Oxopyrido[2,3-d]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00193

In nonsmall cell lung cancer (NSCLC), the threonine⁷⁹⁰-methionine⁷⁹⁰ (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFR^L858R,T790M with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR^L858R,T790M driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR.

Full text of DOI:10.1021/acsmedchemlett.5b00193

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Oxopyrido[2,3-d]pyrimidines as Covalent L858R/T790M Mutant Se-
lective Epidermal Growth Factor Receptor (EGFR) Inhibitors.
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Ryan P. Wurz*,† Liping H. Pettus,† Kate Ashton,† James Brown,† Jian Jeffrey Chen,† Brad Herber-
ich,† Fang-Tsao Hong,† Essa Hu-Harrington,† Tom Nguyen,† David J. St. Jean, Jr.† Seifu Tadesse,†
David Bauer,¡ Michele Kubryk,¡ Jinghui Zhan,‡ Keegan Cooke,‡ Petia Mitchell,‡ Kristin L. An-
drews,§ Faye Hsieh,# Dean Hickman,# Nataraj Kalyanaraman,# Tian Wu,% Darren L. Reid,% Ed-
ward K. Lobenhofer,Φ Dina A. Andrews,¥ Nancy Everds,¥ Roberto Guzman,¥ Andrew T. Parsons,¤
Simon J. Hedley, ⊥ Jason Tedrow, ⊥ Oliver R. Thiel,⊥ Matthew Potter,Ψ Robert Radinsky, ‡ Pedro J.
Beltran,‡ and Andrew S. Tasker.†
†Medicinal Chemistry, ‡Oncology Research, §Molecular Structure, #Pharmacokinetics and Drug Metabolism, %Oral
Delivery − Product and Process Development, ΦDiscovery Toxicology, ¥Pathology, ⊥Chemical Process R&D, Amgen
Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States
¡Medicinal Chemistry , ¤ Chemical Process R&D, ΨAnalytical R&D, Amgen Inc., 360 Binney Ave. Cambridge, MA
02142-1011, United States
KEYWORDS: Epidermal growth factor receptor, EGFR, non-small cell lung cancer, EGFR T790M mutant, irreversible
inhibitor, kinase inhibitor
ABSTRACT: In non-small cell lung cancer (NSCLC), the threonine790−methionine790 (T790M) point mutation of EGFR
kinase is one of the leading causes of acquired resistance to the first genera-
tion tyrosine kinase inhibitors (TKI’s), such as gefitinib and erlotinib. Herein,
we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-
derived irreversible inhibitors of EGFR kinase. This led to the discovery of
compound 24 which potently inhibits gefitnib-resistant EGFRL858R,T790M with
100−fold selectivity over wild-type EGFR. Compound 24 displays strong an-
tiproliferative activity against the H1975 non-small cell lung cancer cell line,
the first line mutant HCC827 cell line, as well as promising antitumor activity
in an EGFRL858R,T790M driven H1975 xenograft model sparing the side-effects
associated with the inhibition of wild-type EGFR.
Aberrant signaling by the epidermal growth factor receptor
(EGFR, ErbB1) kinase is a well validated target in the lung
cancer setting. Several EGFR inhibitors, such as gefitinib and
erlotinib (Figure 1), have achieved significant clinical benefit
in the management of non-small cell lung cancer (NSCLC) in
patients with activating point mutations in exon 21 in the
EGFR kinase domain, such as L858R and in-frame deletions,
such as del(E746−A750), in exon 19. Unfortunately, a sec-
ondary threonine790–methionine790 mutation (T790M) in the
EGFR catalytic domain is responsible for approximately 50%
of the clinical acquired drug resistance in NSCLC patients.1-3
tors (Figure 1). Afatinib (gilotrif) was recently approved by
the US FDA for treatment of late-stage NSCLC patients with
actively mutated EGFR.14 All the second generation inhibi-
tors, however, lack significant selectivity between EGFRT790M
mutants and the wild-type kinase which leads to dose-limiting
toxicities in these patients, including skin rash and diarrhea.
Only limited clinical efficacy was observed for these inhibi-
tors, and this may be due to the fact that the inhibitors at the
tolerated therapeutic concentrations are insufficient to ade-
quately suppress the functions of EGFRT790M mutant.
The desire to identify inhibitors selective for the EGFR mu-
tants (especially T790M) while sparing EGFRWT led to a third
generation of irreversible EGFR inhibitors pioneered by Zhou
and co-workers with the discovery of WZ-4002 (Gatekeeper
Pharmaceuticals Inc.). This generation of inhibitors demon-
strated that much higher levels of selectivity for EGFRT790M
were possible (Figure 2).15 Since that report, a significant
In order to address resistance to the first generation of re-
versible tyrosine kinase inhibitors (TKIs), a second generation
of irreversible4-5 EGFR inhibitors was developed.6-9 CI-1033
(canertinib)10, HKI-272 (neratinib)11,12 and PF-00299804
(dacomitinib)13 advanced into late stage clinical development
and structurally resemble the first generation of EGFR inhibi-
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strate for an aldol-like cyclocondensation reaction with ester
enolates to afford the 7-oxopyridopyrimidinyl scaffold (10).31
In this manner, analogs with the desired substitution patterns
at the 5- and 6-positions of the pyridone ring could be pre-
pared. The Boc-protecting group was removed with TFA and
the acrylamide warhead was introduced using acryloyl chlo-
ride to provide intermediate 11. Oxidation of the me-
thyl(thio)pyrimidinyl motif to the sulfoxide with m-CPBA
furnished sulfoxide (12) which underwent SNAr displacement
with the desired aniline to afford the final product 13.
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2
3
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12
13
14
15
16
17
Figure 1. First and second generation EGFR inhibitors.
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24
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Figure 3. Bicyclic scaffolds resulting from the fusion of het-
erocycles onto the core of WZ-4002.
Figure 2. Third generation EGFR inhibitors.
Scheme 1. Synthesis of EGFR Inhibitor Analogs
number of third generation inhibitors have entered early clini-
cal trials. Recent disclosures by Clovis Oncology and Astra-
Zeneca revealed their clinical candidates CO-168616 and
AZD-929117-19 which possess selectivity for the
EGFRL858R,T790M mutant over EGFRWT 20-22
.
Our efforts in this area began with compound 1 which was
the result of structure-guided design. Inspired by our previous
research efforts on p38 MAP kinase23,24 and the structural sim-
ilarities of the ATP binding pockets of p38 and EGFR we ex-
plored the fusion of a pyridone ring onto the scaffold of WZ-
4002 (Figure 3). The carbonyl oxygen of the pyridone ring
would serve as a hydrogen-bond acceptor to satisfy the coor-
dination requirements of a crystallographically resolved water
molecule in the ATP binding pocket of EGFR protein and help
improve inhibitor potency. Ding and co-workers have also
explored similar approaches and recently communicated their
efforts involving the fusion of cyclic ureas (compounds 2-
3)25,26 a pyrimidino[4,5-d]pyrimidin-4(1H)-one scaffold (com-
pound 4)27 and a pteridine-7(8H)-one scaffold (compound 5)28
onto the WZ-4002 core with varying degrees of selectivity for
Reagents and conditions: (a) K2CO3 (2 equiv.), m-NH2-C6H4-
NHBoc, DMF, 80 °C, 86%; (b) i) LiAlH4 (2.5 equiv.), THF, 0
°C; ii) MnO2, CHCl3, RT, 66% (2 steps); (c) i) RMgBr (3.2
equiv.), THF, 0 °C - RT, 1 h; ii) MnO2, CHCl3, 60 °C, 16 h; (d)
R1CH2CO2Et, LiHMDS, THF, -78 °C – RT; (e) i) TFA, CH2Cl2,
RT 30 min ii) acryloyl chloride, Hunig’s base, CH2Cl2, 0 °C-
RT, 1 h; (f) m-CPBA (1.1 equiv.), CH2Cl2, 0 °C, 1 h; (g) DMAC,
Ar-NH2 (2.2 equiv.), 95 °C, 6 h.
EGFRL858R,T790M over EGFRWT
.
We designed synthesis of the 7-oxopyrido[2,3-
a
An NCI-H1975 adenocarcinoma cell line bearing
d]pyrimidinyl core that would allow for versatile analoging
(Scheme 1).29,30 Commercially available ethyl 4-chloro-2-
(methylthio)-pyrimidine-5-carboxylate (6) underwent an
SNAr-type displacement with mono-Boc protected 1,3-
phenylenediamine to produce diaryl amine 7 in high yield.
Reduction of the ethyl ester with lithium aluminum hydride
afforded the corresponding alcohol, which was subsequently
oxidized with MnO2 to aldehyde 8. Treatment of the aldehyde
with the appropriate Grignard reagent followed by oxidation
of the resulting secondary alcohol to ketone 9 provided a sub-
EGFRL858R,T790M point mutations and a HCC827 adenocarci-
noma cell line bearing the deletion mutation (EGFRΔ746-750
)
were used for potency optimization while a A431 epidermoid
carcinoma cell line bearing over-expressed EGFRWT was used
as a counterscreen, allowing an early assessment of selectivity
over EGFRWT
.
With a synthetic route in hand to facilitate exploration of a
structure-activity relationship for the 7-oxopyrido[2,3-
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d]pyrimidinyl analogs, compound 1 was prepared (via Scheme while maintaining the 5-methyl substitution on the pyridone
1
2
3
4
5
6
7
8
1, omitting step c). Compound 1 displayed single digit nano-
molar activity in both of the EGFR mutant cell lines with good
selectivity against the A431 cell line (Table 1). This com-
pound was further profiled in a biochemical cell signaling
assay by meso scale discovery (MSD)32 and showed reduced
selectivity (ca. 17-fold) against the A431 cell line (Table 3).
Given the large differences in affinities for ATP33 between the
different mutant forms and EGFRWT, we believed it would be
beneficial to further improve upon selectivity over EGFRWT in
order to give a better window of tolerability. These large dif-
ferences in ATP affinity also influenced our decision to use
cell-based assays to direct SAR efforts instead of the tradition-
al enzyme-based assays.
ring. It quickly became apparent that small modifications to
the left-hand side had a significant impact on the properties of
the inhibitor. For example, removal of the ortho-methoxy
group on the aniline resulted in compound 18 which main-
tained potency in the proliferation assays. However, this
compound had a greatly diminished pharmacodynamic (PD)
effect as compared to compound 14 presumably due to a pre-
cipitous drop in exposure. N-acylation of the piperazinyl
group (compound 19), resulted in loss in inhibitor potency as
did removal of the methyl group (compound 20).
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10
11
12
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21
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23
24
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Table 1. Antiproliferation Activities for Compounds
Arising from Modifications to the Pyridone Ringa
R
5
R1
N
6
HN
N
N
O
Modeling studies docking 7-oxopyrido[2,3-d]pyrimidinyl
analogs into the reported crystal structure of WZ-4002 in
EGFRT790M (PDB code: 3IKA) suggested that introduction of
small hydrophobic groups into the gatekeeper pocket would
potentially benefit selectivity. The threonine present in
EGFRWT is more sterically congested and polar in nature while
the EGFRT790M bears a methionine residue which is hydropho-
bic, and given the lack of a β-branch, more accommodating of
small aliphatic substitutions. Given these differences in the
gatekeeper pocket between the T790M mutant and EGFRWT
protein, we investigated a brief, but focused series of substitu-
tion patterns in the 5- and 6-positions on the 7-oxopyrido[2,3-
d]pyrimidinyl ring.
O
O
N
H
N
N
H1975
IC50
(nM)
26
8
HCC827 A431
Cmpd
R
R1
IC50
(nM)
IC50
(nM)
AZD9291 N/A
1
14
15
16
N/A
H
6
4
12
41
61
18
849
5229
1200
1387
201
H
Me
Me
H
H
4
OMe 10
OMe 273
Introduction of a methyl-group in the 5-position of the pyri-
done ring (14) led to a compound which was roughly equipo-
tent to compound 1 in the H1975 (H1975 IC50 = 4 nM) and
HCC827 cell lines (HCC827 IC50 = 12 nM) and also >100-
fold selective against the wild-type expressing cell line (A431
IC50 = 1200 nM). This potency and selectivity profile was
also confirmed by the MSD assay in which the potency in the
A431 cell line remained ca. 100-fold (A431 IC50 = 386 nM,
Table 3). Introduction of a methoxy group to the 6-position
(compound 15) resulted in a very similar profile with slight
erosion in potency in the H1975 cell line (H1975 IC50 = 10
nM) and a small loss in selectivity. Removal of the 5-methyl
group (compound 16) led to a significant loss in potency and a
complete loss in selectivity for the EGFRL858R,T790M mutant
17
H
Bn
4
250
a
Data represents an average of at least two separate de-
terminations. Standard deviations are reported in the Sup-
porting Information and vary by less than ± 50%.
An N-methylpiperidine (compound 21) was a replacement
that was tolerated in terms of potency in the mutant cell lines
but resulted in a loss in selectivity for the EGFRL858R,T790M mu-
tant over EGFRWT. A morphilino-group (compound 22) re-
sulted in complete loss of potency in the proliferation assay
due to a signficant loss in permeability. Ring-opening of the
N-methylpiperazine to
a N,N,N’-trimethylethylenediamine
(compound 23) resulted in a compound that was virtually
equipotent to compound 14 and maintained a robust PD effect.
Compound 23 was found to be slightly less efficacious in a
H1975 xenograft model than compound 14 (90% TGI at 30
mg/kg QD). However, in an exploratory 4-day rat toxicology
study this analogue was devoid of hepatobiliary toxicity. Ef-
forts to prepare this compound on larger scale for additional
toxicology studies led to the realization of a potential stability
liability for this compound. A variable amount of the quater-
nary ammonium salt byproduct (26) was observed during iso-
lation of the final API. Mechanistically, this decomposition
product presumably results from oxidation of the electron rich
aniline ring to aza-quinone intermediate 25 followed by 1,4-
nucleophilic addition to restore aromaticity (eq 1).
over EGFRWT
position (compound 17) was found to adversely influence
selectivity over EGFRWT
.
Introduction of a benzyl group in the 6-
.
Compound 14 was then evaluated for its pharmacodynamic
(PD) effect in female athymic nude mice. These mice were
implanted with matrigel plugs containing H1975 tumor cells
(5 x 106 cells) and dosed orally with compound 14 at 30
mg/kg. At 4 h post dosing, tumor cell plugs and plasma were
harvested. Total p-EGFR levels in the plug were measured
with a quantitative MSD assay resulting in 90% inhibition of
p-EGFR (Table 2). Compound 14 was also efficacious in a
H1975 murine xenograft model resulting in tumor stasis at 30
mg/kg QD.34 However, in an exploratory 4-day rat toxicology
study, hepatobiliary toxicity (moderate to marked increased
serum biomarkers of hepatobiliary perturbation/injury with
histologic evidence of minimal to mild portal inflammation
and hepatocellular necrosis) was observed at low exposure
multiples. Due to the observed toxicity, we decided to explore
structure-activity relationships (SAR) for replacements for the
N-methylpiperazine motif on the left-hand side of the inhibitor
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This mechanistic proposal led to the design of an analog
SNAr displacement with the mono-Boc protected 1,3-
phenylenediamine to furnish 28. Heck-coupling with crotonic
acid followed by intramolecular dehydration of the resulting
adduct with Ac2O afforded the 7-oxopyrido[2,3-d]pyrimidine
core 29 in modest yields. Subsequent Boc-deprotection using
HCl, followed by neutralization with NaOH and treatment
with acryloyl chloride yielded the compound 30. Anilines
could be introduced via SNAr displacement of the chloride in
refluxing 2,2,2-trifluoroethanol. In the case of compound 24,
it is noteworthy that the use of the monohydrochloride salt of
the aniline is critical for the success of this reaction to prevent
displacement of the chloride by the more electron rich dime-
thylamine pendant of this reactant.
1
2
3
4
5
6
7
8
with a slightly less electron-rich aniline. Compound 23, the
corresponding ether linked analogue (of compound 24), was
prepared and found to have slightly improved potency and
greatly improved stability. Compound 24 also maintained the
robust PD effect as observed with compound 23. Its selectivi-
ty was also confirmed in the MSD assay with >100−fold se-
lectivity over EGFRWT
. Hepatobiliary toxicity was also not
observed when this compound was tested in a 4-day rat toxi-
cology study.
9
Table 2. Antiproliferation Activities for Compounds
Arising From Modifications to the Left-Hand-Sidea
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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57
58
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60
Table 3. Cell Signaling: Inhibition of p-EGFR by MSD
A431
H1975
HCC827
Cmpd
IC50
(nM)
IC50 (nM)
IC50 (nM)
PDb
%
Inhib
AZD9291 15
6
3
348
50
H1975
IC50
(nM)
HCC827 A431
Cmpd R3
AZD
R4
IC50
(nM)
IC50
(nM)
1
3
14
23
24
4
6
4
12
12
9
386
521
1054
N/A
N/A
26
6
849
92
9291
a
Data represents an average of at least two separate de-
terminations. Standard deviations are reported in the Sup-
porting Information and vary by less than ± 50%.
14
OMe
H
4
12
1200
90
5
Scheme 2. Second Generation Synthesis of Com-
pound 24
18
4
7
>3000
19
OMe
OMe
30
58
>5000 NT
N
20
68
4
211
33
>1000
NT
N
H
21
OMe
OMe
681
NT
NT
NT
Reagents and conditions: (a) K2CO3 (1.3 equiv.), m-NH2-
C6H4-NHBoc, DMF, RT, 16 h, 98%; (b) PdCl2(PhCN)2, P(o-
tolyl)3, crotonic acid, DIEA, PhMe, 60 °C; (c) Ac2O, 60 °C, 1 h,
60% (2 steps); (d) i) 4N HCl, 1,4-dioxane, 50 °C ii) 10N NaOH,
22
>10,000 Undef.
.
acryloyl chloride, 92% (2 steps); (e) Ar-NH2 HCl (1.1 equiv.),
23
OMe
OMe
8
4
22
28
>1000
2400
80
90
2,2,2-trifluoroethanol, reflux, 16 h, 51%.
Modeling of compound 24 in the EGFRT790M protein sug-
gested a number of key interactions with the mutant EGFR
protein that likely contributed to this compound’s potency.35
The N−H of the aminopyrimidine engages the linker residue in
a pair of hydrogen bonding interactions (2.9, 3.2 Å) with the
Met793 residue of the linker region of the EGFR protein in the
ATP binding pocket. The crystallographically resolved water
molecule between catalytic Lys745/Asp855 residues has an H-
bond satisfied by the pyridone carbonyl. The 5-methyl group
is projected back into the gatekeeper pocket engaging the
Met790 residue with van der Waals contacts. This is sterically
and electrostatically unfavorable in the case of EGFRWT pro-
tein where the gatekeeper residue is a threonine. The acryla-
mide motif is projected with the right trajectory onto the floor
of the ATP binding pocket to allow for a Michael addition
with the Cys797 residue.
24
a
Data represents an average of at least two separate de-
terminations. Standard deviations are reported in the Sup-
porting Information and vary by less than ± 50%. Athymic
nude mice were implanted with matrigel plugs containing 5 x
106 H1975 cells. Animals (n = 3) were treated with control or
compound (30 mg/kg po dose). Plug & plasma samples were
collected 4 hr post sample administration. Results denotes
% p-EGFR/PRAS40 vs control. NT = not tested.
b
As demand for larger quantities of material grew to facili-
tate additional studies, a more efficient synthetic route was
developed (Scheme 2). This alternative route used commer-
cially available 5-bromo-2,4-dichloropyrimidine (27) and a
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The pharmacokinetic (PK) properties of several compounds lular potency in proliferation assays. Compound 24 was orally
1
2
3
4
5
6
7
8
were assessed (Table 4). These were found to have clearance
in excess of liver blood flow in rats and low oral bioavailabil-
ity. This was believed to be related to the high turnover of the
acrylamide motif in the presence of glutathione. Although
these compounds had poor pharmacokinetic properties in
rats,36,37 we believed it would be possible to obtain a sustained
pharmacodynamic (PD) effect due to the covalent nature of
the interaction. Compound 24 was then evaluated in a time
course PD experiment upon oral dosing at 30 mg/kg.38 At 2,
4, 8, 12 and 24 h time points post dosing, tumor cell plugs and
plasma were harvested. Compound 24 showed a >50% inhibi-
tion of phosphorylation of EGFR for >12 h. Compound 24
reached maximal concentration of 0.10 μM at 2 h and system-
ic exposure (AUC0-inf.) was 0.33 μM.h.
efficacious in an H1975 plug PD assay in mice resulting in a
>50% inhibition of p-EGFR for >12 h. In an H1975 xenograft
model, oral dosing of compound 24 at 30 mg/kg daily for two
weeks led to significant tumor growth inhibition with no ob-
served loss in body weight. In an exploratory 4-day rat toxi-
cology study, no hepatobiliary toxicity was observed. Addi-
tional data regarding compound 24 will be reported in due
course.
9
ASSOCIATED CONTENT
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supporting Information. Experimental procedure for the
preparation of compound 24, NMR data for all compounds.
Description of assays. ScanMAX KINOMEscanTM panel data
for compound 24. This material is available free of charge via
the Internet at http://pubs.acs.org.
In a mouse H1975 xenograft model, compound 24 was ad-
ministered orally at 30 mg/kg daily (QD) beginning on day 6.
This resulted in 90% tumor growth inhibition (AUC0-t = 0.56
μM.h). It is noteworthy that no changes in body weights of all
treatment groups were observed.39
AUTHOR INFORMATION
Corresponding Author
*Tel: 805-313-5400. Fax: 805-480-1337. E-mail:
rwurz@amgen.com.
Table 4. Pharmacokinetic properties of selected com-
poundsa
Cmpd
ABBREVIATIONS
ivb
po
AUC, area under the plasma concentration−time curve; CL,
clearance; EC50, molar concentration that produces half
maximal response; F, bioavailability; MRT, mean residence
time; Vss, volume of distribution in steady state.
CL
Vdss
t1/2
(h)
1.4
3.0
2.6
4.5
AUC(0-inf.)
(μM*h)
0.18
0.18
0.16
F
((L/h)/kg) (L/kg)
(%)
6.6c
9.5c
21d
10e
1
8.9
10.6
7.7
19
14
23
24
a
7.8
20
22
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421.
Pharmacokinetic parameters following administration in
male Sprague Dawley rats; mean values from 3 animals per
dosing route. Dosed at 1 mg/kg as a solution in DMSO.
po doses were 10 mg/kg in 1% Pluronic F68, 2% hydroxypro-
pyl methylcellulose (HPMC), 15% hydroxypropyl β-
cyclodextrin (HPBCD), 82% water/methanesulfonic acid pH
b
c
d
4.5. po doses were 10 mg/kg in 20% hydroxypropyl β-
cyclodextrin (HPBCD), 80% water/methanesulfonic acid pH
e
4.0.
po doses were 10 mg/kg in 1% Pluronic F68, 2% hy-
methylcellulose (HPMC), 97% wa-
ter/methanesulfonic acid pH 4.0.
droxypropyl
The selectivity of compound 24 against a panel of 100 pro-
tein and lipid kinases was examined in the ScanMAX KI-
NOMEscanTM panel.40 In this panel, the competitive binding
of 24 at 1 μM was measured as a percentage of control (POC).
Competitive binding (POC <30%) by 24 was observed for
only 2 kinases, BTK (17% POC) and TTK (4% POC). As a
follow-up, the Kd values were measured on a number of ki-
nases including BLK (320 nM), BTK (290 nM), EGFRWT (220
nM), EGFRT790M (0.96 nM), ERBB2 (220 nM), ERBB4 (130
nM), GAK (430 nM), ITK (140 nM), JAK1 (JH2 domain-
pseudokinase, 2800 nM), JAK3 (30 nM), TTK (81 nM) and
TXK (410 nM). Compound 24 possessed moderate permea-
bility and high efflux (10 x 10-6 cm/s and ER = >37 and >37,
respectively in human and rat LLC-PK1 cell line transfected
with a MDR1 gene), but exhibited low plasma protein binding
(Fu = 0.25, 0.27, 0.54 and 0.46 in mouse, rat, human and dog,
respectively).
7) Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.;
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34) Data not shown.
35) See Figure 3 in Supporting Information.
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38) See Figure 1 in Supporting Information for results.
39) See Figure 2 in Supporting Information for results.
40) See Supporting Information for details.
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