Modification of a dihydropyrrolopyrimidine phosphoinositide 3-kinase (PI3K) inhibitor to improve oral bioavailability

Article abstract of DOI:10.1016/j.bmc.2015.11.009

Phosphoinositide 3-kinase (PI3K) is activated in various human cancer cells and well known as a cancer therapy target. We previously reported a dihydropyrrolopyrimidine derivative as a highly potent PI3K inhibitor that has strong tumor growth inhibition in a xenograft model. In this report, we describe further optimization to improve its bioavailability.

Full text of DOI:10.1016/j.bmc.2015.11.009

Bioorganic & Medicinal Chemistry 23 (2015) 7650–7660
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modification of a dihydropyrrolopyrimidine phosphoinositide
3-kinase (PI3K) inhibitor to improve oral bioavailability
b
b
b
a
Hatsuo Kawada a, , Hirosato Ebiike , Masao Tsukazaki , Shun Yamamoto , Kohei Koyama ,
Mitsuaki Nakamura b, Kenji Morikami a, Kiyoshi Yoshinari b, Miyuki Yoshida b, Kotaro Ogawa a,
Nobuo Shinma 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:
Phosphoinositide 3-kinase (PI3K) is activated in various human cancer cells and well known as a cancer
therapy target. We previously reported a dihydropyrrolopyrimidine derivative as a highly potent PI3K
inhibitor that has strong tumor growth inhibition in a xenograft model. In this report, we describe further
optimization to improve its bioavailability.
Received 8 September 2015
Revised 29 October 2015
Accepted 10 November 2015
Available online 11 November 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
PI3K
Cancer
Solubility
Liver microsomal stability
Permeability
Molecular planarity
Pharmacokinetic profile
1. Introduction
phenol, our lead compound was metabolically unstable, and
bioavailability was extremely low in mice (1.6%). To overcome this
Phosphoinositide 3-kinases (PI3K) are a family of intracellular
lipid kinases. Activated by receptor tyrosine kinases and
G-protein-coupled receptors, PI3K converts phosphatidylinositol-
4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-triphos-
phate (PI(3,4,5)P3).1,2 PI(3,4,5)P3 activates serine/threonine kinase
AKT as a second messenger and regulates various cellular func-
tions, including cell cycle, apoptosis, and glucose metabolism.
Oncogenic gene mutations and gene amplifications of PI3K, espe-
cially class IA PI3K, are found in various human cancers, as are loss
of heterozygosity and mutations of the tumor suppressor PTEN,
which controls the cellular concentration of PI(3,4,5)P3 by convert-
ing PI(3,4,5)P3 to PI(4,5)P2.3–5 Therefore, the PI3K pathway is a
promising target for cancer therapeutics and several inhibitors
are now in clinical trials.6
issue, an aminopyrimidine was designed as a bioisostere of phenol
based on a FlexSIS docking simulation and structure–activity rela-
tionship information. Finally we identified compound 1 as a
metabolically stable and potent PI3K inhibitor. The key amino
acids of PI3K
D841 and Y867, but the X-ray structure of PI3K
c
that interact with the phenol-type inhibitor are
and 1 revealed
c
that compound 1 formed hydrogen bonding with K833 and
D836. Compound 1 had oral bioavailability of 41% in mice and
showed strong tumor growth inhibition in a KPL-4 xenograft
model.8
In this paper, we report further optimization of the pyridine
moiety of compound 1 to improve its low oral bioavailability in
monkey (5.6%). Compound 3 was identified by the following opti-
mizations: (1) adding a solubilizing group (2) removing the hydro-
gen bonding acceptor (3) introducing an ortho-substituent (4)
optimizing the solubilizing group. The solubility in FaSSIF (fasted
state simulated intestinal fluid),9 liver microsome (LM) stability,
and permeability of compound 3 were better than in compound
1, and the oral bioavailability of compound 3 in monkey was 8
times better than that of compound 1 (Fig. 1).
We have already reported the design of a dihydropyrrolopyrim-
idine-type PI3K inhibitor by superimposing Piramed’s PI103 and
Chiron’s PI3K inhibitors and have also reported the optimization
of the lead compound.7 Because of the rapid glucuronidation of
⇑
Corresponding author. Tel.: +81 0550 87 8364; fax: +81 0550 87 5326.
E-mail address: kawadahto@chugai-pharm.co.jp (H. Kawada).
http://dx.doi.org/10.1016/j.bmc.2015.11.009
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
7651
Figure 1. Improvement of bioavailability in monkey.
deprotection of PMB groups by TFA afforded the desired pyridine
derivatives 1, 15, 16, and 17.
2. Chemistry
The preparation of compound 24 and its benzamide derivatives
is summarized in Scheme 3. Compound 24 was prepared by the
coupling reaction of 11 with bromobenzene and the subsequent
deprotection of the PMB groups. For the synthesis of 27, 3-bro-
mobenzoic acid methyl ester was coupled with key intermediate
11, and the methyl ester was cleaved under a basic condition. Ami-
dation of the resulting benzoic acid by N-methylpiperazine and
subsequent deprotection of PMB groups afforded 27. Compound
28 was synthesized by the coupling reaction of 11 with 30 and fol-
lowing deprotection of PMB groups. To prepare compound 3, com-
pound 11 was coupled with 4-bromo-3-fluoro-benzoic acid. Amide
bond formation with N-methylpiperazine followed by deprotec-
tion of PMB groups afforded compound 3.
The synthesis of benzylamine derivatives is shown in Scheme 4.
The coupling reaction of 11 with 2-(4-bromophenyl)-1,3-dioxolane
and deprotection under a mild acidic condition gave benzaldehyde
derivative 32. Reductive amination with N-methylpiperazine or
morpholine, followed by deprotection of PMB groups afforded 2
or 33, respectively. To synthesize compound 35, the key intermedi-
ate 11 was coupled with 4-bromo-3-fluorobenzaldehyde to afford
34. Reductive amination with morpholine and subsequent depro-
tection of PMB groups gave 35.
Compound 11 was selected as a key intermediate to prepare
new derivatives, because various aryl functions can be introduced
by the Buchwald–Hartwig reaction. The preparation of this inter-
mediate from the commercially available diester 4 is described in
Scheme 1. Treatment of 4 with morpholinoformamidine under a
basic condition, and subsequent chlorination of the resulting triol
by POCl3 afforded the trichloride 5. Next, 4-methoxybenzylamine
was coupled with 5 as a nitrogen atom source and a dihydropy-
rrolopyrimidine skeleton was constructed by the intramolecular
cyclization of 6 under a basic condition. The p-methoxybenzyl
(PMB) group of 7 was removed by trifluoroacetic acid in the pres-
ence of conc. H2SO4 and replaced with acetyl group to afford 9.
Suzuki-Miyaura coupling with the pinacol ester 14 followed by
the deprotection of the acetyl group afforded the desired key com-
pound 11. This compound was coupled with various aryl halides by
the Buchwald–Hartwig reaction.
The coupling reaction of 11 with halopyridine derivatives is
summarized in Scheme 2. The amidation of 4-chloropicolinic acid
(18), 5-bromopicolinic acid (20), and 5-bromonicotinic acid (22)
with N-methylpiperazine afforded the corresponding derivatives:
19, 21 (picolinamides), and 23 (nicotinamide). The palladium–cat-
alyzed Buchwald–Hartwig reaction with 11 and the subsequent
Scheme 1. Preparation of key intermediate 11. Reagents and conditions: (a) morpholinoformamidine hydrochloride, NaOMe, MeOH, reflux; (b) POCl3, 100 °C, 28% in 2 steps;
(c) 4-methoxybenzylamine, DIPEA, CH3CN, reflux, 68%; (d) Cs2CO3, NaI, CH3CN, reflux; (e) TFA, conc.H2SO4, reflux; (f) AcCl, pyridine, DMAP, CH3CN, 0 °C to rt, 92% in 3 steps;
(g) 14, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 °C, quant.; (h) 5 M NaOH, THF, reflux, 85%; (i) 4-methoxybenzyl chloride, NaH, NaI, THF, rt, 92%; (j) B(OiPr)3, nBuLi, toluene/THF,
ꢀ78 °C; (k) pinacol, MgSO4, DME, rt, 88% in 2 steps.

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H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
Scheme 2. Preparation of pyridine derivatives. Reagents and conditions: (a) 3-bromopyridine, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 °C; (b) TFA, N-acetylcysteine (NAC), 70 °C,
18% in 2 steps; (c) 19, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 °C; (d) TFA, NAC, 70 °C, 38% in 2 steps; (e) 21, Pd2(dba)3, S-Phos, K3PO4, DMF, 100 °C; (f) TFA, NAC, 70 °C, 58% in 2
steps; (g) 23, Pd2(dba)3, X-Phos, K3PO4, DMF, 100 °C; (h) TFA, NAC, 70 °C, 91% in 2 steps; (i) N-methylpiperazine, EDC, HOBt, CH2Cl2, 83%; (j) N-methylpiperazine, EDC, HOBt,
CH2Cl2, 28%; (k) N-methylpiperazine, EDC, HOBt, CH2Cl2, 78%.
Scheme 3. Preparation of benzamide derivatives. Reagents and conditions: (a) bromobenzene, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 °C; (b) TFA, NAC, 70 °C, 80% in 2 steps; (c) 3-
bromobenzoic acid methyl ester, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 °C, 86%; (d) 5 M NaOH, THF, 30% aq. H2O2 reflux; (e) N-methylpiperazine, EDC, HOBt, DIPEA, DMF, 60 °C,
71% in 2 steps; (f) TFA, 70 °C, 96%; (g) 30, Pd(OAc)2, S-Phos, K3PO4, DMF, 100 °C; (h) TFA, NAC, 70 °C, 87% in 2 steps; (i) N-methylpiperazine, EDC, HOBt, CH2Cl2, 97%; (j) 4-
bromo-3-fluoro-benzoic acid, Pd2(dba)3, X-Phos, K3PO4, DMF, 100 °C, 80%; (k) morpholine, EDC, HOBt, CH2Cl2; (l) TFA, reflux, 96% in 2 steps.
exposure in human would be poor. In order to improve this low
oral bioavailability, compound 1 was optimized further.
3. Results and discussion
First, because the solubility of compound 1 in FaSSIF was low
(Table 2), an amide function was introduced as a solubilizing
group. After analyzing the X-ray structure of 1, the solubilizing
group was connected to the pyridine, since this substituent is
We previously reported the lead optimization of a virtually
designed PI3K inhibitor and identified the aminopyrimidine moi-
ety as an alternative to the metabolically unstable phenol.8 As
described in Table 1, oral bioavailability of compound 1 was good
in mouse (F = 41%) but poor in monkey (F = 5.6%), which indicated
located at the solvent-exposed region of PI3Ka 8
. As expected, all

H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
7653
Scheme 4. Preparation of benzylamine derivatives. Reagents and conditions: (a) 2-(4-Bromophenyl)-1,3-dioxolane, Pd2(dba)3, X-Phos, K3PO4, DMF, 100 °C; (b) THF, 1 M HCl,
rt, 96% in 2 steps; (c) N-methylpiperazine, NaBH(OAc)3, AcOH, CH2Cl2, rt, 51%; (d) TFA, NAC, 70 °C, 90%; (e) morpholine, NaBH(OAc)3, AcOH, CH2Cl2, rt; (f) TFA, NAC, 70 °C, 50%
in 2 steps; (g) 4-bromo-3-fluorobenzaldehyde, Pd(OAc)2, X-Phos, K3PO4, DMF, 100 °C, 65%; (h) morpholine, NaBH(OAc)3, AcOH, CH2Cl2, rt; (i) TFA, NAC, 70 °C, 52% in 2 steps.
Table 1
Pharmacokinetic data of compound 1 in mouse and monkey
Species
i.v.
p.o.
F (%)
Dose (mg/kg)
C0 (ng/mL)
AUCinf (ngꢁh/mL)
CL (mL/min/kg)
T1/2 (h)
dose (mg/kg)
Cmax (ng/mL)
AUC0–t (ngꢁh/mL)
Mouse
Monkey
10
5
10,400
3120
4690
3700
35.9
24.4
10.3
1.36
100
10
4190
41.6
19,400
417
41
5.6
Table 2
In vitro and in vivo profiles of pyridine derivatives
O
N
N
N
R
N
N
H2N
N
Compound
R
PI3K
a
IC50
(l
M) FaSSIF (
l
g/ml)
LM stability
PAMPA (10ꢀ6 cm/s)
Mouse PK (50 mg/kg po)
mouse (
l
L/min/mg) human (
l
L/min/mg)
Cmax (ng/mL) AUC0–t (ngꢁmL/h)
1
0.033
0.040
<12
439
11.5
27.3
9.7
1.90
1.13
4250
128
7640
188
N
N
N
N
15
15.3
13.4
O
O
N
16
17
0.025
0.032
29
3.5
1.64
0.73
326
158
1650
455
N
N
N
N
N
O
376
19.7
15.2

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H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
Table 3
In vitro and in vivo profiles of phenyl derivatives
O
N
N
N
R
N
N
H2N
N
Compound
R
PI3K
a
IC50 (
l
M) FaSSIF (
l
g/ml)
LM stability
PAMPA (10ꢀ6 cm/s)
Mouse PK (50 mg/kg po)
mouse (
l
L/min/mg) human (
lL/min/mg)
Cmax (ng/mL) AUC0–t (ngꢁmL/h)
24
27
0.035
0.12
N.D.
454
N.D.
6.2
N.D.
N.D.
1.79
N.D.
N.D.
N
N
N
13.8
1500
4030
O
O
N
28
2
0.014
0.048
331
153
10.8
13.3
12.6
13.3
1.24
0.92
2840
1730
13,200
13,400
N
N
three compounds (15, 16, and 17) had improved solubility in FaS-
SIF, without a significant loss of enzyme inhibitory activity. Since
the pharmacokinetic profiles of these compounds in mice were
much poorer than compound 1, the pharmacokinetics in monkeys
was not tested.
Next, because the number of hydrogen bonding acceptors had
been increased by introducing an amide bond and an amine (the
number of hydrogen bonding acceptors in compounds 15, 16,
and 17 is 11), we replaced the pyridine with a phenyl moiety to
decrease the number of hydrogen bonding acceptors. In our previ-
ous study, this replacement resulted in the significant loss of
enzyme inhibitory activity in the phenol derivatives, and the pres-
ence of the nitrogen atom in the pyridine is critical.8 But Table 3
shows, the nitrogen atom in the pyridine was not important for
the aminopyrimidine derivatives, which suggests that the domi-
nant part for enzyme affinity is the aminopyrimidine. In addition
to their strong inhibitory activity, compounds 27 and 28 also had
good solubility in FaSSIF, and their Cmax and AUC in mice were
much better than those of 15 and 16. The benzylamine derivative
2 also showed a good pharmacokinetic profile, and the results in
monkeys are described in Table 4. The oral bioavailability of 2
(21%) was 4 times greater than that of 1 (5.6%), but its clearance
(CL) was slightly increased, and Cmax and AUC were still low.
Our next goal was to overcome the high CL of compound 2 and
simultaneously improve the oral bioavailability even further by
improving its permeability.10 As shown in Table 5, morpholine
Table 4
Pharmacokinetic data of compound 2 in mouse and monkey
Species
i.v.
p.o.
F (%)
Dose (mg/kg)
C0 (ng/mL)
AUCinf (ngꢁh/mL)
CL (mL/min/kg)
T1/2 (h)
dose (mg/kg)
Cmax (ng/mL)
AUC0–t (ngꢁh/mL)
Mouse
Monkey
10
5*
3940
430
4400
2540
38.0
33.9
5.03
10.5
50
10
1730
95.9
13,400
1080
61
21
*
n = 1.
Table 5
In vitro and in vivo profiles of morpholine derivatives
O
N
N
N
R
N
N
H2N
N
Compound
R
PI3Ka
IC50
(l
M) FaSSIF (
l
g/ml)
LM stability
PAMPA (10ꢀ6 cm/s)
Mouse PK (50 mg/kg po)
mouse (
4.32
l
L/min/mg) human (
lL/min/mg)
Cmax (ng/mL) AUC0–t (ngꢁmL/h)
N
33
35
0.046
0.22
<4
1.97
4.52
4.18
N.D.
N.D.
O
F
F
N
107
35.9
0.14
19.1
2.87
16,900
50,900
O
O
N
3
0.042
37
3.16
10,000
64,500
O

H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
7655
4. Conclusion
We examined various strategies to overcome the low oral
bioavailability of compound 1 in monkey (F = 5.6%). As the solubil-
ity of 1 in FaSSIF was low, a solubilizing group was introduced at
the pyridine, which is located at the solvent-exposed region of
N
N
X = H
X = F
φ
PI3Ka. This method improved water solubility without losing inhi-
N
bitory activity, but the increased number of hydrogen bonding
acceptors resulted in a poor pharmacokinetic profile in mice.
Reducing the number of hydrogen bonding acceptors by replacing
the pyridine with phenyl improved the pharmacokinetic profile,
and the oral bioavailability of compound 2 in monkey was more
than 4 times greater than that of 1. The LM stability and passive
permeability were improved by substituting N-methylpiperazine
with morpholine, but this change lowered the water solubility
(33). This antimony was then resolved by introducing an ortho-flu-
oro atom. Finally, based on the structure–activity relationship,
enzyme inhibitory activity was recovered and compound 3 showed
X
good pharmacokinetic profiles (F = 47%) with strong PI3Ka inhibi-
tion (IC50 = 0.042 lM).
φ
Figure 2. B3LYP/6-31+G(d,p) level calculation of N-phenyl dihydropyrrolopyrim-
idine derivatives. Potential energies were calculated with fixing the torsion /
between 0 and 350 at 10° intervals while other variables were optimized. All
calculations were done by the program Gaussian 09.
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), and
chemical shifts are expressed as d 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 bs, broad singlet peak. High
resolution mass spectra (HRMS) were measured with a Thermo
Fisher Scientific LTQ Orbitrap XL MS spectrometer using an ESI
source coupled to a Waters HPLC system operating in reversed
was the means to this goal. By replacing N-methylpiperazine of 2
with morpholine, the LM stability and passive permeability were
significantly improved, but the solubility in FaSSIF was lost (33).
To recover the water solubility, an ‘ortho-substituent’ was intro-
duced in the phenyl group. The introduction of an ortho-sub-
stituent in a bicyclic compound disrupts the molecular planarity
and improves water solubility, as was demonstrated by Ishikawa
et al., who introduced an ortho-fluoro atom into an N-phenylpiper-
idine derivative to improve the water solubility.11,12 As described
in Fig. 2, B3LYP/6-31 + G(d,p) level calculation13 of the N-phenyl
dihydropyrrolopyrimidine derivatives showed that the most stable
conformation of the unsubstituted one is planar (/ = 0, 180°) and
that of the ortho-fluoro derivative is twisted (/ = 150, 210°). In fact,
ortho-fluorinated compound 35 showed good FaSSIF solubility and
remarkable pharmacokinetic profiles in mice. On the other hand,
phase with an ACQUITY UPLC BEH C18 (1.7
column. Flash column chromatography was performed with Bio-
l
m, 2.1 mm ꢂ 50 mm)
tage SNAP cartridges or SILICYCLE SiliaSep packed columns.
5.1.1. 4-[4,6-Dichloro-5-(2-chloroethyl)-pyrimidin-2-yl]-mor-
pholine (5)
the inhibitory activity of 35 against PI3K
that of 33, and its LM stability was low. As shown in Table 3, the
PI3K IC50 value of benzamide 28 was more than 3 times stronger
a was 5 times weaker than
To a solution of Na (3.19 g, 139 mmol) in MeOH (140 mL) was
added morpholinoformamidine hydrochloride (15.3 g, 92.0 mmol)
and 4 (13.3 g, 92.0 mmol). After refluxing for 2 h, the mixture
was cooled to an ambient temperature and concentrated under
reduced pressure, which afforded 5-(2-hydroxyethyl)-2-mor-
pholin-4-yl-pyrimidine-4,6-diol as a crude product. The residue
was dissolved in POCl3 (90 ml) and the resulting mixture was stir-
red at 100 °C for 10 h. After cooling to ambient temperature, the
mixture was concentrated under reduced pressure. After ice
(100 g) was added at 0 °C, the crude mixture was neutralized with
5 M NaOH and extracted with EtOAc. The combined organic extract
was washed with brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified by flash
column chromatography (n-hexane/EtOAc, 20/1 to 10/1) to afford
the titled compound as a yellow solid (8.4 g, 28% in 2 steps). 1H
a
than that of benzylamine 2, suggesting that the carbonyl group
contributes as a hydrogen bonding acceptor and that this struc-
ture–activity relationship could be applied to 35. As shown in
Table 5, the benzoylmorpholine derivative 3 recovered not only
the enzyme inhibitory activity but also the LM stability. Although
its solubility in FaSSIF decreased, it was 3 times greater than that
of compound 1 and acceptable for further examination. The phar-
macokinetic profiles of compound 3, as summarized in Table 6,
show that Cmax, AUC, and CL were good, and oral bioavailability
in monkeys was greatly improved compared to 1 (F in compound
1 = 5.6% vs F in compound 3 = 47%).
Table 6
Pharmacokinetic data of compound 3 in mouse and monkey
Species
i.v.
p.o.
F (%)
Dose (mg/kg)
C0 (ng/mL)
AUCinf (ngꢁh/mL)
CL (mL/min/kg)
T1/2 (h)
dose (mg/kg)
Cmax (ng/mL)
AUC0–t (ngꢁh/mL)
Mouse
Monkey
10
9180
2660
15,000
5570
11.1
7.54
1.27
4.50
50
10
10,000
1060
64,500
10,400
86
47
2.5*
*
n = 1.

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H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
NMR (400 MHz, DMSO-d6) d: 3.76 (2H, t, J = 7.3 Hz), 3.65 (8H, s),
3.12 (2H, t, J = 7.3 Hz).
was added to afford precipitate. The precipitate was dissolved in
CH2Cl2 and washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified
by flash column chromatography (CH2Cl2/MeOH, 1/0 to 50:1) to
afford the titled compound as a white solid (4.26 g, quant.). 1H
NMR (400 MHz, DMSO-d6) d: 8.99 (2H, s), 7.20 (4H, d, J = 8.8 Hz),
6.88 (4H, d, J = 8.3 Hz), 4.79 (4H, s), 3.93 (2H, t, J = 8.3 Hz), 3.72-
3.68 (14H, m), 3.19 (2H, t, J = 8.3 Hz), 2.58 (3H, s); HRMS (ESI),
m/z calcd for C32H35N7O4 + H: 582.2828, found 582.2842.
5.1.2. 6-Chloro-5-(2-chloroethyl)-N-[(4-methoxyphenyl)-
methyl]-2-morpholino-pyrimidin-4-amine (6)
To a solution of 5 (5.78 g, 19.47 mmol) in CH3CN (80 mL) was
added 4-aminomethyl-anisole (5.04 mL, 38.9 mmol) and DIPEA
(8.50 mL, 48.7 mmol). After being refluxed for 17 h, the reaction
mixture was cooled, and then evaporated under reduced pressure.
The residue was dissolved in EtOAc and washed with saturated
aqueous NH4Cl and then brine. The combined organic layer was
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash column chromatography (n-
hexane/EtOAc, 1/0 to 5.5/1) afforded titled compound as a yellow
solid (68%, 5.28 g). 1H NMR (400 MHz, DMSO-d6) d: 7.76 (1H, t,
J = 5.9 Hz), 7.23 (2H, d, J = 8.8 Hz), 6.86 (2H, d, J = 8.8 Hz), 4.44
(2H, d, J = 5.9 Hz), 3.71 (3H, s), 3.60 (2H, t, J = 7.8 Hz), 3.55 (8H,
s), 2.98 (2H, t, J = 7.6 Hz); HRMS (ESI), m/z calcd for C18H22Cl2N4-
O2 + H: 397.1198, found 397.1195.
5.1.5. N,N-Bis[(4-methoxyphenyl)methyl]-5-(2-morpholino-6,7-
dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrimidin-2-amine
(11)
To a solution of 10 (4.00 g, 6.86 mmol) in THF was added 5 M
NaOH (69 ml) at ambient temperature. After being refluxed for
10 h, the reaction mixture was cooled to ambient temperature
and neutralized by conc. HCl to afford precipitate. The precipitate
was dissolved in CH2Cl2 and washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (CH2Cl2/MeOH, 1/0 to
50:1) to afford the titled compound as a white solid (3.16 g,
85%). 1H NMR (400 MHz, DMSO-d6) d: 8.88 (2H, s), 7.19 (4H, d,
J = 8.3 Hz), 6.88 (4H, d, J = 8.3 Hz), 4.78 (4H, s), 3.73 (6H, s), 3.65-
3.63 (8H, br m), 3.59-3.57 (2H, br m), 3.18 (2H, t, J = 7.8 Hz); HRMS
(ESI), m/z calcd for C30H33N7O3 + H: 540.2723, found 540.2722.
5.1.3. 1-(4-Chloro-2-morpholino-5,6-dihydropyrrolo[2,3-d]
pyrimidin-7-yl)ethanone (9)
To a solution of 6 (5.26 g, 13.24 mmol) in CH3CN (200 ml) was
added Cs2CO3 (12.94 g, 39.7 ml) and NaI (3.97 g, 26.5 mmol). After
being refluxed for 24 h, the reaction mixture was cooled, and then
evaporated under reduced pressure. The residue was dissolved in
EtOAc and washed with H2O and then brine. Combined organic
layer was dried over Na2SO4, filtered, and concentrated under
reduced pressure, affording 4-[4-chloro-7-[(4-methoxyphenyl)
methyl]-5,6-dihydropyrrolo[2,3-d]pyrimidin-2-yl] morpholine (7)
as a crude product. This crude product was used in the next reac-
tion without further purification. To a solution of the crude com-
5.1.6. 5-Bromo-N,N-bis[(4-methoxyphenyl)methyl]pyrimidin-2-
amine (13)
To
a
suspension of 5-bromopyrimidin-2-amine (5.00 g,
28.7 mmol) and NaI (431 mg, 2.87 mmol) in DMF (60 ml) was
added NaH (60% dispersion in mineral oil, 2.87 g, 71.8 mmol) at
0 °C. After stirring for 15 min at 0 °C, 1-(chloromethyl)-4-meth-
oxy-benzene (8.57 ml, 63.2 mmol) was added at 0 °C. After stirring
for 30 min at ambient temperature, the reaction mixture was neu-
tralized 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. The residue was purified by flash column chro-
matography (n-hexane/EtOAc, 1/0 to 9/1) to afford the titled com-
pound as a white solid (10.9 g, 92%). 1H NMR (400 MHz, DMSO-d6)
d: 8.49 (2H, s), 7.16 (4H, d, J = 8.8 Hz), 6.87 (4H, d, J = 8.8 Hz), 4.68
(4H, s), 3.72 (6H, s); HRMS (ESI), m/z calcd for C20H20BrN3O2 + H:
414.0817, found 414.0814.
pound in TFA (14 ml) was added conc. H2SO4 (672 lL,
12.6 mmol). After being refluxed for 5 h, the reaction mixture
was cooled, and then evaporated under reduced pressure. The resi-
due was diluted by CH2Cl2, poured into iced water, and neutralized
by 5 M NaOH and extracted with EtOAc/THF (4/1). The combined
organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure, which afforded 4-(4-
chloro-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine
(8) as a crude product. This crude product was used in the next
reaction without further purification. To a solution of the crude
compound, pyridine (2.42 mL, 29.9 mmol), DMAP (29 mg,
0.239 mmol) in CH3CN (50 ml) was added acethyl chloride
(1.70 mL, 23.9 mmol) dropwise at 0 °C. After being stirred for
15 min at an ambient temperature, the reaction mixture was
diluted with H2O (200 ml) and EtOAc (200 ml), filtered through
Celite. The filtrate was extracted with EtOAc twice, and the com-
bined organic layer was washed with brine, dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (n-hexane/EtOAc, 1/0
to 3/1) to afford the titled compound as a light yellow solid
(3.13 g, 92% in 3 steps). 1H NMR (400 MHz, DMSO-d6) d: 3.92
(2H, t, J = 8.3 Hz), 3.64 (8H, s), 2.85 (2H, t, J = 8.3 Hz), 2.53 (3H, s);
HRMS (ESI), m/z calcd for C12H15ClN4O2 + H: 283.0962, found
283.0962.
5.1.7. N,N-Bis[(4-methoxyphenyl)methyl]-5-(4,4,5,5-tetramet-
hyl-1,3,2-dioxaborolan-2-yl)pyrimidin -2-amine (14)
To a solution of 13 (5.85 g, 14.1 mmol) and triisopropyl borate
(6.48 ml, 28.2 mmol) in toluene/THF = 4/1 (66 ml) was added n-
BuLi (1.6 M in hexane, 10.6 ml, 28.2 mmol) at ꢀ78 °C. After stirring
for 15 min at ꢀ78 °C, the reaction mixture was neutralized by sat-
urated 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-methoxyphenyl)methyl]amino]pyrimidin-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 (6.79 g, 56.4 mmol) in 1,2-
dichloroethane was added pinacol (3.33 g, 28.2 mmol). After stir-
ring for 1.5 h at ambient temperature, the reaction mixture was fil-
tered 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
(5.71 g, 88% in 2 steps). 1H NMR (400 MHz, DMSO-d6) d: 8.53
(2H, s), 7.15 (4H, d, J = 8.6 Hz), 6.86 (4H, d, J = 8.6 Hz), 4.73 (4H,
s), 3.72 (6H, s), 1.28 (12H, s); HRMS (ESI), m/z calcd for C26H32BN3-
O4 + H: 462.2564, found 462.2565.
5.1.4. 1-[4-[2-[Bis[(4-methoxyphenyl)methyl]amino]pyrimidin-
5-yl]-2-morpholino-5,6-dihydro- pyrrolo[2,3-d]pyrimidin-7-yl]
ethanone (10)
A suspension of 9 (2.00 g, 7.07 mmol), 14 (3.92 g, 8.49 mmol),
Pd(OAc)2 (32 mg, 0.141 mmol), 2-dicyclohexylphosphino-2’,6’-
dimethoxy-1,10-biphenyl (S-Phos) (116 mg, 0.283 mmol) and
K3PO4 (3.00 g, 14.2 mmol) in DMF (70 ml) was degassed under
ultrasonic irradiation and stirred for 30 min at 100 °C in a nitrogen
atmosphere. After cooling to ambient temperature, H2O (200 ml)

H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
7657
5.1.8. 5-[2-Morpholino-7-(3-pyridyl)-5,6-dihydropyrrolo[2,3-d]
pyrimidin-4-yl]pyrimidin-2-amine (1)
trated under reduced pressure. The residue was used in the next
reaction without further purification. This crude product and N-
acetyl-cysteine (NAC) (30 mg, 0.185 mmol) was dissolved in TFA
(2 ml) and the resulting reaction mixture was stirred at 70 °C for
3 h. After cooling to ambient temperature, TFA was evaporated
under reduced pressure. The residue was purified by amino silica
gel flash column chromatography (CH2Cl2/MeOH/8 M NH3 in
MeOH, 30/1/0 to 10/1/0, then 50/5/1) to afford the titled compound
as a light yellow solid (42 mg, 91% in 2 steps). 1H NMR (400 MHz,
DMSO-d6) d: 9.18 (1H, s), 8.83 (2H, s), 8.26 (2H, s), 7.13 (2H, s), 4.14
(2H, t, J = 8.0 Hz), 3.71 (8H, s), 3.34 (10H, s), 2.40 (3H, s); HRMS
(ESI), m/z calcd for C25H30N10O2 + H: 503.2631, found 503.2631.
A mixture of 11 (50 mg, 0.093 mmol), 3-bromopyridine (13 ul,
0.14 mmol), Pd(OAc)2 (1.0 mg, 4.6
lmol), S-Phos (3.8 mg,
9.3 mol), K3PO4 (39 mg, 0.19 mmol) in DMF (2 ml) was degassed
l
under ultrasonic irradiation and stirred at 100 °C for 8 h under a
nitrogen atmosphere. The reaction mixture was then cooled to
ambient temperature, diluted with EtOAc, washed with half-brine,
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was used in the next reaction without further
purification. This crude product and N-acetyl-cysteine (NAC)
(30 mg, 0.185 mmol) were dissolved in TFA (1 ml) and the result-
ing reaction mixture was stirred at 70 °C for 3 h. After cooling to
ambient temperature, TFA was evaporated under reduced pres-
sure. The residue was purified by amino silica gel flash column
chromatography (CH2Cl2/MeOH, 1/0 to 9/1) to afford the titled
compound as a white solid (6.3 mg, 18% in 2 steps). 1H NMR
(400 MHz, DMSO-d6) d: 9.08 (1H, s), 8.82 (2H, s), 8.25-8.23 (2H,
br m), 7.41 (1H, dd, J = 8.3, 4.9 Hz), 7.07 (2H, s), 4.12 (2H, t,
J = 9.0 Hz), 3.71 (8H, d, J = 4.4 hz), 3.33 (2H, t, J = 9.0 Hz); HRMS
(ESI), m/z calcd for C19H20N8O + H: 377.1838, found 377.1835.
5.1.12. (4-Chloro-2-pyridyl)-(4-methylpiperazin-1-yl)
methanone (19)
To a solution of 4-chloropyridine-2-carboxylic acid (18, 400 mg,
2.54 mmol), HOBt (686 mg, 5.08 mmol) and 1-methyl piperazine
(0.309 ml, 2.79 mmol) in CH2Cl2 was added EDC (584 mg,
3.05 mmol). After stirring for 30 min at ambient temperature, the
reaction mixture was diluted with saturated NaHCO3 and extracted
with EtOAc (3 times). 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 col-
umn chromatography (CH2Cl2/MeOH, 1/0 to 9/1) to afford the
5.1.9. [4-[4-(2-Aminopyrimidin-5-yl)-2-morpholino-5,6-dihy-
dropyrrolo[2,3-d]pyrimidin-7-yl]-2-pyridyl]-(4-methylpipera-
zin-1-yl)methanone (15)
titled compound as
a
brown oil (506 mg, 83%). 1H NMR
Compound 15 was prepared from 11 and 19 by following the
same procedure as described for 1 (yellow solid, 38% in 2 steps).
1H NMR (400 MHz, CDCl3) d: 8.89 (2H, s), 8.47 (1H, d, J = 6.0 Hz),
8.09 (1H, dd, J = 6.0, 2.1 Hz), 7.79 (1H, d, J = 1H, d, J = 2.1 Hz), 5.28
(2H, s), 4.12 (2H, t, J = 8.8 Hz), 3.85 (10H, m), 3.69 (2H, m), 3.33
(2H, t, J = 8.8 Hz), 2.57 (2H, m), 2.47 (2H, m), 2.36 (3H, s); HRMS
(ESI), m/z calcd for C25H30N10O2 + H: 503.2631, found 503.2628.
(400 MHz, DMSO-d6) d: 8.57 (1H, d, J = 5.4 Hz), 7.71-7.71 (1H, m),
7.65-7.63 (1H, m), 3.64 (2H, t, J = 5.1 Hz), 3.35 (2H, t, J = 5.1 Hz),
2.38 (2H, t, J = 5.1 Hz), 2.28 (2H, t, J = 5.1 Hz), 2.20 (3H, s); HRMS
(ESI), m/z calcd for C11H14ClN3O + H: 240.0903, found 240.0902.
5.1.13. (5-Bromo-2-pyridyl)-(4-methylpiperazin-1-yl)methan-
one (21)
Compound 21 was prepared from 5-bromopyridine-2-car-
boxylic acid by following the same procedure as described for 19
(brown oil, 28%). 1H NMR (400 MHz, DMSO-d6) d: 8.73 (1H, dd,
J = 2.4, 1.0 Hz), 8.19 (1H, dd, J = 8.3, 2.4 Hz), 7.55 (1H, d,
J = 7.8 Hz), 3.63 (2H, t, J = 5.1 Hz), 3.36 (2H, t, J = 5.1 Hz), 2.37
(2H, t, J = 5.1 Hz), 2.27 (2H, t, J = 5.1 Hz), 2.19 (3H, s); HRMS (ESI),
m/z calcd for C11H14BrN3O + H: 284.0398, found 284.0393.
5.1.10. [5-[4-(2-Aminopyrimidin-5-yl)-2-morpholino-5,6-dihy-
dropyrrolo[2,3-d]pyrimidin-7-yl]-2-pyridyl]-(4-methylpipera-
zin-1-yl)methanone (16)
A mixture of 11 (50 mg, 0.093 mmol), 21 (32 mg, 0.11 mmol),
Pd2(dba)3 (4.2 mg, 4.6 lmol), S-Phos (7.6 mg, 19 lmol), K3PO4
(39 mg, 0.19 mmol) in DMF (2 ml) was degassed under ultrasonic
irradiation and stirred at 100 °C for 4 h under a nitrogen atmo-
sphere. The reaction mixture was then cooled to ambient temper-
ature, diluted with CH2Cl2, washed with saturated aqueous NH4Cl,
H2O. The organic layer was dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was used in the next
reaction without further purification. This crude product and N-
acetyl-cysteine (NAC) (30 mg, 0.185 mmol) was dissolved in TFA
(1 ml) and the resulting reaction mixture was stirred at 70 °C for
4 h. After cooling to ambient temperature, TFA was evaporated
under reduced pressure. The residue was purified by amino silica
gel flash column chromatography (CH2Cl2/MeOH, 1/0 to 33/1) to
afford the titled compound as a white solid (27 mg, 58% in 2 steps).
1H NMR (400 MHz, DMSO-d6) d: 9.05 (1H, d, J = 2.0 Hz), 8.83 (2H,
s), 8.38 (1H, dd, J = 9.0, 2.0 Hz), 7.65 (1H, d, J = 9.0 Hz), 7.13 (2H,
s), 4.15 (2H, t, J = 8.1 Hz), 3.71 (8H, br s), 3.59 (4H, m), 3.34 (2H,
t, J = 8.1 Hz), 2.33 (4H, m), 2.20 (3H, s); HRMS (ESI), m/z calcd for
5.1.14. (5-Bromo-3-pyridyl)-(4-methylpiperazin-1-yl)
methanone (23)
Compound 23 was prepared from 5-bromopyridine-3-car-
boxylic acid by following the same procedure as described for 19
(yellow solid, 78%). 1.H NMR (400 MHz, DMSO-d6) d: 8.79 (1H, d,
J = 2.0 Hz), 8.59 (1H, d, J = 1.5 Hz), 8.12 (1H, t, J = 2.2 Hz), 3.62
(2H, br s), 3.34 (2H, br s), 2.37 (2H, br s), 2.28 (2H, br s), 2.20
(3H, s); HRMS (ESI), m/z calcd for C11H14BrN3O + H: 284.0398,
found 284.0395.
5.1.15. 5-(2-Morpholino-7-phenyl-5,6-dihydropyrrolo[2,3-d]
pyrimidin-4-yl)pyrimidin-2-amine (24)
Compound 24 was prepared from 11 and bromobenzene by fol-
lowing the same procedure as described for 1 (yellow solid, 80% in
2 steps). 1H NMR (400 MHz, d-TFA) d: 9.10 (2H, s), 7.59-7.42 (4H,
m), 7.35 (1H, t, J = 7.7 Hz), 4.41 (2H, t, J = 7.4 Hz), 4.08-3.89 (8H,
m), 3.31 (2H, t, J = 7.4 Hz); HRMS (ESI), m/z calcd for C20H21N7O
+ H: 376.1886, found 376.1882.
C25H30N10O2 + H: 503.2631, found 503.2627.
5.1.11. [5-[4-(2-Aminopyrimidin-5-yl)-2-morpholino-5,6-dihy-
dropyrrolo[2,3-d]pyrimidin-7-yl]-3-pyridyl]-(4-methylpipera-
zin-1-yl)methanone (17)
A mixture of 11 (50 mg, 0.093 mmol), 23 (40 mg, 0.14 mmol),
Pd2(dba)3 (4.3 mg, 4.7 lmol), X-Phos (8.9 mg, 19 lmol), K3PO4
5.1.16. Methyl 3-[4-[2-[bis[(4-methoxyphenyl)methyl]amino]
pyrimidin-5-yl]-2-morpholino-5,6-dihydropyrrolo[2,3-d]
pyrimidin-7-yl]benzoate (25)
(39 mg, 0.19 mmol) in DMF (2 ml) was stirred at 100 °C for 18 h
under a nitrogen atmosphere. The reaction mixture was then
cooled to ambient temperature, diluted with CH2Cl2, washed with
saturated aqueous NH4Cl, dried over Na2SO4, filtered, and concen-
A mixture of 11 (100 mg, 0.19 mmol), 3-bromobenzoic acid
methyl ester (48 mg, 0.22 mmol), Pd(OAc)2 (2.1 mg, 9.3
Phos (7.6 mg, 19 mol), K3PO4 (79 mg, 0.37 mmol) in DMF (2 ml)
was degassed under ultrasonic irradiation and stirred at 100 °C
lmol), S-
l

7658
H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
for 3 h under a nitrogen atmosphere. The reaction mixture was
then cooled to ambient temperature and H2O was added. The
resulting filter cake was washed by H2O, n-hexane/EtOAc = 2/1 to
afford the titled compound as a yellow solid (108 mg, 86%). The
residue was used in the next reaction without further purification.
X-Phos (8.83 mg, 18.5 lmol), K3PO4 (118 mg, 0.556 mmol) in
DMF (2 ml) was degassed under ultrasonic irradiation and stirred
at 100 °C for 24 h under a nitrogen atmosphere. The reaction mix-
ture was then cooled to ambient temperature, and then 1 M HCl
(6 ml) was added. After being stirred for 1 hour at ambient temper-
ature, the resulting filter cake was washed by H2O, n-hexane/
EtOAc = 1/1 to afford the titled compound as a light yellow solid
(101 mg, 80%). The residue was used in the next reaction without
further purification.
5.1.17. 3-[4-[2-[Bis[(4-methoxyphenyl)methyl]amino]pyrimi-
din-5-yl]-2-morpholino-5,6-dihydro-pyrrolo[2,3-d]pyrimidin-
7-yl]benzoic acid (26)
A mixture of 25 (60 mg) in THF/5 M NaOH/30%-H2O2 aq = 4/2/1
(1.4 ml) was refluxed for 48 h. The reaction mixture was then
cooled to ambient temperature, acidified with 6 M HCl aq. The
resulting filter cake was washed by H2O, n-hexane/EtOAc = 2/1 to
afford the titled compound as a yellow solid (56 mg, 95%). The resi-
due was used in the next reaction without further purification.
5.1.22. [4-[4-(2-Aminopyrimidin-5-yl)-2-morpholino-5,6-dihy-
dropyrrolo[2,3-d]pyrimidin-7-yl]-3-fluoro-phenyl]-
morpholino-methanone (3)
To
a
solution of 31 (120 mg, 0.18 mmol), HOBt (24 mg,
l, 0.35 mmol) in CH2Cl2 (4 ml)
0.15 mmol) and morpholine (31
l
was added EDC (68 mg, 0.35 mmol) at ambient temperature. After
stirring for 1 hour at ambient temperature, H2O was added. The
organic layer was separated and dried over Na2SO4, filtered, and
concentrated under reduced pressure. This crude product was dis-
solved in TFA (2 ml) and the resulting reaction mixture was
refluxed for 4 h. After cooling to ambient temperature, TFA was
evaporated under reduced pressure. The residue was purified by
silica gel flash column chromatography (CH2Cl2/MeOH, 9/1) to
afford the titled compound as a white solid (86 mg, 96% in 2 steps).
1H NMR (400 MHz, DMSO-d6) d: 8.81(2H, s), 7.75 (1H, t, J = 8.1 Hz),
7.41 (1H, m), 7.29 (1H, m), 7.09 (2H, s), 4.13-4.06 (2H, m), 3.71-
3.45 (10H, m), 3.36-3.33 (8H, m); 13C NMR (d-TFA) d: 171.0,
165.8, 158.0 (d, J = 238.0 Hz), 156.8, 155.4, 154.6, 136.2, 133.6 (d,
J = 6.9 Hz), 127.4, 127.1 (d, J = 12.2 Hz), 123.6 (d, J = 3.1 Hz),
116.4, 114.0 (d, J = 31.3 Hz), 66.1, 65.4, 52.0, 48.0, 44.6, 43.3,
21.6; HRMS (ESI), m/z calcd for C25H27FN8O3 + H: 507.2268, found
507.2280.
5.1.18. [3-[4-(2-Aminopyrimidin-5-yl)-2-morpholino-5,6-dihy-
dropyrrolo[2,3-d]pyrimidin-7-yl]phenyl]-(4-methylpiperazin-
1-yl)methanone (27)
To
0.059 mmol), 1-methyl piperazine (12 mg, 0.117 mmol) and EDC
(17 mg, 0.087 mmol) in DMF (1 ml) was added DIPEA (9.8 l,
a solution of 26 (38 mg, 0.058 mmol), HOBt (8.0 mg,
l
0.056 mmol). After stirring for 90 min at 60 °C, 1-methyl piper-
azine (12 mg, 0.117 mmol) and EDC (17 mg, 0.087 mmol) was
added. After stirring for 90 min at 60 °C, the reaction mixture
was diluted with CH2Cl2 and washed with H2O, then concentrated
under reduced pressure. The residue was purified by silica gel flash
column chromatography (CH2Cl2/MeOH) to afford [3-[4-[2-[bis[(4-
methoxyphenyl)methyl]amino]pyrimidin-5-yl]-2-morpholin-4-yl-
5,6-dihydropyrrolo[2,3-d]pyrimidin-7-yl]phenyl]-(4-methylpiper-
azin-1-yl)methanone (31 mg, 71%). This residue was dissolved in
TFA (1 ml). After being refluxed for 6 h, TFA was evaporated under
reduced pressure. The residue was purified by silica gel flash col-
umn chromatography (CH2Cl2/ MeOH, 30/1 to 10/1) to afford the
5.1.23. 4-[4-[2-[Bis[(4-methoxyphenyl)methyl]amino]pyrimi-
din-5-yl]-2-morpholino-5,6-dihydro-pyrrolo[2,3-d]pyrimidin-
7-yl]benzaldehyde (32)
titled compound as
a
yellow solid (20 mg, 96%). 1H NMR
(400 MHz, DMSO-d6) d: 8.82 (2H, s), 8.02 (1H, d, J = 8.1 Hz), 7.87
(1H, s), 7.50 (1H, dd, J = 8.2, 7.6 Hz), 7.18-7.04 (3H, m), 4.11 (2H,
t, 7.9 Hz), 3.79-3.65 (8H, m), 3.43-2.99 (10H, m), 2.83 (3H, s);
A mixture of 11 (300 mg, 0.556 mmol), 2-(4-boromopheyl)-1,3-
dioxolane (178 ul, 0.778 mmol), Pd2(dba)3 (13 mg, 14
lmol), X-
HRMS (ESI), m/z calcd for
C26H31N9O2 + H: 502.2679, found
Phos (27 mg, 56 mol), K3PO4 (236 mg, 1.11 mmol) in DMF
l
502.2681.
(5 ml) was degassed under ultrasonic irradiation and stirred at
100 °C for 14 h under a nitrogen atmosphere. The reaction mixture
was then cooled to ambient temperature, diluted with EtOAc and
washed with saturated aqueous NH4Cl. The collected organic layer
was washed with brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was used in the next
reaction without further purification. To a solution of the residue
in THF (6 ml) was added 1 M HCl aq. (2 ml). After stirring for 3 h
at ambient temperature, the reaction mixture was diluted with sat-
urated aqueous NH4Cl and extracted with EtOAc twice. The com-
bined 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 (CH2Cl2/
MeOH, 1/0 to 19/1) to afford the titled compound as a brown solid
(345 mg, 96% in 2 steps). 1H NMR (400 MHz, DMSO-d6) d: 9.87 (1H,
s), 8.99 (2H, s), 8.06 (2H, d, J = 8.8 Hz), 7.91 (2H, d, J = 8.8 Hz), 7.21
(4H, d, J = 8.3 Hz), 6.89 (4H, d, J = 8.3 Hz), 4.79 (4H, s), 4.14 (2H, t,
5.1.19. [4-[4-(2-Aminopyrimidin-5-yl)-2-morpholino-5,6-
dihydropyrrolo[2,3-d]pyrimidin-7-yl]phenyl]-(4-
methylpiperazin-1-yl)methanone (28)
Compound 24 was prepared from 11 and 30 by following the
same procedure as described for 1 (white solid, 87% in 2 steps).
1H NMR (400 MHz, DMSO-d6) d: 8.80 (2H, s), 7.89 (2H, d,
J = 8.8 Hz), 7.42 (2H, d, J = 8.8 Hz), 7.04 (2H, s), 4.10 (2H, t,
J = 8.3 Hz), 3.74-3.65 (8H, m), 3.55-3.45 (4H, br m), 3.45-3.42
(2H, m), 2.34-2.27 (4H, br m), 2.18 (3H, s); HRMS (ESI), m/z calcd
for C26H31N9O2 + H: 502.2679, found 502.2683.
5.1.20. (4-Bromophenyl)-(4-methylpiperazin-1-yl)methanone
(30)
Compound 30 was prepared from 4-bromobenzoic acid by fol-
lowing the same procedure as described for 19 (white solid,
97%). 1H NMR (400 MHz, DMSO-d6) d: 7.64 (2H, dt, J = 8.6,
2.1 Hz), 7.34 (2H, dt, J = 8.6, 2.1 Hz), 3.59 (2H, br s), 3.30 (2H, br
s), 2.34 (2H, br s), 2.27 (2H, br s), 2.19 (3H, s); HRMS (ESI), m/z calcd
for C12H15BrN2O + H: 283.0446, found 283.0445.
J = 8.2 Hz), 3.77-3.65 (16H, brm); HRMS (ESI), m/z calcd for C37H37
-
N7O4 + H: 644.2985, found 644.2987.
5.1.24. 5-[7-[4-[(4-Methylpiperazin-1-yl)methyl]phenyl]-2-mor-
pholino-5,6-dihydro-pyrrolo[2,3-d]pyrimidin-4-yl]pyrimidin-2-
amine (2)
5.1.21. 4-[4-[2-[Bis[(4-methoxyphenyl)methyl]amino]pyrimi-
din-5-yl]-2-morpholino-5,6-dihydro-pyrrolo[2,3-d]pyrimidin-
7-yl]-3-fluoro-benzoic acid (31)
The mixture of 32 (76 mg, 0.118 mmol), N-methylpiperazine
(40
(13
l
l
l, 0.354 mmol), NaBH(OAc)3 (100 mg, 472 mmol) and AcOH
l, 0.236 mmol) in CH2Cl2 (2 ml) was stirred for 40 h at ambi-
A mixture of 11 (100 mg, 0.185 mmol), 4-bromo-3-fluoroben-
zoic acid (48.7 mg, 0.222 mmol), Pd2(dba)3 (4.24 mg, 4.63
l
mol),
ent temperature. The mixture was diluted with saturated aqueous

H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
7659
NH4Cl and extracted with EtOAc twice. The combined organic layer
5.3. LM stability assay
of each compound was incubated with human (or
was washed with brine, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified by silica
gel flash column chromatography (CH2Cl2/2 M NH3 in MeOH) to
afford N,N-bis(4-methoxybenzyl)-5-(7-(4-((4-methylpiperazin-1-
yl)methyl)phenyl)-2-morpholino-6,7-dihydro-5H-pyrrolo[2,3-d]
pyrimidin-4-yl)pyrimidin-2-amine (44 mg, 51%). To a solution of
this residue in TFA (1 ml) was added NAC (20 mg, 0.120 mmol).
After being refluxed for 4 h, TFA was evaporated under reduced
pressure. The residue was purified by silica gel flash column chro-
matography (CH2Cl2/2 M NH3 in MeOH, 30/1 to 10/1) to afford the
titled compound as a yellow solid (26 mg, 90%). 1H NMR (400 MHz,
DMSO-d6) d: 8.81 (2H, s), 7.82 (2H, d, J = 8.3 Hz), 7.33 (2H, d,
J = 8.3 Hz), 7.08 (2H, s), 4.08 (2H, t, J = 7.3 Hz), 3.70 (4H, s), 3.56
(2H, s), 3.34 (10 H, br s), 3.00 (4H, s), 2.63 (3H, s); HRMS (ESI),
m/z calcd for C26H33N9O + H: 488.2886, found 488.2881.
1 lM
mouse) LM (0.5 mg protein/mL) in 50 mM phosphate buffer
(pH 7.4) containing 1 mM NADPH (the reduced form of nicoti-
namide adenine dinucleotide phosphate) at 37 °C for 30 min.
After the enzyme reaction was terminated with the addition of
a three-fold 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) LM by measuring the compound in the sample using
LC–MS/MS.
5.4. Permeability assay
10 mM stock solutions of each compounds were diluted with
donor buffer. The resulting mixture was checked for precipitation
and then filtered. The donor plate was filled with the resulting fil-
trate. The filter plate was coated with phosphor-lipids and placed
into the donor plate, and was then filled with acceptor buffer. After
a certain time, donor and acceptor concentration was analyzed by
5.1.25. 5-[2-Morpholino-7-[4-(morpholinomethyl)phenyl]-5,6-
dihydropyrrolo[2,3-d]pyrimidin-4-yl]pyrimidin-2-amine (33)
Compound 33 was prepared from 32 and morpholine by follow-
ing the same procedure as described for 2 (light brown solid, 50% in
2 steps). 1H NMR (400 MHz, DMSO-d6) d: 8.82 (2H, s), 7.95 (2H, d,
J = 8.6 Hz), 7.51 (2H, d, J = 8.6 Hz), 7.09 (2H, s), 4.27 (2H, s), 4.11
(2H, s), 3.68-3.55 (14H, m), 3.35 (2H, s), 3.19 (2H, s); HRMS (ESI),
m/z calcd for C25H30N8O2 + H: 475.2570, found 475.2572.
UV.
A donor buffer of 0.05 M MOPSO (3-(N-morpholino)-2-
hydroxy-1-propanesulfonic acid) containing 0.01 M GCA (glyco-
cholic acid) was used (pH = 6.5). An acceptor buffer of 0.05 M
MOPSO was used (pH = 6.5). Permeability of PAMPA is calculated
by using the following equation:
5.1.26. 4-(4-(2-(Bis(4-methoxybenzyl)amino)pyrimidin-5-yl)-2-
morpholino-5H-pyrrolo[2,3-d]pyri-midin-7(6H)-yl)-3-
fluorobenzaldehyde (34)
ꢀ
ꢁ
VD
A MDð0Þ
DMA
Dt
Pe ¼
A mixture of 11 (50 mg, 0.093 mmol), 4-bromo-3-fluoroben-
Pe = Permeations coefficient (10ꢀ6 cm/s)
VD = Volumes in donor (cm3)
zaldehyde (23 ul, 0.11 mmol), Pd(OAc)2 (1.0 mg, 4.6
lmol), X-Phos
(4.4 mg, 9.3 mol), K3PO4 (39 mg, 0.19 mmol) in DMF (2 ml) was
l
A = Surface area of the film (cm2)
MD(0) = Concentration in donor (mol/cm3)
degassed under ultrasonic irradiation and stirred at 100 °C for 3 h
under a nitrogen atmosphere. The reaction mixture was then
cooled to ambient temperature, diluted with EtOAc, washed with
half-brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel flash col-
umn chromatography (CH2Cl2/MeOH, 1/0 to 19/1) to afford the
D
D
MA = Concentration in acceptor (mol/cm3)
t = Permeation times (s)
titled compound as
a
yellow solid (40 mg, 65%). 1H NMR
5.5. Solubility assay
(400 MHz, DMSO-d6) d: 9.94 (1H, d, J = 1.5 Hz), 8.99 (2H, s), 8.00
(1H, t, J = 7.9 Hz), 7.81 (1H, s), 7.78 (1H, t, J = 1.8 Hz), 7.21 (4H, d,
J = 8.6 Hz), 6.89 (4H, d, J = 8.6 Hz), 4.79 (4H, s), 4.17 (2H, t,
J = 7.8 Hz), 3.73 (6H, s), 3.67-3.60 (8H, m), 3.37 (2H, t, J = 7.8 Hz);
13C NMR (DMSO-d6) d:191.5, 166.4, 161.8, 161.2, 158.9, 158.0,
156.8, 154.3, 153.1, 134.4, 134.3, 133.7, 133.7, 130.2, 129.2,
126.6, 126.5, 125.1, 125.1, 120.6, 117.2, 117.0, 114.4, 105.4, 66.5,
An aliquot of 50
ide (DMSO) was freeze-dried to remove DMSO. To the resulting
residue was added 50 M of FaSSIF (pH = 6.5), which was then irra-
diated ultrasonically for 10 min, shaken for 2 h, centrifuged for
10 min (3000 rpm), and filtered by Whatman Unifilter. Concentra-
tion of the filtrate was analyzed by HPLC-UV based on the calibra-
tion curve of each sample. Composition of FaSSIF: Sodium
taurocholate (1.61 g), Lecithin (0.59 g), KH2PO4 (3.9 g), KCl (7.7 g)
and NaOH (pH 6.5) per 1 L.
ll of 4 mM or 1 mM sample in dimethylsulfox-
l
55.5, 50.9, 50.8, 48.9, 44.8, 25.3; HRMS (ESI), m/z calcd for C37H36
-
FN7O4 + H: 662.2891, found 662.2896.
5.1.27. 5-[7-[2-Fluoro-4-(morpholinomethyl)phenyl]-2-morpho-
lino-5,6-dihydropyrrolo[2,3-d]pyri-midin-4-yl]pyrimidin-2-
amine (35)
5.6. Pharmacokinetic study in mice and monkeys
Compound 35 was prepared from 34 and morpholine by follow-
ing the same procedure as described for 2 (light brown solid, 52% in
2 steps). 1H NMR (400 MHz, DMSO-d6) d: 8.80 (2H, s), 7.58 (1H, t,
J = 8.1 Hz), 7.23 (1H, d, J = 12.2 Hz), 7.17 (1H, d, J = 8.1 Hz), 7.05
(2H, s), 4.02 (2H, t, J = 7.8 Hz), 3.59 (12H, br s), 3.48 (2H, s), 3.31
Pharmacokinetic study: Female BALB/c-nu mice and male
cynomolgus monkeys (n = 2–3 per treatment group) were given
the solutions of test compounds by intravenous (iv) or oral (po)
route at doses of 10 mg/kg (iv) and 50 or 100 mg/kg (po) in mice,
and 2.5 or 5 mg/kg (iv) and 10 mg (po) in monkeys. Blood samples
of each animal were collected with heparin as an anticoagulant at
0.08, 0.25, 1, 2, 4, 7, and 24 h following iv dosing and at 0.5, 1, 2, 4,
7, and 24 h following po dosing. Samples were centrifuged to
obtain plasma and stored at ꢀ80 °C until analysis. Plasma concen-
trations were determined by using LC–MS/MS system. The phar-
macokinetic parameters were calculated by non-compartmental
analysis using WATSON ver. 7.1 (Thermo Fisher Scientific, Wayne,
PA).
(2H, t, J = 7.8 Hz), 2.38 (4H, br s); HRMS (ESI), m/z calcd for C25H29
-
FN8O2 + H: 493.2476, found 493.2472.
5.2. In vitro kinase enzyme assay
The inhibitory activity on PI3Ka (p110a/p85a) (Life Technolo-
gies) was determined by Adapta Universal Kinase Assay Kit with
PIP2:PS Lipid Kinase Substrate (Life Technologies).

7660
H. Kawada et al. / Bioorg. Med. Chem. 23 (2015) 7650–7660
8. Kawada, H.; Ebiike, H.; Tsukazaki, M.; Nakamura, M.; Morikami, K.; Yoshinari,
Acknowledgments
K.; Yoshida, M.; Ogawa, K.; Shimma, N.; Tsukuda, T.; Ohwada, J. Bioorg. Med.
Chem. Lett. 2013, 23, 673.
We thank S. Kuramoto for PK studies. We thank S. Courtney and
W. Trigg at Evotec and Woon Sang Hong and Hoe-Moon Kim 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 spec-
trometry measurements.
9. Galia, E.; Nicolaides, E.; Horter, D.; Lobenberg, R.; Reppas, C.; Dressman, J. B.
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