Synthesis of Novel Asymmertrical Monocarbonyl Analogues of Curcumin

Pekik Wiji Prasetyaningrum1,* ), Anton Bahtiar1), Catur Jatmika1) and Hayun Hayun1,* )
1
Faculty of Pharmacy, Universitas Indonesia, , Depok, 16424, West Java, Indonesia.
*)
Corresponding authors: pekikwiji@gmail.com; hayun.ms@ui.ac.id

ABSTRACT
Novel asymmetrical monocarbonyl analogues of curcumin (asymmetrical MACs) were
synthesized based on Clasein-Schmidt condensation reaction of aldehyde aromatics with
cyclohexanone. The synthesized compounds were purified by column chromatography and the
structure were confirmed on the basis of FTIR, 1H-NMR, 13C-NMR and HR-MS. The yields
obtained from this study were: 50.03%, 2.7%, 47.31%, 6.1%,13.16% and 55.53%, respectively
for 2-benzyliden-6-(4-hydroxy-3-methoxybenzylidene)cyclohexaanone, 2-(4-hydroxy-3-
methoxybenzylidene)6-(4-methoxybenzylidene)cyclohexanone, 2-(4-fluorobenzyliden)6-(4-
hydroxy-3-methoxybenzylidine)cyclohexanone, 2-(4-cholobenzylidene)-6-(4-hydroxy-
3methoxybenzylidene)cyclohexanone, 2-(4-hydroxy-3-methoxybenzylidene)-6-(4-
methylbenzylidene)cyclohexanone, and 2-benzylidene-6-(4-
hydoxybenzylidene/cyclohexanone. Cytotoxicity effect of all the compound have been
analyzed used BSLT bioassay, the result is all compund have potency as anti-carcinogenic
agents, because all of them shown LC50 < 1000 g/mL, and exhibited more active then
curcumin (curcumins LC50 is 210.30 g/mL).

INTRODUCTION
Curcumin modification becomes mono-carbonyl analogues of curcumin (MACs) can
increases the potency of 10-30 times more than curcumin in a number of cell lines and cellular
proteins (1). Monocarbonyl curcumin analogues is much more stable at pH 6.5 and higher,
suggesting better pharmacokinetic profiles and greater tumor regimens on in vivo cancer
xenografts than curcumin (2). Monocarbonyl compounds with cyclohexanone ketones can
inhibit the growth of leukemia, colon, renal, melanaoma, ovarian, CNS, prostate, and breast
cancer cells better than cisplatin (3). Derivatives of asymmetric mono-carbonyl analogues of
curcumin have been known as antioxidant, anti-inflammatory, antimicrobial and anti
carcinogenic effects (4,5,6,7,8,9). Even though, there are a little reasearch to find on asymetrical
MACs and their activities as anti-carciogenic. The synthesis of asymmetric MACs can be done
by the condensation reaction of Clasein-Schmidt (10,11).
2.1 Materials And Experimental
Apparatus

The material used are Vanilin, Benzaldehyde, p-chlorobenzaldehyde, p-

methoxybenzaldehyde, p-fluorobenzaldehyde, p-methylbenzaldehyde, p-methylbenzaldehyde,
cyclohexanone , sodium hydroxide, hydrochloric acid, etanol. The tools used are glassware
for synthesis and purification, , FTIR spectrophotometer (Perkin Elmer 16 PC, FTIR, Prestige-
21 Shimadzu), 1H and 13C spectrophotometer NMR resonance Core Magnet (500 MHz, Jeol)
Liquid chromatography - Mariner Biored Mass Spectrometry (70 ev). BSLT assay kit.
2.2 Chemical Synthesis

Schema 1. The synthesis of asymmetrical MACs

Tabel 1. The chemical structures of the synthetic curcumin analogs

Comp. R1 R2 R3
1a -H -OCH3 -OH
1b - OCH3 -OCH3 -OH
1c -F -OCH3 -OH
1d -Cl -OCH3 -OH
1e -CH3 -OCH3 -OH
1f -H -OH -OH
2a -H -OCH3 -OH
2b -OCH3 -OCH3 -OH
2c -F -OCH3 -OH
2d -Cl -OCH3 -OH
2e -CH3 -OCH3 -OH
2f -H -OH -OH

2.2.1 Synthesis of H/p-methyl/p-methoxy/p-chloro/p-fluoro-benzylidenecyclohexanone

The synthesis were perfomed according to the method of the synthesis of 2-benzyliden
cyclohexanone (15).A mixture of 0.32 mol aromatic aldehyde and 0.88 mol cyclohexanone in
Erlenmeyerwas stirred with a magnetic stirrer, then NaOH solution (10%) was addeddropswise
while stirring for 2 hours. The mixture was neutralized with HCl to pH 7, separated the organic
layer and the existing water layer was extracted with 16 mL of toluene. The toluene layer was
mixed with an organic layer, washed with 16 mL of water, and dried withanhydrous sodium
sulfate. The solvent of dried mixture was evaporated using rotary vacuum evaporator to give
the crude product. The crude products then were used as starting material for the next synthesis
without further purification.
2.2.2 Synthesis of asymmetrical MACs (compound 1a-1f)
The synthesis were performed according to the synthesis methode of asymmetical MACs
reported previously(10,11). 0.005 mol of H/p-methyl/p-methoxy/p-chloro/p-
fluorobenzylidenecyclohexanone and 1.52 g (0.01 mol) vanillin/p-hydroxybenzaldehyde were
mixed in Erlenmeyer containing 10 mL of ethanol, and heated until dissolved, a drop of
concentrated HCL diluted in 1 ml of ethanol was added, and stirred for 30 minutes.The solvent
was evaporated, the solid material obtained was added withcold mixture of glacial acetic acid
and water (1:1), andfiltered with buchner funnel. The solid product was washed back with cold
mixture of glacial acetic acid and water (1: 1), dried, and purified using column chromatigraphy
with mixture of approriate ratio of n-hezane and ethylacetate.
2.2.3 Synthesis of asymmetrical MACs subtitued by Mannich Base (compound 2a-2f)
The method that were adapted from the Geschiker and Meadow method (1969)(15).
Compound 2a-2f 2 mmol were dissolved in ethanol and cooled in an ice bath, then
diethylamine (5 mmol for compound 2a-2e, 7 mmol for compound 2f) were added slowly to
the mixture, then formaldehyde solution 37% with an amount comparable to diethylamine were
added. Then the mixture was stirred for 30 minutes at room temperature. Afther that, the
mixture was heated until boilling point. to know when the reaction time is over, monitoring is
done. After obtaining the desired compound, ethanol was evaporated, then added 40 mL of
methanol and vaporized, then added 50 mL of methanol then warmed, then mixed with cold
aquades. The solution is decanted, the precipitate is filtered off and washed with cold aquades.
The product compound was purified by column chromatography on silica gel, tested its purity
by TLC and its melting point, and elucidated its structure based on FT-IR spectral data, 1H-
NMR, 13C-NMR, and HRMS.

2.3 Cytotoxicity Test

2.3.1 Brine Shrimp Lethality Test
The assay was carried out according to the principle and protocol previously described
by Meyer (21), with slight. Artemina salina L. eggs were inserted in a box that containing
seawater, the box was placed under UV lamp, after 48 hours the eggs hatched into larvae and
ready for the test. The compound (1a-1f, 2a-2f) were diluted in 10 mL seawater that contains
10 larvae (1% DMSO (v/v)) until concentration 20, 200, 500, and 1000 ppm. After 24 hours,
the live and dead shrimp were counted. The mortality rate (%) of mortality is obtained by
comparing the number of total dead larvae and total number of larvae. The LC50 value was
obtained by calculating according to the linear regretion formula y = a + bx (y = % mortality,
x= logC). The compound with LC50 < 1000 g / ml was medium active, LC50 < 100 g / ml
was active. Activities of extract are considered significant when LC50 30 g / ml.
 Rf
Comp. % Yield M.P (C) (E.A:n-
H 1:2)
1a 50,03 149-151 0,8
1b 2,7 133-136 0,55
1c 47,31 129-131 0,52
1d 6,1 150-154 0,75
1e 13,16 130-131 0,75
1f 55,53 214-216 0,81