A novel oxazine ring closure reaction affording (Z)-((E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)acetaldehydes and their anti-osteoclastic bone resorption activity

Article abstract of DOI:10.1016/j.bmcl.2006.08.064

A novel oxazine ring formation method was established using the reaction of 2-acetyl-(E)-3-styrylcarbonylaminobenzo[b]furans (4) with Vilsmeier-Haack-Arnold reagent to afford (E and Z)-((E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)acetaldehydes (5

Full text of DOI:10.1016/j.bmcl.2006.08.064

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5849–5854
A novel oxazine ring closure reaction affording (Z)-((E)-2-
styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)acetaldehydes
and their anti-osteoclastic bone resorption activity
Yuko Ando,a Kumiko Ando,a Mami Yamaguchi,a Jun-ichi Kunitomo,a Masao Koida,b
Ryo Fukuyama,c Hiromichi Nakamuta,c Masayuki Yamashita,d
Shunsaku Ohtad and Yoshitaka Ohishia,*
aDepartment of Medicinal and Synthetic Chemistry, Faculty of Pharmaceutical Sciences,
Mukogawa Women’s University, 11-68 Koshien Kyuban-cho, Nishinomiya 663-8179, Japan
bDepartment of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge cho,
Hirakata, Osaka 573-1010, Japan
cLaboratory of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences,
Hiroshima International University, 5-1-1 Hirokoshinkai, Kure, Hiroshima 737-0112, Japan
dKyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Received 7 June 2006; revised 23 July 2006; accepted 12 August 2006
Available online 1 September 2006
Abstract—A novel oxazine ring formation method was established using the reaction of 2-acetyl-(E)-3-sty-
rylcarbonylaminobenzo[b]furans (4) with Vilsmeier–Haack–Arnold reagent to afford (E and Z)-((E)-2-styrylbenzo[b]furo[3,2-
d][1,3]oxazin-4-ylideno)acetaldehydes (5). (Z)-4-(8-Bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)but-(E)-2-enoic acid
ethyl ester (6b), derived from (Z)-5a, showed significantly potent anti-osteoclastic bone resorption activity comparable to 17b-estra-
diol (E2).
ꢀ 2006 Elsevier Ltd. All rights reserved.
Various oxazine compounds have been found to show
versatile bioactivities.1 This prompted us to establish a
novel oxazine ring formation method to find promising
bioactive oxazine compounds. We chose the Vilsmeier–
(2) were isolated in low yield, and no oxazine derivatives
were obtained. These findings led us to hypothesize that
the 3-amide moiety stabilized by a conjugate system
(3-styrylcarbonylamino group) would be more advanta-
geous for oxazine ring formation than the cyanomethyl-
carbonylamino and ethoxycarbocarbonylamino groups.
We thus prepared four 2-acetyl-(E)-3-styrylcarbonyl-
aminobenzo[b]furans (4a–4d) from 2-acetyl-3-amin-
obenzo[b]furans (3a, 3b)3 by reactions with
trans-cinnamoyl chlorides. To a VM reagent prepared
from POCl3 with dry N,N-dimethylformamide (DMF)
was added 4a at 6 ꢁC. The reaction mixture was stirred
at 25 ꢁC for 30 h and an orange precipitate was formed
but was difficult to purify because of its chemical insta-
bility. A suspension of this precipitate in water was
treated with 10% NaOH aqueous solution or triethyl-
amine with vigorous stirring to give an orange powder.
This orange powder was recrystallized from ethyl ace-
tate–chloroform (5:1) to yield orange needles (5a, mp
213–216 ꢁC, 46% (by treatment with triethylamine)).
1H NMR (HMBC, HMQC), MS, and elemental
Haack–Arnold
reagent
((CH3)2N+@CHClÆClꢀ M
(CH3)2N–C+HClÆClꢀ) (VM reagent) which is extensively
used for the formylation of activated aromatic, heteroa-
romatic, and carbonyl compounds.2 We focused on the
oxazine ring formation using aromatic ortho-keto amide
compounds (2-acetyl-3-alkylcarbonylaminobenzo[b]fur-
ans) under the Vilsmeier reaction conditions. 2-Acetyl-
3-cyanomethylcarbonylaminobenzo[b]furans (1a, 1b)
and 2-acetyl-3-ethoxycarbocarbonylaminobenzo[b]fu-
ran (1c) were treated with VM reagent at 24 ꢁC. Unfor-
tunately, 4-chloro-3-formylbenzo[b]furo[3,2-b]pyridines
Keywords: (Z)-4-(8-Bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-
4-ylideno)but-(E)-2-enoic acid ethyl ester; Oxazine ring closure; Vils-
meier reaction; Anti-osteoclastic bone resorption activity.
*
Corresponding author. Tel.: +798 45 9953; fax: +798 41 2792; e-mail:
yohishi@mukogawa-u.ac.jp
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.064

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Y. Ando et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5849–5854
analysis data suggested 5a to be a novel (E or Z)-(8-bro-
mo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)ac-
etaldehyde with a characteristic exo-formylmethylene
group on the oxazine ring (Scheme 1).
Compounds (4b–4d) were also treated with VM reagent
to afford (Z)-5b, (Z)-5c and (Z)-5d, respectively. These
oxazine ring closure reactions to 5 from 4 afforded mix-
tures consisting of the predominant Z-isomer and the E-
isomer (Z-isomer:E-isomer = 98:2–95:5 by 1H NMR).
Each predominant (Z)-isomer was isolated from the
respective mixture (Scheme 1) (Table 1). We were thus
able to establish a novel oxazine preparation method
based on the reaction of 4 with VM reagent. This oxa-
zine cyclization reaction was markedly dependent on
the chemical property of the 3-carbonylamino function-
al group. 3-Styrylcarbonylamino group was favorable
for the oxazine ring formation.
These findings were not sufficient to confirm the pres-
ence of an oxazine ring in 5a and also did not define
the isomeric form (E or Z) of the exo-formylmethylene
group. Two butadiene derivatives (6a,6b) were prepared
from 5a, because of the difficulty of single crystal prep-
aration of 5a for X-ray analysis. The formyl group of
5a was allowed to react with N,N-diethylphosphono-
acetamide and ethyl diethylphosphonoacetate under
the Horner–Wadsworth–Emmons (HWE) reaction
conditions to give the corresponding butadiene deriva-
tives (6a, 6b) (Scheme 1). The structure of compound
(6a) was confirmed to be (Z)-4-(8-bromo-(E)-2-sty-
rylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)-N,N-dieth-
ylbut-(E)-2-enamide on the basis of its X-ray analysis
as shown in Figure 1.4 This result and the physical data
of 5a demonstrated it to be (Z)-(8-bromo-(E)-2-sty-
rylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)acetaldehyde
((Z)-5a).
(Z)-5a showed isomerization to its E-isomer ((E)-5a) in
dimethylformamide or CHCl3 solution. The more stable
(Z)-5a reached an equilibrium with the less stable (E)-5a
at the ratio of (Z)-5a:(E)-5a = 5:2 in these solutions after
15–48 h.5 The isomerization of (Z)-5a to (E)-5a is con-
sidered to be caused by the formyl group.6 Evidence
for this came from the absence of isomerization of
(Z)-2-(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxa-
zin-4-ylideno)ethanol (7) to its E-isomer. 5-Bromo-2-
Scheme 1. Reagents: (a) NaBH4/THF; (b) (C2H5O)2P(O)CH2CON(C2H5)2 and (C2H5O)2P(O)CH2COOC2H5, NaH/THF.

Y. Ando et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5849–5854
5851
Figure 1. Structure of (Z)-4-(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylideno)-N,N-diethylbut-(E)-2-enamide (6a) and its X-ray
analysis.
Table 1. Physical data and yields of compounds (Z)-5 and 6
Compd Yield Mp
(%)
1H NMR
MS m/z (int.)
Formula
(ꢁC)
HR-MS m/z M+
Calcd (Found)
or Anal. Calcd (Found)
(Z)-5a
(Z)-5b
46
213–216 dH(400 MHz; CDCl3; Me4Si) 5.72 (1H, d, J = 7.7, CHCHO),
6.79 (1H, d, J = 16.2, CH@CHC6H5), 7.43–7.45 (3H, m, 30-,
40-, 50-H), 7.45 (1H, d, J = 8.5, 6-H), 7.59–7.62 (2H, m, 20-, 60-
H), 7.63 (1H, dd, J = 8.8 and 1.8, 7-H) 7.77 (1H, d, J = 16.1,
CH@CHC6H5), 8.05 (1H, d, J = 2.1, 9-H), 10.3 (1H, d, J = 8.1,
CHCHO)
393 (M+, 100.00) C20H12BrNO3
C; 60.93 (60.74)
H; 3.07 (2.88)
N; 3.55 (3.54)
35
39
17
49
215–218 dH(400 MHz; CDCl3; Me4Si) 3.87 (3H, s, OCH3), 5.71 (1H, d, 423 (M+, 99.72)
C21H14BrNO4
C; 59.45 (58.92)
H; 3.33 (3.11)
N; 3.30 (3.10)
J = 8.1, CHCHO), 6.65 (1H, d, J = 16.1, CH@CHC6H4(40-
OCH3)), 6.94–6.97 (2H, m, 20-, 60-H or 30-, 50-H), 7.45 (1H, d,
J = 8.8, 6-H), 7.54–7.57 (2H, m, 20-, 60-H or 30-, 50-H), 7.62
(1H, d, J = 8.8 and 2.2, 7-H), 7.72 (1H, d, J = 16.1,
CH@CHC6H4(40-OCH3)), 8.04 (1H, d, J = 1.8, 9-H), 10.3 (1H,
d, J = 7.7, CHCHO)
425 (100.00)
(Z)-5c
(Z)-5d
6a
239–241 dH(400 MHz; CDCl3; Me4Si) 4.05 (3H, s, OCH3), 5.81 (1H, d, 345 (M+, 100.00) C21H15NO4 Æ 2/3H2O
J = 7.7, CHCHO), 6.82 (1H, d, J = 16.1, CH@CHC6H5), 7.04
(1H, dd, J = 8.1 and 0.8, 7-H or 9-H), 7.34 (1H, dd, J = 8.1 and
8.1, 8-H), 7.41–7.44 (3H, m, 30-, 40-, 50-H), 7.49 (1H, dd,
J = 7.9 and 0.9, 7-H or 9-H), 7.59–7.62 (2H, m, 20-, 60-H), 7.77
(1H, d, J = 16.2, CH@CHC6H5), 10.3 (1H, d, J = 8.1,
CHCHO)
C; 70.58 (70.40)
H; 4.61 (4.01)
N; 3.92 (3.96)
242–246 dH(400 MHz; CDCl3; Me4Si) 3.87 (3H, s, OCH3), 4.05 (3H, s, 375 (M+, 100.00) C22H17NO5
OCH3), 5.79 (1H, d, J = 8.0, CHCHO), 6.68 (1H, d, J = 16.1,
CH@CHC6H4(40-OCH3)), 6.94–6.97 (2H, m, 20-, 60-H or 30-,
50-H), 7.04 (1H, dd, J = 7.9 and 1.0, 7-H or 9-H), 7.33 (1H, t,
J = 8.1, 8-H), 7.48 (1H, dd, J = 7.9 and 1.0, 7-H or 9-H), 7.53–
7.57 (2H, m, 30-, 50-H or 20-, 60-H), 7.72 (1H, d, J = 16.1,
CH@CHC6H4(40-OCH3)), 10.3 (1H, d, J = 7.7, CHCHO)
191–192 dH(400 MHz; CDCl3; Me4Si) 1.21–1.25 (6H, m,
N(CH2CH3)2 · 2), 3.44–3.52 (4H, m, N(CH2CH3)2 · 2), 5.88
(1H, dd, J = 12.1 and 0.7, @CHCH@CHCO), 6.37 (1H, dd,
J = 14.6 and 0.7, @CHCH@CHCO), 6.71 (1H, d, J = 16.1,
CH@CHC6H5), 7.34 (1H, d, J = 8.8, 6-H), 7.37–7.43 (3H, m,
30-, 40-, 50-H), 7.48 (1H, dd, J = 8.8 and 2.2, 7-H), 7.60-7.63
(2H, m, 20-, 60-H), 7.74 (1H, d, J = 16.1, CH@CHC6H5), 7.93
(1H, d, J = 2.2, 9-H), 7.95 (1H, dd, J = 14.6 and 12.1,
CHCH@CHCO)
C; 70.39 (70.24)
H; 4.56 (4.41)
N; 3.73 (3.77)
490 (M+, 76.46),
391 (100.00)
C26H23BrN2O3 Æ 1/2 H2O
C; 62.41 (62.17)
H; 4.83 (4.57)
N; 5.60 (5.59)
6b
71
199–200 dH(400 MHz; CDCl3; Me4Si) 1.35 (3H, t, J = 7.1, OCH2CH3), 463 (M+, 100.00) C24H18BrNO4
4.26 (2H, q, J = 7.2, OCH2CH3), 5.84 (1H, dd, J = 11.8 and
0.8, @CHCH@CHCO), 5.96 (1H, dd, J = 15.4 and 0.7,
@CHCH@CHCO), 6.73 (1H, d, J = 16.1, CH@CHC6H5), 7.36
(1H, d, J = 8.8, 6-H), 7.39–7.45 (3H, m, 30-, 40-, 50-H), 7.51
(1H, dd, J = 8.8 and 2.2, 7-H), 7.59-7.62 (2H, m, 20-, 60-H),
7.72 (1H, d, J = 16.2, CH@CHC6H5), 7.89 (1H, dd, J = 15.4
and 12.1, @CHCH@CHCO), 7.94 (1H, d, J = 1.9, 9-H)
C; 62.08 (62.03)
H; 3.91 (3.71)
N; 3.02 (2.90)

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Y. Ando et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5849–5854
(4-chlorobenzoyl)-(E)-3-styrylcarbonylaminobenzo[b]fu-
ran (9)3 prepared from 83 was treated with VM reagent
under the same reaction conditions as the reaction of 4a
with VM reagent. However, only starting material 9 was
recovered unchanged (Scheme 1). This showed that
reaction of the acetyl group with VM reagent is the
driving force for the oxazine cyclization reaction. The
formation mechanism of (Z)-5 and (E)-5 from 4 is likely
to occur as follows. Both the enol 2-acetyl group and 3-
carbonylamino group of 4 reacted with VM reagent to
form 102 which is converted into two geometrical iso-
mers (11A, 11B). Because the heat of formation of
12A is lower than that of 12B,7 11A is converted faster
to 12A than 11B. Both 12A and 12B produced labile
immonium salts (13) consisting mostly of (Z)-13 with a
little (E)-13. The immonium salts (13) are deposited as
an orange precipitate in the reaction mixture, as
described above. Treatment of 13 with base afforded a
mixture of (Z)-5 (predominant isomer) and (E)-5
(Scheme 2). The predominant formation of (Z)-5 over
(E)-5 can be explained by the lower heat of formation
of 12A compared to 12B.
benzo[b]furan3 which had potent anti-osteoclastic bone
resorption activity in vitro and exhibited an anti-osteo-
porosis effect in vivo in our recent work.8 Therefore, rep-
resentative compounds ((Z)-5a, 6a, 6b) were tested with
an in vitro assay of anti-osteoclastic bone resorption
activity. In coculture of fresh bone marrow preosteo-
clasts expressing the receptor activator of NF-jB
(RANK) with calvarial osteoblasts that express the li-
gand for RANK (RANKL), bone resorbing osteoclasts
developed and formed resorption pits on a dentin slice.
PGE2 stimulated pit formation, and estrogens (e.g., E2)
140
120
100
80
**
**
60
40
20
0
cont. E2
(Z)-
5a
6a
6b
The oxazine ring moiety of compounds 5 and 6 was
similar to the ring type design devised from the (Z)-2-cy-
ano-3-hydroxybut-2-enonylamino group of 2-(4-chloro-
benzoyl)-(Z)-3-(2-cyano-3-hydroxybut-2-enonyl)amino-
Figure 2. Anti-osteoclastic bone resorption activities of the oxazine
derivatives ((Z)-5a, 6a, 6b) cont.: control E2: 17b-estradiol.
Scheme 2. Proposed mechanism of formation of (E and Z)-((E)-2-styrylbenzo[b]furo[3,2-d][1,3]-oxazin-4-ylideno)acetoaldehydes (5) from 4.

Y. Ando et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5849–5854
5853
4. Compound 6a formula: C26H23BrN2O3Æ1/2 H2O, formula
weight: 500.39, crystal color, habit: red, platelet, crystal
dimensions: 0.25 · 0.05 · 0.03 mm, crystal system: mono-
clinic, lattice type: primitive, indexing images: 3 oscilla-
tions at 30.0 s, Detector position: 127.40 mm, Pixel size:
inhibited PGE2-stimulated pit formation by suppressing
the RANKL effect.9 Among the compounds tested, the
butadiene ester derivative (6b) showed significantly po-
tent inhibition activity comparable to E2, but the exo-
formylmethylene compound ((Z)-5a) was inactive
(Fig. 2).10 This suggested that the butadiene moiety hav-
ing particular polar functional groups might play an
important role in inhibiting osteoclasts.
˚
0.100 mm, lattice parameters: a = 16.7921(11) A, b =
˚
˚
11.3491(7) A, c = 25.3011(16) A, b = 109.668(4)ꢁ, V =
3
4540.5(5) A , space group: P21/a (#14), Z value: 8, Dcalc
˚
:
1.464 g/cm3, F000: 2056.00, l(CuKa): 27.447 cmꢀ1. CCDC
Deposit No. 600565.
We found the novel compound (6b) which possesses sig-
nificantly potent anti-osteoclastic bone resorption activi-
ty. It shows promise as a lead compound to develop new
osteoporosis treatment agents, because of its significant
potency and favorable balance of lipophilic and hydro-
philic properties at the molecular level. In this work, we
developed a novel oxazine ring preparation method by
reaction of 2-acetyl-(E)-3-styrylcarbonylaminobenzo
[b]furans (4) with VM reagent. This is a new application
of the VM reaction. The (Z)-5 prepared had the character-
istic exo-formylmethylene group on the oxazine moiety.11
This group enabled further synthesis of versatile oxazine
compounds. The butadiene ester derivative (6b) prepared
from (Z)-5a showed significantly potent anti-osteoclastic
bone resorption activity comparable to E2. The potent
anti-osteoclastic bone resorption activity of oxazine
derivative (6b) encouraged us to synthesize additional
(Z)-benzo[b]furo[3,2-d][1,3]oxazin-4-ylidene derivatives
having various butadiene groups and to evaluate their
anti-osteoclastic bone resorption activity. Work contin-
ues on study of the mechanism of the inhibitory action
of 6b.
5. The heat of formation of (Z)-5a was 0.5 kcal/mol lower
than that of (E)-5a. The calculation was performed by
using Spartan 2004, Wavefunction, Inc., Irvine, CA.
Hehre J. W., ‘A Guide to Molecular Mechanics and
Quantum Chemical Calculations’, Wavefunction, Irvine,
2003.
6. Dubbaka, R. S.; Vogel, P. Tetrahedron 2005, 61, 1523.
7. The heat of formation of 12A was about 2 kcal/mol lower
than that of 12B. The calculation was performed in a
similar manner to that described in Ref. 5.
8. Paper in preparation. A part of this work was presented at
the ‘International Symposium of Maxillofacial and Oral
Regenerative Biology in Okayama (Japan) 2005’. The
proceeding version of the presentation was published in
(Koida, M.; Nakamuta, H.; Ohishi, Y.) J. Hard Tissue
Biol. (special issue) 2005, 14, 160.
9. (a) Nakagawa, N.; Kinosaki, M.; Yamaguchi, K.; Shima,
N.; Yasuda, H.; Yano, K.; Morinaga, T.; Higashio, K.
Biochem. Biophys. Res. Commun. 1998, 253, 395; (b)
Yasuda, H.; Shima, N.; Nakagawa, N.; Mochizuki, S. I.;
Yano, K.; Fujise, N.; Sato, Y.; Goto, M.; Yamaguchi, K.;
Kuriyama, M.; Kanno, T.; Murakami, A.; Tsuda, E.;
Morinaga, T.; Higashio, K. Endocrinology 1998, 139,
1329; (c) Yasuda, H.; Shima, N.; Nakagawa, N.; Yamag-
uchi, K.; Kinosaki, M.; Mochizuki, S. I.; Tomoyasu, A.;
Yano, K.; Goto, M.; Murakami, A.; Tsuda, E.; Morinaga,
T.; Higashio, K.; Udagawa, N.; Takahashi, N.; Suda, T.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3597.
Acknowledgments
10. In vitro assay of anti-osteoclastic bone resorption activ-
ity: preconfluent primary calvarial osteoblasts from 1 to
2-day-old ddY mice (Japan SLC, Shizuoka, Japan) and
fresh bone marrow cells from 5-week-old ddY male mice
were cocultured in a-MEM (pH 7.0; Sigma Chemical
Co., St. Louis, MO, USA) containing 10% fetal calf
serum (FCS, Moregate, Australia and New Zealand),
10 nM calcitriol (Wako Pure Chemical Ind., Osaka,
Japan), and 1.0 lM prostaglandin E2 (PGE2, Sigma
Chemical Co.) on a 100 mm dish (Greiner, Tokyo,
Japan) precoated with collagen (cell matrix Type I-A,
Nitta Gelatin Inc., Osaka, Japan), for 7 days. The cells
were then resuspended by collagenase (Wako Pure
Chemical Ind.) digestion and each 0.30 ml suspension
was plated over a dentin slice (10 mm in diameter and
200 lm in thickness) in 0.50 ml a-MEM (pH 7.0)
containing 10% FCS and 20 mM HEPES on a well of
This work was supported in part by a grant from the
Ministry of Education, Culture, Sports, Science and
Technology for a ‘University–Industry Joint Research’
Project (2004–2008). The authors thank the staff of the
Instrument Analysis Center of Mukogawa Women’s
University for the 1H NMR and MS measurements
and element analyses.
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