Triazole-curcuminoids: A new class of derivatives for 'tuning' curcumin bioactivities?

Article abstract of DOI:10.1016/j.bmc.2015.11.044

Curcumin is a unique blend of pharmacophores responsible for the pleiotropy of this natural pigment. In the present study we have replaced the 1,3-dicarbonyl moiety with a 1,2,3-triazole ring to furnish a new class of triazole-curcuminoids as a possible strategy to generate new compounds with different potency and selectivity compared to curcumin. We obtained a proof-of-principle library of 28 compounds tested for their cytotoxicity (SY-SY5Y and HeLa cells) and for their ability to inhibit NF-κB. Furthermore, we also generated 1,3-dicarbonyl curcuminoids of selected click compounds. Triazole-curcuminoids lost their ability to be Michael's acceptors, yet maintained some of the features of the parent compounds and disclosed new ones. In particular, we found that some compounds were able to inhibit NF-κB without showing cytotoxicity, while others, unlike curcumin, activated NF-κB signalling. This validates the hypothesis that click libraries can be used to investigate the biological activities of curcumin as well as generate analogs with selected features.

Full text of DOI:10.1016/j.bmc.2015.11.044

Bioorganic & Medicinal Chemistry 24 (2016) 140–152
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Triazole-curcuminoids: A new class of derivatives for ‘tuning’
curcumin bioactivities?
Diego Caprioglio y, Simone Torretta y, Maila Ferrari, Cristina Travelli, Ambra A. Grolla, Fabrizio Condorelli,
⇑
⇑
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:
Curcumin is a unique blend of pharmacophores responsible for the pleiotropy of this natural pigment. In
the present study we have replaced the 1,3-dicarbonyl moiety with a 1,2,3-triazole ring to furnish a new
class of triazole-curcuminoids as a possible strategy to generate new compounds with different potency
and selectivity compared to curcumin. We obtained a proof-of-principle library of 28 compounds tested
Received 9 October 2015
Revised 27 November 2015
Accepted 28 November 2015
Available online 30 November 2015
for their cytotoxicity (SY-SY5Y and HeLa cells) and for their ability to inhibit NF-jB. Furthermore, we also
generated 1,3-dicarbonyl curcuminoids of selected click compounds. Triazole-curcuminoids lost their
ability to be Michael’s acceptors, yet maintained some of the features of the parent compounds and
Keywords:
Curcumin
Curcuminoids
Click chemistry
1,3-Dipolar cycloaddition
disclosed new ones. In particular, we found that some compounds were able to inhibit NF-
jB without
showing cytotoxicity, while others, unlike curcumin, activated NF- B signalling. This validates the
j
hypothesis that click libraries can be used to investigate the biological activities of curcumin as well as
generate analogs with selected features.
NF-jB
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
pleiotropic actions make curcumin an interesting scaffold from a
medicinal chemistry point of view.8 On the other hand, the lack
Natural pigments are widely used in the food and textile indus-
try, and besides their chromatic features some of them, like flavo-
noids and carotenoids, have also important biological properties.
Curcuma longa (turmeric) is a perennial plant of the ginger family
and its deep yellow powder is commonly used for flavouring food
in the Southeast Asian and Middle Eastern countries. In India it has
been used for thousand years as a spice for flavouring curry, as a
dye for the holy robes in Hindu religion and it still represents a
common remedy in Ayurvedic medicine for its anti-inflammatory
and antibacterial activities.1 Turmeric owes its colour to a group
of pigments all belonging to the diarylheptanoid family extracted
from the roots. The major constituent of this group of compounds,
called curcuminoids, is curcumin (1) that is also the active phyto-
chemical of turmeric. In the last decades curcumin has emerged as
an important scientific topic for its pleiotropy due to its ability to
of specificity and the low potency of most of its actions call for
analogs to be synthetized with increased potency and higher
specificity. For this to be achieved, enough analogs should be
synthesized to gather a good insight on the structure–activity rela-
tionships for each action.
The structure of curcumin has indeed been widely explored by
different SAR studies.9 In a number of these, the 1,3-dicarbonyl
system has been replaced with success with different cycles or
heterocycles;10–14 in particular the pyrazole derivative of curcumin
has received attention due to its improved potency in different
biological assays compared to the natural dye.15,16
In the present contribution, we decided to proceed with a proof
of principle library to investigate whether the copper-catalyzed
Huigsen cycloaddition reaction could be applied to this end. The
copper-catalyzed 1,3-dipolar cycloaddition of an azide and an
alkyne is an increasingly popular strategy to connect different
structural units through the introduction of a stable permanent
link,17–21 represents a new, powerful tool in the drug discovery
process,22–27 and is the archeotype of click chemistry reactions. It
should be noticed that we have previously reported C5-curcumin
analogs synthetized via the Pabon reaction, which also may be con-
sidered a click reaction.28 Curcumin is a symmetric feruloyl dimer,
and most of the SAR studies carried out on this natural pigment
act directly on more than 100 molecular targets2 (e.g., NF-
j
B,3
Nrf2,4 STAT-3,5 PPAR- 6). Furthermore, from a medicinal chemistry
c
standpoint, the pharmacophore of curcumin is a unique blend:
Michael acceptor, metal chelating, and antioxidant.7 These
⇑
Corresponding authors.
E-mail addresses: armando.genazzani@uniupo.it (A.A. Genazzani), alberto.
minassi@uniupo.it (A. Minassi).
y
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.11.044
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
141
have focused on symmetric analogs,9 despite the increased
potency, in several anti-inflammatory and biochemical assays, of
monodemethoxycurcumin29 (1a). Furthermore, it should be noted
that many tubulin inhibiting compounds have a heterodimeric
structure (Vinca alkaloids, combretastatins and podophyllotoxin).
We therefore postulated that the insertion of a 1,2,3-triazole ring
in place of the 1,3-dicarbonyl moiety, provided us with easy access
to asymmetric derivatives with different aryl substitution patterns,
extending the structure–activity relationship of curcuminoids on
different biological targets. We now report that this strategy would
be amenable to be exploited to generate a large number of analogs
with just selected properties of curcumin.
For the synthesis of the b-azidostyrene moiety we tried differ-
ent methodologies found in the literature,30–32 but we were not
able to reproduce the same results and all efforts to obtain the
vinylazide from ferulic acid failed. As a second approach to obtain
the azide synthon we decided to start from styrene 7A, derived
from O-pivaloyl-vanilline 6A after Wittig olefination, that
undergoes an azoiodination reaction to give the corresponding
2-azido-1-iodoethyl derivative 8A. The latter was obtained in high
yield and with high regioselectivity by using a modified version of
the azoiodination reaction of styrenes published by Sudalai, in
which CH2Cl2 was used as solvent in place of AcOH.33 By using
AcOH as a solvent we obtained a 1:1 mixture of our product and
1-acetyl-2-iodoethyl derivative, probably due to the ability of
AcOH to stabilize the benzylic carbocation and to trap it. By reflux-
ing compound 8A in CH2Cl2 in presence of TEA, we obtained the
protected b-azidostyrene 9A (Scheme 2).
The enyne moiety was obtained starting from O-silylated ferulic
acid 10a using the Hunsdiecker34–38 reaction with triethylammo-
nium bromide (TEAB) and oxone in CH2Cl2 to give the correspond-
ing vinyl bromide 11a that reacting with (trimethylsilyl)-acetylene
under Sonogashira conditions, afforded the bis-protected vanillic-
enyne 12a in good yield (Scheme 3).
The azide 9A and the alkyne 12a were then reacted under the
classical copper-catalyzed [3+2] azide-alkyne cycloaddition to give
click-curcumin 2Aa after complete deprotection with KOH/MeOH
(Scheme 4).
Last, given that a number of triazole-curcuminoids were close
analogs of already reported curcuminoids, we compared the activity
between selected triazole-curcuminoids and the corresponding
1,3-dicarbonyl analogs to evaluate whether the triazole ring on this
scaffold confers novel properties or is only an experimental strategy
to yield libraries in an efficient manner.
2. Results
2.1. Chemistry
In the present study the 1,3-dicarbonyl moiety of curcumin (1)
has been replaced by a 1,2,3-triazole ring to give triazole-curcumin
(2Aa) as the archetypal example of a new class of triazole-curcum-
inoids, by using the Huisgen [3+2] cycloaddition catalyzed by
copper (I) salts. From a first retrosynthetic analysis we identified
ferulic acid (3) as a possible starting material for the synthesis of
both b-azidostyrene 4 and vanillic-enyne 5 (Scheme 1).
Scheme 2. Synthesis of the protected b-azidostyrene 9A.
Scheme 1. Retrosynthetic analysis.
Scheme 3. Synthesis of the bis-protected vanillic-enyne 12a.

142
D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
In analogy to what described above, 4 commercially available
lines, SY-SY5Y and HeLa cells. These cell lines were chosen given
their wide use in the screening of anti-tumoural agents and given
the experience that lab has previously gained on them. As it can be
observed from Table 1, high concentrations of 1 induced an
antiproliferative/cytotoxic effect in both cell lines. The effect on
the two cell lines was comparable. We also proceeded with a
concentration–response curve and the apparent IC50s were
aldehydes were converted to the respective vinylazides (9A–D)
and 8 commercially available cinnamic acids were converted to
the respective enyne moieties (12a–h). Huigsens cycloaddition
then yielded a small library of 28 new triazole-curcuminoids
(2Aa–2Dg) (Table 1).
In addition we synthetized the compounds 13,39 14,39 15,40–42 16,41
as parent compounds of 2Ag, 2Ah, 2Cg and 2Dg derivatives.
16.1 + 0.7 lM and 21.8 + 1.2 lM in SH-SY5Y and HeLa cells,
2.2. Biological results
respectively. This is comparable to IC50s published in the literature
on these cell lines.28,46 For the screening of the synthesized com-
The insertion of a 1,2,3-triazole ring in place of the 1,3-dicar-
bonyl moiety should affect both the conjugation and the reactivity
of curcumin. We therefore first tested this via the thiol trapping
assay.43,44 As expected, triazole-curcuminoids were unreactive,
while 1 and the curcuminoids synthesized were yielded the
respective Michael adducts in the presence of 4 equiv of cysteam-
mine in DMSO (see Suppl. Fig. 1). Therefore, as expected, we can
conclude that triazole-curcuminoids, unlike the parent com-
pounds, are not Michael acceptors.
We then evaluated whether triazole-curcuminoids have an
effect on cell viability, as it has been described that curcumin
and its analogs may have cytotoxic activity that might be exploited
to generate anti-tumoural agents.45 To investigate this aspect, we
studied the antiproliferative actions of our compounds on two cells
pounds, we first proceeded to test a concentration of 40 lM. As it
can be observed in Table 1, at this concentration most of the
compounds showed a modest effect. We therefore proceeded to
select those compounds yielding a reduction in viability of at least
60% in SH-SY5Y cells (2Aa, 2Ag, 2Ah, 2Ce, 2Cd, 2Cg, 2Df) and per-
formed a full concentration–response curve on these compounds
on both HeLa and SH-SY5Y. As it can be observed, most compounds
had a good IC50 correlation between cell lines (i.e., did not display
any selectivity) and one compound (2Cg) showed an increased
potency by one order of magnitude compared to 1 in both cell lines
(Fig. 1).
The mechanism of action of the antiproliferative effect of cur-
cumin is at present not well understood. A possible mechanism
that has been postulated is the inhibition of tubulin polymerization.47

D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
143
normalized over renilla luciferase reporter gene driven by an thy-
midine kinase promoter.
When used alone, no compound, including 1, was able to signif-
icantly modify NF-
cells transfected with the relevant reporter gene (data not shown).
We then tested the effect of the compounds on TNF -induced
NF- B-dependent transcription. Treatment for 6 h with TNF
induced a 10-fold increase of transcription. Compound 1, at a
concentration of 30 M, reduced TNF -induced NF B-dependent
jB-dependent transcription over basal in HeLa
a
j
a
l
a
j
transcription significantly (Table 1; 60.5 4.1% of control). Five
compounds (2Aa, 2Ag, 2Ah, 2Cg, 2Dg) were more potent than 1
(Table 1), as the same concentration induced a greater reduction
of TNF
six compounds (2Ab, 2Ad, 2Ba, 2Bf, 2Cd, 2Cf) were able to poten-
tiate the effect of TNF . Most other compounds were able to reduce
a-induced NF-jB-dependent transcription. To our surprise,
a
transcription but to a lower extent compared to 1.
We then proceeded to explore the concentration–response
curves of selected compounds that appeared more promising in
terms of inhibition or activation. We also evaluated in a compara-
tive the respective curcuminoids (13–16). As shown in Figure 3,
generally triazole-curcuminoids and curcuminoids displayed simi-
lar efficacy, with some exceptions. First, 2Ah was more potent than
the parent curcuminoid 14 and curcumin. Second, triazole-
curcuminoids displayed a biphasic response, which was more
Scheme 4. Synthesis of click-curcumin 2Aa.
To test this hypothesis, we used a commercial fluorescent kit and
investigated the effect on tubulin polymerization in vitro of 1
and on five selected compounds (2Aa, 2Ag, 2Ah, 2Cg, 2Dg). Selec-
tion was based on potency in cytotoxicity or a significant effect on
evident in 2Aa, which at a concentration of 3
lM potentiated
by two-fold TNF -induced NF- B-dependent transcription. The
shape of these curves hampered a correct estimation of the IC50
values.
a
j
the NF-
Compound 1 had no effect on polymerization at the lowest concen-
tration tested (10 M) while it reduced slightly polymerization at
higher concentrations (up to 60 M). 2Aa and 2Ah had no effect
jB assay (see below). Data are presented in Suppl. Figure 3.
l
l
on tubulin polymerization, while 2Ag, 2Cg and 2Dg, at the highest
concentration tested, increased polymerization. We also tested
compounds 13–16, the parent curcuminoids. The four compounds
reduced slightly polymerization. The minute effects observed at
concentrations similar to the IC50 values raise the possibility that
tubulin is not the primary target of these compounds, as com-
pounds that have this mechanism of action (e.g., combretastatin
A4) display a significantly more pronounced effect.48 To confirm
the suspicion that tubulin was not the primary target, we also per-
formed a cellular assay on tubulin polymerization in which cells
are treated with the selected compounds for 16 h, proteins are then
extracted in the presence of paclitaxel and the resultant pelletable
and soluble fractions are run on an SDS–PAGE gel. Again, curcumin
and the triazole-curcuminoids had no effect, while combretastatin
A4 significantly increased the soluble fraction (data not shown).
Last, qualitative analysis of cells stained in immunocytochemistry
with an anti-tubulin antibody did not display abnormal features.
This is in line with other reports.49 To further confirm that the
effect observed in the first screening was not due to an effect on
tubulin, we decided to investigate the effect of these compounds
on the cell cycle, as it is well known that agents that act on tubulin
lead to a G2/M arrest. Compounds were tested at the IC50 value, at
twice the IC50 value and at three times the IC50. All the compounds
tested, including 1, had no effect on the cell cycle after 8 h of
treatment. On the other hand, 16 h treatment led to an increase
of the G1 phase (see Fig. 2). While this effect came unexpected, it
has been previously reported50,51 that in HeLa cells curcumin leads
to a G1 arrest that is then followed by apoptotic cell death. This
seems to be re-conducted to an effect on cyclin D152 and might
or might not be correlated with the anti-proliferative effects
observed.
3. Discussion and conclusions
The scope of the present contribution was to investigate,
through a proof-of-principle protocol, whether click chemistry
could be amenable to (i) generate a large number of analogs of 1
(ii) to generate potent and specific compounds. To this end 28 ana-
logs of 1 were generated that bore a 1,2,3-triazole ring in place of
the 1,3-dicarbonyl moiety. Furthermore, 4 corresponding curcum-
inoids were also generated to explore differences with the parent
triazole-curcuminoids.
Three parameters were arbitrarily chosen in the present study.
When evaluating the ability to act as Michael’s acceptors, triazole-
curcuminoids obviously lost the effect.
When evaluating cytotoxicity and TNFa-induced NF-jB-depen-
dent transcription, the true analog of 1 (2Aa) displayed similar
properties compared to the parent compound, strongly suggesting
that this replacement can be adopted for future screenings. On the
other hand, the triazole replacement also unmasked an activating
property at lower concentrations of TNF
dent transcription.
While the dataset is probably too small to generate meaningful
structure–activity relationships, we were able to generate com-
pounds which were an order of magnitude more potent than 1 in
inducing cytotoxicity (with an apparently similar mechanism of
action; 2Cg) and compounds that were an order of magnitude
a-induced NF-jB-depen-
more potent in inhibiting TNF
scription (2Ah). More importantly, 2Dg was equipotent to 1 in
NF- B inhibition but was devoid of a cytotoxic effect, leading to
a-induced NF-jB-dependent tran-
j
the hypothesis that the plethora of actions of 1 can indeed be
dissected.
In conclusion, we now show that a 1,2,3-triazole can indeed be
a good bioisostere of 1,3-dicarbonyl moiety, at least in the context
of 1, and that a click chemistry library can be used to find com-
pounds that retain some features of 1 while losing others as well
as generate large datasets to further explore the SAR of curcumin
in the future.
Having gathered data on cytotoxicity, we decided to evaluate
another feature of curcumin: its ability to inhibit NF-j
B.53 This
effect is probably linked to its anti-inflammatory properties. To
this end, we used a firefly luciferase reporter gene to investigate
the effect of curcumin on NF-jB-dependent transcription and we

144
D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
Table 1
Synopsis of the triazole-curcuminoids synthetized and their biological activity
Compounds
Viability SH-SY5Y
(% of ctrl)
IC50
(l
M) Viability HeLa
IC50
(lM)
NF
TNF
j
a
B-dependent transcription (% of
-induced transcription)
(% of ctrl)
1
22.6 1.2***
36.1 1.0***
86.7 5.3
16.1 0.7 42.5 0.1***
21.8 1.2
36.4 7.2
n.d.
60.5 4.1***
48.0 8.3***
144.5 12.5**
73.2 17.6
2Aa
2Ab
2Ac
2Ad
2Ae
2Ag
2Ah
2Ba
17.8 7.2 50.5 1.3***
n.d.
84.3 4.4
50.3 5.9***
50.0 3.6***
65.3 1.8***
23.5 3.3***
18.1 1.2***
109.9 3.0
n.d.
83.5 2.0***
67.9 3.0**
38.7 0.6***
23.0 1.1***
7.9 0.3***
79.5 1.8***
n.d.
n.d.
n.d.
145.9 5.1*
n.d.
n.d.
88.9 5.8
7.8 1.7
6.9 1.4
n.d.
13.6 1.7
11.7 1.3
n.d.
27.5 0.7***
15.3 1.4***
137.6 13.6**
2Bb
2Bc
117.8 4.2
70.6 1.4*
n.d.
n.d.
70.0 1.9***
n.d.
n.d.
79.9 11.6
85.4 10.1
39.5 1.1***
2Bd
125.5 7.9
n.d.
69.2 1.4***
n.d.
74.5 3.7*

D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
145
Table 1 (continued)
Compounds
Viability SH-SY5Y
(% of ctrl)
IC50
n.d.
(l
M) Viability HeLa
IC50
n.d.
(lM)
NF
TNF
j
a
B-dependent transcription (% of
-induced transcription)
(% of ctrl)
2Be
2Bf
2Bg
88.7 7.1
93.9 6.2*
87.0 1.0
166.8 7.8***
65.3 4.4**
40.1 2.4***
n.d.
n.d.
18.4 1.2***
n.d.
n.d.
69.2 5.1*
52.1 1.5***
2Ca
55.4 2.7***
n.d.
56.8 0.9***
n.d.
96.0 9.0
2Cb
2Cc
48.0 1.9***
n.d.
n.d.
50.7 1.2***
n.d.
n.d.
88.9 3.6
49.6 2.1***
55.3 4.8***
111.6 7.9
2Cd
2Ce
25.7 0.6***
11.3 1.5 66.3 2.3***
34 7.6
131.1 4.5**
37.9 3.0***
9.0 8.6
30.4 0.8***
60.1 4.8***
19.2 1.7***
17.2 7.2
61.5 3.2**
2Cf
60.4 6.4***
n.d.
n.d.
124.2 1.4*
2Cg
21.3 2.0***
1.7 0.2
1.5 0.3
14.6 2.2***
2Da
2Db
66.3 5.9**
n.d.
n.d.
66.9 2.7***
n.d.
n.d.
57.0 1.8**
107.3 13.9
82.1 1.0*
76.1 7.9*
(continued on next page)

146
D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
Table 1 (continued)
Compounds
Viability SH-SY5Y
(% of ctrl)
IC50
n.d.
(
l
M) Viability HeLa
IC50
n.d.
(
l
M)
NF
TNF
j
a
B-dependent transcription (% of
-induced transcription)
(% of ctrl)
2Dc
2Dd
2De
2Df
2Dg
68.9 1.3*
52.0 2.6***
71.4 7.3**
27.7 2.6***
99.9 8.8
41.9 2.2***
55.1 1.9***
92.8 0.5
94.3 6.4
n.d.
n.d.
n.d.
n.d.
81.3 10.0
71.0 2.2**
97.0 12.1
42.2 6.5***
17 2.0
16.6 0.5***
18.7 4.99
n.d.
77.9 3.1*
n.d.
Viability values represent mean S.E.M. of n = 8 in two separate experiments. Compounds were all tested at a fixed concentration of 40
lM. IC50s for viability were calculated
from 6-point curves (see Fig. 1 for details). Single-point effects at a fixed concentration of 30
separate experiments. n.d. not determined.
lM on NF-jB-dependent transcription represent mean S.E.M. of n = 6 in two
*
p <0.5.
p <0.01.
**
***
p <0.001.
1H NMR (300 MHz, CDCl3) d: 6.98 (d, J = 1.7 Hz, 1H), 6.96 (d,
J = 1.6 Hz, 1H), 6.95 (s, 1H), 6.67 (dd, J1 = 10.9; J2 = 17.5 Hz, 1H),
5.68 (dd, J1 = 0.8; J2 = 17.5 Hz, 1H), 5.23 (dd, J1 = 0.8; J2 = 10.8 Hz,
1H), 3.82 (s, 3H), 1.35 (s, 9H).
4. Experimental section
4.1. Chemistry
1H (300 MHz) and 13C (75 MHz) NMR spectra were measured on
4.1.1.2.
(8A).
4-(2-Azido-1-iodoethyl)-2-methoxyphenyl
To a stirred suspension of sodium azide (3 equiv mol)
pivalate
a JEOL Eclipse 300 instrument. Chemical shifts were referenced to
the residual solvent signal (CDCl3: dH = 7.26, dC = 77.1, CD3OD:
dH = 3.34, dC = 49.0, DMSO: dH = 2.50, dC = 39.5, (CD3)2CO:
dH = 2.05, dC = 206.7, 29.9). Low resolution ESIMS were obtained
on a LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. IR:
Shimadzu DR 8001 spectrophotometer. Silica gel 60 (70–230
mesh) and RP-18 used for gravity column chromatography was
purchased from Merck. 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 before evaporation.
in dry CH2Cl2, KI (1 equiv mol), NaIO4 (1 equiv mol) and acetic acid
(6 equiv mol) were sequentially added. After 10 min. 7A was
added, and the reaction was stirred at room temperature over-
night. The reaction was quenched with NaHCO3 satd sol and
extracted with CH2Cl2. The organic layers were washed with
Na2SO3 satd sol, dried over Na2SO4, filtered and evaporated. The
residue was purified by GCC on silica gel (PE/EtOAc 9:1 as eluant)
to afford 8A as pure compound (80%). 1H NMR (300 MHz, CDCl3) d:
6.98 (br d, 2H), 6.96 (d, J = 1.2 Hz, 1H), 5.11 (t, J = 7.95 Hz, 1H), 3.87
(d, J = 7.89 Hz, 1H), 3.80 (s, 3H), 1.35 (s, 9H).
4.1.1. General procedure for the synthesis of b-azidostyrenes
(9A–D)
4.1.1.3.
(9A).
(E)-4-(2-Azidovinyl)-2-methoxyphenyl
To a stirred solution of 8A (1 equiv mol) in dry CH2Cl2,
pivalate
4.1.1.1. 2-Methoxy-4-vinylphenyl pivalate (7A).
To a cooled
TEA (5 equiv mol) was added and the reaction was heated at
50 °C for 3 h. The reaction is quenched with H2SO4 2 N and
extracted with CH2Cl2. The organic layers were dried over Na2SO4,
filtered and evaporated. The residue was purified by GCC on silica
gel (PE/EtOAc 95:5 as eluant) to afford 9A as pure compound (85%).
1H NMR (300 MHz, CDCl3) d: 6.92 (d, J = 8.1 Hz, 1H), 6.86 (d,
J = 2.3 Hz, 1H), 6.82 (t, J = 1.6 Hz, 1H), 6.56 (d, J = 13.8 Hz, 1H),
6.23 (d, J = 13.8 Hz, 1H), 3.80 (s, 3H), 1.35 (s, 9H).
solution (0 °C) of methyltriphenylphosphonium bromide
(1.2 equiv mol) in dry THF, NaH 60% (6 equiv mol) was slowly
added. After 15 min. O-Pivaloyl-vanilline (6A) (1 equiv mol) was
added and the reaction is stirred at room temperature overnight.
The reaction is quenched with H2SO4 2 N and extracted with
EtOAc. The organic layers were dried over Na2SO4, filtered and
evaporated. The residue was purified by GCC on silica gel
(PE/EtOAc 95:5 as eluant) to afford 7A as pure compound (83%).

D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
147
6.02 (d, J = 16.2 Hz, 1H), 3.79 (s, 3H), 0.99 (s, 9H), 0.20 (s, 3H),
0.15 (s, 3H).
4.1.3. General procedure for the synthesis of triazole-
curcuminoids (2Aa–2Dg)
4.1.3.1. Triazole-curcumin (2Aa).
To a suspension of 12A
(1 equiv mol) and 9a (1 equiv mol) in H2O/t-ButOH (1:1), sodium
ascorbate (0.1 equiv) of freshly prepared 1 M solution in water
was added, followed by the addition of copper (II) sulfate pentahy-
drate (0.05 equiv). The solution is stirred at room temperature
overnight. The reaction is quenched with H2SO4 2 N and extracted
with EtOAc. The organic layers were dried over Na2SO4, filtered and
evaporated. The residue was purified by GCC on silica gel
(PE/EtOAc 8:2 as eluant) to afford bis-protected click curcumin.
The residue was dissolved in MeOH and KOH 5% sol in MeOH
(350 lL of solution for 100 mg of product) was added. The reaction
was stirred at room temperature for 1.5 h, quenched with H2SO4
2 N and extracted with EtOAc. The organic layers were dried over
Na2SO4, filtered and evaporated. The residue was purified by GCC
on silica gel (PE/EtOAc 5:5 as eluant) to afford 2Aa as pale brown
solid (60% over two steps). mp: 195 °C; IR (KBr) cmꢀ1: 3334,
3102, 2921, 2876, 1724, 1706, 1623, 1575, 1436, 1285, 1180,
1062, 1034, 935, 659, 512; 1H NMR (300 MHz, (CD3)2CO) d: 8.36
(s, 1H), 7.92 (d, J = 14.7 Hz, 1H), 7.32 (d, J = 12.1 Hz, 1H), 7.30 (d,
J = 3.9 Hz, 1H), 7.27 (d, J = 12.5 Hz, 1H), 7.23 (d, J = 1.9 Hz, 1H),
7.07 (s, 1H), 7.06 (d, J = 6 Hz, 1H), 7.02 (dd, J1 = 10.1 Hz
J2 = 1.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H),
3.918 (s, 3H), 3.918 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d:
148.0, 147.8, 147.5, 147.0, 146.5, 130.8, 129.1, 126.1, 121.3,
121.0, 120.5, 115.3, 115.2, 114.2, 109.3, 109.1, 55.5, 55.4; CI-EIMS:
m/z [M+Na]+ 388; C20H19N3O4 (365.14): calcd C, 65.74; H, 5.24; N,
11.50. Found: C, 65.60; H, 5.55; N, 11.46.
Figure 1. Effect of selected compounds on cytotoxicity/proliferation. (A) Concen-
tration–response curves in HeLA (left) or SH-SY5Y (right) cells treated with the
indicated compounds for 48 h. n = 12–16 in 3–4 separate experiments.
4.1.2. General procedure for the synthesis of enyne derivatives
(12a–h)
4.1.2.1. (E)-(4-(2-Bromovinyl)-2-methoxyphenoxy)(tert-butyl)
dimethylsilane (11a).
To a stirred solution of O-silylated
ferulic acid (10a) (1 equiv mol) in dry CH2Cl2, triethylammonium
bromide (TEAB) (1.2 equiv mol) and oxone (1.2 equiv mol) were
sequentially added. The reaction is stirred for 4 h at room temper-
ature, quenched with Na2SO3 and washed with CH2Cl2. The organic
layers were washed with NaHCO3 satd sol, dried over Na2SO4,
filtered and evaporated. The residue was purified by GCC on silica
gel (PE/EtOAc 95:5 as eluant) to afford 11a as pure compound
(73%). 1H NMR (300 MHz, CDCl3) d: 7.01 (d, J = 13.7 Hz, 1H), 6.77
(br s, 3H), 6.60 (d, J = 13.7 Hz, 1H), 3.80 (s, 3H), 0.98 (s, 9H), 0.20
(s, 3H), 0.15 (s, 6H).
4.1.2.2. (E)-tert-Butyl(2-methoxy-4-(4-(trimethylsilyl)but-1-en-
3-yn-1-yl)phenoxy)dimethylsilane (12a).
To a degassed
solution of 11a in dry toluene, CuI (3 mol %), Pd(TPP)2Cl2 (3 mol
%), TEA (2 equiv mol) and ethynyltrimethylsilane (2 equiv mol)
were sequentially added. The reaction was heated in a Schlenk
tube at 90 °C overnight. The reaction is quenched with H2SO4 2 N
and extracted with EtOAc. The organic layers were dried over
Na2SO4, filtered and evaporated. The residue was purified by GCC
on silica gel (PE/EtOAc 95:5 as eluant) to afford 9a as pure com-
pound (92%). 1H NMR (300 MHz, CDCl3) d: 6.92 (d, J = 16.2 Hz,
1H), 6.85 (s, 1H), 6.84 (d, J = 6.8 Hz, 1H), 6.78 (d, J = 6.7 Hz, 1H),
Figure 2. Effect of selected compounds on the cell cycle. Pooled analysis of the
effects of the indicated compounds on HeLA cells. n = 6 from
3 separate
experiments.

148
D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
Figure 3. Concentration response curves of the indicated compounds on NF-
jB activation/inhibition in the presence of 20 ng/mL TNFa for 6 h. n = 6–11 from 3 to 6 different
experiments. Each panel contains curcumin/curcuminoids and the respective triazole-curcuminoid.
4.1.3.2. 2-Methoxy-4-((E)-2-(4-((E)-styryl)-1H-1,2,3-triazol-1-yl)
(335.13): calcd C, 68.05; H, 5.11; N, 12.53. Found: C, 68.10; H,
5.09; N, 12.50.
vinyl) phenol (2Ab).
(65% over the last two steps) pale brow
solid mp: 163 °C; IR (KBr) cmꢀ1: 3334, 3102, 2921, 2876, 1724,
1706, 1623, 1575, 1436, 1285, 1180, 1062, 1034, 935, 659, 512;
1H NMR (300 MHz, (CD3)2CO) d: 8.43 (s, 1H), 7.94 (d, J = 14.7 Hz,
4.1.3.4. 2-Methoxy-4-((E)-2-(4-((E)-4-methoxystyryl)-1H-1,2,3-
triazol-1-yl)vinyl)phenol (2Ad).
(62% over the last two steps)
1H), 7.57 (d, J = 7.3 Hz, 2H), 7.18–7.44 (m,
7
H), 7.06 (dd,
brow solid mp: 167 °C; IR (KBr) cmꢀ1: 3539, 3097, 3066, 2936,
2841, 1604, 1509, 1244, 1175, 1025, 809, 569; 1H NMR
(300 MHz, (CD3)2CO) d: 8.37 (s, 1H), 7.93 (d, J = 14.7 Hz, 1H), 7.51
(d, J = 8.5 Hz, 2H), 7.35–7.25 (m, 3H), 7.07 (s, 1H), 7.03 (d,
J = 5.5 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 7.95 Hz, 1H),
3.91 (s, 3H), 3.80 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 159.8,
148.0, 147.5, 146.3, 130.1, 129.6, 127.8, 126.1, 121.4, 121.3,
121.0, 117.8, 115.3, 114.7, 114.2, 109.3, 55.5, 54.8; CI-EIMS: m/z
[M+Na]+ 372; C20H19N3O3 (349.14): calcd C, 68.75; H, 5.48; N,
12.03. Found: C, 68.84; H, 5.39; N, 12.05.
J1 = 8.2 Hz J2 = 1.8 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 3.91 (s, 3H);
13C NMR (75 MHz, (CD3)2CO) d: 148.0, 147.5, 146.0, 137.0, 130.4,
128.8, 127.9, 126.5, 126.0, 121.6, 121.3, 121.1, 118.4, 117.0,
115.3, 109.4, 55.5; CI-EIMS: m/z [M+Na]+ 342. C19H17N3O2
(319.13): calcd C, 71.46; H, 5.37; N, 13.16. Found: C, 71.43; H,
5.32; N, 13.46.
4.1.3.3. 4-((E)-2-(4-((E)-4-Hydroxystyryl)-1H-1,2,3-triazol-1-yl)
vinyl)-2-methoxyphenol (2Ac).
(55% over the last two steps)
pale yellow solid mp: 218 °C; IR (KBr) cmꢀ1: 3365, 3143, 2952,
2801, 1744, 1723, 1623, 1514, 1453, 1418, 1257, 1180, 935, 769,
699, 524; 1H NMR (300 MHz, (CD3)2CO) d: 8.37 (s, 1H), 7.93 (d,
J = 14.6 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.36–7.25 (m, 3H), 7.04
(s, 1H), 7.03 (d, J = 5.5 Hz, 1H), 6.86 (d, J = 8.3 Hz, 3H) 3.88 (s,
3H); 13C NMR (75 MHz, (CD3)2CO) d: 157.6, 148.0, 147.5, 146.5,
130.5, 128.6, 128.0, 127.9, 126.1, 121.3, 121.0, 115.7, 115.6,
115.3, 113.9, 109.3, 55.5.; CI-EIMS: m/z [M+Na]+ 358; C19H17N3O3
4.1.3.5. 2-Methoxy-4-((1E)-2-(4-(3-methoxystyryl)-1H-1,2,3-tri-
azol-1-yl)vinyl)phenol (2AE) (mixture of isomers trans/cis
2:1).
(60% over the last two steps) yellow solid mp: 143 °C;
IR (KBr) cmꢀ1: 3547, 3102, 3035, 3012, 2871, 1656, 1577, 1454,
1275, 1153, 1119, 858, 490; 1H NMR (300 MHz, (CD3)2CO) d: 8.51
(s, 1H), 8.43 (s, 1H), 7.95 (d, J = 14.6 Hz, 1H), 7.94 (d, J = 14.6 Hz,
1H), 7.69 (d, J = 16.5 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.43–7.05

D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
149
(m), 6.86 (d, J = 8.2 Hz, 3H), 3.91 (s), 3.86 (s), 3.83 (s); 13C NMR
(75 MHz, (CD3)2CO) d: 160.2, 159.5, 148.0, 147.6, 145.6, 137.3,
133.7, 130.3, 129.7, 128.4, 126.0, 121.8, 121.5, 121.1, 121.1,
120.2, 119.1, 117.3, 116.0, 115.3, 114.1, 113.8, 111.5, 109.3, 55.5,
55.1, 54.7; CI-EIMS: m/z [M+Na]+ 372; C20H19N3O3 (349.14): calcd
C, 68.75; H, 5.48; N, 12.03. Found: C, 68.80; H, 5.45; N, 12.09.
113.9, 113.7; CI-EIMS: m/z [M+Na]+ 312; C18H15N3O (289.12):
calcd C, 74.72; H, 5.23; N, 14.52. Found: C, 74.64; H, 5.17; N, 14.50.
4.1.3.11. 4-((E)-4-Methoxystyryl)-1-((E)-styryl)-1H-1,2,3-triazole
(2Bd).
(69% cycloaddition reaction) pale yellow solid mp:
195 °C; IR (KBr) cmꢀ1: 3096, 3059, 2974, 2939, 2842, 1656, 1604,
1510, 1245, 1050, 1024, 961, 924, 814, 693, 517; 1H NMR
(300 MHz, (CD3)2CO) d: 8.44 (s, 1H), 8.05 (d, J = 14.7 Hz, 1H), 7.66
(br d, J = 1.5 Hz, 1H), 7.63–762 (m, 1H), 7.53 (dd,
J1 = 8.9 Hz J2 = 0.5 Hz, 2H), 7.45–740 (m, 4H), 7.35 (dd, J1 = 8.9 J
2 = 2.2 Hz, 1H), 7.07 (dd, J1 = 16.4 Hz J2 = 0.4 Hz, 1H), 6.95 (d,
J = 8.7 Hz, 2H), 3.82 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d:
159.7, 134.4, 130.6, 129.6, 129.4, 128.9, 128.4, 127.4, 124.1,
121.3, 115.0, 114.8, 114.5, 55.7; CI-EIMS: m/z [M+H]+ 304;
4.1.3.6. 2-Methoxy-4-((E)-2-(4-((E)-3,4,5-trimethoxystyryl)-1H-
1,2,3-triazol-1-yl)vinyl)phenol (2Ag).
(70% over the last
two steps) yellow solid mp: 131 °C; IR (KBr) cmꢀ1: 3565, 3100,
2998, 2811, 1665, 1525, 1372, 1296, 1275, 1046, 959, 940, 788,
528; 1H NMR (300 MHz, (CD3)2CO) d: 8.38 (1H, s), 7.92 (d,
J = 14.6 Hz, 1H), 7.36–7.10 (m, 5 H), 7.04 (dd, J1 = 8.2 Hz J2 = 1.8 Hz,
1H), 6.89 (s, 2H), 6.86 (d, J = 6.6 Hz, 1H), 3.90 (s, 3H), 3.87 (s, 6H),
3.74 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 153.8, 148.0, 147.5,
146.1, 132.7, 130.6, 126.0, 121.5, 121.3, 121.0, 118.1, 116.3,
115.3, 109.4, 104.0, 104.0, 59.8, 55.6, 55.5; CI-EIMS: m/z [M+Na]+
432; C22H23N3O5 (409.16): calcd C, 64.54; H, 5.66; N, 10.26. Found:
C, 64.60; H, 5.65; N, 10.31.
C19H17N3O (303.14): calcd C, 75.23; H, 5.65; N, 13.85. Found: C,
75.40; H, 5.73; N, 13.66.
4.1.3.12. 4-((E)-3-Methoxystyryl)-1-((E)-styryl)-1H-1,2,3-triazole
(2Be).
(71% cycloaddition reaction) brown solid mp: 185 °C;
IR (KBr) cmꢀ1: 3142, 2951, 2925, 1585, 1454, 1238, 1046, 968,
841, 746, 693, 508; 1H NMR (300 MHz, CDCl3) d: 7.97 (s, 1H),
7.73 (d, J = 14.7 Hz, 1H), 7.58 (d, J = 16.5 Hz, 1H), 7.46–7.32 (m,
5H), 7.16 (br d, 1H), 7.13 (d, J = 10.3 Hz, 1H), 7.06 (d, J = 16.5 Hz,
1H), 6.71 (dd, J1 = 8.8 Hz J2 = 2.7 Hz, 2H), 3.80 (s, 3H); 13C NMR
(75 MHz, CDCl3) d: 159.1, 137.1, 133.7, 133.6, 130.1, 129.1, 128.9,
126.8, 122.9, 121.8, 119.4, 115.8, 114.9, 111.6, 55.6; CI-EIMS:
m/z [M+H]+ 304; C19H17N3O (303.14): calcd C, 75.23; H, 5.65; N,
13.85. Found: C, 75.35; H, 5.70; N, 13.58.
4.1.3.7. 4-((E)-2-(4-((E)-3-Hydroxy-4-methoxystyryl)-1H-1,2,3-
triazol-1-yl)vinyl)-2-methoxyphenol (2Ah).
(62% over the
last two steps) brown solid mp: 183 °C; IR (KBr) cmꢀ1: 3365,
3206, 2915, 2900, 1746, 1674, 1521, 1445, 1285, 1277, 1180,
1135, 1012, 1004, 705, 645, 535; 1H NMR (300 MHz, (CD3)2CO)
d:8.36 (s, 1H), 7.93 (d, J = 14.7 Hz, 1H), 7.31 (d, J = 10.2 Hz, 2H),
7.26 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.07–6.97 (m, 3H),
6.91 (d, J = 8.34 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 3.90 (s, 3H), 3.83
(s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 148.0, 147.8, 147.5,
146.8, 146.3, 130.5, 130.4, 126.1, 121.4, 121.11, 121.10, 119.0,
115.3, 114.7, 112.5, 111.6, 109.3, 55.5, 55.4; CI-EIMS: m/z
[M+Na]+ 388. C20H19N3O4 (365.14): calcd C, 65.74; H, 5.24; N,
11.50. Found: C, 65.57; H, 5.40; N, 11.58.
4.1.3.13. 4-((E)-2-(1-((E)-Styryl)-1H-1,2,3-triazol-4-yl)vinyl)ben-
zene-1,2-diol (2Bf).
(55% over the last two steps) brown solid
mp: 217 °C; IR (KBr) cmꢀ1: 3462, 3132, 2918, 2838, 1601, 1525,
1414, 1285, 1219, 1022, 807, 608, 570; 1H NMR (300 MHz, CD3OD)
d: 8.39 (s, 1H), 7.95 (d, J = 14.4 Hz, 1H), 7.57 (d, J = 6.9 Hz, 2H),
7.41–7.31 (m, 4H), 7.20 (d, J = 15.8 Hz, 1H), 7.02 (s, 1H), 6.90–
6.86 (m, 2H) 6.75 (d, J = 7.8 Hz, 1H); 13C NMR (75 MHz, CD3OD)
d: 146.8, 145.9, 145.3, 134.0, 131.8, 128.8, 128.7, 128.4, 126.6,
122.9, 121.8, 119.1, 117.9, 115.1, 112.5, 112.4; CI-EIMS: m/z [M
+Na]+ 328; C18H15N3O2 (305.12): calcd C, 70.81; H, 4.95; N,
13.76. Found: C, 70.55; H, 5.01; N, 13.68.
4.1.3.8. 2-Methoxy-4-((E)-2-(1-((E)-styryl)-1H-1,2,3-triazol-4-yl)
vinyl)phenol (2Ba).
(67% over the last two steps) brown solid
mp: 167 °C; IR (KBr) cmꢀ1: 3540, 3160, 3006, 2936, 2841, 1646,
1604, 1521, 1372, 1244, 1110, 965, 655, 614, 490; 1H NMR
(300 MHz, CDCl3) d:7.85 (s, 1H), 7.75 (d, J = 14.8 Hz, 1H), 7.45–
7.32 (m, 6H), 7.26 (d, J = 4.4 Hz, 1H), 7.12 (d, J = 14.8 Hz, 1H),
7.03 (s, 1H), 6.98 (d, J = 9.1 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 3.92
(s, 3H); 13C NMR (75 MHz, CDCl3) d: 146.8, 146.8, 146.1, 133.6,
131.7, 129.2, 129.1, 128.8, 126.8, 123.0, 121.5, 120.8, 116.7,
114.7, 113.9, 108.3, 56.0; CI-EIMS: m/z [M+Na]+ 342; C19H17N3O2
(319.13): calcd C, 71.46; H, 5.37; N, 13.16. Found: C, 71.36; H,
5.28; N, 13.35.
4.1.3.14. 1-((E)-Styryl)-4-((E)-3,4,5-trimethoxystyryl)-1H-1,2,3-
triazole (2Bg).
(73% cycloaddition reaction) pale brown solid
mp: 148 °C; IR (KBr) cmꢀ1: 3125, 3064, 3004, 2939, 2834, 1657,
1580, 1504, 1454, 1328, 1232, 1126, 1003, 953, 805, 753, 695; 1H
NMR (300 MHz, (CD3)2CO) d: 8.45 (s, 1H), 8.05 (d, J = 14.8 Hz,
1H), 7.65 (br d, J = 1.5 Hz, 1H), 7.63–762 (m, 1H), 7.45–7.33 (m,
5H), 7.17 (dd, J1 = 16.3 Hz J2 = 0.4 Hz, 1H), 6.92 (s, 2H), 3.88 (s,
6H), 3.74 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 153.1, 134.8,
132.5, 130.7, 129.1, 128.2, 126.7, 123.8, 120.9, 118.1, 116.0,
104.6, 59.2, 55.6; CI-EIMS: m/z [M+H]+ 364; C21H21N3O (363.16):
calcd C, 69.41; H, 5.82; N, 11.56. Found: C, 69.55; H, 5.72; N, 11.57.
4.1.3.9.
1,4-Di((E)-styryl)-1H-1,2,3-triazole
(2Bb).
(70%
cycloaddition reaction) brown solid mp: 133 °C; IR (KBr) cmꢀ1
:
3099, 3006, 2868, 1654, 1588, 1416, 1328, 1236, 1002, 809, 529;
1H NMR (300 MHz, (CD3)2CO) d: 8.73 (s, 1H), 8.19 (d, J = 14.8 Hz,
1H), 7.64 (t, J = 8.1 Hz, 3H), 7.45–7.30 (m, 9H), 7.26 (d,
J = 16.4 Hz, 1H); 13C NMR (75 MHz, (CD3)2CO) d: 146.1, 137.0,
134.3, 130.5, 130.5, 129.0, 128.8, 128.5, 127.9, 126.8, 126.5,
123.5, 121.0, 116.9, CI-EIMS: m/z [M+H]+ 274; C18H15N3 (273.13):
calcd C, 79.10; H, 5.53; N, 15.37. Found: C, 79.16; H, 5.56; N, 15.43.
4.1.3.15. 4-((E)-2-(1-((E)-4-Hydroxystyryl)-1H-1,2,3-triazol-4-yl)
vinyl)-2-methoxyphenol (2Ca).
(58% over the last two steps)
pale yellow solid mp: 184 °C; IR (KBr) cmꢀ1: 3341, 3130, 2966,
2813, 1724, 1701, 1608, 1590, 1515, 1285, 1257, 960, 839, 811,
524; 1H NMR (300 MHz, (CD3)2CO) d: 8.36 (s, 1H), 7.58 (d,
J = 14.7 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.34–7.21 (m, 2H), 7.12–
6.97 (m, 3H), 6.89 (d, J = 8.5 Hz, 2H), 6.84–678 (m, 2H), 3.88 (s,
6H), 3.74 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 158.1, 147.8,
130.8, 130.6, 130.4, 129.1, 128.3, 125.6, 121.1, 120.5, 115.9,
115.6, 115.2, 114.2, 109.2, 109.1, 55.4; CI-EIMS: m/z [M+Na]+
358; C19H17N3O3 (335.13): calcd C, 68.05; H, 5.11; N, 12.53. Found:
C, 68.15; H, 5.02; N, 12.57.
4.1.3.10.
4-((E)-2-(1-((E)-Styryl)-1H-1,2,3-triazol-4-yl)vinyl)
phenol (2Bc).
(65% over the last two steps) brown solid mp:
177 °C; IR (KBr) cmꢀ1: 3139, 2848, 1698, 1651, 1608, 1455, 1236,
1127, 1015, 935, 525; 1H NMR (300 MHz, (CD3)2CO) d: 8.42 (s,
1H), 8.04 (d, J = 15 Hz, 1H), 7.64 (d, J = 7.0 Hz, 2H), 7.49–7.20 (m,
5H), 7.01 (d, J = 16.5 Hz, 1H), 6.85 (dd, J1 = 14.7 Hz J2 = 5.8 Hz,
4H); 13C NMR (75 MHz, (CD3)2CO) d: 157.9, 146.5, 134.6, 130.6,
128.9, 128.8, 128.1, 128.5, 128.0, 127.4, 126.4, 120.7, 115.8,

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D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
4.1.3.16. 4-((E)-2-(4-((E)-Styryl)-1H-1,2,3-triazol-1-yl)vinyl)phe-
nol (2Cb). (55% over the last two steps) yellow solid mp:
J = 16.3 Hz, 1H), 6.91 (d, J = 7.7 Hz, 2H), 6.72 (s, 2H); 13C NMR
(75 MHz, CDCl3) d: 158.1, 153.7, 146.1, 132.7, 130.6, 128.3, 125.6,
121.2, 121.0, 118.2, 116.3, 115.9, 103.9, 59.8, 55.6; CI-EIMS: m/z
[M+Na]+ 402; C21H21N3O4 (379.15): calcd C, 66.48; H, 5.58; N,
11.08. Found: C, 66.29; H, 5.50; N, 11.00.
183 °C; IR (KBr) cmꢀ1: 3111, 3001, 2945, 2821, 1624, 1542, 1500,
1258, 1221, 953, 869, 805, 683; 1H NMR (300 MHz, (CD3)2CO) d:
8.43 (s, 1H), 7.87 (d,=14.6 Hz, 1H), 7.58 (d, J = 7.3 Hz, 2H), 7.48 (d,
J = 8.5 Hz, 2H), 7.44–7.27 (m, 4H), 7.21 (d, J = 16.5 Hz, 2H), 6.90
(d, J = 8.5 Hz, 2H); 13C NMR (75 MHz, (CD3)2CO) d: 158.2, 137.1,
130.3, 128.8, 128.5, 128.3, 127.9, 126.5, 121.0, 117.0, 115.9;
CI-EIMS: m/z [M+Na]+ 312; C18H15N3O (289.12): calcd C, 74.72;
H, 5.23; N, 14.52. Found: C, 74.67; H, 5.33; N, 14.43.
4.1.3.22. 2-Methoxy-4-((E)-2-(1-((E)-3-methoxystyryl)-1H-1,2,3-
triazol-4-yl)vinyl)phenol (2Da).
(55% over the last two steps)
pale yellow solid mp: 130 °C; IR (KBr) cmꢀ1: 3525, 3103, 3035,
2749, 1656, 1607, 1495, 1373, 1275, 1153, 1000, 835, 528; 1H
NMR (300 MHz, CDCl3) d: 8.41 (s, 1H), 8.07 (d, J = 14.9 Hz, 1H),
7.36 (d, J = 3 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.30 (d, J = 4.1 Hz,
1H), 7.24 (d, J = 1.9 Hz, 1H), 7.22 (t, J = 1.6 Hz, 1H), 7.18 (brdt,
1H), 7.05 (d, J = 16.3 Hz, 1H), 7.02 (dd, J1 = 8.1 Hz J2 = 1.9 Hz, 1H),
6.91 (ddd, J1 = 8.1 Hz J2 = 2.5 J3 = 0.9 Hz, 1H), 6.83 (d, J = 8.1 Hz,
1H), 3.91 (s, 3H), 3.84 (s, 3H); 13C NMR (75 MHz, CDCl3) d: 160.3,
147.1, 146.6, 135.8, 131.0, 130.0, 129.1, 123.7, 120.7, 120.5,
119.4, 115.2, 114.3, 114.1, 111.7, 109.1, 55.4, 54.8; CI-EIMS: m/z
[M+Na]+ 372; C20H19N3O3 (349.14): calcd C, 68.75; H, 5.48; N,
12.03. Found: C, 68.82; H, 5.52; N, 12.01.
4.1.3.17. 4-((E)-2-(4-((E)-4-Hydroxystyryl)-1H-1,2,3-triazol-1-yl)
vinyl)phenol (2Cc).
(56% over the last two steps) pale brown
solid mp: 260 °C; IR (KBr) cmꢀ1: 3315, 2814, 2689, 1606, 1538,
1451, 1418, 1251, 1059, 837, 827, 528; 1H NMR (300 MHz, (CD3)2SO)
d: 8.60 (s, 1H), 7.95 (d, J = 14.6 Hz, 1H), 7.44 (t, J = 9.5 Hz, 4H), 7.28
(d, J = 8.2 Hz, 1H), 7.22 (d, J = 9.7 Hz, 1H), 6.98 (d, J = 16.2 Hz, 1H),
6.79 (t, J = 8.2 Hz, 4H); 13C NMR (75 MHz, (CD3)2SO) d: 158.2,
157.7, 146.5, 130.5, 130.4, 130.2, 128.5, 128.3, 127.9, 125.8,
125.5, 121.1, 115.9, 115.7, 115.6, 113.9, 113.8, CI-EIMS: m/z
[M+Na]+ 328; C18H15N3O2 (305.12): calcd C, 70.81; H, 4.95; N,
13.76. Found: C, 70.75; H, 5.04; N, 13.73.
4.1.3.23. 1-((E)-3-Methoxystyryl)-4-((E)-styryl)-1H-1,2,3-triazole
(2Db).
(71% cycloaddition reaction) brown solid mp: 137 °C;
4.1.3.18. 4-((E)-2-(4-((E)-4-Methoxystyryl)-1H-1,2,3-triazol-1-yl)
IR (KBr) cmꢀ1: 3165, 2945, 2877, 2686, 1600, 1589, 1513, 1456,
1058, 975, 862, 680; 1H NMR (300 MHz, CDCl3) d: 7.86 (s, 1H),
7.72 (d, J = 14.7 Hz, 1H), 7.48 (d, J = 16.5 Hz, 2H), 7.36 (d,
J = 7.0 Hz, 1H), 7.35 (d, J = 6.9 Hz, 1H), 7.31 (d, J = 9.0 Hz, 2H),
7.28–7.25 (m, 1H), 7.10 (d, J = 2.8 Hz, 1H), 7.05 (br s, 2H), 6.97
(br t, 1H), 6.87 (dd, J1 = 8.2 Hz J2 = 2.4 Hz, 1H), 3.81 (s, 3H); 13C
NMR (75 MHz, CDCl3) d: 160.1, 136.6, 135.0, 131.6, 130.1, 128.8,
128.2, 126.7, 123.2, 121.3, 119.3, 116.1, 114.4, 112.1, 55.4;
CI-EIMS: m/z [M+H]+ 304; C19H17N3O (303.14): calcd C, 75.23; H,
5.65; N, 13.85. Found: C, 75.34; H, 5.62; N, 13.90.
vinyl)phenol (2Cd).
(68% over the last two steps) pale yellow
solid mp: 205 °C; IR (KBr) cmꢀ1: 3152, 2838, 1608, 1566, 1455,
1282, 1236, 1056, 839, 525; 1H NMR (300 MHz, (CD3)2CO) d: 8.40
(s, 1H), 7.88 (d, J = 14.7 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.49 (d,
J = 8.5 Hz, 2H), 7.35 (d, J = 16.5 Hz, 1H), 7.29 (d, J = 14.9 Hz, 1H),
7.06 (d, J = 16.5 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 3.81 (s, 3H);
13C NMR (75 MHz, (CD3)2CO) d: 158.8, 158.0, 130.0, 129.7, 128.3,
127.8, 125.7, 122.0, 121.0, 115.8, 115.7, 114.7, 114.2, 54.8;
CI-EIMS: m/z [M+Na]+ 342; C19H17N3O2 (319.13): calcd C, 71.46;
H, 5.37; N, 13.16. Found: C, 71.55; H, 5.24; N, 13.13.
4.1.3.24. 4-((E)-2-(1-((E)-3-Methoxystyryl)-1H-1,2,3-triazol-4-yl)
4.1.3.19. 4-((E)-2-(4-((E)-3-Methoxystyryl)-1H-1,2,3-triazol-1-yl)
vinyl)phenol (2Dc).
(59% over the last two steps) pale yellow
vinyl)phenol (2Ce).
(66% over the last two steps) yellow solid
solid mp: 208 °C; IR (KBr) cmꢀ1: 3149, 2960, 2832, 1607, 1581,
1456, 1268, 1172, 1150, 938, 822, 773, 680; 1H NMR (300 MHz,
(CD3)2SO) d:8.61 (s, 1H), 8.18 (d, J = 14.6 Hz, 1H), 7.42 (d,
J = 8.5 Hz, 2H), 7.37–7.17 (m, 5H), 6.99 (d, J = 16.2 Hz, 1H), 6.89
(dd, J1 = 7.9 Hz J2 = 2.1 Hz, 1H), 6.80 (d, J = 8.3 Hz, 2H), 3.80 (s,
3H); 13C NMR (75 MHz, (CD3)2SO) d: 160.2, 158.2, 135.9, 131.0,
130.5, 130.4, 128.4, 128.0, 124.3, 121.1, 120.0, 118.9, 116.1,
114.8, 113.0, 112.1, 55.6, CI-EIMS: m/z [M+Na]+ 342; C19H17N3O2
(319.13): calcd C, 71.46; H, 5.37; N, 13.16. Found: C, 71.49; H,
5.33; N, 13.18.
mp: 201 °C; IR (KBr) cmꢀ1: 3262, 2818, 2683, 1651, 1517, 1354,
1331, 1219, 1013, 863, 810, 703, 652, 458; 1H NMR (300 MHz,
(CD3)2CO) d: 8.51 (s, 1H), 7.88 (d, J = 14.7 Hz, 1H), 7.68 (d,
J = 16.2 Hz, 1H), 7.51 (d, J = 5.7 Hz, 2H), 7.48 (d, J = 5.6 Hz, 2H),
7.36 (t, J = 3.0 Hz, 2H), 7.29 (d, J = 7.4 Hz, 1H), 6.89 (d, J = 8.6 Hz,
2H), 6.83 (dd, J1 = 8.7 J2 = 3.0 Hz, 1H), 3.86 (s, 3H); 13C NMR
(75 MHz, (CD3)2CO) d: 159.6, 158.1, 137.3, 133.7, 128.4, 128.4,
128.3, 125.5, 121.5, 120.9, 120.3, 119.1, 116.0, 115.9, 111.5, 55.1;
CI-EIMS: m/z [M+Na]+ 342; C19H17N3O2 (319.13): calcd C, 71.46;
H, 5.37; N, 13.16. Found: C, 71.50; H, 5.30; N, 13.10.
4.1.3.25. 1-((E)-3-Methoxystyryl)-4-((E)-4-methoxystyryl)-1H-
4.1.3.20. 4-((E)-2-(1-((E)-4-Hydroxystyryl)-1H-1,2,3-triazol-4-yl)
1,2,3-triazole (2Dd).
(70% cycloaddition reaction) brown
vinyl)benzene-1,2-diol (2Cf).
(48% over the last two steps)
solid mp: 157 °C; IR (KBr) cmꢀ1: 3097, 3058, 3004, 2963, 2929,
2840, 1603, 1510, 1244, 1175, 1035, 942, 840, 691, 529; 1H NMR
(300 MHz, CDCl3) d: 7.84 (s, 1H), 7.73 (d, J = 14.7 Hz, 1H), 7.42 (d,
J = 8.2 Hz, 2H), 7.35–7.25 (m, 3H), 7.07 (d, J = 8.2 Hz, 1H),
7.03–6.91 (m, 3H), 6.87 (d, J = 8.2 Hz, 2H), 3.82 (s, 3H), 3.81 (s,
3H); 13C NMR (75 MHz, CDCl3) d: 160.0, 159.7, 135.0, 131.2,
130.1, 129.0, 127.9, 123.3, 121.1, 119.3, 116.8, 114.4, 114.2,
113.9, 112.1, 55.4, 55.3; CI-EIMS: m/z [M+H]+ 334; C20H19N3O2
(333.15): calcd C, 72.05; H, 5.74; N, 12.60. Found: C, 72.15; H,
5.78; N, 12.73.
brown solid mp: 215 °C; IR (KBr) cmꢀ1: 3316, 3139, 2700, 2813,
1603, 1588, 1511, 1428, 1262, 1170, 953, 811, 694, 528; 1H NMR
(300 MHz, (CD3)2CO) d: 8.38 (s, 1H), 7.85 (d, J = 14.6 Hz, 1H), 7.48
(d, J = 8.5 Hz, 2H), 7.28 (d, J = 14.2 Hz, 2H), 7.12 (dd, J1 =
4.7 Hz J2 = 1.9 Hz, 1H), 7.07–6.86 (m, 5H); 13C NMR (75 MHz,
(CD3)2CO) d: 158.0, 148.5, 146.6, 130.5, 130.4, 129.3, 128.3,
128.3, 127.0, 125.68, 125.67, 121.1, 120.2, 118.8, 118.0, 115.9;
CI-EIMS: m/z [M+Na]+ 344; C18H15N3O32 (321.11): calcd C, 67.28;
H, 4.71; N, 13.08. Found: C, 67.37; H, 4.80; N, 13.13.
4.1.3.21. 4-((E)-2-(4-((E)-3,4,5-Trimethoxystyryl)-1H-1,2,3-tria-
4.1.3.26.
(2De).
1,4-Bis((E)-3-methoxystyryl)-1H-1,2,3-triazole
zol-1-yl)vinyl)phenol (2Cg).
(63% over the last two steps)
(74% cycloaddition reaction) brown solid mp: 179 °C;
pale brown solid mp: 186 °C; IR (KBr) cmꢀ1: 3109, 2888, 2611,
1698, 1645, 1517, 1364, 1280, 1127, 1046, 920, 788, 528; 1H
NMR (300 MHz, CDCl3) d: 7.85 (s, 1H), 7.60 (d, J = 14.4 Hz, 1H),
7.33 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 14.7 Hz, 2H), 7.00 (d,
IR (KBr) cmꢀ1: 3102, 3023, 2941, 2910, 2760, 1573, 1510, 1280,
1235, 1005, 942, 840, 794, 781, 717, 529; 1H NMR (300 MHz,
CDCl3) d: 7.95 (s, 1H), 7.69 (d, J = 14.7 Hz, 1H), 7.54
(d, J = 16.5 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.26 (dd,

D. Caprioglio et al. / Bioorg. Med. Chem. 24 (2016) 140–152
151
J1 = 12.8 Hz J2 = 3.0 Hz, 1H), 7.10–6.95 (m, 6H), 6.84 (dd, J1 =
mixture containing both pNF-jB-TA-Luc plasmid and of pRLTK
8.2 Hz J2 = 2.1 Hz, 1H), 6.67 (dd, J1 = 8.8 Hz J2 = 3.0 Hz, 1H), 3.82
(s, 3H), 3.81 (s, 3H); 13C NMR (75 MHz, CDCl3) d: 160.07, 159.10,
137.03, 134.93, 133.75, 130.16, 130.13, 123.09, 121.78, 119.40,
119.26, 117.77, 115.87, 114.90, 114.52, 112.17, 111.59, 55.59,
55.3; CI-EIMS: m/z [M+H]+ 334; C20H19N3O2 (333.15): calcd C,
72.05; H, 5.74; N, 12.60. Found: C, 72.13; H, 5.80; N, 12.75.
vector in a 1:2 ratio for 24 h. Cells were then plated in
96-multiwell plates at a concentration of 5 ꢁ 103 and after 8 h
were treated for 6 h with TNFa (20 ng/mL) in the presence or
absence of the selected compounds. Cells were finally lysed in
luciferase lysis buffer, and the luciferase activity was recorded
and normalized to pRLTK luciferase activity using a dual luciferase
assay system (Promega, Madison, WI, USA).
4.1.3.27. 4-((E)-2-(1-((E)-3-Methoxystyryl)-1H-1,2,3-triazol-4-yl)
vinyl)benzene-1,2-diol (2Df).
(52% over the last two steps)
Supplementary data
brown solid mp: 185 °C; IR (KBr) cmꢀ1: 3465, 3073, 2928, 2838,
1609, 1580, 1455, 1441, 1284, 977, 935, 807, 608; 1H NMR
(300 MHz, (CD3)2CO) d: 8.41 (s, 1H), 8.06 (d, J = 14.6 Hz, 1H),
7.36–7.18 (m, 5H), 7.11 (d, J = 1.5 Hz, 1H), 6.96 (d, J = 16.3 Hz,
1H), 6.92 (dt, J1 = 8.1 Hz J2 = 1.9 Hz, 2H), 6.83 (d, J = 7.9 Hz, 1H),
3.84 (s, 3H); 13C NMR (75 MHz, (CD3)2CO) d: 160.3, 146.6, 145.7,
145.4, 135.8, 131.0, 129.9, 129.3, 123.7, 120.7, 119.4, 119.2,
115.5, 114.3, 113.8, 113.0, 111.7, 54.8; CI-EIMS: m/z [M+Na]+
358; C19H17N3O3 (335.13): calcd C, 68.05; H, 5.11; N, 12.53. Found:
C, 68.13; H, 503; N, 12.61.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.11.044.
References and notes
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4.1.3.28.
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1-((E)-3-methoxystyryl)-4-((E)-3,4,5-trimethoxys-
(78% cycloaddition reaction)
yellow oil; IR (KBr) cmꢀ1: 3087, 3064, 2928, 2840, 1666, 1510,
1494, 1465, 1175, 837, 817, 519; 1H NMR (300 MHz, CDCl3) d:
7.85 (s, 1H), 7.65 (d, J = 14.6 Hz, 1H), 7.21 (t, J = 7.6 Hz, 2H), 7.01
(d, J = 14.7 Hz, 1H), 6.96–6.89 (m, 3H), 6.79 (dd, J1 = 8.2 Hz
J2 = 2.3 Hz, 1H), 6.65 (s, 2H), 3.81 (s, 3H), 3.79 (s, 6H), 3.74 (s,
3H); 13C NMR (75 MHz, CDCl3) d: 160.0, 153.4, 146.3, 138.2,
135.0, 132.3, 131.3, 130.0, 123.1, 121.3, 119.2, 117.5, 115.7,
114.2, 112.2, 103.6, 60.9, 56.0, 55.3; CI-EIMS: m/z [M+Na]+ 394;
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4.2.1. Cell culture, cytotoxicity and tubulin assays
Culture of HeLa (ATCC, LGC Promochem Teddington, UK) and
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toxicity assays were conducted as described previously.54 Tubulin
polymerization was monitored by a fluorescence-based tubulin
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HeLa cells were plated at a concentration of 6 ꢁ 105 cells/well
on 6-wells plates. After an overnight starvation, cells were then
grown in the present or absence of compounds for 8 h or 16 h. At
the end of the incubation period, cells were washed once in PBS
and re-suspended in 1 mL of 70% EtOH and stored at ꢀ20 °C over-
night. Cells were then washed once in PBS and re-suspended in PBS
containing RNAse (100
l
g/mL) for 1 h at 37 °C. DNA was stained
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with a PBS solution containing EDTA 5 mM and 25
ium iodide for 30 min at 4 °C in the dark. Analysis was performed
with a BD Accuri FACS and analyzed via MODFIT.
l
g/mL propid-
4.2.3. IC50 determination
Approximate IC50s for concentration–response curves were
determined via Kaleidagraph software with the equation
m1 + {(m2 ꢀ m1)/[1 + ((m3/m0)m4)]} where m1 is the maximum
value, m2 is the minimum value, m3 is the IC50 and m4 is the slope
of curve.
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4.2.4. Determination of NF-jB transcription activity
1 ꢁ 106 HeLa cells were plated in 10 mm2 dishes and transiently
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