Isoxazole-type derivatives related to combretastatin A-4, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2006.02.001

Novel combretastatin analogues bearing various five-membered heterocycles with consecutive oxygen and nitrogen atoms, in place of the olefinic bridge of CA4, have been synthesized (isoxazole, isoxazoline, oxadiazole, etc). These compounds have been evalua

Full text of DOI:10.1016/j.bmc.2006.02.001

Bioorganic & Medicinal Chemistry 14 (2006) 4067–4077
Isoxazole-type derivatives related to combretastatin A-4,
synthesis and biological evaluation
a
a,
b
b
*
´
`
Julia Kaffy, Renee Pontikis, Daniele Carrez, Alain Croisy,
Claude Monnereta and Jean-Claude Florenta
aCNRS, UMR 176, Institut Curie, Centre de Recherche, 26 rue d’Ulm, 75248 Paris cedex 05, France
bINSERM U 759 Imagerie inte´grative, Institut Curie, Centre de Recherche, Laboratoire Raymond Latarjet,
Centre Universitaire, 91405 Orsay cedex, France
Received 28 November 2005; revised 30 January 2006; accepted 3 February 2006
Available online 28 February 2006
Abstract—Novel combretastatin analogues bearing various five-membered heterocycles with consecutive oxygen and nitrogen
atoms, in place of the olefinic bridge of CA4, have been synthesized (isoxazole, isoxazoline, oxadiazole, etc). These compounds have
been evaluated for cytotoxicity and their ability to inhibit the tubulin assembly. On the basis of the relative position of the aromatic
A- and B-rings on the heterocyclic moiety, they could be split in two classes, the a,c- or a,b-diaryl heterocyclic derivatives. In the first
series, the 3,5-diaryloxadiazole 9a displayed comparable antitubulin activity to that of CA4, but was devoid of cytotoxic effects.
Among the a,b-diaryl heterocyclic derivatives, the 4,5-diarylisoxazole 35 exhibited greater antitubulin activity than that of CA4
(0.75 vs 1.2 lM), but modest antiproliferative activity. These data showed that minor alteration in the chemical structure of the
heterocyclic ring and its relative orientation with regard to the two phenyl rings of CA4 could dramatically influence the tubulin
binding properties.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
vascular effects observed well below its maximum toler-
ated dose rapidly led to extensive haemorrhagic necrosis
in areas that are often resistant to conventional anti-can-
cer treatments.12,13 CA4P, in combination with cytotox-
ic chemotherapy and radiotherapy, is undergoing phase
II trials, for the treatment of solid tumours.14,15 Another
CA4 derivative, AVE8062,16,17 is currently under clini-
cal evaluation as tumour vascular targeting agent.18
Combretastatin A-4 (CA4), a natural product isolated
by Pettit et al. in 1989 from the South African willow
tree Combretum caffrum,1 strongly inhibits tubulin poly-
merization by binding to the colchicine site.2 A disodi-
um phosphate prodrug form (CA4P) was selected for
further preclinical developments.3 CA4, which displayed
potent activity against a broad spectrum of human can-
cer cells, including multidrug-resistant cells,4 has drawn
significant attention due to its potent and selective effect
on the established tumour vasculature.5,6 CA4 has
shown the ability to shut down tumour vasculature,
whereas the blood flow to normal tissues was much less
affected.7 The most likely causes of the rapid vascular
collapse are morphological and functional changes in
endothelial cells,8,9 associated with an increased perme-
ability,10,11 which resulted in the disruption of the tubu-
lin cytoskeleton. In experimental tumours, the anti-
Given the encouraging antivascular/anticancer activity
of CA4P, synthesis of numerous analogues has been
reported in order to have a better understanding of the
structure–activity relationships.19 A key structural fac-
tor for tubulin affinity is the presence of the double
bond, or of a suitable linker, forcing the two aromatic
rings to stay within an appropriate distance.2,20 To over-
come the problem of the isomerization of the active cis
double bond into an inactive form, heterocyclic rings
were used in place of the ethene bridge.19,21,22
We report here, the synthesis and biological activities of
a series of CA4 analogues with a five-membered hetero-
cycle as linker of the two aromatic rings of CA4: isoxaz-
ole and other heterocyclic moieties with adjoined
nitrogen and oxygen atoms (Fig. 1). We intended to
Keywords: Combretastatin; Isoxazole; 1,3-Dipolar cycloaddition tubu-
lin; Cytotoxicity.
*
Corresponding author. Tel.: +33 142346659; fax: +33 142346631;
e-mail: renee.pontikis@curie.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.001

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J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
bearing a carboxylic or an amido group (Scheme 2).
With 1,2-disubstituted alkenes as dipolarophiles27 cyclo-
addition took place with reduced regioselectivity, lead-
ing to a mixture of the two possible regioisomeric d2-
isoxazolines. Thus, trans-ethyl 3,4,5-trimethoxycinna-
mate 11 afforded the 3,5- and 3,4-diarylcycloadducts
13a and 15a in an 85:15 ratio. With the bulky cinnamide
12, cycloadducts 14a and 16a were formed in almost
equal amounts.28
Figure 1.
We next investigated the reversed cycloadditions,
which were performed by reaction of the 3,4,5-tri-
methoxybenzaldehyde oxime30 17 with dipolarophiles
providing the aromatic B-ring (Scheme 3). Reaction
with the protected isovanillin 1 afforded the 3,5-diary-
ldioxazole 18a (10% yield). With arylacetylene 19, pre-
pared31,32 from aldehyde 1 by a Corey–Fuchs reaction,
3,5-diarylisoxazole 20a was obtained in 53% yield.
With cinnamide 23 (synthesized from the acid 21 by
a carbodiimide coupling with Et2NH) a mixture of
two regioisomeric adducts was obtained in modest
investigate the influence of the position of the hetero-
atoms of the bridge ring, with regard to A- and B-aro-
matic rings, on the antitubulin and cytotoxicity
activities of these analogues.
2. Results and discussion
2.1. Chemistry
1
yield (25%) in a 4:6 ratio determinated by H NMR,
but only the major isomer, 3,4-diarylisoxazoline 24a,
could be isolated in a pure form.
We have previously communicated the synthesis of nov-
el isoxazole-type derivatives related to CA4, using 1,3-
dipolar cycloaddition reaction.23 Recently, using the
same approach, Simoni et al.22 described the prepara-
tion of closely related heterocyclic analogues of CA4.
These results prompted us to publish our additional
result in this series.
Deprotection of the phenol function of the cycloadducts
7a–10a, 13a–16a, 18a, 20a and 24a, carried out using
TBAF (yields ranging from 80% to 95%), gave eleven
new heterocyclic CA4 analogues (corresponding deriva-
tives b in Schemes 1–3).33
As we have reported, the nitrile oxide generated in situ
from aldoxime 2 following Lee procedure24 underwent
a [3 + 2] regioselective cycloaddition (Scheme 1) with
3,4,5-trimethoxyphenylacetylene 325 to give 3,5-diarylis-
oxazole 7a (38% yield). In the same manner, oxime 2
was allowed to react with the known olefine 4,26 the
commercially available aldehyde 5 and nitrile 6 to give
isoxazoline 8a, dioxazole 9a and oxadiazole 10a in
62%, 10% and 6% yields, respectively.
Next, we turned to the synthesis of 2,3-diarylisoxazoli-
dines including a nitrogen at a bridgehead, which can
be prepared through the 1,3-dipolar cycloaddition reac-
tions of nitrones with ethylenic dipolarophiles.34 So we
have undertaken the reaction of methylacrylate with
the C,N-diarylnitrone 26 (Scheme 4), which was easily
obtained by one-pot preparation from nitroaryl 25 and
isovanillin under zinc-mediated reductive conditions.35
Condensation of methylacrylate with nitrone 26 was
carried out in toluene under reflux conditions for 18 h.
Along with nitrone which was mostly recovered un-
changed, a less polar derivative was isolated, albeit in
In order to obtain more hydrophilic derivatives, cou-
pling reactions have also been performed with olefines
1
very low yield. On the basis of H NMR its structure
0
0
was assigned to be
a
cis-cycloadduct ðJ3 4
¼
8:9 HzÞ,36,37 the isoxazolidine 27 bearing the ester group
in position 4.
Our following aim was to obtain CA4 analogues hav-
ing the olefinic bond incorporated into the hetero-
cyclic ring (Fig. 2). We first attempted 1,3-dipolar
cycloaddition with the known diarylacetylenic deriva-
tive 28 as dipolarophile (phenolic function protected
by a TBDMS group).25 However, compound 28 failed
to react with nitrile oxide A generated, either from
nitroalkane 29 under Mukaiyama conditions (phenyl-
isocyanide and Et3N),38 or prepared in situ (Et3N)
from chloroaldoxime 30,39 and even in the ionic liquid
[BMIM][BF4].40
Thus, we investigated an alternative strategy based on
the expected regioselective addition of hydroxylamine
on a b-ketoaldehyde species B.41 Such derivatives could
Scheme 1. Reagents and conditions: (i) NH2OHÆHCl, pyridine, EtOH,
reflux, 1 h; (ii) aq NaOCl, Et3N, CH2Cl2, 0 ꢁC, then rt 24 h (additional
reflux 24 h for 5 and 6); (iii) TBAF, THF, rt, 2 h.

J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
4069
Scheme 2. Reagents and conditions: (i) aq NaOCl, Et3N, CH2Cl2, 0 ꢁC, then rt, 24 h; (ii) TBAF, THF, rt, 2 h.
Scheme 3. Reagents and conditions: (i) aq NaOCl, Et3N, CH2Cl2, 0 ꢁC, rt, 24 h (additional reflux 24 h for 1; (ii) TBAF, THF, rt, 2 h; (iii) a—Ref. 31:
CBr4, PPh3, CH2Cl2, 0 ꢁC, 10 min; b—Ref. 32: n-BuLi (2 equiv.), THF, ꢀ78 ꢁC to rt; (iv) EDCI, HOBT, Et2NH, DMF, rt, 3 h; (v) TBDMSCl, i-
Pr2EtN, THF, rt, 24 h.
Figure 2. Considered pathways of access to 4,5-diarylisoxazoles.
Nitrile oxides A were generated from O2NCH2CH2CO2Et 29 or
HONCH(Cl)CO2Et 30.
Scheme 4. Reagents and conditions: (i) isovanillin, Zn, AcOH, EtOH,
0 ꢁC, 2 h then rt, 2 h; (ii) CH2@CHCOOCH3, toluene, reflux, 17 h.
5) base-catalyzed Claisen–Schmidt condensation be-
tween 3,4,5-trimethoxyacetophenone 31 and the O-pro-
tected isovanillin 32,44 afforded the chalcone 33 in good
yield. Its treatment with hydrogen peroxide in alkaline
conditions at 0 ꢁC, according to Jain and Krish-
namurty45 provided the ketoepoxide derivative 34 in
result from the Lewis acid-promoted rearrangement
(pinacol–pinacone type)42,43 of ketoepoxides C, which
could be easily obtained from chalcones. Thus, (Scheme

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J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
Scheme 5. Reagents and conditions: (i) 31, 6 N NaOH, EtOH, 60 ꢁC, 5 h; (ii) H2O2, 2 N NaOH, EtOH, 0 ꢁC to rt, 4 h; (iii) a—BF3ÆEt2O, THF,
reflux, 45 min; b—NH2OHÆHCl, pyridine, EtOH, reflux, 6 h.
83% yield. Epoxide 34 was next stirred with an excess of
BF3ÆEt2O in THF at 40 ꢁC for 45 min and, after usual
workup, the crude mixture was submitted to oximation
with hydroxylamine hydrochloride.42 The expected 4,5-
diarylisoxazole 35, with the phenolic group free, was
isolated in 32% yield from a mixture of other unchar-
acterized products. The structure of 35 was assigned
on the basis of the 1H NMR chemical shift value
(8.29 ppm) for the heterocyclic proton.21b,46 The
modest yield obtained for this two-step reaction could
result from the lack of regioselectivity during the
rearrangement process (hydride, aryl or acyl migra-
tion)43 and the lack of chemoselectivity in the oxima-
tion step (aldehyde > ketone).41
2.2. Biological evaluation
Our main objective was to investigate the importance of
(a) the nature and (b) the substitution pattern of the het-
erocyclic ring for the antitubulin and the antiprolifera-
tive activities of these new CA4 analogues (Table 1).
These derivatives could be classified in two series,
depending on the relative position of the aromatic A-
and B-rings on the heterocyclic moiety (a,c or a,b).
2.2.1. Inhibition of tubulin polymerization (ITP). In
the first series, the 3,5-diaryldioxazole 9b displayed an
interesting inhibition of tubulin polymerization
(IC50 = 5.0 lM), nevertheless 4-fold lower than that of
Table 1. Inhibition of tubulin polymerization (ITP) and cellular proliferation for heterocyclic analogues of CA4
Compound
ITPa IC50 (lM)
HT29b IC50 (lM)
SVECb IC50 (lM)
7b Z = CH
8b Z = CH2
>25
>25
5.0
78 20
NDc
>50
23.5 10
ND
>50
O
N
O
9b Z = O
10b Z = N
Ar(A)
Ar(A)
Ar(B)
Ar(B)
>25
>25
>25
>50
>50
40
Z
13b Z = CHCO2Et
14b Z = CHCONEt2
NEd
NE
NE
N
18b Z = O
20b Z = CH
>25
>25
>50
>50
>50
49
Z
R
O
15b R = CO2Et
16b R = CONEt2
>25
>25
NE
NE
ND
NE
N
Ar(A)
Ar(A)
Ar(A)
Ar(A)
Ar(B)
O
N
O
CONEt2
CO2Et
24b
27
>25
>25
7.1 1.4
9.5 0.5
Ar(B)
N
NE
39
Ar(B)
N
O
35
CA4
0.75
1.2
3
0.47 0.03
8.5
3
0.1
Ar(B)
a Drug concentration needed to inhibit tubulin polymerization by 50%. The compounds were assayed at least twice and the reported IC50 values were
the averages.
b Drug concentration needed to inhibit cell growth by 50% SD. Given values are the mean of two independent experiments in triplicate, with CA4
as positive control.
c ND: not determined.
d NE: no effect was shown at the highest concentration (50 lM) tested.

J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
4071
CA4 (IC50 = 1.2 lM). Switching the position of the two
aryl groups (18b vs 9b) resulted in a dramatic loss in inhi-
bition potency. The other derivatives, with at the 4-posi-
tion a methine (7b, 20b), a methylene (8b), a nitrogen
atom (10b) or an exocyclic carboxylate or amido group
(13b, 14b), were ineffective in the tubulin assay (IC50 val-
ues > 25 lM). From these data it appeared that in this
series of a,c-diaryl heterocyclic analogues, the substitu-
tion pattern on the heterocyclic moiety played an impor-
tant role in the antitubulin activity: a CH group in the
benzylic position of the 3,4,5-trimethoxyphenyl ring
and a sp2 carbon adjacent to the B-ring seemed neces-
sary. Oxygen atom at the 4-position also had a great
influence, since the isoxazoline analogue 8b, with a
CH2 group replacing the heteroatom, did not interact
with the tubulin; These three structural requirements
(Fig. 3) were present in the two potent analogues devel-
oped at Abbott Laboratories, the oxadiazoline
(A105972)47 and the oxazoline (A289099).48
cells, 24b was equipotent to the powerful antitubulin
inhibitor 35 and, surprisingly, the isomeric isoxazoline
16b was inactive. It is noteworthy that the four heterocy-
clic analogues, 7b, 13b, 20b and 27, which were ineffective
in the tubulin assay and in the proliferation test on H29
cell line, displayed weak cytotoxicity (8- to 16-fold less
than that of CA4) on the SVEC cell line. This suggests that
these derivatives may have some specificity for the endo-
thelial cells and another target than tubulin.
3. Conclusion
By using 1,3-dipolar cycloaddition reaction a series of
twelve CA4 analogues had been synthesized (6–62%
yields). The olefinic bond was replaced by five-membered
heterocyclic rings: isoxazole or other moieties with ad-
joined nitrogen and oxygen atoms. On the other hand,
the isoxazole-based CA4 derivative 35 had been obtained
by a concise synthesis starting from an easily accessible
chalcone. All these compounds were evaluated for their
antitubulin and antiproliferative activities. Among the
eight derivatives with A- and B-rings attached to an het-
erocyclic moiety in an a,c-relationship, only the 3,5-diar-
yloxadiazole 9b showed an antitubulin activity
comparable to that of CA4, but was nevertheless devoid
of cytotoxicity. Of the five a,b-diaryl heterocyclic deriva-
tives, the isoxazole 35 displayed higher antitubulin activ-
ity than that of CA4. With this constrained derivative, the
liability of CA4 to isomerize to an inactive form is pre-
vented. So, it seems appropriate to undertake the synthe-
sis of a series of such isoxazole derivatives in which the
natural B-ring would be replaced by other rings, for exam-
ple heteroaromatic moieties, to increase the solubility.
Forthea,b-diarylheterocyclic analogues, the4,5-diarylis-
oxazole 35 exhibited a stronger activity (IC50 = 0.75 lM),
almost twice more potent than that of CA4. The other
analogues, the isoxazoline derivatives 15b, 16b and 24b
and the 2,3-diarylisoxazolidine 27, were essentially inac-
tive. Numerous CA4 analogues with an ethylene bridge
as part of a five-membered heterocyclic ring have been
reported in the literature.19,21b Among them, imidazole
36 and oxazole 3749 displayed, as isoxazole 35, a higher
antitubulin activity than that of CA4 (Fig. 4).
2.2.2. Antiproliferative activity. Compounds were evalu-
ated for their antiproliferative activity against human co-
lon adenocarcinoma cell line HT29 and transformed
murine endothelial cell line SVEC 4–10, using an MTT as-
say.50,51 The most potent tubulin inhibitor 35 showed
some antiproliferative activities on HT29 and SVEC cells,
nevertheless 6- and 3-fold lower than that of CA4, respec-
tively. For its part, the other potent tubulin inhibitor,
dioxazole 9b, was devoid of cytotoxic effects. Prolifera-
tion of HT29 cells was not inhibited by any of the other
derivatives, except the carboxamide group-bearing isox-
azoline 24b, which showed a weak cytotoxicity (7.1 lM,
i.e., 15-fold lower than CA4). Moreover, against SVEC
These results demonstrated that minor alteration in the
chemical structure of the heterocyclic ring and its rela-
tive orientation with regard to the two phenyl rings of
CA4 could dramatically influence the tubulin binding
properties.
4. Experimental
4.1. General methods
NMR spectra were recorded on a Bruker AM-300 spec-
trometer at 300 MHz (1H) and at 75 MHz (13C) using
CDCl3 as the solvent. The residual proton-solvent peak
is used as the internal standard [(d 7.25 (1H) and
77.0 ppm (13C)] and J values are given in hertz. Multi-
plicities are reported using the following abbreviations:
s, singlet; d, doublet; dd, doublet of doublet; br, broad;
and m, multiplet. Chemical ionization (CI) mass spectra
were recorded on a Nermag R10-10-C spectrometer.
High-resolution mass spectra (HRMS) were obtained
on a Jeol-700 spectrometer. Melting points were deter-
mined on an Electrothermal 9200 apparatus and are
uncorrected. Elemental analyses were performed by
the ‘Service de Microanalyses du CNRS’ (Vernaison-
Lyon, France). The thin-layer chromatographic analyses
were performed using pre-coated silica gel (Merck,
60F254) plates and the spots were examined with UV
Figure 3. A = 3,4,5-trimethoxyphenyl; B1 = 4-hydroxy-3-methoxyphe-
nyl; B2 = 4-amino-3-methylphenyl; B3 = 5-(N-methyl)indolyl.
Figure 4. A = 3,4,5-trimethoxyphenyl; B = 4-hydroxy-3-methoxy-
phenyl.

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J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
light and phosphomolybdic acid spray. Preparative col-
umn chromatographies were carried out on Merck silica
gel (230–240 mesh). THF was distilled from sodium ben-
zophenone ketyl prior to use. Derivatives 1,52 3,25 4,26
17,30 19,31,32 and 20b,22 were prepared according to pub-
lished procedures.
aqueous layer was extracted with EtOAc, the combined
organic layers were washed with brine, dried (MgSO4), fil-
tered and concentrated under reduced pressure. Follow-
ing flash chromatography (CH2Cl2/MeOH 97:3),
silylated compound 23 was isolated as a white crystalline
1
solid (2.14 g, 65%). Mp 90–92 ꢁC; H NMR (300 MHz,
CDCl3): d 7.60 (d, 1H, J = 15.3 Hz, ArCH), 7.10 (dd,
1H, J = 8.3 Hz, J = 2.1 Hz, H-6), 7.02 (d, 1H, J = 2.1
Hz, H-2), 6.82 (d, 1H, J = 8.3 Hz, H-5), 6.65 (d, 1H,
J = 15.3 Hz, CHCO), 3.83 (s, 3H, OCH3), 3.45 (m, 4H,
2· CH2), 1.25 (t, 3H, J = 7.1 Hz, CH3), 1.18 (t, 3H,
J = 7.1 Hz, CH3), 0.99 [s, 9H, C(CH3)3], 0.15 [s, 6H,
Si(CH3)2].
Numbering for 1H NMR assignments is usual for the A-
ring, for B-ring and for the heterocyclic ring.
0
00
4.2. 3-(tert-Butyldimethylsilanyloxy)-4-methoxybenzalde-
hyde oxime (2)
To a solution of silylated isovanillin 1 (16.7 g, 62.9 mmol)
in methanol (300 mL) were added, under argon, hydrox-
ylamine hydrochloride (4.4 g, 63.3 mmol) and then pyri-
dine (5.6 mL, 69.1 mmol). After refluxing for 1 h, the
reaction mixture was concentrated to 50 mL, diluted with
water (300 mL) and extracted with EtOAc. Combined
organic extracts were washed with brine, dried (MgSO4),
filtered and concentrated under reduced pressure. The
residual yellow oil was washed with pentane to give oxime
2 (15.54 g, 88%) as a thick oil. 1H NMR (300 MHz,
CDCl3): d 8.02 (s, 1H, CH), 7.14 (d, 1H, J = 2.0 Hz, H-
2), 7.08 (dd, 1H, J = 8.3 Hz, J = 2.0 Hz, H-6), 6.83 (d,
1H, J = 8.3 Hz, H-5), 3.83 (s, 3H, OCH3), 0.99 [s, 9H,
C(CH3)3], 0.15 [s, 6H, Si(CH3)2].
4.5. General procedure for the cycloaddition reactions
To dipolarophile 3, 4, 5, 6, 11, 12, 1, 19 or 23 (1 equiv)
and triethylamine (0.1 equiv) in dichloromethane were
added, under argon atmosphere, a 13% aqueous solu-
tion of NaOCl (1.6 equiv) and dropwise (over a period
of 1 h) at 0 ꢁC, the appropriate oxime 2 or 17 (1 equiv)
in dichloromethane. After being stirred at room temper-
ature for 24 h (an additional reflux period was necessary
for less reactive dipolarophiles 5, 6 and 1), water was
added to the reaction mixture and the aqueous layer
was extracted with dichloromethane. The combined
organic layers were washed with water, brine, dried
(MgSO4), filtered and concentrated under reduced pres-
sure. The residue was chromatographed as indicated.
4.3. N,N-Diethyl-3-(3-hydroxy-4-methoxyphenyl)-acryl-
amide (22)
4.5.1. 3-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-5-(3,4,5-trimethoxyphenyl)isoxazole (7a). From al-
kyne 3 (271 mg, 1.41 mmol) and oxime 2 (398 mg,
1.42 mmol), isoxazole 7a was obtained as a yellow oil
(251 mg, 38% yield) after a flash chromatography (cyclo-
hexane/EtOAc 9:1–8:2). 1H NMR data are in agreement
with those described by Simoni et al.22; 13C NMR
(75 MHz, CDCl3): d 169.9 (C-Het), 162.7 (C-Het), 153.6
(2· C), 152.6 (C), 145.3 (C), 139.8 (C), 123.0 (C), 121.8
(C), 120.6 (CH), 119.3 (CH), 111.9 (CH), 103.1 (2·
CH), 97.0 (CH-Het), 61.0 (CH3), 56.3 (2· CH3), 55.4
(CH3), 25.7 (3· CH3), 18.5 (C), ꢀ4.6 (2· CH3).
To a solution of 3-hydroxy-4-methoxycinnamic acid 21
(2.5 g, 12.9 mmol) in dry DMF (25 mL) were added dieth-
ylamine (1.4 mL, 13.4 mmol), hydroxybenzotriazole
(2.1 g, 15.5 mmol) and EDCI (3.0 g, 15.6 mmol). After
stirring at room temperature for 3 h, the yellow mixture
was partitioned between EtOAc and water. The aqueous
layer was extracted with EtOAc, the combined organic
layers were washed with brine, dried (MgSO4), filtered,
concentrated under reduced pressure and chromato-
graphed (CH2Cl2/MeOH 97:3) to give amide 22 as a white
solid, which was recrystallized from EtOAc (2.10 g, 65%).
Mp 112–114 ꢁC; 1H NMR(300 MHz, CDCl3): d 7.63 (d,
1H, J = 15.3 Hz, ArCH), 7.19 (d, 1H, J = 2.0 Hz, H-2),
6.99 (dd, 1H, J = 8.3 Hz, J = 2.0 Hz, H-6), 6.81 (d, 1H,
J = 8.3 Hz, H-5), 6.67 (d, 1H, J = 15.3 Hz, CHCO), 6.31
(br s, 1H, OH), 3.89 (s, 3H, OCH3), 3.45 (m, 4H, 2·
CH2), 1.23 (t, 3H, J = 7.1 Hz, CH3), 1.17 (t, 3H,
J = 7.1 Hz, CH3); 13C NMR (75 MHz, CDCl3): d 166.0
(CO), 148.1 (C), 145.9 (C), 142.3 (CH), 128.9 (C), 121.3
(CH), 115.6 (CH), 112.9 (CH), 110.6 (CH), 55.9 (CH3),
42.3 (CH2), 41.1 (CH2), 15.0 (CH3), 13.2 (CH3). Anal.
Calcd for C14H19NO3: C, 67.45; H, 7.68; N, 5.62. Found
C: 67.13; H, 7.89; N, 5.51.
4.5.2. 3-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-5-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole (8a).
From alkene 4 (1.56 g, 8.0 mmol) and oxime 2 (2.24 g,
8.0 mmol), dihydroisoxazole 8a was obtained as a yellow
oil (2.34 g, 62% yield) after a flash chromatography (cyclo-
hexane/EtOAc 8:2). 1H NMR data are in agreement with
those described by Simoni et al.22; 13C NMR (75 MHz,
CDCl3): d 155.8 (C-Het), 153.5 (2· C), 152.8 (C), 145.1
(C), 137.7 (C), 136.7 (C), 122.1 (C), 120.9 (CH), 119.0
(CH), 111.5 (CH), 102.6 (2· CH), 82.4 (CH-Het), 60.8
(CH3), 56.1 (2· CH3), 55.4 (CH3), 43.6 (CH2-Het), 25.6
(3· CH3), 18.4 (C), ꢀ4.6 (2· CH3).
4.4. 3-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-N,N-diethyl-acrylamide (23)
4.5.3. 3-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-5-(3,4,5-trimethoxyphenyl)-1,2,4-dioxazole (9a). From
trimethoxybenzaldehyde 5(985 mg, 5.02 mmol)andoxime
2 (1.40 g, 4.98 mmol), dioxazole 9a was obtained as a col-
ourless oil (235 mg, 10% yield) after a flash chromatogra-
Diisopropylethylamine (1.2 mL, 6.87 mmol) was added,
under argon, to a stirred solution of phenol 22 (914 mg,
3.67 mmol) in dry THF followed by tert-butyldimethylsi-
lyl chloride (773 mg, 5.13 mmol). After stirring at room
temperature for 24 h, water was added (100mL). The
1
phy (cyclohexane/EtOAc 9:1–8:2). H NMR (300 MHz,
CDCl3): d 7.43 (dd, 1H, J = 8.4 Hz, J = 2.1 Hz, H-60),

J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
4073
7.33 (d, 1H, J = 2.1 Hz, H-20), 6.88 (d, 1H, J = 8.4 Hz, H-
50), 6.82(s, 2H, H-2, H-6), 6.73(s, 1H, H-500), 3.89(s, 6H, 2·
OCH3), 3.86 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 0.99 [s,
9H, C(CH3)3], 0.16 [s, 6H, Si(CH3)2].
(587 mg, 2.0 mmol) and oxime 2 (562 mg, 2.0 mmol) a
mixture of dihydroisoxazoles 14a and 16a (660 mg, 58%
yield, ratio 45:55 determined by 1H NMR) was obtained
after a flash chromatography (cyclohexane/EtOAc 6:4–
4:6). They could be separated after another two tedious
chromatographies (cyclohexane/EtOAc 7:3) to give 14a
(312 mg, 27%) and 16a (251 mg, 22%) as white foams.
Cycloadduct 14a: TLC (cyclohexane/ EtOAc 3:7) Rf
0.57; 1H NMR (300 MHz, CDCl3): d 7.17 (d, 1H,
J = 2.1 Hz, H-20), 7.13 dd, 1H, J = 8.3 Hz, J = 2.1 Hz,
H-60), 6.81 (d, 1H, J = 8.3 Hz, H-50), 6.62 (s, 2H, H-2,
H-6), 5.65 (d, 1H, J = 9.9 Hz, H-500), 4.59 (d, 1H,
J = 9.9 Hz, H-400), 3.84 (s, 9H, 3· OCH3), 3.81 (s, 3H,
OCH3), 3.41 (q, 2H, J = 7.1 Hz, CH2), 3.19 (m, 2H,
CH2), 1.25 (t, 3H, J = 7.1 Hz, CH3), 1.15 (t, 3H,
J = 7.1 Hz, CH3), 0.99 [s, 9H, C(CH3)3], 0.14 [s, 6H,
Si(CH3)2]. Cycloadduct 16a: TLC (cyclohexane/EtOAc
4.5.4. 3-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-5-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (10a).
From trimethoxybenzonitrile 6 (970 mg, 5.02 mmol)
and oxime 2 (1.59 g, 5.04 mmol), oxadiazole 10a was ob-
tainedas an oil (142 mg, 6% yield) aftera flashchromatog-
raphy (cyclohexane/EtOAc 9:1). 1H NMR (300 MHz,
CDCl3): d 7.76 (dd, 1H, J = 8.5 Hz, J = 2.1 Hz, H-60),
7.63 (d, 1H, J = 2.1 Hz, H-20), 7,44 (s, 2H, H-2, H-6),
6.95 (d, 1H, J = 8.5 Hz, H-50), 4.00 (s, 6H, 2· OCH3),
3.94 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 1.03 [s, 9H,
C(CH3)3], 0.20 [s, 6H, (SiCH3)3].
1
4.5.5. trans-Ethyl-3-[3-(tert-butyldimethylsilanyloxy)-4-
methoxyphenyl]-5-(3,4,5-trimethoxyphenyl)-4,5-dihydrois-
oxazole-4-carboxylate (13a). trans-Ethyl-3-[3-(tert-butyl-
dimethylsilanyloxy)-4-methoxyphenyl]-4-(3,4,5-trimethoxy-
phenyl)-4,5-dihydroisoxazole-5-carboxylate (15a). From
ethyl-3,4,5-trimethoxycinnamate 11 (2.08 g, 7.80 mmol)
and oxime 2 (2.11 g, 7.50 mmol) a mixture of dihydrois-
oxazoles 13a and 15a (2.57 g, 63% yield, ratio 85:15
determined by 1H NMR) was obtained after a flash
chromatography (cyclohexane/EtOAc 8:2). They could
be separated after another tedious chromatography
(CH2Cl2/acetone 99:1) to give 13a (1.99 g, 49%) and
3:7) Rf 0.51; H NMR (300 MHz, CDCl3): d 7.24 (dd,
1H, J = 8.5 Hz, J = 2.1 Hz, H-60), 7.07 (d, 1H,
J = 2.1 Hz, H-20), 6.75 (d, 1H, J = 8.5 Hz, H-50), 6.46 (s,
2H, H-2, H-6), 5.54 (d, 1H, J = 5.7 Hz, H-400), 5.05 (d,
1H, J = 5.7 Hz, H-500), 3.79 (s, 9H, 3· OCH3), 3.77 (s,
3H, OCH3), 3.45 (m, 4H, 2· CH2), 1.27 (t, 3H,
J = 7.0 Hz, CH3), 1.25 (t, 3H, J = 7.0 Hz, CH3); 0.92 [s,
9H, C(CH3)3], 0.05 (s, 3H, SiCH3), 0.02 (s, 6H, SiCH3).
4.5.7. 5-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-3-(3,4,5-trimethoxyphenyl)-1,2,4-dioxazole
(18a).
From silylated isovanillin 1 (2.66 g, 10.00 mmol) and
oxime 17 (2.11 g, 10.0 mmol), dioxazole 18a was ob-
tained as a white foam (472 mg, 10% yield) after a flash
1
15a (139 mg, 3%) as white foams. Cycloadduct 13a: H
NMR (300 MHz, CDCl3): d 7.28 (d, 1H, J = 2.1 Hz,
H-20), 7.23 (dd, 1H, J = 8.4 Hz, J = 2.1 Hz, H-60), 6.83
(d, 1H, J = 8.4 Hz, H-50), 6.58 (s, 2H, H-2, H-6), 5.86
(d, 1H, J = 6.7 Hz, H-500), 4.37 (d, 1H, J = 6.7 Hz, H-
400), 4.23 (q, 2H, J = 7.1 Hz, CH2), 3.86 (s, 6H, 2·
OCH3), 3.83 (s, 6H, 2· OCH3), 1.22 (t, 3H,
J = 7.1 Hz, CH3), 0.99 [s, 9H, C(CH3)3], 0.16 [s, 6H,
Si(CH3)2]; 13C NMR (75 MHz, CDCl3): d 169.3 (CO),
153.6 (2· C), 155.3 (C-Het), 152.9 (C), 145.1 (C), 138.1
(C), 135.3 (C), 121.1 (CH), 121.0 (C), 119.3 (CH),
111.5 (CH), 102.2 (2· CH), 86.7 (CH-Het), 62.3 (CH-
Het), 62.1 (CH2), 60.8 (CH3), 56.1 (2· CH3), 55.3 (CH3),
25.6 (3· CH3), 18.4 (C), 14.0 (CH3), ꢀ4.7 (2· CH3). Cyc-
1
chromatography (cyclohexane/EtOAc 85:15). H NMR
(300 MHz, CDCl3):
d 7.16 (dd, 1H, J = 8.3 Hz,
J = 2.1 Hz, H-60), 7.08 (d, 1H, J = 2.1 Hz, H-20), 7.07
(s, 2H, H-2, H-6), 6.90 (d, 1H, J = 8.3 Hz, H-50), 6.74
(s, 1H, H-500), 3.89 (s, 3H, OCH3), 3.88 (s, 6H, 2·
OCH3), 3.84 (s, 3H, OCH3), 1.00 [s, 9H, C(CH3)3],
0.16 [s, 6H, Si(CH3)2]; 13C NMR (75 MHz, CDCl3): d
159.4 (C-Het), 153.3 (2· C), 152.3 (C), 145.3 (C), 140.8
(C), 126.8 (C), 120.8 (C), 119.4 (CH), 118.0 (C), 111.7
(CH), 108.9 (CH-Het), 104.1 (2· CH), 61.0 (CH3),
56.3 (2· CH3), 55.5 (CH3), 25.7 (3· CH3), ꢀ4.6 (2·
CH3).
1
loadduct 15a: H NMR (300 MHz, CDCl3): d 7.22 (dd,
1H, J = 8.5 Hz, J = 2.1 Hz, H-60), 7.07 (d, 1H,
J = 2.1 Hz, H-20), 6.76 (d, 1H, J = 8.5 Hz, H-50), 6.44 (s,
2H, H-2, H-6), 4.89 (m, 2H, H-400, H-500), 4.29 (m, 2H,
CH2), 3.80 (s, 9H, 3· OCH3), 3.78 (s, 3H, OCH3), 1.33
(t, 3H, J = 7.1 Hz, CH3), 0.92 [s, 9H, C(CH3)3], 0.06 (s,
3H, SiCH3), 0.02 (s, 3H, SiCH3); 13C NMR (75 MHz,
CDCl3): d 170.1 (CO), 157.3 (C-Het), 153.9 (2· C), 152.8
(C), 144.7 (C), 137.6 (C), 133.7 (C), 121.7 (CH), 120.4
(C), 120.0 (CH), 111.6 (CH), 104.2 (2· CH), 86.2 (CH-
Het), 62.1 (CH2), 60.8 (CH3), 58.7 (CH-Het), 56.1 (2·
CH3), 55.4 (CH3), 25.6 (3· CH3), 18.4 (C), 14.1 (CH3),
ꢀ4.8 (2· CH3).
4.5.8. 5-[3-(tert-Butyldimethylsilanyloxy)-4-methoxyphe-
nyl]-3-(3,4,5-trimethoxyphenyl) isoxazole (20a). From al-
kyne 19 (346 mg, 1.32 mmol) and oxime 17 (217 mg,
1.00 mmol), isoxazole 20a was obtained as a yellow oil
(258 mg, 53% yield) after a flash chromatography
(cyclohexane/EtOAc 8:2). Data are in agreement with
those described by Simoni et al.22
4.5.9. trans-N,N-Diethyl-4-[3-(tert-butyldimethylsilanyl-
oxy)-4-methoxyphenyl]-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazole-5-carboxamide (24a). From N,N-dieth-
ylcinnamide 23 (1.52 g, 4.20 mmol) and oxime 17
(0.88 g, 4.20 mmol) and after three tedious chromato-
graphies (cyclohexane/EtOAc 8:2), the cycloadduct 24a
could be isolated (143 mg, 6% yield). 1H NMR
4.5.6. trans-N,N-Diethyl-3-[3-(tert-butyldimethylsilanyl-
oxy)-4-methoxyphenyl]-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydroisoxazole-4-carboxamide (14a). trans-N,N-
Diethyl-3-[3-(tert-butyldimethylsilanyloxy)-4-methoxy-
phenyl]-4-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-
5-carboxamide (16a). From N,N-diethylcinnamide 12
(300 MHz, CDCl3):
d 6.86 dd, 1H, J = 8.3 Hz,
J = 2.1 Hz, H-60), 6.83 (s, 2H, H-2, H-6), 6.79 (d, 1H,
J = 8.3 Hz, H-50), 6.74 (d, 1H, J = 2.1 Hz, H-20), 5.52
(d, 1H, J = 6.2 Hz, H-400), 5.07 (d, 1H, J = 6.2 Hz, H-

4074
J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
500), 3.80 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.72 (s, 3H,
2· OCH3) 3.44 (m, 4H, 2· CH2), 1.22 (t, 3H, J = 7.1 Hz,
CH3), 1.16 (t, 3H, J = 7.1 Hz, CH3), 0.92 [s, 9H,
C(CH3)3], 0.04 (s, 3H, SiCH3), 0.03 (s, 6H, SiCH3).
4.6.4. 3-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimeth-
oxyphenyl)-1,2,4-oxadiazole (10b). Oxadiazole 10b was
obtained from protected compound 10a (123 mg,
0.26 mmol) after a flash chromatography (cyclohexane/
EtOAc 62:38) as a white crystalline solid (65 mg, 70%).
1
4.6. General procedure for removal of the TBDMS-
protecting group
Mp 154–156 ꢁC; H NMR (300 MHz, CDCl3): d 7.74
(d, 1H, J = 2.0 Hz, H-20), 7.7 (dd, 1H, J = 8.3 Hz,
J = 2.0 Hz, H-60), 7.43 (s, 2H, H-2, H-6), 6.95 d, 1H,
J = 8.3 Hz, H-50), 5.76 (br s, 1H, OH), 3.94 (s, 3H,
OCH3), 3.96 (s, 6H, 2· OCH3), 3.94 (s, 3H, OCH3);
13C NMR (75 MHz, CDCl3): d 175.2 (C-Het), 168.6
(C-Het), 153.6 (2· C), 149.0 (C), 145.8 (C), 141.9 (C),
120.1 (C, CH), 119.4 (C), 113.6 (CH), 110.5 (CH),
105.3 (2· C), 61.0 (CH3), 56.4 (2· CH3), 56.0 (CH3);
HRMS (DCI/NH3) m/z 359.1240 [(M + H)+ calcd for
C19H20NO6: 359.1243].
A solution of the appropriate silyl ether 7a-10a, 13a-16a,
18a, 20a or 24a in dry THF (1 equiv) was treated with a
1 M solution of tetrabutylammonium fluoride in THF
(1 equiv). The mixture was stirred at room temperature
during 2 h, then diluted with water and extracted with
EtOAc. The organic layers were washed with water, dried
(MgSO4), filtered and concentrated under reduced pres-
sure. The residue was chromatographed as indicated.
4.6.1. 3-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimeth-
oxyphenyl)isoxazole (7b). Isoxazole 7b was obtained from
protected compound 7a (453 mg, 0.96 mmol) after a flash
chromatography (cyclohexane/EtOAc 6:4–3:7) as a white
4.6.5. trans-Ethyl-3-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazole-4-carboxylate
(13b). Ester 13b was obtained from protected compound
13a (624 mg, 1.10 mmol) after a flash chromatography
(cyclohexane/EtOAc 1:1) as a yellow crystalline solid
1
crystalline solid (302 mg, 88%). H NMR data are in
agreement with those described by Simoni et al.22; 13C
NMR (75 MHz, CDCl3): d 169.9 (C-Het), 162.6 (C-
Het), 153.6 (2· C), 148.0 (C), 145.9 (C), 139.7 (C), 122.9
(C), 122.3 (C), 119.0 (CH), 113.0 (CH), 110.7 (CH),
103.1 (2· CH), 97.1 (CH-Het), 61.0 (CH3), 56.3 (2·
CH3), 56.0 (CH3); MS (DCI/NH3) m/z 358 (M+H)+;
Anal. Calcd for C19H19NO6Æ0.25 H2O: C, 63.06; H,
5.43; N, 3.87. Found C: 63.32; H, 5.31; N, 3.97.
(356 mg, 75%). Mp 111–113 ꢁC; H NMR (300 MHz,
1
CDCl3): d 7.33 (d, 1H, J = 2.1 Hz, H-20), 7.19 (dd, 1H,
J = 8.4 Hz, J = 2.1 Hz, H-60), 6.83 (d, 1H, J = 8.4 Hz,
H-50), 6.57 (s, 2H, H-2, H-6), 5.86 (d, 1H, J = 6.6 Hz,
H-500), 5.78 (br s, 1H, OH), 4.38 (d, 1H, J = 6.6 Hz, H-
400), 4.23 (m, 2H, CH2CH3), 3.92 (s, 3H, OCH3), 3.84 (s,
6H, 2· OCH3), 3.81 (s, 3H, OCH3), 1.22 (t, 1H,
J = 7.0 Hz, CH2CH3); 13C NMR (75 MHz, CDCl3): d
169.2 (CO), 153.6 (2· CH), 153.4 (CH-Het), 148.4 (C),
145.7 (C), 138.2 (C), 135.2 (C), 121.6 (C), 119.6 (CH),
113.0 (CH), 110.4 (CH), 102.2 (2· CH), 86.7 (CH-Het),
62.1 (CH-Het, CH2), 60.7 (CH3), 56.1 (2· CH3), 55.9
(CH3), 13.9 (CH3); MS (DCI/NH3) m/z 432 (M+H)+;
Anal. Calcd for C22H25NO8: C, 61.25; H, 5.84; N, 3.25.
Found C: 60.89; H, 5.91; N, 3.03.
4.6.2. 3-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimeth-
oxyphenyl)-4,5-dihydroisoxazole (8b). Dihydroisoxazole
8b was obtained from protected compound 8a (2.08 g,
4.39 mmol) after a flash chromatography (cyclohexane/
EtOAc 5:5–3:7) as a white crystalline solid (1.40 g,
1
89%). H NMR data are in agreement with those de-
scribed by Simoni et al.22; 13C NMR (75 MHz, CDCl3):
d 155.9 (C-Het), 153.5 (2· C), 148.2 (C), 145.7 (C), 137.6
(C), 136.6 (C), 122.7 (C), 119.3 (CH), 112.8 (CH), 110.5
(CH), 102.6 (2· C), 82.4 (CH-Het), 60.8 (CH3), 56.1 (2·
CH3), 56.0 (CH3), 43.5 (CH2-Het); MS (DCI/NH3) m/z
360 (M+H)+; Anal. Calcd for C19H21NO6 Æ 0.5 H2O:
C, 61.95; H, 6.02; N, 3.80. Found C: 61.98; H, 5.91;
N, 3.72.
4.6.6. trans-Ethyl-3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-
trimethoxyphenyl)-4,5-dihydroisoxazole-5-carboxylate (15b).
Ester 15b was obtained from protected compound 15a
(139 mg, 0.25 mmol) after a flash chromatography (cyclo-
hexane/EtOAc 1:1) as a white crystalline solid (113 mg,
1
95%). Mp 125–127 ꢁC; H NMR (300 MHz, CDCl3): d
7.25 (1H, J = 2.1 Hz, H-20), 7.12 (dd, 1H, J = 8.4 Hz,
J = 2.1 Hz, H-60), 6.76 (d, 1H, J = 8.4 Hz, H-50), 6.45 (s,
2H, H-2, H-6), 5.57 (s, 1H, OH), 4.90 (m, 2H, H-500, H-
400), 4.29 (m, 2H, CH2CH3), 3.88 (s, 3H, OCH3), 3.82 (s,
9H, 3· OCH3), 1.33 (t, 3H, J = 7.1 Hz, CH2CH3), 13C
NMR (75 MHz, CDCl3): d 170.1 (CO), 153.9 (2· C),
157.5 (C-Het), 148.3 (C), 145.5 (C), 137.7 (C), 133.5 (C),
121.2 (C), 120.3 (CH), 113.5 (CH), 110.4 (CH), 104.2
(2· CH), 86.3 (CH-Het), 62.1 (CH2), 60.8 (CH3), 58.5
(CH-Het), 56.2 (2· CH3), 55.9 (CH3), 14.1 (CH3); MS
(DCI/NH3) m/z 432 (M+H)+; Anal. Calcd for
C22H25NO8: C, 61.25; H, 5.84; N, 3.25. Found C: 61.27;
H, 5.84; N, 3.24.
4.6.3. 3-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimeth-
oxyphenyl)-1,2,4-dioxazole (9b). Dioxazole 9b was ob-
tained from protected compound 9a (310 mg,
0.65 mmol) after a flash chromatography (cyclohexane/
EtOAc 6:4) as a white crystalline solid (195 mg, 83%).
1
Mp 118–120 ꢁC; H NMR (300 MHz, CDCl3): d 7.40
(s, 1H, H-20), 7.39 (d, 1H, J = 8.5 Hz, H-60), 6.90 d,
1H, J = 8.5 Hz, H-50), 6.82 (s, 2H, H-2, H-6), 5.76 (s,
1H, H-500), 5.68 (s, 1H, OH), 3.95 (s, 3H, OCH3), 3.89
(s, 6H, 2· OCH3), 3.87 (s, 3H, OCH3); 13C NMR
(75 MHz, CDCl3): d 159.2 (C-Het), 153.4 (2· C), 149.4
(C), 145.7 (C), 139.6 (C), 130.1 (C), 119.9 (CH), 115.7
(C), 113.1 (CH), 110.4 (CH), 108.4 (CH-Het), 103.7
(2· CH), 60.8 (CH3), 56.2 (2· CH3), 56.0 (CH3); MS
(DCI/NH3) m/z 362 (M+H)+; Anal. Calcd for
C18H19NO7: C, 59.83; H, 5.30; N, 3.88. Found C:
59.99; H, 5.33; N, 3.88.
4.6.7.
trans-N,N-Diethyl-3-(3-hydroxy-4-methoxyphe-
nyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-4-
carboxamide (14b). Amide 14b was obtained from pro-
tected compound 14a (248 mg, 0.43 mmol) after a flash
chromatography (CH2Cl2/MeOH 96:4) as a white crys-

J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
4075
talline solid (187 mg, 95%). Mp 144 ꢁC; 1H NMR
(300 MHz, CDCl3): d 7.17 (d, 1H, J = 2.1 Hz, H-20),
7.13 (dd, 1H, J = 8.4 Hz, J = 2.1 Hz, H-60), 6.83 (d,
1H, J = 8.4 Hz, H-50), 6.62 (s, 2H, H-2, H-6), 5.67 (d,
1H, J = 9.8 Hz, H-500), 5.61 (s, 1H, OH), 4.62 (d, 1H,
J = 9.8 Hz, H-400), 3.91 (s, 3H, OCH3), 3.85 (s, 9H, 3·
OCH3), 3.50–3.25 (m, 4H, 2· CH2), 1.15 (t, 3H,
J = 7.1 Hz, CH3), 1.00 (t, 3H, J = 7.1 Hz, CH3); 13C
NMR (75 MHz, CDCl3): d 168.1 (CO), 155.6 (C-Het),
153.7 (2· C), 148.2 (C), 145.7 (C), 138.2 (C), 134.5 (C),
122.1 (C), 119.2 (CH), 112.8 (CH), 110.5 (CH), 103.0
(2· CH), 88.8 (CH-Het), 60.9 (CH3), 60.3 (CH-Het),
56.1 (2· CH3), 55.9 (CH3), 42.4 (CH2), 41.0 (CH2),
14.6 (CH3), 12.7 (CH3); MS (DCI/NH3) m/z 459
(M+H)+; Anal. Calcd for C24H30N2O7Æ0.25 H2O: C,
62.26; H, 6.64; N, 6.05. Found C: 62.30; H, 6.59; N, 6.13.
6.86 (s, 3H, H-Ar), 6.79 (s, 2H, H-Ar), 5.70 (s, 1H,
OH), 5.50 (d, 1H, J = 5.5 Hz, H-400), 5.09 (d, 1H,
J = 5.5 Hz, H-500), 3.85 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 3.73 (s, 6H, 2· OCH3), 3.46 (q, 2H,
J = 7.1 Hz, CH2), 3.40 (q, 2H, J = 7.2 Hz, CH2), 1.23
(t, 3H, J = 7.1 Hz, CH3), 1.15 (t, 3H, J = 7.1 Hz, CH3)
13C NMR (75 MHz, CDCl3): d 166.6 (CO), 158.9 (C-
Het), 152.9 (2· C), 146.2 (2· C), 139.4 (C), 132.0 (C),
123.6 (C), 119.6 (CH), 113.9 (CH), 111.0 (CH), 104.9
(2· CH), 87.1 (CH-Het), 60.8 (CH3), 60.0 (2· CH3),
55.9 (CH3), 55.7 (CH-Het), 42.0 (CH2), 40.7 (CH2),
14.4 (CH3), 12.7 (CH3); HRMS (DCI/NH3) m/z
459.2128 [(M + H)+ calcd for C24H31N2O7 : 459.2131].
4.7. C-(3-Hydroxy-4-methoxyphenyl)-N-(3,4,5-trimeth-
oxyphenyl)nitrone (26)
4.6.8.
trans-N,N-Diethyl-3-(3-hydroxy-4-methoxyphe-
To a stirred solution of isovanillin (1.52 g, 10 mmol) and
3,4,5-trimethoxynitrophenyl 25 (3.2 g, 15 mmol) in EtOH
(200 mL) was added zinc powder (1.46 g) at 0 ꢁC underar-
gon. Glacial acetic acid (2.6 mL) was then added drop-
wise, and the mixture was stirred for 2 h at 0 ꢁC and
then raised to room temperature for 2 h. After evapora-
tion under reduced pressure, the crude material was chro-
matographed (CH2Cl2/MeOH 97:3) to give 2.7 g of
nitrone 26, which was recrystallized from EtOH (2.06 g,
nyl)-4-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-5-
carboxamide (16b). Amide 16b was obtained from
protected compound 16a (320 mg, 0.56 mmol) after a
flash chromatography (CH2Cl2/MeOH 97:3) as a white
crystalline solid (218 mg, 85%). Mp 193–195 ꢁC; 1H
NMR (300 MHz, CDCl3): d 7.27 (d, 1H, J = 2.1 Hz,
H-20), 7.09 (dd, 1H, J = 8.4 Hz, J = 2.1 Hz, H-60), 6.74
(d, 1H, J = 8.4 Hz, H-50), 6.46 (s, 2H, H-2, H-6), 5.57
(s, 1H, OH), 5.54 (d, 1H, J = 5.3 Hz, H-400), 5.05 (d,
1H, J = 5.3 Hz, H-500), 3.87 (s, 3H, OCH3), 3.80 (s,
9H, 3· OCH3), 3.48 (q, 2H, J = 7.1 Hz, CH2), 3.42 (q,
2H, J = 7.2 Hz, CH2), 1.25 (t, 3H, J = 7.1Hz, CH3),
1.16 (t, 3H, J = 7.1 Hz, CH3); 13C NMR (75 MHz,
CDCl3): d 166.7 (CO), 158.6 (C-Het), 153.7 (2· C),
148.1 (C), 145.4 (C), 137.2 (C), 134.4 (C), 121.5 (C),
120.3 (CH), 113.4 (CH), 110.3 (CH), 104.6 (2· CH),
87.0 (CH-Het), 60.8 (CH3), 56.3 (CH-Het), 56.1 (2·
CH3,), 55.8 (CH3), 42.0 (CH2), 40.7 (CH2), 14.3 (CH3),
12.6 (CH3); MS (DCI/NH3) m/z 459 (M+H)+; Anal.
Calcd for C24H30N2O7 : C, 62.87; H, 6.59; N, 6.11.
Found C: 63.04; H, 6.64; N, 6.13.
1
62%). Mp 186–189 ꢁC; H NMR (CDCl3): d 8.28 (d,
1H, J = 1.9 Hz, H-20), 7.84 (dd, 1H, J = 8.5 Hz,
J = 1.9 Hz, H-60), 7.77 (s, 1H, CH=N), 7.02 (s, 2H, H-2,
H-6), 6.93 (d, 1H, J = 8.5 Hz, H-50), 3.95 (s, 3H, OCH3),
3.92 (s, 6H, 2· OCH3), 3.88 (s, 3H, OCH3); MS (DCI/
NH3) m/z 334 (M + H)+, 318 (imine + H)+.
4.8. Methyl-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-tri-
methoxyphenyl)-isoxazolidine-4-carboxylate (27)
To a suspension of nitrone 26 (1.0 g, 3.0 mmol) in tolu-
ene (15 mL) was added methylacrylate (1.1 mL,
12.2 mmol) under argon. After stirring under reflux for
18 h, the resulting mixture was evaporated to dryness.
The residue was chromatographed (cyclohexane/EtOAc
4.6.9. 5-(3-Hydroxy-4-methoxyphenyl)-3-(3,4,5-trimeth-
oxyphenyl)-1,2,4-dioxazole (18b). Dioxazole 18b was
obtained from protected compound 18a (607 mg,
1.28 mmol) after a flash chromatography (cyclohexane/
EtOAc 6:4) as a white crystalline solid (410 mg, 89%).
1
5:5 then 4:6) to give isoxazolidine 27 (75mg, 6%). H
NMR (CDCl3): d , 7.02 (d, 1H, J = 2.1 Hz, H-20), 6.97
(dd, 1H, J = 8.3 Hz, J = 2.1 Hz, H-60), 6.81 (d, 1H,
J = 8.3 Hz, H-50), 6.26 (s, 2H, H-2, H-6), 5.60 (br, 1H,
OH), 4.83 (d, 1H, J = 8.9 Hz, H-300), 4.50 (t, 1H,
J = 8.1 Hz, H-500a),), 4.33 (t, 1H, J = 8.3 Hz, H-500b)
3.87 (s, 3H, OCH3), 3.78 (s, 9H, 3· OCH3), 3.76 (m,
1H, H-400), 3.39 (s, 3H, OCH3); 13C NMR (CDCl3): d
169.6 (CO), 153.2 (2· C), 146.6 (C), 146.2 (C), 145.4
(C), 133.3 (C), 130.5 (C), 119.2 (CH), 114.0 (CH),
110.3 (CH), 93.1 (2· CH), 71.5 (CH-Het), 67.4 (CH2),
60.8 (CH3), 55.8 (2· CH3), 55.7 (2· CH3), 52.8 (CH-
Het), 51.7 (CH3). HRMS (DCI/NH3) m/z 420.1654
[(M + H)+ calcd for C21H26NO8: 420.1658].
1
Mp 121–123 ꢁC; H NMR (300 MHz, CDCl3): d 7.12
(d, 1H, J = 2.0 Hz, H-20), 7.10 (dd, 1H, J = 8.3 Hz,
J = 2.0 Hz, H-60), 7.06 (s, 2H, H-2, H-6), 6.90 (d, 1H,
J = 8.3 Hz, H-50), 6.77 (s, 1H, H-500), 5.74 (sl, 1H,
OH), 3.91 (s, 3H, C-40OCH3), 3.89 (s, 9H, C-3OCH3,
C-5OCH3, C-4OCH3); 13C NMR (75 MHz, CDCl3): d
159.2 (C-Het), 153.3 (2· C), 148.4 (C), 145.5 (C), 140.8
(C), 127.7 (C), 119.2 (CH), 117.9 (C), 112.9 (CH),
110.4 (CH), 108.8 (CH-Het), 104.1 (2· CH), 60.9
(CH3), 56.2 (2· CH3), 56.0 (CH3); HRMS (DCI/NH3)
m/z 361.1157 [M+ calcd for C18H19NO7: 361.1162].
4.9. 1-[(3-p-Methoxybenzyloxy)-4-methoxyphenyl]-3-
(3,4,5-trimethoxyphenyl)-propenone (33)
4.6.10. trans-N,N-Diethyl-4-(3-hydroxy-4-methoxyphe-
nyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole-5-
carboxamide (24b). Amide 24b was obtained from
protected compound 24a (86 mg, 0.15 mmol) after a
flash chromatography (CH2Cl2/MeOH 97:3) as a white
foam (64 mg, 83%). 1H NMR (300 MHz, CDCl3): d
A mixture of 3,4,5-trimethoxyacetophenone (2.10 g,
10 mmol), aldehyde 32 (2.72 g, 10 mmol) and aqueous
NaOH (6N, 0.4 mL) in EtOH (25 mL) was stirred at
60 ꢁC for 5 h. After standing at room temperature, the

4076
J. Kaffy et al. / Bioorg. Med. Chem. 14 (2006) 4067–4077
chalcone crystallized from the solution (4.26 g, 91%).
Mp 86–87 ꢁC; 1H NMR (CDCl3): d 7.71 (d, 1H,
J = 15.6 Hz, CH), 7.38 (d, 2H, J = 8.4 Hz, 2· Ar-H),
7.26–7.18 (5H, m, H-2, H-6, H-20, H-60, CH), 6.91
(3H, m, H-50, 2· Ar-H), 5.12 (s, 2H, CH2), 3.94 (s,
6H, 2· OCH3), 3.82 (s, 3H, OCH3), 3.80 (s, 3H, OCH3).
etry at 350 nM with a Uvikon 931 spectrophotometer
(Kontron) fitted with a thermostatically regulated cuv-
ette holder. The products are dissolved at 10 mM in
DMSO and added at variable concentrations (0.5–
10 lM) to the tubulin solution before polymerization.
The IC50 value is defined as the concentration of prod-
uct which inhibits the rate of polymerization by 50%.
4.10. (E)-2,3-Epoxy-3-[3-(p-methoxybenzyloxy)-4-meth-
oxyphenyl]-1-(3,4,5-trimethoxyphenyl)propenone (34)
4.13. Cytotoxicity assays50
H2O2 (35%, 1.0 mL) was added dropwise to a stirred sus-
pension of chalcone 33 (2.32 g, 5 mmol) in EtOH (30 mL)
and aqueous NaOH (2N, 1.25 mL) at 0 ꢁC. After com-
plete addition, the ice bath was removed and the mixture
was stirred for 4 h at room temperature. The precipitated
white crystals were filtered out, washed with cold EtOH
HT29 cells (human colon adenocarcinoma, ATCC HTB
38) and SVEC 4–10 cells (SV40 transformed endothelial
cells from mouse axillary lymph node, ATCC CRL-
2181) were cultivated in Dulbecco’s MEM supplement-
ed with 10% FCS. Cells from log-phase culture were
seeded in 24-microwell plates (1 mL–5 · 104 cells/well)
and incubated for two days at 37 ꢁC in a water-jacketed
CO2 incubator. Tested compounds, in DMSO solution,
were added under the minimum volume (5 lL) in
increasing concentration. Control cells received 5 lL
DMSO alone. Plates were incubated for 24 h, then medi-
um was removed and cells were washed twice with phos-
phate-buffered saline (PBS) before addition of fresh
medium. Plates were reincubated for three days before
evaluation of the cell survival using the MTT assay51
using 30 min incubation with 100 lg/well of 3-[4,5-di-
methylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
(MTT, Sigma). After removal of the medium, formazan
crystals were taken up with 100 lL DMSO and absor-
bance at 540 nm was measured with a microplate reader
(Model 450 Bio-Rad). Survival was expressed as per-
centage of DMSO-treated controls.
1
and dried in vacuo (1.99 g, 83%). Mp 122–123 ꢁC; H
NMR (CDCl3): d 7.36 (d, 2H, J = 8.5 Hz, 2· Ar-H),
7.26 (s, 2H, H-2, H-6), 6.94–6.88 (m, 5H, H-20, H-50, H-
60, 2· Ar-H), 5.08 (m, 2H, CH2), 4.11 (d, 1H,
J = 1.7 Hz, CH), 3.99 (d, 1H, J = 1.7 Hz, CH), 3.93 (s,
3H, OCH3), 3.87 (s, 9H, 2· OCH3), 3.79 (s, 3H, OCH3);
13C NMR (CDCl3): d 192.0 (CO), 159.4 (C), 153.2 (2·
C), 150.5 (C), 148.6 (C), 143.4 (C), 130.6 (C), 129.2 (2·
CH), 128.7 (C), 127.6 (C), 119.2 (CH), 114.0 (2· CH),
111.7 (CH), 110.9 (CH), 105.9 (2· CH), 70.9 (CH2), 61.2
(CH), 61.0 (CH3), 59.4 (CH), 56.4 (2· CH3), 56.0 (CH3),
55.3 (CH3). Anal. Calcd for C27H28O8: C, 67.49; H,
5.87. Found: C 66.55; H, 5.72.
4.11. 4-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimeth-
oxyphenyl)isoxazole (35)
BF3.Et2O (1.25 mL, 10 mmol) was added to a solution
of ketoepoxide 34 (480 mg, 1.0 mmol) in dry THF
(10 mL) under argon. After reflux for 45 min, the mix-
ture was diluted with Et2O and washed with water.
The combined organic layers were dried, filtered and
concentrated under reduced pressure. The residue was
dissolved in EtOH (6 mL) and then pyridine (160 lL)
and NH2OH.HCl (140 mg, 2 mmol) were added and
the resulting mixture was refluxed for 6 h. The solvent
was removed in vacuo, CH2Cl2 was added and this solu-
tion was washed with water. The combined organic lay-
ers were dried, filtered, concentrated under reduced
pressure and the residue was chromatographed
(CH2Cl2/MeOH 99:1) to give 35 as a white solid, which
was crystallized in EtOAc (114 mg, 32%). Mp 167–
Cytotoxic activities were expressed as IC50, the concen-
tration that reduced by 50% the number of treated cells
relative to controls. IC50 values were extracted from
regression curves obtained with experimental points.
Acknowledgments
The authors are pleased to thank Drs Alain Commerc¸on
and Patrick Mailliet for helpful suggestions and discus-
sions (Aventis Pharma, Medicinal Chemistry Depart-
ment, 94400 Vitry-sur-Seine, France). Laboratories
Aventis Pharma are gratefully acknowledged for a doc-
toral grant to Julia Kaffy and for biological evaluation.
This work was financially supported by the Centre
National de la Recherche Scientifique and the Institut
1
168 ꢁC; H NMR (CDCl3): d 8.29 (s, 1H, H-300), 7.01
(d, 1H, J = 1.3 Hz, H-20), 6.92 (s, 2H, H-2, H-6), 6.89
(m, 2H, H-50, H-60), 5.72 (br, 1H, OH), 3.92 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 3.74 (s, 6H, 2· OCH3);
13C NMR (CDCl3): d 163.3 (C-Het), 153.3 (2· C),
152.0 (CH-Het), 146.5 (C), 145.9 (C), 139.5 (C), 123.3
(C), 122.8 (C), 120.8 (CH), 115.4 (C-Het), 115.1 (CH),
110.9 (CH), 104.5 (2· CH), 60.9 (CH3), 56.0 (3· CH3).
HRMS (DCI/CH4) m/z 358.1287 [(M + H)+ calcd for
C19H20NO6: 358.1291].
´ `
Curie (Programme Incitatif et Cooperatif: angiogenese
tumorale).
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