Lead optimization of a dihydropyrrolopyrimidine inhibitor against phosphoinositide 3-kinase (PI3K) to improve the phenol glucuronic acid conjugation

Article abstract of DOI:10.1016/j.bmcl.2012.11.112

Our lead compound for a phosphoinositide 3-kinase (PI3K) inhibitor (1) was metabolically unstable because of rapid glucuronidation of the phenol moiety. Based on structure-activity relationship (SAR) information and a FlexSIS docking simulation score, aminopyrimidine was identified as a bioisostere of phenol. An X-ray structure study revealed a hydrogen bonding pattern of aminopyrimidine derivatives. Finally, aminopyrimidine derivatives 33 showed strong tumor growth inhibition against a KPL-4 breast cancer xenograft model in vivo.

Full text of DOI:10.1016/j.bmcl.2012.11.112

Bioorganic & Medicinal Chemistry Letters 23 (2013) 673–678
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead optimization of a dihydropyrrolopyrimidine inhibitor against
phosphoinositide 3-kinase (PI3K) to improve the phenol glucuronic acid
conjugation
Hatsuo Kawada a, , Hirosato Ebiike b, Masao Tsukazaki b, Mitsuaki Nakamura b, Kenji Morikami a,
⇑
Kiyoshi Yoshinari b, Miyuki Yoshida b, Kotaro Ogawa a, Nobuo Shimma b, Takuo Tsukuda b, Jun Ohwada b
a Research Division, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
b 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:
Our lead compound for a phosphoinositide 3-kinase (PI3K) inhibitor (1) was metabolically unstable
because of rapid glucuronidation of the phenol moiety. Based on structure–activity relationship (SAR)
information and a FlexSIS docking simulation score, aminopyrimidine was identified as a bioisostere of
phenol. An X-ray structure study revealed a hydrogen bonding pattern of aminopyrimidine derivatives.
Finally, aminopyrimidine derivatives 33 showed strong tumor growth inhibition against a KPL-4 breast
cancer xenograft model in vivo.
Received 14 September 2012
Revised 22 November 2012
Accepted 27 November 2012
Available online 3 December 2012
Keywords:
PI3K
Ó 2012 Elsevier Ltd. All rights reserved.
Cancer
SBDD
Bioisostere
Glucuronic acid conjugation
Phosphoinositide 3-kinase (PI3K) pathway is activated in vari-
ous human cancers and have recently been ꢀ target.1,2 Among
the three PI3K family members (class I, II, III), class I PI3Ks are a
well-known promising target for cancer treatment because, by
converting phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to
phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) which then
activates the protein serine/threonine kinase AKT, they regulate a
range of cellular functions including cell cycle and cell survival
promotion.3 In addition, although PI(3,4,5)P3 is converted back to
PI(4,5)P2 by tumor suppressor PTEN, loss of heterozygosity and
mutations of PTEN are observed in various human cancers.4 Class
I PI3Ks are heterodimers consisting of a p110 catalytic subunit
port details, based on the X-ray study, of the interactions between
PI3K protein and our dihydropyrrolopyrimidine inhibitor with
aminopyrimidine moiety.
As we have already reported,7 we designed and identified the
dihydropyrrolopyrimidine PI3K inhibitor 1 as a lead compound
by superimposing PIramed’s PI103 (2)8 and Chiron’s PI3K inhibitor
(3)9 based on their docking models against the crystallographic
structure of PI3K (Fig. 1) and found that compound 1 inhibited
PI3Ka with IC50 value of 0.0086 l
M.10
First, we modified the 4-pyridyl moiety of compound 1. Because
the modeling suggested 4-pyridyl moiety extends to the solvent-
exposed region of PI3K and no clear interaction between 4-pyridyl
moiety and PI3K was specified, various kinds of heteroaromatic
ring were introduced. As shown in Table 1, removal of the nitrogen
atom significantly decreased activity (6) and several heteroaro-
matic rings, such as benzoimidazole (10) or thiazole (11), showed
moderate inhibitory activity but the most potent substituent was
pyridine. In the case of pyridine, the position of the nitrogen atom
was critical and 4-pyridyl was the best among three pyridine
regioisomers (1, 4, 5).
and a p85 regulatory subunit. Of four known p110 isoforms (
a, b,
c, and, d), significant efforts have focused on developing a p110
a
(PI3K
a
) inhibitor because frequent oncogenic mutations and gene
amplifications of p110
a
are found in human cancers.1,5,6
In this Letter, we describe details of optimizing our lead dihy-
dropyrrolopyrimidine PI3K inhibitor to overcome the issue of rapid
glucuronidation of the phenol moiety. Our approaches to solving
the issue were bioisosteric modifications and molecular design of
the phenol part using a virtual docking study. In addition, we re-
Unfortunately, oral bioavailability of compound 1 in mouse was
extremely low (1.6%). Further investigation suggested 1 is rapidly
metabolized by glucuronidation of phenol and the T1/2 in an
in vitro glucuronidation study was 11 min in human and 6.1 min
⇑
Corresponding author. Tel.: +81 0550 87 8364; fax: +81 0550 87 5326.
E-mail address: kawadahto@chugai-pharm.co.jp (H. Kawada).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.112

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H. Kawada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 673–678
O
N
O
O
N
N
N
O
N
N
N
N
N
N
HO
HO
HO
N
N
N
H
N
2 (PI103)
3 (Chiron’s PI3K inhibitor)
Figure 1. Lead generation.
1
Table 1
Table 2
In vitro activity of heteroaromatic compounds
In vitro activity of phenol derivatives
O
O
N
N
N
N
N
N
HO
N
Ar
Ar
Ar
N
N
Compound
PI3K
0.41
a IC50 (lM)
Compound
Ar
PI3Ka IC50 (lM)
O
N
13
1
0.0086
0.041
HO
HO
N
14
15
>50
>50
4
N
5
6
7
8
>50
1.22
0.17
0.20
N
N
derivatives. 3-Pyridyl derivative (28) was 10 times more potent
than 4-pyridyl (27) and 3,5-pyrimidyl (29) was much stronger
again. In addition, a FlexSIS docking simulation12 of virtual com-
pounds with PI3K
c
that was based on the X-ray structure informa-
good score for
OH
N
tion of PI3K with compound 10 gave a
c
aminopyridyl derivatives, such as 4-amino-2-pyridyl (30), 4-ami-
no-3-pyridyl (31) and 4-amino-3,5-pyrimidyl (32). FlexSIS scores
of 30, 31 and 32 were À27.3, À25.8 and À25.5 kJ/mol, respectively,
all lower than that of 10 (À23.3 kJ/mol). As shown in Table 6, these
9
>50
H
N
compounds showed good inhibitory activity against PI3Ka and 4-
10
0.077
amino-3,5-pyrimidyl was the most potent, with an IC50 value of
N
0.026 M (32).
l
S
The strong potency of 4-amino-3,5-pyrimidyl derivatives was
11
12
0.049
0.23
N
N
confirmed by an X-ray structure analysis of compound 33 with
PI3K
c
(Fig. 2).13 In the case of the hydrogen bonding pattern of
N
compound 10, the hydroxyl group of phenol interacts with
Asp841 and Tyr867. In the case of compound 33, the amino group
of aminopyrimidine is located between Asp836 and Asp841 and
makes a hydrogen bond with Asp836 (the distance between the
nitrogen of the amino group and the oxygen of Asp836 is 3.0 Å)
and an attractive electrostatic interaction with Asp841 (distance
between the nitrogen of the amino group and the oxygen of
Asp841 is 3.7 Å, which is not close enough for a hydrogen bond
to form). In addition, one of the nitrogen atoms of the pyrimidine
in mouse.11 To avoid this rapid metabolism, phenol was protected
as methyl ether (13) but the activity was 50-fold lower than 1
(Table 2). Introducing an ortho methyl group to block glucuronida-
tion also lost activity (14, 15). Based on the X-ray crystal structure
of 10 in PI3K
c (Fig. 2), various phenol bioisosteres that have a
+
hydrogen bond donor and acceptor were evaluated (16–20) but
ring interacts with the side chain NH3 of Lys833, and the other
their inhibitory activity was weak (Table 3).
nitrogen makes a weak attractive electrostatic interaction with
OH of Tyr867 (distance between the oxygen of Tyr867 and the
nitrogen of pyrimidine of 33 is 4.3 Å, which is not close enough
for a hydrogen bond). Notably, in order to make these hydrogen
bonds possible, the dihydropyrrolopyrimidine core structure of
The heteroaromatic ring was also tested (Table 4). Although
indazole derivatives lost inhibitory activity, benzoimidazole (21)
showed moderate activity. As shown in Table 5, further investiga-
tion of the heteroaromatic ring revealed a clear SAR of pyridine

H. Kawada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 673–678
675
O
N
Val882
(Val851)
Tyr867
(Tyr836)
N
N
H
N
HO
N
N
Asp841
(Asp810)
10
O
solvent-exposed
region
N
N
N
N
N
N
Lys833
(Lys802)
Asp836
(Asp805)
H2N
N
33
Figure 2. Crystal structure of 10 (shown in yellow) and 33 (shown in green) in PI3Kc. Numbering of PI3Ka is shown in parentheses.
Table 3
Table 4
In vitro activity of phenol bioisosteres
In vitro activity of benzoimidazole and indazoles
O
O
N
N
N
N
N
N
N
N
Ar
Ar
N
N
Compound
Ar
PI3K
3.7
a
IC50
(
lM)
Compound
Ar
PI3K
a IC50 (lM)
H2N
N
16
21
0.30
N
H
H
O
N
17
18
>50
19
HO
22
23
>50
>50
N
N
O
H2N
N
H
O
S
H
N
19
20
2.4
N
N
H
24
>50
H2N
O
O
N
HN
N
H
>50
25
26
>50
>50
H
N
N
33 shifted closer toward the solvent-exposed region than that of
the phenol compound 10.
As shown in Table 7,14,15 compound 32 and 33 showed strong
in vitro anti-proliferative activity against human cancer cell lines
that are known as PI3K
a mutant or PTEN null. The T1/2 of these
compounds in in vitro glucuronidation was over 60 min both in
human and mouse. Compound 33 showed remarkably improved
bioavailability (41%) in mice compared to lead compound 1
(Table 8).16 As presented in Figure 3, compound 33 showed strong
tumor growth inhibition (166%) against the KPL-417 human breast
sition, we first introduced various aryl groups for c-butyrolactone
34 (Route B: applied for compound 14, 15, 20, 28), and later ap-
plied Suzuki coupling19 to useful key intermediate 43 (Route C: ap-
plied for compound 6, 16–19, 21–27, 29–33). Representative
experimental procedures are available in the Supplementary data.
In summary, based on SARs and a FlexSIS docking simulation, 4-
amino-3,5-pyrimidyl derivative was identified as a bioisostere of
metabolically unstable 3-hydroxyphenyl derivative. 4-amino-3,5-
pyrimidyl derivatives 32 and 33 showed comparable PI3K inhibi-
tory activity to 1 and greatly improved metabolic stability. X-ray
cancer cell line (PI3Ka
H1047R mutant) at a dose of 80 mg/kg.18
Synthetic routes of our PI3K inhibitors are summarized in
Scheme 1. Compound 36 was identified as a key intermediate for
optimizing the substituent at the solvent-exposed region and var-
ious aniline derivatives were coupled with 36 (Route A: applied for
compound 1, 4, 5, 7–13). To optimize the aryl group of phenol po-
structure analysis of PI3K
c with 33 revealed the newly-formed

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H. Kawada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 673–678
Table 5
In vitro activity of pyridine derivatives
O
N
N
N
N
Ar
N
Compound
Ar
PI3K
22.6
a IC50 (lM)
27
N
N
28
29
1.70
0.44
N
N
Table 6
In vitro activity of aminopyridine derivatives
O
N
N
N
N
Ar
Ar
N
Compound
PI3K
0.34
a IC50 (lM)
N
30
H2N
H2N
H2N
N
N
31
32
0.24
0.026
N
Table 7
Improvement of glucuronidation and liver microsome (LM) stability
O
N
N
N
Ar2
Ar1
N
Compound
Ar1
Ar2
IC50
(l
M)
Glucuronidation T1/2 (min)
LM stablility T1/2 (min)
PI3K
a
HCT116a
PC-3b
KPL-4c
0.45
Human
Mouse
Human
Mouse
HO
N
N
1
0.0086
0.026
0.72
0.14
11
6.1
21
20
N
N
32
0.091
0.12
0.42
0.029
0.015
>60
>60
>60
>60
127
71
27
63
H2N
N
N
N
33
0.033
0.17
H2N
a
b
c
CRC, PI3K
Prostate cancer, PTEN negative.
Breast cancer, PI3K H1047R.
a
H1047R.
a

H. Kawada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 673–678
677
Table 8
Mouse PK profile of 33
Mouse PK (10 mg/kg iv)
Mouse PK (100 mg/kg po)
AUC (ng/mL h)
4690
CL (mL/min/kg)
35.9
T1/2 (h)
Cmax (ng/mL)
AUC (ng/mL h)
19400
CL/F (mL/min/kg)
86.1
T1/2 (h)
F (%)
41
10.3
4190
6.58
Pharmacokinetic parameters of 33 in female nude mice following an intravenous (10 mg/kg) and an oral (100 mg/kg) administration of 33. Values represent the mean of two
animals.
Y867
O
H
O
H
N
O
H
D841
N
H
N
OH
NH2
O
D836
OH
K833
Figure 4. Interactions of phenol of 10 and aminopyrimidine of 33.
Figure 3. In vivo antitumor activity of 33 in the KPL-4 mouse xenograft model. KPL-
4 cells were orthotopically injected into female BALB-nu/nu mice (Charles River
Laboratories Japan). The compound was orally administered once daily.
O
O
N
O
N
Route A
HBr
N
O
O
Ar
H2N
NH
H2N
NaSEt
DMF
N
N
POCl3
O
N
N
O
HO
NaOt Bu
t BuOH
neat
sealed tube
Ar
N
Pd catalyst
or NaH
O
150o
C
Cl
35
y. 36%
micro wave 110oC
120oC
37
ArCOCl
for route A and B
Cl
36
y. 28% ( 2 steps)
O
N
O
O
N
Route B
N
O
N
O
O
O
HCl
H2N
POCl3
LHMDS
H2N
NH
N
N
N
N
O
Ar
NaOMe
1,4-dioxane
r.t.
Pd catalyst
Cl
or NaH
neat
X
THF,
Ar
Ar
N
-78oC
sealed tube
Y
34
110o
C
38
39
40
Cl
Pd catalyst
Ar-B(OR)2
(CH3O)2CO
for route C
O
N
O
N
O
Route C
X
Y
N
HBr
O
O
H2N
H2N
NH
POCl3
N
N
N
N
O
O
NaH
THF
reflux
NaOMe
MeOH
reflux
neat
X
Y
Cl
Cl
Cl
100oC
N
42
y. 28% (2 steps)
43
41
y. 82%
Cl
y. 78% (X=Y=C)
y. 88% (X=C, Y=N)
y. 54% (X=N, Y=C)
Scheme 1. Alternative synthetic routes for lead optimization.

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H. Kawada et al. / Bioorg. Med. Chem. Lett. 23 (2013) 673–678
8. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Kawaguchi, K.; Koizumi, T.; Yamano,
M.; Matsuda, K.; Okada, M.; Ohta, M.; WO Patent, 1083456, 2001.
9. Nuss, J M.; Pecchi, S.; Renhowe, P A.; WO Patent, 4048365, 2004.
hydrogen bonds with Asp836 and Lys833 and the slight shift of the
whole inhibitor toward the solvent-exposed region. When modify-
ing the phenol moiety, we first concentrated on the interactions
with Asp841 and Tyr867 (depicted in blue in Fig. 4) based on the
X-ray structure of 10, but the results of this approach were not suc-
cessful. The virtual docking of the whole active site took into con-
sideration not only Asp841 and Tyr867, but also Asp836 and
Lys833 (depicted in red in Fig. 4). Considering the whole active site
by virtual docking in this way resulted in finding the 4-amino-3,5-
pyrimidyl derivative, which has interactions not only between
Asp841 and Tyr867 (a weak electrostatic interaction) but also be-
tween Asp836 and Lys833 (a strong hydrogen bond). Finally, ami-
nopyrimidine derivative 33 showed strong tumor growth
inhibition in a KPL-4 xenograft model in mouse.
10. Kinase assay: The inhibitory activity on PI3K
a
(p110
a
/p85a)
(Life
Technologies) was determined by Adapta Universal Kinase Assay Kit with
PIP2:PS Lipid Kinase Substrate (Life Technologies).
11. UGT–glucuronidation activity assay: Human (or Mouse) liver microsome (1.0 mg
protein/ml) was incubated in 50 mM Tris–HCl (pH 7.5) buffer containing
25
l
g/ml
alamethicin
at
4 °C
for
30 min.
Then,
the
UDP-
glucuronosyltransferases were released from the inside of the endoplasmic
reticulum membranes. Five micromolar of each compound was incubated in
human (or mouse) liver microsome solution with 2 mM of UDPGA (UDP–
glucuronic acid) cofactor at 37 °C for 60 min. After the enzyme reaction was
terminated with the addition of a threefold volume of acetonitrile, the reaction
mixture was centrifuged at 1500 rpm for 10 min. The resultant supernatant
was used as a test sample to measure the glucuronidation activity catalyzed by
human (or mouse) microsome by quantitating the compound in the sample
using LC/MS/MS.
12. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261, 470.
Compounds were docked to the crystal structure of PI3Kc complexed with 10
Acknowledgments
(PDB: 3APF) by the in silico docking tool, FlexSIS, while the
dihydropyrrolopyrimidine moiety was fixed to the position of compound 10 in
the crystal structure. Docking scores were definedby the FlexSIS standard scoring.
13. PDB ID code: 3APD.
14. Cell proliferation assay: The breast cancer KPL-4, colon cancer HCT116 (ATCC)
and prostate cancer PC-3 (ATCC) cells were treated with various concentrations
of assay compounds for 96 h. Cell growth inhibition was determined by Cell
Counting Kit-8 solution (Dojindo Laboratories).
15. Microsomal stability assay: One micromolar of each compound was incubated
with human (or mouse) liver microsome (0.5 mg protein/ml) in 50 mM
phosphate buffer (pH 7.4) containing 1 mM NADPH (the reduced form of
nicotinamide adenine dinucleotide phosphate) at 37 °C for 30 min. After the
enzyme reaction was terminated with the addition of a threefold volume of
acetonitrile, the reaction mixture was centrifuged at 1500 rpm for 10 min. The
resultant supernatant was used as a test sample to measure the stability in
human (or mouse) liver microsome by quantitating the compound in the
sample using LC/MS.
16. Pharmacokinetic study: Female BALB/c-nu mice (n = 2 per treatment group)
were given 33 by intravenous (iv) or oral (po) route at doses of 10 and 100 mg/
kg, respectively. Blood samples of each animal were collected with heparin as
an anticoagulant at 0.08, 0.25, 2, 4, 7, and 24 h following iv dosing and at 0.50,
2, 4, 7, and 24 h following po dosing. Samples were centrifuged to obtain
plasma and stored at À80 °C until analysis. Plasma concentrations were
determined by using LC–MS/MS system. The pharmacokinetic parameters
were calculated by non-compartmental analysis using WATSON ver. 7.1
(Thermo Fisher Scientific, Wayne, PA).
We thank H. Suda for glucuronidation assays. We thank T. Fujii
and K. Sakata for biological assays. We thank S. Kuramoto for PK
studies. We thank S. Courtney and W. Trigg (Evotec) for synthetic
assistance. We thank the staffs of synchrotron beam lines NW12
at PF, and of KEK and BL41XU at SPring-8 for their help in data col-
lection. We are grateful to Dr. Junichi Kurebayashi for providing
KPL-4 cells.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.
112. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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