Structure-based design of novel human Pin1 inhibitors (II)

Article abstract of DOI:10.1016/j.bmcl.2010.02.033

Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the drug design strategy shifted to replacement of the phosphate group with an isostere with potential better pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker charge-charge interactions in the putative phosphate binding pocket and was subsequently recovered by structure-based optimization of ligand-protein interactions in the proline binding site, leading to the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.

Full text of DOI:10.1016/j.bmcl.2010.02.033

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2210–2214
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design of novel human Pin1 inhibitors (II)
*
*
Liming Dong, Joseph Marakovits, Xinjun Hou , Chuangxing Guo , Samantha Greasley, Eleanor Dagostino,
RoseAnn Ferre, M. Catherine Johnson, Eugenia Kraynov, James Thomson, Ved Pathak, Brion W. Murray
Pfizer Global Research and Development, 10770 Science Center Drive, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the
drug design strategy shifted to replacement of the phosphate group with an isostere with potential better
pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker
charge–charge interactions in the putative phosphate binding pocket and was subsequently recovered
by structure-based optimization of ligand–protein interactions in the proline binding site, leading to
the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.
Received 6 November 2009
Revised 8 February 2010
Accepted 8 February 2010
Available online 14 February 2010
Keywords:
Pin1
Ó 2010 Elsevier Ltd. All rights reserved.
Pin1 inhibitors
Phosphate replacement
PPIase
Anti-cancer agents
SBDD
Carboxylates
Tetrazoles
The peptidyl-prolyl isomerase (PPIase) Pin1 is a potential target
for therapeutic intervention and has attracted significant research
efforts1 since its discovery.2 Pin1 catalyzes the cis–trans isomeriza-
tion of prolyl amide bonds (pSer/Thr-Pro) in its substrate proteins
which play critical roles in cell-cycle regulation and are commonly
deregulated in human cancer cells.3 For example, Pin1 is required
for proper progression through mitosis4,5 with depletion of Pin1
causing mitotic arrest and apoptosis in budding yeast and tumor
cell lines.2 Pin1 is up-regulated in wide variety of human tumors
and its over-expression correlates with tumor grades and survival.6
As such, inhibition of Pin1 presents a new opportunity for anti-can-
cer therapy.1
Since Pin1 specifically recognizes a phosphorylated Ser/Thr-Pro
peptide motif in its active site,7 potent Pin1 inhibitors commonly
contain a negatively double-charged phosphate group which ac-
counts for a large proportion of binding energy.8–10 As a phosphate
group can be detrimental to cell permeability, discovering whole-
cell active Pin1 inhibitors is challenging. We have recently reported
a class of novel, potent non-peptide phosphate-containing Pin1
inhibitors.8 While these high affinity Pin1 inhibitors (e.g., 1a–b,
Fig. 1) provide tools to increase our understanding of the structural
elements critical for efficient binding to the Pin1 active site, the
presence of a double-charged phosphate moiety11 is a liability for
achieving cell membrane permeability.12 Recently, moderately
potent (Ki lM-range) non-phosphorylated small molecule inhibi-
tors13–17 as well as prodrugs18 of phosphate-containing inhibitors
have been reported.
Initially, the charge–charge interactions between phosphate
inhibitors and the active site appeared to be essential for Pin1 inhi-
bition because an alcohol analog, such as 1c is inactive in Pin1
enzymatic assay. Therefore, replacement of the phosphate group
with less charged functional groups such as a carboxylic acid or
acid isosteres is investigated. Carboxylic acid and related tetrazole
moieties have been incorporated in many successful drugs in
clinic.19 A simple replacement of the phosphate with either a sul-
fate or carboxylate group (2, Fig. 1)8 diminishes the affinity to
Pin1, presumably due to suboptimal charge–charge interactions.
To shift our chemistry effort toward a new chemical space, we de-
cide to revisit a marginally active compound 3a, which is an
H
H
N
CO2H
N
X
Y
Z
S
O
O
X
Y
Z
Ki (nM)
3a
K =28
1a -PO3H2
1b -PO3H2
1c -H
O
O
O
F
H
F
H
6
M
μ
i
179
inactive
inactive
2
-COOH CH2
* Corresponding authors. Tel.: +1 860 686 0310 (X.H.), +1 858 622 5927 (C.G.).
E-mail addresses: xinjun.hou@pfizer.com (X. Hou), alex.guo@pfizer.com (C. Guo).
Figure 1. Simple replacement of the phosphate diminished binding.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.033

L. Dong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2210–2214
2211
M)
Table 1
SAR of fragment binding to prolyl pocket
No.
R
Linker (n)
Ki (l
3a
3b
3c
3d
H
2-CF3
2-F
2-CN
2,3-di-F
3-F
0
0
0
0
0
0
1
0
1
0
1
0
1
0
1
0
1
0
0
0
0
1
0
28
93
63
351
103
12
5.7
2.8
9.7
3.2
2.3
1.8
13
3e (racemic)
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3-F
3-CH3
3-CH3
3-CN
3-CN
3-CF3
3-CF3
3-Cl
3.4
2
23
3-Cl
3,4-Di-F
3,4-Di-F
3,4-Di-Cl
3,4,5-Tri-F
4-CN
4-CF3
4-CF3
4-CH3
82
2.2
9.0
3.0
11
121
17
Figure 2. A docked model of 3a (orange) in the X-ray co-crystal structure of 1b
(cyan, Ref. 8) with Pin1 K77Q, K82Q construct.
3s
3t
3u
3v
3w
advanced intermediate of the phosphate inhibitors. Although there
is a substantial loss of potency, the binding of compound 3a to the
catalytic site is confirmed by isothermal calorimetric titration and
NMR binding studies (data not shown). This observation is surpris-
ing because the linker length between the central amido carbon
and the negative charged group (the oxygens of the carboxylate)
is substantially shorter than that in the phosphate series (1a vs
3a). With the advantage of low MW leads (<350) and good syn-
thetic accessibility, the phenylalanine template is chosen for fur-
ther optimization.
The model of 3a (Fig. 2) in the co-crystal structure of 1b shows a
slight shift in the binding of benzyl group, and reveals a gap be-
tween the carboxylate and the charge pocket formed by Lys-63,
Arg-68, and Arg-69. One of the carboxylate acid oxygens makes a
hydrogen bond to Arg-69 and Lys-63 and the other oxygen is
unsatisfied. It is evident that the binding of the initial lead (3a)
may be improved by either introducing substitution(s) to fill the
space or inserting a methylene between the amido carbon and
the carboxylate so that the phenyl can reach deeper into the pro-
line pocket. Based on this hypothesis, a number of naphthylamido
acid analogs are synthesized (Scheme 1)20 and tested in Pin1 enzy-
matic assay.8,20
As shown in Table 1, benzene substitutions (R groups) at the 2-
position (3b–d) are less active. As expected, small substitutions at
the 3-position (e.g., 3f) increase the potency, reaching the small
pocket near Phe-134 and His-157 as previously observed in the
non-peptide phosphate Pin1 inhibitors.8 Surprisingly, larger
groups at the 3-position (3h, 3j, 3l and 3n vs 3f) also improve
the inhibitory potency—a trend that was not observed in the phos-
phate series.8 One hypothesis is that the shorter linker to the acid
group in these series pulls the benzyl group away from the bottom
of the Pin1 prolyl pocket, creating additional space between Phe-
134 and the benzyl group. A bulkier substitution on the phenyl
would increase the hydrophobic interaction with the prolyl pocket
and allow closer charge–charge interactions with the phosphate
recognition site consist of Lys-63, Arg-68, and Arg-69. In order to
Figure 3. X-ray co-crystal structure of 3f with Pin1 K77Q, K82Q construct (PDB:
3I6C, Ref. 20).
test this hypothesis, seven b-amino acid analogs (Table 1, linker
n = 1) are designed and synthesized. The observed SAR is generally
in-line with the hypothesis—when substitutions are small (3f vs
3g, 3n vs 3o), there are slight improvements in activity of b-amino
acids over
a-amino acids. But when the substitutions are larger
(3m vs 3l, 3q vs 3p, and 3v vs 3u), b-amino acids are less active,
indicating that extending the carboxylate further into the charge
pocket is no longer beneficial. The analogs with an ‘S’ configuration
(not shown) are synthesized and found to be 10–30 times less ac-
tive than corresponding ‘R’ isomers shown here.
25ºC
H
(R)
H2N
(CH2)nCO2H
O
(R)
N
(CH2)nCO2H
R
1M Na2CO3
Cl
O
+
CH3CN
R
5
3
4
(12-55% yield)
Scheme 1. Syntheses of analogs of 3a.

2212
L. Dong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2210–2214
The co-crystal structure of 3f with Pin1 (Fig. 3) represents our
counterparts while b-tetrazoles such as 14 are less potent than
tetrazoles (9a–c).
a-
first Pin1 protein structure with a non-phosphate small molecular
inhibitor bound. The co-crystal structure confirms the hypothesis
that the carboxylic group does not fully occupy the phosphate
pocket.
Acylaminothiazoles and acylsulfonamides are known isosteres
for carboxylic acids.22 Acylsulfonamide 16 (Fig. 4) has acidity
(pKa 3.73, ACD LAB) comparable to carboxylic acid whereas acyla-
minothiazole 15 (Fig. 4) has a pKa close to 7, which has the poten-
tial to be more cell-permeable. Both acylaminothiazole 15 and
acylsulfonamide 16 are synthesized readily from the correspond-
ing carboxylic acid 3f.20 However, 15 and 16 are found to be weak-
er binders than the corresponding carboxylic acid (3f) likely due to
lower acidity (acylaminothiazole 15) or geometry mis-match for
the charge–charge interactions (acyl-sulfonamide 16).
Tetrazole analogs demonstrate comparable Pin1 inhibitory
activity compared to corresponding carboxylic acids. However,
since the SAR of Pin1 inhibitory activities of these two series tracks
and there is no additional potency gain for tetrazoles, the carbox-
ylic acid series is chosen for further optimization due to better syn-
thetic accessibility.23
To test the second hypothesis, the length of the linker between
the amido carbon and phenyl ring is investigated to optimize
charge interactions while maintaining the dual edge-on interac-
tions with Phe-134 and His-157 in the proline pocket. Accordingly,
several analogs with longer linkers (straight-chain alkane, more-ri-
gid alkene, and alkyne) are synthesized.20 The syntheses of these
compounds are shown in Scheme 3. The naphthamide 18 is pre-
pared from propargylglycine 17 in good yield via esterification fol-
lowed by HATU amide formation. The alkyne (18) is then coupled
with desired aryl iodide using Pd(PPh3)2Cl2 as a catalyst. The
resulting intermediate 19 is saponified to give desired carboxylic
acid product 21 or alternatively the triple bond can be hydroge-
The binding mode of 3f is similar to that of the phosphate-con-
taining inhibitors such as 1a–b,8 as shown in Figure 3. The 3-fluo-
rophenyl ring occupies the prolyl binding pocket and is in close
contact to a hydrophobic region formed by Phe-134, Met-130,
and Leu-122 with an edge-on interaction with the His-157 and
Phe-134. The fluorine is situated in a small pocket near Phe-134
and His-157. Because of the shorter linker between the charged
group and the 3-fluorophenyl ring, the phenyl ring is shifted
slightly outward toward the charge pocket in comparison with
phosphate inhibitor (Fig. 3), similar to the model in Figure 2. This
subtle change in binding conformation of the phenyl ring indicates
that there is some degree of elasticity in fitting the phenyl group to
the prolyl pocket, consistent with the observed SAR (Table 1). Sim-
ilar to a non-peptide phosphate inhibitor,8 the naphthalene ring
contacts the hydrophobic surface region near Leu-122. The most
significant difference from the binding mode of phosphate-con-
taining inhibitors is the network of charged–charged interactions
of the carboxylate. Instead of directly interacting with Arg-68
and Arg-69 side-chains, as observed for phosphate-containing
inhibitors, the carboxylate forms a water-mediated interaction
with Arg-69 and has no interaction with the disordered Arg-68
side-chain. One carboxylate oxygen makes a good H-bond interac-
tion to a water molecule (2.9 Å) and the other oxygen interacts di-
rectly with Lys-63 (2.9 Å). Interestingly, this second carboxylic
oxygen also interacts with a crystal water molecule, which bridges
the interaction with Ser-154 (3.2 Å) and another buried crystal
water molecule in the charged pocket.
N
N
N
NH
While the carboxylate acidic forms a hydrogen bond with Lys-
63 on the edge of the basic cluster, it leaves the rest of the phos-
phate recognition pocket unfilled. With these observations, we
generates two strategies to improve potency: (1) replace the car-
boxylic acid with acid isosteres of different sizes to interact with
the charged pocket, and (2) extend the linker between the amido
carbon and phenyl group to extend the charge interaction and
maintain the dual edge-on interactions with Phe-134 and His-
157 in the proline pocket.
R
n
Ki (μM)
14 H
9a H
9b F
1
0
0
0
39
11
4
H
(R)
N
(CH2)n
O
R
9c CF3
8.5
pKa:7.87
pKa: 3.73
O
O
N
H
H
SO2CH3
N
To test our first hypothesis, we investigated various acid isos-
teres, including tetrazoles, acylaminothiazoles, and acylsulfona-
N
N
H
S
N
H
O
O
mides.21 Tetrazole analogs with an amide at the
a-carbon (‘a-
tetrazoles’) are readily synthesized via a three step sequence from
the corresponding aminoesters (6, Scheme 2) whereas the tetra-
zoles with an amide at the b-carbon (‘b-tetrazoles’) are prepared
from Boc-protected aminoalcohol (10) in five steps.20 In general,
F
F
15 (Ki = 57μM)
16 (Ki = 53μM)
Figure 4. Pin1 inhibitors with a carboxylic acid isostere.
a-tetrazoles show comparable activities with their carboxylic acid
Route to α-tetrazoles:
N
N
H
H
N
R1
N
CONH2
R1
CN
H
NaN3, Et3N*HCl
NMP, 150ºC
N
R1
N
1) NH3/MeOH
H2N
CO2CH3
TFAA
N
H
R1 = 2-naphthyl
2) R1COCl
Et3N/CH2Cl2
O
O
O
Et3N/THF
R
R
R
8
9
6
7
R
(90% yield)
(42%% yield)
(50-70% yield)
Route to β-tetrazoles:
KCN
H
H
H
H
H
N
H
N
1) TFA/CH2Cl2
N
N
N
N
NaN3
MsCl
DMSO
Boc
CN
Boc
OH
Boc
OMs
N
CN
2) 2-napthoyl
chloride
Et3N/CH2Cl2
N
50ºC
O
pyridine
(94% yield)
N
Et3N*HCl
O
13
(56% yield)
14
(45% yield)
12
10
11
(74% yield)
Scheme 2. Route to a-tetrazoles.

L. Dong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2210–2214
2213
H
N
CO2H
THF
/
ArI
H (aq)
O
20
(~90% yield)
O
i
L
Pd(PPh3)2Cl2, CuI
DIEA/DMF
90ºC, 10 min.
1) acetyl chloride
EtOH
H
N
H
N
H2N
CO2H
CO2Et
Ar
CO2Et
1
)
O
H
H
N
18
O
2) 2-naphthoic acid
HATU
DIEA/DMF
O
E
19
(60-70% yield)
2
,
t
1
/
CO2H
17
0
A
(90% yield)
%
H
2
c
)
P
Ar
L
d
i
O
/
C
1) Ipc2BH/THF
2) CH3CHO
3) H2O
O
H
(aq)
T
F
21
(94% yield)
F
OTf
Ar
H
N
CO2H
H
N
1) Pd(PPh3)4,
H
N
I
CO2Et
CO2H
O
Na2CO3(aq), DME, 80ºC
2) LiOH(aq)/THF
O
O
1) Pd(PPh3)4/CsF
DME, 70ºC
2) LiOH(aq)/THF
24
F
23
(2-47% yield from 18)
B(OH)2
(9% yield from 18)
Scheme 3.
Table 2
O
23a
SAR of different linker analogs
MW: 345
NH
H
N
LogP: 2.79 (measured)
H
H
R2
OH
R2
N
R2
N
CO2H
CO2H
R3
CO2H
(R)
Solubility: > 347μM
HLM Extraction Ratio: 0.5
Caco 2Papp (10-6cm/sec): AB 2.98; BA 7.73
O
O
O
O
R3
R3
A
B
C
No.
Core
R2
R3
Ki (lM)
20a
20b
20c
20d
21
23a
23b
23c
24
A
A
A
A
B
C
C
C
C
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
2-Naphthyl
Ph
3-F-Ph
12.8
1.87
4.43
7
2-OH-Ph
2-thienyl
3-F-Ph
Ph
3-F-Ph
Ph
18
1.8
0.89
3.7
2
2-Benzothiophenyl
2-Naphthyl
Cyclohex-1-enyl
nated before saponification to the saturated chain (20). The alkene
analogs (23–24) are synthesized by conversion of the terminal
alkyne naphthamidoester 18 to the
a,b-unsaturated boronic acid
22 which is subsequently subjected to Suzuki coupling and
saponification.
All three different linker designs are tested for Pin1 inhibition
and the results are summarized in Table 2. There is no gain in po-
tency with an inserted saturated alkyl chain (21, Ki = 18 lM), pre-
Figure 5. X-ray co-crystal structure of 23a (magenta) with Pin1 K77Q, K82Q
construct (distance in A, PDB: 3JYJ, Ref. 20) and its alignment with X-ray structure
of 3f (cyan). The properties of 23a are shown above.
sumably due to the increased flexibility of the molecule that leads
to additional entropic penalty. On the other hand, the rigidity of-
fered by the unsaturated linkers increases the binding affinity by
an order of magnitude (e.g., 20, 23). In general, a fluorine in 3-posi-
tion of the phenyl ring seems to confer the greatest benefit in bind-
ing, and in the case of 23b it resulted in the first sub-micromolar
small molecular non-phosphate Pin1 inhibitor. The binding mode
of the styryl analogs (23a–c and 24) is confirmed with a co-crystal
protein structure of 23a, Figure 5. As expected, the binding of 23a
is similar to 3f, but also extends further into the binding pocket.
The phenyl ring moves deeper into the His-157/Phe-134 pocket,
and the carboxylate moves slightly outward. The water (w1) mol-
ecule mediating interaction between Arg-69 and inhibitor carbox-
ylate (in the structure of 3f) moves accordingly, but still interacts
with the carboxylate at 3.5 Å. This water makes a bidentate inter-
action to the second carboxylate oxygen. The partially disordered
Arg-69 no longer interacts with this water. This may explain the
longer interaction distance (3.5 Å) of carboxylate oxygen with the
w1 water molecule. Similar to 3f, the second carboxylate oxygen
interacts directly with Lys-63 (2.9 Å), but it no longer interacts
with Ser-154 via a water molecule (w2) in the charge pocket.
Selected carboxylate Pin1 inhibitors (Ki <10
Pin1 whole cell assay, but none of them show any significant bind-
ing activity (IC50 >50 M). These carboxylate inhibitors have rea-
lM) are tested in
l
sonable cell permeability (e.g., 23a: CACO2 AB = 2.98, Fig. 5),24
suggesting on-target potency instead of intrinsic cell permeability
may be the under-lined problem. Together with other more desir-
able ADME properties, such as reasonable metabolic stability (HLM
extraction ratio)25 and excellent solubility, these compounds vali-
dated our approach for replacing phosphate with a carboxylate.
These studies highlight the need for further improving binding po-
tency to Pin1.
In summary, structure-based design is used to guide the optimi-
zation of the phosphate group replacement, leading to the discov-
ery of a series of non-phosphate small molecular Pin1 inhibitors

2214
L. Dong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2210–2214
7. (a) Daum, S.; Fanghanel, J.; Wildemann, D.; Schiene-Fischer, C. Biochemistry
2006, 45, 12125; (b) Pani, E.; Menigatti, M.; Schubert, S.; Hess, D.; Gerrits, B.;
Klempnauer, K. H.; Ferrari, S. Biochim. Biophys. Acta 2008, 1783, 1121.
8. Guo, C.; Hou, X.; Dong, L.; Dagostino, E.; Greasley, S.; Ferre, R. A.; Marakovits, J.;
Johnson, M. C.; Matthews, D.; Mroczkowski, B.; Parge, H.; VanArsdale, T.;
Popoff, I.; Piraino, J.; Margosiak, S.; Thomson, J.; Los, G.; Murray, B. W. Bioorg.
Med. Chem. Lett. 2009, 19, 5613.
9. Wildemann, D.; Erdmann, F.; Alvarez, B. H.; Stoller, G.; Zhou, X. Z.; Fanghanel,
J.; Schutkowski, M.; Lu, K. P.; Fischer, G. J. Med. Chem. 2006, 49, 2147.
10. Wang, X. J.; Xu, B.; Mullins, A. B.; Neiler, F. K.; Etzkorn, F. A. J. Am. Chem. Soc.
2004, 126, 15533.
with low to sub-micromolar inhibitory activity. This study not only
confirms the feasibility of replacing the phosphate with a carbox-
ylate for Pin1 binding, but it also highlights the challenge of recov-
ering the loss of binding affinity due to significantly weaker
charge–charge interactions. The X-ray co-crystal structures of
these small molecular Pin1 inhibitors reveal the flexibility of Pin1
active site interactions on the molecular level as well as a unique
binding mode of the carboxylate in Pin1 phosphate recognition
pocket. These learnings may be applicable to other related drug
targets such as phosphatases or other PPIases.
11. At pH 7.4, the phosphoric acid of 1a likely exists as a double-charged anion.
ACD Lab predicted its pKa1 = 1.84 and pKa2 = 6.34.
12. Pin1 inhibitors with a phosphate group were found to have low permeability in
our Caco2 assay [i.e., Papp (10À6 cm/s): both A to B and B to A <0.1].
13. Do, Q.-Q. T.; Guo, C.; Humphries, P. S.; Marakovits, J. T.; Dong, L.; Hou, X.;
Johnson, M. C. PCT Int. Appl. WO 2006040646, 2006.
Supplementary data
14. Bayer, E.; Thutewohl, M.; Christner, C.; Tradler, T.; Osterkamp, F.; Waldmann,
H.; Bayer, P. Chem. Commun. (Cambridge) 2005, 516.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.033.
15. Daum, S.; Erdmann, F.; Fischer, G.; Feaux de Lacroix, B.; Hessamian-Alinejad,
A.; Houben, S.; Frank, W.; Braun, M. Angew. Chem., Int. Ed. 2006, 45, 7454.
16. Bao, L.; Kimzey, A. PCT Int. Appl. WO 2004093803, 2004.
17. Uchida, T.; Takamiya, M.; Takahashi, M.; Miyashita, H.; Ikeda, H.; Terada, T.;
Matsuo, Y.; Shirouzu, M.; Yokoyama, S.; Fujimori, F.; Hunter, T. Chem. Biol.
2003, 10, 15.
18. Zhao, S.; Etzkorn, F. A. Bioorg. Med. Chem. Lett. 2007, 17, 6615.
19. Based on retail sells, 26 of the top 200 marketed drugs in year 2008 contain
either carboxylic acids or tetrazoles, http://www.chem.cornell.edu/jn96/
outreach.html.
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