An anthraquinone scaffold for putative, two-face bim BH3 α-helix mimic

Article abstract of DOI:10.1021/jm301504b

Bim BH3 peptide features an α-helix with hotspot residues on multiple faces. Compound 5 (6-bromo-2,3-dihydroxyanthracene-9,10-dione), which adopts a rigid-plan amphipathic conformation, was designed and evaluated as a scaffold to mimic two faces of Bim α-helix. It reproduced the functionalities of both D67 and I65 on two opposing helical sides. Moreover, it maintained the two-faced binding mode during further evolution. A putative BH3 α-helix mimic and nanomolar Bcl-2/Mcl-1 dual inhibitor, 6, was obtained based on the structure of 5.

Full text of DOI:10.1021/jm301504b

Article
pubs.acs.org/jmc
An Anthraquinone Scaffold for Putative, Two-Face Bim BH3 α‑Helix
Mimic
Zhichao Zhang,*,† Xiangqian Li,†,‡ Ting Song,§ Yan Zhao,§ and Yingang Feng∥
†State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116012, People’s Republic of
China
‡State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, People’s Republic of China
§School of Life Science and Technology, Dalian University of Technology, Dalian 116024, People’s Republic of China
∥Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, People’s Republic of
China;
S
* Supporting Information
ABSTRACT: Bim BH3 peptide features an α-helix with hotspot residues on multiple faces.
Compound 5 (6-bromo-2,3-dihydroxyanthracene-9,10-dione), which adopts a rigid-plan
amphipathic conformation, was designed and evaluated as a scaffold to mimic two faces of
Bim α-helix. It reproduced the functionalities of both D67 and I65 on two opposing helical
sides. Moreover, it maintained the two-faced binding mode during further evolution. A
putative BH3 α-helix mimic and nanomolar Bcl-2/Mcl-1 dual inhibitor, 6, was obtained
based on the structure of 5.
To mimic multiple faces of the Bim BH3 α-helix with small
molecules to inhibit both the Bcl-2 and Mcl-1 proteins in tumor
cells, an anthraquinone platform compound 5 was obtained
with a conserved two-face binding mode, on which a functional
and structural Bim mimetic compound 6 was derived.
Compound 6 exhibited nanomolar affinities toward the two
proteins and specific antitumor ability in cells.
INTRODUCTION
■
Protein−protein interaction (PPI) interfaces are attractive
targets for therapeutic agents.1,2 Most multiprotein complexes
feature α-helical segments at their interfaces.3,4 The hotspot
residues, which contribute significantly to the binding energy
between proteins, can be spatially distributed on one, two, or all
three faces of an α-helix.3
The PPIs between anti- and pro-apoptotic Bcl-2 members are
mediated by the α-helix of the BH3-only proteins.2 The six
known Bcl-2-like anti-apoptotic proteins are divided into two
classes, represented by Bcl-2 and Mcl-1.5,6 They can bind to
pro-apoptotic BH3-only proteins through the shared BH3
domain.7,8 Bim is a nonselective BH3-only protein that can
neutralize both arms of the anti-apoptotic Bcl-2 family, Bcl-2
and Mcl-1 proteins.9 Therefore, small molecules that reproduce
the spatial distribution of hotspots in the Bim BH3 peptide
have been investigated for decades as potential antitumor
drugs.10,11
This is a challenge because the Bim BH3 peptide is an α-helix
with hotspots on three faces, while small-molecule BH3
mimetics, including clinical candidates, fail to mimic multiple
Bim faces. Crystallographic results have shown that ABT-737
can mimic hotspot residues only on one face of Bim (L62 and
F69) to interact with the p2 and p4 pockets of the Bcl-XL
protein and cannot mimic D67 that is conserved in all BH3
domains. Additionally, it cannot hit Mcl-1.9,12 The reported
binding modes of (−)-Gossypol are inconsistent, as is
Apogossypol.13−15 No convincing two-face-mimicking features
were shown by NMR-derived structures. Nonpeptide α-helix
mimicry, including terphenyl scaffolds and their related
structures, typically impart functionality from only one face of
the helix.3,16,17
RESULTS AND DISCUSSION
■
Rationale. We sought to understand the hotspot distribu-
tion of Bim in complex with Mcl-1 and Bcl-2. Because the
crystal structure of the Bim/Bcl-2 complex has not been
reported, we used the isogenous protein Bcl-XL, which has a
similar three-dimensional architecture, in its place.18,19 We
found that residues E55, I58, L62, R63, I65, D67, F69, and F73
are hotspots of Bim binding to both Mcl-1 and Bcl-XL. These
residues are located on two faces of the helix (Figure 1a,b;
Figure S1 in Supporting Information). Among them, D67, I65,
and L62 show the highest free energy penalty (ΔΔG bind = 2.0−
3.0 kcal·mol−1). W57 and E61 for Mcl-1 and Bcl- XL,
respectively, reside on the third face of the helix and are
much less important for binding (ΔΔG bind = 1.0 kcal·mol−1).3
We concluded that the mimicking of residues covered by D67
to I65 is important for a Bim mimetic.
In the hMcl-1/hBim complex, D67 can form a hydrogen
bond with R263 of Mcl-1, while L62 and I65 occupy the
hydrophobic p2 and p3 pockets, respectively.5,9 The p3 pocket
located on the opposite side of R263 (or R146 in Bcl-2) and on
Received: October 17, 2012
© XXXX American Chemical Society
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Figure 1. (a, b) Diagrams of hotspots in (a) Bim/Mcl-1 and (b) Bim/Bcl-XL. (c) Surface representation of the BH3 binding domain of Mcl-1 (side
view). (d) Positions of hotspot residues in a ribbon diagram of the Bim/Mcl-1 complex (front view). (e, f) Docking results of Mcl-1 with (e)
compound 5 (front view) and (f) compound 6 (side view). Carbon, oxygen, nitrogen, bromine, and sulfur atoms are colored gray, red, blue, green,
and yellow, respectively. Hydrogen bonds are depicted as green dotted lines.
the edge of the groove. The p2 pocket is located in the bottom
of the groove, and it is larger and deeper than p3 (Figure
1c,d).9,20 Notably, D67 is the “hottest” residue of the Mcl-1
protein, and its mutation to alanine affects its binding affinity
much more significantly than other hotspots.5 Additionally, our
previous study indicated that mimicking D67 can contribute to
Bcl-2/Mcl-1 dual inhibition by a small molecule.21
hydrophobic and hydrophilic is favorable. D67, at one end of
the bridge, was considered first because a specific atom was
needed to mimic a hydrophilic residue and to form a hydrogen
bond. Another conserved residue, N260 in Mcl-1, a neighbor of
R263 in the three-dimensional structure, was also conserved in
the Bcl-2 protein (Figure 1d) and was selected to form an
additional hydrogen bond to further stabilize the bridge.26
Therefore, three adjacent hydroxyl groups were attached to
anthraquinone to mimic the interactions of D67 with both Bcl-
2 and Mcl-1. As the p3 pocket corresponding to I65 is on the
other end of the bridge, a hydrophobic phenyl group was
employed to occupy the p3 pocket.
However, terphenyl-like scaffolds can only mimic the surface
functionality projected along the bottom hydrophobic face,
including L62, I58, and I65. All of these scaffolds lost the
hydrophilic hotspot D67 on the opposite side of the α-
helix.16,17 A main reason is that the width of these scaffolds (2.4
Å) is less than the diameter of the α-helix (4.6 Å), and the
rigidity of these scaffolds might be not sufficient to allow the
substituents to reach the most solvent-exposed residue, D67.
The lack of this very hot residue may result in suboptimal
affinity for the target. In addition, the free rotation between the
phenyl rings of the terphenyl scaffolds always results in multiple
conformations. As such, adaptive changes in conformation are
required for these molecules to bind to the BH3 groove, which
lead to a decrease in conformational entropy that is unfavorable
for binding energy.22,23 Correspondingly, these “one-face” α-
helix mimetics always exhibit weaker affinity in the micromolar
range.2,24 A suitable scaffold for amphiphilic α-helix mimetics
should facilitate the installation of functional groups on both
sides of the scaffold and also ensure rigid conformation by its
preorganized framework.25
On the basis above, compound 1 was synthesized by
Friedel−Craft acylation, by reacting phthalic anhydride with
pyrogallol in a molten homogeneous mixture of aluminum
chloride and sodium chloride (Scheme 1).27 We measured its
a
Scheme 1. Synthesis of Compounds 1−4
a
Reagents and conditions: (a) AlCl3, NaCl, substituted phenols, 160
°C, 4 h; 10% HCl, 100 °C.
Structure-Based Design, Synthesis, and Evaluation.
We then constructed a molecular platform that is different from
the terphenyl scaffolds and mimics the sagittal axis of the helix.
We tried to mimic the helix such that it reproduces the
arrangement of D67 and the opposite residue I65, which cover
approximately 200° of the circle. We developed a rigid
naphthalene ring, whose width (4.8 Å) is similar to the
diameter of natural α-helix peptides. We hope this platform can
serve as a bridge that spans from D67 to I65. We noticed that
there is a small residue, G66, between D67 and I65 in the
structure of the Bim BH3 peptide, corresponding to the
hydrophilic, solvent-exposed residue T266 in Mcl-1 (Figure
1c,d).6 As such, a rigid, planar anthraquinone that is both
binding to Mcl-1 and Bcl-2 by fluorescence polarization assays
(FPAs) and found a Ki of 270 nM for Mcl-1 and 583 nM for
Bcl-2 (Table 1; Figure S2 in Supporting Information). To
identify which hydroxyls of pyrogallol are responsible for this
function, compounds 2−4 were designed and synthesized by
similar methods (Scheme 1). Except for compound 3, which
maintained similar affinities to Mcl-1 and Bcl-2 as 1, the
absence of either the 2- or 3- hydroxyl made compounds 2 and
4 lose most of their affinities (Table 1; Figure S2 in Supporting
Information). This finding highlighted the necessity of these
two neighboring hydroxyl groups, which can mimic a hydrogen-
B
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a
Table 1. Structure and Binding Affinities of Small-Molecule Inhibitors to Mcl-l and Bcl-2
a
As determined by fluorescence polarization assays.
bonding network formed by D67 and R263/N260 as predicted
by the docking study (Figure S3 in Supporting Information).
From the predicted binding mode, the length of 3 is 8.6 Å,
which is shorter than the maximum distance between D67 and
I65 (10.9 Å in Mcl-1/Bim, Figure 1d and Figure S3b in
Supporting Information; 10.4 Å in Bcl-XL/Bim, not shown).
We then extended 3 by attaching a bromine atom at the 6-
position to probe the proper length of the p3 pocket.
Compound 5 was synthesized by Friedel−Craft acylation
following bromination (Scheme 2). The Ki value of 5 toward
Mcl-1 (107 nM) is approximately 3-fold higher than that of 3
(312 nM) (Table 1; Figure S2a in Supporting Information). A
similar improvement was found for Bcl-2 (Table 1; Figure S2b
in Supporting Information). The docking study also suggested
that compound 5 has the proper length (10.2 Å) to span from
R263 to the p3 pocket (Figure 1e).
Next, we performed heteronuclear single quantum coherence
(HSQC) NMR spectroscopy, using 15N labeled Mcl-1 protein
to identify the binding site of 5. Two-dimensional (2D)
1H−15N HSQC NMR spectra of Mcl-1 were recorded without
(black) and with (green) compound 5 (Figure S4, Supporting
Information). Figure 2a shows a plot of the chemical shift
perturbations against the overall Mcl-1 protein residues.
Approximately 70% of the residues that were perturbed above
the threshold value (0.05 ppm) were located in the cleft of the
Mcl-1 protein into which the Bim helix binds (Figure 2b). The
residues of R263 and N260 and the neighboring residues V258
and V253 showed significant chemical shift (ΔCS > 0.05 ppm)
C
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a
evidence that 5 can find its binding position and serve as a
platform from which more substituents can project to mimic
additional residues on other face of the Bim BH3 α-helix. L62,
which is located in the deepest p2 pocket of the BH3 groove,
was the next target. Cumene, benzene, toluene, and
bromobenzene were introduced at the 6-position of compound
5. A rotatable sp3 hybrid sulfur atom, which is one of the most
hydrophobic atoms, was employed as a linker to allow
substituents to turn to the left (C terminal of Bim BH3
peptide) and penetrate into the bottom of BH3 groove (Figure
1f).
Scheme 2. Synthesis of Compounds 5−9
Compounds 6, 7, 8, and 9 were synthesized and evaluated in
our FP-based binding assay (Scheme 2, Table 1). Excitingly,
improved affinities were found for these analogues, and the
greatest improvement was found for 6, which exhibited an 8-
fold enhancement in Ki toward Mcl-1 (13 nM) and a net 6-fold
improvement to Bcl-2 (24 nM) over 5 (Table 1; Figure S2 in
Supporting Information). These improvements can be
attributed to the similarity in shape and hydrophobicity of
cumene with L62. A docking study found that compound 6
maintained the mimicry of D67 and I65 and achieved
functional mimicry of an additional residue, L62, to occupy
the p2 pocket in Mcl-1. Compound 6 reproduced a similar
arrangement with L62, I65, and D67 in the Bim peptide
(Figure 1d,f; Figure S5b in Supporting Information).
a
Reagents and conditions: (a) NaOH, H2O, Br2, 90 °C, 6 h; HCl. (b)
Ac2O, 140 °C, 2 h. (c) AlCl3, NaCl, pyrocatechol, 160 °C, 4 h; 10%
HCl, 100 °C. (d) 4-R-PhSH, K2CO3,CuI, DMF, 130 °C, 8 h, 10%
HCl.
To confirm that 6 maintains the binding mode of 5, we
performed 2D 1H−15N HSQC NMR recorded without (black)
and with (red) compound 6 (Figure S4, Supporting
Information). With the addition of 6, approximately 80% of
the residues that showed chemical shift changes ΔCS > 0.05
ppm were located in the BH3 binding domain of the Mcl-1
protein (Figure 3). In addition to the chemical shifts of R263,
N260, V258, T266, V220, and F228, which are the same for
compound 5, residues M250 and F270, located in the bottom
of the p2 pocket and corresponding to L62, are newly emergent
residues with significant chemical shift perturbations (ΔCS >
0.05 ppm). NMR identified the conserved binding mode of 5
and provided both a two-face Bim mimetic and a scaffold on
which numerous derivatives mimicking multiple faces of Bim
can be designed (Figure S5, Supporting Information).
The disruption of both Bcl-2/Bax and Mcl-1/Bak in
ABT737-resistant Nalm-6 cells was verified by coimmunopre-
cipitation, and the results mirrored its in vitro pan-inhibition
(Figure 4a). Because compound 6 occupies the BH3 groove, its
pure BH3 mimicking property was identified through a cell-
based shRNA assay, which showed that 6 completely killed the
cells through Bax/Bak (Figure 4b). Consistent with its
mechanism-based killing, a micromolar lethal effect was found
in multiple cancer cell lines, while no significant cytotoxicity
was found in the normal HEK293 cell line (Table S1,
Supporting Information). Notably, 6 did not show DNA
damage in vitro (Figure S6, Supporting Information) and
induced apoptosis but not necrosis in cells (Figure S7,
Supporting Information). These results illustrated that 6 is a
functional and structural Bim mimetic.
Figure 2. (a) Chemical shift perturbations of Mcl-1 residues bound to
compound 5. (b) NMR-derived structure of 5 bound to Mcl-1. Mcl-1
residues with chemical shift changes ΔCS > 0.05 ppm are shown,
including R263, N260, and their nearby residues. V220, H224, F228,
and T266 from the p3 pocket are also included.
changes upon the addition of compound 5. V220, H224, V274,
and F228, which are located in the p3 pocket, also exhibited
significant chemical shift perturbations. T266, in the middle of
R263 and p3, was also significantly affected by 5. The NMR
results demonstrated that 5 can mimic two hotspots in Bim,
D67 and I65, which are located on the two faces of α-helix
(Figure S5a, Supporting Information).
The promising compound ABT-737 binds with high affinity
(Ki < 1 nM) to Bcl-XL and Bcl-2. Its IC50 values range from
submicromolar to micromolar depending on cell species.9,12,28
Compound 6 exhibited 3−7 μM IC50 values on certain tumor
cells in the present study (Table S1, Supporting Information).
Together with the docking studies, the modest binding
affinity, exact binding mode, and rigid structure provided
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Figure 3. (a) Chemical shift perturbations of Mcl-1 residues bound to
compound 6. (b) NMR-derived structure of 6 bound to Mcl-1.
Residues with ΔCS > 0.05 ppm are shown. M250 and F270,
representing the p2 pocket, are newly involved in binding.
Figure 4. (a) Compound 6 interferes with Bcl-2/Bax and Mcl-1/Bak
interactions in Nalm-6 cells. Nalm-6 cells were incubated with 5 μM
ABT-737 and 6, respectively, for 12 or 24 h, after which time total cell
lysates were subjected to coimmunoprecipitation with anti-Bcl-2 or
anti-Mcl-1 antibody. The precipitate and cell lysates were separated by
sodium dodecyl sulfate−polyacrylamide gel electrophoresis )SDS−
PAGE) and analyzed by Western blot with anti-Bcl-2, anti-Bax, anti-
Mcl-1, and anti-Bak antibody. (b) MCF-7 cells transfected with NS
shRNA or Bax/Bak shRNA were treated for 48 h with a graded dose of
compound 6, after which cell viability was determined by MTT assay.
CONCLUSIONS
■
In summary, we have constructed a novel rigid-plan small
molecule scaffold 5 to mimic the structural and recognized
binding features on two faces of the Bim BH3 α-helix. This
scaffold is able to maintain its two-face binding mode and
serves as a platform for further molecular evolution, leading to
novel Bcl-2/Mcl-1 dual inhibitors with optimized affinities and
pharmaceutical properties. Compound 6, for example, was
derived from 5 as a putative Bim mimetic and dual inhibitor
with improved nanomolar affinity. The results reported here
expand the range of small-molecule BH3 mimetics previously
considered able to mimic only one face of the α-helix.
product was purified by column chromatography on silica gel with
chloroform and ethyl acetate as the mobile phase.
1,2,3-Trihydroxyanthracene-9,10-dione (1). Yield 690 mg, 45%;
mp 282−284 °C. 1H NMR (400 MHz, in dimethyl sulfoxide, DMSO)
δ 12.70 (s, 1H), 10.85 (s, 1H), 9.95 (s, 1H), 8.20 (t, J = 8.0 Hz, 1H),
8.14 (t, J = 8.0 Hz, 1H), 7.89 (d, J = 12.0 Hz, 2H), 7.27 (s, 1H). 13C
NMR (DMSO) 186.944, 180.962, 151.948, 151.766, 138.931, 134.556,
134.124, 133.214, 133.047, 126.618, 126.254, 124.655, 110.333,
108.809. Time-of-flight mass spectrometry with electron ionization
[TOF MS (EI+)] C14H8O5, found 256.04. HPLC (40/60 to 0/100
H2O/CH3OH) purity = 98.56%, tR = 12.39 min.
1,3-Dihydroxyanthracene-9,10-dione (2). Yield 375 mg, 28%; mp
218−219 °C. 1H NMR (400 MHz, DMSO) δ 12.74 (s, 1H), 11.34 (s,
1H), 8.19 (t, J = 8.0 Hz, 1H), 8.14 (t, J = 8.0 Hz, 1H), 7.91 (d, J = 12.0
Hz, 2H), 7.14 (s, 1H), 6.61 (s, 1H). 13C NMR (DMSO) 185.886,
180.952, 165.457, 164.756, 134.935, 134.675, 134.473, 132.987,
132.870, 126.821, 126.349, 109.358, 108.392. TOF MS (EI+)
C14H8O4, found 240.06. HPLC (40/60 to 0/100 H2O/CH3OH)
purity = 97.32%, tR = 14.63 min.
EXPERIMENTAL SECTION
■
Purity of all final products was determined by analytical HPLC to be
≥95%. HPLC purity of compounds was measured with a normal-phase
HPLC (XBridge C18, 4.6 × 150 mm, 5 μm) with two diverse
wavelength detection systems. Compounds were eluted by gradient
elution of 40/60 to 0/100 H2O/CH3OH over 30 min at a flow rate of
0.3 mL/min.
General Procedure for Preparation of Compounds 1−4. A
mixture of anhydrous AlCl3 (18 g) and prebaked NaCl (4 g) was
heated (110 °C) in an oil bath until molten. A homogeneous mixture
of phthalic anhydrides (888 mg, 6 mmol) and substituted phenols (6
mmol) separately were reacted with the AlCl3/NaCl melt. The
temperature was slowly increased and maintained at 165 °C for 4 h.
The reaction mixture was cooled to 20 °C, 10 mL of 10% HCl was
added, and the mixture was stirred for 15 min at 20 °C and then
refluxed at 100 °C for 30 min. The reaction mixture was cooled to
room temperature and extracted with ethyl acetate. The resulting
2,3-Dihydroxyanthracene-9,10-dione (3). Yield 216 mg, 15%; mp
259−262 °C. 1H NMR (400 MHz, DMSO) δ 10.63 (s, 2H), 8.13 (t, J
= 8.0 Hz, 2H), 7.86 (t, J = 8.0 Hz, 2H), 7.53 (s, 2H). 13C NMR
(DMSO) 181.660, 151.592, 134.003, 133.222, 134.003, 133,222,
E
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126.884, 126.429, 112.941. TOF MS (EI+) C14H8O4, found 240.03.
HPLC (40/60 to 0/100 H2O/CH3OH) purity = 96.98%, tR = 13.42
min.
1,2-Dihydroxyanthracene-9,10-dione (4). Yield 518 mg, 36%; mp
186−189 °C. 1H NMR (400 MHz, DMSO) δ 12.61 (s, 1H), 10.89 (s,
1H), 8.25 (m, J = 8.0 Hz, 1H), 8.19 (m, J = 8.0 Hz, 1H), 7.93 (m, J =
12.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H). 13C
NMR (DMSO) 189.30, 181.10, 153.25, 151.24, 135.63, 134.58,
134.07, 133.37, 127.23, 127.00, 124.30, 121.65, 121.33, 116.78. TOF
MS (EI+) C14H8O4, found 240.08. HPLC (40/60 to 0/100 H2O/
CH3OH) purity = 97.69%, tR = 15.87 min.
General Procedure for Preparation of Compounds 5−9. To
sodium hydroxide (2.4 g, 0.06 mol) dissolved in water (20 mL) was
added phthalic anhydride (4.44 g, 0.03 mol). When complete solution
was obtained, bromine (9.4 g, 3 mL, 0.06 mol) was incrementally
added, while stirring, over 1 h. The reaction mixture was heated to 90
°C and allowed to react under reflux for 6 h. After 10 h of standing, the
white solids that crystallized out of solution were filtered, washed with
cold water, and analyzed as 4-bromophthalic acid salt. The total
product was dissolved in hot water, and the pH was adjusted to about
1.5 by addition of concentrated HCl. The resulting solution was
evaporated to dryness on a rotary evaporator and extracted with
acetone to give 4-bromophthalic acid. Yield 5.0 g, 68%; mp 164−166
°C.
= 12.0 Hz, 3H) 7.56 (m, J = 12.0 Hz, 3H), 7.49 (s, 1H), 7.46 (s, 1H).
13C NMR (DMSO) 181.60, 181.36, 152.21, 151.93, 146.34, 145.97,
134.71, 134.14, 131.88, 131.03. 130.76, 130.58, 130.19, 127.97, 127.40,
127.04, 123.95, 113.41, 113.39. TOF MS (EI+) C20H12O4S, found
348.07. HPLC (40/60 to 0/100 H2O/CH3OH) purity = 97.62%, tR =
14.07 min.
2,3-Dihydroxy-6-(p-tolylthio)anthracene-9,10-dione (8). Yield 67
mg, 37%; mp 256−258 °C. 1H NMR (400 MHz, DMSO) δ 10.63 (d, J
= 12.0 Hz, 2H), 8.00 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.52 (m, J = 8.0
Hz, 4H), 7.45 (s, 1H), 7.37 (m, J = 8.0 Hz, 2H), 2.51 (s, 3H). 13C
NMR (DMSO) 181.174, 180.886, 151.743, 151.432, 146.383, 139.878,
134.746, 133.608, 130.993, 130.280, 127.414, 126.914, 126,542,
126.065, 126.065, 122.744, 112.903, 62.783. TOF MS (EI+)
C21H14O4S, found 362.09. HPLC (40/60 to 0/100 H2O/CH3OH)
purity = 98.16%, tR = 14.45 min.
6-(4-Bromophenylthio)-2,3-dihydroxyanthracene-9,10-dione (9).
Yield 51 mg, 23.9%; mp 258−262 °C. 1H NMR (400 MHz, DMSO) δ
10.62 (d, J = 12.0 Hz, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0
Hz, 2H), 7.49 (m, J = 8.0 Hz, 4H), 7.36 (m, J = 8.0 Hz, 2H). 13C
NMR (DMSO) 181.55, 181.32, 152.24, 151.97, 145.18, 144.87,
137.55, 136.26, 135.27, 134.19, 133.67, 133.29. 133.16, 132.46, 132.36,
131.40, 128.08, 127.04, 123.58, 113.42. TOF MS (EI+) C20H11BrO4S,
found 426.97. HPLC (40/60 to 0/100 H2O/CH3OH) purity =
96.82%, tR = 14.12 min.
A solution of 4-bromophthalic acid (5 g, 0.02 mol) and acetic
anhydride (30 mL) was heated for 2 h at 140 °C. The reaction mixture
was cooled to room temperature and the excess acetic anhydride was
removed under reduced pressure. The residue was washed with
petroleum ether, and 4-bromophthalic anhydride was obtained as
awhite solid. Yield 3.8 g, 84%; mp 104−106 °C.
ASSOCIATED CONTENT
* Supporting Information
Additional text, seven figures, and one table with experimental
procedures and full spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
A mixture of anhydrous AlCl3 (18 g) and prebaked NaCl (4 g) was
heated (110 °C) in an oil bath until molten. A homogeneous mixture
of 5-bromoisobenzofuran-1,3-dione (1.36 g, 6 mmol) and pyroca-
techol (660 mg, 6 mmol) separately were reacted with the AlCl3/NaCl
melt. The temperature was slowly increased and maintained at 165 °C
for 4 h. The reaction mixture was cooled to 20 °C, 10 mL of 10% HCl
was added, and the mixture was stirred for 15 min at room
temperature and refluxed at 100 °C for 30 min. The reaction mixture
was cooled to room temperature and extracted with ethyl acetate. The
resulting product was purified by column chromatography on silica gel
with ethyl acetate/petroleum ether (1:1) as the mobile phase.
A mixture of 6-bromo-2,3-dihydroxyanthracene-9,10-dione (160
mg, 0.5 mmol), corresponding benzenethiol (3 mmol), CuI (10 mg),
and K2CO3 (69 mg, 0.5 mmol) was stirred in N,N-dimethylformamide
(DMF) (20 mL) for 8 h at 140 °C. After being cooled to room
temperature, the solution was poured into 10% HCl (20 mL),
followed by extraction with ethyl acetate (3 × 20 mL). The organic
layer was collected and dried with MgSO4. After evaporation under
reduced pressure, the product was purified by column chromatography
on silica gel with ethyl acetate/petroleum ether (1:1).
AUTHOR INFORMATION
Corresponding Author
*E-mail zczhang@dlut.edu.cn; fax 86-411-84986032; tel 86-
411-84986032.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by the National Natural Science
Foundation of China (81273436 and 81272876).
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