Non-competitive and selective dipeptidyl peptidase IV inhibitors with phenethylphenylphthalimide skeleton derived from thalidomide-related α-glucosidase inhibitors and liver X receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.026

Novel dipeptidyl peptidase IV (DPP-IV) inhibitors with a phenethylphenylphthalimide skeleton were prepared based on α-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide. Representative compounds showed non-competitive i

Full text of DOI:10.1016/j.bmcl.2011.03.026

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3041–3045
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Non-competitive and selective dipeptidyl peptidase IV inhibitors with
phenethylphenylphthalimide skeleton derived from thalidomide-related
a-glucosidase inhibitors and liver X receptor antagonists
⇑
Kazunori Motoshima, Kazuyuki Sugita, Yuichi Hashimoto , Minoru Ishikawa
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel dipeptidyl peptidase IV (DPP-IV) inhibitors with a phenethylphenylphthalimide skeleton were pre-
Received 17 February 2011
Revised 8 March 2011
Accepted 9 March 2011
Available online 16 March 2011
pared based on a-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide.
Representative compounds showed non-competitive inhibition of DPP-IV and 28a exhibited 10-fold
selectivity for DPP-IV over DPP-8. Compound 28a is the first non-competitive, selective DPP-IV inhibitor.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dipeptidyl peptidase IV
Enzyme inhibitor
Phthalimide
Non-competitive inhibition
Subtype selectivity
Dipeptidyl peptidase IV (DPP-IV; E.C. 3.4.14.5) is a 220–240 kDa
homodimeric type II transmembrane glycoprotein catalyzing the
cleavage of Xaa-Pro or Xaa-Ala dipeptides preferentially from the
N-terminus of polypeptides (where Xaa is any amino acid except
for Pro).1–4 DPP-IV is identical with the CD26 T-cell activating anti-
gen found in almost all human organs and tissues.5 It is anchored
to the plasma membrane of endothelia of almost all organs exam-
ined, and is also found in a soluble form in body fluids, such as
blood plasma and cerebrospinal fluid.6 In vivo, this enzyme cleaves
various polypeptides, including chemokines and peptide hor-
mones.4,7 Glucagon-like peptide-1 (GLP-1), an insulin-releasing
hormone, is also cleaved and inactivated by DPP-IV.8 GLP-1 is se-
creted in response to ingestion of food and stimulates insulin
secretion.9 It has been suggested that potentiation and extension
of the action of GLP-1 by DPP-IV inhibition would stimulate insulin
secretion only after a meal,10 and DPP-IV inhibitors have therefore
come to be seen as a potential new type of antidiabetic agent free
of side effects such as hypoglycemia. Some DPP-IV inhibitors such
as alogliptin (1) and sitagliptin (2) are already on the market
(Fig. 1).11 Almost all of the currently known inhibitors show com-
petitive inhibition.12,13
Figure 1. Chemical structures of currently available DPP-IV inhibitors (1 and 2).
that the number of three-dimensional spatial structures (fold struc-
tures) of human proteins is only approximately 1000, which is much
smaller than the number of human proteins, estimated to be
50,000–70,000.20–22 Therefore, ignoring physical/chemical interac-
tions, a template/scaffold structure which is spatially complemen-
tary to one fold structure might serve as a multi-template for
structural development of ligands that would interact specifically
with 50–70 or more different human proteins. We have focused on
thalidomide (3) as a candidate multi-template structure. Thalido-
mide (3) is a hypnotic/sedative drug, which was launched in the
1950’s, but was withdrawn from the market in the 1960’s because
of its severe teratogenicity. In spite of this, thalidomide (3) has been
established to be useful for the treatment of Hansen’s disease and
multiple myeloma. Additionally, many reports have appeared on
its therapeutic potential for the treatment of a range of diseases,
We have been engaged in the creation of bioactive compounds
based on the multi-template approach utilizing thalidomide
(3).14–19 The concept underlying the multi-template approach is
⇑
Corresponding author.
E-mail address: hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.026

3042
K. Motoshima et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3041–3045
including cancers, rheumatoid arthritis and diabetes.14–19,23 We
have developed many biological response modifiers, including
Table 1
DPP-IV-inhibitory activity of phenethylphenylphthalimides (6,9–26)
tumor necrosis factor-
a
(TNF-
a
) production regulators,24,25 nitric
oxide synthase (NOS) inhibitors,26,27 cyclooxygenase (COX) inhibi-
R
X
O
tors,28–30,liver X receptor (LXR) antagonists,31–34
a-glucosidase
2' 3'
X
X
inhibitors34–37 and glycogen phosphorylase inhibitors,38 by employ-
ing the multi-template approach. During these studies, we also dis-
covered novel DPP-IV inhibitors, including PPS-33 (4) and 5APP-33
(5) (Fig. 2).39 However, these compounds possess only weak inhibi-
R
N
4'
O
X
tory activity (4: IC50 = 57.8 lM, 5: IC50 = 65.9 lM), and they also
Compound
X
Position
R
Inhibition ratio at 10 lM (%)
show LXR-antagonistic and
a-glucosidase-inhibitory activity, as
9
—
H
H
H
H
H
H
H
H
—
20
20
20
30
30
30
40
40
40
20
20
20
30
30
30
40
40
40
—
H
14
0
0
4
2
0
7
6
0
7
7
2
12
26
0
5
12
3
well as DPP-IV-inhibitory activity.33 Recently, we have developed
various phenethylphenylphthalimide derivatives, including an
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
6
OMe
OH
H
OMe
OH
H
OMe
OH
H
OMe
OH
H
OMe
OH
H
OMe
OH
a
-glucosidase inhibitor (6), an LXR antagonist (7) and an LXRa-
selective antagonist (8) (Fig. 2).34 The above considerations led us
to speculate that phenethylphenylphthalimide derivatives might
possess DPP-IV-inhibitory activity. Thus, we carried out a screening
assay to search for novel DPP-IV inhibitors among our derivatives.
A classical DPP-IV inhibitor, diprotin A (9) (Fig. 2),40 was
adopted as a positive control; the extent of inhibition by 9 at
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
10 lM was 14% under our experimental conditions. In the initial
screening of phenethylphenylphthalimide derivatives reported
previously,34 compound 22 showed the most potent DPP-IV-inhib-
itory activity (Table 1). Among dimethoxy and dihydroxyl deriva-
tives, dihydroxyl derivatives seem to be more potent than
dimethoxy derivatives [12 > 11, 15 > 14, 18 > 17, 21 > 20, 24 > 23,
26 > 6].
26
11
Next, we tried to improve the inhibitory activity of compound
22. Introduction of two methoxy (23) or hydroxyl groups (24) into
compound 22 resulted in decreased DPP-IV-inhibitory activity
(Table 1). Based on the above structure–activity information, ana-
logs of 22 which possess electron-withdrawing groups were de-
signed as candidates for more potent DPP-IV inhibitors. An
analog of 22 bearing a methyl group was also designed, for com-
parison with the analog bearing the CF3 group.
These analogs were synthesized as shown in Scheme 1. Various
benzyl halides were reacted with triphenylphosphine to generate
triphenylphosphonium salts 30a–d. Diphenylethene derivatives
31a–d were prepared as E/Z mixtures by Wittig reaction of 3-nitro-
benzaldehyde with 30a–d. After reduction of the nitro group and
olefin moiety of compounds 31a–d, phenethylphenylphthalimides
27a–d were obtained by condensation with tetrachlorophthalic
anhydride.
Introduction of a CF3 group at the 400 position of 22 afforded
compound 27b, in which exhibited increased DPP-IV-inhibitory
activity (Table 2). On the other hand, the potency of analogs substi-
tuted with fluorine, that is, compounds 27a and 27c, or a methyl
group, that is, compound 27d, was similar to that of 22 (Table 2).
Hence, there does not seem to be a clear relationship between elec-
tronic character of substituents (electron-donating or -withdraw-
ing) and DPP-IV-inhibitory activity.
Subsequently, we designed 200- and 300-CF3 analogs in order to
examine the effect of the position of the CF3 group on DPP-IV-
inhibitory activity. The analogs were synthesized as shown in
Scheme 2, using a route nearly identical to that in Scheme 1.
Surprisingly, a positional shift of the CF3 group of 27b from the
400-position to the 300- (28a) or 200-position (28b) resulted in a dras-
Figure 2. Chemical structures of thalidomide (3), PPS-33 (4), 5APP-33 (5),
non-competitive DPP-IV inhibitor (29).
a-glucosidase inhibitor (6), LXR antagonist (7), LXRa-selective antagonist (8), diprotin A (9) and

K. Motoshima et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3041–3045
3043
Scheme 1. Synthesis of phenethylphenylphthalimide analogs with electron-withdrawing substituents 27a–d. Reagents and conditions: (a) triphenylphosphine, toluene,
reflux; (b) K2CO3, 18-crown-6, CH2Cl2, reflux; (c) H2, 10% Pd/C, EtOAc, rt; (d) tetrachlorophthalic anhydride, neat, 160 °C.
Table 2
There exist many types of dipeptidylpeptidases with similar
substrate selectivity. Among them, DPP-8, which is a cytoplasmic
aminopeptidase like DPP-IV,41 possesses an almost identical ac-
tive-site amino acid sequence to DPP-4.41,42 The administration
of selective DPP-8 inhibitors to animals results in severe toxic reac-
tions, including alopecia, thrombocytopenia, anemia, enlarged
spleen, multiple histological pathologies and increased mortality.43
Hence, DPP-IV selectivity over DPP-8 would be an advantage to
eliminate side effects. Alogliptin (1) and sitagliptin (2) show very
weak DPP-8-inhibitory activity. That is to say, they are DPP-IV-
selective inhibitors.12,13 Interestingly, although the amino acid se-
quences of DPP-IV and DPP-8 in the active sites are almost identi-
cal, as mentioned above, these proteins show only 51% amino acid
sequence homology overall.41 Therefore, we speculated that non-
competitive DPP-IV inhibitors might show selectivity between
the two enzymes. As far as we know, compound 29 (Fig. 2) is the
only non-competitive DPP-IV inhibitor reported so far, but its
activity against DPP-8 was not evaluated.44 Our phen-
ethylphenylphthalimide derivatives show non-competitive DPP-
IV inhibition (vide supra), so we next evaluated their DPP-8-inhib-
itory activity. We found that compound 28a possessed 10-fold
selectivity for DPP-IV over DPP-8 (Table 4). Compound 28b also
showed three-fold selectivity for DPP-IV. On the other hand, com-
pounds 22 and 27b showed no selectivity for DPP-IV over DPP-8.
These results suggest that the binding site for phenethylphenylph-
thalimide derivatives in DPP-IV might be similar to that in DPP-8.
But, since a positional shift of the CF3 group in phenethylphenylph-
thalimide derivatives affected the selectivity for DPP-IV over DPP-
8, we speculated that the CF3 group might interact with some
hydrophobic amino acids in DPP-IV and/or DPP-8, and therefore
differences in hydrophobic amino acid residues between DPP-IV
and DPP-8 might be important for selectivity.
DPP-IV-inhibitory activity of phenethylphenylphthalimides with electron-withdraw-
ing groups (27a–d)
R1
R2
R1
R1
R1
Cl
Cl
O
Cl
Cl
N
O
Compound
R1
R2
Inhibition ratio at 10 lM (%)
9
22
27a
27b
27c
27d
—
H
H
H
F
—
H
F
CF3
F
14
26
20
38
19
25
H
Me
tic increase of DPP-IV-inhibitory activity; 28a and 28b showed IC50
values of 3.1 M and 4.0 M, respectively (Table 3).
l
l
As mentioned above, almost all currently known DPP-IV inhib-
itors, including alogliptin (1) and sitagliptin (2), show competitive
inhibition12,13 and share similar pharmacophores, that is, they pos-
sess cyano and/or free amino groups. In terms of chemical struc-
ture, compounds 28a and 28b are quite different from the known
competitive DPP-IV inhibitors. In particular, these phen-
ethylphenylphthalimide derivatives do not possess a cyano or free
amino group, unlike the known inhibitors. Therefore, we specu-
lated that the mode of DPP-IV inhibition elicited by 28a and 28b
might be different from that of the known competitive inhibitors.
Indeed, Lineweaver–Burk plot analysis showed that 28a and 28b
In conclusion, we discovered novel DPP-IV inhibitors among our
a-glucosidase inhibitors/LXR antagonists derived from thalido-
mide (3). Structural development led to compound 28a, which is
the first non-competitive and selective DPP-IV inhibitor. Further
investigation of the structure–activity relationships aiming at the
development of superior DPP-IV inhibitors is in progress.
are non-competitive inhibitors (Fig.
respectively).
3 and data not shown,

3044
K. Motoshima et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3041–3045
Scheme 2. Synthesis of phenethylphenylphthalimide analogs with CF3 groups, 28a, b. Reagents and conditions: (a) triphenylphosphine, toluene, reflux; (b) K2CO3, 18-crown-
6, CH2Cl2, reflux; (c) H2, 10% Pd/C, EtOAc, rt; (d) tetrachlorophthalic anhydride, neat, 160 °C.
8000
6000
4000
2000
0
Table 3
DPP-IV-inhibitory activity of phenethylphenylphthalimides with CF3 groups (28a,b)
+28b
(30 µM)
3'' 4''
CF
3
2''
control
Cl
O
- 0.5
0
0.5
1
1.5
2
2.5
Cl
Cl
-2000
1/[S] (1/mM)
N
Figure 3. Lineweaver–Burk plot analysis of the inhibition of DPP-IV by 28b.
O
Cl
Compound
Position
Inhibition ratio at 10 lM (IC50 (lM))
Acknowledgments
5
—
14%
38%
27b
28a
28b
400
300
200
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and the Japan
Society for the Promotion of Science.
87% (3.1
86% (4.0
l
l
M)
M)
Supplementary data
Table 4
DPP-IV- and DPP-8-inhibitory activities of phenethylphenylphthalimides (9, 22, 27b,
28a and 28b)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.026.
3'' 4''
R
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