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Structure-activity relationship…….. Chapter 3
105
Structure-activity relationship of chalcones and their derivatives for
antimalarial and pesticidal activity
Chalcones are 1,3-diphenyl-2-propene-1-ones in which two aromatic rings are linked by an
-unsaturated carbonyl unit. It is an exceptional chemical template having multifarious
biological activities such as antioxidant, antileishmanial, antitumor, antibacterial besides
antimalarial and pesticidal activity. On the other side, the synthesis of chalcones with
different substitution pattern on both aromatic rings further allow to explore a large number
of desired potential analogues. Keeping in view the pharmacological importance of
chalcones and their derivatives, this chapter has been divided into two parts:
i) Synthesis and structure-activity relationship of chalcones for antimalarial activity.
ii) Structure-activity relationship of chalcones for pesticidal activity against diamondback
moth.
3.1 SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIP OF
CHALCONES FOR ANTIMALARIAL ACTIVITY:
3.1.1 Malaria:
Malaria is currently one of the most endemic diseases that affects more than 500 million
people per year, with an associated 2.5 million deaths [Wahlgren and Bejarano (1999);
Fattorusso et al. (2008)]. According to the World Health Organization (WHO), more than
100 tropical and sub-tropical countries including sub-Saharan Africa, Latin America,
Middle and Far East, Indian subcontinent are endemic for malaria [WHO (2009)]. Malaria
is caused by a protozoan parasite Plasmodium, which is transmitted via bite of infected
female Anopheles mosquito, acting as a vector of malaria. In humans, the disease is caused
by four species of Plasmodium namely P. falciparum, P. vivax, P. malariae and P. ovale.
3.1.2 Therapeutic agents for treatment of malaria:
The most successful drug for the treatment of malaria finds its origin in a plant called the
cinchona [Yepuri (2004)]. Various forms of powdered cinchona bark were in use until two
French chemists, Pierre Joseph Pelletier and Joseph Bienaime Caventou got success for the
isolation of its active ingredient which they named quinine [Yepuri (2004)]. This has paved
the way for the development of other antimalarial agents including various derivatives of
quinine. For instance, chloroquine, a derivative of quinine was produced on a large scale for
treatment and prevention of malaria. Similarly, other analogues include mefloquine,
Structure-activity relationship…….. Chapter 3
106
halofantrine, primaquine etc [Vangapandu et al. (2007)]. Examples of various drugs used
for the treatment of malaria infection are shown in Figure 1.
N
NH3CO
HHO
H H
N
HN
N
Cl
N
CF3
CF 3
HONH
Quinine Chloroquine Mefloquine
N
NH2N
NH 2Cl
Pyrimethamine Ferroquine
F3C
Cl Cl
OH
N[(CH 2)3CH 3]2
Halofantrine
NHN
OCH 3
NH 2
Primaquine
N
HNN(C2H5)2
Mepacrine
Cl
OCH 3 Cl
Cl
HN N
NH2 NH2
N
Chlorproguanil
N
HN
Cl
Fe
NCH 3
CH3
Figure 1
However, emergence of resistance by malarial parasite to various classes of above
mentioned drugs is a cause of huge concern. For instance, P. falciparum has developed
resistance to mainstay drugs like chloroquine, mefloquine, pyrimethamine etc
[Wongsrichanalai et al. (2002); Nowakowska (2007)]. In this context, artemisinin has been
a miraculous entry for the treatment of multidrug-resistant P. falciparum malaria.
3.1.3 Artemisinin and its derivatives for treatment of malaria:
Artemisinin (or qinghaosu) is a tetracyclic 1,2,4-trioxane with an endoperoxide linkage and
is a potent antimalarial compound (Figure 2). Artemisinin was isolated as an active
ingredient from traditional Chinese plant Artemisia annua which has been used in the
traditional Chinese medicine for the treatment of fever since A.D. 341 [Agtmael et al.
(1999); Wu (2002)]. Artemisinin was found to have low solubility, short plasma half life,
neurotoxicity besides poor oral bioavailability [Kamchonwongpaisan et al. (1997);
Meshnick (2002); Yepuri (2004)]. Consequently, various derivatives of artemisinin (having
better physiochemical properties) including artemether, arteether and artesunate have been
prepared (Figure 2) and used for the treatment of drug-resistant and drug-sensitive malaria
Structure-activity relationship…….. Chapter 3
107
[Wang and Xu (1985); Lin et al. (1987); Yepuri (2004)], particularly in cases of deadliest
cerebral malaria. These compounds were characterized by their fast action, high efficacy
and good tolerance.
O
O
H
H
H
O
O
O
O
O
H
H
H
OR
O
O
O
O
H
H
H
O
O
O
COONa
OArtemisinin R = OCH3; ArtemetherR = OC2H5; Arteether Sodium artesunate
Figure 2
However, the fear of developing resistance to this vital class of drug has led to grave
apprehensions as no alternative antimalarial medicines will be available in the near future
[WHO (2001)]. Therefore, to avoid or slow down the development of resistance,
combination of artemisinin derivatives with other antimalarial agents also known as
artemisinin based combination therapies (ACTs) are being advocated and promoted by
WHO. For instance, combination of artemether and lumefantrine into a single tablet, known
as “Coartem®” has been reported [Falade et al. (2005)].
However, access to ACTs is still limited in most malaria-endemic countries due to high
fetching prices of such drugs. On the other side, low percentage of artemisinin in plants and
complexities of its total synthesis has still posed a great challenge [Avery et al. (1992);
Yadav et al. (2003)]. Moreover, the emerging clinical resistance against artemisinins
noticed in South Asia [Gogtay et al. (2000); Sahr et al. (2001); Dondorp et al. (2009)] has
motivated the scientists to look for alternate synthetic and cost effective medicines for the
treatment of malaria. In this context, chalcones have attracted much attention due to their
wide ranging biological profiles.
3.1.4 Chalcones:
Chalcones are prominent secondary metabolite precursors of flavonoids or isoflavonoids in
plants [Nowakowska (2007); Patil et al. (2009)]. Chalcones either natural or synthetic have
been found to possess plethora of biological activities [Dimmock et al. (1999);
Modzelewska et al. (2006); Nowakowska (2007)] as also mentioned earlier in the
introduction section 1.2.1.2.3.1 (page 8).
Structure-activity relationship…….. Chapter 3
108
3.1.4.1 Reported methods for synthesis of chalcones and their derivatives:
Chalcones can be synthesized by Claisen-Schmidt condensation between benzaldehydes and
acetophenones [Patil et al. (2009)]. The reaction is generally carried out in the presence of
aqueous alkaline bases such as NaOH, KOH etc. Chalcones have also been synthesized by
using energy efficient microwave (MW) or ultrasonic irradiation. For instance, a MW
assisted methodology involving reaction of aromatic benzaldehydes with various
acetophenones in presence of catalytic amount of zinc chloride has been disclosed (Scheme
1) [Reddy et al. (2001)].
Scheme 1
Dong et al. utilized the ability of SO3H-functionalized acidic ionic liquids (ILs) as catalysts
for the synthesis of chalcones by Claisen–Schmidt condensation [Dong et al. (2008)]. Use
of IL under conventional heating at 140°C provided the chalcones in good yields (83-96%)
within 2-3 h of reaction time (Scheme 2).
OCHO
R1 R2
O
+
R1 = H, OCH 3,C lR2 = H, OCH 3,N O2
NH3C
CH3
CH3
SO3HHSO4
R1 R2
[TMPSA ]HSO 4
140°C, 2-3 h
Scheme 2
3.1.4.1.1 Synthesis of multiconjugated chalcones:
The most common synthetic approach for multiconjugated chalcones such as 1,5
diarylpenta-2,4-dien-1-ones involves the base promoted condensation between
cinnamaldehyde and acetophenone. For instance, Xin et al. utilised activated barium
hydroxide as a condensing agent for an ultrasound promoted synthesis of 1,5 diarylpenta-
2,4-dien-1-ones (Scheme 3) [Xin et al. (2009)].
CHOO
+
O
RR= H, Cl, OMe, Me, NO2
ZnCl 2
M.WR
Structure-activity relationship…….. Chapter 3
109
3.1.4.2 Chalcones as antimalarial agents:
The antimalarial activity of chalcones was first noted when licochalcone A (Figure 3),
isolated from Chinese liquorice roots, was reported
to exhibit potent in vivo and in vitro antimalarial
activity against both chloroquine-susceptible (3D7)
and chloroquine-resistant (Dd2) strains of P.
falciparum [Chen et al. (1997)]. Thereafter,
extensive efforts have been made for its
modification and antimalarial structure-activity
correlation of other novel chalcones. The antimalarial property of chalcones mainly results
from their ability to act against falcipain-2, a malarial cysteine protease (an enzyme used by
the parasite for the degradation of host haemoglobin for nutritional purposes) [Shenai et al.
(2000)], however, involvement of some other mechanism may not be ruled out. A brief
literature demonstrating chalcones as antimalarial agents is described below. For the sake of
discussion, the aromatic rings in the chalcones were designated as A (from aldehydes) and
B (from acetophenones) respectively.
Li et al. carried out a detail study on the in vitro antimalarial activity of chalcone
derivatives, wherein, Cl and F substitutions at ring A of chalcones displayed potent
antimalarial agents [Li et al. (1995)]. Similarly, Liu et al. and Go et al. synthesized a series
of substituted chalcones and
screened them against
chloroquine-susceptible
(3D7) and chloroquine-
resistant (Dd2) strains of P.
falciparum [Liu et al.
(2001); Go et al. (2004)].
From SAR analysis, size and hydrophobicity of substituents on both rings of chalcones were
identified as critical parameters. In addition, most of the hydroxylated chalcones were found
O
R3R2
R1
OCH3OCH3
OCH3
O
R2 R1
OCH3
OCH3
(i). R1 = R2 = H, R3 = CF3(ii). R1 = R3 = Cl, R2 = H
(iii). R1 = R2 = OCH3(iv). R1 = H, R2 = C2H5(v). R1 = H, R2 = CF3(vi). R1 = R2 = F
Figure 4
A B A B
OH
OCH 3
HO
O
Figure 3
Licochalcone A
H
OO
+
O
R R= H, OMe, F, Br, NO2 etc.
Ba( OH)2
EtOH, Ultrasound
Scheme 3
R
Structure-activity relationship…….. Chapter 3
110
less active than the corresponding alkoxylated derivatives. Chalcones i-vi (Figure 4) were
found to have IC50 < 6.5 M.
Later on, Liu et al. studied the physiochemical and structural requirements for
antileishmanial activity (Leishmania donovani) and compared with antimalarial activity
[Liu et al. (2003)]. Thus it was found that antileishmanial activity is favored by hydrophilic
chalcones possessing 4′-hydroxy-substitution, whereas, good antimalarial effects were
found among alkoxylated chalcones as mentioned above.
Wu et al. reported the synthesis and antimalarial potential of ferrocenyl chalcones against
chloroquine resistant strain of P. falciparum [Wu et al. (2002)]. The most active compounds
were 1-(3-pyridyl)-3-ferrocenyl-2-propen-1-one and 1-ferrocenyl-3-(4-nitrophenyl)-2-
propen-1-one with IC50 of 4.6 M and 5.1 M respectively (Scheme 4).
O
RCHO
O
R FeFe
CHO
RFe
O
Fe
KOH,EtOH
KOH , EtOH,R
+
+
r.t.
r.t.
When ring B = 3-pyridinylIC50 = 4.6 µM
When R = 4-nitroIC50 = 5.1 µM
O
Scheme 4
Similarly, Domínguez et al. carried out the synthesis of a series of phenylurenyl chalcone
derivatives [Domínguez et al. (2005)] which were screened for antimalarial activity. The
most active compound, 1-[3′-N-(N′-
phenylurenyl)phenyl]-3(3,4,5-trimethoxy-
phenyl)-2-propen-1-one, has IC50 value of 1.76
M (Figure 5) against chloroquine-resistant
strain of P. falciparum. Further investigation
revealed that chalcones exert their antimalarial activity via multiple mechanisms.
A series of substituted indole derivatives were prepared by the cyclization of chalcones with
imidine hydrochlorides in the presence of sodium isopropoxide (synthesized in situ by
adding sodium metal in isopropanol) (Scheme 5) [Agarwal et al. (2005)]. The study
suggested that presence of N-methyl piperazine group at 2-position of pyrimidine ring
O
NHIC50 = 1.76 µM
NH
O
OMe
OMe
OMe
Figure 5
Structure-activity relationship…….. Chapter 3
111
favors the antimalarial activity, however, when piperazine ring was replaced with
pyrrolidine, piperidine or morpholine moiety the activity dropped significantly.
NH
O
NH
Ar
O
NH
N
N
N
N NMeNH2
NH
HCl, NaO-iPri-PrOH, reflux
Ar = Ph, C6H5OMe etc.
NCH3
Ar
ArCOCH3PiperidineMeOH,reflux (8 h)
Scheme 5
Leon et al. synthesized a series of sulfonamide chalcones and tested them for antimalarial
activity (Scheme 6) [Leon et al. (2007)]. Among all the tested compounds, (E)-1-[4′-(3-
(2,4-difluorophenyl)acryloyl)phenyl]-3-tosylurea showed the most favorable antimalarial
activity with an IC50 value of 1.2 M against P. falciparum.
S NCO+
O
H2N
CH2Cl2
3 hR O
NH
NH
O
S
R
R'MeOH, NaOH
R'CHO, 8-12 h, r.t. O
O
O
NH
NH
O
S
R
O
OO
O
R = Cl, CH3R' = 2,4-di-OMeC6H3, 4-FC6H4, 3,4-OCH2OC6H3, 2,4-di-FC6H3, C5H4N etc.
When R = CH3R' = 2,4 di-FC6H3IC50 = 1.2 µM
Scheme 6
Valla et al. reported the aromatic annelation reaction for the synthesis of a series of
‘retinoid-like chalcones’ using an enaminone synthon [Valla. et al. (2006)]. The 4-hydroxy-
substituted compound (Scheme 7) exhibited a good antimalarial potential with an IC50 value
of 4.93 M and 8.47 M against K1 strain and Thaï strain respectively.
Scheme 7
Similarly, a series of substituted quinolinyl chalcones and pyrimidines was evaluated for
their in vitro antimalarial activity against NF-54 strain of P. falciparum [Sharma et al.
O
N
O
OH
O OH O
OM eCOOEt
IC50 = 4.93 µM (K1 strain)IC50 = 8.47 µM (Thaï strain)
LDA, DME(-20°C)
LDA, DME(-20°C)
Structure-activity relationship…….. Chapter 3
112
(2008)]. In another report, screening of substituted chalcone derivatives for antimalarial
activity suggested that small or medium sized lipophilic groups (containing multiple
nitrogen or amine in acetophenone moiety) favors the antiplasmodial potential (Figure 6)
[Mishra et al. (2008)]. Among all the tested compounds, 1,2,4-triazole substituted chalcone
was found to be the most effective (IC50: 1.52 g/ml) in inhibiting the growth of P.
falciparum.
N N
N N
NNN
IC50 = 2.93 µg/ml IC50 = 2.5 µg/ml IC50 = 1.52 µg/ml
Cl Cl Cl
O O O
Figure 6
Tomar et al. synthesized a series of chalcone derivatives bearing acridinyl moiety (Scheme
8) and screened them for in vitro antimalarial activity against NF-54 strain of P. falciparum
[Tomar et al. (2010)]. Most of the synthesized compounds showed complete inhibition at
concentration of 10 g/ml.
N
Cl
9-Chloroacridine
+
O
H2NR1
O
HNR1
N
2-butanolReflux
R1 =H, OCH 3, CH 3, Cl etc.
Scheme 8
Recently, a number of chalcone derivatives such as acetylenic chalcones [Hans et al.
(2010)], -pyranochalcones [Wanare et al. (2010)] and chalcone-chloroquinoline hybrids
[Guantai et al. (2011)] have been synthesized (Figure 7) and screened for antimalarial
activity by various groups.
Figure 7
O
H3CO
O
R
O
O
R
Chalcone-chloroquinoline hybrid-Pyranochalcone
Acetylenic chalcones
O
R
HO
O OMe
OMe
N
N
N Cl
Structure-activity relationship…….. Chapter 3
113
3.1.4.3 Combination of chalcone with artemisinin:
As mentioned earlier that to decrease the dependence on artemisinin without compromising
on the potency to cure malaria, WHO advocates ACTs. In this context, various chalcone
derivatives in fixed-ratio combination with artemisinin were employed to assess their in
vitro antimalarial activity (Scheme 9) [Bhattacharya et al. (2009)].
O
O
H
H
H
O
O
O
O
F
O
R
+
Cl
H
O O
ClN
O
ClNNN
O
ClNN N
Synergistic or Additiveantimalarial interaction
(ii)
or
or
(iii)
(i)
Scheme 9. Reagent and conditions; (a) Cyclic amines, K2CO3, DMF, 18 h, 110°C; (b) NaOH, methanol,16-20 h, r. t.
(a) (b)
(Artemisinin)
(Chalcones)
Some of the evaluated combinations showed synergistic interaction and resulted in the
decrease of hemozoin formation in parasitized erythrocytes (particularly combination of
chalcone iii with artemisinin, Scheme 9). In addition, it was observed that such combination
do not affect new permeation pathways induced in the host cells.
3.1.5 Aim of the present work:
From the above discussion, it is clear that extensive effort for the introduction of chalcones
as effective and inexpensive antimalarials are in progress. Consequently, various derivatives
of chalcones including heterocyclic analogues (with N, S and O containing compounds)
have been reported to possess antimalarial activity. However, a majority of above
heterocyclic lead candidates involve delicate reaction conditions while there is a vast scope
for exploring the antimalarial potency of core skeleton of chalcones having simple
substituents. In addition, it would be doubly beneficial if such simple chalcone scaffolds
could be derived from easily available natural precursors utilizing principles of green
chemistry. Therefore the aim of the present work is:
(1) To study the effect of various substituents on ring A as well as on ring B of chalcone
and to find out the antimalarial SAR.
Structure-activity relationship…….. Chapter 3
114
(2) Synthesis of lead candidates from abundantly available plant based natural
precursors.
(3) Ecofriendly and expedite synthesis of lead candidates using various tools of green
chemistry such as ILs and MW etc.
3.1.6 Results and Discussion:
3.1.6.1 Chemistry and synthesis of chalcone derivatives:
To find out the potent core skeleton of chalcones having simple substituents, compounds 1-
16 used for preliminary screening (Table 1-2) were prepared by Claisen-Schmidt
condensation between benzaldehydes and acetophenones (acetone in case of 13) using
NaOH as a base (Scheme 10).
CHO
R
O
R' R R'
O
+10% NaOHMeOH, r.t.
R = R' = Cl, Br, OCH3, OH etc. 1-16
Scheme 10
A B A B
From the preliminary screening, we found that 2,4,5-trimethoxy-substitution on ring A
significantly favors the antimalarial activity. With an aim to search for the environmentally
benign synthesis of lead candidates, various chalcones having 2,4,5-trimethoxy substitution
pattern on ring A were accesed from abundantly available natural β-asarone (a natural
phenylpropene, present up to 96% yield in Acorus calamus oil) [Sinha et al. (2002, 2003,
2004, 2005)]. For this, β-asarone was oxidized with NaIO4/OsO4 under MW to provide
asaronaldehyde [Sinha et al. (2003)] which upon condensation with substituted
acetophenones in ionic liquid [MIMBSA]HSO4 [Dong et al. (2008)] under MW provided an
expedite and cost effective route for chalcones 14 (Table 2) and 22-29 (Table 4) (Scheme
11).
CHO
H3CO
OCH 3
OCH 3
OCH 3
H3COOCH 3
-asarone fromAcorus calamus oil
OsO4(cat.)
NaIO4, MW
O
R
[MIMBSA]HSO4, MW
OCH 3
H3COOCH 3
R
O
A B
(14 & 22-29)1,3-diarylprop-2-en-1-one
Asaronaldehyde
R =Cl , I, Br, CN, OCH3 NO2 etc.
Scheme 11. General procedure for the synthesis of 2,4,5-trimethoxy-substituted chalcones from-asarone of Acorus calamus oil.
Structure-activity relationship…….. Chapter 3
115
Chemoselective hydrogenation of above 14 with PdCl2/HCOOH/MeOH/H2O combination
under MW irradiation [Sharma et al. (2006)] yielded a compound 17 which was further
reduced with NaBH4 [Chamberlin and Daniel (1991)] to obtain compound 18 (Scheme 12).
OCH3
H3COOCH3
Cl
O
A B
OCH3
H3COOCH3
Cl
O
A B
OCH3
H3COOCH3
Cl
OH
A B
Scheme 12. General method for the reduction of enone moiety of chalcone 14 to dihydro derivatives.Reagents and conditions: (a) PdCl2/HCOOH/MeOH/H2O, microwave; (b) NaBH4, MeOH, r.t.
(a) (b)
17 1814
Indole-chalcone adduct 19 (Table 3) was prepared by the Michael addition of indole moiety
on the 2,4,5-trimethoxy substituted chalcone 14 using p-toluenesulfonic acid (PTSA) as a
catalyst (Scheme 13) [Ji and Wang (2005)]. Further N-allylation of 19 with allyl bromide
provided 20 (Scheme 13 & Table 3). Compound 21 (Table 3) was prepared by DDQ
assisted dehydrogenation of corresponding N-allylated indole-chalcone hybrid (Scheme 13).
O
H3COOCH3
NH
H3CO
R
O
H3COOCH3
N
H3CO
R
OCH3
H3COOCH3
O
R
O
H3COOCH3
N
R
CH3O
R = Cl or Br
a b c
19 (R = Cl) 20 (R = Cl) 21 (R = Br)
Scheme 13. General procedure for the addition of indole moiety on chalcone. Reagents and conditions:(a) indole, PTSA (cat.), acetonitrile, r.t.; (b) allyl bromide, KOH, TBAB, r.t.; (c) DDQ, dioxane, r.t. overnight
For the synthesis of multiconjugated chalcone 30 (Table 5), initially β-asarone was
oxidatised with DDQ to give 2,4,5-trimethoxy cinnamaldehyde [Joshi et al. (2006)] which
upon condensation with 4-bromoacetophenone using ionic liquid [MIMBSA]HSO4 under
MW provided 30 (Scheme 14).
Similarly, condensation of asaronaldehyde (obtained from oxidation of β-asarone, see
Scheme 11) with acetone under MW using ionic liquid [MIMBSA]HSO4 gave the
DDQ/dioxane
OCH3
H3COOCH3
CHO
OCH3
H3COOCH3
O
Br
O
Br
[MIMBSA]HSO4 , M.W
1,5-diarylpent-2,4-dien-1-one30
OCH3
H3COOCH3
Ultraso und
Scheme 14
-asarone fromAcorus calamus oil
2,4,5-trimethoxycinnamaldehyde
Structure-activity relationship…….. Chapter 3
116
corrseponding enone which was further subjected to Claisen-Schmidt condensation with 4-
bromobenzaldehyde using NaOH as a base to provide 31 (Scheme 15 & Table 5).
CHO
H3CO
OCH3
OCH3[MIMBSA]H SO 4,MW
OCH3
H3COOCH3
O
31
CH3COCH3
CHO
Br
OOCH3
H3COOCH3
Br
Scheme 15
NaOH, MeOH
Synthesis of compounds 32-43 is mentioned in section 3.1.6.2.8 (Scheme 17). All the
synthesized compounds were well characterized by NMR (1H & 13C) spectra.
3.1.6.2 General antimalarial activity:
Screening of the comparative efficacies of the molecules described in this work was based
on the validated, micro titer plate based high through put format SYBR green fluorescence
technique [Smilkstein et al. (2004)]. This method is based on the fact that in the mature
human red blood cells (which lack DNA), the quantitative estimation of SYBR green
fluorescence acts as an index for the growth of malaria parasite allowing precise estimation
of IC50 value for each compound.
3.1.6.2.1 Preliminary screening:
In view of the well known antimalarial potency of various chlorinated compounds like
chloroquine, pyronaridine, acridinedione etc [Fu and Xiao (1991); Kesten et al. (1992);
Manohar et al. (2010)], we initially desired to explore some chlorinated chalcones using 3-
(4-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (1, Table 1) as a representative
compound. However, screening against P. falciparum 3D7 strain revealed that compound 1
with an IC50: 88.0 M lack significant potency. Thereafter, molecules with different
substituents on each of the two rings were synthesized and evaluated for antimalarial
activity. It is clear (Table 1) that introduction of 3,4-methylenedioxy (4), hydroxy (5), allyl
(6), chloro (7), bromo (8) or nitro (9) group at ring B did not lead to significant activity. On
the other side, replacement of 4-chloro on ring A with 3,4-dichloro (10) drastically reduced
the antimalarial activity (IC50: >200 M). Also, chalcones with furan (2) and thiophene ring
(3) proved futile for enhancing the antimalarial activity.
To our surprise, a reversal of substituents between rings A & B of 1 (IC50: 88.0 M) led to a
three fold increase in activity (IC50: 28.8 M) of resulting 11. Thereafter, we thought to
increase the electron density on ring A keeping chloro substitution constant on ring B. To
our pleasure, compounds with 2,4-dimethoxy (12, Table 1) and 2,4,5-trimethoxy groups
(14, Table 2) on ring A exhibited progressively better antimalarial potential with IC50 values
Structure-activity relationship…….. Chapter 3
117
of 6 M and 4 M respectively. These observations clearly indicate that increase in the
electron density on ring A significantly enhanced the antimalarial activity which is
evidently in contrast to earlier reports linking potent antimalarial activity with electron
deficient ring A of chalcones [Li et al. (1995); Liu et al. (2003)]. In addition, 13 (Table 1),
with B ring absent, showed drastic reduction in activity (13, IC50: 48.5 M) as compared to
12, thus underlining the importance of both aryl rings (A & B) of chalcone for activity.
Cl
O
OCH 3
OO
NO 2
SO
NO 2
Cl
O
O
O
Cl
O
OCH 3
Cl
CH 3
O
OCH 3H3CO
H3CO
O
Cl
Cl
O
Cl
Cl
O
Br
Cl
O
OHO
Cl O
O
H3CO ClOCH 3
Compound No. Structure IC 50
2
3
4
5
6
7
8
9
10
11
12
13
O
NO 2Cl
88.0
200
169.9
97.9
30
28.8
48.5
6
>
75
65
50
78
1
Structure IC50Compound No.
200>
(µM)(µM)
Table 1. Screening of chalcones for antimalarial activity against P. falciparum 3D7 strain
(Published in Eur. J. Med. Chem. 2010, 45, 5292-5301)
A
A
B
B
3.1.6.2.2 Effect of changing the position of trimethoxy substitution on ring A:
We next evaluated the positional importance of the
three methoxy groups on ring A for further
optimization of antimalarial activity (Table 2). The
SAR analysis showed that 2,4,5-trimethoxy
substituted chalcone (14, IC50: 4 M) exhibited
significantly to marginally better antimalarial
potency as compared to its 2,4,6 (15, IC50: 8 M) and
3,4,5 (16, IC50: 4.6 M) counterparts. The fact that
activity is markedly affected by changing the position
of trimethoxy substituents at the phenyl ring suggests
that 2,4,5-trimethoxy substitution makes a special
IC 50
H3COOCH 3
O
Cl
H3CO
OCH 3
H3CO
O
ClOCH 3
Compound No. Structure
15
16
8
4.6
(µM)
OOCH 3
H3COOCH 3
Cl
14 4
Table 2. Effect of position of trimethoxysubstitution on antimalarial activity
Structure-activity relationship…….. Chapter 3
118
contribution which may be due to its orientation and binding ability with the malarial
parasite proteins.
3.1.6.2.3 Effect of reduction ofunsaturated ketone unit:
In order to evaluate the role of - unsaturated ketone moiety, the reduction of double
bond of active compound 14 was carried out. However, resulting compound 17 (IC50: 64
M) showed significantly reduced activity. Similarly, reduction of both double bond and
keto group (18, IC50: 50 M) also proved futile (Scheme 16).
OCH 3
H3COOCH 3
O
Cl
OCH 3
H3COOCH 3
O
Cl
OCH 3
H3COOCH 3
OH
Cl
Chemoselectivehydrogenation
18, IC50 = 50 µM
17, IC50 = 64µM
14, IC50 = 4 µM
Scheme 16
3.1.6.2.4 Effect of incorporation of heterocyclic moiety:
Since indole derivatives are well known to be
antimalarials [Agarwal et al. (2005)], we
became interested to see the effect of Michael
addition of indole moiety on the lead structure
(14, Table 2). Thus addition of indole on 14
(Scheme 13) yielded a saturated compound 19
(IC50: 8.6 M, Table 3) which, however, was
found to be less active than the parent 14.
Further, N-allylation of 19 slightly improved the
activity (20, IC50: 8.4 M). Hence, it was
realized that introduction of a double bond on
20 might increase the activity. However,
oxidation of 20 (Cl substituted B ring) with
DDQ to regenerate the double bond was found
problematic due to formation of numerous side
products. Interestingly, the corresponding
IC50 (µM)Compound No. Structure
19 8.6H3CO
OCH3
O
Cl
H3CO
NH
H3COOCH3
O
Cl
H3CO
N
H3COOCH3
O
Br
H3CO
N
21
8.4
17.5
Table 3. Effect of incorporation ofheterocyclic moiety on antimalarial activityof chalcone against P. falciparum 3D7 strain
20
Structure-activity relationship…….. Chapter 3
119
bromo derivative 21 (Table 3) could be easily prepared (Scheme 13) but its antimalarial
activity didn’t improve as compared to 14.
3.1.6.2.5 Effect of substituents on ring B:
After realizing the crucial role of 2,4,5-position and double bond of enone moiety of
chalcone for potent activity, we next ventured to evaluate the effect of various substituents
on ring B keeping 2,4,5-trimethoxy group constant on ring A (22-29, Table 4). The
structure-activity analysis demonstrated that ring B having 4-methoxy (22, IC50: 11.5 M)
or 2,3,4-trimethoxy (23, IC50 : 7.8 M) substitution provided compounds with lower activity
as compared to 14 which indicated the importance of electron deficient ring B.
Consequently, various other chalcones with EWG’s like NO2 (25), I (26), Br (27) or CN
(28) on ring B were prepared and screened for activity.
O
H3CO
OCH3
OCH3
OCH3
OCH3
OCH3 OCH3
H3CO
OCH3
O
Br
OCH3
H3COOCH3
O
I
Compound No. IC50 (µM)
27
25
28
OCH3
H3CO
OCH3
O
Cl
Cl
2
1.8
>200
OCH3
H3COOCH3
O
CN>100
OCH 3
H3COOCH 3
O
OCH 3
OCH 3
H3COOCH 3
O
NO 2
23
22 11.5
7.8
20>24
OOCH 3
H3COOCH 3 Br
29 47
Structure
26
Compound No. Structure
Table 4. Effect of substitution on ring B of chalcones for antimalarial activity against P.falciparum 3D7 strain
(Published in Eur. J. Med. Chem. 2010, 45, 5292-5301)
IC50 (µM)
A B
The compounds 25 and 28 possessing NO2 and CN substituents respectively were clearly
inactive (Table 4). However, a two fold increase in activity was observed with I or Br
substituents as both 26 & 27 showed good antimalarial potential with IC50 value of 2 M
and 1.8 M respectively as compared to 4 M in case of Cl (14, Table 2). Surprisingly, the
presence of multiple electron-withdrawing substituents (3,4-dichloro substituted B ring, 24)
Structure-activity relationship…….. Chapter 3
120
did not lead to significant activity. On the other hand, replacement of ring B of 27 with 4-
bromobiphenyl moiety produced lesser active compound 29 (IC50: 47 M), which
emphasizes that size characteristics of ring B also play an important role in antimalarial
activity.
3.1.6.2.6 Effect of extended conjugation:
After studying a series of chalcones (1-29, Table 1-4), we planned to extend the conjugation
in the most active chalcone 27. Consequently,
multiconjugated chalcone 30 (Table 5) was
synthesized from -asarone (Scheme 14) which
upon antimalarial evaluation showed reduced
activity than 27. Thereafter, another
multiconjugated chalcone derivative 31 (Table 5),
an analogue of bioactive curcumin [Lee et al.
(2009)] was synthesized (Scheme 15). However, the
above compound also depicted reduced activity
(IC50: 18 µM), thereby, suggesting the critical role of basic chalcone nucleus for potent
antimalarial activity.
3.1.6.2.7 Evaluation of Cytotoxicity and Resistant index for identified lead candidates:
The identified lead chalcones (12, 14, 16, 26 & 27) were also tested against chloroquine
resistant Dd2 strain of P. falciparum. Against a Resistance Index (IC50 Dd2/IC50 3D7) of ~ 4
CompoundNo.
SYBR Green 1 AssayIC50 (µM)
Pf 3D7
Resistance IndexIC50 Dd2/IC50 3D7 TC50 HeLa/
IC50 3D7TC50 L929/IC50 3D7
12
14
16
26
27
Chloroquine
6
4
4.6
2
1.8
40 nM
6.8
6.9
11.5
7.5
5
170 nM
1.1
1.7
2.5
3.7
2.8
4.2
>16.6
9
3
50
31.7
>200
8.3
2.8
>50
55.6
>200
14.6
Pf Dd2
Selectivity Index
Table 6. Resistance and Selectivity Indices for potent chalcones
OCH 3
H3COOCH 3
O
Br
O
H3CO Br
OCH 3
OCH 3
Compound No. Structure IC 50 (µ M)
31
30 12.2
18
Table 5. Effect of conjugation on antimalarialactivity against P. falciparum 3D7 strain
Structure-activity relationship…….. Chapter 3
121
for chloroquine, these indices for the potent chalcones were found to be 1.1, 1.7, 2.5, 3.7
and 2.8 respectively (Table 6). Finally, the above compounds were also analyzed for their
cytotoxic behavior against two mammalian cell lines viz. HeLa and fibroblast L929.
Selectivity indices (Table 6) with values ranging between 3 and >50 indicated that the most
active compounds (26 & 27) were also relatively non toxic.
3.1.6.2.8 Modification on identified potent chalcone 27 with emphasis on heterocyclic
analogues:
After finding out the potent antimalarial skeleton of chalcone (27, Table 4), later on it was
planned to modify it with various heterocyclic derivatives to see the effect on antimalarial
activity. Consequently, compound 27 was allowed to react with substituted hydrazines,
hydroxylamine, thiourea and ethylacetoacetate to produce the corresponding cyclized
derivatives i.e. 32-36 (Scheme 17). Similarly, N-benzylated indole-chalcone hybrids (37-43)
were prepared (Scheme 17) and evaluated for antimalarial activity.
Scheme 17. Reagent and conditions; (a) RNHNH2, NaOAc, AcOH, H2O, MW; (b) NH2OH.HCl,
NaOAc, AcOH, H2O, MW; (c) NH2CSNH2, ethanolic solution of NaOH, MW; (d) ethyl
acetoacetate, K2CO3, silica, MW; (e) substituted indole, [MIMBSA]HSO4 (cat.), ACN, r.t.; (f) NaH,
DCM, benzyl bromide, r.t; (g) piperidine, HCHO, AcOH, dioxane, r.t.
[For synthesis of 37-39 and 41-43 steps (e) & (f), whereas, for synthesis of 40, steps (e) & (g)]
OCH3
H3COOCH 3
Br
O N
OCH3
H3COOCH3
Br
N N
H3COOCH3
Br
HN NH
R
O
N
OCH3
H3COOCH3
BrBr
O
O
I
Br
N
R1
H
H
H
BrBr
H
H
H
36
34
(37-43)
R = H; 32R = Ph; 33
S
37
38
41
42
39
43
40
R1 R2
35
OOCH3
H3COOCH3
Br
(a)
(b)
(c)
(d)
(e)
(f) or (g)
CH3OIC50: 1.8 M
O O
OCH3O
Br H3CO OCH3
R2
27
Structure-activity relationship…….. Chapter 3
122
However, compounds 32-36 were found highly sensitive to air and showed lot of
fluctuations in their IC50 values (tested in triplicate; in all cases IC50 were found to be >5
M). Among indole-chalcone hybrids (37-43), only compounds 41-43 showed IC50 values
below 5 M and were also tested against chloroquine resistant Dd2 strain of P. falciparum
(Table 7). The most active compound 43 (IC50: 1.6 M) showed excellent selectivity index
with value of >125 (Table 7).
In order to find the antimalarial target of chalcones, we resorted to examination of the
possible inhibitory effect of the potent chalcones in a hemoglobin degradation assay. In this
assay, freshly prepared parasite extract was used to digest the human hemoglobin and
inhibition of degradation in presence of chalcones (100 M) was monitored using SDS
PAGE. However none of the tested chalcones showed inhibition in this assay suggesting
that these molecules do not exert their antimalarial action via inhibition of the proteolytic
pathways of hemoglobin degradation.
CompoundNo.
SYBR Green 1 AssayIC50 (µM)
Pf 3D7
Resistance IndexIC50 Dd2/IC50 3D7 TC50 HeLa/
IC50 3D7TC50 L929/IC50 3D7
Chloroquine 40 nM 170 nM 4.2
Pf Dd2
Selectivity Index
Table 7. Resistance and Selectivity Indices for potent heterocyclic analogues
414243
2.3 1.2 >43.5 8.3
1.6 0.6 >125 >1253.3 0.5 >60.6 >60.6
>200 >200
2.81.71
Structure-activity relationship…….. Chapter 3
123
3.2 STRUCTURE-ACTIVITY RELATIONSHIP OF CHALCONES FOR
PESTICIDAL ACTIVITY AGAINST DIAMONDBACK MOTH:
3.2.1 Introduction:
The diamondback moth, Plutella xylostella (L.) (Figure 8), is one of the most destructive
pests of crucifers worldwide [Talekar and Shelton (1993); Verkerk and Wright (1996)].
Larvae of P. xylostella, feed on the foliage of the cruciferous plants
from the seedling stage to harvest and greatly reduce the yield and
quality of produce. Control of P. xylostella has largely been
depending on use of various classes of pesticides such as substituted
hydrocarbons, carbamates, organophosphates [Miyata et al. 1986],
pyrethroids [Miyata et al. (1986); Schuler et al. (1998)], benzophenyl ureas [Sun 1990)],
emamectin benzoate [Zhao et al. (2006)] beside biopesticides such as Bacillus thuringiensis
[Sun (1990); Tabashnik et al. (1990)]. Javier et al. estimated that the cost for controlling
this most devastating crucifers pest is approx. 1 billion US dollar annually [Javier (1992)].
Some of the pesticidal agents used against diamondback moth are given in Figure 9.
OO
Br
Br
O
NDeltamethrin
OO
O
Fenvalerate
CNCl
N
N
O
P OS
O
Quinalphos
PO
O
O
O
HN
O
Monocrotophos
Figure 9
However, development of resistance for various classes of pesticides is a major concern for
the effective management of this important crucifer defoliator [Shelton et al. (1993); Zhao
et al. (2006)]. On the other side, adverse effects of some of the above pesticides [Eskenazi
et al. (1999)] to humans, animals and environment beside difficulties associated with their
preparations have fostered the need for developing new pesticidal agents with greater
selectivity, better health and environmental profiles.
Figure 8
Structure-activity relationship…….. Chapter 3
124
Chalcones being structurally simple class of natural products have assumed importance due
to their wide ranging biological profiles [Nowakowska (2007); Patil et al. (2009)].
Chalcones have also been explored for their pesticidal activities as mentioned below.
3.2.2 Chalcone and their derivatives as pesticidal agents:
Das et al. investigated the larvicidal activity of chalcone derivatives against 3rd instar larvae
of Culex quinquefasciatus [Das et al. (2010)]. Among all the compounds tested, 1,3-
diphenyl-2-propen-1-one showed highest toxicity with LC50 value of 19.31 ppm (Figure
10). Replacement of phenyl ring of above chalcone with CH3 group produced 4-phenylbut-
3-en-2-one with lesser activity (LC50: 69.90 ppm, Figure 10). Similarly, conjugated
chalcone, 1,5-diphenylpenta-1,4-dien-3-one and its 2,4-dinitro-phenylhydrazone analogue
was found almost inactive at 100 ppm concentration.
LC50 = 19.31 ppm LC50 = 69.90 ppm
O
LC50 >100.00 ppm
O
LC50 = 55.52 ppm
NO
O
NNH
NO2O2N
LC50 >100.00 ppm
Figure 10
Similarly, cinnoline based chalcones and their pyrazoline derivatives have been evaluated
for insecticidal activity against Periplaneta americana [Gautam and Chourasia (2010)].
Results show that cinnoline chalcones possessing electron-withdrawing substitution (R = 2-
Cl, 3-Br, 4-Cl etc) provided potent insecticidal agents whereas in the pyrazoline series good
activity is obtained in case of hydroxyl substitution (R = 2-OH, 4-OH etc) (Figure 11).
NN
CH3 O
R
NN
CH3
R
N NH
R = 3-NO2, 2-Cl, 3-Cl, 4-Cl, 3-Br, 2-NO2, 4-OMe, 4-NO2, 2-OH, 4-OH,4-OH-3-OMe, 4-N(CH3)2 etc.
(Cinnoline chalcones) (Cinnoline based pyrazoline derivatives)
Figure 11
Structure-activity relationship…….. Chapter 3
125
Recently, Begum et al. synthesized a series of chalcone derivatives and evaluated for their
larvicidal activity in mosquito [Begum et al. (2011]. SAR studies revealed that chalcones
having ERG’s on either ring A or ring B favors the larvicidal activity, whereas, presence of
EWG’s, particularly on ring B, reduced the activity of chalcones. Similarly, extension of
conjugation or blocking of -unsaturated ketone unit of chalcones reduced the activity
(Figure 12).
O O
LC50 = 90.00 µmole/dm3 LC50 = 5.00 µmole/dm3 LC50 = 5.00 µmole/dm3
O
O
O
O
LC50 = 2064.00 µmole/dm3
Cl
O
O HO
LC50 = 19.00 µmole/dm3
NNH
LC50 = 4654.00 µmole/dm3
A B
Figure 12
Moreover, chalcones have also been reported to possess nematicidal activity. For instance,
Gonzalez et al. studied the nematicidal activity against potato-cyst nematodes, wherein, (E)-
chalcone (trans-1,3-diphenylpropenone) was found to be highly toxic for phytoparasitic
nematodes besides acting as potent inhibitor of nematode hatch (HIC50 = 7 M) [Gonzalez
and Braun (1998)].
However, to the best of our knowledge, pesticidal activity of chalcones against one of the
most destructive crucifer defoliator, P. xylostella has not yet been explored. In the present
work, for the first time, we have studied the pesticidal SAR of chalcones against P.
xylostella.
3.2.3 Results and Discussion:
3.2.3.1 Chemistry and synthesis of chalcones:
Compounds 44 & 45 (Table 8, entries 1-2) were prepared from the oxidation of natural β-
asarone and anethole respectively (Scheme 18) [Sinha et al. (2003)]. Chalcones 46-57
(Table 8-9) were prepared by Claisen-Schmidt condensation between benzaldehydes and
acetophenone (acetone in case of 46) under MW irradiation.
Structure-activity relationship…….. Chapter 3
126
O
CH3COCH3, M.WOsO4/NaIO4
R R
CHO
M.W
R = 2,4,5-tri-OCH3; -AsaroneR = 4-OCH3; Anethole
[MIMBSA]HSO4R44-45
Scheme 18. Synthesis of chalcone derivatives 44 & 45 from natural phenylpropenes under MWirradiation
3.2.3.2 General pesticidal activity:
The % mortality for all the synthesized chalcone derivatives were determined using leaf dip
method against 2nd instar larvae of lepidopteron insect, P. xylostella. Initial screening of the
test compounds were carried out at two higher test dosages; 10000 g/ml & 5000 g/ml.
Lethal concentration (LC50) was calculated only for those compounds which showed 100%
mortality at 5000 g/ml.
3.2.3.2.1 Preliminary screening for pesticidal activity:
Preliminary screening of compounds (Table 8, entries 1-7) for pesticidal activity did not
show promising results (≤30% mortality) against the larvae after 48 h of exposure time.
Moreover, presence of an OH group in 46 (Table 8, entry 3) and the naphthalene ring (47,
Table 8, entry 6) created solubility problems. In view of the well known pesticidal potency
of various chlorinated compounds, chalcone 11 having Cl substitution at ring B and OCH3
at ring A was screened which showed 50% larval mortality at 10000 g/ml dosage (Table 8,
entry 8). To our surprise, reversal of the substituents (Cl v/s OCH3) of 11 resulted in
compound 1 (Table 8, entry 9) with 100% mortality even at 5000 g/ml. On the other side,
replacement of Cl on ring A with F (49, Table 8) or Br (50, Table 8) substituents caused
dramatic reduction in the larval mortality. A comparison of the activities of chalcones
(Table 8) indicates that electron-withdrawing substituents (particularly Cl) on ring A and
electron-releasing groups (ERG’s) on ring B of chalcone ( A-CH=CH-CO-B )
increases the pesticidal potential while positional interchange ( B-CH=CH-CO-A ) of
these substituents on both rings causes a decrease. Interestingly, above observation is in
contrast to our recent finding on antimalarial activity [Kumar et al. (2010)] wherein, potent
chalcones were having ERG’s on ring A and EWG’s on ring B. Such findings would draw
the attention of researchers in future while designing chalcone based novel compounds with
desired activities.
Structure-activity relationship…….. Chapter 3
127
3.2.3.2.2 Screening of chloro-substituted chalcones for pesticidal activity:
Among the various tested chalcones (Table 8), compound 1 (LC50: 356.45 g/ml) was
selected as prelude for further modification to improve and understand the pesticidal SAR.
As expected, compounds with more electron-withdrawing substituents such as 2,4-dichloro
(51, Table 9, entry 13) or 3,4-dichloro (10, Table 9, entry 14) on ring A exhibited better
activity compared to single chloro (1, Table 8, entry 9). Although, pesticidal activity of both
CompoundNo
45
46
3
26.6 ± 0.57
solubility problem
30 ± 0
0 ± 0
-
26.6 ± 0.57
10000 µg/ml 5000 µg/ml
O
R R'( a; 44-46) ( b; 2-3) (d; 1, 4, 11, 48-50)
2,4,5-trimethoxy44 a
4-methoxy
4-hydroxy-3-methoxy
a
a
R R'
-
-
-
d 4-methoxy 4-methoxy
2
47
48
11
1
49
50
4
b
b
d
d
d
d
d
sulphur 4-nitro
oxygen 4-nitro
4-methoxy 4-chloro
4-chloro 4-methoxy
4-fluoro 4-methoxy
4-bromo 4-methoxy
4-chloro 3,4-methylenedioxy
c 4-methoxy solubility problem
23.3 ± 0.57
-
23 ± 0.57 16.6 ± 0.57
50 ± 1.15 26.6 ± 0.57
100 ± 0 100 ± 0
33.3 ± 0.57 26.6 ± 0.57
10 ± 0 3.3 ± 0.57
23 ± 0 13.3 ± 0.57
26.6 ± 0 16.6 ± 0.57
30 ± 0
O
R'
O
R R
O
R'( c; 47)
Mortality [%]a ± SD
-
356.45
-
-
-
-
-
-
-
-
-
-
-
LC50(µg/ml)b
Table 8. Preliminary screening of chalcone derivatives against P. xylostella after 48 h
a Data represent the mean values of the three replicates and mortality in control accounted usingAbbott's formula; b Only for those compounds which showed 100% larval mortality at 5000 µg/ml
TypeEntry
2
3
4
1
5
6
7
8
9
10
11
12
(Med. Chem. Res. 2011, DOI: 10.1007/s00044-011-9602-8)
Structure-activity relationship…….. Chapter 3
128
the dichlorinated chalcones 51 & 10 is comparable, however, based on evaluation of dose
response data values, 10 could be a better candidate if 100% kill is considered on
bioefficacy indicator parameter. Therefore, in further study, effect of different substituents
on ring B in association with 3,4-dichloro substitution on ring A was evaluated (52-58,
Table 9, entries 15-21).
51
10
52
53
54
55
56
57
58
Mortality [%]a ± SD
100 ± 0
solubility problem
100 ± 0
83.3 ± 0.57 60 ± 0
63.3 ± 0.57 50 ± 1
68 ± 0.57 46.6 ± 0.57
solubility problem -
97.7 ± 0 60 ± 0
73.3 ± 0.57 46.6 ± 0.57
100 ± 0 100 ± 0
10000 µg/ml 5000 µg/ml
O
Ring A Ring B
Substitution on
2,4-dichloro 4-methoxy
3,4-dichloro 4-methoxy
4-methyl
2-methoxy
3-methoxy
3,4-dimethoxy
3,4,5-trimethoxy
4-chloro
3,4-dichloro
CompoundNo.
LC50 (µg/ml)b
7 4-chloro 4-chloro
4-nitro
4-hydroxy
4-allyl
9
5
6
100 ± 0 100 ± 0
26.6 ± 0.57 20.0 ± 0
100 ± 0 100 ± 0
100 ± 0 100 ± 0
278.50
287.64
170.24
871.05
268.20
-
-
-
-
-
-
-
-
-
Table 9. Pesticidal activity of chloro-substituted chalcones against P. xylostella after 48 h
a Data represent the mean values of the three replicates and mortality in control accountedusing Abbott's formula; bFor those compounds which showed 100% mortality at 5000 µg/ml.
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
3,4-dichloro
4-chloro
4-chloro
4-chloro
Entry
13
14
15
16
17
18
19
20
21
22
23
24
25
Ring A Ring B
(Med. Chem. Res. 2011, DOI: 10.1007/s00044-011-9602-8)
Interestingly, replacement of OCH3 group of compound 10 with another ERG i.e. CH3 (52)
induced less mortality. On the other side, introduction of OCH3 group at ortho (53) position
showed drastic reduction in the activity, whereas, its presence at meta position (54) met
with the solubility problem (Table 9, entries 16-17). Similarly, increase in the electron
Structure-activity relationship…….. Chapter 3
129
density on ring B with 3,4-dimethoxy (55) or 3,4,5-trimethoxy groups (56) provided lower
activity as compared to 10.
From a structure-activity perspective, effect of electron-withdrawing substituents on ring B
was also evaluated. However, compounds 57 and 58 possessing 4-chloro and 3,4-dichloro
substituents respectively were found inactive (Table 9, entries 20-21). A comparison of the
% mortality of compounds 10, 57 & 58 (Table 9) indicated that as we increased the number
of Cl substituents from two to three or four, activity reduced significantly. Hence, it was
realized that chalcone derivatives with two Cl substituents might increase the pesticidal
potential. Consequently, chalcone 7 (Table 9, entry 22) possessing electron-withdrawing Cl
substituent on ring A as well as on ring B was screened and it showed good activity (7,
LC50: 170.24 g/ml), however introduction of polar NO2 group (9, Table 9, entry 23) on
ring B caused drastic reduction in the larval mortality. On the other side, replacement of Cl
of 7 with isosteric polar group i.e. OH provided 5 (Table 9, entry 24) with lower activity
(LC50: 871.05 g/ml). These results indicate the importance of both lipophilic as well as
electron-withdrawing substitution (such as Cl) on ring B for enhanced pesticidal activity.
The above assumption proved right when compound 6 (OCH2CH=CH2; lipophilic and
ERG) showed lesser activity than 7 but significantly improved activity than 5.
In order to develop dosages response lines for identified potent chalcones (1, 5, 6, 7, 10 &
51), their efficacy at different concentration were evaluated. LC50 values and other
statistical parameters generated by linear regression analysis were compared and
represented in Table 10. Overall pesticidal activity of above potential chalcone was found to
follow the order: 7 > 6 > 51 > 10 > 1 > 5.
Table 10. Insecticidal activity (LC50 values and regression parameters of Probitanalysis) of the promising compounds against P. xylostella after 48 h.
SerialNo.
Compound No.
LC50 values and regression parameters of probitanalysis after 48 h
LC50 [FL] in g/ml
2 InterceptLC50LowerLimit
UpperLimit
1
2
3
4
5
6
7
1
51
10
7
5
6
Deltamethrin
356.45
278.50
287.64
170.24
871.05
268.20
6.25
307.8
213.2
221.3
130.2
552.7
197.3
2.21
417.1
367.8
377.8
222.08
1950.8
381.8
11.9
1.442
6.682
3.729
1.644
1.477
3.681
4.189
0.37623
0.07669
0.08344
1.91373
1.33189
1.04128
4.02511
Structure-activity relationship…….. Chapter 3
130
3.2.3.3 Proposed mode of action:
A brief delve in literature demonstrates the presence of glutathione S-transferase (GST)
isoenzymes and GST genes in P. xylostella which helps in detoxification of endogenous and
xenobiotic compounds in vertebrates and invertebrates [Rushmore and Pickett (1993); Sonoda
et al. (2006)]. Mode of action of chalcone may be attributed to the interaction of electrophilic
-position of -unsaturated ketone bridge (-CH=CH-CO-) with reduced glutathione (GSH)
to form its conjugate and consequent inhibition of GST [Miyamoto and Yamamoto (1994)].
In general, EWG’s groups on ring A and ERG’s on ring B of chalcone would help increase
the stability of GSH conjugate (Figure 13) and therefore accounts for their pesticidal
potential.
O
A B
Cl OCH 3
O
A B
Cl OCH 3
GSH GSH
(i) Electron withdrawing groups on ring A(ii) Electron releasing groups on ring B Stable GSH adduct
glutathione S-transferase
Figure 13. Proposed Michael type addition of glutathione with chalcone
The above hypothesis demonstrates that compound 1 (Table 8, entry 9) is more active than
compound 11 (Table 8, entry 8). However, low activities of compounds 55 & 56 (Table 9,
entries 18-19) despite having more ERG’s on ring B may be attributed to the reluctance of
such chalcones towards Michael GSH adduct formation [Jin et al. (2007)]. Beside electronic
consideration, lipophilic and hydrophilic characteristics of various substituents are also
responsible for influencing the activity.
3.3 Conclusions:
We have identified a basic chalcone scaffold (having 2,4,5-trimethoxy substitution on aryl
ring A) which is crucial for antimalarial activity. This is the first SAR study which shows
that chalcones which are methoxylated in their aryl ring A and electron deficient at ring B
are better antimalarials than those in which these groups are interchanged. Moreover,
synthesis of lead candidates from abundantly available natural precursor i.e. β-asarone
(2,4,5-trimethoxyphenyl propene) rich Acorus calamus oil offers a good potential to
develop cost effective antimalarial drugs. The strong dependence of antimalarial activity on
Structure-activity relationship…….. Chapter 3
131
specific substitution of rings A & B as well as possibility of further modification on above
identified unit would be helpful for design of new therapeutic antimalarials.
In addition, for the first time we have explored the pesticidal potential of chalcones against
P. xylostella, wherein, for good activity, electron-withdrawing ring A of chalcone was
found crucial while ring B can bear either electron-withdrawing or electron-releasing
substituents. Particularly, Cl substitution and its position on ring A as well as on ring B was
found vital as compound 1,3-bis(4-chlorophenyl)prop-2-en-1-one (7) showed the maximum
activity with LC50 value of 170.24 g/ml. Although, this compound is 27 times lesser active
than commercial pesticide deltamethrin, however, this study is the first report wherein a
simple moiety like chalcone has provided a promising lead for pesticidal activity against P.
xylostella. The identified potent units can be further modified to exhibit better potency than
commercial pesticides. In addition, the results of present study would be of value in guiding
the design of novel chalcone based economical pesticidal agents against P. xylostella and
related insect pests.
3.4 Experimental Section:
3.4.1 Materials & Instruments:
All the reagents/solvents were obtained from commercial sources (Merck or Sigma Aldrich)
and used without further purification. β-asarone was obtained from natural Acorus calamus
oil following our earlier reported procedure [Sinha et al. (2002)]. The ILs used in this study
were obtained either commercially (Merck & Alfa Aesar) or synthesized ([hmim]Br,
[Hmim]pTSA, [bmim]OH, [MIMBSA]HSO4) according to reported methods [Zhao et al.
(2004); Nockemann et al. (2005); Ranu and Banerjee (2005); Dong et al. (2008)]. Column
chromatography was performed using silica gel (60-120 mesh size). CEM Discover©
focused microwave (2450 MHz, 300W) was used wherever mentioned. 1H (300 MHz) and13C (75.4 MHz) NMR spectra were recorded on a Bruker Avance-300 spectrometer using
tetramethylsilane (TMS) as internal standard. HRMS-ESI spectra were determined using
micromass Q-TOF ultima spectrometer. The melting points were determined on a digital
Barnsted Electrothermal 9100 apparatus.
3.4.2. General procedure for the synthesis of chalcones and their derivatives:
3.4.2.1 General procedure for the synthesis of chalcones 1-5 (Table 1 & Scheme 10):
To a solution of substituted benzaldehyde (3 mmol) and appropriate acetophenone (3 mmol)
in methanol (15 ml), 10% aqueous NaOH (6 mmol) was added. The reaction mixture was
Structure-activity relationship…….. Chapter 3
132
stirred till completion of starting material (3-24 h). The obtained precipitates were washed
with dilute HCl, excess of water, methanol, dried in air and finally recrystallized with
methanol to obtain pure chalcones whose structure were confirmed by NMR spectra as
discussed below:
3-(4-Chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (1, Table 1) [Dong et al.
(2008)]
Cl
O
OCH3 Creamy solid (88%), m.p. 128-131°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.97 (2H, d, J = 9.2 Hz), 7.69 (1H, d, J = 16.1 Hz), 7.50-7.41 (3H, m),
7.31 (2H, d, J = 8.1 Hz), 6.92 (2H, d, J = 8.7 Hz), 3.81 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 188.4, 163.5, 142.4, 136.1, 133.6, 130.8, 129.5, 129.2, 122.3, 113.9 and
55.5.
3-(Furan-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (2, Table 1)
OO
NO2
Yellow solid (76%), m.p. 139-142°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.27 (2H, d, J = 8.4 Hz), 8.09 (2H, d, J = 8.8 Hz), 7.58-7.49 (2H, m), 7.35 (1H, d, J =
15.3 Hz), 6.73 (1H, d, J = 4.0 Hz), 6.48 (1H, s); 13C NMR (75.4 MHz, CDCl3); (ppm)
188.1, 151.2, 150.0, 145.6, 142.9, 132.1, 129.3, 123.8, 118.3, 117.7 and 113.0. HRMS-ESI:
m/z [M+H]+ for C13H9NO4, calculated 244.0604; observed 244.0615.
1-(4-Nitrophenyl)-3-(thiophen-2-yl)prop-2-en-1-one (3, Table 1)
SO
NO2
Yellow solid (73%), m.p. 171-173°C, 1H NMR (300 MHz, DMSO);
(ppm) 8.34-8.30 (4H, m), 7.99 (1H, d, J = 15.0 Hz), 7.83 (1H, s), 7.73 (1H, s), 7.57 (1H, d,
J = 15.0 Hz ), 7.21 (1H, s); 13C NMR (75.4 MHz, DMSO); (ppm) 188.3, 150.2, 142.8,
139.9, 138.7, 134.2, 131.8, 130.2, 129.3, 124.3 and 120.5. HRMS-ESI: m/z [M+H]+ for
C13H9O3NS, calculated 260.0376; observed 260.0372.
1-(3,4-Dioxymethylene)-3-(4-chlorophenyl)prop-2-en-1-one (4, Table 1)
Cl
O
O
O
White solid (85%), m.p. 164-167°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.68 (1H, d, J = 15.7 Hz), 7.57 (1H, d, J = 8.0 Hz), 7.49-7.44 (3H, m), 7.40 (1H, d, J
= 15.7 Hz), 7.31 (2H, d, J = 8.4 Hz ), 6.82 (1H, d, J = 8.0 Hz), 5.98 (2H, s); 13C NMR (75.4
Structure-activity relationship…….. Chapter 3
133
MHz, CDCl3); (ppm) 187.9, 151.8, 148.3, 142.7, 136.2, 133.5, 132.8, 129.5, 129.2, 124.7,
122.1, 108.4, 107.9 and 101.9. HRMS-ESI: m/z [M+H]+ for C16H11ClO3, calculated
287.0470; observed 287.0469.
3-(4-Chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (5, Table 1) [Batovska et al.
(2009)]
Cl
O
OH White solid (54%), m.p. 178-179°C, 1H NMR (300 MHz,
CD3COCD3); (ppm) 9.31(1H, s), 8.10 (2H, s), 7.85-7.73 (4H, m), 7.48 (2H, s), 6.98 (2H,
s); 13C NMR (75.4 MHz, CD3COCD3); (ppm) 188.4, 163.4, 142.7, 136.7, 135.7, 132.5,
131.5, 130.4, 124.2 and 116.7.
3.4.2.2 Procedure for the synthesis of allylated chalcone 6 (Table 1):
To the solution of hydoxychalcone 5 (0.5 g, 1.9 mmol) in dry acetone (20 ml), allyl bromide
(0.46 g, 3.8 mmol) and anhydrous K2CO3 (0.52 g, 3.8 mmol) were added. The reaction
mixture was refluxed for 6 h. After consumption of starting chalcone (monitored on TLC),
reaction mixture was filtered to remove K2CO3. The filtrate was vacuum evaporated and
washed with hexane to remove excess of allyl bromide. The obtained crude solid was
recrystallized with methanol to obtain 6 as a white solid in 69% yield (0.39 g), m.p. 119-
120°C.
3-(4-Chlorophenyl)-1-[4-(prop-2-en-1-yloxy)phenyl] prop-2-en-1-one (6, Table 1)O
Cl O 1H NMR (300 MHz, CDCl3); (ppm) 8.05 (2H, d, J = 8.0 Hz),
7.77 (1H, d, J = 16.2 Hz), 7.58-7.49 (3H, m), 7.40 (2H, d, J = 8.9 Hz), 7.02 (2H, d, J = 8.04
Hz ), 6.14-6.01 (1H, m), 5.48-5.31 (2H, m), 4.64 (2H, d, J = 5.1 Hz); 13C NMR (75.4 MHz,
CDCl3); (ppm) 188.7, 162.9, 142.7, 136.5, 134.0, 132.9, 131.4, 131.2, 129.8, 129.5, 122.7,
118.5, 115.0 and 69.3. HRMS-ESI: m/z [M+H]+ for C18H15ClO2, calculated 299.0833;
observed 299.0833.
3.4.2.3 General procedure for the synthesis of chalcones 7-12 (Table 1 & Scheme 10):
The reactions were performed in the same manner as given in section 3.4.2.1 above using
substituted benzaldehydes and acetophenones. The reactions upon completion were worked
Structure-activity relationship…….. Chapter 3
134
up in the same manner as given in section 3.4.2.1. The NMR spectra of obtained pure
chalcones 7-12 are given below:
Bis(4-chlorophenyl)prop-2-en-1-one (7, Table 1) [Santos et al. (2006)]
Cl
O
Cl Off white solid (91%), m.p. 108-111°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.98 (2H, d, J = 7.6 Hz), 7.79 (1H, d, J = 16.6 Hz), 7.59 (2H, d, J = 8.4 Hz), 7.50-
7.39 (5H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 189.1, 144.1, 139.7, 137.0, 136.7,
133.6, 130.2, 130.0, 129.7, 129.4 and 122.3.
1-(4-Bromophenyl)-3-(4-chlorophenyl)prop-2-en-1-one (8, Table 1)
Cl
O
Br White solid (89%), m.p. 164-168°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.88-7.74 (3H, m), 7.64-7.58 (4H, m), 7.41-6.99 (3H, m); 13C NMR (75.4 MHz,
CDCl3); (ppm) 188.7, 143.5, 136.4, 132.9, 131.7, 129.7, 129.3, 129.0, 128.5, 127.7 and
121.6.
3-(4-Chlorophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (9, Table 1) [Batovska et al.
(2009)]O
NO2Cl Yellow solid (73%), m.p. 158-161°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.37 (2H, d, J = 7.2 Hz), 8.16 (2H, d, J = 7.2 Hz), 7.82 (1H, d, J = 15.6), 7.61 (2H,
d, J = 8.2 Hz), 7.49-7.41 (3H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 188.4, 149.8,
144.9, 142.5, 136.9, 132.5, 130.7, 129.1, 128.8, 123.6 and 121.4. HRMS-ESI: m/z [M+H]+
for C15H10ClNO3, calculated 288.0422; observed 288.0425.
3-(3,4-Dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (10, Table 1)
Cl
O
OCH3
Cl
Pale yellow solid (89%), m.p. 129-131°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.05 (2H, d, J = 9.3 Hz), 7.70-7.64 (2H, m), 7.54-7.42 (3H, m), 7.00 (2H,
d, J = 9.1 Hz ), 3.90 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 188.3, 164.0, 141.3,
135.6, 134.5, 133.6, 131.2, 131.1, 130.0, 127.8, 123.8, 114.3 and 55.9. HRMS-ESI: m/z
[M+H]+ for C16H12Cl2O2, calculated 307.0287; observed 307.0280.
Structure-activity relationship…….. Chapter 3
135
1-(4-Chlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (11, Table 1) [Liu et al.
(2001)]
H3CO
O
Cl Creamy solid (87%), m.p. 120-124°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.02-7.97 (2H, m), 7.86 (1H, d, J = 16.6 Hz), 7.66-7.62 (2H, m), 7.52-7.48
(2H, m), 7.44 (1H, d, J = 16.6 Hz ), 7.00-6.96 (2H, m), 3.89 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 189.4, 162.2, 145.5, 139.3, 137.2, 130.7, 130.2, 129.2, 127.8, 119.5, 114.8
and 55.7.
1-(4-Chlorophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (12, Table 1)O
H3CO ClOCH3 Yellow solid (92%), m.p. 124-126°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.09 (1H, d, J = 15.7 Hz), 7.97-7.93 (2H, m), 7.58 (1H, d, J = 8.4 Hz ), 7.53-7.44
(3H, m), 6.55 (1H, d, J = 7.1 Hz), 6.48 (1H, s), 3.90 (3H, s), 3.86 (3H, s); 13C NMR (75.4
MHz, CDCl3); (ppm) 190.1, 163.6, 160.9, 141.4, 138.9, 137.5, 131.4, 130.2, 129.1, 120.2,
117.3, 105.9, 98.8, 55.9 and 55.8. HRMS-ESI: m/z [M+H]+ for C17H15O3Cl calculated
303.0783; observed 303.0783.
3.4.2.4 General procedure for the synthesis of chalcone 13 (Table 1):
A solution of 2,4-dimethoxybenzaldehyde (0.5 g, 3.0 mmol), acetone (7 ml) and 10%
aqueous NaOH (6.0 mmol) was stirred at room temperature for overnight. The reaction
upon completion followed by work up and purification as given in section 3.4.2.1 provided
13 as a pale yellow solid in 81% yield (0.5 g).
4-(2,4-Dimethoxyphenyl)but-3-en-2-one (13, Table 1)
CH3
O
OCH3H3CO m.p. 62-64°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.73 (1H, d, J =
16.5 Hz), 7.39 (1H, d, J = 8.7 Hz), 6.59 (1H, d, J = 16.5 Hz), 6.42 (1H, d, J = 8.7 Hz), 6.35
(1H, s), 3.76 (3H, s), 3.73 (3H, s), 2.2 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm)
199.0, 163.0, 159.8, 138.7, 129.8, 125.4, 116.3, 105.5, 98.3, 55.5, 55.4 and 27.0. HRMS-
ESI: m/z [M+H]+ for C12H14O3, calculated 207.1016; observed 207.1011.
Structure-activity relationship…….. Chapter 3
136
3.4.2.5 Green synthesis of chalcone 14 from -asarone rich Acorus calamus oil in ionic
liquid under microwave irradiation (Table 2 & Scheme 11):
A mixture of -asarone (0.31 g, 1.5 mmol), NaIO4 (1.17 g, 5.5 mmol), OsO4 (0.0004 g,
0.0015 mmol), and benzyltriethylammonium chloride (0.01 g, 0.04 mmol) were dissolved in
H2O-THF (3 ml, 4:1) and irradiated under focused MW (150W, 100°C) for 2 min. After
completion of starting material, the reaction mixture was extracted with ethyl acetate (3 x 15
ml) and vacuum evaporated to give a crude mixture, which on column chromatography with
silica gel (60-120 mesh size) using hexane-ethyl acetate (9:1), provided 2,4,5-
trimethoxybenzaldehyde in 83% yield (0.24 g) [Sinha et al. (2003)]. Subsequently, the
above benzaldehyde (0.24 g, 1.22 mmol) was taken in methanol (1 ml) and subjected to
Claisen-Schmidt condensation with 4-chloroacetophenone (0.189 g, 1.22 mmol) using
[MIMBSA]HSO4 (1 g) under MW irradiation (100W, 75°C) for 3 minutes. The obtained
precipitates were washed with excess of water and recrystallized from methanol to obtain 14
as a yellow solid in 87% yield (0.35 g).
Various other ILs such as [hmim]Br, [bmim]Cl, [bmim]OH, [Hmim]pTSA etc in place of
[MIMBSA]HSO4 were also screened for above condensation under MW irradiation,
however, in all cases product was obtained in inferior yield.
1-(4-Chlorophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (14, Table 2) [Patil and
Dharmaprakash (2008)]OOCH3
H3COOCH3
Cl
Yellow solid (87%), m.p. 141-142°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.12 (1H, d, J = 15.8 Hz), 7.97 (2H, d, J = 8.6 Hz), 7.49 (2H, d, J =8.4 Hz), 7.45
(1H, d, J = 15.8 Hz), 7.13 (1H, s), 6.54 (1H, s); 3.9 (3H, s), 3.92 (6H, s); 13C NMR (75.4
MHz, CDCl3); (ppm) 190.18, 155.2, 155.1, 143.7, 141.0, 139.0, 137.5, 130.2, 129.1,
120.1, 115.7, 112.0, 97.2, 57.0, 56.7 and 56.4.
The same procedure was also applied for the synthesis of various other 2,4,5-trimethoxy
substituted chalcones 22-29 (Table 4) whose spectral data is discussed in section 3.4.3.1.
3.4.2.6 General procedure for the synthesis of chalcones 15-16 (Table 2 & Scheme 10):
The reactions were performed in the same manner as given in section 3.4.2.1 using
substituted benzaldehydes and acetophenones. The reactions upon completion were worked
Structure-activity relationship…….. Chapter 3
137
up in the same manner as given in section 3.4.2.1. The NMR spectral data of obtained pure
chalcones 15 and 16 are given below:
1-(4-Chlorophenyl)-3-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (15, Table 2)OCH 3
H3CO
O
ClOCH3 Yellow solid (88%), m.p. 155-157°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.29 (1H, d, J = 15.5 Hz), 7.96 (2H, d, J = 8.2 Hz), 7.85 (1H, d, J = 15.5
Hz), 7.45 (2H, d, J = 8.6 Hz), 6.13 (2H, s), 3.91 (6H, s), 3.86 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 191.1, 163.7, 162.2, 138.5, 138.0, 136.9, 130.2, 128.6, 121.6, 106.8, 90.9,
56.2 and 55.7. HRMS-ESI: m/z [M+H]+ for C18H17O4Cl, calculated 333.0888; observed
333.0886.
1-(4-Chlorophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (16, Table 2)
H3COOCH 3
O
Cl
H3CO
Pale yellow solid (91%), m.p. 108-110°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.05 (2H, d, J = 9.1 Hz), 7.82 (1H, d, J = 15.7 Hz), 7.55 (2H, d, J = 8.0
Hz), 7.47 (1H, d, J = 15.7 Hz), 6.94 (2H, s), 4.00 (9H, s); 13C NMR (75.4 MHz, CDCl3);
(ppm) 189.6, 154.2, 146.1, 141.4, 139.7, 137.2, 130.8, 130.6, 129.5, 121.5, 106.6, 61.6 and
56.9. HRMS-ESI: m/z [M+H]+ for C18H17O4Cl, calculated 333.0888; observed 333.0883.
For comparison purpose, synthesis of some of the above chalcones (1-5, 7 and 9-12, Table
1) was also accomplished under MW conditions (150W, 55°C). The yields of the products
were found almost similar to that of room temperature stirring. However, shorter reaction
time (20 min v/s 3 h) beside less amount of solvent is required in case of MW irradiation.
3.4.2.7 General procedure for synthesis of hydrogenated derivatives of chalcones, 17
and 18 (Scheme 12 & Scheme 16):
Compound 17 was prepared by the chemoselective hydrogenation of 14 using silica-
supported PdCl2 as catalyst and a combination of MeOH/HCOOH/H2O [Sharma et al.
(2006)] as a source of hydrogen. Product 17 was isolated in 82% yield as a viscous liquid.
Further, reduction of 17 with NaBH4 [Chamberlin and Daniel (1991)] afforded the
corresponding alcohol (18) in 85% yield. The NMR data (1H & 13C) of compounds 17 & 18
are given below:
Structure-activity relationship…….. Chapter 3
138
1-(4-Chlorophenyl)-3-(2,4,5-trimethoxyphenyl)propan-1-one (17)OCH3
H3COOCH3
O
Cl
Viscous liquid, 1H NMR (300 MHz, CDCl3); (ppm) 7.92 (2H,
d, J = 8.5 Hz), 7.43 (2H, d, J = 8.5 Hz), 6.76 (1H, s), 6.53 (1H, s), 3.89 (3H, s), 3.83 (3H,
s), 3.81 (3H, s), 3.23 (2H, t, J = 7.2 Hz), 3.00 (2H, t, J = 7.60 H); 13C NMR (75.4 MHz,
CDCl3); (ppm) 199.3, 152.1, 148.7, 143.4, 139.8, 135.8, 130.1, 129.3, 121.2, 115.1, 98.3,
57.2, 56.8, 56.7, 39.9 and 25.9. HRMS-ESI: m/z [M+H]+ for C18H19ClO4, calculated
335.1045; observed 335.1041.
1-(4-Chlorophenyl)-3-(2,4,5-trimethoxyphenyl)propan-1-ol (18)OCH3
H3COOCH3
OH
Cl
Viscous liquid, 1H NMR (300 MHz, CDCl3); (ppm) 7.35-7.25
(4H, m), 6.70 (1H, s), 6.53 (1H, s), 4.60-4.55 (1H, m), 3.88 (3H, s), 3.83 (3H, s), 3.82 (3H,
s), 2.79-2.62 (2H, m), 2.01-1.89 (2H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 151.4,
147.9, 143.3, 132.7, 128.3, 127.2, 125.8, 121.3, 114.4, 98.1, 72.5, 56.7, 56.3, 56.2, 39.7 and
25.8.
3.4.2.8 Procedure for synthesis of Michael adduct 19 (Table 3 & Scheme 13):
To a solution of chalcone 14 (1.05 mmol, 0.35 g) in acetonitrile (15 ml), catalytic amount of
P-toluenesulfonic acid (20 mol%, 0.038 g) and indole (1.26 mmol, 0.147 g) were added [Ji
and Wang (2005)]. The reaction mixture was stirred at room temperature for overnight.
After evaporation of acetonitrile, the obtained crude mixture was purified through
recrystallization with methanol and water to afford 19 as a white solid in 81% yield (0.21
g).
1-(4-Chlorophenyl)-3-(1H-indol-3-yl)-3-(2,4,5-trimethoxyphenyl)propan-1-one (19,
Table 3)
H3COOCH3
O
Cl
H3CO
NH
m.p. 149-150°C, 1H NMR (300 MHz, CDCl3); (ppm) 8.05 (1H,
br, s), 7.97 (2H, d, J = 8.2 Hz), 7.45-7.42 (3H, m), 7.36 (1H, d, J = 7.8 Hz), 7.19 (1H, d, J
Structure-activity relationship…….. Chapter 3
139
= 6.7 Hz ), 7.14-7.12 (1H, m), 7.05-7.00 (1H, m), 6.69 (1H, s), 6.56 (1H, s), 5.37 (1H, t, J =
7.3 Hz), 3.90 (3H, s), 3.86 (3H, s), 3.69 (2H, d, J = 7.5 Hz), 3.65 (3H, s); 13C NMR (75.4
MHz, CDCl3); (ppm) 198.2, 151.1, 148.4, 143.2, 139.3, 136.7, 135.5, 129.8, 128.9, 127.0,
124.0, 122.2, 122.0, 119.7, 119.5, 118.5, 113.5, 111.2, 97.9, 56.9, 56.5, 56.3, 44.9 and 32.5.
HRMS-ESI: m/z [M+H]+ for C26H24O4Cl N, calculated 450.1467; observed 450.1467.
3.4.2.9 Procedure for the synthesis of N-allylated Michael adduct 20 (Table 3 &
Scheme 13):
To a solution of 19 (0.3 g, 0.67 mmol) in dry THF (15 ml), KOH (0.112 g, 2 mmol), allyl
bromide (0.161 g, 1.34 mmol) and CTAB (0.048 g, 20 mol%) were added. The reaction
mixture was stirred for overnight. After evaporation of THF, the crude reaction mixture was
washed with hot water and then hexane to remove the excess of allyl bromide. The obtained
crude product on recrystallization with diethyl ether afforded 20 as a white solid in 79%
yield (0.26 g).
1-(4-Chlorophenyl)-3-[1-(prop-2-en-1-yl)-1H-indol-3-yl]-3-(2,4,5-trimethoxyphenyl)-
propan-1-one (20, Table 3)
H3COOCH3
O
Cl
H3CO
N
m.p. 126-127°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.94 (2H, d,
J = 8.2 Hz), 7.42-7.40 (3H, m), 7.28 (1H, d, J = 7.3 Hz), 7.19-7.14 (1H, m), 7.03-7.00 (2H,
m), 6.70 (1H, s), 6.54 (1H, s), 6.02-5.95 (1H, m), 5.35 (1H, t, J = 7.1 Hz), 5.19 (1H, d, J =
10.0), 5.05 (1H, d, J = 17.3 Hz), 4.70 (2H, s), 3.88 (3H, s), 3.84 (3H, s), 3.65 (5H, s); 13C
NMR (75.4 MHz, CDCl3); (ppm) 198.4, 151.3, 148.6, 143.4, 139.5, 137.1, 135.9, 134.1,
130.0, 129.1, 127.8, 126.0, 124.4, 122.1, 120.1, 119.3, 117.6, 117.2, 113.7, 109.8, 98.2,
57.1, 56.7, 56.5, 49.0, 45.2 and 32.7. HRMS-ESI: m/z [M+H]+ for C29H28O4ClN, calculated
490.1780; observed 490.1780.
The above two procedures i.e 3.4.2.8 & 3.4.2.9 were also applied on chalcone 1-(4-
Bromophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (synthesis and spectral data is
discussed in section 3.4.3.1, see compound 27) to provide the Michael adduct i.e. 1-(4-
Bromophenyl)-3-[1-(prop-2-en-1-yl)-1H-indol-3-yl]-3-(2,4,5-trimethoxyphenyl)propan-1-
one (27c) in overall 64% yield. Further, dehydrogenation of 27c into 21 was performed as
mentioned below:
Structure-activity relationship…….. Chapter 3
140
3.4.2.10 DDQ-assisted dehydrogenation for synthesis of unsaturated Michael adduct
21 (Table 3 & Scheme 13):
To a solution of 27c (0.3 g, 0.56 mmol) in dry dioxane (15 ml), DDQ (0.153 g, 0.67 mmol)
was added and stirred at room temperature for overnight. The reaction mixture was
extracted with ethyl acetate (3x15 ml). The combined organic phases were washed
successively with water (2x10 ml), brine (2x5 ml), dried over anhydrous Na2SO4 and finally
evaporated in vacuum. The obtained crude mixture after passing through a small bed of
neutral alumina, recrystallized with diethyl ether to afford 21 as a reddish solid in 71% yield
(0.21 g).
1-(4-Bromophenyl)-3-[1-(prop-2-en-1-yl)-1H-indol-3-yl]-3-(2,4,5-trimethoxyphenyl)-
prop-2-en-1-one (21, Table 3)
H3COOCH3
O
Br
H3CO
N
m.p. 145-147°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.89 (1H, d,
J = 7.6 Hz), 7.82 (2H, d, J = 8.4 Hz), 7.55 (2H, d, J = 8.2 Hz), 7.44 (1H, s), 7.40 (1H, d, J
= 8.6 Hz ), 7.28-7.23 (2H, m), 6.99 (1H, s), 6.73 (1H, s), 6.51 (1H, s), 6.01-5.90 (1H, m),
5.25 (1H, d, J = 10.2 Hz), 5.15 (1H, d, J = 17.2 Hz), 4.71 (2H, d, J = 4.9 Hz), 3.93 (3H, s),
3.75 (3H, s), 3.60 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 190.7, 151.3, 149.8,
146.9, 143.0, 138.8, 137.7, 132.8, 132.7, 131.5, 130.0, 126.6, 126.3, 122.9, 121.4, 121.2,
120.8, 118.8, 118.1, 114.8, 110.6, 98.0, 56.7, 56.1 and 49.3. HRMS-ESI: m/z [M+H]+ for
C29H26O4BrN, calculated 532.1112; observed 532.1115.
3.4.2.11 General procedure for the synthesis of 2,4,5-trimethoxy-substituted chalcones
22-29 from -asarone rich Acorus calamus oil in ionic liquid under microwave
irradiation (Table 4 & Scheme 11):
The reactions were performed in the same manner as given in section 3.4.2.5 using various
substituted acetophenones. The reactions upon completion were worked up in the same
manner as given in section 3.4.2.5. The NMR spectra of obtained pure chalcones 22-29 are
given below:
Structure-activity relationship…….. Chapter 3
141
1-(4-Methoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (22, Table 4)OCH 3
H3COOCH 3
O
OCH 3Yellow solid (84%), m.p. 121-123°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.09-7.99 (3H, m), 7.49 (1H, d, J = 16.6 Hz), 7.11 (1H, s), 6.96 (2H, d, J
= 8.0 Hz), 6.50 (1H, s), 3.91 (3H, s), 3.87 (6H, s), 3.84 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 189.6, 163.4, 154.9, 152.7, 143.6, 139.6, 132.0, 130.8, 120.4, 116.1,
114.0, 111.9, 97.4, 57.3, 56.9 and 56.7. HRMS-ESI: m/z [M+H]+ for C19H20O5, calculated,
329.1384; observed 329.1380.
1-(2,3,4-Trimethoxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (23, Table 4)O
H3CO
OCH3
OCH3
OCH3
OCH3
OCH3
Yellow solid (86%), m.p. 90-94°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.01-7.93 (1H, m), 7.46-7.27 (2H, m), 7.14-7.11 (1H, m), 6.78-6.72 (1H, m), 6.53-
6.51 (1H, m), 3.94-3.83 (18H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 191.5, 156.5,
154.4, 153.3, 152.4, 143.3, 142.1, 138.6, 127.3, 125.4, 124.6, 115.7, 111.2, 107.2, 97.0,
62.0, 60.9, 56.5, 56.3 and 56.0. HRMS-ESI: m/z [M+H]+ for C21H24O7, calculated
389.1595; observed 389.1587.
1-(3,4-Dichlorophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (24, Table 4)OCH 3
H3CO
OCH 3
O
Cl
Cl
Yellow solid (87%), m.p. 131-134°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.13-8.07 (2H, m), 7.83 (1H, d, J = 8.1 Hz), 7.57 (1H, d, J = 6.8 Hz), 7.38 (1H, d,
J = 15.6 Hz), 7.11 (1H, s), 6.52 (1H, s), 3.96 (3H, s), 3.91 (6H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 188.9, 155.4, 153.4, 143.7, 141.7, 138.9, 137.0, 133.4, 130.9, 130.8, 127.9,
119.5, 115.5, 112.1, 97.2, 57.0, 56.7 and 56.5. HRMS-ESI: m/z [M+H]+ for C18H17O4Cl2,
calculated 367.0498; observed 367.0490.
1-(4-Nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (25, Table 4)OCH 3
H3COOCH 3
O
NO 2
Brick red solid (82%), m.p. 187-189°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.35-8.31 (2H, m), 8.13-8.09 (3H, m), 7.44 (1H, d, J = 15.7 Hz), 7.12 (1H,
s), 6.54 (1H, s), 3.98 (3H, s), 3.96 (3H, s), 3.92 (3H, s); 13C NMR (75.4 MHz, CDCl3);
Structure-activity relationship…….. Chapter 3
142
(ppm) 189.4, 154.9, 153.0, 149.5, 143.6, 143.1, 141.9, 129.1, 123.4, 119.2, 114.6, 111.3,
96.4, 56.3, 56.0 and 55.8. HRMS-ESI: m/z [M+H]+ for C18H17O6N, calculated 344.1129;
observed 344.1149.
1-(4-Iodophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (26, Table 4)OCH3
H3COOCH3
O
I
Yellow solid (92%), m.p. 183-185°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.12 (1H, d, J = 15.7 Hz), 7.87 (2H, d, J = 8.7 Hz), 7.74 (2H, d, J =8.7 Hz), 7.42
(1H, d, J = 15.7 Hz), 7.12 (1H, s), 6.53 (1H, s), 3.96 (3H, s), 3.91 (6H, s); 13C NMR (75.4
MHz, CDCl3); (ppm) 190.7, 155.2, 153.2, 143.7, 141.1, 138.5, 138.1, 130.3, 120.1, 115.7,
112.0, 100.2, 97.2, 57.0, 56.7 and 56.4. HRMS-ESI: m/z [M+H]+ for C18H17O4I, calculated
425.0244; observed 425.0239.
1-(4-Bromophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (27, Table 4)OCH3
H3CO
OCH3
O
Br
Fluorescent yellow solid (94%), m.p. 157-158°C, 1H NMR (300
MHz, CDCl3); (ppm) 8.01 (1H, d, J = 15.7 Hz), 7.77 (2H, d, J = 9.15 Hz), 7.52 (2H, d, J
=9.7 Hz), 7.32 (1H, d, J = 15.7 Hz), 7.00 (1H, s), 6.41 (1H, s), 3.84 (3H, s), 3.80 (6H, s);13C NMR (75.4 MHz, CDCl3); (ppm) 190.2, 155.2, 153.2, 143.7, 141.0, 137.9, 132.1,
130.3, 127.6, 120.0, 115.7, 112.0, 97.2, 57.0, 56.7 and 56.4. HRMS-ESI: m/z [M+H]+ for
C18H17O4Br, calculated 377.0383; observed 377.0379.
1-(4-Cynophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (28, Table 4)OCH 3
H3COOCH 3
O
CN
Bright yellow solid (84%), m.p. 197-199°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.13-8.04 (3H, m), 7.79 (2H, d, J = 8.4 Hz), 7.42 (1H, d, J = 15.1 Hz ),
7.11(1H, s), 6.53 (1H, s), 3.92 (3H, s), 3.90 (3H, s), 3.87 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 190.2, 155.7, 153.8, 143.9, 142.8, 142.5, 132.9, 129.4, 119.9, 118.7, 115.9,
115.5, 112.2, 97.3, 57.1, 56.8 and 56.6. HRMS-ESI: m/z [M+H]+ for C19H17O4N, calculated
324.1230; observed 324.1261.
Structure-activity relationship…….. Chapter 3
143
1-[(4-(4-Bromophenyl)phenyl]-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (29, Table
4)OOCH 3
H3COOCH 3 Br Bright yellow solid (96%), m.p. 137-139°C, 1H NMR (300
MHz, CDCl3); (ppm) 8.16-8.07 (3H, m), 7.67-7.48 (7H, m), 7.15 (1H, s), 6.53 (1H, s),
3.95 (3H, s), 3.91 (6H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 190.6, 155.1, 153.1,
144.0, 143.7, 140.6, 139.3, 138.2, 132.4, 129.5, 129.1, 127.3, 122.8, 120.4, 115.9, 112.0,
97.3, 57.0, 56.7 and 56.4. HRMS-ESI: m/z [M+H]+ for C24H21O4Br, calculated 453.0696;
observed 453.0766.
3.4.2.12 Procedure for synthesis of conjugated chalcone 30 from -asarone (Table 5 &
Scheme 14):
To the solution of -asarone (0.4 g, 1.92 mmol) in dry dioxane (20 ml), acetic acid (1 drop)
and DDQ (0.87 g, 3.84 mmol) was added. The reaction mixture was sonicated for 30
minutes. Filter the reaction mixture to remove solid DDQH2. The filtrate was evaporated,
took in ethyl acetate (50 ml), washed with water (3x10 ml), 10% NaOH (2x2 ml), brine
(3x10 ml) and dried over Na2SO4. The filtrate was evaporated to afford a crude yellow
liquid, which was purified on neutral alumina with hexane-ethyl acetate (2 : 3) mixture to
provide the corresponding 2,4,5-trimethoxy cinnamaldehyde [Joshi et al. (2006)] in 85%
yield (0.363 g). Subsequently, above cinnamaldeyde (0.363 g, 1.63 mmol,) was subjected to
Claisen-Schmidt condensation with 4-bromoacetophenone (0.324 g, 1.63 mmol) using
[MIMBSA]HSO4 under MW irradiation (as mentioned in section 3.4.2.5) providing 30 as
orange yellow solid in 70% yield (0.46 g).
1-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)-pent-2,4-dien-1-one (30, Table 5)OCH3
H3COOCH3
O
Br
m.p. 146-149°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.79
(2H, d, J = 8.4 Hz), 7.63-7.54 (3H, m), 7.32 (1H, d, J = 15.3 Hz), 6.99 (1H, s), 6.94-6.85
(2H, m), 6.45 (1H, s), 3.88 (3H, s), 3.85 (3H, s), 3.83 (3H, s); 13C NMR (75.4 MHz, CDCl3);
(ppm) 189.9, 153.7, 152.1, 147.4, 144.0, 138.0, 132.3, 130.4, 127.9, 125.5, 123.7, 117.5,
Structure-activity relationship…….. Chapter 3
144
110.8, 97.8, 57.1, 57.0 and 56.6. HRMS-ESI: m/z [M+H]+ for C20H19O4Br, calculated
403.0539; observed 403.0528.
3.4.2.13 General procedure for synthesis of curcumine like chalcone 31 (Table 5 &
Scheme 15):
2,4,5-trimethoxybenzaldehyde (0.3 g, 1.53 mmol) (obtained from the oxidation of -
asarone, see section 3.4.2.5) was allowed to react with excess of acetone (5 ml) in
[MIMBSA]HSO4 (1.5 g) under focused MW irradiation (100W, 80°C) for 5 min. to obtain
the corresponding 4-(2,4,5-trimethoxyphenyl)but-3-en-2-one as a deep yellow solid in 67%
yield (0.242 g, 1.03 mmol). Subsequently, the above enone (0.242 g, 1.03 mmol) was
further subjected to Claisen-Schmidt condensation with 4-bromobenzaldehyde (0.189 g,
1.03 mmol) using the same procedure as mentioned in section 3.4.2.1 to afford 31 as an
orange yellow solid in 82% yield (0.339 g).
1-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (31, Table 5)O
H3CO Br
OCH3
OCH3 m.p. 152-154°C, 1H NMR (300 MHz, CDCl3); (ppm) 8.10
(1H, d, J = 16.0 Hz), 7.68 (1H, d, J = 15.8 Hz), 7.53-7.48 (4H, m), 7.17-7.10 (2H, m),
6.98 (1H, d, J = 16.0 Hz), 6.52 (1H, s), 3.96 (3H, s), 3.89 (6H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 189.3, 154.8, 153.1, 143.7, 141.3, 139.0, 134.4, 132.5, 130.0, 126.1, 124.7,
124.4, 115.5, 111.1, 97.1, 56.8, 56.7 and 56.4. HRMS-ESI: m/z [M+H]+ for C20H19O4Br,
calculated 403.0539; observed 403.0536.
3.4.2.14 MW-assisted synthesis of pyrazoline derivatives 32-33 (Scheme 17)
A mixture of chalcone 27 (0.5 g, 1.32 mmol), hydrazine hydrate (0.132 g, 2.64 mmol),
NaOAc (0.016 g, 0.20 mmol), AcOH (8 ml) and water (4 ml) was refluxed for 30 min under
MW irradiation (150W, 100°C). The reaction mixture was concentrated by distilling out the
solvent under reduced pressure. The obtained crude mixture was washed with water (10 ml),
hexane (10 ml) and recrystallized with methanol to obtain the pure pyrazoline derivative 32
as white solid in 73% yield (0.378 g).
Using phenylhydrazine hydrochloride in place of hydrazine hydrate in above procedure,
compound 33 (Scheme 17) was obtained as a while solid in 82% yield. The NMR spectra of
compounds 32 and 33 are given below:
Structure-activity relationship…….. Chapter 3
145
3-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (32)
H3CO
OCH 3
Br
HN NCH3O
White solid, m.p. 160-163°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.63-7.53 (4H, m), 6.62 (1H, s), 6.53 (1H, s), 5.77-5.72 (1H, m), 3.87 (3H, s), 3.79
(6H, s), 3.71-3.61 (1H, m), 3.09-3.01(1H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 169.2,
154.0, 151.0, 149.7, 143.6, 132.2, 131.1, 128.3, 124.7, 121.4, 98.7, 57.2, 56.8, 56.5, 41.7
and 30.1. HRMS-ESI: m/z [M+H]+ for C18H19BrN2O3, calculated 391.0652; observed
391.0631.
3-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (33)
OCH3
H3CO
OCH3
Br
N N
White solid, m.p. 153-155°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.62-7.49 (5H, m), 7.20 (2H, d, J = 7.8 Hz), 7.09 (2H, d, J = 7.8 Hz), 6.69 (1H, s),
6.60 (1H, s), 5.57-5.53 (1H, m), 3.93 (3H, s), 3.91 (3H, s), 3.64 (3H, s), 3.41-3.37 (1H, m),
3.04-3.01 (1H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 150.4, 149.3, 146.8, 145.2,
143.8, 132.0, 129.0, 127.5, 124.4, 122.7, 121.8, 119.6, 113.7, 110.8, 97.9, 59.1, 56.9, 56.8,
53.8 and 42.5. HRMS-ESI: m/z [M+H]+ for C24H23BrN2O3, calculated 467.0965; observed
467.0958.
3.4.2.15 MW-assisted synthesis of compound 34 with oxazoline ring (Scheme 17)
The reaction was performed and worked up in the same manner as given in section 3.4.2.14
replacing hydrazine hydrate with hydroxylamine hydrochloride. Compound 34 was
obtained as a white solid in 76% yield.
3-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1,2-oxazole (34)OCH 3
H3CO
OCH 3
Br
O N
White solid, m.p. 140-144 ºC, 1H NMR (300 MHz, CDCl3);
(ppm) 7.58-7.52 (4H, m), 7.00 (1H, s), 6.55 (1H, s), 5.97-5.91 (1H, m), 3.91 (3H, s), 3.85
(6H, s), 3.79-3.69 (1H, m), 3.23-3.14 (1H, m); 13C NMR (75.4 MHz, CDCl3); (ppm)
Structure-activity relationship…….. Chapter 3
146
156.2, 150.7, 149.7, 143.5, 132.3, 129.1, 128.5, 124.6, 120.9, 110.6, 97.9, 78.8, 57.0, 56.6
and 42.6. HRMS-ESI: m/z [M+H]+ for C18H18BrNO4, calculated 392.0492; observed
392.0486.
3.4.2.16 Synthesis of compound 35 (Scheme 17):
To the mixture of chalcone 27 (0.5 g, 1.32 mmol) and thiourea (0.1 g, 1.32 mmol) in ethanol
(5 ml) was added a solution of sodium ethoxide (0.036 g, 0.53 mmol) in absolute ethanol
(10 ml) [Kidwai and Misra (1999)]. The reaction mixture was irradiated under MW (80W,
50°C) for 5 min. Reaction mixture was concentrated under reduced pressure and the
obtained crude solid was washed with water (10 ml), ether (10 ml) and recrystallised from
methanol providing 35 as a white solid in 67% yield (0.387 g).
4-(4-Bromophenyl)-6-(2,4,5-trimethoxyphenyl)-1,2,5,6-tetrahydropyrimidine-2-thione
(35)
NHHN
S
Br
OCH3
H3CO
OCH3 m.p. 223-224°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.80
(2H, d, J = 8.7 Hz), 7.58 (2H, d, J = 8.7 Hz), 7.06 (1H, s), 6.73 (1H, s), 5.78 (1H, d, J = 3.9
Hz), 5.38 (1H, d, J = 3.9 Hz), 4.03 (3H, s), 3.98 (3H, s), 3.94 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 175.7, 151.1, 150.4, 132.8, 132.6, 127.1, 121.1, 112.2, 112.1, 100.0, 97.5,
57.3, 56.6, 56.5, 51.1 and 51.0. HRMS-ESI: m/z [M+H]+ for C19H19BrN2O3S, calculated
435.0373; observed 435.0369.
3.4.2.17 Procedure for synthesis of compound 36 under MW irradiation (Scheme 17):
A slurry of 27 (0.5 g, 1.32 mmol), ethyl acetoacetate (0.34 g, 2.64 mmol), anhydrous K2CO3
(0.73 g, 5.28 mmol) and silica (3 gm) was irradiated under MW (250W, 140°C) for 45 min
[Shakil et al. (2010)]. After completion of reaction as indicated by TLC, the reaction
mixture was extracted with ethyl acetate (3x10 ml) and evaporated under vacuum. The
obtained crude viscous mixture was washed with water (10 ml), hexane (10 ml) and
recrystallized with methanol to obtain the pure chalcone derivative 36 as a white solid in
75% yield (0.486 g).
Structure-activity relationship…….. Chapter 3
147
Ethyl-4-(4-bromophenyl)-2-oxo-6-(2,4,5-trimethoxyphenyl)cyclohex-3-ene-1-
carboxylate (36)H3CO OCH 3
OCH 3
Br
O
O
O White solid, m.p. 150-155°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.56 (2H, d , J = 8.5 Hz), 7.43 (2H, d, J = 8.5 Hz), 6.75 (1H, s), 6.56 (1H, s), 6.51
(1H, d, J = 1.9 Hz), 4.14-4.03 (3H, m), 3.98-3.93 (1H, m), 3.90 (3H, s), 3.85 (3H, s), 3.82
(3H, s), 3.19-3.09 (1H, m), 3.00-2.93 (1H, m), 1.11 ( 3H, t, J = 7.1 Hz); 13C NMR (75.4
MHz, CDCl3); (ppm) 195.2, 169.9, 158.5, 152.3, 149.3, 143.3, 137.2, 132.4, 128.1, 125.1,
124.5, 120.3, 113.4, 98.5, 61.1, 58.1, 57.0, 56.7, 56.5, 40.5, 34.2, 14.4. HRMS-ESI: m/z
[M+H]+ for C24H25BrO6, calculated 489.0907; observed 489.0902.
3.4.2.18 Procedure for synthesis of indole-chalcone hybrids 37-39 (Scheme 17):
To a solution of chalcone 27 (0.5 g, 1.32 mmol) in acetonitrile (15 ml), catalytic amount of
[MIMBSA]HSO4 (0.1 g) and indole (0.183 g, 1.58 mmol) were added. The reaction mixture
was stirred at room temperature for overnight. After evaporation of acetonitrile, the
obtained crude mixture was washed with water (10 ml) and recrystallized with methanol to
afford the corresponding Michael adduct in 82% yield (0.537 g). Subsequently, the solution
of above adducts (0.537 g, 1.08 mmol) in DCM (10 ml) was treated with benzyl bromide
(0.24 g, 1.4 mmol) and 60% NaH (0.05 g, 2.16 mmol) for overnight. After completion of
reaction, the mixture was acidified with dilute HCl, extracted with ethyl acetate (3x15 ml),
washed with brine (2x5 ml), dried over anhydrous sodium sulphate and concentrated under
reduced pressure. The obtained precipitates were washed with hexane (5 ml), ether (10 ml)
and recrystallized with methanol to obtain the pure indole-chalcone hybrid 37 as a white
solid in 76% yield (0.48 g).
The above procedure was also applied for the synthesis of various other indole-chalcone
hybrids 38-39 (Scheme 17) using substituted benzyl bromides. The NMR spectra of
compounds 37-39 are given below:
Structure-activity relationship…….. Chapter 3
148
3-(1-Benzyl-1H-indol-3-yl)-1-(4-bromophenyl)-3-(2,4,5-trimethoxyphenyl)propan-1-
one (37)
O
N
OCH3
H3CO
OCH3
Br
White solid (74%), m.p. 160-162°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.85 (2H, d, J = 8.2 Hz) 7.58 (2H, d, J = 8.2 Hz), 7.44 (1H, d, J = 7.8), 7.28-7.25
(4H, m), 7.15-6.98 (5H, m), 6.66 (1H, s), 6.54 (1 H, s), 5.36-5.29 (3H, m), 3.88 (3H, s), 3.83
(3H, s), 3.64-3.61 (5H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 198.7, 151.2, 148.4,
143.3, 138.2, 137.2, 136.2, 132.1, 130.2, 129.0, 128.2, 127.9, 126.9, 126.3, 124.2, 122.3,
120.1, 119.4, 117.8, 113.3, 109.9, 98.0, 56.8, 56.7, 56.5, 50.2, 45.1, 32.7 and 31.3. HRMS-
ESI: m/z [M+H]+ for C33H30BrNO4, calculated 584.1431; observed 584.1427.
3-[1-(2H-1,3-Benzodioxol-5-ylmethyl)-1H-indol-3-yl]-1-(4-bromophenyl)-3-(2,4,5-
trimethoxyphenyl)propan-1-one (38)
O
N
OCH 3
H3CO
OCH 3
Br
O
O
White solid (69%), m.p. 144-146°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.86 (2H, d, J = 8.0 Hz), 7.59 (2H, d, J = 8.0 Hz), 7.41 (1H, d, J = 7.8
Hz), 7.24 (2H, d, J = 8.1 Hz), 7.15-7.10 (1H, m), 7.04-6.97 (2H, m), 6.74 (1H, d, J = 7.8
Hz), 6.64-6.59 (2H, m), 6.53 (2H, d, J = 7.8 Hz), 5.93 (2H, s), 5.34 (3H, t, J = 7.2 Hz), 5.20
(2H, s), 3.87 (3H, s), 3.84 (3H, s), 3.61-3.58 (5H, m); 13C NMR (75.4 MHz, CDCl3);
(ppm) 198.7, 162.7, 148.5, 143.3, 137.1, 136.2, 132.1, 130.2, 126.2, 124.1, 122.3, 120.3,
120.1, 119.4, 117.7, 113.3, 109.9, 108.6, 107.5, 101.5 , 98.0, 56.9, 56.7, 56.5, 50.1, 45.1
and 32.8. HRMS-ESI: m/z [M+H]+ for C34H30BrNO6, calculated 628.1329; observed
628.1332.
Structure-activity relationship…….. Chapter 3
149
1-(4-Bromophenyl)-3-{1-[(4-iodophenyl)methyl]-1H-indol-3-yl}-3-(2,4,5-trimethoxy-
phenyl)propan-1-one (39)
O
N
OCH3
H3CO
OCH3
Br
I
White solid (83%), m.p. 176-179°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.85 (2H, d, J = 8.3 Hz), 7.60-7.57 (4H, m), 7.44 (1H, d, J = 7.8 Hz),
7.19-7.10 (2H, m), 7.03-6.98 (2H, m), 6.80 (2H, d, J = 7.8 Hz), 6.65 (1H, s), 6.54 (1H, s),
5.35-5.30 (1H, m), 5.23 (2H, s), 3.88 (3H, s), 3.84 (3H, s), 3.62-3.59 (5H, m); 13C NMR
(75.4 MHz, CDCl3); (ppm) 198.6, 151.2, 148.5, 143.3, 138.1, 137.9, 137.1, 136.1, 132.1,
130.2, 128.8, 128.3, 127.9, 126.2, 124.1, 122.4, 120.2, 119.6, 118.1, 113.5, 109.8, 98.0,
93.2, 57.0, 56.7, 56.5, 49.7, 45.0 and 32.7. HRMS-ESI: m/z [M+H]+ for C33H29BrINO4,
calculated 710.0397; observed 710.0397.
3.4.2.19 Mannich type reaction for the synthesis of indole-chalcone hybrid 40 (Scheme
17):
To a solution of 27 (0.5 g, 1.32 mmol) in dioxane-AcOH (4:1, 8 ml) was added a mixture of
piperidine (0.135 g, 1.58 mmol) and 37% aq. HCHO (0.129 g, 1.58 mmol) in dioxane-
AcOH (4:1, 4 ml) [Lindquist et al. (2006)]. Reaction mixture was stirred at room
temperature for overnight. Evaporation of the solvent provided a crude solid which was
washed with excess of water (20 ml), hexane (10 ml) and recrystallised with methanol to
obtain pure compound 40 as a white solid in 88% yield (0.69 g).
1-(4-Bromophenyl)-3-[1-(piperidin-1-ylmethyl)-1H-indol-3-yl]-3-(2,4,5-trimethoxy-
phenyl)propan-1-one (40)
O
NN
OCH 3
H3CO
OCH 3
Br
m.p. 171-172°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.87
(2H, d, J = 6.8 Hz), 7.59 (2H, d, J = 8.5 Hz), 7.41-7.36 (2H, m), 7.18-7.13 (1H, m), 7.08
(1H, s), 7.01-6.96 (1H, m), 6.64 (1H, s), 6.54 (1H, s), 5.34-5.29 (1H, m), 4.81 (2H, s), 3.88 (
3H, s), 3.85 (3H, s), 3.64-3.62 (5H, m), 2.50-2.47 (4H, m), 1.60-1.53 (4H, m), 1.37-1.35
(2H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 198.7, 151.2, 148.5, 143.3, 138.0, 136.2,
Structure-activity relationship…….. Chapter 3
150
132.1, 130.2, 128.2, 127.7, 126.9, 124.2, 122.1, 119.9, 119.4, 117.1, 113.4, 110.3, 98.0,
68.7, 56.8, 56.7, 56.5, 52.1, 45.1, 32.7, 26.2 and 24.3. HRMS-ESI: m/z [M+H]+ for
C32H35BrN2O4, calculated 591.1853; observed 591.1874.
3.4.2.20 Procedure for synthesis of indole-chalcone hybrids 41-43 (Scheme 17):
The procedure mentioned in section 3.4.2.18 was applied for the synthesis of indole-
chalcone hybrids 41-43 using substituted indole and benzyl bromides. The NMR spectra of
compounds 41-43 are given below.
1-(4-Bromophenyl)-3-{1-[(4-bromophenyl)methyl]-1H-indol-3-yl}-3-(2,4,5-trimethoxy-
phenyl)propan-1-one (41)
O
N
OCH 3
H3CO
OCH 3
Br
Br
White solid (81%), m.p. 169-170 °C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.75-7.72 (2H, m,), 7.50-7.46 (2H, m), 7.31-7.28 (3H, m),7.06-7.00 (2H,
m), 6.93 (2H, d, J = 3.9 Hz), 6.83 (2H, s), 6.57 (1H, d, J = 4.6 Hz), 6.45( 1H, d, J = 4.5
Hz), 5.24-5.20 (1H, m),5.14 (2H, s) 3.79 (6H, dd, J = 4.7 Hz), 3.54 (5H, d, J = 4.6 Hz); 13C
NMR (75.4 MHz, CDCl3); (ppm) 197.2, 149.9, 147.2, 141.9, 135.8, 135.7, 134.7, 130.8,
128.7, 127.2, 126.9, 126.6, 124.8, 122.7, 121.0, 120.3, 118.8, 118.2, 117.1, 112.1, 108.4,
96.7, 55.6, 55.3, 55.1, 48.3, 43.6 and 31.4. HRMS-ESI: m/z [M+H]+ for C33H29Br2NO4,
calculated 662.0536; observed 662.0529.
3-{6-Bromo-1-[(4-bromophenyl)methyl]-1H-indol-3-yl}-1-(4-bromophenyl)-3-(2,4,5-
trimethoxyphenyl)propan-1-one (42)
O
N
OCH3
H3CO
OCH3
Br
Br
Br
White solid (79%), m.p. 160-162°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.83 (2H, d, J = 6.6 Hz), 7.59-7.56 (3H, m), 7.40 (2H, d, J = 6.6 Hz),
7.20-7.17 (1H, m), 7.03 (2H, s), 6.89 (2H, d, J = 6.2 Hz), 6.61 (1H, s), 6.55 (1H, s), 5.25-
5.19 (3H, m), 3.88 (3H, s), 3.86 (3H, s), 3.63-3.56 (5H, m); 13C NMR (75.4 MHz, CDCl3);
(ppm) 198.3 151.3, 148.9, 143.5, 136.7, 136.1, 135.8, 132.3, 132.2, 130.1, 129.7, 128.5,
Structure-activity relationship…….. Chapter 3
151
128.4, 127.3, 125.3, 123.7, 122.8, 122.0, 117.9, 113.7, 113.1, 111.3, 98.3, 57.2, 56.6, 49.9,
44.8 and 32.5. HRMS-ESI: m/z [M+H]+ for C33H28Br3NO4, calculated 739.9641; observed
739.9637.
1-(4-Bromophenyl)-3-{1-[(2-bromophenyl)methyl]-1H-indol-3-yl}-3-(2,4,5-trimethoxy-
phenyl)propan-1-one (43)
O
N
OCH 3
H3CO
OCH 3
Br
Br
White solid (74%), m.p. 150-152°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.86 (2H, d, J = 6.8 Hz), 7.62-7.56 (3H, m), 7.46 (1H, d, J = 7.8), 7.20-7.0
(6H, m), 6.69 (1H, s), 6.55 (1H, s), 6.49 (1H, d, J = 7.8 Hz), 5.38-5.33 (3H, m), 3.88 (3H,
s), 3.85 (3H, s), 3.65-3.62 (5H, m); 13C NMR (75.4 MHz, CDCl3); (ppm) 198.6, 151.2,
148.5, 143.4, 137.3, 137.2, 136.2, 133.1, 132.1, 130.2, 129.4, 128.2, 128.0, 127.9, 126.3,
124.1, 122.6, 122.5, 120.2, 119.6, 118.2, 113.4, 109.9, 98.1, 57.0, 56.7, 56.5, 50.5, 45.1 and
32.7. HRMS-ESI: m/z [M+H]+ for C33H29Br2NO4, calculated 662.0536; observed 662.0527.
3.4.2.21 MW-assisted synthesis of compound 44 from natural -asarone rich Acorus
calamus oil (Table 8 & Scheme 18):
Initially, 2,4,5-trimethoxybenzaldehyde was obtained from -asarone of Acorus calamus oil
using the same procedure as mentioned in section 3.4.2.5. Subsequently, the above
benzaldehyde (0.24 g, 1.22 mmol) was allowed to react with excess of acetone (3 ml) in the
presence of MIMBSA]HSO4 (1g) under MW irradiation (100W, 75°C) for 5 min. The
reaction mixture was vacuum evaporated and taken in ethyl acetate (3x15 ml). The
combined organic extract was washed with brine (2x5 ml), dried over anhydrous sodium
sulphate and concentrated under reduced pressure. The obtained crude mixture was purified
through column chromatography (silica gel 60-120 mesh size) and recrystallization with
methanol to obtain the desired pure product 44 as a deep yellow solid in 65% yield (0.187
g).
Structure-activity relationship…….. Chapter 3
152
4-(2,4,5-Trimethoxyphenyl)but-3-en-2-one (44)O
H3CO
OCH3
OCH3 m.p. 98-101°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.94 (1H, d, J
= 16.4 Hz), 7.10 (1H, s), 6.69 (1H, d, J = 16.4 Hz), 6.56 (1H, s), 3.98 (3H, s), 3.94 (6H, s),
2.42 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 199.3, 154.4, 153.0, 143.9, 138.7,
125.7, 115.3, 110.8, 97.3, 56.9, 56.8, 56.5 and 27.4. HRMS-ESI: m/z [M+H]+ for C13H16O4,
calculated 237.1121; observed 237.1109.
3.4.2.22 Synthesis of 45 from anethole (Table 8 & Scheme 18):
Compound 45 was prepared from anethole employing a procedure similar to that described
for compound 44.
4-(4-Methoxyphenyl)but-3-en-2-one (45)O
H3CO Yellow solid (61%), m.p. 67-71°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.48-7.42 (3H, m), 6.90 (2H, d, J = 8.3 Hz), 6.61 (1H, d, J = 16.4 Hz), 3.81 (3H, s),
2.33 (3H, s); 13C-NMR (75.4 MHz, CDCl3); (ppm) 198.1, 161.5, 143.1, 129.8, 127.0,
124.9, 114.3, 55.2 and 27.2.
3.4.2.23 MW-assisted synthesis of compound 46 (Table 8):
Compound 46 was prepared by Claisen-Schmidt condensation of 4-hydroxy-3-
methoxybenzaldehyde (0.456 g, 3 mmol) with excess of acetone (3 ml) using 10% aqueous
NaOH (6 mmol) under MW irradiation (110W, 55°C) for 20 min. The reaction mixture was
concentrated and the obtained residue was subsequently purified by column
chromatography on silicagel (60-120 mesh size) using hexane-ethylacetate (4 : 1) to give a
solid which was further recrystallised in methanol to provide pure 46 as a yellow solid in
56% yield (0.32 g).
4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one (46, Table 8) [Sharma et al. (2006)]O
HO
OCH 3 m.p.128-129°C, 1H NMR (300 MHz, CDCl3); (ppm) 7.48 (1H, d, J =
16.2 Hz), 7.10-7.05 (2H, m), 6.94 (1H, d, J = 8.2 Hz), 6.61 (1H, d, J = 16.2 Hz), 3.92 (3H,
Structure-activity relationship…….. Chapter 3
153
s), 2.37 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 198.9, 148.7, 147.3, 144.2, 127.2,
125.3, 123.9, 115.2, 109.7, 56.3 and 27.6.
3.4.2.24 MW-assisted synthesis of chalcones 47-58 using NaOH as a condensing agent
(Table 8-9):
A mixture of 1-naphthaldehyde (0.47 g, 3 mmol) and 4-methoxy acetophenone (0.45 g, 3
mmol) in MeOH (5 ml) was treated with 10% aqueous NaOH (6 mmol) and the reaction
mixture was irradiated under MW irradiation (110W, 55°C) with continuous stirring for 20
minutes. The obtained precipitates were washed with dilute HCl (10 ml), excess of water
(20 ml), dried in air and finally recrystallized from methanol to obtain 47 as a yellow solid
in 90% yield (0.78 g).
The above MW assisted procedure was also applied for the synthesis of chalcones 48-50
(Table 8) and 51-58 (Table 9). The spectral data of compounds 47-58 are discussed below:
1-(4-Methoxyphenyl)-3-(naphthalene-1-yl)prop-2-en-1-one (47, Table 8) [Geyer et al.
(2009)]O
OCH3 m.p. 127-133°C, 1H NMR (300 MHz, CDCl3); (ppm) 8.62 (1H,
d, J = 15.3 Hz), 8.21 (1H, d, J = 8.4 Hz), 8.04 (2H, d, J = 7.6 Hz), 7.85-7.80 (3H, m), 7.59
(1H, d, J = 15.3 Hz), 7.51-7.42 (3H, m), 6.94 (2H, d, J = 8.4 Hz), 3.81 (3H, s); 13C NMR
(75.4 MHz, CDCl3); (ppm) 188.5, 163.5, 140.9, 133.7, 132.6, 131.8, 131.0, 130.9, 130.6,
128.7, 126.9, 126.3, 125.4, 125.0, 124.6, 123.6, 113.7 and 55.5.
1,3-Bis(4-methoxyphenyl)prop-2-en-1-one (48, Table 8) [Dong et al. (2008)]O
H3CO OCH3 Light yellow solid (93%), m.p. 100-102°C, 1H NMR (300
MHz, CDCl3); (ppm) 8.04 (2H, d, J = 8.7 Hz), 7.80 (1H, d, J = 16.1 Hz), 7.59 (2H, d, J =
8.2 Hz), 7.45 (1H, d, J = 16.1 Hz), 6.97-6.89 (4H, m), 3.84 (3H, s), 3.81 (3H, s); 13C NMR
(75.4 MHz, CDCl3); (ppm) 188.9, 163.5, 161.8, 144.0, 131.6, 130.9, 130.3, 128.1, 119.8,
114.6, 114.0 and 55.7.
Structure-activity relationship…….. Chapter 3
154
3-(4-Fluorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (49, Table 8) [Liu et al.
(2001)]O
F OCH3 White solid, (93%), m.p. 115-118°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.07 (2H, d, J = 8.8 Hz), 7.81 (1H, d, J = 15.6 Hz), 7.67-7.63 (2H, m),
7.52 (1H, d, J = 15.6 Hz), 7.15-7.09 (2H, m), 7.01 (2H, d, J = 8.1 Hz), 3.91 (3H, s); 13C
NMR (75.4 MHz, CDCl3); (ppm) 188.6, 165.7, 163.6, 162.4, 142.7, 131.4, 130.9, 130.4,
121.7, 116.3, 114.0 and 55.6. HRMS-ESI: m/z [M+H]+ for C16H13O2F, calculated 257.0972;
observed 257.0964.
3-(4-Bromophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (50, Table 8)O
Br OCH3 Off white solid (91%), m.p. 149-152°C, 1H NMR (300 MHz,
CDCl3); (ppm) 8.34-8.28 (2H, m), 8.05-7.96 (1H, m), 7.80-7.52 (5H, m), 7.29-7.23 (2H,
m), 4.19 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 189.2, 164.4, 143.3, 134.8, 133.0,
131.7, 130.5, 125.3, 123.2, 114.7 and 56.3. HRMS-ESI: m/z [M+H]+ for C16H13BrO2,
calculated 317.0172; observed 317.0172.
3-(2,4-Dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (51, Table 9)O
Cl OCH3Cl White solid (88%), m.p. 134-137°C, 1H NMR (300 MHz, CDCl3);
(ppm) 8.09-8.00 (3H, m), 7.67 (1H, d, J = 8.4 Hz), 7.49 (1H, d, J = 15.8 Hz), 7.43 (1H,
s), 7.28 (1H, d, J = 8.4 Hz), 6.98 (2H, d, J = 8.2 Hz), 3.88 (3H, s); 13C NMR (75.4 MHz,
CDCl3); (ppm) 188.4, 164.0, 138.6, 136.5, 136.2, 132.4, 131.3, 131.0, 130.4, 128.8, 127.8,
125.2, 114.3 and 55.8.
3-(3,4-Dichlorophenyl)-1-(4-methylphenyl)prop-2-en-1-one (52, Table 9)O
Cl CH3
Cl
Light yellow solid, (86%), m.p. 137-138°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.95 (2H, d, J = 6.9 Hz), 7.71-7.65 (2H, m), 7.53-7.42 (3H, m), 7.32 (2H,
d, J = 7.1 Hz), 2.41 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 189.5, 144.4, 141.7,
135.6, 135.5, 134.6, 133.6, 131.2, 130.1, 129.8, 129.0, 127.8, 123.9 and 22.0. HRMS-ESI:
m/z [M+H]+ for C16H12Cl2O, calculated 291.0338; observed 291.0335.
Structure-activity relationship…….. Chapter 3
155
3-(3,4-Dichlorophenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (53, Table 9)O
Cl
Cl
H3CO Yellow solid, (81%), m.p. 77-78°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.72-7.69 (2H, m), 7.62-7.33 (5H, m), 7.15-7.05 (2H, m), 3.99 (3H, s); 13C NMR
(75.4 MHz, CDCl3); (ppm) 192.4, 158.7, 140.2, 135.7, 134.3, 133.7, 133.5, 131.2, 130.9,
130.1, 129.2, 128.9, 127.7, 121.2, 112.1 and 56.2. HRMS-ESI: m/z [M+H]+ for
C16H12Cl2O2, calculated 307.0287; observed 307.0284.
3-(3,4-Dichlorophenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (54, Table 9)O
Cl
OCH3Cl
Light yellow solid (79%), m.p. 105-107°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.80-7.74 (2H, m), 7.69 (1H, d, J = 7.3 Hz), 7.62-7.59 (2H, m), 7.56-7.47
(3H, m), 7.24 (1H, d, J = 8.6 Hz), 3.97 (3H, s); 13C NMR (75.4 MHz, CDCl3); (ppm)
189.6, 160.1, 141.9, 139.2, 135.0, 134.5, 133.4, 131.0, 129.9, 129.8, 127.6, 123.6, 121.2,
119.7, 113.0 and 55.6. HRMS-ESI: m/z [M+H]+ for C16H12Cl2O2, calculated 307.0287;
observed 307.0272.
3-(3,4-Dichlorophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (55, Table 9)O
Cl
OCH3Cl
OCH3 White solid, (87%), m.p. 122-123°C, 1H NMR (300 MHz, CDCl3);
(ppm) 7.71-7.65 (3 H, m), 7.61 (1H, s), 7.55-7.45 (3H, m), 6.94 (1H, d, J = 8.6 Hz), 3.97
(6H, s); 13C NMR (75.4 MHz, CDCl3); (ppm) 188.3, 154.0, 149.8, 141.5, 135.6, 134.6,
133.7, 131.4, 130.1, 128.0, 123.6, 123.7, 111.2, 110.5, 56.6 and 56.5. HRMS-ESI: m/z
[M+H]+ for C17H14Cl2O3, calculated 337.0393; observed 337.0398.
3-(3,4-Dichlorophenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (56, Table 9)O
Cl
OCH3Cl
OCH3OCH 3 Pale yellow solid (83%), m.p. 139-140°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.72-7.67 (2H, m), 7.51-7.42 (3H, m), 7.20 (2H, s), 3.95 (9H, s); 13C NMR
(75.4 MHz, CDCl3); (ppm) 188.8, 153.6, 143.1, 142.2, 135.3, 134.7, 133.6, 133.4, 131.3,
130.1, 127.9, 123.5, 106.5, 61.4 and 56.8. HRMS-ESI: m/z [M+H]+ for C18H16Cl2O4,
calculated 367.0498; observed 367.0493.
Structure-activity relationship…….. Chapter 3
156
1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)prop-2-en-1-one (57, Table 9)O
Cl Cl
Cl
Off white solid (92%), m.p. 135-137°C, 1H NMR (300 MHz,
CDCl3); (ppm) 7.96-7.92 (2H, m), 7.70-7.63 (2H, m), 7.48-7.40 (5H, m); 13C NMR (75.4
MHz, CDCl3); (ppm) 188.9, 142.8, 140.1, 136.6, 135.3, 135.1, 133.9, 131.5, 130.5, 130.3,
129.6, 128.1 and 123.4. HRMS-ESI: m/z [M+H]+ for C15H9Cl3O, calculated 310.9791;
observed 310.9786.
1,3-Bis(3,4-dichlorophenyl)prop-2-en-1-one (58, Table 9)O
Cl
ClCl
Cl Pale yellow solid (88%), m.p. 164-167°C, 1H-NMR (300 MHz,
CDCl3); (ppm) 8.10 (1H, s), 7.87-7.70 (3H, m), 7.62-7.40 (4H, m); 13C NMR (75.4 MHz,
CDCl3); (ppm) 187.7, 143.5, 138.1, 137.7, 135.2, 134.9, 133.8, 131.4, 131.2, 130.8, 130.3,
128.0, 127.9 and 122.7. HRMS-ESI: m/z [M+H]+ for C15H8Cl4O, calculated 344.9402;
observed 344.4444.
3.4.3 Biological assays for antimalarial activity testing:
3.4.3.1 Measurement of inhibition of P. falciparum growth in culture:
In this study chloroquine sensitive 3D7 and resistant Dd2 strains of P. falciparum were used
in in vitro culture. Parasite strains were cultivated by the method of Trager and Jensenm
[Trager and Jensenm (1976)] with minor modifications. Cultures were maintained in fresh
O+ve human erythrocytes at 4% hematocrit in complete medium (RPMI 1640 with 0.2%
sodium bicarbonate, 0.5% albumax, 45 µg/liter hypoxanthine and 50 µg/liter gentamicin) at
37°C under reduced O2 (gas mixture 5% O2, 5% CO2, and 90% N2) [Kumar et al. (2010)].
Stock solutions of chloroquine were prepared in water (miliQ grade) and test compounds
were dissolved in DMSO. All stocks were then diluted with culture medium to achieve the
required concentrations (In all cases the final concentration contained 0.4% DMSO, which
was found to be non-toxic to the parasite). Drugs and test compounds were then placed in
96-well flat-bottom tissue-culture grade plates to yield triplicate wells with drug
concentrations ranging from 0 to 10-4 M in a final well volume of 100 µL. Chloroquine was
used as a positive control in all experiments. Parasite culture was synchronized at ring stage
with 5% sorbitol. Synchronized culture was aliquoted to drug containg 96 well plate at 2%
hematocrit and 1% parasitemia. After 72 hours of incubation under standard culture
Structure-activity relationship…….. Chapter 3
157
conditions, plates were harvested and read by the SYBR Green I fluorescence-based method
[Smilkstein et al. (2004)] using a 96-well fluorescence plate reader (Victor, Perkin Elmer),
with excitation and emission wavelengths at 497 and 520 nm, respectively. The
fluorescence readings were plotted against drug concentration and IC50 values obtained by
visual matching of the drug concentration giving 50% inhibition of growth.
3.4.3.2 Measurement of cytotoxic activity against mammalian cell lines in culture:
Animal cell lines (Hela and fibroblast L929) were used to determine drug toxicity by using
MTT assay for mammalian cell viability assay as described by Mosmann [Mosmann
(1983)] using Hela and fibroblast L929 cells cultured in complete RPMI containing 10%
fetal bovine serum, 0.2% sodium bicarbonate, 50 g/ml gentamycin. Briefly, cells (104
cells/200l/well) were seeded into 96-well flat-bottom tissue culture plates in complete
culture medium [Kumar et al. (2010)]. Drug solutions were added after overnight seeding
and incubated for 24 hrs in a humidified atmosphere at 37°C and 5% CO2. DMSO (final
concentration 10 %) was added as +ve control. An aliquot of a stock solution of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml in 1X phosphate-
buffered saline) was added at 20 l per well, and incubated for another 4 h. After spinning
the plate at 1500 RPM for 5 min, supernatant was removed and 100 μl of the stop agent
DMSO was added. Formation of formazon, an index of survival was read at 570 nm. The
50% cytotoxic concentration (TC50) was determined by analysis of dose-response curves.
Selectivity index was calculated as TC50 / IC50.
3.4.4 Biological evaluation for pesticidal activity:
3.4.4.1 Test insect:
Insects, P. xylostella, were obtained from infested field crops and reared in the laboratory.
Adult insects were allowed to lay eggs on one week old mustard plants grown in the pots
and encaged inside the wooden boxes. 2nd instar larvae were removed from the mustard
plants and transferred on to the cabbage leaves encaged in other boxes where the insect
completed rest of the stages [Kumar et al. (2011)]. The adults emerged from the pupae were
allowed to lay eggs on the mustard plants placed in the cages. Insects were reared and
maintained at 25 ±1 οC, 65 ±5% RH and a photoperiod of 16:8 (L: D). 2nd instar larvae
obtained from the established colony maintained for >20 generations in the laboratory were
used in the experiments.
Structure-activity relationship…….. Chapter 3
158
3.4.4.2 Bioassays for preliminary screening:
The larvicidal activity of the test compounds (Tables 8-9) was evaluated by the leaf dip
method against 2nd instar larvae [Kumar et al. (2011)]. Larvicidal activity was evaluated on
cabbage leaves (~34 cm2). Known amount of the test compounds was dissolved in acetone
and then diluted with water to obtain a desired range of concentrations. Test compounds
were suspended in distilled water using triton X-100 LR spreader (s.d. Fine-Chem. Ltd;
India) at 0.1 ml/L. Based on our previous experiences, [Tewary et al. (2006)] preliminary
screening of the test compounds were carried out at two higher test dosages; 10000 g/ml &
5000 g/ml. Three leaf disks were separately dipped in each test solution for 30 seconds and
allowed to dry at ambient conditions. 2nd instar larvae (10 larvae in each replicate), starved
for 3-4 h, were transferred individually on treated and control (disks treated with water
mixed with acetone: triton only) leaf disks placed in petri plates. Moistures build up inside
the petri plates was swabbed after 24 h using tissue paper and petri plates were resealed
using parafilm. Mortality was determined at 48 h after larvae were placed on disks. Larvae,
which did not show movements when probed with camel hairbrush, were considered dead.
All treated samples were maintained at 25+1οC, 65+5% RH and a photoperiod of 16:8 (L:
D) in the laboratory.
3.4.4.3 Dose-response experiment:
Based on the review of preliminary screening results, promising test compounds were
selected and subjected to dose-response bioassay. Test compounds of six concentrations
each were prepared to provide dosage in the range 1mg/ml and set for bioassay as
mentioned above. Commercial pesticide, deltamethrin was estimated against P. xylostella as
positive control. Seven concentrations (400, 200, 100, 50, 25, 12.5 and 6.25 g/ml) of
deltamethrin were prepared in the tap water by serial dilutions. For control set, leaf discs
were treated with tap water only.
3.4.4.4 Statistical analysis:
Mortality was corrected by using Abbott formula [Abbott (1925)]. Lethal concentration to
kill 50% of the population relative to control values (LC50) was determined using (EPA
PROBIT ANALYSIS PROGRAM used for calculating LC/EC values version 1.5) [Finney
(1971)].
Structure-activity relationship…….. Chapter 3
159
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Structure-activity relationship…….. Chapter 3
168
Structure-activity relationship…….. Chapter 3
i
NMR spectra of some compounds
1H NMR (in CDCl3) spectrum of 1-(4-Bromophenyl)-3-[1-(prop-2-en-1-yl)-1H-indol-3-yl]-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (21, Table 3)
13C NMR (in CDCl3) spectrum of 1-(4-Bromophenyl)-3-[1-(prop-2-en-1-yl)-1H-indol-3-yl]-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (21, Table 3)
H3COOCH3
O
Br
H3CO
N
3.03.54.04.55.05.56.06.57.07.58.08.5 ppm
220 200 180 160 140 120 100 80 60 40 20 ppm
Structure-activity relationship…….. Chapter 3
ii
1H NMR (in CDCl3) spectrum of 1-(4-Bromophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one
(27, Table 4)
13C NMR (in CDCl3) spectrum of 1-(4-Bromophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one
(27, Table 4)
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
OCH3
H3COOCH3
O
Br
220 200 180 160 140 120 100 80 60 40 20 ppm
Structure-activity relationship…….. Chapter 3
iii
1H NMR (in CDCl3) spectrum of 3-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1,2-oxazole
(34, Scheme 17)
13C NMR (in CDCl3) spectrum of 3-(4-Bromophenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1,2-oxazole
(34, Scheme 17)
OCH 3
H3CO
OCH 3
Br
O N
160 140 120 100 80 60 40 20 0 ppm
3.03.54.04.55.05.56.06.57.07.58.08.5 ppm
Structure-activity relationship…….. Chapter 3
iv
H3CO OCH3
OCH3
Br
O
O
O
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm
1H NMR (in CDCl3) spectrum of Ethyl-4-(4-bromophenyl)-2-oxo-6-(2,4,5-trimethoxyphenyl)cyclohex-3-ene-
1-carboxylate (36, Scheme 17)
220 200 180 160 140 120 100 80 60 40 20 ppm13C NMR (in CDCl3) spectrum of Ethyl-4-(4-bromophenyl)-2-oxo-6-(2,4,5-trimethoxyphenyl)cyclohex-3-ene-
1-carboxylate (36, Scheme 17)
Structure-activity relationship…….. Chapter 3
v
O
N
OCH 3
H3CO
OCH 3
Br
Br
3.03.54.04.55.05.56.06.57.07.58.08.5 ppm1H NMR (in CDCl3) spectrum of 1-(4-Bromophenyl)-3-{1-[(2-bromophenyl)methyl]-1H-indol-3-yl}-3-(2,4,5-
trimethoxyphenyl)propan-1-one (43, Scheme 17)
220 200 180 160 140 120 100 80 60 40 20 ppm13C NMR (in CDCl3) spectrum of 1-(4-Bromophenyl)-3-{1-[(2-bromophenyl)methyl]-1H-indol-3-yl}-3-
(2,4,5-trimethoxyphenyl)propan-1-one (43, Scheme 17)
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