Synthesis of novel curcumin analogues and their evaluation as selective cyclooxygenase-1 (COX-1) inhibitors

Article abstract of DOI:10.1248/cpb.55.64

Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Recent studies have indicated that cyclooxygenase-1 (COX-1) plays an important role in inflammation and carcinogenes

Full text of DOI:10.1248/cpb.55.64

64
Chem. Pharm. Bull. 55(1) 64—71 (2007)
Vol. 55, No. 1
Synthesis of Novel Curcumin Analogues and Their Evaluation as Selective
Cyclooxygenase-1 (COX-1) Inhibitors
a
b
a
a
,a
*
Norbert HANDLER, Walter JAEGER, Helmut PUSCHACHER, Klaus LEISSER, and Thomas ERKER
a Department of Medicinal Chemistry, University of Vienna; and b Department of Clinical Pharmacy and Diagnostics,
University of Vienna; Althanstrasse 14, A-1090 Vienna, Austria. Received July 26, 2006; accepted October 23, 2006
Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-in-
flammatory and anti-cancer activities. Recent studies have indicated that cyclooxygenase-1 (COX-1) plays an im-
portant role in inflammation and carcinogenesis. In order to find more selective COX-1 inhibitors a series of
novel curcumin derivatives was synthesized and evaluated for their ability to inhibit this enzyme using in vitro in-
hibition assays for COX-1 and COX-2 by measuring PGE2 production. All curcumin analogues showed a higher
rate of COX-1 inhibition. The most potent curcumin compounds were (1E,6E)-1,7-di-(2,3,4-trimethoxyphenyl)-
1,6-heptadien-3,5-dione (4) (COX-1: IC50ꢀ0.06 mM, COX-2: IC50ꢀ100 mM, selectivity indexꢀ1666) and (1E,6E)-
methyl 4-[7-(4-methoxycarbonyl)phenyl]-3,5-dioxo-1,6-heptadienyl]benzoate (6) (COX-1: IC50ꢀ0.05 mM, COX-2:
IC50ꢀ100 mM, selectivity indexꢀ2000). Curcumin analogues therefore represent a novel class of highly selective
COX-1 inhibitors and promising candidates for in vivo studies.
Key words curcumin; cancer; cyclooxygenase-1; cyclooxygenase-2
Curcumin (diferuloyl methane, Chart 1) is a major con- discontinued and the drug was withdrawn from the market
stituent found in the spice tumeric, which is a dried powder because its use was associated with an increased incidence of
from the rhizomes of Curcuma longa L. Several in vitro and cardiovascular events, such as heart attack and stroke.11)
in vivo studies demonstrated suppression, retardation, or in-
Very recently, experimental results have also indicated a
version of carcinogenesis.1—3) Furthermore, it also exhibits possible involvement of the other isoform of COX, COX-1,
anti-inflammatory, antioxidant, antiviral, and anti-infectious in angiogenesis, thereby providing the rationale for the devel-
activities and wound healing properties.4—8) Inhibition of opment of selective COX-1 inhibitors.12,13) These data were
arachidonic acid metabolism by curcumin has been sug- also confirmed by in vitro studies in isolated ovine COX-1
gested to be a key mechanism for its anticarcinogenic action. and COX-2 enzymes which showed that curcumin and its
The enzyme cyclooxygenase (COX) catalyzes the first two analogues tetrahydrocurcumin and trimethoxydibenzoyl-
steps in the biosynthesis of prostaglandins (PGs) from the methane had significantly higher inhibitory effects on the
substrate arachidonic acid. At least two forms of this enzyme peroxidase activity of COX-1 than that of COX-2.2) Further-
exist.9,10)
more, a recent report by Gupta et al.14) has also indicated that
One of these forms, COX-1, is constitutively expressed COX-1 is overexpressed in a significant number of ovarian
and is responsible for maintaining normal physiologic func- cancers. We therefore investigated whether novel curcumin
tion and the PGs produced by this enzyme play a protective analogues might achieve an even better and more selective
role. The other known form of the enzyme, cyclooxygenase- COX-1 inhibition than curcumin. Thus, seven analogues of
2 (COX-2), is an inducible form and its expression is af- curcumin were synthesized using standard chemical meth-
fected by various stimuli such as mitogens, oncogenes, tumor ods. Each analogue (except the intermediate 2) was then
promoters, and growth factors.10) COX-2 has been detected tested for COX-1 and COX-2 inhibition in an in vitro model
in various tumors and its role in carcinogenesis and angio- and the resulting inhibition values compared with that of the
genesis has been well documented. Therefore, COX-2 is clinically established selective COX-2 inhibitor celecoxib.
thought to be a promising therapeutic target for cancer. How- Also molecular docking studies were performed to investi-
ever, current clinical studies of a COX-2-selective inhibitor, gate the ligand-protein interactions responsible for the bio-
rofecoxib (Vioxx), for preventing recurrence of colorectal logical data found.
polyps in patients with a history of colorectal adenomas were
Results and Discussion
The chemical synthesis of the curcumin analogues was
carried out following two known pathways. Their basic prin-
ciple is the same, but they differ in technique, reaction time
and temperature. Generally, the first step was the reaction of
acetylacetone with boron oxide building a boron complex,
which inhibited an unpleased Knoevenagel reaction. After
addition of a corresponding benzaldehyde and a base, the
condensation of the acetylacetone–boron complex with the
aldehyde and an additional elimination occured; eventual
heating with dilute acid cleaved the boron complex to give
the desired curcumin analogues (Chart 2).
Chart 1. Two Tautomeric Forms of Curcumin
Method 1, decribed by Mazumder et al.15) was successful
∗ To whom correspondence should be addressed. e-mail: thomas.erker@univie.ac.at
© 2007 Pharmaceutical Society of Japan

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65
Table 1. Inhibitory Effect of 1—7, Curcumin and the Reference Com-
pound Celecoxib on COX-1 and COX-2 Activity (Values Given in mM)
IC50(COX-1)/ IC50(COX-2)/
Compound
IC50(COX-1)
IC50(COX-2)
IC50(COX-2)
IC50(COX-1)
1
2
0.33
n.d.
12.58
n.d.
0.026
n.d.
38
n.d.
3
4
5
6
7
2.68
0.06
0.37
0.05
1.14
50
ꢀ100
ꢀ100
9.92
ꢂ0.026
ꢂ0.0006
0.037
ꢀ37
ꢀ1666
27
ꢀ100
5.13
ꢂ0.0005
0.22
ꢀ2000
4.5
Curcumin2)
Celecoxib
Indomethacin21)
ꢀ100
0.03
0.5
2
13.7
0.018
456.6
0.002
1.444
0.026
0.692
was then analyzed in a cell-free immunoassay system. Puri-
fied ovine enzyme served as the source of COX-1, while the
human recombinant enzyme formed the source of COX-2.
The inhibition of COX-1 by curcumin analogues (compound
2, an intermediate, was not tested) is shown in Table 1. All
compounds tested demonstrated a several-fold higher in-
hibitory activity than curcumin itself (COX-1: IC50ꢁ50 mM,
COX-2: IC50ꢀ100 mM).2)
Chart 2. Basic Steps of the Synthesis of Curcuminoids
In detail, the results showed that all our curcumin deriva-
tives had a preference towards COX-1 isoenzyme not de-
pending on the substituent of the molecule. Even compound
3 bearing a methylsulfonyl group, which can be found in
some COX-2-selective compounds, exhibited an distinct
affinity towards COX-1. Nurfina et al.7) postulated that, be-
sides olefinic double bonds, a 4-hydroxyl group at the phenyl
ring of curcuminoids was essential for an antiinflammatory
effect. Also position 3 of the aromatic ring played an impor-
tant role for the pharmacoloigal profile, since bigger 3-alkyl
groups (like tert-butyl) lead to inactive molecules, whereas
lower alkyl and especially 3,5-dialkyl-substituents showed
high oedema inhibiting activity. Selvam et al.17) calculated
docking studies with curcumin and some derivatives, which
revealed that these compounds could dock into the active site
of only COX-1. In case of the COX-2 enzym complex only
curcumin itself interacted with the enzyme, but no hydrogen
bonding interactions were detected for the other molecules.
Thus we chose derivatives without hydroxy groups but
lipophilic and mainly polar groups to check possible SAR
concerning COX-1 interaction.
The data are presented in this paper (Table 1), where all
novel curcumin derivatives showed a distinct affinity towards
COX-1 isoenzyme. The corresponding IC50 values reached
Chart 3. Novel Curcumin Analogues 1—7
in poor yields for compounds 2 and 7. Method 2, first de- from 2.68 mM (compound 3) to 0.05 mM (compound 6) in-
scribed by Pabon,16) was used for the other molecules ending cluding selectivity indices (IC50(COX-2)/IC50(COX-1)) from
up in poor yields, too. Study of the literature concerning the 4.5 for compound 7 to ꢀ2000 for compound 6. Especially
synthesis of curcuminoids pointed out difficulties, since only the trimethoxy derivative 4 as well as the methyl ester 6 ex-
some derivatives seemed to be accessable quite well. The hibited very pronounced and selective COX-1 inhibition
yields ranged from about 50% shown only for methoxy-de- (IC50ꢁ0.06 and 0.05 mM, selectivity indices ꢀ1666 and
rivatives to poor yields for p-(dimethylamino) (36%) or o- ꢀ2000), whereas the other molecules showed just poor
furyl derivatives (8%), respectively,15,16) and so we tried to affinities and selectivities towards this isoenzym. However,
improve our pathways. Exact analysis of our syntheses re- also compound 3, bearing a methylsulfonyl group, a group
vealed that the volume of n-butylamine plays an important found in some COX-2 inhibiting molecules, had weak effects
role in this kind of reaction and thus, in case of molecules 1 on COX-1 but almost none towards COX-2 (IC50(COX-
and 4, its quantity was reduced to obtain the desired com- 1)ꢁ2.68 mM, IC50 (COX-2)ꢀ100 mM, selectivity index ꢀ37).
pounds in a somehow acceptable yield.
The other molecules exhibited only little COX-1-effects and
Inhibition of COX-1 and COX-2 by curcumin analogues selectivities with IC50 values ranging from 0.33 to 2.68 mM

66
Vol. 55, No. 1
and selectivity indices from 4.5 to 38, respectively.
The active site of 1PGG is considered to be constituted of
In order to better understand the anti-inflammatory activi- the amino acid residues ARG120, SER530, TYR385 and
ties of compounds 1, 3, 4, 6 and 7 we performed a molecular GLU524.18) 1, 6 and 7 exhibited a very good DOCK 6.0 grid
docking analysis. We tried to understand the ligand-protein score showing favorable van der Waals interactions. Com-
interaction responsible for the perceived COX-1/COX-2 in- pound 1 showed two hydrogen bonds. The two carbonyl oxy-
hibitory data. Docking conformations of compounds 1, 3, 4, gens were acceptors for hydrogen bonds forming from
6 and 7 were created by a systematic conformational search ARG83 (NH1). The O–N distance was found to be 3.09 and
with the MMFF94x as implemented in MOE. Each rotate- 2.51 Å; the O–H distance was found to be 2.38 and 1.52 Å,
able bond of the heptanoid part of the compounds was as- respectively (Fig. 1). Although compound 3 and 4 exhibited
signed a 60° rotation increment. Rotateable bonds of sub- only moderate to weak interactions calculated with DOCK,
stituents of the phenyl rings were assigned a 30° rotation in- they show a tight hydrogen bond network. 3 is able to build
crement. All resulting conformers were subjected to full en- three hydrogen bonds to 1PGG. These hydrogen bonds are
ergy minimization using the MMFF94x force field to a gradi- formed by ARG83 and the sulfonyl oxygen of 3 with a bond
ent of 0.05 kcal/mol. Conformers having an energy of length of 1.52 Å, ARG120 NH1 and the carbonyl oxygen of
15 kcal/mol above the lowest energy found were not taken 3 with a bond length of 1.51 and ARG120 NH2 and the car-
into consideration. Duplicate entries in the resulting con-
Table 2. Total Grid Score of Compounds Interacting with 1PGG
former database were discarded using a RMS filter of 0.1 Å.
This resulted in databases of 3000 conformers on average.
Compound
Grid score (Dock 6.0) 1PGG
From these databases 1500 conformers were selected based
on the dissimilarity of their internal coordinates. The result-
ing conformer databases were used as starting structures for
a rigid docking simulation. We used the simple grid energy
scoring function to score the docked conformations as imple-
mented in program Dock 6.0. Partial charges where calcu-
lated semi-empirically using the AM1 hamiltonian. From the
docked conformations only the best scored conformation was
retained. The complexes were subsequently energy mini-
mized using force field MMFF94 to a gradient of 0.05
kcal/mol and were rescored using the same grid. All com-
pounds could be docked into the active site of 1PGG suc-
cessfully. The individual grid scores are shown in Table 2.
All compounds showed hydrogen bond interaction to the
1PGG protein. The exact numbers of hydrogen bonds can be
seen in Table 3.
1
3
4
6
7
ꢃ24.60
ꢃ16.09
ꢃ11.33
ꢃ20.10
ꢃ24.46
Table 3. Hydrogen Bond Interaction with 1PPG
Compound
No. of hydrogen bonds
Bond lengths [Å]
1
3
4
6
7
2
3
3
2
4
2.38, 1.52
1.52, 1.51, 2.32
1.59, 1.43, 1.80
1.52, 2.38
1.33, 1.47, 1.86, 1.99
Fig. 1. Binding of 1 into the Active Site of 1PGG

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67
Fig. 2. Binding of 3 into Active Site of 1PGG
Fig. 3. Binding of 4 into the Active Site of 1PGG
bonyl oxygen of 3 with a bond length of 2.32, respectively tively (Fig. 3). We think that this accounts for the low ob-
(Fig. 2). served IC50 of compound 4. Compound 6 showed an excel-
Compound 4 exhibited three hydrogen bonds to the 1PGG lent electrostatic and steric interaction with 1PGG and could
protein. These hydrogen bonds were formed by ARG120 and build two hydrogen bonds with 1PGG (Fig. 4). These were
the methoxy group of 4, TYR355 and the carbonyl oxygen of formed by TYR355 and one of the carbonyl oxygens and
4 and SER530 and the second methoxy group of 4. The cal- SER530 and the other carbonyl oxygen of the heptanoid part
culated bond length were 1.59 Å, 1.43 Å and 1.79 Å respec- of 6. The bond length were 1.53 Å and 2.38 Å respectively.

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Vol. 55, No. 1
Fig. 4. Binding of 6 into the Active Site of 1PGG
Fig. 5. Binding of 7 into the Active Site of 1PGG
Four hydrogen bonds were formed by compound 7 (Fig. 5). der Waals interaction with 1PGG. One of the phenyl rings of
Three hydrogen bonds were formed with amino acids of the compound 1 was surrounded by GLU524, ARG120 and
active site of 1PGG ARG120, TYR385 and SER530. One PHE470. The heptanoid part of 1 was surrounded by ILE89,
hydrogen bond was formed with TYR355. The hydrogen ILE115 and VAL119. The second phenyl ring was sur-
bond lengths were 1.24 Å, 1.86 Å, 1.99 Å and 1.47 Å respec- rounded by LEU93, TRP100 and LEU112. The deeper
tively.
buried phenyl ring of compound 3 is surrounded by the ac-
All five compounds used for docking showed a good van tive site amino acid residues SER530, TYR385, LEU352 and

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Table 4. Total Grid Score of Compounds Interacting with 4COX
Table 5. Hydrogen Bond Interaction with 4COX
Compound
Grid score (Dock 6.0) 4COX
Compound
No. of hydrogen bonds
Bond lengths [Å]
1
3
4
6
ꢃ16.32
ꢃ46.76
n.d.
ꢃ36.02
ꢃ35.99
ꢃ45.89
1
3
4
6
2
2
n.d.
2
3
3
1.46, 3.00
2.07, 1.64
n.d.
1.49, 1.65
1.54, 1.65, 2.15
1.33, 2.46, 2.26
7
7
Indomethacin
Indomethacin
Fig. 6. Interaction of Indomethacin with 4COX
ILE523. The heptanoid part of 3 was surrounded by selectivity of the compounds under investigation.
GLU524, ARG120 and VAL116; the second ring of 3 was In conclusion, we have demonstrated that our curcumin
surrounded by ILE89 and ARG83. Similar trends were ob- analogues were selective COX-1 inhibitors with submicro-
served for the other compounds under investigation. molar to micromolar IC50 values and promising selectivities.
After docking simulations with 1PGG (COX-1) we tried to Especially lipophilic and polar substituents on the phenyl
dock the compounds considered in the first simulation into ring, like methoxy or methyl ester groups improved the
the active site of 4COX (COX-2). The active site of 4COX is specifity of the compounds. The biological data were also
considered to be formed of the amino acid residues TYR385, confirmed by docking studies which revealed good van der
that is supposed to abstract one hydrogen from the substrate, Waals interaction and hydrogen bonding towards COX-1
SER530, which is acetylated by acetylsalicylic acid and isoenzym performed by our curcuminoid molecules. As this
HEME.19) All compounds except 4 could be docked into the isoform is overexpressed in a significant number of cancer
active site of 4COX with a reasonable steric and electrostatic cells and tissues our present results may yield in new candi-
interaction. As a test compound, we docked the co-crystal- date drugs for the treatment of these tumor entities.
lized ligand indomethacin into the active site of 4COX. The
Experimental
results can bee seen in Table 4. Indomethacin showed the
Chemistry Melting points were determined on a Kofler hot stage appa-
best electrostatic and steric interaction scores and the tightest
ratus and are uncorrected. The 1H- and 13C-NMR spectra were recorded on a
hydrogen bond network with the active site (Fig. 6). In-
Varian UnityPlus-200 (200 MHz). Chemical shifts are reported in d values
domethacin formed hydrogen bonds between ARG120,
TYR355 and SER530 with bond length of 1.33, 2.46 and
2.26 respectively.
The hydrogen bonds formed by the curcumin analogues
complexes considered in this work and 4COX were much
longer than those of the respective 1PGG complexes (Table 5
(ppm) relative to Me4Si line as internal standard and J values are reported in
Hertz. Mass spectra were obtained by a Shimadzu GC/MS QP 1000 EX
using EI method. The elemental analysis obtained were within ꢄ0.4% of the
theoretical values for the formulas given.
The following compounds were synthesized via two different pathways.
Method 115): In a dry three-necked flask acetylacetone (5 mmol, 0.51 ml)
and boron oxide (3.5 mmol, 0.244 g) were solved in absolute ethyl acetate
and Fig. 7). We therefore argue, that this is the reason for the and stirred for 30 min at 40 °C. Then the corresponding aldehyde (10 mmol)

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Vol. 55, No. 1
Fig. 7. Interaction of 1, 3, 6 and 7 with 4COX
183.2. MS (EI) m/z: 368 (Mꢅ), 279, 201, 105, 77. Anal. Calcd for
C21H20O2S2: C, 68.45; H, 5.47. Found: C, 68.20; H, 5.25.
and tributyl borate (10 mmol, 2.4 ml) were added and stirred for another
30 min. N-Butylamine (quantities given below) was solved in dry ethyl ac-
etate and then added over a period of 15 min. The mixture was heated to
40 °C for 24 h. Then 5 ml of HCl (10%) were added and heated to 60 °C for
an additional hour. The aqueous phase was extracted with ethyl acetate sev-
eral times, the organic layers were dried over Na2SO4 and the solvent dis-
tilled off. An unsoluable precipitate (part of the product) was filtered off and
recrystallized with the residue from various solvents.
(1E,6E)-1,7-Di-[4-(methylsulfonyl)phenyl]-1,6-heptadien-3,5-dione (3)
(1E,6E)-1,7-Di-[4-(methylmercapto)phenyl]-1,6-heptadien-3,5-dione (2)
(2.5 mmol, 0.924 g) was solved in acetone; then oxone® (10 mmol, 6.14 g) in
15 ml water was added and the mixture was stirred at room temperature for
24 h. After addition of 20 ml ammonia (10%) and stirring for an additional
hour water was added, the precipitate formed was filtered off and washed
with water. The yellow solid was solved in DMF and ethanol was added to
isolate 0.278 g (25%) of the pure compound; mp ꢀ300 °C. 1H-NMR
(DMSO-d6) d: 3.24 (6H, s), 5.77 (1H, s), 7.02 (2H, d, JEꢁ15.7 Hz), 7.48
(2H, d, JEꢁ15.7 Hz), 7.87—7.98 (8H, m), OH signal not detected; 13C-NMR
(DMSO-d6) d: 43.7, 104.6, 127.7, 128.4, 134.3, 134.4, 140.6, 141.0, 181.1.
MS (EI) m/z: 432 (Mꢅ), 201, 111, 68. Anal. Calcd for C21H20O6S2: C, 58.32;
H, 4.66. Found: C, 58.47; H, 4.34.
Method 216): In a dry three-necked flask acetylacetone (5 mmol, 0.51 ml)
and boron oxide (3.5 mmol, 0.244 g) were solved in 5 ml absolute ethyl
acetate and heated to 75 °C for 1 h. The corresponding benzaldehyde
(10 mmol) and tributyl borate (10 mmol, 2.4 ml) were mixed with ethyl ac-
etate, stirred for 45 min and then added to the solution. The mixture was
heated to 100 °C for 1 h. Then n-butylamine (quantities given below) was
solved in 5 ml ethyl acetate and 3.85 ml of this solution were added drop-by-
drop over a period of 90 min. The reaction was stirred for 18 h at 85 °C and
then cooled to 60 °C. Then 5 ml of a HCl solution (10%) were heated to
50 °C, added and the mixture was stirred at 60 °C for 1 h. The solution was
extracted three times with ethyl acetate, the organic layers were dried over
Na2SO4 and the solvent reduced to 7—8 ml. Two and a half milliliters
ethanol were added, the solution was cooled overnight, then the precipitate
was filtered off and recrystallized to obtain the purified product.
(1E,6E)-1,7-Di-(3,4-difluorphenyl)-1,6-heptadien-3,5-dione (1) The
compound was synthesized from 3,4-difluorobenzaldehyde (10 mmol,
1.16 ml) and n-butylamin (2.5 mmol, 0.25 ml) following method 2. Crystal-
lization from ethyl acetate/water (1ꢅ1) afforded 0.066 g (3.8%) of a yellow
solid; mp 137 °C. 1H-NMR (CDCl3) d: 5.82 (1H, s), 6.52 (2H, d,
JEꢁ15.8 Hz), 7.12—7.42 (6H, m), 7.56 (2H, d, JEꢁ15.8 Hz), 15.76 (1H,
br s). 13C-NMR (CDCl3) d: 102.2, 116.1 (d, JC,Fꢁ18.0 Hz), 117.9 (d,
(1E,6E)-1,7-Di-(2,3,4-trimethoxyphenyl)-1,6-heptadien-3,5-dione (4)
The compound was synthesized from 2,3,4-trimethoxybenzaldehyde
(10 mmol, 1.96 g) and n-butylamine (2.5 mmol, 0.25 ml) following method
2. Purification was carried out with column chromatography (eluent
toluene/ethyl acetate 8ꢅ2) and crystallization from ethanol (80%) to afford
1
0.087 g (3.8%) of a yellow solid; mp 115 °C. H-NMR (DMSO-d6) d: 3.89
(6H, s), 3.91 (6H, s), 3.94 (6H, s), 5.83 (1H, s), 6.63 (2H, d, JEꢁ15.0 Hz),
6.71 (2H, A-part of AB-system, JA,Bꢁ8.8 Hz), 7.30 (2H, B-part of AB-sys-
tem, JA,Bꢁ8.8 Hz), 7.85 (2H, d, JEꢁ15.8 Hz), 16.06 (1H, br s). 13C-NMR
(DMSO-d6) d: 56.1, 60.9, 61.4, 101.3, 107.6, 122.2, 123.2, 123.4, 135.3,
142.4, 153.4, 155.4, 183.6. MS (EI) m/z: 278 (half mass), 247, 235, 204,
131, 43. Anal. Calcd for C25H28O8: C, 65.78; H, 6.18. Found: C, 65.49; H,
6.17.
(1E,6E)-1,7-Diferrocenyl-1,6-heptadien-3,5-dione (5) The compound
was synthesized from ferrocenealdehyde (10 mmol, 2.18 g) and n-butyl-
amine (7.5 mmol, 0.74 ml) following method 2. Crystallization from ethyl
acetate afforded 0.205 g (8.3%) of a purple solid; mp 210 °C. 1H-NMR
(CDCl3) d: 4.17 (10H, s), 4.44 (4H, s), 4.52 (4H, s), 5.62 (1H, s), 6.21 (2H,
d, JEꢁ15.5 Hz), 7.54 (2H, d, JEꢁ15.5 Hz), enol OH signal not detected. 13C-
NMR (CDCl3) d: 68.5, 69.7, 71.0, 79.7, 100.1, 121.3, 141.5, 182.7. MS (EI)
m/z: 492 (Mꢅ), 280, 246, 239, 121, 56. HR-MS m/z: 492.0476 (Calcd for
C27H24O2Fe2: 492.0459).
(1E,6E)-Methyl 4-[7-(4-methoxycarbonyl)phenyl]-3,5-dioxo-1,6-hep-
tadienyl]benzoate (6) The compound was synthesized from (4-formyl)-
methylbenzoate (10 mmol, 1.64 g) and n-butylamine (7.5 mmol, 0.74 ml) fol-
lowing method 2. Crystallization from ethyl acetate afforded 0.334 g
J
C,Fꢁ18.0 Hz), 124.9 (quintett), 132.1 (quartett), 138.3—138.5 (m), 148.5
(d, JC,Fꢁ22.2 Hz), 153.6 (d, JC,Fꢁ39.4 Hz), 182.8; MS (EI) m/z: 348 (Mꢅ),
306, 167, 139, 119. Anal. Calcd for C19H12O2F4: C, 65.52; H, 3.47. Found:
C, 65.41; H, 3.69.
(1E,6E)-1,7-Di-[4-(methylmercapto)phenyl]-1,6-heptadien-3,5-dione
(2) The compound was synthesized from 4-(methylmercapto)benzalde-
hyde (10 mmol, 1.33 ml) and n-butylamine (7.5 mmol, 0.74 ml) following
method 1. Crystallization from ethanol afforded 1.14 g (62%) of a yellow
solid; mp 194 °C. 1H-NMR (CDCl3) d: 2.51 (6H, s), 5.81 (1H, s), 6.58 (2H,
d, JEꢁ15.8 Hz), 7.24 (4H, A-part of AB-system, JA,Bꢁ8.7 Hz), 7.47 (4H, B-
part of AB-system, JA,Bꢁ8.7 Hz), 7.62 (2H, d, JEꢁ15.8 Hz), enol OH signal
not detected; 13C-NMR (CDCl3) d: 15.2, 101.8, 123.1, 126.0, 128.5, 140.0,

January 2007
71
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(16.8%) of a yellow solid; mp 215 °C: H-NMR (CDCl3) d: 3.94 (6H, s),
5.90 (1H, s), 6.71 (2H, d, JEꢁ15.9 Hz), 7.62 (4H, A-part of AB-system,
J
ABꢁ8.2 Hz), 7.69 (2H, d, JEꢁ15.9 Hz), 8.07 (4H, B-part of AB-system,
J
ABꢁ8.2 Hz), 15.74 (1H, br s). 13C-NMR (CDCl3) d: 52.3, 102.5, 126.1,
127.9, 130.1, 131.2, 139.1, 139.4, 166.4, 182.9; MS (EI) m/z: 392 (Mꢅ),
145, 115, 102, 59. Anal. Calcd for C23H20O6: C, 70.40; H, 5.14. Found: C,
70.18; H, 5.04.
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(1E,3E,8E,10E)-1,11-Di-(4-methoxyphenyl)-1,3,8,10-undecatetraen-
5,7-dione (7) The compound was synthesized from 4-methoxycin-
namaldehyde (10 mmol, 1.62 g) and n-butylamine (7.5 mmol, 0.74 ml) fol-
lowing method 1. Crystallization from ethanol (70%) afforded 0.219 g
1
(11.3%) of an orange solid; mp 210 °C. H-NMR (CDCl3) d: 3.83 (6H, s),
7) Nurfina A. N., Reksohadiprodjo M. S., Timmermann H., Jenie U. A.,
Sugiyanto D., van der Goot H., Eur. J. Med. Chem., 32, 321—328
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5.66 (1H, s), 6.12 (2H, d, JEꢁ15.0 Hz), 6.72—6.93 (8H, m), 7.36—7.51
(6H, m), 15.90 (1H, br s). 13C-NMR (CDCl3) d: 55.4, 101.5, 114.3, 125.0,
126.6, 128.7, 129.1, 139.9, 141.1, 160.4, 183.1. MS (EI) m/z: 388 (Mꢅ),
187, 121, 57. Anal. Calcd for C25H24O4·0.25H2O: C, 76.31; H, 6.29. Found:
C, 76.33; H, 6.45.
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Cyclooxygenase Assay The effects of the test compounds on COX-1
and COX-2 were determined by measuring prostaglandin E2 (PGE2) using a
COX Inhibitor Screening Kit (Catalog No 560131) from Cayman Chemi-
cals, Ann Arbor, Michigan, U.S.A. Reaction mixtures were prepared in
100 mM Tris–HCl buffer, pH 8.0 containing 1 mM heme and COX-1 (ovine)
or COX-2 (human recombinant) and pre-incubated for 10 min in a waterbath
(37 °C). The reaction was initiated by the addition of 10 ml arachidonic acid
(final concentration in reaction mixture 100 mM). After 2 min the reaction
was terminated by adding 1 M HCl and finally PGE2 was quantified by an
ELISA method. The test compounds were dissolved in DMSO and diluted to
the desired concentration with 100 mM potassium phosphate buffer (pH 7.4).
Following transfer to a 96 well plate coated with a mouse anti-rabbit IgG,
the tracer prostaglandin acetylcholine esterase and primary antibody (mouse
anti PGE2) were added. Plates were then incubated at room temperature
overnight, reaction mixtures were removed, and the wells were washed with
10 mM potassium phosphate buffer containing 0.05% Tween 20. Ellman’s
reagent (200 ml) was added to each well and the plate was incubated at room
temperature (exclusion of light) for 60 min, until the control wells yielded an
ODꢁ0.3—0.8 at 412 nm. A standard curve with PGE2 was generated from
the same plate, which was used to quantify the PGE2 levels produced in the
presence of test samples. Results were expressed as a percentage relative to
a control (solvent-treated samples). All determinations were performed in
duplicate and values generally agreed within 10%.
Docking Studies All calculations were performed on a SUN Ultra 40
workstation using program DOCK 6.0 (Kuntz, I.D., DOCK, UCSF Box
2240, UCSF, San Francisco, CA 94143-2240) and MOE (Molecular Operat-
ing Environment, Chemical Computing Group Inc., Montreal, Quebec,
Canada). The crystal structures of COX-1 and COX-2 co-crystallized with
indomethacin (1PGG.pdb and 4COX.pdb respectively)19,20) were taken from
the Brookhaven Protein Databank18) and used for docking. Regions with a
7.0 Å radius from the complexed inhibitor were marked as interesting.
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