Synthesis and tubulin-binding properties of non-symmetrical click C5-curcuminoids

Article abstract of DOI:10.1016/j.bmc.2013.05.053

A click-type entry into shortened curcuminoids of the diarylpentanoid type has been developed. The reaction is ideally suited to generate non-symmetrical analogues of curcumin, a class of natural products difficult to access but of growing biomedical rele

Full text of DOI:10.1016/j.bmc.2013.05.053

Bioorganic & Medicinal Chemistry 21 (2013) 5510–5517
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and tubulin-binding properties of non-symmetrical click
C5-curcuminoids
Antonio Caldarelli, Eleonora Penucchini, Diego Caprioglio, Armando A. Genazzani,
⇑
Alberto Minassi
Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Via Bovio 6, Novara 28100, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A click-type entry into shortened curcuminoids of the diarylpentanoid type has been developed. The
reaction is ideally suited to generate non-symmetrical analogues of curcumin, a class of natural products
difficult to access but of growing biomedical relevance and special mechanistic interest to investigate the
unique binding mode of curcumin to tubulin. Investigation of a series of click diarylpentane curcuminoids
and their pyrazole adducts in various cellular tubulin functional assays validated this class of compounds
as a novel type of anti-mitotic agents, evidencing structure–activity relationships, and identifying the
pyrazole adduct 4k as a promising lead.
Received 9 April 2013
Revised 22 May 2013
Accepted 28 May 2013
Available online 13 June 2013
Keywords:
Curcumin
Curcuminoids
Click chemistry
Antimitotic agents
Tubulin
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
stantial lack of toxicity, make it unlikely that any synthetic analogue
will displace natural curcumin in this broad-spectrum, but essen-
No plant better than turmeric (Curcuma longa L.) exemplifies the
versatility of spices in human nutrition and health, flavoring all
dishes of the cuisine of India and, allegedly, also curing all diseases
in its folk medicine.1 Unlike other panaceas of traditional medicine,
preclinical investigations have confirmed the potential of curcumin
(1a), the major phytochemical of turmeric, in the management of a
multitude of conditions that include some of the hottest areas of cur-
rent pharmaceutical research (cancer,2 inflammation,3 infectious
disesase4). Unsurprisingly given its cure-all status, the hallmark of
the biological profile of curcumin is the redundancy of targets, but,
from a mechanistic standpoint, there is a general agreement that
curcumin is mainly an epigenetic agent, acting on key transcription
tially supportive, medical use.
On the other hand, with over 100 different distinct biological
targets,12 curcumin is also a most versatile privileged structure
for the discovery of bioactivity, directly interfering with the func-
tion of a host of enzymes and receptors. In this context, the activity
on tubulin has received considerable attention, since curcumin can
affect microtubule function in cancer cells in a way substantially
different from all known antitubulin agents,13 acting at a binding
site located between the
a and b-subunits of two a, b-dimers,
and clearly distinct from those of colchicine, taxoids and Vinca
alkaloid. Despite compelling evidence of anti-mitotic activity, cur-
cumin shows an extraordinary level of tolerance in humans, possi-
bly because the functional translation of its anti-tubulin activity
involves process(es) differently regulated in normal and in cancer
cells. Taken together, these observations qualify curcumin as an
interesting anticancer lead, worth exploration in terms of struc-
ture–activity relationships.14
factors (NF-kB,5 Nrf2,6 STAT-3,7 PPAR- 8) involved in anti-inflam-
c
matory/anti-oxidant responses and in cancer development and pro-
gression. Curcumin has, indeed, been used with promising results in
a phase II clinical study for pancreatic cancer6 and in some small
anti-inflammatory clinical trials.9 However, its clinical potential re-
mains largely untapped, and megadoses (>5 g/day) are required be-
cause of a combination of moderate potency and very poor oral
bioavailability1,10 Significant clinical progress has recently been ob-
tained with a lecithin formulation of curcumin as a sparing agent for
anti-inflammatory drugs in various chronic diseases.11 From a
medicinal chemistry standpoint, the pleiotropic bioactivity and sub-
O
OH
HO
OH
R1
R2
R1
R2
1a OMe OMe
1b
OMe
H
⇑
Corresponding author. Tel.: +39 0321375849; fax: +39 0321375821.
E-mail address: minassi@pharm.unipmn.it (A. Minassi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.053

A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
5511
Many tubulin ligands have a dimeric structure. Thus, Vinca
alkaloids are heterodimeric structures, and tubulin ligands like
combretastatins and podophyllotoxin share a non-symmetrical
dibenzyl dimeric structure. Conversely, curcumin is a symmetric
feruloyl dimer, and it was therefore interesting to investigate the
tubulin binding properties of non-symmetrical curcuminoids. Ow-
ing to difficulties in the synthesis of these compounds, most struc-
ture–activity studies on this class of compounds have focused on
symmetrical analogues of the natural product, despite the in-
creased potency of demethoxycurcumin (1b)9 in several anti-
inflammatory and biochemical assays. To address this issue and
extend the structure–activity relationships of curcuminoids as
tubulin ligands, we have developed a click-type entry C5-curcumi-
noids of the benzoyl-feruloylmethane type, capitalizing on the
reaction of the borate complex of a series of 1-aryl-2,4-butandi-
ones with various aromatic aldehydes. The stepwise carbonyl
chemistry involved in their synthesis makes it possible to easily ac-
cess curcuminoids with a non-symmetrical aryl substitution pat-
tern. Compared to the C5 curcuminoids of the monocarbonyl-
type, these abridged curcuminoids retain the 1,3-dicarbonyl sys-
tem and its associated rich chemistry, including the conversion
to the corresponding pyrazole derivatives, a validated maneuver
to improve the potency of curcuminoids in many biological
assays.15,16
one single set of signals in their NMR spectra, with two well-differ-
entiated ‘carbonyl’ resonances around d 190 and 180. In curcumin,
there is one single carbonyl resonance at 180 ppm, and, in the ab-
sence of any bias, two close values could be expected. The larger
separation between the ‘carbonyl’ resonances, suggests the preva-
lence of one tautomer, and considerations of calculated chemical
shifts suggest the prevalence of the tautomeric form A compared
to B.
OH
O
O
OH
Ar'
Ar
Ar'
Ar
A
B
2.2. Biological results
Cytotoxicity in a human melanoma cell line (MeWo) was used
as preliminary screening, treating cells with curcumin or its non-
symmetrical analogues 3a–o and 4a–o at a fixed concentration of
10 lM for 48 h (Table 1). In accordance with its low potency, 1a
was unable to significantly affect viability at this concentration,
while four analogues of the pyrazole series (4i, 4k, 4n, 4p) reduce
cell viability below 50% of the control sample that was taken as a
threshold value for cytotoxicity. For these hits, a concentration–re-
sponse curve with built, again using 1a as a positive reference. Cur-
cumin (1a) showed an IC50 of 35.5 4.7
lM, with 4p being the
2. Results
most potent (3.6 0.7 M; Fig. 1). The activity of the lead com-
l
pounds, as well as of curcumin, was then investigated, building a
full concentration–response curve, in a neuroblastoma cell line
(SH-SY5Y) exquisitely sensitive to antitubulin agents.19 The cyto-
toxic activity was confirmed, but the rank of potency was slightly
modified, with only 4k significantly more cytotoxic than curcumin.
2.1. Chemistry
The synthesis of the C5-curcuminoids was carried out by con-
densing a series of aromatic aldehydes with the boric complex of
various 1-aryl-1,3-butanediones,17 in turn obtained from the reac-
tion of the corresponding acetophenones with ethyl acetate
(Scheme 1). By stabilizing its enol tautomer, formation of a boric
complex prevents alkylation of the central methylene of phenylb-
utanedione, quenching the methylene-centered Knoevenagel reac-
tivity at the expenses of the methyl-centered aldol-type
reactivity.18 The reaction was carried out in a click fashion, and
Table 1
Summarization of synthesized compounds and relative cytotoxicity in Mewo cells,
after 48 h administration at 10 lM
Compound
R1
R2
R3
R4
R5
Viability (%)
90
90 11
90 11
1a
3a
3b
3c
3d
3e
3f
3g
3h
3i
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
OCH3
H
H
OCH3
OCH3
OCH3
H
OH
OH
OCH3
H
OCH3
OH
OH
OCH3
H
OCH3
OH
OH
OCH3
H
OCH3
OH
OH
OCH3
H
OCH3
OH
OH
OCH3
H
OCH3
OH
H
H
H
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
the resulting
a,b-unsaturated diketones (3a–o) were simply ob-
tained by precipitation with acetic acid and washing with water.
Purity was evaluated by HPLC, and, when unsatisfactory (<95%),
was improved by re-crystallization to reach the threshold set of
purity, set at least 95%.
The pyrazole analogues were prepared by overnight treatment
with hydrazine hydrate in acetic acid at 50 °C. An aqueous work-
up and crystallization of the crude products afforded pure com-
pounds without any further purification.
90
92
93
89
77
93
90
85
97
98
84
88
89
51
98
92
90
89
69
87
81
4
6
2
5
9
4
2
1
4
2
4
5
2
2
2
4
4
4
2
8
7
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
OCH3
OCH3
OCH3
H
3j
3k
3l
The non-symmetrical dicarbonyl nature of compounds 3a–o
could bias their tautomeric equilibration, favoring one tautomeric
form over the other possible one. All compounds showed only
3m
3n
3o
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
H
OCH3
OCH3
OCH3
H
H
H
H
H
H
O
O
OH
O
RCHO, B2O3,
OCH3
OCH3
OCH3
H
R5
R4
B(OCH3)3,nBuNH2
DMF, 40 o
C
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
R1
R1
3a-o
2
R2
R3
R2
H
OCH3
OCH3
OCH3
H
38 10
H
85
36
95
92
43
75
27
2
3
7
5
1
2
1
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
AcOH
N
NH
OH
OCH3
H
OCH3
H
NH2NH2
R5
R4
50 o
C
4m
4n
4o
4p
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
R1
4a-o
R3
R2
Results relative to the vehicle (DMSO) and representative of 8–12 different data
points AV S.D.
Scheme 1. Synthesis of mono-
unsaturated pyrazoles (4a–o).
a,b-unsaturated b-diketones (3a–o) and mono-a,b-

5512
A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
The anti-tubulinic activity of the hit compounds was next inves-
tigated by FACS analysis in a cell cycle progression assay (Fig. 2).
Blockade in the G2/M phase is a phenotypic signature of antitubu-
linic agents, since impairment of microtubules does not allow cell
cycle progression. The incubation of 1a or compound 4i, 4k, 4n, 4p
at a concentration 3-fold higher than the IC50 induced indeed G2/M
phase block of the cell cycle. Combretastatin CA-4 (CA-4) was used
as a positive control in this assay.
The involvement of tubulin in the cytotoxicity of 4i, 4k, 4n, 4p
was confirmed by direct investigation of the cellular tubulin state
by immunofluorescence. Thus, MeWo cells were treated with cur-
cumin or 4i, 4k, 4n, 4p, for 16 h and next fixed, staining the tubulin
cytoskeleton with a specific antibody. The microtubule network
was clearly observable in control cells but disappeared in all trea-
ted samples (Fig. 3).
Finally, tubulin polymerization was investigated in an in cellu-
lar assay. Briefly, cells were treated with the desired compound for
16 h, and proteins were next extracted in presence of paclitaxel to
prevent subsequent microtubule re-arrangement (Fig. 3). Western
Figure 2. Curcumin and the most potent synthesized analogues induced cell cycle
arrest. Cell cycle analysis of Mewo cells treated for 16 h with vehicle (CTR) or 3 X
IC50 values of indicated compounds. Data are representative of three separate
experiments. The Y-axis represents cell number, and the X-axis represents
fluorescence on a linear scale.
blotting of the pelletable fraction and the soluble fraction made
then possible to distinguish between polymerized and free tubulin.
As expected, in the control cells tubulin was present in similar
amounts in the polymerized (pellet) and free form (supernatant),
while the equilibrium was significantly shifted toward the free
form when cells were treated with curcumin or the hit compounds
4i, 4k, 4n and 4p. CA-4 served also in this case as positive control
for its well-characterized antitubulin action. Taken together, these
experiments provide conclusive evidence that tubulin polymeriza-
tion represents the primary mechanism of action of these cytotoxic
compounds.
3. Discussion
Curcumin, the bioactive ingredient of turmeric, has been used
for multiple medical purpose for thousand of years. Modern re-
search has shown that curcumin can modulate a host of cellular
Figure 1. Concentration–response curves of curcumin and the most potent
compounds in MeWo and SH-SY5Y cells lines after 48 h treatment. Values are
mean + S.D. of 8–16 replicates from 4 separate experiments.

A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
5513
of this type are available in a click fashion from a stepwise carbonyl
chemistry that make it possible to easily access analogues with dif-
ferent substitution at the terminal aryl moieties, something that is
difficult to achieve in curcuminoids of the diarylheptanoid type.
Furthermore, these analogues retain the rich chemistry of curcumi-
noids associated to the presence of the enolized b-dicarbonyl sys-
tem, including the capacity to afford pyrazole derivative, an
important potency-inreasing maneuver in curcuminoids.
Cytotoxicity at a concentration where curcumin shows only
marginal activity was used as a rapid comparative initial screening.
Most C5 curcuminoids outperformed the natural product, with sig-
nificant cytotoxicity being observed in the four pyrazoles 4i, 4k,
4n, 4p, all having non-symmetrically substituted terminal aryl
moieties. These findings are in line with the general potency trend
observed in curcuminoids of the natural C7-type, and these hits
were then investigated for their capacity to disrupt tubulin, one
intriguing property of curcumin. In accordance with the cytotoxic-
ity data on the human melanoma cell line MeWo, all four com-
pounds outperformed curcumin. While the data-set should be
increased to draw extensive structure–activity relationship within
this novel class of C5-curcuminoids, some considerations are evi-
dent, mirroring those observed in curcuminoids regarding the
replacement of the enolized b-dicarbonyl system with a pyrazole
and the non-identical substitution pattern of the peripheral rings.
Formation of pyrazole derivatives impose a conformational con-
straint that is beneficial for tubulin activity, that is seemingly in-
creased by rigidification.15 Regarding the substitution pattern at
the peripheral phenyls, oxygenated functions are important for
activity, with a particularly beneficial role for a p-hydroxyl in ben-
zoyl-type phenyl, and a m-methoxyl in the stiryl-type phenyl.
4. Conclusions
A novel class of non-symmetrical C5-curcuminoids has been
identified, with cytotoxicity being especially remarkable in their
pyrazole analogues. Despite the shortening of the conjugated sys-
tem, these compounds show similar, or even better, bioactivity
compared with curcumin, with a series of four pyrazole analogues
qualifying as lead compounds to develop curcuminoid-based anti-
mitotic agents potentially selective for cancer cells. Due to an in-
creased potency, these compounds also qualify as probe to
investigate the molecular basis of the selectivity of action of
curcuminoids.
Figure 3. Curcumin and the most active compounds affect tubulin polymerization.
Immunofluorescence using an anti -tubulin antibody (green) and DRAQ5 (blue) for
a
nuclear staining of control cells (CTR) or cells treated for 16 h with 3 Â IC50 value of
the indicated compounds. Images have been obtained with a 63Â oil immersion
objective (upper pannel). Western blot of
a-tubulin extracted in the presence of
paclitaxel from Mewo cells treated with the indicated compounds at 3 Â IC50 value
(lower panel). Results are representative of three separate experiments. P = pellet-
able fraction. S = soluble fraction.
5. Experimental section
5.1. Chemistry
processes, probably because of its extended and oxygenated conju-
gated system endows it with Michael acceptor, an antioxidant,20
and a metal chelation properties. Most structure- activity studies
on curcumin have focused on the modification of the diacryloylme-
thane moiety, with simplification to C5-curcuminoids, that is curc-
uminoids of the dienone-type, being expecially popular and often
providing more potent analogueds. Replacement of the b-diketone
system with a pyrazole has also been validated as a potency
increasing maneuver,21 The classic C5 curcuminoid lack the eno-
lized b-dicarbonyl moiety of the natural product, and might behave
as more potent analogue in assays strongly dependant on, since
they can outperform curcumin in assays of thio trapping capac-
ity.22,23 Competitive experiments were carried out by reacting
equimolecular binary mixtures of curcumin and C5 monocarbon-
yl-curcumin with substoichiometric amount of cysteamine. Under
those conditions C5 curcumin showed a higher reactivity than cur-
cumin (unpublished results). We have developed an alternative
class of C5 curcuminoids that retain the hallmark of the curcumin
pharmacophore, namely the presence of an extensively conjugated
IR spectra were taken on a FT-IR Thermo Nicolet equipment. 1H
(300 MHz) and 13C (75 MHz) NMR spectra were measured on a
JEOL Eclipse 300 instrument. Chemical shifts were referenced to
the residual solvent signal (CDCl3: dH = 7.26, dC = 77.0, CD3OD:
dH = 3.34, dC = 49.0). Low- and high-resolution ESIMS were ob-
tained on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer.
Silica gel 60 (70–230 mesh) and RP-18 used for gravity column
chromatography was purchased from Macherey-Nagel. Reactions
were monitored by TLC on Merck 60 F254 (0.25 mm) plates, that
were visualized by UV inspection and/or staining with 5% H2SO4
in ethanol and heating. Organic phases were dried with Na2SO4 be-
fore evaporation.
5.1.1. General procedure for the synthesis of mono-a,b-
unsaturated b-diketones (3a–o)
To a stirred solution of vanillin (1 equiv mol) in dry DMF (2 mL/
enolized b-dicarbonyl system, while differing for a shortened
p-
mmol), 1-phenyl-1,3-butanedione17 (1 equiv mol), B2O3 (1,6 equiv
conjugation between the peripheral aromatic rings. Compounds

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A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
mol), B(OCH3)3 (0,73 equiv mol) and butyl amine (0,08 equiv mol)
were sequentially added. The reaction was stirred at 40 °C over-
night and quenched with a 5% AcOH (2.5 mL/mmol). A precipitate
was formed, and after filtration and washing with water 3a was
obtained as a yellow solid (55%) without further purification. Mp:
163 °C; IR (KBr) cmÀ1: 3438, 3422, 2354, 1866, 1538, 1504, 1470,
1427, 1285,1271, 837, 764, 689, 421, 402; 1H NMR (300 MHz,
CDCl3) d: 7.94 (d, 2H, J = 7.02), 7.62 (d, 1H, J = 15.6), 7.50 (m, 3H),
7.13 (dd, 1H, J1 = 1.3, J2 = 7.9), 7.06 (d, 1H, J = 1.4), 6.94 (dd, 1H,
J1 = 0.8, J2 = 8.1), 6.32 (s, 1H), 3.95 (s, 3H); 13C NMR (75 MHz,
(CD3)2CO) d: 188.22, 181.35, 149.30, 148.01, 140.71, 136.29,
132.61, 128.79, 128.63, 127.36, 127.25, 123.10, 120.69, 115.48,
110.69, 96.83; CI-EIMS: m/z [M+] 295; C18H16O4 (296.31): calcd C
72.96, H 5.44; found: C 72.99, H 5.29.
7.16 (dd, 1H, J1 = 1.8, J2 = 8.3), 6.95 (d, 2H, J = 8.8), 6.88 (d, 1H,
J = 7.9), 6.81 (d, 1H, J = 15.6), 6.49 (s, 1H), 3.91 (s, 9H); 13C NMR
(75 MHz, (CD3)2CO) d: 188.84, 179.16, 161.87, 148.99, 147.97,
139.52, 129.76, 128.01, 127.52, 122.78, 120.74, 115.51, 115.42,
110.55, 96.22, 55.49
(312.10): calcd C 69.22, H 5.16; found: C 69.21, H 5.17.
; CI-EIMS: m/z [ ] 310; C18H16O5
M2+
5.1.7. (E)-1,5-Bis(4-hydroxyphenyl)pent-4-ene-1,3-dione (3g)
43% as a dark yellow solid. Mp: 194 °C; IR (KBr) cmÀ1: 3820,
33730, 3710, 2718, 2507, 2363, 1907, 1728, 1557, 1514, 1435,
1372, 1168, 963, 837, 487, 419, 409; 1H NMR (300 MHz, (CD3)2CO)
d: 7.94 (d, 2H, J = 8.5), 7.60 (d, 1H, J = 15.87), 7.54 (d, 2H, J = 8.8),
6.94 (d, 2H, J = 8.5) 6.89 (d, 2H, J = 8.5), 6.68 (d, 1H, J = 15.6), 6.50
(s, 1H); 13C NMR (75 MHz, (CD3)2CO) d: 188.79, 179.21, 161.87,
159.52, 139.17, 129.94, 129.75, 128.00, 127.06, 120.55, 115.98,
115.52, 96.19; CI-EIMS: m/z [M2+] 280; C17H14O4 (282.09): calcd
C 72.33, H 5.00; found: C 72.34, H 5.01.
5.1.2. (E)-5-(4-Hydroxyphenyl)-1-phenylpent-4-ene-1,3-dione
(3b)
43% as a yellow powder. Mp: 179 °C; IR (KBr) cmÀ1: 3468, 3417,
2961, 2733, 2589, 1618, 1525, 1453, 1364, 1166, 769, 688, 418,
407; 1H NMR (300 MHz, CDCl3) d: 7.94 (d, 2H, J = 8.5), 7.62 (d,
1H, J = 15.7), 7.47 (m, 5H), 6.86 (d, 2H, J = 8.8), 6.52 (d, 1H,
J = 15.6), 6.31 (s, 1H); 13C NMR (75 MHz, (CD3)2CO) d: 190.24,
157.61, 132.91, 130.00, 128.70, 128.57, 127.90, 127.65, 125.38,
115.68, 114.51, 99.38; CI-EIMS: m/z [M+] 265; C17H14O3 (266.29):
calcd C 76.68, H 5.30; found: C 76.70, H 5.31.
5.1.8. (E)-1-(4-Hydroxyphenyl)-5-(4-methoxyphenyl)pent-4-
ene-1,3-dione (3h)
47% as a yellow solid. Mp: 165 °C; IR (KBr) cmÀ1: 3852, 3800,
3627, 2856, 1914, 1716, 1557, 1460, 1211, 845, 682, 545; 1H
NMR (300 MHz, (CD3)2CO) d: 7.93 (d, 2H, J = 8.5), 7.61 (m, 3H),
6.98 (d, 2H, J = 8.8) 6.52 (d, 2H, J = 8.8), 6.73 (d, 1H, J = 15.6), 6.52
(s, 1H), 3.84 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 189.03,
178.83, 161.91, 161.46, 138.74, 129.79, 129.71, 128.05, 121.26,
115.51, 114.49, 96.35, 54.95; CI-EIMS: m/z [M+] 295; C18H16O4
(296.10): calcd C 72.96, H 5.44; found: C 72.95, H 5.43.
5.1.3. (E)-5-(4-Methoxyphenyl)-1-phenylpent-4-ene-1,3-dione
(3c)
49% as an orange powder. Mp: 130 °C; IR (KBr) cmÀ1: 3954,
3800, 3428, 2858, 2589, 1903, 1715, 1505, 1398, 769, 395, 438;
1H NMR (300 MHz, CDCl3) d: 7.94 (d, 2H, J = 8.2), 7.66 (d, 1H,
J = 15.9), 7.51 (m, 5H), 6.92 (d, 2H, J = 8.8), 6.53 (d, 1H, J = 15.6),
6.31 (s, 1H), 3.88 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 188.42,
161.66, 139.77, 136.36, 132.65, 129.93, 128.80, 127.90, 127.31,
121.10, 114.54, 96.98, 54.99; CI-EIMS: m/z [M+] 279; C18H16O3
(280.31): calcd C 77.12, H 5.75; found: C 77.15, H 5.77.
5.1.9. (E)-5-(3,5-Dimethoxyphenyl)-1-(4-hydroxyphenyl)pent-
4-ene-1,3-dione (3i)
51% as a yellow solid. Mp: 139 °C; IR (KBr) cmÀ1: 3900, 3852,
3742, 3648, 3496, 3418, 2839, 2364, 1887, 1539, 1455, 1350,
964, 823, 419; 1H NMR (300 MHz, (CD3)2CO) d: 7.95 (d, 2H,
J = 8.2), 7.57 (d, 1H, J = 15.9), 6.96 (d, 2H, J = 8.2) 6.93 (d, 1H,
J = 10.4), 7.85 (d, 2H, J = 2.1), 6.57 (s, 1H), 6.52 (brt, 1H,) 3.84 (s,
6H); 13C NMR (75 MHz, (CD3)2CO) d: 189.82, 177.57, 162.14,
161.40, 138.78, 137.40, 130.71, 127.97, 124.19, 115.58, 105.82,
102.10, 97.04, 54.96; CI-EIMS: m/z [M+] 325; C19H18O5 (326.11):
calcd C 69.93, H 5.56; found: C 69.90, H 5.55.
5.1.4. (E)-5-(3,5-Dimethoxyphenyl)-1-phenylpent-4-ene-1,3-
dione (3d)
53% as a yellow powder. Mp: 126 °C; IR (KBr) cmÀ1: 3898, 3867,
3805, 3750, 3342, 2837, 2335, 1734, 1518, 1501, 1471, 1323, 437,
418; 1H NMR (300 MHz, CDCl3) d: 7.95 (d, 2H, J = 8.2), 7.61 (d, 1H,
J = 15.9), 7.50 (m, 3H), 6.70 (d, 2H, J = 2.1), 6.60 (d, 1H, J = 15.6),
6.49 (t, 1H, J = 2.1), 6.33 (s, 1H), 3.82 (s, 6H); 13C NMR (75 MHz,
CDCl3) d:189.55, 179.24, 161.14, 140.07, 137.04, 136.31, 132.69,
128.73, 127.45, 123.91, 107.21, 106.03, 102.43, 97.83,55.49; CI-
EIMS: m/z [M+] 309; C19H18O4 (310.34): calcd C 73.53, H 5.85;
found: C 73.55, H 5.88.
5.1.10. (E)-1-(4-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)
pent-4-ene-1,3-dione (3j)
49% as a yellow solid. Mp: 129 °C; IR (KBr) cmÀ1: 3880, 3549,
2847, 2325, 1903, 1683, 1652, 1524, 1348, 1211, 1189, 934, 780,
682; 1H NMR (300 MHz, CDCl3) d: 7.89 (d, 2H, J = 8.5), 7.57 (d,
1H, J = 15.6), 6.89 (d, 2H, J = 8.5) 6.77 (s, 2H), 6.88 (d, 1H, J = 2.1),
6.52 (d, 1H, J = 15.6), 6.27 (s, 1H), 3.90 (s, 6H), 3.88 (s, 3H); 13C
NMR (75 MHz, CDCl3) d: 189.14, 178.12, 160.01, 153.53, 139.89,
139.52, 130.85, 129.87, 129.14, 122.78, 115.63, 105.21, 97.16,
61.10, 56.25; CI-EIMS: m/z [M+] 355; C20H20O6 (356.36): calcd C
67.41, H 5.66; found: C 67.43, H 5.65.
5.1.5. (E)-1-Phenyl-5-(3,4,5-trimethoxyphenyl)pent-4-ene-1,3-
dione (3e)
50% as a yellow powder. Mp: 117 °C; IR (KBr) cmÀ1: 3900, 3868,
3829, 3800, 3768, 3710, 3422, 2839, 2365, 1908, 1715, 1538, 1505,
1455, 1418, 1346, 431, 421, 414; 1H NMR (300 MHz, CDCl3) d: 7.94
(d, 2H, J = 8.2), 7.59 (d, 1H, J = 15.9), 7.50 (m, 3H), 6.79 (s, 2H), 6.55
(d, 1H, J = 15.9), 6.34 (s, 1H), 3.89 (m, 9H); 13C NMR(75 MHz, (CD3)2CO)
d: 189.03, 153.86, 140.43, 140.23, 136.28, 132.78, 130.75, 128.82,
127.33, 122.76, 105.80, 97.28, 59.89, 55.71; CI-EIMS: m/z [M+] 339;
5.1.11. (E)-1,5-Bis(4-hydroxy-3-methoxyphenyl)pent-4-ene-1,3-
dione (3k)
56% as a pale orange solid. Mp: 172 °C; IR (KBr) cmÀ1: 3890,
3831, 3658, 3410, 3387, 3004, 2927, 2843, 2659, 1887, 1715,
1516, 1469, 1310, 1263, 1034, 960, 790, 421; 1H NMR (300 MHz,
(CD3)2CO) d: 8.55 (–OH), 8.20 (–OH), 7.61 (s, 2H), 7.58 (d, 1H,
J = 7.3), 7.31 (d, 1H, J = 1.8), 7.16 (dd, 1H, J1 = 1.8, J2 = 8.2), 6.94 (d,
1H, J = 8.5), 6.88 (d, 1H, J = 8.2), 6.72 (d, 1H, J = 15.9), 6.53 (s, 1H),
3.91 (s, 3H), 3.89 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 189.06,
178.72, 151.42, 149.01, 147.98, 147.70, 139.49, 128.46, 127.53,
122.80, 122.09, 120.68, 115.43, 114.98, 110.54, 110.38, 96.41,
55.55, 55.51; CI-EIMS: m/z [ M2+] 340; C19H18O6 (342.11): calcd C
66.66, H 5.30; found: C 66.68, H 5.33.
C20H20O5 (340.13): calcd C 70.57, H 5.92; found: C 70.58, H 5.95.
5.1.6. (E)-5-(4-Hydroxy-3-methoxyphenyl)-1-(4-hydroxy
phenyl)pent-4-ene-1,3-dione (3f)
40% as a yellow oil purified on silica gel (PE/EtOAc 6:4 as elu-
ant). IR (KBr) cmÀ1: 3799, 3748, 3710, 2923, 2852, 2362, 1866,
1747, 1731, 1567, 1538, 1455, 421, 410; 1H NMR (300 MHz, (CD3)2-
CO) d: 7.94 (d, 2H, J = 8.5), 7.59 (d, 1H, J = 15.6), 7.30 (d, 1H, J = 1.8),

A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
5515
5.1.12. (E)-1-(4-Hydroxy-3-methoxyphenyl)-5-(4-hydroxy
phenyl)pent-4-ene-1,3-dione (3l)
5.1.17. (E)-4-(2-(5-Phenyl-1H-pyrazol-3-yl)vinyl)phenol (4b)
79% as a yellow solid. Mp: 160 °C; IR (KBr) cmÀ1: 3004, 2601,
1897, 1684, 1512, 1235, 1169, 964, 700, 532; 1H NMR (300 MHz,
(CD3)2CO) d: 7.85 (brdd, 2H, J = 7.3), 7.39 (m, 5H), 7.18 (d, 1H,
J = 16.4), 6.99 (d, 1H, J = 16.5), 6.85 (m, 3H); 13C NMR (75 MHz,
(CD3)2CO) d: 157.64, 149.21, 149.95, 132.81, 132.60, 130.11,
128.91, 127.70, 127.26, 125.40, 115.71, 114.46, 99.40 ; CI-EIMS:
m/z [M+] 261; C17H14N2O (262.11): calcd C 77.84, H 5.38, N
10.68; found: C 77.84, H 5.40, N 10.69.
49% as a orange solid. Mp: 124 °C; IR (KBr) cmÀ1: 3780, 3651,
3411, 2999, 2867, 2833, 1890, 1746, 1539, 1475, 1215, 1127,
983, 879, 775, 685; 1H NMR (300 MHz, (CD3)2CO) d: 7.61 (s,
2H), 7.58 (d, 1H, J = 6.7), 7.53 (d, 2H, J = 8.5), 6.94 (d, 1H,
J = 8.5), 6.90 (d, 2H, J = 8.5), 6.67 (d, 1H, J = 15.9), 6.54 (s, 1H),
3.90 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 189.05, 178.75,
159.50, 151.42, 147.70, 139.16, 129.96, 128.46, 127.09, 122.13,
120.48, 116.01, 115.00, 110.40, 96.45, 55.56; CI-EIMS: m/z [
M2+] 310; C18H16O5 (312.10): calcd C 69.22, H 5.16; found: C
69.20, H 5.17.
5.1.18. (E)-3-(4-Methoxystyryl)-5-phenyl-1H-pyrazole (4c)
84% as a pale yellow solid. Mp: 148 °C; IR (KBr) cmÀ1: 3649,
3336, 2943, 2379, 2356, 1987, 1670, 1620, 1416, 832, 700, 551,
533; 1H NMR (300 MHz, CDCl3) d: 7.72 (brdd, 2H, J = 8.2), 7.38
(m, 5H), 7.05 (d, 1H, J = 16.5), 6.88 (m, 3H), 6.71 (m, 1H); 13C
NMR (75 MHz, (CD3)2CO) d: 159.82, 149.23, 146.07, 132.81,
129.71, 128.78, 127.82, 127.74, 125.45, 115.37, 114.28, 99.61,
54.84; CI-EIMS: m/z [M+] 275; C18H16N2O (276.12): calcd C 78.24,
H 5.84, N 10.14; found: C 78.26, H 5.85, N 10.17.
5.1.13. (E)-1-(4-Hydroxy-3-methoxyphenyl)-5-(4-methoxy
phenyl)pent-4-ene-1,3-dione (3m)
45% as a yellow solid. Mp: 157 °C; IR (KBr) cmÀ1: 3780, 3334,
3005, 2967, 2845, 1587, 1643, 1557, 1475, 1310, 1230, 1171,
695; 1H NMR (300 MHz, (CD3)2CO) d: 7.62 (m, 5H), 6.98 (d, 2H,
J = 8.8), 6.93 (d, 1H, J = 8.8), 6.72 (d, 1H, J = 15.9), 6.56 (s, 1H),
3.91 (s, 3H), 3.83 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 189.28,
178.38, 161.46, 151.49, 147.72, 138.69, 129.70, 128.42, 128.06,
122.14, 121.18, 114.97, 114.50, 110.41, 96.57, 55.53, 54.96; CI-
EIMS: m/z [M+] 325; C19H18O5 (326.11): calcd C 69.93, H 5.56;
found: C 69.91, H 5.55.
5.1.19. (E)-3-(3,5-Dimethoxystyryl)-5-phenyl-1H-pyrazole (4d)
77% as an amorphous solid. IR (KBr) cmÀ1: 3587, 3466, 3400,
2965, 2476, 2455, 1740, 1665, 1489, 1402, 1256, 1213, 783, 582,
320; 1H NMR (300 MHz, CDCl3) d: 7.71 (brdd, 2H, J = 7.9), 7.41
(m, 3H), 7.03 (brd, 2H), 6.88 (brs, 1H), 6.63 (brd, 2H), 6.41 (brt,
1H), 3.81 (s, 6H); 13C NMR (75 MHz, (CD3)2CO) d: 161.37, 148.89,
146.23, 139.17, 132.39, 130.16, 128.85, 127.90, 125.49, 118.37,
104.52, 100.18, 100.14, 54.89; CI-EIMS: m/z [M+] 305;
5.1.14. (E)-5-(3,5-Dimethoxyphenyl)-1-(4-hydroxy-3-methoxy
phenyl pent-4-ene-1,3-dione (3n)
47% as a yellow solid. Mp: 141 °C; IR (KBr) cmÀ1: 3890, 3831,
3760, 3288, 2941, 2842, 1635, 1519, 1471, 1438, 1349, 1287,
1204, 1066, 960, 791; 1H NMR (300 MHz, (CD3)2CO) d: 7.57 (m,
3H), 6.94 (d, 1H, J = 7.9), 6.84 (m, 3H), 6.56 (s, 1H), 6.50 (s, 1H),
3.91 (s, 3H), 3.81 (s, 6H); 13C NMR (75 MHz, (CD3)2CO) d: 190.01,
177.15, 161.37, 151.63, 147.72, 138.80, 137.39, 128.45, 124.10,
122.39, 115.01, 110.45, 105.84, 102.10, 97.31, 55.56, 54.97; CI-
EIMS: m/z [M+] 355; C20H20O6 (356.12): calcd C 67.41, H 5.66;
found: C 67.43, H 5.69.
C19H18N2O2 (306.13): calcd C 74.49, H 5.92, N 9.14; found: C
74.48, H 5.90, N 9.15.
5.1.20. (E)-5-Phenyl-3-(3,4,5-trimethoxystyryl)-1H-pyrazole (4e)
75% as a pale yellow solid. Mp: 146 °C; IR (KBr) cmÀ1: 3566,
3487, 3451, 3418, 2838, 2362, 1662, 1506, 1458, 1335, 763, 692,
421; 1H NMR (300 MHz, CDCl3) d: 7.71 (brdd, 2H, J = 7.9), 7.44
(brdd, 2H, J = 7.2), 7.36 (brdd, 1H, J = 7.4), 7.02 (m, 2H), 6.74 (m,
3H), 3.91 (s, 6H), 3.88 (s, 3H); 13C NMR (75 MHz, CDCl3) d:
153.50, 148.75, 147.27, 138.36, 132.39, 131.29, 130.84, 129.00,
128.45, 125.69, 117.29, 103.71, 100.20, 61.06, 56.18; CI-EIMS: m/
z [M+] 335; C20H20N2O3 (336.14): calcd C 71.41, H 5.99, N 8.33;
found: C 71.42, H 5.98, N 8.34.
5.1.15. (E)-1-(4-Hydroxy-3-methoxyphenyl)-5-(3,4,5-tri
methoxyphenyl)pent-4-ene-1,3-dione (3o)
48% as a yellow solid. Mp: 160 °C; IR (KBr) cmÀ1: 3800, 3788,
3309, 3099, 3004, 1975, 1715, 1567, 1473, 1315, 1128, 554; 1H
NMR (300 MHz, (CD3)2CO) d: 7.57 (m, 3H), 6.96 (m, 3H), 6.81 (d,
1H, J = 15.9), 6.53 (s, 1H), 3.90 (s, 3H), 3.86 (s, 6H), 3.77 (s, 3H);
13C NMR (75 MHz, CDCl3) d: 189.47, 177.41, 153.56, 150.28,
146.88, 140.08, 139.34, 130.83, 129.10, 122.65, 122.27, 114.29,
109.69, 105.35, 105.13, 7.17, 61.02, 56.26, 56.15; CI-EIMS: m/z
[M+] 385; C18H16N2O3 (308.11): calcd C 70.12, H 5.23, N 9.09;
found: C 70.09, H 5.17, N 9.07.
5.1.21. (E)-4-(2-(5-(4-Hydroxyphenyl)-1H-pyrazol-3-yl)vinyl)-2-
methoxyphenol (4f)
81% as a pale yellow solid. Mp: 219 °C; IR (KBr) cmÀ1: 3875,
3813, 3710, 2938, 2701, 2363, 1887, 1715, 1519, 1455, 1269,
963, 943, 787; 1H NMR (300 MHz, (CD3)2CO) d: 7.65 (d, 2H,
J = 8.8), 7.19 (d, 1H, J = 1.8), 7.11 (s, 1H), 7.04 (s, 1H), 6.98 (dd,
1H, J1 = 1.8, J2 = 6.3), 6.88 (d, 2H, J = 8.5), 6.82 (d, 1H, J = 8.1), 6.75
(s, 1H), 3.88 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 157.32,
148.47, 147.83, 146.89, 146.65, 129.79, 129.36, 126.74, 124.13,
120.35, 115.68, 115.51, 115.16, 109.04, 98.44, 55.43; CI-EIMS: m/
z [M+] 307; C18H16N2O3 (308.11): calcd C 70.12, H 5.23, N 9.09;
found: C 70.15, H 5.24, N 9.10.
5.1.16. General procedure for the synthesis of pyrazole (4a–o)
To a stirred solution of 3a (1 equiv mol) in AcOH (2.5 mL/
1 mmol.) hydrazine monohydrate (2.3 equiv mol) was added.
The reaction was stirred at 50 °C overnight and quenched with
the addition of brine and extraction with EtOAc, to give 6a as
a yellow solid (80%) without further purification. Mp: 158 °C;
5.1.22. (E)-4-(2-(5-(4-Hydroxyphenyl)-1H-pyrazol-3-yl) vinyl)
phenol (4g)
IR (KBr) cmÀ1
: 3821, 3801, 3711, 2839, 2364, 1898, 1733,
1513, 1460, 1368, 1029, 961, 764, 420; 1H NMR (300 MHz,
(CD3)2CO) d: 7.85 (dd, 2H, J1 = 1.23, J2 = 7.3), 7.41 (t, 2H,
J = 7.6), 7.30 (t, 1H, J = 7.3), 7.21 (d, 1H, J = 2.1), 7.14 (s, 1H),
7.05 (s, 1H), 7.01 (d, 1H, J=), 6.87 (s, 1H), 6.83 (d, 1H, J = 7.9),
3.90 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 147.86, 147.03,
132.89, 130.18, 129.22, 128.69, 128.55, 127.62, 125.33, 120.79,
120.45, 115.20, 114.90, 109.10, 99.35, 55.44; CI-EIMS: m/z [M+]
291; C18H16N2O2 (292.12): calcd C 73.95, H 5.52, N 9.58; found:
C 73.92, H 5.55, N 9.60.
70% as an amorphous solid. IR (KBr) cmÀ1: 3710, 3648, 3417,
2572, 2358, 1867, 1731, 1557, 1455, 1048, 733, 700, 461; 1H
NMR (300 MHz, (CD3)2CO) d: 7.61 (d, 2H, J = 8.5), 7.39 (d, 2H,
J = 8.5), 7.15 (d, 1H, J = 16.5), 7.01 (d, 1H, J = 16.5), 6.88 (d, H,
J = 8.5), 6.81 (d, 2H, J = 8.5), 6.78 (s, 1H); 13C NMR (75 MHz, (CD3)2-
CO) d: 157.52, 157.33, 149.55, 146.98, 129.55, 128.82, 127.81,
126.75, 124.16, 115.65, 115.49, 115.28, 98.45; CI-EIMS: m/z [M+]
277; C17H14N2O2 (278.10): calcd C 73.37, H 5.07, N 10.07; found:
C 73.40, H 5.08, N 10.09.

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A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
5.1.23. (E)-4-(3-(4-Methoxystyryl)-1H-pyrazol-5-yl)phenol (4h)
82% as pale yellow solid. Mp: 187 °C; IR (KBr) cmÀ1: 3833, 3800,
3675, 2987, 2620, 1950, 1716, 1683, 1558, 1530, 1414, 1389, 1151,
1066, 1012, 957, 841, 535; 1H NMR (300 MHz, (CD3)2CO) d: 7.68 (d,
2H, J = 8.5), 7.49 (d, 2H, J = 8.8), 7.09 (q, 2H, J = 16.5), 6.93 (d, H,
J = 8.5), 6.89 (d, 2H, J = 8.5), 6.75 (s, 1H), 3.80 (s, 3H); 13C NMR
(75 MHz, (CD3)2CO) d: 159.73, 157.36, 148.55, 146.57, 142.36,
129.90, 129.17, 128.34, 127.67, 126.77, 124.10, 116.13. 115.53,
(d, 1H, J = 16.4), 6.91 (d, 1H, J = 8.2), 6.90 (d, 2H, J = 8.9), 6.86 (s,
1H), 3.86 (s, 3H), 3.78 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d:
159.74, 148.57, 147.76, 146.66, 145.14, 129.89, 129.20, 127.67,
124.36, 119.92, 118.45, 116.19, 115.35, 115.22, 114.23, 109.00,
98.70, 55.49, 54.79; CI-EIMS: m/z [M+] 321; C19H18N2O3 (322.13):
calcd C 70.79, H 5.63, N 8.69; found: C 70.75, H 5.63, N 8.70.
5.1.29. (E)-4-(3-(3,5-dimethoxystyryl)-1H-pyrazol-5-yl)-2-
methoxyphenol (4n)
114.22, 98.56, 54.79
;
CI-EIMS: m/z [M+] 291; C18H16N2O2
(292.12): calcd C 73.95, H 5.52, N 9.58; found: C 73.93, H 5.49, N
9.55.
82% as a pale yellow solid. Mp: 184 °C; IR (KBr) cmÀ1: 3890,
3801, 3743, 2926, 2835, 2601, 1584, 1415, 1355, 949, 817, 420;
1H NMR (300 MHz, (CD3)2CO) d: 7.41 (brd, 1H), 7.30 (brdd, 1H,
J = 7.9), 7.23 (d, 1H, J = 16.4), 7.15 (d, 1H, J = 16.5), 6.90 (d, 1H,
J = 8.22), 6.87 (s, 1H), 6.71 (brd, 2H), 6.41 (s, 1H), 3.88 (s, 3H),
3.79 (s, 6H); 13C NMR (75 MHz, (CD3)2CO) d: 161.35, 148.21,
147.81, 146.90, 146.76, 139.30, 129.64, 123.93, 119.19, 118.52,
115.31, 109.02, 104.35, 100.02, 99.16, 55.49, 54.83; CI-EIMS: m/z
[M+] 351; C20H20N2O4 (352.14): calcd C 68.17, H 5.72, N 7.95;
found: C 65.20, H 5.70, N 7.96.
5.1.24. (E)-4-(3-(3,5-dimethoxystyryl)-1H-pyrazol-5-yl)phenol
(4i)
78% as a pale yellow solid. Mp: 294 °C; IR (KBr) cmÀ1: 3820,
3800, 3710, 2925, 2840, 2601, 2362, 1907, 1586, 1512, 1457,
1343, 1204, 1151, 1066, 826, 784; 1H NMR (300 MHz, (CD3)2CO)
d: 7.68 (d, 2H, J = 8.5), 7.12 (d, 2H, J = 2.1), 6.89 (d, 2H, J = 8.8),
6.80 (s, 1H), 6.72 (d, 2H, J = 2.1), 6.41 (brt, 1H), 3.80 (s, 6H); 13C
NMR (75 MHz, (CD3)2CO) d: 161.35, 157.42, 148.01, 146.99,
139.33, 129.49, 126.78, 123.99, 119.17, 115.56, 104.32, 99.98,
98.95, 54.82; CI-EIMS: m/z [M+] 321; C19H18N2O3 (322.13): calcd
C 70.79, H 5.63, N 8.69; found: C 70.84, H 5.65, N 8.70.
5.1.30. (E)-2-methoxy-4-(3-(3,4,5-trimethoxystyryl)-1H-
pyrazol-5-yl)phenol (4o)
83% as a pale yellow solid. Mp: 191 °C; IR (KBr) cmÀ1: 3910,
3855, 3821, 3799, 2843, 1598, 1512, 1445, 1434, 1003, 957, 419;
1H NMR (300 MHz, (CD3)2CO) d: 7.46 (d, 1H, J = 1.8), 7.31 (dd, 1H,
J1 = 1.9, J2 = 8.2), 7.15 (s, 2H), 6.90 (d, 1H, J = 7.9), 6.85 (s, 3H),
3.87 (s, 3H), 3.84 (s, 6H), 3.73 (s, 3H); 13C NMR (75 MHz, (CD3)2CO)
d: 154.83, 147.83, 146.78, 132.94, 129.71, 124.09, 118.54, 118.08,
115.29, 109.13, 104.09, 98.91, 59.81, 55.61, 55.49; CI-EIMS: m/z
[M+] 381; C21H22N2O4 (382.15): calcd C 65.96, H 5.80, N 7.33;
found: C 65.98, H 5.79, N 7.35.
5.1.25. (E)-4-(3-(3,4,5-trimethoxystyryl)-1H-pyrazol-5-yl)
phenol (4j)
81% as a pale yellow solid. Mp: 246 °C; IR (KBr) cmÀ1: 3870,
3821, 3765, 3749, 1549, 1533, 1430, 1415, 1394, 1200, 1172,
1151, 892, 450; 1H NMR (300 MHz, CDCl3) d: 7.67 (d, 2H,
J = 8.58), 7.12 (d, 2H, J = 1.1), 6.89 (d, 2H, J = 8.5), 6.86 (s, 2H),
6.77 (s, 1H) 3.86 (s, 6H), 3.73 (s, 3H); 13C NMR (75 MHz, (CD3)2CO)
d: 157.46, 153.79, 148.13, 146.35, 138.46, 132.95, 129.66, 126.78,
123.73, 118.05, 115.60, 103.90, 98.73, 59.79, 55.60; CI-EIMS: m/z
[M+] 351; C20H20N2O4 (352.14): calcd C 68.17, H 5.72, N 7.95;
found: C 68.15, H 5.75, N 7.94.
5.1.31. (E)-2-methoxy-4-(3-(3-methoxystyryl)-1H-pyrazol-5-
yl)phenol (4p)
83% as a brown oil. IR (KBr) cmÀ1: 3981, 3812, 3334, 2758,
2697, 1635, 1601, 1457, 1278, 1003, 988, 565, 461; 1H NMR
(300 MHz, (CD3)2CO) d: 7.49 (d, 1H, J = 1.83), 7.32 (dd, 1H,
J1 = 1.8, J2 = 8.2), 7.26 (d, 1H, J = 7.9), 7.18 (m, 5H), 6.92 (d, 1H,
J = 7.9), 6.85 (brdd, 1H), 3.87 (s, 3H), 3.84 (s, 6H), 3.79 (s, 3H);
13C NMR (75 MHz, (CD3)2CO) d: 172.05, 160.26, 147.91, 146.89,
138.67, 129.88, 129.81, 123.86, 119.04, 118.76, 118.71, 115.46,
113.62, 111.61, 109.16, 55.54, 54.76; CI-EIMS: m/z [M+] 321;
C19H18N2O3 (322.12): calcd C 70.79, H 5.63, N 8.69; found: C
70.81, H 5.64, N 8.66.
5.1.26. (E)-4-(2-(5-(4-hydroxy-3-methoxyphenyl)-1H-pyrazol-3-
yl)vinyl)-2-methoxyphenol (4k)
83% as a yellow solid. Mp: 97 °C; IR (KBr) cmÀ1: 3846, 3458,
3400, 2924, 2853, 2358, 1715, 1515, 1455, 1276, 814, 421; 1H
NMR (300 MHz, CDCl3) d: 7.22 (d, 1H, J = 1.6), 7.18 (dd, 1H,
J1 = 1.8, J2 = 8.1), 6.92 (m, 6H), 6.59 (s, 1H) 3.86 (s, 6H); 13C NMR
(75 MHz, (CD3)2CO) d: 148.46, 148.17, 147.84, 147.75, 146.91,
146.83, 146.63, 129.84, 129.34, 124.35, 120.33, 118.43, 115.74,
115.17, 109.04, 108.97, 98.57, 55.48, 55.43; CI-EIMS: m/z [M+]
337; C19H18N2O4 (338.12): calcd C 67.44, H 5.36, N 8.28; found:
C 67.45, H 5.37, N 8.27.
5.2. Biology
5.2.1. Cell Culture and Cytotoxicity Assay
5.1.27. (E)-4-(3-(4-hydroxystyryl)-1H-pyrazol-5-yl)-2-methoxy
phenol (4l)
The SH-SY5Y human neuroblastoma cell line was obtained from
ATCC (LGC Promochem Teddington, UK) and cultured in 50%
DMEM and 50% F-12 supplemented with 10% foetal bovine serum,
83% as an amorphous solid. IR (KBr) cmÀ1: 3780, 3761, 3317, 2498,
2458, 1961, 1745, 1312, 1168, 993, 567, 490, 461; 1H NMR (300 MHz,
(CD3)2CO) d: 7.46 (d, 1H, J = 2.1), 7.40 (d, 2H, J = 8.5), 7.30 (dd, 1H,
J1 = 1.83, J2 = 7.9), 7.15 (d, 1H, J = 16.5), 6.99 (d, 1H, J = 16.5), 6.88 (d,
1H, J = 7.9), 6.85 (d, 2H, J = 8.5), 6.80 (s, 1H), 3.88 (s, 3H); 13C NMR
(75 MHz, (CD3)2CO) d: 157.61, 148.77, 147.78, 146.81, 146.69,
129.79, 128.75, 127.84, 124.30, 118.52, 115.67, 115.28, 115.21,
108.95, 98.67, 55.45; CI-EIMS: m/z [M+] 307; C18H16N2O3 (308.11):
calcd C 70.12, H 5.23, N 9.09; found: C 70.14, H 5.21, N 9.06.
2 mM l-glutamine, penicillin (100
lg/mL), and streptomycin
(100 g/mL). The MeWo Human melanoma cell line was obtained
l
from ATCC (LGC Promochem Teddington, UK) and cultured in
DMEM supplemented with 10% foetal bovine serum, 2 mM l-gluta-
mine, penicillin (100 lg/mL), and streptomycin (100 lg/mL). For
cytotoxicity assays, cells were plated on 24-well plates and grown
for 48 h in the presence or absence of Curcumin or the synthesized
compounds. On the experimental day, cells were washed twice in
Locke’s solution (134 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 10 mM
HEPES [pH 7.6], 2.3 mM CaCl2, 1 mM MgCl2, 5 mM sucrose) and
5.1.28. (E)-2-methoxy-4-(3-(4-methoxystyryl)-1H-pyrazol-5-yl)
phenol (4m)
incubated for 90 min with MTT (250 lg/mL in Locke’s solution)
74% as a brown solid. Mp: 164 °C; IR (KBr) cmÀ1: 4001, 3765,
3456, 2980, 2748, 2567, 1589, 1503, 1457, 1345, 987, 937, 435;
1H NMR (300 MHz, (CD3)2CO) d: 7.49 (d, 1H, J = 2.1), 7.46 (d, 2H,
J = 8.4), 7.33 (dd, 1H, J1 = 2.1, J2 = 8.2), 7.21 (d, 1H, J = 16.6), 7.03
at 37 °C. Reactions were then stopped and the crystals solubilized
in isopropyl alcohol/HCl before being read at 570 nm in a spectro-
photometer. To determine IC50 values, data were plotted and fitted
using the Kaleidagraph software (Synergy software, Reading, PA).

A. Caldarelli et al. / Bioorg. Med. Chem. 21 (2013) 5510–5517
5517
5.2.2. Immunocytochemistry
the dark. Cell cycle analysis was determined with a FACSVantage
SE DiVa (Becton Dickinson).
MeWo were plated on glass coverslips and grown to subconflu-
ency in the presence or absence of drugs for 24 h. Cover slips were
then fixed in paraformaldehyde (3.7%) for 20 min at RT. Cells were
then washed once in phosphate buffered saline (PBS) and perme-
abilized in ice cold 80% MeOH for 10 min in ice. Cells were then
washed twice in PBS and blocked with 0,2% cold fish gelatine.
Anti-tubulin (mouse) primary antibody (Sigma-Aldrich; 1:1000)
was incubated for 20’ at RT, and after three further washes, cover-
slips were incubated with a goat anti-mouse Alexa fluo 488
(Molecular Probes, 1:2000) and DAPI (Cell Signaling 1:10.000).
Slides were then visualized in a Leica DMI6000B wide field
microscope.
Acknowledgments
Dr. Antonella Vallario is acknowledge for the FACS analysis. A.C.
gratefully acknowledges financial support from Regione Piemonte
(Azione linea B).
References and notes
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5.2.3. Tubulin Polymerization Assay
To measure the degree of tubulin polymerization, we used an
adaptation of the methods described by Minotti et al.15 In brief,
cells were grown in 75 cm2 flasks in the presence or absence of
drugs (3 x IC50) for 16 h. Cells were then tripsinised and centri-
fuged at 600g for 3 min, washed once by PBS at room temperature.
Cells were then re-suspended in 70
Tris-Hcl pH 6.8, 1 mM MgCl2, 2 mM EGTA, protease inhibitors, 0.5%
NP-40) containing 4 g/mL paclitaxel. Lysates were incubated for
10 min at room temperature. Lysates were then corrected for pro-
tein amounts (Bradford assay, Sigma-Aldrich) and 50 g (corre-
sponding to 50 ls) was centrifuged at 13000 rpm for 15 min at
room temperature. Supernatant and pellet were then re-suspended
in equal volumes of SDS-loading buffer and run on 10%
lL of hypotonic buffer (20 mM
l
l
l
a
SDSÀPAGE polyacrylamide gel. After transfer of proteins to nitro-
cellulose (blocked in 5% milk), tubulin was identified with an
anti-tubulin primary antibody (1:1000, Sigma-Aldrich) and anti
mouse secondary antibody peroxidase-conjugated (1:8000, Amer-
sham Bioscience) and visualized by chemiluminescence (Supersig-
nal WestPico, Pierce).
5.2.4. Flow-Cytometric Analysis of Cell-Cycle Status
MeWo grown in the presence or absence of compounds for 16 h
were washed once in PBS and re-suspended in 1 mL of 30:70 ice
cold PBS/EtOH and stored at À20 °C. Cells were then washed twice
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Natl. Acad. Sci. U.S.A. 2001, 98, 3404.
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Petrocellis, L.; Appendino, G. Angew. Chem. Int. Ed. Engl. 2011, 50, 467.
in PBS and re-suspended in PBS containing RNAse (100 lg/mL) for
30’ at r.t. DNA was then stained with a PBS solution containing
5 mM EDTA and 100 g/mL propidium iodide for 30 min at RT in
l