Discovery of novel 5-alkynyl-4-anilinopyrimidines as potent, orally active dual inhibitors of EGFR and Her-2 tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2011.10.103

5-Alkenyl or 5-alkynyl-4-anilinopyrimidines were prepared and evaluated for in vitro inhibition of EGFR/Her-2 kinase activity and the growth of tumor cell lines (BT474 and N87). Several of these compounds inhibited the growth of BT474 and N87 at concentra

Full text of DOI:10.1016/j.bmcl.2011.10.103

Bioorganic & Medicinal Chemistry Letters 22 (2012) 456–460
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 5-alkynyl-4-anilinopyrimidines as potent, orally active
dual inhibitors of EGFR and Her-2 tyrosine kinases
⇑
Naoyuki Suzuki , Takeshi Shiota, Fumihiko Watanabe, Norihiro Haga, Takami Murashi, Takafumi Ohara,
Kenji Matsuo, Naoki Omori, Hiroshi Yari, Keiji Dohi, Makiko Inoue, Motofumi Iguchi, Jyunko Sentou,
Tooru Wada
Shionogi Medicinal Research Laboratories, Shionogi & Co., Ltd, 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Alkenyl or 5-alkynyl-4-anilinopyrimidines were prepared and evaluated for in vitro inhibition of EGFR/
Her-2 kinase activity and the growth of tumor cell lines (BT474 and N87). Several of these compounds
inhibited the growth of BT474 and N87 at concentrations below 200 nM. Structure–activity relationship
studies revealed a critical role for the 5-alkynyl moieties. The representative compound 19 exhibited sig-
nificant antitumor potency in a mouse xenograft model.
Received 21 June 2011
Revised 5 October 2011
Accepted 29 October 2011
Available online 6 November 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Kinase inhibitor
EGFR
Her-2
BT474
N87
Pyrimidine
Antitumor efficacy
Epidermal growth factor receptor (EGFR/ErbB-1) and Her-
2(ErbB-2), members of the Type 1 receptor tyrosine kinase family,
play key roles in cell proliferation and differentiation,1 and have
been clinically validated as rational targets for cancer therapy by
small molecule EGFR inhibitors (erlotinib 12 and gefitinib 23)
(Fig. 1) and Her-2 specific monoclonal antibody (trastuzumab).4
The ErbB receptors can be activated through homo or heterodimer-
ization with other receptors resulting in phosphorylation events
and downstream signaling that produce excessive growth by
inducing cell proliferation and inhibiting apoptotic pathways.1 In
addition, coexpression of EGFR and Her-2 has been found in vari-
ous cancers such as breast, ovarian, colon and prostate cancers,
and is associated with poor prognosis of the patients.5 Therefore,
simultaneous inhibition of EGFR and Her-2 is expected to provide
superior efficacy to single receptor targeting. Lapatinib ditosylate
(Tykerb 3), an oral dual EGFR/Her-2 inhibitor, was recently ap-
proved for treatment of patients with Her-2 over-expressing ad-
vanced or metastatic breast cancer.6 This drug has effective and
highly selective EGFR/Her-2 dual inhibitory activity and shows
good therapeutic efficacy in clinical practice.7
In our effort to develop novel ATP-competitive dual EGFR/Her-2
inhibitors for cancer therapeutics, we have previously reported 5-
alkenyl or 5-alkynyl-4-anilinopyrimidine derivatives to be potent
inhibitors of EGFR and Her-2 (Fig. 2).8 As the 5-alkenyl and alkynyl
substituents can be coplanar with the pyrimidine ring, these scaf-
folds should be able to effectively mimic the well-known quinazo-
line kinase template. Subsequently, we demonstrated that the
introduction of a phenyl ring into the C-5 substituent improved
the inhibitory activity against the Her-2 dependent cell line
(BT474), although their potencies were modest. Figure 3 shows
the molecular model of compounds 5 and 7 overlaid with the crys-
tal structure of Lapatinib in EGFR (PDB code 1XKK).9 Both the phe-
nyl rings and amino groups in the 5-substitutents overlap with the
furan and amino groups of Lapatinib.
In this Letter, we report further characterization of the benzyl-
amine part in these two classes of dual EGFR/Her-2 inhibitors,
leading to identification of a novel 5-alkynyl-4-anilinopyrimidine
derivative 19, a potent inhibitor of tumor cell growth that exhib-
ited significant in vivo antitumor activity.
The synthetic routes to 5-alkynyl and 5-alkenylpyrimidine ana-
logues are shown in Scheme 1. The reaction of 5-iodo-4-chloro-6-
methylpyrimidine (8)8 with the 4-benzyloxy aniline (9)10 provided
the 4-anilino-5-iodopyrimidine analogue 10. Sonogashira coupling
reaction of 10 with alkynes gave 5-alkynyl analogues (11). On the
⇑
Corresponding author. Tel.: +81 6 6458 5861; fax: +81 6 6458 0987.
E-mail address: naoyuki.suzuki@shionogi.co.jp (N. Suzuki).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.103

N. Suzuki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 456–460
457
F
O
F
O
HN
N
Cl
HN
N
Cl
HN
N
H
N
O
H3C
N
O
O
N
S
N
H3CO
H3CO
N
O
O
O
2TsOH
H3CO
1
2
3
Erlotinib (Tarceva)
Gefitinib (Iressa)
0.001 / 0.24
Lapatinib ditosylate (Tykerb)
0.01 /0.009
IC50[EGFR / Her-2 (µM)]
0.001 / 0.76
Figure 1. Examples of EGFR and Her-2 inhibitors in clinical use.
O
N
N
R =
N
N
H
O
F
F
4
6
0.009/0.036/1.13
0.003/0.019/1.13
HN
Cl
R
N
N
O
N
H3C
N
O
IC50 (µM) EGFR/Her-2/BT474
7
5
0.12/0.19/0.64
0.24/0.44/0.39
Figure 2. 5-Alkynylpyrimidine and 5-alkenylpyrimidine based dual EGFR/Her-2 inhibitors.
other hand, Suzuki–Miyaura coupling reaction of 10 with phenyl-
vinylborate yielded compound 12. Conversion of the terminal
methyl ester into aldehyde, followed by reductive amination with
various alkyl amines gave 5-alkenyl analogues 13.
The structure–activity relationships (SARs) of 5-alkynyl pyrim-
idine derivatives are shown in Table 1. Compounds were evaluated
for their inhibitory activities toward tyrosine phosphorylation of
the EGFR and Her-2 kinases by homogeneous time-resolved fluo-
rescence (HTRF) assay.11 IC50 values were calculated using the
XLFit (IDBS, Guildford, United Kingdom) curve-fitting tool from
Microsoft Excel. All molecules were also tested by cell proliferation
assay using the Her-2 overexpressed cell lines BT474 (breast tu-
mor) and N87 (gastric tumor).6
Unfunctionalized phenylacetylene analogue 14 showed de-
creased potency in both the enzyme and the cellular assays com-
pared with compound 5. The mesylethylamime analogue 15 was
a tenfold more potent EGFR inhibitor than 5. However, it did not
Figure 3. Overlay of Lapatinib with compounds 5 and 7. Lapatinib is colored pink.
Compound 5 is shown in orange and compound 7 in green. Atoms are colored by
element with nitrogen in blue, oxygen red, fluorine gray, chlorine dark green and
sulfur yellow.
O
O
Cl
F
F
a
b
I
O
N
+
F
HN
N
Cl
R
HN
N
Cl
I
H3C
N
H2N
Cl
N
N
H3C
H3C
11
8
9
10
a
O
O
O
F
F
MeO
HN
N
Cl
R
HN
N
Cl
c
d, e, f
10
N
N
H3C
H3C
12
13
Scheme 1. Reagents and conditions: (a) EtOH, reflux, 54%. (b) Alkyne (1.5 equiv), PdCl2(PPh3)2 (5 mol %), CuI (10 mol %), Et3N (5 equiv), DMF, 80 °C, 50–90%. (c)
Phenylvinylbronic acid pinacol ester (1.2 equiv), Pd(OAc)2 (10 mol %), PPh3 (30 mol %), 2 M K2CO3 (4 equiv), DMF, 100 °C, 65%. (d) LiAlH4 (1.5 equiv), THF, rt, 77%. (e) MnO2
(10 equiv), THF, rt, 77%. (f) Amine (4 equiv) NaBH(OAc)3 (4 equiv), AcOH (1 equiv), rt to 50 °C, 70–82%.

458
N. Suzuki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 456–460
Table 1
3
Structure–activity relationships for the alkynylpyrimidines
R
2.5
HN
N
O
F
H3C
Cl
N
2
Compound
R
EGFR
IC50
Her-2
IC50
BT474
IC50
N87
IC50
2.5
(l
M)a
(
l
M)a
(l
M)b
(l
M)b
14
15
0.65
0.75
0.22
0.52
0.33
2.09
0.42
SO2CH3
100
200 300 400 500 600 700 800
0.022
N
H
BT474_IC50
CH3
Figure 4. Correlation between SlogP values of the benzylamine part and inhibitory
activities against BT474. Blue: phenyl alkyne analogues, red: heterocyclic
analogues.
16
0.003
0.018
0.17
0.24
H3C
N
H
N
17
18
0.009
0.007
0.041
0.041
0.08
0.08
0.10
0.10
H3C
N
CH3
Table 2
N
Structure–activity relationships for the alkenyl pyrimidines
19
20
0.19
0.062
0.14
0.05
0.81
0.06
1.36
F
R
O
S
S
N
0.079
HN
N
O
F
H3C
Cl
N
N
21
0.030
0.18
0.41
0.69
N
N
22
23
0.074
0.055
0.11
0.45
0.84
0.44
1.14
Compound
R
EGFR
IC50
Her-2
IC50
BT474
IC50
M)b
N87
IC50
CH3
N
(
l
M)a
(
l
M)a
(
l
(l
M)b
0.073
H3C
SO2CH3
CH3
25
26
0.094
0.024
0.12
0.64
0.58
0.78
0.49
H3C
N
H
24
0.086
0.01
0.13
1.83
0.10
2.72
0.09
N
H
O
Lapatinib
0.009
0.030
N
a
H
n = 3.
n = 1.
b
27
0.098
0.01
0.086
0.009
1.07
0.10
1.21
0.09
N
H
Lapatinib
a
n = 3.
n = 1.
exhibit enhancement of inhibitory activities against Her-2 and the
tumor cell lines. Introduction of an alkylamine such as isopropyl-
amine (16), pyrrolidine (17) and dimethylamine (18) at the termi-
nal position led to remarkable improvement in the enzyme and the
cellular potencies, which were comparable to Lapatinib. Moreover,
the incorporation of a meta-fluoro substituent onto the phenyl ring
has been attributed to an increase in potency in the BT474 and N87
assay (19). However, the heterocyclic analogues (20–22), which
were expected to be more effective mimetics of Lapatinib, exhib-
ited decreased potencies against the enzymes and the tumor cell
lines compared with analogue 17. The SARs of these analogues sug-
gested that the inhibitory activities against BT474 and N87 were
improved by an increase in lipophilicity of the benzylamine part.
Figure 4 displayed the relationship between inhibitory potency
against BT474 and the SlogP12 value of the benzylamine part
(the compounds synthesized in our previous work were included
as data8). The analogues whose SlogP values were equal to or more
b
than 2.5 tended to exhibit effective antitumor activity (IC50
<200 nM). On the other hand, most compounds which showed less
effective BT474 potency had less lipophilic benzylamine parts
(SlogP values <2.5).
In terms of the position of the terminal amine group, the meta-
dimethyaminomethyl analogue 23 showed decreased potency in
the enzyme and cellular assays compared with analogue 18, and
the phenoxypropynyl analogue 24 exhibited poorer inhibitory
activities than analogue 16. Since the terminal amine moieties
were speculated to interact with Asp 776 of EGFR and Asp 808 of
Her-2, the positions of the amino groups might play a key role in
the inhibitory potency.

N. Suzuki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 456–460
459
Table 3
are caused by the difference of the membrane permeability or the
difference of protein conformation from the cell free protein. These
results discouraged us from further investigation of the 5-alkenyl-
pyrimidine series.
As a representative pyrimidine-based dual EGFR/Her-2 inhibi-
tor, further characterization of 19 was conducted. Compound 19
was screened against a small panel of tyrosine and serine/threo-
nine kinases. As shown in Table 3, 19 was highly selective for
Her family kinases as demonstrated by its minimal activity against
VEGFR-2, PDGFRb, Aurora-A, InsR and Lck.
Kinase selectivity profile for compound 19
Kinase
IC50 (lM)
EGFR
Her-2
VEGFR2
PDGFRb
AurA
0.19
0.11
>100
>100
>100
>100
>100
InsR
Lck
The solubility of analogue 19 was measured in pH 7.4 aq. buffer
solution (Table 4). Compound 19 had better aqueous solubility
than Lapatinib (24 lM @ in-house), suggesting that the pyrimidine
scaffold might contribute to better aqueous solubility. Compound
19 was also evaluated for stability in human and rat liver micro-
somes (Table 4); it was poor in rat liver microsomes but relativity
good in human liver microsomes. Evaluation of the pharmacoki-
netic properties of 19 in rat (Table 4) showed acceptable oral bio-
availability (35%). The limited bioavailability could be explained by
the low stability in rat liver.
Table 4
Physicochemical and pharmacokinetic properties of compound 19
Parameter
Solubility (
HLM (%)b
RLM (%)b
AUC (PO) (ng/mL h)
CLtot (IV) (mL/min kg)
BA (%)
l
M)a
>50
24
63
361
18
35
Compound 19 was evaluated for inhibition of the growth of
N87C tumor xenograft in
a 19-day study in athymic mice
(Fig. 5).13 Administration of a 20 mg/kg oral dose of 19 exhibited
effective inhibition of the tumor cell growth, comparable to a
20 mg/kg dose of Lapatinib. However, administration of an
80 mg/kg oral dose of 19 did not enhance the antitumor activity.
The limited in vivo efficacy might be attributed to the low meta-
bolic stability of 19 in mouse liver. However, this significant po-
a
Solubility was measured by high-speed solubility screening
assay using DMSO solutions. The solvent is Japanese Pharma-
copoeia XIV (JP) disintegration media JP 2nd-fluid (JP2; pH 6.8)
plus taurocholic acid.
b
Percentage of test compound remaining after 30 min reac-
tion with human liver microsomes (HLM) or rat liver micro-
somes (RLM).
tency
suggested
that
further
development
of
this
alknylpyrimidine series had the potential to reveal effective antitu-
mor agents.
In summary, we have described a SAR study of 5-alkenyl, 5-
alkynyl pyrimidine analogues which led to the identification of a
potent dual EGFR/Her-2 inhibitor 19. The key modification was
the introduction of a pyrrolidine moiety as the terminal hydro-
philic group and meta-fluoro substitution onto the terminal phenyl
ring. Compound 19 was highly selective for the Her family, and
exhibited significant in vivo antitumor potency. Encouraging re-
sults with analogue 19 highlight the potential of this series to pro-
vide novel potent antitumor agents.
The SARs of 5-alkenylpyrimidine analogues are presented in
Table 2. The mesylethylamine analogue 25 exhibited modest inhib-
itory activities in the enzyme and the cellular assays, similar to the
extent of the corresponding alkyne analogue 15. However, the
introduction of isopropylamine (26) or cyclopropylamine (27) at
the benzylic position did not enhance inhibitory activities against
the tumor cell lines. The reason for the difference of these SARs be-
tween the 5-alkynyl and 5-alkenyl series has not been elucidated.
However, we speculate that the differences of the cellular activities
1.00
0.90
0.80
0.70
1400.0
1200.0
1000.0
800.0
600.0
400.0
200.0
0.0
0.60
0.50
0.40
0.30
0.20
0.57
0.56
0.54
0.37
5
9
12
15
19
Day after tumor inoculation
0.10
0.00
Vehicle
Cmpd 19 20mg/kg
Cmpd 19 80mg/kg
Lapatinib 20mg/kg
Lapatinib 80mg/kg
Control
19
19
3
3
20 mg/kg 80 mg/kg 20 mg/kg 80 mg/kg
p<0.05, p<0.01 for no treatment by Welch's t-test
Figure 5. In vivo efficacy of 19 (q.d. Â 14 days) versus N87 human tumor xenografts in athymic mice (N = 6).

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