Synthesis and biological evaluation of new symmetric curcumin derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.07.088

A series of novel curcumin bisacetamides aiming of enriching their biological activities have been synthesized. The synthesized compounds were screened for their in vitro antioxidant, anti-inflammatory and cytotoxic activities. All the compounds exhibited

Full text of DOI:10.1016/j.bmcl.2015.07.088

Accepted Manuscript
Synthesis and biological evaluation of new symmetric curcumin derivatives
Rajendran Sribalan, Maruthan Kirubavathi, Govindharaj Banuppriya,
Vediappen Padmini
PII:
S0960-894X(15)00807-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.088
BMCL 22977
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
22 April 2015
6 July 2015
27 July 2015
Please cite this article as: Sribalan, R., Kirubavathi, M., Banuppriya, G., Padmini, V., Synthesis and biological
evaluation of new symmetric curcumin derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.07.088
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Synthesis and biological evaluation of new symmetric curcumin derivatives
Rajendran Sribalan, Maruthan Kirubavathi, Govindharaj Banuppriya, Vediappen Padmini*
Department of Organic Chemistry, Madurai Kamaraj University, Madurai-625 021, Tamil Nadu, India.
*Corresponding author e-mail: padimini_tamilenthi@yahoo.co.in
Abstract
A series of novel curcumin bisacetamides aiming of enriching their biological activities have been synthesized.
The synthesized compounds were screened for their in vitro antioxidant, anti-inflammatory and cytotoxic activities. All
the compounds exhibited potent to good anti-inflammatory, antioxidant and noteworthy cytotoxic activities.
Keywords: curcumin, acetamides, anti-inflammatory, cytotoxicity, antioxidant.
The biological evaluation of natural products and/or their derivatives are fascinating in medicinal research,
since most of the natural products are zero toxic. Curcumin is familiar bioactive natural product1 and food product
isolated from curcuma longa Linn2. The attention in biomedical research of parent or altered curcumins is due to their
extensive spectrum of biological applications3-10
.
The curcumin derivatives have been demonstrated as in vivo
biologically active with anticancer11, antidiabetic12, anti-inflammatory13 and cytotoxicity14 characteristics. The
significant advantage of the curcumin is that it is nontoxic to human even with high dosage15. The clinical trials
validate that the daily dose up to 12g for 3 months has not shown any effect on the biological system of healthy
people16.
Even though curcumin is the lead molecule, the researchers are focusing to synthesize covalently modified
curcumin. The chemical modification of curcumin can be carried out in several positions (active methylene, aromatic
ring and enones) 17-19. Most of the literature reports has revealed that the phenolic unit of curcumin is responsible for
biological activity like free radical scavenging property 20,21. In addition to that the enolic hydroxyl unit also plays a
major role in radical scavenging activity along with phenolic hydroxyl group22. Wichitnithad et al reported
curcuminoids without free phenolic hydroxyl group and have shown enhanced anticancer activity and stability23. Ohtsu
et al reported the cytotoxicity of dimethoxy curcuminoid24. Similarly, hindering of hydroxyl group with glucosyl unit
in curcumin improved the stability as well as the antioxidant property25. Parvathy et al reported the curcumin amino
acid conjugates on phenolic hydroxyl with enhanced antioxidant activity than the parent drug26. Safavy et al described
1

the blocking of hydroxyl with polyethylene glycol which enriched the cytotoxicity27. From these intelligences, we have
planned to synthesise the curcuminoid blocking the phenolic hydroxyl unit as better bioactive functional group.
One of the well-known simple bioactive functional groups is peptide link which has been demonstrated as a better
bioactive molecule in medicinal chemistry. Especially the acetamide or N-substituted acetamides played an essential
role in many biological activities28. Hence we can anticipate enhanced biological activities by the introduction of
acetamido unit to curcumin. Based on this we have introduced the N-substituted acetamido group to the curcumin
hydroxyl group and their in vitro antioxidant, anti-inflammatory and cytotoxic activities and the details are presented
here.
To synthesise the curcumin bisacetamides (Fig 1), the intermediate curcumin bisacetic acid was
synthesized as per the reported procedure29. Initially the vanillin acetic acid was prepared from vanillin with
chloro acetic acid in presence of sodium hydroxide in water29. This synthon further used to get boron complex
by the conventional method in the presence of acetyl acetone, boron oxide, tri-n-butylborate, n-butyl amine and
DMF. The addition of 5% hot acetic acid cleaves the complex and offered the intermediate curcumin bisacetic
acid30. The coupling of curcumin bisacetic acid with various amines yielded the product31. All the synthesized
compounds were characterized by 1H NMR, 13C NMR, mass and IR spectral techniques. The appearance of the
amide NH peak in 1H NMR spectrum, the appearance of carbonyl carbon of the amide and the disappearance of
acid carbonyl in 13C NMR spectrum indicate the formation of carboxamide. The appearance of a singlet for 6
protons at 4.00 ppm and a singlet for 4 protons at 4.68 ppm indicate the presence of methoxy and methylene
(present in between phenoxy and amide) units. The singlet at 5.84 ppm for 1 proton indicates enolic CH. These
results revealed that the curcumin is present in the enolic form. The doublet at 6.54 ppm represents the presence
of enone CH. Additionally the compound 4a was characterized by one dimensional DEPT-135 and 2D NMR
(1H-1H COSY, 13C-1H COSY and HMBC) experiments. In DEPT-135, 10 positive signals and 1 negative signal
were appeared indicating 10 sets of CH and one CH2 carbons. The correlation of signals at 6.53 & 7.63 in the
1H-1H COSY spectrum clearly indicates the presence of ethylene unit. The signals at 4.00, 4.68 and 5.64 ppm
with the signals at 56.2, 70.0 and 101.6ppm respectively in the 13C-1H COSY indicates the presence of O-CH3,
O-CH2, and enolic CH units. The HMBC correlation of the signals at 4.68ppm with the carbons at 149.00 and
166.4ppm indicates the correlation of acetyl CH with quaternary phenoxy carbon and carbonyl carbon of amide.
The contours connecting signals at 6.54 ppm & 130.77 ppm and 6.54 ppm & 183.25 ppm indicate the presence
of enone unit. The analytical spectra were shown in the supporting information.
2

Fig. 1. Synthetic route for curcumin bisacetamide derivatives (4a-h)
The synthesized curcumin bisacetamides were tested for radical scavenging activity with DPPH free
radical32 These compounds showed potent radical scavenging ability when compared to the curcumin and
.
vitamin C. Especially the compounds 4c, 4e and 4g having p-chloro acetanilide, napthanilide and N-
methylacetanilide moieties have exhibited a better scavenging ability than the curcumin. The compound 4f and
4h bearing the aliphatic substituent such as N,N diethyl and cyclohexyl amide disclosed good activity. The
compounds 4a, 4b and 4d having acetanilide, p-tolylacetanilide and p-fluoro acetanilide fragments showed
3

moderate DPPH radical scavenging activity. Overall, curcumin with bulky substituents has showed better
activity than the parent and standard. The IC50 values of the radical scavenging activity of different compounds
are listed in Table 1. The scavenging of DPPH free radical by the curcumin bisacetamides could be viewed with
naked eye (Fig 2). The synthesized curcumin bisacetamides were tested for H2O2 scavenging activity as well all
compounds showed worthy H2O2 scavenging activity than ascorbic acid33. The compound 4h having the
cyclohexyl amide unit showed potent H2O2 scavenging ability. Similarly compound 4e bearing naphthalene
amide units showed good scavenging activity for which the IC50 value is nearer to curcumin. But most of the
compounds exhibited less scavenging activity than curcumin but nearer to ascorbic acid. The IC50 values of the
compounds are listed in Table 1.
Table. 1 Antioxidant activity of the compounds (4a-h)
S.No Compound DPPH(IC50) µg/mL H2O2(IC50) µg/mL
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
4a
4b
4c
4d
4e
4f
4g
23.77
22.85
12.92
24.26
12.65
19.68
12.05
16.00
14.83
32.74
63.70
61.98
65.40
70.51
41.40
68.16
63.21
26.14
34.71
67.54
4h
Curcumin
Vitamin C
Fig 2. (i) Appearance of curcumin and 4a. (ii) DPPH radical scavenging for the ascorbic acid, curcumin and
4a-h (25µg/mL) with 0.1mM DPPH.
The curcumin bisacetamides were studied for their anti-inflammatory activity (bovine serum albumin
assay)34. The compounds 4f, 4g and 4h having the N,N-diethyl acetamide, N-methyl acetanilide and cyclohexyl
amide showed potent activity than the curcumin, while the compounds 4a, 4b and 4d bearing acetanilide, p-
tolylacetmide and p-fluoro acetanilide exhibited good activity. The percentage inhibitions were almost closer to
the standards curcumin and diclofenac sodium. The compounds 4c and 4e having the p-chloroacetanilide and
napthanilide disclosed reasonable activity. Overall, the N-alkyl/cycloalkyl substituted compounds showed
enhanced activity with bulky substituents retains the inflammatory activity. The percentage inhibition for the
various concentration of synthesized compounds, curcumin and diclofenac sodium are presented in Fig 3.
4

2, 5-di
100
90
%
I
80
70
60
50
40
30
20
10
0
n
h
i
b
i
t
i
o
n
10
50
100
250
500
4a
4b
4c
4d
4e
4f
4g
4h
cur
std
Concentration (µg/mL)
Cur: curcumin, Standard: diclofec sodium
Fig 3. Anti-inflammatory activity of the compounds 4a-h (Bovine serum albumin assay)
The curcumin bisacetamides were tested for cytotoxic activity by MTT ((3-[4,5-dimethylthiozol-2-yl]-
phenyltetrazolium bromide)) assay against A549 cancer cell line35. All the synthesized compounds have
shown good cytotoxic activity. The compounds 4f and 4h having the N,N-diethyl and cyclohexyl acetamide
showed very good activity. Other compounds showed only a moderate cytotoxic activity. The experimental
results revealed that the compounds having the aliphatic substitution showed good cytotoxic activity than
curcumin. The % inhibition for the different concentration of synthesized compounds and curcumins are shown
in Fig 5 and the cell images were represented in Fig 4.
Fig 4. Cell images (A549) of control, 4a-h (100 µg/mL) & Curcumin (100µg/mL)
5

100
90
80
70
1
%
60
50
40
30
20
10
0
10
50
100
500
D
C
4a
4b
4c
4d
4e
4f
4g
4h
cur
Concentration (µg/mL)
% DC: % of death cells
Fig 5. Cytotoxic activity of the compounds 4a-h against A549 cell line
The synthesis of curcumin bisacetamides is reported in this work and it is shown that the introduction
of acetamide on the curcumin phenolic hydroxyl enhanced/retained the in vitro antioxidant, anti-inflammatory
and retained cytotoxic activity. Further work to synthesise water soluble curcumin derivatives with polar units
is in progress.
Acknowledgement
The authors are grateful to DST and UGC for financial support and DST-IRHPA programme for
providing high resolution NMR facility.
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Cancer Ther. Res. 2012, Doi: 10.7243/2049-7962-1-10
12. Abdel Aziz M. T.; El-Asmar, M. F.; Rezq, A. M.; Mahfouz, S. M.; Wassef, M. A.; Fouad, H. H. et al.,
Diabetol. Metob. Syndr. 2013, 5, 75-88.
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14. Liang, G.; Shao, L.; Wang, Y.; Zhao, C.; Chu, Y.; Xiao, J. et al., Bioorg. Med. Chem. 2009, 17, 2623-2631.
15. Sharma, R. A.; Mclelland, H. R.; Hill, K. A. Clin. Cancer Res. 2001, 7, 1894-1900.
16. Goel, A.; Kunnumakkara, A. B.; Aggarwal, B. B. Biochem. Pharmacol. 2008, 75, 787-809.
17. Lin, L.; Shi, Q.; Nyarko, K.; Bastow, K. F.; Wu, C-C.; Su, C-C. et al., J. Med. Chem. 2006, 49, 3963-3972.
18. Qiu, X.; Du, Y.; Lou, B.; Zuo, Y.; Shao, W.; Huo, Y. et al., J. Med. chem. 2010, 53, 8260-8273.
19. Leow, P-C.; Bahety, P.; Boon, C. P.; Lee, C. Y.; Tan, K. L.; Yang, T. et al., Eur. J. Med. Chem. 2014, 71,
67-80.
20. Masuda, T.; Hidaka, K.; Shinohara, A.; Maekawa, T.; Takeda, Y.; Yamaguchi, H. J. Agric. Food Chem.
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21. Jovanovic, S. V.; Boone, C. W.; Steenken, S.; Trinoga, M.; Kaskey, R. B. J. Am Chem. Soc. 2001, 123,
3064-3068.
22. Feng, J-Y.; Liu, Z-Q. J. Agric. Food Chem. 2009, 57, 11041-11046.
7

23. Wichitnithad, W.; Nimmannit, U.; Wacharasindhu, S.; Rajsitthisak, P. Molecules. 2011, 16, 1888-1900.
24. Ohtsu, H.; Xiao, Z.; Ishida, J.; Nagai, M.; Wang, H-K.; Itokawa, H. et al., J. Med. Chem. 2002, 45, 5037-
5042.
25. Parvathy, K. S.; Negi, P. S.; Srinivas, P. Food Chem. 2009, 115, 265-271.
26. Parvathy, K. S.; Negi, P. S.; Srinivas, P. Food Chem. 2010, 120, 523- 530.
27. Safavy, A.; Raisch, K. P.; Mantena, S.; Sanford, L. L.; Sham, S. W.; Krishna, N. R.; Bonner, J. A. J. Med.
Chem. 2007, 50, 6284-6288.
28. Liu, Y.; Feng, Y.; Wang, R.; Gao, Y.; Lai, L. Bioorg. Med. Chem. Lett. 2001, 11, 1631-1641.
29. Synthetic procedure for 2-(4-formyl-2-methoxyphenoxy)acetic acid (2): The vanillin (10g, 0.065 mol) was
dissolved in 1N NaOH (160 mL) solution. To that solution chloro aceticacid (7.5g, 0.078 mol) was added
and it was refluxed for 12 hrs. Then the reaction mixture was diluted with water (100 mL) washed with
ethyl acetate (2 x 75 mL), then the aqueous layer was separated and acidified with Con.HCl. Then the
mixture was extracted with ethyl acetate (3 x 100 mL) (in the ethyl acetate layer the product was dissolved
along with unreacted vanillin). The combined organic layers were extracted with saturated sodium
bicarbonate solution (2 x 75 mL) and separated. The sodium bicarbonate solution was acidified with
Con.HCl gave white precipitate of the product. It was filtered and dried in hot air oven at 60 °C for 24hrs.
1
White Solid. Yield: 86.2%. Mp: 176-178 °C IR (KBr Disc) cm-1: 2968, 2924, 1662, 1030, 965. H NMR
(300 MHz, CDCl3) δ 9.86 (s, 1H), 7.49 – 7.39 (m, 2H), 6.92 (d, J = 8.6 Hz, 1H), 4.76 (s, 2H), 3.95 (s, 3H).
13C NMR (75 MHz, CDCl3) δ 190.52, 169.57, 152.52, 130.57, 125.77, 112.14, 109.76, 65.39, 55.74. ESI-
MS calculated. m/z 210.04. found 211.05 (M++1). Anal.Calcd. for: C10H10O5: C, 57.14; H, 4.80% found:
C, 57.18; H, 4.78%
30. Venkateswarlu, S.; Ramachandra, M. S.; Subbaraju, G. V. Bioorg. Med. Chem. 2005, 13, 6374-6380.
Synthetic
procedure
for
2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-
phenylene))bis(oxy))diacetic acid (3): To a pentadione (0.976 mL, 0.0095 mol), boron oxide (0.662 g,
0.0095mol), tri-n-butyl borate (2.51 mL, 0.0095mol) and vanillin acetic acid (4g, 0.019mol) were added.
The mixture was dissolved in Dimethylformamide (20 mL) and heated to 70 °C. When the reaction
temperature reaches 70 °C, n-butylamine (1.87 mL, 0.019mol) was added drop wise over 5 min. Then the
8

reaction mixture was stirred at 70 °C for 4 hrs. Then the reaction mixture was added to 5% hot acetic acid
solution (40 mL) and stirred for 3 hrs. It was diluted with water (100 mL) and extracted with ethyl acetate
(2 x 150 mL). The combined organic layers were again washed with water (100 mL) and extracted with
10% sodium bicarbonate solution (2 x 75 mL). The aqueous layer was separated and neutralized with 2N
HCl (the PH of the solution should be 6.0-6.5). The yellow precipitate obtained was filtered and dried in
vacuum. The filtered solid was washed with ethyl acetate afforded product 3. Yellow Solid. Yield: 62.5%.
Mp: 126-128 °C. IR(KBr Disc) cm-1: 3513, 3068, 2918, 2284, 1710, 1631, 1601, 1030, 966, 808. 1H NMR
(300 MHz, DMSO-D6) δ 7.64 (d, J = 15.6 Hz, 2H), 7.43 (s, 2H), 7.29 (d, J = 7.8 Hz, 2H), 7.02 – 6.86 (m,
4H), 6.18 (s, 1H), 4.79 (s, 4H), 3.90 (s, 6H). 13C NMR (75 MHz, DMSO-D6) δ 183.12, 169.83, 149.41,
149.14, 140.15, 128.20, 122.39, 113.09, 111.26, 100.93, 65.17, 55.72. ESI-MS calculated. m/z 484.14.
found 485.15 (M++1). Anal.Calcd. for: C25H24O10: C, 61.98; H, 4.99 % found: C, 61.96; H, 4.98;%
31. Christian A. G. N.; Montalbetti; Falque, V. Tetrahedron. 2005, 61, 10827-10852. General procedure to
synthesize curcumin bisacetamide (4a-h): The curcumin bisaceticacid (0.3 g, 0.619 mmol) was dissolved
in dimethylformamide (3 mL). To that solution Dicyclohexylcarbodiimide (0.569g, 1.859 mmol),
hydroxybenzotriazole (0.083g, 0.619 mmol), N,N dimethyl amino pyridine (0.0075g, 0.0619mmol) and
aniline (0.113 mL, 1.239 mmol) was added. The reaction mixture was stirred for 24 hours and diluted with
dichloromethane (10 mL). The white precipitate obtained was filtered and evaporated. Finally the product
was purified by column chromatography with mixture of chloroform and methanol as eluent. (The
appearance of compound 4a was represented in fig 5.).
2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N-
phenylacetamide)(4a): Yellow solid. Yield: 74.3%. Mp: 160-161 °C. IR (KBr Disc) cm-1: 2960, 2920,
2315, 1697, 1690, 1683, 1030, 976. 1H NMR (300 MHz, CDCl3) δ 8.77 (s, 2H), 7.65 – 7.56 (m, 6H), 7.37
(t, J = 7.9 Hz, 4H), 7.20 – 7.12 (m, 6H), 6.98 (d, J = 8.3 Hz, 2H), 6.54 (d, J = 15.8 Hz, 2H), 5.84 (s, 1H),
4.68 (s, 4H), 4.00 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 183.26, 166.38, 150.23, 149.00, 140.02, 137.29,
130.78, 129.24, 124.90, 123.55, 122.25, 120.10, 116.12, 111.29, 101.64, 70.05, 56.21. ESI-MS calculated.
m/z 634.23. found 635.27 (M++1). Anal.Calcd. for: C37H34N2O8: C, 70.02; H, 5.40; N, 4.41% found: C,
70.03; H, 5.39; N, 4.39 %.
2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N-(p-
tolyl)acetamide) (4b): Yellow solid. Yield: 74.9%. Mp: 125-126 °C IR (KBr Disc) cm-1: 2961, 2847,
9

2359, 1683, 1652, 1031, 972. 1H NMR (300 MHz, CDCl3) δ 8.72 (s, 2H), 7.59 (d, J = 15.8 Hz, 2H), 7.48 –
7.45 (m, 6H), 7.16-7.10 (m, 4H), 6.95 (d, J = 8.3 Hz, 2H), 6.52 (d, J = 15.8 Hz, 2H), 5.83 (s, 1H), 4.65 (s,
4H), 3.97 (s, 6H), 2.32 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 183.20, 166.21, 150.01, 148.88, 140.01,
134.54, 130.50, 129.68, 123.32, 122.24, 120.10, 115.75, 111.01, 69.80, 56.09, 20.99. ESI-MS calculated.
m/z 662.24. found 663.05 (M++1). Anal.Calcd. for: C39H38N2O8: C, 70.68; H, 5.78; N, 4.23 % found: C,
70.66; H, 5.77; N, 4.24 %. 2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-
phenylene))bis(oxy))bis(N-(4-chlorophenyl)acetamide) (4c): Yellow solid. Yield: 73.2%. Mp: 172-174 °C.
IR (KBr Disc) cm-1: 2926, 2919, 2849, 1710, 1690, 1652, 1031, 966, 668. 1H NMR (300 MHz, CDCl3) δ
8.84 (s, 2H), 7.63 - 7.55 (m, 6H), 7.33 (d, J = 8.1 Hz, 4H), 7.18 (d, J = 7.7 Hz, 2H), 7.14 (s, 2H), 6.98 (d, J
= 8.4 Hz, 2H), 6.54 (d, J = 15.8 Hz, 2H), 5.85 (s, 1H), 4.68 (s, 4H), 4.00 (s, 6H). 13C NMR (75 MHz,
CDCl3) δ 183.22, 166.47, 150.21, 148.91, 139.96, 135.89, 130.91, 129.93, 129.25, 123.62, 122.26, 121.29,
116.27, 111.38, 70.05, 56.24. ESI-MS calculated. m/z 702.25. found 703.09 (M++1). Anal.Calcd. for:
C37H32Cl2N2O8: C, 63.16; H, 4.58; N, 3.98 % found: C, 63.19; H, 4.60; N, 3.95 %.
2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N-(4-
fluorophenyl)acetamide) (4d): Yellow solid. Yield: 70.2%. Mp: 190-192 °C. IR (KBr Disc) cm-1: 2959,
1
2920, 2849, 2365, 2340, 1689, 1652, 1028, 968. H NMR (300 MHz, CDCl3) δ 8.77 (s, 2H), 7.63 – 7.54
(m, 6H), 7.18 (d, J = 8.5 Hz, 2H), 7.13 (s, 2H), 7.07 (d, J = 8.6 Hz, 4H), 6.98 (d, J = 8.3 Hz, 2H), 6.54 (d,
J = 15.8 Hz, 2H), 5.84 (s, 1H), 4.68 (s, 4H), 3.99 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 183.23, 166.38,
156.48, 150.07, 148.84, 140.02, 133.25, 130.71, 129.73, 123.47, 122.29, 121.91, 121.81, 117.73, 116.07,
115.77, 111.14, 69.86, 56.17. ESI-MS calculated. m/z 670.21. found 671.10 (M++1). Anal.Calcd. for:
C37H32F2N2O8: C, 66.26; H, 4.81; N, 4.18 % found: C, 66.28; H, 4.79; N, 4.19%. 2,2'-((((1E,6E)-3,5-
dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N-(naphthalen-1-yl)acetamide)
(4e): Yellow solid. Yield: 68.7 %. Mp: 195-198°C. IR (KBr Disc) cm-1: 2959, 2922, 2851, 1690, 1631,
1058, 965. 1H NMR (300 MHz, DMSO-D6) δ 10.11 (s, 2H), 8.09 – 7.94 (m, 4H), 7.86 – 7.75 (m, 6H), 7.66
– 7.50 (m, 8H), 7.33 (d, J = 7.6 Hz, 2H), 7.13 (s, 2H), 6.90 (d, J = 15.8 Hz, 2H), 6.14 (s, 1H), 4.96 (s, 4H),
3.92 (s, 6H). 13C NMR (75 MHz, DMSO-D6) δ 183.24, 166.46, 156.47, 149.40, 149.15, 140.15, 134.16,
132.54, 128.68, 128.21, 127.94, 126.30, 126.27, 126.04, 125.68, 122.38, 121.89, 121.23, 113.08, 111.26,
100.94, 70.06, 56.22. ESI-MS calculated. m/z 734.26. found 735.24 (M++1). Anal.Calcd. for: C45H38N2O8:
C, 73.56; H, 5.21; N, 3.81 % found: C, 73.55; H, 5.20; N, 3.84 %.
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2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N,N-
diethylacetamide) (4f): Yellow solid. Yield: 65.0%. Mp: 198-200 °C. IR (KBr Disc) cm-1: 2967, 2924,
1
2851, 1683, 1652, 1029, 966. H NMR (300 MHz, CDCl3) δ 7.59 (d, J = 15.7 Hz, 2H), 7.12 – 7.08 (m,
4H), 6.93 (d, J = 8.1 Hz, 2H), 6.50 (d, J = 15.8 Hz, 2H), 5.83 (s, 1H), 4.80 (s, 4H), 3.93 (s, 6H), 3.44 –
3.38 (m, 8H), 1.18 (t, J = 6.9 Hz, 6H), 1.15 (t, J = 6.9 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 183.38,
166.83, 149.90, 149.82, 140.38, 129.39, 122.75, 122.36, 114.14, 111.05, 101.34, 68.64, 56.14, 41.71,
40.53, 14.37, 12.91. ESI-MS calculated. m/z 594.29. found 595.46 (M++1). Anal. Calcd. for: C33H42N2O8:
C, 66.65; H, 7.12; N, 4.71 % found: C, 66.61; H, 7.10; N, 4.74 %.
2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N-methyl-N-
phenylacetamide) (4g): Yellow solid. Yield: 69.5%. Mp: 90-92 °C. IR (KBr Disc) cm-1: 2958, 2923, 2854,
1
1680, 1623, 1045, 965. H NMR (300 MHz, CDCl3) δ 7.58 – 7.34 (m, 10H), 7.07 - 7.04 (m, 4H), 7.02 –
6.97 (m, 2H), 6.69 (d, J = 8.4 Hz, 2H), 6.47 (d, J = 15.8 Hz, 2H), 5.81 (s, 1H), 4.52 (s, 4H), 3.88 (s, 6H),
3.32 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 183.90, 167.47, 150.02, 149.83, 142.29, 140.64, 130.27,
130.18, 128.56, 127.25, 122.54, 113.81, 110.81, 101.07, 67.22, 56.14, 37.74. ESI-MS calculated. m/z
.662.26 found 663.17 (M++1). Anal.Calcd. for: C39H38N2O8: C, 70.68; H, 5.78; N, 4.23 % found: C, 70.70;
H, 5.75; N, 4.25 %.
2,2'-((((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylene))bis(oxy))bis(N-
cyclohexylacetamide) (4h): Yellow solid. Yield: 72.3%. Mp: 96-98 °C. IR (KBr Disc) cm-1: 2961, 2917,
2924, 1662, 1645, 1030, 965. 1H NMR (300 MHz, CDCl3) δ 7.61 (d, J = 15.9 Hz, 2H), 7.16 (d, J = 8.2 Hz,
2H), 7.10 (s, 2H), 6.91 (d, J = 8.2 Hz, 2H), 6.53 (d, J = 15.8 Hz, 2H), 5.84 (s, 1H), 4.55 (s, 4H), 3.95 (s,
6H), 2.36 – 2.28 (m, 2H), 2.08 – 2.06 (m, 4H), 1.83 – 1.79 (m, 4H), 1.56 – 1.41 (m, 8H), 1.38 – 1.19 (m,
4H). 13C NMR (75 MHz, CDCl3) δ 183.26, 167.23, 150.08, 149.19, 140.08, 130.29, 123.30, 122.20,
115.47, 111.17, 101.49, 69.63, 56.05, 47.91, 41.10, 33.02, 24.74. ESI-MS calculated. m/z 646.33. found
645.58 (M--1). Anal.Calcd. for: C37H46N2O8: C, 68.71; H, 7.17; N, 4.33 % found: C, 68.70; H, 7.16; N,
4.36 %.
32. DPPH radical scavenging activity: The radical scavenging activity of compound can be determined on the
basis of the capacity to scavenge stable 1, 1-diphenyl 2-pieryl hydrazyl (DPPH) radical. The absorbance of
DPPH radical is 517 nm in the UV-visible spectrum. The scavenging capacity measured by this method,
decrease in the absorbance of DPPH radical, because of the formation of stable DPPH molecule. Curcumin
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and ascorbic acid was used as standards. Experiments were repeated as twice. The different concentrations
of the compounds are 10 µg/mL, 20 µg/mL, 30 µg/mL, 40 µg/mL and 50 µg/mL. The stock solution of
DPPH (0.1 mmol) in DMSO has been prepared. 2 mL of the stock solution was added to the 2 mL of
DMSO and the absorbance has recorded at 517 nm. From the all sample solution, 1 mL was diluted to 2 mL
of DMSO and 2 mL of stock solution of DPPH was added and then the absorbance was recorded at 517 nm.
The percentage inhibition has been calculated from the blank and tested solution. (Fig 5. Evidently displays
the scavenging of DPPH radical. The colour of DPPH radical absolutely vanishes with the synthetic
compounds). % Inhibition = [(blank-test)/blank] x 100. From the various concentrations of % inhibition, the
IC50 values are calculated.
33. H2O2 scavenging activity: 2.0 mM solution of H2O2 was prepared in phosphate buffer (0.2 M, PH = 7.4).
The different concentrations of synthesized compounds, curcumin and ascorbic acid were (10 µg/mL, 20
µg/mL, 30 µg/mL, 40 µg/mL and 50 µg/mL) added to the hydrogen peroxide solution (0.6 mL) in phosphate
buffer. The total solution is made up to 4 mL with phosphate buffer. The same solution without the
compound was taken as a negative control. The absorption of hydrogen peroxide recorded at 230 nm and
the phosphate buffer was taken as blank. % Inhibition = [(blank-test)/blank] x 100. From the various
concentrations of % inhibition, the IC50 values are calculated.
34. Anti-inflammatory activity: The synthesized compounds were screened for anti-inflammatory activity by
using inhibition of albumin denaturation technique with minor modification. The standard drug and
synthesized compounds were dissolved in minimum quantity of Dimethyl formamide (DMF) and diluted
with phosphate buffer (0.2 M, PH 7.4). Final concentration of DMF in all solution was less than 2.5%. Test
Solution (4 mL) containing different concentrations of drug was mixed with 1 mL of 1 mM albumin solution
in phosphate buffer and incubated at 37 °C in incubator for 15 min. Denaturation was induced by keeping
the reaction mixture at 70°C in water bath for 15 min. After cooling, the turbidity was measured at 660 nm.
Percentage of Inhibition of denaturation was calculated from control where no drug was added. The
diclofenac sodium and curcumin were used as standard drug. The percentage inhibition of denaturation was
calculated by using following formula. % of Inhibition = 100 x (At - Ac) / At. At = O.D. of test solution. Ac
= O.D. of control.
35. Cell culture: Human lung cancer A549 cells were obtained from NCCS Pune, India. Cells were incubated
with MEM (Hi Media Laboratories) supplemented with 10% Fetal Bovine Serum, Streptomycin (100 U/ml)
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and Penicillin (100U/ml). Cell was maintained in a humidified atmosphere at 37 °C incubator with 5% of
CO2. For treating cells Curcumin bisacetamide were added in DMSO at concentration of 2 mg/ mL and
followed by filter sterilization. Then the solution was diluted in growth medium at different concentration 1
µg/mL, 10 µg/mL, 50 µg/mL, 100 µg/mL and 500 µg/mL. Cytotoxicity: The Human lung cells (A549) were
seed in 96 well plates in different concentration 1, 10, 50, 100, 500 µg/mL and incubated for 24 hrs and each
compound were diluted in 0.1% of DMSO (control). 20 µL of MTT (3-[4,5-dimethylthiozol-2-yl]-2, 5-
diphenyltetrazolium bromide) (5mg/mL in phosphate buffer pH = 7.4) was added in each well. After
incubation 4 hrs media was carefully removed and purple formazan precipitate was dissolved in 100 µL/well
DMSO and kept in incubator for 15 min in dark. Estimation of formazan product was performed at 545 nm
in a microplate reader. The assay was performed in triplicate. The data was plotted against the drug
concentration (vs) Cytotoxicity.
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Synthesis and biological evaluation of new symmetric curcumin derivatives
Rajendran Sribalan, Maruthan Kirubavathi, Govindharaj Banuppriya, Vediappen Padmini*
Department of Organic Chemistry, Madurai Kamaraj University, Madurai-625 021, Tamil Nadu, India.
*Corresponding author e-mail: padimini_tamilenthi@yahoo.co.in
Graphical abstract
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