Discovering novel quercetin-3-O-amino acid-esters as a new class of Src tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.09.051

Quercetin-3-O-amino acid-esters, a new type of quercetin derivatives, were successfully prepared for the first time. Different from quercetin, the novel compounds show higher selectivity as inhibitors against Src tyrosine kinase (IC₅₀ values ra

Full text of DOI:10.1016/j.ejmech.2008.09.051

European Journal of Medicinal Chemistry 44 (2009) 1982–1988
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovering novel quercetin-3-O-amino acid-esters as a new class of Src
tyrosine kinase inhibitors
,
, ,
He Huang a 1, Qi Jia b 1, Jingui Ma a, Guangrong Qin a, Yingyi Chen b, Yonghua Xi b, Liping Lin
,
*
a,**
Weiliang Zhu a, Jian Ding a, Hualiang Jiang a, Hong Liu
a,**
a Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
b School of Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Quercetin-3-O-amino acid-esters, a new type of quercetin derivatives, were successfully prepared for the
Received 28 February 2008
Received in revised form
22 September 2008
Accepted 30 September 2008
Available online 17 October 2008
first time. Different from quercetin, the novel compounds show higher selectivity as inhibitors against
Src tyrosine kinase (IC50 values ranging from 3.2 mM to 9.9 mM) than against EGFR tyrosine kinase.
Molecular docking reveals that both hydrophobic and hydrogen bonding interactions are important to
the selectivity. Therefore, this study provides a new promising scaffold for further development of new
anticancer drugs targeting Src tyrosine kinase.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Quercetin
Amino acid
Src
EGFR
Inhibitor
1. Introduction
PTKs catalyze phosphoryl transfer of the g-phosphoryl group of
ATP to tyrosine residues of proteins, playing a central role in signal
transduction and cellular mechanisms [16]. As one of the important
PTKs, EGFR and its ligands (EGF and TGF-a) have been found to have
Quercetin, a water soluble flavanoid, and its derivatives have
ameliorative effects on a host of disorders including cancer, renal
and cardiovascular diseases, and have inhibitory activity against
SARS-CoV 3CLpro or viral replication.[1–5] Particularly, quercetin is
a well-known protein tyrosine kinase (PTK) inhibitor at micromolar
high expression levels in many tumors of epithelial origin and
proliferative disorders of the epidermis, such as psoriasis [17,18].
Src, a nonreceptor tyrosine kinase which functions as an early
upstream signal transduction protein, is activated in several human
cancers, including carcinomas of the breast, lung, colon, esophagus,
skin, parotid, cervix, as well as gastric tissues [19,20]. Therefore,
they are attractive targets for the discovery of antitumor drugs.
In this paper, we report our first attempt to synthesize a series of
novel quercetin-3-O-amino acid-esters. Remarkably, not only these
compounds can be synthesized, but also they show promising high
selective inhibitory activity against Src tyrosine kinase. The primary
structure–activity relationship of the new compounds is elucidated
by molecular modeling as well.
level. For instance, its IC50 value is 0.9
mM against epidermal growth
factor receptor (EGFR) and 15 M against Src tyrosine kinase
m
respectively, [6,7] indicating that this natural product is more active
against EGFR than Src tyrosine kinase. During the last decade there
has been considerable interest in synthesis, [8–10] functional
elucidation, [11,12] and biological evaluation of quercetin and its
derivatives [13–15]. Most of the studies were focused on the
quercetin O-glycosides, the majority of which have a sugar linkage
at the 3-OH. Up to now, the quercetin-3-O-amino acid-ester was
neither discovered as a natural product, nor reported on synthesis
and bioactivity studies. Therefore, whether this type of compounds
could be synthesized and whether they are still active against PTKs
remain unknown.
2. Chemistry and pharmacology
2.1. Chemistry
* Corresponding author.
To prepare a variety of quercetin derivatives with various O-3
substituents, an efficient and facile synthesis approach is developed
as depicted in Scheme 1. The straightforward three-step synthetic
** Corresponding authors. Tel.: þ86 21 50807042; fax: þ86 21 50807088.
E-mail address: hliu@mail.shcnc.ac.cn (L. Lin).
1
These authors contributed equally to this work.
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.051

H. Huang et al. / European Journal of Medicinal Chemistry 44 (2009) 1982–1988
1983
OH
OBn
OBn
HO
O
O
a
b
BnO
O
O
OH
amino acid
O
rutinosyl
OH
OH
OH
Rutin
1
OH
OH
OBn
OBn
c
HO
O
O
BnO
O
OR
OR
OH
OH
O
2a-n
3a-n
Scheme 1. Reagents and conditions: (a) (i) K2CO3, BnBr, DMF, 60 ꢀC, 3 h, (ii) HCl/ethanol, 70 ꢀC, 2 h; (b) DCC, DMAP, THF, 25 ꢀC, 4 h; (c) H2, Pd/C, 1,4-dioxane/ethanol, 25 ꢀC, 2 h.
route allowed us to diversify position 3 of the quercetin moiety via
the key intermediate 1 at a later stage.
The intermediates before debenzylation were obtained in moderate
yields. However, no desired products were detected in the crude
products after debenzylation.
Beginning with the commercially available rutin, protection of
the hydroxyl groups and subsequent deglycosylation led to the
selective protected quercetin 1 which gave an entry in the series
substituted on the 3 position [21]. Indeed compound 1 still exhibits
two free hydroxyl groups. However the higher reactivity of position
3 allows the selective esterification by protected amino acids in THF
using 1.2 equiv. of DCC (N,N-dicyclohexylcarbodiimide) as
condensing agent and a little DMAP (4-dimethylaminopyridine) as
catalyst. Cleavage of benzyl group was performed with hydro-
genolysis catalyzed by 10% Pd/C. Then, the desired final products
quercetin-3-O-amino acid-esters (3a–o) were obtained in good
yields after purification by chromatography. Substitution at N atom
of amino acid moiety is important. In our first approach to get the
compounds bearing deprotected amino group of amino acid
moiety, trifluoroacetate acid solution of dichloromethane was used
to remove the tertbutyloxycarbonyl group (Scheme 2). However, no
desired product was detected in the crude product. We conjecture
that the target compound was decomposed in the acidic medium.
So an alternative approach performed under mild condition was
then investigated (Scheme 2). Unfortunately, attempts to deben-
zylate the compounds bearing benzylated N atom in amino acid
moiety under H2 atmosphere at ambient temperature was not
successful. Based on above study, we deduced that the compounds
without protective groups of N atom are unstable. Besides, we tried
to synthesize the compounds bearing Boc–Ser, Ac–Gly and Ac–Thr.
2.2. Biological assay
2.2.1. EGFR and Src kinase activity assays by ELISA
The assay was performed in 96-well plates pre-coated with
20
50
m
m
g/mL Poly(Glu, Tyr)4:1 (Sigma) as a substrate. In each well,
L of 8 M ATP solution and 10 L compound were added at
m
m
varying concentrations. 4557W and PP2 were used as positive
controls for EGFR and Src kinase, respectively, and 0.1% (v/v) DMSO
was the negative control. Experiments at each concentration were
performed in triplicate. The kinase reaction was initiated by the
addition of purified EGFR or Src tyrosine kinase proteins diluted in
40
at 37 ꢀC, the plate was washed three times with Phosphate Buffered
Saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 L anti-
mL of kinase reaction buffer solution. After incubation for 60 min
m
phosphotyrosine (PY99) antibody (1:500 dilution) diluted in T-PBS
containing 5 mg/mL BSA was added. After 30 min incubation at
37 ꢀC, the plate was washed three times. Horseradish peroxidase-
conjugated goat anti-mouse IgG 100
containing 5 mg/mL BSA was added. The plate was reincubated at
37 ꢀC for 30 min, and washed as before. Finally, 100
L of a solution
containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M
citrate buffer, pH 5.5, was added and samples were incubated at
room temperature until color emerged. The reaction was
mL diluted 1:2000 in T-PBS
m
OH
OH
HO
O
O
HO
O
O
OH
OH
CF3COOH
CH2Cl2
O
O
OH
NH2
OH
NHBoc
O
O
R
R
OBn
OH
OH
BnO
O
O
HO
O
O
OBn
H2
O
Pd/C
O
OH
NHBn
OH
NH2
O
O
R
R
Scheme 2. Methods to get the compounds bearing deprotected amino group of amino acid moiety.

1984
H. Huang et al. / European Journal of Medicinal Chemistry 44 (2009) 1982–1988
terminated by the addition of 50
mL of 2 M H2SO4, and the plate was
Different conformations have been found for these compounds
read using a multiwall spectrophotometer (VERSAmaxÔ, Molec-
ular Devices, Sunnyvale, CA, USA) at 490 nm. The inhibition rate (%)
was calculated using the following equation: [1 ꢁ (A490/A490
control)] ꢂ 100%. IC50 values were calculated from the inhibition
curves.
in the active pockets of both proteins. For the comparison of the
difference in binding mechanism, the pairs of hydrophobic inter-
action (HI hereinafter) and the number of hydrogen bonds (HB
hereinafter) between the new compounds and the two targets are
analyzed by the program LIGPLOT [23]. The result reveals that there
are, in average, 14 HIs and 3.5 HBs between Src and each of the new
compounds, while there are, in average, only 9 HIs and 2.5 HBs
between EGFR and each compound (Table S1, Figs. S1–S5, Supple-
mentary material). In other words, one third more of these two
kinds of interactions were observed between the compounds and
Src kinase than EGFR kinase. As examples, the interaction details of
the two most active compounds (3i and 3j) are shown in Fig. 1.
There are 12 atoms of 3i forming hydrophobic interactions with 7
residues of Src, of which 5 atoms are from the newly substituted
group of 3i (Fig. 1B); while there are only 5 atoms of 3i forming
hydrophobic interaction with 2 residues of EGFR, of which only 2
atoms are from the substituted group (Fig. 1A). Two hydrogen
bonds form between 3i and Src, while 4 HBs between the
compound and EGFR. Regarding the binding of 3j, the hydrophobic
interaction between 3j and Src is similar to that between 3j and
EGFR, but there are 4 HBs between 3j and Src while only 1 between
3j and EGFR. Therefore, both hydrophobic and hydrogen bonding
interactions are important to the high selectivity of the novel
quercetin-3-O-amino acid-esters against Src kinase.
2.3. Molecular docking
To explore the interaction mechanism between the newly
synthesized quercetin-3-O-amino acid-esters and the two targets,
molecular docking was carried out with the program AutoDock
4.0.1 [22] for the 7 compounds binding to both Src and EGFR,
respectively. The three dimensional (3D) structures of target
proteins of human c-Src and EGFR are from Brookhaven protein
data bank (pdb entry No.: 2SRC and 1M17, respectively). The
Lamarckian genetic algorithm (LGA) is used for docking with the
following settings: a maximum number of 1,500,000 energy eval-
uations, an initial population of 50 randomly placed individuals,
a maximum number of 37,000 generations, a mutation rate of 0.02,
a crossover rate of 0.80 and an elitism value (number of top indi-
viduals that automatically survive) of 1. For the adaptive local
search method, the pseudo-Solis and Wets algorithm was applied
with a maximum of 300 iterations per search. To validate the reli-
ability of our docking approach and parameters used, the binding
free energies of quercetin to EGFR and to Src kinase were calculated
with same parameters.
4. Conclusions
3. Results and discussions
In summary, for the first time fifteen novel quercetin-3-O-amino
acid-ester derivatives are successfully prepared in this study. The
bioassay reveals that the new compounds have higher selectivity as
inhibitors against Src kinase than against EGFR kinase, which is
different from quercetin that is more active against EGFR than
against Src. Molecular docking result reveals that both hydrophobic
and hydrogen bonding interactions are important to the selectivity.
In average, one third more of these two kinds of interactions were
observed between the compounds and Src kinase than EGFR kinase.
Therefore, this study provides a new promising scaffold with
3.1. Biological activities
For the primary assay, the percent inhibitions of the compounds
(3a–o) at 10
mM were measured (data are listed in Table 1).
Remarkably, the newly synthesized quercetin-3-O-amino acid-
esters show low inhibition against EGFR kinase (<43%), whereas
exhibit inhibitory activity as high as 76% against Src kinase
(Table 1). This result suggests that the novel quercetin-3-O-amino
acid-esters have higher inhibitory selectivity against Src kinase
than EGFR kinase. Thus, the introduction of the amino acid group
into quercetin leads to the reverse of the high inhibitory selectivity
from EGFR to Src. To confirm the bioactivity, the IC50 values were
further determined for the compounds with inhibition rate higher
moderate inhibitory activities (IC50 values ranging from 3.2
mM to
9.9 M) for further development of new anticancer drugs targeting
m
Src tyrosine kinase.
5. Experimental protocols
than 50% against Src kinase at 10 mM, namely, compounds 3a–c, 3g,
3–k and 3m (Table 2). The data show that all the 8 compounds have
prominent inhibitory activities with IC50 values ranging from
3.2 mM to 9.9 mM, indicating that some quercetin-3-O-amino acid-
esters are moderately active inhibitors of Src kinase.
The reagents (chemicals) were purchased from commercial
sources (Alfa, GL Biochem Ltd. and Shanghai Chemical Reagent
Company), and used without further purification. Analytical thin
layer chromatography (TLC) was HSGF 254 (0.15–0.2 mm thickness,
Yantai Huiyou Company, China). Column chromatography was
performed with CombiFlashÒ Companion system (Teledyne Isco,
Inc.). NMR spectra were obtained on Varian Mercury-300 spec-
trometers (TMS as IS). Chemical shifts were reported in parts per
3.2. Theoretical structure–activity relationship
The binding free energies of quercetin to EGFR and Src kinase
were calculated to be ꢁ6.6 kcal/mol and ꢁ5.7 kcal/mol, respec-
tively, which are in good agreement with the experimental results
that quercetin is more active against EGFR than Src kinase [6,7].
Therefore, the docking approach and parameters are reasonably
million (ppm, d) downfield from tetramethylsilane. Proton coupling
patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Low- and high-resolution mass
spectra (LRMS and HRMS) were given with electric and electro-
spray ionization (EI. and ESI.) produced by Finnigan MAT-95 and
LCQ-DECA spectrometer.
reliable. The predicted binding free energies (DG) of the 8 new
compounds are listed in Table 2. Noticeably, the predicted
DG
values of the new compounds to Src kinase (ꢁ8.1 kcal/mol in
average) is stronger by 1.4 kcal/mol than that to EGFR (ꢁ6.7 kcal/
mol in average), which is in agreement with our experimental
observation that the 8 new compounds are stronger inhibitors
against Src kinase than against EGFR kinase. Therefore, the selec-
tivity of the newly synthesized quercetin-3-O-amino acid-esters
should be attributed to the specific property of the substituted R
groups, the amino acids (Table 1).
5.1. 7-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3,5-dihydroxy-
4H-chromen-4-one (1)
A mixture of rutin (2.44 g, 4 mmol) and K2CO3 (1.83 g, 10 mmol)
in anhydrous DMF (20 mL) was stirred under nitrogen for 0.5 h.
Then benzyl bromide (1.6 mL, 13.4 mmol) was added. After stirring
for 3 h at 60 ꢀC, the mixture was acidified to pH 5 with 10% AcOH

H. Huang et al. / European Journal of Medicinal Chemistry 44 (2009) 1982–1988
1985
Table 1
Enzyme inhibitory activity of the quercetin-3-O-amino acid-esters.
OH
OH
HO
O
O
O
R
OH
Compound
R
% Inhibition at 10
EGFRa
m
M
Compound
R
% Inhibition at 10
EGFRa
mM
Srcb
Srcb
NHBoc
O
NHBoc
3a
30.4%
55.4%
3i
O
43.2%
76.2%
NHBoc
OH
NHBoc
NHBoc
O
O
O
O
3b
3c
35.9%
21.0%
51.4%
52.5%
3j
36.8%
34.6%
76.1%
71.0%
NHBoc
OH
3k
NHBoc
NH
O
NHBoc
O
3d
29.2%
44.5%
3l
15.6%
44.4%
NHBoc
NH2
O
O
NHBoc
NHAc
O
3e
3f
22.3%
25.9%
40.7%
39.0%
3m
3n
3o
23.8%
19.3%
21.0%
50.8%
27.7%
24.3%
Boc
N
O
O
NHAc
Ac
N
O
O
3g
20.9%
35.9%
60.5%
40.9%
NHBoc
O
3h
a
The percent inhibition rate of 4557 W at 10
m
M is 97.2%.
b
The percent inhibition rate of PP2 at 10
mM is 99.0%.

1986
H. Huang et al. / European Journal of Medicinal Chemistry 44 (2009) 1982–1988
recrystallized with CH2Cl2/EtOH to afford 1 in 86.4% yield. 1H NMR
(DMSO-d6):
1.9 Hz, 1H, –ArH), 7.51–7.32 (m, 15H, –ArH), 7.27 (d, J ¼ 8.7 Hz, 1H,
–ArH), 6.88 (d, J ¼ 1.9 Hz, 1H, –ArH), 6.45 (d, J ¼ 1.9 Hz, 1H, –ArH),
5.24 (s, 4H, –ArCH2), 5.21 (s, 2H, –ArCH2); MS (EI, m/z): 572 [M]þ.
Table 2
The IC50 values (in mM) and predicted binding free energies (DG in kcal/mol) of some
quercetin-3-O-amino acid-esters.
d
7.89 (d, J ¼ 1.9 Hz, 1H, –ArH), 7.83 (dd, J ¼ 8.7 Hz,
Compd.
EGFR Kinase
% Inhibition
Src Kinase
Predicted
DG
% Inhibition
IC50
Predicted DG
at 10
m
M
at 10 mM
3a
3b
3c
3g
3i
3j
3k
3m
30.4%
35.9%
21.0%
20.9%
43.2%
36.8%
34.6%
23.8%
ꢁ6.20
ꢁ6.43
ꢁ7.40
ꢁ7.02
ꢁ7.45
ꢁ5.66
ꢁ7.82
ꢁ6.20
55.4%
51.4%
52.5%
60.5%
76.2%
76.1%
71.0%
50.8%
4.2
6.5
7.4
5.9
3.3
3.5
4.9
9.9
ꢁ7.40
ꢁ8.03
ꢁ8.17
ꢁ7.89
ꢁ7.63
ꢁ7.07
ꢁ8.78
ꢁ10.14
5.2. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-4-methylpentanoate
(3a)
A mixture of compound 1 (100 mg, 0.175 mmol), DCC (43.2 mg,
0.209 mmol), DMAP (3 mg, 0.025 mmol) and Boc–Leu–OH$H2O
(52.4 mg, 0.210 mmol) in THF (15 mL) was stirred at ambient
temperature for 10 h. The mixture was filtered and the filtrate was
purified by flash column chromatography (CH2Cl2/CH3OH) to give
2a. To the formed product (2a) in a mixture of EtOH/dioxane
(10 mL, 3:1) was added 10% palladium on carbon (10 mg), then
the mixture was stirred under H2 atmosphere at ambient temper-
ature for 3 h. The resulting mixture was filtered through Celite,
washed with EtOH and purified by flash column chromatography
solution, and the precipitate was collected. The precipitate was
added to 60 mL EtOH and 9 mL concd. HCl was added in portions.
This reaction mixture was stirred at 70 ꢀC for 2 h. The mixture
was then cooled to ambient temperature, and the precipitate
was filtered and washed with water. The crude product was
Fig. 1. The interaction mechanism between the targets and the two most active ligands. A & B are for compound 3i with EGFR and Src kinase, respectively; C & D are for compound
3j with EGFR and Src kinase, respectively. This image was generated with LIGPLOT program.[23].

H. Huang et al. / European Journal of Medicinal Chemistry 44 (2009) 1982–1988
1987
(CH2Cl2/CH3OH) to give 3a in 39.3% yield. M.p. 85–88 ꢀC; 1H NMR
(DMSO-d6):
13C NMR (DMSO-d6):
d 174.0, 169.6, 165.8, 161.2, 160.3, 156.5, 149.4,
d
7.33 (d, J ¼ 2.1 Hz, 1H, –ArH), 7.30 (dd, J ¼ 8.6 Hz,
146.6, 145.5, 128.7, 120.8, 118.5, 115.3, 115.0, 102.8, 99.3, 94.2, 82.0,
57.3, 30.1, 29.2, 18.9; MS (ESI, m/z): 524.1 [M þ Na]þ. HRMS (ESI):
Calcd. for C25H27NO10Na [M þ Na]þ: 524.1533; Found: 524.16.
2.1 Hz, 1H, –ArH), 6.89 (d, J ¼ 8.6 Hz, 1H, –ArH), 6.49 (d, J ¼ 2.1 Hz,
1H, –ArH), 6.27 (d, J ¼ 2.1 Hz, 1H, –ArH), 4.40–4.29 (m, 1H, –CH),
1.69–1.56 (m, 2H, –CH2), 1.52–1.43 (m, 1H, –CH), 1.37 (s, 9H,
–C(CH3)3), 0.83 (dd, J ¼ 6.7 Hz, 2.3 Hz, 6H, –CH3); 13C NMR (DMSO-
5.7. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-acetamido-4-methylpentanoate (3f)
d6): d 174.1, 169.5, 165.4, 160.6, 160.1,156.4, 149.8,146.3, 145.5, 128.9,
120.4, 119.1, 115.6, 115.0, 102.5, 99.5, 94.0, 81.5, 50.1, 39.2, 29.4, 22.7,
22.2, 21.1; MS (ESI, m/z): 538.2 [M þ Na]þ. HRMS (ESI): Calcd. for
C26H29NO10Na [M þ Na]þ: 538.1689; Found: 538.1680.
The same procedure described for 3a was used starting with Ac–
Leu–OH, Yield 71.7%. M.p. 147–149 ꢀC; 1H NMR (DMSO-d6):
d 7.35 (d,
J ¼ 2.1 Hz, 1H, –ArH), 7.25 (dd, J ¼ 8.6 Hz, 2.1 Hz, 1H, –ArH), 6.88 (d,
J ¼ 8.6 Hz, 1H, –ArH), 6.46 (d, J ¼ 2.1 Hz,1H, –ArH), 6.22 (d, J ¼ 2.1 Hz,
1H, –ArH), 4.64–4.56 (m, 1H, –CH), 1.90 (s, 3H, –CH3), 1.86–1.80 (m,
5.3. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)propanoate (3b)
2H, –CH2), 1.75–1.67 (m, 1H, –CH), 0.91 (d, J ¼ 5.6 Hz, 6H, –CH3); 13
C
The same procedure described for 3a was used starting with
Boc–Ala–OH, Yield 58.2%. M.p. 169–173 ꢀC; 1H NMR (DMSO-d6):
NMR (DMSO-d6): d 174.4, 169.6, 165.8, 160.8, 160.5, 156.5, 149.6,
146.6, 145.6, 129.1, 120.8, 119.0, 115.8, 115.1, 102.8, 99.3, 94.2, 52.7,
39.6, 24.1, 22.7, 22.2, 21.5; MS (ESI, m/z): 480.1 [M þ Na]þ. HRMS
(ESI): Calcd. for C23H23NO9Na [M þ Na]þ: 480.1271; Found: 480.1273.
d
7.36 (d, J ¼ 2.1 Hz,1H, –ArH), 7.31 (dd, J ¼ 8.6 Hz, 2.1 Hz, 1H, –ArH),
6.90 (d, J ¼ 8.6 Hz, 1H, –ArH), 6.48 (d, J ¼ 2.1 Hz, 1H, –ArH), 6.24 (d,
J ¼ 2.1 Hz, 1H, –ArH), 4.40 (q, J ¼ 7.4 Hz, 1H, –CH), 1.39 (s, 9H,
–C(CH3)3), 1.44 (d, J ¼ 7.4 Hz, 3H, –CH3); 13C NMR (DMSO-d6):
5.8. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-acetamido-3-phenylpropanoate (3g)
d
174.8, 169.6, 165.6, 160.6, 160.2, 157.1, 149.5, 147.0, 146.1, 129.6,
120.8, 119.3, 116.0, 115.6, 103.0, 99.3, 94.7, 81.7, 51.0, 29.4, 18.1; MS
(ESI, m/z): 496.0 [M þ Na]þ. HRMS (ESI): Calcd. for C23H23NO10Na
[M þ Na]þ: 496.1220; Found: 496.1234.
The same procedure described for 3a was used starting with Ac–
Phe–OH, Yield 51.9%. M.p. 188–191 ꢀC; 1H NMR (DMSO-d6):
d 7.40–
7.15 (m, 7H, –ArH), 6.93 (d, J ¼ 8.2 Hz, 1H, –ArH), 6.52 (d, J ¼ 2.1 Hz,
1H, –ArH), 6.25 (d, J ¼ 2.1 Hz, 1H, –ArH), 4.62–4.51 (m, 1H, –CH),
3.08–2.86 (m, 2H, –ArCH2), 1.86 (s, 3H, –CH3); 13C NMR (DMSO-d6):
5.4. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-3-methylbutanoate
(3c)
d
174.1, 169.5, 165.7, 160.9, 160.4, 156.9, 149.6, 146.8, 145.3, 143.5,
130.1,129.5, 129.1, 128.8, 128.5, 127.4, 120.5, 118.6,115.8,115.0, 102.4,
99.5, 94.4, 56.1, 37.8, 24.1; MS (ESI, m/z): 514.1 [M þ Na]þ. HRMS
(ESI): Calcd. for C26H21NO9Na [M þ Na]þ: 514.1114; Found: 514.1102.
The same procedure described for 3a was used starting with
Boc–Val–OH, Yield 78.5%. M.p. 174–176 ꢀC; 1H NMR (DMSO-d6):
d
7.35 (d, J ¼ 2.0 Hz,1H, –ArH), 7.32 (dd, J ¼ 7.9 Hz, 2.0 Hz,1H, –ArH),
6.89 (d, J ¼ 7.9 Hz, 1H, –ArH), 6.49 (d, J ¼ 2.0 Hz, 1H, –ArH), 6.27 (d,
J ¼ 2.0 Hz, 1H, –ArH), 3.65 (d, J ¼ 5.9 Hz, 1H, –CH), 1.99–1.88 (m, 1H,
–CH), 1.38 (s, 9H, –C(CH3)3), 0.80 (d, J ¼ 6.5 Hz, 6H, –CH3); 13C NMR
5.9. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-3-phenylpropanoate
(3h)
(DMSO-d6):
d 173.9, 169.3, 165.8, 161.0, 160.5, 156.3, 149.8, 146.9,
145.1, 128.4, 120.8, 118.0, 115.3, 114.9, 102.7, 99.5, 94.2, 81.7, 57.1,
30.3, 29.4, 18.8; MS (ESI, m/z): 524.0 [M þ Na]þ. HRMS (ESI): Calcd.
for C25H27NO10Na [M þ Na]þ: 524.1533; Found: 524.1539.
The same procedure described for 3a was used starting with
Boc–Phe–OH, Yield 31.7%. M.p. 168–171 ꢀC; 1H NMR (DMSO-d6):
d
7.37–7.19 (m, 7H, –ArH), 6.92 (d, J ¼ 8.2 Hz, 1H, –ArH), 6.55 (d,
J ¼ 2.1 Hz,1H, –ArH), 6.30 (d, J ¼ 2.1 Hz,1H, –ArH), 4.60–4.51 (m,1H,
5.5. (R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-4-methylpentanoate
(3d)
–CH), 3.05–2.91 (m, 2H, –ArCH2), 1.30 (s, 9H, –C(CH3)3); 13C NMR
(DMSO-d6):
d 174.3, 169.6, 165.8, 160.8, 160.3, 156.7, 149.6, 146.6,
145.2, 143.5, 130.2, 129.3, 129.0, 128.7, 128.5, 127.5, 120.8, 118.3,
115.9, 115.0, 102.8, 99.3, 94.7, 81.2, 56.0, 38.0, 24.3; MS (ESI, m/z):
572.0 [M þ Na]þ. HRMS (ESI): Calcd. for C29H27NO10Na [M þ Na]þ:
572.1533; Found: 572.1528.
The same procedure described for 3a was used starting with
Boc–D–Leu–OH$H2O, Yield 72.6%. M.p. 134–136 ꢀC; 1H NMR
(DMSO-d6):
d
7.32 (d, J ¼ 2.1 Hz, 1H, –ArH), 7.28 (dd, J ¼ 8.4 Hz,
2.1 Hz, 1H, –ArH), 6.45 (d, J ¼ 8.4 Hz, 1H, –ArH), 6.22 (d, J ¼ 2.1 Hz,
1H, –ArH), 6.27 (d, J ¼ 2.1 Hz, 1H, –ArH), 4.38–4.29 (m, 1H, –CH),
1.66–1.57 (m, 2H, –CH2), 1.54–1.45 (m, 1H, –CH), 1.39 (s, 9H,
–C(CH3)3), 0.91 (dd, J ¼ 6.7 Hz, 2.3 Hz, 6H, –CH3); 13C NMR (DMSO-
5.10. 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-
3-yl 2-(tert-butoxycarbonylamino)acetate (3i)
The same procedure described for 3a was used starting with
Boc–Gly–OH, Yield 58.2%. M.p. 129–132 ꢀC; 1H NMR (DMSO-d6):
d6):
d 174.3, 169.6, 165.5, 160.8, 160.3, 156.5, 149.6, 146.3, 145.6,
128.8, 120.5, 119.0, 115.8, 115.2, 102.5, 99.6, 94.1, 81.7, 50.3, 39.7,
29.6, 22.7, 22.6, 21.4; MS (ESI, m/z): 538.1 [M þ Na]þ. HRMS (ESI):
Calcd. for C26H29NO10Na [M þ Na]þ: 538.1689; Found: 538.1668.
d
7.65 (d, J ¼ 2.4 Hz, 1H, –ArH), 7.53 (dd, J ¼ 8.6 Hz, 2.4 Hz, 1H,
–ArH), 6.91 (d, J ¼ 8.6 Hz, 1H, –ArH), 6.48 (d, J ¼ 2.4 Hz, 1H, –ArH),
6.22 (d, J ¼ 2.4 Hz, 1H, –ArH), 3.14 (s, 2H, –CH2), 1.21 (s, 9H,
–C(CH3)3); 13C NMR (DMSO-d6):
d 174.4, 169.6, 165.2, 160.8, 160.3,
5.6. (R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-3-methylbutanoate
(3e)
156.5, 149.8, 146.5, 145.6, 128.7, 120.5, 119.0, 115.7, 115.1, 102.6, 99.3,
94.3, 80.8, 42.7, 28.8; MS (ESI, m/z): 482.1 [M þ Na]þ. HRMS (ESI):
Calcd. for C22H21NO10Na [M þ Na]þ: 482.1063; Found: 482.1052.
The same procedure described for 3a was used starting with
Boc–D–Val–OH, Yield 76.3%. M.p. 159–162 ꢀC; 1H NMR (DMSO-d6):
5.11. (2S,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-3-hydroxybutanoate
(3j)
d
7.34 (d, J ¼ 2.0 Hz, 1H, –ArH), 7.30 (dd, J ¼ 8.3 Hz, 2.0 Hz, 1H,
–ArH), 6.87 (d, J ¼ 8.3 Hz, 1H, –ArH), 6.45 (d, J ¼ 2.0 Hz, 1H, –ArH),
6.22 (d, J ¼ 2.0 Hz,1H, –ArH), 4.23 (d, J ¼ 6.0 Hz,1H, –CH), 2.32–2.20
(m, 1H, –CH), 1.40 (s, 9H, –C(CH3)3), 0.98 (d, J ¼ 6.7 Hz, 6H, –CH3);
The same procedure described for 3awas used starting with Boc–
Thr–OH, Yield 63.9%. M.p. 183–187 ꢀC; 1H NMR (DMSO-d6):
d 7.36

1988
H. Huang et al. / European Journal of Medicinal Chemistry 44 (2009) 1982–1988
(d, J ¼ 2.1 Hz, 1H, –ArH), 7.33 (dd, J ¼ 8.6 Hz, 2.1 Hz, 1H, –ArH), 6.84
(d, J ¼ 8.6 Hz, 1H, –ArH), 6.37 (d, J ¼ 2.1 Hz, 1H, –ArH), 6.14 (d,
J ¼ 2.1 Hz,1H, –ArH), 4.38–4.32 (m,1H, –CH), 4.28–4.20 (m,1H, –CH),
3.99 (s, 1H,, –OH), 1.40 (s, 9H, –C(CH3)3), 1.14 (d, 3H, –CH3); 13C NMR
–CH2), 2.05–1.73 (m, 4H, –CH2CH2), 1.41 (s, 9H, –C(CH3)3); 13C NMR
(DMSO-d6): 172.9, 169.4, 165.5, 160.7, 160.5, 156.7, 149.3, 146.6,
145.8, 129.0, 120.4, 119.0, 116.2, 115.4, 102.3, 99.1, 94.0, 80.3, 61.8,
46.7, 29.9, 24.5, 22.5; MS (ESI, m/z): 522.2 [M þ Na]þ. HRMS (ESI):
Calcd. for C25H25N2O10Na [M þ Na]þ: 522.1376; Found: 522.1367.
d
(DMSO-d6):
d 174.5, 169.6, 165.6, 160.7, 160.3, 156.6, 149.7, 146.6,
145.3,129.1,120.6,118.8,115.8,115.1,102.6, 99.3, 94.0, 82.0, 68.3, 60.5,
29.3, 20.1; MS (ESI, m/z): 526.1 [M þ Na]þ. HRMS (ESI): Calcd. for
C24H25NO11Na [M þ Na]þ: 526.1304; Found: 526.1325.
5.16. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 1-acetylpyrrolidine-2-carboxylate (3o)
5.12. (2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-3-hydroxybutanoate
(3k)
The same procedure described for 3a was used starting with Ac–
Pro–OH, Yield 63.7%. M.p. 161–164 ꢀC; 1H NMR (DMSO-d6):
d 7.40
(d, J ¼ 2.1 Hz, 1H, –ArH), 7.34 (dd, J ¼ 8.4 Hz, 2.1 Hz, 1H, –ArH), 6.90
(d, J ¼ 8.4 Hz, 1H, –ArH), 6.54 (d, J ¼ 2.2 Hz, 1H, –ArH), 6.28 (d,
J ¼ 2.2 Hz, 1H, –ArH), 4.70–4.59 (m, 1H, –CH), 3.67–3.56 (m, 2H,
–CH2), 2.35–2.25 (m, 4H, –CH2CH2), 2.04 (s, 3H, –CH3); 13C NMR
The same procedure described for 3a was used starting with
Boc–D–Trp–OH, Yield 25.4%. M.p. 158–160 ꢀC; 1H NMR (DMSO-d6):
d
7.38 (d, J ¼ 2.1 Hz,1H, –ArH), 7.34 (dd, J ¼ 8.6 Hz, 2.1 Hz,1H, –ArH),
(DMSO-d6):
d 173.1, 169.6, 165.5, 160.8, 160.6, 156.5, 149.4, 146.8,
6.89 (d, J ¼ 8.6 Hz, 1H, –ArH), 6.45 (d, J ¼ 2.1 Hz, 1H, –ArH), 6.30 (d,
J ¼ 8.9 Hz, 1H, –CONH), 6.20 (d, J ¼ 2.1 Hz, 1H, –ArH), 4.08–4.02 (m,
1H, –CH), 3.88 (dd, J ¼ 9.2 Hz, 3.3 Hz, 1H, –CH), 1.40 (s, 9H,
–C(CH3)3), 1.09 (d, J ¼ 6.2 Hz, 3H, –CH3); 13C NMR (DMSO-d6):
145.7, 129.1, 120.8, 119.0, 115.9, 115.6, 102.3, 99.3, 94.1, 80.1, 61.6,
46.5, 29.3, 24.5, 22.7; MS (ESI, m/z): 464.2 [M þ Na]þ. HRMS (ESI):
Calcd. for C22H19NO9Na [M þ Na]þ: 464.0958; Found: 464.0946.
d
174.4, 169.6, 165.7, 160.8, 160.3, 156.5, 149.8, 146.6, 145.2, 129.1,
Acknowledgments
120.8, 119.0,115.9,115.0,102.8, 99.2, 94.2, 82.0, 68.5, 61.0, 29.4, 20.1;
MS (ESI, m/z): 526.2 [M þ Na]þ. HRMS (ESI): Calcd. for
C24H25NNaO11 [M þ Na]þ: 526.1325; Found: 526.1321.
We gratefully acknowledge financial support from the State Key
Program of Basic Research of China (Grant 2006BAI01B02), the
National Natural Science Foundation of China (Grants 20721003
and 20872153), the 863 Hi-Tech Program of China (Grants
2006AA020602, 2006AA02Z336), and 973 program (Grant
2004CB518901).
5.13. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 2-(tert-butoxycarbonylamino)-3-(1H-indol-3-
yl)propanoate (3l)
The same procedure described for 3a was used starting with
Boc–Trp–OH, Yield 65.4%. M.p. 176–178 ꢀC; 1H NMR (DMSO-d6):
Appendix. Supplementary material
d
7.41–7.29 (m, 3H, –ArH), 7.19 (s, 1H, –ArH), 7.14–6.97 (m, 3H,
Supplementary material associated with this article can be
found in the online version, at doi:10.1016/j.ejmech.2008.09.051.
–ArH), 6.87 (d, J ¼ 8.0 Hz, 1H, –ArH), 6.46 (d, J ¼ 2.1 Hz, 1H, –ArH),
6.23 (d, J ¼ 2.1 Hz, 1H, –ArH), 4.59–4.50 (m, 1H, –CH), 3.19–3.07 (m,
2H, –CH2), 1.31 (s, 9H, –C(CH3)3); 13C NMR (DMSO-d6):
d 175.0,
References
168.9, 165.5, 160.8, 160.2, 156.7, 149.6, 146.8, 145.3, 137.8, 129.3,
128.0, 122.7, 122.5, 121.0, 120.0, 119.4, 118.3, 115.5, 115.1, 112.2, 110.7,
102.6, 99.1, 94.2, 79.8, 56.0, 31.2, 28.2; MS (ESI, m/z): 611.1
[M þ Na]þ. HRMS (ESI): Calcd. for C31H28N2O10Na [M þ Na]þ:
611.1642; Found: 611.1638.
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5.14. (S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl 4-amino-2-(tert-butoxycarbonylamino)-4-
oxobutanoate (3m)
The same procedure described for 3a was used starting with
Boc–Asn–OH, Yield 42.8%. M.p. 187–189 ꢀC; 1H NMR (DMSO-d6):
d
7.45–7.30 (m, 2H, –ArH), 6.85 (d, J ¼ 8.6 Hz, 1H, –ArH), 6.44 (d,
J ¼ 2.1 Hz,1H, –ArH), 6.21 (d, J ¼ 2.1 Hz,1H, –ArH), 4.66–4.57 (m,1H,
–CH), 2.99–2.76 (m, 2H, –CH2), 1.39 (s, 9H, –C(CH3)3); 13C NMR
(DMSO-d6):
d 175.0, 173.0, 169.8, 166.0, 160.8, 160.3, 156.8, 149.4,
146.6, 145.2, 129.5, 120.9, 119.3, 116.0, 115.1, 102.7, 99.1, 94.2, 80.1,
53.1, 37.9, 28.5; MS (ESI, m/z): 539.1 [M þ Na]þ. HRMS (ESI): Calcd.
for C24H24N2NaO11 [M þ Na]þ: 539.1278; Found: 539.1263.
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chromen-3-yl 1-tert-butoxycarbonylpyrrolidine-2-carboxylate (3n)
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The same procedure described for 3a was used starting with
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d
7.37 (d, J ¼ 2.1 Hz, 1H, –ArH), 7.24 (dd, J ¼ 8.3 Hz, 2.1 Hz,1H, –ArH),
6.91 (d, J ¼ 8.3 Hz, 1H, –ArH), 6.52 (d, J ¼ 2.1 Hz, 1H, –ArH), 6.27 (d,
J ¼ 2.1 Hz, 1H, –ArH), 4.65–4.54 (m, 1H, –CH), 3.37–3.25 (m, 2H,
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