Discovery and biological activity of a novel class i PI3K inhibitor, CH5132799

Article abstract of DOI:10.1016/j.bmcl.2011.01.065

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase and a promising therapeutic target for cancer. Using structure-based drug design (SBDD), we have identified novel PI3K inhibitors with a dihydropyrrolopyrimidine skeleton. Metabolic stability of the first lead series was drastically improved by replacing phenol with aminopyrimidine moiety. CH5132799, a novel class I PI3K inhibitor, exhibited a strong inhibitory activity especially against PI3Kα (IC₅₀ = 0.014 μM). In human tumor cell lines with PI3K pathway activation, CH5132799 showed potent antiproliferative activity. CH5132799 is orally available and showed significant antitumor activity in PI3K pathway-activated human cancer xenograft models in mice.

Full text of DOI:10.1016/j.bmcl.2011.01.065

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1767–1772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and biological activity of a novel class I PI3K inhibitor, CH5132799
⇑
Jun Ohwada , Hirosato Ebiike, Hatsuo Kawada, Masao Tsukazaki, Mitsuaki Nakamura, Takuya Miyazaki,
Kenji Morikami, Kiyoshi Yoshinari, Miyuki Yoshida, Osamu Kondoh, Shino Kuramoto, Kotaro Ogawa,
Yuko Aoki, Nobuo Shimma
Research Division, Chugai Pharmaceutical Co., Ltd 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase and a promising therapeutic target for cancer.
Using structure-based drug design (SBDD), we have identified novel PI3K inhibitors with a dihydro-
pyrrolopyrimidine skeleton. Metabolic stability of the first lead series was drastically improved by
replacing phenol with aminopyrimidine moiety. CH5132799, a novel class I PI3K inhibitor, exhibited
Received 25 October 2010
Revised 14 January 2011
Accepted 18 January 2011
Available online 22 January 2011
a strong inhibitory activity especially against PI3K
a (IC50 = 0.014 lM). In human tumor cell lines with
PI3K pathway activation, CH5132799 showed potent antiproliferative activity. CH5132799 is orally
available and showed significant antitumor activity in PI3K pathway-activated human cancer xeno-
graft models in mice.
Keywords:
PI3K
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Phosphatidylinositol 3-kinase (PI3K) plays crucial roles for
tumor progression in the survival signaling pathway (PI3K/Akt
pathway). This pathway is most commonly activated in human
we presumed that each common moiety plays an important role
in their interaction with PI3K. This led us to design a novel dihy-
dropyrrolopyrimidine inhibitor (4 and 5), as depicted in Figure 1,
whose morpholine and phenol moieties interact with valine 882
cancer by various factors such as frequent mutation of PI3Ka,
overexpression of growth factor receptor tyrosine kinases and
inactivation of the PTEN gene. Therefore targeting PI3K is a
promising approach for cancer therapy1 and significant efforts
have been made to develop antitumor PI3K inhibitors already.2
Utilizing the structural information thus made available, we de-
signed and synthesized novel dihydropyrrolopyrimidine deriva-
tives as PI3K inhibitors for cancer therapeutic agents using
and tyrosine 867 of PI3Kc, respectively, and whose pyridine ring
is nearly directed toward that of PI103 by conformational
restriction of dihydropyrrolopyrimidine skeleton, although its
interaction with PI3K is unclear. Compound 5 strongly inhibited
PI3K
of its analog (6) was confirmed by analyzing the X-ray crystal
structure of the PI3K
/inhibitor complex (Fig. 2).7 Hydrogen
a (IC50 = 0.0086 lM) and our prediction of the binding mode
c
structure-based drug design (SBDD).
PI3K was used for molecular design and the binding modes
of several compounds were confirmed by X-ray crystal struc-
ture4 of PI3K
. In this Letter, we wish to report effective SBDD
A
homology model3 of
bonds were observed between morpholine oxygen and valine
882 at the hinge region and between phenol and aspartic acid
841/tyrosine 867 in the affinity pocket.
a
c
However, oral bioavailability (BA) of compound 5 in mouse
was very low (only 1.6%), mainly due to rapid glucuronidation.
The metabolic stability in human liver microsomes was also
low (T1/2 = 21 min) (Fig. 3). To improve the metabolic stability,
we searched for a bioisostere of phenol by docking tools and
found an aminopyrimidine moiety as an alternative, which was
highly prioritized by FlexSIS virtual docking simulations.8 Thus
we prepared the second lead compound (7). It showed 3.8 times
from lead generation to optimization, and the synthesis and
biological activities of CH5132799 (1), a novel potent class I
PI3K inhibitor.
Design and synthesis:LY294002 is a well known PI3K inhibitor
and its crystal structure complex with PI3K
c was reported in
2000, indicating that the morpholine ring is essential for the
interaction with PI3K at the catalytic site.4 Using this 3D infor-
mation, two known PI3K inhibitors possessing a morpholino
group, PIramed’s PI103 (2)5 and Chiron’s inhibitor (3),6 were
superimposed onto the binding pocket by modeling tools. Both
inhibitors have morpholine, phenol, and pyridine moieties and
less inhibitory activity (IC50 = 0.033 lM) against PI3Ka than the
first lead (5), but 30 times stronger in vitro antiproliferative
activity against human breast cancer cell line KPL-4
(IC50 = 0.015
in vivo in
l
a
M). It also exhibited good antitumor activity
human prostate cancer PC-3 xenograft model
(112% tumor growth inhibition (TGI) after oral administration
of 100 mg/kg, q.d., of 7 for 11 days) as a result of improved met-
⇑
Corresponding author. Tel.: +81 467 47 6035; fax: +81 467 47 2248.
E-mail address: ohwadajn@chugai-pharm.co.jp (J. Ohwada).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.065

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J. Ohwada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1767–1772
Figure 1. Lead generation from known PI3K inhibitors by docking study.
Val882
hinge
O
N
H
Tyr867
N
N
N
N
HO
N
Asp841
solvent-exposed
region
affinity pocket
α
μ
Figure 2. Crystal structure of PI3Kc with 6.
Figure 3. Profiles of compounds 5 and 7.

J. Ohwada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1767–1772
1769
11a with sodium thioethoxide. Compounds 5 and 6 were synthe-
sized analogously using 4-aminopyridine and 1H-benzimidazole-
5-ylamine instead of 3-aminopyridine, respectively (Fig. 6). Com-
pound 7 was synthesized as shown in Figure 7. Cyclization of 14
(Fig. 8) with 3-aminopyridine in NaH/THF gave 15 in 88% yield.
Suzuki coupling with boronate 13, derived from 2-amino-5-bro-
mopyrimidine in two steps, followed by deprotection of the 4-
methoxybenzyl group of 16 in TFA, afforded compound 7 as a
white solid in good yield. The synthesis of CH5132799 (1) is out-
lined in Figure 8. A pyrimidine derivative (18) was prepared
from
morpholin-4-carboxamidine
hydrobromide
and
2-
oxotetrahydrofuran-3-carboxylic acid methyl ester11 (17) using
sodium methoxide as a base. Tri-chlorination of 18 with phos-
phoryl chloride in toluene gave pyrimidine trichloride (14) as a
white solid in good yield. 6,7-Dihydro-5H-pyrrolo[2,3-d]pyrimi-
dine derivative (19) was successfully synthesized from 14 and
methanesulfonamide in the presence of potassium carbonate in
NMP. Suzuki–Miyaura cross coupling of 19 and 2-amino-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine was con-
ducted using potassium phosphate and dichlorobis(triphenyl-
phosphine)palladium(II) in DMF to give CH5132799 (1) as a
colorless powder in 93% yield.
Figure 4. Crystal structure of PI3K with 7.
Biological activities of CH5132799:CH5132799 selectively inhib-
its class I PI3Ks, especially PI3K
inhibition of class II PI3Ks, class III PI3K and mTOR and also no
inhibitory activity (IC50 >10
M) against 26 protein kinases12 (Ta-
a (IC50 = 0.014 lM) but showed less
abolic stability and oral BA (41% in mouse) (Fig. 3). It was con-
firmed that compound 7 binds ATP binding sites of the enzyme
by X-ray crystal structure analysis of the PI3Kc/inhibitor com-
l
ble 1). In human tumor cell lines with PI3K pathway activation
plex.9 Hydrogen bonds were observed between morpholine oxy-
gen and valine 882 and between aminopyrimidine and aspartic
acid 836/lysine 833 (Fig. 4). With the aim of further improving
the physicochemical and ADME profiles, we modified the right-
hand side of the molecule (pyrido-3-yl moiety), and finally iden-
tified CH5132799 (1) as a clinical candidate that showed excel-
lent oral BA (101% in mouse), human liver microsomal stability
and in vivo antitumor activity in the PC-3 xenograft model
(TGI: 101% at 25 mg/kg, po, q.d. ꢀ 11 days). Its X-ray crystal
structure showed almost the same binding mode as that of com-
pound 7 (Fig. 5).10 An additional hydrogen bond between amino-
pyrimidine and aspartic acid 841 was observed.
by mutation, CH5132799 showed potent antiproliferative activity
[HCT116(CRC): IC50 = 0.20
l
M, KPL-4(BC):13 IC50 = 0.032
lM, T-
47D(BC): IC50 = 0.056 M, SK-OV-3(Ovarian): IC50 = 0.12
l
lM] as
shown in Table 1. In KPL-4 cells, effective suppression of phosphor-
ylation of AKT was observed.12 CH5132799 exhibited good oral BA
in mouse, rat, monkey and dog (F: 54.2–101%, Table 2). In a human
breast cancer (KPL-4: PI3Ka H1047R) xenograft model in mice, oral
treatment with CH5132799 (12.5 mg/kg, q.d.) showed strong tu-
mor regression as shown in Figure 9. The strong regression was
maintained during the 6 week administration, even in the inter-
mittent dosing schedule (q.d., 2 weeks on/1 week off; q.d., 5 days
on/2 days off), suggesting that a flexible administration schedule
can be applicable in the clinic.
Compound 4 was synthesized from
c-butyrolactone. Conden-
In summary, we successfully designed and discovered novel
PI3K inhibitors by SBDD. CH5132799 is an orally available, potent
class I PI3K inhibitor which showed significant antitumor activity
in PI3K pathway-activated human cancer xenograft models in mice
and promises to provide clinical benefits.
sation with 3-methoxybenzoyl chloride gave ketolactone (8).
Compound 8 was condensed with amidine followed by chlorina-
tion by POCl3 which gave dichloropyrimidine derivative 10. Pal-
ladium catalyzed amination with 3-aminopyridine afforded 11a
as a white solid. Compound 4 was obtained by treatment of
Figure 5. Structure and profiles of CH5132799 (1) and crystal structure with PI3K.

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J. Ohwada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1767–1772
Figure 6. Synthesis of 4, 5 and 6.
Figure 7. Synthesis of 7.

J. Ohwada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1767–1772
1771
Figure 8. Synthesis of CH5132799 (1).
Table 1
Enzyme inhibition profile and antiproliferation activity of CH5132799 (1)
Enzyme inhibition profile
Antiproliferation activity
IC50
(
lM)
IC50(lM)
PI3K class I
PI3K
PI3Kb
PI3Kd
PI3K
PI3K class II
PI3K C2
a
0.014
0.12
0.50
HCT116 (CRC, PI3K
KPL-4 (breast, PI3K
T-47D (breast, PI3K
SK-OV-3 (ovarian, PI3Ka H1047R)
MFE-280 (endometrial, PI3K H1047Y)
ME-180 (cervical, PI3Ka E545K)
a
a
H1047R)
H1047R)
a H1047R)
0.20
0.032
0.056
0.12
c
0.036
a
0.18
0.14
a
>10
5.3
PI3K C2b
PI3K class III
Vps34
>10
PIKK
mTOR
1.6
Protein kinase12
>10
Table 2
Pharmacokinetic parameters of CH5132799 in four animals
Species
Mouse
Dose (mg/kg)
Administration
T1/2 (h)
Tmax (h)
CL (mL/min/kg)
AUCinf (ng h/mL)
Bioavailability (%)
2
1
0.5
1
0.25
1
iv
po
iv
po
iv
po
iv
1.7
3.8
2.6
3.5
1.6
3.3
3.2
6.7
—
0.25
—
0.8
—
4.5
—
4.0
17.5
—
6.3
—
6.4
—
6.2
—
1930
978
1350
1790
660
1430
692
2170
—
101
—
66.2
—
54.2
—
78.4
Rat
Dog
Monkey
0.25
1
po

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J. Ohwada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1767–1772
2. (a) Sutherlin, D. P.; Sampath, D.; Berry, M.; Castanedo, G.; Chang, Z.;
2500
2000
1500
1000
500
KPL-4 (PIK3CAH1047R
)
Chuckowree, I.; Dotson, J.; Folkes, A.; Friedman, L.; Goldsmith, R.; Heffron, T.;
Lee, L.; Lesnick, J.; Lewis, C.; Mathieu, S.; Nonomiya, J.; Olivero, A.; Pang, J.;
Prior, W. W.; Salphati, L.; Sideris, S.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.;
Wiesmann, C.; Wong, S.; Zhu, B. Y. J. Med. Chem. 2010, 53, 1086; (b) Yaguchi, S.;
Fukui, Y.; Koshimizu, I.; Yoshimi, H.; Matsuno, T.; Gouda, H.; Hirono, S.;
Yamazaki, K.; Yamori, T. J. Natl. Cancer Inst. 2006, 98, 545; (c) Maira, S. M.;
Stauffer, F.; Brueggen, J.; Furet, P.; Schnell, C.; Fritsch, C.; Brachmann, S.; Chene,
P.; De Pover, A.; Schoemaker, K.; Fabbro, D.; Gabriel, D.; Simonen, M.; Murphy,
L.; Finan, P.; Sellers, W.; Garcia-Echeverria, C. Mol. Cancer Ther. 2008, 7(7), 1851.
Vehicle
q.d.
q.d. 2 weeks on-1 week off
q.d. 5 days on-2 days off
3. A homology model of PI3K
a
was constructed from the crystal structure of PI3K
c
comlex with staurosporine (PDB:1E8Z).
4. PI3Kc was expressed and purified as described previously Walker, E. H.; Pacold,
M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L.
Mol. Cell 2000, 6, 909. Crystals were grown in 100 mM Tris–HCl pH7.2, 200 mM
ammonium sulfate and 20% PEG 3350 with seeds. Diffraction data were
collected at beamlines NW12 at PF, KEK and BL41XU at SPring-8. Crystal
structures were solved by MR using the coordinates of PDB 1E8Y. 3D structure
complexes of 6, 7 and CH5132799 (1) with PI3Kc were refined to R/Rfree values
of 0.241/0.315 at 2.9 Å resolution, 0.250/0.316 at 2.5 Å, and 0.236/0.290 at
2.5 Å, respectively..
5. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Kawaguchi, K.; Koizumi, T.; Yamano,
M.; Matsuda, K.; Okada, M.; Ohta, M.; WO Patent, 1083456, 2001.
6. Nuss, J M.; Pecchi, S.; Renhowe, P A.; WO Patent, 4048365, 2004.
7. PDB ID code: 3APF
0
25
32
39
46
53
60
67
Days after tumor implantation
8. FlexX with the single interaction scan algorithm. (a) Matthias, R.; Bernd, K.;
Thomas, L.; Gerhard, K. J. Mol. Biol. 1996, 261, 470; (b) Matthias, R.; Stephan,
W.; Thomas, L. J. Comput. Aided Mater. Des. 1996, 10, 41. Virtually synthesized
compounds from 4-chloro-2-morpholin-4-yl-7-pyridin-4-yl-6,7-dihydro-5H-
pyrrolo[2,3-d]pyrimidine and available boronic acids having hydrogen bond
donor/acceptor were scored by FlexSIS docking simulations and compounds
with high FlexSIS scores were actually synthesized by Suzuki coupling. For
example, the score of 2-aminopyrimidine-5-yl was ꢁ25.5 kJ/mol, and this was
more highly prioritized than indoline-5-yl (ꢁ21.5 kJ/mol). We are publishing
these details in the near future. Filed patent: WO Patent, 018426, 2008 (Filing
date: Aug, 8, 2006).
Figure 9. In vivo efficacy of CH5132799 (1) in KPL-4 xenograft model CH5132799
was orally administered at 12.5 mg/kg for 6 weeks in three dosing schedules. Ç:
vehicle; }: q.d; 4: q.d., 2 weeks on/1 week off; h: qd, 5 days on/2 days off.
Acknowledgments
The HER2-positive human inflammatory breast cancer cell line
KPL-4 was kindly provided by Dr. J. Kurebayashi (Kawasaki Medi-
cal School, Kurashiki, Japan). We thank Drs. S. Courtney and W.
Trigg (Evotec) for synthetic assistance. We also thank the staffs
of synchrotron beamlines NW12 at PF, KEK and BL41XU at
SPring-8 for their help in data collection.
9. PDB ID code: 3APD
10. PDB ID code: 3APC
11. Quesada, M. L.; Schlessinger, R. H. J. Org. Chem. 1978, 43, 346.
12. Tanaka, H.; Yoshida, M.; Tanimura, H.; Fujii, T.; Sakata, K.; Tachibana, Y.;
Ohwada, J.; Ebiike, H.; Kuramoto, S.; Morita, K.; Yoshimura, Y.; Yamazaki, T.;
Ishii, N.; Kondoh, O.; Aoki, Y. The selective class I PI3K inhibitor CH5132799
targets human cancers harboring oncogenic PIK3CA mutations; Clin. Cancer
Res., in press.
References and notes
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