Reversibility of the thia-Michael reaction of cytotoxic C5-curcuminoid and structure-activity relationship of bis-thiol-adducts thereof

Article abstract of DOI:10.1039/c6ob01771a

C₅-curcuminoids [a.k.a. bis(arylmethylidene)acetones] are curcumin analogues bearing a reactive cross-conjugated dienone structure essential for eliciting cytotoxicity. To gain insight into the mode of action of C₅-curcuminoids, we investigated the reversibility of the thia-Michael reaction of 1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-one, named GO-Y030 which is the most potent cytotoxic C₅-curcuminoid, using spectroscopic methods. A panel of GO-Y030-bis-thiol-adducts were synthesized and the structure-reactivity relationship regarding the retro thia-Michael reaction as well as the cell growth inhibitory activity against human colon cancer HCT116 were evaluated. Some C₅-curcuminoid thiol-adducts exhibited comparable cytotoxicity with GO-Y030, demonstrating their potential use as prodrugs. These results imply that C₅-curcuminoids elicit cytotoxicity by covalently interacting with various biothiols via a reversible thia-Michael reaction.

Full text of DOI:10.1039/c6ob01771a

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Reversibility of thia-Michael reaction of the cytotoxic C5-
curcuminoid and structure-activity relationship of the bis-thiol-
adducts thereof
0
Received 00th January 20xx,
Accepted 00th January 20xx
Aki Kohyama,a Michihiro Fukuda,a Shunsuke Sugiyama,b Hiroyuki Yamakoshi,a Naoki Kanoh,a
Chikashi Ishioka,b Hiroyuki Shibatac and Yoshiharu Iwabuchi*a
DOI: 10.1039/x0xx00000x
C5-curcuminoids [a.k.a. bis(arylmethylidene)acetones] are curcumin analogues bearing a reactive cross-conjugated
dienone structure essential for eliciting cytotoxicity. To gain insight into the mode of action of C5-curcuminoids, we
investigated the reversibility of the thia-Michael reaction of 1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-
one, named GO-Y030 which is the most potent cytotoxic C5-curcuminoid, using spectroscopic methods. A panel of GO-
Y030-bis-thiol-adducts were synthesized and the structure-reactivity relationship regarding retro thia-Michael reaction
as well as the cell growth inhibitory activity against human colon cancer HCT116 were evaluated. Some C5-curcuminoid
thiol-adducts exhibited comparable cytotoxicity with GO-Y030, demonstrating their potential use as prodrugs. These
results imply that C5-curcuminoids elicit cytotoxicity by covalently interacting with various biothiols via a reversible thia-
Michael reaction.
www.rsc.org/
curcuminoid, to identify that GOꢀY086 covalently binds to
FUBP2 (far upstream element binding protein 2) at C500,
which ultimately resulted in a marked inhibition of the
expression of the cꢀMyc protein.4
Introduction
Owing to their inherent reactivity as Michael acceptors that
would form a covalent bond with multiple nucleophilic
Considering the high reactivity of C5ꢀcurcuminoids as
Michael acceptors, GOꢀY086 should experience instantaneous
Michael reaction with biothiols upon administration,
particularly with glutathione, which is the most abundant (2ꢀ3
mM) nonprotein thiol in eukaryotic cells, to give the
corresponding thiaꢀMichael adducts. Therefore, it was
presumed that GOꢀY086 survives via a retro thiaꢀMichael
reaction before it participates in the crucial irreversible Michael
reaction with binding protein(s). Such a hypothesis prompted us
to obtain evidence that C5ꢀcurcuminoids undergo a reversible
thiaꢀMichael reaction.
biomolecules,
α,βꢀunsaturated carbonyl compounds have
traditionally been research subjects in toxicology and thus have
been avoided in modern medicinal chemistry.1 However,
recently, the druggability of Michael acceptors has substantially
been reviewed in light of accumulating knowledge that the
modulation of the Michael reaction could lead to significant
improvement of pharmacological properties, and a novel design
concept of covalent drugs has been formulated.2
Previously, we disclosed that some bis(arylmethylidene)ꢀ
acetones, which we named C5ꢀcurcuminoids in connection with
naturally occurring C5ꢀcurcumin, exhibit antitumor activities
without any remarkable toxicity in common with curcumin
(Figure 1).3 Thus, we identified 1,5ꢀbis(3,5ꢀbis(methoxyꢀ
methoxy)ꢀphenyl)ꢀ1,4ꢀpentadieneꢀ3ꢀone (GOꢀY030) out of
more than 100 synthetic analogues as the most potent and
promising cytotoxic agent that successfully prevents colorectal
carcinogenesis in familial adenomatous polyposis (FAP) mice
Aside from its promising antitumor activity, GOꢀY030 has a
low water solubility6, which led us to envisage the development
of a prodrug employing the reversibility of the thiaꢀMichael
O
O
OH
R1
R2
R1
R3
in vivo 3b
.
We then explored molecular targets of C5ꢀ
MeO
HO
OMe
OH
curcuminoids using GOꢀY086, a biotinylated analogue of a C5ꢀ
R1
GOꢀY030 : R1=OMOM R2=R3=H
GOꢀY086 : R1=R2=OMe R3=biotin tag
R1
curcumin
O
a.Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba,
Aramaki, Aoba-ku, Sendai 980-8578, Japan.
O
biotin tag =
HN
H
NH
MeO
HO
OMe
OH
H
b.Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-0872,
JAPAN.
H
N
N
O
S
N
9
C5ꢀcurcumin
N
c. Graduate School of Medicine, Akita University, Akita, 010-8643, JAPAN.
†Electronic supplementary information (ESI) available: General synthetic methods,
experimental data, and spectral data are presented in pdf format. See
DOI: 10.1039/x0xx00000x
O
Figure 1 Structure of curcumin and C5-curcuminoid
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reaction.7 In this context, we intended to gain insight into the change in the absorption at 340 nm, the maximum absorption
structureꢀreactivity relationship focusing on the thiol moiety.
wavelength attributed to the crossꢀconjugated dienone moiety
of GOꢀY030, provided a comparable result with the NMR
adducts in aqueous systems. It was found that monitoring the
change in the absorption at 340 nm, the maximum absorption
wavelength attributed to the crossꢀconjugated dienone moiety
of GOꢀY030, provided a comparable result with the NMR
method: upon addition of 24 equiv. of cysteamine, the
absorption at 340 nm derived from GOꢀY030 in DMSOꢀd6 was
instantaneously depleted, and it gradually recovered up to its
original level (Figure 2c). Note that the UV assay could be
conducted in a 96ꢀwell plate, for which less than oneꢀfifth of
the reagents used in the NMR assay was needed.
In this paper, we describe our investigation into the reversibility of
the thiaꢀMichael reaction between the cytotoxic C5ꢀcurcuminoid
GOꢀY030 and thiols using spectroscopic methods. A panel of GOꢀ
Y030ꢀthiolꢀadducts were synthesized to examine the structureꢀ
reactivity relationship in the retro thiaꢀMichael reaction in aqueous
media as well as to probe its potential use in prodrugs.
Results and discussion
Observation of reversible Michael reaction between C5-
curcuminoid and cysteamine
H2N
NH2
H2N
(a)
O
S
O
S
S
O
+ cysteamine
– cysteamine
+ cysteamine
– cysteamine
To observe a reversible thiaꢀMichael reaction of GOꢀY030
Ar
Ar
Ar
Ar
Ar
monoꢀadduct B
Ar
1
(Figure 2a), we conducted a cysteamine assay using a HꢀNMR
bisꢀadduct C
GOꢀY030 A
spectroscopic method,8 devised by Appendino and coworkers.
Upon the addition of 4 equiv. of cysteamine (Figure 2b), the
Ar = 3,5ꢀbis(methoxymethoxy)phenyl
(b)
olefinic protons of GOꢀY030 (
disappeared instantly, giving a spectrum consisting of monoꢀ
adduct and bisꢀadduct (5 min), thereby confirming the thiaꢀ
δ 7.7 and 7.3 ppm in DMSOꢀd6)
B
C
Michael reaction. Then, according to Appendino’s protocol, an
aliquot of the DMSOꢀd6 solution of the inꢀsituꢀgenerated
Michael adducts was diluted 1:20 with CDCl3 to change the
position of equilibrium of the reversible thiaꢀMichael reaction,
however, the spectrum showed only tiny peaks corresponding
to the dienone (See SI pp 96ꢀ98). During our attempts to
monitor a clear retro thiaꢀMichael reaction, we eventually
found that a simple exposure of the DMSOꢀd6 solution of the
inꢀsituꢀgenerated Michael adducts (prepared by mixing
cysteamine with GOꢀY030) tends to shift to the retroꢀMichael (c)
reaction. As shown in Figure 2b, a time dependent restoration
of the olefin resonance was clearly observed, (Figure 2b, from 5
min to 6 hr), confirming that the retroꢀMichael reaction of GOꢀ
Y030ꢀcysteamineꢀadducts (
B or C) occurred after the rapid
Michael reaction between GOꢀY030 and cysteamine. It is
important to point out that the thiaꢀMichael addition proceeded
in a stereoꢀindiscriminating manner to give the bisꢀadduct
C as
an ca. 1:1 diastereomeric mixture, and both diastereomers
exhibited almost the same reactivity in the retro thiaꢀMichael
reaction. Also no retroꢀMichael reaction was observed under
anaerobic conditions, indicating that autoxidation of cysteamine
to give cystamine operates to shift the equilibrium. A negative
control experiment was conducted using maleimide, which is
most widely used Michael acceptor for labelling proteins. 9 As
a result, no reappearance of the olefin resonance was observed,
even after prolonged exposure of the thiaꢀMichael adduct to air,
although a rapid thiaꢀMichael reaction occurred. This result
supports the validation of this modified cysteamine assay to
assess reversibility of the thiaꢀMichael reaction (See SI pp 96ꢀ
102).
Figure 2 (a) Possible thia-Michael/retro thia-Michael reactions between GO-Y030 and
cysteamine. (b) H1-NMR spectra of GO-Y030 after addition of cysteamine (4 eq.) in
DMSO-d6 (c) Time-dependent change of ꢀ Absorbance at 340 nm of GO-Y030 without
and with cysteamine (4 equiv.) in DMSO-d6
Synthesis of GO-Y030-bis-thiol-adducts
With the recognition of the potential of GOꢀY030 to undergo a
reversible thiaꢀMichael reaction, we planned to develop a thiaꢀ
Michael adductꢀtype prodrug of GOꢀY030. To gain a basic
understanding of the structureꢀreactivity relationship of GOꢀ
Y030ꢀthiolꢀadducts in the retro thiaꢀMichael reaction, a panel
of GOꢀY030ꢀthiolꢀadducts were designed using 14 thiols with
different physical and stereoelectronic properties. At this
Having confirmed the usefulness of the modified
cysteamine assay, we examined its adoptability to a UV
spectroscopic method that should enable the highꢀthroughput
screening of the retro thiaꢀMichael reactions of GOꢀY030ꢀthiolꢀ
adducts in aqueous systems. It was found that monitoring the
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Table 1 Structure-reactivity relationships of retro-Michael reaction profiles and antiproliferative activity of of GO-Y030 thiol-adducts
(b) Antiproliferative activity against HCTꢀ116d
(a) Reactivity of GOꢀY030ꢀbisꢀthiolꢀadducts towrard reteo thiaꢀMichael reaction
R
R
R
R
R
S
O
S
O
S
O
S
O
S
buffer
24 h
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
bisꢀadduct
GOꢀY030
bisꢀadduct
monoꢀadduct
Ar = 3,5ꢀbis(methoxymethoxy)phenyl
Yield of GOꢀY030 (%)a
RꢀSH
name
GI50 (ꢁM)
name
GI50 (ꢁM)
pH 3 pH 7.3
pH 8.5
92
GOꢀY135
GOꢀY137
GOꢀY181
GOꢀY136
< 5
< 5
6
5
0.92
0.97
0.84
0.89
HS
HS
78
79
CO2Me
HS
OH
GOꢀY139
GOꢀY138
< 5
< 5
73
95
0.56
0.98
0.72
OH
O
NH2
CO2H
H
N
GOꢀY140b
HO2C
N
100
H
O
HS
NHBoc
NHAc
GOꢀY142
GOꢀY146
GOꢀY141
GOꢀY145
< 5
< 5
5
60
98
0.84
1.00
0.91
0.99
HS
HS
12
HS
HS
GOꢀY174
GOꢀY075c
GOꢀY173
< 5
< 5
< 5
< 5
27
49
0.72
2.00
1.00
0.82
GOꢀY077c
HS
GOꢀY178
GOꢀY177
< 5
9
9
> 40
0.92
GOꢀY144
GOꢀY180
> 40
0.34
0.34
0.42
0.67
GOꢀY143
GOꢀY179
GOꢀY184
GOꢀY186
GOꢀY188
< 5
7
5
6
< 5
61
70
17
11
> 40
0.72
0.76
0.78
> 40
HS
O
HS
O
GOꢀY185
GOꢀY187
< 5
10
9
< 5
10
7
HS
HS
O
HS
O
GOꢀY189
a Yield of GO-Y030 = [Abs thiol-adduct 24 hr – Abs thiol-adduct 5 min] / Abs GO-Y030 (eq. 1). b GO-Y140 was obtained as salts of Et3N. c GO-Y075 and GO-Y077 were reported in
reference 3(d). d GI50 value of GO-Y030 is 0.3 µM.
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Figure 3 UV spectra of GO-Y030, GO-Y139, and GO-Y140 after dilution with pH 7.3 phosphate buffer
juncture, we focused on the bisꢀthiolꢀadducts for their synthetic adducts (Figures S8ꢀS10). The results suggest that lipophilic
accessibility, and we particularly envisioned GOꢀY139 (GOꢀ substituents hinder the retro thiaꢀMichael reaction.
Y030ꢀbis(2,3ꢀdihydroxyꢀpropanethiolꢀadduct) and GOꢀY140
(GOꢀY030ꢀbisꢀglutathioneꢀadduct) to be possible water soluble Biological evaluation of GO-Y030-thiol-adduct
prodrugs of GOꢀY030. On treatment with 4 equiv. of thiol in
the presence of Et3N in an appropriate solvent at rt, GOꢀY030
by cell growth inhibitory testing against HCT116 (Table 1b). In
quantitatively gave GOꢀY030ꢀbisꢀthiolꢀadducts as an ca. 1:1
nseparable mixture of diastereomers. As references, the
thiolꢀadducts that regenerate GOꢀY030 in a buffered solution
corresponding GOꢀY030ꢀmonoꢀthiolꢀadducts were prepared in
a similar manner by decreasing the amount of thiol (1–2 equiv.)
(See SI).
The cytotoxic activity of GOꢀY030ꢀthiolꢀadducts was assessed
general, irrespective of the monoꢀ or bisꢀadducts, GOꢀY030ꢀ
via retro thiaꢀMichael reaction(s) exhibit
a comparable
antiproliferative activity with GOꢀY030. Specifically, GOꢀY143,
GOꢀY144, GOꢀY178, and GOꢀY189, which were inactive
toward retro thiaꢀMichael reaction did not exhibit apparent
cytotoxicity under the assay conditions. These results suggested
Retro thia-Michael reaction of GO-Y030-bis-thiol-adduct
The retro thiaꢀMichael reactions of each GOꢀY030ꢀbisꢀthiolꢀ that the cytotoxic thiolꢀadducts release GOꢀY030 through the
adducts at three different pH (vide infra) were monitored by retroꢀMichael reaction after the treatment of cancer cells with
using a 96ꢀwellꢀplateꢀbased assay system.10 The amount of each analogue. Importantly the glutathione adduct GOꢀY140
GOꢀY030, generated from GOꢀY030ꢀbisꢀthiolꢀadduct via two had high water solubility compared with GOꢀY030 and
retro thiaꢀMichael reactions, was assessed by measuring the exhibited moderate cytotoxicity.
absorbance at 340 nm, where GOꢀY030ꢀmonoꢀthiolꢀadducts
(
λmax ~302 nm) interfere only slightly (Figure S7). GOꢀY030ꢀ Discussion
bisꢀthiolꢀadducts (in 100 µL DMSO) were mixed with 100
ꢁ
L
Focusing on the reversible thiaꢀMichael reaction, fourteen kinds
of GOꢀY030ꢀbisꢀthiolꢀadducts and thirteen kind of GOꢀY030ꢀ
monoꢀthiolꢀadducts were synthesized to investigate the
structureꢀactivity relationship towards the retroꢀMichael
reaction in aqueous medium as well as to evaluate their
potential as prodrugs.
Our results showed that GOꢀY030ꢀbisꢀthiolꢀadducts
exhibited a pHꢀdependent reactivity toward the retro thiaꢀ
Michael reaction, in which the greater the pH of the medium,
the faster the retro thiaꢀMichael reaction. At this juncture, it
of buffer (pH 3 glycine·HCl or pH 7.3 phosphate or pH 8.5
Tris·HCl) in each well, and the yield of GOꢀY030 after 24 hr
was estimated from the changed in absorbance. In acidic
medium (50% DMSO in pH 3 glycineꢀHCl buffer), the yield of
GOꢀY030 for all analogues was ≤10%, indicating that GOꢀ
Y030ꢀbisꢀthiolꢀadducts hardly undergo retro thiaꢀMichael
reaction in acidic medium (Figures S1, S4). In nearly neutral
medium (50% DMSO in pH 7.3 phosphate buffer), two adducts
bearing hydrophilic functionalities, namely, GOꢀY139 and GOꢀ
Y140, exhibited marked reactivity in the retro thiaꢀMichael
reaction to give GOꢀY030 in 73% and 95% yield after 24 hr
(Figure 3), whereas others exhibited ≤10% yield of GOꢀY030,
demonstrating that GOꢀY139 and GOꢀY140 have an
exceptionally high reactivity for the retro thiaꢀMichael reaction
(Figures S2, S5). In basic medium (50% DMSO in pH 8.5 Trisꢀ
HCl buffer), most of the bisꢀthiolꢀadducts showed a moderate to
high reactivity to yield GOꢀY030 in 27% to 100% yield after 24
hr, except for GOꢀY178, GOꢀY144, GOꢀY187, and GOꢀY189,
was shown that the
retro thiaꢀMichael reaction to regenerate the parent drug GOꢀ
Y030: the more hydrophilic or polar functionality the
Sꢀsubstituent exerts a marked impact on the
S
ꢀ
substituent equipped with, the more efficient the retro thiaꢀ
Michael reaction. Note that GOꢀY139 and GOꢀY140, bearing a
highly hydrophilic
Sꢀsubstituent, exhibited a pronounced
reactivity in the retroꢀthiaꢀMichael reaction even in nearly
neutral medium (50% DMSOꢀpH7.3 buffer). In contrast, GOꢀ
Y178, GOꢀY144, GOꢀY187, and GOꢀY189, bearing a highly
which bear a lipophilic Sꢀalkyl chain (Figures S3, S6). The
quite similar structureꢀreactivity profiles were observed in the
retro thiaꢀMichael reaction of a panel of GOꢀY030ꢀmonoꢀthiolꢀ
hydrophobic
Sꢀsubstituent, underwent the retro thiaꢀMichael
reaction only sluggishly to give GOꢀY030 in less than 17%
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yield. It was unexpected to find that GOꢀY178 and GOꢀY185 acceptor and that GOꢀY030ꢀthiolꢀadducts elicited cytotoxicity
showed the marked difference in retro thiaꢀMichael reaction, depending on the structure of ꢀsubstituents and the pH of the
suggesting that even a change (ꢀCH2ꢀ to ꢀOꢀ) at the ꢀposition reaction medium. Our study suggests that a reversible thiaꢀ
S
ε
from sulfur exerted impact on the reactivity, probably by Michael/retro thiaꢀMichael system between C5ꢀcurcuminoid
facilitating solvation of the reactive site. Further implication on and biothiols is operative in cells. Some GOꢀY030ꢀthiolꢀ
the solvation issue would be provided from ClogP values: GOꢀ adducts exhibited identical cytotoxicity to GOꢀY030, indicating
Y030ꢀthiolꢀadducts with Clog P > 10 did not undergo retroꢀ their potential as prodrugs. The results on the retro thiaꢀ
Michael reaction (see SI Table S1).
Michael reaction of GOꢀY030ꢀthiolꢀadducts coupled with the
Our results also indicated that there is a correlation between cytotoxicity should inspire new avenues for the design of
the tendency towards the retro thiaꢀMichael reaction and the prodrugs derived from Michael acceptors.
cytotoxicity of GOꢀY030ꢀthiolꢀadducts: GOꢀY030ꢀthiolꢀ
adducts that are prone to regenerate GOꢀY030 through the
retroꢀMichael reaction exhibit comparable cytotoxicity to GOꢀ
Y030. In this context, GOꢀY187 represents a surprising
exception: comparison with GOꢀY144 illuminates impact of
Experimental
Supplementary data associated with this article can be found, in
the online version, at xxx.
etheric O atom at
ηꢀposition from sulfur atom, which might
affect on the solvation or aggregation. Relating to this issue,
the salient differences observed between particular monoꢀthiolꢀ
adducts and bisꢀthiol adducts, namely, (i) GOꢀY188 and GOꢀ
Y189, and (ii) GOꢀY177 and GOꢀY178, in terms of reactivity
toward retro thiaꢀMichael reaction as well as the
antiproliferative activity against HCTꢀ116 would indicate that
monoꢀthiolꢀadducts undergo a more facile retro thiaꢀMichael
reaction.
That GOꢀY030ꢀthiolꢀadducts bearing a hydrophilic group ꢀ
in particular, GOꢀY140 (bisꢀglutathioneꢀadducts of GOꢀY030)ꢀ
are freely miscible in water (Figure S14), will encourage further
studies for the clinical use. In this context, Snyder and coꢀ
workers should be credited for their pioneering work on the
development of EF24ꢀ(GSH)2 which is, to the best of our
knowledge, the first waterꢀsoluble prodrug of C5ꢀcurcuminoid
Acknowledgements
This research is partially supported by JSPS KAKENHI Grants
Number 22501041, 25670051, and by the Platform Project for
Supporting in Drug Discovery and Life Science Research（Platform
for Drug Discovery, Informatics, and Structural Life Science）from
the Ministry of Education, Culture, Sports, Science and Technology
(MEXT), and Japan Agency for Medical Research and development
(AMED).
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Sato, N. Kanoh, C. Ishioka, H. Shibata and Y. Iwabuchi,
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that employs the reversible thiaꢀMichael reaction.
The
structureꢀactivityꢀrelationship (SAR) information on the
reactivity of GOꢀY030ꢀthiolꢀadducts toward retroꢀthiaꢀMichael
reaction gained in this study will be useful for designing a
prodrug with advanced pharmacological properties based on
retro thiaꢀMichael reactions.
The SAR information of GOꢀY030ꢀthiolꢀadducts also
provides a consistent rationale why C5ꢀcurcuminoids induce
multiple biological activities and interact with various
molecules3a, such as FUBP24, Trxꢀ1, GSH,11 and Keap1,12 in
either an irreversible or reversible manner. C5ꢀcurcuminoids
experience a random thiaꢀMichael reaction with biothiols and a
retro thiaꢀMichael reaction depending on the nature of their
thiol, in which equilibrium dominates its temporal binding until
C5ꢀcurcuminoids lose reactivity. A similar reversible Michael
system was proposed by Suzuki and coworkers to explain
influxꢀefflux phenomena observed for cytotoxic prostaglandin
A (PGA) methyl ester in glutathione in cells.13 The design and
synthesis of thiaꢀMichaelꢀadductꢀtype prodrugs may promote
the clinical use of C5ꢀcurcuminoids for cancer chemotherapy.
2.
3.
4.
H. Yamakoshi, N. Kanoh, C. Kudo, A. Sato, K. Ueda, M.
Muroi, S. Kon, M. Satake, H. Ohori, C. Ishioka, Y. Oshima,
H. Osada, N. Chiba, H. Shibata and Y. Iwabuchi, ACS Med.
Chem. Lett., 2010, 1, 273.
Conclusions
In summary, we have demonstrated that the cytotoxic C5ꢀ
curcuminoid GOꢀY030 is potentially a reversible thiaꢀMichael
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DOI: 10.1039/C6OB01771A
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