Design and synthesis of novel iminothiazinylbutadienols and divinylpyrimidinethiones as ARE inducers

Article abstract of DOI:10.1016/j.bmcl.2013.12.072

Novel iminothiazinylbutadienols and divinylpyrimidinethiones were designed and synthesized as analogues of curcumin with its diketone moiety masked as a heterocyclic adduct with thiourea. The chemical stability of these novel heterocyclic compounds was improved as compared to curcumin. They exhibit longer half-lives and do not react with nucleophilic thiols under physiological conditions. In an ARE-luciferase reporter assay, some of these new curcumin analogues are more effective ARE activators than curcumin and isothiocyanates.

Full text of DOI:10.1016/j.bmcl.2013.12.072

Bioorganic & Medicinal Chemistry Letters 24 (2014) 940–943
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel iminothiazinylbutadienols
and divinylpyrimidinethiones as ARE inducers
Lin Chen a, Sadagopan Magesh a, Hong Wang b, Chung S. Yang b,e,f, Ah-Ng Tony Kong c,e,f, Longqin Hu a,d,e,f,
⇑
a Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
b Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
c Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
d School of Pharmaceutical Sciences, Shanxi Medical University, Taiyuan 030001, PR China
e The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
f Center for Cancer Prevention Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel iminothiazinylbutadienols and divinylpyrimidinethiones were designed and synthesized as ana-
logues of curcumin with its diketone moiety masked as a heterocyclic adduct with thiourea. The chemical
stability of these novel heterocyclic compounds was improved as compared to curcumin. They exhibit
longer half-lives and do not react with nucleophilic thiols under physiological conditions. In an ARE-
luciferase reporter assay, some of these new curcumin analogues are more effective ARE activators than
curcumin and isothiocyanates.
Received 10 October 2013
Revised 15 December 2013
Accepted 17 December 2013
Available online 24 December 2013
Keywords:
Curcumin
Ó 2014 Elsevier Ltd. All rights reserved.
Iminothiazinylbutadienols
Divinylpyrimidinethiones
Anti-inflammatory agents
ARE induction
Curcumin, the yellow principal polyphenol curcuminoid, was
isolated from the popular Indian spice turmeric (Curcuma longa).1
It has been shown to activate antioxidant response element (ARE)
genes. The activation of ARE genes lead to the enhanced expression
of oxidative stress response enzymes that function as a cytoprotec-
tive shield against carcinogens, reactive intermediates, and inflam-
mation.2–6 Curcumin exhibits a variety of biological and cellular
activities including antioxidant, anti-inflammatory, anti-carcino-
genic and hypocholesterolemic properties.6–11 It has been evalu-
ated in clinical trials as therapeutic and chemopreventive agents
for conditions involving inflammation such as multiple myeloma,
pancreatic cancer, colon cancer, psoriasis, and Alzheimer’s dis-
ease.12–18 Unfortunately, the potential clinical utility of curcumin
is limited by its low potency, fast metabolism, poor bioavailability
and selectivity.18–22
During the last decade, many synthetic modifications of curcu-
min have been carried out to enhance its biological activities and to
improve its pharmacokinetic properties. The potential utility of
curcumin is limited partly due to its chemical and metabolic
instability. Curcumin decomposes rapidly under neutral and basic
conditions.22 The presence of the active methylene group and
diketone moiety contributes to the instability of curcumin under
physiological conditions and its fast metabolism.22–24 In vivo,
recent studies indicate that the diketone moiety appears to be a
specific substrate of a series of aldo–keto reductases and can
decompose rapidly.24–26 The stability, metabolic profiles and bio-
logical activities of curcumin could be enhanced by modifying its
diketone moiety.15,27–29 Heterocycles are commonly found in syn-
thetic bioactive small molecules that exert specific biological
effects.30 Curcumin analogues bearing certain heterocycles also
showed improved anticancer and anti-inflammatory activi-
ties.29,31,32 Thus, our design is to mask the chemically reactive
diketone moiety of curcumin through the conjugation with differ-
ent thioureas and the introduction of one heterocyclic ring struc-
ture to curcumin while retaining most of the other curcumin
structure features in efforts to reduce its cytotoxicity and systemic
side effects and to increase chemical stability without adversely
affecting its anti-inflammatory activity. The introduction of basic
heterocyclic nitrogen may provide an opportunity to convert the
target compounds into their salt forms and potentially benefit
the aqueous solubility. The rigidity and hydrogen bonding
potential of the heterocyclic ring introduced might also provide
additional conformational restriction and achieve certain target
selectivity for these curcumin analogues.30,33
Curcumin has two Michael acceptors when it exists in the
keto form. The diketone moiety can accept a variety of nucleo-
philes such as thiols and amines via Michael addition
⇑
Corresponding author.
E-mail address: LongHu@rci.rutgers.edu (L. Hu).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.072

L. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 940–943
941
O
O
O
OH
O
O
O
O
HO
OH
HO
OH
curcumin (diketo form)
curcumin (enol ketone form)
HN
R
S
NH2
thiourea
H
a
b
c
d
e
f
R =
R =
R =
R =
R =
R =
R =
R
N
S
N
OH
O
O
O
N
O
HO
OH
iminothiazinylbutadienol (Ia-g)
O
O
S
R
O
O
N
N
g
O
O
HO
OH
divinylpyrimidinethione (IIa-g)
Figure 1. Design of novel iminothiazinylbutadienols (Ia–g) and divinylpyrimidinethiones (IIa–g) as conjugates of curcumin and thiourea.
reactions.32,34,35 However, the tautomeric enol form is energeti-
cally more stable which means one of the two Michael acceptors
in curcumin is more reactive towards nucleophilic attack. Thus,
we masked as shown in Figure 1 one of the reactive Michael
acceptors via conjugation with thioureas derived from the
chemopreventive isothiocyanates and formed a novel class of
heterocyclic curcumin analogues iminothiazinylbutadienols
(Ia–g), which could rearrange to form the second novel class of
heterocyclic analogues, divinylpyrimidinethiones (IIa–g). The
second series of compounds effectively masks both of the
Michael acceptors in curcumin. We further evaluated the effect
of introducing different substituents to the imino group on the
curcumin was carried out in the presence of 4 N HCl in dioxane.
Nucleophilic attack of the sulfur in thioureas to the b-carbon of
the Michael acceptor in the enol ketone form of curcumin under
the acidic conditions give the Michael adduct intermediates, which
then cyclize with the elimination of water to afford the desired
iminothiazinylbutadienol products (Ia–g). The thiazine ring can
open up in the presence of K2CO3 in DMF and then cyclize through
the addition of the thiourea nitrogen to the carbonyl carbon of the
second Michael acceptor with the elimination of water to give the
second series of desired curcumin analogues, divinylpyrimidin-
ethiones (IIa–g). Generally, the reactions proceed in good yields
ranging from 44% to 79%.
heterocyclic thiazine ring in series
pyrimidine nitrogen in series II analogues to derive valuable
structure–activity relationships (SAR).
The synthesis of the novel heterocyclic curcumin analogues is
shown in Scheme 1. Thioureas are either commercially available
or readily prepared from the reaction between isothiocyanates
and ammonia. The acid-catalyzed addition of thioureas to
I
analogues and to the
The chemical reactivity of curcumin is well known as evidenced
by the adduct formation when it is incubated with glutathione
(GSH).36 Indeed, when curcumin was incubated with 1 mM GSH
in pH 7.4 buffer at 37 °C, it forms a 1:2 adduct with GSH as indi-
cated by LC–MS analysis (data not shown). However, when our no-
vel heterocyclic curcumin analogues were incubated with GSH
under the same conditions, no GSH adducts were detected using
N
R
C
S
isothiocyanate
NH3
R
R
N
R
N
HN
NH2
O
S
N
OH
O
OH
S
OH
NH2
thiourea
S
O
O
O
O
O
O
-H2O
HCl/dioxane
HO
OH
HO
OH
HO
OH
curcumin
iminothiazinylbutadienol (Ia-g)
R
N
N
S
R
S
O
N
N
K2CO3
DMF
-H2O
O
HO
O
O
O
OH
HO
OH
divinylpyrimidinethione (IIa-g)
Scheme 1. Synthesis of novel iminothiazinylbutadienols (Ia–g) and divinylpyrimidinethiones (IIa–g) from curcumin and isothiocyanates.

942
L. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 940–943
the
analogues.
iminothiazinylbutadienol and divinylpyrimidinethione
100
80
60
40
20
0
To investigate the stability of curcumin analogs under cell cul-
ture conditions, we determined the changes in HPLC peak area of
curcumin and its analogues upon incubation in Dulbecco’s Modified
Eagle’s Medium (pH 7.4) containing 5% methanol in the absence or
presence of 1% FBS. Methanol was used to make sure that curcumin
and its analogues were fully soluble under the incubation condi-
tions. As shown in Figure 2, it was found curcumin degraded very
rapidly with a half-life of about 46 min in the absence of FBS. In
the presence of 1% FBS, the stability of curcumin was also poor,
about 50% of the curcumin was degraded after 2 h of incubation.
In contrast, the novel heterocyclic curcumin analogues we prepared
are much more stable. For example, compound Ib and IIb had only
about 25% decomposition after 72 h of incubation under the same
conditions in the presence or absence of FBS (Fig. 2).
t1/2
144.4 min
46.5 min
>72 h
>72 h
>72 h
Cur w/ 1%FBS
Cur
Ib w/ 1% FBS
Ib
IIb w/ 1% FBS
>72 h
IIb
0
10
20
30
40
50
60
70
Time (h)
The new curcumin analogs were submitted to NCI Developmen-
tal Therapeutics Program (DTP) for cell growth inhibition evalua-
tion in the NCI-60 Human Tumor Cell Line Screen.37 The NCI60
screening at NCI is a two-stage process, beginning with the evalu-
ation of all compounds against the 60 cell lines at a single dose of
Figure 2. Stability study of curcumin and representative analogues Ib and IIb as
monitored by changes in peak area at 220 nm using an HPLC assay.
LC–MS. Their lack of reactivity towards GSH suggests that we have
successfully masked the reactive Michael acceptors of curcumin in
10 lM. Compounds that exhibit significant growth inhibition are
Table 1
The cytotoxicity and ARE induction of novel iminothiazinylbutadienols and divinylpyrimidinethiones in the NCI60 Screen
R
N
S
R
S
N
OH
N
N
O
O
O
O
HO
OH
HO
OH
iminothiazinylbutadienols (Ia-g)
divinylpyrimidinethiones (IIa-g)
b
Compd
Ia
R
NSC# (Lab ID)
762482 (LH502)
762483 (LH503)
% Mean cell growtha (% at 10
l
M)
Average GI50
2.17
(lM)
ARE inductionc
507, 3317
H
37.84
3.61
Ib
2.07
131, 1083
O
Ic
762485 (LH505)
762487 (LH507)
70.93
0.50
16.5
2.32
94, 420
N
O
f
Id
—
O
f
Ie
If
762489 (LH510)
762484 (LH504)
60.56
54.9
—
O
d
f
101.57
—
—
O
O
f
Ig
762520 (LH509)
762502 (LH525)
762503 (LH526)
10.85
93.53
36.61
2.41
—
d
H
IIa
IIb
—
93, 77
6.87
125, 1635
O
d
IIc
762505 (LH528)
762507 (LH530)
85.22
—
124, 4217
N
O
f
IId
À18.35
1.67
9.51
—
O
O
f
IIe
762509 (LH532)
36.95
—
d
f
IIf
762504 (LH527)
762508 (LH531)
66.43
—
—
O
O
f
IIg
À5.50
2.88
—
CUR
PEITC
SFN
32982
87868
749790
37.2
52.18
87.25
8.04
1.09
—
198, 2093
—
413, 582
f
e
a
b
c
d
e
f
Mean cell growth (% relative to control) in the NCI60 single dose assay at 10
Average GI50 M) in the NCI60 5-dose testing.
lM.
(
l
ARE-Luciferase reporter assay in SW480 cells at 1
lM and 10 lM (for comparison, the blank control has a luminescence count of 119 11).
Not selected for 5-dose testing.
5-Dose testing data not available in the NCI60 database.
Not tested.

L. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 940–943
943
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.
072.
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and antioxidative assays.
In summary, we have designed and synthesized two series of
novel heterocyclic curcumin analogues combining with the
structure features of isothiocyanates. These novel analogues
were designed to mitigate the chemical reactivity of both curcu-
min and isothiocyanates, both of which are known to react with
biological nucleophiles and be cytotoxic. The new compounds
are chemically more stable with much longer half-lives under
physiological conditions and do not react with nucleophilic thi-
ols such as GSH while some of them demonstrated stronger
ARE induction in an ARE-luciferase reporter assay. Further eval-
uation of these promising heterocyclic curcumin derivatives are
underway.
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This project was funded in part by grants CA133791 and
CA125868 from the National Institutes of Health.