Synthesis and screening of anilides having olefinic and alkyl moiety in the side chain as chemical hybridizing agents for wheat (Triticum aestivum L.)

Article abstract of DOI:10.1021/jf050746m

Induction of male sterility by deployment of chemical hybridizing agents (CHAs) holds immense potential in heterosis breeding of wheat. A total of 21 anilides having different aromatic substitutions and side-chain variation were synthesized and screened as CHAs on three genotypes of wheat viz., PBW 343, HW 2046, and HD 2733, at winter season. Various anilides having vinyl moiety in the acyl side chain were synthesized by condensation between substituted anilines with different esters or acid chlorides. Another lead in the form of N-alkyl anilines also became evident. The percent male sterility data caused by CHAs revealed the significant contribution of anilides containing vinyl double bond incorporated in the form of closed ring structure viz., furyl moiety as the side chain. 4′-Fluoro-furyl anilide (1) and 4′-bromo-furyl anilide (2) are found to be promising lead CHAs for the design of highly active molecules. QSAR analysis revealed a direct relationship of field effect exemplified by the Swain-Lupton constant F_p for the aromatic substitution but an inverse relationship of molar refractivity MR for the side chain. The negative influences of parachor for the acyl domain have been underlined. The real guiding principle for selectivity of CHA action was found to be the π value. The CHAs act by mimicking UDP-glucose, the key substrate in the synthesis of callose, or lead to an imbalance in acid-base equilibrium in pollen mother cells resulting in dissolution of callose wall by premature callase secretion.

Full text of DOI:10.1021/jf050746m

J. Agric. Food Chem. 2005, 53, 5959−5968 5959
Synthesis and Screening of Anilides Having Olefinic and Alkyl
Moiety in the Side Chain as Chemical Hybridizing Agents 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) holds immense
potential in heterosis breeding of wheat. A total of 21 anilides having different aromatic substitutions
and side-chain variation were synthesized and screened as CHAs on three genotypes of wheat viz.,
PBW 343, HW 2046, and HD 2733, at winter season. Various anilides having vinyl moiety in the acyl
side chain were synthesized by condensation between substituted anilines with different esters or
acid chlorides. Another lead in the form of N-alkyl anilines also became evident. The percent male
sterility data caused by CHAs revealed the significant contribution of anilides containing vinyl double
bond incorporated in the form of closed ring structure viz., furyl moiety as the side chain. 4′-Fluoro-
furyl anilide (1) and 4′-bromo-furyl anilide (2) are found to be promising lead CHAs for the design of
highly active molecules. QSAR analysis revealed a direct relationship of field effect exemplified by
the Swain-Lupton constant Fp for the aromatic substitution but an inverse relationship of molar
refractivity MR for the side chain. The negative influences of parachor for the acyl domain have been
underlined. The real guiding principle for selectivity of CHA action was found to be the π value. The
CHAs act by mimicking UDP-glucose, the key substrate in the synthesis of callose, or lead to an
imbalance in acid-base equilibrium in pollen mother cells resulting in dissolution of callose wall by
premature callase secretion.
KEYWORDS: Anilides; chemical hybridizing agents (CHAs); QSAR; wheat
INTRODUCTION
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. Under
situations where CHAs are unable to induce a very high degree
of male sterility, they can still enable a breeder to produce a
sufficient quantity of hybrid seed to test the value of hybrid
population on a limited scale. The search for CHAs, known as
gametocides in the fifties and sixties, was rather random and
from the chemicals already used in agriculture such as plant
growth regulators and herbicides (1). Prominent among the
CHAs used in wheat includes maleic hydrazide, dalapon (2),
DPX 3778 (3), Ethephon (4), LY 195259 (5), RH-531,
RH-532, RH-2956, RH-4667 (6, 7), SC-1058, and SC-1271 (8).
Although the application of these chemicals at a specific plant
growth stage was able to induce partial male sterility, several
associated defects were observed. These include impaired female
fertility in the majority of instances, phytotoxic effects, and
growth inhibition. Ethyl oxanilates (9-11) and Clofencet
(Genesis, MON 21200; 2-(4-chlorophenyl)-3-ethyl-2,5-dihydro-
5-oxo-4-pyridazinecarboxylic acid) (12) were reported to be of
practical value.
Vertical intensification through heterosis breeding is one of
the most practical options for breaking the present yield plateau
of wheat productivity. Through the infusion of hybrid technol-
ogy, which has the potential of enhancing the productivity levels
by 20-25%, one can achieve the projected production target
of wheat. Even though the research efforts on the possibility of
hybrids in wheat were initiated globally in the early fifties using
a three-line system, it suffered major limitations of excessive
height and biomass production because of the use of a tall parent,
fewer seeds in hand pollination for hybrid seed production, and
nonavailability of suitable restorer lines. Chemical hybridizing
agents (CHAs) are important in heterosis breeding of self-
pollinated crops (hermaphrodite) like wheat, wherein the male
and female organs are in the same flower. Selective sterilization
of the male organ (pollen) is of paramount importance in
heterosis breeding. CHAs would be preferred over the existing
cytoplasmic-genetic male sterility (CGMS) method because the
former saves time and labor needed for transferring male sterility
because male sterile and restorer lines are not required and
In earlier studies, we have seen that 6-membered heterocycles
such as 1-aryl-4-pyridones (N-aryl-5-carbethoxy-4,6-dimethyl-
1,2-dihydro-pyrid-2-one), 2-phenyl-4-oxonicotinates, and N-
aralkyl-pyrid-2-ones containing an amido group and olefinic
double bond in a cyclic system are promising as CHAs (13).
* To whom correspondence should be addressed. Telephone: 91-484-
2394867 (ext. 272), 91-484-2392905, 09447084867. Fax: 0091-0484-
2394909. E-mail: kajal_iari@yahoo.com, kajal_cmfri@rediffmail.com.
† Current address: Division PNP, Central Marine Fisheries Research
Institute, Ernakulam North P. O., P. B. no. 1603, Cochin-682018, Kerala,
India.
10.1021/jf050746m CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/29/2005

5960 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Chakraborty and Devakumar
General Procedure of Synthesis of Furyl Anilides (1-3). The
following method illustrates the general scheme of synthesis of the
title compounds using different substituted anilines and various esters.
This idea prompted us to explore the involvement of the olefinic
double bond in the acyl side chain for the activity as CHAs. In
one case, the vinylogous double bond was kept open to
synthesize a series of (ar)-alkenyl anilides. In another case, we
have introduced the N functional group at the third position of
the double bond to synthesize a series of 3-amino alkenyl
anilides. The side chain of anilides containing the vinylogous
double bond has been further incorporated in the form of a furyl
ring to produce a series of furyl anilides. Also, instead of an
olefinic group, the side chain was varied in the form of an aryl
ring and imides to synthesize phthalanilate/salicylanilide and
phthalimides, respectively. Further, to compare the effect of an
increasing number of bonds on the male sterility, a series of
3-methoxycarbonyl propiolanilides containing triple bonds in
the alcoholic moiety have been prepared. In another study, we
varied the N-acyl moiety by the N-alkyl group, and in this
process, two N-alkylanilines [ethyl-(4-fluoroanilino) acetate and
4-fluoro-N-â-cyanoethyl aniline] have also been synthesized.
Quantitative structure-activity relationship (QSAR) analysis
is a useful tool in elucidating essential structural features that
govern the interaction of CHAs with the macromolecular
receptor in wheat-controlling pollen formation and its viability.
In this study, we report the syntheses, spectral data, and QSAR
analyses of 21 anilides having olefinic and alkyl moiety in the
side chain and their ability to induce male sterility on three
genotypes of wheat (PBW 343, HW 2046, and HD 2733) were
evaluated.
To a solution of furoic acid (0.01 mol, 1.2 g) (obtained from furfural
following the standard procedure using the Cannizaro reaction) in
dichloromethane (10 mL) was added thionyl chloride (0.012 mol, 1.43
mL). The reaction mixture was stirred vigorously for 1 h in the presence
of dry pyridine (0.011 mol, 1.11 mL) in cold conditions. The resultant
solid intermediate (furoyl chloride) was solubilized in dichloromethane
(10 mL), and then substituted aniline (0.01 mol) was added to that in
cold conditions. The reaction mixture under an inert atmosphere of
nitrogen was stirred at 0 °C for 2 h. Then, the mixture was diluted
with diethyl ether (30 mL) and water (30 mL) and washed with ice-
cold water (20 mL). The reaction was followed by TLC (hexane/ethyl
acetate (4:1) as a developing medium) until completion. The organic
phase was dried with magnesium sulfate, filtered, and evaporated to
yield a solid residue, which was recrystallized with diethyl ether/hexane
(1:1) to give a white crystalline solid of furyl anilides (Figure 1), which
were homogeneous, by TLC.
4′-Fluoro-furyl Anilide (1). To a stirred cold solution of furyl chloride
(0.01 mol, 1.3 g) in dichloromethane (10 mL), 4-fluoroaniline (0.01
mol, 0.93 mL) was added under an inert atmosphere of nitrogen at 0
°C for 2 h following Figure 1 to obtain white crystalline solid; yield,
1.46 g (71%); mp, 152 °C; TLC Rf, 0.62; GC Rt, 11.54 min; 1H NMR
(CDCl3-DMSO-d6, δ), 6.55 (m, 1H, Hbf), 7.20 (m, 2H, Haf, Hcf), 7.55
(m, 3H, Ha′,Hb, Hb′ aromatic), 7.55 (m, 1H, Ha aromatic), 10.70 (s,
1H, NH); El-MS m/z (rel. int. %), 205 (M+, 17), 111(17), 110(17),
109(4), 96(5), 95(100), 83(31), 75(5), 67(5), 57(12).
4′-Bromo-furyl Anilide (2). To a solution of furyl chloride (0.01 mol,
1.3 g) in dichloromethane (10 mL) at 0 °C was added 4-bromoaniline
(0.01 mol, 1.7 g) following the general procedure to obtain white
crystalline solid; yield, 1.91 g (72%); mp, 182-184 °C; TLC Rf, 0.60;
GC Rt, 10.25 min; 1H NMR (CDCl3-DMSO-d6, δ), 6.60 (m, 1H, Hbf),
7.10 (m, 2H, Haf, Hcf), 7.30 (m, 1H, Hb′ aromatic), 7.80 (m, 3H, Ha′,
Ha, Hb aromatic), 9.95 (s, 1H, NH); El-MS m/z (rel. int. %), 265 (M+,
14), 263(11), 71(5), 156(7), 154(6), 143(22), 141(21), 135(5), 109(9),
95(100), 57(10).
MATERIALS AND METHODS
Substituted anilines, dimethyl acetylene dicarboxylate, O-acetyl
methyl salicylate, methyl cinnamate, methylcrotonate, ethyl 3-amino
crotonate, and acrylonitrile were procured from Aldrich Chemical Co.
Inc. Furfural, acetic anhydride, phthalic anhydride, pyridine, dimethyl
phthalate, and thionyl chloride were procured from E. Merck. The
structures of synthesized compounds were confirmed by 1H NMR and
mass spectroscopy. Melting points (mp’s) were determined by using a
sulfuric acid bath and were uncorrected. Anhydrous reactions were
performed under an inert atmosphere. The setup was assembled and
cooled under dry nitrogen. Unless otherwise noted, the starting material,
reactant, and solvents were obtained commercially and were used as
such or purified and dried by standard means. Organic solutions were
dried over magnesium sulfate (MgSO4), evaporated on a rotatory
evaporator, and under reduced pressure. All reactions were monitored
by UV fluorescence or staining with iodine. 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. Preparative TLC was performed on 1.0 mm silica gel, 60 Å,
20 × 20 plates. GC data were recorded on a HP Series-II GC using a
FID detector and 10 m × 0.53 mm i.d., 0.25 µm, OV-1 megabore
column with the injector temperature maintained at 250 °C and the
oven temperature programmed from 80 to 250 °C at 9.9 °C/min. The
carrier gas used was N2 with a flow rate of 30 mL/min. All solvents
used in chromatography had been distilled. All test compounds gave
the correct elemental analyses using Euro Vector elemental analyzer
(model number EA3011). Mass spectral assays were obtained using a
FISONS TRIO 1000 (HRGC Mega-2 coupled with EI-mass detector)
instrument under electron-impact conditions using an ionization energy
of 70 eV. A capillary column (30 m, HP-1, 0.32 mm i.d.) and helium
(He) as the carrier gas at the flow rate of 2 mL/min were used in the
mass spectrometer. Nuclear magnetic resonance (1H NMR) spectra were
recorded on a Varian EM-360, 60 MHz NMR apparatus. Samples were
dissolved in deuteriochloroform (CDCl3) or deuteriodimethyl sulfoxide
(DMSO-d6) for data acquisition using tetramethylsilane as the internal
standard (TMS, δ 0.0 ppm for 1H NMR). Chemical shifts (δ) are
expressed in parts per million, and the coupling constants (J) are
expressed in hertz. Multiplicities are described by the following
abbreviations: s for singlet, d for doublet, dd for doublet of doublets,
t for triplet, q for quartet, and m for multiplet.
Furyl Acetanilide (3). A stirred solution of furyl chloride (0.01 mol,
1.3 g) in dichloromethane (10 mL) at 0 °C was added to aniline (0.01
mol, 0.81 mL) under an inert atmosphere of nitrogen following Figure
1 to obtain a white crystalline solid; yield, 1.05 g (56%); mp, 128-
129 °C; TLC Rf, 0.69; GC Rt, 11.89 min; 1H NMR (CDCl3-DMSO-d6,
δ), 6.45 (dd, J ) 4 Hz, 1H, Hbf), 7.10 (d, J ) 4 Hz, 2H, Haf, Hcf), 7.70
(m, 1H, Hc aromatic), 7.60 (m, 1H, Hb, Hb′ aromatic), 7.20 (m, 2H,
Ha, Ha′ aromatic), 9.60 (s, 1H, NH); El-MS m/z (rel. int. %), 187 (M+,
12), 110(73), 109(16), 95(100), 78(11), 77(15), 65(18), 57(2).
General Procedure of Synthesis of 3-Methoxycarbonyl Propi-
olanilides (4-6). A solution of aniline (0.012 mol) suspended in
anhydrous benzene (10%, 14 mL) was added over a period of 30 min
to a solution of dimethyl acetylene dicarboxylate (0.015 mol, 2.13 mL)
in dry benzene (40 mL) kept at e10 °C with continuous stirring under
an inert atmosphere of nitrogen. The reaction was followed by TLC
[hexane/ethyl acetate (4:1) as developing medium] until completion.
The residue obtained was cooled, and hexane was added to get the
precipitate. The precipitate was filtered off to get a solid product, which
was diluted with cold ethyl acetate (100 mL) and treated with a saturated
ammonium chloride solution (50 mL). The organic phase was washed
with water (5 mL), dried, and evaporated to give a yellowish solid.
The resulting crude solid product was filtered, washed with water (100
mL), and dried in a desiccator for 1 day. Recrystallization of the residue
with a mixture of diethyl ether/hexane (1:1) yielded the title compound
as a white crystalline solid of the title compound (3-methoxycarbonyl
propiolanilide) (Figure 1), which was homogeneous by TLC.
3-Methoxycarbonyl 4-Fluoro-propiolanilide (4). To a stirred solution
of 4-fluoro-aniline (0.012 mol) and dimethyl acetylene dicarboxylate
(0.015 mol, 2.13 mL) in dry benzene (40 mL) kept at e10 °C were
reacted following Figure 1 to obtain a white crystalline solid of the
title compound; yield, 1.10 g (∼42%); mp, 180 °C (dec.); TLC Rf,
0.72; GC Rt, 12.62 min; 1H NMR (CDCl3, δ), 3.90 (s, 1H, COOCH3),
6.85 (m, 1H, Hb′ aromatic), 7.15 (m, 1H, Hb aromatic), 7.30 (m, 2H,

Synthesis and Screening of Anilides as CHAs
J. Agric. Food Chem., Vol. 53, No. 15, 2005 5961
Figure 1. General procedure of the synthesis of anilides having olefinic moiety in the side chain.
Ha, Ha′ aromatic), 9.80 (s, 1H, NH); El-MS m/z (rel. int. %), 221 (M+,
0.69), 138(64), 111(100), 110(83), 95(17), 83(4), 77(18), 76(9), 65(22),
64(8).
(m, 2H, Hb, Hb′ aromatic), 7.40 (m, 2H, Ha, Ha′ aromatic), 9.30 (s, 1H,
NH); El-MS m/z (rel. int. %), 203 (M+, 11), 119(44), 93(100), 77(45),
76(29), 65(13), 59(8), 31(16).
3-Methoxycarbonyl 4-Bromo-propiolanilide (5). The title compound
(white crystalline solid) was made as described for the synthesis of
3-methoxycarbonyl propiolanilides (Figure 1) by the condensation of
4-bromo-aniline (0.012 mol) and dimethyl acetylene dicarboxylate
(0.015 mol, 2.13 mL) under an inert atmosphere of nitrogen followed
by crystallization; yield, 1.40 g (41%); mp, 186-188 °C (dec.); TLC
Rf, 0.70; GC Rt, 12.39 min; 1H NMR (CDCl3, δ), 3.90 (s, 1H, COOCH3),
7.20 (m, 1H, Hb′ aromatic), 7.45 (m, 3H, Ha, Ha′,Hb aromatic), 9.80 (s,
1H, NH); El-MS m/z (rel. int. %), 282 (M+, 2), 199(51), 197(46),
172(100), 170(89), 156(25), 154(28), 93(18), 77(25), 65(19), 59(38),
31(15).
Anilides Containing Aryl Moiety as a Side Chain (7-10). In this
series, two analogues viz., methyl phthalanilate (7-9) and O-acetyl
salicylanilide (10), were synthesized.
Methyl 4-Fluoro-phthalanilate (7). A stirred solution of dimethyl
phthalate (0.03 mol, 5.82 mg) and 4-fluoro-aniline (0.025 mol, 2.8 mL)
suspended in dry THF (5 mL) was heated to reflux for 2 h under an
inert atmosphere of nitrogen. The reaction was followed by TLC
[hexane/ethyl acetate (4:1) as a developing medium] until completion.
The resulting suspension was filtered with diethyl ether (70 mL), washed
with water (100 mL), and dried in a desiccator for 1 day. Recrystal-
lization of the crude product with diethyl ether/hexane (1:1) furnished
the title compound in the form of white crystals. The solid was further
crystallized with ethanol to furnish white crystals of methyl 4-fluoro-
phthalanilate (Figure 1) (7); yield, 4.33 g (63%); mp, 182-183 °C;
3-Methoxycarbonyl Propiolanilide (6). Aniline (0.012 mol, 1.38 mL)
and dimethyl acetylene dicarboxylate (0.015 mol, 2.13 mL) were reacted
under stirring at e10 °C to provide the title compound as a white
crystalline solid; yield, 1.05 g (43%); mp, 142 °C (dec.); TLC Rf, 0.74;
1
TLC Rf, 0.42; GC Rt, 14.42 min; H NMR (CDCl3, δ), 2.10 (s, 1H,
1
GC Rt, 12.86 min; H NMR (CDCl3, δ), 3.90 (s, 1H, COOCH3), 7.20
COOCH3), 7.00 (m, 1H, H3 aromatic), 7.20 (m, 1H, H2 aromatic), 7.35

5962 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Chakraborty and Devakumar
(m, 1H, H4 aromatic), 7.55 (m, 1H, H1 aromatic), 7.60 (m, 1H, Hb′
aromatic), 7.80 (m, 3H, Ha, Ha′, Hb aromatic), 9.95 (s, 1H, NH);
El-MS m/z (rel. int. %), 273 (M+, 8), 148(100), 136(51), 120(18),
111(67), 95(76), 78(5), 77(28), 66(12), 59(33).
Methyl 4-Bromo-phthalanilate (8). Reaction of dimethyl phthalate
(0.03 mol, 5.82 mg) in dry THF (5 mL) with 4-bromo-aniline (0.025
mol, 2.8 mL) following Figure 1 furnished white crystals of methyl
4-bromo-phthalanilate (8); yield, 5.59 g (67%); mp, 195 °C; TLC Rf,
0.41; GC Rt, 13.39 min; 1H NMR (CDCl3, δ), 3.80 (s, 1H, COOCH3),
7.20 (m, 4H, phthalide aromatic), 7.60 (m, 1H, anilide aromatic), 9.60
(s, 1H, NH); El-MS m/z (rel. int. %), 334 (M+, 12), 199(62), 197(58),
172(39), 170(35), 157(18), 155(14), 148(100), 136(61), 120(19),
115(27), 77(36), 65(9).
0.42; GC Rt, 9.25 min; 1H NMR (CDCl3, δ), 6.35 (s, 1H, dCH), 6.40
(s, 1H, dCHCO), 7.00 (m, 2H, Hb and Hb′ aromatic), 7.25 (m, 2H, Ha
and Ha′ aromatic), 10.30 (s, 1H, NH); El-MS m/z (rel. int. %), 180
(M+, 11), 139(58), 138(46), 110(100), 91(42), 78(61), 41(28), 15(20).
3-Amino Alkenyl Anilides and Analogues (14-16). In this series,
three analogues viz., 3-(N-acetamino)-4-fluoro-crotonanilide (14), 3-(N-
trityl)amino-4-fluoro-crotonanilide (15), and 3-amino-4-bromo-croton-
anilide (16), were synthesized.
3-(N-Acetamino)-4-fluoro-crotonanilide (14). A mixture of ethyl
3-amino crotonate (0.05 mol, 6.45 g), acetic anhydride (0.05 mol), and
pyridine was kept stoppered for overnight at room temperature to be
followed by the addition of 5 mL of HPLC-grade methanol. The
resulting intermediate (ethyl 2-acetamino crotonate) was recrystallized
from ether/hexane (1:1). A stirred solution of the resulting ethyl
2-acetamino crotonate (0.025 mol, 4.28 g) and 4-fluoro-aniline (0.025
mol, 2.8 mL) suspended in dry xylene (5 mL) was heated to reflux for
3.5 h under an inert atmosphere of nitrogen. The reaction was followed
by TLC [hexane/ethyl acetate (4:1) as a developing medium] until
completion. Chilled hexane (20 mL × 2) was added to the mixture to
remove xylene to obtain a solid, which was recrystallized in diethyl
ether/hexane to furnish a buff solid of the title compound 14 (Figure
2); yield, 3.51 g (59%); mp, 178-179 °C; TLC Rf, 0.32; GC Rt, 11.38
Methyl Phthalanilate (9). To a stirred solution of dimethyl phthalate
(0.03 mol, 5.82 mg) was added aniline (0.025 mol, 2.3 mL) dissolved
in dry THF (5 mL) and heated to reflux for 2 h under an inert
atmosphere of nitrogen following the general procedure to furnish white
crystals of the title compound (9); yield, 3.89 g (61%); mp, 143 °C;
1
TLC Rf, 0.35; GC Rt, 15.58 min; H NMR (CDCl3, δ), 3.30 (s, 1H,
COOCH3), 7.15 (m, 4H, phthalide aromatic), 7.50 (m, 1H, anilide
aromatic), 9.50 (s, 1H, NH); El-MS m/z (rel. int. %), 255 (M+, 9),
148(100), 136(74), 115(63), 93(14), 77(41), 76(29), 65(9), 59(5).
Synthesis of O-Acetyl-4′-fluoro-salicylanilide (10). To a solution of
O-acetyl-methyl salicylate (0.025 mol, 4.87 mL) in dichloromethane
(10 mL) was added 4-fluoro-aniline (0.025 mol, 2.8 mL) suspended in
dichloromethane. The reaction mixture was stirred vigorously at reflux
for 2 h. Then, the mixture was diluted with diethyl ether (40 mL) and
water (50 mL) and washed with water (10 mL). The organic phase
was dried with magnesium sulfate, filtered, and evaporated to yield a
solid compound (Figure 1). The residue was recrystallized with boiling
ethanol to furnish white crystals of the title compound in 79% yield
(5.39 g), which was homogeneous by TLC [hexane/ethyl acetate (4:1)
as a developing medium]; mp, 156-158 °C; TLC Rf, 0.51; GC Rt, 13.25
min; 1H NMR (CDCl3, δ), 2.10 (s, 1H, COCH3), 6.90 (m, 4H, aromatic),
9.27 (s, 1H, NH); El-MS m/z (rel. int. %), 273 (M+, 13), 138(79),
137(22), 134(11), 148(100), 111(59), 78(27), 77(13), 65(9).
1
min; H NMR (CDCl3, δ), 2.30 (s, 3H, CH3), 2.55 (s, 3H, COCH3),
5.00 (s, 1H, dCHCO), 7.00 (m, 2H, Hb, Hb′ aromatic), 7.30 (m, 2H,
Ha, Ha′ aromatic), 10.56 (s, 1H, NH), 10.60 (s, 1H, NH); El-MS m/z
(rel. int. %), 236 (M+, 2), 174(28), 173(69), 159(100), 138(72), 110(56),
86(19), 78(27), 77(13), 58(12), 43(31).
3-(N-Trityl)amino-4-fluoro-crotonanilide (15). To a mixture of ethyl
3-amino crotonate (0.05 mol, 6.45 g) in dry CHCl3 (15 mL),
triethylamine (0.05 mol, 5.5 mL), and trityl chloride (0.01 mol, 2.78
g) were added, and the mixture was allowed to react for 6 h at room
temperature with continuous stirring. The reaction was followed by
TLC [hexane/ethyl acetate (4:1) as a developing medium] until
completion. The product was then washed with water and dried with
Na2SO4. The complete removal of CHCl3 was ensured by adding a
few milliliters of methanol, and reconcentration was done in a vacuum.
The residue was recrystallized from methanol to obtain grayish-white
crystals of the intermediate (ethyl 2-tritylamino crotonate). To a solution
of the resultant solid intermediate (ethyl 2-tritylamino crotonate; 0.025
mol, 9.28 g) was added 4-fluoro-aniline (0.025 mol, 2.8 mL) in xylene,
and the reaction mixture was stirred vigorously at reflux for 4.5 h.
Chilled hexane (20 mL × 2) was added to the mixture to remove xylene.
The excess hexane was evaporated off and the mixture was poured
into ice-cold water followed by recrystallization in ether/hexane (1:1)
to obtain a grayish-white solid of 15 (Figure 2), which was homo-
geneous, by TLC; yield, 3.00 g (28%); mp, 216-218 °C; TLC Rf, 0.48;
GC Rt, 9.57 min; 1H NMR (CDCl3, δ), 2.50 (s, 3H, CH3), 5.20 (s, 1H,
dCHCO), 7.00 (m, 2H, Hb, Hb′ aromatic), 7.50 (m, 2H, Ha, Ha′
aromatic), 10.85 (s, 1H, NH); El-MS m/z (rel. int. %), 437 (M+, 6),
243(19), 174(57), 167(21), 159(100), 158(77), 138(69), 110(85), 78(67),
77(52), 64(21).
(Ar)-Alkenyl Anilides (11-13). In this series, three analogues viz.,
cinnamyl anilate (11), methyl crotonyl anilate (12), and 4-fluoro-methyl
crotonyl anilate (13), were synthesized.
Cinnamyl Anilate (11). To a solution of aniline (0.025 mol) in DMF
(1 mL) was added methyl cinnamate (0.03 mol, 4.86 mL) dissolved in
a mixture of DMF/H2O (2:1.5 mL) under an inert atmosphere of
nitrogen. The resulting mixture was stirred under reflux for 3 h, and
methanol was collected as an azeotrope. The dark colored aliquot
produced was cooled below 90 °C to solidify. The resulting solid was
filtered, washed with water (100 mL), and dried in a desiccator for a
day. Recrystallization of the residue with boiling ethanol furnished the
title compound (Figure 1) in the form of a white crystalline solid (3.24
g, 58% yield), which was homogeneous in TLC [hexane/ethyl acetate
(4:1) as a developing medium]; mp, 112-115 °C; TLC Rf, 0.39; GC
1
Rt, 8.64 min; H NMR (CDCl3-DMSO-d6, δ), 6.10 (s, 1H, dCHPh),
3-Amino-4-bromo-crotonanilide (16). 4-Bromo-aniline (0.045 mol)
and ethyl 3-amino crotonate were reacted together for 4 h following
the general procedure (Figure 2) to furnish a white crystalline solid of
the title compound; yield, 4.00 g (35%); mp, 226 °C; TLC Rf, 0.51;
6.40 (s, 1H, dCHCO), 7.25 (m, 10H, aromatic), 7.60 (m, 5H, aromatic),
10.60 (s, 1H, NH); El-MS m/z (rel. int. %), 223 (M+, 6), 146(8),
131(29), 130(11), 103(18), 102(25), 93(100), 78(62), 77(52), 64(25).
Methyl Crotonyl Anilate (12). To a stirred solution of aniline (0.025
mol) was added methylcrotonate in DMF and heated to reflux for 4 h
following the general procedure (Figure 1) to furnish a solid compound.
The resulting solid was recrystallized from ethanol to furnish a white
crystalline solid of the title compound; yield, 1.93 g (48%); mp, 109
°C; TLC Rf, 0.48; GC Rt, 7.69 min; 1H NMR (CDCl3, δ), 6.35 (s, 1H,
dCH), 6.40 (s, 1H, dCHCO), 6.60 (m, 3H, Hb, Hb′, Hc aromatic), 6.80
(m, 2H, Ha and Ha′ aromatic), 10.60 (s, 1H, NH); El-MS m/z (rel. int.
%), 161 (M+, 17), 145(8), 119(69), 93(100), 91(26), 78(65), 69(36),
41(48), 15(56).
4-Fluoro-methyl Crotonyl Anilate (13). A stirred solution of 4-fluoro-
aniline (0.025 mol, 2.8 mL), in DMF (5 mL) and methylcrotonate (0.03
mol, 3.00 mL) was heated to reflux for 5 h under an inert atmosphere
of nitrogen to furnish a suspension, which was filtered, washed with
water (100 mL), and dried in a desiccator. Recrystallization of the resi-
due with boiling ethanol furnished the title compound in the form of a
white crystalline solid; yield, 2.39 g (53%); mp, 112-115 °C; TLC Rf,
1
GC Rt, 12.06 min; H NMR (CDCl3, δ), 1.50 (s, 3H, CH3), 2.30 (s,
2H, NH2), 4.50 (s, 1H, dCHCO), 7.40 (m, 4H, aromatic), 8.90 (s, 1H,
NH); El-MS m/z (rel. int. %), 255 (M+, 13), 253(11), 235(88), 233(85),
220(100), 218(92), 199(42), 197(39), 171(27), 169(25), 155(72),
119(48), 91(55), 78(61), 77(13), 56(17).
General Method of Synthesis of N-Aryl Phthalimides (17-19).
To a suspension of phthalic anhydride (0.025 mol, 3.7 g) in dry toluene
(4 mL) under inert nitrogen atmosphere was slowly added a solution
of substituted aniline (0.025 mol) under stirring. The resulting mixture
was stirred for 1 h to furnish N-aryl phthalimide as a solid product.
The reaction was followed by TLC [hexane/ethyl acetate (4:1) as a
developing medium] until completion. The resultant solid material was
filtered to furnish the crude product, which was recrystallized with
acetone to give solid products of the title compounds (Figure 1).
N-(4′-Fluoro-phenyl)phthalimide (17). The title compound was made
as described for the synthesis of the N-aryl phthalimides by the

Synthesis and Screening of Anilides as CHAs
J. Agric. Food Chem., Vol. 53, No. 15, 2005 5963
Figure 2. Synthesis of 3-amino alkenyl anilides and analogues.
condensation of 4-fluoro-aniline (0.025 mol, 2.8 mL) and phthalic
anhydride (0.025 mol, 3.7 g) followed by recrystallization from acetone
to yield a grayish amorphous solid of the title compound 17; yield,
1
5.59 g (92%); mp, 139-140 °C; TLC Rf, 0.63; GC Rt, 11.22 min; H
NMR (CDCl3, δ), 7.10 (m, 2H, Hb, Hb′ aromatic), 7.35 (m, 2H, Ha,
H′a aromatic), 7.80 (m, 4H aromatic protons in phthalimide ring);
El-MS m/z (rel. int. %), 241(M+, 21), 147(100), 138(73), 120(35),
111(39), 97(64), 91(76), 78(22), 66(35).
N-(4′-Bromo-phenyl)phthalimide (18). To a stirred suspension of
4-bromo-aniline (0.025 mol, 4.28 mL) was made to react with phthalic
anhydride following the general procedure to yield a grayish amorphous
solid; yield, 6.48 g (86%); mp, 168 °C; TLC Rf, 0.60; GC Rt, 10.38
min; 1H NMR (CDCl3, δ), 7.35 (m, 4H, aromatic), 7.75 (m, 4H aromatic
protons in phthalimide ring); El-MS m/z (rel. int. %), 302 (M+, 13),
301(11), 147(100), 199(58), 197(55), 171(29), 169(27), 119(15), 97(31),
91(16), 78(48), 66(25).
N-Phenylphthalimide (19). A stirred suspension of phthalic anhydride
(0.025 mol, 3.7 g) and aniline (0.025 mol, 2.3 mL) in dry toluene (4
mL) was stirred under an inert atmosphere of nitrogen to furnish
N-phenylphthalimide as a solid product of the title compound; yield,
4.58 g (82%); mp, 105 °C; TLC Rf, 0.53; GC Rt, 12.18 min; 1H NMR
(CDCl3, δ), 7.20 (m, 2H, Hc, Hb′ aromatic), 7.40 (m, 3H, Ha, H′a, Hb
aromatic), 7.80 (m, 4H aromatic protons in phthalimide ring); El-MS
m/z (rel. int. %), 224 (M+, 16), 147(100), 119(32), 97(46), 91(68),
77(32).
Figure 3. Synthesis of N-alkylaniline analogues.
acrylonitrile (0.03 mol, 1.3 mL) with iodine as a catalyst. The reaction
mixture was stirred vigorously at reflux for 9 h. The reaction mixture
was cooled, and the residue was recrystallized with boiling ethanol to
give grayish white flakes of the title compound, which was homo-
geneous by TLC [hexane/ethyl acetate (4:1) as a developing medium];
yield, 1.16 g (28%); mp, 132-140 °C; TLC Rf, 0.29; GC Rt, 12.38
min; 1H NMR (CDCl3-DMSO-d6, δ), 2.90 (m, 4H, NHCH2 and
CH2CN), 7.20 (m, 2H, Hb, Hb′ aromatic), 7.55 (m, 2H, Ha, Ha′ aromatic),
8.20 (s, 1H, NH); El-MS m/z (rel. int. %), 164 (M+, 15), 124(39),
111(100), 96(55), 91(67), 77(84), 76(62), 54(20), 52(13), 40(11).
Field Evaluation of CHAs. Three high-yielding varieties of bread
wheat (Triticum aestiVum L.) viz., PBW 343, HW 2046, and HD 2733,
recommended for timely sowing were chosen for evaluation of chemical
induction of male sterility. The experiment was laid out following
randomized block design with three replicates. Seeds of the genotypes
(pollen and female parents) of the wheat varieties were sown at a
100-kg/ha seed rate in November 2001 under drilling at Indian
Agricultural Research Institute (IARI), New Delhi. Other optimum
agronomic practices were also followed, which included recommended
fertilizer schedule, timely weeding, and other cultural operations. Five
irrigations were given at different stages of crop growth viz., crown
root initiation (CRI), late tillering, late jointing, flowering, and dough
N-Alkylanilines (20-21). In this series, two analogues viz., ethyl-
(4-fluoroanilino)acetate (20) and 4-fluoro-N-â-cyanoethyl-aniline (21),
were synthesized.
Ethyl-(4-fluoroanilino)acetate (20). To a stirred solution of ethyl
bromo-acetate (0.03 mol, 5.01 mL), 4-fluoro-aniline (0.025 mol, 2.8
mL), and triethylamine (0.03 mol, 3.03 mL) in dichloromethane (12
mL) was heated to reflux for 45 min at 100 °C (Figure 3). The product
was cooled to 70 °C to solidify, recrystallized with boiling ethanol,
and refrigerated to allow recrystallization of the title compound as a
white crystalline solid, which was homogeneous by TLC [hexane/ethyl
acetate (4:1) as a developing medium]; yield, 3.87 g (78%); mp,
92-93 °C; TLC Rf, 0.54; GC Rt, 7.18 min; 1H NMR (CDCl3, δ), 1.20
(t, J ) 6 Hz, 3H, CH3), 1.75 (s, 2H, CH2CO), 4.20 (q, J ) 6 Hz, 2H,
OCH2), 6.70 (m, 3H, Ha′, Hb, Hb′ aromatic), 7.00 (m, 1H, Ha aromatic),
9.65 (s, 1H, NH); El-MS m/z (rel. int. %), 197 (M+, 4), 124(18),
111(100), 91(25), 87(31), 78(64), 73(15), 66(49), 45(38).
4-Fluoro-N-â-cyanoethyl-aniline (21). To a solution of 4-fluoroa-
niline (0.025 mol, 2.8 mL) in DMSO (10 mL) was added excess

5964 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Chakraborty and Devakumar
stage. Row-row distance was kept at 30 cm. Five rows of 2 m in
length were taken as a plot in which the outermost two rows were
treated as the pollen parents and the inner three rows as the female
parents. The incoming foreign pollens were prevented to enter the plot
by planting two border rows of oat (AVena satiVa L.) on the
surroundings of the experimental plot. An emulsifiable concentrate (5
EC) of test CHAs (1.5 g) in cyclohexanone (7 mL) using polyoxyethyl-
ene sorbitan monooleate (FW ∼ 1200) (Tween-80) (1 g), as an
emulsifier, was prepared after trial and error with different solvents.
To ensure phytosafety of cyclohexanone, a blank solvent emulsion
without CHAs was sprayed on the crop well in advance. No serious
phytotoxic symptoms were visible 2-3 days after the spray. The
synthetic compounds were sprayed at premeiotic stage in the winter of
2001-2002 at a concentration of 1500 ppm when the length of the
spike emerging out from the first node was about 6-9 mm in length
(60 days after sowing (14). While spraying, utmost precaution was taken
not to spill the chemicals on the tillers of the pollinator lines. Crossing
of the female parents was done by generous dusting of the pollens
from HW 2045. To ensure cross pollination, pollens from the male
parent were also manually dusted on the sterilized female spike and
bagging was done immediately to prevent any further cross pollination
by any undesirable foreign pollens. Because the degree of synchrony
of flowering varied with the variety, care was taken to tag the treated
tillers of an appropriate stage. A total of 10 spikes of each treated plants
were covered immediately after emergence. Remaining ear heads were
left uncovered. The efficacy of the test chemicals was studied using
pollen sterility and seed set under bagged (male sterility) conditions.
Anthers from three to four florets were smeared together over a drop
of acetocarmine (1%) or KI (2%) in iodine and examined under a light
microscope. Pollen sterility was calculated in percentages. A total of
10 of each bagged and unbagged spikes from each treatment including
one control were harvested at maturity. Male sterility was calculated
as the percent inhibition of seed set in bagged spikes of treated plants.
Data were recorded on pollen sterility and spikelet sterility under
bagged/unbagged conditions as detailed elsewhere (10, 11).
autocorrelation using the SPSS program (version 10.0). None of the
independent variables appearing in the equations was ensured to be
orthogonal.
RESULTS AND DISCUSSION
Syntheses and Spectral Analyses. The synthesis of different
series of CHAs belonging to anilides having olefinic and alkyl
moiety in the side chain involves condensation of anilines with
esters, acid chlorides, anhydrides, and nitrile as detailed under
the earlier section. The substituent fluoro at the para position
in furyl anilide caused deshielding of the Ha and H′ aromatic
a
protons by about 0.90 and 0.35 ppm, respectively. The effect
of bromine on the deshielding of the same is comparatively less
being 0.60 ppm for both Ha and H′ as compared to that of the
a
unsubstituted furyl anilide (δ 7.20 ppm) (Figure 1). It was
observed that there was an overall deshielding effect of halogen
atoms on the chemical shifts of furyl protons (Haf, Hbf, and Hcf).
The R-olefinic proton in cinnamyl anilate appeared as a singlet
at δ 5.56 ( 0.84 ppm, with the highest deshielding effect being
experienced both by the olefinic proton geminal to the methyl
or phenyl group in methyl crotonyl and cinnamyl anilides (δ )
6.40 ppm). In N-aryl phthalimide, the anilido protons experi-
enced a marked variation in the chemical shift because of their
proximity with the halogen substituents. In the 4′-fluoro
analogue, the Ha and Hb protons were shielded by 0.05 and 0.30
ppm, respectively, perhaps because of the predominant +M
effect of the fluorine atom. The NH proton in N-â-cyanoethyl
aniline appeared upfield at δ 4.40 ppm in contrast to the same
in ethyl-4-(fluoroanilino)acetate, which appeared significantly
downfield at δ 9.65 ppm. The methylene protons (CH2CO) in
ethyl-(4-fluoroanilino)acetate appeared at δ 1.75 ppm as a sharp
singlet. In 4-fluoro-N-â-cyanoethylaniline, the CH2CN protons
appeared upfield at about δ 2.90 ppm.
In the mass fragmentation pattern of furyl anilides, the base
peak was found to be the furyl acylium moiety (m/z 95). The
parent aniline was assigned to be the base peak in the mass
fragmentation of 3-methoxycarbonyl propiolanilide. Methyl
phthalanilates gave the mass fragmentation pattern similar to
O-acetyl-(4-fluoro)salicylanilde, and the base peak was assigned
to be the indane dione moiety. The spectra further contained
lower m/z fragment viz., m/z 111, 78, 77, 65, etc., characteristic
of the aromatic skeleton. In the mass fragmentation pattern of
3-amino alkenyl anilides and analogues, fission of ethoxycar-
bonyl moiety gave rise to the protonated aryl isocyanate moiety.
The mass fragment at m/z 159 (4′-fluoro-phenyl-pyridone
moiety) was assigned to be the base peak in this series. In N-aryl
phthalimides, phthalimide (m/z 147) was assigned to be the base
peak.
Effect of CHAs on Spikelet Sterility. As may be seen from
Table 1, the maximum male sterility was induced by 4-fluoro-
furyl anilide (1) followed by 4-bromo-furyl anilide (2) on three
genotypes of wheat. In HD 2733, the trend of activity was the
same as in the other two genotypes, but the magnitude of the
effect was found to be more pronounced. Incorporation of the
acetylinic bridge in the amide side chain did not improve the
activity as compared to the olefinic double bond. Among aryl-
substituted propiolanilides, 4-fluoro propiolanilide (4) induced
greater male sterility than the other analogues (i.e., 4-bromo
and unsubstituted), with the trend of activity being 4F (4) >
4Br (5) . H (6). It was observed that 4-fluoro-phthalanilate
(7) induced greater activity as compared to O-acetyl-4-fluoro-
salicylanilide (10). A series of compounds of N-acylanilines with
an (ar)-alkenyl side chain were prepared among which cinnamyl
analogues (12 and 13) were found to be more active than the
Analysis of variance (ANOVA) of factorial randomized block design
(RBD) was performed with all treatments. On the basis of the
significance of the treatments, critical differences (CDs) at a 5% level
of significance (p ) 0.05) were computed.
QSAR Study. 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 a CHA mode of
action. The QSAR method applied to two families of CHAs in the
anilides containing olefinic moiety in the side chain and N-alkyl aniline
class of chemistry. To observe the variability in the olefinic and alkyl
side chain, the physicochemical descriptors, namely, πa, molecular
weight (MWa), molar refractivity (MRa), parachor (P), and polarizability
(Pl), were generated using ACD Chemsketch (version 2) software. The
following descriptive variables were used for aromatic substituents viz.,
electronic parameters: Swain-Lupton field constant or inductive
parameter (F) (15), Hammett constants (σm and σp) to define electron-
donating and -withdrawing tendencies, partial atomic charges and
electrostatic field densities, and Swain-Lupton resonance constant R
(16); steric parameters: Taft steric parameter (Es), molecular weight
(MWb), and Verloop-Hoogenstraaten multidimensional steric param-
eters, L and B4 (17, 18); hydrophobic parameter: πb; and other param-
eters such as molar refractivity (MRb), and index variable (D).
A total of 15 independent variables were used in constructing the
correlation matrix. The independent variables, which were found
orthogonal to each other in the correlation matrix, were minimized.
The “agrophore” data viz., percent induction of spikelet sterility caused
by test CHAs tested at 1500 ppm, were transferred into sin arc and
used as the dependent variable (PS % sin arc). The descriptor variables
were used to generate multiple linear regression equations (MLR) by

Synthesis and Screening of Anilides as CHAs
J. Agric. Food Chem., Vol. 53, No. 15, 2005 5965
Table 1. Effect of Side-Chain Variation in Anilides on the Percent Induction of Spikelet Sterility at 1500-ppm Test Concentrations on Three
Genotypes of Wheat on Winter 2001 2002
−
% spikelet sterility
PBW 343
compound number
X
R
HW 2046
HD 2733
1
2
3
4
5
6
7
8
4-F
4-Br
H
4-F
4-Br
H
4-F
4-Br
H
4-F
H
COC4H3O
COC4H3O
COC4H3O
82.81
82.45
54.53
67.47
65.42
49.72
70.87
65.01
26.17
40.48
55.97
48.51
52.99
39.41
23.66
21.29
74.21
69.38
51.50
41.84
59.29
0.46
79.68
79.46
50.82
66.79
64.58
48.65
69.99
63.32
22.67
35.16
51.37
45.95
50.16
34.70
20.48
18.07
71.89
65.82
48.09
41.43
56.69
0.33
83.48
83.22
55.66
68.54
66.76
52.61
73.64
67.50
27.78
42.28
57.69
51.17
55.07
39.59
24.42
22.31
74.83
71.87
52.73
47.33
61.23
0.49
COC
COC
COC
tCCOOMe
tCCOOMe
tCCOOMe
COC6H4COOMe (ortho)
COC6H4COOMe (ortho)
COC6H4COOMe (ortho)
COC6H4OCOCH3 (ortho)
9
10
11
12
13
14
15
16
17
18
19
20
21
COCH
COCH
COCH
COCH
COCH
COCH
d
d
d
d
d
d
CHPh
CHMe
CHMe
C(Me)NHAc
C(Me)NHTri
C(Me)NH2
H
4-F
4-F
4-F
4-Br
4-F
4-Br
H
CONCOC6H4CO
CONCOC6H4CO
CONCOC6H4CO
NHCH2COOEt
NHCH2CH2CN
4-F
4-F
emulsion control
CD (p 0.05)
)
1.02
2.21
0.95
crotonate analogue (11). Even the unsubstituted cinnamyl anilate
induced a higher (55.97) percent of male sterility in PBW 343
than crotonyl anilate, which induced 52.99%. Also, function-
alization of the third carbon position in a series of 3-amino
alkenyl anilides and analogues did not improve their perfor-
mance as CHAs (male sterility < 40%). Among 3-(N-trityl-
amino)-4-fluoro-crotonanilide (15) and 3-(N-acetamino)croton-
anilide (14), the later had shown comparatively higher activity
than the former. Among the N-aryl phthalimides, N-(4-fluo-
rophenyl)phthalimides (17) had shown greater induction of male
sterility on three genotypes of wheat (Table 1). Among
N-alkylanilines, 4-fluoro-N-â-cyanoethyl aniline (21) exhibited
comparatively higher activity than ethyl-(4-fluoro-anilino)acetate
(20) (Table 1). It is significant that the CHAs belonging to furyl
anilides not only induced a very high degree of male sterility
but also modify the reproductive biology in such a fashion to
ensure cross pollination in the cleistogamous wheat flowers and
increasing the probability for the development of hybrids. Wheat
is a self-pollinated crop having a closed floret. Florets of male
sterile wheat opened twice to facilitate the cross fertilization.
First floret opening with the action of lodicules lasted only for
a shorter period. Second floret opening started after the lodicules
have collapsed, and the carpels in the sterile floret continued to
grow palea and lemma apart. This second opening lasted for
more periods (5-6 days) and was normally sufficient for cross
pollination to take place by the pollen source. The extent of
cross pollination without cutting of palea and lemma is indicative
of natural floret opening, stigma receptivity, and out-crossing
percentage. To ensure cross pollination, there is no need to cut
the palea and lemma, because second floret opening is sufficient
for cross pollination to occur. Further, peak period for stigma
receptivity lasted for about 3-4 days, which helped in planning
for hybrid seed production. The CHA technology was optimized
in terms of variation in genotype, choice of CHA, stage of spray,
number of spray, type of formulation, and dose. The premeiotic
stage (6-8 mm of spike length) was found to be very ideal.
The single spray was adequate and safe. Oil in water emulsion
was used. Cyclohexanone was found to be the best among the
solvents tested. As far as the aspect of toxicity of the CHAs is
concerned, no residue of CHA has been noticed in the F2 seeds
of wheat, and thus, it has no outreach to the consumer in the
form of crop produce.
From the stain test, it was seen that the sterile grains obtained
from the treatment were transparent, thereby confirming the
disintegration of cytoplasm and nucleus in the sterile pollen. In
contrast, fertile pollen from control plots stained uniform deep
red or blue color depending on the stain, confirming the
induction of male sterility in various treatments. In some cases,
the sterile pollen grains became shriveled and the mass of
cytoplasm and nucleus contracted in the pollen keeping the
external pollen wall (exine) intact.
QSAR Study. Results of the multiple regression analysis
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%.
In this study, the acyl side chain has been varied with
various chemical groups such as C4H3O, -CCOOMe,
-C(Cl)2CH3, -C6H4COOCH3, -C6H4OCOCH3, -CHdCHPh,
-CHdCHCH3, -CHdC(CH3)NHAc, -CHdC(CH3)NHTri,
and -CHdC(NH2)CH3 with concurrent variation in the aromatic
substitution mostly by F, Br, or unsubstituted as well. Replacing
the alcohol moiety from furoyl (-C4H3O) (1-3) to methoxy-
carbonyl propiolanilide (CtCCOOCH3) (4-6) led to a decrease
in the target activity. Inspired by the enhancing effect of the
electronegative groups (F and Br) in our earlier studies (10,
11), O-acetyl-(4′-fluoro)salicylanilide (10) and methyl phthal-
anilides (7-9) containing different substituents in the aromatic
ring (4′-F, 4′-Br, and unsubstituted) were synthesized. While

5966 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Chakraborty and Devakumar
methyl 4′-fluoro-phthalanilide (7) induced 70.87% male sterility,
O-acetyl-(4′-fluoro)salicylanilide (10) induced remarkably low
male sterility, i.e., 40.48%. It therefore appears that methoxy-
carbonyl rather than the acetoxy group in the ortho position of
the side-chain aromatic ring had an activity-enhancing effect.
Functionalization of the ethoxycarbonyl group in the side chain
by way of cinnamyl and crotonyl groups has caused a severe
reduction in the male sterility percentage. Functionalization of
the amino group in 16 with acetyl (-NHAc) and trityl (-NHTri)
groups as in 14 and 15 led to the enhancement of activity as
compared to that of the parent compound (16).
QSAR analysis of the 21 analogues thus generated was carried
out by using a combination of chemical descriptors both for
aromatic substitution and side-chain variation, and the correla-
tion matrix was constructed. The models for each CHA family
gave a good correlation between the variations in log percentages
of spikelet sterility and the steric-electrostatic properties of the
sets. In the anilides synthesized, the observed bioactivity could
be collectively explained in the form of two multiple linear
regression (MLR) equations using a total of 15 independent
variables:
Figure 4. Sketch model of closed vis-a`-vis open vinylogous double bond
of anilides.
binding at the bioreceptor site, a negative MR term in the best
equation could mean unfavorable conformational changes in the
enzyme-inhibitor complex as compared to favorable confor-
mational changes caused by the enzyme-substrate complex
(19). It is known that, for representing bulk rather than
directional effects, molar refractivity would explain better than
Taft’s steric constant and sterimol parameters. The negative sign
of the coefficient in MR indicated that less hydrophobicity of
the olefinic or alkyl moiety would enhance the target activity.
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 or energetic metabolism
disruption (uncoupling effect on stamen mitochondria). The
observed effects were the result of leaf transcuticular uptake
and possible phloem transport. The compounds in the anilide
series having olefinic moiety in the side chain are esters or
substituted amides, which very likely are immediately trans-
formed in the leaves to give the free acids. All of the free acids
studied here 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. These products can therefore be suspected to
concentrate inside the stamen through that way. Moreover, the
acidic pH generated by the hydrolysis of anilides lead to an
imbalance of acid-base equilibria in the pollen mother cells
resulting in dissolution of microsporocyte callose wall during
the meiotic I prophase by premature callase (1,3-â glucanase)
secretion. The high polarity of the acids (viz., furoic acid
obtained from the hydrolysis of 4-fluoro-furyl anilide) results
in easy water solubility and phloem transport, which add to the
efficiency of the active CHAs. Therefore, chemicals with high
water solubility would be preferred as active CHAs. The
equation essentially highlights the negative effect of the steric
parameter viz., parachor of the side chain containing the
vinylogous double bonds. Interestingly, in eq 1, the sign of the
coefficient of the steric variable is reversed unlike in N-
acylaniline derivatives (11). It implies that acyl linkage in the
side chain is directly proportional to their bulk, unlike in open
vinylogous side chains where the unsaturation hinders the proper
fitting in the macromolecular receptor site (Figure 4). There is
ample scope for the development of potent CHA analogues
based on the leads postulated and predicted in the present
studies.
spikelet sterility (sin arc %) ) 0.32Fp - 2.19P + 99.52 (1)
where N ) 21, r ) 0.73, s ) 15.63, and F ) 6.480
spikelet sterility (sin arc %) )
34.71F - 8.44 MR - 5.27P + 99.517 (2)
∑
p
b
where N ) 21, r ) 0.92, s ) 6.14, and F ) 28.63.
The best equation (eq 2) was the one that combined the
independent variables Fp for aromatic substitution and parachor
(P) and ∑MRb for the side chain with r values of 0.92. The
direct involvement of Swain-Lupton field constant for para
substitution (Fp) with the target bioactivity in the best equation
implied that inductive (field or polar) rather than resonance
effect (R) of the substituent is the key factor influencing the
induction of male sterility. It can be inferred that para
substitution with highly electronegative groups such as F or 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. Swain and Lupton studied the inverse correlation
of Fp and R (19) in the form of the equation viz., σp ) RF +
R, where σp is the Hammett constant for para substitution and
R is a constant. Parachor in the olefinic side chain appeared to
influence the bioactivity in a significant manner and was found
to be the most dominant descriptor, which could guide us to
devise better CHAs. It is apparent from the study that the closed
ring structure in the side chain as in furyl analogues has a
comparatively lesser surface area resulting in a lower value of
parachor than 3-amino alkenyl anilides and (ar)-alkenyl anilides,
having an open olefinic side chain of comparatively more
surface area. It is hypothesized that the extended olefinic
structure at the acyl side chain has a less efficient macro-
molecular receptor-fit at the enzyme active site than the closed
ring structure viz., furyl anilides. It has generally been assumed
that a positive coefficient with a molar refractivity (MR) term
in a correlation equation suggests a binding action via dispersion
forces. Such binding could produce a concomitant conformation
change in a macromolecular binding site. If the conformational
change favored the process under study, one would certainly
expect a positive coefficient with the detrimental; a negative
coefficient could result from the MR term. In competitive
Two distinct types of histological changes (type 1 and type
2) have been established in the induction of male sterility (20).
In type 1, callose synthesis is inhibited, and because of
nonsecretion of callose, the sporogenous cells did not undergo
meiosis to form tetrads and microspores, leading to the absence

Synthesis and Screening of Anilides as CHAs
J. Agric. Food Chem., Vol. 53, No. 15, 2005 5967
Figure 6. Comparison between UDP-glucose and furyl anilides with the
binding of different regions bound to UDP-glucose pyrophosphorylase thus
inhibiting the enzyme.
Table 2. Performance of Fluoroaryl Substituted Anilides for Important
Traits of Wheat (PBW 343) Tested in Winter 2001−2002 at 1500-ppm
Test Concentration
Figure 5. Sterile pollens of wheat because of the treatment of CHAs
vis-a`-vis fertile pollens as observed in KI−I2 stain test.
compound
number
female
1000-grain
spike
fertility (%)
weight (g)
length (cm)
1
4
7
10
13
14
15
17
20
21
92.60
89.15
47.70
58.54
79.07
52.80
45.26
92.11
93.01
94.29
99.88
1.39
37.20
34.72
25.15
33.29
33.41
28.32
26.01
36.97
37.25
38.78
39.82
0.86
10.42
8.46
7.53
8.10
9.04
of a pollen grain. In type 2, alterations in the timing of callase
(endo-â-1,3-glucanase) expression leads to abnormal dissolution
of the tetrad callose walls, which has been shown to be a primary
cause of male sterility in cytoplasmic male sterile lines of petunia
(21) and sorghum (22). In light of the importance of callose
deposition vis-a`-vis callase activity it is likely that the CHA
may either block the synthesis of callose inducing type 1 action
or accelerate the premature release of callase causing type 2
disruption. The absence of any starch material in the sterile
pollen grains as shown by the KI-I2 pollen stain test could be
indicative of either of the processes leading to starch depletion
or the blocking of its synthesis (Figure 5). The first step in the
synthesis of cell-wall polysaccharides is catalyzed by UDP-
glucose pyrophosphorylase (EC 2.7.7.9; monomer of an ap-
proximately 55-kDa polypeptide), and this synthesis is inhibited
by the substrates such as UDP-glucose (22) and the most active
CHA in the study. 4-Fluoro-furyl anilide (2) of the present
invention has a dissociation constant for the reversible associa-
tion with the enzyme, KI, of 1.3 ( 0.1 µM. Because 4-fluoro-
furyl anilide has a very low KI value, it is an efficient inhibitor
of UDP-glucose pyrophosphorylase. A comparison of CHAs
vis-a`-vis UDP-glucose is depicted with the labeling of different
regions in Figure 6. The CHAs containing the heterocyclic furyl
ring and N-aryl moiety can possibly mimic UDP-glucose that
also contains similar groups viz., uridinyl group, and isosteric
glucose moiety. Incidentally, the molar volumes of bromophenyl
(105.6 cm3) and glucosyl moieties (106.2 cm3) are almost
similar. Only the field effect (Fp) rather than the resonance effect
of aromatic substitution can give an appropriate isosteric effect
with the glucosyl group as predicted by QSAR analysis.
Besides high and selective induction of male sterility, the most
potent CHAs had neither shown any adverse effects on growth
parameters such as plant height and germination percentage nor
on yield parameters viz., spike length, female fertility, and
thousand-grain weight, and thus have a great potential as CHAs
for wheat. It was found that anilides with closed vinyl double
bond viz., furyl anilides and N-alkyl aniline analogues, are most
selective CHAs for wheat (Table 2). The real guiding principle
for selectivity was found to be the π value. For the less selective
3-(N-trityl)amino-4-fluoro-crotonanilide (15), and 3-(N-acetamino)-
4-fluoro-crotonanilide (14) analogues, the π values are negative
7.81
7.26
10.38
10.59
10.85
10.99
1.42
emulsion control
CD (p 0.05)
)
(π ) -0.69 and -0.48, respectively) for the olefinic side chain
as compared to the positive value in the more selective furyl
analogues [π (furyl) ) 0.31]. Therefore, it can be concluded
that more hydrophobicity in the olefinic side chain of the
molecule lead to higher selectivity. The study concludes how
an array of compounds can be brought down to a narrow range
of active compounds using QSAR analyses. The leads derived
from the present study can be valuable in exploring the primary
site and mode of action of these CHAs.
ACKNOWLEDGMENT
The authors are thankful to the Director, IARI, New Delhi for
providing necessary facilities to carry out the work. The senior
author gratefully acknowledges the ICAR award of Senior
Research Fellowship. Thanks are also due to Dr. S. M. S. Tomar,
Principal Scientist, Division of Genetics, IARI, New Delhi for
useful discussion and help in the execution of the study.
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