Exploration of the SAR of anti-invasive chalcones: Synthesis and biological evaluation of conformationally restricted analogues

Article abstract of DOI:10.1016/j.bmc.2012.05.069

In order to get a clearer view on the active geometry of anti-invasive chalcones, we have prepared a number of isoxazoles and related substances as conformationally restrained mimics of 1,3-diarylpropenones, and also of (Z)-stilbenes. In vitro anti-invasi

Full text of DOI:10.1016/j.bmc.2012.05.069

Bioorganic & Medicinal Chemistry 20 (2012) 4812–4819
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of the SAR of anti-invasive chalcones: Synthesis and biological
evaluation of conformationally restricted analogues
Bart I. Roman a, , Tine De Ryck b, Laura Dierickx a, Barbara W. A. Vanhoecke b, Alan R. Katritzky c,d
,
Marc Bracke b, Christian V. Stevens a,
⇑
a Research Group SynBioC, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University,
Coupure Links 653, B-9000 Ghent, Belgium
b Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital,
De Pintelaan 185, B-9000 Ghent, Belgium
c Center for Heterocyclic Compounds, Department of Chemistry, 127 Chemistry Research Building, University of Florida, Gainesville, FL32611-7200, United States
d Department of Chemistry, King Abdulaziz University, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to get a clearer view on the active geometry of anti-invasive chalcones, we have prepared a num-
ber of isoxazoles and related substances as conformationally restrained mimics of 1,3-diarylpropenones,
and also of (Z)-stilbenes. In vitro anti-invasive activity data for 3,5-isoxazoles and 4,5-isoxazoles, together
with an in silico geometrical comparison, point towards an active conformation for chalcones more
resembling their s-trans geometry than the s-cis counterpart.
Received 5 January 2012
Revised 25 April 2012
Accepted 29 May 2012
Available online 7 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Invasion
Cancer
Isoxazole
SAR
Chalcone
1. Introduction
tackling this hallmark of malignant disease represent a high unmet
need in the management of cancer.
In the second half of the 20th century, a major leap in the battle
against cancer was made due to the advent of cytotoxic anticancer
drugs. Most available therapies, however, induce little or no
response in advanced stages of the disease, when cancer cells have
disseminated throughout the body. This development of so-called
metastases is the foremost treat of malignancy and accordingly
the major cause of death in cancer patients.1,2 The process of inva-
sion is a requisite first step in the development of metastases, and
can be defined as the protrusion of the basement membrane. In the
ensuing steps, cancer cells will move into surrounding tissues,
spread throughout the body, and establish colonies in permissive
tissues.
In striking contrast to the array of antiproliferative chemother-
apeutics targeting primary tumor growth, no effective treatments
are available that can tackle the sequelae of metastasis. Our
laboratories therefore seek novel agents that can interfere in the
first and requisite step of the metastatic cascade: invasion. Drugs
In our quest for such substances, our attention was primarily
drawn towards chalcones, which emerged as promising leads from
the Indo-Belgian anti-invasive screening program.3,4 As the molec-
ular target of these substances has not yet been revealed, we devel-
oped and validated a predictive quantitative structure-activity
relationship (QSAR)-model for chalcones as a rationale behind
our work. During these investigations, (E)-3-(4-fluorophenyl)-1-
(3,4,5-trimethoxyphenyl)propenone 1 and (E)-3-(4-chlorophenyl)-
1-(4-methoxyphenyl)propenone
2 surfaced as remarkably
interesting molecules (Fig. 1). Accordingly, a patent application cov-
ering these findings has been filed.5
Given the extraordinary in vitro anti-invasive potential of these
chalcones, we decided to delve deeper into their structure-activity
⇑
Corresponding author. Tel.: +32 9 264 5957; fax: +32 9 264 6243.
E-mail address: chris.stevens@ugent.be (C.V. Stevens).
URL: http://www.synbioc.ugent.be (C.V. Stevens).
Figure 1. Two highly potent anti-invasive chalcones that serve as a starting point
B.I.R. is a PhD fellow of the Research Foundation—Flanders (FWO—Vlaanderen).
for the present SAR-study.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.069

B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
4813
relationship (SAR) by synthesizing close analogues. We were par-
ticularly interested in acquiring more information on the active
geometry of these substances, and therefore pointed our attention
towards the design, synthesis and assessment of conformationally
restrained congeners.
Chalcones can adopt different conformations by rotating around
the sigma bond of the enone moiety, leading to two main geome-
tries: the s-cis conformer 3a and the s-trans counterpart 3b
(Scheme 1). The antimitotic activity of chalcones is, for example,
exerted by their s-trans conformers, which bear close resemblance
to colchicine and combretastatin and hence bind at the colchicine
site of tubulin.6 It was therefore of fundamental interest to verify
whether the molecular target of our anti-invasive chalcones
discerns between these two conformers.
For this reason, we decided to prepare isoxazoles 4, for which
the conformation is locked into the s-cis form (Scheme 1, A). The
extra heteroatom introduces an additional hydrogen bond acceptor
into these chalcone analogues. Still, these ring systems possess ste-
ric and stereoelectronic resemblance to the original enone moiety
in chalcones and can accordingly provide information on the con-
formation-activity relationship of these propenones. Hence, if isox-
azoles 4 would fail to exert anti-invasive effects, this would
provide an initial indication that the active conformer of the
chalcones may be more s-trans like.
In addition, we were intrigued to evaluate the potential of
4,5-diarylisoxazoles 5 as well. These substances bear—to some
extent—resemblance to s-trans chalcones (Scheme 1, B). More
importantly, they can be considered as stable analogues of cis-
stilbenes 6 (C), a substance class we have recently evaluated as
anti-invasive agents (data unpublished). Cis-stilbenes tend to
isomerize to the thermodynamically more stable trans-isomers
both during storage and in vivo.7
Due to their interest as antitubulin agents, a lot of stable ring-
closed analogues have accordingly been proposed (Fig. 2). Exam-
ples of evaluated cis-1,2-substituted ethylene mimics encompass
the cyclopropyl group8 (7) and five-membered heterocyclic rings
such as dioxolanes,9 isoxazoles (8),10 imidazoles (9)11 and thiadiaz-
oles.12 Hence, given our previous investigations on anti-invasive
cis-stilbenes, we were interested to evaluate 4,5-diarylisoxazoles
5 as stable analogs of the latter compounds. Furthermore, compar-
ison with the identically decorated 3,5-diarylisoxazole congeners 4
might bring about additional SAR-insights.
Figure 2. Examples of stable analogues of cis-stilbenes (7, 8, 9).
1,3-dipoles to alkynes, or condensations of b-diketone equivalents
with hydrazine or hydroxylamine. We chose to evaluate two clo-
sely related routes belonging to the latter category, as depicted
in Scheme 2 (via A, or B–C). These transformations are versatile,
straightforward and make use of readily available starting materi-
als. In route A, the a-substituent X of chalcone 12 is selected to be a
good leaving group, resulting in in situ aromatization by elimina-
tion of HX. When proceeding through route B, isoxazolines 13
are isolated at first, and can then be oxidized to the desired isoxaz-
ole 4 (C).
One problem often encountered when conducting such
condensation reactions is the competition between an initial 1,
2- and 1,4-addition of the dinucleophile, thereby resulting in
isomeric mixtures of isoxazoles when R1 differs from R2. However,
Katritzky and co-workers encountered regioselectivity for route A
when the benzotriazolyl moiety was chosen as the
a-substituent
(X = Bt).13 Hence, we evaluated their protocol in an initial attempt
at the synthesis of our desired isoxazoles.
In a first step, 1-(trimethylsilylmethyl)benzotriazole 15 was
prepared from benzotriazole 14 and trimethylsilylmethylchloride
(Scheme 3a).14 The rather moderate yield for this transformation
may be partly explained by the formation of the 2-substituted ana-
logue in a 1:4-ratio to the main product 15, hence encumbering the
isolation of the latter.15 Next, the benzotriazolylacetophenones 16
were prepared by reaction of 15 with a suitable acid chloride.
Although not mentioned in the original protocol,14 we found the
use of a catalytic amount of DMAP in this transformation beneficial
as it enhanced chemoselectivity.
Benzotriazolylacetophenone 16a was subsequently reacted
with an appropriately functionalized benzaldehyde in a piperi-
dine-catalyzed Claisen–Schmidt condensation using microwave
heating, which furnished (Z)-a-benzotriazolylchalcone 12a as a
precipitate. In contrast, despite the evaluation of numerous bases
(LiOH, piperidine, NaH, DIPEA, LDA), the analogous reaction of ace-
tophenone 16b with 4-fluorobenzaldehyde failed.
2. Results and discussion
2.1. Chemistry
Subsequently, we tried to convert a-benzotriazolylchalcone 12a
into the corresponding isoxazole via treatment with NaOEt and
hydroxylammonium chloride in EtOH (Scheme 4).13 Although,
the desired isoxazole 4a was indeed present in the reaction mix-
ture, as indicated by LC/MS analysis, an appreciable amount of side
2.1.1. Synthesis of 3,5-diarylisoxazoles
Most strategies for the preparation of 3,5-diarylisoxazoles
can be considered as either intermolecular cycloadditions of
α
β
a
Scheme 1. Left: Potential energy scan along the dihedral angle O@C-C @Cb of chalcone 3 (AM1 level of theory, HyperChem 8.0). Right: Synthesis of 3,5-diarylisoxasoles 4 as
conformationally restrained analogues of s-cis chalcones 3a (A); preparation of 4,5-diarylisoxazoles 5 as stable mimics of (Z)-stilbenes 6 (C), and analogues of s-trans
chalcones 3b (B).

4814
B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
Scheme 4. 10 equiv NaOEt (10 M in EtOH), 2 equiv NH2OH.HCl, rt to
D, overnight.
Scheme 2. Synthesis of isoxazoles 4 from 2-substituted 1,3-diarylpropenones 12
via an annulation-elimination sequence (route A), or from chalcones 3 through the
corresponding isoxazolines 13 (route B–C).
products had also been formed which would render product isola-
tion difficult. Due to this observation, and given the failure of the
synthesis of 12b, we abandoned the benzotriazolyl approach, as
this elegant route proved inapplicable to our compounds.
Next, we turned our focus to pathway b–c from Scheme 2. Syn-
thesis of the isoxazoles through the isoxazolines requires an oxida-
tion step, but starts from readily available chalcones. A further
advantage of proceeding via this route is that it enables to evaluate
the isoxazolines in our anti-invasive screening program as well.
We chose the straightforward protocol of Sinisterra and co-work-
ers for the conversion of substituted chalcones into isoxazolines
13a–c, which were indeed formed using the reported conditions.16
Nevertheless, we altered the prescribed work-up procedure, as it
failed to remove many impurities. In contrast, extraction with
EtOAc and recrystallization in absolute EtOH furnished the desired
isoxazoles in high purity.
Scheme 5. Regioselective 1,2-addition of hydroxylamine leads to isoxazoline 13 as
a sole product (route x); no 1,4-addition was observed (route y). (a) Synthesis of
isoxazolines 13a–c (X@O): 6 equiv NH2OH.HCl, 7 equiv KOH, abs EtOH,
N2; (b) Synthesis of pyrazoline 20 (X@NH): 6 equiv NH2NH2.H2O, 7 equiv KOH,
abs EtOH, , 24 h, N2.
D, 24 h,
D
According to Sinisterra, their protocol selectively proceeds
through an initial 1,2-addition of hydroxylamine across the enone,
thereby allowing for the regiocontrolled formation of a single isox-
azoline isomer (Scheme 5, route x). We were able to confirm this
selectivity by means of NMR analysis of isoxazoline 13a. The alter-
native possibility, involving an initial 1,4-addition, would give rise
to isoxazoline 19 (route y). All NMR-data clearly point towards
structure 13a: (i) HSQC analysis confirmed the presence of a CH2
group. (ii) In the 1H spectrum, chemical shift, signal integration
and coupling constants indicate the presence of an HaHb-system
and a vicinal aliphatic CH (see Scheme 5). (iii) Furthermore, chem-
ical shift values in the 13C spectrum agree with the presence of an
imino group and two aliphatic carbon atoms. (iv) Finally, HMBC
Scheme 6. Oxidation of isoxazolines 13 towards isoxazoles 4.
sical conditions, resulted in isolation of the starting material for all
three substrates. Activated MnO2, however, proved capable of fur-
nishing the desired isoxazoles 4 at elevated temperatures. For
compounds 4a and 4b, full conversion was obtained faster in the
presence of MgSO4 and under microwave irradiation. It should fur-
ther be noted that the degree of conversion dropped severely on
scale-up (above 1 mmol).
0
0
data indicates a coupling between H2 and H6 , which clearly bear
a fluorine coupling, on the one hand and an aliphatic CH on the
other hand.
In order to increase the molecular diversity in our set, we also
prepared an analogous pyrazoline 20 via the same procedure,
which was obtained in excellent yield.
Having the isoxazolines 13 in hand, we proceeded with the aro-
matization towards the corresponding isoxazoles 4 (Scheme 6).
Our first attempts, employing DDQ as an oxidant under fairly clas-
2.1.2. Synthesis of 4,5-diarylisoxazoles
In a second part of the synthetic work, we envisaged preparing
4,5-diarylisoxazoles 5 as stable analogues of cis-stilbenes, and to a
lesser extent of s-trans chalcones. Since we disposed of appropri-
ately decorated benzaldehydes 21, we opted for a synthetic
route involving an initial umpolung of these electrophiles via
Scheme 3. (a) (1) 1 equiv NaOEt (1 M in EtOH); (2) removal of solvent; (3) 50 °C, 16 h, vacuum; (4) 1 equiv ClCH2SiMe3, DMF, rt, 24 h; (b) 1.2 equiv Ar1C(O)Cl, 0.1 equiv
DMAP, dry THF, , 24 h, N2; (c) 1.02 equiv 3,4,5-trimethoxybenzaldehyde, 1 equiv piperidine, EtOH, 70 °C, 2 h, MW.
D

B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
4815
transformation to their corresponding O-protected
a
-hydrox-
from 9-day-old chick embryos, are confronted with aggregates of
human invasive MCF-7/6 mammary carcinoma cells in the pres-
ence of a certain concentration of a test compound. After eight days
of incubation at 37 °C, the interaction between the cancer cells and
the PHF is evaluated histologically and classified along a 5-grades
scale (Fig. 3).
yphosphonates 23, followed by a Horner–Wadsworth–Emmons
(HWE)-type reaction, yielding deoxybenzoins 26 (Scheme 7). These
intermediates could then furnish the desired isoxazoles upon con-
densation with dimethylformamide dimethylacetal (DMFDMA)
and hydroxylammonium chloride.
In an initial step, we reacted the appropriately decorated benz-
aldehydes 21 with dimethyl phosphite in the presence of a cata-
lytic amount of n-BuNH2 and using Et2O as a solvent (Scheme
7).17 These mild reaction conditions provided us with the
Compounds are coined ‘active’ or ‘anti-invasive’ when the inva-
sion grade is I or II, and ‘inactive’ when the grade is III or IV. For
substances that exhibit selective toxicity versus MCF-7/6 cells, de-
noted as invasion grade 0, a judgment on their anti-invasive po-
tency cannot be made. A notable advantage of this type of
confronting culture is the involvement of living host tissue. The
contributions of such normal tissue to the micro-ecosystem that
governs tumor behavior should indeed not be neglected.3 More-
over, the design of the chick heart assay enables the observer to
discern the effect of a substance on the tumor cells from effects
on the normal host tissue.
a-hydroxyphosphonates 22 as a pure precipitate in nearly quanti-
tative yield. Trials employing other base/solvent pairs, such as
NaOMe/MeOH18 or CaO/neat,19 had an inferior outcome. Next,
the hydroxyl group was converted into its THP ether under stan-
dard conditions. The protected phosphonate was not isolated, but
in situ submitted to a Horner-Wadsworth-Emmons reaction with
a suitable second benzaldehyde. The resulting protected enols 24
were immediately hydrolyzed, furnishing the corresponding
deoxybenzoins 26, which were accompanied by homocoupling
product 27b (13–20%).18
For the compounds depicted in Table 1, the invasion grade in
the CHI assay was scored at concentrations ranging from 100 to
0.1 l
mol Lꢀ1 (Table 1). In order to translate the resulting assay data
Subsequently, the deoxybenzoins 26 were condensed with
DMFDMA under solventless conditions, yielding b-aminoenones
28.20 These molecules were not isolated, but underwent immediate
regioselective ring closure upon treatment with hydroxylammo-
nium chloride, furnishing the desired 4,5-diarylisoxazoles 5 in
modest yields (Scheme 7).
into one anti-invasive potency value per compound, we introduced
‘Logcmin’, the logarithm of the ‘lowest active concentration cmin’ for
each compound, being the lowest concentration at which a sub-
stance exhibits an anti-invasive effect (invasion grades I or II).
Hence, six activity levels were defined, ranging from class-2 for
the most active compounds (invasion grade
0.01
mol Lꢀ1 level) to class 3 (compounds with no apparent effect
at 100
mol Lꢀ1
I or II at the
l
2.2. Biological activity and SAR
l
.
Unfortunately, all but one of the evaluated molecules com-
In the light of our SAR program, we submitted the newly syn-
thesized, conformationally restrained heterocyclic chalcone mim-
ics to the in vitro chick heart invasion assay.21 In this screening
technique, precultured heart tissue fragments (PHFs), dissected
pletely lacked anti-invasive potency (class P1, Table 1). Isoxazole
4b inhibited invasion at 10
ing score. 4,5-Diarylisoxazole 5a, however, proved potent up to
mol Lꢀ1 (class 0), and thereby distinguished itself clearly from
l
mol Lꢀ1, which is a rather dissapoint-
1
l
Scheme 7. (a) 2 equiv HP(O)(OMe)2, n-Bu2NH, Et2O, rt, 3 h; (b) 2.2 equiv 3,4-dihydro-2H-pyran, 2.6 mol % para- TsOHꢁH2O, benzene, 50 °C, 2 h; (c) 50 °C to rt; (d) 4 equiv NaH,
1 equiv substituted benzaldehyde, EtOH, rt, 18 h, N2; (e) 0.95 equiv 10% aq HCl, MeOH, rt, 1 h; (f) dimethylformamide dimethylacetal, , 14 h, N2; (g) solvent evaporation;
(h) 1.96 equiv NH2OH.HCl, 1 equiv Na2CO3, MeOH/H2O (2:1), adjust pH to 4–5 with AcOH, then , 2 h, N2.
D
D

4816
B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
Figure 3. This illustration of the chick heart invasion assay displays 7 lm thick sections from confronting cultures between precultured heart fragments (PHF) and invasive
human mammary carcinoma cells (MCF-7/6). Panel A results from an untreated culture, in which the confronting cells have occupied the PHF, and was attributed an invasion
grade of III. In Panel B, addition of an anti-invasive agent resulted in a clear histological separation between the confronting partners; the invasion grade is denoted as I/II.
Panel C displays the outcome for a product with selective toxicity for MCF-7/6 cells with respect to the PHF. In this case, no judgment of the anti-invasive potency can be
made; this pattern is coined grade 0.
Table 1
In vitro anti-invasive activity data against MCF7/6 cells, obtained via the CHI-assay (0 = toxic for MCF7/6; I and II = no invasion, the compound is coined active; III and IV: invasion,
the compound is inactive)
Structure
Invasion grade at different concentrations (
l
mol Lꢀ1
)
Anti-invasive activity classa
100
10
1
0.1
IV
4b
5a
IIb/IIb
II/II
III/III/IV
1
IIb/IIIb
III/III
Ic
II/II
III/III
0
13c
4c
II/III
III/IV
III/IV
P2
P2
IV/IV
26a
IIb/IIb
III/III
III/III
III/III
2
a
b
c
Logcmin
Toxic.
Slightly toxic for PHF, few MCF7/6 cells left.
.
the rest of the set. The observed cytotoxicity of compounds 4b, 5a
mation of their parent chalcones, compounds which proved active
up to 10 nmol Lꢀ1 (class-2). This geometrical similarity was con-
firmed by flexible superposition of s-cis conformer 1a of 3-(4-fluo-
rophenyl)-1-(30,40,50-trimethoxyphenyl)propenone(Fig. 4, above,
green) and its isoxazole analogue 4b (yellow). Both molecules
are planar and a good overlap of methoxy and fluorine substitu-
and 26a at 100 l
mol Lꢀ1 can be considered normal at this elevated
dose.
At 10 l
mol Lꢀ1, on the other hand, isoxazole 5a exhibits an
interesting behavior: few MCF7-6 cells were left upon histological
examination, while only a slight toxicity vis-à-vis the PHF was
observed. Compound 5a thus exerts—to a certain extent—selective
cytotoxicity versus the malignant cells.
Although only one of the assessed compounds proved reason-
ably active, the acquired data is surely valuable in the broader per-
spective of our quest for anti-invasive molecules. Indeed, these
results provides us with important information on the conforma-
tion-activity relationship of the highly potent chalcones on which
this study was based.
ents, aromatic rings and the
a,b-unsaturated enone or its imine
equivalent can be appreciated, resulting in an RMS of 0.7466. Gi-
ven the absence of activity for isoxazole 4b, the active geometry
of chalcone 1 may therefore clearly deviate from that of s-cis con-
former 1a.
In a similar way, we have analyzed the geometrical similarity
between the most active of the tested compounds, 4,5-diarylisox-
azole 5a, and the s-trans conformer of chalcone 1. A good match
between these two geometries is apparent from their three-dimen-
sional representations in Figure 4, and from the RMS of 0.7038.
Firstly, both isoxazoles completely lack potency (class P1).
These molecules should, however, be mimics of the s-cis confor-

B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
4817
Figure 4. Geometrical comparisons of various compounds using flexible superposition. Above: Isoxazole 4b (yellow) and s-cis conformer 1a of 3-(4-fluorophenyl)-1-(30,40,50-
trimethoxyphenyl)propenone (green). Below: Isoxazole 5a (red) and s-trans conformer 1b of 3-(4-fluorophenyl)-1-(30,40,50-trimethoxyphenyl)propenone (cyan). Root mean
square (RMS) values were calculated for the atom–atom distances between the 3D-structures of atoms indicated by colored arrows.
This adds value to the assumption that the active conformer of the
potent chalcones is more s-trans than s-cis like. Such a conforma-
tion furthermore better resembles that of (Z)-stilbenes, for which
we earlier observed a high anti-invasive potency. Indeed, isoxazole
5a was the only molecule in the test set designed as a (Z)-stilbene
mimic.
The above corollaries concerning the active conformation of po-
tent anti-invasive chalcones seem plausible, but surely warrant
further investigation in order to obtain a more complete view of
the SAR-landscape of chalcones. Hence, we are currently synthesiz-
ing further conformationally restricted analogues of the most po-
tent 1,3-diarylpropenones in our library, for which the results
will be communicated in due course.
4.1.1. Synthesis of 1-trimethylsilylmethyl-1H-benzotriazole (15)
In a flame-dried round-bottomed flask, 49.6 mmol (5.95 g) of
benzotriazole is dissolved in 50 mL of a freshly prepared 1 M solu-
tion of NaOEt in EtOH (1.15 g of Na). Then, the solvent is removed
under reduced pressure and the residue is dried overnight at 50 °C
in a vacuum oven. Subsequently, the flask is cooled to room tem-
perature, and 50 mL of DMF is added, followed by 1 equiv
(6.95 mL) of chlorimethyltrimethylsilane. The mixture is stirred
for one hour at 50 °C, and then for 23 h at room temperature.
Afterwards, 40 mL of water is added, and the resulting mixture
is extracted with Et2O (7 ꢂ 50 mL). The combined organic layers
are subsequently dried over MgSO4. Solvent removal affords a
red oil, which is taken up in hexanes. Overnight storage at
ꢀ20 °C affords crystals of 15, which are isolated in a yield of 40%
after washing and thorough drying.
3. Conclusion
Although further exploration of their SAR-landscape is neces-
sary, the present study provides initial indications that the active
conformation of anti-invasive chalcones resembles the s-trans
geometry more closely than the s-cis counterpart. This observation
furthermore correlates with the interesting anti-invasive activity
profile of (Z)-stilbenes.
4.1.2. Synthesis of 2-benzotriazol-1-yl-1-arylethanones (16)
To a flame-dried round-bottomed flask, kept under an N2-atmo-
sphere, 4.87 mmol (1 g) of 1-trimethylsilylmethyl-1H-benzotria-
zole 15 and 25 mL of dry THF are added, followed by 0.1 equiv
(0.06 g) of DMAP and 1 equiv of a suitable benzoyl chloride. The
resulting mixture is stirred at reflux and under an N2-atmosphere
for 24 h. The volatiles are subsequently removed under reduced
pressure, upon which crystallization of the resulting crude in EtOH
affords the desired 2-benzotriazol-1-yl-1-arylethanone 16 in high
purity.
4. Experimental part
4.1. Chemistry
All reagents were purchased from commercial sources (Aldrich,
Acros) and used without further purification. Solvents were pur-
chased from commercial sources (Aldrich) and employed as is.
Microwave reactions were carried out in a CEM Benchmate appa-
ratus using a standard temperature program (maximum power:
300 W). Crude reaction mixtures were analyzed on LC/MS/UV. Thin
layer chromatography was carried out on silica gel 60F254 plates
(Merck).
4.1.3. 2-Benzotriazol-1-yl-1-(3,4,5-trimethoxyphenyl) ethanone
(16b)
Yield: 65%, as white crystals; mp: 151.5–153.0 °C; IR (ATR,
cmꢀ1
) mmax: 1002, 1123, 1160, 1229, 1316, 1415, 1456, 1586,
1694; 1H NMR (CDCl3, 300 MHz, ppm): d 3.93 (6H, s, 2x OMe),
0
0
3.94 (3H, s, 1x OMe), 6.07 (2H, s, CH2), 7.32 (2H, s, H2 , H6 ),
7.33–7.55 (3H, m, 3x CH), 8.11 (1H, d, Jvic = 8.3 Hz, CH); 13C NMR
(CDCl3, 75 MHz, ppm): d 55.97 (CH2), 58.31 (2x OMe), 62.95
High resolution 1H (300 MHz), 13C (75 MHz) and 19F (282 MHz)
magnetic resonance (NMR) spectra were recorded on a Jeol
Eclipse+300 FT NMR spectrometer in deuterated solvents. Chemi-
cal shifts are reported using TMS and/or CFCl3 as an internal refer-
ence, unless otherwise indicated. Peak assignments were obtained
with the aid of DEPT, HSQC, HMBC and COSY spectra. Attenuated
total reflection (ATR) IR spectra were recorded on a Perkin Elmer
Spectrum BX spectrometer, equipped with a ZnSe-crystal, at room
temperature. Low-resolution mass spectra were recorded on an
Agilent 1100 series VL mass spectrometer (ES, 70 eV). Melting
points were measured with a Büchi B-540 apparatus and are
uncorrected.
0
0
(OMe), 107.82 (C2 , C6 ), 111.53 (CH), 122.02 (CH), 126.04 (CH),
129.82 (CH), 130.94 (Cq), 135.66 (Cq), 145 (Cq), 147.99 (Cq)
0
0
155.26 (C3 , C5 ), 191.34 (C@O); ESI-MS (+) m/z: 318.1 ([M+H]+,
100).
4.1.4. Synthesis of 2-benzotriazol-1-yl-1-(4-fluorophenyl)-3-(3,4,
5-trimethoxyphenyl) propenone (12a)
0.85 mmol (0.22 g) of 2-benzotriazol-1-yl-1-(4-fluorophenyl)eth-
anone 16a is loaded into a 10 mL microwave vial, upon which of
7 mL abs EtOH, 1 equiv (75 mg) of piperidine and 0.9 mmol
(0.176 g) of 3,4,5-trimethoxybenzaldehyde are added. The head-
space is flushed with nitrogen, the vial is subsequently sealed with

4818
B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
a
a TeflonÒ ‘snap-on’ cap, and the mixture is heated at 70 °C under
microwave irradiation for 3 h. The resulting precipitate is the desired
propenone 12a, which can be isolated by filtration, washing and
thorough drying.
d 43.51 (C ), 56.33 (2x OCH3), 60.98 (OCH3), 77,16 (t, CDCl3),
0
0
82.90 (Cb), 102.81 (C2, C6), 116.07 (d, JCF = 21.9 Hz, C3 , C5 ),
0
0
0
125.80 (d, JCF = 3.5 Hz, C1 ), 128.82 (d, JCF = 8.1 Hz, C2 , C6 ), 136.49
(C4), 137.97 (C1), 153.71 (C3, C5), 155.44 (C@N), 163.96 (d,
0
JCF = 251.5 Hz, C4 ); ESI-MS (+) m/z: 332.1 ([M+H]+, 100), 333.1
4.1.5. 2-Benzotriazol-1-yl-1-(4-fluorophenyl)-3-(3,4,5-trime-
thoxyphenyl) propenone (12a)
([M+H+1]+, 22), 457.2 (73), 458.2 (23).
Yield: 51%, as white crystals; mp: 179.1–180.7 °C; IR (ATR,
4.1.9. 5-(4-Fluorophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihyd-
roisoxazole (13b)
cmꢀ1
)
m
max: 998, 1051, 1070, 1130, 1148, 1241, 1261, 1333,
1505; 1H NMR (CDCl3, 300 MHz, ppm): d 3.42 (6H, s, 2x OMe),
Yield: 34%, as white crystals; mp: 115.8–117.4 °C; IR (ATR,
00
00
3.79 (3H, s, OMe), 5.98 (2H, s, H2 , H6 ), 7.12 (2H, dd, Jvic = 8.6 Hz,
cm ꢀ1 max = 826, 838, 914, 1005, 1132, 1215, 1374; 1H NMR
) m
0
0
JHF = 8.6 Hz, H3 , H5 ), 7.25 (1H, td, Jvic = 7.2 Hz, Jallyl = 1.1 Hz, H5 or
H6), 7.39 (1H, td, Jvic = 7.2 Hz, Jallyl = 1.1 Hz, H5 or H6), 7.46 (1H,
dd, Jvic = 7.2 Hz, Jallyl = 1.1 Hz, H4 or H7), 7.76 (1H, s, Hb), 7.84 (2H,
(CDCl3, 300 MHz, ppm): d 3.20 (1H, dd, Jgem = 16.5 Hz, Jvic = 8.3 Hz,
CHaHb), 3.78 (1H, dd, Jgem = 16.5 Hz, Jvic = 10.9 Hz, CHaHb), 3.88
(3H, s, OCH3), 3.89 (6H, s, 2x OCH3), 5.73 (1H, dd, Jvic.1 = 10.9 Hz,
00
00
0
0
dd, Jvic = 8.8 Hz, JHF = 5.5 Hz, H2 , H6 ), 8.11 (1H, dd, Jvic = 7.2 Hz,
Jallyl = 1.1 Hz, H4 or H7); 13C NMR (CDCl3, 75 MHz, ppm): d 55.77
Jvic.2 = 8.3 Hz, Hb), 6.92 (2H, s, H2 , H6 ), 7.07 (2H, dd, Jvic = 8.8 Hz,
JHF = 8.8 Hz, H3, H5), 7.37 (2H, dd, Jvic= 8.8 Hz, JHF = 5.5 Hz, H2, H6);
00
00
(2x OMe), 60.98 (OMe), 77.16 (t, CDCl3), 107.71 (C2 , C6 ), 110.20
13C NMR (CDCl3, 75 MHz, ppm): d 43.31 (C ), 56.26 (2x OCH3),
a
0
0
0
0
(C5 or C6), 115.97 (d, JCF = 21.9 Hz, C3 , C5 .), 120.14 (C4 or C7),
60.93 (OCH3), 77.16 (t, CDCl3) 82.08 (Cb), 104.14 (C2 , C6 ), 115.67
0
124.67 (C5 or C6), 126.05 (Cq), 128.72 (C4 or C7), 130.16 (Cq),
(d, JCF = 21.9 Hz, C3, C5), 124.75 (C1 ), 127.69 (d, JCF = 8.1 Hz, C2,
0
0
0
0
0
0
131.79 (d, JCF = 9.2 Hz, C2 , C6 ) 133.09 (d, JCF = 2.3 Hz, C1 ), 133.32
C6), 136.71 (d, JCF = 2.3 Hz, C1), 139.99 (C4 ), 153.39 (C3 , C5 ),
156.00 (C@N), 162.59 (d, JCF = 246.9 Hz, C4); ESI-MS (+) m/z:
332.1 ([M+H]+, 100), 333.0 ([M+H+1]+, 22).
(Cq), 141.09 (C4), 142.21 (Cb), 145.92 (Cq), 153.12 (C3, C5), 165.51
0
(d, JCF = 255.0 Hz, C4 ), 189.66 (C@O); ESI-MS (+) m/z: 434.1
([M+H]+, 100), 435.3 ([M+H+1]+, 28).
4.1.10. 5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihyd-
roisoxazole (13c)
4.1.6. General synthetic procedure for chalcones (1–3)
15 mmol of an acetophenone, 0.1 equiv of LiOH.H2O and 20 mL
of absolute EtOH are brought into a round bottomed flask, and the
resulting suspension is stirred for 15 min. Subsequently, 1 equiv of
the appropriate benzaldehyde is added and the mixture is stirred
until conversion has reached its maximum, as monitored by TLC
and/or LC/MS. The reaction is quenched with a 10 mL of a 1% aque-
ous solution of HCl, followed by 10 mL of H2O.
When a precipitate is obtained, it is isolated by filtration and
thoroughly washed with water until the filtrate turns clear. Recrys-
tallization of the precipitate in absolute EtOH furnishes crystals of
chalcone in high purity. If, upon quenching, the product separates
as a thick oily layer at the bottom of the bulb, it is extracted with
Et2O (2 ꢂ 20 mL). The combined organic layers are subsequently
washed with brine (2 ꢂ 20 mL) and dried over MgSO4. Solvent
evaporation under reduced pressure furnishes the crude chalcone
3 as a solid, which can be purified by recrystallization in absolute
EtOH.
Spectral data are in full accordance with those reported earlier
by Mizuno.22
Yield: 94%, as white crystals; mp: 128.3–129.4 °C; IR (ATR,
cmꢀ1
) mmax = 808, 834, 862, 892, 1021, 1091, 1177, 1254, 1491,
1606, 2853, 2922.03, 2955; 1H NMR (CDCl3, 300 MHz, ppm): d
3.27 (1H, dd, Jgem = 16.7 Hz, Jvic = 7.8 Hz, CHaHb), 3.76 (1H, dd,
Jgem = 16.7 Hz, Jvic = 11.0 Hz, CHaHb), 3.84 (3H, s, OCH3), 5.68 (1H,
0
0
dd, J1 = 11.0 Hz, J2 = 7.8 Hz, Hb), 6.93 (2H, d, Jvic = 9.2 Hz, H3 , H5 ),
0
0
7.34 (4H, br. m, H2, H6, H3, H5), 7.62 (2H, d, Jvic = 9.2 Hz, H2 , H6 );
a
13C NMR (CDCl3, 75 MHz, ppm): d 42.46 (C ), 54.34 (OCH3), 80.48
0
0
0
0
(Cb), 113.17 (C3 , C5 ), 120.74 (C1 ), 126.24 (C2, C6), 127.27 (C2 ,
0
C6 ), 127.87 (C3, C5), 132.91, 138.63 (C1, C4), 154.62 (C@N), 160.16
0
(C4 ); ESI-HRMS (+) m/z: Anal. Calcd for C16H14ClNO2 (M+H)+:
288.0786. Found: 288.0790.
4.1.10.1. Synthesis of 5-(4-chlorophenyl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazole (20).
In a round-bottomed flask
kept under a nitrogen atmosphere, 0.01 mol (3.15 g) of chalcone
2 is dissolved in 20 mL of absolute EtOH. Subsequently, 6 equiv
(3 g) of NH2NH2.H2O and 7 equiv (3.91 g) of KOH are added. The
resulting mixture is stirred at reflux for 24 h. Upon cooling, water
(20 mL) is added and the pyrazole is extracted with EtOAc
(2 ꢂ 15 mL). The combined organic layers are dried over MgSO4;
solvent removal by rotary evaporation furnishes the desired pyraz-
oline 20 as a solid residue.
4.1.7. Synthesis of 3,5-diaryl-4,5-dihydroisoxazoles (13a–c)
In a round-bottomed flask containing 20 mL of absolute ethanol
and kept under a nitrogen atmosphere, 0.1 mmol of a chalcone,
6 equiv (41.7 mg) of NH2OH.HCl and 7 equiv (39.3 mg) of KOH
are added respectively. The resulting mixture is stirred at reflux
and under a nitrogen atmosphere for 24 h. Upon cooling of the con-
tents to ambient temperature, an equal volume of H2O is added
and the resulting suspension is extracted with ethyl acetate
(2 ꢂ 20 mL). The combined organic layers are subsequently dried
over MgSO4, and the solvent is removed under reduced pressure.
The resulting solid can be recrystallized in absolute ethanol, fur-
nishing the pure isoxazoline 13.
4.1.11. Synthesis of isoxazoles (4a–c)
A microwave vial is loaded with 0.9 mmol of an isoxazoline 13,
6 mL of benzene, 10 equiv (0.89 g) of activated MnO2 and 2.4 equiv
(0.26 g) of MgSO4. Electrolytically precipitated, active MnO2 (88%)
from Acros was used in this reaction. Next, the vial is sealed and
heated at 100 °C under microwave irradiation during 1.5 h. Subse-
quently, the reaction mixture is filtered over CeliteÒ, and the resi-
due is washed with CH2Cl2. Solvent removal from the filtrate
furnishes the pure isoxazole 4 as a solid residue.
4.1.8. 3-(4-Fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihyd-
roisoxazole (13a)
Yield: 30%, as white crystals; mp: 125.4–126.9 °C; IR (ATR,
cmꢀ1
) mmax: 832, 896, 1007, 1123, 1218, 1236, 1324, 1426, 1509,
1592; 1H NMR (CDCl3, 300 MHz, ppm): d: 3.33 (1H, dd,
Jgem = 16.5 Hz, Jvic = 8.3 Hz, CHaHb), 3.75 (1H, dd, Jgem = 16.5 Hz,
Jvic = 10.0 Hz, CHaHb), 3.84 (3H, s, OCH3), 3.87 (6H, s, 2x OCH3),
5.69 (1H, dd, Jvic,1 = 10.0 Hz, Jvic,2 = 8.3 Hz, Hb), 6.61 (2H, s, H2, H6),
4.1.12. 5-(4-Fluorophenyl)-3-(3,4,5-trimethoxyphenyl) isoxazole
(4b)
Yield: 65%, as white crystals; 1H NMR (CDCl3, 300 MHz, ppm):
0
0
a
7.11 (2H, dd, Jvic = 8.5 Hz, JHF = 8.5 Hz, H3 , H5 ), 7.69 (2H, dd,
d = 3.91 (3H, s, OCH3), 3.95 (6H, s, 2x OCH3), 6.75 (1H, s, H ), 7.08
0
0
00
00
0
0
Jvic = 8.8 Hz, JHF = 5.5 Hz, H2 , H6 ); 13C NMR (CDCl3, 75 MHz, ppm):
(2H, s, H2 , H6 ), 7.19 (2H, dd, Jvic = 8.5 Hz, JHF = 8.5 Hz, H3 , H5 ),

B. I. Roman et al. / Bioorg. Med. Chem. 20 (2012) 4812–4819
4819
0
0
0
0
7.84 (2H, ddd, Jvic = 8.5 Hz, JHF = 5.1 Hz, Jallyl = 1.9 Hz, H2 , H6 ); 13C
NMR (CDCl3, 75 MHz, ppm): d = 56.20 (2x OCH3), 61.07 (OCH3),
Jvic = 8.5 Hz, JHF = 5.2 Hz, H2 , H6 ), 8.32 (1H, s, H3); 19F NMR (CDCl3,
282 MHz, ppm): d (ꢀ)112.90–(ꢀ)112.75 (m); ESI-HRMS (+) m/z:
Anal. Calcd for C18H16FNO4 (M+H)+: 330.1136. Found: 330.1145.
00
00
a
77.16 (t, CDCl3), 97.31 (C ), 103.96 (C2
,
C6 ), 116.18 (d,
0
0
0
00
JCF = 21.9 Hz, C3 , C5 ), 123.70 (d, JCF = 3.5 Hz, C1 ), 124.35 (C1 ),
0
0
00
00
00
127.84 (d, JCF = 8.1 Hz, C2 , C6 ), 139.61 (C4 ), 153.59 (C3 , C5 ),
4.2. Molecular modeling
0
162.89 (C@N), 163.73 (d, JCF = 251.5 Hz, C4 ), 169.41 (Cb); ESI-HRMS
(+) m/z: Anal. Calcd for C18H16FNO4 (M+H)+: 330.1136. Found:
Two-dimensional drawings of compounds were made using
ChemDraw Ultra 7.0.1.24 These structures were subsequently
loaded into HyperChem 8.0.325 and their refined equilibrium
molecular geometry was obtained by (i) 2D–3D conversion using
parameters included in HyperChem, (ii) pre-optimization using
the molecular mechanics force field (MM+) method and (iii) final
optimization at the semi-empirical AM1-level of theory using a
Polak-Ribière conjugated gradient and an RMS gradient of
0.01 kcal Åꢀ1 molꢀ1 as the termination condition for optimized
structures. These lowest energy conformers were used to generate
molecular alignments via the ‘RMS fit and overlay’ algorithm,
embedded in HyperChem 8.0.3. The potential energy scan, for
which the result is shown in Scheme 1, was obtained in HyperChem
8.0.3 at the AM1 level of theory.
330.1144.
4.1.13. 5-(4-Chlorophenyl)-3-(4-methoxyphenyl)isoxazole (4c)
Spectral data were in full accordance with those reported earlier
by Upadhyay.23
ESI-HRMS (+) m/z: Anal. Calcd for C16H12ClNO2 (M+H)+:
286.0629. Found: 286.0634.
4.1.14. Synthesis of dimethyl [hydroxyl(3,4,5-trimethoxyphenyl)-
methyl]phosphonate (22a) and dimethyl [(4-fluorophenyl)-
hydroxymethyl]phosphonate (22b)
25 mmol of an appropriate benzaldehyde is dissolved in 25 mL
of Et2O, upon which 2 equiv (4.6 mL) of dimethyl phosphite and
14 mol % (0.6 mL) of n-Bu2NH are added. The mixture is stirred at
ambient temperature for 3 h, and the resulting precipitate is iso-
lated by filtration. The residue is subsequently washed with Et2O,
Acknowledgments
yielding the pure
a
-hydroxyphosphonate 22.
The authors thank the Research Foundation – Flanders (Fonds
voor Wetenschappelijk Onderzoek – Vlaanderen) for financial sup-
port for this research, and would like to express their sincere
appreciation to Marleen De Meulemeester for meticulously con-
ducting the CHI-assay on all compounds investigated in this study.
4.1.15. Synthesis of 2-(4-fluorophenyl)-1-(3,4,5-trimethoxy-
phenyl)ethanone (26a) and 1-(4-fluorophenyl)-2-(3,4,5-
trimethoxyphenyl)ethanone (26b)
In a round-bottomed flask kept under a nitrogen atmosphere,
10 mmol of phosphonate 22, 2.2 equiv (2 mL) of 3,4-dihydro-2H-
pyran and 2.5 mol % (50 mg) of para-TsOHꢁH2O are dissolved in
50 mL of benzene. The resulting mixture is heated to reflux tem-
perature for 2 h. Upon cooling to room temperature, 1 equiv of
an appropriate aldehyde, 4 equiv (0.6 g) of NaH and 1.7 equiv
(1 mL) of dry EtOH are added. The contents of the flask is stirred
during 18 h under a nitrogen atmosphere, after which water is
added (30 mL). The organic phase is separated, washed with water
(30 mL) and dried over MgSO4. Upon solvent removal via rotary
evaporation, the residue is taken up in a mixture of 30 mL of MeOH
and 0.95 equiv aq HCl (3 ml of a 10% solution) and stirred at room
temperature for 1 h. Next, the flask is stored at ꢀ20 °C to allow the
ethanone to precipitate as a solid. Pure 26 can be obtained from
this crude mixture by chromatographic separation, employing a
solvent mixture of EtOAc and petroleum ether in a 2:1 ratio.
References and notes
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4.1.16. Synthesis of 4-(4-fluorophenyl)-5-(3,4,5-trimethoxy-
phenyl)isoxazole (5a) and 5-(4-fluorophenyl)-4-(3,4,5-
trimethoxyphenyl)isoxazole (5b)
11. Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.; Hannick, S. M.;
Gherke, L.; Credo, R. B.; Hui, Y.-H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-
Mozng, N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J. Med. Chem. 2002, 45, 1697.
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A solution 1.61 mmol of ethanone 26 in 10 mL of DMFDMA,
kept under a nitrogen atmosphere, is stirred at reflux for 14 h.
Next, the solvent is removed under reduced pressure, and the res-
idue is taken up in a mixture of MeOH and H2O in a 2:1 ratio. Sub-
sequently, 1.96 equiv (0.22 g) of NH2OH.HCl and 1 equiv (0.17 g) of
Na2CO3 are added. The pH of the reaction mixture is adjusted to 4–
5 by addition of AcOH, whereupon the contents of the flask, still
under a nitrogen atmosphere, is heated to reflux for 2 h. Afterward,
NH4OH is added until the pH of the mixture equals 8, and the isox-
azole is extracted with CH2Cl2. Upon drying over MgSO4, rotary
evaporation of the solvent furnishes the crude isoxazole 5, which
can be recrystallized from MeOH.
13. Katritzky, A. R.; Wang, M.; Zhang, S.; Voronkov, M. V. J. Org. Chem. 2001, 66,
6787.
14. Katritzky, A. R.; Lam, J. N. Heteroat. Chem. 1990, 1, 21.
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4.1.17. 4-(4-Fluorophenyl)-5-(3,4,5-trimethoxyphenyl)
isoxazole (5a)
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Yield: 28%, as white crystals; 1H NMR (CDCl3, 300 MHz, ppm):
24. ChemDraw
(www.cambridgesoft.com).
25. HyperChem 8.0.3. Hypercube Inc., 2007 (www.hyper.com).
Ultra
7.0.1.
CambrigdeSoft
Corporation,
2002
00
00
d = 3.72 (6H, s, 2x OCH3), 3.88 (3H, s, OCH3), 6.84 (2H, s, C2 , C6 ),
0
0
7.13 (2H, dd, Jvic = 8.5 Hz, JHF = 8.5 Hz, H3 , H5 ), 7.40 (2H, dd,