Development of cyclic peptomer inhibitors targeting the polo-box domain of polo-like kinase 1

Article abstract of DOI:10.1016/j.bmc.2013.02.020

The polo-box domain (PBD) of polo-like kinase 1 (Plk1) is essentially required for the function of Plk1 in cell proliferation. The availability of the phosphopeptide-binding pocket on PBD provides a unique opportunity to develop novel protein-protein interaction inhibitors. Recent identification of a minimal 5-residue-long phosphopeptide, PLHSpT, as a Plk1 PBD-specific ligand has led to the development of several peptide-based inhibitors, but none of them is cyclic peptide. Through the combination of single-peptoid mimics and thio-ether bridged cyclization, we successfully demonstrated for the first time two cyclic peptomers, PL-116 and PL-120, dramatically improved the binding affinity without losing mono-specificity against Plk1 PBD in comparison with the linear parental peptide, PLHSpT. These cyclic peptomers could serve as promising templates for future drug designs to inhibit Plk1 PBD.

Full text of DOI:10.1016/j.bmc.2013.02.020

Bioorganic & Medicinal Chemistry 21 (2013) 2623–2634
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of cyclic peptomer inhibitors targeting the polo-box
domain of polo-like kinase 1
Ravichandran N. Murugan a, , Jung-Eun Park b, , Dan Lim c, Mija Ahn a, Chaejoon Cheong a,
Taeho Kwon b,d,e,f, Ky-Youb Nam g, Sun Ho Choi b,h, Bo Yeon Kim f, Do-Young Yoon e, Michael B. Yaffe c,
a,
Dae-Yeul Yu d, Kyung S. Lee b, , Jeong Kyu Bang
⇑
⇑
a Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Cheongwon, Republic of Korea
b Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 3118, Bethesda, MD 20892,
United States
c Department of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
d Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
e Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
f World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea
g Bioinformatics and Molecular Design Research Center, B128A Yonsei University Research Complex, Shinchon-dong, Seoul 120-749, Republic of Korea
h Dong-A Pharmaceutical Co., Ltd., Research Laboratories, Yongin 449-905, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The polo-box domain (PBD) of polo-like kinase 1 (Plk1) is essentially required for the function of Plk1 in
cell proliferation. The availability of the phosphopeptide-binding pocket on PBD provides a unique oppor-
tunity to develop novel protein–protein interaction inhibitors. Recent identification of a minimal 5-res-
idue-long phosphopeptide, PLHSpT, as a Plk1 PBD-specific ligand has led to the development of several
peptide-based inhibitors, but none of them is cyclic peptide. Through the combination of single-peptoid
mimics and thio-ether bridged cyclization, we successfully demonstrated for the first time two cyclic
peptomers, PL-116 and PL-120, dramatically improved the binding affinity without losing mono-specific-
ity against Plk1 PBD in comparison with the linear parental peptide, PLHSpT. These cyclic peptomers
could serve as promising templates for future drug designs to inhibit Plk1 PBD.
Received 11 January 2013
Revised 22 January 2013
Accepted 6 February 2013
Available online 27 February 2013
Keywords:
Polo-box domain (PBD)
Cyclic peptomers
Kinase inhibitors
Solid phase peptide synthesis
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
from a lack of target specificity. Therefore, to achieve a high degree
of specific inhibition, while avoiding the competition with a high
Protein kinases comprise a large family of enzymes that phos-
phorylate specific protein substrates using the terminal phosphoryl
group of adenosine triphosphate (ATP). A large body of evidence
suggests that phosphorylation frequently alters normal cellular
functions, such as cell metabolism and proliferation, which are
associated with various disease states in humans.1 As a result, pro-
tein kinases constitute the second largest group of attractive drug
targets behind membrane-associated receptors, including G pro-
tein-coupled receptors. Over the years, various attempts have been
made to generate potent inhibitors against clinically important ki-
nases by inhibiting their catalytic activity. However, largely due to a
significant level of structural similarities among the ATP-binding
sites of the catalytic domains, these efforts have greatly suffered
concentration of intracellular ATP, targeting specific protein–pro-
tein interaction has emerged as an attractive alternative strategy.2
Polo-like kinase 1 (Plk1) is a member of the polo-like kinases
family (Plk1–5 in humans; collectively termed Plks) that plays a
critical role in regulating mitotic progression and cell proliferation.
Plk1 is overexpressed in a broad spectrum of human cancers. A ele-
vated Plk1 activity is thought to promote tumorigenesis, and is
considered an attractive anti-cancer drug target. On the other
hand, Plk2 and Plk3 appear to play a role in checkpoint-mediated
cell cycle arrest to maintain genetic stability and prevent onco-
genic transformation.3 Therefore, specific inhibition of Plk1, but
not Plk2 or Plk3, could be important for anti-Plk1 cancer therapy.4
Like other Plks, Plk1 contains a characteristic polo-box domain
(PBD) within its C-terminal non-catalytic region. A large body of
evidence suggests that PBD interacts with phosphoserine/phos-
phothreonine (pS/pT)-containing motifs and directs the N-terminal
catalytic activity to specific subcellular localizations.3 Extensive
studies on the interaction between Plk1 and its centromere/kineto-
chore-associated binding target, called polo-box-interacting
⇑
Corresponding authors. Tel.: +1 301 496 9635; fax: +1 301 496 8419 (K.S.L.);
tel.: +82 43 240 5023; fax: +82 43 240 5059 (J.K.B.).
E-mail addresses: kyunglee@mail.nih.gov (K.S. Lee), bangjk@kbsi.re.kr
(J.K. Bang).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.020

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R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
protein 1 (PBIP1),5 led to the discovery of a 5-residue-long se-
quence, PLHSpT (residues 74–78 of PBIP1), as a minimal peptide
motif that binds to Plk1 PBD with high affinity and specificity.6
Since then, several attempts have been made to generate the linear
PLHSpT derivatives with enhanced anti-Plk1 PBD except the one to
synthesize the cyclic peptides with improved binding affinity.7–11
In this study, using PLHSpT-based peptide–peptoid hybrids
(peptomers), we generated a group of cyclized peptomer deriva-
tives that potently inhibit Plk1 PBD with a high degree of selectiv-
ity. Given the advantages offered by cyclized peptomers in terms of
binding affinity and mono-specificity, these inhibitors may serve as
a template for developing clinically valuable anti-Plk1 inhibitors.
In addition, our study reveals for the first time structural elements
that are crucial to providing cyclic constraints for thioether-
bridged cyclic peptomers and to achieving potent inhibition of
Plk1 PBD. The structure-based approach described here may pro-
vide valuable insights into the future design of selective small-mol-
ecule Plk1 inhibitors.
Figure 1. First-phase cyclic peptide or peptomer derivatives (categories I–III)
generated using solid-phase peptide synthesis (SPPS).
groups to replace the N-terminal Pro of PLHSpT.12 This was
accomplished by substituting the Pro-4 residue with a peptoid
backbone via a submonomer method13 that utilizes a vast number
of aromatic amines as starting materials, mimicking the side
chains of amino acid residues. This method eventually led to the
generation of diverse (aromatic, charge, size, heteroaryl, length,
etc.) structural scaffolds that may target the broader pyrrolidine
binding region.14
2. Results and discussion
2.1. Structure-guided design of cyclic peptomers
To design conformationally constrained cyclic peptomers that
might best mimic the phosphopeptide-binding pocket of Plk1
PBD, we first closely examined the binding nature of Plk1 PBD in
complex with phosphopeptide ligands. In particular, analysis of
the structure of Plk1 PBD in complex with PLHSpT (PDB ID:
3HIK) revealed that the C-terminal pT residue is essential for
high-affinity binding, while the N-terminal Pro residue is crucial
in conferring the specificity by docking its side chain into a hydro-
phobic core surrounded by the Trp414, Phe535, and Arg516 resi-
dues. The specificity appears to stem from the fact that the N-
terminal Pro residue fails to interact with the PBDs of Plk2 and
Plk3, which possess the Lys and Tyr residues at positions corre-
sponding to the Arg516 and Phe535 residues, respectively, in
Plk1 PBD.6 In addition, the ‘broader’ pyrrolidine-binding region of
PBD surrounded by Trp414 and Phe535 suggests the presence of
an unexplored binding surface on PBD (Scheme 1).
By exploiting the broader pyrrolidine-binding pocket described
above, we generated cyclic peptomer inhibitors against Plk1 PBD
that exhibit an improved binding affinity while retaining Plk1
PBD specificity. To obtain diverse cyclic derivatives, a library
was made by employing both head-to-tail and side chain-to-tail
cyclization methods (Figs. 1 and 3). These methods allowed us
to achieve skeletal diversity by forming amide-, thioether-, or tri-
azole-bridged cyclic analogues. To explore the above broader pyr-
rolidine-binding region surrounded by the Trp414 and Phe535
residues of Plk1 PBD, we utilized the pi–pi mediating functional
2.2. Peptide synthesis and preliminary evaluation
All the linear precursors were prepared with the standard
Fmoc-based solid-phase peptide protocol using either Rink amide
or Chloro-trityl resin. Our first-phase derivatization efforts for cyc-
lic peptides or peptomer (Figs. 1 and 3) were divided into four cat-
egories. The first category contained the amide-bridged cyclic
peptides (PL-1 to PL-3) either bearing all the key residues of the
parent PLHSpT peptide (PL-1), bearing Gly in replacement of Pro
(PL-2), or lacking the Pro residue (PL-3). To facilitate selective
cleavage and amide cyclization on resin, the Fmoc-Glu-OAll amino
acid was attached to Rink amide resin via its side-chain acid func-
tionality, followed by the sequential attachment of the Fmoc-pro-
tected amino acids on the growing chain (PL-1 to PL-3).
Subsequent deprotection of the allyl group using Pd(PPh3)4, fol-
lowed by head-to-tail cyclization in the presence of a peptide-cou-
pling agent, PyBrOP, yielded the cyclic peptides PL-1 to PL-3. For
the second thioether-bridged category, Fmoc-Cys(Trt)-OH, instead
of Fmoc-Glu-OAll, was attached to the C-terminus of the 5-mer
LHSpTA (PL-4) or PLHSpT (PL-5) and acylation was performed at
the N-terminus using bromoacetic acid in the presence of DIC
(N,N0-Diisopropylcarbamide) to facilitate the side chain-to-tail
cyclization. Our third category contained a single triazole-bridged
Scheme 1. Binding nature of linear peptide, PLHSpT with Plk1 PBD, and illustration of the peptidomimetic cyclization.

R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
2625
cyclic peptide (PL-50) that was generated through the condensa-
tion (i.e., 1,3-dipolar cycloaddition reaction) between the
N-terminal 5-azidopropanoic acid (Scheme 3) and the C-terminal
propargylamine of the linear peptide in the presence of CuI and
2,6-lutidine. Finally, for the fourth category, we utilized the power
of peptoid mimics to conduct an extensive structure–activity rela-
tionship (SAR) investigation on the chosen bioactive cyclic scaffold
from the above three different categories (amide, thioether, and
triazole-bridged peptides). The competitive inhibitory activity of
the above cyclic peptides was evaluated using the ELISA-based
Plk1 PBD inhibition assay described previously.6
Using the thioether template (Supplementary Scheme S1), we
generated cyclic peptide or peptomers that either containing the
key LHSpTC residues (PL-12), N-shifted Leu (Nleu) substitution
on PL-12 (PL-11, Scheme 2), different lengths of the aromatic
substituted side chains (PL-7, 8, and 9) diverse electronegative
atoms (PL-6, 10, and 22), or aromatic groups (PL-8, 21, and 23).
Similarly extensive structural variation was also made using dou-
ble-peptoid mimics of cyclic peptomers (PL-13 to PL-20, Fig. 3).
From these efforts, we obtained eight confirmed hits (7–10, 15,
19, 22, and 23) when compared to the parental PLHSpT peptide
(IC50 = 32.03 lM) (Fig. 4 and Supplementary Table S1). As ex-
From the results of the cyclic peptide derivatives (PL-1 to PL-5
and PL-50; see Fig. 1), we found that the thioether-bridged cyclic
analogue PL-5 (IC50 = 9.80 lM) inhibited Plk1 PBD more efficiently
pected, cyclic peptomers, PL-11 and PL-12, lacking the Pro residue
at their N-termini failed to inhibit Plk1 PBD. Among the cyclic
peptomers from the first phase of derivatization, PL-23
than other cyclized peptides (PL-1–PL-4 and PL-50) (Fig. 2a).
Molecular modeling studies on PL-5 suggested that the pThr resi-
due of PL-5 coordinated with the two side chains of the His538
and Lys540 residues through ionic interactions, and the N-terminal
Pro residue interacted with the Arg516 residue through an H-
bonding interaction (Fig. 2b). In contrast to PL-5, both amide-
and triazole-bridged cyclic peptides (PL-1–PL-3 and PL-50, respec-
tively) failed to dock into the phosphopeptide-binding region of
Plk1 PBD with the electrostatic (pThr) and hydrophobic (Pro) inter-
action-mediating residues (Supplementary Fig. S1), resulting in the
loss of inhibitory activity. Therefore, we utilized the thioether scaf-
fold in our fourth category as a core skeleton for further modifica-
tions (Fig. 3).
(IC50 = 8.50 lM; Supplementary Table S1) containing an indole
moiety showed the highest binding affinity (Fig. 4). Additionally,
a gain in the inhibitory activity of PL-7 was revealed by testing
alkylated phenyl groups with different lengths of methylene spac-
ers (PL-7, PL-8, and PL-9; Figs. 3 and 4a). Interestingly, the two
most potent cyclic peptomers, PL-23 and PL-7, closely resembled
each other in terms of possessing aromaticity at the Pro-4 position.
These findings support our hypothesis that the occupation of the
border pyrrolidine-binding region using the pi–pi stacking inter-
acting moieties is important to improve the binding affinity, and
further suggest that optimizing both carbon tethering and aroma-
ticity is critical to achieving a potent inhibition against Plk1 PBD.
Another interesting finding was that single peptoid–substituted
cyclic peptomers were more active than double peptoid–substi-
tuted peptomers (compare PL-23 with PL-15 and PL-10 with PL-
16). This observation supports the argument that, in comparison
to highly flexible double peptoid–substituted derivatives, single
peptoid–substituted derivatives may provide optimum flexibility,
which is important for assuming a favorable conformation for
Plk1 PBD inhibition.15 The SAR analyses also suggested that a free
OH-substituted aromatic derivative (PL-22) has greater inhibitory
activity than a chloro-substituted cyclic peptomer (PL-6, Fig. 3).
Among the cyclic peptomers containing two peptoids, PL-20 exhib-
ited a significantly increased level of Plk1 PBD inhibition, whereas
PL-13 displayed inhibitory activity similar to PLHSpT (Fig. 4b). This
binding difference led us to speculate that the methyl substitution
in PL-13 may have prevented its phenyl group from being properly
positioned to have pi–pi interactions with the Trp414 and Phe535
residues. Since PL-23 showed the highest binding affinity, we per-
formed a more detailed SAR study with PL-28, PL-29, and PL-30. To
our surprise, PL-28, bearing Trp in replacement of NTrp, signifi-
cantly lost its binding affinity in comparison to cyclic peptomer
PL-23 (Supplementary Fig. S2a). This finding suggests that confer-
ring optimum flexibility using peptoid mimics is crucial to attain-
ing potent inhibition of Plk1 PBD. Additionally, deleting either Leu
(PL-29) or both His and Leu (PL-30) from PL-23 dramatically de-
creased the binding affinity (Supplementary Fig. S2a). Taken to-
gether, our results provided above show that the broader
pyrrolidine-binding region can interact with diverse aromatic
groups, and this interaction can provide additional affinity to
Plk1 PBD binding.
2.3. Second-phase library design, synthesis, and screening
On the basis of our first phase of cyclic peptomer derivatization,
we found that both the four methylene linker (PL-7) and indole
group (PL-23) are important for Plk1 PBD binding (Fig. 4). Hence,
for the second phase of derivatization, we decided to further diver-
sify the Pro position (Fig. 5a) to try to determine other elements
critical for the binding of cyclic peptomers. We observed that com-
pound PL-24, bearing an indole group with a four methylene
spacer, exhibited an approximately twofold increase in Plk1 PBD
Figure 2. Quantification and binding nature of cyclic peptomers. (a) An ELISA-
based assay was used to determine the efficiency of Plk1 PBD inhibition by the
indicated first-phase cyclic peptide or peptomer derivatives. Representative graphs
are shown from three independent experiments. (b) PL-5 was docked into the
ligand binding site and energy minimized with respect to pThr residue pointing
toward the PBD of Plk1. This model predicts that the residues, such as pThr and Pro,
contribute to the high binding affinity and specificity against Plk1 PBD.

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R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
Figure 3. First-phase cyclic peptomers (category IV) generated using solid-phase peptide synthesis (SPPS).
Scheme 2. Reagents and conditions: (a) isobutylamine (2.2 equiv), THF, 0 °C, 2.5 h,
97%; (b) (1) 4 N NaOH, MeOH/1,4-dioxane (v/v, 1/4), rt, 0.5 h; (2) Fmoc-OSu, pH
8.5–9.0, 1,4-dioxane, rt, 1 h, 60% for two steps.
Scheme 3. Reagents and conditions: (a) sodium azide (1.4 equiv), DMSO, 45 °C,
24 h, quan; (b) (1) 2 N NaOH, MeOH, rt, 48 h.
binding affinity (IC50 = 4.75
lM) in comparison to that of the par-
ent cyclic peptide (PL-23, IC50 = 8.50
lM) (Fig. 5b, Supplementary
Fig. S2a and Supplementary Table S1). This finding suggests that
lengthening the methylene spacer from two (PL-23) to four (PL-
24, Scheme 4) is likely important for enhancing Plk1 PBD binding.
Next, we investigated whether the acetylation of the indole ring
in PL-24 (PL-113) influences its inhibitory activity against Plk1
PBD. We observed that the activity of PL-113 was very similar to
that of PL-24, although PL-113 exhibited a much higher synthetic
yield than PL-24. Intriguingly, however, lengthening the carbon
tethering from four (PL-113) to six (PL-119) or seven (PL-116) sub-
stantially improved Plk1 PBD inhibitory activity (Scheme 4 and
Supplementary Fig. S2b). Notably, PL-116, containing the longest
methylene spacer conjugated to the acetylated indole group, dis-
Figure 4. An ELISA-based assay was used to determine the efficiency of Plk1 PBD
inhibition by the indicated first-phase cyclic peptide or peptomer derivatives.
Representative graphs are shown from three independent experiments. ⁄p-13mer:
Ac-ETFDPPLHSpTAIY-NH2.
played one of the highest inhibitory activities (IC50 = 2.56
against Plk1 PBD, which was 3.3-fold higher than the parental cyc-
lic peptide PL-23 (IC50 = 8.50 M) (Fig. 5b and Supplementary
lM)
l
Table S1). A recent study showed that the C6H5(CH2)8– moiety con-
jugated to the His residue of PLHSpT conferred a substantially in-
creased binding affinity to Plk1 PBD by tightly binding to the
Tyr-rich hydrophobic channel residing across the well-character-
ized SpT dipeptide-binding pocket.7 Therefore, we investigated
whether adding the C6H5(CH2)8– moiety (Scheme 5) enhances
the ability of PL-116 to bind to Plk1 PBD as well. Remarkably, the
resulting compound, PL-120, exhibited approximately 2.5-fold
lower IC50 value (IC50 = 1.08
lM; Supplementary Table S1) than
PL-116 (IC50 = 2.56 M). These findings suggest that both the pep-
l

R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
2627
that of parent PLHSpT, we carried out a specificity test for these
derivatives against PBDs from closely related Plk1, 2, and 3. Plk4
PBD was not included because of the distinct binding nature of this
protein.17 The results showed that both of these cyclic peptomers
exhibited mono-specificity against Plk1 PBD (Fig. 6), even though
their binding affinities were ꢀ30-fold higher than parent PLHSpT.
To the best of our knowledge, PL-116 and PL-120 were the first
cyclic peptomers to exhibit isoform-selective inhibition against
Plk1 PBD.
2.5. Modeling studies on Plk1 PBD:ligand complex
To better understand the binding nature of two selected cyclic
peptomers, PL-116 and PL-120, in complex with Plk1 PBD, we used
the previously reported crystal structure of the Plk1 PBD–PLHSpT
complex6 as a template and performed molecular dynamics simu-
lation studies. These modeling studies suggested that the acety-
lated indole peptoid fragment was engaged in the cation–pi
interaction with the Arg579 residue present in the extended pyr-
rolidine binding region surrounded by Phe534, Arg579, and
Tyr582 residues. In addition, a strong hydrogen bond could be
formed between the amide bond close to the indole ring and the
backbone carbonyl group of Arg518 (Fig. 7a and Supplementary
Fig. S3a). This model also suggested that the peptoid fragment from
PL-116 failed to fit into the Tyr-rich hydrophobic channel, which
was previously disclosed by Burke and co-workers.7 These predic-
tions are consistent with the molecular models obtained with PL-
120 (Fig. 7b and Supplementary Fig. S3b) that comprises two dis-
crete binding elements for the Plk1 PBD—the His-2-dependent
Tyr-rich hydrophobic channel and the Pro-4-dependent hydropho-
bic region.
In this study, we synthesized two cyclic peptomers, PL-116
and PL-120, containing either an indole fragment alone as a pep-
toid mimic (PL-116) or both an indole fragment and the previ-
ously described His7 (PL-120), and compared their potency with
that of the parental linear PLHSpT in vitro. The cyclic peptomer,
PL-120, bearing two pharmacophoric groups binding to the oppo-
site sides of the PLHSpT backbone, exhibited a twofold increase in
Plk1 PBD inhibition activity over PL-116. Single peptoid mimics
and thioether-bridged cyclization appeared to be the two ele-
ments that are critical to imposing a highly potent inhibition
against Plk1 PBD. We successfully demonstrated that our two
mono-specific cyclic peptomers, PL-116 and PL-120, dramatically
improved binding affinity (ꢀ30 times) when compared with the
linear parental peptide, PLHSpT. These cyclic peptomers could
serve as promising templates for future drug designs to inhibit
Plk1 PBD.
Figure 5. Compounds generated from the second-phase diversification and quan-
tification of their inhibitory activities against Plk1 PBD. (a) Chemical structures of
second-phase cyclic peptomer derivatives. (b) ELISA-based Plk1 PBD-binding IC50
graph. O.D., optical density.
A representative graph from three independent
experiments is shown. ⁄p-13mer: Ac-ETFDPPLHSpTAIY-NH2.
toid moiety and the C6H5(CH2)8-conjugated His adduct are critical
for the binding of PL-120 to Plk1 PBD (Fig. 5b).
2.4. Impact on selectivity
Restricting an inhibitor’s conformation to a structure recog-
nized by a target protein(s) is thought to increase potency by low-
ering the entropic barrier to complex formation. This restriction
could also potentially enhance the specificity of the inhibitor by
limiting its interaction modes with the target protein.16 To exam-
ine the binding specificity of PL-116 and PL-120 in comparison to
Scheme 4. Reagents and conditions: (a) acid (1.05 equiv), DCC (2 equiv), DCM, rt, overnight, 90–99%; (b) TFA/CH2Cl2 (v/v, 1/1), rt, 1 h; (c) DMAP (0.2 equiv), Et3N (1.5 equiv),
Ac2O (1.9 equiv), DMF, 80 °C, 24 h, 69–76%.

2628
R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
Scheme 5. Reagents and conditions: (a) HOBt (1.5 equiv), DCC (1.5 equiv), THF, rt, overnight, quan; (b) (1) Tf2O (1.1 equiv), 8-phenyl-1-octanol (1.1 equiv), DIEA (1.1 equiv),
CH2Cl2, ꢁ75 °C to rt, 16 h; (2) TFA (10 equiv), TIPS (1.1 equiv), CH2Cl2, rt, 2 h, quan for two steps; (c) 2 N HCl/1,4-dioxane (v/v, 1/1), 100 °C, 3 h, 64%.
Figure 6. Comparison of fluorescence polarization-based assays showing the selective inhibition of Plk1 PBD over Plk2 and 3 PBDs by PL-116 and PL-120.
Figure 7. The binding nature of cyclic peptomers. (a) A molecular model of PL-116 docked into the ligand binding site and energy-minimized with respect to the pThr residue
pointing toward the PBD of Plk1. (b) A molecular modeling of Plk1-PBD in complex with PL-120. The modeling was done using Discover 2.98/Insight II with CVFF force field.

R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
2629
of 1.0 mL/min, detection at 230 nm or Vydac HPLC columns (C18
,
3. Experimental
250 ꢂ 10 mm, 5
l
) with a linear gradient from 10% aqueous aceto-
nitrile (0.05% trifluoroacetic acid) to 90% acetonitrile (0.05% triflu-
oroacetic acid) over 30 min at a flow rate of 2.5 mL/min, detection
at 230 nm.
3.1. General experimental
All reactions requiring anhydrous conditions were conducted in
flame-dried reaction vessels under a positive pressure of Ar. All re-
agents were purchased from Aldrich Company. For the chromatog-
raphy analysis, HPLC-grade solvents such as hexane, ethyl acetate,
methylene chloride, and methanol were used. Thin-layer chroma-
tography (TLC) was performed on analytical Merck silica gel 60
F254, and were visualized under UV light first, then by using a nin-
hydrin solution (approx. 0.1% w/v in ethanol) followed by warm-
ing. Flash chromatography was performed on Merck silica gel 60
(230–400 mesh). NMR spectra were recorded on a Bruker Avance
300 spectrometer at 300 and 75.5 MHz for 1H and 13C, respectively,
with CDCl3. All organic extracts were dried using magnesium sul-
fate and concentrated under reduced pressure by rotary evapora-
tion. Reverse-phase HPLC analysis (RP-HPLC) was carried out on
an Agilent HPLC system equipped with a C18 analytical column
3.5. Peptide synthesis using Fmoc-Glu-OAll
Peptides were synthesized by a three-step synthesis method in
a 0.061 mmol scale with Rink Amide Resin to produce an amide C-
terminus using manual solid phase peptide synthesis. An example
for synthesizing designed cyclo [PLHSpTQ], PL-1 was shown below.
Standard Fmoc strategy as described above was used in Step 1. Re-
moval of the Allyl group in Step 2 was performed manually by
2.0 equiv of Pd(PPh3)4 in a mixture solvent of AcOH/CHCl3/NEM
(2/37/1) for 12 h, followed by washing using MeOH, DCM and
ether. Then, 20% piperidine in DMF was transferred to the reaction
vessel to remove the Fmoc. Peptides were cleaved by TFA at room
temperature for 2 h, followed by filtration, precipitation in cold
ether, centrifugation, and drying under vacuum. The resulting lin-
ear peptides were cyclized in Step 3 using 5.0 equiv of PyBrop in
DIEA at 50 °C. Crude peptides were further purified by reverse
phase-HPLC.
(250 ꢂ 10 mm, 5
l). Two different linear gradients of 0.05% aq
TFA (eluent A) and 0.05% TFA in CH3CN (eluent B) were used with
a flow rate of 2.5 mL/min at 25 °C.
Amines (6–10, 13, 15, 18, and 22) were purchased from Sigma–
Aldrich. Fmoc-NLeu-OH,18 5-azidopropanoic acid,19 amines20 (24,
113, 116 & 119), and Fmoc-[N(p
)-(CH2)8C6H5]-His-OH21 were pre-
3.5.1. First phase cyclic peptide or peptomer derivatives–amide-
bridged cyclic peptides
pared according to the indicated literature procedures (Supple-
Cyclic peptide, PL-1. Synthesis on 0.062 mmol scale. Purity of
crude product 43% (C18 RP-HPLC). Yield after purification: 11 mg,
24%; white powder; RP-HPLC tR = 17.07 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 745.8 [M+H]+.
mentary Fig. S4)
3.2. General procedures for peptide synthesis
Cyclic peptide, PL-2. Synthesis on 0.058 mmol scale. Purity of
crude product 42% (C18 RP-HPLC). Yield after purification: 9 mg,
22%; white powder; RP-HPLC tR = 13.75 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 702.1 [Mꢁ1].
All peptides were prepared by Fmoc SPPS methods using Rink
amide or Chloro-trityl resin with an initial loading of 0.61 mmol/
g, unless otherwise noted. Fmoc-Thr(PO(OBzl)OH)-OH and other
Fmoc(fluorenylmethoxycarbonyl) protected amino acids were pur-
chased from Novabiochem. Resins were swollen in N,N0-dimethyl-
foramide (DMF) for 45 min prior to synthesis. For sequence
extension, the Fmoc-protected amino acid (5.0 equiv) was acti-
vated by treatment with 1-O-benzotriazole-N,N,N0,N0-tetramethyl-
uronium-hexafluoro-phosphate (HBTU) (5.0 equiv) and hydroxy-
benzotriazole (HOBt) (5.0 equiv) in DMF (2 mL) for 2 min. This
solution was added to the free amine on resin along with N,N-
diisopropylethylamine (10.0 equiv) and the coupling reaction was
allowed to proceed for 1 h with Vortex stirring. After washing with
DMF, Fmoc deprotection was achieved with 20% piperidine in DMF
(1 ꢂ 10 min, 2 ꢂ 3 min). The resin was washed once again, and the
process was repeated for the next amino acid and finally the resin
was washed with DMF, methanol, dichloromethane and ether, and
then dried under vacuum.
Cyclic peptide, PL-3. Synthesis on 0.062 mmol scale. Purity of
crude product 11% (C18 RP-HPLC). Yield after purification: 2 mg,
5%; white powder; RP-HPLC tR = 13.53 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 647.52 [M+H]+.
3.6. Peptide synthesis by thioether-bridged bond formation
As mentioned above, using Fmoc protection strategy, the linear
side-chain protected peptide was synthesized on the Rink amide
resin (0.61 mmol/g) coupled with the respective amino acids,
and the Fmoc group in the N-terminal of the resin-bound pro-
tected peptide was removed with 20% piperidine/DMF. Then, the
resin-bound protected peptide was N-terminally bromoacetylated
by (BrCH2CO)2O which was prepared by mixing 10.0 equiv of
BrCH2COOH and 5.0 equiv of DIC for 3 h at rt. The peptide
(0.061 mmol) was cleaved from the resin and dissolved in 10 mL
of water/CH3CN (1:1) mixture followed by the dropwise addition
of triethylamine to adjust the pH 8–9, repeatedly. Under the basic
conditions the N-bromoacetylated linear peptide cyclized sponta-
neously by intramolecular nucleophilic displacement of the bro-
mo group by cysteine thiol. The cyclization process was
monitored by HPLC. After 1 h at rt, the solution was acidified with
30% AcOH aq solution and lyophilized. The product was purified
by RP-HPLC.
3.3. Peptide cleavage
Linear peptides were cleaved from the resin with 5% triisopro-
pylsilane (TIS) and 5% H2O in trifluoroacetic acid (TFA, approxi-
mately 2 mL of TFA per 100 mg of resin) for 2 h. The cleavage
mixture was mixed with cold ether to precipitate the peptide
and then filtered. The filtrate was washed with cold ether, and
the peptide was used for further cyclization step.
In case of peptoid substitution in cyclic peptomers, after the
bromoacetylation step, the linear peptides on resin was coupled
with the respective primary amine aromatic derivatives (Supple-
mentary Fig. S4). Then, one more bromoacetylation step was car-
ried out on the secondary amine in order to facilitate the
cyclization in solution phase using cysteine thiol.
3.4. Analytical HPLC conditions
The purities of peptides were determined by using either Phe-
nomenex column (C18, 250 ꢂ 4.60 mm, 5
l) with a linear gradient
from 10% aqueous acetonitrile (0.05% trifluoroacetic acid) to 90%
acetonitrile (0.05% trifluoroacetic acid) over 30 min at a flow rate

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R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
3.6.1. Thioether-bridged cyclic peptides or peptomers having
single peptoid
Cyclic peptide, PL-4. Synthesis on 0.061 mmol scale. Purity of
crude product 25% (C18 RP-HPLC). Yield after purification: 5.5 mg,
12%; white powder; RP-HPLC tR = 15.52 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 750.26 [M+H]+.
(silica, eluent: ether) afforded H-NLeu-OEt (7.7 g, 48.4 mmol) as an
oil in 97% yield (eluent: ether); 1H NMR (300 MHz, CDCl3): d 4.19
(q, J = 7.2 Hz, 2H), 3.39 (s, 2H), 2.41 (d, J = 6.7 Hz, 2H), 1.74 (quintet,
1H), 1.56 (br s, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.93 (d, J = 6.7 Hz, 6H).
3.7.2. N-[(9H-Fluoren-9-yl)methoxycarbonyl]-N-
Cyclic peptide, PL-5. Synthesis on 0.061 mmol scale. Purity of
crude product 23% (C18 RP-HPLC). Yield after purification: 5.2 mg,
11%; white powder; RP-HPLC tR = 15.52 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 775.67 [M].
Cyclic peptomer, PL-6. Synthesis on 0.061 mmol scale. Purity of
crude product 44% (C18 RP-HPLC). Yield after purification:
11.2 mg, 21%; white powder; RP-HPLC tR = 18.25 (linear gradient,
10–90% B, 30 min); MS (MALDI-TOF) m/z = 874.91 [M+H]+.
Cyclic peptomer, PL-7. Synthesis on 0.055 mmol scale. Purity of
crude product 21% (C18 RP-HPLC). Yield after purification: 5.2 mg,
11%; white powder; RP-HPLC tR = 18.97 (linear gradient, 10–90%
B, 30 min); MS (MALDI-TOF) m/z = 868.51 [M+H]+.
Cyclic peptomer, PL-8. Synthesis on 0.061 mmol scale. Purity of
crude product 30.1% (C18 RP-HPLC). Yield after purification:
7.2 mg, 14%; white powder; RP-HPLC tR = 16.65 (linear gradient,
10–90% B, 30 min); MS (MALDI-TOF) m/z = 838.67 [Mꢁ1].
Cyclic peptomer, PL-9. Synthesis on 0.061 mmol scale. Purity of
crude product 17.5% (C18 RP-HPLC). Yield after purification: 4 mg,
8%; white powder; RP-HPLC tR = 21.08 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 826.62 [M+H]+.
Cyclic peptomer, PL-10. Synthesis on 0.062 mmol scale. Purity of
crude product 53.6% (C18 RP-HPLC). Yield after purification: 13 mg,
25%; white powder; RP-HPLC tR = 16.67 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 844.72 [M+H]+.
(isobutyl)glycine (11, Fmoc-NLeu-OH)
NaOH (4 N, 4.71 mL) was added to a solution of H-NLeu-OEt
(18.84 mmol, 3 g) in dioxane (66 mL) and MeOH (24 mL). After
stirring for 30 min at room temperature the reaction mixture
was concentrated in vacuo to give H-NLeu-ONa.
The sodium salt was dissolved in water (18 mL) and the pH ad-
justed to 9–9.5 with 2 N HCl. Then, Fmoc-OSu (19.78 mmol,
1.05 equiv, 6.67 g) in 1,4-dioxane solution was added dropwise,
and the reaction was continued with stirring at pH 8.5–9.0. After
1 h of stirring, the reaction was concentrated and worked up as fol-
lows: Excess Fmoc-OSu was washed out with ether, and the aque-
ous phase was neutralized using 2 N HCl to pH 2–3. Ethyl acetate
was used for extracting the resulting Fmocpeptoid. After back-
washing with saturated NaCl and drying over Mg2SO4, evaporation
of the solvent by a rotary evaporator gave the crude Fmoc-NLeu-OH
as an oil. Crystallization (ether/hexanes) afforded Fmoc-NLeu-OH,
11(4 g, 11.3 mmol) as a white solid in 60% yield (eluent: DCM/
MeOH/HOAc, 90/10/0.5, v/v).
Fmoc-NLeu-OH, 11: The NMR spectra clearly show the presence
of both rotamers.
1H NMR (300 MHz, CDCl3): d 7.73 (t, 2H), 7.51–7.58 (4 lines, 2H),
7.23–7.41 (m, 4H), 4.46, 4.54 (two d, J = 6.0 Hz, 2H), 4.16–4.25 (m,
1H), 3.87, 3.99 (two s, 2H), 2.93, 3.13 (two d, J = 7.5 Hz, 2H), 1.61,
1.82 (two septets, 1H), 0.72, 0.87 (two doublets, J = 6.8 Hz 6H). All
data were in agreement with the reported values.18
Cyclic peptomer, PL-21. Synthesis on 0.041 mmol scale. Purity of
crude product 31% (C18 RP-HPLC). Yield after purification: 2.9 mg,
8%; white powder; RP-HPLC tR = 12.88 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 870.18 [M+H]+.
3.7.3. Thioether-bridged cyclic peptomers having double
peptoid
Cyclic peptomer, PL-22. Synthesis on 0.036 mmol scale. Purity of
crude product 14% (C18 RP-HPLC). Yield after purification: 1 mg,
3%; white powder; RP-HPLC tR = 11.182 (linear gradient, 10–90%
B, 30 min); MS (MALDI-TOF) m/z = 872.20 [M+H]+.
Cyclic peptomer, PL-11. Synthesis on 0.041 mmol scale. Purity of
crude product 21.6% (C18 RP-HPLC). Yield after purification: 2.2 mg,
8%; white powder; RP-HPLC tR = 12.39 (linear gradient, 0–70% B,
30 min); MS (MALDI-TOF) m/z = 679.57 [M+H]+.
Cyclic peptomer, PL-23. Synthesis on 0.061 mmol scale. Purity of
crude product 13% (C18 RP-HPLC). Yield after purification: 2.5 mg,
5%; white powder; RP-HPLC tR = 12.48 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 877.31 [Mꢁ1].
Cyclic peptomer, PL-12. Synthesis on 0.061 mmol scale. Purity of
crude product 29.22% (C18 RP-HPLC). Yield after purification:
3.2 mg, 8%; white powder; RP-HPLC tR = 11.59 (linear gradient,
0–70% B, 30 min); MS (MALDI-TOF) m/z = 679.56 [M+H]+.
Cyclic peptomer, PL-13. Synthesis on 0.041 mmol scale. Purity of
crude product 30% (C18 RP-HPLC). Yield after purification: 3.2 mg,
9%; white powder; RP-HPLC tR = 13.72 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 838.30 [Mꢁ1].
Cyclic peptomer, PL-28. Synthesis on 0.061 mmol scale. Purity of
crude product 7% (C18 RP-HPLC). Yield after purification: 3.3 mg,
6%; white powder; RP-HPLC tR = 8.18 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 863.30 [Mꢁ1].
Cyclic peptomer, PL-29. Synthesis on 0.061 mmol scale. Purity of
crude product 10% (C18 RP-HPLC). Yield after purification: 0.5 mg,
1%; white powder; RP-HPLC tR = 10.20 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 766.30 [M+H]+.
Cyclic peptomer, PL-30. Synthesis on 0.061 mmol scale. Purity of
crude product 13% (C18 RP-HPLC). Yield after purification: 4.7 mg,
9%; white powder; RP-HPLC tR = 12.48 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 877.31 [Mꢁ1].
Cyclic Peptomer, PL-15. Synthesis on 0.031 mmol scale. Purity of
crude product 12% (C18 RP-HPLC). Yield after purification: 1.5 mg,
6%; white powder; RP-HPLC tR = 13.80 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 877.31 [Mꢁ1].
Cyclic peptomer, PL-16. Synthesis on 0.031 mmol scale. Purity of
crude product 26% (C18 RP-HPLC). Yield after purification: 3.6 mg,
14%; white powder; RP-HPLC tR = 13.84 (linear gradient, 10–90%
B, 30 min); MS (MALDI-TOF) m/z = 844.72 [M+H]+.
Cyclic peptomer, PL-18. Synthesis on 0.033 mmol scale. Purity of
crude product 31% (C18 RP-HPLC). Yield after purification: 4.9 mg,
17%; white powder; RP-HPLC tR = 12.56 (linear gradient, 10–90%
B, 30 min); MS (MALDI-TOF) m/z = 870.18 [M+H]+.
3.7. Synthesis of (N-[(9H-fluoren-9-yl)methoxycarbonyl]-N-
(isobutyl)glycine) (11, Fmoc-NLeu-OH) from 1
3.7.1. Synthesis of ethyl 2-(isobutyl amino)acetate (H-NLeu-OEt)
Ethyl bromoacetate (50 mmol, 5.54 mL) in THF (25 mL) was
added dropwise to a cooled (ice bath) solution of isobutylamine
(110 mmol, 10.9 mL) in THF (25 mL). After stirring for 2.5 h at room
temperature, the reaction mixture was concentrated in vacuo to re-
move THF and resuspended in ether. The mixture was filtered to re-
move isobutylaminehydrobromide, the residue washed with ether,
and the filtrate concentrated in vacuo. Column chromatography
Cyclic peptomer, PL-19. Synthesis on 0.031 mmol scale. Purity of
crude product 26% (C18 RP-HPLC). Yield after purification: 0.8 mg,
3%; white powder; RP-HPLC tR = 16.13 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 868.51 [M+H]+.
Cyclic peptomer, PL-20. Synthesis on 0.025 mmol scale. Purity of
crude product 11% (C18 RP-HPLC). Yield after purification: 0.6 mg,
3%; white powder; RP-HPLC tR = 13.93 (linear gradient, 10–90% B,
30 min); MS (MALDI-TOF) m/z = 838.67 [Mꢁ1].

R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
2631
3.8. Peptide synthesis using click chemistry
added to the flask and the resulting mixture was stirred at room
temperature overnight. The suspension was evaporated and fil-
tered off using ethyl acetate. The resulting solution was washed
with water and extracted with ethyl acetate. The combined organic
layers were combined and dried with anhydrous magnesium sul-
fate, filtered and concentrated by rotary evaporation to give brown
oil, 1.5 g, 4.67 mmol, 89%. The Boc group was then quantitatively
removed by treatment with 8 mL of 1:1 trifluoroaceticacid: CH2Cl2
for 1 h at rt. The final compound, 24 was obtained by removal of
solvents in vacuo.
Macrocyclization using ring-closing [1,3] dipolar cycloaddition
was undertaken using the protected monomeric sequences as de-
scribed below. Chloro-trityl resin (0.061 mmol) was used to syn-
thesize the fully side-chain-protected linear peptides which in
turn cleaved from the resin under mild acidic conditions (TFA,
DCM) affording compounds soluble in organic solvents.
The alkyne group was usually incorporated as an N-propargyl-
glycine unit. The symmetric anhydride of 2-bromoacetic acid
(5 equiv of anhydride relative to free amine) was prepared by mix-
ing the acid (10 equiv) with N,N0-diisopropylcarbodiimide (DIC,
5.0 equiv) in DMF at room temperature for 15 min. This mixture
was then added to the resin and allowed to react for 3 h with stir-
ring. The resin was washed and then treated with propargylamine
(20.0 equiv) in DMF for 12–16 h. After washing, coupling of the
next residue to the resultant secondary amine was achieved by
usual procedure using HBTU, HOBt and DIEA for 1 h with stirring.
Azide was incorporated by the attachment of 5-azidopropanoic
acid by the standard peptide coupling procedure.
3.10.1.1.
(24A).
tert-Butyl-5-(1H-indol-3-yl)-3-oxopentylcarbamate
lH NMR (300 MHz, CDCl3) d 8.82 (s, 1H), 7.57 (d,
J = 7.80 Hz, 1H), 7.35 (d, J = 8.10 Hz, 1H), 7.18 (t, J = 8.80 Hz, 1H),
7.10 (t, J = 6.90 Hz, 1H), 6.97 (s, 1H), 6.07 (s,1H), 5.22 (s, 1H), 3.55
(q, J = 6.60 Hz, 2H), 3.35 (s, 2H), 2.94 (t, J = 6.60 Hz, 2H), 2.26 (t,
J= 6.30 Hz, 2H), 1.46 (s, 9H).
3.10.1.2. 1-Amino-5-(1H-indol-3-yl)-pentan-3-one (24).
NMR (300 MHz, CDCl3) 7.21 (d, J = 7.62 Hz, 1H), 6.99 (d,
lH
d
Click chemistry proceeded smoothly in the presence of CuI and
2,6-lutidine and at room temperature for 16 h. Removal of the side-
chain protecting groups under acidic conditions followed by puri-
fication by preparatory HPLC to give a white powder.
J = 7.95 Hz, 1H), 6.77–6.64 (m, 2H), 6.72 (s, 1H), 3.16 (t,
J = 7.20 Hz, 2H), 2.78 (t, J = 6.33 Hz, 2H), 2.61(t, J = 7.14 Hz, 2H),
2.2 (t, J = 6.42 Hz, 2H).
Cyclic peptomer, PL-24. Synthesis on 0.061 mmol scale. Purity of
crude product 10% (C18RP-HPLC). Yield after purification: 1.3 mg,
2.2%; white powder; RP-HPLC tR = 17.85 (linear gradient, 10–90%
B, 30 min); MS (MALDI-TOF) m/z = 953.40 [M+3].
3.9. Synthesis of 3-azidopropanoic acid (50)
3.9.1. Synthesis of 3-azidopropanoate
Methyl 3-bromopropanoate (1.09 mL, 10 mmol) and sodium
azide (0.91 g, 14 mmol) were dissolved in DMSO (4.6 mL) in a
25 mL round bottom flask. The mixture solution was heated to
45 °C and stirred for 24 h. After the mixture was cooled down to
room temperature, and extracted with diethyl ether. The combined
organic layer was dried over anhydrous magnesium sulfate, fil-
tered, and concentrated on a rotary evaporator to give azidopro-
panoate as colorless liquid (1.29 g, quan).
3.11. Synthesis of 1-(1-acetyl-1H-indol-3-yl)-5-aminopentan-3-
one (113)
tert-Butyl-5-(1H-indol-3-yl)-3-oxopentylcarbamate,
24A
(0.309 g, 0.93 mmol) and DMAP (0.022 g, 0.18 mmol) were taken
in a round bottom flask fitted with reflux condenser under argon
atmosphere. DMF (2.2 mL) was added to the flask via syringe fol-
lH NMR (300 MHz, CDCl3) d 3.62 (s, 3H), 3.47 (t, J = 6.3 Hz, 2H),
2.87 (t, J = 6.6 Hz, 2H).
lowed by triethylamine (195
lL, 1.40 mmol), and acetic anhydride
(168 L, 1.77 mmol). The reaction was heated at 80 °C for 24 h.
l
Upon cooling to room temperature the reaction was diluted with
ethyl acetate and washed with a saturated solution of ammonium
chloride, and the aqueous layer was further extracted with ethyl
acetate. The combined organic layers were dried over magnesium
sulfate and the solvent evaporated. The brown residue was purified
by flash chromatography on silica gel with ethyl acetate/hexanes
as the solvent to give pale yellow solid, 0.240 g, 0.643 mmol,
69%. The Boc group was then quantitatively removed by treatment
with 1 mL of 1:1 trifluoroaceticacid/CH2Cl2 for 1 h at rt. The final
compound, 113 was obtained by removal of solvents in vacuo.
3.9.2. 3-Azidopropanoic acid (50)
In
a
50 mL flash, methyl 3-azidopropanoate (0.640 g,
4.96 mmol) was dissolved in 2 N NaOH (4.95 mL) solution. A small
amount of MeOH was added slowly to make it homogeneous solu-
tion. The mixture was stirred at room temperature for 48 h, and
then MeOH was removed under vacuo. The aqueous layer was
washed with Et2O, and then acidified with conc. HCl to pH 1. The
compound was extracted with diethyl ether, and the combined or-
ganic fractions were dried over anhydrous magnesium sulfate, fil-
tered, and concentrated on a rotary evaporator to give 50 as
brownish liquid.
3.11.1. tert-Butyl-5-(1H-indol-3-yl)-3-oxopentylcarbamate
(113B)
lH NMR (300 MHz, CDCl3) d 11.55 (s, 1H), 3.57 (t, J = 6.6 Hz, 2H),
2.63 (t, J = 6.6 Hz, 2H). All data were in agreement with the re-
ported values.19
Cyclic peptide 50. Synthesis on 0.061 mmol scale. Purity of crude
product 7% (C18 RP-HPLC). Yield after purification: 1.5 mg, 3%; RP-
HPLC tR = 12.82 min; (linear gradient, 10–90% B, 30 min) MS (MAL-
DI-TOF) m/z 838.97 [Mꢁ3].
lH NMR (300 MHz, CDCl3) d 8.40 (d, J = 7.74 Hz, 1H), 7.53 (d,
J = 7.26 Hz, 1H), 7.35–7.38 (m,1H), 7.33–7.30 (m, 1H), 7.26 (s,
1H), 6.19 (s, 1H), 5.21 (s, 1H), 3.60 (q, J = 6.84 Hz, 2H), 3.38 (q,
J = 6.24 Hz, 2H), 2.91 (t, J = 7.02 Hz, 2H), 2.55 (s, 3H) 2.36 (t,
J = 6.0 Hz, 2H), 1.41 (s, 9H).
3.11.2. 1-(1-Acetyl-1H-indol-3-yl)-5-aminopentan-3-one (113)
lH NMR (300 MHz, CDCl3) d 8.27–8.24 (m, 1H), 7.52–7.49 (m,
1H), 7.40 (s, 1H), 7.25–7.16 (m, 2H), 3.46 (t, J = 7.20 Hz, 2H), 3.09
(t, J = 6.51 Hz, 2H), 2.84 (t, J = 7.17 Hz, 2H), 2.52 (t, J = 6.53 Hz,
2H), 2.51 (s, 3H).
3.10. General synthetic procedure for the Indole analogues (24–
116)
3.10.1. Synthesis of 1-amino-5-(1H-indol-3-yl)-pentan-3-one
Cyclic peptomer, PL-113. Synthesis on 0.041 mmol scale. Purity
of crude product 20% (C18 RP-HPLC). Yield after purification:
5 mg, 12%; white powder; RP-HPLC tR = 14.95 (solvent gradient,
10–90% B, 30 min); MS (MALDI-TOF) m/z = 992.38 [M+H]+.
(24)
A
solution of N,N0-dicyclohexylcarbodiimide (DCC, 2.06 g,
10 mmol) and Boc-b-Ala-OH (0.993 g, 5.25 mmol) was stirred in
DCM (12 mL) for 10 min. Tryptamine (0.801 g, 5 mmol) was then

2632
R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
3.12. Synthesis of 1-(1-acetyl-1H-indol-3-yl)-7-aminoheptan-3-
one (119)
13C NMR (75 MHz, CDCl3) d 172.9, 156.2, 136.6, 127.3, 122.4,
122.0, 119.2, 118.6, 112.5, 111.4, 79.2, 40.4, 39.6, 36.5, 29.8, 28.4,
26.3, 25.3, 25.2.
3.12.1. Synthesis of tert-butyl-7-(1H-indol-3-yl)-5-
oxoheptylcarbamate (119A)
3.14.2. Synthesis of 1-(1-acetyl-1H-indol-3-yl)-8-aminooctan-3-
one (116)
The title compound is synthesized in a manner identical to com-
pound 113, absent of 24A; instead 116A (0.754 g, 2.02 mmol).
The title compound is synthesized in a manner identical to com-
pound 24A, absent of Boc-b-Ala-OH; instead Boc-5-Ava-OH (1.14 g,
5.25 mmol). Materials: Tryptamine (0.801 g, 5 mmol), and DCC
(2.06 g, 10 mmol) in 13 mL DCM. The compound is purified by col-
umn chromatography on silica gel, eluted with 4% EtOAc–Hexane.
Yield: Product 119A (1.7 g, 4.95 mmol, 99%).
Materials: DMAP (0.047 g, 0.386 mmol), triethylamine (423
lL,
3.03 mmol), and acetic anhydride (363 L, 3.84 mmol) in4.6 mL
l
DMF. The compound is purified by column chromatography on sil-
ica gel, eluted with 2% EtOAc–Hexane. Yield: Product 116B
(0.637 g, 1.53 mmol, 76%). The Boc group was then quantitatively
removed by treatment with 2.6 mL of 1:1 trifluoroaceticacid/
CH2Cl2 for 1 h at rt. The final compound, 116 was obtained by re-
moval of solvents in vacuo.
lH NMR (300 MHz, CDCl3) d 8.29 (s, 1H), 7.61 (d, J = 7.80 Hz, 1H),
7.39 (d, J = 8.04 Hz, 1H), 7.24–7.19 (m, 1H), 7.16–7.10 (m, 1H), 7.04
(d, J = 2.28 Hz, 1H), 5.59 (s, 1H), 4.59 (s, 1H), 3.61 (q, J = 6.06 Hz,
2H), 3.08 (q, J = 6.36 Hz, 2H), 2.99 (t, J = 6.69 Hz, 2H), 2.12 (t,
J = 7.32 Hz, 2H), 1.59 (q, J = 7.98 Hz, 2H), 1.48–1.41 (m, 2H) 1.43
(s, 9H).
3.14.2.1. tert-Butyl-8-(1-acetyl-1H-indol-3-yl)-6-oxooctylcarba-
3.13. 1-(1-Acetyl-1H-indol-3-yl)-7-aminoheptan-3-one (119)
mate (116B).
lH NMR (300 MHz, CDCl3) d 8.39 (d, J = 7.92 Hz,
1H), 7.54 (d, J = 7.32 Hz, 1H), 7.40–7.24 (m, 3H), 6.12 (s, 1H), 4.68
(s, 1H), 3.60 (q, J = 6.84 Hz, 2H), 3.06 (q, J = 6.36 Hz, 2H), 2.55 (s,
3H), 2.14 (t, J = 7.32 Hz, 2H), 1.69–1.54 (m, 2H), 1.51–1.44 (m,
2H), 1.42 (s, 9H), 1.36–1.23 (m, 4H).
The title compound is synthesized in a manner identical to com-
pound 113, absent of 24A; instead 119 A (1.11 g, 3.23 mmol).
Materials: DMAP (0.075 g, 0.62 mmol), triethylamine (675
lL,
4.85 mmol), and acetic anhydride (580 L, 6.14 mmol) in 7.5 mL
l
13C NMR (75 MHz, CDCl3) d 173.2, 168.5, 156.1, 135.9, 130.5,
125.3, 123.6, 122.6, 119.8, 118.8, 116.6, 79.1, 40.3, 38.9, 36.5,
29.7, 28.4, 26.3, 25.3, 25.2, 23.9.
DMF. The compound is purified by column chromatography on sil-
ica gel, eluted with 2% EtOAc–Hexane. Yield: Product 119B
(0.905 g, 2.25 mmol, 70%). The Boc group was then quantitatively
removed by treatment with 3.6 mL of 1:1 trifluoroaceticacid/
CH2Cl2 for 1 h at rt. The final compound, 119 was obtained by re-
moval of solvents in vacuo.
3.14.2.2.
(116).
1-(1-Acetyl-1H-indol-3-yl)-8-aminooctan-3-one
lH NMR (300 MHz, CDCl3) d 8.40 (d, J = 7.32 Hz, 1H),
7.69–7.63 (m, 1H), 7.58 (s, 1H), 7.41–7.29 (m, 2H), 3.59 (t,
J = 7.07 Hz, 2H), 2.98 (t, J = 7.04 Hz, 2H), 2.90 (t, J = 7.5 Hz, 2H),
2.68 (s, 3H), 2.23 (t, J = 7.31 Hz, 2H), 1.72–1.57 (m, 4H), 1.42–
1.32 (m, 2H).
Cyclic peptomer, PL-116. Synthesis on 0.041 mmol scale. Purity
of crude product 29% (C18 RP-HPLC). Yield after purification:
6.5 mg, 15%; white powder; RP-HPLC tR = 15.55 (solvent gradient,
10–90% B, 30 min); MS (MALDI-TOF) m/z = 1034.49 [M+H]+.
3.13.1. tert-Butyl-7-(1-acetyl-1H-indol-3-yl)-5-
oxoheptylcarbamate (119B)
lH NMR (300 MHz, CDCl3) d 8.43 (d, J = 8.16 Hz, 1H), 7.56 (d,
J = 7.11 Hz, 1H), 7.42–7.29 (m, 3H), 7.27 (s, 1H), 5.77 (s, 1H), 4.60
(s, 1H), 3.62 (q, J = 6.62 Hz, 2H), 3.11 (q, J = 6.45 Hz, 2H), 2.93 (t,
J = 6.86 Hz, 2H), 2.62 (s, 3H) 2.18 (t, J = 7.40 Hz, 2H), 1.71–1.59
(m, 2H), 1.44–1.54 (m, 2H), 1.43 (s, 9H).
3.15. Synthesis of N(a)-[(9H-fluoren-9-ylmethoxy)carbonyl-
3.13.2. 1-(1-Acetyl-1H-indol-3-yl)-7-aminoheptan-3-one (119)
lH NMR (300 MHz, CDCl3) d 7.61–7.38 (m, 1H), 7.37–7.13 (m,
1H), 7.12–6.85 (m, 3H), 3.43–3.28 (m, 2H), 3.06–2.90 (m, 2H),
2.88–2.61 (m, 4H), 2.18–2.04 (m, 2H), 2.57 (s, 3H), 1.35–1.21 (m,
2H).
Cyclic peptomer, PL-119. Synthesis on 0.041 mmol scale. Purity
of crude product 23% (C18 RP-HPLC). Yield after purification:
4.8 mg, 11%; white powder; RP-HPLC tR = 18.01 (solvent gradient,
10–90% B, 30 min); MS (MALDI-TOF) m/z = 1020.41 [M+H]+.
[N( )-(8-phenyl octyl)]-L
p
-histidine (120)
3.15.1. Synthesis of N(
[N( )-(trityl)]- -histidine methyl ester (C)
A 100 mL Schlenk flask is charged with solid Fmoc-His(Trt)-OH
a)-[(9H-fluoren-9-ylmethoxy)carbonyl-
s
L
(10 g, 16.14 mmol), HOBt (3.27 g, 24.2 mmol) and degassed by vac-
uum-Ar purge cycle. Dry THF (81 mL) is added to dissolve all solids
and the solution is cooled to ꢁ13 °C with an ice-acetone bath. To this
mixture, a dry THF solution (81 mL) of DCC (3.33 g, 16.14 mmol) is
added along with 12.4 mL of MeOH dropwise over ꢀ30 min. The
reaction is allowed to warm slowly to room temperature while stir-
ring overnight (ꢀ16 h), during which time solid dicyclohexylurea is
observed to precipitate. This is removed by vacuum filtration
through a medium-porosity frit and the solvent is removed by ro-
tary evaporation. The oily residue is redissolved in dichlorometh-
ane, washed three times with saturated NaHCO3 (aq), three times
with water, and the organic layer is dried over Mg2SO4. The solvent
is removed by rotary evaporation and a crude yellow solid is iso-
lated after drying. The compound is purified by column chromatog-
raphy on silica gel, eluted with 1% MeOH–DCM (Yield: 10.34 g,
quan.). The compound is purified by column chromatography on sil-
ica gel, eluted with 1% MeOH/DCM. (Yield: 6.89 g, 83.6%).
3.14. Synthesis of 1-(1-acetyl-1H-indol-3-yl)-8-aminooctan-3-
one (116)
3.14.1. Synthesis of tert-butyl-8-(1H-indol-3-yl)-6-
oxooctylcarbamate (116A)
The title compound is synthesized in a manner identical to com-
pound 24A, absent of Boc-b-Ala-OH; instead Boc-6-Ahx-OH (1.5 g,
6.49 mmol). Materials: Tryptamine (0.990 g, 6.17 mmol), and DCC
(2.55 g, 12.34 mmol) in 40 mL DCM. The compound is purified by
column chromatography on silica gel, eluted with 3% EtOAc–Hex-
ane. Yield: Product 116A (2.2 g, 5.90 mmol, 90%).
lH NMR (300 MHz, CDCl3) d 9.27 (s, 1H), 7.57 (d, J = 7.74 Hz, 1H),
7.35 (d, J = 8.07 Hz, 1H), 7.17 (t, J = 7.05 Hz, 1H), 7.08 (t, J = 7.86 Hz,
1H), 6.97 (d, J = 1.90 Hz, 1H) 5.98 (t, J = 5.44 Hz, 1H), 4.84 (s, 1H),
3.56 (q, J = 6.37 Hz, 2H), 3.10–2.99 (m, 2H), 2.95 (t, J = 6.78 Hz,
2H), 2.09–2.0 (m, 2H), 1.47 (s, 9H), 1.44–1.32 (m, 2H),1.27–1.17
(m, 2H).
lH NMR (300 MHz, CDCl3) d 7.75 (d, J = 7.5 Hz, 2H), 7.62 (t,
J = 7.0 Hz, 2H), 7.42–7.24 (m, 17H), 7.14–7.09 (m, 7H), 6.57 (s,
1H), 6.51 (d, J = 8.2 Hz, 1H), 4.38–4.22 (m, 2H), 4.12 (m, 1H), 3.63
(s, 3H), 3.06 (m, 2H).

R. N. Murugan et al. / Bioorg. Med. Chem. 21 (2013) 2623–2634
2633
3.15.2. Synthesis of N(
[N( )-(8-phenyloctyl)-
To a stirred solution of triflic anhydride (0.443 mL, 2.63 mmol)
a
)-[(9H-fluoren-9-ylmethoxy) carbonyl-
of the competitor peptides, and then incubated with constant rock-
ing for 1 h at 25 °C. Next, the plates were washed four times with
PBST, and to detect the bound HA-EGFP-Plk1, the plates were incu-
p
L-histidinemethylester (D)
in DCM (17 mL) under Ar at ꢁ75 °C was added a solution of 8-phe-
nyl-1-octanol (0.578 mL, 2.63 mmol) and diisopropylethylamine
(DIEA) (0.459 mL, 2.63 mmol) in DCM (12 mL) dropwise over
10 min. Stirring was continued at ꢁ75 °C (20 min), then a solution
of Fmoc-His(Trt)-OMe (1.5 g, 2.37 mmol) in DCM (20 mL) was
added dropwise, and the mixture was allowed to gradually warm
to room temperature over a period of 16 h. The mixture was
quenched using aqueous NaHCO3 and stirred vigorously (30 min).
The organic layer was diluted with DCM and washed with aqueous
NaHCO3 and brine, then dried (Mg2SO4), and concentrated to vis-
cous oil. To a solution of the resulting gum in DCM (13 mL) was
added trifluoroacetic acid (1.77 mL, 23.7 mmol) and triisopropylsi-
lane (TIS) (0.534 mL, 2.61 mmol), and the mixture was stirred at
room temperature until reaction was complete as shown by TLC
(2 h). The solvent was removed in vacuo, and the residue was puri-
fied by silica gel column chromatography using 1–5% MeOH in
DCM provided product as colorless gum (1.45 g, quan).
bated for 2 h with 100
tion of 0.5 g/mL in the blocking buffer and then washed five
times. Further, the plates were incubated with 100 L/well of sec-
lL/well of anti-HA antibody at a concentra-
l
l
ondary antibody at a 1:1000 dilution in the blocking buffer. After-
ward, the plates were washed five times with PBST and incubated
with 100 l
L/well of 3,30,5,50-tetramethylbenzidine (TMB) substrate
solution (Sigma, St. Louis, MO) until a desired absorbance was
achieved. The reactions were terminated by the addition of 1 N
H2SO4, and the optical densities were measured at 450 nm using
an ELISA plate reader (Molecular Device, Sunnyvale, CA). Data are
shown in Figure. 2, 4 and 5; Supplementary Figure S2.
3.17. Fluorescence polarization assays
5-Carboxyfluorescein-labeled peptides (5-carboxyfluorescein-
Ahx-DPPLHS-pT-AI-NH2 for Plk1 PBD, 5-carboxyfluorescein-Ahx-
GPMQTS-pT-PKNG for Plk2 PBD, and 5-carboxyfluorescein-Ahx-
GPLATS-pT-PKNG for Plk3 PBD; final concentrations: 25 nM) were
incubated with the bacterially-expressed purified PBDs of Plk1,
Plk2 and Plk3 (final concentrations: 15 nM, 50 nM, and 500 nM,
respectively) in a binding buffer containing 10 mM Tris (pH 8.0),
1 mM EDTA, 50 mM NaCl, and 0.01% Nonidet P-40. Indicated grad-
ual concentration of compounds was provided into the reaction.
Fluorescence polarization was analyzed 10 min after mixing of all
components in the 384-well format using a Molecular Devices
Spectra Max Paradigm Multi-Mode Microplate Detection Platform.
All experiments were performed in triplicate
lH NMR (300 MHz, CDCl3) d 8.37 (s, 1H), 7.77 (d, J = 7.41 Hz, 2H),
7.58 (m, J = 7.47 Hz, 1H), 7.38 (t, J = 7.14 Hz, 2H), 7.36–7.22 (m,
4H), 7.21–7.13 (m, 4H), 5.77–5.67 (m, 1H), 4.62–4.50 (m, 1H),
4.48–4.37 (m, 2H), 4.20 (t, J = 6.49 Hz, 1H), 4.04–3.93 (m, 2H)
3.78 (s, 3H), 3.27–3.13 (m, 2H), 2.58 (t, J = 7.77 Hz, 2H), 1.85–
1.71 (m, 2H), 1.67–1.49 (m, 2H) 1.29 (s, 8H). MS (MALDI-TOF) m/
z 580.17 [M+H]+.
3.15.3. N(
a
)-[(9H-Fluoren-9-ylmethoxy) carbonyl-[N(
p)-(8-
phenyloctyl)-
L
-histidine (120)
1:1 Mixture of 2 N HCl (48 mL) and 1,4-dioxane (48 mL) solu-
tion were added to the D (1.45 g, 2.5 mmol) and reflux at 100 °C
for 3 h. The mixture was brought to room temperature, and the sol-
vent was removed by Rota vapor. The resulting aqueous mixture
was extracted with DCM, washed with brine, dried (Mg2SO4), con-
centrated to viscous oil, and then the residue was purified by silica
gel flash chromatography from 5% to 20% MeOH in DCM to provide
acid, 120 as a light yellow gum (0.903 g, 1.6 mmol, 64% yield.
lH NMR (300 MHz, CDCl3) d 9.98 (br s, 1H), 8.37 (s, 1H), 7.74 (d,
J = 7.41 Hz, 2H), 7.56 (t, J = 6.42 Hz, 2H), 7.37 (t, J = 7.32 Hz, 2H),
7.28 (t, J = 6.57 Hz, 3H), 7.17 (t, J = 8.12 Hz, 4H), 6.50 (br s, 1H),
4.61–4.47 (m, 1H), 4.35 (d, J = 6.15 Hz, 2H), 4.17 (t, J = 6.57 Hz,
1H), 4.08–3.85 (m, 2H), 3.35–3.17 (m, 2H), 2.56 (t, J = 7.47 Hz,
2H), 1.62–1.48 (m, 2H), 1.21 (s, 10H).
3.18. Molecular modeling methods
Theoretical calculations were performed using Discover 2.98/
Insight II with CVFF force field as described previously.22 The syn-
thetic peptides such as PL-1, PL-5, PL-50, PL-116, and PL-120 were
built from the coordinates of the pThr mimetic-containing PLHSpT
as found in the crystal structure using the Builder module in In-
sight II. The computational complex model was solvated using a
solvent sphere of water extending 30.0 ÅA around the phosphate
atom of pThr. The system was initially minimized using 1000 steps
0
of steepest decent and 3000 steps of conjugated gradient with a
0
14.0 ÅA nonbonded cutoff distance. Data are shown in Figures 2
and 7; Supplementary Figure S1.
13C NMR (75 MHz, CDCl3) d 172.4, 156.2, 144.2, 143.8, 142.9,
141.8, 134.2, 130.0, 128.8, 128.7, 128.2, 127.5, 126.1, 125.3,
120.4, 119.4, 66.8, 53.0, 47.1, 46.9,, 35.8, 31.3, 30.0, 29.1, 29.0,
28.8, 26.3, 25.8.. MS (MALDI-TOF) m/z 566.37 [M+H]+. All data were
in agreement with the reported values.21
Acknowledgments
This work was supported in part by the Korea Basic Science
Institute’s research Grant T33418 (J. K. B.), the National Cancer
Institute’s Intramural Research Grant (K.S.L.), and the National Re-
search Foundation of Korea (NRF)’s basic research program (2010-
0019306) (D.Y.Y.).
Cyclic peptomer, PL-120. Synthesis on 0.040 mmol scale. Purity
of crude product 16% (C18 RP-HPLC). Yield after purification:
1.1 mg, 2.2%; White powder; RP-HPLC tR = 22.54 min (linear gradi-
ent, 5–95% B, 35 min); MS (MALDI-TOF) m/z 1222.63 [M+H]+.
Supplementary data
3.16. ELISA-based PBD-binding inhibition assay
Supplementary data (additional figures and table that corre-
sponds to the modeling and Plk1 PBD-binding assay studies, IC50
values and compounds structures) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2013.02.020.
A biotinylated p-T78 peptide was diluted with coating solution
(KPL, Inc., Gaithersburg, MD) to the final concentration of 0.3
lM,
and then 50 L of the resulting solution was immobilized onto a
l
96-well streptavidin-coated plate (Nalgene Nunc, Rochester, NY).
The wells were washed once with PBS + 0.05% Tween 20 (PBST)
and incubated with 200
1 h to prevent the unoccupied sites. Mitotic 293A lysates express-
ing HA-EGFP-Plk1 were prepared in TBSN buffer (60 g total ly-
sates in 100 L) and applied onto the biotinylated peptide-coated
ELISA wells immediately after mixing with the indicated amount
lL of PBS + 1% BSA (blocking buffer) for
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