Peptidyl prolyl isomerase Pin1-inhibitory activity of d-glutamic and d-aspartic acid derivatives bearing a cyclic aliphatic amine moiety

Article abstract of DOI:10.1016/j.bmcl.2015.10.033

Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so

Full text of DOI:10.1016/j.bmcl.2015.10.033

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5619–5624
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and
D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety
a
a
a
a
Hidehiko Nakagawa a, , Suguru Seike , Masatoshi Sugimoto , Naoya Ieda , Mitsuyasu Kawaguchi ,
⇑
Takayoshi Suzuki b, Naoki Miyata a
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
b Graduate School of Medical Sciences, Kyoto Prefectural University of Medicine, Kyoto, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis–trans isomerization of phosphorylated
Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular
processes, including cancer progression, so it is a potential target of cancer therapy. We designed and
synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing
an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity
for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthia-
zole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1.
The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues
plays a key role in enhancing Pin1-inhibitory activity.
Received 16 September 2015
Revised 8 October 2015
Accepted 13 October 2015
Available online 22 October 2015
Keywords:
cis–trans isomerization
Proline
Phosphoserine
Phosphothreonine
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Pin1 (protein interacting with never in mitosis A-1) is a member
of the peptidyl prolyl isomerase (PPIase) family, and specifically
catalyzes cis–trans isomerization of phosphorylated Thr-Pro or
phosphoryl Ser-Pro peptide bonds in its substrate proteins and
peptides. There are three subfamilies of PPIase: cyclophilins (Cyps),
FK506-binding proteins (FKBPs), and purvulins. Pin1 is a member
of the purvulin family and is the only enzyme that catalyzes
isomerization of phosphorylated substrates in humans.1,2 Pin1 is
reported to be involved in regulation of kinase signaling processes
by mediating change in the ratio of cis-/trans-conformers of phos-
phorylated proteins.3 For example, signal transduction pathways
involving cyclin-dependent kinases and MAP kinases, as well as
cell cycle controllers, are known to be regulated by Pin1 activity.4
Substrates of Pin1 includes cancer-related signaling proteins such
as cyclin D1, NF-kappaB, and p53.5–8 Furthermore, Pin1 is overex-
pressed in various types of cancer cells, including prostate cancer,
rectal cancer, hepatic cancer, and esophageal cancer.2 It was also
reported that the prognosis of prostate cancer is related to the
expression level of Pin1 in the cancer cells.9 Based on these reports,
Pin1 may be a new therapeutic target for these cancers. Pin1 is also
involved in the pathogenesis of Alzheimer’s disease by isomerizing
phosphorylated tau proteins, resulting in a reduction of tau-
dependent fibril formation. Thus, Pin1 catalyzes a unique reaction,
Figure 1. Structure of Pin1 catalytic domain with D-peptide (PDB 2ITK).
and it is also considered to contribute to the temporal regulation of
protein phosphorylation, acting like a ‘molecular timer’.10 The cat-
alytic domain of Pin1 enzyme, containing the cation-recognition
site, consists of Lys63, Arg68, and Arg69, which serve to stabilize
the phosphoryl moiety of the substrate peptide via electrostatic
effect11 (Fig. 1).
Several Pin1 inhibitors (1–7) have been reported to date, as illus-
trated in Figure 2.11–19 Very recently, it was reported that all-trans
retinoic acid (ATRA) (8) is also an inhibitor of Pin1.19 Among these
inhibitors, compound 4 has a very high affinity for the Pin1 catalytic
⇑
Corresponding author. Tel./fax: +81 528363407.
E-mail address: deco@phar.nagoya-cu.ac.jp (H. Nakagawa).
http://dx.doi.org/10.1016/j.bmcl.2015.10.033
0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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H. Nakagawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5619–5624
Figure 2. Previously reported Pin1 inhibitors.
site, but is not applicable to cell-based systems due to its high
hydrophilicity arising from the presence of the phosphonate group.
On the other hand, inhibitors bearing a carboxyl group, instead of a
phosphonate group, such as VER1 (6) and VER2 (7) are applicable to
cells, and show cell growth-inhibitory activity.
Structural analysis of VER1–Pin1 complexes has shown that
VER1 binds to Pin1 mainly through three interactions: ionic inter-
action of the carboxylate group of VER1 with the cationic pocket in
the Pin1 catalytic site, hydrophobic interaction of the aromatic
group of VER 1 with the proline-binding pocket of Pin1, and inter-
action of another aryl group of VER1 with the hydrophobic surface
of the Pin1 catalytic site (Figs. 3 and S1).
Figure 3. Schematic illustration of the interaction of Pin1 catalytic site and VER1.
Figure 4. Molecular design for Glu/Asp-scaffold Pin1 inhibitor candidates.

H. Nakagawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5619–5624
5621
Scheme 1. Synthesis of compounds 9 and 10. Reagents and conditions: (a) amine a–j, EDCI. HOBt, DMF; (b) HCl, dioxane; (c) 19, EDCI, HOBt, NEt3, DMF; (d) LiOH, H2O, MeOH,
THF.
To develop new Pin1 inhibitors, we focused on interactions
Table 1
within the proline pocket of Pin1. In the complex of VER1 and
Pin1, the pocket interacts with the naphthyl group of VER1, but
it is not completely filled by the planar aromatic group. Therefore,
we explored sterically bulkier aliphatic groups, in place of the
naphthyl group, and designed potential inhibitors, 9a–j and
10a–j, bearing various cyclic or cycloalkylamine moieties (Fig. 4).
The synthetic procedures are summarized in Scheme 1. Com-
Pin1-inhibitory effect of the synthesized compounds 9 and 10
O
R
O
O
O
R
N
N
N
H
COOH
N
H
COOH
N
N
pounds 9a–j were derived from
D-glutamic acid, and 10a–j were
derived from -aspartic acid. Boc-
D
D
-Glu-OBzl (11) or Boc- -Asp-
D
OBzl (12) was subjected to condensation reaction with various ami-
nes in DMF, using EDCI and HOBt. The Boc moiety of the resulting
amide (13a–j, 14a–j) was deprotected with HCl-containing dioxane.
The obtained amine (15a–j, 16a–j) was condensed with 1-methyl-
3-phenyl-1H-pyrazole-5-carboxylic acid (19), and then hydrolyzed
with LiOH to afford compounds 9a–j and 10a–j (Table S1).
9a-j
10a-j
Compd
R
IC50 (lM)
9a
9b
9c
9d
9e
9f
9g
9h
Pyrrolidin-1-yl
Piperidin-1-yl
Homopiperidin-1-yl
>100
>100
>100
39
>100
10
The Pin1-inhibitory activity of the synthesized compounds was
evaluated by means of the proteinase-coupled assay method.20
3-Methylpiperidin-1-yl
4-Methylpiperidin-1-yl
Morpholin-1-yl
Cyclopentylamino
Cyclohexylamino
Cycloheptylamino
Adamant-1-ylamino
Pyrrolidin-1-yl
Briefly, 250
pNA) was preincubated with 0.2 mM DTT, 100
22 nM Pin1 in 150 L of 35 mM HEPES-KOH (pH 7.8) for 10 min
lM synthetic substrate peptide (suc-Ala-Glu-Pro-Phe-
lg/mL BSA, and
>100
77
l
9i
9j
>100
>100
>100
>100
>100
>100
81
>100
>100
67
at 10 °C after adding the indicated concentration of test compound
(as a DMSO solution; the final DMSO concentration was 5% v/v).
C-terminal hydrolysis of the substrate peptide was initiated by
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
VER1 (6)
Piperidin-1-yl
addition of an excess amount of
a-chymotrypsin (150 lL of
Homopiperidin-1-yl
3-Methylpiperidin-1-yl
4-Methylpiperidin-1-yl
Morpholin-1-yl
Cyclopentylamino
Cyclohexylamino
Cycloheptylamino
Adamant-1-ylamino
0.8 mg/mL protease in 35 mM HEPES-KOH, pH 7.8). The absor-
bance of the released p-nitroaniline (pNA) at 390 nm was recorded
for 10 min with a spectrophotometer. Initially,
a-chymotrypsin
rapidly digested the substrate peptides present in the trans form
(rapid phase), and then subsequently hydrolyzed the peptides as
they were slowly isomerized from cis- to trans-form by Pin1
(isomerization phase). The observed reaction rate of the isomeriza-
tion phase was thus taken as the Pin1 activity. The inhibitory activ-
ity was then expressed as ((k(inh) À k0)/(k(noinh) À k0)) Â 100 (%),
where k(inh) is the observed pseudo-first order rate in the presence
of an inhibitor, k(noinh) is that without inhibitor, and k0 is that in the
absence of Pin1.
18
91
4.2 (3.9a)
a
Ref. 16.
bearing small cyclic amines or large cycloalkylamines were ineffec-
tive (9a–c, e, g, i, j). As for Asp-derived compounds, those bearing
smaller amines (10a–d, g) showed poor activity, and the mor-
pholino derivative (10f) was also ineffective.
The synthesized compounds were tested for Pin1-inhibitory
activity in the range of 1 to 200 lM, and IC50 values were calcu-
lated (Table 1). As the positive control, VER1 was also synthesized
as reported16 and evaluated. Among the compounds with various
amine moieties, 9f and 10i showed moderate to good inhibitory
activity. Among the Glu-derived compounds, the compounds
Structural analysis of the VER1 and Pin1 complex indicates that
the distance between the proline pocket and the cation site is
important, suggesting that the cyclic amine or cycloalkylamine
moiety on the Glu scaffold would be too long to interact with the

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H. Nakagawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5619–5624
Table 2
Table 3
Pin1-inhibitory effect of derivatives of 9f and 10j
Inhibitory effect of the derivatives of 20c and 21c
Compd
R
IC50 (lM)
Compd
R
IC50 (lM)
20a
20b
20c
21a
21b
21c
Naphth-2-yl
83
91
5.4
>100
>100
4.3
22a
22b
22c
23a
23b
23c
23d
3-Methylpiperidin-1-yl
Azocan-1-yl
Cyclooctylamino
Cyclohexylamino
4-Methylpiperidin-1-yl
Azocan-1-yl
19
>50
>50
>100
14
3.0
Benzofuran-2-yl
2-Phenylthiazol-4-yl
Naphth-2-yl
Benzofuran-2-yl
2-Phenylthiazol-4-yl
Cyclooctylamino
>100
ATRA showed good inhibitory activity toward Pin1, considering
the distance between its carboxylic acid and aliphatic ring
structures.19
The proteinase-coupled assay revealed that the compounds
bearing a phenylthiazole moiety (20a–c and 21a–c) showed
inhibitory activity comparable to that of VER1 (Table 2). The
phenylthiazole moiety was assumed to interact favorably with
the hydrophobic surface of the enzyme in the case of compounds
bearing an alicyclic moiety. This aryl moiety has not previously
been used as a hydrophobic interacting group in small-molecular
Pin1 inhibitors, so this result means that phenylthiazole com-
pounds may be useful tools to explore the motif interacting with
the hydrophobic surface of the enzyme pocket, in combination
with appropriate proline-pocket-binding groups. Although the
benzofuran moiety was expected to interact with residues such
as Cys113 or Ser114, it did not appear to be effective. The naphthyl
group has been used for hydrophobic interaction in compound 5,
another effective Pin1 inhibitor, but seems ineffective in our
inhibitor scaffold (Fig. 5).
Figure 5. Synthesized derivatives of 9f and 10i.
two important binding sites, and those with the Asp scaffold would
be too short. As for the ring size, a 7-membered cyclic amine or
cycloalkylamine moiety was more effective than a 6- or 5-mem-
bered one, suggesting that it may match well the steric size of
the proline pocket. This is consistent with a recent report that
Next we explored combinations of proline pocket-binding
aliphatic groups with the phenylthiazole moiety to examine the
structure–activity relationship. We synthesized derivatives of 20c
Figure 6. Synthesized derivatives of 20c and 21c.

H. Nakagawa et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5619–5624
5623
Figure 7. Docking model of compound 23c with Pin1 catalytic site (Docking stimulation was conducted with CLC Drug Discovery Workbench (Filgen)) based on PDB 3KAI.16
and 21c with different ring size and scaffold length (22a–c and
23a–d, Fig. 6). These compounds were obtained by the same
method shown in Scheme 1, using corresponding cyclic amines
or cycloalkylamines. The proteinase-coupled assay revealed that
the combination of a 1-azocanyl group and the Asp scaffold (23c)
was as potent as VER1 (Table 3). It seems likely that appropriate
folding of the azocane moiety enabled it to fill the bulky proline
pocket (Fig. 7). In the Glu scaffold series, cycloalkylamino and
cycloalkylimino moiety (22a–c) resulted in less active than
morpholino moiety (20c) unlike the case of Asp scaffold. The
morpholino moiety may have additional interaction(s) with polar
residues in the relatively hydrophobic pocket.
In conclusion, we explored aliphatic cyclic amines and
cycloalkylamines as proline-pocket binding motifs on a glutamic
acid or aspartic acid scaffold, and discovered unique Pin1 inhibitors
bearing morpholino, cycloheptylamino, and azocanyl moieties
instead of the simple aromatic groups present in previously
reported small-molecular inhibitors. Inhibitors containing these
new moieties are expected to have improved logP values and
decreased molecular planarity, which might be favorable for prac-
tical application. Among the various drug properties, the logP
value is one of the importance parameters because the appropriate
logP value is considered to elucidate the good efficiency and also
provide good clearance from the body. The logP value of a drug
is better to be less than 5 in accordance with Lipinsky’s rule of
five21, and in general the value of 1 to 3 is more suitable.22 Consid-
ering these guidance, compound 23c showed better logP value
(1.59) than VER1 (3.02). Furthermore, compound 23c is bearing
balkier functional group of aliphatic imine which is expected to
provide a positive effect on the logP value through inhibiting the
molecular self-stacking via planer molecular structures.23,24 The
introduction of steric groups is also expected to interact with larger
surface of the proteins. The aspartic acid scaffold with a steric
imide group might have good balkiness for Pin1 binding pocket.
Further work is needed to evaluate the cellular effects of these
compounds, and to extend our knowledge of the structure–activity
relationships.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
033.
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Acknowledgment
This work was supported by JSPS KAKENHI Grant Numbers
25670059 (H.N.) and 26670060 (H.N.).

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