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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,


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


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).


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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


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


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


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)


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


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.


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.


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


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


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


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


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)


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


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


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


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


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


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.


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.


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)


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


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


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


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


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


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.


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


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.


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.


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


(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.


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


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,


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:


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


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:


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:


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


(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


(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);


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


(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,


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.


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,


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,


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:


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)


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).


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:


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,


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.


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


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,


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-


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,


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,


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-


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,


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


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,


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-


O

N

OCH 3

H3CO

OCH 3

Br

Br


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).


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,


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,


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.


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,


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.


155


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.


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,



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


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.


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)].


159

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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


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


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


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)


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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