Synthesis and biological evaluation of arylidene analogues of Meldrum's acid as a new class of antimalarial and antioxidant agents

Article abstract of DOI:10.1016/j.bmc.2010.06.033

A series of arylidene analogues of Meldrum's acid were synthesized and evaluated for in vitro antimalarial and antioxidant activities for the first time. The influence of various physico-chemical parameters such as dielectric constant (), donor number (DN

Full text of DOI:10.1016/j.bmc.2010.06.033

Bioorganic & Medicinal Chemistry 18 (2010) 5626–5633
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of arylidene analogues of Meldrum’s acid
as a new class of antimalarial and antioxidant agents
Harmeet S. Sandhu a, Sameer Sapra a, Mukesh Gupta a, Kunal Nepali a, Raju Gautam b, Sunil Yadav a,
b
c
a
a
a
Raj Kumar a, , Sanjay M. Jachak , Manoj Chugh , Manish K. Gupta , Om P. Suri , K. L. Dhar
*
a Laboratory for Drug Design and Synthesis, Department of Pharmaceutical Chemistry, Indo-Soviet Friendship (ISF) College of Pharmacy, Moga 142 001, Punjab, India
b Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, SAS Nagar, Mohali 160 062, Punjab, India
c National Institute of Pharmaceutical Education and Research (NIPER), C/O B. V. Patel PERD Centre, Ahmedabad, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of arylidene analogues of Meldrum’s acid were synthesized and evaluated for in vitro antimalar-
ial and antioxidant activities for the first time. The influence of various physico-chemical parameters such
as dielectric constant (e), donor number (DN), acceptor number (AN), hydrogen bond donor (HBD), hydro-
gen bond acceptor (HBA), and solubilizing power of the solvents on Meldrum’s acid anion generation and
thus on promoting the Knoevenagel condensation of Meldrum’s acid with aryl aldehydes has been dis-
cussed. Five compounds 9l, 9m, 9n, 9r, and 9s were found to be most active against Plasmodium falcipa-
Received 4 May 2010
Revised 10 June 2010
Accepted 11 June 2010
Available online 17 June 2010
Keywords:
Antimalarial
Antioxidant
Arylidene analogues of Meldrum’s acid
Knoevenagel condensation
rum with IC50 values in the range of 9.68–16.11
activity (IC50 9.68 M). The compounds were also found to possess antioxidant activity when tested
against DPPH and ABTS free radicals.
lM. Compound 9l exhibited the most potent antimalarial
l
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
parum malaria infection oxidative stress is increased and relates to
severity of disease and anemia.9,10 Therefore, the antimalarial
drugs with antioxidant action are of considerable interest due to
their additional benefit to the treatment.
Malaria is a tropical parasitic disease and one of the top three
killers among communicable diseases.1 It is a public health prob-
lem in more than 90 countries inhabited by about 40% of the
world’s population. According to recent WHO reports, it affects
approximately 250 million people and kills about one million peo-
ple every year.2 Among four species of human malarial parasite,
Plasmodium falciparum is considered most fatal due to high mortal-
ity rate particularly in children. Several drugs are being used in ma-
laria-endemic regions of the world to control, treat, and prevent
malaria; however, the emergence and spread of antimalarial drug
resistance has made the malaria treatments ineffective and is
therefore, a global problem.3 Drug resistance can be defined as
the ability of a parasite strain to survive and/or multiply in the
presence of a drug administered in doses equal to or higher than
those usually recommended.4 Resistance of P. falciparum has
emerged to all classes of antimalarial drugs (chloroquine (1), pri-
maquine, sulfadoxine, pyrimethamine, etc.) except artemisinin
(2)5–8 (Fig. 1). The emergence of resistance of P. falciparum has pro-
vided a fresh impetus to the researchers for the development of a
better and safe antimalarial drug. Recently, the use of antioxidants
in the malaria as an adjunct treatment has generated a renewed
interest. Several reports have evidenced that in patients with falci-
The various classes such as pyrimidines,11 indole,12 chalcone,13
xanthone,14 pyrazoline,15 propenone,16 etc., are reported to exhibit
antimalarial activity. Licochalcone (3),17 chalcones-like retinoid
H
Et
Et
HN
N
O
H
O
O
O
Cl
N
2
1
O
O
O
O
N
HO
HO
OH
3
4
O
O
O
O
OH
O
O
O
O
6
5
* Corresponding author. Tel./fax: +91 1636 236564.
E-mail address: raj.khunger@gmail.com (R. Kumar).
Figure 1. Chemical structures of antimalarial molecules.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.033

H. S. Sandhu et al. / Bioorg. Med. Chem. 18 (2010) 5626–5633
5627
(4),18 and various other chalcones (5 and 6)16,19 (Fig. 1) are re-
ported to possess promising antimalarial activity. It was noticed
that chalcones with higher number of methoxyl groups exhibited
potent antimalarial activity.
O
O
O
O
R
R
O
O
H
+
O
O
O
O
In view of the difficulty in making peroxides besides being more
toxic it was thought worthwhile to design a compound which
could react with Fe+2 of heme or free Fe+2 in a similar way, we con-
ceptualized a molecule with structural features where important
functionalities are equally placed as in artemisinin. The most suit-
able molecule we could design is in starting with an arylidene ana-
logue of Meldrum’s acid where two oxygen and one carbon atoms
of the six membered ring of Meldrum’s acid are exactly positioned
with two oxygen and one carbon atoms of the six membered ring
of artemisinin where one has to ignore the structural features
while emphasizing on the shape of the molecule. We could visual-
ize the ease of a ketal to bind with Fe+2 and proved to be correct
(Fig. 2). The novelty of this molecule appears to be that site of
the molecule where it binds with Fe+2 is symmetrical with the re-
sult that only one type radical is capable of interacting with Fe+2
while in artemisinin two types of radicals are possible.7
8
7
9
O
R
O
O
O
10
Figure 3. Proposed synthetic route of arylidene and epoxide derivatives of
Meldrum’s acid.
benzene,22c,d piperidine22e, pyrrolidine/acetic acid in dry ben-
zene,22f NAP22g (3-aminopropylated silica gel), a potassium ex-
changed zirconium hydrogen phosphate Zr(O3POK)2
a
heterogeneous basic catalyst22h or by Lewis acid such as anhydrous
ZnCl2,23 or in ionic liquids,24 or in solvents such as water25 under
heating for 2 h, DMF under heating at 80 °C26,27 and DMSO,27 or
using surfactants28 or under microwave irradiation,29 or melt con-
dition.30 However, these methodologies have one or more disad-
vantages such as the use of high boiling solvent (e.g., DMF,
DMSO, and benzene) that are difficult to recover, use of acid or
base catalyst, special efforts required to prepare the catalysts
(e.g., NAP and Zr(O3POK)2) or to prepare the polar medium (e.g., io-
nic liquids), need to use special apparatus (e.g., microwave irradi-
ation) and heating conditions. Thus, there is necessity to develop a
more effective and convenient synthetic procedure.
In the present communication the synthesis of various arylid-
ene analogues of Meldrum’s acid, the corresponding epoxides
(Fig. 3) and their biological evaluation as antimalarial and antioxi-
dant agents are reported for the first time.
2. Results and discussion
2.1. Optimization of reaction conditions for the synthesis of
arylidene analogues of Meldrum’s acid
Arylidene analogues (9) of Meldrum’s acid are reported as the
key intermediates for cycloaddition reactions and for the synthesis
of heterocyclic compounds of biological importance such as cardio-
tonic20a and HIV integrase inhibitory activities.20b They are also re-
ported to serve as the versatile substrates for various kinds of
reactions.21
While planning for the synthesis of 9, it was realized that a con-
venient synthetic protocol is lacking. The synthesis of 9 via Knoe-
venagel condensation of 7 with aldehydes has been documented
either catalyzed by bases such as pyridine under reflux and water
removal by Dean–Stark,22a,b piperidine/glacial acetic acid in
It is known that 7 exists in a boat form and its corresponding
anion formed due to ease of deprotonation of one of the more
acidic (flagpole) methylene hydrogens in a polar medium, is stabi-
lized by extensive conjugation.31 The reported condensation in po-
lar medium24–27,31 encouraged us to screen various solvents of
different dielectric constants32
In
(e
), (Scheme 1, Table 1).
a
model reaction, 3,4,5-trimethoxybenzaldehyde (8i,
1 mmol) was treated with 7 (1 mmol) under solvent-free condi-
tions at rt (ꢀ30–35 °C) for 12 h that did not result in the significant
formation (5%) of Knoevenagel product 2,2-dimethyl-5-(3,4,5-tri-
Figure 2. (a) Chemical structures of arylidene analogue of Meldrum’s acid and artemisinin; (b) energy minimized structures of arylidene analogue of Meldrum’s acid (blue)
and artemisinin (yellow); and (c) overlay of arylidene analogue of Meldrum’s acid and artemisinin (carbon: black, oxygen: red, overlay atoms are shown in ball). Hydrogens
are removed for clarity.

5628
H. S. Sandhu et al. / Bioorg. Med. Chem. 18 (2010) 5626–5633
have higher
e
, higher DN but lesser AN, comparable HBA, and nil
, higher AN, com-
O
O
O
O
OMe
OMe
OMe
OMe
HBD than MeOH; whereas the water has highest
e
i
H
O
O
+
O
parable HBD but lesser DN (data not reported), lesser HBA and poor
solubility of starting materials at rt and hence retarded the rate of
reaction. This was further supported by the fact that the formation
of the condensed product was enhanced from 35% to 66% and 68%
while carrying out the reaction under reflux for 2 h or in surfactant
(sodium dodecyl sulfate; 100 mol %) at rt for 2 h, respectively, indi-
cating the influence of solubility. The results were found in good
agreement with the previously reported studies.25,28
O
O
OMe
8i
OMe
7
9i
Scheme 1. Reagents and conditions: (i) solvent, rt, 15 min.
Table 1
Knoevenagel condensation of 7 with 8ia
Thus we can anticipate that the high e of MeOH makes it a polar
medium with the ability to donate hydrogen bond (HBD), accept
hydrogen bond (HBA), optimum Lewis acidity (AN 41.5), Lewis ba-
sicity (DN 20.0) and solubility of the starting materials at rt during
Knoevenagel condensation make it an ideal coordinating solvent
with the 7 and that results into Meldrum’s acid anion generation
11 via a polar six membered transitions state TS (Scheme 2) fol-
lowed by condensation with aldehyde 8 to yield the dehydrated
product 9 as the target compound.
Entry Solvent
eb
DNc
ANc
HBD
HBA
Yieldf (/
%)
d
(
a
)
(b)e
1
2
3
4
5
6
7
8
9
Neat
5 (15)g
10
13
17
19
23
21
32
18
PhMe
Hexane
CHCl3
DCM
2.4
1.9
4.8
—
—
—
—
—
—
—
—
—
—
—
—
0.481
—
0.0
23.1 Nil
20.4
16.7
9.3
8.9
—
—
—
DCE
10.3
0.0
EtOAc
MeCN
1,4-
Dioxane
Et2O
6.0 17.1
36.6 14.1
2.2 14.8
18.9 0.29
10.8
—
0.386
2.2. Synthesis of arylidenes and the corresponding epoxides
10
11
12
13
14
15
16
17
18
4.3 19.2
7.6 20.0
38.3 26.6
47.2 29.8
3.9
8.0
16.0
19.3
—
—
—
—
0.488
0.523
0.710
0.752
0.70
15
0.73
0.80
0.47
24
37
By using optimized procedure, we synthesised compounds 9a
and 9d–w (85–96%) via Knoevenagel condensation of 7 (1 equiv)
with aldehydes (8a and 8d–w; 1 equiv) in MeOH at rt (Scheme 3,
Fig. 4) in 15–45 min. Various aromatic and heteroaromatic alde-
hydes condensed smoothly. The crude products were purified by
column chromatography and were characterized by using spectro-
scopic techniques such as IR, NMR, and mass spectrometry. The
reaction was found to be compatible with diverse functional
groups such as ether, nitro, hydroxyl and halogens.
It was noticed that the reaction of 7 with aldehydes bearing
electron withdrawing group(s) irrespective of their position on
aromatic ring (8b and 8c) in MeOH yielded a mixture of Knoevena-
gel product (9b and 9c), aldol product (12b and 12c) and Michael
adduct (13b and 13c) (Scheme 2). Similar results were obtained
in carrying out the reaction with water under heating for 2 h which
were in accordance with earlier observations reported by Bigi
et al.25 The reason why electron donating substituents on aromatic
ring accelerate the last step of Knoevenagel condensation and elec-
tron withdrawing groups accelerate the first step of Knoevenagel
condensation is still unclear and is a question of study.22e,25
Though the various approaches such as methoxide,36 amines,37
and thiols38 are utilized to minimize the formation of Michael ad-
duct 13 but this is an unsatisfactory solution in terms of atom and
step economy. However we succeeded to synthesize the desired
compounds 9b and 9c with excellent selectivity (>96) when the
reaction of 7 (1 equiv) with 8b and 8c (1 equiv each) was carried
out separately under the catalytic influence of type 3 Å MS
(50 mol %) in MeOH under reflux for 3 h. Two epoxide derivatives
10a and 10b (Scheme 4, Fig. 4) and one 2-benzylidenecyclohex-
ane-1,3-dione (14), respectively, were synthesized as per the re-
ported procedures.39,40
THF
DMF
48 (65)g
51 (71)g
96
DMSO
MeOH
Dry MeOH
EtOH
iPrOH
H2O
32.7 20.0b 41.5 0.990
93
88
24.6 19.0b 37.1 0.850
9.9
80
—
—
33.5 0.687
54.8 1.017
82
35 (66)g
a
8i (1 mmol) was treated with 7 (1 mmol) in the solvent (1 mL) at room tem-
perature (30–35 °C).
b
Values were taken from Ref. 32a.
Values were taken from Ref. 33.
Values were taken from Ref. 34.
Values were taken from Ref. 35.
Determined by IR and NMR.
The figure in parenthesis is the yield obtained after heating the reaction mixture
c
d
e
f
g
for 2 h at 80 °C.
methoxybenzylidene)-[1,3]-dioxane-4,6-dione (9i) (Scheme 1),
indicating the requirement of a solvent for the reaction.
The reactions were performed in solvents of different polarity.
The importance of solvent polarity was demonstrated by the obser-
vations that excellent results were obtained in MeOH having the
highest value of
the solvents used at rt after 15 min (96%) (Table 1). The product
yield decreased with the decrease of of the alcohols and a similar
e (except water, MeCN, DMF and DMSO) of all
e
trend was also observed for the ethereal solvents. The Knoevenagel
adduct 9i was formed in 18%, 24%, and 37% yields after 15 min in
1,4-dioxane, Et2O, and THF, respectively. The poor yields were ob-
tained using the hydrocarbon and halogenated solvents due to
their low
e values and low solubility of starting materials in them.
The use of MeOH under non-anhydrous and anhydrous conditions
resulted in comparable yields and provided an added advantage to
this new methodology in eliminating additional efforts required in
drying the solvent.
2.3. Biological evaluation of synthesized compounds for
antimalarial activity
Lesser yields obtained with the use of MeCN, DMF, DMSO, and
water signified the role of various factors other than e in promoting
In vitro screening of all the compounds (9a–x, 10a, 10b, and 14)
was carried out for antimalarial activity using histidine-rich pro-
tein 2 (HRP-2) double site sandwich enzyme-linked immunosor-
bent assay (ELISA) with the previously reported method.41 Each
compound was tested in triplicate. None of the compounds showed
any cytotoxic activity (IC50 value higher than 100 lM after 48 h).
Among the series of 27 compounds, five compounds 9l, 9m, 9n,
9r, and 9s were found to be most active against the resistant strain
the condensation (Table 1). The involvement of various factors
such as donor number33 (DN), acceptor number33 (AN), hydrogen
bond donor34 (HBD, ), and hydrogen bond acceptor35 (HBA, b) of
a
the solvents and their influence in Meldrum’s acid anion genera-
tion and hence on Knoevenagel condensation was also considered.
Solvent MeCN, in spite of having higher
e (36.6) than MeOH (32.7),
afforded only 32% product which could be probably due to its low
DN, poor AN, poor HBD, and nil HBA. Similarly, DMF and DMSO
of P. falciparum with IC50 ranging from 9.68 lM to 16.11 lM

H. S. Sandhu et al. / Bioorg. Med. Chem. 18 (2010) 5626–5633
5629
H
O
Me
O
H
Me
R
Me
H
ROH
H
Me
O
O
O
O
O
O
H
O
O
H
O
TS
O
O
7
anion
11
R = H or any alkyl group
O
H
A
8
A
O
O
OH
O
O
O
8
O
O
O
A
O
A
O
O
O
O
H2O
O
O
13
O
9
12
Scheme 2. Knoevenagel condensation of 7 with 8.
O
O
O
O
O
O
O
R
R
O
i
O
i
+
O
O
O
H
A
O
A
O
O
7
O
O
O
9a-9x
8a-8x
(9a and 9b)
R = H; 10a
10b
R = NO2;
Scheme 3. Reagents and conditions: (i) For 9a and 9d–9w: MeOH, rt, 15–45 min,
85–96% and for 9b and 9c: MeOH, 3 Å MS, reflux, 3 h, 77–81%.
Scheme 4. Reagents and conditions: (i) H2O2, MeCN, rt, 10 min to 1 h, 58–62%.
(Table 2). Compound 9l exhibited the most potent antimalarial
activity (IC50 9.68 lM).
(9b, 9c, and 9d–p). Replacement of phenyl group with more bulky
groups such as 1-naphthyl (9s) and 2-naphthyl (9r), resulted in
enhancement of antimalarial activity (except 2-methoxy-1-naph-
thyl; 9u and anthranyl; 9t). The compounds with hydroxyl group
on phenyl ring at p-position (9l) showed better activity than those
with m-position (9n). The replacement of the phenyl ring with
2-theinyl (9v) or 3-indolyl (9w) did not result in increase of the
activity. The conversion of the arylidene analogues into their
2.4. Structure–activity relationship (SAR)
The compounds bearing electron withdrawing substituents on
phenyl moiety of ring A irrespective of their position, were found
to be inactive or less active than electron donating substituents
O
O
O
O
R
O
O
O
O
R1
R4
R1
R2
R2
R3
O
O
O
O
O
R
O
O
O
9a
, R = H
9c
, R = Cl
9g
, R1, R2 = OCH3
9k
, R1, R2, R3 = OCH3, R4 = H
R1 = H, R2, R3, R4 = OCH3
R1, R3, R4 = OCH3, R2 = H
9b
9d
, R = OH
, R = NO2
9l
,
R1 = OCH3, R2 = OH
9i
,
9e
, R = OCH3
9m
, R1 = OCH2CH3, R2 = OH
, R1 = OH, R2 = OCH3
9j
,
9f
, R = N(CH3)2
9n
9o
9p
9q
9h
, R1, R4 = OCH3, R2, R3 = H
, R1 = OCH3, R2 = OCH2Ph
, R1 = OCH2Ph, R2 = OCH3
, R1 + R2 = O-CH2-O
O
O
O
O
O
O
O
O
O
O
O
A
O
O
A
O
9x
14
10a
S
9r
9t
9v
9s
9u
O
O
NO2
O
O
O
O
9w
N
H
10b
MeO
Figure 4. Chemical structures of the synthesized compounds.

5630
H. S. Sandhu et al. / Bioorg. Med. Chem. 18 (2010) 5626–5633
Table 2
Table 3
In vitro screening of the synthesized compounds (9a–9x, 10a–10b
and 14) for antimalarial activity
Antioxidant activity of synthesized compoundsa
Compound
Antioxidant activity
(% Inhibition at 100
M)a
Compound Antimalarial
activity
P. falciparuma (IC50
M)
,
l
(IC50
DPPH
,
l
M)
l
DPPH
ABTS
ABTS
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
37.82
39.63
>100
34.64
36.37
35.21
26.05
33.20
20.21
33.94
34.68
9.68
15.54
12.44
37.21
36.06
28.09
14.34
16.11
>100
31.84
>100
41.72
24.31
>100
>100
>100
1.03
9b
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9q
9r
6.41 1.11
5.12 0.76
5.81 0.37
10.32 1.45
4.74 0.38
12.17 0.12
3.24 0.89
6.23 0.47
5.72 1.79
76.02 0.12
66.15 1.11
40.25 1.03
10.68 1.58
8.20 0.51
20.62 1.41
8.88 0.37
8.76 0.45
9.48 2.82
6.32 0.42
1.79 0.77
53.07 0.97
99.48 1.05
99.43 0.12
1.30 0.26
14.40 1.46
9.26 1.64
13.89 0.62
6.84 0.61
13.79 1.30
5.33 0.82
9.86 0.53
12.99 1.66
92.59 1.60
95.56 0.78
52.76 2.54
3.62 0.90
8.25 0.70
17.01 1.02
7.55 0.52
10.67 0.56
4.73 0.66
3.77 1.60
3.12 0.78
20.24 1.84
99.12 0.09
98.03 1.17
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
23.99
39.10
80.03
—
—
—
—
—
—
—
30.48
30.65
72.16
—
—
—
—
—
—
—
9s
9t
9u
9v
9w
9x
Trolox
AA
9s
9t
—
—
15.72
22.44
—
—
16.23
25.52
9u
9v
9w
9x
10a
10b
14
a
Values are expressed as means SEM of three independent experiments.
1
2
Chloroquine
Artemisinin
2.8 nM
Table 4
Antiradical power and stoichiometry from the DPPH assay
a
Values are means of three experiments.
Compound
Parameters of DPPH assay
ED50 ARP
0.39
Stoichiometric value H atoms per molecule
Olefinic linkage
9l
9m
9n
2.56 0.78
1.23
0.76
0.37
1.92
1.35
0.65
1.33
0.26
1.53 1.30
O
Phenyl ring with electron
0.75 2.66
3.84 0.52
2.70 0.74
donating
substituents
O
Trolox
A
(prefferably OH group at p-
Ascorbic acid 0.37
O
O
position)
Two oxygen atoms in the ring
2.5.1. Free radical scavenging effects in DPPH assay
Figure 5. Essential structural requirements for antimalarial activity.
The free radical scavenging effect of the synthesized com-
pounds were tested in DPPH assay.45,46 Initially, all the compounds
corresponding epoxides (9a and 9b) resulted in loss of the antima-
larial activity indicating the importance of olefinic linkage. Further
no activity was observed with 14 highlighting the significance of
two oxygen atoms. These observations helped us to formulate a ba-
sic pharmacophore with the structural features required for anti-
malarial activity (Fig. 5).
were tested at 100 lM concentration and their results are shown in
Table 3. Three compounds 9l, 9m, and 9n were found to be strong
free radical scavengers with 76.0%, 66.1%, and 40.2% scavenging of
the DPPH activity. All other compounds were found to have very
weak radical scavenging activity. The study was further extended
to determine the scavenging effects of 9l, 9m, and 9n at different
concentrations and to calculate the IC50 values and various other
parameters (Table 3 and Table 4).
2.5. Biological evaluation for antioxidant activity
Compound 9l was found to exhibit comparable IC50 and ED50
values (Table 4) with that of ascorbic acid while it was less active
than that of trolox. Compounds 9m and 9n were less active than 9l.
Compound 9l was found to exhibit the reduction of more than one
molecule of DPPH per molecule of 9l; whereas 9m and 9n demon-
strated the reduction of less than one molecule of DPPH/molecule.
The standard trolox demonstrated the reduction of about two mol-
ecules of DPPH/molecule. Thus, it can be concluded that com-
pounds 9l showed the comparable activity as that of ascorbic acid.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azino bis(3-eth-
ylbenzothiazoline-6-sulfonic acid) (ABTS) radicals42,43 are com-
monly used for assessing the antioxidant property of substances
of natural or synthetic origin because of their good reproducibility.
Both assays measure the decrease in absorbance of radicals at a
characteristic wavelength during the reaction with antioxidant;
which reverses the formation of ABTS radical cation and DPPH
radical:
DPPHꢁ þ AH ! DPPH þ Aꢁ
2.5.2. Free radical scavenging effects in ABTS assay
ABTSÅ+ cation radical is commonly used to determine the hydro-
ABTSꢁþ þ AH ! ABTSþ þ Aꢁ
gen-donating ability of antioxidants.47,48 Initially, all the com-
It is well known that ABTS activity is closely related with
DPPH44 because both are responsible for the same chemical prop-
erty of hydrogen or electron donation.
pounds were tested at 100 lM concentration and their results are
shownin Table 3. Thesimilar results were also obtained in this assay.
Compound 9l, 9m, and 9n showed the 92.5%, 95.5%, and 52.7%

H. S. Sandhu et al. / Bioorg. Med. Chem. 18 (2010) 5626–5633
5631
scavenging of the ABTS free radical, respectively; while the other
compounds were weak radical scavengers. The IC50 values of 9l,
9m, and 9n were found as 30.4, 30.6, and 72.1 lM, respectively
(Table 4).
5-(1H-Indol-3-ylmethylene)-2,2-dimethyl-[1,3]dioxane-4,6-dione
(9w),20b 2,2-dimethyl-5-(3-phenyl-allylidene)-[1,3]dioxane-4,6-dione
(9x),25 6,6-dimethyl-2-phenyl-1,5,7-trioxaspiro[2.5]octane-4,8-dione
(10a),39 6,6-dimethyl-2-(2-nitro-phenyl)-1,5,7-trioxaspiro[2.5]octane-
4,8-dione (10b),39 2-benzylidenecyclohexane-1,3-dione (14),40
were found to be identical with those reported in the literature.
The physical data of new compounds are provided below.
3. Conclusions
The present study was initiated with the aim of providing an
efficient and convenient method for the synthesis of arylidene ana-
logues of Meldrum’s acid. The optimization of the synthetic proce-
dure was carried out and the effect of the various factors such as
donor number (DN), acceptor number (AN), hydrogen bond donor
4.1.1. 5-Anthracen-9-ylmethylene-2,2-dimethyl-[1,3]dioxane-
4,6-dione (9t)
Yellow solid; mp 182–184 °C; IR (KBr): mmax 1732, 1596,
1363 cmꢂ1 1H NMR (400 MHz, CDCl3): d 1.91 (s, 6H), 7.52 (m,
;
4H), 7.82 (m, 2H), 8.06 (m, 2H), 8.55 (s, 1H), 9.47 (s, 1H); 13C
NMR (100 MHz, CDCl3): d 28.15, 104.94, 121.03, 124.50, 125.56,
125.71, 126.80, 127.06, 128.63, 129.32, 130.25, 130.94, 157.72;
MS-ESI: m/z 355.07 [M+Na]+. Anal. Calcd for C21H16O4: C, 75.89;
H, 4.85. Found: C, 76.12; H, 5.01.
(HBD, a), and hydrogen bond acceptor (HBA, b) and the solubility
on the reaction conditions was also studied. The MeOH emerged
as the best suitable solvent for carrying out the Knoevenagel con-
densation of Meldrum’s acid with aldehydes. Following the opti-
mized procedure twenty four analogues of Meldrum’s acid were
synthesized out of which six were found as new. The synthesized
compounds were evaluated for antimalarial (against P. falciparum)
and free radical (DPPH and ABTS) scavenging activities. SAR study
revealed that: (i) nature of substituents present on phenyl ring, (ii)
an olefinic double bond, and (iii) two oxygen atoms are critical
requirements for high antimalarial activity. Further the determina-
tion of mode of action of arylidene analogues of Meldrum’s acid
and a detailed target analysis is currently in progress.
4.1.2. 5-(2,5-Dimethoxybenzylidene)-2,2-dimethyl-1,3-dioxane-
4,6-dione (9h)
Yellow solid; mp 104–106 °C; IR (KBr): mmax 1726, 1585,
1377 cmꢂ1 1H NMR (400 MHz, CDCl3): d 1.81 (s, 6H), 3.80 (s,
;
3H), 3.85 (s, 3H), 6.87 (d, 1H, J = 9.12 Hz), 7.08 (dd, 1H,
J12 = 3.12 Hz, J13 = 9.12 Hz), 7.68 (d, 1H, J = 3.12 Hz), 8.73 (s, 1H);
13C NMR (100 MHz, CDCl3): d 27.51, 55.89, 56.08, 104.43, 112.03,
115.04, 116.14, 121.49, 122.12, 152.38, 152.87, 154.41, 160.10,
163.32; MS-ESI: m/z 314.93 [M+Na]+. Anal. Calcd for C15H16O6: C,
61.64; H, 5.52. Found: C, 61.39; H, 5.87.
4. Experimental
The reagents were purchased from Sigma–Aldrich, Loba and
CDH, India and used without further purification. All yields refer
to isolated products after purification. Products were characterized
by comparison with authentic samples and by spectroscopic data
(IR, 1H NMR, 13C NMR spectra). The NMR spectra were recorded
on a Bruker Avance DEX 400 MHz instrument. The spectra were
measured in CDCl3 relative to TMS (0.00 ppm). IR (KBr pellets)
spectra were recorded on a Fourier transform infrared (FT-IR) Ther-
mo spectrophotometer. Melting points were determined in open
capillaries and were uncorrected.
4.1.3. 5-(3-Ethoxy-4-hydroxybenzylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (9m)
Yellow solid; mp 84–87 °C; IR (KBr): mmax 3366, 1748, 1698,
1546 cmꢂ1 1H NMR (400 MHz, CDCl3): d 1.5 (t, 3H, J = 6.92 Hz),
;
1.79 (s, 6H), 4.23 (q, 2H, J = 6.92 Hz), 6.45 (OH, D2O exchangeable,
1H), 7.01 (d, 1H, J = 8.24 Hz), 7.54 (d, 1H, J = 8.24 Hz), 8.35 (s, 1H),
8.39 (s, 1H); 13C NMR (100 MHz, CDCl3): d 14.63, 27.47, 64.88,
104.16, 110.14, 114.52, 116.05, 124.73, 133.41, 145.59, 152.22,
158.66, 160.80, 164.23; MS-ESI: m/z 315.20 [M+Na]+. Anal. Calcd
for C15H16O6: C, 61.64; H, 5.52. Found: C, 61.33; H, 5.76.
4.1. Typical experimental procedure for the synthesis of 9a and
9d–w: 2,2-Dimethyl-5-(3,4,5-trimethoxybenzylidene)-
[1,3]dioxane-4,6-dione (9i)21d
4.1.4. 5-(2-Methoxy-naphthalen-1-ylmethylene)-2,2-dimethyl-
[1,3]dioxane-4,6-dione (9u)
Yellow solid; mp 177–179 °C; IR (KBr): mmax 1732, 1597,
1364 cmꢂ1 1H NMR (400 MHz, CDCl3): d 1.91 (s, 6H), 3.97 (s,
;
A mixture of 7 (1 mmol, 1 equiv, 0.14 g) and 8i (1 equiv, 0.19 g)
in methanol (1 mL) was stirred at room temperature. The progress
of the reaction was monitored by TLC (15 min). After the comple-
tion of reaction, the precipitate was filtered-off and dried to afford
9i (0.30 g, 96%). The remaining reactions were carried out follow-
ing these general procedures. In each occasion, the spectral data
(IR, 1H NMR and 13C NMR) of known compounds such as
5-benzylidene-2,2-dimethyl-[1,3]dioxane-4,6-dione (9a),28 5-ben-
zylidene-2,2-dimethyl-[1,3]dioxane-4,6-dione (9a),28 5-(4-hydrox-
ybenzylidene)-2,2-dimethyl-[1,3]dioxane-4,6-dione (9d),25 5-(4-
methoxybenzylidene)-2,2-dimethyl-[1,3]dioxane-4,6-dione(9e),25
5-(4-dimethylaminobenzylidene)-2,2-dimethyl-[1,3]dioxane-4,6-
dione (9f),24a 5-(3,4-dimethoxybenzylidene)-2,2-dimethyl-[1,3]
dioxane-4,6-dione (9g),24a 5-(4-hydroxy-3-methoxybenzylidene)-
2,2-dimethyl-[1,3]dioxane-4,6-dione (9l),22f 5-(3-hydroxy-4-meth-
oxybenzylidene)-2,2-dimethyl-[1,3]dioxane-4,6-dione (9n),20b 5-
benzo[1,3]dioxol-5-ylmethylene-2,2-dimethyl-[1,3]dioxane-4,6-
dione (9q),22c 5-(2,3,4-trimethoxybenzylidene)-2,2-dimethyl-
[1,3]dioxane-4,6-dione (9k),21e 5-(2,4,5-trimethoxybenzylidene)-
2,2-dimethyl-[1,3]dioxane-4,6-dione (9j),21e 2,2-dimethyl-5-naph-
thalen-2-ylmethylene-[1,3]dioxane-4,6-dione (9r),22f 2,2-dimethyl
-5-naphthalen-1-ylmethylene-[1,3]dioxane-4,6-dione (9s),22f 2,2-
dimethyl-5-thiophen-2-ylmethylene-[1,3]dioxane-4,6-dione (9v),21d
3H), 7.29 (d, 1H, J = 9.16 Hz), 7.44 (m, 1H), 7.57 (m, 1H), 7.83 (m,
2H), 7.98 (d, 1H, J = 9.16 Hz), 8.84 (s, 1H); 13C NMR (100 MHz,
CDCl3): d 27.48, 56.04, 104.67, 112.66, 115.61, 119.56, 123.39,
124.60, 128.22, 128.52, 128.74, 132.36, 134.06, 149.64, 155.72,
159.99, 162.91; MS-ESI: m/z 335.00 [M+Na]+. Anal. Calcd for
C18H16O5: C, 69.22; H, 5.16. Found: C, 69.41; H, 5.51.
4.1.5. 5-(3-Methoxy-4-benzyloxybenzylidene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (9o)
Yellow solid; mp 186–188 °C; IR (KBr): mmax 1701, 1596,
1364 cmꢂ1 1H NMR (400 MHz, CDCl3): d 1.79 (s, 1H), 3.99 (s, 3H),
;
5.23 (s, 2H), 6.97 (d, 1H, J = 8.60 Hz), 7.32–7.51 (m, 5H), 7.65 (dd, 1H,
J12 = 2.12 Hz, J13 = 8.60 Hz), 8.30 (d, 1H, J = 2.12 Hz), 8.32 (s, 1H); 13
C
NMR (100 MHz, CDCl3): d 27.48, 56.23, 70.81, 104.18, 110.61,
110.87, 117.68, 124.52, 127.66, 128.08, 128.57, 132.77, 136.33,
147.79, 155.32, 158.23, 160.64, 164.15; MS-ESI: m/z 391.00 [M+Na]+.
Anal. Calcd for C21H20O6: C, 68.47; H, 5.47. Found: C, 68.24; H, 5.74.
4.1.6. 5-(3-Benzyloxy-4-methoxybenzylidene)-2,2-dimethyl-
1,3-dioxane-4,6-dione (9p)
Yellow solid; mp 174–176 °C; IR (KBr): mmax 1703, 1594,
;
1363 cmꢂ1 1H NMR (400 MHz, CDCl3): d 1.79 (s, 6H), 3.97

5632
H. S. Sandhu et al. / Bioorg. Med. Chem. 18 (2010) 5626–5633
(s, 3H), 5.29 (s, 2H), 6.95 (d, 1H, J = 8.52 Hz), 7.34–7.45 (m, 5H),
7.59 (dd, 1H, J12 = 2.12 Hz, J13 = 8.52 Hz), 8.30 (d, 1H, J = 2.12 Hz),
8.35 (s, 1H); 13C NMR (100 MHz, CDCl3): d 27.49, 56.07, 70.85,
104.19, 110.70, 112.38, 116.07, 125.23, 127.19, 128.30, 128.78,
132.33, 135.76, 149.14, 153.87, 158.22, 160.64, 164.17; MS-ESI:
m/z 391.10 [M+Na]+. Anal.Calcd for C21H20O6: C, 68.47; H, 5.47.
Found: C, 68.31; H, 5.82.
fined as the concentration of the test compound that causes 50%
scavenging of DPPH radical and was calculated from the regression
analysis obtained by plotting the scavenging effect of compounds
at five different concentrations. ED50, efficient dose is defined as
micromoles of compound able to consume half the amount of free
radical divided by micromoles of initial DPPH and the value is ob-
tained by dividing IC50 value by 60. The inverse of ED50 is the mea-
sure of the antiradical power (ARP). By multiplying the ED50 by
two, the stoichiometric value (theoretical concentration of antiox-
idant to reduce 100% of the DPPH) is obtained. The inverse of this
value represents the moles of DPPH reduced by one mole of antiox-
idant and gives an estimate of the number of hydrogen atoms in-
volved in the process.
4.2. Typical experimental procedure for the synthesis of 9b and
9c
To a mixture of 7 (1 mmol, 1 equiv, 0.14 g) and 8b (1 equiv,
0.15 g) in methanol (1 mL) was added MS 3 Å (50 mol %). The reac-
tion mixture was refluxed for 3 h (TLC) The reaction mixture was
filtered, dried under reduced pressure and washed with ether
and hexane mixture to afford pure 9b (0.22 g, 81%). Mp 116–
118 °C (Lit.24a 117–118 °C).
4.3.2.2. ABTS radical scavenging assay.
2,20-Azino-bis (3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging
activity of the compounds were determined using an ABTS radical
cation decolorization assay.47,48 ABTS was dissolved in water to a
concentration of 7 mM. The ABTSÅ+ cation radical was produced
by reacting ABTS stock solution with 2.45 mM potassium persul-
fate and by allowing the mixture to stand in the dark for 12 h. To
4.3. Biology
4.3.1. Antimalarial assay
4.3.1.1. P. falciparum in vitro culture.
Parasites were main-
an aliquot (5 lL) of ethanol solution of test compounds was added
tained in in vitro culture using the method of Trager and Jensen.41a
Briefly, an equal volume of sterile 3.5% sodium chloride was added
to the infected blood samples, and the mixture was centrifuged at
1000g for 5 min. The pellet was washed three times and re-sus-
pended with RPMI-1640 medium supplemented with 10% human
serum and placed in a 25 mL tissue culture flask in a total volume
of 10 mL containing a 5% RBC suspension. The flask was flushed
with a gas mixture of 5% CO2, 5% O2, and 90% N2 for 30 s and incu-
bated at 37 °C. The culture medium was changed once a day; group
O red blood cells were added to maintain the 5% cell suspension.
Parasite growth was monitored by thin smear examination with
Field stain (A and B).
ABTS solution (0.1 mL) and the absorbance was read at 734 nm
after mixing up to 6 min. All the determinations were carried out
in triplicate and the results are expressed as the% of the ABTS rad-
ical scavenged by the test compounds. The IC50 values were calcu-
lated by regression analysis in the similar manner as described
above.
Acknowledgment
We thank Mr. Parveen Garg, Chairman of the College for provid-
ing facilities to carry out the present research work.
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