Design and synthesis of a new, conformationally constrained, macrocyclic small-molecule inhibitor of STAT3 via 'click chemistry'

Article abstract of DOI:10.1016/j.bmcl.2007.04.096

STAT3 is a promising molecular target for the design of new anticancer drugs. In this paper, we report the design and synthesis of a conformationally constrained macrocyclic peptidomimetic 2 via click chemistry. Compound 2 was determined to bind to STAT3

Full text of DOI:10.1016/j.bmcl.2007.04.096

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3939–3942
Design and synthesis of a new, conformationally
constrained, macrocyclic small-molecule inhibitor of
STAT3 via ‘click chemistry’
Jianyong Chen,a,b Zaneta Nikolovska-Coleska,a,b Chao-Yie Yang,a,b Cindy Gomez,a,b
Wei Gao,a,b Krzysztof Krajewski,c Sheng Jiang,c Peter Rollerc and Shaomeng Wanga,b,
*
aUniversity of Michigan Comprehensive Cancer Center, Department of Internal Medicine,
University of Michigan, Ann Arbor, MI 48109, USA
bUniversity of Michigan Comprehensive Cancer Center, Department of Pharmacology and Medicinal Chemistry,
University of Michigan, Ann Arbor, MI 48109, USA
cLaboratory of Medicinal Chemistry, National Cancer Institute-Frederick, National Institutes of Health, Frederick, MD 21702, USA
Received 10 January 2007; revised 26 April 2007; accepted 30 April 2007
Available online 3 May 2007
Abstract—STAT3 is a promising molecular target for the design of new anticancer drugs. In this paper, we report the design and
synthesis of a conformationally constrained macrocyclic peptidomimetic 2 via click chemistry. Compound 2 was determined to bind
to STAT3 with a Ki value of 7.3 lM in a competitive fluorescence-polarization-based binding assay, representing a promising initial
lead compound for further optimization.
Ó 2007 Elsevier Ltd. All rights reserved.
Constitutive activation of the signal transducers and
activators of transcription 3 (STAT3) is frequently de-
tected in human cancer specimens from patients with ad-
vanced disease and cancer cell lines, but not in normal
epithelial cells.1,2 Persistent activation of STAT3 signal-
ing has been demonstrated to contribute directly to
oncogenesis by stimulating cell proliferation and pre-
venting apoptosis in human cancer cells.1,2 STAT3 acti-
vation may not only provide a growth advantage,
allowing accumulation of tumor cells, but also confer
resistance to conventional therapies that rely on apopto-
tic machinery to eliminate tumor cells.1,2 STAT3 is an
important and specific molecular target for the design
of an entirely new molecularly targeted therapy for hu-
man cancer with constitutively active STAT3. Such ther-
apeutic agents should have low toxicity to the normal
cells without constitutive STAT3 signaling.1–4
docking sites on different cytokine receptors. STAT3
then becomes phosphorylated on a carbonyl terminal
tyrosine (Tyr705).1,2 Tyrosine phosphorylation of
STAT3 causes it to dimerize and translocate to the
nucleus and bind to specific promoter sequences on its
target genes.1–6 Dimerization of STAT3 is a decisive
event for its activation1–5 and blocking this dimerization
with a small-molecule antagonist is a very attractive
therapeutic approach to the development of a molecu-
larly targeted therapy for the treatment of human can-
cers in which STAT3 is constitutively activated.7–10
Two approaches are currently being pursued for the de-
sign of small-molecule antagonists to block STAT3
dimerization.7–10 The first is to design peptide-based
antagonists and peptidomimetics7,8 and the second is
to discover non-peptidic small-molecules.9,10 While a
number of peptide-based ligands can achieve quite high
binding affinities to STAT3, they are generally not
cell-permeable due to their peptidic nature and the
negatively charged phosphotyrosine group in the
ligands. For non-peptide small-molecule inhibitors,
the major advantage is their good cell permeability,7,8
but the inhibitors reported to date still have relatively
poor binding affinities to STAT3. Although they may
STAT3, recruited from cytosol, makes specific interac-
tions through its SH2 domain with phosphotyrosine
Keywords: STAT3; Conformationally; Constrained mimetic; Click
chemistry.
*
Corresponding author. Tel.: +1 734 615 0362; fax: +1 734 647
9647; e-mail: shaomeng@umich.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.096

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J. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3939–3942
be considered as potential lead compounds, their bind-
ing affinity and specificity to STAT3 must be signifi-
cantly improved. In this paper, we wish to present our
structure-based design, synthesis, and biochemical
evaluation of a conformationally constrained macrocy-
clic peptidomimetic as a new inhibitor of STAT3. The
present study represents our initial but an important
step toward our ultimate goal for the design of potent,
cell-permeable, conformationally constrained peptidomi-
metics that block STAT3 dimerization.
degradation and may also achieve higher binding affini-
ties to the target protein than its linear counterparts by
reducing the entropic cost associated with loss of con-
formational degrees of freedom upon binding to the tar-
get protein.
Based upon these considerations, we have designed com-
pound 2, in which the ring cyclization was accomplished
via ‘click chemistry’,11 a highly efficient synthetic meth-
od for coupling. Modeling showed that compound 2
interacts with STAT3 in a similar fashion as the linear
peptide 1 (Fig. 2) and predicted that compound 2 would
bind to STAT3 with a good affinity.
The phosphopeptide, acetyl-pY*LKTKF-amide
1
(Fig. 1), has been found to inhibit STAT3 dimerization8
and was the starting point in our design. The corre-
sponding peptide segment (pY*LKTKF) in a complex
with STAT3 (Fig. 2) in the crystal structure of the di-
meric STAT36 showed that the phosphotyrosine has
extensive electrostatic interactions with the side chains
of Lys591, Arg609, Ser611, and Ser613, the hydrophobic
side chains of leucine and phenylalanine residues inter-
act with several hydrophobic residues or hydrophobic
side chains of polar residues (Trp623 with leucine and
Cys712, Y640, Met648, Asn647 with phenylalanine) in
the protein, and the side chain of the threonine residue
forms a hydrogen bond with Y640 in STAT3. However,
the side chains of the two lysines are exposed to solvent
(Fig. 2). This provided us with an opportunity to cyclize
these two lysine residues to form a macrocyclic
ring, obtaining conformationally constrained, macrocy-
clic, new STAT3 inhibitors. Compared to linear
peptides, conformationally constrained, macrocyclic
peptidomimetics can be much more resistant to protease
The synthesis of compound 2 is shown in Scheme 1.
Boc-L-6-hydroxynorleucine (3) was condensed with
O-t-butyl-L-threonine methyl ester (4) to afford com-
pound 5. Compound 5 was treated with methanesulfo-
nyl chloride to give the methanesulfonate ester, which
was then converted to the azide by the treatment with
sodium azide in DMF at 90 °C. The methyl ester was
hydrolyzed to the carboxylic acid 6, condensation of
which with (S)-propargylglycine methyl ester furnished
compound 7. This was converted in 80% yield into the
key intermediate 812 via click chemistry11 in the presence
of CuSO4Æ5H2O/sodium ascorbate. Compound 8, whose
structure was confirmed by NMR spectroscopy,11 was
hydrolyzed to the acid 9, which was coupled with L-phe-
nylalaninamide to afford compound 10. Removal of the
Boc group gave 11 and coupling with N-Fmoc-L-leucine
furnished compound 12. Removal of the Fmoc in 12 fol-
lowed by coupling with Cbz-phosphotyrosine di-t-butyl
HO
HO
O
O
O
O
O
O
H
N
H
N
H
N
H
N
AcHN
AcHN
N
H
N
H
N
H
CONH
2
N
N
N
CON H
2
H
H
H
O
O
O
O
N
N
H O PO
N
2
3
OPO H
3 2
NH
NH
2
2
1
2
Figure 1. Design of a conformationally constrained, macrocyclic STAT3 inhibitor.
Figure 2. The STAT3 protein monomer is drawn in ribbon representation. STAT3 residues interacting with the ligand are also displayed in the stick
model. The peptide segment (pY*LKTKF) is colored in green (a), while the designed compound (2) is colored in yellow (b). Hydrogen bond
interactions are drawn as cyan dashed lines. The linkage sites are depicted as red lines.

J. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3939–3942
3941
O
O
O
O
BocHN
CO2H
BocHN
N
H
CO2H
BocHN
O
a
b,c,d
N
H
CO2CH3
+
H2N
CO2CH3
OH
N3
OH
3
4
5
6
O
O
O
O
H
H
N
e
f
BocHN
N
CO2CH3
BocHN
CO2CH3
N
N
H
H
O
O
N
N
N
N3
7
8
O
O
O
O
O
h
g
H
H
i
BocHN
N
CO2H
N
BocHN
N
H
N
H
N
H
CONH2
O
O
N
N
N
N
N
N
9
10
HO
HO
O
O
O
O
H
H
H
k, l
j
N
N
N
H2N
FmocHN
N
H
N
H
CONH2
N
H
N
CONH2
H
O
O
O
N
N
N
N
N
N
11
12
HO
HO
O
O
O
O
O
O
H
N
H
N
H
H
N
m, n, o
Cb zHN
AcHN
N
N
N
H
N
H
CONH2
N
N
H
N
CONH2
H
H
H
O
O
O
O
N
N
N
N
N
OPO3H2
N
OPO3ditBu
2
13
Scheme 1. Reagents and conditions: (a) EDCI/HOBt/i-Pr2NEt/DCM; (b) MsCl/i-Pr2NEt/DCM; (c) NaN3/DMF/90 °C; (d) 2 N LiOH/dioxane, then
1 M HCl; (e) (S)-propargylglycine methyl ester, EDCI/HOBt/i-Pr2NEt/DCM; (f) CuSO4Æ5H2O/Na ascorbate/H2O/t-BuOH; (g) 2 N LiOH/dioxane,
then 1 M HCl; (h) L-phenylalaninamide, EDCI/HOBt/i-Pr2Net/DCM; (i) 4 M HCl; (j) N-Fmoc-L-leucine, EDCI/HOBt/i-Pr2NEt/DCM; (k)
diethylamine/acetonitrile; (l) Cbz-phosphotyrosine di-t-butyl ester, EDCI/HOBt/i-Pr2NEt/DCM; (m) H2/10% Pd–C/MeOH; (n) acetic anhydride/
i-Pr2NEt/DCM; (o) TFA/TES/DCM, rt, 30 min.
ester yielded compound 13. Hydrogenation of 13 to
remove the Cbz group, followed by acetylation, and
removal of the two t-butyl groups afforded the designed
target compound 2.
sequence labeled with a fluorescence tag, 5-carboxy-
fluorescein (5-FAM) at the N-terminus and expressed
and purified a recombinant STAT3 protein (residues
127–722). It was determined that this fluorescently
tagged peptide binds to the recombinant STAT3 with
a Kd value of 50 nM, consistent with its high affinity
reported in the previous study.13 Using this FP-based
assay, it was determined that compounds 1 and 2 are
capable of competing with the binding of the fluores-
cently tagged peptide to STAT3 in a dose-dependent
manner with Ki values of 25.9 and 7.3 lM, respec-
tively (Fig. 3). Hence, compound 2 is 3 times more
potent than the lead peptide 1.
In order to determine quantitatively the binding affin-
ity of compounds 1 and 2 to STAT3, we have devel-
oped a fluorescence-polarization-based binding assay.
A previous study13 showed that a peptide with the
GpY*LPQTV sequence derived from gP120 protein
binds to STAT3 with a much higher affinity than
the native STAT3 peptide. We have therefore synthe-
sized a peptide with the b(Ala)-b(Ala)-G-pY*LPQTV

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J. Chen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3939–3942
References and notes
Compound # (Ki SD (μM))
Compound 2 (7.3 0.9)
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100
75
50
25
0
Compound 1 (25.9 2.5)
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Zhao, Y.; Pestell, R. G.; Albanese, C.; Darnell, J. E., Jr.
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1
2
[Inhibitor] (log [μM])
9. Song, H.; Wang, R.; Wang, S.; Lin, J. Proc. Natl. Acad.
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Figure 3. Competitive binding curves of compounds 1 and 2 to STAT3
as determined using a fluorescence-polarization-based binding assay.
10. Schust, J.; Sperl, B.; Hollis, A.; Mayer, T. U.; Berg, T.
Chem. Biol. 2006, 13, 1235.
11. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
K. B. Angew. Chem. Int. Ed. 2002, 41, 2596.
12. 1H NMR of key intermediate 8: 1H NMR (300 MHz,
DMSO-d6) d ppm 8.55 (d, J = 9.0 Hz, 1H), 7.89 (d,
J = 9.4 Hz, 1H), 7.51 (s, 1H), 6.49 (d, J = 7.8 Hz, 1H),
4.87–4.80 (m, 1H), 4.32–4.09 (m, 4H), 3.72–3.69 (m, 1H),
3.67 (s, 3H), 3.20–3.15 (m, 1H), 3.01–2.92 (m, 1H), 1.80–
1.30 (m, 4H), 1.35 (s, 9H), 1.01 (s, 9H), 1.02 (d, J = 6.0 Hz,
3H), 0.92–0.60 (m, 2H).
In summary, a new, conformationally constrained
macrocyclic compound 2 has been designed and syn-
thesized using intramolecular ‘click chemistry’ as an
inhibitor of STAT3. Compound 2 binds to STAT3
with a Ki value of 7.3 lM, and is a promising initial
lead compound for further optimization for the de-
sign and development of potent, cell-permeable,
small-molecule inhibitors of STAT3 as a new class
of anticancer drugs.
13. Schust, J.; Berg, T. Anal. Biochem. 2004, 330, 114.