Synthesis and characterization of N-acylaniline derivatives as potential chemical hybridizing agents (CHAs) for wheat (Triticum aestivum L.)

Article abstract of DOI:10.1021/jf061390x

Induction of male sterility by deployment of chemical hybridizing agents (CHAs) are important in heterosis breeding of self-pollinated crops like wheat, wherein the male and female organs are in the same flower. Taking a lead from the earlier work on rice, a total of 25 N-acylanilines comprising of malonanilates, acetoacetanilides, and acetanilides (including halogenated acetanilides) were synthesized and screened as CHAs on three genotypes of wheat, viz., PBW 343, HD 2046, and HD 2733 at 1500 ppm in the winter of 2001-2002. The N-acylanilines containing variations at the acyl and aromatic domain were synthesized by condensation of substituted anilines with appropriate diesters, acid chlorides, or monoesters. The test compounds with highly electronegative groups such as F/Br at the para position of the aryl ring were identified as the most potent CHAs, causing higher induction of male sterility. A variation of N-substitution at the side chain generally furnished analogues like 4′-fluoroacetoacetanilide (7) and ethyl 4′-fluoromalonanilate (1), which induced 89.12 and 84.66% male sterility, respectively, in PBW 343. Among halogenated acetanilides, the increasing number of chlorine atoms in the side chain led to an increase in the activity of 4′-fluoro (23) and 4′-bromo (24) derivatives of trichoroacetanilides, which induced >87% male sterility. Quantitative structure-activity relationship (QSAR) models indicated the positive contributions of the field effect exemplified by the Swain-Lupton constant (Fp) and negative contributions of the Swain-Lupton resonance constant (R) for the aromatic substitution. The positive influences of parachor (P) for the acyl domain have been underlined. These leads will be significant in explaining the CHA fit in the macromolecular receptor site. The CHAs appeared to act by causing an imbalance in the acid-base equilibrium in pollen mother cells resulting in dissolution of the callose wall by premature callase secretion.

Full text of DOI:10.1021/jf061390x

6800 J. Agric. Food Chem. 2006, 54, 6800−6808
Synthesis and Characterization of N-Acylaniline Derivatives as
Potential Chemical Hybridizing Agents (CHAs) for Wheat
(Triticum aestivum L.)
KAJAL CHAKRABORTY*,† AND C. DEVAKUMAR
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110012, India
Induction of male sterility by deployment of chemical hybridizing agents (CHAs) are important in
heterosis breeding of self-pollinated crops like wheat, wherein the male and female organs are in the
same flower. Taking a lead from the earlier work on rice, a total of 25 N-acylanilines comprising of
malonanilates, acetoacetanilides, and acetanilides (including halogenated acetanilides) were syn-
thesized and screened as CHAs on three genotypes of wheat, viz., PBW 343, HD 2046, and HD
2733 at 1500 ppm in the winter of 2001-2002. The N-acylanilines containing variations at the acyl
and aromatic domain were synthesized by condensation of substituted anilines with appropriate
diesters, acid chlorides, or monoesters. The test compounds with highly electronegative groups such
as F/Br at the para position of the aryl ring were identified as the most potent CHAs, causing higher
induction of male sterility. A variation of N-substitution at the side chain generally furnished analogues
like 4′-fluoroacetoacetanilide (7) and ethyl 4′-fluoromalonanilate (1), which induced 89.12 and 84.66%
male sterility, respectively, in PBW 343. Among halogenated acetanilides, the increasing number of
chlorine atoms in the side chain led to an increase in the activity of 4′-fluoro (23) and 4′-bromo (24)
derivatives of trichoroacetanilides, which induced >87% male sterility. Quantitative structure-activity
relationship (QSAR) models indicated the positive contributions of the field effect exemplified by the
Swain-Lupton constant (Fp) and negative contributions of the Swain-Lupton resonance constant
(R) for the aromatic substitution. The positive influences of parachor (P) for the acyl domain have
been underlined. These leads will be significant in explaining the CHA fit in the macromolecular
receptor site. The CHAs appeared to act by causing an imbalance in the acid-base equilibrium in
pollen mother cells resulting in dissolution of the callose wall by premature callase secretion.
KEYWORDS: Chemical hybridizing agents; N-acylanilines; QSAR; malonanilates; anilides; wheat
INTRODUCTION
any pollinator, which is heterotic in combination with the female
parent, irrespective of the presence of the fertility restorer gene
(Rf), can be used for developing a hybrid. Thus, it broadens
the choice of combinations that can be used for producing
commercial hybrids. Because the system does not use sterile
cytoplasm, the negative effect of the male sterile cytoplasm is
also absent. In addition, the long and cumbersome method of
developing a male sterile line can be overcome. The risks of a
narrow genetic base for cytoplasmic-genetic male sterility
(CGMS) being experienced in a three-line hybrid breeding
program would cease to be a problem in this system. The hybrids
based on a particular CGMS source are essentially half-sibs
(have 50% of the genome in common) and therefore are
generally more uniform, while in the case of CHA-based
hybrids, one has the freedom to diversify hybrid combinations.
CHAs would be preferred over CGMS because the former saves
time and labor needed for transferring male sterility and
sufficiently large quantity of seeds can be produced. An easy,
economical, effective, and environment-friendly CHA technol-
ogy would permit facile production of hybrid seeds (2, 3). Under
In the self-pollinated (hermaphrodite) crops like wheat,
selective induction of male sterility by deployment of chemical
hybridizing agents (CHAs) facilitating a “two-line” approach
holds immense potential in heterosis breeding. The CHAs
facilitate cross-breeding in plant species with perfect flowers
by selectively sterilizing male sex cells or by interrupting
microsporogenesis to prevent self-pollination and promote
fertilization by an outside pollen source (1). When CHAs are
used to induce male sterility, the maintainer line is not required,
because the sterility is induced only when the chemical is
sprayed on the plant to make its male sterile for hybrid seed
production. When the chemical is not sprayed, the line is fully
fertile and its seed can be multiplied by selfing. In this system,
* To whom correspondence should be addressed. Telephone: 91-484-
2394867, 91-484-4069936, and 009447084867. Fax: 0091-0484-2394909.
E-mail: kajal_iari@yahoo.com and kajal_cmfri@rediffmail.com.
† Current address: Division PNP, Central Marine Fisheries Research
Institute, Ernakulam North, Post Office Box 1603, Cochin 682018, Kerala,
India.
10.1021/jf061390x CCC: $33.50
© 2006 American Chemical Society
Published on Web 08/04/2006

N-Acylanilines as CHAs for Wheat
J. Agric. Food Chem., Vol. 54, No. 18, 2006 6801
used as such or purified and dried by standard means. Organic solutions
were dried over Na2SO4/MgSO4, evaporated on a rotatory evaporator,
and were under reduced pressure. Anhydrous reactions were performed
under an inert atmosphere, with the setup assembled and cooled under
dry N2. Samples were evaporated under N2 and redissolved in CHCl3
at 50 µg/mL for analyses by GC/MS.
situations where male sterility induction is not 100% or female
fertility is slightly reduced, CHA could still enable a breeder to
produce a sufficient quantity of hybrid seed to test the value of
the hybrid population on a limited scale. The search for CHAs
in the 1950s and 1960s was rather random and from the
chemicals already used in agriculture such as plant-growth
regulators and herbicides (2). Of the many chemicals investi-
gated since the 1950s, ethephon was prominent in earlier work,
with a series of products from Rohm and Hass and Shell
dominating the field in the mid-1980s. A number of CHAs have
been reported for cereals viz, rice and wheat. The azetidine
group of CHAs rendered wheat male sterile (4). The fenridazon
group of CHAs induced 95-100% male sterility in various
wheat genotypes at application rates of 0.2-2.0 kg/ha (5). Ethrel
is an effective CHA for wheat (6, 7), but it induced high female
sterility at application rates required for male sterility. Pyridine
monocarboxylates were reported as potential CHAs for wheat
(8). Prominent among other CHAs used in wheat includes MH,
dalapon (9), DPX 3778 (10), LY 195259 (11), RH-531, RH-
532, RH-2956, RH-4667, and SC-1058 (12, 13).
The development of an agrophore model can serve as a
powerful tool in discovering new leads based on existing active
chemistry. The agrophore strategy involves identifying critical
structural elements responsible for activity via a hypothetical
mode of action. There has been very little progress in the mode
of action of CHAs. This situation makes CHA-agrophore
development difficult, but at the same time, it provides an
opportunity to build a discovery program focused on developing
new CHAs and on elucidating the CHA mode of action. It is
therefore necessary to explore molecular information on the
possible site and mode of action of CHAs. Quantitative
structure-activity relationship (QSAR) analysis is a powerful
tool in elucidating essential structural features that govern the
interaction of CHAs with the macromolecular receptor in the
crop plants controlling pollen formation and its viability (14-
16).
In a program of design and development of CHAs for crop
plants, we have reported earlier the deployment of ethyl
oxanilates in rice (17), wheat (18-21), and chickpea (22). It
was therefore of interest to investigate a broader group of
potential CHAs based on the leads and predictions from the
earlier studies. In the present study, a total of 25 N-acylanilines
having different aromatic substitutions and acyl side-chain
variation were synthesized, characterized by different spectro-
scopic techniques [1H nuclear magnetic resonance (NMR) and
electronic impact gas chromatography/mass spectroscopy (EI-
GC/MS)], and screened as CHAs on three genotypes of wheat,
viz., PBW 343, HW 2046, and HD 2733 in the winter of 2001-
2002. The current QSAR modeling focuses on discerning
whether agrophores represented by N-acylanilines are active via
related mechanisms. With this view in mind, we tried to get an
insight into the structural features governing the activity and
possible mode of action of the N-acylanilines as CHAs.
Instrumentation. The structures of synthesized compounds were
1
confirmed by infrared (IR) spectroscopy, H NMR, and MS. Melting
points (mp) were determined by using a sulfuric acid bath and were
uncorrected. 1H NMR spectra were recorded on a Varian EM-360, 60
MHz NMR apparatus. Samples were dissolved in deuterochloroform
(CDCl3) or deuterodimethylsulfoxide (DMSO-d6) for data acquisition
using tetramethylsilane as an internal standard (Me4Si, 0.0 ppm for 1H
NMR). Thin-layer chromatography (TLC) was performed on 250 µm
(60 Å) silica gel G plates, preactivated at 100 °C for 2 h and using
hexane/ethyl acetate (4:1) as a developing medium. All reactions were
monitored by UV fluorescence or staining with iodine. GC data were
recorded on a Hewlett-Packard Series-II gas chromatograph equipped
with an OV-1 (nonpolar) megabore column (10 m × 0.53 mm i.d.,
0.25 µm film thickness) using a flame ionization detector (FID). The
samples were injected (1 µL) into the GC using an initial column
temperature of 80 °C, followed by 9.9 °C/min ramp to 250 °C. Ultrahigh
purity N2 (IOLAR grade-I, 99.999% purity) was used as the carrier
gas at a flow rate of 30 mL/min. The injector temperature was
maintained at 250 °C. The detector was isothermal at 290 °C. Peak
retention times and areas were calculated by the integration of areas
under the peaks. The qualitative GC-MS analyses were performed
using EI ionization mode using a FISONS TRIO 1000 (HRGC Mega-2
coupled with an EI-mass detector). The GC apparatus was equipped
with a capillary column of intermediate polarity (HP-1; 30 m × 0.32
mm i.d., 0.39 mm o.d., and 0.25 µm film thickness). The carrier gas
was ultrahigh purity He (IOLAR grade-I, 99.999% purity) with a
constant flow rate of 2 mL/min. The injector and detector temperature
were maintained at 300 °C. The injection volume was 1 µL. Samples
were injected in splitless mode at 290 °C into the capillary column
similar to that used for the GC analyses, and the oven was identically
programmed. The ion source and transfer line were kept at 300 °C.
Electron ionization was produced by accelerating electrons from a hot
filament through a potential difference at the standard value of 70 eV.
All test compounds gave correct elemental analyses using Euro Vector
elemental analyzer (model number EA3011).
General Methods of Syntheses and Purification of N-Acylanilines.
A total of 25 N-acylanilines having variation at the acyl and aromatic
domain were synthesized by condensation of substituted anilines (0.025
mol) with appropriate diesters or acid chlorides or monoesters (0.03
mol) as the case might be (Table 1). A large variation in the
substitutions in both aryl and acyl side chains has been achieved to
synthesize malonanilates, acetoacetanilides, and acetanilides (including
halogenated acetanilides) (Figure 1). The duration of reaction, yield,
and physicochemical characteristics of the products thus prepared are
listed in Table 1. The 1H NMR and mass spectral data of the test CHAs
are listed in Table 2.
Malonanilates (1-6). In this series, five analogues having different
aromatic substitutions (4-F, 4-Br, 2-OMe, 3-OMe, and 2-NO2) and one
unsubstituted malonanilate were synthesized. To a solution of aniline/
substuted aniline (0.025 mol) in toluene/xylene was added diethyl ester
of malonate (0.03 mol), and the reaction mixture was refluxed for 2-4
h over an oil bath. Ethanol was collected as an azeotrope. The reaction
was followed by TLC [hexane/ethyl acetate (4:1) as a developing
medium] until completion. The resultant solid product was cooled to
90 °C to let the product solidify, triturated with boiling ethanol, and
refrigerated to allow for recrystallization of the title compounds. For
example, ethyl 4′-bromomalonanilate (2) was obtained as white
crystalline solids by thermal condensation between 4-bromoaniline
(0.025 mol) and diethyl malonate (0.03 mol). 2-Anisidine (0.025 mol)
and 3-anisidine (0.025 mol) in toluene (10 mL) were used as the starting
anilines to furnish ethyl 2′-methoxymalonanilate (4) and ethyl 3′-
methoxymalonanilate (5), respectively. Other analogues in this series
[ethyl 4′-fluoromalonanilate (1) and ethyl 2′-nitromalonanilate (6)] were
synthesized in a similar way. TLC and GC monitored the course of
MATERIALS AND METHODS
Chemicals and Reagents. Analar grade substituted anilines, diethyl
malonate, and ethyl acetoacetate was procured from Aldrich Chemical
Co., Inc. 2-Chloroacetyl chloride, 2,2-dichloroacetyl chloride, and 2,2,2-
trichloroacetyl chloride were procured from E-Merck (Darmstadt,
Germany). All solvents used for the sample preparation were of
analytical grade, and the solvents used for MS analyses were of liquid
chromatography (LC) grade from E-Merck. Double-distilled water was
used throughout this work, while all reagents used were of analytical
grade and purchased from E-Merck. Unless otherwise stated, starting
material, reactants, and solvents were obtained commercially and were

6802 J. Agric. Food Chem., Vol. 54, No. 18, 2006
Table 1. Physicochemical Parameters of N-Acylanilines
Chakraborty and Devakumar
Similarly, 3-nitroaniline and ethyl acetoacetate were thermally con-
densed using xylene as the solvent on an oil bath for 3 h to furnish
deep yellow crystals of 3′-nitroacetoacetanilide (13). The reaction of
4-chloroaniline (0.025 mol) and ethyl acetoacetate (0.03 mol) in dry
toluene following Figure 1 furnished white crystals of 4′-chloroac-
etoacetanilide (12), which was further purified by column chromatog-
raphy (using silica gel as an adsorbent), using 10% acetone in benzene
as an eluant. Other chloro-substituted derivatives of acetoacetanilide
[2′-chloroacetoacetanilide (10) and 3′-chloroacetoacetanilide (11)] were
synthesized in a similar way. The physicochemical characteristics and
spectroscopic data of the compounds are listed in Tables 1 and 2,
respectively.
Acetanilides (Including Halogenated Acetanilides) (15-25). In this
series, four series of analogues, viz., 4-fluoroacetanilide (15), chloro-
acetanilides (16-18), dichloroacetanilides (19-22), and trichloroac-
etanilides (23-25), comprising a total of 11 compounds were synthe-
sized. A solution of substituted aniline (0.025 mol) in dry pyridine (0.05
mol, 3.96 mL) was added over a period of 30 min to a solution of acid
chlorides (2-chloroacetyl chloride, 2,2-dichloroacetyl chloride, and
2,2,2-trichloroacetyl chloride) (0.03 mol) in dry toluene kept at e10
°C with continuous stirring under an inert atmosphere of nitrogen. The
reaction mixture was kept under stirring until the product was formed
as a solid. Toluene was removed under vacuum, and the residue
obtained was recrystallized in diethyl ether/hexane to obtain acetanilides
(including halogenated derivatives), which were homogeneous by TLC
(Figure 1). For example, 4-bromochloroacetanilide (17) was synthesized
by the reaction between 4-bromoaniline (0.025 mol) and 2-chloroacetyl
chloride (0.03 mol) under stirring at e10 °C. Similarly, in dichloro-
acetanilide and trichloroacetanilide series, the analogues having different
aromatic substitution [4-F, 4-Br, and 2,4-(NO2)2] were synthesized
following the general procedure. The physicochemical characteristics
and spectroscopic data of the compounds are listed in Tables 1 and 2,
respectively.
compd
no.
melting point
C)
Rt
(min)
X
R
(
°
Rf
1
2
3
4
5
6
7
8
4-F
4-Br
H
2-OMe
3-OMe
2-NO2
4-F
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
CH2COOEt
CH2COOEt
CH2COOEt
CH2COOEt
CH2COOEt
CH2COOEt
CH2COCH3
CH2COCH3
CH2COCH3
CH2COCH3
CH2COCH3
CH2COCH3
CH2COCH3
CH2COCH3
CH3
CH2Cl
CH2Cl
CH2Cl
CHCl2
CHCl2
CHCl2
CHCl2
CCl3
72
92
65
−
−
−
103
109
89
73
93
67
6.12
7.80
9.37
8.56
8.57
0.58
0.40
0.41
0.63
0.67
0.78
0.58
0.59
0.63
0.50
110
128
6.11
10.13
9.85
4-Br
H
9
86−88
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2-Cl
3-Cl
4-Cl
3-NO2
3-CH3
4-F
4-F
4-Br
H
4-F
4-Br
H
2,4-(NO2)2
4-F
4-Br
H
112
106
107
137
58
152
6.70
8.25
7.19
8.28
9.03
9.02
9.17
6.99
9.09
10.30
11.27
0.47
0.62
135−
56−
0.62
0.49
0.51
0.59
0.32
0.43
0.53
0.27
0.29
0.31
0.43
109-112
136 137
92 94
120
−
−
119
142
−
−
144
102
160
128
175
110
158−
13.42
13.59
12.82
CCl3
CCl3
172
109
−
−
Biological Activity. Three high-yielding common wheat (Triticum
aestiVum L.) cultivars, viz, PBW 343, HW 2046, and HD 2733,
recommended in the northwestern plain zone of India were chosen for
evaluation of spikelet sterility by the test CHAs in the winter of 2001-
2002. The experiment was laid out in randomized block design (RBD)
in three replicates. All of the details of agronomic practices mentioned
in our earlier papers were followed (18-21). The test chemicals were
sprayed at 1500 ppm as an oil-in-water emulsion containing cyclohex-
anone (1%) as the solvent and polyoxyethylene sorbitan monooleate
(formula weight ∼ 1200) (Tween-80) (0.02%) as the emulsifier at a
premeiotic stage. The spraying was carried out on three replicate plots
of about 2 m lengths containing about 400 tillers, keeping the outermost
two lines as pollinator lines (HW 2045). At the first signs of flower
opening, 10 spikes were cross-pollinated, using the approach method,
with the pollinator parent. A total of 10 spikes of each treated plants
were covered immediately after the emergence. Remaining ear heads
were left uncovered. Anthers from 3-4 florets of the sprayed genotypes
were smeared together on a glass slide over a drop of acetocarmine
(1%) and/or KI-I2 (2%), and examined under a light microscope. Pollen
sterility was calculated as a percentage. A total of 10 each of bagged
and unbagged spikes including one control were harvested at maturity.
As soon as the seeds became plainly visible, the number of seeds per
spikelet was counted in both bagged and crossed spikes. To study the
spikelet sterility, the number of fertile (filled) and sterile (unfilled)
spikelets was counted and percent male sterility was computed as the
percent inhibition of seed set in bagged spikes of treated plants as
compared to that of the untreated control using the formula: percent
male sterility ) (Sc - Sf)/Sc × 100, where Sc ) seeds per spike in
control plants and Sf ) seeds per spike in bagged and treated plants.
QSAR Study. The QSAR method applied to three families of
N-acylanilines in the malonanilate, acetoacetanilide, and acetanilide
(including halogenated acetanilide) classes of chemistry. The following
descriptor variables were used for aromatic substituents, viz., electronic
parameters, Swain-Lupton field constant for para substitution (Fp) (23),
Swain-Lupton resonance constant (ΣR), Taft steric parameter (Es),
Verloop-Hoogenstraaten multidimensional steric parameters, L and B4
(24, 25), descriptor variable (D); hydrophobic parameter π, and other
parameters, such as molar refractivity (MR) (26). To observe the
Figure 1. General synthetic scheme of N-acylanilines (1−25). The reaction
conditions are as described in the Materials and Methods, and the details
of the acyl (R) and aromatic substitution (X) patterns were described in
Table 1.
the reaction, wherever required. The physicochemical characteristics
and spectroscopic data of the compounds are listed in Tables 1 and 2,
respectively.
Acetoacetanilides (7-14). In this series, seven analogues having
different aromatic substitutions (4-F, 4-Br, 2-Cl, 3-Cl, 4-Cl, 3-NO2,
and 3-CH3) and one unsubstituted acetoacetanilide were synthesized.
In a stirred solution of aniline/substituted anilines (0.025 mol) in dry
toluene/xylene (50 mL), ethyl acetoacetate (0.03 mol) was added under
reflux for 2-4 h in a round-bottomed flask fitted with a Dean-Stark
apparatus, over an oil bath. The reaction was followed by TLC [hexane/
ethyl acetate (4:1) as a developing medium] until completion. The
resulting product was cooled to 90 °C to let the products solidify and
dried in a desiccator for a 1 day. Recrystallization of the crude product
with boiling ethanol furnished the title compounds (7-14). For example,
4-bromoaniline and ethyl 3-oxobutanoate were refluxed in toluene for
2 h to furnish a white crystalline solid of 4′-bromoacetoacetanilide (8).

N-Acylanilines as CHAs for Wheat
J. Agric. Food Chem., Vol. 54, No. 18, 2006 6803
Table 2. Spectral Data (1H NMR and EI
−MS) of N-Acylanilines
compd no.a
1H NMR (
δ, ppm)
EI−MS m/z (relative intensity, %)
+
1
1.23 (t, J
)
6 Hz, 3H, CH3), 3.46 (s, 2H, CH2), 4.15 (q, J
)
6 Hz, 2H,
225 (M , 30), 180 (4), 111 (100), 110 (11),
109 (4), 95 (4), 83 (12), 75 (2), 69 (2)
OCH2), 7.11 (t, J 6 Hz, 2H, Hb and Hb , aromatic), 7.64 (dd,
6 Hz, 2H, Ha and Ha aromatic), 9.70 (s, 1H, NH)
1.40 (t, J 6 Hz, 3H, CH3), 3.45 (s, 2H, CH2), 4.40 (q, J
OCH2), 7.35 (m, 1H, Hb aromatic), 7.50 (m, 2H, Ha , Hb aromatic),
7.60 (m, 1H, Ha aromatic), 9.80 (s, 1H, NH)
)
′
J
)
′
+
2
)
)
6 Hz, 2H,
286 (M , 9), 241 (82), 240 (4), 201 (10),
′
′
200 (3), 199 (11), 198 (3), 174 (96),
172 (100), 171 (3), 156 (2), 146 (4),
145 (5), 75 (3), 69 (2), 65 (6), 64 (3)
+
3
4
1.20 (t, J
OCH2), 6.90 (t, J
6 Hz, 2H, Ha and Ha
1.21 (t, J 6 Hz, 3H, OCH2CH3), 3.89 (s, 3H, OCH3), 3.56 (s, 2H,
CH2), 4.0 (q, J 6 Hz, 2H, OCH2CH3), 6.95 (m, 1H, Hb aromatic),
7.01 (m, 1H, Hc aromatic), 7.07 (m, 1H, Hb aromatic), 8.34 (dd, 1H,
Ha aromatic), 9.52 (s, 1H, NH)
0.75 (t, J 6 Hz, 3H, OCH2CH3), 3.23 (s, 3H, OCH3), 3.15 (s, 2H,
)
6 Hz, 3H, CH3), 3.45 (s, 2H, CH2), 4.13 (q, J
6 Hz, 2H, Hb and Hb , aromatic), 7.55 (dd,
aromatic), 9.90 (s, 1H, NH)
)
6 Hz, 2H,
208 (M , 7), 162 (7), 121 (13), 94 (100),
)
′
93 (28), 78 (7), 65 (10)
J
)
′
+
)
237 (M , 38), 192 (7), 150 (6), 135 (4),
)
124 (9), 123 (100), 120 (7), 108 (67),
92 (7), 80 (11), 6.5 (14)
′
′
+
5
)
237 (M , 37.4), 192 (8.5), 149 (10.8),
CH2), 3.75 (q, J
6.55 (m, 1H, Hb
7.50 (m, 1H, Ha
)
6 Hz, 2H, OCH2), 6.40 (m, 1H, Hc aromatic),
aromatic), 6.90 (m, 1H, Ha aromatic),
aromatic), 8.55 (s, 1H, NH)
135 (4.6), 123 (100), 120 (9.2), 108 (70.0),
80 (11.5), 77 (4.6), 65 (13.8)
′
′
′
6
7
8
1.50 (t, J
OCH2), 7.40 (m, 1H, Hc aromatic), 7.80 (m, 1H, Hb
(t, 1H, Hb aromatic), 9.10 (t, 1H, Ha aromatic), 10.30 (s, 1H, NH)
2.45 (s, 3H, COCH3), 3.70 (s, 2H, CH2CO), 7.25 (dd, J 6 Hz, 2H,
b , Hb aromatic), 7.70 (dd, J 6 Hz, 2H, Ha, Ha aromatic),
9.50 (s, 1H, NH)
)
6 Hz, 3H, CH3), 4.20 (s, 2H, CH2), 4.55 (q, J
)
6 Hz, 2H,
unstable
′
aromatic), 8.50
′
+
)
195 (M , 1), 123 (11), 110 (3), 98 (8),
H
′
)
′
97 (100), 83 (4), 69 (12)
+
3.00 (s, 3H, COCH3), 3.00 (s, 2H, CH2CO), 7.40 (m, 3H, Hb, Hb
′
, Ha
′
256 (M , 2), 222 (21), 221 (7), 199 (3),
aromatic), 7.60 (m, 1H, Ha aromatic), 10.05 (s, 1H, NH)
172 (100), 156 (13), 138 (22), 93 (8),
66 (8), 64 (3)
+
9
2.20 (s, 3H, COCH3), 3.40 (s, 2H, CH2CO), 6.95 (m, 3H, Hb, Hb
′
, Hc
177 (M , 1), 143 (9), 142 (2), 120 (6),
aromatic), 7.55 (m, 2H, Ha, Ha
′
aromatic), 9.90 (s, 1H, NH)
93 (100), 92 (6), 77 (23), 65 (1)
+
10
2.45 (s, 2H, CH2CO), 3.75 (s, 2H, CH2CO), 7.35 (m, 1H, Hc aromatic),
211 (M , 8), 177 (5), 176 (38), 129 (31),
7.60 (m, 2H, Hb, Hb
′
aromatic), 8.60 (m, 1H, Ha aromatic), 9.93
127 (100), 99 (8), 92 (6), 91 (5), 85 (7),
75 (4), 71 (4), 69 (6), 65 (7), 63 (8)
(s, 1H, NH)
11
2.35 (s, 3H, COCH3), 3.50 (s, 2H, CH2CO), 7.40 (m, 1H, Hc aromatic),
unstable
7.50 (m, 1H, Hb′, Ha aromatic), 7.65 (m, 1H, Ha aromatic), 9.40
(s, 1H, NH)
+
12
13
2.50 (s, 3H, COCH3), 3.72 (s, 2H, CH2CO), 7.60 (m, 2H, Hb, Hb
aromatic), 7.80 (m, 2H, Ha, Ha aromatic), 9.60 (s, 1H, NH)
2.40 (s, 3H, COCH3), 3.70 (s, 2H, CH2CO), 7.40 (m, 1H, Hc aromatic),
7.55 (m, 2H, Hb , Ha aromatic), 7.90 (m, 1H, Ha aromatic),
9.60 (s, 1H, NH)
1.35 (s, 3H, ArCH3), 2.45 (s, 3H, COCH3), 3.70 (s, 2H, CH2CO), 7.20
(m, 1H, Hc aromatic), 7.40 (m, 1H, Hb aromatic), 7.50 (m, 2H, Ha,
Ha aromatic), 9.60 (s, 1H, NH)
2.12 (s, 3H, COCH3), 7.10 (t, 2H, Hb, Hb
Ha aromatic), 10.00 (s, 1H, NH)
4.05 (s, 2H, CH2Cl), 7.10 (m, 2H, Hb, Hb
a aromatic), 9.95 (s, 1H, NH)
4.05 (s, 2H, CH2Cl), 7.15 (m, 2H, Hb, Hb
a aromatic), 10.05(s, 1H, NH)
4.10 (s, 2H, CH2Cl), 7.20 (m, 2H, Hb, Hb
a aromatic), 8.30 (s, 1H, NH)
5.80 (s, 1H, CHCl2), 6.80 (m, 2H, Hb and Hb
′
211 (M , 0.29), 196 (5), 154 (5), 153 (6),
′
127 (100), 111 (4), 93 (6), 92 (6), 65 (10)
+
222 (M , 2), 138 (100), 122 (2), 121 (13),
′
′
108 (14), 104 (13), 92 (2), 65 (12)
+
14
191 (M , 1), 157 (13), 156 (7), 147 (9),
′
134 (11), 107 (100), 92 (16), 91 (22),
73 (5), 65 (9)
′
+
15
16
17
18
19
20
21
22
′
′
′
′
aromatic), 7.65 (dd, 2H, Ha,
aromatic), 7.50 (m, 2H, Ha,
aromatic), 7.50 (m, 2H, Ha,
aromatic), 7.40 (m, 2H, Ha,
153 (M , 12), 138 (29), 111 (100), 110 (9),
′
78 (26), 77 (67), 65 (58)
+
187 (M , 2), 138 (30), 124 (12), 111 (100),
H
′
110 (69), 78 (11), 77 (75), 65 (81), 64 (15)
+
248 (M , 6), 199 (21), 185 (8), 172 (100),
H
′
171 (71), 79 (5), 78 (59), 66 (63), 65 (6)
+
169 (M , 1), 120 (25), 106 (14), 93 (100),
H
′
92 (61), 78 (18), 77 (85), 65 (95), 63 (28)
+
′
′
′
aromatic), 7.20 (m, 2H,
aromatic), 7.20 (m, 2H,
aromatic), 7.30 (m, 2H,
222 (M , 5), 159 (5), 151(7), 139 (28),
H
a and Ha
′
aromatic), 8.10 (s ,1H, NH)
111 (80), 110 (8), 84 (100), 77 (28)
+
5.85 (s, 1H, CHCl2), 6.85 (m, 2H, Hb and Hb
282 (M , 3), 171 (80), 170 (12), 156 (118),
H
a and Ha
5.90 (s, 1H, CHCl2), 6.70 (m, 2H, Hc and Hb
b and Ha
4.40 (s, 1H, CHCl2), 6.60 (m, 2H, Hb and Hb
Ha aromatic), 8.10 (s, 1H, NH)
7.00 (m, 4H, aromatic), 9.80 (s, 1H, NH)
′
aromatic), 8.15 (s ,1H, NH)
146 (21), 144 (100), 137 (30), 136 (11)
+
204 (M , 3), 120 (12), 93 (62), 92 (8),
H
′
aromatic), 7.80 (m, 1H, Ha aromatic), 8.65 (s, 1H, NH)
aromatic), 7.25 (m, 1H,
78 (9), 68 (21), 66 (100), 59 (29), 58 (7)
unstable
′
′
+
23
24
257 (M , 7), 169 (22), 119 (37), 111 (100)
+
7.00 (m, 2H, Hb and Hb
′
aromatic), 7.05 (m, 2H, Ha and Ha
′
aromatic),
317 (M , 6), 283 (18), 248 (15), 201 (41),
9.85 (s, 1H, NH)
199 (8), 190 (100), 189 (10), 110 (17),
77 (12), 65 (3)
25
6.95 (m, 4H, aromatic), 8.90 (s, 1H, NH
unstable
a The identity of the compound number was mentioned under Table 1.
variability in the acyl side chain, the descriptor variables, viz., π,
molecular weight (MW), MR, and parachor (P) (ACD Chemsketch,
version 2.0) were taken (16). The “agrophore” data, viz., percent
induction of spikelet sterility (SS) caused by test CHAs tested at 1500
ppm were transferred into sin arc and used as the dependent variable
(SS % sin arc). The descriptor variables were used to generate multiple
regression equations (MLR) by autocorrelation using the computer
software package SPSS (version 10.0) program. None of the indepen-
dent variables appearing in the equations was ensured to be orthogonal.
Statistical Analyses. Analysis of variance (ANOVA) of factorial

6804 J. Agric. Food Chem., Vol. 54, No. 18, 2006
Chakraborty and Devakumar
RBD was performed with all treatments. On the basis of the significance
of the treatments, critical differences (CD) at a 5% level of significance
(p ) 0.05) were computed.
Table 3. Change in Chemical-Shift Values (∆δ) of Aromatic Protons
of N-Acylanilines as Compared to Unsubstituted Analogues
RESULTS AND DISCUSSION
Chemical Syntheses and Spectroscopic Analyses. The
compounds synthesized numbering 25 belong to three broad
chemical classes, viz., malonanilates, acetoacetanilides, and
acetanilides (including halogenated acetanilides). The CHAs
were synthesized by condensation with appropriate diesters/
monoesters or acid chlorides with substituted anilines. Several
N-acylanilines containing variations at the acyl domain were
prepared having various substituents in the aromatic ring (1-
25). The compounds were purified using physical and chro-
matographic separation methods. Their structures were con-
Unsubstituted Malonanilate (Compd No. 3)
Ha
)
7.50 ppm, Hb
)
6.90 ppm, and Hc ) 6.20 ppm
∆δ (ppm)
compd no.
X
Ha
H′
Hb
H
′
Hc
a
b
1
1
2
4
5
6
4-F
4-Br
2-OCH3
3-OCH3
2-NO2
0.14
0.00
0.14
0.00
0.84
0.60
1.60
0.21
0.60
0.05
0.21
0.45
0.17
0.35
0.90
firmed using H NMR and MS.
In N-acylanilines, the effect of different side chains on the
chemical shifts of different protons was investigated by keeping
the aromatic moiety constant. In malonanilate series, the signal
corresponding to the methylene group (-CH2-) flanked by two
carbonyl groups appeared as a sharp singlet centered at δ 3.54
( 0.32 ppm. The presence of the ethyl ester group was
confirmed by the signal, viz., a triplet centered at δ 1.22 (
0.24 ppm and a quartet centered at δ 4.16 ( 0.26 ppm for
-OCH2-. A broad singlet corresponding to the -NH proton
appeared at δ 9.56 ( 0.52 ppm. The 4′-fluoro and 4′-bromo
analogues of malonanilate caused substantial deshielding of the
-NH proton (Table 3). Ethyl 4′-bromomalonanilate also had a
strong deshielding effect on Hb and H′b aromatic protons by
about 0.60 and 0.45 ppm, respectively, as understood from its
-I effect. Ethyl 4′-fluoromalonanilate followed the same trend
as its bromo counterpart. In acetoacetanilides, the methyl group
appeared as a sharp singlet at δ 2.48 ( 0.22 ppm. The chemical-
shift values of the 4′-bromo analogue appeared downfield by
0.52 ppm, whereas those in unsubstituted acetoacetanilide (9)
appeared upfield by about 0.28 ppm for COCH3 protons in the
side chain. The -NH proton appeared as a broad singlet at δ
9.59 ( 0.29 ppm in acetoacetanilides. The 4′-bromo substituent
caused marked deshielding of the highly exchangeable -NH
protons by 0.46 ppm because of its electron-withdrawing -I
effect. The unsubstituted acetoacetanilide recorded substantial
shielding by 0.54 ppm. The substituent nitro at the meta position
caused substantial deshielding by 0.45 ppm (∆δ) of the Ha aryl
proton as compared to the same in acetoacetanilide (9). All of
the substituents, namely, F, Cl, and Br groups having a -I effect,
exhibited a deshielding effect on the Ha aryl proton. In
comparison, the methyl group having a +I effect exerted a
comparative shielding effect and its chemical shift (δ 7.50 ppm)
value matches that of the unsubstituted analogue. The 2′- and
4′-chloro analogues exerted a strong deshielding effect on the
Hb and H′b aromatic protons by about 0.65 ppm as compared
to that of the unsubstituted one. The 3′-nitro and 3′-chloro
analogues, by virtue of their -M and -I effects, exerted a strong
deshielding effect on Hc aryl protons by 0.45 ppm, as compared
to that of acetoacetanilide (Table 3). In unsubstituted acetanilide,
the methylene protons (CH2Cl) appeared at δ 4.00 ppm. The
methylene protons in 4-fluorochloroacetanilide (16) and 4-bro-
mochloroacetanilide (17) were deshielded by 0.05 ppm as
compared to the unsubstituted one. Because of the halogen
substitution (F/Br) in the aryl moeity, it withdraws the electron
cloud by virtue of their -I effect operating in them, from the
aryl as well as the methylene protons resulting in the deshielding
of the same. In unsubstituted dichloroacetanile (18), the methine
(CHCl2) proton appeared at δ 5.90 ppm. In 4-bromodichloro-
0.81
0.20
1.20
0.00
−
1.60
Unsubstituted Acetoacetanilide (Compd No. 9)
7.45 ppm, Hb 6.95 ppm, and Hc 6.95 ppm
Ha
)
)
)
∆δ (ppm)
compd no.
R
Ha
0.25
H
′
Hb
H′
Hc
a
b
7
8
4-F
4-Br
2-Cl
3-Cl
4-Cl
3-NO2
3-CH3
0.25
0.05
1.15
0.05
0.35
0.10
0.05
0.30
0.45
0.65
0.30
0.45
0.65
0.55
0.65
0.60
0.45
0.15
−
10
11
12
13
14
0.40
0.45
0.20
0.35
0.45
0.05
0.65
0.45
0.25
acetanilide (20), substantial deshielding by about 0.30 ppm of
the CHCl2 proton as compared to the same in the unsubstituted
one was observed. In 4-fluorodichloroacetanilide (19), the
methine proton was shielded by 0.10 ppm perhaps because of
the very small size of the electronegative fluorine atom. The
amido proton in trichloroacetanilide was more deshielded (δ
10.45 ppm) as compared to that of dichloroacetanilide (δ 8.65
ppm) and chloroacetanile (δ 9.90 ppm) because of the presence
of more electronegative chlorine atoms in the side chain. The
amido (NH) proton in 4-fluorotrichloroacetanilide (23) and
4-bromotrichloroacetanilide (24) was shielded by 0.65 and 0.60
ppm, respectively, perhaps because of the halogen substitution
in the aromatic moiety. In regard to the influence of substituents
on the chemical shift of aromatic protons, the δ aryl of
chloroacetanile was taken as the base value. It was seen that
there was no difference in the chemical shift of Ha and H′a
aromatic protons (δ 7.50 ppm) in 4-fluorochloroacetanilide (16)
and 4-bromochloroacetanilide (17) as compared to their unsub-
stituted counterpart. The substituent bromo and fluoro at the
para position caused deshielding respectively by about 0.10 and
0.05 ppm (∆δ) of Hb and H′b aryl protons as compared to the
same in chloroacetanilide (δ 7.50 ppm). It was observed that
the halogen substitution at the aromatic moiety caused shielding
of the Ha aryl proton by 0.30 and 0.60 ppm in 4-fluorodichlo-
roacetanilide (19) and 4-bromodichloroacetanilide (20) as

N-Acylanilines as CHAs for Wheat
J. Agric. Food Chem., Vol. 54, No. 18, 2006 6805
substitution with highly electronegative groups, i.e., F/Br, can
give rise to analogues having a high level of activity in
malonanilates. Ethyl 4′-fluoromalonanilate (1) and ethyl 4′-
bromomalonanilate (2) containing F and Br, respectively, at the
para position of the aromatic ring were found to be the best in
that order when considered across three genotypes of wheat.
The other aromatic substituents influenced the activity in the
following order: H (3) > 2-OMe (4) > 3-OMe (5) > 2-NO2
(6). In all cases, the influence of aromatic substituents on the
induction of spikelet sterility was in the order: para > ortho
> meta (Table 4). Among anilides, two types of compounds
were synthesized, viz., acetoacetanilides and chloroacetanilides.
Among acetoacetanilides, 4-fluoro (7) and 4-bromo derivatives
(8) induced 88.5 ( 1.3 and 82.5 ( 2.2% spikelet sterility,
respectively, in the genotypes, which was significantly higher
as compared to other members in this series. The other
substituents influenced the activity in the order Cl > NO2 >
CH3. Among chloro-substituted acetoacetanilides, para substitu-
tion was found to be more effective followed by ortho and meta
substitution. It can be inferred that para substitution with highly
electronegative groups such as F or Br can give rise to analogues
having a high level of activity. The steric effect seems to be
operating for the higher activity of para and not ortho
substituents. Increasing the number of chlorine atoms in the side
chain of chloroacetanilides (16-18) led to an increase in the
activity. 4-Fluorotrichloroacetanilide (23) exhibited the highest
induction of male sterility (88.6 ( 2.6%).
In a program of design and development of CHAs for crop
plants, we earlier reported the deployment of ethyl oxanilates
in rice, wheat, and chickpea. Incidentally, the highly active
compounds have F/Br as substituents at the para position of
the aromatic ring inducing >98% spikelet sterility when
considered across two test concentrations, three genotypes of
wheat, and 2 year trial data. Earlier results with rice (variety
Pusa 150) indicated similar trends governing structure-activity
relationship inducing >80% male sterility (17). The ethyl
oxanilate class of CHAs mostly having F/Br/CF3 at the para
position of the aryl ring were identified as the most potent
compounds, causing g99% induction of pollen sterility at the
1000 ppm test concentration on chickpea (variety BG 1088)
(22). On the basis of the leads obtained in the earlier studies,
we have synthesized the CHAs with electronegative groups (F/
Br) in the para position of aryl rings and varying the acyl side
chain. It is interesting to note that elongation of the acyl side
chain in ethyl 4′-fluoro-oxanilate by introducing a methylene
bridge (-CH2-) as in ethyl 4′-fluoromalonanilate (1) has caused
a reduction in the spikelet sterility apparently because of the
induction of steric hindrance in the macromolecular receptor
site. Earlier, ethyl 4′-fluoro-oxanilate was screened on 29
genotypes of wheat at a 0.15% test concentration and was
observed to induce 99.76 ( 0.37% male sterility (20), whereas
ethyl 4′-fluoromalonanilate (1) exhibited 83.14 ( 2.46% spikelet
sterility in the wheat genotypes in the present study.
Table 4. Effect of N-Acylanilines on the Percent Induction of Spikelet
Sterility at 1500 ppm Spray Concentrations on Three Genotypes of
Wheat in the Winter of 2001
−2002
spikelet sterility (%)
compd
no.
X
R
PBW 343
HW 2046
HD 2733
Malonanilates
CH2 COOEt
1
2
3
4
5
6
4-F
4-Br
H
2-OMe
3-OMe
2-NO2
−
−
−
−
−
−
84.66
83.40
62.97
25.51
20.54
12.53
80.30
79.15
57.73
24.18
18.07
10.42
84.46
83.48
64.62
29.16
27.51
14.66
CH2 COOEt
CH2 COOEt
CH2 COOEt
CH2 COOEt
CH2 COOEt
Acetoacetanilides
7
8
9
10
11
12
13
14
4-F
4-Br
H
2-Cl
3-Cl
4-Cl
3-NO2
3-CH3
−
−
−
−
−
−
−
−
CH2 COCH3
89.12
83.65
64.52
34.98
15.53
58.65
22.13
2.94
87.00
80.01
62.47
32.39
10.57
50.57
14.04
2.29
89.36
83.82
66.42
36.69
16.76
61.04
23.09
3.40
CH2 COCH3
CH2 COCH3
CH2 COCH3
CH2 COCH3
CH2 COCH3
CH2 COCH3
CH2 COCH3
Acetanilides/Halogenated Acetanilides
15
16
17
18
19
20
21
22
23
24
25
4-F
4-F
4-Br
H
4-F
−
−
−
−
−
−
−
−
−
−
−
CH3
61.35
39.92
39.45
32.16
63.75
62.38
34.85
24.86
89.61
88.09
46.87
0.46
59.33
38.17
38.26
26.11
56.05
57.13
29.71
22.49
85.00
84.35
42.47
0.33
64.13
41.28
40.73
33.83
65.10
62.75
36.97
26.81
91.20
89.12
49.22
0.49
CH2Cl
CH2Cl
CH2Cl
CHCl2
CHCl2
CHCl2
CHCl2
CCl3
4-Br
H
2,4-(NO2)2
4-F
4-Br
H
emulsion control
CD (p 0.05)
CCl3
CCl3
)
0.84
1.28
1.07
compared to dichloroacetanile (δ 7.80 ppm). In 4-fluorotrichlo-
roacetanilide (23) and 4-bromotrichloroacetanilide (24), the
aromatic protons (Ha, H′a, and Hb) were comparatively more
shielded than those in trichloroacetanilide (25).
In the mass spectroscopic analysis of malonanilates and
acetoacetanilides, the base peak was dominated by the aryl side
chain in one form or another. Apparently, the molecular ion
(M+) loses the ethoxy radical (EtO‚) to give rise to the acylium
ion radical (M-45), which upon cyclization became 4-hydroxy
carbostyril. The loss of CH2COOEt or CH2COCH3 as in
malonanilates and acetoacetanilides, respectively, led to the
formation of the protonated aryl isocyanate moiety (ArNCO+‚
), which, very likely in turn, eliminated CO from the side chain
to give rise to azatropylium ion species. The formation of aryl
isocyanate (M-88) can be visualized as a result of the McLafferty
rearrangement in malonanilates. The formation of aniline (M-
114) stems from the loss of CO from aryl isocyanate. In the
mass fragmentation pattern of acetanilides, fission of the -CH2-
Cl, -CH3, -CHCl2, or -CCl3 moieties led to the azatropylium
ion species via protonated aryl isocyanate. Further fragmentation
could explain the formation of substituted aniline species, which
in most cases was found to be the base peak.
The percent pollen sterility was found to have a high
correlation (r ) 0.98) with the spikelet sterility. Thus, pollen
sterility appears to be the direct cause of spikelet sterility. From
the stain test, it was seen that the sterile grains were transparent,
thereby confirming the disintegration of the cytoplasm and
nucleus in the sterile pollen. In contrast, fertile pollens from
control plots were stained a uniform deep blue color in the KI-
I2 stain test, thus confirming the induction of male sterility in
various treatments. The negative color in the KI-I2 stain test
shown by sterile pollens was indicative of the absence of starch.
The absence of any starch material in the sterile pollen grains
Effect of CHAs on Spikelet Sterility. The results of
induction of spikelet sterility on bread wheat caused by test
chemicals at 1500 ppm on three genotypes of wheat (PBW 343,
HW 2046, and HD 2733) are given in Table 4. There was no
marked variation in the response of three genotypes. Para

6806 J. Agric. Food Chem., Vol. 54, No. 18, 2006
Chakraborty and Devakumar
Figure 3. Sketch model of N-acylanilines. Induction of male sterility is
directly proportional to the inductive (field or polar, Fp) effect and inversely
related to the resonance effect (R) of the aromatic substituent. The positive
influences of the steric parameter (P) for the acyl domain have been
underlined.
where N ) 25, r ) 0.84, r2 ) 0.71, s ) 16.12, and F
(probability) ) 11.56 (0.0000).
The best equation (eq 2) was the one that combined the
independent variables Fp, ΣR, and D for aromatic substitution
and parachor (P) for the acyl side chain with r ) 0.84. Both
electronic and steric effects have a significant influence on the
bioactivity. The direct involvement of the Swain-Lupton field
constant for para substitution (Fp) with the target bioactivity in
the best equation implied that the inductive (field or polar) (Fp)
rather than the resonance (R) effect of the aromatic substituent
appears to be the key factor influencing the induction of male
sterility. 4-Bromotrichloroacetanilide (24) found to be highly
active because of bromine has a very high Fp value (Fp ) 0.44).
It can be inferred that para substitution with highly electro-
negative groups (F/Br) withdraw the electron cloud by the
inductive effect (-I effect) from the aromatic ring as well as
substituted amide moiety (-CO-NH) of the most active CHAs,
thus acting as the nucleophilic center of the molecules resulting
in a high level of activity.
In competitive binding at the bioreceptor site, a negative R
term could mean unfavorable conformational changes in the
enzyme-inhibitor complex as compared to favorable confor-
mational changes caused by the enzyme-substrate complex.
The equation essentially highlights the positive effect of the
steric parameter, viz., parachor of the side chain. It implies that
bulk in acyl side chain is directly proportional to the induction
of male sterility (Figure 3). There is ample scope for the
development of potent CHA analogues based on the leads
postulated and predicted in the present studies. These leads will
be significant in explaining the CHA fit in the macromolecular
receptor site and exploring the primary site and mode of action
of these CHAs.
The mode of action of these substances is supposed to be
due to specific inhibition in the meiosis mechanism, leading to
degeneration of pollen mother cells. The observed effects were
the result of leaf transcuticular uptake and possible phloem
transport. The compounds in malonanilate series have ethyl ester
moiety in the side chain, which are very likely immediately
transformed in the leaves to give the free acids. Similarly,
hydrolysis of 4-fluorotrichloroacetanilide (23) in pholoem sap
may likely result in N-(4-fluorophenyl)oxanilic acid through the
intermediates, viz., 2,2-dichloro-2-hydroxy-4-fluoroacetanilide
and 2-(4-fluoroanilino)-2-oxoethanoyl chloride (Figure 4). The
high hydrophilicity of the free acids results in an easy water
solubility and phloem transport, which add to the efficiency of
the active CHAs. The free acids are very likely able to move
and concentrate inside the phloem sieve tubes and be transported
to the organs presenting a high metabolic activity, as is the case
of the stamen at this stage. Moreover, N-phenyl oxanilic acid
results to an imbalance in the pH equilibrium in the pollen
mother cells, leading to the dissolution of the microsporocyte
callose wall during early stages of meiosis (meiotic I prophase)
by premature callase (1,3-â glucanase) secretion. Alterations
Figure 2. Sterile pollens of wheat because of the treatment of CHA vis-
a`-vis fertile pollens as revealed from KI−I2 stain test.
could be indicative of either of the processes leading to starch
depletion or blocking of its synthesis (Figure 2). This could
provide an important lead in unraveling the mode of action of
the CHAs.
QSAR and Mode of Action of CHAs. QSAR is a useful
tool in elucidating essential structural features governing the
induction of male sterility. Apparently, CHAs exert their
biological effects (inducing male sterility) by participating in a
series of events, which include transport binding with the
macromolecular receptor and metabolism. Because the interac-
tion mechanisms between the molecule and the putative receptor
are unknown in most cases (i.e., no bound crystal structure),
one is reduced to making inferences from properties, which can
easily be obtained (molecular properties and descriptors), to
explain these interactions for known molecules. Once the
relationship is defined, it can be used to aid in the prediction of
new or unknown molecules.
No significant variation in the response of different genotypes
for CHAs was apparent in this study (Table 4). Therefore, the
variety PBW 343, sprayed at a 1500 ppm concentration was
taken for QSAR analysis. Results of the multiple regression
analyses carried out are given along with the statistical values
(N ) number of compounds; r ) multiple regression coefficient;
s ) standard deviation, and F ) Fisher’s ratio of significance
index with respect to the equation). All of the equations were
found to be statistically significant at p < 0.01%. Using a
combination of chemical descriptors both for aromatic substitu-
tion and side-chain variation as detailed in the earlier section
carried out QSAR analysis of the 25 analogues of N-acylanilines
thus generated, and the correlation matrix was constructed. The
models for each CHA family gave a good correlation between
the variations in sin arc percent of the spikelet sterility and the
steric-electrostatic properties of the sets. In the CHAs synthe-
sized, the observed bioactivity could be collectively explained
in the form of two MLR equations containing two and three
descriptor variables, respectively, using a total of 12 independent
variables
spikelet sterility (sin arc %) ) 108.71Fp - 10.53D + 44.52
(1)
where N ) 25, r ) 0.82, r2 ) 0.68, s ) 16.21, and F
(probability) ) 21.77 (0.0000), and
spikelet sterility (sin arc %) ) 108.54Fp - 0.83ΣR -
9.66D + 1.52P + 14.68 (2)

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ACKNOWLEDGMENT
The authors are thankful to the Director, IARI, New Delhi for
providing necessary facilities to carry out the work. Thanks are
also due to Dr. S. M. S. Tomar, Principal Scientist, Division of
Genetics, IARI, for useful discussion and help.
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Received for review May 17, 2006. Revised manuscript received July
3, 2006. Accepted July 6, 2006.
(26) Hansch, C.; Leo, A.; Unger, S. H.; Kim, K. H.; Nikaitani, D.;
JF061390X