Synthesis of novel 1,4-benzoxazin-3-one derivatives as inhibitors against tyrosine kinases

Article abstract of DOI:10.1016/j.bmc.2008.11.060

We designed and synthesized a novel 1,4-benzoxazin-3-one derivative 4 which would have inhibitory activities against tyrosine kinases. They could be synthesized easily from various carboxylic acids 10 and commercially available amines using TFP resin with

Full text of DOI:10.1016/j.bmc.2008.11.060

Bioorganic & Medicinal Chemistry 17 (2009) 699–708
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 1,4-benzoxazin-3-one derivatives as inhibitors
against tyrosine kinases
b
Takahiro Honda a,b, , Takahiro Terao , Hiroyuki Aono a,b, Masakazu Ban a,b
*
a Research and Development Center, Santen Pharmaceutical Co. Ltd, 8916-16 Takayama-cho, Ikoma-shi, Nara 630-0101, Japan
b Graduate School of Materials Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma-shi, Nara 630-0192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized a novel 1,4-benzoxazin-3-one derivative 4 which would have inhibitory
activities against tyrosine kinases. They could be synthesized easily from various carboxylic acids 10
and commercially available amines using TFP resin without purification. In this article, we will report
the design and synthesis of a novel 1,4-benzoxazin-3-one chemical library 4 and the inhibitory activities
against KDR and ABL which are closely related to chronic diseases such as cancer.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 6 October 2008
Revised 18 November 2008
Accepted 19 November 2008
Available online 6 December 2008
Keywords:
1,4-Benzoxazin-3-one chemical library
Tyrosine kinase inhibitor
TFP resin
KDR
ABL
Intramolecular S–O non-bonded interaction
1. Introduction
2. Chemistry
Protein kinases play a very important role in the cell signaling
process, affecting cell proliferation, differentiation, apoptosis and
secretion.1 Indolinone derivatives, SU5416 (semaxanib, 1) and
SU11248 (sunitinib malate, 2) are reported to be potent inhibitors
against vascular endothelial growth factor receptor 2 tyrosine ki-
nase (KDR).2,3 SU11248 shows especially potent antiangiogenic ef-
fects in vivo, by introducing a substituent group in R2 to increase
aqueous solubility. This compound inhibits other tyrosine kinases
as well as the KDR, so the structure of indolinone is thought to
be a good framework as a kinase inhibitor. On the other hand,
1,4-benzoxazin-3-one derivatives 3, having a similar structure to
these indolinone derivatives, are also reported in a patent to be
anti-proliferative agents due to inhibition against tyrosine ki-
nases.4 However, 1,4-benzoxazin-3-one derivatives, which have a
hydrophilic group like an amide moiety of SU11248 (2), have not
been synthesized. Thus, we designed and synthesized novel 1,4-
benzoxazin-3-one derivatives 4 which would have inhibitory
activities against tyrosine kinases. They could be synthesized easily
from various carboxylic acids 10 and commercially available
amines 12 using TFP resin without purification5 (Fig. 1).
Syntheses of novel 1,4-benzoxazin-3-one derivative chemical li-
brary 4 are illustrated in Scheme 1. The Knoevenagel reaction of
1,4-benzoxazin-3-one 5 with various haloarylcarboxamide 6a–i
afforded the 2-bromoarylyliden-1,4-benzoxazin-3-one 7a–i. The
palladium catalyzed C–C bond formation of various bromides 7a–
i with tert-butyl acrylate gave acrylate 8a–i in good yields except
in the case using 7b. The acrylate 7b, which was derived from bro-
mides 6b, did not react with tert-butyl acrylate in this condition. So
in order to obtain the desired acrylate 8b, the reaction of 1,4-benz-
oxazin-3-one 5 with aldehyde 13, which was derived from m-bro-
mobenzaldehyde 6b, was investigated (Scheme 2). Fortunately, the
desired acrylate 8b was obtained, although in low yield. The N-
alkylated acrylates 9 were prepared from various 8a–i by usual
methods (alkylhalide, NaH, DMF), and the tert-butyl group of acry-
lates 8 and 9 were removed by the hydrogen chloride solution of
1,4-dioxane. The loading TFP resin with acid 10a–i were carried
out following the reported procedure.5 The reaction of the acrylat-
ed TFP resin 11a–i with various amines 12a–m afforded the de-
sired amides in good to moderate yield without purification to
achieve the preparation of the chemical library consisting of vari-
ous 1,4-benzoxazin-3-one derivatives 4 (Fig. 2).
The double bond geometries of tri-substituted alkenes of vari-
ous 1,4-benzoxazin-3-one derivatives 4 were determined by mea-
surement of the coupling constants JC–H between H1 and C1 on
non-decoupling 13C NMR of compounds 4a–d and, 6f and 6g
* Corresponding author. Tel./fax: +81 743 72 6145.
E-mail address: hondat@ms.naist.jp (T. Honda).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.060

700
T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
R2
Me
O
NR2R3
Ar
O
Me
N
H
R1
Ar
O
O
O
R
N
H
O
N
H
1 R1=R2 = H (SU5416)
N
2
R
1 =F, R2 = CONHCH2CH2NEt2
R1
(SU11248)
3
4
Figure 1. Structures of indolinone derivatives 1 (SU5416) and 2 (SU11248), and 1,4-benzoxazin-3-one derivatives 3 and 4.
O-t-Bu
Knoevenagel
Reaction
Mizoroki-Heck
Reaction
Ar
O
Br
O
Ar
O
O
b
a
O
O
N
N
H
O
1
N
H
8 R = H
c
OHC
Br
1
9 R = Me
Ar
1
7
5
R
6
F
F
F
HN
O
O
O
OH
O
Ar
Ar
O
F
d
e
f
O
N
O
N
4
2
3
12
R R NH (
)
O
1
1
R
R
10
11
Scheme 1. Reagents and conditions: (a) Ac2O/Et3N (2/1), reflux, y. 7–30%; (b) tert-butyl acrylate (5 equiv), 10 mol % Pd(OAc)2, 20 mol% P(o-tol)3, i-Pr2EtN (10 equiv), DMF,
100 °C, y. 43–94%; (c) R1–X, NaH, DMF, rt, y. 59–88%; (d) 4 M HCl–1,4-dioxane, rt, y. 72–100%; (e) TFP resin, DIC, cat. DMAP, Et3N, DMF, rt, percent loading 48–83%; (f) R2R3NH,
DMF, rt–80°C, y. 9–98%.
O
Knoevenagel
Reaction
Mizoroki-Heck
Reaction
O-t-Bu
O
h
g
OHC
Br
OHC
O
O-t-Bu
N
H
O
6b
13
8b
Scheme 2. Reagents and conditions: (g) tert-butyl acrylate (5 equiv), 10 mol % Pd(OAc)2, 20 mol % P(o-tol)3, i-Pr2EtN (10 equiv), 100 °C, y. 74% (h) Ac2O/Et3N (2/1), reflux, y.
18%.
(Fig. 3).6 Based on these results, it was supposed that the tri-substi-
tuted alkenes of all compounds were Z configuration (Table 1).
Moreover, X-ray crystal structure analysis of 4l revealed that the
tri-substituted alkene was of Z configuration in its structure, and
the S–O distance (2.81 Å) were shorter than the sum of van der
Waals radii of sulfur and oxygen (3.32 Å).7 The result suggested
that the intramolecular S–O non-bonded interaction was found
in 4l and it had a stable conformation with the pseudo 5-mem-
bered ring (Fig. 4).8,9
are illustrated in Figure 5. The average of inhibitory activities of
4 against KDR was relatively high. In particular, the derivatives
synthesized from carboxylic acid 10i gave good results, including
4l which had an IC50 value of 1.48 lM (Table 2). On the other hand,
though the average of inhibitory activities against ABL of 4 was not
so high, the derivatives synthesized from carboxylic acid 10e and
10i showed good efficacy for ABL (Table 2), including 4r (IC50
0.29
4l showed potent inhibitory activity against not only KDR (IC50
1.48 M) but also ABL (IC50 1.90 M). The X-ray co-crystal struc-
lM) which was the most potent ABL inhibitor of all, while
l
l
ture of indolinone derivative SU5402 and FGFR1, which has high
homology with KDR and ABL for amino acid sequences of the
ATP binding site, revealed that the amide moiety of indolinone
was a hinge binder confirming the potent inhibitory activity.11 So
the amide moiety of indolinone was thought to bind to the hinge
region of KDR. However the results of inhibitory activities against
3. Results and discussion
The evaluation of kinase inhibitory activities of novel 1,4-benz-
oxazin-3-one derivative chemical library 4 was carried out at 4 lg/
ml concentrations of compounds using commercially available ki-
nase assay kits.10 Their inhibitory activities against KDR and ABL

T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
701
HO
O
O
OH
O
O
OH
OH
S
O
O
O
O
O
N
H
O
N
H
O
N
H
O
N
H
10a
10b
10c
10d
OH
S
OH
HO
OH
O
O
O
O
O
O
OH
S
O
O
N
O
N
O
O
O
O
O
N
H
O
N
H
O
N
H
O
CH3
CH3
10e
10f
10g
10h
10i
OH
H2N
O
N
H2N
N
HN
HN
N
H2N
H2N
12b
HN
N
12a
12h
12c
12d
12e
12g
12f
OMe
O
OMe
N
H2N
H2N
H2N
N
H2N
H2N
H2N
Cl
N
N
F
12i
12j
12k
12l
12m
Figure 2. Structures of acid 10a–i and amines 12a–m.
p
R
5'
S
4'
R
m
o
H
O
N
N
O
O
JC-H = 11.0 Hz
H1
H1
C1
C1
H3C
N
H
N
H
O
O
1H
trans
4a (p-)
4b (m-)
4c (o-)
4d (4'-)
4e (5'-)
CH3
N
H
O
C1
O
H1
N
5'
4'
H
C1
R =
N
H
Br
O
cis
O
O
O
1
JC-H = 3.1 Hz
N
H
H1
4a
N
C1
N
O
7f (4'-)
7g (5'-)
H
Figure 3. The coupling constants JC–H between H1 and C1 of compounds 4a–d, 7f and 7g.
KDR or ABL of 4l or 4r, which are N-methyl derivatives of the amide
moiety of 1,4-benzoxazin-3-one, suggest that their interactions at
the ATP binding site are different from that of indolinone deriva-
tives. Figure 7 shows a docking model of compound 4r at the
ATP binding site of ABL.12 In this proposed binding mode, 1,4-benz-
oxazin-3-one core of compound 4r forms a hydrogen bond with
residue Thr315 (Gatekeeper) (Table 3).
4. Conclusion
In summary, we synthesized 1,4-benzoxazin-3-one derivative
chemical library 4 using TFP resin without purification and evalu-
ated the inhibitory activities against KDR and ABL. The results sup-
posed that a 1,4-benzoxazin-3-one framework was favorable
structure for inhibiting KDR. Compound 4l showed relatively

702
T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
NH
N
O
2.81 Å
O
S
N
CH3
O
4l
Figure 4. ORTEP drawing and structure of compound 4l.
Table 2
Inhibitory activities against KDR of selected compounds from library 4a
Compd
Carboxylic acid
Amine
% Inhibition @ 4 g/ml
IC50 (M)
4f
10b
10g
10h
10i
10i
10i
10i
10i
12a
12e
12m
12a
12c
12e
12g
12h
70.8
76.7
79.9
83.9
79.2
88.3
76.7
77.1
N.D.b
N.D.b
N.D.b
N.D.b
N.D.b
N.D.b
1.48
4g
4h
4i
4j
4k
4l
4m
N.D.b
a
Inhibitory activities were measured with a kinase assay development kit pur-
chased from CARNA BIOSCIENCE Co. Ltd.
b
IC50 value (M) of compound was more than 0.4 g/ml.
potent inhibitory activities against both KDR and ABL and the X-ray
crystal structure analysis of 4l revealed that the S–O non-bonded
interaction was found in its structure and had the conformation
stabilized by the psuedo 5-membered ring. On the other hand this
framework is not so favorable as a ABL inhibitor, but the IC50
against ABL of compound 4r was 0.29 lM, so it would be an ABL
inhibitor as potent as Gleevec in in vitro assay (Fig. 6).13
TM
5. Experimental
5.1. Synthesis
Unless otherwise noted, all materials were obtained from com-
mercial suppliers and used without further purification. 1H NMR
spectra were recorded on a JEOL GSX400 or on a JEOL JMTC-300/
54/SS spectrometer at ambient temperature. Chemical shifts are
reported as ppm downfield from the tetramethylsilane (TMS). Data
are reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), cou-
pling constants and number of protons.
5.1.1. (Z)-2-(4-Bromobenzylidene)-4H-benzo[b][1,4]oxazin-3-
one (7a)
To
a mixture of 2H-1,4-benzoxazin-3(4H)-one 5 (12.2 g,
82.1 mmol) and 4-bromobenzaldehyde 6a (22.0 g, 120 mmol), ace-
tic anhydride (50 ml) and triethylamine (25 ml) were added, and
the reaction mixture was stirred at 110 °C for 5 h. The mixture
was cooled to rt and stirred for an additional 12 h. The mixture
was poured into aqueous 1.0 M NaOH solution with ice and then
the precipitate was collected by filtration. The precipitate was
washed with water, acetonitrile and AcOEt, and dried in vacuo to
give the desired product (7a) (7.90 g, 24.9 mmol, y. 30 %).
1H NMR (300 MHz, DMSO-d6) d: 11.18 (s, 1H), 7.85 (d, J = 8.8 Hz,
2H), 7.62 (d, J = 8.6 Hz, 2H), 7.31–7.28 (m, 1H), 7.07–7.03 (m, 2H),
7.01–6.98 (m, 1H), 6.76 (s, 1H); IR (cmÀ1, KBr) 3025, 2978, 2895,
2864, 2767, 1687, 1638, 1598, 1586, 1394, 1286, 1267, 1234,
1112, 1072, 1008, 928, 815, 748; HRMS (EI) calcd for C15H10BrNO2
314.989, found 314.990.
Figure 5. Kinase inhibitory activities of 1,4-benzoxazin-3-one chemical library 4.
Table 1
The JC-H between H1 and C1 of compounds 4a-d, 7f and 7g
Compd
JC–H (Hz)
E-/Z-
SU5416 (1)
11.0
3.1
3.6
3.0
3.5
3.0
3.5
3.3
E-
Z-
Z-
Z-
Z-
Z-
Z-
Z-
4a
4b
4c
4d
4e
7f
7g

T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
703
O
N
CH3
NH
N
NH
NH
O
N
O
O
O
O
O
S
S
4l
4r
Figure 6. Structure of 1,4-benzoxazin-3-ones 4l and 4r.
Table 3
Inhibitory activities against ABL of selected compounds from library 4a
1466, 1437, 1397, 1268, 1232, 1113, 752; HRMS (EI) calcd for
15H10BrNO2 314.989, found 314.990.
C
Compd
Carboxylic acid
Amine
% Inhibition @ 4
lg/ml
IC50 (lM)
5.1.4. (Z)-2-(4-Bromothiophen-2-ylmethylene)-4H-
benzo[b][1,4]oxazin-3-one (7d)
4i
4l
10i
10i
10i
10b
10d
10d
10e
10e
10g
12a
12g
12h
12f
12a
12c
12f
90.8
76.7
82.6
74.8
72.3
76.5
90.7
92.6
81.1
0.82
1.90
1.76
1H NMR (300 MHz, DMSO-d6) d: 11.20 (s, 1H), 7.83 (s, 1H), 7.51
(s, 1H), 7.25–7.22 (m, 1H), 7.11 (s, 1H), 7.08–7.04 (m, 2H), 7.02–
6.98 (m, 1H); IR (cmÀ1, KBr) 3032, 2980, 2900, 1683, 1637, 1500,
1419, 1389, 1346, 1266, 1235, 1107, 871, 746; HRMS(EI) calcd
for C13H8BrNO2S 320.946, found 320.945.
4m
4n
4o
4p
4q
4r
N.D.b
N.D.b
N.D.b
N.D.b
0.29
12m
12c
4s
N.D.b
a
5.1.5. (Z)-2-(5-Bromothiophen-2-ylmethylene)-4H-
benzo[b][1,4]oxazin-3-one (7e)
Inhibitory activities were measured with a kinase assay development kit pur-
chased from CARNA BIOSCIENCE Co. Ltd.
b
1H NMR (300 MHz, DMSO-d6) d: 11.16 (s, 1H), 7.32 (d, J = 3.9 Hz,
1H), 7.28 (d, J = 3.9 Hz, 1H), 7.10 (s, 1H), 7.07–7.03 (m, 2H), 7.01–
6.97 (m, 1H); IR (cmÀ1, KBr) 3036, 2980, 2909, 1682, 1635, 1501,
1427, 1234, 1335, 1266, 1234, 1104, 781, 742; HRMS (EI) calcd
for C13H8BrNO2S 320.946, found 320.946.
IC50 value (lM) of compound was more than 0.4 lg/ml.
5.1.6. (Z)-2-(4-Bromofruran-2-ylmethylene)-4H-
benzo[b][1,4]oxazin-3-one (7f)
1H NMR (300 MHz, DMSO-d6) d: 11.25 (s, 1H), 8.03 (s, 1H),
7.47–7.44 (m, 1H), 7.24 (s, 1H), 7.07–7.03 (m, 2H), 7.01–6.97 (m,
1H), 6.64 (s, 1H); IR (cmÀ1, KBr) 3032, 2981, 2898, 1686, 1636,
1501, 1389, 1350, 1268, 1230, 921, 746; HRMS (EI) calcd for
C13H8BrNO3 304.9688, found 304.9688.
5.1.7. (Z)-2-(5-Bromofruran-2-ylmethylene)-4H-
benzo[b][1,4]oxazin-3-one (7g)
1H NMR (300 MHz, DMSO-d6) d: 11.23 (s, 1H), 7.36–7.33 (m,
1H), 7.14 (d, J = 3.5 Hz, 1H), 7.06–7.02 (m, 2H), 7.00–6.96 (m,
1H), 6.80 (d, J = 3.5 Hz, 1H), 6.64 (s, 1H); 13C NMR (125 MHz,
DMSO-d6) d: 156.26, 140.95, 140.49, 138.15, 130.99, 130.88,
126.02, 124.30, 123.85, 116.33, 116.03, 115.02, 105.89; IR (cmÀ1
,
KBr) 3032, 2978, 2909, 2864, 1685, 1632, 1500, 1479, 1387,
1266, 1233, 1212, 1189, 917, 788, 745; HRMS (EI) calcd for
C13H8BrNO3 304.969, found 304.982.
Figure 7. Compound 4r docked at the ATP binding site of ABL. The protein structure
is taken from Brookhaven Protein Data Bank, entry 1M52. Hydrogen bonds are
shown as green dotted lines.
5.1.8. tert-Butyl (E)-3-(4-((Z)-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidenemethyl)phenyl) acrylate (8a)
5.1.2. (Z)-2-(3-Bromobenzylidene)-4H-benzo[b][1,4]oxazin-3-
one (7b)
To a mixture of 7a (7.88 g, 24.9 mmol), palladium acetate
(510 mg, 2.28 mmol) and tri(o-tryl)phosphine (1.36 g, 4.47 mmol),
N,N-dimethylformamide (120 ml), diisopropylethylamine (36 ml,
207 mmol) and tert-butyl acrylate (15.0 ml, 102 mmol) were added
under nitrogen atmosphere. The mixture was stirred at 100 °C over
night. The reaction mixture was cooled to rt and filtered through
Celite with AcOEt. The filtrate was washed with brine and dried
over anhydrous sodium sulfate. After concentration in vacuo the
resultant was collected by filtration with hexane–AcOEt (1:1) and
washed with AcOEt to give the desired product (8a) (7.88 g,
21.7 mmol, y. 87%).
1H NMR (300 MHz, DMSO-d6) d: 11.23 (s, 1H), 8.04 (s, 1H), 7.94
(d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1 H), 7.40 (t, J = 7.9 Hz, 1H),
7.26–7.23 (m, 1H), 7.08–7.04 (m, 2H), 7.01–6.98 (m, 1H), 6.77 (s,
1H); IR (cmÀ1, KBr) 3180, 3148, 3084, 3054, 2977, 2907, 1694,
1640, 1501, 1389, 1236, 1111, 886, 781, 745; HRMS (EI) calcd for
C15H10BrNO2 314.99, found 314.99.
5.1.3. (Z)-2-(2-Bromobenzylidene)-4H-benzo[b][1,4]oxazin-3-
one (7c)
1H NMR (300 MHz, DMSO-d6) d: 11.31 (s, 1H), 8.29 (dd, J = 8.0,
1.6 Hz, 1H), 7.73 (dd, J = 8.0, 1.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H),
7.26 (dd, J = 14.9, 9.2 Hz, 2H), 7.07 (s, 1H), 7.06–6.98 (m, 3H); IR
(cmÀ1, KBr) 3032, 2977, 2896, 2858, 2769, 1688, 1638, 1501,
1H NMR (300 MHz, DMSO-d6) d: 8.77 (s, 1H), 7.87 (d, J = 8.3 Hz,
2H), 7.60 (d, J = 15.2 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.26 (s, 1H),
7.21–7.19 (m, 1H), 7.09–7.04 (m, 2H), 6.95 (s, 1H), 6.91–6.88 (m,

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T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
1H), 6.41 (d, J = 15.8 Hz, 1H), 1.53 (s, 9H); IR (cmÀ1, KBr) 3033,
2974, 2914, 2866, 2765, 1715, 1685, 1634, 1502, 1386, 1365,
1332, 1320, 1281, 1146, 1109, 870, 827, 751; HRMS (EI) calcd for
C22H21NO4 363.147, found 363.146.
(d, J = 16.0 Hz, 1H), 1.50 (s, 9H); IR (cmÀ1, KBr) 3392, 2979, 2933,
2837, 2732, 1705, 1639, 1601, 1580, 1478, 1457, 1392, 1368,
1325, 1234, 1152, 980, 851, 797; HRMS (EI) calcd for C14H16O3
232.11, found 232.11.
5.1.9. tert-Butyl (E)-3-(2-((Z)-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidenemethyl)phenyl) acrylate (8c)
5.1.15. tert-Butyl (E)-3-(3-((Z)-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidene-methyl)phenyl) acrylate (8b)
1H NMR (300 MHz, DMSO-d6) d: 8.13 (s, 1H), 7.93 (d, J = 6.1 Hz,
1H), 7.91 (d, J = 10.3 Hz, 1H), 7.61 (d, J = 6.8 Hz, 1H), 7.43 (t,
J = 7.4 Hz, 1H), 7.34 (t, J = 7.3 Hz, 1H), 7.19 (s, 1H), 7.04–6.99 (m,
3H), 6.87–6.82 (m, 1H), 6.31 (d, J = 15.8 Hz, 1H), 1.49 (s, 9H); IR
(cmÀ1, KBr) 3438, 3047, 2980, 2920, 1702, 1686, 1632, 1502,
1389, 1292, 1268, 1237, 1161, 1112, 977, 763, 741; HRMS (EI)
calcd for C22H21NO4 363.147, found 363.147.
To a mixture of 2H-1,4-benzoxazin-3(4H)-one 5 (1.17 g,
7.91 mmol) and 13 (2.37 g, 10.2 mmol), acetic anhydride (4 ml)
and triethylamine (2 ml) were added, and the reaction mixture
was stirred at 120 °C for 5 h. The mixture was cooled to r.t. and
stirred for an additional 12 h. The mixture was poured into aque-
ous 1.0 M NaOH solution with ice and then the precipitate was col-
lected by filtration. The precipitate was washed with water,
acetonitrile and AcOEt, and dried in vacuo to give the desired prod-
uct (8b) (506 mg, 1.39 mmol, y. 18 %).
5.1.10. tert-Butyl (E)-3-(5-((Z)-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidenemethyl) thiophen-3-yl)acrylate (8d)
1H NMR (300 MHz, DMSO-d6) d: 11.18 (s, 1H), 8.11 (s, 1H), 7.83
(s, 1H), 7.53 (d, J = 15.8 Hz, 1H), 7.27–7.24 (m, 1H), 7.09–7.04 (m,
2H), 7.05 (s, 1H), 7.02–6.98 (m, 1H), 6.37 (d, J = 15.8 Hz, 1H), 1.48
(s, 9H); IR (cmÀ1, KBr) 3423, 3040, 2979, 2912, 2864, 1702, 1631,
1503, 1439, 1368, 1312, 1282, 1151, 979, 859, 746; HRMS (EI)
calcd for C20H19NO4S 369.1035, found 369.1033.
1H NMR (300 MHz, DMSO-d6) d: 11.18 (s, 1H), 7.85 (d, J = 8.8 Hz,
2H), 7.62 (d, J = 8.6 Hz, 2H), 7.31–7.28 (m, 1H), 7.07–7.03 (m, 2H),
7.01–6.98 (m, 1H), 6.76 (s, 1H); IR (cmÀ1, KBr) 3025, 2978, 2895,
2864, 2767, 1687, 1638, 1598, 1586, 1394, 1286, 1267, 1234,
1112, 1072, 1008, 928, 815, 748; HRMS (EI) calcd for C15H10BrNO2
314.989, found 314.990.
5.1.16. tert-Butyl (E)-3-((Z)-3-(4-methyl-3-oxo-3,4dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl) phenyl)acrylate (9h)
To a THF solution (3 ml) of 8b (548 mg, 1.51 mmol), sodium hy-
dride (60% oil dispersion, 90 mg, 2.25 mmol) was added and stirred
5.1.11. tert-Butyl (E)-3-(5-((Z)-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidenemethyl) thiophen-2-yl) acrylate (8e)
1H NMR (300 MHz, DMSO-d6) d: 11.23 (s, 1H), 7.72 (d,
J = 15.8 Hz, 1H), 7.52 (d, J = 3.9 Hz, 1H), 7.45 (d, J = 3.9 Hz, 1H),
7.45–7.43 (m, 1H), 7.12 (s, 1H), 7.09–7.05 (m, 2H), 7.03–6.99 (m,
1H), 6.33 (d, J = 15.6 Hz, 1H), 1.49 (s, 9H); IR (cmÀ1, KBr) 3036,
2979, 2913, 2861, 1688, 1632, 1501, 1440, 1386, 1154, 967, 844,
745; HRMS (EI) calcd for C20H19NO4S 363.14706, found 369.10297.
rt for 15 min. methyl iodide (0.20 ll, 3.20 mmol) was added to the
mixture at rt, and then the reaction mixture was stirred at rt for
1 h. The reaction mixture was diluted with AcOEt, washed with
brine (2 times) and dried over anhydrous sodium sulfate. After
evaporation, the residue was triturated with hexane–AcOEt (1:1),
then the indissolved matter was collected by filtration and washed
with AcOEt to give the desired product (9h) (447 mg, 1.33 mmol, y.
88%).
5.1.12. tert-Butyl (E)-3-(5-((Z)-3-oxo-3,4-
dihydrobenzo[b][1,4]oxazin-2-ylidene-methyl)furan-3-
yl)acrylate (8f)
1H NMR (300 MHz, DMSO-d6) d: 8.07 (s, 1H), 8.06 (d, J = 7.2 Hz,
1H), 7.69 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 16.0 Hz, 1H), 7.49 (t,
J = 8.1 Hz, 1H), 7.39–7.36 (m, 1H), 7.28–7.25 (m, 1H), 7.18–7.14
(m, 2H), 6.87 (s, 1H), 6.59 (d, J = 16.0 Hz, 1H), 3.42 (s, 3H), 1.50
(s, 9H); IR (cmÀ1, KBr) 3051, 2984, 2941, 1708, 1673, 1640, 1593,
1505, 1473, 1422, 1384, 1323, 1292, 1450, 1154, 978, 857, 789,
741; HRMS (EI) calcd for C23H23NO4 377.16, found 377.16.
1H NMR (300 MHz, DMSO-d6) d: 11.24 (s, 1H), 8.18 (s, 1H),
7.64–7.61 (m, 1H), 7.47 (d, J = 15.8 Hz, 1H), 7.46 (s, 1H), 7.08–
6.97 (m, 3H), 6.63 (s, 1H), 6.55 (d, J = 16.0 Hz, 1H), 1.49 (s, 9H);
IR (cmÀ1, KBr) 3033, 2979, 2909, 2861, 1686, 1638, 1501, 1395,
1314, 1139, 975, 924, 815, 749; HRMS (EI) calcd for C20H19NO5
353.13, found 353.13.
5.1.13. tert-Butyl (E)-3-(5-((Z)-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidene-methyl) furan-2-yl)acrylate (8g)
1H NMR (300 MHz, DMSO-d6) d: 11.28 (s, 1H), 7.40–7.35 (m,
2H), 7.24 (d, J = 3.5 Hz, 1H), 7.13 (d, J = 3.5 Hz, 1H), 7.08–7.04 (m,
2H), 7.03–6.98 (m, 1H), 6.71 (s, 1H), 6.23 (d, J = 15.8 Hz, 1H), 1.48
(s, 9H); IR (cmÀ1, KBr) 3036, 2979, 2915, 1685, 1631, 1503, 1387,
1280, 1231, 1157, 1099, 847, 797, 749; HRMS (EI) calcd for
C20H19NO5 353.13, found 353.13.
5.1.17. tert-Butyl (E)-3-(5-((Z)-4-methyl-3-oxo-3,4-
dihydrobenzo[b][1,4]oxazin-2-ylidene-methyl) thiophen-2-
yl)acrylate (9i)
1H NMR (300 MHz, DMSO-d6) d: 7.72 (d, J = 15.8 Hz, 1H), 7.52
(d, J = 3.9 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 7.29–7.25 (m, 1H),
7.21–7.13 (m, 3H), 6.34 (d, J = 15.6 Hz, 1H), 3.41 (s, 3H), 1.49 (s,
9H); IR (cmÀ1, KBr) 1714, 1667, 1617, 1504, 1443, 1420, 1378,
1337, 1209, 1148, 1258, 1209, 1148, 962, 842, 788, 741; HRMS
(EI) calcd for C21H21NO4S 383.12, found 383.12.
5.1.14. tert-Butyl (E)-3-(3-formylphenyl)acrylate (13)
To a mixture of m-bromobenzaldehyde 6b (35 ml, 30.0 mmol),
palladium acetate (339 mg, 1.51 mmol) and tri(o-tolyl)phosphine
(913 mg, 3.00 mmol), N,N-dimethylformamide (130 ml), diisopro-
pylethylamine (60 ml, 345 mmol) and tert-butyl acrylate (20 ml,
137 mmol) were added under nitrogen atmosphere. The mixture
was stirred at 110 °C over night. The reaction mixture was cooled
to rt and filtered through Celite with AcOEt. The filtrate was
washed with brine and dried over anhydrous sodium sulfate. After
concentration in vacuo the resultant was collected by filtration
with hexane–AcOEt (1:1) and washed with AcOEt to give the de-
sired product (13) (5.17 g, 22.3 mmol, y. 87%).
5.1.18. (E)-3-(4-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)phenyl)acrylic acid (10a)
To 8a (6.80 g, 18.7 mmol), a dioxane solution of 4 M hydrogen
chloride (150 ml, 600 mmol) and chloroform (50 ml) were added
and stirred at rt over night. The reaction mixture was concentrated
in vacuo to give the desired product (10a) (6.78 g, 18.7 mmol, y.
quant.).
1H NMR (300 MHz, DMSO-d6) d: 12.43 (s, 1H), 11.21 (s, 1H),
7.93 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 16.0 Hz,
1H), 7.31 (q, J = 3.1 Hz, 1H), 7.09–7.04 (m, 2H), 7.01–6.99 (m,
1H), 6.81 (s, 1H), 6.57 (d, J = 16.0 Hz, 1H); IR (cmÀ1, KBr) 3033,
2979, 2909, 2853, 1686, 1630, 1596, 1214, 1110, 747; HRMS (EI)
calcd for C18H13NO4 307.300, found 307.085.
1H NMR (300 MHz, DMSO-d6) d: 10.03 (s, 1H), 8.25 (s, 1H), 8.05
(d, J = 7.7 Hz, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.68–7.61 (m, 2 H), 6.67

T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
705
5.1.19. (E)-3-(3-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
5.1.26. (E)-3-(5-((Z)-4-Methyl-3-oxo-3,4-dihydrobenzo[b]
ylidenemethyl)phenyl)acrylic acid (10b)
[1,4]oxazin-2-ylidene-methyl) thiophen-2-yl)acrylic acid (10i)
1H NMR (300 MHz, DMSO-d6) d: 8.32 (s, 1H), 7.76 (d, J = 15.8 Hz,
1H), 7.50 (dt, J = 9.8,3.1 Hz, 3H), 7.22 (tt, J = 16.2,5.4 Hz, 4H), 6.36
(d, J = 15.8 Hz, 1H), 3.41 (s, 3H); IR (cmÀ1, KBr) 3068, 2925, 1675,
1629, 1610, 1504, 1418, 1383, 1215, 743; HRMS (EI) calcd for
C17H13NO4S 327.06, found 327.06.
1H NMR (300 MHz, DMSO-d6) d: 12.48 (s, 1H), 11.20 (s, 1H), 8.08
(s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.66 (d,
J = 16.0 Hz, 1H), 7.50 (dd, J = 7.7, 7.7 Hz, 1H), 7.32 (t, J = 4.8 Hz,
1H), 7.07–7.04 (m, 2H), 7.02–6.97 (m, 1H), 6.82 (s, 1 H), 6.59 (d,
J = 16.1 Hz, 1H); IR (cmÀ1, KBr) 3040, 2979, 2905, 2861, 1688,
1632, 1503, 1437, 1390, 1281, 1227, 1110, 979, 742; HRMS (EI)
calcd for C18H13NO4 307.08, found 307.09.
5.1.27. Polymeric TFP 4-((E)-3-(4-((Z)-3-Oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)phenyl) acryl)benzoate
ester (11a)
Polymeric TFP ester 11cwas prepared by adding TFP resin (2.37 g,
calcd 1.35 mmol/g) to a polypropylene reaction vessel at rt. The resin
was swelled with N,N-dimethylformamide (30 ml) for 10 min with
mild agitation. Acid 10a (1.10 g, 3.50 mmol) was added to the resin
mixture and agitated gently until all of the acid dissolved. 4-Dimeth-
5.1.20. (E)-3-(2-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)phenyl)acrylic acid (10c)
1H NMR (300 MHz, DMSO-d6) d: 12.51 (s, 1H), 11.29 (s, 1H),
8.02 (d, J = 7.0 Hz, 1H), 7.85 (d, J = 15.8 Hz, 1H), 7.80 (d, J = 7.9 Hz,
1H), 7.51 (t, J = 7.1 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.12–6.98 (m,
4H), 6.98 (s, 1H), 6.46 (d, J = 15.8 Hz, 1H); IR (cmÀ1, KBr) 3036,
2980, 2902, 1587, 1634, 1503, 1476, 1399, 1315, 1279, 1216,
1112, 746; HRMS (EI) calcd for C18H13NO4 307.300, found 307.085.
ylaminopyridine (10 mg, 81.8 lmol) was added to the reaction. The
reaction was agitated for 5 min, then N,N-diisopropyl carbodiimide
(815 mg, 6.45 mmol) was added to the mixture. The polypropylene
reaction vessel was capped and agitated over night. The resin was
washed with N,N-dimethylformamide, THF and dichloromethane
and dried in vacuo to give desired Polymeric TFP ester 11a (3.11 g,
calcd 1.01 mmol/g). Percent loading 75%.
5.1.21. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)thiophen-3-yl)acrylic acid (10d)
1H NMR (300 MHz, DMSO-d6) d: 11.17 (s, 1H), 8.09 (s, 1H),
7.82 (s, 1H), 7.56 (d, J = 16.0 Hz, 1H), 7.25–7.23 (m, 1H), 7.08–
6.97 (m, 3H), 7.06 (s, 1H), 6.36 (d, J = 16.0 Hz, 1H); IR (cmÀ1
,
IR (cmÀ1, ATR) 3026, 2920, 1751, 1685, 1634, 1597, 1491, 1367,
1314, 1101, 992, 745, 698.
KBr) 3033, 2979, 2913, 2861, 1685, 1632, 1502, 1387, 1281,
1233, 1104, 744; HRMS (EI) calcd for C16H11NO4S 313.0409,
found 313.0424.
5.1.28. Polymeric TFP 4-((E)-3-(3-((Z)-3-oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)phenyl)acryl)benzoate
ester (11b)
5.1.22. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)thiophen-2-yl)acrylic acid (10e)
Percent loading 83%.
1H NMR (300 MHz, DMSO-d6) d: 12.40 (s, 1H), 11.23 (s, 1H),
7.76 (d, J = 15.6 Hz, 1H), 7.51 (d, J = 3.9 Hz, 1H), 7.46 (d, J = 3.9 Hz,
1H), 7.44–7.41 (m, 1H), 7.12 (s, 1H), 7.09–7.05 (m, 2H), 7.03–7.00
(m, 1H), 6.35 (d, J = 15.6 Hz, 1H); IR (cmÀ1, KBr) 3021, 2470,
1680, 1631, 1502, 1403, 1351, 1301, 1258, 1232, 1110, 959, 930,
849, 804, 746; HRMS (EI) calcd for C16H11NO4S 313.04, found
313.04.
5.1.29. Polymeric TFP 4-((E)-3-(2-((Z)-3-oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)phenyl)acryl)benzoate
ester (11c)
Percent loading 82%.
5.1.30. Polymeric TFP 4-((E)-3-(5-((Z)-3-oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)thiophen-3-
yl)acryl)benzoate ester (11d)
5.1.23. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)furan-3-yl) acrylic acid (10f)
Percent loading 76%.
1H NMR (300 MHz, DMSO-d6) d: 12.32 (s, 1H), 11.25 (s, 1H),
8.18 (s, 1H), 7.62–7.59 (m, 1H), 7.51 (d, J = 16.0 Hz, 1H), 7.46
(s, 1H), 7.10–6.97 (m, 3H), 6.64 (s, 1H), 6.55 (d, J = 15.8 Hz,
1H); IR (cmÀ1, KBr) 3143, 3084, 3027, 2909, 2853, 2624, 2519,
1683, 1640, 1504, 1407, 1280, 1260, 1218, 1150, 749; HRMS
(EI) calcd for
5.1.31. Polymeric TFP 4-((E)-3-(5-((Z)-3-oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)thiophen-2-yl)acryl)
benzoate ester (11e)
Percent loading 65%.
5.1.32. Polymeric TFP 4-((E)-3-(5-((Z)-3-oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)furan-3-yl)acryl)
benzoate ester (11f)
5.1.24. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)furan-2-yl) acrylic acid (10g)
1H NMR (300 MHz, DMSO-d6) d: 11.28 (s, 1H), 7.41 (d,
J = 15.6 Hz, 1H), 7.38–7.35 (m, 1H), 7.22 (d, J = 3.7 Hz, 1H), 7.11
(d, J = 3.5 Hz, 1H), 7.08–7.05 (m, 2H), 7.02–6.98 (m, 1H), 6.71 (s,
1H), 6.26 (d, J = 15.6 Hz, 1H), 3.57 (s, 1H); IR (cmÀ1, KBr) 3033,
2977, 2910, 2865, 2571, 1685, 1627, 1557, 1501, 1388, 1308,
1270, 1219, 1097, 1035, 965, 925, 849, 797; HRMS (EI) calcd for
C16H11NO5 297.064, found 279.064.
Percent loading 48%.
5.1.33. Polymeric TFP 4-((E)-3-(5-((Z)-3-oxo-3,4-dihydro-
benzo[b][1,4]oxazin-2-ylidene-methyl)furan-2-yl)acryl)
benzoate ester (11g)
Percent loading 65%.
5.1.34. Polymeric TFP 4-((E)-3-(3-((Z)-4-Methyl-3-oxo-3,4-
dihydrobenzo[b][1,4]oxazin-2-ylidenemethyl)phenyl)acryl)
benzoate ester (11h)
5.1.25. (E)-3-((Z)-3-(4-Methyl-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidene-methyl) phenyl)acrylic acid (10h)
1H NMR (300 MHz, DMSO-d6) d: 12.48 (s, 1H), 8.09 (s, 1H), 8.03
(d, J = 8.3 Hz, 1H), 7.68 (t, J = 3.9 Hz, 1H), 7.63 (s, 1H), 7.50 (t,
J = 7.7 Hz, 1H), 7.39–7.36 (m, 1H), 7.28–7.25 (m, 1H), 7.21–7.12
(m, 2H), 6.87 (s, 1H), 6.59 (d, J = 16.0 Hz, 1H), 3.42 (s, 3H); IR
(cmÀ1, KBr) 3072, 2977, 2829, 2702, 2590, 1674, 1631, 1599,
1511, 1419, 1376, 1312, 1384, 978, 746; HRMS (EI) calcd for
C19H15NO4 321.10, found 321.10.
Percent loading 74%.
5.1.35. (E)-3-(4-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide (4a)
The amide was prepared by adding TFP 4-benzoate ester 11a
(103 mg, 1.01 mmol/g) to a 5 ml polypropylene vessel. The resin
was swelled with N,N-dimethylformamide (3 ml) for 10 min with

706
T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
gentle agitation. 3-Aminomethylpyridine (16.8 mg, 155
l
mol) was
7.37 (dd, J = 7.7, 4.8 Hz, 1H), 7.25–7.21 (m, 1H), 7.10 (s, 1H),
7.08–7.01 (m, 3H), 6.57 (d, J = 15.6 Hz, 1H), 4.43 (d, J = 3.9 Hz,
2H); 13C NMR (125 MHz, DMSO-d6) d: 165.46, 156.31, 149.45,
148.70, 142.21, 141.13, 141.02, 137.87, 135.81, 135.47, 132.55,
131.48, 126.36, 124.33, 124.05, 123.71, 121.57, 116.60, 115.81,
105.87; IR (cmÀ1, KBr) 3024, 2960, 2900, 2842, 2750, 2148, 1629,
1560, 1502, 1436, 1374, 1275, 1233, 1104, 849, 796, 746; HRMS
(EI) calcd for C22H17N3O3S 403.09906, found 403.09866.
added, and the mixture was capped. The reaction was agitated over
night. The mixture was filtered and washed with N,N-dimethyl-
formamide and dichloromethane. The washes were collected and
evaporated in vacuo. The product 4a (45.3 mg, 114 lmol, y. 63%)
was obtained as a solid.
1H NMR (300 MHz, DMSO-d6) d: 11.21 (s, 1H), 8.78 (t, J = 5.7 Hz,
1H), 8.58 (s, 1H), 8.52 (d, J = 4.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H),
7.80 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 15.6 Hz,
1H), 7.46 (dd, J = 7.7, 5.1 Hz, 1H), 7.34–7.31 (m, 1H), 7.08–7.03
(m, 2H), 7.02–6.98 (m, 1H), 6.80 (s, 1H), 6.73 (d, J = 16.0 Hz, 1H),
4.46 (d, J = 5.9 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) d: 165.19,
156.03, 147.66, 146.99, 142.36, 140.35, 128.58, 136.53, 135.57,
134.43, 134.31, 130.13, 127.86, 125.36, 123.94, 123.52, 123.14,
122.06, 115.60, 115.56, 109.79, 109.76; IR (cmÀ1, KBr) 3285,
3192, 3140, 3052, 1678, 1647, 1322, 1591, 1551, 1503, 1422,
1378, 1336, 1319, 1223, 1110, 1033, 972, 763; HRMS (EI) calcd
for C24H19N3O3 397.14264, found 397.14249.
5.1.40. (E)-N-((2-Dimethylamino)ethyl)-3-(3-((Z)-3-oxo-3,4-
dihydrobenzo[b][1,4]oxazin-2-ylidenemethyl)-
phenyl)acrylamide (4f)
1H NMR (300 MHz, DMSO-d6) d: 11.20 (s, 1H), 8.13 (s, 1H), 8.02
(s, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.56–7.45 (m, 3H), 7.34 (d,
J = 9.2 Hz, 1H), 7.07–6.99 (m, 3H), 6.80 (s, 1H), 6.73 (d,
J = 15.8 Hz, 1H), 3.33–3.31 (m, 4H), 2.24 (s, 6H).
5.1.41. (Z)-2-(5-((E)-3-(4-(2-Hydroxyethyl)piperazin-1-yl)-3-
oxo-propenyl)fran-2-ylmethylene)-4H-benzo[b][1,4]oxazin-3-
one (4g)
5.1.36. (E)-3-(3-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide (4b)
1H NMR (300 MHz, DMSO-d6) d: 11.20 (s, 1H), 8.77 (t, J = 6.0 Hz,
1H), 8.55 (d, J = 1.7 Hz, 1H), 8.47 (dd, J = 4.8,1.7 Hz, 1H), 8.05 (s,
1H), 7.91 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.58–7.47 (m,
3H), 7.40–7.32 (m, 2H), 7.07–7.04 (m, 2H), 7.02–6.98 (m, 1H),
6.80 (s, 1H), 6.74 (d, J = 15.8 Hz, 1H), 4.45 (d, J = 5.9 Hz, 2H); 13C
NMR (125 MHz, DMSO-d6) d: 165.11, 156.00, 148.85, 148.07,
142.28, 140.30, 138.89, 135.19, 134.97, 133.85, 130.67, 129.27,
128.64, 127.08, 125.33, 123.48, 123.43, 123.19, 122.33, 115.74,
115.62, 109.77; IR (cmÀ1, KBr) 3281, 3033, 2980, 2898, 2861,
1690, 1640, 1542, 1503, 1398, 1243, 1111, 1032, 966, 855, 783,
744; HRMS (EI) calcd for C24H19N3O3 397.14, found 397.14.
1H NMR (500 MHz, DMSO-d6) d: 11.23 (s, 1H), 7.38–7.33 (m,
2H), 7.22 (d, J = 3.7 Hz, 1H), 7.09–7.03 (m, 3H), 7.01–6.99 (m,
2H), 6.73 (s, 1H), 4.45 (t, J = 5.4 Hz, 1H), 3.65–3.50 (m, 8H), 3.31
(br s, 2H), 2.42 (t, J = 6.1 Hz, 2H).
5.1.42. (E)-N-(4-(Morpholin-4-yl)phenyl)-3-(3-((Z)-4-methyl-3-
oxo-3,4-dihydrobenzo[b] [1,4]oxazin-2-ylidenemethyl)-
phenyl)acrylamide (4h)
1H NMR (300 MHz, DMSO-d6) d: 10.07 (s, 1H), 8.07 (s, 1H), 7.95
(d, J = 7.5 Hz, 1H), 7.64–7.58 (m, 4H), 7.52 (t, J = 7.8 Hz, 1H), 7.42
(dd, J = 7.1, 2.5 Hz, 1H), 7.29–7.26 (m, 1H), 7.21–7.14 (m, 2H),
6.94 (d, J = 9.0 Hz, 2H), 6.87 (s, 1H), 6.86 (d, J = 15.6 Hz, 1H), 3.74
(t, J = 4.7 Hz, 4H), 3.43 (s, 3H), 3.07 (t, J = 4.7 Hz, 4H).
5.1.37. (E)-3-(2-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide (4c)
1H NMR (300 MHz, DMSO-d6) d: 8.75 (t, J = 6.0 Hz, 1H), 8.52 (d,
J = 1.8 Hz, 1H), 8.45 (dd, J = 4.8, 1.7 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H),
7.76 (d, J = 15.8 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.61 (d, J = 6.8 Hz,
1H), 7.45 (t, J = 7.1 Hz, 1H), 7.38–7.33 (m, 2H), 7.06 (d, J = 7.9 Hz,
1H), 6.98–6.92 (m, 3H), 6.93 (s, 1H), 6.59 (d, J = 15.6 Hz, 1H), 4.41
(d, J = 5.9 Hz, 2H); IR (cmÀ1, KBr) 3282, 3066, 2977, 2894, 2845,
2762, 1696, 1652, 1546, 1503, 1428, 1376, 1265, 1228, 1110,
1033, 761, 743; HRMS (EI) calcd for C24H19N3O3 397.14264, found
397.14243.
5.1.43. (E)-N-((2-Dimethylamino)ethyl)-3-(3-((Z)-4-methyl-3-
oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-ylidenemethyl)-
thiophen-2-yl)acrylamide (4i)
1H NMR (300 MHz, DMSO-d6) d: 8.05 (t, J = 5.5 Hz, 1H), 7.57 (d,
J = 15.4 Hz, 1H), 7.46 (d, J = 3.9 Hz, 1H), 7.38 (d, J = 3.9 Hz, 1H),
7.32–7.26 (m, 2H), 7.22–7.17 (m, 3H), 6.56 (d, J = 15.6 Hz, 1H),
3.41 (s, 3H), 3.28 (q, J = 6.2 Hz, 2H), 2.35 (t, J = 6.4 Hz, 2H), 2.18
(s, 6H).
5.1.44. (Z)-2-(3-((E)-4-Methyl-3-oxo-3-(pyrrolidin-1-
yl)propenyl)thiophen-2-ylmethylene)-4H-benzo[b][1,4]oxazin-
3-one (4j)
5.1.38. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)thiophen-3-yl)-N-(pyridin-3-ylmethyl)
acrylamide (4d)
1H NMR (300 MHz, DMSO-d6) d: 7.65 (d, J = 15.2 Hz, 1H), 7.49–
7.47 (m, 2H), 7.46–7.43 (m, 1H), 7.29–7.25 (m, 1H), 7.22–7.14 (m,
3H), 6.78 (d, J = 15.2 Hz, 1H), 3.67 (t, J = 6.8 Hz, 2H), 3.41 (t,
J = 6.6 Hz, 5H), 1.93 (dd, J = 12.9, 6.7 Hz, 2H), 1.83 (dd, J = 13.1,
6.3 Hz, 2H).
1H NMR (300 MHz, DMSO-d6) d: 11.15 (s, 1H), 8.68 (t, J = 6.1 Hz,
1H), 8.52 (s, 1H), 8.46 (d, J = 4.2 Hz, 1H), 7.95 (s, 1H), 7.69 (t,
J = 3.8 Hz, 2H), 7.45 (d, J = 15.8 Hz, 1H), 7.36 (dd, J = 7.9, 4.8 Hz,
1H), 7.25–7.22 (m, 1H), 7.11 (s, 1H), 7.07–7.05 (m, 2H), 7.01–6.97
(m, 1H), 6.50 (d, J = 15.6 Hz, 1H), 4.41 (d, J = 5.9 Hz, 2H); 13C NMR
(150 MHz, DMSO-d6) d: 165.30, 155.76, 148.85, 148.12, 140.41,
140.37, 137.51, 136.79, 135.18, 132.99, 130.08, 127.19, 125.44,
5.1.45. (Z)-4-Methyl-2-(3-((E)-3-(4-(2-hydroxyethyl)piperazin-
1-yl)-3-oxo-propenyl)thiophen-2-ylmethylene)-4H-
benzo[b][1,4]oxazin-3-one (4k)
123.68, 123.48, 123.26, 121.68, 115.75, 115.36, 105.16; IR (cmÀ1
,
1H NMR (300 MHz, DMSO-d6) d: 7.68 (d, J = 15.0 Hz, 1H), 7.50–
7.46 (m, 3H), 7.29–7.25 (m, 1H), 7.22–7.17 (m, 3H), 7.05 (d,
J = 15.2 Hz, 1H), 4.47 (t, J = 5.0 Hz, 1H), 3.69–3.50 (m, 8H), 3.41 (s,
3H), 2.47–2.40 (m, 4H).
KBr) 3149, 3054, 2982, 2914, 1688, 1626, 1560, 1503, 1434,
1369, 1317, 1280, 1260, 1236, 1213, 1101, 967, 743; HRMS (EI)
calcd for C22H17N3O3S 403.0991, found 403.0988.
5.1.39. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)thiophen-2-yl)-N-(pyridin-3-
5.1.46. (E)-3-(5-((Z)-4-Methyl-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidenemethyl)thiophen-2-yl)-N-(pyridin-3-
ylmethyl)acrylamide (4l)
ylmethyl)acrylamide (4e)
1H NMR (300 MHz, DMSO-d6) d: 8.78 (s, 1H), 8.54 (d, J = 2.2 Hz,
1H), 8.48 (d, J = 4.8 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.64 (d,
J = 15.4 Hz, 1H), 7.44 (d, J = 3.7 Hz, 1H), 7.40 (d, J = 3.3 Hz, 1H),
1H NMR (300 MHz, DMSO-d6) d: 8.59 (d, J = 2.0 Hz, 1H), 8.55
(dd, J = 4.9, 1.6 Hz, 1H), 7.80 (d, J = 15.2 Hz, 1H), 7.71 (td, J = 7.9,
2.0 Hz, 1H), 7.31–7.00 (m, 7H), 6.30 (d, J = 15.2 Hz, 1H), 6.01 (t,

T. Honda et al. / Bioorg. Med. Chem. 17 (2009) 699–708
707
J = 6.1 Hz, 1H), 4.61 (d, J = 5.9 Hz, 3H), 3.48 (d, J = 4.8 Hz, 3H); 13C
NMR (125 MHz, DMSO-d6) d: 164.76, 155.30, 148.85, 148.13,
141.61, 140.89, 139.68, 137.21, 135.18, 130.70, 127.13, 123.80,
123.60, 123.44, 120.80, 115.33, 115.18, 106.03; IR (cmÀ1, KBr)
3434, 3248, 3042, 1665, 1628, 1560, 1504, 1423, 1384, 1338,
1222, 745; HRMS (EI) calcd for C23H19N3O3S 417.11, found 417.12.
(m, 3H), 7.01–7.00 (m, 1H), 6.76 (d, J = 15.3 Hz, 1H), 6.73 (s, 1H),
3.64 (t, J = 6.9 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 1.93 (tt, J = 6.9,
6.9 Hz, 2H), 1.82 (tt, J = 6.9, 6.9 Hz, 2H).
5.2. X-ray crystallographic analysis
A yellow block crystal of C23H19N3O3S having approximate
dimensions of 0.66 Â 0.10 Â 0.09 mm was mounted on a glass fi-
ber. All measurements were made on a Rigaku RAXIS RAPID imag-
5.1.47. (E)-3-(3-((Z)-4-Methyl-3-oxo-3,4-dihydrobenzo[b]
[1,4]oxazin-2-ylidenemethyl)thiophen-2-yl)-N-(pyridin-4-
ylmethyl)acrylamide (4m)
ing plate area detector with graphite monochromated MoK
radiation.
a
1H NMR (300 MHz, DMSO-d6) d: 8.75 (t, J = 6.1 Hz, 1H), 8.52 (dd,
J = 4.5, 1.6 Hz, 2H), 7.65 (d, J = 15.4 Hz, 1H), 7.48 (d, J = 3.9 Hz, 1H),
7.42 (d, J = 3.9 Hz, 1H), 7.31–7.14 (m, 7H), 6.59 (d, J = 15.4 Hz, 1H),
4.44 (d, J = 6.1 Hz, 2H), 3.41 (s, 3H).
5.2.1. Summary of X-ray crystallographic analysis of compound
4l
Formula: C23H19N3O3S; formula weight: 417.48; crystal color,
habit: yellow, block; crystal system: monoclinic; space group:
P21/n (#14); lattice constants: a = 7.7010 Å, b = 22.3165(12) Å,
5.1.48. (E)-3-(3-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)phenyl)-N-(pyridin-2-ylmethyl)acrylamide (4n)
1H NMR (300 MHz, DMSO-d6) d: 11.20 (s, 1H), 8.80 (t, J = 5.9 Hz,
1H), 8.53 (d, J = 4.0 Hz, 1H), 8.07 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H),
7.78 (td, J = 7.77 Hz, 1H), 7.57–7.47 (m, 3H), 7.37–7.26 (m, 3H),
7.07–6.98 (m, 3H), 6.82 (d, J = 15.6 Hz, 1H), 6.81 (s, 1H), 4.52 (d,
J = 6.1 Hz, 2H).
c = 11.8450(6) Å,
b = 95.4915(14)°,
volume = 2026.33(18) Å3;
Z = 4; density (calcd): 1.368 g/cm3; residual R1 = 0.0344,
wR2 = 0.0684.
5.3. Kinase assay
5.1.49. (E)-N-((2-Dimethylamino)ethyl)-3-(5-((Z)-3-oxo-3,4-
dihydrobenzo[b][1,4]oxazin-2-ylidenemethyl)thiophen-3-
yl)acrylamide (4o)
KDR and ABL inhibitory activity assays were performed by the
ELISA method by measuring the phosphorylation level of biotinyl-
ated peptide including Tyr in the sequence. Kinase reactions were
carried out in a streptavidin-coated 96-well plate (DelfiaÒ Strepta-
vidin Microtitration Strips, PerkinElmerÒ 4009-0010) and con-
sisted of baculovirus-expressed kinase (KDR or ABL), 250 nM
substrate, 15 mM Tris–HCl (pH 7.5), 0.01% Tween 20, 2 nM DTT,
1H NMR (500 MHz, DMSO-d6) d: 11.18 (s, 1 H), 8.01 (t, J = 5.7 Hz,
1H), 7.93 (s, 1H), 7.67 (d, J = 1.5 Hz, 1H), 7.39 (d, J = 15.6 Hz, 1H),
7.27–7.24 (m, 1H), 7.11 (s, 1H), 7.09–7.03 (m, 2H), 7.02–6.99 (m,
1H), 6.51 (d, J = 15.6 Hz, 1H), 3.26 (t, J = 6.1 Hz, 2H), 2.33 (t,
J = 6.7 Hz, 2H), 2.16 (s, 6H).
100
rt and stopped by aspirating and washing them three times with
200 L of wash buffer (50 mM Tris–HCl (pH 7.5) containing
150 mM NaCl and 0.02% Tween 20). Then 200 M of blocking buf-
lM ATP and 40 mM Mg. Reactions were incubated for 1 h at
5.1.50. (Z)-2-(4-((E)-3-Oxo-3-(pyrrolidin-1-yl)propenyl)-
thiophen-2-ylmethylene)-4H-benzo[b][1,4]oxazin-3-one (4p)
1H NMR (500 MHz, DMSO-d6) d: 11.17 (s, 1H), 8.03 (s, 1H), 7.86
(s, 1H), 7.46 (d, J = 15.4 Hz, 1H), 7.27–7.24 (m, 1H), 7.09–7.04 (m,
2H), 7.03–6.99 (m, 1H), 6.84 (d, J = 15.4 Hz, 1H), 3.63 (t,
J = 6.7 Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H), 1.93 (tt, J = 6.8, 6.8 Hz, 2H),
1.81(tt, J = 6.8, 6.8 Hz, 2H).
l
l
fer (wash buffer containing 0.1% BSA) was added to each well in or-
der to block the plate. After incubating the plate for 30 min, the
blocking buffers were discarded and 100 lM of Detection Mixture
(HRP-conjugated anti-phosphotyrosine (pY) antibody was added
to each well. After incubating the plate for 30 min, detection mix-
tures were discarded and the plate was washed three times with
250 lL of the Wash Buffer. To each well 100 lL of Color Reagent
(TMBE-1000; TMB Peroxidase substrate elisa, Moss, Inc.) was
added and the plate was incubated for 5 min. To each well
100 lL of 0.1 M H2SO4 was then added and the plate was tapped
gently to ensure thorough mixing. The absorbance of each well
was measured immediately using a microplate reader set to
450 nM. Staurosporine was used as a positive control in both ki-
nase assay.
5.1.51. (E)-3-(5-((Z)-3-Oxo-3,4-dihydrobenzo[b][1,4]oxazin-2-
ylidenemethyl)thiophen-2-yl)-N-(pyridin-2-ylmethyl)
acrylamide (4q)
1H NMR (500 MHz, DMSO-d6) d: 11.19 (s, 1H), 8.70 (t, J = 6.0 Hz,
1H), 8.53 (dq, J = 4.7, 0.9 Hz, 1H), 7.78 (td, J = 7.8, 1.8 Hz, 1H), 7.64
(d, J = 15.3 Hz, 1H), 7.46 (d, J = 3.7 Hz, 1H), 7.40 (d, J = 4.0 Hz, 1H),
7.33 (d, J = 7.9 Hz, 1H), 7.30–7.24 (m, 2H), 7.12 (s, 1H), 7.09–7.06
(m, 2H), 7.04–7.01 (m, 1H), 6.64 (d, J = 15.6 Hz, 1H), 4.50 (d,
J = 5.5 Hz, 2H).
Acknowledgments
5.1.52. (E)-N-(4-(Morpholin-4-yl)phenyl)-3-(5-((Z)-3-oxo-3,4-
dihydrobenzo[b][1,4]oxazin-2-ylidenemethyl)thiophen-2-
yl)acrylamide (4r)
We greatly thank Shouhei Katao of Nara Institute of Science and
Technology for X-ray crystallographic analysis of compound 4l and
Eiko Nishioka the Research and Development Center at Santen
Pharmaceutical Co. Ltd for measurements of non-decoupling 13C
NMR of compounds 4a–d and, 6f and 6g. Also, we thank Dr. Koushi
Fujisawa and Dr. Ken-ichi Fujimura of the Research and Develop-
ment Center at Santen Pharmaceutical Co. Ltd for their helpful
suggestions.
1H NMR (500 MHz, DMSO-d6) d: 11.20 (s, 1H), 10.02 (s, 1H), 7.71
(d, J = 15.3 Hz, 1H), 7.58 (d, J = 9.2 Hz, 2H), 7.48 (d, J = 3.7 Hz, 1H),
7.46–7.41 (m, 1H), 7.28–7.25 (m, 1H), 7.13 (s, 1H), 7.11–7.07 (m,
1H), 7.04–7.01 (m, 1H), 6.93 (d, J = 8.9 Hz, 2H), 6.66 (d,
J = 15.3 Hz, 1H), 3.75–3.68 (m, 4H), 3.07–3.01 (m, 4H); IR (cmÀ1
KBr) 3315, 3030, 2972, 2900, 2855, 1675, 1627, 1523, 1389,
1352, 1266, 1230, 1119; HRMS (EI) calcd for C26H23N3O4S 473.14,
found 473.15.
,
References and notes
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ylmethylene)-4H-benzo[b][1,4]oxazin-3-one (4s)
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708
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TM
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BIOSCIENCE Co. Ltd.