Synthesis of a novel peptidic photoaffinity probe for the PTP-1B enzyme

Article abstract of DOI:10.1016/j.bmcl.2004.01.101

The synthesis of a novel radioactive peptidic photoaffinity probe for the PTP-1B enzyme as well as some SAR leading to the choice of this compound as a photoaffinity probe are presented.

Full text of DOI:10.1016/j.bmcl.2004.01.101

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2319–2322
Synthesis of a novel peptidic photoaffinity probe for the PTP-1B
enzyme
a,
b
a
a
a
*
ꢀ
Michel Therien, Kathryn Skorey, Robert Zamboni, Chun Sing Li, Cheuk K. Lau,
Tammy LeRiche,a Vouy Linh Truong,a Deena Waddletonb and
Chidambaram Ramachandranb
aDepartment of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, PO Box 1005, Quebec, Pointe Claire––Dorval,
Canada H9R 4P8
bDepartment of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, PO Box 1005, Quebec,
Pointe Claire––Dorval, Canada H9R 4P8
Received 5 January 2004; revised 28 January 2004; accepted 29 January 2004
Abstract—The synthesis of a novel radioactive peptidic photoaffinity probe for the PTP-1B enzyme as well as some SAR leading to
the choice of this compound as a photoaffinity probe are presented.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Upon binding of insulin to its receptor (IR), the latter is
activated and its regulatory domain tyrosine residues are
phosphorylated, resulting in the insulin signaling path-
way.1 The protein tyrosine phosphatase (PTPase)
enzymes are known to be responsible for the dephos-
phorylation of tyrosine residues.1 In particular, and of
significant interest to us, the PTP-1B enzyme has been
implicated in negative regulation of the insulin signaling
pathway by dephosphorylating the IR.2–4 It is believed
that this negative regulation of insulin signaling could be
a contributing factor to the insulin resistance observed
in type 2 diabetes, noninsulin dependent diabetes mell-
itus.5 Furthermore, studies with PTP-1B knock-out mice
have demonstrated that they are resistant to diet-in-
duced obesity and possess increased insulin sensitivity.6
These results prompted us to pursue the preparation of a
specific PTP-1B inhibitor as potential treatment for type
2 diabetes.
PTP-1B7 and decided to prepare analogs of those pep-
tides, which would contain a phenyl group substituted
with a trifluoromethyldiazirine8 unit as the photoactive
group and an iodo as a potential radioactive group,9 as
shown in Figure 1.
The phenyl trifluoromethyldiazirine unit was prepared
as described in the literature.8;9 Where methyl 3,5-di-
iodobenzoate or methyl 3-bromo-5-iodobenzoate is used
as starting material, at the benzyl alcohol stage either a
bromination or an oxidation is performed to obtain the
desired benzyl bromide or benzoic acid. For consider-
ations of practicality, some compounds were originally
prepared as the bromo substituted trifluoromethyldiaz-
irine instead of the iodo, assuming that the SAR would
be similar between the two compounds and that any
potent compound could be synthesized as the iodo
analog in order to prepare a radioactive version.
We felt that in order to better characterize the action of
our specific PTP-1B inhibitors, such as their ability to
penetrate the cell, the preparation of a radiolabeled
photoaffinity probe would be quite useful. We had
already prepared some very potent peptidic inhibitors of
The chiral o-bromo difluoromethylphosphonophenyl-
alanine (o-BrF2PMP) was prepared as illustrated in
R
CF3
X
N
N
X = Br, I
R = CH2Br,COOH
Keywords: Photoaffinity probe; Protein tyrosine phosphatase PTP-1B.
* Corresponding author. Tel.: +1-5144283082; fax: +1-5144284939;
e-mail: michel_therien@merck.com
Figure 1.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.101

2320
M. Thꢀerien et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2319–2322
1) LiN(TMS)2/THF/-78oC
Br
TentaGel sRam from RAPP
F
F
X
F
F
OEt
OEt
OH
N
O
P
O
OEt
OEt
P
O
Br
F
F
2)
OtBu
20% piperidine
DMF,45 min.
OEt
OEt
OH
P
O
OtBu
N
NH2
O
OH
FMOCNH
Br
F
TentaGel sRam
O
Br
Br
F
F
F
HATU, DIEA
DMF
OEt
OEt
OEt
OEt
P
O
P
O
90% TFA
HCl/THF
X= H, Br
OH
X
OH
OH
F
F
H2N
N
X
F
F
OEt
OEt
O
O
OEt
OEt
HCl
P
O
P
O
20% piperidine
DMF,45 min.
F
F
Br
F
O
F
FMOCNH
FMOCNH
HN
X
F
OEt
OEt
HN
F
H2N
O
P
O
O
O
F
O
F
F
X
OH
FMOCNH
F
F
OEt
OEt
NaHCO3/H2O/acetone
OEt
OEt
O
P
O
P
O
O
Scheme 1. Synthesis of chiral o-bromo difluoromethylphosphono-
phenylalanine.
OH
HN
N
H
FMOCNH
O
O
F
F
HATU, DIEA
DMF
Scheme 1, utilizing a bicyclic Schiff base chiral auxil-
iary.10 Using the same chiral auxiliary an analog of
tyrosine with a trifluoromethyldiazirine unit was pre-
pared as in Scheme 2.
O
X
P
EtO OEt
O
N
N
OH
F3C
20% piperidine
90% TFA
TMSBr, CHCl3
Scheme 3 shows the synthesis of a representative peptide
substituted with the required trifluoromethyldiazirine-
benzoic acid capping the N-terminus using standard
procedures.11–13 Typically, TentaGel sRam from RAPP
was deprotected using piperidine in DMF for 30 min,
then washed with DMF (5·), and CH2Cl2 (5 ·). A
Fmoc-protected amino acid was then coupled to the
resin using HATU and diisopropylethylamine in DMF,
deprotection with piperidine then affords the free amine
ready for coupling with another carboxylic acid using
the same procedure. The peptide was cleaved from the
resin by treatment with trifluoroacetic acid and water,
subsequent treatment with TMSBr in CHCl3 afforded
the phosphonic acid directly or the ammonium salt after
reverse-phase HPLC purification using an ammonium
acetate buffer.
DMF,45 min.
Z
HATU, DIEA
DMF
Z= I, Br
+
X
F
NH4
F
O-
O-
P
O
+
NH4
O
O
N
N
F3C
H
N
N
H
H2N
O
F
Z
F
O
X
P
-O O-
+
+
NH4
NH4
Scheme 3. Representative synthesis of peptides.
Br
Figure 2 lists all the analogs that were prepared. We
considered two factors for what would constitute a
desirable photoaffinity probe. The most important cri-
terion was the compoundsÕ ability to inhibit the PTP-1B
enzyme in the fluorescein diphosphate (FDP) assay.14
Secondly, the probe had to be irreversibly bound to the
enzyme. The co-valent binding of the photoprobe to
the enzyme was assessed by irradiating a solution of the
photoprobe and the PTP1B enzyme with a 40 W lamp
and the amount of adduct formed was measured by
mass spectrometry. The results are summarized in Table
1. First, we noted that there was a significant increase in
potency in going from a monopeptide/monophosphonic
acid, 8, to a dipeptide/diphosphonic acid 1, as indicated
in Table 1. As expected, having two trifluoromethyl-
diazirine units, 9, did not improve the potency. The
introduction of a glutamic acid to form a tripeptide, 7,
did not improve the potency over the dipeptide where
1) LiN(TMS)2/THF/-78oC
OH
N
O
CF3
N
Br
OtBu
2)
OH
Br
N
OtBu
N
CF3
N
O
Br
N
Br
CF3
N
CF3
N
TFA/H2O
Citric acid
OH
N
N
H2N
OtBu
F
H2N
TFA
O
O
F
Br
O
F
O
O
CF3
N
F
F
OH
N
FMOCNH
O
NaHCO3/H2O/acetone
Scheme 2. Synthesis of chiral trifluoromethyldiazirine phenylalanine.

M. Thꢀerien et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2319–2322
2321
Br
X
F
F
F
F
+
NH4
OEt
OEt
O-
O-
P
O
P
O
+
NH4
O
O
N
Y
O
N
H
N
N
H
N
1, Z = Br, Y = =O, X = H
2, Z = I, Y = =O, X = H
3, Z = I, Y = =O, X = Br
4, Z = Br, Y = H2, X = H
5, Z = Br, Y = =O, X = Br
N
H
N
H2N
N
H
NH2
F3C
F3C
O
F
O
F
I
F
Z
F
O
Br
P
O
X
P
EtO OEt
+
+-O O-
NH4
NH4
10
1) Pd(Ph3P)4,[ (CH3)3Sn]2
Dioxane
X
F
F
+
2) pH 7 phosphate buffer,
Na125I,ChloramineT, DMF
HO
NH4
O-
O-
P
O
O
O
+
NH4
3) TMSBr, CHCl3
Y
O
N
N
F3C
H
N
H
N
N
H
NH2
+
Br
F
NH4
F
O-
O-
O
F
6, Z = Br, Y = H2, X = H
7, Z = Br, Y = =O, X = H
P
O
+
Z
NH4
F
O
O
N
H
N
O
N
X
P
N
H
H2N
F3C
+
+-O O-
NH4
NH4
O
F
125I
F
O-
P
F
F
+
NH4
O
Br
P
+
O-
NH4
-O O-
+
O
+
NH4
O
NH4
N
N
F3C
11
8
N
NH2
H
O
Scheme 4.
Br
O
Br
F
F
+
NH4
compound. The introduction of an ortho bromo sub-
stituent on the phenyl ring, 3 and 5 versus 1 and 2 had a
marked improvement on the potency against the PTP-
1B enzyme. We therefore opted to prepare compound 3
as a radioactive photoaffinity probe for the PTP-1B
enzyme.
O-
O-
P
O
+
NH4
O
N
N
F3C
H
N
N
NH2
9
H
O
Br
Br
As illustrated in Scheme 4, treatment of compound 10,
with 10 equivof hexamethylditin and 0.1 equivof tetra-
kistriphenylphosphine palladium in dioxane at 50 ꢁC
for 90 min afforded the tin intermediate, which was
treated (1 mg) with a 50 lL (2.5 mCi) solution of Na125I
in water, 0.2 M pH 7 phosphate buffer (38 lL), and
Chloramine T (27 lL of a 10 mg/mL aqueous solution)
in DMF for 1 h, and quenched with 2 M NaHSO3
(100 lL) to afford 11 after reverse-phase HPLC purifi-
cation followed by cleavage of the ester in a 1:2 mixture
of TMSBr–CHCl3.
CF3
N
N
Figure 2. Peptidic analogs synthesized.
Table 1. Photoprobe results summary
Compound
PTP-1B inhibition
IC50, (lM)
% Photolysis % Adduct
after 5 mina
formation
1
2
3
4
5
6
7
8
9
0.012
0.009
0.004
1.7
52
45
33
44
58
50
40
50
64b
8
8
In summary, we have prepared a novel photoaffinity
probe for the PTP-1B enzyme utilizing a dipeptide
substituted with a trifluoromethyldiazirine unit and an
iodo substituent giving us the ability to make the
radioactive analog. By adding an ortho bromo sub-
stituent on the phenyl difluoromethylphosphonate unit
we were able to increase the intrinsic potency of the
compound. This probe binds irreversibly to the PTP-1B
enzyme upon irradiation by light, further studies with
this probe will be published at a later date.
0.0003
0.003
0.014
9.5
6
5
8
0.015
<5
a 50 lL of 25 lM photoprobe in water, irradiated with 40 W lamp for
5 min, product formation measured by MS, loss of N2, gain of H2O.
b 64% mono, 24% di.
the trifluoromethyldiazirine is attached as an amide, 1.
However, tripeptide 6 did show an increase in potency
from the dipeptide 4 where the trifluoromethyldiazirine
is attached as an amine. However, because of the low
(5%) amount, of adduct formation (which is a measure
of irreversibility) of 6, it was not considered a suitable
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