Discovery and evaluation of novel inhibitors of mycobacterium protein tyrosine phosphatase B from the 6-hydroxy-benzofuran-5-carboxylic acid scaffold

Article abstract of DOI:10.1021/jm301781p

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (mPTPB) is a virulence factor secreted by the pathogen and mediates mycobacterial survival in macrophages by targeting host cell immune responses. Consequently, mPTPB represents an exciting n

Full text of DOI:10.1021/jm301781p

Article
pubs.acs.org/jmc
Discovery and Evaluation of Novel Inhibitors of Mycobacterium
Protein Tyrosine Phosphatase B from the 6‑Hydroxy-benzofuran-5-
carboxylic Acid Scaffold
Yantao He,† Jie Xu,† Zhi-Hong Yu,† Andrea M. Gunawan,‡ Li Wu,†,‡ Lina Wang,†
and Zhong-Yin Zhang*,†
†Department of Biochemistry and Molecular Biology and ‡Chemical Genomics Core Facility, Indiana University School of Medicine,
635 Barnhill Drive, Indianapolis, Indiana 46202, United States
ABSTRACT: Mycobacterium tuberculosis (Mtb) protein tyro-
sine phosphatase B (mPTPB) is a virulence factor secreted by
the pathogen and mediates mycobacterial survival in macro-
phages by targeting host cell immune responses. Conse-
quently, mPTPB represents an exciting new target to combat
tuberculosis (TB) infection. We describe a medicinal chemistry
oriented approach that transforms a benzofuran salicylic acid
scaffold into a highly potent (IC50 = 38 nM) and selective
mPTPB inhibitor (>50 fold against a large panel of PTPs).
Importantly, the inhibitor is capable of reversing the altered
host immune responses induced by the bacterial phosphatase and restoring the macrophage’s full capacity to secrete IL-6 and
undergo apoptosis in response to interferon-γ stimulation, validating the concept that chemical inhibition of mPTPB may be
therapeutically useful for novel TB treatment. The study further demonstrates that bicyclic salicylic acid pharmacophores can be
used to deliver PTP inhibitors with high potency, selectivity, and cellular efficacy.
lung macrophages. Although macrophages play a central role in
host defense, recognizing and destroying potential invaders,
Mtb has evolved several strategies that enable it to survive and
replicate within the macrophages.7 Traditional antibiotics act by
inhibiting essential processes in Mtb that prevent bacterial
growth and replication. This approach results, inevitably, in the
selection of mutant strains that developed drug resistance. An
emerging and alternative approach is to target pathogen
virulence factors to compromise infection process and
persistence.8
Mtb protein tyrosine phosphatase B (mPTPB) is a virulence
factor secreted by Mtb into the host macrophage.9−11 Deletion
of mPTPB has no effect on the growth of the pathogen itself
but severely attenuates intracellular survival of Mtb in infected
macrophages and reduces the bacterial load in a guinea pig
model of TB infection.9 These findings suggest that mPTPB
may mediate mycobacterial survival in macrophages by
targeting host cell processes.11 Recent studies reveal that
mPTPB suppresses the innate immune responses by blocking
the extracellular signal-regulated protein kinase (ERK)1/2 and
p38 mediated interleukin-6 (IL-6) production and preventing
host cell apoptosis by activating the Akt pathway.12,13
INTRODUCTION
■
Protein tyrosine phosphorylation-mediated signal transduction
is a major post-translational mechanism required for a wide
range of cellular functions, including proliferation, differ-
entiation, migration, metabolism, apoptosis, and the immune
responses. Perturbation of the delicate balance between the
opposing activities of protein tyrosine kinases (PTKs) and
protein tyrosine phosphatases (PTPs) causes aberrant tyrosine
phosphorylation, which has been implicated in several human
diseases including cancer, diabetes/obesity, and autoimmune
disorders.1−3 Hence, PTKs and PTPs are emerging as high-
value targets for therapeutic intervention. Notable success has
been achieved in targeting signaling pathways regulated by
protein tyrosine phosphorylation with more than a dozen of
small molecule kinase inhibitors already in the clinic.4 However,
the therapeutic benefits of modulating PTPs are still underex-
plored due to lack of suitable chemical probes.
Tuberculosis (TB) is the leading cause of worldwide
mortality, especially among patients coinfected with HIV and
TB. Although several drug regimens are available, current
treatments for TB take approximately six to nine months, which
makes them difficult for compliance, leading to the appearance
of multidrug resistant (MDR) and the virtually untreatable
extensively drug-resistant (XDR) TB. Therefore, there is urgent
need for new anti-TB agents with novel mechanisms of action
to reduce the possibility of a return to an era in which existing
drugs are no longer effective.5,6 TB is a bacterial infection
caused by Mycobacterium tuberculosis (Mtb) that primarily
affects the lung. The predominant host cells for Mtb are the
Given the requirement of mPTPB for host cell survival and
the importance of macrophage apoptosis as a defense
mechanism to counter Mtb infection,14−18 specific inhibition
of mPTPB may augment intrinsic host signaling pathways to
Received: September 11, 2012
Published: January 10, 2013
© 2013 American Chemical Society
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Figure 1. Discovery of mPTPB inhibitors from 6-hydroxybenzofuran-5-carboxylic acid scafford.
eradicate TB infection. Indeed, small molecule inhibitors of
mPTPB are capable of reducing intracellular mycobacteria in
infected macrophages.12,13,19 Thus, mPTPB represents an
exciting new target for TB with a completely novel mechanism
of action. Because mPTPB inhibitors function within the
cytosol of macrophages and have no mechanistic overlap with
current anti-TB agents, they target the intracellular pool and
may compliment/synergize with existing therapeutic ap-
proaches to shorten standard TB treatment. Furthermore,
since mPTPB acts outside of the bacterium, mPTPB inhibitors
need not cross the thick and impenetrable Mtb cell wall, which
presents a huge barrier to efficient delivery of antibacterial
compounds. Understandably, there is intense interest in
targeting mPTPB for therapeutic development.13,19−26 We
previously described a benzofuran salicylic acid-based mPTPB
inhibitor (I-A09) with highly efficacious cellular activity.12
However, I-A09 displays a rather modest potency (IC50 = 1.26
μM) and selectivity (∼10-fold against a panel of mammalian
PTPs), which may not be sufficient for therapeutic develop-
ment. Here we present a medicinal chemistry oriented
approach that transforms the benzofuran salicylic acid core
into a highly potent and selective mPTPB inhibitor with
excellent in vivo efficacy.
explore the impact of substitutions at the terminal alkynyl
group, we initially synthesized compounds 2, 3, and 4a (Table
1), which were prepared via a common key intermediate 11
(Scheme 1) using a previously described method.12 Commer-
cially available 4-hydroxysalicylic acid reacted with ICl to afford
compound 7 in good yield.27 Upon treatment with acetone and
TFAA/TFA, 7 was regioselectively protected to provide
dioxanone 8 in modest yield.28 Dioxanone 8 then reacted
with MeI at room temperature in the presence of K2CO3/
DMSO to give the methylation product 9 with excellent yield.
Compound 9 coupled with phenylacetylene in the presence of
catalytic amounts of Pd(PPh3)2Cl2 and CuI furnished 1029,30
which was then subjected to I2 induced cyclization.12,31
Cyclization of 10 proceeded exceedingly well and afforded
key compound 11 in high yield on a multigram scale (Scheme
1). The iodo-substituted 11 was utilized to prepare compounds
2, 3, and 4a under Sonogashira coupling conditions (Scheme
2).
The ability of compounds 2, 3, and 4a to inhibit the mPTPB-
catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) was
assessed at pH 7 and 25 °C. The IC50 values were 5.1, 22, and
0.7 μM, respectively, for compounds 2, 3, and 4a (Table 1).
Thus no improvement in potency was observed when
ethisterone or methanol was introduced to the terminal alkyne
position in Core 1. In contrast, when a benzene ring was
attached to the terminal alkyne position, the resulting
compound 4a experienced a 10-fold increase in binding affinity
(IC50 = 0.7 μM) relative to the parent Core 1 (IC50 = 7.3 μM).
Encouraged by this result, we decided to investigate whether
substitutions at the β-benzene ring could further improve the
potency and specificity of compound 4a. A small series of
analogues of compound 4a were prepared via Sonogashira
coupling (Scheme 2). Among the various compound 4a
derivatives 4b−i (Table 1), 3-phenylethynyl-substitued 4b
with a fluorine at the meta position of the β-benzene ring was
found to be 4-fold more potent than its parent 4a for mPTPB.
Compound 4c with a chlorine at the same position showed
similar activity as 4b. Interestingly, substitution at the meta
position with a trifluoromethyl group (4g) enhanced the
binding affinity by more than 18-fold whereas a methoxy
substitution (4d) at the same position did not exert any
significant impact on mPTPB inhibition. Compared to the
monosubstituted 4b, the 3,5-difluorine substituted 4f had a
further 3.3-fold decrease in IC50 although the 2.4-difluorine
substituted 4e showed no improvement. Substitution at the
para-position by a trifluoromethoxy (4h) or a phenoxy (4i)
group caused a 7.4- and 16.3-fold increase in binding affinity
respectively. Finally, the triple bond in compounds 4b, 4f, 4g,
4h, and 4i were reduced to a double bond (5a,b) or a single
RESULTS
■
Chemistry. The PTP active sites (i.e., pTyr-binding
pockets) are highly positively charged, which poses a significant
challenge for the development of PTP inhibitors possessing
favorable pharmacological properties. Indeed, most current
PTP inhibitors are negatively charged and unable to penetrate
cell membranes. To address the bioavailability issue, we sought
to discover novel nonhydrolyzable pTyr mimetics from natural
products because they are evolutionarily selected and validated
for interfering and interacting with biological targets in vivo.
Benzofuran salicylic acid Core 1 (Figure 1) was found to be an
excellent pTyr surrogate for mPTPB with an IC50 value of 7.3
μM.12 Click chemistry was employed to tether Core 1 with 80
azide-containing amines and hydrazines in order to target
adjacent secondary binding sites in addition to the active site.
This led to the identification of I-A09 (Figure 1), which has an
IC50 of 1.26 μM for mPTPB and greater than 10-fold
preference for mPTPB versus a panel of mammalian PTPs.12
Despite its highly efficacious cellular activity, the potency and
selectivity displayed by I-A09 are relatively modest, and
therefore may not be adequate for chemical biological
investigation and therapeutic development.
Given the prominent contribution of Core 1 to the binding
affinity of I-A09, we sought to improve Core 1’s potency and
selectivity by further modifications on the 3-ethynyl group. To
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Table 1. IC50 (μM) Values of 1, 2, 3, 4a−i, 5a−b, and 6a−d against mPTPB and a Panel of Mammalian PTPs
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Table 1. continued
a
The results indicated that compound 6-hydroxy-2-phenyl-3-
((3-trifluoromethyl)phenyl)benzofuran-5-carboxylic acid (4g)
was the most potent mPTPB inhibitor in this series with an
IC50 value of 38 nM (Table 1). Kinetic analysis revealed that
compound 4g is a reversible and noncompetitive inhibitor for
mPTPB with a Ki of 44 1.5 nM (Figure 2). To determine the
specificity of compound 4g, its inhibitory activity toward
mPTPA and a panel of mammalian PTPs including cytosolic
PTPs, PTP1B, TC-PTP, SHP1, SHP2, FAP1, Lyp, PTP-MEG2,
and HePTP, the receptor-like PTPs, PTPα, LAR, CD45, and
PTPγ, the dual specificity phosphatases VHR, Laforin, VHX,
and Cdc14A, and the low molecular weight PTP (LMWPTP)
were measured. For comparison, the specificity of compounds
4f and 4h were also measured. As shown in Table 2, 4g is
highly selective for mPTPB, exhibiting at least 47-fold
selectivity over all other PTPs examined. Together, the results
show that 4g is the most potent and specific mPTPB inhibitors
reported to date.
Scheme 1. Synthesis of Key Intermediate 11
a
Reagents and conditions: (a) ICl, AcOH, rt; (b) acetone, TFA,
TFAA, rt; (c) MeI, K2CO3, DMSO, rt; (d) phenylacetylene,
Pd(PPh3)2Cl2, CuI, DMF, rt; (e) I2, NaHCO3, MeCN; 70 °C.
Cellular Activity of Compound 4g. Our ultimate goal is
to develop mPTPB-based small molecule therapeutics as new
weapons to combat TB infections. Given the excellent potency
and selectivity of compound 4g toward mPTPB, we proceeded
to evaluate its ability to inhibit mPTPB activity inside the cell.
To define the biochemical mechanism used by mPTPB to
evade host immune responses for the benefit of Mtb survival in
the macrophages, we have established a murine macrophage
bond (6a−d) by selective hydrogenation (Scheme 3) in order
to evaluate the requirement of the alkyne group. As shown in
Table 1, the potency of the triple bond reduced compounds
dramatically decreased, indicating that the rigidity and
directionality of substitutions at the 3-position of the
benzofuran salicylic core are important for optimal interaction
with mPTPB.
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a
Scheme 2. Synthesis of Compounds 2, 3, and 4a−i
a
Reagents and conditions: (a) Pd(PPh3)2Cl2, CuI, alkyne, DMF, rt; (b) KOH, THF/H2O, reflux.
a
Scheme 3. Synthesis of Compounds 5a,b and 6a−d
Table 2. Selectivity of 4g, 4f, and 4h against a Panel of PTPs
PTP
IC50 (μM) for 4g IC50 (μM) for 4f IC50 (μM) for 4h
mPTPB
mPTPA
PTP1B
TC-PTP
SHP2
0.038 0.002
2.5 0.2
0.054 0.004
1.6 0.1
4.5 0.2
6.1 0.5
2.2 0.1
2.3 0.1
1.9 0.04
1.3 0.1
2.3 0.08
3.3 0.5
>30
0.090 0.017
2.8 0.3
5.1 0.3
6.7 0.4
1.8 0.6
2.5 0.1
1.6 0.1
2.0 0.1
4.0 0.2
1.9 0.3
6.7 0.7
13
1
1.8 0.2
2.3 0.3
1.3 0.2
2.0 0.1
4.3 0.5
2.1 0.3
40 10
1.4 0.2
1.9 0.4
4.3 0.6
6.0 1.0
>10
SHP1
FAP1
Lyp
PTP-MEG2
HePTP
Laforin
VHX
31
4
2.9 0.3
5.3 0.6
2.9 0.3
1.9 0.1
2.1 0.2
3.0 0.1
6.0 1.0
>10
VHR
a
LMWPTP
Cdc14A
PTPα
Reagents and conditions: (a) Lindlar catalysis, H2, THF/MeOH, rt;
21
2
>10
>10
(b) H2, Pd/C, THF/MeOH, rt.
LAR
>10
>10
CD45
4.2 0.5
2.6 0.6
4.1 0.5
5.0 0.3
3.6 0.2
1.8 0.1
PTPγ
mPTPB inhibits the responses of mPTPB-containing macro-
phages to IFN-γ by reducing the IFN-γ stimulated IL-6
production through down-regulating the ERK1/2 and p38
pathways. In addition, we also obtained evidence that mPTPB
activity is responsible for increased phospho-Akt and decreased
apoptosis of macrophages when exposed to IFN-γ. As a result,
the Raw264.7 cell line serves as a very convenient model system
to evaluate the cellular efficacy of mPTPB inhibitors. We
predict that inhibition of mPTPB activity should reverse the
effects of mPTPB on ERK1/2, p38, and Akt signaling and
restore macrophage’s full capacity to secrete IL-6 and undergo
apoptosis in response to IFN-γ stimulation.
As shown in Figure 3, Raw264.7 cells expressing mPTPB
displayed decreased IFN-γ stimulated ERK1/2 and p38
activation and IL-6 production when compared to the vector
control. Moreover, Raw264.7 macrophages experienced in-
creased apoptosis under continuous IFN-γ stimulation and
mPTPB expression more than tripled the amount of viable cells
(as evidenced by the increased annexin V-negative/PI-negative
cells from 7.5 to 24.5%) by activating Akt and blocking caspase
3 activity (Figure 4). Consistent with compound 4g being an
Figure 2. Compound 4g is a reversible and noncompetitive inhibitor
of mPTPB with pNPP as a substrate. Line weaver-Burk plot for 4g-
mediated mPTPB inhibition. Compound 4g concentrations were 0
■
○
▼
▽
(•), 10 ( ), 20 ( ), 30 ( ), and 40 nM ( ), respectively. The Ki
value of 44
1.5 nM was determined from three independent
measurements.
Raw264.7 cell line ectopically expressing mPTPB.12 Interferon-
γ (IFN-γ), the predominant activator of microbicidal functions
of macrophages,32 is essential for macrophage’s antimicrobial
activity against diverse intracellular pathogens. We showed that
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Figure 3. Compound 4g restores ERK1/2 and p38 activity (A) and
IL-6 production (B) in activated macrophages. Cells overexpressing
mPTPB have decreased ERK1/2 and p38 activity and secrete lower
levels of IL-6, and these can be reversed by treatment with the mPTPB
inhibitor 4g but not with a structurally related inactive compound 6c.
Data in panel B are expressed as means SD of three independent
experiments.
mPTPB inhibitor, treatment of mPTPB expressing Raw264.7
macrophages with compound 4g restored the IFN-γ induced
activation of ERK1/2, p38, as well as IL6 production in a dose
dependent manner (Figure 3). In addition, compound 4g
normalized Akt and caspase 3 activities and rescued the IFFN-γ
induced apoptosis in mPTPB cells to the same extend as the
vector control cells (Figure 4). To ensure that the cellular
activity exerted by compound 4g is not due to nonspecific
effects, we also evaluated a structurally related but inactive
compound 6c (IC50 = 3.4 μM for mPTPB), which should not
inhibit mPTPB and the mammalian PTPs at 240 nM
concentration (Table 1). As expected, compound 6c was
unable to reverse the biochemical and functional perturbations
introduced by mPTPB (Figures 3 and 4). Moreover, the
observed cellular activity by compound 4g also phenocopied
those of several structurally unrelated small molecule mPTPB
inhibitors.12,13 Thus the ability of compound 4g to block the
mPTPB-mediated cellular signaling is unlikely due to off-target
effects. Taken together, the results demonstrate that compound
4g is highly efficacious in cell-based assays and capable of
blocking mPTPB activity inside the cell.
Figure 4. Inhibition of mPTPB with 4g blocks Akt activation (A) and
promotes caspase 3 activation (B) and macrophage apoptosis (C).
Cells overexpressing mPTPB have increased Akt activity, lower caspase
3 activity, and higher propensity for cell survival, and these can be
reversed by treatment with the mPTPB inhibitor 4g but not with a
structurally related inactive compound 6c. Data in panel B are
expressed as means SD of three independent experiments.
CONCLUSION
■
Inhibitor specificity for the desired PTP is clearly an important
goal in a PTP drug discovery program in order to avoid
unintended off-target effect on other family members.
Obtaining PTP inhibitors with optimal potency and selectivity
has been difficult, due primarily to the high homology of the
catalytic domains shared by all PTPs. In this study, we describe
a medicinal chemistry optimization strategy that led to the
discovery of a group of benzofuran salicylic acid-based mPTPB
inhibitors that exhibit extremely high potency and selectivity.
This is significant as it demonstrates the feasibility of acquiring
specific PTP inhibitors by targeting unique areas within the
active site pocket. Besides selectivity, bioavailability is another
challenge in the development of PTP-based small-molecule
therapeutics, because active site-directed inhibitors tend to
mimic the pTyr substrate with substantial negative charges.
Such molecules are generally not drug-like, with limited cell
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membrane permeability. Our current and previous work12,33,34
demonstrate that bicyclic salicylic acid pharmacophores are
sufficiently polar to bind the PTP active site, yet remain capable
of efficiently crossing cell membranes, offering PTP inhibitors
with both high affinity and selectivity and excellent cellular
efficacy. To this end, compound 4g possesses highly efficacious
cellular activity and is capable of reversing the altered host cell
immune responses induced by the bacterial phosphatase.
Overall, this class of compounds offers further opportunities
to evaluate mPTPB inhibition as an anti-TB strategy.
concentrated. The crude material was then purified by column
chromatography on silica gel to give the compound 9 (34.2 g, 95%).
1H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 6.40 (s, 1H), 3.93 (s,
3H), 1.73 (s, 6H). Mass spectra (ESI): m/e 335 (M + H)+.
7-Methoxy-2,2-dimethyl-6-(phenylethynyl)-4H-benzo[d]-
[1,3]dioxin-4-one (10). A Schlenk flask was charged with 6-iodo-7-
methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one 9 (10.00 g, 29.9
mmol), phenylacetylene (1.2 equiv, 35.9 mmol), Pd(PPh3)2Cl2 (3.0
mol %, 0.90 mmol), and CuI (6.0 mol %, 1.79 mmol). DMF (100 mL)
was added and the flask was evacuated and backfilled with N2 three
times, and then Et3N (4.0 equiv, 120 mmol) was added. The resulting
reaction was stirred overnight at ambient temperature under N2
overnight. The mixture was then diluted with 100 mL of water and
300 mL of ethyl acetate. After the phase cut, the organic layer was
washed with water and brine. The organic layer was dried over Na2SO4
and filtered. Evaporation and chromatography on silica gel with the
hexanes/ethyl acetate afforded the product 10 (7.46 g, 81%). 1H NMR
(500 MHz, CDCl3) δ 8.08 (s, 1 H), 7.52 (m, 2H), 7.32−7.34 (m, 3H),
6.44 (s, 1H), 3.93 (s, 3H), 1.73 (s, 6H). 13C NMR (125 M, CDCl3) δ
165.91, 159.95, 157.72, 134.57, 131.50, 128.20, 123.07, 108.39, 106.58,
105.87, 98.95, 93.22, 83.67, 56.33, 25.72. Mass spectra (ESI): m/e 309
(M + H)+.
EXPERIMENTAL SECTION
■
Materials. Recombinant mouse IFN-γ was purchased from
PeproTech Inc. Anti-ERK1/2, antiphospho-ERK1/2, anti-p38, anti-
phospho-p38, and antiphospho-Akt473 antibodies were purchased
from Cell Signaling. p-Nitrophenyl phosphate (pNPP) was purchased
from Fluke Co. Dithiothreitol (DTT) was provided by Fisher (Fair
Lawn, NJ). All reagents and solvents for organic synthesis were
purchased from commercially available sources (FisherSci, Aldrich,
Acros, Alfa Aesar, TCI).
General Procedures for Chemistry. 1H NMR spectra were
obtained on a Bruker 500 MHz NMR instrument. The chemical shifts
were reported as δ ppm relative to TMS, using residual solvent peak as
the reference unless otherwise noted. The following abbreviations
were used to express the multiplicities: s = singlet; d = doublet; t =
triplet; q = quartet; m = multiplet; br = broad. High-performance
liquid chromatography (HPLC) purification was carried out on a
Waters Delta 600 equipped with a Sunfire Prep C18 OBD column (30
× 150 mm, 5 μM) with methanol−water (both containing 0.1% TFA)
as mobile phase (gradient: 50−100% methanol, flow 10 mL/min). All
final compounds were obtained in highly purified form (>95%). Low-
resolution mass spectra were obtained on an Agilent Technologies
6130 Quadrupole LC/MS. HRMS data were obtained at the Mass
Spectrometry Facility at Indiana University Chemistry Department
(http://msf.chem.indiana.edu) on a Waters/Macromass LCT (electro-
spray ionization ESI). All reactions were monitored by thin layer
chromatography (TLC) carried out on Dynamic Adsorbents silica gel
plates (0.25 mm thick, 60F254), visualized by using UV (254 nm). All
compounds used for biological assays were purified by HPLC and are
at least of 95% purity based on HPLC analytical results monitored
with 254 nm wavelengths.
6-Iodo-2,2-dimethyl-7-phenyl-4H-[1,3]dioxino[5,4-f ]-
benzofuran-4-one (11). The following procedure is a modification
of our earlier procedure.12 To a solution of 7-methoxy-2,2-dimethyl-6-
(phenylethynyl)-4H-benzo[d][1,3]dioxin-4-one 10 (5 g, 16.22 mmol)
in acetonitrile (100 mL) were added iodine (8.24 g, 32.44 mmol) and
NaHCO3 (2.7 g, 32.4 mmol). After the resulting mixture was stirred at
70 °C for 12 h, additional iodine (4.12 g, 16.22 mmol) and NaHCO3
(1.36 g, 16.22 mmol) were added and the resulting reaction was
allowed to stir for additional 12 h at 70 °C. Then the solvent was
removed under reduced pressure and the residue was purified by
column chromatography on silica gel using 6:1 hexanes/ethyl acetate
to afford compound 11 (5.59 g, 82%). 1H NMR (500 MHz, CDCl3) δ
8.13 (m, 2H), 8.10 (s, 1H), 7.40−7.52 (m, 3H), 7.06 (s, 1H), 1.76 (s,
1H); 13C NMR (125 M, CDCl3) δ 161.11, 158.04, 154.74, 129.69,
129.13, 128.84, 128.61, 127.31, 123.93, 110.69, 106.52, 99.75, 60.50,
30.87, 25.86. Mass spectra (ESI): m/e 421 (M + H)+.
General Procedure for Preparation of Compounds 14a−i via
Sonogashira Coupling. A Schlenk flask was charged with 6-iodo-
2,2-dimethyl-7-phenyl-4H-[1,3]dioxino[5,4-f ]benzofuran-4-one (11)
(1.0 mmol), Pd(PPh3)2Cl2 (3.0 mol %, 0.03 mmol), and CuI (6.0
mol %, 0.06 mmol). DMF (5 mL) was added and the flask was
evacuated and backfilled with N2 three times. The alkyne (1.2 mmol)
and Et3N (4.0 equiv, 4.0 mmol) were then successively added. The
resulting reaction was stirred at 40 °C under N2 for the corresponding
reaction times by TLC, after which time the reaction mixture was
diluted with water (10 mL) and extracted with ethyl acetate (3 × 20
mL). The combined ethyl acetate layers were dried over Na2SO4,
filtered, concentrated, and purified by silica gel flash chromatography
using hexanes/ethyl acetate to elute the corresponding desired
coupling product 14a−i.
2,4-Dihydroxy-5-iodobenzoic Acid (7).27 To a solution of 2,4-
dihydroxybenzoic acid (20 g, 130 mmol) in acetic acid (150 mL) was
added a solution of iodine monochloride (8.0 mL, 160 mmol) in acetic
acid (100 mL) dropwise for 1 h at room temperature. The reaction
mixture was stirred for another 4 h at room temperature. Acetic acid
was removed in vacuum and water was added. The resulting
precipitate was collected by filtration, washed with water and dried
1
in vacuo to give compound 7 (33.1 g, 91%). H NMR (500 MHz,
CDCl3) δ 12.45 (br, 1H), 11.31 (s, 1H), 8.10 (s, 1 H), 6.43 (s, 1H).
Mass spectra (ESI): m/e 281 (M + H)+.
2,2-Dimethyl-7-phenyl-6-(phenylethynyl)-4H-[1,3]dioxino-
7-Hydroxy-6-iodo-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-
one (8).28 To a suspension of 2,4-dihydroxy-5-iodobenzoic acid 7
(16.71 g, 59.7 mmol) in TFA (300 mL) were added TFAA (41.5 mL,
298 mmol) and dry acetone (35 mL, 478 mmol) dropwise at room
temperature, and the mixture was stirred overnight. Then the
homogeneous solution was concentrated to dry, and 500 mL of
ethyl acetate was subsequently added. The organic layers were washed
with water, the aqueous saturated NaHCO3, water, and brine in stated
order and dried over anhydrous Na2SO4, filtered, and concentrated.
This crude material 8 (10.0 g, 52%) was then used directly in the next
step.
1
[5,4-f ]benzofuran-4-one (14a). 351 mg, yield 89%. H NMR (500
MHz, CDCl3) δ 8.37 (s, 1H), 8.28 (m, 2H), 7.62 (m, 2H), 7.50 (t, J =
7.8 Hz, 2H), 7.40 (m, 4H), 7.08 (s, 1H), 1.77 (s, 6H). 13C NMR (125
M, CDCl3) δ 161.38, 157.70, 157.43, 154.47, 131.61, 129.64, 129.34,
128.80, 128.78, 128.54, 125.90, 122.80, 122.38, 110.53, 106.51, 99.90,
99.33, 97.87, 79.79, 30.92, 25.86. Mass spectra (ESI): m/e 395 (M +
H)+.
6-((3-Fluorophenyl)ethynyl)-2,2-dimethyl-7-phenyl-4H-
[1,3]dioxino[5,4-f ]benzofuran-4-one (14b). 296 mg, yield 72%.
1H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 8.25 (d, J = 7.6 Hz, 2H),
7.51 (t, J = 7.65 Hz, 2H), 7.44 (m, 1H), 7.40 (m, 2H), 7.31 (m, 1H),
7.12 (m, 1H), 7.09 (s, 1H), 1.78 (s, 6H). 13C NMR (125 M, CDCl3) δ
162.58 (d, J = 245.6 Hz), 161.47, 157.99, 157.80, 154.64, 130.30 (d, J
= 8.4 Hz), 129.96, 129.32, 128.97, 127.65, 127.64, 126.07, 125.84,
124.70 (d, J = 9.2 Hz), 122.44, 118.43 (d, J = 22.9 Hz), 116.29 (d, J =
21.0 Hz), 110.73, 106.68, 100.09, 99.00, 96.54 (d, J = 3.5 Hz), 80.90,
25.99. Mass spectra (ESI): m/e 413 (M + H)+.
6-Iodo-7-methoxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-
one (9). To a solution of 7-hydroxy-6-iodo-2,2-dimethyl-4H-benzo-
[d][1,3]dioxin-4-one 8 (34.66 g, 108 mmol) in DMSO (300 mL) were
added K2CO3 (29.8 g, 216 mmol) and MeI (6.7 mL, 15.3 g, 108
mmol) at 0 °C. The reaction mixture was allowed to stir for 4 h at 25
°C and then diluted in a large amount of ether and washed with water
and brine. The ether layer was then dried over NaSO4, filtered,and
838
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Journal of Medicinal Chemistry
Article
6-((3-Chlorophenyl)ethynyl)-2,2-dimethyl-7-phenyl-4H-
[1,3]dioxino[5,4-f ]benzofuran-4-one (14c). 342 mg, yield 80%.
1H NMR (500 MHz, CDCl3) δ 8.35 (s, 1H), 8.23 (m, 2H), 7.59 (t, J =
1.8 Hz, 1H), 7.50 (m, 3H), 7.45 (m, 1H), 7.36 (m, 2H), 7.09 (s, 1H),
1.85 (s, 6H). 13C NMR (125 M, CDCl3) δ 161.31, 157.86, 157.66,
154.52, 134.40, 131.32, 129.83, 129.78, 129.74, 129.17, 129.03, 128.84,
125.94, 125.70, 124.47, 122.29, 110.61, 106.55, 99.96, 98.84, 96.26,
81.04, 25.86. Mass spectra (ESI): m/e 429 (M + H)+.
6-((3-Methoxyphenyl)ethynyl)-2,2-dimethyl-7-phenyl-4H-
[1,3]dioxino[5,4-f ] benzofuran-4-one (14d). 301 mg, yield 71%.
1H NMR (500 MHz, CDCl3) δ 8.39 (s, 1H), 8.28 (m, 2H), 7.53 (t, J =
7.4 Hz, 2H), 7.45 (tt, J = 7.4 and 1.2 Hz, 1H), 7.33 (t, J = 8 Hz, 1H),
7.22 (dt, J = 7.5 and 1.2 Hz, 1H), 7.14 (m, 1H), 7.10 (s, 1H), 6.96
(ddd, J = 8.3, 2.55, and 0.85 Hz, 1H), 3.88 (s, 3H), 1.78 (s, 6H). 13C
NMR (125 M, CDCl3) δ 161.55, 159.62, 157.85, 157.72, 154.62,
129.81, 129.75, 129.47, 128.93, 126.06, 126.01, 124.34, 123.92, 122.55,
116.42, 115.62, 110.68, 106.66, 100.05, 99.42, 97.88, 79.75, 55.56,
26.00. Mass spectra (ESI): m/e 425 (M + H)+.
6-((2,4-Difluorophenyl)ethynyl)-2,2-dimethyl-7-phenyl-4H-
[1,3]dioxino[5,4-f ]benzofuran-4-one (14e). 297 mg, yield 69%.
1H NMR (500 MHz, CDCl3) δ 8.35 (s, 1H), 8.30 (m, 2H), 7.58 (m,
1H), 7.50 (m, 2H), 7.43 (m, 1H), 7.08 (s, 1H), 6.93 (m, 2H), 1.76 (s,
6H). 13C NMR (125 M, CDCl3) δ 163.19 (dd, J = 253.4 and 12.3 Hz),
163.07 (dd, J = 251.6 and 11.5 Hz), 161.33, 157.91, 157.67, 154.52,
134.08 (dd, J = 9.7 and 2.3 Hz), 129.83, 129.10, 128.80, 125.94,
125.63, 122.29, 111.87 (dd, J = 21.8 and 3.6 Hz), 110.63, 107.92 (dd, J
= 16.0 and 3.8 Hz), 106.55, 104.49 (t, J = 25.5 Hz), 99.96, 98.80,
90.30, 84.82, 25.87. Mass spectra (ESI): m/e 431 (M + H)+.
filtered, and concentrated under reduced pressure. The residue was
purified by HPLC to furnish the corresponding products 4a−i. Yield
70−90%. Compounds 2 and 3 were prepared from compound 11
directly via two steps.
6-Hydroxy-2-phenyl-3-(phenylethynyl)benzofuran-5-car-
boxylic Acid (4a). 1H NMR (500 MHz, DMSO-d6) δ 8.17 (d, J = 7.6
Hz, 2H), 8.08 (s, 1H), 7.68 (m, 2H), 7.58 (t, J = 7.6 Hz, 2H), 7.50 (m,
4H), 7.20 (s, 1H). 13C NMR (125 M, DMSO-d6) δ 171.99, 160.58,
156.94, 155.98, 131.41, 129.69, 129.18, 129.08, 128.88, 128.75, 125.23,
121.95, 121.80, 121.37, 110.50, 99.08, 98.30, 97.31, 79.88. Mass
spectra (ESI): m/e 355 (M + H)+.
3-((3-Fluorophenyl)ethynyl)-6-hydroxy-2-phenylbenzofur-
1
an-5-carboxylic Acid (4b). H NMR (500 MHz, DMSO-d6) δ 8.15
(d, J = 7.7 Hz, 2H), 8.09 (s, 1H), 7.58 (t, J = 7.7 Hz, 2H), 7.50 (m,
4H), 7.33 (m, 1H), 7.21 (s, 1H). Mass spectra (ESI): m/e 373 (M +
H)+.
3-((3-Chlorophenyl)ethynyl)-6-hydroxy-2-phenylbenzofur-
1
an-5-carboxylic Acid (4c). H NMR (500 MHz, DMSO-d6) δ 8.11
(d, J = 7.6 Hz, 2H), 8.05 (s, 1H), 7.70 (s, 1H), 7.55 (m, 4H), 7.46 (m,
2H), 7.15 (s, 1H). 13C NMR (125 M, DMSO-d6) δ 171.99, 160.60,
156.89, 156.41, 133.46, 130.69, 130.66, 130.14, 129.82, 129.21, 129.11,
128.60, 125.33, 123.89, 121.90, 121.18, 110.61, 99.06, 97.91, 95.66,
81.22. Mass spectra (ESI): m/e 389 (M + H)+.
6-Hydroxy-3-((3-methoxyphenyl)ethynyl)-2-phenylbenzo-
1
furan-5-carboxylic Acid (4d). H NMR (500 MHz, DMSO-d6) δ
8.17(d, J = 7.5 Hz, 2H), 8.09 (s, 1H), 7.58 (t, J = 7.5 Hz, 2H), 7.48 (t,
J = 7.4 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.22 (m, 3H), 7.05 (dd, J =
8.2 and 2.0 Hz, 1H), 3.82 (s, 3H). 13C NMR (125 M, DMSO-d6) δ
171.99, 160.58, 159.30, 156.98, 156.17, 130.08, 129.80, 129.15, 128.74,
125.29, 123.88, 122.96, 121.89, 121.34, 116.15, 115.67, 110.58, 99.17,
98.27, 97.25, 79.70, 55.31. Mass spectra (ESI): m/e 385 (M + H)+.
3-((2,4-Difluorophenyl)ethynyl)-6-hydroxy-2-phenylbenzo-
6-((3,5-Difluorophenyl)ethynyl)-2,2-dimethyl-7-phenyl-4H-
[1,3]dioxino[5,4-f ]benzofuran-4-one (14f). 314.2 mg, yield 73%.
1H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H), 8.24 (m, 2H), 7.53 (t, J =
1
7.85 Hz, 2H), 7.47 (m, 1H), 7.12 (m, 2H), 7.10 (s, 1H), 6.87 (m, 1H),
1.78 (s, 6H). 13C NMR (125 M, CDCl3) δ 163.01 (dd, J = 248.1 and
13.3 Hz), 161.42, 158.46, 157.82, 154.73, 130.17, 129.21, 129.04,
126.15, 125.66, 125.51 (t, J = 11.7 Hz), 122.40, 114.67 (dd, J = 20.3
and 6.8 Hz), 110.86, 106.74, 105.11 (t, J = 25.2 Hz), 98.60, 95.40 (t, J
= 3.7 Hz), 82.06, 26.01. Mass spectra (ESI): m/e 431 (M + H)+.
2,2-Dimethyl-7-phenyl-6-((3-(trifluoromethyl)phenyl)-
ethynyl)-4H-[1,3]dioxino[5,4-f ]benzofuran-4-one (14g). 300
furan-5-carboxylic Acid (4e). H NMR (500 MHz, DMSO-d6) δ
8.17 (d, J = 7.4 Hz, 2H), 8.06 (s, 1H), 7.82 (m, 1H), 7.54 (t, J = 7.4
Hz, 2H), 7.46 (m, 2H), 7.20 (m, 2H). 13C NMR (125 M, DMSO-d6)
δ 171.92, 162.58 (dd, J = 248.8 and 23.0 Hz), 162.49 (dd, J = 248.4
and 24.1 Hz), 160.63, 156.90, 156.26, 134.55 (d, J = 10.1 Hz), 129.88,
129.10, 128.54, 125.21, 121.76, 121.12, 112.49 (d, J = 21.9 Hz),
110.66, 107.12 (d, J = 15.5 Hz), 104.80 (t, J = 25.6 Hz), 99.52, 99.15,
97.84, 89.83, 84.87. Mass spectra (ESI): m/e 391 (M + H)+.
1
mg, yield 65%. H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 8.26
3-((3,5-Difluorophenyl)ethynyl)-6-hydroxy-2-phenylbenzo-
1
(m, 2H), 7.85 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 7.7 Hz,
1H), 7.52 (m, 3H), 7.46 (m, 1H), 7.09 (s, 1H), 1.77 (s, 6H). 13C
NMR (125 M, CDCl3) δ 161.32, 158.07, 157.69, 154.57, 134.70,
131.23 (q, J = 32.5 Hz), 129.93, 129.15, 129.13, 128.88, 128.26 (q, J =
4.0 Hz), 126.00, 125.65, 125.31 (q, J = 3.7 Hz), 124.76, 123.74,
122.59, 110.67, 106.59, 100.01, 98.71, 96.03, 81.45, 25.87. Mass
spectra (ESI): m/e 463 (M + H)+.
furan-5-carboxylic Acid (4f). H NMR (500 MHz, DMSO-d6) δ
8.11(d, J = 7.5 Hz, 2H), 8.06 (s, 1H), 7.56 (t, J = 7.5 Hz, 2H), 7.47 (m,
1H), 7.38 (m, 3H), 7.15 (s, 1H). 13C NMR (125 M, DMSO-d6) δ
171.99, 162.38 (d, J = 245.7 Hz), 162.27 (d, J = 245.7 Hz), 160.61,
156.84 (d, J = 9.8 Hz), 129.93, 129.17, 128.47, 125.40, 124.74 (t, J =
12.0 Hz), 121.98, 121.05, 114.68 (dd, J = 20.3 and 7.0 Hz), 110.64,
105.36 (t, J = 25.7 Hz), 99.09, 97.55, 94.80, 82.00. Mass spectra (ESI):
m/e 391 (M + H)+.
6-Hydroxy-2-Phenyl-3-((3-(trifluoromethyl)phenyl)ethynyl)-
benzofuran-5-carboxylic Acid (4g). 1H NMR (500 MHz, DMSO-
d6) δ 8.11 (d, J = 7.5 Hz, 2H), 8.06 (s, 1H), 7.95 (s, 1H), 7.90 (d, J =
7.7 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.54 (t,
J = 7.4 Hz, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.14 (s, 1H). 13C NMR (125
M, DMSO-d6) δ 171.99, 160.60, 156.89, 156.55, 135.30, 130.01,
129.84, 129.74 (q, J = 32.0 Hz), 129.08, 128.58, 127.68 (q, J = 3.7 Hz),
125.54 (q, J = 3.7 Hz), 125.37, 123.70 (q, J = 271 Hz), 123.09, 121.95,
121.14, 110.59, 99.05, 97.83, 95.49, 81.56. Mass spectra (ESI): m/e
423 (M + H)+.
2,2-Dimethyl-7-phenyl-6-((4-(trifluoromethoxy)phenyl)-
ethynyl)-4H-[1,3]dioxino[5,4-f ]benzofuran-4-one (14h). 426
1
mg, yield 89%. H NMR (500 MHz, CDCl3) δ 8.35 (s, 1H), 8.25
(m, 2H), 7.64 (dt, J = 8.8 and 2.0 Hz, 2H), 7.50 (t, J = 7.9 Hz, 2H),
7.44 (m, 1H), 7.27 (d, J = 6.8 Hz, 2H), 7.09 (s, 1H), 1.78 (s, 6H). 13C
NMR (125 M, CDCl3) δ 161.37, 157.78, 157.69, 154.53, 149.28,
149.26, 133.14, 129.82, 129.23, 128.84, 125.94, 125.74, 122.30, 121.56,
121.07, 120.41 (q, J = 256.3 Hz), 110.61, 106.58, 99.98, 98.94, 96.26,
80.73, 25.87. Mass spectra (ESI): m/e 478 (M + H)+.
2,2-Dimethyl-6-((4-phenoxyphenyl)ethynyl)-7-phenyl-4H-
[1,3]dioxino[5,4-f ]benzofuran-4-one (14i). 462 mg, yield 95%. 1H
NMR (500 MHz, CDCl3) δ 8.38 (s, 1H), 8.28 (d, J = 7.3 Hz, 2H),
7.57 (dt, J = 8.7 and 2.0 Hz, 2H), 7.39 (m, 3H), 7.17 (t, J = 8.7 Hz,
1H), 7.08 (m, 3H), 7.03 (tt, J = 8.7 and 2.0 Hz, 2H), 1.77 (s, 6H). 13C
NMR (125 M, CDCl3) δ 161.40, 158.15, 157.71, 157.26, 156.27,
154.46, 133.27, 129.95, 129.59, 129.39, 128.78, 125.95, 125.86, 124.03,
122.39, 119.58, 118.44, 117.24, 110.51, 106.51, 99.89, 99.44, 97.50,
79.15, 25.87. Mass spectra (ESI): m/e 487 (M + H)+.
6-Hydroxy-2-phenyl-3-((4-(trifluoromethoxy)phenyl)-
ethynyl)benzofuran-5-carboxylic Acid (4h). 1H NMR (500 MHz,
DMSO-d6) δ 8.15 (d, J = 7.5 Hz, 2H), 8.07 (s, 1H), 7.79 (d, J = 8.8
Hz, 2H), 7.56 (t, J = 7.5 Hz, 2H), 7.46 (m, 3H), 7.19 (s, 1H). Mass
spectra (ESI): m/e 439 (M + H)+.
6-Hydroxy-3-((4-phenoxyphenyl)ethynyl)-2-phenylbenzo-
1
furan-5-carboxylic Acid (4i). H NMR (500 MHz, DMSO-d6) δ
11.70 (br, 1H), 8.16 (d, J = 7.5 Hz, 2H), 8.06 (s, 1H), 7.67 (d, J = 8.7
Hz, 2H), 7.56 (t, J = 7.5 Hz, 2H), 7.45 (m, 3H), 7.22 (m, 2H), 7.10 (d,
J = 7.8 Hz, 2H), 7.04 (d, J = 8.7 Hz, 2H). 13C NMR (125 M, DMSO-
d6) δ 171.99, 160.56, 157.75, 156.97, 155.80, 155.61, 133.45, 130.26,
129.68, 129.12, 128.79, 125.19, 124.31, 121.83, 121.43, 119.48, 118.37,
General Procedure for Preparation of Compounds 2, 3, and
4a−i. To a solution of 14a−i (0.5 mmol) in THF (3 mL), KOH (400
mg, 7.14 mmol) in water (1 mL) was added. The obtained mixture
was refluxed for 2 h, then acidified with 3 M HCl and extracted with
ethyl acetate. The organic layers were dried over anhydrous Na2SO4,
839
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Journal of Medicinal Chemistry
Article
116.48, 110.49, 99.13, 98.44, 97.01, 79.23. Mass spectra (ESI): m/e
447 (M + H)+.
6-Hydroxy-2-phenyl-3-(4-(trifluoromethoxy)phenethyl)-
1
benzofuran-5-carboxylic Acid (6c). H NMR (500 MHz, DMSO-
d6) δ 11.65 (br, 1H), 7.97 (s, 1H), 7.61 (d, J = 7.3 Hz, 2H), 7.48 (t, J =
7.7 Hz, 2H), 7.40 (m, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.0
Hz, 2H), 7.11 (s, 1H), 3.17 (t, J = 7.5 Hz, 2H), 3.00 (t, J = 7.5 Hz,
2H). Mass spectra (ESI): m/e 443 (M + H)+.
3-Ethisterone-6-hydroxy-2-phenylbenzofuran-5-carboxylic
1
Acid (2). H NMR (500 MHz, DMSO-d6) δ 11.7 (br, 1H), 8.16 (m,
2H), 8.09 (s, 1H), 7.51 (t, J = 7.8 Hz, 2H), 7.46 (t, J = 7.8 Hz, 1H),
7.22 (s, 1H), 5.62 (s, 1H), 2.56 (s, 2H), 2.39−2.2 (m, 4H), 2.14−2.02
(m, 2H), 1.92 (m, 1H), 1.83 (m, 1H), 1.76 (m, 3H), 1.57 (m, 4H),
1.38 (m, 2H), 1.14 (s, 3H), 0.90 (m, 2H), 0.87 (s, 3H). 13C NMR
(125 M, DMSO-d6) δ 197.93, 172.08, 170.60, 160.51, 156.92, 155.05,
129.52, 128.87, 128.80, 125.00, 123.21, 121.93, 121.86, 110.34, 103.48,
99.06, 98.69, 78.96, 74.77, 53.43, 50.21, 46.74, 40.36, 38.11, 35.56,
35.03, 33.53, 32.74, 31.84, 31.41, 22.78, 20.34, 16.88, 12.75.
6-Hydroxy-3-(4-phenoxyphenethyl)-2-phenylbenzofuran-5-
1
carboxylic Acid (6d). H NMR (500 MHz, DMSO-d6) δ 11.65 (br,
1H), 7.97 (s, 1H), 7.65 (d, J = 7.4 Hz, 2H), 7.50 (t, J = 7.7 Hz, 2H),
7.41 (t, J = 7.4 Hz, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.17 (d, J = 8.5 Hz,
2H), 7.11 (s, 1H), 7.08 (t, J = 7.4 Hz, 1H), 6.84 (m, 1H), 3.17 (t, J =
7.4 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H). 13C NMR (125 M, DMSO-d6) δ
172.39, 159.82, 157.49, 157.24, 154.56, 150.66, 136.20, 130.09, 129.95,
129.86, 128.94, 128.46, 126.11, 122.91, 122.80, 122.11, 119.00, 117.83,
115.26, 109.28, 98.44, 34.12, 25.54. Mass spectra (ESI): m/e 451 (M +
H)+.
Cell Culture and Transfection. Raw264.7 mouse macrophages
were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% FBS (Invitrogen), penicillin (50 units/mL),
and streptomycin (50 μg/mL) under a humidified atmosphere
containing 5% CO2 at 37 °C. Transfected Raw264.7 cells (Vector,
WT-mPTPB) were seeded in a 12-well plate at a density of 4 × 104
cells/well. The following day cells were treated with mPTPB inhibitor
4g for 1 h and stimulated with IFN-γ (200 U/ml) for 1 h. Cells were
then washed with ice-cold phosphate buffered saline, and lysed with
lysis buffer on ice for 30 min. Cell lysates were cleared by
centrifugation at 13 000 rpm for 15 min. The phosphorylation of
ERK1/2, p38, and Akt was detected by Western blotting.
IL-6 Enzyme-Linked Immunosorbent Assay (ELISA). Trans-
fected Raw264.7 cells (Vector, WT-mPTPB) were seeded in a 12-well
plate at a density of 4 × 104 cells/well. The following day cells were
treated with mPTPB inhibitor 4g for 1 h and then were left
unstimulated or stimulated for 24 h with IFN-γ (200 U/ml). After 24
h incubation, supernatants were collected, cleared by centrifugation,
and assayed for IL-6 release using mouse IL-6 ELISA kit (eBioscience)
and a plate reader.
Caspase Activity Assay. mPTPB transfected Raw264.7 cells were
treated with either compound 4g or the negative control 6c for 1 h,
then stimulated with IFN-γ (200U/ml). After 24 h, about 4 × 105 cells
were collected. The substrate Ac-DEVD-pNA (Sigma) was used to
measure caspase-3 activity following the described procedures.35
Briefly, the collected cells were washed in ice-cold PBS, and lysed in 50
μL of caspase lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 5
mM MgCl2, 10 mM DTT, 10 μg/mL each of pepstatine and leupeptin,
0.5% TX-100, and 2 mM PMSF). Cells were kept on ice for 20 min
with occasional mixing. The lysates were then centrifuge at 13 000g for
15 min at 4 °C. Protein concentration of the supernatant was
determined using the Bradford method (Bio-Rad). Subsequently, the
supernatant containing 50 μg of protein was diluted into a final volume
of 200 μL with the assay buffer (50 mM HEPES, 10% sucrose, 0.1%
CHAPS, and 10 mM DTT) containing 100 μM of the substrate Ac-
DEVD-pNA and incubated for 2hr in a 96-well plate at 37 °C. The
cleavage of the substrate by caspase-3 in the lysate released pNA,
which was determined by measuring the absorbance at 405 nm using a
plate reader spectrophotometer.
6-Hydroxy-3-(3-hydroxyprop-1-yn-1-yl)-2-phenylbenzofur-
1
an-5-carboxylic Acid (3). H NMR (500 MHz, DMSO-d6) δ 8.18
(d, J = 7.4 Hz, 2H), 8.07 (s, 1H), 7.57 (t, J = 7.4 Hz, 2H), 7.49 (t, J =
7.4 Hz, 1H), 7.24 (s, 1H), 4.50 (s, 2H). 13C NMR (125 M, DMSO-d6)
δ 161.0, 157.37, 156.15, 130.18, 129.58, 129.21, 125.60, 122.27,
100.00, 99.62, 99.22, 98.77, 74.50, 50.26.
(Z)-3-(3,5-Difluorostyryl)-6-hydroxy-2-phenylbenzofuran-5-
carboxylic Acid (5a). To a stirred solution of 3-((3,5-
difluorophenyl)ethynyl)-6-hydroxy-2-phenylbenzofuran-5-carboxylic
acid 4f (126 mg, 0.32 mmol) in MeOH/THF (1/1, 2 mL) was added
5% Pd/BaSO4 (15 mg), and the mixture was allowed to stir for 12 h at
room temperature under H2 atmosphere. The mixture was filtered
through Celite pad, and the filtrate was concentrated under reduced
pressure. The residue was purified by HPLC to furnish compound 5a
1
(110 mg, 88%). H NMR (500 MHz, DMSO-d6) δ 7.82 (d, J = 7.35
Hz, 2H), 7.52 (t, J = 7.45 Hz, 2H), 7.44 (m, 1H), 7.30 (s, 1H), 7.16 (s,
1H), 7.03 (m, 1H), 6.98 (d, J = 12 Hz, 1H), 6.92 (m, 2H), 6.86 (d, J =
12 Hz, 1H). 13C NMR (125 M, DMSO-d6) δ 171.99, 162.25 (d, J =
244.3 Hz), 161.14 (d, J = 244.4 Hz), 159.80, 157.84, 152.03, 140.26 (t,
J = 9.9 Hz), 131.23, 129.48, 129.15, 129.04, 126.37, 123.21, 121.81,
119.46, 112.43, 111.38 (dd, J = 20.4 and 7.0 Hz), 109.54, 102.99 (t, J =
26.0 Hz), 98.79. Mass spectra (ESI): m/e 393 (M + H)+.
(Z)-6-Hydroxy-2-phenyl-3-(4-(trifluoromethoxy)styryl)-
benzofuran-5-carboxylic Acid (5b). To a stirred solution of 6-
hydroxy-2-phenyl-3-((4-(trifluoromethoxy)phenyl)ethynyl)-
benzofuran-5-carboxylic acid 4h (98 mg, 0.22 mmol) in MeOH/THF
(1/1, 2 mL) was added 5% Pd/BaSO4 (15 mg), and the mixture was
allowed to stir for 12 h at room temperature under H2 atmosphere.
The mixture was filtered through Celite pad, and the filtrate was
concentrated under reduced pressure. The residue was purified by
1
HPLC to furnish compound 5b (90 mg, 92%). H NMR (500 MHz,
DMSO-d6) δ 11.6 (br, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.53 (t, J = 8.0
Hz, 2H), 7.45 (t, J = 7.4 Hz, 1H), 7.37 (t, J = 8.7 Hz, 2H), 7.25 (s,k
1H), 7.16 (m, 3H), 7.05 (d, J = 12 Hz, 1H), 6.80 (d, J = 12 Hz, 1H).
13C NMR (125 M, DMSO-d6) δ 172.03, 159.76, 157.88, 151.67,
147.48, 135.95, 132.08, 130.25, 129.63, 129.02, 128.99, 126.22, 123.41,
120.83, 120.17, 119.96 (q, J = 255.1 Hz), 119.54, 112.91, 109.37,
98.71. Mass spectra (ESI): m/e 441 (M + H)+.
General Procedure for Preparation of Compounds 6a−d. To
a stirred solution of appropriate carboxylic acid (0.30 mmol) in
MeOH/THF (1/1, 2 mL) was added 10% Pd/C (15 mg), and the
mixture was allowed to stir for 12 h at room temperature under H2
atmosphere. The mixture was filtered through Celite pad, and the
filtrate was concentrated under reduced pressure. The residue was
purified by HPLC to furnish desired compounds 6a−d in 90−95%
yield.
Flow Cytometric Analysis. mPTPB transfected Raw264.7 cells
were treated with 4g or the negative control 6c for 1 h, and then
stimulated with IFN-γ (200U/ml). After 24 h, about 4 × 105 cells were
harvested by resuspending in the Cell Dissociation Buffer
(GIBCOTM, Invitrogen Corporation) and gently scraped into a 15
mL centrifuge tube. The collected cells were stained either with
annexinV-APC and PI proceeding with the protocol of Annexin V-
APC Apoptosis Detection Kit (BD PharmingenTM). Briefly, cells
were washed twice with cold PBS and then were resuspended in 500
μL of 1x binding buffer. A totalof 100 μL of solution was transferred to
a 5 mL culture tube. Five microliters of Annexin V-APC and 5 μL of PI
were added into the solution. The solution was gently vortexed and
incubated for 15 min at room temperature in the dark. Then 400 μL of
1x binding buffer was added into each tube. Each group of samples was
3-(3-Fluorophenethyl)-6-hydroxy-2-phenylbenzofuran-5-
1
carboxylic Acid (6a). H NMR (500 MHz, DMSO-d6) δ 11.70 (br,
1H), 8.03 (s, 1H), 7.64 (d, J = 7.35 Hz, 2H), 7.50 (t, J = 7.7 Hz, 2H),
7.40 (t, J = 7.4 Hz, 1H), 7.25 (m, 1H), 7.11 (s, 1H), 7.09 (m, 1H),
7.01 (d, J = 7.7 Hz, 1H), 6.97 (m, 1H), 3.17 (t, J = 7.4 Hz, 2H), 2.98
(t, J = 7.4 Hz, 2H). Mass spectra (ESI): m/e 377 (M + H)+.
6-Hydroxy-2-phenyl-3-(3-(trifluoromethyl)phenethyl)-
1
benzofuran-5-carboxylic Acid (6b). H NMR (500 MHz, DMSO-
d6) δ 11.65 (br, 1H), 7.96 (s, 1H), 7.61 (d, J = 7.3 Hz, 2H), 7.54 (s,
1H), 7.51 (m, 1H), 7.47 (m, 4H), 7.40 (m, 3H), 7.10 (s, 1H), 3.20 (t,
J = 7.4 Hz, 2H), 3.07 (t, J = 7.4 Hz, 2H). Mass spectra (ESI): m/e 427
(M + H)+.
840
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Journal of Medicinal Chemistry
Article
analyzed using a BD FACSArray Bioanalyzer. The data were analyzed
using CellQuest software (BD Bioscences).
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mptpB impairs the ability of Mycobacterium tuberculosis to survive in
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Kinetic Analysis of mPTPB Inhibition. The phosphatase activity
of mPTPB was assayed using p-nitrophenyl phosphate (pNPP) as a
substrate at 25 °C in 50 mM 3,3-dimethylglutarate buffer, pH 7.0,
containing 1 mM EDTA with an ionic strength of 0.15 M adjusted by
NaCl. Compounds exhibiting more than 50% of inhibitory activity
against mPTPB were selected for IC50 measurement. The reaction was
started by the addition of 5 μL of the enzyme to 195 μL of reaction
mixture containing 2.5 mM (the Km value) of pNPP and various
concentrations of the inhibitor. The reaction was quenched after 5 min
by the addition of 50 μL of 5N NaOH, and then 200 μL of reaction
mixture was transferred to a 96-well plate. The nonenzymatic
hydrolysis of pNPP was corrected by measuring the control without
the addition of enzyme. The amount of product p-nitrophenol was
determined from the absorbance at 405 nm detected by a Spectra
MAX340 microplate spectrophotometer (Molecular Devices) using a
molar extinction coefficient of 18 000 M−1 cm−1. IC50 values were
calculated by fitting the absorbance at 405 nm versus inhibitor
concentration to the following equation:
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AI/A0 = IC50/(IC50 + [I])
where AI is the absorbance at 405 nm of the sample in the presence of
inhibitor; A0 is the absorbance at 405 nm in the absence of inhibitor;
and [I] is the concentration of the inhibitor.
For selectivity studies, the PTPs, including mPTPA, PTP1B, TC-
PTP, SHP1, SHP2, FAP1, Lyp, PTP-MEG2, HePTP, PTPα, LAR,
CD45, PTPγ, VHR, Laforin, VHX, Cdc14A, and LMWPTP PTP were
expressed and purified from E. coli. The inhibition assay for these PTPs
were performed under the same conditions as mPTPB except using a
different pNPP concentration corresponding to the Km of the PTP
studied. Inhibitor concentrations used for IC50 measurements cover
the range from 0.2 to 5× of the IC50 value.
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tuberculosis-Infected Macrophages. J. Cell Death 2010, 3, 1−11.
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defense function of macrophages against Mycobacterium tuberculosis.
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Johnson, M. E.; Fordham-Skelton, A.; Abad-Zapatero, C.; Cavet, J. S.;
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: (317) 274-8025. E-mail: zyzhang@iupui.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
Grants R56 AI090829 and CA126937.
(21) Noren-Muller, A.; Reis-Correa, I.; Prinz, H.; Rosenbaum, C.;
Saxena, K.; Schwalbe, H.; Vestweber, D.; Cagna, G.; Schunk, S.;
Schwarz, O.; Schiewe, H.; Waldmann, H. Discovery of protein
phosphatase inhibitor classes by biology-oriented synthesis. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 10606−10611.
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