Synthesis and biological evaluation of pyrimidine-based dual inhibitors of human epidermal growth factor receptor 1 (HER-1) and HER-2 tyrosine kinases

Article abstract of DOI:10.1021/jm201758g

A novel series of N⁴-(3-chlorophenyl)-5-(oxazol-2-yl)pyrimidine- 4,6-diamines were synthesized and evaluated as dual inhibitors of HER-1/HER-2 tyrosine kinases. In contrast to the currently approved HER-2-targeted agent (lapatinib, 1), our irreversible HER-1/HER-2 inhibitors have the potential to overcome the clinically relevant and mutation-induced drug resistance. The selected compound (19a) showed excellent inhibitory activity toward HER-1/HER-2 tyrosine kinases with selectivity over 20 other kinases and inhibited the proliferation of both cancer cell types: lapatinib-sensitive cell lines (SK-Br3, MDA-MB-175, and N87) and lapatinib-resistant cell lines (MDA-MB-453, H1781, and H1975). The excellent pharmacokinetic profiles of 19a in mice and rats led us to further investigation of a novel therapeutic agent for HER-2-targeting treatment of solid tumors, especially HER-2-positive breast/gastric cancer and HER-2-mutated lung cancer.

Full text of DOI:10.1021/jm201758g

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Pyrimidine-Based Dual
Inhibitors of Human Epidermal Growth Factor Receptor 1 (HER-1)
and HER-2 Tyrosine Kinases
Mi Young Cha,†,‡ Kwang-Ok Lee,† Seok-Jong Kang,† Young Hee Jung,† Ji Yeon Song,† Kyung Jin Choi,†
Joo Yun Byun,† Han-Jae Lee,‡ Gwan Sun Lee,† Seung Bum Park,*,‡,§ and Maeng Sup Kim*,†
†Department of Drug Discovery, Hanmi Research Center, 377-1 Yeongcheon-ri, Dongtan-myeon, Hwaseong, Gyeonggi-do 445-813,
Korea
‡Department of Chemistry, Seoul National University, Seoul 151-747, Korea
§Department of Biophysics and Chemical Biology/Bio-MAX Institute, Seoul National University, Seoul 151-747, Korea
S
* Supporting Information
ABSTRACT: A novel series of N4-(3-chlorophenyl)-5-
(oxazol-2-yl)pyrimidine-4,6-diamines were synthesized and
evaluated as dual inhibitors of HER-1/HER-2 tyrosine kinases.
In contrast to the currently approved HER-2-targeted agent
(lapatinib, 1), our irreversible HER-1/HER-2 inhibitors have
the potential to overcome the clinically relevant and mutation-
induced drug resistance. The selected compound (19a)
showed excellent inhibitory activity toward HER-1/HER-2
tyrosine kinases with selectivity over 20 other kinases and inhibited the proliferation of both cancer cell types: lapatinib-sensitive
cell lines (SK-Br3, MDA-MB-175, and N87) and lapatinib-resistant cell lines (MDA-MB-453, H1781, and H1975). The excellent
pharmacokinetic profiles of 19a in mice and rats led us to further investigation of a novel therapeutic agent for HER-2-targeting
treatment of solid tumors, especially HER-2-positive breast/gastric cancer and HER-2-mutated lung cancer.
As shown in Figure 1, several small-molecule inhibitors of
HER-2 tyrosine kinases have been developed.16,17 The first
INTRODUCTION
■
HER-2 is a transmembrane oncoprotein encoded by the Her-2/
neu gene and is a member of HER/ErbB/EGFR tyrosine
kinases family, which is composed of HER-1 (ErbB-1, EGFR),
HER-2 (neu, ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4).
The signaling cascade of EGFRs plays a crucial role in
regulation of cell proliferation and differentiation in many tissue
types, and the dysregulation of EGFR signaling pathway may
contribute to malignant transformation.1−3 In particular, HER-
2 is overexpressed in 20−25% of breast or gastric cancer, which
is associated with poor prognosis.4,5 Therefore, HER-2 has
been recognized as a therapeutic target with good clinical
outcomes.6 For example, trastuzumab7,8 (a humanized
monoclonal antibody targeting the extracellular domain of
HER-2) was approved for HER-2-positive metastatic breast
cancer and gastric cancer by U.S. Food and Drug Admin-
istration (FDA). Lapatinib9,10 (reversible small-molecule
tyrosine kinase inhibitor of HER-1 and HER-2) was also
approved for HER-2-positive metastatic breast cancer. Even
though these therapeutic agents are effective for patients with
HER-2-positive breast or gastric cancer, trastuzumab has
potential cardiotoxicity when combined with chemotherapy
(especially with anthracyclines) and lapatinib did not show the
overall survival advantage in metastatic breast cancer.11,12 In
addition, the intrinsic or acquired resistance against trastuzu-
mab or lapatinib has been an important issue in HER-2-
targeted cancer therapy.13−15
Figure 1. Representative inhibitors of HER-2 tyrosine kinase.
HER-2-targeting small-molecule agent is lapatinib (1), the
quinazoline-based HER-1/HER-2 dual inhibitor. Since HER-2
tyrosine kinase binds either HER-1 or HER-2 tyrosine kinases
to form a hetero- or homodimer for the signal transduction of
Received: December 30, 2011
Published: February 28, 2012
© 2012 American Chemical Society
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EGFR signal cascade, HER-1/HER-2 dual inhibition can be
more effective than just HER-1 or HER-2 inhibition.11 The
next class includes the irreversible EGFR inhibitors cyanoqui-
noline-based pelitinib (2) and quinazoline-based afatinib
(3).18−20 Among these HER-2 inhibitors, we focused on
irreversible EGFR inhibitors because of their interesting mode
of action; both HER-1 and HER-2 tyrosine kinases were
inhibited by the irreversible modification of a Cys residue
(Cys773 of HER-1 and Cys805 of HER-2) at their active sites.
Their strong inhibitory activity toward both HER-1 and HER-2
and the unique mode of action can provide the potential to
overcome the limited efficacy of current HER-2-targeted
therapy. Therefore, we designed new HER-2-targeting small-
molecule inhibitors with a distinct scaffold for improved
antitumor efficacy to address the limited activity profile of the
currently approved HER-2-targeting small molecular therapy,
lapatinib.
can influence the orientation of N-acryloyl group as a Michael
acceptor for the alkylation to desired cysteine residues.
Therefore, we synthesized a series of N-4-((3-chloro-4-
(pyridin-2-ylmethoxy)phenyl)amino)pyrimidin-5-linked acryla-
mide analogues 10a−d to introduce various moieties at the X
position of pyrimidine scaffold as an irreversible HER-1/HER-2
dual inhibitor. As shown in Scheme 1, the synthesis of
a
Scheme 1
RESULTS AND DISCUSSION
■
Chemical Design. To overcome the drug resistance and
the limited activity of the current anti-HER-2 therapy, we
pursued the discovery of novel HER-1/HER-2-targeting
therapeutic agents with enhanced potency. We selected
pyrimidine as a novel core skeleton, inspired by quinazoline-
or cyanoquinoline-based irreversible EGFR inhibitors (2, 3, and
their general molecular framework 4 shown in Figure 2).
a
Reagents and conditions: (i) N-iodosuccinimide, AcOH, 100 °C; (ii)
(a) POCl3, reflux, (b) 8, 2-propanol, reflux; (iii) (a) N-Boc-
propargylamine, PdCl2(PPh3)2, CuI, TEA, THF, rt; (b) TFA, DCM,
rt; (c) acryloyl chloride, NaHCO3, aq THF (THF/H2O = 5:1), 0 °C;
(iv) (a) 3-aminophenylboronic acid, Pd(OAc)2, K2CO3, MeOH, 70
°C; (b) acryloyl chloride, NaHCO3, aq THF (THF/H2O = 5:1), 0 °C;
(v) (a) trimethylsilyl acetylene, PdCl2(PPh3)2, CuI, TEA, THF, rt; (b)
TBAF, THF, 0 °C; (c) 2-(1,3-dioxoisoindolin-2-yl)-N-hydroxyaceti-
midoyl chloride, TEA, THF, reflux; (d) hydrazine monohydrate,
EtOH, 65 °C; (e) acryloyl chloride, NaHCO3, aq THF (THF/H2O =
5:1), 0 °C.
analogues 10a−c was initiated using 4(3H)-pyrimidinone 6
treated with N-iodosuccinimide at 100 °C in acetic acid to yield
5-iodopyrimidin-4(3H)-one 7. The resulting compound 7 was
chlorinated with phosphorus oxychloride under reflux con-
ditions and subjected to substitution with aniline 8 in refluxing
2-propanol to yield N-(3-chloro-4-(pyridin-2-ylmethoxy)-
phenyl)-5-iodopyrimidin-4-amine 9 as a key intermediate.
This intermediate 9 was reacted with N-Boc-propargylamine
under Sonogashira coupling conditions in the presence of
bis(triphenylphosphine)palladium(II) dichloride and copper(I)
iodide. Subsequently, the Boc group was removed upon
treatment with 30% trifluoroacetic acid in dichloromethane,
followed by amide coupling with acryloyl chloride in the
presence of sodium bicarbonate at 0 °C to yield propargyl
analogue 10a. Phenyl analogue 10b was synthesized through
Suzuki coupling reaction of the key intermediate 9 with 3-
aminophenylboronic acid in the presence of palladium(II)
acetate, followed by N-acryloylation. For the preparation of
isoxazolylmethyl analogue 10c, the key intermediate 9 was
treated with trimethylsilyl (TMS) acetylene under the identical
Sonogashira coupling conditions used above. The desired
analogue 10c was synthesized through a series of reactions
including TMS deprotection using tetrabutylammonium
fluoride (TBAF), 1,3-dipolar cycloaddition with 2-(1,3-
dioxoisoindolin-2-yl)-N-hydroxyacetimidoyl chloride, phthali-
mide deprotection using hydrazine, and subsequent N-
acryloylation.
Figure 2. Design of novel HER-1/HER-2 dual inhibitors.
According to previous reports,21 N1 and N3 (if Z = N;
quinazoline) or 3-carbonitrile (if Z = C−CN; cyanoquinoline)
in the molecular framework 4 is essential for the critical
hydrogen bonding interaction with Met769 and Thr766 (or
Thr830) of EGFR, respectively. In addition, the introduction of
substituents at the C8 position of compound 4 resulted in the
loss of EGFR inhibitory activity.21 In contrast, the aniline
moiety at the C4 position of compound 4 is essential to achieve
bioactive conformation. Bulky substituents such as pyridin-2-
ylmethoxy or substituted benzyloxy moiety, as shown in 1, at
the R1 position increase HER-2 inhibition, and small electron-
withdrawing lipophilic groups such as chloride or fluoride at the
R2 position increase HER-1 and HER-2 inhibitory activity. On
the basis of this structure−activity relationship (SAR)
information, we designed pyrimidine-based analogues 5 with
an N-acryloyl moiety at the C5 position and bulky aniline
moiety at the C4 position to secure irreversible HER-2
inhibitory activity. Herein, we report the identification of N4-
(3-chlorophenyl)-5-(oxazol-2-yl)pyrimidine-4,6-diamines with
N-acryloyl moiety as potential candidates and compound 19a
as a novel HER-1/HER-2 dual inhibitor for HER-2-targeting
anticancer therapy.
Selection of Active Scaffold. The modification at the X
position of compound 5 provides the important element that
For the synthesis of oxazolylmethyl analogue 10d, ethyl 4-
((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)pyrimidine-
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a
Scheme 2
a
Reagents and conditions: (i) (a) formamidine hydrochloride, 160 °C; (b) POCl3, SOCl2, DIPEA, reflux; (c) 8, 2-propanol, reflux; (ii) (a) 2 N
NaOH aq solution, EtOH, 80 °C; (b) DL-serine methyl ester hydrochloride, EDCI, HOBt, DIPEA, DMF, rt; (iii) (a) DAST, DCM, −78 to 0 °C; (b)
DBU, CBrCl3, DCM, −40 °C to rt; (ix) (a) LAH, THF, 0 °C; (b) NaN3, PPh3, DMF/CCl4 (4:1), 90 °C; (c) PPh3, H2O, THF, 60 °C; (d) acryloyl
chloride, NaHCO3, aq THF (THF/H2O = 5:1), 0 °C.
a
5-carboxylate (12) was prepared by condensation of diethyl
ethoxymethylenemalonate (11) with formamidine, chlorination
using thionyl chloride and phosphorus oxychloride in the
presence of diisopropylethylamine, and substitution with
aniline 8 in the refluxing 2-propanol (Scheme 2). Then the
resulting ethyl carboxylate 12 was hydrolyzed in 2 N sodium
hydroxide aqueous solution and coupled with DL-serine methyl
ester using EDCI and HOBt to provide β-hydroxyamide 13,
which was converted to oxazole 14 via the mild cyclo-
dehydration reaction using diethylaminosulfur trifluoride
(DAST) and oxidation using bromotrichloromethane and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU). Finally, oxazolylmethyl
analogue 10d was prepared by the reduction of methyl ester of
oxazole 14, substitution of hydroxyl group to azide, Staudinger
reduction of azide to amine, and subsequent N-acryloylation.
All synthesized analogues 10a−d were initially tested for
their ability to inhibit cellular proliferation in two representative
HER-1 and HER-2-positive cell lines: A431 (HER-1-over-
expressing human vaginal epidermoid cancer cell line)22 and
SK-Br3 (HER-2-overexpressing human breast cancer cell
line).23 As shown in Table 1, analogues containing N-acryloyl
moiety with phenyl group (10b, IC50 = 6.1 nM) or
oxazolylmethyl group (10d, IC50 = 9.7 nM) at the C5 position
of pyrimidine showed stronger growth inhibition potency in
SK-Br3 cell-based proliferation assay, compared to analogues
with propargyl group (10a, IC50 > 1000 nM) or isoxazo-
lylmethyl group (10c, IC50 = 122 nM). In addition, the
oxazolylmethyl derivative 10d (IC50 = 105 nM) showed better
potency compared to the phenyl derivative 10b (IC50 = 593
nM) in A431 cell lines. It was quite exciting to see that the
growth inhibitory activity of analogue 10d is comparable to that
of 1, which is an approved HER-1/HER-2 dual inhibitor for the
treatment of patients with HER-2-positive metastatic breast
cancer.
Table 1. In Vitro Cellular Activity of R-Modified Analogues
a
All biological data are mean values of three independent experiments
performed in duplicate. A431 is HER-1-overexpressing human vaginal
epidermoid cancer cell line. SK-Br3 is HER-2-overexpressing human
breast cancer cell line. 1 was synthesized using the synthetic protocol
b
c
d
reported in WO99/35146.
hydrogen bonding interaction with Gly796 (Grid score, which
is not considered for H-bond: −53.65 for 10d vs −57.04 for
amine (Y) containing analogue of 10d). Therefore, we aimed to
introduce the amine moiety at the Y position of pyrimidine 5 to
enhance its binding affinity at the active site of EGFR. The
synthesis of the amine-containing analogue of 10d at the Y
position is outlined in Scheme 3. 4,6-Dichloropyrimidine-5-
carbaldehyde 15 was chlorinated under radical conditions,
followed by amide coupling with DL-serine methyl ester to give
β-hydroxyamide 16. The C6-amine was introduced to 16 using
ammonia gas in 50% ethanolic toluene in a sealed tube,
followed by oxazoline formation using DAST. The C4-chloride
of oxazoline 17 was substituted with various anilines (R-NH2)
under acidic conditions and oxidized to oxazole using
Optimization of Active Analogue 10d. On the basis of
the results of cell-based proliferation assay against 10a−d, the
N-acryloyl moiety with oxazolylmethyl group was selected for
position X of pyrimidine. For the further optimization of active
analogue 10d, we performed a series of docking studies using
the EGFR crystal structure (PDB code 1XKK) cocrystallized
with 1. As shown in Figure 3, the modification at the Y position
of compound 5 with amine group allows the additional
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Figure 3. Binding mode of 10d (a) and its analogue with amine at the Y position (b) to the active site of EGFR crystal structure cocrystallized with 1
(PDB entry 1XKK).
a
a
Scheme 3
Table 2. In Vitro Cellular Activity of Analogues 19a and 21
a
Reagents and conditions: (i) (a) SOCl2, AIBN, CCl4, 80 °C; (b) DL-
serine methyl ester hydrochloride, DIPEA, THF, 0 °C to rt; (ii) (a)
NH3 (gas), toluene/EtOH (1:1), sealed tube, 60 °C; (b) DAST,
DCM, −78 to 0 °C; (iii) (a) R-NH2, 4 N HCl in 1,4-dioxane, t-BuOH,
60 °C; (b) DBU, CBrCl3, DCM, −40 °C to rt; (c) LAH, THF, 0 °C;
(d) NaN3, PPh3, DMF/CCl4 (4:1), 90 °C; (iv) (a) PPh3, H2O, THF,
60 °C; (b) acryloyl chloride, NaHCO3, aq THF (THF/H2O = 5:1), 0
°C.
a
All biological data are mean values of three independent experiments
b
performed in duplicate. A431 is HER-1-overexpressing human vaginal
epidermoid cancer cell line. SK-Br3 is HER-2-overexpressing human
breast cancer cell line. 1 was synthesized using the synthetic protocol
of WO99/35146.
c
d
bromotrichloromethane and DBU. After the reduction of
methyl ester in 17 and the substitution of alcohol to azide, the
resulting azide moiety in azidomethyloxazole 18 was reduced to
amine in the presence of PPh3 and subsequently N-acryloylated
to obtain the desired analogue 19.
Compound 19a, an analogue of 10d with an amine moiety at
the C6 position of pyrimidine, showed 10-fold and 14-fold
enhancement in its antiproliferative activity compared to 10d in
A431 (IC50 of 10.8 vs 105 nM) and SK-Br3 (IC50 of 0.7 vs 9.7
nM) cell lines, respectively (Table 2). Compound 21 with an
amide moiety as a bioisostere of oxazole was also prepared to
ensure the significance of oxazole’s conformation (see Scheme
S1). As shown in Table 2, amide analogue 21 showed no
antiproliferative activity in both cancer cell lines (entry 2, IC50
> 1000 nM).
After significant enhancement of its antiproliferative activity
via the introduction of an amine moiety at the C6 position of
the pyrimidine scaffold, we turned our attention to the
structure−activity relationship study of substituents (R1, R2,
and R3 moieties) on the aryl ring of compound 19. As stated
earlier, the introduction of small electron-withdrawing lip-
ophilic groups such as chloride or fluoride at the R2 position
increases HER-1/HER-2 inhibitory activity and the introduc-
tion of bulkier moieties such as pyridin-2-ylmethoxy or
substituted benzyloxy at the R1 position increases HER-2
inhibitory activity. In addition, we aimed to develop an orally
available anticancer agent with respect to the patients’
convenience. Because the first-pass metabolism by liver is one
of the major causes of poor oral bioavailability and short in vivo
half-life, the in vitro metabolic stability using liver microsome is
one of the efficient in vitro guidelines for the evaluation of oral
bioavailability and used in a prospective manner to choose the
potential candidates for further development with desirable
pharmacokinetic properties including long half-life time and
low oral clearance.24,25 Therefore, we focused on the
improvement of HER-1/HER-2 potency and liver microsomal
stability through the substituent modification at the R1, R2, and
R3 positions of compound 19. Analogues 19b−r were
synthesized in a similar manner as 19a for the systematic
introduction of various substituents such as halogen, alkyl,
pyridylmethyloxy, benzyloxy, phenyloxy, or heteroaryloxy on
phenyl moiety and subjected to the cell-based growth inhibition
assay using the HER-1-positive A431 cell line and the HER-2-
positive SK-Br3 cell line, along with the microsomal stability.
As shown in Table 3, the HER-1/HER-2 cellular inhibitory
activity was decreased by the replacement of 3-chloro (19a,
IC50 = 11 nM for A431 and IC50 = 0.7 nM for SK-Br3) with 3-
fluoro (19b, IC50 = 63 nM for A431 and IC50 = 6.1 nM for SK-
Br3) at the R2 position or the replacement of 4-(2-pyridine)
(19a) with 4-(3-fluorophenyl) (19c, IC50 = 63 nM for A431
and IC50 = 6.1 nM for SK-Br3) at the R1 position, which shares
the same structure−activity relationship pattern of phenyl
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a
Table 3. In Vitro Cellular Activity and Microsomal Stability of Analogues 19
a
b
All biological data are mean values of three independent experiments performed in duplicate. A431 is HER-1-overexpressing human vaginal
c
d
epidermoid cancer cell line. SK-Br3 is HER-2-overexpressing human breast cancer cell line. Half-life (HL) of metabolism by incubation of parent
molecule with liver microsomes of mouse, rat, dog, and human (duplicate). NT means “not tested”. The asterisk (∗) means very stable with a HL
of >100 min.
e
f
substituents in the case of 1. The reduced anticancer activities
were observed in a series of analogues 19d−p substituted with
4-phenyloxy or 4-heteroaryloxy at the R1 position. Analogue
19e containing the 3-trifluoromethyl-1H-pyrazol-5-yloxy moi-
ety had excellent microsomal stability, but this compound also
showed extremely high affinity to plasma proteins (PPB =
100%, data not shown). Compound 19k showed relatively low
growth inhibitory activity in A431 cells, but it showed
comparable SK-Br3 cellular activity with improved microsomal
stability. Analogues 19q−r with small anilines containing only
fluoro or chloro substituents at the R1 position showed
relatively low cellular HER-2 inhibitory activity (for SK-Br3:
IC50(19q) = 34.1 nM and IC50(19r) = 92.6 nM vs IC50(19a) =
0.7 nM). Among these analogues, 19a showed the highest
potency in growth inhibition of A431 and SK-Br3 cells with
acceptable microsomal stability.
Pharmacokinetic Studies of 19a. Prior to further
evaluation, our leading compound 19a was subjected to
pharmacokinetic studies in ICR (imprinting control region)
mice and SD (Sprague−Dawley) rats. As shown in Table 4,
pharmacokinetic parameters of 19a were determined after a
single oral administration (10 mg/kg for mouse and 5 mg/kg
for rat) and intravenous injection (2 mg/kg for mouse and 1
mg/kg for rat). Compound 19a demonstrated fair oral
bioavailability with >20% (21.6% for mouse and 21.9% for
rat) and desirable exposure levels (Cmax and AUC) in mice and
rats.
Enzymatic Activity and EGFR Irreversible Inhibition of
19a. We examined the specificity of 19a conducting in vitro
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Table 4. Pharmacokinetic Profile of 19a in Mouse and Rat
Article
a
confirmed by in vitro enzymatic assays (IC50 > 3 μM except for
Blk).
b
c
parameters (po)
mouse
rat
19a is an oxazolylpyrimidine-based HER-1/HER-2 dual
inhibitor containing a functional α,β-unsaturated amide as
Michael acceptor. This moiety is responsible for the irreversible
inhibition of 19a via the covalent modifications at the active site
(Cys773 of EGFR and Cys805 of HER-2) of EGFR tyrosine
kinase domains in a manner similar to that with other
irreversible EGFR inhibitors, such as 2 and 3. To validate
this hypothesis, the phosphorylation levels (% relative to
positive control) of EGFR tyrosine kinases (EGFR and HER-2)
in EGFR-overexpressing A431 and HER-2-overexpressing SK-
Br3 cells were measured immediately or in 8 h after the removal
of 19a from the medium. We observed the prolonged
inhibitory effect on the phosphorylation of EGFR (97.5%
inhibition at 0 h and 87.5% at 8 h after medium washing) and
HER-2 (87.2% inhibition at 0 h and 68.2% at 8 h after medium
washing) upon treatment of 19a in both A431 and SK-Br3
cells, which might be caused by the irreversible modification of
19a to EGFR and HER-2 (see Figure S1).
Cellular Activity of 19a. On the basis of the results of the
cellular growth inhibition assays in A431 and SK-Br3 cell lines
along with in vitro/in vivo pharmacokinetic study, we selected
compound 19a for further biological evaluation as a potential
HER-1/HER-2-targeting therapeutic agent. The effects of 19a
were evaluated using a panel of cancer cell lines, which express
various levels of HER-2 and other genetic information with
sensitivity or resistance to 1. 19a showed excellent growth
inhibitory activity in lapatinib-sensitive breast (MDA-MB-175,
IC50 = 1.6 nM) and gastric (NCI-N87, IC50 = 3.0 nM) cancer
cell lines with 12- to 27-fold superiority to 1.
dose (mg/kg)
AUClast (ng·h/mL)
Cmax (ng/mL)
T1/2 (h)
10
5
454.8
282.5
27.7
21.6
439.7
189.0
3.23
F (%)
21.9
a
19a was treated as HCl salt in a vehicle as a solution of 30% PEG400
and 5% ethanol in distilled water. The pharmacokinetic parameters
were determined after a single oral administration (10 mg/kg, n = 3)
and intravenous injection (2 mg/kg, n = 3) in ICR mice. The
pharmacokinetic parameters were determined after a single oral
administration (5 mg/kg, n = 3) and intravenous injection (1 mg/kg, n
= 3) in SD rats.
b
c
assays against purified tyrosine kinases of the EGFR family
(Table 5) and against a panel of other protein kinases (Table
a
Table 5. In Vitro Enzymatic Activity of 19a
enzyme assay, IC50 (nM)
EGFR
compd
WT
T790M
HER-2
HER-4
19a
b
3.3
3.2
22.4
36.3
4.0
1
52.2
>1000
>1000
a
All biological data are mean values of three independent experiments
performed in duplicate. 1 was synthesized using the synthetic
protocol of WO99/35146.
b
HER-2-directed therapy including 1 is an important
treatment for breast cancer. However, some tumors do not
respond or develop resistance to this agent.26 Although the
overexpression of HER-2 correlates with sensitivity to the
growth inhibition by 1, some cancer cells that overexpress
HER-2 do not respond to 1.27 Even though the factors that
confer primary or acquired resistance to 1 are not well
characterized, several hypotheses including low PTEN
expression, PIK3CA mutations, or ER (estrogen receptor)
activation are reported to be associated with resistance in HER-
2-positive breast cancer. Analogue 19a showed excellent growth
inhibitory activity with IC50 of 5.0 nM in lapatinib-resistant
MDA-MB-453 cell line (IC50(1) = 555 nM) (Table 7), which is
conferred by low PTEN expression and PIK3CA mutations
with high expression of HER-2. But 19a did not induce the
growth inhibitory effect in JIMT-1 cells with low HER-2
expression level (IC50(19a) = 1880 nM and IC50(1) > 10000
nM) and MDA-MB-361 cells with ER activation (IC50(19a) =
562 nM and IC50(1) = 1029 nM). In addition, analogue 19a
induced the growth inhibition in HER-2-mutated
(G776insV_G/C, NCI-H1781, IC50 = 22 nM) and EGFR-
mutated (L858R/T790M, NCI-H1975, IC50 = 40 nM) NSCLC
cell lines with resistance to 1 (IC50(NCI-H1781) = 3047 nM
and IC50(NCI-H1975) > 1000 nM).
a
Table 6. Selectivity Profile of 19a against 20 Kinases
kinase
% inhibition at 3 μM
Abl
15
0
AMPKα1
Aurora-A
Blk
42
77
0
CDK1/cyclinB
cKIT
0
FAK
0
FGFR1
Flt1
0
15
0
Flt3
IGF-1R
IR
0
0
JAK2
0
LKB1
0
MEK1
Met
14
13
0
PDGFRα
PDGFRβ
Pim-1
Syk
0
2
15
a
This test was conducted by Millipore Kinase Profiling Service.
SUMMARY
■
6). 19a effectively inhibited EGFR family kinases with IC50
values of 3.3, 3.2, 22.4, and 4.0 nM against EGFRWT
EGFRT790M, HER-2, and HER-4, respectively. In particular,
19a showed excellent inhibitory activity against mutated EGFR
(EGFRT790M) and HER-4, both of which have been resistant to
1. 19a also showed excellent selectivity over other 20 kinases
HER-2 is overexpressed in 20−25% of metastatic breast or
gastric cancers and is associated with an aggressive tumor type
and reduced survival rate. Therefore, HER-2-targeted therapy
such as trastuzumab or lapatinib is beneficial for cancer patients
with high HER-2 expression level. However, most cancers that
initially respond to HER-2-targeted therapy begin to progress
,
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a
Table 7. In Vitro Cellular Activities of 19a
IC50 (nM)
b
tissue
cell line
character
Src++, Ret+++, low HER-2
19a
1.6
1
breast cancer
MDA-MB-175
MDA-MB-361
MDA-MB-453
JIMT-1
44.4
Her-2++, Src+++, PIK3CA mutation, ER activation
HER-2++, PTEN deficiency, PIK3CA mutation
low HER-2
562.0
5.0
1029
555.0
>10000
36
1,880
3.0
gastric cancer
N87
HER-2+++
c
NSCLC
H1781
HER-2 G776insV_G/C
22.0
40.0
3047
>1000
H1975
EGFR L858R/T790M
a
b
All biological data are mean values of three independent experiments performed in duplicate. 1 was synthesized using the synthetic protocol of
c
WO99/35146. NSCLC: non-small-cell lung cancer.
MHz, CDCl3) δ 8.95 (s, 1H), 8.83 (s, 1H), 8.62−8.60 (m, 1H), 7.79−
7.76 (m, 1H), 7.68−7.65 (m, 1H), 7.47−7.46 (m, 1H), 7.17−7.14 (m,
1H), 6.62 (s, 1H), 6.31−6.25 (m, 1H), 6.11−6.07 (m, 1H), 5.75 (m,
1H), 5.71−5.68 (m, 1H), 5.36 (s, 2H), 4.59−4.57 (d, 2H). MS (ESI+):
m/z = 420.7 [M + H]+.
again within 1 year. The several mechanisms by HER-2 therapy
contribute to address this resistance. Herein, we have described
the synthesis of a novel series of N4-(3-chlorophenyl)-5-
(oxazol-2-yl)pyrimidine-4,6-diamines which are explored as
HER-1/HER-2 dual inhibitors for the treatment of resistant
patients to the current HER-2-targeted therapy, lapatinib. The
selected compound (19a) showed excellent HER-1 and HER-2
inhibition activity and also showed excellent selectivity in an
enzyme-based inhibition assay with a panel of 20 kinases. We
confirmed that our leading HER-1/HER-2 dual inhibitor (19a)
has an irreversible binding mode to HER-1 and HER-2
confirmed by a cell washout test in A431 and SK-Br3 cell lines.
In contrast to the currently approved HER-2-targeted agent,
our selected compound (19a) is an irreversible small-molecule
inhibitor of HER-1 and HER-2 tyrosine kinases with the
potential to overcome clinically relevant, mutation-induced
drug resistance. 19a showed excellent inhibitory activity toward
cellular growth of lapatinib-sensitive or resistant cancer cells.
The excellent pharmacokinetic profiles of 19a in rats and mice
clearly demonstrate its merit for further investigation as a novel
therapeutic agent for HER-2-targeting treatment of solid
tumors, especially HER-2-positive breast or gastric cancer.
N-(3-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)phenylamino)-
pyrimidin-5-yl)phenyl)acrylamide (10b). Step 1. Preparation of
5-(3-Aminophenyl)-N-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-
pyrimidin-4-amine. 300 mg of compound 9 and 142 mg of 3-
aminophenylboronic acid hydrochloride were dissolved in 6 mL
of methanol, and 7.7 mg of palladium acetate and 284 mg of
potassium carbonate were added thereto. The mixture was
stirred at 70 °C for 2 h. After the reaction was completed, the
reaction mixture was condensed under a reduced pressure, and
the resulting residue was subjected to flash column
chromatography (CHCl3/CH3OH = 15: 1) to obtain the title
compound (120 mg, yield 44%). 1H NMR (300 MHz, CDCl3)
δ 8.70 (s, 1H), 8.60 (d, 1H), 8.21 (s, 1H), 7.76 (t, 2H), 7.69 (d,
1H), 7.65 (d, 1H), 7.34 (m, 2H), 7.24 (d, 1H), 6.97 (d, 1H),
6.80 (m, 1H), 6.73 (s, 1H), 5.28 (s, 2H), 3.88 (s, 2H).
Step 2. Preparation of N-(3-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)-
phenylamino)pyrimidin-5-yl)phenyl)acrylamide (10b). 51 mg of the
compound obtained in step 1 and 32 mg of sodium bicarbonate were
dissolved in a mixture of 2 mL of tetrahydrofuran (THF) and 0.5 mL
of distilled water at 0 °C. Then 10 μL of acryloyl chloride was added to
the mixture and stirred for 30 min. After the reaction was completed,
the resulting mixture was added to a saturated aqueous solution of
sodium bicarbonate and extracted twice with chloroform. The
separated organic layer was washed with water and saturated saline,
dried over anhydrous sodium sulfate, filtered, and distilled under a
reduced pressure. The resulting residue was subjected to flash column
chromatography (CHCl3/CH3OH = 15: 1) to obtain the title
compound (12 mg, yield 21%). 1H NMR (300 MHz, CDCl3) δ
8.68 (s, 1H), 8.59 (d, 1H), 8.19 (s, 1H), 8.13 (s, 1H), 7.74 (m, 3H),
7.65 (t, 2H), 7.50 (t, 1H), 7.35 (m, 1H), 7.22 (m, 2H), 6.95 (s, 1H),
6.94 (d, 1H), 6.48 (d, 1H), 6.33 (m, 1H), 5.82 (d, 2H), 5.26 (s, 2H).
MS (ESI+): m/z = 458.1 [M + H]+.
N-((5-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)phenylamino)-
pyrimidin-5-yl)isooxazol-3-yl)methyl)acrylamide (10c). Step
1. Preparation of N-(3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)-5-
((trimethylsilyl)ethinyl)pyrimidin-4-amine. 5.67 g of the com-
pound 9, 181 mg of dichlorobis(triphenylphosphine)palladium-
(II), and 98 mg of copper(I) iodide were dissolved in 60 mL of
THF, and 7.2 mL of triethylamine and 2.15 mL of
trimethylsilylacetylene were added to the mixture. The mixture
was stirred at room temperature for 24 h. After the reaction was
completed, a saturated aqueous solution of sodium bicarbonate
was added thereto, and the resulting mixture was extracted
twice with ethyl acetate. The separated organic layer was
washed with water and saturated saline, dried over anhydrous
sodium sulfate, filtered, and distilled under a reduced pressure.
The resulting residue was subjected to column chromatography
(ethyl acetate/hexanes = 1:10) to obtain the title compound
(1.8 g, yield 34%). 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H),
8.60 (d, 1H), 8.41 (s, 1H), 7.80 (d, 1H), 7.75 (m, 1H), 7.65 (d,
EXPERIMENTAL SECTION
■
Chemistry. General Information. All commercial chemicals and
solvents were reagent grade and were used without further purification
unless otherwise specified. All reported yields are isolated yields after
flash column chromatography or crystallization. 1H spectra were
recorded on a Bruker DRX-300 [Bruker Biospin, Germany]. Chemical
shifts are reported in ppm relative to the residual solvent peak (CDCl3,
TMS: 0.00). Multiplicity was indicated as follows: s (singlet); d
(doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of
doublet); dt (triplet of doublet); td (doublet of triplet); br s (broad
singlet); etc. Products were purified by flash column chromatography
on silica gel (230−400 mesh). The eluent used for purification is
reported in parentheses. The purity of all synthesized compounds
(>95% area) was analyzed by high performance liquid chromatography
using a Shimadzu HPLC instrument equipped with a reverse phase
column (XDB C18, 5 μm, 4.6 mm × 150 mm) and a UV detector
(254 nm). HPLC column was equilibrated with 5% acetonitrile
(CAN) in H2O (0.1% trifluoroacetic acid, TFA) for 5 min. After the
injection of 10 μL of individual samples, the eluent was changed from
5% ACN in H2O (0.1% TFA) to 100% ACN (0.1% TFA) over 45 min
with a flow rate of 1.0 mL/min. Thin-layer chromatography (TLC)
was performed on precoated glass-backed plates (silica gel 60 F254
,
0.25 mm), and the components were visualized under UV light (254
and 365 nm) or by treating the plates with anisaldehyde, KMnO4, and
phosphomolybdic acid, followed by heating. Distilled water was
treated using ion exchange and filtration.
N-(3-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-
1
pyrimidin-5-yl)prop-2-yn-1-yl)acrylamide (10a). H NMR (300
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1H), 7.37 (m, 1H), 7.23 (m, 2H), 7.00 (d, 1H), 5.29 (s, 2H),
1.77 (s, 1H), 0.32 (s, 9H).
room temperature for 10 min. 200 mg of the compound obtained in
step 4 was added to the mixture, and the mixture was stirred at room
temperature for 3 h. After the reaction completion monitored by TLC,
the resulting mixture was added to a saturated aqueous solution of
sodium bicarbonate and extracted twice with chloroform. The
combined organic layer was dried over anhydrous sodium sulfate,
filtered, and condensed under a reduced pressure. The resulting
residue was subjected to silica gel flash column chromatography
(CHCl3/CH3OH = 50:1) to obtain the title compound (86 mg, yield
38%). 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 8.59 (s, 1H), 8.57
(s, 1H), 7.74 (m, 2H), 7.67 (d, 1H), 7.38 (m, 1H), 7.27 (m, 1H), 7.01
(m, 2H), 6.71 (s, 1H), 6.37 (m, 1H), 6.20 (m, 1H), 5.74 (m, 1H), 5.28
(s, 2H), 4.64 (d, 2H). MS (ESI+): m/z = 463.1 [M + H]+.
N-((2-(4-(3-Chloro-4-(pyridin-2-ylmethoxy)phenylamino)-
pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide (10d). 1H NMR
(300 MHz, CDCl3) δ 10.56 (s, 1H), 8.95 (s, 1H), 8.72 (s, 1H), 8.59
(d, 1H), 7.86 (s, 1H), 7.76−7.67 (m, 3H), 7.53 (dd, 1H), 7.00 (d,
1H), 6.40 (s, 1H), 6.19 (s, 1H), 5.73 (d, 1H), 5.30 (s, 2H), 4.56 (d,
2H). MS (ESI+): m/z = 463.3 [M + H]+.
Step 2. Preparation of N-(3-Chloro-4-(pyridin-2-ylmethoxy)-
phenyl)-5-ethinyl pyrimidin-4-amine. 1.8 g of the compound
obtained in step 1 was dissolved in 30 mL of THF, and 8.8 mL of
1.0 M tetrabutylammonium fluoride solution in THF was added
thereto. The mixture was stirred at room temperature for 1 h. After the
reaction completion monitored by TLC, the reaction was quenched by
the addition of saturated aqueous solution of sodium bicarbonate. The
reaction mixture was extracted twice with ethyl acetate, and the
combined organic layer was dried over anhydrous sodium sulfate,
filtered, and distilled under a reduced pressure. The resulting residue
was subjected to flash column chromatography (ethyl acetate/hexanes
= 1:1) to obtain the title compound (1.4 g, yield 94%). 1H NMR (300
MHz, CDCl3) δ 8.65 (s, 1H), 8.60 (m, 1H), 8.45 (s, 1H), 7.76 (m,
2H), 7.64 (d, 1H), 7.41 (m, 1H), 7.24 (m, 2H), 6.99 (d, 1H), 5.29 (s,
2H), 3.67 (s, 1H).
Step 3. Preparation of 2-((5-(4-(3-Chloro-4-(pyridin-2-
ylmethoxy)phenylamino)pyrimidin-5-yl)isooxazol-3-yl)methyl)-
isoindoline-1,3-dione. 6.8 mL of oxalylchloride was dissolved in 250
mL of CH2Cl2, and the mixture was cooled to −78 °C. Then 8.9 mL of
dimethyl sulfoxide was added to the mixture. The mixture was stirred
for 10 min. 10 g of N-(2-hydroxyethyl)phthalimide was gradually
added to the mixture, and the mixture was stirred for 30 min, and 36.5
mL of triethylamine was added thereto. The resulting mixture was
heated to 0 °C and stirred for 1 h. After the reaction was completed,
the resulting mixture was added to a saturated aqueous solution of
NH4Cl and extracted twice with CH2Cl2. The combined organic layer
was dried over anhydrous sodium sulfate, filtered, and condensed
under a reduced pressure. 5.5 g of the solid thus obtained was
dissolved in 50 mL of pyridine, and 6.1 g of hydroxylamine
hydrochloride was added thereto, followed by stirring at 65 °C for 5
h. After the reaction was completed, the reaction mixture was
condensed under a reduced pressure, ethyl acetate was added thereto,
and the mixture was washed three times with water. The combined
organic layer was dried over anhydrous sodium sulfate, filtered, and
condensed under a reduced pressure. The resulting residue was
crystallized in diethyl ether. The obtained solid was filtered, washed
with diethyl ether, and dried under a reduced pressure. 588 mg of the
solid was dissolved in 10 mL of THF, and 595 mg of N-
chlorosuccinimide and 36 μL of pyridine were added thereto, followed
by stirring at 70 °C for 2 h. 500 mg of the compound obtained in step
2 and 621 μL of triethylamine were added to the mixture, and the
mixture was stirred at 70 °C for 12 h. After the reaction completion
monitored by TLC, the reaction mixture was condensed under a
reduced pressure and the resulting residue was subjected to flash
column chromatography (ethyl acetate/hexanes = 1:1) to obtain the
desired compound (450 mg, yield 56%). 1H NMR (300 MHz, DMSO-
d6) δ 8.84 (s, 1H), 8.61 (s, 1H), 8.59 (s, 1H), 8.56 (d, 1H), 7.90 (m,
5H), 7.67 (d, 1H), 7.54 (d, 1H), 7.40 (m, 1H), 7.35 (m, 1H), 7.18 (d,
1H), 7.05 (s, 1H), 5.25 (d, 2H), 4.92 (d, 2H).
Methyl 2-(4,6-Dichloropyrimidin-5-carboxamido)-3-hydrox-
ypropanoate (16). 4,6-Dichloropyrimidin-5-carbaldehyde 15 (10 g)
was dissolved in 100 mL of carbon tetrachloride, and 7.8 mL of
sulfuryl chloride and 0.51 g of 2,2-azobis(2-methylpropionitrile) were
added thereto, followed by stirring under reflux conditions at 80 °C for
1.5 h. After the reaction completion monitored by TLC, the reaction
mixture was cooled to room temperature and filtered under a reduced
pressure. The filtrate was condensed under a reduced pressure to
obtain 4,6-dichloropyrimidin-5-carbonyl chloride without further
purification (11.4 g, yield 95%). The resulting 4,6-dichloropyrimidin-
5-carbonyl chloride (8.6 g) and DL-serine methyl ester (12.6 g) were
dissolved in 100 mL of THF, and 14.2 mL of N,N-diisopropylethyl-
amine was added thereto dropwise at 0 °C. Then the reaction mixture
was heated to room temperature, followed by stirring for 30 min. After
the reaction completion monitored by TLC, the reaction mixture was
added to water and extracted twice with chloroform. The combined
organic layer was washed with water and saturated saline and dried
over anhydrous sodium sulfate, followed by filtration and condensation
under a reduced pressure. The obtained residue was subjected to silica
gel flash column chromatography (ethyl acetate/CH2Cl2/CH3OH =
1
30:30:1) to obtain the title compound (10.2 g, yield 85%). H NMR
(300 MHz, DMSO-d6) δ 9.31 (d, 1H), 8.96 (s, 1H), 5.15 (s, 1H), 4.58
(m, 1H), 3.85 (m, 1H), 3.68 (s, 3H), 3.67 (m, 1H).
Methyl 2-(4-Amino-6-chloropyrimidin-5-yl)-4,5-dihydrooxa-
zol-4-carboxylate (17). Compound 16 (7.0 g) was dissolved in 50
mL of toluene and 50 mL of ethanol in a sealed tube, and ammonia gas
was bubbled through the solution, followed by stirring at 60 °C for 2 h.
Ammonia gas was further bubbled into the solution until the reaction
was substantially completed, and the reaction mixture continued to be
stirred at the same temperature for an additional 1 h. After the reaction
completion monitored by TLC, the reaction mixture was condensed
under a reduced pressure to obtain methyl 2-(4-amino-6-chloropyr-
imidin-5-carboxamido)-3-hydroxypropanoate (6.54 g). The resulting
compound (5.0 g) was dissolved in 50 mL of CH2Cl2, and 2.65 mL of
(diethylamino)sulfur trifluoride was added thereto dropwise at −78
°C. The reaction mixture was slowly warmed up to 0 °C and stirred for
2.5 h. After the reaction was completed, the reaction mixture was
basificated (pH 8) with a saturated aqueous solution of sodium
bicarbonate and extracted twice with CH2Cl2. The combined organic
layer was dried over anhydrous sodium sulfate, filtered, and condensed
under a reduced pressure. The obtained residue was crystallized with a
mixed solvent of ethyl acetate and diethyl ether (v/v, 3:1), followed by
filtering and drying under a reduced pressure to obtain the desired
Step 4. Preparation of 5-(3-(Aminomethyl)isooxazol-5-yl)-N-(3-
chloro-4-(pyridin-2-ylmethoxy)phenyl)pyrimidin-4-amine. 450 mg
of the compound obtained in step 3 was dissolved in 10 mL of ethanol,
and 203 μL of hydrazine hydrate was added thereto, followed by
stirring at 70 °C for 1 h. After the reaction completion monitored by
TLC, the reaction mixture was cooled to 0 °C and the solid thus
obtained was filtered and washed with chloroform. The filtrate
therefrom was condensed under a reduced pressure. The resulting
residue was subjected to flash column chromatography (CHCl3/
CH3OH = 15:1) to obtain the title compound (200 mg, yield 59%).
1H NMR (300 MHz, DMSO-d6) δ 8.79 (m, 1H), 8.61 (s, 1H), 8.57
1
compound (2.38 g, yield 51%). H NMR (300 MHz, CDCl3) δ 8.70
(d, 1H), 7.86 (t, 1H), 7.71 (d, 1H), 7.55 (d, 1H), 7.45 (m, 1H), 7.35
(t, 1H), 7.20 (d, 1H), 7.04 (s, 1H), 5.26 (d, 2H), 3.79 (s, 2H).
Step 5. Preparation of N-((5-(4-(3-Chloro-4-(pyridin-2-
ylmethoxy)phenylamino)pyrimidin-5-yl)isooxazol-3-yl)methyl)-
acrylamide (10c). 42 mg of acrylic acid was dissolved in 10 mL of
CH2Cl2, and 140 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide hydrochloride, 20 mg of N-hydroxybenzotriazole, and 255 μL of
N,N-diisopropylethylamine were added thereto, followed by stirring at
(s, 1H), 8.30 (s, 1H), 6.10 (s, 1H), 4.97 (m, 1H), 4.63 (m, 2H), 3.82
(s, 3H).
N-((2-(4-Amino-6-(3-chloro-4-(pyridin-2-ylmethoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19a). Step 1. Preparation of Methyl 2-(4-Amino-6-(3-chloro-4-
(pyridin-2-ylmethoxy)phenylamino)pyrimidin-5-yl)-4,5-dihydrooxa-
zol-4-carboxylate. Compound 17 (1.5 g) and 3-chloro-4-
(pyridin-2-ylmethoxy)benzenamine 8 (1.37 g) were dissolved
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in 25 mL of tert-butanol, and 0.73 mL of 4 N hydrochloric acid
solution in 1,4-dioxane was added thereto dropwise. The
mixture was heated to 70 °C and stirred for 1.5 h, followed by
condensation under a reduced pressure when the reaction was
completed. After removal of solvents, 25 mL of 2-propanol was
added thereto, followed by stirring at 0 °C for 2 h. The
resulting solid was washed with 2-propanol, followed by
filtering and drying under a reduced pressure to obtain the
mg of sodium bicarbonate were dissolved in 2 mL of THF and 0.5 mL
of distilled water at 0 °C, and 8.6 μL of acryloyl chloride was added
thereto, followed by stirring at the same temperature for 10 min. After
the reaction was completed, a saturated aqueous solution of sodium
bicarbonate was added thereto and the mixture was extracted twice
with chloroform. The isolated layer was washed with water and
saturated saline, dried over anhydrous sodium sulfate, followed by
filtering and condensing under a reduced pressure. The resulting
residue was subjected to silica gel flash column chromatography
(CHCl3/CH3OH = 15:1) to obtain the desired compound 19a (20
mg, yield 39%). 1H NMR (300 MHz, CDCl3) δ 10.65 (s, 1H), 8.59 (d,
1H), 8.18 (s, 1H), 7.72 (m, 3H), 7.65 (d, 1H), 7.41 (m, 1H), 7.24 (m,
1H), 6.96 (d, 1H), 6.35 (m, 1H), 6.13 (m, 2H), 5.70 (m, 2H), 5.27 (s,
2H), 4.53 (d, 2H). MS (ESI+): m/z = 478.2 [M + H]+.
N-((2-(4-Amino-6-(3-fluoro-4-(pyridin-2-ylmethoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19b). The similar procedure of 19a was repeated to obtain the
compound 19b (40 mg, final yield 51%). 1H NMR (300 MHz,
CDCl3) δ 10.69 (s, 1H), 8.59 (d, 1H), 8.20 (s, 1H), 7.74 (m, 1H),
7.71 (s, 1H), 7.64 (m, 2H), 7.22 (m, 2H), 6.99 (t, 1H), 6.36 (m, 1H),
6.14 (m, 1H), 6.00 (m, 1H), 5.71 (m, 1H), 5.27 (s, 2H), 4.54 (m, 2H).
MS (ESI+): m/z = 462.2 [M + H]+.
1
title compound (2.10 g, yield 79%). H NMR (300 MHz,
DMSO-d6) δ 11.24 (s, 1H), 8.63 (d, 1H), 8.26 (s, 1H), 8.10
(m, 1H), 7.95 (m, 1H), 7.75 (d, 1H), 7.61 (d, 1H), 7.43 (m,
1H), 7.32 (m, 1H), 7.25 (d, 1H), 5.31 (s, 2H), 5.05 (m, 1H),
4.63 (m, 2H), 3.71 (s, 3H).
Step 2. Preparation of Methyl 2-(4-Amino-6-(3-chloro-4-(pyridin-
2-ylmethoxy)phenylamino)pyrimidin-5-yl)oxazol-4-carboxylate. 2.0
g of the compound obtained in step 1 was dissolved in 40 mL of
dichloromethane, and 1.9 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene
was added thereto at −40 °C, followed by adding thereto 1.3 mL of
bromotrichloromethane dropwise for 20 min. The mixture was
warmed to room temperature and stirred for 2 h. After the reaction
completion monitored by TLC, the reaction mixture was condensed
under a reduced pressure and then crystallized with ethyl acetate. The
resulting solid was washed with ethyl acetate, followed by filtering and
drying under a reduced pressure to obtain the desired compound (0.4
N-((2-(4-Amino-6-(3-chloro-4-(3-fluorobenzyloxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19c). The similar procedure of 19a was repeated to obtain the
compound 19c (9 mg, final yield 47%). 1H NMR (300 MHz,
CD3OD) δ 8.04 (s, 1H), 7.88 (m, 1H), 7.80−7.79 (d, 1H), 7.64−7.03
(m, 6H), 6.30 (s, 1H), 6.28−6.27 (d, 1H), 5.70−5.66 (m, 1H), 5.18 (s,
2H), 4.48 (s, 2H). MS (ESI+): m/z = 495.3 [M + H]+.
1
g, yield 20%). H NMR (300 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.88
(s, 1H), 8.58 (m, 1H), 8.11 (m, 1H), 7.85 (m, 2H), 7.54 (m, 3H), 7.37
(m, 2H), 7.22 (d, 1H), 5.26 (s, 2H), 3.87 (s, 3H).
Step 3. Preparation of (2-(4-Amino-6-(3-chloro-4-(pyridin-2-
ylmethoxy)phenylamino)pyrimidin-5-yl)oxazol-4-yl)methanol. 0.35
g of the compound obtained in step 2 was dissolved in 10 mL of
dichloromethane at 0 °C, and 1.6 mL of 1.0 M lithium aluminum
hydride ether solution was added dropwise thereto. The reaction
mixture was stirred at the same temperature for 1 h. After the reaction
completion monitored by TLC, an aqueous solution of Rochelle salt
(potassium sodium tartrate, NaKC4H4O6) was added thereto. The
resulting mixture was stirred for 1 h and extracted twice with a mixed
solvent of chloroform and 2-propanol (v/v, 3:1). The combined
organic layer was washed with distilled water and saturated saline and
dried over anhydrous sodium sulfate, followed by filtering and
distilling under a reduced pressure. The obtained residue was
subjected to silica gel flash column chromatography (ethyl acetate/
CH2Cl2/CH3OH = 10:10:1) to obtain the title compound (0.27 g,
N-((2-(4-Amino-6-(3-chloro-4-(1-methyl-1H-pyrazol-5-
yloxy)phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)-
acrylamide (19d). The similar procedure of 19a was repeated to
1
obtain the compound 19d (68 mg, final yield 31%). H NMR (300
MHz, CDCl3 + CD3OD) δ 10.96 (bs, 1H), 8.21 (s, 1H), 7.93−7.91
(m, 1H), 7.74 (s, 1H), 7.57−7.53 (m, 1H), 7.14−7.11 (d, 1H), 6.50
(m, 1H), 6.40−6.33 (m, 1H), 6.20−6.11 (m, 1H), 5.73−5.69 (m, 1H),
5.52 (m, 1H), 4.54 (d, 2H), 3.80 (s, 3H). MS (ESI+): m/z = 467.2 [M
+ H]+.
N-((2-(4-Amino-6-(3-chloro-4-(1-methyl-3-(trifluoromethyl)-
1H-pyrazol-5-yloxy)phenylamino)pyrimidin-5-yl)oxazol-4-yl)-
methyl)acrylamide (19e). The similar procedure of 19a was
1
repeated to obtain the compound 19e (25 mg, final yield 67%). H
NMR (300 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.64 (t, 1H), 8.19 (d,
1H), 8.16 (s, 1H), 8.05 (s, 1H), 7.72 (m, 1H), 7.49 (s, 2H), 7.41 (d,
1H), 6.25 (m, 2H), 6.02 (s, 1H), 5.62 (m, 1H), 4.39 (d, 2H), 3.84 (s,
3H). MS (ESI+): m/z = 535.3 [M + H]+.
1
yield 82%). H NMR (300 MHz, CDCl3) δ 10.70 (s, 1H), 8.58 (d,
1H), 8.15 (s, 1H), 7.72 (m, 3H), 7.64 (d, 1H), 7.33 (m, 1H), 7.26 (m,
1H), 6.92 (d, 1H), 5.25 (s, 2H), 4.72 (s, 2H).
Step 4. Preparation of (5-(4-(Aminomethyl)oxazol-2-yl)-N4-(3-
chloro-4-(pyridin-2-ylmethoxy)phenyl)pyrimidin-4,6-diamine. 0.27
g of the compound obtained in step 3 was dissolved in 10 mL of
N,N-dimethylformamide and 2 mL of carbon tetrachloride, and 50 mg
of sodium azide and 400 mg of triphenylphosphine were added
thereto. The reaction mixture was heated to 90 °C and stirred for 30
min. Subsequently, the mixture was condensed under a reduced
pressure to remove carbon tetrachloride, and distilled water was added
thereto, followed by the extraction twice with ethyl acetate. The
combined organic layer was washed with water and saturated saline
and dried over anhydrous sodium sulfate, followed by filtering and
condensing under a reduced pressure. The obtained residue was dried
under a reduced pressure, dissolved in 10 mL of THF, and 437 mg of
triphenylphosphine and 0.1 mL of distilled water were added thereto,
followed by stirring at 60 °C for 2 h. After the reaction completion
monitored by TLC, the reaction mixture was condensed under a
reduced pressure and then subjected to silica gel flash column
chromatography (CHCl3/CH3OH = 1:1) to obtain the desired
compound (50 mg, yield 19%). 1H NMR (300 MHz, CDCl3) δ 10.71
(s, 1H), 8.58 (d, 1H), 8.19 (s, 1H), 7.72 (m, 2H), 7.64 (d, 2H), 7.41
(m, 1H), 7.24 (m, 1H), 6.98 (d, 1H), 5.28 (s, 2H), 3.90 (s, 2H). MS
(ESI+): m/z = 424.3 [M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-phenoxyphenylamino)-
pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide (19f). The similar
procedure of 19a was repeated to obtain the compound 19f (29 mg,
final yield 67%). 1H NMR (300 MHz, CDCl3) δ 10.86 (bs, 1H), 8.25
(s, 1H), 7.92 (d, 1H), 7.74 (s, 1H), 7.55−7.52 (m, 1H), 7.37−7.31 (m,
2H), 7.10−6.97 (m, 4H), 6.41−6.35 (m, 1H), 6.19 (m, 1H), 5.73 (m,
1H), 4.57 (d, 2H). MS (ESI+): m/z = 463.3 [M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-(2,5-dichlorophenoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19g). The similar procedure of 19a was repeated to obtain the
compound 19g (14 mg, final yield 40%). 1H NMR (300 MHz, CDCl3
+ CD3OD) δ 8.37 (s, 1H), 7.97 (m, 1H), 7.75 (s, 1H), 7.50 (m, 1H),
7.40−7.37 (m, 1H), 7.05−6.75 (m, 2H), 6.60 (m, 1H), 6.17 (m, 1H),
5.73 (m, 1H), 4.55 (m, 2H). MS (ESI+): m/z = 531.3 [M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-(2,4-dichlorophenoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19h). The similar procedure of 19a was repeated to obtain the
compound 19h (14 mg, final yield 41%). 1H NMR (300 MHz, CDCl3
+ CD3OD) δ 8.24 (s, 1H), 7.96 (m, 1H), 7.75 (s, 1H), 7.55 (m, 1H),
7.49 (m, 1H), 7.18−7.14 (m, 1H), 6.98−6.95 (d, 1H), 6.78−6.75 (d,
1H), 6.41−6.35 (m, 1H), 6.20−6.17 (m, 1H), 5.75−5.71 (m, 1H),
4.58−4.56 (m, 2H). MS (ESI+): m/z = 531.3 [M + H]+.
Step 5. Preparation of N-((2-(4-Amino-6-(3-chloro-4-(pyridin-2-
ylmethoxy)phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)-
acrylamide (19a). 45 mg of the compound obtained in step 4 and 27
N-((2-(4-Amino-6-(3-chloro-4-(2,3-dichlorophenoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19i). The similar procedure of 19a was repeated to obtain the
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compound 19i (11 mg, final yield 39%). 1H NMR (300 MHz,
CDCl3+CD3OD) δ 8.20 (s, 1H), 7.94−7.93 (m, 1H), 7.40 (m, 1H),
7.20 (m, 1H), 7.10 (m, 1H), 6.98 (m, 1H), 6.60 (m, 1H), 6.34 (m,
1H), 6.10 (m, 1H), 5.80 (m, 1H), 4.56−4.54 (m, 2H). MS (ESI+): m/
z = 531.3 [M + H]+.
1H), 6.16−6.09 (m, 1H), 4.35 (s, 2H). MS (ESI+): m/z = 423.2 [M +
H]+.
N-(2-Acrylamidoethyl)-4-amino-6-((3-chloro-4-(pyridin-2-
ylmethoxy)phenyl)amino)pyrimidine-5-carboxamide (21).
Compound 21 was synthesized in 14.4% yield over three steps from
1
20. H NMR (300 MHz, CDCl3) δ 8.58 (s, 1H), 8.56 (s, 1H), 8.08−
N-((2-(4-Amino-6-(3-chloro-4-(2-fluorophenoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19j). The similar procedure of 19a was repeated to obtain the
compound 19j (7 mg, final yield 21%). 1H NMR (300 MHz, CDCl3 +
CD3OD) δ 8.20 (s, 1H), 7.87 (m, 1H), 7.73 (s, 1H), 7.49 (m, 1H),
7.19 (m, 1H), 7.07 (m, 2H), 6.94 (m, 2H), 6.36 (m, 2H), 6.16 (m,
1H), 5.70 (m, 1H), 4.53 (d, 2H). MS (ESI+): m/z = 481.3 [M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-(3-fluorophenoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19k). The similar procedure of 19a was repeated to obtain the
compound 19k (14 mg, final yield 38%). 1H NMR (300 MHz,
CDCl3) δ 8.24 (s, 1H), 7.95 (m, 1H), 7.72 (s, 1H), 7.59−7.56 (m,
1H), 7.25 (m, 1H), 7.09−7.06 (m, 1H), 6.77−6.71 (m, 2H), 6.67−
6.64 (m, 1H), 6.63 (m, 1H), 6.19−6.15 (m, 1H), 5.73−5.69 (m, 1H),
4.56 (s, 2H). MS (ESI+): m/z = 481.3 [M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-(3-fluorophenoxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19l). The similar procedure of 19a was repeated to obtain the
compound 19l (21 mg, final yield 44%). 1H NMR (300 MHz, CDCl3)
δ 8.24 (s, 1H), 7.90 (m, 1H), 7.73 (s, 1H), 7.51 (m, 1H), 7.02−6.93
(m, 5H), 6.40−6.34 (m, 1H), 6.19−6.10 (m, 1H), 5.74−5.70 (m, 1H).
MS (ESI+): m/z = 481.2 [M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-(pyridin-2-yloxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19m). The similar procedure of 19a was repeated to obtain the
compound 19m (7 mg, final yield 30%). 1H NMR (300 MHz, CDCl3
+ CD3OD) δ 10.93 (s, 1H), 8.20 (s, 1H), 7.90 (s, 1H), 7.73−7.71 (m,
2H), 7.54 (dd, 1H), 7.18 (d, 2H), 7.01−6.96 (m, 2H), 6.37 (d, 1H),
6.18 (t, 1H), 5.68 (d, 1H), 4.53 (d, 2H). MS (ESI+): m/z = 464.3 [M
+ H]+.
N-((2-(4-Amino-6-(3-chloro-4-(pyridin-3-yloxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19n). The similar procedure of 19a was repeated to obtain the
compound 19n (20 mg, final yield 44%). 1H NMR (300 MHz,
CDCl3) δ 11.10 (s, 1H), 8.64 (t, 1H), 8.33 (m, 2H), 8.17 (m, 2H),
8.05 (s, 1H), 7.70 (m, 1H), 7.49 (s, 1H), 7.39 (m, 1H), 7.25 (m, 2H),
6.20 (m, 2H), 5.62 (m, 2H), 4.39 (d, 2H). MS (ESI+): m/z = 464.3
[M + H]+.
N-((2-(4-Amino-6-(3-chloro-4-(6-methylpyridin-3-yloxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19o). The similar procedure of 19a was repeated to obtain the
compound 19o (1 mg, final yield 2%). 1H NMR (300 MHz, CDCl3) δ
8.23 (d, 1H), 8.21 (s, 1H), 7.92 (d, 1H), 7.75 (s, 1H), 7.55 (m, 1H),
7.16 (m, 2H), 7.02 (m, 2H), 6.50 (m, 1H), 6.37 (m, 1H), 6.15 (m,
1H), 5.71 (m, 1H), 4.55 (d, 2H), 2.54 (s, 3H). MS (ESI+): m/z =
478.2 [M + H]+.
N-((2-(4-Amino-6-(3-methyl-4-(6-methylpyridin-3-yloxy)-
phenylamino)pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide
(19p). The similar procedure of 19a was repeated to obtain the
compound 19p (34 mg, final yield 76%). 1H NMR (300 MHz,
CDCl3) δ 10.65 (bs, 1H), 8.28−8.27 (d, 1H), 8.23 (s, 1H), 7.74 (s,
1H), 7.53 (d, 1H), 7.48−7.44 (m, 1H), 7.10 (m, 2H), 6.92−6.89 (d,
1H), 6.40−6.34 (m, 1H), 6.20−6.11 (m,1H), 5.74−5.70 (m, 1H), 4.57
(d, 2H), 2.54 (s, 3H), 2.28 (s, 3H). MS (ESI+): m/z = 458.4 [M +
H]+.
7.87 (m, 1H), 7.75−7.69 (m, 2H), 7.38−7.32 (m, 2H), 7.02 (d, 1H),
6.23 (dd, 2H), 5.68 (d, 1H), 5.28 (s, 2H), 3.43 (t, 2H), 3.36 (t, 2H).
MS (ESI+): m/z = 468.8 [M + H]+.
General Procedures for the Cell Growth Inhibitory Assay. A
human vaginal epidermoid cancer cell line A431 (ATCC, CRL-1555)
and a human breast cancer cell line SK-Br3 (ATCC, HTB-30) were
used to measure the inhibitory activities of synthesized compounds
toward cancer cell growth. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) with 4.5 g/L glucose and 1.5 g/L sodium
bicarbonate and were supplemented with 10% fetal bovine serum
(FBS). In addition, MDA-MB-175, MDA-MB-361, MDA-MB-453,
NCI-N87, NCI-H1781, and NCI-H1975 cells were purchased from
the American Type Culture Collection (ATCC). JIMT-1 cell line was
purchased from DSMZ (Deutsche Sammlung von Mikroorganismen
and Zellkulturen GmbH) and was incubated in the same conditions as
above. MDA-MB-175, MDA-MB-361, and MDA-MB-453 cells were
incubated in L-15 medium, and NCI-N87, NCI-H1781, and NCI-
H1975 cells were incubated in RPMI medium containing 1 mM
sodium pyruvate. All culture media were supplemented with 1%
penicillin−streptomycin and 10% fetal bovine serum (FBS). Cells were
incubated in a humidified atmosphere under 5% CO2 except for MDA-
MB-175, MDA-MB-361, and MDA-MB-453 cells (CO2 free) at 37 °C.
Cell growth inhibition assays and GI50 determinations were performed
as previously described.28
General Procedures for the Microsomal Stability Assay. All
assays were conducted in duplicate. The incubation mixtures were
prepared in E-tube and were contained with 5 μM synthesized
analogue, 1 mg/mL liver microsomes (from mouse, rat, dog, and
human), and NADPH regeneration solution (1.3 mM NADP+, 3.3
mM glucose 6-phosphate, 3.3 mM MgCl2, and 0.4 U/mL glucose 6-
phosphate dehydrogenase) in 100 mM potassium phosphate buffer
solution. Reactions were initiated by the addition of NADPH and kept
in a shaking water bath at 37 °C. At the each sampling time, aliquots
were removed and added to termination solvent (acetonitrile). The
samples were centrifuged for 4−5 min at 13 000 rpm, and the
supernatant was subjected to HPLC analysis (HPLC/Agilent 1200
series). In the determination of the in vitro t1/2 (half-life, HL), the
analyte peak areas were converted to percentage of drug remaining,
using the T = 0 peak area values as 100%. The slope of the linear
regression from log percentage remaining versus incubation time
relationships (−k) was used in the conversion to the in vitro t1/2
,
values by the in vitro t1/2= −0.693/k. The percent remaining of test
compound is calculated compared to the initial quantity at 0 time.
Pharmacokinetic Studies. Male mice (imprinting control region
mice, body-weight range of 27 5 g, iv, n = 3, po, n = 3) and male rats
(Sprague−Dawley rats, body-weight range of 250 10 g, iv, n = 3, po,
n = 3) were administered analogue 19a intravenously via the tail vein
at 2 and 1 mg/kg, respectively, or orally at 10 and 5 mg/kg,
respectively, by gavage in a solution of 30% PEG400 and 5% ethanol in
distilled water. For the in vivo study, 19a was prepared as HCl salt. At
predetermined times 24 h or more after dosing, 0.3 mL blood was
collected from the jugular vein using a tube containing anticoagulant
(1000 IU/mL, heparin, 3 μL). The plasma was separated by
centrifugation (12 000 rpm, 2 min, Eppendorf). The concentrations
of the compound were measured in the plasma using LC/MS/MS
after protein precipitation with acetonitrile. The relevant estimated
pharmacokinetic parameters for plasma were derived using WinNon-
lin, version 5.2 (Pharsight).
N-((2-(4-Amino-6-(3-chloro-4-fluorophenylamino)pyrimidin-
5-yl)oxazol-4-yl)methyl)acrylamide (19q). The similar procedure
of 19a was repeated to obtain the compound 19q (19 mg, final yield
1
16%). H NMR (300 MHz, CDCl3) δ 8.07 (s, 1H), 7.75 (dd, 1H),
7.69 (s, 1H), 7.42−7.37 (m, 1H), 7.06 (t, 1H), 6.28 (d, 1H), 6.20 (d,
1H), 5.63 (d, 1H), 4.42 (s, 2H). MS (ESI+): m/z = 389.2 [M + H]+.
N-((2-(4-Amino-6-(3,4-dichloro-2-fluorophenylamino)-
pyrimidin-5-yl)oxazol-4-yl)methyl)acrylamide (19r). The similar
procedure of 19a was repeated to obtain the compound 19r (26 mg,
General Procedures for the EGFR Enzyme Assay. An amunt of
10 μL of EGFR enzyme (EGFRWT, EGFRT790M, HER-2, or HER-4
kinase, Upstate) was added to each well of a 96-well microplate. As an
EGFR inhibitor, 10 μL of serially diluted solution of synthesized
compounds was added to the individual wells, and the plate was
incubated at room temperature for 10 min. Then 10 μL of Poly (Glu,
1
final yield 45%). H NMR (300 MHz, DMSO-d6) δ 8.38−8.30 (m,
1H), 8.13 (s, 1H), 8.04 (s, 1H), 7.61−7.48 (m, 3H), 6.32−6.23 (m,
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Tyr 4:1, Sigma) and 10 μL of ATP were successively added to initiate
a kinase reaction, and the resulting mixture was incubated at room
temperature for 1 h. 10 μL of 100 mM EDTA was added to each well,
and the mixture was stirred for 5 min to terminate the kinase reaction.
Then 10 μL of 10× anti-phosphotyrosine antibody (Pan Vera), 10 μL
of 10× protein tyrosine kinase (PTK) green tracer (Pan Vera), and 30
μL of fluorescence polarization (FP)-diluted buffer were added to the
reacted mixture, followed by incubation in the dark at room
temperature for 30 min. The FP value of each well was determined
using a VICTORIII fluorescence meter (Perkin-Elmer) at 488 nm.
The IC50, i.e., the concentration at which 50% inhibition was observed,
was determined by setting the maximum value (0% inhibition) to the
polarized light value for a well untreated with EGFR inhibitor and the
minimum value (100% inhibition). IC50 calculations and analysis were
carried out using Microsoft Excel.
Prolongation of Phosphorylation Inhibition. Cells were plated
at a density of 5 × 105/well in six-well plates under normal culture
conditions (10% FBS and 1% penicillin−streptomycin). After 24 h, the
medium was changed to 0.1% FBS medium and cells were incubated
for 16 h. Cells were then treated with 1 μM analogue 19a for 4 h. Each
set was washed 4 times with warmed compound-free medium and
incubated for 0 and 8 h. Each set was stimulated with EGF (100 ng/
mL) for 5 min. The phosphorylation rates of EGFR or HER-2 were
measured by Western blotting.
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ASSOCIATED CONTENT
* Supporting Information
Scheme showing the synthesis of compound 21 and figure
showing prolonged inhibitory results on the phosphorylation of
EGFR for compound 19a. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
(15) Koninki, K.; Barok, M.; Tanner, M.; Staff, S.; Pitkanen, J.;
̈
̈
Hemmila, P.; Ilvesaro, J.; Isola, J. Multiple molecular mechanisms
̈
underlying trastuzumab and lapatinib resistance in JIMT-1 breast
cancer cells. Cancer Lett. 2010, 294, 211−219.
AUTHOR INFORMATION
Corresponding Author
*For S.B.P.: phone, +82-2-880-9090; fax, +82-2-884-4025; e-
mail, sbpark@snu.ac.kr. For M.S.K.; phone, +82-31-371-5001;
fax, +82-31-371-5006; e-mail, kims@hanmi.co.kr.
■
(16) Adjei, A. A. Epidermal growth factor receptor tyrosine kinase
inhibitors in cancer therapy. Drugs Future 2001, 26, 1087−1092.
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B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.;
Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; Nilakantan, R.;
Shen, R.; Wang, Y.; Greenberger, L. M.; Tsou, H. Synthesis and
structure−activity relationships of 6,7-disubstituted 4-anilinoquinoline-
3-carbonitriles: the design of an orally active, irreversible inhibitor of
the tyrosine kinase activity of the epidermal growth factor receptor
(EGFR) and the human epidermal growth factor receptor-2 (HER-2).
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Wang, Y. F.; Upeslacis, J.; Wissner, A. Optimization of 6,7-
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BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in
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Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
HER, human epidermal growth factor receptor; EGFR,
epidermal growth factor receptor; IC50, half-maximal inhibitory
concentration; po, per os; AUClast, area under the curve from 0
h to last time; Cmax, maximum plasma concentration; T1/2, drug
half-life; F, oral bioavailability
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