Preparation of novel (Z)-4-ylidenebenzo[b]furo[3,2-d][1,3]oxazines and their biological activity

Article abstract of DOI:10.1016/j.bmc.2009.04.017

A reaction of 2-acetyl-3-acylaminobenzo[b]furans (9d-o) with Vilsmeier (VM) reagent afforded a mixture of (E)- and (Z)-{(E)-2-aralkenylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene}acetaldehydes (5) with a characteristic exo-formylmethylene group on the oxazin

Full text of DOI:10.1016/j.bmc.2009.04.017

Bioorganic & Medicinal Chemistry 17 (2009) 3959–3967
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preparation of novel (Z)-4-ylidenebenzo[b]furo[3,2-d][1,3]oxazines
and their biological activity q
Yukako Tabuchi a, Yuko Ando a, Hidemi Kanemura a, Ikuo Kawasaki a, Takahiro Ohishi b, Masao Koida c,
a,f,
Ryo Fukuyama d, Hiromichi Nakamuta d, Shunsaku Ohta e, Kiyoharu Nishide a, , Yoshitaka Ohishi
*
*
a School of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien Kyubancho, Nishinomiya, Hyogo 663-8179, Japan
b Science of Environment and Mathematical Modeling, Graduate School of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan
c Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-1010, Japan
d Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Hiroshima International University, 5-1-1 Hirokoshinkai,
Kure, Hiroshima 737-0112, Japan
e Kyoto Pharmaceutical University, Misasagi, Yamashinaku, Kyoto 607-8412, Japan
f Taisho Pharm. Ind., Ltd, Research & Development Dept., 3, Oharaichiba, Koka-cho, Koka, Shiga 520-3433, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2009
Revised 6 April 2009
Accepted 7 April 2009
Available online 12 April 2009
A reaction of 2-acetyl-3-acylaminobenzo[b]furans (9d–o) with Vilsmeier (VM) reagent afforded a mix-
ture of (E)- and (Z)-{(E)-2-aralkenylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene}acetaldehydes (5) with a
characteristic exo-formylmethylene group on the oxazine ring. The Z-isomer was more stable than the
E-isomer. The Z-isomers ((Z)-5) were reacted with phosphonate reagents under two different conditions
to obtain various butadiene derivatives (12) containing benzo[b]furo[3,2-d][1,3]oxazine skeleton. Typical
compounds (5 and 12) were evaluated for their anti-osteoclastic bone resorption activity, antagonistic
activity for the cysLT1 receptor and growth inhibitory activity for MIA PaCa-2 and MCF-7. Compounds
12f and 12j showed potent anti-osteoclastic bone resorption activity comparable to E2 (17b-estradiol).
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Benzo[b]furo[3,2-d][1,3]oxazine
Anti-osteoclastic bone resorption activity
Antagonistic activity for the cysLT1 receptor
Growth inhibitory activity for MIA PaCa-2
and MCF-7
1. Introduction
What was lacking was the examination of oxazine cyclization
from aromatic carbonylamines (1b) having a ketone group at the
4H-3,1-Benzoxazine derivatives with fused aromatic rings
showed various bioactivities such as anti-human coronavirus
activity,2 ICAM-1 expression inhibition activity,2 inhibition of hu-
man leukocyte elastase,3 inhibition of human cathepsin G,4 inhibi-
tion of chymotrypsin,4,5 inhibition of C1r serine protease of the
complement system,6 inhibition of thrombin7 and inhibition of hu-
man cytomegalovirus protease.8 Studies have been advancing on
several oxazine ring cyclization reactions and the preparation of
oxazine derivatives.9 Over the past decade, oxazine ring cyclization
has been examined for aromatic carbonylamines (1) having a car-
bonyl functional group at the ortho(o)-position,10 representative
example one shown in Scheme 1.3b,11 Thus, these aromatic carbon-
ylamines (1a) having carboxylic acid, ester, amide, and alcohol
groups at the o-position were subjected to cyclization between
these adjacent group pairs to form aromatic ring fused oxazines.
o-position. Here we report a novel oxazine cyclization reaction of
2-acetyl-3-acylaminobenzo[b]furans, as representative of 1b, un-
der the Vilsmeier–Haack–Arnold reaction conditions and the prep-
aration of novel (Z)-4-ylidene-benzo[b]furo[3,2-d][1,3]oxazine
derivatives.
Recently, substantial efforts have been made toward the discov-
ery of selective estrogen receptor modulators (SERMs). Several are
currently on the market (tamoxifen for treatment of breast can-
cer12 and raloxifene for the prevention and treatment of osteoporo-
sis13a,b
) or are in advanced clinical trials (lasofoxifene and
bazedoxifene). SERMs are characterized by at least two common
structural features, a phenolic hydroxyl group and a phenoxyeth-
ylamino group (phenyl-OCH2CH2N–).14a,b The phenoxyethylamino
group has been postulated to be important for binding to the cen-
tral core of the estrogen receptor.14c It has also been suggested to
influence the endometrial properties of SERMs in women by the
antiestrogenic side chain.14a,b,d We recently reported that the com-
pound 415 prepared in our current studies displayed very potent
q
See Ref. 1.
anti-bone resorption activity in vitro and exhibits
a potent
* Corresponding authors. Tel.: +81 798 45 9953; fax: +81 798 41 2792 (Y.O.).
E-mail addresses: ikuo_k@mukogawa-u.ac.jp (I. Kawasaki), nishide@mukogawa-u.
ac.jp (K. Nishide), ymohishi@kcc.zaq.ne.jp (Y. Ohishi).
anti-osteopenic effect in vivo. This compound showed equivalent
activity in vitro and in vivo assays for estrogen 2 and raloxifene.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.017

3960
Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
NHCOR3
R2
R1
1
1a : R1= COOH, COOR4, CONR5R6, CH(OH)R7
1b : R1 = COR8 (R8= alkyl, aryl)
ref. 3b,11)
NHCONR1R2
COR3
N
O
NR1R2
H+
R4
R4
O
73 - 86%
2
3
R3 = OH, OCH3, NR5R6
HO
CN
R
O
NH
N
Br
O
O
R
CHO
O
O
Cl
4
5
Scheme 1.
It is currently being examined for SERM activity and possible
development as a new medicine to treat osteoporosis.16 Both the
compound 415 and (E)-(8-bromo-(E)-2-aralkenylbenzo[b]furo[3,2-
d][1,3]oxazin-4-ylidene)acetaldehydes (5) possess the phenoxy-
ethenylamino moiety through the furan oxygen atom and the
nitrogen atom. This suggests the value of preparing derivatives
from 5 and evaluating their inhibition activity for osteoclasts. We
therefore planed preparation of derivatives of 5 to evaluate their
biological activities.
carbonyl group on the nitrogen at 3-position would be favorable
for cyclization between the 2-acetyl group and the 3-acylamino
group. Some kinds of 2-acetyl-3-acylaminobenzo[b]furans (9d,15
9e–g, 9h,15 9i–o) were prepared by reactions of 8a and 8b with
various acid chlorides such as (E)-5-phenylpenta-2-enonyl chlo-
ride,19 cinnamoyl chlorides,20 (E,E)-5-phenylpenta-2,4-dienoyl
chlorides,21 crotonyl chloride, 2-hexenoyl chloride and (E)-2-meth-
ylbut-2-enoyl chloride. The physical and spectral data of 2-acetyl-
3-acylaminobenzo[b]furans (9) were listed in Table S1, see Supple-
mentary data. To a VM reagent prepared from POCl3 with dry N,N-
dimethylformamide (DMF) at 6 °C was added 2-acetyl-5-bromo-
(E)-3-cinnamoylaminobenzo[b]furan (9d). The reaction mixture
was stirred at 25 °C for 30 h, and an orange precipitate was depos-
ited. Purification of the orange precipitate (presumed to be the
immonium salt)1 was difficult because of its chemical instability.
An orange suspension of this precipitation in water was treated
with 10% NaOH aqueous solution with vigorous stirring, and an or-
ange powder was obtained (Method A). The orange powder could
also be obtained by treating the orange suspension with triethyl-
amine (Method B). Recrystallization of each orange powder from
CHCl3–ethyl acetate (5:1) gave orange needles (representative 5a,
mp 213–216 °C, 46% (Method B)) (Scheme 3). 1H NMR (HMBC,
HMQC), MS and elemental analysis data of 5a supported cycliza-
tion of oxazine ring fused at the 2- and 3-position of the benzo[b]-
furan ring. Compound 5a was presumed to be a novel (E or Z)-(8-
bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)acet-
aldehyde with a characteristic exo-formylmethylene group on the
oxazine ring. These data were, however, insufficient to confirm
the structure of 5a. Attempts to prepare single crystals of 5a for
X-ray analysis were unsuccessful. Thus, 5a was treated with
diethyl 2-(diethylamino)-2-oxoethylphosphonate (11a) under
Horner–Wadsworth–Emmons (HWE) reaction conditions to afford
a butadiene derivative (12a) of which single crystals were success-
fully prepared. X-ray analysis of 12a demonstrated it to be (Z)-4-
2. Results and discussion
Halomethyleniminium salts (VM Reagent) have found extensive
use as formylating, halogenating and dehydroxylating reagents.17
In addition, many kinds of heterocyclic compounds such as pyri-
dines, quinolines, thienopyridines, quinolones, isoquinolones,
naphthyridines, pyrans and furans can be efficiently prepared by
ring closure reaction from acylamides under the VM conditions.18
Although N-(2-acetylphenyl)acetamides (6) afforded 4-chloro-3-
formylquinolines (7), not oxazine compounds, by reaction
with VM reagent at 90 °C,18a we expected the reaction of 2-acet-
yl-3-cyanomethylcarbonylaminobenzo[b]furans (9a, 9b) and
2-acetyl-3-ethoxycarbonylaminobenzo[b]furan (9c) prepared from
2-acetyl-3-aminobenzo[b]furans (8a, 8b)15 to give some oxazine
compounds. However, both reactions of 9a and 9c with VM reagent
at low temperature (24 °C) afforded 8-bromo-4-chloro-3-form-
ylbenzo[b]furo[3,2-b]pyridine (10a), accompanied by loss of
cyanomethylcarbonyl and ethoxycarbonyl groups, respectively, as
shown in Scheme 2. The reaction of 9b with VM reagent also affor-
ded
4-chloro-3-formyl-6-methoxybenzo[b]furo[3,2-b]pyridine
(10b). These results were similar to the case of benzene derivative
6.
We predicted that the 2-acetyl-3-(2-aralkenylcarbonylami-
no)benzo[b]furan derivatives (9d–o) having a stable conjugating

Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
3961
R1
NHCOCH3
COCH3
R1
N
ref. 18a)
POCl3 - DMF
90°C
CHO
R2
R2
Cl
6
7
NHCOR3
COCH3
N
NH2
R1
R1
CHO
R1
R3COCl
POCl3 - DMF
24°C
COCH3
Cl
O
O
O
THF or CHCl3
R2
R2
R2
9
10
8
10a : R1 = Br, R2 = H (12% from 9a, 3% from 9c)
10b : R1 = H, R2 = OCH3 (14% from 9b)
9a : R1 = Br, R2 = H, R3 = CH2CN
9b : R1 = H, R2 = OCH3, R3 = CH2CN
9c : R1 = Br, R2 = H, R3 = COOC2H5
8a : R1 = Br, R2 = H
8b : R1= H, R2 = OCH3
Scheme 2.
(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)-
N,N-diethylbut-(E)-2-enamide (Fig. 1).1 Consequently, a novel oxa-
zine compound 5a was determined to be (Z)-(8-bromo-(E)-2-sty-
rylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)acetaldehyde ((Z)-5a)
on the basis of physical data of 5a and X-ray analysis of 12a.
The reaction of 9d with VM reagent gave a mixture of (Z)-5a and
(E)-5a in a ratio of (Z)-5a:(E)-5a = 98:2 (by 1H NMR). Both the
oxazine ring closure mechanism to 5 from 9 and the reason for
predominant production of the Z-isomer (5) were discussed in
the preliminary communication.1 Also, eleven 2-acetyl-(E)-3-ara-
lkenylcarbonylaminobenzo[b]furans (9e–o) afforded correspond-
ing mixtures of (Z)- and (E)-{(E)-2-aralkenylbenzo[b]furo[3,2-
d][1,3]oxazin-4-ylidene}acetaldehydes (5b–l) under the same
reaction conditions. The Z-isomer was preferentially produced in
all of these reactions. Five predominant Z-isomers ((Z)-5d, 5e, 5i,
5k, 5l) were isolated. Results and the physical data of 5e–l were
listed in Table S2, see Supplementary data.
bromo-(E)-3-cinnamoylaminobenzo[b]furan15 occurs by treatment
with the VM reagent under the above reaction conditions.
Isomerization of the Z-isomers ((Z)-5) to corresponding the E-
isomer occurred in their solution. The (Z)-isomer ((Z)-5a) in
DMSO-d6 solution isomerized to (E)-5a in a time-dependent man-
ner reaching a constant ratio of (Z)-5a:(E)-5a = 5:2 after 48 h
according to 1H NMR analysis. The isomerization of two mixtures,
A ((Z)-5a:(E)-5a = 92:8) and B ((Z)-5a:(E)-5a = 62:38), was also
examined in toluene solution and found to reach a constant equi-
librium at the ratio of (Z)-5a:(E)-5a = 5:2 after 15 h by HPLC anal-
ysis (Fig. 2).23 These results suggested that (Z)-5a would be more
thermodynamically stable than (E)-5a. The heat of formation of
(Z)-5a was calculated to be 0.5 kcal/mol lower than that of (E)-5a.1
The isomerization between (Z)-5a and (E)-5a would be caused
by the formyl group which conjugated with the exo-methylene.
This was supported by the absence of isomerization of (Z)-2-((E)-
2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)ethanols
((Z)-
The ring closure reaction of 2-acetyl-(E)-3-aralkenylcarbonyl-
aminobenzo[b]furans (9) with VM reagent generated two different
fused-rings, that is, the oxazine ring of 5 and the pyridine ring of
10, depending on the functional group at the 3-position. The
2-acetyl group of 9 is indispensable for these ring closure reac-
tions,22 because no cyclization reaction of 2-(4-chlorobenzoyl)-5-
13a and (Z)-13b) prepared by NaBH4 reduction of the respective
(Z)-5a and (Z)-5c.
We prepared thirty derivatives using the exo-formylmethylene
group of 5 by Method C (NaH) and Method D (TiCl4), as shown in
Scheme 4. The compounds (5) were reacted with 11 phosphonate
reagents (11)24 in the presence of NaH under HWE reaction condi-
H
H
N
H
N
Br
Br
O
O
O
O
CHO
Z
O
N
E
H
Selected HMBC data of (Z )-5a
X-ray analysis and structure of 12a
Figure 1. Selected HMBC data of (Z)-(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene) acetaldehyde ((Z)-5a) and X-ray analysis of (Z)-(8-bromo-(E)-2-
styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)-N,N-diethylbut-(E)-2-enamide (12a).

3962
Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
R3
O
NHCOR3
N
R1
25°C
1) POCl3 - DMF
R1
R3
COCl
COCH3
8a or 8b
2) Method A (NaOH aq. sol.)
O
THF or CHCl3
15 - 92%
CHCHO
O
or
R2
R2
H
H
OCH3
OCH3
H
Method B (Et3N)
R2
R2
9
5
16 - 46%
R1
Br
Br
H
R3
CH=CHC6H5
R1
R3
9d
5a
Br
Br
H
H
H
OCH3
OCH3
H
CH=CHC6H5
CH=CHC6H4(4-OCH3)
CH=CHC6H5
CH=CHC6H4(4-OCH3)
CH=CHCH=CClC6H4(4-F)
9e
9f
9g
CH=CHC6H4(4-OCH3)
5b
5c
5d
5e
CH=CHC6H5
H
CH=CHC6H4(4-OCH3)
CH=CHCH=CClC6H4(4-F)
H
9h
Br
H
Br
H
9i
9j
9k
9l
CH=CHCH=CClC6H4(4-F)
5f
5g
5h
5i
CH=CHCH=CClC6H4(4-F)
OCH3
H
OCH3
H
OCH3
H
OCH3
H
Br
H
CH=CHCH=CClC6H4(4-Cl)
CH=CHCH=CClC6H4(4-Cl)
Br
H
CH=CHCH=CClC6H4(4-Cl)
CH=CHCH=CClC 6H4(4-Cl)
Br
Br
Br
Br
CH=CHCH=CHC6H5
CH=C(CH3)C6H4(4-OCH3)
CH=C(C6H5)2
Br
Br
Br
Br
CH=CHCH=CHC6H5
CH=C(CH3)C6H4(4-OCH3)
CH=C(C6H5)2
9m
9n
9o
H
5j
H
H
5k
5l
H
H
CH=CHCH3
H
CH=CHCH3
N
N
R1
NaBH4
THF
R1
O
O
O
O
CHCHO
CH2OH
R2
R2
H
5
13
5a: R1 = Br, R2 = H
13a : R1 = Br, R2 = H (60%)
5b: R1= H, R2= OCH3
13b : R1 = H, R2 = OCH3 (77%)
Scheme 3.
100
80
60
40
20
0
2-enoic acid esters (12e–g, 12o). Reactions of (Z)-5a with diethyl
cyanomethylphosphonate (11h) and tetraethyl methylenedi-
phosphonate (11i) gave the butadiene derivatives (12h and 12i),
respectively. Only a NOE correlation between Ha and Hc was
observed among the three olefinic H of 12a–i (Scheme 4).
A
B
This suggested that the carbon–carbon double bond introduced
by the HWE reaction has an E-form and the two carbon–carbon
double bonds of the butadiene moiety are oriented with the s-trans
conformation. This result was compatible with the structure of 12a
identified by X-ray analysis (Fig. 1). Physical and spectral data of
the butadiene derivatives (12) were listed in Table S3, see Supple-
mentary data. An aldehyde (Z)-5c was treated with 11b, 11e, 11f,
11g, 11h, 11i and 11k to afford the butadiene compounds 12p,
12q, 12r, 12s, 12t, 12u and 12v, respectively. Reaction of (Z,E)-5g
with 11f and reaction of (Z)-5i with 11i gave 12w and 12x, respec-
tively. Reaction of (Z)-5a with diethyl-4-chlorobenzylphosphonate
(11j) afforded the unexpected phosphonate 12j which appeared
likely to have been formed by dehydration instead of dephospho-
nation, similar to the Knoevenagel condensation mechanism. The
terminal carbon–carbon double bond of 12j has the E-form because
of the observation of only NOE between Ha and the hydrogen of the
phenyl ring (Scheme 4). Because we expected enhancement of the
binding to bone hydroxyapatite, the phosphonate group was intro-
duced to the molecule of 12. An aldehyde (Z)-5a was reacted with
11b, 11f, 11h, 11i in the presence of titanium tetrachloride (TiCl4)
and N-methylmorpholine (Method D) to produce the correspond-
ing butadiene derivatives (12k–n) bearing a phosphonate group
2
4
6
8
10
12
14
16
Time (h)
Isomeric
mixture comp.
Ratio of
:
(Z)-5a (E )-5a
original ratio ratio after 15h
A
B
92 : 8
5 : 2
5 : 2
62 : 38
Figure 2. Isomerization progress of mixture of (Z)-5a and (E)-5a.
tions to afford the corresponding butadiene derivatives (12)
(Method C). The reactions of (Z)-5a with N-substituted dialkyl 2-
amino-2-oxoethylphosphonates (11a–d) gave the corresponding
(Z)-4-(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-yli-
dene)-N- or N,N-(mono- or di)substituted but-(E)-2-enamides
(12a–d).
a-Substituted diethyl methylphosphonates (11e–g, 11k)
were reacted with (Z)-5a to afford the corresponding (Z)-4-(8-bro-
mo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)but-(E)-

Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
3963
R3
N
R1
O
R1CHR 2PO(OR3)2
+
Z
O
CHO
11
R2
H
5
(
Z
)-5a R1 = Br, R2 = H, R3 = C6H5
11a R1 = CON(C2H5)2, R2 = H, R3 = C2H5
11b R1 = CONCH2CH2OCH2CH2, R2 = H, R3 = C2H5
(Z )-5c R1 = H, R2 = OCH3, R3 = C6H5
(Z,E)-5g R1= Br, R2 = H, R3= CH=CClC6H4(4-Cl)
(Z )-5i R1 = Br, R2= H, R3 = CH=CHC6H5
11c R1 = CONHC6H4(3-OCH3), R2= H, R3 = C2H5
11d R1 = CONH(CH2)2C6H3(3, 4-OCH3), R2= H, R3 = C2H5
11e R1 = COOCH3, R2= H, R3 = CH3
11f R1= COOC2H5, R2 = H, R3 = C2H5
11g R1 = COOC(CH3)3, R2 = H, R3 = C2H5
11h R1 = CN, R2 = H, R3 = C2H5
11i R1= PO(OC2H5)2, R2 = H, R3 = C2H5
11j R1 = C6H4(4-Cl), R2 = H, R3 = C2H5
11k R1 = COOC2H5, R2 = CH3, R3 = C2H5
R3
R3
N
R1
N
R1
s-trans
Method C (NaH / THF, 6 - 79%)
O
O
Hb
H
Z
O
Method D (TiCl4, H3C
N
O / THF, 4 - 60%)
O
R2
Ha
R4
E
R2
H
R4
R5
(Hc)
12a-12j, 12n-12x, 12ac
R5
: NOE
12k-12m, 12y-12ab, 12ad
12a R1 = Br, R2= H, R3 = C6H5, R4 =CON (C2H5)2, R5= H
12b R1 = Br, R2 = H, R3 = C6H5, R4 = CONCH2CH2OCH2CH2, R5= H
12c
R
1 = Br, R2= H, R3 = C6H5, R4 = CONHC6H4(3-OCH3), R5 = H
12d R1 = Br, R2= H, R3 = C6H5, R4 = CONH(CH2)2C6H3(3, 4-OCH3), R5 = H
12e R1 = Br, R2= H, R3 = C6H5, R4 = COOCH3, R5 = H
12f R1= Br, R2 = H, R3= C6H5, R4 = COOC2H5, R5 = H
12g R1 = Br, R2 = H, R3 = C6H5, R4 = COOC(CH3)3, R5 = H
12h R1 = Br, R2 = H, R3 = C6H5, R4 = CN, R5 = H
12i
R
1 = Br, R2 = H, R3 = C6H5, R4 = PO(OC2H5)2, R5 = H
12j R1 = Br, R2 = H, R3 = C6H5, R4 = PO(OC2H5)2, R5 = C6H4(4-Cl)
12k R1 = Br, R2 = H, R3 = C6H5, R4 and R5 = PO(OC2H5)2, CONCH2CH2OCH2CH2
12l R1 = Br, R2 = H, R3 = C6H5, R4 and R5 = PO(OC2H5)
H5
2, COOC2
12m R1 = Br, R2 = H, R3 = C6H5, R4 and R5 = PO(OC2H5)2, CN
12n R1 = Br, R2 = H, R3 = C6H5, R4 = R5 = PO(OC2H5)2
12o R1 = Br, R2 = H, R3 = C6H5, R4 = COOC2H5, R5 = CH3
12p R1 = H, R2 = OCH3, R3 = C6H5, R4 = CONCH2CH2OCH2CH2, R5 = H
12q R1 = H, R2 = OCH3, R3 = C6H5, R4 = COOCH3, R5 = H
12r
12s
R
1 = H, R2 = OCH3, R3 = C6H5, R4 = COOC2H5, R5 = H
c. HCl
/ THF
R
1 = H, R2 = OCH3, R3 = C6H5, R4 = COOC(CH3)3, R5 = H
12t R1 = H, R2 = OCH3, R3 = C6H5, R4 = CN, R5 = H
12u R1 = H, R2 = OCH3, R3 = C6H5, R4 = PO(OC2H5)2, R5 = H
12v
R
1 = H, R2 = OCH3, R3 = C6H5, R4 = COOC2H5, R5 = CH3
12w R1 = Br, R2 = H, R3 = CH=CClC6H4(4-Cl), R4 = COOC2H5, R5 = H
12x
12y
R
R
1 = Br, R2 = H, R3 = CH=CHC6H5, R4 = PO(OC2H5)2, R5 = H
1 = H, R2 = OCH3, R3 = C6H5, R4 and R5 = PO(OC2H5)2, CONCH2CH2OCH2CH2
12z R1 = H, R2 = OCH3, R3 = C6H5, R4 and R5 = PO(OCH3)2, COOCH3
12aa R1 = H, R2 = OCH3, R3 = C6H5, R4 and R5 = PO(OC2H5)2, COOC2H5
12ab R1= H, R2= OCH3, R3= C6H5, R4 and R5= PO(OC2H5)2, CN
12ac
R
1 = H, R2 = OCH3, R3 = C6H5, R4= R5 = PO(OC2H5)2
12ad R1= Br, R2 = H, R3= C6H5, R4 and R5 = PO(OC2H5)2, COOH
Scheme 4.

3964
Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
at the terminal carbon.25 An aldehyde (Z)-5c was also reacted with
11b, 11e, 11f, 11h and 11i to afford the butadiene derivatives 12y,
12z, 12aa, 12ab, 12ac, respectively, under the conditions of Meth-
od D. Tetraethyl methylenediphosphonate (11i) produced diphos-
phonate compounds 12n and 12ac slowly by reaction with 5a
and 5c, respectively, while the reaction of ethyl diethylphosphono-
acetate 11f with 5a and 5c proceeded smoothly to give monopho-
sphonate 12l and 12aa, respectively. These reactions might
proceed via a cyclic titanium complex.25e
4H-3,1-Benzoxazines showed various kinds of physiology
activity.2–8 Therefore we performed two kinds of small scale bioac-
tive assays to find novel bioactivity for compounds prepared here.
First, we evaluated the anti-osteoclastic bone resorption in vitro of
seven representative compounds ((Z)-5a, (Z)-5l, 12a, 12b, 12d, 12f,
12j) prepared in this work. By coculture of fresh bone marrow
both types of cancer cells. Thus, two series of selective MIA Paca-
2 inhibitory new compounds were found, and examinations of
their inhibition mechanism of these compounds is preparing.
In conclusion, we established a new oxazine ring formation
method using the reaction of 2-acetyl-3-(2-aralkenylcarbonylami-
no)benzo[b]furans with VM reagent. This led to a novel application
of the Vilsmeier reaction for heterocyclization. (E and Z-(8-bromo-
(E)-2-aralkenylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene)acetalde-
hydes (5) bearing the characteristic exo-formylmethylene group at
the 4-position were prepared by this reaction.30 Unsaturated alde-
hydes (Z)-5 were reacted with several phosphonate reagents under
two reaction conditions to afford the butadiene derivatives (12)
having an ester or phosphonate or amide group on the terminal
carbon in the butadiene moiety. The butadiene ester and phospho-
nate compound (12f, 12j) showed potent anti-osteoclastic bone
resorption activity comparable to E2 (17b-estradiol), and the
evaluation of these activities of most of all prepared compounds
is under way. These results including detail mechanism of biolog-
ical activities will be reported elsewhere in due course. The exo-
formylmethylene compound (Z)-5f and the butadiene amide 12p
inhibited cell growth of MIA PaCa-2 and MCF-7, respectively. In
vivo studies for two kinds of biological activities are in progress,
aimed at developing new drugs for osteoporosis and pancreatic
cancer.
preosteoclasts expressing the receptor activator of NF-jB (RANK)
with calvarial osteoblasts that express the ligand for RANK (RANKL),
bone resorbing osteoclasts developed and formed resorption pits on
a dentin slice. PGE2 stimulated pit formation, and estrogens (e.g.,
estrogen 2 (E2)) inhibited PGE2-stimulated pit formation by sup-
pressing the RANKL effect.26 Among the compounds tested, the ethyl
ester 12f and phosphonate 12j showed potent inhibition compara-
ble to E2, whereas the amide (12a, 12b, 12d) and exo-formylmethyl-
ene compounds ((Z)-5a, (Z)-5l) were inactive (Fig. 3).27 The
butadiene moiety with an ester or a phosphonate functional group
might play an important role in inhibiting osteoclasts.
3. Experimental
Several estrogenic agents such as 2-methoxyestradiol and E2
were reported their growth inhibitory activity against human
pancreatic carcinoma (MIA PaCa-2) and breast cancer (MCF-7).28
Therefore, the aldehyde 5f and butadiene derivatives 12p and
12w were selected as representative compounds and evaluated
growth inhibitory activity in vivo against MIA PaCa-2 and MCF-7,
and the results are shown in Table 1.29 The butadiene amide
(12p) inhibited MIA PaCa-2 more than 5-FU. The exo-formylmeth-
ylene compound ((Z)-5f) showed more inhibitory activity against
MCF-7 than 5-FU. The butadiene ester (12w) was inactive against
All melting points were determined using a Yanaco microscopic
hot-stage apparatus and are uncorrected. 1H NMR, 13C NMR and
HMBC, HMQC spectra were obtained on a JEOL JNM-ECP400, JEOL
JNM-ECP500 or JEOL PMX60FT spectrometer with tetramethylsil-
ane as an internal standard. MS spectra (MS, HRMS) were obtained
using a JEOL JMS-700 EIMS spectrometer. Elemental analyses were
performed on a CHN CORDER MT-3 (Yanaco). All organic extracts
were dried over anhydrous MgSO4. Column chromatography was
carried out on Wakogel C-200. Thin layer chromatography was
performed on an E. Merck silica gel plate (0.5 mm, 60F-254).
160
140
120
100
3.1. 2-Acetyl-5-bromo-(E)-3-(4-methoxycinnamoylamino)-
benzo[b]furan (9e) and general procedure for 9a–d, 9f–o
To a solution of 8a (5.0 g, 19.7 mmol) in dry THF (120 ml) was
added 4-methoxycinnamoyl chloride (7.72 g, 39.3 mmol) in dry
THF (45 ml). The mixture was stirred at 68 °C for 6.0 h. The solution
was poured into water and a brown precipitate was deposited. The
precipitate was dissolved in chloroform. The organic layer was
washed with a saturated sodium bicarbonate solution, brine, and
dried. The solvent was evaporated to give a residue. The residue
was washed with hexane–ethyl acetate (5:1) and recrystallized
from ethyl acetate–chloroform (5:1) to afford 9e (3.94 g, 48%) as
pale brown needles.
80
60
40
**
**
**
20
0
12a 12b 12d 12f
12j
(Z)-
5a
(Z)-
5l
E2
cont.
Figure 3. Anti-osteoblastic bone resorption activities of the oxazine derivatives
((Z)-5a,(Z)-5l, 12a, 12b, 12d, 12f, 12j). All data were expressed as the means and
SEs(n = 5). cont.: control, E2: 17b-estradiol,
**
: significant difference (p < 0.05)
versus control.
3.2. 8-Bromo-4-chloro-3-formylbenzo[b]furo[3,2-b]pyridine
(10a) from 9a
Table 1
A mixture of phosphoryl chloride (0.79 ml, 8.48 mmol) and dry
DMF (2 ml) was stirred at 6 °C for 0.5 h under an N2 atmosphere.
The mixture was added dropwise to a solution of 9a (0.69 g,
2.15 mmol) in dry DMF (20 ml) at 6 °C and then stirred at 24 °C
for 2.5 h. Additional phosphoryl chloride (0.79 ml, 8.48 mmol)
was added to the mixture and stirred at 25 °C for 43 h. A solution
was poured into ice water and extracted with ethyl acetate. The or-
ganic layer was washed with brine and dried. The solvent was
evaporated off. The residue was purified with silica gel column
chromatography [CHCl3–ethyl acetate (5:1)] and [hexane–ethyl
In vitro cell growth inhibitory activities of 12p, 5f, 12w and 5-FU
a
Compd
GI50
(l
M)
MIA PaCa-2
MCF-7
12p
5f
12w
5-FU
5.34
>10
>10
>10
>10
7.14
>10
>10
a
GI50 shows the concentration of the compound which affords 50% inhibition in
cell growth compared to the negative control.

Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
3965
acetate (5:2)] and then recrystallized from ethyl acetate to give 10a
(0.08 g, 12%) as colourless plates: mp 214–215 °C. Calcd for
C12H5BrClNO2: C, 46.41; H, 1.62; N, 4.51. Found: C, 46.26; H,
1.53; N, 4.41. dY (400 MHz; CDCl3) 7.61 (1H, d, J = 8.8 Hz, 6-H),
7.80 (1H, dd, J = 8.8 and 2.2 Hz, 7-H), 8.41 (1H, d, J = 1.9 Hz, 9-H),
9.10 (1H, s, 2-H), 10.59 (1H, s, CHO); dC (125 MHz; CDCl3) 114.32,
117.94, 124.59, 125.28, 125.88, 129.41, 134.33, 146.61, 147.58,
147.79, 157.67, 187.61; m/z (EI) 313 (M+4, 25), 311 (M+2, 100),
309 (M+, 76), 284 (3), 282 (9), 280 (7), 247 (4), 245 (4).
3.6. Ethyl (Z)-4-(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]-
oxazin-4-ylidene)but-(E)-2-enoate (12f): (Method C) and
general procedure for 12aꢀj, 12oꢀx
To a mixture of ethyl diethylphosphonoacetate (11f) (0.3 ml,
1.50 mmol) and NaH (60% in oil, 0.076 g, 1.90 mmol) in anhy-
drous THF (2.0 ml) with stirring was added dropwise a solution
of (Z)-5a (0.5 g, 1.27 mmol) in anhydrous THF (90 ml) at 3 °C un-
der a N2 atmosphere. The mixture was stirred at 27 °C for 2 h.
The mixture was quenched with H2O and concentrated under re-
duced pressure. The residue was added to a saturated NH4Cl
solution and extracted with chloroform. The organic layer was
washed with brine and dried. The solvent was evaporated off
to give a residue which was recrystallized from ethyl acetate–
hexane (5:1) to give 12f1 (0.42 g, 71%) as red needles. Physical
and spectral data of 12bꢀ12e, 12gꢀ12j, 12oꢀ12x are shown in
Supplementary data.
3.3. 8-Bromo-4-chloro-3-formylbenzo[b]furo[3,2-b]pyridine
(10a) from 9c
A similar reaction to that described above using 9c gave 10a
(0.042 g, 3%): mp 210–212 °C.
3.4. 4-Chloro-3-formyl-6-methoxybenzo[b]furo[3,2-b]pyridine
(10b)
3.7. Diethyl 3-{(Z)-4-(8-bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]
oxazin-4-ylidene)-1-(N-morpholino-carbonyl)-(E)-1-propenylphos-
phonate (12k): (Method D) and general procedure for 12kꢀn, 12y,
12z, 12aa, 12ab, 12ac
Phosphoryl chloride (1.03 ml, 11.1 mmol) was added to dry
DMF (2 ml) under a N2 atmosphere at 6 °C with stirring. The mix-
ture was added dropwise to a solution of 9b (1.0 g, 3.67 mmol) in
dry DMF (30 ml) at 6 °C and then stirred at 24 °C for 24 h. Addi-
tional phosphoryl chloride (0.5 ml, 5.36 mmol) was added to the
mixture, which was stirred at 25 °C for 24 h. A solution was poured
into ice water and extracted with ethyl acetate. The organic layer
was washed with brine and dried. The solvent was evaporated
off. The residue was purified with silica gel column chromatogra-
phy [hexane–ethyl acetate (7:1)] and then recrystallized from hex-
ane–ethyl acetate (1:5) to give 10b (0.13 g, 14%) as colourless
needles: mp 191–193 °C. Calcd for C13H8ClNO3: C, 59.67; H, 3.08;
N, 5.35. Found: C, 59.65; H, 2.92; N, 5.27. dH (400 MHz; CDCl3)
4.11 (3H, s, OCH3), 7.20 (1H, dd, J = 8.0 and 0.7 Hz, 7-H or 9-H),
7.44 (1H, t, J = 7.9 Hz, 8-H), 7.84 (1H, dd, J = 8.0 and 1.1 Hz, 7-H
or 9-H), 9.10 (1H, s, 2-H), 10.59 (1H, s, CHO); dC (100 MHz; CDCl3)
56.46, 113.27, 113.97, 124.27, 125.46, 125.56, 129.43, 146.03,
146.09, 147.36, 148.61, 149.23, 187.87; m/z (EI) 263 (M+2, 33),
261 (M+, 100), 248 (3), 246 (9), 232 (2), 219 (3), 217 (5).
To a yellow suspension of TiCl4 (0.11 ml, 1.00 mmol) in carbon
tetrachloride (10 ml) was added dropwise a mixture of (Z)-5a
(0.2 g, 0.51 mmol) and 11b (0.12 g, 0.45 mmol) in anhydrous THF
(40 ml) at ꢀ10 to ꢀ4 °C, and the mixture was stirred at the same
temperature for 0.5 h. N-Methylmorpholine (0.45 ml, 4.09 mmol)
was added to the solution at ꢀ7 to ꢀ4 °C. The mixture was stirred
at ꢀ10 °C for 2 h and 25 °C for 12 h and poured into water. A chlo-
roform extraction was washed with brine and dried over MgSO4.
The solvent was evaporated off to give a residue which was puri-
fied with silica gel column chromatography [CHCl3–ethyl acetate
(20:1)] and recrystallized from hexane–ethyl acetate (5:1) to give
12k (0.07 g, 22%) as red needles. Physical and spectral data of
12kꢀn, 12y, 12z, 12aa, 12ab, 12ac are shown in Supplementary
data.
3.8. 2-{(Z)-(8-Bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-
3.5. (Z)-(8-Bromo-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-4-
ylidene)acetaldehyde ((Z)-5a) andgeneral procedure for (Z) -5d, -5e,
-5i, -5k, -5l; (E/Z)-5b, -5c, -5f, -5g, -5h, -5j, -5k
4-ylidene)}ethanol (13a)
To a solution of (Z)-5a (3.0 g, 7.61 mmol) in THF (320 ml) was
added sodium borohydride (0.35 g, 9.25 mmol), and the mixture
was stirred for 30 min at 45 °C. The mixture was poured into
water and concentrated by evaporation under reduced pressure
to give a precipitate. The precipitate was filtrated off and recrys-
tallized from THF–chloroform (1:1) to afford 13a (1.82 g, 60%) as
yellow needles: mp 200–203 °C. Calcd for C20H14BrNO3ꢁ1/2 H2O:
C, 59.28; H, 3.73; N, 3.46. Found: C, 59.44; H, 3.37; N, 3.43. dH
(400 MHz; DMSO-d6) 4.34 (2H, dd, J = 7.1 and 5.7 Hz, CH2OH),
4.83(1H, t, J = 5.7 Hz, CH2OH), 5.16 (1H, t, J = 7.2 Hz, @CHCH2OH),
6.85 (1H, d, J = 16.1 Hz, CH@CHC6H5), 7.40–7.47 (3H, m, 30-, 40-,
50-H), 7.59 (1H, dd, J = 8.8 and 2.2 Hz, 7-H), 7.61 (1H, d,
J = 16.1 Hz, CH@CHC6H5), 7.68 (1H, d, J = 8.8 Hz, 6-H), 7.75–7.78
(2H, m, 20-, 60-H), 7.85 (1H, d, J = 2.2 Hz, 9-H); m/z (EI) 397
(M+2, 24), 395 (M+, 26), 380 (12), 378 (12), 299 (2), 131 (100),
103 (68), 77 (50).
A mixture of phosphoryl chloride (1.2 ml, 12.9 mmol) and dry
DMF (2.0 ml) was stirred under a N2 atmosphere at 6 °C for
40 min. The mixture was added dropwise to a solution of 9d
(2.5 g, 6.51 mmol) in dry DMF (40 ml) at 6 °C and stirred at 25 °C
for 30 h. The orange precipitate deposited in the mixture was fil-
trated off. The precipitate was treated by Method A or B. Method
A: To a suspension of the orange precipitate in water (250 ml)
was added dropwise 10% NaOH aqueous solution at 25 °C to adjust
the pH at 10–11, and the mixture was stirred at 25 °C for 30 min to
obtain an orange precipitate as a powder. Method B: A suspension
of the orange precipitate in water (250 ml) was added dropwise to
a solution of triethylamine (1.82 ml, 13.1 mmol) in chloroform. The
mixture was vigorously stirred at 25 °C for 25 min and then ex-
tracted with chloroform. The organic chloroform layer was washed
with brine, then dried. The solvent was evaporated off to afford an
orange powder. Recrystallization of each orange powder from ethyl
acetate–chloroform (5:1) gave (Z)-5a as orange needles. It was very
difficult to isolate pure (Z)- 5b, -5c, -5f, -5g, -5h, -5j, -5k: dC
(125 MHz; CDCl3) 98.7 (OHC–CH@), 114.1 (C-5a), 118.0 (C-8),
118.1 (Ph–CH@CH), 124.0 (C-9a), 128.2 (C-20, 60), 129.2 (C-30, 50),
130.7 (C-40), 132.2 (C-7), 132.4 (C-9b), 132.9 (C-9), 134.6 (C-10),
138.0 (C-4a), 142.5 (Ph–CH@CH), 153.1 (C-4), 155.8 (C-5a), 156.7
(C-2), 185.8 (CHO).
3.9. 2-{(Z)-(6-Methoxy-(E)-2-styrylbenzo[b]furo[3,2-d][1,3]oxazin-
4-ylidene)}ethanol (13b)
Aldehyde (Z)-5c was treated with sodium borohydride in a
similar manner to (Z)-5a to afford pale yellow prisms (13b):
mp 186–190 °C. Calcd for C21H17NO4: C, 72.61; H, 4.93; N,
4.03. Found: C, 72.44; H, 4.89; N, 4.04. dH (400 MHz; DMSO-d6)
3.97 (3H, s, OCH3), 4.33 (2H, d, J = 6.6 Hz, CH2OH), 4.81 (1H, br

3966
Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
s, CH2OH), 5.12 (1H, t, J = 7.2 Hz, @CHCH2OH), 6.87 (1H, d,
J = 16.1 Hz, CH@CHC6H5), 7.10 (1H, dd, J = 7.3 and 1.5 Hz, 7-H),
7.27–7.34 (2H, m, 8-, 9-H), 7.39–7.47 (3H, m, 30-, 40-, 50-H),
7.58 (1H, d, J = 16.2 Hz, CH@CHC6H5), 7.75–7.77 (2H, m, 20-, 60-
H); m/z (EI) 347 (M+, 100), 330 (86), 303 (17), 244 (4), 217
(11), 131 (31), 103 (36).
Acknowledgments
This work was supported in part of a grant from the Ministry of
Education, Culture, Sports, Science and Technology for the ‘Univer-
sity–Industry Joint Research’ Project (2004–2008). We thank the
staff of the instrument analysis center of Mukogawa Women’s Uni-
versity for the NMR and MS measurements and elemental
analyses.
4. Evaluation of anti-bone resorption activity
Calvarial osteoblasts precultured to preconfluent from 1 to
2 day old ddY mice (Shizuoka Laboratory Animal Center, Hamama-
tsu, Japan) and fresh bone marrow cells from 5 week-old ddY male
mice (Shizuoka Laboratory Animal Center) were cocultured in
Supplementary data
Supplementary data (physical and spectral data of 5, 9 and 12)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.04.017.
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)
References and notes
and 1.0 lM prostaglandin E2 (PGE2, Sigma Chemical Co.)(13) for
1. Part of this work has been published as a preliminary communication: Ando,
Y.; Ando, K.; Yamaguchi, M.; Kunitomo, J.; Koida, M.; Fukuyama, R.; Nakamuta,
H.; Yamashita, M.; Ohta, S.; Ohishi, Y. Bioorg. Med. Chem. Lett. 2006, 16, 5849.
2. Hsieh, P.-W.; Chang, F.-R.; Chang, C.-H.; Cheng, P.-W.; Chiang, L.-C.; Zeng, F.-L.;
Lin, K.-H.; Wu, Y.-C. Bioorg. Med. Chem. Lett. 2004, 14, 4751.
7 days on a 100 mm dish (Greiner, Tokyo, Japan) precoated with
collagen (Cell matrix Type I-A, Nitta Gelatin Inc., Osaka, Japan)
for the development of osteoclasts. Cells were then resuspended
by collagenase digestion and plated over dentin slices (10 mm in
3. (a) Gutschow, M.; Kuerschner, L.; Neumann, U.; Pietsch, M.; Loeser, R.; Koglin,
N.; Eger, K. J. Med. Chem. 1999, 42, 5437; (b) Krantz, A.; Spencer, R. W.; Tam, T.
F.; Liak, T. J.; Copp, L. J.; Thomas, E. M.; Rafferty, S. P. J. Med. Chem. 1990, 33, 464.
4. Gutschow, M.; Neumann, U. Bioorg. Med. Chem. 1997, 5, 1935.
5. (a) Neumann, U.; Gutschow, M. Bioorg. Chem. 1995, 23, 72; (b) Hedstrom, L.;
Moorman, A. R.; Dobbs, J.; Abeles, R. H. Biochemistry 1984, 23, 1753.
6. Hays, S. J.; Caprathe, B. W.; Gilmore, J. L.; Amin, N.; Emmerling, M. R.; Michael,
W.; Nadimpalli, R.; Nath, R.; Raser, K. J.; Stafford, D.; Watson, D.; Wang, K.; Jaen,
J. C. J. Med. Chem. 1998, 41, 1060.
diameter and 0.64 mm in height) in
a-MEM containing 10 nM E2
or the oxazine derivatives on a 24-well plate (Greiner) for 2 days
pit formation. Slices were dipped in 0.01 N NaOH, treated with
ultrasonic waves to remove the cells and then dried and stained
with 0.1% toluidine blue in 1.0% sodium borate for pit counting.
The decrease of the number of pits on slice indicates anti-bone
resorption activity of test compound.
7. Brown, A. D.; Powers, J. C. Bioorg. Med. Chem. 1995, 3, 1091.
8. Abood, N. A.; Schretzman, L. A.; Flynn, D. L.; Houseman, K. A.; Wittwer, A. J.;
Dilworth, V. M.; Hippenmeyer, P. J.; Holwerda, B. C. Bioorg. Med. Chem. Lett.
1997, 7, 2105.
5. Materials and methods for measurement of growth
inhibitory activity on cancer cell lines
9. Eckstein, Z.; Urbanski, T. Adv. Heterocycl. Chem. 1978, 23, 1.
10. (a) Messeri, T.; Pentassuglia, G.; Fabio, R. D. Tetrahedron Lett. 2001, 42, 3227; (b)
Wiley, M. R.; Weir, L. C.; Briggs, S.; Bryan, N. A.; Buben, J.; Campbell, C.;
Chirgadze, N. Y.; Conrad, R. C.; Craft, T. J.; Ficorilli, J. V.; Franciskovich, J. B.;
Froelich, L. L.; Gifford-Moore, D. S.; Goodson, T., Jr.; Herron, D. K.; Klimkowski,
V. J.; Kurz, K. D.; Kyle, J. A.; Masters, J. J.; Ratz, A. M.; Milot, G.; Shuman, R. T.;
Smith, T.; Smith, G. F.; Tebbe, A. L.; Tinsley, J. M.; Towner, R. D.; Wilson, A.; Yee,
Y. K. J. Med. Chem. 2000, 43, 883; (c) Hart, D. J.; Magomedov, N. Tetrahedron Lett.
1999, 40, 5429; (d) Hernandez, F.; Avendano, C.; Sollhuber, M. Tetrahedron Lett.
2003, 44, 3367.
5.1. Reagents
5-Fluorouracil (5-FU) and dimethyl sulfoxide (DMSO) were pur-
chased from Sigma Chemical Co. Stock solutions of the prepared
compounds or 5-FU were prepared by dissolving each compound
into DMSO at 10 lM. Some of the dilutions were subsequently pre-
pared in growth medium (D-MEM or E-MEM). The final concentra-
11. Gutschow, M. J. Org. Chem. 1999, 64, 5109.
12. Miller, C. P. Curr. Pharm. Des. 2002, 8, 2089.
13. (a) Hochner-Celnikier, D. Eur. J. Obstet. Gynecol. Reprod. Biol. 1999, 85, 23; (b)
Maricic, M.; Gluck, O. Exp. Opin. Pharmacother. 2002, 3, 767.
tion of DMSO in growth medium was made to be 0.25% or less.
14. (a) Renaud, J.; Bischoff, S. F.; Buhl, T.; Floersheim, P.; Fournier, B.; Geiser, M.;
Halleux, C.; Kallen, J.; Keller, H.; Ramage, P. J. Med. Chem. 2005, 48, 364; (b)
Grese, T. A.; Sluka, J. P.; Bryant, H. U.; Cullinan, G. J.; Glasebrook, A. L.; Jones, C.
D.; Matsumoto, K.; Palkowitz, A. D.; Sato, M.; Termine, J. D.; Winter, M. A.;
Yang, N.-N.; Dodge, J. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14105; (c)
Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.; Bonn, T.; Engstrom, O.;
Ohman, L.; Greene, G. L.; Gustafsson, J.-A.; Carlquist, M. Nature 1997, 389, 753;
(d) Jordan, V. C. J. Med. Chem. 2003, 46, 883.
5.2. Cell Lines
MIA Paca-2 ‘human pancreatic carcinoma’ and MCF-7 ‘human
adenocarcinoma of the breast’ were purchased from the Japan
Health Sciences Foundation. MCF-7 was grown in E-MEM. MIA
Paca-2 was grown in D-MEM. Each medium was supplemented
with 10% of fetal calf serum (MultiSerTM) and 6 ml of antibiotic-anti-
mycotic 100ꢂ (GIBCO).
15. Ando, K.; Tsuji, E.; Ando, Y.; Kuwata, N.; Kunitomo, J.; Yamashita, M.; Ohta, S.;
Kohno, S.; Ohishi, Y. Org. Biomol. Chem. 2004, 2, 625.
16.
A
part of this work was presented at the ‘International Symposium of
Maxillofacial Oral Regenerative Biology in Okayama (Japan) 2005’. The
&
proceedings version of the presentation was published in (Koida, M.;
Nakamuta, H.; Ohishi, Y.) J. Hard Tissue Biological, (Special Issue) 2005, 14.
160. Paper in preparation.
TM
5.3. AlamarBlue assay for cell cytotoxicity
TM
17. Jutz, C. Adv. Org. Chem. 1976, 9, 225.
An alamarBlue (Biosource) assay was used to measure cell
cytotoxicity. The human cells were seeded at 1 ꢂ 104 cells in
18. (a) Amaresh, R. R.; Perumal, P. T. Indian J. Chem., Sect. B 1997, 36, 541; (b) Meth-
Cohn, O.; Tarnowski, B. Adv. Heterocycl. Chem. 1982, 31, 207; (c) Meth-Cohn, O.
Heterocycles 1993, 35, 539; (d) Meth-Cohn, O.; Taylor, D. L. J. Chem. Soc. Chem.
Commun. 1995, 1463; (e) Jackson, A.; Meth-Cohn, O. J. Chem. Soc. Chem.
Commun. 1995, 1319.
19. (E)-5-Phenyl-penta-2-enoic acid was prepared according to the Ashton
method. 1H NMR: dH (60 MHz, CDCl3, Me4Si) 2.61ꢀ3.45 (4H, m, C6H5CH2,
C6H5CH2CH2) 5.84 (1H, d, J = 14.6 Hz, CH@CHCOOH or CH@CHCOOH),
6.90ꢀ7.25 (6H, m, phenyl-H, CH@CHCOOH or CH@CHCOOH). m/z (EI) 176
(M+, 10), 158 (4), 130 (8), 91 (100), 65 (8). Ashton, M. J.; Hills, S. J.; Newton, C.
G.; Taylor, J. B.; Tondu, S. C. D. Heterocycles 1989, 28, 1015. All of the acid
chlorides were prepared in the usual manner.
200 ll of growth medium/well in 96-well flat bottom tissue cul-
ture plates (Nunc). The cells were incubated for 24 h at 37 °C in a
humidified atmosphere of 5% CO2 in air. Next, the growth media
in plates were eliminated, and then 180
taining drug was added to triplicate wells. The cells were incubated
ll of growth medium con-
continuously for 72 h. Following incubation of the plates, 20 ll of
TM
alamarBlue was added to all wells, and the plates were set in an
incubator for an additional 3 h. The live cells were counted on a
microplate reader (Spectra Max M5, Molecular Devices), using an
excitation wavelength of 530 nm and emission wavelength of
590 nm.
20. Fukuyama, T.; Arai, M.; Matsubara, H.; Ryu, I. J. Org. Chem. 2004, 69, 8105.
21. Clough, S. C.; Gupton, J. T.; Driscoll, D. R.; Griffin, K. A.; Hewitt, A. M.; Hudson,
M. S.; Ligali, S. A.; Mulcahy, S. P.; Roberts, M. N.; Miller, R. B.; Belachew, T. T.;
Kamenova, I. D.; Kanters, R. P. F.; Norwood, B. K. Tetrahedron 2004, 60, 10165.

Y. Tabuchi et al. / Bioorg. Med. Chem. 17 (2009) 3959–3967
3967
22. Marson, C. M. Tetrahedron 1992, 48, 3659.
Morinaga, T.; Higashio, K. Endocrinology 1998, 139, 1329; (c) Yasuda, H.; Shima,
N.; Nakagawa, N.; Yamaguchi, 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. 1998, 95, 3597.
27. These compounds were evaluated by the procedure reported in Ref. 1.
28. (a) Schumacher, G.; Kataoka, M.; Roth, J. A.; Mukhopadhyay, T. Clin. Cancer Res.
1999, 5, 493; (b) Brady, H.; Desai, S.; Gayo-Fung, L. M.; Khammungkhune, S.;
McKie, J. A.; O’leary, E.; Pascasio, L.; Sutherland, M. K.; Anderson, D. W.;
Bhagwat, S. S.; Stein, B. Cancer Res. 2002, 62, 1439.
29. Kuramoto, M.; Sakata, Y.; Terai, K.; Kawasaki, I.; Kunitomo, J.; Ohishi, T.;
Yokomizo, T.; Takeda, S.; Tanaka, S.; Ohishi, Y. Org. Biomol. Chem. 2008, 6, 2772.
30. A few compounds somewhat similar to the oxazine derivatives prepared here
have been reported. However, the starting materials and reaction conditions
were quite different from those of our preparation method; (a) Costa, M.; Della
Ca, N.; Gabriele, B.; Massera, C.; Salerno, G.; Soliani, M. J. Org. Chem. 2004, 69,
2469; (b) Echavarren, A. M. J. Org. Chem. 1990, 55, 4255.
23. HPLC conditions, column: Inertsil ODS-2, 4.6 mm ꢂ 15 cm, UV detector:
325 nm, solvent: 0.05 M KH2PO4/CH3CN = 30: 70, flow rate: 1.0 ml/min, 25 °C
24. Four phosphonates (11a–d) were prepared according to our previously
reported procedure (Ando k.; Tsuji E.; Ando Y.; Kunitomo J.; Kobayashi R.;
Yokomizo T.; Shimizu T.; Yamashita M.; Ohta S.; Nabe T.; Kohno S.; Ohishi Y.
Org. Biomol, Chem., 2005, 3, 2129–2139).
25. (a) Nagy, I.; Hajos, G.; Riedl, Z. Heterocycles 2004, 63, 2287; (b) Begum, S.;
Rahman, M. M.; Takagi, R.; Ohkata, K. Heterocycles 2004, 62, 251; (c)
Steinmeyer, A.; Schwarz, K.; Haberey, M.; Langer, G.; Wiesinger, H. Steroids
2001, 66, 257; (d) Reetz, M. T.; Peter, R.; Von Itzstein, M. Chem. Ber. 1987, 120,
121; (e) Lehnert, W. Tetrahedron 1974, 30, 301.
26. (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.; Yano, K.; Fujise, N.; Sato,
Y.; Goto, M.; Yamaguchi, K.; Kuriyama, M.; Kanno, T.; Murakami, A.; Tsuda, E.;