Optimization of the phenylurea moiety in a phosphoinositide 3-kinase (PI3K) inhibitor to improve water solubility and the PK profile by introducing a solubilizing group and ortho substituents

Article abstract of DOI:10.1016/j.bmc.2016.04.060

Phosphoinositide 3-kinase (PI3K) is a promising anti-cancer target, because various mutations and amplifications are observed in human tumors isolated from cancer patients. Our dihydropyrrolopyrimidine derivative with a phenylurea moiety showed strong PI3

Full text of DOI:10.1016/j.bmc.2016.04.060

Accepted Manuscript
Optimization of the phenylurea moiety in a phosphoinositide 3kinase (PI3K)
inhibitor to improve water solubility and the PK profile by introducing a solu-
bilizing group and ortho substituents
Hatsuo Kawada, Hirosato Ebiike, Masao Tsukazaki, Shun Yamamoto, Kohei
Koyama, Mitsuaki Nakamura, Kenji Morikami, Kiyoshi Yoshinari, Miyuki
Yoshida, Kotaro Ogawa, Nobuo Shimma, Takuo Tsukuda, Jun Ohwada
PII:
S0968-0896(16)30316-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.060
BMC 12975
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2016
26 April 2016
28 April 2016
Please cite this article as: Kawada, H., Ebiike, H., Tsukazaki, M., Yamamoto, S., Koyama, K., Nakamura, M.,
Morikami, K., Yoshinari, K., Yoshida, M., Ogawa, K., Shimma, N., Tsukuda, T., Ohwada, J., Optimization of the
phenylurea moiety in a phosphoinositide 3kinase (PI3K) inhibitor to improve water solubility and the PK profile
by introducing a solubilizing group and ortho substituents, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.04.060
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Optimization
of the
phenylur
ea m
oiety in a phosphoin
ositide 3-kin
as
e (
PI3K
)
inhibitor to
improve
water solubility and the PK profile by introducing a solubilizing group and ortho substituents
Hatsuo Kawada a,*, Hirosato Ebiike b, Masao Tsukazaki b, Shun Yamamoto b, Kohei Koyama a,
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
*Corresponding author. Tel.: +81 0550 87 8364; fax: +81 0550 87 5326
E-mail address: kawadahto@chugai-pharm.co.jp (H. Kawada).
ABSTRACT
Phosphoinositide 3-kinase (PI3K) is a promising anti-cancer target, because various mutations and
amplifications are observed in human tumors isolated from cancer patients. Our
dihydropyrrolopyrimidine derivative with a phenylurea moiety showed strong PI3K enzyme inhibitory
activity, but its pharmacokinetic property was poor because of lack of solubility. Herein, we report how we
improved the solubility of our PI3K inhibitors by introducing a solubilizing group and ortho substituents to
break molecular planarity.
1. Introduction
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that control various functions, including cell
survival, gene expression, and metabolism.1 The PI3K family comprises classes IA, IB, II, and III, and
class IA PI3Ks phosphorylate phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to generate
phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3),2 which is an important lipid second messenger
that activates serine/threonine kinase AKT, by which many cellular processes are regulated.1 In addition,
numerous cases of oncogenic gene mutations and gene amplification of class IA PI3Ks have been
reported in human cancer.2, 3, 4 Furthermore, efficient inhibition of PI3Kβ (an isozyme of class IA) in
PTEN-negative cancer cell lines was also reported.5 Thus, PI3Ks are promising cancer therapeutic
targets; several small molecule inhibitors are now being developed in clinical trials,6 and idelalisib was
approved as a selective inhibitor of PI3Kδ(another isozyme of class IA) in 2014.7
We have previously reported lead identification and optimization of our dihydropyrrolopyrimidine
PI3K inhibitors.8, 9 A lead compound was identified by superimposing Piramed’s PI3K inhibitor PI103 and
Chiron’s inhibitor. Based on information from a docking simulation, the metabolically unstable phenol of
our lead compound was replaced by aminopyrimidine, and strong in vivo antitumor efficacy was
observed. During our lead optimization process, we found that the phenylurea derivative (1) also shows
strong PI3K enzyme inhibitory activity, but its solubility was quite low and pharmacokinetic profiles were
poor. In this report, we describe how we improved the low solubility by introducing a solubilizing group
and breaking the molecular planarity.10 After the optimization, compound 2 showed much better AUC
(p.o.) in mice and showed strong tumor growth inhibition in vivo against PC-3 xenograft model.
Figure 1. PI3K inhibitors of phenylurea derivatives
2. Chemistry
11
Compound 1 was prepared by the urea formation of dihydropyrrolopyrimidine derivative 3 with
phenyl isocyanate in the presence of triethylamine (TEA), followed by deprotection of
paramethoxybenzyl (PMB) under an acidic condition (Scheme 1). Compounds 4a–4d were synthesized
from the same key intermediate 3. First, compound 3 was treated with triphosgene to afford a carbamoyl
chloride intermediate. The resulting crude mixture was treated with four different aniline derivatives to
give corresponding phenylurea derivatives. Deprotection of PMB by trifluoroacetic acid (TFA) in the
presence of N-acetylcysteine as a cation trap reagent gave compounds 4a–4d, respectively.

Scheme 1. Preparation of phenyl urea derivatives. Reagents and conditions: (a) phenyl isocyanate, TEA, DCE, reflux,
51%; (b) TFA, conc.H2SO4, 40 ˚C, 82%; (c) triphosgene, pyridine, DCM, rt; (d) aniline, DCM, rt; (e) TFA, N-acetylcysteine,
reflux, 49% in 3 steps (4a), 68% in 3 steps (4b), 56% in 3 steps (4c), 57% in 3 steps (4d).
Synthesis of ortho methyl aminopyrimidine derivative 10 is described in Scheme 2. Based on our
knowledge of preparing another aminopyrimidine derivative,11 2-amino-5-bromo-4-methylpyrimidine (5)
was protected by PMB. After the boronic acid formation using nBuLi and triisopropyl borate, the resulting
boronic acid was converted to boronic acid pinacol ester 7 by treatment with pinacol and Mg2SO4. A
coupling partner 9 was prepared from trichloride 8 7. 3-aminopyridine was treated with NaH and coupled
with trichloride 8. In situ cyclization afforded the desired dihydropyrrolopyrimidine derivative 9.
Suzuki-Miyaura coupling of 7 and 9 under conventional conditions, and the subsequent deprotection of
PMB gave 10.
Scheme 2. Preparation of compound 10. Reagents and conditions: (a) 4-methoxybenzyl chloride, NaH, NaI, THF, rt, 79%;
(b) B(OiPr)3, nBuLi, toluene/THF, −78 ˚C; (c) pinacol, MgSO4, DME, rt, 78% in 2 steps; (d) 3-aminopyridine, NaH, THF,
reflux, 67%; (e) 7, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 ˚C; (f) TFA, conc.H2SO4, 40 ˚C, 43% in 2 steps.
Preparation of ortho-substituted phenylurea derivatives with a solubilizing group is shown in
Scheme 3. Amidation of 3-methyl-4-nitrobenzoic acid 11 with N-ethylpiperazine in the presence of a
condensation reagent gave 12. Following hydrogenation of the nitro group by Pd/C under a H2 gas
atmosphere afforded the desired aniline derivative 13. 4-Fluoro-2-methyl-nitrobenzene 15 was treated
with N-ethylpiperazine in the presence of K2CO3 in DMSO at 80 ˚C to afford 16. Then, the nitro group
was reduced by zinc to give 17. 4-Bromo-2,6-difluoro-aniline was treated with (Boc)2O and a catalytic
amount of DMAP to protect the amino group by Boc. Following the Buchwald-Hartwig reaction using
N-ethylpiperazine under conventional coupling conditions gave 20. Deprotection of Boc under an acidic
condition afforded the desired aniline derivative 21. These aniline derivatives 13, 17, and 21 were
treated with carbamoyl chloride of 3, which was prepared by a method similar to that described above,
and deprotection of PMB gave 14, 18, and 2, respectively.

Scheme 3. Preparation of phenyl urea derivatives with
a solubilizing group. Reagents and conditions: (a)
N-ethylpiperazine, EDC•HCl, DMAP, DCM, rt, 79%; (b) Pd/C, H2 gas, MeOH, rt, 96%; (c) triphosgene, pyridine, DCM, rt;
(d) 13, DCM, rt; (e) TAF, N-acetylcysteine, reflux, 19% in 3 steps; (f) N-ethylpiperazine, K2CO3, DMSO, 80 ˚C; (g) Zn,
NH4Cl, MeOH, rt, 90% in 2 steps; (h) phosgene, DCM/sat.NaHCO3 aq. = 1/1, rt; (i) 17, TEA, DCE, rt; (j) TFA, reflux, 76% in
3 steps; (k) (Boc)2O, DMAP, DMF, rt; (l) N-ethylpiperazine, Pd(OAc)2, S-Phos, tBuOK, toluene, 60 ˚C, 20% in 2 steps; (m)
6M HCl/EtOAc, rt, 91%; (o) triphosgene, pyridine, DCM, rt; (p) 21, DCM, rt; (q) TAF, N-acetylcysteine, reflux, 52% in 3
steps.
3. Result and Discussion
Previously, we have reported our dihydropyrrolopyrimidine PI3K inhibitor 22 (IC50 for PI3Kαis 0.033
μM) and described its hydrogen bonding patterns with PI3Kαaccording to an X-ray crystal structure of
PI3Kγ 6,7. We have revealed that morpholine of compound 22 interacts with Val851 of the hinge region,
that aminopyrimidine makes a key interaction with both Asp805 and Lys802, and that pyridine is located
at the solvent-exposed region of PI3Kα, which allows the pyridine part to be used for various structural
modifications without significant loss of inhibitory activity 11. On the other hand, the phenylurea derivative
1 inhibited PI3Kαwith an IC50 value of 0.013 μM (Table 1). This enhanced inhibitory activity could be
explained from the docking simulation of compound 1 with the PI3KαX-ray structure. As shown in Figure
2, phenyl group can be expected to make a favorite lipophilic interaction with W780 (the distance is
somewhat far for good interaction) and cation-πinteraction with R770. We could find the side chain of
R770 near the phenyl ring of compound 1 in two PI3KαX-ray structures (PDB: 3ZIM, 4A55) but we could
not find the side chain of R770 at the phenyl ring side in the other two PI3KαX-ray structures (PDB: 4L23,
4L2Y). Thus the amino acid side chain of R770 is flexible and induced fit of this side chain could be
expected for our phenylurea derivatives. Compound 22 can also make these interactions using the
pyridine part, but the pyridine of compound 22 is relatively far from R770. Thus, the urea linker would
place the aromatic ring close enough to R770. The importance of the aromatic ring position is also
suggested by compound 4a, which has a methylated nitrogen atom in the urea. As shown in Table 1, the
enzyme inhibitory activity of 4a was 10 times weaker than that of compound 1, indicating that the phenyl
ring of 4a is not positioned suitably to interact with R770 and W780. This methyl group was introduced in
an attempt to improve the quite low solubility and poor pharmacokinetics profiles of 1. As described in
Table 1, the solubility of compound 1 in FaSSIF (fasted state simulated intestinal fluid)12 was extremely
low and pharmacokinetic profiles in mice were poor. One reason for this low water solubility would be the
highly planar structure of compound 1. As shown in Figure 3, intramolecular hydrogen bonding of urea
NH with the pyrimidine hetero atom would induce a flat configuration that increases the crystallization
energy and results in poor solubility. Although we tried breaking this intramolecular hydrogen bond to
reduce the crystallization energy in N-methylated compound 4a, this methylation did not improve the
solubility, as Table 1 shows.
Table 1
In vitro and in vivo profiles of phenylurea derivatives

PI3Kα
IC50 (μM)
F
aSSIF
Mouse PK
(50 mg/kg po)
Compound
R
(μg/ml)
AUC
Cmax
(ng/mL)
(ng• h/mL)
1
H
0.013
0.13
<3
<3
104
437
4a
Me
N.T.
N.T.
Figure 2. Docking simulation of phenylurea compound 1 with PI3Kα. Based on the X-ray structure information of PI3Kα(PDB: 3ZIM) and
the X-ray structure of our dihydropyrrolopyrimidine PI3K inhibitor with PI3Kγ (PDB: 3APD), compound 1 (green) was docked with PI3Kα
X-ray structure (ribbon). V851 and amino acid side chains of R770 and W780 were shown in stick form.
Figure 3. Estimated intramolecular hydrogen bonding
Ishikawa and Hashimoto reported that disrupting the molecular planarity by introducing an ortho
substituent can improve the solubility because its decreases the efficiency of the crystal packing.10 Thus,
we introduced an ortho methyl group on the biaryl moiety of our inhibitor, and compound 10 showed
good FaSSIF solubility (Table 2). But the IC50 value of compound 10 against PI3Kαwas decreased 8-fold
compared to compound 1, because (as mentioned above) both aminopyrimidine and morpholine are
essential for making key interactions with the PI3K enzyme, and disrupting the planarity at this point was
not acceptable for keeping these interactions. As changes in the dihedral angle at the aminopyrimidine
part resulted in a loss of inhibitory activity, ortho subsutituents were introduced on the aromatic ring of
the phenylurea. In the case of phenylurea compounds, alkyl substituents at the ortho position could be

placed opposite the urea carbonyl to avoid steric repulsion. As shown in Figure 4a, the most stable
structure of ortho methyl phenylurea derivatives without morpholine, as calculated at
B3LYP/6-31+G(d,p) level, 13 is a flat conformation; however, the most stable structure in the ortho methyl
phenylurea derivatives with morpholine (φ=33˚, Figure 4b) was a twisted conformation that avoided the
steric repulsion between ortho methyl and the methylene of morpholine. As summarized in Table 3, the
enyzme inhibitory activity of ortho methylated compound 4b was decreased 4-fold (IC50=0.052 μM). In
the case of an ethyl group, which is larger than a methyl group, the IC50 value was 0.064 μM (4c). A more
bulky isopropyl group was also tested, and compound 4d inhibited PI3Kαwith an IC50 value of 0.081 μM.
These results indicate that the ortho substituent at this point gradually disrupts the molecular planarity,
and the reduced inhibitory activity would depend on the position of the aromatic ring or the bulk of the
introduced alkyl substituents. Unfortunately, solubility in FaSSIF was still low even in compound 4d, so
we attempted further derivatizations to compound 4b, which had shown the most potent IC50 value out of
4a–4d.
Table 2
In vitro profiles of ortho substituted compounds (1)
O
N
R
N
N
N
N
N
H N
2
N
PI3Kα
FaSSIF
(μg/ml)
Compound
R
H
IC50 (μM)
0.033
0.26
22
10
<12
56
Me
Table 3
In vitro profiles of ortho substituted compounds (2)
PI3Kα
IC50 (μM)
FaSSIF
(μg/ml)
Compound
R
4b
4c
4d
Me
Et
iPr
0.052
0.064
0.081
<14
N.T.
13
As described in Table 4, solubilizing groups and other ortho substituents were introduced to 4b. First,
N-ethylpiperazine was introduced through an amide linker (14), which increased the enzyme inhibitory
activity to 0.026 μM, but solubility in FaSSIF was not improved. Next, N-ethylpiperazine was directly
connected to the phenyl ring, and the IC50 value of compound 18 was stronger than with an amide linker,
but solubility was not enough. Simply introducing amines as a solubilizing group was not effective, so
other ortho substituents were tested. We evaluated ortho difluorinated compound 2, in which
electrostatic repulsion between one fluoro atom and the urea carbonyl can be expected. As shown in
Figure 4c, the most stable conformation of the mono fluorinated compound was flat, because of a
favorable electrostatic interaction of the fluoro atom with urea NH; however, the most stable difluorinated
compound had the twisted conformation that avoided, as expected, the electrostatic repulsion of the
second fluoro atom with urea carbonyl (φ=55˚, Figure 4d). In fact, FaSSIF solubility of 2 was slightly
improved to 20 μg/ml, indicating that the molecular planarity had been broken more than compound 18.

The PI3Kαenzyme inhibitory activity of compound 2 was lower than the ortho methyl compound 18, but
its IC50 value was still potent (IC50=0.022 μM). Compound 2 was an inhibitor that balanced both inhibitory
activity and solubility, which was achieved by appropriately disrupting the molecular planarity and
introducing a solubilizing group. In addition, liver microsomal stability of compound 2 was good in both
mouse (7.6 μL/min/mg) and human (2.2 μL/min/mg), and permeability was also acceptable (1.1 x 10-6
cm/sec in PAMPA). Following these results, the pharmacokinetic profile of compound 2 in mice was also
evaluated. Compound 2 was orally administered at a dose of 50 mg/kg, and its AUC value was more
than 10 times better than the non-substituted phenylurea lead compound 1. As shown in Table 4,
compound 2 inhibited PI3Kβwith an IC50 value of 7 nM. PI3Kβis an antitumor target in PTEN-negative
cancer cell lines,5 and compound 2 showed antiproliferative activity in vitro against the PC-3 prostate
cancer cell lines, which are known to be PTEN-negative. Finally, the antitumor efficacy of compound 2
was evaluated in a PC3 xenograft model. As described in Figure 5, compound 2 showed strong tumor
growth inhibition (82%) at the low dose (6.25 mg/kg) without significant body weight loss.
Table 4
In vitro and in vivo profiles of ortho substituted compounds with a solubilizing group
Mouse PK
(50 mg/kg po)
PI3Kα
IC50 (μM)
PI3Kβ
IC50 (μM)
PC31
FaSSIF
Compound
R
IC50 (μM)
(μg/ml)
AUC
Cmax
(ng/mL) (ng• h/mL)
14
0.026
0.010
0.022
N.T.
N.T.
0.25
0.21
<18
11
N.T.
N.T.
460
N.T.
N.T.
N
N
18
2
0.007
0.058
20
5680
1 Prostate cancer, PTEN negative
Figure 4a
Figure 4b

Figure 4c
Figure 4d
Figure 4. B3LYP/6-31+G(d,p) level calculation of phenylurea derivatives. The most stable conformations and torsions of
each compound are shown. All calculations were done by the program Gaussian 09.

Figure 5. In vivo antitumor effect of 2 in PC-3 xenograft mice models. Mice (four mice per group) bearing PC-3 were
administered compound 2 orally once daily for 11 days at 6.25 mg/kg (82% TGI).
4. Conclusion
By the combination of disrupting molecular planarity and introducing a solubilizing group, the
solubility and pharmacokinetic profiles of a phenylurea derivative were improved. Ortho methylation at
the biaryl part was effective in improving solubility, but it was not acceptable in our case, because of
significant loss of inhibitory activity (10). On the other hand, ortho alkylation at the phenylurea moiety
slightly decreased inhibitory activity but did not improve solubility (4b, 4c, and 4d). Combinations of ortho
substitutions at the phenylurea moiety that introduced N-ethylpiperazine as a solubilizing group were
also tested. Although ortho methylated compound 18 did not improve the solubility, ortho difluorinated
compound 2 showed moderate solubility. In the case of the ortho difluorinated compound, one fluoro
atom is arranged in the carbonyl site of urea, which increases the dihedral angle to avoid electrostatic
repulsion between the fluoro atom and urea carbonyl. As a result, solubility in FaSSIF of compound 2
was slightly improved to 20 μg/ml and the mouse AUC was more than 10 times better than 1.
Furthermore, compound 2 showed strong tumor growth inhibition in vivo against a PC3 xenograft model
without significant body weight loss.
5. Experimental
5.1 Chemistry
All solvents and reagents were obtained commercially. 1H and 13C NMR spectra were recorded on
a JEOL JNM-EX270 (270 MHz), JEOL JNM-EX400 (400 MHz), or JNM-GSX400 (400 MHz), or Bruker
Ascend400 (400 MHz) and chemical shifts are expressed as δ units using tetramethylsilane as an
internal standard. The spectral splitting patterns are described as follows: s, singlet; d, doublet; dd,
double doublet; t, triplet; q, quartet; m, multiplet; and brs, broad singlet peak. High resolution mass
spectra (HRMS) were measured with a I-Class/Xevo G2S TOF (Waters) using an ESI source. Flash
column chromatography was performed with Biotage SNAP cartridges or SILICYCLE SiliaSep packed
columns.
5.1.1
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid phenylamide (1)
To a solution of 3 (54.0 mg, 0.100 mmol) and TEA (41.7 μL, 0.300 mmol) in DCE (1 mL) was added
phenyl isocyanate (33.0 μL, 0.300 mmol). After refluxing for 2 hours, the mixture was cooled to an
ambient temperature and washed with saturated aqueous NH4Cl and then brine. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica
gel
flash
column
chromatography
(n-hexane/EtOAc,
1/0
to
1/1)
to
afford
the

4-(2-(bis(4-methoxybenzyl)amino)pyrimidin-5-yl)-2-morpholino-N-phenyl-5,6-dihydro-7H-pyrrolo[2,3-d]p
yrimidine-7-carboxamide (33.7 mg, 51%). To a solution of this intermediate in TFA (1 mL) was added
conc. H2SO4 (0.53 μL, 10.0 μmol), and the mixture was stirred at 70 ˚C for 3 hours. After cooling to
ambient temperature, the mixture was diluted with H2O (1 mL) and neutralized by 5M NaOH. The
resulting filter cake was washed with MeOH to afford the titled compound as a light brown powder (17.1
mg, 82%). 1H-NMR (400 MHz, DMSO-d6) δ: 8.85 (2H, s), 7.53 (2H, d, J = 7.8 Hz), 7.36 (2H, t, J = 8.1 Hz),
7.19 (2H, s), 7.07 (1H, t, J =7.6 Hz), 4.05 (2H, t, J = 8.5 Hz), 3.79-3.73 (8H, m), 3.23 (2H, t, J = 8.5 Hz);
13C-NMR (400 MHz, DMSO-d6) δ: 164.1, 163.6, 159.9, 158.4, 155.0, 151.0, 138.7, 129.7, 123.6, 120.0,
119.4, 106.3, 66.4, 46.4, 45.0, 23.5 ; HRMS (ESI), m/z Calcd for C21H22N8O2+H: 419.1944, Found
419.1943.
5.1.2
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid methyl-phenyl-amide (4a)
To a solution of 3 (54.0 mg, 0.100 mmol) and pyridine (16 μL, 0.200 mmol) in DCM (1 mL) was added
triphosgene (59.4 mg, 0.200 mmol) at 0 ˚C. After stirring for 1 hour at an ambient temperature, DCM was
evaporated under reduced pressure. The residue was dissolved in DCM (1 mL) again and
N-methylaniline (13 μL, 0.120 mmol) was added to the mixture. After stirring for 4 hours, the mixture was
directly charged on silica gel and purified (DCM/MeOH, 1/0 to 95/5) to afford
4-[2-[bis(4-methoxybenzyl)amino]pyrimidin-5-yl]-N-methyl-2-morpholino-N-phenyl-5,6-dihydro-7H-pyrrol
o[2,3-d]pyrimidine-7-carboxamide (52.2 mg, 78% in 2 steps). This residue and NAC (25.2 mg, 0.155
mmol) was dissolved in TFA (1 mL) and the mixture was refluxed for 6 hours. After cooling to an ambient
temperature, H2O (1 mL) and 5M NaOH (1 mL) was added. The resulting filter cake was washed by
MeOH and purified by silica gel flash column chromatography (DCM/MeOH, 1/0 to 95/5) to afford titled
compound as a white powder (21.1 mg, 63%). 1H-NMR (270 MHz, DMSO-d6) δ: 8.71 (2H, s), 7.32-7.27
(4H, m), 7.14-7.11 (1H, m), 7.11-7.08 (2H, m), 3.87 (2H, t, J = 8.2 Hz), 3.64-3.59 (8H, brm), 3.35 (3H, s),
3.09 (2H, t, J =8.2 Hz); 13C-NMR (400 MHz, DMSO-d6) δ: 164.9, 163.9, 160.7, 158.1, 155.5, 153.5,
144.8, 129.1, 125.7, 125.0, 120.3, 105.1, 66.6, 48.4, 44.7, 38.8, 24.5 ; HRMS (ESI-TOF), m/z Calcd for
C22H24N8O2+H: 433.2100, Found 433.2094.
5.1.3
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid o-tolylamide (4b)
Compound 4b was prepared from 3 and 2-methylaniline by following the same procedure as
1
described for 4a (white powder, 68% in 3 steps). H-NMR (400 MHz, DMSO-d6) δ: 10.29 (1H, s), 8.84
(2H, s), 7.59 (1H, d, J = 8.1 Hz), 7.26 (1H, d, J = 7.0 Hz), 7.23-7.18 (3H, m), 7.09 (1H, t, J = 7.5 Hz), 4.05
(2H, t, J = 8.3 Hz), 3.74-3.65 (8H, m), 3.24 (2H, t, J = 8.3 Hz), 2.27 (3H, s); 13C-NMR (400 MHz,
DMSO-d6) δ: 164.1, 163.9, 160.1, 158.4, 154.8, 151.6, 136.3, 130.8, 130.4, 126.6, 125.1, 124.9, 120.0,
106.5, 66.4, 46.5, 45.1, 23.5, 18.1 ; HRMS (ESI-TOF), m/z Calcd for C22H24N8O2+H: 433.2100, Found
433.2097.
5.1.4
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid (2-ethyl-phenyl)-amide (4c)
Compound 4c was prepared from 3 and 2-ethylaniline by following the same procedure as
described for 4a (white powder, 56% in 3 steps). 1H-NMR (DMSO-d6) δ: 10.29 (1H, s), 8.85 (2H, s), 7.52
(1H, d, J = 7.5 Hz), 7.29 (1H, d, J = 7.5 Hz), 7.24-7.13 (4H, m), 4.05 (2H, t, J = 8.3 Hz), 3.72-3.65 (8H, m),
3.24 (2H, t, J = 8.3 Hz), 2.63 (2H, q, J = 7.5 Hz), 1.16 (3H, t, J = 7.5 Hz); 13C-NMR (400 MHz, DMSO-d6)
δ: 164.1, 163.9, 160.0, 158.4, 154.8, 151.9, 136.9, 135.5, 128.8, 126.6, 126.1, 125.8, 120.0, 106.5, 66.3,
46.5, 45.1, 24.1, 23.5, 14.5 ; HRMS (ESI-TOF), m/z Calcd for C23H26N8O2+H: 447.2257, Found
447.2255.
5.1.5
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid (2-isopropyl-phenyl)-amide (4d)
Compound 4d was prepared from 3 and 2-isopropylaniline by following the same procedure as
1
described for 4a (white powder, 57% in 3 steps). H-NMR (DMSO-d6) δ: 10.33 (1H, s), 8.85 (2H, s),
7.46-7.33 (2H, m), 7.24-7.17 (4H, m), 4.05 (2H, t, J = 8.3 Hz), 3.67 (8H, brs s), 3.24 (2H, t, J = 8.3 Hz),
3.18 (1H, q, J = 7.0 Hz), 1.17 (6H, d, J = 7.0 Hz); 13C-NMR (400 MHz, DMSO-d6) δ: 164.1, 163.9, 160.0,
158.4, 154.8, 152.1, 142.2, 134.5, 126.9, 126.4, 126.3, 126.1, 120.0, 106.5, 66.3, 46.5, 45.0, 28.0, 23.7,
23.5 ; HRMS (ESI-TOF), m/z Calcd for C24H28N8O2+H: 461.2413, Found. 461.2413

5.1.6 5-Bromo-N,N-bis(4-methoxybenzyl)-4-methylpyrimidin-2-amine (6)
To a suspension of 5-bromo-4-methylpyrimidin-2-amine (500 mg, 2.66 mmol) in DMF (5 mL) was
added NaH (60% dispersion in mineral oil, 266 mg, 6.65 mmol) at 0 ˚C. After stirring for 30 minutes at
0 °C, 4-methoxybenzyl chloride (0.797 ml, 5.85 mmol) and NaI (39.9 mg, 0.266 mmol) was added at
0 °C. After stirring for 4 hours at ambient temperature, 4-methoxybenzyl chloride (0.400 ml, 2.94 mmol)
and NaH (60% dispersion in mineral oil, 80 mg, 2.00 mmol) was added at 0 ˚C. After stirring for 1 hour
at ambient temperature, the reaction mixture was neutralized by saturated aqueous NH4Cl. The resulting
mixture was extracted with EtOAc/n-hexane=2/1. The combined organic layer was washed with brine
twice, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified
by flash column chromatography (n-hexane/EtOAc, 1/0 to 9/1) to afford the titled compound as a
colorless oil (898 mg, 79%). 1H-NMR (CDCl3) δ: 8.24 (1H, d, J = 2.7 Hz), 7.16 (4H, dd, J = 8.6, 2.7 Hz),
6.84 (4H, dd, J = 8.6, 2.1 Hz), 4.73 (4H, d, J = 3.2 Hz), 3.79 (6H, s), 2.45 (3H, d, J = 2.1 Hz) ; HRMS
(ESI-TOF), m/z Calcd for C21H22BrN3O2+H: 428.0974, Found 428.0959.
5.1.7
N,N-Bis(4-methoxybenzyl)-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-a
mine (7)
To a solution of 6 (400 mg, 0.934 mmol) and triisopropyl borate (0.431 ml, 1.87 mmol) in
toluene/THF=4/1 (10 ml) was added n-BuLi (2.6 M in n-hexane, 0.718 ml, 1.87 mmol) at − 78 °C. After
stirring for 30 minutes at −78 °C, the reaction mixture was neutralized by saturated aqueous NH4Cl.
The resulting mixture was extracted with EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure affording
[2-[bis(4-methoxybenzyl)amino]-4-methylpyrimidin-5-yl]boronic acid as a crude product. This crude
product was used in the next reaction without further purification. To a suspension of the crude
compound and MgSO4 (450 mg, 3.74 mmol) in 1,2-dichloroethane (10 mL) was added pinacol (221 mg,
187 mmol). After stirring for 15 hours at ambient temperature, the reaction mixture was filtered through
Celite and concentrated under reduced pressure. The residue was purified by flash column
chromatography (n-hexane/EtOAc, 4/1) to afford the titled compound as a yellow solid (346 g, 78% in 2
steps). 1H-NMR (400 MHz, DMSO-d6) δ: 8.43 (1H, s), 7.16 (4H, d, J = 6.8 Hz), 6.86 (4H, dt, J = 9.2, 2.4
Hz), 4.72 (4H, s), 3.72 (6H, s), 2.46 (3H, s), 1.28 (12H, s) ; HRMS (ESI-TOF), m/z Calcd for
C27H34BN3O4+H: 476.2721, Found 476.2739.
5.1.8 4-(4-Chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (9)
NaH (60% dispersion in mineral oil, 2.00 g, 50.0 mmol) was washed with n-hexane twice.
3-aminopyridine (4.70 g, 50.0 mmol) in THF (80 mL) was added to the NaH above in one portion at
ambient temperature. The mixture was stirred at 50 ˚C for 1 hour and the resulting purple solution was
cooled to ambient temperature. A solution of 8 (3.00 g, 10.1 mmol) in THF (15 mL) was added to the
mixture and the resulting mixture was stirred at ambient temperature for 6.5 hours. The mixture was
poured onto saturated aqueous NH4Cl (250 mL) at 0 ˚C. The resulting filter cake was washed with
1
n-hexane to afford the titled compound as a light yellow powder (2.12 g, 67%). H-NMR (270 MHz,
CDCl3) δ: 9.06 (1H, d, J = 2.6 Hz), 8.31 (1H, dd, J = 4.7, 1.4 Hz), 8.06 (1H, dq, J = 8.5, 1.4 Hz), 7.31 (1H,
dd, J = 8.5, 4.7, 0.7 Hz), 4.09 (2H, t, J = 8.3 Hz), 3.81-3.73 (8H, m), 3.11 (2H, t, J = 8.3 Hz) ; HRMS
(ESI-TOF), m/z Calcd for C15H16ClN5O+H: 318.1122, Found 318.1115.
5.1.9
4-Methyl-5-(2-morpholin-4-yl-7-pyridin-3-yl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-pyrimidin
-2-ylamine (10)
A suspension of 9 (64.0 mg, 0.201 mmol), 7 (105 mg, 0.221 mmol), Pd(OAc)2 (2.3 mg, 10.2 μmol),
2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl (S-Phos) (8.2 mg, 20.0 μmol) and K3PO4 (85.0 mg,
0.400 mmol) in DMF (2 ml) was degassed under ultrasonic irradiation and stirred for 10 hours at 100 °C
in a nitrogen atmosphere. After cooling to ambient temperature, the reaction mixture was diluted with
EtOAc/THF=4/1 (80 mL), washed with saturated aqueous NaHCO3, 1% aqueous NAC and then brine.
The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography (DCM/MeOH, 1/0 to 50/1) to afford
Bis-(4-methoxy-benzyl)-[4-methyl-5-(2-morpholin-4-yl-7-pyridin-3-yl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimi
din-4-yl)-pyrimidin-2-yl]-amine as a yellow amorphous (78 mg). This residue and NAC (78.0 mg, 0.478
mmol) was dissolved in TFA (5 mL) and the mixture was refluxed for 3 hours. After cooling to ambient
temperature, TFA was evaporated under reduced pressure. The residue was purified by amino silica gel
flash column chromatography (DCM/MeOH, 1/0 to 50/1) to afford the titled compound as a light yellow
powder (34 mg, 43% in 2 steps). 1H-NMR (270 MHz, DMSO-d6) δ: 9.07 (1H, d, J = 2.3 Hz), 8.23 (1H, s),

8.23 (1H, dd, J = 8.7, 3.0 Hz), 7.67 (1H, td, J = 5.4, 2.7 Hz), 7.42 (1H, dd, J = 8.7, 4.8 Hz), 6.77 (2H, s),
4.08 (2H, t, J = 8.2 Hz), 3.67 (8H, brs), 3.04 (2H, t, J = 8.2 Hz), 2.33 (3H, s); 13C-NMR (400 MHz,
DMSO-d6) δ: 166.0, 165.6, 163.2, 161.3, 157.9, 156.5, 143.0, 139.9, 138.1, 124.9, 124.0, 120.4, 108.9,
66.5, 48.6, 45.0, 23.3, 23.1 ; HRMS (ESI-TOF), m/z Calcd for C20H22N8O+H: 391.1995, Found 391.1991.
5.1.10 (4-Ethylpiperazin-1-yl)(3-methyl-4-nitrophenyl)methanone (12)
To a solution of 3-methyl-4-nitrobenzoic acid (500 mg, 2.76 mmol), EDC•HCl (794 mg, 4.14 mmol)
and DMAP (33.7 mg, 0.276 mmol) in DCM (10 mL) was added 1-ethylpiperazine (0.420 mL, 3.31 mmol).
After stirring for 2 hours at ambient temperature, the reaction mixture was diluted with saturated NH4Cl
and extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash column
chromatography (DCM/MeOH, 1/0 to 9/1) to afford the titled compound as a light yellow powder (606 mg,
79%). 1H-NMR (400 MHz, CDCl3) δ: 8.00 (1H, d, J = 8.3 Hz), 7.39 (1H, s), 7.35 (1H, dd, J = 8.3, 1.2 Hz),
3.81 (2H, brs), 3.40 (2H, brs), 2.63 (3H, s), 2.54 (2H, brs), 2.46 (2H, q, J = 7.2 Hz), 2.39 (2H, brs), 1.10
(3H, t, J = 7.2 Hz) ; HRMS (ESI-TOF), m/z Calcd for C14H19N3O3+H: 278.1505, Found 278.1505.
5.1.11 4-Amino-3-methylphenyl)(4-ethylpiperazin-1-yl)methanone (13)
A suspension of 12 (300 mg, 1.08 mmol) and palladium (10% on carbon, 30.0 mg, 28.0 μmol) in
MeOH (5.40 mL) was stirred under H2 atmosphere for 3.5 hours at ambient temperature. The reaction
mixture was filtered through Celite and concentrated under reduced pressure. The residue was purified
by flash column chromatography (DCM/MeOH, 1/0 to 9/1) to afford the titled compound as a white
amorphous (245 mg, 96%). 1H-NMR (400 MHz, DMSO-d6) δ: 7.00 (1H, s), 6.97 (1H, dd, J = 8.2, 2.0 Hz),
6.58 (1H, d, J = 8.2 Hz), 5.24 (2H, s), 3.49-3.44 (4H, m), 2.36-2.29 (6H, m), 2.05 (3H, s), 1.00 (3H, t, J =
7.2 Hz) ; HRMS (ESI-TOF), m/z Calcd for C14H21N3O+H: 248.1754, Found 248.1754.
5.1.12
4-(2-Aminopyrimidin-5-yl)-N-(4-(4-ethylpiperazine-1-carbonyl)-2-methylphenyl)-2-morpholino-5,6-
dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide (14)
Compound 14 was prepared from 3 and 13 by following the same procedure as described for 4a
(white powder, 19% in 3 steps). 1H-NMR (400 MHz, DMSO-d6) δ: 10.35 (1H, s), 8.85 (2H, s), 7.71 (1H, d,
J = 8.1 Hz), 7.29 (1H, s), 7.25-7.19 (3H, m), 4.06 (2H, t, J = 8.3 Hz), 3.75-3.65 (8H, m), 3.61-3.40 (2H, br
m), 3.28-3.20 (4H, m), 2.39-2.32 (6H, m), 2.30 (3H, s), 1.00 (3H, t, J = 7.3 Hz); 13C-NMR (400 MHz,
DMSO-d6) δ: 169., 164.1, 163.8, 160.1, 158.4, 154.9, 151.4, 137.5, 132.2, 130.0, 129.7, 125.5, 124.0,
120.0, 106.5, 66.4, 52.8, 52.0, 46.6, 45.2, 23.5, 18.1, 12.3 ; HRMS (ESI-TOF), m/z Calcd for
C29H36N10O3+H: 573.3050, Found 573.3065.
5.1.13 1-Ethyl-4-(3-methyl-4-nitrophenyl)piperazine (16)
To a solution of 4-fluoro-2-methylnitrobenzene (0.394 mL, 3.22 mmol) in DMSO (8 mL) was added
1-ethylpiperazine (0.612 mL, 4.82 mmol) and K2CO3 (668 mg, 4.83 mmol). The resulting mixture was
stirred at 80 ˚C for 90 minutes. After cooling to ambient temperature, the mixture was diluted with H2O
(16 mL) and extracted with EtOAc. The combined organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. This crude product was used in the next
reaction without further purification.
5.1.14 4-(4-Ethylpiperazin-1-yl)-2-methylaniline (17)
To a solution of 16 (803 mg, 3.22 mmol) in MeOH (15 mL) was added NH4Cl (517 mg, 9.66 mmol)
and zinc (1.05 g, 16.1 mmol). After stirring for 2 hours, the reaction mixture was filtered through Celite
and concentrated under reduced pressure. The residue was purified by flash column chromatography
(DCM/MeOH, 1/0 to 9/1) to afford the titled compound as a brown powder (638 mg, 90% in 2 steps).
1H-NMR (400 MHz, CDCl3) δ: 6.75-6.67 (2H, m), 6.62 (1H, d, J = 8.3 Hz), 3.36 (2H, brs), 3.10-3.06 (4H,
m), 2.64-2.59 (4H, m), 2.48 (2H, q, J = 7.2 Hz), 2.16 (3H, s), 1.13 (3H, t, J = 7.2 Hz) ; HRMS (ESI-TOF),
m/z Calcd for C13H21N3+H: 220.1814, Found 220.1811.
5.1.15
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid [4-(4-ethyl-piperazin-1-yl)-2-methyl-phenyl]-amide (18)
To a solution of 3 (150 mg, 0.278 mmol) in DCM (4 mL) was added saturated aqueous NaHCO3 (3.5
mL). After cooling to 0 ˚C, phosgene (20% in toluene, 0.45 mL, 0.834 mmol) was added to the mixture
dropwise. After stirring for 40 minutes at 0 ˚C, the organic layer was separated, washed with brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in
DCM (4 mL) and TEA (58.0 μl, 0.417 mmol) was added. To this mixture was added 17 (67 mg, 0.306

mmol) in DCM (1 mL) dropwise and stirred overnight at ambient temperature. The mixture was
quenched by 1M HCl, then extracted with DCM/MeOH (50/1). The combined organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (DCM/MeOH, 50/1 to 20/1) to afford
4-(2-(bis(4-methoxybenzyl)amino)pyrimidin-5-yl)-N-(4-(4-ethylpiperazin-1-yl)-2-methylphenyl)-2-morphol
ino-5,6-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide as a yellow powder (205 mg). This residue
was dissolved in TFA (1.5 mL) and the mixture was refluxed for 4 hours. After cooling to ambient
temperature, TFA was evaporated under reduced pressure. The residue was purified by amino silica gel
flash column chromatography (DCM/MeOH, 100/1 to 50/1) to afford the titled compound as a light yellow
powder (110 mg, 76% in 3 steps). 1H-NMR (270 MHz, CDCl3) δ: 10.20 (1H, s), 8.90 (2H, s), 7.43 (1H, d,
J = 8.8 Hz), 6.82 (2H, m), 5.32 (2H, s), 4.22 (2H, t, J = 8.5 Hz), 3.77 (8H, m), 3.22 (6H, m), 2.62 (4H, m),
2.49 (2H, q, J = 7.2 Hz), 1.14 (3H, t, J = 7.2 Hz); 13C-NMR (400 MHz, DMSO-d6) δ: 163.5, 163.4, 159.4,
157.7, 154.1, 151.3, 148.4, 131.3, 127.2, 125.8, 119.5, 116.9, 113.2, 105.8, 65.7, 52.3, 51.5, 48.4, 45.9,
44.4, 40.1, 39.9, 39.7, 39.4, 39.2, 39.0, 38.8, 22.9, 18.0, 11.9 ; HRMS (ESI-TOF), m/z Calcd for
C28H36N10O2+H: 545.3101, Found 545.3119.
5.1.16 N,N-Di-Boc-4-bromo-2,6-fluoro-phenylamine
To a solution of 4-bromo-2,5-difluoroniline (10.0 g, 4.81 mmol) in DMF (10 mL) was added (Boc)2O
(2.31 g, 10.6 mmol) and DMAP (29 mg, 0.240 mmol). After stirring overnight at ambient temperature, the
mixture was diluted with H2O (20 mL) and extracted with EtOAc. The combined organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. This crude
product was used in the next reaction without further purification.
5.1.17 tert-Butyl(4-(4-ethylpiperazin-1-yl)-2,6-difluorophenyl)carbamate (20)
A
suspension of N,N-di-boc-4-bromo-2,6-fluoro-phenylamine (220 mg, 0.539 mmol),
1-ethylpiperazine (0.273 mL, 2.16 mmol), Pd(OAc)2 (12.1 mg, 54.0 μmol), S-Phos (44.2 mg, 108 μmol)
and Cs2CO3 (351 mg, 1.08 mmol) in toluene (5 ml) was degassed under ultrasonic irradiation and
refluxed for 2 hours in a nitrogen atmosphere. After cooling to ambient temperature, the reaction mixture
was diluted with EtOAc, washed with saturated aqueous NH4Cl and then brine. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash
column chromatography (DCM/MeOH, 1/0 to 95/5). The resulting residue was washed with n-hexane to
1
afford the titled compound as a light brown powder (37.7 mg, 20% in 2 steps). H-NMR (270 MHz,
CDCl3) δ: 6.42 (2H, dt, J = 17.4, 3.3 Hz), 5.74 (1H, s), 3.21-2.15 (4H, m), 2.60-2.54 (4H, m), 2.46 (2H, q,
J = 7.3 Hz), 1.49 (9H, s), 1.12 (3H, t, J = 7.3 Hz) ; HRMS (ESI-TOF), m/z Calcd for C17H25F2N3O2+H:
342.1993, Found 342.2002.
5.1.18 4-(4-ethylpiperazin-1-yl)-2,6-difluoroaniline (21)
To a solution of 20 (30 mg, 88.0 μmol) in THF (343 μL) was added 6M HCl (117 μL). After stirring for
30 minutes at ambient temperature, the mixture was added 8M NaOH and extracted with EtOAc. The
combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel flash column chromatography (DCM/MeOH, 1/0
to 95/5) to afford the titled compound as a colorless oil (19.2 mg, 91%). 1H-NMR (270 MHz, CDCl3) δ:
6.44 (2H, ddd, J = 20.4, 10.7, 2.9 Hz), 3.37 (2H, s), 3.07-3.04 (4H, m), 2.61-2.55 (4H, m), 2.46 (2H, q,
7.2 Hz), 1.12 (3H, t, J = 7.2 Hz); HRMS (ESI-TOF), m/z Calcd for C12H17F2N3+H: 242.1469, Found
242.1478.
5.1.19
4-(2-Amino-pyrimidin-5-yl)-2-morpholin-4-yl-5,6-dihydro-pyrrolo[2,3-d]pyrimidine-7-carboxylic
acid [4-(4-ethyl-piperazin-1-yl)-2,6-difluoro-phenyl]-amide (2)
Compound 2 was prepared from 3 and 21 by following the same procedure as described for 4a
(white powder, 52% in 3 steps). 1H-NMR (270 MHz, DMSO-d6) δ: 10.15 (1H, s), 8.84 (2H, s), 7.22 (2H, s),
6.73 (2H, d, J = 11.7 Hz), 4.01 (2H, t, J = 8.0 Hz), 3.67 (8H, s), 3.26 (2H, t, J = 8.0 Hz), 3.22-3.16 (4H, m),
2.48-2.44 (4H, m), 2.36 (2H, q, J = 7.1 Hz), 1.03 (3H, t, J = 7.1 Hz); 13C-NMR (400 MHz, DMSO-d6) δ:
164.1, 163.7, 159.8, 158.4, 157.7, 155.1, 151.7, 128.5, 126.0, 120.0, 106.1, 98.5, 98.2, 66.3, 52.2, 51.9,
47.5, 46.4, 44.8, 23.7, 12.1 ; HRMS (ESI-TOF), m/z Calcd for C27H32F2N10O2+H: 567.2756, Found
567.2773.
5.2 In vitro kinase enzyme assay
Inhibitory activities on human PI3Kα (p110α/p85α) or PI3Kβ (p110β/p85α) (expressed in baculovirus
and purified) were determined by AlphaScreen competition assay. Test compound solutions were mixed
with PI3K, and incubated for 20 minutes at room temperature. Substrate solution 4 μM PI(4,5)P2

(echelon) and 10 μM ATP solution (5 mM Hepes pH7.5, 2.5 mM MgCl2) were added, and incubated for
15 minutes at 37 ˚C. Detection of the reaction product PI(3,4,5)P3 was done using AlphaScreen GST
Detection Kit (PerkinElmer) according to the manufacture’s recommendation. GST-GRP1 (expressed in
E.coli and purified), Anti-GST Acceptor Beads, Streptavidin Donor Beads, biotin-PI(3,4,5)P3 (echelon)
(10 mM Tsir-HCl pH7.4, 150 mM NaCl, 7.5 mM EDTA, 1 mM DTT, 0.1% Tween20) were added, and
incubated for 1 hour at room temperature. Signal was measured with an EnVision plate reader
(PerkinElmer).
5.3 In vitro cell proliferation assay
The prostate cancer PC-3 (ATCC) cells were treated with various concentrations of assay
compounds for 96 hours. Cell growth inhibition was determined by Cell Counting Kit-8 solution (Dojindo
Laboratories).
5.4 Solubility assay
An aliquot of 50 μl of a 4-mM or 1-mM sample in dimethylsulfoxide (DMSO) was freeze-dried to
remove DMSO. To the resulting residue was added 50 μM of FaSSIF (pH=6.5), which was then irradiated
ultrasonically for 10 minutes, shaken for 2 hours, centrifuged for 10 minutes (3000 rpm), and filtered by
Whatman Unifilter. Concentration of the filtrate was analyzed by HPLC-UV based on the calibration
curve of each sample. Composition of FaSSIF: Sodium taurocholate (1.61g), Lecithin (0.59 g), KH2PO4
(3.9 g), KCl (7.7 g) and NaOH (pH 6.5) per 1 L.
5.5 Pharmacokinetic study in mice
Pharmacokinetic study: Female BALB/c-nu mice (n=2 per treatment group) were given a solution of
the test compounds by oral (po) route at doses of 50 mg/kg (po) in mice. Vehicle was 10% DMSO, 10%
Cremophor EL, 15% PEG400, and 15% HPCD (2-hydroxypropyl-β-cyclodextrin). Blood samples of each
animal were collected with heparin as an anticoagulant at 0.25, 2, 4, 7, and 24 hours following po dosing.
Samples were centrifuged to obtain plasma and stored at −80 °C until analysis. Plasma concentrations
were determined using a LC–MS/MS system. The pharmacokinetic parameters were calculated by
non-compartmental analysis using WATSON ver. 7.1 (Thermo Fisher Scientific, Wayne, PA).
5.6 In vivo antitumor effect on PC-3 in nude mice
A cell line (PC-3) was used to evaluate antitumor activity of 2 in vivo. It was grown as sc tumors in
nude mice (Charles River Laboratories Japan, Inc.). Therapeutic experiments were started (day 0) when
the tumor reached around 130 mm3. Mice (four mice per group) were randomized to treatment groups to
receive vehicle or 2 (oral, QD) on days 0 to 10. Vehicle was 10% DMSO, 10% Cremophor EL, 15%
PEG400, and 15% HPCD (2-hydroxypropyl-β-cyclodextrin). Compound 2 was given to mice by forced
oral administration using a sonde tube. The length (L) and width (W) of the tumor mass were measured,
and the tumor volume (TV) was calculated as: TV = (L W2)/2. Body weight change rate (BW) was
calculated using the following formula: BW = W/W0 x 100, where W and W0 are the body weight on a
specific experimental day and on the first day of treatment, respectively. Tumor growth inhibition (TGI)
was calculated using the following formula: TGI = [1 - (T - T0)/(C - C0)] x 100, where T and T0 are the
mean tumor volumes on a specific experimental day and on the first day of treatment, respectively, for
the experimental groups and, likewise, where C and C0 are the mean tumor volumes for the control
group.
Acknowledgments
We thank S. Kuramoto for PK studies. We thank T. Fujii and K. Sakata for biological assays. We
thank Nam Woong-Hyun at C&C for synthetic assistance. We thank Y. Itezono and K. Nii for NMR
measurement. We thank Y. Furuta and M. Arai for mass spectrometry measurements.
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*Graphical Abstract (for review)
2
1
PI3K IC : 0.013 M
Solubility in FaSSIF: < 3 g/ml
Cmax: 104 ng/mL
PI3K IC : 0.022 M
a m
a
m
50
50
Solubility in FaSSIF: 20 g/ml
m
m
Disruption of molecular planarity
Cmax: 460 ng/mL
AUC: 437 ng x h/mL
AUC: 5680 ng x h/mL
82% TGI in PC-3 xenograft model