Discovery of aminopyridine-containing spiro derivatives as EGFR mutations inhibitors

Article abstract of DOI:10.1080/14756366.2019.1634704

Neratinib is an oral pan HER inhibitor, that irreversibly inhibits EGFR and HER2 and was proven to be effective against multiple EGFR mutations. In previous study, we reported spiro [indoline-3, 4′-piperidine]-2-ones as anticancer agents. In this study, we designed aminopyridine-containing spiro [indoline-3,4′-piperidine] derivatives A1-A4 using Neratinib and spiro [indoline-3, 4′-piperidine]-2-one compound patented as lead structure, then replaced piperidine with cyclopropane to obtain B1-B7 and replaced indoline with benzmorpholine to get C1-C4 and D1-D2. We synthesized these compounds and evaluated their residual activities under 0.5 M drug concentration on EGFR and ERBB2. Most of compounds showed stronger inhibition on EGFR-wt and ERBB2, in which A1-A4 showed excellent inhibitory activity with inhibition percentage on EGFR-wt kinase of 7%, 6%, 19%, 27%, respectively and 9%, 5%, 12%, 34% on ERBB2 kinase compared with 2% and 6% of Neratinib.

Full text of DOI:10.1080/14756366.2019.1634704

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of aminopyridine-containing spiro
derivatives as EGFR mutations inhibitors
Lianbao Ye, Tao Zhao, Wenjun Du, Anhu Li, Wei Gao, Jingrong Li, Ling Wang &
Weiqiang Chen
To cite this article: Lianbao Ye, Tao Zhao, Wenjun Du, Anhu Li, Wei Gao, Jingrong Li, Ling
Wang & Weiqiang Chen (2019) Discovery of aminopyridine-containing spiro derivatives as EGFR
mutations inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1233-1246, DOI:
10.1080/14756366.2019.1634704
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1233–1246
https://doi.org/10.1080/14756366.2019.1634704
RESEARCH PAPER
Discovery of aminopyridine-containing spiro derivatives as EGFR
mutations inhibitors
a
a
a
b
a
b
d
ꢀ
ꢀ
Lianbao Ye , Tao Zhao , Wenjun Du , Anhu Li , Wei Gao , Jingrong Li , Ling Wang
and Weiqiang Chenc
aSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China; Esa Biotech Co., LTD., Guangzhou, China; School of Basic
Courses, Guangdong Pharmaceutical University, Guangzhou, China; Joint International Research Laboratory of Synthetic Biology and Medicine,
b
c
d
Guangdong Provincial Engineering and Technology Research Center of Biopharmaceuticals, School of Biology and Biological Engineering, South
China University of Technology, Guangzhou, China
ABSTRACT
ARTICLE HISTORY
Received 27 February 2019
Revised 26 May 2019
Accepted 13 June 2019
Neratinib is an oral pan HER inhibitor, that irreversibly inhibits EGFR and HER2 and was proven to be
effective against multiple EGFR mutations. In previous study, we reported spiro [indoline-3, 40-piperidine]-
2-ones as anticancer agents. In this study, we designed aminopyridine-containing spiro [indoline-3,40-
piperidine] derivatives A1-A4 using Neratinib and spiro [indoline-3, 40-piperidine]-2-one compound
patented as lead structure, then replaced piperidine with cyclopropane to obtain B1-B7 and replaced
indoline with benzmorpholine to get C1-C4 and D1-D2. We synthesized these compounds and evaluated
their residual activities under 0.5 M drug concentration on EGFR and ERBB2. Most of compounds showed
stronger inhibition on EGFR-wt and ERBB2, in which A1-A4 showed excellent inhibitory activity with inhib-
ition percentage on EGFR-wt kinase of 7%, 6%, 19%, 27%, respectively and 9%, 5%, 12%, 34% on ERBB2
kinase compared with 2% and 6% of Neratinib.
KEYWORDS
EGFR/HER2 inhibitors;
aminopyridine; spiro
derivatives; EGFR mutations;
molecular docking
1. Introduction
being tested in a number of clinical trials for its safety and efficacy
in lung cancer, and colorectal, bladder, and breast cancers13–15
.
Overexpression of epidermal growth factor receptor (EGFR or
HER1) and human epidermal growth factor receptor (HER2) is fre-
quently found in different solid tumors. Coexpression of EGFR and
HER2 has been reported in different tumors. They are also accom-
panied with postoperative adverse, radiotherapy, and chemother-
apy resistance1–3. Therefore, it is more effective to dual target
EGFR/HER2 rather than just EGFR inhibition4. Targeting of EGFR
and HER2 is a proven anti-cancer strategy5. Both kinases have
been an attractive therapeutic targets for cancer therapy. A variety
of ATP-competitive EGFR/HER2 RTK dual inhibitors related to dif-
ferent scaffolds have been reported and many of them are cur-
In previous study, we reported spiro [indoline-3, 40-piperidine]-
2-ones as anticancer agents and several compounds showed
stronger inhibition on tyrosine kinase16. In this study, we designed
aminopyridine-containing spiro [indoline-3, 40-piperidine] deriva-
tives A1-A4 using neratinib and spiro [indoline-3, 40-piperidine]-2-
one compound (a) patented as lead structure, then replaced
piperidine with cyclopropane to obtained B1-B7 and replaced
indoline with benzmorpholine to get C1-C4 (Figure 1 and Tables
1–3). We synthesized these compounds and evaluated their
residual activities under 0.5 M drug concentration on EGFR-wt and
ERBB2. Then, we performed head to head comparative trial with
neratinib to investigate inhibitory effects of compounds (A1-A2)
with the best activity on EGFR-wt, HER2 and EGFR mutations.
Molecular docking was adopted for all the synthesized com-
pounds to confirm their mechanism of action.
rently in market or clinical trials for the treatment of cancer6–8
.
Neratinib (HKI-272) is a highly selective inhibitor of HER2 and
EGFR that leads to reduced phosphorylation and activation of
downstream signaling pathways9,10. Neratinib was proven to be
effective against multiple EGFR mutations including T790 muta-
tion, L858R mutation and T790/L858. In complex with Neratinib,
the EGFR kinase adopts an inactive conformation in which the
regulatory C-helix is displaced from its active position. The
enlarged hydrophobic pocket created by the outward rotation of
the C-helix appears to be required to accommodate the bulky
2. Results and discussions
2.1. Chemistry
In previous study, we reported spiro [indoline-3, 40-piperidine]-2-
aniline substituent found in Neratinib, which contain additional ones as anticancer agents. In this study, we designed aminopyri-
aromatic groups appended to the 2-pyridinyl group and binds the dine-containing spiro [indoline-3,40-piperidine] derivatives A1-A4
inactive conformation of the kinase11,12. Neratinib is currently using Neratinib and spiro[indoline-3, 40-piperidine]-2-one
CONTACT Weiqiang Chen
These authors contributed equally to this work.
cwq2187@126.com
School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou, Guangdong, 510006, China
ꢀ
Supplemental data for this article is available online at here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1234
L. YE ET AL.
Figure 1. Design of target compounds.
compound patented as lead structure. The synthetic route of nitration, reduction reaction and methylation. The piperidine of
compounds A1-A4 was shown in Figure 2. The 1-f6-amino-5- A1-A4 was replaced by cyclopropane to obtain B1-B7, which
methoxy-10-methyl-1,2-dihydrospiro[indole-3,40-piperidine]-1-ylg-
were synthesized according to Figure 3. Compounds B1-B7 were
2,2,2-trifluoroethan-1-one (6) reacted with 2-chloro-4-aminopyrimi- obtained by acylation of 15a-15d, which was synthesized by the
dine-5-carbonitriles with different substituents on amino nitrogen reaction of 14a-14d and zincdicarbonitrile using tris(dibenzylide-
to obtain 7a-7d using p-toluenesulfonic acid as catalyst with the neacetone)dipalladium and 1,10-ferrocenebis(diphenylphosphine)
yield of about 70% and compounds 7a-7d were hydrolyzed to get as catalyst and N, N-dimethylformamide and water as solvents.
8a-8d, which were transferred to target compounds A1-A4 by Compounds 14a-14d were prepared by the reaction of the spiro
acylation of acryloyl chloride. The compound 6 was prepared compound
50-methoxy-10,
20-dihydrospiro[cyclopropane-1,
using (4-methoxyphenyl)hydrazine hydrochloride as starting 30-indole]-60-amine (13) and 2-chloro-4-aminopyrimidine-5-carboni-
material through condensation reaction, acylation reaction, triles with different substituents on amino nitrogen. The

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1235
Table 1. Novel aminopyridine-containing spiro derivatives A1-A4.
Compound
R
Compound
R
A1
A3
A2
A4
compound 13 was synthesized using 5-methoxy-2, 3-dihydro-1H- 2.2.2. EGFR wt, HER2, and seven EGFR mutants protein kin-
indol-2-one (9) as starting material through alkylation, nitration,
reduction reaction. The indoline structure of A1-A4 and B1-B7
was replaced with benzmorpholine to obtain C1-C4. The synthetic
route of compounds C1-C4 was shown in Figure 4. Firstly, spiral
ase assay
Based on good inhibitions of A1-A2 on EGFR-wt and HER2, we
performed head to head comparative trial with neratinib to inves-
tigate inhibition of A1-A2 on EGFR-wt, HER2 and seven EGFR
mutations (EGF-R d747-749/A750P, EGF-R d752-759, EGF-R G719S,
EGF-R L858R, EGF-R L861Q, EGF-R T790M,EGF-R T790M/L858R).
intermediates
7-methoxy-3,4-dihydrospiro[1,4-benzoxazine-2,10-
cyclopropane]-6-amine (19) and 7-methoxy-10-methyl-3,4-dihydro-
spiro[1,4-benzoxazine-2,40-piperidine]-6-amine (26) were prepared The IC50 values for three compounds in 9 protein kinase assays
using 1-fluoro-5-methoxy-2,4-dinitrobenzene as starting material. are shown in Table 5. The corresponding IC50 curves of three com-
pounds in 9 protein kinase assays are shown in Supporting
Information. The results confirmed that A1 and A2 were dual
EGFR/HER2 inhibitors, biochemical activities on ERBB2 were about
the same to neratinib with the IC50 values of 0.18 lM, 0.26 lM,
and 0.31 lM, respectively, biochemical activities on EGFR wt were
not more than that of neratinib but within five times. However,
A1 and A2 had stronger inhibitory effect on various EGFR
mutants, especially on T790M and L858R that were main mutants
resistant to EGFR inhibitors in lung cancer. The inhibitory activities
of A1 and A2 on EGFR mutants containing both T790M and
L858R were 31 times stronger with IC50 values of 0.09 lM and 0.
08 lM than neratinib of 2.5 lM. These results showed that A1 and
A2could be developed as potential inhibitors to therapy breast
cancer and drug-resistant lung cancer.
Then, the compound 19 was used to synthesize C1-C4 by taking
a similar approach to prepare A1-A4 and B1-B7, respectively.
2.2. Biological evaluation
2.2.1. EGFR-wt/HER2 protein kinase assay
The inhibitory profile of 17 compounds was determined using two
protein kinases EGFR-wt and HER2. All values are residual activities
(% of control activity) were shown in Table 4. The testing of 17
compounds in singlicate at one concentration (5 ꢁ 1 0ꢂ7 M) with
two protein kinases showed
a differential inhibitory profile.
Residual activity of compounds as followed: Neratinib and A1-
A3 ꢃ 20%, A4 > 20% and ꢃ60%, C1,C3, B5 > 60% and ꢃ80%, the
other compounds >80%. As can be seen from the results, com-
pounds A1-A4 were lucky to show stronger inhibition on EGFR-wt
and HER2 with inhibition percentage on EGFR-wt kinase of 7%,
6%, 19%, 27%, respectively and 9%, 5%, 12%, 34% on ERBB2 kin-
ase, in which A1 and A2 was pretty much to neratinib with
residual activities of 2% and 6%. Since A1 and A2 had superior
activity on EGFR-wt, they will almost certainly be effect on various
types of EGFR mutations, and was the strong possibility that A1
and A2 could therapy both HER2-positive breast cancer and lung
cancer-resistance to the first and the second line EGFR inhibitors
compared to neratinib. The compounds revealed that spiro[indo-
line-3, 40-piperidine] could increase the activity. Further research
2.3. Docking study
The main structural difference between the active and inactive
states is a structural change in the TKD activation loop and move-
ment of the N-lobe helix, both located near the adenosine tri-
phosphate (ATP) binding site. Unlike the active and inactive
conformations present in most RTKs and EGFR, in HER2 the con-
formation as experimentally identified is in the middle of these
active–inactive conformations, and has been named the “active-
like conformation”, due to the orientation of the helix-aC-ac, the
DFG-in and the unformed secondary structure of the activation
works are currently under investigation and will be reported in loop17,18.Structural analysis showed that the drugs impact differ-
due course. ently the conformational space of active and inactive EGFR and

1236
L. YE ET AL.
Table 2. Novel aminopyridine-containing spiro derivatives B1-B7.
Compound
R1
R
B1
B2
B3
B4
B5
B6
B7
energetic analysis pointed out that some ligands have better affin-
ity for the inactive EGFR than the active EGFR state19–21
In this study, docking experiments were performed to eluci-
date the binding model of the compounds A1, A2 and neratinib
.
The reports show that in complex with neratinib, the EGFR kin- with two mutant proteins 2jiv and 3w2q which was the crystal
ase adopts an inactive conformation in which the regulatory structure of EGFR kinase domain T790M mutation in compex
C-helix is displaced from its active position and the enlarged with neratinib and EGFR kinase domain T790M/L858R mutant
hydrophobic pocket created by the outward rotation of the C- with neratinib, respectively. A1 and A2 could adopt the same
helix appears to be required to accommodate the bulky aniline combination mode as neratinib and bound to active sites of two
substituent found in neratinib, neratinib contain additional aro- proteins (shown in Figures 5 and 6). The binding modes of A1,
matic groups appended 2-pyridinyl group and binds the inactive A2 and neratinib with 2jiv showed that their binding energies
conformation of the kinase, the quinoline core of neratinib forms (kcal/mol) is followed as Neratinib(–7.4)ꢄA1(–8.1)ꢄA2(–7.4), which
a single hydrogen bond with the hinge region of the kinase. The is consistent with our further experimental activity with IC50 val-
2-pyridinyl group of neratinib is surrounded by hydrophobic resi- ues of 0.17 lM, 0.089 lM and 0.13 lM. the 2-pyridinyl group of
dues in the expanded pocket, including Met-766 in the C-helix, compounds were surrounded by hydrophobic residues in the
Phe-856, and Met-790, the mutant gatekeeper residue, the nitrile expanded pocket, including Met-766 in the C-helix, Phe-856, and
substituent of neratinib also approaches the gatekeeper Met-790, the mutant gatekeeper residue, the nitrile substituent
residue11,12
.
of neratinib also approaches the gatekeeper residue, and the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1237
Table 3. Novel aminopyridine-containing spiro derivatives C1-C4.
Compound
C1
R
Compound
R
C2
C4
C3
Figure 2. Synthesis of A1-A4, Reagents and conditions: (a) (i) TFA, Toluene, rt, (ii) NaBH4, MeOH, rt; (b) TEA, DCM, 0 ꢅC; (c) Cu(NO3)2.3H2O, Ac2O; (d)Pd/C, H2, rt; (e)
MeI, TEA, THF, rt; (f) PTSA, 2- propanol, dioxane, 80ꢅC; (g) K2CO3, MeOH, rt; (h) TEA, DCM, –20 ꢅC.
covalent bond was formed between Cys-797 at the edge of the mainly pointed to the hydrophilic surface, which is far from the
active site cleft and the crotonamide Michael-acceptor group on hinge region. These effecte could explain that the EGFR kinase
the inhibitor, it can not match the active site owing to the ten- adopted an inactive conformation (Figure 5). The binding modes
sion of the spiral ring, the piperidine ring on the spiral ring of A1, A2 and neratinib with 3w2q showed that their binding

1238
L. YE ET AL.
Figure 3. Synthesis of B1-B7, Reagents and conditions:(a) (i) iPr2NH, nBuLi, THF, –60 ꢅC, (ii) BrCH2CH2Br, 60 ꢅC; (b) HNO3, Ac2O, DCM, rt; (c) Fe, NH4Cl, MeOH, H2O;
(d) LiAlH4, THF, 70ꢅC; (e) pTSA, Dioxane,100 ꢅC, 2 h; (f) Zn(CN)2, DMF, H2O, 120 ꢅC(MW)/90 min; (g) TEA, DCM, 0 ꢅC, 2 h.
Figure 4. Synthesis of C1-C4, Reagents and conditions: (a) Cs2CO3, MeCN, rt, 2 h; (b) Fe/NH4Cl, MeOH, H2O,70 ꢅC,12h; (c) LAH, THF, 70 ꢅC, 2 h; (d) pTSA, Dioxane,
100 ꢅC, 2 h; (e) Zn(CN)2, Pd2(dba)3.CHCl3/dppf, DMF, H2O, 120ꢅC(MW), 1.5 h; (f) (E)-4- (dimethylamino)but-2-enoyl chloride, TEA, 40 ꢅC, 2 h.
energies (kcal/mol) is followed as Neratinib(–7.2)<A1(–8.3)
ꢄA2(–7.7), which is consistent with our further experimental
activity with IC50 values of 2.5 lM, 0.09 lM and 0.08 lM, A1 and
3. Experimental part
3.1. General methods of synthesis
A2 could bind in the ATP-binding cleft with a covalent to Cys- The solvents and reagents were purchased from commercial ven-
797 in a fashion similar to neratinib and the Cys-797 side-chain dors and were dried and purified by conventional methods prior
orientation wasrelevant to the position and length of the to use. Nuclear magnetic resonance (NMR) spectra were recorded
acceptor substituent to accommodate covalent binding, a dis- on a Bruker AC-300P spectrometer with TMS as the internal stand-
tinct conformation change for Met790 was observed in the vicin- ard and DMSO as solvent. ESI mass spectra were performed on an
ity of these compounds, the side-chain rearrangement of Met790 Shimadzu LCMS-2020. TLC analysis was carried out on silica gel 60
was necessary to accommodate binding of the cyano group for F254 silica plates (Merck, KGaA, Germany).
A1, A2 and neratinib, The moiesty of 3-chloro-4-(pyridin-2-ylme-
Synthesis of benzyl5-methoxy-1,2-dihydrospiro[indole-3,40-piperi-
solution of (4-methoxyphenyl)
thoxy)benzenamine in A1, A2 and neratinib had similar binding dine]-10-carboxylate (2). To
a
mode with Val726, Leu718, Leu788, and it can not match the hydrazine hydrochloride (9.85 g, 71.29 mmol) and benzyl 4-formyl-
active site owing to the tension of the spiral ring, the piperidine piperidine-1-carboxylate (7 g, 28.31 mmol) in toluene/acetonitrile
ring on the spiral ring mainly pointed to the hydrophilic surface, (100 ml/2 ml) was added trifluoroacetic acid (9.68 g, 85.63 mmol).
which is far from the hinge region (Figure 6).
The mixture was stirred for 12 h at 35 ꢅC. The reaction was cooled

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1239
to 0 ꢅC and methanol (100 ml) was added followed by the slow 3.67 (s, 3H), 3.34 (s, 2H), 3.01–2.95 (m, 2H), 1.71–1.51 (m, 4H). MS
(ESI) calculated for (C21H24N2O3) [M þ 1]þ, 353; found, 353.
addition of sodium borohydride (1.6 g, 42.29 mmol). After stirring
for 3 h at 0–5 ꢅC, the reaction was quenched by the addition of
Synthesis of benzyl 5-methoxy-1-(trifluoroacetyl)-1,2-dihydrospir-
o[indole-3,40- piperidine]-10-carboxylate (3). To a solution of 2 (4.3 g,
water (200 ml) and extracted with ethyl acetate (4 ꢁ 100 ml). The
combined organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated under vacuum. The residue was purified
by flash column chromatography with 0–30% ethyl acetate in pet-
roleum ether to afford 2 (4.5 g, 45% yield). 1H NMR (300 MHz,
DMSO-d6) d 7.39–7.35 (m, 5H), 6.67 (d, J ¼ 2.5 Hz, 1H), 6.57 – 6.49
(m, 1H), 6.42 (d, J ¼ 8.4 Hz, 1H), 5.11 (m, 3H), 4.13 – 3.86 (m, 2H),
12.20 mmol) and triethylamine (2.44 g, 24.11 mmol) in dichlorome-
thane (50 ml) was added trifluoroacetyl 2,2,2-trifluoroacetate
(3.34 g, 15.90 mmol) slowly with stirring at 0 ꢅC. The resulting solu-
tion was stirred for 12 h at room temperature under nitrogen. The
reaction mixture was diluted with water (50 ml) and extracted
with dichloromethane (3 ꢁ 40 ml). The combined organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated
under vacuum. The residue was purified by flash column chroma-
tography with 0–35% ethyl acetate in petroleum ether to afford 3
Table 4. Residual activities under 0.5 drug concentration of compounds on
EGFR-wt and HER2.
1
as a yellow solid (3.8 g, 66% yield). H NMR (300 MHz, DMSO-d6) d
7.96 (d, J ¼ 8.9 Hz, 1H), 7.44 – 7.27 (m, 5H), 7.05 (d, J ¼ 2.6 Hz, 1H),
6.86 (dd, J ¼ 8.9, 2.6 Hz, 1H), 5.11 (s, 2H), 4.20 (s, 2H), 4.06 – 3.99
(m, 2H), 3.76 (s, 3H), 3.01 (s, 2H), 1.85 (td, J ¼ 13.2, 4.7 Hz, 2H), 1.65
(d, J ¼ 13.0 Hz, 2H). MS (ESI) calculated for (C23H23F3N2O4)
[M þ 1]þ, 449; found, 449.
Residual activity under 0.5 lM drug
concentration (%)
Compound
EGF-R wt
HER2
Neratinib
A1
A2
2
7
6
6
9
5
Synthesis of benzyl 5-methoxy-6-nitro-1-(trifluoroacetyl)-1,2-dihy-
drospiro[indole-3, 40-piperidine]-10-carboxylate (4). To a solution of
3(4.1 g, 9.14 mmol) in acetic anhydride (50 ml) was added cupric
nitrate trihydrate (2.43 g, 10 mmol) at 0 ꢅC. After stirring for 3 h at
room temperature, the reaction was quenched by the addition of
water (50 ml) and extracted with dichloromethane (3 ꢁ 40 ml). The
combined organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated under vacuum. The residue was purified
by flash column chromatography with 0–80% ethyl acetate in pet-
roleum ether to afford 4as a yellow solid (3.9 g, 86% yield). 1H
NMR (300 MHz, DMSO-d6) d 8.47 (s, 1H), 7.57 (s, 1H), 7.52 – 7.23
(m, 5H), 5.12 (s, 2H), 4.29 (s, 2H), 4.16 – 4.03 (m, 2H), 4.00 (s, 3H),
3.05–2.96 (m, 2H), 1.99 – 1.91 (m, 2H), 1.73 (d, J ¼ 13.2 Hz, 2H).
Synthesis of 1-f6-amino-5-methoxy-1,2-dihydrospiro[indole-3,40-
piperidine]-1-ylg-2, 2,2-trifluoroethan-1-one (5). To a solution of 4
(3.9 g, 7.90 mmol) in methanol (50 ml) was added palladium on
activated carbon (500 mg, 10%). The mixture was stirred for 5 h at
room temperature under hydrogen (2 atm). The solids were fil-
tered out, and the filtrate was concentrated under vacuum to
afford 5 (2.4 g, 92% yield). 1H NMR (300 MHz, DMSO-d6) d 7.47 (s,
1H), 6.79 (s, 1H), 4.88 (s, 2H), 4.04 (s, 2H), 3.79 (s, 3H), 2.99 – 2.85
(m, 2H), 2.63 – 2.43 (m, 3H), 1.75 (td, J ¼ 12.8, 4.3 Hz, 2H), 1.52 –
1.42 (m, 2H). MS (ESI) calculated for (C15H18F3N3O2) [M þ 1]þ, 330;
found, 330.
A3
A4
B1
B2
B3
B4
B5
B6
B7
C1
C2
C3
19
27
92
97
95
87
68
94
89
86
76
79
85
100
88
105
12
34
103
100
102
99
102
113
135
100
108
111
100
113
123
110
C4
DMSO
DMSO
DMSO
Table 5. Biochemical IC50 values of compounds against EGFR wt, HER2, and
seven EGFR mutants protein kinase.
IC50 values lM
Protein kinase
Neratinib
A1
A2
EGF-R d747-749/A750P
EGF-R d752-759
EGF-R G719S
EGF-R L858R
EGF-R L861Q
EGF-R T790M
EGF-R T790M/L858R
EGF-R wt
0.67
0.17
0.019
0.08
0.017
0.17
2.5
0.053
0.21
0.2
0.086
0.076
0.089
0.09
0.061
0.26
0.054
0.2
0.24
0.081
0.081
0.13
0.08
0.059
0.31
Synthesis of 1-f6-amino-5-methoxy-10-methyl-1,2-dihydrospiro[in-
dole-3,40-piperidine]-1-ylg-2,2,2-trifluoroethan-1-one (6). To a solution
of 5 (2.4 g, 7.29 mmol) and triethylamine (2.2 g, 21.74 mmol) in
tetrahydrofuran (40 ml) was added iodomethane (1.35 g, 9.5 mmol).
After stirring for 6 h at room temperature, the mixture was
0.014
0.18
HER2
Figure 5. Docking of compounds the active site of 2JIV: (A) Docking of A1 to the active site of 2JIV. (B) Docking of A2 to the active site of 2JIV. (C) Docking of
Neratinib to the active site of EGFR.

1240
L. YE ET AL.
Figure 6. Docking of compounds the active site of 3W2Q: (A) Docking of A1 to the active site of 3W2Q. (B) Docking of A2 to the active site of 3W2Q (C) Docking of
Neratinib to the active site of 3W2Q.
concentrated under vacuum. The residue was purified by flash col- Mobile Phase B: acetonitrile; Flow rate: 30 ml/min; Gradient: 10% B
umn chromatography with 0–10% methanol in dichloromethane to 60% B in 13 min; 254 nm] to afford 8a as a yellow solid (64 mg,
to afford 6 as a light yellow solid (1.4 g, 58% yield). 1H NMR 88.9% purity, 26% yield). 1H NMR (300 MHz, DMSO-d6) d 9.25 (s,
(300 MHz, DMSO-d6) d 7.46 (s, 1H), 6.81 (s, 1H), 4.89 (s, 2H), 4.01 (s, 1H), 8.58 (ddd, J ¼ 4.9, 1.8, 1.0 Hz, 1H), 8.45 (s, 1H), 8.39 (s, 1H),
2H), 2.77 (d, J ¼ 8.0 Hz, 2H), 2.21 (s, 3H), 1.96 – 1.79 (m, 4H), 1.53 7.84 (td, J ¼ 7.7, 1.8 Hz, 1H), 7.50–7.37 (m, 3H), 7.35 (ddd, J ¼ 7.6,
(d, J ¼ 10.5 Hz, 2H). MS (ESI) calculated for (C16H20F3N3O2) 4.8, 1.3 Hz, 1H), 7.00 – 6.91 (m, 2H), 6.83 (s, 1H), 6.79 (s, 1H), 5.15
[M þ 1]þ, 344; found, 344.
(s, 2H), 4.91 (s, 1H), 3.68 (s, 3H), 3.24 (s, 2H), 2.78 – 2.60 (m, 2H),
2.20 (s, 3H), 1.98 (t, J ¼ 11.7 Hz, 2H), 1.90 – 1.73 (m, 2H), 1.61 –
Synthesis of 2-f[5-methoxy-10-methyl-1-(trifluoroacetyl)-1,2-dihy-
drospiro[indole-3,
40-piperidine]-6-yl]aminog-4-f[4-(pyridin-2-ylme- 1.44 (m, 2H).MS (ESI) calculated for (C31H32N8O2) [M þ 1]þ, 549;
thoxy)phenyl]aminogpyrimidine-5-carbonitrile (7a). To a solution of found, 549.
2-chloro-4-[[4-(pyridin-2-ylmethoxy)phenyl]amino]pyrimidine
-5-
Compound 8b-8d was prepared in analogy to 8a.
Synthesis of 4-f[3-chloro-4-(pyridin-2-ylmethoxy)phenyl]aminog-2-
carbonitrile (300 mg, 0.89 mmol) and 6 (200 mg, 0.58 mmol) in 2-
propanol (20 ml) was added p-toluenesulfonic acid (153 mg,
0.89 mmol) at room temperature. After was stirring for 2 h at
75 ꢅC, the reaction mixture was diluted with saturated sodium
bicarbonate aqueous solution (50 ml) and extracted with ethyl
acetate (3 ꢁ 50 ml). The combined organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated under vac-
uum to afford 7a as a yellow crude solid (370 mg, 72% purity,
71% yeild). MS (ESI) calculated for (C33H31F3N8O3) [M þ 1]þ, 645;
found, 645.
(f5-methoxy-10
-methyl-1,2-dihydrospiro[indole-3,40-piperidine]-6-
ylgamino)pyrimidine-5-carbonitrile (8b).
a yellow solid (46 mg,
85.6% purity, 29% yield). 1H NMR (300 MHz, DMSO-d6) d 9.31 (s,
1H), 8.69 – 8.54 (m, 2H), 8.41 (s, 1H), 7.87 (td, J ¼ 7.7, 1.8 Hz, 1H),
7.64 (s, 1H), 7.58 – 7.54 (m, 2H), 7.42 – 7.30 (m, 1H), 7.14 (d,
J ¼ 9.0 Hz, 1H), 6.81 (s, 1H), 6.74 (s, 1H), 5.25 (s, 2H), 4.94 (s, 1H),
3.68 (s, 3H), 3.24 (s, 2H), 2.67 (d, J ¼ 10.5 Hz, 2H), 2.17 (s, 3H), 2.00
– 1.71 (m, 4H), 1.52 (d, J ¼ 11.8 Hz, 2H).MS (ESI) calculated for
(C31H31ClN8O2) [M þ 1]þ, 583; found, 583.
Synthesis
of
4-f[4-(benzyloxy)-3-chlorophenyl]aminog-2-(f5-
Compound 7b-7d was prepared in analogy to 7a.
Synthesis of 4-f[3-chloro-4-(pyridin-2-ylmethoxy)phenyl]aminog-2- methoxy-10-methyl-1,2
-dihydrospiro[indole-3,40-piperidine]-6-
f[5-methoxy-10- methyl-1-(trifluoroacetyl)-1,2-dihydrospiro[indole-3,40- ylgamino)pyrimidine-5-carbonitrile (8c). a yellow solid (70 mg, 60%
1
piperidine]-6-yl]aminogpyrimidine-5-carbonitrile (7b) . a yellow crude yield). H NMR (300 MHz, DMSO-d6) d 9.30 (s, 1H), 8.60 (s, 1H), 8.41
solid (290 mg, 65% purity, 63% yield). MS (ESI) calculated for (s, 1H), 7.63 (s, 1H), 7.59 – 7.51 (m, 1H), 7.50 – 7.30 (m, 5H), 7.15
(C33H30ClF3N8O3) [M þ 1]þ, 679; found, 679.
(d, J ¼ 9.0 Hz, 1H), 6.81 (s, 1H), 6.75 (s, 1H), 5.17 (s, 2H), 4.94 (s,
Synthesis
of
4-f[4-(benzyloxy)-3-chlorophenyl]aminog-2-f[5- 1H), 3.68 (s, 3H), 3.28 – 3.19 (m, 2H), 2.68 (d, J ¼ 10.7 Hz, 2H), 2.17
-(trifluoroacetyl)-1,2-dihydrospiro[indole-3,40- (s, 3H), 2.02 – 1.75 (m, 4H), 1.53 (d, J ¼ 12.0 Hz, 2H).MS (ESI) calcu-
methoxy-10-methyl-1
piperidine]-6-yl]aminogpyrimidine-5-carbonitrile (7c). a yellow solid lated for (C32H32ClN7O2) [M þ 1]þ, 582; found, 582.
(210 mg, 70% purity, 53% yield). MS (ESI) calculated for
Synthesis of 2-(f5-methoxy-10-methyl-1,2-dihydrospiro[indole-3,40-
(C34H31ClF3N7O3) [M þ 1]þ, 678; found, 678.
piperidine]-6-ylg amino)-4-f[1-(pyridin-2-ylmethyl)-1H-pyrazol-4-yl]a-
Synthesis of 2-f[5-methoxy-10-methyl-1-(trifluoroacetyl)-1,2-dihy- minogpyrimidine-5-carbonitrile (8d). a yellow solid (67 mg, 34%
1
drospiro[indole-3,
40-piperidine]-6-yl]aminog-4-f[1-(pyridin-2- yield). H NMR (300 MHz, DMSO-d6) d 9.64 (s, 1H), 8.81 (s, 1H), 8.51
ylmethyl)-1H-pyrazol-4-yl]aminogpyrimidine-5-carbonitrile (7d). a yel- (dd, J ¼ 5.0, 1.6 Hz, 1H), 8.36 (s, 1H), 7.80 – 7.55 (m, 2H), 7.34 –
low solid (360 mg, 70% purity, 93% yield). MS (ESI) calculated for 7.22 (m, 1H), 6.86 (s, 1H), 6.79 (s, 1H), 6.74 (s, 1H), 5.31 (s, 2H), 5.15
(C30H29F3N10O2) [M þ 1]þ, 619; found, 619.
(s, 1H), 3.65 (s, 3H), 3.20 (s, 2H), 2.63 (d, J ¼ 10.9 Hz, 2H), 2.17 (s,
3H), 1.90 (t, J ¼ 11.6 Hz, 2H), 1.75 (t, J ¼ 12.5 Hz, 2H), 1.43 (d,
J ¼ 12.4 Hz, 2H). MS (ESI) calculated for (C28H30N10O) [M þ 1]þ, 523;
found, 523.
Synthesis of 2-(f5-methoxy-10-methyl-1,2-dihydrospiro[indole-3,40-
piperidine]-6-ylg
amino)-4-f[4-(pyridin-2-ylmethoxy)phenyl]ami-
nogpyrimidine-5-carbonitrile (8a). To a solution of 7a (370 mg,
0.40 mmol, 72% purity) in methanol (10 ml) was added potassium
Synthesis of 50-methoxy-10, 20-dihydrospiro [cyclopropane-1, 30-
carbonate (280 mg, 2.03 mmol). After stirring for 2 h at room tem- indole]-20-one (10). To a solution of 5-methoxy-2, 3-dihydro-1H-
perature, the mixture was diluted with water (50 ml) and extracted indol-2-one (9, 5 g, 30.64 mmol,) and diisopropylamine (6.14 g,
with ethyl acetate (30 ml ꢁ 3). The combined organic layer was 60.79 mmol) in dry tetrahydrofuran (50 ml) was added n-butyl-
dried over anhydrous sodium sulfate, filtered and concentrated lithium (49 ml, 122.68 mmol, 2.5 M in hexane) slowly at -60 ꢅC. The
under vacuum. The crude product was purified by Prep-HPLC mixture was stirred for 1 h at –60 ꢅC. This was followed by the
ꢀ
[Column: XBridge Shield RP18 OBD Column, 5um, 19 150 mm; addition of 1, 2-dibromoethane (6.89 g, 36.68 mmol) slowly at
Mobile Phase A:Water with 10 mmol/L ammonium bicarbonate, –60 ꢅC. The resulting solution was warmed to room temperature

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1241
for 14 h. The reaction was then quenched by the addition of 3H), 0.94 (d, J ¼ 3.0 Hz, 4H). MS (ESI) calc’d for (C27H25BrN6O2)
[M þ 1]þ, 545, 547; found, 545, 547.
100 ml of water and extracted with ethyl acetate (3 ꢁ 300 ml).
The combined organic layer was dried over anhydrous sodium sul-
fate, filtered and concentrated under vacuum. The residue was
purified by flash column chromatography with 0 ꢆ 50% ethyl acet-
ate in petroleum ether to afford 10 as an off-white solid (2.5 g,
43% yield). 1H NMR (300 MHz, CDCl3) d 8.89 (s, 1H), 6.89 (d,
J ¼ 9.6 Hz, 1H), 6.86 (dd, J1 ¼ 8.7 Hz, J2 ¼ 2.7 Hz, 1H), 6.47 (d,
J ¼ 2.4 Hz, 1H), 3.77 (s, 3H), 1.78–1.74 (m, 2H), 1.53–1.49 (m, 2H).
MS (ESI) calc’d for (C11H11NO2) [M þ 1]þ, 190; found, 190.
Compound 14b-14d was prepared in analogy to 14a.
Synthesis
of
5-bromo-4-N-[3-chloro-4-(pyridin-2-ylmethoxy)-
phenyl]-2-N-f50-methoxy -10,20-dihydrospiro[cyclopropane-1,30-indole]-
60-ylgpyrimidine-2,4-diamine (14 b). an off-white solid (11.5 mg, 8%
yield). 1H NMR (400 MHz, DMSO-d6) d 8.65–8.60 (m, 2H), 8.23 (s,
1H), 7.91–7.87 (m, 2H), 7.61–7.54 (m, 2H), 7.39–7.36 (m, 1H),
7.26–7.24 (m, 1H), 6.30 (s, 1H), 5.28 (s, 2H), 4.20 (br, 1H), 3.99 (s,
2H), 3.69 (s, 3H), 0.95 (d, J ¼ 3.0 Hz, 4H). MS (ESI) calc’d for
(C27H24BrClN6O2) [M þ 1]þ, 579, 581; found, 579, 581.
Synthesis of 50-methoxy-60-nitro-10,20-dihydrospiro[cyclopropane-
1,30-indole]-20-one (11). To a solution of 10 (2.5 g, 13.21 mmol) and
acetyl acetate (2.0 g, 19.59 mmol) in dichloromethane (30 ml) was
added nitric acid (1.5 g, 23.80 mmol). The resulting solution was
stirred for 2 h at room temperature. The reaction was quenched
by the addition of saturated sodium bicarbonate aqueous solution
(20 ml). The solids were collected by filtration and dried in an
oven under reduced pressure to afford 11 as a yellow solid (1.7 g,
55% yield). H1 NMR (300 MHz, DMSO-d6) d 10.68 (s, 1H), 7.37 (s,
1H), 7.13 (s, 1H), 3.87 (s, 3H), 1.79–1.71 (m, 2H), 1.61–1.58 (m,
2H).MS (ESI) calc’d for (C11H10N2O4) [M þ 1] þ, 235; found, 235.
Synthesis of 60-amino-50-methoxy-10,20-dihydrospiro[cyclopropane-
Synthesis of 4-N-[4-(benzyloxy)-3-chlorophenyl]-5-bromo-2-N-f50-
methoxy-10,20-
dihydrospiro[cyclopropane-1,30-indole]-60-
ylgpyrimidine-2,4-diamine (14c). An off-white solid (14.6 mg, 10%
1
yield). H NMR (400 MHz, DMSO-d6) d 8.63 (s, 1H), 8.22 (s, 1H), 7.85
(s, 1H), 7.54 (d, J ¼ 8.3 Hz, 1H), 7.49 (d, J ¼ 7.5 Hz, 2H), 7.42 (t,
J ¼ 7.5 Hz, 2H), 7.35 (t, J ¼ 7.2 Hz, 1H), 7.25 (d, J ¼ 8.9 Hz, 1H), 6.29
(s, 1H), 5.20 (s, 2H), 4.32 (s, 3H), 3.97 (s, 2H), 3.68 (s, 3H), 0.95 (d,
J ¼ 9.8 Hz, 4H).MS (ESI) calc’d for (C28H25BrClN5O2) [M þ 1]þ, 578,
580; found, 578, 580.
Synthesis of 5-bromo-2-N-f50-methoxy-10, 20-dihydrospiro [cyclo-
propane-1, 30-indole] -60-ylg-4-N-[1-(pyridin-2-ylmethyl)-1H-pyrazol-4-
yl] pyrimidine-2, 4-diamine (14d). An off-white solid (260 mg, 32%
yield) of. 1H NMR (300 MHz, DMSO-d6) d 8.80 (s, 1H), 8.54 (d,
J ¼ 4.8 Hz, 1H), 8.14 (s, 1H), 8.09 (s, 1H), 7.79–7.73 (m, 2H), 7.62 (s,
1H), 7.34–7.29 (m, 1H), 7.07 (d, J ¼ 7.8 Hz, 1H), 6.28 (s, 1H), 5.41 (s,
2H), 4.46 (br, 2H), 4.00 (s, 2H), 3.68 (s, 3H), 0.94–0.93 (m, 4H). MS
(ESI) calc’d for (C24H23BrN8O) [M þ 1]þ, 519, 521; found, 519, 521.
Synthesis of 2-(f50-methoxy-10, 20-dihydrospiro [cyclopropane-1,
30-indole]-60-ylg amino)-4-f[4-(pyridin-2-ylmethoxy) phenyl] aminog
1,30-indole]-20-one (12).
A
degassed solution of 11 (1.7 g,
7.26 mmol), iron dust (1.6 g, 28.57 mmol) and ammonium chloride
(1.9 g, 35.85 mmol) in methanol and water (1:1) (10 ml) was stirred
for 6 h at 70 ꢅC. The reaction mixture was diluted with water
(20 ml) and extracted with ethyl acetate (3 ꢁ 50 ml). The combined
organic layer was dried over anhydrous sodium sulfate, filtered
and concentrated under vacuum to afford 12 as a yellow solid
(1.1 g, 74% yield). H1 NMR (400 MHz, DMSO-d6) d 10.07 (s, 1H),
6.50 (s, 1H), 6.34 (s, 1H), 4.68 (s, 2H), 3.69 (s, 3H), 1.38–1.35 (m,
2H), 1.32–1.30 (m, 2H). MS (ESI) calc’d for (C11H12N2O2) [M þ 1]þ,
205; found, 205.
pyrimidine-5-carbonitrile (15a).
A degassed solution of 14a
(200 mg, 0.37 mmol), zincdicarbonitrile (214 mg, 1.82 mmol), tris(di-
benzylideneacetone)dipalladium (39 mg, 0.04 mmol) and 1,10-ferro-
Synthesis
of
50-methoxy-10,20-dihydrospiro[cyclopropane-1,30-
cenebis(diphenylphosphine)
(41 mg,
0.07 mmol)
in
N,N-
indole]-60-amine (13). To a solution of 12 (500 mg, 2.45 mmol) in
dry tetrahydrofuran (10 ml) was added lithium aluminum hydride
(375 mg, 9.87 mmol) at room temperature. The resulting solution
was stirred for 4 h at 70 ꢅC. The reaction was then quenched by
the addition of water (20 ml) and extracted with ethyl acetate
(3 ꢁ 50 ml). The combined organic layer was dried over anhydrous
sodium sulfate filtered and concentrated under vacuum. The resi-
due was purified by flash column chromatography with 0 ꢆ 50%
ethyl acetate in petroleum ether to afford 13 as a brown solid
(260 mg, 56% yield). 1H NMR (300 MHz, DMSO-d6) d 6.14 (s, 1H),
5.94 (s, 1H), 4.92 (s, 1H), 4.04 (s, 2H), 3.61 (s, 3H), 3.31 (s, 2H),
0.80–0.72 (m, 4H). MS (ESI) calc’d for (C11H14N2O) [M þ 1]þ, 191;
found, 191.
dimethylformamide and water (10:1) (2 ml) was stirred for 3 h at
110 ꢅC in microwave reactor. The reaction mixture was diluted
with water (25 ml) and extracted with ethyl acetate (3 ꢁ 15 ml).
The combined organic layer was dried over sodium sulfate, filtered
and concentrated under vacuum. The residue was purified by
flash column chromatography with 0 ꢆ 50% ethyl acetate in pet-
roleum ether to afford 15a as a brown solid (110 mg, 55% yield).
1H NMR (300 MHz, DMSO-d6) d 9.35 (br, 1H), 8.59 (d, J ¼ 4.5 Hz,
1H), 8.46 (s, 1H), 7.87 (dd, J1¼ 7.8, 1.5 Hz, 1H), 7.55(br, 2H),
7.37–7.33 (m, 2H), 7.16–7.01 (m, 3H), 6.29 (s, 1H), 5.18 (s, 2H), 4.65
(s, 1H), 4.04–3.97 (m, 2H), 3.75 (br, 1H), 3.69 (s, 3H), 1.00–0.95 (m,
4H). MS (ESI) calc’d for (C28H25N7O2) [M þ 1]þ, 492; found, 492.
Compound 15b-15d was prepared in analogy to 15a
Synthesis of 5-bromo-2-N-f50-methoxy-10, 20-dihydrospiro [cyclo-
propane-1, 30-indole]-60-ylg-4-N-[4-(pyridin-2-ylmethoxy) phenyl] pyr-
imidine-2,4-diamine (14a). To a solution of 13 (450 mg, 2.37 mmol)
and 5-bromo-2-chloro-N-[4-(pyridin-2-ylmethoxy)phenyl]pyrimidin-
4-amine (647 mg, 1.65 mmol) in dioxone (10 ml) was added 4-
methylbenzene-1-sulfonic acid monohydrate (408 mg, 2.37 mmol).
The resulting solution was stirred for 6 h at 100 ꢅC. The reaction
mixture was diluted water (20 ml) and extracted with ethyl acetate
(3 ꢁ 30 ml). The combined organic layer was dried over sodium
sulfate, filtered and concentrated under vacuum. The residue was
purified by flash column chromatography with 0 ꢆ 50% ethyl acet-
ate in petroleum ether to afford 14a as an off-white solid
(260 mg, 20% yield). 1H NMR (300 MHz, DMSO-d6) d 8.59 (d,
J ¼ 4.5 Hz, 1H), 8.51 (s, 1H), 8.17 (s, 1H), 7.87 (dd, J1¼ 7.5 Hz, 1.8 Hz,
1H), 7.56–7.47 (m, 3H), 7.37–7.33 (m, 2H), 7.07 (d, J ¼ 8.7 Hz, 1H),
Synthesis of 4-[[3-chloro-4-(pyridin-2-ylmethoxy)phenyl]amino]-2-
([50-methoxy-10,20
-dihydrospiro[cyclopropane-1,30-indole]-60-yl]ami-
no)pyrimidine-5-carbonitrile (15b).
1
a yellow solid (5.3 mg, 12% yield). H NMR (300 MHz, DMSO-d6)
d 8.60–8.50 (m, 2H), 7.96–7.36 (m, 6H), 7.24–7.10 (m, 1H), 6.33 (s,
1H), 5.27 (s, 2H), 4.17–4.07 (m, 2H), 3.71 (br, 3H), 3.69 (s, 3H),
1.04–0.98 (m, 4H). MS (ESI) calc’d for (C28H24ClN7O2) [M þ 1]þ, 526;
found, 526.
Synthesis
methoxy-10,20-
of
4-f[4-(benzyloxy)-3-chlorophenyl]aminog-2-(f50-
dihydrospiro[cyclopropane-1,30-indole]-60-
ylgamino)pyrimidine-5-carbonitrile (15c). A yellow solid(4.2 mg, 9%
1
yield). H NMR (300 MHz, DMSO-d6) d 9.15 (s, 1H), 8.46 (s, 1H), 7.75
(s, 1H), 7.49–7.22 (s, 8H), 6.30 (s, 1H), 5.20 (s, 2H), 4.17–4.07 (m,
2H), 4.04 (s, 2H), 3.71 (s, 3H), 0.97 (s, 4H). MS (ESI) calc’d for
6.26 (s, 1H), 5.18 (s, 2H), 4.18–4.01 (br, 1H), 3.93 (s, 2H), 3.67 (s, (C29H25ClN6O2) [M þ 1]þ, 525; found, 525.

1242
L. YE ET AL.
Synthesis of 2-(f50-methoxy-10, 20-dihydrospiro [cyclopropane-1, 30- washed with brine (50 ml ꢁ 3), then dried over anhydrous magne-
indole]-60-ylg amino)-4-f[1-(pyridin-2-ylmethyl)-1H-pyrazol-4-yl] aminog sium sulfate, filtered and the filtrate was concentrated under
pyrimidine-5-carbonitrile (15d). a yellow solid (110 mg, 68% yield). reduced pressure. The residue was purified by flash column chro-
1H NMR (300 MHz, DMSO-d6) d 9.67 (s, 1H), 8.54 (d, J ¼ 3.6 Hz, 1H), matography with 0–10% ethyl acetate in methanol to afford 20a
8.46 (s, 1H), 8.09 (s, 1H), 7.79–7.74 (m, 3H), 7.34–7.30 (m, 1H), as an off-white solid (59.2 mg, 22% yield). 1H NMR (300 MHz,
7.07–7.05 (m, 1H), 6.32 (s, 1H), 5.42 (s, 2H), 4.66 (br, 2H), 4.08 (s, 2H), DMSO-d6) d 8.59 (d, J ¼ 4.5 Hz, 1H), 8.35 (s, 1H), 8.07 (s, 1H), 7.87
3.71 (s, 3H), 0.97 (s, 4H). MS (ESI) calc’d for (C25H23N9O) [M þ 1]þ, (dd, J1¼ 7.5 Hz, J2 ¼ 1.8 Hz, 1H), 7.70 (s, 1H), 7.55–7.45 (m, 3H),
466; found, 466.
7.39–7.31 (m, 1H), 6.99–6.93 (m, 3H), 6.36 (s, 1H), 5.16–5.12 (m,
3H), 3.62 (s, 3H), 3.13 (d, J ¼ 2.8 Hz, 2H), 0.88–0.84 (m, 2H),
0.69–0.65 (m, 2H) MS (ESI) calc’d for (C27H25BrN6O3) [M þ 1]þ, 561,
563; found, 561, 563.
Synthesis of methyl 1–(5-methoxy-2,4-dinitrophenoxy)cyclopropa-
necarboxylate (17). To a solution of 1-fluoro-5-methoxy-2,4-dinitro-
benzene (16, 20 g, 92.54 mmol) and methyl 1-hydroxycyclopropane-
1-carboxylate (11.8 g, 101.62 mmol) in acetonitrile (400 ml) was
added cesium carbonate (45.14 g, 138.9 mmol) at room tempera-
Compound 20b-20d was prepared in analogy to 20a
Synthesis
of
5-bromo-4-N-[3-chloro-4-(pyridin-2-ylmethoxy)-
ture. The resulting solution was stirred for 12 h at room tempera- phenyl]-2-N-f7-methoxy -3,4-dihydrospiro[1,4-benzoxazine-2,10-cyclo-
ture. The reaction mixture was diluted with water (400 ml) and propane]-6-ylgpyrimidine-2,4-diamine (20 b). a gray solid (57.7 mg,
extracted with ethyl acetate (200 ml ꢁ 3). The combined organic 20% yield). 1H NMR (300 MHz, DMSO-d6) d 8.59 (d, J ¼ 2.8 Hz, 1H),
phase was washed with brine (200 ml ꢁ 3), then dried over 8.44 (s, 1H), 8.10 (s, 1H), 7.91–7.84 (m, 2H), 7.71 (s, 1H), 7.64–7.55
anhydrous magnesium sulfate, filtered and concentrated under (m, 2H), 7.39–7.35 (m, 1H), 7.17 (d, J ¼ 6.0 Hz, 1H), 6.92 (s, 1H), 6.37
reduced pressure. The residue was purified by flash column chro- (s, 1H), 5.27–5.15 (m, 3H), 3.61 (s, 3H), 3.12 (d, J ¼ 2.8 Hz, 2H),
matography with 0 ꢆ 50% ethyl acetate in petroleum ether to 0.88–0.84 (m, 2H), 0.68–0.64 (m, 2H). MS (ESI) calc’d for
afford 17 as yellow solid (11 g, 38% yield). 1H NMR (300 MHz, (C27H24BrClN6O3) [M þ 1]þ, 595, 597; found, 595, 597.
Synthesis of 4-N-[4-(benzyloxy)-3-chlorophenyl]-5-bromo-2-N-f7-
DMSO-d6) d 8.70 (s, 1H), 6.88 (s, 1H), 4.11 (s, 3H), 3.71 (s, 3H), 1.73
(t, J ¼ 5.8 Hz, 2H), 1.53 (t, J ¼ 5.8 Hz, 2H). MS (ESI) calc’d for methoxy-3,4- dihydrospiro[1,4-benzoxazine-2,10-cyclopropane]-6-ylg
(C12H12N2O8) [M þ 1]þ, 313; found, 313.
pyrimidine-2,4-diamine (20c). An off-white solid(28.9 mg, 20%
Synthesis of 6-amino-7-methoxy-3,4-dihydrospiro[1,4-benzoxazine- yield). H NMR (300 MHz, DMSO-d6) d 8.41 (s, 1H), 8.10 (s, 1H), 7.83
1
2,10- cyclopropane]-3-one (18). A mixture of 17 (10 g, 32.03 mmol), (s, 1H), 7.71 (s, 1H), 7.62–7.60 (m, 1H), 7.48–7.33 (m, 5H), 7.15 (d,
iron dust (5.4 g, 96.70 mmol) and ammonium chloride (10.1 g, J ¼ 6.0 Hz, 1H), 6.93 (s, 1H), 6.38 (s, 1H), 5.20–5.18 (m, 3H), 3.62 (s,
188.8 mmol) in methanol (150 ml) and water (150 ml) was stirred 3H), 3.14 (d, J ¼ 2.8 Hz, 2H), 0.88–0.84 (m, 2H), 0.68–0.64 (m, 2H).
for 12 h at 70 ꢅC. The resulting solution was extracted with ethyl MS (ESI) calc’d for (C28H25BrClN5O3) [M þ 1]þ, 594, 596; found,
acetate (300 ml ꢁ 2). The combined organic phase was washed 594, 596.
with brine (200 ml ꢁ 3), then dried over anhydrous magnesium
Synthesis of 5-bromo-2-N-f7-methoxy-3,4-dihydrospiro[1,4-ben-
sulfate, filtered and the filtrate was concentrated under reduced zoxazine-2,10 -cyclopropane]-6-ylg-4-N-[1-(pyridin-2-ylmethyl)-1H-pyr-
pressure. The residue was purified by flash column chromatog- azol-4-yl]pyrimidine-2,4-diamine (20d). A gray solid (42.9 mg, 24%
raphy with 20 ꢆ 50% ethyl acetate in petroleum ether to afford 18 yield). 1H NMR (300 MHz, DMSO-d6) d 8.78 (s, 1H), 8.52–8.50 (m,
as a yellow solid (4 g, 57% yield). 1H NMR (400 MHz, DMSO-d6) d 1H), 8.16–8.12 (m, 1H), 7.98 (s, 1H), 7.93 (s, 1H), 7.76–7.69 (s, 2H),
10.38 (s, 1H), 6.44 (s, 1H), 6.28 (s, 1H), 4.53 (s, 2H), 3.68 (s, 3H), 7.31–7.27 (m, 1H), 6.89–6.87 (m, 2H), 6.34 (s, 1H), 5.37–5.31 (m,
1.16 (t, J ¼ 3.0 Hz, 2H), 1.06 (t, J ¼ 3.0 Hz, 2H). MS (ESI) calc’d for 3H), 3.64 (s, 3H), 3.11 (d, J ¼ 2.8 Hz, 2H), 0.88–0.84 (m, 2H),
(C11H12N2O3) [M þ 1]þ, 221; found, 221.
0.69–0.65 (m, 2H). MS (ESI) calc’d for (C24H23BrN8O2) [M þ 1]þ, 535,
537; found, 535, 567.
Synthesis of 7-methoxy-3,4-dihydrospiro[1,4-benzoxazine-2,10-
cyclopropane]-6-amine (19). To a solution of 18 (1 g, 4.54 mmol) in
Synthesis of 2-(f7-methoxy-3,4-dihydrospiro[1,4-benzoxazine-2,10-
dry tetrahydrofuran (20 ml) was added lithium aluminum hydride cyclopropane]
-6-ylgamino)-4-f[4-(pyridin-2-ylmethoxy)phenyl]ami-
(690 mg, 18.18 mmol,) at 0 ꢅC. Then the resulting solution was nogpyrimidine-5-carbonitrile (21a). A degassed solution of 20a
heated to 70 ꢅC for 2 h. The reaction was then quenched by the (30 mg, 0.05 mmol) tris(dibenzylideneacetone)dipalladium (11.1 mg,
addition of 50 ml of water at 0 ꢅC and extracted with ethyl acetate 0.01 mmol), zinc cyanide (22.7 mg, 0.19 mmol) and 1,10-bis(diphe-
(100 ml ꢁ 2). The combined organic phase was washed with brine nylphosphino)ferrocene (11.9 mg, 0.02 mmol) in N,N-dimethylfor-
(100 ml ꢁ 2), then dried over anhydrous magnesium sulfate, fil- mamide (1 ml) and water (0.1 ml) was stirred for 3 h at 110 ꢅC in
tered and the filtrate was concentrated under reduced pressure. microwave reactor. The resulting solution was diluted with water
The residue was purified by flash column chromatography with (20 ml) and extracted with ethyl acetate (30 ml ꢁ 2). The com-
1
ethyl acetate to afford 19 as brown oil (0.5 g, 53% yield). H NMR bined organic phase was washed with brine (50 ml ꢁ 2), then
(300 MHz, DMSO-d6) d 6.17 (s, 1H), 6.00 (s, 1H), 5.14 (s, 1H), 4.11 (s, dried over anhydrous magnesium sulfate, filtered and the filtrate
2H), 3.59 (s, 3H), 3.06 (d, J ¼ 2.1 Hz, 2H), 0.79–0.77 (m, 2H), was concentrated under reduced pressure. The residue was puri-
0.63–0.59 (m, 2H). MS (ESI) calc’d for (C11H14N2O2) [M þ 1]þ, 207; fied by flash column chromatography with 0–10% methanol in
found, 207.
ethyl acetate to afford 21a as a yellow solid(13.9 mg, 51% yield).
1H NMR (400 MHz, DMSO-d6) d 9.16 (br, 1H), 8.70–8.60 (m, 2H),
8.36 (s, 1H), 7.87 (dd, J1¼ 7.8 Hz, J2 ¼ 1.5 Hz, 1H), 7.55–7.43(m,
3H), 7.39–7.31 (m, 1H), 6.97–6.93 (m, 2H), 6.80–6.71 (m, 1H), 6.38
(s, 1H), 5.23–5.14 (m, 3H), 3.59 (s, 3H), 3.15 (s, 2H), 0.89–0.87 (m,
2H), 0.70–0.67 (m, 2H). MS (ESI) calc’d for (C28H25N7O3) [M þ 1]þ,
508; found, 508.
Synthesis of 5-bromo-2-N-f7-methoxy-3,4-dihydrospiro[1,4-ben-
zoxazine-2,10- cyclopropane]-6-ylg-4-N-[4-(pyridin-2-ylmethoxy)phe-
nyl]pyrimidine-2,4-diamine (20a). To
a solution of 5-bromo-2-
chloro-N-[4-(pyridin-2-ylmethoxy)phenyl]pyrimidin
-4-amine
(151.5 mg, 0.39 mmol) and 19 (100 mg, 0.48 mmol) in dioxane
(5 ml) was added 4-Toluene sulfonic acid monohydrate (83.5 mg,
0.49 mmol) at room temperature. The resulting solution was
stirred for 2 h at 90 ꢅC. The reaction mixture was diluted with satu-
Compound 21 b-21d was prepared in analogy to 21a
Synthesis of 4-[[3-chloro-4-(pyridin-2-ylmethoxy)phenyl]amino]-2-
rated sodium bicarbonate solution (30 ml) and extracted with ([7-methoxy-3,4 -dihydrospiro[1,4-benzoxazine-2,10-yclopropane]-6-yl]
ethyl acetate (50 ml ꢁ 3). The combined organic phase was amino)pyrimidine-5-carbonitrile (21 b). A yellow solid (20.7 mg, 23%

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1243
yield). 1H NMR (300 MHz, DMSO-d6) d 9.23 (br, 1H), 8.74 (br, 1H), 2.81 (s, 3H), 2.19 – 1.97 (m, 2H), 1.85 (d, J ¼ 13.9 Hz, 2H).MS (ESI)
calculated for (C35H34ClN7O3) [M þ 1]þ, 636; found, 636.
8.58 (d, J ¼ 2.1 Hz, 1H), 8.39 (s, 1H), 7.90–7.85 (m, 1H), 7.72–7.50
(m, 3H), 7.38–7.32 (m, 1H), 7.13–7.09 (m, 1H), 6.79–6.71 (m, 1H),
6.40 (s, 1H), 5.40–5.24 (m, 3H), 3.60 (s, 3H), 3.13 (s, 2H), 0.89–0.87
(m, 2H), 0.69–0.67 (m, 2H). MS (ESI) calc’d for (C28H24ClN7O3)
[M þ 1]þ, 542; found, 542.
Synthesis of 2-f[5-methoxy-10-methyl-1-(prop-2-enoyl)-1,2-dihydro-
spiro[indole-3,40
-piperidine]-6-yl]aminog-4-f[1-(pyridin-2-ylmethyl)-
1H-pyrazol-4-yl]aminogpyrimidine-5-carbonitrile (A4). a white solid
(14 mg, 32% yield). 1H NMR (300 MHz, DMSO-d6) d 9.65 (s, 1H),
9.12 (s, 1H), 8.56 – 8.44 (m, 1H), 8.36 (s, 1H), 8.22 (s, 1H), 7.71–7.67
(m, 2H), 7.27 (dd, J ¼ 7.4, 4.9 Hz, 1H), 7.04 (s, 1H), 6.77 (dd,
J ¼ 16.5, 10.2 Hz, 1H), 6.22 (dd, J ¼ 16.5, 2.3 Hz, 1H), 5.75 (dd,
J ¼ 10.2, 2.3 Hz, 1H), 5.16 (s, 2H), 3.98 (s, 2H), 3.73 (s, 3H), 2.81 –
2.57 (m, 2H), 2.20 (s, 3H), 2.07 – 1.76 (m, 4H), 1.48 (d, J ¼ 11.7 Hz,
2H). MS (ESI) calculated for (C31H32N10O2) [M þ 1]þ, 577;
found, 577.
Synthesis
of
4-[[4-(benzyloxy)-3-chlorophenyl]amino]-2-([7-
methoxy-3,4- dihydrospiro[1,4-benzoxazine-2,10-cyclopropane]-6-yl]a-
mino)pyrimidine-5-carbonitrile (21c). A yellow solid (21.6 mg, 24%
yield). 1H NMR (400 MHz, DMSO-d6) d 8.82 (br, 2H), 8.39 (s, 1H),
7.69 (br, 1H), 7.55 (s, 1H), 7.48–7.36 (m, 5H), 7.13–7.09 (m, 1H),
6.72–6.69 (m, 1H), 6.41 (s, 1H), 5.30–5.16 (m, 3H), 3.60 (s, 3H), 3.15
(s, 2H), 0.89–0.87 (m, 2H), 0.69–0.67 (m, 2H). MS (ESI) calc’d for
(C29H25ClN6O3) [M þ 1]þ, 541; found, 541.
Synthesis
of
(2E)-4-f60-[(5-cyano-4-f[4-(pyridin-2-
ylmethoxy)phenyl]aminog pyrimidin-2-yl)amino]-50-methoxyspiro[cy-
clopropane-1,30-indole]-10-ylg-1-(dimethylamino)-4-oxobut-2-en-2-yl
(B1). To a solution of 15a (40 mg, 0.08 mmol) and N,N-diisopropy-
lethylamine (31.53 mg, 0.24 mmol) in dry tetrahydrofuran (2 ml)
Synthesis of 2-([7-methoxy-3,4-dihydrospiro[1,4-benzoxazine-2,10-
cyclopropane]-6-
yl]amino)-4-[[1-(pyridin-2-ylmethyl)-1H-pyrazol-4-
yl]amino]pyrimidine-5-carbonitrile (21d). A yellow solid (21.8 mg,
48% yield). 1H NMR (400 MHz, DMSO-d6) d 9.62 (br, 1H), 8.84 (s,
1H), 8.51 (d, J ¼ 2.1 Hz, 1H), 8.33 (s, 1H), 7.80–7.70(m, 2H),
7.65–7.50 (m, 1H), 7.32–7.29 (m, 1H), 6.90–6.82 (m, 1H), 6.80–6.73
(m, 1H), 6.37 (s, 1H), 5.54 (s, 1H), 5.28 (br, 2H), 3.56 (s, 3H), 3.07 (s,
2H), 0.89–0.87 (m, 2H), 0.70–0.67 (m, 2H). MS (ESI) calc’d for
(C25H23N9O2) [M þ 1]þ, 482; found, 482.
was added
a
solution of (2E)-4-bromobut-2-enoyl chloride
(14.83 mg, 0.08 mmol) in dry tetrahydrofuran (0.5 ml) at 0 ꢅC. The
resulting solution was stirred for 1 h at 0 ꢅC. This was followed by
the addition of a solution of dimethylamine in tetrahydrofuran
(2 ml, 2 M). The resulting solution was allowed to react, with stir-
ring, for an additional 18 h at room temperature. The reaction mix-
ture was diluted with saturated sodium bicarbonate solution
(30 ml) and extracted with ethyl acetate (50 ml ꢁ 3). The com-
bined organic phase was washed with saturated sodium chloride
solution (50 ml ꢁ 3), then dried over anhydrous magnesium sul-
fate, filtered and the filtrate was concentrated under reduced
pressure. The residue was purified with Prep-HPLC [Column:
Xbridge RP18, 5 um, 19 ꢁ 150 mm; mobile phase: water (0.05%
ammonium bicarbonate) and acetonitrile (35% acetonitrile up to
65% in 12 min); Detector, UV 220 and 254 nm] to afford B1 as an
off-white solid(1.3 mg, 3% yield). 1H NMR (300 MHz, DMSO-d6) d
8.97 (s, 1H), 8.81 (s, 1H), 8.56 (d, J ¼ 4.2 Hz, 1H), 8.48–8.44 (m, 2H),
7.81 (dd, J1¼ 7.8 Hz, J2 ¼ 1.8 Hz), 7.53–7.42 (m, 3H), 7.33–7.31 (m,
1H), 6.98 (d, J ¼ 8.7 Hz, 2H), 6.70–6.65 (m, 1H), 6.51 (s, 1H), 6.32
(dd, J1 ¼ 17.1 Hz, J ¼ 2.1 Hz, 1H), 5.14 (s, 2H), 4.09 (s, 2H), 3.75 (s,
3H), 2.16 (s, 6H), 1.09 (s, 4H).MS (ESI) calc’d for (C34H34N8O3)
[M þ 1]þ, 603; found, 603.
Synthesis of 2-f[5-methoxy-10-methyl-1-(prop-2-enoyl)-1,2-dihydro-
spiro[indole-3,40 -piperidine]-6-yl]aminog-4-f[4-(pyridin-2-ylmethoxy)-
phenyl]aminogpyrimidine-5-carbonitrile (A1). To a solution of 8a
(42 mg, 0.08 mmol) and triethylamine (80 mg, 0.79 mmol) in
dichloromethane (5 ml). This was followed by the addition of a
solution of prop-2-enoyl chloride (5.6 mg, 0.06 mmol) in dichloro-
methane (0.2 ml) at –20 ꢅC under nitrogen. After stirring for 1 h at
–20 ꢅC, the mixture was concentrated under vacuum. The crude
product was purified by Prep-HPLC [Column: XBridge Shield RP18
ꢀ
OBD Column, 5um, 19 150 mm;: Water with 10 mmol/L ammo-
nium bicarbonate, mobile phase B: acetonitrile. Gradient: 25.0%B
up to 48.0%B in 10 min; Detector: UV 220 nm] to afford A1 as a
white solid (14.4 mg, 31% yield). 1H NMR (300 MHz, DMSO-d6) d
10.17 (s, 1H), 9.19 (s, 1H), 9.00 (s, 1H), 8.60 (d, J ¼ 4.2 Hz, 1H), 8.40
(s, 1H), 8.24 (s, 1H), 7.84 (td, J ¼ 7.8, 1.8 Hz, 1H), 7.50–7.34 (m, 4H),
6.81 – 6.71 (m, 3H), 6.30 (d, J ¼ 18.0 Hz, 1H), 5.84 (d, J ¼ 11.1 Hz,
1H), 5.09 (s, 2H), 4.23 (s, 2H), 3.74 (s, 3H), 3.47 – 3.43 (s, 2H), 3.20 –
3.13 (m, 2H), 2.80 (s, 3H), 2.24 (t, J ¼ 12.0 Hz, 2H), 1.89 – 1.85 (m,
2H).MS (ESI) calculated for (C34H34N8O3) [M þ 1]þ, 603; found, 603.
Compound A2-A4 was prepared in analogy to A1.
Compound B2-B7 was prepared in analogy to B1.
Synthesis
of
(2E)-4-f60-[(4-f[3-chloro-4-(pyridin-2-
ylmethoxy)phenyl]aminog-5- cyanopyrimidin-2-yl)amino]-50-methoxy-
spiro[cyclopropane-1, 30-indole]-10-ylg-1-(dimethylamino)-4-oxobut-2-
en-2-yl (B2). An off-white solid (1.2 mg, 2% yield). 1H NMR
(300 MHz, DMSO-d6) d 8.81 (s, 1H), 8.58–8.56 (m, 2H), 8.51 (s, 1H),
7.87–7.52 (m, 5H), 7.35–7.31 (m, 1H), 7.16–7.13 (m, 1H), 6.73–6.62
(m, 1H), 6.51 (s, 1H), 6.41–6.31 (m, 1H), 5.22 (s, 2H), 4.12 (s, 2H),
3.76 (s, 3H), 2.16 (s, 6H), 1.08 (s, 4H).MS (ESI) calc’d for
(C34H33ClN8O3) [M þ 1]þ, 637; found, 637.
Synthesis of 4-f[3-chloro-4-(pyridin-2-ylmethoxy)phenyl]aminog-2-
f[5-methoxy-10- methyl-1-(prop-2-enoyl)-1,2-dihydrospiro[indole-3,40-
piperidine]-6-yl]aminogpyrimidine-5-carbonitrile (A2). A white solid
1
(4.2 mg, 12% yield). H NMR (300 MHz, Methanol-d4) d 8.64 (s, 1H),
8.57 (dt, J ¼ 4.8, 1.4 Hz, 1H), 8.35 (s, 1H), 7.91 (dd, J ¼ 8.5, 6.7 Hz,
1H), 7.73 – 7.64 (m, 2H), 7.46 – 7.29 (m, 2H), 6.97– 6.94 (m, 2H),
6.77 (dd, J ¼ 16.7, 10.3 Hz, 1H), 6.30 (d, J ¼ 16.8 Hz, 1H), 5.77 (d,
J ¼ 10.6 Hz, 1H), 5.16 (s, 2H), 4.12 (s, 2H), 3.90 (s, 3H), 2.93 (d,
J ¼ 12.0 Hz, 2H), 2.39 (s, 3H), 2.23 (t, J ¼ 12.3 Hz, 2H), 2.04 (dt,
J ¼ 14.3, 7.2 Hz, 2H), 1.72 (d, J ¼ 13.4 Hz, 2H).MS (ESI) calculated for
(C34H33ClN8O3) [M þ 1]þ, 637; found, 637.
Synthesis of 4-f[4-(benzyloxy)-3-chlorophenyl]aminog-2-(f10-[(2E)-
4-(dimethylamino) but-2-enoyl]-50-methoxy-10,20-dihydrospiro[cyclo-
propane-1,30-indole]-60-ylgamino)pyrimidine-5-carbonitrile (B3). An
off-white solid(3.8 mg, 6% yield). 1H NMR (300 MHz, DMSO-d6) d
8.80 (s, 1H), 8.60 (s, 1H), 8.51 (s, 1H), 7.80 (s, 1H), 7.53–7.37 (m,
7H), 7.14–7.12 (m, 1H), 6.71–6.61 (m, 1H), 6.52 (s, 1H), 6.35–6.27
(m, 1H), 5.16 (s, 2H), 4.11 (s, 2H), 3.76 (s, 3H), 2.15 (s, 6H),
1.12–1.08 (m, 4H).MS (ESI) calc’d for (C35H34ClN7O3) [M þ 1]þ, 636;
found, 636.
Synthesis
of
4-f[4-(benzyloxy)-3-chlorophenyl]aminog-2-f[5-
methoxy-10-methyl-1
-(prop-2-enoyl)-1,2-dihydrospiro[indole-3,40-
piperidine]-6-yl]aminogpyrimidine-5-carbonitrile (A3). A white solid
1
(TFA salt, 23.1 mg, 38% yield). H NMR (300 MHz, DMSO-d6) d 8.41
Synthesis of 3-f60-[(5-cyano-4-f[4-(pyridin-2-ylmethoxy)phenyl]a-
(s, 1H), 8.25 (s, 1H), 7.70 (d, J ¼ 2.6 Hz, 1H), 7.54 – 7.24 (m, 6H),
6.98 (d, J ¼ 9.3 Hz, 1H), 6.92 (s, 1H), 6.72 (dd, J ¼ 16.6, 10.3 Hz, 1H),
6.25 (d, J ¼ 16.6 Hz, 1H), 5.81 (d, J ¼ 10.4 Hz, 1H), 5.13 (s, 2H), 4.17
minogpyrimidin -2-yl) amino]-50-methoxyspiro [cyclopropane-1, 30-
indole]-10-ylg-3-oxoprop-1-en-2-yl (B4). An off-white solid (21.9 mg,
1
(s, 2H), 3.76 (s, 3H), 3.43 (d, J ¼ 12.7 Hz, 2H), 3.22 – 3.03 (m, 2H), 35% yield). H NMR (300 MHz, DMSO-d6) d 8.84 (s, 1H), 8.78 (s, 1H),

1244
L. YE ET AL.
8.56 (d, J ¼ 4.2 Hz, 1H), 8.48–8.44 (m, 2H), 7.83 (dd, J1¼ 7.8 Hz, J2 ¼ 2H), 6.98 (d, J ¼ 3.2 Hz, 1H), 6.62–6.55 (m, 2H), 6.45–6.37 (m, 1H),
1.8 Hz), 7.77–7.46 (m, 3H), 7.32–7.28 (m, 1H), 6.98 (d, J ¼ 8.7 Hz, 5.20 (s, 2H), 3.88 (s, 2H), 3.72 (s, 3H), 2.99–2.87 (m, 2H), 2.04 (s,
1H), 6.54–6.45 (m, 2H), 6.19 (dd, J1 ¼ 17.1 Hz, J ¼ 2.1 Hz, 1H), 5.62 6H), 0.96–0.94 (m, 2H), 0.78–0.76(m, 2H). MS (ESI) calc’d for
(C34H33ClN8O4) [M þ 1]þ, 653; found, 653.
(dd, J1 ¼ 10.5 Hz, J ¼ 2.1 Hz, 1H), 5.14 (s, 2H), 4.10 (s, 2H), 3.75 (s,
3H), 1.09 (s, 4H). MS (ESI) calc’d for (: C31H27N7O3) [M þ 1]þ, 546;
found, 546.
Synthesis of (2E)-4-f6-[(4-f[4-(benzyloxy)-3-chlorophenyl]aminog-
5-cyanopyrimidin -2-yl)amino]-7-methoxyspiro[1,4-benzoxazine-2,10-
Synthesis
of
3-f60-[(4-f[3-chloro-4-(pyridin-2-
cyclopropane]-4-ylg-1-(dimethylamino)-4-oxobut-2-en-2-yl
(C3).an
1
ylmethoxy)phenyl]aminog-5- cyanopyrimidin-2-yl)amino]-50-methoxy-
spiro[cyclopropane-1,30-indole]-10-ylg-3-oxoprop-1-en-2-yl (B5). An
off-white solid (1.6 mg, 1% yield). 1H NMR (300 MHz, DMSO-d6) d
8.91 (s, 1H), 8.56 (d, J ¼ 4.2 Hz, 2H), 8.51 (s, 1H), 7.85–7.82 (m, 2H),
7.56–7.51 (m, 2H), 7.34–7.28 (m, 1H), 7.16–7.12 (m, 1H), 6.56–6.51
(m, 2H), 6.19 (d, J ¼ 10.5 Hz, 1H), 5.62 (d, J ¼ 10.5 Hz, 1H), 5.22 (s,
2H), 4.12 (s, 2H), 3.77 (s, 3H), 1.10 (d, J ¼ 6.0 Hz, 4H). MS (ESI) calc’d
for (C31H26ClN7O3) [M þ 1]þ, 580; found, 580.
off-white solid (14.5 mg, 15% yield). H NMR (300 MHz, DMSO-d6) d
9.02 (br, 1H), 8.46 (s, 1H), 8.37 (s, 1H), 7.64 (s, 2H), 7.47–7.32 (m,
6H), 7.02 (d, J ¼ 3.2 Hz, 1H), 6.63–6.57 (m, 2H), 6.44–6.39 (m, 1H),
5.14 (s, 2H), 3.88 (s, 2H), 3.72 (s, 3H), 2.93–2.87 (m, 2H), 2.05 (s,
6H), 0.96–0.94 (m, 2H), 0.78–0.76(m, 2H). MS (ESI) calc’d for
(C35H34ClN7O4) [M þ 1]þ, 652; found, 652.
Synthesis of (2E)-4-f6-[(5-cyano-4-f[1-(pyridin-2-ylmethyl)pyrazol-4-
yl]aminog pyrimidin-2-yl)amino]-7-methoxyspiro[1,4-benzoxazine-2,10-
cyclopropane]-4-ylg-1-(dimethylamino)-4-oxobut-2-en-2-yl (C4). An off-
white solid (23.2 mg, 19% yield). H NMR (300 MHz, DMSO-d6) d 9.41
(s, 1H), 8.76 (s, 1H), 8.50 (d, J¼ 3.2 Hz, 1H), 8.32 (s, 1H), 7.90–7.80(m,
1H), 7.70–7.53 (m, 3H), 7.28–7.24 (m, 1H), 6.90 (d, J ¼ 3.2 Hz, 1H),
6.70–6.47 (m, 3H), 5.19 (s, 2H), 3.85 (s, 2H), 3.68 (s, 3H), 2.91 (br, 2H),
2.06 (s, 6H), 0.96–0.94 (m, 2H), 0.78–0.76(m, 2H). MS (ESI) calc’d for
(C31H32N10O3) [M þ 1]þ, 593; found, 593.
Synthesis of (2E)-4-f60-[(5-cyano-4-f[1-(pyridin-2-ylmethyl)pyrazol-
4-yl]aminog pyrimidin-2-yl)amino]-50-methoxyspiro[cyclopropane-1,30-
indole]-10-ylg-1-(dimethylamino)-4-oxobut-2-en-2-yl trifluoroacetate
(B6). A yellow solid (3.8 mg, 6% yield). 1H NMR (300 MHz, DMSO-
d6) d 9.35 (s, 1H), 9.14 (s, 1H), 8.70 (s, 1H), 8.52 (d, J ¼ 8.4 Hz, 1H),
8.44 (s, 1H), 8.03 (s, 1H), 7.78–7.72 (m, 2H), 7.32–7.28 (m, 1H),
7.12–7.09 (m, 1H), 6.76–6.55 (m, 3H), 5.37 (s, 2H), 4.16 (s, 2H), 3.90
(d, J ¼ 2.8 Hz, 2H), 3.78 (s, 3H), 2.82 (s, 6H), 1.13 (t, J ¼ 6.7 Hz, 4H).
MS (ESI) calc’d for: (C31H32N10O2) [M þ 1]þ, 577; found, 577.
3.2. Biological activity evaluation
Synthesis of 3-f60-[(5-cyano-4-f[1-(pyridin-2-ylmethyl)pyrazol-4-
yl]aminog pyrimidin-2-yl)amino]-50-methoxyspiro[cyclopropane-1,30-
indole]-10-ylg-3-oxoprop-1-en-2-yl (B7). An off-white solid (12 mg,
3.2.1. EGFR-wt/HER2 protein kinase assay
The inhibitory profile of 16 compounds was determined using
two protein kinases. The compounds were tested at a concentra-
tion of 5 ꢁ 1 0ꢂ7 M, in singlicate. The compounds were solved
and diluted to 5 ꢁ 10ꢂ5 M stock solutions in 100% DMSO accord-
ing to the customer-filled CSF. 10 lL of each of the stock solu-
tions were transferred into column 3–5 of a 96-well, Column 1
and 2 of the plate were filled with 10 lL100% DMSO. The plate
is in the following referred to as “copy plate”. In the process,
90 lL H2O were added to each well of the copy plate. To minim-
ize potential compound precipitation, the H2O was added to
each well only a few minutes before the transfer of the com-
pound solutions into the assay plates. The plate was shaken
thoroughly, resulting in a “compound dilution plate” with a
compound concentration of 5 ꢁ 10ꢂ6 M/10% DMSO. This plate
was used for the transfer of 5 lL compound solution into the
assay plates. The final volume of the assay was 50 lL. All com-
pounds were tested at 5 ꢁ 1 0ꢂ7 M in singlicate. The final DMSO
concentration in the reaction cocktails was 1% in all cases. The
compound dilution plate was disposed.
1
27% yield). H NMR (300 MHz, DMSO-d6) d 9.74 (s, 1H), 9.32 (s, 1H),
8.80 (s, 1H), 8.55 (d, J ¼ 8.4 Hz, 2H), 8.11 (s, 1H), 7.80–7.77 (m, 2H),
7.34–7.30 (m, 1H), 7.06 (d, J ¼ 8.4 Hz, 1H), 6.63–6.58 (m, 2H), 6.24
(d, J ¼ 17.7 Hz, 1H), 5.72 (d, J ¼ 8.4 Hz, 1H), 5.42 (s, 2H), 4.17 (s, 2H),
3.78 (s, 3H), 1.13 (t, J ¼ 8.7 Hz, 4H). MS (ESI) calc’d for: (C28H25N9O2)
[M þ 1]þ, 520; found, 520.
Synthesis
of
(2E)-4-f6-[(5-cyano-4-f[4-(pyridin-2-
ylmethoxy)phenyl]aminog pyrimidin-2-yl)amino]-7-methoxyspiro[1,4-
benzoxazine-2,10-cyclopropane]-4-ylg-1-(dimethylamino)-4-oxobut-2-
en-2-yl (C1). To a solution of 21a (100 mg, 0.20 mmol) and triethyl-
amine (298.8 mg, 2.95 mmol) in dichloromethane (10 ml) was
added a solution of (2E)-4-(dimethylamino)but-2-enoyl chloride
hydrochloride (360.9 mg, 1.96 mmol) in dichrolomethane (3 ml) at
0 ꢅC. The resulting solution was stirred for 2 h at 40 ꢅC. The reac-
tion was quenched with saturated sodium bicarbonate solution
(30 ml) and extracted with dichloromethane (30 ml ꢁ 3). The com-
bined organic phase was washed with brine (50 ml ꢁ 3), then
dried over anhydrous magnesium sulfate, filtered and the filtrate
was concentrated under reduced pressure. The residue was puri-
R
V
A radiometric protein kinase assay (33PanQinase Activity Assay)
ꢀ
fied by preparative HPLC [Column: X Bridge C18, 19 150 mm, 5
was used for measuring the kinase activity of the two protein kin-
ases. All kinase assays were performed in 96-well FlashPlatesTM from
PerkinElmer (Boston, MA, USA) in a 50 lL reaction volume. The reac-
tion cocktail was pipetted in 4 steps in the following order: 20 lL of
assay buffer, 5lL of ATP solution (in H2O), 5 lL of test compound
(in 10% DMSO), 20 lL enzyme/subtrate mix. The assay for both pro-
tein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2,
3 mM MnCl2, 3lM Na-orthovanadate, 1.2 mM DTT, ATP. The assay
for ERBB2 additionally contained 50 lg/mLPEG20000. The reaction
cocktails were incubated at 30 ꢅC for 60min. The reaction was
stopped with 50lL of 2% (v/v) H3PO4, plates were aspirated and
washed two times with 200 lL 0.9% (w/v) NaCl. Incorporation of
um; Mobile Phase A:Water with 10 mM ammonium bicarbonate,
Mobile Phase B: acetonitrile; Flow rate: 20 ml/min; Gradient: 20% B
to 45% B in 10.0 min; 254 nm] to afford C1 as an off-white solid
(8.8 mg, 7% yield). 1H NMR (300 MHz, DMSO-d6) d 9.25 (br, 1H),
8.73–8.61 (m, 1H), 8.56 (d, J ¼ 4.2 Hz, 1H), 8.40 (s, 1H), 7.83 (dd,
J1¼ 7.8 Hz, J2 ¼ 1.8 Hz, 1H), 7.55–7.38 (m, 5H), 6.90–6.50 (m, 5H),
5.13 (s, 2H), 3.89 (s, 2H), 3.72 (s, 3H), 2.90 (br, 2H), 2.02 (br, 6H),
0.96–0.94 (m, 2H), 0.78–0.76(m, 2H). MS (ESI) calc’d for
(C34H34N8O4) [M þ 1]þ, 619; found, 619.
Compound C2-C7 was prepared in analogy to C1.
Synthesis
of
(2E)-4-f6-[(4-f[3-chloro-4-(pyridin-2-
ylmethoxy)phenyl]aminog-5- cyanopyrimidin-2-yl)amino]-7-methoxy-
33Pi was determined with
a microplate scintillation counter
spiro[1,4-benzoxazine-2,10-cyclopropane]-4-ylg-1-(dimethylamino)-4-
(Microbeta, Wallac). All assays were performed with
a
oxobut-2-en-2-yl(C2). An off-white solid (15.3 mg, 25% yield). 1H BeckmanCoulter/SAGIANTMCore System. The median value of the
NMR (300 MHz, DMSO-d6) d 9.04 (br, 1H), 8.56 (s, 1H), 8.47 (s, 1H), counts in column 1 (n¼ 8) of each assay plate was defined as “low
8.37 (s, 1H), 7.86–7.80 (m, 1H), 7.65–7.55 (m, 3H), 7.41–7.28 (m, control”. This value reflects unspecific binding of radioactivity to the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1245
plate in the absence of a protein kinase but in the presence of the values for residual activities per kinase. Based on each 10 corre-
substrate. The median value of the counts in column 2 of each assay
plate (n¼ 8) was taken as the “high control”. As part of the data
evaluation the low control value from a particular plate was sub-
tracted from the high control value as well as from all 80
“compound values” of the corresponding plate. The residual activity
(in %) for each well of a particular plate was calculated by using the
following formula:
sponding residual activities, IC50 values were calculated using
Prism 5.04 for Windows. The mathematical model used was
“Sigmoidal response (variable slope)” with parameters “top” fixed
at 100% and “bottom” at 0%. The fitting method used was a
least-squares fit.
3.3. Molecular docking
ð
Þ
¼ 100 ꢁ
Res: Activity
%
The docking was performed with MOE (version 2019)22. The crystal
structures of EGFR mutations (T790M/L858R and T790M) with ner-
atinib (PDB code: 3W2Q and 2JIV) were downloaded from Protein
Data Bank (PDB: http://www.rcsb.org/). Only A chain of EGFR and
neratinib were kept, and then the complexes were prepared by
using MOE QuickPrep module in MOE (Molecular Operating
Environment, version 2019.01) with default parameters. The com-
pounds A1 and A2 were optimized based on MMFF94X force field.
The MOE covalent docking was employed to predict the covalent
binding modes of EGFR protein with A1, A2 and neratinib. In our
covalent docking study, this method relies on a reaction/trans-
formation placement methodology to match the reactive groups
(ethylene bond) on the A1, A2 and neratinib and the cysteine resi-
due followed by the formation of the covalent bond between
them. Covalent ligands were modeled and minimized in the prere-
action form generated with the Builder panel module. The default
postprocessing protocol using minimization by the Rigid Receptor
refinement scheme and rescoring by the GBVI/WSA dG scoring
function was used to estimate the binding free energy (Kcal/mol)
of docked pose and EGFR protein. A total of 30 covalent docking
poses for each compound were ranked by the estimated binding
free energy.
Â
ðcpm of compound – low controlÞ=
ðhigh control ꢂ low controlÞꢇ
3.2.2. Seven EGFR mutants protein kinase assay
The compounds were serially diluted in semi-log steps with 100%
DMSO in a 96 well microtiter plate. The concentration range of
this serial dilution was 1 ꢁ 10ꢂ3 M to 3 ꢁ 10ꢂ8 M. Directly before
use the test compounds were further diluted 1:10 with water,
resulting in a concentration range from 1 ꢁ 10ꢂ4 M to 3 ꢁ 1 0ꢂ9 M
in 10% DMSO. For the assays, 5 lL from each concentration were
transferred into the assay. The final volume of the assay was
50 lL. The compounds were tested at 10 final assay concentra-
tions in the range from 1 ꢁ 10ꢂ5 M to 3 ꢁ 10ꢂ10 M. The final DMSO
concentration in the reaction cocktails was 1% in all cases.
R
A radiometric protein kinase assay (33PanQinaseV Activity
Assay) was used for measuring the kinase activity of the 9 protein
kinases. All kinase assays were performed in 96-well FlashPlatesTM
from Perkin Elmer (Boston, MA, USA) in a 50 lL reaction volume.
The reaction cocktail was pipetted in 4 steps in the following
order: 10 lL of non-radioactive ATP solution (in H2O), 25 lL of
assay buffer/[c-33P]-ATP mixture, 5 lL of test sample in 10%
DMSO, 10 lL of enzyme/substrate mixture. The assay for all pro-
tein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2,
3 mM MnCl2, 3 lM Na-orthovanadate, 1.2 mM DTT, ATP, [c-33P]-
ATP, protein kinase, and substrate.The reaction cocktails were
incubated at 30 ꢅC for 60 min. The reaction was stopped with
50 lL of 2% (v/v) H3PO4, plates were aspirated and washed two
times with 200 lL 0.9% (w/v) NaCl. Incorporation of 33Pi (counting
of “cpm”) was determined with a microplate scintillation counter
Acknowlegements
The work was supported by Special Innovation Project of
Guangdong
Education
Department
(Natural
Science)
(2017KTSCX107), Guangdong Natural Science Foundation
(2015A03031356, 2015A030310176), Science and Technology
Planning Project of Guangdong Province (2017ZC0199), National
Natural Science Foundation of China (81872740), Medical Scientific
Research Foundation of Guangdong province (B2019004), Key
laboratory of new drug discovery and evaluation of ordinary uni-
versities of Guangdong province(2017KSYS002), Guangzhou key
laboratory of construction and application of new drug screening
model systems(201805010006), Guangdong province precise medi-
(Microbeta, Wallac). All assays were performed with
BeckmanCoulter Biomek 2000/SL robotic system.
a
For each kinase, the median value of the cpm of three wells
with complete reaction cocktails, but without kinase, was defined
as “low control” (n ¼ 3). This value reflects unspecific binding of
radioactivity to the plate in the absence of protein kinase but in
the presence of the substrate. Additionally, for each kinase the cine and big data engineering technology research center for
median value of the cpm of three other wells with the complete
reaction cocktail, but without any compound, was taken as the
“high control”, i.e. full activity in the absence of any inhibitor
(n ¼ 3). The difference between high and low control was taken as
100% activity for each kinase. As part of the data evaluation the
low control value of each kinase was subtracted from the high
control value as well as from their corresponding “compound val-
ues”. The residual activity (in %) for each compound well was cal-
culated by using the following formula:
traditional Chinese medicine, the open project foundation of the
Fujian Provincial Key Laboratory of Theoretical and Computational
Chemistry (201502), Guangdong province engineering technology
center for molecular probes & biomedical imaging. The authors
are grateful to Biochemical Screening Systems ProQinase GmbH
for biological evaluation.
Ethics approval and consent to participate
The current study was not required to complete an eth-
ical assessment.
ð
Þ
¼ 100 ꢁ
Res: Activity
%
Â
ðcpm of compound – low controlÞ=
ðhigh control – low controlÞꢇ
Consent for publication
Since 10 distinct concentrations of each compound were tested
against each kinase, the evaluation of the raw data resulted in 10 Not applicable.

1246
L. YE ET AL.
11. Yun CH, Mengwasser KE, Toms AV, et al. The T790M muta-
tion in EGFR kinase causes drug resistance by increasing the
affinity for ATP. Proc Natl Acad Sci USA 2008;105:2070–5.
12. Satoshi S, Youichi K, Shigeru I, et al. Structure-based
approach for the discovery of Pyrrolo[3,2-d]pyrimidine-Based
EGFR T790M/L858R mutant inhibitors. ACS Med Chem Lett
2013;4:201–5.
Competing interests
The authors declare that they have no competing interests.
ORCID
Ling Wang
http://orcid.org/0000-0001-5116-7749
13. Schwab CL, English DP, Roque DM, et al. Neratinib shows
efficacy in the treatment of HER2/neu amplified uterine ser-
ous carcinoma in vitro, and in vivo. Gynecol Oncol 2014;135:
142–8.
14. Saura C, Garcia-Saenz JA, Xu B, et al. Safety and efficacy of
neratinib in combination with capecitabine in patients with
metastatic human epidermal growth factor receptor 2eposi-
tive breast cancer. J Clin Oncol 2014;32:3626–33.
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