Synthesis of [difluoro-(3-alkenylphenyl)-methyl]-phosphonic acids on non-crosslinked polystyrene and their evaluation as inhibitors of PTP1B.

Article abstract of DOI:10.1016/S0960-894X(02)00768-0

A series of [difluoro-(3-alkenylphenyl)-methyl]-phosphonates were prepared on non-crosslinked polystyrene, a soluble polymer support. After cleavage from the support, the resulting phosphonic acids were examined for inhibition with protein tyrosine phosphatase 1B. Compound 20, bearing an alpha,beta-unsaturated allyl ester moiety, was the most potent of this series of compounds, being a reversible, competitive inhibitor with a K(i) of 8.0+/-1.4 microM.

Full text of DOI:10.1016/S0960-894X(02)00768-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3471–3474
Synthesis of [Difluoro-(3-alkenylphenyl)-methyl]-phosphonic
Acids on Non-crosslinked Polystyrene and Their Evaluation as
Inhibitors of PTP1B
Gabriel Hum, Jason Lee and Scott D. Taylor*
Department of Chemistry, University of Waterloo, 200 University Avenue West, Ontario, Canada N2L 3G1
Received 29 May 2002;accepted 12 August 2002
Abstract—A series of [difluoro-(3-alkenylphenyl)-methyl]-phosphonates were prepared on non-crosslinked polystyrene, a soluble
polymer support. After cleavage from the support, the resulting phosphonic acids were examined for inhibition with protein tyro-
sine phosphatase 1B. Compound 20, bearing an a,b-unsaturated allyl ester moiety, was the most potent of this series of compounds,
being a reversible, competitive inhibitor with a Ki of 8.0ꢀ1.4 mM.
# 2002 Elsevier Science Ltd. All rights reserved.
Recently, there has been considerable interest in the
development of inhibitors of protein tyrosine phospha-
tase 1B (PTP1B).1 This stems for the relatively recent
discovery that PTP1B knock-out mice are insulin sensi-
tive and are resistant to obesity.2 These studies suggest
that PTP1B is involved in the down regulation of insulin
signaling and that inhibitors of PTP1B may be useful
for the treatment of type II diabetes and obesity.2 As
part of our program on the development of non-pepti-
dyl PTP1B inhibitors,3 we became interested in devising
methods that would allow for the rapid synthesis of
potential inhibitors bearing the difluoromethylenephos-
phonic acid group (DFMP), a highly effective non-
hydrolyzable phosphate mimetic for obtaining PTP1B
inhibtors.4 Towards this end we developed an approach
to the synthesis of biaryl derivatives of type 1 on non-
crosslinked polystyrene (NCPS), a soluble polymer
support.5a,b Biaryl derivatives were specifically targeted
since we had previously demonstrated that substitution
of 2 with a phenyl group at the meta position, 3
(IC50=15 mM), significantly increased inhibitory
potency by 17-fold compared to 2.3a Quite recently,
Shibuya et al. reported that the ethynyl compound 4
(IC50=19 mM) was also a good inhibitor. These results
indicate that certain unsaturated moieties at the meta
position of 2 can significantly increase inhibitory
potency which suggests that the vinyl derivative 5 and
compounds of type 6 might also be effective inhibitors.
However, Shibuya reported that they were unable to
construct compound 5 in pure form.6 Nevertheless,
since the styryl derivative (6, R=Ph) was reported,6 we
reasoned that other substituted vinyl derivatives of type
6 should be readily obtainable. Here we report the
polymer-supported synthesis of alkenes of type 6 and
their evaluation as inhibitors of PTP1B.
Our approach to compounds of type 6 involved a Wittig
reaction between NCPS-bound aldehyde 7 and a series
of phosphorus ylids (Scheme 1). Although compounds
of type 6 could potentially be prepared using Stille cou-
plings,6 we chose to use a Wittig reaction7 since a wide
*Corresponding author. Tel.: +1-519-888-4567x3325;fax: +1-519-
746-0435;e-mail: s5taylor@sciborg.uwaterloo.ca
Scheme 1. Proposed route to alkenes from polymer-bound 7.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00768-0

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G. Hum et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3471–3474
variety of phosphonium salts are commercially available
and these reactions could be performed at room tem-
perature. Polymer-supported chemistry would facilitate
the removal of triphenylphosphine oxide from the reac-
tion mixtures. NCPS is soluble in many aprotic organic
solvents yet is insoluble in polar protic solvents such as
MeOH or water. This approach has certain advantages
over the more conventional solid phase methodologies
since reactions are carried out under homogeneous
conditions, which results in faster reaction rates8 and
facile monitoring of the reaction using conventional
solution NMR. Pure polymer-bound product is
obtained by precipitation followed by filtration.
17 as outlined in Scheme 3. Thus, a,a0-dibromo-meta-
xylene was reacted with allyl alcohol in the presence of
NaH and 15-C-5 to give a mixture of the mono and
diallylated compounds 13 and 14. These compounds
were not separated but instead were subjected to excess
triethytlphosphite in refluxing benzene to give the
phosphonate 15 in 54% yield (two steps). Electrophilic
fluorination10 using N-fluorobenzenesulfonimide (NFSi)
gave compound 16 in 80% yield. Basic hydrolysis
followed by acidification gave acid 17 in 97% yield.
Initially, we wished to prepare aldehyde 7 by lithiation
of polymer bound aryl bromide 85a followed by for-
mylation (Scheme 2). However, such reactions with
model compound 9 in solution indicated that this would
not be a suitable approach due to the formation of
numerous impurities. An alternative approach was to
prepare the aldehyde 10 (or its acetal equivalent), fol-
lowed by attachment to functionalized polymer 115a by
a Mitsonobu reaction (Scheme 2).5a We initially exam-
ined this approach using the known aldehyde 129 as a
model substrate. However, we were unable to attach
this compound to polymer 11 cleanly and in good yield
under a wide variety of conditions. Attempts to convert
acetal derivatives of 12 to the acid chloride and attach
them to 11 were also unsuccessful. Our third approach
was by oxidizing a polymer-bound alcohol to the alde-
hyde. This involved first preparing the protected alcohol
Compound 17 was attached to polymer 11 (0.4 mmol
alcohol moiety/gram of polymer)5a in quantitative
yields (as determined by NMR) by a Mitsonobu reac-
tion (Scheme 4).5a Pure polymer-bound 18 was obtained
by addition of the reaction mixture to MeOH–water
(9:1) followed by filtration. Polymer recovery was
approximately 97%. Using the conditions developed by
Honda et al.11 for removal of allyl groups, polymer
bound alcohol 19 was obtained in near quantitative
yields as determined by 19F NMR. By subjecting 19 to
the Dess–Martin periodane in methylene chloride at room
temperature for 2 h, aldehyde 7 was obtained in quantita-
tive yields as determined by 19F NMR with the only loss of
material being due to the precipitation/filtration step (3%
for the deprotection and oxidation).
Wittig reactions using 400 mg of 7 were performed at
room temperature using an excess of phosphorus ylid
Scheme 2. Potential routes to 7.
Scheme 3. Synthesis of 17.
Scheme 4. Synthesis of alkenes 20–39.

G. Hum et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3471–3474
3473
(generated from commercially available phosphonium
salts and KH) in the presence of 20 mol% 18-C-6 in
THF (Scheme 4). The reactions were quenched by add-
ing the reaction mixture to a stirred 9:1 MeOH–water
solution followed by filtration. With these smaller scale
reactions, recovery of the polymer ranged from 80 to
95%. Simultaneous cleavage from the support and
phosphonate deprotection was achieved using
TMSBr.5a The reactions were concentrated by rotary
evaporation and the residue redissolved in methylene
chloride which was then added to a 9:1 solution of
MeOH and water and stirred for 12 h. The polymer was
removed by filtration and the filtrate concentrated to
give the phosphonic acids. The acids were applied to an
LC-NH2 weak anion exchange resin12 and washed with
THF, MeOH and water. The compounds were removed
from the column as their diammonium salts by elution
with 0.5 M NH4OH followed by repeated lyophilization.
20 different alkenes were constructed (20–39, Table 1).
1
Compounds were characterized by H, 31P, 19F NMR
and negative FABMS.13 Yields ranged from 28 to 83%.
The purities for the majority of the compounds were
Table 1. Yields and purities of alkenes 20–39
R
Crude yield
Purity
NMR (19F, 31P)
R
Crude yield
Purity
NMR (19F, 31P)
HPLC
100
HPLC
95
83
96, 100
88, 100
90, 97
68
98, 100
98, 98
99, 98
96, 100
98, 97
92, 98
98, 95
100, 98
100, 98
97, 85
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
64
44
70
41
82
82
60
82
71
92
95
56
62
62
61
65
83
28
38
31
90
99
88, 97
93
97
97, 98
97
99
95, 99
91
95
96, 100
99, 93
80
CI
36
65
100
100
88
CH3CH2
37
100
100
72
100, 100
98, 85
38
39

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G. Hum et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3471–3474
Table 2. Percent Inhibition of PTP1B with 50 mM compounds 20–26
with Lys 116 and Lys 120.17 It is possible that similar
interactions are occurring with some of the compounds
reported here.
In summary, polymer-supported organic synthesis was
used to prepare a series of alkenes of type 6. Inhibition
studies reveal that an a,b-unsaturated allyl ester at the
meta-position on the aryl ring significantly increases
potency in that compound 20 is a 76-fold more potent
inhibitor than the parent compound 2. Further studies
to increase compound diversity by utilizing other reac-
tions on polymer bound aldehyde 7, as well as alcohol
19, and their para analogues, are in progress.
R
% Inhibition
84
R
% Inhibition
53
20
21
22
23
24
25
26
71
70
60
45
23
Acknowledgements
We thank the Natural Sciences and Engineering
Research Council of Canada and MerckFrosst for
financial support and for PTP1B.
References and Notes
greater than 85% as judged by 19F and 31P NMR and
HPLC (Table 1). The exceptions to this were com-
pounds 26, 36 and 39 which were obtained in purities of
80%, 65 and 72% as judged by HPLC. 19F and 31P
NMR of these three compounds suggested that their
purity was considerably greater than that determined by
HPLC.
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A rapid screen of 20–39 for PTP1B inhibition was per-
formed by determining the percent inhibition in the
presence of 50 mM 20–39 and using fluorescein diphos-
phate as substrate at Km concentration (20 mM) at pH
6.5.14 Compounds 20–26 were the most potent inhibi-
tors (Table 2). Compounds 35–39 showed no inhibition.
Compounds 27–34 showed little or no inhibition and
these results are consistent with the studies of Shibuya
et al. who reported that the styryl derivative (6, R=-Ph)
was a weak inhibitor (IC50=386 mM).6 Interestingly,
compound 22, which has an additional double bond
exhibited 70% inhibition (Table 2) while its more satu-
rated analogue, 38, showed no inhibition. The data in
Table 2 reveals that an a,b-unsaturated ester moiety
aids inhibition and that an unsaturated group in the
alcohol portion of the ester further enhance inhibition.
The IC50 of the most potent inhibitor, 20, was deter-
mined to be 12.2ꢀ1.0 mM indicating that it is slightly
more potent than compounds 3 and 4. Further kinetic
studies revealed that 20 was a reversible competitive
inhibitor with a Ki of 8.0ꢀ1.4 mM. We also prepared 20
in solution from difluoro-(3-formylphenyl)-methyl]-
phosphonic acid diethyl ester15 using the room tem-
perature Wittig coupling conditions of Belluci et al.16
which are known to give exclusively the E isomer. This
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12. Available from Supelco.
13. No attempt was made to identify the proportions of geo-
metric isomers formed.
14. Specific assay conditions were: 0.2 mg/mL PTP1B in 50 mM
Bis–Tris (pH 6.5), 2 mM EDTA, 5 mM DTT, 0.01% triton X-
100 and 5% DMSO. Assays were carried out in 96-well plates
using a SPECTRAmax (Molecular Devices) microplate spec-
trofluorometer, (excitation at 485 nm, emission at 538 nm).
15. Prepared from 16 by alcohol deprotection and oxidation
followed by reaction with TMSBr.
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1
compound exhibited identical 19F, 31P and H NMR
spectra to that prepared on the polymer which is con-
sistent with the product being formed from a stabilized
ylid. The compound prepared in solution also exhibited
identical inhibition properties. Recent theoretical stud-
ies on compound 3 and PTP1B suggest that the meta-
phenyl ring increases potency by pi-cation interaction