Structure activity relationship, cytotoxicity and evaluation of antioxidant activity of curcumin derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.12.013

Series of curcumin derivatives/analogues were designed and efficient method for synthesis thereof is described. All the synthesized compounds have been screened for their cytotoxicity and evaluated their antioxidant activity. Cytotoxicity effect has been evaluated against three cell lines Hep-G2, HCT-116 and QG-56 by MTT assay method. Structure activity relationship has revealed that particularly, compound 3c, (IC₅₀ value 6.25 μM) has shown better cytotoxicity effect against three cell lines. According to results of SAR study, it was found that 4H-pyrimido[2,1-b]benzothiazole derivatives (2e and 2f), pyrazoles (3a, 3b, 3c and 3d) benzylidenes (4d) exhibited better antioxidant activity than curcumin. A correlation of structure and activities relationship of these compounds with respect to drug score profiles and other physico-chemical properties of drugs are described and verified experimentally.

Full text of DOI:10.1016/j.bmcl.2015.12.013

Accepted Manuscript
Structure activity relationship, cytotoxicity and evaluation of antioxidant activ-
ity of curcumin derivatives
Pramod K. Sahu, Praveen K. Sahu, Puran L. Sahu, Dau D. Agarwal
PII:
S0960-894X(15)30326-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.013
BMCL 23374
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 June 2015
16 November 2015
4 December 2015
Please cite this article as: Sahu, P.K., Sahu, P.K., Sahu, P.L., Agarwal, D.D., Structure activity relationship,
cytotoxicity and evaluation of antioxidant activity of curcumin derivatives, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.013
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1
Graphical abstract
R1
O
O
O
OCH3
N
HO
OH
S
N
OH
OCH3
OCH3
R2
OCH3
(4a-4d)
R2 = 4-OH-3-OCH3
Derivatives (4a-4d)
OH
(2a-2h)
(4d)
R1 = 4-OH-3-OCH3, 4-OH,
R1 = 4-OH
( )
(2e, 2f)
2f
Antioxidant activity
Cytotoxicity activity
Activity between
50.8-90.2%
IC50 value 12.5 µM/mL
Antioxidant activity
Cytotoxicity activity
IC50 value 12.5 µM/mL
Activity between
46.6-90.2%
R3
N
N
HO
OH
OCH3
OCH3
(3a-3d)
R3 = -COC5H4N
R3 = -CSNH2, -COC5H4N
(3c)
(
)
3b, 3c
Cytotoxicity activity
Antioxidant activity
IC50 value 6.25 µM/mL
Activity between
57.7-84.4%
2
3
4
5
1

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Structure activity relationship, cytotoxicity and evaluation of antioxidant
activity of curcumin derivatives
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Pramod K. Sahu*a,b, Praveen K. Sahub, Puran L. Sahu,c Dau D. Agarwala,b
aSchool of Studies in Chemistry, Jiwaji University, Gwalior-474011, Madhya Pradesh, India
bDepartment of Industrial Chemistry, Jiwaji University, Gwalior-474011, Madhya Pradesh, India
cIndian Pharmacopoeia Commission Ministry of Health and Family Welfare, Sector-23, Raj Nagar, Ghaziabad-
201002, Government of India.
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*To whom correspondence should be addressed; Email: sahu.chemistry@gmail.com, researchdata6@gmail.com (Pramod
K. Sahu)
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Abstract
Series of curcumin derivatives/analogues were designed and efficient method for
synthesis thereof is described. All the synthesized compounds have been screened for their
cytotoxicity and evaluated their antioxidant activity. Cytotoxicity effect has been evaluated
against three cell lines Hep-G2, HCT-116 and QG-56 by MTT assay method. Structure activity
relationship has revealed that particularly, compound 3c, (IC50 value 6.25 µM) has shown better
cytotoxicity effect against three cell lines. According to results of SAR study, it was found that
4H-pyrimido[2,1-b]benzothiazole derivatives (2e and 2f), pyrazoles (3a, 3b, 3c and 3d)
benzylidenes (4d) exhibited better antioxidant activity than curcumin. A correlation of structure
and activities relationship of these compounds with respect to drug score profiles and other
physico-chemical properties of drugs are described and verified experimentally.
Keywords: Antioxidant Activity, Cytotoxicity, Structure-activity Relationship, Curcumin
Derivatives/Analogues.
Curcuminoids are the major constituents of turmeric (Curcuma longa L.), originated from
India and Southeast Asia. The powdered rhizome of turmeric is widely used as spice and
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coloring agent in food by virtue of its yellowish-orange color and pleasant aroma.1 Yellowish
orange color present in turmeric is chemically 1,6-heptadiene-3,5-dione-1,7-bis(4-hydroxy-3-
methoxy phenyl)-(1E,6E) or curcumin (Figure 1). Curcumin is a naturally occurring
phytochemical which is used for centuries in a variety of pharmaceutical applications.2,3
Curcumin and its derivatives exhibited many interesting biological activity such as antiviral,4
anti-inflammatory,5 antimicrobial,6 antioxidant,7 anti-HIV,8 cancer preventive properties,9, 10 anti-
parkinson,11 anti-Alzheimer’s,12 anti-angiogenesis,13 free radical scavenging activity,14 and
anticancer.15
Figure 1. Structure of curcumin used in experiment
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40
Various curcumin analogs/derivatives have been designed and synthesized in order to
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45
46
47
48
49
50
enhance metabolic stability and antiproliferative activity against human cancer cells.16,
Recently, many structural modification efforts were carried out by looking at the variations on
carbonyl moiety and active methylene group and it was found that some of the active methylene
and carbonyl substituted curcumin derivatives/analogues showed better antioxidant activity than
curcumin.18-26 One of the most important aspects of curcumin is its effectiveness against various
types of cancer with both chemopreventive and chemotherapeutic properties.27, 28 Unfortunately,
the potential utility of curcumin is somewhat limited due to poor bioavailability29 and stability in
physicological media.30 It is believed that the presence of the active methylene group and β-
diketone moiety contributes to the instability of curcumin under physiological conditions, poor
absorption, and fast metabolism.31
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Recently, synthetic modifications on carbonyl and active methylene moiety of curcumin has
been studied intensively in order to develop the molecules with enhanced properties and
stability.32, 33 From these studies it has been shown that compounds synthesized using carbonyl
and active methylene moiety of curcumin have enhanced activity and stability in biological
medium compared to curcumin.31-33 However, pyrimido[2,1-b]benzothiazole and pyrazole
(derived from isoniazide, semicarbazide and thiosemicarbazide) derivatives of curcumin have
not been reported so far for their antioxidant activity and cytotoxicity. In an attempt to better
understand the curcumin pharmacophore and to improve its pharmacodynamic profile, we
designed molecules retaining the E,E-1,7-diarylhepta-1,6-diene-3,5-dione backbone and
synthesized curcumin analogues/derivatives on carbonyl and active methylene moiety of
curcumin. Further, synthesized compounds have been evaluated for their cytotoxicity against
human cancer lines (hepato carcinoma, colon carcinoma and lung carcinoma) using standard
MTT assay method and antioxidant activity by adopting DPPH,34 superoxide35 and nitric oxide
radical36 scavenging activity evaluation.
4H-Pyrimido[2,1-b]benzothiazole derivatives of curcumin (2a-2h) were synthesized with
good yield as outlined in Scheme 1 by condensation of curcumin (5 mmol), aldehydes (5 mmol)
and 2-aminobenzothiazole (5 mmol) in the presence of piperidine using conventional heating
(Table 1). The structures of 4H-pyrimido[2,1-b]benzothiazole derivatives of curcumin were
confirmed by IR, NMR, Mass spectra and elemental analysis.
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Scheme 1. Synthesis of 4H-pyrimido[1,2-b]benzothiazole derivatives of curcumin (2a-2h).
O
O
CHO
N
S
NH2
+
+
HO
OH
OCH3
OCH3
R1
Reflux, 60-65 oC
Piperidine (0.001 mol%)
Methanol as solvent
R1
O
OCH3
N
S
N
OH
OCH3
OH
(2a-2h)
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Methodology for the synthesis of pyrazole derivative (3a-3d), involves reaction of curcumin
o
(5 mmol), and hydrazines (5 mmol) in acetic acid at 60-65 C temperature (Scheme 2, see
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supporting information). Under the above optimized conditions, benzylidene derivatives (4a-4d)
of curcumin were synthesized (Scheme 3) using curcumin (5 mmol) and substituted aromatic
aldehydes (5 mmol); see supporting information.
Scheme 2. Preparation of pyrazoles of curcumin.
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Scheme 3. Synthesis of benzylidene derivatives of curcumin.
86
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Various aromatic aldehydes containing electron-withdrawing and electron-donating
substituents at ortho, meta or para positions show equal ease towards the product formation
(Table 1). There was no significant effect of electron donating and electron withdrawing
88
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90
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92
substituents on yield and reaction time.
Table 1. One-pot synthesis of 4H-pyrimido[2,1-b]benzothiazole, pyrazole and benzyliden derivatives of
curcumin.
Entry
R1, R2, R3
H
Product Time (hrs)/ Yield (%)
M.P.(oC)
97-98
1
2
16/81
16/81
2a
2b
2-OH
155-156
3
4
5
4-Cl
18/89
18/86
18/81
112-113
85-86
2c
2d
2e
4-NO2
4-OH-3-OCH3
80-81
6
4-OH
20/89
20/84
20/85
6/87
101-102
108-109
90-92
2f
7
4-CH3
2g
2h
3a
3b
3c
3d
4a
8
2,6-Cl2
-CONH2
-CSNH2
-COC5H4N
-C6H3(NO2)2
H
9
204-206
100-101
105-106
118-119
119-120
10
11
12
13
7/81
7/83
6/86
12/87
14
15
16
4-N(CH3)2
2-OH
13/86
12/89
14/81
104-106
92-94
4b
4c
4d
4-OH-3-OCH3
98-100
93
94
The in vitro cytotoxicity of the synthesized curcumin derivatives (2a-2h, 3a-3d, 4a-4d)
95
were evaluated by MTT assay method37, 38 using three selected human tumor cell lines Hep-G2,
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HCT-116 and QG-56. Inhibitory activities (IC50) are being presented in µM concentrations of the
synthesized curcumin derivatives as shown in Table 2. As we can see among pyrimido
bezohiazole derivatives of curcumin (2a-2h), derivatives 2c, 2d and 2e showed good activity
with 25 µM IC50 against HCT-116, Hep-G2 and QG-56 respectively. Introduction of methyl
substituent at para position (2g) showed much better activity against two cell lines i.e. HCT-116
and QG-56 with 25 µM IC50. Results of cytotoxicity activity have been demonstrated that
electron withdrawing and electron releasing groups at para position enhanced the activity. As far
as compounds 3c (6.25 µM), 3d (6.25 µM) and 2f (12.5 µM) are concerned, they showed better
activity than curcumin against all of the tested cancer cell lines, indicating a wide anticancer
spectrum. Particularly, cytotoxicity of isoniazide moiety containing pyrazole (3c), has sharply
increased against Hep-G2, HCT-116 (6.25 µM). Anticancer activity of derivatives 3c might be
due to isoniazide moiety because isoniazide itself shown anticancer activity which increased the
overall activity by linking with curcumin molecule, because of derivatives 3c is a hybrid of
isoniazide and curcumin.
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Compound 3d showed very potent activity against Hep-G2 (6.25 µM) is over 8-folds
higher than curcumin (50 µM), while weak activity towards the other cell lines, indicating
selective inhibition effect. However, the positive control, adriamycin demonstrated the IC50 in
the range of <2.5 to 5.0 µM. In other hand, derivatives 3a exhibited four folds cytotoxicity
against HCT-116 and QG-56 than curcumin. As far as other derivatives are concerned, they
showed comparable cytotoxicity activity to curcumin. Interestingly, compound 3c showed potent
activity against both Hep-G2 and HCT-116 cancer cell lines, it may be due to isoniazide moiety
in curcumin derivatives.
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Table 2. Cytotoxicity of curcumin derivatives against carcinoma cell lines (IC50 µM)a
Compound
Cytotoxicity (IC50)
HCT-116c
Hep-G2b
100
100
50
QG-56d
100
50
50
2a
50
2b
25
50
2c
25
50
50
2d
50
100
12.5
25
25
2e
25
12.5
25
2f
50
2g
100
25
50
100
12.5
25
2h
12.5
12.5
6.25
12.5
100
100
50
3a
12.5
6.25
6.25
50
3b
12.5
12.5
50
3c
3d
4a
25
50
4b
100
50
25
4c
50
12.5
100
2.5
4d
Curcumin
Adriamycine
50
50
2.5
5.0
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a IC50, concentration of drug that decreases the cell viability by 50% compared to non-treated control cells.
b Hep-G2: human hepato carcinoma.
c HCT-116: human colon carcinoma.
d QG-56: human lung carcinoma.
e Control drug.
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Structure activity relationship has revealed that pyrazole having isoniazide moiety has
showed better influence on cytotoxicity. Isoniazide derived pyrazole derivative of curcumin 3c
showed the potent activity against all tested cancer cell lines, indicating a wide anticancer
spectrum while other pyrazole derivatives showed selective cytotoxic activity (4-10 folds better
than curcumin). Among benzylidene derivatives of curcumin, only para methoxy derived (4d)
derivative showed much better activity against QG-56 (12.5 µM) in series. Furthermore, para
hydroxyl derivatives (2f, 12.5 µM) of 4H-pyrimido[2,1-b]benzothiazole had the 2-8 folds better
activity than curcumin while active methylene linked compounds (4a-4d, range between 12.5-
100 µM) showed weakest activity. It seem to have no influence on the cytotoxicity, as no
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significant difference of activity was found in benzylidenes derivatives which have not possess
pyrazole moieties in their structure like pyrazole derivatives (3a-3d). Overall structure activity
relationship studies demonstrated that pyrazole moiety is significant for cytotoxicity activity.
Table 3. Antioxidant activity by DPPH. method of compounds synthesized.
DPPH. FRSA%
Compound
50 µM
52.2
47.8
87.2
58.0
86.4
88.2
62.6
60.3
64.1
86.8
89.2
81.2
65.0
83.1
58.2
62.2
50.2
20 µM
42.4
41.2
82.0
52.1
84.2
87.2
60.3
58.1
62.2
82.1
88.1
78.0
62.0
80.8
52.0
60.3
42.2
10 µM 2 µM
33.0
32.2
71.2
42.2
72.3
72.2
49.0
44.5
49.7
68.2
70.2
61.9
51.2
76.6
39.8
44.2
33.2
17.2
19.9
25.5
20.0
35.0
28.6
15.2
18.2
20.0
29.9
31.2
28.3
16.2
22.4
13.3
12.5
6.0
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
4a
4b
4c
4d
Curcumin
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143
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145
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147
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It is believed that curcumin moiety has responsible for antioxidant effect as biological
activity. In order to investigate whether target compounds maintain the antioxidant activity,
synthesized curcumin derivatives were evaluated for their antioxidant activity by DPPH.,
superoxide and nitric oxide radical scavenging activity evaluation methods. The present study
identified the structure activity relationship in curcumin derivatives by DPPH method, which
underscores the important chemical feature for this class of molecules to increased antioxidant
activity. All of the tested compounds showed moderate to strong free radical scavenging activity
(FRSA). From structure activity relationship, it can be seen in Table 3 that antioxidant activity of
compound 3c showed the higher FRS activity (89.2%) against DPPH, all other tested compounds
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have lower percentage of activity as compared to compound 3c. However all the compounds
have higher FRS activity (between 52.2-88.2%) than curcumin (50.2%). It is obvious from
antioxidant activity, thiosemicarbazide (3b) and 2,4-dinitro phenyl hydrazine (3d) derived
pyrazole derivatives also showed better activity with 86.8% and 81.2% respectively.
It may be produced due to combining effect of pyrazole moiety instead of diketone of
curcumin. FRSA of 4H-pyrimido[2,1-b]benzothiazole derivatives could be enhanced by the
introduction of hydroxyl group (derivatives 2c, 87.2%; 2e, 86.4% and 2f, 88.2%) at ortho and
para position. It is due to the involvement of hydroxyl moiety in free radical mechanism.
Literature survey also shows that phenolic hydroxyl group is responsible for antioxidant
activity.39
This result indicates that, in addition to the phenolic hydroxyl group, the adjacent
methoxy (2e) or hydroxyl groups (2b and 2f) is also required for formation of stable phenoxy
radical which is believed to play the critical role in radical scavenging activity of the
curcuminoids.40-42 It is also noticeable that DPPH. scavenging activity of benzylidene derivatives
of curcumin is significantly better than curcumin. All the benzylidenes (4a-4d, range between
58.2%-83.1%) having two phenolic hydroxy groups, and consequently their FRSA were in
magnitude at the same order. There was not most significant effect of aryl moiety of aldehydes
used.
In nitric oxide radical scavenging assay, curcumin derivatives with 4-methoxy-3-hydroxy
moieties (2e, 35-86.4% and 4d, 80.4%) and hydroxyl derived (2b 90.2 % and 2f, 89.2%) showed
better activity due to involvement of electron donating substituents as methoxy and hydroxyl
groups in FRSA activity, whereas derivatives have not hydroxyl moiety as substituent failed to
reduced the amount of FRSA comparatively. Form table 4 and 5, it is interestingly that curcumin
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derivatives (3a, 3b and 3c) also showed the higher scavenging ability as compare to curcumin
due to participation of pyrazole moieties in mechanism of FRSA. Among all derivatives, six
derivative showed promising effect in nitric oxide scavenging property which follow the order,
2f>3b>2e>3c>3a>4d. The structure-activity relationship presented above indicates that the
phenolic hydroxyl group is prerequisite, but alone insufficient, for radical-scavenging effect of
the curcuminoids.
Table 4. Antioxidant activity of compounds (2a-4d) synthesized and curcumin
NO. FRSA%
Compound
50 µM
48.0
90.2
52.2
50.0
86.4
89.2
47.2
55.5
88.8
90.2
82.2
50.8
65.0
57.7
70.2
80.4
59.9
20 µM
38.2
78.8
36.6
33.3
84.2
84.2
35.5
38.3
72.6
76.1
74.2
33.0
49.2
36.6
52.2
72.5
36.5
10 µM 2 µM
21.2
62.2
29.9
21.0
72.3
70.8
20.2
21.2
49.7
52.5
50.2
20.8
21.2
22.2
38.0
38.2
22.2
11.2
25.5
16.8
14.2
35.0
26.6
10.2
17.5
29.9
32.3
31.2
15.5
16.2
12.8
24.2
28.8
12.2
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
4a
4b
4c
4d
Curcumin
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180
181
182
183
184
185
The results clearly revealed that the hydroxyl derivatives and pyrazole moieties enhanced
the activity by involvement in the mechanism of scavenging activity. Therefore it could be
inferred that 4H-pyrimido[1,2-b]benzothiazole and pyrazole ring structure have considerable role
in enhancing the activities. The superoxide scavenging capacity decreases in the order
3c>3b>3a>2f>2e>4c>4a>4d>2c>2b>4b>3d>2a>2d>2g>2h. The above mentioned structure-
activity relationship studies revealed that derivatives possess hydroxyl and electron donating
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groups (3c, 3b, 3a, 2f, 2e and 4c) demonstrated considerable superoxide radical scavenging and
antioxidant capacity. In comparison with curcumin, the FRSA of the derivatives/analogues are
much higher. This is probably due to the introduction of the substituent which is an electron
donating group and may subsequently enhance the free radical-capturing ability of the phenolic
hydroxy group.
Table 5. Antioxidant activity of curcumin derivatives (2a-4d) by superoxide radical method
Compound
FRSA%
20 µM
32.2
48.2
36.6
32.0
76.6
78.0
35.5
32.2
49.0
78.8
76.6
33.0
55.5
52.9
71.9
44.9
30.2
50 µM
49.8
68.8
65.2
50.0
89.2
88.9
56.6
46.6
62.9
90.0
89.2
58.9
70.8
66.1
84.4
69.9
42.2
10 µM
20.2
31.2
29.9
21.0
65.5
62.3
21.2
19.9
30.2
48.8
50.2
19.9
30.8
34.2
50.2
31.2
21.2
2 µM
11.2
16.6
17.0
14.8
35.0
33.5
13.2
12.2
20.2
30.2
32.2
14.9
20.8
19.9
34.0
14.9
13.9
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
4a
4b
4c
4d
Curcumin
192
193
194
195
196
197
198
199
Above structure activity relationship studies also revealed that isoniazide derivative 3c
showed the better FRSA over other derivatives in series. Derivatives synthesized using carbonyl
(2a-2h) moiety of curcumin, hydroxyl derivatives (2e) and (2f) enhanced the FRSA activity due
to involvement of hydroxyl moiety in FRSA mechanism. Curcumin derivative derived using
active methylene moiety (4a-4d) did not change the nature of radical-scavenging activity. In
comparison with curcumin, the FRSA of curcumin derivative/analogues is much higher. This is
probably due to introduction of additional moieties as pyrazole and hydroxyl derived pyrimido
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205
206
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209
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benzothiazole in curcumin molecule which may be enhancing the free radical-capturing ability of
the phenolic hydroxyl group. Structure-activity relationship presented above indicates that the
phenolic hydroxyl group is prerequisite, but alone insufficient for radical scavenging activity
effect of the curcuminoids.
Physico-chemical properties of synthesized compounds has been calculated and showed
in table 6. The method is very robust and is able to process practically all organic molecules.
Molecular Polar Surface Area TPSA is calculated based on the methodology previously
published.43 TPSA has been shown to be a very good descriptor characterizing drug absorption,
including intestinal absorption, bioavailability and blood–brain barrier penetration. Prediction
results of compounds 2a-2h, 3a-3d and 4a-4d molecular properties (TPSA, GPCR ligand and
ICM) are valued (Table 6).
From the data evaluated in Table 6 indicates that, all structures are supposed to be non
mutagenic when run through the mutagenicity assessment system and as far as irritating and
reproductive effects are concerned, all the compounds are at low risk comparable (except 3d and
4b). The clogP value of a compound, which is the logarithm of its partition coefficient between
n-octanol and water, is a well-established measure of the compound’s hydrophilicity. Low
hydrophilicity and therefore high cLogP values may cause poor absorption or permeation. It has
been shown for compounds to have a reasonable probability of being well absorb their cLogP
value must not be greater than 5.0.
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220
221
222
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Table 6. Physico-chemical properties and drug score of synthesized curcumin derivatives
Physico-chemical properties
TPSA O/NH VIOL Volume
Drug-score
Compound
CLP
5.89
5.89
6.50
5.76
5.48
5.59
6.20
7.11
3.09
3.94
4.14
4.32
4.59
4.59
4.29
4.19
2.97
S
DL
4.85
4.53
6.03
-5.18
4.47
5.15
3.52
5.01
1.13
0.30
1.70
-10.3
1.27
-1.61
2.50
2.21
-3.95
DS
93
113
93
2
3
2
2
3
3
2
2
4
4
2
2
2
2
3
3
2
2
2
2
2
2
2
2
2
0
0
0
3
0
0
0
1
0
516
524
529
539
549
524
532
543
360
369
416
448
414
460
422
447
332
-7.61
-7.31
-8.34
-8.07
-7.33
-7.31
-7.95
-9.08
-4.87
-3.25
-5.84
-6.04
-5.02
-5.05
-4.72
-4.74
-3.62
0.22
0.22
0.18
0.10
0.22
0.23
0.20
0.16
0.55
0.54
0.41
0.04
0.43
0.09
0.50
0.47
0.39
2a
2b
2c
139
123
113
93
2d
2e
2f
2g
93
2h
120
103
107
168
93
3a
3b
3c
3d
4a
96
4b
4c
113
122
93
4d
Curcumin
224
225
TPSA: Total polar surface area, O/NH: O---HN interraction, VIOL: number of violation;
CLP: cLogP; Log P calculated by Molinspiration, S: Solubility, DL: Drug likness, DS: Drug-Score.
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227
On this basis, all the series of compounds 3a–4d is having clogP values under the
acceptable criteria should be active. The geometrical parameter and the aqueous solubility of a
compound significantly affect its absorption, distribution characteristics and bioactivity.
Typically, a low solubility goes along with a bad absorption and therefore the general aim is to
avoid poorly soluble compounds. Further, the Table 6 shows drug-likeness of compounds 2a-4d
is 0.10-0.23; 0.04-0.55 and 0.09-0.50 respectively for series 2a-2h, 3a-3d and 4a-4d. We have
calculated overall drug-score (DS) for the compounds 2a-4d and compared with that of curcumin
used. The DS combines drug-likeness and clogP in one handy value that may be used to judge
228
229
230
231
232
233
234
235
the compound’s overall potential to qualify for a drug. The reported compounds showed six
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236
237
238
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242
compounds have good DS but the rest of series have low to moderate DS as compared with
curcumin.
All statistical analysis was performed using graph pad prism 6.0 version. All the
experiments were conducted in triplicates and the results were calculated as mean standard
deviation (SD) in this study. P-values less than 0.05 were considered to be statistically
significant (Figure 2).
Figure 2. Data presented are the means SD of results from three independent experiments
243
244
15

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In conclusion, we have designed and synthesized 4H-pyrimido[2,1-b]benzothiazole (2a-2h),
pyrazole (3a-3d) and benzylidene (4a-4d) derivatives of curcumin. The results of cytotoxic
activity by MTT assay exhibited that compounds 3c (6.25 µM) and 3d (6.25 µM) showed much
better activity against all tested three human cancer cell lines. In vitro antioxidant activity results
revealed all of the target compounds have higher FRSA than curcumin toward DPPH, superoxide
and nitric oxide radical. In addition, hydroxyl substituted pyrimido benzothiazole derivatives (2e,
and 2f) and pyrazoles (3b and 3c) exhibited significant antioxidant activity due to its
involvement in FRSA. Present study showed curcumin derivatives possess electron donating
group, enhanced the free radical scavenging activity due to involvement of electron in free
radical capturing ability of molecules by phenolic hydroxyl group. The compounds showed
suitable drug like properties and are expected to present good bioavailability profile. Thus, from
the data obtained from virtual and practical screening, it is concluded that the compounds were
varied to possess a broad range of lipophilic character, revealed by Log P values. These
observations may promote further development of our research in this field and activity make
these curcumin derivatives/analogues as promising antioxidant and anti-cancer drug candidates.
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Acknowledgements
We gratefully acknowledge to Chandigarh and SAIF Punjab University, Chandigarh for
spectral analytical data.
Supplementary data: Supplementary data associated with this article can be found, in the online
version.
References and notes
1. Wichitnithad, W.; Jongaroonngamsang, N.; Pummuangura, S.; Rojsitthisak, P. Phyto
Chem. Anal. 2009, 40, 314.
2. Chattopadhyay, I.; Biswas, K.; Bandyopdhyay, U.; Banerjee, R. K. Curr. Sci. 2004, 87,
44.
3. Chopra, R. N.; Chopra, J. C.; Handa, K. L.; Kapur, L. D. Indigenous Drugs of India,
second ed. Dhur, Calcutta, 1958.
4. Abraham, S. K.; Sharma, L.; Keshvan, P. C. Mutat. Res. 1993, 303, 109.
5. Jayaprakash, G. K.; Rao, L. J.; Gakariah, K. K. Food Chem. 2006, 98, 720.
6. Han, S.; Yang, Y. Dyes and Pigment 2005, 64, 157.
7. Sharma, O. P. Biochem. Biopharmacol. 1976, 25, 1811.
8. Mazumdar A.; Neamate, N.; Sunder, S.; Sehulz, J.; Eich, H. E.; Pommier, Y. J. Med.
Chem. 1997, 40, 3057.
9. Adams, B. K.; Ferstl, E. M.; Davis, M. C.; Herold, M.; Kustkaya, S.; Camalier, R. F.;
Hollingshea, M. G.; Kaur, G.; Sausville, E. A.; Rickles, F. R.; Snyder, J. P.; Liottr, D. C.;
Shoji, M. Bioorg. Med. Chem. 2004, 12, 3871.
17

283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
10. Ohtsu, H.; Xiao, Z. Y.; Ishida, J.; Nagai, M.; Wang, H. K.; Itokawa, H.; Su, C. Y.; Shih,
C.; Chiang, T. Y.; Chang, E.; Lee, Y. F.; Tsai, M. Y.; Chang, C. S.; Lee, K. H. J. Med.
Chem. 2002, 45, 5037.
11. Wang, M. S.; Boddapati, S.; Emadi, S.; Sierks, M. R. BMC Neurosci. 2010, 11, 57.
12. Narlawar, R.; Pichardt, S.; Leuchtenberger, K.; Baumann, S.; Krause, T.; Dryks, S.;
Weggen, E.; Mandelkow, B.; Schmidt, B. Chem. Med. Chem. 2008, 3, 165.
13. Folkman, J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 398.
14. Kim, M. K.; Jeong, W.; Kang, J.; Chong, Y. Bioorg. Med. Chem. 2011, 19, 3793.
15. Ku, B.; Zhou, W.; Yu, F.; Yao, H.; Yao, G. U. S. Patent 2011, 018,3945.
16. Aiello, E.; Aiello, S.; Mingoia, F.; Bacchi, A.; Pelizzi, G.; Musiu, C.; Setzu, M. G.; Pani,
A.; Colla, P. L.; Marongiu, M. E. Bioorg. Med. Chem. 2000, 8, 2719.
17. El-Gaby, M. S. A.; Atalla, A. A.; Gaber, A. M.; Abd Al- Wahab, K. A. II Farmaco 2000,
55, 596.
18. Kim, M. K.; Jeong, W.; Kang, J.; Chong, Y. Bioorg. Med. Chem. 2011, 19, 3793.
19. Weber, W. M.; Hunsaker, L. A.; Abcouwer, S. F.; Decka, L. M.; Jagt, D. L. V. Med.
Chem. 2005, 13, 3811.
20. Dutta, S.; Murugkar, A.; Gandhe, N.; Padhye, S. Metal Based Drugs 2001, 8, 183.
21. Bhullar, K. S.; Jha, A.; Youssef, D.; Rupasinghe, H. P. V. Molecules 2013, 18, 5389.
22. Chakraborti, S.; Dhar, G.; Dwivedi, V.; Das, A.; Poddar, A.; Chakraborti, G.; Basu, G.;
Chakrabarti, P.; Surolia, A.; Bhattacharyya, B. Biochemistry, 2013, 52, 7449.
23. Das, J.; Pany, S.; Panchal, S.; Majhi, A.; Rahman, G. M. Bioorg. Med. Chem. 2011, 19,
6196.
18

305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
24. Claramunt, R. M.; Bouissane, L.; Cabildo, M. P.; Cornago, M. P.; Elguero, J.; Radziwon,
A.; Medina, C. Bioorg. Med. Chem. 2009, 17, 1290.
25. Sahu, P. K.; Sahu, P. K.; Gupta, S. K.; Thavaselvam, D.; Agarwal, D. D. Eur. J. Med.
Chem. 2012, 54, 366.
26. Endo, H.; Nikaido, Y.; Nakadate, M.; Ise, S.; Konno, H. Bioorg. Med. Chem. Lett. 2014,
24, 5621.
27. Shishodia, S.; Chaturvedi, M. M.; Aggarwal, B. B. Curr. Prob. Cancer 2007, 31, 243.
28. Duvoix, A.; Blasius, R.; Delhalle, S.; Schnekenburger, M.; Morceau, F.; Henry, E.;
Dicato, M.; Diederich, M. Cancer Lett. 2005, 223, 181.
29. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Mol. Pharm. 2007, 4,
807.
30. Cheng, A. L.; Hsu, C. H.; Lin, J. K.; Hsu, M. M.; Ho, Y. F.; Shen, T. S.; Ko, J. Y.; Lin, J.
T.; Lin, B. R.; Wu, M. S.; Yu, H. S.; Jee, S. H.; Chen, G. S.; Chen, T.-M.; Chen, C. A.;
Lai, M. K.; Pu, Y. S.; Pan, M. H.; Wang, Y.-J.; Tsai, C. C.; Hsieh, C. Y. Anticancer Res.
2001, 21, 2895.
31. Sardjiman, S. S.; Reksohadiprodjo, M. S.; Hakim, L.; van der Goot, H.; Timmerman, H.
Eur. J. Med. Chem. 1997, 32, 625.
32. Fang, L.; Gou, S.; Liu, X.; Cao, F.; Cheng, L. Bioorg. Med. Chem. Lett. 2014, 24, 40.
33. Fang, X.; Fang, L.; Gou, S.; Cheng, L. Bioorg. Med. Chem. Lett. 2013, 5, 1297.
34. George, S.; Parameswaran, M.; Chakraborty, A. R.; Ravi, T. K. Acta. Pharm. 2008, 58,
119.
35. Meir, S.; Kanner, J.; Akiri, B.; Hadas, S. P. J. Agric. Food. Chem. 1995, 43, 1813.
19

327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
36. Mirkov, S. M.; Djordjevic, A. N.; Andric, N. L.; Andric, S. A.; Kostic, T. S.;
Bogdanovic, G. M.; Vojinovic-Miloradov, M. B.; Kovacevic, R. Z. Nitric Oxide 2004,
11, 201.
37. Mosman, T. J. Immunolog. Methods 1983, 65, 55.
38. Heilmann, J.; Wasescha, M. R.; Schmidt, T. J. Bioorg. Med. Chem. 2001, 9, 2189.
39. Priyadarsini, K. I.; Maity, D. K.; Naik, G. H.; Kumar, M. S.; Unnikrishnan, M. K.; Satav,
J. G.; Mohan, H. Free Rad. Biol. Med. 2003, 35, 475.
40. Barclay, L. R. C.; Vinqvist, M. R. Org. Lett. 2000, 2, 2841.
41. Sreejavan, N.; Rao, M. N. Prog. Drug Res. 1996, 46, 169.
42. Portes, E.; Gardrat, C.; Castellan, A. Tetrahedron 2007, 63, 9092.
43. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
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Table Captions
Table 1. One-pot synthesis of 4H-pyrimido[2,1-b]benzothiazole, pyrazoles and benzylidene derivatives
of curcumin.
Table 2. Cytotoxicity of curcumin derivatives against carcinoma cell lines (IC50 µM).
Table 3. Antioxidant activity by DPPH. method of compounds synthesized.
Table 4. Antioxidant activity of compound (2a-4d) synthesized and curcumin.
Table 5. Antioxidant activity of curcumin derivatives (2a-4d) by superoxide radical method.
Table 6. Physico-chemical properties and drug score of synthesized curcumin derivatives
Figure 1. Structure of curcumin used in experiment
Figure 2. Data presented are the means SD of results from three independent experiments
Scheme Captions
Scheme 1. Synthesis of 4H-pyrimido[1,2-b]benzothiazole derivatives of curcumin (2a-2h).
Scheme 2. Preparation of pyrazoles of curcumin.
Scheme 3. Synthesis of benzylidene derivatives of curcumin.
21