Design, synthesis, and preliminary biological evaluation of 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.03.013

We synthesized a series of novel small molecules, 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives, by tandem reduction-oxirane opening of 2-nitroaroxymethyloxiranes in moderate or excellent yields. We investigated the effects of all of the compounds on HUVEC apoptosis and A549 cell growth. The results showed that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine was the most effective small molecule in promoting HUVEC apoptosis and inhibiting A549 cell proliferation, but 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine could remarkably inhibit HUVEC apoptosis and might induce the formation of microvessel.

Full text of DOI:10.1016/j.bmcl.2006.03.013

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2862–2867
Design, synthesis, and preliminary biological evaluation of
2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives
Pei-Fu Jiao,a Bao-Xiang Zhao,a,* Wei-Wei Wang,b Qiu-Xia He,b Mao-Sheng Wan,a
Dong-Soo Shinc and Jun-Ying Miaob,*
aInstitute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
bInstitute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
cDepartment of Chemistry and Research Institute of Natural Sciences, Changwon National University,
Changwon 641-773, Republic of Korea
Received 24 January 2006; revised 3 March 2006; accepted 6 March 2006
Available online 24 March 2006
Abstract—We synthesized a series of novel small molecules, 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives, by tandem
reduction-oxirane opening of 2-nitroaroxymethyloxiranes in moderate or excellent yields. We investigated the effects of all of the
compounds on HUVEC apoptosis and A549 cell growth. The results showed that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-
benzoxazine was the most effective small molecule in promoting HUVEC apoptosis and inhibiting A549 cell proliferation, but
6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine could remarkably inhibit HUVEC apoptosis and might induce the
formation of microvessel.
Ó 2006 Elsevier Ltd. All rights reserved.
The use of small molecules to affect biological phenom-
ena, also known as chemical genetics, has made a signif-
icant impact in diverse areas of biology.1–3 The design of
the library is the first and a very crucial step in the for-
ward chemical genetics process, and this step determines
the success of the library.4 In addition to natural prod-
ucts library and known drug-like library, the design
and synthesis of novel small molecules library with
valuable chemical diversity has been shown to be
challenging.5
promoters and inhibitors will be very useful for us to
provide new insights into the mechanisms of angiogenesis,
and to advance the development of new antiangiogenic
and anti-cancer agents.7 Cancer is the second leading
cause of mortality in developed countries. The discovery
and development of new treatments is urgently needed
due to problems with currently available treatments,
such as toxicities and drug-resistance. It has been report-
ed that the antitumor efficacy of chemotherapeutic
agents correlated with their growth-inhibiting, differenti-
ation-inducing or apoptosis-inducing abilities.8
Vascular endothelial cells (VECs) play important roles
in angiogenesis that is critical for normal physiological
processes such as embryonic development and wound
repair. Angiogenesis also promotes tumor growth. The
regulation of endothelial cell apoptosis was as a poten-
tial therapeutic target to blood vessel disease.6 There-
fore, the current research focuses on the utilization of
chemical genetics to discover novel endothelial cell
apoptosis promoters or inhibitors. The new candidate
In our effort to discover and develop apoptosis
inducers as potential new anti-cancer agents, we have
identified several classes of molecules as novel
apoptosis inducers, including safrole oxide, 1-alkoxy-
3-(30,40-methylenedioxy)phenyl-2-propanol, c-lactone,
and morpholinone derivatives.9–20 In an ongoing study
in our laboratory on the design and synthesis of the
small molecule, we are interested in extending our
small molecules library to meet the requirement of
our research.
Keywords: 2,3-Dihydro-3-hydroxymethyl-1,4-benzoxazines; Vascular
endothelial cells; A549 cells; Apoptosis.
The 2,3-dihydro-1,4-benzodioxine system has been
widely used as a substructure of several biologically
interesting agents and hence, a variety of reports have
been presented for their synthesis and biological
*
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531
88564464; e-mail addresses: bxzhao@sdu.edu.cn; miaojy@sdu.
edu.cn
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.013

P.-F. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2862–2867
2863
evaluation of compounds including this ring.21–25 The
2,3-dihydro-1,4-benzoxazine derivatives, replacement
of the one oxygen atom by nitrogen in 2,3-dihydro-
1,4-benzodioxine, have been synthesized so far and var-
ious pharmacological activities have been reported with
this class of molecules.26–28 However, to the best of our
knowledge, only few of 2,3-dihydro-3-substituted-1,4-
benzoxazine derivatives, especially 2,3-dihydro-3-
hydroxymethyl-1,4-benzoxazine derivatives, have been
described in the literature.29–34
Effects of the compounds 3a–g on the morphology41 of
HUVEC. Morphological changes are associated with
the physiological and pathological processes in
HUVECs. When the cells detach from the culture dish
bottom and become round, they will enter into apopto-
sis processes.39 If HUVECs elongate and bend into
rings, they will form microvessels.42 In our experiments,
we found that compounds 3a–f first promoted the
detachment of HUVECs from the dish bottom and then
triggered them becoming round and forming apoptotic
bodies (Figs. 2D and E). Compound 3g induced
HUVEC to elongate and form into loop-like structures
(Fig. 2F). The data suggested that compound 3g might
be capable of promoting the formation of microvessel.
Herein, we report the discovery of 2,3-dihydro-3-hy-
droxymethyl-1,4-benzoxazine derivatives and the find-
ings of their biological activities in controlling
HUVEC apoptosis and A549 cell growth.
Effects of compounds 3a–g on the viability of A549 cells.
The data obtained by MTT assay showed that exposure
of A549 cells to compound 3c 400 lM for 24 h resulted
in cell viability decrease from 100% to 77.8% (p < 0.05)
(Fig. 3A). When the exposure continued on to 48 h,
compared with the vehicle group, cell viability reduced
more significantly from 100% to 59.5% (p < 0.01)
(Fig. 3A). If the cells were treated with compound 3c
200 lM for 48 h, cell viability could be also decreased
to 72.6% (p < 0.01) (Fig. 3A). The data suggested that
compound 3c inhibited cell growth in a dose dependent
manner from 200 to 400 lM. Compound 3d also obvi-
ously inhibited the cell growth at 200 lM after 48 h of
the treatment and its growth inhibitory effect was more
obvious (p < 0.05) at 400 lM (Fig. 3B). When A549 cells
were exposed to compound 3f at 50 lM for 48 h or
200 lM for 24 and 48 h, the viability of the cells was
decreased obviously (p < 0.05) (Fig. 3C). When the cells
were treated with compound 3f 400 lM for 24 or 48 h,
the cell growth was remarkably suppressed (p < 0.01).
Surprisingly, the apoptosis-promoting activity of
compound 3f at a concentration of 50 lM was higher
than that at 100 lM (Fig. 3C). The results showed that
the apoptosis-promoting activity of a compound was
not always linear to concentration. Compounds 3a, 3b,
3e and 3g had no effect on A549 cell growth
(p > 0.05). Taken together, the results showed that
compounds 3c, 3d, and 3f had obviously inhibitory
effects on A549 cell growth at 200 and 400 lM, but
the compounds 3a, 3b, 3e, and 3g had no effects on
Chemistry. The method used to synthesize the 2,3-dihy-
dro-3-hydroxymethyl-1,4-benzoxazine derivatives (3) is
shown in Scheme 1. Substituted 2-nitrophenoxymethyl-
oxiranes (1) were prepared according to the known
method.35 The tandem reduction-oxirane opening of
substituted 2-nitrophenoxymethyloxiranes (1) in the
presence of iron powder and acid gave substituted
2,3-dihydro-3-hydroxymethyl-1,4-benzoxazines (3) in
moderate or excellent yields. All the seven synthesized
compounds gave satisfactory spectral data. Representa-
1
tively, the structure of 3a was confirmed by H NMR
13
and C NMR data,36 which showed the presence of
two CH2–O signals at 75.2 and 68.4 ppm, and a
CH–N signal at 52.2 ppm (assigned to C-3), confirming
the formation of only one regioisomer.37 The one-pot
synthesis provides a novel and facile method of prepar-
ing the 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine
derivatives.
Effects of the compounds 3a–g on HUVEC apoptosis. In
order to evaluate the effects of the compounds on
HUVEC38 apoptosis, we used the given model of
HUVEC apoptosis induced by deprivation of growth
factors.39,40 The results showed that compounds 3a–f
promoted HUVEC apoptosis at the concentrations of
25–200 lM to
a certain extent. Among the six
compounds, the most effective one was compound 3f
(Figs. 1A–F). Contrarily, compound 3g inhibited the
apoptosis of HUVECs from 50 to 200 lM (Fig. 1G).
R1
R1
R1
O
Fe/AcOH
EtOH/H2O
O
O
O
O
OH
R2
N
R2
NO2
R2
NH2
reflux
H
1
3
2
1a: R1 = R2 = H
3a: R1 = R2 = H
1b: R1 = H, R2 = CH3
1c: R1 = H, R2 = C(CH3)3
1d: R1 = H, R2 = Cl
1e: R1 = Cl, R2 = H
1f : R1 = R2 = Cl
3b: R1 = H, R2 = CH3
3c: R1 = H, R2 = C(CH3)3
3d: R1 = H, R2 = Cl
3e: R1 = Cl, R2 = H
3f : R1 = R2 = Cl
1g: R1 = H, R2 = NO2
3g: R1 = H, R2 = NH2
Scheme 1. Synthesis of 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives.

2864
P.-F. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2862–2867
120
100
80
60
40
20
0
A
B
120
Compound 3a
24h
48h
Compound
*
24h
48h
3b
100
80
60
40
20
0
*
**
**
**
**
**
**
normal
0
DMSO
25
50
100
200
normal
0
DMSO 25
50
100
200
Concentration ( M)
Concentration ( M)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
D
C
24h
48h
Compound 3c
Compound 3d
24h
48h
*
*
*
**
**
**
**
**
**
**
**
**
**
**
normal
0
DMSO
25
50
100
200
normal
0
DMSO 25
50
100
200
Concentration ( M)
Concentration ( M)
120
100
E
F
120
100
80
60
40
20
0
24h
48h
Compound 3e
Compound 3f
24h
48h
80
60
40
20
0
**
**
**
**
**
**
**
**
**
**
**
normal
0
DMSO 25
50
100
200
normal
0
DMSO
25
50
100
200
Concentration ( M)
Concentration ( M)
120
G
Compound 3g
24h
48h
100
80
60
40
20
0
**
**
**
**
0
normal
DMSO 25
50
100
200
0
Concentration ( M)
Figure 1. Effects of compounds 3a–g on apoptosis of HUVECs. Data are means SE from three independent experiments. ( P < 0.05 vs the control
*
group; P < 0.01 vs the control group).
**

P.-F. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2862–2867
2865
Figure 2. Morphology image of HUVEC treated with compounds 3d,
3f, and 3g (200 lM) for 24 h. (A) normal; (B) control; (C) DMSO; (D)
3d; (E) 3f; and (F) 3g.
Figure 4. Morphology image of A549 treated with compounds 3c, 3d,
and 3f (200 lM) for 48 h. (A) DMSO; (B) 3c; (C) 3d; and (D) 3f.
the cell growth. Among compounds 3c, 3d and 3f,
compound 3f was the most effective one.
4B and D, when exposed to compound 3c and 3f
200 lM for 48 h, A549 cells became slender and longer,
the effect of compound 3f was much stronger than that
of 3c. The data suggested that compounds 3c and 3f not
only could inhibit A549 cell growth, but also might
induce the cell differentiation to type I lung epithelial
cells in morphology. When A549 cells were treated with
compound 3d, the cells vacuolated gradually as the
Effects of the compounds 3a–g on the morphology of A549
cells. Concomitant with cell growth inhibition, com-
pounds 3c, 3d, and 3f induced the changes of A549 cell
morphology. Compounds 3a, 3b, 3e, and 3g had no
effects on the cell morphology. As shown in Figures
140
140
120
100
80
A
B
Compound 3d
24h 48h
24h 48h
Compound 3c
120
100
80
60
40
20
0
60
40
20
0
ctr
DMSO 50
100
200
400
ctr
DMSO
50
100
200
400
Concentration (µM)
Concentration (µM)
140
120
100
80
C
24h
48h
Compound 3f
60
40
20
0
ctr DMSO
50
100
200
400
Concentration (µM)
Figure 3. Inhibition of compounds 3c, 3d, and 3f on A549 cell growth. Data are means SE from three independent experiments. ( P < 0.05 vs the
*
control group; P < 0.01 vs the control group).
**

2866
P.-F. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2862–2867
18. Su, L.; Zhao, J.; Zhao, B.-X.; Miao, J.-Y.; Yin, D.-L.;
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In summary, we have described a facile approach to
prepare 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine
derivatives by tandem reduction-oxirane opening of
2-nitroaroxymethyloxiranes, and we found two very
interesting compounds. 6,8-Dichloro-2,3-dihydro-3-hy-
droxymethyl-1,4-benzoxazine 3f was the most effective
small molecule in promoting HUVEC apoptosis
and inhibiting A549 cell proliferation. 6-Amino-2,3-
dihydro-3-hydroxymethyl-1,4-benzoxazine 3g could
remarkably inhibit HUVEC apoptosis and might induce
the formation of microvessel. The findings lead us
to find their targets and to investigate the mechanisms
of the small molecules acting in controlling cell
proliferation, differentiation and apoptosis in our next
researches.
28. Sanchez, I.; Pujol, M. D. Tetrahedron 1999, 55, 5593.
29. Zhou, Y.-G.; Yang, P.-Y.; Han, X.-W. J. Org. Chem.
2005, 70, 1679.
30. Yang, S.-C.; Lai, H.-C.; Tsai, Y.-C. Tetrahedron Lett.
2004, 45, 2693.
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M. L. J. Org. Chem. 2000, 65, 2847.
Acknowledgments
32. Ciske, F. L.; Barbachyn, M. R.; Genin, M. J.; Grega, K.
C.; Lee, C. S.; Dolak, L. A.; Seest, E. P.; Watt, W.;
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Bioorg. Med. Chem. Lett. 2003, 13, 4235.
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Romeo, M. R.; Massarelli, P.; Teramoto, S.; Fujiki, H.;
Mori, T. Eur. J. Med. Chem. 1999, 34, 903.
This study was supported by the Foundation of the
Ministry of Education (104112) and the Natural Science
Foundation of Shandong Province (Z2002D05).
34. Largeron, M.; Lockhart, B.; Pfeiffer, B.; Fleury, M.-B.
J. Med. Chem. 1999, 42, 5043.
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A.; Abad, J. L.; Claveria, C.; Gomez, L.; Bernad, A.;
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Vinella, A.; Prevosto, C.; Dell’Erba, C.; Petrillo, G.; Novi,
M. Invest. New Drugs 2004, 22, 359.
9. Du, A.-Y.; Zhao, B.-X.; Yin, D.-L.; Zhang, S.-L.; Miao,
J.-Y. Bioorg. Med. Chem. Lett. 2006, 16, 81.
10. Zhao, J.; Miao, J.-Y.; Zhao, B.-X.; Zhang, S.-L. FEBS
Lett. 2005, 579, 5809.
36. 2,3-Dihydro-3-hydroxymethyl-[1,4]benzoxazine (3a): light
yellow solid, mp 97–99 °C; IR (KBr) m: 3360, 3282, 2960,
2925, 1501, 1289, 1059 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d: 3.19 (dd, J = 2.4, 12.9 Hz, 1H), 3.23 (br s, 2H), 3.36 (dd,
J = 4.8, 12.9 Hz, 1H), 3.88 (dd, J = 2.0, 12.8 Hz, 1H),
3.91–3.97 (m, 1H), 4.26 (dd, J = 3.8, 12.8 Hz, 1H), 6.76
(dd, J = 1.7, 7.6 Hz, 1H), 6.83 (td, J = 1.7, 7.6 Hz, 1H),
6.90 (td, J = 1.5, 7.6 Hz, 1H), 7.0 (dd, J = 1.5, 7.6 Hz, 1H);
13C NMR (CDCl3, 100 MHz) d: 52.2 (CH, C-3), 68.4
(CH2, CH2–O), 75.2 (CH2, CH2–O), 119.7 (CH, C-5),
121.8 (CH, C-8), 122.0 (CH, C-7), 123.9 (CH, C-6), 141.8
(C, C-10), 150.5 (C, C-9); EIMS (m/z, %): 165 (M+, 54),
121 (56), 120 (100), 93 (15), 65 (15); Anal. Calcd for
C9H11NO2: C, 65.44; H, 6.71; N, 8.48. Found: C, 65.16; H,
6.76; N, 8.23.
2,3-Dihydro-3-hydroxymethyl-6-methyl-[1,4]benzoxazine
(3b): white solid, mp 122–123 °C; IR (KBr) m: 3361, 3272,
2952, 2920, 1520, 1305, 1289, 1066 cmꢀ1 1H NMR
;
(CDCl3, 400 MHz) d: 2.24 (s, 3H), 3.14 (dd, J = 1.9,
12.8 Hz, 1H), 3.28 (br s, 2H), 3.37 (dd, J = 3.9, 12.8 Hz,
1H), 3.80 (dd, J = 1.5, 12.3 Hz, 1H), 3.89–3.95 (m, 1H),
4.25 (dd, J = 2.7, 12.3 Hz, 1H), 6.60 (s, 1H), 6.64 (d,
J = 8.0 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H); EIMS (m/z, %):
179 (M+, 48), 135 (54), 134 (100), 107 (13), 77 (13); Anal.
Calcd for C10H13NO2: C, 67.02; H, 7.31; N, 7.82. Found:
C, 66.80; H, 7.36; N, 7.49.
11. Zhao, J.; Miao, J.-Y.; Zhao, B.-X.; Zhang, S.-L.; Yin,
D.-L. Vasc. Pharmacol. 2005, 43, 69.
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Med. Chem. 2005, 13, 4176.
13. Du, A.-Y.; Zhao, B.-X.; Zhang, S.-L.; Miao, J.-Y. Exp.
Lung Res. 2004, 30, 1.
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C.-Q. Vasc. Pharmacol. 2003, 40, 183.
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J.-Y. Chin. J. Org. Chem. 2003, 23, 1026.
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J.-Y. Endothelium 2004, 11, 267.
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S.-L. Bioorg. Med. Chem. 2006, 14, 2438.
2,3-Dihydro-3-hydroxymethyl-6-tert-butyl-[1,4]benzox-
azine (3c): white solid, mp 152–154 °C; IR (KBr) m: 3441,
1
3342, 2961, 2924, 1520, 1491, 1306, 1258, 1052 cmꢀ1; H
NMR (CDCl3, 400 MHz) d: 1.27 (s, 9H), 3.18 (dd, J = 2.2,
12.8 Hz, 1H), 3.36 (br s, 2H), 3.39 (dd, J = 4.7, 12.8 Hz,
1H), 3.84 (dd, J = 1.9, 12.3 Hz, 1H), 3.89–3.97 (m, 1H),

P.-F. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2862–2867
2867
4.25 (dd, J = 3.7, 12.3 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H),
6.86 (dd, J = 2.2, 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H);
EIMS (m/z, %): 221 (M+, 67), 206 (24), 177 (64), 162 (100),
133 (30), 105 (16), 77 (14); Anal. Calcd for C13H19NO2: C,
70.56; H, 8.65; N, 6.33. Found: C, 69.83; H, 8.63; N, 6.18.
2,3-Dihydro-3-hydroxymethyl-6-chloro-[1,4]benzoxazine
(3d): white solid, mp 108–110 °C; IR (KBr) m: 3353, 3275,
C9H9Cl2NO2: C, 46.18; H, 3.88; N, 5.98. Found: C, 46.76;
H, 4.16; N, 5.56.
2,3-Dihydro-3-hydroxymethyl-6-amino-[1,4]benzoxazine
(3g): light yellow solid, mp 154–156 °C; IR (KBr) m: 3399,
1
3337, 2955, 2923, 1561, 1414, 1213, 1053 cmꢀ1; H NMR
(DMSO, 400 MHz) d: 2.88 (dd, J = 7.7, 12.6 Hz, 1H), 3.20
(d, J = 12.6 Hz, 1H), 3.58 (dd,J = 5.6, 11.8 Hz, 1H), 3.72–
3.81 (m, 1H), 4.17 (dd, J = 4.0, 11.8 Hz, 1H), 4.50 (br s,
3H), 5.00 (br s, 1H), 5.82 (dd, J = 2.6, 8.3 Hz, 1H), 5.94 (d,
J = 2.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H); EIMS (m/z, %):
180 (M+, 80), 137 (10), 136 (86), 135 (100), 108 (15), 95
(21), 80 (28).
2962, 2923, 1493, 1288, 1060 cmꢀ1 1H NMR (CDCl3,
;
400 MHz) d: 3.19 (br s, 2H), 3.21 (dd, J = 2.5, 13.0 Hz,
1H), 3.39 (dd, J = 4.8, 13.0 Hz, 1H), 3.87 (dd, J = 2.1,
12.3 Hz, 1H), 3.93–4.00 (m, 1H), 4.25 (dd, J = 3.8,
12.3 Hz, 1H), 6.76 (d, J = 2.5 Hz 1H), 6.77 (dd, J = 2.5,
8.2 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H); EIMS (m/z, %): 199
(M+, 45), 157 (22), 155 (58), 154 (100), 127 (10), 92 (14);
Anal. Calcd for C9H10ClNO2: C, 54.15; H, 5.05; N, 7.02.
Found: C, 53.86; H, 5.02; N, 6.90.
37. Henry, N.; Guillaumet, G.; Pujol, M. D. Tetrahedron Lett.
2004, 45, 1465.
38. Araki, S.; Shimada, Y.; Kaji, K.; Hayashi, H. Biochem.
Biophys. Res. Commun. 1990, 68, 1194.
2,3-Dihydro-3-hydroxymethyl-8-chloro-[1,4]benzoxazine
(3e): light yellow oil; IR (film) m: 3371, 2924, 1593, 1476,
1221, 1083 cmꢀ1; 1H NMR (CDCl3, 400 MHz) d: 3.22 (dd,
J = 2.5, 13.1 Hz, 1H), 3.33 (br s, 2H), 3.41 (dd, J = 4.8,
13.1 Hz, 1H), 3.93 (dd, J = 2.2, 12.3 Hz, 1H), 3.93–4.05
(m, 1H), 4.37 (dd, J = 3.7, 12.3 Hz, 1H), 6.68 (dd, J = 1.5,
8.0 Hz, 1H), 6.81 (t, J = 8.0 Hz, 1H), 6.93 (dd, J = 1.5,
8.0 Hz, 1H); EIMS (m/z, %): 199 (M+, 60), 157 (37), 156
(52), 155 (67), 154 (100), 127 (18), 99 (19), 92 (17); Anal.
Calcd for C9H10ClNO2: C, 54.15; H, 5.05; N, 7.02. Found:
C, 54.22; H, 5.06; N, 6.78.
2,3-Dihydro-3-hydroxymethyl-6,8-dichloro-[1,4]benzox-
azine (3f): light yellow oil; IR (film) v: 3380, 2964, 2925,
1589, 1472, 1303, 1055 cmꢀ1; 1H NMR (CDCl3, 400 MHz)
d: 2.90 (br s, 1H), 3.25 (dd, J = 2.8, 13.2 Hz, 1H), 3.40 (dd,
J = 4.9, 13.2 Hz, 1H), 3.65 (br s, 1H), 3.95 (dd, J = 2.4,
12.3 Hz, 1H), 3.98-4.20 (m, 1H), 4.34 (dd, J = 3.7, 12.3 Hz,
1H), 6.65 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H);
EIMS (m/z, %): 233 (M+, 55), 193 (12), 192 (22), 189 (72),
188 (100), 161 (12), 126 (15), 99 (11); Anal. Calcd for
39. Human umbilical vein endothelial cells (HUVECs) were
grown in MCDB131 medium (Sigma) supplemented with
10% (v/v) fetal calf serum and 80 mg/L bFGF. All
experiments were performed on cells from 10 to 20
passages. HUVEC apoptosis was induced by deprivation
of growth factors.
40. Miao, J.-Y.; Zhao, B.-X.; Li, H.-H.; Zhang, S.-L.; Du,
C.-Q. Acta Pharmacol. Sin. 2002, 23, 323.
41. HUVECs were cultured in the medium with or without the
compounds 3a–g 25–200 lM for 24 and 48 h, respectively.
A549 cells were cultured in the medium with or without
the compounds 3a–g 50–400 lM for 24 and 48 h, respec-
tively. Then, the morphological changes of the cells were
observed under phase contrast microscope (Nikon,
Japan).
42. Oishi, K.; Kobayashi, A.; Fujii, K.; Kanehira, D.;
Ito, Y.; Uchida, M. K. J. Pharm. Sci. 2004, 96,
208.
43. Ono, K.; Wang, X.; Han, J. Mol. Cell. Biol. 2001, 21,
8276.