anti-HCC activity
HCC, and attributes to almost 50% of all HCC cases in vitro.[15] With these in minds, and in view of that
globally.[9][10] Considerable attempts, such as lamivu- the substitutes at position-7 on ring A of MTS might
dine and 5-fluorouracil (5-Fu), have been used to com- affect the anti-HBV and cytotoxic activities,[14 – 16]
bat HBV infection and HBV-mediated HCC, twenty new MTS derivatives with alkoxy substitutes at
respectively, however, until now, a multi-purpose position-7 on ring A were synthesized, and their inhi-
drug, which could inhibit HBV infection and HCC bitory effects on HBV DNA replication and HCC in vitro
simultaneously, has not been discovered yet.[11] There- were subsequently evaluated in the present work.
fore, there is a clear and urgent need requirement for
developing novel drugs with dual therapeutic effects Results and Discussion
not only on HBV infection but also on HBV-induced
HCC.
Chemistry
Matijin-Su (MTS, (2S)-2-{[(2S)-2-benzamido-3-phen- The synthetic procedure of the target compounds was
ylpropanoyl]amino}-3-phenylpropyl acetate, Fig. 1), a illustrated in Scheme 1. Briefly, L-tyrosine methyl ester
hydrochloride was condensed with benzoic acid to
give intermediate 1, which was then hydrolyzed to
yield intermediate
2
as described previously.[17]
1
These two authors contributed equally to this work.
© 2016 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2016, 13, 1584 – 1592 DOI: 10.1002/cbdv.201600113

Chem. Biodiversity 2016, 13, 1584 – 1592
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IC50 value for inhibition of HBV DNA replication and
the selectivity index (SI).
As shown in Table 1, some MTS derivatives could
significantly inhibit the replication of HBV DNA in
HepG2 2.2.15 cells. Among them, compounds 4g, 4j,
5c, 5g, 5h and 5j (IC50 8.55, 5.21, 8.14, 7.22, 2.18,
5.65 lM, respectively) displayed more potent inhibitory
effect on HBV DNA replication than positive control
lamivudine (82.42 lM). Clearly, compound 5h showed
the most potence in vitro anti-HBV activity within all of
the tested compounds, for its IC50 value (2.18 lM) was
almost 38-folds lower than that of the control lamivu-
dine (82.84 lM). Interestingly, the SI value of 5h
(151.59) was about four times higher than that of lami-
vudine (41.59), indicating that compound 5h, as a can-
didate of anti-HBV agents, has a relatively safety profile.
Figure 1. Chemical structure of MTS.
Intermediate 3 was obtained by the condensation of
2 with L-phenylalaninol, which was then alkylated with
a variety of iodoalkane or bromoalkane to provide
alkoxy substituted derivatives 4a – 4j.[18] Finally,
4a – 4j were acetylated with Ac2O to provide the tar-
get compounds 5a – 5j.
In vitro Assessment of Cytotoxic Activity in Three Human
In vitro Virological Assays of Anti-HBV Activities
Cancer Cell Lines
The target compounds 4a – 4j and 5a – 5j were
firstly examined for the inhibitory effect on HBV DNA
replication and cytotoxicities in HBV-infected HepG2
2.2.15 cells according to the previous method,[12] and
the well-known anti-HBV drug lamivudine was used as
the positive control. The anti-HBV activity of each
compound was evaluated by the combination of the
Encouraging by results of the anti-HBV activities, we
next evaluated all of the target compounds for the
inhibitory effect on QGY-7701, SMMC-7721 and A549
cells proliferation in vitro by MTT assay.[15] A well-
known anticancer drug 5-fluorouracil (5-Fu) was used
as the positive control. The cytotoxic activities of indi-
vidual compounds against each cancer cell line
Scheme 1: Synthetic route of the target compounds. a) Benzoic acid, CH2Cl2, IBCF, NMM, 0 °C. b) NaOH, DMF, r.t. c)
L-Phenylalaninol, CH2Cl2, IBCF, NMM, 0 °C. d) Iodoalkane or bromoalkane, K2CO3, DMF, 35 °C. e) Ac2O, pyridine, r.t.
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Chem. Biodiversity 2016, 13, 1584 – 1592
Table 1. Anti-HBV activity of target compounds 4a – 4j and
5a – 5j
Table 2. In vitro cytotoxic activity of analogues 4a – 4j and
5a – 5j
Compound
R
TC50 [lM]a
DNA replication
Compound
Cytotoxicity IC50 [lM]a
IC50 [lM]b
SIc
QGY-7701
SMMC-7721
A549
e
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
5e
5f
Me
Et
Pr
i-Pr
N/Ad
77.14
388.05
N/A
–
N/A
4a
4b
4c
4d
4e
4f
4g
4h
4i
> 100
26.44
16.076
10.79
> 100
> 100
> 100
> 100
> 100
8.84
> 100
> 100
> 100
> 100
68.12
> 100
> 100
> 100
57.37
23.21
16.81
> 100
19.89
23.98
12.09
> 100
> 100
> 100
> 100
> 100
6.66
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
59.01
22.75
13.23
> 100
17.85
> 100
8.21
16.61
> 100
> 100
> 100
> 100
3.42
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
45.15
16.92
5.43
12.20
13.31
–
6.33
29.15
N/A
Bu
N/A
–
N/A
Me2CHCH2
Me(CH2)4
Me(CH2)5
NꢀCCH2
CH2=CHCH2
Me
Et
Pr
i-Pr
Bu
Me2CHCH2
Me(CH2)4
Me(CH2)5
NꢀCCH2
CH2=CHCH2
284.46
61.42
N/A
N/A
58.25
N/A
N/A
268.6
N/A
28.40
8.55
–
–
5.21
–
–
8.14
–
10.02
7.18
N/A
N/A
11.18
N/A
N/A
33.00
N/A
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
N/A
–
N/A
261.31
72.12
330.47
N/A
289.08
120.18
3427.82
38.69
7.22
2.18
–
5.65
11.16
82.42
6.75
9.85
151.59
N/A
51.16
10.78
41.59
5g
5h
5i
5j
5j
5-Fu
MTS
Lamivudine
a
IC50 Values are expressed as means of triplicate experiments.
a TC50, 50% Toxic concentration in HepG2 2.2.15 cells. b IC50,
50% Inhibitory concentration. c SI = TC50/IC50. d Not available.
e Means no antiviral activity at a concentration lower than
50 lM.
Disappointingly, the rest compounds had no in vitro
cytotoxic effect on both two hepatocellular carcinoma
cell lines. Notably, besides the significant antihepato-
cellular carcinoma effect in vitro, compounds (4b, 4d,
4e, 4j, 5j) also displayed potent in vitro cytotoxic
activity against human lung carcinoma cell line A549
with IC50 values in range of 3.42 – 17.85 lM. Clearly,
4j (IC50 3.42 lM) was the best one as the novel anti-
lung cancer lead, and was worthwhile for further
study and optimization.
in vitro were represented as IC50 values, defined as
the compound concentration causing a 50% cell
growth inhibition as compared to the control cells,
and summarized in Table 2.
As shown in Table 2, several compounds displayed
definite cytotoxic activity in vitro against all the tree
human cancer cell lines. Towards to hepatocellular
carcinoma cell lines (QGY-7701 and SMMC-7721),
three derivatives displayed relatively high inhibitory
effect on cells proliferation with IC50 values < 20 lM,
respectively. Obviously, 4j exhibited the most promi-
nent in vitro cytotoxic activity with IC50 values lower
than 10 lM (IC50 values on QGY-7701 and SMMC-7721
cells were 8.84 and 6.66 lM, respectively), which was
about two-fold less than that of positive control 5-Fu
(IC50 values on QGY-7701 and SMMC-7721 cells were
16.81 and 13.23 lM, respectively). In addition, com-
pounds 4b, 5e, 5i, 5j (IC50 26.44, 68.12, 57.37,
23.21 lM, respectively) showed comparatively weak
inhibitory activity on QGY-7701 cells proliferation
in vitro, and compounds 4c, 5i, 5j (IC50 23.98, 59.01,
22.75 lM, respectively) exhibited relatively poor
antiproliferation activities on SMMC-7721 cells in vitro.
Structure–Activity Relationships
According to the above bioactivity results, structure-
anti-HBV activity relationship of MTS derivatives could
be drawn as follows. Alkoxy substituent group at posi-
tion-7 on ring A of the MTS derivatives could signifi-
cantly affect their anti-HBV activities. Generally, when
the number of total C-atoms in alkoxy group is more
than two, increasing the length of the carbon chain was
benefit for the anti-HBV activity (5h > 5g > 5c > MTS),
except for 5e. Compound with six C-atoms substituted
on ring A of MTS displayed the highest inhibitory activi-
ties on HBV DNA replication (5h vs. MTS). Interestingly,
when propoxy at position-7 changed into isopropoxy,
the anti-HBV activity was significantly decreased (5c vs.
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Chem. Biodiversity 2016, 13, 1584 – 1592
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1
5d). However, when propoxy changed into allyloxy, the a model XT-4 apparatus and are uncorrected. H (400
anti-HBV activity was slightly increased, indicating that MHz) and 13C (100 MHz) NMR spectra were recorded
the conformation of the carbon chain in alkoxy might on a Varian Unit INOVA-400 spectrometer in CDCl3 or
affect the anti-HBV activities. One plausible explanation (D6)DMSO; d in ppm rel. to Me4Si as internal standard,
is that the transformation of conformation might affect J in Hz. Mass spectra (MS) were run on a Agilent MS-
the binding between compound and the potential anti- 5973 spectrometer; in m/z.
HBV receptor. In addition, when AcO substituent at
position-10 of MTS derivatives was hydrolyzed into OH,
General Procedure for the Synthesis of Compounds
the anti-HBV activity has no obvious improvement (5c
4a – 4j
vs. 4c; 5f vs. 4f; 5g vs. 4g; 5j vs. 4j), except for 5b, while
SI was significantly decreased (5c vs. 4c; 5g vs. 4g; 5j vs. Isobutyl chloroformate (IBCF) (1.1 mmol) was added
4j), except for 5f. However, the precise SARs remain fur- dropwise to the mixture of benzoic acid (1.0 mmol),
ther investigation when more MTS derivatives with vari- L-tyrosine methyl ester hydrochloride (1.1 mmol) and
ous substituents at 7-position on A ring will be N-methylmorpholine (NMM) (2.3 mmol) in CH2Cl2
obtained in the future research. Towards structure–anti- (50 ml) at 0 °C within 60 min. The mixture was stirred
cancer activity relationship of MTS derivatives, the cyto- for 30 min and the bulk of CH2Cl2 was removed
toxic activities were significantly increased (5b vs. 4b, in vacuo. The residue was dissolved in AcOEt and
5c vs. 4c, 5d vs. 4d), except for 5i, when AcO sub- washed sequentially with H2O, 5% HCl, sat. aq.
stituent at position-10 of MTS derivatives was hydro- NaHCO3 soln., and brine, dried (Na2SO4). Removal of
lyzed into OH. However, due to the bioactive data and the solvent gave a residue which was recrystallized
the number of the compounds are relatively few, an from AcOEt to afford target compound 1. 2M NaOH (1
objective structure–anticancer activity relationship ml) was added to the soln. of compound 1 (1.0 mmol)
could be summarized only when more MTS derivatives in DMF (5 ml). After stirring at r.t. for 2 h, the mixture
with cytotoxic activities will be available in the near was acidified to pH 6 – 7 with conc. HCl, and parti-
future.
tioned between AcOEt and H2O. The org. phase was
separated and washed with brine, dried (Na2SO4), and
evaporated in vacuo to give compound 2. Compound
3 was prepared with a similar procedure to that
Conclusions
In summary, a new series of MTS derivatives were syn- described for the synthesis of compound 1. Com-
thesized and evaluated for their anti-HBV and cytotoxic pound 3 was followed by dissolved in DMF under Ar
activities in vitro. Most of the synthesized compounds atmosphere, and then iodoalkane (or bromoalkane)
exhibited significant inhibitory effect on HBV DNA repli- and K2CO3 were added. The mixture was stirred at
cation and cancer cells proliferation. Notably, com- 35 °C overnight, then poured into H2O. The solid was
pounds 4g, 4j, 5c, 5g, 5h and 5j exhibited more potent filtered and recrystallized from AcOEt to give com-
inhibitory effect on HBV DNA replication than positive pounds 4a – 4j.
control lamivudine. Among them, compounds 5h and N-[(2S)-1-{[(2S)-1-Hydroxy-3-phenylpropan-2-yl]amino}-
5j displayed relatively low cytotoxicities towards HepG2 3-(4-methoxyphenyl)-1-oxopropan-2-yl]benzamide (4a).
2.2.15 cells, resulting in high SI values. Interestingly, White needle crystals. Yield: 65%. M.p. 196 – 198 °C
besides the significant anti-HBV effect, compound 4j (AcOEt). IR (KBr): 1654 (C=O), 1633 (C=O), 1541 (NH), 1514
1
showd more potent in vitro antihepatocellular carci- (NH), 1248 (=C–O). H-NMR ((D6)DMSO, 400 MHz): 8.46
noma effect than classic anticancer drug 5-fluorouracil, (d, J = 8.8, NHCO, 1 H); 7.90 (d, J = 8.0, NHCO, 1 H); 7.80 (d,
indicating that 4j may be a promising lead for the J = 8.0, H–C(3″, 7″), 2 H); 7.51 (t, J = 8.0, H–C(5″), 1 H); 7.42
exploration of drugs with dual therapeutic effects on (t, J = 8.0, H–C(4″, 6″), 2 H); 7.23 – 7.09 (m, H–C(50 – 90, 5,
HBV infection and HBV-induced HCC.
9), 7 H); 6.80 (d, J = 8.8, H–C(6, 8), 2 H); 4.85(t, J = 4.0, OH, 1
H); 4.62 – 4.60 (m, H–C(2), 1 H); 3.86 – 3.83 (m, H–C(20), 1
H); 3.66 (s, MeO, 3 H); 3.66 – 3.26 (m, CH2(10), 2 H);
2.97 – 2.63 (m, H–C(3, 30), 4 H). 13C-NMR ((D6)DMSO, 100
MHz): 171.2 (C(1)); 166.2 (C(1″)); 157.8 (C(7)); 139.1 (C(40));
Experimental Section
General
All solvents were reagent grade and, when necessary, 134.1 (C(2″)); 131.4 (C(5″)); 130.3 (C(5, 9, 4)); 129.3 (C(60, 80));
were purified and dried by standard methods. All of 128.3 (C(4″, 6″)); 128.2 (C(50, 90)); 127.5 (C(3″, 7″)); 126.0
the synthesized compounds were purified by recrystal- (C(70)); 113.5 (C(6, 8)); 62.3 (C(10)); 55.2 (C(20)); 55.0 (MeO);
lization or column chromatography (CC) on SiO2 60 52.6 (C(2)); 36.5 (C(30, 3). ESI-MS: 433.2 ([M + H]+). TOF-ES-
(200 – 300 mesh). Melting points were measured with MS: 455.1951 ([M + Na]+, C26H28N2NaO4+; calc. 455.1947).
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Chem. Biodiversity 2016, 13, 1584 – 1592
N-[(2S)-3-(4-Ethoxyphenyl)-1-{[(2S)-1-hydroxy-3-phe- Me2CH–O,
1 1 H);
H); 3.92 – 3.87 (m, H–C(20),
nylpropan-2-yl]amino}-1-oxopropan-2-yl]benzamide 3.35 – 3.22 (m, CH2(10), 2 H); 2.99 – 2.67 (m, H–C(3, 30),
(4b). White powder. Yield: 63%. M.p. 198 – 199 °C 4 H); 1.31 (d, J = 6.4, Me2CH–O, 6 H). 13C-NMR ((D6)
(AcOEt). IR (KBr): 1654 (C=O), 1633 (C=O), 1541 (NH), DMSO, 100 MHz): 171.0 (C(1)); 166.0 (C(1″)); 157.1
1508 (NH), 1248 (=C–O). 1H-NMR ((D6)DMSO, 400 (C(7)); 139.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); 130.2
MHz): 8.46 (d, J = 8.8, NHCO, 1 H); 7.87 (d, J = 8.4, (C(5, 9)); 130.1 (C(4)); 129.2 (C(60, 80)); 128.2 (C(4″, 6″));
NHCO, 1 H); 7.80 (d, J = 8.0, H–C(3″, 7″), 2 H); 7.52 (t, 128.1 (C(50, 90)); 127.4 (C(3″, 7″)); 125.9 (C(70)); 113.9
J = 7.6, H–C(5″), 1 H); 7.43 (t, J = 8.0, H–C(4″, 6″), 2 H); (C(6, 8)); 71.6 (Me2CH–O); 62.2 (C(10)); 55.1 (C(20)); 52.5
7.23 – 7.10 (m, H–C(50 – 90, 5, 9), 7 H); 6.78 (d, J = 8.8, (C(2)); 36.4 (C(30, 3)); 21.7 (Me2CH–O). ESI-MS: 461.2
H–C(6, 8), 2 H); 4.82 (t, J = 5.6, OH, 1 H); 4.67 – 4.61 ([M + H]+). TOF-ES-MS: 461.2441 ([M + H]+, C28H33
(m, H–C(2), 1 H); 3.95 – 3.89 (m, H–C(20), MeCH2O, 3 N2O4+; calc. 461.2440).
H); 3.34 – 3.25 (m, CH2(10), 2 H); 2.99 – 2.65 (m, H–C(3, N-[(2S)-3-(4-Butoxyphenyl)-1-{[(2S)-1-hydroxy-3-phe-
30), 4 H); 1.26 (t, J = 4.0, MeCH2O, 3 H). 13C-NMR ((D6) nylpropan-2-yl]amino}-1-oxopropan-2-yl]benzamide
DMSO, 100 MHz): 171.1 (C(1)); 166.1 (C(1″)); 157.0 (4e). White powder. Yield: 64%. M.p. 199 – 200 °C
(C(7)); 139.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); 130.2 (AcOEt). IR (KBr): 1658 (C=O), 1633 (C=O), 1535 (NH),
1
(C(5, 9)); 130.1 (C(4)); 129.2 (C(60, 80)); 128.2 (C(4″, 6″)); 1512 (NH), 1242 (=C–O). H-NMR ((D6)DMSO, 400 MHz):
128.1 (C(50, 90)); 127.5 (C(3″, 7″)); 125.9 (C(70)); 113.9 8.47 (d, J = 8.8, NHCO, 1 H); 7.90 (d, J = 8.4, NHCO, 1 H);
(C(6, 8)); 62.8 (MeCH2O)); 62.2 (C(10)); 55.1 (C(20)); 52.5 7.81 (d, J = 8.4, H–C(3″, 7″), 2 H); 7.52 (t, J = 8.0, H–
(C(2)); 36.5 (C(30, 3)); 14.7 (MeCH2O). ESI-MS: 447.5 C(5″), 1 H); 7.45 (t, J = 8.0, H–C(4″, 6″), 2 H); 7.24 – 7.11
([M + H]+). TOF-ES-MS: 447.2286 ([M + H]+, C27H31 (m, H–C(50 – 90, 5, 9), 7 H); 6.79 (d, J = 8.8, H–C(6, 8), 2
N2O4+; calc. 447.2284).
H); 4.83 (t, J = 5.6, OH, 1 H); 4.65 – 4.59 (m, H–C(2), 1 H);
H);
N-[(2S)-1-{[(2S)-1-Hydroxy-3-phenylpropan-2-yl]amino}- 3.91 – 3.86 (m, H–C(20), MeCH2CH2CH2O,
3
1-oxo-3-(4-propoxyphenyl)propan-2-yl]benzamide (4c). 3.35 – 3.26 (m, CH2(10), 2 H); 2.99 – 2.67 (m, H–C(3, 30), 4
White powder. Yield: 65%. M.p. 202 – 203 °C (AcOEt). IR H); 1.68 – 1.63 (m, MeCH2CH2CH2O, 2 H); 1.44 – 1.35
(KBr): 1654 (C=O), 1635 (C=O), 1541 (NH), 1508 (NH), 1250 (m, MeCH2CH2CH2O, 2 H); 0.90 (t, J = 7.2, MeCH2CH2-
1
(=C–O). H-NMR ((D6)DMSO, 400 MHz): 8.49 (d, J = 8.4, CH2O, 3 H). 13C-NMR ((D6)DMSO, 100 MHz): 171.0 (C(1));
NHCO, 1 H); 7.93 (d, J = 8.4, NHCO, 1 H); 7.80 (d, J = 8.4, 166.0 (C(1″)); 157.1 (C(7)); 139.0 (C(40)); 134.1 (C(2″));
H–C(3″, 7″), 2 H); 7.50 (t, J = 8.0, H–C(5″), 1 H); 7.43 (t, 131.3 (C(5″)); 130.2 (C(5, 9)); 130.1 (C(4)); 129.2 (C(60, 80));
J = 8.4, H–C(4″, 6″), 2 H); 7.23 – 7.10 (m, H–C(50 – 90, 5, 9), 128.2 (C(4″, 6″)); 128.1 (C(50, 90)); 127.4 (C(3″, 7″)); 125.9
7 H); 6.78 (d, J = 8.8, H–C(6, 8), 2 H); 4.83 (t, J = 5.6, OH, 1 (C(70)); 113.9 (C(6, 8)); 66.9 (MeCH2CH2CH2O); 62.2 (C(10));
H); 4.65 – 4.50 (m, H–C(2), 1 H); 3.90 – 3.89 (m, H–C(20), 1 55.1 (C(20)); 52.5 (C(2)); 36.4 (C(30, 3)); 30.8 (MeCH2CH2-
H); 3.82 (t, J = 6.8, MeCH2CH2O, 2 H); 3.34 – 3.27 (m, CH2O); 18.8 (MeCH2CH2CH2O); 13.7 (MeCH2CH2CH2O).
CH2(10), 2 H); 2.97 – 2.64 (m, H–C(3, 30), 4 H); 1.68 – 1.65 ESI-MS: 475.5 ([M + H]+). TOF-ES-MS: 497.2421
(m, MeCH2CH2O, 2 H); 0.92 (t, J = 7.6, MeCH2CH2O, 3 H). ([M + Na]+, C29H34N2NaO4+; calc. 497.2416).
13C-NMR ((D6)DMSO, 100 MHz): 171.0 (C(1)); 166.0 (C(1″)); N-{(2S)-1-{[(2S)-1-Hydroxy-3-phenylpropan-2-yl]amino}-
157.1 (C(7)); 139.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); 130.2 3-[4-(2-methylpropoxy)phenyl]-1-oxopropan-2-yl}ben-
(C(5, 9)); 130.1 (C(4)); 129.2 (C(60, 80)); 128.2 (C(4″, 6″)); 128.1 zamide (4f). White needle crystals. Yield: 59%. M.p.
(C(50, 90)); 127.4 (C(3″, 7″)); 125.9 (C(70)); 113.9 (C(6, 8)); 68.7 191 – 193 °C (AcOEt). IR (KBr): 1654 (C=O), 1633 (C=O),
1
(MeCH2CH2O)); 62.2 (C(10)); 55.1 (C(20)); 52.5 (C(2)); 36.4 1541 (NH), 1508 (NH), 1245 (=C–O). H-NMR ((D6)DMSO,
(C(30, 3)); 22.1 (MeCH2CH2O); 10.4 (MeCH2CH2O). ESI-MS: 400 MHz): 8.44 (d, J = 8.8, NHCO, 1 H); 7.85 (d, J = 8.4,
461.2 ([M + H]+). TOF-ES-MS: 461.2441 ([M + H]+, C28H33 NHCO, 1 H); 7.79 (d, J = 8.4, H–C(3″, 7″), 2 H); 7.51 (t,
N2O4+; calc. 461.2440).
J = 8.4, H–C(5″), 1 H); 7.45 (t, J = 8.0, H–C(4″, 6″), 2 H);
N-{(2S)-1-{[(2S)-1-Hydroxy-3-phenylpropan-2-yl]amino}- 7.22 – 7.09 (m, H–C(50 – 90, 5, 9), 7 H); 6.78 (d, J = 8.8,
1-oxo-3-[4-(propan-2-yloxy)phenyl]propan-2-yl}ben- H–C(6, 8), 2 H); 4.80 (t, J = 5.6, OH, 1 H); 4.65 – 4.59 (m,
zamide (4d). White powder. Yield: 60%. M.p. H–C(2), 1 H); 3.92 – 3.89 (m, H–C(20), 1 H); 3.64 (d,
198 – 199 °C (AcOEt). IR (KBr): 1654 (C=O), 1635 J = 7.2, Me2CHCH2O, 2 H); 3.33 – 3.27 (m, CH2(10), 2 H);
(C=O), 1541 (NH), 1508 (NH), 1244 (=C–O). 1H-NMR 2.98 – 2.63 (m, H–C(3, 30), 4 H); 1.97 – 1.91 (m, Me2CH-
((D6)DMSO, 400 MHz): 8.50 (d, J = 8.8, NHCO, 1 H); CH2O, 1 H); 0.92 (d, J = 5.6, Me2CHCH2O, 6 H). 13C-NMR
7.94 (d, J = 8.0, NHCO, 1 H); 7.81 (d, J = 8.4, H–C(3″, ((D6)DMSO, 100 MHz): 171.1 (C(1)); 166.1 (C(1″)); 157.3
7″), 2 H); 7.52 (t, J = 8.0, H–C(5″), 1 H); 7.45 (t, J = 8.0, (C(7)); 139.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); 130.2 (C(5,
H–C(4″, 6″), 2 H); 7.24 – 7.11 (m, H–C(50 – 90, 5, 9), 7 9, 4)); 129.2 (C(60, 80)); 128.2 (C(4″, 6″)); 128.1 (C(50, 90));
H); 6.79 (d, J = 8.8, H–C(6, 8), 2 H); 4.84 (t, J = 5.6, OH, 127.4 (C(3″, 7″)); 125.9 (C(70)); 114.0 (C(6, 8)); 73.6
1 H); 4.64 – 4.60 (m, H–C(2), 1 H); 4.39 – 4.34 (m, (Me2CHCH2O); 62.2 (C(10)); 55.1 (C(20)); 52.5 (C(2)); 36.5
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Chem. Biodiversity 2016, 13, 1584 – 1592
1589
(C(30, 3)); 27.7 (Me2CHCH2O); 19.1 (Me2CHCH2O). ESI-MS: 171 – 173 °C (AcOEt). IR (KBr): 1652 (C=O), 1633 (C=O),
1
475.5 ([M + H]+). TOF-ES-MS: 497.2421 ([M + Na]+, 1541 (NH), 1508 (NH), 1236 (=C–O). H-NMR ((D6)DMSO,
C29H34N2NaO4+; calc. 497.2416).
400 MHz): 8.47 (d, J = 8.8, NHCO, 1 H); 7.88 (d, J = 8.4,
N-{(2S)-1-{[(2S)-1-Hydroxy-3-phenylpropan-2-yl]amino}- NHCO, 1 H); 7.78 (d, J = 6.8, H–C(3″, 7″), 2 H); 7.51 (t,
1-oxo-3-[4-(pentyloxy)phenyl]propan-2-yl}benzamide J = 8.0, H–C(5″), 1 H); 7.43 (t, J = 8.0, H–C(4″, 6″), 2 H);
(4g). White needle crystals. Yield: 63%. M.p. 7.31 – 7.10 (m, H–C(50 – 90, 5, 9), 7 H); 6.94 (d, J = 8.8,
194 – 196 °C (AcOEt). IR (KBr): 1652 (C=O), 1635 (C=O), H–C(6, 8), 2 H); 5.09 (s, OCH2, 2 H); 4.82 (t, J = 5.6, OH,
1
1541 (NH), 1508 (NH), 1245 (=C–O). H-NMR ((D6)DMSO, 1 H); 4.68 – 4.62 (m, H–C(2), 1 H); 3.91 – 3.89 (m, H–C
400 MHz): 8.45 (d, J = 8.4, NHCO, 1 H); 7.89 (d, J = 8.4, (20), 1 H); 3.36 – 3.27 (m, CH2(10), 2 H); 3.02 – 2.64 (m,
NHCO, 1 H); 7.77 (d, J = 8.4, H–C(3″, 7″), 2 H); 7.49 (t, H–C(3, 30), 4 H). 13C-NMR ((D6)DMSO, 100 MHz): 170.9
J = 8.4, H–C(5″), 1 H); 7.42 (t, J = 8.4, H–C(4″, 6″), 2 H); (C(1)); 166.1 (C(1″)); 154.8 (C(7)); 139.0 (C(40)); 134.1 (C
7.20 – 7.06 (m, H–C(50 – 90, 5, 9), 7 H); 6.73 (d, J = 8.8, (2″)); 131.3 (C(5″)); 130.4 (C(5, 9, 4)); 129.2 (C(60, 80));
H–C(6, 8), 2 H); 4.83 (t, J = 5.6, OH, 1 H); 4.62 – 4.59 128.2 (C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″)); 125.9 (C(70));
(m, H–C(2), 1 H); 3.89 – 3.81 (m, H–C(20), Me(CH2)3CH2O, 116.7 (CN); 114.4 (C(6, 8)); 62.2 (C(10)); 54.9 (C(2)); 53.4
3 H); 3.31 – 3.23 (m, CH2(10), 2 H); 2.95 – 2.61 (m, H–C (OCH2); 52.5 (C(20)); 36.4 (C(30, 3)). ESI-MS: 458.5
(3, 30), 4 H); 1.64 – 1.58 (m, Me(CH2)2CH2CH2O, 2 H); ([M + H]+). TOF-ES-MS: 480.1903 ([M + Na]+, C27H27N3
1.34 – 1.23 (m, Me(CH2)2CH2CH2O, 4 H); 0.82 (t, J = 7.2, NaO4+; calc. 480.1899).
Me, 3 H). 13C-NMR ((D6)DMSO, 100 MHz): 171.2 (C(1)); N-{(2S)-1-{[(2S)-1-Hydroxy-3-phenylpropan-2-yl]amino}-
166.2 (C(1″)); 157.2 (C(7)); 139.1 (C(40)); 134.1 (C(2″)); 1-oxo-3-[4-(prop-2-en-1-yloxy)phenyl]propan-2-yl}ben-
131.4 (C(5″)); 130.2 (C(5, 9, 4)); 129.3 (C(60, 80)); 128.3 zamide (4j). White needle crystals. Yield: 58%. M.p.
(C(4″, 6″)); 128.2 (C(50, 90)); 127.5 (C(3″, 7″)); 126.0 216 – 217 °C (AcOEt). IR (KBr): 1654 (C=O), 1635 (C=O),
1
(C(70)); 114.0 (C(6, 8)); 67.3 (Me(CH2)3CH2O); 62.3 (C(10)); 1541 (NH), 1508 (NH), 1251 (=C–O). H-NMR ((D6)DMSO,
55.2 (C(20)); 52.6 (C(2)); 36.5 (C(30, 3)); 28.5 400 MHz): 8.44 (d, J = 8.4, NHCO, 1 H); 7.86 (d, J = 8.4,
(Me(CH2)2CH2CH2O); 27.8 (MeCH2CH2CH2CH2O); 22.0 NHCO, 1 H); 7.78 (d, J = 7.2, H–C(3″, 7″), 2 H); 7.51 (t,
(MeCH2); 14.0 (Me). ESI-MS: 489.5 ([M + H]+). J = 8.4, H–C(5″), 1 H); 7.44 (t, J = 8.0, H–C(4″, 6″), 2 H);
TOF-ES-MS: 511.2577 ([M + Na]+, C30H36N2NaO4 ; calc. 7.22 – 7.10 (m, H–C(50 – 90, 5, 9), 7 H); 6.81 (d, J = 8.8, H–C
+
511.2573).
(6, 8), 2 H); 6.03 – 5.90 (m, OCH2CH, 1 H); 5.36 – 5.31 (m,
N-[(2S)-3-[4-(Hexyloxy)phenyl]-1-{[(2S)-1-hydroxy-3- OCH2CH=CH2, 2 H); 4.80 (t, J = 5.6, OH, 1 H); 4.66 – 4.60
phenylpropan-2-yl]amino}-1-oxopropan-2-yl]benza- (m, H–C(2), 1 H); 4.46 (d, J = 5.2, OCH2, 2 H); 3.92 – 3.87
mide (4h). White needle crystals. Yield: 61%. M.p. (m, H–C(20), 1 H); 3.33 – 3.25 (m, CH2(10), 2 H); 2.98 – 2.64
184 – 186 °C (AcOEt). IR (KBr): 1654 (C=O), 1633 (m, H–C(3, 30), 4 H). 13C-NMR ((D6)DMSO, 100 MHz): 171.0
(C=O), 1541 (NH), 1508 (NH), 1246 (=C–O). 1H-NMR (C(1)); 166.1 (C(1″)); 156.6 (C(7)); 139.0 (C(40)); 134.1 (C(2″));
((D6)DMSO, 400 MHz): 8.45 (d, J = 8.4, NHCO, 1 H); 133.9 (C–OCH2CH); 131.3 (C(5″)); 130.4 (C(4)); 130.2 (C(5, 9));
8.11 (d, J = 8.4, NHCO, 1 H); 7.79 (d, J = 6.8, H–C(3″, 129.2 (C(60, 80)); 128.1 (C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″));
7″), 2 H); 7.50 (t, J = 8.4, H–C(5″), 1 H); 7.43 (t, J = 8.0, 125.9 (C(70)); 117.3 (OCH2CH=CH2); 114.2 (C(6, 8)); 68.0
H–C(4″, 6″), 2 H); 7.22 – 7.14 (m, H–C(50 – 90, 5, 9), (OCH2); 62.2 (C(10)); 55.1 (C(2)); 52.5 (C(20)); 36.5 (C(30, 3).
7 H); 6.77 (d, J = 8.4, H–C(6, 8), 2 H); 4.62 – 4.59 (m, ESI-MS: 459.5 ([M + H]+). TOF-ES-MS: 481.2107 ([M + Na]+,
H–C(2), 1 H); 4.18 – 4.17 (m, H–C(20), 1 H); 3.87 – 3.83 C28H30N2NaO4+; calc. 481.2103).
(m, CH2(10), OCH2, 2 H); 2.90 – 2.76 (m, H–C(3, 30), 4
H); 1.66 – 1.61 (m, OCH2CH2, 2 H); 1.36 – 1.27 (m,
General Procedure for the Synthesis of Compounds 5a – 5j
MeCH2CH2CH2CH2, 6 H); 0.85 (t, J = 7.2, Me, 3 H). 13C-
NMR ((D6)DMSO, 100 MHz): 171.3 (C(1)); 170.3 (OCO)); Ac2O (1.2 mmol) was added to the soln. of compound
166.1 (C(1″)); 157.2 (C(7)); 138.0 (C(40)); 134.1 (C(2″)); 4a – 4j (1 mmol) in pyridine (5 ml). The mixture was
131.3 (C(5″)); 130.2 (C(5, 9)); 130.0 (C(4)); 129.2 (C(60, stirred at r.t. for 5 h. The solvent was removed
80)); 128.2 (C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″)); 126.2 (C in vacuo and the residue was dispersed in H2O (100
(70)); 114.0 (C(6, 8)); 67.2 (OCH2); 64.6 (C(10)); 55.2 (C ml) and acidified to pH 2 – 3 with conc. HCl. The
(2)); 52.5 (C(20)); 36.5 (C(30, 3)); 28.4 (CH2); 27.7 (CH2); resulting precipitate was collected by filtration,
21.9 (CH2); 20.6 (CH2); 13.9 (Me). ESI-MS: 503.5 washed with H2O (30 ml), and dried to give the title
([M + H]+). TOF-ES-MS: 525.2735 ([M + Na]+, C31H38 compounds 5a – 5j, resp.
N2NaO4+; calc. 525.2729).
(2S)-2-{[(2S)-2-Benzamido-3-(4-methoxyphenyl)pro-
N-[(2S)-3-[4-(Cyanomethoxy)phenyl]-1-{[(2S)-1-hydroxy- panoyl]amino}-3-phenylpropyl Acetate (5a). White
3-phenylpropan-2-yl]amino}-1-oxopropan-2-yl]benza- powder. Yield: 83%. M.p. 202 – 203 °C (AcOEt). IR
mide (4i). White needle crystals. Yield: 54%. M.p. (KBr): 1730 (C=O), 1660 (C=O), 1629 (C=O), 1541 (NH),
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1590
Chem. Biodiversity 2016, 13, 1584 – 1592
1508 (NH), 1259 (=C–O). 1H-NMR ((D6)DMSO, 400 6″)); 127.4 (C(3″, 7″)); 126.2 (C(70)); 113.9 (C(6, 8)); 68.7
MHz): 8.46 (d, J = 8.0, NHCO, 1 H); 8.11 (d, J = 7.8, (OCH2); 64.6 (C(10)); 55.2 (C(20)); 49.1 (C(2)); 36.5 (C(30,
NHCO, 1 H); 7.80 (d, J = 6.8, H–C(3″, 7″), 2 H); 7.49 (t, 3)); 22.1 (MeCH2); 20.6 (MeCO); 10.4 (Me). ESI-MS: 503.5
J = 8.4, H–C(5″), 1 H); 7.45 (t, J = 8.0, H–C(4″, 6″), 2 H); ([M + H]+). TOF-ES-MS: 525.2372 ([M + Na]+, C30H34N2
7.38 – 7.02 (m, H–C(50 – 90, 5, 9), 7 H); 6.79 (d, J = 8.0, NaO5+; calc. 525.2365).
H–C(6, 8),
2 H); 4.68 – 4.59 (m, H–C(2), 1 H); (2S)-2-({(2S)-2-Benzamido-3-[4-(propan-2-yloxy)phe-
4.21 – 4.15 (m, H–C(20), 1 H); 3.67 (s, MeO, 3 H); nyl]propanoyl}amino)-3-phenylpropyl Acetate (5d).
2.90 – 2.78 (m, H–C(3, 30), 4 H); 1.97 (s, Me, 3 H). 13C- White needle crystals. Yield: 74%. M.p. 186 – 188 °C
NMR ((D6)DMSO, 100 MHz): 171.3 (C(1)); 170.3 (C(10)); (AcOEt). IR (KBr): 1726 (C=O), 1660 (C=O), 1629 (C=O),
166.1 (C(1″)); 157.7 (C(7)); 138.0 (C(40)); 134.1 (C(2″)); 1541 (NH), 1508 (NH), 1255 (=C–O). 1H-NMR ((D6)DMSO,
131.3 (C(5″)); 130.1 (C(5, 9, 4)); 129.1 (C(60, 80)); 128.2 400 MHz): 8.46 (d, J = 8.4, NHCO, 1 H); 8.11 (d, J = 8.4,
(C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″)); 126.2 (C(70)); 113.9 NHCO, 1 H); 7.80 (d, J = 7.2, H–C(3″, 7″), 2 H); 7.51 (t,
(C(6, 8)); 64.6 (C(10)); 55.2 (C(2)); 54.9 (MeO); 49.1 (C J = 8.0, H–C(5″), 1 H); 7.43 (t, J = 8.4, H–C(4″, 6″), 2 H);
(20)); 36.5 (C(30, 3)); 20.6 (MeCO). ESI-MS: 475.5 7.22 – 7.13 (m, H–C(50 – 90, 5, 9), 7 H); 6.77 (d, J = 9.2,
([M + H]+). TOF-ES-MS: 497.2057 ([M + Na]+, C28H30N2 H–C(6, 8), 2 H); 4.65 – 4.59 (m, H–C(2), 1 H); 4.53 – 4.47
NaO5+; calc. 497.2052).
(m, OCH, 1 H); 4.19 – 4.16 (m, H–C(20), 1 H); 4.03 – 3.83
(2S)-2-{[(2S)-2-Benzamido-3-(4-ethoxyphenyl)propa- (m, CH2(10), 2 H); 2.93 – 2.77 (m, H–C(3, 30), 4 H); 1.97 (s,
noyl]amino}-3-phenylpropyl Acetate (5b). White Me, 3 H); 1.19 (t, J = 6.0, Me2CH–O, 6 H). 13C-NMR ((D6)
powder. Yield: 83%. M.p. 189 – 190 °C (AcOEt). IR DMSO, 100 MHz): 171.3 (C(1)); 170.3 (C(10)); 166.1 (C(1″));
(KBr): 1726 (C=O), 1660 (C=O), 1629 (C=O), 1541 (NH), 155.9 (C(7)); 138.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″));
1508 (NH), 1257 (=C–O). 1H-NMR ((D6)DMSO, 400 130.2 (C(5, 9)); 129.9 (C(4)); 129.1 (C(60, 80)); 128 2 (C(50,
MHz): 8.47 (d, J = 8.4, NHCO, 1 H); 8.12 (d, J = 8.4, 90, 4″, 6″)); 127.4 (C(3″, 7″)); 126.2 (C(70)); 115.1 (C(6, 8));
NHCO, 1 H); 7.80 (d, J = 6.8, H–C(3″, 7″), 2 H); 7.51 (t, 68.9 (OCH); 64.6 (C(10)); 55.1 (C(2)); 49.1 (C(20)); 36.5 (C
J = 8.0, H–C(5″), 1 H); 7.44 (t, J = 8.0, H–C(4″, 6″), 2 (30, 3)); 21.8 (Me2CH–O); 20.6 (Me). ESI-MS: 503.5
H); 7.24 – 7.14 (m, H–C(50 – 90, 5, 9), 7 H); 6.77 (d, ([M + H]+).
J = 7.8, H–C(6, 8), 2 H); 4.64 – 4.58 (m, H–C(2), 1 H); C30H34N2NaO5+; calc. 525.2365).
TOF-ES-MS:
525.2371
([M + Na]+,
4.18 – 4.15 (m, H–C(20), 1 H); 4.02 – 3.83 (m, CH2(10), (2S)-2-{[(2S)-2-Benzamido-3-(4-butoxyphenyl)propa-
OCH2, 4 H); 2.93 – 2.76 (m, H–C(3, 30), 4 H); 1.97 (s, noyl]amino}-3-phenylpropyl Acetate (5e). White
MeCO, 3 H); 1.26 (t, Me, 3 H). 13C-NMR ((D6)DMSO, needle crystals. Yield: 84%. M.p. 189 – 190 °C (AcOEt).
100 MHz): 171.3 (C(1)); 170.3 (MeCO); 166.1 (C(1″)); IR (KBr): 1731 (C=O), 1658 (C=O), 1635 (C=O), 1541
1
157.0 (C(7)); 138.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); (NH), 1508 (NH), 1244 (=C–O). H-NMR ((D6)DMSO, 400
130.2 (C(5, 9)); 130.0 (C(4)); 129.1 (C(60, 80)); 128.2 MHz): 8.46 (d, J = 8.4, NHCO, 1 H); 8.11 (d, J = 8.4,
(C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″)); 126.2 (C(70)); 113.9 NHCO, 1 H); 7.80 (d, J = 7.2, H–C(3″, 7″), 2 H); 7.49 (t,
(C(6, 8)); 64.6 (C(10)); 62.8 (OCH2); 55.2 (C(20)); 49.1 J = 8.0, H–C(5″), 1 H); 7.42 (t, J = 8.0, H–C(4″, 6″), 2 H);
(C(2)); 36.5 (C(30, 3)); 20.6 (MeCO); 14.7 (Me). ESI-MS: 7.23 – 7.14 (m, H–C(50 – 90, 5, 9), 7 H); 6.79 (d, J = 8.4,
489.5 ([M + H]+). TOF-ES-MS: 511.2214 ([M + Na]+, H–C(6, 8),
C29H32N2NaO5+; calc. 511.2209).
2
H); 4.62 – 4.59 (m, H–C(2),
1 H);
4.19 – 4.17 (m, H–C(20), 1 H); 3.88 – 3.85 (m, CH2(10),
(2S)-2-{[(2S)-2-Benzamido-3-(4-propoxyphenyl)propa- OCH2, 4 H); 2.91 – 2.76 (m, H–C(3, 30), 4 H); 1.97 (s,
noyl]amino}-3-phenylpropyl Acetate (5c). White nee- Me, 3 H); 1.64 – 1.59 (m, OCH2CH2, 2 H); 1.41 – 1.35
dle crystals. Yield: 80%. M.p. 186 – 187 °C (AcOEt). IR (m, OCH2CH2CH2, 2 H); 0.89 (t, J = 7.2, MeCH2, 3 H).
(KBr): 1730 (C=O), 1660 (C=O), 1629 (C=O), 1541 (NH), 13C-NMR ((D6)DMSO, 100 MHz): 171.3 (C(1)); 170.3
1
1508 (NH), 1257 (=C–O). H-NMR ((D6)DMSO, 400 MHz): (C(10)); 166.1 (C(1″)); 157.2 (C(7)); 138.0 (C(40)); 134.1
8.48 (d, J = 8.4, NHCO, 1 H); 8.13 (d, J = 8.4, NHCO, 1 (C(2″)); 131.3 (C(5″)); 130.1 (C(5, 9)); 130.0 (C(4)); 129.1
H); 7.81 (d, J = 7.2, H–C(3″, 7″), 2 H); 7.51 (t, J = 8.0, H– (C(60, 80)); 128 2 (C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″));
C(5″), 1 H); 7.44 (t, J = 8.0, H–C(4″, 6″), 2 H); 7.22 – 7.15 126.2 (C(70)); 113.9 (C(6, 8)); 66.9 (OCH2); 64.6 (C(10));
(m, H–C(50 – 90, 5, 9), 7 H); 6.78 (d, J = 8.4, H–C(6, 8), 2 55.2 (C(2)); 49.1 (C(20)); 36.5 (C(30, 3)); 30.8 (OCH2CH2);
H); 4.65 – 4.59 (m, H–C(2), 1 H); 4.19 – 4.16 (m, H–C(20), 20.6 (Me); 18.8 (OCH2CH2CH2); 13.7 (Me). ESI-MS: 517.5
1 H); 4.03 – 3.81 (m, CH2(10), OCH2, 4 H); 2.94 – 2.77 ([M + H]+).
TOF-ES-MS:
539.2528
([M + Na]+,
+
(m, H–C(3, 30), 4 H); 1.97 (s, MeCO, 3 H); 1.69 – 1.63 (m, C31H36N2NaO5 ; calc. 539.2522).
MeCH2, 2 H); 0.92 (t, Me, 3 H). 13C-NMR ((D6)DMSO, 100 (2S)-2-({(2S)-2-Benzamido-3-[4-(2-methylpropoxy)phe-
MHz): 171.3 (C(1)); 170.3 (MeCO); 166.1 (C(1″)); 157.2 nyl]propanoyl}amino)-3-phenylpropyl Acetate (5f).
(C(7)); 138.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); 130.2 (C White needle crystals. Yield: 73%. M.p. 175 – 177 °C
(5, 9)); 130.0 (C(4)); 129.1 (C(60, 80)); 128.2 (C(50, 90, 4″, (AcOEt). IR (KBr): 1731 (C=O), 1660 (C=O), 1631 (C=O),
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Chem. Biodiversity 2016, 13, 1584 – 1592
1591
1
1541 (NH), 1514 (NH), 1247 (=C–O). H-NMR ((D6)DMSO, NMR ((D6)DMSO, 100 MHz): 171.4 (C(1)); 170.4 (MeCO);
400 MHz): 8.46 (d, J = 8.8, NHCO, 1 H); 8.13 (d, J = 8.4, 166.2 (C(1″)); 157.2 (C(7)); 138.1 (C(40)); 134.1 (C(2″));
NHCO, 1 H); 7.80 (d, J = 6.8, H–C(3″, 7″), 2 H); 7.50 (t, 131.4 (C(5″)); 130.2 (C(5, 9)); 130.0 (C(4)); 129.2 (C(60, 80));
J = 8.4, H–C(5″), 1 H); 7.43 (t, J = 8.0, H–C(4″, 6″), 2 H); 128.3 (C(4″, 6″, 50, 90)); 127.5 (C(3″, 7″)); 126.3 (C(70));
7.23 – 7.13 (m, H–C(50 – 90, 5, 9), 7 H); 6.78 (d, J = 7.8, 114.0 (C(6, 8)); 67.3 (Me(CH2)4CH2O); 64.7 (C(10)); 55.2 (C
H–C(6, 8), 2 H); 4.63 – 4.57 (m, H–C(2), 1 H); 4.18 – 4.14 (20)); 49.2 (C(2)); 36.5 (C(30, 3)); 31.1 (MeCH2CH2); 28.8
(m, H–C(20), 1 H); 4.02 – 3.82 (m, CH2(10), 2 H); 3.64 – 3.62 (OCH2CH2); 25.3 (OCH2CH2CH2); 22.2 (MeCH2);
(m, OCH2, 2 H); 2.93 – 2.76 (m, H–C(3, 30), 4 H); 1.96 (s, 20.7 (MeCO); 14.0 (Me). ESI-MS: 545.5 ([M + H]+).
MeCO, 3 H); 1.97 – 1.88 (m, OCH2CH, 2 H); 0.92 (d, TOF-ES-MS: 567.2841 ([M + Na]+, C33H40N2NaO5+; calc.
J = 7.8, Me2CH, 6 H). 13C-NMR ((D6)DMSO, 100 MHz): 567.2835).
171.3 (C(1)); 170.3 (MeCO); 166.1 (C(1″)); 157.3 (C(7)); (2S)-2-({(2S)-2-Benzamido-3-[4-(cyanomethoxy)phe-
138.0 (C(40)); 134.1 (C(2″)); 131.3 (C(5″)); 130.2 (C(5, 9)); nyl]propanoyl}amino)-3-phenylpropyl Acetate (5i).
130.1 (C(4)); 129.2 (C(60, 80)); 128.3 (C(50, 90, 4″, 6″)); 127.5 White needle crystals. Yield: 79%. M.p. 161 – 162 °C
(C(3″, 7″)); 126.3 (C(70)); 114.0 (C(6, 8)); 73.6 (OCH2); 64.6 (AcOEt). IR (KBr): 1731 (C=O), 1660 (C=O), 1629 (C=O),
(C(10)); 55.2 (C(20)); 49.1 (C(2)); 36.5 (C(30, 3)); 27.8 (CH); 1541 (NH), 1508 (NH), 1257 (=C–O). 1H-NMR ((D6)
20.6 (MeCO); 19.1 (Me). ESI-MS: 517.5 ([M + H]+). TOF-ES- DMSO, 400 MHz): 8.50 (d, J = 8.4, NHCO, 1 H); 8.14
MS: 539.2528 ([M + Na]+, C31H36N2NaO5+; calc. 539.2522).
(d, J = 8.0, NHCO, 1 H); 7.80 (d, J = 8.4, H–C(3″, 7″), 2
(2S)-2-({(2S)-2-Benzamido-3-[4-(pentyloxy)phenyl]pro- H); 7.52 (t, J = 8.4, H–C(5″), 1 H); 7.43 (t, J = 8.4, H–C
panoyl}amino)-3-phenylpropyl Acetate (5g). White (4″, 6″), 2 H); 7.29 (d, J = 7.8, H–C(5, 9), 2 H);
needle crystals. Yield: 75%. M.p. 172 – 173 °C (AcOEt). IR 7.22 – 7.14 (m, H–C(50 – 90), 5 H); 6.94 (d, J = 8.0, H–
(KBr): 1731 (C=O), 1660 (C=O), 1633 (C=O), 1541 (NH), C(6, 8), 2 H); 5.08 (s, OCH2, 2 H); 4.66 – 4.60 (m, H–C
1
1508 (NH), 1247 (=C–O). H-NMR ((D6)DMSO, 400 MHz): (2), 1 H); 4.18 – 4.15 (m, H–C(20), 1 H); 4.03 – 3.83 (m,
8.45 (d, J = 8.4, NHCO, 1 H); 8.11 (d, J = 8.4, NHCO, 1 H); CH2(10), 2 H); 2.97 – 2.76 (m, H–C(3, 30), 4 H); 1.96 (s,
7.79 (d, J = 6.8, H–C(3″, 7″), 2 H); 7.50 (t, J = 8.0, H–C(5″), MeCO, 3 H). 13C-NMR ((D6)DMSO, 100 MHz): 171.3
1 H); 7.43 (t, J = 8.0, H–C(4″, 6″), 2 H); 7.22 – 7.14 (m, H–C (C(1)); 170.4 (MeCO); 166.2 (C(1″)); 154.9 (C(7)); 138.0
(50 – 90, 5, 9), 7 H); 6.77 (d, J = 8.4, H–C(6, 8), 2 H); (C(40)); 134.1 (C(2″)); 132.3 (C(4)); 131.4 (C(5″)); 130.5
4.63 – 4.59 (m, H–C(2), 1 H); 4.19 – 4.14 (m, H–C(20), 1 H); (C(5, 9)); 129.2 (C(60, 80)); 128.3 (C(4″, 6″, 50, 90)); 127.5
3.86 – 3.81 (m, CH2(10), OCH2, 2 H); 2.90 – 2.76 (m, H–C(3, (C(3″, 7″)); 126.3 (C(70)); 116.8 (CN); 114.4 (C(6, 8));
30), 4 H); 1.96 (s, MeCO, 3 H); 1.66 – 1.59 (m, OCH2CH2, 2 64.7 (C(10)); 55.0 (C(20)); 53.4 (Ar-OCH2)); 49.2 (C(2));
H); 1.36 – 1.22 (m, MeCH2CH2, 4 H); 0.85 (t, J = 7.2, Me, 3 36.5 (C(30, 3)); 20.7 (MeCO). ESI-MS: 500.5 ([M + H]+).
H). 13C-NMR ((D6)DMSO, 100 MHz): 171.3 (C(1)); 170.3 TOF-ES-MS: 522.2010 ([M + Na]+, C29H29N3NaO5+; calc.
(OCO); 166.1 (C(1″)); 157.2 (C(7)); 138.0 (C(40)); 134.1 522.2005).
(C(2″)); 131.3 (C(5″)); 130.2 (C(5, 9)); 130.0 (C(4)); 129.2 (C (2S)-2-({(2S)-2-Benzamido-3-[4-(prop-2-en-1-yloxy)phe-
(60, 80)); 128.2 (C(50, 90, 4″, 6″)); 127.4 (C(3″, 7″)); 126.2 (C nyl]propanoyl}amino)-3-phenylpropyl Acetate (5j).
(70)); 114.0 (C(6, 8)); 67.2 (OCH2); 64.6 (C(10)); 55.2 (C(2)); White needle crystals. Yield: 98%. M.p. 171 – 173 °C
52.5 (C(20)); 36.5 (C(30, 3)); 28.4 (CH2); 27.7 (CH2); 21.9 (AcOEt). IR (KBr): 1733 (C=O), 1652 (C=O), 1629 (C=O),
1
(CH2); 20.6 (CH2); 13.9 (Me). ESI-MS: 531.5 ([M + H]+). TOF- 1541 (NH), 1508 (NH), 1257 (=C–O). H-NMR ((D6)DMSO,
ES-MS: 553.2683 ([M + Na]+, C32H38N2NaO5 ; calc. 553. 400 MHz): 8.46 (d, J = 8.4, NHCO, 1 H); 8.11 (d, J = 8.4,
+
2678).
NHCO, 1 H); 7.79 (d, J = 8.4, H–C(3″, 7″), 2 H); 7.52 (t,
(2S)-2-({(2S)-2-Benzamido-3-[4-(hexyloxy)phenyl]pro- J = 8.0, H–C(5″), 1 H); 7.42 (t, J = 8.0, H–C(4″, 6″), 2 H);
panoyl}amino)-3-phenylpropyl Acetate (5h). White 7.22 – 7.15 (m, H–C(50 – 90, 5, 9), 7 H); 6.81 (d, J = 8.8, H–
needle crystals. Yield: 73%. M.p. 177 – 179 °C (AcOEt). C(6, 8), 2 H); 6.02 – 5.95 (m, CH=CH2, 1 H); 5.37 – 5.18 (dd,
IR (KBr): 1731 (C=O), 1662 (C=O), 1635 (C=O), 1541 (NH), CH=CH2, 2 H); 4.63 – 4.60 (m, H–C(2), 1 H); 4.47 (s, OCH2, 2
1
1508 (NH), 1255 (=C–O). H-NMR ((D6)DMSO, 400 MHz): H); 4.18 – 4.16 (m, H–C(20), 1 H); 4.02 – 3.83 (m, CH2(10), 2
8.47 (d, J = 8.4, NHCO, 1 H); 8.13 (d, J = 8.4, NHCO, 1 H); H); 2.92 – 2.76 (m, H–C(3, 30), 4 H); 1.97 (s, MeCO, 3 H).
7.79 (d, J = 8.4, H–C(3″, 7″), 2 H); 7.51 (t, J = 8.0, H–C(5″), 13C-NMR ((D6)DMSO, 100 MHz): 171.3 (C(1)); 170.4 (MeCO);
1 H); 7.42 (t, J = 8.4, H–C(4″, 6″), 2 H); 7.22 – 7.14 (m, H– 166.2 (C(1″)); 156.7 (C(7)); 138.0 (C(40)); 134.1 (C(2″)); 133.9
C(50 – 90, 5, 9), 7 H); 6.77 (d, J = 8.8, H–C(6, 8), 2 H); (CH=CH2); 131.3 (C(5″)); 130.3 (C(4)); 130.2 (C(5, 9)); 129.1
4.63 – 4.59 (m, H–C(2), 1 H); 4.18 – 4.15 (m, H–C(20), 1 (C(60, 80)); 128.2 (C(4″, 6″, 50, 90)); 127.4 (C(3″, 7″)); 126.2
H); 3.84 (t, J = 8.0, Me(CH2)4CH2O, 2 H); 3.46 – 3.39 (m, (C(70)); 117.3 (CH=CH2); 114.2 (C(6, 8)); 68.0 (Ar-OCH2); 64.6
CH2(10), 2 H); 2.92 – 2.75 (m, H–C(3, 30), 4 H); 1.96 (s, (C(10)); 55.1 (C(20)); 49.1 (C(2)); 36.5 (C(30, 3)); 20.6 (MeCO).
MeCO, 3 H); 1.59 – 1.66 (m, OCH2CH2, 1 H); 1.36 – 1.23 ESI-MS: 501.5 ([M + H]+). TOF-ES-MS: 523.2214 ([M + Na]+,
(m, MeCH2CH2CH2, 6 H); 0.84 – 0.85 (m, Me, 3 H). 13C- C30H32N2NaO5 ; calc. 523.2209).
+
© 2016 Wiley-VHCA AG, Zurich, Switzerland
www.cb.wiley.com

1592
Chem. Biodiversity 2016, 13, 1584 – 1592
and the National Natural Science Foundation of China
Biological Assay
(NSFC No. 81360472). 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.
In vitro anti-HBV assays. HepG2 2.2.15 cells were
seeded in 24 well culture plates at a density of
1 9 105 cells per well and allowed to recover for
24 h. Culture medium was replaced by assay medium
containing the compound that is being tested or 3TC.
Every 3 days, medium was changed. After 8 days, the
levels of intracellular HBV DNA were measured by
quantitative Southern blot hybridization.
Cytotoxicity induced by the target compounds in
cultures of HepG2 2.2.15 cells was also determined.
Briefly, HepG2 2.2.15 cells were grown to confluence
in 96-well culture plates, and then treated with test
compounds (in 0.2 ml culture medium/well), resp.
Untreated control cultures were maintained on each
96-well plate. The cytotoxicity was determined by
measuring neutral red dye uptake, as determined
from the absorbance at 510 nm relative to untreated
cells, at 24 h following 6 days of treatment.
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Acknowledgements
Received April 19, 2016
Accepted June 23, 2016
This work was supported by the grants from the Natural
Science Foundation of Jiangsu Province (BK20140223)
www.cb.wiley.com
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