Synthesis, characterization and biological evaluation of succinate prodrugs of curcuminoids for colon cancer treatment

Article abstract of DOI:10.3390/molecules16021888

A novel series of succinyl derivatives of three curcuminoids were synthesized as potential prodrugs. Symmetrical (curcumin and bisdesmethoxycurcumin) and unsymmetrical (desmethoxycurcumin) curcuminoids were prepared through aldol condensation of 2,4-pentanedione with different benzaldehydes. Esterification of these compounds with a methyl or ethyl ester of succinyl chloride gave the corresponding succinate prodrugs in excellent yields. Anticolon cancer activity of the compounds was evaluated using Caco-2 cells. The succinate prodrugs had IC₅₀ values in the 1.8-9.6 μM range, compared to IC₅₀ values of 3.3-4.9 μM for the parent compounds. Curcumin diethyl disuccinate exhibited the highest potency and was chosen for stability studies. Hydrolysis of this compound in phosphate buffer at pH 7.4 and in human plasma followed pseudo first-order kinetics. In phosphate buffer, the κ_obs and t_1/2 for hydrolysis indicated that the compound was much more stable than curcumin. In human plasma, this compound was able to release curcumin, therefore our results suggest that succinate prodrugs of curcuminoids are stable in phosphate buffer, release the parent curcumin derivatives readily in human plasma, and show anti-colon cancer activity.

Full text of DOI:10.3390/molecules16021888

Molecules 2011, 16, 1888-1900; doi:10.3390/molecules16021888
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Characterization and Biological Evaluation of
Succinate Prodrugs of Curcuminoids for Colon
Cancer Treatment
Wisut Wichitnithad 1, Ubonthip Nimmannit 1,2, Sumrit Wacharasindhu 3 and
Pornchai Rojsitthisak 4,*
1
Pharmaceutical Technology (International) Program, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand; E-Mail: wisutrd@yahoo.com
2
National Nanotechnology Center (Nanotec), National Science and Technology Development
Agency, Pathumthani 12120, Thailand; E-Mail: ubonthip.n@chula.ac.th
3
Natural Products Research Unit, Department of Chemistry, Faculty of Sciences, Chulalongkorn
University, Bangkok 10330, Thailand; E-Mail: sumrit.w@chula.ac.th
4
Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand
* Author to whom correspondence should be addressed; E-Mail: pornchai.r@chula.ac.th;
Tel.: +662-218-8310; Fax: +662-254-5195.
Received: 11 January 2011; in revised form: 25 January 2011 / Accepted: 17 February 2011 /
Published: 22 February 2011
Abstract: A novel series of succinyl derivatives of three curcuminoids were synthesized as
potential prodrugs. Symmetrical (curcumin and bisdesmethoxycurcumin) and
unsymmetrical (desmethoxycurcumin) curcuminoids were prepared through aldol
condensation of 2,4-pentanedione with different benzaldehydes. Esterification of these
compounds with a methyl or ethyl ester of succinyl chloride gave the corresponding
succinate prodrugs in excellent yields. Anticolon cancer activity of the compounds was
evaluated using Caco-2 cells. The succinate prodrugs had IC50 values in the 1.8–9.6 μM
range, compared to IC50 values of 3.3–4.9 μM for the parent compounds. Curcumin diethyl
disuccinate exhibited the highest potency and was chosen for stability studies. Hydrolysis
of this compound in phosphate buffer at pH 7.4 and in human plasma followed pseudo
first-order kinetics. In phosphate buffer, the kobs and t1/2 for hydrolysis indicated that the
compound was much more stable than curcumin. In human plasma, this compound was

Molecules 2011, 16
able to release curcumin, therefore our results suggest that succinate prodrugs of
1889
curcuminoids are stable in phosphate buffer, release the parent curcumin derivatives
readily in human plasma, and show anti-colon cancer activity.
Keywords: curcumin; prodrug; succinylation; stability; hydrolysis
1. Introduction
Curcumin (1), bisdesmethoxycurcumin (2) and desmethoxycurcumin (3) (Figure 1) are well known
curcuminoids that are found in turmeric (Curcuma longa L., Zingiberaceae). Curcuminoids are dietary
polyphenolic compounds that are of interest from the use of turmeric plants as herbal medicines. Since
their initial isolation in 1870 [1], the pharmacological activities of curcuminoids have been widely
investigated, and the antioxidative, anti-inflammatory, and antimicrobial properties of these molecules
are well established [2-7]. Evidence has also been found for antiproliferative properties [8], an
inhibitory effect against HIV-I integrase as a promising HIV treatment strategy [9], and a cholesterol-
lowering effect [10]. The most attractive feature of curcuminoids is the lack of significant toxicity, as
shown in several animal and human studies [11-14]. Thus, the various pharmacological effects and
excellent safety profile make these molecules of interest as lead compounds for treatment of human
diseases.
Figure 1. Curcuminoid structures.
The major obstacle in clinical trials has been the low bioavailability of curcuminoids due to their
instability in a biological environment, and inadequate absorption and fast metabolism resulting in
rapid systematic elimination. These drawbacks require administration at a high dose to achieve a
significant intracellular concentration [15]. Phenolic groups and the 1,3-dicarbonyl moiety are the sites
of in vitro and in vivo degradation through pathways including oxidation and hydrolysis [16]. Many
strategies have been used to enhance the bioavailability of curcumin. Protection of ionization of
phenolic groups and elimination of electron delocalization on the structure have been shown to
improve curcuminoid stability and result in increased bioavailability. This has been accomplished by
conjugation of small endogenous molecules such as amino acids, acetic acid, glucose and nucleic acids
at the phenolic groups [6,17,18]. Succinic acid derivatives may also be useful in this approach, but
conjugation of these molecules with curcuminoids has not been investigated.
Attachment of a succinic acid derivative to a drug molecule (succinylation) is usually carried out by
formation of an ester bond between a hydroxyl group of the target molecule and one of the carboxylic
acids of the succinic acid. Since succinic acid contains two carboxylic acids, the remaining free
carboxylic group can cause autocatalysis that results in instability of the succinyl products. Therefore,

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the free carboxylic acid is usually protected by esterification with a simple alcohol (a succinic acid
monoester derivative). Succinic acid derivatives are attached to target drugs to produce more desirable
physical and biological properties, with the goal that the resulting conjugates are degraded by cellular
enzymes to release the target drugs in vivo. Therefore, succinylation falls into the prodrug category,
and has the advantage of safety because succinic acid, the by-product after cleavage of the conjugate,
is an endogenous substance. Succinylation has been carried out successfully for several drugs on the
market, such as methylprednisolone [19], hydrocortisone [20], chloramphenicol [21], metronidazole
[22] and primaquine [23]. Therefore, succinylation is suitable for development of therapeutic agents,
including those based on phenolic compounds such as curcuminoids.
Herein, we report the synthesis of six novel curcuminoid prodrugs 4-9 (Figure 2) carrying succinyl
ester moieties with enhanced stability and anticolon cancer activity. The anticancer activity of
compounds 1-9 was evaluated in colon cancer cell lines. Curcumin diethyl disuccinate (5) that
exhibited the highest potency was chosen for further investigation of stability in phosphate buffer and
curcumin release in human plasma.
Figure 2. Structures of succinate prodrugs of curcuminoids.
2. Results and Discussion
2.1. Synthesis of Curcuminoids 1-3 and Succinyl Derivatives 4-9
Symmetrical curcuminoids [curcumin (1) and bisdesmethoxycurcumin (2)] were synthesized
according to the Pabon method, as described in Scheme 1 [24]. To prevent Knovenagel condensation
at the C-3 methylene group, boric anhydride was first reacted with acetylacetone to generate an
acetone-boric oxide complex. The less active methyl moieties were then reacted further with an
appropriate ratio of vanillin (3-methoxy-4-hydroxybenzaldehyde) to ensure double aldol condensation
at both ends of the 2,4-pentanedione moiety. The reaction was performed in ethyl acetate, which
provided the proper solubility for both reactants and intermediates and can be easily removed from the
product, resulting in low contamination with solvent. Because the reaction is water-sensitive, tributyl
borate was used as a desiccant. Tributyl borate also increases the partial positive charge at the carbonyl
carbon of vanillin, resulting in an increased reaction rate. After decomplexation with 1N hydrochloric
acid, compound 1 was obtained in excellent yield. NMR and other data support a curcumin purity of

Molecules 2011, 16
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>99% after recrystallization in methanol. Similarly, reaction of the acetyl acetone-boric oxide complex
with 4-hydroxybenzaldehyde produced 2 in excellent yield and with high purity.
Scheme 1. Syntheses of curcumin (1) and bisdesmethoxycurcumin (2).
A synthetic method for the asymmetrical curcuminoid, desmethoxycurcumin (3), is presented in
Scheme 2. The monosubstituted intermediate 10 were first prepared by reacting vanillin with acetyl
acetone. Subsequently, 10 was subjected to a second aldol condensation with 4-hydroxybenzaldehyde
to give 3 after column chromatography. The synthesized curcuminoids were fully characterized by IR,
NMR and MS and the results were in agreement with previous data [25,26].
Scheme 2. Synthesis of desmethoxycurcumin (3).
O
OH
O
O
MeO
HO
1. B2O3, B(OBu)3
2. BuNH2, vanillin
3. 1N HCl
10, 45%
1. B2O3, B(OBu)3
3, 68%
2. BuNH2, 4-hydroxybenzaldehyde
3. 1N HCl
With all three curcuminoids in hand, we next began synthesis of the curcuminoid-succinate
prodrugs, as shown in Scheme 3. Curcumin (1) was first reacted with methyl-4-chloro-4-oxobutyrate
or ethyl-4 chloro-4-oxobutyrate in the presence of 4-(N,N-dimethylamino)pyridine (DMAP) as the
activator to obtain 4 and 5, respectively. In the same manner, bisdesmethoxycurcumin (2) and
desmethoxycurcumin (3) were converted into the desired prodrugs 6-9 upon treatment with the
corresponding acid chlorides. All ester prodrugs 4-9 were obtained in excellent yields as yellow solids
after recrystallization from methanol. The synthesized prodrugs were fully characterized by IR, NMR
and MS.

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Scheme 3. Synthesis of succinate prodrugs of curcuminoids.
2.2. Cytotoxicity Evaluation
The anticancer activity of curcumin-succinate prodrugs 4–9 against human epithelial colorectal
adenocarcinoma cells (Caco-2 cells) was compared with that of the parent curcuminoids 1–3. The
cytotoxicity values expressed as IC50 values are shown in Table 1. Compounds 4–9 were all active,
with IC50 values in the 1.8–9.6 μM range. Curcumin diethyl disuccinate (5) exhibited the highest
potency and desmethoxycurcumin diethyl disuccinate (9) had the lowest potency. The other prodrugs
inhibited cell growth with potency comparable to that of their parent compounds. Therefore,
compound 5 was chosen for further investigation of stability in phosphate buffer and curcumin release
in human plasma.
Table 1. In vitro cytotoxicity of curcuminoids 1-3 and curcuminoid-succinate prodrugs 4-9
against human epithelial colorectal adenocarcinoma (Caco-2) cells (n = 3).
Compounds
IC50 (μM) ± SD
3.31 ± 0.16
4.93 ± 0.21
3.36 ± 0.26
3.84 ± 0.19
1.84 ± 0.11
3.78 ± 0.31
5.97 ± 0.28
4.40 ± 0.15
9.60 ± 0.31
1
2
3
4
5
6
7
8
9
2.3. Chemical Stability Study
The chemical stability of curcumin (1) and curcumin diethyl disuccinate (5) in 0.1 M phosphate
buffer (pH 7.4) at 37 °C was determined for 12 h using reversed-phase HPLC with UV-VIS detection
at 400 nm. No curcumin was found after 2 h. Semi-logarithmic plots of the concentrations of 1 and 5 in
the buffer vs. time (Figure 3) were linear, indicating that degradation followed pseudo first-order
kinetics. The overall degradation rate constants (kobs) and half-lives (t1/2) were 1.239 h−1 and 0.56 h,
respectively, for 1 and 0.0795 h−1 and 7.66 h for 5, respectively. These results indicate that
succinylation significantly enhanced the chemical stability of curcumin against hydrolytic degradation
at pH 7.4. This suggests that delivery of the prodrug via an intravenous route may be viable. To
examine the potential oral bioavailability of the curcuminoid-succinate prodrugs, we plan to perform a
chemical stability study in acidic pH conditions similar to those in the gastrointestinal tract.

Molecules 2011, 16
1893
We note that there are two ester groups in compound 5: a phenolate ester and an alkoxyl (methyl or
ethyl) ester. The phenolate ester is more prone to hydrolysis since phenolate anion is a better leaving
group than methoxylate or ethoxylate anions. Therefore, cleavage of prodrug 5 is likely to take place
mainly at the phenolate ester, which is the closer ester to the parent curcumin.
Figure 3. Pseudo first-order plots for degradation of curcumin (1; ○) and curcumin diethyl
disuccinate (5; ●) in 0.1 M phosphate buffer (pH 7.4) at 37 °C.
10
1
.1
.01
0
2
4
6
8
10
12
14
Time (h)
2.4. Release Study
Release of curcumin from curcumin diethyl disuccinate (5) in human plasma was monitored by
measuring the depletion of 5 and the increase of curcumin using reversed-phase HPLC with UV-VIS
detection at 400 nm. The amount of curcumin released was consistent with the decrease in 5
(Figure 4). All curcumin was released within 2 h. The release of curcuminoids may be mainly due to
enzymatic hydrolysis by esterases in human plasma. The ability of 5 to release the parent curcumin
suggests that succinylation could be useful as a potential prodrug approach for curcuminoids.
Figure 4. Release profiles of curcumin (1; ○) from curcumin diethyl disuccinate (5; ●) in
human plasma at 37 °C.
120
100
80
60
40
20
0
0
2
4
6
8
10
12
Time (h)

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3. Experimental
3.1. General
All reagents and solvents were purchased from commercial sources and used without further
purification. Reactions were performed under nitrogen and monitored by TLC. Flash chromatography
1
13
was performed on 230–400 mesh silica gel with technical grade solvents. H- and C-NMR spectra
were recorded on a Varian Inova Fourier Transform NMR 500 MHz spectrometer operated with the
VnmrJ software. NMR spectra were obtained in deuterated chloroform (CDCl3) or deuterated
dimethylsulfoxide (DMSO). Chemical shifts are reported as δ values in parts per million (ppm)
relative to the residual solvent peak and coupling constants are reported as J values in Hertz (Hz). IR
spectra were recorded with a Perkin Elmer 1760 X. High resolution mass spectra were obtained on a
Reflex IV Bruker time-of-flight High-Resolution Mass Spectrometer (HRMS). Melting points were
determined using a Differential Scanning Calorimeter (DSC823e, Mettler Toledo). The amount of
curcumin (1) and curcumin diethyl disuccinate (5) were determined using an Agilent 1200 HPLC
system (Agilent, CA, USA) equipped with an Alltech Alltima C18 column (150 × 4.6 mm i.d., 5μ,
Grace, IL, USA).
3.2. General Method for the Synthesis of Curcumin (1) and Bisdesmethoxycurcumin (2)
Acetylacetone (1.03 mL, 10 mmol) was added to a solution of boric anhydride (0.35 g, 5.0 mmol) in
ethyl acetate (30 mL), followed by addition of vanillin (3.04 g, 20 mmol) or 4-hydroxybenzaldehyde
(2.44 g, 20 mmol) and tributyl borate (10.8 mL, 40 mmol). The reaction mixture was stirred at 50 °C
for 5 min. Subsequently, n-butylamine (0.4 mL, 5.0 mmol) in ethyl acetate (5 mL) was added dropwise
over 15 min at 50 °C and additionally stirred for 4 hours at 80 °C. Hydrochloric acid (1 N, 30 mL) was
added and the mixture was stirred for another 30 min. The organic layers were separated and extracted
with ethyl acetate (3 × 30 mL). The combined organic layer was washed with water. The organic layer
was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product
was recrystallized from methanol to give the corresponding curcuminoids as yellow solids.
(1E,6E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (1, curcumin). Yield 81%;
m.p. 187–188 °C (lit. 184–185 °C) [25]; IR (KBr) 3,500, 1,626, 1,601, 1,504, 1,427, 1,261, 1,026
1
cm−1; H- NMR (CDCl3) δ 3.95 (s, 6H), 5.80 (s, 1H) 6.48 (d, J = 15.7 Hz, 2H), 6.94 (d, J = 8.1 Hz,
2H), 7.05 (d, J = 1.8 Hz, 2H), 7.13 (dd, J = 8.1 and 1.8 Hz, 1H), 7.59 (d, J = 15.7 Hz, 2H); 13C-NMR
(CDCl3) 183.3, 147.8, 146.8, 140.5, 127.6, 122.8, 121.8, 114.8, 109.6, 101.2, 56.0; HRMS calcd. for
C21H21O6 [M+H+]: 369.1338; found 369.1335.
(1E,6E)-1,7-bis(4-Hydroxyphenyl)hepta-1,6-diene-3,5-dione (2, bisdesmethoxycurcumin). Yield 85%;
m.p. 233–234 °C (lit. 231–232 °C) [25]; IR (KBr) 3,184, 1,626, 1,620, 1,509, 1,431, 1,235, 1,103
cm−1; 1H-NMR (DMSO-d6) δ 6.07 (s, 1H), 6.69 (d, J = 15.9 Hz, 2H), 6.85 (d, J = 8.6 Hz, 4H), 7.56 (d,
13
J = 8.6 Hz, 4H), 7.56 (d, J = 15.9 Hz, 2H), 10.25 (s, 2H); C-NMR (CDCl3) 183.6, 160.1, 140.8,
130.7, 126.2, 121.1, 116.3, 101.4; HRMS calcd. for C19H16O4 [M+H+]: 309.1127; found 309.1125.

Molecules 2011, 16
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3.3. Synthesis of Desmethoxycurcumin (3).
A complex of 2,4-pentanedione (acetylacetone) (4.12 mL, 40 mmol) with boric anhydride (0.69 g,
10 mmol) was formed in ethyl acetate (30 mL) by reaction at 80 °C for 30 min. A mixture of vanillin
(1.52 g, 10 mmol) and tributyl borate (5.38 mL, 20 mmol) in dried ethyl acetate (10 mL) was then
added to the acetylacetone-boron complex mixture. The solution was stirred vigorously for 15 min at
50 °C, with dropwise addition of n-butylamine (0.73 mL, 10 mmol) in ethyl acetate (5 mL) over
15 min. The reaction mixture gradually turned yellow. Stirring was continued overnight at room
temperature. 1N HCl (30 mL) was added and the mixture was stirred for another 30 min. The organic
layers were separated and the aqueous fraction was extracted with ethyl acetate (3 × 30 mL). The
combined organic layer was washed with water. The organic layer was dried over anhydrous sodium
sulfate as desiccant and the organic solvent was then removed under reduced pressure. After removal
of the solvent under vacuum, the crude products were purified by flash column chromatography with
elution with an hexane-ethyl acetate gradient to afford 4-Hydroxy-6-(4-hydroxy-3-methoxyphenyl)-
hexa-3,5-dien-2-one (furoylacetone, 10) as a pale yellowish solid. Yield 45%; m.p. 144–146 °C (lit.
146–147 °C) [26]; IR (KBr) 3,335, 1,632, 1,561, 1,511, 1,419, 1,247, 1,029 cm-1; 1H-NMR (CDCl3) δ
2.14 (s, 3H), 3.91 (s, 3H), 5.61 (s, 1H), 6.30 (d, J = 15.8 Hz, 1H), 6.90 (d, J = 2.1 Hz, 1H), 7.00 (d,
13
J = 1.5 Hz, 1H), 7.06 (dd, J = 8.2 and 1.5 Hz, 1H), 7.51 (d, J = 15.8 Hz, 1H); C-NMR (CDCl3)
196.9, 178.0, 147.7, 146.8, 140.0, 127.7, 122.6, 120.3, 114.8, 109.5, 100.6, 55.9, 26.8. A complex of
furoylacetone 10 (0.40 g, 1.71 mmol) and boric anhydride (0.60 g, 0.85 mmol) was formed in ethyl
acetate (15 mL). The mixture was stirred constantly at 50 °C for 30 min until the solution was milky.
Subsequently, a mixture of 4-hydroxybenzaldehyde (0.21 g, 1.71 mmol) and tributyl borate (460 µL,
1.71 mmol) was added and stirred at 50 °C for 30 min. n-Butylamine (169 µL) in ethyl acetate (5 mL)
was added dropwise over 15 min. The reaction mixture gradually turned yellowish. Stirring was
continued overnight at room temperature. 1N HCl (30 mL) was added and the mixture was further
stirred for 30 min. The organic layer was separated and the aqueous fraction was extracted with ethyl
acetate (3 × 30 mL). The combined organic layer was washed with water. The organic layer was dried
over anhydrous sodium sulfate as desiccant and the organic solvent was then removed under reduced
pressure. After removal of the solvent under vacuum, the crude products were purified by flash column
chromatography with elution with a hexane-ethyl acetate gradient to afford (1E,6E)-1-(4-Hydroxy-3-
methoxyphenyl)-7-(4-hydroxyphenyl)hepta-1,6-diene-3,5-dione (desmethoxycurcumin, 3) as
a
yellowish solid. Yield 68%; m.p. 174–176 °C (lit 175–176 °C) [25]; IR (KBr) 3,154, 1,630, 1,618,
1
1,501, 1,433, 1,225, 1,100 cm-1; H-NMR (CDCl3) δ 3.93 (s, 3H), 5.78 (s, 1H), 6.46 (d, J = 15.8 Hz,
2H), 6.84 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 1.5 Hz, 1H), 7.10 (dd, J = 8.2 and
1.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 15.8 Hz, 1H), 7.59 (d, J = 15.8 Hz, 1H); 13C-NMR
(CDCl3) 183.6, 157.6, 147.9, 147.0, 146.9, 146.8, 140.6, 140.1, 129.9, 127.9, 127.7, 122.9, 121.8,
115.9, 114.8, 109.7, 101.2, 55.9; HRMS calcd. for C20H18O5 [M+H+]: 361.1052; found 361.1053.
3.4. General Method for Synthesis of Curcuminoid Succinate Ester Derivatives 4–9
A round-bottomed flask (50 mL) was charged with curcuminoids 1, 2 or 3 (0.54 mmol) and 4-(N,N-
dimethylamino)pyridine (DMAP) (1.2 mmol), followed by addition of dichloromethane or 50%

Molecules 2011, 16
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dichloromethane in diethyl ether (30 mL). The solution was slowly added to a solution of methyl-4-
chloro-4-oxobutyrate (1.2 mmol) or ethyl-4-chloro-4-oxobutyrate (1.2 mmol) in dichloromethane
(2 mL) under nitrogen at room temperature. After stirring for 2 h, the solvent was removed and the
residue was diluted with ethyl acetate (20 mL). The reaction mixture then was filtered, washed with 1
M sodium bicarbonate (20 mL) and water (20 mL), dried over sodium sulfate, and concentrated under
reduced pressure. The crude product was recrystallized from cold methanol to give the curcuminoid
succinate ester derivatives 4-9 as pale yellowish solids.
4,4'-((1E,6E)-3,5-Dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-1,4-phenylene)dimethyl disuccinate (4,
curcumin dimethyl disuccinate). Yield 90%; m.p. 143–145 °C, IR (KBr) 2,985, 1,756, 1,732, 1,630,
1
1,598, 1,511, 1,415, 1,254, 1,204, 1,123, 1,029 cm−1; H-NMR (CDCl3) δ 2.76 (t, J = 6.8 and 7.0 Hz,
4H), 2.94 (t, J = 6.7 and 7.0 Hz, 4H), 3.73 (s, 6H), 3.87 (s, 6H), 5.86 (s, 1H), 6.56 (d, J = 15.9 Hz,
2H), 7.08 (d, J = 8.1 Hz, 2H) 7.09 (d, J = 1.8 Hz, 2H), 7.16 (dd, J = 8.1 and 1.8 Hz, 2H), 7.62 (d,
13
J = 15.9 Hz, 2H). C-NMR (CDCl3) 183.1, 172.4, 170.3, 151.4, 141.2, 139.9, 133.9, 124.3, 123.3,
121.0, 111.5, 101.8, 55.9, 51.9, 28.9; HRMS calcd. for C31H32O12 [M+H+]: 597.1972; Found 597.1979.
4,4'-((1E,6E)-3,5-Dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-1,4-phenylene)diethyl disuccinate (5,
curcumin diethyl disuccinate). Yield 95%; m.p. 95–96 °C, IR (KBr) 2,985, 1,746, 1,723, 1,633, 1,598,
1
1,509, 1,412, 1,252, 1,034 cm−1; H-NMR (CDCl3) δ 1.25 (t, J = 7.0 and 7.2, 6H), 2.72 (t, J = 6.9 and
7.0 Hz, 4H), 2.91 (t, J = 6.9 and 7.0 Hz, 4H), 3.84 (s, 6H), 4.16 (m, J = 7.2 Hz, 4H), 5.83 (s, 1H), 6.54
(d, J = 15.7 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H) 7.09 (d, J = 1.7 Hz, 2H), 7.16 (dd, J = 8.3 and
13
1.7 Hz, 2H), 7.58 (d, J = 15.7 Hz, 2H). C-NMR (CDCl3) 183.1, 171.9, 170.3, 151.3, 141.2, 139.9,
133.9, 124.3, 123.3, 121.0, 111.5, 101.8, 60.79, 55.9, 29.2, 28.9, 14.2; HRMS calcd. for
C33H36O12[M+H+]: 625.2285; Found 625.2281.
4,4'-((1E,6E)-3,5-Dioxohepta-1,6-diene-1,7-diyl)bis(1,4-phenylene)dimethyl disuccinate (6, bis-
desmethoxycurcumin dimethyl disuccinate). 95% yield, m.p. 192–193 °C, IR (KBr) 2,935, 1,746,
1
1,734, 1,623, 1,560, 1,502, 1,420, 1,308, 1,115, 1,035 cm−1; H-NMR (CDCl3) δ 2.75 (t, J = 6.5 and
6.8 Hz, 4H), 2.89 (t, J =6.5 and 6.8 Hz, 4H), 3.72 (s, 6H), 5.82 (s, 1H), 6.55 (d, J = 15.8 Hz, 2H), 7.12
13
(d, J = 8.5 Hz, 4H), 7.54 (d, J = 8.6 Hz, 4H), 7.62 (d, J = 15.8 Hz, 2H). C-NMR (MHz, CDCl3)
183.1, 172.5, 170.6, 151.9, 139.5, 132.8, 129.2, 124.2, 122.1, 120.9, 101.8, 51.9, 29.3, 28.8; HRMS
calcd. for C29H28O10 [M+H+]: 537.1760; Found 537.1764.
4,4'-((1E,6E)-3,5-Dioxohepta-1,6-diene-1,7-diyl)bis(1,4-phenylene)
diethyl
disuccinate
(7,
bis-desmethoxycurcumin diethyl disuccinate). Yield 97%; m.p. 155–158 °C, IR (KBr) 2,983, 2,940,
1
1,750, 1,724, 1,631, 1,567, 1,508, 1,416, 1,313, 1,131, 1,021 cm−1; H-NMR (CDCl3) δ 1.25 (t, J = 7.1
and 7.1, 6H), 2.70 (t, J = 6.4 and 6.8 Hz, 4H), 2.89 (t, J =6.4 and 6.8 Hz, 4H), 4.16 (m, J = 7.1, 4H),
5.81 (s, 1H), 6.55 (d, J = 15.8 Hz, 2H), 7.12 (d, J = 7.3 Hz, 4H), 7.54 (d, J = 7.4 Hz, 4H), 7.62 (d,
13
J = 15.8 Hz, 2H). C-NMR (CDCl3) 183.1, 172.0, 170.7, 151.9, 139.5, 132.8, 129.2, 124.2, 122.1,
120.9, 101.8, 60.9, 29.4, 29.1, 14.2; HRMS calcd for C31H32O10 [M+H+]: 565.2073; Found 565.2081.

Molecules 2011, 16
1897
4-((1E,6E)-7-(4-(4-Methoxy-4-oxobutanoyloxy)-3-methoxyphenyl)-3,5-dioxohepta-1,6-dienyl)phenyl
methyl succinate (8, desmethoxycurcumin dimethyl disuccinate). Yield 98%; m.p. 105–109 °C, IR
1
(KBr) 2,947, 1,740, 1,734, 1,627, 1,596, 1,509, 1,410, 1,255, 1,044, 1,029 cm−1; H-NMR (CDCl3) δ
2.74 (m, 4H), 2.90 (m, 4H), 3.71 (s, 6H), 3.85 (s, 3H), 5.82 (s, 1H), 6.54 (d, J = 15.8 Hz, 1H), 6.55 (d,
J = 15.8 Hz, 1H), 7.06 (d, J = 8.6 Hz, 2H) 7.04-7.14 (m, 3H), 7.55 (d, J = 8.6 Hz, 2H), 7.55 (d,
13
J = 15.8 Hz, 1H), 7.56 (d, J = 15.8 Hz, 1H). C-NMR (CDCl3) 183.1, 183.0, 172.4, 170.6, 170.2,
151.9, 151.4, 141.3, 139.9, 139.5, 134.0,132.8, 129.2, 124.3, 124.2, 123.3, 122.1, 121.0, 111.5, 101.8,
55.9, 51.9, 51.8, 29.3, 29.0, 28.8; HRMS calcd. for C30H30O11[M+H+]: 567.1866; Found 567.1862.
4-((1E,6E)-7-(4-(4-Ethoxy-4-oxobutanoyloxy)-3-methoxyphenyl)-3,5-dioxohepta-1,6-dienyl)phenyl
ethyl succinate (9, desmethoxycurcumin diethyl disuccinate). Yield 96%; m.p. 95–97 °C, IR (KBr)
1
2,980, 2,936, 1,758, 1,735, 1,623, 1,584, 1,508, 1,420, 1,221, 1,070, 1,020 cm−1; H-NMR (CDCl3) δ
1.26 (t, J = 7.1 and 7.1 Hz, 6H), 2.72 (m, 4H), 2.89 (m, 4H), 3.85 (s, 3H), 4.16 (m, 4H), 5.82 (s, 1H),
6.54 (d, J = 15.8 Hz, 1H), 6.55 (d, J = 15.8 Hz, 1H), 7.06 (d, J = 8.6 Hz, 2H) 7.04–7.14 (m, 3H), 7.54
13
(d, J = 8.6 Hz, 2H), 7.54 (d, J = 15.8 Hz, 1H), 7.56 (d, J = 15.8 Hz, 1H). C-NMR (CDCl3) 183.2,
183.1, 172.0, 170.7, 170.3, 151.9, 151.4, 141.3, 139.9, 139.6, 134.0, 132.7, 129.2, 124.3, 124.2, 123.3,
122.1, 121.1, 111.5, 101.8, 60.9, 60.8, 55.9, 29.3, 29.2, 29.1, 28.9, 14.2; HRMS calcd for
C32H34O11[M+H+]: 595.2179; Found 595.2188.
3.5. Cytotoxicity Evaluation
The anticancer activity of curcuminoids and their succinate prodrugs was evaluated in a colon
cancer cell line: Caco-2 (human colon adenocarcinoma, ATCC Cat. No. HTB-37). Cytotoxicity was
determined using a MTT microplate assay [27]. Stock solutions of curcuminoids or succinate prodrugs
were prepared in dimethylsulfoxide (DMSO) at 1,000 µM. The solutions were serially diluted with the
culture medium of the cells to obtain sample solutions at appropriate concentrations. The cells were
exposed to the sample at a concentration range of 10 to 0.078 µM. The incubation time was 24 h, as
used in previous studies of the cytotoxicity of curcumin prodrugs [28,29]. After incubation, the
samples were removed from the cell cultures prior to performance of the MTT assay. Doxorubicin was
used as a positive control and 0.5% DMSO as a negative control. Results are expressed as the
concentration of compound required to kill 50% of the cells (IC50) in comparison with the control.
Experiments were performed in triplicate.
3.6. Chemical Stability Study
Stock methanolic solutions of curcumin (1) and curcumin diethyl disuccinate (5) were separately
prepared in 0.1 M potassium phosphate buffer (pH 7.4) and then diluted with the same buffer to give a
final concentration of 1.5 μM. The solution was left to stand at 37 °C for 12 h. The amount of
curcumin (1) or curcumin diethyl disuccinate (5) was determined at appropriate time intervals using a
previously reported HPLC method with modifications [30]. Chromatography was performed using a
gradient system with an autosampler temperature of 15 °C, a column temperature of 33 °C, a flow rate
of 2.0 mL/min, and a detection wavelength of 400 nm. Gradient elution was performed with eluent A

Molecules 2011, 16
1898
(2% v/v aqueous acetic acid) and B (acetonitrile). The elution program was optimized as follows:
initial 0–4 min, isocratic elution A-B (70:30, v/v); 4–5 min, linear gradient to A-B (50:50, v/v);
5–8 min, isocratic elution A-B (50:50, v/v); 8–9 min, linear gradient to A-B (45:55, v/v); 9-10 min,
isocratic elution A-B (45:55, v/v); 10–11 min, linear gradient to A-B (30:70, v/v); 11–12 min, isocratic
elution A-B (30:70, v/v); 12–13 min, linear gradient to A-B (70:30, v/v); 13–15 min, isocratic elution
A-B (70:30, v/v). The injection volume was 20 μL. Experiments were performed in triplicate. Kinetic
parameters were determined by a semi-logarithmic plot of concentration vs. time and calculated using
a linear least-squares regression analysis.
3.7. Release Study
Plasma was spiked with a stock aqueous solution of curcumin diethyl disuccinate (5) to give a final
concentration of 1.5 μM. The spiked plasma was left to stand at 37 °C for 12 h. The release profile of
curcumin was determined at appropriate time intervals by extraction with acetonitrile and analysis
using the HPLC conditions described above. Experiments were performed in triplicate.
4. Conclusions
In conclusion, curcuminoid-succinate prodrugs 4–9 were prepared via succinylation of the parent
curcuminoids 1–3. The six derivatives were readily synthesized in high purity and good yields. A
hydrolytic study indicated improved stability of the prodrug in comparison with the parent drug. The
observation of significantly increased curcumin release in human plasma suggests that succinylation of
curcuminoids is an effective prodrug strategy. Therefore, prodrugs 4–9 may be promising candidates
for future treatment of colon cancer.
Acknowledgements
The authors express their gratitude to the Faculty of Pharmaceutical Sciences, Chulalongkorn
University for providing research funds to P. Rojsitthisak. Financial support from the Thailand
Research Fund through the Royal Golden Jubilee Ph.D Program (Grant No. PHD/0217/2548) to W.
Wichitnithad and U. Nimmannit is also acknowledged. The authors thank Ian S. Haworth (University
of Southern California) for his suggestions and comments on the manuscript.
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Sample Availability: Samples of compounds 1–9 are available from the authors.
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