Potent and Nontoxic Chemosensitizer of P-Glycoprotein-Mediated Multidrug Resistance in Cancer: Synthesis and Evaluation of Methylated Epigallocatechin, Gallocatechin, and Dihydromyricetin Derivatives

Article abstract of DOI:10.1021/acs.jmedchem.5b00085

We are interested in developing novel natural product-derived P-gp inhibitors to reverse cancer drug resistance. Here, we have synthesized 55 novel derivatives of methylated epigallocatechin (EGC), gallocatechin (GC), and dihydromyricetin (DHM). Three EGC derivatives (23, 35, and 36) and three GC derivatives (50, 51, and 53) are significantly better than epigallocatechin gallate (EGCG) with a relative fold (RF) ranging from 31.4 to 53.6. The effective concentration (EC₅₀) of 23 and 51 ranges from 102 to 195 nM. Compounds 23 and 51 are noncytotoxic to fibroblasts with IC₅₀ > 100 μM. Compound 23 is specific for P-gp without modulating activity toward MRP1 or BCRP. Compounds 23 and 51 are non-P-gp substrates. Important pharmacophores for P-gp modulation were identified. In summary, methylated EGC and GC derivatives represent a new class of potent, specific, noncytotoxic, and nonsubstrate P-gp modulators.

Full text of DOI:10.1021/acs.jmedchem.5b00085

Article
pubs.acs.org/jmc
Potent and Nontoxic Chemosensitizer of P‑Glycoprotein-Mediated
Multidrug Resistance in Cancer: Synthesis and Evaluation of Methylated
Epigallocatechin, Gallocatechin, and Dihydromyricetin Derivatives
Iris L. K. Wong,†,§ Bao-Chao Wang,‡,§ Jian Yuan,‡ Liang-Xing Duan,‡ Zhen Liu,‡ Tao Liu,‡ Xue-Min Li,‡
Xuesen Hu,† Xiao-Yu Zhang,‡ Tao Jiang,‡ Sheng-Biao Wan,*,‡ and Larry M. C. Chow*,†
†Department of Applied Biology and Chemical Technology, and State Key Laboratory of Chirosciences, The Hong Kong Polytechnic
University, Hung Hom, Hong Kong SAR, China
‡Key Laboratory of Marine Drugs, Ministry of Education, Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology,
School of Medicine and Pharmacy, Ocean University of China, Qingdao, China
S
* Supporting Information
ABSTRACT: We are interested in developing novel natural product-derived P-gp inhib-
itors to reverse cancer drug resistance. Here, we have synthesized 55 novel derivatives of
methylated epigallocatechin (EGC), gallocatechin (GC), and dihydromyricetin (DHM).
Three EGC derivatives (23, 35, and 36) and three GC derivatives (50, 51, and 53) are
significantly better than epigallocatechin gallate (EGCG) with a relative fold (RF) rang-
ing from 31.4 to 53.6. The effective concentration (EC50) of 23 and 51 ranges from 102
to 195 nM. Compounds 23 and 51 are noncytotoxic to fibroblasts with IC50 > 100 μM.
Compound 23 is specific for P-gp without modulating activity toward MRP1 or BCRP.
Compounds 23 and 51 are non-P-gp substrates. Important pharmacophores for P-gp
modulation were identified. In summary, methylated EGC and GC derivatives represent a
new class of potent, specific, noncytotoxic, and nonsubstrate P-gp modulators.
of them have been used in clinical trials but without much
success.21,30 There is an urgent need to discover new non-
cytotoxic and potent P-gp modulators.
INTRODUCTION
■
Multidrug resistance (MDR) is a major clinical problem in
cancer chemotherapy. One important cause of MDR is the
overexpression of the ATP-binding cassette (ABC) transporters
which can efflux different kinds of drugs across the cell mem-
brane.1−3 Fifteen ABC transporters have been reported to extrude
anticancer drugs in vitro.4,5 Among them, P-glycoprotein (P-gp;
MDR1; ABCB1) has been widely studied and is known to be
the major contributor to clinical MDR.2−4 Modulation of P-gp
can potentially reverse clinical MDR.2,4−8
P-gp was the first identified and best characterized mam-
malian ABC protein.9−11 In the past two decades, considerable
effort has been spent in the discovery of P-gp modulators. The
first-generation P-gp inhibitors, such as the calcium channel
blocker verapamil12,13 and immunosuppressive agent cyclo-
sporin A,14,15 were found to be competitive inhibitors.2−5,16
Their low potency, however, precluded them from further de-
velopment for clinical use. Some of the first generation
modulators, e.g., verapamil, have side effects and were
undesirable for clinical use. Second generation P-gp modulators
include dexverapamil,17 dexniguldipine,18 and valspodar (an
analogue of cyclosporin A; also known as PSC833).19,20
Although they were less toxic and more potent, their uses
were limited by their unpredictable pharmacokinetic inter-
actions with the anticancer drugs.2−5,21 Third generation P-gp
modulators including tariquidar,22−24 elacridar,25,26 zosuquidar,27,28
and laniquidar29 were potent and safe in in vitro studies. Some
Noncytotoxic natural phenolic compounds like flavo-
noids,31−38 catechins,39−41 curcumin,42 lamallarines,43 and
ningalins44−47 are potential candidates for good MDR
modulators.35,48 We have previously demonstrated that
methylation can improve the P-gp modulating activity of
flavonoid. Examples include methylated quercetin with side
chain modifications (compounds 1 and 2 shown in Chart 1)36
and permethyl ningalin B and its synthetic analogues (com-
pounds 3 and 4 shown in Chart 1).49,50 They displayed sig-
nificantly better P-gp modulating activity than the parental
compounds. These results suggest that the methylation of poly-
phenolic compounds is a reasonable and important modifica-
tion to improve P-gp modulating activity. We also found that
methylated quercetin derivatives containing O-3 substituents
(compounds 5, 6, and 7 shown in Chart 1) exhibited promising
P-gp- and/or BCRP-modulating activity in cancer cell lines
and did not possess any inherent cytotoxicity toward cancer or
normal mouse fibroblast cells.51 We also found that permethyl
epigallocatechin gallate (permethyl EGCG, shown in Chart 1)
is a good P-gp inhibitor.52 Herein, we report the design, synthesis,
and evaluation of a novel series methylated epigallocatechin (EGC),
Received: January 16, 2015
© XXXX American Chemical Society
A
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Chart 1. P-gp Inhibitors of Methylated Phenolic Compounds
methylated gallocatechin (GC), and methylated dihydromyricetin
(DHM) derivatives in reversing P-gp-mediated drug resis-
tance.35,36,48−50
Synthesis of this series of methylated GC derivatives with
the 2R,3S configuration is shown in Scheme 3. The Mitsunobu
method was adopted in the configuration inversion at C3,
which is more efficient than that reported.53 (−)-(2R,3R)-5,7-
Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-ol 8 (pen-
tamethyl EGC)54 was reacted with 3,4,5-trimethoxybenzoic acid
in the presence of PPh3 and DIAD to produce (+)-(2R,3S)-5,7-
dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl 3,4,5-trime-
thoxybenzoate 43 (permethyl GCG). Hydrolysis of permethyl
GCG 43 gave (−)-(2R, 3S)-5,7-dimethoxy-2-(3,4,5-
trimethoxyphenyl)chroman-3-ol 44 (pentamethyl GC). Pen-
tamethyl GC 44 was reacted with 4-methoxybenzoic acid or
3,4-dimethoxybenzoic acid catalyzed by EDCI to give the target
compounds 45 or 46, respectively. Similarly, target compounds
47−53 were obtained using pentamethyl GC 44 as the starting
material.
To investigate analogues of permethyl EGCG with a similar
scaffold, a series of methylated dihydromyricetin55 derivatives
with a chromanone scaffold was synthesized as shown in
Scheme 4. Pentamethyl dihydromyricetin 54 was acetylated to
provide 55. Pentamethyl dihydromyricetin 54 was reacted with
4-methoxybenzoic acid, 3,4-dimethoxybenzoic acid, 3,4,5-
trimethoxybezoic acid, or 3,4,5-triallyloxybezoic acid catalyzed
by EDCI to give target compounds 56−59, respectively. Using
a similar method, target compounds 60−66 were obtained.
RESULTS AND DISCUSSION
■
Chemistry. A series of methylated EGC derivatives with
various substituents at the C3 position were synthesized
using the synthetic route shown in Scheme 1. Pentamethyl
epigallocatechin 8 was reacted with methoxy-, ethoxy-, allyoxy-
fluoro-, or acetamido-substituted benzoic acid catalyzed by EDCI
to give the target compounds 9−17, respectively, with 80−88%
yield. Similarly, compounds 18−23 were also prepared from
compound 8 with 81−90% yield. Hydrogenation of compounds
21, 22, and 23 afforded compounds 24, 25, and 26, respectively.
Seven methylated EGC derivatives with various substituents
at C3, containing a 3-amino-4-fluorobenzoyloxy linker, were syn-
thesized as shown in Scheme 2. Coupling of methyl 3-amino-4-
fluorobenzoate 27 with (E)-3-(3,4-dimethoxyphenyl)acrylic
acid or (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, followed
by hydrolysis, provided intermediates 28 and 29, respectively.
Using a similar method, intermediates 32−34 and 37−39 were
prepared from methyl 3-amino-4-fluorobenzoate and 3,4,5-
trimethoxybenzoic acid, 3,4-dimethoxybenzoic acid, or 4-
methoxybenzoic acid, respectively. Catalyzed by EDCI,
pentamethyl (−)-EGC 8 was reacted either with compounds
28 and 29 to generate the target compounds 30 and 31 or with
compounds 33 and 34 to give 35 and 36 or with compounds
37, 38, and 39 to produce 40, 41, and 42, respectively.
BIOLOGICAL EVALUATION
■
Structure−Activity Relationship Study of the P-gp
Modulating Activity of Methylated EGC, Methylated GC,
and Methylated DHM Derivatives. In this study, the P-gp-
transfected breast cancer cell line (MDA435/LCC6MDR) and
To investigate the importance of the chiral center C3, a series
of methylated GC derivatives were made which have 2R,3S
configuration instead of the 2R,3R in EGC derivatives.
B
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a
Scheme 1. Synthetic Route of Compounds 9−26
a
Reagents and conditions: (a) EDCI/DMAP/DCM, rt; (b) EDCI/DMAP/DCM, 4-methoxyphenylacetic acid, 3,4-dimethoxyphenylacetic acid,
3,4,5-trimethoxyphenylacetic acid, rt; (c) EDCI/DMAP/DCM, (E)-3-(4-methoxyphenyl) acrylic acid, (E)-3-(3,4-dimethoxyphenyl)acrylic acid, or
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, rt; (d) Pd/C, H , CH3OH, rt.
2
a
Scheme 2. Synthetic Route of Compounds 28−42
a
Reagents and conditions: (a) DMAP/DCM, (E)-3-(3,4-dimethoxyphenyl)acryloyl chloride or (E)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride, rt;
(b) LiOH, MeOH/H2O, rt; (c) DMAP/EDCI/DCM, 4-methoxybenzoic acid, 3,4-dimethoxybenzoic acid, or 3,4,5-trimethoxybenzoic acid, rt; (d)
DMAP/EDCI/DCM, methyl 3-amino-4-fluorobenzoate, rt; (e) DMAP/EDCI/DCM, compound 8.
its parent (MDA435/LCC6) were used. LCC6MDR cells were
about 90.4-fold more resistant to paclitaxel (PTX) than the
parental LCC6 cells (Table 1). P-gp modulating activity of
compounds was evaluated by a parameter known as relative
C
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a
Scheme 3. Synthetic Route of Compounds 43−53
a
Reagents and conditions: (a) Ph3P/DIAD, 0 °C−rt; (b) K2CO3/MeOH/DME, rt; (c) DMAP/EDCI/DCM, 3,4-dimethoxybenzoic acid, or
3,4,5-trimethoxybenzoic acid, rt; (d) EDCI/DMAP/DCM, (E)-3-(4-methoxyphenyl)acrylic acid, (E)-3-(3,4-dimethoxyphenyl)acrylic acid, or
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, rt; (e) EDCI/DMAP/DCM, compounds 33 or 34, rt; (f) EDCI/DMAP/DCM, compounds 28 or 29, rt.
a
Scheme 4. Synthetic Route of Compounds 55−66
a
Reagents and conditions: (a) Ac2O/Py; (b) DMAP/EDCI/DCM, 4-methoxybenzoic acid, 3,4-dimethoxybenzoic acid, 3,4,5-trimethoxybenzoic
acid, or 3,4,5-triallyloxybenzoic acid, rt; (d) EDCI/DMAP/DCM, (E)-3-(4-methoxyphenyl)acrylic acid, (E)-3-(3,4-dimethoxyphenyl)acrylic acid, or
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, rt; (e) EDCI/DMAP/DCM, compounds 33 or 34, rt; (f) EDCI/DMAP/DCM, compounds 28 or 29, rt.
fold (RF), which is defined as the ratio of IC50 toward PTX
in LCC6MDR cells in the absence of 1 μM of modulators
relative to that with modulator. Higher RF means better P-gp
modulating activity (Table 1). Verapamil, a first-generation
P-gp modulator, displayed a moderate activity with a RF of 3.8
(Table 1).
D
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Table 1. Cytotoxicity, P-gp-Modulating Activity, Calculated LogP, and Calculated PSA of Methylated EGC, Methylated GC, and
Methylated Dihydromyricetin Derivatives*
E
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A total of 55 methylated EGC, methylated GC, and methylated
DHM derivatives were synthesized. They were divided into 12
series and studied for their P-gp modulating activity. These new
derivatives were designed by (1) making substitutions at ring D, (2)
changing linker length and rigidity between rings C and D at the C3
position, and (3) using various scaffolds with stereoselectivity.
Substituents at Phenyl Ring D of Methylated EGC
Derivatives. In series I, EGCG (RF = 1.2) was a weak P-gp
Table 1. continued
F
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modulator. Even at the highest concentration tested (10 μM),
the RF of EGCG remained 1.2. Peracetylation of EGCG at all
OH groups of A, B, and D rings (peracetyl EGCG) showed no
improvement (RF = 0.8). In contrast, permethylation of EGCG
(permethyl EGCG; grouped in series II) resulted in significant
improvement to RF = 6.9, suggesting that methoxy rather than
acetyl groups on rings A, B, and D are preferred. Removal
of ring D from permethyl EGCG abolished all activity in 8
(RF = 0.9), suggesting that ring D is critical.
derivatives have ring D attached to C3 via an oxycarbonyl
linker. Monosubstituted compounds with either methoxy at the
meta-position (compound 9 with RF = 3.2) or para-position
(compound 10 with RF = 1.0), or fluoro at the meta-position
(compound 14 with RF = 1.5) or para-position (compound 15
with RF = 2.7) yielded relatively weak P-gp modulators.
Disubstitution at meta- and para-positions in general yielded
better P-gp modulators. These include compound 11 with two
methoxys at the meta- and para-positions (RF = 7.4), com-
pound 16 with a para-fluoro and meso-dimethylamino (RF = 4.0),
or compound 17 with a para-fluoro and meso-acetylamino at
In view of the critical importance of ring D, we investigated
the effect of substituents in ring D. In series II, all EGC
Table 1. continued
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Table 1. continued
*
Methylated EGC, GC, and DHM derivatives are divided into 12 series with their R group indicated in the Table. P-gp modulating activity was
measured by determining IC50 toward PTX in LCC6MDR cells with or without 1.0 μM of the modulator. RF reflects P-gp modulating activity and is
calculated as [IC50 of PTX without modulator/IC50 with 1.0 μM modulator]. All modulators were dissolved in DMSO and used at 1 μM
concentration unless indicated otherwise. Each experiment was repeated at least three times independently, and the IC50 value is presented as the
mean standard error of the mean. In each experiment, each concentration of PTX with or without modulators was done in triplicate. The
a
cytotoxicity of these modulators toward LCC6, LCC6MDR, and normal fibroblast L929 cells was determined. RF values of compounds 5, 6, and 7
b
have been reported. Cytotoxicity of PTX alone toward LCC6 and LCC6MDR in the absence of modulators was determined. Verapamil, a first
generation P-gp inhibitor, was included for comparison. L929: mouse fibroblasts. cLogP and PSA were calculated as described in Experimental
Section. /: Not determined.
ring D (RF = 9.8). Addition of the third methoxy (permethyl
EGCG with RF = 6.9) cannot further improve the activity of
compound 11 (RF = 7.4), which already has disubstitution.
Trisubstitution with other functional groups yields the fol-
lowing compounds with the rank order of P-gp modulating
activity: allyloxy (compound 13; RF = 12.1) > methoxy
(permethyl EGCG; RF = 6.9) ≫ ethoxy (compound 12;
RF = 1.3). We have previously observed a similar structural require-
ment in the study of ningalin B analogues.49 The above results
suggest that tri- and disubstitutions are in general better than
monosubstitution in ring D, and allyloxy or acetyl amino at ring
D may improve the P-gp modulating activity of methylated
EGC derivatives.
Linker between C3 and Ring D of Methylated EGC
Derivatives. As ring D was critical to the P-gp modulating
activity of methylated derivatives, we investigated the structural
requirements for the linker between rings D and C3. All linkers
used were attached to C3 via an oxycarbonyl group. Each series
has either one, two, or three methoxy groups at the meta- or
para-position of ring D. They were oxycarbonyl (series II: 9, 11,
and permethyl EGCG), oxycarbonylmethylene (series III:
18, 19, and 20), oxycarbonylvinyl (series IV: 21, 22, and 23),
oxycarbonylethylene (series V: 24, 25, and 26), oxycarbonyl-
phenylcarbamoylvinyl (series VI: 30 and 31), and oxy-
carbonylphenylcarbamoyl (series VII: 35 and 36).
First, we found that di- or trimethoxy groups at ring D
generally displayed stronger P-gp modulating activity than
those with the monomethoxy group irrespective of the linkers
used. Second, we found that linker length did not have a sig-
nificant effect on P-gp modulating activity. Activity was equally
mild when the length of the flexible linkers was increased from
oxycarbonyl (series II: 9, 11, and permethyl EGCG with RF =
3.2, 7.4, and 6.9) to oxycarbonylmethylene (series III: 18, 19,
and 20 with RF = 4.2. 4.5, and 5.8) and to oxycarbonylethylene
(series V: 24, 25, and 26 with RF = 3.6, 7.6, and 8.0). Third,
when the flexible oxycarbonylethylene linker (series V: 24, 25,
and 26 with RF = 3.6, 7.6, and 8.0) was replaced with a rigid
oxycarbonylvinyl linker of the same length (series IV: 21, 22,
and 23 with RF = 6.6, 16.6, and 39.1), P-gp modulating activity
was significantly improved. The requirement of a rigid linker
was further exemplified when the rigid oxycarbonylphenylcar-
bamoyl linker was used and found to give the most potent
activity (series VII: 35 and 36 with RF = 48.2 and 43.8).
However, the advantage of a rigid linker was compromised
when the length of the rigid linker was further increased
from oxycarbonylphenylcarbamoyl (series VII: 35 and 36 with
RF = 48.2 and 43.8) to oxycarbonylphenylcarbamoylvinyl
(series VI: 30 and 31 with RF = 17.9 and 23.3) to the longest
oxycarbonylphenylcarbamoylphenylcarbamoyl linker (series
VIII: 40, 41, and 42 with RF = 9.2, 10.6, and 10.8). This
H
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Figure 1. Comparison of cLogP or PSA with the RF values of methylated EGC, methylated GC, and methylated dihydromyricetin derivatives. (A)
RF versus cLogP, (B) RF versus PSA, and (C) cLogP versus PSA. RF > 30 is in red, RF = 20−30 is in green, RF = 10−20 is in blue, and RF <10 is in
black in (C).
result suggests that having a rigid linker between rings D and
C3 is important for the P-gp modulating activity of these
methylated EGC analogues. The optimal linker length was
between 7.75 and 13.37 Å, which corresponds to the length of
oxycarbonylvinyl and oxycarbonylphenylcarbamoyl.
RF = 31.4 and 53.6) have the same linker (oxycarbonylphe-
nylcarbamoyl) as methylated EGC derivatives (35 and 36 with
RF = 48.2 and 43.8), but the methylated GC derivatives prefer
trimethoxy ring D, whereas the methylated EGC derivative
slightly prefer dimethoxy ring D. Overall, the activity of GC
derivatives was in general slightly better than that of EGC
derivatives. Another interesting observation is that dimethoxy
or trimethoxy substitutions in ring D were always preferred
over monomethoxy substitution in EGC derivatives; however,
it was not the case in GC derivatives. Compound 43 with
trimethoxy substitution in ring D (RF = 3.0) was equally
inactive as monomethoxyl 45 (RF = 2.2) and much less active
than dimethoxyl substituted 46 (RF = 12.1). Overall, the
pharmacophore for the GC derivative is similar to that of EGC
derivatives with GC being slightly more potent.
Chromanone Scaffold of Methylated Dihydromyricetin
(DHM) Derivatives. Here, we studied the structure−activity
relationship of the P-gp modulating activity in methylated
dihydromyricetin (DHM). DHM belongs to flavonolol (3-hydroxy-
2,3-dihydro-2-phenylchromen-4-one). We have previously reported
that methylated quercetin derivatives with a chromene scaffold have
potent P-gp and BCRP modulating activities.51 As shown in
Chart 1 and Table 1, compounds 5 (RF = 2.1) and 7 (RF =
11.2) (quercetin derivatives with a chromene scaffold) possess
weaker P-gp inhibitory activity than cis-methylated EGC
derivatives containing a chromanol scaffold, permethyl EGCG
(RF = 6.9), and 23 (RF = 39.1). However, trans-methylated
GC derivatives, 43 (RF = 3.0) and 49 (RF = 12.5), showed
P-gp modulating activity similar to that of compounds 5 and 7
even though they also have the chromanol scaffold. There is no
particular rule governing the structural requirement for
quercetin derivatives and methylated DHM derivatives in
terms of their P-gp modulating activity.
Chiral Configuration at C3 of Methylated GC Derivatives.
EGC and GC differ from each other at how ring D attaches to
the chiral center C3. In EGC, ring D is in cis-configuration with
ring B (2R,3R) whereas in GC, ring D is in trans-configuration
(2R,3S). After testing the EGC series above, we studied the GC
series here (series IX and X). Each series has one, two, or three
methoxy groups at the meta- or para-position in ring D. All
linkers used were attached to C3 via oxycarbonyl groups. Ring
D was important in GC derivatives as compound 44 only has
OH at C3, and it displayed no P-gp modulating activity (RF = 1.1),
whereas all other GC derivatives with different forms of ring D
(compounds 43 and 45−53) all have higher levels of P-gp
modulating activity (RF = 2.2 to 53.6).
We then investigated the effect of linker length and rigidity
on P-gp modulating activity. Similar to EGC derivatives, we
found that rigid linkers were more desirable than flexible
linkers. In general, when the short and flexible oxycarbonyl
linker was used (45, 46, and 43 with RF = 2.2, 12.1, and 3.0),
their P-gp modulating activity was weaker than the longer and
more rigid linkers (47−53 with RF = 7.8 to 53.6). Among the
rigid linkers (47 to 53), P-gp modulating activity improved
when the rigid linker length was increased from oxy-
carbonyvinyl (47, 48, and 49 with RF = 7.8, 12.3, and 12.5)
to oxycarbonylphenylcarbamoyl (50 and 51 with RF = 31.4
and 53.6) but further increase in length resulted in a drop of
activity in oxycarbonylphenylcarbamoylvinyl (52 and 53 with
RF = 21.9 and 34.4). This requirement for linker rigidity with
optimal linker length was identical to that observed in EGC
derivatives.
A group of methylated DHM derivatives with a chromanone
scaffold similar to that of permethyl EGCG (series XI and XII
of Table 1) were studied. Linkers with different lengths were
introduced into DMH derivatives and compared for their P-gp
When GC derivatives were compared with EGC derivatives
with the same linker and substituents in ring D, we found
that the best two methylated GC derivatives (50 and 51 with
I
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Table 2. Effective Concentration EC50 (nM) of Methylated EGC and Methylated GC Derivatives for Reversing the Multidrug
a
Resistance of LCC6MDR
cytotoxicity toward
EC50 (nM) for reversing drug resistance of LCC6MDR
Cpds
L929 (IC50, μM)
PTX
vinblastine
vincristine
167 47
DOX
selective index
22
>100
>100
>100
>100
>100
>100
>100
>100
157 13
127 30
159 23
214 25
171 11
123 19
195 50
193 29
/
160 30
130 24
139 48
/
>638
>786
>698
>467
>585
>714
>357
>444
200
23
123
181
/
2
7
35
36
50
173 12
147 19
208 81
51
140
280 30
225
446 41
32
0
108
/
4
102
/
6
107
/
4
52
53
5
/
/
/
verapamil
cyclosporin A
89
30
8
6
503 92
/
385 35
/
245 23
43
1
4
934
a
EC50 values were presented as the mean standard error of the mean. N = 3−4 independent experiments. Each experiment was done in triplicate.
Verapamil and cyclosporin A were used as positive controls. Selective index value = (IC50 of modulators toward L929 fibroblasts)/(EC50 of
modulators for reversing PTX resistance in LCC6MDR cells). /: not determined.
modulating activity. These linkers include oxycarbonyl (56−59),
oxycarbonylvinyl (60−62), oxycarbonylphenylcarbamoyl (63−64),
and oxycarbonylphenylcarbamoylvinyl (65−66). In contrast to
methylated EGC and GC derivatives, no obvious linker length
effect was observed among these dihydromyricetin derivatives.
Overall, they showed moderate P-gp modulating activity with
RF values ranging from 3.0 to 15.4. This result suggests that the
chromanol scaffold of permethyl EGCG derivatives, rather than
the chromanone scaffold of methylated dihydromyricetin
derivatives, is important in conferring P-gp modulating activity.
It was reported that P-gp substrates are usually amphipathic
and lipid soluble. Fifty-two percent of P-gp substrates have a
topological polar surface area (tPSA) over 90 Å2.56 LogP and
the polar surface area (PSA) may reflect the P-gp modulating
activity.49,57 Here, we have calculated the PSA and cLogP value
of the 55 compounds (Figure 1 and Table 1). Most compounds
have cLogP values of 3.8−7.0 and PSA values of 65.3−191.7 Å2.
For the potent P-gp modulators (RF > 20), the PSA values fall
between 108.9 and 169.0 Å2, while cLogP values fall between
4.7−6.4.
EC50 and Selective Index Values of Methylated EGC
and Methylated GC Derivatives for Reversing Multidrug
Resistance in LCC6MDR. We have chosen four potent
methylated EGC derivatives (compounds 22, 23, 35, and 36)
and four potent methylated GC derivatives (compounds
50−53) for further characterization in terms of their effective
concentration (EC50) in reversing P-gp and their selective index
(Table 2). All eight compounds were noncytotoxic toward
L929 normal fibroblasts with IC50 greater than 100 μM (Table 2).
Their EC50 for reversing P-gp mediated resistance toward PTX,
vinblastine, vincristine, and DOX resistance in LCC6MDR cells
ranged from 102 to 280 nM (Table 2). Their selective indices
ranged from >357 to >786. Verapamil was less potent with
EC50 ranging from 245 to 503 nM and a selective index of 200
(Table 2). Cyclosporin A was the most potent modulator with
EC50 ranging from 32 to 43 nM. It is, however, slightly toxic toward
L929 with an IC50 of 30 μM, giving it a selective index of 934.
MRP1- and BCRP-Modulating Activity of Methylated
EGC and Methylated GC Derivatives. We have also deter-
mined the specificity of methylated EGC and methylated GC
toward P-gp, MRP1, and BCRP transporters. All of them are
ABC transporters with two transmembrane domains and two
nucleotide binding domains. They can mediate drug efflux in an
ATP-dependent manner. MRP1 activity was measured as
DOX resistance in MRP1-transfected ovarian cancer cell line
2008/MRP1 when compared to its parent 2008 cell line. BCRP
activity was measured as topotecan resistance in BCRP-
transfected human kidney embryonic cell line HEK293/R2
when compared to the empty vector-transfected HEK293/
pcDNA3.1 cell line. 2008/MRP1 was about 5.8-fold more
resistant to DOX than 2008/P cells, whereas HEK293/R2 was
about 29.6-fold more resistant to topotecan than HEK293/
pcDNA3.1 cells. Compound 4e is a flavonoid homodimer
previously reported to have potent MRP1-modulating activity
with a RF of 8.9.58 As shown in Table 3, compounds 23, 35,
Table 3. Specificity of Methylated EGC and Methylated GC
a
Derivatives toward MRP1 and BCRP Transporters
MRP1-modulating activity BCRP-modulating activity
IC50 of DOX
(nM)
IC50 of topotecan
(nM)
Cpds
RF
RF
23
322 80
434 139
418 127
1.1
0.8
0.9
2.6
2.6
8.9
357 141
176 43
207 73
1.3
35
2.7
36
2.3
50
142
141
4
6
45
38
/
1
4
10.4
12.3
51
4e
41 13
/
Ko143
2008/MRP1
2008/P
HEK293/R2
24
/
2
19.5
365 80
1.0
5.8
63
/
5
/
468 33
16
1.0
HEK293/
pcDNA3.1
/
2
29.6
a
MRP1- and BCRP-modulating activity of 23, 35, 36, 50, 51, 4e, and
Ko143 (all at 1.0 μM) were investigated using 2008/MRP1 and
HEK293/R2 cells, respectively (N = 2−4 independent experiments,
and the values are presented as the mean
standard error of the
mean. Each experiment was done in triplicate). IC50 toward DOX and
topotecan in these two cell lines were determined with or without
modulators to determine RF. IC50 values were also determined for
their parental cell lines (2008/P and HEK293/pcDNA3.1) for
reference.
and 36 displayed no MRP1-modulating activity, whereas 50
and 51 showed moderate MRP1 inhibition (RF = 2.6). Ko143
is a known specific BCRP modulator (RF = 19.5). Compounds
23, 35, and 36 displayed no or very low BCRP-modulating
activity (RF = 1.3 to 2.7), whereas compounds 50 and 51
J
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Figure 2. Effect of methylated EGC derivatives on DOX accumulation in LCC6MDR cells. Effect of compounds 23 and 35 or verapamil on
intracellular DOX accumulation was studied. (A) LCC6 or LCC6MDR cells were incubated with 20 μM DOX with or without 2 μM of modulators
(23 and 35 or verapamil) for 150 min at 37 °C. DMSO (0.2%) was used as the negative control. After the incubation period, cells were lysed, and
the supernatant was saved for measuring the DOX level by spectrofluorometry. (B, C, and D) LCC6MDR cells were incubated with 20 μM DOX
and with increasing doses (0, 0.05, 0.1, 0.5, 1, 3, and 8 μM) of 23 (B), 35 (C), or verapamil (D). After incubation, cells were lysed, and their
intracellular DOX level was measured by spectrofluorometry. N = 2−5 independent experiments. The results are presented as the mean standard
error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.005 relative to the LCC6MDR negative control.
surprisingly exhibited very promising BCRP-modulating activity
(RF = 10.4 and 12.3). All in all, cis-methylated EGC derivatives
are likely monospecific toward the P-gp transporter, especially
compound 23. Trans-methylated GC derivatives, 50 and 51,
are multispecific toward P-gp, MRP1, and BCRP transporters.
Modulators with different transporter specificity (mono-, dual-,
and multi-) are helpful for treating multidrug resistant cancers
caused by the overexpression of ABC transporters.
Methylated EGC Derivatives Increase DOX Accumu-
lation by Inhibiting P-gp-Mediated Efflux in LCC6MDR
Cells. DOX is a known fluorescent P-gp substrate, and its
fluorescence can be used for monitoring the intracellular DOX
level. We found that LCC6 cells accumulated about 2.9-fold
more DOX than LCC6MDR cells (Figure 2A). Treatment of
LCC6MDR cells with 2 μM of 23, 35, 36, or verapamil can
inhibit P-gp and increase DOX accumulation by 2.4- to 2.7-fold,
to a level similar to that of LCC6 cells (Figure 2A). Restora-
tion of DOX accumulation in LCC6MDR cells by 23, 36,
and verapamil was dosage-dependent (Figure 2B, C, and D).
Three micromolar 23 and 36 was sufficient to restore DOX
accumulation of LCC6MDR to the parental LCC6’s level
(Figure 2B and C). For verapamil, the highest concentration
of 8 μM was not enough to restore DOX accumulation of
LCC6MDR to the parental LCC6’s level (Figure 2D). Com-
pounds 23 and 36 are therefore roughly at least 2.7-fold more
potent than verapamil in inhibiting P-gp’s DOX transport activity.
Is reduced DOX accumulation in LCC6MDR due to de-
creased DOX influx or increased DOX efflux? Here, we studied
the effect of 23 and 35 on P-gp-mediated DOX influx and
efflux. In the DOX efflux studies, cells were first loaded with
DOX, followed by incubation in DOX-free medium. In LCC6
cells, there is no difference in the DOX efflux rate among 23,
35, and the DMSO control (Figure 3A). On the contrary, 23
and 35 can reduce DOX efflux significantly compared to that
of the DMSO control (Figure 3B). In the DOX influx studies,
there is no significant difference in influx rate for both LCC6
K
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Figure 3. Effect of methylated EGC derivatives on DOX influx and DOX efflux. Effect of methylated EGC derivatives on DOX influx and DOX
efflux was studied. For the efflux study, LCC6 (A) or LCC6MDR cells (B) were incubated with 20 μM DOX for 60 min. Cells were then washed
with ice cold PBS and resuspended with DMEM media with or without 1.5 μM 23 or 35 to inhibit efflux if there is any. After incubation for 30, 60,
90, 120, and 150 min, cells were collected by centrifugation, and the intracellular DOX level was determined by spectrofluorometry. For the influx
study, LCC6 (C) or LCC6MDR cells (D) were incubated with 5 μM of DOX. After incubation for 10, 20, 30, and 40 min, cells were collected, and
the intracellular DOX level was determined by spectrofluorometry. N = 3−4 independent experiments. The values are presented as the mean
standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.005 relative to the LCC6MDR negative control at each time point.
(Figure 3C) and LCC6MDR (Figure 3D) cells treated with
compounds 23, 35, or the DMSO control (Figure 3C and D).
The above data suggests that the methylated EGC derivatives
can inhibit P-gp-mediated DOX efflux.
substitution at ring D, (2) rigid linkers of oxycarbonylvinyl and
oxycarbonylphenylcarbamoyl with the optimal linker length
ranging from 7.75 to13.37 Å between rings D and C3, (3) chiral
configuration at C3, and (4) the chromanol scaffold of permethyl
EGCG derivatives. Among the four potent cis-methylated EGC
derivatives (22, 23, 35, and 36), compound 23 with trimethoxy
substituents at ring D and an oxycarbonylvinyl linker between
ring D and the C3 position was the most potent with EC50
ranging from 123 nM to 195 nM. Among the four potent trans-
methylated GC derivatives (50, 51, 52, and 53), compound 51
with a linker containing an oxycarbonylphenylcarbamoyl and
trimethoxy groups at ring D was the most potent with EC50
values ranging from 102 to 140 nM. The mechanism of
methylated EGC derivatives in reversing P-gp mediated drug
resistance is by virtue of inhibiting the active drug efflux of P-gp
transporter (Figure 3B). Importantly, compounds 23 and 51 are
not substrates of the P-gp transporter (Figure 4). They will be
predicted to stay inside P-gp overexpressing cells for a longer
time and result in a longer-lasting P-gp modulating effect. In
summary, our study demonstrates that methylated EGC or
methylated GC derivatives are noncytotoxic, effective, and
specific P-gp modulators that can be used in the future for
reversing P-gp mediated clinical cancer drug resistance.
Intracellular Accumulation of Methylated EGC and
Methylated GC Derivatives in LCC6 and LCC6MDR Cells.
Some P-gp modulators are competitive inhibitors of anticancer
drugs to bind P-gp, but they are also substrates of P-gp and are
effluxed out of the cells. Here, we study whether methylated
EGC or methylated GC derivatives are P-gp substrates. We
found that both LCC6 and LCC6MDR cells accumulated a
similar level of methylated EGC 23 and methylated GC 51 at
20 or 200 μM (Figure 4A and B), indicating that compounds
23 and 51 are not substrates of P-gp. Here, we used DOX as a
positive control because it is a known P-gp substrate. At 20 μM,
parental cells LCC6 accumulated 2.9-fold more DOX than P-gp
overexpressed cells LCC6MDR (Figure 4C), indicating that
DOX can bind the P-gp transporter and was exported out by
P-gp in LCC6MDR cells.
CONCLUSIONS
■
In the present study, a total of 55 novel methylated EGC,
methylated GC, and methylated dihydromyricetin derivatives
were synthesized and evaluated for their P-gp modulating activity
in a P-gp overexpressing breast cancer cell line LCC6MDR. The
SAR study illustrates several important pharmacophores for
modulating P-gp, including (1) methoxy, allyloxy, or acetylamino
EXPERIMENTAL SECTION
■
General. All moisture sensitive reactions were conducted under a
nitrogen atmosphere in anhydrous, freshly distilled solvents. Starting
L
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EtOAc (20 mL). Insoluble substance in the reaction mixture was
removed by filtration. The filtrate was evaporated under reduced
pressure and purified by flash chromatography on silica gel (EtOAc/
PE = 1/3, v/v) to afford the desired compound 8 (300 mg, 90.0%
yield): mp 157−159 °C; [α]20D = −57.6 (c = 0.1, CH2Cl2); 1H NMR
(CDCl3, 600 MHz) δ 6.74 (s, 2 H), 6.20 (d, J = 2.2 Hz, 1 H), 6.12 (d,
J = 2.2 Hz, 1 H), 4.92 (bs, 1 H), 4.28 (bs, 1 H), 3.88 (s, 6 H), 3.85 (s,
3 H), 3.79 (s, 3 H), 3.77 (s, 3 H), 2.97 (A of ABX, J = 17.1, 1.2 Hz,
1 H), 2.90 (B of ABX, J = 17.1,4.3 Hz,1 H); 13C NMR (CDCl3,
150 MHz) δ 159.8, 159.3, 155.1, 153.5, 137.7, 134.1, 103.3, 103.4,
93.4, 92.3, 78.8, 66.5, 60.9, 56.3, 55.6, 55.5, 28.2; HRMS calcd for
(C20H24O7 + H)+ 377.1600; found, 377.1588.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-methoxybenzoate (9). A mixture of compound 8 (200 mg,
0.53 mmol), 3-methoxybenzoic acid (100 mg, 0.66 mmol), 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·
HCl, 200 mg, 1.04 mmol), and 4-dimethylaminopyridine (DMAP,
130 mg, 1.06 mmol) in anhydrous CH2Cl2 (20 mL) was stirred
at room temperature overnight under a nitrogen atmosphere. Then
the solution was washed by diluted hydrochloric acid two times, and
brine in sequence. The organic layer was dried by anhydrous MgSO4.
The solvent was evaporated, and the residue was purified by flash
chromatography to afford the title compound 9 (242 mg, 89.4% yield);
1
mp 131−134 °C; [α]20 = −182.1 (c = 0.06, CH2Cl2); H NMR
D
(CDCl3, 600 MHz) δ 7.53 (d, J = 7.7 Hz, 1 H), 7.45 (d, J = 1.1 Hz,
1 H), 7.24 (d, J = 7.7 Hz, 1 H), 7.03 (m, 1 H), 6.69 (s, 2 H), 6.23 (d,
J = 2.2 Hz, 1 H), 6.10 (d, J = 2.2 Hz, 1 H), 5.64 (bs, 1 H), 5.05 (bs,
1 H), 3.78 (m, 12 H), 3.69 (m, 6 H), 3.04 (d, J = 3.3 Hz, 2 H); 13C
NMR (CDCl3, 150 MHz) δ 165.4, 159.7, 159.5, 158.9, 155.5, 153.2,
137.9, 133.4, 129.3, 122.0, 119.1, 114.7, 104.0, 100.2, 93.5, 92.0, 77.9,
68.7, 65.8, 60.7, 56.0, 55.4, 26.0; HRMS calcd for (C28H30O9 + H+
511.1968; found, 511.1957.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
4-methoxybenzoate (10). Following the procedure for the prepara-
tion of compound 9, but with 4-methoxybenzoic acid as starting
Figure 4. Intracellular accumulation of methylated EGC and methyl-
ated GC derivatives in LCC6 and LCC6DR cells. LCC6 or
LCC6MDR cells (3.0 × 106) were incubated with 20 or 200 μM of
compound 23 (A), 51 (B), or DOX (C) at 37 °C for 150 min. After
incubating with compounds 23 and 51, the cell pellet was resuspended
with 100 μL of 100% methanol and vortexed for 5 min. The lysate was
spinned down, and the supernatant was used to measure the level of
23 or 51 by HPLC. After incubating with DOX, the cell pellet was
lysed with 100 μL of lysis buffer, and the intracellular DOX fluo-
rescence level was measured using a spectrofluorometer. N = 2−3
independent experiments. The data are represented as the mean
standard error of the mean.
material, compound 10 was obtained. Yield 89.8%; mp 67−69 °C;
1
[α]20 = −179.3 (c = 0.10, CH2Cl2); H NMR (CDCl3, 600 MHz) δ
D
7.91 (d, J = 8.8 Hz, 2 H), 6.84 (d, J = 8.8 Hz, 2H), 6.70 (s, 2 H), 6.25
(d, J = 2.2 Hz, 1 H), 6.11 (d, J = 2.2 Hz, 1 H), 5.65 (bs, 1 H), 5.06 (bs,
1 H), 3.80 (m, 12 H), 3.71 (m, 6 H), 3.04 (d, J = 2.8 Hz, 2 H); 13C
NMR(CDCl3, 150 MHz) δ 165.3, 163.6, 159.7, 159.0, 155.6, 153.2,
137.8, 133.5, 131.9, 131.8, 122.5, 113.6, 104.0, 93.5, 92.0, 78.1, 68.2,
56.0, 56.0,55.5, 26.2; HRMS calcd for (C28H30O9 + H)+ 511.1968;
found, 511.1954.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3,4-dimethoxybenzoate (11). Following the procedure for the
preparation of compound 9, but with 3,4-dimethoxybenzoic acid as
starting material, compound 11 was obtained. Yield 88.9%; mp 146−
materials and reagents, unless otherwise stated, were of commercial
grades and were used without further purification. Solvents were dried
according to standard procedures. TLC chromatography was
1
148 °C; [α]20 = −170.4 (c = 0.03, CH2Cl2); H NMR (CDCl3, 600
D
MHz) δ 7.60 (dd, J = 1.9, 8.5 Hz, 1 H), 7.42 (d, J = 1.9 Hz, 1 H), 6.80
(d, J = 8.5 Hz, 1 H), 6.70 (s, 2 H), 6.24 (d, J = 2.2 Hz, 1 H), 6.11 (d,
J = 2.2 Hz, 1 H), 5.66 (bs, 1 H), 5.06 (bs, 1 H), 3.88 (m, 15 H), 3.71
(s, 6 H), 3.04 (d, J = 3.3 Hz, 2 H); 13C NMR (CDCl3, 150 MHz) δ
165.3, 159.7, 158.9, 155.6, 153.2, 153.1, 148.7, 137.9, 133.4, 123.7,
122.6, 112.3, 110.2, 104.1, 100.3, 93.4, 91.9, 78.0, 68.3, 65.8, 60.8, 56.0,
55.4, 26.0; HRMS calcd for (C29H32O10 + H)+ 541.2073; found,
541.2085.
1
performed on aluminum sheets (Silica gel 60-F254, E. Merck). H
(600 or 500 MHz) and 13C (150 or 125 MHz) NMR experiments
were determined on an instrument when CDCl3 was used as a solvent.
Chemical shifts are given in ppm (δ) values with TMS as an internal
standard and coupling constants (J) in Hz. High-resolution (ESI) MS
spectra were performed with a QTOF-2 micromass spectrometer. In
addition to NMR and high-resolution (ESI) MS, HPLC analysis was
used to determine the purity (>95%) of the compounds. Compounds
were dissolved in methanol (1.5 mL). A reversed phase Diamonsil C18
(2) (4.6 × 150 mm) column attached to a Gilson 322 pump coupled
to a Gilson UV−vis-152 detector was used. Each sample was injected
at a volume of 20 μL and eluted with methanol, and the flow rate was
1 mL/min. Melting points were determined with a micromelting point
apparatus MP-500D and were uncorrected.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3,4,5-triethoxybenzoate (12). Following the procedure for the pre-
paration of compound 9, but with 3,4,5-triethoxybenzoic acid as starting
material, compound 12 was obtained. Yield 88.5%; mp 47−51 °C; [α]20
=
D
−129.8 (c = 0.05, CH2Cl2); 1H NMR (CDCl3, 600 MHz) δ 7.13 (s, 2 H),
6.68 (s, 2 H), 6.23 (d, J = 1.1 Hz, 1 H), 6.10 (d, J = 1.1 Hz, 1 H), 5.62 (bs,
1 H), 5.06 (bs, 1 H), 4.06 (q, J = 7.1 Hz, 2 H), 4.00 (q, J = 7.1 Hz,
4 H), 3.78 (m, 9 H), 3.69 (s, 6 H), 3.02 (d, J = 2.8 Hz, 2 H), 1.38 (t, J =
7.1 Hz, 6 H), 1.31 (t, J = 7.1 Hz, 3 H); 13C NMR (CDCl3, 150 MHz)
δ 165.3, 159.8, 159.0, 155.6, 153.2, 152.7, 142.5, 137.9, 133.5, 124.9,
108.7, 108.7,104.0, 100.3, 93.3, 91.9, 77.9, 69.0, 68.6, 64.9, 56.0, 55.5,
26.0, 15.6, 14.9; HRMS calcd for (C33H40O11 + H)+ 613.2649; found,
613.2661.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-
ol(8). Permethyl EGCG (500 mg, 1.09 mmol) was dissolved in
methanol (10 mL) and dimethoxyethane (10 mL), then K2CO3
(243 mg, 1.76 mmol) was added. The mixture was stirred for 2 h
until TLC showed the reaction had been completed. The solvent was
removed under reduced pressure. The residue was dissolved with
M
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(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxy phenyl)chroman-3-yl
3,4,5-tris(allyloxy)benzoate (13). Following the procedure for the
preparation of compound 9, but with 3,4,5-tris(allyloxy)benzoic acid as
(s, 3 H), 3.43 (A of AB, J = 15.0 Hz, 1H), 3.39 (B of AB, J = 15.0 Hz,
1H), 2.91 (d, J = 2.3 Hz, 1H); 13C NMR (CDCl3, 150 MHz) δ 170.9,
159.5, 158.7, 158.3, 155.1, 152.9, 137.5, 133.2, 129.7, 125.4, 113.6,
103.3, 99.8, 93.2, 91.7, 67.9, 60.5, 55.8, 55.1, 54.8, 40.0, 25.6; HRMS
calcd for (C29H32O9 + Na)+ 517.1939; found, 517.1934.
starting material, compound 13 was obtained. Yield 87.9%; [α]20
=
D
1
−138.2 (c = 0.10, CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.17 (s,
2 H), 6.67 (s, 2 H), 6.24 (d, J = 2.8 Hz, 1 H), 6.12 (d, J = 2.8 Hz, 1 H),
6.03 (m, 1 H), 5.97 (m, 2 H), 5.65 (bs, 1H), 5.35 (A of ABX, J = 17.3,
1.3 Hz, 2 H), 5.28 (A of ABX, J = 17.3, 1.4 Hz, 1H), 5.24 (B of ABX,
J = 10.5, 1.3 Hz, 2 H), 5.15 (B of ABX, J = 10.5, 1.4 Hz, 1H)., 5.06 (bs,
1 H), 4.57 (d, J = 6.0 Hz, 2 H), 4.52 (d, J = 5.3 Hz, 4 H), 3.79 (m, 9
H), 3.68 (s, 6 H), 3.03 (d, J = 2.6 Hz, 2 H); 13C NMR (CDCl3, 150
MHz) δ 164.9, 159.8, 158.9, 155.5, 153.2, 152.2, 142.1, 138.1, 134.1,
133.4, 133.0, 133.0, 125.0, 118.0, 117.9, 117.8, 109.2, 109.1, 104.1,
100.2, 93.4, 91.9, 77.8, 74.0, 70.0, 68.5, 60.7, 56.0, 55.4, 55.3, 26.0;
HRMS calcd for (C36H40O11 + H)+ 649.2649; found, 649.2637.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-fluorobenzoate(14). Following the procedure for the preparation of
9, but with 3-fluorobenzoic acid as starting material, compound 14 was
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
2-(3,4-dimethoxyphenyl)acetate (19). Following the procedure for
the preparation of 9, but with 3,4-dimethoxyphenylacetic acid as starting
material, compound 19 was obtained. Yield 88.4%; mp 51−53 °C; [α]20
=
D
1
−56.2 (c = 0.1, CH2Cl2); H NMR (CDCl3, 600 MHz) δ 6.66 (s, 2
H),6.65 (d, J = 8,2 Hz, 1H), 6.59 (d, J = 1.9 Hz, 1H), 6.51 (dd, J = 8.2, 1.9
Hz, 1H), 6.21 (d, J = 2.3 Hz, 1H), 6.10 (d, J = 2.3 Hz, 1H), 5.50 (bs, 1H),
4.99 (bs, 1H), 3.83 (s, 3H), 3.81 (s, 6 H), 3.81 (s, 3H), 3.78 (s, 3H), 3.75
(s, 3H), 3.70 (s, 3H), 3.43 (A of AB, J = 15.2 Hz, 1 H), 3.39 (B of AB, J =
15.2 Hz, 1H), 2.93 (A of ABX, J = 17.9, 4.3 Hz, 1H), 2.88 (B of ABX, J =
17.9, 1.8 Hz, 1H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 159.6, 158.8,
155.2, 153.0, 148.7, 147.9, 137.6, 133.2, 125.9, 121.1, 112.1, 110.9, 103.5,
99.9, 93.3, 91.9, 68.0, 60.7, 56.0, 55.7, 55.5, 55.3, 40.7, 25.8; HRMS calcd for
(C30H34O10 + Na)+ 577.2024; found, 577.2030.
obtained. Yield 89.5%; mp 59−62 °C; [α]20 = −22.7 (c = 0.1,
D
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.74 (d, J = 7.7 Hz, 1 H),
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
2-(3,4,5-trimethoxyphenyl)acetate (20). Following the procedure
for the preparation of 9, but with 3,4,5-trimethoxyphenylacetic acid
7.63 (d, J = 9.3 Hz, 1 H), 7.35 (m, 1 H), 7.21 (m, 1 H), 6.70 (s, 2 H),
6.26 (d, J = 2.2 Hz, 1 H), 6.13 (d, J = 2.2 Hz, 1 H), 5.66 (bs, 1 H),
5.08 (bs, 1 H), 3.80 (m, 9 H), 3.74 (s, 6 H), 3.07 (d, J = 3.3 Hz, 2 H);
13C NMR (CDCl3, 150 MHz) δ164.4, 163.3, 161.6, 159.8, 158.9,
as starting material, compound 20 was obtained. Yield 89.7%; mp
1
56−58 °C; [α]20 = −54.3 (c = 0.1, CH2Cl2); H NMR (CDCl3,
D
600 MHz) δ 6.68 (s, 2H), 6.29 (s, 2H), 6.21 (d, J = 2.2 Hz, 1H), 6.10 (d,
J = 2.2 Hz, 1H), 5.52 (bs, 1H), 5.00 (bs, 1H), 3.83 (s, 9 H), 3.75 (m,
9 H), 3.70 (s, 6H), 3.43 (A of AB, J = 15.2 Hz, 1 H), 3.39 (B of AB, J =
15.2 Hz, 1H), 2.93 (A of ABX, J = 17.9, 4.5 Hz, 1H), 2.89 (B of ABX,
J = 17.9, 1.8 Hz, 1H); 13C NMR (CDCl3, 150 MHz) δ 170.7, 159.8,
158.9, 155.3, 153.2, 137.8, 137.0, 133.3, 129.1, 106.1, 103.7, 100.0,
93.4, 92.1, 68.2, 60.8, 56.2, 55.9, 55.4, 41.4, 26.0; HRMS calcd for
(C31H36O11 + Na)+ 607.2150; found, 607.2144.
155.5, 153.2, 138.0, 133.3, 130.0, 129.9, 125.5, 120.2, 120.0,116.6,
116.5, 103.8, 100.0, 93.6, 92.1, 77.8, 69.1, 60.8, 56.0, 55.4, 26.0; HRMS
calcd for (C27H27FO8 + H)+ 499.1768; found, 499.1776.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
4-fluorobenzoate (15). Following the procedure for the preparation
of 9, but with 4-fluorobenzoic acid as starting material, compound 15
was obtained. Yield 88.6%; mp 55−57 °C; [α]20 = −10.8 (c = 0.2,
D
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.96 (m, 2 H), 7.03 (m,
2 H), 6.69 (s, 2 H), 6.25 (d, J = 1.9 Hz, 1H), 6.12 (d, J = 1.9 Hz, 1 H),
5.67 (bs, 1 H), 5.06 (bs, 1 H), 3.79 (m, 9 H), 3.71 (s, 6 H), 3.04 (d,
J = 2.8 Hz, 2 H); 13C NMR (CDCl3, 150 MHz) δ 166.6, 164.9, 164.5,
159.7, 158.9, 155.5, 153.2, 137.9, 133.3, 132.2, 126.3, 115.5, 115.4,
100.1, 93.5, 92.0, 77.8, 68.7, 60.7, 55.9, 55.4, 26.1; HRMS calcd for
(C27H27FO8 + H)+ 499.1768; found, 499.1779.
(E)-(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl 3-(4-methoxyphenyl)acrylate (21). Following the procedure for
the preparation of compound 9, but with (E)-3-(4-methoxyphenyl)-
acrylic acid as starting material, compound 21 was obtained. Yield
1
85.4%; mp77−81 °C; [α]20 = −186.2 (c = 0.05, CH2Cl2); H NMR
D
(CDCl3, 600 MHz) δ 7.55 (d, J = 16.0 Hz, 1 H), 7.39 (d, J = 8.5 Hz,
2 H), 6.86 (d, J = 8.5 Hz, 2 H), 6.73 (s, 2 H), 6.25 (d, J = 16.0 Hz, 1 H),
6.24 (bs, 1 H), 6.12 (bs, 1 H), 5.61 (bs, 1 H), 5.03 (bs, 1 H), 3.82 (m,
18 H), 2.99 (bs, 2 H); 13C NMR (CDCl3, 150 MHz) δ 166.5, 161.6,
159.7, 159.0, 155.5, 153.2, 145.1, 137.9, 133.4, 129.8, 127.0, 115.3,
114.4, 104.0, 100.4, 93.5, 92.1, 77.9, 67.6, 60.9, 56.2, 55.5, 55.4, 26.2;
HRMS calcd for (C30H32O9 + H)+ 537.2124; found, 537.2113.
(E)-(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman
−3-yl 3-(3,4-dimethoxyphenyl)acrylate (22). Following the procedure
for the preparation of compound 9, but with (E)-3-(3,4-dimethoxyphenyl)-
acrylic acid as starting material, compound 22 was obtained.Yield
87.5%: mp 76−79 °C; [α]20D = −175.0 (c = 0.03, CH2Cl2); 1H NMR
(CDCl3, 600 MHz) δ 7.53 (d, J = 15.9 Hz, 1 H), 7.01 (d, J = 8.2 Hz,
1 H), 6.95 (s, 1 H), 6.81 (d, J = 8.2 Hz, 1 H), 6.72 (s, 2 H), 6.25 (d,
J = 15.9 Hz, 1 H), 6.24 (d, J = 1.7 Hz, 1 H), 6.12 (d, J = 1.7 Hz, 1 H),
5.63 (bs, 1 H), 5.03 (bs, 1 H), 3.82 (m, 21 H), 2.99 (bs, 2 H); 13C
NMR (CDCl3, 150 MHz) δ 166.3, 159.6, 158.9, 155.3, 153.1, 151.2,
149.1, 145.2, 137.8, 133.2, 127.1, 122.7, 115.4, 110.9, 109.3, 103.8,
100.3, 93.3, 92.0, 77.8, 67.4, 60.8, 56.1, 55.9, 55.8, 55.4, 55.3, 26.1;
HRMS calcd for (C31H34O10 + H)+ 567.2230; found, 567.2241.
(E)-(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl 3-(3,4,5-trimethoxyphenyl)acrylate (23). Following the proce-
dure for the preparation of compound 9, but with (E)-3-(3,4,5-
trimethoxyphenyl)acrylic acid as starting material, compound 23 was
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(dimethylamino)-4-fluorobenzoate (16). Following the procedure
for the preparation of 9, but with 3-(dimethylamino)-4-fluorobenzoic
acid as starting material, compound 16 was obtained. Yield 87.8%; mp
1
56−58 °C; [α]20 = −17.6 (c = 0.1, CH2Cl2); H NMR (CDCl3,
D
600 MHz) δ 7.47 (m, 2 H), 6.96 (m, 1 H), 6.69 (s, 2 H), 6.25 (d, J =
2.2 Hz, 1 H), 6.11 (d, J = 2.2 Hz, 1 H), 5.65 (m, 1 H), 5.06 (bs, 1 H),
3.79 (m, 9 H), 3.71 (s, 6 H), 3.04 (d, J = 3.3 Hz, 2 H), 2.81 (s, 6 H);
13C NMR (CDCl3, 150 MHz) δ 165.1, 159.8, 159.0, 158.8, 155.6,
153.2, 153.2, 140.9, 137.9, 133.5, 126.4, 122.9, 119.9, 115.9,103.9,
103.9, 100.2, 93.4, 92.0, 78.0, 68.6, 60.9, 56.0, 55.5, 42.6, 26.1; HRMS
calcd for (C29H32FNO8 + H)+ 542.2190; found, 542.2178.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-acetamido-4-fluorobenzoate (17). Following the procedure for the
preparation of 9, but with 3-acetamido-4-fluorobenzoic acid as starting
material, compound 17 was obtained. Yield 86.9%; mp 93−96 °C;
1
[α]20 = −106.8 (c = 0.04, CH2Cl2); H NMR (CDCl3, 600 MHz) δ
D
8.84 (bs, 1 H), 7.67 (bs, 1 H), 7.33 (bs, 1 H), 7.06 (m, 1 H), 6.72 (s,
2 H), 6.27 (d, J = 2.2 Hz, 1 H), 6.11 (d, J = 2.2 Hz, 1 H), 5.64 (bs, 1 H),
5.07 (bs, 1 H), 3.80 (m, 9 H), 3.75 (s, 6 H), 3.05 (d, J = 3.3 Hz, 2 H),
2.19 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 168.6, 164.5, 159.7,
158.9, 156.5, 155.5, 153.1, 137.7, 133.4, 126.6, 126.4, 124.4, 114.9,
114.8, 103.9, 100.1, 93.6, 92.0, 77.7, 69.0, 60.7, 56.0, 55.3, 25.9, 24.0;
HRMS calcd for (C29H30FNO9 + H)+ 556.1983; found, 556.1998.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
2-(4-methoxyphenyl)acetate (18). Following the procedure for the
preparation of 9, but with 4-methoxyphenylacetic acid as starting
obtained. Yield 86.5%; mp111−113 °C; [α]20 = −177.8 (c = 0.1,
D
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.50 (d, J = 16.0 Hz, 1 H),
6.72 (s, 2 H), 6.66 (s, 2 H), 6.28 (d, J = 16.0 Hz, 1 H), 6.25 (d, J = 2.2
Hz, 1H), 6.14 (d, J = 2.2 Hz, 1H), 5.66 (bs, 1 H), 5.05 (bs, 1 H), 3.84
(m, 18 H), 3.83 (s, 6 H), 3.00 (bs, 2 H); 13C NMR (CDCl3, 150
MHz) δ 166.2, 159.8, 159.1, 155.4, 153.5, 153.3, 145.4, 140.3, 137.8,
133.3, 129.8, 117.1, 105.3, 104.0, 100.4, 93.5, 92.1, 77.8, 67.6, 61.0,
60.9, 56.2, 55.5, 26.2; HRMS calcd for (C32H36O11 + H)+ 597.2336;
found, 597.2339.
material, compound 18 was obtained. Yield 88.5%; mp 46−48 °C;
1
[α]20 = −63.7 (c = 0.1, CH2Cl2); H NMR (CDCl3, 600 MHz) δ
D
6.89 (d, J = 8.4 Hz, 2 H), 6.70 (d, J = 8.4 Hz, 2 H),6.68 (s, 2 H), 6.24
(d, J = 2.0 Hz, 1H), 6.12 (d, J = 2.0 Hz, 1H), 5.49 (bs, 1H), 4.98 (bs,
1H), 3.85 (s, 3 H), 3.81 (s, 6 H), 3.78 (s, 3 H), 3.75 (s, 3 H), 3.72
N
DOI: 10.1021/acs.jmedchem.5b00085
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(E)-(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl3-(4-methoxyphenyl)propionate (24). Compound 21 (300 mg,
0.56 mmol) was dissolved in CH3OH (20 mL) and carefully added to
10% palladium on carbon. Hydrogen was introduced to the flask, and
the mixture was allowed to stir under a positive hydrogen atmosphere
for 12 h. The catalyst was filtered from the reaction mixture, and
the filtrate was evaporated to dryness to give the title compound 24
127.9, 127.8, 127.3, 126.6, 124.8, 122.5, 119.7, 116.2, 116.1, 112.2,
110.5, 56.1, 55.9; HRMS calcd for (C18H16O5NF + H)+ 346.1085;
found, 346.1082.
(E)-4-Fluoro-3-(3-(3,4,5-trimethoxyphenyl)acrylamido)benzoic
Acid (29). Following the procedure for the preparation of compound
28, but with 3-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-4-fluoroben-
zoic acid as starting material, compound 29 was obtained.Yield 73.6%.
(236 mg, 78.5% yield): mp 52−54 °C; [α]20 = −72.3 (c = 0.1,
1
mp 186−188 °C; H NMR (DMSO-d6, 600 MHz) δ 8.82 (d, J = 7.2
D
1
CH2Cl2); H NMR (CDCl3, 600 MHz)δ 6.90 (d, J = 8.4 Hz, 2 H),
Hz, 1 H), 7.76 (d, J = 2.7 Hz, 1 H), 7.60 (d, J = 15.4 Hz, 1 H), 7.44
(m, 1 H), 7.14 (d, J = 15.4 Hz, 1 H), 7.01 (s, 3 H), 3.87 (s, 6 H), 3.73
(s, 3 H); 13C NMR (DMSO-d6, 150 MHz) δ 167.0, 164.7, 157.0,
155.4, 153.6, 141.7, 139.6, 130.8, 127.8, 127.3, 126.8, 124.9, 121.6,
116.3, 116.2, 105.8, 60.7, 56.4; HRMS calcd for (C19H18O6NF + H)+
376.1191; found, 376.1185.
6.72 (d, J = 8.4 Hz, 2 H), 6.71 (s, 2 H), 6.23 (d, J = 2.0 Hz, 1 H), 6.12
(d, J = 2.0 Hz, 1H), 5.46 (bs, 1H), 4.98 (bs, 1H), 3.85 (s, 6 H), 3.84
(s, 3 H), 3.77 (s, 6 H), 3.73 (s, 3 H), 2.96 (A of ABX, J = 17.8, 4.5 Hz,
1H), 2.88 (B of ABX, J = 17.8, 1.8 Hz, 1H), 2.67 (m, 2 H), 2.42 (m,
2 H); 13C NMR (CDCl3, 150 MHz)δ 172.0, 159.6, 158.8, 157.9,
155.2, 153.1, 137.6, 133.4, 132.1, 128.9, 113.7, 103.5, 100.0, 93.3,
91.9, 67.8, 60.7, 56.1, 55.2, 55.0, 36.0, 29.8, 25.8; HRMS calcd for
(C30H34O9 + Na)+ 561.2095; found, 561.2089.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-4-fluorobenzoate (30).
Following the procedure for the preparation of compound 9, but
with 3-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-4-fluorobenzoic acid
(E)-(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl3-(3,4-dimethoxyphenyl)propionate (25). Following the proce-
dure for the preparation of compound 24, but with compound 22
as starting material, compound 25 was obtained. Yield 79.3%; mp
as starting material, compound 30 was obtained. Yield 85.8%; mp 93−
1
95 °C; [α]20 = −159.6 (c = 0.1, CH2Cl2); H NMR (CDCl3, 600
D
MHz) δ 7.68 (d, J = 15.3 Hz, 1H), 7.66 (s, 1 H), 7.46 (d, J = 2.7 Hz,
1 H), 7.12 (dd, J = 8.2, 2.2 Hz, 1 H), 7.07 (m, 2 H), 6.87 (d, J = 8.2
Hz, 1 H), 6.74 (s, 2 H), 6.43 (d, J = 15.3 Hz, 1 H), 6.28 (d, J = 2.2 Hz,
1 H), 6.11 (d, J = 2.2 Hz, 1 H), 5.65 (t, J = 2.7 Hz, 1 H), 5.08 (s, 1 H),
3.91 (d, J = 2.2 Hz, 6 H), 3.80 (s, 3 H), 3.79 (s, 3 H), 3.77 (s, 3 H),
3.76 (s, 6 H), 3.06 (d, J = 3.3 Hz, 2 H); 13C NMR (CDCl3, 150
MHz)δ 164.6, 163.9, 159.7, 158.9, 155.5, 153.2, 151.7, 149.2, 143.3,
143.2, 137.7, 133.4, 127.3, 126.9, 122.6, 122.5, 111.2, 111.1, 109.8,
109.7, 103.8, 103.7, 100.2, 69.1, 69.0, 60.9, 60.8, 56.1, 56.0, 55.9, 55.5,
55.4, 26.1; HRMS calcd for (C38H38O11NF + H)+ 704.2502; found,
704.2504.
85−87 °C; [α]20 = −71.2 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600
D
MHz) δ 6.70 (s, 2 H), 6.68 (d, J = 8.2 Hz, 1 H), 6.58 (d, J = 1.6 Hz,
1 H), 6.52 (dd, J = 8.2, 1.6 Hz, 1 H), 6.21 (d, J = 2.2 Hz, 1 H), 6.11 (d,
J = 2.2 Hz, 1 H), 5.47 (bs, 1 H), 4.99 (bs, 1 H), 3.85 (s, 6 H), 3.83 (s,
3 H), 3.81 (s, 3 H), 3.79 (s, 3 H), 3.78 (s, 6 H), 2.95 (A of ABX,
J = 17.9, 4.7 Hz, 1H), 2.87 (B of ABX, J = 17.9, 1.7 Hz, 1H), 2.72 (m,
2 H), 2.52 (m, 2 H); 13C NMR (CDCl3, 150 MHz) δ 172.2, 159.7,
158.9, 155.3, 153.2, 148.8, 147.4, 137.7, 133.4, 132.8, 119.9, 111.4,
111.2, 103.6, 100.1, 93.4, 92.1, 67.9, 60.9, 56.2, 55.9, 55.5, 55.4, 36.0,
30.3, 26.0; HRMS calcd for (C31H36O10 + H)+ 569.2381; found,
569.2388; (C31H36O10+Na)+ 591.2201; found, 591.2206.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-((E)-3-(3,4,5-trimethoxyphenyl)acrylamido)-4-fluorobenzoate
(31). Following the procedure for the preparation of compound 9, but
with 3-((E)-3-(3,4,5-trimethoxyphenyl) acrylamido)-4-fluorobenzoic
acid as starting material, compound 31 was obtained. Yield 85.2%; mp
96−98 °C; [α]20D= −141.0 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600
MHz) δ 7.69 (m, 1 H), 7.66 (d, J = 15.4 Hz, 1 H), 7.54 (d, J = 2.7 Hz,
1 H), 7.07 (m, 1 H), 6.76 (s, 2 H), 6.74 (s, 2 H), 6.49 (d, J = 15.4 Hz,
1 H), 6.28 (d, J = 2.2 Hz, 1 H), 6.11 (d, J = 2.2 Hz, 1 H), 5.64 (bs,
1 H), 5.08 (bs, 1 H), 3.87 (s, 9 H), 3.80 (s, 3 H), 3.79 (s, 3 H), 3.76 (s,
3 H), 3.75 (s, 6 H), 3.06 (d, J = 3.3 Hz, 2 H); 13C NMR (CDCl3, 150
MHz) δ164.5, 163.7, 159.7, 158.9, 155.5, 153.5, 153.2, 143.3, 140.1,
137.7, 133.4, 129.9, 126.9, 126.8, 126.7, 126.3, 123.8, 119.3, 114.9,
114.7, 105.3, 103.8, 100.2, 93.6, 92.0, 69.1, 61.0, 60.8, 56.2, 56.1, 55.5,
42.0, 26.1; HRMS calcd for (C39H40O12NF +Na)+ 756.2427; found,
756.2427.
3-(3,4-Dimethoxybenzamido)-4-fluorobenzoic Acid (33). A mix-
ture of methyl 3-amino-4-fluorobenzoate (150 mg, 0.89 mmol), 3,4-
dimethoxybenzoic acid (200 mg, 1.07 mmol), 1-(3-(dimethylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 400 mg, 2.12
mmol), and 4-dimethylaminopyridine (DMAP, 260 mg, 2.12 mmol) in
anhydrous CH2Cl2 (20 mL) was stirred at room temperature over-
night under a nitrogen atmosphere. Then, the solution was washed by
diluted in hydrochloric acid two times and in brine in sequence. The
organic layer was dried by anhydrous MgSO4. The solvent was
evaporated, and the residue was purified by flash chromatography to
give a yellow solid. The yellow solid obtained above was dissolved
in methanol (30 mL) and water (1 mL), then LiOH·H2O (500 mg,
12.0 mmol) was added. The solution was stirred for 10 h at room
temperature, then the solvent was evaporated. Water (20 mL) was
added into the mixture and acidified with dilute hydrochloric acid until
the pH test paper showed that it is neutral, and a copious amount of
white solid was precipitated. The product was isolated by filtration and
dried in vacuo to afford compound 33 (200 mg, 71.2% yield); mp
169−171 °C; 1H NMR (DMSO-d6, 600 MHz) δ 10.16 (s, 1 H), 8.24
(d, J = 6.6 Hz, 1 H), 7.89 (m, 1 H), 7.69 (d, J = 6.6 Hz, 1 H), 7.62 (s,
1H), 7.48 (m, 1 H), 7.14 (d, J = 9.1 Hz, 1H), 3.88 (s, 6 H); 13C NMR
(DMSO-d6, 150 MHz) δ 166.8, 165.4, 159.8, 1525, 148.9, 128.7,
(E)-(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl3-(3,4,5-trimethoxyphenyl)propionate (26). Following the pro-
cedure for the preparation of compound 24, but with compound
23as starting material, compound 26 was obtained. Yield 78.2%; mp
52−54 °C; [α]20 = −64.6 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600
D
MHz) δ: 6.69 (s, 2 H), 6.26 (s, 2 H), 6.19 (d, J = 2.2 Hz, 1 H), 6.09
(d, J = 2.2 Hz, 1 H), 5.48 (bs, 1 H), 4.98 (bs, 1 H), 3.82 (m, 24 H),
2.96 (A of ABX, J = 17.9, 4.7 Hz, 1H), 2.87 (B of ABX, J = 17.9, 1.7
Hz, 1H), 2.69 (m, 2 H), 2.51 (m, 2 H); 13C NMR (CDCl3, 150 MHz)
δ 172.1, 159.7, 158.9, 155.3, 153.2, 137.7, 136.3, 136.0, 133.4, 104.9,
103.6, 100.0, 93.4, 92.0, 68.0, 60.9, 56.2, 56.0, 55.5, 35.8, 31.1, 26.0;
HRMS calcd for (C32H38O11 + Na)+ 621.2306; found, 621.2300.
(E)-3-(3-(3,4-Dimethoxyphenyl)acrylamido)-4-fluorobenzoic Acid
(28). (E)-3-(3,4-Dimethoxyphenyl)acrylic acid (300 mg, 1.4 mmol)
was suspended in anhydrous CH2Cl2 (20 mL). The mixture was
cooled to 0 °C under a nitrogen atmosphere. One drop of DMF and
oxalyl chloride (330 mg, 2.6 mmol) was added dropwise to the
reaction mixture. When the addition was completed, the ice bath was
removed, and the reaction mixture was stirred at room temperature for
8 h. The solvent was evaporated to give a yellow solid, and the yellow
solid was dissolved in dry CH2Cl2. A solution of methyl 3-amino-
4-fluorobenzoate (compound 27, 210 mg, 1.24 mmol) and DMAP
(310 mg, 2.5 mmol)) in anhydrous CH2Cl2 (5 mL) was then added
dropwise to the reaction mixture. The reaction mixture was stirred at
room temperature for 12 h, and the solvent was evaporated to give a
yellow solid. The yellow solid obtained above was dissolved in
methanol (30 mL) and water (1 mL), then LiOH·H2O (500 mg,
12.0 mmol) was added. The solution was stirred for 10 h at room
temperature, and the solvent was evaporated. Water (30 mL) was
added into the mixture and acidified with dilute hydrochloric acid until
the pH test paper showed that it is neutral; a copious amount white
solid was precipitated. The product was isolated by filtration, dried in
vacuo to afford compound 28 (300 mg, 68.9% yield). mp 198−
200 °C; 1H NMR (DMSO-d6, 600 MHz) δ 9.97 (s, 1 H), 8.81 (d, J =
7.6 Hz, 1 H), 7.73 (m, 1 H), 7.57 (d, J = 15.4 Hz, 1 H), 7.40 (m, 1 H),
7.23 (d, J = 2.1 Hz, 1 H), 7.20 (d, J = 8.2 Hz, 1 H), 7.02 (d, J = 8.2 Hz,
1 H), 6.99 (d, J = 15.4 Hz, 1 H), 3.82 (s, 3 H), 3.80 (s, 3 H); 13C
NMR (DMSO-d6, 150 MHz) δ 167.0, 164.9, 151.1, 149.4, 141.7,
O
DOI: 10.1021/acs.jmedchem.5b00085
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Journal of Medicinal Chemistry
Article
128.6, 127.7, 126.7, 126.2, 121.9, 116.8, 116.7, 111.5, 56.2, 56.1;
HRMS calcd for (C16H14O5NF + H)+ 320.0929; found, 320.0924.
3-(3,4,5-Trimethoxybenzamido)-4-fluorobenzoic acid (34). Fol-
lowing the procedure for the preparation of compound 33, but with
3,4,5-trimethoxybenzoic acids material, compound 34 was obtained.
Yield 72.6%; mp 213−215 °C; 1H NMR (DMSO-d6, 600 MHz) δ
10.26 (s, 1 H), 8.21 (dd, J = 2.2, 8.3 Hz, 1 H), 7.90 (m, 1H), 7.49 (m,
1 H), 7.37 (s, 2 H), 3.94 (s, 6 H), 3.78 (s, 3 H); 13C NMR (DMSO-d6,
150 MHz) δ 166.8, 165.3, 159.8, 158.1, 153.2, 141.3, 129.2, 128.8,
127.9, 126.5, 116.9, 116.8, 105.9, 105.9, 60.7, 56.6; HRMS calcd for
(C17H16O6NF + H)+ 350.1034; found, 350.1028.
7.51 (m, 1H), 7.43 (m, 1H), 7.38 (s, 2H), 3.88 (s, 6H), 3.75 (s, 3H);
13C NMR (DMSO-d6, 125 MHz) δ 166.4, 165.0, 164.4, 159.4, 159.2,
157.4, 157.2, 152.8, 140.8, 130.1, 128.7, 128.2, 127.5, 127.4, 126.1,
126.0, 116.3, 105.5, 60.2, 56.2; HRMS calcd for (C24H20O7F2N2 + H)+
487.1311; found, 487.1305.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3-(4-methoxybenzamido)-4-fluorobenzamido)-4-fluorobenzoate
(40). Following the procedure for the preparation of compound 9, but
with compound 37 as starting material, compound 40 was obtained.
Yield 84.4%; mp 116−118 °C; [α]20D = −174.9 (c = 0.1, CH2Cl2); 1H
NMR (CDCl3, 600 MHz) δ 8.89 (d, J = 7.1 Hz, 1H), 8.84 (d, J = 7.4
Hz, 1H), 8.28 (s, 1H), 8.13 (s, 1H), 7.83 (d, J = 8.7 Hz, 2 H), 7.71 (m,
1H), 7.68−7.63 (m, 1H), 7.16 (m, 1H), 7.10−7.07 (m, 1H), 6.95 (d,
J = 8.7 Hz, 2H), 6.74 (s, 2H), 6.27 (d, J = 2.1 Hz, 1H), 6.09 (d, J = 2.1
Hz, 1H), 5.65 (s, 1H), 5.07 (s, 1H), 3.84 (s, 3 H), 3.76 (m, 15H), 3.06
(s, 2 H); 13C NMR (CDCl3, 125 MHz) δ 165.3, 164.6, 164.4, 163.2,
159.8, 159.0, 155.6, 153.3, 137.9, 133.5, 131.0, 129.2, 127.1, 126.6,
126.2, 124.7, 120.0, 115.8, 115.6, 115.3, 115.1, 114.3, 103.9, 100.3,
93.7, 92.2, 78.0, 69.2, 60.9,58.6, 56.2, 55.7, 55.5,26.2, 18.6; HRMS
(ESI) calcd for (C42H38F2N2O11 + Na)+ 807.2336; found, 807.2339.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3-(3,4-dimethoxybenzamido)-4-fluorobenzamido)-4-fluoroben-
zoate (41). Following the procedure for the preparation of compound
9, but with compound 38 as starting material, compound 41 was ob-
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3,4-dimethoxybenzamido)-4-fluorobenzoate (35). Following the
procedure for the preparation of compound 9, but with3-(3,4-
dimethoxybenzamido)-4-fluorobenzoic acid as starting material,
compound 35 was obtained. Yield 85.8%; mp 103−105 °C; [α]20
=
D
−168.1 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600 MHz) δ 8.96 (d, J =
6.1 Hz, 1 H), 7.94 (d, J = 2.8 Hz, 1 H), 7.71 (m, 1 H), 7.47 (d, J = 1.7
Hz, 1 H), 7.37 (m, 1 H), 7.09 (m, 1 H), 6.91 (d, J = 8.3 Hz, 1 H), 6.74
(s, 2 H), 6.28 (d, J = 2.2 Hz, 1 H), 6.11 (d, J = 2.2 Hz, 1 H), 5.66 (bs,
1 H), 5.08 (bs, 1 H), 3.95 (m, 6 H), 3.79 (m, 15 H), 3.07 (d, J = 3.3
Hz, 2 H); 13C NMR (CDCl3, 150 MHz) δ 164.9, 164.6, 159.7, 158.9,
155.6, 154.9, 153.2, 152.5, 149.4, 137.7, 133.4, 127.0, 126.7, 126.5,
124.0, 119.7, 115.0, 114.8, 110.8, 110.5, 103.8, 100.2, 93.6, 92.1, 77.9,
69.1, 60.9, 56.2, 56.1, 26.1; HRMS calcd for (C36H36FNO11 + H)+
678.2350; found, 678.2364.
tained. Yield 83.7%; mp 122−124 °C; [α]20 = −188.7 (c = 0.1,
D
1
CH2Cl2); H NMR (CDCl3, 500 MHz) δ 8.96 (d, J = 6.7 Hz, 1H),
8.88 (d, J = 7.2 Hz, 1H), 8.13 (s, 2H), 7.66 (m, 2H), 7.50 (s, 1H), 7.42
(d, J = 8.2 Hz, 1H), 7.22 (d, J = 9.1 Hz, 1H), 7.10 (m, 1H), 6.92 (d,
J = 8.3 Hz, 1H), 6.74 (s, 2H), 6.28 (s, 1H), 6.10 (s, 1H), 5.66 (bs,
1H), 5.08 (bs, 1H), 3.96 (s, 3 H), 3.94 (s, 3H), 3.79 (s, 6 H), 3.77 (s, 9
H), 3.06 (bs, 2H); 13C NMR (CDCl3, 125 MHz) δ 165.3, 164.6,
164.4, 159.8, 159.0, 155.6, 153.3, 152.8, 149.5, 137.9, 133.5, 131.0,
127.1, 126.5, 124.6, 120.1, 119.8, 115.8, 115.6, 115.2, 115.1, 110.9,
110.6, 103.9, 100.2, 93.7, 92.2, 78.0, 69.2, 60.9, 56.2, 55.5, 26.2; HRMS
calcd for (C43H40F2N2O12 + Na)+ 837.2442; found, 837.2436.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3,4,5- trimethoxybenzamido)-4-fluorobenzoate (36). Following
the procedure for the preparation of compound 9, but with 3-(3,4,5-
trimethoxybenzamido)-4-fluorobenzoic acid as starting material,
compound 36 was obtained. Yield 83.8%; mp 98−100 °C; [α]20
=
D
−172.1 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600 MHz) δ 8.89 (d, J =
7.2 Hz, 1 H), 7.93 (s, 1 H), 7.73 (m, 1 H), 7.10 (m, 1 H), 7.06 (s, 2
H),6.74 (s, 2 H), 6.27 (s, 1 H), 6.11 (s, 1 H), 5.66 (bs, 1 H), 5.08 (bs,
1 H), 3.91 (m, 9 H), 3.79 (m, 15 H), 3.06 (bs, 2 H); 13C NMR
(CDCl3, 150 MHz) δ 165.1, 164.5, 159.7, 158.9, 155.6, 155.0, 153.5,
153.2, 141.7, 137.7, 133.4, 129.6, 127.0, 126.8, 124.2, 115.7, 114.9,
104.9, 104.6, 104.6, 103.8, 103.8, 100.2, 93.6, 92.1, 77.9, 69.1, 61.1,
60.9, 56.5, 56.1, 55.5, 26.1; HRMS calcd for (C37H38FNO12 + H)+
708.2456; found, 708.2468.
(2R,3R)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3-(3,4,5-trimethoxybenzamido)-4-fluorobenzamido)-4-fluoro-
benzoate (42). Following the procedure for the preparation of
compound 9, but with compound 39 as starting material, compound
42 was obtained. Yield 84.9%; mp 125−127 °C; [α]20D = −116.6 (c =
1
0.1, CH2Cl2); H NMR (CDCl3, 600 MHz) δ 8.77 (dd, J = 7.4, 1.9
4-Fluoro-3-(4-fluoro-3-(4-methoxybenzamido)benzamido)-
benzoic Acid (37). Following the procedure for the preparation of
compound 33, but with 3-(4-methoxybenzamido)-4-fluorobenzoic
acid (compound 32) as starting material, compound 37 was obtained.
Hz, 1H), 8.71 (d, J = 7.0 Hz, 1H), 8.32 (s, 1H), 8.26 (s, 1H), 7.70 (m,
1H), 7.60−7.58 (m, 1H), 7.26 (s, 1H), 7.11 (d, J = 9.9 Hz, 1H), 7.09
(s, 2H), 7.02 (m, 1H), 6.72 (s, 2H), 6.24 (d, J = 2.2 Hz, 1H), 6.07 (d,
J = 2.3 Hz, 1H), 5.62 (bs, 1H), 5.06 (d, J = 17.7 Hz, 1H), 3.87 (s, 3H),
3.86 (s, 6H), 3.76 (s, 3 H), 3.74 (s, 3 H), 3.73 (s, 3 H), 3.72 (s, 6 H),
3.03 (bs, 2 H); 13C NMR (CDCl3, 150 MHz) δ 165.5, 164.5, 164.4,
159.7, 158.9, 157.2, 156.1, 155.5, 154.4, 153.3, 153.1, 141.6, 137.7,
133.4, 130.7, 129.1, 127.1, 126.7, 126.6, 126.3, 124.8, 121.2, 115.7,
115.1, 104.7, 103.8, 100.1, 93.6, 92.0, 77.8, 69.1, 60.9, 60.8, 56.3, 56.0,
55.4, 26.0; HRMS calcd for (C44H42F2N2O13 + Na)+ 867.2547; found,
867.2548.
1
Yield 78.6%; mp 238−240 °C; H NMR (DMSO-d6, 600 MHz) δ
10.34 (s, 1H), 10.16 (s, 1H), 8.22 (m, 2H), 8.00 (d, J = 8.8 Hz, 1H),
7.92 (m, 1H), 7.85 (m, 1H), 7.48 (dd, J = 9.8, 8.8 Hz, 1H), 7.45 (dd,
J = 9.8, 8.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H);13C NMR
(DMSO-d6, 125 MHz) δ 166.4, 165.0, 164.4, 162.3, 159.3, 159.1,
157.3, 157.0, 130.0, 129.8, 127.2, 126.7, 126.2, 126.1, 125.9, 125.7,
116.2, 113.8, 55.5.HRMS calcd for (C22H16O5F2N2 − H)+ 425.0944;
found, 425.0955.
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3,4,5-trimethoxybenzoate (43). Compound 8 (300.mg, 0.80 mmol),
3,4,5-trimethoxybenzoic acid (200 mg, 0.94 mmol), and Ph3P (840 mg,
3.20 mmol) were dissolved in anhydrous THF (20 mL), then DIAD
(650 mg, 3.20 mmol) was added at 0 °C. The mixture was stirred at
room temperature for 6 h under a nitrogen atmosphere. The solvent
was evaporated, and the residue was dissolved in EtOAc (50 mL), then
the mixture was washed by diluted hydrochloric acid two times and
in brine in sequence. The organic layer was dried with anhydrous
MgSO4. The solvent was evaporated, and the residue was purified by
3-(3-(3,4-Dimethoxybenzamido)-4-fluorobenzamido)-4-fluoro-
benzoic Acid (38). Following the procedure for the preparation of
compound 33, but with 3-(3,4-dimethoxybenzamido)-4-fluorobenzoic
acid as starting material, compound 38 was obtained. Yield 81.4%; mp
246−248 °C; 1H NMR (, DMSO-d6, 600 MHz) δ 10.33 (s, 1H), 10.19
(s, 1H), 8.22 (dd, J = 7.3, 2.0 Hz, 1H), 8.18 (d, J = 5.9 Hz, 1H), 7.91
(m, 1H), 7.84 (s, 1H), 7.66 (dd, J = 8.3, 2.1 Hz, 1H), 7.60 (d, J = 2.0
Hz, 1H), 7.46 (m, 1H), 7.38 (m, 1H), 7.11 (d, J = 8.6 Hz, 1H), 3.84
(s, 6H); 13C NMR (DMSO-d6, 125 MHz) δ 165.1, 164.4, 159.2, 157.2,
152.0, 148.5, 130.1, 128.2, 127.4, 126.2, 125.7, 121.5, 116.2, 111.1,
111.0, 55.8, 55.7. HRMS calcd for (C23H18O6F2N2 + Na)+ 479.1025;
found, 479.1017.
flash chromatography to afford the title compound 43 (240 mg, 52.2%
1
yield); mp 61−63 °C; [α]20 = +49.5 (c = 0.1, CH2Cl2); H NMR
D
(CDCl3, 600 MHz) δ7.10 (s, 1H), 6.65 (s, 1H), 6.19 (d, J = 2.3 Hz,
1H), 6.11 (d, J = 2.2 Hz, 1H), 5.49 (d, J = 7.4 Hz, 1H), 5.09 (d, J = 7.4
Hz, 1H), 3.86 (s, 3 H), 3.82 (s, 6 H), 3.81−3.74 (m, 15 H), 3.14 (A of
ABX, J = 16.5, 5.4 Hz, 1H), 2.80 (B of ABX, J = 16.5, 7.6 Hz, 1H); 13C
NMR (CDCl3, 150 MHz) δ 165.3, 160.0, 158.7, 155.0, 153.4, 153.0,
142.5, 138.1, 133.4, 125.1, 107.0, 104.1, 101.0, 100.0, 93.1, 92.0, 79.1,
3-(3-(3,4,5-Trimethoxybenzamido)-4-fluorobenzamido)-4-fluoro-
benzoic Acid (39). Following the procedure for the preparation of
compound 33, but with 3-(3,4,5-trimethoxybenzamido)-4-fluoroben-
zoic acid as starting material, compound 39 was obtained. Yield
81.8%; mp 250−252 °C; 1H NMR (DMSO-d6, 500 MHz) δ 10.40 (s,
1H), 10.30 (s, 1H), 8.25 (m, 2H), 8.00−7.95 (m, 1H), 7.86 (m, 1H),
P
DOI: 10.1021/acs.jmedchem.5b00085
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
70.3, 60.9, 56.3, 55.5, 25.0; HRMS calcd for (C30H34O11 + H)+
571.2174; found, 571.2161.
5.3 Hz, 1H), 2.76 (B of ABX, J = 16.8, 6.2 Hz, 1H); 13C NMR
(CDCl3, 150 MHz) δ 166.4, 159.9, 158.7, 154.7, 153.3, 151.3, 149.2,
145.3, 137.8, 133.6, 127.2, 122.9, 115.4, 111.0, 109.5, 103.6, 100.8,
93.0, 91.9, 78.5, 69.0, 60.9, 56.2, 56.0, 55.5, 23.8; HRMS calcd for
(C23H34O10 + H)+567.2225; found, 567.2211.
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-ol
(44). Following the procedure for the preparation of compound 9, but
with compound 4358 as starting material, compound 44 was obtained.
1
Yield 87.1%; mp 152−154 °C; [α]20 = −17.4 (c = 0.1, CH2Cl2); H
(E)-(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl 3-(3,4,5-trimethoxyphenyl)acrylate (49). Following the proce-
dure for the preparation of compound 45, but with (E)-3-(3,4,5-
trimethoxyphenyl)acrylic acid as starting material, compound 49 was
D
NMR (CDCl3, 600 MHz) δ 6.67 (s, 2 H), 6.14 (d, J = 2.1 Hz, 1H),
6.11 (d, J = 2.1 Hz, 1H), 4.62 (d, J = 8.5 Hz, 1H), 4.16−3.98 (m, 1H),
3.86 (s, 6 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.75 (s, 3 H), 3.09 (A of
ABX, J = 16.2, 5.8 Hz, 1H), 2.59 (B of ABX, J = 16.2, 9.3 Hz, 1H); 13C
NMR (CDCl3, 150 MHz) δ 159.8, 158.8, 155.3, 153.6, 138.2, 133.6,
104.2, 101.8, 93.1, 92.1, 82.3, 68.4, 60.9, 56.2, 55.6, 55.5, 27.9; HRMS
calcd for (C20H24O7 + H)+ 377.1595; found, 377.1588.
obtained. Yield 84.7%; mp 78−80 °C; [α]20 = +30.7 (c = 0.1,
D
1
CH2Cl2); H NMR (CDCl3, 500 MHz) δ 7.52 (d, J = 15.9 Hz, 1H),
6.70 (s, 2H), 6.62 (s, 2H), 6.28 (d, J = 15.9, 1H), 6.22 (s, 1H), 6.11 (s,
1H), 5.51 (m, 1H), 5.15 (d, J = 6.1 Hz, 1H), 3.87 (s, 9H), 3.81 (s,
9H), 3.78 (s, 6 H), 2.92 (A of ABX, J = 16.9, 5.1 Hz, 1H), 2.77 (B of
ABX, J = 16.9, 6.1 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 166.2,
160.0, 158.8, 154.8, 153.5, 153.4, 145.4, 140.4, 138.0, 133.6, 129.8,
117.1, 105.4, 103.7, 100.8, 93.1, 92.0, 78.5, 69.2, 61.1, 60.9, 56.3, 55.6,
55.5, 23.7; HRMS calcd for (C32H36O11 + H)+ 597.2330; found,
597.2321.
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
4-methoxybenzoate (45). Compound 44 (400 mg, 1.06 mmol),
4-methoxybenzoic acid (0.20 g, 1.31 mmol), 1-(3-(dimethylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl,400 mg, 2.12
mmol), and 4-dimethylaminopyridine (DMAP, 260 mg, 2.12 mmol)
were dissolved in anhydrous CH2Cl2 (30 mL), then the mixture was
stirred at room temperature overnight under a nitrogen atmosphere.
The solution was washed in diluted hydrochloric acid two times and in
brine in sequence. The organic layer was dried in anhydrous MgSO4.
The solvent was evaporated, and the residue was purified by flash
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3,4 - dimethoxybenzamido)-4-fluorobenzoate (50). Following the
procedure for the preparation of compound 45, but with 3-(3,4-
dimethoxybenzamido)-4-fluorobenzoic acid as starting material,
compound 50 was obtained. Yield 83.2%; mp 99−101 °C; [α]20
=
chromatography to afford the title compound 45 (480 mg, 89.5%
D
1
yield); mp 69−71 °C; [α]20 = +25.4 (c = 0.1, CH2Cl2); H NMR
1
+64.5 (c = 0.1, CH2Cl2); H NMR (CDCl3, 500 MHz) δ 8.99 (d, J =
6.6 Hz, 1H), 8.01 (s, 1H), 7.69 (m, 1H), 7.48 (s, 1H), 7.40 (d, J = 7.7
Hz, 1H), 7.11 (m, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.70 (s, 2H), 6.19 (d,
J = 1.7 Hz, 1H), 6.11 (d, J = 1.7 Hz, 1H), 5.49 (dd, J = 14.0, 7.9 Hz,
1H), 5.08 (d, J = 8.1 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 3.82−3.73
(m, 15H), 3.20 (A of ABX, J = 16.5, 5.7 Hz, 1H), 2.80 (B of ABX, J =
16.5, 8.2 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ 165.0, 164.4, 160.0,
158.7, 156.6, 155.1, 154.6, 153.3, 152.6, 149.4, 138.0, 133.4, 127.8,
123.5, 119.8, 115.0, 110.7, 104.1, 101.0, 93.2, 92.1, 79.1, 70.6, 60.8,
56.2, 55.5, 25.4; HRMS calcd for (C36H36FNO11 + H)+ 678.2345;
found, 678.2337.
D
(CDCl3, 500 MHz) δ 7.88 (d, J = 8.8 Hz, 2 H), 6.86 (d, J = 8.8 Hz,
2 H), 6.65 (s, 2 H), 6.21 (d, J = 2.2 Hz, 1H), 6.12 (d, J = 2.2 Hz, 1H),
5.51 (dd, J = 12.6, 7.0 Hz, 1H), 5.13 (d, J = 7.0 Hz, 1H), 3.83 (s, 3 H),
3.79 (s, 3 H), 3.78 (s, 12 H), 3.07 (A of ABX, J = 16.7, 5.5 Hz, 1H),
2.79 (B of ABX, J = 16.7, 7.1 Hz, 1H); 13C NMR (CDCl3, 125 MHz)
δ 165.3, 163.5, 159.8, 158.6, 154.8, 153.2, 137.8, 133.5, 131.6 131.0,
122.3, 113.6, 103.7, 100.9, 93.0, 91.9, 78.8, 69.6, 60.8, 56.1, 55.3, 24.5,
21.4; HRMS calcd for (C28H30O9 + H)+ 511.1963; found, 511.1948.
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3,4-dimethoxybenzoate (46). Following the procedure for the pre-
paration of compound 45, but with 3,4-dimethoxybenzoic acid as start-
ing material, compound 46 was obtained. Yield 87.4%; mp 70−72 °C;
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-(3,4,5- trimethoxybenzamido)-4-fluorobenzoate (51). Following
the procedure for the preparation of compound 45, but with 3-(3,4,5-
trimethoxybenzamido)-4-fluorobenzoic acid as starting material,
1
[α]20D=+59.1 (c = 0.1, CH2Cl2); H NMR (CDCl3, 600 MHz)δ 7.54
(dd, J = 8.4, 2.0 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 6.82 (d, J = 8.4 Hz,
1H), 6.65 (s, 2H), 6.21 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H),
5.51 (m, 1H), 5.12 (d, J = 7.1 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.77
(m, 15H), 3.10 (A of ABX, J = 16.6, 5.5 Hz, 1H), 2.80 (B of ABX, J =
16.6, 7.3 Hz, 1H); 13C NMR (CDCl3, 150 MHz) δ 165.5, 159.9, 158.7,
154.9, 153.3, 153.2, 148.7, 137.9, 133.6, 123.7, 122.5, 112.1, 110.2,
103.8, 101.0, 93.1, 92.0, 78.9, 69.9, 60.9, 56.2, 55.5, 24.7; HRMS calcd
for (C29H32O10 + H)+ 541.2068; found, 541.2055.
compound 51 was obtained. Yield 84.6%; mp 99−101 °C; [α]20
=
D
1
+64.5 (c = 0.1, CH2Cl2); H NMR (CDCl3, 500 MHz) δ 8.94 (d, J =
6.8 Hz, 1H), 7.97 (s, 1H), 7.72−7.64 (m, 1H), 7.13 (m, 1H), 7.08 (s,
2H), 6.69 (s, 2H), 6.19 (d, J = 1.2 Hz, 1H), 6.11 (d, J = 1.2 Hz, 1H),
5.50 (dd, J = 14.0, 8.0 Hz, 1H), 5.08 (d, J = 8.0 Hz, 1H), 3.92 (s, 6H),
3.91 (s, 3H), 3.81−3.74 (m, 15H), 3.19 (A of ABX, J = 16.5, 5.7 Hz,
1H), 2.80 (B of ABX, J = 16.5, 8.1 Hz, 1H); 13C NMR (CDCl3, 125
MHz) δ 165.3, 164.4, 160.0, 158.7, 156.7, 155.0, 154.7, 153.5, 153.3,
141.7, 137.9, 133.4, 129.5,126.9, 126.5, 123.6, 115.1, 104.6, 104.0,
101.0, 93.1, 92.1, 79.1, 70.6, 61.0, 56.3, 55.5, 25.4; HRMS calcd for
(C37H38FNO12 + H)+ 708.2451; found, 708.2443.
(E)-(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl3-(4-methoxyphenyl)acrylate (47). Following the procedure for
the preparation of compound 45, but with (E)-3-(4-methoxyphenyl)-
acrylic acid as starting material, compound 47 was obtained. Yield
1
84.3%; mp 66−68 °C; [α]20D= +36.1 (c = 0.1, CH2Cl2); H NMR
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-4-fluorobenzoate (52).
Following the procedure for the preparation of compound 45, but
with (E)-3-(3-(3,4-dimethoxyphenyl)acrylamido)-4-fluorobenzoic acid
(CDCl3, 500 MHz)δ 7.57 (d, J = 16.0 Hz, 1H), 7.42 (d, J = 8.6 Hz,
2H), 6.87 (d, J = 8.6 Hz, 2H), 6.63 (s, 1H), 6.23 (d, J = 16.0 Hz,
1 H),6.21 (d, J = 2.3 Hz, 1H), 6.11 (d, J = 2.3 Hz, 1H), 5.48 (m, 1H),
5.13 (d, J = 6.5 Hz, 1H), 3.81 (s, 12 H), 3.78 (s, 6 H), 2.96 (A of ABX,
J = 16.8, 5.3 Hz, 1H), 2.76 (B of ABX, J = 16.8, 6.5 Hz, 1H); 13C NMR
(CDCl3, 125 MHz) δ 166.5, 161.6, 159.9, 158.7, 154.8, 153.3,
145.1, 137.8, 133.6, 132.3, 129.9, 127.0, 115.2, 114.4, 113.5, 103.7,
100.8, 93.0, 91.9, 78.6, 69.0, 60.9, 56.2, 55.5, 24.0; HRMS calcd for
(C30H32O9 + H)+ 537.2117; found, 537.2107.
as starting material, compound 52 was obtained. Yield 82.8%; mp
1
112−114 °C; [α]20 = +64.6 (c = 0.09, CH2Cl2); H NMR (CDCl3,
D
500 MHz) δ 9.05 (d, J = 6.2 Hz, 1H), 7.70 (d, J = 15.4 Hz, 1H), 7.67−
7.63 (m, 1H), 7.50 (bs, 1H), 7.14 (dd, J = 8.3, 1.7 Hz, 1H), 7.12−7.07
(m, 2 H), 6.88 (d, J = 8.3 Hz, 1H), 6.71 (s, 2H), 6.45 (d, J = 15.4 Hz,
1H), 6.20 (d, J = 2.0 Hz, 1H), 6.12 (d, J = 2.0 Hz, 1H), 5.49 (m, 1H),
5.08 (d, J = 8.1 Hz, 1H), 3.92 (m, 6H), 3.79 (m, 15 H), 3.21 (A of
ABX, J = 16.5, 5.8 Hz, 1H), 2.81 (B of ABX, J = 16.5, 8.3 Hz, 1H); 13C
NMR (CDCl3, 125 MHz) δ 164.5, 164.1, 160.0, 158.8, 155.1, 153.4,
151.4, 149.4, 143.5, 133.4, 127.4, 123.3, 122.8, 117.8, 115.0, 111.3,
109.9, 104.2, 101.2, 93.3, 92.2, 79.2, 70.7, 60.9, 57.6, 55.6, 25.6; HRMS
calcd for (C38H38FNO11 + H)+ 704.2502; found, 704.2488.
(E)-(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman
−3-yl 3-(3,4-dimethoxyphenyl)acrylate (48). Following the proce-
dure for the preparation of compound 45, but with (E)-3-(3,4-
dimethoxyphenyl)acrylic acid as starting material, compound 48 was
obtained. Yield 85.1%; mp 73−76 °C; [α]20 = +37.1 (c = 0.1,
D
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.55 (d, J = 15.9 Hz, 1H),
7.04 (dd, J = 8.4, 1.9 Hz, 1H), 6.99 (d, J = 1.9 Hz, 1H), 6.83 (d, J = 8.4
Hz, 1H), 6.62 (s, 2H), 6.24 (d, J = 15.9 Hz, 1H), 6.21 (d, J = 2.2 Hz,
1H), 6.10 (d, J = 2.2 Hz, 1H), 5.50 (m, 1H), 5.15 (d, J = 6.2 Hz, 1H),
3.89 (s, 3H), 3.88 (s, 3H), 3.80 (m, 15 H), 2.92 (A of ABX, J = 16.8,
(2R,3S)-5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-yl
3-((E)-3-(3,4,5-trimethoxyphenyl)acrylamido)-4-fluorobenzoate
(53). Following the procedure for the preparation of compound 45,
but with (E)-3-(3-(3,4,5-trimethoxyphenyl)acrylamido)-4-fluorobenzoic
Q
DOI: 10.1021/acs.jmedchem.5b00085
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
acid as starting material, compound 52 was obtained. Yield 83.0%; mp
104.6, 93.8, 81.4, 74.2, 61.1, 60.9, 56.4, 56.3, 55.8; HRMS calcd for
(C30H33O12 + H)+ 585.1967; found, 585.1972.
1
111−113 °C; [α]20 = +80.2 (c = 0.1, CH2Cl2); H NMR (CDCl3,
D
500 MHz) δ 9.03 (d, J = 7.2 Hz, 1H), 7.66 (m, 3 H), 7.08 (t, J = 9.3
Hz, 1H), 6.77 (s, 2 H), 6.70 (s, 2H), 6.56−6.48 (m, 1H), 6.19 (s, 1H),
6.12 (s, 1H), 5.57−5.43 (m, 1H), 5.08 (d, J = 8.1 Hz, 1H), 3.88 (s, 9
H), 3.81−3.74 (m, 15 H), 3.20 (A of ABX, J = 16.5, 5.6 Hz, 1H), 2.80
(B of ABX, J = 16.5, 8.2 Hz, 1H); 13C NMR (CDCl3, 125 MHz) δ
164.5, 163.9, 160.0, 158.8, 155.1, 153.6, 153.4, 143.6, 140.4, 138.0,
133.4, 129.9, 123.4, 119.4, 115.1, 114.9, 105.5, 104.2, 101.1, 93.2, 92.2,
79.2, 70.7, 61.2, 60.9, 56.3, 56.2, 55.6, 55.5, 25.5; HRMS calcd for
(C39H40FNO12 + H)+ 734.2607; found, 734.2598.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 3,4,5-tris(allyloxy)benzoate (59). Following the
procedure for the preparation of compound 56, but with 3,4,5-
tris(allyloxy)benzoic acid as starting material, compound 59 was
obtained. Yield 78.7%; mp 122−124 °C; [α]20 = +28.1 (c = 0.1,
D
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.20 (s, 2H), 6.72 (s, 2H),
6.19 (d, J = 2.2 Hz, 1H), 6.13 (d, J = 2.2 Hz, 1H), 6.03 (m, 3H), 5.85
(d, J = 12.1 Hz, 1H), 5.44 (A1 of A1B1X1, J = 12.0 Hz, 1.4 Hz, 1H),
5.39 (A2 of A2B2X2, J = 17.3, 1.2 Hz, 2H), 5.30 (B1 of A1B1X1, J = 17.2,
1.4 Hz, 1H), 5.26 (B2 of A2B2X2, J = 10.6, 1.2 Hz, 2H), 5.18 (d, J=12.1
Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H), 4.55 (d, J = 5.1 Hz, 4H), 3.88 (s,
3H), 3.84 (s, 3 H), 3.81 (s, 6 H), 3.80 (s, 3 H); 13CNMR (CDCl3, 150
MHz) δ 184.1, 165.9, 164.1, 163.4, 161.9, 152.7, 151.5, 141.6, 138.0,
133.4, 132.2, 130.4, 123.5, 117.3, 117.1, 108.6, 103.8, 92.9, 80.6, 73.4,
69.4, 60.2, 55.5, 55.1. HRMS calcd for (C36H39O12H)+ 663.2436;
found, 663.2446.
(2R,3R)-5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl Acetate (55). Acetic anhydride (1 mL) and pyridine
(1.5 mL) were stirred at 0 °C, and (2R,3R)-3-hydroxy-5,7-dimethoxy-
2-(3,4,5-trimethoxyphenyl)chroman-4-one54 (200 mg, 0.51 mmol)59
was added. The mixture was stirred at room temperature for 36 h and
then poured into ice water (5 mL), and a copious amount of white
solid was precipitated. The solid was dissolved in EtOAc (30 mL).
Then, the solution was washed in diluted hydrochloric acid two times
and in brine in sequence. The organic layer was dried with anhydrous
MgSO4. The solvent was evaporated, and the residue was purified by
flash chromatography to afford the title compound 55 (190 mg, 85.4%
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl (E)-3-(4-methoxyphenyl)acrylate (60). Following the
procedure for the preparation of compound 56, but with (E)-3-(4-
methoxyphenyl)acrylic acid as starting material, compound 60 was
obtained. Yield 81.2%; mp 123−125 °C; [α]20 = +6.1 (c = 0.1,
1
yield); mp187−189 °C; [α]20D= +6.6 (c = 0.1, CH2Cl2); HNMR
D
CH2Cl2); 1H NMR (CDCl3, 600 MHz) δ 7.58 (d, J = 16.4 Hz,
1H),6.73 (s, 2H), 6.69 (s, 2H), 6.31 (d, J = 16.4 Hz, 1H), 6.16 (d, J =
2.2 Hz, 1H), 6.12 (d, J = 2.2 Hz, 1H), 5.85 (d, J = 12.1 Hz, 1H), 5.46
(d, J = 12.1 Hz, 1H), 3.85 (m, 18 H); 13C NMR (CDCl3, 150 MHz)
δ185.0, 166.7, 165.4, 164.2, 162.6, 153.5, 146.1, 140.3, 138.8, 131.1,
129.8, 116.3, 105.4, 104.7, 104.3, 93.7, 81.4, 73.5, 61.0, 56.2, 55.8;
HRMS calcd for (C30H31O10 + H)+ 551.1912; found, 551.1919;
(C30H31O10 + Na)+ 573.1731; found, 573.1740.
(CDCl3, 600 MHz)δ6.69 (s, 2H), 6.19 (d, J = 2.2 Hz, 1H), 6.13 (d, J =
2.2 Hz, 1H), 5.67 (d, J = 12.1 Hz, 1H), 5.27 (d, J = 12.1 Hz, 1H), 3.85
(m, 15 H), 2.21(s, 3H); 13C NMR (CDCl3, 150 MHz) δ184.9, 169.6,
166.6, 164.1, 162.6, 153.5, 138.7, 131.1, 104.6, 104.5, 104.4, 93.6, 81.3,
73.5, 56.3, 56.2, 55.8; HRMS calcd for (C22H24O9 + H)+ 433.1493;
found, 433.1498; (C17H16O6NF+Na)+ 455.1313; found, 455.1314.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 4-methoxybenzoate (56). (2R,3R)-3-hydroxy-5,7-
dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-4-one 54 (200 mg,
0.51 mmol), 4-methoxybenzoic acid (150 mg, 0.99 mmol), EDC·
HCl (200 mg, 1.06 mmol), and DMAP (130 mg, 1.06 mmol) were
dissolved in anhydrous CH2Cl2 (20 mL), then the mixture was stirred
at room temperature overnight under a nitrogen atmosphere. Then,
the solution was washed with diluted hydrochloric acid two times and
in brine in sequence. The organic layer was dried with anhydrous
MgSO4. The solvent was evaporated, and the residue was purified by
flash chromatography to afford the title compound 56 (230 mg, 85.4%
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl (E)-3-(3,4-Dimethoxyphenyl)acrylate (61). Following
the procedure for the preparation of compound 56, but with (E)-3-
(3,4-dimethoxyphenyl)acrylic acid as starting material, compound 61
was obtained. Yield 80.6%; mp 121−123 °C; [α]20D = +12.7 (c = 0.1,
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.61 (d, J = 15.9 Hz, 1H),
7.05 (dd, J = 8.3, 1.9 Hz, 1H), 7.00 (d, J = 1.9 Hz, 1H), 6.84 (d, J = 8.3
Hz, 1H), 6.73 (s, 2H), 6.28 (d, J = 15.9 Hz, 1H), 6.16 (d, J = 1.7 Hz,
1H), 6.13 (d, J = 1.7 Hz, 1H), 5.83 (d, J = 11.9 Hz, 1H), 5.39 (d, J =
11.9 Hz, 1H), 3.90−3.83 (m, 21 H). 13C NMR (CDCl3, 150 MHz) δ:
185.1, 166.6, 165.7, 164.2, 162.6, 153.5, 151.3, 149.2, 146.1, 138.7,
131.1, 127.3, 122.9, 114.7, 111.0, 109.7, 104.7, 104.4, 93.6, 81.4, 73.4,
60.9, 56.3, 56.1, 55.8; HRMS calcd for (C31H33O11 + H)+ 581.2017;
found, 581.2021.
1
yield); mp 124−126 °C; [α]20D= +10.7 (c = 0.1, CH2Cl2); H NMR
(CDCl3, 600 MHz) δ 7.93 (s, 1 H), 7.91 (s, 1 H), 6.86 (s, 1 H), 6.84
(s, 1 H), 6.73 (s, 2H), 6.19 (d, J = 2.2 Hz, 1H), 6.13 (d, J = 2.2 Hz,
1H), 5.86 (d, J = 12.1 Hz, 1 H), 5.44 (d, J = 12.1 Hz, 1 H), 3.82(s,
3H), 3.81 (s, 6 H), 3.80 (s, 9 H); 13C NMR (CDCl3, 150 MHz) δ
185.1, 166.6, 164.9, 163.7, 162.6, 153.4, 138.6, 132.1, 131.3, 121.8,
113.7, 104.5, 93.7, 81.4, 73.8, 60.9, 56.2, 55.8, 55.5; HRMS calcd for
(C29H33O9 + H)+ 525.1755; found, 525.1764.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl (E)-3-(3,4,5-Trimethoxyphenyl)acrylate (62). Follow-
ing the procedure for the preparation of compound 56, but with (E)-3-
(3,4,5-trimethoxyphenyl)acrylic acid as starting material, compound
62 was obtained. Yield 83.6%; mp 116−119 °C; [α]20D = +6.5 (c = 0.1,
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 3,4-dimethoxybenzoate (57). Following the procedure
for the preparation of compound 56, but with 3,4-dimethoxybenzoic
acid as starting material, compound 57 was obtained. Yield 83.3%; mp
125−127 °C; [α]20D = +5.2 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600
MHz) δ 7.61 (dd, J = 2.1, 8.3 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 6.82
(d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 6.19 (d, J = 2.2 Hz, 1H), 6.13 (d, J =
2.2 Hz, 1H), 5.87 (d, J = 12.1 Hz, 1H), 5.46 (d, J = 12.1 Hz, 1H), 3.89
(s, 3H), 3.88 (s, 3 H), 3.87 (s, 3 H), 3.84 (s, 3 H), 3.81 (s, 9 H); 13C
NMR (CDCl3, 150 MHz) δ 185.1, 166.6, 164.2, 162.6, 153.4, 148.6,
138.7, 131.3, 124.1, 121.9, 112.4, 110.2, 104.5, 93.7, 81.4, 73.9, 60.9,
56.2, 56.1, 55.8; HRMS calcd for (C29H31O11 + H)+ 555.1861; found,
555.1866.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)chroman-
3-yl 3,4,5-trimethoxybenzoate (58). Following the procedure for the
preparation of compound 56, but with 3,4,5-trimethoxybenzoic acid
as starting material, compound 58 was obtained. Yield 84.3%; mp
126−128 °C; [α]20D = +4.1 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600
MHz) δ 7.21 (s, 2H), 6.74 (s, 2H), 6.19 (d, J = 2.2 Hz, 1H), 6.13 (d,
J = 2.2 Hz, 1H), 5.84 (d, J = 12.1 Hz, 1H), 5.46 (d, J = 12.1 Hz, 1H),
3.84 (m, 24 H); 13C NMR (CDCl3, 150 MHz) δ 184.9, 166.7, 165.0,
164.3, 162.7, 153.5, 152.9, 142.7, 141.4, 138.8, 131.2, 124.5, 107.4,
1
CH2Cl2); H NMR (CDCl3, 600 MHz) δ 7.61 (d, J = 15.9 Hz, 1H),
7.41 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.73 (s, 2H), 6.26
(d, J = 15.9 Hz, 1H), 6.15 (d, J = 1.8 Hz, 1H), 6.12 (d, J = 1.8 Hz,
1H), 5.80 (d, J = 12.1 Hz, 1H), 5.38 (d, J = 12.1 Hz, 1H), 3.85 (m, 24
H); 13CNMR (CDCl3, 150 MHz) δ 185.0, 166.6, 165.8, 164.2, 162.6,
161.6, 153.4, 145.8, 138.7, 131.2, 129.9, 127.1, 114.4, 104.6, 104.4,
93.6, 81.4, 73.4, 60.9, 56.3, 55.8, 55.5. HRMS calcd for (C32H35O12
+
H)+ 611.2123; found, 611.2134; (C30H31O10 + Na)+ 633.1942; found,
633.1953.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 3-(3,4-dimethoxybenzamido)-4-fluorobenzoate (63).
Following the procedure for the preparation of compound 56, but with
3-(3,4-dimethoxybenzamido)-4-fluorobenzoic acid as starting material,
compound 63 was obtained. Yield 82.3%; mp 127−129 °C; [α]20
=
D
+16.7 (c = 0.1, CH2Cl2); 1H NMR (CDCl3, 600 MHz) δ 9.05 (s, 1H),
8.04 (s, 1H), 7.75 (s, 1H), 7.47 (s, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.12
(t, J = 8.8 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H), 6.79 (s, 2H),6.18 (d, J =
2.2 Hz, 1H), 6.12 (d, J = 2.2 Hz, 1H), 5.86 (d, J = 12.1 Hz, 1H), 5.47
(d, J = 12.1 Hz, 1H), 3.94 (s, 6H), 3.93 (s, 9 H), 3.83 (s, 3 H), 3.80 (s,
3 H); 13CNMR (CDCl3, 150 MHz) δ 184.6, 166.7, 164.2, 164.1,
R
DOI: 10.1021/acs.jmedchem.5b00085
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Journal of Medicinal Chemistry
Article
162.6, 156.6, 154.9, 153.4, 152.5, 149.3, 138.6, 131.3, 126.8, 123.8,
119.8, 115.1, 110.7, 110.5, 104.4, 104.3, 93.7, 81.3, 74.4, 60.9, 56.2,
55.8; HRMS calcd for (C36H35O12NF + H)+ 692.2138; found,
692.2141.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 3-(3,4,5-trimethoxybenzamido)-4-fluorobenzoate
(64). Following the procedure for the preparation of compound 56,
but with 3-(3,4,5-trimethoxybenzamido)-4-fluorobenzoic acid as
confluent monolayer was formed. To split cells, the plate was washed
briefly with phosphate-buffered saline (PBS), treated with 0.05%
trypsin-EDTA, and harvested by centrifugation.
Cell Proliferation Assay. Six thousand cells of LCC6 or
LCC6MDR and PTX (concentrations including 400, 133, 44, 15, 5,
1.6, and 0 nM) were mixed with or without modulators to a final
volume of 200 μL in each well of 96-well plates. Four thousand cells of
2008/P or 2008/MRP1 and DOX (concentrations including 2000,
667, 222, 74, 25, 8, and 0 nM) were coincubated with or without
modulators to a final volume of 200 μL. Four thousand five hundred
cells of HEK293/pcDNA3.1 or HEK293/R2 and topotecan (con-
centrations including 2000, 667, 222, 74, 25, 8, and 0 nM) were
coincubated with or without modulators to a final volume of 200 μL.
The plates were then incubated for 5 days at 37 °C. The cell viability
was determined using the CellTiter 96 AQueous Assay (Promega) as
reported previously.58,59 The IC50 of the anticancer drugs of the cell
line was determined using nonlinear regression dose−response cuve
analysis of the Prism software.
EC50 and Selective Index Value Determination. Six thousand
LCC6MDR cells were incubated with different concentrations of PTX
(400, 133, 44, 15, 5, 1.6, and 0 nM), vinblastine (20, 6.7, 2.2, 0.7, 0.2,
0.08, and 0 nM), vincristine (50, 16.7, 5.6, 1.9, 0.6, 0.2, and 0 nM) or
DOX (10, 3.3, 1, 0.4, 0.1, 0.04, and 0 μM) with different con-
centrations of modulators (1000, 500, 250, 125, 62.5, 31.3, and 0 nM)
for 5 days at 37 °C in a volume of 200 μL. The percentage of survival
was determined using the MTS assay as mentioned previously.58,59
IC50 (drug) was determined using Prism 5.0 (GraphPad). EC50 was
defined as the concentration of modulator needed to reduce the
IC50 (drug) by 50%. Selective index value is an indicator of compound
safety. It is determined by dividing the IC50 of the modulator toward
mouse fibroblast cells L929 by the EC50 of the modulator.
starting material, compound 64 was obtained. Yield 81.4%; mp117−
1
119 °C; [α]20 = +10.2 (c = 0.1, CH2Cl2); H NMR (CDCl3, 600
D
MHz) δ 8.97 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.14 (m, 1H), 7.08 (s,
2H), 6.78 (s, 2H), 6.18 (d, J = 2.2 Hz, 1H), 6.12 (d, J = 2.2 Hz, 1H),
5.86 (d, J = 12.1 Hz, 1H), 5.47 (d, J = 12.1 Hz, 1H), 3.85 (m, 24 H);
13CNMR (CDCl3, 150 MHz) δ 184.6, 166.7, 164.2, 164.2, 162.4,
156.9, 155.4, 153.7, 141.8, 138.9, 131.3, 129.8, 127.4, 127.3, 124.1,
115.3, 115.1, 104.9, 104.2, 104.3, 93.8, 81.5, 74.2, 61.1, 56.8, 56.2.
HRMS calcd for (C37H37O13NF + H)+ 722.2243; found, 722.2260.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 3-((E)-3-(3,4-dimethoxyphenyl)acrylamido)-4-fluoro-
benzoate (65). Following the procedure for the preparation of
compound 56, but with (E)-3-(3-(3,4-dimethoxyphenyl)acrylamido)-
4-fluorobenzoic acid as starting material, compound 65 was obtained.
1
Yield 84.7%; mp 153−155 °C; [α]20 = +26.2 (c = 0.1, CH2Cl2); H
D
NMR (CDCl3, 600 MHz) δ 9.06 (d,J = 6.1 Hz, 1H), 7.71 (m, 1H),
7.68 (s, 1H), 7.64 (d, J = 15.4 Hz, 1H), 7.08 (m, 1H), 7.06 (s, 2H),
6.86 (d, J = 8.2 Hz, 1H), 6.79 (s, 2H), 6.47 (d, J = 15.4 Hz, 1H), 6.19
(d, J = 2.2 Hz, 1H), 6.13 (d, J = 2.2 Hz, 1H), 5.88 (d, J = 12.1 Hz,
1H), 5.49 (d, J = 12.1 Hz, 1H), 3.85 (m, 21H); 13CNMR (CDCl3, 150
MHz) δ184.9, 166.8, 164.4, 164.3, 162.8, 153.8,151.2, 149.4, 143.4,
138.7, 131.2, 127.6, 126.8, 126.3, 123.8, 122.9, 118.4, 115.1, 114.8,
111.3, 109.9, 104.2, 93.8, 81.4, 74.2, 60.8, 56.4, 56.2. HRMS calcd for
(C38H37O12NF + H)+ 718.2294; found, 718.2311.
Cytotoxicity Assay. Ten thousand cells of LCC6 or LCC6MDR
or L929 were mixed with different concentrations (100, 33.3, 11.1, 3.7,
1.2, 0.4, and 0 μM) of modulators to a final volume of 100 μL in each
well of 96-well plates. The plates were then incubated for 3 days at
37 °C. The percentage of survival was determined using the MTS
assay, and the IC50 of the modulators was determined using Prism
software.
(2R,3R)-5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-
chroman-3-yl 4-fluoro-3-((E)-3-(3,4,5-trimethoxyphenyl)-
acrylamido)benzoate (66). Following the procedure for the pre-
paration of compound 56, but with (E)-3-(3-(3,4,5-trimethoxyphenyl)-
acrylamido)-4-fluorobenzoic acid as starting material, compound 66 was
obtained. Yield 80.7%; mp 169−172 °C; [α]20 = +14.7 (c = 0.1,
D
CH2Cl2); 1H NMR (CDCl3, 600 MHz) δ 9.05 (d, J = 6.6 Hz, 1H), 7.71
(s, 2H), 7.62 (d, J = 15.4 Hz, 1H), 7.08 (t, J = 8.8 Hz, 1H), 6.79 (s, 2H),
6.77 (s, 2H), 6.52 (d, J = 15.4 Hz, 1H), 6.20 (d, J = 2.2 Hz, 1H), 6.14
(d, J = 2.2 Hz, 1H), 5.86 (d, J = 12.1 Hz, 1H), 5.49 (d, J = 12.1 Hz, 1H),
3.85 (m, 24 H); 13CNMR (CDCl3, 150 MHz) δ 184.7, 166.8, 164.3,
164.2, 162.7, 153.5, 143.1, 140.1, 138.6, 131.2, 130.1, 126.8, 126.1, 123.7,
119.7, 115.1, 114.9, 106.5, 105.3, 104.4, 93.8, 81.4, 74.4, 61.1, 60.9, 56.3,
55.9; HRMS calcd for (C39H39O13NF + H)+ 748.2400; found, 748.2421.
Materials for Biological Studies. DMSO, verapamil, doxorubicin
(DOX), topotecan, and paclitaxel (PTX) were purchased from Sigma-
Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), trypsin-
ethylenediaminetetracetic acid (EDTA), and penicillin/streptomycin
were from Gibco BRL. Fetal bovine serum (FBS) was from Hyclone
Laboratories. 2-(4,5-Dimethylthiazol-2-yl-)-5-[3-(carboxymethoxy)
phenyl]- 2-(4-sulfophenyl)-2H-tetrazolium (MTS), and phenazine
methosulfate (PMS) were purchased from Promega. Human breast
cancer cell lines MDA435/LCC6 and MDA435/LCC6MDR were
kindly provided by Dr. Robert Clarke (Georgetown University,
Washington, DC). The human ovarian carcinoma cell lines 2008/P
and 2008/MRP1 were generous gifts from Professor P. Borst (The
Netherlands Cancer Institute, Amsterdam, The Netherlands). The
HEK293/pcDNA3.1 and HEK293/R2 cell lines were kindly provided
by Dr. Kenneth To (The Chinese University of Hong Kong, Hong
Kong). The L929 cell line was purchased from ATCC.
Intracellular DOX Accumulation. LCC6 or LCC6MDR cells
(1 ×106 cells) were mixed with 20 μM DOX and 2 μM of the
modulator at 37 °C for 150 min. DMSO (0.2%) was used as a negative
control. After incubation, the cells were spun down and washed with
cold PBS, pH 7.4, and lysed with lysis buffer (0.75 M HCl and 0.2%
Triton-X100 in isopropanol). The lysate was spun down, and the
supernatant was saved. The fluorescence level of DOX was determined
as reported previously.58,59
DOX Influx and DOX Efflux. For the influx, 5 μM DOX with or
without 1.5 μM of the modulator was added to LCC6 or LCC6MDR
cells. After 0, 10, 20, 30, and 40 min, 1 × 106 cells were taken out and
chilled on ice. For the efflux, LCC6 and LCC6MDR cells were
preincubated with 20 μM oDOX for 1 h at 37 °C. After 1 h, the cells
were washed once with 1× PBS. The washed cells were resuspended
with supplemented DMEM media with or without 1.5 μM of the
modulator. After 0, 30, 60, 90, 120, and 150 min, the cells were taken
out and chilled on ice. The intracellular DOX level at each time point
was measured as described previously.58,59
Intracellular Accumulation of Methylated EGC (23) and
Methylated GC (51). LCC6 or LCC6MDR (3 ×106 cells) were
incubated with 20 or 200 μM 23 or 51 at 37 °C for 150 min. The cell
lysis and determination methods were according to a previous
report.49 A reversed phase Diamonsil C18 (4.6 × 150 mm) column
attached to a Gilson 322 pump coupled to a Gilson UV−vis-152
detector was used. Each sample was injected at a volume of 20 μL and
eluted with methanol, and the flow rate was 1 mL/min. The peak
wavelength of 23 was detected at 308 nm, and the retention time was
3.26 min. The peak wavelength of 51 was detected at 270 nm, and the
retention time was 3.23 min.
Cell Culture. MDA435/LCC6, MDA435/LCC6MDR, and L929
cell lines were cultured in supplemented DMEM media with 10%
heat inactivated FBS and 100 U/mL penicillin and 100 μg/mL of
streptomycin. 2008/P, 2008/MRP1, HEK293/pcDNA3.1, and
HEK293/R2 cells were cultured in RPMI 1640 medium containing
heat inactivated 10% FBS and 100 U/mL penicillin and 100 μg/mL
of streptomycin. They were maintained at 37 °C in a humidified
atmosphere with 5% CO2. The cells were split constantly after a
Calculated Molecular Descriptors. Calculated descriptors such
as cLogP and PSA were determined by SYBYL-X 2.0. The structures
of compounds 35−38 were built and energy minimized under the
S
DOI: 10.1021/acs.jmedchem.5b00085
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Tripos force field with 0.05 kcal/(mol Å). The Gasteiger−Huckel
method was used to calculate the charges. Energy minimization was
performed by the Powell method with 2000 iterations. Then, the
distance of the linkers was calculated.
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42.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra and 13C NMR spectra of all compounds. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00085.
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AUTHOR INFORMATION
Corresponding Authors
*(S.B.W.) Tel: 86-532-82031087. Fax: 86-532-82033054.
E-mail: biaowan@ouc.edu.cn.
*(L.M.C.C.) Tel: 852-34008662. Fax: 852-23649932. E-mail:
bclchow@polyu.edu.hk.
■
Author Contributions
§I.L.K.W. and B-C.W. contributed equally to this work.
Compounds 30, 35, 36, 43, 50, and 51 were synthesized by J.Y.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was funded by National Natural Science Foundation
of China (NSFC 81172926), NSFC-Shandong Joint Fund for
Marine Science Research Centers (U1406402), General
Research Fund (PolyU 5602/09M) of the Research Grant
Council of Hong Kong, and Special Fund for Marine Scientific
Research in the Public Interest of China (201005024). We
acknowledge the support of the Research Projects of Strategic
Importance (1-ZE22) by The Hong Kong Polytechnic
University and the State Key Laboratory of Chirosciences.
We acknowledge Professor Tak-Hang Chan for his useful
advice on this study.
ABBREVIATIONS USED
■
MDR, multidrug resistance; ABC, ATP-binding cassette; P-gp,
P-glycoprotein; RF, relative fold; DOX, doxorubicin; PTX,
paclitaxel; PSA, polar surface area; cLogP, calculated log P
values
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