α-Bromoacetophenone derivatives as neutral protein tyrosine phosphatase inhibitors: Structure-activity relationship

Article abstract of DOI:10.1016/S0960-894X(02)00681-9

A series of α-haloacetophenone derivatives was tested for inhibition of protein tyrosine phosphatases SHP-1 and PTP1B. The results show that the bromides are much more potent than the corresponding chlorides, whereas the phenyl ring is remarkably tolerant to modifications. Derivatization of the phenyl ring with a tripeptide Gly-Glu-Glu resulted in a potent, selective inhibitor against PTP1B.

Full text of DOI:10.1016/S0960-894X(02)00681-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3047–3050
ꢀ-Bromoacetophenone Derivatives as Neutral Protein Tyrosine
Phosphatase Inhibitors: Structure–Activity Relationship
Gulnur Arabaci, Tian Yi, Hua Fu, Mary E. Porter, Kirk D. Beebe and Dehua Pei*
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue,
Columbus, OH 43210, USA
Received 19 July 2002; accepted 6 August 2002
Abstract—A series of a-haloacetophenone derivatives was tested for inhibition of protein tyrosine phosphatases SHP-1 and PTP1B.
The results show that the bromides are much more potent than the corresponding chlorides, whereas the phenyl ring is remarkably
tolerant to modifications. Derivatization of the phenyl ring with a tripeptide Gly-Glu-Glu resulted in a potent, selective inhibitor
against PTP1B.
# 2002 Elsevier Science Ltd. All rights reserved.
Protein tyrosine phosphatases (PTPs) are a diverse
family of enzymes that catalyze the hydrolysis of phos-
photyrosine (pY) residues in proteins. Together with
protein tyrosine kinases, they control the level of intra-
cellular tyrosine phosphorylation and regulate a variety
of cellular functions.1 Malfunction of PTPs can lead to
human diseases and conditions.2 Therefore, inhibitors
against PTPs provide potential therapeutic agents as
well as useful probes for studying their physiological
functions. For example, specific inhibitors of PTP1B are
expected to provide effective treatment for type II dia-
betes.3
none core and report herein the initial findings of this
study.
Figure 2 shows the compound series used in this study.
Compounds 1, 6, and 13 were synthesized as previously
described.8 Compounds 9, 15, and 16 were prepared
from the corresponding methyl ketones by treatment
with Br2 in acetic acid. Compound 14 was obtained by
treating 2-acetylpyridine with pyridine hydrobromide
perbromide in acetic acid.9 Compound 20 was prepared
from 2-biphenylcarboxylic acid (22), which was first
protected as the methyl ester and then acetylated by
Friedel–Crafts reaction to give ketone 23 (Scheme 1).
Hydrolysis of the methyl ester by NaOH followed by
bromination with Br2 resulted in acid 20, which was
further converted into its N-hydroxysuccinimide ester
24 using dicyclohexylcarbodiimide (DCC). Treatment of
a support-bound tripeptide Gly-Glu-Glu (on Wang
resin) with 24 followed by cleavage with trifluoroacetic
acid afforded compound 21. Peptidyl derivatives 18 and
19 were similarly prepared from acid 6. The rest of
compounds were obtained from commercial suppliers.
Virtually all of the reported PTP inhibitors contain a
negatively charged, nonhydrolyzable pY mimetic such
as phosphonates,4 malonates,5 aryl carboxylates,6 or
cinnamates7 as the core structure. The poor membrane
permeability of these inhibitors may compromise their
potential for further development. We reported pre-
viously8 that several a-bromoacetophenone derivatives
act as fairly potent PTP inhibitors, by covalently alky-
lating the conserved catalytic cysteine in the PTP active
site (Fig. 1). Because they are neutral, these agents
readily diffuse into human B cells and inhibit the intra-
cellular PTPs. In an attempt to improve both the
potency and selectivity of these molecules as PTP inhib-
itors, we undertook an SAR study of the haloacetophe-
The SAR study was performed with the catalytic
domain of phosphatase SHP-1, SHP-1(ÁSH2).10 All of
the compounds exhibited time-dependent inactivation
of SHP-1, consistent with the mechanism in Figure 1.
Their potency is described by an equilibrium constant
KI, which represents the dissociation constant of the
noncovalent enzyme–inhibitor complex (EÃI), and the
first-order rate constant (kinact) for conversion of the EÃI
*Corresponding author. Tel.:+1-614-688-4068; fax:+1-614-292-1532;
e-mail: pei@chemistry.ohio-state.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00681-9

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G. Arabaci et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3047–3050
complex into the covalent adduct, E–I. The KI and kinact
values were determined using p-nitrophenyl phosphate
as substrate as previously described.8
We noted previously8 that a-bromoacetophenone 6 is
13-fold more potent than the corresponding chloride 1
against the catalytic domain of SHP-1 (Table 1). To
determine whether this is a general trend, we tested four
other a-chloroacetophenones (2–5) containing various
substituents on the phenyl ring and the corresponding
bromides (6–8). The overall potency of the bromides
(kinact/KI) are consistently 13- to 20-fold more potent
than the corresponding chlorides. This difference is pri-
marily due to the higher affinity of the bromides to the
PTP active site (lower KI values), whereas the kinact
values are remarkably similar for all of the compounds.
We speculate that the larger a-bromoacetyl group is
perhaps a closer match (in size) to the phosphate group.
Figure 1. Mechanism of inhibition of PTPs by a-haloacetophenone
derivatives. X=Br or Cl.
We next examined the effect of phenyl substitution on
inhibitor potency. From the comparison between com-
pounds 6–16 and a-bromoacetic acid (17), it is clear that
an aromatic ring is essential for high-affinity binding to
the PTP active site (Table 2). However, the exact nature
of the aromatic ring is less critical. Attachment of either
electron-withdrawing (8–10) or electron-donating
groups (6 and 11–13) to the para position causes only
small changes (<6-fold) in the overall potency (kinact
/
KI). Even the addition of a fused ring (15 and 16) or
substitution of a ring nitrogen (14) has little effect. The
only exception is substitution at the ortho position,
which substantially decreases the inhibitor potency as a
Table 1. Comparison of a-bromo and chloro derivatives
Compd
KI, (mMa)
kinact, (minÀ1 a
)
kinact/KI, (MÀ1minÀ1
)
1
2
3
4
5
6
7
8
2500Æ600b
480Æ25
1.8Æ0.4b
0.21Æ0.04
0.22Æ0.07
2.1Æ0.1
710b
430
580
120
380Æ48
17,500Æ2300
1400Æ990
193Æ38b
81Æ2
0.59Æ0.24
1.8Æ0.3b
0.58Æ0.02
0.51Æ0.03
420
9300b
7100
9100
Figure 2. Structures of PTP inhibitors used in this work.
56Æ1
aValues are meansÆSD from three independent sets of experiments.
bData from ref 8.
Table 2. Effect of substitution on the phenyl ring
Compd
KI, (mM)a
kinact, (minÀ1 a
)
kinact/KI, (MÀ1minÀ1
)
6
7
8
9
10
11
12
13
14
15
16
17
193Æ38b
81Æ2
1.8Æ0.3b
0.58Æ0.02
0.51Æ0.03
0.37Æ0.01
2.9Æ1.5
9300b
7100
9100
26,000
14,800
41,000
18,000b
9300b
7500
56Æ1
14Æ1
195Æ150
14Æ8
0.59Æ0.16
2.4Æ0.2b
0.40Æ0.10b
1.3Æ0.2
128Æ10b
43Æ10b
173Æ19
25Æ9
0.48Æ0.04
0.86Æ0.03
1.4Æ0.2b
20,000
9400
18b
92Æ4
77,000Æ14,000b
Scheme 1. (i) MeOH/H2SO4; (ii) CH3COCl, AlCl3, 84% (2 steps); (iii)
NaOH/H2O; (iv) Br2/CHCl3, 75% (2 steps); (v) NHS, DCC, 91%; (vi)
H2N-GEE-resin; (vii) 9:1 TFA/H2O.
aValues are meansÆSD from three independent sets of experiments.
bData from ref 8.

G. Arabaci et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3047–3050
3049
result of reduced binding affinity (compare compounds
3 and 4 in Table 1). The reason behind the reduced
potency is not entirely clear but does not appear to be
steric in origin, since a fluorine atom is not substantially
larger than a hydrogen atom.
The data in Table 2 suggest that it may be difficult to
further improve the inhibitor potency by modifying the
structure of the phenyl ring. We thus decided to assess
the possibility of appending additional binding domains
to the para (or meta) position of the phenyl ring. Our
rationale is that the additional binding domain would
interact with the substrate-binding surfaces near the
active site. These interactions should increase both the
binding affinity and selectivity of the inhibitor. As a
proof of principle, we attached a tripeptide, Gly-Glu-
Glu, to the carboxyl group of inhibitor 6 to produce the
peptidyl bromoacetophenone 19 (Fig. 2). This tripep-
tide, when attached to the para position of cinnamic
acid, results in a highly potent inhibitor against PTP1B
(KI=79 nM).7a As expected, compound 19 is a highly
potent inactivator of PTP1B, with a KI of 2.8 mM, a
kinact of 1.2 minÀ1, and an overall inhibition constant
(kinact/KI) of 4.3Â105 MÀ1 minÀ1 (Table 3). The corre-
sponding values of inhibitor 13 against PTP1B are 42
mM, 0.57 minÀ1, and 1.4Â104 MÀ1 minÀ1, respectively.8
Compound 19 also shows drastically improved potency
(25-fold) against SHP-1, when compared to the parent
molecule (6). In contrast, attachment of a positively
charged tripeptide Gly-Arg-Arg to inhibitor 6 decreased
the overall potency by 1.5-fold (compared 6 and 20 in
Table 3). It is known that PTP1B and SHP-1 prefer
substrates that contain acidic residues N-terminal to the
pY and disfavor pY peptide containing positively
charged residues at these positions.11
Figure 3. Comparison of the inactivation kinetics of PTP1B and SHP-
1 by 21. The apparent inactivation rate kobs was determined as descri-
bed.8 The lines represent the best fits to the data according to equa-
ꢀ
tion: kobs=kinact [I]/(KI + [I]).
PTPs. While perturbation of the electronic properties of
the phenyl ring does not significantly improve their
overall potency against PTPs, attachment of a proper
peptidyl moiety to the para position can substantially
improve both the potency and the selectivity toward a
given PTP. It should be possible to develop membrane
permeable and metabolically stable inhibitors by
attaching peptidomimetics or small molecules to the
para position of a-bromoacetophenone. Furthermore,
since the covalent PTP inhibitor complex can be photo-
lytically cleaved to regenerate the PTP activity,8 these
molecules may provide a novel class of photolytic
switches for controlling cellular signaling processes.
Acknowledgements
To determine whether it is possible to generate inhibi-
tors with selectivity toward a particular PTP, we
attached Gly-Glu-Glu to acid 18 via a rigid biphenyl
linker and tested the resulting compound (21 in Fig. 2)
against both PTP1B and SHP-1. While inhibitor 21 has
a KI value of 9.9 mM and a kinact/KI value of 3.6Â104
MÀ1 minÀ1 against PTP1B, it does not show saturation
at 200 mM and has a kinact/KI value of 666 MÀ1 minÀ1
against SHP-1 (Fig. 3 and Table 3). This represents a
54-fold selectivity toward PTP1B.
This work was supported by National Institutes of
Health (AI 40575).
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Table 3. Effect of derivatization with peptides
Compd
KI, (mM)a
kinact, (minÀ1 a
)
kinact/KI, (MÀ1minÀ1
)
6
18
193Æ38b
220Æ64
29Æ4
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36,000
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