Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: Improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy

Article abstract of DOI:10.1016/j.bmc.2012.08.002

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC ₅₀: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI₅₀: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).

Full text of DOI:10.1016/j.bmc.2012.08.002

Bioorganic & Medicinal Chemistry 20 (2012) 6171–6180
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Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors:
Improvement of the physicochemical and pharmacokinetic profiles
for potent in vivo anti-tumor efficacy
Youichi Kawakita a, , Kazuhiro Miwa b, Masaki Seto a, Hiroshi Banno a, Yoshikazu Ohta a,
⇑
Toshiya Tamura a, Tadashi Yusa a, Hiroshi Miki a, Hidenori Kamiguchi a, Yukihiro Ikeda a,
Toshimasa Tanaka a, Keiji Kamiyama a, Tomoyasu Ishikawa a,
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Co. Ltd, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b Chemical Development Laboratories, CMC Center Pharmaceutical Production Division, Takeda Pharmaceutical Co. Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative
potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this
compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically
modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent
HER2/EGFR kinase inhibitory activity (IC50: 11/11 nM) and cellular growth inhibitory activity (BT-474 cell
GI50: 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant
in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer
cell lines (mouse, T/C = 0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 June 2012
Revised 2 August 2012
Accepted 3 August 2012
Available online 25 August 2012
Keywords:
HER2
EGFR
Pyrrolo[3,2-d]pyrimidine
1. Introduction
activity. These events activate downstream signaling pathways
and eventually promote tumor cell growth.3 Therefore, HER2/EGFR
In recent years, molecular targeted cancer therapies have at-
tracted the attention of researchers because they are more effec-
tive and safer than conventional cytotoxic chemotherapies, due
to these therapies having well-defined targets and mechanisms
of action (MOAs).1 The epidermal growth factor receptor (EGFR
or erbB1) and human epidermal growth factor receptor 2 (HER2
or erbB2) are members of the erbB family of receptor tyrosine
kinases, which have been validated as target molecules for cancer
therapy.2 The stimulation of ligands such as EGF causes the erbB
receptors to either homodimerize or heterodimerize with the other
erbB family receptors, thus inducing elevation of tyrosine kinase
inhibitors can inhibit tyrosine kinase phosphorylation and block
elevated intracellular signaling pathways in cancer cells, resulting
in loss of regulatory function of the tumor. The significant clinical
efficacy of small-molecule EGFR inhibitors, such as gefitinib4 (Ires-
saTM) and erlotinib5 (TarcevaTM), provided the proof of concept
(POC) for their efficacy in lung cancer therapy. More recently, a
small-molecule HER2/EGFR dual inhibitor, lapatinib6 (TykerbTM
)
was launched for the treatment of advanced metastatic breast
cancer.7
Most reported small-molecule inhibitors of HER2/EGFR contain
a quinazoline ring as the core molecular scaffold.4–6,8 In a previous
study, we reported the discovery of a novel pyrrolo[3,2-d]pyrimi-
dine derivative compound A (TAK-285) as a potent and orally
active HER2/EGFR dual inhibitor (Fig. 1).9 During our research on
A, we also searched for another compound that could replace A if
any unpredictable issues occurred during the clinical studies. Here,
we describe the discovery of an alternative preclinical candidate,
compound 2cb, with an improved physicochemical and pharmaco-
kinetic (PK) profile.
Abbreviations: HER2 (erbB2)/EGFR (erbB1), human epidermal growth factor
receptor 2/epidermal growth factor receptor; PK profile, pharmacokinetic profile;
IC, inhibitory concentration; GI, growth inhibitiory; DMPK, drug metabolism and
pharmacokinetic profile; MOA, mechanism of action; GCDC, sodium glycocheno-
deoxycholic acid; FISA, hydrophilic component of solvent accessible surface area;
POC, proof of concept; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride; HOBT, 1-hydroxybenzotriazole monohydrate; NMM, 4-methylmor-
pholine; m-CPBA, m-chloroperbenzoic acid; BsOH, benzenesulfonic acid; TsOH,
p-toluenesulfonic acid.
Although compound 1a showed potent cellular antiproliferative
activity (BT-474 cell GI50: 5.2 nM), we did not select it as a candi-
date compound based on its poor cell membrane permeability and
PK profile (see below, Table 1). Therefore, we initiated program to
⇑
Corresponding authors. Tel.: +81 466 32 1249; fax: +81 466 29 4450 (Y.K.);
tel.: +81 466 32 1155; fax: +81 466 29 4449 (T.I.).
E-mail addresses: youichi.kawakita@takeda.com (Y. Kawakita), tomoyasu.
ishikawa@takeda.com (T. Ishikawa).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.002

6172
Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
N
to the corresponding sulfones 1d–f by oxidation in two steps with
m-chloroperbenzoic acid (m-CPBA) followed by titanium tetraiso-
propoxide/tert-butylperoxide (Kagan’s method11) in 42–58% over-
all yield.
F
N
F
F
N
H
N
H3C
H3C OH
HN
Cl
O
O
A (TAK-285)
3. Results and discussion
Figure 1. Structure of compound A (TAK-285).
To analyze effects of modification of the N-5 side chain of pyr-
rolo[3,2-d]pyrimidine, we replaced the 3-hydroxy-3-methylbu-
identify alternative compounds with both potent cellular growth
inhibitory (GI) activity and good oral bioavailability. On the basis
of our prior experience during research on A, we conducted studies
focused on (a) introduction of alkyl groups into the N-5 sulfonyla-
mide side chain (R2, X) and (b) optimization of substituents on the
4-anilino group (R1 = CF3, Cl) to enhance the membrane permeabil-
ity and PK profile of 1a (Fig. 2). Further, we expected that the con-
version of 1a into its respective salts after chemical modification
could lead to the preparation of candidate compounds with high
solubility and enhanced PK profiles.
tanamide moiety of A with a 2-(methylsulfonyl)acetamide
moiety (1a). Compound 1a showed potent HER2/EGFR kinase
activity and inhibition of cell growth (HER2/EGFR IC50: 6.3/
15 nM, BT-474 cell GI50: 5.2 nM), results which were superior to
those obtained for A. However, the PK of 1a in mice and the low
A-B permeability of 1a across Caco-2 membrane (B-A/A-B: 82.9/
5.2) were issues that required further evaluation. We assumed that
1a was a P-glycoprotein (P-gp) substrate, whereas A is not. We
envisioned that masking of the polar sulfonyl group of 1a by intro-
ducing R2 into the vicinal position should improve the A-B perme-
ability, because it is known that molecules with a highly polar
substituent such as sulfonamide, phenol, or a secondary amine
group have a tendency to behave as P-gp substrates.12 In addition,
a preliminary physicochemical study indicated that introduction of
various alkyl groups into 1a could increase the lipophilicity of the
molecule (cLogD) and improve its physicochemical profile. All the
compounds thus synthesized (1d–f) showed potent HER2/EGFR
kinase inhibitory activities, although their cell growth inhibitory
activities were weaker than that of 1a (Table 1). The HER2/EGFR
kinase and cell growth inhibitory activities of 1d–f show a trend
towards lower potencies for compounds with larger R2 substituents.
Furthermore, it was apparent that the introduction of alkyl groups
at R2 would not improve the PK profile, although the cLogD (3.32–
4.02) values of 1d–f were close to that of A. On the other hand, cal-
2. Chemistry
Synthesis of compounds 1a–c and 2a–c was conducted as out-
lined in Scheme 1. Condensation of compound 39 with anilines
was carried out in 2-propanol, followed by removal of the terminal
Boc group of the resulting compound 4 or 5 with 10% w/w HCl/
MeOH solution to afford key intermediates (6 or 7) in greater than
94% yield. Amidation of 6 or 7 with a variety of carboxylic acids in
the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) and 1-hydroxybenzotriazole monohydrate
(HOBT) provided the corresponding desired carboxamides (1a, 2a
and 2c) in approximately 70–80% yield. Conversion of 2-methyl-
2-(methylsulfonyl)propanoic acid10 to the corresponding acid
chloride using thionyl chloride followed by treatment with 6 pro-
vided 1c in 99% yield. In addition, mono-methylated derivatives
(1b, 2b) were prepared by condensation of 6 or 7 with 2-chloro-
propionic acid in the presence of EDC and HOBT followed by
nucleophilic displacement of the chloro group with sodium
methanesulfinate in 68% and 43% yield over two steps,
respectively.
culation of the hydrophilic component of the solvent-accessible
13
surface area (FISA: See Table 1 footnote)
values for this series
suggested that we should seek compounds with lower FISA values,
as well as higher cLogD.
Next, we examined the effect of introducing alkyl groups into
the linker (X) between the sulfonyl and carbonyl groups of 1a.
Methyl (1b) and dimethyl (1c) groups were selected as representa-
tive alkyl substituents (Table 2). In addition, the corresponding
derivatives 2a–c were prepared because they were expected to
exhibit good HER2/EGFR inhibitory activity according to our previ-
ous research on A. We also performed a physicochemical calcula-
tion, which suggested that replacement of the 3-trifluoromethyl
Chemical modification of the terminal sulfonyl alkyl groups was
carried out as shown in Scheme 2. Reaction of 6 with chloroacetyl-
chloride in the presence of 4-methylmorpholine (NMM) in THF and
subsequent displacement with the appropriate sodium alkylthio-
lates provided 9–11. The resulting sulfides 9–11 were converted
Table 1
Evaluation of N-5-substituted pyrrolo [3,2-d] pyrimidine derivatives
R2
O
CF3
S
O
NH
O
HN
N
Cl
O
N
N
Compound
R2
Enzyme
IC50 (nM)
Cell growth
GI50 (nM)
Metabolic stability
L/min/mg)
Caco-2
(nm/sec)
Mouse PK
AUC0–8 h
FISAa
Å2
(
l
(
lg h/mL)
cLogD
HER2
EGFR
BT-474
Human
Mouse
B–A/A–B
(10 mg/kg)
1a
1d
1e
1f
A
Me
Et
6.3
8.5
11
14
17
15
31
53
56
23
5.2
36
48
36
17
18
26
42
45
35
11
22
35
31
28
82.9/5.2
NT
NT
NT
68.7/50.5
0.947
0.647
1.275
1.07
2.79
3.32
3.67
4.02
3.47
147
135
131
127
97
iPr
tBu
1.923
a
FISA is the hydrophilic component of the solvent accessible surface area using a probe with a 1.4-Å radius. Compounds computationally found to be outside of the range
for 95% of all drugs based on QikProp 2.1 are indicated in boldface.

Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
6173
(a) Introduction of alkyl group to R2 and X
R2
S
O
O
X
O
R1 (b) Optimization of substitutent R1
H
N
O
HN
N
Cl
(c)
Conversion to their salt
4
N
5
N
: R1 = CF3, R2 = CH3, X = CH2
1a
2a
: R1 = Cl, R2 = CH3, X = CH2
Figure 2. Synthetic strategies for improvement of membrane permeability and PK.
O
R1
BocHN
BocHN
Cl
N
HN
N
Cl
b
a
N
N
N
N
3
R
R
1 = CF3: 85%
4:
5:
1 = Cl : 87%
R1
R1
Me
O
O
O
S
O
X
H2N
NH
HN
Cl
HN
N
Cl
c, d or e
O
N
N
N
N
.2HCl
N
1 = CF3: quant.
1 = Cl : 94%
1 = CF3, X = -CH2-: 79%
6:
7:
R
R
1a:
1b:
1c:
2a:
2b:
2c:
R
1
R = CF3, X = -CH(Me)-: 68%
R
R
1 = CF3, X = -C(Me)2-: 99%
1 = Cl, X = -CH2- : 69%
1
R = Cl, X = -CH(Me)- : 43%
R
1 = Cl, X = -C(Me)2- : 74%
Scheme 1. Reagents: (a) 3-Chloro-4-[3-(trifluoromethyl)phenoxy]aniline or 3-chloro-4-(3-chlorophenoxy)aniline, 2-propanol; (b) 10% HCl/MeOH; (c) MeSO2-X-CO2H, EDC,
HOBT, Et3N, DMF; (d) (i) MeCH(Cl)CO2H, EDC, HOBT, Et3N, DMF, (ii) MeSO2Na, pyridine, DMF; (e) (i) MeSO2C(Me)2COOH, SOCl2, cat.DMF, (ii) Et3N, THF.
R2
S
O
CF3
O
CF3
NH
H2N
HN
N
Cl
a
HN
N
O
Cl
N
N
N
N
.2HCl
R2 = Et
9 :
10:
11:
6
R2 = iso-Pr
R2 = tert-Bu
R2
O
CF3
S
O
NH
O
HN
N
Cl
b
O
N
N
1d: R2 = Et: 58% from 6
1e: R2 = iso-Pr: 48% from 6
1f: R2 = tert-Bu: 42% from 6
Scheme 2. Reagents: (a) (i) ClCH2COCl, NMM, THF, (ii) R2SNa, DMF/THF; (b) (i) m-CPBA, CH2Cl2, (ii) Ti(OPri)4, tert-butylperoxide, MeOH/CH2Cl2.
moiety with a 3-chloro moiety could provide more favorable pro-
files for 2a–c with higher cLogD and lower FISA values than those
of the corresponding derivatives of A (1a–c).
The mono- and dimethylated compounds (1b and 1c, respec-
tively) exhibited good in vitro HER2/EGFR kinase activity and BT-
474 cell growth inhibitory activity, although their activities were

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Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
Table 2
Evaluation of C4- and N5-substituted pyrrolo[3,2-d]pyrimidine derivatives
Me
O
O
R1
S
X
NH
O
HN
N
Cl
O
N
N
Compound
X
R1
Enzyme
IC50 (nM)
Cell growth
GI50 (nM)
Metabolic stability
L/min/mg)
Caco-2
(nm/sec)
Mouse PK
AUC0–8 h (lg h/mL)
In vivo efficacyb
T/C (%)
cLogD
FISAa
(
l
HER2
EGFR
BT-474
Human
Mouse
B–A/A–B
(10 mg/kg)
Mice
1a
1b
1c
2a
2b
2c
A
CH2
CF3
CF3
CF3
Cl
Cl
Cl
6.3
7.1
17
2.9
4.1
11
15
18
47
6.7
9.8
11
23
5.2
34
55
3.9
32
56
17
18
30
38
11
4
11
33
23
NT
ꢀ12
ꢀ3
28
82.9/5.2
NT
42.6/28.7
67.9/6.7
NT
0.947
0.882
2.289
0.936
1.547
3.506
1.923
80
NT
43
NT
NT
24
29
2.79
3.14
3.49
3.52
3.86
4.21
3.47
147
133
122
148
111
78
CH(Me)
C(Me)2
CH2
CH(Me)
C(Me)2
21
35
76.9/28.5
68.7/50.5
17
97
a
See Table 1 footnote.
b
Compounds suspended in 0.5% (w/v) methylcellulose solution, administered po b.i.d. (100 mg/kg) in BT-474 xenograft mice for 14 days.
Table 3
explained by the optimal combination of c LogD and FISA values.
As a reflection of the good PK profile of 2c, the compound showed
potent efficacy (T/C: 24%) in a BT-474 tumor xenograft model in
mice, efficacy that was comparable to that observed for A.
Physicochemical and pharmacokinetics properties of 2c
2c (FormA)
2c (FormB)
2ca (BsOH salt)
2cb (TsOH salt)
A
55
28
620
620
>1000
0.56
0.33
9.47
7.58
5.80
1.46
—
1.71
3.39
2.87
Next, we examined the polymorphism of 2c and identified mul-
tiple crystal forms (forms A–C): the most stable form was B,
whereas form A was used for PK evaluation in mouse and other
in vivo studies. We presumed that form B may yield a poor PK pro-
file due to its low solubility and solubility rate compared to form A
(Table 3). Therefore, to improve these physical properties, we initi-
ated a study on the conversion of the salt-free form of 2c into its
salt form. Although we examined a variety of acid salts, only two
salts of 2c were obtained in crystalline form: 2ca, which was
obtained using benzenesulfonic acid (BsOH), and 2cb, which was
obtained using p-toluenesulfonic acid (TsOH). The physicochemical
and PK properties of 2ca and 2cb are given in Table 3. As expected,
2ca and 2cb showed higher solubility and solubility rate than the
salt-free form B of 2c; the AUC value of the tosylate form (2cb),
in particular, was better than that of the salt-free form A of 2c in
rats. Since we did not observe the formation of polymorphic forms
for 2cb, we selected 2cb as the best salt form of inhibitor 2c.
The in vivo antitumor efficacy of 2cb in mouse and rat xenograft
models with transplanted 4-1ST gastric cancer cell lines is shown
a or b 2caðBsOH saltÞ
2c ƒƒƒ!
2cbðTsOH saltÞ
Reagents: (a) benzenesulfonic acid monohydrate, EtOAc/EtOH; (b) p-toluenesul-
fonic acid monohydrate, EtOAc/EtOH.
weaker than those of 1a, without a substituent at X. On the other
hand, in mouse models, the introduction of a dimethylmethylene
group (1c) at X was found to be the most effective for improving
the PK profile and the Caco-2 membrane A-B permeability (B–A/
A–B: 42.6/28.7). In addition, as anticipated, the 3-chloro anilino
derivatives (2) showed similar structure–activity relationships
(SAR) to the 3-trifluoromethyl derivatives (1). In particular, 2c
exhibited good in vitro activity (HER2/EGFR IC50: 11/11 nM, BT-
474 cell GI50: 56 nM) and improved A-B permeability across
Caco-2 membrane (B-A/A-B: 76.9/28.5). Furthermore, 2c showed
favorable PK properties (AUC: 3.506 lg h/mL), which can be
4-1ST xenograft models(rats)b
4-1ST xenograft models(mice)a
8000
1800
1600
1400
1200
1000
800
600
400
200
0
6000
4000
#
#
2000
0
14 16 18 20 22 24 26 28 30
10
15
20
25
30
Days after inoculation
Days after inoculation
Figure 3. Anti-tumor activity of 2cb in 4–1ST xenograft models. aValues are means S.D. of five mice. (d): Control, (s): 65.3 mg/kg and (
D): 130.6 mg/kg of 2cb twice daily
from 15 to 28 days after tumor inoculation. Anti-tumor activity of 2cb was evaluated by comparison of the T/C (%) values at 29 days after tumor inoculation. #: p 60.025
versus control (one-tailed Shirley–Williams test). bValues are means S.D. of eight rats. (d): Control, (s): 16.4 mg/kg and (
24 days after tumor inoculation. #: p 60.025 versus control (one-tailed Shirley–Williams test).
D): 32.8 mg/kg of 2cb twice daily from 11 to

Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
6175
Table 4
In vivo anti-tumor efficacy of 2cb and compound A in mouse and rat
Compound
In vivo anti-tumor efficacy in mouse 4–1ST xenograft model T/C (%)
In vivo anti-tumor efficacy in rat 4-1ST xenograft model T/C (%)
100 mg/kg, bid (as salt free)
50 mg/kg, bid (as salt free)
25 mg/kg, bid (as salt free)
12.5 mg/kg, bid (as salt free)
2cb
A
0
11
41
44
ꢀ1
41
14
ꢀ12
Table 5
Kinase inhibition profile of 2cb
an L-column 2 ODS (3.0 ꢁ 50 mm I.D., CERI, Japan) with a temper-
ature of 40 °C and a flow rate of 1.2 mL/min. Mobile phase A was
0.05% TFA in ultrapure water. Mobile phase B was 0.05% TFA in ace-
tonitrile which was increased linearly from 5% to 90% over 2 min,
90% over the next 1.5 min, after which the column was equili-
brated to 5% for 0.5 min. Column chromatography was carried
out on a silica gel column (Kieselgel 60, 63–200 mesh, Merck or
ChromatorexÒ NH-DM1020, 100–200 mesh, Fuji Silysia chemical).
Yields were not optimized.
Enzyme
IC50 (nM)
Enzyme
IC50 (nM)
HER2
EGFR
HER4
AuroraB
MEK1
LCK
11
11
160
500
980
1300
Lyn B
Lyn A
c-met
3300
3600
3900
5100
CSK
28 Other kinasesa
>10,000
a
Other kinases: VEGFR1, VEGFR2, PDGFRalpha, PDGFRbeta, InsR, TIE2, c-kit, IGF-
Commercial reagents and solvents were used without addi-
tional purification. Abbreviations are used as follows: CDCl3, deu-
terated chloroform; DMSO-d6, dimethyl sulfoxide-d6; AcOEt,
ethyl acetate; DMF, N,N-dimethylformamide; MeOH, methanol;
THF, tetrahydrofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide;
NMP, N-methylpyrrolidone.
1R, FAK, ASK1, MEK5, MEKK, TTK, TAK1, JNK1, IKKbeta, ERK1, P38alpha, PKA,
PKCtheta, PLK, GSK3beta, B-rafw, FGFR1, FGFR3, Src, ZAP70, BMX.
in Fig. 3. As a reflection of the good PK profile of 2cb, we observed
significant in vivo antitumor efficacy in both mouse and rat models
(mouse T/C: 0%, 2cb po b.i.d at 100 mg/kg as free salt form; rat T/C:
ꢀ1%, 2cb po b.i.d at 25 mg/kg as free salt form); these efficacies
were comparable to those obtained with A (Table 4).
We also investigated the kinase selectivity of 2cb against 30 dif-
ferent kinases. Except for EGFR/HER2/HER4 (IC50 = 11/11 nM/
155 nM, respectively), no other tyrosine and serine/threonine ki-
nases were significantly inhibited by 2cb. Thus, we demonstrated
that 2cb is a HER family-selective inhibitor and is as potent as A
(Table 5).
5.1. tert-Butyl{2-[4-({3-chloro-4-[3-(trifluoromethyl)phenoxy]
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-
yl]ethyl}carbamate (4)
A mixture of tert-butyl [2-(4-chloro-5H-pyrrolo[3,2-d]pyrimi-
din-5-yl)ethyl]carbamate9 (3, 0.71 g, 2.39 mmol) and 3-chloro-
4-[3-(trifluoromethyl)phenoxy]aniline9 (0.83 g, 2.79 mmol) in
2-propanol (7.1 mL) was stirred at 80 °C for 12 h. To the reaction
mixture was added saturated sodium hydrogen carbonate (50 mL)
under ice-cooling, and the mixture was extracted with ethyl
acetate (200 mL). The organic layer was washed with brine and
dried over anhydrous magnesium sulfate. The solvent was concen-
trated in vacuo, and the residue was purified by silica gel column
chromatography (AcOEt/hexane eluent, 1:1 to 1:0) to give 1.12 g
(85%) of 4 as white solid. 1H NMR (CDCl3) d: 1.49 (9H, s), 3.43–
3.54 (2H, m), 4.43–4.51 (2H, m), 5.10 (1H, t, J = 5.6 Hz), 6.60 (1H,
d, J = 3.3 Hz), 7.07 (1H, m), 7.09–7.14 (1H, m), 7.16–7.22 (2H, m),
7.25–7.30 (1H, m), 7.37–7.45 (1H, m), 7.89 (1H, dd, J = 8.7,
2.4 Hz), 8.02 (1H, d, J = 2.4 Hz), 8.50 (1H, s), 8.64 (1H, br s).
4. Conclusion
We performed chemical modification of 1a to obtain a com-
pound with both high HER2/EGFR inhibitory potency and a good
PK profile. Introduction of a dimethyl group into the N-5 linker
(X) of the pyrrolo[3,2-d]pyrimidine ligand was found to be the
most effective method to improve the A–B permeability and PK
profile with good cLogD and FISA values. We then converted 2c
into its tosylate salt (2cb) to enhance the solubility and PK profile.
The salt 2cb exhibited significant in vivo antitumor efficacy in both
mouse and rat models (mouse T/C: 0%, 2cb po b.i.d at 100 mg/kg;
rat T/C: -1%, 2cb po b.i.d at 25 mg/kg), with results comparable
to those obtained for A. Consequently, we selected 2cb as an alter-
native candidate for preclinical development.
The following compound 5 was prepared from 3 by a method
similar to that described for
4
using 3-chloro-4-(3-
chlorophenoxy)aniline.9
5.2. 2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)ethylcarbamate (5)
5. Experimental
Melting points were determined on a Yanagimoto micro melt-
ing point apparatus or SRS OptiMelt melting point apparatus, and
are uncorrected. Proton nuclear magnetic resonance (1H NMR)
Yield 87%, white solid. 1H NMR (CDCl3) d: 1.50 (9H, s), 3.40–3.60
(2H, m), 4.40–4.60 (2H, m), 5.00–5.10 (1H, m), 6.61 (1H, d,
J = 2.6 Hz), 6.85–7.05 (2H, m), 7.07 (2H, d, J = 8.8 Hz), 7.18 (1H, d,
J = 2.6 Hz), 7.20–7.30 (1H, m), 7.85–7.95 (1H, m), 8.00–8.05 (1H,
m), 8.52 (1H, s), 8.62 (1H, br s).
spectra were recorded on
a Varian Gemini-200 (200 MHz)
spectrometer or Varian Mercury-300 (300 MHz) spectrometer.
Chemical shifts are given in parts per million (ppm) with tetra-
methylsilane as an internal standard, and coupling constants (J val-
ues) are given in Hertz (Hz). Splitting patterns and apparent
multiplicities are designated as s (singlet), d (doublet), dd (double
doublet), t (triplet), dt (double triplet), q (quartet), m (multiplet), br
s (broad singlet). Elemental analyses were carried out by Takeda
Analytical Research Laboratories, Ltd., and the results obtained
were within 0.4% of the theoretical values. MS spectra were col-
lected with a Waters LC–MS system (ZMD-1) and were used to
confirm P95% purity of each compound. The column used was
5.3. 5-(2-Aminoethyl)-N-{3-chloro-4-[3-(trifluoromethyl)
phenoxy]phenyl}-5H-pyrrolo[3,2-d]pyrimidin-4-amine
dihydrochloride (6)
A mixture of 4 (1.12 g, 2.04 mmol), 2 N HCl (15 mL) and THF
(30 mL) was stirred at 60 °C for 20 h. After the mixture was con-
centrated in vacuo, EtOH (50 mL) was added to the concentrate,
and the mixture was further concentrated in vacuo. The residual
solid was collected by filtration and washed with AcOEt (30 mL)

6176
Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
to give 1.07 g (quant.) of 6 as pale-yellow solid. 1H-NMR (DMSO-
d6) d: 3.21–3.35 (2H, m), 4.92–5.02 (2H, m), 6.71–6.76 (1H, m),
7.24–7.32 (2H, m), 7.37 (1H, d, J = 9.0 Hz), 7.50–7.56 (1H, m),
7.64–7.71 (2H, m), 7.91–7.97 (1H, m), 7.98–8.06 (1H, m), 8.13–
8.26 (3H, m), 8.71 (1H, br s), 9.90 (1H, br s).
the residue in DMF (5.0 mL) were added sodium methanesulfinate
(85 mg, 0.833 mmol) and pyridine (67 L, 0.828 mmol) at room
l
temperature, and the mixture was stirred at 70 °C for 48 h. Water
(20 mL) was added to the reaction mixture and the mixture was
extracted with AcOEt (50 mL). The organic layer was washed with
saturated brine (20 mL) and dried over anhydrous magnesium sul-
fate. After concentration under reduced pressure, the residue was
purified by basic silica gel column chromatography (AcOEt/MeOH,
1:0 to 9:1) and recrystallized from AcOEt/diisopropyl ether
(10:2 mL) to give 114 mg (68%) of 1b as colorless crystals. mp
169–171 °C. 1H NMR (CDCl3) d: 1.71 (3H, d, J = 7.2 Hz), 2.98 (3H,
s), 3.63–3.75 (2H, m), 3.81 (1H, q, J = 7.2 Hz), 4.44–4.55 (2H, m),
6.64 (1H, d, J = 3.0 Hz), 7.09 (1H, d, J = 8.7 Hz), 7.11–7.18 (2H, m),
7.19–7.25 (2H, m), 7.30–7.36 (1H, m), 7.40–7.47 (1H, m), 7.85
(1H, dd, J = 8.7, 2.7 Hz), 8.01 (1H, d, J = 2.7 Hz), 8.30 (1H, s), 8.54
(1H, s). Anal. Calcd for C25H23ClF3N5O4S: C, 51.59; H, 3.98; N,
12.03. Found: C, 51.66; H, 3.98; N, 12.17.
The following compound 7 was prepared from 5 by a method
similar to that described for 6.
5.4. 5-(2-Aminoethyl)-N-[3-chloro-4-(3-chlorophenoxy)
phenyl]-5H-pyrrolo[3,2-d]pyrimidin-4-amine dihydrochloride
(7)
Yield 94%, pale-yellow solid. 1H NMR (95%CDCl3 + 5%DMSO-d6)
d: 3.30–3.60 (4H, m), 5.00–5.15 (2H, m), 6.71 (1H, d, J = 3.2 Hz),
6.90–7.00 (2H, m), 7.10–7.20 (1H, m), 7.22 (1H, d, J = 8.8 Hz),
7.30–7.45 (1H, m), 7.60–7.70 (1H, m), 7.87 (1H, d, J = 2.6 Hz),
8.05 (1H, d, J = 2.4 Hz), 8.20–8.40 (2H, m), 8.71 (1H, s).
The following compound 2b was prepared from 7 by a method
similar to that described for 1b.
5.5. N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-
(methylsulfonyl)acetamide (1a)
5.8. N-[2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]-2-(methylsulfonyl)
propanamide (2b)
A mixture of 6 (150 mg, 0.288 mmol), 2-(methylsulfonyl)acetic
acid (79.6 mg, 0.576 mmol), 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDC) (166 mg, 0.870 mmol), 1-
hydroxybenzotriazole monohydrate (HOBt) (133 mg, 0.870 mmol)
and triethylamine (0.40 mL) in N,N-dimethylformamide (DMF)
(5.0 mL) was stirred at room temperature for 20 h. Water (50 mL)
was added to the reaction mixture and the mixture was extracted
with AcOEt (100 mL). The organic layer was washed with water
(30 mL) and brine (30 mL), dried over anhydrous magnesium sul-
fate and concentrated in vacuo. The residue was purified by silica
gel column chromatography (MeOH/AcOEt eluent, 0:1 to 1:4) to
give 129 mg (79%) of 1a as colorless crystals. mp 177–178 °C. 1H
NMR (CDCl3) d: 3.12 (3H, s), 3.64–3.75 (2H, m), 3.98 (2H, s),
4.43–4.53 (2H, m), 6.62 (1H, d, J = 3.0 Hz), 7.07 (1H, d, J = 9.0 Hz),
7.09–7.15 (1H, m), 7.18–7.33 (4H, m), 7.40–7.45 (1H, m), 7.77
(1H, dd, J = 9.0, 2.7 Hz), 7.96 (1H, d, J = 2.7 Hz), 8.19 (1H, s), 8.51
(1H, s). Anal. Calcd for C24H21ClF3N5O4: C, 50.75; H, 3.73; N,
12.33. Found: C, 50.85; H, 3.71; N, 12.38.
Yield 43%, colorless crystals. Mp 169–171 °C. 1H NMR (CDCl3) d:
1.71 (3H, d, J = 7.2 Hz), 2.98 (3H, s), 3.65–3.75 (2H, m), 3.81 (1H, q,
J = 7.2 Hz), 4.45–4.55 (2H, m), 6.61 (1H, d, J = 3.3 Hz), 6.85–6.90
(1H, m), 6.90–6.95 (1H, m), 7.00–7.10 (2H, m), 7.20–7.30 (1H, m),
7.30–7.40 (1H, m), 7.75–7.85 (1H, m), 7.97 (1H, d, J = 2.4 Hz),
8.28 (1H, s), 8.51 (1H, s). Anal. Calcd for C24H23Cl2N5O4Sꢂ0.2H2O:
C, 52.22; H, 4.27; N, 12.69. Found: C, 52.06; H, 4.24; N, 12.72.
5.9. N-{2-[4-({3-Chloro-4-[3-
(trifluoromethyl)phenoxy]phenyl}amino)-5H-pyrrolo[3,2-
d]pyrimidin-5-yl]ethyl}-2-methyl-2-
(methylsulfonyl)propanamide (1c)
To a solution of 2-methyl-2-(methylsulfonyl)propanoic acid
(115 mg, 0.690 mmol) and DMF (20 lL) in THF (5.0 mL) was added
thionyl chloride (0.10 mL, 1.37 mmol) at room temperature. After
being stirred at room temperature for 3 h, the mixture was concen-
trated in vacuo. The residue was dissolved in THF (10 mL) and the
solution was added dropwise to a suspension of 6 (180 mg,
0.346 mmol) and triethylamine (0.48 mL, 3.44 mmol) in THF
(10 mL) at room temperature. After being stirred at room temper-
ature for 20 h, water (30 mL) was added to the reaction mixture.
The mixture was extracted with AcOEt (50 mL). The organic layer
was washed with saturated brine (30 mL), dried over anhydrous
magnesium sulfate and concentrated in vacuo. The residue was
purified by basic silica gel column chromatography (AcOEt/MeOH
eluent, 1: 0 to 9: 1) to give 205 mg (99%) of 1c as colorless crystals.
Mp 167–168 °C. 1H NMR (CDCl3) d: 1.70 (6H, s), 2.93 (3H, s), 3.63–
3.73 (2H, m), 4.43–4.52 (2H, m), 6.64 (1H, d, J = 3.3 Hz), 7.09 (1H, d,
J = 8.7 Hz), 7.10–7.16 (1H, m), 7.18–7.24 (2H, m), 7.27–7.35 (2H,
m), 7.40–7.47 (1H, m), 7.90 (1H, dd, J = 8.7, 2.7 Hz), 8.05 (1H, d,
J = 2.7 Hz), 8.38 (1H, s), 8.54 (1H, s). Anal. Calcd for
The following compound 2a was prepared from 7 by a method
similar to that described for 1a.
5.6. N-[2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]-2-
(methylsulfonyl)acetamide (2a)
Yield 69%, pale-yellow crystals. Mp 206–207 °C. 1H NMR
(CDCl3) d: 3.13 (3H, s), 3.60–3.80 (2H, m), 3.99 (2H, s), 4.40–4.60
(2H, m), 6.62 (1H, d, J = 3.4 Hz), 6.85–6.95 (2H, m), 7.00–7.10
(2H, m), 7.20–7.30 (2H, m), 7.70–7.80 (1H, m), 7.95–8.00 (1H, m),
8.19 (1H, s), 8.52 (1H, s). Anal. Calcd for C23H21Cl2N5O4S: C,
51.69; H, 3.96; N, 13.10. Found: C, 51.72; H, 3.92; N, 13.01.
5.7. N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-
(methylsulfonyl)propanamide (1b)
C26H25ClF3N5O4S: C, 52.39; H, 4.23; N, 11.75. Found: C, 52.33; H,
4.18; N, 11.85.
To
(0.39 mL, 2.80 mmol) and THF (5.0 mL) was added 2-chloropropio-
nyl chloride (54 L, 0.550 mmol) at room temperature. The mix-
ture was stirred at room temperature for 72 h, and then water
(20 mL) was added to the mixture at room temperature. The mix-
ture was extracted with AcOEt (50 mL). The organic layer was
washed with saturated brine (20 mL) and dried over anhydrous
magnesium sulfate. After concentration in vacuo, to a solution of
a mixture of 6 (150 mg, 0.288 mmol), triethylamine
5.10. N-[2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-
5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]-2-methyl-2-
(methylsulfonyl)propanamide (2c)
l
A mixture of 7 (974 mg, 2 mmol), 2-methyl-2-(methylsulfo-
nyl)propanoic acid (499 mg, 3 mmol), EDC (633 mg, 3.3 mmol),
HOBt (450 mg 3.33 mmol) and triethylamine (1.39 mL, 10 mmol)

Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
6177
in DMF (10 mL) was stirred at room temperature for 15 h. Water
5.12. N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-
(isopropylsulfonyl)acetamide (1e)
(100 mL) was added to the reaction mixture and the mixture was
extracted with AcOEt (200 mL). The organic layer was washed with
water (50 mL) and brine (50 mL), dried over anhydrous magnesium
sulfate and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (AcOEt/MeOH eluent, 1:0 to 20:1)
and recrystallized from AcOEt/EtOH-diisopropyl ether (30 mL/
6 mL/30 mL) to give 838 mg (74%) of 2c as colorless crystals. mp
178–180 °C. 1H NMR (CDCl3) d: 1.70 (6H, s), 2.93 (3H, s), 3.60–
3.80 (2H, m), 4.40–4.60 (2H, m), 6.46 (1H, d, J = 2.8 Hz), 6.85–
7.00 (2H, m), 7.00–7.15 (2H, m), 7.15–7.30 (2H, m), 7.30–7.40
(1H, m), 7.85–7.95 (1H, m), 8.00–8.05 (1H, m), 8.36 (1H, br s),
8.54 (1H, s). Anal. Calcd for C25H25Cl2N5O4S: C, 53.38; H, 4.48; N,
12.45. Found: C, 53.40; H, 4.54; N, 12.36.
Yield 48%, colorless crystals. Mp 188–191 °C. 1H NMR (DMSO-
d6) d: 1.24 (6H, d, J = 7.0 Hz), 3.41–3.59 (3H, m), 4.03 (2H, s),
4.48–4.61 (2H, m), 6.51 (1H, d, J = 3.0 Hz), 7.16–7.25 (2H, m),
7.31 (1H, d, J = 8.9 Hz), 7.47 (1H, d, J = 7.7 Hz), 7.57–7.69 (2H, m),
7.78 (1H, dd, J = 8.9, 2.5 Hz), 7.98 (1H, d, J = 2.5 Hz), 8.36 (1H, s),
8.72 (2H, s). Anal. Calcd for C26H25ClF3N5O3S: C, 52.39; H, 4.23;
N, 11.75. Found: C, 52.54; H, 4.20; N, 11.71.
5.13. 2-(tert-Butylsulfonyl)-N-{2-[4-({3-chloro-4-[3-
(trifluoromethyl)phenoxy]phenyl}amino)-5H-pyrrolo[3,2-
d]pyrimidin-5-yl]ethyl}acetamide (1f)
5.11. N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-
(ethylsulfonyl)acetamide (9)
Yield 42%, colorless crystals. Mp 172–173 °C. 1H NMR (DMSO-
d6) d: 1.30 (9H, s), 3.42–3.51 (2H, m), 3.96 (2H, s), 4.50–4.61 (2H,
m), 6.51 (1H, d, J = 3.0 Hz), 7.12–7.36 (3H, m), 7.45–7.55 (1H, m),
7.58–7.67 (2H, m), 7.74 (1H, dd, J = 8.9, 2.5 Hz), 8.00 (1H, d,
J = 2.5 Hz), 8.35 (1H, s), 8.53–8.63 (1H, m), 8.71 (1H, s). Anal. Calcd
for C27H27ClF3N5O4S: C, 53.16; H, 4.46; N, 11.48. Found: C, 53.16; H,
4.38; N, 11.42.
Under ice-cooling, to a solution of 6 (222 mg, 0.497 mmol) and
4-methylmorpholine (3.0 mL, 27.3 mmol) in THF (7.0 mL) was
added chloroacetyl chloride (0.7 mL, 8.78 mmol), and the mixture
was stirred at 0 °C for 5 h. To the reaction mixture was added sat-
urated aqueous sodium hydrogen carbonate (100 mL) under ice-
cooling, and the mixture was extracted with AcOEt (100 mL). The
extract was dried over anhydrous magnesium sulfate and concen-
trated in vacuo, and then the residue was dissolved in a mixed sol-
vent of DMF (3.5 mL) and THF (6.0 mL). To the mixture was added
sodium ethanethiolate (630 mg, 7.49 mmol), and the mixture was
stirred at room temperature for 2 h. To the reaction mixture was
added saturated aqueous sodium hydrogen carbonate (100 mL)
under ice-cooling and the mixture was extracted with AcOEt
(100 mL). The extract was dried over anhydrous magnesium sul-
fate and concentrated in vacuo, and the residue was purified by sil-
ica gel column chromatography (AcOEt/MeOH eluent, 100:0 to
90:10) to give 9 as oil. 9 was dissolved in dichloromethane
(7.0 mL), and to the solution were added m-chloroperbenzoic acid
(162 mg, 0.939 mmol) at room temperature. The mixture was stir-
red at room temperature for 1 h. Saturated aqueous sodium thio-
sulfate solution (100 mL) was added to the reaction mixture
under ice-cooling, and then the mixture was stirred at room tem-
perature for 1 h. The mixture was extracted with dichloromethane
(100 mL) and the extract was dried over anhydrous magnesium
sulfate and concentrated in vacuo. The residue was dissolved in
dichloromethane (5.0 mL), and to the solution were added tita-
nium tetraisopropoxide (0.9 mL, 3.06 mmol), MeOH (0.50 mL)
and 70% aqueous tert-butyl hydroperoxide solution (10 mL,
72.2 mmol), and the mixture was stirred at room temperature for
1 h. Saturated aqueous sodium thiosulfate solution (100 mL) was
added to the reaction mixture under ice-cooling, and the mixture
was stirred at room temperature for 1 h. The mixture was ex-
tracted with dichloromethane (100 mL) and the extract was dried
over anhydrous magnesium sulfate and concentrated in vacuo.
The residue was purified by silica gel column chromatography
AcOEt/MeOH eluent, 100:0 to 90:10) and crystallization from
diethyl ether–AcOEt–hexane to give 167 mg (58%) of 1d as color-
less crystals. Mp 196–198 °C. 1H NMR (DMSO-d6) d: 1.22 (3H, t,
J = 7.5 Hz), 3.25 (2H, q, J = 7.5 Hz), 3.40–3.51 (2H, m), 4.03 (2H, s),
4.48–4.63 (2H, m), 6.51 (1H, d, J = 3.0 Hz), 7.17–7.27 (2H, m),
7.31 (1H, d, J = 8.9 Hz), 7.47 (1H, d, J = 7.9 Hz), 7.58–7.68 (2H, m),
7.78 (1H, dd, J = 8.9, 2.5 Hz), 7.99 (1H, d, J = 2.5 Hz), 8.36 (1H, s),
8.72 (2H, s). Anal. Calcd for C25H23ClF3N5O4S: C, 51.59; H, 3.98;
N, 12.03. Found: C, 51.72; H, 3.99; N, 12.11.
5.14. N-[2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-
5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]-2-methyl-2-
(methylsulfonyl)propanamide benzenesulfonate monohydrate
(2ca)
Compound 2c (400 mg, 0.711 mmol) was dissolved in AcOEt
(12 mL) and EtOH (4 mL) by heating at 60 °C, and to the solution
was added benzenesulfonic acid monohydrate (132 mg,
0.747 mmol). The mixture was left at room temperature for 17 h
under light shielding, and then concentrated in vacuo. To the resi-
due was added AcOEt (10 mL), and the mixture was stood at room
temperature for 17 h under light shielding. The resulting crystals
were collected by filtration, washed with diisopropyl ether to give
447 mg (87%) of 2ca as colorless crystals. Mp 142–144 °C. 1H NMR
(DMSO-d6) d: 1.41 (6H, s), 2.93 (3H, s), 3.50–3.60 (2H, m), 4.65–
4.75 (2H, m), 6.65 (1H, d, J = 3.0 Hz), 6.95–7.00 (1H, m), 7.00–
7.05 (1H, m), 7.20–7.25 (1H, m), 7.25–7.35 (3H, m), 7.35 (1H, d,
J = 8.4 Hz), 7.45 (1H, t, J = 8.4 Hz), 7.55–7.65 (2H, m), 7.67 (1H,
dd, J = 8.7, 2.4 Hz), 7.88 (1H, d, J = 3.0 Hz), 7.93 (1H, d, J = 2.4 Hz),
8.20–8.25 (1H, m), 8.73 (1H, s), 9.74 (1H, br s). Anal. Calcd for
C
31H31Cl2N5O7S2ꢂ1.0H2O: C, 50.41; H, 4.50; N, 9.48. Found: C,
50.53; H, 4.43; N, 9.48.
5.15. N-[2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-
5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]-2-methyl-2-
(methylsulfonyl)propanamide p-toluenesulfonate (2cb)
Compound 2c (9.0 g, 16.0 mmol) was dissolve in ethyl acetate
(200 mL) and EtOH (70 mL) by heating at 65 °C, and to the solution
was added p-toluenesulfonic acid monohydrate (3.04 g,
16.0 mmol). The mixture was left at room temperature under light
shielding for 23 h and the resulting crystals were collected by fil-
tration, washed with AcOEt (30 mL) and diisopropyl ether
(30 mL) to give 11.5 g (98%) of 2cb as colorless crystals. mp 217–
218 °C. 1H NMR (DMSO-d6) d: 1.40 (6H, s), 2.28 (3H, s), 2.93 (3H,
s), 3.50–3.60 (2H, m), 4.65–4.75 (2H, m), 6.65 (1H, d, J = 3.0 Hz),
6.90–7.00 (1H, m), 7.00–7.05 (1H, m), 7.10 (2H, d, J = 7.8 Hz),
7.20–7.25 (1H, m), 7.35 (1H, d, J = 9.0 Hz), 7.40–7.50 (3H, m),
7.60–7.70 (1H, m), 7.89 (1H, d, J = 3.0 Hz), 7.91 (1H, d, J = 1.8 Hz),
8.15–8.25 (1H, m), 8.74 (1H, s), 9.80 (1H, br s). Anal. Calcd for
The following compounds (1e, 1f) were prepared from 6 and the
corresponding sodium thiolate by a method similar to that de-
scribed for 1d.
C32H33Cl2N5O7S2: C, 52.32; H, 4.53; N, 9.53. Found: C, 52.35; H,
4.54; N, 9.49.

6178
Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
5.16. HER2 and EGFR kinase assay
and allowed to develop to measurable size. On day 0 and day 7
after inoculation, each mouse was administered intramuscularly
with 0.25 mg estradiol dipropionate (ASKA Pharmaceutical Co.,
Ltd.). The tumor-bearing animals were inspected for physical
abnormalities and tumor growth two or three times per week,
and animals bearing tumor volume between 200 and 300 mm3
were used for the further study. At 21 days after the tumor cell
inoculation, selected mice were separated into 3 groups and com-
parisons between the volumes of each group were carried out
using the one-way analysis of ANOVA for values of P <0.05.
The cytoplasmic domain (amino acids 676–1255) of human
HER2 and the cytoplasmic domain (amino acids 669–1210) of hu-
man EGFR were expressed as N-terminal peptide (DYKDDDD)-
tagged protein using baculovirus expression system. The expressed
HER2 kinase and EGFR kinase were purified by using anti-FLAG M2
affinity gel (Sigma–Aldrich, USA).
The HER2 and EGFR kinase assays were performed using radio
labeled [c-
32P] ATP (GE Healthcare, USA) in 96 well plates. The ki-
nase reactions were performed in 50 mmol/L Tris–HCl, pH7.5,
5 mmol/L MnCl2, 0.01% Tween-20 and 2 mmol/L DTT containing
5.21. Inoculation of 4–1ST in nude mice or rats
0.9
Glu-Tyr (4:1) and 0.25
plasmic domain in a total volume of 50
l
Ci of [
c
-
32P] ATP per reaction, 50
l
mol/L ATP, 5
g/mL of the purified Her2 or EGFR cyto-
L. To measure the IC50 va-
lg/mL poly-
l
Anti-tumor studies were conducted according to the following
procedures. Human gastric cancer, 4-1ST tumor tissues were ex-
cised from donor nude mice or rats bearing tumors and minced
to small pieces. The tumor fragments were implanted subcutane-
ously and allowed to develop to measurable size. Mice or rats bear-
ing tumor volume between 200 and 300 mm3 were used for the
further study. At 16 days after tumor inoculation, selected mice
or rats were separated into 3 groups and comparisons between
the volumes of each group were carried out using the one-way
analysis of ANOVA for values of p <0.05.
l
lue for enzyme inhibition, the compounds were incubated with the
enzyme for 5 min prior to the reaction at room temperature. The
kinase reactions were initiated by adding ATP. After the kinase
reaction for 10 min at room temperature, the reactions were termi-
nated by the addition of 10% (final concentration) trichloroacetic
acid. The [
plate (Millipore, USA) with a Cell harvester (PerkinElmer, USA) and
washed free of [
32P] ATP with 3% phosphoric acid. The plates
were dried, followed by the addition of 25 L of MicroScint0 (Perk-
c-
32P]-phosphorylated proteins were filtrated in Harvest
c-
l
inElmer, USA). The radioactivity was counted by a Topcount scintil-
lation counter (PerkinElmer, USA). IC50 values and 95% confidence
intervals were calculated by nonlinear regression analysis.
5.22. Antitumor activity in vivo
Selected compounds suspended in a 0.5% (w/v) methylcellulose
solution were orally twice daily administered to the mice or rats at
the indicated dose (10 mL/kg) for 14 days. For control group, 0.5%
(w/v) solution was administered. Tumor volume was assessed in
a blind manner and measured at two or three times per week
throughout the dosing period using digital calipers. The volume
was calculated using the following formula.
5.17. Cell lines and culture
The cell lines BT-474 (HER2 over-expressing human breast can-
cer) were obtained from American Type Culture Collection. BT-474
cell was cultured in RPMI 1640, and mediums supplemented with
10% heat-inactivated fetal bovine serum (FBS).
Human gastric cancer, 4-1ST was obtained from Central Insti-
tutes for Experimental Animals (Kawasaki, Japan). These tumors
are maintained in vivo in mice.
2
Tumor volume ðmm3Þ ¼ ðaÞ ꢁ ðbÞ =2
(a) longer diameter and (b) shorter diameter.
Drug effects were expressed as (each growth volume of treated
tumor/average of growth volume of control tumor) ꢁ 100 (T/C (%)).
Anti-tumor activities of selected compounds were evaluated by
comparison of the T/C (%) values at Day 31, the day following the
last dosing day.
5.18. Cell growth assay
HER2 over-expressing human cancer cell (BT-474) was used for
the cell growth assays. BT-474 cells were seeded into 48-multiwell
plates (3 ꢁ 104 cells/well) and allowed to attach overnight. The
cells were treated continuously with compounds for 5 days and
then the live cell numbers were counted with a particle analyzer
(CDA-500; Sysmex Corporation).
5.23. Mouse cassette dosing study
The animals used in the study were female BALB/cAJcl mice (7-
weeks old; CLEA Japan, Inc.). Mixture of 5 test compounds were
suspended in 0.5 w/v% methylcellulose solution for oral adminis-
tration at a dose of 10 mg each/10 mL/kg. The concentrations of
compounds in the plasma were determined by LC/MS/MS.
5.19. Animals
Female BALB/c nu/nu mice, 5 weeks old, were obtained from
Clea Japan Inc. All animals were housed in rooms maintained at
24 1 °C with a 12-h light/12-h dark photo-cycle. Food (CE-2, Clea
Japan Inc.) and tetracyclin including water (5% w/v) was provided
ad libitum during the experiment period.
Female nude rats (F344/N Jcl-rnu), 5 weeks old, were obtained
from Clea Japan Inc. All animals were housed in rooms maintained
at 24 °C with a 12-h light/12-h dark photo-cycle. Food (CE-2, Clea
Japan Inc.) and water was provided ad libitum.
5.24. Metabolic stability assay
Human hepatic microsomes were purchased from Xenotech,
LLC (Lenexa, KS). An incubation mixture with a final volume of
0.1 mL consisted of microsomal protein in 50 mmol/L phosphate
buffer (pH 7.4) and 1 lmol/L test compound. The concentration
of hepatic microsomal protein was 0.2 mg/mL. An NADPH-generat-
ing system containing 50 mmol/L MgCl2, 50 mmol/L glucose-6-
phosphate, 5 mmol/L b-NADP+ and 15 unit/mL glucose-6-phos-
phate dehydrogenase was prepared and added to the incubation
mixture with a 10% volume of the reaction mixture. After the addi-
tion of the NADPH-generating system, the mixture was incubated
at 37 °C for 0 and 20 min. The reaction was terminated by the addi-
tion of acetonitrile equivalent to the volume of the reaction mix-
ture. All incubations were made in duplicate. The test compound
5.20. Inoculation of BT-474 cells in nude mice
Anti-tumor studies were conducted according to the following
procedures. BT-474 xenografts were initiated by subcutaneous
implantation of 1 ꢁ 107 cells, suspended in 100
lL of MATRIGEL
(Becton Dickinson, USA) solution, into the right flank of nude mice

Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
6179
in the reaction mixture was measured by HPLC. The Waters Alli-
ance HPLC system was equipped with a separation module, a
thermostatted column compartment, and a photodiode array
detector (Waters, Milford, MA). The column was a Capcell Pak
MG (75 ꢁ 4.6 mm I.D., Shiseido, Japan). The column temperature
and the flow-rate were 40 °C and 1.2 mL/min, respectively. The
mobile phase A was 0.01 mol/L ammonium acetate and the mobile
phase B was methanol. The time program for the gradient elution
was as follows: the concentration of mobile phase B was linearly
increased from 42% to 90% over a period of 8.5 min, 90% over a per-
iod of the next 4 min, after which the column was equilibrated to
42% for 5 min. For metabolic stability determinations, chromato-
grams were analyzed for parent compound disappearance from
the reaction mixtures.
FGFR3, VEGFR1(Flt1), PDGFR
a
, PDGFRb, TIE2, c-Met, c-kit, Src,
Lck, ZAP70, and InsR were purchased from Upstate (USA). FGFR1
was purchased from ProQinase (Germany). IGF-1R and CSK were
purchased from BIOMOL (USA). Lyn A and Lyn B were purchased
from Invitrogen (USA). The reaction conditions were optimized
for each kinase: VEGFR2 (19 ng/mL of enzyme, 10
10 min reaction, PY-100 conjugated acceptor beads [PY-100]);
VEGFR1 (20 ng/mL of enzyme, 0.5 mol/L ATP, 5 min reaction,
PT66 conjugated acceptor beads [PT-66]); FGFR1 (10 ng/mL of en-
zyme, 0.2 mol/L ATP, 10 min reaction, PY-100); FGFR3 (20 ng/mL
of enzyme, 20 mol/L ATP, 10 min reaction, PY-100); PDGFR
(50 ng/mL of enzyme, 10 mol/L ATP, 30 min reaction, PT66);
PDGFRb (50 ng/mL of enzyme, 20 mol/L ATP, 60 min reaction,
PT66); InsR (100 ng/mL of enzyme, 10 mol/L ATP, 60 min reac-
tion, PT66); TIE2 (20 ng/mL of enzyme, 2 mol/L ATP, 10 min reac-
tion, PT66); c-Met (1 ng/mL of enzyme, 2 mol/L ATP, 10 min
reaction, PT66); c-kit(10 ng/mL of enzyme, 20 mol/L ATP,
lmol/L ATP,
l
l
l
a
l
l
l
l
5.25. Permeability of test compounds across Caco-2 monolayers
l
l
Caco-2 monolayers were grown to confluence on collagen-
coated, microporous, polycarbonate membranes in 12-well Costar
TranswellÒ plates. The permeability assay buffer was Hanks’ Bal-
anced Salt Solution containing 10 mmol/L HEPES, 15 mmol/L glu-
cose, and 1% bovine serum albumin at a pH of 7.3–7.5. The test
20 min reaction, PT66); IGF-1R (10 ng/mL of enzyme, 10
ATP, 20 min reaction, PT66); Src (0.33 ng/mL of enzyme, 2
l
l
mol/L
mol/L
ATP, 10 min reaction, PY-100); Lck (100 ng/mL of enzyme,
mol/L ATP, 30 min reaction, PY-100); BMX (6.6 ng/mL of en-
zyme, 2 mol/L ATP, 10 min reaction, PY-100); ZAP70 (30 ng/mL
of enzyme, 2 mol/L ATP, 10 min reaction, PY-100); CSK(3.2 ng/
mL of enzyme, 2 mol/L ATP, 10 min reaction, PY-100); FAK
(62 ng/mL of enzyme, 2 mol/L ATP, 60 min reaction, PT66); Lyn
A (2 ng/mL of enzyme, 2 mol/L ATP, 10 min reaction, PY-100);
Lyn B (2.7 ng/mL of enzyme, 2 mol/L ATP, 10 min reaction, PY-
2
l
l
compounds dosing concentrations were 10
lmol/L in the assay
l
buffer. The cells were dosed on the apical side (A-to-B) or basolat-
eral side (B-to-A) and incubated at 37 °C with 5% CO2 in a humid-
l
l
ified chamber. At each time point, 1 and 2 h, 200
from the A-to-B receivers, and 50 L were taken from the B-to-A
receivers. Fresh assay buffer was added to the receivers after the
1-hour sampling. Also, at 2 h, 50 L of the donors were taken. Each
lL were taken
l
l
l
100). The tyrosine kinase reactions were performed in 50 mmol/L
Tris–HCl, pH 7.5, 5 mmol/L MnCl2, 5 mmol/L MgCl2, 0.01%
l
determination was performed in duplicate. The permeability
through a cell-free (blank) membrane was studied to determine
non-specific binding and free diffusion of the test compounds
through the device. The lucifer yellow flux was also measured for
each monolayer after being subjected to the test compounds to en-
sure no damage was inflicted to the cell monolayers during the flux
period. All samples were assayed by LC/MS/MS using electrospray
ionization. The apparent permeability, Papp, and percent recovery
were calculated as follows:
Tween-20 and 2 mmol/L DTT, 0.1
Tyr (4:1) containing optimized concentration of enzyme, ATP as
described above in a total volume of 25 L. To determine IC50 val-
ues, the remaining kinase activities at 7 concentrations (0.01, 0.1,
1, 10, 100, 1000, and 10000 nmol/L) of compound were measured.
Prior to the kinase reaction, test compound and enzyme were incu-
bated for 5 min at room temperature. The reactions were initiated
by adding ATP. After the reaction period as described above at
room temperature, reactions were stopped by the addition of
lg/mL biotinylated poly-Glu-
l
2t
l
L of 100 mmol/L EDTA, 10
lg/mL Alphascreen streptavidine do-
Papp ¼ ðdCr=dtÞ ꢁ Vr=ðA ꢁ C0Þ
ð1Þ
nor beads and 10
lg/mL acceptor beads described above in
62.5 mM HEPES, pH7.4, 250 mmol/L NaCl, and 0.1% BSA. The plates
were incubated in the dark for more than 12 h and read by an EnVi-
Percent Recovery ¼ 100 ꢁ ððVr ꢁ CfinalÞ þ ðVd ꢁ CfinalÞÞ=ðVd ꢁ C0Þ
r
d
ð2Þ
sion 2102 Multilabel Reader (PerkinElmer, USA) or a Fusion
a Plate
Reader (Packard, USA). Wells containing the substrate and the en-
zyme without the compound were used as total reaction control.
Wells containing the biotinylated poly-Glu-Tyr (4:1) and the en-
zyme without ATP were used as basal control.
where, dCr /dt is the slope of the cumulative concentration in the
receiver compartment versus time in
l
mol/L sꢀ1; Vr is the volume
of the receiver compartment in cm3; Vd is the volume of the donor
compartment in cm3; A is the area of the cell monolayer (1.1 cm2
Assays for 16 serine/threonine kinases were performed using
for 12-well TranswellÒ); C0 is the nominal concentration of the dos-
radiolabeled [
c-33P] ATP (GE Healthcare, USA) in 96-well plates.
final
ing solution in
tion in
l
mol/L; Cr
is the cumulative receiver concentra-
p38 , ERK1, TAK1, ASK1, PKCh, JNK1, MEK5, GSK3b, B-raf, PLK1,
a
final
l
mol/L at the end of the incubation period; Cd
is the
and TTK were expressed as N-terminal FLAG tagged protein using
a baculovirus expression system. IKKb and MEKK1 were expressed
as C-terminal FLAG tagged protein using a baculovirus expression
system. Aurora-B was expressed as N-terminal 6ꢁ His tagged pro-
tein using a baculovirus expression system. MEK1 was expressed
as N-terminal GST fusion protein using freestyle293 (Invitrogen,
USA) expression. PKA was expressed using E.coli expression. The
concentration of the donor in
period.
lmol/L at the end of the incubation
5.26. Kinase selectivity assay
The HER4 kinase assay was performed in the same method as
the HER2 and EGFR kinase assays described above using
reaction conditions were optimized for each kinase: p38
(100 ng/well of enzyme, 1 g/well of MBP (Wako, Japan), 0.1 Ci/
well of [ -33P] ATP, 60 min reaction at 30 °C); ERK1 (100 ng/well
of enzyme, 2 g/well of MBP, 0.1 Ci/well of [ -33P] ATP, 60 min
reaction at 30 °C); MEKK1 (25 ng/well of enzyme, 1 g/well of
MBP, 0.1 Ci/well of [ -33P] ATP, 60 min reaction at 30 °C); TAK1
(30 ng/well of enzyme, 1 g/well of MBP, 0.1 Ci/well of [ -33P]
ATP, 60 min reaction at 30 °C); ASK1 (30 ng/well of enzyme,
g/well of MBP, 0.1 Ci/well of [ -33P] ATP, 60 min reaction at
a
0.125
lg/mL of HER4 cytoplasmic domain purchased from Upstate
l
l
(USA).
c
Assays for the other 17 tyrosine kinases were performed using
the AlphascreenÒ system (Perkin Elmer, USA) in 384-well plates.
The cytoplasmic domains of VEGFR2 were expressed as N-terminal
FLAG-tagged proteins using a baculovirus expression system. The
full-length proteins of FAK and BMX were expressed as N-terminal
FLAG-tagged proteins using a baculovirus expression system.
l
l
c
l
l
c
l
l
c
1
l
l
c

6180
Y. Kawakita et al. / Bioorg. Med. Chem. 20 (2012) 6171–6180
2. (a) Berchuck, A.; Kamel, A.; Whitaker, R.; Kerns, B.; Olt, G.; Kinney, R.; Soper, J.
T.; Dodge, R.; Clarke-Pearson, D. L.; Marks, P.; McKenzie, S.; Yin, S.; Bast, R. C., Jr.
Cancer Res. 1990, 50, 4087; (b) Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W.
J.; Ullrich, A.; McGuire, W. L. Science 1987, 235, 177; (c) Gschwind, A.; Fischer,
O. M.; Ullrich, A. Nat. Rev. Cancer 2004, 4, 361; (d) Reid, A.; Vidal, L.; Shaw, H.;
de Bono, J. Eur. J. Cancer 2007, 43, 481.
3. Hynes, N. E.; Stern, D. F. Biochim. Biophys. Acta 1994, 1198, 165.
4. (a) Woodburn, J. R.; Barker, A. J.; Gilson, K. H.; Ashton, S. E.; Wakeling, A. E.;
Curry, B. J.; Scarlett, L.; Henthom, L. R. Proc. Am. Assoc. Cancer Res. 1997, 38, 633;
(b) Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.; Barlow, J. J.; Healy, M.
P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Scarlett, L.; Henthorn, L.; Richards,
L. Bioorg. Med. Chem. Lett. 1911, 2001, 11; (c) Wakeling, A. E.; Guy, S. P.;
Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Barker, A. J.; Gilson, K. H. Cancer Res.
2002, 62, 5749; (d) Matar, P.; Rojo, F.; Cassia, R.; Moreno-Bueno, G.; Cosimo, S.
D.; Tabernero, J.; Guzman, M.; Rodriguez, S.; Arribas, J.; Palacios, J.; Baselga, J.
Clin. Cancer Res. 2004, 10, 6487.
5. (a) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. J. Biol. Chem. 2002, 277, 46265;
(b) Sorbera, L. A.; Castañer, J.; Silvestre, J. S.; Bayes, M. Drugs Future 2002, 27,
923; (c) Dai, Q.; Ling, Y.-H.; Lie, M.; Zou, Y.-Y.; Kroog, G.; Iwata, K. K.; Perez-
Soler, R. Clin. Cancer Res. 2005, 11, 1572; (d) Melosky, B.; Agulnik, J.; Assi, H.
Curr. Oncol. 2008, 6, 279.
6. (a) Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.; Alligood, K. J.; Rhodes, N.;
Keith, B. R.; Murray, D. M.; Glennon, K.; Knight, W. B.; Mullin, R. J.; Gilmer, T. M.
Mol. Cancer Ther. 2001, 1, 85; (b) Wood, E. R.; Truesdale, A. T.; McDonald, O. B.;
Yuan, D.; Hassell, A.; Dickerson, S. H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.;
Alligood, K. J.; Rusnak, D. W.; Gilmer, T. M.; Shewchuk, L. Cancer Res. 2004, 64,
6652; (c) Petrov, K. G.; Zhang, Y. M.; Carter, M.; Cockerill, G. S.; Dickerson, S.;
Gauthier, C. A.; Guo, Y.; Mook, R. A., Jr.; Rusnak, D. W.; Walker, A. L.; Wood, E.
R.; Lackey, K. E. Bioorg. Med. Chem. Lett. 2006, 16, 4686.
30 °C); PKCh (25 ng/well of enzyme, 2 lg/well of MBP, 0.1 lCi/well
of [
zyme, 1
tion at 30 °C); MEK5 (3 ng/well of enzyme, 1
ERK5(K83 M), 0.3 Ci/well of [ -33P] ATP, 30 min reaction at
30 °C); GSK3b (100 ng/well of enzyme, 0.2 g/well of pGS peptide,
0.1 Ci/well of [ -33P] ATP, 30 min reaction at room temperature);
IKKb (20 ng/well of enzyme, 1 g/well of I , 0.1 Ci/well of [
33P] ATP, reaction at room temperature); B-raf (25 ng/well of en-
zyme, 1 g/well of GST-MEK1(K96R), 0.1 Ci/well of [ -33P] ATP,
20 min reaction at room temperature); MEK1 (100 ng/well of en-
zyme, 0.3 g/well of GST-ERK1(K71A) 0.2 Ci/well of [ -33P]
ATP, 20 min reaction at room temperature); Aurora-B (50 ng/well
of enzyme, 30 mol/L of Aurora substrate peptide, 0.2 Ci/well of
-33P] ATP, 60 min reaction at room temperature); PLK1 (80 ng/
well of enzyme, 3 g/well of -casein (usb, USA), 0.2 Ci/well of
-33P] ATP, 40 min reaction at room temperature); TTK (120 ng/
well of enzyme, 0.3 g/well of GST-MOBK1B, 0.2 Ci/well of [
33P] ATP, 10 min reaction at room temperature); PKA (3 nmol/L
of enzyme, 1 mol/L of PKA substrate peptide (Upstate, USA),
0.2 Ci/well of [ -33P] ATP, 10 min reaction at room temperature).
c
-33P] ATP, 60 min reaction at 30 °C); JNK1 (10 ng/well of en-
g/well of c-Jun, 0.1 Ci/well of [ -33P] ATP, 60 min reac-
g/well of GST-
l
l
c
l
l
c
l
l
c
l
jBa
l
c-
l
l
c
l
l
c
l
l
[c
l
a
l
[c
l
l
c-
l
l
c
Except for the PKCh reaction, the serine/threonine kinase reactions
7. (a) Iqbal, S.; Goldman, B.; Fenoglio-Preiser, C. M.; Lenz, H. J.; Zhang, W.;
Danenberg, K. D.; Shibata, S. I.; Blanke, C. D. Ann. Oncol 2011, Epub ahead of
print.; (b) Whang, Y. E.; Armstrong, A. J.; Rathmell, W. K.; Godley, P. A.; Kim, W.
Y.; Pruthi, R. S.; Wallen, E. M.; Crane, J. M.; Moore, D. T.; Grigson, G.; Morris, K.;
Watkins, C. P.; George, D. J. Urol. Oncol. 2011, Epub ahead of print.; (c) Ross, H.
J., ; Blumenschein, G. R., Jr.; Aisner, J.; Damjanov, N.; Dowlati, A.; Garst, J.; Rigas,
J. R.; Smylie, M.; Hassani, H.; Allen, K. E.; Leopold, L.; Zaks, T. Z.; Shepherd, F. A.
Clin. Cancer Res. 1938, 2010, 16; (d) Ooi, A.; Takehana, T.; Li, X.; Suzuki, S.;
Kunitomo, K.; Iino, H.; Fujii, H.; Takeda, Y.; Dobashi, Y. Mod. Pathol. 2004, 17,
895–904.
8. (a) Eskens, F. A. L. M.; Mom, C. H.; Planting, A. S. T.; Gietema, J. A.; Amelsberg,
A.; Huisman, H.; van Doorn, L.; Burger, H.; Stopfer, P.; Verweij, J.; de Vries, E. G.
Br. J. Cancer 2008, 98, 80–85; (b) Bhattacharya, S. K.; Cox, E. D.; Kath, J. C.;
Mathiowetz, A. M.; Morris, J.; Moyer, J. D.; Pustilnik, L. R.; Rafidi, K.; Richter, D.
T.; Su, C.; Wessel, M. D. Biochem. Biophys. Res. Commun. 2003, 307, 267; (c) Jani,
J. P.; Finn, R. S.; Campbell, M.; Coleman, K. G.; Connell, R. D.; Currier, N.;
Emerson, E. O.; Floyd, E.; Harriman, S.; Kath, J. C.; Morris, J.; Moyer, J. D.;
Pustilnik, L. R.; Rafidi, K.; Ralston, S.; Rossi, A. M.; Steyn, S. J.; Wagner, L.;
Winter, S. M.; Bhattacharya, S. K. Cancer Res. 2007, 67, 9887; (d) Ripin, D. H. B.;
Bourassa, D. E.; Brandt, T.; Castaldi, M. J.; Frost, H. N.; Hawkins, J.; Johnson, P. J.;
Massett, S. S.; Neumann, K.; Phillips, J.; Raggon, J. W.; Rose, P. R.; Rutherford, J.
L.; Sitter, B.; Stewart, A. M., III; Vetelino, M. G.; Wei, L. Org. Process Res. Dev.
2005, 9, 440; (e) Lippa, B.; Kauffman, G. S.; Arcari, J.; Kwan, T.; Chen, J.;
Hungerford, W.; Bhattacharya, S.; Zhao, X.; Williams, C.; Xiao, J.; Pustilnik, L.;
Su, C.; Moyer, J. D.; Ma, L.; Campbell, M.; Steyn, S. Bioorg. Med. Chem. Lett. 2007,
17, 3081.
were performed in 25 mmol/L HEPES, pH 7.5, 10 mmol/L magne-
sium acetate, 1 mmol/L DTT and 0.5
mized concentration of enzyme, substrate and radiolabeled ATP
as described above in a total volume of 50 L. For the PKCh reac-
lmol/L ATP containing opti-
l
tion, enzyme reactions were performed in 25 mmol/L HEPES, pH
7.5, 10 mmol/L magnesium acetate, 1 mmol/L DTT, lipid activator
(Upstate, USA) and 0.5
tration of enzyme, substrate and radiolabeled ATP as described
above in a total volume of 50 L. To determine IC50 values, the
lmol/L ATP containing optimized concen-
l
remaining kinase activities at 5 concentrations (1, 10, 100, 1000,
and 10000 nmol/L) of compound were measured. Prior to the ki-
nase reaction, compound and enzyme were incubated for 5 min
at reaction temperature. The kinase reactions were initiated by
adding ATP. After the reaction period as described above, the reac-
tions were terminated by the addition of 10% (final concentration)
trichloroacetic acid. The [
filtrated in Harvest Plate (Millipore, USA) with a Cell Harvester
(PerkinElmer, USA) and then free of [ -33P] ATP was washed out
with 3% phosphoric acid. The plates were dried, followed by the
addition of 40 L of MicroScintO (PerkinElmer, USA). Radioactivity
c-33P]-phosphorylated proteins were
c
l
9. (a) Ishikawa, T.; Seto, M.; Banno, H.; Kawakita, Y.; Oorui, M.; Taniguchi, T.;
Ohta, Y.; Tamura, T.; Nakayama, A.; Miki, H.; Kamiguchi, H.; Tanaka, T.; Habuka,
N.; Sogabe, S.; Aertgeerts, K.; Kamiyama, K. J. Med. Chem. 2011, 54, 8030; (b)
Kawakita, Y.; Banno, H.; Ohashi, T.; Tamura, T.; Yusa, T.; Nakayama, A.; Miki, H.;
Iwata, H.; Kamiguchi, H.; Tanaka, T.; Habuka, N.; Sogabe, S.; Ohta, Y.; Ishikawa,
T. J. Med. Chem. 2012, 55, 3975; (c) Doi, T.; Takiuchi, H.; Ohtsu, A.; Fuse, N.;
Goto, M.; Yoshida, M.; Dote, N.; Kuze, Y.; Jinno, F.; Fujimoto, M.; Takubo, T.;
Nakayama, N.; Tsutsumi, R. Br. J. Cancer 2012, 106, 1.
was counted by a TopCount scintillation counter (PerkinElmer,
USA). Wells containing the substrate and the enzyme without the
compound were used as total reaction control. Wells containing
the substrate and radiolabeled ATP without the enzyme were used
as basal control. IC50 values were calculated by nonlinear regres-
sion analysis.
10. Sasse, K.; Fischer, R.; Santel, H.-J.; Schmidt, R. R. PCT Int. US 4898608 (A), Feb.
1990.
11. (a) Pitchen, P.; Kagan, H. B. Tetrahedron Lett. 1984, 25, 1049; (b) Donnoli, M. I.;
Superchi, S.; Rosini, C. J. Org. Chem. 1998, 63, 9392; (c) Komatsu, N.;
Nishibayashi, Y.; Sugita, T.; Uemura, S. Tetrahedron Lett. 1992, 33, 5391.
12. Raub, J. T. Mol. Pharm. 2005, 3, 3.
References and notes
1. (a) Folkman, J. New Engl. J. Med. 1971, 285, 1182; (b) Yamamoto, T.; Nishida, T.;
Miyajima, N.; Kawai, S.; Ooi, T.; Toyoshima, K. Cell 1983, 35, 71; (c) Traxler, P.
Expert Opin. Ther. Targets 2003, 7, 215; (d) Nagar, B.; Bornmann, W. G.;
Pellicena, P.; Schindler, T.; Veach, D. R.; Miller, W. T.; Clarkson, B.; Kuriyan, J.
Cancer Res. 2002, 62, 4236.
13. Wang, D.-F.; Wiest, O.; Helquist, P.; Lan-Hargest, H.-Y.; Wiech, L. N. Bioorg. Med.
Chem. Lett. 2004, 14, 707.