Synthesis of thiophene and NO-curcuminoids for antiinflammatory and anti-cancer activities

Article abstract of DOI:10.3390/molecules18021483

In search of better NSAIDs four novel nitric oxide donating derivatives of curcumin (compounds 9a-d), and four thiophene curcuminoids (compounds 10a-c, 11) have been synthesised. The cytotoxic effects of these compounds along with the lead compound curcumin (7) and their effect on the production of the reactive oxygen species nitric oxide and pro-inflammatory cytokines IL-1β, TNF-α and chemokine CXCL-8 were evaluated using human monocytic THP-1 and colon adenocarcinoma CACO-2 cell lines. All of the nitric oxide donating curcuminoids 9a-d and the thiophene curcuminoids 10a-c and 11 were non-cytotoxic to THP-1 cells over a concentration range of 10-100 μM and compared with curcumin compounds 10b and 10c, were more toxic. In CACO-2 cells, 10b and 11 appeared to be non-toxic at 10 to 50 μM, whereas 10a and 10c were non-cytotoxic at 10 μM only. These results clearly indicate that the introduction of a nitroxybutyl moiety to curcumin and replacement of phenyl rings with thiophene units reduces the cytotoxic effect of the parent curcumin, whereas a methyl substituted thiophene increases the cytotoxic effects. In THP-1 cells, drugs 10a and 11 significantly decreased IL-1-β production at their non-cytotoxic concentrations, whereas, they did not decrease TNF-α production in CACO-2 cells. Compound 11 showed a significant decrease in CXCL-8 production.

Full text of DOI:10.3390/molecules18021483

Molecules 2013, 18, 1483-1501; doi:10.3390/molecules18021483
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Thiophene and NO-Curcuminoids for
Antiinflammatory and Anti-Cancer Activities
Mahera M. Ahmed, M. Akram Khan * and Kim Drummond Rainsford
Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S11WB, UK
* Author to whom correspondence should be addressed; E-Mail: m.a.khan@shu.ac.uk;
Tel.: +44-0-1142254451; Fax: +44-0-1142253066.
Received: 13 November 2012; in revised form: 7 January 2013 / Accepted: 15 January 2013 /
Published: 25 January 2013
Abstract: In search of better NSAIDs four novel nitric oxide donating derivatives of
curcumin (compounds 9a–d), and four thiophene curcuminoids (compounds 10a–c, 11)
have been synthesised. The cytotoxic effects of these compounds along with the lead
compound curcumin (7) and their effect on the production of the reactive oxygen species
nitric oxide and pro-inflammatory cytokines IL-1β, TNF-α and chemokine CXCL-8 were
evaluated using human monocytic THP-1 and colon adenocarcinoma CACO-2 cell lines.
All of the nitric oxide donating curcuminoids 9a–d and the thiophene curcuminoids 10a–c
and 11 were non-cytotoxic to THP-1 cells over a concentration range of 10-100 μM and
compared with curcumin compounds 10b and 10c, were more toxic. In CACO-2 cells, 10b
and 11 appeared to be non-toxic at 10 to 50 μM, whereas 10a and 10c were non-cytotoxic
at 10 μM only. These results clearly indicate that the introduction of a nitroxybutyl moiety
to curcumin and replacement of phenyl rings with thiophene units reduces the cytotoxic
effect of the parent curcumin, whereas a methyl substituted thiophene increases the
cytotoxic effects. In THP-1 cells, drugs 10a and 11 significantly decreased IL-1-β production
at their non-cytotoxic concentrations, whereas, they did not decrease TNF-α production in
CACO-2 cells. Compound 11 showed a significant decrease in CXCL-8 production.
Keywords: NO-NSAIDs; synthesis; curcuminoids; cytotoxicity; cytokines; chemokines;
anti-cancer

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1. Introduction
In the last two decades curcumin and its derivatives have received widespread interest for their
therapeutic effects for various indications [1] for example, in treating inflammatory conditions and
cancer [1–7]. We recently reported the anti-inflammatory activities, determined in vivo and in vitro, for
a range of novel aromatic and heterocyclic aromatic curcuminoids [8]. Some of our novel
curcuminoids may form the basis for development of new low gastro-toxicity anti-inflammatory agents
with selective activity as inhibitors of cytokine inflammatory mediators.
Here we report the anti-inflammatory properties of some nitric oxide donating curcuminoids 9a–d
analogous to the nitric oxide donating non-steroidal anti-inflammatory drugs NO-NSAIDs. Nitrate esters
such as cellulose nitrate and glycerol nitrate are esters of nitric acid that have been known for over 150
years. However, recently the synthesis of nitrate esters and in particular 4-nitroxy-1-butylcarboxylates
has received a renewed interest as antiinflammatory agents [9–14]. The development of NO-NSAIDs
is based on various findings whereby NO compliments some of the properties of the prostaglandins
(PGs) within the gastric mucosa, thus the NO-coupled NSAIDs might deliver NO to the site where
NSAIDs induce micosal damage, thereby decreasing the gastric toxicity, which is often a consequence
of diminished PG levels in the gastric mucosa [15]. NO-aspirin and other NSAIDs 1–4 with NO
donating activity have been reported to suppress platelet aggregation and thereby inflammation with
much reduced gastrointestinal tract injury. These NO-NSAIDs (Figure 1) are reported as stable in
aqueous solutions and well absorbed after oral administration [15]. Though a number of studies have
shown NO-NSAIDs to have enhanced anti-inflammatory activity with lesser side-effects, their major
limitation is the rapid release of NO with relatively small quantities being produced in the systemic
circulation [16–18]. The NO donating curcuminoids 6–9 were developed seeking potentially prolonged
production of NO in vivo.
Figure 1. NO-NSAIDs reported in the literature.
ONO2
O
O
O
OH
O
O
O
ONO2
1
2
NO-salicylate
NO-aspirin
CH3
O
NO2
O
Cl
O
H3CO
3
NO-naproxen
N
H
Cl
O
S
NO2
S
O
4
O
NO-diclofenac
Here we report the synthesis of NO-curcuminoids 9a–d, all containing the 4-nitroxybut-1-yl group
as a nitric oxide donating group. Their cytotoxicity and potential anti-inflammatory and anti-cancer
activities are reported in terms of effects on NO, the cytokines IL-1, TNF- and chemokine CXCL-8

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(IL-8) production relative to the naturally occurring curcumin (7). Some novel thiophenyl
curcuminoids 10a–c, 11 [8] (Figure 2) were also investigated in human monocytic THP-1 and
adenocarcinoma-derived CACO-2 cell lines. The effects on the cytokines were studied as chemokine
CXCL-8 has similarly been reported to be involved in the pathogenesis of a number of disorders
including inflammatory bowel diseases [19–21]. The cytotoxic effects of thiophene derivatives 10a–c,
11 complexed with HP-γ-CD were also assessed using the THP-1 cells. IL-1β and TNF-α, initiates a
cascade of events leading to inflammation and tissue destruction in various inflammatory diseases [22–24].
Figure 2. Synthesised thiophene and NO-curcuminoids.
O
O
H3CO
HO
OCH3
OH
7
curcumin
O2NO
ONO2
O
O
O
O
9a
O
O
O
O
ONO2
ONO2
O2NO
9b
O
O
O2NO
O
O
9c
O
O
H3CO
OCH3
O
ONO2
O2NO
O
9d
O
O
R3
R2
R3
R2
O
O
R1
R1
S
S
11
S
S
10
a. R1=R2=R3=H
b. R1=R2=H, R3=CH3
c. R1=CH3, R2=R3=H

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2. Results and Discussion
2.1. Chemistry
The NO-curcuminoids were synthesized in three steps as shown in Scheme 1. The synthesis of
curcuminoids from the aldehydes 5a–d followed by alkylation with excess dibromobutane to produce
the 4-bromobutoxy curcuminoids failed, and the best method for making the target compounds 9a–d
was that shown in Scheme 1. It is important to note that the synthesised NO-curcuminoids 9a–d are
phenolic ethers of NO as compared with the carboxylate esters of conventional NO-NSAIDs reported
in the literature and shown in Figure 1. We have recently reported that 2-methoxycurcuminoid has
higher anti-cancer activity compared with other derivatives [25] and in this study we have made a
systematic attempt to examine the activity profile of butoxynitrate group in positions 2, 3 and 4 of the
aromatic ring in curcuminoids 9a–d.
Scheme 1. Synthesis of NO-curcuminoids 9a–d.
Step 1 : Synthesis of bromobutoxyaldehydes 6a - d
CHO
CHO
R1
R2
R1
R2
(i) NaOEt/EtOH
Br
(ii)
Br
R3
R3
reflux, 12 hr
6
5
a : R1 = (-O-(CH2)4-Br), R2 = R3 = (-H)
b : R1 = R3 = (-H), R2 = (-O-(CH2)4-Br)
c : R1 = R2 = (-H), R3 = (-O-(CH2)4-Br)
d : R1 = (-H), R2 = (-OCH3), R3 = (-O-(CH2)4-Br)
a : R1 = (-OH), R2 = R3 = (-H)
b : R1 = R3 = (-H), R2 = (-OH)
c : R1 = R2 = (-H), R3 = (-OH)
d : R1 = (-H), R2 = (-OCH3), R3 = (-OH)
Step 2 : Synthesis of curcumin 7 and bromobutoxy curcuminoids 8a - d
O
O
H3CO
HO
OCH3
OH
(iii) 5d
(i) B2O3/Acetylacetone
(ii) tri-(sec)-butylborate
(iv) n-BuNH2, 30 min.
O
O
Curcumin 7
H3C
CH3
(v) EtOAc, 60 oC
(vi) HCl ( 0.4 M )
R1
O
O
R1
acetylacetone
R2
R3
R2
R3
(iii) 6a-d
8
a : R1 = (-O-(CH2)4-Br), R2 = R3 = (-H)
b : R1 = R3 = (-H), R2 = (-O-(CH2)4-Br)
c : R1 = R2 = (-H), R3 = (-O-(CH2)4-Br)
d : R1 = (-H), R2 = (-OCH3), R3 = (-O-(CH2)4-Br)

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Scheme 1. Cont.
All the synthesized compounds (Figure 2 and Scheme 1) were purified by flash column
chromatograpy and characterized spectroscopically. Thiophene and furan curcuminoids possess potent
anti-inflammatory properties [8], and the pharmacological activities of these curcuminoids was
compared with our NO-curcuminoids 9a–d and the natural curcumin (7).
2.2. Pharmacology
Curcumin (7), its nitric oxide donating derivatives 9a–d and thiophene derivatives 10a–d, 11 were
evaluated for cytotoxicity, production of NO and pro-inflammatory cytokines (IL-1, TNF- and
chemokine CXCL-8) using the human monocytic leukamia, THP-1 and the human Caucasian colon
adenocarcinoma-2, CACO-2 cell lines. For stimulating the cells we used the two activating agents LPS
and L-methionine sulfoximine (MS). LPS acts by binding to receptors on cell membranes whilst MS
acts as a membrane active agent that opens up cell membranes to release enzymes. Our aim was to
compare the effects of the active compounds with the parent compound, curcumin (7), and to
determine the structure-activity relationships of the active compounds as a guide to design future
derivatives of curcumin with enhanced biological potency.
2.2.1. Cytotoxicity
At 10 µM, all compounds were as non-toxic, as was curcumin (7), to THP-1 cells. However, at
concentrations of 50 and 100 µM, the NO donating compounds 10a–c and 11 were significantly less
toxic than curcumin (7) (Figure 3).
From the structure-activity relationships, it appears that the replacement of both the phenolic
hydrogens of curcumin (7) with the nitroxybutyl ether moiety enhances the non-cytotoxic properties of
these compounds. However, the presence of the methoxy group (-OCH3) at the meta positions of the
phenyl rings, as is found in compounds 7 and 9d, does not seem to be crucial for the cytotoxic effects
since compounds 9a–c do not possess any -OCH3 groups and these were as non-cytotoxic as 9d at 50
and 100 μM (Figure 3). Furthermore, change in the position of the nitroxybutyl moiety in structures of
the compounds 9a–c also did not have any effect on cell viability. Thus, it appears that all of the

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nitroxybutyl curcuminoids 9a–d are non-toxic to THP-1 cells and are less cytotoxic than curcumin (7)
at 10, 50 and 100 μM concentrations.
Figure 3. The effects of curcumin (7) and nitric oxide donating curcuminoids 9a–d on the
viability of THP-1 cells as determined by the MTS assay. Results are presented as mean ± SD.
* p < 0.05 compared with DMSO control, * p > 0.05 compared with curcumin control (7).
The replacement of both of the phenyl rings of the curcumin (7) with thiophene rings resulted in
lesser cytotoxic effects on THP-1 cells. Amongst all four thiophene curcuminoids, compounds 10b and
10c which are the methyl substituted derivatives of 10a at the 3 and 5 positions, respectively, show
significant (p < 0.05) cytotoxic effects in concentration-dependent manner, suggesting the methyl
group as being responsible for the cytotoxic effects (Figure 4). On the other hand compound 11 which
is a positional isomer of 10a also appeared to be less cytotoxic than 10b and 10c, which further
confirms that there is a possibility of a methyl group being involved in the induction of cytotoxic
effects associated with these derivatives. In comparison with the curcumin (7) the thiophene
curcuminoids 10a and 11 at 10 M, have similar cytotoxicity to curcumin 7 whereas the methyl
substituted curcuminoids 10b and 10c appeared to be more cytotoxic than curcumin (7). Since 10b and
10c at their lowest concentration (10 M) are more toxic than curcumin (7), these could serve as
potential anti-cancer drugs (Figure 4). In CACO-2 cells all the compounds 10a–c, 11 had a similar
toxicity profile as curcumin (7) but at 50 M concentration only the 5-methyl derivative 10c showed
increased cytotoxicity that was similar to that of curcumin (7). The toxicity profile of the
5-methylthiophene derivative 10c appears to be very similar in both THP-1 and CACO-2 cells (Figure 5).

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Figure 4. The effects of curcumin (7) and thiophene curcuminoids 10a–c, 11 on the
1489
viability of THP-1 cells as determined by the MTS assay. Results are presented as mean ± SD.
* p < 0.05 compared with DMSO control, * p > 0.05 compared with curcumin control (7).
Figure 5. The effects of curcumin (7) and thiophene curcuminoids 10a–c, 11 on the viability
of CACO-2 cells as determined by the MTS assay. Results are presented as mean ± SD.
* p < 0.05 compared with DMSO control, * p > 0.05 compared with curcumin control (7).
2.2.2. Nitric Oxide Production
In THP-1 cells all NO-derivatives of curcumin enhanced the production of NO in a concentration-
dependent manner, except for 9c (Figures 6 and 7). From the structure activity relationships it appears
that the replacement of the phenolic hydrogen of curcumin (7) with the nitroxybutyl ether moiety
significantly enhances nitric oxide production. Compared to the DMSO control a concentration
dependent increase in the production of nitrite was observed with compounds 9a, 9b and 9d at all the
three concentrations but the effect was significant at 50 and 100 μM. In comparison to the cells alone
control, DMSO + LPS control significantly reduced the nitrite production, whereas the LPS control
caused a non-significant effect in nitrite production (Figure 7). Compared with curcumin (7) at 10 μM
concentration a significant increase in nitrite production was observed in THP-1 cells that were

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stimulated with LPS for compound 9b. Compounds 9a, 9b and 9d significantly increased the nitrite
production at 50 and 100 μM, whereas curcumin was cytotoxic at these concentrations (Figure 7). When
compared with the DMSO + LPS control a concentration dependent increase in nitrite production was
observed with 9a whilst with 9b the effect was equally significant at all the three concentrations studied.
Figure 6. The effects of synthesized curcumin (7) and nitric oxide donating curcuminoids
9a–d on the production of nitric oxide in THP-1 cells using the Griess reagent. Results are
presented as mean ± SD. * p < 0.05 compared with DMSO control, * p > 0.05 compared
with curcumin control (7).
Figure 7. The effects of synthesized curcumin (7) and nitric oxide donating curcuminoids
9a–d on the production of nitric oxide in LPS-induced THP-1 cells using the Griess
reagent. Results are presented as mean ± SD. * p < 0.05 compared with DMSO + LPS
control, * p > 0.05 compared with curcumin control (7).

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2.2.3. Cytokine Production
The results show that curcumin (7) at 10 M, 10a and 11 significantly decreased IL-1 production
in a concentration-dependent manner in THP-1 cells stimulated with LPS. In the case of compounds
10b and 10c the decreased effect in IL-1β production exhibited at 10 µM was also significant and this
may be attributed to cytotoxicity. In comparison, all compounds were as potent as curcumin (7) at
10 µM. The thiophene curcuminoids 10a from 10 to 100 μM and 11 at 10 to 50 μM range may be
potential future anti-inflammatory drugs as they inhibit IL-1β production in THP-1 leukaemia cells and
have low cytotoxicity (Figure 8).
Figure 8. The effects of curcumin (7) and thiophene curcuminoids 10a–c, 11 on the
production of IL-1 in LPS-induced THP-1 cells using ELISA assay. Results are presented
as mean ± SD. * p < 0.05 compared with DMSO + LPS control, * p > 0.05 compared with
curcumin control (7).
Curcumin (7) inhibits the production of pro-inflammatory cytokines LPS-induced IL-1, IL-6 and
TNF- in THP-1 cells [20]. None of the thiophene curcuminoids 10a–c, 11 or curcumin (7) alone
affected production of TNF-α at all the three concentration 10, 50 and 100 μM. However, treatment of
LPS stimulated cells with curcumin (7) for 24 h resulted in a concentration-dependent decrease in
TNF-α production albeit the effects being significant only at 50 and 100 μM. This inhibitory effect
might be due to cytotoxic effects (Figure 9). Furthermore, none of the thiophene curcuminoids 10a–c, 11
at their non-cytotoxic concentrations inhibited TNF-α production in LPS stimulated cells. Curcumin
(7) and all its synthesised thiophene derivatives 10a–d, 11 appeared to be non-cytotoxic at 10 μM
concentration (Figure 9).

Molecules 2013, 18
Figure 9. The effects of synthesized curcumin (7) and thiophene curcuminoids 10a–c, 11
1492
on the production of TNF-α in LPS-induced THP-1 cells using ELISA assay. Results are
presented as mean ± SD. * p < 0.05 compared with DMSO + LPS control, * p > 0.05
compared with curcumin control (7).
2.2.4. Chemokine Production
Curcumin (7) did not affect the LPS-induced production of the CXCL-8 chemokine compared with
the solvent control (Figure 10). However, the thiophene compounds 10a–c and 11 reduced the
LPS-induced production of CXCL-8 chemokine in a concentration-dependent manner compared with
the DMSO solvent control. Thus at 10 µM, compounds 10b and 10c significantly reduced the
production of CXCL-8 whereas 10a and 11, like curcumin (7), did not affect the production of CXCL-8.
At 50 µM all the compounds significantly reduced the CXCL-8 production except 10c. At the highest
concentration of 100 µM, a significant decrease in CXCL-8 production was observed with all the
compounds. A significant increase in the production of CXCL-8 was observed when the cells were
treated with MS in the presence of LPS and DMSO. In comparison, the combined treatments with the
(DMSO + LPS + MS) control, curcumin (7), as well as compounds 10a and 10b were effective inhibitors.
2.2.5. Discussion of Pharmacology and Conclusions
The cytotoxicity studies showed that all of the nitroxybutyl curcuminoids 9a–d and thiophene
curcuminoid 10a appeared in general to be non or less toxic to THP-1 cells than curcumin (7). Thus,
addition of the nitrobutoxy moiety has the effect of reducing cytotoxicity of curcumin. These
nitroxybutyl curcuminoids 9a–d all produce nitric oxide, but this was not evident with curcumin (7).
High concentrations of some of the nitroxybutyl curcuminoids variously reduces production of IL-1β
and TNFα and in the case of thiophene compounds reduces production of the CXCL-8 chemokine. The
thiophene compounds 10a–c, 11 and some of the nitroxybutyl curcuminoinds 9a–d may have potential
in the future as anti-inflammatory agents.

Molecules 2013, 18
Figure 10. The effects of curcumin (7) and thiophene curcuminoids 10a–c, 11 on the
1493
production of CXCL-8 in MS and LPS-induced CACO-2 cells using ELISA assay. Results
are presented as mean ± SD. * p < 0.05 compared with DMSO control, * p > 0.05
compared with curcumin control (7).
3. Experimental
3.1. Chemistry: General
All apparatus was oven-dried overnight prior to use. Ethanol and ethyl acetate were dried over
molecular sieves, other solvents and chemicals were used as received without further purification.
1H-and 13C- Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AC 250
spectrometer operating at 250 and 62.9 MHz, respectively, for solutions in deuterated chloroform,
unless otherwise stated. Chemical shifts (δ) were recorded in parts per million (ppm) relative to the
reference, Tetramethylsilane (TMS) and coupling constants (J) were calculated in Hz. Electron impact
mass spectra (EIMS) were recorded on a VG 7070 Analytical Mass spectrometer. Electrospray mass
spectra (ESMS) were obtained on Micromass platform single quadrupole mass spectrometer fitted with
a Harvard syringe driver. The accurate mass of the compounds was detected using an Applied
Biosystems/MDS Sciex Hybrid quadrupole time-of-flight instrument (Q-Star Pulsar-i) fitted with an
orthogonal MALDI ion source and an ND:VAG Laser. Infrared spectra were recorded on ATI Mattson
Genesis Series FTIR spectrophotometer using either potassium bromide pellets as a support for solid
samples or sodium chloride disc for liquid samples. Melting points (°C) were recorded on Stuart SMP
3 digital Electrothermal melting point apparatus and are uncorrected. The compounds/hydroxypropyl-
γ-cyclodextrin complexes were freeze-dried using a Thermo ModulyoD freeze-dryer. Curcumin (7)
and curcuminoids 10a–c have been previously reported [8].
3.1.1. Preparation of tri-sec-Butyl Borate
A mixture of powdered boric acid (12.4 gm; 0.2 mol) and 2-butanol (44.4 gm; 0.6 mol) in toluene
(120 mL) was refluxed with azeotropic removal of water using a Dean-Stark apparatus. The toluene

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was evaporated on a rotary evaporator at 60 °C to give tri-sec-butyl borate as a clear liquid which was
stored in a tightly sealed bottle. Acetyl acetone/boron oxide complex was prepared according to the
method of Pabon [26].
3.1.2. General Procedure for the Synthesis of Bromobutoxybenzaldehydes 6a–d
All four compounds 6a–d were synthesized by a typical procedure which is illustrated for the
formation of compound 2-(4-bromobutoxy)benzaldehyde (6a). A three-neck round bottom flask fitted
with a dropping-funnel, and a double surface condenser having a calcium chloride drying-tube was
charged with dry EtOH (60 mL). Freshly cut sodium metal (2.3 gm; 0.1 mol) pre-washed in toluene
was added slowly to EtOH with gentle stirring under reflux, until all the sodium had reacted.
2-Hydroxybenzaldehyde (0.1 mol) was added and the reaction mixture was heated at 80 °C for 30 min.
1, 4-Dibromobutane (65 gm; 0.3 mol) was added dropwise to the reaction mixture through a dropping
funnel over a period of 35 min. The reaction mixture was refluxed for 12 h after which the mixture was
allowed to settle and then filtered by suction filtration on a Buchner flask. To the filtrate was added
water (60 mL) and extracted with EtOAc (2 × 60 mL). The organic layers were combined and dried
over MgSO4, filtered and the solvent was evaporated on a rotary evaporator. The residue was distilled
under reduced pressure (B.P. 45–50 °C/8mmHg) to remove the excess 1,4-dibromobutane and the
crude product was purified by silica-gel flash chromatography using [pet. Ether–EtOAc (8:1 v/v)] as
eluent, to yield product 2a, (58%) as a light yellow oil, Rf 0.45 [pet. ether-EtOAc, 5:1 v/v]. IR (ν) 3075
(aromatic C-H stretch), 2932 (aliphatic C-H stretch), 2759 (aldehyde C-H stretch), 1687 (conjugated
>C=O stretch), 1598 and 1577 (aromatic C=C stretch), 1242 (asymmetric C-O-C stretch), 1042
(symmetric C-O-C stretch), 758 cm−1 (ortho di-substituted out of plane C-H stretch); 1H-NMR δ 1.89–2.17
(4H, m, -CH2-CH2-), 3.50 (2H, t, J = 6.2 Hz, -CH2-Br), 4.11 (2H, t, J = 5.6 Hz, -O-CH2-), 6.95–7.05
(2H, m, Ar H-3 and H-5), 7.53 (1H, dt, J = 6.9 and 1.5 Hz, Ar H-4), 7.82 (1H, dd, J = 7.7 and 1.5 Hz,
Ar H-6), 10.5 (1H, s, -CHO); EIMS m/z 256 [M 79Br]+ (12%), 258 [M 81Br]+. (12%), 227 [M−CHO]+.
(3%), 135 [C4H8 79Br]+ (62%), 137 [C4H8 81Br]+ (59%), 121 [C7H5O2]+ (85%); Accurate mass found:
m/z 256.0093 (Br 79), calculated for C11H13O2 79Br: 256.0099.
3-(4-Bromobutoxy)benzaldehyde (6b). Yield: 79%, as a pale yellow oil, Rf 0.50 [pet. Ether–EtOAc, 5:1
v/v]. IR (ν) 3067 (aromatic C-H stretch), 2945 (aliphatic C-H stretch), 2729 (aldehyde C-H stretch),
1696 (conjugated C=O stretch), 1596 and 1585 (aromatic C=C stretch), 1262 (asymmetric C-O-C
stretch), 1043 cm−1 (symmetric C-O-C stretch); 1H-NMR δ 1.90–2.16 (4H, m, -CH2-CH2-), 3.48 (2H, t,
J = 6.4 Hz, -CH2-Br), 4.04 (2H, t, J = 5.9 Hz, -O-CH2-), 7.11–7.46 (4H, m, Ar H), 9.95 (1H, s, -CHO);
EIMS m/z: 256 [M 79Br]+. (7%), 258 [M 81Br]+. (7%), 135 [C4H8 79Br]+ (71%), 137 [C4H8 81Br]+ (69%),
121 [C7H5O2]+ (69%); Accurate mass found: m/z 256.0089, calculated for C11H13O2 79Br: 256.0099.
4-(4-Bromobutoxy)benzaldehyde (6c). Yield: 81%, as golden yellow oil, Rf 0.47 [pet. Ether–EtOAc,
5:1 v/v]. IR (ν) 3074 (aromatic C-H stretch), 2945 (aliphatic C-H stretch), 2738 (aldehyde C-H stretch),
1690 (conjugated C=O stretch), 1600 and 1577 (aromatic C=C stretch), 1255 (asymmetric C-O-C
stretch), 1040 (symmetric C-O-C stretch), 832 cm−1 (para disubstituted out of plane C-H stretch);
1H-NMR δ 1.85–2.13 (4H, m, -CH2-CH2-), 3.49 (2H , t, J = 6.4 Hz, -CH2-Br), 4.08 (2H, t, J = 5.9 Hz,
-O-CH2-), 6.98 (2H, d, J = 8.8 Hz, Ar H-3 and H-3'), 7.82 (2H, d, J = 8.3 Hz, Ar H-2 and H-2'), 9.87

Molecules 2013, 18
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(1H, s, -CHO); EIMS m/z 256 [M 79Br]+ (6%), 258 [M 81Br]+. (6%), 135 [C4H8 79Br]+ (58%), 137
79
[C4H8 81Br]+ (56%); Accurate mass found: m/z 257.0176, calculated for C12H14O2 Br: 257.0171.
4-(4-Bromobutoxy)-3-methoxybenzaldehyde (6d). Yield: 50%, as a white solid, Rf 0.33 [pet. Ether–
EtOAc, 3:1 v/v], m.p. 49.7 °C. IR (ν) 3081 (aromatic C-H stretch), 2872 and 2933 (aliphatic C-H
stretch), 2756 and 2821 (aldehyde C-H stretch), 1679 (conjugated C=O stretch), 1596 and 1584
(aromatic C=C stretch), 1263 (asymmetric C-O-C stretch), 1046 cm−1 (symmetric C-O-C stretch);
1H-NMR δ 2.06–2.08 (4H, m, -CH2-CH2-), 3.51 (2H, t, J = 6.4 Hz, -CH2-Br), 3.92 (3H, s, -O-CH3),
4.14 (2H, t, J = 5.9 Hz, -O-CH2-), 6.96 (1H, d, J = 7.7 Hz, Ar H-5), 7.40–7.46 (2H, m, Ar H-2 and
H-6), 9.85 (1H, s, -CHO); EIMS m/z 286 [M 79Br]+. (9%), 288 [M 81Br]+. (8%), 135 [C4H8 79Br]+ (54%), 151
[C4H8 81Br]+ (54%); Accurate mass found: m/z 286.0200 (79Br), calculated for C12H15O379Br: 286.0205.
3.1.3. Synthesis of Curcuminoids
Method A: In this procedure the synthesis of curcuminoids was carried out according to Pabon [8,21] in
which separately prepared acetyl acetone-boron oxide complex was used.
Method B: In this procedure the synthesis of curcuminoids was carried out by the in-situ formation
of acetyl-acetone-boron oxide complex.
The crude products 8a–d obtained were all purified by flash column chromatography using [pet.
Ether-EtOAc 8:1 v/v] as eluent.
(1E,4Z,6E)-1,7-Bis(2-94-bromobutoxy)phenyl)-5-hydroxyhepta-1,4,6-trien-3-one (8a). Yield: 34% (method
A), 21% (method B), a dark brown gum, Rf 0.44 [pet. ether-EtOAc, 3:1 v/v]. IR (KBr pellet) ν 3032
(aromatic C-H stretch), 2924 (aliphatic C-H stretch), 1620 (H-bonded >C=O stretch), 1595 and 1570
(aromatic C=C stretch), 1244 (asymmetric C-O-C stretch), 1046 (symmetric C-O-C stretch), 750 cm−1
(ortho disubstituted out of plane C-H stretch); 1H-NMR δ 1.98–2.20, (8H, m, -CH2-CH2-), 3.55 (4H, t,
J = 5.9 Hz, -CH2-Br), 4.10 (4H, t, J = 5.6 Hz, -O-CH2-), 5.87 (1H, s, enol H), 6.75 (2H, d, J = 16.0 Hz,
Ar-CH=CH-), 6.91 (2H, d, J = 8.2 Hz, Ar H-3), 6.98 (2H, t, J = 7.7 Hz, Ar H-5), 7.33 (2H, dt, J = 8.7
and J = 1.7 Hz, Ar H-4), 7.57 (2H, dd, J = 7.7 and J = 1.5 Hz, Ar H-6), 7.97 (2H, d, J = 16.0 Hz,
13
-CO-CH=CH-Ar); C-NMR δ 28.1, 29.8, 33.7, 67.7, 101.9, 112.4, 121.2, 124.5, 125.2, 129.1, 131.5,
79
81
136.0, 157.9, 184.0; EIMS m/z 576 [M Br]+. (4%), 580 [M Br]+. (6%), 577 [M+H]+., (2%), 578,
[M+2H]+., (7%), 135 [C4H8 79Br]+ (39%), 137 [C4H8 81Br]+ (37%); Accurate mass found: m/z
577.0583, calculated for C27H31O4 79Br2: 577.0589.
(1E,4Z,6E)-1,7-Bis(3-(4-bromobutoxy)phenyl)hepta-1,6-dien-3,5-dione (8b). Yield: 24% (method A)
21% (method B), as a dark brown solid, Rf 0.41 [pet. ether-EtOAc, 3:1 v/v], m.p. 74.6–75.9 °C. IR (ν)
3426 (OH stretch), 3052 (aromatic C-H stretch), 2947 (aliphatic C-H stretch), 1625 (H-bonded >C=O
stretch), 1596 and 1579 (aromatic C=C stretch), 1508 (enol), 1243 (asymmetric C-O-C stretch), 1044
(symmetric C-O-C stretch), 885, 791, 677 cm−1 (meta disubstituted out of plane C-H stretch); 1H-NMR
δ 1.95–2.18 (8H, m, -CH2-CH2-), 3.51 (4H, t, J = 6.45 Hz, -CH2-Br), 4.04 (4H, t, J = 5.9 Hz, -O-CH2-),
5.85 (1H, s, enol H), 6.62 (2H, d, J = 16.0 Hz, Ar-CH=CH-), 6.92 (2H, d, J = 7.7 Hz, Ar H-4), 7.07
(2H, s, Ar H-2), 7.16 (2H, d, J = 7.7 Hz, Ar H-6), 7.31 (2H, t, J = 7.9 Hz, Ar H-5), 7.63 (2H, d,
13
J = 16.0 Hz, -CO-CH=CH-Ar); C-NMR δ 28.1, 29.8, 33.6, 67.2, 102.0, 113.9, 116.7, 121.2, 124.7,

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79
79
79
130.2, 136.7, 140.8, 159.5, 183.5; ESMS m/z 576 [M Br]+., 577 [M Br+H]+., 578 [M Br+2H]+.,
580 [M 81Br]+., 581 [M 81Br+H]+., 582, [M 81Br+2H]+..
(1E,6E)-1,7-Bis(4-(4-bromobutoxy)phenyl)hepta-1,6-dien-3,5-dione (8c). Yield: 66% (method A),
41% (method B), as a bright yellow solid, Rf 0.42 [pet. ether-EtOAc, 3:1 v/v], m.p. 145–146.9 °C. IR
(ν) 3433 (OH stretch), 3035 (aromatic C-H stretch), 2945 (aliphatic C-H stretch), 1628 (H-bonded
>C=O stretch), 1600 (aromatic C=C stretch), 1510 (enol), 1420 (olefinic in plane bending vibration),
1257 (asymmetric C-O-C stretch), 1046 (symmetric C-O-C stretch), 837 cm−1 (para disubstituted out
1
of plane C-H stretch); H NMR δ 1.92–2.14 (8H, m, -CH2-CH2-), 3.50 (4H, t, J = 6.4 Hz, -CH2-Br),
4.04 (4H, t, J = 5.6 Hz, -O-CH2-), 5.78 (1H, s, enolic CH=C), 6.50 (2H, d, J = 15.5 Hz, Ar-CH=CH-),
6.90 (4H d, J = 8.8 Hz, Ar H-3 and H3'), 7.50 (4H, d, J = 8.7 Hz, Ar H-2 and H2'), 7.62 (2H, d,
J = 15.5 Hz, -CO-CH=CH-Ar); 13C-NMR δ 29.7, 28.1, 67.3, 101.6, 115.1, 122.1, 128.3, 130.0, 140.3,
79
79
160.8, 183.6; EIMS m/z 576 [M Br]+ (3%), 578 [M Br+2H]+ (8%), 135, [C4H8 79Br]+(100%), 137
[C4H8 81Br]+ (98%); Accurate mass found: m/z 576.0535, calculated for C27H30O4 79Br2 : 576.0511).
(1E,4Z,6E)-1,7-Bis(4-(4-bromobutoxy)-3-methoxyphenyl)hepta-1,6-dien-3,5-dione (8d). Yield: 57%
(method A), as a dark yellow solid between EtOAc and aqueous layers during work-up and no further
purification was required; Rf 0.38 [pet. ether-EtOAc, 2:1 v/v], m.p. 124.4–125.4 °C. IR (ν) 3548-3235
(OH enolic), 3003 (aromatic C-H stretch), 2955 and 2870 (aliphatic C-H stretch), 1620 (H-bonded
>C=O), 1597 and 1581 (aromatic C=C stretch), 1508 (enol), 1422 cm−1 (olefinic in plane bending
1
vibration); H-NMR δ 2.00–2.17 (8H, m, -CH2-CH2-), 3.51 (4H t, J = 6.4 Hz, -CH2-Br), 3.92 (6H, s,
-OCH3), 4.10 (4H, t, J = 5.9 Hz, -O-CH2-), 5.82 (1H, s, enol H), 6.50 (2H, d, J = 16.0 Hz,
Ar-CH=CH-), 6.87 (2H, d, J = 8.2 Hz, Ar H-5), 7.08 (2H, d, J = 2.0 Hz, Ar H-2), 7.13 (2H, dd,
J1,3 = 8.2 and J1,2 = 1.5 Hz, Ar H-6), 7.61 (2H, d, J = 15.5 Hz, -CO-CH=CH-Ar); EIMS m/z 637
[M+H]+ (18%), 619 [M+H−H2O]+ (31%), 324 [C15H18O3 79Br+H]+ (31%), 326 [C15H18O3 81Br+H]+
(29%), 324 [C14H16O3 79Br]+ (20%), 313 [C14H16O3 81Br]+ (18%), 135 [C4H8 79Br]+ (100%), 137 [C4H8
81Br]+ (99%); Accurate mass found: m/z 636.0714 (Br79), calculated for C29H34O6 79Br2 : 636.0722.
3.1.4. Synthesis of Nitroxybutyl Curcuminoids 9a–d
The general procedure for the synthesis of butoxy-nitrate curcuminoids 9a–d is illustrated for the
formation of (1E,6E)-1,7-bis(2-(4-butoxynitrato)hepta-1,6-diene-3,5-dione (9a). In a one-neck round
bottom flask, a mixture of silver nitrate (1.17 gm, 6.9 mmol) in acetonitrile (3 mL) was stirred for 30
min. and then a solution of 8a (0.86 mmol) in acetonitrile (2 mL) was added. The reaction mixture was
refluxed under stirring for 5 h at 80 °C. An aluminium sheet was wrapped round the flask to protect it
from light and the mixture was allowed to stand overnight. Water (5 mL) was added and after filtration
the mixture was extracted with EtOAc (2 × 30 mL). The organic layers were combined, dried over
MgSO4, filtered under gravity and the solvent was removed on the rotary evaporator to yield pure 9a
(90%) as a dark brown gum, Rf 0.43 [pet. ether-EtOAc, 2:1 v/v], m.p. 136.0–136.8 °C. IR (KBr pellet)
ν 3035 (aromatic C-H stretch), 2926 and 2878 (aliphatic C-H stretch), 1625 (conjugated >C=O
stretch), 1596 and 1488 (aromatic C=C stretch), 1472 and 1280 (aliphatic NO2 stretch), 1455 (CH2
bending absorption), 1244 (asymmetric C-O-C stretch), 1049 (symmetric C-O-C stretch), 753 cm−1
1
(ortho disubstituted out of plane C-H stretch); H-NMR δ 1.99–2.04 (8H, m, -CH2-CH2-), 4.10 (4H, t,

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J = 5.4 Hz, -O-CH2-), 4.58 (4H, t, J = 5.9 Hz, -CH2-ONO2), 5.83 (1H, s, enol H), 6.74 (2H, d, J = 16.0 Hz,
Ar-CH=CH-), 6.91 (2H, d, J = 8.2 Hz, Ar H-3), 6.99 (2H, t, J = 7.4 Hz, Ar H-7.97 (2H, d, J = 16.0 Hz,
13
-CO-CH=CH-Ar); C-NMR δ 24.2, 25.9, 67.8, 73.1, 102.1, 112.4, 121.3, 124.6, 125.2, 129.0, 131.5,
135.8, 157.7, 183.9; ESMS m/z 543 [M+H]+., 332 [C17H18O6N]+; Accurate mass found: m/z 543.1964,
calculated for C27H31N2O10: 543.1973.
(1E,6E)-1,7-Bis(3-(4-butoxy-nitrate)hepta-1,6-diene-3,5-dione (9b). Yield: 58% as a dark brown solid,
Rf 0.46 [pet. ether-EtOAc, 2:1 v/v], m.p. 133.9–134.9 °C. IR (ν) 3414 (OH stretch), 3020 (aromatic
C-H stretch), 2929 and 2875 (aliphatic C-H stretch), 1638 (conjugated >C=O stretch), 1596 and 1489
(aromatic C=C stretch), 1473 and 1279 (aliphatic NO2 stretch), 1458 (CH2 bending absorption), 1247
(asymmetric C-O-C stretch), 1041 cm−1 (symmetric C-O-C stretch); 1H-NMR δ 1.95 (8Hz, t, J = 2.8 Hz,
-CH2-CH2-), 4.04 (4H, t, J = 5.1 Hz, -O-CH2-), 4.56 (4H, t, J = 5.9 Hz, -CH2-ONO2), 5.85 (1H, s, enol
H), 6.61 (2H, d, J = 16.0 Hz, Ar-CH=CH-), 6.91 (2H, dd, J = 7.9 and 1.7 Hz, Ar H-4), 7.06 (2H, s, Ar
H-2), 7.16 (2H, d, J = 7.7 Hz, Ar H-6), 7.31 (2H, t, J = 7.7 Hz, Ar H-5), 7.62 (2H, d, J = 15.5 Hz,
-CO-CH=CH-Ar); 13C-NMR: δ 24.2, 25.9, 67.3, 73.1, 102.1, 113.9, 116.6, 121.3, 124.7, 130.3, 136.7,
140.7, 159.4, 183.5; ESMS m/z 543 [M+H]+., 565 [M+Na]+; Accurate mass found: m/z 543.1985,
calculated for C27H31N2O10: 543.1973.
(1E,6E)-1,7-Bis(4-(4-Butoxy-nitrate)hepta-1,6-diene-3,5-dione (9c). Yield: 95%, golden yellow solid,
Rf 0.35 (pet. ether-EtOAc, 2:1 v/v), m.p. 112.6–113 °C. IR (ν) 3412 (OH stretch), 3040 (aromatic C-H
stretch), 2936 (aliphatic C-H stretch), 1620 (conjugated >C=O stretch), 1603 and 1511 (aromatic C=C
stretch), 1472 and 1287 (aliphatic NO2 stretch), 1256 (asymmetric C-O-C stretch), 1056 (symmetric
C-O-C stretch), 837 cm−1 (para disubstituted out of plane C-H stretch); 1H-NMR δ 1.95 (8H, t, J = 2.2 Hz,
-CH2-CH2-), 4.06 (4H, t, J = 5.6 Hz, -O-CH2-), 4.56 (4H, t, J = 5.3 Hz, -CH2-ONO2), 5.79 (1H, s, enol
H), 6.51 (2H, d, J = 15.5 Hz, Ar-CH=CH-), 6.90 (4H, d, J = 8.8 Hz, Ar H-3 and H-3'), 7.51 (4H, d,
13
J = 8.8 Hz, Ar H-2 and H-2'), 7.63 (2H, d, J = 16.0 Hz, -CO-CH=CH-Ar); C-NMR: δ 24.1, 25.8,
67.4, 73.1, 101.6, 115.1, 122.2, 128.3, 130.0, 140.3, 160.7, 183.6; EIMS m/z 543 [M+H]+, (42%).
(1E,6E)-1,7-Bis(4-(4-butoxy-nitrate)-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (9d). Yield: 92% as
an orange solid, Rf 0.43 [pet. ether-EtOAc, 1:3 v/v], m.p. 101.6–102.6 °C. IR (ν) 3436 (OH stretch),
2954 (aromatic C-H stretch), 2931 and 2873 (aliphatic C-H stretch), 1621 (conjugated >C=O stretch),
1458 (CH2 bending absorption), 1512 (aromatic C=C stretch), 1257 (asymmetric C-O-C stretch), 1027
1
(symmetric C-O-C stretch). H-NMR δ 1.63 (8H, s, br. -CH2-CH2-), 3.91 (6H, s, -O-CH3), 4.10 (4H, t,
J = 5.1 Hz, -O-CH2-), 4.58 (4H, t, J = 5.9 Hz, -CH2-ONO2), 5.83 (1H, s, enol H), 6.50 (2H, d, J = 15.5 Hz,
Ar-CH=CH-), 6.87 (2H, d, J = 8.2 Hz, Ar H-5), 7.09 (2H, s, Ar H-2 ), 7.13 (2H, d, J = 8.2 Hz, Ar
H-6), 7.61 (2H, d, J = 15.5 Hz, -CO-CH=CH-Ar); EIMS m/z 603 [M]+ (38%), 604 [M+H]+., (12%);
Accurate mass found: m/z 603.2162, calculated for C29H35N2O10: 603.2184.
1,7-Bis(thiophen-3-yl-)-1,6-heptadien-3,5,dione (11). Yield: 36% (method A) purified by column
chromatography (pet. ether-EtOAc, 6:1 v/v), 23% yield (method B), yellow solid, Rf 0.48 (pet. ether-
EtOAc, 5:1 v/v), m.p. 140.1–141.3 °C.; IR (ν) 3435 (OH stretch), 3092 (aromatic C-H stretch), 1624
(H-bonded >C=O), 1584 (conjugated C=C), 1507 (enol), 1412 cm−1 (olefinic in plane bending
vibration); 1H-NMR δ 5.79 (1H, s, enol H), 6.45 (2H, d, J = 15.5 Hz, Ar-CH=CH-), 7.33–7.38 (4H, m, Ar

Molecules 2013, 18
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H), 7.52 (2H, d, J = 2.6 Hz, Ar H-2), 7.66 (2H, d, J = 16.0 Hz, -CO-CH=CH-Ar); EIMS m/z 288 [M]+
(60%), 270 [M−H2O]+. (8%), 192 [C10H8O2S]+ (4%), 179 [C9H7O2S]+ (5%), 151 [C8H7OS]+ (21%),
137 [C7H5OS]+ (100%), 109 [C6H5S]+ (43%), 97 [C5H4S]+ (21%); Accurate mass found: m/z 288.0277,
calculated for C15H12O2S2: 288.0279.
3.2. Pharmacology
3.2.1. General Procedures for Cell Cultures And Compound Preparations
Human monocytic leukemia (THP-1) cell line and human caucasian colon adenocarcinoma
(CACO-2) cell lines were obtained from the European Collection of Cell Cultures (ECACC)
(Salisbury, Wiltshire, UK). Preparation of drug treatments and general procedures for cell culture were
as previously described [27–30]. All cell culture techniques, i.e., thawing, passaging, plating as well as
respective compound treatments applied to the cells and cytotoxicity using 3-(4,5-dimethylthiazol-
2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays were
performed under sterile conditions using a laminar flow cabinet. In these experiments the colorimetric
assay CellTiter 96® AQueous one solution cell proliferation in vitro was used to examine the effects of
the synthesized curcumin (7) and curcuminoids on cell viability [27]. For experimental work, samples
were prepared in media, without serum (serum-free RPMI) for THP-1 cells and CACO-2 cells
respectively. Lipopolysaccharide (LPS) Escherichia coli O127:B8 (cell culture tested) and
L-methionine sulfoximine (MS) were obtained from Sigma-Aldrich (St. Louis, MO, USA)). All
incubations were carried out in a humidified incubator maintained at 37 °C with 5% (v/v) CO2 and
95% air. Experiments were performed in duplicate wells of the 96-well plates and were repeated at
least four times unless otherwise stated in the figure legend.
For all MTS, nitric oxide and ELISAs, the stock solutions (33 mM) of all the compounds were
prepared in DMSO (100%) followed by appropriate dilutions. For the determination of cytotoxic
effects of the compounds 9a–d these were complexed with hydroxypropyl-γ-cyclodextrin (HP-γ-CD)
for improved solubilisation. A 100 mM stock solution was prepared using DMSO (100%). The drug/
HP-γ-CD complex (1:2 molar ratio) was diluted to 10, 50 and 100 µM (relative to the drug
concentration) [27].
3.2.2. Nitric Oxide Assay
The determination of nitrite in biological samples was measured by the spectrophotometric method
using Griess reagent. NO levels are quantified by estimating its stable end product (nitrite anion)
which is formed as a result of nitric oxide oxidation [31]. Experimental samples (50 µL/well) were
added to duplicate wells of a 96-well plate, 50 µL of Griess reagent was then added and samples
incubated for 5–10 min at room temperature protected from light for colour development. Absorbance
was measured at 570 nm using a plate reader (Wallac Victor 2 multi-label plate reader). Nitrite
concentrations were determined from a sodium nitrite standard curve.

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3.2.3. Sandwich ELISA Assay
Sandwich ELISAs are a widely used standard method for the quantitative detection of cytokines
and other specific proteins in serum samples. Substrate was added which reacted with the enzyme
producing a colour change, the intensity of which is directly proportional to the concentration of
antigen in incubation mixture allowing its spectrophotometric detection and quantification. Standard
proteins of known concentrations are incorporated into the assay to allow quantification of specific
proteins to be determined [32–34]. The capture antibodies, mouse anti-human IL-1β or mouse anti-human
TNF-α were diluted to a working concentration of 4.0 µg/ml in PBS (without carrier proteins). ELISA
flat-bottom 96-well plates were prepared according to the manufacturer’s protocol for the detection of
IL-1β or TNF-α in cell supernatants of THP-1, CXCL-8 and CACO-2 cells. An ELISA flat-bottom 96-well
plate was immediately coated with 100 µL/well of the diluted capture antibody, sealed and incubated
overnight at room temperature. Next day, each well of the 96-well micro plate was aspirated and
washed six times with wash buffer (WB, 0.05% Tween® 20 in PBS). After the last wash, the plate was
dried by blotting against clean paper towel, blocked by adding 300 µL/well of reagent diluent (RD) (1%).
The 96-well microtitre plate is coated with capture antibody (1st antibody) and the target protein or
antigen is added. Detection antibody (2nd antibody) is added followed by the addition of enzyme,
horseradish peroxidase (HRP)-linked antibody. Substrate solution containing colorimetric substrate,
3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) is added. TMB reacts with H2O2 in
the presence of HRP enzyme to produce a water-soluble, blue coloured by-product the intensity of which
is proportional to the amount of HRP activity, which in turn is related to the levels of target analyte in the
experimental sample. Upon acidification with sulphuric-acid (stop solution) the colour changes from
blue to yellow, enabling accurate measurement of the intensity at 450 nm using a plate-reader.
Acknowledgements
We thank Nicola Woodroofe for use of facilities in the BMRC and some financial support. We also
thank Rowena Bunning for proof reading the paper and for her valuable suggestions. There is no
conflict of interest to disclose.
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Sample Availability: Samples of the compounds 9a–d, 10a–c and 11 are available from the authors.
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