Synthesis and evaluation of antimicrobial activity of 4H-pyrimido[2,1-b] benzothiazole, pyrazole and benzylidene derivatives of curcumin

Article abstract of DOI:10.1016/j.ejmech.2012.05.020

A novel, one-pot, simple, efficient procedure for 4H-pyrimido[2,1-b] benzothiazole (4a-h), pyrazole (6a-d) and benzylidene (7a-d) derivatives of curcumin under solvent and solvent free conditions in microwave with good yield is have been synthesized. The synthesized compounds were evaluated for their antibacterial activity against gram-positive and gram-negative bacteria viz. Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Bacillus cereus and Providencia rettgeri and antifungal activity against fungi viz Aspergillus niger, Aspergillus fumigates, Aspergillus flavus. Detailed mechanistic study shows reaction proceeds through Knoevenagel type intermediate 3a which has been suggested as key intermediate for reaction (Fig. 3).

Full text of DOI:10.1016/j.ejmech.2012.05.020

European Journal of Medicinal Chemistry 54 (2012) 366e378
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of antimicrobial activity of 4H-pyrimido[2,1-b]
benzothiazole, pyrazole and benzylidene derivatives of curcumin
,
b
,
b
,
,b,c
Pramod K. Sahu a , Praveen K. Sahu c, S.K. Gupta a, D. Thavaselvam d, D.D. Agarwal a
*
a School of Studies in Chemistry, Jiwaji University, Gwalior 474011, Madhya Pradesh, India
b Department of Industrial Chemistry, Jiwaji University, Gwalior 474011, Madhya Pradesh, India
c Jagdishprasad Jhabarmal Tibrewala University, Jhunjhunu 333001, Rajasthan 333001, India
d Microbiology Division, Defence Research Development Establishment, Jhansi Road, Gwalior 474002, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< One-pot syntheses of novel curcu-
min derivatives have good biological
activities.
< First example of three component
reaction of curcumin using pyridine
as catalyst.
< First time that we have isolated and
characterized
intermediates.
the
reaction
< Easy synthesis of pyrazoles and
benzylidenes of curcumin which
possesses
activity.
good
antimicrobial
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel, one-pot, simple, efficient procedure for 4H-pyrimido[2,1-b]benzothiazole (4aeh), pyrazole (6aed)
and benzylidene (7aed) derivatives of curcumin under solvent and solvent free conditions in microwave
with good yield is have been synthesized. The synthesized compounds were evaluated for their antibac-
terial activity against gram-positive and gram-negative bacteria viz. Staphylococcus aureus, Pseudomonas
aeruginosa, Salmonella typhi, Escherichia coli, Bacillus cereus and Providencia rettgeri and antifungal activity
against fungi viz Aspergillus niger, Aspergillus fumigates, Aspergillus flavus. Detailed mechanistic study shows
reaction proceeds through Knoevenagel type intermediate 3a which has been suggested as key interme-
diate for reaction (Fig. 3).
Received 30 October 2011
Received in revised form
11 May 2012
Accepted 11 May 2012
Available online 22 May 2012
Keywords:
Antimicrobial activity
SAR
Ó 2012 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Pyrazoles
Benzylidene
4H-Pyrimido[2,1-b]benzothiazole
derivatives of curcumin
1. Introduction
Multi-component reactions (MCRs) are powerful tool and have
attracted much attention of synthetic organic chemists because of
the building of complex molecules with diverse range of
complexity which can easily be achieved from readily available
starting material [1e3]. The synthesis of heterocycles using MCRs is
* Corresponding author. School of Studies in Chemistry, Jiwaji University, Gwalior
474011, Madhya Pradesh, India. Tel.: þ91 9993932425.
E-mail addresses: sahu.chemistry@gmail.com, researchdata6@gmail.com
(P.K. Sahu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.020

P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
367
a domain of classic carbonyl condensation chemistry. Pyrimidines
have attracted considerable interest because of their wide range of
biological activities. Some of which have anti-viral, anti-tumour,
anti-inflammatory, antihypertensive activities [4e6], calcium
channel modulators [7] and antimicrobial agents [8e10]. Pharma-
cologically, pyrazoles and its derivatives represent one of the most
important class of heterocyclic compounds and have attracted
considerable attention from both synthetic and medicinal chemists
due to their biological activities covering a wide ranges of appli-
cation of pyrazole moiety as potential antimicrobial [11], anti-viral
[12], anticancer [13], herbicide [14]. Some of its derivatives have
been reported to exhibit significant cyclooxygenase (COX-2)
inhibitors [15], molluscicidal activity [16], antiglaucoma activity
[17], anti-inflammatory [18], antioxidant [19] and anti-HCV
activity [20].
benzothiazole (4aeh) derivatives of curcumin has been synthe-
sized by three component reaction of curcumin, substituted
aromatic aldehydes and 2-amino benzothiazole using pyridine
under solvent and solvent free conditions (Scheme 1). Benzylidene
derivatives of curcumin (7aed) have also been synthesized under
optimized reaction conditions using curcumin and substituted
aromatic aldehydes (Scheme 3). Our synthetic method for pyrazole
derivatives of curcumin (6aed) has been developed in acetic acid
(Scheme 4).
3. Pharmacology
3.1. To determine MIC by the micro dilution broth susceptibility test
Different concentrations 20, 10, 5, 2.5, 1.25 and 0.625 mM/mL of
Curcumin possess antimicrobial [21], antioxidant [22], anti-HIV
protease activity [23], and cancer preventive properties [24].
Extensive research has revealed that this polyphenol can both
prevent and treat cancer [25]. Phenolic OeH and methylene
hydrogen of curcumin revealed antioxidant activity by free radical
reactions [26]. Elias et al. [27] have reported the dihydropyridones
synthesis by microwave assisted reaction of curcumin with either
primary amines or the amine acetate in the presence of montmo-
rillonite K-10 as a catalyst but with poor yield. Various substituted
thiazoles have been synthesized and examined for their antifungal
and antibacterial activities [28]. Objective of the present MCR is to
use biologically important component viz. curcumin, 2-amino
benzothiazole, thiosemicarbazide, semicarbazide, isoniazide to
convert it into other potential antimicrobial agents. Our target
molecules have shown good antibacterial and antifungal activity.
We have developed the pyrazoles of curcumin using acetic acid as
potential active antimicrobial agents. The synthesis of 4H-pyrimido
[2,1-b]benzothiazole and benzylidene derivatives of curcumin was
tried with acetic acid which resulted in 55% yield. The use of
pyridine (base) as catalyst led to formation of 67% yield of 4H-
pyrimido[2,1-b]benzothiazole. Pyridine has not been reported as
catalyst, hence this paper also report this new catalyst for multi-
component reactions. The intermediate of this reaction has been
isolated and characterized for the first time, hence mechanism
could be established.
the compound (4aeh, 6aed and 7aed) were prepared in sterile
microwell to determine minimum inhibitory concentration (MIC).
Nutrient broth was adjusted to pH 7.0 used for the determination of
MIC. The inoculum of the test microorganisms was prepared by
using 16 hold cultures adjusted by reference to the 0.5 McFarland
standards (1.5 ꢀ 108 CFU/mL). Brain heart infusion broth was
prepared and 150
compound was added in broth with different concentrations, then
10 L of bacterial culture broth was added. The plate was shaken to
uniformly mix the inoculum with the broth. Optical density was
taken by photo spectrometer ( Quant, Biotek Ltd. USA) and incu-
mL of it was taken in each well and 10 mL of each
m
m
bated for 24 h at 37 ꢁC. Appearance of any turbidity shows that the
compound is not able to inhibit the growth of the bacteria, while no
turbidity indicates the inhibition of microorganism by the sample
(Table 1).
3.2. In-vitro antifungal test [29]
For antifungal testing the pyrimidine derivatives of curcumin,
curcumin and fluconazole (standard drug) solution was prepared in
acetone at initial concentration (120
make effective concentration of 20, 10, 5.0, 2.5, 1.25, 0.625 and
0.315 M/mL. Isolated fungal species A. niger, A. fumigates and
A. flavus were selected. Subourad dextrose agar was prepared and
150 L of it was taken in each well and 10 L of each compound was
added in broth with different concentrations, then 10 L of fungi
mM/mL) and serially diluted to
m
m
m
m
2. Chemistry
culture broth was added. The plate was shaken to uniformly mix
the inoculum with the broth and note the optical density. The well
incubated for 72 h at 28 ꢁC. Appearance of any turbidity shows that
the compound is not able to inhibit the growth of the bacteria,
In this study we have developed a simple and efficient synthetic
method for novel derivatives of curcumin. 4H-Pyrimido[2,1-b]
CHO
O
O
OCH3
OH
N
H3CO
HO
+
R
NH2
+
S
2
1
7
(i)
OCH3
2
15
16
3
R
1
7
O
14
OH
17
4
10
12
5
9
18
6
8
13
OCH3
N
S
11
N
OH
6
Scheme 1. Conditions (i) refluxing in methanol at 60e65 ꢁC using pyridine as catalyst.

368
P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
OH
+.
OH
O
O
HO
OCH
Fragmentation
3
HO
OCH
3
N
i
N
N
S
OCH
OH
3
S
N
455.3
M++NH 4 Peak (623.1)
ii
+.
OCH
3
OH
151
Scheme 2. Mass fragmentation patterns of product 4b.
while no turbidity indicates the inhibition of microorganism by the
sample. To ensure that solvent had no effect on fungal growth
a control test was performed with test medium supplemented with
acetone at the same dilutions as used in the experiment. The results
incorporated in Table 2.
The multi-component reaction of curcumin (5 mmol), 2-amino
benzothiazole (5 mmol) and substituted aromatic aldehydes
(5 mmol) dissolved in methanol using pyridine (1 mL), resulted
product formation within 18e20 h with yield ranges from 66 to 76%
(Scheme 1). The structure of 4H-pyrimido[2,1-b]benzothiazole
derivatives of curcumin, was confirmed by IR, NMR, Mass spectral
analysis. The 1H NMR spectra of the product 4aeh were charac-
To determine zone of inhibition, sterilized filter discs were
dipped in these solutions and subsequently dried to remove
acetone. Subourad dextrose agar was prepared and allowed to
solidify. One of these discs was kept free from antifungal drug
(fluconazole) and served as growth control. Fungi were selected viz
A. niger, A. fumigates, A. flavus and 1 mL of each fungi culture were
added in the Subourad dextrose agar plates and spread with the
help of sterile spreader. The filter paper discs soaked in above
mentioned dilutions of curcumin, fluconazole and curcumin
derivatives were placed aseptically over the inoculated plates using
sterile forceps. The plates were incubated at 28 ꢁC for 72 h, in
upright position. The zone of inhibition was measured using scale
(Table 2).
terized by singlet around 7.26
d
due to asymmetric CeH hydrogen
and the multiplet at 7.1e7.61
d with unsymmetrical pattern for
aromatic hydrogens of benzothiazole and benzene ring of aldehyde,
which is not shown by curcumin. Mass spectra of product 4b shows
the [M þ NH4]þ molecular ion peak at 623.1 and other fragments
peak at 455.3, 151 for corresponding fragments which are shown in
Scheme 2. This supports the probable structure of product 4b. A
variety of electron donating and electron withdrawing groups on
aromatic aldehydes have been studied (4a-4h) (Table 3).
Optimized reaction conditions in hand lead us to synthesize the
benzylidene derivatives of curcumin using curcumin (5 mmol) and
substituted aromatic aldehydes (5 mmol) in the presence of pyri-
dine in methanol at 60e65 ꢁC (Scheme 3). Methodology involves
for synthesis of pyrazole derivative, curcumin (5 mmol), and
hydrazines (5 mmol) were refluxed in acetic acid at 60e65 ꢁC
(Scheme 4). Synthesized benzylidene and pyrazole derivatives
have been characterized by 1H NMR, 13C NMR and mass spectral
study.
The reaction in solvent took longer time (6e20 h), hence there is
need to find methods to reduce reaction time. Therefore the reac-
tion was carried out in microwave under solvent free condition. The
reaction was completed in 8e10 min with good yield (Table 3). Thus
synthesis under microwave is very simple, giving high yield
(69e89%) in shorter duration and greatly reducing the environ-
mental pollution by eliminating volatile organic solvent. Various
substituted aromatic aldehydes containing electron withdrawing
and electron donating substituents at ortho, meta or para positions
show equal ease towards the product formation in high yields.
4. Result and discussion
4.1. Chemistry
Reaction of curcumin, 2-amino benzothiazole and benzaldehyde
at 60e65 ꢁC in the absence of pyridine as a catalysts fail to give
product. This shows that pyridine is needed to get the product. In
order to evaluate the appropriate catalyst loading, a model reaction
using curcumin (5 mmol), 2-amino benzothiazole (5 mmol) and
substituted aromatic aldehydes (5 mmol) was carried out using
0.25 mL, 0.5 mL, 0.75 mL and 1 mL of pyridine at 60e65 ꢁC with
methanol as solvent (Fig. 1). It was found that 1 mL of pyridine
resulted in the product formation. Higher quantity of pyridine
(1.5 mL and 2 mL) neither increases the yield nor improves the
conversion time. So, 1 mL of pyridine was found to be the optimal
quantity.
R
OH
OH
HO
H3CO
HO
H3CO
CHO
Pyridine, 60-65 oC
Methanol
+
OCH3
OCH3
R
O
O
O
O
(7a-7d )
Scheme 3. Synthesis of benzylidene derivatives of curcumin.

P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
369
5
OH
HO
OH
6
HO
4
Reflux at 60-65 o
Acetic acid
C
RNHNH 2
7
+
3
1
H3CO
OCH3
10
H3CO
OCH3
2
11
9
8
N
N
O
O
R
(6a-6d )
Scheme 4. Preparation of pyrazoles of curcumin.
There was no significant effect of electron donating and electron
withdrawing substituents.
which gave target product but author have reported that target
product was formed with low yield (15%) in toluene; hence there is
minimum possibility of this type of intermediate in reaction
because this reaction does not proceed well in toluene. Interme-
diate 9 proposed by Shen et al. also was prepared in benzene but
author have reported that reaction was well tolerated in protic
solvent, so there is also minimum possibility to follow the mech-
anistic pathway by this type of intermediate.
The mechanism of three component reaction has been studied
in the present paper (Scheme 5). For this, three set of reactions of
two components each were carried out. The intermediate formed
was reacted with third component and the product formed was
analyzed by mp, IR, NMR and mass spectral studies. It was found
that only one set of reaction gave the product i.e. the reaction
proceeds in two steps: condensation of benzaldehyde 1 and cur-
cumin 2 according to Knoevenagel type reaction. Then 2-amino
benzothiazole 3 is reacted with compound 3a through Michael
addition (3a characterized by IR, NMR and mass spectra) to afford
pyrimidine derivative of curcumin having 4H-pyrimido[2,1-b]ben-
zothiazoles ring system in structure 4.
4.2. Mechanism
The mechanistic pathway of three component reaction using
aldehyde, dicarbonyl and 2-amino benzothiazole has not been
reported so far. In a previous report, Kappe proposed that Lewis
acid or protic acid-mediated Biginelli reaction proceeded via the
formation of an iminium ion (formed by acid-catalyzed conden-
sation of aldehyde with urea) as a key intermediate rather than
carbenium ion intermediate (derived from the acid-catalyzed aldol
reaction of aldehyde and ethyl acetoacetate) [30]. Literature survey
revealed that till now the intermediate of these three component
condensation reaction has not been isolated and characterized.
Shen et al. [31] have suggested that mechanism of Biginelli reaction
proceeded with urea and thiourea in different ways when t-BuOK
was used. But they failed to isolate and characterize the interme-
diate. They have synthesized compound 6 using literature proce-
dure (currently, there is no report on the successful isolation of
intermediate 6). Further, they have synthesized the intermediates 9
(derived from the acid-mediated condensation of 4-chloro benz-
aldehyde and 2-phenyl acetophenone) intermediates 7 and 8 under
acidic conditions by the well known method documented in liter-
ature [32,33] (Fig. 2), and further reacts with third component
which gives the target product. Shen et al. have synthesized the
intermediate 8 in toluene solvent using acid-mediated conditions
The mass fragmentation of intermediate 3a (prepared by
reacting of benzaldehyde with curcumin) shows [M þ H] ion peak
at 457.7. NMR spectra of intermediate 3a shows the singlet at 10.03
d
(]CHeAr), and the multiplet between 7.32 and 7.90 unsym-
metrical pattern due to hydrogen of benzene ring of benzaldehyde,
which is not shown by the NMR spectra of the curcumin and
multiplet between 6.67 and 7.15 due to 6 hydrogen of two aromatic
Table 1
Minimum inhibitory concentration (MIC95 in m
M/mL)a of curcumin derivatives against bacteria strain.
Compounds
Bacteria species
S. aureus
S. typhi
P. aeruginosa
E. coli
Bacillus cereus
Providencia rettgeri
(ATCC 11632)
(ATCC 15499)
(ATCC 23564)
(ATCC 35218)
(MTCC 7350)
(DRDE strain)
4a
4b
4c
20
20
d
20
d
10
20
20
d
5
5
5
20
d
d
d
10
d
4d
d
20
10
5
2.5
10
4e
4f
4g
4h
6a
6b
6c
6d
d
10
20
d
5
10
d
10
5
10
20
2.5
d
2.5
2.5
5
5
d
10
d
1.25
20
d
20
10
2.5
1.25
d
d
20
5
2.5
1.25
d
d
d
1.25
1.25
1.25
2.5
5
2.5
0.625
d
2.5
d
1.25
d
7a
7b
1.25
5
2.5
2.5
d
1.25
d
0.625
0.625
0.625
d
1.25
7c
7d
1.25
2.5
d
5
5
d
1.25
2.5
d
2.5
d
1.25
1.25
d
1.25
2.5
d
Acetone
Curcumin
Ciprofloxacin
Inoculum control
d
20
1.25
Growth in all
concentration
No growth
20
2.5
d
20
1.25
Growth in all
concentration
No growth
10
0.625
Growth in all
concentration
No growth
20
1.25
Growth in all
concentration
No growth
1.25
Growth in all
concentration
No growth
Growth in all
concentration
No growth
Broth control
(d) Resistant.
a
MIC (
mM) ¼ Minimum inhibitory concentration.

370
P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
Table 2
Antifungal activity (zone of inhibition and MIC95 in mM/mL) of curcumin derivatives and their cytotoxicity.
Compounds
Fungal strains
Cytotoxicity activity
IC50 (in mM/mL) (L123 cell)
Zone of inhibition (in mm)
MIC95 (in mM/mL)
A. niger
A. fumigates
A. flavus
A. niger
A. fumigates
A. flavus
4a
4b
4c
4d
4e
4f
4g
4h
d
d
d
d
d
d
10
d
d
d
d
5
d
d
>100
50
>100
>100
ND
ND
ND
>100
ND
d
d
d
d
d
d
d
d
d
d
10
d
15
d
d
20
d
d
11
d
12
d
d
12
d
5
d
d
d
d
d
d
d
d
d
6a
15
20
d
1.25
d
6b
6c
6d
7a
7b
7c
7d
22
18
17
d
25
d
20
22
20
d
2.5
2.5
2.5
d
5
2.5
d
1.25
2.5
1.25
d
ND
>100
>100
ND
>100
50
d
d
15
20
20
d
2.5
1.25
2.5
d
15
17
d
d
d
d
2.5
d
d
20
1.25
>100
Acetone
Curcumin
Fluconazole
Broth control
No activity
9
18
No activity
12
22
No activity
d
No activity
20
2.5
No activity
20
1.25
No activity
d
25
1.25
No growth
No growth
No growth
No growth
No growth
No growth
(d) Resistant.
ND ¼ Not determined.
ring of curcumin. IR spectra of intermediate 3a show peaks
between 2864 and 2916 cmꢂ1 due to CeHstr of benzene ring, peak
around 3736 cmꢂ1 due to stretching of hydroxyl group, peak
around 1600 cmꢂ1 can be assigned to carbonyl group. Mass frag-
ments, IR and NMR spectra of 3a support its structure. This is first
time that we have isolated the intermediate 3a which has been
characterized and confirmed by comparing the data published in
literature [34], hence it can be suggested that it may be the key
intermediate for mechanistic study (Fig. 3). We have successfully
isolated the intermediate 5a (prepared by reacting benzaldehyde
and 2-amino benzothiazole). The mass fragmentation of interme-
diate 5a show [M ꢂ H]þ ion peak at 387. 1H NMR spectra of inter-
mediate shows the triplet at 6.99 (eCH), doublet at 7.19 due to two
similar hydrogen of eNH and multiplet are observed between 7.37
and 8.02 due to hydrogen of aromatic rings. This intermediate fail to
produce the target product, hence it involvement as intermediate
can be ruled out.
method. The stock solution of curcumin derivatives (4aeh, 6aed,
7aed) and curcumin 2 were prepared in acetone. The stock solu-
tion of each of these derivatives was serially diluted (20, 10, 5, 2.5,
1.25, 0.625, 0.312
after which a standardized bacterial suspension was added. The
lowest concentration of curcumin derivatives in M/mL that pre-
mM/mL) and added to Brain Heart Infusion broth,
m
vented in-vitro growth of microorganism has been represented as
MIC95 (minimum inhibitory concentration) shown in Table 1.
4.3.2. Antibacterial activity (interpretation)
Susceptibility test in-vitro was done on bacteria Staphylococcus
aureus (ATCC 11632), Pseudomonas aeruginosa (ATCC 15499),
Salmonella typhi (ATCC 23564), Escherichia coli (ATCC 35218),
Bacillus cereus (MTCC 7350) and Providencia rettgeri. Each test was
performed in triplicate and the MICs reported represent the best of
at least two repetitions.
4.3.3. Structure activity relationships
We have investigated curcumin derivatives in order to access
their potential effect against different bacterial strains. The effects
of various substituents in the phenyl group attached and
substituted hydrazines in order to find out their structure activity
relationship. As evident from Table 1, substitution at para position
as nitro and hydroxyl group (derivatives 4def) demonstrated good
activity on microorganism. Particularly the derivatives 4f was
active on all species of bacteria tested and the MIC95 value was as
4.3. Antimicrobial activity
4.3.1. Dilution
The antibacterial activity of curcumin derivatives was compared
with curcumin 2 based on micro dilution broth susceptibility test
low as 1.25
mM/mL for S. aureus followed by 2.5 mM/mL for
B. cereus. Electron releasing group at para and meta positions
particularly as hydroxy and methoxy substituents shows good
activity against P. aeruginosa and B. cereus with 5 and 2.5 mM/mL
respectively. SAR study showed that introduction of hydroxyl
group at para position only (compound 4f) showed equipotent
activity as ciprofloxacin against S. aureus. Electron withdrawing
group as nitro group at the para position of C-4 aryl ring
exhibited good activity against P. aeruginosa, E. coli and B. cereus.
SAR also showed that any other substitution at the meta and para
position of the C-4 aryl ring retarded the efficiency of the
resulting compounds as can be seen in the derivative 4h, 4g, 4c
and 4b. Overall structure activity relationship results show that
all curcumin derivatives effectively work as antibacterial agents
Fig. 1. Optimization of catalyst loading. Three component reaction of curcumin,
benzaldehyde and 2-amino benzothiazole proceeds in methanol in the presence of
pyridine as catalyst at 60e65 ꢁC.

P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
371
Table 3
One-pot synthesis of 4H-pyrimido[2,1-b]benzothiazole, pyrazoles and benzylidene derivatives of curcumin.
Entry
R
Product
Time (h)/yield (%)a,b
Time (min)/yield (%)a,c
M.P. (ꢁC)
OH
O
OCH3
OCH3
1
H
18/67
8/69
97e98
N
S
N
OH
OH
4a
O
OCH3
OCH3
HO
2
2-OH
18/69
8/73
155e156
N
N
S
OH
OH
4b
Cl
O
OCH3
OCH3
3
4-Cl
20/66
8/77
112e113
N
S
N
OH
OH
4c
NO2
O
OCH3
OCH3
4
5
6
4-NO2
20/71
18/74
20/75
10/73
10/79
8/71
85e86
N
S
N
OH
4d
4-OHe3-OCH3
80e81
OH
OH
O
OCH3
OCH3
4-OH
101e102
N
S
N
OH
(continued on next page)
4f

372
P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
Table 3 (continued )
Entry
R
Product
Time (h)/yield (%)a,b
Time (min)/yield (%)a,c
M.P. (ꢁC)
OH
CH3
O
OCH3
OCH3
7
4-CH3
20/76
8/70
108e109
N
S
N
OH
OH
4g
O
Cl
OCH3
Cl
8
2,6-Cl2
20/72
8/71
90e92
N
S
OCH3
OH
N
4h
NH2
O
NH2
O
N
N
9
6/85
10/89
204e206
OCH3
OH
H3CO
NHNH2
HO
6a
NH2
N
S
NH2
N
10
7/82
10/83
100e101
S
OCH3
OH
H3CO
NHNH2
HO
6b
N
N
N
C
O
11
7/78
10/80
105e106
N
O
OCH3
OH
H3CO
NHNH2
HO
6c
NO2
O2N
NO2
N
N
12
6/86
10/88
118e119
OCH3
OH
H3CO
NO2
NHNH2
HO
6d

P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
373
Table 3 (continued )
Entry
R
Product
Time (h)/yield (%)a,b
Time (min)/yield (%)a,c
M.P. (ꢁC)
O
O
OCH3
OH
H3CO
HO
CHO
13
12/72
8/76
119e120
7a
O
O
OCH3
OH
H3CO
HO
14
13/76
8/77
104e106
N
CH3
CH3
7b
O
O
OCH3
OH
H3CO
HO
CHO
15
12/78
8/78
92e94
OH
HO
O
7c
O
OCH3
OH
H3CO
HO
CHO
16
14/76
10/78
98e100
OH
H3CO
OH
OCH3
7d
a
Isolated yield.
b
c
Reaction carried out in solvent.
Reaction carried out under solvent free conditions in microwave.
against B. cereus (Fig. 4 and Table 1). The data also revealed that the
activity of all compounds follow the order
4f > 4e > 4d > 4a>4h > 4g > 4c > 4b.
The structural activity data suggests the derivatives 6a having
amide moiety at hydrazine scaffold don’t show significant activity
but replacement of oxygen atom from sulphur atom showed
immense antibacterial activity as 6b was effective against all
species except E. coli. It may be due to difference between atomic
size of sulphur and oxygen atoms. SAR study also revealed that 6c
has isoniazide moiety shows equipotent antibacterial activity as
excellent activity against S. aureus and B. cereus. SAR study indicates
that presence of pyrazole moiety is additive towards the antibac-
terial activity. Furthermore, SAR exhibited electron releasing
substituents at ortho para positions (benzylidene derivatives 7b
and 7c) showed immense activity against S. aureus and P. aeur-
uginosa. Literature survey however suggests that pyrimidine, ben-
zylidene and pyrazole derivatives of curcumin have not been
evaluated so far for their antibacterial activity.
4.4. Antifungal activity (interpretation)
ciprofloxacin against S. aureus (1.25
(1.25 M/mL). Electron withdrawing substituent at ortho and para
positions as 2,4-dinitro phenyl group (derivatives 6d) have shown
mM/mL) and P. aeruginosa
m
Literature survey however suggests that synthesized curcumin
derivatives so far have not been evaluated for their antifungal
Fig. 2. Proposed intermediate for mechanistic study. Suggested by Shen et al.

374
P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
O
O
O
O
CHO
CHO
N
S
N
S
NH
+
NH
2
2
+
+
OH
O
OCH
OH
OH
OCH
OH
OCH
3
OCH
3
3
3
1
3
1
3
2
2
Pyridine
O
Pyridine
Pyridine
No Intermediate Formed
N
H
N
OCH
H
CO
HO
3
3
S
N
H
OH
N
H
S
3a
5a
N
S
NH
2
OCH
3
3
O
OH
OCH
3
N
N
OH
S
4
Scheme 5. Proposed mechanism for synthesis of 4H-pyrimido[2,1-b]benzothiazole derivatives of curcumin 4.
activity. We have now evaluated the antifungal activity of curcumin
derivatives against isolated fungi viz. A. niger, A. fumigates, A. flavus.
A broad spectrum of antifungal activity of derivatives 4d (p-nitro),
4e (4-hydroxy-3-methoxy) and 4f (4-hydroxy) were obtained
against tested fungal strain. The antifungal activity of curcumin
derivatives was compared with the standard antifungal drugs flu-
conazole. Regarding the SAR study from the analysis data, it is
suggested that p-substitution by electron withdrawing (4d)
exhibited significant activity against A. niger and A. fumigates.
Electron releasing substituents at para positions (4e and 4f) also
revealed good spectrum against derivatives show the antifungal
activity against A. fumigates and A. flavus respectively. SAR study
confirmed that excellent activity was demonstrated by pyrazole
derivative 6b against A. niger, A. fumigates and A. flavus with MIC
antifungal screening. However, this approach of combining
different active molecules present in nature, resulting in
enhancement of their bioavailability, is certainly encouraging.
These derivatives of curcumin are more hydrophilic than the parent
molecule. The hydrophilic nature of pyrimidines, pyrazoles and
benzylidenes may also help in their active transport across the
cellular membrane.
4.5. Cytotoxicity against L123 (human lung cells)
Cytotoxicity was performed by MTT assay method [35,36]. A 96
well flat bottom tissue culture plate was seeded with 2 ꢀ 103 cells
in 0.1 mL of MEM medium supplemented with 10% FBS and allowed
to attach for 24 h. After 24 h of incubation, cells were treated with
2.5, 2.5 and 1.25
mM/mL which is quite comparable with standard
test compounds to get a concentration of 5, 10, 20, 50 and 100 mM/
drug fluconazole (MIC 2.5, 1.25 and 1.25
m
M/mL) tested under
mL incubated for 48 h. The cells in the control group received only
the medium containing the 0.2% DMSO. Each treatment was per-
formed in duplication. After the treatment, drug containing media
similar conditions. It is interesting that electron releasing substit-
uents at para and ortho positions (benzylidene derivatives 7b and
7c) was effective against A. niger and A. fumigates, but decreased the
in-vitro activity by introducing the electron releasing group at meta
position (compound 7d). Analysis of result showed that substitu-
tion at para position by electron releasing group increased the
antifungal activity. It opens a new era for exploring suitably
designed curcumin derivatives as potential antibacterial/antifungal
drugs.
was removed and washed with 200 mL of PBS. To each well of the 96
well plate, 100 mL of MTT reagent (stock: 1 mg/mL in serum free
medium) was added and incubated for 4 h at 37 ꢁC. After 4 h of
incubation the plate was inverted on tissue paper to remove the
MTT reagent. To solubilize formazan crystals in the wells, 100 mL of
100% DMSO was added to each well. The optical density was
measured by microtiter plate reader at 590 nm. Compound
Overall structure activity relationship showed that a minor
alteration in the molecular configuration of investigated
compounds may have a pronounced effect on antibacterial and
concentration (mM) required to reduce the viability of mock-
infected cells by 50% as determined by MTT method which
summarized in Table 2. Results of MTT assay mentioned on the cell
14
9
N
13
H
N
O
O
2
7
8
3
10
OCH3
1
H3CO
HO
8
12
2
S
N
4
10
9
11
3
13
1
14
11
6
N
H
4
5
OH
7
12
17
5
6
S
15
16
3a
5a
Fig. 3. Key intermediate for mechanistic study. Suggested by present study.

P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
375
Fig. 4. Comparable chart for MIC95 of curcumin and its derivatives against tested bacteria strain.
viability based on ability to metabolize the compounds. The results
show that derivatives 4b and 7c are comparatively more cytotoxic
than 4a, 4c, 4g, 6c, 4d, 7d and 7d as these derivatives showed IC50
values more than 100 mM/mL. It means para chloro (4c) and ortho
hydroxyl (7c) substituted derivatives showed the higher cytotox-
(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 (cmꢂ1). MS 1927 microwave starter kit was
used for microwave reactions. Reaction was carried out under
microwave conditions at 300 W in open to air conditions. The
output power can be controlled by controller button. 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 was uncorrected. Curcumin, hydrazines,
ciprofloxacin and substituted aromatic aldehydes were purchased
from Hi media Laboratory Ltd., Mumbai, India. 2-Amino benzo-
thiazole was purchased from Sigma Aldrich. Brain heart infusionÔ,
MuellereHinton agar, Subourad dextrose agar and Subourad
dextrose broth were purchased from OXOID LTD., Basingstoke,
Hampshire, England. 96 Microwell with lid was purchased from
IWAKI Brand SCITECH DIV. Asahi Techno Glass, Japan.
icity that is doses related.
5. Conclusion
It can be concluded that reaction of substituted aromatic alde-
hydes, curcumin and 2-amino benzothiazole in presence of catalyst
under solvent free conditions in microwave produces novel deriv-
atives of curcumin. A rapid and environmentally benign method for
synthesizing 4H-pyrimido[2,1-b]benzothiazole, pyrazole and ben-
zylidene derivatives of curcumin has been developed, the short
reaction times and ease of work-up make the method advanta-
geous. During the present work we have concluded that the
synthesized curcumin derivatives are more effective than curcumin
itself against many common strains of bacteria and fungal. Struc-
ture activity relationship studies revealed that para substituted
derivatives play important role in antimicrobial activity. The
present work suggests that there is need to develop nonantibiotic
drugs which may overcome antibiotic resistance. Such highly
bioactive drugs preferably herbal preparations with least toxicity. A
little structure variation can cause immense difference in the
activity of the drug. This approach can open new vistas in the
chemotherapy of the infective disease. The field is further open for
pharmacokinetics and clinic studies to establish these molecules as
drugs in the market. In addition, Knoevenagel type intermediate 3a
is suggested as key intermediate for this reaction.
6.2. Typical procedure for preparation of 4H-pyrimido[2,1-b] [1,3]
benzothiazole curcumin (4aeh)
A mixture of curcumin (5 mmol), substituted aromatic alde-
hydes (5 mmol) and 2-amino benzothiazole (5 mmol) was refluxed
at 60e65 ꢁC in presence of pyridine (1 mL) as catalysts in methanol
(10 mL). The reaction was monitored by TLC. After completion the
reaction, mixture was then poured in cold water and filtered. The
residue was purified via column chromatography on silica gel using
EtOAc/MeOH (3:1). The pyrimidine derivatives were collected as
yellow crystals.
6. Experimental protocols
6.3. Microwave synthesis of 4H-pyrimido[2,1-b] [1,3]benzothiazole
curcumin (4aeh)
6.1. General
A mixture of curcumin (5 mmol), substituted aromatic alde-
hydes (5 mmol) and 2-amino benzothiazole (5 mmol) was irradi-
ated in microwave under solvent free conditions using pyridine
(1 mL) as catalyst. After completion of the reaction, the contents
The 1H NMR spectra were measured by BRUKER AVANCE II 400
NMR spectrometer with tetramethylsilane as an internal standard
at 20e25 ꢁC; data for 1H NMR are reported as follow: chemical shift

376
P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
were transferred in water with the help of methanol and filtered.
The residue was purified via column chromatography on silica gel
using EtOAc/MeOH (3:1). The pyrimidine derivatives of curcumin
were collected as yellow crystals.
6.9. 4-(2-Hydroxy phenyl)-4H-pyrimido[2,1-b] [1,3]benzothiazole
curcumin (4b)
Yellow crystal, mp 155e156 ꢁC, Rf
¼
0.54 (DCM/
Toluene ¼ 3:2); IR (KBr) (nmax, cmꢂ1): 3504 (OHstr), 2941 (CeHstr),
6.4. Synthesis of pyrazoles of curcumin (6aed)
1627 (C]Ostr), 1508 (CeHstr), 1280 (CeHstr) 1153 (CeSstr),
962e813 (CeHdef); 1H NMR (400 MHz, CDCl3): dH 3.93 (6H, s,
OCH3), 5.78 (1H, d, 6-H, J ¼ 5.8 Hz), 6.49 (1H, d, 5-H, J ¼ 5.76 Hz),
6.87 (1H, d, 8-H, J ¼ 8.52 Hz), 7.05 (1H, d, 7-H, J ¼ 3.04 Hz), 7.1e7.6
(14H, m, arom), 7.26 (1H, s, CeH), 8.83 (1H, s, OH); 13C NMR
(100 MHz, CDCl3): 182.89 (C-9), 149.11 (C-1), 147.74 (C-3), 140.36
(C-4), 129.80 (C-7), 126.22 (C-5), 122.70 (C-11), 120.79 (C-6),
115.77 (C-7), 115.53 (C-8), 110.63 (C-2), 100.79 (C-10), 55.47
(OCH3); ESI-MS: m/z calculated for C35H28N2O6S 604.62, found
[M þ NH4]þ 623.1.
A mixture of curcumin (5 mmol) and hydrazines (5 mmol) was
refluxed at 65e70 ꢁC in presence of acetic acid as solvent. The
reaction was monitored by TLC. After completion the reaction
mixture was then poured in cold water and the acetic acid was
neutralized using sodium carbonate till then effervescences came
from the solution. The solid mass was filtered and recrystallized by
ethyl acetate. The pyrazole derivatives of curcumin were collected
as yellow crystals (Scheme 4).
6.5. General procedure for synthesis of Knoevenagel condensate of
curcumin (7aed)
6.10. 4-(4-Chloro phenyl)-4H-pyrimido[2,1-b] [1,3]benzothiazole
curcumin (4c)
A mixture of curcumin (5 mmol) and substituted aromatic
aldehydes (benzaldehyde, 4-dimethylamino benzaldehyde, 2-
hydroxy benzaldehyde and 4-hydroxy-3-methoxy benzaldehyde)
(5 mmol) was refluxed at 60e65 ꢁC in methanol using pyridine.
After completion the reaction (judged by TLC analysis), the reaction
mixture was cooled at room temperature and poured in cold water.
The solid mass was filtered. It was dissolved in ethanol and filtered
again. The residue was purified via column chromatography on
silica gel using EtOAc/MeOH (3:1). The solid was recrystallized by
ethyl acetate to give the product (Scheme 3).
Yellow crystal, mp 112e113 ꢁC, Rf ¼ 0.49 (DCM/Toluene ¼ 3:2);
IR (KBr) (nmax, cmꢂ1): 3510 (OHstr), 3012 (CeHstr), 2922 (CeHstr),
1625 (C]Ostr), 1500 (CeHdef), 1278 (CeHdef), 1155 (CeSstr),
960e812 (CeHdef); 1H NMR (400 MHz, CDCl3): dH 3.93 (6H, s,
OCH3), 5.78 (1H, d, 6-H, J ¼ 6.76 Hz), 6.49 (1H, d, 5-H, J ¼ 5.76 Hz),
6.94 (1H, d, 8-H, J ¼ 8.12 Hz), 7.05 (1H, d, 7-H, J ¼ 1.8 Hz), 7.1e7.6
(14H, m, arom), 7.26 (1H, s, CeH); 13C NMR (100 MHz, CDCl3):
182.86 (C-9), 149.10 (C-1), 147.73 (C-3), 140.35 (C-4), 140.09 (C-12),
129.77 (C-13), 129.39 (C-18 and C-14), 129.34 (C-17 and C-15),
126.21 (C-7), 125.61 (C-5), 122.63 (C-11), 120.77 (C-6), 120.44 (C-
16), 115.76 (C-7), 115.51 (C-8), 110.59 (C-2), 100.78 (C-10), 55.46
(OCH3); ESI-MS: m/z calculated for C35H27N2ClO5S 623.15, found
[M þ NH4]þ 641.1.
6.6. Synthesis of intermediate 3a
A mixture of curcumin (5 mmol) and benzaldehyde (5 mmol)
was refluxed at 60e65 ꢁC for 15 h in presence of pyridine (1 mL) as
catalysts in methanol (10 mL). The reaction was monitored by TLC.
After completion the reaction, mixture was cooled to room
temperature and poured in cold water and filtered. The residue was
purified via column chromatography on silica gel using EtOAc/
MeOH (3:1). The solid was recrystallized by ethyl acetate. Inter-
mediate 3a was collected as yellow crystals.
6.11. 4-(4-Nitro phenyl)-4H-pyrimido[2,1-b] [1,3]benzothiazole
curcumin (4d)
Yellow crystal, mp 112e113 ꢁC, Rf ¼ 0.49 (DCM/Toluene ¼ 3:2);
IR (KBr) (nmax, cmꢂ1): 3007 (CeHstr), 2922 (CeHstr), 1625 (C]Ostr),
1510 (CeHdef), 1267 (CeHdef), 1122 (CeSstr), 962e812 (CeHstr); 1H
NMR (400 MHz, CDCl3): dH 3.94 (6H, s, OCH3), 5.78 (1H, d, 6-H,
J ¼ 6.36 Hz), 6.49 (1H, d, 5-H, J ¼ 5.76 Hz), 6.93 (1H, d, 8-H,
J ¼ 3.4 Hz), 7.04 (1H, d, 7-H, J ¼ 1.76 Hz), 7.1e7.61 (14H, m, arom),
7.26 (1H, s, CeH); 13C NMR (100 MHz, CDCl3): 182.89 (C-9), 149.12
(C-1), 147.75 (C-3), 140.37 (C-4), 130.34 (C-13), 129.81 (C-18 and C-
15), 126.21 (C-7), 123.90 (C-5), 123.46 (C-11), 122.90 (C-6), 120.79
(C-16), 115.77 (C-7), 115.52 (C-8), 110.66 (C-2), 100.78 (C-10), 55.47
(OCH3); ESI-MS: m/z calculated for C35H27N3O7S 633.7, found
[M þ NH4]þ 652.1.
6.7. Synthesis of intermediate 5a
A mixture of benzaldehyde (5 mmol) and 2-amino benzothia-
zole (5 mmol) was refluxed at 60e65 ꢁC in presence of pyridine
(1 mL) as catalysts in methanol (10 mL) for 5 h. The reaction was
monitored by TLC. After completion the reaction, mixture was then
poured in cold water and filtered. The residue was purified via
column chromatography on silica gel using Hexane/MeOH (1:1).
The solid was recrystallized by ethyl alcohol. Intermediate 5a was
collected as yellow crystal.
6.12. 4-(4-Hydroxy-3-methoxy phenyl)-4H-pyrimido[2,1-b] [1,3]
6.8. 4-Phenyl-4H-pyrimido[2,1-b] [1,3]benzothiazole curcumin (4a)
benzothiazole curcumin (4e)
Yellow crystal, mp 97e98 ꢁC, Rf ¼ 0.52 (DCM/Toluene ¼ 3:2); IR
(KBr) (nmax, cmꢂ1): 3348 (CeHstr), 2933 (CeHstr), 1625 (C]Ostr),
1510 (CeHdef), 1267 (CeHstr), 1122 (CeSstr), 962e812 (CeHdef); 1H
NMR (400 MHz, CDCl3): dH 3.93 (6H, s, OCH3), 5.80 (1H, d, 6-H,
J ¼ 6.44 Hz), 6.49 (1H, d, 5-H, J ¼ 5.76 Hz), 6.87 (1H, d, 8-H,
J ¼ 8.56 Hz), 7.05 (1H, d, 7-H, J ¼ 1.8 Hz), 7.1e7.6 (15H, m, arom), 7.26
(1H, s, CeH); 13C NMR (100 MHz, CDCl3): 178.06 (C-9), 149.74 (C-1),
142.66 (C-3), 141.59 (C-4), 135.38 (C-7), 123.61 (C-5), 123.51 (C-6),
122.39 (C-11), 117.67 (C-7), 116.46 (C-8), 109.64 (C-2), 104.42 (C-10),
50.71 (OCH3); ESI-MS: m/z calculated for C35H28N2O5S 588.62,
found [M þ NH4]þ 607.2.
Yellow crystal, mp 80e81 ꢁC, Rf ¼ 0.49 (DCM/Toluene ¼ 3:2); IR
(KBr) (nmax, cmꢂ1): 3504 (OHstr), 1627 (C]Ostr); 1H NMR (400 MHz,
CDCl3): dH 3.13 (3H, s, OCH3), 3.97 (6H, s, OCH3), 5.80 (1H, d, 6-H,
J ¼ 5.6 Hz), 6.49 (1H, d, 5-H, J ¼ 5.76 Hz), 6.88 (1H, d, 8-H,
J ¼ 8.52 Hz), 7.05 (1H, d, 7-H, J ¼ 1.76 Hz), 6.92e7.62 (13H, m, arom)
7.26 (1H, s, CeH), 7.85 (1H, s, OH); 13C NMR (100 MHz, CDCl3):
182.91 (C-9), 149.15 (C-1), 147.77 (C-3), 140.38 (C-4), 126.21 (C-7),
125.08 (C-5), 122.76 (C-6), 120.82 (C-18 and C-14), 120.59 (C-16),
120.36 (C-17), 117.66 (C-16), 115.77 (C-7), 115.53 (C-8), 115.14 (C-14),
110.71 (C-2), 99.49 (C-10), 55.50 (OCH3); ESI-MS: m/z calculated for
C36H30N2O7S 634.65, found [M þ NH4]þ 652.2.

P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
377
6.13. 4-(4-Hydroxy phenyl)-4H-pyrimido[2,1-b] [1,3]benzothiazole
6.18. N-Amido pyrazole curcumin (6a)
curcumin (4f)
Yellow Powder, mp 204e206 ꢁC, Rf ¼ 0.70 (DCM/Toluene ¼ 3:1);
1H NMR (400 MHz, CDCl3): dH 1.56 (2H, s, NH2), 3.95 (6H, s, OCH3),
5.80 (2H, s, 2-H), 6.49 (2H, d, 5-H, J ¼ 15.76 Hz), 6.89e7.86 (8H, m,
AreH); 13C NMR (100 MHz, CDCl3): 182.86 (C-9), 147.56 (C-1),
146.55 (C-3), 129.42 (C-4), 128.25 (C-5), 127.29 (C-6), 119.76 (C-7),
115.46 (C-8), 115.37 (C-11), 108.89 (C-2), 98.87 (C-10), 55.38 (OCH3);
ESI-MS: m/z calculated for C22H21O5N3 407.41, found [M þ H]þ
408.3.
Yellow crystal, mp 101e102 ꢁC, Rf ¼ 0.58 (DCM/Toluene ¼ 3:2);
IR (KBr) (n
, cmꢂ1): 3504 (OHstr), 1724 (C]Ostr); 1H NMR (400 MHz,
CDCl3): dH 3.92 (6H, s, OCH3), 5.82 (1H, d, 6-H, J ¼ 8.48 Hz), 6.48 (1H,
d, 5-H, J ¼ 5.76 Hz), 6.88 (1H, d, 8-H, J ¼ 8.52 Hz), 7.05 (1H, d, 7-H,
J ¼ 1.76 Hz), 6.85e7.58 (14H, m, arom), 7.07 (1H, s, CeH), 9.73 (1H, s,
OH); 13C NMR (100 MHz, CDCl3): 182.89 (C-9), 149.11 (C-1), 147.74
(C-3), 140.36 (C-4), 129.80 (C-16), 126.22 (C-7), 122.70 (C-5), 120.79
(C-18 and C-14), 115.77 (C-7), 115.53 (C-8), 110.63 (C-2), 100.79 (C-
10), 55.47 (OCH3); ESI-MS: m/z calculated for C35H28N2O6S 604.62,
found [M þ NH4]þ 623.1.
6.19. N-Thiamido pyrazole curcumin (6b)
Yellow crystal, mp 100e101 ꢁC, Rf ¼ 0.69 (DCM/Toluene ¼ 3:1);
1H NMR (400 MHz, CDCl3): 2.04 (2H, s, NH2), 3.95 (6H, s, OCH3),
5.80 (2H, s, 2-H), 6.49 (2H, d, 5-H, J ¼ 15.76 Hz), 6.91e7.61 (6H, m,
AreH); 13C NMR (100 MHz, CDCl3): 149.32 (C-1), 147.65 (C-3),
146.65 (C-4), 129.44 (C-5), 128.25 (C-6), 127.38 (C-7), 119.84 (C-8),
115.45 (C-11), 109.08 (C-10), 55.42 (OCH3); ESI-MS: m/z calculated
for C22H21O4N3S 423.12, found [M þ H]þ 424.2.
6.14. 4-(4-Methyl phenyl)-4H-pyrimido[2,1-b] [1,3]benzothiazole
curcumin (4g)
Yellow crystal, mp 108e109 ꢁC, Rf ¼ 0.59 (DCM/Toluene ¼ 3:2);
IR (KBr) (nmax, cmꢂ1): 3292 (CeHstr), 3007 (CeHstr), 1720 (C]Ostr);
1H NMR (400 MHz, CDCl3): dH 3.13 (3H, s, CH3), 3.97 (6H, s, OCH3),
5.80 (1H, d, 6-H, J ¼ 5.6 Hz), 6.50 (2H, d, 5-H, J ¼ 5.76 Hz), 6.88 (2H,
d, 8-H, J ¼ 8.96 Hz), 7.05 (1H, d, 7-H, J ¼ 5.24 Hz), 6.92e7.85 (14H, m,
arom); 13C NMR (100 MHz, CDCl3): 182.86 (C-9), 149.08 (C-1),
147.72 (C-3), 147.57 (C-13), 146.55 (C-11), 140.33 (C-4), 129.39 (C-
16), 128.25 (C-12), 126.20 (C-7), 122.67 (C-5), 120.75 (C-6), 119.76
(C-17 and C-15), 115.75 (C-7), 115.50 (C-8), 115.37 (C-14 and C-18),
110.53 (C-2), 100.92 (C-10), 55.47 (OCH3); ESI-MS: m/z calculated
for C36H30N2O5S 602.82, found [M þ NH4]þ 623.1.
6.20. N-Carbonyl pyridine pyrazole curcumin (6c)
Yellow crystal, mp 105e106 ꢁC, Rf ¼ 0.68 (DCM/Toluene ¼ 3:1);
1H NMR (400 MHz, CDCl3): 3.91 (6H, s, OCH3), 5.88 (2H, s, 2-H), 6.56
(2H, d, 5-H, J ¼ 15.76 Hz), 6.79e7.89 (10H, m, AreH); 13C NMR
(100 MHz, CDCl3): 182.86 (C-9),149.08 (C-1),147.72 (C-3),147.57 (C-
4), 146.55 (C-pyridine ring), 140.33 (C-C-pyridine ring), 129.39 (C-
5), 128.25 (C-6), 126.20 (C-pyridine ring), 122.67 (C-pyridine ring),
120.75 (C-7), 119.76 (C-8), 115.75 (C-11), 110.53 (C-2), 108.92 (C-10),
53.47 (OCH3); ESI-MS: m/z calculated for C27H23O5N3 469.22, found
[M þ 2]þ 471.9.
6.15. 4-(2,6-Dichloro phenyl)-4H-pyrimido[2,1-b] [1,3]
benzothiazole curcumin (4h)
Yellow crystal, mp 90e92 ꢁC, Rf ¼ 0.54 (DCM/Toluene ¼ 3:2); IR
(KBr)
(nmax,
cmꢂ1): 3078 (CeHstr), 1716 (C]Ostr); 1H NMR
6.21. N-(2,4-Dinitro phenyl) pyrazole curcumin (6d)
(400 MHz, CDCl3): dH 3.90 (6H, s, OCH3), 5.87 (1H, d, 6-H, J ¼ 5.6 Hz),
6.53 (1H, d, 5-H, J ¼ 5.88 Hz), 6.77e7.02 (7H, m, arom), 7.06e7.59
(6H, m, arom), 7.74 (1H, s, CeH), 13C NMR (100 MHz, CDCl3):
182.78 (C-9), 148.91 (C-1), 147.59 (C-3), 140.26 (C-4), 133.25 (C-11),
129.57 (C-12), 127.70 (C-15), 126.22 (C-17), 125.09 (C-16), 122.54 (C-
5), 120.84 (C-6), 120.24 (C-13), 117.78 (C-7), 115.71 (C-8), 115.71 (C-
18), 115.39 (C-14), 110.11 (C-2), 100.72 (C-10), 55.42 (OCH3); ESI-MS:
m/z calculated for C35H26N2O5Cl2S 657.67, found [M þ NH4]þ 675.6.
Yellow powder, mp 118e119 ꢁC, Rf ¼ 0.68 (DCM/Toluene ¼ 3:1);
1H NMR (400 MHz, CDCl3): 3.89 (6H, s, OCH3), 5.80 (2H, s, 2-H), 6.49
(2H, d, 5-H, J ¼ 15.56 Hz), 6.86e7.53 (9H, m, AreH); 13C NMR
(100 MHz, CDCl3): 149.11 (C-1), 147.73 (C-3), 147.03 (C-4), 129.50 (C-
5), 122.71 (C-6), 120.97 (C-7), 120.43 (C-8), 115.51 (C-11), 110.60 (C-
2), 109.10 (C-10), 55.48 (OCH3); ESI-MS: m/z calculated for
C27H22O8N4 530.14, found [M þ H]þ 531.5.
6.16. 4-Benzylidene curcumin (Intemediate 3a)
6.22. 4-(4-N-Dimethyl benzylidene) curcumin (7b)
Yellow crystal, mp 120e122 ꢁC, Rf ¼ 0.54 (DCM/Toluene ¼ 3:2);
1H NMR (400 MHz, CDCl3): dH 3.93 (6H, s, OCH3), 5.80 (2H, s, C2),
6.50 (2H, d, 5-H, J ¼ 15.8 Hz), 6.67e7.15 (6H, m, arom), 7.32e7.90
(5H, m, arom), 10.03 (1H, s, ]CHeAr); 13C NMR (100 MHz,
CDCl3): 182.81 (C-9), 149.04 (C-1), 147.68 (C-3), 145.96 (C-4), 140.30
(C-11), 139.88 (C-12), 134.13 (C-13), 129.83 (C-17), 128.69 (C-14 and
C-16), 129.21 (C-15), 126.18 (C-5), 125.87 (C-6), 123.38 (C-7), 122.64
(C-8), 115.74 (C-2), 110.36 (C-10), 55.45 (OCH3); ESI-MS: m/z
calculated for C28H24O6 456.5, found [M þ H]þ 457.7.
Yellow powder, mp 104e106 ꢁC, Rf ¼ 0.67 (DCM/Toluene ¼ 3:2);
1H NMR (400 MHz, CDCl3): dH 3.13 (6H, s, N(CH3)2), 3.95 (6H, s,
OCH3), 5.80 (2H, s, 2-H), 6.50 (2H, d, 5-H, J ¼ 15.8 Hz), 6.85e7.15
(6H, m, AreH), 7.45e7.85 (4H, m, AreH), 9.84 (1H, s, ]CHeAr);
13C NMR (100 MHz, CDCl3): 182.79 (C-9), 148.76 (C-1), 147.63 (C-
3), 140.27 (C-11), 126.19 (C-12), 122.57 (C-17 and C-13), 120.69 (C-
15), 115.92 (C-14 and C-16), 110.21 (C-2), 100.72 (C-10), 55.43
(OCH3); ESI-MS: m/z calculated for C30H29O6N 499.58, found
[M þ H]þ 500.2.
6.17. Intermediate 5a
6.23. 4-(2-Hydroxy benzylidene) curcumin (7c)
Off white crystal, mp 117e119 ꢁC, Rf
¼
0.61 (Hexane/
Methanol ¼ 1:1); 1H NMR (400 MHz, CDCl3): dH 6.99 (t, 1H, eCH,
J ¼ 6.96 Hz), 7.19 (d, 2H, NH, J ¼ 6.72 Hz), 7.37e8.02 (13H, m,
AreH); 13C NMR (100 MHz, CDCl3): 166.72 (C-1), 152.51 (C-8),
132.41 (C-2), 130.86 (C-3), 128.15 (C-7), 125.25 (C-5), 120.77 (C-6),
120.52 (C-4), 117.69 (C-9), 78.97 (C-10), 78.64 (C-11 and C-14), 78.31
(C-12 and C-13); ESI-MS: m/z calculated for C21H16N4S2 388.52,
found [M ꢂ H]þ 387.2.
Yellow crystal, mp 92e94 ꢁC, Rf ¼ 0.65 (DCM/Toluene ¼ 3:2); 1H
NMR (400 MHz, CDCl3): dH 2.55 (3H, s, CH3), 3.89 (6H, s, OCH3), 5.92
(2H, s, 2-H), 6.57 (2H, d, 5-H, J ¼ 12.12 Hz), 6.72 (2H, d, 8-H,
J ¼ 8.0 Hz), 6.97e7.77 (12H, m, AreH); 13C NMR (100 MHz, CDCl3):
188.10 (C-9), 154.32 (C-1), 152.96 (C-3), 145.57 (C-11), 134.98 (C-12),
131.45 (C-13 and C-17), 127.91 (C-5), 126.00 (C-6), 121.00 (C-7),
120.74 (C-8), 115.77 (C-14 and C-16), 110.21 (C-2), 105.99 (C-10),

378
P.K. Sahu et al. / European Journal of Medicinal Chemistry 54 (2012) 366e378
R. Snoeck, J. Balzarini, A.A. Rashad, Eur. J. Med. Chem. 44 (2009)
3746e3753.
60.71 (OCH3); ESI-MS: m/z calculated for C29H26O6 470.53, found
[M þ 2]þ 472.3.
[13] (a) J. Ishida, H. Ohtsu, Y. Tachi Bana, Y. Nakanishi, K.F. Bastow, M. Nagai,
H.K. Wang, H. Itokawa, K.H. Lee, Bioorg. Med. Chem. 10 (2002) 3481e3487;
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(d) Y. Xia, C.D. Fan, B. Zhao, X. Zhao, D.S. Shin, J.Y. .Miao, Eur. J. Med. Chem. 43
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245e249.
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R. Cirilli, Eur. J. Med. Chem. 45 (2010) 6135e6138.
[16] A.A. Fadda, E. Abdel-Latif, R.E. El-Mekawy, Eur. J. Med. Chem. 44 (2009)
1250e1256.
[17] R. Kasimogullari, M. Bulbul, B.S. Arslan, B. Gokce, Eur. J. Med. Chem. 45 (2010)
4769e4773.
6.24. 4-(4-Hydroxy-3-methoxy benzylidene) curcumin (7d)
Yellow crystal, mp 98e100 ꢁC, Rf ¼ 0.64 (DCM/Toluene ¼ 3:2);
1H NMR (400 MHz, CDCl3): dH 3.25 (3H, s, OCH3), 3.93 (6H, s, OCH3),
5.80 (2H, s, 2-H), 6.49 (2H, d, 5-H, J ¼ 15.76 Hz), 6.87 (2H, d, 8-H,
J ¼ 8.52 Hz), 6.92e7.61 (11H, m, AreH), 9.84 (1H, s, OH); 13C NMR
(100 MHz, CDCl3): 182.85 (C-9), 149.05 (C-1), 147.70 (C-3), 140.34
(C-11), 126.21 (C-12), 122.65 (C-7), 120.78 (C-8), 115.49 (C-14 and C-
16), 110.49 (C-10), 55.45 (OCH3); ESI-MS: m/z calculated for
C29H26O8 502.53, found [M þ NH4]þ 521.1.
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We gratefully acknowledge to Mr. Yogesh Sharma, Parabolic
drugs Ltd., Chandigarh and SAIF Punjab University, Chandigarh for
spectral analytical data.
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