Design, structure activity relationship, cytotoxicity and evaluation of antioxidant activity of curcumin derivatives/analogues

Article abstract of DOI:10.1016/j.ejmech.2016.05.037

New fourteen 3,4-dihydropyrimidine derivatives/analogues of curcumin (2a-2n) were designed, synthesized and biologically evaluated for their cytotoxicity and antioxidant activity. Cytotoxicity effect has been evaluated against three cell lines HeLa, HCT-116 and QG-56 by MTT assay method. From SAR study, it has been revealed that particularly, compound 2e and 2j (IC₅₀ value 12.5 μM) have shown better cytotoxicity effect against three cell lines. According to results of SAR study, it was found that 3,4-dihydropyrimidines of curcumin, 2c, 2d, 2j and 2n 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. Therefore, we conclude that physico-chemical analyses may prove structural features of curcumin analogues with their promising combined cytotoxicity/antioxidant activity and it is also concluded from virtual and practical screening that the compounds were varied to possess a broad range of lipophilic character, revealed by Log P values.

Full text of DOI:10.1016/j.ejmech.2016.05.037

Accepted Manuscript
Design, structure activity relationship, cytotoxicity and evaluation of antioxidant
activity of curcumin derivatives/analogues
Pramod K. Sahu
PII:
S0223-5234(16)30435-4
10.1016/j.ejmech.2016.05.037
EJMECH 8629
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 March 2016
Revised Date: 17 May 2016
Accepted Date: 19 May 2016
Please cite this article as: P.K. Sahu, Design, structure activity relationship, cytotoxicity and evaluation of
antioxidant activity of curcumin derivatives/analogues, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.05.037.
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Design, structure activity relationship, cytotoxicity and evaluation of
antioxidant activity of curcumin derivatives/analogues
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Pramod K. Sahu*a,b
aSchool of Studies in Chemistry, Jiwaji University, Gwalior-474011, Madhya Pradesh, India
bDepartment of Industrial Chemistry, Jiwaji University, Gwalior-474011, Madhya Pradesh, India .
*To whom correspondence should be addressed; Email: sahu.chemistry@gmail.com, researchdata6@gmail.com (Pramod
K. Sahu)
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Abstract
New fourteen 3,4-dihydropyrimidine derivatives/analogues of curcumin (2a-2n) were
designed, synthesized and biologically evaluated for their cytotoxicity and antioxidant activity.
Cytotoxicity effect has been evaluated against three cell lines HeLa, HCT-116 and QG-56 by
MTT assay method. From SAR study, it has been revealed that particularly, compound 2e and 2j
(IC50 value 12.5 µM) have shown better cytotoxicity effect against three cell lines. According to
results of SAR study, it was found that 3,4-dihydropyrimidines of curcumin, 2c, 2d, 2j and 2n
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. Therefore, we conclude that
physico-chemical analyses may prove structural features of curcumin analogues with their
promising combined cytotoxicity/antioxidant activity and it is also concluded from virtual and
practical screening that the compounds were varied to possess a broad range of lipophilic
character, revealed by Log P values.
Keywords: Structure-activity Relationship, Drug Design, Curcumin Derivatives/Analogues,
Antioxidant Activity, Physico-Chemical Analyses.
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1. Introduction
Curcumin is well-known chemical constituents abundantly found in turmeric (Curcuma
longa L.), is an herbaceous plant of Zingiberaceae family, originated from India and Southeast
Asia. The curcuminoids complex is also referred to as Indian saffron, ukon, yellow root, yellow
ginger, kacha haldi or natural yellow, curcuminoids are present in 3-5% of turmeric. The
powdered rhizome of turmeric is widely used as spice and 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]. Literature survey reveals that curcumin, and its
derivatives have various pharmacological activities such as antiviral [4], anti-inflammatory [5],
antimicrobial [6,7], antioxidant [8-10], anti-HIV [11], cancer preventive properties [12.13], anti-
parkinson [14], anti-Alzheimer’s [15], anti-angiogenesis [16], free radical scavenging activity
[17], and anticancer [18].
Figure 1. Structure of curcumin
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Number of curcumin analogs/derivatives have been designed and synthesized in order to
enhance metabolic stability and antiproliferative activity against human cancer cells [19,20]. By
looking at variation on carbonyl moiety and active methylene group, recently many structural
modification efforts were carried out and it was found that some of the active methylene and
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carbonyl substituted curcumin derivatives/analogues showed better antioxidant activity than
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curcumin [21-29]. One of the most important aspects of curcumin is its effectiveness against
various types of cancer with both chemopreventive and chemotherapeutic properties [30,31].
Unfortunately, due to poor bioavailability [32], and stability in physicological media [33], the
potential utility of curcumin is somewhat limited. 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 [34].
In order to develop the molecules with enhances properties and stability, recently synthetic
modifications on carbonyl and active methylene moiety of curcumin has been studied intensively
[35,36]. It seems from these studies that compounds synthesized using carbonyl and active
methylene moiety of curcumin have enhanced activity and stability in biological medium
compared to curcumin [34-36]. However, 3,4-dihydropyrimidine derivatives of curcumin have
not been reported so far for their antioxidant activity, cytotoxicity, drug scores and physico-
chemical analyses. 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 using standard MTT assay method and antioxidant
activity by adopting DPPH [37], and nitric oxide radical [38] scavenging activity evaluation.
2. Results and discussion
2.1 Chemistry
3,4-Dihydropyrimidine derivatives/analogues of curcumin (2a-2n) were synthesized with
good yield as outlined in Scheme 1 by condensation of curcumin (5 mmol), aldehydes (5 mmol)
and urea/thiourea (5 mmol) in the presence of piperidine using conventional heating (Table 1).
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The structures of 3,4-dihydropyrimidines of curcumin were confirmed by IR, NMR, Mass
spectra and elemental analysis. 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 substituents on yield and reaction time.
Scheme 1. Synthesis of 3,4-dihydropyrimidine derivatives of curcumin (2a-2n).
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Table 1. Synthesis of dihydropyrimidine derivatives/analogues of curcumin
Entry
R
H
4-CH3
X
O
O
Product Time (hrs)/ Yield (%)a
1
2
16/76
16/81
2a
2b
3
4
5
2-OH
4-OH
O
O
O
18/75
18/62
18/55
2c
2d
2e
4-N(CH3)2
6
7
2,6-Cl2
3-NO2
2-Cl
O
O
O
S
20/78
20/72
20/80
16/76
18/78
16/86
16/80
2f
2g
2h
2i
8
9
H
10
11
12
4-CH3
2-OH
4-N(CH3)2
S
S
S
2j
2k
2l
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2,6-Cl2
2-Cl
S
S
18/86
16/87
2m
2n
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90
91
aIsolated yield
2.2 Antioxidant activity
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It is believed that curcumin moiety is responsible for antioxidant effect as biological
activity. To facilitate the explore whether target compounds maintain the antioxidant activity,
synthesized curcumin derivatives were evaluated for their antioxidant activity by DPPH. and
nitric oxide radical scavenging activity evaluation methods. Preliminary evaluation for DPPH
inhibitory activity was conducted at 50 µM test concentration. Four bioactive compounds (2c,
2d, 2l and 2k) were found to exhibit strong DPPH inhibitory activity giving 88.9%, 90.9%,
89.5% and 86.3% respectively. Meanwhile, nitro derived derivative (2g) displayed moderate
inhibitory activity. Rest of compounds showed lower activity. From structure activity
relationship, it can be seen in Table 2 that antioxidant activity of derivatives 2c, 2d and 2k
showed the higher free radical scavenging activity due to attachment of hydroxyl moiety in the
benzene ring which may be involved in free radical mechanism. Literature survey also shows
that phenolic hydroxyl group is responsible for antioxidant activity [39].
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Table 2. Antioxidant activity by DPPH. method
.
Compound
X
DPPH FRSA%
50 µM 20 µM
10 µM
26.4
29.9
73.6
75.0
37.6
19.2
51.2
40.0
26.5
43.2
60.2
72.0
2 µM
15.2
17.2
33.6
25.0
17.9
11.2
17.0
20.2
15.5
15.5
25.0
30.2
O
O
O
O
O
O
O
O
S
40.2
46.0
88.9
90.3
56.0
43.2
71.0
55.5
48.0
62.0
86.3
89.5
38.2
38.2
81.0
84.6
48.0
31.9
66.3
46.0
38.0
51.0
72.3
83.0
2a
2b
2c
2d
2e
2f
2g
2h
2i
S
2j
S
S
2k
2l
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50.2
60.0
50.2
43.2
56.6
42.2
29.0
43.2
33.2
17.7
16.2
6.0
2m
2n
Curcumin
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112
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From structure activity relationship, it is clear that phenolic group is essential for
antioxidant activity. Besides, it has also been found that the substitution at para position as
dimethylammino group (2l) does effect the DPPH activity due to strong effect of electron
donating moiety. However, 2c, 2d and 2k are hydroxyl derived dihydropyrimidone derivatives of
curcumin but 2k showed slightly lower DPPH activity as compared to 2c and 2d derivatives, it
may be due to atomic size of oxygen and sulphur which may be also involved in the activity
because oxygen atom is smaller than sulphur. Along with all derivatives, four 3,4-
dihydropyrimidines of curcumin showed promising effect in DPPH scavenging property which
follow the order; 2d>2c>2l>2k>2g>2j>2n>2e>2h>2m>2i>2b>2f>2a.
Synthesized curcumin dihydropyrimidines have been evaluated for their nitric oxide
(NO) inhibitory activity radical scavenging assay. Nitric oxide is an important mediator involved
in the inflammatory process. It is biosynthesized endogenously from L-arginine, oxygen and
NADPH, and catalyzed by various nitric oxide synthase (NOS) enzymes. Appropriate level of
NO produced is responsible for body’s defense mechanism against abnormalities [40].
Therefore, pharmacological intervention of NO production is a rapid and promising strategy in
the search for new potent drugs against inflammatory related degenerative diseases.
In term of NO inhibitory activity, hydroxyl derived 3,4-dihydropyrimidine drivatives of
curcumin (2c, 2d and 2k) demonstrated excellent activity. In this case, hydroxyl in the ortho and
para position seems to be required. Structure activity relationship also revealed that para
substitution as dimethylamino group (2e and 2l) displayed better activity. It may be strongly
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effect of more electrons density (two electron donating –CH3 groups attached at para position)
which may be involved in radical scavenging activity. SAR studies showed that when aromatic
hydrogen is replaced with electron withdrawing group (2g) on meta position and electron
releasing groups (rest of compounds) on ortho and para position decreases the scavenging
activity. Among all derivatives, five 3,4-dihydropyrimidine derivative of curcumin showed
promising effect in nitric oxide scavenging property which follow the order;
2e>2k>2c>2d>2l>2h>2n>2b>2i>2a>2m>2g>2f>2j
Table 3. Antioxidant activity by NO. method
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Compound
NO FRSA%
X
O
O
O
O
O
O
O
O
S
50 µM 20 µM
10 µM 2 µM
50.2
52.2
88.0
86.2
89.2
44.2
48.0
56.0
50.4
41.0
88.5
84.0
50.0
55.6
59.9
31.0
36.3
80.9
80.1
82.2
33.0
33.0
38.7
36.6
30.0
79.9
72.1
33.9
39.2
36.5
20.0
23.9
71.0
72.0
69.9
20.0
19.9
23.0
22.0
19.9
70.8
59.0
20.2
21.2
22.2
10.5
15.5
29.9
33.3
28.2
11.2
10.2
16.6
16.6
10.2
34.0
32.3
15.0
15.5
12.2
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
Curcumin
S
S
S
S
S
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All result indicates that, in addition to the phenolic hydroxyl groups (2c, 2d and 2k) is
also required for formation of stable phenoxy radical which is believed to play the critical role in
radical scavenging activity of the curcuminoids [41-43]. Above, structure activity relationship
studies also revealed that substitution on meta potion decreased the activity due to strong nature
of nitro as electron withdrawing group and strong releasing group (dimethylamino group) on
para position (2e and 2l) enhanced the scavenging activity of 3,4-dihydropyrimidines of
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curcumin. Structure-activity relationship presented above indicates that the phenolic hydroxyl
group is prerequisite, but alone insufficient for radical scavenging activity effect of the
curcuminoids.
2.3 Cytotoxicity
In vitro cytotoxicity of the synthesized curcumin derivatives (2a-2n) were evaluated by
MTT assay method [44,45] using three selected human tumor cell lines HeLa, HCT-116 and
QG-56. Inhibitory activities (IC50) are being presented in µM concentrations of the synthesized
3,4-dihydropyrimidines of curcumin as shown in Table 4. Structure activity relationship showed
good result from molecule design point of view. As we can see among 3,4-dihydropyrimidines
(2a-2n), derivatives 2b, 2c, 2h, 2m and 2n showed more or less equal cytotoxicity with range of
50-100 µM IC50 against HeLa, HCT-116 and Hep-G2. Introduction of dimethylamino substituent
at para position (2e and 2l) as electron releasing group showed excellent activity against two cell
lines i.e. HCT-116, QG-56 and HeLa, HCT-116 with 12.5 µM IC50 which is eight fold than
curcumin. It may be due to strong electronic effect of two methyl groups attached on aryl ring
which make more activate aryl ring. 3, 4-Dihydropyrimidines 2e and 2l also showed better
activity against HeLa and QG-56 with 25 µM IC50 value which is also four folds of curcumin.
Results of cytotoxicity activity have been demonstrated that electron releasing groups (2b and
2j) at para position good cytotoxicity with 12.5 µM IC50 against HCT-116 and QG-56, eight
folds more active than curcumin. Structure activity relationship also revealed that strong electron
releasing groups at para position drastically enhanced the cytotoxicity against all cell lines. As
far as rest of 3,4-dihydropyrimidines of curcumin are concerned, their activity were near about
curcumin.
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Structure activity relationship demonstrated that atomic size of oxygen (2a-2h) and
sulphur (2i-2n) could not affect the cytotoxicity of all compounds. However, the positive control,
adriamycin demonstrated the IC50 in the range of <2.5 to 5.0 µM. Interestingly, compound 2e
and 2l showed potent activity against cancer cell lines, it may be due to involvement of electron
in activity. However we have synthesized curcumin derivatives using ortho substitution with
electron releasing group (2c, 2d and 2k) but these derivatives didn’t show significant cytotoxicity
effect against cancer cell lines. Overall structure activity relationship studies demonstrated that
electron releasing moiety is significant for cytotoxicity activity.
Table 4. Cytotoxicity of curcumin derivatives/analogues by MTT assay
Compound
Cytotoxicity (IC50)
X
O
O
O
O
O
O
O
O
S
HeLa
100
100
50
25
25
HCT-116
25
QG-56
50
100
100
50
12.5
100
25
100
100
12.5
50
25
100
50
100
2.5
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
12.5
100
100
12.5
50
25
100
100
25
25
25
12.5
100
50
50
2.5
50
50
100
50
100
12.5
50
100
50
5.0
S
S
S
S
S
Curcumina
Adriamycinb
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a Isolated from Curcuma Longa
b Control drug.
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2.4 Physico-chemical analyses
The in silico physico-chemical properties of synthesized dihydropyrimidones of
curcumin has been calculated and showed in table 5. Molecular Polar Surface Area TPSA is
calculated based on the methodology previously published [46] as a sum of fragment
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contribution, O- and N- centred polar fragments are considered. All the tested compounds
displayed TPSA values within the satisfactory range (100-163 Ao).Results clearly indicated that
most of the compounds showed excellent lipophilicity to cross blood–brain barrier. Prediction
results of compounds 2a-2n, molecular properties (TPSA, G protein-coupled receptor ligand and
ion channel modulator) are valued (Table 5).
Table 5. Physico-chemical properties of curcumin derivatives/analogues
Physico-chemical properties[a]
Compd.
MW (g/mole)
TPSA
O/NH
VIOL
ROTB
VOL
CLP
498
512
117
117
4
4
0
1
8
8
445
462
4.31
4.75
2a
2b
2c
514
514
542
567
543
532
515
529
531
558
583
549
368
137
137
120
117
163
121
100
100
120
103
100
100
93
5
5
4
4
4
3
4
4
5
4
4
4
2
1
1
1
2
2
2
1
2
1
1
2
2
0
8
8
9
8
9
8
8
8
8
9
8
8
8
453
453
490
472
462
453
454
470
462
500
481
467
332
4.25
3.83
4.41
5.57
4.24
4.94
4.85
5.30
4.79
5.95
6.11
5.48
2.30
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
Curcumin
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TPSA: Total polar surface area, O/NH: O---HN interraction, VIOL: number of violation; ROTB: Number of rotation; VOL:
volume, CLP: cLogP
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The in silico physiochemical properties of the target compounds (2a-2n) are listed in
Table 5. Synthesized curcumin analogues/derivatives 2a-n showed log P value ranges from 2.30
to 4.94 except compounds 2f, 2l, 2j and 2m. This result clearly indicates that most of the
compounds showed excellent lipophilicity to cross blood brain barrier (BBB) easily. TPSA
(topological polar surface area) is an important descriptor that was shown to correlate well with
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molecular transport through membranes. All the tested compounds (2a-n) displayed TPSA values
within satisfactory range (93–163 A °). Moreover, the number of rotatable bonds (n-ROTB),
molecular weight, hydrogen bond donor/acceptor were also in acceptable range.
A number of important points emerged concerning the electronic and steric factors which
have direct impacts on bioactivity properties. The positive results have recorded, while
encouraging for purposes of new drug design confirm that very likely most of these compounds
could be used as potential antioxidant agents after major modifications. The future flexible O,O/
O,N-pharmacophore site (s) geometric conformation enables us to prepare molecules for multi-
therapeutic materials with high antioxidant activity. 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. On this basis, all
the series of compounds 2a-2n is having clogP values under the acceptable criteria (except
compounds 2f, 2l, 2j, 2m and 2n) 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.
3. Conclusion
In conclusion, we have designed and synthesized a series of 3,4-dihydropyrimidine
derivatives/analogues of curcumin (2a-2n). Cytotoxicity results exhibited that derivatives 2e and
2l (dimethylamino derived) showed much better activity than curcumin. It is due to strong effect
of electron donating nature of two methyl group on para position. Our previous and current
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finding suggested that hydroxyl substituents is a crucial moiety for antioxidant activity of
curcuminoids and might have promising therapeutic potential as antioxidant agents. Results of
antioxidant activity demonstrated that hydroxyl substituted 3,4-dihydropyrimide derivatives of
curcumin (2c, 2d and 2k) exhibited significant antioxidant activity due to its involvement in
FRSA mechanism. Strong electron donating group (2e and 2l), enhanced the free radical
scavenging activity due to involvement of electron in free radical capturing ability of molecules
by phenolic hydroxyl group. It is also concluded from virtual and practical screening 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.
4. Materials and Methods
4.1 Experimental
1
The H NMR spectra were measured by BRUKER AVANCE II 400 NMR spectrometer with
o
1
tretramethylsilane as an internal standard at 20-25 C; data for H-NMR are reported as follow:
chemical shift (ppm), integration, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet and br, broad), coupling constant (Hz). IR spectra were recorded by SHIMADZU; IR
spectrometer of sample dispersed in KBr pellet or nujol and is reported in terms of frequency of
absorption (cm-1). E-Merck pre-coated TLC plates, RANKEM silica gel G for preparative thin-
layer chromatography were used. Melting points were determined in open capillaries and are
uncorrected. Curcumin, urea, thiourea, and aldehydes were purchased from Hi media Laboratory
Ltd., Mumbai, India. All the solvents and reagents used for the synthesis were of analytical
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grade. 2,2-Diphenyl-1-picrylhydrazyl (DPPH.) was procured from Sigma Chemical Co. (St.
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Louis, MO, USA).
4.2 Typical procedure for curcumin derivatives (2a-2n)
A mixture of curcumin (5 mmol), substituted aromatic aldehydes (5 mmol) and urea/thiourea (5
o
mmol) were refluxed at 60-65 C in the presence of piperidine as catalysts in methanol solvent.
The reaction was monitored by TLC. After completion the reaction, mixture was then poured in
cold water and filtered. Product was purified by column chromatography with ethyl acetate and
hexane (3:1). The 3,4-dihydropyrimidine derivatives of curcumin were collected as yellow
crystals.
4.3 Pharmacological activity
4.3.1 Radical scavenging activity
Radical-scavenging activity of synthesized curcumin derivatives was analyzed by the DPPH. and
nitric oxide radical scavenging methods. Individual samples were prepared by dissolving each
compound in DMSO. The samples were assayed at different concentrations, making them up to 1
mL with 100 mM Tris–HCl buffer (pH 7.4), followed by addition of 4 mL of 2,2-diphenyl-1-
picrylhydrazyl (0.1 mM solution in methanol) and mixing of the contents by vigorous shaking. A
control in these experiments was prepared by same protocol except that the compound was not
included. The tubes were incubated in the dark at room temperature for 20 min. The absorbance
was then recorded at 517 nm using DMSO for baseline correction. The experiments were carried
out in triplicates. Free radical scavenging is one of the best known method by which antioxidant
inhibit lipid peroxidation. Antioxidant activities viz. DPPH, and nitric oxide radical scavenging
activity evaluation are standard assays and offer rapid techniques for screening the radical
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scavenging activity (RSA). Radical-scavenging assay (RSA) was evaluated for their antioxidant
behavior in comparison with curcumin and the results are shown in the Table 2 and 3.
4.3.2 Cytotoxicity against cell lines
Cytotoxicity was performed by MTT assay method.44, 45 A 96-well flat bottom tissue
culture plate was seeded with 2 x 103 cells in 0.1 mL of MEM medium supplemented with 10%
FBS and allowed to attach for 24 h. After 24 hrs of incubation, cells were treated with test
compounds to get a concentration of 5, 10, 20, 50 100 and 200 ꢀM/mL incubated for 48 hrs. The
cells in the control group received only the medium containing the 0.2% DMSO. Each treatment
was performed in duplication. After the treatment, drug containing media was removed and
washed with 200 ꢀL of PBS. To each well of the 96 well plate, 100 ꢀL of MTT reagent (Stock: 1
mg/mL in serum free medium) was added and incubated for 4 hour at 37 ºC. After 4 hours of
incubation the plate was inverted on tissue paper to remove the MTT reagent. To solubilize
formazan crystals in the wells, 100 ꢀL of 100% DMSO was added to each well. The optical
density was measured by microtiter plate reader at 590 nm. Compound concentration required to
reduce the viability of mock-infected cells by 50% as determined by MTT method is summarized
in Table 4.
4.3.3 Characterization data
Compound 2a: 4-Phenyl-3,4-dihydropyrimidine-2(1H)-one curcumin
Yellow crystal, mp 206-208 oC, Rf = 0.65 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3510 (N-
Hstr), 2922 (C-Hstr), 2848 (C-Hstr), 1625 (C=Ostr), 1500 (C-Hben), 1427 (C-Nstr), 1382 (C-Hben),
1080-812 (C-Hdef); 1H NMR (400 MHz, DMSO): δH 3.88 (6H, s, OCH3), 5.90 (2H, s, 2-H), 6.52
(2H, d, 5-H, J = 15..64 Hz), 6.87 (2H, d, arom, J = 8.16 Hz), 7.04-7.07 (4H, dd, arom), 7.45 (2H,
d, arom, J = 8.56 Hz), 8.06 (2H, t, arom, J = 13.96 Hz), 8.57 (1H, t, arom, J = 15.64 Hz), 8.92
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(2H, s, NH); 13C NMR (100 MHz, DMSO): 182.75, 148.43, 147.54, 140.24, 129.52, 126.22,
122.52, 120.58, 120.26, 115.68, 115.33, 109.97, 100.70, 55.71; ESI-MS: m/z Calculated for
C29H26N2 O6 498.54 Found [M+2]+ 501.2; C, H and N analyses Calculated for C 69.87, H 5.26,
N 5.62, Found C 69.91, H 5.32, N 5.55.
Compound 2b: 4-(4-Methyl phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
Yellow crystal, mp 154-158 oC, Rf = 0.58 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3562 (N-
Hstr), 1627 (C=Ostr), 1508 (C-Hben), 1429 (C-Nstr), 1280 (C-Hben), 1153 (C-Hdef), 808-962 (C-
Hdef); 1H NMR (400 MHz, CDCl3): δH 2.17 (3H, s, CH3), 3.96 (6H, s, OCH3), 5.78 (2H, s, 2-H),
6.48 (2H, d, 5-H, J = 3.36 Hz), 6.85-6.95 (2H, m, arom), 7.04 (2H, d, arom, J = 1.6 Hz), 7.10-
7.13 (2H, dd, arom, J = 1.76 Hz), 7.26 (1H, s, -CH), 7.44 (1H, d, arom, J = 8.56 Hz), 7.56-7.61
(2H, dd, arom, J = 3.92 Hz), 8.08 (1H, d, arom, J = 2.0 Hz), 8.36 (2H, s, NH); 13C NMR (100
MHz, DMSO): 182.89, 149.11, 147.74, 140.36, 129.80, 126.22, 122.70, 120.79, 115.77, 115.53,
110.63, 100.79, 98.92, 55.47; ESI-MS: m/z Calculated for C30H28N2O6 512.55 Found [M+NH4]+
530.5; C, H and N analyses Calculated for C 70.30, H 5.51, N 5.47, Found C 70.41, H 5.49, N
5.60.
Compound 2c: 4-(2-Hydroxy phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
o
Yellow powder, mp >250 C, Rf = 0.67 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3504 (C-
Nstr), 2941 (C-Hstr), 1627 (C=Ostr), 1508 (C-Hben), 1429 (C-Nstr), 1208 (C-Hben), 813-962 (C-
Hdef); 1H NMR (400 MHz, CDCl3): δH 3.92 (6H, s, OCH3), 5.82 (2H, s, 2-H), 6.45 (2H, d, 5-H, J
13
= 15..68 Hz), 6.64-7.58 (10H, arom, m), 8.38 (2H, s, NH), 9.93 (1H, s, OH); C NMR (100
MHz, DMSO): 182.82, 149.38, 147.73, 147.57, 146.35, 140.33, 129.29, 128.95, 126.20, 122.67,
120.75, 119.76, 115.75, 115.50, 110.53, 110.53, 108.92, 55.47; ESI-MS: m/z Calculated for
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C29H26N2O7 514.58 Found [M]+ 514.4; C, H and N analyses Calculated for C 67.70, H 5.09, N
5.44, Found C 67.79, H 5.17, N 5.41.
Compound 2d: 4-(4-Hydroxy phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
o
Yellow powder, mp 148-150 C, Rf = 0.62 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3321
(OHstr), 3043 (C-Hstr), 2968 (C-Hstr), 1670 (C=Ostr), 1589 (C-Hben), 1516 (C-Nstr), 1330 (C-Hben),
1244 (C-Hdef), 744-975 (C-Hdef); 1H NMR (400 MHz, CDCl3): δH 3.94 (6H, s, OCH3), 5.80 (2H,
s, 2-H), 6.45 (2H, d, 5-H, J = 15..68 Hz), 6.85-6.95 (2H, m, arom),7.04 (2H, d, arom, J = 1.6
Hz), 7.10-7.13 (2H, dd, arom, J = 1.76 Hz), 7.26 (1H, s, -CH), 7.44 (1H, d, arom, J = 8.56 Hz),
7.56-7.61 (2H, dd, arom, J = 3.92 Hz), 8.08 (1H, d, arom, J = 2.0 Hz), 8.38 (2H, s, NH), 10.16
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(1H, s, OH); C NMR (100 MHz, DMSO): 182.84, 149.34, 147.89, 147.66, 146.63, 140.39,
129.43, 128.27, 127.34, 126.07, 122.77, 120.72, 119.81, 115.58, 115.43, 110.66, 109.02, 98.92,
55.47, 13.97; ESI-MS: m/z Calculated for C29H26N2O7 514.58 Found [M]+ 514.5; C, H and N
analyses Calculated for C 67.70, H 5.09, N 5.44, Found C 67.68, H 5.11, N 5.41.
Compound 2e: 4-(4-Dimethyamino phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
o
Yellow powder, mp 130-132 C, Rf = 0.52 (DCM / Toluene 3:2); 1H NMR (400 MHz, CDCl3):
δH 2.52 (6H, s, -N(CH3)2), 3.93 (6H, s, OCH3), 5.79 (2H, s, 2-H), 6.49 (2H, d, 5-H, J = 8.0 Hz),
6.83-6.91 (2H, m, arom),7.05 (2H, d, arom, J = 1.6 Hz), 7.11-7.16 (2H, dd, arom, J = 1.76 Hz),
7.26 (1H, s, -CH), 7.44 (1H, d, arom, J = 8.56 Hz), 7.58-7.60 (2H, dd, arom, J = 3.92 Hz), 8.09
13
(1H, d, arom, J = 2.0 Hz), 8.48 (2H, s, NH); C NMR (100 MHz, DMSO): 181.84, 148.34,
145.89, 145.66, 144.63, 140.30, 128.43, 127.27, 126.34, 125.07, 121.77, 120.72, 118.71, 113.50,
113.40, 109.60, 108.02, 98.92, 55.45, 13.97; ESI-MS: m/z Calculated for C31H31N2O6 541.59
Found [M]+ 541.5; C, H and N analyses Calculated for C 68.75, H 5.77, N 7.76, Found C 68.80,
H 5.81, N 7.69.
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Compound 2f: 4-(2,6-Dichloro phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
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Yellow powder, mp 106-108 C, Rf = 0.62 (DCM / Toluene 3:2); H NMR (400 MHz, DMSO):
δH 3.81 (6H, s, OCH3), 5.91 (2H, s, 2-H), 6.54 (2H, d, 5-H, J = 15..64 Hz), 6.88 (2H, d, arom, J
= 8.16 Hz), 7.09-7.12 (3H, dd, arom, J = 7.4 Hz), 7.49 (2H, d, arom, J = 8.56 Hz), 8.06 (2H, t,
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arom, J = 13.96 Hz), 8.57 (1H, t, arom, J = 15.64 Hz), 8.90 (2H, s, NH); C NMR (100 MHz,
DMSO): 182.70, 148.41, 147.44, 140.40, 129.51, 126.00, 121.20, 120.58, 120.16, 115.38,
115.12, 109.80, 100.50, 55.69; ESI-MS: m/z Calculated for C29H24Cl2N2O6 567.42 Found
[M+2]+ 570; C, H and N analyses Calculated for C 61.39, H 4.26, N 4.94, Found C 61.44, H
4.31, N 5.02.
Compound 2g: 4-(3-Nitro phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
Yellow crystal, mp 160-162 oC, Rf = 0.69 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 1H NMR
(400 MHz, CDCl3): δH 3.87 (6H, s, OCH3), 5.95 (2H, s, 2-H), 6.40 (2H, d, 5-H, J = 15..68 Hz),
6.59-7.89 (11H, m, arom), 9.45 (2H, s, NH); 13C NMR (100 MHz, DMSO): 181.63, 147.91,
146.60, 144.50, 140.42, 139.27, 128.00, 125.90, 121.11, 119.15, 114.70, 114.40, 109.21, 99.70,
54.04, 30.48; ESI-MS: m/z Calculated for C29H25N3O8 543.52 Found [M]+ 543.5; C, H and N
analyses Calculated for C 64.08, H 4.64, N 7.73, Found C 64.10, H 4.68, N 7.78.
Compound 2h: 4-(2-Chloro phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
o
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Yellow powder, mp 144-146 C, Rf = 0.64 (DCM / Toluene 3:2); H NMR (400 MHz, CDCl3):
δH 3.90 (6H, s, OCH3), 5.88 (2H, s, 2-H), 6.40 (2H, d, 5-H, J = 15..68 Hz), 6.82-7.78 (11H, arom,
m), 8.38 (2H, s, NH); 13C NMR (100 MHz, DMSO): 182.80, 149.10, 147.10, 147.17, 146.15,
140.03, 129.59, 128.05, 126.00, 122.07, 120.50, 119.15, 115.50, 115.35, 110.33, 110.03, 108.42,
55.17; ESI-MS: m/z Calculated for C29H25ClN2O6 532.97 Found [M]+ 533; C, H and N analyses
Calculated for C 65.63, H 4.73, N 5.26, Found C 65.57, H 4.79, N 5.31.
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Compound 2i: 4-Phenyl-3,4-dihydropyrimidine-2(1H)-thione curcumin
Yellow crystal, mp 215-217 oC, Rf = 0.67 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3491 (N-
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Hstr), 3007 (C-Hstr), 1720 (C=Ostr); H NMR (400 MHz, CDCl3): δH 3.89 (6H, s, OCH3), 5.92
(2H, s, 2-H), 6.57-8.00 (10H, m, arom), 9.45 (2H, s, NH); 13C NMR (100 MHz, DMSO): 182.79,
148.99, 147.65, 145.57, 141.42, 140.27, 129.60, 126.96, 122.59, 120.65, 115.73, 115.44, 110.25,
100.72, 55.44, 30.48; ESI-MS: m/z Calculated for C29H26N2O5S 514.54 Found [M+NH4]+ 531.5;
C, H and N analyses Calculated for C 67.69, H 5.09, N 5.44, Found C 67.71, H 5.13, N 5.41.
Compound 2j: 4-(4-Methyl phenyl)-3,4-dihydropyrimidine-2(1H)-thione curcumin
Yellow crystal, mp 120-123 oC, Rf = 0.68 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3028 (C-
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Hstr),1668 (C=Ostr), 1575 (C-Hdef), 1519 (C-Nstr), 1240 (C-Hdef), 725-1016 (C-Hdef); H NMR
(400 MHz, CDCl3): δH 2.04 (3H, s, CH3), 3.96 (6H, s, OCH3), 5.80 (2H, s, 2-H), 6.54 (2H, d, 5-
H, J = 15.68 Hz), 6.85-7.61 (10H, s, arom), 9.30 (2H, s, NH); 13C NMR (100 MHz, DMSO):
178.06, 162.38, 148.92, 147.60, 142.66, 141.59, 135.38, 123.61, 122.39, 117.67, 116.46, 109.64,
104.42, 50.71; ESI-MS: m/z Calculated for C30H28N2O5S 528.62 Found [M+NH4]+ 547.1; C, H
and N analyses Calculated for C 68.16, H 5.34, N 5.30, Found C 68.20, H 5.30, N 5.26.
Compound 2k: 4-(2-Hydroxy-phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
o
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Yellow powder, mp 130-132 C, Rf = 0.67 (DCM / Toluene 3:2); H NMR (400 MHz, CDCl3):
δH 3.89 (6H, s, OCH3), 5.78 (2H, s, 2-H), 6.55 (2H, d, 5-H, J = 15..68 Hz), 6.69-7.67 (10H, arom,
m), 8.38 (2H, s, NH), 9.99 (1H, s, OH); 13C NMR (100 MHz, DMSO): 182.83, 149.08, 147.73,
147.07, 146.15, 140.23, 129.39, 128.45, 126.50, 122.60, 120.75, 119.86, 115.95, 115.50, 110.53,
110.53, 108.92, 55.89; ESI-MS: m/z Calculated for C29H26N2O7S 530.59 Found [M]+ 531; C, H
and N analyses Calculated for C 65.65, H 4.95, N 5.28, Found C 65.76, H 5.02, N 5.31.
Compound 2l: 4-(4-Dimethylamino-phenyl)-3,4-dihydropyrimidine-2(1H)-thione curcumin
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Yellow crystal, mp 135-137 C, Rf = 0.65 (DCM / Toluene 3:2); IR (KBr) (υmax, cm-1): 3288
(OHstr), 2897 (C-Hstr), 1703 (C=Ostr), 1581 (C-Hdef), 1531 (C-Nstr), 1377 (C-Hdef), 1165 (C-Hdef),
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754-977 (C-Hdef); H NMR (400 MHz, CDCl3): δH 2.36 (6H, s, N(CH3)2), 3.40 (6H, s, OCH3),
5.34 (2H, s, 2-H), 6.99 (2H, s, 5-H), 7.18-7.56 (10H, s, arom), 7.94 (1H, s, arom), 9.64 (2H, s,
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NH); C NMR (100 MHz, DMSO): 182.69, 162.80, 148.96, 146.99, 140.27, 129.80, 126.89,
122.57, 120.64, 115.42, 110.41, 100.22, 55.43; ESI-MS: m/z Calculated for C31H31N3O5S 557.54
Found [M+NH4]+ 575.0; C, H and N analyses Calculated for C 66.77, H 5.60, N 7.54, Found C
66.69, H 5.64, N 7.48.
Compound 2m: 4-(2,6-Dichloro-phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
o
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Yellow powder, mp 126-129 C, Rf = 0.62 (DCM / Toluene 3:2); H NMR (400 MHz, DMSO):
δH 3.89 (6H, s, OCH3), 5.85 (2H, s, 2-H), 6.64 (2H, d, 5-H, J = 15..64 Hz), 6.84 (2H, d, arom, J
= 8.16 Hz), 7.11-7.15 (3H, dd, arom, J = 7.4 Hz), 7.51 (2H, d, arom, J = 8.56 Hz), 8.09 (2H, t,
13
arom, J = 13.96 Hz), 8.57 (1H, t, arom, J = 15.64 Hz), 8.90 (2H, s, NH); C NMR (100 MHz,
DMSO): 182.98, 148.53, 147.12, 140.39, 129.11, 126.89, 121.23, 120.40, 120.20, 115.38,
115.25, 109.71, 100.08, 55.49; ESI-MS: m/z Calculated for C29H24Cl2N2O5S 583.48 Found
[M+2]+ 585; C, H and N analyses Calculated for C 59.70, H 4.14, N 4.80, Found C 59.68, H
4.21, N 4.89.
Compound 2n: 4-(2-Chloro phenyl)-3,4-dihydropyrimidine-2(1H)-one curcumin
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Yellow powder, mp 150-152 C, Rf = 0.65 (DCM / Toluene 3:2); H NMR (400 MHz, CDCl3):
δH 3.87 (6H, s, OCH3), 5.90 (2H, s, 2-H), 6.46 (2H, d, 5-H, J = 15..68 Hz), 6.812-7.79 (11H,
arom, m), 8.38 (2H, s, NH); 13C NMR (100 MHz, DMSO): 182.81, 149.10, 147.10, 147.00,
146.21, 140.13, 129.49, 128.25, 126.21, 122.17, 120.20, 119.15, 115.52, 115.35, 110.31, 110.43,
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108.42, 55.87; ESI-MS: m/z Calculated for C29H25ClN2O5S 549.04 Found [M]+ 549; C, H and N
analyses Calculated for C 63.44, H 4.59, N 5.10, Found C 63.49, H 4.69, N 5.11.
Acknowledgements
We gratefully acknowledge to Chandigarh and SAIF Punjab University, Chandigarh for
spectral analytical data.
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Table Captions
Table 1. Synthesis of dihydropyrimidine derivatives/analogues of curcumin
Table 2. Antioxidant activity by DPPH. method
Table 3. Antioxidant activity by NO. method
Table 4. Cytotoxicity of curcumin derivatives/analogues by MTT assay
Table 5. Physico-chemical properties of curcumin derivatives/analogues
Figure 1. Structure of curcumin
Scheme Captions
Scheme 1. Synthesis of 3,4-dihydropyrimidine derivatives of curcumin (2a-2n).
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Research highlights
ꢀ Antioxidant activity and cytotoxic agents.
ꢀ One-pot reaction of curcumin using piperidine.
ꢀ Structure activity relationship of curcumin derivatives.
ꢀ Good yield and better biological profile.
ꢀ In silico and physico-chemical study.