Pyrrole-Based Macrocyclic Small-Molecule Inhibitors That Target Oocyte Maturation

Article abstract of DOI:10.1002/cmdc.201700048

Polo-like kinase 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents.

Full text of DOI:10.1002/cmdc.201700048

Accepted Article
Title: Pyrrole-Based, Macrocyclic, Small-Molecule Inhibitors Targeting
Oocyte Maturation
Authors: Jeong Kyu Bang, Pethaiah Gunasekaran, So Rim Lee,
Seung-min Jeong, Jeong-woo Kwon, Toshiki Takei, Yuya
Asahina, Geul Bang, Seongnyeon Kim, Mija Ahn, Eun Kyung
Ryu, Hak Nam Kim, Ki-Yub Nam, Song Yub Shin, Hironobu
Hojo, Suk Namgoong, and Nam-Hyung Kim
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To be cited as: ChemMedChem 10.1002/cmdc.201700048
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Pyrrole-Based, Macrocyclic, Small Molecule Inhibitors Targeting
Oocyte Maturation
Pethaiah Gunasekaran,[a]¶ , So-Rim Lee,[a]¶ Seung-min Jeong,[a] Jeong-Woo Kwon,[a] Toshiki Takei,[b]
Yuya Asahina,[b] Geul Bang,[c] Seongnyeon Kim,[c] Mija Ahn,[d] Eun Kyung Ryu,[d,g] Hak Nam Kim,[d] Ki-
Yub Nam,[e] Song Yub Shin,[f] Hironobu Hojo,[b] Suk Namgoong,*[a] Nam-Hyung Kim,*[a] Jeong Kyu
Bang* [d,g]
[a]
Dr. P. Gunasekaran, S. Lee, S .M. Jeong, J.W. Kwon, Dr. S. Namgoong, Dr. N.H. Kim.
Molecular Embryology Laboratory, Department of Animal Sciences, Chungbuk National University, Chung-Buk 361-763, Republic of Korea
E-mail address: suknamgoong@chungbuk.ac.kr and nhkim@chungbuk.ac.kr
[b] T. Takei, Dr. Y. Asahina, Dr. H. Hojo,
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan
[c] G. Bang, S. Kim
Biomedical Omics Group, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea
[d] Dr. M. Ahn, Dr. E. K. Ryu, H. N. Kim, Dr. J. K. Bang
Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea
E-mail:bangjk@kbsi.re.kr
[e] Dr. K.Y. Nam
Pharos I&BT Co., Ltd., Gyeonggi-do 14059, Republic of Korea
[f] Dr. S.Y. Shin,
Department of Medical Science, Graduate School and Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-
759, Republic of Korea
[g] Dr. E. K. Ryu, Dr. J. K. Bang
Department of Bio-analytical Science, University of Science & Technology, Daejeon, 34113, Republic of Korea.
¶ These authors are contributed equally
evident that disrupting the specific interaction between PBD and
Abstract: Polo-like kinase 1 (Plk1) plays crucial roles in various
Emi2 may be an attractive strategy for the development of novel
contraceptive agents. In addition to lead peptidomimetics,
AB103-8 was found to be successful in blocking oocyte
maturation; however, the peptidic character of AB103-8 results
in poor membrane permeability and protease stability, thereby
hindering its development into a novel therapeutic.[5] The
enhancement of cell permeability can be achieved by converting
them into conjugated peptides, such as a cell-penetrating
peptide, or by PEGylation. However, these processes have
complex routes of synthesis and can sometimes result in a great
loss of activity.[6] To overcome these problems, the design and
synthesis of cell permeable, proteolytic non-hydrolysable, drug-
like small-molecule inhibitors are highly imperative. The
investigation of the structural parameters of AB103 series
compounds (PDB ID 5DMJ) and related Plk1 PBD crystal
structures (PDB ID 3RQ7) showed that the following three key
functional moieties are required for effective interaction with Plk1
PBD: i) a broad pyrrolidine binding pocket surrounded by
Trp414, Phe535, and Arg516, the binding of which determines
the Plk1 selectivity among Plk2 and Plk3; ii) a deep and narrow
tyrosine-rich channel, surrounded by Tyr417, Tyr481, and
Tyr485, which are key for achieving a high binding affinity >1000
fold; and iii) a phospho binding pocket that interacts with His538
and Lys540 (Figure 1).[7] Therefore, we envisioned the design
and synthesis of a novel class of pyrrole-based, non-peptidic,
cell-permeable, protease stable macrocyclic molecules for the
efficient inhibition of oocytes.
stages of oocyte maturation. Recently, we reported that the
peptidomimetic AB103-8 that targets polo box domain (PBD) of Plk1
affected oocyte meiotic maturation and meiosis resumption.
However, to overcome the peptidic drawbacks, we designed and
synthesized a series of pyrrole-based small-molecule inhibitors and
screened against porcine oocyte maturation rates. Among them,
macrocyclic compound 4 showed the highest inhibitory activity with
enhanced inhibition against the embryos blastocyst formation.
Furthermore, the addition of this compound to culture medium
efficiently blocked the maturation of porcine and mouse oocytes,
indicating that the lead compound could penetrate zona pellucida
and cell membrane. To prove the Plk1 inhibition, we investigated this
compound treated mouse oocytes which confirmed the Plk1
inhibition by showing impaired spindle formation. Finally, molecular
modeling studies with Plk1 PBD also confirmed the presence of
significant interactions between compound 4 and Plk1 PBD binding
pockets, including phosphate, tyrosine-rich and pyrrolidine binding
pockets. Collectively, these results suggest that the macrocyclic
compound 4 may serve as a promising template for the development
of novel contraceptive agents.
Introduction
Intracellular protein-protein interactions (PPIs) are playing
essential roles in almost every cellular process, including DNA
replication, transcription, translation, splicing, secretion, cell
cycle control, and signal transduction.[1-3] Similarly, such
interactions serve a crucial role in the oocyte maturation
process, for example, they are involved in the recognition and
phosphorylation of Emi2 by polo-like kinase 1 (Plk1), and are
crucial in controlling the cell cycle resumption. Recently, we
demonstrated that one of our synthesized peptidomimetics,
AB103-8, targeting the polo-box domain (PBD) of Plk1, inhibited
the interaction between PBD and Emi2 that affected oocyte
meiotic maturation and meiosis resumption.[4] Hence, it is
The present work emerges as part of a research program that
recently embarked on the development of oocyte maturation
blocking studies[4] and Plk1 PBD inhibitor synthesis.[8-11] In this
work, we have designed and synthesized an N-phenylalkyl
1
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pyrrole-based macrocyclic inhibitor featuring phosphate residues
on the alkyl side chain. To mimic AB103-8, we closely examined
its binding in the crystal structure of P Plk 1 PBD in complex with
702 (PDB ID 5DMJ). The complex structure reveals that its long
phenylalkyl residue was directed towards deep and narrow
tyrosine-rich binding pockets,[4] and that the phosphate group
underwent a polar interaction with His538 and Lys540 at the
phosphate binding pocket. We chose pyrrole residue as a core
moiety, which can hold a flexible phenylalkyl chain linked at the
NH position, and a variable alkyl chain bearing a phosphate
moiety. Besides, deriving the macrocyclic structures in core
moiety often result in the enhancement of protease stability.[12]
Based
on the aforementioned
preliminary structural
prerequisites, we designed the N-phenylalkyl pyrrole-based
macrocyclic molecule 8, which features an alkyl phosphate
group for binding with the phosphate binding pocket, and an N-
phenylalkyl unit for targeting the deep and conserved tyrosine-
rich channel, as shown in Figure 1. Retrosynthetic analysis
suggested that macrocyclic small molecule 8 could be obtained
from two terminal alkenic precursors, N-phenylalkyl pyrrole
carboxylic acid (10) and the secondary amine (11) anchoring the
phosphonate
group,
as
outline
in
Scheme
1.
The terminal diene 9 could be generated by amide coupling
reaction of precursors 10 and 11. Then, the ring-closing
metathesis (RCM) reaction was performed for further cyclization,
and subsequent benzylic deprotection of the phosphonate group
would ultimately lead to the desired final compound.
Scheme 1. Retro synthetic pathway for the synthesis of macrocyclic
compounds.
Our synthesis of 10 began with the incorporation of
phenylalkyls at the nitrogen of pyrrole-2-methylester. N-
alkylation of 12 was successfully carried out using various
phenylalkyl bromides with different alkyl chain lengths, in the
presence of NaH in dimethylformamide (DMF) (Scheme 2). To
introduce the long alkenic chain at position 4 of the pyrrole
residue, the Friedel-Crafts reaction was selected as a key tool to
Figure 1. Binding nature of peptidomimetic 702 with Plk1 PBD and
illustration of the cyclic molecule with all the synthesized compounds.
Thus, to the best of our knowledge, this is the first study to
report
a novel class of macrocyclic, non-peptidic, small
achieve the formation of 15 using 10-undecenoyl chloride and
molecules that effectively inhibit oocyte maturation. We
synthesized a series of macrocyclic inhibitors by manipulating
the alkyl lengths between the phenyl ring and pyrrole residue,
and changing the alkyl chain length of the phosphate attached
linker. These macrocyclic compounds were microinjected into
immature porcine oocytes, and their potential to block in vitro
oocyte maturation was compared with previously developed
compound AB103-8 (positive control) and Bg34 (negative
control). Among the synthesized compounds, the macrocyclic
inhibitor compound 4 displayed the highest inhibition potential.
To verify the Plk1 inhibition, spindle informations of compound 4
treated mouse oocytes were studied. Then, to analyze the
binding nature of compound 4, molecular models of Plk1 PBD
[13]
Yb(OTf)3.
Finally, the ester hydrolysis reaction was performed
using KOH to yield the desired precursor 10 in good yields.
Scheme 2. Synthesis of 10. Reagents and conditions: (i) NaH, DMF, 0 oC -
rt ; (ii) 10-undecenoyl chloride, Yb(OTf)3, CH3NO2, 0 oC - rt; (iii) KOH,
THF/EtOH/H2O (9:1:1), reflux.
and compound
4 complexes were generated by docking
simulation and studied further.
Results and Discussion
Chemistry
Next, the synthetic route for precursor 11 was developed using
the initial coupling reaction of 4-bromo-1-butene (16) and
respective amino alcohols 17 in the presence of NaI (Scheme 3)
to yield 18. Prior to achieving the phosphorylation of the hydroxyl
group, the secondary amine must be protected to prevent
undesirable reactions. Accordingly, the initial silyl protection of
2
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the primary alcohol 19 was achieved in good yields using tert-
butyldiphenylchlorosilane (TBDPSCl) in the presence of
imidazole. Then, Boc-protection of the secondary amine was
performed using (Boc)2O in the presence of triethylamine and 4-
dimethylaminopyridine (DMAP). To recover the alcohol
functional group, deprotection of the primary alcohol was
effected in the presence of tetrabutylammonium fluoride (TBAF)
in tetrahydrofuran (THF) to yield alcohol 21.[14]
Scheme 4. Synthesis of compounds 1-7
Reagents and conditions: (i) HBTU, DIEA, DMF, rt; (ii) Grubbs 2nd gen. DCM,
45 oC; (iii) TFA/TIS/H2O (10:1:1), rt.
Biological evaluations
Having developed the synthetic protocol for the facile synthesis
of the targeted molecule, we then tested the effect of our
macrocyclic compounds on porcine oocyte maturation rates, as
shown in Figure 2A. The synthesized macrocyclic inhibitors 1-7
were screened for porcine oocyte maturation rates along with
the peptidomimetics AB103-8 (positive control) and Bg34
(negative control), and subjected to in vitro maturation for 44h.
The majority of the macrocyclic compounds exhibited
significant effect on the maturation rate of porcine oocytes.
a
Scheme 3. Synthesis of 11. Reagents and conditions: (i) NaI, MeOH, reflux;
(ii) TBDPSCl, Imidazole, THF, rt; (iii) (Boc)2O, DMAP, Et3N, CH2Cl2, rt;
(iv) TBAF, THF, rt; (v) dibenzyl N,N-diisopropyl phosphoramidite, 1H-tetrazole,
rt; (vi) m-CPBA, rt; (vii) TFA/ CH2Cl2 (1:6), rt.
Then, the alcohol was phosphorylated using dibenzyl-N,N-
diisopropylphosphoramidite in the presence of 1H-tetrazole, and
the subsequent oxidation in the presence of m-
chloroperoxybenzoic acid (m-CPBA) resulted in the benzyl-
protected phosphoryl ester 22.[11] Finally, TFA-mediated Boc (t-
butyloxycarbonyl) deprotection of the secondary amine resulted
in the formation of desired precursor 11. After the successful
synthesis of precursors 10 and 11, our next objective was to
enable the amide coupling reaction using 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and
N,N-diisopropylethylamine (DIEA) in DMF to achieve diene 23 in
good yields (Scheme 4). Then, the two terminal alkenes of 23
underwent the RCM reaction in order to enable the cyclization
process using the Grubbs 2nd generation catalyst.[15] Finally, the
removal of the benzyl group from the phosphonate ester was
effected in the presence of TFA/TIS/H2O (10:1:1) to obtain the
desired final compounds. Following the above methodology,
further derivatization of macrocyclic compounds, 1-7 was
achieved by manipulating the alkyl chain lengths of the phenyl
and phosphate linkers.
Figure 2. Microinjection of macrocyclic compounds on porcine oocytes impairs
oocyte maturations and pathernogenic embryo developments.
A. Inhibition of porcine oocyte maturation by microinjections of macrocyclic
compounds (1-7). Percentage of oocytes matured to metaphase II (MII)
against total oocytes were plotted. Numbers of oocytes per each treatment
groups were indicated on the top of the graph. Oocyte failed to reach MII stage
were defined as ‘MI arrested’, while oocyte extruded polar body were counted
as ‘MII’. Statistically significant differences (p < 0.05, ANOVA) in oocyte
maturation rates are indicated by a different superscript letters (a,b and c).[16]
B. Failure of pathernogenic embryo developments caused by microinjection of
macrocyclic compounds: Oocytes injected with macrocyclic compounds (3 or
4), control chemicals (AB103-8) and non-injected control reached MII stages
were subjected to pathernogenetic activations and their developments to
blastocyst stages were monitored and plotted. Embryo developed until
blastocyst stages were counted as ‘BL Developments’ and embryos did not
reach to blastocyst was classified as ‘Development Failure’. Number of
embryos per each treatment groups was indicated on the top of the plot.
Significant differences between the treatment groups and the control group are
indicated by an asterisk (***Pꢀ≤ꢀ0.001, **Pꢀ≤ꢀ0.01, *Pꢀ≤ꢀ0.05). Arrows indicate
blastocysts developed. Scale bar: 120 μm.
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Firstly, compounds, 1 and 2, were attached with butylphenyl and
propylphenyl, respectively, and the phosphate linker was
connected with ethyl chain. During the screening of porcine
oocyte maturation rates, these compounds showed a slight
decrease in inhibition rates (1: 59.6%, and 2: 56.9%) compared
with that of the positive control, AB103-8 (55.0%). The extension
of the phosphate linkers in 1 and 2, from ethyl to a propyl chain,
resulted in the formation of 4 and 3, respectively. Further
examination of these compounds on porcine oocyte maturation
rates reveals that compound 4 was found to exhibit effective
inhibitory activity (maturation rate, 43.8%, 105/240), which was
significantly higher compared with that of the negative control
Bg34 (83.2%, 148/178, p<0.001). Meanwhile, compound 3
showed an inhibition rate similar to that of the peptidomimetic
AB103-8 (55.2 %). It is evident from the above results that
variation in the chain lengths of the phosphate linker alters the
inhibition rate of the macrocyclic compounds in porcine oocyte
maturation.
macrocyclic compounds significantly decreased the porcine
oocyte maturation rate, similar to the results obtained using
direct microinjections of compounds in cytoplasm. In this
screening, compound 4 was found to be the most effective
compound at inhibiting oocyte maturation rates (30.3%, 61/201,
p<0.001) compared with that of the control Bg34 (84.0%,
278/331), and AB103-8 (65%, 118/179). This result proves that
the most potent compound 4 is highly permeable than that of the
peptidomimetics AB103-8. And also this consistency in the
results of 4 indicates that macrocyclic compounds can penetrate
zona pellucida and oocyte cell membranes, and consequently
negatively affect oocyte maturation.
Next, to investigate the effect of the phenylheptyl attached to
AB103-8 that targets the tyrosine-rich channel, we modified 4 by
attaching the phenylheptyl chain at the pyrrole residue leading to
the formation of the inhibitor, 5. Unfortunately, the change in
phenylalkyl chain length did not result in any improvements in
the porcine oocyte maturation inhibition rate (50%). Instead, it
was found to display reduced inhibition rates compared with that
of the most effective compound, 4. To further determine the
effect of increasing the chain lengths of the phosphate linker on
the inhibition rate, we further extended the chain lengths from
propyl to hexyl chains, resulting in compounds 6 and 7. In these
compounds, the pyrrole remained connected to the phenylheptyl
and phenylbutyl chains, respectively. In contrast to the previous
results, macrocyclic compounds 6 and 7 resulted in reduced
oocyte maturation rates, at 62.5% and 70.2%, respectively.
Thus, compound 4 was found to be most effective inhibitor of
porcine oocytes maturation rates.
Although the injection of macrocyclic compounds decreased
oocyte maturation efficiency, significant proportions of oocytes
still managed to extrude the polar body to evolve as a matured
oocyte. Therefore, we tested the embryonic developmental
potential of these oocytes, which managed to develop until
metaphase II (MII). Following the injections of a macrocyclic
compound (3 or 4) or AB103-8, the oocytes were subjected to in
vitro maturation. Then, the MII-stage oocytes were subjected to
parthenogenetic activation by electric shock in order to induce
embryonic development. As shown in Figure 2B, the blastocyst
formation rate of parthenogenetically activated oocytes injected
with 3 or 4 macrocyclic compounds was nearly abolished (3:
2/27, p<0.01; 4: 0/15, p<0.05), which was in contrast to that of
oocytes injected with the control (37%, 40/108) or AB103-8
(23%, 5/22). These results prove that macrocyclic compounds
inhibit porcine oocyte maturation and further affect embryonic
development potential.
Figure 3. The inclusion of macrocyclic compounds in culture media impairs
porcine and mouse oocyte maturation.
A. Inhibition of porcine oocyte maturation by macrocyclic compounds was
supplemented in culture medium at 10 μM of concentration. Percentage of
oocytes matured to metaphase II (MII) against total oocytes were plotted.
Numbers of oocytes per each treatment groups were indicated on the top of
the graph. Oocyte failed to reach MII stage were defined as ‘MI arrested’,
while oocyte extruded polar body were counted as ‘MII’. Statistical differences
(p < 0.05, ANOVA) between different treatments are indicated by a different
superscript letters (a, b, c, d and e). B. Failure of mouse oocyte maturation by
macrocyclic compound, 4 treatments: 10 μM of macrocycles and control
chemicals were supplemented in the medium during in vitro maturation.
Oocyte growth stages after 12 h of in vitro maturation were classified and
counted; GV:Oocytes failed to undergo germinal vesicle (GV) breakdown; MI:
Oocytes had undergone to GV breakdown, but failed to extrude polar body;
Symmetric: Oocytes had undergone cytokinesis, but have unusually bigger
polar body (classified with polar body which has more than 50% of diameter of
that of oocyte); MII: Oocytes had undergone polar body extrusion and had
normal polar body. Significant differences between the treatment groups and
the control group are indicated by an asterisk (***Pꢀ≤ꢀ0.001, **Pꢀ≤ꢀ0.01,
*Pꢀ≤ꢀ0.05). C. Meiotic spindles in oocytes treated with 4 were stained with
alpha-tubulin (Green) and DAPI for DNA (Blue). Oocytes failed with polar body
extrusion (MI arrest) and oocytes protruded polar body (MII) were classified
In contrast to the peptidomimetic AB103-8, macrocyclic
compounds have hydrophobic properties, so we tested the
hypothesis that the macrocyclic compounds could penetrate the
zona pellucida and oocyte membrane of porcine. To facilitate the
screening, we added each macrocyclic compound, at
a
concentration of 10 μM, to the culture media for the in vitro
maturation of porcine oocytes and assessed their effects on
oocyte maturation by comparing with peptidomimetic AB103-8
and Bg34. As shown in Figure 3A, the addition of 10 μM
4
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separately. Each oocyte was classified as wild type (WT), non-polar spindle
(Non-Polar), multipolar spindle (MultiPolar), and Segregation Failure and
counted.
mouse oocyte maturation. Considering previously described
roles of Plk1 in meiotic spindle formation,[17] the results from the
present study strongly suggest that the inhibitory effect of
macrocyclic compound 4 results in the inhibition of Plk1, and
therefore cause depletion of MTOC formation.
Then, in order to investigate the ability of macrocyclic
compounds in inhibiting other mammalian oocyte maturation, we
tested the inhibitory potential of 4 in mouse oocyte maturation.
As presented in Figure 3B, the addition of 4 to in vitro culture
medium at 10 μM concentration significantly decreased mouse
oocyte maturation rates (48.5%, n=95/196; p<0.001) compared
with that of the control (83.3%, n=100/120). Treatment of 4
compound also increases the fraction of oocytes with the large
polar body (13.7%, n=27/196), which have polar body bigger
than 50% of oocyte’s diameter, while none of oocyte without
chemical treatments showed abnormally large polar body (0%,
n=0/120). Considering that bigger polar body or failure of
asymmetric division is caused by spindle migration defect,[17]
these results suggest that treatment with 4 negatively affects
spindle migration during oocyte maturation.
In order to examine the Plk1 inhibiting ability of the lead
compound, we analyzed the meiotic spindles in compound 4
treated mouse oocytes. It is previously reported that the
inhibition of Plk1 in mouse oocytes causes the impairment of the
microtubule organization center (MTOC), and therefore results in
problems regarding meiotic spindle formation.[18] It is pertinent to
note that the meiotic spindles in 4-treated mouse oocytes are
consisted of major portions of abnormal meiotic spindles
compared with that of wild-type oocytes (Figure 3C). For
instance, the majority of control MII-stage oocytes had typically
normal bipolar spindles (91%, 49/54). However, 4 treated oocyte
displayed only a small fraction of bipolar spindles (14%, 2/14 in
MI-arrested oocytes; 13%, 6/47 in MII-stage oocytes). Moreover,
most of the 4 treated meiotic spindles in mouse oocytes
displayed non-polar morphology (86%, 12/14 in MI arrested
oocytes; 62%, 29/47 in MII-stage oocytes). In addition, 4 treated
oocytes in MII-stage also displayed the multipolar spindles
(19%, 9/47). These results firmly suggest that treatment with
compound 4 impair the formation of meiotic spindles during
Molecular Modelling
To gain molecular insight into the ligand binding site interactions
responsible for the inhibitory effect of macrocyclic compound 4
in oocyte maturation studies, we investigated the binding
interactions of molecular modeling structure of the Plk1 PBD and
compound 4 complexes (Figure 4). The molecular modeling
results of the Plk1 PBD and compound 4 complexes revealed
that three primary binding interactions were present between the
macrocyclic compound, 4, and Plk1 PBD. The initial inference of
molecular docking results revealed that there were tight charge-
charge interactions between the phosphate group of 4 and the
two positively charged residues, His538 and Lys540, in the
phosphate-binding region. Secondly, the four carbon tethered
phenyl alkyl unit of 4 were projected towards the narrow
passage of the tyrosine-rich hydrophobic channel to exert
hydrophobic interactions, as shown in Figure 4. These two
interactions are identical to that of the AB103-8-related
compound 702 binding the Plk1 PBD complex (PDB ID 5DMJ).
In addition to the above interactions, the pyrrole ring in
compound 4 is bound to the pyrrolidine binding pocket through
an effective π-π-stacking interaction that exists between the
pyrrole ring of compound 4 and the side-chain of Trp414.
Furthermore, the ethylene moiety present in the macrocyclic ring
of compound 4 showed contact with the side chain of Phe535.
Finally, the carbonyl group that connected directly with the fourth
position of the pyrrole residue in the macrocyclic compound, 4,
showed weak hydrogen bonding interactions, with a backbone
N-H of Asp416. It is expected that the additional interactions
exhibited by compound 4 may be instrumental factors in its
superior activity in oocyte maturation, compared with AB103-8.
Figure 4. Modeling structure of the Plk1 PBD and 4 complexes, which shows the presence of the phosphate binding pocket, pyrrolidine-binding pocket and Tyr-
rich hydrophobic channel. Modeling structure was generated using Pymol (http://www.pymol.org)
5
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and stirred for 4 h. Then the reaction mixture was treated with saturated
aqueous NH4Cl (10 mL) and extracted with ethyl acetate (30 mL). The
combined organic extracts were washed with water (3x15 mL) and brine,
dried over Na2SO4, and evaporated. The crude product was purified by
silica gel column chromatography (hexane-ethyl acetate, 20:1) to afford
14a as colourless oil (248 mg, 64%).
Conclusions
This study demonstrates the efficient design and synthesis of
pyrrole-based, macrocyclic, small molecule inhibitors that are
more effective than previously reported peptidomimetics, such
as AB103-8, in inhibiting oocyte maturation. The synthesized
molecules were successfully screened for their effects on
porcine oocyte maturation rates, and the macrocyclic inhibitor 4
was found to have good inhibitory potential against oocyte
maturation. In addition, this inhibitor was found to prevent
embryonic development, indicating that compound, 4 treatment
could block mammalian early embryogenesis in addition to
oocyte maturation. It has been reported that the inhibition of
Plk1, by kinase-domain inhibitor BI2536 or morpholino antisense
oligonucleotide, can block the early embryogenesis of
zebrafish,[19] indicating the importance of Plk1 in the early
embryogenesis of vertebrae. Moreover, 4 treated mouse oocyte
was found to show the impaired mouse spindles, suggesting that
there is Plk1 inhibition. These results suggest that Plk1 would be
an effective target for the development of novel
methyl 1-(4-phenylbutyl)-1H-pyrrole-2-carboxylate (14b)
Compound 14b was synthesized from methyl 1H-pyrrole-2-carboxylate
(12) and 4-phenylbutyl bromide (13b) following the general procedure A
(colourless oil, 69%)
methyl 1-(7-phenylheptyl)-1H-pyrrole-2-carboxylate (14c)
Compound 14c was synthesized from methyl 1H-pyrrole-2-carboxylate
(12) and 7-phenylheptyl bromide (13c) following the general procedure A
(colourless oil, 62%)
General Procedure B for the synthesis of compound 15a-15c
methyl1-(3-phenylpropyl)-4-undec-10-enoyl-1H-pyrrole-2-
carboxylate (15a)
To a stirred solution of 10-undecenoyl chloride (728 mg, 2.12 mmol) and
Yb(OTf)3 (101.6 mg, 0.164 mmol) in nitromethane at 0 °C was added 14a
(400 mg, 1.64 mmol) slowly. The resulting reaction mixture was stirred at
room temperature for 18 h. The reaction was quenched with saturated
aqueous NaHCO3 (15 mL) and the mixture was extracted with diethyl
ether (3x15 mL). The organic layers were washed with water and brine,
dried over Na2SO4 and volatiles were removed under vacuo. The
resultant residue was purified by silica gel column chromatography
(hexane-ethyl acetate, 20:1) to afford 15a as colourless oil (281 mg,
42%).
anticontraceptive drugs. We also confirmed that
4 could
effectively block oocyte maturation when it was added to oocyte
culture media at 10 μM. The hydrophobic nature of this inhibitor
may explain its ability to penetrate the zona pellucida and cell
membrane. Finally, molecular modeling of Plk1 PBD and 4
complexes revealed that the primary binding interactions in the
regions, including the phospho binding pocket, tyrosine-rich
channel, and pyrrolidine binding pocket, along with additional
interactions, may be a critical factor in its ability to more
effectively inhibit oocyte maturation, in comparison with the
peptidomimetic AB103-8. Hence, the macrocyclic compound 4
could represent a model for developing novel contraceptive
agents.
methyl 1-(4-phenylbutyl)-4-undec-10-enoyl-1H-pyrrole-2-carboxylate
(15b)
Compound 15b was synthesized from compound 14b and 10-
undecenoyl chloride in the presence of Yb(OTf)3 following the general
procedure B (colourless oil, 44 %).
methyl1-(7-phenylheptyl)-4-undec-10-enoyl-1H-pyrrole-2-
carboxylate (15c)
Compound 15c was synthesized from compound 14c and 10-undecenoyl
chloride in the presence of Yb(OTf)3 following the general procedure B
(colourless oil, 41 %).
.
Experimental Section
Chemistry
All reagents and starting materials were obtained from commercial
chemical suppliers (Sigma-Aldrich or TCI) and used as received.
Anhydrous organic solvents were purchased from Aldrich and used
directly. Thin layer chromatography (TLC) was performed on Merck
aluminum sheets with silica gel 60 F254 and were visualized by
ultraviolet light and staining with KMnO4. Column chromatography was
performed on Merck silica gel 60 (70-230 mesh or 230-400 mesh). 1H
and 13C NMR spectra were recorded on a Bruker DRX-400 and DRX-500
spectrometer. Chemical shifts (δ) are reported in parts per million (ppm)
measured relative to an internal standard and coupling constants (J) are
expressed in hertz (Hz). All final compounds (1-7) were purified by
preparative reverse-phase high performance liquid chromatography (RP-
HPLC) and judged to be ≥ 95% pure by analytical HPLC equipped with
C18 column (4.6 × 250 mm, 10 microns). Two different linear gradients of
0.05% aq. TFA (eluent A) and 0.05% TFA in CH3CN (5-95/30 min,eluent
B) were used with a flow rate of 1.0 mL/min at 25 °C. The synthesized
compounds were confirmed by ESI-MS, HRMS, and MALDI-TOF mass
spectrometer (Shimadzu).
General Procedure C for the synthesis of compound 10a-10c
1-(3-phenylpropyl)-4-undec-10-enoyl-1H-pyrrole-2-carboxylic
(10a)
To a solution of 15a (650 mg, 1.59 mmol) in THF/EtOH (20 mL (6:1)) was
added potassium hydroxide (711 mg, 12.71 mmol) in water (3 mL). The
resulting reaction mixture was refluxed for 12 h and cooled to room
temperature. Then the reaction mixture solvents were removed under
vacuo, and the residue in water was acidified to pH 1 using 32% aqueous
HCl. The resultant precipitate was filtered and washed with water to
obtain the carboxylic acid as a yellow sticky solid 10a (464 mg, 74%).
acid
1-(4-phenylbutyl)-4-undec-10-enoyl-1H-pyrrole-2-carboxylic acid
(10b)
Compound 10b was synthesized from compound 15b using potassium
hydroxide following the general procedure C (yellow solid, 79 %).
1-(7-phenylheptyl)-4-undec-10-enoyl-1H-pyrrole-2-carboxylic
(10c)
acid
Compound 10c was synthesized from compound 15c using potassium
hydroxide following the general procedure C (yellow solid, 79 %).
General Procedure A for the synthesis of 14a-14c
methyl 1-(3-phenylpropyl)-1H-pyrrole-2-carboxylate (14a)
General Procedure D for the synthesis of compound 19a-19c
N-[2-(tert-butyldiphenylsilyloxy)ethyl]but-3-en-1-amine (19a)
To a stirred solution of 16 (1.00 g, 7.40 mmol) and 2-aminoethanol (17a)
(1.58 mL, 22.2 mmol) in MeOH (30 mL) was added NaI (0.27 g, 1.85
mmol). The resultant reaction mixture was refluxed for 3 h. Then the
reaction mixture was cooled to room temperature and evaporated under
NaH (60% w/w in mineral oil, 102 mg, 2.55 mmol) was added to a
stirred solution of methyl 1H-pyrrole-2-carboxylate (12) (200 mg, 1.59
mmol) in DMF (20 mL) at 0 °C. After stirring at room temperature for 30
min, 3-phenylpropyl bromide (13a) (324 mg, 1.63 mmol) was added
slowly at 0 °C. The temperature was slowly raised to room temperature
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700048
ChemMedChem
FULL PAPER
vacuo. The residue was treated with saturated aqueous NH4Cl solution
and ethyl acetate. After the separation of the two layers, the aqueous
layer was basified with 40% sodium hydroxide and extracted with ethyl
acetate. The combined organic layers were dried over Na2SO4 and
concentrated under vacuo to afford 18 (0.84 g, 100% yield) as a
colourless viscous oil. This crude oil was used for the next step by
dissolving in THF (50 mL), to which TBDPSCl (4.06 g, 1.48 mmol) and
imidazole (1.1 g, 16.3 mmol) were added, and stirred for 14 h at room
temperature. The reaction mixture was evaporated under vacuo, and the
residue was purified by silica gel column chromatography (hexane-ethyl
acetate, 3:7) to provide 19a (2.45 g, 94 %) as a colourless oil.
diisopropylphosphoramidite (5.2 g, 15.3 mmol) at 0 °C. The resulting
solution was stirred at room temperature for 4 h. Then, m-CPBA (1.98 g,
11.5 mmol) was added to the reaction mixture at 0 °C, and the reaction
mixture was stirred for further 1 h at room temperature. Then the
precipitate was filtered and washed with dichloromethane (30 mL). The
filterate was concentrated under vacuo and resuspended in
dichloromethane (50 mL). The combined dichloromethane solutions were
washed with a saturated aqueous NaHCO3 and brine. The organic
layer was dried over sodium sulfate, filtered, and concentrated under
vacuo. The residue was purified by flash column chromatography (diethyl
ether-hexane, 1:1) to provide 22a (3.2 g, 87 % over two steps) as a
colourless oil.
N-(3-(tert-butyldiphenylsilyloxy)propyl)but-3-en-1-amine (19b)
Compound 19b was synthesized from 16 and 17b in the presence of NaI
following the general procedure D (colourless oil, 76 %).
tert-butyl 3-(bis(benzyloxy)phosphoryloxy)propyl(but-3-enyl)
carbamate (22b)
Compound 22b was synthesized from 21b by following the general
procedure G (colourless oil, 95 %).
N-(but-3-enyl)-6-(tert-butyldiphenylsilyloxy)hexan-1-amine (19c)
Compound 19c was synthesized from 16 and 17c in the presence of NaI
following the general procedure D (colourless oil, 76 %).
tert-butyl 6-(bis(benzyloxy)phosphoryloxy)hexyl(but-3-enyl)
carbamate (22c)
General Procedure E for the synthesis of compound 20a-20c
Compound 22c was synthesized from 21c by following the general
procedure G (colourless oil, 95 %).
tert-butyl
but-3-enyl(2-(tert-butyldiphenylsilyloxy)ethyl)carbamate
(20a)
General Procedure H for the synthesis of compound 11a-11c
dibenzyl 2-(but-3-enylamino)ethyl phosphate (11a)
(Boc)2O (0.495 g, 2.27 mmol) was added to a solution of 19a (0.4 g, 1.13
mmol), triethylamine (3.8 mL, 2.66 mmol), and 4-dimethylaminopyridine
(DMAP) (0.05 g, 0.44 mmol) in dichloromethane (DCM) (100 mL) at
0 °C. The reaction mixture was allowed to stir at room temperature for 14
h. The resultant reaction mixture was washed with brine, dried (Na2SO4)
and concentrated under vacuo. The crude product was purified by flash
column chromatography (hexane-ethyl acetate, 20:1) to afford 20a (0.451
g, 88 %) as a colourless oil.
50% TFA in DCM (5 mL) was added slowly to a stirred solution of 22a
(1.0 g, 2.10 mmol) in dichloromethane (10 mL) at 0 °C. The reaction
mixture temperature was increased and the resulting solution was stirred
at room temperature for 2 h. Then the reaction mixture was diluted
with the addition of DCM (20 mL) and treated with 30% NaHCO3
solution (20 mL). The organic layer was washed with water and
brine, dried over Na2SO4 and evaporated under vacuo. The crude
residue
was
purified
with
flash
column
chromatography
tert-butyl but-3-enyl(3-(tert-butyldiphenylsilyloxy)propyl)carbamate
(20b)
Compound 20b was synthesized from 19b using (Boc)2O, triethylamine,
and DMAP following the general procedure E (colourless oil, 94 %).
(dichloromethane-methanol (100:4)) to provide 11a (0.34 g, 43 %) as a
colourless oil.
dibenzyl 3-(but-3-enylamino)propyl phosphate (11b)
Compound 11b was synthesized from 22b using trifluoroacetic acid
following the general procedure H (colourless oil, 37 %).
tert-butyl but-3-enyl(6-(tert-butyldiphenylsilyloxy)hexyl)carbamate
(20c)
Compound 20c was synthesized from 19c using di-tert-butyldicarbonate,
triethylamine, and DMAP following the general procedure E (colourless
oil, 84 %).
dibenzyl 6-(but-3-enylamino)hexyl phosphate (11c)
Compound 11c was synthesized from 22c using trifluoroacetic acid
following the general procedure H (colourless oil, 39 %).
General Procedure F for the synthesis of compound 21a-21c
tert-butyl but-3-enyl(2-hydroxyethyl)carbamate (21a)
General Procedure I for the synthesis of compound 23a-23g
dibenzyl 2-(N-(but-3-enyl)-1-(4-phenylbutyl)-4-undec-10-enoyl-1H-
pyrrole-2-carboxamido)ethyl phosphate (23a)
To a stirred solution of 20a (0.5 g, 1.10 mmol) in THF (50 mL) at 0 °C
was added n-tetrabutylammonium fluoride (TBAF) (1.0 M in THF) (1.33
mL, 1.33 mmol). The reaction mixture was warmed to room temperature
and stirred for 14 h. Then the reaction mixture was concentrated under
vacuo. The resulting residue was re-suspended in diethyl ether (80 mL)
and washed with water (40 mL). The aqueous layer was extracted again
with diethyl ether. The combined organic extracts were dried (Na2SO4)
and concentrated. The crude residue was purified by flash column
chromatography (hexane-ethyl acetate, 6:4) to afford 21a (0.18 g, 76 %)
as a colourless oil.
To a stirred solution of 10b (0.50 g, 1.2 mmol) and 11a (0.69 g, 1.4
mmol) in DMF (10 mL) was added HBTU (0.64 g, 1.7 mmol) and DIEA
(8.6 mL, 4.8 mmol). The reaction was stirred at room temperature for 14
h. Then reaction mixture was diluted with ethyl acetate (20 mL) and
washed with water (15 mL x 3) and the combined ethyl acetate layers
were washed with brine and dried over Na2SO4. The crude product was
purified with flash column chromatography (hexane-ethyl acetate, (10:4))
to yield 23a (0.73 g, 79 %) as a colourless oil.
dibenzyl 2-(N-(but-3-enyl)-1-(3-phenylpropyl)-4-undec-10-enoyl-
1H-pyrrole-2-carboxamido)ethyl phosphate (23b)
Compound 23b was synthesized from 10a and 11a by following the
general procedure I (colourless oil, 66 %)
tert-butyl but-3-enyl(3-hydroxypropyl)carbamate (21b)
Compound 21b was synthesized from 20b using n-tetrabutylammonium
fluoride(TBAF) following the general procedure F (colourless oil, 95 %).
tert-butyl but-3-enyl(6-hydroxyhexyl)carbamate (21c)
Compound 21c was synthesized from 20c using n-tetrabutylammonium
fluoride(TBAF) following the general procedure F (colourless oil, 92 %).
dibenzyl 3-(N-(but-3-enyl)-1-(3-phenylpropyl)-4-undec-10-enoyl-
1H-pyrrole-2-carboxamido)propyl phosphate (23c)
Compound 23c was synthesized from 10a and 11b by following the
general procedure I (colourless oil, 92 %).
General Procedure G for the synthesis of compound 22a-22c
tert-butyl 2-(bis(benzyloxy)phosphoryloxy)ethyl(but-3-enyl)
carbamate (22a)
To a mixture of 21a (1.65 g, 7.67 mmol) and 0.45 M acetonitrile solution
of tetrazole (51 mL, 23 mmol) was added dibenzyl N,N-
dibenzyl 3-(N-(but-3-enyl)-1-(4-phenylbutyl)-4-undec-10-enoyl-1H-
pyrrole-2-carboxamido)propyl phosphate (23d)
Compound 23d was synthesized from 10b and 11b by following the
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700048
ChemMedChem
FULL PAPER
general procedure I (colourless oil, 79 %).
(2:0.2:0.2 mL) and stirred for 2 h. The reaction mixture was concentrated
under vacuo and residue was washed with cold diethyl ether. The
resultant crude product was purified by RP-HPLC to give final compound
1 (11 mg. 26 %) as a colourless oil.
dibenzyl 3-(N-(but-3-enyl)-1-(7-phenylheptyl)-4-undec-10-enoyl-1H-
pyrrole-2-carboxamido)propyl phosphate (23e)
Compound 23e was synthesized from 10c and 11b by following the
general procedure I (colourless oil, 62 %).
(E/Z)-2-(2,16-dioxo-19-(3-phenylpropyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)ethyl dihydrogen phosphate (2)
Compound 2 was synthesized from 24b following the general procedure
K (colourless oil, 31 %)
dibenzyl 6-(N-(but-3-enyl)-1-(7-phenylheptyl)-4-undec-10-enoyl-1H-
pyrrole-2-carboxamido)hexyl phosphate (23f)
Compound 23f was synthesized from 10c and 11c by following the
general procedure I (colourless oil, 72 %).
(E/Z)-3-(2,16-dioxo-19-(3-phenylpropyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (3)
Compound 3 was synthesized from 24c following the general procedure
K (colourless oil, 26 %).
dibenzyl 6-(N-(but-3-enyl)-1-(4-phenylbutyl)-4-undec-10-enoyl-1H-
pyrrole-2-carboxamido)hexyl phosphate (23g)
Compound 23g was synthesized from 10b and 11c by following the
general procedure I (colourless oil, 79 %)
(E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4)
Compound 4 was synthesized from 24d following the general procedure
K (colourless oil, 28 %).
General Procedure J for the synthesis of compound 24a-24g
(E/Z)-dibenzyl2-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)ethyl phosphate (24a)
To the diene 23a (0.25 g, 0.33 mmol) in anhydrous dichloromethane (20
mL) was added Grubb’s 2nd generation catalyst (0.065 mmol). The
mixture was heated to 45 °C and stirred for 30 min under a nitrogen
atmosphere. An additional portion of Grubb’s 2nd generation catalyst
(0.065 mmol) was added and the solution was stirred for 10 h at 45 °C.
Further, an additional portion of Grubb’s 2nd generation catalyst (0.065
mmol) was added and stirred for 12 h. The reaction mixture was
quenched by addition of activated charcoal and stirred for 6 h at room
temperature. The reaction mixture was filtered through a pad of celite
and concentrated under vacuo. The crude product was purified with flash
column chromatography (hexane-ethyl acetate, (1:1)) to yield 24a (0.137
g, 57 %) as a brown color oil.
(E/Z)-3-(2,16-dioxo-19-(7-phenylheptyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (5)
Compound 5 was synthesized from 24e following the general procedure
K (colourless oil, 32 %).
(E/Z)-6-(2,16-dioxo-19-(7-phenylheptyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)hexyl dihydrogen phosphate (6)
Compound 6 was synthesized from 24f following the general procedure
K (colourless oil, 37 %).
(E/Z)-6-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)hexyl dihydrogen phosphate (7)
Compound 7 was synthesized from 24g following the general procedure
K (colourless oil, 55 %).
(E/Z)-dibenzyl 2-(2,16-dioxo-19-(3-phenylpropyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)ethyl phosphate (24b)
Compound 24b was synthesized from 23b following the general
procedure J (brown colour oil, 45 %).
In vitro porcine oocyte maturation
Prepubertal porcine ovaries were obtained from
a
local
slaughterhouse(Farm Story dodram B&F, um-sung, chungbuk, Korea).
Cumulus-oocyte complexes (COCs) were obtained from follicles that
were 3-6 mm in diameter using 18-gauge microneedles. Oocytes with
evenly granulated cytoplasm and a compact surrounding cumulus mass
were collected and washed three times with TL-HEPES-PVA medium
(Tyrode’s lactate–HEPES medium supplemented with 0.01% polyvinyl
alcohol). After washing, 70–80 COCs were transferred into 500 mL of
IVM medium (TCM-199; Invitrogen, Carlsbad, CA) supplemented with 20
ng/mL epidermal growth factor, 1 g/mL insulin, 75 g/mL kanamycin, 0.91
mM sodium pyruvate, 0.57 mM l-cysteine, and 10% (v/v) porcine
follicular fluid. After 22 h of culture, the COCs were transferred into IVM
medium without hormones and cultured for an additional 22 h at 38.5°C
in an atmosphere containing 5% CO2 and 100% humidity.
(E/Z)-dibenzyl 3-(2,16-dioxo-19-(3-phenylpropyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl phosphate (24c)
Compound 24c was synthesized from 23c following the general
procedure J (brown colour oil, 46 %).
(E/Z)-dibenzyl 3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl phosphate (24d)
Compound 24d was synthesized from 23d following the general
procedure J (colourless oil, 37 %).
(E/Z)-dibenzyl3-(2,16-dioxo-19-(7-phenylheptyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl phosphate (24e)
Compound 24e was synthesized from 23e following the general
procedure J (brown colour oil, 44 %).
Parthenogenetic activation and in vitro culture (IVC)
After maturation (44 h), cumulus cells were removed by repeated
pipetting in the presence of 1 mg/mL hyaluronidase for 2–3 min.
Denuded oocytes were activated by an electric pulse (1.0 kV/cm for 60
ms) in activation medium (280 mM mannitol, 0.01 mM CaCl2, and 0.05
mM MgCl2), followed by 3 h of incubation in PZM3 medium containing 2
mM cytochalasin B. About 70–80 post-activation oocytes were cultured in
4-well dishes containing 500 mL of PZM3 for 168 h. Embryo culture
conditions were maintained at 38.5°C in an atmosphere containing 5%
CO2 and 100% humidity.
(E/Z)-dibenzyl 6-(2,16-dioxo-19-(7-phenylheptyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)hexyl phosphate (24f)
Compound 24f was synthesized from 23f following the general procedure
J (brown oil, 49 %) .
(E/Z)-dibenzyl 6-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)hexyl phosphate (24g)
Compound 24g was synthesized from 23g following the general
procedure J (colourless oil, 58 %)
General Procedure K for the synthesis of compound 1 to 7
(E/Z)-2-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo
[15.2.1]icosa-1(20),6,17-trien-3-yl)ethyl dihydrogen phosphate (1)
Compound 24a (54 mg, 0.074 mmol) was dissolved in TFA/TIS/H2O
Mouse Oocyte collection and the in vitro maturation procedure
All procedures with mice were conducted according to the Animal
Research Committee guidelines of Chungbuk National University, Korea,
and all animal manipulations and experimental protocols were approved
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700048
ChemMedChem
FULL PAPER
by the Animal Research Committee, Chungbuk National University,
Korea. The 4 to 6 week-old imprinting control region (ICR) mice were
sacrificed by cervical dislocation at 48 h after injected with 10 IU of
pregnant mare’s serum gonadotropin (PMSG, Daesung Biochemical,
Daejun, Korea) and germinal vesicle (GV) stage oocytes were collected
from ovaries. Cumulus-free and intact-GV follicular oocytes were
released from large antral follicles via puncture with a needle in the M2
medium (Sigma-Aldrich, St. Louis, MO, USA) with 60 μg/mL of penicillin
and 50 μg/mL of streptomycin. All cell culture were maintained in the M16
medium (Sigma-Aldrich) at 37 °C in a humidified atmosphere containing
5% of CO2. Cumulus cell-enclosed metaphase II-arrested eggs were
obtained from mice of the same strain. The cumulus cell masses
surrounding the eggs were removed by brief exposure to 300 IU/mL
hyaluronidase in the M2 medium.
Korea Basic Science Institute Grant T36412 (J.K.B.) and the
Next-Generation BioGreen 21 Program (PJ011126 and
PJ011206), Rural Development Administration (N.H.K. and
S.N.). The authors would like to thank Professor Kyoung Tai No
for accessing the computational resources in his group.
Keywords: Oocyte maturation• Polo-like kinase 1• Macrocyclic
small molecule inhibitor• Contraceptive agent
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300 μM in
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upon Thames, Surrey, UK) equipped with
a Narishige MM0-202N
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Molecular modeling methods
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All calculations were performed using Discover 2.98/InsightII with CVFF
force field as described by Hagler, A. T.; Lifson, S.; Dauber, P. J. Am.
Chem. Soc., 1979, 101, 5122. The crystal structure of PBD of Plk1
having a pThr mimetic-containing C6H5(CH2)8- group bound was used as
the computational model (3RQ7.pdb). The synthetic inhibitor 4 was built
from the coordinates of this pThr mimetic-containing C6H5(CH2)8- group
as found in the crystal structure using the Builder module in Insight II.
The computational complex model was solvated using a solvent sphere
of water extending 30.0 Å around the phosphate atom. The system was
initially minimized using 1000 steps of steepest decent and 3000 steps of
conjugated gradient with a 19.0 Å non-bonded cutoff distance.
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Chrpova, E. Chmelikova, L. Tumova, F. Jilek, PLoS One 2015, 10,
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Notes
This manuscript was written with the contribution of all authors. All the
authors have approved the final version of the manucript. The authors
declare no competing financial interest.
[19] K. Jeong, J. Y. Jeong, H. O. Lee, E. Choi, H. Lee, Dev. Biol. 2010, 345,
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Acknowledgements
This work was partly supported by International Collaborative
Research Program of Institute for Protein Research, Osaka
University, ICR-15-05 (J.K.B.), National Research Council of
Science & Technology (NST) grant CAP-16-03-KRIBB, (J.K.B.),
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10.1002/cmdc.201700048
ChemMedChem
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Entry for the Table of Contents
Text for Table of Contents: Plk1 plays vital roles in fertilization and the inhibition of Plk1 hinders the oocyte maturation. We
synthesized pyrrole based macro cyclic compounds which inhibited the oocyte maturation and affected the embryonic development
potential of porcine oocytes. Besides, the most potent macrocyclic compound, 4 displayed enhanced cell penetration towards cell
membrane. Further, investigation of this compound treated mouse oocytes showed impaired spindles, confirmed the inhibition of
Plk1.
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