Design, synthesis, and pharmacological evaluation of glutamate carboxypeptidase II (GCPII) inhibitors based on thioalkylbenzoic acid scaffolds

Article abstract of DOI:10.1021/jm300488m

A series of thiol-based glutamate carboxypeptidase II (GCPII) inhibitors have been synthesized with either a 3-(mercaptomethyl)benzoic acid or 2-(2-mercaptoethyl)benzoic acid scaffold. Potent inhibitors were identified from each of the two scaffolds with IC₅₀ values in the single-digit nanomolar range, including 2-(3-carboxybenzyloxy)-5-(mercaptomethyl)benzoic acid 27c and 3-(2-mercaptoethyl)biphenyl-2,3'-dicarboxylicacid 35c. Compound 35c was found to be metabolically stable and selective over a number of targets related to glutamate-mediated neurotransmission. Furthermore, compound 35c was found to be orally available in rats and exhibited efficacy in an animal model of neuropathic pain following oral administration.

Full text of DOI:10.1021/jm300488m

Article
pubs.acs.org/jmc
Design, Synthesis, and Pharmacological Evaluation of Glutamate
Carboxypeptidase II (GCPII) Inhibitors Based on Thioalkylbenzoic
Acid Scaffolds
Doris Stoermer,† Dilrukshi Vitharana,† Niyada Hin,†,‡ Greg Delahanty,‡ Bridget Duvall,‡
Dana V. Ferraris,‡ Brian S. Grella,† Randall Hoover,† Camilo Rojas,†,‡ Megan K. Shanholtz,‡
Kyle P. Smith,‡ Marigo Stathis,†,‡ Ying Wu,†,‡ Krystyna M. Wozniak,†,‡ Barbara S. Slusher,†,‡,§
and Takashi Tsukamoto*,†,‡,§
†Eisai Inc., Baltimore, Maryland 21224, United States
‡Brain Science Institute, Johns Hopkins University, Baltimore, Maryland 21205, United States
§Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21205, United States
ABSTRACT: A series of thiol-based glutamate carboxypeptidase II
(GCPII) inhibitors have been synthesized with either a 3-
(mercaptomethyl)benzoic acid or 2-(2-mercaptoethyl)benzoic acid scaffold.
Potent inhibitors were identified from each of the two scaffolds with IC50
values in the single-digit nanomolar range, including 2-(3-carboxybenzy-
loxy)-5-(mercaptomethyl)benzoic acid 27c and 3-(2-mercaptoethyl)-
biphenyl-2,3′-dicarboxylic acid 35c. Compound 35c was found to be
metabolically stable and selective over a number of targets related to
glutamate-mediated neurotransmission. Furthermore, compound 35c was
found to be orally available in rats and exhibited efficacy in an animal model
of neuropathic pain following oral administration.
INTRODUCTION
■
One of the major sources of glutamate in the nervous system is
believed to be from hydrolysis of the neuropeptide N-
acetylaspartylglutamate (NAAG), catalyzed by a membrane-
bound extracellular metalloprotease glutamate carboxypepti-
dase II (GCPII).1 In theory, inhibition of GCPII would prevent
the production of extracellular glutamate and, thereby, provide
neuroprotective effects against excitotoxicity caused by
excessive synaptic glutamate. Thus, substantial efforts have
been made to identify potent and selective GCPII inhibitors,
some of which have shown efficacy in a variety of animal
models of neurological diseases.2−4
Nearly all potent GCPII inhibitors possess a zinc-binding
group that interacts with the GCPII active site catalytic zinc
atoms. Phosphonate,5 phosphinate,6 urea,7,8 thiol,9 and
hydroxamate10 were found to serve as an effective zinc binding
group for GCPII inhibitors. Among these classes of GCPII
inhibitors, those containing a thiol group offer a distinct
Figure 1. Thiol-based inhibitors of glutamate carboxypeptidase II
pharmacological advantage over other series, particularly in
chronic neurological disorders, because of their oral bioavail-
ability. The first reported orally available GCPII inhibitor 2-(3-
mercaptopropyl)pentanedioic acid 1 (2-MPPA, IC50 = 90 nM,
Figure 1)9 exhibited efficacy in various preclinical models of
neurological disorders following oral administration including
neuropathic pain,9 amyotrophic lateral sclerosis (ALS),11
diabetic neuropathy,12 cocaine addiction,13 and schizophrenia.14
Compound 1 was subsequently tested in healthy subjects,
(GCPII).
which represented the first administration of a GCPII inhibitor
in humans.15 Overall, 1 was safe and generally well-tolerated at
plasma exposures that were effective in animal models of
Received: April 6, 2012
Published: May 29, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of 9 and 13
a
Reagents and conditions: (a) potassium thioacetate, acetone, 67 °C, 66%; (b) sodium hydroxide, water−dioxane, 100 °C, 46%; (c) methyl iodide,
potassium carbonate, acetone, rt, 89%; (d) thioacetic acid, AIBN, toluene, 60 °C, 84%; (e) sodium hydroxide, water−dioxane, 100 °C, 96%.
a
Scheme 2. Synthesis of 19a−d
a
Reagents and conditions: (a) Pd(PPh3)4, potassium bicarbonate, DMF, 85 °C, 59−84%; (b) N-bromosuccinimide, AIBN, carbon tetrachloride, 77
°C, 78−100%; (c) potassium thioacetate, acetone, 67 °C, 23−71%; (d) sodium hydroxide, water−dioxane, reflux, 33−92%.
neuropathic pain. More importantly, no clinically significant
adverse CNS effects normally associated with glutamate
receptor antagonism were observed. The relatively higher oral
doses (300 mg or higher) of 1 required for therapeutically
relevant plasma concentrations, however, posed a risk for thiol-
related adverse effects inducing immune reactions when
conjugates are formed with endogenous proteins. Indeed,
some of the adverse reactions reported for captopril are
believed to be due in large part to its thiol group.16
Following the discovery of 1, further efforts have been
directed toward identifying new generations of thiol-based
GCPII inhibitors with improved potency so that much lower
doses can be sufficient for efficacy. For example, our group
investigated the effect of P1′ side chain modifications on GCPII
inhibitory potency using 1 as a template and identified 3-(2-
carboxy-5-mercaptopentyl)benzoic acid 2 (CMBA, IC50 = 15
nM, Figure 1), bearing a 3-carboxybenzyl group at the P1′ side
chain (Figure 1).17 This represented the first successful attempt
to improve the potency by modifying the glutarate moiety of
the GCPII inhibitors. In a preclinical model of neuropathic
pain, the effective dose (1.0 mg kg−1 day−1) for 2 was found to
be lower by an order of magnitude compared to that of 1 (10
mg kg−1 day−1) presumably because of its improved potency
toward GCPII. Interestingly, the benefit of introducing a 3-
carboxybenzyl group to the P1′ side chain appears unique to the
thiol-based GCPII inhibitors, as similar modifications to
phosphonate-, phosphinate-, and hydroxamate-based GCPII
inhibitors failed to enhance the inhibitory potency.17 Further
modification at the P1′ side chain resulted in the identification
of GCPII inhibitors based on an indole scaffold.18 1-(3-
Carboxyphenyl)-3-(2-mercaptoethyl)-1H-indole-2-carboxylic
acid 3 potently inhibited GCPII with an IC50 of 22 nM (Figure
1). The common structural feature of all these thiol-based
inhibitors is a 5-mercaptopentanoic acid backbone serving as
the key pharmacophore for GCPII inhibition. Indeed, 5-
mercaptopentanoic acid 4 itself is a low micromolar GCPII
inhibitor (IC50 = 1400 nM) despite the absence of the P1′ side
chain (Figure 1).9 These findings prompted us to take
advantage of the greater degree of structural diversity tolerated
for thiol-based GCPII inhibitors and to further explore the
possibility of incorporating other ring systems into the thiol-
based pharmacophore.
Here we present a new class of thiol-based GCPII inhibitors
5 and 6 containing benzoic acid as a core ring system where the
5-mercaptopentanoic acid pharmacophore is embedded (Figure
1). Like the indole series, the new scaffolds possess a reduced
number of rotatable bonds and increased lipophilicity in an
attempt to achieve enhanced oral bioavailability. With an
additional substituent at the ortho position of the benzoate
moiety, a number of compounds were identified as potent
GCPII inhibitors with IC50 values in the low nanomolar range.
One of these compounds showed improved oral pharmacoki-
netic profiles in rats and exhibit in vivo efficacy superior to the
existing thiol-based GCPII inhibitors in an animal model of
neuropathic pain following oral administration.
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a
Scheme 3. Synthesis of 27a−d
a
Reagents and conditions: (a) Ac2O, potassium carbonate, DMAP, dichloromethane, rt, 92%; (b) N-bromosuccinimide, AIBN, carbon tetrachloride,
77 °C, 84%; (c) TrSH, potassium carbonate, acetone, rt, 44%; (d) sodium methoxide, methanol, rt, 100% crude yield; (e) RC6H4CH2Br, potassium
carbonate, acetone, reflux, 57−70%; (f) sodium hydroxide, water−dioxane, reflux, 41−100%; (g) triisopropylsilane, TFA, dichloromethane, rt, 17−
75%.
a
Scheme 4. Synthesis of 35a−d and 37a−d
a
Reagents and conditions: (a) Pd(PPh3)4, potassium carbonate, vinylboronic anhydride pyridine complex, 1,2-dimethoxyethane, 80 °C, 83%; (b)
thioacetic acid, AIBN, toluene, 80 °C, 55%; (c) sodium hydroxide, TCEP, methanol−water, 65 °C, 93% crude yield; (d) p-TsOH, toluene, 110 °C,
71%; (e) Tf2O, pyridine, dichloromethane, rt, 67%; (f) arylboronic acid, Pd(PPh3)4, cesium carbonate, toluene, 110 °C, 49−88%; (g) sodium
hydroxide, water−dioxane, 110 °C, 53−68%; (h) RC6H4CH2Br, potassium carbonate, acetone, 55 °C, 31−61%; (i) sodium hydroxide, water−
dioxane, 100 °C, 13−61%.
Synthesis of 27a−d is summarized in Scheme 3. Methyl 2-
hydroxy-5-methylbenzoate 20 was converted into methyl 2-
hydroxy-5-(tritylthiomethyl)benzoate 24 in four steps. This key
intermediate was coupled with various benzyl bromides to form
the 2-benzyloxybenzoates 25a−d. Base-mediated hydrolysis of
the methyl ester group(s) followed by reductive cleavage of the
trityl group with triisopropylsilane/trifluoroacetic acid afforded
the desired compounds 27a−d.
As shown in Scheme 4, synthetic routes to 35a−d and 37a−
d share the first four steps starting from the triflate 28.19 Suzuki
coupling of 28 with vinylboronic anhydride catalyzed by
tetrakis(triphenylphosphine)palladium20 afforded the substi-
tuted styrene 29. Radical-mediated addition of thioacetic acid
to 29 gave the thioacetate 30, and subsequent base-mediated
hydrolysis provided compound 31. Thiolactonization of 31 was
carried out by heating the solution of 31 in toluene in the
RESULTS AND DISCUSSION
■
As shown in Scheme 1, compound 9 was synthesized from
methyl 3-(bromomethyl)benzoate 7 by reacting with potassium
thioacetate followed by base-mediated hydrolysis of both
methyl ester and thioester. The synthesis of 13 started with
methyl esterification of 2-vinylbenzoic acid 10 to 11 with
methyl iodide. Radical-mediated addition of thioacetic acid to
11 gave the thioacetate 12, and subsequent base-mediated
hydrolysis afforded the desired compound 13.
Scheme 2 illustrates the synthesis of compounds 19a−d.
Palladium-catalyzed Suzuki coupling of methyl 2-bromo-5-
methylbenzoate 14 with phenylboronic acids 15a−d provided
biphenyl compounds 16a−d. Bromination of 16a−d with N-
bromosuccinimide followed by the treatment with potassium
thioacetate afforded 18a−d. Subsequent base-mediated hydrol-
ysis of 18a−d gave the desired compounds 19a−d.
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a
Table 1. Inhibition of GCPII by Thiol-Based Inhibitors
a
Values are the mean SD of three or more independent experiments.
presence of p-toluenesulfonic acid to give the common key
intermediate 32.
positively charged residues (Arg463, Arg534, and Arg536) at
this site, which might explain the increased potency of
compounds 27b−d bearing a carboxyl group in the benzyloxy
moiety over 27a. A similar mode of “flipped” binding was
proposed for sulfonamide-based GCPII inhibitors.22 Among
this series of GCPII inhibitors, 2-(3-carboxybenzyloxy)-5-
(mercaptomethyl)benzoic acid 27c was found to be a
particularly potent GCPII inhibitor with an IC50 of 2 nM.
Introduction of a phenyl ring onto 13 led to an equally
potent GCPII inhibitor 35a with an IC50 of 1400 nM. In a
fashion similar to compounds 19b−d, addition of a carboxyl
group to the phenyl ring provided more potent inhibitors 35b−
d. Among them, 2-(3-carboxyphenyl)-6-(2-sulfanylethyl)-
benzoic acid 35c exhibited extremely potent inhibition of
GCPII with an IC50 of 2 nM. Like compound 19c, the entire
skeletal frame of 2 is embedded in compound 35c. Nearly 10-
fold improvement in potency of 35c over 2 suggests that the
rigid conformation provided by the biphenyl backbone of 35c
allows optimal interaction with GCPII. Benzyloxy-substituted
derivative 37a exhibited more potent GCPII inhibition than the
parent compound 13. Addition of a carboxyl group, however,
did not significantly enhanced GCPII inhibitory potency in this
series (compounds 37b−d).
The overall SAR analysis indicates a reasonable correlation
among the different scaffolds with respect to the effects of
substitution pattern on GCPII inhibitory potency. In all cases,
compounds containing a 3-carboxyphenyl group produced the
most potent GCPII inhibitors as in compounds 19c, 27c, 35c,
and 37c. In particular, compounds 27c and 35c represent the
most potent thiol-based GCPII inhibitors reported to date. The
two benzyloxy-substituted derivatives 27a and 37a exhibited
IC50 values in the nanomolar range despite their lack of a
second carboxyl group. While these compounds are not nearly
as potent as 27c and 35c, their favorable druglike properties
should warrant new SAR efforts strictly on monocarboxylate
compounds within and beyond the benzyloxy substituted
derivatives.
For the synthesis of 35a−d, the intermediate 32 was first
converted into the corresponding triflate 33 with triflic
anhydride. Palladium-catalyzed Suzuki coupling of 33 with
phenylboronic acids provided biphenyl compounds 34a−d.
Subsequent base-mediated hydrolysis of 34a−d gave the
desired compounds 35a−d. Synthesis of 37a−d involved
Williamson reaction of 32 with various benzyl bromides to
yield the benzyloxy derivatives 36a−d. Subsequent base-
mediated hydrolysis of 36a−d gave the desired compounds
37a−d.
Inhibitory potencies of the thiol-based compounds were
measured using N-acetyl-L-aspartyl-[3H]-L-glutamate as a
substrate and purified human recombinant GCPII.21 The
results are summarized in Table 1. Both compounds 9 (IC50
1300 nM) and 13 (IC50 = 930 nM) exhibited inhibitory
potencies comparable to 5-mercaptopentanoic acid 4 (IC50
=
=
1400 nM). Introduction of a phenyl ring into 9 resulted in
nearly 5-fold reduction in potency as exemplified by compound
19a with an IC50 of 5700 nM. Incorporation of a carboxyl
group at any position on the phenyl ring, however, dramatically
improved potency as in compounds 19b−d. The significant
improvement in potency by the addition of a carboxyl group
suggests that these compounds take advantage of the positively
charged residues located at the S1′ pocket of GCPII. Among
them, the most potent compound 19c inhibited GCPII activity
with an IC50 of 38 nM comparable to that of 2 (IC50 = 15 nM)
which shares its core skeletal frame.
Interestingly, benzyloxy-substituted derivatives 27a−d ex-
hibited GCPII inhibitory potency superior to that of their
phenyl counterparts 19a−d. As is the case with 19b−d,
increased potency was obtained by adding a carboxyl group into
the phenyl ring of the benzyloxy moiety as in compounds 27b−
d. It is questionable, however, whether the bulky benzyloxy
moiety of compounds 27a−d occupies the S1′ site given the
limited space available within the pocket. Although speculative,
it is conceivable that the benzyloxy group of compounds 27a−d
is oriented toward the S1 pocket. Indeed, there are a few
Further pharmacological characterization was conducted with
one of the most potent GCPII inhibitors, compound 35c. No
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evidence of mutagenicity was observed in the Ames test using
Salmonella typhimurium strains (TA98, TA100, TA1535, and
TA1537) in the presence or absence of metabolic activation by
S9 mixture. Compound 35c was also tested in a panel of in
vitro assays including those related to glutamate-mediated
neurotransmission. As shown in Table 2, compound 35c
Table 2. In Vitro Pharmacological Characterization of 35c
assay
results
hERG channel
no inhibition up to 100 μM
<5% inhibition at 10 μM
12% inhibition at 10 μM
no inhibition at 10 μM
no inhibition at 10 μM
no inhibition at 10 μM
no inhibition at 10 μM
no inhibition at 10 μM
no inhibition up to 100 μM
NMDA receptor (agonism)
NMDA receptor (glycine)
NMDA receptor (phencyclidine)
NMDA receptor (polyamine)
AMPA receptor
kainate receptor
glycine receptor (strychnine sensitive)
glutaminase (kidney-type)
Figure 2. Antinociceptive effects of compound 35c in the rat chronic
constriction injury (CCI) model of neuropathic pain. Oral
administration of 35c at 0.1 mg kg−1 day−1 significantly attenuated
CCI-induced hyperalgesic state relative to the vehicle-treated control
(∗, p < 0.05; ∗∗, p < 0.005).
showed no significant activity in any of these assays at 10 μM
(>1000-fold greater than IC50 of 35c) or higher concentration.
In vitro metabolic stability of 35c was examined by using liver
microsomes of mouse, rat, dog, monkey, and human. No
significant metabolism of 35c was detected across all species
during a 2 h incubation period. In vivo pharmacokinetic studies
of 35c were carried out in fasted rats by intravenous and oral
administration (10 mg/kg). The plasma pharmacokinetic
parameters are summarized in Table 3. The oral bioavailability
of 35c was approximately 40%. Absorption of 35c was relatively
rapid and the plasma peak concentration was generally
observed within the first hour with a terminal half-life of 3−4
h. It is worth noting that the AUC followed by 10 mg/kg oral
administration of 35c (33.8 μg·h/mL) is nearly 20-fold higher
than that of 1 (1.82 μg·h/mL)9 as a result of the higher Cmax
and lower clearance.
Compound 35c was subsequently tested for its antinoci-
ceptive effects following oral administration (0.1 mg kg−1
day−1) using the rat chronic constriction injury model of
neuropathic pain.23 As shown in Figure 2, 35c significantly
reduced thermal hyperalgesia relative to the vehicle-treated
control on days 8 and 12. The effective dose for 35c is lower by
2 orders of magnitude compared to that of 1 (10 mg kg−1
day−1)9 and by 1 order of magnitude compared to that of 2 (1.0
mg kg−1 day−1),17 presumably because of its enhanced GCPII
inhibitory potency coupled with the improved pharmacokinetic
properties.
captopril, it is vital to identify highly potent thiol-based GCPII
inhibitors with low efficacious doses in order to minimize the
thiol-related side effects. We synthesized a series of compounds
based on either 3-(mercaptomethyl)benzoic acid 9 or 2-(2-
mercaptoethyl)benzoic acid 13 scaffold. Some of these
compounds were found to be very potent inhibitors of
GCPII. 2-(3-Carboxyphenyl)-6-(2-sulfanylethyl)benzoic acid
35c was found to be orally available and efficacious in an
animal model of peripheral neuropathy by oral administration.
Given the low dose (0.1 mg/kg) required for efficacy,
compound 35c and other potent inhibitors reported herein
may represent a new class of GCPII inhibitors with significant
therapeutic potential. Further dose−response pharmacological
evaluations of 35c are currently underway in various animal
models of neurological disorders associated with glutamate
excitotoxicity.
Furthermore, the synthetic routes developed herein will
allow us to utilize widely available arylboronic acids and benzyl
bromides, leading to more extensive SAR studies on derivatives
based on scaffolds 9 and 13. In addition, very potent GCPII
inhibitors emerging from present and future SAR efforts should
serve as valuable templates into which other zinc-binding
groups can be incorporated to further expand the structural
diversity of GCPII inhibitors.
CONCLUSIONS
■
EXPERIMENTAL SECTION
■
Unlike other zinc-binding groups, the thiol group is
nucleophilic and prone to oxidation. These chemical properties
are linked to the risk of inducing immune reactions when
conjugates are formed with endogenous proteins. Indeed, some
of the adverse reactions reported for captopril are believed to
be due in large part to its thiol group.16 Since more serious
reactions can be managed by decreasing doses, as in the case of
General. All solvents were reagent grade or HPLC grade. Unless
otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. All reactions were
performed under nitrogen. Preparative HPLC purification was
performed on an Agilent 1200 series HPLC system equipped with a
multiwavelength detector using a Phenomenex Luna 5 μm C18 (2)
column (250 mm × 4.6 mm) with a flow rate of 15 mL/min. The
Table 3. Pharmacokinetic Profile of 35c in Fasted Rats
dose
Cmax (μg/mL)
Tmax (h)
AUCinf (μg·h/mL)
CL/F (mL h−1 kg−1
)
T1/2 (h)
F (%)
10 mg/kg iv (n = 4)
10 mg/kg po (n = 9)
90.6 9.0
33.8 9.9
267 120
318 90
3.6 0.6
3.2 0.6
7.10 5.55
1.1 0.6
37
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solvent system consisted of distilled water with 0.1% formic acid
(solvent A) and acetonitrile with 0.1% formic acid (solvent B). 40% B
was used from 0 to 5 min followed by a linear gradient from 40% to
85% B over 35 min. Melting points were obtained on a Mel-Temp
apparatus and are uncorrected. 1H NMR spectra were recorded at 400
MHz. 13C NMR spectra were recorded at 100 MHz. Elemental
analyses were performed by Atlantic Microlab (Norcross, GA) and
were within 0.4% of calculated values.
J = 7.3, 15.2 Hz, 2H), 3.36 (t, J = 7.8 Hz, 2H), 7.35 (m, 2H), 7.54 (m,
1H), 8.12 (dd, J = 1.3, 7.8 Hz, 1H); 13C NMR (CDCl3) δ 25.9, 39.3,
126.8, 127.9, 131.9, 132.0, 133.2, 142.7, 172.8; MS ES− m/z 181.1 (M
−
− H) ; MS ES+ m/z 183.0 (M + 1)+. Anal. Calcd for C9H10O2S: C,
59.32; H, 5.53; S, 17.60. Found: C, 59.02; H, 5.71; S, 17.30.
Methyl 5-Methyl-2-phenylbenzoate (16a). To a solution of
methyl 2-bromo-5-methylbenzoate 14 (1.00 g, 4.37 mmol) in DMF
(10 mL) were added phenylboronic acid 15a (0.64 g, 5.25 mmol),
potassium bicarbonate (1.99 g, 19.9 mmol), and tetrakis-
(triphenylphosphine)palladium (0.15 g, 0.13 mmol). The mixture
was heated at 85 °C for 3.5 h. The solvent was removed in vacuo, and
the residue was taken up by ethyl acetate (40 mL). The solution was
subsequently washed with water, aqueous 10% KHSO4, and brine. The
solution was then dried over MgSO4 and concentrated. The residue
was purified by silica gel chromatography (5% EtOAc in hexanes) to
provide 0.83 g of 16a as a clear oil (84% yield): 1H NMR (DMSO-d6)
δ 2.38 (s, 3H), 3.57 (s, 3H), 7.25 (m, 2H), 7.33 (m, 2H), 7.39−7.44
(m, 3H), 7.54 (s, 1H).
Methyl 3-(Acetylsulfanylmethyl)benzoate (8). To a solution of
methyl 3-(bromomethyl)benzoate 7 (1.00 g, 4.37 mmol) in acetone
(60 mL) was added potassium thioacetate (0.60 g, 5.24 mmol), and
the resulting mixture was stirred at 67 °C for 1.5 h. The mixture was
filtered, and the filtrate was concentrated in vacuo. The residue was
purified by silica gel chromatography (9:1 hexanes/EtOAc) to provide
1
0.64 g of 8 as clear oil (66% yield): H NMR (DMSO-d6) δ 2.36 (s,
3H), 3.85 (s, 3H), 4.19 (s, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.57 (m, 1H),
7.83 (d, J = 7.8 Hz, 1H), 7.90 (m, 1H).
3-(Sulfanylmethyl)benzoic Acid (9). To a solution of 8 (0.30 g,
1.34 mmol) in H2O−dioxane (1:1, 6 mL) was added sodium
hydroxide (0.21 g, 5.25 mmol, 4.0 equiv). The resulting mixture was
heated at 100 °C for 1 h. The mixture was cooled to room
temperature, and one spatula tip of tris(2-carboxyethyl)phosphine was
added. After the mixture was stirred for 15 min, the solvents were
removed in vacuo. The resulting residue was acidified with aqueous
10% KHSO4 and the product extracted with ethyl acetate. The organic
layer was washed with brine, dried over MgSO4, and concentrated in
vacuo. The resulting solid was purified by reverse-phase HPLC to
provide 0.103 g of 9 as a white crystal (46% yield): mp 104−106 °C;
1H NMR (DMSO-d6) δ 3.00 (t, J = 8.0 Hz, 1H), 3.81 (d, J = 7.8 Hz,
2H), 7.45 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 7.1 Hz, 1H), 7.80 (d, J = 8.1
Hz, 1H), 7.94 (s, 1H), 12.99 (s, 1H). Anal. Calcd for C8H8O2S: C,
57.12; H, 4.79; S, 19.06. Found: C, 57.27; H, 4.98; S, 18.94.
Methyl 2-Vinylbenzoate (11). To a solution of 2-vinylbenzoic
acid 10 (0.16 g, 1.08 mmol) in acetone (10 mL) was added potassium
carbonate (0.30 g, 2.16 mmol). The mixture was allowed to stir for 5
min, after which methyl iodide (0.10 mL, 1.62 mmol, 1.5 equiv) was
added. After 24 h at room temperature, the solvent was removed in
vacuo and the residue was taken up by ethyl acetate and washed with
water and brine. The organic layer was dried over sodium sulfate and
concentrated to yield 0.156 g of 11 as a colorless oil (89% crude yield).
The crude material was used as is without purification: 1H NMR
(CDCl3) δ 3.92 (s, 3H), 5.37 (dd, J = 1.3, 10.9 Hz, 1H), 5.64 (dd, J =
1.3,17.4 Hz, 1H), 7.32 (dt, J = 1.3, 7.6 Hz, 1H), 7.43 (d, J = 11.1 Hz,
1H), 7.48−7.53 (m, 1H), 7.58 (m, 1H), 7.89 (dt, J = 0.8, 7.8 Hz, 1H).
Methyl 2-(2-(Acetylthio)ethyl)benzoate (12). To a solution of
11 (0.05 g, 0.31 mmol) in toluene (0.5 mL) was added thioacetic acid
(0.44 mL, 6.16 mmol). The mixture was heated at 60 °C, after which
AIBN (0.005 g, 0.031 mmol) was added in one portion. After 1 h at 60
°C, the mixture was cooled to room temperature. Solvent was
removed under reduced pressure, and excess thioacetic acid was
removed by coevaporation with toluene (25 mL × 2). The crude
product was purified by a Biotage flash chromatography apparatus
(SNAP 10 g cartridge) using 5−40% gradient of ethyl acetate in
hexanes to yield 0.061 g of 12 as a colorless oil (84% yield): 1H NMR
(CDCl3) δ 2.34 (s, 2H), 3.17 (m, 2H), 3.23 (m, 2H), 3.92 (s, 3H),
7.31−7.34 (m, 2H), 7.47 (dt, J = 1.56, 7.6 Hz, 1H), 7.93 (dd, J = 1.1,
7.7 Hz, 1H); 13C NMR (CDCl3) δ 30.2, 30.6, 34.5, 52.1, 126.7, 129.3,
130.9, 131.6, 132.2, 141.6, 167.7, 195.8.
Methyl 2-(2-Methoxycarbonylphenyl)-5-methylbenzoate
(16b). Compound 16b was prepared as described for the preparation
of 16a except (2-methoxycarbonylphenyl)boronic acid 15b was used
1
in place of phenylboronic acid 15a: clear oil (59% yield); H NMR
(DMSO-d6) δ 2.40 (s, 3H), 3.51 (s, 3H), 3.52 (s, 3H), 7.11 (d, J = 7.8
Hz, 1H), 7.19−7.24 (m, 1H), 7.41 (m, 1H), 7.51 (m, 1H), 7.59 (m,
1H), 7.69 (s, 1H), 7.84−7.89 (m, 1H).
4-Methyl-[1,1′-biphenyl]-2,3′-dicarboxylic Acid Dimethyl
Ester (16c). Compound 16c was prepared as described for the
preparation of 16a except (3-methoxycarbonylphenyl)boronic acid
15c was used in place of phenylboronic acid 15a: colorless oil (82%
1
yield); H NMR (CDCl3) δ 2.44 (s, 3H), 3.64 (s, 3H), 3.93 (s, 3H),
7.28 (t, J = 3.9 Hz, 1H), 7.36 (m, 1H), 7.46−7.49 (m, 2H), 7.71 (m,
1H), 7.99−8.04 (m, 2H); 13C NMR (CDCl3) δ 21.0, 51.9, 52.1, 127.9,
128.2, 129.5, 130.6, 130.7, 131.5, 132.3, 133.0, 137.6, 138.8, 140.4,
141.6, 167.0, 168.7.
Methyl 2-(4-Methoxycarbonylphenyl)-5-methylbenzoate
(16d). Compound 16d was prepared as described for the preparation
of 16a except (4-methoxycarbonylphenyl)boronic acid 15d was used
1
in place of phenylboronic acid 15a: clear oil (65% yield); H NMR
(DMSO-d6) δ 2.39 (s, 3H), 3.58 (s, 3H), 3.87 (s, 3H), 7.33 (d, J = 7.8
Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H), 7.44 (m, 1H), 7.61 (m, 1H), 7.97
(d, J = 8.3 Hz, 2H).
Methyl 5-(Bromomethyl)-2-phenylbenzoate (17a). To a
solution of 16a (0.81 g, 3.58 mmol) in CCl4 (10 mL) were added
N-bromosuccinimide (0.70 g, 3.93 mmol) and AIBN (0.12 g, 0.73
mmol). The mixture was heated at 77 °C for 3.5 h and then cooled to
room temperature. The white precipitate was filtered off and the
filtrate was concentrated in vacuo to give 0.85 g of 17a as a brown oil
(78% crude yield). The crude material was used for the next reaction
1
without further purification: H NMR (DMSO-d6) δ 3.60 (s, 3H),
4.82 (s, 2H), 7.28 (m, 2H), 7.36−7.46 (m, 4H), 7.68 (dd, J = 2.0, 7.8
Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H).
Dimethyl 4-(Bromomethyl)biphenyl-2,2′-dicarboxylate
(17b). Compound 17b was prepared as described for the preparation
of 17a except methyl 2-(2-methoxycarbonylphenyl)-5-methylbenzoate
16b was used in place of 16a: brown oil (100% crude yield); 1H NMR
(DMSO-d6) δ 3.54 (s, 6H), 4.83 (s, 2H), 7.21 (m, 2H), 7.49 (t, J = 7.6
Hz, 1H), 7.61 (t, J =, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.8
Hz, 1H), 7.96 (s, 1H).
2-(2-Mercaptoethyl)benzoic Acid (13). To a solution of 12
(0.047 g, 0.20 mmol) in water−dioxane mixture (1:1, 0.5 mL) was
added sodium hydroxide (0.035 g, 0.88 mmol) in one portion. The
reaction mixture was heated at 100 °C for 1 h, followed by the
addition of one spatula tip of TCEP. After the mixture was stirred for
an additional 20 min at room temperature, solvents were removed in
vacuo and the residue was taken up by ethyl acetate and water. The
aqueous layer was then acidified to pH ∼4 with aqueous 10% KHSO4
and extracted with ethyl acetate three times. The combined organic
extracts were washed with brine, dried over sodium sulfate, and
concentrated to give 0.035 g of 13 as an off-white solid (96% yield):
mp 84−85 °C; 1H NMR (CDCl3) δ 1.45 (t, J = 8.1 Hz, 1H), 2.85 (dd,
Dimethyl 4-(Bromomethyl)biphenyl-2,3′-dicarboxylate
(17c). Compound 17c was prepared as described for the preparation
of 17a except methyl 2-(3-methoxycarbonylphenyl)-5-methylbenzoate
16c and benzoyl peroxide were used in place of 16a and AIBN,
1
respectively: brown oil (60% yield); H NMR (DMSO-d6) δ 3.61(s,
3H), 3.88 (s, 3H), 4.84 (s, 2H), 7.48−7.50 (d, J = 8.0 Hz, 1H), 7.59−
7.60 (m, 2H), 7.72−7.75 (m, 1H), 7.85 (s, 1H), 7.90 (m, 1H), 7.97−
7.99 (m, 1H).
Dimethyl 4-(Bromomethyl)biphenyl-2,4′-dicarboxylate
(17d). Compound 17d was prepared as described for the preparation
of 17a except methyl 2-(4-methoxycarbonylphenyl)-5-methylbenzoate
16d was used in place of 16a: brown oil (94% crude yield); 1H NMR
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Journal of Medicinal Chemistry
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1
(DMSO-d6) δ 3.60 (s, 3H), 3.88 (s, 3H), 4.83 (s, 2H), 7.43 (d, J = 8.1
Hz, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.72 (dd, J = 1.8, 7.8 Hz, 1H), 7.90
(m, 1H), 7.99 (d, J = 8.1 Hz, 2H).
solid (92% yield); H NMR (DMSO-d6) δ 3.10 (t, J = 8.0 Hz, 1H),
3.89 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.58−7.65 (m, 3H),
7.83 (d, J = 2.0 Hz, 1H), 7.92 (s, 1H), 7.97−8.00 (m, 1H). Anal. Calcd
for C15H12O4S·0.5AcOH: C, 60.37; H, 4.43; O, 25.13; S, 10.07.
Found: C, 60.28; H, 4.45; S, 10.15.
Methyl 5-(Acetylsulfanylmethyl)-2-phenylbenzoate (18a).
To a solution of 17a (0.59 g, 1.93 mmol) in acetone (30 mL) was
added potassium thioacetate (0.27 g, 2.36 mmol), and the mixture was
heated at 67 °C for 1.5 h. The reaction mixture was cooled to room
temperature, and excess potassium carbonate was filtered. The filtrate
was concentrated in vacuo. The residue was purified by reverse-phase
HPLC (method, 40−100% acetonitrile−water, 0.1% formic acid) to
give 0.41 g of 18a as white powder (71% yield): 1H NMR (DMSO-d6)
δ 2.37 (s, 3H), 3.57 (s, 3H), 4.20 (s, 2H), 7.25 (m, 2H), 7.35−7.44
(m, 4H), 7.52 (dd, J = 2.0, 8.1 Hz, 1H), 7.65 (d, J = 1.8 Hz, 1H).
M e t h y l 5 - ( A c e t y l s u l f a n y l m e t h y l ) - 2 - ( 2 -
methoxycarbonylphenyl)benzoate (18b). Compound 18b was
prepared as described for the preparation of 18a except dimethyl 4-
(bromomethyl)biphenyl-2,2′-dicarboxylate 17b was used in place of
17a: white powder (23% yield); 1H NMR (DMSO-d6) δ 2.40 (s, 3H),
3.52 (s, 3H), 3.53 (s, 3H), 4.23 (s, 2H), 7.15 (d, J = 7.8 Hz, 1H), 7.21
(dd, J = 1.3, 8.1 Hz, 1H), 7.48 (dt, J = 1.5, 7.6, 1H), 7.50 (dd, J = 1.8,
7.8 Hz, 1H), 7.60 (dt, J = 1.3, 7.3 Hz, 1H), 7.81 (d, J = 2.02 Hz, 1H),
7.87 (dd, J = 1.5, 7.8 Hz, 1H).
4-(Mercaptomethyl)biphenyl-2,4′-dicarboxylic Acid (19d).
Compound 19d was prepared as described for the preparation of
19a except dimethyl 4-(acetylthiomethyl)biphenyl-2,4′-dicarboxylate
18d was used in place of 18a: white powder (53% yield): mp 236−238
1
°C; H NMR (DMSO-d6) δ 3.06 (t, J = 8.1 Hz, 1H), 3.83 (d, J = 8.1
Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.57 (dd, J
= 2.0, 7.8 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.94 (d, J = 8.1 Hz, 2H),
12.92 (bs, 2H). Anal. Calcd. for C15H12O4S·0.4EtOAc: C, 61.62; H,
4.73; S, 9.91. Found: C, 62.02; H, 4.37; S, 9.55.
Methyl 2-Acetoxy-5-methylbenzoate (21). To a solution of
methyl 2-hydroxy-5-methylbenzoate 20 (10 mL, 70 mmol), potassium
carbonate (14.5 g, 105 mmol), and DMAP (50 mg, 0.41 mmol) in
dichloromethane (50 mL) was dropwise added acetic anhydride (7.2
mL, 77 mmol). The reaction mixture was stirred for 72 h at room
temperature. The reaction was quenched by the addition of 300 mL of
10% KHSO4. The aqueous phase was extracted with dichloromethane
twice (300 and 150 mL). The combined organic phases were dried
over MgSO4, filtered, and concentrated in vacuo to afford 13.3 g of 21
as a light yellow oil (92% crude yield). This material was used without
Dimethyl 4-(Acetylthiomethyl)biphenyl-2,3′-dicarboxylate
(18c). Compound 18c was prepared as described for the preparation
of 18a except dimethyl 4-(bromomethyl)biphenyl-2,3′-dicarboxylate
1
further purification: H NMR (CDCl3) δ 2.34 (s, 3H), 2.37 (s, 3H),
1
17c was used in place of 17a (76% yield): H NMR (DMSO-d6) δ
3.86 (s, 3H), 6.98 (d, J = 8.3 Hz, 1H), 7.35 (m, 1H), 7.82 (m, 1H).
Methyl 2-Acetoxy-5-(bromomethyl)benzoate (22). To a
solution of 21 (9.13 g, 43.9 mmol) in CCl4 (65 mL) were added N-
bromosuccinimide (9.77 g, 54.9 mmol) and AIBN (1.3 g, 7.9 mmol).
The reaction mixture was heated at 77 °C for 4 h. The reaction
mixture was poured into 10% KHSO4 (60 mL) and extracted with
dichloromethane (60 mL × 2). The combined organic extracts were
dried over MgSO4, filtered, and concentrated. The crude material was
purified by a silica gel chromatography (10% ethyl acetate/hexanes) to
2.39 (s, 3H), 3.60 (s, 3H), 3.88 (s, 3H), 4.23 (s, 2H), 7.42−7.44 (d, J
= 8.0 Hz, 1H), 7.57−7.60 (m, 3H), 7.74 (s 1H), 7.83 (s, 1H), 7.96−
7.99 (m, 1H).
Dimethyl 4-(Acetylthiomethyl)biphenyl-2,4′-dicarboxylate
(18d). Compound 18d was prepared as described for the preparation
of 18a except dimethyl 4-(bromomethyl)biphenyl-2,4′-dicarboxylate
1
17d was used in place of 17a (33% yield): H NMR (DMSO-d6) δ
2.39 (s, 3H), 3.59 (s, 3H), 3.88 (s, 3H), 4.22 (s, 2H), 7.42 (d, J = 8.1
Hz, 3H), 7.58 (dd, J1 = 2.0, 8.1 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.99
(d, J = 8.6 Hz, 2H).
1
afford 10.6 g of 22 as a white solid (84% yield): H NMR (CDCl3) δ
2.35 (s, 3H), 3.88 (s, 3H), 4.48 (s, 2H), 7.09 (d, J = 8 Hz, 1H), 7.58
(dd, J = 8.4, 2.3 Hz, 1H), 8.04 (d, J = 2.0 Hz, 1H).
4-(Mercaptomethyl)biphenyl-2-carboxylic Acid (19a). To a
solution of 18a (0.41 g, 1.36 mmol) in H2O/dioxane (6 mL, 1:1) was
added sodium hydroxide (0.22 g, 5.50 mmol), and the resulting
mixture was stirred at 100 °C for 1 h. The reaction mixture was cooled
to room temperature, and one spatula tip of tris(2-carboxyethyl)-
phosphine was added. After the mixture was stirred for an additional
15 min, the solvents were removed in vacuo. The resulting residue was
acidified with aqueous 10% KHSO4 and extracted with ethyl acetate.
The organic layer was washed with brine, dried over MgSO4, and
concentrated. The resulting solid was recrystallized in EtOAc/hexanes
to give 0.11 g of 19a as a white solid (33% yield): mp 179−181 °C; 1H
NMR (DMSO-d6) δ 3.03 (t, J = 7.8 Hz, 1H), 3.81 (d, J = 7.8 Hz, 2H),
7.30−7.34 (m, 3H), 7.35−7.42 (m, 3H), 7.52 (m, 1H), 7.69 (d, J = 1.8
Hz, 1H), 12.77 (bs, 1H); 13C NMR (DMSO-d6) δ 27.01, 127.1, 127.2,
128.1, 128.26, 128.29, 128.8, 130.0, 130.5, 130.6, 139.2, 140.6, 140.9,
169.6. Anal. Calcd. for C14H12O2S: C, 68.83; H, 4.95; S, 13.12. Found:
C, 68.56; H, 5.16; S, 12.85.
4-(Mercaptomethyl)biphenyl-2,2′-dicarboxylic Acid (19b).
Compound 19b was prepared as described for the preparation of
19a ex cept methyl 5-(a cetylsulfa nylmethyl) -2-(2-
methoxycarbonylphenyl)benzoate 18b was used in place of 18a:
white powder (38% yield): mp 203−205 °C; 1H NMR (DMSO-d6) δ
3.03 (t, J = 8.1 Hz, 1H), 3.82 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 7.8 Hz,
2H), 7.13 (dd, J = 1.1, 7.6 Hz, 1H), 7.42 (dt, J = 1.5, 7.6 Hz, 1H), 7.51
(m, 2H), 7.86 (m, 2H), 12.46 (s, 2H); 13C NMR (DMSO-d6) δ 27.1,
126.9, 129.3, 129.5, 130.4, 130.4, 130.4, 130.5, 130.8, 131.0, 140.3,
141.4, 142.8, 167.7, 167.9. Anal. Calcd. for C15H12O4S·0.36EtOAc: C,
61.70; H, 4.69; S, 10.02. Found: C, 62.07; H, 4.54; S, 9.64.
Methyl 2-Acetoxy-5-(tritylthiomethyl)benzoate (23). To a
solution of 22 (5.52 g, 19.2 mmol) in acetone (30 mL) was added
potassium carbonate (4.0 g, 28.5 mmol) followed by triphenylmetha-
nethiol (5.76 g, 20.9 mmol). The mixture was stirred for 14 h at room
temperature. The excess potassium carbonate was removed by
filtration, and the filtrate was concentrated in vacuo. The resultant
residue was reconstituted in EtOAc (100 mL). The organic layer was
washed with 10% KHSO4 (75 mL). The aqueous phase was extracted
again with EtOAc (100 mL). The combined organic extracts were
dried over MgSO4, filtered, and concentrated in vacuo. The crude
material was purified by trituration with a mixture of MeOH and
1
EtOAc to give 4.07 g of 23 as a light yellow crystal (44% yield): H
NMR (CDCl3) δ 2.32 (s, 3H), 3.35 (s, 2H), 3.85 (s, 3H), 6.96 (d, J =
8.4 Hz, 1H), 7.21−7.32 (m, 10H), 7.43−7.46 (m, 6H), 7.72 (d, J = 2.3
Hz, 1H).
Methyl 2-Hydroxy-5-(tritylthiomethyl)benzoate (24). To a
solution of 23, (4.07 g, 8.4 mmol) in MeOH (80 mL) was added
sodium methoxide (684 mg, 12.7 mmol) portionwise at 0 °C. The
mixture was warmed to room temperature overnight. The reaction was
quenched with 10% KHSO4 (30 mL), and then the mixture was
concentrated to dryness. The residue was dissolved in EtOAc, and the
organic layer was washed with 10% KHSO4. The organic layer was
dried over MgSO4, filtered, and concentrated to give 3.62 g of 24
(100% crude yield). The crude material was used in the subsequent
1
step without further purification: H NMR (CDCl3) δ 3.27 (s, 2H),
3.94 (s, 3H), 6.86 (d, J = 8.5 Hz, 1H), 7.19−7.25 (m, 4H), 7.28−7.33
(m, 6H), 7.44−7.47 (m, 6H), 7.58 (d, J = 2.3 Hz, 1H), 10.67 (s, 1H).
Methyl 2-(Benzyloxy)-5-(tritylthiomethyl)benzoate (25a). To
a solution of 24 (0.500 g, 1.13 mmol) in acetone (10 mL) was added
potassium carbonate (0.188 g, 1.36 mmol) followed by benzyl
bromide (0.233 g, 1.36 mmol). The mixture was heated at reflux for 72
h. The excess potassium carbonate was removed by filtration. The
filtrate was concentrated in vacuo, and the residue was partitioned
4-(Mercaptomethyl)biphenyl-2,3′-dicarboxylic Acid (19c).
Compound 19c was prepared as described for the preparation of
19a except dimethyl 4-(acetylthiomethyl)biphenyl-2,3′-dicarboxylate
18c was used in place of 18a. A silica gel column chromatography
(dichloromethane/EtOAc, 9:1 with 1% acetic acid) was used instead
of recrystallization for the purification of the crude material: white
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Journal of Medicinal Chemistry
Article
between EtOAc (50 mL) and 10% KHSO4 (50 mL). The organic layer
was separated, and the aqueous layer was extracted with EtOAc (50
mL). The combined organic extracts were dried over MgSO4, filtered,
and concentrated. The crude material was purified by a silica gel
column chromatography (20% EtOAc in hexanes) to give 0.320 g of
25a as a white solid (53% yield): 1H NMR (DMSO-d6) δ 3.28 (s, 2H)
3.80 (s, 3H) 5.18 (s, 2H) 7.02−7.54 (m, 23H).
a silica gel column chromatography (50% EtOAc in hexanes) to give
0.094 g of 27a as a white solid (71% yield): mp 132−138; H NMR
(CDCl3) δ 1.79 (t, J = 7.8 Hz, 1H), 3.74 (d, J = 7.8 Hz, 2H), 5.30 (s,
2H), 7.10 (d, J = 8.6 Hz, 1H), 7.37−7.49 (m, 5H), 7.55 (dd, J = 8.59,
2.27 Hz, 1 H), 8.14 (d, J = 2.3 Hz, 1H). Anal. Calcd for
C15H14SO3·0.3H2O: C, 64.40; H, 5.26; S, 11.46. Found: C, 64.73;
H, 5.25; S, 11.25.
2-(2-Carboxybenzyloxy)-5-(mercaptomethyl)benzoic Acid
(27b). Compound 27b was prepared as described for the preparation
of 27a except 26b was used in place of 26a. The crude material was
purified by trituration with hot EtOAc to give 27b as a white solid
(75% yield): mp 196−203 °C; 1H NMR (DMSO-d6) δ 2.91 (t, J = 7.8
Hz, 1H), 3.72 (d, J = 7.8 Hz, 2H), 5.51 (s, 2H), 7.03 (d, J = 8.6 Hz,
1H), 7.46 (m, 2H), 7.61 (dt, J = 1.4, 7.6 Hz, 1H), 7.68 (d, J = 2.4 Hz,
1H), 7.90 (dd, J = 0.9, 7.8 Hz, 1H), 7.96 (dd, J = 1.3, 7.8 Hz, 1H),
12.93 (br s, 2H); 13C NMR (DMSO-d6) δ 26.8, 68.2, 113.6, 121.3,
127.2, 127.4, 128.4, 130.6, 130.7, 132.4, 132.9, 133.7, 138.8, 155.9,
167.2, 167.9. Anal. Calcd for C16H14O5S·1.7NaCl: C, 46.01; H, 3.38; S,
7.68. Found: C, 46.06; H, 3.37; S, 7.65.
1
Methyl 2-(2-(Methoxycarbonyl)benzyloxy)-5-
(tritylthiomethyl)benzoate (25b). Compound 25b was prepared
as described for the preparation of 25a except methyl 2-
(bromomethyl)benzoate was used in place of benzyl bromide: white
1
solid (66% yield); H NMR (CDCl3) δ 3.29 (s, 2H), 3.91 (s, 3H),
3.93 (s, 3H), 5.55 (s, 2H), 6.96 (d, J = 8.0 Hz, 1H), 7.18−7.25 (m,
4H), 7.28−7.32 (m, 6H), 7.38 (dt, J = 8.1, 1.6 Hz, 1H), 7.46 (m, 6H),
7.59−7.63 (m, 2H), 8.04 (dd, J = 8.0, 2.1 Hz, 2H).
Methyl 2-(3-(Methoxycarbonyl)benzyloxy)-5-
(tritylthiomethyl)benzoate (25c). Compound 25c was prepared
as described for the preparation of 25a except methyl 3-
(bromomethyl)benzoate was used in place of benzyl bromide: white
1
solid (70% yield); H NMR (CDCl3) δ 3.28 (s, 2H), 3.92 (s, 3H),
2-(3-Carboxybenzyloxy)-5-(mercaptomethyl)benzoic Acid
(27c). Compound 27c was prepared as described for the preparation
of 27a except 26c was used in place of 26a: white solid (17% yield);
3.93 (s, 3H), 5.17 (s, 2H), 6.87 (d, J = 8.0 Hz, 1H), 7.18−7.25 (m,
4H), 7.28−7.32 (m, 6H), 7.44−7.48 (m, 7H), 7.60 (d, J = 4.0 Hz,
1H), 7.70 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H).
Methyl 2-(4-(Methoxycarbonyl)benzyloxy)-5-
(tritylthiomethyl)benzoate (25d). Compound 25d was prepared
as described for the preparation of 25a except methyl 4-
(bromomethyl)benzoate was used in place of benzyl bromide: white
1
mp 215−217 °C; H NMR (DMSO-d6) δ 2.89 (t, J = 7.7 Hz, 1H),
3.71 (d, J = 7,6 Hz, 2H), 5.26 (s, 2H), 7.14 (d, J = 8.5 Hz, 1H), 7.44
(dd, J = 2.4, 8.6 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.64 (d, J = 2.3 Hz,
1H), 7.74 (d, J = 7.8 Hz, 1H), 7.88 (dt, J = 7.7, 1.3 Hz, 1H), 8.07 (s,
1H), 12.92 (br s, 2H); 13C NMR (DMSO-d6) δ 26.7, 69.2, 114.1,
121.8, 127.9, 128.6, 128.7, 130.5, 131.0, 131.4, 132.6, 133.8, 137.7,
155.7, 167.2 (2C). Anal. Calcd for C16H14S1O5·0.4H2O: C, 59.03; H,
4.58; S, 9.85. Found: C, 58.81; H, 4.21; S, 9.84.
1
solid (63% yield): H NMR (CDCl3) δ 3.29 (s, 2H), 3.92 (s, 3H),
3.94 (s, 3H) 5.17 (s, 2H), 6.87 (d, J = 8.5 Hz, 1H), 7.20−7.33 (m, 10
H), 7.44−7.47 (m, 7H), 7.60 (d, J = 2.3 Hz, 1H), 7.70 (dt, J = 7.7, 1.7
Hz, 1H), 7.98 (dt, J = 7.8, 1.2 Hz, 1H), 8.15 (s, 1H).
2-(4-Carboxybenzyloxy)-5-(mercaptomethyl)benzoic Acid
(27d). Compound 27d was prepared as described for the preparation
of 27a except 26d was used in place of 26a: white solid (69% yield);
2-(Benzyloxy)-5-(tritylthiomethyl)benzoic Acid (26a). To a
solution of 25a (0.300 g, 0.57 mmol) in dioxane (10 mL) and water
(10 mL) was added sodium hydroxide (0.090 g, 2.26 mmol). The
mixture was heated at reflux for 3 h. The reaction was quenched with
10% KHSO4, and then the mixture was evaporated to dryness. The
residue was dissolved in EtOAc (50 mL), and the organic layer was
washed with 10% KHSO4. The organic layer was washed with water
(50 mL), dried over MgSO4, filtered, and concentrated. The residue
was purified by a silica gel column chromatography (20% EtOAc in
hexanes) to give 0.26 g of 26a as a white solid (89% yield): 1H NMR
(DMSO-d6) δ 3.26 (s, 2H) 5.16 (s, 2H) 7.07 (d, J = 8.8 Hz, 1H)
7.14−7.23 (m, 1H) 7.15−7.43 (m, 19H) 7.43−7.58 (m, 2H).
2-(2-Carboxybenzyloxy)-5-(tritylthiomethyl)benzoic Acid
(26b). Compound 26b was prepared as described for the preparation
of 26a except 25b was used in place of 25a: white powder (100%
yield); 1H NMR (DMSO-d6) δ 3.26 (s, 2H), 5.49 (s, 2H), 6.97 (d, J =
8.6 Hz, 1H), 7.21−7.45 (m, 18H), 7.60 (dt, J = 1.4, 7.7 Hz, 1H), 7.87
(d, J = 8.0 Hz, 1H), 7.96 (dd, J = 1.1, 8.0 Hz, 1H), 12.88 (br s, 2H).
2-(3-Carboxybenzyloxy)-5-(tritylthiomethyl)benzoic Acid
(26c). Compound 26c was prepared as described for the preparation
of 26a except 25c was used in place of 25a: off-white solid (88%
yield); 1H NMR (DMSO-d6) δ 3.26 (s, 2H), 5.24 (s, 2H), 7.07 (d, J =
8.5 Hz, 1H), 7.19 (dd, J = 2.3, 8.7 Hz, 1H), 7.24−7.30 (m, 3H), 7.34−
7.39 (m, 13H), 7.51 (t, J = 7.7 Hz, 1H), 7.72 (dt, J = 7.8, 1.4 Hz, 1H),
7.87 (dt, J = 7.8, 1.4 Hz, 1H), 8.06 (s, 1H), 12.88 (br s, 2H).
2-(4-Carboxybenzyloxy)-5-(tritylthiomethyl)benzoic Acid
(26d). Compound 26d was prepared as described for the preparation
of 26a except 25d was used in place of 25a. Instead of column
chromatography, the crude material was recrystallized from hot
methanol: white powder (41% yield); 1H NMR (DMSO-d6) δ 3.26 (s,
2H), 5.25 (s, 2H), 7.06 (d, J = 8.6 Hz, 1H), 7.20 (dd, J = 2.4, 8.6 Hz,
1H), 7.27 (m, 3H), 7.37 (m, 12H), 7.40 (d, J = 2.4 Hz, 1H), 7.59 (d, J
= 8.5 Hz, 2H), 7.95 (d, J = 8.5 Hz, 2H), 12.87 (brs, 2H).
1
mp 181−193 °C; H NMR (DMSO-d6) δ 2.89 (t, J = 7.7 Hz, 1H),
3.72 (d, J = 7.5 Hz, 2H), 5.27 (s, 2H), 7.12 (d, J = 8.6 Hz, 1H), 7.43
(dd, J = 2.4, 8.6 Hz, 1H), 7.60 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 2.3 Hz,
1H), 7.95 (d, J = 8.4 Hz, 2H), 12.87 (br s, 2H); 13C NMR (DMSO-d6)
δ 26.8, 69.2, 114.2, 122.0, 127.1, 129.6, 130.4, 130.7, 132.8, 134.0,
142.4, 155.8, 167.2, 167.3. Anal. Calcd for C16H14SO5·0.3H2O: C,
59.36; H, 4.55; S, 9.90. Found: C, 59.45; H, 4.64; S, 9.66.
2,2-Dimethyl-5-vinyl-4H-benzo[d][1,3]dioxin-4-one (29). To
a solution of 2,2-dimethyl-4-oxo-4H-benzo[d][1,3]dioxin-5-yl trifluor-
omethanesulfonate 28 (16.3 g, 50 mmol) in 1,2-dimethoxyethane (200
mL) and water (50 mL) were added tetrakis(triphenylphosphine)
palladium (5.77 g, 5.00 mmol), potassium carbonate (13.9 g, 100
mmol), and vinylboronic anhydride pyridine complex (14.4 g, 60.0
mmol) under argon atmosphere. The reaction mixture was heated at
80 °C for 5 h. The reaction mixture was diluted with EtOAc (200 mL).
The organic layer was separated, washed with water (75 mL × 2),
dried over Na2SO4, filtered, and concentrated in vacuo. The crude
residue was purified by silica gel column chromatography (3% EtOAc
1
in hexanes) to afford 8.50 g of 29 as a yellow syrup (83% yield): H
NMR (CDCl3) δ 1.72 (s, 6H), 5.43 (d, J = 8.8 Hz, 1H), 5.71 (d, J =
13.6 Hz, 1H), 6.89 (d, J = 6.0 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.47
(t, J = 6.4 Hz, 1H), 7.73 (dd, J = 8.8, 13.6 Hz, 1H).
S-2-(2,2-Dimethyl-4-oxo-4H-benzo[d][1,3]dioxin-5-yl)ethyl
Ethanethioate (30). To a solution of 29 (23.0 g, 113 mmol) and
thioacetic acid (25.7 g, 338 mmol) in toluene (350 mL) was added
AIBN (18.5 g, 113 mmol) at 80 °C. After the mixture was stirred for 4
h, saturated aqueous NaHCO3 (100 mL) and EtOAc (300 mL) were
poured into the reaction mixture. The organic layer was washed with
saturated aqueous NaHCO3 (100 mL × 3), water, and brine, dried
over Na2SO4, and concentrated in vacuo. The crude material was
purified by silica gel column chromatography (1% EtOAc in hexanes)
1
2-(Benzyloxy)-5-(mercaptomethyl)benzoic Acid (27a). To a
solution of 26a (0.250 g, 0.48 mmol) in dichloromethane (2 mL) was
dropwise added triisopropylsilane (0.12 mL, 0.58 mmol) followed by
TFA (0.5 mL). The reaction mixture slowly turned colorless over the
next 0.5 h and was allowed to continue stirring for an additional 3 h.
The solvent was evaporated to dryness and the residue was purified by
to afford 17.5 g of 30 as a low melting solid (55% yield): H NMR
(CDCl3) δ 1.71 (s, 6H), 2.30 (s, 3H), 3.18 (t, J = 7.2 Hz, 2H), 3.34 (t,
J = 7.2 Hz, 2H), 6.86 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H),
7.43 (t, J = 8.0 Hz, 1H).
2-Hydroxy-6-(2-mercaptoethyl)benzoic Acid (31). To a
solution of 30 (10.0 g, 35.6 mmol) in a mixture of methanol and
5929
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water (200 mL, 1:1) were added sodium hydroxide (5.71 g, 143
mmol) and one spatula amount of tris(2-carboxyethyl)phosphine
(TCEP). The mixture was heated at 65 °C for 3.5 h. The solvents
were removed in vacuo. The resulting residue was dissolved in EtOAc
and acidified with aqueous 10% KHSO4. The organic phase was
washed with brine, dried over Na2SO4, and concentrated to give 6.55 g
of 31 as a brown oil (93% crude yield). The material was used in
4H), 3.93 (s, 3H), 7.28−7.31 (m, 2H), 7.34 (m, 1H), 7.36 (m, 1H),
7.49 (t, J = 7.6 Hz, 1H), 8.05 (m, 1H), 8.07 (m, 1H).
3-(2-Mercaptoethyl)biphenyl-2-carboxylic Acid (35a). To a
solution of 34a (0.111 g, 0.47 mmol) in a mixture of 1,4-dioxane and
water (5 mL, 1:1) was added 1 M NaOH (1 mL), and the resulting
mixture was stirred at 100 °C for 2 h. The mixture was subsequently
treated with a spatula of TCEP until complete disappearance of the
corresponding disulfide byproduct. The solvents were removed, and
the residual material was taken up in aqueous 10% KHSO4 and EtOAc.
The organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The resulting residue was triturated with 10%
EtOAc in hexanes to give 0.068 g of 35a as a white solid (56% yield):
mp 153−155 °C; 1H NMR (CDCl3) δ 1.47 (t, J = 8.1, 1H), 2.86 (m,
2H), 3.06 (m, 2H), 7.29 (m, 1H), 7.31 (m, 1H), 7.39−7.42 (m, 5H),
7.46 (t, J = 7.6 Hz, 1H). Anal. Calcd for C15H14O2S: C, 69.74; H, 5.46;
S, 12.41. Found: C, 69.87; H, 5.47; S, 12.23.
3-(2-Mercaptoethyl)biphenyl-2,2′-dicarboxylic Acid (35b).
Compound 35b was prepared as described for the preparation of
35a except 34b was used in place of 34a. The crude material was
purified by reverse phase preparative HPLC: white solid (61% yield);
mp 185−187 °C; 1H NMR (CDCl3) δ 1.43 (t, J = 8.1, 1H), 2.83 (m,
2H), 3.04 (m, 2H), 7.04 (dd, J = 1.3, 7.6 Hz, 1H), 7.28 (m, 1H), 7.40
(t, J = 7.7 Hz, 1H), 7.47 (m, 1H), 7.56 (m, 1H), 7.91 (dd, J = 1.3, 7.6
Hz, 1H). Anal. Calcd for C16H14O4S·0.2H2O: C, 62.81; H, 4.74; S,
10.48. Found: C, 62.60; H, 4.61; S, 10.53.
3-(2-Mercaptoethyl)biphenyl-2,3′-dicarboxylic Acid (35c).
Compound 35c was prepared as described for the preparation of
35a except 34c was used in place of 34a. The crude material was
purified by recrystallization from EtOAc/hexanes: white powder (68%
yield); mp 165−167 °C; 1H NMR (CDCl3) δ 1.52 (t, J = 8.1 Hz, 1H),
2.90 (m, 2H), 3.11 (m, 2H), 7.38 (m, 2H), 7.49−7.58 (m, 2H), 7.70
(m, 1H), 7.97 (m, 1H), 8.30 (s, 1H). Anal. Calcd for
C16H14O4S·0.25AcOH: C, 62.45; H, 4.76; S, 10.10. Found: C,
62.33; H, 4.85; S, 10.17.
3-(2-Mercaptoethyl)biphenyl-2,4′-dicarboxylic Acid (35d).
Compound 35d was prepared as described for the preparation of
35a except 34d was used in place of 34a. The crude material was
purified by reverse phase preparative HPLC: white powder (53%
yield); mp 245−246 °C; 1H NMR (DMSO-d6) δ 2.45 (t, J = 7.8, 1H),
2.73 (m, 2H), 2.93 (m, 2H), 7.29 (dd, J = 1.3, 7.6 Hz, 1H), 7.40 (dd, J
= 1.3, 7.6 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.49 (m, 1H), 7.52 (m,
1H), 7.97 (m, 1H), 7.99 (m, 1H), 13.04 (bs, 1H), 13.19 (bs, 1H).
Anal. Calcd for C16H14O4S: C, 63.56; H, 4.67; S, 10.61. Found: C,
63.28; H, 4.66; S, 10.38.
1
subsequent reaction as is without purification: H NMR (CDCl3) δ
1.45 (t, J = 8.0 Hz, 1H), 2.83 (t, J = 7.8 Hz, 2H), 3.30 (t, J = 7.8 Hz,
2H), 6.81 (dd, J = 1.0, 7.4 Hz, 1H), 6.93 (dd, J = 1.1, 8.4 Hz, 1H), 7.42
(dd, J = 7.6, 8.4 Hz, 1H), 10.49 (bs, 1H).
8-Hydroxyisothiochroman-1-one (32). A solution of 31 (6.51 g,
32.8 mmol) in toluene (200 mL) was heated at 110 °C for 24 h in the
presence of p-TsOH (1.70 g, 9.85 mmol). The reaction mixture was
concentrated, and the residue was taken up by EtOAc and saturated
aqueous NaHCO3. The organic layer was subsequently washed with
10% KHSO4 and brine, dried over Na2SO4, and concentrated in vacuo.
The resulting brown oil was purified by silica gel column
chromatography (20% EtOAc in hexanes) to provide 4.23 g of 32
as a brown semisolid (71% yield): 1H NMR (CDCl3) δ 3.23 (m, 2H),
3.27 (m, 2H), 6.69 (m, 1H), 6.88 (m, 1H), 7.33 (dd, J = 7.6, 8.6 Hz,
1H), 11.59 (s, 1H).
1-Oxoisothiochroman-8-yl Trifluoromethanesulfonate (33).
To a solution of 32 (3.02 g, 16.8 mmol) in dichloromethane (30 mL)
was added pyridine (3.00 mL, 36.9 mmol), followed by the addition of
1.0 M solution of trifluoromethanesulfonic anhydride in dichloro-
methane (18.4 mL, 18.4 mmol) at 0 °C. The reaction mixture was
stirred at 0 °C for 2.5 h, and an additional 0.3 equiv of 1.0 M of triflic
anhydride and 0.3 equiv of pyridine were added. After 1.5 h, more
triflic anhydride (1 mL) was added and stirring continued for 30 min.
The reaction was quenched with 1 N HCl. The organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo. The
crude material was recrystallized from EtOAc/hexanes to give 3.08 g of
33 as the first crop. The mother liquor was purified by silica gel
column chromatography (20−50% EtOAc in hexanes) to give 0.41 g
of 33 as the second crop: light brown solid (3.49 g, 67% combined
1
yield); H NMR (CD3OD) δ 3.29 (s, 4H), 7.27 (d, J = 8.1 Hz, 1H),
7.34 (d, J = 7.1 Hz, 1H), 7.56 (t, J = 7.9, 1H).
8-Phenylisothiochroman-1-one (34a). To a solution of 33
(0.500 g, 1.60 mmol) in toluene (10 mL) were added cesium
carbonate (1.04 g, 3.19 mmol), phenylboronic acid 15a (0.235 g, 1.92
mmol), and tetrakis(triphenylphosphine)palladium (0.092 g, 0.08
mmol). The mixture was heated at 110 °C for 2.5 h. After cooling, the
mixture was filtered through a pad of Celite and concentrated in vacuo.
Trituration of the residual solid with a mixture of EtOAc and hexanes
gave 0.220 g of 34a as a yellow solid (57% yield): 1H NMR (CDCl3) δ
3.28 (m, 4H), 7.25−7.30 (m, 4H), 7.34−7.41 (m, 3H), 7.47 (t, J = 7.6
Hz, 1H).
Methyl 2-(1-Oxoisothiochroman-8-yl)benzoate (34b). Com-
pound 34b was prepared as described for the preparation of 34a
except 2-(methoxycarbonyl)phenylboronic acid 15b was used in place
of 15a. The crude material was purified by Biotage Isolera One using
EtOAc/hexanes as eluent: viscous oil (51% yield); 1H NMR (CDCl3)
δ 3.27 (m, 4H), 3.68 (s, 3H), 7.13 (dd, J = 1.3, 7.6 Hz, 1H), 7.18 (m,
1H), 7.26 (m, 1H), 7.41 (m, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.53 (m,
1H), 8.00 (m, 1H).
Ethyl 3-(1-Oxoisothiochroman-8-yl)benzoate (34c). Com-
pound 34c was prepared as described for the preparation of 34a
except 3-(ethoxycarbonyl)phenylboronic acid was used in place of 15a.
The crude material was purified by Biotage Isolera One using EtOAc/
hexanes as eluent: yellow oil (88% yield); 1H NMR (CDCl3) δ 1.39 (t,
J = 7.1 Hz, 3H), 3.29 (m, 4H), 4.37 (q, J = 7.1 Hz, 2H), 7.28−7.30 (m,
2H), 7.44−7.46 (m, 2H), 7.49 (t, J = 7.6 Hz, 1H), 7.97 (m, 1H), 8.02
(m, 1H).
8-(Benzyloxy)isothiochroman-1-one (36a). To a solution of 32
(0.220 g, 1.22 mmol) in acetone (10 mL) were added benzyl bromide
(0.310 g, 1.83 mmol) and potassium carbonate (0.250 g, 1.83 mmol).
The mixture was heated at 55 °C overnight. Solids were removed by
filtration, and the filtrate was concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography (50% EtOAc
in hexanes, 1% acetic acid) to provide 0.100 g of 36a as an off-white
1
solid (31% yield): H NMR (CDCl3) δ 3.19 (s, 4H), 5.20 (s, 2H),
6.84 (d, J = 6.6 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 7.28−7.33 (m, 1H),
7.33−7.42 (m, 3H), 7.53 (d, J = 7.1 Hz, 2H).
Methyl 2-((1-Oxoisothiochroman-8-yloxy)methyl)benzoate
(36b). Compound 36b was prepared as described for the preparation
of 36a except methyl 2-(bromomethyl)benzoate was used in place of
benzyl bromide. The resulting residue was recrystallized from EtOAc/
1
hexanes: yellow crystals (40% yield); H NMR (CDCl3) δ 3.22 (s,
4H), 3.93 (s, 3H), 5.59 (s, 2H), 6.86 (d, J = 6.8 Hz, 1H), 7.12 (d, J =
8.3 Hz, 1H), 7.34−7.46 (m, 2H), 7.67 (t, J = 7.0 Hz, 1H), 8.05 (d, J =
6.8 Hz, 1H), 8.23 (d, J = 7.8 Hz, 1H).
Methyl 3-((1-Oxoisothiochroman-8-yloxy)methyl)benzoate
(36c). Compound 36c was prepared as described for the preparation
of 36a except methyl 3-(bromomethyl)benzoate was used in place of
Methyl 4-(1-Oxoisothiochroman-8-yl)benzoate (34d). Com-
pound 34d was prepared as described for the preparation of 34a
except 4-(methoxycarbonyl)phenylboronic acid 15d was used in place
of 15a. The compound was purified by recrystallization from EtOAc/
1
benzyl bromide: clear oil (61% yield); H NMR (CDCl3) δ 3.20 (s,
3H), 3.93 (s, 4 H), 5.22 (s, 2H), 6.86 (d, J = 7.6 Hz, 1H), 6.97 (d, J =
8.6 Hz, 1H), 7.33−7.41 (m, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.87 (d, J =
7.6 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 8.10 (s, 1H).
1
hexanes: off-white solid (49% yield); H NMR (CDCl3) δ 3.30 (m,
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Methyl 4-((1-Oxoisothiochroman-8-yloxy)methyl)benzoate
(36d). Compound 36d was prepared as described for the preparation
of 36a except methyl 4-(bromomethyl)benzoate was used in place of
benzyl bromide. The crude material was recrystallized from EtOAc/
hexanes: beige powder (39% yield); 1H NMR (CDCl3) δ 3.21 (s, 3H),
3.92 (s, 4H), 5.24 (s, 2H), 6.87 (d, J = 7.1 Hz, 1H), 6.96 (d, J = 8.3
Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H), 8.07 (d, J =
8.3 Hz, 2H).
ACKNOWLEDGMENTS
■
We thank Elizabeth Travis, an American Chemical Society
Project SEED student, for her technical assistance in the
synthesis of GCPII inhibitors. This work was in part supported
by the Johns Hopkins Brain Science Institute through its
NeuroTranslational Drug Discovery program.
2-(Benzyloxy)-6-(2-mercaptoethyl)benzoic Acid (37a). To a
solution of 36a (0.090 g, 0.34 mmol) in H2O/dioxane (1.0 mL, 1:1)
was added sodium hydroxide (0.040 g, 1.01 mmol). The mixture was
heated at 100 °C for 2.5 h. A spatula of TCEP was added, and the
mixture was stirred for 15 min at room temperature. Solvents were
removed in vacuo, and the residual material was taken up in aqueous
10% KHSO4 and EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated. The resulting residue was
purified by preparative HPLC to give 0.024 g of 37a as white crystals
ABBREVIATIONS USED
■
GCPII, glutamate carboxypeptidase II; NAAG, N-acetylaspar-
tylglutamate; 2-MPPA, 2-(3-mercaptopropyl)pentanedioic acid;
ALS, amyotrophic lateral sclerosis; CMBA, 3-(2-carboxy-5-
mercaptopentyl)benzoic acid; AUC, area under the curve
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1
(25% yield): mp 54−56 °C; H NMR (CDCl3) δ 1.45 (t, J = 8.2 Hz,
1H), 2.82 (q, J = 7.6, 15.2 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 5.19 (s,
2H), 6.95 (d, J = 8.6 Hz, 2H), 7.33−7.43 (m, 6H). Anal. Calcd for
C16H16O3S·0.2H2O: C, 65.82; H, 5.66; S, 10.98. Found: C, 65.94; H,
5.57; S, 10.70.
2-(2-Carboxybenzyloxy)-6-(2-mercaptoethyl)benzoic Acid
(37b). Compound 37b was prepared as described for the preparation
of 37a except 36b was used in place of 36a: white powder (13% yield);
mp 171−173 °C; 1H NMR (CD3OD) δ 2.75 (m, 2H), 2.93 (m, 2H),
5.55 (s, 2H), 6.93 (dd, J = 2.5, 7.6 Hz, 2H), 7.30 (t, J = 8.3 Hz, 1H),
7.40 (t, J = 7.8 Hz, 1H), 7.57 (dt, J = 1.3, 7.8 Hz, 1H), 7.82 (d, J = 7.8
Hz, 1H), 8.06 (dd, J = 1.0, 7.8 Hz, 1H). Anal. Calcd for
C17H16O5S·0.2H2O: C, 60.77; H, 4.92; S, 9.54. Found: C, 60.86; H,
4.85; S, 9.32.
2-(3-Carboxybenzyloxy)-6-(2-mercaptoethyl)benzoic Acid
(37c). Compound 37c was prepared as described for the preparation
of 37a except 36c was used in place of 36a. The crude material was
recrystallized from EtOAc/hexanes: yellow powder (61% yield); mp
142−147 °C; 1H NMR (CD3OD) δ 2.74 (t, J = 7.6 Hz, 2H), 2.90 (dd,
J = 5.6, 8.3 Hz, 2H), 5.19 (s, 2H), 6.93 (d, J = 7.6 Hz, 1H), 7.01 (d, J =
8.1 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.71 (d,
J = 8.1 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 8.12 (s, 1H). Anal. Calcd for
C17H16O5S: C, 61.43; H, 4.85; S, 9.65. Found: C, 61.31; H, 4.86; S,
9.36.
2-(4-Carboxybenzyloxy)-6-(2-mercaptoethyl)benzoic Acid
(37d). Compound 37d was prepared as described for the preparation
of 37a except 36d was used in place of 36a. The crude material was
recrystallized from EtOAc/dichloromethane: off-white powder (13%
1
yield); mp 193−196 °C; H NMR (CD3OD) δ 2.75 (t, J = 8.2 Hz,
2H), 2.92 (t, J = 7.7 Hz, 2H), 5.23 (s, 2H), 6.93 (d, J = 7.6 Hz, 1H),
6.98 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.57 (d, J = 7.6 Hz,
2H), 8.03 (d, J = 8.1 Hz, 2H). Anal. Calcd for C17H16O5S·0.4H2O: C,
60.13; H, 4.99; S, 9.44. Found: C, 60.03; H, 4.80; S, 9.16.
Pharmacological Studies. The GCPII assay was carried out as
outlined previously.21 The Ames test was performed by Covance
Laboratories, Vienna, VA. The hERG assay was conducted by Zenas
Technologies, New Orleans, LA. Receptor binding assays were
performed by MDS Pharma Services, Bothell, WA. Details of the
pharmacokinetics study will be reported elsewhere. The chronic
constrictive injury models were performed following the procedure
reported by Bennett’s group23 and will be detailed elsewhere.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (410) 614-0982. Fax: (410) 614-0659. E-mail:
ttsukamoto@jhmi.edu.
Notes
The authors declare no competing financial interest.
The Johns Hopkins University has a collaborative agreement
with Eisai Inc. on the discovery of new GCPII inhibitors.
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