Substrate-Based fragment identification for the development of selective, nonpeptidic inhibitors of striatal-enriched protein tyrosine phosphatase

Article abstract of DOI:10.1021/jm401037h

High levels of striatal-enriched protein tyrosine phosphatase (STEP) activity are observed in a number of neuropsychiatric disorders such as Alzheimer's disease. Overexpression of STEP results in the dephosphorylation and inactivation of many key neuronal

Full text of DOI:10.1021/jm401037h

Article
pubs.acs.org/jmc
Substrate-Based Fragment Identification for the Development of
Selective, Nonpeptidic Inhibitors of Striatal-Enriched Protein
Tyrosine Phosphatase
Tyler D. Baguley,†,∥ Hai-Chao Xu,†,∥,⊥ Manavi Chatterjee,‡ Angus C. Nairn,§ Paul J. Lombroso,‡
and Jonathan A. Ellman*,†
†Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
‡Departments of Psychiatry and Neurobiology, Yale University, New Haven, Connecticut 06520, United States
§Department of Psychiatry, Yale University, New Haven, Connecticut 06508, United States
S
* Supporting Information
ABSTRACT: High levels of striatal-enriched protein tyrosine
phosphatase (STEP) activity are observed in a number of
neuropsychiatric disorders such as Alzheimer’s disease. Over-
expression of STEP results in the dephosphorylation and
inactivation of many key neuronal signaling molecules, including
ionotropic glutamate receptors. Moreover, genetically reducing
STEP levels in AD mouse models significantly reversed cognitive
deficits and decreased glutamate receptor internalization. These
results support STEP as a potential target for drug discovery for
the treatment of Alzheimer’s disease. Herein, a substrate-based
approach for the discovery and optimization of fragments called substrate activity screening (SAS) has been applied to the
development of low molecular weight (<450 Da) and nonpeptidic, single-digit micromolar mechanism-based STEP inhibitors
with greater than 20-fold selectivity across multiple tyrosine and dual specificity phosphatases. Significant levels of STEP
inhibition in rat cortical neurons are also observed.
increased receptor levels on synaptic membranes.11 These
INTRODUCTION
■
results support STEP as a potential target for drug discovery for
Synaptic connections provide the physical basis for communi-
the treatment of AD.
cation within the brain, and synaptic plasticity, the ability for
We have previously reported on the use of a fragment-based
synapses to strengthen or weaken between neurons as a result
inhibitor discovery and optimization approach termed substrate
of molecular signals, is critical to maintaining proper cognitive
activity screening (SAS) for the identification of low molecular
function. Therefore, disruptions in synaptic function can lead to
weight inhibitors of the tyrosine phosphatases of the bacterium
impairments in cognition. Synaptic dysregulation has been
Mycobacterium tuberculosis, PtpA and PtpB.12 To date, there is a
implicated in a range of neuropsychiatric disorders,1 including
single reported ancillary example of a STEP inhibitor having
Alzheimer’s disease (AD),2 schizophrenia,3 depression,4 fragile
modest activity and without selectivity or cell data.13 Herein, we
X syndrome,5 and drug addiction.6
have applied the SAS method to identify low molecular weight
One protein that has been implicated in the dysregulation of
(<450 Da) nonpeptidic STEP inhibitors with single-digit
synaptic plasticity is striatal-enriched protein tyrosine phospha-
micromolar inhibition, 20-fold selectivity over multiple human
tase (STEP), which is encoded by the PTPN5 gene and is
PTPs, and significant activity in rat cortical neurons.
found in striatum, hippocampus, cortex, and related regions.
High levels of STEP activity result in the dephosphorylation
and inactivation of several neuronal signaling molecules,
RESULTS AND DISCUSSION
■
In the SAS fragment approach as applied to PTPs,12 a library of
including extracellular signal-regulated kinases 1 and 2
(ERK1/2),7 proline-rich tyrosine kinase 2 (Pyk2),8 mitogen-
low molecular weight O-aryl and -heteroaryl phosphates are
activated protein kinase p38,9 and the GluN2B subunit of the
screened as potential PTP substrates using a simple and
N-methyl-D-aspartate receptor (NMDAR).10,11 Dephosphory-
continuous coupled assay with purine nucleoside phosphorylase
used to detect the release of inorganic phosphate. Active
lation of the kinases inactivates them, while dephosphorylation
substrates are then converted to inhibitors through the
of GluN2B results in internalization of NMDA receptors. To
replacement of the phosphate group with a nonhydrolyzable
test the hypothesis that overexpression of STEP might
contribute to cognitive deficits in AD mouse models, STEP
levels were reduced genetically in AD mice. Progeny null for
Received: July 9, 2013
STEP exhibited significant cognitive improvements and
© XXXX American Chemical Society
A
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Figure 1. Selected initial substrate hits obtained against STEP.
phosphate mimetic. Key advantages of this approach for the
identification of mechanism-based inhibitors are (1) the ease of
preparing a library of diverse low molecular weight phosphate
substrates, (2) the inherent “turn on” nature of a substrate
screen, which minimizes the high percentage of false positives
typical to PTP inhibitor screens,14,15 and (3) the flexibility to
explore different phosphate mimetics after the identification of
substrate fragments.
Identification of Substrates. A library of O-aryl phosphate
substrate fragments was previously generated and screened to
identify Mycobacterium tuberculosis PtpB and PtpA inhibitors.12
Screening this library of phosphates against STEP yielded
several promising fragment substrates (Figure 1). Of note,
fragment substrates 6−10 had much improved KM values
relative to the phosphotyrosine derivative 4, which much more
closely resembles naturally occurring PTP substrates.
Conversion of Substrates to Inhibitors. The two
substrate scaffolds 6 and 8 were identified as initial starting
points for further optimization because the biphenyl scaffold
has been regarded as a “privileged scaffold” with druglike
properties and because analogue preparation is straightforward
using cross-coupling methodology.16 Inhibitors 11 and 12
(Figure 2) were first prepared by replacing the phosphate group
of each substrate with the nonhydrolyzable phosphate mimetic
difluoromethylphosphonic acid (DFMP).17 The Ki values of
the two inhibitors were measured using a standard, continuous
in vitro inhibition assay, with p-nitrophenyl phosphate (pNPP)
serving as the chromogenic substrate.18 Triton-X 100, 0.01% v/
v, was added to the assay buffer to prevent nonproductive
inhibition by micelle formation.19 Importantly, the relative Ki
values of inhibitors 1120 and 1212b were determined to be 337
and 120 μM, respectively, which correlate reasonably well with
the KM values of the corresponding substrates 6 and 8.21
Optimization of Inhibitor Potency. Introduction of
diverse substitution onto the biphenyl cores of inhibitors 11
and 12 was next performed. For fragment 11, a series of
substitutions was first introduced on the distal aromatic ring
(Table 1). Although substitution at the para position of the
distal ring was beneficial for inhibition (11a), any substitution
larger than a methyl group resulted in decreased potency
(11b). Alkyl substitution at the meta position also led to an
increase in potency of the inhibitors, with the α-branched and
more bulky isopropyl group outperforming the methyl group
(11d versus 11c). The presence of an oxygen atom at the ortho
position was also beneficial to the potency of the inhibitors,
with the free hydroxyl resulting in greater inhibition than the
methoxy derivative (11e and 11f). Combining a meta alkyl
group and an ortho hydroxyl group in a 2,3-orientation (11g)
led to an increase in potency, while the 2,5-substitution was not
advantageous (11h).
We next investigated the effect of altering the m-alkyl group
(11i−n) on inhibitory potency. The ethyl group (11i) resulted
in 2-fold reduction in inhibitory potency, while the tert-butyl
group provided a modest enhancement (11j). A more
significant increase in potency was achieved by introducing
cycloalkyl groups, with cyclopentyl (11l) and cyclohexyl (11m)
providing the optimal ring size. Decreasing the ring size to
cyclobutyl (11k) or increasing it to cycloheptyl (11n) was met
with diminished potency compared to the optimal cyclohexyl
substituted derivative (11m, Ki = 20 μM). Finally, introduction
of the hydroxymethylphosphonic acid phosphate mimetic
(11o) in place of difluoromethylphosphonic acid resulted in
an approximate 2-fold reduction in potency.
The effect of substitution on the distal aromatic ring of
inhibitor 12 was also evaluated (Table 2). The electron
Figure 2. DFMP inhibitors 11 and 12 based on privileged substrate
scaffolds 6 and 8.
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and meta (12e) positions. Although tolerated, a modest
decrease in potency was observed with simple alkyl substitution
at the meta (12f) and para (12g) positions. Introduction of H-
bond donors were detrimental when placed at the ortho (12h)
and para (12k) positions but were tolerated at the meta
position (12i, 12j, and 12l), with the hydroxyethyl group (12j)
providing modestly increased inhibition. However, the greatest
increase in potency was observed for benzyl substitution at the
meta position (12m), which resulted in a 2-fold enhancement.
Further modification of the benzyl substituted inhibitor 12m
was next performed (Figure 3). The introduction of halogens
on the benzyl group was found to be beneficial for the potency
of the inhibitors (12n and 12o), with the 3,4-dichlorobenzyl
group being optimal (12p). The hydroxyl group, which was
previously observed to be well tolerated at the benzylic position
(see 12j, Table 2), increased the solubility of the inhibitor with
only a moderate reduction in potency (12q). Replacing the
difluoromethylphosphonic acid group with a α-hydroxyphos-
phonic acid group maintained the desired potency (12r).
We next explored the effect of combining the hydroxyl
groups in inhibitors 12q and 12r by the preparation and assay
of the four possible stereoisomers, 12s−v. While the potency is
not affected by the stereochemistry of the distal biarylmethanol
(12s versus 12t), the (S)-configuration at the α-hydroxyphos-
phonic acid is crucial for inhibitory activity (see 12u and 12v).
Further evidence of the importance of this hydroxyl group was
the dramatic loss in potency with its absence as demonstrated
by compound 12w.
Table 1. Optimization of Distal Aryl Ring Substitution for
Inhibitor 11
a
X, X
F, F
R1
R2
R3
R4
Ki (μM)
11
H
H
H
H
337 60
11a
11b
11c
11d
11e
11f
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
F, F
OH, H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
Me
i-Pr
H
205
222
137
81
1
1
8
H
H
H
Me
i-Pr
H
H
H
2
OH
OMe
OH
H
H
168 17
271 17
H
H
11g
11h
11i
i-Pr
i-Pr
Et
H
69
95
124
63
45
25
20
62
36
9
1
H
OH
OH
OH
OH
OH
OH
OH
H
3
11j
t-Bu
H
1
2
4
3
1
1
11k
11l
cyclobutyl
cyclopentyl
cyclohexyl
cycloheptyl
cyclohexyl
H
H
11m
11n
11o
H
H
H
a
Ki values were determined using at least two independent
measurements.
Inhibitor Selectivity. There is high structural homology
among PTP active sites, which often leads to difficulty in
achieving high selectivity. We therefore tested the best
inhibitors from the two biaryl series, inhibitors 11m, 12s, and
12t, against a panel of tyrosine and dual-specificity
phosphatases (Table 3). Notably, our most potent inhibitors
from the 1,4-biphenyl series 12s and 12t displayed greater than
20-fold selectivity against all phosphatases tested.
Table 2. Optimization of Distal Aryl Ring Substitution for
Inhibitor 12
a
STEP Inhibition in Neuronal Cultures. The most
promising inhibitors, 12s and 12t, were also evaluated for
their ability to inhibit STEP in rat cortical neurons by
monitoring the phosphorylation levels of the known STEP
substrates, GluN2B, Pyk2 and ERK1/2.7,8,10,11 Clear increases
in the phosphorylation levels of each of the substrates were
observed for inhibitor 12s with more modest effects observed
for 12t (Figure 4).
Blood−Brain Barrier Permeability. Given the promising
cell data, the best compound, 12s, was evaluated for its ability
to passively permeate the blood−brain barrier (BBB) using the
well validated parallel artificial membrane permeability assay
(PAMPA) technique (Pion, Inc., Billerica, MA).22 Although the
compound can cross cell membranes, as evidenced in the above
cell data, compound 12s did not possess the ability to passively
cross the BBB in this model system (data in Supporting
Information). As such, BBB permeability remains a real
challenge for future work. The highly polar nature of the α-
hydroxyphosphonic acid motif is likely to be one of the key
factors limiting BBB permeability. Replacement of this motif
with less polar, nonhydrolyzable phosphate mimetics might
overcome this problem.15 Alternatively, effective prodrug
strategies have also been developed to mask polar functionality
in order to enable BBB permeability.23
R1
R2
R3
Ki (μM)
12
H
H
H
120
7
12a
12b
12c
12d
12e
12f
12g
12h
12i
CN
H
H
H
375 30
400 48
617 45
CN
H
H
H
CN
H
H
O-i-Pr
200
231
132
1
6
3
H
H
O-i-Pr
H
i-Pr
H
H
H
i-Pr
185 33
NHMs
H
H
H
673
113
88
5
1
NHMs
H
12j
H
CH(OH)CH3
H
3
12k
12l
H
H
CH(OH)CH3
450 183
H
NHPh
Bn
H
H
112
73
9
12m
H
3
a
Ki values were determined using at least two independent
measurements.
deficient cyano group proved to be detrimental to inhibition at
the ortho (12a), meta (12b), and para (12c) sites. Alkoxy
groups also reduced inhibition when placed at the ortho (12d)
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Figure 3. Additional analogues based upon inhibitor 12m.
a
Table 3. Selectivity of Most Potent Inhibitors against a Panel of PTPs
STEP
TC-Ptp
71
CD45
>500
LAR
>900
MKP5
11m
12s
12t
Ki, μM
20
3
2
150 20
7.5
selectivity
Ki, μM
3.6
164
18
>25
>45
8.9 1.2
7.8 0.7
9
340 40
38
360 30
40
340 45
41
selectivity
Ki, μM
170 20
22
270 20
35
390 110
50
>500
selectivity
>65
a
Ki values were determined using at least two independent measurements.
Figure 4. Rat cortical neurons were treated with vehicle or 12s (a) or 12t (b) (concentrations of 0.1, 1, or 10 μM) for 1 h and analyzed by Western
blotting: (∗) p < 0.05; (∗∗) p < 0.01; (∗∗∗) p < 0.001 one-way ANOVA, Dunnett’s post hoc. Data represent the phospho signal normalized to the
total protein signal and GAPDH SEM (n = 3−5 each group).
For the preparation of 3-biphenyl inhibitors 11g,i−j,l−m,o,
the synthesis of 2-hydroxyphenylboronic acids was required
(Scheme 2). Selective ortho-bromination of the 2-alkylphenols
15a−e gave the aryl bromides 16a−e,24 upon which
ortholithiation, treatment with B(OMe)3, and aqueous workup
provided the desired arylboronic acids 17a−e.25
Starting phenols 15f and 15g were not commercially
available and were synthesized from 2-bromophenol in a
three-step sequence (Scheme 3). Dilithiation was followed by
CHEMISTRY
■
The biaryl difluoromethylphosphonic acid (DFMP) inhibitors
could be conveniently prepared by relying on the Suzuki−
Miyaura cross-coupling reaction (Scheme 1). The commercial
availability of many diverse arylboronic acids enabled rapid
access to the initial set of biaryl inhibitors. However, further
optimization of the inhibitor structures required that we
prepare the requisite more complex arylboronic acid inputs.
D
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Scheme 1. Inhibitor Synthesis through Suzuki−Miyaura
Scheme 4. Synthesis of Diarylmethane-Based Boron
Reagents 22 and 24
a
a
Cross-Coupling
a
Reagents: (a) ArBLn, PdCl2, CyJohnPhos, K2CO3, dioxane/H2O; (b)
ArBLn, Pd(PPh3)4, Na2CO3, DME/EtOH/H2O.
a
Scheme 2. Synthesis of Arylboronic Acids 17
a
Reagents: (a) PhMgBr, Et2O or 1-bromo-3-iodobenzene, n-
butyllithium, THF; aldehyde; (b) Et3SiH, F3CCOOH, CH2Cl2; (c)
Pd(dppf)Cl2, KOAc, B2pin2, DMSO; (d) n-butyllithium, THF, 3,4-
dichlorobenzaldehyde.
a
Reagents: (a) N-bromosuccinimide, N,N-diisopropylamine, CH2Cl2;
(b) n-butyllithium, Et2O, B(OMe)3, aqueous HCl.
Scheme 5. Synthesis of α-Hydroxymethylphosphonic Acid
Inhibitors 11o and 12r
a
a
Scheme 3. Synthesis of Aryltrifluoroborates 18
a
Reagents: (a) Pd(PPh3)4, Na2CO3, DME/EtOH/H2O; (b) NaBH4,
a
Reagents: (a) n-butyllithium, Et2O; (b) ketone, aqueous NH4Cl; (c)
MeOH.
Et3SiH, F3CCOOH, CH2Cl2; (d) Et2SiH2, [Ir(cod)Cl]2, benzene,
B2pin2, HBpin, [Ir(cod)Cl]2, dtbpy, THF, aqueous KHF2.
The four stereoisomeric α-hydroxymethylphosphonic acids
12s−v were prepared from the enantiomerically pure α-
hydroxymethylphosphonic acids 30 and the enantiomerically
enriched boronic esters 32 by Suzuki−Miyaura cross-coupling
(Scheme 6). The synthesis of the enantiomerically pure
phosphonic acids 30 started with the addition of tris-
[(1R,2S,5R)-menth-2-yl]phosphite to 4-bromobenzaldehyde
to give a mixture of diastereomers 29a and 29b, which were
separated by recrystallization according to literature procedures
for analogous compounds.30 Removal of the menthyl groups by
treatment with TMSCl and NaI afforded phosphonic acids 30a
and 30b. The absolute stereochemistry of the α-hydroxyphos-
phonic acids was confirmed by chemical correlation through
hydrodebromination of 30b to the corresponding α-hydroxy-
phenylmethylphosphonic acid, for which the absolute config-
uration had previously been determined.31 The catalytic
asymmetric addition of a 3,4-dichlorophenylzinc reagent to 3-
bromobenzaldehyde using each enantiomer of 3-exo-
(morpholino)isoborneol (MIB)32 gave the enantiomerically
enriched diarylmethanols 31a and 31b in 90% ee. Subsequent
Miyaura borylation led to the boronic esters 32a and 32b.33
addition of the desired ketones, resulting in benzylic alcohols,
which upon treatment with Et3SiH under acidic conditions
afforded the 2-alkylphenols 15. The phenols were then
converted to the aryltrifluoroborates 18 utilizing an iridium-
catalyzed one-pot silyl-directed ortho borylation approach.26
The synthesis of the arylboron reagents needed to prepare
inhibitors 12m−p and 12r started with the addition of PhMgBr
or in situ generated 3-bromophenyllithium to aldehydes 19 to
give diarylmethanols 20 (Scheme 4). Acid mediated reductive
removal of the hydroxyl group to give 21 was followed by
Miyaura borylation reactions to afford boronic esters 22.27
Alternatively, boronic acid 24 was conveniently prepared from
the previously reported intermediate 23.28
The α-hydroxymethylphosphonic acid inhibitors 11o and
12r were also prepared by Suzuki cross-coupling reaction
(Scheme 5). Ketones 26 and 28 were first obtained by cross-
coupling ketophosphonic acids 2529 and 27 with arylboronic
acids 17e and 22d, respectively. Subsequent reduction then led
to the α-hydroxymethylphosphonic acid inhibitors 11o and
12r.
E
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a
Scheme 6. Synthesis of the Four Stereoisomeric α-Hydroxymethylphosphonic Acid Inhibitors 12s−v
a
Reagents (a) P(OMnt)3, TMSCl, 0 °C, separation of diastereomers by recrystallization; (b) TMSCl, NaI, CH3CN; (c) (i) n-butyllithium, t-
BuOMe; (ii) ZnCl2; (iii) n-butyllithium; (iv) tetraethylethylenediamine, toluene; (v) (+)-MIB or (−)-MIB (10% mol); (vi) 3-bromobenzaldehyde;
(d) Pd(dppf)Cl2, KOAc, B2Pin2, DMSO; (e) Pd(PPh3)4, Na2CO3, DME/EtOH/H2O.
400 mesh silica gel or by utilizing a Biotage SP1 flash purification
Final Suzuki−Miyaura cross-coupling afforded the four
system (Biotage model SP1-B1A) or a Teledyne Isco CombiFlash Rf
system. Reversed-phase purifications were conducted with a Teledyne
diastereomeric inhibitors 12s−v.
Isco CombiFlash Rf system equipped with HP C18 gold cartridges.
Product yields are not optimized. Enzymatic assays were carried out on
a BioTek Synergy 2 multimode microplate reader. All of the tested
substrates and inhibitors displayed ≥95% purity as determined by
HPLC or UPLC. BBB PAMPA studies were conducted by Pion, Inc.
(Billerica, MA).
CONCLUSION
■
Although high STEP activity has been observed in many
neuropsychiatric disorders, such as Alzheimer’s disease, there
have been no selective and potent inhibitors reported for
potential treatment and study of these diseases. This report
describes the first dedicated effort for the identification of
selective small-molecule mechanism-based inhibitors of STEP.
A library of low molecular weight O-aryl and -heteroaryl
phosphate fragments were screened, which identified both the
4- and the 3-biaryl scaffolds as promising templates for inhibitor
development. After conversion to inhibitors, SAR of the
scaffolds was explored to identify potent inhibitors from each
series, with the most potent inhibitors (11m, 12s, and 12t) all
showing promising selectivity over other human phosphatases
tested. Importantly, the most selective inhibitors 12s and 12t
were able to inhibit STEP in rat cortical neurons as indicated by
the significant increase in phosphorylation levels of STEP
substrates. Future efforts will focus on increasing the inhibitory
activity and enhancing the blood−brain barrier permeability of
the STEP inhibitors.
General Procedures for Determination of Substrate KM. To
facilitate screening in a high-throughput fashion, 96-well plates were
used with reaction volumes of 100 μL. An amount of 30 μL of water
was added to each well, followed by 5 μL of 20× buffer (1.0 M
imidazole, 1.0 M NaCl, 0.2% Triton-X 100, pH 7.0), 10 μL of 10×
DTT (50 mM, 5 mM in assay), 40 μL of 2-amino-6-mercapto-7-
methylpurine riboside (MESG) solution (1 mM, 400 μM in assay),
and 5 μL of purine nucleotide phosphorylase (PNP) solution (20 U/
mL, 1 U/mL in assay). An amount of 5 μL of STEP phosphatase (2
μM, 100 nM in assay) was added, and the 96-well assay plate was
incubated at 27 °C for 3 min. The coupled assay was started by
addition of 5 μL of the appropriate substrate dilution in DMSO
(typically 3.00, 1.20, 0.480, 0.192, 0.077, 0.031, 0.012, 0 mM in assay).
The plate was then immediately placed into a spectrophotometric
plate reader, and 20 min of kinetic data was obtained (360 nm, 27 °C).
The initial rate data collected were used for Michaelis−Menten kinetic
analysis where the KM was obtained using the substrate−velocity data
with the equation V = (Vmax[S])/(KM + [S]).
General Procedures for Determination of Inhibitor Ki.
Reaction volumes of 100 μL were used in 96-well plates. An amount
of 65 μL of water was added to each well, followed by 5 μL of 20×
buffer (1.0 M imidazole, 1.0 M NaCl, 0.2% Triton-X 100, pH 7.0), 10
μL of 10× DTT (50 mM, 5 mM in assay), 5 μL of STEP phosphatase
(2 μM, 100 nM in assay), and 5 μL of the appropriate inhibitor
dilution in DMSO (with 2- or 3-fold serial dilutions). The assay plate
was incubated for 5 min at 27 °C, at which point the reaction was
started by addition of 10 μL of a 10× pNPP substrate (5 mM, 500 μM
in assay). The plate was then immediately placed into a
spectrophotometric plate reader, and 20 min of kinetic data was
obtained (405 nm, 27 °C). The initial rate data collected were used for
determination of Ki values. For Ki determination, the kinetic values
were obtained directly from nonlinear regression of substrate−velocity
EXPERIMENTAL SECTION
■
Previously Reported Substrates and Inhibitors. Substrates 1−
1012a and inhibitors 1120 and 1212b have been previously reported.
General Methods. Unless otherwise noted, all reagents were
obtained from commercial suppliers and used without further
purification. Tetrahydrofuran (THF), dioxane, CH2Cl2, and diethyl
ether were passed through a column of activated alumina (type A2, 12
× 32, Purify Co.) under nitrogen pressure immediately prior to use. All
1H, 19F, and 31P NMR spectra were obtained at room temperature on a
Bruker AVB-400 or AVB-500 spectrometer. NMR chemical shifts are
reported in ppm relative to TMS (0.00), CHCl3 (7.26), or CH3OH
(3.31) for 1H, trifluoroacetic acid (−76.55) for 19F, and H3PO4 (0.00)
for 31P. Mass spectrometry (HRMS, ESI) are reported in m/z.
Chromatography was performed with SiliCycle SiliaFlash P60 230−
F
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curves in the presence of various concentrations of inhibitor. Assays
were run in at least duplicate using the same inhibitor stock solutions.
For the selectivity assays, the KM of pNPP toward each of the
enzymes was determined in the above assay buffer and used for data
analysis. For the assays with the dual-specificity MKP5, due to poor
turnover of pNPP, the chromogenic substrate 6,8-difluoro-4-
methylumbelliferyl phosphate (DiFMUP) was used instead. In
addition, the assay buffer used was a 10× buffer (0.2 M Tris-HCl,
0.2% Triton-X 100, pH 8.0) and 5 mM DTT was used as in the other
assays.
Cell Culture and Western Blotting. The Yale University
Institutional Animal Care and Use Committee approved all
procedures. Primary cortical neurons were isolated from Sprague−
Dawley rat embryos (E18) (Charles River Laboratories, Wilmington,
MA) as previously described.34 Briefly, cells were dissociated with
trypsin, resuspended in Hanks’ balanced salt solution, and then plated
on poly-D-lysine-coated plates (1 × 106 cells/well) in neurobasal
medium supplemented with 2% B27 (Invitrogen, San Diego, CA).
Neurons were allowed to grow for 18−21 days at 37 °C in a CO2
incubator. Compounds to be tested were diluted in DMSO and added
to the medium at final concentrations of 0, 0.1, 1, and 10 μM and
incubated for 1 h at 37 °C in a CO2 incubator. The percentage of
DMSO remained constant (0.1%) in all wells. After incubation,
neurons were lysed in radioimmuno precipitation assay (RIPA) buffer
supplied with protease inhibitor cocktail (Roche Applied Science,
Indianapolis, IN) and phosphatase inhibitors (NaF and Na3VO4).
Protein concentration of the samples were estimated using the BCA
assay kit (Pierce, Thermo Scientific).
Samples were prepared and resolved by SDS−PAGE, transferred to
nitrocellulose membrane, and incubated with phospho-specific anti-
bodies (anti-phospho-pY204/187 ERK1/2, anti-pY402 Pyk2, anti-pY1472
GluN2B) or total protein antibodies, Pyk2, anti-GluN2B, anti-ERK1/
2) overnight at 4 °C. All antibodies used in this study are listed in the
Supporting Information. Membranes were washed and incubated in
peroxidase-conjugated secondary antibodies (GE Healthcare, Wauke-
sha, WI). The immunoreactivity was visualized using a chemilumi-
nescent substrate kit (Pierce Biotechnology, Rockford, IL) and
detected using a G:BOX with the image program GeneSnap (Syngene,
Cambridge, U.K.). All densitometric quantifications were performed
using the Image J (NIH) software.
the reaction mixture was stirred at ambient temperature for 18 h. The
reaction mixture was quenched with 0.5 volumes of 1 N HCl. The
layers were separated, and the organic layer was dried over MgSO4.
The volatile material was removed with a rotary evaporator under
vacuum to afford the crude products as orange to brown viscous oils or
sticky solids. Although crude products show multiple sets of peaks in
the aromatic region (potentially the hydrolytic boroxine species), the
crude products show only one major peak on LCMS which
corresponds to the mass of the boronic acids, and the products were
used without further purification.
General Procedure for Suzuki−Miyaura Cross-Coupling (C).
A 1 dram oven-dried vial was charged with a stir bar, the appropriate
aryl bromide (1.0 equiv), organoboron species (1.5 equiv), potassium
carbonate (5 equiv), palladium(II) chloride or palladium(II) acetate (5
mol %), and (2-biphenyl)dicyclohexylphosphine (10 mol %). After
addition of all reagents, solvent (4:1 dioxane/water) was added to the
vial (0.25 M in aryl bromide). The vial was sealed with a screw top
containing a Teflon septum and placed in a preheated heating block to
80 °C, and the mixture was vigorously stirred for 18 h. The vial was
then removed from the heating block and allowed to cool to room
temperature, followed by addition of 0.3 volumes of 10 N HCl open to
air. The reaction mixture was diluted with 1 volume of water and 1
volume of methanol, filtered through a Kimwipe, and purified by
reversed-phase gradient column chromatography (5−100% acetoni-
trile in water with 0.1% trifluoroacetic acid). Volatile components were
removed, and the resulting water solutions were lyophilized to afford
the products.
General Procedure for Suzuki−Miyaura Cross-Coupling (D).
A 1 dram oven-dried vial was charged with a stir bar, the appropriate
aryl bromide (1.0 equiv), organoboron species (1.5 equiv), sodium
carbonate (6 equiv), and tetrakis(triphenylphosphine)palladium(0)
(10 mol %). After addition of all reagents, solvent (4:1:1 dimethoxy-
ethane/ethanol/water) was added to the vial (0.10−0.25 M in aryl
bromide). The vial was sealed with a screw top containing a Teflon
septum and placed in a preheated heating block to 80 °C to stir for 4−
18 h with vigorous stirring. The vial was then removed from the
heating block and allowed to cool to room temperature, followed by
addition of 0.3 volumes of 10 N HCl open to air. The reaction mixture
was diluted with 1 volume of water and 1 volume of methanol, filtered
through a Kimwipe, and purified by reversed-phase gradient column
chromatography (5−100% acetonitrile in water with 0.1% trifluoro-
acetic acid). Volatile components were removed, and the resulting
water solutions were lyophilized to afford the products.
All data are presented as the mean
SEM. Differences among
multiple groups were evaluated using one-way ANOVA with
Dunnett’s post hoc test using Graph Pad Prism 6 software. For all
analyses, a p value of <0.05 indicated a statistically significant
difference.
((3-Bromophenyl)difluoromethyl)phosphonic Acid (13). Io-
dotrimethylsilane (5.22 mL, 36.6 mmol, 2.2 equiv) was added to a
stirred solution of diethyl ((3-bromophenyl)difluoromethyl)-
phosphonate21b (5.71 g, 16.6 mmol) in CH2Cl2 (50 mL, 0.3 M).
The reaction solution was stirred at ambient temperature for 14 h. The
volatile components were then removed by a rotary evaporator under
vacuum. Sodium hydroxide (664 mg, 16.6 mmol) in methanol (20
mL) was added to the resulting residue, and this solution was stirred at
ambient temperature for 1 h, allowing the monosodium salt of the
desired compound to precipitate from the solution. The solvent was
removed by filtration. LCMS showed the presence of an undesired
byproduct in the collected crude product; thus, it was purified via
reversed-phase gradient column chromatography (5−100% acetoni-
trile in water with 0.1% trifluoroacetic acid buffer). Volatile
components were removed, and the resulting water solution was
lyophilized to afford the product as an off-white powder (2.75 g, 9.44
General Procedure for Selective Ortho Bromination of
Phenols (A). Selective ortho-bromination of alkylphenols was
accomplished via a modified literature procedure.24 A solution of N-
bromosuccinimide (1.0 equiv) in CH2Cl2 (0.2 M) was added dropwise
via cannula to a stirred solution of the phenol in CH2Cl2 (0.5 M)
containing catalytic N,N-diisopropylamine (0.1 equiv), the reaction
flask having been placed in an ambient temperature water bath. The
reaction mixture was stirred for 90 min at which point it was acidified
with 1 N HCl to pH < 2.0. The reaction mixture was diluted with 1
volume of water. The layers were separated, and the organic layer was
dried over MgSO4. The volatile material was removed with a rotary
evaporator under vacuum to afford the crude products, generally as
yellow to colorless liquids, which were typically used without further
purification.
General Procedure for Boronic Acid Formation from o-
Bromophenols (B). Borylation of the phenols was accomplished
following a literature procedure.25 Butyllitium (2.5 M in hexanes, 2.15
equiv) was added dropwise via syringe to a stirred solution of the
starting o-bromophenol in diethyl ether (0.2 M) in a dry ice−acetone
cold temperature bath. The cold bath was removed, and the reaction
solution was allowed to warm to ambient temperature in air for 2.5 h.
The reaction flask was resuspended in the dry ice−acetone cold
temperature bath. After the mixture was stirred for 10 min in the cold
bath, a solution of trimethyl borate (1.67 equiv, 1.5 M in diethyl ether)
was added. After the mixture was stirred in the cold bath for 30 min,
1
mmol, 57%). H NMR (400 MHz, CD3OD) δ 7.41 (t, J = 7.9 Hz,
1H), 7.58 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H).
19F NMR (376 MHz, CD3OD) δ −110.13 (d, JFP = 110.2 Hz). 31P
NMR (162 MHz, CD3OD) δ 6.76 (br). HRMS (ESI) m/z: calcd
284.9133 (M − H+); found 284.9129.
(Difluoro(4′-methyl-[1,1′-biphenyl]-3-yl)methyl)phosphonic
Acid (11a). For the Suzuki coupling, general procedure C was
followed with 51 mg (0.375 mmol) of 4-methylphenylboronic acid and
71 mg (0.25 mmol) of aryl bromide 13. The procedure yielded 42 mg
1
(56%) of the desired compound as a white powder. H NMR (400
MHz, CD3OD) δ 2.38 (s, 3H), 7.28 (d, J = 8.0 Hz, 2H), 7.49−7.60
G
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Journal of Medicinal Chemistry
Article
(m, 4H), 7.72 (d, J = 7.2 Hz, 1H), 7.82 (s, 1H). 19F NMR (376 MHz,
CD3OD) δ −109.69 (d, J = 112.9 Hz). 31P NMR (162 MHz, CD3OD)
δ 7.05 (t, J = 113.0 Hz). HRMS (ESI) m/z: calcd 297.0498 (M − H+);
found 297.0500.
of aryl bromide 13. The procedure yielded 43 mg (25%) of the desired
compound as a white, hygroscopic powder. 1H NMR (400 MHz,
CD3OD) δ 1.26 (d, J = 6.9 Hz, 6H), 3.38 (hept, J = 6.9 Hz, 1H), 6.92
(t, J = 7.6 Hz, 1H), 7.04 (dd, J = 7.6, 1.7 Hz, 1H), 7.19 (dd, J = 7.8, 1.7
Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 7.1 Hz, 1H), 7.62 (d, J =
7.5 Hz, 1H), 7.72 (s, 1H). 19F NMR (376 MHz, CD3OD) δ −109.81
(Difluoro(4′-isopropyl-[1,1′-biphenyl]-3-yl)methyl)-
phosphonic Acid (11b). For the Suzuki coupling, general procedure
C was followed with 62 mg (0.375 mmol) of 4-isopropylphenylbor-
onic acid and 71 mg (0.25 mmol) of aryl bromide 13. The procedure
(d, JFP = 114.0 Hz). 31P NMR (162 MHz, CD3OD) δ 7.27 (t, JPF
=
113.5 Hz). HRMS (ESI) m/z: calcd 341.0760 (M − H+); found
341.0755.
1
yielded 45 mg (55%) of the desired compound as a yellow solid. H
NMR (400 MHz, CD3OD) δ 1.28 (d, J = 6.9 Hz, 6H), 2.95 (hept, J =
6.9 Hz, 1H), 7.33 (d, J = 8.3 Hz, 2H), 7.48−7.60 (m, 4H), 7.73 (d, J =
7.3 Hz, 1H), 7.83 (s, 1H). 19F NMR (376 MHz, CD3OD) δ −109.67
(d, J = 112.9 Hz). 31P NMR (162 MHz, CD3OD) δ 7.11 (br t).
HRMS (ESI) m/z: calcd 325.0811 (M − H+); found 325.0816.
(Difluoro(3′-methyl-[1,1′-biphenyl]-3-yl)methyl)phosphonic
Acid (11c). For the Suzuki coupling, general procedure C was
followed with 51 mg (0.375 mmol) of 3-methylphenylboronic acid and
71 mg (0.25 mmol) of aryl bromide 13. The procedure yielded 51 mg
(Difluoro(2′-hydroxy-5′-isopropyl-[1,1′-biphenyl]-3-yl)-
methyl)phosphonic Acid (11h). General procedure A was followed
for the ortho-bromination of 4-isopropylphenol starting with 1.36 g of
the phenol (10.0 mmol). To remove the dibrominated byproduct, the
crude product was purified by column chromatography (9:1 hexanes/
ethyl acetate, product Rf = 0.33) to afford the product as a yellow oil
(760 mg, 35%). This bromophenol (625 mg) was converted to the
boronic acid through general procedure B, yielding 437 mg (84%) of
an off white solid with molecular mass corresponding to the boronic
acid. The crude boronic acid was used without further purification. For
the final Suzuki coupling, general procedure C was followed with 68
mg (0.375 mmol) of this boronic acid and 71 mg (0.25 mmol) of aryl
bromide 13. The procedure yielded 34 mg (40%) of the desired
1
(68%) of the desired compound as a colorless oil. H NMR (400
MHz, CD3OD) δ 2.40 (s, 3H), 7.18 (d, J = 7.5 Hz, 1H), 7.32 (t, J =
7.6 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.45 (s, 1H), 7.53 (t, J = 7.7 Hz,
1H), 7.58 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.83 (s, 1H).
19F NMR (376 MHz, CD3OD) δ −109.64 (d, J = 113.4 Hz). 31P NMR
1
compound as a brown, hygroscopic powder. H NMR (400 MHz,
CD3OD) δ 1.24 (d, J = 6.9 Hz, 6H), 2.87 (hept, J = 6.9 Hz, 1H), 6.83
(d, J = 8.3 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz, 1H), 7.12 (d, J = 2.3 Hz,
1H), 7.48 (t, J = 7.7 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.5
Hz, 1H), 7.78 (s, 1H). 19F NMR (376 MHz, CD3OD) δ −109.48 (d,
JFP = 114.1 Hz). 31P NMR (162 MHz, CD3OD) δ 7.39 (t, JPF = 114.7
Hz). HRMS (ESI) m/z: calcd 341.0760 (M − H+); found 341.0756.
((3′-Ethyl-2′-hydroxy-[1,1′-biphenyl]-3-yl)difluoromethyl)-
phosphonic Acid (11i). General procedure A was followed for the
ortho-bromination of 2-ethylphenol starting with 1.20 mL of the
phenol (10.0 mmol). The procedure yielded 1.86 g (93%) of the
brominated phenol, 16a, as a yellow liquid which was used without
purification. This bromophenol (1.86 g) was converted to the boronic
acid through general procedure B, yielding 870 mg (57%) of an orange
sticky solid with molecular mass corresponding to the boronic acid,
17a. The crude boronic acid was used without further purification. For
the final Suzuki coupling, general procedure C was followed with 62
mg (0.375 mmol) of 17a and 71 mg (0.25 mmol) of aryl bromide 13.
The procedure yielded 14 mg (17%) of the desired compound as a
white, hygroscopic powder. 1H NMR (400 MHz, CD3OD) δ 1.23 (t, J
= 7.5 Hz, 3H), 2.70 (q, J = 7.5 Hz, 2H), 6.89 (t, J = 7.6 Hz, 1H), 7.06
(dd, J = 7.6, 1.7 Hz, 1H), 7.12 (dd, J = 7.5, 1.7 Hz, 1H), 7.51 (t, J = 7.6
Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 7.4 Hz, 1H), 7.73 (s,
1H). 19F NMR (376 MHz, CD3OD) δ −109.75 (d, JFP = 113.6 Hz).
31P NMR (162 MHz, CD3OD) δ 7.25 (t, JPF = 113.6 Hz). HRMS
(162 MHz, CD3OD) δ 7.09 (t, J = 113.3 Hz). HRMS (ESI) m/z: calcd
297.0498 (M − H+); found 297.0498.
(Difluoro(3′-isopropyl-[1,1′-biphenyl]-3-yl)methyl)-
phosphonic Acid (11d). For the Suzuki coupling, general procedure
C was followed with 62 mg (0.375 mmol) of 3-isopropylphenylbor-
onic acid and 71 mg (0.25 mmol) of aryl bromide 13. The procedure
yielded 13 mg (16%) of the desired compound as a yellow viscous oil.
1H NMR (400 MHz, CD3OD) δ 1.29 (d, J = 7.0 Hz, 6H), 2.97 (hept,
J = 6.9 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.43
(d, J = 7.6 Hz, 1H), 7.48 (s, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.59 (d, J =
7.8 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.83 (s, 1H). 19F NMR (376
MHz, CD3OD) δ −109.67 (d, JFP = 113.4 Hz). 31P NMR (162 MHz,
CD3OD) δ 7.16 (br t). HRMS (ESI) m/z: calcd 325.0811 (M − H+);
found 325.0805.
(Difluoro(2′-hydroxy-[1,1′-biphenyl]-3-yl)methyl)-
phosphonic Acid (11e). For the Suzuki coupling, general procedure
C was followed with 52 mg (0.375 mmol) of 2-hydroxyphenylboronic
acid and 71 mg (0.25 mmol) of aryl bromide 13. The procedure
yielded 35 mg (47%) of the desired compound as an off white
1
hygroscopic powder. H NMR (400 MHz, CD3OD) δ 6.87−6.94 (m,
2H), 7.17 (td, J = 7.7, 1.6 Hz, 1H), 7.27 (dd, J = 7.5, 1.7 Hz, 1H), 7.48
(t, J = 7.7 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H),
7.79 (s, 1H). 19F NMR (376 MHz, CD3OD) δ −109.53 (d, JFP = 113.8
Hz). 31P NMR (162 MHz, CD3OD) δ 7.24 (t, JPF = 113.9 Hz). HRMS
(ESI) m/z: calcd 299.0290 (M − H+); found 299.0285.
(ESI) m/z: calcd 327.0603 (M − H+); found 327.0599.
((3′-(tert-Butyl)-2′-hydroxy-[1,1′-biphenyl]-3-yl)-
difluoromethyl)phosphonic Acid (11j). General procedure A was
followed for the ortho-bromination of 2-tert-butylphenol starting with
1.52 mL of the phenol (10.0 mmol). The procedure yielded 2.30 g
(99%) of the brominated phenol, 16c, as a light yellow solid which was
used without purification. This bromophenol (2.30 g) was converted
to the boronic acid through general procedure B, yielding 1.09 g
(56%) of a very viscous brown oil with molecular mass corresponding
to the boronic acid, 17c. The crude boronic acid was used without
further purification. For the final Suzuki coupling, general procedure C
was followed with 73 mg (0.375 mmol) of 17c and 71 mg (0.25
mmol) of aryl bromide 13. The procedure yielded 11 mg (12%) of the
(Difluoro(2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)-
phosphonic Acid (11f). For the Suzuki coupling, general procedure
C was followed with 57 mg (0.375 mmol) of 2-methoxyphenylboronic
acid and 71 mg (0.25 mmol) of aryl bromide 13. The procedure
1
yielded 35 mg (45%) of the desired compound as a yellow solid. H
NMR (400 MHz, CD3OD) δ 3.79 (s, 3H), 7.02 (t, J = 7.4 Hz, 1H),
7.08 (d, J = 8.2 Hz, 1H), 7.30 (d, J = 9.1 Hz, 1H), 7.35 (d, J = 7.9 Hz,
1H), 7.47 (t, J = 7.7 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.7
Hz, 1H), 7.73 (s, 1H). 19F NMR (376 MHz, CD3OD) δ −109.54 (d,
JFP = 114.3 Hz). 31P NMR (162 MHz, CD3OD) δ 7.18 (t, JPF = 114.0
Hz). HRMS (ESI) m/z: calcd 313.0447 (M + H+); found 313.0445.
(Difluoro(2′-hydroxy-3′-isopropyl-[1,1′-biphenyl]-3-yl)-
methyl)phosphonic Acid (11g). General procedure A was followed
for the ortho-bromination of 2-isopropylphenol starting with 1.35 mL
of the phenol (10.0 mmol). The procedure yielded 1.49 g (69%) of the
brominated phenol, 16b, as a liquid which was used without
purification. This bromophenol (495 mg) was converted to the
boronic acid through general procedure B, yielding 315 mg (76%) of a
very viscous brown oil with molecular mass corresponding to the
boronic acid, 17b. The crude boronic acid was used without further
purification. For the final Suzuki coupling, general procedure C was
followed with 136 mg (0.75 mmol) of 17b and 142 mg (0.50 mmol)
1
desired compound as a white, hygroscopic powder. H NMR (400
MHz, CD3OD) δ 1.44 (s, 9H), 6.87 (t, J = 7.7 Hz, 1H), 7.02 (d, J =
7.5 Hz, 1H), 7.26 (d, J = 7.7 Hz, 1H), 7.50−7.63 (m, 3H), 7.68 (s,
1H). 19F NMR (376 MHz, CD3OD) δ −110.00 (d, JFP = 113.0 Hz).
31P NMR (162 MHz, CD3OD) δ 6.96 (t, JPF = 113.0 Hz). HRMS
(ESI) m/z: calcd 355.0916 (M − H+); found 355.0913.
((3′-Cyclobutyl-2′-hydroxy-[1,1′-biphenyl]-3-yl)-
difluoromethyl)phosphonic Acid (11k). For the Suzuki coupling,
general procedure D was followed with 90 mg (0.35 mmol) of the
potassium trifluoroborate 18a and 100 mg (0.35 mmol) of aryl
H
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Journal of Medicinal Chemistry
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bromide 13. The procedure yielded 18 mg (15%) of the desired
compound as a white hygroscopic powder. 1H NMR (400 MHz,
CD3OD) δ 1.75−1.93 (m, 1H), 1.96−2.23 (m, 3H), 2.32−2.43 (m,
2H), 3.82 (p, J = 8.6 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 7.05 (dd, J =
7.6, 1.7 Hz, 1H), 7.20 (dd, J = 7.4, 1.6 Hz, 1H), 7.49 (t, J = 7.7 Hz,
1H), 7.54 − 7.61 (m, 2H), 7.71 (s, 1H). 19F NMR (376 MHz,
CD3OD) δ −109.20 (d, JFP = 109.4 Hz). 31P NMR (162 MHz,
CD3OD) δ 6.97 (br t). HRMS (ESI) m/z: calcd 353.0760 (M − H+);
found 353.0754.
removed, and the resulting water solution was lyophilized to afford the
desired product as a white hygroscopic powder (5 mg, 50%). 1H NMR
(400 MHz, CD3OD) δ 1.15−1.44 (m, 5H), 1.63−1.72 (m, 1H), 1.73−
1.86 (m, 4H), 2.86−3.01 (m, 1H), 4.84 (d, J = 13.2 Hz, 1H), 6.78 (t, J
= 7.5 Hz, 1H), 6.91 (dd, J = 7.6, 1.7 Hz, 1H), 7.04 (dd, J = 7.6, 1.8 Hz,
1H), 7.26−7.35 (m, 2H), 7.40 (d, J = 7.3 Hz, 1H), 7.51 (s, 1H). 31P
NMR (162 MHz, CD3OD) δ 22.45 (s). HRMS (ESI) m/z: calcd
361.1210 (M − H+); found 361.1215
((2′-Cyano-[1,1′-biphenyl]-4-yl)difluoromethyl)phosphonic
Acid (12a). Compound 12a was prepared using general procedure D.
Hence, by use of 80 mg (0.28 mmol) of ((4-bromophenyl)-
difluoromethyl)phosphonic acid 14 and 61 mg (0.42 mmol) of (2-
cyanophenyl)boronic acid, the title compound was obtained in 23%
((3′-Cyclopentyl-2′-hydroxy-[1,1′-biphenyl]-3-yl)-
difluoromethyl)phosphonic Acid (11l). General procedure A was
followed for the ortho-bromination of 2-cyclopentylphenol starting
with 406 mg of the phenol (2.5 mmol). The procedure yielded 590 mg
(98%) of the brominated phenol, 16d, as a liquid which was used
without purification. This bromophenol (590 mg) was converted to
the boronic acid through general procedure B, yielding 420 mg (83%)
of a very viscous brown oil with molecular mass corresponding to the
boronic acid, 17d. The crude boronic acid was used without further
purification. For the final Suzuki coupling, general procedure C was
followed with 77 mg (0.375 mmol) of 17d and 71 mg (0.25 mmol) of
aryl bromide 13. The procedure yielded 10 mg (11%) of the desired
1
yield (23 mg) as a white solid. H NMR (400 MHz, DMSO-d6) δ
7.54−7.59 (m, 1H), 7.60−7.62 (m, 1H), 7.67 (d, J = 8.0 Hz, 2H),
7.74−7.84 (m, 3H), 7.85−7.87 (m, 1H). 31P NMR (162 MHz,
DMSO-d6) δ 5.39 (t, J = 110.5 Hz). 19F NMR (376 MHz, DMSO-d6)
δ −111.15 (d, J = 110.8 Hz). HRMS (ESI) (m/z) [M − H]−
calculated for C14H9F2NO3P, 308.0294; found, 308.0298.
((3′-Cyano-[1,1′-biphenyl]-4-yl)difluoromethyl)phosphonic
Acid (12b). Compound 12b was prepared using general procedure C.
Hence, by use of 100 mg (0.35 mmol) of 14 and 77 mg (0.52 mmol)
of (3-cyanophenyl)boronic acid, the title compound was obtained in
35% yield (38 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
7.63−7.67 (m, 1H), 7.71−7.78 (m, 5H), 7.96−8.04 (m, 2H). 31P
NMR (162 MHz, DMSO-d6) δ 5.81 (t, J = 112.2 Hz). 19F NMR (376
MHz, DMSO-d6) δ −111.49 (d, J = 112.0 Hz). HRMS (ESI) (m/z)
[M − H]− calculated for C14H9F2NO3P, 308.0294; found, 308.0296.
((4′-Cyano-[1,1′-biphenyl]-4-yl)difluoromethyl)phosphonic
Acid (12c). Compound 12c was prepared using general procedure C.
Hence, by use of 100 mg (0.35 mmol) of 14 and 77 mg (0.52 mmol)
of (4-cyanophenyl)boronic acid, the title compound was obtained in
25% yield (27 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
7.73 (d, J = 8.0 Hz, 2H), 7.78−7.87 (m, 6H). 31P NMR (162 MHz,
DMSO-d6) δ 5.61 (t, J = 111.7 Hz). 19F NMR (376 MHz, DMSO-d6)
δ −111.49 (d, J = 111.7 Hz). HRMS (ESI) (m/z) [M − H]−
calculated for C14H9F2NO3P, 308.0294; found, 308.0297.
(Difluoro(3′-isopropoxy-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12d). Compound 12d was prepared using general
procedure C. Hence, by use of 100 mg (0.35 mmol) of 14 and 94 mg
(0.52 mmol) of (3-isopropoxyphenyl)boronic acid, the title compound
was obtained in 20% yield (24 mg) as a white solid.1H NMR (400
MHz, DMSO-d6) δ 1.33 (d, J = 6.0 Hz, 6H), 4.67 (sept, J = 6.0 Hz,
1H), 6.92 (ddd, J = 8.4 Hz, 2.4, 0.8 Hz, 1H), 7.13−7.19 (m, 2H), 7.34
(t, J = 8.4 Hz, 1H), 7.65−7.70 (m, 4H). 31P NMR (162 MHz, DMSO-
d6) δ 5.88 (t, J = 112.9 Hz). 19F NMR (376 MHz, DMSO-d6) δ
−111.15 (d, J = 112.9 Hz). HRMS (ESI) (m/z) [M − H]− calculated
for C16H16F2O4P, 341.0760; found, 341.0760.
1
compound as an off white hygroscopic powder. H NMR (400 MHz,
CD3OD) δ 1.56−1.78 (m, 4H), 1.78−1.92 (m, 2H), 2.01−2.12 (m,
2H), 3.33−3.46 (m, 1H), 6.91 (t, J = 7.6 Hz, 1H), 7.03 (dd, J = 7.6, 1.7
Hz, 1H), 7.20 (dd, J = 7.6, 1.7 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.57
(d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.72 (s, 1H). 19F NMR
(376 MHz, CD3OD) δ −109.57 (d, JFP = 111.9 Hz). 31P NMR (162
MHz, CD3OD) δ 7.19 (br t). HRMS (ESI) m/z: calcd 367.0916 (M −
H+); found 367.0912.
((3′-Cyclohexyl-2′-hydroxy-[1,1′-biphenyl]-3-yl)-
difluoromethyl)phosphonic Acid (11m). For the Suzuki coupling,
general procedure C was followed with 106 mg (0.375 mmol) of the
potassium trifluoroborate, 17e, and 71 mg (0.25 mmol) of aryl
bromide 13. The procedure yielded 22 mg (23%) of the desired
compound as a white, hygroscopic powder. 1H NMR (400 MHz,
CD3OD) δ 1.23−1.56 (m, 5H), 1.73−1.82 (m, 1H), 1.82−1.93 (m,
4H), 2.95−3.08 (m, 1H), 6.91 (t, J = 7.6 Hz, 1H), 7.03 (dd, J = 7.6, 1.7
Hz, 1H), 7.17 (dd, J = 7.6, 1.7 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.57
(d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.72 (s, 1H). 19F NMR
(376 MHz, CD3OD) δ −109.61 (d, JFP = 113.3 Hz). 31P NMR (162
MHz, CD3OD) δ 7.29 (t, JPF = 112.4 Hz). HRMS (ESI) m/z: calcd
381.1073 (M − H+); found 381.1068.
((3′-Cycloheptyl-2′-hydroxy-[1,1′-biphenyl]-3-yl)-
difluoromethyl)phosphonic Acid (11n). For the Suzuki coupling,
general procedure D was followed with 90 mg (0.30 mmol) of the
potassium trifluoroborate 18b and 87 mg (0.30 mmol) of aryl bromide
13. The procedure yielded 20 mg (17%) of the desired compound as a
white hygroscopic powder in a 1:0.4 ratio with residual trifluoroacetic
1
acid (as determined by 19F NMR). H NMR (400 MHz, CD3OD) δ
(Difluoro(4′-isopropoxy-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12e). Compound 12e was prepared using general
procedure C. Hence, by use of 100 mg (0.35 mmol) of 14 and 94 mg
(0.52 mmol) of (4-isopropoxyphenyl)boronic acid, the title compound
1.49−2.00 (m, 12H), 3.16−3.29 (m, 1H), 6.87 (t, J = 7.6 Hz, 1H),
6.99 (dd, J = 7.4, 1.7 Hz, 1H), 7.13 (dd, J = 7.6, 1.7 Hz, 1H), 7.44−
7.68 (m, 3H), 7.72 (s, 1H). 19F NMR (376 MHz, CD3OD) δ −109.61
(d, J = 111.5 Hz). 31P NMR (162 MHz, CD3OD) δ 7.01 (br t).
HRMS (ESI) m/z: calcd 395.1229 (M − H+); found 395.1225.
((3′-Cyclohexyl-2′-hydroxy-[1,1′-biphenyl]-3-yl)(hydroxy)-
methyl)phosphonic Acid (11o). For the Suzuki coupling, general
procedure D was followed with 85 mg (0.30 mmol) of the potassium
trifluoroborate 17e and 72 mg (0.25 mmol) of monosodium (3-
bromobenzoyl)phosphonate, 25.35 The procedure yielded 21 mg
(23%) of the α-ketophosphate, 26, as an off-white hygroscopic
powder. To accomplish reduction of the ketone, a 1 dram vial was
charged with a stir bar, 10 mg of this α-ketophosphate, and 0.3 mL of
MeOH. The vial was cooled in an ice−water bath, and 10 mg of
NaBH4 was added as a solid. The mixture was stirred for 10 min in the
ice bath after which time another portion of 10 mg of NaBH4 was
added. After 10 additional minutes stirring, the reaction was quenched
with addition of 50 μL of glacial acetic acid. Following removal of the
volatile components on a rotary evaporator, the residue was purified by
reversed-phase gradient column chromatography (5−100% acetoni-
trile in water with 0.1% trifluoroacetic acid). Volatile components were
1
was obtained in 16% yield (19 mg) as a white solid. H NMR (400
MHz, DMSO-d6) δ 1.33 (d, J = 6.0 Hz, 6H), 4.65 (sept, J = 6.0 Hz,
1H), 6.98 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.63, 7.66
(ABq, J = 8.8 Hz, 4H). 31P NMR (162 MHz, DMSO-d6) δ 5.93 (t, J =
104.0 Hz). 19F NMR (376 MHz, DMSO-d6) δ −111.02 (d, J = 113.3
Hz). HRMS (ESI) (m/z) [M − H]− calculated for C16H16F2O4P,
341.0760; found, 341.0763.
(Difluoro(3′-isopropyl-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12f). Compound 12f was prepared using general
procedure C. Hence, by use of 80 mg (0.28 mmol) of 14 and 86 mg
(0.52 mmol) of (3-isopropylphenyl)boronic acid, the title compound
was obtained in 32% yield (29 mg) as a sticky oil. 1H NMR (400 MHz,
DMSO-d6) δ 1.30 (d, J = 6.4 Hz, 6H), 2.98 (sept, J = 6.4 Hz, 1H),
7.24−7.27 (m, 1H), 7.37−7.45 (m, 3H), 7.66−7.72 (m, 4H). 31P
NMR (162 MHz, DMSO-d6) δ 5.95 (t, J = 113.4 Hz). 19F NMR (376
MHz, DMSO-d6) δ −111.15 (d, J = 113.6 Hz). HRMS (ESI) (m/z)
[M − H]− calculated for C16H16F2O3P, 325.0811; found, 325.08114.
I
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(Difluoro(4′-isopropyl-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12g). Compound 12g was prepared using general
procedure C. Hence, by use of 80 mg (0.28 mmol) of 14 and 86 mg
(0.52 mmol) of (4-isopropylphenyl)boronic acid, the title compound
(m, 4H), 7.18−7.22 (m, 2H), 7.27−7.34 (m, 2H), 7.58 (d, J = 8.0 Hz,
2H), 7.68 (d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, DMSO-d6) δ 2.91
(t, J = 107.3 Hz). 19F NMR (376 MHz, DMSO-d6) δ −108.01 (d, J =
107.3 Hz). HRMS (ESI) (m/z) [M − H]− calculated for
C19H15F2NO3P, 374.0763; found, 374.0764.
1
was obtained in 15% yield (13 mg) as a white solid. H NMR (400
MHz, DMSO-d6) δ 1.28 (d, J = 6.4 Hz, 6H), 2.94 (sept, J = 6.4 Hz,
1H), 7.33 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0
Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, DMSO-d6) δ
5.99 (t, J = 113.9 Hz). 19F NMR (376 MHz, DMSO-d6) δ −111.19 (d,
J = 114.0 Hz). HRMS (ESI) (m/z) [M − H]− calculated for
C16H16F2O3P, 325.0811; found, 325.0815.
((3′-Benzyl-[1,1′-biphenyl]-4-yl)difluoromethyl)phosphonic
Acid (12m). Compound 12m was prepared using general procedure
D. Hence, by use of 80 mg (0.28 mmol) of 14 and 123 mg (0.42
mmol) of 22a, the title compound was obtained in 22% yield (25 mg)
1
as a white solid. H NMR (400 MHz, DMSO-d6) δ 4.01 (s, 2H),
7.15−7.20 (m, 1H), 7.24−7.30 (m, 5H), 7.39 (t, J = 8.0 Hz, 1H), 7.51
(d, J = 8.0 Hz, 1H), 7.57−7.60 (m, 3H), 7.32 (d, J = 8.0 Hz, 2H). 31P
NMR (162 MHz, DMSO-d6) δ 2.90 (t, J = 106.9 Hz). 19F NMR (376
MHz, DMSO-d6) δ −107.98 (d, J = 106.8 Hz). HRMS (ESI) (m/z)
[M − H]− calculated for C20H16F2O3P, 373.0811; found, 373.0803.
(Difluoro(3′-(4-fluorobenzyl)-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12n). Compound 12n was prepared using general
procedure D. Hence, by use of 80 mg (0.28 mmol) of 14 and 131 mg
(0.42 mmol) of 22b, the title compound was obtained in 34% yield
(Difluoro(2′-(methylsulfonamido)-[1,1′-biphenyl]-4-yl)-
methyl)phosphonic Acid (12h). Compound 12h was prepared
using general procedure D. Hence, by use of 80 mg (0.28 mmol) of 14
and 124 mg (0.42 mmol) of 2-(methanesulfonylamino)phenylboronic
acid pinacol ester, the title compound was obtained in 12% yield (13
mg) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.74 (s, 3H),
7.29−7.46 (m, 4H), 7.53, 7.57 (ABq, J = 8.0 Hz, 4H), 9.01 (s, 1H). 31P
NMR (162 MHz, DMSO-d6) δ 2.83 (t, J = 106.3 Hz). 19F NMR (376
MHz, DMSO-d6) δ −107.44 (d, J = 106.3 Hz). HRMS (ESI) (m/z)
[M − H]− calculated for C14H13F2NO5PS, 376.0226; found, 376.0232.
(Difluoro(3′-(methylsulfonamido)-[1,1′-biphenyl]-4-yl)-
methyl)phosphonic Acid (12i). Compound 12i was prepared using
general procedure D. Hence, by use of 80 mg (0.28 mmol) of 14 and
90 mg (0.42 mmol) of 3-(methanesulfonylamino)phenylboronic acid,
the title compound was obtained in 15% yield (16 mg) as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ 3.03 (s, 3H), 7.23−7.25 (m,
1H), 7.39−7.47 (m, 3H), 7.61 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz,
2H), 9.86 (s, 1H). 31P NMR (162 MHz, DMSO-d6) δ 2.86 (t, J =
106.6 Hz). 19F NMR (376 MHz, DMSO-d6) δ −108.08 (d, J = 106.7
Hz). HRMS (ESI) (m/z) [M − H]− calculated for C14H13F2NO5PS,
376.0226; found, 376.0230.
1
(37 mg) as a white solid. H NMR (400 MHz, DMSO-d6) δ 4.00 (s,
2H), 7.06−7.12 (m, 2H), 7.24 (d, J = 8.0 Hz, 1H), 7.29−7.34 (m,
2H), 7.39 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.51−7.60 (m,
3H), 7.72 (d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, DMSO-d6) δ 2.90
(t, JFP = 106.92 Hz). 19F NMR (376 MHz, DMSO-d6) δ −107.78 (d, J
= 106.7 Hz), −117.29 (m). HRMS (ESI) (m/z) [M − H]− calculated
for C20H15F3O3P, 391.0716; found, 391.0719.
((3′-(4-Chlorobenzyl)-[1,1′-biphenyl]-4-yl)difluoromethyl)-
phosphonic Acid (12o). Compound 12o was prepared using general
procedure D. Hence, by use of 80 mg (0.28 mmol) of 14 and 138 mg
(0.42 mmol) of 22c, the title compound was obtained in 24% yield (27
mg) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 3.99 (s, 2H),
7.22 (d, J = 7.6 Hz, 1H), 7.28−7.33 (m, 4H), 7.38 (t, J = 7.6 Hz, 1H),
7.51−7.53 (m, 1H), 7.56−7.58 (m, 3H), 7.69 (d, J = 8.0 Hz, 2H). 31P
NMR (162 MHz, DMSO-d6) δ 2.59 (t, JFP = 103.7 Hz). 19F NMR
(376 MHz, DMSO-d6) δ −108.01 (d, JFP= 105.3 Hz). HRMS (ESI)
(m/z) [M − H]− calculated for C20H15F2ClO3P, 407.0421; found,
407.0418.
(Difluoro(3′-(1-hydroxyethyl)-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12j). Compound 12j was prepared using general
procedure D. Hence, by use of 80 mg (0.28 mmol) of 14 and 69 mg
(0.42 mmol) of 3-(1-hydroxyethyl)phenylboronic acid, the title
1
compound was obtained in 16% yield (22 mg) as a white solid. H
NMR (400 MHz, DMSO-d6) δ 1.34 (d, J = 6.4 Hz, 3H), 4.77 (q, J =
6.4 Hz, 1H), 7.32−7.37 (m, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.48−7.53
(m, 1H), 7.56−7.65 (m, 3H), 7.73 (d, J = 8.4 Hz, 2H). 31P NMR (162
MHz, DMSO-d6) δ 2.91 (t, J = 107.1 Hz). 19F NMR (376 MHz,
DMSO-d6) δ −107.96 (d, J = 107.1 Hz). HRMS (ESI) (m/z) [M −
H]− calculated for C15H14F2O4P, 327.0603; found, 327.0602.
((3′-(3,4-Dichlorobenzyl)-[1,1′-biphenyl]-4-yl)-
difluoromethyl)phosphonic Acid (12p). Compound 12p was
prepared using general procedure D. Hence, by use of 80 mg (0.28
mmol) of 14 and 152 mg (0.42 mmol) of 22d, the title compound was
1
obtained in 28% yield (34 mg) as a white solid. H NMR (400 MHz,
DMSO-d6) δ 4.01 (s, 2H), 7.25−7.29 (m, 2H), 7.40 (t, 8.0 Hz, 1H),
7.51−7.54 (m, 2H), 7.58−7.62 (m, 4H), 7.74 (d, J = 8.0 Hz, 2H). 31P
NMR (162 MHz, DMSO-d6) δ 2.66 (t, JFP = 104.2 Hz). 19F NMR
(376 MHz, DMSO-d6) δ −107.62 (d, JFP = 104.2 Hz). HRMS (ESI)
(m/z) [M − H]− calculated for C20H14F2Cl2O3P, 441.0031; found,
441.0030.
(Difluoro(4′-(1-hydroxyethyl)-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12k). Compound 12k was prepared from 14 in
two steps. Suzuki−Miyaura cross-coupling of 14 (100 mg, 0.35 mmol)
and 4-acetylphenylboronic acid (86 mg, 0.52 mmol) using general
procedure C afforded ((4′-acetyl-[1,1′-biphenyl]-4-yl)difluoromethyl)-
phosphonic acid (S1) as a yellow solid (18 mg, 20% yield). S1 (10 mg,
0.030 mmol) was dissolved in methanol (0.3 mL) and treated with
NaBH4 (10 mg) at 0 °C. The reaction mixture was stirred at 0 °C for
20 min. More NaBH4 (10 mg) was added, and the reaction mixture
was stirred for an additional 20 min. Acetic acid (0.1 mL) was added.
The reaction mixture was purified by automated reversed-phase C-18
column chromatography (liquid injection, 50 g C18 column, column
volume = 42.6 mL, 40 mL/min, linear gradient of 5−100%
acetonitrile/water with 0.1% trifluoroacetic acid over 20 min) to
((3′-((3,4-Dichlorophenyl)(hydroxy)methyl)-[1,1′-biphenyl]-
4-yl)difluoromethyl)phosphonic Acid (12q). Compound 12q was
prepared using general procedure D. Hence, by use of 80 mg (0.28
mmol) of 14 and 124 mg (0.42 mmol) of 24, the title compound was
1
obtained in 36% yield (46 mg) as a white solid. H NMR (400 MHz,
DMSO-d6) δ 6.04 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz,
1H), 7.46 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.56
(d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.65 (s, 1H), 7.70 (d, J =
8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 1H). 31P NMR (162 MHz, DMSO-
d6) δ 2.91 (t, JFP = 106.9 Hz). 19F NMR (376 MHz, DMSO-d6) δ
−108.09 (d, JFP = 107.0 Hz). HRMS (ESI) (m/z) [M − H]−
calculated for C20H14Cl2F2O4P, 456.9980; found, 456.9979.
((3′-(3,4-Dichlorobenzyl)-[1,1′-biphenyl]-4-yl)(hydroxy)-
methyl)phosphonic Acid (12r). To a solution of 28 (35 mg, 0.083
mmol) in MeOH (0.2 mL) at 0 °C was added NaBH4 (16 mg, 0.41
mmol). The reaction mixture was stirred at 0 °C for 20 min. More
NaBH4 (16 mg) was added, and the reaction mixture was stirred for an
additional 20 min. Acetic acid (0.1 mL) was added. The reaction
mixture was purified by automated reversed-phase C-18 column
chromatography (liquid injection, 50 g C18 column, column volume =
42.6 mL, 40 mL/min, linear gradient of 5−100% acetonitrile/water
with 0.1% trifluoroacetic acid over 20 min) to give the title compound
1
give the title compound as a white solid (5 mg, 50% yield). H NMR
(400 MHz, DMSO-d6) δ 1.32 (d, J = 6.4 Hz, 3H). 4.73 (q, J = 6.4 Hz,
1H), 7.41 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0
Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, DMSO-d6) δ
2.62 (t, J = 103.6 Hz). 19F NMR (376 MHz, DMSO-d6) δ −104.57 (d,
J = 103.6 Hz). HRMS (ESI) (m/z) [M − H]− calculated for
C15H14F2O4P, 327.0603; found, 327.0608.
(Difluoro(3′-(phenylamino)-[1,1′-biphenyl]-4-yl)methyl)-
phosphonic Acid (12l). Compound 12l was prepared using general
procedure D. Hence, by use of 80 mg (0.28 mmol) of 14 and 124 mg
(0.42 mmol) of 3-(phenylamino)phenylboronic acid pinacol ester, the
title compound was obtained in 10% yield (10 mg) as a white solid. 1H
NMR (400 MHz, DMSO-d6) δ 6.82 (t, J = 7.2 Hz, 1H), 7.05−7.12
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as a white solid (20 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6) δ
4.00 (s, 2H), 4.70 (d, J = 14.0 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.28
(dd, J = 7.6, 2.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.45−7.58 (m, 8H).
31P NMR (162 MHz, DMSO-d6) δ 18.03. HRMS (ESI) (m/z) [M −
solution was allowed to warm to ambient temperature in air for 1.5 h.
The reaction flask is resuspended in the dry ice−acetone cold
temperature bath. After the mixture was stirred for 10 min in the cold
bath, a solution of trimethyl borate (0.84 mL, 7.5 mmol, 1.67 equiv) in
THF (5 mL, 1.5 M) was added via syringe. After being stirred in the
cold bath for 30 min, the reaction mixture was stirred at ambient
temperature for 18 h. The reaction flask was placed in an ice−water
bath, and the reaction was quenched by addition of 7 mL of saturated
potassium hydrogen difluoride (4.5 M, 7 equiv). The reaction mixture
was allowed to come to ambient temperature and stirred for 24 h. The
volatile material was removed with a rotary evaporator under vacuum
to afford the crude product. The crude products were dissolved in 150
mL of hot acetone, and the insoluble salts were filtered. The volatile
components of the filtrate were removed with a rotary evaporator
under vacuum until 5 mL of acetone was remaining. Approximately 30
mL of pentane was added to the acetone solution to precipitate the
product. Filtration and washing with pentane afforded the purified
product as a white solid (500 mg, 39%). 1H NMR (400 MHz, acetone-
d6) δ 1.18−1.46 (m, 5H), 1.67−1.75 (m, 1H), 1.75−1.86 (m, 4H),
2.86−2.96 (m, 1H), 6.57 (t, J = 7.3 Hz, 1H), 6.84 (dd, J = 7.5, 1.7 Hz,
1H), 7.11 (dd, J = 7.2, 1.9 Hz, 1H), 7.60 (q, J = 11.8 Hz, 1H). HRMS
(ESI) m/z: calcd 219.1198 (boronic acid M − H+); found 219.1202.
2-Cyclobutylphenol (15f). Following a previous report, dilithia-
tion of 2-bromophenol was accomplished.25 2-Bromophenol (3.5 g, 20
mmol, 1.0 equiv) was dissolved in diethyl ether (55 mL) and cooled in
a dry ice−acetone cold bath. After the mixture was stirred for 10 min,
n-butyllithium (1.6 M in hexanes, 26.5 mL, 42 mmol, 2.1 equiv) was
added dropwise by syringe. The cold bath was removed and the
reaction mixture was stirred at ambient temperature for 2 h, resulting
in the dilithiated reagent. LCMS of an acid quenched aliquot
confirmed that there was no starting bromide remaining. An amount
of 19 mL (approximately 5 mmol) of the above reagent was
transferred to a reaction flask charged with a stir bar and was
suspended in the dry ice−acetone cold bath. Next, 392 μL of
cyclobutanone (5.5 mmol, 1.1 equiv) was added neat via syringe. The
reaction mixture was allowed to warm to ambient temperature and was
stirred for 14 h. The reaction was quenched by addition of 10 mL of
saturated ammonium chloride. The layers were separated, and the
water layer was washed with 15 mL of diethyl ether. The combined
organic layers were dried over MgSO4, and the volatile material was
removed with a rotary evaporator under vacuum to afford the crude
benzyl alcohol product as a yellow liquid (880 mg). The crude product
(∼5 mmol) was dissolved in 5 mL of CH2Cl2 in a 20 mL scintillation
vial open to the atmosphere, and triethylsilane (2.4 mL, 15 mmol, ∼3
equiv) was added followed by 2.5 mL of trifluoroacetic acid (1:0.5 by
volume). The reaction mixture was stirred at ambient temperature for
16 h. The volatile material was removed with a rotary evaporator
under vacuum to afford the crude product. The crude product was
purified via gradient column chromatography (2−20% ethyl acetate in
hexanes) to afford the product as a clear oil (550 mg, 67%). 1H NMR
(400 MHz, CDCl3) δ 1.80−1.95 (m, 1H), 2.00−2.12 (m, 1H), 2.12−
2.26 (m, 2H), 2.30−2.46 (m, 2H), 3.58−3.75 (m, 1H), 4.63 (s, 1H),
6.76 (dd, J = 7.9, 1.2 Hz, 1H), 6.92 (td, J = 7.4, 1.1 Hz, 1H), 7.09 (td, J
= 7.7, 1.7 Hz, 1H), 7.18 (dt, J = 7.6, 1.3 Hz, 1H). MS (ESI) m/z 149
(M + H+).
H]− calculated for C20H16Cl2O4P, 421.0169; found, 421.0155.
((S)-(3′-((R)-(3,4-Dichlorophenyl)(hydroxy)methyl)-[1,1′-bi-
phenyl]-4-yl)(hydroxy)methyl)phosphonic Acid (12s). Inhibitor
12s was prepared using general procedure D. Hence, by use of 80 mg
(0.30 mmol) of 30b and 150 mg (0.40 mmol) of 32b, the title
compound was obtained in 12% yield (16 mg) as a white solid.
1
Reaction time = 4 h. H NMR (400 MHz, DMSO-d6) δ 4.70 (d, J =
14.0 Hz, 1H), 5.79 (s, 1H), 6.17 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H),
7.36−7.41 (m, 2H), 7.45−7.51 (m, 3H), 7.54−7.56 (m, 3H), 7.67−
7.69 (m, 2H). 31P NMR (162 MHz, DMSO-d6) δ 17.97. HRMS (ESI)
(m/z) [M − H]− calculated for C20H16Cl2O5P, 437.0118; found,
437.0110.
((S)-(3′-((S)-(3,4-Dichlorophenyl)(hydroxy)methyl)-[1,1′-bi-
phenyl]-4-yl)(hydroxy)methyl)phosphonic Acid (12t). Inhibitor
12t was prepared using general procedure D. Hence, by use of 80 mg
(0.30 mmol) of 30b and 150 mg (0.40 mmol) of 32a, the title
compound was obtained in 10% yield (13 mg) as a white solid.
1
Reaction time = 4 h. H NMR (400 MHz, DMSO-d6) δ 4.70 (d, J =
14.0 Hz, 1H), 5.79 (s, 1H), 6.17 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H),
7.36−7.41 (m, 2H), 7.45−7.51 (m, 3H), 7.54−7.56 (m, 3H), 7.67−
7.69 (m, 2H). 31P NMR (162 MHz, DMSO-d6) δ 18.00. HRMS (ESI)
(m/z) [M − H]− calculated for C20H16Cl2O5P, 437.0118; found,
437.0111.
((R)-(3′-((R)-(3,4-Dichlorophenyl)(hydroxy)methyl)-[1,1′-bi-
phenyl]-4-yl)(hydroxy)methyl)phosphonic Acid (12u). Inhibitor
12u was prepared using general procedure D. Hence, by use of 80 mg
(0.30 mmol) of 30a and 150 mg (0.40 mmol) of 32b, the title
compound was obtained in 10% yield (13 mg) as a white solid.
Reaction time = 4 h. The 1H NMR and 31P NMR spectra are the same
as those reported for the enantiomer, 12t. HRMS (ESI) (m/z) [M −
H]− calculated for C20H16Cl2O5P, 437.0118; found, 437.0111.
((R)-(3′-((S)-(3,4-Dichlorophenyl)(hydroxy)methyl)-[1,1′-bi-
phenyl]-4-yl)(hydroxy)methyl)phosphonic Acid (12v). Inhibitor
12v was prepared using general procedure D. Hence, by use of 80 mg
(0.30 mmol) of 30a and 150 mg (0.40 mmol) of 32a, the title
compound was obtained in 15% yield (19 mg) as a white solid.
Reaction time = 4 h. The 1H NMR and 31P NMR spectra are the same
as those reported for the enantiomer, 12s. HRMS (ESI) (m/z) [M −
H]− calculated for C20H16Cl2O5P, 437.0118; found, 437.0112.
((3′-((3,4-Dichlorophenyl)(hydroxy)methyl)-[1,1′-biphenyl]-
4-yl)methyl)phosphonic Acid (12w). Compound 12w was
prepared using general procedure D. Hence, by use of 70 mg (0.28
mmol) of 4-bromobenzylphosphonic acid and 124 mg (0.42 mmol) of
24, the title compound was obtained in 14% yield (16 mg) as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 2.97 (d, J = 21.2 Hz, 2H),
5.78 (s, 1H), 6.15 (br, 1H), 7.30−7.32 (m, 3H), 7.35−7.39 (m, 2H),
7.48 (d, J = 8.0 Hz, 1H), 7.50−7.55 (m, 3H), 7.65 (d, J = 4.0 Hz,
2H).31P NMR (162 MHz, DMSO-d6) δ 20.79 (s). HRMS (ESI) (m/
z) [M − H]− calculated for C20H16Cl2O4P, 421.0169; found,
421.0162.
2-Bromo-6-cyclohexylphenol (16e). General procedure A was
followed for the ortho-bromination of 2-cyclohexylphenol starting with
880 mg of the phenol (5.0 mmol). The crude product was purified by
column chromatography (9:1 hexanes/ethyl acetate, product Rf =
Potassium ((2-Hydroxy-3-cyclobutyl)phenyl)trifluoroborate
(18a). Borylation of 2-cyclobutylphenol, 15f, was accomplished by
closely following a previous report.26 A 1 dram oven-dried vial was
charged with a stir bar and brought into an inert atmosphere glovebox
where it was charged with 2-cyclobutylphenol (222 mg, 1.5 mmol, 1.0
equiv), diethylsilane (290 μL, 2.25 mmol, 1.5 equiv), bis(1,5-
cyclooctadiene)diiridium(I) dichloride (10 mg, 0.015 mmol, 0.01
equiv, 2 mol % Ir), and 3 mL of benzene (0.5 M). The vial was capped,
removed from the inert atmosphere glovebox, and the reaction mixture
was stirred at ambient temperature for 1 h. Solvent was removed with
a rotary evaporator under vacuum, and the reaction vial was brought
back into the inert atmosphere glovebox where it was charged with
bis(pinacolato)diboron (381 mg, 1.5 mmol, 1.0 equiv), pinacolborane
(21.8 μL, 0.15 mmol, 10 mol %), bis(1,5-cyclooctadiene)diiridium(I)
dichloride (10 mg, 0.015 mmol, 0.01 equiv, 2 mol % Ir), 4,4-di-tert-
1
0.35) to afford the product as a yellow oil (1.16 g, 91%). H NMR
(400 MHz, CDCl3) δ 1.19−1.52 (m, 5H), 1.71−1.80 (m, 1H), 1.80−
1.93 (m, 4H), 2.86−3.04 (m, 1H), 5.56 (s, 1H), 6.77 (t, J = 7.8 Hz,
1H), 7.13 (dd, J = 7.7, 1.4 Hz, 1H), 7.29 (dd, J = 8.0, 1.5 Hz, 1H). MS
(ESI) m/z 255 (M + H+).
Potassium ((2-Hydroxy-3-cyclohexyl)phenyl)trifluoroborate
(17e). Borylation of the phenol was accomplished by a modified
version of general procedure B. Butyllithium (2.5 M in hexanes, 3.90
mL, 9.75 mmol, 2.15 equiv) was added dropwise over 5 min via
syringe to a stirred solution of the o-bromophenol 16e (1.15 g, 4.5
mmol) in tetrahydrofuran (THF, 25 mL, 0.18 M) in a dry ice−acetone
cold temperature bath. The cold bath was removed, and the reaction
K
dx.doi.org/10.1021/jm401037h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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butylbipyridine (8 mg, 0.03 mmol, 2 mol %), and 3 mL of THF (0.5
M). The vial was capped, removed from the inert atmosphere
glovebox, and placed in a heating block preheated to 80 °C, and the
mixture was stirred vigorously for 2 h. The vial was cooled to ambient
temperature, and saturated potassium hydrogen difluoride (4.5 M, 3
mL, 13.5 mmol, 9 equiv) was added. The resulting reaction mixture
was stirred vigorously for 18 h. The volatile material was removed with
a rotary evaporator under vacuum to afford the crude product. The
crude product was dissolved in 5 mL of hot acetone, and the insoluble
salts were filtered. The volatile components of the filtrate were
removed with a rotary evaporator under vacuum until 1 mL of acetone
remained. The solution was transferred to a 15 mL conical centrifuge
tube, and approximately 5 mL of pentane was added to the acetone
solution to precipitate the product. The suspension was centrifuged to
pellet the solid product, and the black liquid was removed. Additional
washes were accomplished by adding 5 mL of pentane, resuspending
the solid followed by centrifugation to pellet the solid products for a
total of three additional washes. Removal of residual solvent under
2-(3-Benzylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(22a). To a Schlenk flask equipped with a stirring bar was added 21a
(0.80 g, 3.23 mmol), bis(pinacolato)diboron (B2Pin2, 1.64 g, 6.48
mmol), KOAc (1.27 g, 12.95 mmol), and DMSO (13 mL). Nitrogen
was bubbled through the reaction mixture for 0.5 h. Pd(dppf)Cl2 (0.13
g, 0.16 mmol) was added. Nitrogen was bubbled through the reaction
mixture for an additional 0.5 h. The reaction mixture was then stirred
at 80 °C for 6 h, cooled to ambient temperature, and poured into
water (130 mL). The resulting suspension was extracted with Et2O (2
× 100 mL). The combined organic extracts was dried over anhydrous
MgSO4, concentrated, and the residue was purified by automated silica
gel flash column chromatography (80 g flash column, column volume
= 125 mL, 60 mL/min, linear gradient of 0−20% ether in hexanes over
25 min) to give the title compound as a colorless oil (0.75 g, 79%
1
yield). H NMR (400 MHz, CD3OD) δ 1.32 (s, 12H), 3.94 (s, 2H),
7.12−7.17 (m, 3H), 7.22−7.29 (m, 4H), 7.56 (d, J = 8.0 Hz, 1H), 7.59
(s, 1H). 13C NMR (101 MHz, CD3OD) δ 23.8, 41.4, 83.6, 125.6,
127.5, 128.0, 128.4, 131.6, 132.0, 134.7, 140.6, 141.2.
1
(3-Bromophenyl)(4-fluorophenyl)methanol (20b). To a sol-
ution of 3-bromoiodobenzene (2.0 g, 7.1 mmol) in THF (15 mL) at
−78 °C under nitrogen atmosphere was added dropwise n-BuLi (2.5
M in hexanes, 2.8 mL, 7.0 mmol). The reaction mixture was stirred at
the same temperature for 0.5 h. 4-Fluorobenzaldehyde (0.75 mL, 7.1
mmol) was added. The reaction mixture was kept at −78 °C for 2 h.
Saturated NH4Cl (50 mL) and ether (50 mL) were added, and the
reaction mixture was allowed to warm to ambient temperature. The
phases were separated. The aqueous phase was extracted with ether
(100 mL). The combined organic solution was dried over anhydrous
MgSO4, filtered, and concentrated to give a viscous oil, which was used
without purification and characterization.
vacuum afforded the product as a light gray solid (200 mg, 52%). H
NMR (400 MHz, acetone-d6) δ 1.71−1.84 (m, 1H), 1.86−2.03 (m,
2H), 2.03−2.15 (m, 1H), 2.18−2.33 (m, 2H), 3.74 (p, J = 8.5 Hz,
1H), 6.60 (t, J = 7.3 Hz, 1H), 6.90 (dd, J = 7.5, 1.8 Hz, 1H), 7.15 (d, J
= 7.0 Hz, 1H), 7.62 (q, J = 11.8 Hz, 1H). MS (ESI) m/z 191 (boronic
acid M − H+).
2-Cycloheptylphenol (15g). Synthesis was achieved analogously
to that of 15f using 19 mL of the dilithiated reagent (approximately 5
mmol) and 650 μL of cycloheptanone (5.5 mmol, 1.1 equiv) to afford
the crude benzyl alcohol product as a yellow liquid (1.072 g). The
crude product (∼5 mmol) was treated with triethylsilane (2.4 mL, 15
mmol, ∼3 equiv) and trifluoroacetic acid and was purified via gradient
column chromatography (2−20% ethyl acetate in hexanes) to afford
the product as a clear oil (422 mg, 44%). 1H NMR (400 MHz, CDCl3)
δ 1.48−1.77 (m, 8H), 1.76−1.86 (m, 2H), 1.87−1.99 (m, 2H), 2.92−
3.02 (m, 1H), 4.71 (s, 1H), 6.74 (dd, J = 8.1, 1.2 Hz, 1H), 6.89 (td, J =
7.5, 1.2 Hz, 1H), 7.05 (td, J = 7.6, 1.7 Hz, 1H), 7.18 (dd, J = 7.7, 1.6
Hz, 1H). MS (ESI) m/z 191 (M + H+).
1-Bromo-3-(4-fluorobenzyl)benzene (21b). The crude product
of 20b was dissolved in dichloromethane (7 mL). Et3SiH (TES, 3.3
mL, 20.6 mmol) and trifluoroacetic acid (TFA, 3.5 mL) were added.
The resulting solution was stirred at ambient temperature for 15 h.
Volatiles were removed under reduced pressure. The residue was
purified by automated silica gel flash column chromatography (80 g
flash column, column volume = 125 mL, 60 mL/min, linear gradient of
0−20% ether in hexanes over 25 min) to give the title compound as a
Potassium ((2-Hydroxy-3-cycloheptyl)phenyl)-
trifluoroborate (18b). Borylation of 2-cycloheptylphenol, 15g, was
accomplished in the same manner as 18a, starting with 285 mg of 2-
cycloheptylphenol (1.5 mmol). The procedure yielded 90 mg of a gray
solid (20%), which is taken directly into the next reaction without
further purification.
1
colorless oil (1.18 g, 64% yield for two steps). H NMR (400 MHz,
CDCl3) δ 3.92 (s, 2H), 6.97−7.02 (m, 2H), 7.08−7.18 (m, 4H),
7.31−7.36 (m, 2H).
2-(3-(4-Fluorobenzyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-diox-
aborolane (22b). Compound 22b (0.58 g) was obtained as a
colorless oil in 67% yield starting from 21b (0.75 g, 2.8 mmol) by
following the procedure described for the preparation of 22a. 1H
NMR (400 MHz, CDCl3) δ 1.35 (s, 12H), 3.95 (s, 2H), 6.93−6.98
(m, 2H), 7.12−7.15 (m, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 8.0
Hz, 1H), 7.66−7.68 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 24.8,
41.0, 83.8, 115.0 (d), 128.0, 130.2(d), 131.8, 132.7, 135.1, 136.9 (d),
140.1, 160.1, 162.6. 19F NMR (376 MHz, CDCl3) δ −117.58 (m).
(3-Bromophenyl)(4-chlorophenyl)methanol (20c). Com-
pound 20c was prepared from 4-chlorobenzaldehyde (1.09 g, 7.78
mmol) by following the procedure described for the preparation of
20b. The crude material was used in the following step without
purification and characterization.
(3-Bromophenyl)(phenyl)methanol (20a). To a solution of 3-
bromobenzaldehyde (1.42 g, 7.67 mmol) in THF (15 mL) at −78 °C
under nitrogen atmosphere was added PhMgBr (3.0 M in Et2O, 2.56
mL, 7.68 mmol) dropwise. The reaction solution was stirred at −78
°C for 2 h. Saturated aqueous NH4Cl solution (20 mL) and Et2O (40
mL) were added, and the reaction mixture was allowed to warm to
ambient temperature. The layers were separated, and the aqueous layer
was extracted with Et2O (40 mL). The combined organic solution was
dried over anhydrous MgSO4, concentrated, and the residue was
purified by automated silica gel flash column chromatography (linear
gradient of 0−70% ethyl acetate in hexanes over 25 min) to give 20a
1
as a colorless oil (1.72 g, 85% yield). H NMR (400 MHz, CDCl3) δ
2.43 (d, J = 3.2 Hz, 1H), 5.76 (d, J = 3.2 Hz, 1H), 7.18−7.21 (m, 1H),
7.27−7.41 (m, 7 H), 7.56−7.57 (m, 1H). 13C NMR (101 MHz,
CDCl3) δ 75.6, 122.6, 125.1, 126.5, 127.9, 128.6, 129.4, 130.0, 130.5,
143.1, 145.9.
1-Bromo-3-(4-chlorobenzyl)benzene (21c). Compound 21c
(1.22 g) was obtained as a colorless oil in 56% yield starting from 4-
chlorobenzaldehyde (1.09 g, 7.78 mmol) by following the procedure
described for the preparation of 21b. 1H NMR (400 MHz, DMSO) δ
3.92 (s, 2H), 7.21−7.27 (m, 4H), 7.31−7.37 (m, 3H), 7.43−7.44 (m,
1H).
2-(3-(4-Chlorobenzyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-di-
oxaborolane (22c). The title compound (1.26 g) was obtained as a
white solid in 72% yield starting from 21c (1.50 g, 5.33 mmol) by
following the procedure described for the preparation of 22a. 1H
NMR (400 MHz, CDCl3) δ 1.34 (s, 12H), 3.95 (s, 2H), 7.10 (d, J =
8.0 Hz, 2H), 7.21−7.26 (m, 3H), 7.30 (t, J = 8.0 Hz, 1H), 7.66−7.68
(m, 2H).
1-Benzyl-3-bromobenzene (21a). Compound 20a (1.70 g, 6.50
mmol) was dissolved in dichloromethane (6 mL). Et3SiH (TES, 3.0
mL, 18.7 mmol) was added at ambient temperature, followed by
trifluoroacetic acid (TFA, 3.0 mL). The resulting solution was stirred
at ambient temperature for 15 h. Volatiles were removed under
reduced pressure. The residue was purified by automated silica gel
flash column chromatography (80 g flash column, column volume =
125 mL, 60 mL/min, linear gradient of 0−20% ether in hexanes over
25 min) to the title compound as a colorless oil (1.43 g, 89% yield).
1H NMR (400 MHz, CDCl3) δ 3.97 (s, 2H), 7.13−7.38 (m, 9H). 13
C
NMR (101 MHz, CDCl3) δ 41.6, 122.6, 126.4, 127.6, 128.6, 128.9,
(3-Bromophenyl)(3,4-dichlorophenyl)methanol (20d). The
129.2, 130.0, 131.9, 140.2, 143.5.
title compound (1.85 g) was obtained as a viscous oil in 72% yield
L
dx.doi.org/10.1021/jm401037h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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starting from 3,4-dichlorobenaldehyde (1.36 g, 7.77 mmol) by
following the procedure described for the preparation of 20b. 1H
NMR (400 MHz, CDCl3) δ 2.31 (d, J = 3.2 Hz, 1H), 5.74 (d, J = 3.2
Hz, 1H), 7.16−7.27 (m, 3H), 7.40−7.44 (m, 2H), 7.48 (m, 1H), 7.51
(m, 1H).
CDCl3) δ 0.69 (d, J = 7.2 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H), 0.80 (d, J
= 7.2 Hz, 3H), 0.85−1.41 (m, 19 H), 1.59−1.65 (m, 4H), 1.77−1.81
(m, 1H), 1.97−2.06 (m, 2H), 2.14−2.19 (m, 1H), 3.42 (br, 1H),
4.15−4.24 (m, 2H), 4.92 (dd, J = 11.2, 5.2 Hz, 1H), 7.37 (dd, J = 8.0,
4.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, CDCl3) δ
1
4-(3-Bromobenzyl)-1,2-dichlorobenzene (21d). The title
compound (1.57 g) was obtained as a white solid in 92% yield
starting from 20d (1.80 g, 5.42 mmol) by following the procedure
18.75. Compound 29b: H NMR (400 MHz, CDCl3) δ 0.72 (d, J =
6.8 Hz, 3H), 0.76 (d, J = 7.2 Hz, 3H), 0.79−1.42 (m, 22H), 1.59−1.70
(m, 4H), 1.91−1.99 (m, 3H), 2.13−2.16 (m, 1H), 3.36 (dd, J = 9.2,
4.8 Hz, 1H), 4.16−4.26 (m, 2H), 4.91 (dd, J = 11.2, 5.2 Hz, 1H), 7.35
(dd, J = 8.0, 4.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H). 31P NMR (162
MHz, CDCl3) δ 19.06.
1
described for the synthesis of 21a. H NMR (400 MHz, CDCl3) δ
3.89 (s, 2H), 7.00 (dd, J = 8.0, 2.0 Hz, 1H), 7.06−7.09 (m, 1H), 7.17
(t, J = 8.0 Hz, 1H), 7.25 (d, J = 2.0 Hz, 1H), 7.30−7.31 (m, 1H),
7.35−7.38 (m, 2H).
(S)-((4-bromophenyl)(hydroxy)methyl)phosphonic Acid
(30b). To a suspension of 29b (1.8 g, 3.3 mmol) and NaI (2.0 g,
13.3 mmol) in acetonitrile (20 mL) under nitrogen atmosphere was
added chlorotrimethylsilane (1.7 mL, 13.5 mmol). The reaction
mixture was refluxed for 10 h and then cooled to ambient temperature.
The precipitates were filtered off. The filtrate was concentrated under
reduced pressure. The residue was purified by automated reversed-
phase C-18 column chromatography (150 g C18 column, column
volume = 130 mL, 85 mL/min, liquid injection, linear gradient of 5−
30% acetonitrile in water with 0.1% trifluoroacetic acid over 25 min) to
give the title compound as a white solid (0.52 g, 59%). 1H NMR (400
MHz, DMSO-d6) δ 4.62 (d, J = 12.0 Hz, 1H), 7.33 (dd, J = 8.0, 4.0 Hz,
2H), 7.48 (d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, DMSO-d6) δ
17.43.
(S)-(Hydroxy(phenyl)methyl)phosphonic Acid. A mixture of
30b (43 mg, 0.16 mmol) and 10% Pd/C (dry, 16 mg) in MeOH (4
mL) was stirred under hydrogen atmosphere (1 atm) for 3 h. The
reaction mixture was filtered through a syringe filter (0.2 μm). The
filtrate was concentrated to give the title compound (28 mg, 93%). 1H
NMR (400 MHz, DMSO-d6) δ 4.64 (d, J = 14.0 Hz, 1H), 7.17−7.39
(m, 5H). 31P NMR (162 MHz, DMSO-d6) δ 18.14. The phosphonic
acid was dissolved in ethanol (0.5 mL), and excess of cyclohexylamine
(100 μL, 0.87 mmol) was added. The white precipitate was collected
by filtration. [α]D −10.6 (c 0.77, 50% MeOH/H2O at 20 °C) (lit.31a
−13.8, c 0.77, 50% MeOH/H2O).
2-(3-(3,4-Dichlorobenzyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (22d). The title compound (1.60 g) was obtained as
a white solid in 82% yield starting from 21d (1.70 g, 5.38 mmol) by
1
following the procedure described for the synthesis of 22a. H NMR
(400 MHz, CDCl3) δ 1.35 (s, 12H), 3.92 (s, 2H), 7.0 (d, J = 8.0 Hz,
1H), 7.22−7.26 (m, 2H), 7.32 (t, J = 8.0 Hz, 2H), 7.64 (s, 1H), 7.69
(d, J = 8.0 Hz, 1H).
(3-((3,4-Dichlorophenyl)(hydroxy)methyl)phenyl)boronic
Acid (24). To a solution of compound 23 (1.5 g, 5.3 mmol) in THF
(45 mL) was added n-BuLi (2.5 M in hexanes, 2.1 mL, 5.3 mmol)
dropwise at −78 °C under nitrogen atmosphere. The resulting
solution was stirred at the same temperature for 0.5 h. 3,4-
Dichlorobenzaldehyde (0.93 g, 5.3 mmol in THF (3 mL) was
added. The reaction mixture was stirred at −78 °C for 1 h. 1 N HCl
(16.5 mL) and ether (100 mL) were added. The reaction mixture was
allowed to warm to ambient temperature. The layers were separated,
and the aqueous layer was extracted with diethyl ether (2 × 100 mL).
The combined organic solution was dried over anhydrous MgSO4 and
concentrated under reduced pressure to give the title compound,
which was used in the following step without purification and
characterization.
(3′-(3,4-Dichlorobenzyl)-[1,1′-biphenyl]-4-carbonyl)-
phosphonic Acid (28). Compound 28 was prepared using general
procedure D. Hence, by use of 80 mg (0.28 mmol) of 27 and 152 mg
(0.42 mmol) of 22d, the title compound was obtained in 42% yield
(R)-((4-Bromophenyl)(hydroxy)methyl)phosphonic Acid
(30a). Compound 30a (0.22 g) was obtained as a white solid in
45% yield starting from 29a (1.0 g, 1.8 mmol) by following the
procedure described for the synthesis of compound 30b. The 1H
NMR and 31P NMR spectra are the same as those of 30b.
1
(49 mg) as a white solid. H NMR (400 MHz, DMSO-d6) δ 4.03 (s,
2H), 7.28−7.32 (m, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz,
1H), 7.58−7.60 (m, 2H), 7.70 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 8.26
(d, J = 8.0 Hz, 2H). 31P NMR (162 MHz, DMSO-d6) δ −3.07. HRMS
(ESI) (m/z) [M − H]− calculated for C20H14Cl2O4P, 419.0012;
found, 419.0020.
(R)-(3-Bromophenyl)(3,4-dichlorophenyl)methanol (31a).
Compound 31a was prepared by adapting a literature procedure.32a
To a solution of 3-bromo-1,2-dichlorobenzene (1.00 g, 4.42 mmol) in
tert-butyl methyl ether (5.50 mL) at −78 °C was added dropwise n-
BuLi (2.5 M in hexanes, 1.77, 4.42 mmol). The reaction mixture was
stirred at −78 °C for 1 h. The dry ice−acetone cooling bath was
replaced with an ice−water cooling bath. Anhydrous ZnCl2 (0.63 g,
4.64 mmol) was added. The resulting suspension was stirred at 0 °C
for 1 h. Additional n-BuLi (2.5 M in hexanes, 1.77 mL, 4.42 mmol)
was added. The cooling bath was removed, and the reaction mixture
was stirred at ambient temperature for 4.5 h. Toluene (anhydrous, 22.5
mL) and tetraethylethylenediamine (0.37 mL, 1.77 mmol) were
added. The reaction mixture was stirred at ambient temperature for an
additional 1 h. (+)-3-exo-(Morpholino)isoborneol ((+)-MIB,32 27 mg,
0.11 mmol) was added. The reaction mixture was cooled to 0 °C and
stirred for 0.5 h. 3-Bromobenzaldehyde (0.78 g, 2.21 mmol) was
added, and the reaction mixture was stirred at 0 °C for 17 h. Water (30
mL) and ether (30 mL) were added. The phases were separated, and
the aqueous layer was extracted with ether (2 × 50 mL). The
combined organic solution was dried over anhydrous MgSO4, filtered,
and concentrated. The residue was purified by automated silica gel
column chromatography (80 g flash column, column volume = 125
mL, 60 mL/min, linear gradient of 0−50% ethyl acetate in hexane over
25 min) to afford the title compound as a colorless oil (0.51 g, 70%).
The enantiomeric excess was determined by HPLC (AS-H column,
isopropanol/hexane = 5:95, flow rate = 0.5 mL/min, tr,major = 32.3 min,
Bis((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) ((R)-(4-
Bromophenyl)(hydroxy)methyl)phosphonate (29a) and Bis-
((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl) ((S)-(4-
Bromophenyl)(hydroxy)methyl)phosphonate (29b). Tris-
[(1R,2S,5R)-menth-2-yl]phosphite is prepared according to literature
procedure.30 Hence, to a solution of phosphorus trichloride (1.7 mL,
20.0 mmol) in anhydrous toluene (30 mL) at −20 °C was added
slowly a solution of D-menthol (9.4 g, 60.0 mmol) and triethylamine
(10 mL) in toluene (50 mL). Upon complete addition, the cooling
bath was removed and the reaction mixture was stirred at ambient
temperature for 5 h. The white precipitate was filtered off and the
filtrate was concentrated under reduced pressure to give tris-
[(1R,2S,5R)-menth-2-yl]phosphite (9.2 g), which was treated with 4-
bromobenzaldehyde (3.4 g, 18.4 mmol) and chlorotrimethylsilane (5.4
mL, 42.6 mmol) at 0 °C. The suspension was stirred at 0 °C for 2 h
and then at ambient temperature for an additional 1 h. Volatiles were
removed under reduced pressure. The residue was purified by
automatic silica gel column chromatography (220 g flash column,
column volume = 334 mL, 150 mL/min, linear gradient of 0−10%
hexane/ethyl acetate over 35 min) to give the product as a mixture of
diastereoisomers in a ratio of 1:0.7 (8.8 g, 88%). The diastereoisomeric
mixture was dissolved in acetonitrile (1 g/100 mL) at 60 °C and
cooled to room temperature. The solid was collected by filtration and
recrystallized in acetonitrile one more time to give diastereomerically
pure 29b. The filtrate solution was cooled to 0 °C. The solid was
collected and recrystallized in acetonitrile one more time to give
1
minor enantiomer tr,minor = 35.4 min, 90% ee). H NMR (400 MHz,
1
diastereomerically pure 29a. Compound 29a: H NMR (400 MHz,
CDCl3) δ 2.30 (d, J = 3.2 Hz, 1H), 5.75 (d, J = 3.2 Hz, 1H), 7.18 (dd,
M
dx.doi.org/10.1021/jm401037h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J = 8.4. 2.0 Hz, 1H), 7.20−7.27 (m, 2H), 7.40−7.44 (m, 2H), 7.48 (d,
J = 2.0 Hz, 1H), 7.52 (s, 1H).
REFERENCES
■
(1) For a general overview of synaptic plasticity in neuropsychiatric
disorders, see the following: (a) Luscher, C.; Issac, J. T. The synapse:
(S)-(3-Bromophenyl)(3,4-dichlorophenyl)methanol (31b).
Compound 31b (0.49 g) was obtained as a colorless oil in 67%
yield starting from 3-bromo-1,2-dichlorobenzene (1.00 g, 4.42 mmol)
by following the procedure used for the synthesis of 31a but replacing
the catalyst (+)-MIB with (−)-MIB. The enantiomeric excess was
determined by HPLC (AS-H column, isopropanol/hexane = 5:95,
flow rate = 0.5 mL/min, tr,major = 35.4 min, tr,minor = 32.3 min, 90% ee).
̈
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1
The H NMR spectrum is the same as that of 31a.
(S)-(3,4-Dichlorophenyl)(3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenyl)methanol (32a). Compound 32a (0.28 g)
was obtained as a colorless oil in 64% yield starting from 31a (0.38 g,
1.14 mmol) by following the procedure described for the synthesis of
22a. 1H NMR (400 MHz, CDCl3) δ 1.35 (s, 12H), 2.23 (d, J = 3.2 Hz,
1H), 5.80 (d, J = 3.2 Hz, 1H), 7.20 (ddd, J = 8.4, 2.4, 0.8 Hz, 1H),
7.33−7.44 (m, 3H), 7.51 (dd, J = 2.4, 0.8 Hz, 1H), 7.75 (dt, J = 6.8,
1.2 Hz, 1H), 7.79−7.80 (m, 1H).
(S)-(3,4-Dichlorophenyl)(3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenyl)methanol (32b). Compound 32b was pre-
pared in 58% yield starting from 31b (0.50 g, 1.51 mmol) by following
the procedure described the synthesis of 32a. The 1H NMR spectrum
of 32b is the same as that of 32a.
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ASSOCIATED CONTENT
■
S
* Supporting Information
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 203-432-3647. E-mail: jonathan.ellman@yale.edu.
Present Address
⊥H.-C.X.: Department of Chemistry, College of Chemistry and
Chemical Engineering, Xiamen University, Xiamen, Fujian
361005, China.
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Author Contributions
∥T.D.B. and H.-C.X. contributed equally.
The manuscript was written with contributions from all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
J.A.E. gratefully acknowledges the support of Yale University
setup funds and NIH Grant R01 GM054051. P.J.L. acknowl-
edges support from NIMH Grants R01 MH052711 and R01
MH091037.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; B2pin2, bis(pinacolato)diboron; BBB,
blood−brain barrier; cod, 1,5-cyclooctadiene; dppf, 1,1′-bis-
(diphenylphosphino)ferrocene; LAR, leukocyte common anti-
gen-related; DFMP, difluoromethylphosphonic acid; dtbpy,
4,4′-di-tert-butyl-2,2′-bipyridine; HBpin, pinacolborane; MKP5,
mitogen-activated protein (MAP) kinase phosphatase 5; MIB,
3-exo-(morpholino)isoborneol; PTP, protein tyrosine phospha-
tase; STEP, striatal-enriched protein tyrosine phosphatase; TC-
Ptp, T-cell protein tyrosine phosphatase
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