The development of potent non-peptidic PTP-1B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.11.048

The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of non-insulin dependent diabetes mellitus (NIDDM).

Full text of DOI:10.1016/j.bmcl.2003.11.048

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1039–1042
The development of potent non-peptidic PTP-1B inhibitors
Claude Dufresne,a,* Patrick Roy,a Zhaoyin Wang,a Ernest Asante-Appiah,b
Wanda Cromlish,b Yves Boie,b Farnaz Forghani,b Sylvie Desmarais,b Qingping Wang,b
Kathryn Skorey,b Deena Waddleton,b Chidambaram Ramachandran,b
Brian P. Kennedy,b Lijing Xu,c Robert Gordon,c Chi Chung Chanc and Yves Leblanca,*
aDepartment of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, PO Box 1005, Pointe-Claire,
Dorval, Canada H9R 4P8
bDepartment of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, PO Box 1005,
Pointe-Claire, Dorval, Canada H9R 4P8
cDepartment of Pharmacology, Merck Frosst Centre for Therapeutic Research, PO Box 1005, Pointe-Claire,
Dorval, Canada H9R 4P8
Received 16 May 2003; accepted 14 November 2003
Abstract—The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The
introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency
compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of
non-insulin dependent diabetes mellitus (NIDDM).
# 2004 Elsevier Ltd. All rights reserved.
When insulin binds to the insulin receptor (IR), the
intrinsic tyrosine kinase activity of the b-subunit
becomes activated. This results in autophosphorylation
of critical tyrosine residues in the regulatory domain
which fully activates the tyrosine kinase of the receptor.
The insulin receptor (IR) is then capable of phosphoryl-
ating its various substrates to propagate the insulin sig-
nal.1 For the past 10 years since the identification of the
protein tyrosine phosphatases (PTPs), it has been
speculated that a specific PTP is involved in the
dephosphorylation and inactivation of the insulin
receptor, thus attenuating insulin signaling.2 It has also
been hypothesized that disequilibrium between the
insulin receptor and the PTP could be a contributing
factor to the insulin resistance observed in type 2 or
non-insulin dependent diabetes mellitus (NIDDM).3
Inhibition of the PTP specific for the insulin receptor
would be beneficial in the treatment of this disease as it
would result in maintenance of an activated insulin
receptor and hence prolong insulin signaling.
PTP-1B is an intracellular PTP that has been implicated
in the negative regulation of insulin signaling. Recently,
we and others have shown that mice lacking PTP-1B
have enhanced insulin sensitivity.4,5 These mice require
only half the amount of insulin to maintain normal
blood glucose in the fed state. They also show increased
sensitivity in glucose and insulin tolerance tests. In
addition, animal models treated with the specific PTP-
1B anti-sense oligonucleotide ISIS 113715 show nor-
malization of both blood glucose and insulin levels.6
Thus, inhibitors of PTP-1B may enhance insulin sensi-
tivity, making them ideal for the treatment of type 2
diabetes.
Heretofore, a few research groups have shown interest
in developing PTP-1B inhibitors.7À16 PTPs have a high
affinity for phosphotyrosine. As for PTP-1B inhibitors,
there was an interest to develop compounds containing
non-hydrolyzable phosphate mimetics. Burke et al. has
shown that difluorophosphonomethyl-phenylalanine or
(F2PMP) is a mimetic of phosphotyrosine with similar
binding affinity.17 Our research group has embarked on
the synthesis of peptide libraries leading to the discovery
of peptide I.
* Corresponding author. Tel.: +1-514-428-3037; fax: +1-514-428-
4900; e-mail: claude_1_dufresne@merck.com
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.048

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C. Dufresne et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1039–1042
Table 1. In vitro potency data18À21 for 1–8
The X-ray crystal structure of peptide I in complex with
PTP-1B shows that the C-terminal (F2PMP) is located
in the active site while the second phosphonic group
interacts with R 47.20 Also, the SAR results from these
peptide libraries has revealed that monophosphonate-
containing peptide inhibitors can be equipotent to
bisphosphonate inhibitors.21a
Entry
R1
R2
X
(IC50 mM)
FDP assay
PTP-1B CD-45 Sf9 (PTP-1B)
1
2
3
4
5
6
7
8
CO2Bn CO2Me CF2PO3H2 0.19 >50.0
CO2Bn CO2Bn CF2PO3H2 0.06 19.0
CO2iPr CO2iPr CF2PO3H2 0.11 >50.0
CO2Me CO2H CF2PO3H2 0.39 >50.0
CH2OBn CH2OBn CF2PO3H2 0.16 >50.0
CO2Bn CO2Bn CF2CO2H 0.04 >50.0
1.00
0.54
1.65
3.80
1.30
1.90
1.70
7.20
Surprisingly, even though these peptidic inhibitors are
highly charged molecules they also show cellular activ-
ity. For example the tripeptide I has an IC50 of 200 nM
in an Sf9 cell-based assay.21b
CO2Bn CO2Bn SO2NHAc 0.09 25.0
CO2Bn CO2Bn SO2Me 0.94 >50.0
With cell permeable peptide inhibitors in hand, we then
decided to investigate the in vivo efficacy of peptide I in
animal models. Despite its short half-life (<30 min), I
was found to reduce the AUC in ob/ob mice by 40% in
the oral glucose tolerance test22 following a single IP
dose (10 mg/kg, 30 min pre-treatment). This result was
consistent with the observation made in the knockout
(KO) mice and established the proof of concept for the
hypothesis that PTP-1B inhibitors will prolong insulin
signaling. At this point our efforts became focused on
the design of low molecular weight, non-peptidic inhi-
bitors based on the peptide SAR and structural infor-
mation from the enzyme–peptide I complex.20 Malonate
esters (1) was chosen to be the first template to replace
the peptide backbone. Alkylation with a benzyl bromide
such as (2) and (3) was achieved as demonstrated in
Scheme 1, to provide (4).
nM in FDP assay18À21 and an IC50 of 1.9 mM in Sf9 cell-
based assay thereby supporting our previous observation
in the peptide series that monophosphonate inhibitors
can be equipotent to bis-phosphonate inhibitors. Unfor-
tunately, the malonate esters were predicted to be
unstable to blood esterases presumably due to hydrolysis
of the ester group resulting in the poor pharmacokinetics
in rats when dosed PO or IV. Thus, we have turned our
attention to deoxybenzoin as a possible template to
introduce the phosphonate warhead (Fig. 1). Deoxy-
benzoin (5) (Scheme 2) was deprotonated with sodium
hydride or potassium tert-butoxide and alkylated with
benzyl bromide (6) and (7) giving the tetrapode (8). This
This approach has produced potent, selective (except
for TC-PTP)19 and reversible PTP-1B inhibitors as
exemplified in Table 1 with entry 1–8. The most potent
bisphosphonate inhibitors are entry 2 and entry 3 based
on their potency in the FDP assay and the Sf9 cell-based
assay.21b Of the monophosphonates, entry 6 was identi-
fied to be the most potent inhibitor with an IC50 of 40
Figure 1.
Scheme 1.
Scheme 2.

C. Dufresne et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1039–1042
1041
series has provided potent inhibitors as shown in Table
2, such as entry 9. Entry 9 has an IC50 of 60 nM in the
FDP assay and 0.58 nM in the Sf9 cell based assay. As
found for peptide I, a statistically significant glucose
lowering effect was attained in animals. A 43% AUC
reduction at 10 mg/kg I.P. dosing in ob/ob mice and a
63% AUC reduction at 20 mg/kg I.P. dosing in fa/fa rat
models (Table 3)22,23 was observed.
The next step was to develop orally bioavailable inhibi-
tors. We decided to further optimize the deoxybenzoin
series. The monophosphonic analogue of entry 9, entry
13 was found to have good bioavailability in rats. This
compound, however, was found to be poorly active in
the enzyme assay with an IC50 of 2 mM (Table 2). We
had observed that the introduction of a halogen ortho to
the difluoromethylphosphonate group had the ability to
increase inhibitor potency. For example entry 15 is
more potent than the desbromo analogue entry 13 by a
factor of 20. The tripode 15 containing the ortho bromo
warhead was prepared using Scheme 2 with deoxy-
benzoin (5) and benzyl intermediate (12) (Scheme 3)
Scheme 3.
which in turn was prepared from 2-bromo-4-methyl
benzoic acid (9). The benzoic acid (9) was transformed
in 3 steps to benzaldehyde (10). Reaction of 10 with
the lithium salt of diethyl phosphite followed by a
Swern oxidation gave ketophosphonate (11). The last
step required the reaction of 11 with DAST to give
diethyl[2-bromo-4-(bromomethyl) phenyl] (difluoro)
methylphosphonate (12).
Table 2. In vitro potency data18À21 for 9–16
The ortho bromo analogue entry 15 was found to be
orally bioavailable (F=13%) in rats with a Cmax of 35
mM. In the oral glucose tolerance test (OGTT) in fa/fa
Zucker rats, a reduction of AUC by 50% was observed
after a single PO dose of 30 mg/kg. A similar effect was
observed in diet induced obesity mice (DIO mice) where
the AUC decreased by 50% at a dose of 10 mg/kg
(Table 3).
Entry
X
Y(IC
mM)
50
FDP assay
PTP-1B CD-45 Sf9 (PTP-1B)
In summary, we have reported the design and the
development of a series of deoxybenzoin PTP-1B inhi-
bitors. The introduction of a bromine atom ortho to the
warhead has allowed the preparation of potent analo-
gues such as entry 15. This inhibitor contains a single
phosphonate group and showed an increase of potency
by a factor of 20 compared to the desbromo analogue.
This PTP-1B inhibitor 15 is both bioavailable and active
in the animal models of NIDDM. The present work has
served as a platform to elaborate very potent PTP-1B
inhibitors using structure-based design.24
9
H
H
H
H
H
Cl
Br
Ph
p-CF2PO3H2benzyl
p-CF2PO3H2benzyl
p-CO2Hbenzyl
Cinnamyl
0.06
0.19
0.80
0.19
2.00
0.36
0.12
>50.0
>50.0
>50.0
0.58
1.50
6.63
2.00
10
11
12
13
14
15
16
5.50
H
H
H
H
>50.0
>50.0
>50.0
>50.0
16.0
2.40
1.22
NA
16.0
Table 3. In vivo efficacy data22,23 for 9 and 15
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