Design and synthesis of 4-anilinoquinazolines as Raf kinase inhibitors. Part 1. Selective B-Raf/B-RafV600E and potent EGFR/VEGFR2 inhibitory 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines

Article abstract of DOI:10.1016/j.bioorg.2021.104715

This paper presents the design and synthesis of 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines of scaffold 9 as selective B-Raf/B-Raf^V600E and potent EGFR/VEGFR2 kinase inhibitors. Total 14 compounds of scaffold 9 having different side chains at the triazolyl group with/without fluoro substituents at the anilino group were synthesized and investigated. Among them, 9m with a 2-carbamoylethyl side chain and C-4′/C-6′ difluoro substituents was the most potent, which selectively inhibited B-Raf (IC₅₀: 57 nM) and B-Raf^V600E (IC₅₀: 51 nM) over C-Raf (IC₅₀: 1.0 μM). Compound 9m also actively inhibited EGFR (IC₅₀: 73 nM) and VEGFR2 (IC₅₀: 7.0 nM) but not EGFR^T790M and PDGFR-β (IC₅₀: >10 μM). Despite having good potency for B-Raf and B-Raf^V600E in the enzymatic assays, 9m was less active to inhibit melanoma A375 cells which proliferate due to constitutively activated B-Raf^600E. The inferior activity of 9m for A375 was similar to that of sorafenib (6), suggesting that 9m might bind to the inactive conformations of B-Raf and B-Raf^V600E. Docking simulations could thus be performed to reveal the binding poses of 9m in B-Raf, B-Raf^V600E, and VEGFR2 kinases.

Full text of DOI:10.1016/j.bioorg.2021.104715

Bioorganic Chemistry 109 (2021) 104715
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of 4-anilinoquinazolines as Raf kinase inhibitors. Part
1. Selective B-Raf/B-RafV600E and potent EGFR/VEGFR2 inhibitory
4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines
,
Cheng-I Leea b, Chu-Bin Liaoc, Chih-Shang Chend, Fen-Ying Chengd, Yu-Hsuan Chungd,
Yu-Chuan Wangd, Sian-Yi Ciouc, Wen-Yun Hsuehd, Tzu-Hao Lod, Guan-Ru Huangd,
,e,*
Hsin-Yi Huangd, Chia-Shen Tsaid, Yu-Jung Lua, Shih-Hsien Chuangc, Jiann-Jyh Huangd
a Department of Biomedical Sciences, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102, Taiwan
b Center for Nano Bio-detections, Advanced Institute of Manufacturing with High-tech Innovations (AIM-HI), National Chung Cheng University, Min-Hsiung, Chia-Yi
62102, Taiwan
c Development Center for Biotechnology, National Biotechnology Research Park, Taipei City 11571, Taiwan
d Department of Applied Chemistry, National Chiayi University, No. 300, Syuefu Rd., Chiayi City 60004, Taiwan
e The Training and Research Institute of Food and Agriculture, National Chiayi University, No. 300, Syuefu Rd., Chiayi City 60004, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
4-Anilinoquinazoline
Raf
This paper presents the design and synthesis of 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines of
scaffold 9 as selective B-Raf/B-RafV600E and potent EGFR/VEGFR2 kinase inhibitors. Total 14 compounds of
scaffold 9 having different side chains at the triazolyl group with/without fluoro substituents at the anilino group
were synthesized and investigated. Among them, 9m with a 2-carbamoylethyl side chain and C-4′/C-6′ difluoro
substituents was the most potent, which selectively inhibited B-Raf (IC50: 57 nM) and B-RafV600E (IC50: 51 nM)
EGFR
VEGFR
Kinase
over C-Raf (IC50: 1.0 μM). Compound 9m also actively inhibited EGFR (IC50: 73 nM) and VEGFR2 (IC50: 7.0 nM)
but not EGFRT790M and PDGFR-β (IC50: >10
μ
M). Despite having good potency for B-Raf and B-RafV600E in the
enzymatic assays, 9m was less active to inhibit melanoma A375 cells which proliferate due to constitutively
activated B-Raf600E. The inferior activity of 9m for A375 was similar to that of sorafenib (6), suggesting that 9m
might bind to the inactive conformations of B-Raf and B-RafV600E. Docking simulations could thus be performed
to reveal the binding poses of 9m in B-Raf, B-RafV600E, and VEGFR2 kinases.
1. Introduction
mutation of EGFR is observed in various cancers and prevalent in non-
small-cell lung cancer (NSCLC) [7]. Activation of VEGFR is respon-
Inhibition of the oncogenic protein kinases [1,2] has been proved as
a successful anticancer strategy [3]. As of 1 March 2019, the U.S. FDA
has approved total 43 small-molecule kinase inhibitors for the treatment
of various cancers [4]. The majority of the approved kinase inhibitors is
developed for receptor tyrosine kinases, among which EGFR (epidermal
growth factor receptor) [5] and VEGFR (vascular endothelial growth
factor receptor) [6] kinases are the best validated. Amplification or
sible for tumor angiogenesis/metastasis [8,9] and correlates with the
poor prognosis of cancer patients [10]. Treatment of NSCLC with EGFR
inhibitors [11] as well as kidney and thyroid cancers with VEGFR in-
hibitors [12] are generally practiced. 4-Anilinoquinazoline (see the blue
scaffold of 1–3, Fig. 1) is a privileged structure versatile for EGFR and
VEGFR inhibitions [13,14]. By introduction of distinct substituents to
the anilino group, 4-anilinoquinazolines can be EGFR selective, VEGFR
Abbreviations: CuAAC, copper(I)-catalyzed alkyne–azide cycloaddition; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; dppf, 1,1′-bis(diphenylphosphino)
ferrocene; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; FDA, Food and Drug Administration; GI50, the concentration for 50%
of maximal inhibition of cell proliferation; H-bond, hydrogen bond; IC50, the half maximal inhibitory concentration; MAPK, mitogen-activated protein kinase; MEK,
mitogen-activated protein kinase kinase; NaAsc, sodium ascorbate; NSCLC, non-small-cell lung cancer; PDB, Protein Data Bank; PDGFR, platelet-derived growth
factor receptor; Raf, rapid accelerated fibrosarcoma; Ras, rat sarcoma viral oncogene homolog; TBAF, tetrabutylammonium fluoride; TMSN3, trimethylsilyl azide;
VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.
* Corresponding author at: Department of Applied Chemistry, National Chiayi University, No. 300, Syuefu Rd., Chiayi City 60004, Taiwan.
E-mail address: lukehuang@mail.ncyu.edu.tw (J.-J. Huang).
https://doi.org/10.1016/j.bioorg.2021.104715
Received 1 November 2020; Received in revised form 29 January 2021; Accepted 30 January 2021
Available online 8 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
selective, or both. EGFR inhibitors gefitinib (1) [15] and lapatinib (2)
[16] as well as VEGFR inhibitor vandetanib (3) [17] are the represen-
tative examples of kinase inhibitory 4-anilinoquinazolines. In addition,
several modified quinazolines also show EGFR [18], VEGFR [19], and
Aurora kinase [20] inhibitory activities.
Serine/threonine protein kinase Raf (rapid accelerated fibrosar-
coma) [21], comprising A-Raf, B-Raf, and C-Raf, plays a central role in
the Ras/Raf/MEK/ERK (MAPK, mitogen-activated protein kinase) signal
transduction pathway [22]. Growth signals from cell surface receptors
(e.g., EGFR and VEGFR) through this pathway to nucleus lead to cell
proliferation, differentiation, and survival. Among Raf isoforms, B-Raf is
more frequently mutated in cancers [23]. Constitutively activated B-
RafV600E accounts for the majority of B-Raf mutations (~90%) [24] and
is found in hairy cell leukemia (100%), melanoma (66%) [23], thyroid
cancer (38–69%) [25,26], colorectal cancer (20%), and a variety of
cancers [27]. Selective B-RafV600E inhibitors vemurafenib (4) [28] and
dabrafenib (5) [29] have been approved for the treatment of advanced
melanoma.
Fig. 2. Design of the Raf/EGFR/VEGFR inhibitors of scaffold 9 from C-Raf
inhibitor 7 through virtual 7a and 8.
anilino group was added to the C-4 position of 7a, the 4-anilinoquinoline
structure was formed in 8. The rest o,o’-dimethylphenyl and methyl-
cabamoyloxy (R’O–) groups in 7a were converted to its bioisosteric
triazolyl group bearing various side chains (R–) which could be readily
constructed by established methods. Virtual 8 was further transformed
by replacing its C-3 carboxyl group with a nitrogen atom to form the 4-
anilinoquinazoline scaffold in 9, similar to the structures of EGFR and
VEGFR inhibitors 1–3 (Fig. 1). As the carboxyl group is essential to the
Raf inhibitory activity of 7 [41], the presence of a phenolic hydroxyl
group at the C-3′ position of 9 could compensate the missing carboxyl.
Further addition of fluoro substituents to the anilino group could make 9
more resemble to structures of drugs 1 (4′-F) and 3 (2′-F) shown in Fig. 1,
thus increasing its EGFR and VEGFR inhibitory activities. The potency of
9 toward Raf kinases might also be enhanced as the electron-
withdrawing fluoro substituents could make the phenolic hydroxyl
more acidic, similar to the carboxyl group in 7. Although the structure of
9 (having a C-6 triazolyl group) is similar to that of drug 2 (having a C-6
furanyl group, Fig. 1), drug 2 as well as drug 1 are reported not active to
inhibit Raf kinases [42].
In addition to deactivating oncogenic B-Raf signaling, Raf inhibition
provides an alternative way to treat the most frequently mutated (~20%
in all cancers) yet elusive Ras protein [30]. C-Raf inhibitor sorafenib (6),
also potently inhibiting VEGFR and PDGFR (platelet-derived growth
factor receptor) families, is thus developed and approved for clinical
uses [31–33]. Signals from receptor tyrosine kinases (e.g., EGFR and
VEGFR) can also be blocked by the downstream Raf inhibition. On the
other hand, resistance from the current B-RafV600E therapy is found
associated with EGFR signaling pathway [34–36] or VEGF-A upregula-
tion [37]. Using EGFR antibody cetuximab with drug 4 shows clinical
benefits in refractory B-RafV600E metastatic colorectal cancers [38].
Combination of B-RafV600E inhibitor PLX4720 with VEGF antibody
bevacizumab shows synergistic effects in vivo [39]. These findings
suggested that a small-molecule Raf inhibitor with EGFR/VEGFR
inhibitory activity could be useful for refractory cancers. A leading study
is a new class of dual B-Raf/EGFR inhibitors reported by Ding and his
colleagues [40]. The optimized compound is active to melanoma and/or
colorectal cancers resistant to 4.
We surmised that a 4-anilinoquinazoline, through the introduction of
proper substituents, could inhibit Raf kinases and retain its inherent
activity toward EGFR and VEGFR families. Aiming at this, 4-(3-
hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines of scaffold 9 was
rationally designed (Fig. 2). Although the structure of 9 does not
resemble to those of the approved Raf inhibitors 4–6 (Fig. 1), it was a
cyclic analog of azastilbene 7, a C-Raf inhibitor reported by McDonald
et al. (Fig. 2) [41]. When the C-6 at the pyridinyl group of 7 was cyclized
with the distal carbon of the alkene moiety (see virtual 7a, Fig. 2) and an
Herein, we reports the synthesis and optimization of scaffold 9 as our
first approach to discover 4-anilinoquinazolines as Raf and EGFR/
VEGFR inhibitors. We first selected proper side chains to promote the
Raf inhibitory activity of 9. Subsequently, we added fluoro substituents
to the anilino group of 9 to further enhance its potency toward Raf in-
hibition. The activity of 9 toward EGFR, VEGFR, and PDGFR was also
evaluated to explore the inhibition profile of 9 and validate our design.
Fig. 1. Structures of FDA-approved EGFR/VEGFR inhibitory 4-anilinoquinazolines 1–3 and Raf inhibitors 4–6.
2

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
2. Results and discussion
2.1. Compound synthesis
Scheme 1 presents the synthesis of 4-anilinoquinazolines 9a–j of
scaffold 9 (Fig. 2) bearing different side chains at the triazolyl group. 6-
Bromo-4-chloroquinazoline (10), prepared by use of the published
method [43], served as the starting material. The chloro group in 10 was
substituted by 3-aminophenol to give 6-bromo-4-(3-hydroxyanilino)qui-
nazoline (11) in 89% yield. Compound 11 was ethynylated by trime-
thylsilylacetylene using Pd(OAc)2/CuI/dppf/Et3N/DMF catalytic
system (Sonogashira reaction) followed by desilylation with TBAF,
giving 6-ethynylquinazoline 12 in 96% yield. Copper(I)-catalyzed
alkyne–azide cycloaddition (CuAAC) was subsequently used to convert
the C-6 ethynyl group in 12 to a triazolyl group. For the preparation of
9a having an unsubstituted triazolyl group, 12 was cyclized with TMSN3
by means of CuI in DMF/MeOH [44]. Compound 9a was obtained in
53% yield. For 9b–j, 12 was reacted with a series of alkyl azides in the
CuSO4/NaAsc/PhCO2H catalytic system. The reaction gave the corre-
sponding 9b–j with a triazolyl group connected with different side
chains in 40–95% yields.
Scheme 2. Synthesis of 9k–n. Reagents and conditions: (i) fluorinated 3-ami-
nophenol, EtOH, reflux, 75–98%. (ii) (a) trimethylsilylacetylene, Pd(OAc)2,
CuI, dppf, Et3N, DMF, 100 ◦C; (b) TBAF, MeOH, 38–65% (two steps). (iii)
NH2COCH2CH2N3, CuSO4, sodium ascorbate, PhCO2H, t-BuOH, H2O, 27–42%.
Scheme 2 illustrates the synthesis of 9k–n bearing a fluorinated 4-
anilino group and 2-carbamoylethyl side chain at the triazolyl moiety.
Compound 10 (Scheme 1) served as the starting material and was
reacted with a mono- or di-fluorinated 3-aminophenol. The reaction
gave the corresponding 13a–d in 75–98% yields. Compounds 13a–d
were ethynylated by trimethylsilylacetylene followed by desilylation to
give the corresponding 14a–d in 38–65% yields. Reaction of 14a–d with
3-azidopropanamide using the CuAAC method gave the target 4-anilino-
quinazolines 9k–n having a fluoro group (9k and 9l) or two fluoro
groups (9m and 9n) with a 2-carbamoylethyl side chain in 27–42%
yields.
RafV600E). Compound 9c, homologous to 9b by an extra methylene,
showed slightly improved potency (IC50: 0.13 μM for B-Raf, 0.24 μM for
B-RafV600E). Replacement of the hydroxyl terminus at the side chain of
9b by a morpholino group generated 9d with decreased potency (IC50
:
0.98
μM for B-Raf, 0.70
μ
M for B-RafV600E). The carboxylmethyl side
chain strongly deactivated 9e toward the two B-Raf kinases (IC50: 2.2,
1.6 μM). However, replacement of the carboxyl terminus in 9e by a
carbamoyl group generated 9f with ~3-fold enhanced potency (IC50
:
0.70
μ
M for B-Raf, 0.53
μ
M for B-RafV600E).
Elongation of the carboxylmethyl side chain in 9e by a methylene
generated 9g with ~8–10-fold enhanced potency (IC50: 0.21 μM for both
B-Raf kinases, Table 1). Similarly, 9h carrying a 2-carbamoylethyl side
chain showed significant increased potency compared to its lower ho-
molog 9f. Compound 9h, respectively inhibiting B-Raf and B-RafV600E
2.2. Potencies of 9a–n of scaffold 9 to inhibit Raf and receptor tyrosine
kinases
with IC50 of 0.086 and 0.12 μM, was the most potent analog among the
Table 1 presents the potencies of 4-(3-hydroxyanilino)-6-(1H-1,2,3-
triazol-4-yl)quinazolines 9a–j bearing different side chains (R) at the
triazolyl group toward Raf inhibition. The potencies of clinically
approved Raf inhibitors 4 and 6 as well as EGFR inhibitor 1 were also
measured for comparison. Compound 9a, having a C-6 unsubstituted
triazolyl group (R = H), inhibited B-Raf and B-RafV600E at sub-
unfluorinated 9a–j. The higher potencies of 9c, 9g, and 9h implied that
the hydroxyl or amino terminus at their side chains would interact with
B-Raf kinases by H-bonds through a three-carbon linker. When the
cabamoyl group in 9h was mono-methylated, the resulting analog 9i
was ~4–6-times less potent (IC50: 0.53, 0.48 μM) than 9h. Compound 9j,
the dimethylated analog of 9h, was further deactivated (IC50: 1.5, 1.3
M). As a result, the hydroxyl or amino terminus at the side chain of 9c,
micromolar concentrations (IC50: 0.94 μM for B-Raf, 0.80 μM for B-
μ
RafV600E). Compound 9b, bearing a 2-hydroxyethyl side chain, dis-
9g, and 9h possibly served as an H-bond donor to interact with B-Raf
and B-RafV600E
.
played increased potency (IC50: 0.23
μM for B-Raf, 0.33 μM for B-
Unlike their good potencies toward B-Raf and B-RafV600E, com-
pounds 9a–j listed in Table 1 were less active to inhibit C-Raf (IC50: 0.52
to >10 μM). Among them, 9j was more potent for C-Raf (IC50: 0.52 μM)
than B-Raf (IC50: 1.5, 1.3
μM). The most potent B-Raf inhibitor 9h
merely inhibited C-Raf with an IC50 of 2.8 μM, which was ~33 and ~23
less potent than inhibiting B-Raf and B-RafV600E, respectively. On the
contrary, clinically used drugs 4 and 6 were very active for all the three
Raf kinases with IC50 of 0.0070–0.040 μM. Drug 4 was slightly more
potent for B-RafV600E (IC50: 0.026
μM), and drug 6 was more potent for
C-Raf (IC50: 0.0070 μM). The potencies for both drugs obtained in-house
were similar to those from literatures [45,46]. As a result, 9h was a more
selective inhibitor for B-Raf and B-RafV600E than 4 and 6.
We then investigated the potencies of compounds 9k–n (Table 2), the
fluorinated analogs of the most potent and selective B-Raf inhibitory 9h
that has a 2-carbamoylethyl side chain. The potencies of 9h and 9k–n for
EGFR, EGFRT790M, VEGFR2 (as a representative for VEGFR family), and
PDGFR-β kinases were also determined to prove our design. For B-Raf
inhibition, compound 9k, the 4′-fluoro analog of 9h, was less potent
Scheme 1. Synthesis of 9a–j. Reagents and conditions: (i) 3-aminophenol,
EtOH, reflux, 89%. (ii) (a) trimethylsilylacetylene, Pd(OAc)2, CuI, dppf, Et3N,
DMF, reflux; (b) TBAF, MeOH, 96% (two steps). (iii) For 9a, TMSN3, CuI, DMF,
MeOH, 53%. (iv) For 9b–j, RN3, CuSO4, sodium ascorbate, PhCO2H, t-BuOH,
H2O, 40–95%.
(IC50: 0.25, 0.19 μM) than 9h (IC50: 0.086, 0.12 μM). Compound 9l,
3

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
Table 1
Inhibitory potencies of 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines 9a–j toward Raf kinases.a
compound
R
kinase inhibitory IC50
B-RafV600E
(μM)
B-Raf
C-Raf
9a
9b
H
0.94 ± 0.05
0.23 ± 0.02
0.80 ± 0.06
0.33 ± 0.04
8.2 ± 1.0
5.8 ± 0.3
9ca
9d
0.13 ± 0.01
0.98 ± 0.14
0.24 ± 0.03
0.70 ± 0.06
5.3 ± 0.1
>10
9e
9f
2.2 ± 0.1
0.70 ± 0.05
0.21 ± 0.03
0.086 ± 0.005
0.53 ± 0.04
1.6 ± 0.2
0.53 ± 0.02
0.21 ± 0.04
0.12 ± 0.01
0.48 ± 0.05
>10
>10
9g
9h
9i
4.4 ± 0.3
2.8 ± 0.3
2.2 ± 0.1
9j
1.5 ± 0.1
1.3 ± 0.2
0.52 ± 0.06
4
6
1
–
–
–
0.038 ± 0.005
0.040 ± 0.003
>10
0.026 ± 0.004
0.029 ± 0.004
>10
0.039 ± 0.006
0.0070 ± 0.0005
>10
a
The inhibitory potency of 9c for EGFR, EGFRT790M, VEGFR2, and PDGFR-β was 3.2 ± 0.4, >10, 0.043 ± 0.005, and > 10
μ
M, respectively.
having a C-6′ fluoro group, showed improved potency (IC50: 0.14, 0.11
and B-RafV600E. For VEGFR2, 9m was more active (IC50: 0.0070
μ
M)
μ
M) compared to 9k that has a C-4′ fluoro group. Nevertheless, both 9k
than drugs 4 (IC50: 0.35 μM), 6 (IC50: 0.017 μM), and 1 (IC50: >10 μ
M).
and 9l were less potent than 9h for B-Raf and B-RafV600E inhibitions. For
the di-fluorinated analogs, 9m with C-4′/C-6′ difluoro groups was more
As a result, we successfully added the Raf inhibitory capability to a
EGFR/VEGFR inhibitory 4-anilinoquinazoline in this study. As few
compounds were reported to potently inhibit Raf/EGFR/VEGFR
[47,48], the lead compounds (e.g., 9h and 9m) from this study could be
further optimized to evaluate its spectrum of anticancer activity. Adding
that 9h and 9m was far more selective for B-Raf and B-RafV600E over C-
Raf than 4 and 6, they might be useful as a biological tool to study the
cross-interactions and paradox of Raf kinases [49], especially in the field
of oncology.
potent than unfluorinated 9h, having IC50 of 0.057 and 0.051 μM for B-
Raf and B-Raf600E, respectively. Compound 9n with C-2′/C-4′ difluoro
groups was less potent (IC50: 0.18 and 0.15 μM) than 9m and parent 9h.
Similar to 9a–i (Table 1), 9k–n were less active (IC50: 1.0–3.7 μM) for C-
Raf inhibition. Compared with drugs 4 and 6, 9m was ~1.5–2-fold less
potent for B-Raf and B-RafV600E but far less potent for C-Raf. Similar to
9h, 9m was more selective for B-Raf and B-RafV600E inhibitions than 4
and 6.
As anticipated, 9h and 9k–n were active for EGFR (IC50: 0.073–0.42
2.3. In vitro anticancer activity of 9m
μ
M) and VEGFR2 (IC50: 0.0070–0.70 μM). Nevertheless, they were
inactive for EGFRT790M and PDGFR-β (IC50: >10
μM). Compound 9m
Nevertheless, 9m that showed the best potency for B-Raf (IC50: 57
nM) and B-RafV600E (IC50: 51 nM) among the compounds listed in
with the best B-Raf inhibitory activity was also the most potent for EGFR
(IC50: 0.073 μM) and VEGFR2 (IC50: 0.0070 μM). Same inhibition profile
Table 1 and 2 was not active (GI50: >10 μM, Table 3) to inhibit mela-
was also observed in 9c having a 3-hydroxylpropyl side chain (Table 1).
However, 9m was ~10-fold less potent compared to its structurally
noma A375 cells that proliferate due to constitutively activated B-
RAFV600E (in the absence of ligands). Compounds 9a–l and 9n that had
inferior potency to inhibit B-Raf and B-RafV600E were also not active to
similar drug 1 (IC50: 0.0070 μM) for EGFR inhibition. The EGFR inhib-
itory profile of 9m was similar to that of 1: they were potent for EGFR
inhibit A375 (GI50: >10 μM). Drug 6, a type II inhibitor preferentially
and relatively not potent for EGFRT790M. On the contrary, 1 was inactive
binding to the inactive conformation of B-Raf and B-RafV600E, was also
for the three Raf kinases (IC50: >10 μM) and 9m was potent for B-Raf
found less active for A375 (GI50: 6.3 μM). In contrast, the type I inhibitor
4

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
Table 2
Inhibitory potencies of 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazolines 9h and 9k–n toward Raf, EGFR, EGFRT790M, VEGFR2, and PDGFR-β kinases.a
compound
Ar
kinase inhibitory IC50
EGFR
(μM)
B-Raf
B-RafV600E
C-Raf
EGFRT790M
VEGFR2
PDGFR-β
>10
9h
9k
0.086 ± 0.005
0.12 ± 0.01
2.8 ± 0.3
0.18 ± 0.04
>10
0.070 ± 0.009
0.25 ± 0.02
0.14 ± 0.02
0.057 ± 0.004
0.18 ± 0.01
0.19 ± 0.02
0.11 ± 0.02
0.051 ± 0.003
0.15 ± 0.02
3.7 ± 0.4
2.3 ± 0.2
1.0 ± 0.1
2.8 ± 0.2
0.18 ± 0.03
0.14 ± 0.02
0.073 ± 0.004
0.42 ± 0.03
>10
>10
>10
>10
0.064 ± 0.005
0.012 ± 0.001
0.0070 ± 0.0005
0.70 ± 0.05
>10
>10
>10
>10
9l
9m
9n
4
6
1
–
–
–
0.038 ± 0.005
0.039 ± 0.003
>10
0.026 ± 0.004
0.032 ± 0.004
>10
0.039 ± 0.006
0.0090 ± 0.0005
>10
>1.0
>10
>1.0
>10
0.35 ± 0.02
0.017 ± 0.002
3.3 ± 0.1
2.3 ± 0.3
0.065 ± 0.008
>10
0.0070 ± 0.0005
0.46 ± 0.03
could further deactivate its cell activity. Another possibility for the
Table 3
inactivity of 9m was that the activity of C-Raf might also contribute the
In vitro anticancer activity of 9m, 9h, 6, 4, and 1.
proliferation of A375. Although 9m was active for B-Raf and B-RafV600E
,
compound
anticancer GI50
(
μ
M)
its low potency for C-Raf (IC50: 1.0 μM) retarded its cell activity. Finally,
9m might not have good cell permeability, thus reducing its cell activity.
A375
PC-9
HCC827
H1975
Drug 1 without Raf inhibitory activity (IC50: >10 μM, Table 2) was
9m
9h
6
>10
>10
6.0 ± 0.2
6.4 ± 0.6
>10
1.3 ± 0.3
3.2 ± 0.2
>10
>10
>10
>10
>10
>10
inactive for A375 proliferation.
6.3 ± 0.4
0.19 ± 0.02
>10
For 1-sensitive/EGFR-mutant NSCLC cells PC-9 and HCC827, 9m
4
>10
>10
only showed moderate potency (GI50: 6.0
HCC827, Table 3). It was far less potent than drug 1 (GI50: 0.053, 0.043
M) to inhibit these two cells. This would result from ~10 times inferior
enzymatic EGFR inhibitory activity of 9m (IC50: 0.073 M) relative to
that of 1 (IC50: 0.0070 M). Also, the cell permeability would affect the
μM for PC-9, 1.3 μM for
1
0.053 ± 0.006
0.043 ± 0.002
μ
4 which binds to the active conformation of B-RafV600E, inhibited A375
μ
μ
potently (IC50: 0.19 μM). As a result, 9m might act on the inactive
conformations of B-Raf and B-RafV600E as 6 does. The lower enzymatic
activity of 9m than 6 for B-Raf and B-RafV600E (~1.5-fold less potent)
activity of 9m in cell. Both 9m and 1 were inactive to NSCLC H1975 cells
that harbor EGFRL858R/T790M mutation (GI50: >10
μM). This should
Fig. 3. Docking simulations of 9m in the kinase domains of B-Raf, B-RafV600E, and VEGFR2. (a) Compound 9m in B-Raf (PDB entry 1UWH). (b) Compound 9m in B-
RafV600E (PDB entry 1UWJ). (c) Overlapping of the binding poses of 9m and drug 3 in VEGFR2 (4AGD). Compounds 9m and 3 are shown in yellow and light blue
sticks, respectively; H-bonds are shown as dashed green line; and the nitrogen, oxygen, and fluorine atoms are colored blue, red, and light blue, respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
triazolyl group could thus be replaced with other substituents. In our
other study [43], we had synthesized and evaluated a series of C-6
phenyl analogs of 9 with more diverse substituents at the 4-anilino
group. Results from that study suggested that the C-3′ hydroxyl group
was essential for the Raf inhibitory potency of 9 as the C-3′ methoxy
analog showed >10-fold decreased potency. Fluoro substituent at the 4-
anilino group also improved the potency while chloro substituent did
not. On the contrary, electron-donating methyl or methoxy group
reduced the Raf inhibitory potency significantly. Some of C-6 phenyl
analogs of 9 in that study [43] was more active than 9m to inhibit Raf
Table 4
Calculated binding energy of 9m in B-Raf and B-RafV600E with inactive or active
conformation.
PDB entry
B-Raf
conformation
calculate binding energy
(Kcal/mol)
1UWH
4KSP
wt
wt
inactive
inactive
inactive
inactive
active
–10.35
–10.30
–9.79
–9.88
–8.68
–7.24
–8.53
–8.25
1UWJ
4G9R
3C4C
4MNE
3OG7
4XV2
V600E
V600E
wt
wt
active
and EGFR kinases and thus inhibited the proliferation of A375 (GI50
<1.0 M) and PC-9 (GI50: <0.2 M) more potently.
:
V600E
V600E
active
active
μ
μ
The interaction of 9m with VEGFR2 (PDB entry 4ADG) [52] was also
explored by docking simulation as 9m actively inhibited VEGFR2 at low
nanomolar concentrations (IC50: 7.0 nM, Table 2) and was more potent
than drug 6 (IC50: 17 nM). The binding pose of 9m in VEGFR2 is over-
lapped with that of 4-anilinoquinazoline drug 3 and shown in Fig. 3c.
The 4-anilinoquinazoline scaffolds of 9m and 3 aligned well in the ki-
nase domain of VEGFR2, both occupying the ATP-binding cleft by their
quinazolinyl moiety and forming an H-bond with the hinge residue
Cys919. The anilino groups of 9m and 3 buried in the back pocket of
result from the low potency of 9m (IC50: >10 μM) and 1 (IC50: 0.46 μM)
for EGFRT790M. Similar anticancer profile was also observed in
unfluorinated 9h to inhibit PC-9 (GI50: 6.4 μM), HCC827 (GI50: 3.2 μM),
and H1975 (GI50: >10 μM). Compound 9c (Table 1), less active for EGFR
(IC50: 3.2
μ
M) and EGFRT790M (IC50: >10
μ
M), was not active to inhibit
PC-9, HCC827, and H1975 (GI50: >10
μM). Similarly, Raf inhibitors 4
and 6 did not inhibit the proliferation of the three cancer cells (GI50
:
>10
μ
M) as they were inactive for EGFR and EGFRT790M (IC50: >1.0
μM,
VEGFR2 and contacted with Lys868 through a π-cation interaction. The
Table 2). Cumulatively, compound 9m showed its anticancer activity in
phenolic hydroxyl of 9m formed an additional H-bond with Asp1046
than 3 in the back pocket. On the other hand, the C-6 side chain of 9m
and the C-7 side chain of drug 3 headed to the different regions in
VEGFR2. The carbamoyl terminus of 9m formed two H-bonds with
Phe921 and Asn923, and the triazolyl group formed one H-bond with
Asn923. These H-bond interactions could account for the high potency
of 9m for VEGFR2 inhibition.
PC-9 and HCC827 cells through EGFR inhibition.
2.4. Interaction of 9m with B-Raf, B-RafV600E, and VEGFR2
To explore the interaction of scaffold 9 with B-Raf kinases, we
docked the most potent 9m into the kinase domains of B-Raf (PDB entry
1UWH) [50] and B-RafV600E (PDB entry 1UWJ) [50] both having the
inactive DFG-out conformations using Autodock [51] software. In B-Raf
(Fig. 3a), 9m was found to occupy the ATP-binding pocket by forming an
H-bond with the hinge residue Cys532 through its quinazoline N-1 atom
3. Conclusions
We have developed 4-(3-hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)
quinazolines of scaffold 9 as selective B-Raf/B-RafV600E and potent
EGFR/VEGFR2 kinase inhibitors. For Raf kinases, the most potent and
selective 9m inhibited B-Raf/B-RafV600E at low nanomolar concentra-
tions (IC50: 57 and 50 nM) and was less potent toward C-Raf (IC50: 1.0
and stacked with Trp531 through its bicyclic
π system. The phenolic
hydroxyl group in 9m served as an H-bond donor to interact with
Phe595 in the DFG motif. The carbamoyl terminus at the side chain of
9m formed two H-bonds with Asp594 in the DFG motif and one H-bond
with Glu501. In B-RafV600E (Fig. 3b), 9m also formed an H-bond with
Cys532 and stacked with Trp531 in the ATP-binding pocket. An intra-
molecular H-bond formed between the phenolic hydroxyl and the
carbonyl of the carbamoyl of 9m, which assisted the NH2 terminus
donating two H-bonds to Asp594 and Gly596 in the DGF motif. The
multiple H-bond interaction of 9m with B-Raf and B-RafV600E should be
important for its enzymatic activity as the secondary amide 9i and ter-
tiary amide 9j were less active than primary amide 9h (Table 1).
Compound 9m was also docked into a series of B-Raf and B-RafV600E
crystal structures with inactive DFG-out or active DFG-in conformation
by Autodock. The calculated binding energy of the most stable pose of
9m in these crystals was shown in Table 4. Compound 9m was found to
have stronger interactions with DFG-out B-Raf (1UWH and 4KSP) and B-
RafV600E (1UWJ and 4G9R) with binding energy of –9.79 to –10.35 Kcal/
mol. The interaction of 9m with DFG-in B-Raf (3C4C and 4MNE) and B-
RafV600E (3OG7 and 4XV2) was weaker (–7.24 to –8.68 Kcal/mol). The
difference in binding energy (~1–2 Kcal/mol) supported the preference
of 9m to interact with B-Raf and B-RafV600E having inactive DFG-out
conformations. Furthermore, the interaction of 9n with B-Raf and B-
RafV600E was also explored by docking simulations to account for its
inferior potency than that of its isomeric 9m (Supplementary Fig. S1).
The triazolyl group at the C-6 position of 9 was originally designed to
imitate the phenyl group of azastilbene 7 as it can be readily constructed
by the well-established Sonogashira and CuACC reactions (Fig. 2).
However, results from docking simulations revealed that the triazolyl
group only interacted with the alkyl groups of the proximal residues in
μ
M). For receptor tyrosine kinases, 9m was potent for EGFR (IC50: 73
nM) and VEGFR2 (IC50: 7.0 nM) but inactive (IC50: >10
μM) for
EGFRT790M and PDGFR-β. Compound 9m inhibited cell proliferation
attributed to EGFR inhibition. Docking simulations were performed to
analyze the binding poses of 9m in B-Raf, B-RafV600E, and VEGFR2.
4. Experimental section
4.1. Compound synthesis
Merck Reagents Silica Gel 60 (particle size of 0.063–0.200 mm,
70–230 mesh ASTM) was used for column chromatography. Melting
points were measured on STUARTTM SMP3 melting point apparatus. 1H
(400 MHz) and 13C (100 MHz) NMR spectra were recorded on Agilent
400-MR spectrometer using DMSO‑d6 as the solvent. High-resolution
mass spectra were measured on LTQ Orbitrap XL mass spectrometer
(Thermo Fisher Scientific). The purities of the compounds for biological
evaluations (>95%) were determined from an Agilent 1100 series HPLC
equipped with Agilent ZORBAX SB-C18 column (5 μm, 4.6 mm × 150
mm) and a UV detector (254 nM) using gradient of 35–100% CH3CN in
H2O for 20 min and 100% CH3CN for 10 min.
4.1.1. Standard procedure for the synthesis of 4-(3-hydroxyanilino)-6-
bromoquinazolines 11 and 13a–d
6-Bromo-4-chloroquinazoline (10, ~5.0 g, 1.0 equiv) and the cor-
responding 3-aminophenol (1.5 equiv) were mixed in EtOH (25 mL).
The reaction mixture was heated under reflux for 12 h. The solution was
cooled to room temperature and the resulting precipitate was collected
by filtration. The solids were washed with EtOH (20 mL) and air-dried to
B-Raf and B-RafV600E through its
π system (π–alkyl interactions). The
three nitrogen atoms of the triazolyl group did not form energy-favored
bonds (e.g., H-bond) with B-Raf and B-RafV600E (Fig. 3a and 3b). The
6

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
give the targets 11 and 13a–d as solids in 75–98% yields.
14a–d as solids in 38–96% yields.
4.1.1.1. 3-[(6-Bromoquinazolin-4-yl)amino]phenol (11). Yield: 89%;
mp 233.1–234.8 ◦C; 1H NMR: δ 11.10 (brs, 1H), 9.72 (brs, 1H), 9.07 (s,
1H), 8.88 (s, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H),
7.28–7.12 (m, 3H), 6.71 (d, J = 7.7 Hz, 1H); 13C NMR: δ 157.8, 157.5,
152.9, 138.5, 137.5, 129.3, 126.4, 125.7, 119.9, 115.7, 114.2, 112.6,
110.6, 109.5; HRMS calcd for [C14H10BrN3O + H]+ 316.0080, found
316.0101.
4.1.2.1. 3-[(6-Ethynylquinazolin-4-yl)amino]phenol (12). Yield: 96%;
mp 148.9–150.5 ◦C; 1H NMR: δ 9.75 (s, 1H), 9.44 (s, 1H), 8.80 (d, J =
1.4 Hz, 1H), 8.60 (s, 1H), 7.85 (dd, J = 8.6, 1.4 Hz, 1H), 7.74 (d, J = 8.6
Hz, 1H), 7.41 (t, J = 2.0 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.15 (t, J =
7.8 Hz, 1H), 6.54 (dd, J = 7.8, 2.0 Hz, 1H), 4.39 (s, 1H); 13C NMR: δ
157.9, 157.6, 155.8, 149.9, 140.4, 135.7, 129.5, 128.7, 127.4, 119.7,
115.5, 113.5, 111.5, 109.9, 83.5, 82.3; HRMS calcd for [C16H11N3O +
H]+ 262.0975, found 262.1001.
4.1.1.2. 5-[(6-Bromoquinazolin-4-yl)amino]-2-fluorophenol
(13a).
Yield: 83%; mp 279.6–280.9 ◦C; 1H NMR: δ 11.47 (s, 1H), 10.28 (s, 1H),
9.15 (s, 1H), 8.91 (s, 1H), 8.22 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 8.9 Hz,
1H), 7.38 (dd, J = 8.0, 2.5 Hz, 1H), 7.24 (dd, J = 11.0, 8.8 Hz, 1H),
7.15–7.10 (m, 1H); 13C NMR: δ 159.4, 158.7, 151.0, 149.5 (d, J = 241.6
Hz), 144.9 (d, J = 13.0 Hz), 138.8, 137.6, 132.6 (d, J = 2.8 Hz), 127.4,
121.0, 115.9 (d, J = 5.1 Hz), 115.7 (d, J = 7.6 Hz), 114.9, 114.5 (d, J =
2.7 Hz); HRMS calcd for [C14H9BrFN3O + H]+ 333.9986, found
334.0013.
4.1.2.2. 5-[(6-Ethynylquinazolin-4-yl)amino]-2-fluorophenol
(14a).
Yield: 45%; mp 296.1–298.2 ◦C; 1H NMR: δ 9.95 (s, 1H), 9.79 (s, 1H),
8.76 (s, 1H), 8.57 (s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.5 Hz,
1H), 7.57 (dd, J = 8.2, 2.4 Hz, 1H), 7.24–7.19 (m, 1H), 7.13 (dd, J =
11.0, 8.9 Hz, 1H), 4.41 (s, 1H); 13C NMR: δ 157.6, 155.8, 149.9, 148.2
(d, J = 238.0 Hz), 144.9 (d, J = 12.9 Hz), 135.7, 135.7 (d, J = 2.7 Hz),
128.7, 127.4, 119.7, 115.9 (d, J = 19.0 Hz), 115.4, 113.8 (d, J = 6.3 Hz),
112.7 (d, J = 2.4 Hz), 83.5, 82.3; HRMS calcd for [C16H10FN3O + H]+
280.0881, found 280.0854.
4.1.1.3. 3-[(6-Bromoquinazolin-4-yl)amino]-4-fluorophenol
(13b).
Yield: 98%; mp 282.9–284.2 ◦C; 1H NMR: δ 11.64 (s, 1H), 9.81 (s, 1H),
9.11 (s, 1H), 8.90 (s, 1H), 8.24 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 9.0 Hz,
4.1.2.3. 3-[(6-Ethynylquinazolin-4-yl)amino]-4-fluorophenol
(14b).
Yield: 38%; mp >350 ◦C (decomposed); 1H NMR: δ 9.84 (s, 1H), 9.52 (s,
1H), 8.67 (s, 1H), 8.49 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.6
Hz, 1H), 7.14–7.04 (m, 1H), 6.93–6.89 (m, 1H), 6.69–6.63 (m, 1H), 4.39
(s, 1H); 13C NMR: δ 158.1, 155.6, 153.4 (d, J = 1.7 Hz), 150.2 (d, J =
237.6 Hz), 149.4, 135.4, 128.2, 127.1, 126.0 (d, J = 13.9 Hz), 119.3,
116.1 (d, J = 21.5 Hz), 114.7, 114.2, 113.4 (d, J = 7.3 Hz), 82.9, 81.9;
HRMS calcd for [C16H10FN3O – H]– 278.0735, found 278.0717.
1H), 7.18 (t, J = 9.4 Hz, 1H), 6.94–6.89 (m, 1H), 6.83–6.77 (m, 1H); 13
C
NMR: δ 160.2, 154.2 (d, J = 1.6 Hz), 151.7, 150.4 (d, J = 239.4 Hz),
139.5, 138.4, 127.9, 124.3 (d, J = 14.2 Hz), 122.5, 121.6, 116.9 (d, J =
21.2 Hz), 116.0 (d, J = 7.1 Hz), 115.0, 114.8; HRMS calcd for
[C14H9BrFN3O + H]+ 333.9986, found 334.0011.
4.1.1.4. 5-[(6-Bromoquinazolin-4-yl)amino]-2,4-difluorophenol (13c).
Yield: 79%; mp 225.1–226.9 ◦C; 1H NMR: δ 11.60 (brs, 1H), 10.29 (brs,
1H), 9.08 (s, 1H), 8.90 (s, 1H), 8.24 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 8.9
Hz, 1H), 7.42 (t, J = 9.4 Hz, 1H), 7.13 (t, J = 8.3 Hz, 1H); 13C NMR: δ
160.3, 151.8, 150.0 (dd, J = 245.1, 10.9 Hz), 149.3 (dd, J = 242.4, 10.7
Hz), 142.0 (dd, J = 12.7, 2.6 Hz), 139.5, 138.5, 127.9, 122.6, 121.6,
119.9 (dd, J = 13.9, 3.6 Hz), 116.6 (d, J = 2.8 Hz), 115.0, 105.6 (t, J =
24.4 Hz); HRMS calcd for [C14H8BrF2N3O + H]+ 351.9892, found
351.9881.
4.1.2.4. 5-[(6-Ethynylquinazolin-4-yl)amino]-2,4-difluorophenol (14c).
Yield: 38%; mp 286.2–287.6 ◦C; 1H NMR: δ 9.98 (s, 1H), 9.84 (s, 1H),
8.65 (s, 1H), 8.49 (s, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 8.5 Hz,
1H), 7.32 (t, J = 10.5 Hz, 1H), 7.08 (t, J = 8.3 Hz, 1H), 4.41 (s, 1H); 13C
NMR: δ 158.2, 155.6, 149.4, 149.1 (dd, J = 241.2, 10.8 Hz), 148.4 (dd, J
= 242.9, 11.0 Hz), 141.0 (dd, J = 12.7, 2.7 Hz), 135.5, 128.2, 127.1,
121.5 (dd, J = 13.6, 3.4 Hz), 119.4, 116.2, 114.6, 104.8 (t, J = 24.4 Hz),
82.9, 81.9; HRMS calcd for [C16H9F2N3O – H]– 296.0641, found
296.0673.
4.1.1.5. 3-[(6-Bromoquinazolin-4-yl)amino]-2,6-difluorophenol (13d).
Yield: 75%; mp 299.0–300.8 ◦C; 1H NMR: δ 12.00 (s, 1H), 10.52 (s, 1H),
9.25 (s, 1H), 8.91 (s, 1H), 8.24 (d, J = 8.9 Hz, 1H), 7.97 (d, J = 8.9 Hz,
1H), 7.14 (t, J = 9.5 Hz, 1H), 6.98–6.90 (m, 1H); 13C NMR: δ 160.3,
152.1 (dd, J = 242.3, 5.3 Hz), 151.9, 148.2 (dd, J = 246.5, 6.7 Hz),
139.5, 138.8, 135.0 (dd, J = 16.8, 14.7 Hz), 127.9, 122.8, 121.5, 121.4
(d, J = 3.1 Hz), 117.4 (d, J = 8.5 Hz), 115.1, 111.6 (dd, J = 19.8, 3.1 Hz);
HRMS calcd for [C14H8BrF2N3O + H]+ 351.9892, found 351.9912.
4.1.2.5. 3-[(6-Ethynylquinazolin-4-yl)amino]-2,6-difluorophenol (14d).
Yield: 65%; mp 274.8–276.0 ◦C; 1H NMR: δ 10.28 (s, 1H), 9.89 (s, 1H),
8.67 (s, 1H), 8.48 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.6 Hz,
1H), 7.10–7.03 (m, 1H), 6.95–6.88 (m, 1H), 4.38 (s, 1H); 13C NMR: δ
158.8, 156.1, 151.1 (dd, J = 240.3, 5.4 Hz), 149.8, 148.4 (dd, J = 244.6,
6.6 Hz), 135.9, 134.7 (dd, J = 16.5, 15.4 Hz), 128.7, 127.5, 123.3 (dd, J
= 10.9, 3.1 Hz), 119.8, 117.4 (d, J = 8.2 Hz), 115.0, 111.1 (dd, J = 19.4,
3.3 Hz), 83.4, 82.4; HRMS calcd for [C16H9F2N3O – H]– 296.0641, found
296.0675.
4.1.2. Standard procedure for the synthesis of 6-ethynyl-4-(3-hydroxyani-
lino)quinazolines 12 and 14a–d
Compound 11 or 13a–d (~10.0 mmol, 1.0 equiv) was mixed with
CuI (0.10 equiv), Pd(OAc)2 (0.010 equiv), and 1,1′-bis(diphenylphos-
phino)ferrocene (dppf, 0.060 equiv) in anhydrous DMF (50 mL). The
solution was added with Et3N (10 mL) and trimethylsilylacetylene (10
equiv). After purged with nitrogen to remove oxygen, the reaction
mixture was heated under reflux for 12 h. The solution was cooled to
room temperature, added with EtOAc, and stirred for 30 min. The so-
lution was passed through a short column packed with silica gel, and the
filtrate was washed with water and aqueous NH4Cl. The organic layer
was collected, dried over anhydrous MgSO4, and concentrated under
reduced pressure. The residue was re-dissolved in MeOH, added with
TBAF (3.0 equiv), and stirred at room temperature for 60 min under N2.
The solution was concentrated under reduced pressure to remove most
solvent. The residue was added with water and stirred for 10 min. The
resulting solids were collected by filtration to give the targets 12 or
4.1.3. 4-(3-Hydroxyanilino)-6-(1H-1,2,3-triazol-4-yl)quinazoline (9a)
A solution of 12 (149.7 mg, 0.5729 mmol, 1.0 equiv), trimethylsilyl
azide (131.4 mg, 1.141 mmol, 2.0 equiv), and CuI (8.6 mg, 0.045 mmol,
0.079 equiv) in anhydrous DMF (0.90 mL) and MeOH (0.10 mL) was
purged with nitrogen to remove oxygen. The reaction mixture was
heated at 100 ◦C for 12 h. The solution was cooled to room temperature
and diluted with water. The resulting solids were collected by filtration
and re-dissolved in MeOH (5.0 mL). The solution was filtered, and the
filtrate was concentrated under reduced pressure. The residue was pu-
rified by column chromatography to give 9a (93.2 mg, 0.306 mmol) as
yellow solids in 53% yield: mp 266.4–268.8 ◦C; 1H NMR: δ 9.80 (s, 1H),
9.45 (brs, 1H), 9.00 (s, 1H), 8.56 (s, 1H), 8.43 (s, 1H), 8.30 (d, J = 8.6
Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.39 (s, 1H), 7.24 (d, J = 8.0 Hz, 1H),
7.15 (t, J = 8.0 Hz, 1H), 6.54 (d, J = 8.0 Hz, 1H); 13C NMR: δ 158.2,
157.9, 155.0, 149.9, 146.3, 140.5, 130.9, 129.5, 128.9, 128.8, 120.0,
7

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
115.9, 113.7, 111.5, 110.1, 109.8; HRMS calcd for [C16H12N6O + H]+
4.1.4.6. 3-(4-{4-[(3-Hydroxyphenyl)amino]quinazolin-6-yl}-1H-1,2,3-
triazol-1-yl)propanoic acid (9g). Yield: 50%; mp 266.7–268.1 ◦C; 1H
NMR: δ 12.50 (s, 1H), 9.83 (s, 1H), 9.42 (s, 1H), 9.02 (s, 1H), 8.61 (s,
1H), 8.28 (s, 1H), 7.84 (brs, 1H), 7.39 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H),
7.16 (t, J = 7.8 Hz, 1H), 6.54 (d, J = 7.8 Hz, 1H), 4.66 (brs, 2H), 2.98
(brs, 2H); 13C NMR: δ 158.0, 157.9, 146.1, 140.5, 140.2, 130.6, 129.5,
129.2, 122.7, 119.6, 118.2, 113.7, 113.5, 111.7, 111.5, 110.1, 109.9,
46.3, 29.5; HRMS calcd for [C19H16N6O3 + H]+ 377.1357, found
377.1377.
305.1145, found 305.1132.
4.1.4. Standard procedure for the synthesis of 4-(3-hydroxyanilino)-6-
(1H-1,2,3-triazol-4-yl)quinazolines 9b–n
Compound 12 or 14a–d (~0.30 mmol, 1.0 equiv) and the corre-
sponding alkyl azide (1.5 equiv) were added to a solution of CuSO4⋅H2O
(0.020 equiv), sodium ascorbate (0.010 equiv), and benzoic acid (0.20
equiv) in t-BuOH (2.0 mL) and water (1.0 mL). The reaction mixture was
stirred at room temperature for 12 h. The solution was concentrated
under reduced pressure to remove t-BuOH and then added with water
(5.0 mL). The resulting precipitate was collected by filtration and pu-
rified by column chromatography to provide the targets 9b–n in
27–95% yields.
4.1.4.7. 3-(4-{4-[(3-Hydroxyphenyl)amino]quinazolin-6-yl}-1H-1,2,3-
triazol-1-yl)propanamide (9h). Yield: 95%; mp 208.8–210.2 ◦C; 1H
NMR: δ 9.82 (s, 1H), 9.42 (s, 1H), 8.98 (s, 1H), 8.55 (s, 1H), 8.54 (s, 1H),
8.27 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.45 (s, 1H), 7.38 (t, J
= 2.1 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.95 (s,
1H), 6.53 (dd, J = 7.6, 2.1 Hz, 1H), 4.63 (t, J = 6.6 Hz, 2H), 2.76 (t, J =
6.6 Hz, 2H); 13C NMR: δ 171.5, 158.1, 157.8, 154.8, 146.0, 141.7, 140.5,
130.6, 129.6, 129.5, 129.2, 129.1, 122.8, 119.5, 113.7, 111.5, 110.1,
46.5, 35.6; HRMS calcd for [C19H17N7O2 + H]+ 376.1516, found
376.1537.
4.1.4.1. 3-({6-[1-(2-Hydroxyethyl)-1H-1,2,3-triazol-4-yl]quinazolin-4-
yl}amino)phenol (9b). Yield: 54%; mp 285.2–287.0 ◦C; 1H NMR: δ 9.86
(s, 1H), 9.44 (s, 1H), 9.05 (s, 1H), 8.64 (s, 1H), 8.56 (s, 1H), 8.32 (d, J =
8.7 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.26 (d, J = 8.0
Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 5.17 (t, J =
5.3 Hz, 1H), 4.50 (t, J = 5.2 Hz, 2H), 3.87–3.78 (m, 2H); 13C NMR: δ
157.7, 157.3, 154.3, 149.2, 145.6, 140.0, 130.1, 128.9, 128.8, 128.4,
122.5, 118.9, 115.5, 113.2, 110.9, 109.6, 59.8, 52.5; HRMS calcd for
[C18H16N6O2 + H]+ 349.1408, found 349.1406.
4.1.4.8. 3-(4-{4-[(3-Hydroxyphenyl)amino]quinazolin-6-yl}-1H-1,2,3-
triazol-1-yl)-N-methylpropanamide (9i). Yield: 55%; mp 121.5–124.0 ◦C;
1H NMR: δ 9.84 (s, 1H), 9.44 (s, 1H), 9.00 (s, 1H), 8.60 (brs, 1H), 8.54 (s,
1H), 8.27 (d, J = 6.1 Hz, 1H), 7.94 (d, J = 6.1 Hz, 1H), 7.80 (brs, 1H),
7.38 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.54 (d, J
= 8.0 Hz, 1H), 4.65 (t, J = 6.6 Hz, 2H), 2.76 (t, J = 6.6 Hz, 2H), 2.54 (d,
J = 4.6 Hz, 3H); 13C NMR: δ 169.9, 158.1, 157.8, 146.0, 140.5, 130.6,
129.5, 129.2, 125.8, 122.8, 119.5, 116.2, 113.8, 113.6, 111.5, 110.2,
110.0, 46.6, 35.8, 26.0; HRMS calcd for [C20H19N7O2 + H]+ 390.1673,
found 390.1650.
4.1.4.2. 3-({6-[1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl]quinazolin-4-
yl}amino)phenol (9c). Yield: 40%; mp 235.0–236.7 ◦C; 1H NMR: δ 9.83
(s, 1H), 9.45 (s, 1H), 9.01 (s, 1H), 8.62 (s, 1H), 8.55 (s, 1H), 8.27 (d, J =
8.6 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.39 (s, 1H), 7.24 (d, J = 7.8
Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.53 (d, J = 7.8 Hz, 1H), 4.73 (s, 1H),
4.50 (t, J = 6.8 Hz, 2H), 3.43 (t, J = 6.8 Hz, 2H), 2.02 (p, J = 6.8 Hz, 2H);
13C NMR: δ 157.7, 157.3, 154.4, 149.2, 145.7, 140.0, 130.2, 129.0,
128.7, 128.4, 122.0, 118.9, 115.4, 113.2, 110.9, 109.5, 57.3, 46.8, 32.8;
HRMS calcd for [C19H18N6O2 + H]+ 363.1564, found 363.1555.
4.1.4.9. 3-(4-{4-[(3-Hydroxyphenyl)amino]quinazolin-6-yl}-1H-1,2,3-
triazol-1-yl)-N,N-dimethylpropanamide
(9j). Yield:
45%;
mp
1
◦
241.9–243.1 C; H NMR: δ 9.85 (s, 1H), 9.45 (brs, 1H), 9.06 (s, 1H),
8.61 (s, 1H), 8.55 (s, 1H), 8.28 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 8.7 Hz,
1H), 7.38 (s, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.15 (t, J = 8.4 Hz, 1H), 6.53
(d, J = 8.4 Hz, 1H), 4.64 (t, J = 6.6 Hz, 2H), 3.03 (t, J = 6.6 Hz, 2H), 2.94
(s, 3H), 2.81 (s, 3H); 13C NMR: δ 169.6, 158.2, 157.8, 154.9, 149.7,
146.0, 140.5, 130.6, 129.5, 129.2, 128.9, 123.0, 119.4, 116.0, 113.7,
111.4, 110.1, 46.4, 36.9, 35.3, 33.3; HRMS calcd for [C21H21N7O2 + H]+
404.1829, found 404.1844.
4.1.4.3. 3-({6-[1-(2-Morpholinoethyl)-1H-1,2,3-triazol-4-yl]quinazolin-
4-yl}amino)phenol (9d). Yield: 74%; mp 190.8–193.2 ◦C; 1H NMR: δ
9.86 (s, 1H), 9.44 (s, 1H), 9.02 (s, 1H), 8.62 (s, 1H), 8.56 (s, 1H), 8.26
(d, J = 8.6 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.39 (s, 1H), 7.24 (d, J =
8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 4.58 (t, J
= 6.3 Hz, 2H), 3.54 (t, J = 4.0 Hz, 4H), 2.81 (t, J = 6.3 Hz, 2H), 2.44 (t, J
= 4.0 Hz, 4H); 13C NMR: δ 158.2, 157.8, 146.1, 140.5, 131.8, 130.8,
129.5, 129.4, 129.2, 122.7, 119.4, 113.7, 113.5, 111.4, 110.0, 109.8,
66.6, 57.9, 53.4, 47.3; HRMS calcd for [C22H23N7O2 + H]+ 418.1986,
found 418.1972.
4.1.4.10. 3-(4-{4-[(4-Fluoro-3-hydroxyphenyl)amino]quinazolin-6-yl}-
1H-1,2,3-triazol-1-yl)propanamide
(9k). Yield:
30%;
mp
274.8–276.0 ◦C; 1H NMR: δ 10.03 (s, 1H), 9.99 (s, 1H), 9.16 (s, 1H), 8.75
(s, 1H), 8.54 (s, 1H), 8.31 (d, J = 8.7 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H),
7.64 (dd, J = 8.2, 2.4 Hz, 1H), 7.58 (s, 1H), 7.32–7.23 (m, 1H), 7.12 (dd,
J = 11.0, 8.9 Hz, 1H), 6.98 (s, 1H), 4.65 (t, J = 6.6 Hz, 2H), 2.80 (t, J =
6.6 Hz, 2H); 13C NMR: δ 171.1, 157.8, 154.4, 149.2, 147.7 (d, J = 237.8
Hz), 145.7, 144.4 (d, J = 12.8 Hz), 135.4 (d, J = 2.4 Hz), 130.0, 128.7,
128.4, 122.5, 119.4, 115.5 (d, J = 1.4 Hz), 115.3, 113.7 (d, J = 6.2 Hz),
112.6 (d, J = 2.1 Hz), 46.1, 35.2; HRMS calcd for [C19H16FN7O2 + H]+
394.1422, found 394.1410.
4.1.4.4. 2-(4-{4-[(3-Hydroxyphenyl)amino]quinazolin-6-yl}-1H-1,2,3-
triazol-1-yl)acetic acid (9e). Yield: 67%; mp 244.3–246.8 ◦C; 1H NMR: δ
9.88 (s, 1H), 9.42 (s, 1H), 9.06 (s, 1H), 8.61 (s, 1H), 8.31 (d, J = 7.2 Hz,
1H), 7.96–7.75 (m, 2H), 7.39 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.16 (t, J
= 7.8 Hz, 1H), 6.54 (d, J = 7.8 Hz, 1H), 5.39 (s, 2H); 13C NMR: δ 158.1,
157.9, 154.6, 146.2, 140.4, 133.3, 130.7, 129.5, 129.2, 129.0, 123.9,
119.8, 113.8, 113.6, 111.6, 110.2, 110.0, 49.4; HRMS calcd for
[C19H17N7O2 + H]+ 376.1516, found 376.1537.
4.1.4.5. 2-(4-{4-[(3-Hydroxyphenyl)amino]quinazolin-6-yl}-1H-1,2,3-
triazol-1-yl)acetamide (9f). Yield: 54%; mp 255.9–258.0 ◦C; 1H NMR: δ
9.90 (s, 1H), 9.42 (s, 1H), 8.58 (s, 1H), 8.44 (d, J = 6.8 Hz, 1H), 7.81 (d,
J = 6.8 Hz, 1H), 7.45 (s, 1H), 7.39 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.14
(t, J = 7.8 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 5.18 (s, 2H); 13C NMR: δ
167.7, 160.2, 158.0, 157.3, 145.7, 139.9, 133.3, 130.4, 130.0, 129.7,
129.1, 124.3, 120.6, 114.2, 112.1, 112.0, 110.7, 52.2; HRMS calcd for
[C18H14N6O3 + H]+ 363.1200, found 363.1190.
4.1.4.11. 3-(4-{4-[(2-Fluoro-5-hydroxyphenyl)amino]quinazolin-6-yl}-
1H-1,2,3-triazol-1-yl)propanamide (9l). Yield: 27%; mp 285.5–287.0 ◦C;
1H NMR: δ 9.88 (s, 1H), 9.49 (s, 1H), 8.95 (s, 1H), 8.54 (s, 1H), 8.48 (s,
1H), 8.31 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.48 (s, 1H), 7.10
(t, J = 9.5 Hz, 1H), 7.00–6.94 (m, 2H), 6.69–6.63 (m, 1H), 4.66 (t, J =
6.5 Hz, 2H), 2.78 (t, J = 6.5 Hz, 2H); 13C NMR: δ 171.0, 158.6, 154.7,
153.4 (d, J = 1.6 Hz), 150.2 (d, J = 237.2 Hz), 149.2, 145.5, 130.3,
128.7, 128.4, 126.4 (d, J = 13.8 Hz), 122.3, 119.0, 116.0 (d, J = 21.6
Hz), 115.1, 114.3, 113.2 (d, J = 7.3 Hz), 46.1, 35.2; HRMS calcd for
[C19H16FN7O2 + H]+ 394.1422, found 394.1403.
8

C.-I. Lee et al.
Bioorganic Chemistry 109 (2021) 104715
4.1.4.12. 3-(4-{4-[(2,4-Difluoro-5-hydroxyphenyl)amino]quinazolin-6-
4.4. Computational method
yl}-1H-1,2,3-triazol-1-yl)propanamide
(9m). Yield:
42%;
mp
298.7–300.3 ◦C; 1H NMR: δ 10.05 (brs, 1H), 9.99 (s, 1H), 9.02 (s, 1H),
8.60 (s, 1H), 8.46 (s, 1H), 8.31 (d, J = 8.7 Hz, 1H), 7.83 (d, J = 8.7 Hz,
1H), 7.52 (s, 1H), 7.29 (t, J = 10.6 Hz, 1H), 7.15 (t, J = 8.3 Hz, 1H), 6.97
(s, 1H), 4.65 (t, J = 6.6 Hz, 2H), 2.78 (t, J = 6.6 Hz, 2H); 13C NMR: δ
171.0, 158.7, 154.7, 149.2, 149.1 (dd, J = 240.5, 10.4 Hz), 148.3 (dd, J
= 242.5, 11.1 Hz), 145.5, 141.1 (dd, J = 12.6, 2.7 Hz), 130.2, 128.7,
128.4, 122.3, 121.8 (dd, J = 13.6, 3.6 Hz), 119.2, 116.3, 115.0, 104.7 (t,
J = 24.3 Hz), 46.1, 35.2; HRMS calcd for [C19H15F2N7O2 + H]+
412.1328, found 412.1350.
The crystal structures of B-Raf, B-RafV600E, and VEGFR2 were
retrieved from Protein Data Bank. The entries of these crystal structures
are shown in Fig. 3 and Table 4. The structures of 9m and drug 3 were
constructed by Chemdraw and optimized by the MM2 method in
Chem3D. The macromolecules were processed by removal of the co-
crystallized ligand and waters, addition of hydrogens, and assignment
of Gasteiger–Marsili charges using AutoDockTools 1.5.6. For the li-
gands, the root of torsion trees and the active torsions were generated
automatically. The grid box size was set to 60 × 60 × 60 (spacing 0.375
Å) and centered at the cavity of the removed ligand. The grid map was
calculated by Autogrid. Docking was performed using Autodock 4.2.6
with the default parameters. The interaction of 9m with 1UWH (B-Raf),
1UWJ (B-RafV600E), and 4AGD (VEGFR2) was analyzed by Discovery
Studio Visualizer (BIOVIA software, Inc.) and are shown in Fig. 3. The
calculated binding energy of the most stable poses of 9m in the kinases
was retrieved from the docking results and shown in Table 4.
4.1.4.13. 3-(4-{4-[(2,4-Difluoro-3-hydroxyphenyl)amino]quinazolin-6-
yl}-1H-1,2,3-triazol-1-yl)propanamide
(9n). Yield:
34%;
mp
248.1–249.9 ◦C; 1H NMR: δ 10.29 (brs, 1H), 10.04 (s, 1H), 9.02 (s, 1H),
8.59 (s, 1H), 8.45 (s, 1H), 8.31 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.7 Hz,
1H), 7.52 (s, 1H), 7.07 (t, J = 9.6 Hz, 1H), 7.02–6.86 (m, 2H), 4.65 (t, J
= 6.6 Hz, 2H), 2.78 (t, J = 6.6 Hz, 2H); 13C NMR: δ 171.0, 158.8, 154.7,
150.6 (dd, J = 240.2, 5.1 Hz), 149.2, 148.1 (dd, J = 244.4, 6.6 Hz),
145.5, 134.3 (t, J = 15.7 Hz), 130.2, 128.7, 128.4, 123.1 (dd, J = 10.9,
2.9 Hz), 122.3, 119.2, 116.9 (d, J = 7.9 Hz), 115.0, 110.6 (dd, J = 19.5,
2.8 Hz), 46.1, 35.2; HRMS calcd for [C19H15F2N7O2 + H]+ 412.1328,
found 412.1344.
Author contribution
C.-I.L. designed the compounds and revised the manuscript; C.-B.L.
designed the biological study and analyzed the biological data; C.-S.C.,
F.-Y.C., Y.-H.C., Y.-C.W., W.-Y.H., T.-H.L., G.-R.H., H.-Y.H., and C.-S.T.
synthesized the compounds; S.-Y.C. screened the compounds and per-
formed the biological study; Y.-J.L. analyzed the spectroscopic data; S.-
H.C. and J.-J.H. designed the compounds and performed the docking
simulations; J.-J.H. prepared the manuscript.
4.2. Kinase assays
The kinase inhibitory activity of 9a–n and reference standards (1, 4,
and 6) was determined on the radiometric assays by measuring the
amount of 33P incorporated into the substrate in the presence of a
Funding sources
compound. For Raf kinase assays, a reaction mixture (25 μL, final vol-
ume) containing recombinant B-Raf (5 ng, Upstate) or recombinant B-
RafV600E (5 ng, Upstate) or recombinant C-Raf (2 ng, Upstate) with 500
ng MEK1K97R (Millipore), 50 mM Tris/HCl (pH 7.5), 1 mM EGTA, 1 mM
Na3VO4, 1% 2-mercaptoethanol, 0.1% Brij 35 (Millipore), 0.2 mg/mL
This research was supported by the Ministry of Science and Tech-
nology of the Republic of China (Taiwan, MOST 102–2113-
Mꢀ 415ꢀ 005ꢀ MY2).
BSA (Sigma), 8
μ
M ATP (Sigma), 0.5
μ
Ci [33P]ATP (specific activity
3000 Ci/mmol, PerkinElmer), and a compound (or 4% DMSO) was
incubated at 30 ◦C. After 30 min, the reaction was stopped by adding 3%
phosphoric acid. The solution was subsequently harvested onto a 96-
well GF/B UniFilter (PerkinElmer), and the radioactivity of the filter
was counted on a TopCount microplate scintillation counter (Perki-
nElmer). The compounds were 3-fold serially diluted in triplication for
the assays. Software GraphPad Prism 4 (GraphPad Software Inc.) was
used to analyze the results.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
For EGFR assays, a reaction mixture (25 μL, final volume) of kinase
We thank Prof. Cheng-Liang Huang for helpful discussions.
Appendix A. Supplementary data
domain of EGFR (25 ng, Millipore) or EGFRT790M (25 ng, Millipore) with
5 μg poly(Glu-Tyr) (Sigma), 10 mM MOPS (pH 7.0), 0.3 mM EDTA, 0.5%
glycerol, 0.001% Brij-35 (Sigma), 10 mM MnCl2, 0.1 mg/mL BSA
(Sigma), 100
μ
M ATP (Sigma), 0.1
μ
Ci [33P]ATP (specific activity 3000
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104715.
Ci/mmol, PerkinElmer), and a compound (or 4% DMSO) was incubated
at 30 ◦C. For EGFR assay, the incubation time was 30 min. For
EGFRT790M assay, the incubation time was 60 min. The reaction was
stopped, processed, and analyzed following the same procedures for the
Raf kinase assays. For VEGFR2 and PDGFR-β kinase assays, the com-
pounds were assayed following our published methods [53].
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