Antiangiogenic versus cytotoxic activity in analogues of aeroplysinin-1

Article abstract of DOI:10.1016/j.bmc.2007.05.011

A series of analogues of the potentially angiogenic inhibitor aeroplysinin-1 1 were synthesized and their in vitro antiangiogenic and cytotoxic activities evaluated. In the case of epoxy ketone 6 and azlactone 36 the relationship sprouting inhibition assay/cytotoxicity in BAE cells was enhanced by one order and two orders of magnitude, respectively, with respect to the reference. These results imply more specific antiangiogenic properties for the synthesized derivatives.

Full text of DOI:10.1016/j.bmc.2007.05.011

Bioorganic & Medicinal Chemistry 15 (2007) 5300–5315
Antiangiogenic versus cytotoxic activity in analogues
of aeroplysinin-1
a,
b
*
´
´
´
Ruben Cordoba, Nelida Salvador Tormo,
b
a,
*
´
´
Antonio Fernandez Medarde and Joaquın Plumet
aDepartamento de Quı´mica Orga´nica, Facultad de Quı´micas, Universidad Complutense, 28040 Madrid, Spain
bInstituto Biomar, S.A., Polı´gono Industrial, Edificio CEEI, 24231 Onzonilla, Leo´n, Spain
Received 7 March 2007; revised 25 April 2007; accepted 2 May 2007
Available online 6 May 2007
´
Dedicated to Professor Miguel Angel Yus (Universidad de Alicante) on occasion of his 60th birthday.
Abstract—A series of analogues of the potentially angiogenic inhibitor aeroplysinin-1 1 were synthesized and their in vitro antian-
giogenic and cytotoxic activities evaluated. In the case of epoxy ketone 6 and azlactone 36 the relationship sprouting inhibition assay/
cytotoxicity in BAE cells was enhanced by one order and two orders of magnitude, respectively, with respect to the reference. These
results imply more specific antiangiogenic properties for the synthesized derivatives.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Aeroplysinin-1 1 (Fig. 1a) is a natural product isolated,
in both enantiomeric forms, as a secondary metabolite
present in different marine sponges.5,6
Cancer is a group of diseases characterized by the exces-
sive and uncontrolled growth of cells that leads to the
formation of tumours. In the evolutionary process of a
tumour three differentiated phases can exist: (1) neopla-
sic transformation. (2) Tumour growth. (3) Metastasis.
Many biological properties of this compound have been
described, such as antibacterial activity,7 inhibition of
the EGFR tyrosine kinase in in vitro test systems8 and
cytotoxic activity against several tumour cell lines and
antileukaemic activity in both in vivo and in vitro test
systems.9 Aeroplysinin-1 1 has also shown antiangio-
genic properties10 as it inhibits certain functions of the
endothelial cells (these cells are necessary for the forma-
tion of new blood vessels) in both in vivo and in vitro
assays, using less dose than the ones required for other
angiogenic inhibitors previously reported.11
Angiogenesis, the formation of new blood vessels from
the pre-existing vascular bed of the guest,1 is a necessary
process induced by the tumour for its growth due to the
distance limit of 100–200 lm that the original tumoral
cells can stand to be from a blood vessel in order to
achieve their metabolic needs.2 Therefore, the process
of angiogenesis is an important target in order to inhibit
the tumour growth and, consequently, the harm of the
disease.3 The possibility to cultivate endothelial cells
in vitro and the development of in vivo assays to confirm
the in vitro results have led to an important increase of
the angiogenesis studies in the last years.4
We have initiated a study on the sprouting inhibition assay/
cytotoxicity relationship of aeroplysinin-1 1 analogues in
order to select structures having specific antiangiogenic
properties without cytotoxic activities. For the synthesis
Br
Br
MeO
Br
MeO
Br
OR1
R2
O
OH
OH
R
O
CN
Keywords: Antiangiogenic compounds; Cytotoxicity; Aeroplysinin-1;
Spiroepoxycyclohexadienones.
1
b) Spiroepoxycyclo-
hexadienones( )
a) Aeroplysinin-1
c) Aromatic analogues
*
Corresponding authors. Tel.: +34 91 8466160; fax: +34 91 8466001
(R.C.); Tel.: +34 91 3944100; fax: +34 913944100 (J.P.); e-mail
addresses: rcordoba@pharmamar.com; plumety@quim.ucm.es
Figure 1. Structures of aeroplysinin-1 1 and the two types of analogues.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.011

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5301
and biological test of analogues of 1, we have chosen two
types of compounds: (1) spiroepoxycyclohexadienones as
structural analogues of the non-aromatic cyclic skeleton
(Fig. 1b). (2) Aromatic compounds with analogue cyclic
substitution (Fig. 1c). This selection has been done mainly
for three reasons: (a) the simplicity of the synthetic meth-
ods used for the preparation of compounds 1b and 1c
(Fig. 1). That allows us for the synthesis of a significant
number of compounds by means of versatile and not elab-
orated synthetic procedures; (b) the spiroepoxycyclo-
hexadienone analogues can undergo ring-opening
reactions of the epoxide moiety under ‘physiological’ con-
ditions to yield molecules with structural similarity to
aeroplysinin-1 1; (c) regarding the aromatic analogues,
these compounds could be the final products of the aro-
matization process of the natural product, also under
‘physiological’ conditions, in the cell environment with
concomitant ring-opening of the epoxide substructure.
In consequence, we report in this paper the synthesis of
several compounds with structure 1b and 1c (Fig. 1) and
the biological evaluation of the structure-specific antian-
giogenic activity relationship of these compounds.
Compounds 6 (33% overall yield) and 713 (39% overall
yield) were synthesized following this procedure using,
respectively, 2,4-dimethoxybenzaldehyde 2 and 2,4,6-tri-
methoxybenzaldehyde as starting materials. In the case
of epoxy ketone 8, the ortho monobromination of com-
pound 3 using PHPB (pyridinium hydrobromide perbr-
omide)14,15 took place with poor regioselectivity because
the formation of the brominated by-products 14 and 4
(Scheme 2). Nevertheless, compound 13 was easily iso-
lated by column chromatography (Hex/AcOEt, 1:1).
Lithium aluminium hydride reduction of 13 and Beck-
er–Adler oxidation of the resulting product 15 afforded
spiroepoxycyclohexadienone 8.
In order to achieve the synthesis of compounds 9 and
1016 (Scheme 3) the synthetic protocol was slightly mod-
ified due to the instability of the salicylic alcohols 17 and
18. Thus, the reduction and oxidation processes were
done in a single step without isolating those alcohols.
In addition, synthesis of compound 14 was achieved
by reaction of 2,4-dimethoxybenzaldehyde 2 with pyrid-
inium tribromide and, subsequently, selective cleavage
of the phenolic ether in position ortho respect to the
aldehyde moiety. Due to the presence of the methoxy
groups in 2 and 4 positions, no aromatic bromination
of position 3 took place.
2. Results and discussions
2.1. Chemistry
Spiroepoxycyclohexadienone 1117 was directly obtained
by oxidation of commercially available salicylic alcohol
19 (Scheme 4), whereas epoxy ketone 12 was synthesized
using three reaction steps with salicylic aldehyde as
starting material. It is worthy of note that in the
2.1.1. Spiroepoxycyclohexadienones. For the synthesis of
epoxy ketone analogues 6–12 (Fig. 2) the methodology
described by K. Hinterding et al.8d,12 to obtain com-
pound 6 was used (Scheme 1).
Compound
R1
R2
R3
R4
R1
number
R2
R3
O
O
6
7
Br
Br
Br
H
MeO
MeO
MeO
MeO
MeO
H
Br
Br
H
H
MeO
H
R4
8
9
Br
H
H
10
11
12
H
H
H
Br
Br
H
Br
H
H
Figure 2. Spiroepoxycyclohexadienones synthesized.
Br
MeO
Br
OH
MeO
OH
OH
MeO
OMe
H
Br2.HBrPy / Py
BCl3 / CH2Cl2
r.t,99%
H
H
50ºC,80%
4
O
3
O
O
2
Br
Br
MeO
MeO
Br
O
O
NaBH4 / THF
r.t,75%
NaIO4
OH
HCl-H2O
THF,50%
Br
5
6
Scheme 1. Previously reported synthesis of epoxy ketone 6.

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5302
Br
Br
Br2.HBrPy
MeO
Br
OH
MeO
Br
OH
MeO
OH
MeO
OH
Py
H
H
1.0 Eq.
H
H
O
O
O
O
14
4
13
(31%)a)
3
(19%)a)
(28%)a)
LAH
THF
45 %
MeO
Br
Br
NaIO4
MeO
O
OH
OH
HCl/H2O
THF
O
50 %
15
8
Scheme 2. Synthesis of epoxy ketone 8. (a) Product ratio determined by 1H NMR. 22% of starting material was recovered.
3
MeO
OMe
H
MeO
Br
OMe
H
MeO
Br
OH
Br2.HBrPy / Py
91 %
BCl3 / CH2Cl2
86 %
H
O
O
16
14
O
2
MeO
OH
OH
MeO
R2
OH
H
MeO
O
iii) NaIO4
HCl-H2O
i) LAH/THF
ii) H3O+
O
R2
R2
9 R2=Br
O
17 R2=Br
18 R2=H
14 R2=Br
3 R2=H
(53 % Overall yield)
10 R2=H
(63 % overall yield)
Scheme 3. Synthesis of epoxy ketones 9 and 10.
treatment of alcohol 22 with sodium periodate the
temperature must be controlled under 25 ꢁC in order
to avoid the dimerization process giving 23.18
2.1.2. Aromatic compounds. Analogues 24, 25 and 26, with
different phenolic protection group, were obtained by
simple transformations of salicylic aldehyde 4 (Scheme
5). The subsequent reduction of 24 and 25 with NaBH4
led to the isolation of 27, 28 and 29, whereas the treatment
of 25 with LiAlH4 led to the isolation of 29 and 31.
NaIO4
H2O
O
OH
OH
The formation of compound 29 can be explained as an
intramolecular transesterification of the intermediary
reaction product 30 followed by a reduction process of
the carbonylic moiety.
O
MeOH
55 %.
Br
Br
11
19
Br
The Wittig reaction was also used for the synthesis of aro-
matic analogues. Thus, the treatment of salicylic alde-
hydes 3 and 4 with Ph3P@CHCO2Me as fosforane led
to the formation of alkenes 32 and 33, both compounds
as a unique (E) isomer (Scheme 6). In order to attempt a
selective reduction of the alkenyl moiety, these com-
pounds were treated with magnesium in methanolic solu-
tion.19 In the case of the non-brominated compound 32
the reaction took place with high selectivity and high
yield, whereas in the case of 33 a complex mixture of
debrominated products, with and without reduction of
the carbon–carbon double bond, was obtained. There-
fore, in order to obtain compound 35, the non-bromi-
nated compound 34 was synthesized in first place and,
subsequently, treated with 2.12 equivalents of pyridinium
tribromide.
OH
LAH/THF
61 %
OH
Br2.HBrPy/Py
83 %
H
H
Br
O
O
21
20
Br
Br
NaIO4
HCl/H2O
O
OH
OH
O
THF
50 %
Br
Br
12
22
O
O
O
H
Br
Br
O
Br
Br
23
Finally, we focussed the synthesis of aromatic analogues
towards the azlactone20 36 (Scheme 7) due to the high
Scheme 4. Synthesis of epoxy ketones 11 and 12.

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5303
Br
Br
MeO
Br
OBn
H
NaBH4
MeO
Br
OBn
THF
64%
OH
24
O
27
(i)
93 %
Br
Br
Br
Br
O
H+
MeO
Br
OH
MeO
Br
OCH2COOMe
MeO
Br
OCH2COOMe
MeO
Br
O
OMe
(ii)
76 %
NaBH4
THF
H
OH
H
O
O
28
46%
O
25
30
4
LiAlH4
(iii)
62 %
THF
Br
Br
Br
Br
H-
O
MeO
Br
MeO
Br
OAc
H
MeO
Br
OCH2CH2OH
O
MeO
Br
29
27%
OH
O
OH
O
O
7%
47%
O
26
29
31
Scheme 5. Synthesis of aromatic analogues. Reagents and conditions: (i) BrBn, K2CO3, DMF, 95 ꢁC, 3 h; (ii) BrCH2CO2Me, K2CO3, 18-crown-6,
acetone, rt, 3 h; (iii) AcCl, TBAH, NaOH, dioxane, rt, 32h.
MeO
MeO
OH
Mg/ MeOH
MeO
OH
Ph3P=CHCO2Me
THF
, 5 h
Cuant.
3 h, r. t.
70 %
H
CO2Me
OH
O
32
3
PHPB (2,12 Eq.)
Py
Br
MeO
OH
2 h., r. t.
93 %
Br
CO2Me
CO2Me
34
35
Br
Br
MeO
Br
OH
Ph3P=CHCO2Me
Mg/ MeOH
3 h, r. t.
MeO
Br
OH
complex
mixture
THF
Δ, 5 h
Cuant.
H
CO2Me
O
33
4
Scheme 6. Synthesis of aromatic analogues.
Br
36
Br
reactivity of aromatic aldehydes with hipuric acid (N-
benzoylglycine) as previously described for the non-bro-
minated compound in the Andersen and Faulkner6a,21
synthesis of aeroplysinin-1 1.
MeO
OBn
MeO
Br
OBn
H
N-benzoylglycine
Ph
N
AcONa anh.
Ac2O
3 h, 85 ºC
52 %
Br
H
O
O
O
24
Thus, the treatment of benzaldehyde 24 with hipuric
acid (N-benzoylglycine) and AcONa in Ac2O led to
the isolation of azlactone 36 as a (Z) diastereomer.22
Another analogue was isolated by methanolic treatment
of azlactone 36 rendering compound 37 in an oxazolone
ring opening process.
MeOH/ MeONa
30 min., r. t.
82%
Br
MeO
Br
OBn
NHCOPh
COOMe
At this stage, the biological results of the aeroplysinin-1
1 analogues synthesized were evaluated (see Section 2.2).
Azlactone 36 showed, significantly, the best sprouting
inhibition assay/cytotoxicity relationship.
37
Scheme 7. Synthesis of aromatic analogues.

´
R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5304
Br
2.2. Biological results and discussion
MeO
Br
OBz
H
67 %
The biological evaluation of the synthesized analogues
(Table 1) has been done using bovine aortic endothelial
cells (BAEC) in an in vitro assay system and taking
aeroplysinin-1 1 as a reference. Two types of tests were
carried out: (a) cytotoxicity assay: IC50, lM (the drug
concentration inhibiting the growth value of the BAE
cells by 50%); (b) sprouting inhibition assay: MIC, lM
(the minimum inhibition concentration at which no tube
formation is observed). In this test, the endothelial cells
are supported in pearls for a 3-D angiogenic event sim-
ulation (Fig. 3).
BzCl/Et3N
Dioxane
r.t.
O
38
Br
N
Br
MeO
Br
O
MeO
Br
OH
4-Me2N-BzCl
O
CHO
51 %
H
Et3N, r.t.
Dioxane
39
O
4
K2CO3, DMF
75 ºC
Br
Br
Br
N
MeO
Br
O
N
Spiroepoxycyclohexadienones 8, 12 and 23 showed the
same magnitude values for the inhibition assay (Table
1) as the reference (aeroplysinin-1 1). As the reference it-
self, these compounds also showed cytotoxic activity
against the endothelial cells, therefore, having a low ra-
tio between angiogenesis inhibition and cytotoxicity. It
is worthy of note that compounds 8 and 12 are suitable
for dimerization in mild conditions, whereas 23 is a di-
meric compound. The cytotoxicity of this type of di-
meric spiro-epoxy compounds has been previously
reported in the literature.26
CHO
Br
87 %
40
Scheme 8. Phenolic protection for the synthesis of azlactone 36
derivatives.
Consequently, the next part of this study was focussed in
the synthesis of azlactone 36 derivatives that could im-
prove the water solubility and, therefore, the biological
evaluation. For this purpose was selected in order to fi-
nally obtain a hydrochloride. In this context, the pheno-
lic group of compound 4 was protected using benzoyl
chloride, 4-N,N-dimethylbenzoyl chloride and 2-bro-
mo-5-N,N-dimethylbenzyl bromide23 to obtain com-
pounds 38 (this one was synthesized in order to
evaluate the effect of the N,N-dimethyl moiety in the
activity), 39 and 40, respectively (Scheme 8). The treat-
ment of these aldehydes with hipuric acid led to the for-
mation of azlactones 41, 42, 43 and 44 (Scheme 9), being
42 a partial benzoyl ester cleavage24 by-product isolated
in the synthesis of azlactone 41. Finally, hydrochloride25
45 was synthesized by treatment of azlactone 44 with
gaseous hydrogen chloride in dioxane.
Nevertheless, spiroepoxycyclohexadienone 6, with two
bromine atoms and a methoxylic group in the dienic sys-
tem substitution, showed a significant increase in the
sprouting inhibition assay/cytotoxicity relationship com-
pared with the aeroplysinin-1 1 values.
In the case of the aromatic analogues synthesized (Table
1) only the azlactone 36 showed interesting values in the
biological evaluation, exhibiting a 103 value for the
sprouting inhibition assay/cytotoxicity ratio. This
value is about 102-fold higher than the ratio for the
Br
Br
Br
MeO
Br
OH
(i)
MeO
Br
OBz
MeO
Br
OBz
H
N
N
O
O
O
15%
38
O
O
18%
41
42
Br
Br
N
N
MeO
Br
O
MeO
Br
O
(i)
O
CHO
O
O
N
19%
O
39
43
Br
Br
Br
Br
Br
Br
MeO
Br
O
MeO
Br
O
MeO
Br
O
(i)
(ii)
N
Cl
NH
Ph
N
Ph
N
N
51%
CHO
O
O
21%
O
O
40
45
44
Scheme 9. Synthesis of azlactone 36 derivatives. Reagents and conditions: (i) N-benzoylglycine, anhydrous sodium acetate, acetic anhydride, 90 ꢁC,
3 h; (ii) HCl (g), dioxane, rt, 30 min.

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5305
Table 1. Cytotoxicity and tube inhibition assays of bovine aortic endothelial cells for aeroplysinin-1 1 analogues in an in vitro assay system (for the
description of the assays, see Section 4)
MICb,d
a,d
c,d
/
Compound
Structure
IC50
Br
MeO
OH
OH
CN
1
29
2
14
1
Br
MeO
O
10
9
33
>22
161
33
>22
3
O
MeO
Br
O
—
54
O
Br
Br
MeO
Br
O
6
O
MeO
Br
O
7
>15
15
—
O
OMe
Br
MeO
O
8
11
12
3
2
4
6
O
Br
O
12
O
Br
O
O
O
H
Br
23
4
2
2
Br
O
Br
Br
O
11
3
>25
>33
806
36
25
>33
>16
71
—
O
Br
MeO
OH
—
H
O
Br
MeO
Br
OH
4
—
H
O
Br
OH
21
5
0,5
—
H
Br
O
Br
MeO
Br
OH
OH
>16
>16
(continued on next page)

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5306
Table 1 (continued)
a,d
IC50
c,d
/
Compound
Structure
Br
MICb,d
OH
OH
22
26
24
>35
71
—
0,7
—
Br
Br
MeO
Br
OAc
H
23
33
O
Br
Br
MeO
Br
OBn
H
>12.5
>12.5
O
MeO
Br
OCH2COOMe
25
27
28
32
29
40
151
>12
>26
>28
>28
>19
52
>12
>26
28
3
H
O
Br
Br
Br
Br
MeO
Br
OBn
OH
—
—
—
—
—
MeO
Br
OCH2COOMe
OH
MeO
Br
OCH2CH2OH
OH
MeO
Br
O
>28
>38
OH
O
Br
Br
MeO
Br
O
N
CHO
Br
Br
N
MeO
Br
O
39
33
35
>22
>14
136
>44
>14
>27
—
—
—
O
CHO
MeO
Br
OH
CO2Me
Br
Br
MeO
Br
OH
COOMe
MeO
Br
OBn
36
]
921
1
921
Ph
N
O
O

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5307
Table 1 (continued)
a,d
IC50
c,d
/
Compound
Structure
MICb,d
Br
MeO
Br
OBn
OH
37
42
>17
>35
—
—
NHCOPh
COOMe
Br
MeO
Br
>22
>18
>22
Ph
Ph
N
N
O
O
O
Br
Br
MeO
Br
OBz
41
43
27
—
—
O
N
MeO
Br
O
O
O
83
>17
Ph
N
O
Br
Br
MeO
Br
O
N
44
>15
>30
—
Ph
N
O
O
Br
Br
MeO
Br
O
45
71
>28
—
Cl
NH
Ph
N
O
a Drug concentration inhibiting the growth value of the BAE cells by 50% (IC50, lM).
b Minimum inhibition concentration at which no tube formation is observed (MIC, lM).
c Sprouting inhibition assay/cytotoxicity ratio.
d Symbol > means that IC50 and MIC were not found at the biggest concentration assayed. From our criteria compounds with a sprouting inhibition
tube value <10 lM are not interesting as antiangiogenic candidates.
aeroplysinin-1 1. The biological activity comparison be-
tween compounds 36, 24, 41, 42, 43 and 44 shows the
necessity of a benzylic group, as a protector group of
the phenolic moiety, and the oxazolone ring in order
to have a high sprouting inhibition assay/cytotoxicity ra-
tio. The formation of the hydrochloride azlactone 45 did
not improve the mentioned ratio.
3. Conclusions
In conclusion, two series of aeroplysinin-1 1 analogues
have been synthesized, one with the same non-aromatic
cyclic skeleton and the other as aromatic derivatives with
the same substitution in the ring. Their in vitro cytotoxic-
ity and their tube inhibition activity have been evaluated
Figure 3. Tube formation between endothelial cells supported in
pearls.

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5308
using in vitro assays with bovine aortic endothelial cells
(BAEC). Epoxy ketone 6 and azlactone 36 have shown
a relationship value of sprouting inhibition assay/cytotox-
icity enhanced by one order and two orders of magnitude,
respectively, with respect to the reference (aeroplysinin-1
1). This result implies the attribution of specific antiangio-
genic properties for compounds 6 and 36, whereas in the
case of the reference the observable inhibition of tubes
can be attributed either to antiangiogenic properties or
to cytotoxicity. Therefore, epoxy ketone 6 and azlactone
36 seem to be attractive compounds as angiogenesis inhib-
itors. Further studies, including in vivo evaluations, on
this interesting type of antiangiogenic compounds are
underway in our laboratories.
25%. 1H NMR (200 MHz, CDCl3) d 3.95 (3H, s,
OMe), 6.55 (1H, d, J = 8.7 Hz, ArH, H-5), 7.45 (1H,
3
d, 3J = 8.7 Hz, ArH, H-6), 9.69 (1H, s, CHO), 11.88
(1H, s, OH) ppm. 13C NMR (50 MHz, CDCl3) d 56.90
(OMe), 99.76 (ArCABr), 103.84 (ArCH, C-5), 116.22
(ArCACHO), 134.60 (ArCH, C-6), 160.25 (ArCAOH),
162.85 (ArCAOMe), 194.33 (CHO) ppm. MS (70 eV,
EI) m/z (%) 230/232 (82/86) (M+Å), 229/231 (100/100)
(MꢀH), 79 (22), 77 (8), 65 (24), 63 (16), 52 (25), 50
(19), 39 (24). Anal. Calcd for C8H7BrO3: C, 41.59; H,
3.05. Found: C, 41.71; H, 3.09.
4.2.2. 5-Bromo-2,4-dimethoxybenzaldehyde (16). White
crystals, mp 140–142 ꢁC: 1.34 g, 5.47 mmol, 91%. 1H
NMR (300 MHz, CDCl3) d 3.92 (3H, s, OMe), 3.95
(3H, s, OMe), 6.41 (1H, s, ArH, H-3), 7.95 (1H, s,
ArH, H-6), 10.20 (1H, s, CHO) ppm. 13C NMR
(50 MHz, CDCl3) d 56.0 (OMe), 56.5 (OMe), 95.6
(ArCH, C-3), 103.5 (ArCABr), 119.4 (ArCACHO),
132.9 (ArCH, C-6), 161.7 (ArCAOMe), 163.0
(ArCAOMe), 187.1 (CHO) ppm. MS (70 eV, EI) m/z
(%) 244/246 (92/100) (M+Å), 243/245 (40/50) (MꢀH),
227/229 (34/39) (MꢀOH), 226/228 (23/35) (MꢀH2O),
198/200 (30/38) (MꢀH2OꢀCO), 148 (60), 79 (19), 77
(18), 63 (34), 51 (25), 39 (10). Anal. Calcd for
C9H9BrO3: C, 44.11; H, 3.70. Found: C, 44.00; H, 3.62.
4. Experimental
4.1. General
All reactions were carried out under argon atmosphere.
Column chromatography was performed on silica gel
Merck 230–400 mesh. Melting points are uncorrected
and were determined using a Gallenkamp instrument.
NMR spectra were recorded on Bruker 200-AM
(200 MHz), Bruker AM300 (300 MHz) and on Bruker
AM500 (500 MHz) instruments, using CDCl3 and ace-
tone-d6 as solvents. Chemical shifts are in ppm relative
to TMS. Mass spectra were recorded on a mass spectrom-
eter HP-5890. Electrospray ionization (ESI) spectra were
recorded on a Bruker Esquire-LC 00126 by direct method
and using positive polarity.
4.2.3. 3,5-Dibromo-2-hydroxybenzaldehyde (21). Yellow
crystals, mp 79–81 ꢁC: 1.90 g, 6.79 mmol, 83%. 1H
4
NMR (200 MHz, CDCl3) d 7.61 (1H, d, J = 2.3 Hz,
4
ArH, H-4), 7.85 (1H, d, J = 2.3 Hz, ArH, H-6), 9.78
(1H, s, CHO), 11.47 (1H, s, OH) ppm. 13C NMR
(50 MHz, CDCl3) d 111.5 (ArCABr), 112.5 (ArCABr),
121.9 (ArCACHO), 134.9 (ArCH, C-6), 142.0 (ArCH,
C-4), 157.3 (ArCAOH), 194.9 (CHO) ppm. Anal. Calcd
for C7H4Br2O2: C, 30.04; H, 1.44. Found: C, 30.19; H,
1.39.
Spiroepoxycyclohexadienone 6 was synthesized using
the methodology described by Hinterding, K. et al.8d
Compound 7 was obtained13 following the same syn-
thetic route and using 2,4,6-trimethoxybenzaldehyde as
the starting material.
4.2.4. 3-Methyl (3,5-dibromo-2-hydroxy-4-methoxyphenyl)
propanoate (35). White crystals, mp 76–78 ꢁC: 815 mg,
Compound 11 was obtained using the methodology de-
scribed by V. Bonnarme et al.17 Epoxy ketone 10 has
been previously synthesized by Corey, E. J. et al.16 and
compounds 3, 4 and 5 have been previously described
by Hinterding, K. et al.8d The rest of chemicals were ob-
tained from commercial sources and were used without
further purification. Solvents were distilled and dried
over molecular sieves.
1
2.23 mmol, 93%. H NMR (200 MHz, CDCl3) d 2.59
(2H, t, 3J = 7.2 Hz, CH2, H-2), 2.85 (2H, t,
3J = 7.2 Hz, CH2, H-3), 3.66 (3H, s, COOMe), 3.81
(3H, s, OMe), 6.46 (1H, s, OH), 7.21 (1H, s, ArH)
ppm. 13C NMR (50 MHz, CDCl3) d 25.4 (CH2, C-3),
33.6 (CH2, C-2), 51.8 (CH3, COOMe), 60.4 (CH3,
OMe), 107.2 (2· ArCABr), 125.0 (C, ArCACH2),
132.5 (ArCH), 150.9 (ArCAOH), 152.7 (ArCAOMe),
173.9 (COO) ppm. Anal. Calcd for C11H12Br2O4: C,
35.90; H, 3.29. Found: C, 35.93; H, 3.44.
4.2. Aromatic bromination: general procedure
A solution of pyridinium hydrobromide perbromide
(2.12 equiv) (in the case of compound 13 only 1 equiv
was used) in pyridine (1 mL/mmol) was added dropwise
to a solution of the aldehyde (1 equiv) in pyridine (2 mL/
mmol) at 50 ꢁC. The reaction mixture was stirred for 2 h
at 50 ꢁC and then hydrolysed with ice-water (100 mL/g
aldehyde). After filtration of the precipitate, this was
purified by column chromatography (AcOEt/hex-
ane = 1:2 v/v) (in the case of compound 13 a 1:1 ratio
was used, Rf = 0.37).
4.3. Selective cleavage of phenolic ethers
4.3.1. 5-Bromo-2-hydroxy-4-methoxybenzal-dehyde (14).
A 1M solution of BCl3 in CH2Cl2 (5 mL, 5 mmol) was
added dropwise to a solution of the aldehyde 16
(740 mg, 3.02 mmol) in 5 mL of dry CH2Cl2 at 0 ꢁC.
After stirring at room temperature for 16 h the mixture
was cooled to 0 ꢁC and 5 mL of 1 N HCl followed by
10 mL of H2O was added. The aqueous phase was
extracted with CH2Cl2 (3· 5 mL) and the combined or-
ganic layers were washed with brine (2· 10 mL). Drying
of the organic phase with MgSO4 was followed by
4.2.1. 3-Bromo-2-hydroxy-4-methoxybenzal-dehyde (13).
White crystals, mp 115–117 ꢁC: 605 mg, 2.62 mmol,

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5309
evaporation of the solvent under vacuum. The product
14 was purified by chromatography on silica gel
(AcOEt/hexane = 1:1 v/v). White crystals, mp 118–
120 ꢁC: 740 mg, 3.02 mmol, 86%. H NMR (200 MHz,
CDCl3) d 3.91 (3H, s, OMe), 6.42 (1H, s, ArH-3), 7.61
(1H, s, ArH-6), 9.64 (1H, s, CHO), 11.38 (1H, s, OH)
ppm. 13C NMR (50 MHz, CDCl3) d 56.6 (OMe), 100.2
(ArCH, C-3), 102.0 (ArCABr), 115.6 (ArCACHO),
137.1 (ArCH, C-6), 162.4 (ArCAOMe), 163.5
(ArCAOH), 193.5 (CHO) ppm. Anal. Calcd for
C8H7BrO3: C, 41.59; H, 3.05. Found: C, 41.39; H, 3.01.
4.4.3. 2-[2,4-Dibromo-6-(hydroxymethyl)-3-methoxy-
phenoxy] ethanol (31) and 7,9-dibromo-8-methoxy-2,3-
dihydro-5-H-1,4-benzodioxepin-3-ol (29). A solution of
compound 25 (16 mg, 0.04 mmol) in THF (0.5 mL) was
added, dropwise, to a solution of LiAlH4 (3 mg,
0.08 mmol) in THF (1 mL) at 0 ꢁC. The mixture was stir-
red at 0 ꢁC for 30 min. The reaction was quenched by the
dropwise addition of water, and the mixture was extracted
with Et2O (3· 2 mL). Drying of the combined organic
phases with MgSO4 was followed by evaporation of the
solvent under vacuum. The chromatography on silica
gel (AcOEt/hexane = 1:1 v/v) yielded compound 31
(white crystals, mp 109–113 ꢁC: 7 mg, 0.02 mmol, 47%)
and compound 29 (white crystals, mp 106–107 ꢁC: 4 mg,
1
4.4. Treatment of aromatic aldehydes with reductive
agents
1
0.01 mmol, 27%). Compound 31: H NMR (300 MHz,
4.4.1. 2-Bromo-6-(hydroxymethyl)-3-methoxy phenol (15).
A solution of compound 13 (580 mg, 2.51 mmol) in THF
(2.5 mL) was added, dropwise, to a solution of LiAlH4
(95 mg, 2.51 mmol) in THF (4 mL) at 0 ꢁC. The mixture
was stirred at 0 ꢁC for 30 min. The reaction was quenched
by the dropwise addition ofwater, andthe mixture was ex-
tracted with Et2O (3· 30 mL). Drying of the combined or-
ganic phases with MgSO4 was followed by evaporation of
the solvent under vacuum. Compound 15 was purified by
chromatography on silica gel (AcOEt/hexane = 1:1 v/v).
White crystals, mp 106–109 ꢁC: 263 mg, 1.13 mmol,
45%. 1H NMR (200 MHz, CDCl3) d 2.28 (1H, br s,
HOACH2), 3.82 (3H, s, OCH3), 4.66 (2H, s, CH2AOH),
6.39 (1H, d, 3J = 8.4 Hz, ArH, H-4), 6.61 (1H, s,
acetone-d6) d 3.88 (3H, s, OMe), 3.90 (2H, c,
3
3J = 5.3 Hz, CH2, H-1), 4.15 (2H, t, J = 5.3 Hz, CH2,
H-2), 4.22 (1H, t, 3J = 5.3 Hz, CH2CH2AOH), 4.56 (1H,
t, 3J = 6.0 Hz, ArCACH2AOH), 4.72 (2H, d,
3J = 6.0 Hz, ArCACH2AOH), 7.68 (1H, s, ArH) ppm.
Anal. Calcd for C10H12Br2O4: C, 33.74; H, 3.40. Found:
1
C, 33.53; H, 3.40. Compound 29: H NMR (200 MHz,
3
CDCl3) d 2.93 (1H, d, J = 5.4 Hz, OH, exchangeable
with D2O), 3.84 (3H, s, OMe), 4.02 (1H, dd,
2J = 13.0 Hz, 3J = 6.3 Hz, CH2, H-2), 4.19 (1H, dd,
2J = 13.0 Hz, 3J = 2.8 Hz, CH2, H-2), 4.37 (1H, d,
2J = 14.4 Hz, CH2, H-5), 5.09 (1H, d, 2J = 14.4 Hz,
CH2, H-5), 5.31 (1H, m, CHAOH; with D2O: dd,
3
3J = 6.3 Hz, J = 2.8 Hz), 7.22 (1H, s, ArH) ppm. MS
HOAPh), 7.02 (1H, d, 3J = 8.4 Hz, ArH, H-5) ppm. 13
C
(70 eV, EI) m/z (%) 352/354/356 (19/38/18) (M+Å), 306/
308/310 (41/50/51) (MꢀH2OACO), 291/293/295 (13/29/
19) (M-61), 263/265/267 (12/24/10) (M-89), 212/214 (38/
34), 148 (34), 77 (29), 75 (24).
NMR (50 MHz, CDCl3) d 56.5 (OCH2), 62.3 (OCH3),
100.4 (ArCABr), 103.3 (ArCH, C-4), 120.1 (ArCA-
CH2OH), 127.9 (ArCH, C-5), 152.4 (ArCAOH), 156.5
(ArCAOMe) ppm. MS (70 eV, EI) m/z (%) 232/234 (20/
19) (M+Å), 215/217 (24/31) (MꢀOH), 214/216 (81/100)
(MꢀH2O), 186/188 (14/8) (MꢀH2OACO), 135 (39), 124
(12), 105 (17), 77 (28), 65 (14), 51 (16). Anal. Calcd for
C8H9BrO3: C, 41.23; H, 3.89. Found: C, 41.48; H, 3.98.
Anal. Calcd for C10H10Br2O4: C, 33.93; H, 2.85. Found:
C, 34.19; H, 2.97.
4.4.4. Methyl [2,4-dibromo-6-(hydroxy-methyl)-3-meth-
oxyphenoxy] acetate (28) and 7,9-dibromo-8-methoxy-
4.4.2. 2,4-Dibromo-6-hydroxymethylphenol (22). A solu-
tion of compound 21 (900 mg, 3.21 mmol) in THF
(5 mL) was added, dropwise, to a solution of LiAlH4
(126 mg, 3.21 mmol) in THF (10 mL) at 0 ꢁC. The mix-
ture was stirred at 0 ꢁC for 30 min. The reaction was
quenched by the dropwise addition of water, and the mix-
ture was extracted with Et2O (3· 30 mL). Drying of the
combined organic phases with MgSO4 was followed by
evaporation of the solvent under vacuum. Compound
22 was purified by chromatography on silica gel
(AcOEt/hexane = 1:1 v/v). White crystals, mp 130–
2,3-dihydro-5-H-1,4-benzodioxepin-3-ol (29). To
a
solution of compound 25 (50 mg, 0.13 mmol) in THF
(1.3 mL) was added NaBH4 (3.8 mg, 0.13 mmol) at
room temperature. After stirring for 20 h the reaction
was quenched by the addition of 1 N HCl (0.5 mL)
and the aqueous phase was extracted with Et2O
(3· 1 mL). Drying of the combined organic phases with
MgSO4 was followed by evaporation of the solvent un-
der vacuum. The chromatography on silica gel (AcOEt/
hexane = 1:1 v/v) yielded compound 28 (white crystals:
28 mg, 0.06 mmol, 46%) and compound 29 (white crys-
tals, mp 100–103 ꢁC: 4 mg, 0.01 mmol, 7%). Compound
28: 1H NMR (300 MHz, CDCl3) d 3.28 (1H, br s,
HOACH2), 3.85 (3H, s, COOMe), 3.89 (3H, s, OMe),
4.70 (2H, s, CH2AOH), 4.77 (2H, s, OACH2ACOOMe),
7.54 (1H, s, ArH) ppm. 13C NMR (50 MHz, CDCl3) d
52.4 (CH3, COOMe), 60.6 (OMe), 60.6 (HOACH2),
69.9 (OACH2ACOOMe), 113.2 (ArCABr), 113.3
(ArCABr), 132.5 (ArCH), 133.1 (ArCACH2OH), 154.6
(ArCAOCH2), 162.6 (ArCAOMe), 169.8 (COO) ppm.
MS (70 eV, EI) m/z (%) 382/384/386 (3/7/3) (M+Å), 350/
352/354 (8/16/8) (MꢀMeOH), 309/311/313 (66/100/45)
(MꢀCH2COOMe), 308/310/313 (18/28/17) (M-74),
293/295/297 (9/17/11), 292/294/296 (25/47/26) (M-90),
1
133 ꢁC: 550 mg, 1.95 mmol, 61%. H NMR (200 MHz,
CDCl3) d 2.40 (1H, br s, HOACH2), 4.82 (2H, s,
CH2AOH), 6.74 (1H, s, HOAPh), 7.30 (1H, d,
4
4J = 2.3 Hz, ArH, H-5), 7.61 (1H, d, J = 2.3 Hz, ArH,
H-3) ppm. 13C NMR (50 MHz, CDCl3) d 62.4 (OCH2),
111.1 (ArCABr), 112.4 (ArCABr), 128.8 (ArCA-
CH2OH), 130.3 (ArCH, C-3), 133.7 (ArCH, C-5), 150.4
(ArCAOH) ppm. MS (70 eV, EI) m/z (%) 280/282/284
(6/14/8) (M+Å), 262/264/266 (41/100/48) (MꢀH2O), 234/
236/238 (3/6/2) (MꢀH2OACO), 183/185 (16/21)
(MꢀH2OABr), 155/157 (9/8) (MꢀH2OABrACO), 77
(12), 76 (19), 75 (43), 63 (24), 39 (13). Anal. Calcd for
C7H6Br2O2: C, 29.82; H, 2.15. Found: C, 29.83; H, 2.18.

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5310
204 (16), 202 (19), 75 (13), 74 (28), 45 (18). Anal. Calcd
for C11H12Br2O5: C, 34.40; H, 3.15. Found: C, 34.29; H,
3.21.
with MgSO4 and filtered. To the solution obtained [17]
a solution of NaIO4 (1.11 g,) in H2O (8.3 mL) and 1 N
HCl (2 mL) at room temperature was added. After stir-
ring for 90 min the aqueous phase was extracted with
Et2O (2· 10 mL). Drying of the combined organic
phases with MgSO4 was followed by evaporation of
the solvent under vacuum. Compound 9 was purified
by chromatography on silica gel (AcOEt/hexane = 1:1
v/v). Yellow crystals, mp 114–116 ꢁC: 1.59 g, 0.69 mmol,
53%. 1H NMR (200 MHz, CDCl3) d 3.11 (1H, d,
2J = 7.8 Hz AB system, CH2O), 3.33 (1H, d,
2J = 7.8 Hz AB system, CH2O), 3.82 (3H, s, OMe),
5.76 (1H, s, H-5) 6.53 (1H, s, H-8) ppm. 13C NMR
(50 MHz, CDCl3) d 56.0 (CAO), 57.2 (OMe), 57.7
(OCH2), 101.3 (CH, C-5), 118.5 (CBr), 139.9 (CH, C-
8), 166.2 (CAOMe), 191.3 (C@O) ppm. MS (70 eV,
EI) m/z 230/232 (28/28) (M+Å), 215/217/218/219/220
(12/27/10/13/8), 124 (19) (M-106), 86 (49), 84 (100), 53
(22), 51 (45), 49 (94), 45 (55). Anal. Calcd for
C8H7BrO3: C, 41.59; H, 3.05. Found: C, 41.43; H, 3.20.
4.4.5. [2-(Benzyloxy)-3,5-dibromo-4-methoxy-phenyl] meth-
anol (27). To a solution of compound 24 (100 mg,
0.25 mmol) in THF (2.5 mL) was added NaBH4 (9.5 mg,
0.25 mmol) at room temperature. After stirring for
90 min the reaction was quenched by the addition of 1 N
HCl (1 mL) and the aqueous phase was extracted with
Et2O (3· 1.5 mL). Drying of the combined organic phases
with MgSO4 was followed by evaporation of the solvent
under vacuum. Compound 27 was purified by chromatog-
raphy on silica gel (AcOEt/hexane = 1:1 v/v). White crys-
1
tals, mp 85–87 ꢁC: 64 mg, 0.16 mmol, 64%. H NMR
(200 MHz, CDCl3) d 1.85 (1H, br s, HOACH2), 3.92
(3H, s, OMe), 4.57 (2H, s, CH2AOH), 5.09 (2H, s,
CH2AOAPh), 7.31–7.55 (5H, m, 5· ArCH), 7.58 (1H, s,
ArH, H-6) ppm. 13C NMR (50 MHz, CDCl3) d 60.6, 60.8
(OMe, HOACH2), 75.9 (PhAOACH2), 113.2 (ArCABr),
114.5 (ArCABr), 128.6 (2· ArCH), 128.79 (2· ArCH),
128.82 (ArCH), 131.9 (ArCH, C-6), 133.3, 136.4 (ArCA
CH2OH, ArCACH2OPh), 154.2, 154.8 (ArCAOBn,
ArCAOMe) ppm. MS (70 eV, EI) m/z (%) 400/402/404
(1/2/1) (M+Å), 292/294/296 (6/12/6) (MꢀPhCH2OH), 92
(8), 91 (100) ðPhCHþ2 Þ, 65 (7). Anal. Calcd for
C15H14Br2O3: C, 44.81; H, 3.51. Found: C, 44.69; H, 3.37.
4.5.3. 5,7-Dibromo-1-oxa-spiro[2.5]octa-5,7-dien-4-one
(12) and 10,30,70,8a0-tetrabromo-20,40,4a0, 80-tetrahydro-
di-spiro-[oxiran-2-50(60H)-[1,4]ethanenaphthalen-90,200-
oxiran]-60,100-dione (23). To a solution of compound 22
(250 mg, 0.89 mmol) in THF (4.5 mL) and 1 N HCl
(0.6 mL) was added, dropwise, a solution of NaIO4
(1.52 g, 7.1 mmol) in H2O (8.3 mL) and 1 N HCl
(2.8 mL) at room temperature. After stirring for
90 min the aqueous phase was extracted with Et2O
(3· 20 mL). Drying of the combined organic phases with
MgSO4 was followed by evaporation of the solvent un-
der vacuum. Compound 12 was purified by chromatog-
raphy on silica gel (AcOEt/hexane = 1:5 v/v). Yellow
crystals: 124 mg, 0.44 mmol, 50%. 1H NMR (CDCl3,
200 MHz) d 3.21 (1H, d, 2J = 8.0 Hz AB system,
CH2AO), 3.36 (1H, d, 2J = 8.0 Hz AB system, CH2AO),
6.32 (1H, d, 4J = 2.2 Hz, CH, H-8), 7.60 (1H, d,
4J = 2.2 Hz, CH, H-6) ppm. 13C NMR (CDCl3,
75 MHz) d 58.7 (CAO), 60.3 (CH2AO), 117.3 (CABr,
C-7), 124.2 (CABr, C-5), 138.0 (CH, C-6), 146.8 (CH,
C-8), 187.0 (C@O) ppm. MS (70 eV, EI) m/z (%) 278/
280/282 (52/100/42) (M+Å), 277/279/281 (29/70/32)
(MꢀH), 262/264/266 (24/33/17) (M-16), 63 (62), 62
(38), 45 (25), 43 (23). Anal. Calcd for C7H4Br2O2: C,
30.04; H, 1.44. Found: C, 30.30; H, 1.21. When the
treatment of the reaction was carried out at T > 25 ꢁC
a progressive dimerization of epoxy ketone 12 to render
compound 23 was observed. The dimer 23 was purified
by chromatography on silica gel (AcOEt/hexane = 1:5
4.5. Synthesis of ( )-spiroepoxycyclohexa-dienones12b
4.5.1. 5-Bromo-6-methoxy-1-oxa-spiro[2.5]octa-5,7-dien-
4-one (8). To a solution of compound 15 (200 mg,
0.86 mmol) in THF (4.3 mL) and 1 N HCl (0.53 mL)
was added, dropwise, a solution of NaIO4 (1.48 g,
6.9 mmol) in H2O (8.1 mL) and 1 N HCl (2.7 mL) at
room temperature. After stirring for 90 min the aqueous
phase was extracted with Et2O (3· 15 mL). Drying of
the combined organic phases with MgSO4 was followed
by evaporation of the solvent under vacuum. Com-
pound 8 was purified by chromatography on silica gel
(AcOEt/hexane = 1:5 v/v). Yellow crystals, mp 121–
1
126 ꢁC: 99 mg, 0.43 mmol, 50%. H NMR (200 MHz,
2
CDCl3) d 3.19 (1H, d, J = 8.2 Hz AB system, CH2O),
2
3.36 (1H, d, J = 8.2 Hz AB system, CH2O), 4.02 (3H,
s, OMe), 6.28 (1H, d, 3J = 10.3 Hz, CH, H-7), 6.73
(1H, d, 3J = 10.3 Hz, CH, H-8) ppm. 13C NMR
(50 MHz, CDCl3) d 57.0 (CAO), 57.7 (OMe), 58.9
(OCH2), 103.6 (CABr), 120.1 (CH, C-7), 141.2 (CH,
C-8), 166.7 (CAOMe), 194.3 (C@O) ppm. MS (70 eV,
EI) m/z (%) 230/232 (95/100) (M+Å), 229/231 (96/100)
(MꢀH), 215/217 (39/40) (MꢀCH3), 214/216 (29/42)
(M-16), 135 (17), 79 (24), 77 (22), 63 (19), 57 (25), 55
(29), 51 (32), 43 (26), 41 (20), 39 (19). Anal. Calcd for
C8H7BrO3: C, 41.59; H, 3.05. Found: C, 41.60; H, 3.11.
1
v/v). White crystals, mp 160–162 ꢁC. H NMR (CDCl3,
3
4
200 MHz) d 3.08 (1 H, dd, J = 2.0, J = 2.6 Hz, H-4a),
2
3.10 (1H, d, J = 5.9 Hz AB system, CH2AO, oxirane-
2
5), 3.15 (1H, d, J = 5.9 Hz AB system, CH2AO, oxi-
2
rane-5), 3.20 (1H, d, J = 6.4 Hz AB system, CH2AO,
4.5.2. 7-Bromo-6-methoxy-1-oxa-spiro[2.5] octa-5,7-dien-
4-one (9). A solution of compound 14 (300 mg,
1.30 mmol) in THF (2 mL) was added, dropwise, to a
solution of LiAlH4 (51 mg, 1.30 mmol) in THF (4 mL)
at 0 ꢁC. The mixture was stirred at 0 ꢁC for 30 min.
The reaction was quenched by the dropwise addition
of water, and the mixture was extracted with Et2O
(3· 10 mL). The combined organic phases were dried
oxirane-9), 3.34 (1H, d, 2J = 6.4 Hz AB system, CH2AO,
3
oxirane-9), 3.45 (1H, d, J = 2.0 Hz, H-4), 6.48 (1H, d,
4J = 2.6 Hz, H-8), 7.47 (1H, s, H-10) ppm. 13C NMR
(CDCl3, 50 MHz) d 51.1 (CH, C-4 or C-4a), 53.4 (CH,
C-4 or C-4a), 54.2 (CH2AO), 56.2 (CAO), 56.5
(CAO), 59.3 (CH2AO), 61.2 (CBr, C-8a), 76.8 (CBr,
C-1), 124.4 (CBr, C-3 or 7), 125.1 (CBr, C-3 or 7),
132.8 (CH, C-8 or 2), 146.1 (CH, C-2 or 8), 190.3

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5311
(2· C@O) ppm. ESI m/z (%) 579/581/583/585/587 (15/
35/62/40/11) (M+Na)+. Anal. Calcd for C14H8Br4O4:
C, 30.04; H, 1.44. Found: C, 30.23; H, 1.50.
uum. White solid, mp 77–81 ꢁC: 70 mg, 0.20 mmol,
1
62%. H NMR (200 MHz, CDCl3) d 2.47 (3 H, s, Ac),
3.98 (3H, s, OMe), 8.05 (1H, s, ArH), 9.91 (1H, s, CHO)
ppm. 13C NMR (50 MHz, CDCl3) d 20.3 (CH3, Ac),
60.8 (OMe), 115.31 (ArCABr), 115.36 (ArCABr), 126.5
(ArCACHO), 133.8 (ArCH), 149.9 (ArCAOC@O),
159.8 (ArCAOMe), 167.7 (COOMe), 185.7 (CHO) ppm.
MS (70 eV, EI) m/z (%) 307/309/311 (18/39/29) (MꢀAc),
308/310/312 (34/74/29) (M-42), 86 (59), 84 (78), 51 (38),
49 (15), 43 (31).
4.6.Phenolichydroxylprotectionofthesalicylicaldehyde(4)
4.6.1. 2-(Benzyloxy)-3,5-dibromo-4-methoxy benzalde-
hyde (24). To a solution of aldehyde 4 (2.05 g, 6.61 mmol)
in DMF (1.55 mL) were added, successively, K2CO3
(823 mg, 5.95 mmol) and BnCl (0.84 mL, 7.27 mmol).
The reaction mixture was heated up to 95 ꢁC and allowed
to react for 3 h. The reaction mixture was then cooled to
room temperature and diluted with H2O. After filtration
of the brown precipitate formed, compound 24 was puri-
fied by chromatography on silica gel (AcOEt/hex-
ane = 1:10 v/v). Green crystals, mp 91–93 ꢁC: 2.45 g,
4.6.4. 2,4-Dibromo-6-formyl-3-methoxy-phenyl benzoate
(38). To a solution of aldehyde 4 (100 mg, 0.32 mmol) in
dioxane (0.4 mL) were added, successively, Et3N
(0.11 mL, 0.81 mmol) and BzCl (0.05 mL, 0.39 mmol).
The reaction mixture was stirred at room temperature
for 30 min. The reaction mixture was filtered washing,
several times, with Et2O. Drying of the resulting solution
with MgSO4 was followed by evaporation of the solvent
undervacuum. Compound 38 was purified by chromatog-
raphy on silica gel (AcOEt/hexane = 1:5 v/v). White crys-
1
6.12 mmol, 93%. H NMR (200 MHz, CDCl3) d 4.00
(3 H, s, OMe), 5.12 (2H, s, CH2), 7.30–7.60 (5H, m, Ph),
8.01 (1H, s, ArH), 9.94 (1H, s, CHO) ppm. 13C NMR
(50 MHz, CDCl3) d 61.1 (OMe), 78.2 (CH2O), 114.6
(ArCABr), 115.5 (ArCABr), 128.5 (ArCACHO), 128.92
(2· ArCH), 128.96 (2· ArCH), 129.2 (ArCH), 131.6
(ArCH, C-6), 159.4 (ArCAO), 160.5 (ArCAO), 187.2
(CHO). MS (70 eV, EI) m/z (%) 398/400/402 (1/2/1)
(M+Å), 369/371/373 (2/4/2) (MꢀCHO), 307/309/311 (1/2/
1) (MꢀCH2Ph), 92 (7), 91 (100) ðC7 Hþ7 Þ, 65 (8). Anal.
Calcd for C15H12Br2O3: C, 45.03; H, 3.02. Found: C,
44.83; H, 3.12.
1
tals, mp 104–107 ꢁC: 90 mg, 0.22 mmol, 67%. H NMR
(300 MHz, CDCl3) d 4.02 (3H, s, OMe), 7.58 (2H, t,
3J = 7.8 Hz, 2· ArCH, H-20 and 60), 7.73 (1H, t,
3J = 7.8 Hz, ArCH, H-40), 8.14 (1H, s, ArH, H-5), 8.27
(2H, d, 3J = 7.8 Hz, 2· ArCH, H-30 and 50), 9.49 (1H, s,
CHO) ppm. 13C NMR (50 MHz, CDCl3) d 61.1 (OMe),
115.5 (ArCABr), 116.2 (ArCABr), 127.2 (ArCAC@O),
127.8 (ArCAC@O), 128.9 (2· ArCH), 130.7 (2· ArCH),
133.0 (ArCH), 134.6 (ArCH), 150.9 (ArCAOC@O),
160.1 (ArCAOMe), 163.9 (COO), 185.8 (CHO) ppm.
Anal. Calcd for C15H10Br2O4: C, 43.51; H, 2.43. Found:
C, 43.51; H, 2.41.
4.6.2. Methyl (2,4-dibromo-6-formyl-3-methoxyphenoxy)
acetate (25). To a solution of aldehyde 4 (300 mg,
0.93 mmol) in acetone (2.80 mL) were added, succes-
sively, K2CO3 (115 mg, 0.84 mmol), ether-18-crown-6
(26 mg, 0.09 mmol) and BrCH2CO2Me (0.10 mL,
1.02 mmol). The reaction mixture was allowed to react
for 3 h at room temperature. The inorganic salt was elim-
inated by filtration and the resulting solution was dried
with MgSO4 followed by evaporation of the solvent under
vacuum. Compound 25 was purified by chromatography
on silica gel (AcOEt/hexane = 1:1 v/v). White crystals, mp
114–116 ꢁC: 280 mg, 0.73 mmol, 76%. 1H NMR
(200 MHz, CDCl3) d 3.74 (3H, s, COOMe), 3.89 (3H, s,
OMe), 4.76 (2H, s, CH2), 7.97 (1H, s, ArH), 10.35 (1H,
s, CHO) ppm. 13C NMR (50 MHz, CDCl3) d 52.5 (CH3,
COOMe), 61.1 (OMe), 70.5 (CH2O), 114.1 (ArCABr),
114.8 (ArCABr), 128.4 (ArCACHO), 132.0 (ArCH),
158.5 (ArCAOCH2), 161.3 (ArCAOMe), 168.6 (COO),
187.7 (CHO) ppm. MS (70 eV, EI) m/z (%) 380/382/384
(12/25/13) (M+Å), 348/350/352 (9/18/9) (MꢀMeOH), 321/
323/325 (16/27/13) (MꢀCOOMe), 307/309/311 (50/100/
49) (MꢀCH2COOMe), 306/308/310 (18/38/28), 47 (64),
45 (29), 43 (17). Anal. Calcd for C11H10Br2O5: C, 34.59;
H, 2.64. Found: C, 34.57; H, 2.72.
4.6.5. 4-(Dimethylamino) 2,4-dibromo-6-formyl-3-meth-
oxyfenyl benzoate (39). To a solution of aldehyde 4
(310 mg, 0.99 mmol) in dioxane (1.1 mL) were added, suc-
cessively, Et3N (0.33 mL, 2.44 mmol) and 4-(dimethylam-
in) benzoyl chloride (198 mg, 1.08 mmol). The reaction
mixture was stirred at room temperature for 1 h. The reac-
tion mixture was filtered washing, several times, with
Et2O. Drying of the resulting solution with MgSO4 was
followed by evaporation of the solvent under vacuum.
Compound 39 was purified by chromatography on silica
gel (AcOEt/hexane = 1:5 v/v). White crystals, mp 140–
1
142 ꢁC: 230 mg, 0.50 mmol, 51%. H NMR (300 MHz,
CDCl3) d 3.11 (6H, s, N(CH3)2), 4.00 (3H, s, OMe), 6.73
(2H, d, 3J = 8.9 Hz, 2· ArCH, H-30 and 50), 8.11 (2H, d,
3J = 8.8 Hz, 2· ArCH, H-20 and 60), 8.14 (1H, s, ArH,
H-5), 10.02 (1H, s, CHO) ppm. 13C NMR (50 MHz,
CDCl3) d 40.1 (N(CH3)2), 61.0 (OMe), 111.2 (2· ArCH,
C-30 and 50), 113.6 (ArCABr), 115.6 (ArCACOO), 115.8
(ArCABr), 127.6 (ArCACHO), 131.9 (ArCH, C-5), 132.7
(2· ArCH, C-20 and 60), 152.0 (ArC-N), 154.4
(ArCAOC@O), 159.9 (ArCAOMe), 164.1 (COO), 186.2
(CHO) ppm. MS (70 eV, EI) m/z (%) 455/3457/459 (1/2/
1) (M+Å), 149 (11), 148 (100), 120 (3), 105 (3), 104 (2), 79
(3), 42 (4). Anal. Calcd for C17H15Br2NO4: C, 44.67; H,
3.31. Found: C, 44.50; H, 3.58.
4.6.3. 2,4-Dibromo-6-formyl-3-methoxy-phenyl acetate
(26). A solution of AcCl (0.03 mL, 0.38 mmol) in dioxane
(0.32 mL) was added, dropwise, during 30 min to a mix-
ture of aldehyde 4 (100 mg, 0.32 mmol) in dioxane
(0.39 mL), TBAH (0.4 mg) and NaOH (32 mg,
0.81 mmol). The mixture was stirred at room temperature
for 32 h. The reaction mixture was filtered washing with
dioxane. Drying of the resulting solution with MgSO4
was followed by evaporation of the solvent under vac-
4.6.6. 3,5-Dibromo-2-{[2-bromo-5-(dimethyl-amino)ben-
zyl]oxy}-4- methoxybenzaldehyde (40). (a) Synthesis
of 4-bromo-3-(bromomethyl)-N,N-dimethylaniline.
A

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5312
3
suspension of NBS (1.72 g, 9.66 mmol) in anhydrous
CH2Cl2 (33 mL) was cooled to 0 ꢁC and SMe2
(0.87 mL, 10.63 mmol) was added dropwise during
5 min. The reaction mixture was then cooled to
ꢀ20 ꢁC and a solution of 3-(hydroxymethyl)-N,N-dime-
thylaniline (710 mg, 4.70 mmol) in anhydrous CH2Cl2
(2.5 mL) was added. After the addition, the temperature
was raised to 0 ꢁC and the mixture stirred for 3 h. The
reaction mixture was then diluted with pentane and
50 mL of a water/ice mixture was added. The organic
phase was separated, washed with cold brine
(5· 30 mL), dried with MgSO4 and the solvent evapo-
rated under vacuum. Purification by chromatography
on silica gel (AcOEt/hexane = 1:5 v/v) gave yellow crys-
3J = 8.3 Hz, ArH, H-50), 6.97 (1H, d, Jtrans = 15.9 Hz,
3
CH, H-2), 7.31 (1H, d, J = 8.3 Hz, ArH, H-60), 7.92
(1H, d, Jtrans = 15.9 Hz, CH, H-3) ppm. 13C NMR:
3
(50 MHz, CDCl3) d 51.7 (MeOOC), 55.4 (OMe), 101.9
(ArCH, C-30 or 50), 107.0 (ArCH, C-30 or 50), 115.0
(ArCACH), 115.1 (CH, C-2), 130.6 (ArCH, C-60), 140.9
(CH, C-3), 157.3 (ArCAOH), 162.6 (ArCAOMe), 169.3
(COOMe) ppm. Anal. Calcd for C11H12O4: C, 63.45; H,
5.81. Found: C, 63.33; H, 5.84.
4.7.2. Methyl (2E)-3-(3,5-dibromo-2-hydroxy-4-methoxy-
phenyl) acrylate (33). To a solution of aldehyde 4 (5.08 g,
16.40 mmol) in THF (165 mL) was added Ph3P@CH2
COOMe (5.49 g, 16.44 mmol). The reaction mixture was
refluxed at 95 ꢁC for 5 h. After evaporation of the solvent
under vacuum, compound 33 was purified by chromatog-
raphy on silica gel (AcOEt/hexane = 1:1 v/v). White crys-
tals, mp 177–179 ꢁC: 6.02 g, 16.40 mmol, quantitative. 1H
NMR (200 MHz, CDCl3) d 3.71 (3H, s, COOMe), 3.85
(3H, s, OMe), 5.98 (1H, s, OH), 6.45 (1H, d,
3Jtrans = 16.1 Hz, CH, C-2), 7.58 (1H, s, ArH), 7.73 (1H,
d, 3Jtrans = 16.1 Hz, CH, C-3) ppm. 13C NMR (50 MHz,
CDCl3) d 51.9 (MeOOC), 60.9 (OMe), 107.9 (ArCABr),
108.2 (ArCABr), 119.8 (ArCACH), 119.9 (CH, C-2),
131.8 (ArCH), 137.9 (CH, C-3), 151.7 (ArCAOH),
155.4 (ArCAOMe), 167.2 (COOMe) ppm. MS (70 eV,
EI) m/z (%) 364/366/368 (13/27/14) (M+Å), 332/334/336
(50/100/46) (MꢀMeOH), 304/306/308 (15/32/15)
(MꢀMeOHACO), 289/291/293 (21/40/23) (M-75), 75
(16). Anal. Calcd for C11H10Br2O4: C, 36.10; H, 2.75.
Found: C, 37.25; H, 2.64.
1
tals, mp 87–90 ꢁC: 751 mg, 2.56 mmol, 55%. H NMR
(300 MHz, CDCl3) d 2.96 (6H, s, N(CH3)2), 4.57 (2H,
s, CH2Br), 6.55 (1H, dd, 3J = 8.9 Hz, 4J = 3.2 Hz,
4
ArH-6), 6.77 (1H, d, J = 3.2 Hz, ArH-2), 7.37 (1H, d,
3J = 8.9 Hz, ArH-5) ppm. 13C NMR (75 MHz, CDCl3)
d 34.9 (CH2Br), 40.9 (N(CH3)2), 110.5 (ArCABr),
114.7 (ArCH), 115.0 (ArCH), 133.8 (ArCH, C-5),
137.3 (ArCACH2), 150.4 (ArC-N) ppm. MS (70 eV,
EI) m/z (%) 291/293/295 (36/78/32) (M+Å), 212/214 (97/
100) (MꢀBr), 133 (52) (MꢀBr2), 132 (30), 118 (27), 91
(33). Anal. Calcd for C9H11Br2N: C, 36.89; H, 3.78.
Found: C, 37.05; H, 3.72.
(b) Phenolic group protection. To a solution of aldehyde 4
(298 mg, 0.93 mmol) in DMF (0.5 mL) were added, suc-
cessively, K2CO3 (116 mg, 0.84 mmol) and 4-bromo-
3-(bromomethyl)-N,N-dimethylaniline (300 mg, 1.03
mmol) in DMF (0.5 mL).The reaction mixture was heated
up to 75 ꢁC and allowed to react for 2 h. The reaction mix-
ture was then cooled to room temperature and diluted
with H2O. Filtration of the yellow precipitate formed
yielded compound 40. Yellow crystals, mp 140–142 ꢁC:
4.8. Selective reduction of the alkenyl moiety
4.8.1. Methyl 3-(2-hydroxy-4-methoxyphenyl) propionate
(34). To a solution of ester 32 (3.69 g, 17.74 mmol) in
dry MeOH (83 mL) was added metallic Mg (4.31 g,
177.40 mmol). The reaction mixture was stirred at room
temperature for 3 h. The reaction was hydrolysed by the
dropwise addition of HCl (0.5 N) and the mixture was
extracted with Et2O (3· 25 mL). Drying of the resulting
solution with MgSO4 was followed by evaporation of
the solvent under vacuum. Compound 34 was purified
by chromatography on silica gel (AcOEt/hexane=1:1 v/
v). White crystals, mp 55–57 ꢁC: 2.61 g, 12.42 mmol,
70%. 1H NMR (200 MHz, CDCl3) d 2.62 (2H, t,
1
425 mg, 0.81 mmol, 87%. H NMR (200 MHz, CDCl3)
d 2.95 (6H, s, N(CH3)2), 3.98 (3H, s, OMe), 5.19 (2H, s,
CH2O), 6.58 (1H, dd, 3J = 8.9 Hz, 4J = 3.1 Hz, ArH-30),
6.86 (1H, d, 4J = 3.1 Hz, ArH-60), 7.38 (1H, d,
3J = 8.9 Hz, ArH-40), 8.01 (1H, s, ArH-6), 10.00 (1H, s,
CHO) ppm. 13C NMR (50 MHz, CDCl3) d 40.5
(N(CH3)2), 61.3 (OMe), 77.5 (CH2O), 109.1 (ArCABr),
114.2 (ArCH, C-40 or 60), 114.4 (ArCH, C-40 or 60),
114.4 (ArCABr), 115.3 (ArCABr), 128.7 (ArCACHO),
0
0
´
131.4 (ArCH, C-3 o 6), 133.2 (ArCH, C-3 or 6), 134.5
(ArCACH2), 150.1 (ArC-N), 159.5 (ArCAOCH2), 160.2
(ArCAOMe), 187.4 (CHO) ppm. MS (70 eV, EI) m/z
(%) 519/521/523/525 (8/23/22/7) (M+Å), 212/214 (100/96),
133 (30), 91 (21).
3J = 7.0 Hz, CH2, H-2), 2.80 (2H, t, J = 7.0 Hz, CH2,
3
H-3), 3.62 (3H, s, COOMe), 3.70 (3H, s, OMe), 6.35
3
(1H, d, J = 7.9 Hz, ArH, H-50), 6.38 (1H, s, ArH, H-
3
30), 6.91 (1H, d, J = 7.9 Hz, ArH, H-60), 7.33 (1H, s,
OH) ppm. Anal. Calcd for C11H14O4: C, 62.85; H,
6.71. Found: C, 62.86; H, 6.84.
4.7. Wittig reactions
4.7.1. Methyl (2E)-3-(2-hydroxy-4-methoxy-phenyl) pro-
pionate (32). To a solution of aldehyde 3 (1.00 g,
6.58 mmol) in THF (65 mL) was added Ph3P@CH2
COOMe (2.20 g, 6.60 mmol). The reaction mixture was
refluxed at 95 ꢁC for 5 h. After evaporation of the solvent
under vacuum, compound 32 was purified by chromatog-
raphy on silica gel (AcOEt/hexane = 1:1 v/v). White crys-
tals, mp 142–145 ꢁC: 1.38 g, 6.58 mmol, quantitative. 1H
NMR (200 MHz, CDCl3) d 3.71 (3H, s, COOMe), 3.74
(3H, s, OMe), 6.36 (1H, s, ArH, H-30), 6.40 (1H, d,
4.9. Methanolysis of the oxazolone ring
4.9.1. Methyl (2Z)-2-(benzoylamino)-3-[2-(benzyloxy)-
3,5-dibromo-4-methoxyphenyl]acrylate (37). A solution
of azlactone 36 (38 mg, 0.07 mmol) in dry MeOH
(1.2 mL) was stirred at room temperature for 90 min.
The reaction mixture was filtered followed by evapora-
tion of the solvent under vacuum. Finally, cold water
was added until the formation of a yellow precipitate.
Compound 37 was purified by chromatography on silica

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R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5313
3
gel (AcOEt/hexane = 1:5 v/v). White crystals, mp 109–112
ꢁC: 33 mg, 0.06 mmol, 82%. 1H NMR (200 MHz, CDCl3)
d 3.90 (3H, s, OMe), 3.91 (3H, s, OMe), 4.88 (2H, s,
CH2O), 7.05 (1H, s, ArCACH@C), 7.10–7.60 (10H, m,
10· ArH, 2· Ph), 8.54 (1H, s, ArH, H-60) ppm. 13C
NMR (50 MHz, CDCl3) d 52.95 (COOMe), 60.81
(OMe), 76.81 (CH2AO), 113.70 (ArCABr), 115.32
(ArCABr), 121.03 (CH, ArCACH@C), 127.05 (ArC),
127.43 (2· ArCH), 128.23 (ArCH), 128.68 (2· ArCH),
128.81 (2· ArCH), 129.15 (ArCH), 129.60 (2· ArCH),
132.15 (ArCH), 132.80 (ArCH), 133.32 (CH@CAN),
135.26 (ArC), 153.22, 155.62 (ArCAOCH2, ArCAOMe),
165.25, 165.46 (OAC@N, OAC@O) ppm. MS (70 eV, EI)
m/z (%) 573/575/577 (1/2/1) (M+Å), 541/543/545 (1/2/1)
(MꢀMeOH), 494/496 (1/1) (MꢀBr), 452/454/456 (7/12/
6) (MꢀH2NCOPh), 436/438/440 (3/6/3), 420/422/424 (2/
4/2), 106 (7), 105 (100) (PhCO+), 91 (6), 77 (28). Anal.
Calcd for C25H21Br2NO5: C, 52.20; H, 3.68. Found: C,
22.43; H, 654.
H-20 and 60 or H-200and 600), 8.28 (2H, d, J = 8.6 Hz,
2· ArH, H-20 and 60 or H-200 and 600), 9.29 (1H, s, ArH,
H-5) ppm. 13C NMR (50 MHz, CDCl3) d 61.2 (OMe),
115.0 (ArCABr), 116.0 (ArCABr), 121.5 (CH,
ArCACH@C), 125.2, 126.2, 128.1 (3· ArC, C-100 or
ArCACH@C or ArCAC@O), 128.8 (2· ArCH), 129.1
(2· ArCH), 129.2 (2· ArCH), 130.9 (ArCH), 134.7
(ArCH), 135.4 (ArCH), 135.4 (CH@CAN), 149.5
(ArCAOAC@O), 156.5 (ArCAOMe), 163.9 (OAC@N),
165.2 (COO), 167.2 (COO) ppm. MS (70 eV, EI) m/z
(%) 555/557/559 (3/6/3) (M+Å), 106 (8), 105 (100) (PhCO+),
77 (24). Anal. Calcd for C24H15Br2NO5: C, 51.73; H, 2.71;
N, 2.51. Found: C, 51.87; H, 2.61; N, 2.60. Compound 42:
Yellow crystals, mp 184–187 ꢁC: 40 mg, 0.09 mmol, 18%.
1H NMR (200 MHz, CDCl3) d 3.92 (3H, s, OMe), 7.35–
7.75 (3H, m, 3· ArH, H-30,40 and 50), 7.78 (1H, s,
4
ArCACH@C), 7.86 (2H, d, 3J = 8.1 Hz, J = 1.5 Hz,
2· ArH, H-20 and 60), 8.72 (1H, s, ArH, H-600), 8.76 (1H,
s ancho, ArH, HO) ppm. 13C NMR (50 MHz, CDCl3) d
61.1 (OMe), 106.9 (ArCABr), 114.2 (ArCABr), 118.4
(ArCACH@C), 121.3 (CH, ArCACH@C), 124.2 (ArC,
C-10), 127.2 (2· ArCH), 129.0 (2· ArCH), 130.0 (ArCH),
132.7 (ArCH), 133.2 (CH@CAN), 147.3 (ArCAOH),
155.6 (ArCAOMe), 157.9 (OAC@N), 166.1 (COO)
ppm. MS (70 eV, EI) m/z (%) 451/453/455 (8/17/9)
(M+Å), 106 (8), 105 (100) (PhCO+), 77 (29). Anal. Calcd
for C17H11Br2NO4: C, 45.07; H, 2.45; N, 3.09. Found:
C, 45.29; H, 2.65; N, 3.09.
4.10. Synthesis of azlactones: general procedure
To a solution of the corresponding aldehydes (1 equiv) in
Ac2O (2.3 mL/mmol) were added, successively, anhy-
drous AcONa (2.94 equiv) and BzGly (2 equiv). The reac-
tion mixture was stirred at 85–90 ꢁC for 3 h. The reaction
mixture was filtered washing, several times, with Ac2O.
The corresponding azlactones were purified by chroma-
tography on silica gel (AcOEt/hexane = 1:5 v/v).
4.10.3. Phenyl 2,4-Dibromo-3-methoxy-6-[(Z)-(5-oxo-2-
phenyl-1,3-oxazol-4(5H)-yliden)methyl]4-(dimethyl-
amino) benzoate (43). Yellow crystals, mp >200 ꢁC:
4.10.1. (4Z)-4-[2-(benzyloxy)-3,5-dibromo-4-methoxy-
benzyliden]-2-phenyl-1,3-oxazol-5(4H)-one (36). Yellow
1
1
crystals, mp 191–192 ꢁC: 200 mg, 0.37 mmol, 52%. H
20 mg, 0.03 mmol, 19%. H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3) d 3.99 (3H, s, OMe), 5.05
(2H, s, CH2O), 7.38 (1H, s, ArCACH@C), 7.30–7.80
(8H, m, 8· ArH, H-30,40,50,200,300,400,500 and 600), 8.19
d 3.13 (6H, s, N(CH3)2), 4.01 (3H, s, OMe), 6.77 (2H,
3
d, J = 9.1 Hz, 2· ArCH, H-30 and 50), 7.23 (1H, s,
3
ArCACH=C), 7.58 (2H, t, J = 7.5 Hz, 2· ArCH, H-300
4
3
(2H, dd, 3J = 6.8 Hz, J = 1.6 Hz, 2· ArH, H-20 and
and 500), 7.67 (1H, t, J = 7.5 Hz, 2· ArCH, H-400), 8.13
3
60), 9.10 (1H, s, ArH, 6000) ppm. 13C RMN (50 MHz,
(2H, d, J = 9.1 Hz, 2· ArH, H-20 and 60), 8.19 (2H, d,
CDCl3)
(ArCABr),
d
60.92 (OMe), 77.33 (CH2AO), 113.92
114.78 (ArCABr), 123.11 (CH,
3J = 7.5 Hz, 2· ArCH, H-200 and 600), 9.28 (1H, s, ArH,
H-5) ppm. 13C NMR (75 MHz, CDCl3) d 40.51
(N(CH3)2), 61.41 (OMe), 111.37 (2· ArCH, C-3 and
5), 114.24 (ArCABr), 115.24, 115.77 (ArCABr,
ArCACH@C), 122.60 (ArCACH@C), 125.26, 126.79
(ArCACOO, ArCAC@N), 133.07 (2· ArCH, C-20 and
60), 134.26, 135.26, 135.54 (ArCH, ArCH, CH@CAN),
150.32 (ArCAN), 154.67 (ArCAOAC@O), 157.31
(ArCAOMe), 164.38 (OAC@N), 165.03 (COO), 167.16
(COO) ppm. MS (70 eV, EI) m/z (%) 598/600/602 (1/2/
1) (M+Å), 149 (11), 148 (100) ((Me)2NAPhCO+), 120
(2), 106 (2), 105 (21) (PhCO+), 79 (2), 77 (11). Anal.
Calcd for C26H20Br2N2O5: C, 52.02; H, 3.36; N, 4.67.
Found: C, 52.18; H, 3.56; N, 4.85.
ArCACH@C), 125.28 (C, (ArC) C-10 or ArCACH@C),
126.76 (C, (ArC) C-10 or ArCACH@C), 128.55
(2· ArCH), 128.79 (2· ArCH), 128.82 (ArCH, C-40),
129.00 (2· ArCH), 129.04 ( 2· ArCH), 133.72 (ArCH,
C-400 or C-6000), 134.03 (C, (ArC) C-1000 or CH@CAN),
135.19 (ArCH, C-400 or C-6000), 135.24 (C, (ArC) C-1000
or CH@CAN), 156.63 (ArCAOCH2 or ArCAOMe),
157.24 (ArCAOCH2 or ArCAOMe), 164.37 (OAC@N),
166.73 (OAC@O) ppm. MS (70 eV, EI) m/z (%) 541/543/
545 (1/2/1) (M+Å), 436/438/440 (7/13/16) (MꢀPhACO),
308/310/312 (3/6/3), 106 (6), 105 (68), 92 (18), 91 (100)
ðPhCHþ2 Þ, 77 (31), 65 (6), 47 (8), 43 (8). Anal. Calcd
for C24H17Br2NO4: C, 53.07; H, 3.15; N, 2.58. Found:
C, 52.81; H, 3.21; N, 2.57.
4.10.4. (4Z)-4-(3,5-Dibromo-2-{[2-bromo-5-(dimethyla-
mino)benzyl]oxy}-4-methoxy-benzyliden)-2-phenyl-1,3-
oxazol-5(4H)-one (44). Orange crystals, mp >200 ꢁC:
4.10.2. Phenyl 2,4-dibromo-3-methoxy-6-[(Z)-(5-oxo-2-phe-
nyl-1,3-oxazol-4(5H)-yliden)-methyl]benzoate (41) and (4Z)-
4-(3,5-dibromo-2-hydroxy-4-methoxybenzyliden)2-phenyl-
1,3-oxazol-4(5H)-one (42). Compound 41: Yellow
crystals, mp >200 ꢁC: 40 mg, 0.07 mmol, 15%. 1H NMR
(200 MHz, CDCl3) d 3.99 (3H, s, OMe), 7.16 (1H, s,
ArCACH@C), 7.45–7.85 (6H, m, 8· ArH, H-
1
105 mg, 0.16 mmol, 21%. H NMR (300 MHz, CDCl3)
d 2.95 (6H, s, N(CH3)2), 3.99 (3H, s, OMe), 5.11 (2H,
s, CH2O), 6.56 (1H, dd, J = 8.9 Hz, J = 2.9 Hz, ArH,
3
4
4
H-400), 6.95 (1H, d, J = 2.9 Hz, ArH, H-600), 7.37 (1H,
3
d, J = 8.9 Hz, ArH, H-300), 7.46 (1H, s, ArCACH@C),
3
7.56 (2H, t, J = 6.7 Hz, 2· ArH, H-30 and 50), 7.67
3
30,40,50,300,400 and 500), 8.16 (2H, d, J = 8.3 Hz, 2· ArH,
(1H, t, 3J = 6.7 Hz, ArH, H-40), 8.18 (2H, d,

´
R. Cordoba et al. / Bioorg. Med. Chem. 15 (2007) 5300–5315
5314
3J = 6.7 Hz, 2· ArH, H-20 and 60), 9.10 (1H, s, ArH, H-
6000) ppm. MS (70 eV, EI) m/z (%) 662/664/666/668 (2/8/8/
3) (M+Å), 582/584/586/588 (2/5/5/2), 583/585/587 (5/11/6)
(MꢀBr), 555/557/559 (3/5/3) (MꢀBrACO), 504/506 (3/
3) (MꢀBr2), 451/453/455 (4/7/3), 212/214 (64/63)
((Br)((Me)2N)AC6H3(CH2)+), 133 (21), 132 (11), 118
(11), 106 (10), 105 (100) (PhCO+), 77 (41). Anal. Calcd
for C26H20Br2N2O5: C, 52.02; H, 3.36; N, 4.67. Found:
C, 52.18; H, 3.56; N, 4.85. Anal. Calcd for
C26H21Br3N2O4: C, 46.95; H, 3.18; N, 4.21. Found: C,
47.12; H, 3.08; N, 4.25.
and read at 490 nm. All determinations were carried
out in triplicate. IC50 value was calculated as the concen-
tration of drug yielding a 50% of cell survival.
4.12.3. Sprouting inhibition assay. BAE cells were mixed
with Cytodex 3 microcarriers beads (Amersham Phar-
macia Bioech) and the mixture was incubated for 48 h
at 37 ꢁC and 5% CO2. Following incubation cell-coated
beads were washed and resuspended in fresh medium at
a concentration of 250 beads per millilitre of media. 96-
Well tissue culture plates with the compound to be
tested were coated with 40 lL Matrigel Basement Mem-
brane Matrix (BD Biosciences) and cell-coated beads
(10 lL) were seeded on. Plates were incubated in 37 ꢁC
and 5% CO2 incubator and 30 min later 50 lL of culture
medium was added. After 48 h the extent and the length
of the sprouting formed is assessed by observation at
TMS-Nikon microscope and compared with non-trea-
ted well cells.
4.11. Synthesis of hydrochlorides
4.11.1.
(4Z)-4-(3,5-dibromo-2-{[2-bromo-5-(dimethyl-
amonium chloride) benzyl]oxy}4-methoxybenzyliden)-2-
phenyl-1,3-oxazol-5(4H)-one (45). A solution of the
azlactone 44 (70 mg, 0.11 mmol) in dioxane (30 mL)
was treated with gaseous HCl at room temperature for
30 min. Filtration of the yellow precipitate washing with
dioxane yielded hydrochloride 45. Yellow crystals, mp
1
>200 ꢁC: 40 mg, 0.06 mmol, 51%. H NMR (300 MHz,
Acknowledgments
CDCl3) d 3.22 (6H, s, N(CH3)2), 4.00 (3H, s, OMe),
5.12 (2H, s, CH2O), 7.42 (1H, s, ArCACH@C), 7.57
´
We thank Dr. Jesu´s Angel de la Fuente (Instituto Bio-
3
(2H, t, J = 7.4 Hz, 2· ArH, H-30 and 50), 7.69 (1H, t,
mar S.A., Leon, Spain) for valuable suggestions. This
´
work has been supported by Instituto Biomar S.A.
3J = 7.4 Hz, ArH, H-40), 7.73 (1H, br d, 3J = 8.7 Hz,
3
ArH, H-300), 7.83 (1H, dd, J = 8.7 Hz, ArH, H-400),
and Ministerio de Educacion y Ciencia (Spain, Grant
No. PTR1995-0651-OP).
´
3
7.98 (1H, br s, ArH, H-600), 8.20 (2H, d, J = 7.4 Hz,
2· ArH, H-20 and 60), 9.17 (1H, s, ArH, H-6000) ppm.
ESI m/z (%) 663/665/667/669 (30/90/75/25) (Mꢀ Cl)+.
References and notes
4.12. Biological assays
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´
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20% FBS) were incubated in each well with serial dilu-
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tion (37 ꢁC, 5% CO2 in a humid atmosphere) 50 mL of
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resulting formazan was dissolved in 100 mL DMSO

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(Dd = +1.03, +1.27, +0.36 and +0.27 for H-60, H-40,
H-30 and N(CH3)3, respectively) compared with the data
of the precursor 44.
´
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