Design and Synthesis of Novel EGFR Inhibitors with L-Rhamnose–Benzoxazinone Hybrids

Article abstract of DOI:10.1002/jhet.2463

Four L-rhamnose–benzoxazinone compounds as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were designed and synthesized. All structures of the compounds were characterized by¹H-NMR,¹³C-NMR, and high-resolution mas

Full text of DOI:10.1002/jhet.2463

Month 2015
Design and Synthesis of Novel EGFR Inhibitors with
L-Rhamnose–Benzoxazinone Hybrids
Renshuai Zhang,a,b Shaopeng Chen,a,b Guanzhao Wu,a,b Shengbiao Wan,a,b and Tao Jianga,b
*
aKey Laboratory of Marine Drugs, Chinese Ministry of Education, Shandong Provincial Key Laboratory of Glycoscience
and Glycotechnology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
bDongying Tiandong Pharmaceutical Co., Ltd, No.1236, Nan-er road, Dongying, Shandong, China
*
E-mail: jiangtao@ouc.edu.cn
Additional Supporting Information may be found in the online version of this article.
Received March 13, 2015
DOI 10.1002/jhet.2463
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
Four L-rhamnose–benzoxazinone compounds as epidermal growth factor receptor (EGFR) tyrosine kinase
inhibitors were designed and synthesized. All structures of the compounds were characterized by 1H-NMR,
13C-NMR, and high-resolution mass spectrometry. The inhibition activities of the target compounds for the
EGFR tyrosine kinase activity in vitro were determined. Compounds 6a–d displayed moderate activity in
targeting EGFR.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
modifying the quinazoline, with focus on the C-6 or C-7
position, is the reasonable strategy that developed many
potent EGFR inhibitors [6]. H.R. Tsou and other workers
reported that the core framework lied in a hydrophobic
pocket [11]. A previous research by our group demonstrated
that modifying benzoxazinone can improve its biological
activity against EGFR [9]. However, most of the
benzoxazinone derivatives exhibited poor solubility in
water, which led to considerable difficulty in further
research. To improve the water solubility and the biological
activity, four novel benzoxazinone derivatives that
incorporate water-solubilizing groups and bear different
substituents at the N-3 position were designed and synthe-
sized (Fig. 1).
Glycosides of physiologically active compounds (i.e.,
vitamin glycosides) have been reported [12]. Glycosylation
allows water-insoluble and unstable organic compounds
to be converted into their corresponding water-soluble
and stable compounds, which could probably improve
their bioavailability and pharmacological properties. In
addition, the carbohydrate moiety may have an important
function in their biological activities because saccharides
contain a high density of functional groups. In this study,
L-rhamnose ring was introduced into the target molecule.
The effect on activity of different substituents at the
3-position, including methyl and benzyl, was then investi-
gated. The resulting L-rhamnose–benzoxazinone compounds
were evaluated in vitro using enzyme-linked immunosorbent
assay.
Receptor protein tyrosine kinases perform critical func-
tions in the signal transduction pathways that regulate cell
differentiation and proliferation [1]. The human epidermal
growth factor receptor (HER) tyrosine kinase family con-
sists of four structurally related cellular receptors: the epi-
dermal growth factor receptor [epidermal growth factor
receptor (EGFR), erbB1], erbB2/HER2, erbB3/HER3,
and erbB4/HER4 [2]. Overexpression of EGFR has been
associated with oncogenic activity, such as unregulated
cell growth, proliferation, differentiation, and survival,
and is also a marker for poor prognosis in many human
cancers [3]. For this reason, EGFR has been an attractive
target for the design of novel anticancer drugs. Inhibition
of EGFR has been achieved using small-molecule inhibi-
tors that interact at the adenosine-triphosphate-binding site
[4]. Further development in this class has led to the discov-
ery of gefitinib and lapatinib (Fig. 1), which have been ap-
proved for the chemotherapeutic treatment of patients with
advanced non-small cell lung cancer [5–7].
In the previous years, various compounds, such as
4-anilinoquinazolines, 4-anilinopyrazolo[3,4-d]pyrimidines,
benzoxazinone, and 4-anilinopyrroloquinazolines, have
been reported as EGFR tyrosine kinase inhibitors [8]. Our
group is active in the design and synthesis of benzoxazinone
derivatives [9]. Benzoxazinone compounds with potential
anticancer activity have certain similarities with the
quinazoline ring [10]. Many studies have realized that
© 2015 HeteroCorporation

R. Zhang, S. Chen, G. Wu, S. Wan, and T. Jiang
Vol
Figure 1. Structures of gefitinib, lapatinib, and compounds 6a–d. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
RESULTS AND DISCUSSION
reported [13]. 1-O-bromoethy-2,3,4,6,-tetra-O-acetyl-β-L-
rhamnopyranose (1) was synthesized as described in
Experimental section. The key intermediates 2a, 2b, 3c, 3d,
4c, and 4d were obtained as described in the literature [9].
Synthesis of the target compounds 6a–d. 2,3,4,6-Tetra-O-
acetyl-L-rhamnopyranosyl was synthesized as previously
Scheme 1. Reagents and conditions: (a) BF.3Et2O, CH2Cl2, 0°C, 20 h, 44%; (b) CH3I, K2CO3, DMF, 70°C, 3 h, 69–78%; (c) concentrated ammonia
liquor/MeOH, 91–96%; (d) CHCl3, K2CO3, tetrabutylammonium bromide, DMF, 40°C, 4 h, 85–92%; (e) CH3ONa/CH3OH, 3 h, 66–95%.
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EGFR Inhibitors with L-Rhamnose–Benzoxazinone Hybrids
The synthetic route of the target compounds is outlined in
Scheme 1. First, benzylation was carried out through the
reaction of compounds 2a and 2b with BnBr in
dimethylformamide to afford 3a and 3b, respectively.
Compounds 3a and 3b were then deacetylated by
concentrated ammonia liquor (28%) in methanol to obtain
4a and 4b. Subsequently, intermediates 5a–d were
synthesized using tetrabutyl ammonium bromide as phase-
transfer catalyst, which facilitated the migration of 4a–d
from the aqueous phase into the organic phase that contains
1 in chloroform. For the last step, compounds 5a–d were
deacetylated by a catalytic amount of CH3ONa in methanol
to obtain the target compounds 6a–d, with yields of 66–95%
after recrystallization in methanol. All the compounds were
enzyme. The inhibition rate of the target compound 6c
against EGFR in vitro was 28.0% at 10.0 μM
concentration. The four analogs exhibited lower EGFR
inhibition activity compared with lapatinib.
During the preparation of compounds, we found that the
compounds 4a–d could be easily dissolved in methanol but
it is difficult to dissolve in water. In contrast, the com-
pounds 6a–d with sugar moiety are more soluble in water
compared with methanol. Therefore, the clogP values of
compounds 4a–d and 6a–d were calculated using
SYBYL-X (Tripos Associates, St. Louis, MO). As shown
in Table 1, compounds 6a–d, with sugar moiety, have
smaller clogP values than compounds 4a–d, without the
L-rhamnose moiety, again indicating that glycosylation
improved the water solubility.
1
characterized by H-NMR, 13C-NMR, and high-resolution
mass spectrometry (HRMS) spectrometry.
Binding mode analysis.
To investigate whether the
Evaluating the activity of compounds 3a–b, 4a–b, 5a–d,
and 6a–d against epidermal growth factor receptor and
the clogP values. The inhibition activities of compounds
added saccharide ring can impact the bonding between
compounds and EGFR, molecular docking was adopted
to predict the binding modes of 6a-6d. The docking
simulations were carried out by means of the SYBYL-X
2.0 software, using the publicly available coordinate of
the EGFR (PDB code: 1XKK). The compound 6a-6d
was docked into the EGFR model using standard default
settings.
It can be seen from Figure 3A that 6a-6d with sugar
moiety and lapatinib adopts a completely opposite binding
mode. The aniline groups of lapatinib lied in the hydropho-
bic pocket; in contrast, the aniline groups of 6a-6d were
positioned in the entrance of the binding pocket, while
the sugar moiety lied in the same hydrophobic pocket.
Figure 3B showed some main interactions (H-bond) be-
tween 6a and the receptor. The oxygen atom of linker
and the saccharide moiety of 6a established H-bonds with
Phe 856, Asp 855, and Thr 854, which may be the reasons
why 6a-6d adopts different binding mode. The H-bond
could increase the binding affinities and the improving
activity; however, the saccharide moiety as hydrophilic
group was positioned in the hydrophobic pocket may be
unfavorable to the activity.
3a–b, 4a–b, 5a–d, and 6a–d toward EGFR in vitro were
determined by enzyme-linked immunosorbent assay [14]
(Fig. 2). Our group evaluated the inhibition (%) of
compounds 3c–d and 4c–d, as reported by Shaopeng Chen
et al. [9] Previous studies have demonstrated that most
synthesized benzoxazinone compounds showed low or
moderate inhibition effect against EGFR in vitro at 10.0μM
concentration. Among the novel compounds, compounds
3a–d and 4a–d showed small inhibition rates (<40%).
The activities of compounds 5a, 5b, and 5d were better
than that of 5c, which is a hydroxyl-protected L-
rhamnose–benzoxazinone compound. Compound 6a,
which had a benzyl substituted at the 3-position,
displayed the best activity against EGFR (inhibition%
= 55.1%), whereas compounds 6b and 6d showed
moderate inhibition effects against the isolated EGFR
Table 1
cLogP of compounds 4a–d, 6a–d, and lapatinib.
Compounds
cLogP
4a
4b
4c
4d
6a
6b
6c
6d
5.07
6.66
3.30
4.89
4.41
6.01
2.65
4.24
5.90
Figure 2. Epidermal growth factor receptor (EGFR) inhibition activ-
ities of the compounds (10 μM) and lapatinib. The data on inhibi-
tion (%) of compounds 3c–d and 4c–d were from Shaopeng Chen
et al. [9]. [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
Lapatinib
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R. Zhang, S. Chen, G. Wu, S. Wan, and T. Jiang
Vol
Figure 3. A. Overlay poses between Lapatinib (stick model with colored in orange) and 6a-6d (stick model with colored by atom type). The binding site
of the EGFR is shown in the transparent molecular surface colored in green. B. Low-energy docking model of the EGFR-TK/6a complex. The EGFR
kinase is shown in a cartoon representation (green) with the inhibitor 6a in a ball-and-stick representation with colored by atom type. H-bond interactions
are shown as yellow dotted lines. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
CONCLUSIONS
for 1.5 h at 0°C and for a further 20h at root temperature.
After completion of the reaction, the mixture was poured
into ice water (37mL) and extracted with CH2Cl2
(15mL×2). The organic lay was combined and washed
with saturated NaHCO3 (15mL×2) and H2O (15mL×2)
and dried over MgSO4 for 4h. The solvent was removed
under reduced pressure, and the residue was purified
through column chromatography on silica gel (eluent ethyl
acetate/petroleum ether, 1/3) to give the compound 1 as a
white powder. (44% yield); mp 69–71°C; 1H-NMR
(CDCl3, 600MHz): δ 5.31-5.29 (dd, J=10.1, 3.2Hz, 1H),
5.27-5.26 (m, 1H), 5.10-5.08 (t, J=10.1Hz, 1H), 4.79-
4.78 (d, J=1.4Hz, 1H), 4.00-3.96 (m, 2H), 3.86-3.83 (m,
1H), 3.51-3.49 (t, J=5.9Hz, 2H), 2.16 (s, 3H, OAc), 2.06
(s, 3H, OAc), 2.00 (s, 3H, OAc), 1.24-1.22 (d, J=5.9Hz,
3H); 13C-NMR (CDCl3, 150MHz): δ 170.3, 170.2, 170.1,
97.6, 71.0, 69.8, 69.1, 68.2, 66.9, 29.8, 21.0, 20.9, 20.8,
In conclusion, four target L-rhamnose–benzoxazinone
compounds and eight novel intermediates were synthe-
sized and characterized by NMR and HRMS spectrometry.
The preliminary evaluation demonstrated that the com-
pounds with L-rhamnose displayed moderate inhibition ef-
fect against EGFR at 10.0μM concentration. The benzyl
substituent at the 3-position was better than methyl. A strat-
egy for the modification of benzoxazinone with carbohydrate
was proposed, with the hope that it can be used to synthesize
other inhibitors. Further modification and evaluation of
L-rhamnose–benzoxazinone conjugates are still needed to
study their therapeutic efficacy and potential medicinal use.
EXPERIMENTAL
General. All reagents used in the experiments were
commercially available and purified in a conventional
manner. Thin-layer chromatography was performed on Merck
silica-gel 60 F254 plates (Darmstadt, Germany). Flash column
chromatography was performed on silica gel (200–300mesh,
Qing dao, China). Melting points were measured on a
WRX-1S melting-point apparatus (Taike Ltd., Beijing, China)
17.4; ESI-MS 419.0 (M+Na)+.
4-(3-chloro-4-fluorophenylimino)-3-benzyl-2-oxo-3,4-dihydro-1,
3-benzoxazine-6-yl acetate (3a). A suspension of 2a (500mg,
1.43mmol ) and a hydrous potassium carbonate (400mg,
2.89mmol ) in dry DMF (40mL) was stirred at 40°C for
20 min; then BnBr (0.23 mL) was added, and the mixture
was stirred at 70°C for 8h under a N2 atmosphere. After
completion, the solvent was removed under reduced
pressure and then purified by chromatography on silica gel
to give 3a (504 mg, 83% yield) as a yellow powder. mp
1
and were uncorrected. H-NMR and 13C-NMR spectra were
taken on JNM-ECP-600 spectrometer (Jeol Ltd, Tokyo, Japan)
with tetramethylsilane (Me4Si) as the internal standard. Mass
spectra were recorded on a Q-TOF Global mass spectrometer
(Micromass UK Ltd., Manchester, UK).
The data for compounds 2a, 2b, 3c, 3d, 4c, and 4d are
the same as previously reported [9].
1
136–138°C; H-NMR (DMSO-d6, 600 MHz): δ 7.48-7.38
(overlap, 5H, ArH), 7.34-7.31 (t, J= 7.1Hz, 2H, ArH),
7.26-7.23 (t, J= 7.0 Hz, 1H, ArH), 7.04-7.03 (dd, J=6.6,
2.2 Hz, 1H, ArH), 6.82-6.80 (m, 1H, ArH), 6.70 (s, 1H,
ArH), 5.24 (s, 2H), 2.15 (s, 3H, OAc); 13C-NMR (DMSO-d6,
150MHz): δ 169.3, 154.9 (d, J=180Hz), 153.3, 149.3,
148.0, 146.1, 145.6, 144.0, 137.0, 128.8, 128.5, 128.0, 127.6,
121.1 (d, J= 36 Hz), 120.9, 120.7, 120.6, 119.8 (d, J=53Hz),
119.2, 118.5, 118.4, 47.5, 21.1; ESI-MS 439.0 (M+ H)+;
HRMS (ESI): Anal. Calcd. for C23H17N2O4FCl+, 439.0861.
Found: 439.0846.
Synthesis of 1-O-bromoethy-2,3,4,6,-tetra-O-acetyl–β-D-L-
rhamnose (1). Full acety-L-rhamnose (0.33 g, 1.0 mmol)
and 2-bromoethanol (1.1 mL, 1.2 mmol) were dissolved in
dry CH2Cl2 (22mL), stirring at 0°C under N2 atmosphere.
Then boron trifluoride ethyl ether (8.0mL) was added
dropwisely to the solution at 0°C in the darkness over a
period of 20min. The reaction mixture was allowed to stir
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EGFR Inhibitors with L-Rhamnose–Benzoxazinone Hybrids
4-(3-chloro-4-(3-fluorobenzyloxy)phenylimino)-3-benzyl-2-oxo-3,
4-dihydro-1,3-benzoxazine-6-yl acetate (3b). The same method that
was used in getting 3a was adopted to prepare 3b. (80% yield);
mp 114–116°C; 1H-NMR (DMSO-d6, 600MHz): δ 7.48-7.40
(overlap, 5H, ArH), 7.34-7.30 (overlap, 4H, ArH), 7.26-7.24 (t,
J=7.7 Hz, 1H, ArH), 7.21-7.20 (d, J=8.8Hz, 1H, ArH), 7.18-
7.16 (dd, J= 7.8, 2.1 Hz, 1H, ArH), 6.94-6.93 (d, J=2.8Hz,
1H, ArH), 6.76-6.74 (dd, J=8.8, 2.2Hz, 1H, ArH), 6.72 (br
s, 1H, ArH), 5.24 (s, 2H), 5.22 (s, 2H), 2.13 (s, 3H, ÀOAc);
13C-NMR (DMSO-d6, 150 MHz): δ 169.3, 163.6, 162.0,
150.1, 149.2, 148.1, 146.1, 142.6, 140.3, 137.1, 131.1, 131.0,
128.8, 128.5, 128.0, 127.6, 123.7, 123.5, 120.8, 120.7, 119.1
(d, J=20Hz), 118.9, 116.7, 115.3, 115.2 (d, J=21Hz),
114.5, 114.4, 70.3, 47.3, 21.1; ESI-MS 545.1 (M + H)+;
HRMS (ESI): Anal. Calcd. for C30H23N2O5FCl+, 545.1280.
Found: 545.1263.
(2R,3R,4R,5S,6S)-2-(2-(((Z)-3-benzyl-4-((3-chloro-4-fluorophenyl)
imino)-2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)oxy)
ethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate
(5a).
A mixture of 10 mL H2O and 10 mL CHCl3
was stirred at 40°C, and tetrabutylammonium bromide
(484mg, 1.5mmol) was added. Then a solution of K2CO3
(622mg, 4.5mmol) and compound 4a (1.5mmol) in 25mL
H2O and 10mL DMF was added to the mixture. After that,
a solution of compound 1 (2.0mmol) in CHCl3 (25mL)
was added dropwisely at 40°C over a period of 30min to
the solution. The reaction mixture was allowed to stir
overnight at 40°C. After completion of the reaction, the
mixture was extracted with CH2Cl2 (50mL×3). The
organic layer was combined and washed with H2O(50 mL× 3)
and dried over MgSO4 for 4 h. Then the solution was
removed under reduced pressure and purified by
chromatography on silica gel to give product 5a. mp
4-(3-chloro-4-fluorophenylimino)-6-hydroxy-3-benzyl-3,4-dihydro-1,
3-benzoxazine-2-one (4a). Compound 3a (381 mg, 0.87 mmol)
1
was dissolved in methanol (30 mL), and then
concentrated ammonia liquor (28%, 0.5 mL ) was
added. The reaction mixture was stirred at room
temperature for 3 h. After completion of the reaction,
the solvent was evaporated under reduced pressure. The
yellow precipitate formed was filtered and washed
with diethylether (50 mL) to give 4a. (94% yield); mp
215–217°C; 1H-NMR (DMSO-d6, 600MHz): δ 9.77
(br s, ArOH), 7.41-7.38 (overlap, 3H, ArH), 7.34-7.31
(t, J = 7.1 Hz, 2H, ArH), 7.25-7.24 (t, J=8.8Hz, 1H,
ArH), 7.23 (s, 1H, ArH), 7.03-7.01 (d, J= 9.4, 2.8 Hz,
2H, ArH), 6.77-6.76 (m, 1H, ArH), 6.44-6.43
(d, J=2.8Hz, 1H, ArH), 5.23 (s, 2H); 13C-NMR
(DMSO-d6 150MHz): δ 153.8 (d, J=180Hz), 148.3,
145.9, 144.5, 144.2, 143.0, 137.3, 128.8, 128.6, 128.0,
127.6, 127.1, 127.0, 122.5 (d, J=36Hz), 120.9, 119.5,
118.9 (d, J=54Hz), 118.1, 113.2, 112.5, 56.6; ESI-MS
397.1 (M+H)+; HRMS(ESI): Anal. Calcd. for
55–58°C; H-NMR (CDCl3, 600 MHz): δ 7.52-7.51 (d,
J = 6.8 Hz, 2H, ArH), 7.34-7.32 (t, J = 7.8 Hz, 2H, ArH),
7.30-7.29 (d, J = 7.3 Hz, 1H, ArH), 7.20-7.16 (q,
J = 8.7 Hz, 2H, ArH), 7.08-7.06 (dd, J = 8.5, 4.4 Hz, 1H,
ArH), 6.88-6.87 (dd, J = 6.4, 2.8 Hz, 1H, ArH), 6.68-6.66
(m, 1H, ArH), 6.58-6.57 (d, J= 2.8 Hz, 1H, ArH), 5.38 (s,
2H), 5.19-5.18 (d, J= 1.9 Hz, 1H), 5.17-5.15 (m, 1H),
5.73-5.72 (d, J=1.4Hz, 1H), 4.24-4.23 (m, 1H), 3.81-3.79
(m, 2H), 3.63-3.62 (m, 3H), 2.15 (s, 3H, ÀOAc), 2.05 (s,
3H, ÀOAc), 2.00 (s, 3H, ÀOAc), 1.20-1.19 (d, J=6.4Hz,
3H); 13C-NMR (CDCl3, 150MHz): δ 170.2, 170.1, 170.0,
154.4, 154.3 (d, J=180Hz), 148.5, 145.8, 145.3, 143.3,
136.5, 129.1, 128.5, 127.7, 123.2, 121.9 (d, J=36Hz),
120.8, 118.8, 118.6 (d, J=54Hz), 118.5, 117.7, 117.6,
111.9, 111.0, 97.6, 71.1, 69.7, 69.0, 66.9, 66.6, 66.0, 63.2,
21.0, 20.9, 20.8, 17.5; ESI-MS 713.2 (M+H)+; HRMS
(ESI): Anal. Calcd. for C35H35N2O11FCl+, 713.1913.
Found: 713.1932.
C21H15N2O3FCl+, 397.0755. Found: 397.0755.
4-(3-chloro-4-(3-fluorobenzyloxy)phenylimino)-6-hydroxy-3-
benzyl-3,4-dihydro-1,3-benzoxazine-2-one (4b). The same method
(2R,3R,4R,5S,6S)-2-(2-(((Z)-3-benzyl-4-((3-chloro-4-((3-fluorobenzyl)
oxy)phenyl)imino)-2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)oxy)
ethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (5b). The
same method that was used in getting 5a was adopted to
prepare 5b. (85% yield); mp 65–68°C; H-NMR (CDCl3,
that was used in getting 4a was adopted to prepare 4b.
(97.5% yield); mp 189–191°C; 1H-NMR (DMSO-d6,
600MHz): δ 9.74 (br s, 1H, ArOH), 7.47-7.45 (t,
J =6.1 Hz, 1H, ArH), 7.39-7.38 (d, J= 7.7 Hz, 2H, ArH),
7.34-7.31 (t, J= 7.7 Hz, 4H, ArH), 7.26-7.24 (t, J= 7.1 Hz,
1H, ArH), 7.22-7.21 (d, J =6.6 Hz, 2H, ArH), 7.20-7.16
(dt, J = 8.8, 2.3 Hz, 1H, ArH), 7.03-7.01 (dd, J = 8.8,
2.8Hz, 1H, ArH), 6.92-6.91 (d, J = 2.8 Hz, 1H, ArH),
6.72-6.71 (dd, J= 8.8, 2.7 Hz, 1H, ArH), 6.52-6.51 (d,
J =2.8 Hz, 1H, ArH), 5.24 (s, 2H), 5.22 (s, 2H); 13C-NMR
(DMSO-d6, 150 MHz): δ 163.6, 161.9, 153.7, 149.9,
148.4, 144.5, 143.8, 142.9, 140.2, 137.4, 131.1, 131.0,
128.8, 128.0, 127.5, 123.8, 123.2, 122.2, 120.6
(d, J= 20Hz), 118.8, 116.3, 115.3, 114.6, 114.5
(d, J = 21 Hz), 113.4, 112.7, 70.1, 47.1; ESI-MS 503.1 (M
+ H)+; HRMS (ESI): Anal. Calcd. for C28H21N2O4FCl+,
503.1174. Found: 503.1192.
1
600 MHz): δ 7.52-7.51 (d, J = 7.3 Hz, 2H, ArH), 7.38-7.36
(q, J= 7.7 Hz, 1H, ArH), 7.34-7.31 (t, J= 7.3Hz, 2H,
ArH), 7.28-7.27 (d, J = 7.3 Hz, 1H, ArH), 7.25-7.24 (d,
J = 7.8 Hz, 1H, ArH), 7.22-7.21 (d, J =9.1 Hz, 1H, ArH),
7.15-7.13 (d, J =9.1 Hz, 1H, ArH), 7.06-7.03 (overlap,
2H, ArH), 7.00-6.98 (d, J = 8.7 Hz, 1H, ArH), 6.92-6.91
(d, J= 2.7 Hz, 1H, ArH), 6.64-6.63 (t, J =2.3 Hz, 2H),
5.38 (s, 2H), 5.18-5.17 (t, J = 1.9 Hz, 1H), 5.15 (s, 2H),
5.13-5.12 (d, J =3.7 Hz, 1H), 5.04-5.02 (t, J= 10.1 Hz,
1H), 4.70 (s, 1H), 3.80-3.78 (m, 1H), 3.74-3.70 (m, 1H),
3.59-3.56 (m, 3H), 2.13 (s, 3H, ÀOAc), 2.03 (s, 3H,
ÀOAc), 1.96 (s, 3H, ÀOAc), 1.15-1.14 (d, J =6.4 Hz,
3H); 13C-NMR (CDCl3, 150MHz): δ 170.2, 170.1, 170.0,
154.3, 150.0, 148.6, 145.8, 143.1, 139.3, 136.7, 130.4,
129.1, 129.0, 128.7, 128.5, 128.4, 127.7, 124.7, 123.1,
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122.5, 121.1, 118.7, 118.1, 116.0, 115.2, 115.0, 114.1,
114.0, 111.0, 97.6, 71.0, 70.9, 69.7, 69.0, 66.9, 66.5,
66.0, 47.1, 21.0, 20.9, 20.8, 17.4; ESI-MS 819.3 (M
+H)+; HRMS (ESI): Anal. Calcd. for C42H41N2O12FCl+,
resin was added to the reaction solution, and the mixture
was stirred at root temperature for 5 min. Then the solvent
was filtered and removed under reduced pressure. The
crude products were recrystallized from cold ethanol to
give product 6a. (85% yield); mp 71–73°C; 1H-NMR
(DMSO-d6, 600 MHz): δ 7.45-7.40 (overlap, 3H, ArH),
7.35-7.32 (t, J =7.3 Hz, 3H, ArH), 7.27-7.23 (overlap, 2H,
ArH), 7.09-7.08 (dd, J = 6.9, 1.4 Hz, 1H, ArH), 6.85-6.84
(m, 1H, ArH), 6.50 (s, 1H, ArH), 5.26 (s, 2H), 4.76
(br s, 3H), 4.60-4.54 (m, 2H), 3.71-3.68 (s, 2H),
3.64-3.60 (m, 2H), 3.58 (s, 1H), 3.19-3.17 (m, 2H),
1.09-1.08 (d, J =6.4 Hz, 3H); 13C-NMR (DMSO-d6,
600 MHz): δ 154.8, 153.8 (d, J = 180Hz), 148.2, 146.1,
145.8, 144.4, 137.2, 128.9, 128.8, 128.0, 127.6, 122.7,
121.0, 119.9 (d, J = 36Hz), 119.2, 118.4 (d, J = 54Hz),
112.5, 111.8, 100.7, 100.6, 72.4, 71.1, 70.9, 69.0, 67.7,
65.0, 60.7, 18.4; ESI-MS 587.1 (M + H)+; HRMS (ESI):
Anal. Calcd. for C29H29N2O8FCl+, 587.1596. Found:
819.2332. Found: 819.2347.
(2R,3R,4R,5S,6S)-2-(2-(((Z)-4-((3-chloro-4-fluorophenyl)imino)-3-
methyl-2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)oxy)ethoxy)-6-
methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (5c). The same
method that was used in getting 5a was adopted to
1
prepare 5c. (92% yield); mp 61–64°C; H-NMR (CDCl3,
600MHz): δ 7.22-7.19 (t, J = 8.7 Hz, 1H, ArH), 7.18-7.16
(d, J = 8.7Hz, 1H, ArH), 7.09-7.07 (dd, J = 8.7, 2.8 Hz,
1H, ArH), 6.97-6.96 (dd, J =6.4, 2.8 Hz, 1H, ArH),
6.77-6.75 (m, 1H, ArH), 6.59 (br s, 1H, ArH), 5.20-5.19
(dd, J= 3.2, 1.4 Hz, 1H), 5.16-5.14 (dd, J= 10.1, 2.8 Hz, 1H),
5.06-5.03 (t, J= 10.1 Hz, 1H), 4.74-4.73 (d, J=1.4Hz, 1H),
3.85-3.81 (m, 2H), 3.65 (br s, 3H), 3.54 (br s, 3H), 2.15
(s, 3H, ÀOAc), 2.05 (s, 3H, ÀOAc), 1.98 (s, 3H, ÀOAc),
1.20-1.19 (d, J =6.4 Hz, 3H); 13C-NMR (CDCl3,
150MHz): δ 170.2, 170.1, 170.0, 155.2, 154.5, 148.5,
145.8, 145.6, 123.1, 123.0, 121.2, 121.1 (d, J= 36Hz),
118.9, 118.7, 117.7, 117.5, 110.9, 97.6, 71.0, 69.7, 69.0,
66.9, 66.6, 66.0, 63.1, 21.0, 20.9, 20.8, 17.4; ESI-MS
637.1 (M + H)+; HRMS (ESI): Anal. Calcd. for
587.1603.
(Z)-3-benzyl-4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)imino)-6-
(2-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-
pyran-2-yl)oxy)ethoxy)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one
(6b). The same method that was used in getting 6a was
adopted to prepare 6b. (95% yield); mp 76–78°C; 1H-NMR
(DMSO-d6, 600MHz): δ 7.48-7.45 (q, J=5.9Hz, 1H, ArH),
7.42-7.40 (t, J=5.9Hz, 2H, ArH), 7.34-7.30 (overlap, 5H,
ArH), 7.27-7.24 (overlap, 2H, ArH), 7.24-7.22 (m, 1H,
ArH), 7.20-7.17 (m, 1H, ArH), 6.98-6.97 (d, J=2.3Hz, 1H,
ArH), 6.78-6.76 (dd, J=8.7, 2.3Hz, 1H), 6.55 (s, 1H), 5.25
(s, 2H), 5.22 (s, 2H), 4.77 (br s, 3H), 4.58 (br s, 1H), 4.52
(s, 1H), 3.64 (s, 2H), 3.62-3.58 (m, 2H), 3.55 (s, 1H), 3.50-
3.48 (m, 1H), 3.16-3.15 (m, 1H), 1.05-1.04 (d, J=6.6Hz,
3H); 13C-NMR (DMSO-d6, 600MHz): δ 163.6, 162.0,
154.2, 150.0, 148.3, 145.8, 144.0, 143.1, 140.3, 137.3,
131.1, 128.9, 128.8, 128.0, 127.9, 127.6, 123.8, 123.4,
122.7, 120.9, 119.1 (d, J= 20Hz), 116.5, 115.3, 115.1,
114.5 (d, J=21 Hz), 111.7, 100.6, 72.4, 71.1, 70.9, 70.2,
69.0, 67.6, 65.0, 47.2, 18.3; ESI-MS 693.2 (M+ H)+;
HRMS (ESI): Anal. Calcd. for C36H35N2O9FCl+, 693.2015.
C29H31N2O11FCl+, 637.1600. Found: 637.1621.
(2R,3R,4R,5S,6S)-2-(2-(((Z)-4-((3-chloro-4-((3-fluorobenzyl)oxy)
phenyl)imino)-3-methyl-2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-
6-yl)oxy)ethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate
(5d). The same method that was used in getting 5a was
1
adopted to prepare 5d. (88% yield); mp 64–67°C; H-NMR
(CDCl3, 600MHz): δ 7.38-7.36 (m, 1H, ArH), 7.37-7.25 (d,
J=10.5 Hz, 1H, ArH), 7.24-7.23 (d, J= 7.8 Hz, 1H, ArH),
7.16-7.14 (d, J= 9.1 Hz, 1H, ArH), 7.07-7.05 (d, J=9.2,
2.8 Hz, 1H, ArH), 7.03-7.00 (overlap, 3H, ArH), 6.72-6.70
(dd, J=8.3, 2.3Hz, 1H), 6.65 (br s, 1H), 5.18 (s, 1H), 5.16
(s, 2H), 5.14-5.12 (dd, J= 10.1, 3.7 Hz, 1H), 5.04-5.01 (t,
J=9.6Hz, 1H), 4.71 (s, 1H), 3.81-3.76 (m, 2H), 3.60 (br s,
3H), 3.53 (br s, 3H), 2.13 (s, 3H, ÀOAc), 2.04 (s, 3H,
ÀOAc), 1.97 (s, 3H, ÀOAc), 1.16-1.15 (d, J=6.4Hz, 3H);
13C-NMR (CDCl3, 150 MHz): δ 170.2, 170.1, 170.0, 150.1,
148.6, 145.7, 143.3, 139.3, 139.2, 130.4, 130.3, 124.7,
123.0, 122.5, 121.4, 118.6, 118.3, 115.9, 115.2, 115.0,
114.1, 114.0, 110.8, 97.6, 71.0, 70.8, 69.7, 69.0, 67.7, 66.9,
66.5, 66.1, 21.0, 20.9, 20.8, 17.4; ESI-MS 743.2 (M + H)+;
HRMS (ESI): Anal. Calcd. C36H37N2O12FCl+, 743.2019.
Found: 693.2043.
(Z)-4-((3-chloro-4-fluorophenyl)imino)-3-methyl-6-(2-(((2R,3R,
4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)
ethoxy)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one (6c).
The
same method that was used in getting 6a was adopted to
prepare 6c. (91% yield); mp 75–77°C; 1H-NMR (DMSO-d6,
600MHz): δ 7.46-7.43 (t, J=9.2Hz, 1H, ArH), 7.32-7.31
(d, J= 8.7 Hz, 1H, ArH), 7.23-7.21 (dd, J=8.7, 2.8Hz, 1H,
ArH), 7.14-7.13 (d, J= 4.6Hz, 1H, ArH), 6.92-6.91 (t,
J=3.9Hz, 1H, ArH), 6.48 (br s, 1H, ArH), 4.76-4.75 (t,
J= 3.7 Hz, 2H), 4.55-4.54 (t, J= 6.4 Hz, 2H), 3.70-3.64 (m,
3H), 3.56-3.50 (m, 3H), 3.45-3.42 (m, 1H), 3.34 (s, 3H),
3.19-3.16 (m, 1H), 1.09-1.08 (d, J=5.9Hz, 3H); 13C-NMR
(DMSO-d6, 600MHz): δ 154.7, 153.7 (d, J=180Hz), 148.3,
146.5, 145.7, 122.6, 122.5, 121.2, 120.2 (d, J=36Hz),
119.0, 118.5 (d, J= 54 Hz), 118.4, 118.3, 111.6, 100.6, 72.4,
Found: 743.2038.
(Z)-3-benzyl-4-((3-chloro-4-fluorophenyl)imino)-6-(2-(((2R,3R,
4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)
ethoxy)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one (6a).
To a
round-bottomed flask (100 mL), compound 5a (0.5 mmol)
was dissolved in 30mL anhydrous CH3OH, and then 2mL
0.5M CH3ONa/CH3OH was added to the solution. The
mixture was allowed to stir at root temperature for 3 h.
When the reaction was completed (monitored by thin-
layer chromatography), 5 g 732-type cation exchange
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
EGFR Inhibitors with L-Rhamnose–Benzoxazinone Hybrids
REFERENCES AND NOTES
71.1, 70.9, 69.0, 68.9, 67.7, 65.0, 18.4; ESI-MS 511.1 (M
+H)+; HRMS (ESI): Anal. Calcd. for C23H25N2O8FCl+,
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(Z)-4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)imino)-3-methyl-6-
(2-(((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-
pyran-2-yl)oxy)ethoxy)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one
(6d). The same method that was used in getting 6a was
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adopted to prepare 6d. (66% yield); mp 80–82°C; 1H-NMR
(DMSO-d6, 600MHz): δ 7.49-7.45 (q, J=7.8Hz, 1H,
ArH), 7.33-7.26 (overlap, 3H, ArH), 7.26-7.25 (d,
J=8.7Hz, 1H, ArH), 7.20-7.17 (overlap, 2H, ArH), 7.04-
7.03 (d, J=1.9Hz, 1H, ArH), 6.85-6.84 (dd, J=8.7, 2.3Hz,
1H, ArH), 6.51 (br s, 1H), 5.22 (s, 2H), 4.76 (br s, 2H),
4.56 (br s, 1H), 4.52 (s, 1H), 3.63-3.61 (d, J=13.7Hz, 3H),
3.55 (s, 1H), 3.50-3.43 (m, 2H), 3.37 (s, 3H), 3.29-3.27 (m,
1H), 3.19-3.14 (m, 1H), 1.05-1.04 (d, J=6.0Hz, 3H); 13C-
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134.5, 140.2, 131.1, 131.0, 123.8, 123.7, 123.3, 122.4,
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114.6, 114.5, 100.6, 72.4, 71.1, 70.9, 70.2, 69.0, 68.6, 67.6,
65.1, 18.3; ESI-MS 617.2 (M+ H)+; HRMS (ESI): Anal.
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Acknowledgments. This research was supported by the Natural
Science Foundation of China (Grant No. 81373322). We are
also grateful to Meiyu Geng from Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, for the biological assay.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet