CHAPTER 3 SYNTHESIS AND CHARACTERIZATION OF BENZOYL...

Ph. D. Thesis (Chemistry), Umesh D. Pete Chapter 3 2013

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CHAPTER 3

SYNTHESIS AND CHARACTERIZATION OF BENZOYL CARVACRYL THIOUREA AND UREA DERIVATIVES &

EFFECT OF INSECT GROWTH REGULATOR ACTIVITY ON AGRICULTURAL PESTS

3.1 INTRODUCTION

Hybrid molecules with dual mode of action are an emerging novel strategy

being employed for the generation of new drug candidates for various human

diseases.1 In present study; the strategy has been used for the generation of new and

more effective agrochemicals with insecticidal and antifungal properties.

Accordingly, a known antifungal monoterpenoid carvacrol was coupled with

benzoyl urea & thiourea moiety as present in commercially available benzoyl phenyl

urea (BPU) class insect growth regulator (IGR) for the synthesis of hybrid molecules

and their synthesis, characterization, insecticidal and antifungal activity is discussed

in this chapter.

Insect pests and plant pathogenic fungi are major causes for crop losses. Many

synthetic organic compounds are in use for their control; however, these traditional

pesticides have drawbacks such as development of resistance, unwanted side-effects,

persistence in the environment, toxicity, etc. Insect Growth Regulators (IGRs) are

receiving more practical attention to provide safer foods and cleaner environment.2

IGRs inhibit different developmental stages in the lifecycle of an insect by specific

action such as inhibition of chitin biosynthesis, metamorphosis or breeding.

Benzoylphenyl urea (BPU) is one such class of IGR compounds which mainly

inhibits chitin synthesis and thus interferes with the formation of insect cuticle.3

Lufenuron, diflubenzuron, penfluzuron, noveluron, flufenoxuron, tefluenzuron,

chlorfuazuron, hexaflumuron are few examples of BPU compounds currently in use

for the control of a wide range of leaf-eating insects and their larvae in vegetables,

fruits and mushrooms (Fig. 1).4 BPU compounds bind to the sulphonyl urea receptors

(SUR), a group of ABC-transporters and inhibit the exocytotic movement of the

vesicles, depolarizes the vesicle membrane through inhibition of K+ channel, which

leads to inhibition of N-acetylglucosamine deposition and subsequent chitin synthesis

in the cuticle.5 Chitin is also an important constituent of fungal cell wall; however,


43

BPU compounds do not affect chitin synthesis in fungi and have been shown to be

ineffective in vivo and in vitro for the control of fungal pathogens.6,7

Noveluron, Lufenuron, Diflubenzuron and Penfluzuron (Fig. 3.1) are the

benzoyl phenyl urea (BPU) compounds which are marketed chitin synthesis inhibitors

PenfluzuronDiflubenzuron

Novaluron Lufenuron

NH

NH

O O F

F

Cl

Cl

O

H

FF

FF

F

FNH

NH

O O F

F

Cl

O

H

FF

FF

F

F

Cl

NH

NH

O O F

F

Cl

NH

NH

O O F

F

F

FF

Fig. 3.1 IGRs: Representative Benzoyl Phenyl Urea chitin synthesis inhibitors

Carvacrol (2-methyl-5-[1-methylethyl] phenol), a phenolic monoterpenoid is a

constituent of essential oils produced by numerous aromatic plants and spices such as

black cumin (Nigella sativa L.), marjoram (Origanum majorana L.), oregano

(Origanum vulgare L.) summer savory (Satureja hortensis L.) and thyme (Thymus

vulgaris L.).8-10 The antifungal activity of carvacrol has been demonstrated against

many phytopathogens11,12 and human pathogenic fungi.13 It causes cytoplasmic

membrane damage through lesion formation and lowering of ergesterol content.14 Rao

et al. have suggested calcium burst and inhibition of TOR pathway as a mode of

action for Carvacrol.15 It is also insecticidal (less effective than BPU) and has been

proved effective against different insect pests like Thecodiplosis Japonensis, Aphis

craccivora, and Leucania separata.16,17

For development of better crop protection agents we envisaged hybrid

molecules of carvacrol and benzoylphenyl ureas which will have dual (insecticidal

and antifungal) biological activity. For this, 4-nitroso carvacrol and 4-amino carvacrol

were synthesized according to previously reported procedures.18,19 Using these


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derivatives, two series of compounds Benzoyl Carvacryl Thiourea’s (BCTU, 4a-f)

and Benzoyl Carvacryl Urea’s (BCU, 5a-f) were synthesized (Scheme 3.2.1).

3.2 EXPERIMENTAL

3.2.1 Reaction Scheme

OH OH

NH2

NaNO2

HClEtOH

OH

NO

28 % NH3

H2O

H2S

OH

NH

NH

S OOH

NH

NH

O OHCOOHH2O2

NH4SCN

AcetoneClOC

R

RR

R = a) H, b) 2-Cl, c) 4-Cl, d) 2-F, e) 4-F and f) 2,6-diF

[1] [2] [3]

[5 a-f] [4 a-f]

3.2.2 Synthesis of 2-isopropyl-5-methyl-4-nitrosophenol (2) (4-Nitrosocarvacrol)20-23

To a solution of Carvacrol (3.00 g, 0.02 moles) in 95% ethyl alcohol (20 ml),

concentrated hydrochloric acid (20 ml) was added. The mixture was cooled to 0-5°C

and sodium nitrite (1.08 g, 0.015 moles) was added slowly in small lots in one hour.

The mixture was stirred well after each addition. The solution first became brown in

colour and a green precipitate soon began to form. After 0.5 g of nitrite had been

added the mixture became pasty; so the intervals between the additions were

lengthened and the stirring was made more vigorous. When all the nitrite had been

added, the bulk of the product was transferred to a 250 ml beaker containing 120 ml

of cold water and the remainder was washed with water. After agitation with water the

product became a light yellow, fluffy solid. It was filtered off by suction, washed well

with water and crystallized from ethanol, Yield 75%.


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3.2.3 Synthesis of 4-amino-2-isopropyl-5-methylphenol (3) (4-Aminocarvacrol)22, 23

The crude wet 4-nitrosocarvacrol (2) (3.1 g, 0.0174 moles) obtained as above

was dissolve in a mixture of 28% aqueous ammonia (30 ml) and water (50 ml). The

brown solution thus obtained was filtered to remove resinous matter and hydrogen

sulphide was passed into it. The brown colour disappeared and a white precipitate of

4-aminocarvacrol was obtained, yield 82%. Due to less stability the product was used

for next step without purification.

3.2.4 General procedure for the synthesis of 4-[N-(N-substitutedbenzoyl

thiocarbomyl)amino]-5-isopropyl-2-methylphenol (4a-f) [Substituted Benzoyl

Carvacryl Thiourea’s (BCTU)]24

To a 100 ml round-bottom flask containing ammonium thiocyanate (0.84 g,

0.011 M), in dry acetone (20 ml), substituted benzoyl chloride (0.011 moles) in dry

acetone (10 ml) was added drop wise. The reaction mixture was stirred and heated at

refluxed for 1.5 hrs. Then 4-aminocarvacrol (0.01 moles) in dry acetone (10 ml) was

added it. The mixture was refluxed for 5-6 hrs at 55°C. The solvent was then removed

under reduced pressure and the reaction mixture was diluted with ice cold water (50

ml) to afford the product. The separated solid was purified by recrystallization from

hexane-ethyl acetate mixture with good yields (Table 3.1).

3.2.5 General procedure for the synthesis of 4-[N-(N-substitutedbenzoyl

oxocarbomyl)amino]-5-isopropyl-2-methylphenol (5a-f) [Substituted Benzoyl

Carvacryl Ureas (BCU)].24

4-[N-(N-substitutedbenzoylthiocarbomyl)amino]-5-isopropyl-2-methyl-phenol

(4a-f) (0.010 moles) was dissolved in 85% (30 ml) formic acid. After addition of 30%

hydrogen peroxide (100 ml), the solution was stirred overnight at room temperature

and poured onto crushed ice. The precipitate was collected, dried and recrystallized

from hexane-ethyl acetate mixture to afford the product (5a-f) with good yield (Table

3.2).

The synthesized compounds (4a-f) and (5a-f) were characterised by IR, NMR,

elemental analysis, LC-MS and single X-ray crystal structure data.


46

3.3 RESULTS AND DISCUSSION

3.3.1 Analytical Studies

The IUPAC name, molecular formula, molecular weight, appearance, Rf

value, % yield, melting point and partial elemental analysis of the prepared benzoyl

Carvacryl thiourea (4a-f) and urea compounds (5a-f) are mentioned in Table 3.1 and

3.2, respectively. Elemental analyses for all compounds are in agreement with

calculated value.


Table 3.1: Analytical data of benzoyl carvacryl thiourea derivatives (4a-f)

Compound No. IUPAC Name of Compound

Molecular Formula (Weight)

Appearance Rf

value*

Yield (%)

Melting point [°C]

CHNS (Theoretical) [%]

C H N S

4a 4-[N-(N-benzoylthio

carbomyl)amino]-5-isopropyl-2-methylphenol

C18H20N2O2S (328.44)

colourless crystal

0.68 81.12 208 66.22

(65.83) 6.17

(6.14) 8.49

(8.53) --

(9.76)

4b 4-{N-[N-(2-chlorobenzoyl) thiocarbomyl] amino}-5-isopropyl-2-methylphenol

C18H19ClN2O2S (362.88)

colourless crystal

0.65 89.95 188 58.27

(59.58) 5.17

(5.28) 7.62

(7.72) 9.78

(8.84)

4c 4-{N-[N-(4-chlorobenzoyl) thiocarbomyl] amino}-5-isopropyl-2-methylphenol

C18H19ClN2O2S (362.88)

colourless crystal 0.75 84.50 164

57.11 (59.58)

5.07 (5.28)

7.59 (7.72)

9.44 (8.84)

4d 4-{N-[N-(2-fluorobenzoyl)

thiocarbomyl]amino} -5-isopropyl-2-methylphenol

C18H19FN2O2S (346.43)

colourless crystal

0.67 78.85 200 61.81

(62.41) 4.79

(5.53) 6.32

(8.09) 12.31 (9.26)

4e 4-{N-[N-(4-fluorobenzoyl)

thiocarbomyl]amino} -5-isopropyl-2-methylphenol

C18H19FN2O2S (346.43)

colourless crystal

0.73 90.00 198 61.41

(62.41) 4.93

(5.53) 6.65

(8.09) 11.31 (9.26)

4f 4-{N-[N-(2,6-difluorobenzoyl)

thiocarbomyl]amino}-5-isopropyl-2-methylphenol

C18H18F2N2O2S (364.42)

colourless crystal

0.64 76.00 212 58.98

(59.33) 4.32

(4.98) 6.278 (7.69)

11.49 (8.80)

* System - Hexane:Ethylacetate (70:30) [compounds were dissolved in ethyl acetate]


Table 3.2: Analytical data of benzoyl carvacryl urea derivatives (5a-f)

Compound No.

IUPAC Name of Compound Molecular Formula (Weight)

Appearance Rf

value*

Yield (%)

Melting point [°C]

CHNS (Theoretical) [%]

C H N S

5a 4-{N-[N-(benzoyl)oxocarbomyl]

amino}-5-isopropyl-2-methylphenol C18H20N2O3

(312.37) Reddish 0.54 84.70 242 68.05

(69.21) 5.55

(6.45) 7.57

(8.97) --

5b 4-{N-[N-(2-chlorobenzoyl)

oxocarbomyl]amino} -5-isopropyl-2-methylphenol

C18H19ClN2O3 (346.82)

colourless crystal

0.50 76.56 270 60.90

(62.34) 4.68

(5.52) 6.54

(8.08) --

5c 4-{N-[N-(4-chlorobenzoyl)


C18H19ClN2O3 (346.82)

Light brown crystal 0.63 78.40 216

62.23 (62.34)

5.53 (5.52)

7.93 (8.08) --

5d 4-{N-[N-(2-fluorobenzoyl)


C18H19FN2O3 (330.36)

Light yellow crystal 0.54 80.55 230

64.27 (65.44)

4.76 (5.80)

6.91 (8.48) --

5e 4-{N-[N-(4-fluorobenzoyl)

oxocarbomyl] amino} -5-isopropyl-2-methylphenol

C18H19FN2O3 (330.36) Brown crystal 0.60 75.48 162

64.43 (65.44)

5.00 (5.80)

7.19 (8.48) --

5f 4-{N-[N-(2,6-difluorobenzoyl)

oxocarbomyl]amino}-5-isopropyl-2-methylphenol

C18H18F2N2O3 (348.35)

Off white powder 0.52 66.25 168

-- (62.06)

-- (5.21)

-- (8.04) --

* System - Hexane:Ethylacetate (70:30) [Compounds were dissolved in ethyl acetate]


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3.3.2 IR Spectral Data

The significant absorption bands in IR spectra of Benzoyl Carvacryl Thiourea

(4a-f) and Urea compounds (5a-f) are summarized in the Table 3.3 and a

representative IR spectrum of 4-{N-[N-(2,6-difluorobenzoyl) thiocarbomyl]amino}-

5-isopropyl-2-methylphenol (4f) is shown in Fig. 3.2.

Table 3.3: Significant absorption bands (cm-1) in IR spectra of Benzoyl Carvacryl, Thiaurea (4a-f) and urea compounds (5a-f)

Compound No. N-H CH O-H C=C C=S C=O

4a 3154 2925 3436 1605 1677 1600

4b 3213 2956 3445 1593 1665 1623

4c 3160 2924 3450 1592 1672 1618

4d 3157 2923 3435 1533 1674 1612

4e 3260 2922 3462 1590 1668 1601

4f 3159 2921 3436 1523 1678 1623

5a 3240 2924 3460 1535 -- 1670, 1610

5b 3240 2924 3450 1531 -- 1664, 1610

5c 3235 2929 3413 1599 -- 1672, 1600

5d 3257 2925 3445 1536 -- 1668, 1614

5e 3254 2925 3454 1534 -- 1665, 1600

5f 3240 2925 3460 1591 -- 1676, 1605


4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0

0.1

5

10

15

20

25

30

35

40

45

50

55

61.5

cm-1

%T

3436.98

3159.50

2921.66

2727.891678.26

1623.97

1536.27

1460.11

1377.48

1287.111253.87

1204.27

1158.93

1094.51

1044.21

1013.02

890.95

868.80

844.16

799.99

769.98

722.36

616.97593.28

474.36

Fig. 3.2 IR spectrum of 4-{N-[N-(2,6-difluorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4f)

OH

NH

NH

S O F

F


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3.3.3 NMR Shift Measurements

The 1H peaks in NMR spectra of benzoyl carvacryl thiourea (4a-f) and urea

compounds (5a-f) are summarized in the Table 3.4 and the representative spectra are

shown in Fig. 3.3 to 3.8. While 13C NMR peaks recorded for two benzoyl carvacryl

urea derivatives (4e) and (5d) are also summarized in Table 3.4 and the spectra for

(4e) and (5d) are represented in Fig. 3.9 and 3.10 respectively.

Table 3.4: NMR data of benzoyl carvacryl thiourea (4a-f) and urea (5a-f)

derivatives

6

1 23

4

5

7

899

1011

12

1314

15 16

17

1819

OH

NH

NH

S/O O

R

Compound No. (Recorded in solvent) NMR (δ ppm)

4a (CDCl3)

1H NMR: 1.22 (6H, d, J = 6.87 Hz, C-9H), 2.24 (3H, s, C-7H), 3.05 (1H, m, C-8H), 5.06 (1H, s, O-H), 6.77 (1H, s, C-6H), 7.27 (1H, s, C-3H), 7.55 (1H, t, C-17H),7.64 (2H, t, C-16 & 18H), 7.91 (2H, d, C-15 & 19H), 9.26 (1H, bs, N-10H), 11.98 (1H, bs, N-12H)

4b

(CDCl3)

1H NMR: 1.23 (6H, d, J = 6.88 Hz, C-9H), 2.23 (3H, s, C-7H), 3.03 (1H, m, C-8H), 4.94 (1H, bs, O-H), 6.77 (1H, s, C-6H), 7.27 (1H, s, C-3H), 7.40 (1H, d, J = 7.40 Hz, C-16H), 7.46 (H, t, J = 7.90 Hz, C-17 ), 7.78 (1H, d, J = 7.70 Hz, C-19H), 7.81 (1H, t, C-18H), 9.36 (1H, bs, N-10H), 11.78 (1H, bs, N-12H)

4c

(CDCl3)

1H NMR: 1.21 (6H, d, J = 6.88 Hz, C-9H), 2.23 (3H, s, C-7H), 3.03 (1H, m, C-8H), 5.25 (1H, bs, O-H), 6.77 (1H, s, C-6H), 7.23 (1H, s, C-3H), 7.51 (2H, dd, J = 8.53, C-16 & 18H), 7.89 (2H, dd, J = 8.80 Hz, C-15 & 19H), 9.29 (1H, bs, N-10H), 11.94 (1H, bs, N-12H)

4d

(CDCl3)

1H NMR: 1.21 (6H, d, C-9H), 2.23 (3H, s, C-7H), 3.04 (1H, m, C-8H), 4.98 (1H, s, O-H), 6.77 (1H, s, C-6H), 7.24 (1H, s, C-3H), 7.33 (1H, d, J = 7.77 Hz, C-16H), 7.38 (H, t, C-17 ), 7.65 (1H, d, J = 7.86 Hz, C-19H), 8.14 (1H, t, C-18H), 9.77 (1H, s, N-10H), 11.92 (1H, s, N-12H)

4e (DMSO-d6)

1H NMR: 1.14 (6H, d, C-9H), 2.07 (3H, s, C-7H), 2.93 (1H, m, C-8H), 6.73 (1H, s, C-6H), 7.02 (1H, s, C-3H), 7.36 (2H, dd, J = 7.6 Hz, C-16 & 18H), 8.06 (2H, dd, C-15 & 19H),


52

9.37 (1H, bs, O-H), 11.59 (1H, bs, N-10H), 11.99 (1H, bs, N-12H) 13C NMR: 15.51 (C-7), 23.18 (C-9), 27.71 (C-8), 111.34 (C-6), 115.32 (C-3), 115.62 (C-4), 121.54 (C-14), 126.55 (C-15 & 19), 129.59 (C-16 & 18), 131.68 (C-5), 131.81 (C-2), 142.24 (C-17), 163.24 (C-1), 167.25 (C-11), 181.14 (C-13)

4f (DMSO-d6)

1H NMR: 1.16 (6H, d, J = 6.60 Hz, C-9H), 2.07 (3H, s, C-7H), 3.16 (1H, m, C-8H), 6.71 (1H, s, C-6H), 7.21 (1H, s, C-3H), 7.24 (2H, d, J = 8.25 Hz C-16 & 18H), 7.61 (1H, t, J = 7.69 Hz, C-17H), 9.21 (1H, bs, O-H), 9.26 (1H, bs, N-10H), 11.98 (1H, bs, N-12H)

5a (DMSO-d6)

1H NMR: 1.13 (6H, d, J = 6.86 Hz, C-9H), 2.05 (3H, s, C-7H), 2.98 (1H, m, C-8H), 6.69 (1H, s, C-6H), 7.30 (1H, s, C-3H), 7.50 (1H, t, C-17H), 7.59 (2H, t, C-16 & 18H), 7.99 (2H, d, C-15 & 19H), 9.13 (1H, bs, O-H), 10.36 (1H, bs, N-10H), 10.93 (1H, bs, N-12H)

5b (DMSO-d6)

1H NMR: 1.17 (6H, d, J = 6.88 Hz, C-9H), 2.08 (3H, s, C-7H), 3.02 (1H, m, C-8H), 6.72 (1H, s, C-6H), 7.31 (1H, s, C-3H), 7.44 (1H, dd, J = 8.53 Hz, C-16H), 7.52 (H, t, J = 7.98 Hz, C-17 ), 7.55 (1H, d, J = 7.98 Hz, C-19H), 7.60 (1H, t, J = 7.70 Hz, C-18H), 9.17 (1H, bs, O-H), 10.00 (1H, bs, N-10H), 11.14 (1H, bs, N-12H)

5c (DMSO-d6)

1H NMR: 1.24 (6H, d, J = 6.80, C-9H), 2.23 (3H, s, C-7H), 3.07 (1H, m, C-8H), 4.94 (1H, bs, O-H), 6.74 (1H, s, C-6H), 7.26 (1H, s, C-3H), 7.45 (2H, dd, J = 7.00 Hz, C-16 & 18H), 7.90 (2H,dd, J = 7.60 Hz, C-15 & 19H), 9.04 (1H, bs, N-10H), 10.38 (1H, bs, N-12H)

5d (DMSO-d6)

1H NMR: 1.14 (6H, d, J = 6.88, C-9H), 2.07 (3H, s, C-7H), 3.01 (1H, m, C-8H), 6.71 (1H, s, C-6H), 7.29 (1H, s, C-3H), 7.32 (1H, dd, J = 7.15, C-16H), 7.34 (1H, dd, J = 7.43 Hz, C-19H), 7.60 (H, t, J = 7.16 Hz, C-17 ), 7.70 (1H, t, J = 7.43, C-18H), 9.22 (1H, bs, O-H), 10.05 (1H, bs, N-10H), 10.96 (1H, bs, N-12H) 13C NMR: 15.67 (C-7), 22.99 (C-9), 27.48 (C-8), 111.35 (C-6), 116.17 (C-3), 116.46 (C-4), 121.41 (C-14), 122.69 (C-15), 124.66 (C-19), 124.91 (C-16), 127.00 (C-18), 130.23 (C-5), 133.99 (C-2), 139.37 (C-17), 151.44 (C-1), 153.29 (C-11), 166.71 (C-13)

5e (DMSO-d6)

1H NMR: 1.14 (6H, d, J = 6.88 Hz, C-9H), 2.08 (3H, s, C-7H), 3.02 (1H, m, C-8H), 6.71 (1H, s, C-6H), 7.31 (1H, s, C-3H),7.38 (2H, dd, J = 8.80 Hz, C-16 & 18H), 8.01 (2H, dd, J = 7.15 Hz, C-15 & 19H), 9.17 (1H, bs, O-H), 10.34 (1H, bs, N-10H), 10.98 (1H, bs, N-12H)

5f (CDCl3)

1H NMR: 1.24 (6H, d, J = 6.88 Hz, C-9H), 2.22 (3H, s, C-7H), 3.02 (1H, m, C-8H), 4.96 (1H, bs, O-H), 6.76 (1H, s, C-6H), 7.07 (1H, s, C-3H), 7.26 (2H, d, J = 6.88 Hz, C-16 & 18H), 7.46 (1H, t, J = 8.25 Hz, C-17H), 9.14 (1H, bs, N-10H), 11.63 (1H, bs, N-12H)


Fig. 3.3 1H NMR spectrum of 4-[N-(N-benzoylthiocarbomyl)amino]-5-isopropyl-2-methylphenol (4a)

OH

NH

NH

S O


Fig. 3.4 1H NMR spectrum of 4-{N-[N-(4-chlorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4c)

OH

NH

NH

S O

Cl


Fig. 3.5 1H NMR spectrum of 4-{N-[N-(4-fluorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4e)

OH

NH

NH

S O

F


Fig. 3.6 1H NMR spectrum of 4-{N-[N-(2,6-diflulorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4f)

OH

NH

NH

S O F

F


Fig. 3.7 1H NMR spectrum of 4-{N-[N-(2-fluorobenzoyl)oxocarbomyl]amino}-5-isopropyl-2-methylphenol (5d)

OH

NH

NH

O O F


Fig. 3.8 1H NMR spectrum of 4-{N-[N-(4-flurobenzoyl)oxocarbomyl]amino}-5-isopropyl-2-methylphenol (5e)

OH

NH

NH

O O

F


Fig. 3.9 13C NMR spectrum of 4-{N-[N-(4-flurobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4e)

OH

NH

NH

S O

F


Fig. 3.10 13C NMR spectrum of 4-{N-[N-(2-flurobenzoyl)oxocarbomyl]amino}-5-isopropyl-2-methylphenol (5d)

OH

NH

NH

O O

F


61

3.3.4 Mass Spectrometry

The major LC-MS peaks in Mass spectra of benzoyl carvacryl thiourea (4a-f)

and urea compounds (5a-f) are summarized in the Table 3.5 and the representative

spectra are shown in Fig. 3.11 to 3.13.

Table 3.5: LC-MS data of benzoyl carvacryl thiourea (4a-f) and urea compounds

(5a-f)

Compound No. Molecular Weight m/z

[M]+ [M]-

4a 328 329.1 --

4b 362 363.2 361.2

4c 362 363.0 361.0

4d 346 347.0 --

4e 346 347.0 --

4f 364 365.1 363.2

5a 312 313.2 --

5b 346 347.0 345.0

5c 346 347.0 --

5d 330 331.1 --

5e 330 331.0 329.0

5f 348 349.0 347.0


Fig. 3.11 LC-MS spectrum of 4-{N-[N-(4-chlorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (5c )

OH

NH

NH

S O

Cl

Molecular weight = 362


Fig. 3.12 LC-MS spectrum of 4-{N-[N-(4-flurobenzoyl)oxocarbomyl]amino}-5-isopropyl-2-methylphenol (5e)

OH

NH

NH

O O

F



Fig. 3.13 LC-MS spectrum of 4-{N-[N-(2,6-difluorobenzoyl)oxocarbomyl]amino}-5-isopropyl-2-methylphenol (5f)

OH

NH

NH

O O

F

F



65

3.3.5 Single Crystal X-ray Crystallography

X-ray diffraction data were collected on an Oxford Xcalibur Eos Mova

diffractometer26 equipped with a CCD detector utilizing MoKα radiation (λ = 0.71073

Å). The structures were solved by direct methods and refined with full matrix least-

squares technique. All non-hydrogen atoms were refined anisotropically whereas the

positions were geometrically fixed and refined isotropically for all the hydrogen

atoms except for the ones on the nitrogen atoms which were located from the

difference maps and refined isotropically. All calculations were performed using the

WinGX software package.27 Crystal data and experimental details for the crystals are

summarized in Table 3.6. Intramolecular and intermolecular hydrogen bonds are

given in Table 3.7. The ORTEP diagram of 4-{N-[N-(benzoyl) thiocarbomyl]amino}-

5-isopropyl-2-methylphenol (4a), 4-{N-[N-(4-chlorobenzoyl) thiocarbomyl]amino}-

5-isopropyl-2-methylphenol (4c), 4-{N-[N-(2-fluorobenzoyl) thiocarbomyl]amino}-

5-isopropyl-2-methylphenol (4d), 4-{N-[N-(4-fluorobenzoyl) thiocarbomyl]amino}-

5-isopropyl-2-methylphenol (4e), and 4-{N-[N-(2,6-difluoro benzoyl)thiocarbomyl]

amino}-5-isopropyl-2-methylphenol (4f) are shown in Fig. 3.14, 3.16, 3.18, 3.20 and

3.22 respectively. Whereas the packing diagrams of above compounds are shown in

Fig 3.15, 3.17, 3.19, 3.21 and 3.23 respectively.


Table 3.6: Crystallographic details of BCTU (4a), 4Cl-BCTU (4c), 2F-BCTU (4d), 4F-BCTU (4e) and 2,6-diF-BCTU (4f)

DATA BCTU 4Cl-BCTU 2F-BCTU 4F-BCTU 2,6-di-FBCTU CCDC 870460 805770 870458 941951 870457

Formula C18H20O2S1N2 C18H19O2N2S1Cl1. C18H19O2 F1S1N2 C18H19N2O2F1S1 C18H18O2 F2S1N2 Formula weight 328.4 380.9 346.4 346.4 364.4

Color Colourless Colourless Colourless Colourless Colourless Crystal morphology Block block Plate Block Block Crystal size (mm) 0.20 0.30 0.30 0.30 0.20 0.20 0.09 0.25 0.30 0.30 0.25 0.20 0.20 0.30 0.30

Temperature/K 110(1) 295(1) 90(1) 295(1) 110(1) Radiation Mo Kα Mo Kα Mo Kα Mo Kα Mo Kα

Wavelength/Å 0.71073 0.71073 0.71073 0.71073 0.71073 Crystal system Monoclinic Monoclinic Monoclinic Orthorhombic Monoclinic Space group C2/c P21/c P21 P212121 C2/c

a (Å) 23.4872(11) 11.834(5) 10.0148(18) 10.3260(14) 23.4200(19) b (Å) 10.7385(5) 9.982(4) 14.7799(27) 14.8608(14) 10.7880(9) c (Å) 13.6376(8) 16.968(4) 11.6010(21) 22.7346(39) 13.5978(12) α (°) 90 90 90 90 90 β (°) 98.421(5) 108.28(1) 90.796(3) 90 95.991(3) γ (°) 90 90 90 90 90

Volume (Å3) 3402.54(27) 1903.2(3) 1716.99(5) 3488.69(8) 3416.8(5) Z 8 4 4 8 8

Density (g/ml) 1.28 1.33 1.34 1.32 1.42 µ (1/mm) 0.201 0.329 0.211 0.208 0.224 F (000) 1391.8 799.9 727.9 1455.8 1519.8

θ (min, max) 2.5, 25.0 2.4, 25.0 2.0, 25.0 2.4, 25.0 1.8, 25.0 No. Unique Reflns 2963 3352 6040 5959 3005 No. of parameters 214 239 450 441 238

hmin,max -27, 18 -14, 14 -11, 11 -12, 7 -27, 26 kmin,max -12, 11 -11, 11 -17, 17 -17, 15 -8, 12 lmin,max -16, 15 -20, 10 -13, 13 -16, 27 -16, 15

R_all, wR2_all 0.067, 0.103 0.127, 0.220 0.072, 0.084 0.115, 0.144 0.047, 0.112 R_obs, wR2_obs 0.045, 0.093 0.069, 0.175 0.046, 0.075 0.068, 0.116 0.042, 0.109

∆ρmin, ∆ρmax (eÅ-3) -0.298, 0.334 -0.441, 0.571 -0.269, 0.293 -0.223, 0.428 -0.249, 0.648 GooF 1.01 1.00 0.99 0.96 1.04


67

Table 3.7: Intramolecular and intermolecular hydrogen bonds in the crystal structures of BCTU (4a), 4Cl-BCTU (4c), 2F-BCTU (4d), 4F-BCTU (4e) and 2,6-diF-BCTU (4f)

D–HּּּA r(D–H)/Å r(D–A)/Å r(H···A)/Å ∠∠∠∠ D–HּּּA/º Symmetry

BCTU (at 110 K) N1−H1N⋅⋅⋅S1 0.98(1) 3.397(2) 2.461(1) 159.7(1) -x+1,-y+1,-z O2−H2⋅⋅⋅S1 0.82 3.349(2) 2.620(1) 148.9(1) -x+1/2,-y+1/2+1,-z

N2−H2N⋅⋅⋅O1 0.95(1) 2.660(3) 1.935(2) 131.1(1) x,y,z N2−H2N⋅⋅⋅O1 0.95(2) 3.108(2) 2.303(1) 141.8(1) -x+1,-y+2,-z

C16−H16B⋅⋅⋅O1 0.96 3.430(3) 2.739(2) 129.4(2) -x+1,+y,-z+1/2 C16−H16B⋅⋅⋅O1 0.96 3.596(3) 2.815(1) 139.0(2) x,-y+2,+z+1/2 C2−H2A⋅⋅⋅O2 0.93 3.566(3) 2.784(2) 142.4(1) -x+1,-y+2,-z C3−H3⋅⋅⋅O2 0.93 3.291(3) 2.624(2) 129.1(2) x+1/2,-y+1/2+1,+z-1/2

C15−H15⋅⋅⋅N2 0.98 2.954(3) 2.464(2) 110.5(1) x,y,z C1−H1⋅⋅⋅Cg(2) 0.93 3.385(2) 2.80 122 -x,1-y,1-z

C18−H18C⋅⋅⋅Cg(2) 0.96 3.487(2) 2.67 143 1/2-x,1/2-y,1-z 4Cl-BCTU

O1-H1…O1w 0.82 2.636(6) 1.829(4) 168.1(2) x,y,z N2–H2···O1 0.86 3.110(4) 2.284(3) 160.8(2) x,+y-1,+z

C14–H14···O1 0.93 3.287(5) 2.811(3) 112.9(2) x,+y-1,+z C18–H18c···Cl1 0.96 3.886(5) 2.963(2) 161.8(2) -x+1/2+2,+y+1/2+1,-z+1/2 C11–H11···S1 0.93 3.706(6) 2.962(1) 137.9(3) x+1/2,-y+1/2+1,+z+1/2

O1w–H1wa···S1 0.87 3.332(4) 2.588(9) 143.6(9) -x+1/2+1,+y+1/2,-z+1/2 2FBCTU (at 90 K)

N3−H3N⋅⋅⋅S1 1.02(1) 3.291(3) 2.272(1) 170.8(1) x,y,z N1−H1N⋅⋅⋅S2 0.99(1) 3.293(3) 2.306(1) 173.2(1) x,y,z O2−H2⋅⋅⋅S2 0.82 3.437(3) 2.648(1) 162.0(2) -x+1,+y+1/2,-z+1

C11−H11⋅⋅⋅S2 0.93 3.400(4) 2.570(1) 148.8(2) -x+1,+y+1/2,-z+1 O4−H4⋅⋅⋅O2 0.82 2.709(3) 1.952(2) 153.0(2) x-1,+y-1,+z

N4−H4N⋅⋅⋅O3 0.92(1) 2.707(3) 1.952(2) 138.0(2) x,y,z N2−H2N⋅⋅⋅O1 0.94(1) 2.699(4) 1.972(2) 132.0(2) X,y,z N4−H4N⋅⋅⋅O1 0.92(1) 3.040(4) 2.347(2) 131.9(2) x-1,+y,+z N2−H2N⋅⋅⋅O3 0.94(1) 2.957(3) 2.196(2) 136.8(2) x+1,+y,+z

C36−H36A⋅⋅⋅O3 0.96 3.572(4) 2.844(2) 133.3(2) -x,+y-1/2,-z C22−H22⋅⋅⋅O4 0.93 3.487(4) 2.563(2) 172.5(2) x,+y+1,+z

C18−H18A⋅⋅⋅O4 0.96 3.398(4) 2.689(3) 131.1(2) x+1,+y+1,+z C5−H5⋅⋅⋅Cg(4) 0.93 3.656(3) 2.75 164 1-x,-1/2+y,1-z

C23−H23⋅⋅⋅Cg(2) 0.93 3.628(3) 2.71 168 1-x,1/2+y,1-z 4F-BCTU

O4–H4O···S1 0.82 3.495(5) 2.710(2) 160.7(3) -x+2,+y+1/2,-z+1/2+1 N1–H1N···S2 0.86 3.377(4) 2.543(2) 163.8(2) x,y,z N3–H3N···S1 0.86 3.403(4) 2.573(1) 162.3(2) x,y,z N2–H2N···O1 0.86 2.643(4) 1.984(3) 132.5(2) x,y,z N2–H2N···O3 0.86 3.043(5) 2.342(4) 138.9(2) x-1,+y,+z N4–H4N···O3 0.86 2.658(5) 1.982(3) 134.7(25) x,y,z N4–H4N···O1 0.86 2.951(5) 2.282(4) 134.8(2) x+1,+y,+z C5–H5···S2 0.93 3.475(6) 2.980(2) 114.8(3) x,y,z

C23–H23···S1 0.93 3.361(6) 2.894(2) 112.4(3) x,y,z C29–H29···S1 0.93 3.453(6) 2.593(2) 154.0(3) -x+2,+y+1/2,-z+1/2+1 C2–H2···O2 0.93 3.577(6) 2.693(3) 159.0(3) x,+y+1,+z

C22–H22···O3 0.93 3.328(8) 2.780(4) 118.7(3) x-1/2,-y+1/2,-z+2 C36–H36B···O2 0.96 3.457(8) 2.726(4) 133.5(3) x+1,+y+1,+z C15–H15···N2 0.98 2.914(7) 2.420(4) 110.6(3) x,y,z C18–H18B···F1 0.96 3.340(7) 2.442(3) 155.6(3) x,+y-1,+z

2,6-di-FBCTU (at 110 K) N1−H1N⋅⋅⋅S1 0.94(1) 3.351(.002) 2.447(.001) 160.2(1) -x,-y+1,-z+2 O2−H2⋅⋅⋅S1 0.82 3.325(.002) 2.605(.001) 147.3(1) -x+1/2,-y+1/2+1,-z+2

N2−H2N⋅⋅⋅O1 0.93(1) 2.660(.003) 1.981(.002) 128.3(1) x,y,z N2−H2N⋅⋅⋅O1 0.93(1) 3.208(.003) 2.421(.002) 142.3(1) -x,-y+2,-z+2 C2−H2A⋅⋅⋅O2 0.93 3.471(.003) 2.772(.002) 132.6(1) -x,-y+2,-z+2 C4−H4⋅⋅⋅O2 0.93 3.271(.004) 2.575(.002) 132.0(1) x-1/2,-y+1/2+1,+z+1/2

C18−H18C⋅⋅⋅Cg(2) 0.96 3.398(2) 2.55 148 1/2-x,1/2-y,1-z C1−F1⋅⋅⋅Cg(2) 1.341(3) 3.549(2) 3.042 100.9(1) -x,-y,1-z


68

Fig. 3.14 ORTEP diagram of 4-{N-[N-(benzoyl)thiocarbomyl]amino}-5-isopropyl-

2-methylphenol (4a) drawn at 50% probability ellipsoids, hydrogen atoms are omitted for clarity.

Fig. 3.15 Packing diagram of compound 4-{N-[N-(benzoyl)thiocarbomyl]amino}-

5-isopropyl-2-methylphenol (4a), showing the arrangement of molecules in the crystal lattice, viewed down the a-axis.


69

Fig. 3.16 ORTEP diagram of compound 4-{N-[N-(4-chlorobenzoyl)thiocarbomyl] amino}-5-isopropyl-2-methylphenol (4c) drawn at 30% probability ellipsoids. Intramolecular N-H…O hydrogen bond is shown by the dotted lines, water molecule is not shown.

Fig. 3.17 Packing diagram of compound 4-{N-[N-(4-chlorobenzoyl)thiocarbomyl] amino}-5-isopropyl-2-methylphenol (4c), showing the arrangement of molecules in the crystal lattice, viewed down the b-axis, water oxygen’s are shown by red balls.


70

Fig. 3.18 ORTEP diagram (molecular dimer build with N-H...O hydrogen bonds and C-H...ΠΠΠΠ interactions) of 4-{N-[N-(2-fluorobenzoyl) thiocarbomyl] amino}-5-isopropyl-2-methylphenol (4d) drawn at 30% probability ellipsoids, other hydrogen atoms are omitted for clarity.

Fig. 3.19 Packing diagram of 4-{N-[N-(2-fluorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4d), showing the arrangement of molecules in the crystal lattice, symmetry independent molecules are colour coded.


71

Fig. 3.20 ORTEP diagram of 4-{N-[N-(4-fluorobenzoyl)thiocarbomyl] amino}-5-isopropyl-2-methylphenol (4e), showing the arrangement of molecules in the crystal lattice at 30% ellipsoidal probability (only one of the two symmetry independent molecules is shown) Hydrogen atoms are omitted for clarity.

Fig. 3.21 Packing diagram of 4-{N-[N-(4-fluorobenzoyl)thiocarbomyl]amino}-5-isopropyl-2-methylphenol (4e), showing the arrangement of molecules in the crystal lattice, viewed down the b-axis.


72

Fig. 3.22 ORTEP diagram of 4-{N-[N-(2,6--difluorobenzoyl) thiocarbomyl] amino}-5-isopropyl-2-methylphenol (4f), showing the arrangement of molecules in the crystal lattice at 50% ellipsoidal probability, hydrogen atoms are omitted for clarity.

Fig. 3.23 Packing diagram of 4-{N-[N-(2,6-difluorobenzoyl)thiocarbomyl]amino}

-2-isopropyl-5-methylphenol (4f), showing the arrangement of molecules in the crystal lattice.


73

3.3.6 BIOLOGICAL ASSAY

3.3.6.1 Insect Growth Regulator activity of benzoyl carvacryl thiourea (4a-f) and

urea (5a-f) derivatives against Spodoptera litura

Insects:

Spodoptera litura (Lepidoptera: Noctuidae) was reared for successive

generations on the leaves of its natural host, Ricinus communis (castor), and on an

artificial diet28 at 26°C±2°C and 70%±5% relative humidity and with 18 hrs/6 hrs

photo and scotophase at the Division of Entomology, Indian Agricultural Research

Institute, New Delhi, India.29-31

Bioassay

The growth inhibition effect of various urea and thiourea derivatives as new

IGRs litura was studied on Spodoptera by incorporating them in artificial diet. The

use of an artificial diet allows easy testing of small quantities of synthetic compounds

to oral exposure under controlled conditions. In addition, this technique is simple,

fast, and inexpensive, and is especially suitable for short-term studies involving the

effects of toxins on the test insects. It may also be used to study the effects of growth

factors, hormones and special nutrients on growth by feeding habits of the insects.

The overall concept was to make the bioassay miniature, easy-to handle and

standardized. Using this technique the potency of a selection of novel biorational

insecticides was evaluated.

The Insect Growth Regulator Activity of the title compounds (4a-f), (5a-f),

were evaluated against the Spodoptera litura (larvae, Pupae, adults) and compared

with the parent compound carvacrol and standard compound Noveluron. The

larvicidal activity was tested against the Spodoptera litura by feeding third instar

larva on artificial diet. Synthesized derivatives were dissolved in acetone and were

homogeneously mixed in mixer with the diet.32 For the IGR activity, individual larva

was placed on artificial diet containing 10, 100, 300, 500, 1000 and 5000 ppm

concentration of each derivatives inside the small cage. Controls were treated with the

solvent alone. Every day diet was changed to provide fresh food for the larval stage.

Ten sets of six concentrations per compound were used in the experiment. After

treatment, larvae were fed with this prepared diet and observations were recorded on

different parameters such as larval mortality, larval-pupal intermediates, pupal


74

mortality, pupal-adult intermediates, abnormal adults and normal adults. Larval

weights were recorded before treatment and after 3rd day (3DAT) and 7th day of

treatment and per cent weight reduction were compared with control and standard

compound Noveluron. After larval treatment, experimental observations were

continued for pupal weight reduction, larval-pupal intermediate, Pupal-adult

intermediates, normal and abnormal adults. Percent weight reduction in larvae/pupae

was calculated as follows.

Formula:

(Weight gain in control - Weight gain in treatment) Weight Reduction = -------------------------------------------------------------- X 100

Weight gain in control

The growth inhibitory effects of the synthesized compounds were expressed as

GI50 values (Growth inhibition for 50% population; ppm) for synthesized compounds

tested in-vitro against larvae of S. litura by feeding method. Evaluations are based on

a percentage scale of 0-100 and the activity results are summarized in Table 3.8-3.12.

The overall results revealed that all the 12 derivatives exhibited better results than the

starting compound, Carvacrol. The activity trend was found to be almost consistent

as far as the larval growth inhibition (3rd DAT & 7th DAT), pupal growth inhibition

and normal adult emergence (I50) was concerned.

3.3.6.1.1. Results and discussion

Effect of (4a-f) and (5a-f) on the development of larvae to adult of S. litura is

given in (Table 3.8 - 3.12) and represented in diagrams (Figure 3.24-3.26) as well as

photographs (Figure 3.27-3.28). Results demonstrated that larval and pupal weight

reduction were dose dependent. Highest larval growth inhibition on 3DAT (Table

3.8) and 7DAT (Table 3.9, Figure 3.28, 3.29) was observed for 5f derivative with

GI50 101 and 31 ppm respectively and lowest larval growth inhibition was observed

for 4a derivative with GI50 408 and 171 ppm respectively. Larval growth inhibition

for carvacrol on 3DAT and 7DAT was observed with GI50 4214 & 4416 ppm

respectively while for standard compound Noveluron with GI50 110 & 57 ppm

respectively.

Highest pupal growth inhibition observed for 5f derivative was with GI50 105

ppm and lowest for 4a derivative with GI50 896 ppm (Table 3.10, Figure 3.29). In


75

case of adult inhibition highest activity was exhibited by 5f derivative with I50 20 ppm

which is better than the standard compound Noveluron with I50 73 ppm. On the other

hand lowest adult inhibition was observed for 4a derivative with I50 299 ppm which is

still better than parent compound Carvacrol with I50 8222 (Table 3.11).

As far as total larval mortality (Table 3.12 and Figure 3.24, 3.27) is

concerned BCTU (4a-f) and BCU (5a-f) were found to have less mortality than

carvacrol and Noveluron. Depending on concentration highest total larval mortality

on 7th day after treatment was found to be 30% for 4c at 1000 and 5000 ppm and 30%

for 5f at 300 ppm concentration. Derivatives 4d, 4e, 4f, 5d, 5e and 5f showed 10%

mortality for lowest concentration 10 ppm. Carvacrol demonstrated 60% larval

mortality at 5000 ppm and 10% at 300 ppm concentration. Noveluron was fond to be

most effective on the basis of total larval mortality; lowest concentration 10 ppm

showed 30% total larval mortality and highest concentration 5000 ppm showed 70%.

Results on larval-pupal (Figure 3.24) and pupal-adult (Figure 3.25, 3.28)

intermediates (Table 3.12) exhibited more potency for BCTU (4a-f) and BCU (5a-f)

than carvacrol and standard compound Noveluron. Derivatives 4d, 4f, 5d, 5e and 5f

showed 20% larval pupal intermediates at the lowest concentration 10 ppm while 50%

larval-pupal intermediates were observed at highest concentration 5000 ppm for 4f

and 5f. Similarly, at lowest concentration 10 ppm 4e, 4f, 5d, 5e, and 5f showed 10%

pupal-adult intermediates and at highest concentration 5000 ppm 4d, 4e, 5b, 5e and 5f

displayed 30% pupal-adult intermediates (Figure 3.29). Carvacrol and Noveluron

were found to be far less effective and showed 10% pupal-adult intermediates at 1000

ppm concentration.

The scrutiny of overall results of IGR activity exhibited that the synthesized

derivatives have better growth inhibitory activity than mortality in comparison with

both standard compound Noveluron and parent compound Carvacrol. The compounds

showing more mortality are toxic to all the organisms whereas the ones exhibiting

more intermediate stages are having potency to imbalance the hormones required for

completion of life cycle. In Case of Growth inhibition activity is more selective than

mortality, and hence our compounds can be more effective in Integrated Pest

Management (IPM). Based on results growth inhibitory activity of S. Litura

following structural activity relationship can be established in case of BCTU (4a-f)

and BCU (5a-f) derivatives.


76

Structural activity relationship

� Derivatisation of Carvacrol into thiourea BCTU (4a-f) and urea BCU (5a-f)

resulted in enhanced IGR activity (larval growth inhibition, pupal growth

inhibition, and adult inhibition) but reduced mortality (Larvicidal activity)

� Carvacrol and Noveluron showed higher larvicidal activity than BCTU (4a-f)

and BCU (5a-f) derivatives. Although Carvacrol exhibits

� Substitution by halo substituent’s on phenyl ring enhances the activity.

� Substitution at ortho position of phenyl ring shows better IGR activity than para

position.

� Fluro substitution demonstrates more IGR activity than chloro substitution

� Urea derivative are more active than thiourea derivatives

� 2,6-di-F-derivative from both the series, urea and thiourea illustrated highest

IGR activity.


Table 3.8: Larval Growth Inhibition (GI50) at 3DAT by (4a-f and 5a-f) derivatives, Carvacrol and Noveluron against 3rd instar larvae of

Spodoptera litura

Derivative Heterogeneity

Regression equation b±SE GI50 (ppm) Fiducial limit

df χχχχ2 Min Max

4a 4 1.9455 5.7409 + 0.5334 x 0.5334 ± 0.0785 408 0.0226 0.0768

4b 4 3.0609 5.7627 + 0.5006 x 0.5006 ± 0.0777 300 0.0169 0.0532

4c 4 1.2407 5.7994 + 0.5376 x 0.5376 ± 0.0782 326 0.0188 0.0565

4d 4 2.9301 6.0410 + 0.6047 x 0.6047 ± 0.0786 190 0.0124 0.0291

4e 4 2.7005 5.9383 + 0.5809 x 0.5809 ± 0.0785 243 0.0152 0.0388

4f 4 1.3407 6.0853 + 0.5869 x 0.5869 ± 0.0781 141 0.0093 0.0215

5a 4 4.0523 5.8260 + 0.5210 x 0.5210 ± 0.0778 260 0.0153 0.0442

5b 4 1.6900 5.8839 + 0.5528 x 0.5528 ± 0.0781 252 0.0153 0.0414

5c 4 1.7502 5.7521 + 0.5298 x 0.5298 ± 0.0783 381 0.0212 0.0683

5d 4 3.2849 6.0522 + 0.5959 x 0.5959 ± 0.0784 172 0.0112 0.0262

5e 4 2.6449 5.9941 + 0.5905 x 0.5905 ± 0.0784 207 0.0133 0.0324

5f 4 3.3539 6.3021 + 0.6527 x 0.6527 ± 0.0792 101 0.0070 0.0146

Carvacrol 4 4.7063 5.1403 + 0.3738 x 0.3758 ± 0.0650 4214 0.1226 1.1190

Noveluron 4 5.7248 6.2116 + 0.6184 x 0.6148 ± 0.0786 110 0.0075 0.0162

DAT: Days After Treatment


Table 3.9: Larval Growth Inhibition (GI50) at 7DAT by (4a-f and 5a-f) derivatives, Carvacrol and Noveluron against 3rd instar larvae of

Spodoptera litura


Regression equation b±SE GI50

(ppm) Fiducial limit


4a 4 0.7176 5.7168 + 0.4054 x 0.4054 ± 0.7610 171 0.0092 0.0315

4b 4 0.9818 5.8928 + 0.4609 x 0.4609 ± 0.0766 116 0.0069 0.0193

4c 4 1.3632 5.7771 + 0.3193 x 0.3193 ± 0.0763 159 0.0090 0.0281

4d 4 4.1584 6.0958 + 0.5202 x 0.5202 ± 0.0774 078 0.0050 0.0124

4e 4 2.3714 6.0428 + 0.5221 x 0.5221 ± 0.0773 101 0.0064 0.0159

4f 4 3.7694 6.2988 + 0.5813 x 0.5813 ± 0.0787 058 0.0038 0.0089

5a 4 1.8253 5.9074 + 0.4781 x 0.4781 ± 0.0768 127 0.0077 0.0209

5b 4 1.9456 5.3948 + 0.4504 x 0.4504 ± 0.0766 084 0.0050 0.0142

5c 4 2.4828 5.8524 + 0.4268 x 0.4268 ± 0.0763 101 0.0058 0.0175

5d 4 4.0734 6.4915 + 0.6613 x 0.6613 ± 0.0799 056 0.0038 0.0081

5e 4 0.7818 6.3042 + 0.5997 x 0.5997 ± 0.0787 067 0.0045 0.0100

5f 4 4.3274 6.7829 + 0.7097 x 0.7097 ± 0.0829 031 0.0021 0.0046

Carvacrol 4 5.4680 5.1280 + 0.3605 x 0.3605 ± 0.0650 4416 0.1211 1.6097

Noveluron 4 1.1976 6.4131 + 0.6297 x 0.6297 ± 0.0795 057 0.0038 0.0085



Table 3.10: Pupal Growth Inhibition (GI50) by (4a-f and 5a-f) derivatives, Carvacrol and Noveluron against 3rd instar larvae of Spodoptera

litura

Derivative Heterogeneity Regression equation b±SE GI50

(ppm)

Fiducial limit


4a 4 3.0445 5.3898 + 0.3719 x 0.3719 ± 0.0763 896 0.0303 0.2644

4b 4 2.5247 5.5516 + 0.4169 x 0.4169 ± 0.0766 474 0.0214 0.1046

4c 4 2.2955 5.4496 + 0.3928 x 0.3928 ± 0.7610 717 0.0275 0.1868

4d 4 3.3916 5.8059 + 0.4811 x 0.4811 ± 0.0774 211 0.0123 0.0364

4e 4 3.4021 5.7511 + 0.4757 x 0.4757 ± 0.0773 264 0.0147 0.0473

4f 4 2.7197 5.8771 + 0.4879 x 0.4879 ± 0.0787 159 0.0096 0.0265

5a 4 3.5922 5.4994 + 0.4058 x 0.4058 ± 0.0768 588 0.0246 0.1404

5b 4 2.5903 5.6145 + 0.4381 x 0.4381 ± 0.0766 396 0.0194 0.0807

5c 4 2.5365 5.5747 + 0.4311 x 0.4311 ± 0.0763 465 0.0217 0.0995

5d 4 3.2182 5.9411 + 0.5128 x 0.5128 ± 0.0799 146 0.0091 0.0235

5e 4 3.8667 5.9118 + 0.5153 x 0.5153 ± 0.0787 170 0.0105 0.0277

5f 4 2.9312 6.1587 + 0.5852 x 0.5852 ± 0.0829 105 0.0070 0.0157

Carvacrol 4 5.0514 5.0192 + 0.3655 x 0.3655 ± 0.0631 8862 0.2120 3.7055

Noveluron 4 3.0481 6.0435 + 0.5398 x 0.5398 ± 0.0776 117 0.0075 0.0182



Table 3.11: Adult Inhibition (I50) by (4a-f and 5a-f) derivatives, Carvacrol and Noveluron against Spodoptera litura


Regression equation b±SE GI50

(ppm) Fiducial limit


4a 4 2.4915 6.6936 + 1.1106 x 1.1106 ± 0.0924 299 0.0228 0.0391

4b 4 0.1740 6.9349 + 1.0875 x 1.0875 ± 0.1066 166 0.0129 0.0214

4c 4 5.8137 6.3745 + 0.8131 x 0.8131 ± 0.1018 204 0.0146 0.0284

4d 4 4.5087 6.4475 + 0.7611 x 0.7611 ± 0.0810 125 0.0091 0.0173

4e 4 2.0628 6.1835 + 0.6533 x 0.6533 ± 0.0792 154 0.0105 0.0226

4f 4 5.0182 6.8531 + 0.7716 x 0.7716 ± 0.0835 040 0.0028 0.0056

5a 4 7.2465 6.6332 + 0.9169 x 0.9169 ± 0.0842 165 0.0125 0.0220

5b 4 1.8750 6.8082 + 0.9396 x 0.9396 ± 0. 1104 119 0.0087 0.0164

5c 4 3.7008 6.8524 + 1.0011 x 1.0011 ± 0.1120 141 0.0103 0.0194

5d 4 3.5836 7.2218 + 0.9410 x 0.9410 ± 0.1570 044 0.0033 0.0057

5e 4 1.4406 6.4311 + 0.6423 x 0.6423 ± 0.1043 059 0.0040 0.0089

5f 4 3.8454 7.8723 + 1.0652 x 1.0652 ± 0.1578 020 0.0015 0.0028

Carvacrol 4 5.0684 5.1892 + 0.3558 x 0.3558 ± 0.0631 8222 0.7420 3.7415

Noveluron 4 7.4649 6.9043 + 0.8909 x 0.8909 ± 0.1088 073 0.0054 0.0098



Table 3.12: Effect of derivatives (4a-f and 5a-f), Carvacrol and Noveluron on growth and development of Spodoptera litura

Concentration→→→→

Derivative ↓

Total Larval Mortality (%) Larval-Pupal Intermediates Pupal-Adult Intermediates

10 ppm

100 ppm

300 ppm

500 ppm

1000 ppm

5000 ppm

10 ppm

100 ppm

300 ppm

500 ppm

1000 ppm

5000 ppm

10 ppm

100 ppm

300 ppm

500 ppm

1000 ppm

5000 ppm

4a 00 00 00 00 20 20 00 10 10 10 20 20 00 00 10 10 20 20

4b 00 00 10 10 20 20 00 10 10 20 20 30 00 00 10 20 20 20

4c 00 00 10 10 30 30 00 10 10 20 20 30 00 00 10 10 20 20

4d 10 10 10 10 20 20 20 20 20 20 30 40 00 00 10 10 10 30

4e 10 10 10 10 20 20 10 10 20 20 20 30 10 10 10 20 20 30

4f 10 20 20 20 30 20 20 30 30 30 30 50 10 20 20 20 30 20

5a 00 00 00 10 10 20 00 10 20 20 30 40 00 10 10 00 20 10

5b 00 10 00 20 10 20 10 10 20 20 30 30 00 00 10 20 30 30

5c 00 10 20 10 20 20 10 10 20 20 20 30 00 10 10 10 20 20

5d 10 10 10 10 10 20 20 20 20 30 30 40 10 10 10 20 30 20

5e 10 10 10 10 10 20 20 20 30 30 30 30 10 10 20 20 20 30

5f 10 20 30 20 20 20 20 30 30 30 40 50 10 20 10 30 20 30

Carvacrol 00 00 10 20 40 60 00 00 00 10 20 30 00 00 00 00 10 00

Noveluron 30 50 60 50 60 70 00 10 10 20 20 20 00 10 10 10 10 00


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Fig. 3.24 Graphical representation of effect of benzoyl carvacryl thiourea (4a-f) and urea (5a-f) derivatives showing total larval mortality on 3rd and 7th DAT of Spodoptera litura

Fig. 3.25 Graphical representation of effect of benzoyl carvacryl thiourea (4a-f) and urea (5a-f) derivatives on larval-pupal intermediates of Spodoptera

litura


83

Fig. 3.26 Graphical representation of effect of benzoyl carvacryl thiourea (4a-f) and urea (5a-f) derivatives on pupal-adult intermediates of Spodoptera

litura

Fig. 3.27 Pictorial presentation of results of benzoyl carvacryl thiourea (4a-f) and urea (5a-f) derivatives on larval development of Spodoptera litura


84

Fig. 3.28 Effect of benzoyl carvacryl thiourea (4a-f) and urea (5a-f) derivatives on larval and pupal development showing larval-pupal intermediates of Spodoptera litura

Fig. 3.29 Photographs showing effect of benzoyl carvacryl thiourea (4a-f) and urea (5a-f) derivatives on pupal and adult development showing pupal-adult intermediates of Spodoptera litura.


85

3.3.6.2 Insect Growth Regulator activity of benzoyl carvacryl thiourea (4a-f) and

urea (5a-f) derivatives on Dysdercus koenigii and antifungal susceptibility

All the newly synthesized BCTU (4a-f) and BCU (5a-f) derivatives were

screened for insect growth regulatory (IGR) activity against red cotton bug,

Dysdercus koenigii. Simplicity of rearing in the laboratory and sensitivity to

morphogenic compounds makes D. koenigii, the insect of choice for the investigation.

The test compounds were dissolved in acetone (1 mg/ml). The required volume of the

test solutions were then topically applied with the help of microlitre syringe to the

dorsal abdominal region of same aged 5th instar nymphs. The concentrations of the

compounds tested were 10, 15, 20, 30, 40 and 50 µg/nymph. The treated nymphs were

placed back in the jars after the acetone had evaporated. At least three replicates

(10 insects per replicate) were used for each dose of a compound. A parallel control

group of nymphs treated only with acetone was set up. The bioactivities of test

compounds were determined by its effects on mortality (toxic/insecticidal effect),

moulting and growth (growth inhibiting/regulating activity).33 Based on the %

mortality data, LD50 values (lethal dose µg/nymph) were calculated using statistical

computer program (Indostat Services, Hyderabad).

Table 3.13: Insecticidal activity of BCTU and BCU derivatives against Dysdercus

koenigii

Compound Chi Square

value (χ2)

Regression

equation LD50

Fiducial limit

Min Max

4a 2.72 0.345+3.389x 23.6 21.7 25.7 4b 3.55 3.136+1.675x 13.0 10.9 15.3 4c 1.91 2.458+1.928x 20.8 18.0 24.0 4d 2.73 3.511+1.354x 12.6 10.2 15.5 4e 2.73 3.510+1.354x 12.6 10.2 15.5 4f 7.32 3.347+1.569x 11.3 09.4 13.7 5a 3.33 2.090+2.186x 21.5 18.8 24.4 5b 5.45 2.518+2.046x 16.4 14.3 18.7 5c 3.49 2.497+1.996x 18.0 15.7 20.7 5d 0.75 3.339+1.469x 12.5 10.3 15.1 5e 5.85 2.601+2.045x 14.9 13.0 17.1 5f 1.37 3.898+1.269x 09.5 07.2 12.6

Lufenuron 1.90 3.507+1.566x 09.0 07.5 10.7 Carvacrol 5.88 2.701+1.510x 33.3 26.7 41.4 Acetone At highest concentration mortality was <15%


86

The LD50 of BCTU derivatives for 5th instar nymphs were found to be in the

range 11.3-23.6 µg/nymph, whereas for BCU derivatives the LD50 range was 9.5-21.5

µg/nymph (Table 3.13). All chi-square values were not significant (α=0.05)

indicating good fit of regression lines. The percentage of abnormal adults was found

to be increased with increase in the treatment dose. High doses between 20 to 50

µg/nymph showed deformities such as smaller body size, crumpled wings and

deformed legs (abnormal adults) in some of the emerging adult population. 2,6-

difluoro derivatives (5f) showed the highest activity amongst respective series. Fluoro

substitutes BCTU, BCU compounds exhibited better IGR activity than corresponding

chloro substitutes. For both the series, chloro or floro substitutions at ortho position

conferred better activity than its substitution at para position. The LD50 of carvacrol

was 33.3 µg/nymph, while its incorporation in a BPU had significant effect as evident

from the comparable activity (<13 µg/nymph) of the derivatives 4b, 4d, 4e, 4f and 5d,

5f with the standard BPU lufenuron (9.0 µg/nymph).

3.3.6.3 Antifungal susceptibility of BCTU (4a-f) and BCU (5a-f) derivatives

The purified final compounds were evaluated for antifungal susceptibility

testing by microbroth dilution method according to the recommendations of the

National Committee for Clinical Laboratory standards (NCCLS).34 The antifungal

activity was tested using the plant pathogenic fungal strains Magnporthe grisea,

Fusarium oxysporum, Dreschlera oryzae and food spoilage yeasts Debaromyces

hansenii, Pichia membranifaciens. Rice is the host for M. grisea and D. oryzae,

whereas F. oxysporum infests diverse plants including tomato, tobacco, legumes,

cucurbits, sweet potatoes and banana. D. hansenii and P. Membranifaciens occur on

grapes and are common wine spoilage yeasts. The antifungal activities of the tested

compounds are given in Table 3.14 as Minimum Inhibitory Concentration (MIC)

values. MIC was defined as the lowest concentration exhibiting >90% inhibition of

visible growth compared to growth of the control.

As seen from Table 3.14, BCTU 4c, 4d, 4e, 4f and BCU 5b, 5c showed

antifungal activity against these fungi. BCTU derivatives 4a, 4b and BCU 5a, 5d, 5e,

5f were ineffective in controlling the growth of the phytopathogens and food spoilage

yeasts at the highest concentration tested. Secondly, the thiourea derivatives were

more effective than urea derivatives. The results of 4c, 5c indicated that presence of

chloride group at para position enhanced the antifungal activity. No in vitro antifungal


87

activity was observed for lufenuron, which is in agreement with previous reports.6,7

As expected, carvacrol showed good antifungal activity against all the tested

organisms. The hydroxyl group of Carvacrol has been shown to have a special role in

the antimicrobial action of Carvacrol.35

Table 3.14: Antifungal Susceptibility Testing of BCTU and BCU derivatives against

phytopathogenic fungi

Derivatives Minimum Inhibitory Concentration (MIC in µg/ml)

Phytopathogenic fungi Food spoilage yeasts

M. grisea F. oxysporum D. oryzae D. hansenii P. membranifaciens

4a >512 >512 >512 >512 512 4b >512 >512 >512 >512 512 4c 256 128 >512 256 512 4d 256 128 >512 256 >512 4e 256 128 256 256 512 4f >512 >512 256 128 >512 5a >512 >512 >512 >512 >512 5b >512 512 256 128 256 5c 512 256 128 128 256 5d >512 >512 >512 >512 >512 5e >512 >512 >512 >512 >512 5f >512 >512 >512 >512 >512

Lufenuron >512 >512 >512 >512 >512 Carvacrol 128 64 64 128 128

There are many reports on the use of carvacrol for the control of human fungal

pathogens such as Candida albicans, Aspergillus niger, Microsporum canis etc.13,36

Carvacrol is a US Food and Drug Administration approved safe food additive, and

used as a flavouring agent in different foods.37 Though initially developed as

insecticides, benzoylphenyl urea compounds were reported to possess potent

antitumor activity and are in clinical development for cancer treatment.38,39 Few

reports have indicated the potential of Lufenuron for the control of fungal pathogens

in animals.40,41 Therefore, the BCTU and BCU derivatives were checked for

antifungal activity against different strains of human pathogens Candia albicans,

Candida glabrata and Cryptococcus neoformans (Table 3.15). All the BCTU

derivatives showed potent antifungal activity against these human pathogens. From

BCU series, 5d, 5e and 5f were most effective, whereas compounds 5a and 5b

showed weak or no antifungal activity against the tested strains. For most of the

compounds the activity was better than carvacrol against human pathogens.


Table 3.15: Antifungal Susceptibility Testing of BCTU and BCU derivatives against human fungal pathogens

Compound

Growth Inhibitory concentration in µg/ml

C. albicans

NCIM 3557 C.

albicans C. albicans

NCIM 3471 C. glabrata NCIM 3237

C.

neoformans C. neoformans

NCIM 3541 C. neoformans

NCIM 3542

C. neoformans

NCIM 3378

4a 128 32 >512 32 64 16 32 128

4b 64 64 >512 64 128 32 64 >512

4c 64 16 32 32 64 8 32 32

ad 128 128 >512 128 32 64 128 512

4e 64 64 >512 64 16 32 64 128

4f 256 64 >512 64 512 32 64 128

5a 512 >512 >512 >512 >512 >512 256 >512

5b 128 256 >512 >512 256 256 128 128

5c 64 32 >512 32 64 32 32 32

5d >512 16 32 16 >512 <4 16 16

5e >512 16 32 16 >512 <4 16 16

5f >512 16 32 16 >512 <4 16 16

Lufenuron >512 >512 >512 >512 >512 >512 >512 >512

Carvacrol 128 128 256 128 128 128 128 128


89

3.4 Cellular Toxicity Assay

As stated earlier, carvacrol affects the cell membranes and results in depletion

of sterols. Therefore, major concern of employing these newly synthesized

compounds as crop protection or antifungal agents is their potential toxicity to

mammalian cells (Sheep Red Blood Cells). Hence, cellular toxicity of the compounds

was checked by haemolysis assay as described by Sajjad et al.42 The concentrations

tested were in the range of 4-1000 µg/ml. The concentration causing 50% haemolysis

(HC50) for all the BCTU, BCU compounds and Lufenuron was >1000 µg/ml.

Maximum haemolysis observed was 17% for compound 4e at 1000 µg/ml

concentration. At MIC concentrations for all the derivatives, the haemolysis was

negligible (<2%). The HC50 values for carvacrol and a similarly acting antifungal

drug Amphoterecin B were 250 and 8 µg/ml, respectively (Table 3.16). The

antifungal activity and haemolysis results indicated that the synthesized compounds

are better and safer than BPU’s and Carvacrol.

Table 3.16: Cellular toxicity testing of BCTU and BCU derivatives on mammalian cells (Sheep Red Blood Cells)

Conc →→→→ Derivative↓

% haemolysis 125 mg /ml 250 mg /ml 500 mg /ml 1000 mg /ml

4a 0.19 7.56 12.04 14.88 4b 0.20 5.55 10.59 13.52 4c 0.19 8.22 12.76 15.80 4d 5.22 7.85 13.70 17.58 4e 2.70 8.54 14.44 17.92 4f 2.71 5.32 09.66 15.74 5a 7.51 7.78 10.40 17.51 5b 7.89 6.22 11.18 16.55 5c 6.43 8.25 10.79 17.04 5d 7.26 7.56 09.66 14.97 5e 6.20 7.85 10.65 15.22 5f 6.34 7.12 09.56 15.39

Lufenuron 6.75 8.32 09.90 16.13 Carvacrol 9.67 43.07 93.25 100.0

Conc →→→→

Amphoterecin B

% haemolysis 128 mg /ml 64 mg /ml 32 mg /ml 16 mg 8 mg /ml

100 97.44 96.98 94.51 91.94


90

3.5 CONCLUSIONS

The present study carried out to evaluate the impacts of twelve insect growth

regulators benzoyl phenyl thiourea (4a-f) and urea (5a-f) groups in a parent

compound have exhibited significant effect on IGR activity of Spodoptera litura and

D. koenigii. BCTU (4a-f) and BCU (5a-f) compounds used in a management of

Spodoptera litura elicit their primary action on insect metabolism, ultimately

interfering and disrupting the process of growth, development and metamorphosis of

the target insects, particularly when applied during larval stage of the development.

The pattern of results is comparable with the standard IGR Noveluron. Therefore, on

similar grounds to Noveluron, it can be said that Spodoptera litura and D. koenigii

might be working as per the mechanism of benzoyl phenyl urea (Chitin syntheses

inhibitors). Presence of halo substituent on benzoyl group particularly at ortho

position was found to be more effective than para position against both the test

species. In both the series 4-{N-[N-(2,6-difluorobenzoyl)oxocarbomyl]amino}-5-

isopropyl-2-methylphenol (5f) compound has the highest activity and thus can

contribute to the improvement of the techniques for the control of this pest by using

compounds of natural origin.

Significant formation deformities like abnormal adults, larval-pupal

intermediates and pupal-adult intermediates were observed with any of the derivative

at different concentrations with synthetic analogs (agonists or antagonists) would

result in the disruption or abnormal growth and development of the target insect.

Similarly, any interference in the various hormone-dependent steps involved in the

synthesis and/or resorption of the cuticle would be detrimental to the survival of the

affected developmental stage. The disruptive effects of benzoyl phenyl ureas on

cuticle are produced differently from those produced by benzoyl phenyl ureas, which

specifically inhibit chitin synthesis.

The mode of action of the title compounds (4a–f) and (5a-f) is very

interesting. Toxicity assays indicated that at higher concentrations, the title

compounds could kill the larval stage of Spodoptera litura but less than the parent

compound and standard compound, from these results it is clear that our synthesized

compounds are less toxic to larval stage than the parent compound and Noveluron.

Results from these studies clearly confirm the improved potential insect-control

properties of the BCTU (4a-f) and BCU (5a-f), as observable from the reduction in


91

growth rate, formation of larva-pupal intermediates, larval and pupal mortality and the

adult malformation than parent compound Carvacrol and comparable with standard

compound Noveluron. From this discussion it is clear that the involvement of natural

moiety in benzoyl phenyl urea has resulted in the safer and more active insect growth

regulator.

In conclusion, two series of BCTU (4a-f) and BCU (5a-f) derivatives were

synthesized by structurally modifying carvacrol and introducing benzoylphenyl urea

linkage. Derivatives 4b, 4d, 4e, 4f and 5d, 5f showed comparable Insect growth

regulator activity with the standard BPU noveluron against Spodoptera litura and

Lufenuron against D. koenigii. Most of the compounds demonstrated potent

antifungal activity against human pathogens and potent to moderate activity against

different phytopathogens and food spoilage yeasts. All the compounds were non-

haemolytic. The synthesized compounds have a potential application in agriculture as

safer and broad spectrum crop protection agents. After comprehensive evaluation,

they may also be used for the control of fungal pathogens in veterinary and human

healthcare.

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