Synthesis and anti-inflammatory properties of some aromatic and heterocyclic aromatic curcuminoids

Article abstract of DOI:10.1016/j.bioorg.2011.11.004

A variety of novel aromatic and heterocyclic aromatic curcuminoids were synthesised, characterised and their anti-inflammatory activities (AIA) determined in vivo. Some of these compounds also were tested for inflammatory mediator production. The AIA of t

Full text of DOI:10.1016/j.bioorg.2011.11.004

Bioorganic Chemistry 40 (2012) 30–38
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and anti-inflammatory properties of some aromatic
and heterocyclic aromatic curcuminoids
a
M. Akram Khan a, , Riyad El-Khatib , K.D. Rainsford a,b, M.W. Whitehouse b
⇑
a Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S11WB, UK
b Therapeutics Research Unit, Department of Medicine, University of Queensland, Princess Alexandra Hospital, Buranda, Queensland 4102, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2011
Available online 25 November 2011
A variety of novel aromatic and heterocyclic aromatic curcuminoids were synthesised, characterised and
their anti-inflammatory activities (AIA) determined in vivo. Some of these compounds also were tested
for inflammatory mediator production. The AIA of the main representatives of these compounds were
assessed by oral administration to female Wistar rats using (a) acute carrageenan-induced paw oedema,
(b) chronic adjuvant arthritis (therapeutic mode), and (c) anti-pyretic activity assessed in the yeast pyr-
exia. Gastric ulceration was determined in pre-inflamed rats. Natural curcumin showed modest aspirin-
like anti-inflammatory activity which was enhanced when co-administered with the PGE1 analogue
misoprostol as a synergist. In contrast, four novel curcuminoids (RK-97, RK-103, RK-104 and RK-106)
in which the bis-methoxy-phenyl group of curcumin was replaced with bis-dimethoxybutenolidyl-
(ascorbate), bis-naphthyl, and bis-furanyl derivatives, respectively, had potent activity in the anti-
arthritic assay with little gastric or systemic toxicity, compared with the vehicle-treated controls. Of
Keywords:
Anti-inflammatory
Curcumin
Curcuminoids
Heterocyclic
Ascorbic acid
In vivo assays
Organic synthesis
Spectroscopy
the curcuminoids the furan RK-106 was the only compound to inhibit production of TNF
a and IL-1b in
a monocytic cell-line THP-1 in vitro. The inactivity of RK-106 on the production of PGE2 may be related
to its absence of gastrotoxicity. None of the curcuminoids exhibited anti-pyretic activity and this may
also be related to its insensitivity to PGE2. Thus, these novel curcuminoids, such as RK-106, may warrant
the development of new low gastro-toxic anti-inflammatory agents with selective inhibitory activity of
cytokine inflammatory mediators.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
[7,9–16]. These activities may be potentially important for the con-
trol of osteoarthritis (OA) [17] and pro-apoptotic and growth inhib-
Curcumin (1) is the yellow pigment and main biologically active
component of turmeric, a spice that comes from the Curcuma longa
rhizome (Zingaberaceae) [1,2]. Tumeric has been long used as a sea-
soning ingredient, the main ingredient in Indian curry, and for its
diverse therapeutic activities such as, inflammation and cancer
[1–8]. Tumeric contains approximately 5% curcumin and two other
compounds demethoxycurcumin (DMC), and bisdemethoxycurcu-
min (BDMC) [5]. The metabolism of curcumin involves reduction to
tetrahydrocurcumin (THC), hexahydrocurcumin (HHC) and hexa-
hydrocurcuminol (HCC) with demethylation and conjugation with
glucuronic acid and sulphate [1].
itory effect in OA synoviocytes [18].
Curcumin and its synthetic derivatives (curcuminoids) have
been investigated for their anti-inflammatory and anti-arthritic
activities [5,6,10,11,13,19,20]. Amongst these THC, HHC, HCC and
some demethoxy- and diacetyl-derivatives of curcumin have been
investigated [5,6,10,11,13,19]. Except THC, these derivatives show
variable in vivo anti-inflammatory activities but diminished inhib-
itory activity than curcumin [5,6]. One of the studies has reported
that an Ar-para hydroxyl group is essential for anti-inflammatory
activity of curcumin [5]. Many of the reported curcuminoids have
simple ring substitutions (e.g. diacetyl groups) or without methyl,
methoxy group or hexahydro-reductions of the diethylene compo-
nents. To our knowledge the effects on anti-inflammatory activity
of substitution of benzene rings with furans, thiophenes, naphthyl,
or ascorbate derivatives of curcumin have not been previously
reported.
The anti-inflammatory actions of curcumins include anti-oxi-
dant activity, inhibition of signal transduction via NFjB and AP-
1, inhibition of phospholipase A2, cyclo-oxygenases (COX), lipoxy-
genases (LOX) involved in leukotriene synthesis and nitric oxide
The aim of this work was to synthesise a number of curcumi-
noids as well as some variants of substituted methoxy- or demeth-
oxy-derivatives of curcumin. Selected biological activities of these
compounds are also being reported (Tables 1–3).
⇑
Corresponding author. Fax: +44 1142253066.
E-mail address: m.a.khan@shu.ac.uk (M.A. Khan).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.11.004

M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
31
RK 97 and RK 103 showed an additional one proton singlet reso-
nating at d 7.27 at room temperature which we interpreted as eno-
lic proton resonance.
2. Results and discussion
2.1. Chemistry
In all of the mass spectra for the curcuminoids the molecular
ion was evident and its accurate mass was measured, Tables 1–3.
The prominent fragmentation, observed in all cases is shown in
Fig. 1. The prominent ion at m/z [Ar–CH@CH–CO]+ resulting from
molecular cleavage in EI mode was observed as base peak. Many
of these curcuminoids showed the same fragmentation forming
the base peak which could be used as a marker ion for the identi-
fication of curcuminoids structures.
Synthesis of curcumin was first reported by Lampe and co-
workers in 1913 [21] and later a modified procedure was published
by Pabon [22] and Pederson et al. [23,24]. All our curcuminoids
were synthesised by Pabon procedure involving a reaction be-
tween the appropriate aldehyde and acetylacetone–boric anhy-
dride complex as shown in Scheme1[25]. In this reaction the
active a-methylene group in acetylacetone is protected in the form
Curcuminoid RK 97 involved an eight-step synthesis starting
of an enolate complex with boron which consequently allows the
desired condensation reaction to occur between the free methyl
groups of acetylacetone and the appropriate aldehyde.
from
the synthesis of
recognition that the anti-oxidant activity of
L
-ascorbic acid [27,28] (Fig. 2). The twofold rationale behind
-ascorbic acid derivative RK97 was based on the
-ascorbic acid is con-
L
L
The compounds RK78, RK 87 [5,25], RK 88-RK 91 [5], RK 92
[25,26], RK 107 [20], RK 99 and RK 106 [26] have already been re-
ported in the literature and we have synthesised them by Pabon
method [25]. All the synthesised curcuminoids were characterised
spectroscopically using 1H and 13C nuclear magnetic resonance and
mass spectrometry data. The proton magnetic resonance spectra
indicated that all the curcuminoids existed entirely in the keto-
enol form rather than the diketo form in deuterochloroform solu-
tion. This was supported by the fact that the H NMR spectra of
fined to the ene-diol system [28] and secondly ascorbic acid would
enhance the water solubility of the curcuminoid molecule possibly
enhancing its bioavailability. It is known that curcumin is poorly
absorbed in the biosystem and therefore its water soluble ascor-
bate derivative may be an added advantage for its absorption
[29]. The RK 97 is a pro-drug with an ene-diol system in its methyl
ether form which in vivo could degrade to the free 1,2-dihydroxy
system.
Table 1
Physical data of substituted aromatic curcuminoids.
R2
R2
R3
R1
R1
R3
3'
6'
3'
4'
5'
4'
5'
2'
1'
2'
1'
6'
3
7
1
5
6
2
4
O
O
H
1,7-bis(aryl)-1,6-hepadiene-3,5-dione (enol form)
Code RK
R1
R2
R3
H
OCH3
H
OCH3
CH3
H
Yield (%)
Rf (solvent)h
Mp (°C)
Formula
High resolution MS (M+)
Found (calculated)
78
87
88
89
90
91
H
H
OCH3
H
H
H
H
H
OCH3
H
78.3
57.6
58.3
59
73
37
0.86
0.62
0.64
0.12
0.80
0.62
138–140a
C19H16O2
C21H20O4
C21H20O4
C23H24O6
C21H20O2
C21H20O4
276.1142 (276.1150)
336.1336 (336.1362)
336.1368 (336.1362)
396.1564 (396.1573)
304.1466 (304.1463)
336.1347 (336.1362)
164–167b,c
110–112d
134–136e
198–202f
Gumg
H
OCH3
a
b
c
Lit 140–142 °C [3].
Lit154–155 °C [3].
Lit 164–165 °C [12].
Lit 121–122 °C [3].
Lit 128–130 °C [3].
Lit 205–206 °C [3].
Lit 72–74 °C [3].
d
e
f
g
h
Ethyl acetate:petroleum ether, (1:2).

32
M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
Table 2
Physical data for additional curcuminoids.
H
Hc
O
O
Hc
2
4
6
Ring
5
3
7
1
Ring
Hb
Ha
Hb
Ring
Ring
Ring
O
O
3'
4'
2'
2'
3'
4'
1'
1'
6'
O
1'
O
2'
3'
5'
4'
5'
9'
10'
5'
8'
RK107
6' 7'
H3CO
OCH3
RK 97
RK103
Code RK
Yield (%)
Rf (solvent)a
Mp (°C)
Formula
High resolution MS (M+)
Found (calculated)
97
103
107
50
79
61.5
0.30
0.83
0.82
>200 dec.
167–169
193–195
C
C
C
21H20O10
27H20O2
21H16O6
432.1048 (432.1058)
376.1453 (376.1464)
364.0943 (364.0948)
a
Ethyl acetate:petrol (1:2).
Table 3
Physical data of heterocyclic aromatic curcuminoids.
1,7-bis(heterocyclyl)-1,6-heptadiene-3,5-dione (enol form)
Code RK
R1
R2
X
Yield (%)
Rf (solvent)a
Mp (°C)
Formula
High resolution MS (M+)
Found (calculated)
92
99
104
105
106
H
H
H
CH3
H
H
H
CH3
H
CH3
O
S
S
S
O
26
28
40
12
63
0.73
0.81
0.83
0.85
0.83
Oil
C
C
15H12O4
15H12O2S2
256.0739 (256.0736)
288.0278 (288.0279)
316.0594 (316.0592)
316.0605 (316.0592)
284.1044 (284.1049)
184–186
130–132
109–111
144–146
C17H16O2S2
C17H16O2S2
C17H16O4
a
Ethyl acetate:petroleum (1:2).
2.2. Pharmacology
some disease-modifying anti-rheumatic drugs (DMARDs) such as
levamisole and gold salts [30,31]. The time-course of inflammation
is shown in Fig. 3. The most potent anti-inflammatory activity of
RK92 and RK 99 was observed at 1 and 2 h after induction of
inflammation. Curcumin itself showed minimal nonsteroidal
anti-inflammatory drug (NSAID) activity unless co-administered
with the PGE1 analogue, misoprostol, as a synergist [32]. The use
of misoprostol was intended to replicate conditions where inflam-
2.2.1. In vivo activities
In the acute anti-inflammatory assays in carrageenan hind paw
oedema compounds RK-92, AK-99 and RK-97 showed inhibitory
activity in order of decreasing potency (Table 4). Paradoxically,
RK-104 and RK-107 showed enhanced paw swelling indicating
their proinflammatory activity. This observation is a feature of

M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
33
Scheme 1. Synthesis of curcuminoids from acetylacetone by the Pabon method [21].
Fig. 1. Synthesis of RK 97 from L-ascorbic acid.
mation could be induced with enhanced anti-inflammatory activ-
ity of the drug.
RK-107, RK-97 as well as curcumin itself (Table 4). These
compounds showed little immediate toxicity, evidenced by
normal weight gain and improved physical condition of the ani-
mals, compared with the vehicle-treated controls. After with-
drawal of the drug a slow rebound of arthritis was observed
indicating that the treated animals had responded to the arthriti-
gen, i.e. this was not a false-positive result for the applied test
compounds.
In contrast to aspirin and some enolic anti-inflammatory drugs
(phenylbutazone, piroxicam), none of these curcuminoids caused
gastric irritation sufficient to induce local bleeding into the gastric
mucosa (data not shown).
Curcumin did not show anti-pyretic activity at doses up to
300 mg/kg, in contrast to some phenols e.g. paracetamol, salicylic
acid/alcohol/amide and some enolic drugs (phenylbutazone, pirox-
icam) which rapidly reduced pre-established fever within 60 min
after oral administration.
At 24 h after oral administration many of the curcuminoids as
well as aspirin showed a rebound in hindpaw inflammationwhich
is a typical effect of anti-inflammatory agents.
By contrast, the most potent analogues in the chronic
anti-arthritic assay were RK-103 and AK-104, followed by

34
M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
Fig. 2. Synthesis of RK 97 from L-ascorbic acid.
Table 4
Anti-inflammatory activities of some curcuminoids in female Wistar rats.
Compound
Dose mg/kg p.o.
Anti-oedemic activitya
Dose mg/kg p.o.
Anti-arthritic activityb
1 h
39%
2 h
Arthritis score
Rear paw swelling
Fore paw inflamm.
DWt gm
RK78
100
37%
RK88
RK89
RK92
RK97
RK99
RK103
RK104
RK-106
RK107
40
100
100
100
100
100
100
100
100
100
32%
10
40
55
05
78
78
37
65
75
18%
36
32
103
08
70
85
27
52
79
17%
28
28
80
0
30
84
21
72
36
+11
+10
+06
+08
+11
+17
+12
+15
+17
+13
100
100
100
100
100
100
100
100
12
51
37
41
17
45
55
37
52
0
ꢀ75c
ꢀ24
ꢀ61
18
ꢀ55
14
ꢀ36
32
64
Curcumin (Cmn)
Cmn and MPLd
Aspirin
100
100 and 0.6
150
61
44
62
Phenylbutazone
80
24
36
Data = mean percentage inhibition of signs of inflammation, compared to vehicle-treated control animals (n 6 4).
a
Measured in the carrageenan paw assay.
b
Measured in animals with pre-established adjuvant-arthritis at time of early onset and compared with untreated animals (see below).
Negative data indicate increase in paw oedema compared to controls.
MPL = Misoprostol co-administered as a synergist; this dose (0.6 mg/kg) alone being inactive.
c
d
Fig. 3. Effects of curcumin and its derivatives on carrageen – an-induced paw swelling in rats. Values are mean SEM.

M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
35
Fig. 4. TNF-a production in curcumin treated THP-1 monocytes (n = 4).
2.2.2. Reference data and in vivo inflammatory mediators
30 min. After stirring for 4 h the reaction mixture was left overnight
at room temperature. Dilute hydrochloric acid (0.4 M, 300 mL) was
added and the mixture was stirred in an oil bath at 60 °C for 1 h.
After cooling the organic layer was separated and the aqueous layer
was extracted several times with ethyl acetate or dichloromethane.
The combined organic layer was washed with water, dried over
anhydrous MgSO4 and evaporated to yield a gummy product which
crystallised from cold methanol. The crude curcuminoid was ana-
lysed by TLC [ethyl acetate:petroleum ether; 1:2] and further puri-
fied by flash chromatography using the same eluents.
Untreated controls (n = 12) had mean arthritis score (AS) = 2.7+
(SE 0.2+), mean rear paw swelling = 1.13 mm (SE 0.17) and mean
forepaw inflammation index (FI) = 2.5+ (SE = 0.31+). Normal rat
rear paws = 7.24 (SE 0.12) mm with zero AS and mean FI 6 0.2+.
[AS and FI were scored for each animal on a scale 0 to 4+].
Plasma concentrations of PGE2 and IL-1b were unaltered in ar-
thritic rats that were orally given curcumin and the other curcumi-
noids (data not shown). However, plasma levels of PGE2 were
reduced in the plasma of rats treated with aspirin (data not shown)
thus showing positive effects of this NSAID in the arthritic animals.
The characteristic spectroscopic properties for all the synthes-
ised curcuminoids RK78–RK107 were as follows:
2.2.3. In vitro production of inflammatory mediators
Of the curcuminoids assayed in THP-1 monocytic cells, only RK-
106 inhibited production of basal and LPS stimulated TNFa (Fig. 4).
This curcuminoid also inhibited LPS-stimulated, but not basal pro-
duction of IL-1b (data not shown). None of the compounds affected
production of PGE2, even though NSAIDs were effective in reducing
production of PGE2 in the cell line used in this study [37].
1,7-Diphenyl-1,6-heptadiene-3,5-dione (RK 78), light-yellow
powder; 1H NMR: (d, ppm, CDCl3), 5.87 (1H, s, @CHa), 6.65 (2H,
d, J = 15.5 Hz, 2x CHb@C), 7.27–7.42 (6H, m, 4x ArH3 and 2x
ArH4), 7.50–7.60 (4H, m, 4x ArH2), 7.68 (2H, d, J = 15.5 Hz, 2x
C@CHc); 13C NMR: (d, ppm,CDCl3), 183.34 (C-3 and C-5), 140.65
(C-1 and C-7), 135.00 (C-10), 130.14 (C-30), 128.95 (C-20), 128.14
(C-40), 124.08 (C-2 and C-6), 101.82 (C-4); MS (EI, m/z), 288.1
(M+, C19H16O2, 100%), 131.1 (Ph–CH@CH–CO, 54%).
1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione
(RK 79), orange powder; 1H NMR: (d, ppm, d6-DMSO/CDCl3), 3.47
(6H, s, 2x OCH3), 5.41 (1H, s, 2x@CHa), 6.03 (2H, d, J = 15.0 Hz, 2x
CHb@C), 6.42 (2H, d, J = 8.3 Hz, 2x ArH3), 6.60 (2H, d, J = 8.3 Hz,
2x ArH2), 6.65 (2H, s, 2x OH), 7.18 (2H, d, J = 15.0 Hz, 2x C@CHc),
7.26 (1H, s, enolic OH); 13C NMR: (d, ppm, d6-DMSO + CDCl3),
183.32 (C-3 and C-5), 149.48 (C-30), 148.21 (C-40), 140.84 (C-1),
126.83 (C-10), 123.00 (C-2), 121.18 (C-20), 115.87 (C-30), 110.62
(C-60), 101.21 (C-4), 55.94 (OCH3); MS (EI, m/z), 368.1 (M+,
3. Materials and methods
3.1. Chemistry
Melting points were recorded on Stuart SMP3 digital apparatus;
1H NMR spectra were recorded on a Brucker AC 250 MHz and 13C
NMR spectra were recorded on a Brucker Avance III 400 MHz spec-
trometers. Mass spectra (MS) were obtained on VG 770E spectrom-
eter in EI mode at 70 eV. TLC analyses were done using pre-coated
Merck silica gel-coated aluminium sheets and flash chromatogra-
phy was performed on BDH flash silica gel 300–400 meshand the
eluents are indicated in parenthesis for each compound. The 13C
NMR spectral interpretation was done using numbering system
indicated on the structure for curcumin (1) and the generalised
structures in Tables 1 and 2.
C21H20O6, 31%), 177.1 (Ar–CH@CH–CO, 100%).
1,7-Bis(4-methoxyphenyl)-1,6-heptadiene-3,5-dione (RK 87), or-
ange powder; 1H NMR: (d, ppm, CDCl3), 3.97 (6H, s, 2x OCH3),
5.98 (1H, s,=CHa), 6.65 (2H, d, J = 15.2 Hz, 2x CHb@C), 6.92 (8H, d,
2x ArH AB system), 7.36 (2H, d, J = 15.2 Hz, 2x C@CHc); 13C NMR:
(d, ppm, d6-DMSO + CDCl3), 183.31 (C-3 and C-5), 161.33 (C-40),
140.11 (C-1 and C-7), 129.88 (C-20), 127.60 (C-10), 121.81 (C-2
and C-6), 114.48 (C-30), 101.47 (C-4), 55.47 (OCH3); MS (EI, m/z),
336.1 (M+, C21H20O4, 62%), 161.0 (Ar–CH@CH–CO, 100%).
3.1.1. General procedure for curcuminoid synthesis [22,23]
The aromatic or heterocyclic aromatic aldehyde (0.4 mol) and
tributylborate [prepared by heating 2-butanol (44.4 g, 0.6 mol)
and boric acid (12.4 g, 0.2 mol) in dry toluene using a Dean–Stark
water separator] (210 mL, 184 g, 0.8 mol) were dissolved in dry
ethyl acetate (200 mL). Acetylacetone–boric acid complex [pre-
pared from acetyl acetone (20 g, 0.2 mol) and boric anhydride
(10 g, 0.14 mol)] was added and after stirring the reaction mixture
for 5 min, n-butylamine (4 mL) was added dropwise over a period
1,7-Bis(2-methoxyphenyl)-1,6-heptadiene-3,5-dione (RK 88), light
orange powder; 1H NMR: (d, ppm, CDCl3), 3.92 (6H, s, 2x OCH3),
5.89 (1H, s,=CHa), 6.74 (2H, d, J = 16.0 Hz, 2x CHb@C), 6.92–7.01
(4H, m, 2x ArH3 and 2x ArH4), 7.32 (2H, t, J = 7.8 Hz, 2x ArH5),
7.56 (2H, d, J = 7.8 Hz, 2x ArH6), 8.00 (2H, d, J = 16.0 Hz, 2x C@CHc);
13C NMR: (d, ppm, CDCl3), 55.53 (OCH3), 101.54 (C-4); 111.19 (C-
30), 120.76 (C-50), 124.07 (C-2 and C-6), 124.80 (C-60) 128.63

36
M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
13
(C-40), 131.30 (C-60), 135.75 (C-1 and C-7), 158.40 (C-20), 183.81 (C-
3 and C-5); MS (EI, m/z), 336.2 (M+, C21H20O4, 67%), 161.1 (Ar–
CH@CH–CO, 100%).
C@CHc); C NMR: (d, ppm, CDCl3), 102.26 (C-4), 123.49 (C-20),
124.93 (C-2 and C-6), 125.51 (C-80), 126.28 (C-60), 126.56 (C-70),
126.91 (C-30), 128.78 (C-50), 130.50 (C-40), 131.60 (C-90), 132.37
(C-100), 133.77 (C-10), 137.53 (C-1 and C-7), 183.32 (C-3 and C-
5); MS (EI, m/z), 376.3 (M+, C27H20O2, 3%), 195 (C14H12O, 2%), 181
(Ar–CH@CH–CO, 3%).
7-Bis(3,4-di-methoxyphenyl)-1,6-heptadiene-3,5-dione (RK 89),
golden brown powder. 1H NMR: (d, ppm, CDCl3), 3.92 (12H, m,
4x OCH3), 5.82 (1H, s,=CHa), 6.50 (2H, d, J = 15.5 Hz, 2x CHb@C),
6.88 (2H, d, J = 8.2 Hz, 2x ArH5), 7.08 (2H, s, 2x ArH2), 7.14 (2H, d,
J = 8.2 Hz, 2x ArH6), 7.61 (2H, d, J = 15.5 Hz, 2x C@CHc); 13C NMR:
(d, ppm, CDCl3),55.90 (OCH3), 55.97 (OCH3), 101.33 (C-4), 109.75
(C-20), 111.12 (C-50), 122.01 (C-2 and C-6), 122.63 (C-60), 128.07
(C-10), 140.38 (C-1 and C-7), 149.21 (C-40), 151.17 (C-30), 183.25
(C-3 and C-5); MS (EI, m/z), 396.2 (M+, C23H24O6, 1%), 191.0 (Ar–
CH@CH–CO, 17%).
1,7-Bis(4-methylphenyl)-1,6-heptadiene-3,5-dione (RK 90), light
mustard yellow powder. 1H NMR: (d, ppm, CDCl3), 2.39 (6H, s, 2x
CH3), 5.83 (1H, s,=CHa), 6.60 (2H, d, J = 16.0 Hz, 2x CHb@C), 7.20
(4H, d, J = 7.8 Hz, AB system 2x ArH3), 7.46 (4H, d, J = 7.8 Hz, AB
system 2x ArH2), 7.65 (2H, d, 16.0 Hz, 2x C@CHc); 13C NMR: (d,
ppm, CDCl3), 183.40 (C-3 and C-5), 140.56 (C-1 and C-7), 140.52
(C-40), 132.32 (C-10), 129.68 (C-30), 128.13 (C-20), 123.12 (C-2 and
C-6), 101.57 (C-4), 21.50 (CH3); MS (EI, m/z), 304.3 (M+, C21H20O2,
46%), 145.1 (Ar–CH@CH–CO, 100%).
1,7-Bis(3-methoxyphenyl)-1,6-heptadiene-3,5-dione (RK 91), red-
dish orange powder. 1H NMR: (d, ppm, CDCl3), 3.84 (6H, s, 2x
OCH3), 5.83 (1H, s,=CHb), 6.59 (2H, d, J = 16.0 Hz, 2x CHb@C),
7.39–6.89 (8H, m, ArH), 7.61 (2H, d, J = 16.0 Hz, C@CHc); 13C
NMR: (d, ppm, CDCl3), 55.28 (OCH3), 101.94 (C-4), 113.16 (C-20),
115.95 (C-40), 120.83 (C-60), 124.35 (C-2 and C-6), 129.94 (C-50),
136.43 (C-10), 140.60 (C-1 and C-7), 159.93 (C-30), 183.28 (C-3
and C-5); MS (EI, m/z), 336.3 (M+, C21H20O4, 39%), 161.1 (Ar–
CH@CH–CO, 100%).
1,7-Bis(2-furanyl)-1,6-heptadiene-3,5-dione (RK 92), dark red oil.
1H NMR: (d, ppm, d6-DMSO), 6.20 (1H, s,=CHa), 6.57 (2H, d,
J = 15.8 Hz, CHb@C), 6.67 (2H, dd, J = 1.5 Hz and 3.1 Hz, 2x ArH4),
6.97 (2H, d, J = 3.1 Hz, 2x ArH5), 7.45 (2H, d, J = 15.8 Hz, 2x C@CHc),
7.88 (2H, s, 2x ArH3); 13C NMR: (d, ppm, CDCl3), 183.30 (C-3 and C-
5), 151.65 (C-20), 143.75 (C-50), 139.05 (C-1 and C-7), 129.40 (C-2
and C-6), 113.88 (C-30), 112.82 (C-40), 101.08 (C-4); MS (EI, m/z),
256.1 (M+, C15H12O4, 77%), 175 (C10H7O3, 15%), 135 (C8H8O2,
14%), 121 (Ar–CH@CH–CO, 100%), 107 (C7H7O, 25%), 94 (C6H6O,
31%), 81 (C5H5O, 41%) .
1,7-Bis[2-(5-methylthiophenyl)]-1,6-heptadiene-3,5-dione
(RK
104), firebrick powder. 1H NMR: (d, ppm, CDCl3), 2.51 (6H, s, 2x
CH3), 5.70 (1H, s,=CHa), 6.27 (2H, d, J = 15 Hz, 2x CHb@C), 6.73
(2H, d, J = 3.6 Hz, 2x ArH4), 7.06 (2H, d, J = 3.6 Hz, 2x ArH3), 7.68
(2H, d, J = 15 Hz, 2x C@CHc); 13C NMR: (d, ppm, CDCl3), 15.92
(CH3), 101.56 (C-4), 121.77 (C-2 and C-6), 126.76 (C-40), 131.64
(C-1 and C-7), 133.42 (C-30), 138.66 (C-20), 144.09 (C-50), 182.74
(C-3 and C-5); MS (EI, m/z), 316.3 (M+, C17H16O2S2, 42%), 298.3
(M-H2O, 31%), 218.3 (C12H10O2S, 17%), 191.2 (C10H7O2S, 15%), 165
(C9H9OS, 32%), 151.2 (Ar–CH@CH–CO, 86%), 111.1 (C6H7S, 40%),
45 (100%).
1,7-Bis[2-(3-methylthiophenyl)]-1,6-heptadiene-3,5-dione
(RK
105), reddish brown powder. 1H NMR: (d, ppm, CDCl3), 2.36 (6H,
s, 2x CH3), 5.70 (1H, s,=CHa), 6.34 (2H, d, J = 15.5 Hz, 2x CHb@C),
6.87 (2H, d, J = 5.1 Hz, 2x ArH4), 7.26 (2H, d, J = 5.1 Hz, 2x ArH5),
7.83 (2H, d, J = 15.5 Hz, 2x C@CHc); 13C NMR: (d, ppm, CDCl3),
14.24 (CH3), 102.06 (C-4), 122.36 (C-2 and C-6), 127.10 (C-20),
131.47 (C-40), 131.58 (C-50), 133.15 (C-1 and C-7), 141.45 (C-30),
182.80 (C-3 and C-5); MS (EI, m/z), 316 (M+, C17H16O2S2, 53%),
165 (C9H9OS, 16%), 151.0 (Ar–CH@CH–CO, 100%), 111 (C6H7S, 59%).
1,7-Bis[2-(5-methyfuranyl)]-1,6-heptadiene-3,5-dione (RK 106),
maroon powder. 1H NMR: (d, ppm, CDCl3), 2.38 (6H,s, 2x CH3),
5.71 (1H, s,=CHa), 6.09 (2H, d, J = 3.6 Hz, 2x ArH4), 6.42 (2H, d,
J = 15.5 Hz, 2x CHb@C), 6.51 (2H, d, J = 3.1 Hz, 2x ArH3), 7.33 (2H,
d, J = 15.5 Hz, 2x C@CHc); 13C NMR: (d, ppm, CDCl3), 13.97 (CH3),
102.08 (C-4), 109.19 (C-40), 116.72 (C-30), 120.15 (C-2 and C-6),
126.75 (C-1 and C-7), 150.39 (C-20), 155.55 (C-50), 182.84 (C-3
and C-5); MS (EI, m/z), 284.4 (M+, C17H16O4, 33%), 266.2 (M-H2O,
6%), 149 (C9H10O2, 8%), 135 (Ar–CH@CH–CO, 67%), 108.2 (C7H8O,
12%), 95.2 (C6H7O, 32%), 45 (100%).
1,7-Bis(3,4-methylenedioxyphenyl)-1,6-heptadiene-3,5-dione (RK
107), golden yellow powder. 1H NMR: (d, ppm, d6-DMSO/CDCl3),
5.73 (5H, s, 2x –OCH2– and @CHa), 6.18 (2H, d, J = 15.8 Hz, 2x
CHb@C), 6.53 (2H, d, J = 8.1 Hz, 2x ArH5), 6.74 (2H, d, J = 8.1 Hz,
2x ArH6), 6.76 (2H, d, J = 15.8 Hz, 2x C@CHc), 7.27 (2H, s, 2x
ArH2); 13C NMR: (d, ppm, d6-DMSO), 183.62 (C-3 and C-5),
149.76 (C-30), 148.62 (C-40),140.61 (C-1 and C-7), 129.67 (C-10),
125.52 (C-2 and C-6), 122.81 (C-60), 109.09 (C-50), 107.00 (C-20),
102.10 (O–CH2–O), 101.94 (C-4); MS (EI, m/z), 364 (M+, C21H16O6,
5%), 232 (C8H4O2, 33%), 189 (C11H9O3, 19%), 175 (Ar–CH@CH–CO,
37%), 149 (C9H9O2, 62%), 99 (73%), 57 (100%).
1,7-Bis(5-methylidenebutenolide)-1,6-heptadiene-3,5-dione (RK
97), sienna powder. 1H NMR: (d, ppm, CDCl3), 3.99 (3H, s, OCH3),
4.17 (3H, s, OCH3), 5.80 (1H, s,=CHa), 5.98 (2H, d, J = 11.3 Hz, 2x
CHd@C<), 6.18 (2H, d, J = 16.0 Hz, 2x CHb@C), 7.27 (1H, s, OH),
7.61 (2H, dd, J = 11.3 Hz and 16.0 Hz, 2x C@CHc); 13C NMR: (d,
ppm, CDCl3), 102.43 (C-5), 105.77 (C-1 and C-9), 125.85 (C-30),
130.19 (C-3 and C-7), 131.36 (C-40), 145.64 (C-50), 148.17 (C-2
and C-8), 163.02 (C-20), 182.45 (C-4 and C-6); MS (EI, m/z), 432
(M+, C21H20O10, 25%), 223 (C11H11O5, 11%), 209 (Ar@CH–CH@CH–
CO, 79%), 43 (100%).
3.2. Pharmacology
3.2.1. In vivo and in vitro activities
1,7-Bis(2-thiophenyl)-1,6-heptadiene-3,5-dione (RK 99), reddish
brown powder. 1H NMR: (d, ppm, CDCl3), 5.75 (1H, s,=CHa), 6.41
(2H, d, J = 15.5 Hz, 2x CHb@C), 7.07 (2H, s, 2x ArH3), 7.27 (2H, s,
2x ArH4), 7.39 (2H, s, 2x ArH5), 7.77 (2H, d, J = 15.5 Hz, 2x C@CHc);
13C NMR: (d, ppm, CDCl3), 182.85 (C-3 and C-5), 140.74 (C-20),
133.36 (C-1 and C-7), 131.06 (C-50), 128.39 (C-30), 128.30 (C-40),
123.04 (C-2 and C-6), 101.89 (C-4); MS (EI, m/z), 288.1 (M+,
Anti-inflammatory activity (AIA) was assessed after oral feeding
in female Wistar rats by two methods:
(A) Acute AIA was determined using the standard carrageenan-
induced paw oedema assay [30,31]. Female rats were employed as
these express greater inflammation in paws and joints than in male
animals of the same strain. Moreover, it is well-recognised that
rheumatoid arthritis (of which AIA attempts to replicate) is ex-
pressed predominantly in females. Changes in paw thickness were
measured with a micrometer 1, 2, and 24 h after inoculating Na
carrageenan (0.6 mg in 0.1 ml physiological saline) into each rear
paw. Test agents in reference drugs were administered orally sus-
pended in 0.02% Tween-20 and 5% (v/v) ethanol 45 min before
inoculating carrageenan.
C15H12O2S2, 49%), 270.1 (M-H2O, 24%), 204.1 (M-C4H4S, 14%), 177
(M-C6H7S, 19%), 151 (C8H7OS, 32%), 137 (Ar–CH@CH–CO, 100%),
109 (C6H5S, 55%), 97 (C5H5S, 52%).
1,7-Bis(1-naphthyl)-1,6-heptadiene-3,5-dione (RK 103), golden
yellow powder. 1H NMR: (d, ppm, CDCl3), 5.97 (1H, s,=CHa), 6.77
(2H, d, J = 15.0 Hz, 2x CHb@C), 7.27 (1H, s, OH), 7.52–7.61 (6H, m,
2x ArH3, ArH6 and ArH7), 7.82–7.90 (6H, m, 2x ArH4, ArH5 and
ArH8), 8.28 (2H, d, J = 7.7 Hz, 2x ArH2), 8.56 (2H, d, J = 15 Hz, 2x
(B) Chronic AIA was determined by suppression of arthritis in
rats developing adjuvant-induced polyarthritis [31,32]. Test agents

M.A. Khan et al. / Bioorganic Chemistry 40 (2012) 30–38
37
and reference drugs were administered once daily for four days only
beginning on day-10; day-0 = day of inoculating adjuvant into tail-
base. In some rats the prostaglandin analogue, misoprostol, was
co-administered with curcuminoid in order to enhance the expres-
sion of anti-inflammatory activity of the latter [32]. The following
signs of arthritis were measured on days-10, 14 and 17 (the latter
measurement indicated rebound after ceasing dosing on day-13):
(a) rear paw thickness, (b) fore paw inflammation, (c) an overall
arthritis score (these last two measurements made subjectively on
a scale 0 ? 4+), and (d) weight changes over days 10 through to 14.
Gastro-irritancy was measured in both normal and arthritic rats
fasted overnight, dosed orally with test compounds ( 0.13 M HCl
in water), then sacrificed 2.5 h later [34]. The stomachs were ex-
cised, lightly rinsed with water and point haemorrhagic lesions
enumerated, and the gastric lesion index was then calculated
[33,34].
significant effect since it is the target of modern anti-cytokine
biologics [38], but have disadvantages because of some risks of
stimulating infections (e.g. tuberculosis, hepatitis) [39]. The anti-
inflammatory effects of RK-106 may represent a lead for the devel-
opment of prototype curcuminoids with anti-TNFa activity for the
therapy of chronic rheumatic diseases.
The negative results with curcumin itself in the acute anti-
inflammatory and anti-pyretic assays suggest that any ability of
curcuminoids to inhibit cyclooxygenase/lipoxygenase or proin-
flammatory cytokines systems in vitro [35,36], may not be rapidly
expressed, if at all, in vivo. This may reflect slow absorption, rapid
detoxification, binding to endogenous ligands for beta-diketones
e.g. thiol groups or yet other factors reducing/slowing bioavailabil-
ity [40,41].
Acknowledgments
Anti-pyretic activity was measured in yeast-fevered rats [33].
These studies were supported by the Anti-inflammatory Re-
search Fund of Professor K D Rainsford and the Biomedical Re-
search Centre, Sheffield Hallam University. We are grateful to
Professor M.S. Roberts (Department of Medicine, University of
Queensland) for providing animal facilities. Our thanks to Mr. Alex-
ander Rainsford for his expert preparation of the figures.
3.2.2. Ethical approval
The animal studies were conducted under protocols approved
by the University of Queensland Animal Ethics Committee-5.
3.2.3. In vitro production of PGE2 and cytokine inflammatory
mediators
Since previous reports have shown that curcumin may affect
production of prostaglandins and pro-inflammatory cytokines
[35,36] it was decided to measure production of the mediators in
response to inflammatory stimuli in THP-1 monocytes in vitro.
The procedures employed were those as described by Rainsford
et al. [37]. The production of prostaglandinE2 (PGE2), interleukin-
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