Design and synthesis of fused bicyclic inhibitors targeting the L5 loop site of centromere-associated protein E

Article abstract of DOI:10.1016/j.bmcl.2016.07.038

Centromere-associated protein-E (CENP-E) is a mitotic kinesin which plays roles in cell division, and is regarded as a promising therapeutic target for the next generation of anti-mitotic agents. We designed novel fused bicyclic CENP-E inhibitors starting from previous reported dihydrobenzofuran derivative (S)-(+)-1. Our design concept was to adjust the electron density distribution on the benzene ring of the dihydrobenzofuran moiety to increase the positive charge for targeting the negatively charged L5 loop of CENP-E, using predictions from electrostatic potential map (EPM) analysis. For the efficient synthesis of our 2,3-dihydro-1-benzothiophene 1,1-dioxide derivatives, a new synthetic method was developed. As a result, we discovered 6-cyano-7-trifluoromethyl-2,3-dihydro-1-benzothiophene 1,1-dioxide derivative (+)-5d (Compound A) as a potent CENP-E inhibitor with promising potential for in vivo activity. In this Letter, we discuss the design and synthetic strategy used in the discovery of (+)-5d and structure–activity relationships for its analogs possessing various fused bicyclic L5 binding moieties.

Full text of DOI:10.1016/j.bmcl.2016.07.038

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of fused bicyclic inhibitors targeting the L5 loop
site of centromere-associated protein E
⇑
⇑
Takaharu Hirayama , Masanori Okaniwa , Hiroshi Banno, Hiroyuki Kakei, Akihiro Ohashi,
Momoko Ohori, Tadahiro Nambu, Kenichi Iwai, Tomohiro Kawamoto, Akihiro Yokota, Maki Miyamoto,
Tomoyasu Ishikawa
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Centromere-associated protein-E (CENP-E) is a mitotic kinesin which plays roles in cell division, and is
regarded as a promising therapeutic target for the next generation of anti-mitotic agents. We designed
novel fused bicyclic CENP-E inhibitors starting from previous reported dihydrobenzofuran derivative
(S)-(+)-1. Our design concept was to adjust the electron density distribution on the benzene ring of the
dihydrobenzofuran moiety to increase the positive charge for targeting the negatively charged L5 loop
of CENP-E, using predictions from electrostatic potential map (EPM) analysis. For the efficient synthesis
of our 2,3-dihydro-1-benzothiophene 1,1-dioxide derivatives, a new synthetic method was developed. As
a result, we discovered 6-cyano-7-trifluoromethyl-2,3-dihydro-1-benzothiophene 1,1-dioxide derivative
(+)-5d (Compound A) as a potent CENP-E inhibitor with promising potential for in vivo activity. In this
Letter, we discuss the design and synthetic strategy used in the discovery of (+)-5d and structure–activity
relationships for its analogs possessing various fused bicyclic L5 binding moieties.
Received 26 May 2016
Accepted 19 July 2016
Available online xxxx
Keywords:
CENP-E
Electrostatic potential map (EPM)
2,3-Dihydro-1-benzothiophene 1,1-dioxide
derivatives
Anti-cancer agent
Anti-mitotic agent
Ó 2016 Elsevier Ltd. All rights reserved.
Centromere-associated protein-E (CENP-E) is a mitotic spindle
motor protein of the kinesin superfamily1–4 and is localized at
the kinetochores of chromosomes.1,5 CENP-E plays a crucial role
in chromosome congression at the equator by capturing the micro-
tubule.6–11 Dysfunction of CENP-E causes the pole-proximal chro-
mosomes by chromosomal misalignment, resulting in prolonged
mitotic arrest and the consequent apoptosis. Based on these find-
ings, CENP-E attracts as a drug discovery target for the next gener-
ation of cancer therapeutic drugs.12–14 Recently, several small
molecule CENP-E inhibitors have been reported to induce antipro-
liferative activities in cancer cells in preclinical studies, while the
clinical potency of the CENP-E inhibitor is being evaluated in the
early phase of the clinical trials.15–20 In our previous work, we
developed a novel slow-binding type of CENP-E inhibitor, (+)-5d
(Compound A).21 Compound A displays antiproliferative activity
in various tumor cell lines as well as antitumor activity in a human
cancer xenograft model in mice. In this study, we demonstrate the
chemical profiles of Compound A and its derivatives. This informa-
tion will be helpful to develop the chemical optimization of CENP-E
inhibitors.
To date, we have reported the results of our drug discovery
efforts on the imidazo[1,2-a]pyridine class of CENP-E inhibitors
represented by (S)-(+)-1 (CENP-E IC50: 3.6 nM).22 With the aim of
further lead optimization starting from (S)-(+)-1, we applied EPM
analysis to the fused bicyclic moiety binding to the L5 loop site
of CENP-E. Here, we report the design and synthetic strategy
employed in the discovery of compound (+)-5d as well as SAR for
its analogs possessing various fused bicyclic L5 binding moieties.
A previously reported structure–activity relationships (SAR)
study based on our hypothetical modeling pointed out the struc-
turally significant molecular characteristics for potent CENP-E
inhibition (Fig. 1): (1) introduction of a basic N,N-dimethy-
laminoethyl group achieves strong interaction with carboxylate
of Glu186,24 (2) a methoxy group inserted into the imidazo[1,2-
a]pyridine ring provides high affinity for the core scaffold with
Ile182 of CENP-E in a hydrophobic interaction manner by adjusting
electron density distribution on the pyridine ring,22 and (3) dihy-
drobenzofuran moiety of 1 is also quite important because bicyclic
ring formation at the benzylic position produces a rigid molecular
conformation and the two consequent enantiomers which are dis-
tinctively recognized by the ligand binding site, providing effective
Abbreviations: DIPEA, N,N-diisopropylethylamine; HATU, hexafluorophosphate
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium; PD, pharmacodynamic.
⇑
Corresponding authors. Tel.: +81 466 32 1160; fax: +81 466 29 4448 (T.H.); tel.:
+81 466 32 1158 (M.O.).
E-mail addresses: takaharu.hirayama@takeda.com (T. Hirayama), masanori.
okaniwa@takeda.com (M. Okaniwa).
http://dx.doi.org/10.1016/j.bmcl.2016.07.038
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hirayama, T.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.038

2
T. Hirayama et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
N,N-dimethylaminoethyl group is
essential for interaction with protein.
adjust electron density distribution on the benzene ring to be pos-
itively charged by chemical modification of the fused bicyclic ring
(Fig. 2B). Designed structures and their EPM views are described in
Figure 2C: (1) selecting the optimal X atom of the fused bicyclic
scaffold to induce positive charge on the benzene ring (compounds
2–5a), (2) discovering the optimal combination of the electron
withdrawing R1 and R2 groups on the benzene ring in a manner
that maintains a favorable electrostatic profile (compounds 5b–
d) (Fig. 2C). According to EPM analysis of these newly designed
compounds, 2,3-dihydro-1-benzothiophene derivative 4 (X = S),
but not compounds 2 (X = NH) and 3 (X = CH2), exhibited similar
electrostatic potential to 1 (X = O). Furthermore, compound 5a
(X = SO2) and its analogs (5b–d) clearly showed positively charged
profiles, suggesting that this series of compounds could be antici-
pated to be optimal.
For the efficient synthesis of the promising series (+)-5a–d,
robust synthetic methods were developed (Fig. 3). We applied
two different approaches to prepare key intermediates I; (1) a con-
ventional synthetic method via conjugate addition reaction25 of
benzothiophene 1,1-dioxide derivatives II with amines in Route
A, and (2) newly developed synthetic method via one-pot imina-
tion and the following cyclization reaction of o-methanesulfonyl
substituted benzaldehyde V in Route B.
The preparation methods for compounds 5a–d are shown in
Scheme 1. The condensation of 3-(4-fluoro-3-methylphenyl)-5-
methoxyimidazo[1,2-a]pyridine-2-carboxylic acid 622 with the
key intermediate diamine derivatives ( )-7a–d using HATU in the
presence of DIPEA or pyridine to give the corresponding racemic
derivative ( )-5a–d in 68–80% yields. The optical resolution of
racemic ( )-5a–d by a chiral HPLC gave two corresponding (+)-
and (ꢀ)-enantiomers. Absolute configurations of the (+)-enan-
tiomers were determined to be (S) by the single X-ray crystallo-
graphic analysis of representative compound (+)-5d (Fig. S1).
For the synthesis of amine derivatives (I) as the key intermedi-
ates, we initially adopted route A to prepare ( )-7a–b. The reduc-
tion of ketone derivatives 8a,b with sodium borohydride
provided alcohols 9a,b. Alcohols 9a,b were treated with boron tri-
fluoride etherate (BF3ꢁEt2O) to give benzothiophene derivative 10a,
b in good yields. Subsequent oxidation of 10a,b with hydrogen per-
oxide under the existence of a catalyst, sodium tungstate dihy-
drate, in AcOH and MeOH afforded benzothiophene 1,1-dioxide
derivatives 11a,b (precursor II) in high yields. The conjugate addi-
tion reaction of 11a,b with N,N-dimethylethane-1,2-diamine pro-
vided desired diamine derivatives ( )-7a,b in 43% and 62% yields,
respectively. Regarding ( )-7c,d, the preparation of 11c,d (precur-
sor II) was achieved in 4 steps from commercially available 1,2-
difluoro-3-(trifluoromethyl)benzene 12c and 2-fluoro-6-(trifluo-
romethyl)benzonitrile 12d. Lithiation and subsequent formylation
reaction assisted by neighboring group effect of fluorine atoms of
12c,d successfully gave formylated derivatives 13c,d in high yields.
The reaction of 13c,d with sodium methanesulfinate in DMSO pro-
vided methanesulfonyl derivatives 14c,d in moderate yields. Next,
the intramolecular cyclization reaction of 14c,d by using sodium
ethoxide in EtOH proceeded to give 15c,d, followed by dehydration
using MsCl under the existence of triethylamine in THF to give 11c,
d in good yields. Compound ( )-7d was synthesized in 43% yield in
a synthetic method similar to that for ( )-7a (route A). On the other
hand, conjugate addition of 11c (R1 = F, R2 = CF3) with N,N-
dimethylethane-1,2-diamine unexpectedly did not provide the
mixture of ( )-7c, but rather byproduct resulting from displace-
ment of the fluorine group (R1) of 11c and additional byproduct
containing N,N-dimethylethane-1,2-diamine introduced at the R1
position of ( )-7c. This result motivated us to develop a one-pot
imination-cyclization reaction (route B) to prepare ( )-7c. The
one-pot imination of 14c (precursor V) with N,N-dimethylethane-
1,2-diamine in the presence of AcOH followed by intramolecular
Glu186
CH3
H3C
N
Cl
Cl
Putative interaction
with L5 loop.
N
N
N
Ile182
Me
O
L5 loop
Glu100
O
O
Asymmetric carbon is well
recognized by protein.
5-Methoxy imidazo[1,2-a]pyridine
is most active scaffold, adjusting
electrostatic potential on scaffold
based on EPM analysis.
Me
F
(S)-(+)-1
CENP-E IC50 = 3.6 nM
Cell HeLa GI50 = 130 nM
Figure 1. Chemical structure of lead compound (S)-(+)-1 and SAR summary for
imidazo[1,2-a]pyridine series.22
interaction of eutomer (S)-(+)-1 with the L5 loop of CENP-E
protein.
In this Letter, we thoroughly investigated chemical modification
at the dihydrobenzofuran moiety. Our docking model of CENP-E
indicated that the benzene ring of the dihydrobenzofuran moiety
in (S)-(+)-1 is located near the negatively charged carboxylate moi-
ety of Glu100 in the L5 loop (shown in red, Fig. 2A). Therefore, our
design concept for identifying an optimal CENP-E inhibitor was to
Figure 2. (A) Docking model of dihydrobenzofuran moiety of compound (S)-(+)-1
with L5 Loop of CENP-E protein. (B) Plausible key interaction with negatively
charged Glu100 of L5 Loop. (C) Design of compounds 2–5 and the EPM surfaces for
the fused bicyclic scaffolds. The EPMs were calculated with MOE (version
2011.10).23 Blue, red and white color indicate positive, negative and neutral
charges, respectively. The benzene ring of the fused bicyclic scaffold is outlined by a
white box.
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T. Hirayama et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Route A
Conjugate
addition
O
R2
S
O
R2
S
O
R1
R1
Me
N
II
III
Me
Route B
HN
One-pot imination
and cyclization
Me
R2
S
O
N
O
OHC
Me
R1
N
Me
M
I
R2
R2
S
F
H2C
O
O
R1
VI
O
R1
R2
S
O
V
R1
IV
Figure 3. Retrosynthetic analysis of key intermediate diamine derivatives (I) via route A and our newly developed route B.
Me
Me
N
N
R2
R1
Me
N
N
N
Me
OH
N
N
HN
(a)
S
O
O
O
O
O
Me
Me
O
R2
S
Me
O
O
Me
R1
Optical resolution
by chiral HPLC
(+)-5a
F
F
6
( )-7a R1 = Cl, R2 = Cl
( )-7b R1 = Cl, R2 = CF3
( )-7c R1 = F, R2 = CF3
( )-7d R1 = CN, R2 = CF3
( )-5a R1 = Cl, R2 = Cl (68%)
( )-5b R1 = Cl, R2 = CF3 (80%)
( )-5c R1 = F, R2 = CF3 (71%)
( )-5d R1 = CN, R2 = CF3 (78%)
(+)-5b
(+)-5c
(+)-5d (Compound A)
Me
N
Me
Route A
HN
O
HO
(b)
(d)
(e)
(c)
R2
R2
S
S
R2
R2
R2
S
S
S
O
O
O
R1
R1
O
R1
1 = Cl, R2 = Cl
1 = Cl, R2 = CF3
R1
R1
R
1 = Cl, R2 = Cl (82%)
1 = Cl, R2 = Cl (93%)
( )-
( )-
1 = Cl, R2 = Cl (43%)
7a
R
7b
11a
11b
R
8a
8b
9a
9b
10a
10b
R
R
R
R
1 = Cl, R2 = Cl (98%)
1 = Cl, R2 = CF3 (98%)
R
1 = Cl, R2 = CF3 (96%)
R
1 = Cl, R2 = CF3 (62%)
R
1 = Cl, R2 = CF3 (80%)
Me
N
Me
HN
O
O
S
HO
(f)
(g)
(h)
(i)
(j)
H
H
Me
R2
S
R2
R2
S
O
S
F
R2
F
R1
R2
O
O
O
O
R1
O
R1
R1
O
R1
1 = F, R2 = CF3
1 = CN, R2 = CF3
R1
1 = F, R2 = CF3 (98%)
R1
O
15c R1 = F, R2 = CF3 (82 %)
15d
11c R1 = F, R2 = CF3 (90%)
( )-7c R1 = F, R2 = CF3 (mixture)
7d
1 = F, R2 = CF3 (56%)
14c
R
12c
12d
13c
13d
R
R
R
R
1 = CN, R2 = CF3 (quant)
R
1 = CN, R2 = CF3 (57%)
R
1 = CN, R2 = CF3 (97%) ( )-
R
1 = CN, R2 = CF3 (43%)
14d
R
1 = CN, R2 = CF3 (96%) 11d
Route B
Me
N
Me
O
HN
H
Me
O
(k)
S
O
R2
R2
S
O
O
R1
R1
14c R1 = F, R2 = CF3
14d R1 = CN, R2 = CF3
( )-7c R1 = F, R2 = CF3 (63%)
( )-7d R1 = CN, R2 = CF3 (16%)
Scheme 1. Synthesis of (+)-5a–d, via route A or one-pot route B. Reagents and conditions: (a) HATU, pyridine, THF (for ( )-5a) or DIPEA, DMF (for ( )-5b–d), room
temperature to 60 °C, 3 h–6 h. (b) Sodium borohydride, MeOH, 0 °C to room temperature, 1 h–4 h; (c) BF3ꢁEt2O, AcOH, room temperature, 0.25 h, then 100–120 °C, 0.3 h; (d)
hydrogen peroxide, sodium tungstate dihydrate, AcOH, MeOH, 60–80 °C, 3 h–4 h; (e) N,N-dimethylethane-1,2-diamine, EtOH, 40 °C, 4 h then room temperature, 3 days (for
i
( )-7a) or PrOH, 60 °C, 12 h (for ( )-7b). (f) 2,2,6,6-Tetramethylpiperidine, 1.6 M butyllithium in hexane, THF, ꢀ60 to ꢀ50 °C, 0.5 h, then N,N-dimethylformamide, ꢀ50 °C,
0.25 h, then to ꢀ10 °C, 0.5 h–1 h; (g) sodium methanesulfinate, DMSO, room temperature, 1.5 h–2 h; (h) NaOEt, EtOH, 0 °C to room temperature, 0.7 h–1 h; (i) MsCl, Et3N, THF,
0 °C to room temperature, 0.5–4 h; (j) N,N-dimethylethane-1,2-diamine, EtOH, 70 °C, 4.5 h. (k) N,N-Dimethylethane-1,2-diamine, AcOH, THF, room temperature, 0.3 h–2 h,
then 1.11 M LDA in hexane, 0 °C or ꢀ50 °C, 0.3 h–1 h.
cyclization using lithium diisopropylamide (LDA) was successfully
conducted to yield the desired ( )-7c in 63% yield. Even when 14d
(R1 = CN, R2 = CF3) was used as a starting material, this one-pot
method proceeded to afford the desired ( )-7d in 16% yield. We
expect that this facile one-pot synthetic method would be useful
for constructing the 3-amino-2,3-dihydro-1-benzothiophene 1,1-
dioxide scaffold as a pharmaceutical building block.
The results from evaluating the CENP-E ATPase inhibitory activ-
ity (IC50) and cellular growth inhibitory activity (GI50) in HeLa cells
for compounds 1–5d are summarized in Table 1. Compound ( )-2
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T. Hirayama et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
dates for further in vivo evaluation. We measured the drug concen-
SAR for fused bicyclic compounds 1–4 and 5a–5d
trations of tested compounds in both plasma and tumor, as well as
accumulation levels of p-HH3 as an in vivo PD marker in a tumor
xenograft model. After intraperitoneal (ip) administration with
100 mg/kg once a day (qd) in a human colorectal cancer Colo205
xenograft mouse model, (+)-5d showed higher levels of p-HH3
accumulation compared with (+)-5a. Drug concentration measure-
ments found that compound (+)-5d showed better drug exposure
in tumor than (+)-5a (Table 2). Based on these results, compound
(+)-5d was selected as our CENP-E inhibitor candidate for further
development.
Me
Me
N
R2
R1
N
∗
N
N
X
O
Me
O
Me
F
R1 R2 CENP-E IC50 (nM) HeLa GI50 (nM)
In this Letter, we have described the rational design of fused
bicyclic moiety targeting the L5 loop moiety of CENP-E using
EPM analysis. A newly developed one-pot method for the prepara-
tion of 3-amino-2,3-dihydro-1-benzothiophene 1,1-dioxide scaf-
fold proved to be facile and robust. Because compound (S)-(+)-5d
demonstrated promising in vivo activity in single ip administration
in a human Colo205 xenograft model in mice, we selected com-
pound (+)-5d as our candidate possessing promising in vivo poten-
tial. As we have reported elsewhere, compound (+)-5d was used as
an in vivo chemical tool designated as ‘Compound A’ to investigate
the unique molecular biology of cell death induced by CENP inhibi-
tion, which exhibits a different mechanism of action from that of
another reported mitotic kinesin inhibitor, ispinesib.20 Compound
(+)-5d analogs are characterized by an ATP-competitive like mode
of action, but with time-dependent inhibition profiles in which 1 h
pre-incubation of compounds and CENP-E protein prior to ATP
addition enhances inhibitory activities even at high ATP concentra-
tion in enzymatic assay.22 This feature is different from the previ-
ously reported CENP-E inhibitor GSK923295A. Further
characterization and in vivo pharmacology studies of (+)-5d were
recently published.21
a
b
Compound
X
( )-1
(+)-1
( )-2
( )-3
O
Cl Cl
4.8 (3.1–7.5)
3.6 (3.1–4.2)
24 (16–37)
19 (15–24)
14 (11–17)
2.7 (2.0–3.8)
1.6 (1.2–2.1)
270
130
870
>1000
850
160
81
110
130
80
NH Cl Cl
CH2 Cl Cl
S
( )-4
Cl Cl
( )-5a
(+)-5a
(+)-5b
(+)-5c
SO2 Cl Cl
SO2 Cl CF3 1.7 (1.3–2.1)
SO2 CF3 1.5 (1.3–1.8)
F
(+)-5d (Compound A) SO2 CN CF3 2.2 (1.9–2.6)
a
n = 2. 95% confidence interval (CI) value.
Cell proliferation of HeLa cells was determined by measuring cellular ATP
amount (n = 1). Cells were collected 3 days after drug treatment.
b
(X = NH, IC50 = 24 nM) and ( )-3 (X = CH2, 19 nM) showed
decreased CENP-E inhibitory activities compared to ( )-1 (X = O,
4.8 nM). On the other hand, oxidized derivative ( )-5a (X = SO2)
showed potent CENP-E inhibition (IC50 = 2.7 nM) in contrast to
unoxidized derivative ( )-4 (X = S, IC50 = 14 nM). Consequently,
the eutomer (+)-5a (IC50 = 1.6 nM, GI50 = 81 nM) was chosen as a
lead compound for next SAR studies in which compounds with
combinations of electron withdrawing R1 and R2 groups on the
benzene ring were evaluated. Compounds (+)-5b–5d showed
potent CENP-E inhibition (IC50 = 1.7 nM, 1.5 nM and 2.2 nM,
respectively) comparable with (+)-5a. Among these compounds,
(+)-5a (R1, R2 = Cl) and (+)-5d (R1 = CN, R2 = CF3) demonstrated
potent anti-proliferative activity against HeLa cells (GI50 = 81 nM
and 80 nM, respectively).
We also performed cell cycle analysis by FACS to demonstrate if
prolonged mitotic arrest was induced in response to (+)-5a and (+)-
5d treatment, both of which exhibited a potent antiproliferative
activity in HeLa cells. The FACS analysis revealed that treatments
with these compounds was indeed induced phosphorylated his-
tone H3 (p-HH3), a mitotic index, in a dose-dependent manner,
and p-HH3 EC50 values of (+)-5a and (+)-5d were 79 and 39 nM,
respectively (Table 2). Given that p-HH3 EC50 values are close to
GI50 values in each compound, prolonged mitotic arrest could be
responsible for antiproliferation in the cells treated with these
compounds. Because (+)-5a and (+)-5d showed sufficient in vitro
cellular potency, we selected these compounds as promising candi-
Acknowledgments
We thank to Hitoshi Kandori and Kentaro Iwata for toxicology
and physicochemical evaluation; Yusuke Nakayama and Daisuke
Morishita for the biological characterization of test compounds;
Natsumi Fujii, Masako Nishimura, and Taeko Hattori for their assis-
tance in chiral separation; and Mitsuyoshi Nishitani, Motoo Iida,
and Yasumi Kumagai for their assistance in structure determina-
tions (X-ray single crystal study along with deposition of data to
the Cambridge Crystallographic Data Centre, and NMR study).
Supplementary data
Supplementary data (chemical synthesis procedures, biological
enzymatic and cellular assay methods, in vivo assay method, calcu-
lation method for electrostatic potential map (EPM) analysis, and
single-crystal X-ray structure analysis of (+)-5d (Fig. S1)) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2016.07.038.
Table 2
In vitro p-HH3 levels in HeLa cells, and pharmacokinetic profiles and p-HH3 levels in tumors in a Colo205 xenograft mouse model after treatment with (+)-5a and (+)-5d
Doseb (mg/kg)
Drug concentration
Accumulation of p-HH3 levels in tumord,e
a
Compound
In vitro p-HH3 EC50 (nM)
Plasmac,d
(
lg/mL)
Tumorc,d
(lg/g)
(+)-5a
(+)-5d (Compound A)
79
39
100
100
0.003
0.047
0.623
9.121
6.9
22.2
a
b
c
% of p-HH3 positive cells were determined using FACS in HeLa cells (n = 3).
Dose of (+)-5a and (+)-5d is described. Compounds were treated by ip administration.
Compound concentration in a Colo205 xenograft model in mice, 24 hours after the administration of (+)-5a and (+)-5d.
Mean (n = 2).
d
e
Phosphorylated HH3 in tumors was detected by Western blotting, 24 h after administration of (+)-5a and (+)-5d. Accumulation of p-HH3 levels were evaluated as band
intensities based on vehicle.
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T. Hirayama et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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Donatelli, C. A.; Darcy, M. G.; Ridgers, L. H.; Newlander, K. A.; Schmidt, S. J.;
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