Synthesis of curcumin and ethylcurcumin bioconjugates as potential antitumor agents

Article abstract of DOI:10.1007/s00044-011-9587-3

Some new curcumin and ethylcurcumin bioconjugates with various functionalities supported on the curcumin skeleton were synthesized and evaluated for antitumor activity. Most of the newly synthesized compounds are more active than curcumin and ethyl curcum

Full text of DOI:10.1007/s00044-011-9587-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:874–890
DOI 10.1007/s00044-011-9587-3
ORIGINAL RESEARCH
Synthesis of curcumin and ethylcurcumin bioconjugates
as potential antitumor agents
•
Reem I. Al-Wabli Omaima M. AboulWafa
•
Khairia M. Youssef
Received: 28 December 2009 / Accepted: 28 January 2011 / Published online: 20 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Some new curcumin and ethylcurcumin bio-
conjugates with various functionalities supported on the
curcumin skeleton were synthesized and evaluated for
antitumor activity. Most of the newly synthesized com-
pounds are more active than curcumin and ethyl curcumin
but are less cytotoxic than the reference compound doxo-
rubicin. Surprisingly, many of these compounds are not
cytotoxic to noncancer cells. Compounds 5c, 5e, 5g, 5j, 6b,
and 6g having 5-methylthiadiazole, 6-methoxy-benzothia-
zole, diethylaminoethyl and the usual alkylating bis(2-
chloroethyl)amino moieties showed the highest cytotoxic
activity against SK-MEL cancer cells. Compounds 5k, 6c,
and 6g are less cytotoxic to KB cancer cells. Moreover,
compounds 5c, 5e, 5j, 5k, 6d, 6e, 6f, and 6g showed
cytotoxicity against BT-549 cancer cells with 5j being the
most active compound. Curcumin and the new intermediate
di-O-chloroacetylcurcumin (3a) were also cytotoxic
against the same cell line but are less active than the target
compounds. Compound 6b is the only one exhibiting
cytotoxicity against SK-OV-3 cancer cells.
Introduction
Curcumin (1), [diferuloylmethane, 1,7-bis(4-hydroxy-3-
methoxyphenyl)-1,6-heptadi-ene-3,5-dione], is well-
a
known acyclic diarylheptanoid which has been identified as
the major constituent of turmeric powder extracted from
the rhizome of the plant curcuma longa (Anderson et al.,
2000). Recently, numerous studies have demonstrated the
remarkable antioxidant and free radical scavenging activ-
ities of curcumin (Mukhopadhyay et al., 1982; Sharmam,
1976; Srimal and Dhawan, 1973; Rao et al., 1982; Kunc-
handy and Rao, 1990; Soudamini and Kuttan, 1989; You-
ssef et al., 2004; Al-Omar et al., 2005). Also, it has long
been used as a natural occurring medicine for the treatment
of inflammatory diseases (Toda et al., 1985; Jovanovic
et al., 2001). In addition, several studies have shown that
curcumin and curcumin derivatives possess antiprolifera-
tive activities against tumor cells in vitro (Mehta et al.,
1997; Al-Omar et al., 2005). Curcumin is a potent inhibitor
of tumor initiation in vivo (Huang et al., 1988, 1994, 1997)
and is also a potent chemopreventive agent inhibiting
tumor promotion in skin, oral, intestinal, and colon carci-
nogenesis (Huang et al., 1994; Kelloff et al., 1994).
Besides, it possesses several other biological activities
including antibacterial (Kumar et al., 2001), antiviral
(Mazumder et al., 1995), antihepatotoxic (Kiso et al., 1983;
Lin et al., 1998), hypotensive (Rajakrishnan et al., 1999),
and anticholesterolemic (Masuda et al., 2001) activities.
Curcumin, having an unique conjugated structure
including two methoxylated phenols (1a) and an enol form
of b-diketone (1b), shows a typical radical trapping ability as
a chain breaking antioxidant (see Scheme 1). The antioxi-
dant mechanism of curcumin and curcumin-related phenols,
also found in edible or medicinal plants, has attracted much
attention (Roughley and Whiting, 1973; Rao et al., 1982;
Keywords Curcumin derivatives ꢀ
Curcumin and ethylcurcumin bioconjugates ꢀ
Antitumor activity
R. I. Al-Wabli
Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia
O. M. AboulWafa ꢀ K. M. Youssef (&)
Department of Pharmaceutical Chemistry,
Faculty of Pharmaceutical Sciences and Pharmaceutical
Industries, Future University, Cairo 12311, Egypt
e-mail: khairia_m@yahoo.com
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Med Chem Res (2012) 21:874–890
875
OH
O
O
O
H
3CO
HO
OCH3
OH
H
3CO
HO
OCH3
OH
(a)
(b)
(1)
Scheme 1
Ruby et al., 1995; Sreejayan and Rao, 1994; Khopde et al.,
1999; Jovanovic et al., 1999; Snyder and Arnone, 2002);.
Accordingly, it became of interest to synthesize some new
bioconjugates with various functionalities supported on the
curcumin skeleton to be evaluated for anticancer activity.
Curcumin bioconjugates can serve a dual purpose of sys-
temic delivery as well as therapeutic agents against cancer
diseases. This design would allow enzyme-mediated trans-
formation of the bioconjugate within the target organ. The
selected moieties involved in such structural modification
featured sulfonamide, alkyl, cycloalkyl, and heterocyclic
amino functionalities attached to curcumin or ethyl curcu-
min through an acetyl or propionyl bridge to investigate the
effect of molecular modification on the biological activity of
compounds (A) (see Structure 1). Furthermore, we were
motivated to select adamantoyl chloride, heptanoyl chloride,
and 2-thienoyl chloride and directly attach them through an
ester function to the curcumin and ethyl curcumin core to
furnish compounds (B). The conjugate bonds reported
herein are ester or amino ester linkages which are enzyme
sensitive to produce the expected systemic delivery.
Recently, curcumin, having a planar topology, has been
shown to inhibit topoisomerase II in a similar fashion to the
antineoplastic agent etoposide (Snyder et al., 2002). Results
pointed to DNA damage induced by topoisomerase II poi-
soning as a possible mechanism by which curcumin initiated
apoptosis. With the hope to go a step forward in the field of
anticancer agents, the synthesized compounds were
screened for their cytotoxic activity as well as topoisomerase
inhibitory activity. The synthesis of the target compounds is
outlined in Schemes 2, 3, and 4.
Experimental part
Synthesis
Melting points were determined in open glass tubes on a
Branstead/Electrothermal IA9100 melting point apparatus
and are uncorrected. Infrared (IR) spectra were recorded,
for potassium bromide disks, m (cm-1) on Perkin Elmer
1430 spectrophotometer. 1H and 13C nuclear magnetic
resonance (NMR) spectra were determined on Jeol
(300 MHz, 500 MHz) and Ultrashield Bruker Biospin
(500 MHz) spectrometers. Chemical shifts are expressed as
d values (ppm) using tetramethylsilane (TMS) as internal
reference. Signals are indicated by the following letters:
s = singlet,
d = doublet,
t = triplet,
q = quartet,
m = multiplet, br = broad. Mass spectra (MS) were
obtained on GC/MS QP 5000 (Ver. 2), Class-5000 (Ver.
1.2) Shimadzu apparatus. Follow up of the reaction and
checking the homogeneity of the compounds were made by
ascending thin layer chromatography (TLC) run on pre-
coated (0.25 mm) (GF 254) silica gel plates. The ratio of the
solvent systems used as eluents were volume to volume.
Visualization of the spots was performed by exposure to UV
lamp at 254 nm. Silica gel (60-230 mesh E. Merck) was
employed for routine column chromatography separations.
According to Scheme 2, the key starting materials,
commercially available, curcumin (1) and ethyl curcumin
(2) were either purchased from Sigma-Aldrich (St. Louis,
MO) or prepared according to previously reported methods
(Mohri et al., 2003).
alkyl, cycloalkyl or heterocyclic amine
O
O
R
O
1O
OR1
O
R2
R3
R2
R3
N-(CH2
)
x
O
(CH2)x-N
O
carbonyl group
curcumin skeleton
methyl or ethyl bridge
(a)
O
O
Di-O-chloroacetylcurcumin (3a) and di-O-
chloropropionylcurcumin (3b)
R1
O
OR1
O
O
R2
O
O
R2
Method A
carbonyl group
curcumin skeleton
Adamantyl or thienyl
(b)
Sodium carbonate (2 g) was added to a stirred ice-cold
solution of curcumin (1) (3.68 g, 0.01 mol) in dry acetone
Structure 1
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876
Med Chem Res (2012) 21:874–890
O
O
R1O
HO
R1O
HO
OR1
OH
CHO
O
O
(BuO)3B /
n-BuNH2
2
+
B2O3
(1 ) R 1 = CH3
(2 ) R 1 = C2H5
O
O
Cl
anh.K2CO3 /
/RT
Cl
(CH2
)
n
O
O
OR1
R1O
O
O
Cl
Cl
n(H2C)
O
O
(CH2)n
(3 a-d)
O
O
n-BuNH2
(BuO)3B /
B O
/
2
3
R1O
O
CHO
R1O
CHO
O
1N NaOH/CHCl3 or
2
Cl
Cl
+
O
Cl
(CH2)n
n(H2C)
O
HO
anh.K2CO3
/
(4 a-d)
Scheme 2 Preparation of Di-O-chloroacylcurcumin and di-O-chloroacylethylcurcumin (3a–d)
O
O
R1O
OR1
O
O
R3R2N
NR2R3
n(H2C)
O
O
(CH2)n
O
(5 a-n, 6a-n)
R2R3NH
NEt3 /
O
O
R1O
O
OR1
O
O
O
Cl
Cl
n(H2C)
(CH2)
n
(3 a-d)
-SO2NHR4
NH2-
O
O
R1O
O
OR1
O
O
NH-
-SO2NHR4
R4NHSO 2
-
-NH
n(H2C)
O
(CH2 )
n
(7a-f)
Scheme 3 Preparation of 1,7-Bis(4-alkyl(cycloalkyl or heteroaryl)-
aminoacyloxy)-3-(methoxyphenyl)-1,6-hepta-diene-3,5-dione (5a–n)
and 1,7-bis(4-alkyl (cycloalkyl or heteroaryl)aminoacyloxy)-3-(eth-
oxyphenyl)-1,6-hepta- diene-3,5-dione (6a–n) and preparation of 1,
7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-(methoxyphenyl)-1,
6-heptadiene-3,5-dione (7a, b) and 1,7-bis(4-(4-substituted sulfani-
lamido)acyloxy)-3-(ethoxy-phenyl)-1,6-heptadiene-3,5-dione (7c–f)
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Med Chem Res (2012) 21:874–890
877
O
O
R1
O
OR1
O
CO-
O-CO
COCl
(8a, b)
O
Na2CO3/
O
O
O
O
R1
O
R
1O
OR1
OR1
CH3(CH2)5COCl
O
HO
OH
H
3C(H 2C)5OCO
OCO(CH 2)5CH3
Na2CO3/
(8c, d)
(1), (2)
O
COCl
S
O
O
Na2CO3/
R
1O
OR1
COO-
OO- C
S
S
(8e, f)
Scheme 4 Preparation of Di-O-adamantoylcurcumin (8a), di-O-adamantoylethyl curcumin (8b, Di-O-heptanoylcurcumin (8c) and di-O-
heptanoylethyl curcumin (8d) and Di-O-(2-thienoyl)curcumin (8e) and di-O-(2-thienoyl)ethyl curcumin (8f)
Table 1 Physicochemical data of di-O-chloroacetylcurcumin (3a) and di-O-chloropropionylcurcumin (3b)
Compds no.
Structures
Yield%
43
M.p. °C
Molecular formula
C25H22Cl2O8
M. wt.
521.5
3a
136
O
O
H3CO
OCH3
ClH2COCO
OCOCH2Cl
3b
38
143
C27H26Cl2O8
549.5
O
O
H3CO
OCH3
ClH2CH2COCO
OCOCH2CH2Cl
(50 ml) for 15 min. Chloroacetyl chloride or chloropro-
pionyl chloride (0.025 mol) was dropwise added and the
mixture was stirred for 3 days at RT. The reaction
mixture was filtered, evaporated, and extracted with
EtOAc (3 9 30 ml). The organic layer was dried over
anhydrous MgSO4 (5 g) and evaporated under reduced
pressure to give an orange oil. Addition of EtOH (20 ml)
gave a yellow precipitate which was filtered off. The
product was purified by column chromatography using
toluene/MeOH (97.5:2.5 v/v) as eluent (Table 1). IR of
di-O-chloroacetylcurcumin (3a) m (cm-1): 3414.8 (OH,
intramolecularly H-bonded), 1768.7 (C=O, ester), 1635.7
(C=O, ketone), 1599.9, 1507.6 (C=C Ar), 1254.8, 1122.4
(mas and ms C–O–C). 1H-NMR of 3a (CDCl3) d ppm
(300 MHz): 3.88 (s, 6H, 2 OCH3), 4.10 (s, 4H, 2 CH2),
5.86 (s, 2H, Ha), 6.57 (d, dist, 2H, 2Hb, J = 15.9 Hz),
7.08–7.26 (m, 6H, 2 (3 Ar–H)), 7.61 (d, dist, 2H, 2Hc,
J = 15.7 Hz). IR of di-O-chloropropionylcurcumin (3b) m
(cm-1): 3414.2 (OH, intramolecularly H-bonded), 1746.6,
(C=O, ester), 1635.1 (C=O, ketone), 1607.4, 1506.4 (C=C
1
Ar), 1253, 1129.3 (mas and ms C–O–C). H-NMR of 3b
(DMSO-d6) d ppm (300 MHz): 3.12 (t, 4H, 2 CH2–Cl,
J = 6.1 Hz), 3.84 (s, 6H, 2 OCH3), 3.89 (t, 4H, 2
COCH2, J = 6.33 Hz), 6.2156 (s, 2H, Ha), 7.0095 (d,
dist, 2H, 2 9 Hb, J = 15.9 Hz), 7.15–7.53 (m, 6H, 2 9 3
Ar–H), 7.65 (d, dist, 2H, 2 9 Hc, J = 15.9 Hz); MS of
3b: m/z (% relative abundance): M? 549.5 (absent), 73
(100).
123

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Med Chem Res (2012) 21:874–890
Table 2 Physicochemical data of the synthesized compounds 4a–d
Compds. no. Structures
Method A reaction Method B reaction Yield% Yield% M.p. °C Molecular
M. wt.
time (h)
time (h)
A
B
formula
CHO
4a
3
24
43
78
68–71
C10H9ClO4
228.5
ClH2COCO
OCH3
4b
24
3
41
72
65–69
C11H11ClO4 242.5
CHO
ClH2CH2COCO
OCH3
CHO
4c
24
24
3
3
42
39
67
61
61–64
55–59
C11H11ClO4 242.5
ClH2COCO
OCH2CH3
CHO
4d
C12H13ClO4 256.5
ClH2CH2COCO
OCH2CH3
Method B
(300 MHz): 1.31 (t, 3H, OCH2CH3, J = 6.87 Hz), 4.16 (q,
2H, OCH2CH3, J = 6.87 Hz), 4.74 (s, 2H, CH2Cl), 7.41 (d,
1H, HA, JAB = 7.98 Hz, ortho coupling), 7.58, 7.6051 (dd,
1H, HB, J = 1.65 and 8.07 Hz, meta and ortho coupling),
7.63 (d, 1H, HX, JAX = 1.63 Hz, para coupling), 9.97 (s,
1H, CHO).
3-Alkoxy-4-chloroacyloxybenzaldehyde
(4a–d) The
appropriate acid chloride (0.01 mol) in CHCl3 (20 ml) was
added dropwise over a period of 5 min to an ice-cold
stirred solution of the aldehyde (0.01 mol) and 1 N NaOH
(2 ml) in chloroform (5 ml). The mixture was stirred at RT
for 1 day and then evaporated to dryness. The residue was
extracted (3 9 20 ml) with EtOAc and 0.1 N NaOH. The
combined organic layers were washed with water
(3 9 20 ml), dried over anhydrous MgSO4 (5 g) and
removed under reduced pressure to give a colorless oil.
Trituration of the crude product with EtOH (20 ml) gave
colorless crystals of 4a–d which were crystallized from
EtOH (Table 2).
Di-O-chloroacylcurcumin (3a, b) and di-O-chloroacyleth-
ylcurcumin (3c, d) To a stirred solution of the appropriate
3-alkoxy-4-chloroacyloxybenzaldehyde (4a–d) (0.04 mol)
in dry EtOAc (20 ml) was added tri-(n-butyl) borate
(21 ml, 0.08 mol) and the mixture was stirred at RT for
10 min. The previously prepared complex formed by stir-
ring for 1 h acetylacetone (2 g, 0.02 mol) with boric
anhydride (1 g, 0.014 mol) was then added to this solution.
After stirring for 5 min at RT, n-butylamine (0.1 ml) was
dropwise added every 10 min (total amount 0.4 ml) and the
reaction mixture was stirred at RT for an overnight. 0.4 N
HCl (30 ml, 60sC) was then added and the mixture was
further stirred for 2 h followed by extraction with EtOAc
(3 9 25 ml). The combined organic layers were washed
with H2O, dried over anhydrous MgSO4 (5 g) and con-
centrated to ^15 ml. MeOH (15 ml) was added and the
mixture was allowed to stand in the refrigerator for 3 h to
give a yellow precipitate of 3a–d which was filtered off,
The selected acid chloride (0.01 mol) was dropwise
added to an ice-cold stirred solution of the appropriate
aldehyde (0.01 mol) in dry acetone and anhydrous K2CO3.
The mixture was further stirred in an ice bath for 3 h, filtered,
and concentrated. Dilution with EtOH (20 ml) precipitated
colorless crystals of 4a–d which were filtered and crystal-
lized from EtOH (Table 2). IR of 4a m (cm-1): 2933.4, 2847
(CHO), 1765 (C=O ester), 1698 (C=O aldehyde), 1600, 1505
(C=C aromatic), 1180, 1150, 1100, 1020 (mas and ms C–O–C);
1H-NMR of 4a (DMSO-d6) d ppm (500 MHz): 3.84 (s, 3H,
OCH3), 4.72 (s, 2H, CH2–Cl), 7.39 (d, 1H, HA, JAB = 8.40,
ortho coupling), 7.56, 7.58 (dd, 1 H, HB, J = 1.55 and
7.65 Hz, meta and ortho coupling), 7.61 (d, 1H, HX,
JAX = 1.55 Hz, para coupling), 9.95 (s, 1H, CHO). IR of 4b
m (cm-1): 3073, 2940 (CHO), 1760 (C=O ester), 1698 (C=O
aldehyde), 1601, 1495 (C=C aromatic), 1154, 1120, 1068,
1025 (mas and ms C–O–C). 1H-NMR of 4c (DMSO-d6) d ppm
1
washed with cold MeOH, and dried (Table 3). IR and H-
NMR spectra of compounds 3a, b were coinciding with
those obtained from method A. IR of 3c m (cm-1): 3435.6
(OH, intramolecularly H-bonded), 1746 (C=O, ester),
1630.9 (C=O, ketone), 1558.1, 1496.3 (C=C aromatic),
1
1270, 1157 (mas and ms C–O–C); H-NMR of 3c (CDCl3) d
ppm (300 MHz): 1.42 (t, 6H, 2 OCH2CH3, J = 6.87 Hz),
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Med Chem Res (2012) 21:874–890
879
Table 3 Physicochemical data of the synthesized compounds 3a–d
O
O
R O
OR
1
1
O
O
Cl
Cl
O
n(H C)
O
(CH )n
2
2
(3a-d)
(3a–d)
Compds. no.
R1
n
Yield%
M. p. °C
Molecular formula
M. wt.
3a
3b
3c
3d
CH3
1
2
1
2
61
56
59
54
136
143
130
145
C25H22Cl2O8
C27H26Cl2O8
C27H26Cl2O8
C29H30Cl2O8
521.5
549.5
549.5
577.5
CH3
C2H5
C2H5
4.11 (q, 4H, 2OCH2CH3, J = 6.87 Hz), 4.33 (s, 4H, 2
CH2–Cl), 5.85(s, 2H, Ha), 6.56 (d, 2H, 2Hb, J = 15.93 Hz),
7.08–7.18 (m, 6H, 2 (3-Ar–H)), 7.61 (d, 2H, 2Hc,
J = 15.93 Hz); MS of 3c m/z (% relative abundance): M?
of 5b m (cm-1): 3550, 3414.4 (NH ? OH intramolecular
H-bonded), 1733.7 (C=O, ester), 1637.4 (C=O, ketone),
1618 (C=N mixed with C=C aromatic), 1510.2 (C=C,
1
aromatic), 1272.1 1122.9 (mas and ms C–O–C); H-NMR of
1
548 (0.24), M? ?2 550 (0.21), M? ?4 552, 83 (100). H-
5b (DMSO-d6) d ppm (300 MHz): 2.09 (s, 4H, 2 CH2),
3.84 (s, 12H, 4 OCH3), 6.06 (s, 2H, Ha), 6.77 (d, 2H, 2 Hb,
J = 15.66 Hz), 6.82 (d, 2H, 2 HA, JAB = 8.25 Hz, ortho
coupling), 7.14, 7.17 (dd, 2H, 2 HB, J = 1.65 and 8.25 Hz,
meta and ortho coupling), 7.33 (d, 2H, HX, JAX = 1.65 Hz,
para coupling), 7.55 (d, 2H, 2 Hc, J = 15.66 Hz, over-
lapping with benzothiazole multiplet), 7.53–7.61 (m, 6H, 2
(3-benzothiazole-H)), 9.69 (s, 2H, 2 NH, D2O-exchange-
able); MS of 5b m/z (% abundance): M? 808 (absent), 125
(100). IR of 5c m (cm-1): 3425 (NH ? OH intramolecu-
larly H-bonded), 1727.3 (C=O, ester), 1630 (C=O, ketone
mixed with C=N), 1575, 1506 (C=C aromatic), 1272.5,
NMR of 3d (CDCl3) d ppm (500 MHz): 1.44 (t, 6H,
2 9 OCH2CH3, J = 7 Hz), 3.10 (t, 4H, 2 COCH2,
J = 7 Hz), 3.90 (t, 4H, 2 CH2–Cl, J = 7 Hz), 4.13 (q, 4H,
2 OCH2CH3, J = 7 Hz), 5.87 (s, 1H, Ha), 6.57 (d, 2H, 2
Hb, J = 16 Hz), 7.09 (d, 2H, 2 HA, JAB = 8 Hz, ortho
coupling), 7.17 (d, 2H, 2 HB, JAB = 8.5 Hz, ortho cou-
pling), 7.28 (s, 2H, 2 HX), 7.55 (d, 2H, 2 Hc, J = 16 Hz),
9.96 (s, 1H, enol OH).
1,7-Bis(4-alkyl(cycloalkyl or heteroaryl)aminoacyloxy)-3-
(methoxyphenyl)-1,6-hepta-diene-3,5-dione (5a–
n) and 1,7-bis(4-alkyl (cycloalkyl
1
1120.7 (mas and ms C–O–C); H-NMR of 5c (DMSO-d6) d
or heteroaryl)aminoacyloxy)-3-(ethoxyphenyl)-1,6-hepta-
diene-3,5-dione (6a–n)
ppm (300 MHz): 2.50 (s, 4H, 2 9 CH2), 3.35 (s, 6H, 2
CH3), 3.84 (s, 6H, 2 OCH3), 6.06 (s, 2H, Ha), 6.76 (d, 2H, 2
Hb, J = 15.93 Hz), 6.82 (d, 2H, 2 HA, JAB = 8.22 Hz,
ortho coupling), 7.14, 7.17 (dd, 2H, 2 HB, J = 1.65 and
8.01 Hz, meta and ortho coupling), 7.33 (d, 2H, 2 HX,
JAX = 1.65 Hz), 7.55 (d, 2H, 2 Hc, J = 15.66 Hz), 9.69 (s,
2H, 2 9 NH, D2O-exchangeable). 1H-NMR of 5d
(CD3OD) d ppm (500 MHz): 2.29 (s, 2H, 2 NH), 2.31 (t,
dist, 4H, 2 9 CH2–N), 3.23 (t, 4H, 2 CH2–Cl, J = 3.5 Hz),
3.33 (s, 4H, 2 9 COCH2–N), 3.92 (s, 6H, 2 9 OCH3),
4.90 (s, 2H, 2 9 Ha), 6.64 (d, 2H, 2 Hb, J = 15.5 Hz), 6.84
(d, 2H, 2 HA, JAB = 8 Hz, ortho coupling), 7.12 (d, 2H, 2
HB, 7.7 Hz, ortho coupling), 7.23 (s, 2H, 2 HX), 7.58 (d,
2H, 2 9 Hc, J = 15.5 Hz). IR of 5e m (cm-1): 3414.8 (OH
intramolecularly H-bonded), 1728.4 (C=O, ester), 1637.6
(C=O, ketone), 1618.1, 1511 (C=C aromatic), 1272.6,
NEt3 (five drops) were added to a solution of di-O-chlo-
roacylcurcumin 3a, b or di-O-chloroacylethyl curcumin 3c,
d (0.005 mol) in EtOH (30 ml). The appropriate amine
(0.01 mol) was then added and the mixture was heated
under reflux for the specified time (Table 4). EtOH was
evaporated under reduced pressure and the residue was
crystallized from CHCl3 (15 ml) giving compounds 5a–n
1
and 6a–n (Table 4). H-NMR of 5a (DMSO-d6) d ppm
(300 MHz): 1.16–1.99 (hump, br, 30H, 2 Ad-H), 2.09 (s,
4H, 2 CO–CH2), 3.83 (s, 6H, 2 OCH3), 6.03 (s, br, 2H, Ha),
6.74 (d, 2H, 2 Hb, J = 15.66 Hz), 6.80 (d, 2H, 2 HA,
JAB = 8.25 Hz, ortho coupling), 7.13, 7.16 (dd, dist, 2H,
2 9 HB, J = 1.5 and 8.22 Hz, meta and ortho coupling),
7.31 (s, dist, 2H, 2 NH), 7.17 (d, 2H, 2 HX, JAX = 0.24 Hz,
para coupling), 7.53 (d, 2H, 2 9 Hc, J = 15.66 Hz); MS
of 5a m/z (% abundance): M? 750 (absent), 135 (100). IR
1
1122.9 (mas and ms C–O–C); H-NMR of 5e (DMSO-d6) d
ppm (300 MHz): 2.09 (s, 4H, 2 COCH2–N), 2.50 (t, 8H,
4N-CH2CH2–Cl, J = 3.57 Hz), 3.48 (t, 8H, 4 NCH2CH2–
123

880
Med Chem Res (2012) 21:874–890
Table 4 Physicochemical data of the synthesized compounds 5a–n and 6a–n
O
O
R1O
OR1
O
O
R3R2N
NR2R3
n(H2C)
O
O
NH
H3CO
S
NH
N
N
N
NH
H3C
S
Cl
HN
N
Cl
Cl
H3C
NCH 2CH 2NH
H3C
C2H5
NCH 2CH 2NH
C2H5
NH
H3CO
S
N
NH
N
N
NH
H3C
S
Cl
HN
Cl
N
Cl
123

Med Chem Res (2012) 21:874–890
881
Table 4 continued
H3C
NCH 2CH 2NH
H3C
C2H5
C2H5
NCH 2CH 2NH
NH
H3CO
S
N
NH
N
N
NH
H3C
S
Cl
HN
Cl
N
Cl
H3C
NCH 2CH 2NH
H3C
C2H5
NCH 2CH 2NH
C2H5
NH
H3CO
S
N
NH
N
N
NH
H3C
HN
S
Cl
Cl
N
Cl
H3C
NCH 2CH 2NH
NCH 2CH 2NH
H3C
C2H5
C2H5
123

882
Med Chem Res (2012) 21:874–890
Cl, overlapping with solvent signal), 3.84 (s, 6H, 2 OCH3),
6.06 (s, 1H, Ha), 6.77 (d, 2H, 2 Hb, J = 15.9 Hz), 6.83 (d,
2H, 2 HA, JAB = 8.25 Hz, ortho coupling), 7.14, 7.16 (dd,
2H, 2 HB, J = 1.65 and 8.37 Hz, meta and ortho coupling),
7.33 (d, 2H, 2 HX, JAX = 1.38 Hz, para coupling), 7.57 (d,
C-10), 127.07 (C-2 ? C-6), 129.77 (C-1 ? C-7), 140.94 (2
C-30), 148.06 (2 C-40), 149.25 (C-3 ? C-5), 183.23 (2 CO);
MS of 5h m/z (% relative abundance): M? at 778 (absent),
45 (100). IR of 5j m (cm-1): 3435.7 (NH ? OH intramo-
lecularly H-bonded), 1790.6 (C=O, ester), 1658.9 (C=O,
ketone), 1558 (C=N mixed with C=C aromatic), 1498.8
1
2H, 2 Hc, J = 15.9 Hz), 9.69 (s, br, 1H, enol OH). H-
1
NMR of 5f (DMSO-d6) d ppm (300 MHz): 3.17 (s, 12H,
2N(CH3)2), 3.54 (t, dist, 4H, 2 CH2–N), 3.67 (t, 4H, 2 CH2–
NH, J = 5.49 Hz), 3.80 (s, dist, 2H, 2 NH, overlapping
with OCH2), 3.83 (s, 4H, 2 COCH2–N), 4.06 (s, 6H, 2
OCH3), 6.06 (s, 1H, Ha), 6.76 (d, 2H, 2 Hb, J = 15.75 Hz),
6.83 (d, 2H, 2 HA, JAB = 8.07 Hz, ortho coupling), 7.15
(d, 2H, 2 HB, J = 8.07 Hz, ortho coupling), 7.32 (s, 2H, 2
HX), 7.54 (d, 2H, 2 Hc, J = 15.75 Hz), 9.74 (s, 1H, enol
H). 1H-NMR of 5h (CD3OD) d ppm (500 MHz): 1.18–2.17
(hump, br, 30H, 2 Ad-H), 2.631 (t, dist, 4H, 2 9 CH2–N,
J = 6.2 Hz), 3.064 (t, 4H, 2 CO–CH2, J = 6.5 Hz), 3.88
(s, 2H, 2 NH overlapping with OCH3 signal), 3.92 (s, 6H, 2
OCH3), 4.94 (s, 2H, Ha), 6.63 (d, 2H, 2 Hb, J = 15.7 Hz),
6.84 (d, 2H, 2 HA, JAB = 8 Hz, ortho coupling), 7.11 (d,
2H, 2 HB, J = 7.8 Hz, ortho coupling), 7.22 (d, 2H, 2 HX),
7.58 (d, 2H, 2 Hc, J = 15.8 Hz); 13C-NMR of 5h (CD3OD)
d ppm (500 MHz): 39.51 (2 CH2N), 40.48 (2 CH2CO),
47.10, 47.27, 47.44, 47.61, 47.78, 47.90, 47.95, 48.07,
48.12, 48.24 (2 (10 Ad-C)), 55.07 (2 OCH3), 101.37 (C-4),
110.27 (2 C-20), 115.04 (2 C-50), 120.79 (2 C-60), 122.77 (2
(C=C aromatic), 1273.2, 1169.9 (mas and ms C–O–C); H-
NMR of 5j (CD3COCD3) d ppm (500 MHz): 3.61 (s, 6H,
2 9 CH3), 3.91 (s, 6H, 2 OCH3), 4.08 (t, dist, 4H, 2 CH2–
N), 4.61 (t, dist, 4H, 2 CH3–CO), 5.79 (s, br, 2H, Ha), 6.70
(d, 2H, 2 Hb, J = 15.5 Hz), 6.89–7.68 (m, 6H, 2 Ar-H),
7.74 (d, 2H, 2 Hc, J = 15.5 Hz), 8.32 (s, 2H, 2 NH); 13C-
NMR of 5j (CD3COCD3) d ppm (500 MHz): 45.35 (2
CH2N), 47.25 (2 CH2CO), 60.85 (2 OCH3), 105.75 (C-4),
111.80 (2 C-20), 116.85 (2 C-50), 120.0 (2 9 C-60), 123.75
(2 9 C-10), 128.82 (C-2 ? C-6), 134.93 (C-1 ? C-7),
142.00 (2 C-30), 147.85 (2 C-40), 148.95 (C-3 ? C-5),
170.40 (2 9 thiazole C-2), 180.85 (2 9 thiazole C-5),
192.75 (2 CO); MS of 5j m/z (% relative abundance): M?
1
?1 707 (0.7), 43 (100). H-NMR of 6a (CDCl3) d ppm
(300 MHz): 1.25–2.36 ((hump, br, 30H, 2 9 Ad-H), 1.48
(t, 6H, 2 9 OCH2CH3, J = 6.87 Hz), 2.35 (s, 4H,
2 9 CH2CO), 2.63 (s, 2H, 2 9 NH), 4.15 (q, 4H,
2 9 OCH2CH3, J = 6.87 Hz), 5.78 (s, 1H, Ha), 6.45 (d,
2H, 2 9 Hb, J = 15.66 Hz), 6.93 (d, 2H, 2 9 HA,
JAB = 8.25 Hz, ortho coupling), 7.03 (s, 2H, 2 9 HX),
Table 5 Physicochemical data of the synthesized compounds (7a–f)
O
O
R O
OR
O
1
1
O
O
R4HNO 2S
HN
NH
SO2NHR 4
(CH2)n
O
(CH 2)n
(7a-f)
(7a–f)
Cpds. No
R1
n
R4
Reaction time (h)
Yield%
m.p. °C
Molecular formula
M. wt.
7a
7b
CH3
CH3
1
1
H
H
H
16
12
69
50
155–60
175–78
C37H36N4O12S2
C45H40N8O12S2
792
948
N
N
7c
C2H5
C2H5
1
1
18
24
63
60
143–48
185–90
C39H40N4O12S2
C47H44N8O12S2
820
976
N
N
7d
7e
7f
C2H5
C2H5
2
2
18
24
78
57
115–20
115–20
C41H44N4O12S2
C49H48N8O12S2
848
N
N
1004
123

Med Chem Res (2012) 21:874–890
883
7.11 (d, 2H, 2 9 HB, J = 8.25 Hz, ortho coupling), 7.57
(d, 2H, 2 9 Hc, J = 15.66 Hz), 9.81 (s, 1H, enol OH).
combined organic layer was dried over anhydrous MgSO4
(5 g), filtered, evaporated, and crystallized from EtOH
(15 ml) to give a yellow precipitate of 8a, b (Table 6). IR
of 8a m (cm-1): 3435.8 (OH intramolecularly H-bonded),
1750.4 (C=O, ester), 1630.9 (C=O, ketone), 1599, 1507.4
1,7-Bis(4-(4-substituted sulfanilamido)acyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (7a, b) and 1,7-
bis(4-(4-substituted sulfanilamido)acyloxy)-3-(ethoxy-
phenyl)-1,6-heptadiene-3,5-dione (7c–f)
1
(C=C aromatic), 1255.2, 1122.8 (mas and ms C–O–C); H-
NMR of 8a (CDCl3) d ppm (300 MHz): 1.23–2.19 (hump,
br, 30H, 2 Ad-H), 3.85 (s, 6H, 2 OCH3), 5.85 (s, 2H, Ha),
6.55 (d, 2H, 2 Hb, J = 15.9 Hz), 7.01 (d, 2H, 2 9 HA,
JAB = 8.25 Hz, ortho coupling), 7.10 (s, 2H, 2 HX), 7.13,
7.16 (dd, 2H, 2 9 HB, J = 1.65 and 8.04 Hz, meta and
ortho coupling), 7.61 (d, 2H, 2 Hc, J = 15.9 Hz); 13C-
NMR of 8a (CDCl3) d ppm (500 MHz): 56.01 (2 OCH3),
27.88, 27.94, 36.48, 38.73, 38.81, 41.14, 76.79, 77.04,
77.29 (2 Ad-C), 101.75 (C-4), 111.48 (2 C-20), 121.16 (2
C-50), 123.32 (2 C-60), 124.03 (2 C-10), 129.18 (C-2 ? C-
6), 140.13 (C-1 ? C-7), 141.95 (2 9 C-30), 151.52
(2 9 C-40), 175.60 (C-3 ? C-5), 183.16 (2 9 CO); MS of
8a m/z (% relative abundance): M? 692 (2.1), 135 (100.
1H-NMR of 8b (CDCl3) d ppm (300 MHz): 1.25–2.36
(hump, br, 30H, 2 9 Ad-H overlapping with t of
OCH2CH3), 1.48 (t, 6H, 2 9 OCH2CH3, J = 6.87 Hz),
4.16 (q, 4H, 2 9 OCH2CH3, J = 6.97 Hz), 5.78 (s, 1H,
Ha), 6.45 (d, 2H, 2 9 Hb, J = 15.66 Hz), 6.93 (d, 2H,
2 9 HA, JAB = 8.25 Hz, ortho coupling), 7.03 (s, 2H,
2 9 HX), 7.11 (d, 2H, 2 9 HB, JAB = 8.25 Hz, ortho
coupling), 7.57 (d, 2H, 2 9 Hc, J = 15.66 Hz); 9.81 (s,
1H, enol OH).
Following the same procedure as for the preparation of
compounds 5a–n and 6a–n, the appropriate sulfonamide
derivative (0.01 mol) was added to a solution of di-O-
chloroacylcurcumin 3a, b or di-O-chloroacylethyl curcu-
min 3c, d (0.005 mol) in EtOH (30 ml) containing trieth-
ylamine (five drops). The mixture was heated under reflux
for the specified time (Table 5), EtOH was evaporated
under reduced pressure and the residue was purified by
elution on a column of silica gel using a mixture of tolu-
ene–EtOH (97:3 v/v) to give compounds 7a–f (Table 5).
Di-O-adamantoylcurcumin (8a) and di-O-adamantoylethyl
curcumin (8b)
Adamantoyl chloride (0.03 mol) dissolved in the least
amount of EtOH (10 ml) was added to a mixture of cur-
cumin (1) or ethyl curcumin (2) (0.01 mol) and Na2CO3
(2 g) in acetone (50 ml). The mixture was heated under
reflux for 9–19 h, filtered, and evaporated to dryness. The
residue was extracted with EtOAc (3 9 25 ml). The
Table 6 Physicochemical data of the synthesized compounds 8a–f
O
O
R O
OR
1
1
O
O
O
O
R2
R2
(8a-f)
(8a–f)
Cpds. no.
R1
R2
Reaction time (h)
Yield%
47
m.p.
215
Molecular formula
C43H48O8
M. wt.
692
8a
8b
CH3
9
C2H5
16
36
220
C45H52O8
720
8c
CH3
C6H13
C6H13
4–5
4–5
63
77
99.5
C35H44O8
C37H48O8
606
620
8d
C2H5
128–30
8e
8f
CH3
4
81.5
199–203
217–19
C31H24O8S2
588
S
C2H5
6
51
C33H28O8S2
616
S
123

884
Med Chem Res (2012) 21:874–890
Di-O-heptanoylcurcumin (8c) and di-O-heptanoylethyl
curcumin (8d)
Ha), 7.051 (d, 2H, 2 9 Hb, J = 16 Hz), 7.32, 7.34 (dd, 2H,
2 9 HB, J = 3.5 and 7.8 Hz, meta and ortho coupling),
7.39 (d, 2H, 2 9 HA, JAB = 7.5 Hz, ortho coupling), 7.59
(s, 2H, 2 9 HX), 7.69 (d, 2H, 2 9 Hc, J = 16 Hz), 7.86 (d,
2H, 2 9 thienyl-5-H, J = 8 Hz), 8.04, 8.06 (dd, 2H,
2 9 thienyl-4-H), J = 1 and 4 Hz ortho and meta cou-
pling), 8.11 (d, 2H, 2 9 thienyl-3-H, J = 4 Hz). IR of 8f m
(cm-1): 3431.1 (OH intramolecularly H-bonded), 1727
(C=O, ester), 1626.5 (C=O, ketone), 1596.7, 1508.7 (C=C
aromatic), 1250.7, 1117.2 (mas and ms C–O–C); 1H-NMR of
8d (DMSO-d6) d ppm (300 MHz): 1.23 (t, 6H, 2 9 3
OCH2CH3, J = 7.00 Hz), 4.14 (q, 4H, 2 9 OCH2CH3,
J = 7.04 Hz), 6.21 (s, 2H, Ha), 7.02 (d, 2H, 2 9 Hb,
J = 15.93 Hz), 7.32 (d, 2H, 2 9 HA, JAB = 8.52 Hz,
ortho coupling), 7.33 (s, 2H, 2 9 HX), 7.67 (d, 2H,
2 9 Hc, J = 15.93 Hz), 7.38 (d, dist, 2H, 2 9 HB,
JAB = 8.52 Hz, ortho coupling), 7.56 (d, dist, 2H,
2 9 thienyl-H-4), 8.02 (br, 2H, 2 9 thienyl-H-5,
J = 3.57 Hz), 8.10 (d, 2H, 2 9 thienyl-H-3, J = 3.57 Hz);
13C-NMR of 8f (DMSO) d ppm (500 MHz): 40.13
(2 9 CH3), 64.95 (2 9 OCH2), 102.31 (C-4), 113.79
(2 9 C-20), 121.94 (2 9 C-50), 123.94 (2 9 C-60), 125.22
(2 9 C-10), 129.26 (C-2 ? C-6), 131.84 (thienyl-C-4),
134.46 (thienyl-C-3), 135.72 (thienyl-C-5), 135.83 (thie-
nyl-C-2), 140.30 (C-1 ? C-7), 141.37 (2 9 C-30), 151.02
(2 9 C-40), 159.88 (C-3 ? C-5), 183.69 (2 9 CO); MS of
8f m/z (% relative abundance): M? 616 (0.7), 43 (100).
Heptanoyl chloride (0.03 mol) was added dropwise to an
ice-cold mixture of curcumin (1) or ethyl curcumin (2)
(0.015 mol) and Na2CO3 (2 g) in acetone (50 ml). The
mixture was stirred at room temperature for 4–6 h, evap-
orated to dryness and the residue was extracted with EtOAc
(2 9 20 ml). The combined organic extracts were dried
over anhydrous MgSO4, evaporated, and crystallized from
EtOH to give a yellow precipitate of 8c, d (Table 6). IR of
8c m (cm-1): 1768 (C=O, ester), 1629 (C=O, ketone), 1598,
1512 (C=C aromatic), 1120, 1010 (mas and ms C–O–C); 1H-
NMR of 8c (CDCl3) d ppm (300 MHz): 0.91 (t, 6H,
2 9 (CH2)5-CH3, J = 6.96 Hz), 1.30–1.79 (m, 16H,
2 9 (CH2)4), 2.58 (t, 4H, 2 9 CH2CO, J = 7.32 Hz), 3.87
(s, 6H, 2 9 OCH3), 5.85 (s, 2H, Ha), 6.55 (d, 2H, 2 9 Hb,
J = 15.75 Hz), 7.05 (d, 2H, 2 9 HA, JAB = 8.04 Hz,
ortho coupling), 7.11, 7.15 (dd, 2H, 2 9 HB, J = 1.8 and
9 Hz, meta and ortho coupling), 7.27 (s, 2H, 2 9 HX), 7.62
(d, 2H, 2 9 Hc, J = 15.75 Hz). IR of 8d m (cm-1): 1769
(C=O, ester), 1629 (C=O, ketone), 1590, 1500 (C=C aro-
1
matic), 1115, 1020 (mas and ms C–O–C); H-NMR of 8d
(CDCl3) d ppm (300 MHz): 0.90 (t, 6H, 2 9 (CH2)5-CH3,
J = 6.96 Hz), 1.33–1.57 (m, 16H, 2 9 (CH2)4), 1.77 (t,
6H, 2 9 OCH2CH3, J = 7.3 Hz), 2.57 (t, 4H, 2 9 CH2–
CO, J = 7.3 Hz), 4.09 (q, 4H, 2 9 OCH2CH3,
J = 6.96 Hz), 5.84 (s, 2H, Ha), 6.54 (d, 2H, 2 9 Hb,
J = 15.75 Hz), 7.04 (d, 2H, 2 9 HA, JAB = 8.4 Hz, ortho
coupling), 7.11 (d, 2H, 2 9 HB, JAB = 7.5 Hz, ortho
coupling), 7.26 (s, 2H, 2 9 HX), 7.60 (d, 2H, 2 9 Hc,
Anticancer screening
J = 15.75 Hz); 13C-NMR of 8d (CDCl3)
d
ppm
Materials and methods
(300 MHz): 14.164, 14.813, 22.592, 25.157, 28.874 (5
peaks for 5Cs of side chain), 31.569 (2 9 OCH2CH3),
34.157 (2 9 –OCOCH2C5H11), 64.454 (2 9 OCH2CH3),
101.874 (C-4), 112.477 (2 9 C-20), 121.034 (2 9 C-50),
123.316 (2 9 C-60), 124.133 (2 9 C-10), 133.812 (C-
2 ? C-6), 140.171 (C-1 ? C-7), 141.728 (2 9 C-30),
150.911 (2 9 C-40), 171.743 (C-3 ? C-5), 183.216
Cytotoxicity to mammalian cells
The cytotoxic activity of the tested compounds 1, 2, 3a, 3c,
3d, 5a–k, 6a–h, 6m, n, 7a, 8a, and 8c–f was determined
against human cancer cell line SK-MEL (malignant, mel-
anoma), KB (epidermal carcinoma, oral), BT-459 (ductal
carcinoma, breast), and SK-OV-3 (ovary carcinoma). Vero
cells, derived from monkey kidney fibroblasts, and LLC-
PK1, from pig kidney epithelial tissue, were used repre-
senting noncancerous cells. The assay was performed in
96-well tissue culture treated microplates according to the
neutral red staining procedure as modified by Lin et al.,
1998. The results are reported in Table 7 and represented in
Figs. 1–4.
1
(2 9 CO); Examination of 2D H-NMR (COSY) (CDCl3)
and 1H, 13C-NMR (C, H correlation) (CDCl3) provided
further confirmation for the structure of the compound 8d.
Di-O-(2-thienoyl)curcumin (8e) and di-O-(2-thienoyl)ethyl
curcumin (8f)
Compounds 8e, f (Table 6) were prepared by adding thi-
ophene-2-carbonyl chloride (0.03 mol) adopting the pre-
viously mentioned procedure. IR of 8e m (cm-1): 3339.6
(OH intramolecularly H-bonded), 1746.7 (C=O, ester),
1672.9 (C=O, ketone), 1606.8, 1494.5 (C=C aromatic),
1278.3, 1122 (mas and ms C–O–C); 1H-NMR of 7c (DMSO)
d ppm (500 MHz): 3.86 (s, 6H, 2 9 OCH3), 6.23 (s, 2H,
Interaction with topoisomerases
Measurement of the catalytic activity of topoisomerase I
(topo I) was based on the conversion of supercoiled DNA
to relaxed DNA. Cleavage complex stabilization was
123

Med Chem Res (2012) 21:874–890
885
substrate. Enzyme activity was assayed in a total volume of
20 ll containing 250 ng of DNA, test compound, 2–4 U of
purified enzyme, 10 mM EDTA, 0.15 mM NaCl, 0.1%
bovine serum albumin (BSA), 0.1 mM spermidine, and 5%
glycerol. The reaction mixture was incubated at 37°C for
30 min. Reactions were then terminated by the addition of
1% sodium dodecyl sulfate (SDS) followed by treatment
with proteinase K (50 lg/ml) at 37°C for 30 min. DNA
was extracted with chloroform–isoamyl alcohol (24:1 v/v)
and analyzed by electrophoresis on a 1% agarose gel in
TAE buffer (40 mM Tris acetate, 2 mM EDTA, pH 8.5).
The gel was stained with ethidium bromide, destained in
water, and photographed on a UV transilluminator fol-
lowed by a densitometric analysis using NIH image soft-
ware 1.52. Enzyme activity was measured as a percentage
of substrate DNA converted to product. The concentration
of test compound that prevented 50% of the substrate from
being converted into product (IC50) was calculated.
Table 7 Cytotoxicity of the synthesized compounds to mammalian
cells
IC 50 value (lM)a
Compds.
no.
Cancer cells
Noncancer cells
SK-
MEL
KB
BT-
549
SK-OV-3 VERO LLC-
PK1
1
13.75 [25
16 [25
12.5 [25
10.5
18.0
10.0
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
22.5
15.5
NC
NC
NC
NC
NC
NC
NC
23.0
NC
[25
NA
19.5
NC
NC
15.0
12.5
NC
NC
11.5
11.0
15.0
NC
5.0
NC
NC
NC
NC
NC
NC
22.5
22.5
NC
20.0
NC
NC
NC
NC
15.5
16.8
NC
NC
13.0
6.8
2
3a
3c
3d
5a
5b
5c
5d
5e
5f
NA
NA
NA
NA
NA
15.0 [25
19.4
18.1
8.75
NA
15.0
23
7.5
22.5
NA
19.5
NA
24.0
NA
7.0
6.75
20.0
16.3
15.0
8.5
For the determination of cleavage complex formation
activity with topo I, the assay was performed with a min-
imum of 4 U of purified enzyme as described above, except
that ethidium bromide was included in both the agarose gel
and running buffer to resolve nicked DNA from super-
coiled or relaxed species. Drug-induced stabilization of the
cleaved complex was determined in terms of the percent
nicked DNA produced. The relaxation activity of Topo II
was analyzed in the same manner, except that the reaction
mixture contained 10 mM Tris–HCl (pH 7.9), 50 mM
NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 lg/
ml BSA, 1 mM ATP, and 4 units Topo IIa. Decatenation of
kDNA (TopoGEN Inc.) was performed in the same con-
dition except that supercoiled plasmid DNA was substi-
tuted by 400 ng kDNA. All compounds tested 1, 2, 3a, 3c,
3d, 5a–k, 6a–e, 6m, 6n, 7a, 8a, and 8c–f were dissolved in
DMSO provided that the DMSO concentration did not
exceed 2.5% in all the assays and a DMSO control was
always included. The results are reported in Table 8.
5g
5h
5i
18.5 [25
23.8 [25
20.50 NA
23.75 NA
5j
7.5
16.3
11.5
NA
NA
11.5
12.5
21.5
NA
11.5
15
4.25
NA
5k
6a
6b
6c
6d
6e
6f
15.0
NA
4.75
12.5
13.3
14.5
NA
11.5
NA
NA
NA
18.8
NA
NA
NA
NA
NA
11.00 23.8
NA
NA
NA
2.8
12.00 15.8
9.00
11.0
NA
13.5
13.8
22.0
5.8
11.50 NA
10.00 14.8
6g
6h
6m
6n
7a
8a
8c
8d
8e
8f
NA
NA
NA
10.5
NA
NA
NA
NA
NA
NA
NA
NA
22.5
NA
NA
NA
NA
NA
NC
NC
NC
11
NC
NC
NC
15.75
NC
NC
NC
NC
NC
0.75
NA
NA
14
NA
NA
NA
NA
NA
NC
NC
NC
NC
NC
[5.0
Results and discussion
Doxorubicin 0.55 \0.55 \0.55 0.8
Chemistry background
NA not active up to 25 lM. NC not cytotoxic up to 25 lM. SK-MEL
human malignant, melanoma. KB human epidermal carcinoma, oral.
BT-549 ductal carcinoma, breast. SK-OV-3 human ovary carcinoma.
Vero monkey kidney fibroblasts. LLC-PK1 pig kidney epithelial
In this investigation, the synthetic method of preparation of
curcumin (1) and ethyl curcumin (2) involved an one step
condensation of the appropriate aromatic aldehyde with
acetylacetone–boric oxide complex. This method was
originally reported (Crabbe et al., 1971) and modified by
Roughley and Whiting (1973). The method founds large
application because of its versatility, wide application
for the preparation of symmetrical and unsymmetrical
curcuminoids and other diketones, high yield and purity
(Nurfina et al., 1997; Mohri et al., 2003). Curcumin (1) and
a
Stock solution = 5 mM in DMSO; test concentrations = 25, 8.33,
and 2.78 lM
assayed by measuring the nicked DNA produced in the
presence of the tested compounds. Human topo I and the
topo I drug kit were purchased from Topogen Inc.
(Columbus, Ohio). A supercoiled plasmid DNA (pHOT1)
with a high affinity topo I recognition element was used as
123

886
Med Chem Res (2012) 21:874–890
Fig. 1 Cytotoxic effect of the
synthesized compounds
to SK-Mel cells
30
25
20
15
10
5
IC 50
SK-MEL
0
c
b
h
n
6
1
2
7
5f
5i 5j
8f
6f
3a 3c
5c
5g 5h
5k
6
8c
8e
3d 5a
5
5d 5e
6a 6b
6d 6e
6g
6
8a
8d
6m
: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds
doxorubicin
30
Fig. 2 Cytotoxic effect of the
synthesized compounds to KB
Cells
IC 50
KB
25
20
15
10
5
0
n
c
c
i
1
2
a
e
a
7
5i
5j
5f
6f
8f
3c
5c
5k
6
8
3
3d 5a 5b
5d
5
5g 5h
6
6b
6d 6e
6g 6h
6n
8a
8d 8e
6m
: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds
xorubic
do
ethyl curcumin (2) were prepared as shown in Scheme 2.
Di-O-chloroacetylcurcumin (3a), di-O-chloropropionyl-
curcumin (3b), di-O-chloroacetylethyl curcumin (3c), and
di-O-chloropropionylethyl curcumin (3d) were prepared by
two methods: (1) treating the appropriate curcumin and
ethylcurcumin with acid chlorides in the presence of Na2CO3
(Crabbe et al., 1971; Nurfina et al., 1997; Ishida et al., 2002,
(2) preparation of the target intermediates 3a–d, through
a two-step reaction involving the treatment of 3-alkoxy-
4-chloroacyloxybenzaldehyde (4a–d) with acetylacetone
(Scheme 2) (Roughley and Whiting, 1973). 1,7-Bis(4-alkyl-
(cycloalkyl or heteroaryl)-aminoacyloxy)-3-(methoxy-phe-
nyl)-1,6-heptadiene-3,5-dione (5a–n) and 1,7-bis(4-alkyl
(cycloalkyl or heteroaryl)aminoacyloxy)-3-(ethoxyphenyl)-
1,6-heptadiene-3,5-dione (6a–n) were synthesized by heat-
ing under reflux an ethanolic solution of di-O-chloroacyl-
curcumin (3a,b) or di-O-chloroacylethylcurcumin (3c, d)
and the appropriate alkyl, cycloalkyl, or heteroarylamine
in the presence of triethylamine (Scheme 3). 1,7-Bis(4-
(4-substituted sulfanilamido)acyloxy)-3-(methoxyphenyl)-1,
123

Med Chem Res (2012) 21:874–890
887
30
25
20
15
10
5
Fig. 3 Cytotoxic effect of the
synthesized compounds to BT-
549 Cells
BT-549
IC 50
0
f
f
c
c
k
c
6
a
e
a
b
e
g
n
6
1
2
7
in
5f
5i
5j
6
8
3
5
5
8c
3
3d 5a 5b
5d
5
5g 5h
6
6
6d
6
6
6h
8a
8d 8e
6m
: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds
doxorubic
30
Fig. 4 Cytotoxic effect of the
synthesized compounds to SK-
OV-3 cells
SK-OV-3
IC 50
25
20
15
10
5
0
i
j
5
n
i
k
a
c
e
h
c
8
d
e
8
a
d
b
d
h
1
2
7
5
5f
6f
8f
3c
5c
5
6
3
3
5a
5
5
5e
5g
5
6
6b
6d
6
6g
6
6n
8a
8
6m
: Most Cytotoxic Compounds
: Less cytotoxic Compounds
: Inactive Compounds
doxorubic
6-heptadiene-3,5-dione (7a, b) and 1,7-bis(4-(4-substituted
sulfanilamido)acyloxy)-3-(ethoxyphenyl)-1,6-hepta-diene-3,
5-dione (7c–f) (Scheme 3) were synthesized using the same
general amination procedure as for the preparation of the
compounds 5a–n and 6a–n. Di-O-adamantoylcurcumin (8a)
and di-O-adamantoylethylcurcumin (8b) were prepared by
esterification of curcumin (1) or ethyl curcumin (2) as pre-
viously mentioned (Luo et al., 2003; Bischoll and Hoffmann,
2002. A solution of either 1 or 2 in acetone containing
anhydrous Na2CO3 was treated with 2 molar equivalents of
adamantoyl chloride (Scheme 4). Di-O-heptanoylcurcumin
(8c) and di-O-heptanoylethylcurcumin (8d) were prepared
by esterification of curcumin 1 or ethyl curcumin 2 in
acetone containing anhydrous Na2CO3 (2 g) with 2
molar equivalents of 2-heptanoyl chloride at rt (Scheme 4).
Di-O-(2-thienoyl)curcumin (8e) and di-O-(2-thienoyl)-
ethylcurcumin (8f) were prepared following the previous
procedure.
123

888
Med Chem Res (2012) 21:874–890
cells, derived from monkey kidney fibroblasts, and LLC-
PK1, from pig kidney epithelial tissue, were used repre-
senting noncancerous cells. The overall results (Figs. 1–4,
Tables 7, 8) demonstrated that, in general, some of the
synthesized compounds showed cytotoxic activity against
the tested cancerous cell lines less then doxorubicin, which
was used as a reference. While the cytotoxic mechanism of
doxorubicin remains somewhat controversial, there is
substantial evidence to suggest that the following events
play a role (Burke and Koch 2004): (1) intercalation and
alkylation of DNA, (2) induction of topo II mediated strand
breaks, (3) interference with DNA unwinding and helicase
activity, (4) lipid peroxidation, and (5) direct membrane
effects at low concentrations resulting in modification of
membrane function and related cytotoxicity (Foye and
Sengupta, 1995).
Table 8 Anticancer activity of the synthesized compound (inhibition
of topoisomerase activity)
IC 50 value (lM)
Samples
Topoisomerase I
Topoisomerase II
Cleavagea
Catalyticb
Cleavagea
Catalyticb
1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
15
7.5
20
12
20
4
2
3a
3c
3d
5a
5b
5c
5d
5e
5f
15
15
0.35
5
Although curcumin was documented (Snyder et al., 2002)
to damage DNA by catalytic topo II poisoning, many of the
tested compounds were more active than curcumin 1 and
ethyl curcumin 2 as cytotoxic agents. The most active com-
pound 6b was tenfold less potent than the reference doxo-
rubicin in various cancer cell lines. Curcumin showed only
moderate activity against BT-549 while being completely
nontoxic to the noncancerous Vero and LLC-PK1 cells. Some
of the synthesized compounds, namely 1,7-bis(4-(5-meth-
ylthiadiazol-2-yl)aminoacetyloxy)-3-(methoxy-phenyl)-1,
6-heptadiene-3,5-dione (5c), 1,7-bis(4-(bis(2-chloroethyl)-
amino-acetyloxy)-3-(methoxyphenyl)-1,6-heptadiene-
3,5-dione (5e), 1,7-bis(4-(2-diethylaminoethyl)amino-acet-
yloxy)-3-(methoxyphenyl)-1,6-heptadiene-3,5-dione (5g),
1,7-bis(4-(5-methylthia-diazol-2-yl)aminopropionyloxy)-3-
(methoxyphenyl)-1,6-heptadiene-3,5-dione (5j), 1,7-bis(4-
(6-methoxybenzothiazol-2-yl)aminoacetyloxy)-3-(ethoxy-
phenyl)-1,6-heptadiene-3,5-dione (6b) and 1,7-bis(4-(2-dieth-
ylaminoethyl)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-hept-
adiene-3,5-dione (6g) showed cytotoxic activity against
SK-MEL cancerous cell lines (Fig.1) with no or little effect
on the noncancerous cells. Compound 6b was the most active
against SK-MEL cells with IC50 = 4.75 lM followed by
compound 5e with an IC50 = 7 lM. Compounds 5c and 5j
were also cytotoxic to the same cell line with IC50 = 7.5 lM
while compounds 5g had IC50 = 8.5 lM and 6g had IC50 =
11.5 lM. Compounds 5j, 5e, and 5c and 1,7-bis(4-(2-chlo-
roethyl)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-
3,5-dione (6d) exhibited cytotoxic activity against BT-549
cells with IC50 = 4.25, 6.75, 8.75, and 9 lM, respectively
(Fig. 3). Di-O-chloroacetylcurcumin (3a), 6g and 1,7-bis(4-
(4-sulfanilamido)acetyloxy)-3-(methoxy-phenyl)-1,6-hept-
adiene-3,5-dione (7a) were almost as active as curcumin
against BT-549 exhibiting IC50 = 10 lM. 1,7-Bis(4-(bis(2-
chloroethyl)aminoacetyloxy)-3-(ethoxy-phenyl)-1,6-heptadi-
ene-3,5-dione (6e) and 1,7-bis(4-(2-dimethylaminoethyl)amino-
acetyloxy)-3-(ethoxyphenyl)-1,6-heptadiene-3,5 dione (6f)
16
3
5g
5h
5i
18
19
3
5j
5k
6a
6b
6c
6d
6e
6f
2.9
3.4
12
2.7
4.1
3.4
4
6g
6h
6m
6n
7a
8a
8c
8d
8e
8f
10
2.1
NA
7.1
3
4.5
2.9
3
8
23
ND not determined, NA not active up to 25 lM
a
Cleavage complex stabilization activity (similar to camptothecin for
topo I and etoposide for topo II)
b
Inhibition of catalytic activity of topoisomerase i.e., relaxation of
supercoiled DNA by topoisomerase I and decatenation of KDNA by
topoisomerase II
Cytotoxic activity
The cytotoxic activity of the tested compounds 1, 2, 3a, 3c,
3d, 5a–k, 6a–h, 6m, 6n, 7a, 8a, and 8c–f was determined
against human cancer cell line SK-MEL (malignant, mel-
anoma), KB (epidermal carcinoma, oral), BT-459 (ductal
carcinoma, breast), and SK-OV-3 (ovary carcinoma). Vero
123

Med Chem Res (2012) 21:874–890
889
were slightly less active than curcumin exhibiting IC50 of 11
and 11.5 lM, respectively (Fig. 3). Moreover, 1,7-bis(4-(2-
chloroethyl)amino-propionyloxy)-3-(methoxyphenyl)-1,6-
heptadiene-3,5-dione (5k), 1,7-bis(4-(5-methyl-thiadiazol-
2-yl)aminoacetyloxy)-3-(ethoxyphenyl)-1,6-hepta-diene-3,
5-dione (6c) and 6g were the most cytotoxic against KB cells
with IC50 = 11.5 lM (Fig. 2). Regarding SK-OV-3 cell line
(Fig. 4), compound 6b was the most cytotoxic among these
series with IC50 = 2.8 lM. It was also not cytotoxic to any
of the noncancer cells tested up to 25 lM. All other com-
pounds were inactive against this type of cancer cells. It was
also noted that none of the tested compounds having an
adamantoyl, heptanoyl, and thienoyl functions directly
esterified to the phenolic hydroxyl groups (compounds 8a–
f), showed any activity against all cell lines used indicating
that this linkage is not appropriate for activity.
diethylaminoethyl and the usual alkylating bis(2-chloro-
ethyl)amino moieties showed the highest cytotoxic activity
against SK-MEL cancer cells (Fig. 1). Compounds 5k, 6c,
and 6g are less cytotoxic to KB cancer cells (Fig. 2). More-
over, compounds 5c, 5e, 5j, 5k, 6d, 6e, 6f, and 6g showed
cytotoxicity against BT-549 cancer cells (Fig. 3) with 5j
being the most active compound. Curcumin and our inter-
mediate di-O-chloroacetylcurcumin (3a) are also cytotoxic
against the same cell line but are less active than the syn-
thesized compounds. Finally, compound 6b is the only one
exhibiting cytotoxicity against SK-OV-3 cancer cells (Fig.
4). Therefore, these compounds and especially compound 6b
should be very promising candidates for further studies.
Acknowledgments We appreciate very much the help of Dr. Abeer
El-Alfy, Pharmacology Department, King Saud University and Dr.
Shabana Khan, National center for Natural Products Research, Uni-
versity of Mississipi, USA, for performing the Cytotoxicity Tests.
Inhibition of topoisomerases
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